Antibodies to Siglec-15 and methods of use thereof

Abstract

To provide compositions and methods for modulating Siglec-15 and signaling initiated therefrom.SOLUTION: A Siglec-15 binding molecule is provided. Typically, the molecule is an antibody or antigen-binding fragment thereof that immunospecifically binds to Siglec-15. Also provided is a Siglec-15 ligand binding molecule. The molecule is typically a Siglec-15 polypeptide or a fusion protein. Also provided is a method for using the molecules in a subject in need thereof to reduce immunosuppression mediated by siglec-15.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application Nos. 62 / 500,578, filed May 3, 2017, 62 / 451,271, filed January 27, 2017, and 62 / 397,794, filed September 21, 2017, all of which are incorporated by reference in their entirety where permissible.

[0002] FIELD OF THE INVENTION The present invention relates generally to the field of immunomodulation, and more particularly to compositions and methods for modulating Siglec-15 and signal transduction initiated therefrom. [Background technology]

[0003] Background of the Invention Sialic acid-binding Ig-like lectins ("Siglecs") are members of the Ig superfamily. These type 1 transmembrane proteins contain an N-terminal V-set domain that binds sialic acid, a variable number of C2-set Ig domains, a transmembrane region, and a cytosolic tail, and specifically bind sialic acid attached to the terminal regions of cell surface glycoconjugates. Two major subsets of Siglecs have been identified: one subset includes CD-33 and CD33-related Siglecs, such as Siglecs-5, -6, -7, -8, -9, 10, -11, -14, and -16 in humans, and CD33 and Siglecs-E, -F, -G, and -H in mice (Crocker and Redelinghuys, Biochemical Society Transactions, 36(6):1467-1471 (2008)). The second subset consists of Sn (sialoadhesin) (Siglec-1), CD22 (Siglec-2), MAG (myelin-associated glycoprotein) (Siglec-4), and Siglec-15, all of which are well conserved in mammals. With the exception of MAG, which is expressed in the nervous system, Siglecs are differentially expressed on various subsets of leukocytes, where they play a role in the positive or negative regulation of immune and inflammatory responses (McMillan and Crocker, Carbohydr. Res., 343:2050-2056 (2008) (Non-Patent Document 2) and Crocker, et al., Nat. Rev. Immunol., 7:255-266 (2007) (Non-Patent Document 3)).

[0004] Studies have shown that many Siglecs are expressed on immune cells and have immunosuppressive properties. However, a subset of Siglecs, including Siglec-15, associates with the signal adapter molecule 12kDa DNAX-activation protein (DAP12), which contains an immunoreceptor tyrosine-based activation motif (ITAM) and is associated with immune cell activation (Takamiya, et al., Glycobiology, 23(2):178-87(2013) (Non-Patent Document 4)). Siglec-15 is specifically expressed in macrophages and dendritic cells in the spleen and lymph nodes and preferentially recognizes the sTn antigen (Angata, et al., Glycobiology, 17(8):838-46(2007) Epub 2007 May 4 (Non-Patent Document 5)). H157 cells overexpressing sTn (H157 / ST6GalNAc-I) stimulated TGF-β secretion from M-CSF-induced macrophages expressing Siglec-15 (Takamiya, et al., Glycobiology, 23(2):178-87(2013)). In addition, TGF-β secretion from THP-1 cells was enhanced by overexpression of Siglec-15 in THP-1 cells and ST6GalNAc-I (an enzyme involved in the biosynthesis of sTn structures) in H157 cells, respectively, in a manner that may depend at least in part on Siglec-15-DAP12-induced signaling as well as one or more DAP12-independent, Sky-dependent, or possibly Sky-independent pathways. TGF-β is produced by both tumor cells and tumor-infiltrating leukocytes, including macrophages, and contributes to tumor progression and metastasis by enhancing tumor cell infiltration and inhibiting immune cell function (Flavell, et al., Nat Rev Immunol, 10:554-567 (2010) (Non-Patent Document 6)).These data suggest that recognition of tumor-associated sTn by Siglec-15 activates the DAP12-Syk pathway, a signaling pathway that enhances TGF-β production from myeloid cells, ultimately altering the tumor microenvironment to favor tumor cells (Takamiya, et al., Glycobiology, 23(2):178-87(2013)). Siglec-15 contains a typical ITIM domain in its cytoplasmic domain. TIFF0007720375000001.tif6128. Its function remains uncharacterized.

[0005] However, there remains a need for tools and techniques for modulating Siglec-15 and the signaling initiated by it.

[0006] It is therefore an object of the present invention to provide compositions for detecting and modulating Siglec-15.

[0007] It is also an object of the present invention to provide methods for modulating Siglec-15 and the signaling initiated by it to enhance immune responses or reduce or reverse immunosuppression.

[0008] It is also an object of the present invention to provide methods for modulating osteoclast differentiation to decrease bone resorption or increase bone formation.

[0009] It is also an object of the present invention to provide methods for treating diseases and disorders by modulating Siglec-15 and the signaling initiated by it. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Crocker and Redelinghuys, Biochemical Society Transactions, 36(6):1467-1471(2008)

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

[0011] Siglec-15 binding molecules are provided, which are typically antibodies or antigen-binding fragments thereof that immunospecifically bind to Siglec-15. For example, in some embodiments, a Siglec-15 binding molecule comprises six complementarity determining regions (CDRs), wherein the CDRs are three light chain CDRs of a polypeptide selected from the group consisting of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107, and a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107. and wherein the Siglec-15 binding molecule comprises three heavy chain CDRs of a polypeptide selected from the group consisting of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, and the Siglec-15 binding molecule binds to Siglec-15. In some embodiments, the Siglec-15 binding molecule comprises the light chain and / or heavy chain CDRs of one of the mouse anti-human monoclonal antibodies designated herein as 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A.

[0012] In some embodiments, the Siglec-15 binding molecule comprises a light chain variable region comprising an amino acid sequence polypeptide selected from the group consisting of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107; and / or and a heavy chain variable region comprising an amino acid sequence polypeptide selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or a variant thereof having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In some embodiments, the Siglec-15 binding molecule comprises the light chain and / or heavy chain variable region of one of the mouse anti-human monoclonal antibodies designated herein as 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A.

[0013] In some embodiments, the Siglec-15 binding molecule that binds to Siglec-15 is (I) arranged on the surface of a cell (preferably a living cell); (II) arranged at endogenous concentrations on the surface of cells (preferably living cells); (III) arranged on the surface of a living cell and modulating the binding of Siglec-15 (e.g., SEQ ID NO:1, SEQ ID NO:2, etc.) to Neu5Acα2-6GalNAcα, LRRC4C, Siglec-15 counter-receptor (S15-CR), or a combination thereof; (IV) arranged on the surface of living cells to reduce, prevent, or inhibit TGF-β secretion; (V) arrayed on the surface of a living cell; or (IV) Combining them.

[0014] Cells that endogenously express Siglec-15 include macrophages, dendritic cells, and cancer cells.

[0015] The Siglec-15 binding molecule can comprise one or more constant domains derived from an immunoglobulin constant region (Fc). The constant domain can be a human constant domain, such as an IgA, IgD, IgE, IgG, or IgM domain. In certain embodiments, the human IgG constant domain is an IgG1, IgG2, IgG3, or IgG4 domain. The Siglec-15 binding molecule can be detectably labeled or contain a conjugated toxin, drug, receptor, enzyme, or receptor ligand. The Siglec-15 binding molecule can be a monoclonal antibody, a human antibody, a chimeric antibody, a humanized antibody, or a single-chain antibody, or an antigen-binding fragment thereof. The antibody can be monospecific, bispecific, trispecific, or multispecific.

[0016] One embodiment provides a humanized anti-Siglec-15 antibody having one or more variable light chains having the amino acid sequence of SEQ ID NO:195, 197, 199, 201, or 209.

[0017] Another embodiment provides a humanized anti-Siglec-15 antibody having one or more variable heavy chains having the amino acid sequence of SEQ ID NO:203, 206, or 207.

[0018] Another embodiment provides a humanized anti-Siglec-15 antibody having one or more variable light chains having the amino acid sequence of SEQ ID NO:195, 197, 199, 201, or 209, and one or more variable heavy chains having the amino acid sequence of SEQ ID NO:203, 206, and 207.

[0019] One embodiment provides an antibody having the light chain CDRs of SEQ ID NO:209, 195, 207, 199, or 201, and the heavy chain CDRs of SEQ ID NO:203, 206, or 207, and combinations thereof.

[0020] Another embodiment provides an antibody having a light chain amino acid sequence of SEQ ID NO:209, 210, or 211.

[0021] Another embodiment provides an antibody having a heavy chain amino acid sequence of SEQ ID NO:212, 213, 215, or 216.

[0022] Another embodiment provides an antibody having a light chain amino acid sequence of SEQ ID NO:209, 210, or 211 and a heavy chain amino acid sequence of SEQ ID NO:212, 213, 215, or 216.

[0023] In some embodiments, the Siglec-15 binding molecule is modified so that it exhibits reduced or no Fc receptor (FcR) binding activity, hi some embodiments, the Siglec-15 binding molecule is modified so that it exhibits enhanced antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity.

[0024] One embodiment provides a fusion protein that is at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:193 or 194.

[0025] Pharmaceutical compositions comprising a Siglec-15 binding molecule and a physiologically acceptable carrier or excipient are also provided. In some embodiments, the Siglec-15 binding molecule reduces or prevents binding of Siglec-15 to its ligand and / or counterreceptor, reduces or prevents Siglec-15-mediated signal transduction, or a combination thereof. The ligand may be a sialylated glycoprotein. The ligand may be expressed on the surface of tumor cells. The following examples demonstrate that leucine-rich repeat-containing protein 4C (LRRC4C) is a ligand for Siglec-15 and may be expressed by cancer cells. Siglec-15 counterreceptors may also be expressed on the surface of immune cells, such as T cells, which, upon binding by Siglec-15, lead to T cell inhibition.

[0026] Methods of treating a subject in need thereof are provided, typically comprising administering to the subject an effective amount of a Siglec-15 binding molecule, e.g., in a pharmaceutical composition.

[0027] In some embodiments, the antagonistic Siglec-15 binding molecule enhances immune responses, delays or prevents tumor growth, inhibits tumor-mediated immunosuppression, eliminates tumors, depletes or blocks the activity of tumor-associated macrophages (TAMs) to alter their activity, reduces TAM-mediated immunosuppression, reduces or reverses T cell suppression, increases T cell proliferation, or a combination thereof. In some embodiments, the cancer or tumor contains macrophages that express Siglec-15. The Siglec-15 binding molecule can be administered to a subject in an amount effective to reduce the expression and / or secretion of TGF-β by the macrophages. In some embodiments, the subject has cancer or an infectious disease. The cancer may contain cells that express or overexpress a ligand for Siglec-15.

[0028] Also provided are methods for reducing osteoclast differentiation, reducing bone resorption, increasing bone formation, and combinations thereof, by administering to a subject an effective amount of an antagonistic Siglec-15 binding molecule.

[0029] In some embodiments, the antagonistic Siglec-15 binding molecule reduces an immune response, increases or enhances T cell suppression, increases T cell proliferation, or a combination thereof. The Siglec-15 binding molecule can be administered to a subject in an amount effective to increase TGF-β expression and / or secretion by macrophages. In some embodiments, the subject has inflammation, an autoimmune disease, or is a transplant recipient.

[0030] Some embodiments include administering to the subject a second therapeutic agent.

[0031] Detection and diagnostic methods are also provided. Any of the detection and diagnostic methods can be coupled with a therapeutic method. For example, a method for detecting or diagnosing a disease, disorder, or infection can include (a) assaying expression of Siglec-15 in a cell or tissue sample of a subject using a disclosed Siglec-15-binding molecule, and (b) comparing the level of Siglec-15 to a control level, wherein an increase in the assayed level of Siglec-15 compared to the control level is indicative of the disease, disorder, or infection.

[0032] A method for monitoring the progression of a disease, disorder, or infection can include (a) assaying the expression of Siglec-15 in cells or tissue samples from a subject obtained at an initial time point and at a subsequent time point using a disclosed Siglec-15 binding molecule; and (b) comparing the level of expression of Siglec-15 in cells or tissue samples from the subject at the initial time point and at the subsequent time point, wherein an increase in the assayed level of Siglec-15 at the subsequent time point compared to the initial time point is indicative of the progression of the disease, disorder, or infection.

[0033] Methods for monitoring response to treatment are provided, comprising: (a) assaying expression of Siglec-15 in cells or tissue samples of a subject before and after treatment using the disclosed Siglec-15 binding molecules; and (b) comparing the levels of Siglec-15 over time, whereby a decrease in the assayed level of Siglec-15 after treatment compared to the level of Siglec-15 before treatment is indicative of a favorable response to treatment. [The present invention 1001] An antibody or antigen-binding fragment thereof comprising six complementarity-determining regions (CDRs), the CDRs are SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107, or and variants thereof, including those having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107. and three light chain CDRs of a polypeptide selected from the group consisting of: SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or and variants thereof, including those having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. the three heavy chain CDRs of a polypeptide selected from the group consisting of Including, the antibody or antigen-binding fragment thereof binds to Siglec-15; The antibody or antigen-binding fragment thereof. [The present invention 1002] SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107, or and variants thereof, including those having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107. and / or a light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or and variants thereof, including those having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. a heavy chain variable region comprising an amino acid sequence polypeptide selected from the group consisting of 1001. The antibody or antigen-binding fragment thereof of the present invention, comprising: [The present invention 1003] (I) arranged on the surface of cells (especially living cells); (II) arranged at endogenous concentrations on the surface of cells (especially living cells); (III) arranged on the surface of living cells and modulating the binding of Siglec-15 (SEQ ID NO:1 or SEQ ID NO:2) to Neu5Acα2-6GalNAcα, LRRC4C, Siglec-15 counter-receptor (S15-CR), or a combination thereof; (IV) arranged on the surface of living cells to reduce, prevent, or inhibit TGF-β secretion; (V) arrayed on the surface of a living cell; or (IV) performing a combination thereof; An antibody or an antigen-binding fragment thereof according to any one of the antibodies 1001 to 1002 or 1046 to 1055 of the present invention that binds to Siglec-15. [The present invention 1004] The antibody or antigen-binding fragment thereof of any one of 1001 to 1003 or 1046 to 1055 of the present invention, wherein the cell is a macrophage or a dendritic cell. [The present invention 1005] 10. The antibody or antigen-binding fragment thereof of any of claims 1001 to 1004 or 1046 to 1055, comprising one or more constant domains from an immunoglobulin constant region (Fc). [The present invention 1006] The antibody or antigen-binding fragment thereof of any one of 1005 and 1046 to 1055 of the present invention, wherein the constant domain is a human constant domain. [The present invention 1007] The antibody or antigen-binding fragment thereof of any one of 1006 and 1046 to 1055 of the present invention, wherein the human constant domain is an IgA, IgD, IgE, IgG, or IgM domain. [The present invention 1008] The antibody or antigen-binding fragment thereof of any one of 1007 or 1046 to 1055 of the present invention, wherein the human IgG constant domain is an IgG1, IgG2, IgG3, or IgG4 domain. [The present invention 1009] 1008. The antibody or antigen-binding fragment thereof of any of claims 1001 to 1008, comprising a detectably labeled or conjugated toxin, drug, receptor, enzyme, or receptor ligand. [The present invention 1010] The antibody or antigen-binding fragment thereof according to any one of claims 1001 to 1009 or 1046 to 1055 of the present invention, wherein the antibody is a monoclonal antibody, a human antibody, a chimeric antibody, a humanized antibody, or a single-chain antibody. [The present invention 1011] The antibody or antigen-binding fragment thereof of any one of claims 1001 to 1010, wherein the antibody is a monospecific, bispecific, trispecific, or multispecific antibody. [The present invention 1012] 10. The antibody or antigen-binding fragment thereof of any one of claims 1001 to 1011, wherein the antibody has been modified so that the molecule exhibits reduced or no Fc receptor (FcR) binding activity. [The present invention 1013] Any of the antibodies or antigen-binding fragments thereof of claims 1001 to 1011, wherein the antibody has been modified to exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity. [The present invention 1014] A pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments thereof of the present invention 1001 to 1013 or 1046 to 1053 and a physiologically acceptable carrier or excipient, wherein the antibody reduces or prevents binding of Siglec-15 to its ligand and / or reduces or prevents Siglec-15-mediated signal transduction. [The present invention 1015] 1014. The composition of claim 10, wherein said ligand is a sialylated glycoprotein, leucine-rich repeat-containing protein 4C (LRRC4C), Siglec-15 counterreceptor, or a combination thereof. [The present invention 1016] The composition of any one of claims 1014 to 1015, wherein said ligand is expressed on the surface of a tumor cell. [The present invention 1017] A method for treating a subject in need of treatment, comprising administering to the subject an effective amount of any of the pharmaceutical compositions of the present inventions 1014 to 1016. [The present invention 1018] The method of claim 1017, wherein said subject has cancer or an infectious disease. [The present invention 1019] The method of claim 1018, wherein said subject has a cancer comprising cells that express or overexpress a ligand for Siglec-15. [The present invention 1020] Any of the methods of claims 1017 to 1019, wherein the antibody or antigen-binding fragment thereof augments an immune response, delays or prevents tumor growth, inhibits tumor-mediated immunosuppression, eliminates tumors, depletes or blocks the activity of tumor-associated macrophages (TAMs) to alter their activity, reduces TAM-mediated immunosuppression, reduces or reverses T-cell suppression, or a combination thereof. [The present invention 1021] The method of any of claims 1018 to 1020, wherein the cancer or tumor comprises macrophages that express Siglec-15. [The present invention 1022] 1021. The method of claim 1021, wherein the antibody or antigen-binding fragment thereof is administered to the subject in an amount effective to reduce expression and / or secretion of TGF-β by the macrophages. [The present invention 1023] The method of any of claims 1017 to 1022, further comprising administering to said subject a second therapeutic agent. [The present invention 1024] 1. A method for detecting or diagnosing a disease, disorder, or infection, comprising: (a) assaying expression of Siglec-15 in cells or a tissue sample of a subject using any of antibodies or antigen-binding fragments thereof of the present invention 1001 to 1013 or 1046 to 1053; and (b) comparing the level of Siglec-15 with that of a control, The method, wherein an increase in the assay level of Siglec-15 compared to the control level is indicative of the disease, disorder, or infection. [The present invention 1025] 1. A method for monitoring the progression of a disease, disorder, or infection, comprising: (a) assaying the expression of Siglec-15 in cells or tissue samples of a subject obtained at an initial time point and at a subsequent time point using an antibody or antigen-binding fragment thereof of any of 1001 to 1013 or 1046 to 1055 of the present invention; and (b) comparing the level of expression of Siglec-15 in the cells or tissue samples of the subject at the initial time point and at the subsequent time point, The method, wherein an increase in the assay level of Siglec-15 at the subsequent time point compared to the initial time point is indicative of progression of the disease, disorder, or infection. [The present invention 1026] 1. A method for monitoring response to treatment, comprising: (a) assaying expression of Siglec-15 in cells or tissue samples of a subject before and after treatment using any of antibodies or antigen-binding fragments thereof of the present invention 1001-1013 or 1046-1053; and (b) comparing the levels of Siglec-15 over time, Whereby, a decrease in the assay level of Siglec-15 after said treatment compared to the level of Siglec-15 before said treatment is indicative of a favorable response to said treatment. [The present invention 1027] A method for reducing T cell suppression in a subject in need thereof, comprising administering to the subject an effective amount of any of the pharmaceutical compositions of the present inventions 1014 to 1016. [The present invention 1028] A method for increasing cytokine production in T cells in a subject in need thereof, comprising administering to the subject an effective amount of any one of the pharmaceutical compositions of the present inventions 1014 to 1016. [The present invention 1029] the cytokine is IFNγ, TNF-α, or a combination thereof; the T cells are CD4+ T cells or CD4+ T cells, or a combination thereof; or It is a combination of these, The method of the present invention 1028. [The present invention 1030] A method for increasing bone growth or reducing bone resorption in a subject in need thereof, comprising administering to the subject an effective amount of any of the pharmaceutical compositions of present inventions 1014 to 1016. [The present invention 1031] A method for reducing osteoclast differentiation in a subject in need thereof, comprising administering to the subject an effective amount of any of the pharmaceutical compositions of present inventions 1014 to 1016. [The present invention 1032] A pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments thereof of the present invention 1001 to 1013 or 1046 to 1053 and a physiologically acceptable carrier or excipient, wherein the antibody increases the binding of Siglec-15 to its ligand and / or increases or enhances Siglec-15-mediated signal transduction. [The present invention 1033] A method for reducing an immune response, reducing inflammation, reducing an inflammatory reaction, treating an autoimmune disease or disorder, reducing graft or transplant rejection, or treating graft-versus-host disease (GVHD) in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition of the present invention 1032. [The present invention 1034] A fusion protein comprising a first fusion partner comprising a Siglec-15 polypeptide or a fragment or variant thereof, and a second fusion partner. [This invention 1035] The fusion protein of the present invention 1034, wherein said second fusion partner is an Fc domain. [The present invention 1036] The fusion protein of the present invention 1035, wherein the Fc domain is derived from IgG1. [This invention 1037] The fusion protein of any one of 1034 to 1036, wherein the first fusion partner and the second fusion partner are linked by a linker. [The present invention 1038] The fusion protein of the present invention 1037, wherein the linker is a hinge domain or a fragment thereof. [The present invention 1039] The fusion protein according to any one of the present inventions 1034 to 1038, wherein the Siglec-15 polypeptide comprises the Siglec-15 extracellular domain. [The present invention 1040] The fusion protein according to any one of the present inventions 1034 to 1039, wherein the Siglec-15 polypeptide consists of the Siglec-15 extracellular domain or a fragment thereof. [The present invention 1041] The fusion protein of the present invention 1040, wherein the fragment comprises the IgV domain, IgC, or a combination thereof of Siglec-15 [The present invention 1042] The fusion protein according to any one of the present inventions 1034 to 1041, further comprising a leader sequence. [The present invention 1043] A fusion protein comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 193 or 194. [The present invention 1044] A pharmaceutical composition comprising the fusion protein according to any one of the present inventions 1034 to 1043 and a physiologically acceptable carrier or excipient. [The present invention 1045] A method of treating a subject in need of treatment, comprising administering to the subject an effective amount of the pharmaceutical composition of the present invention 1044 to promote an immune response. [The present invention 1046] The humanized anti-Siglec-15 antibody, wherein the humanized antibody is derived from clone 5G12 or 1H3. [The present invention 1047] A humanized anti-Siglec-15 antibody having one or more variable light chains having the amino acid sequence of SEQ ID NO: 195, 197, 199, 201, or 209. [The present invention 1048] A humanized anti-Siglec-15 antibody having one or more variable heavy chains having the amino acid sequence of SEQ ID NO:203, 206, or 207. [This invention 1049] A humanized anti-Siglec-15 antibody having one or more variable light chains having the amino acid sequences of SEQ ID NOs: 195, 197, 199, 201, or 209, and one or more variable heavy chains having the amino acid sequences of SEQ ID NOs: 203, 206, and 207. [The present invention 1050] An antibody having a light chain CDR of SEQ ID NO: 209, 195, 207, 199, or 201 and a heavy chain CDR of SEQ ID NO: 203, 206, or 207, and combinations thereof. [This invention 1051] An antibody having a light chain amino acid sequence of SEQ ID NO:209, 210, or 211. [This invention 1052] An antibody having a heavy chain amino acid sequence of SEQ ID NO:212, 213, 215, or 216. [This invention 1053] An antibody having a light chain amino acid sequence of SEQ ID NO:209, 210, or 211 and a heavy chain amino acid sequence of SEQ ID NO:212, 213, 215, or 216. [This invention 1054] A method for inhibiting osteoclast formation, comprising administering to a subject in need thereof an effective amount of any of the anti-Siglec antibodies 1001 to 1013 or 1046 to 1053 of the present invention, or an antigen-binding fragment thereof. [This invention 1055] A method for inhibiting osteoclast formation, comprising administering to a subject in need thereof an effective amount of an anti-Siglec antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 226, 227, and 228, and the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 220, 221, 223, and 225. [This invention 1056] 1056. The method of any of claims 1054 to 1055, wherein said antibody or antigen-binding fragment thereof comprises a therapeutic or cytotoxic agent. [This invention 1057] A cell that constitutively or inducibly expresses an antibody or an antigen-binding fragment thereof that specifically binds to Siglec-15, the cell comprising one or more types of nucleic acids comprising a sequence encoding a light chain variable domain comprising any one of SEQ ID NOs: 120 to 132 and a heavy chain variable domain selected from the group consisting of any one of SEQ ID NOs: 133 to 145. [This invention 1058] A cell that constitutively or inducibly expresses an antibody or an antigen-binding fragment thereof that specifically binds to Siglec-15, the cell comprising one or more types of nucleic acids comprising a sequence encoding any one of the antibodies 1001 to 1013 and 1044 to 1053 of the present invention. [This invention 1059] The antibody or antigen-binding fragment thereof of any one of 1001 to 1013 and 1044 to 1053 of the present invention, wherein the antibody comprises a therapeutic agent or a cytotoxic agent. [Brief explanation of the drawings]

[0034] [Figure 1] 1 is a curve showing plasma antibody titers of two immunized and two non-immunized Siglec-15 knockout mice. [Figure 2A]FIG. 1 is an alignment showing the sequence light chain variable regions of 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, and 105A, highlighting the first complementarity determining region (CDR). [Figure 2B] FIG. 1 is an alignment showing the sequence light chain variable regions of 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, and 105A, highlighting the second complementarity determining region (CDR). [Figure 2C] FIG. 1 is an alignment showing the sequence light chain variable regions of 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, and 105A, highlighting the third complementarity determining region (CDR). [Figure 3A] FIG. 1 is an alignment showing the heavy chain variable region sequences of 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, and 105A, highlighting the first complementarity determining region (CDR). [Figure 3B] FIG. 1 is an alignment showing the heavy chain variable region sequences of 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, and 105A, highlighting the second complementarity determining region (CDR). [Figure 3C] FIG. 1 is an alignment showing the heavy chain variable region sequences of 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, and 105A, highlighting the third complementarity determining region (CDR). [Figure 4A] Figure 4A is a schematic diagram of an assay for measuring direct binding of anti-Siglec-15 antibodies to human or mouse Siglec-15-expressing cells. Figure 4B (1B2, 1C3, 1C12, 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9) and Figure 8C (6A (NC6), 28A (NC28), 63A (NC63), 77A (NC77), 80A (NC80), 82B (NC82), 83B (NC83), 92A (NC92), 93B (NC93), 99B (NC99), 104B (NC104), and 105A (NC105)) are bar graphs showing binding of anti-Siglec-15 antibodies to Siglec-15-expressing cells (% positive cells) in the assay shown in Figure 4A. Figure 4C is a bar graph showing the binding (%) of anti-Siglec-15 antibodies (1B2, 1C3, 1C12, 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A (NC6), 28A (NC28), 63A (NC63), 77A (NC77), 80A (NC80), 82B (NC82), 83B (NC83), 92A (NC92), 93B (NC93), 99B (NC99), 104B (NC104), and 105A (NC105)) to formalin-fixed Siglec-15-expressing cells. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5] Figures 5A-5B are line graphs showing the binding of purified 1C12, 8H8, 5G12, 3H10, 9A5, 6F8, 8C8, 1H3, 10G9, 1B2, and 1C3 Abs to K562.hS15 cells (9A, background binding subtracted) and 293T.mS15 cells (9B) (mean fluorescence intensity (MFI) as a function of primary antibody concentration (μg / ml)). Figure 5C is a dot plot of the binding of purified 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9 Abs to K562.hS15 cells versus the binding (MFI) to 293T.mS15. [Figure 6] Figure 6A is a bar graph showing the percentage of hS15+ U87 cells detected by each of 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, and 10G9 Abs. Figure 6B is a bar graph showing the MFI of hS15+ U87 cells detected by each of 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, and 10G9 Abs. Figure 6C is a bar graph showing the percentage of S15+ U87 cells detected by each of 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9 Abs. [Figure 7A] Figure 7A shows an antibody blocking assay: 293T cells expressing the ligand LRRC4C are treated with soluble receptor (hS15.hG1) and anti-S15 antibody, followed by detection of bound receptor with PE anti-hFc antibody. [Figure 7B] Figure 7B is a bar graph showing hS15.G1 binding (%) for hS15.hG1-1 and the following antibodies: 10G9, 5G12, 6F8, 8C8, 8H8, 9A5, 1C3, 1C12, 1H3, 3H10, 1B2, NC1, NC5, NC7, 28A (NC28), NC38, NC41, NC53, 63A (NC63), NC73, NC74, NC76, 77A (NC77), 82B (NC82), NC84, NC87, NC90, 92A (NC92), CI3-33, and CI1-33. [Figure 7C] FIG. 7C is a bar graph showing % blocking of S15 / LRRC4C for the control mAb and the 1B2, 1C3, 1H3, 8H8, 6F8, 8C8, 9A5, 1C12, 3H10, 10G9, and 5G12 antibodies. [Figure 7D] FIG. 7D is a bar graph showing the blocking (%) of S15 / LRRC4C for 1B2, 1C3, 1C12 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9. [Figure 7E]Figure 7E is a bar graph showing the blocking (%) of S15 / LRRC4C for 6A (NC6), 28A (NC28), 63A (NC63), 77A (NC77), 80A (NC80), 82B (NC82), 83B (NC83), 92A (NC92), 93B (NC93), 99B (NC99), 104B (NC104), and 105A (NC105). [Figure 8A] FIG. 8A is a schematic representation of the T cell suppression assay. [Figure 8B] Figures 8B and 8C are bar graphs showing the reversal of hS15.hG1-mediated human T cell suppression by S15 mAbs for antibodies 1B2, 1C3, 1C12 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9 as percentage division of CD8+ T cells (12B) and CD4+ T cells (12C), and also showing comparative assays performed with (e.g., +hS15.hG1) (left bar of each pair) and without (e.g., -hS15.hG1) (right bar of each pair) hS15.hG1. [Figure 8C] See legend to Figure 8B. [Figure 8D] Figures 8D-8G show the role of S15 in the suppression of human T cells mediated by hS15.hG1 for antibodies 1B2, 1C3, 1C12 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9 (Figures 8D and 12E), and for 6A (NC6), 28A (NC28), 63A (NC63), 77A (NC77), 80A (NC80), 82B (NC82), 83B (NC83), 92A (NC92), 93B (NC93), 99B (NC99), 104B (NC104), and 105A (NC105) (Figures 8F and 8G) for assays performed with hS15.hG1. 8D and 8F) and CD4+ T cell (FIGS. 8E and 8G) division (%) by mAb. [Figure 8E] See legend to Figure 8D. [Figure 8F] See legend to Figure 8D. [Figure 8G] See legend to Figure 8D. [Figure 8H] FIG. 8H is a dot plot showing CD8 T cell proliferation as a function of hS15_LRRC4C blocking activity (%). [Figure 8I] FIG. 8I is a dot plot showing CD8 T cell proliferation as a function of hS15_LRRC4C blocking activity (%). [Figure 9] Figure 9A is a schematic diagram of an assay for measuring changes in INFγ secretion, and Figure 9B is a bar graph showing the results of the assay shown in Figure 13A for antibodies 1B2, 1C3, 1C12 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9. [Figure 10] FIG. 1 is a bar graph showing TRAP (tartrate-resistant acid phosphatase) (absorbance at 540 nm) in the presence of antibodies 1C12, 8H8, 5G12, 3H10, 9A5, 6F8, 8C8, 1H3, 10G9, 1B2, and 1C3 in an osteoclastogenesis assay of fresh PBMCs from which monocytes were isolated and enriched by two methods: MACS column sorting (left panel); or binding to plastic in serum-free medium (right panel). [Figure 11] This diagram shows a model of Siglec-15's negative immune regulation in the tumor microenvironment (TME) (e.g., Siglec-15 (S15):Siglec-15-counterreceptor (S15-CR) >>> directs T cells toward proliferation and cytokine inhibition, and / or Siglec-15:LRRC4C >>> directs macrophages to produce TGF-β, suppressing immunity in the TME). The diagram shows the expression of Siglec-15 and its ligands in myeloid cells, T cells, and cancer cells, signaling from them, and interactions between anti-Siglec-15 (blocking and targeting) and LRRC4C antibodies and Siglec-15 fusion protein (non-crosslinking / blocking) molecules. [Figure 12] Figure 12A is a table showing the amino acid sequences of humanized 5G12 variable light chains L1 to L5, and Figure 12B is a diagram showing the amino acid sequences of humanized 5G12 variable heavy chains H1 to H3. [Figure 13A]FIG. 1 shows the sequence alignment of humanized 5G12 variable light chains VL1 to V15 to mouse VL. [Figure 13B] FIG. 1 shows a sequence alignment of humanized 5G12 variable heavy chains VH1 to VH3. [Figure 14A] FIG. 14A is a line graph of % T cell proliferation versus human S15 Fc (μg / mL), showing that increasing concentrations of S15 Fc decrease the % T cell proliferation. [Figure 14B] FIG. 14B is a bar graph of IFN-γ (pg / ml) in conditioned supernatants from cells treated with 0 or 5 μg / mL of S15 Fc. [Figure 14C] FIG. 14C is a bar graph of TNF-α (pg / ml) in conditioned supernatants from cells treated with 0 or 5 μg / mL of S15 Fc. [Figure 14D] FIG. 14D is a bar graph of IL-6 (pg / ml) in conditioned supernatants from cells treated with 0 or 5 μg / mL of S15 Fc. [Figure 15] 15A and 15B are bar graphs showing the percentage of positive cells for binding of S15 mAbs purified from hybridomas to human S15 or mouse S15 expressing cells. [Figure 16A] 16A and 16B are bar graphs of the percentage of divided CD8+ T cells treated with the indicated antibodies. [Figure 16B] 16A and 16B are bar graphs of the percentage of divided CD8+ T cells treated with the indicated antibodies. [Figure 16C] Figures 16C and 16D are bar graphs of the percentage of divided CD4+ T cells treated with the indicated antibodies. [Figure 16D] Figures 16C and 16D are bar graphs of the percentage of divided CD4+ T cells treated with the indicated antibodies. [Figure 17A] 17A and 17B are line graphs showing percent survival versus days post tumor cell inoculation in animals treated with 5G12. [Figure 17B]17A and 17B are line graphs showing percent survival versus days post tumor cell inoculation in animals treated with 5G12. [Figure 17C] FIG. 17C is a line graph showing the percent weight gain versus days after ID8.OVA inoculation. [Figure 18] 1 is a line graph showing the weight gain rate (%) versus the number of days after inoculation with ID8.OVA. [Figure 19A] FIG. 19A is a schematic diagram showing that human CD14+ monocytes were harvested from human PBMCs using Mitenyi monocyte magnetic beads and then seeded into 96-well plates with the indicated antibodies in the presence of human M-CSF and human RANKL. [Figure 19B] FIG. 19B is a photomicrograph showing osteoclasts treated with 1H3 as indicated. [Figure 19C] FIG. 19C is a bar graph of absorbance (540 nm) of supernatants collected for tartrate-resistant acid phosphatase assay after 7 days. [Figure 20] 1 is a bar graph showing cytokines (pg / mL) for IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF-α. [Figure 21] 1 is a bar graph of absorbance (540 nm) of mouse RAW 264.7 macrophage cells cultured with the indicated antibodies in the presence of RANKL. [Figure 22] 1 shows a comparison of exemplary humanized amino acid sequences of the 1H3 variable light chain. [Figure 23] 1 shows a comparison of exemplary humanized amino acid sequences of the 1H3 variable heavy chain. [Figure 24] FIG. 24 is a diagram of the proposed mode of action for Siglec-15. [Figure 25A] FIG. 25A is a FACS histogram of counts against Siglec-15 PE showing that M2 macrophages express Siglec-15. [Figure 25B] FIG. 25B is a FACS histogram of counts for Siglec-15 PE for M1 macrophages. [Figure 25C] FIG. 25C is a FACS histogram of counts for Siglec-15 PE for myeloid cells derived from mouse bone marrow treated with macrophage colony-stimulating factor (M-CSF). [Figure 25D] FIG. 25D is a FACS histogram of counts for Siglec-15 PE for myeloid cells derived from mouse bone marrow treated with M-CSF and interleukin-10 (IL-10). [Figure 25E] FIG. 25E is a bar graph of MFI-PE for myeloid cells from mouse bone marrow treated with M-CSF or M-CSF+IL10 and stained with isotype-PE (gray box) or anti-Siglec-15 PE. [Figure 26A] Figure 26A is a bar graph of absorbance (450 nm) for supernatants from human CD14+ monocytes from donor number 1603 plated in plates coated with Siglec-15 Fc (left vertical bar of concentration points) or soluble Siglec-15 (right vertical bar of each concentration point). [Figure 26B] Figure 26B is a bar graph of absorbance (450 nm) for supernatants from human CD14+ monocytes from donor number 1704 plated in plates coated with Siglec-15 Fc (left vertical bar of concentration points) or soluble Siglec-15 (right vertical bar of each concentration point). [Figure 26C] FIG. 26C is a bar graph of TNF-α (pg / mL) versus Siglec-15 Fc (μg / mL) for cells from donor #1603. [Figure 26D] FIG. 26D is a bar graph of IL-6 (pg / mL) versus Siglec-15 Fc (μg / mL) for cells from donor no. 1603. [Figure 26E] FIG. 26E is a bar graph of IL-1β (pg / mL) versus Siglec-15 Fc (μg / mL) for cells from donor #1603. [Figure 26F] FIG. 26F is a bar graph of TNF-α (pg / mL) versus Siglec-15 Fc (μg / mL) for cells from donor #1704. [Figure 26G] FIG. 26G is a bar graph of IL-6 (pg / mL) versus Siglec-15 Fc (μg / mL) for cells from donor no. 1704. [Figure 26H] FIG. 26H is a bar graph of IL-1β (pg / mL) versus Siglec-15 Fc (μg / mL) for cells from donor #1704. [Figure 27A] FIG. 27A is a schematic representation of the experimental protocol for Example 19. [Figure 27B] Figure 27B is a bar graph of CD8 proliferation (%) of S15 Fc-pretreated human bone marrow cells; M2φ, M1φ, and immature DCs co-cultured with negatively selected CFSE-labeled autologous pan-T cells (at a cell ratio of 1 myeloid cell:2 T cells) and anti-CD3 / CD28 beads (1 pan-T cell:2 beads). [Figure 27C] For Figures 27C to 27G, the vertical bars are from left to right: T cells only, T cells + beads, S15 Fc-treated, M2φ, M1φ, and imDC. Figure 27C is a bar graph of IFN-γ (pg / mL) for cells treated as above. [Figure 27D] For Figures 27C to 27G, the vertical bars are from left to right: T cells only, T cells + beads, S15 Fc-treated, M2φ, M1φ, and imDC. Figure 27D is a bar graph of TNF-α (pg / mL) for cells treated as above. [Figure 27E] For Figures 27C to 27G, the vertical bars are from left to right: T cells only, T cells + beads, S15 Fc-treated, M2φ, M1φ, and imDC. Figure 27E is the CD4 proliferation (%) for cells treated as above. [Figure 27F] For Figures 27C to 27G, the vertical bars are from left to right: T cells only, T cells + beads, S15 Fc-treated, M2φ, M1φ, and imDC. Figure 27F is a bar graph of IL-6 (pg / mL) for cells treated as above. [Figure 27G]For Figures 27C to 27G, the vertical bars are from left to right: T cells only, T cells + beads, S15 Fc-treated, M2φ, M1φ, and imDC. Figure 27G is a bar graph of IL-10 (pg / mL) for cells treated as above. [Figure 28] Figure 28A is a bar graph of absorbance (450 nm) versus mAb (μg / mL) for cells from donor 1709. The top line is the control mAb and the bottom line is the S15 mAb. Figure 28B is the same as Figure 28A but for cells from donor 1713. [Figure 29] Schematic representation of the role of Siglec-15 in osteoclastogenesis. [Figure 30] Figure 30A is a FACS histogram of human CD14+ monocytes treated with S15 Fc and stained with anti-αvβ3 integrin mAb on day 0. Figure 30B is a FACS histogram of human CD14+ monocytes treated with S15 Fc and stained with anti-αvβ3 integrin mAb on day 6. DETAILED DESCRIPTION OF THE INVENTION

[0035] I. Definition As used herein, a molecule is said to be capable of "immunospecifically binding" to a second molecule if such binding exhibits the specificity and affinity of an antibody for its cognate antigen. An antibody is said to be capable of immunospecifically binding to a target region or conformation of an antigen ("epitope") if such binding involves the antigen recognition portion of an immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind other antigens with lower affinity if the other antigens share certain sequence or conformational similarities recognized by the antigen recognition portion as determined, for example, by immunoassays, BIACORE® assays, or other assays known in the art, but should not bind to completely unrelated antigens. However, antibodies (and their antigen-binding fragments) preferably do not cross-react with other antigens. Antibodies may also bind other molecules in a non-immunospecific manner, such as binding to FcR receptors, via binding domains in other regions / domains of the molecule not involved in the antigen recognition portion, such as the Fc region.

[0036] As used herein, a molecule is said to "physically bind" to a second molecule if such binding exhibits the specificity and affinity of a receptor for its cognate binding ligand. A molecule may be capable of physically binding to more than one other molecule.

[0037] As used herein, the term "antibody" is intended to mean an immunoglobulin molecule having an antigen recognition site that is a "variable region." The term "variable region" is intended to distinguish such domains of an immunoglobulin from domains broadly shared by antibodies (such as the antibody Fc domain). The variable region contains the "hypervariable region," the residues of which are responsible for antigen binding. The hypervariable regions are amino acid residues in the "complementarity determining regions" or "CDRs" (i.e., typically located at about residues 24-34 (L1), 50-56 (L2), and 89-93 (L3) in the light chain variable domain and at about residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.The term antibody includes monoclonal antibodies, polyspecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (see, e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25; International Publication Nos. WO 94 / 04678 and WO 94 / 25591; U.S. Pat. No. 6,005,079), single-chain Fvs (scFvs) (see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, 1999), and the like. New York, pp. 269-315 (1994)), single-chain antibodies, disulfide-linked Fvs (sdFvs), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies and anti-anti-Id antibodies directed against an antibody). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0038] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that contain the antibody's complementarity-determining regions ("CDRs") and, optionally, framework residues comprising the antibody's "variable region" antigen recognition site, and that exhibit immunospecific binding to an antigen. Such fragments include Fab', F(ab')2, Fv, single-chain (ScFv), and mutants and naturally occurring variants thereof, as well as fusion proteins comprising the antibody's "variable region" antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor, or a receptor ligand).

[0039] As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0040] As used herein, the term "modulate" refers to the ability to change an effect, outcome, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuating but not eliminating) or can completely eliminate such activity (e.g., neutralizing). Modulation can include reducing receptor internalization or receptor expression on target cells after antibody binding. Agonistic modulation can enhance, otherwise increase, or potentiate, an activity (e.g., signal transduction). In yet further embodiments, such modulation can alter the nature of the interaction between a ligand and its cognate receptor, so as to change the nature of the signal transduction induced. For example, a molecule, by binding to a ligand or receptor, can alter the ability of such molecule to bind to other ligands or receptors, thereby altering its overall activity. Preferably, such modulation results in at least a 10% change in a measurable immune system activity, more preferably at least a 50% change in such activity, or at least a 2-fold, 5-fold, or 10-fold change, or even more preferably at least a 100-fold change in such activity.

[0041] The term "substantially," when used in reference to binding or an exhibited effect, is intended to mean that the observed effect is physiologically or therapeutically significant. Thus, for example, a molecule can substantially block the activity of a ligand or receptor if the degree of blockage is physiologically or therapeutically significant (e.g., if such degree is greater than 60% complete, greater than 70% complete, greater than 75% complete, greater than 80% complete, greater than 85% complete, greater than 90% complete, greater than 95% complete, or greater than 97% complete). Similarly, a molecule can be said to have substantially the same immunospecificity and / or properties as another molecule if such immunospecificity and properties are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical.

[0042] As used herein, a "costimulatory" signal encompasses positive costimulatory signals (eg, signals that result in an enhancement of an activity) and negative costimulatory signals (eg, signals that result in an inhibition of an activity).

[0043] As used herein, the term "derivative" refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to the same target as a parent or reference antibody but differs in amino acid sequence from the parent or reference antibody or antigen-binding fragment thereof by containing one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications relative to the parent or reference antibody or antigen-binding fragment thereof. Such derivatives preferably have substantially the same immunospecificity and / or properties as the parent or reference antibody or antigen-binding fragment thereof, or the same immunospecificity and properties. The amino acid substitutions or additions of such derivatives can include naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" encompasses, for example, chimeric or humanized variants, as well as variants with altered CH1, hinge, CH2, CH3, or CH4 regions to form, for example, antibodies with mutated Fc regions exhibiting enhanced or weakened effector or binding properties.

[0044] As used herein, a "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region.

[0045] As used herein, the term "humanized antibody" refers to an immunoglobulin containing a human framework region and one or more CDRs derived from a non-human (usually mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "acceptor." The constant region need not necessarily be present, but if present, it should be substantially identical to a human immunoglobulin constant region, i.e., at least about 85-99%, preferably about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly for the CDRs, are substantially identical to corresponding parts of a natural human immunoglobulin sequence. A humanized antibody is an antibody containing a humanized light chain and a humanized heavy chain immunoglobulin. For example, humanized antibodies are not considered to encompass typical chimeric antibodies, for example, because the entire variable region of a chimeric antibody is non-human.

[0046] As used herein, the term "endogenous concentration" refers to the level at which a molecule is naturally expressed (i.e., in the absence of an expression vector or recombinant promoter) by a cell (which may be a normal cell, a cancer cell, or an infected cell).

[0047] As used herein, the terms "treat," "treating," "treatment," and "therapeutic use" refer to the elimination, reduction, or amelioration of one or more symptoms of a disease or disorder exacerbated by Siglec-15 or its ligands.

[0048] As used herein, a "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to mediate a clinically meaningful elimination, reduction, or improvement of such symptoms. An effect is clinically significant if its magnitude is sufficient to affect the health or prognosis of the recipient subject. A therapeutically effective amount can refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of a disease, for example, to delay or minimize the spread of cancer. A therapeutically effective amount can also refer to an amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0049] As used herein, the term "prophylactic agent" refers to an agent that can be used to prevent a disorder or disease before any symptoms of such disorder or disease are detected. A "prophylactically effective" amount is the amount of a prophylactic agent sufficient to mediate such prevention. A prophylactically effective amount may also refer to the amount of a prophylactic agent that provides a prophylactic benefit in disease prevention.

[0050] As used herein, the term "cancer" refers to a neoplasm or tumor resulting from uncontrolled, abnormal cell growth. As used herein, cancer explicitly includes leukemia and lymphoma. The term "cancer" refers to a disease involving cells that have the potential to metastasize to distant sites and exhibit phenotypic traits distinct from those of non-cancerous cells, for example, the formation of colony(s) in a three-dimensional matrix such as soft agar, or the formation of a tubular network or reticular matrix in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancerous cells do not form colonies in soft agar, but rather form distinct spheroid structures in a three-dimensional basement membrane or extracellular matrix preparation.

[0051] As used herein, "immune cell" refers to any cell of hematopoietic origin, including, but not limited to, T cells, B cells, monocytes, dendritic cells, and macrophages.

[0052] As used herein, "valency" refers to the number of available binding sites per molecule.

[0053] As used herein, the terms "immunologic," "immunological," or "immune" response are the development in a recipient patient of a beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretions) response directed against a peptide. Such responses can be active responses induced by administration of an immunogen, or passive responses induced by administration of antibodies or primed T cells. A cellular immune response is one in which a polypeptide epitope is presented in association with class I or class II MHC molecules to elicit antigen-specific CD4 + Helper T cells and / or CD8 + It is elicited by the activation of cytotoxic T cells. This response may also involve the activation of monocytes, macrophages, natural killer cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, or the activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immune response can be determined by proliferation assays (CD4 + The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

[0054] As used herein, an "immunogenic agent" or "immunogen" is capable of inducing an immune response against itself when administered to a mammal, optionally in conjunction with an adjuvant.

[0055] As used herein, the terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans, rodents such as mice and rats, and other laboratory animals.

[0056] As used herein, the term "polypeptide" refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. Polypeptides may be "exogenous," meaning that they are "heterologous," or foreign, to the host cell in which they are utilized, such as human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as amino acid residue sequences. These sequences are written from left to right in the amino- to carboxy-terminal direction. In accordance with standard nomenclature, amino acid residue sequences are designated by either three-letter or single-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0057] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference and variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A polypeptide variant may be naturally occurring, such as an allelic variant, or a variant that is not known to occur naturally.

[0058] Modifications and changes can be made to the structure of the polypeptides of the present disclosure and still obtain molecules with similar properties to the polypeptides (e.g., conservative amino acid substitutions). For example, certain amino acids can be substituted for other amino acids in the sequence without significant loss of activity. Because what defines the biological functional activity of a polypeptide is its ability and properties to interact, certain amino acid sequence substitutions can be made in a polypeptide sequence to obtain polypeptides with similar properties.

[0059] When making such changes, the hydrophilicity index of an amino acid can be taken into consideration. The importance of the hydrophilicity amino acid index in conferring interactive biological function to a polypeptide is generally understood in the art. It is known that substitution of an amino acid with another amino acid having a similar hydrophilicity index or score can result in a polypeptide having similar biological activity. Each amino acid is assigned a hydrophilicity index based on its hydrophobicity and charge characteristics. These indicators are isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0060] The relative hydrophilicity index properties of amino acids are believed to determine the secondary structure of the resulting polypeptide, which in turn determines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that the substitution of an amino acid with another amino acid having a similar hydrophilicity index can result in a functionally equivalent polypeptide. In such changes, substitution of amino acids with hydrophilicity indices within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0061] Substitutions of similar amino acids can also be made on the basis of hydrophilicity, particularly when the resulting biologically functional equivalent polypeptide or peptide is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5±1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that substitution of an amino acid with another amino acid having a similar hydrophilicity value will still result in a biologically equivalent, and particularly an immunologically equivalent, polypeptide. In such changes, substitution of amino acids having hydrophilicity values within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0062] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Representative substitutions that take into consideration the various aforementioned characteristics are well known to those of skill in the art and include the following (original residue: representative substitution): (Ala:Gly, Ser), (Arg:Lys), (Asn:Gln, His), (Asp:Glu, Cys, Ser), (Gln:Asn), (Glu:Asp), (Gly:Ala), (His:Asn, Gln), (Ile:Leu, Val), (Leu:Ile, Val), (Lys:Arg), (Met:Leu, Tyr), (Ser:Thr), (Thr:Ser), (Tip:Tyr), (Tyr:Trp, Phe), and (Val:Ile, Leu). Thus, embodiments of the present disclosure contemplate functional or biological equivalents of the polypeptides as described above. In particular, polypeptide embodiments may include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide of interest.

[0063] The term "percentage of sequence identity (%)" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in a reference nucleic acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity.The alignment for determining the percentage of sequence identity can be achieved in various ways within the art, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.The appropriate parameters for measuring alignment, including any algorithms required to achieve the maximum alignment over the entire length of the sequences being compared, can be determined by known methods.

[0064] For purposes herein, the percent sequence identity of a given nucleotide or amino acid sequence C to or relative to a given nucleic acid sequence D (alternatively, it may be expressed as a given sequence C having or containing a certain percent sequence identity to or relative to a given sequence D) is calculated as follows: 100 times the fraction W / Z, where W is the number of nucleotides or amino acids scored as a perfect match by a sequence alignment program in aligning C with D, and Z is the total number of nucleotides or amino acids in D. It will be understood that if the length of sequence C is not equal to the length of sequence D, then the % sequence identity of C to D will not be equal to the % sequence identity of D to C.

[0065] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents.

[0066] As used herein, the terms "antigenic determinant" and "epitope" are used interchangeably and refer to a structure recognized by an antibody.

[0067] As used herein, a "conformational epitope" is an epitope that comprises a discontinuous stretch of the amino acid sequence of an antigen. Antibodies bind to conformational epitopes based on the three-dimensional surface features, shape, or tertiary structure of the antigen.

[0068] As used herein, a "linear epitope" is an epitope formed by a continuous amino acid sequence from an antigen. A linear epitope typically contains about 5 to about 10 consecutive amino acid residues. An antibody binds to a linear epitope based on the primary sequence of the antigen.

[0069] As used herein, a "paratope," also called an "antigen-binding site," is the portion of an antibody that recognizes and binds to an antigen.

[0070] II. Composition A. Siglec-15 sequence Sialic acid-binding Ig-like lectin 15 (also known as "Siglec-15," CD33 antigen-like 3, and CD33L3) is a type 1 transmembrane protein expressed on macrophages and / or dendritic cells in human spleen and lymph nodes (Angata, et al., Glycobiology, 17(8):838-46 (2007), which is expressly incorporated herein by reference in its entirety). The extracellular domain of Siglec-15 binds to sialylated glycoproteins and preferentially recognizes the Neu5Acα2-6GalNAcα structure.

[0071] Siglec-15 associates with the activating adaptor proteins DNAX-activating protein (DAP) 12 and DAP10 via its lysine residue (residue K274) in the transmembrane domain, indicating that it functions as an activating signaling molecule. Siglec-15 orthologs exist not only in mammals but also in other vertebrate branches, and are thought to play a conserved regulatory role in the vertebrate immune system.

[0072] Siglec-15 directly regulates T cell function by inhibiting T cell proliferation and proinflammatory cytokine production. Siglec-15 indirectly influences T cell function through myeloid cells. Siglec-15, expressed on tumor cells or M2 macrophages, interacts with binding partners on myeloid cells to provide survival and differentiation signals, resulting in a unique myeloid cell population that produces TNF-α, IL-6, and IL-1β. The secreted cytokines further promote tumor growth. This subset of myeloid cells can affect T cell function by reducing IFN-γ production in T cells.

[0073] The amino acid sequence of human Siglec-15 is known in the art and includes, for example: TIFF0007720375000002.tif46136 (SEQ ID NO:1), UniProtKB-Q6ZMC9 (SIG15_HUMAN), which is expressly incorporated by reference in its entirety.

[0074] Human Siglec-15 contains a signal peptide sequence, amino acids 1-19 of SEQ ID NO:1; an extracellular domain, amino acids 20-263 of SEQ ID NO:1 (shown in bold and italicized text); a transmembrane domain, amino acids 264-284 of SEQ ID NO:1; and a cytoplasmic domain, amino acids 285-328 of SEQ ID NO:1. A predicted Ig-like V-type domain is located at amino acids 40-158 of SEQ ID NO:1 (shown in single underline), and a predicted Ig-like C2-type domain is located at amino acids 168-251 of SEQ ID NO:1 (shown in double underline). Disulfide bonds are thought to form at residues 64-142, 95-104, and 187-237, with glycosylation predicted at residue 172. The amino acids 276 to 279 are called the polyleucine domain. A known variant is the F273L substitution variant.

[0075] The amino acid sequence of mouse Siglec-15 is known in the art and includes, for example: TIFF0007720375000003.tif47136 (SEQ ID NO:2), UniProtKB-A7E1W8 (A7E1W8_MOUSE), which is expressly incorporated by reference in its entirety.

[0076] Mouse Siglec-15 contains a signal peptide sequence, amino acids 1 to 23 of SEQ ID NO:2, an extracellular domain, amino acids 24 to 262 of SEQ ID NO:2 (shown in bold and italicized text), a transmembrane domain, amino acids 263 to 283 of SEQ ID NO:2, and a cytoplasmic domain, amino acids 284 to 342 of SEQ ID NO:2. A predicted Ig-like V-type domain is amino acids 40 to 145 of SEQ ID NO:2 (shown in single underline), and a predicted Ig-like C2-type domain is amino acids 169 to 250 of SEQ ID NO:2 (shown in double underline).

[0077] B. Siglec-15 binding molecules Siglec-15 binding molecules, such as antibodies and antigen-binding fragments thereof, and other polypeptides that bind to Siglec-15 are provided. Sequences of heavy and light chain variable regions and their CDRs derived from a murine anti-Siglec-15 antibody are provided below. Antibodies, antigen-binding fragments, and other polypeptides, as well as variants thereof, that include one or more of the following sequences are provided. For example, antibodies, antigen-binding fragments, and polypeptides that bind to Siglec-15 are provided that include one, two, or three CDRs of an anti-Siglec-15 antibody light chain variable region and / or one, two, or three CDRs of an anti-Siglec-15 antibody heavy chain variable region. In some embodiments, the antibodies, antigen-binding fragments, and polypeptides include an anti-Siglec-15 antibody light chain variable region, an anti-Siglec-15 heavy chain variable region, or a combination thereof, and are capable of binding to Siglec-15.

[0078] For example, the disclosed molecules can immunospecifically bind to Siglec-15 (e.g., SEQ ID NO:1, SEQ ID NO:2, etc.). (I) arranged on the surface of a cell (preferably a living cell); (II) arranged at endogenous concentrations on the surface of cells (preferably living cells); (III) arranged on the surface of a living cell and modulating the binding of Siglec-15 (e.g., SEQ ID NO:1, SEQ ID NO:2, etc.) to Neu5Acα2-6GalNAcα, LRRC4C, Siglec-15 counter-receptor (S15-CR), or a combination thereof; (IV) arranged on the surface of living cells to reduce, prevent, or inhibit TGF-β secretion; (V) arranged on the surface of a living cell, which is a myeloid cell, e.g., a macrophage or dendritic cell, or a cancer cell (e.g., a brain cancer cell, a renal cell carcinoma cell (RCC), a Ewing's sarcoma cell, a breast cancer cell, or an ovarian cancer cell); (VI) performing a combination thereof; Molecules capable of immunospecifically binding to human Siglec-15 are provided.

[0079] 1. Mouse anti-human Siglec-15 antibody sequence As described in the Examples below, a panel of mouse anti-human Siglec-15 mAbs was generated by immunizing Siglec-15 knockout mice (n=2) with hS15.mIg (human Siglec-15 extracellular domain [ECD] fused to mouse IgG2a) emulsified in CFA (Freund's complete adjuvant).

[0080] The sequences of the light and heavy chain variable regions of the monoclonal antibodies produced by the 24 hybridomas, referred to herein as follows, are set forth below: 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A (also referred to as NC6 and no. 6), 28A (also referred to as NC28 and no. 28), 63A (also referred to as NC63 and no. 63), 71A (also referred to as NC71 and no. 71), 77A (also referred to as NC77 and 77), 80A (also referred to as NC80 and 80), 82B (also referred to as NC82 and 82), 83B (also referred to as NC83 and 83), 92A (also referred to as NC92 and 92), 93B (also referred to as NC93 and 93), 99B (also referred to as NC99 and 99), 104B (also referred to as NC104 and 104), and 105A (also referred to as NC105 and 105). For the light and heavy chain sequences, the CDRs are underlined and in bold. The sequences and CDRs are also shown in the alignments in Figures 2A-3C.

[0081] a.1B2 sequence: i. Light chain The amino acid sequence of the light chain variable region of 1B2 is: TIFF0007720375000004.tif17135 (SEQ ID NO: 3), which sequence is 1B2 light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 24) 1B2 light chain CDR2: KVSNRFS (SEQ ID NO: 32) 1B2 light chain CDR3: FQGSHVPWT (SEQ ID NO: 39) It has.

[0082] The nucleic acid sequence encoding the light chain variable region of 1B2 is TIFF0007720375000005.tif46135 (SEQ ID NO: 74).

[0083] ii. Heavy chain The heavy chain variable region amino acid sequence of 1B2 is TIFF0007720375000006.tif19135 (SEQ ID NO: 13), which sequence is 1B2 heavy chain CDR1: GFTFSDYGMH (SEQ ID NO: 46) 1B2 heavy chain CDR2: YISSGSSIIYYADTVKG (SEQ ID NO: 56) 1B2 heavy chain CDR3: DHYHGNGSDY (SEQ ID NO: 67) It has.

[0084] The nucleic acid sequence encoding the heavy chain variable region of 1B2 is TIFF0007720375000007.tif46135 (SEQ ID NO: 85).

[0085] b.1C3 sequence i. Light chain The amino acid sequence of the light chain variable region of 1C3 is: TIFF0007720375000008.tif17135 (SEQ ID NO: 4), which sequence is 1C3 light chain CDR1: RSSKSLLHSNGNTYLY (SEQ ID NO::25) 1C3 light chain CDR2: RMSNLAS (SEQ ID NO: 33) 1C3 light chain CDR3: MQHLEYPYT (SEQ ID NO: 40) It has.

[0086] The nucleic acid sequence encoding the 1C3 light chain variable region is TIFF0007720375000009.tif46135 (SEQ ID NO: 75).

[0087] ii. Heavy chain The amino acid sequence of the heavy chain variable region of 1C3 is: TIFF0007720375000010.tif18135 (SEQ ID NO: 14), which sequence is 1C3 heavy chain CDR1: GYIFTDYYVN (SEQ ID NO: 47) 1C3 heavy chain CDR2: KIGPGSVSIYYNEKFKG (SEQ ID NO: 57) 1C3 heavy chain CDR3: YYYGFAY (SEQ ID NO: 68) It has.

[0088] The nucleic acid sequence encoding the heavy chain variable region of 1C3 is TIFF0007720375000011.tif46135 (SEQ ID NO: 86).

[0089] c.1H3 sequence i. Light chain The amino acid sequence of the light chain variable region of 1H3 is: TIFF0007720375000012.tif17135 (SEQ ID NO: 5), which sequence is 1H3 light chain CDR1: KASDHINNWLA (SEQ ID NO: 26) 1H3 light chain CDR2: GATSLET (SEQ ID NO: 34) 1H3 light chain CDR3: QQYWSSPLT (SEQ ID NO: 41) It has.

[0090] The nucleic acid sequence encoding the 1H3 light chain variable region is TIFF0007720375000013.tif46135 (SEQ ID NO: 76).

[0091] ii. Humanized light chain One embodiment has the following variable light chain amino acid sequence: TIFF0007720375000014.tif19137, which has the sequence 1H3 light chain CDR1: KASDHINNWLA (SEQ ID NO: 26) 1H3 light chain CDR2: GATSLET (SEQ ID NO: 34) 1H3 light chain CDR3: QQYWSSPLT (SEQ ID NO: 41) The underlined amino acids are changed relative to the parent sequence.

[0092] Another embodiment has the following variable light chain amino acid sequence: TIFF0007720375000015.tif19136, which has the sequence 1H3 light chain CDR1: KASDHINNWLA (SEQ ID NO: 26) 1H3 light chain CDR2: GATSLET (SEQ ID NO: 34) 1H3 light chain CDR3: QQYWSSPLT (SEQ ID NO: 41) The underlined amino acids are changed relative to the parent sequence.

[0093] Yet another embodiment has the following variable light chain amino acid sequence: TIFF0007720375000016.tif19136, which has the sequence 1H3 light chain CDR1: KASDHINNWLA (SEQ ID NO: 26) 1H3 light chain CDR2: GATSLET (SEQ ID NO: 34) 1H3 light chain CDR3: QQYWSSPLT (SEQ ID NO: 41) The underlined amino acids are changed relative to the parent sequence.

[0094] iii. Heavy chain The heavy chain variable region amino acid sequence of 1H3 is: TIFF0007720375000017.tif19135 (SEQ ID NO: 15), which sequence is 1H3 heavy chain CDR1: NYGVH (SEQ ID NO: 48) 1H3 Heavy Chain CDR2: LIWSDGSTTYNSALKS (SEQ ID NO: 58) 1H3 heavy chain CDR3: HPYDDYSGYYYTMDY (SEQ ID NO: 69) It has.

[0095] The nucleic acid sequence encoding the heavy chain variable region of 1H3 is TIFF0007720375000018.tif46135 (SEQ ID NO: 87).

[0096] iv. Humanized Heavy Chain One embodiment has the following variable heavy chain amino acid sequence: TIFF0007720375000019.tif19137, which has the sequence 1H3 heavy chain CDR1: NYGVH (SEQ ID NO: 48) 1H3 Heavy Chain CDR2: LIWSDGSTTYNSALKS (SEQ ID NO: 58) 1H3 heavy chain CDR3: HPYDDYSGYYYTMDY (SEQ ID NO: 69) The underlined amino acids are changed relative to the parent sequence.

[0097] Another embodiment has the following variable heavy chain amino acid sequence: TIFF0007720375000020.tif19137, the sequence of which is 1H3 heavy chain CDR1: NYGVH (SEQ ID NO: 48) 1H3 Heavy Chain CDR2: LIWSDGSTTYASALKS (SEQ ID NO: 214) 1H3 Heavy chain CDR3: HPYDDYSGYYYTMDY (SEQ ID NO: 69). The underlined amino acids are changed relative to the parent sequence.

[0098] Yet another embodiment has the following variable heavy chain amino acid sequence: TIFF0007720375000021.tif19137, which has the sequence 1H3 heavy chain CDR1: NYGVH (SEQ ID NO: 48) 1H3 heavy chain CDR2: LIWSDGSTTYNPSLKS (SEQ ID NO: 218) 1H3 heavy chain CDR3: HPYDDYSGYYYTMDY (SEQ ID NO: 69) The underlined amino acids are changed relative to the parent sequence.

[0099] Another embodiment has the following variable heavy chain amino acid sequence: TIFF0007720375000022.tif19137, which has the sequence 1H3 heavy chain CDR1: NYGVH (SEQ ID NO: 48) 1H3 Heavy Chain CDR2: LIWSEGSTTYASALKS (SEQ ID NO: 217) 1H3 heavy chain CDR3: HPYDDYSGYYYTMDY (SEQ ID NO: 69) It has.

[0100] d.1C12 sequence: i. Light chain The amino acid sequence of the 1C12 light chain variable region is TIFF0007720375000023.tif18135 (SEQ ID NO: 3), which sequence is 1C12 light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 24) 1C12 light chain CDR2: KVSNRFS (SEQ ID NO: 32) 1C12 light chain CDR3: FQGSHVPWT (SEQ ID NO: 39) It has.

[0101] The nucleic acid sequence encoding the 1C12 light chain variable region is TIFF0007720375000024.tif46135 (SEQ ID NO: 77).

[0102] ii. Heavy chain The heavy chain variable region amino acid sequence of 1C12 is: TIFF0007720375000025.tif19135 (SEQ ID NO: 16), which sequence is 1C12 heavy chain CDR1: GFSFSDYGMH (SEQ ID NO: 49) 1C12 heavy chain CDR2: YISSGSSILYYADIVK (SEQ ID NO: 59) 1C12 heavy chain CDR3: DHYHGNGSDY (SEQ ID NO: 67) It has.

[0103] The nucleic acid sequence encoding the heavy chain variable region of 1C12 is TIFF0007720375000026.tif46135 (SEQ ID NO: 88).

[0104] e.3H10 sequence i. light chain The amino acid sequence of the light chain variable region of 3H10 is: TIFF0007720375000027.tif11135 (SEQ ID NO: 6), which sequence is 3H10 light chain CDR1: SASSSTSFMH (SEQ ID NO: 27) 3H10 light chain CDR2: DTSKLA (SEQ ID NO: 35) 3H10 light chain CDR3: HQRSAYPWT (SEQ ID NO: 42) It has.

[0105] The nucleic acid sequence encoding the light chain variable region of 3H10 is TIFF0007720375000028.tif39135 (SEQ ID NO: 78).

[0106] ii. Heavy chain The heavy chain variable region amino acid sequence of 3H10 is: TIFF0007720375000029.tif19135 (SEQ ID NO: 17), which sequence is 3H10 heavy chain CDR1: GFNIKDYYMH (SEQ ID NO: 50) 3H10 heavy chain CDR2: RIDPEDGDIEYDPKFQG (SEQ ID NO: 60) 3H10 heavy chain CDR3: DYDYDGGWFAY (SEQ ID NO: 70) It has.

[0107] The nucleic acid sequence encoding the 3H10 heavy chain variable region is TIFF0007720375000030.tif46135 (SEQ ID NO: 89).

[0108] f.5G12 array i. light chain The amino acid sequence of the light chain variable region of 5G12 is: TIFF0007720375000031.tif17135 (SEQ ID NO: 7), which sequence is 5G12 light chain CDR1: KASQDINSYLS (SEQ ID NO: 28) 5G12 light chain CDR2: RANRLVD (SEQ ID NO: 36) 5G12 light chain CDR3: LQYDEFPYT (SEQ ID NO: 43) It has.

[0109] The nucleic acid sequence encoding the 5G12 light chain variable region is TIFF0007720375000032.tif46135 (SEQ ID NO: 79).

[0110] ii. Heavy chain The heavy chain variable region amino acid sequence of 5G12 is TIFF0007720375000033.tif19135 (SEQ ID NO: 18), which sequence is 5G12 heavy chain CDR1: GYTFTSYWIT (SEQ ID NO: 51) 5G12 heavy chain CDR2: DIYCGSDTMHYNEKFKN (SEQ ID NO: 61) 5G12 heavy chain CDR3: WWDYGSSYDYFDY (SEQ ID NO: 71) It has.

[0111] The nucleic acid sequence encoding the heavy chain variable region of 5G12 is TIFF0007720375000034.tif46135 (SEQ ID NO: 90).

[0112] g.6F8 sequence i. light chain The amino acid sequence of the light chain variable region of 6F8 is TIFF0007720375000035.tif18135 (SEQ ID NO: 8), which sequence is 6F8 light chain CDR1: RSSKSLLHSNGNTYLY (SEQ ID NO: 25) 6F8 light chain CDR2: RMSNLAS (SEQ ID NO: 33) 6F8 light chain CDR3: MQHLEYPYT (SEQ ID NO: 40) It has.

[0113] The nucleic acid sequence encoding the light chain variable region of 6F8 is TIFF0007720375000036.tif46135 (SEQ ID NO: 80).

[0114] ii. Heavy chain The heavy chain variable region amino acid sequence of 6F8 is TIFF0007720375000037.tif18135 (SEQ ID NO: 19), which sequence is 6F8 heavy chain CDR1: GYTFTDYYVN (SEQ ID NO: 52) 6F8 heavy chain CDR2: KIGPGSVSIYYNEKFKD (SEQ ID NO: 62) 6F8 heavy chain CDR3: YYYGFAY (SEQ ID NO: 68) It has.

[0115] The nucleic acid sequence encoding the heavy chain variable region of 6F8 is TIFF0007720375000038.tif46135 (SEQ ID NO: 91).

[0116] h.8C8 array i. light chain The 8C8 light chain variable region amino acid sequence is TIFF0007720375000039.tif18135 (SEQ ID NO: 9), which sequence is 8C8 light chain CDR1: RSSKSLLHSNGNTYLY (SEQ ID NO: 25) 8C8 light chain CDR2: RMSNLAS (SEQ ID NO: 33) 8C8 light chain CDR3: MQHLEYPYT (SEQ ID NO: 40) It has.

[0117] The nucleic acid sequence encoding the light chain variable region of 8C8 is TIFF0007720375000040.tif46135 (SEQ ID NO: 81).

[0118] ii. Heavy chain The heavy chain variable region amino acid sequence of 8C8 is: TIFF0007720375000041.tif18135 (SEQ ID NO: 20), which sequence is 8C8 heavy chain CDR1: GYTFTDYYVN (SEQ ID NO: 52) 8C8 heavy chain CDR2: KIGPESVSIYYSEKFKA (SEQ ID NO: 63) 8C8 heavy chain CDR3: YYYGFAY (SEQ ID NO: 68) It has.

[0119] The nucleic acid sequence encoding the heavy chain variable region of 8C8 is TIFF0007720375000042.tif46135 (SEQ ID NO: 92).

[0120] i.8H8 sequence i. light chain The 8H8 light chain variable region amino acid sequence is TIFF0007720375000043.tif18135 (SEQ ID NO: 10), which sequence is 8H8 light chain CDR1: RSSSGAVTTGNFAN (SEQ ID NO: 29) 8H8 light chain CDR2: GTNNRAP (SEQ ID NO: 37) 8H8 light chain CDR3: ALWYSNHWV (SEQ ID NO: 44) It has.

[0121] The nucleic acid sequence encoding the light chain variable region of 8H8 is TIFF0007720375000044.tif46135 (SEQ ID NO: 82).

[0122] ii. Heavy chain The heavy chain variable region amino acid sequence of 8H8 is: TIFF0007720375000045.tif19135 (SEQ ID NO: 21), which sequence is 8H8 heavy chain CDR1: GFTFSGFWMS (SEQ ID NO: 53) 8H8 heavy chain CDR2: DINSDGSAINYAPSIKD (SEQ ID NO: 64) 8H8 heavy chain CDR3: YDDYGYFDV (SEQ ID NO: 72) It has.

[0123] The nucleic acid sequence encoding the heavy chain variable region of 8H8 is TIFF0007720375000046.tif46135 (SEQ ID NO: 93).

[0124] j.9A5 sequence i. Light chain The amino acid sequence of the light chain variable region of 9A5 is: TIFF0007720375000047.tif18135 (SEQ ID NO: 11), which sequence is 9A5 light chain CDR1: KSSQSLLDSDGKTYLN (SEQ ID NO: 30) 9A5 light chain CDR2: LVSKLDS (SEQ ID NO: 38) 9A5 light chain CDR3: WQGTHFPFT (SEQ ID NO: 45) It has.

[0125] The nucleic acid sequence encoding the light chain variable region of 9A5 is TIFF0007720375000048.tif46135 (SEQ ID NO: 83).

[0126] ii. Heavy chain The heavy chain variable region amino acid sequence of 9A5 is: TIFF0007720375000049.tif18135 (SEQ ID NO: 22), which sequence is 9A5 heavy chain CDR1: GYTFTSYGLI (SEQ ID NO: 54) 9A5 heavy chain CDR2: EIYPRSGNTYYNEKFKG (SEQ ID NO: 65) 9A5 heavy chain CDR3: SSPHGDY (SEQ ID NO: 73) It has.

[0127] The nucleic acid sequence encoding the heavy chain variable region of 9A5 is TIFF0007720375000050.tif46135 (SEQ ID NO: 94).

[0128] k.10G9 array i. light chain The amino acid sequence of the light chain variable region of 10G9 is: TIFF0007720375000051.tif18135 (SEQ ID NO: 12), which sequence is 10G9 light chain CDR1: RSSTGAVTTSNYAN (SEQ ID NO: 31) 10G9 light chain CDR2: GTNNRAP (SEQ ID NO: 37) 10G9 light chain CDR3: ALWYSNHWV (SEQ ID NO: 44) It has.

[0129] The nucleic acid sequence encoding the light chain variable region of 10G9 is TIFF0007720375000052.tif46135 (SEQ ID NO: 84).

[0130] ii. Heavy chain The heavy chain variable region amino acid sequence of 10G9 is: TIFF0007720375000053.tif19135 (SEQ ID NO: 23), which sequence is 10G9 heavy chain CDR1: GFTFSDFWMS (SEQ ID NO: 55) 10G9 heavy chain CDR2: DINSDGSAVNYAPSIKD (SEQ ID NO: 66) 10G9 heavy chain CDR3: YDDYGYFDV (SEQ ID NO: 72) It has.

[0131] The nucleic acid sequence encoding the heavy chain variable region of 10G9 is: TIFF0007720375000054.tif46135 (SEQ ID NO: 95).

[0132] I.6A sequence i. Light chain The amino acid sequence of the light chain variable region of 6A is: TIFF0007720375000055.tif18135 (SEQ ID NO: 96), which sequence is 6A light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 24) 6A light chain CDR2: KVSNRFS (SEQ ID NO: 32) 6A light chain CDR3: FQGSHVPLT (SEQ ID NO: 157) It has.

[0133] The nucleic acid sequence encoding the light chain variable region of 6A is TIFF0007720375000056.tif46135 (SEQ ID NO: 120).

[0134] ii. Heavy chain The heavy chain variable region amino acid sequence of 6A is TIFF0007720375000057.tif18135 (SEQ ID NO: 108), which sequence is 6A heavy chain CDR1: DDYMH (SEQ ID NO: 162) 6A heavy chain CDR2: CIDPENGDTEYASKFQD (SEQ ID NO: 170) 6A heavy chain CDR3: YVGFAY (SEQ ID NO: 182) It has.

[0135] The nucleic acid sequence encoding the heavy chain variable region of 6A is TIFF0007720375000058.tif46135 (SEQ ID NO: 133).

[0136] m.28A array i. Light chain The amino acid sequence of the light chain variable region of 28A is: TIFF0007720375000059.tif17135 (SEQ ID NO: 97), which sequence is 28A light chain CDR1: KSSQSLLDSDGKTYLN (SEQ ID NO: 30) 28A light chain CDR2: LVSELDS (SEQ ID NO: 153) 28A light chain CDR3: WQGTHFPFT (SEQ ID NO: 45) It has.

[0137] The nucleic acid sequence encoding the light chain variable region of 28A is TIFF0007720375000060.tif46135 (SEQ ID NO: 121).

[0138] ii. Heavy chain The heavy chain variable region amino acid sequence of 28A is TIFF0007720375000061.tif18135 (SEQ ID NO: 109), which sequence is 28A heavy chain CDR1: SYGIT (SEQ ID NO: 163) 28A heavy chain CDR2: EIHPRSGNTYYNENFKD (SEQ ID NO: 171) 28A heavy chain CDR3: GGPGDY (SEQ ID NO: 183) It has.

[0139] The nucleic acid sequence encoding the heavy chain variable region of 28A is TIFF0007720375000062.tif46135 (SEQ ID NO: 134).

[0140] No.63A arrangement i. Light chain The amino acid sequence of the light chain variable region of 63A is TIFF0007720375000063.tif18135 (SEQ ID NO: 98), which sequence is 63A light chain CDR1: KSSQSLLDSDGKTYLN (SEQ ID NO: 30) 63A light chain CDR2: LVSKLDS (SEQ ID NO: 38) 63A light chain CDR3: WQGTHFPFT (SEQ ID NO: 45) It has.

[0141] The nucleic acid sequence encoding the light chain variable region of 63A is TIFF0007720375000064.tif46135 (SEQ ID NO: 122).

[0142] ii. Heavy chain The heavy chain variable region amino acid sequence of 63A is TIFF0007720375000065.tif18135 (SEQ ID NO: 110), which sequence is 63A heavy chain CDR1: SYGIS (SEQ ID NO: 164) 63A heavy chain CDR2: QIYPRSDNTYYNERFKGK (SEQ ID NO: 172) 63A heavy chain CDR3: EGGPDY (SEQ ID NO: 184) It has.

[0143] The nucleic acid sequence encoding the heavy chain variable region of 63A is TIFF0007720375000066.tif46135 (SEQ ID NO: 135).

[0144] o.71A arrangement i. Light chain The amino acid sequence of the light chain variable region of 71A is: TIFF0007720375000067.tif18135 (SEQ ID NO: 99), which sequence is 71A light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 24) 71A light chain CDR2: KVSNRFS (SEQ ID NO: 32) 71A light chain CDR3: FQGSHVPLT (SEQ ID NO: 157) It has.

[0145] The nucleic acid sequence encoding the light chain variable region of 71A is TIFF0007720375000068.tif46135 (SEQ ID NO: 123).

[0146] ii. Heavy chain The heavy chain variable region amino acid sequence of 71A is TIFF0007720375000069.tif18135 (SEQ ID NO: 111), which sequence is 71A heavy chain CDR1: DDYMH (SEQ ID NO: 162) 71A heavy chain CDR2: CIDPENGDIEYASRFQG (SEQ ID NO: 173) 71A heavy chain CDR3: YVGFGY (SEQ ID NO: 185) It has.

[0147] The nucleic acid sequence encoding the heavy chain variable region of 71A is TIFF0007720375000070.tif46135 (SEQ ID NO: 136).

[0148] p.77A sequence i. Light chain The amino acid sequence of the light chain variable region of 77A is: TIFF0007720375000071.tif17135 (SEQ ID NO: 100), which sequence is 77A Light Chain CDR1: RSSQNIVHSNGNTYLE (SEQ ID NO: 146) 77A light chain CDR2: KVSNRFS (SEQ ID NO: 32) 77A light chain CDR3: FQGSHVPLT (SEQ ID NO: 157) It has.

[0149] The nucleic acid sequence encoding the light chain variable region of 77A is TIFF0007720375000072.tif46135 (SEQ ID NO: 124).

[0150] ii. Heavy chain The heavy chain variable region amino acid sequence of 77A is TIFF0007720375000073.tif18135 (SEQ ID NO: 112), which sequence is 77A heavy chain CDR1: DDYMH (SEQ ID NO: 162) 77A heavy chain CDR2: CIDPENGDTEYASKFQG (SEQ ID NO: 174) 77A heavy chain CDR3: YVGFGY (SEQ ID NO: 185) It has.

[0151] The nucleic acid sequence encoding the heavy chain variable region of 77A is TIFF0007720375000074.tif46135 (SEQ ID NO: 137).

[0152] q.80A arrangement i. Light chain The amino acid sequence of the light chain variable region of 80A is TIFF0007720375000075.tif18135 (SEQ ID NO: 101), which sequence is 80A light chain CDR1: KSNQSLLNSGDQKNYLT (SEQ ID NO: 147) 80A light chain CDR2: WASTRES (SEQ ID NO: 154) 80A light chain CDR3: QNDYSYPLT (SEQ ID NO: 158) It has.

[0153] The nucleic acid sequence encoding the light chain variable region of 80A is TIFF0007720375000076.tif46135 (SEQ ID NO: 125).

[0154] ii. Heavy chain The heavy chain variable region amino acid sequence of 80A is TIFF0007720375000077.tif18135 (SEQ ID NO: 113), which sequence is 80A heavy chain CDR1: DFYIN (SEQ ID NO: 165) 80A heavy chain CDR2: RIYPGSDETYYNEKFKD (SEQ ID NO: 175) 80A heavy chain CDR3: WFFDV (SEQ ID NO: 186) It has.

[0155] The nucleic acid sequence encoding the heavy chain variable region of 80A is TIFF0007720375000078.tif46135 (SEQ ID NO: 138).

[0156] r.82B arrangement i. Light chain The amino acid sequence of the light chain variable region of 82B is: TIFF0007720375000079.tif18135 (SEQ ID NO: 102), which sequence is 82B light chain CDR1: KSSQSLLDSDGNTYLN (SEQ ID NO: 148) 82B light chain CDR2: LVSELDS (SEQ ID NO: 153) 82B light chain CDR3: WQGTHFPFT (SEQ ID NO: 45) It has.

[0157] The nucleic acid sequence encoding the light chain variable region of 82B is TIFF0007720375000080.tif46135 (SEQ ID NO: 126).

[0158] ii. Heavy chain The heavy chain variable region amino acid sequence of 82B is: TIFF0007720375000081.tif18135 (SEQ ID NO: 114), which sequence is 82B heavy chain CDR1: SDGIT (SEQ ID NO: 166) 82B heavy chain CDR2: QIHPRSGNTYYNGKFKG (SEQ ID NO: 176) 82B heavy chain CDR3: TGTGDY (SEQ ID NO: 187) It has.

[0159] The nucleic acid sequence encoding the heavy chain variable region of 82B is TIFF0007720375000082.tif46135 (SEQ ID NO: 139).

[0160] s.83B sequence i. Light chain The amino acid sequence of the light chain variable region of 83B is: TIFF0007720375000083.tif18135 (SEQ ID NO: 103), which sequence is 83B light chain CDR1: QATQDIVKNLN (SEQ ID NO: 149) 83B light chain CDR2: YATELAE (SEQ ID NO: 155) 83B light chain CDR3: LQFYEFPYT (SEQ ID NO: 159) It has.

[0161] The nucleic acid sequence encoding the light chain variable region of 83B is TIFF0007720375000084.tif46135 (SEQ ID NO: 127).

[0162] ii. Heavy chain The heavy chain variable region amino acid sequence of 83B is TIFF0007720375000085.tif18135 (SEQ ID NO: 115), which sequence is 83B heavy chain CDR1: DYNMH (SEQ ID NO: 167) 83B heavy chain CDR2: YINPNNGGTSYNQKFKD (SEQ ID NO: 177) 83B heavy chain CDR3: SDWEDC (SEQ ID NO: 188) It has.

[0163] The nucleic acid sequence encoding the heavy chain variable region of 83B is TIFF0007720375000086.tif46135 (SEQ ID NO: 140).

[0164] t.92A arrangement i. Light chain The amino acid sequence of the light chain variable region of 92A is TIFF0007720375000087.tif11135 (SEQ ID NO: 104), which sequence is 92A light chain CDR1: SASSSVSYMH (SEQ ID NO: 150) 92A light chain CDR2: RTSNLAS (SEQ ID NO: 156) 92A light chain CDR3: HQWSSWT (SEQ ID NO: 160) It has.

[0165] The nucleic acid sequence encoding the light chain variable region of 92A is TIFF0007720375000088.tif39135 (SEQ ID NO: 128).

[0166] ii. Heavy chain The heavy chain variable region amino acid sequence of 92A is TIFF0007720375000089.tif19135 (SEQ ID NO: 116), which sequence is 92A heavy chain CDR1: SGYYWN (SEQ ID NO: 168) 92A heavy chain CDR2: YIRHDGSNNYNPSLKN (SEQ ID NO: 178) 92A heavy chain CDR3: EIYDGSSGYFDVWGT (SEQ ID NO: 189) It has.

[0167] The nucleic acid sequence encoding the heavy chain variable region of 92A is TIFF0007720375000090.tif46135 (SEQ ID NO: 141).

[0168] u.93B sequence i. Light chain The amino acid sequence of the light chain variable region of 93B is: TIFF0007720375000091.tif18135 (SEQ ID NO: 105), which sequence is 93B light chain CDR1:KSSQSLLNSGNQKNYLT(SEQ ID NO:151) 93B light chain CDR2: WASTRES (SEQ ID NO: 154) 93B light chain CDR3: QNDYSFPFT (SEQ ID NO: 161) It has.

[0169] The nucleic acid sequence encoding the light chain variable region of 93B is TIFF0007720375000092.tif46135 (SEQ ID NO: 129).

[0170] ii. Heavy chain The heavy chain variable region amino acid sequence of 93B is: TIFF0007720375000093.tif18135 (SEQ ID NO: 117), which sequence is 93B heavy chain CDR1:DYYIN(SEQ ID NO:169) 93B heavy chain CDR2:RIYPGNGNTDYNEKFKD(SEQ ID NO:179) 93B heavy chain CDR3: WYFDV (SEQ ID NO: 190) It has.

[0171] The nucleic acid sequence encoding the heavy chain variable region of 93B is TIFF0007720375000094.tif46135 (SEQ ID NO: 142).

[0172] v.99B array i. Light chain The amino acid sequence of the light chain variable region of 99B is: TIFF0007720375000095.tif18135 (SEQ ID NO: 106), which sequence is 99B light chain CDR1: KSSQSLLDSDGKTYLN (SEQ ID NO: 30) 99B light chain CDR2: LVSKLDS (SEQ ID NO: 38) 99B light chain CDR3: WQGTHFPFT (SEQ ID NO: 45) It has.

[0173] The nucleic acid sequence encoding the light chain variable region of 99B is TIFF0007720375000096.tif46135 (SEQ ID NO: 130).

[0174] ii. Heavy chain The heavy chain variable region amino acid sequence of 99B is: TIFF0007720375000097.tif18135 (SEQ ID NO: 118), which sequence is 99B heavy chain CDR1: SDGIT (SEQ ID NO: 166) 99B heavy chain CDR2: QIHPRSGNTYYNEKFKG (SEQ ID NO: 180) 99B heavy chain CDR3: TGTGDY (SEQ ID NO: 187) It has.

[0175] The nucleic acid sequence encoding the heavy chain variable region of 99B is TIFF0007720375000098.tif46135 (SEQ ID NO: 143).

[0176] w.104B sequence i. Light chain The amino acid sequence of the light chain variable region of 104B is: TIFF0007720375000099.tif17135 (SEQ ID NO: 107), which sequence is 104B light chain CDR1: KSSLSLLDSDGKTYLN (SEQ ID NO: 152) 104B light chain CDR2: LVSKLDS (SEQ ID NO: 38) 104B light chain CDR3: WQGTHFPFT (SEQ ID NO: 45) It has.

[0177] The nucleic acid sequence encoding the light chain variable region of 104B is TIFF0007720375000100.tif46135 (SEQ ID NO: 131).

[0178] ii. Heavy chain The heavy chain variable region amino acid sequence of 104B is: TIFF0007720375000101.tif18135 (SEQ ID NO: 119), which sequence is 104B heavy chain CDR1: SYGIS (SEQ ID NO: 164) 104B heavy chain CDR2: QIHPRSGNTYYNENFKG (SEQ ID NO: 181) 104B heavy chain CDR3: EGGPDY (SEQ ID NO: 184) It has.

[0179] The nucleic acid sequence encoding the heavy chain variable region of 104B is TIFF0007720375000102.tif46135 (SEQ ID NO: 144).

[0180] x.105A array i. Light chain The amino acid sequence of the light chain variable region of 105A is: TIFF0007720375000103.tif17135 (SEQ ID NO: 99), which sequence is 105A Light Chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 24) 105A light chain CDR2: KVSNRFS (SEQ ID NO: 32) 105A light chain CDR3: FQGSHVPLT (SEQ ID NO: 157) It has.

[0181] The nucleic acid sequence encoding the light chain variable region of 105A is TIFF0007720375000104.tif46135 (SEQ ID NO: 132).

[0182] ii. Heavy chain The heavy chain variable region amino acid sequence of 105A is TIFF0007720375000105.tif18135 (SEQ ID NO: 111), which sequence is 105A heavy chain CDR1: DDYMH (SEQ ID NO: 162) 105A heavy chain CDR2: CIDPENGDIEYASRFQG (SEQ ID NO: 173) 105A heavy chain CDR3: YVGFGY (SEQ ID NO: 185) It has.

[0183] The nucleic acid sequence encoding the heavy chain variable region of 105A is TIFF0007720375000106.tif46135 (SEQ ID NO: 145).

[0184] 2. Anti-Siglec-15 antibodies and their antigen-binding fragments Disclosed herein are Siglec-15 binding molecules, including antibodies and antigen-binding fragments thereof, that bind to one or more Siglec-15 polypeptides or fusion proteins, or fragments or variants thereof. The antibodies disclosed herein are typically monoclonal antibodies or antigen-binding fragments thereof that bind to an epitope present on a Siglec-15 polypeptide, or a fragment or fusion thereof. In some embodiments, the antibodies bind to a conformational epitope. In some embodiments, the antibodies bind to a linear epitope. A linear epitope can be 4, 5, 6, 7, 8, 9, 10, 11, or more consecutive amino acids in length. The epitope can include one or more non-amino acid elements, post-translational modifications, or a combination thereof. Examples of post-translational modifications include, but are not limited to, glycosylation, phosphorylation, acetylation, citrullination, and ubiquitination. For example, the antibody can bind to an epitope formed at least in part by one or more sugar groups.

[0185] The antibody or antigen-binding fragment thereof can bind to an epitope present in endogenous Siglec-15 polypeptide, or recombinant Siglec-15 polypeptide, or a combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof binds to the extracellular domain of Siglec-15 or a fragment thereof, or an epitope formed therefrom. In some embodiments, the antibody or antigen-binding fragment thereof is a function-blocking antibody that reduces or prevents Siglec-15 from binding to one or more of its ligands, reduces intracellular signaling regulated by Siglec-15, or a combination thereof.

[0186] As discussed above, Siglec-15 binds to sialylated glycoproteins and preferentially recognizes the Neu5Acα2-6GalNAcα structure. The examples provided below illustrate that Siglec-15 binds to leucine-rich repeat-containing protein 4C (LRRC4C) (also known as netrin-G1 ligand and NGL-1), which may or may not be dependent on the Neu5Acα2-6GalNAcα structure. Nucleic acid and polypeptide sequences for LRRC4C are known in the art and include, for example: TIFF0007720375000107.tif83145 (SEQ ID NO: 192, UniProtKB-Q9HCJ2LRC4C_HUMAN, which is expressly incorporated herein by reference in its entirety).

[0187] Siglec-15 can bind to a counter-receptor (S15-CR) on immune cells such as T cells.

[0188] Thus, in some embodiments, a function-blocking (antagonistic) Siglec-15 binding molecule reduces, blocks, or prevents the interaction between Siglec-15 and its ligand, e.g., a glycoprotein having the Neu5Acα2-6GalNAcα structure, LRRC4C, or a Siglec-15 counterreceptor.

[0189] In some embodiments, the binding of the antibody or antigen-binding fragment thereof to Siglec-15 results in increased immune activation, decreased immune suppression, or a combination thereof. For example, in certain embodiments, the antibody or antigen-binding fragment thereof binds to the Ig-like V-type domain or the Ig-like C2-type domain of Siglec-15. In some embodiments, the epitope comprises the sialic acid binding site of Siglec-15 (e.g., the epitope comprises residue 143 of SEQ ID NO:1).

[0190] In some embodiments, the antibody binds to some or all of the same epitope as monoclonal antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A. The epitope can be a linear or conformational epitope. In some embodiments, the antibody has the same epitope specificity as monoclonal antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A. This can be achieved by generating recombinant antibodies that contain the same paratope as monoclonal antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A. In some embodiments, the Siglec-15 binding molecule comprises some or all of the light chain CDRs, the entire light chain variable region, some or all of the heavy chain CDRs, the entire heavy chain variable region, or a combination thereof, of any of the murine anti-human Siglec-15 antibodies 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A.

[0191] Siglec-15 binding molecules can comprise CDRs that are 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 at least 99% identical in amino acid sequence to the CDRs of the clones listed above, and exhibit immunospecific binding to Siglec-15.

[0192] For example, the disclosed molecules can comprise one or more light chain CDRs having the amino acid sequence of any of SEQ ID NOs: 24-45 and 146-161. The molecules can comprise at least one light chain CDR1, one light chain CDR2, and one light chain CDR3. For example, the molecules can comprise a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-31 and 146-152. The molecules can comprise a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-38 and 153-156. The molecules can comprise a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-45 and 157-161.

[0193] In certain embodiments, the molecule comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprise the following amino acid sequences: TIFF0007720375000108.tif186128

[0194] The disclosed molecules can comprise one or more heavy chain CDRs comprising the amino acid sequence of any of SEQ ID NOs: 46-73 and 162-190. The molecules can comprise at least one heavy chain CDR1, one heavy chain CDR2, and one heavy chain CDR3. The molecules can comprise a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-55 and 162-169. The molecules can comprise a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-66 and 170-181. The molecules can comprise a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-73 and 182-190.

[0195] In certain embodiments, the molecule comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 comprise the following amino acid sequences: TIFF0007720375000109.tif186128

[0196] The Siglec-15 binding molecule can comprise a variable heavy chain and / or variable light chain amino acid sequence that is 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%, at least 99%, or 100% identical to the variable heavy chain and / or light chain amino acid sequence of an antibody produced by any of the above clones, and exhibits immunospecific binding to human Siglec-15.

[0197] For example, the disclosed Siglec-15 binding molecules can comprise a light chain variable region having the amino acid sequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107, or a variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, or 107, and exhibit immunospecific binding to Siglec-15.

[0198] Additionally or alternatively, the disclosed Siglec-15 binding molecules have the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or a sequence similar to that of SEQ ID NO: The antibody can include a heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119, or a variant thereof, which exhibits immunospecific binding to Siglec-15.

[0199] A Siglec-15 binding molecule can be an immunoglobulin molecule (e.g., an antibody, diabody, fusion protein, etc.) that comprises one, two, or three light chain CDRs and one, two, or three heavy chain CDRs (e.g., in some embodiments, three light chain CDRs and three heavy chain CDRs), where the light chain CDRs include: (1) the light chain CDR1 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (2) the light chain CDR2 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (3) the light chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (4) the light chain CDR1 and light chain CDR2 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (5) the light chain CDR1 and light chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (6) the light chain CDR2 and light chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; or (7) The light chain CDR1, light chain CDR2, and light chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof.

[0200] The molecule can be an immunoglobulin molecule that includes one, two, or three light chain CDRs and one, two, or three heavy chain CDRs (e.g., in some embodiments, three light chain CDRs and three heavy chain CDRs), where the heavy chain CDRs include: (1) the heavy chain CDR1 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (2) the heavy chain CDR2 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (3) the heavy chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (4) the heavy chain CDR1 and heavy chain CDR2 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (5) the heavy chain CDR1 and heavy chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (6) the heavy chain CDR2 and heavy chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; or (7) The heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof.

[0201] The molecule can be an immunoglobulin molecule comprising one, two, or three light chain CDRs and one, two, or three heavy chain CDRs (e.g., in some embodiments, three light chain CDRs and three heavy chain CDRs), where the light chain CDRs include: (1) the light chain CDR1 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (2) the light chain CDR2 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (3) the light chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (4) the light chain CDR1 and light chain CDR2 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (5) the light chain CDR1 and light chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (6) the light chain CDR2 and light chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; or (7) the light chain CDR1, light chain CDR2, and light chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; The heavy chain CDRs include: (1) the heavy chain CDR1 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (2) the heavy chain CDR2 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (3) the heavy chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (4) the heavy chain CDR1 and heavy chain CDR2 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (5) the heavy chain CDR1 and heavy chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; (6) the heavy chain CDR2 and heavy chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof; or (7) The heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of mouse anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or a humanized variant thereof.

[0202] For example, the antibody may be mouse 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A, or chimeric antibodies thereof, or mouse anti-human Siglec-15 antibodies 1B2, 1C3, , 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, or 105A.

[0203] One embodiment provides a humanized monoclonal antibody having a variable light chain amino acid sequence 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%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 227, 228, and 229, and / or a variable heavy chain amino acid sequence 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%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 230, 231, 233, and 235.

[0204] 3. Antibody composition The disclosed Siglec-15 binding molecules can be antibodies or antigen-binding fragments thereof. The disclosed antibodies and antigen-binding fragments thereof include whole immunoglobulins of any class (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. In some embodiments, the disclosed molecules contain both an antibody light chain and at least the variable domain of an antibody heavy chain. In other embodiments, such molecules can further comprise one or more of the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain (e.g., the CH1 and hinge regions, or the CH1, hinge, CH2, and CH3 regions, among others). The antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and from any isotype, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, the constant domain is a complement-fixing constant domain when it is desired that the antibody exhibit cytotoxic activity, and the class is typically IgG1. In other embodiments, when such cytotoxic activity is not desired, the constant domain may be of the IgG2 or IgG4 class. An antibody can contain sequences from more than one class or isotype, and it is within the ordinary skill in the art to select a particular constant domain to optimize desired effector functions.

[0205] Variable domains vary in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not usually evenly distributed throughout the variable domains of antibodies. It is typically concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy- and light-chain variable domains each contain four FR regions, which largely adopt a β-sheet structure connected by three CDRs that form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in each chain are held in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.

[0206] Also disclosed are biologically active antibody fragments, which may or may not be linked to other sequences, and which contain insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acid residues, so long as the activity of the fragment is not significantly altered or weakened compared to the unmodified antibody or antibody fragment.

[0207] Several techniques can be adapted to generate single-chain antibodies specific to the antigenic proteins of the present disclosure. Methods for generating single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be made by fusing heavy and light chain variable domains together using a short peptide linker, thereby reconstituting an antigen-binding site on a single molecule. Single-chain antibody variable fragments (scFvs) have been developed in which the C-terminus of one variable domain is connected to the N-terminus of the other variable domain via a 15-25 amino acid peptide or linker without significantly perturbing antigen binding or binding specificity. The linker is selected to allow the heavy and light chains to bind together in their proper conformational orientation.

[0208] Bivalent single-chain variable fragments (di-scFvs) can be created by linking two scFvs. This can be done by generating a single peptide chain with two VH and two VL regions, resulting in tandem scFvs. scFvs can also be engineered to force dimerization using a linker peptide (approximately five amino acids) that is too short to allow the two variable regions to fold together. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40-fold lower than the corresponding scFvs, meaning that diabodies have much higher affinity for their targets. Even shorter linkers (one or two amino acids) result in the formation of trimers (triabodies, or triabodies). Tetrabodies have also been generated, which exhibit even higher affinity for their targets than diabodies.

[0209] Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., each individual antibody within the population is identical except for possible naturally occurring mutations that may exist in a small subset of antibody molecules. Monoclonal antibodies include "chimeric" antibodies (in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass), as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity.

[0210] a. Chimeric and humanized antibodies Chimeric antibodies and antigen-binding fragments thereof comprising one or more of the disclosed sequences and functional variants thereof are also provided.

[0211] Methods for producing chimeric antibodies are known in the art (see, e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397). Chimeric antibodies comprising one or more CDRs from a non-human species and a framework region from a human immunoglobulin molecule can be produced by, for example, CDR-grafting (EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7:805; and Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969), and chain shuffling (US Pat. No. 5,565,332).

[0212] The disclosed molecule can be a human or humanized antibody, or an antigen-binding fragment thereof. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and therefore can induce undesired immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the present method helps to reduce the probability that antibodies administered to humans will induce undesired immune responses.

[0213] Transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production can be used. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J(H)) gene in chimeric and germ-line mutant mice completely prevents endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge.

[0214] Optionally, antibodies are generated in other species and "humanized" for administration in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from the complementarity-determining regions (CDRs) of the recipient antibody are replaced by residues from a CDR of a non-human species, such as mouse, rat, or rabbit (donor antibody), having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are human immunoglobulin consensus sequences. Optimally, the humanized antibody will also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0215] Methods for humanizing non-human antibodies are well known in the art and are described, for example, in European Patent Nos. EP 239,400, EP 592,106, and EP 519,596; International Publication Nos. WO 91 / 09967 and WO 93 / 17105; U.S. Patent Nos. 5,225,539, 5,530,101, 5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska et al., 1994, PNAS 91:969-973; Tan et al. al.,2002,J.Immunol.169:1119-1125;Caldas et al.,2000,Protein Eng.13:353-360;Morea et al.,2000,Methods 20:267-79;Baca et al. al.,1997,J.Biol.Chem.272:10678-10684;Roguska et al.,1996,Protein Eng.9:895-904;Couto et al.,1995,Cancer Res.55(23 Supp):5973s-5977s;Couto et al.,1995,Cancer Res.55:1717-22;Sandhu,1994,Gene 150:409-10;Pedersen et al. al., 1994, J. Mol. Biol. 235:959-973; Jones et al., 1986, Nature 321:522-525; Reichmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596).

[0216] Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Antibody humanization techniques generally involve manipulating the DNA sequence encoding one or more polypeptide chains of an antibody molecule using recombinant DNA technology. Humanization can essentially be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, humanized forms of non-human antibodies (or fragments thereof) are chimeric antibodies or fragments in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0217] The choice of human variable domains (both light and heavy chains) to be used in making a humanized antibody can be crucial to reducing antigenicity. According to the "best fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent is accepted as the human framework (FR) for the humanized antibody. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies.

[0218] It is further important that antibodies be humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that can display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0219] A human, humanized, or chimeric antibody derivative can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are human immunoglobulin consensus sequences. Such antibodies also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The constant domain of such antibodies can be selected with regard to the antibody function to be presented, particularly the effector functions that may be required. In some embodiments, the constant domain of such antibodies can be or comprise a human IgA, IgD, IgE, IgG, or IgM domain. In certain embodiments, when a humanized antibody derivative is intended for therapeutic use and antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity are required, human IgG constant domains, particularly those of the IgG1 and IgG3 isotypes, are used. In alternative embodiments, IgG2 and IgG4 isotypes are used when the antibody is intended for therapeutic purposes and antibody effector function is not required. Fc constant domains containing one or more amino acid modifications that alter antibody effector function, such as those disclosed in U.S. Patent Application Publication Nos. 2005 / 0037000 and 2005 / 0064514.

[0220] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences; for example, the donor CDR or consensus framework can be altered by substitution, insertion, or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or donor antibody. In some embodiments, such alterations are not extensive. Typically, at least 75%, more often 90%, or even more than 95% of the humanized antibody residues correspond to those of the parental framework region (FR) and CDR sequences.Humanized antibodies can be produced using various techniques known in the art, such as CDR-grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al. al., 1994, Proc. Natl. Acad. Sci. 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and, for example, U.S. Pat. al.,2002,J.Immunol.169:1119-25, Caldas et al.,2000,Protein Eng. 13:353-60,Morea et al.,2000,Methods 20:267-79,Baca et al.,1997,J.Biol.Chem.272:10678-84,Roguska et al. al.,1996,Protein Eng. 9:895-904; Couto et al., 1995, Cancer Res. 55(23 Supp):5973s-5977s; Couto et al., 1995, Cancer Res. 55:1717-22; Sandhu, 1994, Gene 150:409-10; Pedersen et al., 1994, J. Mol. Biol. 235:959-73; Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596.

[0221] In many cases, framework residues in the framework regions are substituted with corresponding residues from the CDR donor antibody to alter, e.g., improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as by modeling the interactions between CDRs and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unique framework residues at specific positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; U.S. Patent Application Publication Nos. 2004 / 0049014 and 2003 / 0229208; U.S. Patent Nos. 6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101; and Riechmann et al., 1988, Nature 332:323.)

[0222] Human, chimeric, or humanized derivatives of the disclosed mouse anti-human Siglec-15 antibodies can be used in in vivo methods in humans. Mouse antibodies or antibodies from other species can be advantageously used in many applications (e.g., in vitro or in situ detection assays, acute in vivo applications, etc.). Such human or humanized antibodies can contain substitutions, deletions, or additions of amino acid residues in one or more non-human CDRs. Humanized antibody derivatives can have substantially the same binding, stronger binding, or weaker binding compared to non-derivatized humanized antibodies. In certain embodiments, one, two, three, four, or five amino acid residues in the CDRs are substituted, deleted, or added (i.e., mutated). Fully human antibodies are particularly desirable for therapeutic treatment of human subjects.

[0223] Such human antibodies can be made by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences (see U.S. Pat. Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741). Such human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.

[0224] For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately from or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, J HHomozygous deletion of the region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to generate homozygous offspring that express human antibodies. The transgenic mice are immunized using conventional techniques with a selected antigen, e.g., all or a portion of a polypeptide. Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology (see, e.g., U.S. Pat. No. 5,916,771). The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B-cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such techniques, it is possible to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for generating human antibodies, see Lonberg and See Huszar (1995, Int. Rev. Immunol. 13:65-93, which is incorporated herein by reference in its entirety. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Pat. Nos. 5,413,923 and 5,625,126. , 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated herein by reference in their entireties. Additionally, in partnership with companies such as Abgenix, Inc. (Freemont, CA) and Medarex (Princeton, NJ), human antibodies directed against a selected antigen can be obtained using technology similar to that described above.

[0225] DNA sequences encoding human acceptor framework sequences include, but are not limited to, FR segments from human germline VH segments VH1-18 and JH6 and human germline VL segments VK-A26 and JK4. In certain embodiments, one or more of the CDRs are inserted within the framework regions using conventional recombinant DNA techniques. The framework regions may be naturally occurring or consensus framework regions, and human framework regions (see, e.g., Chothia et al., 1998, "Structural Determinants in the Sequences of Immunoglobulin Variable Domain," J. Mol. Biol. 278:457-479, for a list of human framework regions).

[0226] i. Humanized 5G12 One embodiment provides a humanized 5G12 antibody or antigen-binding fragment thereof.

[0227] b. Single chain antibody The Siglec-15 binding molecule can be a single-chain antibody. Methods for producing single-chain antibodies are well known in the art. Single-chain antibodies are made by fusing heavy and light chain variable domains together using a short peptide linker, thereby reconstituting an antigen-binding site on a single molecule. Single-chain antibody variable fragments (scFvs), in which the C-terminus of one variable domain is connected to the N-terminus of the other variable domain via a 15-25 amino acid peptide or linker, have been developed without significantly perturbing antigen binding or binding specificity. The linker is selected to allow the heavy and light chains to bind together in their proper conformational orientation. These Fvs lack the constant regions (Fc) present in the heavy and light chains of natural antibodies.

[0228] C. monovalent antibody In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using conventional techniques known in the art. For example, digestion can be performed using papain. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. Treatment with pepsin produces a fragment called an F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0229] The Fab fragment produced in antibody digestion also contains the constant domain of the light chain and the first constant domain of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain, including one or more cysteines from the antibody hinge region. The F(ab')2 fragment is a bivalent fragment containing two Fab' fragments linked by a disulfide bridge at the hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine residue(s) in the constant domains bear a free thiol group. Antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

[0230] d. Antibody fragment conjugates or fusions The targeting function of an antibody can be used therapeutically by coupling the antibody or fragment thereof with a therapeutic agent. Such coupling of the antibody or fragment (e.g., at least a portion of an immunoglobulin constant region (Fc)) to a therapeutic agent can be achieved by creating an immunoconjugate or by creating a fusion protein comprising the antibody or antibody fragment and the therapeutic agent.

[0231] Such coupling of the antibody or fragment to a therapeutic agent can be achieved by creating an immunoconjugate, or by creating a fusion protein, comprising the antibody or antibody fragment and the therapeutic agent, or by linking the antibody or fragment to a nucleic acid such as an siRNA.

[0232] In some embodiments, the antibody is modified to alter its half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that it remains in the circulation or at the site of treatment for a longer period of time. For example, it may be desirable to maintain the antibody's titer in the circulation or at the site of treatment for an extended period of time. The antibody can be engineered with Fc variants that extend its half-life, for example, using Xtend™ Antibody Half-Life Extension Technology (Xencor, Monrovia, CA). In other embodiments, the half-life of an anti-DNA antibody is shortened to reduce potential side effects. The disclosed conjugates can be used to modify a given biological response. The drug moiety should not be construed as limited to classical chemical therapeutics. For example, the drug moiety can be a protein or polypeptide possessing a desired biological activity. Such proteins can include toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin.

[0233] e. Mono- and multispecific antibodies In some embodiments, the disclosed antibodies are monospecific and bind only to Siglec-15. Bispecific, trispecific, or even more multispecific derivative antibodies are also provided that exhibit specificity for different immune system targets in addition to their specificity for human Siglec-15. For example, such antibodies can bind both human Siglec-15 and an antigen important for targeting the antibody to a particular cell type or tissue (e.g., an antigen associated with the cancer antigen of the tumor being treated). In another aspect, to enhance immunomodulatory effects and combine multiple mechanisms of action, such as ligand blocking, immune cell activation, and direct tumor targeting, into a single molecule, such multispecific antibodies bind to molecules (receptors or ligands) involved in alternative immunomodulatory pathways, e.g., B7-H1, PD-1, CTLA4, TIM3, TIM4, OX40, CD40, GITR, 4-1-BB, LIGHT, or LAG3.

[0234] f. Derivatives The production and use of "derivatives" of any of the disclosed Siglec-15 binding molecules are also disclosed. Derivative molecules, e.g., antibodies or antibody fragments, can be altered by chemical modification using techniques known to those of skill in the art. Such chemical modifications include, but are not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. The term derivative encompasses non-amino acid modifications, such as amino acids that can be glycosylated (e.g., with altered content of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylated, pegylated, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytic cleavage, linked to cellular ligands or other proteins, and the like. In some embodiments, altered carbohydrate modifications modulate one or more of antibody solubilization, enhanced intracellular trafficking and secretion of the antibody, facilitated antibody assembly, conformational integrity, and antibody-mediated effector function.

[0235] In certain embodiments, the altered carbohydrate modification enhances antibody-mediated effector function relative to an antibody lacking the carbohydrate modification. Carbohydrate modifications that result in altered antibody-mediated effector function are well known in the art (see, e.g., Shields, R. Let al. (2002) "Lack of Fucose on Human IgG N-Linked Oligosaccharide Improves Binding to Human Fc gamma RIII and Antibody-Dependent Cellular Toxicity," J. Biol. Chem. 277(30):26733-26740; Davies J. et al. (2001) "Expression of GnTIII in a Recombinant Anti-CD20 CHO Production Cell Line: Expression of Antibodies with Altered Glycoforms Leads to an Increase in ADCC Through Higher Affinity for Fc gamma RIII," Biotechnology & Bioengineering 74(4):288-294).Methods for altering carbohydrate content are known to those skilled in the art, and include, for example, Wallick, SC et al. (1988) "Glycosylation of a VH residue of a monoclonal antibody against alpha(1-6) dextran increases its affinity for antigen," J. Exp. Med. 168(3):1099-1109; Tao, MH et al. (1989) "Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region," J. Immunol. 143(8):2595-2601; Routledge, E. G. et al. (1995) "The effect of aglycosylation on the immunogenicity of a humanized therapeutic CD3 monoclonal antibody," Transplantation 60(8):847-53; Elliott, S. et al. al.(2003)“Enhancement Of Therapeutic Protein In Vivo Activities Through Glycoengineering,”Nature Biotechnol.21:414-21;Shields,RLet al.(2002)“Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity.,”J.Biol.Chem.277(30):26733-26740).

[0236] The disclosed antibodies can be modified by recombinant means to increase the effectiveness of the antibody in mediating a desired function. Thus, antibodies can be modified by substitution using recombinant means. Typically, the substitutions are conservative. For example, at least one amino acid in the antibody constant region can be replaced with a different residue. See, e.g., U.S. Pat. Nos. 5,624,821, 6,194,551, International Publication No. WO 9958572; and Angal, et al., Mol. Immunol. 30:105-08 (1993). Amino acid modifications include amino acid deletions, additions, and substitutions. In some cases, such changes are made to reduce undesirable activities, such as complement-dependent cytotoxicity. Antibodies are often labeled by covalently or noncovalently attaching a substance that provides a detectable signal. A wide variety of labeling and conjugation techniques are known and are widely reported in both the scientific and patent literature. These antibodies can be screened against Siglec-15 polypeptides, or fragments or fusions thereof. See, for example, Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[0237] In some embodiments, an antibody derivative will possess similar or identical functions as the parent antibody. In other embodiments, an antibody derivative will exhibit altered activities compared to the parent antibody. For example, a derivative antibody (or fragment thereof) may bind to its epitope more tightly than the parent antibody or may be more resistant to proteolysis. The substitutions, additions, or deletions in the derivatized antibody may be in the Fc region of the antibody and thereby function to alter the binding affinity of the antibody to one or more FcγRs. Methods for modifying antibodies by altering their binding to one or more FcγRs are known in the art, see, e.g., PCT Publication Nos. WO04 / 029207, WO04 / 029092, WO04 / 028564, WO99 / 58572, WO99 / 51642, WO98 / 23289, WO89 / 07142, WO88 / 07089, and U.S. Pat. Nos. 5,843,597 and 5,642,821.

[0238] In some embodiments, the antibody has its Fc region deleted (e.g., Fab or F(ab)2) or the molecule has been modified to exhibit reduced or no Fc receptor (FcR) binding activity, or enhanced antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity. In some embodiments, the antibody has altered affinity for an activating FcγR, such as FcγRIIIA. Such modifications may also have altered Fc-mediated effector function. Modifications that affect Fc-mediated effector function are well known in the art (see U.S. Pat. No. 6,194,551 and WO 00 / 42072). In a specific embodiment, modifications to the Fc region result in the antibody having altered antibody-mediated effector function, altered binding to other Fc receptors (e.g., Fc activating receptors), altered antibody-dependent cellular cytotoxicity (ADCC) activity, altered C1q binding activity, altered complement-dependent cytotoxicity (CDC), altered phagocytic activity, or a combination thereof.

[0239] Derivatized antibodies can be used to alter the half-life (e.g., serum half-life) of the parent antibody in a mammal, such as a human. For example, such alterations can result in a half-life of greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. Increasing the half-life of a humanized antibody or fragment thereof in a mammal, such as a human, increases the serum titer of the antibody or antibody fragment in the mammal, thereby reducing the number of administrations and / or the concentration of the antibody or antibody fragment to be administered. Antibodies or fragments thereof with increased in vivo half-lives can be generated by techniques known to those skilled in the art. For example, antibodies or fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting, or adding) amino acid residues identified to be involved in the interaction between the Fc domain and the FcRn receptor. Humanized antibodies can be engineered to increase biological half-life (see, e.g., U.S. Patent No. 6,277,375). For example, humanized antibodies can be engineered in the Fc-hinge domain to increase in vivo or serum half-life.

[0240] Antibodies or fragments thereof with increased in vivo half-lives can be generated by attaching polymer molecules such as high molecular weight polyethylene glycol (PEG) to the antibody or antibody fragment. PEG can be attached to the antibody or antibody fragment via site-specific conjugation of PEG to the N- or C-terminus of the antibody or antibody fragment, or via epsilon-amino groups present on lysine residues, with or without a multifunctional linker. Linear or branched polymer derivatization that results in minimal loss of biological activity is expected to be used. The degree of conjugation is closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate, for example, by size exclusion or ion exchange chromatography.

[0241] Antibodies can also be modified by the methods and coupling agents described by Davis et al. (See U.S. Pat. No. 4,179,337) to obtain compositions that can be injected into the circulatory system of a mammal without substantial immunogenic response.

[0242] Framework residues of humanized antibodies can be modified. Residues in framework regions can be substituted with corresponding residues from the CDR donor antibody to alter, e.g., improve, antigen binding. These framework substitutions can be identified by methods well known in the art, such as by modeling the interactions between CDRs and framework residues to identify framework residues important for antigen binding and by sequence comparison to identify unique framework residues at specific positions. (See, e.g., U.S. Pat. No. 5,585,089; and Riechmann, L. et al. (1988) "Reshaping Human Antibodies For Therapy," Nature 332:323-327.) The disclosed Siglec-15 binding molecules can be recombinantly fused or chemically conjugated (including both covalent and noncovalent conjugation) to heterologous molecules (i.e., unrelated molecules). The fusion need not necessarily be direct, but can occur via a linker sequence.

[0243] In some embodiments, such a heterologous molecule is a polypeptide having at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids. Alternatively, such heterologous molecules may be enzymes, hormones, cell surface receptors, drug moieties, for example: macrophage-specific targeting reagents (e.g., intracellular carboxylesterase, hCE1 (Needham, L.A. et al. (2011) "Drug Targeting To Monocytes And Macrophages Using Esterase-Sensitive Chemical Motif," J. Pharmacol. Exp. Ther. DOI: 10.1124 / jpet.111.183640), chitin and chitosan (Muzzarelli, R.A. (2010) "Chitins And Chitosans As Immunoadjuvants And Non-Allergenic Drug Carriers," Mar Drugs 8(2):292-312), galactosylated low-density lipoprotein (Wu, F. et al. (2009) "Galactosylated LDL Nanoparticles: A Novel Targeted Delivery System To Deliver Antigen To Macrophages And Enhance Antigen Specific T Cell Responses,” Molec. Pharm. 6(5):1506-1517), N-formyl-Met-Leu-Phe (fMLF), a macrophage-specific chemoattractant (Wan, L. et al. (2008) “Optimizing Size and Copy Number For PEG-Fmlf (N-Formyl-Methionyl-Leucyl-Phenylalanine) Nanocarrier Uptake By Macrophages,” Bioconjug. Chem. 19(1):28-38), maleylated or mannosylated proteins, such as maleylated albumin (Anatelli, F. et al.al.(2006)“Macrophage-Targeted Photosensitizer Conjugate Delivered By Intratumoral Injection,”Mol Pharm.3(6):654-664;Bansal,P.et al.(1999)“MHC Class I-Restricted Presentation Of Maleylated Protein Binding To Scavenger Receptors,”J.Immunol.162(8):4430-4437);Mukhopadhyay,A.et al.(2003)“Intracellular Delivery Of Drugs To Macrophages,” Adv. Biochem. Eng. Biotechnol. 84:183-209), toxins (e.g., abrin, ricin A, Pseudomonas exotoxin (i.e., PE-40), diphtheria toxin, ricin, gelonin, or pokeweed antiviral protein), proteins (e.g., tumor necrosis factor, interferons (e.g., α-interferon, β-interferon), nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, or apoptotic agents (e.g., tumor necrosis factor-α, tumor necrosis factor-β)), biological response modifiers (e.g., lymphokines (e.g., interleukin-1 (“I”)), L-1), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), etc.), granulocyte macrophage colony-stimulating factor ("GM-CSF"), granulocyte colony-stimulating factor ("G-CSF"), or macrophage colony-stimulating factor ("M-CSF"), or growth factors (e.g., growth hormone ("GH")), cytotoxins (e.g., cell growth inhibitors or cytocidal drugs, e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione), mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and their analogs or congeners), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa The therapeutic agent may be chlorambucil, melphalan, BiCNU® (carmustine; BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin), and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), or mitotic inhibitors (e.g., vincristine and vinblastine).

[0244] In another embodiment, the molecule is conjugated to a second antibody to form an antibody heteroconjugate, as described in U.S. Patent No. 4,676,980 to Segal. Such heteroconjugate antibodies can additionally bind to a hapten (e.g., fluorescein, etc.), or a cell marker (e.g., 4-1-BB, B7-H1, PD-1, CD4, CD8, CD14, CD25, CD27, CD40, CD68, CD163, CTLA4, GITR, LAG-3, OX40, TIM3, TIM4, TLR2, LIGHT, etc.), or a cytokine (e.g., IL-4, IL-7, IL-10, IL-12, IL-15, IL-17, TGF-beta, IFNg, Flt3, BLy), or a chemokine (e.g., CCL21).

[0245] The Fc portion of the fusion protein may be of a different isotype or subclass, chimeric or hybrid, and / or modified to, for example, improve effector function, control half-life, tissue accessibility, enhance biophysical properties such as stability, and improve production efficiency (and reduce costs). Many modifications useful in constructing the disclosed fusion proteins and methods for making them are known in the art; see, for example, Mueller, JP et al. (1997) "Humanized Porcine VCAM-Specific Monoclonal Antibodies With Chimeric IgG2 / G4 Constant Regions Block Human Leukocyte Binding to Porcine Endothelial Cells," Mol. Immun. 34(6):441-452; Swann, PG (2008) "Considerations For The Development Of Therapeutic Monoclonal Antibodies," Curr. Opin. Immun. 20:493-499 (2008); and Presta, LG (2008) "Molecular Engineering And Design Of Therapeutic Antibodies," Curr. Opin. Immun. 20:460-470. In some embodiments, the Fc region is a native IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a hybrid, e.g., a chimera composed of the Fc constant regions of IgG2 / IgG4. Modifications to the Fc region include, but are not limited to, an IgG4 modified to prevent binding to Fc gamma receptors and complement, an IgG1 modified to improve binding to one or more Fc gamma receptors, an IgG1 modified (amino acid changes) to minimize effector function, an IgG1 with altered / absent glycans (typically by changing the expression host), and an IgG1 with altered pH-dependent binding to FcRn. The Fc region may include the entire hinge region or a region less than the entire hinge region.Treatment outcomes in patients treated with rituximab (a chimeric mouse / human IgG1 monoclonal antibody against CD20) for non-Hodgkin's lymphoma or Waldenstrom's macroglobulinemia have been correlated with individual expression of allelic variants of Fcγ receptors that differ in their inherent affinity for the Fc domain of human IgG1. In particular, patients with high-affinity alleles of the low-affinity activating Fc receptor CD16A (FcγRIIIA) have shown higher response rates and, in the case of non-Hodgkin's lymphoma, improved progression-free survival. Thus, the Fc domain of the disclosed antibodies and fragments can contain one or more amino acid insertions, deletions, or substitutions that reduce binding to the low-affinity inhibitory Fc receptor CD32B (FcγRIIB) and retain or enhance wild-type levels of binding to the low-affinity activating Fc receptor CD16A (FcγRIIIA).

[0246] Another embodiment includes IgG2-4 hybrids and IgG4 mutants with increased half-life due to reduced binding to FcγR. Representative IgG2-4 hybrids and IgG4 mutants are described in Angal, S. et al. (1993) "A Single Amino Acid Substitution Abolishes the Heterogeneity of Chimeric Mouse / Human (Igg4) Antibody," Molec. Immunol. 30(1):105-108; Mueller, J.P. et al. (1997) "Humanized Porcine VCAM-Specific Monoclonal Antibodies With Chimeric IgG2 / G4 Constant Regions Block Human Leukocyte Binding to Porcine Endothelial Cells," Mol. Immun. 34(6):441-452; and U.S. Patent No. 6,982,323. In some embodiments, the IgG1 and / or IgG2 domains are modified; for example, Angal, S. et al. (1993) describe IgG1 and IgG2 mutants in which serine 241 is replaced with proline.

[0247] In some embodiments, the Fc domain of such molecules contains an amino acid insertion, deletion, or substitution that enhances binding to CD16A. Numerous substitutions in the Fc domain of human IgG1 that increase binding to CD16A and decrease binding to CD32B are known in the art and are described in Stavenhagen, JB et al. (2007) "Fc Optimization of Therapeutic Antibodies Enhances Their Ability to Kill Tumor Cells In Vitro and Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors," Cancer Res. 57(18):8882-8890. Exemplary mutants of the Fc domain of human IgG1 that decrease binding to CD32B and / or increase binding to CD16A contain the substitutions F243L, R929P, Y300L, V305I, or P296L. These amino acid substitutions may be present in any combination in the Fc domain of human IgG1. In one embodiment, the human IgG1 Fc domain mutant contains F243L, R929P, and Y300L substitutions. In another embodiment, the human IgG1 Fc domain mutant contains F243L, R929P, Y300L, V305I, and P296L substitutions. In another embodiment, the human IgG1 Fc domain mutant contains N297Q substitution, as this mutation abolishes FcR binding.

[0248] Techniques for conjugating therapeutic moieties to antibodies are well known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.; Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), 1987, pp. 623-53, Marcel Dekker, Inc.; Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), 1985, pp. 475-506; "Analysis, Results, And Future Prospects Of The Therapeutic See "Use Of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), 1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev. 62:119-158.

[0249] Any of the disclosed molecules can be fused to a marker sequence, such as a peptide, to facilitate purification. In some embodiments, the marker amino acid sequence is a hexa-histidine peptide, a hemagglutinin "HA" tag (corresponding to an epitope derived from the influenza hemagglutinin protein) (Wilson, I. A. et al. (1984) "The Structure of an Antigenic Determinant in a Protein," Cell, 37:767-778), or a "flag" tag (Knappik, A. et al. (1994) "An Improved Affinity Tag Based on the FLAG Peptide for the Detection and Purification of Recombinant Antibody Fragments," Biotechniques 17(4):754-761).

[0250] The disclosed Siglec-15 binding molecules can be conjugated to a diagnostic or therapeutic agent, or to another molecule for which it is desired to increase serum half-life. The antibodies can be used diagnostically (in vivo, in situ, or in vitro) to monitor the development or progression of a disease, disorder, or infection, for example, as part of a clinical trial procedure, to determine the effectiveness of a given treatment regimen, or to select patients likely to respond to a particular therapy (such as those expressing high levels of Siglec-15).

[0251] Detection can be facilitated by coupling a molecule such as an antibody or its antigen-binding fragment to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. Detectable substances can be linked or conjugated to antibodies directly or indirectly via intermediates (e.g., linkers known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents. Such diagnosis and detection can be accomplished by coupling the antibody to a detectable substance, including various enzymes, including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic group complexes, such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as, but not limited to, bismuth ( 213 Bi), carbon ( 14 C), chromium ( 51 Cr), Cobalt ( 57 Co), fluorine ( 18 F), gadolinium ( 153 Gd, 159 Gd), gallium ( 68 Ga, 67 Ga), germanium ( 68 Ge), holmium ( 166 Ho), Indium ( 115 In, 113 In, 112 In, 111 In), iodine ( 131I, 125 I, 123 I, 121 I), Lanthanum ( 140 La), lutetium ( 177 Lu), manganese ( 54 Mn), molybdenum ( 99 Mo), palladium ( 103 Pd), phosphorus ( 32 P), praseodymium ( 142 Pr), promethium ( 149 Pm), rhenium ( 186 Re, 188 Re), rhodium ( 105 Rh), ruthenium ( 97 Ru), samarium ( 153 Sm), Scandium ( 47 Sc), Selenium ( 75 Se), strontium ( 85 Sr), sulfur ( 35 S), technetium ( 99 Tc), thallium ( 201 Ti), tin ( 113 Sn, 117 Sn), tritium ( 3 H), xenon ( 133 Xe), Ytterbium ( 169 Yb, 175 Yb), yttrium ( 90 Y), zinc ( 65Zn); positron-emitting metals using various positron emission tomography methods, as well as non-radioactive paramagnetic metal ions. The disclosed molecules can be bound to solid supports, which are particularly useful for immunoassays or purification of target antigens or other molecules capable of binding to target antigens immobilized on the support via binding to antibodies or antigen-binding fragments. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. Nucleic acid molecules (DNA or RNA) encoding any such antibodies, fusion proteins, or fragments, as well as vector molecules (such as plasmids) capable of transmitting or replicating such nucleic acid molecules, are also disclosed. The nucleic acids can be single-stranded, double-stranded, or contain both single- and double-stranded portions.

[0252] 3. Preparation method Siglec-15 binding molecules can be produced by any method known in the art useful for producing polypeptides, such as in vitro synthesis, recombinant DNA production, etc. Humanized antibodies are typically produced by recombinant DNA technology. Antibodies can be produced using recombinant immunoglobulin expression techniques. Recombinant production of immunoglobulin molecules, including humanized antibodies, is described in U.S. Pat. No. 4,816,397 (Boss et al.), U.S. Pat. Nos. 6,331,415 and 4,816,567 (both Cabilly et al.), British Patent No. GB 2,188,638 (Winter et al.), and British Patent No. GB 2,209,757. Techniques for the recombinant expression of immunoglobulins, including humanized immunoglobulins, can also be found in Goeddel et al., Gene Expression Technology Methods in Enzymology Vol. 185, Academic Press (1991), and Borreback, Antibody Engineering, W.H. Freeman (1992). Additional information regarding the generation, design, and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies, Academic Press, San Diego (1993).

[0253] A typical method for producing a recombinant chimeric antibody can include: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody heavy chain in which the CDRs and variable region of an anti-Siglec-15 antibody are fused to an Fc region derived from human immunoglobulin, thereby generating a vector for expressing the chimeric antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses the antibody light chain of a mouse anti-human Siglec-15 monoclonal antibody, thereby generating a vector for expressing the chimeric antibody light chain; c) transferring the expression vector into a host cell by conventional molecular biology methods to generate a transfected host cell for expressing the chimeric antibody; and d) culturing the transfected cell to produce the chimeric antibody by conventional cell culture techniques.

[0254] A typical process for producing a recombinant humanized antibody is to: a) construct, by conventional molecular biology methods, an expression vector encoding and expressing an anti-human Siglec-15 heavy chain in which the CDRs and the minimum portion of the variable region framework required to retain the binding specificity of the donor antibody are derived from a humanized variant of anti-human Siglec-15 antibody(ies), with the remainder of the antibody being derived from a human immunoglobulin, thereby producing a vector for expressing the humanized antibody heavy chain; b) construct, by conventional molecular biology methods, an expression vector encoding and expressing an anti-human Siglec-15 heavy chain in which the CDRs and the minimum portion of the variable region framework required to retain the binding specificity of the donor antibody are derived from a humanized variant of anti-human Siglec-15 antibody(ies), with the remainder of the antibody being derived from a human immunoglobulin; A minimum portion of the work can include constructing an expression vector that encodes and expresses an antibody light chain derived from a non-human immunoglobulin, such as the disclosed mouse anti-human Siglec-15 antibody, with the remainder of the antibody derived from a human immunoglobulin, thereby producing a vector for expressing the humanized antibody light chain; c) transferring the expression vector into a host cell by conventional molecular biology methods to produce a transfected host cell for expressing the humanized antibody; and d) culturing the transfected cell by conventional cell culture techniques to produce the humanized antibody.

[0255] For either representative method, host cells can be co-transfected with expression vectors that may contain different selectable markers but that may be identical except for the heavy and light chain coding sequences. This procedure results in co-expression of heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used. The heavy and light chain coding sequences can comprise cDNA, genomic DNA, or both. Host cells used to express recombinant antibodies can be either bacterial cells, such as Escherichia coli, or eukaryotic cells (e.g., Chinese hamster ovary (CHO) cells or HEK-293 cells). The choice of expression vector depends on the choice of host cell and can be selected to have the desired expression and regulatory characteristics in the selected host cell. Other cell lines that can be used include, but are not limited to, CHO-K1, NSO, and PER.C6 (Crucell, Leiden, Netherlands).

[0256] Any of the disclosed antibodies can be used to generate anti-idiotypic antibodies using techniques well known to those skilled in the art (see, e.g., Greenspan, N.S. et al. (1989) "Idiotypes: Structure And Immunogenicity," FASEB J. 7:437-444; and Nisinoff, A. (1991) "Idiotypes: Concepts And Applications," J. Immunol. 147(8):2429-2438).

[0257] C. Siglec-15 Ligand Binding Molecule Molecules that bind to a ligand of Siglec-15, such as Siglec-15 proteins, Siglec-15 fusion proteins, and fragments and variants thereof, are also provided. Siglec-15 ligand-binding molecules can bind to a ligand of Siglec-15, such as sialylated glycoproteins, LRRC4C, Siglec-15 counterreceptors, etc. In some embodiments, the Siglec-15 ligand-binding molecules can induce signaling through a ligand of Siglec-15. In some embodiments, the Siglec-15 ligand-binding molecules block or otherwise reduce the interaction between Siglec-15 and its ligand without inducing signaling through Siglec-15 or its ligand. As discussed in more detail below and exemplified in the Examples, Siglec-15 ligand-binding molecules can be used to modulate the activity of Siglec-15 and can also be used therapeutically to treat subjects in need thereof.

[0258] 1. Siglec-15 Polypeptide In some embodiments, the Siglec-15 ligand binding molecule is Siglec-15, or a fragment or variant thereof. For example, in some embodiments, the Siglec-15 ligand binding molecule includes a polypeptide at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 1 or 2, or a fragment thereof, e.g., an extracellular domain or a subdomain thereof, e.g., an IgV domain, an IgC domain, or a combination thereof. In some embodiments, the Siglec-15 polypeptide is soluble or otherwise cell-free. For example, in some embodiments, the Siglec-15 lacks one or more of the transmembrane domain, the cytoplasmic domain, or the leader sequence.

[0259] 2. Siglec-15 fusion protein In some embodiments, the Siglec-15 ligand-binding molecule is a Siglec-15 fusion protein. Fusion proteins are provided that contain a Siglec-15 polypeptide coupled to another polypeptide to form a fusion protein. The Siglec-15 fusion polypeptide can include a first fusion partner, comprising all or part of a Siglec-15 protein, either (i) directly fused to a second polypeptide or (ii) fused to a linker peptide sequence, optionally fused to the second polypeptide. The fusion protein optionally contains a domain that functions to dimerize or multimerize two or more fusion proteins. In some embodiments, the fusion protein is neither dimerized nor multimerized. The peptide / polypeptide linker domain can be a separate domain or can be contained within one of the other domains (Siglec-15 polypeptide or second polypeptide) of the fusion protein. Similarly, the domain that functions to dimerize or multimerize the fusion protein may be a separate domain or may be contained within one of the other domains of the fusion protein (the Siglec-15 polypeptide, the second polypeptide, or the peptide / polypeptide linker domain). In some embodiments, the dimerization / multimerization domain and the peptide / polypeptide linker domain are the same.

[0260] The fusion proteins disclosed herein have the formula I: N-R1-R2-R3-C where "N" represents the N-terminus of the fusion protein, "C" represents the C-terminus of the fusion protein, "R1" is the Siglec-15 polypeptide, "R2" is an optional peptide / polypeptide linker domain, and "R3" is the second polypeptide. Alternatively, R3 may be the Siglec-15 polypeptide and R1 may be the second polypeptide.

[0261] Fusion proteins can be dimerized or multimerized. Dimerization or multimerization can occur through the dimerization or multimerization of domains between two or more fusion proteins. Alternatively, the dimerization or multimerization of fusion proteins can occur through chemical crosslinking. The dimer or multimer formed can be a homodimer / homomultimer or a heterodimer / heteromultimer. As discussed above, in some embodiments, the fusion protein is neither dimerized nor multimerized, nor is it one of them.

[0262] In some embodiments, the second polypeptide comprises one or more domains of an immunoglobulin heavy chain constant region, such as the hinge, Cγ1, or Cγ2 domain of the human immunoglobulin Cγ1 chain. H 2 and / or C H 3 region, mouse immunoglobulin Cγ2a chain hinge, C H 2 and / or C H 3 region, C of human immunoglobulin Cγ1 H 2 and / or C H It contains amino acid sequences corresponding to three regions, etc.

[0263] The Fc portion of the fusion protein may be of a different isotype or subclass, chimeric or hybrid, and / or modified to, for example, improve effector function, control half-life, tissue delivery, enhance biophysical properties such as stability, and improve production efficiency (and reduce costs). Many modifications useful in constructing the disclosed fusion proteins and methods for making them are known in the art; see, e.g., Mueller, et al. Mol. Immun., 34(6):441-452 (1997); Swann, et al., Cur. Opin. Immun., 20:493-499 (2008); and Presta, Cur. Opin. Immun. 20:460-470 (2008). In some embodiments, the Fc region is a native IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a hybrid, e.g., a chimera composed of the Fc constant regions of IgG2 / IgG4. Modifications to the Fc region include, but are not limited to, an IgG4 modified to prevent binding to Fc gamma receptors and complement, an IgG1 modified to improve binding to one or more Fc gamma receptors, an IgG1 modified (amino acid changes) to minimize effector function, an IgG1 with altered / absent glycans (typically by changing the expression host), and an IgG1 with altered pH-dependent binding to FcRn. The Fc region may include the entire hinge region or less than the entire hinge region.

[0264] Treatment outcomes in patients treated with rituximab (a chimeric mouse / human IgG1 monoclonal antibody against CD20) for non-Hodgkin's lymphoma or Waldenstrom's macroglobulinemia correlated with individual expression of allelic variants of Fcγ receptors that differ in their inherent affinity for the Fc domain of human IgG1. In particular, patients with high-affinity alleles of the low-affinity activating Fc receptor CD16A (FcγRIIIA) exhibited higher response rates and, in the case of non-Hodgkin's lymphoma, improved progression-free survival. In another embodiment, the Fc domain may contain one or more amino acid insertions, deletions, or substitutions that reduce binding to the low-affinity inhibitory Fc receptor CD32B (FcγRIIB) and retain wild-type levels of binding to or enhance binding to the low-affinity activating Fc receptor CD16A (FcγRIIIA).

[0265] Another embodiment includes IgG2-4 hybrids and IgG4 mutants that have reduced binding to FcRs and therefore increased half-lives. Representative IgG2-4 hybrids and IgG4 mutants are described in Angal, S. et al., Molecular Immunology, 30(1):105-108 (1993); Mueller, J. et al., Molecular Immonology, 34(6):441-452 (1997); and U.S. Patent No. 6,982,323 by Wang et al. In some embodiments, the IgG1 and / or IgG2 domains are deleted; for example, Angal, S. et al. describe IgG1 and IgG2 domains in which serine 241 is replaced with proline.

[0266] In some embodiments, the Fc domain contains an amino acid insertion, deletion, or substitution that enhances binding to CD16A. Numerous substitutions in the Fc domain of human IgG1 that enhance binding to CD16A and reduce binding to CD32B are known in the art and are described in Stavenhagen, et al., Cancer Res., 57(18):8882-90 (2007). Exemplary mutants of the Fc domain of human IgG1 that have reduced binding to CD32B and / or increased binding to CD16A contain F243L, R929P, Y300L, V305I, or P296L substitutions. These amino acid substitutions may be present in any combination in the Fc domain of human IgG1. In one aspect, the human IgG1 Fc domain mutant contains F243L, R929P, and Y300L substitutions. In another embodiment, the human IgG1 Fc domain variant contains the F243L, R929P, Y300L, V305I, and P296L substitutions. In another embodiment, the human IgG1 Fc domain variant contains the N297Q substitution, as this mutation abolishes FcR binding.

[0267] The disclosed fusion proteins optionally contain a peptide or polypeptide linker domain separating the Siglec-15 polypeptide from a second polypeptide. In some embodiments, the linker domain contains an immunoglobulin hinge region. In preferred embodiments, the hinge region is derived from a human immunoglobulin. Suitable human immunoglobulins from which the hinge may be derived include IgG, IgD, and IgA. In preferred embodiments, the hinge region is derived from a human IgG. The amino acid sequences of immunoglobulin hinge regions and other domains are well known in the art.

[0268] A representative fusion protein is the Siglec-15 ECD-IgG1 Fc fusion protein (L234F / L235E / P331S). TIFF0007720375000110.tif69136

[0269] The mouse leader sequence is underlined. The Siglec-15 extracellular domain (ECD) is italicized. The hinge region is double underlined. The remaining sequence is derived from the Fc of IgG1. The L234F / L235E / P331S mutations in the IgG1 Fc domain are in bold with dotted underlining.

[0270] In some embodiments, the leader sequence is truncated or otherwise missing from the fusion protein. For example, the fusion protein may have the following sequence: You can have TIFF0007720375000111.tif70136.

[0271] In some embodiments, the fusion protein is at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:193 or 194.

[0272] In some embodiments, the leader sequence, linker (e.g., hinge region), second fusion partner (e.g., Fc domain of IgG1), or combinations thereof are replaced with another sequence(s) (e.g., alternative leader sequences, hinges, Fc domains, etc.). Suitable substitutions are well known in the art. See, e.g., U.S. Patent No. 9,005,616, which is expressly incorporated by reference in its entirety.

[0273] 3. Nucleic Acids and Cells of Siglec-15 Vectors encoding Siglec-15 polypeptides, fragments, and fusions thereof are also provided. Nucleic acids such as those described above can be inserted into vectors and expressed in cells. Thus, cells containing and expressing Siglec-15 polypeptides, fragments, and fusions thereof are also provided. As used herein, a "vector" is a replicon, such as a plasmid, phage, virus, or cosmid, into which another DNA segment can be inserted to enable replication of the inserted segment. A vector can be an expression vector. An "expression vector" is a vector containing one or more expression control sequences, and an "expression control sequence" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0274] The nucleic acid in the vector can be operably linked to one or more expression control sequences. As used herein, "operably linked" means that the expression control sequence is incorporated into a genetic construct so as to effectively control the expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence consisting of a region of a DNA molecule within 100 nucleotides upstream of the point where transcription begins (generally near the initiation site of RNA polymerase II). To place a coding sequence under the control of a promoter, the translation initiation site of the translational reading frame of the polypeptide must be located 1 to approximately 50 nucleotides downstream of the promoter. Enhancers confer expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. Enhancers can also be located downstream from the transcription initiation site. A coding sequence is "operably linked" and "under the control" of expression control sequences in a cell when RNA polymerase is capable of transcribing the coding sequence into mRNA, which can then be translated into the protein encoded by the coding sequence.

[0275] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0276] The expression vector may contain a tag sequence. The tag sequence is typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted at any position in the polypeptide, including the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E-binding protein, and protein A. In some embodiments, the nucleic acid molecule encoding the Siglec-15 fusion polypeptide contains one or more domains of an Ig heavy chain constant region, such as the hinge, Cγ1, or Cγ2 domain of the human immunoglobulin Cγ1 chain. H 2, and C H The amino acid sequences corresponding to the three regions are present in a vector containing nucleic acid encoding the three regions.

[0277] A vector containing a nucleic acid to be expressed can be transferred into a host cell. The term "host cell" is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, "transformed" and "transfected" encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of several techniques. Without being limited to a particular technique, several of these techniques are well established in the art. Prokaryotic cells can be transformed with nucleic acid, for example, by electroporation or calcium chloride-mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Host cells (e.g., prokaryotic cells or eukaryotic cells such as CHO cells) can be used to produce the Siglec-15 fusion polypeptides described herein.

[0278] The vectors described can be used to express Siglec-15 in cells. Representative vectors include, but are not limited to, adenoviral vectors. One approach involves transferring the nucleic acid into primary cultured cells and then transplanting the ex vivo transformed cells into a host, either systemically or autologously, into a specific organ or tissue. Ex vivo methods can include, for example, harvesting cells from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the encoded polypeptide. These methods are known in the art of molecular biology. The transduction step can be accomplished by any standard means used in ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and gene gun-mediated gene transfer. Alternatively, liposomes or polymeric microparticles can be used. Successfully transduced cells can then be selected, for example, for expression of the coding sequence or a drug resistance gene. The cells can then be lethally irradiated (if desired) and injected or transplanted into the subject. In one embodiment, an expression vector containing a nucleic acid encoding the fusion protein is transfected into cells that are administered to a subject in need thereof.

[0279] In vivo nucleic acid therapy can be achieved by directly transferring functionally active DNA into mammalian somatic tissue or organ in vivo.For example, the nucleic acid encoding the polypeptide disclosed herein can be directly administered to lymphatic tissue or tumor.Alternatively, lymphatic tissue-specific targeting can be achieved by using lymphatic tissue-specific transcriptional regulator (TRE), such as B lymphocyte-specific TRE, T lymphocyte-specific TRE, or dendritic cell-specific TRE.Lymphatic tissue-specific TRE is known in the art.

[0280] The nucleic acid may also be administered in vivo by viral means. The nucleic acid molecule encoding the fusion protein may be packaged into a retroviral vector using a packaging cell line that produces replication-defective retrovirus, as is well known in the art. Other viral vectors that can be made non-replicating may also be used, including recombinant adenovirus and vaccinia virus. In addition to naked DNA or RNA, or viral vectors, engineered bacteria may also be used as vectors.

[0281] Nucleic acids may also be delivered by other carriers, including liposomes, polymeric microparticles and nanoparticles, and polycations such as asialoglycoprotein / polylysine.

[0282] In addition to in vivo viral and carrier-mediated gene transfer, physical means well known in the art can be used to directly transfer DNA, including administration of plasmid DNA and biolistic-mediated gene transfer.

[0283] D. Pharmaceutical Compositions Pharmaceutical compositions comprising the disclosed Siglec-15 binding molecules are provided. The pharmaceutical compositions containing Siglec-15 binding molecules can be intended for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), transdermal (passively, or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration, or by using bioerodible inserts, and can be formulated into dosage forms suitable for each administration route.

[0284] In some in vivo methods, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage that is sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated, or otherwise produce the desired pharmacological and / or physiological effect. The exact dosage will vary depending on various factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being performed.

[0285] As further research is conducted on the disclosed Siglec-15 binding molecules, information regarding appropriate dosage levels for treating various conditions in various patients will become apparent. Those skilled in the art will be able to determine appropriate dosages taking into account the treatment context, the age and general health of the recipient. The selected dosage will depend on the desired therapeutic effect, the route of administration, and the desired duration of treatment. Generally, dosages can be lowered for intravenous injection or infusion.

[0286] The dosage administered to a patient is typically 0.01 mg / kg to 100 mg / kg of the patient's body weight. The dosage administered to a patient can be, for example, 0.01 mg / kg to 20 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.01 mg / kg to 5 mg / kg, 0.01 to 2 mg / kg, 0.01 to 1 mg / kg, 0.01 mg / kg to 0.75 mg / kg, 0.01 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 0.25 mg / kg, 0.01 to 0.15 mg / kg, 0.01 to 0.10 mg / kg, 0.01 to 0.05 mg / kg, or 0.01 to 0.025 mg / kg of the patient's body weight. Representative specific dosages include, but are not limited to, 0.2 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 6 mg / kg, or 10 mg / kg. Doses as low as 0.01 mg / kg are believed to be sufficient to produce adequate pharmacodynamic effects. Dosage levels of 0.10 to 1 mg / kg are expected to be most appropriate. Higher doses (e.g., 1 to 30 mg / kg) are also expected to be active.

[0287] Generally, human antibodies have a longer half-life in the human body than antibodies from other species, due to the immune response to the foreign polypeptides. Therefore, human antibodies often allow for lower dosages and less frequent administration. Furthermore, the dosage and frequency of administration of the antibodies or fragments thereof of the present invention can be reduced by enhancing antibody uptake and tissue penetration through modifications such as lipidation.

[0288] In certain embodiments, the Siglec-15 binding molecule is administered locally, e.g., by injection directly into the area to be treated. Typically, the injection results in an increased local concentration of the Siglec-15 binding molecule composition, which is higher than can be achieved by systemic administration. Combining the Siglec-15 binding molecule composition with a matrix, as described below, can help produce an increased local concentration by reducing passive diffusion of the polypeptide from the area to be treated.

[0289] 1. Parenteral administration preparations In some embodiments, the compositions disclosed herein are administered in aqueous solution form via parenteral injection or infusion. Formulations may also be in the form of suspensions or emulsions. Generally, pharmaceutical compositions are provided containing an effective amount of a Siglec-15-binding molecule, and optionally contain pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions optionally contain one or more of the following: diluents, sterile water, buffered saline solutions of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; and additives, such as surfactants and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), and fillers (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.The preparation can be lyophilized and redissolved / resuspended immediately before use.The preparation can be sterilized, for example, by filtering through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0290] 2. Polymer matrices for controlled delivery The Siglec-15 binding molecules disclosed herein can also be administered in controlled-release formulations. Controlled-release polymeric devices can be fabricated for extended systemic release after implantation or injection (microparticles) of the polymeric device (rods, cylinders, films, discs). The matrix can be in the form of microparticles, such as microspheres, in which the agent is dispersed within a solid polymer matrix or microcapsules, the core of which is of a different material than the polymer shell, and the peptide is dispersed or suspended within the core (which may be liquid or solid in nature). Unless otherwise defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer can be cast as a thin slab or film ranging from nanometers to 4 centimeters in size, a powder produced by milling or other standard techniques, or even a gel, such as a hydrogel.

[0291] Either non-biodegradable or biodegradable matrices can be used to deliver the fusion polypeptide or the nucleic acid encoding the fusion polypeptide. These can be natural or synthetic polymers. Synthetic polymers typically have better characterized degradation and release profiles. The polymer is selected based on the desired release period. In some cases, linear release may be most useful, while in other cases, pulsed or "bulk release" may provide more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% water by weight), optionally crosslinked with multivalent ions or polymers.

[0292] The matrix can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed to make microspheres for drug delivery, such as those described by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

[0293] The devices can be formulated for local release to treat the implanted or injected area (which typically delivers a much smaller dosage than would be required for systemic treatment) or for systemic delivery. They can be implanted or injected subcutaneously, intramuscularly, into fat, or swallowed.

[0294] III.How to use Methods of using the disclosed Siglec-15-binding molecules and Siglec-15 ligand-binding molecules are also provided. Use of such molecules to enhance immune responses, delay or prevent tumor growth, inhibit tumor-mediated immunosuppression, eliminate tumors, and / or deplete or block the activity of tumor-associated macrophages (TAMs), reduce TAM-mediated immunosuppression, and reduce or reverse T cell suppression are disclosed. Use of such molecules in the diagnosis and treatment of cancer and other diseases is also provided.

[0295] TAMs link inflammation and cancer. Macrophages are immune system cells derived from activated blood monocytes. They are primarily recognized as participating in inflammatory responses induced by pathogens or tissue damage by acting to remove (i.e., phagocytose) pathogens, dead cells, cellular debris, and various components of the extracellular matrix (ECM). Macrophages constitute a critical component within the tumor microenvironment and have been found to account for up to 50% of the tumor burden.

[0296] In addition to mediating phagocytosis, macrophages secrete proangiogenic growth factors and matrix-remodeling proteases, thus playing a role in the development of the vascular infrastructure (i.e., angiogenesis) necessary for tumor development and growth (Pollard, JW (2009), Nat. Rev. Immunol. 9:259-270). The presence of macrophages within tumors appears to support tumor growth. Several studies have provided evidence that the presence of tumor-associated macrophages within tumors is a negative prognostic factor for survival (Farinha, P. et al. (2005), Blood 106:2169-2174; Dave, S. et al. (2004), N. Engl. J. Med. 351:2159-2169; Solinas, G. et al. (2009), J. Leukoc. Biol. 86(5):1065-1073). TAMs, as well as neutrophils, fibroblasts, and other cells, cooperate with tumor cells to promote angiogenesis in tumors (Nucera, S. et al. (2011), Int. J. Dev. Biol, doi:10.1387 / ijdb.103227sn; Zamarron, BF et al. (2011), Int. J. Biol. Sci. 7(5):651-658; Liu, J. et al. (2011), PLoS One. 6(4):e19495; Rigo, A. et al. (2010), Molec. Cancer 9(273):1-13; Lin, JY et al. (2011), Chin. J. Cancer 30(4):280-286;Vergati, M. (2011), J. Biomed.Biotechnol.2011:182413).

[0297] Studies have shown that Siglec-15 is inducibly expressed on TAMs and enhances TGF-β secretion by linking tumor cell recognition to the DAP12-Syk signaling pathway (Takamiya, et al., Glycobiology, 23(2):178-87(2013)). In a specific model presented by Takamiya, M-CSF secreted by tumor cells induces monocyte differentiation into macrophages, accompanied by Siglec-15 expression. The interaction between sialyl-Tn antigen and Siglec-15 enhances TGF-β production from macrophages via the DAP12-Syk pathway, thereby tilting the tumor microenvironment toward immunosuppression, leading to tumor progression and even metastasis.

[0298] Binding of a function-blocking or function-reducing anti-Siglec-15 antibody to Siglec-15 can reduce, block, antagonize, attenuate, or incompletely eliminate the ability of Siglec-15 to bind to one or more of its ligands, thereby reducing or preventing Siglec-15-mediated inhibitory immune signaling (including, but not limited to, TGF-β secretion). Increased expression of TGF-β correlates with the aggressiveness of many cancers and the lack of cell growth inhibition in response to TGF-β, often leading to tumorigenesis due to immune suppression. Reducing Siglec-15-mediated TGF-β secretion increases the overall immune response in a subject and directly or indirectly reduces tumor progression.

[0299] The following examples demonstrate that function-reducing and function-blocking anti-Siglec-15 antibodies, including those disclosed herein, can reverse Siglec-15-mediated suppression of CD4+ and CD8+ T cell proliferation in a peripheral blood mononuclear cell (PBMC) proliferation assay.

[0300] Thus, provided are methods for reducing immunosuppression and / or increasing immune responses, most typically by administering to a subject in need thereof an effective amount of an anti-Siglec-15 function-blocking antibody.

[0301] Suitable antibodies, polypeptides, and fusion proteins are disclosed herein and can be further selected by in vitro assays including, but not limited to, proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, trafficking, signal transduction, and inhibition of tumor growth. Representative assays for testing the functionality of the disclosed antibodies are provided in the Examples below.

[0302] The antibodies provided herein may also be useful in diagnostic and research applications. For example, non-neutralizing antibodies can bind to specific antigens without inhibiting receptor binding or biological activity of the antigen and can be used in capture assays and other pull-down methods (e.g., ELISA). Neutralizing (e.g., function-blocking) antibodies can be useful in competitive binding assays.

[0303] The antibodies can be used to quantify Siglec-15 polypeptides or their ligands.

[0304] A. Immune Response-Enhancing Molecules 1. Therapeutic and prophylactic uses Therapeutic and / or prophylactic uses are provided for molecules (e.g., antagonist molecules) that immunospecifically bind to human Siglec-15 and Siglec-15 ligand-binding molecules and are capable of reducing the binding between Siglec-15 and one or more of its ligands and / or counterreceptors (e.g., antibodies or antigen-binding fragments thereof).

[0305] In some embodiments, the molecules reduce or prevent binding between Siglec-15 and a sialylated glycoprotein ligand, e.g., a sialylated glycoprotein endogenously expressed by a subject's cells, thereby reducing or preventing Siglec-15-mediated signaling. Additionally or alternatively, the molecules can reduce or prevent binding between Siglec-15 and its counterreceptor, thereby reducing or preventing Siglec-15-mediated signaling and / or counterreceptor-mediated signaling. For example, the disclosed molecules can bind to an antigen at one or more sites on Siglec-15 that disrupt the sialic acid binding site (e.g., an epitope including residue 143 of SEQ ID NO:1) and / or at a binding site important for binding to a Siglec-15 counterreceptor. Representative compositions are illustrated in the Examples below. For example, in some embodiments, 5G12, 6F8, 8C8, 1C3, 1C12, 3H10, and 1B2 are ("strong blockers"), and 10G9, 8H8, and 9A5 are ("partial blockers"). Thus, in some embodiments, a molecule used to upregulate an immune response comprises one, two, three, four, five, or all six CDRs of 5G12, 6F8, 8C8, 1C3, 1C12, 3H10, 1B2, 10G9, 8H8, or 9A5. In some embodiments, the molecule comprises the light chain variable region and / or the heavy chain variable region of 5G12, 6F8, 8C8, 1C3, 1C12, 3H10, 1B2, 10G9, 8H8, or 9A5. In some embodiments, the molecule is a mouse anti-human 5G12, 6F8, 8C8, 1C3, 1C12, 3H10, 1B2, 10G9, 8H8, or 9A5, or a chimeric or humanized variant thereof.

[0306] As discussed above, interaction between Siglec-15 and sialylated glycoprotein ligands and / or Siglec-15 counterreceptors can inhibit T cell proliferation and increase cytokine production, such as TGF-β. Thus, in some embodiments, administration of the disclosed Siglec-15 binding molecules to a subject upregulates the subject's immune system by blocking or otherwise antagonizing the binding / interaction of Siglec-15 ligands and / or counterreceptors. In another aspect, the avidity and / or affinity of the anti-Siglec-15 antibody can be such that it binds only to cells expressing extremely high levels of Siglec-15, e.g., tumor-associated macrophages (TAMs) or cancer cells, thereby enabling specific targeting of this cell population. Thus, in some embodiments, the molecule reduces TGF-β production, for example, in the tumor microenvironment. In some embodiments, the Siglec-15-expressing cells are monocytes. In a more particular embodiment, the cell expressing Siglec-15 is a macrophage, such as a TAM.

[0307] As shown above, the disclosed antibodies and antigen-binding fragments can bind to and substantially block TAMs so as to modulate the immunosuppressive activity of TAMs. Furthermore, such antibodies can be used to deplete Siglec-15 TAMs in the tumor microenvironment or to deplete the concentration of TAMs in peripheral blood. In one embodiment, such modulation or depletion is achieved using a Siglec-15 antibody that binds to a site that attenuates or disrupts normal Siglec-15 function. Such disruption results in reduced (modulated) TAM activity and / or depletion of the actual or effective (functional) concentration of macrophages in the tumor. Alternatively, such modulation or depletion can be achieved using an anti-Siglec-15 antibody conjugated to a toxin that kills macrophages upon binding to TAMs.

[0308] In addition, the disclosed antagonist molecules can be used to induce, increase, or enhance T cell proliferation. In some embodiments, T cell responses are induced by reducing or preventing Siglec-15 from binding to counterreceptors on T cells. Upregulation of the immune system is particularly desirable in the treatment of cancer and chronic infections, and thus the disclosed compositions can be used to treat such disorders.

[0309] 2. Subjects to be treated Cancer treatment The disclosed function-reducing compositions and methods can be used to treat cancer. Generally, the methods involve stimulating or enhancing an immune response against cancer in a subject, reducing or preventing tumor growth or progression, or a combination thereof, by administering an amount of a Siglec-15 binding agent to the subject. The methods can reduce one or more symptoms of cancer.

[0310] During their development, cancer cells acquire a characteristic set of functional capabilities, albeit through various mechanisms. These capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion / metastasis, limitless replicative potential, and persistent angiogenesis. The term "cancer cell" is meant to encompass both pre-malignant and malignant cancer cells. In some embodiments, cancer refers to a benign tumor that remains localized. In other embodiments, cancer refers to a malignant tumor that invades and destroys adjacent body structures and spreads to distant sites. In still other embodiments, cancer is associated with a specific cancer antigen (e.g., pan-carcinoma antigen (KS1 / 4), ovarian cancer antigen (CA125), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2 / neu, etc.).

[0311] The methods and compositions disclosed herein are useful for treating or preventing a variety of cancers or other abnormal proliferative disorders, including carcinomas, such as those of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, and skin; squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, such as leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloid lineage, such as acute and chronic myeloid leukemia. , and promyelocytic leukemia; tumors of mesenchymal origin, such as fibrosarcoma and rhabdomyosarcoma; other tumors, such as melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous system, such as astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin, such as fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, such as melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and teratocarcinoma.

[0312] Cancers caused by abnormalities in apoptosis can also be treated by the disclosed methods and compositions. Such cancers include, but are not limited to, follicular lymphoma, carcinomas with p53 mutations, hormone-dependent tumors of the breast, prostate, and ovary, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes. In certain embodiments, the methods and compositions treat or prevent malignant or dysproliferative changes (such as metaplasia and dysplasia) or hyperproliferative disorders in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments, the methods and compositions treat or prevent sarcoma, melanoma, or leukemia.

[0313] The disclosed compositions and methods are particularly useful for treating cancers associated with cells that express abnormally high levels of Siglec-15 itself or its glycoprotein ligands, cancers with multiple tumors associated with macrophages (particularly where macrophages express Siglec-15), and / or other cancers in which another cell type(s) express high levels of Siglec-15 or Siglec-15 ligands.

[0314] Specific cancers and related disorders that may be treated or prevented by the methods and compositions disclosed herein include, but are not limited to, leukemias, including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, including, but not limited to, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, and myelodysplastic syndromes; chronic leukemias, including, but not limited to, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, including, but not limited to, and / or other conditions, including, but not limited to, Hodgkin's disease or non-Hodgkin's disease lymphoma (e.g., diffuse anaplastic lymphoma kinase (ALK)-negative, large B-cell lymphoma (DLBCL); diffuse anaplastic lymphoma kinase (ALK)-positive, large B-cell lymphoma (DLBCL); anaplastic lymphoma kinase (ALK)-positive, ALK+ anaplastic large cell lymphoma (ALCL), acute myeloid lymphoma (AML)); multiple myeloma, including, but not limited to, smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; severe undetermined monoclonal gammopathy; benign monoclonal gammopathy; heavy chain disease; sarcomas of bone and connective tissue, including, but not limited to, osteosarcoma, osteogenic sarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangioendothelioma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, including, but not limited to, glioma, astrocytic sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma, tumors, brain stem glioma, ependymoma, oligodendroglioma, non-glial tumors, acoustic neuroma, craniopharyngeal duct tumor, medulloblastoma, meningioma, pinealocytoma, pineoblastoma, primary brain lymphoma; breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, ductal carcinoma, medullary breast carcinoma, mucinous breast carcinoma, tubular breast carcinoma, papillary breast carcinoma, Paget's disease, and inflammatory breast carcinoma; adrenal gland cancer, including but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer, including but not limited to papillary or follicular thyroid carcinoma, medullary thyroid carcinoma, and histoplastic thyroid carcinoma;Pancreatic cancers, including, but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumors, and carcinoid or islet cell tumors; pituitary cancers, including, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancers, including, but not limited to, intraocular melanoma, e.g., iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancers, including, but not limited to, vasculitis, urinary tract cancer ... cervical cancer, including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancer, including, but not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancer, including, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancer, including, but not limited to, epithelial ovarian carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancer, including, but not limited to, squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancer, including, but not limited to, adenocarcinoma, fungus-like carcinoma, (polypoid), ulcerative, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, for example, but not limited to, hepatocellular carcinoma and hepatoblastoma, gallbladder cancer, for example, but not limited to, adenocarcinoma; cholangiocarcinoma, for example, but not limited to, papillary, nodular, and diffuse; lung cancer, for example, but not limited to, non-small cell lung carcinoma, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung carcinoma; testicular cancer, for example, but not limited to, germinal tumor, seminoma, undifferentiated, classic (typical), spermatozoon cellular, non-seminomatous, embryonal carcinoma, teratocarcinoma, choriocarcinoma (yolk sac tumor), prostate cancer, including, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; renal carcinoma; oral cancer, including, but not limited to, squamous cell carcinoma; basal carcinoma; salivary gland cancer, including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, including, but not limited to, squamous cell carcinoma and verrucous; skin cancer, including, but not limited to, basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentigo melanoma;Kidney cancers include, but are not limited to, renal cell carcinoma, adenocarcinoma, adrenal nephroma, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter); Wilms' tumor; bladder cancers include, but are not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Additionally, cancers include myxosarcoma, osteosarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovium, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma (for reviews of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia, and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America);

[0315] b. Treating infections The disclosed function-reducing compositions and methods can be used to treat infectious diseases and infectious disorders. Generally, the methods involve stimulating or enhancing an immune response to an infectious agent in a subject, reducing or preventing the progression of an infectious disease, or a combination thereof, by administering an amount of a Siglec-15 binding molecule to the subject. The methods can reduce one or more symptoms of an infectious disease.

[0316] Infection or disease can be caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens that enter cells and are attacked, i.e., by cytotoxic T lymphocytes.

[0317] Infection or disease can be acute or chronic.Acute infection is typically a short-term infection.During acute microbial infection, immune cells begin to express immunomodulatory receptors.Therefore, in some embodiments, the method comprises increasing the immune stimulatory response to acute infection.

[0318] The infection may be caused by, for example, but not limited to, Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, or Mycobacterium.

[0319] In some embodiments, the disclosed compositions are used to treat chronic infections, for example, infections in which T cell exhaustion or T cell anergy has occurred, allowing the infection to persist in the host for extended periods of time.

[0320] Typical infections to be treated are chronic infections caused by hepatitis viruses, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes viruses, Epstein-Barr virus, or human papillomavirus.

[0321] Because viral infections are primarily eliminated by T cells, increasing T cell activity would be therapeutically useful in situations where more rapid or complete clearance of infectious viral agents would be beneficial to animals or human subjects. Thus, the disclosed compositions can be administered to treat local or systemic viral infections, including but not limited to immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), and the common cold (e.g., human rhinovirus), as well as other viral infections, such as those caused by HTLV, hepatitis virus, respiratory syncytial virus, vaccinia virus, and rabies virus. The molecules can be administered locally to treat viral skin diseases, such as herpes lesions, or shingles, or genital warts. The molecules can be administered systemically to treat systemic viral diseases, including but not limited to AIDS, influenza, the common cold, or encephalitis.

[0322] Exemplary infections that can be treated include, but are not limited to, infections caused by microorganisms such as Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Haemophilus influenzaeInfluenza type B (HIB), Hyphomicrobium, Legionella, Leptospirosis, Listeria, Meningococcus A, B, and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochlorous, Pro The genera Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and the genera Treponema, Vibrio, Yersinia, and Cryptococcus neoformans are all species that are not resistant to antibiotics. neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia rickettsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparumfalciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni.

[0323] Other microorganisms that can be treated using the disclosed compositions and methods include bacteria, such as bacteria of the genera Klebsiella, Serratia, and Pasteurella; pathogens associated with cholera, tetanus, botulism, anthrax, plague, and Lyme disease; or fungal or parasitic pathogens, such as Candida (e.g., albicans, krusei, glabrata, tropicalis), Cryptococcus, Aspergillus (e.g., fumigatus, niger), Mucorales, and the like. Mucorales (mucor, absidia, rhizophus), Sporothrix (schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis) and Histoplasma (capsulatuma), Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp.), Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondii, Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba histolytica, Balantidium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia, or Trypanosoma.

[0324] B. Immune Response-Reducing Molecules 1. Therapeutic and prophylactic uses Therapeutic and / or prophylactic uses are provided for molecules (e.g., antibodies or antigen-binding fragments thereof) that bind to human Siglec-15 or its ligands and are capable of increasing or enhancing the binding of Siglec-15 to one or more of its ligands and / or counterreceptors (e.g., agonist molecules), or for molecules that are capable of directly increasing or enhancing signal transduction mediated by Siglec-15 or Siglec-15 counterreceptors.

[0325] As discussed above, interaction between Siglec-15 and sialylated glycoprotein ligands and / or Siglec-15 counterreceptors can inhibit T cell proliferation and increase cytokine production, such as TGF-β. Thus, in some embodiments, administration of a functionally activating Siglec-15-binding molecule or a functionally activating Siglec-15 ligand-binding molecule to a subject downregulates the subject's immune system by inducing or otherwise agonizing Siglec-15-ligand and / or counterreceptor binding / interaction, or by directly stimulating Siglec-15 or Siglec-15 counterreceptor signaling. In some embodiments, the molecule increases TGF-β production and / or secretion, e.g., from monocytes, such as macrophages.

[0326] Additionally, the disclosed agonist Siglec-15 binding and Siglec-15 ligand binding molecules can be used to reduce or decrease T cell proliferation. In some embodiments, T cell responses are induced by increasing or enhancing Siglec-15 binding to counterreceptors on T cells. Down-regulation of the immune system is particularly desirable for the treatment of inflammatory and autoimmune diseases and disorders, and for treating or preventing transplant rejection and / or graft-versus-host disease, and thus the disclosed compositions can be used to treat such disorders.

[0327] A inflammatory response In some embodiments, Siglec-15 agonist molecules are used to treat or alleviate one or more symptoms of inflammation, eg, acute, chronic, or persistent inflammation.

[0328] Immune responses, including inflammation, can be inhibited or reduced in a subject, preferably a human, by administering an amount of a Siglec-15 binding molecule effective to inhibit or reduce the biological activity of immune cells (e.g., T cells or B cells) or reduce the amount of pro-inflammatory molecules at the site of inflammation. Exemplary pro-inflammatory molecules include, but are not limited to, IL-1β, TNF-α, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.

[0329] b. Excessive inflammatory response In some embodiments, the Siglec-15 binding molecule slows down the immune system. For example, the Siglec-15 binding molecule can be used to control the immune response to an infection that damages healthy tissue through an excessive inflammatory response. Thus, in some embodiments, the agent is administered to a subject with an infection that also causes an excessive inflammatory response. In such cases, controlling the excessive immune response can be beneficial to the subject.

[0330] c. Inflammatory and autoimmune diseases / disorders The disclosed compositions can also be used to treat inflammatory or autoimmune diseases and disorders. Exemplary inflammatory or autoimmune diseases / disorders that can be treated include rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, and cicatricial pemphigoid. Acne, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), Iga nephropathy, insulin-dependent diabetes mellitus (type 1), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome syndrome), polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0331] In some embodiments, the inflammatory or autoimmune disease is caused by a pathogen or is the result of an infection.

[0332] d.Transplantation Siglec-15 binding molecules can be used to reduce or inhibit transplant rejection in a subject, preferably a human subject, by administering to the subject an amount of an agonist Siglec-15 binding molecule effective to inhibit or reduce the biological activity of immune cells or to reduce the amount of proinflammatory cytokines or other molecules associated with or promoting inflammation at the transplant site.

[0333] The transplanted material can be a cell, tissue, organ, limb, finger, or part of a body, preferably a human body. The transplant is typically allogeneic or xenogeneic. The agonist Siglec-15 molecule is typically administered to a subject in an amount effective to reduce or inhibit transplant rejection. The molecule can be administered systemically or locally by an acceptable administration route. In some embodiments, the molecule is administered to the transplant site before, during, or after transplantation.

[0334] The molecule can be administered ex vivo directly to the cell, tissue, or organ to be transplanted. In one embodiment, the transplant material is contacted with the Siglec-15 binding molecule before transplantation, after transplantation, or both.

[0335] In other embodiments, the Siglec-15 binding molecule is administered to an immune tissue or organ, for example, a lymph node or spleen.

[0336] i.Cells Any type of cell population can be transplanted into a subject. Cells can be allogeneic or xenogeneic. Xenogeneic means that the cell population contains two or more types of cells. Representative cells include progenitor cells such as stem cells and pluripotent cells, which can be collected from a donor and transplanted into a subject. Cells can optionally be treated ex vivo before transplantation. Cells can be autologous or xenogeneic.

[0337] ii.Organization Any tissue can be used as a graft. Representative tissues include skin tissue, adipose tissue, cardiovascular tissue (e.g., veins, arteries, capillaries, valves), nerve tissue, bone marrow tissue, lung tissue, eye tissue (e.g., cornea and lens), cartilage tissue, bone tissue, and mucosal tissue. The tissue can be modified as discussed above.

[0338] iii. Organs Representative organs that can be used for transplantation include, but are not limited to, kidney, liver, heart, spleen, bladder, lung, stomach, eye, tongue, pancreas, intestine, etc. The organ to be transplanted can be modified prior to transplantation as discussed above.

[0339] One embodiment provides a method of inhibiting or reducing chronic transplant rejection in a subject by administering a Siglec-15 binding molecule in an amount effective to inhibit or reduce chronic transplant rejection compared to a control.

[0340] e. Graft-versus-host disease (GVHD) The molecule can also be used to treat graft-versus-host disease (GVHD) by administering an amount of the Siglec-15 binding molecule effective to alleviate one or more symptoms associated with GVHD. GVHD is a major complication associated with allogeneic hematopoietic stem cell transplantation, in which functional immune cells in the transplanted bone marrow recognize the recipient as "foreign" and launch an immune attack. Under certain circumstances, it can also occur with blood transfusions. Symptoms of GVD include rash or changes in skin color or texture, diarrhea, nausea, abnormal liver function, yellowing of the skin, increased susceptibility to infection, dry eyes, eye irritation, and oral sensitivity or dry mouth.

[0341] f. Diabetes Agonistic Siglec-15 binding molecules can also be used to treat diabetes. The method includes transplanting insulin-producing cells into a subject and administering to the subject an amount of the molecule effective to reduce or inhibit transplant rejection. Preferably, the insulin-producing cells are beta cells or islet cells. In certain embodiments, the insulin-producing cells are recombinant cells engineered to produce insulin.

[0342] The insulin-producing cells can be encapsulated within a matrix, such as a polymeric matrix, using a suitable polymer, including, but not limited to, alginate, agarose, hyaluronic acid, collagen, synthetic monomers, albumin, fibrinogen, fibronectin, vitronectin, laminin, dextran, dextran sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, chitin, chitosan, heparan, heparan sulfate, or combinations thereof.

[0343] C. Therapeutic Inhibition of Osteoclastic Bone Resorption Studies have shown that the expression and function of Siglec-15 are important for osteoclastogenesis (Stuible, et al., J. Biol. Chem., 289(10):6498-6512 (2014); Ishida-Kitagawa, J. Biol. Chem., 287, 17493-17502 (2012), each of which is expressly incorporated by reference in its entirety). Siglec-15 protein is highly upregulated during osteoclast differentiation, but this protein is undetectable in undifferentiated cells. Results indicate that Siglec-15 and DAP12 form a complex at endogenous expression levels in osteoclasts. Siglec-15(- / -) knockout mice are mildly osteoporotic.

[0344] In vivo studies have also shown that Siglec-15 antibodies can inhibit osteoclast activity in physiological situations and may offer a therapeutic strategy for reducing bone loss. Activation of cell surface receptors and their downregulation by endocytosis are often coupled as a means of limiting the strength and duration of signaling, but for Siglec-15, signaling and endocytosis appear to occur mutually exclusive, depending on whether antibody ligation induces receptor clustering or simply dimerization.

[0345] Siglec-15 is an osteoclast-specific receptor with a very restricted expression pattern, making Siglec-15-targeted therapy selective. Siglec-15 antibodies inhibit osteoclast differentiation at a relatively late stage, preserving the communication between osteoclasts and osteoblasts and avoiding the complications of existing osteoclast-targeted therapies, which induce osteoclast cell death (bisphosphonates) or prevent their differentiation at an early stage (denosumab), resulting in undesirable side effects, including osteonecrosis of the jaw and atypical fractures of the femur.

[0346] Thus, in some embodiments, the disclosed molecules (e.g., antibodies or antigen-binding fragments thereof) that immunospecifically bind to human Siglec-15 and are capable of reducing or blocking binding between Siglec-15 and one or more of its ligands and / or counterreceptors (e.g., agonist molecules) can also be administered to a subject in need thereof in an amount effective to reduce or inhibit osteoclast differentiation, function, or a combination thereof. In some embodiments, the molecules are administered in an amount effective to reduce bone loss, increase bone formation, increase bone mineral density, or a combination thereof.

[0347] D. Targeting and Detection The disclosed Siglec-15 binding and Siglec-15 ligand binding molecules can be used to deliver therapeutic cargo and / or detect the presence of Siglec-15 or its ligand on cells or tissues, respectively, regardless of their effect on Siglec-15 function. For example, Siglec-15 binding and Siglec-15 ligand binding molecules can be conjugated with a biomolecule of interest to form a conjugate. Cargoes including pharmacologically active molecules, such as inorganic and organic molecules, pharmaceuticals, drugs, peptides, proteins, genetic material, etc., can be conjugated to Siglec-15 binding molecules or Siglec-15 ligand binding molecules, which can then target the cargo to cells or tissues expressing Siglec-15 or its ligand, respectively. Siglec-15 molecules can be chemically linked to polypeptides by peptide bonds or by chemical or peptide linker molecules. Methods for attaching drugs or other small molecule pharmaceuticals to antibody fragments are well known and include bifunctional chemical linkers, such as N-succinimidyl(4-iodoacetyl)-aminobenzoate; sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate; 4-succinimidyl-oxycarbonyl-.A-inverted.-(2-pyridyldithio)toluene; sulfosuccinimidyl-6-[.alpha.-methyl-.A-inverted.-(pyridyldithio)toluene]; N-succinimidyl-3-(-2-pyridyldithio)-proprionate; succinimidyl-6-[3(-(-2-pyridyldithio)-proprionmido]hexanoate; sulfosuccinimidyl-6-[3(-(-2-pyridyldithio)-propionamido]hexanoate; 3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent, dichlorotriazine acid, S-(2-thiopyridyl)-L-cysteine, and the like.

[0348] Fusion proteins can be designed to place the protein of interest at the amino or carboxy terminus of either the heavy or light chain of an antibody, although the entire heavy chain may not be required. Possible configurations include the use of truncated portions of the heavy and light chains, with or without spacer sequences as necessary to maintain the functional integrity of the combined proteins.

[0349] Alternatively, a universal carrier system can be devised. For example, various proteins or DNA can be conjugated to common carriers, such as protein A, poly-L-lysine, hexahistidine, etc. The conjugated carrier then forms a complex with a Siglec-15 binding molecule or a Siglec-15 ligand binding molecule. A small portion of the carrier molecule involved in immunoglobulin binding could be used as the carrier. Another similar configuration involves designing a carrier that interacts with a protein engineered into the heavy or light chain of an antibody.

[0350] In some embodiments, Siglec-15-binding molecules or Siglec-15 ligand-binding molecules are conjugated or otherwise incorporated onto or into nanocarriers to target the nanocarriers to Siglec-15-positive cells or Siglec-15 ligand-positive cells. Nanocarriers, such as micro- or nano-polymer particles, liposomes, nanotubes, etc., can contain and deliver active agents to Siglec-15-positive cells or Siglec-15 ligand-positive cells or their microenvironments.

[0351] Similarly, Siglec-15 binding molecules or Siglec-15 ligand binding molecules can be conjugated to a detectable marker or can be unconjugated and detected with a secondary reagent to detect expression of Siglec-15 or Siglec-15 ligand in vitro or in vivo, respectively, and can thus be used for inferential, immunohistochemical, and other assays.

[0352] IV. Combination Therapy The disclosed Siglec-15 binding and Siglec-15 ligand binding molecules can be administered to a subject in need thereof alone or in combination with one or more additional therapeutic agents. In some embodiments, the Siglec-15 binding molecule or Siglec-15 ligand binding molecule and the additional therapeutic agent are administered separately but simultaneously. The Siglec-15 binding molecule or Siglec-15 ligand binding molecule and the additional therapeutic agent can also be administered as part of the same composition. In other embodiments, the Siglec-15 binding molecule or Siglec-15 ligand binding molecule and the second therapeutic agent are administered separately at different times but as part of the same treatment regimen.

[0353] The subject can be administered the second therapeutic agent 1, 2, 3, 4, 5, 6 hours or more, or 1, 2, 3, 4, 5, 6, 7 days or more after administration of the first therapeutic agent. In some embodiments, the subject can be administered one or more doses of the first agent every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, or 48 days before receiving an initial dose of the second agent. A Siglec-15 binding molecule or a Siglec-15 ligand binding molecule can be the first or second therapeutic agent. In some embodiments, one or more Siglec-15 binding molecules and one or more Siglec-15 ligand binding molecules are administered in combination.

[0354] The Siglec-15 binding and / or Siglec-15 ligand binding molecule and additional therapeutic agents can be administered as part of a treatment regimen. For example, if a first therapeutic agent can be administered to a subject every four days, the second therapeutic agent can be administered on day 1, day 2, day 3, or day 4, or a combination thereof. The first therapeutic agent or second therapeutic agent may be administered repeatedly throughout the treatment regimen.

[0355] Representative molecules include, but are not limited to, cytokines, chemotherapeutics, radionuclides, other immunotherapeutics, enzymes, antibiotics, antivirals (protease inhibitors alone or in combination with nucleosides to treat HIV or hepatitis B or C, among others), antiparasitic agents (helminths, protozoans), growth factors, growth inhibitory agents, hormones, hormone antagonists, antibodies and biologically active fragments thereof (e.g., humanized, single-chain, and chimeric antibodies), antigens and vaccine formulations (e.g., adjuvants), peptide drugs, anti-inflammatory drugs, ligands that bind to Toll-like receptors to activate the innate immune system (e.g., but not limited to, polyinosinic:polycytidylic acid (polyI:C) and CpG oligonucleotides), molecules that recruit and optimize the adaptive immune system, other molecules that activate or upregulate the action of cytotoxic T lymphocytes, natural killer cells, and helper T cells, and other molecules that inactivate or downregulate regulators or regulatory T cells.

[0356] The additional therapeutic agent is selected based on the condition, disorder, or disease to be treated. For example, the Siglec-15 binding molecule can be co-administered with one or more additional agents that function to enhance or promote an immune response or to reduce or inhibit an immune response.

[0357] A. Chemotherapy drugs Siglec-15 binding and Siglec-15 ligand binding molecules can be combined with one or more chemotherapeutic agents and pro-apoptotic agents. Representative chemotherapeutic agents include amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, and ribozyme. Exemplary pro-apoptotic agents include, but are not limited to, posomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. Exemplary pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

[0358] B. Other immunomodulatory drugs 1.PD-1 antagonist In some embodiments, the Siglec-15 binding molecule or Siglec-15 ligand binding molecule is co-administered with a PD-1 antagonist. Programmed death-1 (PD-1) is a member of the CD28 family of receptors that, when induced on T cells, delivers a negative immune response. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces T cell proliferation and / or the strength and / or duration of the T cell response. Suitable PD-1 antagonists are described in U.S. Pat. Nos. 8,114,845, 8,609,089, and 8,709,416 (which are expressly incorporated by reference herein in their entireties), and include compounds or agents that bind to or block the ligand of PD-1, interfering with or inhibiting binding of the ligand to the PD-1 receptor, or that directly bind to and block the PD-1 receptor without inducing inhibitory signaling through the PD-1 receptor.

[0359] In some embodiments, the PD-1 receptor antagonist binds directly to the PD-1 receptor without triggering inhibitory signaling, and also binds to a ligand of the PD-1 receptor, reducing or preventing the ligand from triggering signaling through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and trigger inhibitory signaling, fewer cells are attenuated by the negative signals delivered by PD-1 signaling, resulting in a more robust immune response.

[0360] PD-1 signaling is thought to be promoted by binding of PD-1 ligands (such as B7-H1 or B7-DC) in close proximity to peptide antigens presented by the major histocompatibility complex (MHC) (see, e.g., Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Thus, proteins, antibodies, or small molecules that prevent PD-1 from co-engaging with the TCR on the T cell membrane are also useful PD-1 antagonists.

[0361] In some embodiments, the PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or interferes with PD-1 receptor signaling by binding to a ligand of PD-1 or PD-1 itself (particularly in the absence of subsequent co-ligation of PD-1 with a TCR), thereby preventing the triggering of inhibitory signaling through the PD-1 receptor. Other PD-1 antagonists contemplated by the methods of the invention include antibodies that bind to PD-1 or a ligand of PD-1, as well as other antibodies.

[0362] Suitable anti-PD-1 antibodies include, but are not limited to, those described in U.S. Patent Nos. 7,332,582, 7,488,802, 7,521,051, 7,524,498, 7,563,869, 7,981,416, 8,088,905, 8,287,856, 8,580,247, 8,728,474, 8,779,105, 9,067,999, 9,073,994, 9,084,776, 9,205,148, 9,358,289, 9,387,247, 9,492,539, and 9,492,540, all of which are incorporated by reference in their entireties.

[0363] See also Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[0364] Representative anti-B7-H1 (also known as anti-PD-L1) antibodies include, but are not limited to, those described in U.S. Patent Nos. 8,383,796, 9,102,725, 9,273,135, 9,393,301, and 9,580,507, all of which are expressly incorporated by reference in their entirety.

[0365] For anti-B7-DC (also known as anti-PD-L2) antibodies, see U.S. Patent Nos. 7,411,051, 7,052,694, 7,390,888, 8,188,238, and 9,255,147, all of which are expressly incorporated by reference in their entireties.

[0366] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides (including homologs and variants thereof), and active fragments of any of the foregoing, and fusion proteins incorporating any of the foregoing. In some embodiments, the fusion protein comprises a soluble portion of B7-DC linked to the Fc portion of an antibody, such as human IgG, without incorporating all or part of the transmembrane portion of human B7-DC.

[0367] The PD-1 antagonist can be a fragment of mammalian B7-H1, e.g., derived from a mouse or primate, e.g., a human, where the fragment binds to and blocks PD-1 but does not result in inhibitory signaling through PD-1. The fragment can also be part of a fusion protein, e.g., an Ig fusion protein.

[0368] Other useful polypeptide PD-1 antagonists include those that bind to PD-1 receptor ligands. These include PD-1 receptor proteins or soluble fragments thereof that can bind to PD-1 ligands, such as B7-H1 or B7-DC, preventing binding to endogenous PD-1 receptors and thereby preventing inhibitory signaling. B7-H1 has also been shown to bind to the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include soluble ECD portions of PD-1 proteins containing mutations, such as the A99L mutation, that enhance binding to natural ligands (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or soluble fragments thereof that bind to B7-H1 ligands and prevent binding to endogenous PD-1 receptors and thereby prevent inhibitory signaling are also useful.

[0369] Antisense nucleic acids (both DNA and RNA) and siRNA molecules of PD-1 and B7-H1 can also be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on T cells and the production of T cell ligands such as B7-H1, PD-L1, and / or PD-L2. For example, siRNA (e.g., approximately 21 nucleotides in length, specific to the gene encoding PD-1 or the PD-1 ligand, and readily available commercially) complexed with a carrier such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8):2231-2244 (2009)) is readily taken up by cells expressing PD-1 and its ligand, reducing the expression of these receptors and ligands, thereby reducing inhibitory signaling in T cells and activating them.

[0370] 2. CTLA4 antagonists Other molecules useful for mediating the effects of T cells in immune responses are also contemplated as additional therapeutic agents. In some embodiments, the molecule is a CTLA4 antagonist, such as an antagonistic anti-CTLA4 antibody. Examples of anti-CTLA4 antibodies contemplated for use in the methods of the present invention include antibodies such as those described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0371] Dosages for anti-PD-1, anti-B7-H1, and anti-CTLA4 antibodies are known in the art and may range, for example, from 0.1 to 100 mg / kg, or more narrowly from 1 to 50 mg / kg, or from 10 to 20 mg / kg. Suitable doses for human subjects may be from 5 to 15 mg / kg, with 10 mg / kg of antibody (e.g., human anti-PD-1 antibody) being a specific embodiment.

[0372] Specific examples of anti-CTLA4 antibodies useful in the methods of the invention are the human anti-CTLA4 antibody ipilimumab, administered at a dose of, for example, about 10 mg / kg, and the human anti-CTLA4 antibody tremelimumab, administered at a dose of, for example, about 15 mg / kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2):135-137 (2010), published online in December 2009.

[0373] In another embodiment, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand and prevent its binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002)). Such small organic compounds, administered alone or in combination with anti-CTLA4 antibodies, may reduce the inhibitory signaling of T cells.

[0374] 3. Enhancers In some embodiments, the additional therapeutic agent includes a potentiator, which likely acts to increase the effectiveness of the immune response upregulator by more than one mechanism, although the precise mechanism of action is not critical to the broad practice of the invention.

[0375] In some embodiments, the potentiating agent is cyclophosphamide. Cyclophosphamide (CTX, Cytoxan®, or Neosar®) is an oxazaphosphorine drug and analogs, such as ifosfamide (IFO, Ifex), perfosfamide, trofosfamide (trofosfamide; Ixoten), and their pharmaceutically acceptable salts, solvates, prodrugs, and metabolites (U.S. Patent Application Publication No. 20070202077, which is incorporated in its entirety). Ifosfamide (MITOXANA®) is a structural analog of cyclophosphamide, and its mechanism of action is believed to be the same as or substantially similar to that of cyclophosphamide. Perfosfamide (4-hydroperoxycyclophosphamide) and trofosfamide are also alkylating agents, and are structurally related to cyclophosphamide. For example, perfosfamide alkylates DNA, thereby inhibiting DNA replication and RNA and protein synthesis. In an effort to improve selectivity and response while reducing host toxicity, novel oxazaphosphorine derivatives have been designed and evaluated (Liang J, Huang M, Duan W, Yu XQ, Zhou S. Design of new oxazaphosphorine anticancer drugs. Curr Pharm Des. 2007;13(9):963-78. Review). These include mafosfamide (NSC 345842), glufosfamide (D19575, beta-D-glucosylisophosphoramide mustard), S-(-)-bromophosphamide (CBM-11), NSC 612567 (aldofosphamide perhydrothiazine), and NSC 613060 (aldofosphamide thiazolidine). Mahofosfamide is an oxazaphosphorine analog that is a chemically stable 4-thioethanesulfonate salt of 4-hydroxy-CPA. Glufosfamide is an IFO derivative in which isophosphoramide mustard, an alkylated metabolite of IFO, is glycosidically linked to a beta-D-glucose molecule.Additional cyclophosphamide analogs are described in US Pat. No. 5,190,929, entitled "Cyclophosphamide analogs useful as anti-tumor agents," which is incorporated herein by reference in its entirety.

[0376] Although CTX itself is nontoxic, some of its metabolites are cytotoxic alkylating agents that induce DNA cross-linking and strand breaks at high doses. Many cells are resistant to CTX because they express high levels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). CTX targets proliferating lymphocytes because lymphocytes express only low levels of ALDH (but hematopoietic stem cells do not), and cycling cells are most sensitive to DNA alkylating agents.

[0377] Low doses of CTX (<200 mg / kg) can have immunostimulatory effects, including stimulation of antitumor immune responses in human and mouse models of cancer (Brode & Cooke Crit Rev. Immunol. 28:109-126 (2008)). These low doses are subtherapeutic and lack direct antitumor activity. In contrast, high doses of CTX inhibit antitumor responses. Several mechanisms can explain the role of CTX in enhancing antitumor immune responses: (a) depletion of CD4+CD25+FoxP3+ Tregs (specifically proliferating Tregs, which may be particularly suppressive); (b) depletion of B lymphocytes; (c) induction of nitric oxide (NO), which suppresses tumor cell proliferation; and (d) recruitment and proliferation of CD11b+Gr-1+MDSCs. These primary effects have numerous secondary effects. For example, macrophages produce more IFN-γ and less IL-10 after Treg depletion. CTX has also been shown to induce type I IFN expression and promote homeostatic lymphocyte proliferation.

[0378] Treg depletion is the most commonly cited mechanism by which CTX enhances antitumor immune responses. This conclusion is based in part on the results of adoptive transfer experiments. In the AB1-HA tumor model, CTX treatment on day 9 resulted in a 75% cure rate. Transfer of purified Tregs on day 12 almost completely inhibited the CTX response (van der Most et al. Cancer Immunol. Immunother. 58:1219-1228 (2009)). Similar results were observed in the HHD2 tumor model, where adoptive transfer of CD4+CD25+ Tregs after pretreatment with CTX eliminated the therapeutic response to the vaccine (Taieb, JJ Immunol. 176:2722-2729 (2006)).

[0379] Numerous human clinical trials have demonstrated that low-dose CTX is a safe, well-tolerated, and effective agent for promoting anti-tumor immune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47:1-12 (1998)).

[0380] The optimal dose of CTX to enhance anti-tumor immune responses is one that reduces the total number of T cells by reducing Tregs to levels below the normal range but not to therapeutic levels (see Machiels et al. Cancer Res. 61:3689-3697 (2001)).

[0381] In human clinical trials using CTX as an immune enhancer, 300 mg / m 2 is typically used for an average male (6 feet, 170 pounds (78 kg), body surface area 1.98 m) 2 ), 300 mg / m 2The dose is 8 mg / kg, or 624 mg of total protein. In mouse models of cancer, efficacy has been seen at doses ranging from 15 to 150 mg / kg, which corresponds to 0.45 to 4.5 mg of total protein in a 30 g mouse (Machiels et al. Cancer Res. 61:3689-3697 (2001), Hengst et al. Cancer Res. 41:2163-2167 (1981), Hengst Cancer Res. 40:2135-2141 (1980)).

[0382] In the case of large mammals, such as primates, e.g., human patients, such mg / m 2 Although doses may be used, unit doses administered over a finite period of time may also be used. Such unit doses may be administered daily for a finite period of time, such as up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days, or up to 20 days, or up to 25 days (all of which are expressly contemplated by the present invention). The same regimen may be applied to the other potentiators listed herein.

[0383] In other embodiments, the potentiating agent is an agent that reduces the activity and / or number of regulatory T cells (T-reg), such as sunitinib (SUTENT®), anti-TGFβ, or imatinib (GLEEVAC®). The listed therapeutic regimens may also include administering an adjuvant.

[0384] Useful potentiators also include antimitotic agents such as paclitaxel, aromatase inhibitors (e.g., letrozole), and angiogenesis inhibitors (VEGF inhibitors, e.g., Avastin, VEGF-trap) (see, e.g., Li et al., ClinCancer Res. 2006 Nov 15;12(22):6808-16.), anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18 antagonists.

[0385] C. Antibiotics In one embodiment, the Siglec-15 binding molecule or Siglec-15 ligand binding molecule can be used in a preventative or prophylactic role in the treatment or prevention of diseases such as those discussed above, and can also be used in the setting of severe traumatic injuries such as large burns, open fractures, accidental amputations, or other wounds. Thus, the Siglec-15 binding molecule or Siglec-15 ligand binding molecule can be administered to a subject in combination with an antimicrobial agent, e.g., an antibiotic, an antifungal, an antiviral, an antiparasitic, or an essential oil.

[0386] In some embodiments, subjects are administered Siglec-15 binding molecules and / or antibiotics upon hospitalization to prevent further bacterial, fungal, or viral complications. The antibiotics target pathogens, and the Siglec-15 binding molecules or Siglec-15 ligand binding molecules can stimulate the immune system to produce an enhanced response to treat or prevent further infection or disease.

[0387] D. Immunosuppressive agents In some embodiments, an immune response or an inflammatory / autoimmune disease / disorder is treated by administering to a subject a Siglec-15 binding molecule or ligand binding molecule and a second agent that is an immunosuppressant. Immunosuppressive agents include antibodies against other lymphocyte surface markers (e.g., CD40, alpha-4 integrin) or cytokines), fusion proteins (e.g., CTLA-4-Ig (Orencia®), TNFR-Ig (Enbrel®)), TNF-α blockers such as Enbrel, Remicade, Cimzia, and Humira, cyclophosphamide (CTX) (i.e., Endoxan®, Cytoxan®, N, These include, but are not limited to, other immunosuppressive drugs (e.g., cyclosporine A, FK506-like compounds, rapamycin compounds, or steroids), antiproliferative agents, cytotoxic agents, or other compounds that may promote immunosuppression.

[0388] The therapeutic agent can be a CTLA-4 fusion protein, such as CTLA-4-Ig (abatacept). CTLA-4-Ig fusion proteins compete with the costimulatory receptor CD28 on T cells for binding to CD80 / CD86 (B7-1 / B7-2) on antigen-presenting cells, thereby functioning to inhibit T cell activation. In another embodiment, the therapeutic agent is the CTLA-4-Ig fusion protein known as belatacept. Belatacept contains two amino acid substitutions (L104E and A29Y) that significantly increase its binding activity to CD86 in vivo. In another aspect, the therapeutic agent is Maxy-4.

[0389] In another embodiment, the therapeutic agent is cyclophosphamide (CTX). Cyclophosphamide (generic name for Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), also known as cytophosphan, is a nitrogen mustard alkylating agent from the oxazofurin group. It is used to treat various types of cancer and some autoimmune disorders. Cyclophosphamide (CTX) is the primary drug used to treat diffuse proliferative glomerulonephritis in patients with lupus nephritis.

[0390] Therapeutic agents can be administered in an amount effective to result in a reduction in blood or serum levels of anti-double stranded DNA (anti-dsDNA) autoantibodies and / or a reduction in proteinuria in a patient in need thereof.

[0391] In another embodiment, the therapeutic agent increases the amount of adenosine in serum (see, e.g., WO08 / 147482). For example, the second therapeutic agent can be CD73-Ig, recombinant CD73, or other agent (e.g., cytokine, monoclonal antibody, or small molecule) that increases expression of CD73 (see, e.g., WO04 / 084933). In another embodiment, the therapeutic agent is interferon-beta.

[0392] The therapeutic agent can be Tysabri or another MS treatment. In another embodiment, the second therapeutic agent preferentially treats chronic inflammation, thereby targeting both acute and chronic inflammation. In a preferred embodiment, the second therapeutic agent is a TNF-α blocker.

[0393] A therapeutic agent can be a small molecule that inhibits or reduces the differentiation, proliferation, activity, and / or cytokine production and / or secretion by Th1, Th17, Th22, and / or other cells that secrete or cause other cells to secrete inflammatory molecules (including, but not limited to, IL-1β, TNF-α, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs). In another embodiment, a therapeutic agent is a small molecule that interacts with Tregs, enhances the activity of Tregs, promotes or enhances the secretion of IL-10 by Tregs, increases the number of Tregs, increases the suppressive capacity of Tregs, or a combination thereof.

[0394] In some embodiments, the composition increases the activity or production of Tregs. Exemplary Treg enhancers include, but are not limited to, the glucocorticoids fluticasone, salmeterol; antibodies to IL-12, IFN-γ, and IL-4, vitamin D3, and dexamethasone, and combinations thereof.

[0395] In some embodiments, the therapeutic agent is an antibody, for example, a function-blocking antibody against a pro-inflammatory molecule such as IL-6, IL-23, IL-22, or IL-21.

[0396] As used herein, the term "rapamycin compounds" includes the neutral tricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs, and other macrolide compounds that appear to have the same mechanism of action as rapamycin (e.g., inhibition of cytokine function). The term "rapamycin compounds" also includes compounds that have structural similarity to rapamycin, e.g., compounds with a similar macrocyclic structure that have been modified to enhance their therapeutic efficacy. Representative rapamycin compounds are known in the art (see, e.g., WO95122972, WO95116691, WO95104738, U.S. Patent Nos. 6,015,809; 5,989,591; 5,567,709; 5,559,112; 5,530,006; 5,484,790; 5,385,908; 5,202,332; 5,162,333; 5,780,462; 5,120,727).

[0397] The term "FK506-like compounds" includes FK506 as well as FK506 derivatives and analogs, e.g., compounds having structural similarity to FK506, e.g., compounds having a similar macrocyclic structure, which have been modified to enhance their therapeutic efficacy. Examples of FK506-like compounds include, for example, those described in WO00101385. Preferably, the term "rapamycin compounds," as used herein, does not include FK506-like compounds.

[0398] E. Anti-inflammatory drugs Other suitable therapeutic agents include, but are not limited to, anti-inflammatory agents. The anti-inflammatory agent may be non-steroidal, steroidal, or a combination thereof. One embodiment provides an oral composition containing about 1% (w / w) to about 5% (w / w), typically about 2.5% (w / w), of an anti-inflammatory agent. Representative examples of nonsteroidal anti-inflammatory agents include, but are not limited to, oxicams such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates such as aspirin, disalcid, benorylate, trilisate, safapryn, solprin, diflunisal, and fendosal; acetic acid derivatives such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acemetacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, and Ketorolac; fenamic acids such as mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid, and tolfenamic acid; propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, and tiaprofenic acid; pyrazoles such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethasone. Mixtures of these nonsteroidal anti-inflammatory drugs may also be used.

[0399] Representative examples of steroidal anti-inflammatory agents include, but are not limited to, corticosteroids such as hydrocortisone, hydroxyltriamcinolone, alpha-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, desoximetasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluchlorolone acetonide, fludrocortisone, flumethasone pivalate, fluocinolone acetonide, fluocinonide, fluocorticolone aceton ... butyl ester, fluocortolone, fluprednidene acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolon, fludrocortisone, diflurosone diacetate, fluradrenolon acetonide, medrysone, amcinafide, amcinafide, betamethasone and the remainder of its esters, chloroprednisone, chlorprednisone acetate acetate), clocortolone, clesinolone, dichlorisone, difluprednate, fluclonide, flunisolide, fluorometholone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof.

[0400] V. Diagnostic Methods Siglec-15 binding molecules, particularly antibodies and antigen-binding fragments thereof, can be used for diagnostic purposes, e.g., to detect, diagnose, or monitor diseases, disorders, or infections associated with Siglec-15 expression, or to determine or assist in the determination or identification of appropriate patient populations or profiles, any of which can be coupled with a method of treating a subject, e.g., by administering to the subject an effective amount of one or more therapeutic Siglec-15 binding molecules.

[0401] Detection or diagnosis of a disease, disorder, or infection, including but not limited to cancer, can include (a) assaying the expression of Siglec-15 or a derivative thereof in a subject's cells, serum, plasma, blood, or tissue sample (e.g., a tumor sample) using one or more antibodies (or fragments thereof) that immunospecifically bind to such antigen; and (b) comparing the level of the antigen to a control level, e.g., the level in a normal tissue sample, whereby an increase in the assay level of the antigen compared to the control level of the antigen is indicative of the disease, disorder, or infection. Such antibodies and fragments can be used in immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence-activated cell sorting (FACS).

[0402] In some embodiments, the antibodies or fragments are used in IHC analysis in vitro or in cells of in situ tissue samples or in vivo. Thus, the antibodies and fragments can be used for the detection and diagnosis of diseases, disorders, or infections in humans. In one embodiment, such diagnosis involves: a) administering an effective amount of such labeled antibody or antigen-binding fragment to a subject (e.g., parenterally, subcutaneously, or intraperitoneally); b) allowing a time interval after administration to allow the labeled molecule to preferentially concentrate at sites of Siglec-15 expression in the subject (and allow unbound labeled molecule to clear to background levels); c) determining the background level; and d) detecting the labeled antibody in the subject, such that local detection of the labeled antibody above or below the background level indicates that the subject has a disease, disorder, or infection and / or indicates the location and relative expression level of Siglec-15+ tissue. According to this embodiment, the antibody can be labeled with an imaging moiety that can be detected in vivo using imaging systems known to those skilled in the art. Background levels can be determined by a variety of methods, including comparing the amount of labeled molecule detected to a predetermined standard value for a particular system.

[0403] Other methods include, for example, (a) assaying the expression of Siglec-15 in cells or tissue samples from a subject obtained at an initial time point and at a subsequent time point using a Siglec-15 binding molecule, and (b) monitoring the progression of the disease, disorder, or infection by comparing the expression levels of Siglec-15 in cells or tissue samples from the subject at the initial time point and at the subsequent time point, wherein an increase in the level of Siglec-15 assayed at the subsequent time point compared to the initial time point is indicative of the progression of the disease, disorder, or infection.

[0404] A method for monitoring response to treatment can include (a) assaying expression of Siglec-15 in cells or tissue samples of a subject before and after treatment using a Siglec-15 binding molecule; and (b) comparing the level of Siglec-15 over time, whereby a decrease in the assayed level of Siglec-15 after treatment compared to the level of Siglec-15 before treatment is indicative of a favorable response to treatment.

[0405] It will be understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image.

[0406] Depending on several variables, including the type of label used and the mode of administration, the time interval after administration during which the labeled molecule can be preferentially concentrated at a site within the subject and unbound labeled molecule can be cleared to background levels is 6 to 48 hours, or 6 to 24 hours, or 6 to 12 hours, hi another embodiment, the time interval after administration is 5 to 20 days, or 5 to 10 days.

[0407] In one embodiment, monitoring the disease, disorder, or infection is performed by repeating the method for diagnosing the disease, disorder, or infection, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

[0408] The presence of labeled molecule can be detected in a subject by using methods known in the art for in vivo scanning.These methods depend on the type of label used.Those skilled in the art will be able to determine the appropriate method for detecting specific label.Methods and devices that can be used for diagnostic methods include, but are not limited to, computed tomography (CT), whole body scanning (e.g., positron emission tomography (PET)), magnetic resonance imaging (MRI) and ultrasound.

[0409] In certain embodiments, the molecule is labeled with a radioisotope and is detected in the patient using a surgical instrument that is responsive to radiation (Thurston et al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a scanning instrument that is responsive to fluorescence. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patient using positron emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in the patient using magnetic resonance imaging (MRI).

[0410] VI. Kit The disclosed Siglec-15 binding molecules or Siglec-15 ligand binding molecules can be packaged in a sealed container, such as an ampoule or sachet, indicating the amount. The molecules can be supplied in the sealed container as a dry, sterile, lyophilized powder or anhydrous concentrate, which can be reconstituted, for example, with water or saline, to the appropriate concentration for administration to a subject. For example, the molecules can be supplied as a dry, sterile, lyophilized powder in a sealed container in a unit dosage of at least 5 mg, or at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized molecules can be stored in their original container at 2-8°C and are typically administered within 12 hours, or within 6 hours, or within 5 hours, or within 3 hours, or within 1 hour after reconstitution.

[0411] In alternative embodiments, the molecule is provided in liquid form in a sealed container indicating the quantity and concentration, hi some embodiments, the molecule in liquid form is provided in a sealed container containing at least 1 mg / ml, or at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 100 mg / ml, at least 150 mg / ml, or at least 200 mg / ml of the molecule.

[0412] Pharmaceutical packs and kits comprising one or more containers filled with Siglec-15 binding molecules or Siglec-15 ligand-binding molecules are also provided. In addition, the pharmaceutical pack or kit can also comprise one or more other prophylactic or therapeutic agents useful for treating disease. The pharmaceutical pack or kit can also comprise one or more containers filled with one or more of the ingredients of the disclosed pharmaceutical compositions. Optionally, such container(s) can be accompanied by a notice, in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, reflecting approval by the governmental agency for manufacture, use, or sale for human administration.

[0413] Kits designed for the above methods are also provided. Embodiments typically include one or more Siglec-15 binding molecules or Siglec-15 ligand-binding molecules. In certain embodiments, the kits also include, in one or more containers, one or more other prophylactic or therapeutic agents useful for treating cancer. [Example]

[0414] Example 1: Siglec-15 antibodies and their heavy and light chain sequences Materials and Methods Mouse anti-human Siglec-15 monoclonal antibody Siglec-15 knockout mice (n = 2) were immunized with hS15.mIg (human Siglec-15 extracellular domain [ECD] fused to mouse IgG2a) emulsified in CFA (Freund's complete adjuvant). Mice were also injected with GM-CSF and anti-CD40. Mice were challenged with the same immunogen two weeks later. Antiserum titers were assessed by testing serum collected from tail bleeds at various dilutions, from 1:1000 to a maximum of 1:100,000,000, in ELISA plates coated with hS15.hIg (human Siglec-15 ECD fused to human IgG1). Figure 1 shows that anti-hS15 antibodies were detected at dilutions >1:100,000. Mice received a third challenge two weeks later. Three days after the final boost, mouse splenocytes were harvested, resuspended in RPMI supplemented with 10% FBS and glutamine, and then fused to form hybridomas.

[0415] Electrofusion of Siglec-15 knockout (S15 KO) splenocytes The fused cells were plated in methylcellulose gel / medium. The remaining fused cells were cryopreserved and could be thawed for another round of cloning. Single clones were selected and placed in 10 x 96-well plates (960 clones). Supernatants were collected after 2 weeks.

[0416] RACE RACE (rapid amplification of cDNA ends) identification of heavy and light chains was performed according to the following protocol: (1) mRNA denaturation, (2) cDNA synthesis, (3) 5' RACE reaction, (4) PCR analysis (on an agarose gel to visualize the amplified DNA fragments - the correct antibody variable region DNA fragments should be 500-700 base pairs in size), (5) TOPO cloning of PCR-positive bands, (6) PCR amplification of TOPO clones followed by gel electrophoresis and recovery from agarose gel, (7) sequencing of a total of 218 clones, and (8) CDR analysis using the sequencing data (CDR regions were defined using VBASE2, available at vbase2.org).

[0417] result The antibodies were cloned using the RACE method. After sequencing 218 cloned DNA fragments, antibody sequence analysis identified one heavy chain and one light chain in 24 antibody samples, designated herein as 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, 80A, 82B, 83B, 92A, 93B, 99B, 104B, and 105A. These sequences are shown below or above. The sequences and CDRs of the heavy and light chains are shown above, below, and in Figures 2A-3C.

[0418] 1B2 sequence: 1B2 VL amino acid sequence (FASTA format) TIFF0007720375000112.tif191351B2 VL nucleotide sequence (FASTA format) TIFF0007720375000113.tif481351B2 VH amino acid sequence (FASTA format) TIFF0007720375000114.tif191351B2 VH nucleotide sequence (FASTA format) TIFF0007720375000115.tif48135

[0419] 1C3 sequence: 1C3 VL amino acid sequence (FASTA format) TIFF0007720375000116.tif191351C3 VL nucleotide sequence (FASTA format) TIFF0007720375000117.tif481351C3 VH amino acid sequence (FASTA format) TIFF0007720375000118.tif191351C3 VH nucleotide sequence (FASTA format) TIFF0007720375000119.tif48135

[0420] 1H3 sequence 1H3 VL amino acid sequence (FASTA format) TIFF0007720375000120.tif191351H3 VL nucleotide sequence (FASTA format) TIFF0007720375000121.tif471351H3 VH amino acid sequence (FASTA format) TIFF0007720375000122.tif191351H3 VH nucleotide sequence (FASTA format) TIFF0007720375000123.tif55135

[0421] 1C12 sequence: 1C12 VL amino acid sequence (FASTA format) TIFF0007720375000124.tif191351C12 VL nucleotide sequence (FASTA format) TIFF0007720375000125.tif471351C12 VH amino acid sequence (FASTA format) TIFF0007720375000126.tif191351C12 VH nucleotide sequence (FASTA format) TIFF0007720375000127.tif48135

[0422] 3H10 sequence: 3H10 VL amino acid sequence (FASTA format) TIFF0007720375000128.tif191353H10 VL nucleotide sequence (FASTA format) TIFF0007720375000129.tif471353H10 VH amino acid sequence (FASTA format) TIFF0007720375000130.tif191353H10 VH nucleotide sequence (FASTA format) TIFF0007720375000131.tif55135

[0423] 5G12 array: 5G12 VL amino acid sequence (FASTA format) TIFF0007720375000132.tif191355G12 VL nucleotide sequence (FASTA format) TIFF0007720375000133.tif471355G12 VH amino acid sequence (FASTA format) TIFF0007720375000134.tif191355G12 VH nucleotide sequence (FASTA format) TIFF0007720375000135.tif55135

[0424] 6F8 sequence: 6F8 VL amino acid sequence (FASTA format) TIFF0007720375000136.tif191356F8 VL nucleotide sequence (FASTA format) TIFF0007720375000137.tif481356F8 VH amino acid sequence (FASTA format) TIFF0007720375000138.tif191356F8 VH nucleotide sequence (FASTA format) TIFF0007720375000139.tif48135

[0425] 8C8 sequence: 8C8 VL amino acid sequence (FASTA format) TIFF0007720375000140.tif191358C8 VL nucleotide sequence (FASTA format) TIFF0007720375000141.tif481358C8 VH amino acid sequence (FASTA format) TIFF0007720375000142.tif191358C8 VH nucleotide sequence (FASTA format) TIFF0007720375000143.tif48135

[0426] 8H8 sequence: 8H8 VL amino acid sequence (FASTA format) TIFF0007720375000144.tif191358H8 VL nucleotide sequence (FASTA format) TIFF0007720375000145.tif481358H8 VH amino acid sequence (FASTA format) TIFF0007720375000146.tif191358H8 VH nucleotide sequence (FASTA format) TIFF0007720375000147.tif48135

[0427] 9A5 sequence: 9A5 VL amino acid sequence (FASTA format) TIFF0007720375000148.tif191359A5 VL nucleotide sequence (FASTA format) TIFF0007720375000149.tif471359A5 VH amino acid sequence (FASTA format) TIFF0007720375000150.tif191359A5 VH nucleotide sequence (FASTA format) TIFF0007720375000151.tif47135

[0428] 10G9 array: 10G9 VL amino acid sequence (FASTA format) TIFF0007720375000152.tif1913510G9 VL nucleotide sequence (FASTA format) TIFF0007720375000153.tif4813510G9 VH amino acid sequence (FASTA format) TIFF0007720375000154.tif1913510G9 VH nucleotide sequence (FASTA format) TIFF0007720375000155.tif47135

[0429] 6A sequence: 6A VL amino acid sequence (FASTA format) TIFF0007720375000156.tif191356A VL nucleotide sequence (FASTA format) TIFF0007720375000157.tif481356A VH amino acid sequence (FASTA format) TIFF0007720375000158.tif191356A VH nucleotide sequence (FASTA format) TIFF0007720375000159.tif48135

[0430] 28A sequence: 28A VL amino acid sequence (FASTA format) TIFF0007720375000160.tif1913528A VL nucleotide sequence (FASTA format) TIFF0007720375000161.tif4813528A VH amino acid sequence (FASTA format) TIFF0007720375000162.tif1913528A VH nucleotide sequence (FASTA format) TIFF0007720375000163.tif48135

[0431] 63A sequence: 63A VL amino acid sequence (FASTA format) TIFF0007720375000164.tif1913563A VL nucleotide sequence (FASTA format) TIFF0007720375000165.tif4713563A VH amino acid sequence (FASTA format) TIFF0007720375000166.tif1913563A VH nucleotide sequence (FASTA format) TIFF0007720375000167.tif48135

[0432] 71A sequence: 71A VL amino acid sequence (FASTA format) TIFF0007720375000168.tif1913571A VL nucleotide sequence (FASTA format) TIFF0007720375000169.tif4813571A VH amino acid sequence (FASTA format) TIFF0007720375000170.tif1913571A VH nucleotide sequence (FASTA format) TIFF0007720375000171.tif48135

[0433] 77A sequence: 77A VL amino acid sequence (FASTA format) TIFF0007720375000172.tif1913577A VL nucleotide sequence (FASTA format) TIFF0007720375000173.tif4813577A VH amino acid sequence (FASTA format) TIFF0007720375000174.tif1913577A VH nucleotide sequence (FASTA format) TIFF0007720375000175.tif48135

[0434] 80A Array: 80A VL amino acid sequence (FASTA format) TIFF0007720375000176.tif1913580A VL nucleotide sequence (FASTA format) TIFF0007720375000177.tif4713580A VH amino acid sequence (FASTA format) TIFF0007720375000178.tif1913580A VH nucleotide sequence (FASTA format) TIFF0007720375000179.tif47135

[0435] 82B sequence: 82B VL amino acid sequence (FASTA format) TIFF0007720375000180.tif1913582B VL nucleotide sequence (FASTA format) TIFF0007720375000181.tif4813582B VH amino acid sequence (FASTA format) TIFF0007720375000182.tif1913582B VH nucleotide sequence (FASTA format) TIFF0007720375000183.tif47135

[0436] 83B sequence: 83B VL amino acid sequence (FASTA format) TIFF0007720375000184.tif1913583B VL nucleotide sequence (FASTA format) TIFF0007720375000185.tif4813583B VH amino acid sequence (FASTA format) TIFF0007720375000186.tif1913582B VH nucleotide sequence (FASTA format) TIFF0007720375000187.tif48135

[0437] 92A sequence: 92A VL amino acid sequence (FASTA format) TIFF0007720375000188.tif1913592A VL nucleotide sequence (FASTA format) TIFF0007720375000189.tif4713792A VH amino acid sequence (FASTA format) TIFF0007720375000190.tif1913592A VH nucleotide sequence (FASTA format) TIFF0007720375000191.tif55135

[0438] 93B sequence: 93B VL amino acid sequence (FASTA format) TIFF0007720375000192.tif1913593B VL nucleotide sequence (FASTA format) TIFF0007720375000193.tif4813593B VH amino acid sequence (FASTA format) TIFF0007720375000194.tif1913593B VH nucleotide sequence (FASTA format) TIFF0007720375000195.tif48135

[0439] 99B sequence: 99B VL amino acid sequence (FASTA format) TIFF0007720375000196.tif1913599B VL nucleotide sequence (FASTA format) TIFF0007720375000197.tif4813599B VH amino acid sequence (FASTA format) TIFF0007720375000198.tif1913599B VH nucleotide sequence (FASTA format) TIFF0007720375000199.tif48135

[0440] 104B sequence: 104B VL amino acid sequence (FASTA format) TIFF0007720375000200.tif19135104B VL nucleotide sequence (FASTA format) TIFF0007720375000201.tif48135104B VH amino acid sequence (FASTA format) TIFF0007720375000202.tif19135104B VH nucleotide sequence (FASTA format) TIFF0007720375000203.tif48135

[0441] 105A Array: 105A VL amino acid sequence (FASTA format) TIFF0007720375000204.tif19135105A VL nucleotide sequence (FASTA format) TIFF0007720375000205.tif47135105A VH amino acid sequence (FASTA format) TIFF0007720375000206.tif19135105A VH nucleotide sequence (FASTA format) TIFF0007720375000207.tif47135

[0442] Example 2: Anti-huS15 antibodies bind to cells expressing human S15 or mouse S15 Materials and Methods Human 293T and mouse CRC MC38 tumor cell lines were transduced with lentiviral vectors carrying human Siglec-15 or mouse Siglec-15. Cells were sorted to establish human S15 and mouse S15 stable cell lines. 293T.hS15 and MC38.mS15 stable cells were resuspended in FACS buffer, and Fc receptors were blocked with purified Siglec-15 mAb before incubation. 1E05 cells in 100 μL of FACS buffer were aliquoted into separate tubes, and 1 μg of purified mAb was added. The cells were incubated at 4°C for 30 minutes and then washed twice with excess FACS buffer. The cells were resuspended in 100 μL of FACS buffer, and 0.005 μg of anti-mouse IgG-PE secondary antibody was added to the sample, incubated for 30 minutes, and washed twice with excess FACS buffer. The cells were fixed in fixation buffer and then analyzed by flow cytometry.

[0443] The assay is shown in Figure 4A.

[0444] result Anti-huS15 antibodies, including 1B2, 1C3, 1C12, 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9 (Figure 4B), as well as 6A (NC6), 28A (NC28), 63A (NC63), 71A (NC71), NC74, 77A (NC77), 80A (NC80), 82B (NC82), 83B (NC83), NC87, 92A (NC92), 93B (NC93), 99B (NC99), 104B (NC104), and 105A (NC105) (Figure 4C), were tested for binding to cells expressing human S15 or mouse S15. The results are shown in Figures 4B-4C.

[0445] Anti-huS15 antibodies, including 1B2, 1C3, 1C12, 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, and 6A (NC6), 28A (NC28), 63A (NC63), 77A (NC77), 80A (NC80), 82B (NC82), 83B (NC83), 92A (NC92), 93B (NC93), 99B (NC99), 104B (NC104), and 105A (NC105), were also tested for binding to formalin-fixed cells. The results are shown in Figure 4D.

[0446] Example 3: Purified antibodies bind to mouse and human Siglec-15 Materials and Methods 293T cells transiently transfected with S15-TM (Operetta) K562 cells transiently transfected with S15-TM (FACS) 293T cells were transiently transfected with mouse Siglec-15 plasmid DNA using the Lipofectamine system, and K562 cells were transfected with human Siglec-15 plasmid DNA by electroporation. 1e5 transfected cells in 100µl of FACS buffer (PBS containing 0.5% serum) were sorted into separate tubes, and 1µg of purified mAb was added. The cells were incubated at 4°C for 30 minutes and then washed twice with excess FACS buffer. The cells were resuspended in 100µl of FACS buffer, and 0.005µg of anti-mouse IgG-PE secondary antibody was added to the sample, incubated for 30 minutes, and washed twice with excess FACS buffer. The cells were fixed in fixation buffer (2% formaldehyde in PBS) and then analyzed by flow cytometry.

[0447] U87 cells were resuspended in FACS buffer, and Fc receptors were blocked with purified Siglec-15 mAb before incubation. 1e5 cells in 100µl of FACS buffer were sorted into separate tubes, and 1µg of purified mAb was added. The cells were incubated at 4°C for 30 minutes and then washed twice with excess FACS buffer. The cells were resuspended in 100µl of FACS buffer, and 0.005µg of anti-mouse IgG-PE secondary antibody was added to the sample, incubated for 30 minutes, and washed twice with excess FACS buffer. The cells were fixed in fixation buffer and then analyzed by flow cytometry.

[0448] result Purified antibodies 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9 were tested for binding to mouse Siglec-15 (mS15) expressed by 293T cells (a highly transfectable derivative of human embryonic kidney 293 cells) and human Siglec-15 (hS15) expressed by K-562 cells (pleural effusion of a 53-year-old woman with chronic myeloid leukemia in terminal blast crisis). The results are shown in Figures 5A-5C.

[0449] Purified antibodies 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, and 5F10 were also tested for binding to human U87 glioma cells, which endogenously express human Siglec-15 (hS15). The results are shown in Figures 6A-6C, which show the percentage of hS15+ cells (Figures 6A and 6C) and mean fluorescence intensity (MFI) (Figure 6B) for each antibody, respectively.

[0450] Example 4: Anti-Siglec-15 antibodies can block the function of Siglec-15 Materials and Methods Production of hS15.hG1 Monomeric S15 fusion proteins containing the complete S15 extracellular domain fused to an IgG backbone were prepared for affinity and competition analysis. The thrombin-cleavable hS15.hFc cDNA was subcloned into pEE17.4. At 37°C, fragmented fusion proteins were observed in transiently transfected CHO cells but not in 293 cells. The intact fusion protein was more efficiently expressed at 31°C. The fusion protein has the following sequence: TIFF0007720375000208.tif69136

[0451] The mature fusion protein with the signal sequence cleaved is as follows: TIFF0007720375000209.tif70136

[0452] The mouse leader sequence is underlined. The Siglec-15 extracellular domain (ECD) is italicized. The hinge region is double underlined. The remaining sequence is derived from IgG1 Fc. The L234F / L235E / P331S mutations in the IgG1 Fc domain are in bold with dotted underlining.

[0453] Blocking analysis 293T.LRRC4C cells were established by two rounds of lenti-LRRC4C transduction.

[0454] 293T.LRRC4C stable cells were resuspended in FACS buffer and Fc receptors were blocked before incubation. 1E05 cells in 100 μL of FACS buffer were aliquoted into separate tubes, and 2 μg of purified mAb was added first, followed by 0.2 μg of hSiglec-15hFc. The cells were incubated at 4°C for 30 minutes and then washed twice with excess FACS buffer. The cells were resuspended in 100 μL of FACS buffer, and 0.005 μg of anti-human IgG-PE secondary antibody was added to the sample, incubated for 30 minutes, and washed twice with excess FACS buffer. The cells were fixed with fixation buffer and then analyzed by flow cytometry to determine binding of Siglec-15 to LRRC4C.

[0455] 1 μg / mL hS15.hG1 + 100 μL or 33 μg / mL Cl#3, #1 supernatant (sup)

[0456] This assay is shown in Figure 7A.

[0457] result The results are shown in Figures 7B to 7E. Complete blockers of the S15 / LRRC4C interaction include (but are not limited to): o 5G12, 6F8, 8C8, 1C3, 1C12, 3H10, 1B2 Partial blockers include (but are not limited to): 10G9, 8H8, 9A5

[0458] Example 5: S15 mAb reverses hS15.hG1-mediated suppression of human T cells Materials and Methods PBMC proliferation assay Coating with anti-CD3 0.05μg / mL Total human PBMC + / - 5 μg / mL hS15.hFc and 12 μg / mL Siglec-15 mAb, or controls as indicated Anti-human CD3 (clone OKT3) was coated overnight at 4°C at 0.03 μg / mL per well (in 100 μL of PBS) in 96-well flat-bottom tissue culture plates. PBS and unbound CD3 were aspirated immediately before adding assay components. Whole PBMCs from healthy human donors were labeled with 5 μM CFSE in RPMI-complete medium (containing 10% FBS) at 37°C for 10 minutes, washed twice, and resuspended for addition to wells at a concentration of 2.5E05 cells / well. Purified Siglec-15 mAb or control was added to wells at a final concentration of 12 μg / ml, while soluble hSiglec-15hFc or control was added to wells at a final concentration of 5 μg / ml. Plates were incubated in a CO2 incubator at 37°C for 72 hours. After removing a small amount of supernatant for cytokine analysis, the cells were transferred to round-bottom plates, Fc receptors were blocked, and the cells were stained with CD4 and CD8 fluorescent mAbs for 30 minutes at 4°C. The cells were washed twice in FACS buffer and then fixed for analysis by flow cytometry. IFN-gamma levels in the conditioned supernatants were assessed by MSD ELISA kit.

[0459] This assay is shown in Figures 8A and 9A.

[0460] result Purified antibodies 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9 (Figures 8B, 8C, 8D, and 8E), as well as 6A (NC6), 28A (NC28), 63A (NC63), 77A (NC77), 80A (NC80), 82B (NC82), 83B (NC83), 92A (NC92), 93B (NC93), 99B (NC99), 104B (NC104), and 105A (NC105) (Figures 8F and 8G) were tested in PBMC proliferation assays. CD4+ and CD8+ T cells were gated and analyzed for CFSE dilution as a measure of cell division (proliferation). Increasing dilution indicated increased cell proliferation. The results indicate that specific S15 mAbs can reverse hS15.hG1-mediated suppression of human T cells.

[0461] However, blocking the interaction of hS15 with LRRC4C did not correlate with enhanced T cell function, the results of which are shown in Figures 8H and 8I.

[0462] S15 mAb was confirmed to reverse the hS15.hG1-mediated decrease in IFNγ production in human T cells, and the results are shown in Figure 9B.

[0463] Example 6: S15 mAb can block osteoclast formation Materials and Methods Fresh PBMCs were isolated and monocytes were enriched in two ways: by MACS column sorting (FIG. 10, left panel); or by binding to plastic in serum-free medium (FIG. 10, right panel).

[0464] Cells were cultured with M-CSF and RANKL for 8 days with 25E9 or S15 mAb and assayed for TRAP (tartrate-resistant acid phosphatase).

[0465] result The results shown in Figure 15 demonstrate that S15 mAb can block osteoclast formation.

[0466] Example 7: Humanized 5G12 antibody Clone 5G12 was humanized, resulting in three humanized heavy chains and five humanized light chains (Figures 12A and 12B). 5G12 hVLl TIFF0007720375000210.tif191375G12 CDR1 of hVL1 TIFF0007720375000211.tif41285G12 CDR2 of hVL1 RANRLVD (SEQ ID NO: 36) CDR3 of 5G12 hVL1 LQYDEFPYT (SEQ ID NO: 43) 5G12 hVL2 TIFF0007720375000212.tif191375G12 CDR1 of hVL2 TIFF0007720375000213.tif41285G12 CDR2 of hVL2 RANRLVD (SEQ ID NO: 36) CDR3 of 5G12 hVL2 LQYDEFPYT (SEQ ID NO: 43) 5G12 hVL3 TIFF0007720375000214.tif191375G12 CDR1 of hVL3 TIFF0007720375000215.tif41285G12 CDR2 of hVL3 RANRLVD (SEQ ID NO: 36) CDR3 of 5G12 hVL3 LQYDEFPYT (SEQ ID NO: 43) 5G12 hVL4 TIFF0007720375000216.tif191375G12 CDR1 of hVL4 TIFF0007720375000217.tif41285G12 CDR2 of hVL4 RANRLVD (SEQ ID NO: 36) CDR3 of 5G12 hVL4 LQYDEFPYT (SEQ ID NO: 43) 5G12 hVL5 TIFF0007720375000218.tif191375G12 CDR1 of hVL5 TIFF0007720375000219.tif41285G12 CDR2 of hVL5 RANRLTS (SEQ ID NO: 202) CDR3 of 5G12 hVL5 LQYDEFPYT (SEQ ID NO: 43) 5G12 hVH1 TIFF0007720375000220.tif191365G12 CDR1 of hVH1 SYWIT (SEQ ID NO: 204) CDR2 of 5G12 hVH1 TIFF0007720375000221.tif41285G12 CDR3 of hVH1 TIFF0007720375000222.tif51285G12 hVH2 TIFF0007720375000223.tif19137hVH2 CDR1 SYWIT (SEQ ID NO: 204) CDR2 of hVH2 TIFF0007720375000224.tif4128hCDR3 of VH2 TIFF0007720375000225.tif41285G12 hVH3 TIFF0007720375000226.tif191375G12 CDR1 of hVH3 SYWIS (SEQ ID NO: 208) CDR2 of 5G12 hVH3 TIFF0007720375000227.tif41285G12 CDR3 of hVH3 TIFF0007720375000228.tif4128

[0467] Example 8: Membrane-bound S15 is immunosuppressive Materials and Methods Method for assaying T cell suppression of PMBCs in vitro: Peripheral blood mononuclear cells (PBMCs) were collected from apheresis-derived polyclonal leukocyte products (KeyBiologics, Memphis, TN) of healthy donors by standard Ficoll gradient procedures and then cryopreserved. On the day of the assay, frozen PBMCs were thawed, washed in RPMI complete medium (RPMI-C, RPMI [ThermoFisher] + 10% FetalClone III serum [HyClone]), and counted. Cells were labeled with 5 μM CFSE (ThermoFisher) in RPMI-C for 10 minutes at 37°C and then washed twice with RPMI-C. Total PBMCs (3E05 cells / well) were plated into 96-well flat-bottom Corning Costar plates pre-coated overnight at 4°C with anti-human CD3 (OKT3, 50 ng / mL; eBioScience). Siglec-15 hG1 Fc fusion protein was added to the wells at the final concentrations indicated. Cells were cultured at 37°C for 72 hours. At 72 hours, 50 μL of supernatant was removed from each well and immediately frozen for analysis of cytokine levels. Cells were then pipetted from each well and transferred to round-bottom plates for flow cytometry staining and analysis. Fc receptors were blocked with TruStain FcX (2 μL / well; Biolegend) and subsequently stained with antibodies against CD4 (APC-eFluor 780 (2 μL / well; ThermoFisher)) and CD8 (eFluor 450 (2 μL / well; ThermoFisher)) for 1 hour at 4°C. After incubation, plates were washed twice with FACS buffer (PBS containing 1% FetalClone III serum). Cells were resuspended in 150 μL of fixation buffer (3% formaldehyde in PBS) and analyzed on a YETI flow cytometer (Propel Labs). Data were analyzed using FlowJo. Data are based on CFSE dilution of divided cells and unstimulated CD4 + and CD8 +The results are expressed as percentages compared to T cells. Supernatants were analyzed for IFN-γ, TNF-α, and IL-6 using the U-PLEX kit (Meso Scale Diagnostics [MSD]) according to the manufacturer's instructions. U-PLEX was read using a Meso QuickPlex SQ 120 instrument.

[0468] result Membrane-bound S15 is immunosuppressive. Figure 14A is a line graph of T cell proliferation (%) in response to human S15 Fc (μg / mL), showing that T cell proliferation (%) decreases with increasing concentrations of S15 Fc. Human PBMA were labeled with CFSE, added to anti-CD3 (OKT3)-coated 96-well plates, and cultured with the indicated concentrations of human S15 Fc fusion protein for 3 days. Figure 14B is a bar graph of IFN-γ (pg / mL) in conditioned supernatants from cells treated with 0 or 5 μg / mL of S15 Fc. Figure 14C is a bar graph of TNF-α (pg / mL) in conditioned supernatants from cells treated with 0 or 5 μg / mL of S15 Fc. Figure 14D is a bar graph of IL-6 (pg / mL) in conditioned supernatants from cells treated with 0 or 5 μg / mL of S15 Fc.

[0469] Example 9: Binding of s15 mAb purified from hybridomas to cells expressing human S15 or mouse S15 Materials and Methods 293T cells stably expressing human S15 (293T.hS15) and mouse MC38 cells stably expressing mouse S15 (MC38.mS15) were harvested from tissue culture. After washing once with PBS, cells were seeded into 96-well U-bottom plates (5E04 cells / well) for staining. Cells were first mixed with 1 μg of the indicated antibody purified from mouse hybridomas in FACS staining buffer (PBS containing 1% FetalClone III serum) and incubated on ice for 30 minutes. Cells were then washed once with FACS buffer and subsequently incubated wi...

Claims

1. three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable region (HCVR) sequence comprising the amino acid sequence of SEQ ID NO: 16, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in a light chain variable region (LCVR) sequence comprising the amino acid sequence of SEQ ID NO: 3; and (a) an HCDR1 having the amino acid sequence of SEQ ID NO: 49; (b) an HCDR2 having the amino acid sequence of SEQ ID NO: 59; (c) an HCDR3 having the amino acid sequence of SEQ ID NO: 67; (d) an LCDR1 having the amino acid sequence of SEQ ID NO: 24; (e) an LCDR2 having the amino acid sequence of SEQ ID NO: 32; and (f) an LCDR3 having the amino acid sequence of SEQ ID NO:

39. A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to Siglec-15.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 16 and an LCVR comprising the amino acid sequence of SEQ ID NO:

3.

3. 3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising an HCVR encoded by the nucleic acid sequence of SEQ ID NO: 88 and an LCVR encoded by the nucleic acid sequence of SEQ ID NO:

77.

4. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 3 in combination with an excipient.

5. A pharmaceutical composition for treating a tumor in a subject in need thereof, comprising the monoclonal antibody of any one of claims 1 to 3, wherein the pharmaceutical composition reduces the tumor burden in the subject.

6. The pharmaceutical composition of claim 5 , wherein the tumor is a colon tumor, a lymphoma tumor, or an ovarian tumor.

7. A pharmaceutical composition for promoting an immune response in a subject in need thereof, comprising the monoclonal antibody of any one of claims 1 to 3.

8. 8. The pharmaceutical composition of claim 7, wherein the enhanced immune response delays or prevents tumor growth, inhibits tumor-mediated immunosuppression, eliminates tumors, depletes or blocks the activity of tumor-associated macrophages (TAMs), reduces TAM-mediated immunosuppression, reduces or reverses T-cell suppression, increases T-cell proliferation, or a combination thereof.

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