Production and use of siglec-15 binding proteins

Abstract

Preparation of a siglec-15 binding protein and uses thereof, in particular to an isolated antigen binding protein comprising a HCDR3. Applications of the antigen binding protein in the prevention and treatment of diseases are provided.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to the preparation and use of a Siglec-15 binding protein. Background Technology

[0002] Siglec is a classic class of immunoglobulin-like lectins that plays a regulatory role in immune homeostasis. The sialic acid-binding immunoglobulin (Siglec) lectin family comprises over 30 members, located on the cell surface. Abnormalities in several molecules within this family are associated with numerous diseases, and Siglec-15 is one such member. The intracellular region of Siglec-15 lacks immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or ITIM-like sequences. Experimental data indicate that intracellular Siglec-15 is associated with DAP12 or DAP10, both of which contain immunoreceptor tyrosine-based activating motifs (ITAMs) signaling. However, most Siglec intracellular domains contain immunoreceptor tyrosine inhibitory motifs, thereby exerting immunosuppressive functions. Existing data indicate that Siglec-15 can bind to sialyl-Tn and Neu5Aca2–6GalNAca, suggesting they can be ligands for Siglec-15. In normal humans, Siglec-15 is primarily expressed in myeloid cells. Siglec-15 exhibits abnormally high expression in various solid tumors, and preclinical in vitro experiments and animal tumor models have demonstrated that Siglec-15 antibodies can activate T cells and inhibit tumors. However, suitable Siglec-15 antibodies for cancer therapy are currently lacking, and there is an urgent need in this field to develop more therapeutically effective Siglec-15 binding proteins. Summary of the Invention

[0003] This application provides a Siglec-15 binding protein. The Siglec-15 binding protein of this application may have one or more effects selected from the group consisting of: (1) having a Kc of about 1.56E-10M or lower. D (1) The ability to bind Siglec-15 or its functional fragments; (2) Having an EC value of about 3.25 nM or lower. 50(3) It has good physicochemical properties, such as thermal stability; (4) It has the ability to affect the proliferation of immune cells, such as reversing the inhibition of immune cell proliferation caused by Siglec-15; (5) It has the ability to inhibit tumors, such as inhibiting tumors with a tumor volume inhibition rate of about 23% or higher; (6) It has the ability to affect the secretion of cytokines.

[0004] In one aspect, this application provides an isolated antigen-binding protein comprising HCDR3, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.

[0005] In some embodiments, the isolated antigen-binding protein comprises HCDR2, which contains the amino acid sequence shown in SEQ ID NO:33.

[0006] In some embodiments, the isolated antigen-binding protein comprises HCDR2, which comprises the amino acid sequence shown 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 HCDR1, which contains the amino acid sequence shown in SEQ ID NO:34.

[0008] In some embodiments, the isolated antigen-binding protein comprises HCDR1, which contains the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8.

[0009] In some embodiments, the isolated antigen-binding protein comprises LCDR3, which contains the amino acid sequence shown in SEQ ID NO:9.

[0010] In some embodiments, the isolated antigen-binding protein comprises LCDR2, which contains the amino acid sequence shown in SEQ ID NO:35.

[0011] In some embodiments, the isolated antigen-binding protein comprises LCDR2, which contains the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0012] In some embodiments, the isolated antigen-binding protein comprises LCDR1, which contains the amino acid sequence shown in SEQ ID NO:36.

[0013] In some embodiments, the isolated antigen-binding protein comprises LCDR1, which contains the amino acid sequence shown 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 which is directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:15.

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

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

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

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

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

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

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

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

[0023] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown 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 shown in SEQ ID NO:7 or SEQ ID NO:8.

[0024] In some embodiments, isolated antigen-binding proteins, wherein LCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR2 contains the amino acid sequence shown in SEQ ID NO:35, and LCDR1 contains the amino acid sequence shown in SEQ ID NO:36.

[0025] In some embodiments, the isolated antigen-binding protein, wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:9, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and LCDR1 comprises the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:14.

[0026] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:33, HCDR1 contains the amino acid sequence shown in SEQ ID NO:34, LCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR2 contains the amino acid sequence shown in SEQ ID NO:35, and LCDR1 contains the amino acid sequence shown in SEQ ID NO:36.

[0027] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown 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 shown in SEQ ID NO:7 or SEQ ID NO:8, LCDR3 comprises the amino acid sequence shown in SEQ ID NO:9, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and LCDR1 comprises the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:14.

[0028] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:2, HCDR1 contains the amino acid sequence shown in SEQ ID NO:7, LCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR2 contains the amino acid sequence shown in SEQ ID NO:10, and LCDR1 contains the amino acid sequence shown in SEQ ID NO:13.

[0029] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:2, HCDR1 contains the amino acid sequence shown in SEQ ID NO:8, LCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR2 contains the amino acid sequence shown in SEQ ID NO:10, and LCDR1 contains the amino acid sequence shown in SEQ ID NO:13.

[0030] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:3, HCDR1 contains the amino acid sequence shown in SEQ ID NO:7, LCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR2 contains the amino acid sequence shown in SEQ ID NO:10, and LCDR1 contains the amino acid sequence shown in SEQ ID NO:13.

[0031] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:4, HCDR1 contains the amino acid sequence shown in SEQ ID NO:7, LCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR2 contains the amino acid sequence shown in SEQ ID NO:10, and LCDR1 contains the amino acid sequence shown in SEQ ID NO:13.

[0032] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:5, HCDR1 contains the amino acid sequence shown in SEQ ID NO:7, LCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR2 contains the amino acid sequence shown in SEQ ID NO:10, and LCDR1 contains the amino acid sequence shown in SEQ ID NO:13.

[0033] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:6, HCDR1 contains the amino acid sequence shown in SEQ ID NO:7, LCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR2 contains the amino acid sequence shown in SEQ ID NO:10, and LCDR1 contains the amino acid sequence shown in SEQ ID NO:13.

[0034] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:2, HCDR1 contains the amino acid sequence shown in SEQ ID NO:7, LCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR2 contains the amino acid sequence shown in SEQ ID NO:10, and LCDR1 contains the amino acid sequence shown in SEQ ID NO:14.

[0035] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:3, HCDR1 contains the amino acid sequence shown in SEQ ID NO:7, LCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR2 contains the amino acid sequence shown in SEQ ID NO:11, and LCDR1 contains the amino acid sequence shown in SEQ ID NO:13.

[0036] In some embodiments, the isolated antigen-binding protein, wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:3, HCDR1 contains the amino acid sequence shown in SEQ ID NO:7, LCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR2 contains the amino acid sequence shown in SEQ ID NO:12, and LCDR1 contains the amino acid sequence shown in SEQ ID NO:13.

[0037] In some embodiments, the isolated antigen-binding protein includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO:37.

[0038] In some embodiments, the isolated antigen-binding protein comprises VH, which contains the amino acid sequence shown 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 includes a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:38.

[0040] In some embodiments, the isolated antigen-binding protein comprises a VL containing the amino acid sequence shown 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 VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:37 and the VL comprises the amino acid sequence shown in SEQ ID NO:38.

[0042] In some embodiments, the isolated antigen-binding protein comprises VH and VL, wherein the VH comprises the amino acid sequence shown 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 the VL comprises the amino acid sequence shown 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 VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:23 and the VL comprises the amino acid sequence shown in SEQ ID NO:29.

[0044] In some embodiments, the isolated antigen-binding protein comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:24, and the VL comprises the amino acid sequence shown in SEQ ID NO:29.

[0045] In some embodiments, the isolated antigen-binding protein comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:25 and the VL comprises the amino acid sequence shown in SEQ ID NO:29.

[0046] In some embodiments, the isolated antigen-binding protein comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:26, and the VL comprises the amino acid sequence shown in SEQ ID NO:29.

[0047] In some embodiments, the isolated antigen-binding protein comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:27 and the VL comprises the amino acid sequence shown in SEQ ID NO:29.

[0048] In some embodiments, the isolated antigen-binding protein comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:28, and the VL comprises the amino acid sequence shown in SEQ ID NO:29.

[0049] In some embodiments, the isolated antigen-binding protein comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:23 and the VL comprises the amino acid sequence shown in SEQ ID NO:30.

[0050] In some embodiments, the isolated antigen-binding protein comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:25 and the VL comprises the amino acid sequence shown in SEQ ID NO:31.

[0051] In some embodiments, the isolated antigen-binding protein comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:25 and the VL comprises the amino acid sequence shown in SEQ ID NO:32.

[0052] In some embodiments, the isolated antigen-binding protein contains a constant region of the antibody heavy chain.

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

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

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

[0056] In some embodiments, the isolated antigen-binding protein contains a constant region of the antibody light chain.

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

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

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

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

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

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

[0063] In some embodiments, the isolated antigen-binding protein is capable of specifically binding to sialic acid-binding immunoglobulin agglutinin (Siglec) or its functionally active fragments.

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

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

[0066] In some embodiments, the isolated antigen-binding protein has a K0 of about 1.56E-10M or lower. D The ability to combine Siglec-15 or its functional segments.

[0067] In some embodiments, the isolated antigen-binding protein has an EC50 of about 3.25 nM or lower. 50 The ability to combine Siglec-15 or its functional segments.

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

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

[0070] In some embodiments, the isolated antigen-binding protein, which affects immune cell proliferation, includes reducing Siglec-15-related immune cell proliferation inhibition.

[0071] In some embodiments, the isolated antigen-binding protein is contained in immune cells derived from humans.

[0072] In some embodiments, the isolated antigen-binding protein is present, and the immune cells include lymphocytes.

[0073] In some embodiments, the isolated antigen-binding protein is used, and the immune cells include peripheral blood lymphocytes.

[0074] In some embodiments, the isolated antigen-binding protein is present, and the immune cells include T cells.

[0075] In some embodiments, the isolated antigen-binding protein, wherein the immune cells contain CD4 + Cells and / or CD8 + cell.

[0076] In some embodiments, the isolated antigen-binding protein has tumor-suppressive capabilities.

[0077] In some embodiments, the isolated antigen-binding protein has a tumor-suppressing ability that includes influencing the volume of the tumor.

[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 higher.

[0079] In some embodiments, the isolated antigen-binding protein has the ability to influence the secretion of cytokines.

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

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

[0082] In some embodiments, the isolated antigen-binding protein, the cytokine includes inflammatory factors.

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

[0084] On the other hand, this application also provides a polypeptide comprising the isolated antigen-binding protein of this application.

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

[0086] On the other hand, this application also provides nucleic acid molecules that encode the isolated antigen-binding protein of this application and / or the polypeptide of this application.

[0087] On the other hand, this application also provides a vector containing the nucleic acid molecules of this application.

[0088] On the other hand, this application also provides immune conjugates comprising the isolated antigen-binding protein of this application and / or the polypeptide of this application.

[0089] In another aspect, this application also provides cells containing and / or expressing the isolated antigen-binding protein of this application, containing and / or expressing the polypeptide of this application, containing the nucleic acid molecule of this application, containing the vector of this application, and / or containing the immunoconjugate of this application.

[0090] In another aspect, this application also provides pharmaceutical compositions comprising the isolated antigen-binding protein of this application, the polypeptide of this application, the nucleic acid molecule of this application, the carrier of this application, the immunoconjugate of this application, and / or the cell of this application, and optionally a pharmaceutically acceptable carrier.

[0091] On the other hand, this application also provides pharmaceutical compositions comprising the isolated antigen-binding protein of this application, the polypeptide of this application, the nucleic acid molecule of this application, the carrier of this application, the immunoconjugate of this application, the cell of this application, and / or the pharmaceutical composition of this application.

[0092] On the other hand, this application also provides a kit comprising the isolated antigen-binding protein of this application, the polypeptide of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cell of this application, the pharmaceutical composition of this application, and / or the pharmaceutical combination of this application.

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

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

[0095] In another aspect, this application also provides a method for preparing the antigen-binding protein and / or the polypeptide of this application, comprising culturing the cells of this application under conditions that cause the antigen-binding protein and / or the polypeptide to be expressed.

[0096] In some embodiments, the method includes culturing the cells under conditions that cause the antigen-binding protein to be expressed.

[0097] In some embodiments, the method includes culturing the cells under conditions that cause the polypeptide to be expressed.

[0098] On the other hand, this application also provides a method for detecting Siglec in a sample, comprising administering the isolated antigen-binding protein of this application, administering the polypeptide of this application, administering the nucleic acid molecule of this application, administering the carrier of this application, administering the immunoconjugate of this application, administering the cell of this application, administering the pharmaceutical composition of this application, administering the pharmaceutical combination of this application, and / or using the kit of this application.

[0099] In some implementations of the method, the Siglec includes Siglec-15.

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

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

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

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

[0104] In another aspect, this application also provides a method for influencing the proliferation of immune cells, comprising administering the isolated antigen-binding protein of this application, administering the polypeptide of this application, administering the nucleic acid molecule of this application, administering the carrier of this application, administering the immune conjugate of this application, administering the cells of this application, administering the pharmaceutical composition of this application, administering the pharmaceutical combination of this application, and / or using the kit of this application.

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

[0106] In some implementations, the method of influencing immune cell proliferation includes reducing Siglec-15-related immune cell proliferation inhibition.

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

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

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

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

[0111] In some embodiments of the method, the immune cells contain CD4. + and / or CD8 + cell.

[0112] In another aspect, this application also provides a method for influencing the secretion of cytokines, comprising administering the isolated antigen-binding protein of this application, administering the polypeptide of this application, administering the nucleic acid molecule of this application, administering the carrier of this application, administering the immunoconjugate of this application, administering the cells of this application, administering the pharmaceutical composition of this application, administering the pharmaceutical combination of this application, and / or using the kit of this application.

[0113] In some embodiments of the method, the secretion includes in vivo, in vitro, and / or extracorporeal secretions.

[0114] In some embodiments of the method, the secretion of the cytokines includes the secretion of lipopolysaccharide-related cytokines.

[0115] In some embodiments of the method, the cytokines comprise inflammatory factors.

[0116] In some embodiments of the method, the cytokines comprise TNF-α and / or IL-6.

[0117] On the other hand, this application also provides the use of the isolated antigen-binding protein of this application, the polypeptide of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cell of this application, the pharmaceutical composition of this application and / or the pharmaceutical combination of this application in the preparation of a kit.

[0118] In some embodiments, the kit is used to detect the presence and / or content of Siglec in a sample.

[0119] In some implementations, the Siglec includes Siglec-15.

[0120] On the other hand, this application also provides the use of the isolated antigen-binding protein of this application, the polypeptide of this application, the nucleic acid molecule of this application, the carrier of this application, the immunoconjugate of this application, the cell of this application, the pharmaceutical composition of this application, the pharmaceutical combination of this application and / or the kit of this application in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0121] In some embodiments, the disease and / or condition includes a tumor.

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

[0123] In some implementations, the Siglec includes Siglec-15.

[0124] In some embodiments, the disease and / or symptom comprises a solid tumor.

[0125] In some embodiments, the disease and / or condition includes colon cancer.

[0126] On the other hand, this application also provides the isolated antigen-binding protein of this application, the polypeptide of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cell of this application, the pharmaceutical composition of this application, the pharmaceutical combination of this application, and / or the kit of this application, for the prevention and / or treatment of diseases and / or conditions.

[0127] In some implementations, the disease and / or symptom includes 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 implementations, the disease and / or symptom includes a solid tumor.

[0131] In some implementations, the disease and / or condition includes colon cancer.

[0132] On the other hand, this application also provides a method for preventing and / or treating diseases and / or conditions, the method comprising administering to a subject in need the isolated antigen-binding protein of this application, the polypeptide of this application, the nucleic acid molecule of this application, the carrier of this application, the immunoconjugate of this application, the cell of this application, the pharmaceutical composition of this application, the drug combination of this application, and / or the kit of this application.

[0133] In some implementations, the disease and / or symptom includes 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 implementations, the disease and / or symptom includes a solid tumor.

[0137] In some implementations, the disease and / or condition includes colon cancer.

[0138] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0139] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is as follows:

[0140] Figure 1 The diagram shown illustrates the construction method of yeast as described in this application.

[0141] Figure 2 The results shown are the detection results of the binding of the Siglec-15 binding protein of this application to human Siglec-15 on the cell surface.

[0142] Figure 3 The results shown are the detection results of the binding of the Siglec-15 binding protein of this application to monkey Siglec-15 on the cell surface.

[0143] Figure 4 The results shown are the detection results of the binding of the Siglec-15 binding protein of this application to mouse Siglec-15 on the cell surface.

[0144] Figure 5 This application demonstrates how the Siglec-15 binding protein reverses the Siglec-15 protein's effect on CD4. + Results of the detection of T cell proliferation inhibition.

[0145] Figure 6 This application demonstrates how the Siglec-15 binding protein reverses the Siglec-15 protein's effect on CD8. + Results of the detection of T cell proliferation inhibition.

[0146] Figure 7 The results shown are the detection results of the inhibitory effect of Siglec-15 binding protein on tumor growth in this application.

[0147] Figure 8 The results show the detection results of the effect of Siglec-15 binding protein on the secretion level of TNF-α (TNF-alpha) 2 hours after LPS injection.

[0148] Figure 9 The results show the detection results of the effect of Siglec-15 binding protein on the secretion level of TNF-α (TNF-alpha) 6 hours after LPS injection.

[0149] Figure 10 The results show the detection results of the effect of Siglec-15 binding protein on the secretion level of IL-6 2 hours after LPS injection.

[0150] Figure 11 The results show the detection results of the effect of Siglec-15 binding protein on IL-6 secretion level 6 hours after LPS injection. Detailed Implementation

[0151] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0152] Terminology Definition

[0153] In this application, the terms "sialic acid-binding immunoglobulin lectin," "Sialic acid-binding Ig-like lectin," or "Siglec" generally refer to an immunoglobulin-like lectin capable of recognizing glycostructures containing sialic acid. In vitro, recombinant Siglec-15 protein inhibits the proliferation of human and mouse T cells and the secretion of IFN-γ, and also exhibits inhibitory effects on OT-1 T cells activated by chicken ovalbumin (OVA) antigen. Siglec-15 is expressed at low levels in macrophages, and its expression is significantly upregulated after stimulation with macrophage colony-stimulating factor M-CSF. Compared with normal macrophages, Siglec-15 gene knockout macrophages further induce T cell proliferation, indicating that Siglec-15 can directly inhibit T cell activity. Preclinical in vitro experiments and animal tumor models demonstrate that Siglec-15 antibody or Siglec-15 binding protein can activate T cells and inhibit tumors. For example, Siglec can be Siglec-15, and the Uniprot accession number for Siglec-15 can be Q6ZMC9. The Siglec protein of this application may also encompass its functionally active fragments, not limited to proteins containing functionally active fragments of the Siglec protein resulting from processing and / or modification occurring in the cell.

[0154] In this application, the terms "leucine-rich repeat-containing protein 4C," "Leucine-rich repeat-containing protein 4C," or "LRRC4C" are used interchangeably and generally refer to a protein rich in repeating leucine sequences. LRRC4C can act as a ligand for Siglect-15, binding to and activating Siglect-15. The Uniprot accession number for LRRC4C may be Q9HCJ2. The LRRC4C protein in this application may also encompass its functionally active fragments, not limited to proteins containing functionally active fragments of LRRC4C proteins resulting from cellular processing and / or modifications.

[0155] In this application, the term "sialyl-Tn" or "sialyl-Tn" generally refers to a Tn antigen containing a sialic acid group. The Tn antigen can be an N-acetylgalactosamine (GalNAc) oligosaccharide linked to serine or threonine via a glycosidic bond. Sialyl-Tn expression is caused by activation of an aberrant glycosylation pathway, which can generally occur in tumor cells. The sialyl-Tn protein of this application may also encompass its functionally active fragments.

[0156] In this application, the terms "lipopolysaccharide," "Lipopolysaccharide," or "LPS" generally refer to substances composed of lipids and polysaccharides. Lipopolysaccharides are typically components of the outer wall of Gram-negative bacterial cell walls. When lipopolysaccharides act on the human body, they can promote the synthesis and / or secretion of various cytokines by human cells, thus inducing an immune response.

[0157] In this application, the term "functionally active fragment" generally refers to a fragment that has a partial region of a full-length protein or nucleic acid but retains or partially retains 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 a full-length protein to bind to another molecule. For example, a functionally active fragment of Siglec may retain or partially retain the function of the full-length Siglec in recognizing sugar structures containing sialic acid and / or suppressing the immune system.

[0158] In this application, the term "secretion" generally refers to the transfer of expressed polypeptides or proteins from cells to the extracellular environment. For example, in this application, it can refer to the transfer of expressed cytokines from immune cells to the extracellular environment.

[0159] In this application, the term "cytokine" generally refers to proteins released by a cell population that act as intercellular regulators in another cell. The cytokines in this application may be interleukins (ILs), such as IL-6, tumor necrosis factors such as TNF-α, and other polypeptide factors. In this application, the cytokines may encompass their functionally active fragments, and may also include proteins from natural sources or from recombinant cell cultures, as well as bioactive equivalents of natural sequence cytokines.

[0160] In this application, the term "inflammatory cytokine" generally refers to cytokines that promote inflammation. The inflammatory cytokines of this application may include interleukin IL-6 and / or tumor necrosis factor TNF-α. In this application, the inflammatory cytokines may encompass their functionally active fragments, and may also include proteins from natural sources or from recombinant cell cultures, as well as bioactive equivalents of natural sequence cytokines.

[0161] In this application, the term "IL-6" generally refers to an interleukin. The IL-6 of this application can promote inflammation. The Uniprot accession number for IL-6 may be P05231. The IL-6 protein of this application may also encompass its functionally active fragments, not limited to proteins containing functionally active fragments of the IL-6 protein resulting from processing and / or modification occurring in the cell.

[0162] In this application, the term "TNFα" or "TNF-α" generally refers to tumor necrosis factor α. TNF-α can be a major mediator of inflammatory, immunological, 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., humans, mice, and monkeys). The Uniprot accession number for TNF-α can be P01375. The TNF-α protein in this application can also encompass its functionally active fragments, not limited to proteins containing functionally active fragments of TNF-α proteins resulting from processing and / or modification occurring in cells.

[0163] In this application, 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 cells.

[0164] In this application, the term "immune cell" generally refers to cells involved in the immune response. Immune cells may include T cells, B cells, natural killer (NK) cells, mast cells, and / or phagocytes derived from the bone marrow.

[0165] In this application, the term "peripheral blood lymphocytes" generally refers to mature lymphocytes that circulate in the blood, rather than those located in organs such as the spleen or lymph nodes. Peripheral blood lymphocytes may contain T cells, NK cells, and / or B cells.

[0166] In this application, the term "T cell" generally refers to thymus-derived cells. T cells can participate in various cell-mediated immune responses, including thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell population may include, but is not limited to, helper T cells (Th;CD4+). + T cells, cytotoxic T cells (CTLs; CD8) + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other subset of T cells.

[0167] In this application, the term "CD4" generally refers to a differentiation cluster 4 protein expressed by cells. CD4 can be a co-receptor for T cells to recognize antigens and can bind to the non-peptide region 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, which can regulate immune responses. The accession number for CD4 in Uniprot may be P01730. The CD4 protein in this application may also encompass its functionally active fragments, not limited to proteins containing functionally active fragments of the CD4 protein resulting from processing and / or modification occurring in the cell.

[0168] In this application, the term "CD8" generally refers to a differentiation cluster 8 protein expressed by cells. CD8 can be a co-receptor for T cells to recognize antigens and can bind to the non-peptide region 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, which can specifically kill target cells. The accession number for CD8 in Uniprot may be P10966. The CD8 protein in this application may also encompass its functionally active fragments, not limited to proteins containing functionally active fragments of the CD8 protein resulting from processing and / or modification occurring in the cell.

[0169] In this application, the term "in vivo" generally refers to an event that occurs within the body of a subject.

[0170] In this application, the term "in vitro" generally refers to an event that occurs outside the subject.

[0171] In this application, the term "ex vivo" generally refers to an event involving treatment or surgery on cells, tissues, and / or organs that have been removed from a subject's body. In one embodiment, the cells, tissues, and / or organs can be returned to the subject's body via surgery or a treatment method.

[0172] In this application, the term "EC" 50 The "EC50 value" or "EC50 value" typically refers to the half-maximum effective concentration (WMC) of a bound substance (e.g., antibody) that induces a response between 50% of the baseline and maximum values ​​in an in vitro or in vivo analytical context. A decreased EC50 value indicates a lower effective concentration. 50 The value can indicate a high drug affinity and efficacy.

[0173] In this application, the term "K" D The "KD value" or "dissociation constant" typically refers to the dissociation constant, which can be determined via surface plasmon resonance (SPR). Typically, SPR analysis uses the BIAcore system (Pharmacia Biosensor, Piscataway, NJ) to measure the real-time binding interaction between ligands (substances immobilized on the biosensor matrix) and analytes (substances in solution) via SPR. SPR analysis can also be performed by immobilizing both the analyte (substances on the biosensor matrix) and the presenting ligand.

[0174] In this application, the term "tumor suppression capacity" generally refers to any measurable inhibitory capacity against in vitro tumor cell proliferation or in vivo tumor growth, such as a reduction of at least 5% in in vitro tumor cell proliferation or in vivo tumor growth, which can 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, such as at least 100%.

[0175] In this application, the term "isolated" generally refers to something obtained artificially from its natural state. For example, a certain unisolated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state can be called isolated. The term "isolated" may not exclude the presence of artificial or synthetic substances, nor may it exclude the presence of other impurities that do not affect the activity of the substance.

[0176] In this application, the term "isolated antigen-binding protein" generally refers to a protein that has antigen-binding ability but has been removed from its native state. The "isolated antigen-binding protein" of this application may include a portion that binds to an antigen, and optionally, allow the antigen-binding portion to employ a framework or structural portion that promotes the antigen-binding portion's conformation for binding the antigen. The antigen-binding protein may include, for example, an antibody-derived protein framework region (FR) or an alternative protein framework region or artificial framework region having a transplanted CDR or CDR derivative. Such frameworks may include, but are not limited to, antibody-derived framework regions containing, for example, mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic framework regions containing, for example, biocompatible polymers. 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.

[0177] In this application, the term "CDR," also known as "complementarity-determining region," generally refers to a region within the variable structural domain of an antibody whose sequence can be highly variable and / or form a structurally defining loop. For example, an antibody may include six CDRs; three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In some embodiments, naturally occurring camel antibodies consisting only of the heavy chain can function normally and stably even in the absence of the light chain. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at www.bioinf.org.uk / abs / index.html#kabatnum. For example, the amino acid sequence number of the antigen-binding protein can be in accordance with the IMGT numbering scheme (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 CDR of the antigen-binding protein can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242).

[0178] In this application, the term "FR" generally refers to a more conserved portion of the antibody variable domain, which is referred to as a frame region. For example, the variable domains of the natural heavy chain and light chain may each contain 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).

[0179] In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of the antibody heavy chain and / or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL" (or "V" respectively). H " and "V L These domains are typically the most variable parts of an antibody (relative to other antibodies of the same type) and may contain antigen-binding sites. In this application, the term "variable" generally refers to the fact that certain segments of the variable domain may differ significantly in sequence between antibodies. Variable domains mediate antigen binding and determine the specificity of a particular antibody for its specific antigen. However, variability may not be uniformly distributed across the entire variable domain. It can typically be concentrated in three segments within the light and heavy chain variable domains, referred to as hypervariable regions (CDRs or HVRs). More highly conserved portions of the variable domain may be referred to as frame regions (FRs). The variable domains of the native heavy and light chains may each contain four FR regions, mostly in a β-sheet configuration, linked by three CDRs forming a ring link, and in some cases forming part of a β-sheet structure. The CDRs in each chain can be held together closely by the FR regions, and CDRs from the other chain together promote the formation of the antibody's antigen-binding site.

[0180] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment thereof or a derivative thereof, encompassing any polypeptide including an antigen-binding site, whether it is produced in vitro or in vivo. The term may include, but is not limited to, polyclonal, monoclonal, single-specific, multi-specific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies. Unless otherwise modified by the term "complete," such as in "complete antibody," for the purposes of this invention, the term "antibody" may also include antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, VHH, dAb, and other antibody fragments that maintain antigen-binding function (e.g., specific binding to human Siglec). Typically, such fragments may include an antigen-binding domain. The basic 4-chain antibody unit can be a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies can consist of five basic heterotetrameric units and another polypeptide called the J chain, containing 10 antigen-binding sites, while IgA antibodies can include 2-5 basic four-chain units that can bind to the J chain and polymerize to form a multivalent combination. For IgG, the four-chain unit is typically about 150,000 Daltons. Each L chain can be linked to an H chain via a covalent disulfide bond, and two H chains can be linked to each other via one or more disulfide bonds depending on the H chain isoform. Each H and L chain can also have regularly spaced intrachain disulfide bridges. Each H chain can have a heavy chain variable region (VH) at its N-terminus, followed by three constant domains (CH) for α and γ chains, and four CH domains for μ and ε isoforms. Each L chain can have a light chain variable region (VL) at its N-terminus and a constant domain at its other end. VL and VH can correspond, and the light chain constant region (CL) can correspond to the first constant domain (CH1) of the heavy chain. Specific amino acid residues can be considered to form interfaces between the variable domains of the light and heavy chains. VH and VL can pair together to form a single antigen-binding site. L chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domains. Immunoglobulins can be classified into different classes or isotypes based on the amino acid sequence of the constant domain of the heavy chain (CH). Currently, there are five classes of immunoglobulins: IgA, IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and / or IgG4), and IgM, with heavy chains named α, δ, ε, γ, and μ, respectively.

[0181] In this application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind an antigen (e.g., Siglec). In this application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH, and / or dAb.

[0182] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, an intact antibody can be digested using papain. After digestion with papain, the antibody produces two identical antigen-binding fragments, namely the "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region). The Fab fragment may consist of a complete L chain with a variable region of a heavy chain and a first constant region (CH1) of the H chain (VH).

[0183] In this application, the term "F(ab)2" generally refers to the antigen-binding fragment of an antibody. For example, F(ab)2 can be composed of two Fab fragments linked together.

[0184] In this application, the term "Fab'" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which is slightly larger than the Fab fragment. For example, the Fab' fragment may include all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, the Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

[0185] In this application, the term "F(ab')2" generally refers to an antibody fragment produced by digesting an intact antibody with pepsin. The F(ab')2 fragment contains two Fab fragments held together by disulfide bonds and a partial hinge region. The F(ab')2 fragment exhibits bivalent antigen-binding activity and is capable of cross-linking antigens.

[0186] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy chain variable region and light chain variable region, and lacking the heavy chain constant region and light chain constant region. The heavy chain variable region and light chain variable region include, for example, CDRs. For example, an Fv fragment comprises all or part of the amino-terminal variable region of about 110 amino acids of the heavy and light chains.

[0187] In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment including a variable region comprising a light chain and at least one antibody fragment including a variable region comprising a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used in this application, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0188] In this application, the term "di-scFv" generally refers to a divalent scFv, such as a molecule in which two scFv molecules are joined together by a short connector.

[0189] In this application, the term “dAb” generally refers to an antigen-binding fragment consisting of a VH domain or a VL domain, for example, Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11): 484-490.

[0190] In this application, the term "VHH" generally refers to an antibody containing a variable antigen-binding domain of a heavy chain antibody (see Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH may also be referred to as a nanobody (Nb).

[0191] In this application, the term "monoclonal antibody" generally refers to an antibody molecule preparation consisting of a single molecule. Monoclonal antibodies typically exhibit high specificity against a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually contain different antibodies targeting different determinants), each monoclonal antibody can target a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" can indicate the characteristics of an antibody obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in this application can be prepared in hybridoma cells or by recombinant DNA methods.

[0192] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody from a laboratory animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody is less likely to elicit an adverse immune response in human individuals compared to parental (e.g., mouse-derived) antibodies.

[0193] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions, or modifications of amino acids in the CDR region are also permissible, as long as they retain the antibody's ability to bind to a specific antigen. A humanized antibody may optionally contain at least a portion of the constant region of a human immunoglobulin. A "humanized antibody" may retain antigen specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may minimally contain a chimeric antibody with a sequence derived from a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (receptor antibody) may be replaced with CDR region residues from a non-human species (donor antibody) (such as a mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and / or capabilities. In some cases, FR region residues in a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies can contain amino acid modifications not found in receptor antibodies or in donor antibodies. These modifications can be made to further improve antibody performance, such as binding affinity.

[0194] In this application, the term "fully human antibody" generally refers to an antibody that contains only the protein sequence of human immunoglobulins. If it is produced in mice, in mouse cells, or in hybridomas derived from mouse cells, then a fully human antibody may contain mouse glycans. Similarly, "mouse antibody," "mouse antibody," or "rat antibody" respectively refers to an antibody that contains only mouse or rat immunoglobulin sequences. Fully human antibodies can be generated in humans or in transgenic animals with human immunoglobulin germline sequences via phage display or other molecular biology methods. Exemplary techniques that can be used to manufacture antibodies are known in the art.

[0195] In this application, the term "antigen-binding protein" generally refers to a protein containing a portion that binds to an antigen, and optionally, a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes the 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), immunoconjugates, antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, as long as they exhibit the desired antigen-binding activity. The "isolated antigen-binding protein" of this application may contain a portion that binds to an antigen and optionally, allow the antigen-binding portion to adopt a scaffold or backbone portion that promotes the binding of the antigen-binding portion to the antigen.

[0196] In this application, the terms "peptide molecule," "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 parts covalently linked together. Each part can be a polypeptide with different properties. These properties can be biological properties, such as in vitro or in vivo activity. They can also be simple chemical or physical properties, such as binding to target molecules, catalysis of reactions, etc. The two parts can be directly linked by a single peptide bond or through a peptide linker.

[0197] In this application, the term "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in an isolated form, or an analogue isolated from its natural environment or synthesized artificially.

[0198] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted, thereby enabling the protein to be expressed. A vector can be transformed, transduced, or transfected into host cells, allowing the genetic material it carries to be expressed within the host cells. A vector may contain multiple elements controlling expression. Additionally, a vector may contain a replication origin site. A vector may also include components that facilitate its entry into the cell.

[0199] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that may be or is already a recipient of a subject plasmid or vector, including the nucleic acid molecules or vectors described in this application. Cells may include the progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in the morphology of total DNA complements or in the genome). Cells may include cells transfected in vitro using the vectors described in this application.

[0200] In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating the other reagent (e.g., chemotherapeutic agents, radioactive elements, cell growth inhibitors, and cytotoxic agents) with the antibody or its antigen-binding fragment (e.g., covalently linked by a linker molecule), which can deliver the other reagent to the target cell (e.g., tumor cells) by specifically binding the antibody or its antigen-binding fragment to the antigen on the target cell.

[0201] In this 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 described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable adjuvant. Furthermore, the pharmaceutical composition may also comprise one or more suitable formulations such as a (pharmaceutically effective) carrier. The acceptable components of the composition are non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0202] In this application, the term "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed to it at the doses and concentrations used. Physiologically acceptable carriers may include suitable substances. It should be noted that pharmaceutically acceptable carriers are generally not the same type of substance as vectors used in genetic engineering for inserting nucleic acids.

[0203] In this application, the term "solid tumor" generally refers to a tangible tumor that can be detected by clinical examination (e.g., X-ray, CT scan, ultrasound, or palpation). The tumor may include growths or solid lesions formed by abnormal cell growth.

[0204] In this application, the term "colon cancer" generally refers to a tumor of the digestive tract that occurs in or originates from the colon. For example, colon cancer may occur in or originate from the junction of the rectum and sigmoid colon. For example, colon cancer may be a cancer of the digestive tract that occurs in or originates from the colon.

[0205] In this application, the term "directly linked" is used in contrast to the term "indirectly linked," with "directly linked" generally referring to a direct connection. For example, direct linking can refer to a situation where substances are directly linked without a spacer. The spacer can be a linker. For example, the linker can be a peptide linker. The term "indirectly linked" generally refers to a situation where substances are not directly linked. For example, indirect linking can refer to a connection via a spacer. For example, in the isolated antigen-binding protein described in this application, the C-terminus of L-FR1 and the N-terminus of LCDR1 can be directly or indirectly linked.

[0206] In this application, the terms "specific binding" or "specific" generally refer to measurable and reproducible interactions, such as binding between a target and an antibody, where the presence of the target is determined 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 the target with greater affinity, strength, ease, and / or duration than it binds to other targets. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across proteins of different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding.

[0207] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0208] In this application, the term "tumor" generally refers to any new pathological growth of tissue. Tumor cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. In this application, the tumor can include benign tumors and malignant tumors. In this application, the tumor can include solid tumors and / or hematomas. In this application, the tumor can include cancer. In this application, examples of tumors include, but are not limited to, colon cancer.

[0209] In this application, the proteins, peptides and / or amino acid sequences involved should also be understood to include at least the following range: variants or homologs that have the same or similar functions as the said protein or peptide.

[0210] In this application, the variant can be, for example, a protein or polypeptide that has undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to Siglec). For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the protein or polypeptide prior to the alteration. For example, the substitution may be a conserved substitution. For example, the variant may also be a polypeptide encompassing its functionally active fragment, not limited to polypeptides containing the functionally active fragment of the protein resulting from processing and / or modification occurring in the cell.

[0211] In this application, the homolog can be a protein or polypeptide having at least about 85% (e.g., having 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 higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody that specifically binds to Siglec or a fragment thereof).

[0212] In this application, homology generally refers to the similarity, resemblance, or association between two or more sequences. The "sequence homology percentage" can be calculated by comparing two sequences to be aligned within a comparison window, determining the number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence homology percentage. Alignments performed to determine the sequence homology percentage can be performed 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 suitable parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region. Homology can also be determined using FASTA and BLAST. A description of the FASTA algorithm can be found in WRPearson and DJ Lipman, “An Improved Tool for Biological Sequence Alignment,” Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988; and DJ Lipman and WRPearson, “A Fast and Sensitive Search for Protein Similarity,” Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, EW Myers, and D. Lipman, “A Basic Tool for Local Alignment Search,” Journal of Molecular Biology, 215: 403-410, 1990.

[0213] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0214] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 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 specified value. Invention Details

[0216] On the one hand, this application provides an isolated antigen-binding protein.

[0217] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems known in the art, for example, www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ using different coding systems. In this application, the term CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants thereof, including CDRs whose amino acid sequences have been substituted, deleted, and / or added one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; homologs are also included, which can be amino acid sequences having at least about 85% (e.g., having 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 higher) sequence homology with the amino acid sequence of the CDR. In some embodiments, the CDR is determined by the Kabat numbering scheme. In some embodiments, the CDR is determined by the Chothia numbering scheme.

[0218] On one hand, this application provides an isolated antigen-binding protein that may contain HCDR3, wherein HCDR3 may contain the amino acid sequence shown in AIGSSWYSDAFDL (SEQ ID NO:1). For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR3 from Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the isolated antigen-binding protein may contain HCDR3 in the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the CDR may be partitioned according to Chothia rules.

[0219] In this application, the isolated antigen-binding protein may further comprise HCDR2. For example, HCDR2 may comprise the sequence shown in X1GGGX2X3, wherein X1, X2, and X3 may each be independently selected from any amino acid, such as a natural amino acid. For example, HCDR2 may comprise the sequence shown in X1GGGX2X3 (as shown in SEQ ID NO:33), wherein X1 may be S, T, W, or Y, X2 may be E, G, or V, and X3 may be S or Y. In some cases, compared to the amino acid sequence shown in SEQ ID NO:2, HCDR2 may contain at least amino acid substitutions selected from the group consisting of amino acid substitutions at X1, X2, and X3. For example, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:33. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability.

[0220] For example, the HCDR2 may contain amino acid sequences such as 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 this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR2 from Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the isolated antigen-binding protein may contain HCDR2 in the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the CDR may be partitioned according to Chothia rules.

[0221] In this application, the isolated antigen-binding protein may further comprise HCDR1. For example, HCDR1 may comprise the sequence shown in GFTX4SSY, wherein X4 can be selected from any amino acid, such as a natural amino acid. For example, HCDR1 may comprise the sequence shown in GFTX4SSY (as shown in SEQ ID NO:34), wherein X4 can be F or S. In some cases, compared to the amino acid sequence shown in SEQ ID NO:7, HCDR1 may contain at least the amino acid substitution at X4. For example, HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:34. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability.

[0222] For example, HCDR1 may contain amino acid sequences such as GFTFSSY (SEQ ID NO:7) or GFTSSSY (SEQ ID NO:8). For example, the isolated antigen-binding protein of this application may have Siglec-15 binding capability. For example, the isolated antigen-binding protein may contain HCDR1 from Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the isolated antigen-binding protein may contain HCDR1 in the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the CDR may be partitioned according to Chothia rules.

[0223] In this application, the isolated antigen-binding protein may comprise HCDR3, HCDR2, and HCDR1. For example, HCDR3 may comprise the sequence shown in SEQ ID NO:1, HCDR2 may comprise the sequence shown in X1GGGX2X3, wherein X1, X2, and X3 may each be independently selected from any amino acid, such as a natural amino acid, and HCDR1 may comprise the sequence shown in GFTX4SSY, wherein X4 may be selected from any amino acid, such as a natural amino acid. For example, the isolated antigen-binding protein HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:33, and HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:34. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability.

[0224] For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:2 for HCDR2, and the amino acid sequence shown in SEQ ID NO:7 for HCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from Ab0 or Ab9 of this application. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from the heavy chain variable region of Ab0 or Ab9 of this application. For example, the CDR may be partitioned according to Chothia's rules.

[0225] For example, the isolated antigen-binding protein HCDR3 may contain the amino acid sequence shown in SEQ ID NO:1, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:2, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:8. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from Ab2 of this application. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from the heavy chain variable region of Ab2 of this application. For example, the CDRs may be partitioned according to Chothia's rules.

[0226] For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:3 for HCDR2, and the amino acid sequence shown in SEQ ID NO:7 for HCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from Ab3, Ab13, or Ab15 of this application. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from the heavy chain variable region of Ab3, Ab13, or Ab15 of this application. For example, the CDR may be partitioned according to Chothia's rules.

[0227] For example, the isolated antigen-binding protein HCDR3 may contain the amino acid sequence shown in SEQ ID NO:1, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:4, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:7. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from Ab5 of this application. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from the heavy chain variable region of Ab5 of this application. For example, the CDRs may be partitioned according to Chothia's rules.

[0228] For example, the isolated antigen-binding protein HCDR3 may contain the amino acid sequence shown in SEQ ID NO:1, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:5, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:7. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from Ab6 of this application. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from the heavy chain variable region of Ab6 of this application. For example, the CDRs may be partitioned according to Chothia rules.

[0229] For example, the isolated antigen-binding protein HCDR3 may contain the amino acid sequence shown in SEQ ID NO:1, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:6, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:7. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from Ab7 of this application. For example, the isolated antigen-binding protein may contain HCDR1, HCDR2, and HCDR3 from the heavy chain variable region of Ab7 of this application. For example, the CDRs may be partitioned according to Chothia's rules.

[0230] In this application, the isolated antigen-binding protein may contain LCDR3, which may contain the amino acid sequence shown in QQSYSIPYT (SEQ ID NO: 9). For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain LCDR3 from Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the isolated antigen-binding protein may contain LCDR3 in the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the CDR can be partitioned according to Chothia rules.

[0231] In this application, the isolated antigen-binding protein may further comprise LCDR2. For example, LCDR2 may comprise the sequence shown in VASX5X6X7X8, wherein X5, X6, X7, and X8 may each be independently selected from any amino acid, such as a natural amino acid. For example, LCDR2 may comprise the sequence shown in VASX5X6X7X8 (as shown in SEQ ID NO:35), wherein 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. In some cases, compared to the amino acid sequence shown in SEQ ID NO:10, LCDR2 may contain at least amino acid substitutions selected from the following group of positions: amino acid substitutions at X5, X6, X7, and X8. For example, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:35. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability.

[0232] For example, the LCDR2 may contain amino acid sequences such as those 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 this application may have Siglec-15 binding capability. For example, the isolated antigen-binding protein may contain LCDR2 from Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the isolated antigen-binding protein may contain LCDR2 in the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the CDR may be partitioned according to Chothia rules.

[0233] In this application, the isolated antigen-binding protein may further comprise LCDR1. For example, LCDR1 may comprise the sequence shown in RASQDISX9WLA, wherein X9 may be selected from any amino acid, such as a natural amino acid. For example, LCDR1 may comprise the sequence shown in RASQDISX9WLA (as shown in SEQ ID NO:36), wherein X9 may be D or S. In some cases, compared to the amino acid sequence shown in SEQ ID NO:13, LCDR1 may contain at least an amino acid substitution at X9. For example, LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:36. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability.

[0234] For example, the LCDR1 may contain the amino acid sequence shown in RASQDISSWLA (SEQ ID NO: 13) or RASQDISDWLA (SEQ ID NO: 14). For example, the isolated antigen-binding protein of this application may have Siglec-15 binding capability. For example, the isolated antigen-binding protein may contain LCDR1 from Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the isolated antigen-binding protein may contain LCDR1 in the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application. For example, the CDR may be partitioned according to Chothia rules.

[0235] In this application, the isolated antigen-binding protein may comprise LCDR3, LCDR2, and LCDR1. For example, LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9, LCDR2 may comprise the sequence shown in VASX5X6X7X8, wherein X5, X6, X7, and X8 may each be independently selected from any amino acid, such as a natural amino acid, and LCDR1 may comprise the sequence shown in RASQDISX9WLA, wherein X9 may be selected from any amino acid, such as a natural amino acid. For example, the isolated antigen-binding protein may have LCDR3 comprising the amino acid sequence shown in SEQ ID NO:9, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:35, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO:36. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding capability.

[0236] For example, the isolated antigen-binding protein's LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:10, and LCDR1 may contain the amino acid sequence shown in SEQ ID NO:13. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding capability. For example, the isolated antigen-binding protein may contain LCDR1, LCDR2, and LCDR3 from Ab0, Ab2, Ab3, Ab5, or Ab7 of this application. For example, the isolated antigen-binding protein may contain LCDR1, LCDR2, and LCDR3 from the light chain variable region of Ab0, Ab2, Ab3, Ab5, or Ab7 of this application. For example, the CDR can be partitioned according to Chothia's rules.

[0237] For example, the isolated antigen-binding protein's LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:10, and LCDR1 may contain the amino acid sequence shown in SEQ ID NO:14. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding capability. For example, the isolated antigen-binding protein may contain LCDR1, LCDR2, and LCDR3 from Ab9 of this application. For example, the isolated antigen-binding protein may contain LCDR1, LCDR2, and LCDR3 from the light chain variable region of Ab9 of this application. For example, the CDR can be partitioned according to Chothia's rules.

[0238] For example, the isolated antigen-binding protein's LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:11, and LCDR1 may contain the amino acid sequence shown in SEQ ID NO:13. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding capability. For example, the isolated antigen-binding protein may contain LCDR1, LCDR2, and LCDR3 from Ab13 of this application. For example, the isolated antigen-binding protein may contain LCDR1, LCDR2, and LCDR3 from the light chain variable region of Ab13 of this application. For example, the CDR can be partitioned according to Chothia's rules.

[0239] For example, the isolated antigen-binding protein's LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:12, and LCDR1 may contain the amino acid sequence shown in SEQ ID NO:13. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding capability. For example, the isolated antigen-binding protein may contain LCDR1, LCDR2, and LCDR3 from Ab15 of this application. For example, the isolated antigen-binding protein may contain LCDR1, LCDR2, and LCDR3 from the light chain variable region of Ab15 of this application. For example, the CDR can be partitioned according to Chothia's rules.

[0240] In this application, the isolated antigen-binding protein may comprise HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1. For example, HCDR3 may comprise the sequence shown in SEQ ID NO:1, HCDR2 may comprise the sequence shown in X1GGGX2X3, wherein X1, X2, and X3 may each be independently selected from any amino acid, such as a natural amino acid, and HCDR1 may comprise the sequence shown in GFTX4SSY, wherein X4 may be selected from any amino acid, such as a natural amino acid, LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9, LCDR2 may comprise the sequence shown in VASX5X6X7X8, wherein X5, X6, X7, and X8 may each be independently selected from any amino acid, such as a natural amino acid, and LCDR1 may comprise the sequence shown in RASQDISX9WLA, wherein X9 may be selected from any amino acid, such as a natural amino acid. For example, HCDR3 may contain the sequence shown in SEQ ID NO:1, HCDR2 may contain the sequence shown in X1GGGX2X3 (as shown in SEQ ID NO:33), wherein X1 can be S, T, W or Y, X2 can be E, G or V, and X3 can be S or Y, HCDR1 may contain the sequence shown in GFTX4SSY (as shown in SEQ ID NO:34), wherein X4 can be F or S, LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9, LCDR2 may contain the sequence shown in VASX5X6X7X8 (as shown in SEQ ID NO:35), wherein X5 can be F, S or Y, X6 can be E, I or L, X7 can be H or Q, and X8 can be R or S, and LCDR1 may contain the sequence shown in RASQDISX9WLA (as shown in SEQ ID NO:36), wherein X9 can be D or S. In some cases, compared to the amino acid sequence shown in SEQ ID NO:2, HCDR2 may contain at least amino acid substitutions selected from the following positions: amino acid substitutions at X1, X2, and X3; compared to the amino acid sequence shown in SEQ ID NO:7, HCDR1 may contain at least amino acid substitutions at X4; compared to the amino acid sequence shown in SEQ ID NO:10, LCDR2 may contain at least amino acid substitutions selected from the following positions: amino acid substitutions at X5, X6, X7, and X8; and compared to the amino acid sequence shown in SEQ ID NO:13, LCDR1 may contain at least amino acid substitutions at X9.For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:33 for HCDR2, the amino acid sequence shown in SEQ ID NO:34 for HCDR1, the amino acid sequence shown in SEQ ID NO:9 for LCDR3, the amino acid sequence shown in SEQ ID NO:35 for LCDR2, and the amino acid sequence shown in SEQ ID NO:36 for LCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability.

[0241] For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:2 for HCDR2, the amino acid sequence shown in SEQ ID NO:7 for HCDR1, the amino acid sequence shown in SEQ ID NO:9 for LCDR3, the amino acid sequence shown in SEQ ID NO:10 for LCDR2, and the amino acid sequence shown in SEQ ID NO:13 for LCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 from Ab0 of this application. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, and HCDR1 in the heavy chain variable region of Ab0 of this application, and LCDR3, LCDR2, and LCDR1 in its light chain variable region. For example, the CDRs may be partitioned according to Chothia's rules.

[0242] For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:2 for HCDR2, the amino acid sequence shown in SEQ ID NO:8 for HCDR1, the amino acid sequence shown in SEQ ID NO:9 for LCDR3, the amino acid sequence shown in SEQ ID NO:10 for LCDR2, and the amino acid sequence shown in SEQ ID NO:13 for LCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 from Ab2 of this application. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, and HCDR1 in the heavy chain variable region of Ab2 of this application, and LCDR3, LCDR2, and LCDR1 in its light chain variable region. For example, the CDRs may be partitioned according to Chothia's rules.

[0243] For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:3 for HCDR2, the amino acid sequence shown in SEQ ID NO:7 for HCDR1, the amino acid sequence shown in SEQ ID NO:9 for LCDR3, the amino acid sequence shown in SEQ ID NO:10 for LCDR2, and the amino acid sequence shown in SEQ ID NO:13 for LCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 from Ab3 of this application. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, HCDR1 from the heavy chain variable region and LCDR3, LCDR2, and LCDR1 from the light chain variable region of Ab3 of this application. For example, the CDRs may be partitioned according to Chothia's rules.

[0244] For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:4 for HCDR2, the amino acid sequence shown in SEQ ID NO:7 for HCDR1, the amino acid sequence shown in SEQ ID NO:9 for LCDR3, the amino acid sequence shown in SEQ ID NO:10 for LCDR2, and the amino acid sequence shown in SEQ ID NO:13 for LCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 from Ab5 of this application. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, and HCDR1 in the heavy chain variable region of Ab5 of this application, and LCDR3, LCDR2, and LCDR1 in its light chain variable region. For example, the CDRs may be partitioned according to Chothia's rules.

[0245] For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:5 for HCDR2, the amino acid sequence shown in SEQ ID NO:7 for HCDR1, the amino acid sequence shown in SEQ ID NO:9 for LCDR3, the amino acid sequence shown in SEQ ID NO:10 for LCDR2, and the amino acid sequence shown in SEQ ID NO:13 for LCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 from Ab6 of this application. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, and HCDR1 in the heavy chain variable region of Ab6 of this application, and LCDR3, LCDR2, and LCDR1 in its light chain variable region. For example, the CDRs may be partitioned according to Chothia's rules.

[0246] For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:6 for HCDR2, the amino acid sequence shown in SEQ ID NO:7 for HCDR1, the amino acid sequence shown in SEQ ID NO:9 for LCDR3, the amino acid sequence shown in SEQ ID NO:10 for LCDR2, and the amino acid sequence shown in SEQ ID NO:13 for LCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 from Ab7 of this application. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, and HCDR1 in the heavy chain variable region of Ab7 of this application, and LCDR3, LCDR2, and LCDR1 in its light chain variable region. For example, the CDRs may be partitioned according to Chothia's rules.

[0247] For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:2 for HCDR2, the amino acid sequence shown in SEQ ID NO:7 for HCDR1, the amino acid sequence shown in SEQ ID NO:9 for LCDR3, the amino acid sequence shown in SEQ ID NO:10 for LCDR2, and the amino acid sequence shown in SEQ ID NO:14 for LCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 from Ab9 of this application. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, and HCDR1 in the heavy chain variable region of Ab9 of this application, and LCDR3, LCDR2, and LCDR1 in its light chain variable region. For example, the CDRs may be partitioned according to Chothia's rules.

[0248] For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:3 for HCDR2, the amino acid sequence shown in SEQ ID NO:7 for HCDR1, the amino acid sequence shown in SEQ ID NO:9 for LCDR3, the amino acid sequence shown in SEQ ID NO:11 for LCDR2, and the amino acid sequence shown in SEQ ID NO:13 for LCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 from Ab13 of this application. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, and HCDR1 in the heavy chain variable region of Ab13 of this application, and LCDR3, LCDR2, and LCDR1 in its light chain variable region. For example, the CDRs can be partitioned according to Chothia's rules.

[0249] For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 for HCDR3, the amino acid sequence shown in SEQ ID NO:3 for HCDR2, the amino acid sequence shown in SEQ ID NO:7 for HCDR1, the amino acid sequence shown in SEQ ID NO:9 for LCDR3, the amino acid sequence shown in SEQ ID NO:12 for LCDR2, and the amino acid sequence shown in SEQ ID NO:13 for LCDR1. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 from Ab15 of this application. For example, the isolated antigen-binding protein may contain HCDR3, HCDR2, and HCDR1 from the heavy chain variable region of Ab15 of this application, and LCDR3, LCDR2, and LCDR1 from its light chain variable region. For example, the CDRs may be partitioned according to Chothia's rules.

[0250] In this application, the isolated antigen-binding protein may include H-FR1, the C-terminus of which may be directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 may contain an amino acid sequence as shown in SEQ ID NO:15.

[0251] In this application, the isolated antigen-binding protein may include H-FR2, which may be located between HCDR1 and HCDR2, and H-FR2 may contain an amino acid sequence as shown in SEQ ID NO:16.

[0252] In this application, the isolated antigen-binding protein may contain H-FR3, which may be located between HCDR2 and HCDR3, and the H-FR3 may contain an amino acid sequence as shown in SEQ ID NO:17.

[0253] In this application, the isolated antigen-binding protein may include H-FR4, the N-terminus of which may be linked to the C-terminus of HCDR3, and the H-FR4 may contain an amino acid sequence as shown in SEQ ID NO:18.

[0254] In this application, the antigen-binding protein may comprise H-FR1, H-FR2, H-FR3, and H-FR4. For example, the isolated antigen-binding protein's H-FR1, H-FR2, H-FR3, and H-FR4 may respectively comprise the amino acid sequences shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0255] In this application, the isolated antigen-binding protein may include L-FR1, the C-terminus of which may be directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 may contain an amino acid sequence as shown in SEQ ID NO:19.

[0256] In this application, the isolated antigen-binding protein may include L-FR2, which may be located between LCDR1 and LCDR2, and L-FR2 may contain an amino acid sequence as shown in SEQ ID NO:20.

[0257] In this application, the isolated antigen-binding protein may include L-FR3, which may be located between LCDR2 and LCDR3, and L-FR3 may include an amino acid sequence as shown in SEQ ID NO:21.

[0258] In this application, the isolated antigen-binding protein may include L-FR4, the N-terminus of which may be directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 may contain an amino acid sequence as shown in SEQ ID NO:22.

[0259] In this application, the isolated antigen-binding protein may comprise L-FR1, L-FR2, L-FR3, and L-FR4. For example, L-FR1, L-FR2, L-FR3, and L-FR4 of the isolated antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0260] In this application, the isolated antigen-binding protein may contain a VH, which may contain the amino acid sequence shown in SEQ ID NO:37. For example, the VH may contain:

[0261] The sequence shown in SEQ ID NO: 37 (e.g., EVQLLESGGGLVQPGGSLRLSCAASGFTX4SSYAMSWVRQAPGKGLEWVSTIX1GGGX2X3TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAIGSSWYSDAFDLWGQGTMVTVSS) is 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. In some cases, compared to the amino acid sequence shown in SEQ ID NO: 23, the VH may contain at least amino acid substitutions selected from the group consisting of amino acid substitutions at X1, X2, X3, and X4. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability.

[0262] In this application, the isolated antigen-binding protein may contain a VH, which may contain the amino acid sequence shown 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 this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application.

[0263] In this application, the isolated antigen-binding protein may comprise VL, which may contain the amino acid sequence shown in SEQ ID NO:38. For example, VH may comprise:

[0264] The sequence shown in SEQ ID NO:38 (as shown in SEQ ID NO:38) is as follows: 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 shown in SEQ ID NO:29, the VH may contain at least amino acid substitutions selected from the group consisting of amino acid substitutions at X5, X6, X7, X8, and X9. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability.

[0265] In this application, the isolated antigen-binding protein may contain a variable region (VL), which may contain the amino acid sequence shown 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 this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of this application.

[0266] In this application, the isolated antigen-binding protein may comprise the VH and VL. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:37, and the VL may comprise the amino acid sequence shown in SEQ ID NO:38. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability.

[0267] For example, the VH may contain the amino acid sequence shown in SEQ ID NO:23, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain VH and VL from Ab0 of this application.

[0268] For example, the VH may contain the amino acid sequence shown in SEQ ID NO:24, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain VH and VL from Ab2 of this application.

[0269] For example, the VH may contain the amino acid sequence shown in SEQ ID NO:25, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain VH and VL from Ab3 of this application.

[0270] For example, the VH may contain the amino acid sequence shown in SEQ ID NO:26, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain VH and VL from Ab5 of this application.

[0271] For example, the VH may contain the amino acid sequence shown in SEQ ID NO:27, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain VH and VL from Ab6 of this application.

[0272] For example, the VH may contain the amino acid sequence shown in SEQ ID NO:28, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain VH and VL as shown in Ab7 of this application.

[0273] For example, the VH may contain the amino acid sequence shown in SEQ ID NO:23, and the VL may contain the amino acid sequence shown in SEQ ID NO:30. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain VH and VL from Ab9 of this application.

[0274] For example, the VH may contain the amino acid sequence shown in SEQ ID NO:25, and the VL may contain the amino acid sequence shown in SEQ ID NO:31. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain the VH and VL from Ab13 of this application.

[0275] For example, the VH may contain the amino acid sequence shown in SEQ ID NO:25, and the VL may contain the amino acid sequence shown in SEQ ID NO:32. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may contain VH and VL from Ab15 of this application.

[0276] In this application, the isolated antigen-binding protein may contain at least one CDR from any of the VH types in this application. In this application, the isolated antigen-binding protein may contain at least one CDR from any of the VL types in this application. The CDR can be obtained according to any classification method, such as CCG, Kabat, Chothia, IMGT, or a combination of Kabat / Chothia, etc.

[0277] In this application, the isolated antigen-binding protein may include HCDR1, HCDR2, and HCDR3 from the VH described in this application, and the VH may contain the amino acid sequence shown in SEQ ID NO:37. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability.

[0278] In this application, the isolated antigen-binding protein may include HCDR1, HCDR2, and HCDR3 from the VH described in this application, and the VH may contain the amino acid sequence shown 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 this application may have Siglec-15 binding ability.

[0279] In this application, the isolated antigen-binding protein may include LCDR1, LCDR2, and LCDR3 in the VL described in this application, and the VL may contain the amino acid sequence shown in SEQ ID NO:38. For example, the isolated antigen-binding protein of this application may have Siglec-15 binding ability.

[0280] In this application, the isolated antigen-binding protein may include LCDR1, LCDR2, and LCDR3 in the VL described in this application, and the VL may contain the amino acid sequence shown 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 this application may have Siglec-15 binding ability.

[0281] In this application, the isolated antigen-binding protein may include an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from the heavy chain constant regions of human IgG, IgA, IgD, IgE, and / or IgM. The antibody heavy chain constant region may be derived from the heavy chain constant region of human IgG. In some embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from the heavy chain constant regions of human IgG1, IgG2, IgG3, and / or IgG4. In some embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from the heavy chain constant region of human IgG1.

[0282] In this application, the isolated antigen-binding protein may include an antibody light chain constant region. The antibody light chain constant region may be derived from the human Igκ constant region.

[0283] In this application, the isolated antigen-binding protein may contain an antibody or an antigen-binding fragment thereof.

[0284] In some embodiments, the antigen-binding fragment may include Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, VHH and / or dAb.

[0285] In some embodiments, the antibody may include a monoclonal antibody. In some embodiments, the antibody may include a murine antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0286] For example, the VH of the chimeric antibody may contain the amino acid sequence shown in SEQ ID NO:23, and the VL of the chimeric antibody may contain the amino acid sequence shown in SEQ ID NO:29.

[0287] In some embodiments, the VH of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:37, and the VL of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:38. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:23, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:24, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:25, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:26, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:27, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:28, and the VL may contain the amino acid sequence shown in SEQ ID NO:29. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:23, and the VL may contain the amino acid sequence shown in SEQ ID NO:30. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:25, and the VL may contain the amino acid sequence shown in SEQ ID NO:31. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:25, and the VL may contain the amino acid sequence shown in SEQ ID NO:32.

[0288] In some embodiments, the antibody comprises a light chain constant region. For example, the light chain constant region may be a light chain constant region derived from a human antibody; for example, the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO:40.

[0289] In some embodiments, the antibody comprises a heavy chain constant region. For example, the heavy chain constant region may be a heavy chain constant region derived from a human antibody; for example, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO:39.

[0290] Furthermore, it should be noted that the isolated antigen-binding protein described in this application may contain heavy chain and / or light chain sequences with one or more conserved sequence modifications. "Conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter antibody binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding protein described in this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conserved amino acid substitution involves replacing an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described in this application may be replaced with other amino acid residues from the same side chain group. Those skilled in the art will know that some conserved sequence modifications do not cause the antigen to lose its binding ability. For example, see 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.

[0291] The Siglec-15 antigen-binding protein described in this application can be identified, screened, or characterized by various assays known in the art.

[0292] For example, the antigen-binding activity of the antigen-binding protein or fusion protein of this application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0293] In this application, the isolated antigen-binding protein is capable of specifically binding to Siglec or its functionally active fragments, such as Siglec-15 or its functionally active fragments.

[0294] In some embodiments, Siglec-15 or its functionally active fragment can be full-length Siglec-15 or a fragment that exerts the functional activity of Siglec-15. For example, the functional activity of Siglec-15 can be the ability to activate downstream pathways of Siglec-15. In some embodiments, Siglec-15 or its functionally active fragment can be isolated Siglec-15 or a mixture of various forms of Siglec-15 or its functionally active fragments. For example, Siglec-15 or its functionally active fragment can be human Siglec-15 or its functionally active fragment, mouse Siglec-15 or its functionally active fragment, or monkey Siglec-15 or its functionally active fragment; for example, Siglec-15 or its functionally active fragment can be Siglec-15 or its functionally active fragment expressed on the cell surface; for example, Siglec-15 or its functionally active fragment can be Siglec-15 or its functionally active fragment bound to a solid medium.

[0295] In some embodiments, the isolated antigen-binding protein can be subjected to affinity determination using a molecular interaction analyzer. For example, the isolated antigen-binding protein can be determined with a K0 of 1.56E-10M or lower. D The value binds to the Siglec-15 protein. For example, the isolated antigen-binding protein may have a K value of 1.56E-10M or lower, 1.5E-10M or lower, 1.3E-10M or lower, 1.1E-10M or lower, 1.0E-10M or lower, 9.0E-11M or lower, 8.0E-11M or lower, 7.0E-11M or lower, 6.0E-11M or lower, 5.0E-11M or lower, 4.0E-11M or lower, 3.0E-11M or lower, 2.0E-11M or lower, 1.0E-11M or lower, 9.0E-12M or lower, 8.0E-12M or lower, 7.0E-12M or lower, 6.0E-12M or lower, or 5.0E-12M or lower. D The value binds to the Siglec-15 protein.

[0296] In some embodiments, the antigen-binding protein isolated in this application can be expressed at a high level. For example, the expression level of the antigen-binding protein isolated in this application can be 50 mg / L or higher. For example, the expression level of the antigen-binding protein isolated in this application can be 50 mg / L or higher, 53 mg / L or higher, 55 mg / L or higher, 60 mg / L or higher, 70 mg / L or higher, 80 mg / L or higher, 90 mg / L or higher, 100 mg / L or higher, 130 mg / L or higher, 140 mg / L or higher, 150 mg / L or higher, 160 mg / L or higher, 200 mg / L or higher, 210 mg / L or higher, 220 mg / L or higher, or 230 mg / L or higher.

[0297] In some embodiments, the antigen-binding protein isolated in this application can be analyzed by FACS method to determine the binding activity of the antigen-binding protein with the cell surface Siglec-15 protein. For example, it could be human Siglec-15 protein, monkey Siglec-15 protein, and / or mouse Siglec-15 protein on the cell surface. For example, the antigen-binding protein isolated in this application can be detected at an EC50 of approximately 3.25 nM or lower. 50 The value is combined with Siglec-15 or its functional fragments. For example, the antigen-binding protein isolated in this application may be at an EC value of about 3.25 nM or lower, about 3.0 nM or lower, about 2.0 nM or lower, about 1.9 nM or lower, about 1.8 nM or lower, about 1.7 nM or lower, about 1.6 nM or lower, about 1.5 nM or lower, about 1.4 nM or lower, about 1.3 nM or lower, about 1.2 nM or lower, about 1.1 nM or lower, about 1.0 nM or lower, about 0.9 nM or lower, about 0.8 nM or lower, about 0.7 nM or lower, about 0.6 nM or lower, about 0.5 nM or lower, about 0.4 nM or lower, about 0.3 nM or lower, about 0.2 nM or lower, or about 0.1 nM or lower. 50 The value is combined with Siglec-15 or its functional fragments.

[0298] In some embodiments, the antigen-binding proteins isolated in this application can affect the proliferative capacity of immune cells. For example, the antigen-binding proteins isolated in this application can affect the function of the immune system; for example, the antigen-binding proteins isolated in this application can affect the proportion of immune cell subsets and / or the number of immune cells. For example, immune cells may include lymphocytes, peripheral blood lymphocytes, peripheral blood mononuclear cells, peripheral blood monocytes, T cells, B cells, and / or NK cells. For example, the antigen-binding proteins isolated in this application can increase the proportion of immune cell subsets; for example, the antigen-binding proteins isolated in this application can increase CD8+. +The proportion of T cells; for example, the isolated antigen-binding protein of this application can increase CD4. + The proportion of T cells. For example, the isolated antigen-binding protein of this application can increase CD8+ in peripheral blood lymphocytes. + The proportion of T cells; for example, the isolated antigen-binding protein of this application can increase CD4+ in peripheral blood lymphocytes. + The proportion of T cells. For example, compared to the blank control alone, the isolated antigen-binding protein of this application can increase CD8. + The number of T cells is approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 7% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, or approximately 90% or more. For example, compared to administering only a blank control, the isolated antigen-binding protein of this application can increase CD4... + The number of T cells is approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 7% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, or approximately 90% or more.

[0299] In some embodiments, the antigen-binding protein isolated in this application can reverse the inhibitory effect of Siglec-15 protein on immune system function. For example, when Siglec-15 protein is administered in vivo or in vitro and / or when Siglec-15 protein is highly expressed in vivo or in vitro, Siglec-15 protein can inhibit immune system function, for example, by affecting the proportion of immune cell subsets, inhibiting immune cell proliferation, and / or reducing the number of immune cells. For example, when immune system function is inhibited by Siglec-15 protein, the antigen-binding protein isolated in this application can enhance, reverse, restore, and / or activate immune system function. For example, after Siglec-15 protein inhibits immune system function and / or the number of immune cells, the antigen-binding protein isolated in this application can increase CD8 levels compared to a blank control. +The number of T cells is approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 7% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, or approximately 90% or more. For example, after the Siglec-15 protein inhibits the function of the immune system and / or the number of immune cells, the isolated antigen-binding protein of this application can increase CD4+. + The number of T cells is approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 7% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, or approximately 90% or more.

[0300] In some embodiments, the antigen-binding protein isolated in this application can inhibit tumor growth. For example, the isolated antigen-binding protein can inhibit tumor growth, such as in colon cancer, with a tumor volume inhibition rate of about 23% or higher. For example, compared to a blank control administered only, the isolated antigen-binding protein can inhibit tumor growth with a tumor volume inhibition rate of about 20% or higher, about 21% or higher, about 22% or higher, about 23% or higher, about 24% or higher, about 25% or higher, about 26% or higher, about 27% or higher, about 28% or higher, about 29% or higher, about 30% or higher, about 40% or higher, or about 50% or higher.

[0301] In some embodiments, the antigen-binding protein isolated in this application can affect the ability of cytokines to be secreted. For example, the secretion of cytokines in this application can include in vivo, in vitro, and / or extracorporeal secretion. For example, the secretion of cytokines in this application can include lipopolysaccharide-induced cytokine secretion. For example, lipopolysaccharide-induced cytokine secretion can refer to an increase in the number of cytokines secreted in vivo and / or in vitro, an increase in the concentration of cytokines in the culture medium, and / or an increase in the concentration of cytokines in peripheral blood after administration of lipopolysaccharide in vivo and / or in vitro. For example, when the antigen-binding protein of this application is administered to a subject first, followed by the administration of lipopolysaccharide, the number of cytokines in the subject increases compared to administration of lipopolysaccharide alone or administration of a blank control followed by the administration of lipopolysaccharide. For example, compared to applying lipopolysaccharide alone or applying a blank control followed by lipopolysaccharide, the antigen-binding protein of this application can increase cytokine secretion by approximately 1%, 2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 350%, and 400%. For example, the cytokines of this application may include inflammatory factors, such as inflammatory response-related factors or pro-inflammatory factors TNF-α and / or IL-6. For example, compared to applying lipopolysaccharide alone or applying a blank control first and then applying lipopolysaccharide, the antigen-binding protein of this application can increase TNF-α secretion by more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 7%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 350%, and more than 400%. For example, compared to applying lipopolysaccharide alone or applying a blank control followed by lipopolysaccharide, the antigen-binding protein of this application can increase IL-6 secretion by approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 7% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, approximately 100% or more, approximately 150% or more, approximately 200% or more, approximately 250% or more, approximately 300% or more, approximately 350% or more, and approximately 400% or more.

[0302] On the one hand, this application provides a polypeptide molecule, nucleic acid molecule, carrier, immunoconjugate, cell and drug composition.

[0303] On the other hand, this application provides polypeptide molecules that may contain the isolated antigen-binding protein of this application.

[0304] 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 this application may comprise structures other than amino acids; for example, the polypeptide molecule of this application may comprise nucleic acids, polysaccharides, lipids, small molecules, and any combination thereof.

[0305] On the other hand, this application provides isolated nucleic acid molecules that may encode the isolated antigen-binding protein and / or the polypeptide of this application. For example, they may be generated or synthesized by: (i) in vitro amplification, for example by polymerase chain reaction (PCR); (ii) clonal recombination; (iii) purification, for example by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, for example by chemical synthesis.

[0306] On the other hand, this application provides a vector that may contain the nucleic acid molecule of this application. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed in an appropriate 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 regulating gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells to express the genetic material elements it carries within the host cells. The vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. Furthermore, the vector may include components that facilitate its entry into cells, such as viral particles, liposomes, or protein coats, but not only these substances.

[0307] On the other hand, this application also provides immunoconjugates, which may comprise the isolated antigen-binding protein described in this application and / or the polypeptide of this application. In some embodiments, the isolated antigen-binding protein or fragment thereof of this application may be linked to another reagent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, etc. This linking may be through one or more covalent bonds, or non-covalent interactions, and may include chelation. A variety of adapters (which may be known in the art) may be used to form immunoconjugates. Furthermore, immunoconjugates may be provided in the form of fusion proteins, which may be expressed by polynucleotides encoding the immunoconjugate. The immunoconjugates may also comprise, for example, antibody-drug conjugates (ADCs). In an ADC, the antibody and the therapeutic agent may be cross-linked via an adapter, which may be a cleavable adapter, such as an in vivo and / or in vitro enzymatically cleavable adapter, such as a peptide adapter, a disulfide adapter, or a hydrazone adapter.

[0308] On the other hand, this application provides a cell that may contain the isolated antigen-binding protein of this application, the polypeptide molecule of this application, the immunoconjugate of this application, the nucleic acid molecule of this application, or the vector of this application. In some embodiments, each or every host cell may contain one or more of the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain multiple (e.g., two or more) or more types (e.g., two or more) of the nucleic acid molecules or vectors described in this application. For example, the vectors described in this application may be introduced into the host cell, such as eukaryotic cells, such as cells derived from plants, fungi, or yeast cells. In some embodiments, the cell may be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2 / 0 cells, NSO cells, or myeloma cells. The vector described in this application can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0309] On the other hand, this application also provides pharmaceutical compositions that may comprise antigen-binding proteins isolated by this application, polypeptide molecules of this application, immunoconjugates of this application, nucleic acid molecules of this application, carriers of this application, and / or cells of this application, and optionally pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition may be non-toxic to the recipient at the doses and concentrations used. The pharmaceutical compositions of the present invention may include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0310] In some embodiments, the pharmaceutical composition may also contain more than one active compound, typically those with complementary activities that do not adversely affect each other. The type and effective amount of such a drug may depend, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.

[0311] In some embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents that are compatible with drug administration and are generally safe and non-toxic.

[0312] In some embodiments, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally. In some embodiments, the pharmaceutical composition may be administered continuously. This continuous (or uninterrupted) administration may be achieved using a small pump system worn by the patient to measure the amount of therapeutic agent flowing into the patient's body.

[0313] In some embodiments, this application provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition of this application may comprise the isolated Siglec-15 binding protein of this application, a polypeptide comprising the Siglec-15 binding protein, an immunoconjugate of this application, a nucleic acid molecule of this application, a carrier of this application, a cell of this application, and / or a pharmaceutical composition of this application, and may comprise suitable active substances having preventive and / or therapeutic effects.

[0314] In some embodiments, the pharmaceutical combination may comprise a first formulation and a second formulation, the first formulation comprising the isolated Siglec-15 binding protein of the present application and / or a polypeptide comprising the Siglec-15 binding protein and a pharmaceutically acceptable first carrier, and the second formulation comprising a suitable therapeutically active substance and a pharmaceutically acceptable second carrier.

[0315] On one hand, this application provides a kit. For example, the kit may comprise the isolated Siglec-binding protein of this application, a polypeptide containing the Siglec-binding protein, a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, a carrier containing the nucleic acid molecule encoding the isolated Siglec-binding protein of this application, an immunoconjugate containing the isolated Siglec-binding protein of this application, cells containing the above substances, a pharmaceutical composition of this application, and / or a drug combination of this application. For example, Siglec in this application may be Siglec-15. In some embodiments, the kit may be used to detect the presence and / or content of Siglec-15 in a sample.

[0316] On the one hand, this application provides a preparation method.

[0317] On the other hand, this application provides a method for preparing the isolated Siglec-15 binding protein and / or peptides containing the Siglec-15 binding protein of this application. The method may include culturing the host cells described in this application under conditions that cause expression of the isolated Siglec-15 binding protein and / or peptides containing the Siglec-15 binding protein of this application. For example, this can be achieved by using appropriate culture media, appropriate temperatures, and culture times, methods known to those skilled in the art.

[0318] Any method suitable for producing monoclonal antibodies can be used to produce the antigen-binding protein of this application. For example, animals can be immunized with Siglec-15 or fragments thereof, either conjugated or naturally occurring. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, or one or more routes. For example, hybridoma preparation methods can be used to obtain spleen cells from immunized mice, fuse them with SP2 / 0 myeloma cells, and screen for hybridoma cell lines using HAT.

[0319] Any suitable form of Siglec-15 can be used as an immunogen (antigen) to generate non-human antibodies specific to Siglec-15 and to screen for the biological activity of said antibodies. For example, the stimulating immunogen can be full-length mature human Siglec-15, which may include a natural homodimer or a peptide containing one or more epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0320] Chimeric human antibodies can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. In this application, the antibody can be an IgG antibody, and IgG1, IgG2, IgG3, or IgG4 subtypes can be used. The necessary constant domain sequence can be optimized to produce the desired biological activity by screening antibodies using biological assays in the art. Similarly, any class of light chains can be used in the compounds and methods of this application. For example, the κ chain or a variant thereof can be used in the compounds and methods of this application.

[0321] On the one hand, this application provides a method and a use.

[0322] For example, this application provides a method for detecting Siglec in a sample, which may include administering the isolated Siglec-binding protein of this application, administering a polypeptide containing the Siglec-binding protein, administering a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, administering a carrier containing a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, administering an immunoconjugate containing the isolated Siglec-binding protein of this application, administering cells containing the above substances, administering a pharmaceutical composition of this application, administering a pharmaceutical combination of this application, and / or using a kit of this 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 not be a diagnostic method. For example, the Siglec in this application may be Siglec-15.

[0323] For example, this application provides a method for influencing the binding of Siglec or its functionally active fragment to its ligand, which may include administering the isolated Siglec-binding protein of this application, administering a polypeptide containing the Siglec-binding protein, administering a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, administering a carrier containing a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, administering an immunoconjugate containing the isolated Siglec-binding protein of this application, administering cells containing the above substances, administering a pharmaceutical composition of this application, administering a pharmaceutical combination of this application, and / or using a kit of this application. In some cases, the method for influencing the binding of Siglec or its functionally active fragment to its ligand may be an in vitro method. In some cases, the method for influencing the binding of Siglec or its functionally active fragment to its ligand may be a non-therapeutic method. In some cases, the method for influencing the binding of Siglec or its functionally active fragment to its ligand may not be a diagnostic method. For example, the Siglec in this application may be Siglec-15. For example, the ligand of Siglec in this application may comprise leucine-rich repeat-containing protein 4C and sialyl-Tn. For example, influencing the binding of Siglec or its functionally active fragment to its ligand may result in a reduction, relative to the blank control, of the inhibitory effect of Siglec on immune system function by approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 7% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, approximately 100% or more, approximately 150% or more, approximately 200% or more, approximately 250% or more, approximately 300% or more, approximately 350% or more, or approximately 400% or more.

[0324] For example, this application provides a method for influencing immune cell proliferation, which may include administering the isolated Siglec-binding protein of this application, administering a polypeptide containing the Siglec-binding protein, administering a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, administering a carrier containing a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, administering an immunoconjugate containing the isolated Siglec-binding protein of this application, administering cells containing the above substances, administering a pharmaceutical composition of this application, administering a pharmaceutical combination of this application, and / or using a kit of this application. For example, Siglec in this application may be Siglec-15. In some cases, the method for influencing immune cell proliferation may be an in vitro method. In some cases, the method for influencing immune cell proliferation may be a non-therapeutic method. In some cases, the method for influencing immune cell proliferation may not be a diagnostic method. For example, the effect of this application on immune cell proliferation may manifest as reducing the inhibition of immune cell proliferation induced by Siglec-15. For example, it may affect the proportion of immune cell subsets and / or the number of immune cells. For example, immune cells can include lymphocytes, peripheral blood lymphocytes, peripheral blood mononuclear cells, and / or peripheral blood monocytes. For example, the proportion of immune cell subsets can be increased; for example, CD8+ can be increased. + The proportion of T cells; for example, increasing CD4. + The proportion of T cells. For example, increasing CD8+ in peripheral blood lymphocytes. + The proportion of T cells; for example, increasing CD4+ in peripheral blood lymphocytes. + The proportion of T cells. For example, compared to the blank control, CD8 can be increased. + The number of T cells is approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 7% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, or approximately 90% or more. For example, compared to the blank control, it can increase CD4... + The number of T cells is approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 7% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, or approximately 90% or more.

[0325] For example, this application provides a method for influencing cytokine secretion, which may include administering the isolated Siglec-binding protein of this application, administering a polypeptide containing the Siglec-binding protein, administering a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, administering a carrier containing a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, administering an immunoconjugate containing the isolated Siglec-binding protein of this application, administering cells containing the above substances, administering a pharmaceutical composition of this application, administering a pharmaceutical combination of this application, and / or using a kit of this application. For example, Siglec in this application may be Siglec-15. In some cases, the method for influencing cytokine secretion may be an in vitro method. In some cases, the method for influencing cytokine secretion may be a non-therapeutic method. In some cases, the method for influencing cytokine secretion may not be a diagnostic method. For example, influencing cytokine secretion may manifest as influencing lipopolysaccharide-induced cytokine secretion, such as TNF-α and / or IL-6 secretion. For example, compared to the blank control, it can increase TNF-α secretion by more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 7%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 350%, and more than 400%. For example, compared to the blank control, it can increase IL-6 secretion by more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 7%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 350%, and more than 400%.

[0326] On the other hand, this application provides the use of the isolated Siglec-binding protein of this application, a polypeptide containing the Siglec-binding protein, a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, a carrier containing a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, an immunoconjugate containing the isolated Siglec-binding protein of this application, cells containing the above substances, the drug composition of this application, and / or the pharmaceutical combination of this application in the preparation of a kit. For example, the kit can be used to detect the presence and / or content of Siglec in a sample. For example, the Siglec in this application can be Siglec-15.

[0327] On one hand, this application provides a preventive and / or therapeutic use. Siglec-15 can serve as a novel target, and antibodies targeting Siglec-15 can reverse the suppression of immune system function by Siglec-15. Existing data suggest that antibodies targeting Siglec-15 can activate an immune response, and antibodies against Siglec-15 can be developed into a drug for the prevention and / or treatment of diseases and / or conditions, or antibodies against Siglec-15 can be used as a method for the prevention and / or treatment of diseases and / or conditions.

[0328] On the other hand, this application provides the use of the isolated Siglec-binding protein of this application, a polypeptide containing the Siglec-binding protein, a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, a carrier containing a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, an immunoconjugate containing the isolated Siglec-binding protein of this application, a cell containing the above substances, a pharmaceutical composition of this application, a pharmaceutical combination of this application, and / or a kit of this application in the preparation of a medicament, which can be used to prevent and / or treat diseases and / or conditions. For example, the diseases and / or conditions of this application may be immune-related disorders and / or proliferative disorders. For example, the diseases and / or conditions of this application may be diseases related to abnormal Siglec protein expression or dysfunction. For example, the diseases and / or conditions of this application may be Siglec-positive tumors. For example, the diseases and / or conditions of this application may be tumors with high Siglec expression. In this application, the diseases and / or conditions may be Siglec-related diseases and / or conditions. For example, the Siglec in this application may be Siglec-15.

[0329] In this application, the disease and / or condition may include tumors. In this application, the disease and / or condition may include conditions of excessive cell growth or proliferation. In this application, the disease and / or condition may include proliferative diseases, neoplastic diseases, and / or immune diseases. For example, the tumor in this application may be a tumor and / or infection related to abnormal expression or function of the Siglec protein. For example, the tumor in this application may be a Siglec-positive tumor and / or a tumor with high Siglec expression. In this application, the tumor may include a solid tumor. In this application, the disease and / or condition may include gastrointestinal tumors. In this application, the disease and / or condition may include intestinal tumors. In this application, the disease and / or condition may include colon cancer. For example, Siglec in this application may be Siglec-15.

[0330] On the other hand, this application provides an isolated Siglec-binding protein, a polypeptide containing the Siglec-binding protein, a nucleic acid molecule encoding the isolated Siglec-binding protein, a carrier containing a nucleic acid molecule encoding the isolated Siglec-binding protein, an immunoconjugate containing the isolated Siglec-binding protein, a cell containing the above substances, a pharmaceutical composition, a drug combination, and / or a kit, which can be used to prevent and / or treat diseases and / or conditions. For example, the diseases and / or conditions of this application may be immune-related disorders and / or proliferative disorders. For example, the diseases and / or conditions of this application may be diseases related to abnormal Siglec protein expression or function. For example, the diseases and / or conditions of this application may be Siglec-positive tumors. For example, the diseases and / or conditions of this application may be tumors with high Siglec expression. In this application, the diseases and / or conditions may be Siglec-related diseases and / or conditions. For example, the Siglec in this application may be Siglec-15.

[0331] In this application, the disease and / or condition may include tumors. In this application, the disease and / or condition may include conditions of excessive cell growth or proliferation. In this application, the disease and / or condition may include proliferative diseases, neoplastic diseases, and / or immune diseases. For example, the tumor in this application may be a tumor and / or infection related to abnormal expression or function of the Siglec protein. For example, the tumor in this application may be a Siglec-positive tumor and / or a tumor with high Siglec expression. In this application, the tumor may include a solid tumor. In this application, the disease and / or condition may include gastrointestinal tumors. In this application, the disease and / or condition may include intestinal tumors. In this application, the disease and / or condition may include colon cancer. For example, Siglec in this application may be Siglec-15.

[0332] On the other hand, this application provides a method for preventing and / or treating diseases and / or conditions, which may include administering to a subject in need the isolated Siglec-binding protein of this application, a polypeptide containing the Siglec-binding protein, a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, a carrier containing a nucleic acid molecule encoding the isolated Siglec-binding protein of this application, an immunoconjugate containing the isolated Siglec-binding protein of this application, cells containing the above substances, a pharmaceutical composition of this application, a drug combination of this application, and / or a kit of this application. For example, the disease and / or condition of this application may be an immune-related disorder and / or a proliferative disorder. For example, the disease and / or condition of this application may be a disease related to abnormal Siglec protein expression or dysfunction. For example, the disease and / or condition of this application may be a Siglec-positive tumor. For example, the disease and / or condition of this application may be a tumor with high Siglec expression. In this application, the disease and / or condition may be a Siglec-related disease and / or condition. For example, the Siglec in this application may be Siglec-15.

[0333] In this application, the disease and / or condition may include tumors. In this application, the disease and / or condition may include conditions of excessive cell growth or proliferation. In this application, the disease and / or condition may include proliferative diseases, neoplastic diseases, and / or immune diseases. For example, the tumor in this application may be a tumor and / or infection related to abnormal expression or function of the Siglec protein. For example, the tumor in this application may be a Siglec-positive tumor and / or a tumor with high Siglec expression. In this application, the tumor may include a solid tumor. In this application, the disease and / or condition may include gastrointestinal tumors. In this application, the disease and / or condition may include intestinal tumors. In this application, the disease and / or condition may include colon cancer. For example, Siglec in this application may be Siglec-15.

[0334] In this application, the application can be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal application.

[0335] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the binding protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0336] Example

[0337] Example 1: Preparation of Recombinant Cell Lines

[0338] An expression plasmid was constructed by codon optimization of the extracellular region of human Siglec-15 (amino acid sequence described in Uniprot, Q6ZMC9, 20-263) and the transmembrane region of platelet-derived growth factor receptor (PDGFR) (amino acid sequence described in P09619, 513-561). Lentiviral cells were packaged and infected with 293T cells. After antibiotic-induced selection, Siglec-15 expression was detected, and positive clones were selected for amplification culture to obtain the recombinant cell line human S15ECD-293T overexpressing the amino acid sequence of the human Siglec-15 extracellular region. A similar method can be used for the preparation of monkey S15ECD-293T.

[0339] The full-length mouse Siglec-15 amino acid sequence (which can be described in Uniprot, A7E1W8) was linked with the full-length DAP12 amino acid sequence (O54885) using T2A (the amino acid sequence can be shown as GSGEGRGSLLTCGDVEENPGP in SEQ ID NO: 41). After codon optimization, an expression plasmid was constructed using conventional methods, packaged with lentivirus, and used to infect CT26 cells to prepare the recombinant cell line mouse S15-CT26 that overexpresses the full-length mouse Siglec-15.

[0340] Example 2: Siglec-15 binding protein screening and maturation

[0341] Phage screening for Siglec-15 binding protein

[0342] Two rounds of liquid-phase screening were performed using an antibody library targeting the human Siglec-15 antigen. 1×10⁻⁶ antibodies were used. 13 CFU-bound phages were pre-bound with 50 ml of streptavidin magnetic beads for 30 minutes. Non-specifically bound phages were removed using a magnetic rack. This process was repeated three times. The background-removed phages were then incubated with 10 μg of biotinylated human Siglec-15 protein (HS-15 Biotin or human S-15 Biotin, Acrobiosystems, catalog number: SG5-H82E9) and 150 μg of streptavidin magnetic beads at 37°C for 15 minutes. The phages were washed 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 then infected with logarithmic-phase *E. coli* SS320. After 1 hour of infection, a final concentration of 1.5 × 10⁻⁶ ppm was added. 9 CFU helper phage M13KO7 was cultured overnight at 32°C. The next day, the supernatant was collected by centrifugation at 15000g for 10 minutes, and 1 / 5 volume of 20% PEG / NaCl solution was added to precipitate at 4°C for 1 hour. The precipitate was then centrifuged at 15000g for 20 minutes, and the precipitate was resuspended in 1 ml of 1×PBS for a second round of panning. The second round was the same as the first round, except that the amount of antigen was halved in each subsequent round. Enrichment was determined using an enzyme-linked immunosorbent assay (ELISA) of phages. Human Siglec-15 protein at a concentration of 2 ng / μl was added to each well at 100 μl and incubated overnight at 4°C. Then, 1×10⁻⁶ phages enriched from the second round of panning were added. 11 CFU / well, incubated at 37°C for 40 minutes. Unbound phages were washed away with PBST. 100 μL of 1:5000 diluted HRP-conjugated mouse anti-FLAG monoclonal antibody (Sigma, 8592) was added to each well, and incubated at 37°C for 30 minutes. Unbound phages were washed away with PBST, and alkaline phosphatase chromogenic solution was added. The absorbance was read at 450 nm using an ELISA instrument. Results are shown in Table 1.

[0343] Table 1: ELISA binding data of bacteriophage and Siglec-15

[0344] Human Siglec-15 0.4845 irrelevant proteins 0.0666

[0345] Yeast display screening of Siglec-15 binding protein

[0346] like Figure 1 The method for constructing the yeast display shown in this application involves using a plasmid selected from a phage antibody library as a template, designing primers to amplify the single-chain antibody gene (scFv) via polymerase chain reaction (PCR). The PCR-amplified scFv gene fragment is recovered and co-transformed with the yeast display plasmid into *Saccharomyces cerevisiae* strain EBY100 (purchased from ATCC). Homologous recombination in *Saccharomyces cerevisiae* allows the scFv gene to be inserted into the yeast display plasmid, thereby achieving the display of single-chain antibodies on the yeast cell wall surface. The yeast display single-chain antibody library is named JYYDL020. After electroporation, the JYYDL020 library was cultured overnight in 100 mL of SD-Trp medium (Clontech, catalog number: 630308) at 30°C and 225 rpm. 1.0 × 10⁻⁶ cells were then collected. 8 The bacterial culture was 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℃ and 225 rpm for 24 hours, and then stored at 4℃ for later use.

[0347] After library induction, the bacterial culture was used to determine the OD value of the bacterial culture. 600 1OD is 1.0 × 10 7 Cell count was calculated using a value of 4.0 × 10⁻⁶. 8 Cells underwent a first round of sorting to enrich the positive cell population that bound HS-15 Biotin. 2.0 × 10⁶ cells were collected. 7 Cells underwent a second round of negative selection using irrelevant antigens to remove non-specifically bound cell populations. After sorting, an appropriate amount of cells was plated on SD-Trp solid medium and incubated at 30°C for 3 days.

[0348] Yeast monoclonal identification of Siglec-15 binding protein

[0349] From the first round of screening products in the JYYDL020 antibody library, one 48-well plate was sequenced and numbered Y11. From the second round of screening products in the JYYDL020 library, two 48-well plates were sequenced and numbered Y14-Y15. After sequence analysis, single-chain antibodies with single sequences were obtained, and the corresponding yeast monoclonal clones were analyzed by flow cytometry according to Table 2.

[0350] Table 2: Flow Cytometry Staining Protocol for Identifying Monoclonal Yeast Colonies

[0351]

[0352]

[0353] The strength of the cell population bound to HS-15 Biotin in Scheme 1 was reflected by the mean fluorescence intensity (MFI) of the PE. Similarly, Schemes 2, 3, and 4 can be used to evaluate the binding levels of mouse S-15 Biotin (Acrobiosytems, catalog number: SG5-M52H7, which can be biotin-labeled) and monkey S-15-Fc (Acrobiosytems, catalog number: SG5-C5253) as well as non-specific binding levels. The results are shown in Table 3.

[0354] Table 3: Flow cytometry staining results and cell numbering of monoclonal yeast colonies

[0355]

[0356] Based on the staining results, clones that did not bind to humans, monkeys, or mice were excluded. The final single-clone sequence was used to construct a plasmid for expression of the Siglec-15 binding protein.

[0357] Expression of Siglec-15 binding protein

[0358] The full-length light and heavy chain protein sequences of the binding protein obtained above were codon-optimized separately. The codon-optimized DNA fragment was synthesized (Genewiz), and the cloned gene fragment was inserted into the expression vector pcDNA3.4 (Life Technologies) in IgG1 form. After plasmid amplification and extraction, the expression plasmid was co-transformed into ExpiCHO cells (ThermoFisher Scientific, A29133). Transient antibody expression was performed according to the supplier's ExpiCHO expression system method, as follows: ExpiCHO cells were cultured in 25 mL of culture medium at 36.5°C and 8% CO2 concentration until a density of 6 × 10⁶ cells / mL was reached. 6 / mL, use ExpiFecta transfection reagent to transfect 10μg of antibody light and heavy chain expression plasmids into cells; one day after transfection, take 150μL and 4mL of ExpiCHO enhancer and ExpiCHO adjuvant and add them to the cultured cells, continue to culture for 9 days, centrifuge at 3500 rpm at 4℃ and collect the supernatant. Mix the magnetic beads (AmMag™ Protein A, Genscript, L00695) with the supernatant of the Siglec-15 binding protein expression in this application, incubate at room temperature for 2 hours, wash twice with PBS and discard the supernatant, add an appropriate amount of elution buffer (Protein G or A Sefinose™ Elution buffer, Sangon, C600481), mix thoroughly and place on a test tube rack for static incubation for 5 minutes, resuspend the magnetic beads 2-3 times during incubation, repeat the elution twice, immediately add an appropriate amount of neutralization solution 1M Tris-HCl, pH 7.5 (Sangon, B548124) for neutralization and use. The purified Siglec-15 binding protein information is shown in Table 4.

[0359] Table 4: Expression data of fully human binding proteins of Siglec-15

[0360]

[0361] After purification, the Siglec-15 binding protein underwent affinity maturation after affinity assays, physicochemical property analysis, cell binding assays, and in vitro functional activity identification.

[0362] Construction of Affinity Maturation Mutant Library

[0363] The obtained Siglec-15 binding protein underwent affinity maturation to enhance its affinity for human Siglec-15. Amino acids at the antigen-binding determinant (CDR) sites of the Siglec-15 binding protein were randomly mutated to construct mutant libraries for each CDR region. Yeast display technology was used for high-throughput screening of sequences with strong antigen-specific binding affinity. The amino acid sequences of the light and heavy chain variable regions of the Siglec-15 binding protein were encoded according to Chothia coding rules, and the CDR regions were defined according to Chothia. For the light chain variable regions CDR1, CDR1, and CDR3, and the heavy chain variable regions CDR1, CDR2, and CDR3, NNK (N represents any nucleotide, K represents G or T) mutation primers were designed to amplify the gene fragments of each CDR mutant library using polymerase chain reaction (PCR). The gene fragments of each CDR mutant library and the yeast display plasmid were then transformed into *Saccharomyces cerevisiae* strain EBY100, allowing each CDR mutant library to be displayed on the yeast surface in Fab form. Meanwhile, the parental sequence of the Siglec-15 binding protein of this application was displayed on the surface of yeast in Fab form as a control.

[0364] Screening of Affinity Maturation Mutant Library

[0365] After cultivation and induction, 1×10⁻⁶ samples were taken from each library. 9 Cells were first enriched using a magnetic bead sorting system. Cells from each library were resuspended in 1×PBSA containing 1 nM HS-15 Biotin and incubated for 30 minutes. After washing, antibiotin beads were added, mixed, and incubated for 10 minutes. Positive cells were collected by passing through a magnetic column. After further culture and induction, 3.0 × 10⁶ cells were collected from each positive cell. 7 Cells were sorted a second time using 1 nM HS-15 Biotin to collect cell populations exhibiting high levels of antigen binding. Following the second round of sorting, cells were incubated with 0.1 nM HS-15 Biotin antigen, washed with 10 mL of 1×PBSA at room temperature for 2 hours, and then sorted a third time. After sorting, cells were plated on SD-Trp, Leu solid medium and incubated at 30°C for 3 days. Single clones were picked and sequenced to obtain single sequences of mutations in each CDR region of the light and heavy chains.

[0366] The obtained CDR region mutant plasmids of the light and heavy chains were mixed with the parental plasmids and co-transformed into a yeast strain to construct a combined library of light and heavy chain mutants, designated JYYDL062. After culture and induction, 2.0 × 10⁻⁶ cells were collected from the library. 7 Cells were sorted by flow cytometry using 0.1 nMHS-15 Biotin, and cell populations exhibiting high levels of antigen binding were collected. After sorting, cells were plated for growth, and single clones were selected for sequencing analysis.

[0367] Identification of monoclonal antibodies

[0368] Sequencing analysis was performed, and single clones with unique sequences were identified by flow cytometry staining. Each clone 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 signal intensity of different clones binding to the antigen was compared. Based on the staining results, clones with significantly increased binding strength to HS-15 Biotin were selected for further evaluation. The evaluation results and sequence information are shown in Tables 5A, 5B, 5C, and 5D.

[0369] Table 5A: Affinity Mature Cloning Assessment and Expression Numbers of Siglec-15 Binding Protein in this Application

[0370]

[0371]

[0372] Table 5B: Sequence number of the Siglec-15 binding protein in this application (SEQ ID NO)

[0373] Ab0 7 2 1 13 10 9 23 29 Ab2 8 2 1 13 10 9 24 29 Ab3 7 3 1 13 10 9 25 29 Ab5 7 4 1 13 10 9 26 29 Ab6 7 5 1 13 10 9 27 29 Ab7 7 6 1 13 10 9 28 29 Ab9 7 2 1 14 10 9 23 30 Ab13 7 3 1 13 11 9 25 31 Ab15 7 3 1 13 12 9 25 32

[0374] Table 5C: Sequence number of the Siglec-15 binding protein in this application (SEQ ID NO)

[0375] Ab0 15 16 17 18 19 20 21 22 Ab2 15 16 17 18 19 20 21 22 Ab3 15 16 17 18 19 20 21 22 Ab5 15 16 17 18 19 20 21 22 Ab6 15 16 17 18 19 20 21 22 Ab7 15 16 17 18 19 20 21 22 Ab9 15 16 17 18 19 20 21 22

[0376] Ab15 15 16 17 18 19 20 21 22

[0377] Table 5D: Sequence number of the Siglec-15 binding protein in this application (SEQ ID NO)

[0378] Light chain constant region sequence 40

[0379] Siglec-15 binding protein affinity maturation antibody expression, affinity assay

[0380] The Siglec-15 binding protein of this application was expressed and purified using the ExpiCHO expression system method described above in this application. Information on the purified Siglec-15 binding protein is shown in Table 6.

[0381] Table 6: Expression data of Siglec-15 binding protein in this application

[0382]

[0383] Example 3: Affinity determination of Siglec-15 binding protein

[0384] The affinity of the Siglec-15 binding protein of this application to human Siglec-15 (Acro, catalog number: SG5-H52H3) was determined using Octet RED96e (Fortebio). Both the antigen and the Siglec-15 binding protein of this application were diluted with 1xPBST (1xPBS: Sangon Biotech, B548117-0500; 0.02% Tween 20: Sigma-Aldrich, P1379). The concentration of the antigen used was 30 nM, and the concentration of the Siglec-15 binding protein used was 33.3 nM.

[0385] Sample testing (Octet Data Acquisition 11.1.0.11)

[0386] First, samples were added to a 96-well plate (Greiner bio-one, 655209) at a concentration of 200 μL / well. Software parameters were then set, with the plate temperature at 30°C and the standard kinetic signal collection frequency at 5.0 Hz. Next, the AHC sensor (Fortébio, catalog number: 18-0015) was pre-wetted with 1xPBST for 10 minutes before detection. Each cycle included the following steps: 1) immersion in buffer for 60 seconds; 2) detection of non-specific binding of the antigen to the sensor; 3) regeneration with 10 mM pH 1.7 glycine solution; 4) immersion in buffer for 60 seconds; 5) immobilization of the Siglec-15 binding protein onto the sensor for 20 seconds; 6) immersion of the sensor in buffer for 180 seconds; 7) binding of the antigen to the Siglec-15 binding protein for 180 seconds; 8) dissociation of the antigen from the Siglec-15 binding protein for 10 minutes; and 9) sensor regeneration.

[0387] Data Analysis

[0388] Using Fortebio's Data Analysis 12.0 software, the binding rate constant (kon) and dissociation rate constant (kdis) of the antigen-Siglec-15 binding protein of this application were measured in a 1:1 binding ratio. Based on this, the equilibrium dissociation constant (K) of the Siglec-15 binding protein of this application was calculated. D The results are shown in Tables 7A, 7B, and 7C below:

[0389] Table 7A: First determination of Siglec-15 binding protein affinity

[0390] Ab0 0.2435 1.09E-10 2.98E+05 3.24E-05

[0391] Table 7B: Second determination of Siglec-15 binding protein affinity

[0392] Ab2 0.2615 1.554E-10 4.48E+05 6.96E-05 Ab3 0.288 4.44E-11 4.40E+05 1.95E-05 Ab5 0.2768 7.60E-12 3.38E+05 2.57E-06 Ab6 0.2851 8.434E-12 3.40E+05 2.87E-06 Ab7 0.2839 5.084E-12 2.82E+05 1.44E-06 Ab9 0.2592 8.735E-11 4.45E+05 3.88E-05 Ab13 0.3054 6.807E-11 5.56E+05 3.79E-05 Ab15 0.3612 7.298E-11 5.38E+05 3.93E-05

[0393] Table 7C: Third determination of Siglec-15 binding protein affinity

[0394] Ab15 0.3426 9.161E-11 4.75E+05 4.35E-05 Ab9 0.315 6.914E-11 5.19E+05 3.59E-05

[0395] Example 4: Assay of the cell-binding activity of Siglec-15 binding protein

[0396] The function of the purified Siglec-15 binding protein of this application in binding to human S15ECD-293T cells was identified by flow cytometry. Human S15ECD-293T cells prepared by the method described in this application were cultured in 15 cm cell culture dishes to 90% confluence. The culture medium was aspirated, and the cells were washed once with PBS buffer (Thermo Fisher, 10010049), then treated with enzyme-free cell dissociation solution (Versene solution, Thermo Fisher, 15040066) and collected. The cells were washed twice with PBS buffer and counted. The required number of cells were collected, centrifuged, and resuspended in FACS buffer (PBS containing 2% FBS) to a concentration of 2 × 10⁶ cells / mL. 6 Cells were incubated at room temperature for 15 minutes, and then the cell suspension was added at 50 μL / well to a U-shaped 96-well plate (Corning, 3798). The Siglec-15 binding protein of this application was serially diluted with FACS buffer, and the diluted solution was added to the above 96-well plates at 50 μL / well, and incubated at 4°C for 1 hour. After centrifugation and discarding 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 the cells were 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. After 10 minutes, the cells were washed twice with FACS buffer. After resuspending the cells with 30 μL / well of FACS buffer, the results were detected and analyzed using an Intellyciteplus (Sartorius) flow cytometer. A similar method was used to identify the function of purified anti-human Siglec-15 binding protein in binding mouse S15-CT26 and monkey S15ECD-293T cells.

[0397] like Figure 2 , Figure 3 and Figure 4 As shown, the Siglec-15 binding protein of this application can bind to Siglec-15 on the cell surface. Figure 2 The results shown are the detection results of the binding of the Siglec-15 binding protein of this application to human Siglec-15 on the cell surface. Figure 3 The results shown are the detection results of the binding of the Siglec-15 binding protein of this application to monkey Siglec-15 on the cell surface. Figure 4 The results show the detection of the binding of the Siglec-15 binding protein of this application to mouse Siglec-15 on the cell surface. The Ab9, Ab15, and isotype control of this application were bound to human S15ECD-293T cells. 50The values ​​(nM) were 1.516, 0.4750, and NA (no data available); This application used Ab9, Ab15, and isotype controls bound to monkey S15ECD-293T cells for ECMO. 50 The values ​​(nM) were 0.9224, 0.4746, and NA (no available data); EC50 of Ab9, Ab15, and isotype control mice S15-CT26 cells were used in this application. 50 The values ​​(nM) are 3.247, 1.827 and NA (no data available).

[0398] Example 5: Physicochemical property evaluation of Siglec-15 binding protein

[0399] The physicochemical properties of the Siglec-15 binding protein in this application can be detected by referring to the following methods.

[0400] EC-HPLC purity analysis

[0401] (1) Dilute the sample to 1 mg / mL, mix well, centrifuge at 12000 rpm for 5 min, transfer the supernatant to a sample vial, and place it in the HPLC sample tray. Set the chromatographic conditions: the column can be a TSK G3000SWxl; the detection wavelength can be 280 nm; the column temperature can be 25℃; the sample chamber temperature can be 5℃; and the flow rate can be 0.5 mL / min.

[0402] (2) After the column was equilibrated with the mobile phase (200mM phosphate buffer, pH 6.8), the sample was injected for analysis. Data analysis was performed using chromatographic software, and the peak area percentage of each peak was calculated using the peak area normalization method.

[0403] HIC-HPLC analysis

[0404] (1) Dilute the sample to 1 mg / ml, centrifuge and collect the supernatant for analysis. Set the chromatographic conditions as follows: the column can be MAbPac™ HIC-10; the detection wavelength can be 214 nm; the column temperature can be 30 ℃; the sample chamber temperature can be 5 ℃; and the flow rate can be 0.8 mL / min.

[0405] (3) Gradient elution was performed using mobile phase A (50mM phosphate buffer / 1M ammonium sulfate, pH 7.0) and mobile phase B (50mM phosphate buffer, pH 7.0), and the retention time of the main peak was recorded.

[0406] Analysis of melting temperature (Tm)

[0407] Dilute the test sample to 1 mg / mL with sample buffer. Then, following the instructions of the Protein Thermal Shift Starter Kit (ThermoFisher), add 13 μL of the test sample solution to a PCR tube, add 5 μL of Protein Thermal Shift Buffer, and add 2 μL of 10× staining solution to make a reaction volume of 20 μL. Mix well and centrifuge at 12000 rpm for 5 minutes to remove air bubbles. Place the test sample in a PCR instrument for sample analysis and record the Tm value of the sample.

[0408] iCIEF Analysis

[0409] 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 Pharmalyte pH 3-10 amphoteric electrolyte, and 2 μl each of pI markers 5.5 and 9.5. An appropriate volume of ultrapure water was added to bring the total volume to 200 μl, and the mixture was thoroughly mixed. The supernatant was collected by centrifugation and injected for analysis. After analysis, the results were imported into ChromPerfect software for spectral integration and calculation of the isoelectric point and percentage of each peak. The Siglec-15 binding protein of this application has a purity greater than or equal to 98%, a melting temperature (Tm) between 86°C and 92°C, and exhibits good thermal stability, making it suitable for further development.

[0410] Example 6: Inhibitory effect of Siglec-15 binding protein of this application on T cell proliferation in human PBMCs

[0411] CD3 Monoclonal Antibody (OKT3) (Thermo Fisher, catalog number: 16-0037-85) was diluted to 0.03 μg / mL and coated onto cell culture plates (Corning, catalog number 3599) using PBS, 100 μl / well, and incubated overnight at 4°C. The next day, the Siglec-15 binding protein of this application was diluted with culture medium (RPMI 1640 + 10% FBS) to a maximum final concentration of 12 μg / mL (prepared concentration 48 μg / mL), using a 4-fold serial dilution (3 concentration points + 1 0 concentration). Subsequently, human Siglec-15 Fc protein (Sino Biological Inc., catalog number: 13976-H02H) was prepared with culture medium to a final concentration of 5 μg / mL (prepared concentration 20 μg / mL). Aspirate the coating solution from the coated plate, wash the plate twice with PBS, then add 50 μL of the prepared Siglec-15 binding protein of this application, followed by 50 μL of the prepared human Siglec-15 Fc protein. Next, label fresh human peripheral blood lymphocytes (Auscells, catalog number: PB004-C), centrifuge at 400 x g for 10 min, resuspend the cell pellet in PBS, and adjust the cell density to 1 × 10⁻⁶ cells / mL. 7 Cells / mL were collected, followed by the addition of an equal volume of 2 μM CFSE (Thermo Fisher, catalog number: C34554). The mixture was immediately stirred and incubated at 37°C for 5 min. The incubation was then terminated with 10 volumes of PBS + 10% FBS. The cells were then centrifuged at 400 x g for 10 min, washed twice with PBS + 10% FBS, and resuspended in culture medium to a volume of 2 × 10⁶ cells / mL. 6 Cells / mL. Then, CFSE-labeled cells were added to the culture plate at a density of 100 μL per well, resulting in a cell count of 2 × 10⁶ cells per well. 5Cells / well; place the cell culture plate in a 37°C, 5% CO2 cell culture incubator. Three days later, the cells were centrifuged at 300 x g for 5 min, washed once with pre-chilled PBS + 2% FBS, and the cell pellet was resuspended in 100 μl of PBS + 2% FBS. 2 μl of Fc blocking reagent was added to each well, and the cells were incubated at 4°C for 30 min. After thorough mixing, each well was divided into two aliquots. 5 μl of APC-labeled mouse anti-human CD4 antibody (APC Mouse Anti-Human CD4 antibody, BD, catalog number: 555349) was added to each well in one aliquot, and 5 μl of APC-labeled mouse anti-human CD8 antibody (APC Mouse Anti-Human CD8 antibody, BD, catalog number: 555369) was added to each well in the other aliquot. After incubation at 4°C for 1 hour, the cells were washed twice with pre-chilled PBS + 2% FBS, and then resuspended in 30 μl of PBS + 2% FBS. The cells were then analyzed using an iQue flow cytometer (Intellyciteplus, Sartorius). The flow cytometry results were analyzed using Flowjo V10, and the data were processed using GraphPad Prism software.

[0412] Figure 5 This application demonstrates how the Siglec-15 binding protein reverses the Siglec-15 protein's effect on CD4. + Results of the detection of T cell proliferation inhibition. Figure 6 This application demonstrates how the Siglec-15 binding protein reverses the Siglec-15 protein's effect on CD8. + Results of the detection of T cell proliferation inhibition. For example... Figure 5 and Figure 6 As shown, the Siglec-15 binding protein of this application can reverse the effect of Siglec-15 protein on CD4. + and / or CD8 + Inhibitory effect on T cell proliferation.

[0413] Example 7: Evaluation of the antitumor activity of Siglec-15 binding protein in a mouse tumor model.

[0414] CT-26.WT (purchased from ATCC) mouse colon cancer cells were expanded and cultured in vitro, while BMDM (bone marrow-derived macrophages) were cultured simultaneously. BMDM was obtained from the femur and tibia of BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., 7-8 weeks old, female). The bone marrow was cultured in RPMI-1640 medium with 10% fetal bovine serum and 20 ng / ml macrophage colony-stimulating factor (M-CSF) for 7 days, then collected and mixed with CT-26.WT cells. This mixture was then subcutaneously inoculated into BALB / c mice at a dose of 1 × 102 CT-26.WT and BMDM per mouse. 5 Twelve days after inoculation, when the average tumor volume in the mice reached 100 mm... 3 Mice were randomly divided into groups of 8 mice each, based on tumor volume and body weight. The mice were administered the drug on the same day. G1 mice were given isotype control human IgG, G2 mice were given Ab9 of this application, and G3 mice were given Ab15. The dosage for each group of mice was 10 mg / kg, administered intraperitoneally, twice a week.

[0415] like Figure 7 As shown, on day 11 after grouping, the mean tumor volume in group G1 was 2170±269 mm. 3 The mean tumor volume in group G2 was 1677±191 mm. 3 The tumor inhibition rate was 23%; the average tumor volume in group G3 was 1540±437 mm. 3 The tumor inhibition rate was 29%, indicating that the Siglec-15 binding protein of this application has the effect of inhibiting tumor growth.

[0416] Example 8: Enhancement of LPS-induced cytokine secretion by Siglec-15 binding protein according to this application.

[0417] BALB / c mice (purchased 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 their body weight. Group G1 received isotype control human IgG, G2 received Ab9 (as per this application), and G3 received Ab15. The dosage for each group was 10 mg / kg, administered intraperitoneally, and the time interval was recorded as 0 hours. Two hours after administration, lipopolysaccharide (LPS) dissolved in physiological saline was injected intraperitoneally at a dose of 200 μg / kg. Two hours after LPS modeling, all animals were anesthetized with 2-5% isoflurane inhalation, and 200 μL of whole blood was collected through the orbital cavity. Six hours after LPS modeling, all animals were anesthetized with 2-5% isoflurane inhalation, and the maximum amount of peripheral blood was collected through the orbital cavity. After incubation at room temperature for 30-60 minutes, serum was separated by centrifugation at 2000g for 10 minutes at 4°C to detect cytokines TNF-α and IL-6. All experimental animals were euthanized using excessive carbon dioxide.

[0418] Figure 8 The results show the detection results of the effect of Siglec-15 binding protein on the secretion level of TNF-α (TNF-alpha) 2 hours after LPS injection. Figure 9 The results show the detection results of the effect of Siglec-15 binding protein on the secretion level of TNF-α (TNF-alpha) 6 hours after LPS injection. Figure 10 The results show the detection results of the effect of Siglec-15 binding protein on the secretion level of IL-6 2 hours after LPS injection. Figure 11 The results show the effects of the Siglec-15 binding protein on IL-6 secretion levels 6 hours after LPS injection. Figure 8 , Figure 9 , Figure 10 and Figure 11 As shown, TNF-α (TNF-alpha) levels were significantly increased in the G2 group compared to the isotype control group two hours after LPS injection, but returned to near-baseline levels after six hours. Similarly, IL-6 levels were significantly increased in all treatment groups compared to the isotype control group two hours after LPS injection, but returned to baseline levels after six hours. This indicates that the Siglec-15 binding protein described in this application can promote the release of inflammatory factors in an LPS-induced acute inflammation model.

[0419] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments listed herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents.

Claims

1. An anti-Siglec-15 antibody or its antigen-binding fragment, comprising HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1, wherein HCDR3 is the amino acid sequence shown in SEQ ID NO: 1, HCDR2 is the amino acid sequence shown in SEQ ID NO: 2, HCDR1 is the amino acid sequence shown in SEQ ID NO: 7, LCDR3 is the amino acid sequence shown in SEQ ID NO: 9, LCDR2 is the amino acid sequence shown in SEQ ID NO: 10, and LCDR1 is the amino acid sequence shown in SEQ ID NO: 14; or wherein HCDR3 is the amino acid sequence shown in SEQ ID NO: 1, HCDR2 is the amino acid sequence shown in SEQ ID NO: 3, HCDR1 is the amino acid sequence shown in SEQ ID NO: 7, LCDR3 is the amino acid sequence shown in SEQ ID NO: 9, LCDR2 is the amino acid sequence shown in SEQ ID NO: 12, and LCDR1 is the amino acid sequence shown in SEQ ID NO:

13.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, and L-FR4, wherein H-FR1 is the amino acid sequence shown in SEQ ID NO: 15, H-FR2 is the amino acid sequence shown in SEQ ID NO: 16, H-FR3 is the amino acid sequence shown in SEQ ID NO: 17, H-FR4 is the amino acid sequence shown in SEQ ID NO: 18, L-FR1 is the amino acid sequence shown in SEQ ID NO: 19, L-FR2 is the amino acid sequence shown in SEQ ID NO: 20, L-FR3 is the amino acid sequence shown in SEQ ID NO: 21, and L-FR4 is the amino acid sequence shown in SEQ ID NO:

22.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH is the amino acid sequence shown in SEQ ID NO: 23 and the VL is the amino acid sequence shown in SEQ ID NO: 30; or wherein the VH is the amino acid sequence shown in SEQ ID NO: 25 and the VL is the amino acid sequence shown in SEQ ID NO:

32.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, comprising a constant region of the antibody heavy chain. The antibody heavy chain constant region includes a heavy chain constant region derived from IgG1.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, comprising a constant region of the antibody light chain, wherein the constant region of the antibody light chain comprises a constant region derived from human Igκ.

6. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A cell comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5, the nucleic acid molecule of claim 6, and / or the vector of claim 7.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-5, and a pharmaceutically acceptable carrier.

10. A method for preparing an antibody or an antigen-binding fragment thereof according to any one of claims 1-5, comprising culturing the cells of claim 8 under conditions that cause the antibody or the antigen-binding fragment thereof to be expressed.

11. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5, the nucleic acid molecule of claim 6, the carrier of claim 7, the cell of claim 8, and / or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating a disease and / or condition, said disease and / or condition being colon cancer.

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