Monoclonal antibody or its antigen-binding fragment, pharmaceutical composition, and use of a monoclonal antibody.

Abstract

Siglec-15 binding molecules are provided. These molecules are typically an antibody or its antigen-binding fragment that binds immunospecifically to siglec-15. Siglec-15 ligand-binding molecules are also provided. These molecules are typically a polypeptide or siglec-15 fusion protein. Methods for using these molecules to reduce siglec-15-mediated immunosuppression in a subject in need are also provided.

Description

Monoclonal antibody or its antigen-binding fragment, pharmaceutical composition, and use of a monoclonal antibody. CROSS-REFERENCE TO RELATED REQUESTS

[001] This application claims benefit and priority to U.S. Provisional Patent Applications 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 permitted. FIELD OF THE INVENTION

[002] The invention is generally related to the field of immunomodulation and, more particularly, to compositions and methods for modulating Siglec-15 and signaling initiated therefrom. FUNDAMENTALS OF THE INVENTION

[003] Sialic acid-binding Ig-like lectins (“Siglecs”) are members of the Ig superfamily. These type 1 transmembrane proteins include an N-terminal sialic acid-binding V-assembly domain, variable numbers of C2-assembly Ig domains, a transmembrane region, and a cytosolic tail, and specifically bind to sialic acids attached to the terminal regions of cell surface glycoconjugates. Two primary 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 (sialoadesin) (siglec-1), CD22 (siglec-2), MAG (myelin-associated glycoprotein) (siglec-4), and siglec-15, all well conserved in mammals.With the exception of MAG, which is expressed in the nervous system, Siglecs are differentially expressed in various leukocyte subsets, where... Petition 870200066611, dated 05 / 28 / 2020, p. 8 / 280 / 217 play a role in the positive and negative regulation of immune and inflammatory responses (McMillan and Crocker, Carbohydr. Res., 343: 2050-2056 (2008) and Crocker, et al., Nat. Rev. Immunol. 7: 255 266 (2007)).

[004] Research indicates that many Siglecs are expressed in immune system cells and have immunosuppressive properties. However, a subset of Siglecs, including Siglec-15, is associated with the DNAX activation protein of the 12 kDa signal adapter molecule (DAP12), which has an immunoreceptor tyrosine-based activation motif (ITAM) and is involved in the activation of immune cells (Takamiya et al., Glycobiology, 23 (2): 178-87 (2013)). Siglec-15 is specifically expressed in macrophages and dendritic cells of the spleen and lymph nodes and preferentially recognizes the sTn antigen (Angata, et al., Glycobiology, 17 (8): 838-46 (2007) Epub 2007, May 4). H157 cells overexpressing sTn (H157 / ST6GalNAc-I) stimulated TGF-β secretion from M-CSF-induced macrophages expressing Siglec15 (Takamiya, et al., Glycobiology, 23 (2): 178-87 (2013)).Additionally, TGF-β secretion from THP-1 cells is enhanced by Siglec-15 overexpression in THP-1 cells and ST6GalNAc-I (the enzyme responsible for sTn structure biosynthesis) in H157 cells, respectively, in a manner that may be at least partially dependent 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 tumor cells and tumor-infiltrating leukocytes, including macrophages, and contributes to tumor progression and metastasis, for example, by increasing tumor cell invasion and inhibiting immune cell function (Flavell et al., Nat Rev. Immunol, 10: 554-567 (2010)). These data may indicate that recognition of tumor-associated sTn by Siglec-15 activates the DAP12-Syk pathway of the signal transduction pathway, which increases TGF-β production from myeloid cells and... Petition 870200066611, dated 05 / 28 / 2020, page 9 / 280 / 217 eventually modifies the tumor microenvironment which is advantageous for tumor cells (Takamiya, et al., Glycobiology, 23 (2): 178-87 (2013)). Siglec 15 has a typical ITIM domain (SNYENL (SEQ ID NO: 191)) in its cytoplasmic domain. Its function remains to be characterized.

[005] However, the need remains for tools and techniques to modulate Siglec-15 and signaling initiated from there.

[006] Thus, it is an objective of the invention to provide compositions for detecting and modulating Siglec-15.

[007] It is also an object of the invention to provide methods of modulating Siglec-15 and the signaling initiated therefrom to enhance an immune response or to reduce or reverse immune suppression.

[008] It is also an object of the invention to provide a method for modulating osteoclast differentiation to reduce bone resorption or increase bone formation.

[009] It is also an objective of the invention to provide methods of treating diseases and disorders through the modulation of Siglec-15 and the signaling initiated therefrom. SUMMARY OF THE INVENTION

[0010] Siglec-15 binding molecules are also provided. The molecules are typically an antibody or its antigen-binding fragment that binds immunospecifically to Siglec-15. For example, in some embodiments, the Siglec-15 binding molecule includes six complementarity-determining regions (CDRs), wherein the CDRs include the 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 possessing at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with 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 the three heavy chain CDRs of Petition 870200066611, dated 05 / 28 / 2020, p. 10 / 280 / 217 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 including at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity with 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 wherein the Siglec-15 binding molecule binds to Siglec-15. In some embodiments, the Siglec-15 binding molecule includes the light and / or heavy chain CDRs of one of the mouse anti-human monoclonal antibodies referred to in this document 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.

[0011] In some embodiments, the Siglec-15 binding molecule includes a variable light chain region comprising the amino acid sequence 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 with 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 a variable heavy chain region comprising the amino acid sequence 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 having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity with 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. In some embodiments, the Siglec-15 binding molecule includes the variable light and / or heavy chain regions of one of the mouse anti-human monoclonal antibodies referred to in this document as 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A, 28A, 63A, 71A, 77A, Petition 870200066611, dated 05 / 28 / 2020, p. 11 / 280 / 217 80A, 82B, 83B, 92A, 93B, 99B, 104B or 105A.

[0012] In some embodiments, the Siglec-15 binding molecule binds to Siglec-15 (I) located on the surface of a cell (preferably a living cell); (II) placed on the surface of a cell (preferably a living cell) at an endogenous concentration; (III) located on the surface of a living cell, and modulates the binding between Siglec-15 (e.g., SEQ ID NO:1 or SEQ ID NO:2 etc.) and Neu5Aca2-6GalNAca, LRRC4C, a Siglec-15 counterreceptor (S15-CR), or a combination thereof; (IV) located on the surface of a living cell, and reduces, prevents, or inhibits the secretion of TGF-β; (V) located on the surface of a living cell; or (IV) a combination of these.

[0013] Cells that endogenously express Siglec-15 include macrophages, dendritic cells, and cancer cells.

[0014] The Siglec-15 binding molecule may include one or more constant domains of an immunoglobulin (Fc) constant region. The constant domains may be human constant domains, for example, IgA, IgD, IgE, IgG, or IgM domains. In particular embodiments, the human IgG constant domains are IgG1, IgG2, IgG3, or IgG4 domains. The Siglec-15 binding molecule may be detectably labeled or comprise a conjugate toxin, drug, receptor, enzyme, or receptor ligand. The Siglec-15 binding molecule may 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 may be a monospecific, bispecific, trispecific, or multispecific antibody. Petition 870200066611, dated 05 / 28 / 2020, p. 12 / 280 / 217

[0015] One embodiment provides a humanized anti-SIGLEC-15 antibody having one or more variable light chains with an amino acid sequence of SEQ ID NO: 195, 197, 199, 201, or 209.

[0016] Another embodiment provides a humanized anti-SIGLEC-15 antibody that has one or more variable heavy chains with an amino acid sequence of SEQ ID NO:203, 206, or 207.

[0017] Another embodiment provides a humanized anti-SIGLEC-15 antibody that has one or more variable light chains with an amino acid sequence of SEQ ID NO: 195, 197, 199, 201, or 209 and one or more variable heavy chains with an amino acid sequence of SEQ ID NO: 203, 206, and 207.

[0018] One embodiment provides an antibody that has light chain CDRs with SEQ ID NO:209, 195, 207, 199, or 201 and heavy chain CDRs with SEQ ID NO:203, 206, or 207 and combinations thereof.

[0019] Another embodiment provides an antibody possessing a light chain amino acid sequence according to SEQ ID NO: 209, 210 or 211.

[0020] Another embodiment provides an antibody having an amino heavy chain sequence according to SEQ ID NO: 212, 213, 215 or 216.

[0021] Another embodiment provides an antibody possessing a light chain amino acid sequence according to SEQ ID NO: 209, 210 or 211 and a heavy chain amino acid sequence according to SEQ ID NO: 212, 213, 215 or 216.

[0022] In some embodiments, the Siglec-15 binding molecule is modified so that the molecule will exhibit decreased or no Fc receptor (FcR) binding activity. In some embodiments, the Siglec-15 binding molecule is modified to exhibit enhanced antibody-dependent cell-mediated cytotoxicity activities. Petition 870200066611, dated 05 / 28 / 2020, p. 13 / 280 / 217 (ADCC) or complement-dependent cytotoxicity (CDC).

[0023] 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.

[0024] Pharmaceutical compositions including 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 the binding of Siglec-15 to a ligand and / or counter-receptor thereof, 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 a tumor cell. The examples below show that leucine-rich repeat-containing protein 4C (LRRC4C) is a ligand for Siglec-15 and may be expressed by cancer cells. A Siglec-15 counter-receptor may also be expressed on the surface of immune cells, such as T cells, which, when engaged by Siglec-15, lead to T cell inhibition.

[0025] Treatment methods for patients in need of them are also provided. Typically, methods involve administering an effective amount of a Siglec-15 binding molecule to the subject, for example, in a pharmaceutical composition. In some embodiments, the antagonistic Siglec-15 binding molecule enhances an immune response, slows or prevents tumor growth, inhibits tumor-mediated immunosuppression, eliminates tumors, depletes or blocks the activity of tumor-associated macrophages (TAMs) so as to alter their activity, decreases TAM-mediated immunosuppression, reduces or reverses T-cell suppression, increases T-cell proliferation, or a combination thereof. In some embodiments, the cancer or tumor includes macrophages that express Siglec-15. The Siglec-15 binding molecule can be administered to the subject in an amount effective to reduce expression. Petition 870200066611, dated 05 / 28 / 2020, page 14 / 280 / 217 and / or secretion of TGF-β by macrophages. In some modalities, the subject has cancer or an infectious disease. The cancer may include cells expressing or overexpressing a Siglec-15 ligand.

[0026] Methods for reducing osteoclast differentiation, reducing bone resorption, increasing bone formation, and combinations thereof by administering an effective amount of Siglec-15 antagonist molecules are also provided.

[0027] In some modalities, the Siglec-15 agonist-binding molecule decreases 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 the subject in an amount effective to increase the expression and / or secretion of TGF-β by macrophages. In some modalities, the individual has inflammation, an autoimmune disease, or is a transplant recipient.

[0028] Some modalities include administering a second therapeutic agent to the subject.

[0029] Detection and diagnostic methods are also provided. Any of the detection and diagnostic methods may be a treatment method. For example, a method for detecting or diagnosing a disease, disorder, or infection may include (a) testing the expression of Siglec-15 in cells or in a tissue sample from an individual using the disclosed Siglec-15 binding molecules and (b) comparing the level of Siglec-15 with a control level, where an increase in the analyzed level of Siglec-15 compared to the control level is indicative of the disease, disorder, or infection.

[0030] One method of monitoring the progression of a disease, disorder, or infection may include (a) testing the expression of Siglec-15 in cells or in a tissue sample from a subject obtained at an initial and subsequent time point using disclosed Siglec-15 binding molecules and Petition 870200066611, dated 28 / 05 / 2020, page 15 / 280 / 217 (b) compare the expression level of Siglec-15 in the subject’s cells or tissue sample at the earliest and latest time points, wherein an increase in the assayed level of Siglec-15 later, compared to the first time point, is indicative of disease, disorder or infection progression.

[0031] A method for monitoring a response to a treatment is provided and includes (a) testing the expression of Siglec-15 in cells or a tissue sample from a subject before and after treatment using the disclosed Siglec-15 binding molecules; and (b) comparing the level of Siglec-15 over time, wherein 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. BRIEF DESCRIPTION OF THE FIGURES

[0032] Figure 1 is a curve showing the antibody titer in the plasma of two immunized and two non-immunized Siglec-15 knockout mice.

[0033] Figures 2A-2C are alignments showing the variable region sequences of the light chain 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 and highlighting the first (2A), second (2B) and third (2C) complementarity determining regions (CDRs).

[0034] Figures 3A-3C are alignments showing the variable region sequences of the heavy chain 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 and highlighting the first (3A), second (3B) and third (3C) complementarity determining regions (CDRs).

[0035] Figure 4A is a diagram of an assay to measure the 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 Petition 870200066611, dated 05 / 28 / 2020, p. 16 / 280 / 217 (NC77), 80A (NC80), 82B (NC82), 83B (NC83), 92A (NC92), 93B (NC93), 99B (NC99), 104B (NC104) and 105A (NC105)) are bar graphs showing the binding of anti-Siglec-15 antibodies (% of positive cells) to cells expressing Siglec-15 in the assay illustrated 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)) (% of positive cells) to formalin-fixed Siglec-15 expressing cells.

[0036] Figures 5A-5B are linear plots showing binding of purified 1C12, 8H8, 5G12, 3H10, 9A5, 6F8, 8C8, 1H3, 10G9, 1B2, and 1C3 to K562.hS15 cells (9A, background binding subtracted) and 293T.mS15 cells (9B) (Mean fluorescence intensity (MFI) as a function of primary antibody concentration (qg / ml)). Figure 5C is a dot plot (MFI) of purified 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9 Ab to K562.hS15 cells relative to 293T.mS15 cells. mS15.

[0037] Figure 6A is a bar chart showing the percentage of hS15+ U87 cells detected by each of 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, and 10G9Ab. Figure 6B is a bar chart showing the MFI of hS15+ U87 cells detected by each of 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, and 10G9Ab. Figure 6C is a bar graph showing the percentage of S15+ U87 cells detected by each of the 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9Ab.

[0038] Figure 7A is a drawing illustrating an antibody blocking assay. 293T cells expressing the LRRC4C ligand are treated with the soluble receptor (hS15.hG1) and anti-S15 antibody, and subsequently the bound receptor is detected with the PE-anti-hFc antibody. Figure 7B is a bar graph showing the % of hS15. G1 binding to the antibodies. Petition 870200066611, dated 05 / 28 / 2020, page 17 / 280 / 217 hS15.hG1-1 and 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, CI1-33). Figure 7C is a bar graph showing the S15 / LRRC4C blockade (%) for Control mAb antibodies and 1B2, 1C3, 1H3, 8H8, 6F8, 8C8, 9A5, 1C12, 3H10, 10G9, and 5G12. Figure 7D is a bar graph showing the S15 / LRRC4C blockade (%) for 1B2, 1C3, 1C12, 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9. Figure 7E is a bar graph showing the S15 / LRRC4C blockage (%) 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)).

[0039] Figure 8A is a diagram of a T cell suppression assay. Figures 8B and 8C are bar graphs showing mAb S15 reversal of hS15.hG1-mediated suppression of Human T cells as the % split of CD8+ T cells (12B) and CD4+ T cells (12C) and comparison of assays performed with hS15.hG1 (e.g., +hS15.hG1) (left bar in each pair) and without (e.g., -hS15.hG1) (right bar in each pair) for antibodies 1B2, 1C3, 1C12, 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9. Figures 8D-8G are bar graphs showing the reversal of mAb S15 suppression mediated by hS15.hG1 of human T cells as the percentage split between CD8+ T cells (Figures 8D and 8F) and CD4+ T cells (Figures 8E and 8G) for assays performed with hS15.hG1, for antibodies 1B2, 1C3, 1C12, 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9 (Figures 8D and 12E) 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) (Figures 8F and 8G). Figures 8H and 8I are scatter plots showing CD8 T cell proliferation as a function of hS15_LRRC4C blocking activity. Petition 870200066611, dated 05 / 28 / 2020, page 18 / 280 / 217

[0040] Figure 9A is a diagram of an assay to measure the change in INFγ secretion. Figure 9B is a bar graph showing the assay results, schematized in Figure 13A, for antibodies 1B2, 1C3, 1C12, 1H3, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, and 10G9.

[0041] Figure 10 is a bar graph showing TRAP (tartrate-resistant acid phosphatase) (Abs 540 nm) in the presence of antibodies 1C12, 8H8, 5G12, 3H10, 9A5, 6F8, 8C8, 1H3, 10G9, 1B2 and 1C3 in an osteoclast formation assay of fresh isolated and monocyte-enriched PBMCs in 2 ways: MACS column separation (left panel) or fixation in plastic in serum-free medium (right panel).

[0042] Figure 11 is a diagram illustrating a model for the Siglec-15 downregulation of immunity in the tumor microenvironment (TME) including Siglec-15 (S15): Siglec-15 Counter-receptor (S15-CR) >>> targeted inhibition of T cell proliferation and cytokines, and / or Siglec-15: LRRC4C >>> macrophage production of TGF-β and immunosuppression in the TME. The diagram shows myeloid cells, T cells, and cancer cells expression of Siglec-15 and its ligands, and signaling from them, as well as interactions between anti-Siglec-15 molecules (blocking and targeting) and LRRC4C antibodies and Siglec-15 fusion proteins (non-crosslinking / blocking).

[0043] Figure 12A is a table showing the amino acid sequence of the humanized 5G12 variable light chains L1-L5. Figure 12B is a table showing the amino acid sequence of the humanized 5G12 variable heavy chains H1-H3.

[0044] Figure 13A shows the sequence alignment of humanized 5G12 variable light chains VL1-VL5 versus murine VL. Figure 13B shows the sequence alignment of humanized 5G12 variable heavy chains VH1-VH3.

[0045] Figure 14A is a line graph of % Proliferation of Petition 870200066611, dated 05 / 28 / 2020, page 19 / 280 / 217 T cells versus human S15 Fc (pg / mL) showing that the % of T cell proliferation is reduced as the concentration of S15 Fc increases. Figure 14B is a bar graph of pg / ml of IFN-γ in conditioned supernatants of cells treated with 0 or 5 pg / mL of S15 Fc. Figure 14C is a bar graph of pg / ml of TNF-α in conditioned supernatants of cells treated with 0 or 5 pg / mL of S15 Fc. Figure 14D is a bar graph of pg / ml of IL-6 in conditioned supernatants of cells treated with 0 or 5 pg / mL of S15 Fc.

[0046] Figures 15A and 15B are bar graphs showing the percentage of cells positive for binding of purified mAb S15 from hybridoma to cells expressing human S15 or mouse S15.

[0047] Figures 16A and 16B are bar graphs of the percentage of CD8+ T cells divided and treated with the indicated antibodies. Figures 16C and 16D are bar graphs of the percentage of CD4+ T cells divided and treated with the indicated antibodies.

[0048] Figures 17A and 17B are line graphs showing percent survival versus days after inoculation of tumor cells in animals treated with 5G12. Figure 17C is a line graph of percent weight gain versus days after ID8. OVA inoculation.

[0049] Figure 18 is a line graph of percentage weight gain versus days after ID8. OVA inoculation.

[0050] Figure 19A is a schematic diagram showing human CD14+ monocytes harvested from human PBMCs using Mitenyi monocyte magnetic beads, followed by seeding in 96-well plates in the presence of human M-CSF and human RANKL along with indicated antibodies. Figure 19B is a micrograph showing osteoclasts treated as indicated with 1H3. Figure 19C is a bar graph of absorbance at 540 nm of the supernatant collected after 7 days for Petition 870200066611, dated 05 / 28 / 2020, page 20 / 280 / 217 analysis of tartrate-resistant acid phosphatase.

[0051] Figure 20 is a bar graph showing cytokine (pg / mL) for INF-γ, IL-2, IL-4, IL-6, IL-10, IL-17A and TNF-α.

[0052] Figure 21 is a bar graph of 540 nm absorbance RAW 264.7 mouse macrophage cells cultured in the presence of RANKL together with the indicated antibodies.

[0053] Figure 22 shows a comparison of humanized amino acid sequences exemplifying variable 1H3 light chains.

[0054] Figure 23 shows a comparison of exemplary humanized amino acid sequences of variable 1H3 heavy chains.

[0055] Figure 24 is an illustration of a proposed mode of action for Siglec-15.

[0056] Figure 25A is a FACS histogram of count versus Siglec15 PE showing M2 macrophages expressing Siglec-15. Figure 25B is a FACS histogram of count versus Siglec-15 PE for M1 macrophages. Figure 25C is a FACS histogram of count versus Siglec-15 PE for mouse bone marrow-derived myeloid cells treated with Macrophage Colony-Stimulating Factor (M-CSF). Figure 25D is a FACS histogram of count versus Siglec-15 PE for mouse bone marrow-derived myeloid cells treated with M-CSF and interleukin-10 (IL-10). Figure 25E is an MFI-PE bar graph for mouse bone marrow-derived myeloid cells treated with MCSF or M-CSF + IL10 and stained with isotype-PE (gray box) or anti-Siglec15 PE.

[0057] Figure 26A is a bar graph of absorbance at 450 nm for human CD14+ monocyte supernatants from donor #1603 seeded onto plates coated with Siglec-15 Fc (left column of each concentration point) or soluble Siglec-15 (right column of each concentration point). Figure 26B is a bar graph of absorbance at 450 nm Petition 870200066611, dated 05 / 28 / 2020, page 21 / 280 / 217 for human CD14+ monocyte supernatants from donor #1704 seeded onto plates coated with Siglec-15 Fc (left column of concentration point) or soluble Siglec-15 (right column of each concentration point). Figure 26C is a bar graph of TNF-α (pg / mL) versus Siglec-15 Fc (pg / mL) for cells from donor #1603. Figure 26D is a bar graph of IL-6 (pg / mL) versus Siglec-15 Fc (pg / mL) for cells from donor #1603. Figure 26E is a bar graph of IE-1β (pg / mL) versus Siglec-15 Fc (pg / mL) for cells from donor #1603. Figure 26F is a bar graph of TNF-α (pg / mL) versus Siglec-15 Fc (pg / mL) for cells from donor #1704. Figure 26G is a bar graph of IL-6 (pg / mL) versus Siglec-15 Fc (pg / mL) for donor cells #1704. Figure 26H is a bar graph of IL-1e (pg / mL) versus Siglec-15 Fc (pg / mL) for donor cells #1704.

[0058] Figure 27A is a diagram of an experimental protocol for example 19. Figure 27B is a bar graph of the proliferation of % CD8 cells from human myeloid cells pre-treated with S15 Fc; M2φ, M1φ, and immature DCs co-cultured with negatively labeled autologous pan-T cells and CFSE-labeled cells at a cell ratio of 1 myeloid cell:2 T cells together with anti-CD3 / CD28 beads (1 pan-T cell: 2 beads). The columns from left to right are: T cells only, T cells + beads, S15Fc treated, M2φ, M1φ, and imDC for Figures 27C to 27G. Figure 27C is a bar graph of IFN-γ (pg / mL) for the cells as treated above. Figure 27D is a bar graph of TNF-α (pg / mL) for the cells as treated above. Figure 27E is a percentage of CD4 proliferation for the cells as treated above. Figure 27F is a bar graph of IL-6 (pg / mL) for the cells as treated above.Figure 27G is a bar graph of IL-10 (pg / mL) for the cell as described above.

[0059] Figure 28A is a line graph of absorbance at 450 Petition 870200066611, dated 05 / 28 / 2020, p. 22 / 280 / 217 nm versus mAb (pg / mL) for donor cells 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 donor cells 1713.

[0060] Figure 29 is a diagram illustrating the role Siglec-15 plays in osteoclast formation.

[0061] Figure 30A is a FACS histogram of human CD14+ monocytes treated with S15Fc and stained with anti-ave3 integrin mAb on day 0. Figure 30B is a FACS histogram of human CD14+ monocytes treated with S15Fc and stained with anti-ave3 integrin mAb on day 6. DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0062] As used in this document, a molecule is said to be capable of immunospecific binding to a second molecule if that binding exhibits the specificity and affinity of an antibody to its cognate antigen. Antibodies are said to be capable of immunospecific binding to a target region or conformation (epitope) of an antigen if that binding involves the antigen recognition site of the immunoglobulin molecule. An antibody that binds immunospecifically to a specific antigen may bind to other antigens with lower affinity if the other antigen has some sequence or conformational similarity that is recognized by the antigen recognition site, such as determined, for example, by immunoassays, BIACORE® assays, or other assays known in the art, but would not bind to a completely unrelated antigen.Preferably, however, antibodies (and their antigen-binding fragments) do not cross-react with other antigens. Antibodies can also bind to other molecules in a non-immunospecific way, such as to FcR receptors, by virtue of binding domains in other regions / domains of the molecule that do not involve the antigen recognition site, such as the Fc region. Petition 870200066611, dated 05 / 28 / 2020, page 23 / 280 / 217

[0063] As used in this document, a molecule is said to bind physiologically to a second molecule if that binding exhibits the specificity and affinity of a receptor to its cognate binding ligand. A molecule may be capable of binding physiologically to more than one other molecule.

[0064] As used in this document, the term antibody is intended to denote an immunoglobulin molecule that possesses a variable region antigen recognition site. The term variable region is intended to distinguish this immunoglobulin domain from domains that are largely shared by antibodies (such as an antibody Fc domain). The variable region includes a “hypervariable region” whose residues are responsible for antigen binding. The hypervariable region includes amino acid residues of a “Complementarity Determination Region” or “CDR” (i.e., generally at residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the variable light chain domain and approximately at residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the variable heavy chain domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.(1991)) and / or the residues of a hypervariable circuit, that is, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable light chain domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable heavy chain domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). The residues of the framework region or FR are those variable domain residues that are not the residues of the hypervariable region as defined in this document. The term antibody includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (see, for example, Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann & Petition 870200066611, dated 05 / 28 / 2020, page 24 / 280 / 217 Muyldermans, 1999, J. Immunol. Meth. 231: 25; International Publications Nos. WO 94 / 04678 and WO 94 / 25591; US ​​Patent No. 6,005,079), single-chain Fvs (scFv) (see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg & Moore eds. Springer-Verlag, New York, pp. 269-315 (1994)), single-chain antibodies, disulfide-linked Fvs (sdFv), intracellular antibodies, and anti-idiotypic antibodies (anti-Id) (including, for example, anti-Id and anti-anti-Id antibodies to antibodies). 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.

[0065] As used in this document, 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 that include the antibody's variable region antigen recognition site and exhibit an ability to immunologically bind the antigen. Such fragments include Fab', F(ab') 2, Fv, single-chain (ScFv) and their mutants, naturally occurring variants, and fusion proteins, including 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 receptor ligand, etc.).

[0066] As used in this document, the term “fragment” refers to a peptide or polypeptide that includes 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 Petition 870200066611, dated 05 / 28 / 2020, page 25 / 280 / 217 contiguous amino acids, 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 at least 250 contiguous amino acid residues.

[0067] As used in this document, the term modulation refers to an ability to alter an effect, outcome, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (attenuating but not abolishing) or can completely abolish that activity (e.g., neutralization). Modulation can include the internalization of a receptor after antibody binding or a reduction in the expression of a receptor on the target cell. Agonistic modulation can potentiate or enhance or otherwise enhance an activity (e.g., signal transduction). In yet another modulation, such modulation can alter the nature of the interaction between a ligand and its cognate receptor, thus altering the nature of the resulting signal transduction.For example, molecules can, by binding to a ligand or receptor, alter the ability of such molecules to bind to other ligands or receptors and thereby alter their overall activity. Preferably, this modulation will provide at least a 10% change in the activity of a measurable immune system, more preferably at least a 50% change in that activity, or a change of at least 2-fold, 5-fold, 10-fold, or even more preferably at least 100-fold in that activity.

[0068] The term substantially, as used in the context of linkage or effect displayed, is intended to denote that the observed effect is physiologically or therapeutically relevant. Thus, for example, a Petition 870200066611, dated 05 / 28 / 2020, p. 26 / 280 / 217 a molecule is capable of substantially blocking the activity of a ligand or receptor if the extent of the blockade is physiologically or therapeutically relevant (for example, if such extent is more than 60% complete, more than 70% complete, more than 75% complete, more than 80% complete, more than 85% complete, more than 90% complete, more than 95% complete or more than 97% complete). Similarly, a molecule is said to have substantially the same immunospecificity and / or characteristics as another molecule if such immunospecificities and characteristics are more than 60% identical, more than 70% identical, more than 75% identical, more than 80% identical, more than 85% identical, more than 90% identical, more than 95% identical, or more than 97% identical.

[0069] As used in this document, “co-stimulatory” signals encompass both positive co-stimulatory signals (e.g., signals that result in an increase in activity) and negative co-stimulatory signals (e.g., signals that result in the inhibition of activity).

[0070] As used in this document, the term derivative refers to an antibody or its antigen-binding fragment that binds immunospecifically to the same target as a parental or reference antibody, but differs in amino acid sequence from the parental or reference antibody or its antigen-binding fragment, including one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to the parental or reference antibody or its antigen-binding fragment. Preferably, such derivatives will have substantially the same immunospecificity and / or characteristics or the same immunospecificity and characteristics as the parental or reference antibody or its antigen-binding fragment. Amino acid substitutions or additions of such derivatives may include naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. Petition 870200066611, dated 05 / 28 / 2020, page 27 / 280 / 217 natural. The term derivative encompasses, for example, chimeric or humanized variants, as well as variants having altered CH1, hinge, CH2, CH3 or CH4, forming, for example, antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics.

[0071] As used in this document, a chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such as antibodies with a variable region derived from a non-human antibody and a constant region from human immunoglobulin.

[0072] As used in this document, the term humanized antibody refers to an immunoglobulin that includes a human framework region and one or more CDRs from a non-human immunoglobulin (usually from a mouse or rat). The non-human immunoglobulin that provides the CDRs is called the donor, and the human immunoglobulin that provides the framework is called the acceptor. The constant regions need not be present, but if they are, they must be substantially identical to the constant regions of the human immunoglobulin, i.e., at least about 85–99%, preferably about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of the natural human immunoglobulin sequences. A humanized antibody is an antibody that includes a humanized light chain and a humanized heavy chain immunoglobulin.For example, a humanized antibody would not encompass a typical chimeric antibody, for instance, because the entire variable region of a chimeric antibody is non-human.

[0073] As used in this document, the term endogenous concentration refers to the level at which a molecule is natively expressed. Petition 870200066611, dated 05 / 28 / 2020, p. 28 / 280 / 217 (i.e., in the absence of recombinant expression vectors or promoters) by a cell (a cell that may be a normal cell, a cancerous cell, or an infected cell).

[0074] As used in this document, the terms treat, treating, treatment and therapeutic use refer to the elimination, reduction or improvement of one or more symptoms of a disease or disorder exacerbated by a Siglec-15 or respective ligand.

[0075] As used in this document, the term therapeutically effective amount refers to the amount of a therapeutic agent sufficient to mediate a clinically relevant elimination, reduction, or improvement of such symptoms. An effect is clinically relevant if its magnitude is sufficient to affect the health or prognosis of a recipient individual. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, for example, delaying or minimizing the spread of cancer. A therapeutically effective amount may also refer to the amount of therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0076] As used in this document, the term prophylactic agent refers to an agent that can be used in the prevention of a disorder or disease before the detection of any symptoms of that disorder or disease. A prophylactically effective amount is the amount of prophylactic agent sufficient to mediate such protection. A prophylactically effective amount may also refer to the amount of prophylactic agent that provides a prophylactic benefit in preventing the disease.

[0077] As used in this document, the term cancer refers to a neoplasm or tumor resulting from uncontrolled abnormal cell growth. As used in this document, cancer explicitly includes leukemias and lymphomas. The term cancer refers to a disease involving Petition 870200066611, dated 05 / 28 / 2020, page 29 / 280 / 217 cells that have the potential to metastasize to distal sites and exhibit phenotypic characteristics that differ from those of non-cancerous cells, for example, colony formation on a three-dimensional substrate such as soft agar, or the formation of tubular networks or web-like matrices on a three-dimensional basement membrane or extracellular matrix preparation. Non-cancerous cells do not form colonies on soft agar and form distinct sphere-like structures on three-dimensional basement matrix or extracellular matrix preparations.

[0078] As used in this document, the term immune cell refers to any cell of hematopoietic origin, including, but not limited to, T cells, B cells, monocytes, dendritic cells, and macrophages.

[0079] As used in this document, valence refers to the number of binding sites available per molecule.

[0080] As used in this document, the terms “immunological,” “immune,” or “immune” response refers to the development of a beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) response directed at a peptide in a recipient patient. This response may be an active response induced by administration of the immunogen or a passive response induced by administration of the antibody or initiated T cells. An immune cellular response is triggered by the presentation of polypeptide epitopes in association with MHC Class I or Class II molecules to activate antigen-specific CD4+ helper T cells and / or CD8+ cytotoxic T cells. The response may also involve the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils, or other components of innate immunity.The presence of a cell-mediated immune response can be determined by assays of... Petition 870200066611, dated 05 / 28 / 2020, page 30 / 280 / 217 proliferation of cells (CD4+ T cells) or CTL assays (cytotoxic T lymphocytes). The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by isolating antibodies and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second individual.

[0081] As used in this document, an immunogenic agent or immunogen is capable of inducing an immune response against itself upon administration to a mammal, optionally in conjunction with an adjuvant.

[0082] As used in this document, the terms individual, host, subject, and patient are used interchangeably and refer to a mammal, including but not limited to humans, rodents such as mice and rats, and other laboratory animals.

[0083] As used in this document, 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 their fragments. Polypeptides can be exogenous, meaning they are heterologous, i.e., foreign to the host cell to be used, such as the human polypeptide produced by a bacterial cell. Polypeptides are disclosed in this document in the form of amino acid residue sequences. These sequences are written from left to right, in the direction from the amino to the carboxy terminal.According to standard nomenclature, amino acid residue sequences are named by a three-letter code or a single letter, as indicated below: 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). Petition 870200066611, dated 05 / 28 / 2020, p. 31 / 280 / 217 Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).

[0084] As used in this document, 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, the differences are limited so that the sequences of the reference and variant polypeptides are globally similar 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 that is encoded by the genetic code. A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not naturally occurring.

[0085] Modifications and alterations can be made to the structure of disseminating polypeptides and still obtain a molecule with characteristics similar to those of the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Since it is the interactive capacity and nature of a polypeptide that defines the biological functional activity of the polypeptide, certain amino acid sequence substitutions can be made in the sequence of a polypeptide and still obtain a polypeptide with similar properties.

[0086] When making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function in a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted by other amino acids with a similar hydropathic index or score and still result in a polypeptide with biological activity. Petition 870200066611, dated 05 / 28 / 2020, page 32 / 280 / 217 similar. Each amino acid was assigned a hydropathic index based on its hydrophobicity and charge characteristics. These indices 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).

[0087] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that one amino acid can be substituted for another amino acid with a similar hydropathic index and still obtain a functionally equivalent polypeptide. In these changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0088] The substitution of similar amino acids can also be done based on hydrophilicity, particularly when the resulting biologically equivalent polypeptide or peptide is intended for use in immunological modalities. The following hydrophilicity values ​​were assigned to these 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); and tryptophan (-3.4). It is understood that one amino acid can be replaced by another with a similar hydrophilicity value and still obtain a biologically equivalent and, in particular, immunologically equivalent polypeptide. In these Petition 870200066611, dated 05 / 28 / 2020, p. 33 / 280 / 217 amendments, the substitution of amino acids whose hydrophilicity values ​​are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0089] As highlighted above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take several of the preceding characteristics into consideration are well known to those skilled in the art and include (original residue: exemplary 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). The modalities of this disclosure therefore encompass functional or biological equivalents of a polypeptide, as defined above.In particular, polypeptide variants may include variants with approximately 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the polypeptide of interest.

[0090] 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 sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining sequence identity can be achieved in several ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring the Petition 870200066611, dated 05 / 28 / 2020, p. 34 / 280 / 217 alignment, including any algorithms necessary to achieve maximum alignment along the entire length of the sequences being compared, can be determined by known methods.

[0091] For the purposes described in this document, the % sequence identity of a given nucleotide or amino acid sequence C with or against a given nucleic acid sequence D (which may alternatively be expressed as a given sequence C having or comprising a certain % sequence identity with or against a given sequence D) is calculated as follows: 100 times the W / Z ratio, where W is the number of nucleotides or amino acids marked as identical matches by the sequence alignment program in the C and D alignment of that program, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that when the length of sequence C is not equal to the length of sequence D, the % sequence identity from C to D will not be equal to the % sequence identity from D to C.

[0092] As used in this document, the term pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents.

[0093] As used in this document, the terms “antigenic determinant” and “epitope” are used interchangeably and refer to the structure recognized by an antibody.

[0094] As used in this document, a conformational epitope is an epitope that includes discontinuous sections of the antigen's amino acid sequence. Antibodies bind to a conformational epitope based on 3-D surface features, shape, or Petition 870200066611, dated 05 / 28 / 2020, p. 35 / 280 / 217 tertiary structure of the antigen.

[0095] As used in this document, a linear epitope is an epitope that is formed by a continuous sequence of amino acids from the antigen. Linear epitopes typically include about 5 to about 10 continuous amino acid residues. Antibodies bind to a linear epitope based on the primary sequence of the antigen.

[0096] As used in this document, a “paratope”, also called an “antigen-binding site”, is a part of an antibody that recognizes and binds to an antigen. II. Compositions A. Siglec-15 Sequences

[0097] Sialic acid-binding Ig-like lectin 15 (“Siglec-15”, also referred to as CD33 3 antigen and CD33L3) is a type 1 transmembrane protein expressed on macrophages and / or dendritic cells of the human spleen and lymph nodes (Angata, et al., Glycobiology, 17 (8): 838-46 (2007), which is specifically incorporated herein by reference in its entirety). The extracellular domain of Siglec-15 binds to sialylated glycoproteins and preferentially recognizes the Neu5Aca2-6GalNAca- structure.

[0098] Siglec-15 associates with DNAX activating protein adaptor proteins (DAP) 12 and DAP10 via its lysine residue (K274 residue) in the transmembrane domain, indicating that it functions as an activating signaling molecule. Orthologs of Siglec-15 are present not only in mammals but also in other branches of vertebrates, and are believed to play a regulatory and conservative role in the vertebrate immune system.

[0099] Siglec-15 directly regulates T cell function by inhibiting T cell proliferation and the production of pro-inflammatory cytokines. Siglec15 indirectly affects T cell function through myeloid cells. Petition 870200066611, dated 05 / 28 / 2020, page 36 / 280 / 217 Siglec-15 expressed on tumor cells or M2 macrophages interacts with its binding partner on myeloid cells, providing a survival and differentiation signal, resulting in a unique population of myeloid cells that produce 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 on T cells.

[00100] The amino acid sequences for human Siglec-15 are known in the art and include, for example, MEKSIWLLACLAWVLPTGS FVRTKIDTTENLLNTEVHSSPAQR wsmqvppevsaeagdaavlpctfthphrhydgpltaiwragepyag PQVFRCAAARGSELCQTALSLHGRFRLLGNPRRNDLSLRVERLALAD DRRYFCRVEFAGDVHDRYESRHGVRLHVTAAPRIVNISVLPSPAHAF RALCTAEGEPPPALAWSGPALGNSLAAVRSPREGHGHLVTAELPALT HDGRYTCTAANSLGRSEASVYLFRFHGA SGA STVALLLGALGFKALL LLGVLAARAARRRPEHLDTPDTPPRSQAQESNYENLSQMNPRSPPAT MCSP (SEQ ID NO: 1), UniProtKB - Q6ZMC9 (SIG15_HUMAN) and which is specifically incorporated by reference in its entirety.

[00101] Human Siglec-15 includes a signal peptide sequence of amino acids 1-19 of SEQ ID NO: 1, an extracellular domain of amino acids 20-263 of SEQ ID NO: 1 (illustrated with bold and italic letters), a transmembrane domain of amino acids 264-284 of SEQ ID NO: 1, and a cytoplasmic domain of amino acids 285-328 of SEQ ID NO: 1. The Ig-like V-type domain is expected to be of amino acids 40-158 of SEQ ID NO: 1 (illustrated with single underline) and the Ig-like C2-type domain is predicted to be of amino acids 168-251 of SEQ ID NO: 1 (illustrated with double underline). Disulfide bonds are believed to form between residues 64 and 142; 95 and 104; and 187 and 237, and glycosylation is predicted at residue 172. Amino acids 276-279 have been referred to as a Petition 870200066611, dated 05 / 28 / 2020, p. 37 / 280 / 217 polyleucine domain. A known variant is a substitution variant F273L.

[00102] The amino acid sequences for mouse Siglec-15 are known in the art and include, for example, MEGSLQLLACLACVLQMGSLVKTRRDASGDLLNTEAHSAPAQ rwsmqvpaevnaeagdaavlpctfthphrhydgpltaiwrsgepya GPQVFRCTAAPGSELCQTALSLHGRFRLLGNPRRNDLSLRVERLALA DSGRYFCRVEFTGDAHDRYESRHGVRLRVTAAAPRIVNISVLPGPAH AFRALCTAEGEPPPALAWSGPAPGNSSAALQGQGHGYQVTAELPALT RDGRYTCTAANSLGRAEASVYLFRFHGAPGTSTL .ALLLGALGLKALL LLGILGARATRRRLDHLVPQDTPPRSQAQESNYENLSQMSPPGHQLPR VCCEELLSHHHLVIHHEK (SEQ ID NO: 2), UniProtKB - A7E1W8 (A7E1W8_MOUSE), and which is specifically incorporated by reference in its entirety.

[00103] Mouse Siglec-15 includes a signal peptide sequence of amino acids 1-23 of SEQ ID NO: 2, an extracellular domain of amino acids 24-262 of SEQ ID NO: 2 (illustrated with bold and italic letters), a transmembrane domain of amino acids 263-283 of SEQ ID NO: 2, and a cytoplasmic domain of amino acids 284-342 of SEQ ID NO: 2. The Ig-like V-type domain is expected to be of amino acids 40-145 of SEQ ID NO: 2 (illustrated with single underline) and the Ig-like C2-type domain is predicted to be of amino acids 169-250 of SEQ ID NO: 2 (illustrated with double underline). B. Molecules Binding to Siglec-15

[00104] Siglec-15 binding molecules are provided, such as antibodies and their antigen-binding fragments and other polypeptides that bind to Siglec-15. The sequences of the variable regions of the heavy and light chains, and their CDRs, of mouse anti-Siglec-15 antibodies are provided below. Antibodies, antigen-binding fragments Petition 870200066611, dated 05 / 28 / 2020, page 38 / 280 / 217 antigen and other polypeptides, including one or more of the sequences below, and their variants are provided. For example, antibodies, antigen-binding fragments and polypeptides, including one, two or three CDRs of a variable region of the light chain of the anti-Siglec-15 antibody and / or one, two or three CDRs of a variable region of the heavy chain of the anti-Siglec-15 antibody that bind to Siglec-15 are provided. In some embodiments, the antibodies, antigen-binding fragments and polypeptides include the variable region of the light chain of an anti-Siglec-15 antibody, the variable region of the heavy chain of an anti-Siglec-15 antibody, or a combination thereof and can bind to Siglec-15.

[00105] For example, disclosed molecules can bind immunospecifically to Siglec-15 (e.g., SEQ ID NO: 1, SEQ ID NO: 2, etc.). For example, molecules that can bind immunospecifically to human Siglec-15 are provided: (I) placed on the surface of a cell (preferably a living cell); (II) placed on the surface of a cell (preferably a living cell) at an endogenous concentration; (III) located on the surface of a living cell, and modulates the binding between Siglec-15 (e.g., SEQ ID NO:1 or SEQ ID NO:2 etc.) and Neu5Aca2-6GalNAca, LRRC4C, a Siglec-15 counter-receptor (S15-CR), or a combination thereof; (IV) located on the surface of a living cell, and reduces, prevents, or inhibits the secretion of TGF-β; (V) arranged on the surface of a living cell, wherein the cell is a myeloid cell, such as a macrophage or dendritic cell, or a cancerous cell (for example, brain cancer cell, carcinoma cell (RCC), Ewing sarcoma cell, breast cancer cell or ovarian cancer cell); Petition 870200066611, dated 05 / 28 / 2020, page 39 / 280 / 217 (VI) combinations thereof. 1. Mouse Anti-Human Siglec-15 Antibody Sequences

[00106] As described in the Examples below, Siglec-15 knockout mice (n = 2) were immunized with hS15.mIg (extracellular domain [ECD] of human Siglec 15 fused with mouse IgG2a) emulsified with CFA (Freund's complete adjuvant) to generate a mouse anti-human Siglec-15 mAb panel.

[00107] The variable light and heavy chain region sequences for monoclonal antibodies produced by twenty-four hybridomas, referred to in this document as 1B2, 1C3, 1H3, 1C12, 3H10, 5G12, 6F8, 8C8, 8H8, 9A5, 10G9, 6A (also NC6 and #6), 28A (also NC28 and #28), 63A (also referred to as NC63 and #63), 71A (also referred to as NC71 and #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) are provided below. CDRs are underlined and in bold within the context of the light and heavy chain sequences. Sequences and CDRs are also illustrated in the alignments of Figures 2A-3C. a. Sequences 1B2: Me. Light Chain

[00108] The amino acid sequence of the variable region of the 1B2 light chain is: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQ KPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY YCFQGSHVPWTFGGGTKLEIK Petition 870200066611, dated 05 / 28 / 2020, p. 40 / 280 / 217 (SEQ ID NO: 3), with CDR1 of Light Chain 1B2: RSSQSIVHSNGNTYLE (SEQ ID NO: 24) CDR2 Light Chain 1B2: KVSNRFS (SEQ ID NO: 32) CDR3 Light Chain 1B2: FQGSHVPWT (SEQ ID NO:39).

[00109] A nucleic acid sequence that encodes the variable region of the 1B2 light chain is: GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTC TTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGT ACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACC AGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT TTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTT TATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAG GCACCAAGCTGGAAATCAAG (SEQ ID NO:74). ii. Heavy Chain

[00110] The amino acid sequence of the variable region of the 1B2 heavy chain is: EVQLVESGGGFVKPGGSLKLSCAASGFTFSDYGMHWVRQAPE KGLEWVAYISSGSSIIYYADTVKGRFTISRDNAKNTLFLQMTSLRSED TAMYYCARDHYHGNGSDYWGQGTTLTVSS (SEQ ID NO: 13), with Heavy Chain CDR1 1B2: GFTFSDYGMH (SEQ ID NO: 46) Heavy Chain CDR2 1B2: YISSGSSIIYYADTVKG (SEQ ID NO: 56) CDR3 of Heavy Chain 1B2: DHYHGNGSDY (SEQ ID NO:67).

[00111] A nucleic acid sequence that encodes the variable region of the heavy chain of 1B2 is: Petition 870200066611, dated 05 / 28 / 2020, page 41 / 280 / 217 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTCGTGAAGCC TGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTC AGTGACTATGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGG CTGGAGTGGGTTGCATACATTAGTAGTGGCAGTAGTATCATCTACT ATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATG CCAAGAACACCCTGTTCCTGCAAATGACCAGTCTGAGGTCTGAGG ACACGGCCATGTATTACTGTGCAAGGGACCACTACCATGGTAACG GGTCCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:85). b. 1C3 SEQUENCES: Me. Light Chain

[00112] The amino acid sequence of the variable region of the 1C3 light chain is: DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQR PGQSPQLLIYRMSNLASGVPDRFGGSGSGTAFTLRISRVEAEDVGFYY CMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 4), with CDR1 of Light Chain 1C3: RSSKSLLHSNGNTYLY (SEQ ID NO: 25) 1C3 Light Chain CDR2: RMSNLAS (SEQ ID NO: 33) CDR3 Light Chain 1C3: MQHLEYPYT (SEQ ID NO: 40).

[00113] A nucleic acid sequence that encodes the variable region of the 1C3 light chain is: GATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACT CCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCC TGCATAGTAATGGCAACACTTACTTATATTGGTTCCTGCAGAGGCC AGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCC TCAGGAGTCCCAGACAGGTTCGGTGGCAGTGGGTCAGGAACTGCT TTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTTTTT Petition 870200066611, dated 05 / 28 / 2020, p. 42 / 280 / 217 ATTACTGTATGCAACATCTAGAATATCCGTACACGTTCGGAGGGG GGACCAGGCTGGAAATAAAA (SEQ ID NO:75). ii. Heavy Chain

[00114] The amino acid sequence of the variable region of the 1C3 heavy chain is: QVQLKQSGAELVKPGASVKISCKASGYIFTDYYVNWVKQRPG QGLEWIGKIGPGSVSIYYNEKFKGKATLTADKSSSTAYMQLSSLTSE DSAVYFCASYYYGFAYWGQGTLVTVSA (SEQ ID NO: 14), with Heavy Chain CDR1 1C3: GYIFTDYYVN (SEQ ID NO: 47) 1C3 Heavy Chain CDR2: KIGPGSVSIYYNEKFKG (SEQ ID NO:57) CDR3 of Heavy Chain 1C3: YYYGFAY ​​​​(SEQ ID NO: 68).

[00115] A nucleic acid sequence that encodes the variable region of the heavy chain 1C3 is: CAGGTCCAGCTGAAGCAGTCTGGAGCTGAGCTGGTGAAGCC TGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACATCTTC ACTGACTATTATGTAAACTGGGTGAAGCAGAGGCCTGGACAGGGC CTTGAGTGGATTGGAAAGATTGGTCCTGGAAGTGTTAGTATTTACT ACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAAT CCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGG ACTCTGCAGTCTATTTCTGTGCAAGTTATTACTACGGGTTTGCTTAC TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:86). c. 1H3 SEQUENCES: Me. Light Chain

[00116] The amino acid sequence of the variable region of the 1H3 light chain is: Petition 870200066611, dated 05 / 28 / 2020, p. 43 / 280 / 217 DIQMTQASSSLSVSLGGRVTITCKASDHINNWLAWYQQKPGNA PRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQY WSSPLTFGAGTKLELK (SEQ ID NO: 5), with Light Chain CDR1 1H3: KASDHINNWLA (SEQ ID NO: 26) Light Chain CDR2 1H3: GATSLET (SEQ ID NO:34) CDR3 Light Chain 1H3: QQYWSSPLT (SEQ ID NO:41).

[00117] A nucleic acid sequence that codes for the variable region of the 1H3 light chain is: GACATCCAGATGACAGGCTTCATCCTCCTTGTCTGTATCTC TAGGAGGCAGAGTCACCATTACTTGCAAGGCAAGTGACCACATTA ATAATTGGTTGGCCTGGTATCAGCAGAAACCAGGAAATGCTCCTA GGCTCTTAATATCTGGTGCAACCAGTTTGGAAACTGGGGTTCCTTC AAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCAT TACCAGTCTTCAGACTGAAGATGTTGCTACTTATTACTGTCAACAG TATTGGAGTTCTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGC TGAAA (SEQ ID NO:76). ii. Humanized Light Chain

[00118] One embodiment provides a humanized anti-SIGLEC15 antibody with a variable light chain amino acid sequence of diqmtqspsslsasvgdrvtitckasdhinnwlawyqqkpgka PKLLISGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYW SSPLTFGGGTKVEIK (SEQ ID NO:209) with Light Chain CDR1 1H3: KASDHINNWLA (SEQ ID NO: 26) Light Chain CDR2 1H3: GATSLET (SEQ ID NO:34) CDR3 Light Chain 1H3: QQYWSSPLT (SEQ ID NO:41).

[00119] The underlined amino acids are altered from the precursor sequence. Petition 870200066611, dated 05 / 28 / 2020, p. 44 / 280 / 217

[00120] Another embodiment provides a humanized antiSIGLEC-15 antibody with a variable light chain amino acid sequence of DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKV PKLLISGATSLETGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQY WSSPLTFGGGTKVEIK (SEQ ID NO: 210) with Light Chain CDR1 1H3: KASDHINNWLA (SEQ ID NO: 26) Light Chain CDR2 1H3: GATSLET (SEQ ID NO:34) CDR3 Light Chain 1H3: QQYWSSPLT (SEQ ID NO:41).

[00121] The underlined amino acids are altered from the precursor sequence.

[00122] Another additional embodiment provides a humanized anti-SIGLEC-15 antibody with a variable light chain amino acid sequence of DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKA PKLLISGATSLETGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQY WSSPLTFGGGTKVEIK (SEQ ID NO: 211) with Light Chain CDR1 1H3: KASDHINNWLA (SEQ ID NO: 26) Light Chain CDR2 1H3: GATSLET (SEQ ID NO:34) CDR3 Light Chain 1H3: QQYWSSPLT (SEQ ID NO:41).

[00123] The underlined amino acids are altered in relation to the precursor sequence. iii. Heavy Chain

[00124] The amino acid sequence of the variable region of the 1H3 heavy chain is: QVQLKESGPGLVAPSQSLSITCTVSGFSLSNYGVHWVRQPPGKG LEWLVLIWSDGSTTYNSALKSRLSISKDNSKSQVFLKMNSLQTGDTA MYYCARHPYDDYSGYYYTMDYWGQGTSVTVSS (SEQ ID NO: 15), with Petition 870200066611, dated 05 / 28 / 2020, p. 45 / 280 / 217 Heavy Chain CDR1 1H3: NYGVH (SEQ ID NO:48) Heavy Chain CDR2 1H3: LIWSDGSTTYNSALKS (SEQ ID NO:58) Heavy Chain CDR3 1H3: HPYDDYSGYYYTMDY (SEQ ID NO:69).

[00125] A nucleic acid sequence that codes for the variable region of the 1H3 heavy chain is: CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCC CTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTA AGCAATTATGGTGTACACTGGGTTCGCCAGCTCCAGGAAAGGGT CTGGAGTGGCTGGTACTGATATGGAGTGATGGAAGCACAACCTAT AATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCC AAGAGCCAAGTTTTCTTAAAAATGAACAGTCTCCAAACTGGTGAC ACAGCCATGTACTACTGTGCCAGACATCCCTATGATGATTATTCCG GCTATTACTATACTATGGACTACTGGGGTCAAGGAACCTCAGTCAC CGTCTCCTCA (SEQ ID NO:87). iv. Humanized Heavy Prisons

[00126] One embodiment provides a humanized anti-SIGLEC15 antibody with a variable heavy chain amino acid sequence of QVQLQESGPGLVKPSETLSLTCTVSGFSLSNYGVHWVRQPPGK glewivliwsdgsttynsalksrvtiskdtsknqvslklssvtaadt AVYYCARHPYDDYSGYYYTMDYWGQGTLVTVSS (SEQ ID NO: 212) com Heavy Chain CDR1 1H3: NYGVH (SEQ ID NO:48) Heavy Chain CDR2 1H3: LIWSDGSTTYNSALKS (SEQ ID NO:58) Heavy Chain CDR3 1H3: HPYDDYSGYYYTMDY (SEQ ID Petition 870200066611, dated 05 / 28 / 2020, p. 46 / 280 / 217 NO:69).

[00127] The underlined amino acids are altered in relation to the precursor sequence.

[00128] Another embodiment provides a humanized antiSIGLEC-15 antibody with a variable heavy chain amino acid sequence of QVQLQESGPGLVKPSETLSLTCTVSGFSLSNYGVHWVRQPPGK glewigliwsdgsttyasalksrvtiskdtsknqvslklssvtaadt AVYYCARHPYDDYSGYYYTMDYWGQGTLVTVS (SEQ ID NO: 213) com Heavy Chain CDR1 1H3: NYGVH (SEQ ID NO:48) Heavy Chain CDR2 1H3: LIWSDGSTTYASALKS (SEQ ID NO:214) Heavy Chain CDR3 1H3: HPYDDYSGYYYTMDY (SEQ ID NO:69).

[00129] The underlined amino acids are altered in relation to the precursor sequence.

[00130] Another additional embodiment provides a humanized anti-SIGLEC-15 antibody with a variable heavy chain amino acid sequence of QVQLQESGPGLVKPSETLSLTCTVSGFSLSNYGVHWVRQPPGK GLEWIGLIWSDGSTTYNPSLKSRVTISKDTSKNQVSLKLSSVTAADT AVYYCARHPYDDYSGYYYTMDYWGQGTLVTVS (SEQ ID NO: 215) with Heavy Chain CDR1 1H3: NYGVH (SEQ ID NO:48) Heavy Chain CDR2 1H3: LIWSDGSTTYNPSLKS (SEQ ID NO:218) Heavy Chain CDR3 1H3: HPYDDYSGYYYTMDY (SEQ ID NO:69). Petition 870200066611, dated 05 / 28 / 2020, p. 47 / 280 / 217

[00131] The underlined amino acids are altered from the precursor sequence.

[00132] Another embodiment provides a humanized antiSIGLEC-15 antibody with a variable heavy chain amino acid sequence of QVQLQESGPGLVKPSETLSLTCTVSGFSLSNYGVHWVRQPPGK GLEWIGLIWSEGSTTYASALKSRVTISKDTSKNQVSLKLSSVTAADT AVYYCARHPYDDYSGYYYTMDYWGQGTLVTVS (SEQ ID NO: 216) with Heavy Chain CDR1 1H3: NYGVH (SEQ ID NO:48) Heavy Chain CDR2 1H3: LIWSEGSTTYASALKS (SEQ ID NO:217) Heavy Chain CDR3 1H3: HPYDDYSGYYYTMDY (SEQ ID NO:69). d. SEQUENCES 1C12: i. Lightweight Chain

[00133] The amino acid sequence of the variable region of the 1C12 light chain is: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQ KPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY YCFQGSHVPWTFGGGTKLEIK (SEQ ID NO: 3), with CDR1 of Light Chain 1C12: RSSQSIVHSNGNTYLE (SEQ ID NO: 24) CDR2 Light Chain 1C12: KVSNRFS (SEQ ID NO: 32) CDR3 of Light Chain 1C12: FQGSHVPWT (SEQ ID NO:39).

[00134] A nucleic acid sequence that encodes the variable region of the 1C12 light chain is: GATGTTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTC Petition 870200066611, dated 05 / 28 / 2020, p. 48 / 280 / 217 TTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGT ACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACC AGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT TTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTT TATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAG GCACCAAGCTGGAAATCAA (SEQ ID NO:77). ii. Heavy Chain

[00135] The amino acid sequence of the variable region of the 1C12 heavy chain is: EVQLVESGGGLVKPGGSLKLSCAASGFSFSDYGMHWVRQAPE KGLEWVAYISSGSSILYYADIVKGRFTISRDNAKNTLFLQMTSLRSED TAMYYCARDHYHGNGSDYWGQGTTLTVSS (SEQ ID NO: 16), with Heavy Chain CDR1 1C12: GFSFSDYGMH (SEQ ID NO:49) Heavy Chain CDR2 1C12: YISSGSSILYYADIVK (SEQ ID NO:59) CDR3 of Heavy Chain 1C12: DHYHGNGSDY (SEQ ID NO:67).

[00136] A nucleic acid sequence that encodes the variable region of the heavy chain 1C12 is: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCC TGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGTTTCTCTTTC AGTGACTATGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGG CTGGAGTGGGTTGCATACATTAGTAGTGGCAGTAGTATCCTCTACT ATGCAGACATAGTGAAGGGCCGATTCACCATCTCCAGAGACAATG CCAAGAACACCCTGTTCCTGCAAATGACCAGTCTGAGGTCTGAGG ACACGGCCATGTATTACTGTGCAAGGGACCACTACCATGGTAACG GGTCCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA Petition 870200066611, dated 05 / 28 / 2020, p. 49 / 280 / 217 (SEQ ID NO:88). e. 3H10 SEQUENCES: Me. Light Chain

[00137] The amino acid sequence of the variable region of the 3H10 light chain is: QIILTQSPAIMSASPGEKVTMTCSASSSTSFMHWYQQKPGTSPK RWIFDTSKLASGVPGRFIGSGSGTSYSLTISTMEAEDAATYYCHQRSA YPWTFGGGTKLEIK (SEQ ID NO: 6), with Light Chain CDR1 3H10: SASSSTSFMH (SEQ ID NO:27) CDR2 Light Chain 3H10: DTSKLA (SEQ ID NO: 35) CDR3 Light Chain 3H10: HQRSAYPWT (SEQ ID NO: 42).

[00138] A nucleic acid sequence that codes for the variable region of the 3H10 light chain is: CAAATTATTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTC CAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTACAA GTTTCATGCACTGGTACCAGCAGAAGCCAGGCACCTCCCCCAAAA GATGGATTTTTGACACATCCAAACTGGCTTCTGGAGTCCCTGGTCG CTTCATTGGTAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGC ACCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCATCAGCGG AGTGCTTACCCATGGACGTTCGGTGGAGGCACCAAGCTGGAAATC AAA (SEQ ID NO:78). ii. Heavy Chain

[00139] The amino acid sequence of the variable region of the 3H10 heavy chain is: EVQLQQSGAELVRPGASVKLSCTASGFNIKDYYMHWVKERPE QGLEWIGRIDPEDGDIEYDPKFQGKATMTADTSSNTAYLQFSSLTSE DTAVYYCVTDYDYDGGWFAYWGQGTLVTVSA Petition 870200066611, dated 05 / 28 / 2020, p. 50 / 280 / 217 (SEQ ID NO: 17), with Heavy Chain CDR1 3H10: GFNIKDYYMH (SEQ ID NO:50) Heavy Chain CDR2 3H10: RIDPEDGDIEYDPKFQG (SEQ ID NO: 60) CDR3 of Heavy Chain 3H10: DYDYDGGWFAY (SEQ ID NO: 70).

[00140] A nucleic acid sequence that encodes the variable region of the heavy chain 3H10 is: GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAGGCC AGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATT AAAGACTACTATATGCACTGGGTGAAAGAGAGGCCTGAACAGGGC CTGGAGTGGATTGGAAGGATTGATCCTGAGGATGGTGATATTGAA TATGACCCGAAGTTCCAGGGCAAGGCCACTATGACTGCAGATACA TCCTCCAACACAGCCTACCTGCAGTTCAGCAGCCTGACATCTGAGG ACACTGCCGTCTATTATTGTGTCACGGACTATGATTACGACGGAGG CTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:89). f. 5G12 SEQUENCES: Me. Light Chain

[00141] The amino acid sequence of the variable region of the 5G12 light chain is: DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSP KTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYD EFPYTFGGGTKLEIKR (SEQ ID NO: 7), with CDR1 Light Chain 5G12: KASQDINSYLS (SEQ ID NO: 28) CDR2 Light Chain 5G12: RANRLVD (SEQ ID NO: 36) CDR3 Light Chain 5G12: LQYDEFPYT (SEQ ID NO:43).

[00142] A nucleic acid sequence that encodes the variable region of the 5G12 light chain is: Petition 870200066611, dated 05 / 28 / 2020, p. 51 / 280 / 217 GACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCT CTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATT AATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCT AAGACCCTGATCTATCGTGCAAACAGATTGGTAGATGGGGTCCCA TCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCA TCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACA GTATGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGA AATAAAA (SEQ ID NO:79). ii. Heavy Chain

[00143] The amino acid sequence of the variable region of the 5G12 heavy chain is: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYWITWVIQRPG QGLEWIGDIYCGSDTMHYNEKFKNKATLTVDTSSSTAYMQLSSLTS EDSAVYYCARWWDYGSSYDYFDYWGQGTTLTVSS (SEQ ID NO: 18), with Heavy Chain CDR1 5G12: GYTFTSYWIT (SEQ ID NO: 51) Heavy Chain CDR2 5G12: DIYCGSDTMHYNEKFKN (SEQ ID NO:61) Heavy Chain CDR3 5G12: WWDYGSSYDYFDY (SEQ ID NO:71).

[00144] A nucleic acid sequence that encodes the variable region of the 5G12 heavy chain is: CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCT GGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCA CCAGCTACTGGATAACCTGGGTGATACAGAGGCCGGGACAAGGCC TTGAGTGGATTGGAGATATTTATTGTGGTAGTGATACTATGCACTA CAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACACATC CTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGA Petition 870200066611, dated 05 / 28 / 2020, p. 52 / 280 / 217 CTCTGCGGTCTATTACTGTGCAAGATGGTGGGACTACGGTAGTAGC TACGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCT CCTCA (SEQ ID NO:90). g. 6F8 SEQUENCES: Me. Light Chain

[00145] The amino acid sequence of the variable region of the 6F8 light chain is: DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQR PGQSPQLLIYRMSNLASGVPDRFGGSGSGTAFTLRISRVEAEDVGVYY CMQHLEYPYTFGGGTKLEIKR (SEQ ID NO: 8), with CDR1 of Light Chain 6F8: RSSKSLLHSNGNTYLY (SEQ ID NO: 25) CDR2 Light Chain 6F8: RMSNLAS (SEQ ID NO: 33) CDR3 Light Chain 6F8: MQHLEYPYT (SEQ ID NO: 40).

[00146] A nucleic acid sequence that codes for the variable region of the 6F8 light chain is: GATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACT CCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCC TGCATAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCC AGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCC TCAGGAGTCCCAGACAGGTTCGGTGGCAGTGGGTCAGGAACTGCT TTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATTGTGGGTGTT TATTATTGTATGCAACATCTAGAATATCCGTACACGTTCGGAGGGG GGACCAAGCTGGAAATAAAA (SEQ ID NO:80). ii. Heavy Chain

[00147] The amino acid sequence of the variable region of the chain Petition 870200066611, dated 05 / 28 / 2020, p. 53 / 280 / 217, heavy 6F8 is: QVQLKQSGPELVRPGASVKISCEASGYTFTDYYVNWVKQRPG RGLEWIGKIGPGSVSIYYNEKFKDKATLTADKSSSTAYMQLSGLTSE DSAVYFCASYYYGFAYWGQGTLVTVSA (SEQ ID NO: 19), with Heavy Chain CDR1 6F8: GYTFTDYYVN (SEQ ID NO: 52) 6F8 Heavy Chain CDR2: KIGPGSVSIYYNEKFKD (SEQ ID NO:62) Heavy Chain CDR3 6F8: YYYGFAY ​​(SEQ ID NO: 68).

[00148] A nucleic acid sequence that codes for the variable region of the 6F8 heavy chain is: CAGGTCCAGCTGAAGCAGTCTGGACCTGAACTGGTGAGGCCT GGGGCTTCAGTGAAGATATCCTGCGAGGCTTCTGGCTACACCTTCA CTGACTATTATGTAAACTGGGTGAAGCAGAGGCCTGGACGGGCC TTGAGTGGATTGGAAAGATTGGTCCTGGAAGTGTTAGTATTTACTA CAATGAGAAGTTCAAGGACAAGGCCACACTGACTGCAGACAAATC CTCCAGCACAGCCTACATGCAGCTCAGCGGCCTGACATCTGAGGA CTCTGCAGTCTATTTCTGTGCAAGTTATTACTACGGTTTTGCTTACT GGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:91). h. 8C8 SEQUENCES: Me. Light Chain

[00149] The amino acid sequence of the variable region of the 8C8 light chain is: DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQR PGQSPQLLIYRMSNLASGVPDRFGGSGSGTAFTLRISRVEAEDVGVYY CMQHLEYPYTFGGGTKLEIK (SEQ ID NO: 9), with CDR1 of Light Chain 8C8: RSSKSLLHSNGNTYLY (SEQ ID NO: Petition 870200066611, dated 05 / 28 / 2020, p. 54 / 280 / 217 25) CDR2 Light Chain 8C8: RMSNLAS (SEQ ID NO: 33) CDR3 Light Chain 8C8: MQHLEYPYT (SEQ ID NO: 40).

[00150] A nucleic acid sequence that encodes the variable region of the 8C8 light chain is: GATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACT CCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCC TGCATAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCC AGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCC TCAGGAGTCCCAGACAGGTTCGGTGGCAGTGGGTCAGGAACTGCT TTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTT TATTACTGTATGCAACATCTAGAATATCCGTACACGTTCGGAGGGG GGACCAAGCTGGAAATAAAA (SEQ ID NO:81). ii. Heavy Chain

[00151] The amino acid sequence of the variable region of the 8C8 heavy chain is: QVQLKQSGAELVKPGASVKISCKASGYTFTDYYVNWVKQRPG QGLEWIGKIGPESVSIYYSEKFKAKATLTADKSSSTAYMQLSSLTSED SAVYFCASYYYGFAYWGQGTLVTVSA (SEQ ID NO: 20), with Heavy Chain CDR1 8C8: GYTFTDYYVN (SEQ ID NO: 52) Heavy Chain CDR2 8C8: KIGPESVSIYYSEKFKA (SEQ ID NO: 63) Heavy Chain CDR3 8C8: YYYGFAY ​​(SEQ ID NO: 68).

[00152] A nucleic acid sequence that codes for the variable region of the 8C8 heavy chain is: CAGGTCCAGCTGAAGCAGTCTGGAGCTGAGCTGGTGAAGCC TGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTC Petition 870200066611, dated 05 / 28 / 2020, p. 55 / 280 / 217 ACTGACTATTATGTAAACTGGGTGAAGCAGAGGCCTGGACAGGGC CTTGAGTGGATTGGAAAGATTGGTCCTGAAAGTGTTAGTATTTATT ACAGTGAGAAGTTCAAGGCCAAGGCCACACTGACTGCAGACAAAT CCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGG ACTCTGCAGTCTATTTCTGTGCAAGTTATTACTACGGGTTTGCTTAC TGGGGCCAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:92). I. 8H8 SEQUENCES: Me. Light Chain

[00153] The amino acid sequence of the variable region of the 8H8 light chain is: QAVVTQESALTTSPGETVTLTCRSSSGAVTTGNFANWVQEKPD HLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCA LWYSNHWVFGGGTKLTVL (SEQ ID NO: 10), with CDR1 of Light Chain 8H8: RSSSGAVTTGNFAN (SEQ ID NO: 29) CDR2 Light Chain 8H8: GTNNRAP (SEQ ID NO: 37) CDR3 Light Chain 8H8: ALWYSNHWV (SEQ ID NO: 44).

[00154] A nucleic acid sequence that codes for the variable region of the 8H8 light chain is: CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCT GGTGAAACAGTCACACTCACTTGTCGCTCAAGTTCTGGGGCTGTTA CAACTGGTAACTTTGCCAACTGGGTCCAAGAAAAACCAGATCATT TATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGT TCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTC ACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGT GCTCTATGGTACAGCAACCACTGGGTGTTCGGTGGAGGAACCAAA CTGACTGTCCTA (SEQ ID NO:82). Petition 870200066611, dated 05 / 28 / 2020, p. 56 / 280 / 217 ii. Heavy Chain

[00155] The amino acid sequence of the variable region of the 8H8 heavy chain is: EVQLLETGGGLVQPGGSRGLSCEGSGFTFSGFWMSWVRQTPG KTLEWIGDINSDGSAINYAPSIKDRFTIFRDNDKNTLYLQMNNVRSED TATYFCVRYDDYGYFDVWGTGTTVTVSS (SEQ ID NO: 21), with Heavy Chain CDR1 8H8: GFTFSGFWMS (SEQ ID NO: 53) Heavy Chain CDR2 8H8: DINSDGSAINYAPSIKD (SEQ ID NO: 64) CDR3 of 8H8 Heavy Chain: YDDYGYFDV (SEQ ID NO: 72).

[00156] A nucleic acid sequence that encodes the variable region of the 8H8 heavy chain is: GAAGTGCAGCTGTTGGAGACTGGAGGAGGCTTGGTGCAACC GGGGGGGTCACGGGGACTCTCTTGTGAAGGCTCAGGGTTCACTTTT AGTGGCTTCTGGATGAGCTGGGTTCGACAGACACCTGGGAAGACC CTGGAGTGGATTGGAGACATTAATTCTGATGGCAGTGCAATAAAC TACGCACCATCCATAAAGGATCGATTCACTATCTTCAGAGACAATG ACAAGAACACCCTGTACCTGCAGATGAACAATGTGCGATCGGAGG ACACAGCCACGTATTTCTGTGTGAGATATGATGATTACGGGTACTT CGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO:93). j. SEQUENCES 9A5: Me. Light Chain

[00157] The amino acid sequence of the variable region of the 9A5 light chain is: DVVMTQTPLTLSVTIGQSASISCKSSQSLLDSDGKTYLNWLLQ RPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVY YCWQGTHFPFTFGSGTKLEIK Petition 870200066611, dated 05 / 28 / 2020, p. 57 / 280 / 217 (SEQ ID NO: 11), with CDR1 of light chain 9A5: KSSQSLLDSDGKTYLN (SEQ ID NO: 30) CDR2 Light Chain 9A5: LVSKLDS (SEQ ID NO: 38) CDR3 Light Chain 9A5: WQGTHFPFT (SEQ ID NO:45).

[00158] A nucleic acid sequence that encodes the variable region of the 9A5 light chain is: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACC ATTGGACAGTCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCT TAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGC CAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGA CTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGA TTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGT TTATTATTGCTGGCAAGGTACACATTTTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAAAA (SEQ ID NO:83). ii. Heavy Chain

[00159] The amino acid sequence of the variable region of the 9A5 heavy chain is: HVQLQQSGAELARPGASVKLSCKASGYTFTSYGLIWVKQRTG QGLEWIGEIYPRSGNTYYNEKFKGKATLTADISSSTAYMELRSLTSE DSAVYFCASSSPHGDYWGQGTTLTVSS (SEQ ID NO: 22), with Heavy Chain CDR1 9A5: GYTFTSYGLI (SEQ ID NO: 54) Heavy Chain CDR2 9A5: EIYPRSGNTYYNEKFKG (SEQ ID NO:65) CDR3 heavy chain 9A5: SSPHGDY (SEQ ID NO:73).

[00160] A nucleic acid sequence that codes for the variable region of the 9A5 heavy chain is: Petition 870200066611, dated 05 / 28 / 2020, p. 58 / 280 / 217 CACGTTCAGCTGCAGCAGTCTGGAGCTGAGTTGGCGAGGCCT GGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCA CAAGCTATGGTTTAATCTGGGTGAAGCAGAGAACTGGACAGGGCC TTGAGTGGATTGGAGAGATTTATCCTAGAAGTGGTAATACTTACTA CAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACATATC CTCCAGCACAGCGTACATGGAGCTCCGCAGCCTGACATCTGAGGA CTCTGCGGTCTATTTCTGTGCAAGTTCCTCTCCTCACGGGGACTACT GGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:94). k. 10G9 SEQUENCES: Me. Light Chain

[00161] The amino acid sequence of the variable region of the 10G9 light chain is: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPD HLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCA LWYSNHWVFGGGTKLTVL (SEQ ID NO: 12), with Heavy chain CDR1 10G9: RSSTGAVTTSNYAN (SEQ ID NO:31) Heavy chain CDR2 10G9: GTNNRAP (SEQ ID NO:37) CDR3 Light Chain 10G9: ALWYSNHWV (SEQ ID NO: 44).

[00162] A nucleic acid sequence that encodes the variable region of the 10G9 light chain is: CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCT GGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTA CAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATT TATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGT TCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTC ACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGT GCTCTATGGTACAGCAACCACTGGGTGTTCGGTGGAGGAACCAAA Petition 870200066611, dated 05 / 28 / 2020, p. 59 / 280 / 217 CTGACTGTCCTA (SEQ ID NO:84). ii. Heavy Chain

[00163] The amino acid sequence of the variable region of the 10G9 heavy chain is: EVQLLETGGGLVQPGGSRGLSCEGSGFTFSDFWMSWVRQTPG KTLEWIGDINSDGSAVNYAPSIKDQFTIFRDNDKRTLHLQMINVRSED TATYFCVRYDDYGYFDVWGTGTTVTVSS (SEQ ID NO: 23), with Heavy Chain CDR1 10G9: GFTFSDFWMS (SEQ ID NO:55) Heavy Chain CDR2 10G9: DINSDGSAVNYAPSIKD (SEQ ID NO: 66) CDR3 of 10G9 Heavy Chain: YDDYGYFDV (SEQ ID NO:72).

[00164] A nucleic acid sequence that encodes the variable region of the 10G9 heavy chain is: GAAGTGCAGCTGTTGGAGACTGGAGGAGGCTTAGTGCAACC TGGGGGGTCACGGGGACTCTCTTGTGAAGGCTCAGGGTTCACTTTT AGTGACTTCTGGATGAGCTGGGTTCGACAGACACCTGGGAAGACC CTGGAGTGGATTGGAGACATTAATTCTGATGGCAGTGCAGTTAACT ACGCACCATCCATAAAGGATCAATTCACTATCTTCAGAGACAATG ACAAGAGGACCCTGCACCTGCAGATGATCAATGTTCGATCGGAGG ACACAGCCACGTATTTCTGTGTGAGATATGATGATTACGGGTACTT CGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO:95). I. SEQUENCES 6A Me. Light Chain

[00165] The amino acid sequence of the variable region of the 6A light chain is: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQ Petition 870200066611, dated 05 / 28 / 2020, p. 60 / 280 / 217 KPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGVY YCFQGSHVPLTFGAGTKLELK (SEQ ID NO: 96), with CDR1 of Light Chain 6A: RSSQSIVHSNGNTYLE (SEQ ID NO: 24) CDR2 Light Chain 6A: KVSNRFS (SEQ ID NO: 32) CDR3 Light Chain 6A: FQGSHVPLT (SEQ ID NO:157).

[00166] A nucleic acid sequence that encodes the variable region of the 6A light chain is: GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTC TTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGTATTGT ACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAGCC AGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT TTCACACTCAGGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTT TATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTG GGACCAAGCTGGAGCTGAAA (SEQ ID NO:120). ii. Heavy Chain

[00167] The amino acid sequence of the variable region of the 6A heavy chain is: EVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMHWVKQRPE QGLEWIGCIDPENGDTEYASKFQDKATITTDTSSNTAYLQLSSLTSED TAVYYCTTYVGFAYWGQGTLVTVST (SEQ ID NO: 108), with Heavy Chain CDR1 6A: DDYMH (SEQ ID NO:162) Heavy Chain CDR2 6A: CIDPENGDTEYASKFQD (SEQ ID NO:170) Heavy Chain CDR3 6A: YVGFAY (SEQ ID NO:182).

[00168] A nucleic acid sequence that encodes the region Petition 870200066611, dated 05 / 28 / 2020, p. 61 / 280 / 217 heavy chain variable 6A is: GAGGTTCAGCTGCAGCAGTCTGGGGCTGAACTTGTGAGGCCA GGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTTAACATTA AAGACGACTATATGCACTGGGTGAAACAGAGGCCTGAACAGGGCC TGGAGTGGATTGGATGCATTGATCCTGAGAATGGTGATACTGAAT ATGCCTCGAAATTCCAGGACAAGGCCACTATAACAACAGACACAT CCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG ACACTGCCGTCTATTACTGTACTACATACGTTGGATTTGCTTACTG GGGCCAAGGGACTCTGGTCACTGTCTCTACA (SEQ ID NO:133). m. SEQUENCES 28A Me. Light Chain

[00169] The amino acid sequence of the variable region of the 28A light chain is: DVVMTQTPLTLSIPIGQPASISCKSSQSLLDSDGKTYLNWLLQR PGQSPKRLIYLVSELDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYY CWQGTHFPFTFGSGTKLEIK (SEQ ID NO: 97), with CDR1 of Light Chain 28A: KSSQSLLDSDGKTYLN (SEQ ID NO:30) 28A Light Chain CDR2: LVSELDS (SEQ ID NO: 153) CDR3 Light Chain 28A: WQGTHFPFT (SEQ ID NO:45).

[00170] A nucleic acid sequence that codes for the variable region of the 28A light chain is: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGATTCCC ATTGGACAACCAGCCTCCATCTCTTGTAAGTCAAGTCAGAGCCTCT TAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGC CAGGCCAGTCTCCAAAGCGCCTCATCTATCTGGTGTCTGAACTGGA CTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGA Petition 870200066611, dated 05 / 28 / 2020, p. 62 / 280 / 217 TTTCACACTGAAAATCAGCAGAGTGGAGGCTGAAGATTTGGGAGT TTATTATTGTTGGCAAGGTACACATTTTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAAAA (SEQ ID NO:121). ii. Heavy Chain

[00171] The amino acid sequence of the variable region of the 28A heavy chain is: QVQLQQSGAELARPGASVKLSCKASGYTFISYGITWVKQRTGQ GLEWIGEIHPRSGNTYYNENFKDRASLTADKSSSTAYMEVRSLTSED SAVYFCARGGPGDYWGQGTTLTVSS (SEQ ID NO: 109), with Heavy Chain CDR1 28A: SYGIT (SEQ ID NO: 163) Heavy Chain CDR2 28A: EIHPRSGNTYYNENFKD (SEQ ID NO: 171) Heavy Chain CDR3 28A: GGPGDY (SEQ ID NO:183).

[00172] A nucleic acid sequence that codes for the variable region of the 28A heavy chain is: CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGGCGAGGCCT GGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCA TAAGCTATGGTATAACCTGGGTGAAGCAGAGAACTGGACAGGGCC TTGAGTGGATTGGAGAGATTCATCCTAGAAGTGGTAATACTTACTA CAATGAGAATTTCAAGGACAGGGCCTCACTGACTGCAGACAAATC CTCCAGCACAGCGTACATGGAGGTCCGCAGCCTGACATCTGAGGA CTCTGCGGTCTATTTCTGTGCAAGGGGTGGGCCGGGGACTACTGG GGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:134). n. SEQUENCES 63A Me. Light Chain

[00173] The amino acid sequence of the variable region of the light chain Petition 870200066611, dated 05 / 28 / 2020, p. 63 / 280 / 217 63A is: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQ RPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVY YCWQGTHFPFTFGSGTKLEIK (SEQ ID NO: 98), with CDR1 of Light Chain 63A: KSSQSLLDSDGKTYLN (SEQ ID NO:30) CDR2 Light Chain 63A: LVSKLDS (SEQ ID NO: 38) CDR3 Light Chain 63A: WQGTHFPFT (SEQ ID NO:45).

[00174] A nucleic acid sequence that encodes the variable region of the 63A light chain is: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACC ATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCT TAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGC CAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGA CTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGA TTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGT TTATTATTGTTGGCAAGGTACACATTTTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAAAA (SEQ ID NO:122). ii. Heavy Chain

[00175] The amino acid sequence of the variable region of the 63A heavy chain is: QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQ GLEWIGQIYPRSDNTYYNERFKGKATLTADKSSSTAYMALRSLTSED SAVYFCAREGGPDYWGQGTTLTVSS (SEQ ID NO: 110), with Heavy Chain CDR1 63A: SYGIS (SEQ ID NO:164) Heavy Chain CDR2 63A: QIYPRSDNTYYNERFKGK (SEQ ID Petition 870200066611, dated 05 / 28 / 2020, p. 64 / 280 / 217 NO:172) Heavy chain CDR3 63A: EGGPDY (SEQ ID NO:184).

[00176] A nucleic acid sequence that encodes the variable region of the 63A heavy chain is: CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGGCGAGGCCT GGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCA CAAGCTATGGTATAAGCTGGGTGAAGCAGAGAACTGGACAGGGCC TTGAGTGGATTGGACAGATTTATCCTAGAAGTGACAATACTTACTA CAATGAGAGGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATC CTCCAGCACAGCGTACATGGCGCTCCGCAGCCTGACATCTGAGGA CTCTGCGGTCTATTTCTGTGCAAGAGAGGGGGGTCCCGACTACTGG GGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 135). o. SEQUENCES 71A i. Lightweight Chain

[00177] The amino acid sequence of the variable region of the 71A light chain is: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQ KPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY YCFQGSHVPLTFGAGTKLELK (SEQ ID NO: 99), with CDR1 of Light Chain 71A: RSSQSIVHSNGNTYLE (SEQ ID NO: 24) CDR2 Light Chain 71A: KVSNRFS (SEQ ID NO: 32) CDR3 Light Chain 71A: FQGSHVPLT (SEQ ID NO:157).

[00178] A nucleic acid sequence that codes for the variable region of the 71A light chain is: GACGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTC TTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGTATTGT Petition 870200066611, dated 05 / 28 / 2020, page 65 / 280 / 217 ACATAGTAATGGAAACACCTATTTAGAATGGTACCTACAGAAACC AGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT TTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTT TATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTG GGACCAAGCTGGAGCTGAAA (SEQ ID NO:123). ii. Heavy Chain

[00179] The amino acid sequence of the variable region of the heavy chain 71A is: EVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMHWVKQRPE QGLEWIGCIDPENGDIEYASRFQGKATMTADTSSNTAYLQLTSLTSA DTAVYYCTTYVGFGYWGQGTLVTVSA (SEQ ID NO: 111), with Heavy Chain CDR1 71A: DDYMH (SEQ ID NO:162) 71A Heavy Chain CDR2: CIDPENGDIEYASRFQG (SEQ ID NO:173) Heavy Chain CDR3 71A: YVGFGY (SEQ ID NO:185).

[00180] A nucleic acid sequence that codes for the variable region of the 71A heavy chain is: GAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCA GGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTTAACATTA AAGACGACTATATGCACTGGGTGAAACAGAGGCCTGAACAGGGCC TGGAGTGGATTGGATGCATTGATCCTGAGAATGGTGATATTGAATA TGCCTCGAGGTTCCAGGGCAAGGCCACTATGACAGCAGACACATC CTCCAACACAGCCTACCTGCAGCTCACCAGCCTGACATCTGCGGAC ACTGCCGTCTATTACTGTACTACATACGTTGGATTTGGTTACTGGG GCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:136). Petition 870200066611, dated 05 / 28 / 2020, p. 66 / 280 / 217 p. SEQUENCES 77A Me. Light Chain

[00181] The amino acid sequence of the variable region of the 77A light chain is: DVLMTQSPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLK KPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGMY YCFQGSHVPLTFGAGTKLELK (SEQ ID NO: 100), with CDR1 of Light Chain 77A: RSSQNIVHSNGNTYLE (SEQ ID NO: 146) CDR2 Light Chain 77A: KVSNRFS (SEQ ID NO: 32) CDR3 Light Chain 77A: FQGSHVPLT (SEQ ID NO:157).

[00182] A nucleic acid sequence that codes for the variable region of the 77A light chain is: GATGTTTTGATGACCCAAAGTCCACTCTCCCTGCCTGTCAGTC TTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATAGT ACATAGTAATGGTAACACCTATTTAGAATGGTACCTGAAGAAACC AGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTCTCCAACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT TTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATG TATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGAGCTG GGACCAAGCTGGAGCTGAAA (SEQ ID NO:124). ii. Heavy Chain

[00183] The amino acid sequence of the variable region of the heavy chain 77A is: EVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMHWVKQRPE QGLEWIGCIDPENGDTEYASKFQGKATITADTSSNTVYLQLSSLTSE DTAVYYCTTYVGFGYWGQGTLVTVSA Petition 870200066611, dated 05 / 28 / 2020, p. 67 / 280 / 217 (SEQ ID NO: 112), with Heavy Chain CDR1 77A: DDYMH (SEQ ID NO:162) Heavy chain CDR2 77A: CIDPENGDTEYASKFQG (SEQ ID NO:174) Heavy Chain CDR3 77A: YVGFGY (SEQ ID NO:185).

[00184] A nucleic acid sequence that codes for the variable region of the 77A heavy chain is: GAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCA GGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTTAACATTA AAGACGACTATATGCACTGGGTGAAACAGAGGCCTGAACAGGGCC TGGAGTGGATTGGATGTATTGATCCTGAGAATGGTGATACTGAATA TGCCTCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATC CTCCAACACAGTCTACCTGCAGCTCAGCAGCCTGACATCTGAGGA CACTGCCGTCTATTACTGTACTACATACGTTGGATTTGGTTACTGG GGCCAGGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:137). q. SEQUENCES 80A Me. Light Chain

[00185] The amino acid sequence of the variable region of the 80A light chain is: DIVMTQSPSSLTVTAGEKVTMSCKSNQSLLNSGDQKNYLTWY QQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAI YYCQNDYSYPLTFGAGTKLELK (SEQ ID NO: 101), with CDR1 Light Chain 80A: KSNQSLLNSGDQKNYLT (SEQ ID NO: 147) CDR2 Light Chain 80A: WASTRES (SEQ ID NO: 154) CDR3 Light Chain 80A: QNDYSYPLT (SEQ ID NO: 158).

[00186] A nucleic acid sequence that encodes the region Petition 870200066611, dated 05 / 28 / 2020, p. 68 / 280 / 217, variable of the 80A light chain is: GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACA GCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAATCAGAGTCTG TTAAACAGTGGAGATCAAAAGAACTACTTGACCTGGTACCAGCAG AAACCAGGGCAGCCTCCTAAACTATTGATCTACTGGGCATCCACTA GGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAA CAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGC AATTTATTACTGTCAGAATGATTATAGTTATCCACTCACGTTCGGT GCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO:125). ii. Heavy Chain

[00187] The amino acid sequence of the variable region of the 80A heavy chain is: QVQLKQSGAELVRPGASVKLSCRASGYTFTDFYINWVKQRPGQ GLEWIARIYPGSDETYYNEKFKDKVTLTAEKSSSTAYMQLSSLTSED SAVYFCALWFFDVWGTGTTVTVSS (SEQ ID NO: 113), with Heavy Chain CDR1 80A: DFYIN (SEQ ID NO:165) Heavy Chain CDR2 80A: RIYPGSDETYYNEKFKD (SEQ ID NO:175) Heavy Chain CDR3 80A: WFFDV (SEQ ID NO:186).

[00188] A nucleic acid sequence that codes for the variable region of the 80A heavy chain is: CAGGTCCAACTGAAGCAGTCTGGGGCTGAACTGGTGAGGCC TGGGGCTTCAGTGAAGCTGTCCTGCAGGGCTTCTGGCTACACTTTC ACTGACTTCTACATAAACTGGGTGAAGCAGAGGCCTGGACAGGGA CTTGAGTGGATTGCAAGGATTTATCCTGGAAGTGATGAGACTTACT ACAATGAGAAGTTTAAGGACAAGGTCACACTGACTGCAGAAAAAT CCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGG Petition 870200066611, dated 05 / 28 / 2020, p. 69 / 280 / 217 ACTCTGCTGTCTATTTCTGTGCCCTCTGGTTCTTCGATGTCTGGGGC ACAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO:138). r. SEQUENCES 82B Me. Light Chain

[00189] The amino acid sequence of the variable region of the 82B light chain is: DVVMTQTPLTLSVTIGQSASISCKSSQSLLDSDGNTYLNWLLQR PGQSPKRLIYLVSELDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYY CWQGTHFPFTFGSGTKLEIK (SEQ ID NO: 102), with CDR1 of light chain 82B: KSSQSLLDSDGNTYLN (SEQ ID NO: 148) 82B Light Chain CDR2: LVSELDS (SEQ ID NO: 153) CDR3 Light Chain 82B: WQGTHFPFT (SEQ ID NO:45).

[00190] A nucleic acid sequence that encodes the variable region of the 82B light chain is: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACT ATTGGACAATCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCC TAGATAGTGATGGAAACACATATTTGAATTGGTTGTTACAGAGGCC AGGCCAGTCTCCAAAGCGCCTAATCTATTTGGTGTCTGAACTGGAC TCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGAT TTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTT TATTATTGCTGGCAAGGTACACATTTTCCATTCACGTTCGGCTCGG GGACAAAGTTGGAAATAAAA (SEQ ID NO:126). ii. Heavy Chain

[00191] The amino acid sequence of the variable region of the 82B heavy chain is: Petition 870200066611, dated 05 / 28 / 2020, p. 70 / 280 / 217 QVQLQQSGAELARPGASVKLSCKASGYTFTSDGITWVKQRTGQ GLEWIGQIHPRSGNTYYNGKFKGKATLTADRSSSTTYMELRSLTSED SAVYFCAKTGTGDYWGQGTTLTVSS (SEQ ID NO: 114), with Heavy Chain CDR1 82B: SDGIT (SEQ ID NO:166) Heavy Chain CDR2 82B: QIHPRSGNTYYNGKFKG (SEQ ID NO:176) Heavy Chain CDR3 82B: TGTGDY (SEQ ID NO:187).

[00192] A nucleic acid sequence that codes for the variable region of the 82B heavy chain is: CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGTTGGCGAGGCCT GGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCGGGCTACACCTTCA CAAGCGATGGTATTACCTGGGTGAAGCAGAGAACTGGACAGGGCC TTGAGTGGATTGGACAGATTCATCCTAGAAGTGGTAATACCTACTA CAATGGGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAGATC CTCCAGCACAACGTACATGGAACTCCGCAGCCTGACATCTGAGGA CTCTGCGGTCTATTTCTGTGCAAAAACTGGGACGGGGGACTACTGG GGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:139). s. SEQUENCES 83B Me. Light Chain

[00193] The amino acid sequence of the variable region of the 83B light chain is: EIQMTQSPSSMSASLGDRITITCQATQDIVKNLNWYQQKPGKPP SFLIYYATELAEGVPSRFSGSGSGSDYSLTISNLESEDFADYYCLQFYE FPYTFGGGTKLEIK (SEQ ID NO: 103), with 83B Light Chain CDR1: QATQDIVKNLN (SEQ ID NO: 149) CDR2 Light Chain 83B: YATELAE (SEQ ID NO: 155) Petition 870200066611, dated 05 / 28 / 2020, p. 71 / 280 / 217 CDR3 of Light Chain 83B: LQFYEFPYT (SEQ ID NO: 159).

[00194] A nucleic acid sequence that encodes the variable region of the light chain 83B is: GAAATCCAGATGACCCAGTCTCCATCCTCTATGTCTGCATCT CTGGGAGACAGAATAACCATCACTTGCCAGGCAACTCAAGACATT GTTAAGAATTTAAACTGGTATCAGCAGAAACCAGGGAAACCCCCT TCATTCCTGATCTATTATGCAACTGAACTGGCAGAAGGGGTCCCAT CAAGGTTCAGTGGCAGTGGGTCTGGGTCAGACTATTCTCTGACAAT CAGCAACCTGGAGTCTGAAGATTTTGCAGACTATTACTGTCTACAG TTTTATGAATTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAA ATAAAA (SEQ ID NO:127). ii. Heavy Chain

[00195] The amino acid sequence of the variable region of the 83B heavy chain is: EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSH GKSLEWIGYINPNNGGTSYNQKFKDKATLTVNKSSSTAFMELRSLAS EDSAVYYCARSDWEDCWGQGTTLTVSS (SEQ ID NO: 115), with Heavy Chain CDR1 83B: DYNMH (SEQ ID NO: 167) Heavy Chain CDR2 83B: YINPNNGGTSYNQKFKD (SEQ ID NO: 177) Heavy Chain CDR3 83B: SDWEDC (SEQ ID NO:188).

[00196] A nucleic acid sequence that codes for the variable region of the 83B heavy chain is: GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCT GGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCA CTGACTACAACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCC TTGAGTGGATTGGATATATTAACCCTAACAATGGTGGTACTAGCTA Petition 870200066611, dated 05 / 28 / 2020, p. 72 / 280 / 217 CAACCAGAAGTTCAAGGACAAGGCCACATTGACTGTAAACAAGTC CTCCAGCACAGCCTTCATGGAGCTCCGCAGCCTGGCATCGGAGGA TTCTGCAGTCTATTACTGTGCAAGGTCTGACTGGGAAGACTGCTGG GGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:140). t. SEQUENCES 92A Me. Light Chain

[00197] The amino acid sequence of the variable region of the 92A light chain is: QIVLTQSPAIMSASLGEEITLICSASSSVSYMHWYQQKSGTSPKL LIYRTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSSW TFGGGTQLEIK (SEQ ID NO: 104), with CDR1 of Light Chain 92A: SASSSVSYMH (SEQ ID NO: 150) CDR2 Light Chain 92A: RTSNLAS (SEQ ID NO: 156) CDR3 of the 92A light chain: HQWSSWT (SEQ ID NO:160).

[00198] A nucleic acid sequence that codes for the variable region of the 92A light chain is: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTC TAGGGGAGGAGATCACCCTAATTTGCAGTGCCAGCTCGAGTGTAA GTTACATGCACTGGTACCAGCAGAAGTCAGGCACTTCTCCCAAACT CTTGATTTATCGCACATCCAACCTGGCTTCTGGAGTCCCTTCTCGCT TCAGTGGCAGTGGGTCTGGGACCTTTTATTCTCTTACAATCAGCAG TGTGGAGGCTGAAGATGCTGCCGATTATTACTGCCATCAGTGGAGT AGTTGGACGTTCGGTGGAGGCACCCAGCTGGAAATCAAA (SEQ ID NO:128). ii. Heavy Chain

[00199] The amino acid sequence of the variable region of the 92A heavy chain is: Petition 870200066611, dated 05 / 28 / 2020, p. 73 / 280 / 217 DVQLQESGPGLVKFSQSLSLTCSVTGYSITSGYYWNWIRQFPGN KLEWMGYIRHDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVITEDTA TYYCVREIYDGSSGYFDVWGTGTTVTVSS (SEQ ID NO: 116), with Heavy Chain CDR1 92A: SGYYWN (SEQ ID NO:168) CDR2 of the heavy chain 92A: YIRHDGSNNYNPSLKN (SEQ ID NO:178) Heavy Chain CDR3 92A: EIYDGSSGYFDVWGT (SEQ ID NO:189).

[00200] A nucleic acid sequence that codes for the variable region of the 92A heavy chain is: GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAATTT TCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCAC CAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAA ACTGGAATGGATGGGCTACATAAGACACGATGGTAGCAATAACTA CAACCCGTCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCT AAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGATTACTGAGGACA CAGCCACATATTACTGTGTAAGAGAGATCTATGATGGTTCCTCCGG GTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO:141). u. SEQUENCES 93B Me. Light Chain

[00201] The amino acid sequence of the variable region of the 93B light chain is: DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWY QQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISNVQPEDLA VYYCQNDYSFPFTFGGSGTELEMK (SEQ ID NO: 105), with 93B Light Chain CDR1: KSSQSLLNSGNQKNYLT (SEQ ID NO: Petition 870200066611, dated 05 / 28 / 2020, p. 74 / 280 / 217 151) CDR2 Light Chain 93B: WASTRES (SEQ ID NO: 154) CDR3 Light Chain 93B: QNDYSFPFT (SEQ ID NO: 161).

[00202] A nucleic acid sequence that encodes the variable region of the 93B light chain is: GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACA GCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTG TTAAACAGTGGAAATCAAAAGAATTACTTGACCTGGTACCAGCAG AAACCAGGACAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTA GGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAA CAGATTTCACTCTCACCATTAGCAATGTGCAGCCTGAAGACCTGGC AGTTTATTACTGTCAGAATGATTATAGTTTTCCATTCACGTTCGGCT CGGGGACAGAGTTGGAAATGAAA (SEQ ID NO:129). ii. Heavy Chain

[00203] The amino acid sequence of the variable region of the 93B heavy chain is: QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPG QGLEWIARIYPGNGNTDYNEKFKDKATLTAEKSSTTAYIQLSSLTSE DSAVYFCCLWYFDVWGTGTTVTVSS (SEQ ID NO: 117), with Heavy Chain CDR1 93B: DYYIN (SEQ ID NO: 169) 93B heavy chain CDR2: RIYPGNGNTDYNEKFKD (SEQ ID NO: 179) Heavy Chain CDR3 93B: WYFDV (SEQ ID NO:190).

[00204] A nucleic acid sequence that codes for the variable region of the 93B heavy chain is: CAGGTCCAGCTGAAGCAGTCTGGGGCTGAACTGGTGAGGCC TGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACTTTC Petition 870200066611, dated 05 / 28 / 2020, p. 75 / 280 / 217 ACTGACTACTATATAAACTGGGTGAAGCAGAGGCCTGGACAGGGA CTTGAGTGGATTGCAAGGATTTATCCTGGAAATGGTAATACTGACT ACAATGAGAAGTTCAAGGACAAGGCCACACTGACTGCAGAAAAAT CCTCCACCACTGCCTACATACAACTCAGCAGTCTGACATCTGAGGA CTCTGCTGTCTATTTCTGTTGCCTCTGGTACTTCGATGTCTGGGGCA CAGGAACCACGGTCACCGTCTCCTCA (SEQ ID NO:142). v. SEQUENCES 99B Me. Light Chain

[00205] The amino acid sequence of the variable region of the 99B light chain is: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQ RPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYY CWQGTHFPFTFGSGTKLEIK (SEQ ID NO: 106), with CDR1 of Light Chain 99B: KSSQSLLDSDGKTYLN (SEQ ID NO:30) CDR2 Light Chain 99B: LVSKLDS (SEQ ID NO: 38) CDR3 Light Chain 99B: WQGTHFPFT (SEQ ID NO:45).

[00206] A nucleic acid sequence that encodes the variable region of the 99B light chain is: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACC ATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCT TAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGC CAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGA CTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGA TTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAAT TTATTATTGCTGGCAAGGTACACATTTTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAAAA Petition 870200066611, dated 05 / 28 / 2020, p. 76 / 280 / 217 (SEQ ID NO:130). ii. Heavy Chain

[00207] The amino acid sequence of the variable region of the 99B heavy chain is: QVQLQQSGAELARPGASVKLSCKASGYTFTSDGITWLKQRTGQ GLEWIGQIHPRSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSED SAVYFCAKTGTGDYWGQGTTLTVSS (SEQ ID NO: 118), with Heavy Chain CDR1 99B: SDGIT (SEQ ID NO:166) Heavy Chain CDR2 99B: QIHPRSGNTYYNEKFKG (SEQ ID NO:180) Heavy Chain CDR3 99B: TGTGDY (SEQ ID NO:187).

[00208] A nucleic acid sequence that codes for the variable region of the 99B heavy chain is: CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGGCGAGGCCT GGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCGGGCTACACCTTCA CAAGCGACGGTATAACCTGGCTGAAACAGAGAACTGGACAGGGCC TTGAGTGGATTGGACAGATTCATCCTAGAAGTGGTAATACCTACTA CAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATC CTCCAGCACAGCGTACATGGAACTCCGCAGCCTGACATCTGAGGA CTCTGCGGTCTATTTCTGTGCAAAAACTGGGACGGGGGACTACTGG GGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:143). w. SEQUENCES 104B Me. Light Chain

[00209] The amino acid sequence of the variable region of the 104B light chain is: DVVMTQTPLTLSVTIGQPASISCKSSLSLLDSDGKTYLNWLLQR PGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKIIRVEAEDLGIYYC Petition 870200066611, dated 05 / 28 / 2020, p. 77 / 280 / 217 WQGTHFPFTFGSGTKLEVK (SEQ ID NO: 107), with CDR1 of Light Chain 104B: KSSLSLLDSDGKTYLN (SEQ ID NO: 152) CDR2 Light Chain 104B: LVSKLDS (SEQ ID NO: 38) CDR3 Light Chain 104B: WQGTHFPFT (SEQ ID NO:45).

[00210] A nucleic acid sequence that encodes the variable region of the 104B light chain is: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACC ATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCTGAGCCTCT TAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGC CAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGA CTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGA TTTCACACTGAAAATCATCAGAGTGGAGGCTGAGGATTTGGGAAT TTATTATTGCTGGCAAGGTACACATTTTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAGTAAAA (SEQ ID NO:131). ii. Heavy Chain

[00211] The amino acid sequence of the variable region of the 104B heavy chain is: QVQLQQSGPELARPGASVKLSCKASGYTFTSYGISWVKQRTGQ GLEWIGQIHPRSGNTYYNENFKGKATLTAAKSSSTAYLELRSLTSED SAVYFCAREGGPDYWGQGTTLTVSS (SEQ ID NO: 119), with Heavy Chain CDR1 104B: SYGIS (SEQ ID NO:164) Heavy Chain CDR2 104B: QIHPRSGNTYYNENFKG (SEQ ID NO:181) Heavy chain CDR3 104B: EGGPDY (SEQ ID NO:184).

[00212] A nucleic acid sequence that encodes the region Petition 870200066611, dated 05 / 28 / 2020, p. 78 / 280 / 217, states that the heavy chain variable of 104B is: CAGGTTCAGCTGCAGCAGTCTGGGCCTGAGCTGGCGAGGCCT GGGGCCTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCA CAAGCTATGGTATAAGCTGGGTGAAGCAAAGAACTGGACAGGGCC TTGAGTGGATTGGACAGATTCATCCTAGAAGTGGTAATACTTACTA CAATGAGAACTTCAAGGGCAAGGCCACACTGACTGCAGCCAAATC CTCCAGCACAGCGTACCTGGAGCTCCGCAGCCTGACATCTGAGGA CTCTGCGGTCTATTTCTGTGCAAGAGAGGGGGGTCCCGACTACTGG GGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:144). x. SEQUENCES 105A Me. Light Chain

[00213] The amino acid sequence of the variable region of the 105A light chain is: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQ KPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY YCFQGSHVPLTFGAGTKLELK (SEQ ID NO: 99), with CDR1 of Light Chain 105A: RSSQSIVHSNGNTYLE (SEQ ID NO: 24) CDR2 Light Chain 105A: KVSNRFS (SEQ ID NO: 32) CDR3 Light Chain 105A: FQGSHVPLT (SEQ ID NO:157).

[00214] A nucleic acid sequence that codes for the variable region of the 105A light chain is: GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTC TTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGTATTGT ACATAGTAATGGAAACACCTATTTAGAATGGTACCTACAGAAACC AGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT Petition 870200066611, dated 05 / 28 / 2020, p. 79 / 280 / 217 TTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTT TATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTG GGACCAAGCTGGAGCTGAAA (SEQ ID NO:132). ii. Heavy Chain

[00215] The amino acid sequence of the variable region of the 105A heavy chain is: EVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMHWVKQRPE QGLEWIGCIDPENGDIEYASRFQGKATMTADTSSNTAYLQLTSLTSA DTAVYYCTTYVGFGYWGQGTLVTVSA (SEQ ID NO: 111), with Heavy Chain CDR1 105A: DDYMH (SEQ ID NO:162) Heavy Chain CDR2 105A: CIDPENGDIEYASRFQG (SEQ ID NO:173) CDR3 of Heavy Chain 105A: YVGFGY (SEQ ID NO:185).

[00216] A nucleic acid sequence that encodes the variable region of the heavy chain 105A is: GAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCA GGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTTAACATTA AAGACGACTATATGCACTGGGTGAAACAGAGGCCTGAACAGGGCC TGGAGTGGATTGGATGCATTGATCCTGAGAATGGTGATATTGAATA TGCCTCGAGGTTCCAGGGCAAGGCCACTATGACAGCAGACACATC CTCCAACACAGCCTACCTGCAGCTCACCAGCCTGACATCTGCGGAC ACTGCCGTCTATTACTGTACTACATACGTTGGATTTGGTTACTGGG GCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:145). 2. Anti-Siglec-15 Antibodies and Their Antigen-Binding Fragments

[00217] Siglec-15 binding molecules, including antibodies and their Petition 870200066611, dated 05 / 28 / 2020, p. 80 / 280 / 217 Antigen-binding fragments that bind to one or more Siglec-15 polypeptides or fusion proteins, or fragments or variants thereof, are disclosed. The antibodies disclosed in this document are typically monoclonal antibodies, or their antigen-binding fragments, that bind to an epitope present on a Siglec-15 polypeptide, or its fragment or fusion. In some embodiments, the antibody binds to a conformational epitope. In some embodiments, the antibody binds to a linear epitope. A linear epitope may be 4, 5, 6, 7, 8, 9, 10, 11 or more continuous amino acids in length. The epitope may include one or more non-amino acid elements, post-translational modifications, or a combination thereof. Examples of post-translational modifications include, among others, glycosylation, phosphorylation, acetylation, citrullination, and ubiquitination.For example, antibodies can bind to an epitope that is formed at least in part by one or more sugar groups.

[00218] The antibody or its antigen-binding fragment may bind to an epitope that is present in an endogenous Siglec-15 polypeptide, or in a recombinant Siglec-15 polypeptide, or in a combination thereof. In some embodiments, the antibody or its antigen-binding fragment binds to the extracellular domain, or to its fragment, or to an epitope formed from it of Siglec-15. In some embodiments, the antibody or its antigen-binding fragment is a function-blocking antibody that reduces or prevents Siglec-15 from binding to one or more of its ligands, reduces Siglec-15-modulated intracellular signaling, or a combination thereof.

[00219] As discussed above, Siglec-15 siallylates glycoproteins and preferentially recognizes the Neu5Aca2-6GalNAca- structure. The experimental examples below illustrate that Siglec-15 binds to leucine-rich repeat-containing protein 4C (LRRC4C) (also referred to as Netrin-G1 and NGL-1 ligands), which can be dependent or independent of a Petition 870200066611, dated 05 / 28 / 2020, page 81 / 280 / 217 structure Neu5Aca2-6GalNAca-. The nucleic acid and polypeptide sequences for LRRC4C are known in the art and include, for example, MLNKMTLHPQQIMIGPRFNRALFDPLLVVLLALQLLVVAGLVRAQTC PSVCSCSNQFSKVICVRKNLREVPDGISTNTRLLNLHENQIQIIKVNSFK HLRHLEILQLSRNHIRTIEIGAFNGLANLNTLELFDNRLTTIPNGAFVYL SKLKELWLRNNPIESIPSYAFNRIPSLRRLDLGELKRLSYISEGAFEGLS NLRYLNLAMCNLREIPNLTPLIKLDELDLSGNHLSAIRPGSFQGLMHL QKLWMIQSQIQVIERNAFDNLQSLVEINLAHNNLTLLPHDLFTPLHHL ERIHLHHNPWNCNCDILWLSWWIKDMAPSNTACCARCNTPPNLKGR YIGELDQNYFTCYAPVIVEPPADLNVTEGMAAELKCRASTSLTSVSWI TPNGTVMTHGAYKVRIAVLSDGTLNFTNVTVQDTGMYTCMVSNSVG NTTASATLNVTAATTTPFSYFSTVTVETMEPSQDEARTTDNNVGPTPV VDWETTNVTTSLTPQSTRSTEKTFTIPVTDINSGIPGIDEVMKTTKIIIGC FVAITLMAAVMLVIFYKMRKQHHRQNHHAPTRTVEIINVDDEITGDT PMESHLPMPAIEHEHLNHYNSYKSPFNHTTTVNTINSIHSSVHEPLLIR MNSKDNVQETQI (SEQ ID NO: 192, UniProtKB - Q9HCJ2 LRC4C_HUMAN and which is specifically incorporated herein by reference in its entirety).

[00220] Siglec-15 can also bind to a counter-receptor (S15CR) on immune cells, such as T cells.

[00221] Thus, in some embodiments, a Siglec-15 binding molecule that blocks function (antagonist) reduces, inhibits, or prevents the interaction between Siglec-15 and its ligand, such as a glycoprotein possessing the structure Neu5Aca2-6GalNAca-, LRRC4C, or a Siglec-15 counterreceptor.

[00222] In some embodiments, the binding of the antibody or its antigen-binding fragment to Siglec-15 may increase immune activation, reduce immunosuppression, or a combination thereof. Petition 870200066611, dated 05 / 28 / 2020, p. 82 / 280 / 217 For example, in particular embodiments, the antibody or its antigen-binding fragment binds to the Ig-like V-type domain or the Ig-like C2-type domain of Siglec-15. In some embodiments, the epitope includes the sialic acid binding site of Siglec-15 (e.g., the epitope includes residue 143 of SEQ ID NO: 1).

[00223] In some embodiments, the antibody binds to part or the same epitope of the 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 may be a linear epitope or a conformational epitope. In some embodiments, the antibody has the same epitope specificity as the 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 producing a recombinant antibody containing the paratope of the 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 includes some or all of the CDRs of the light chain, the entire variable region of the light chain, some or all of the CDRs of the heavy chain, the entire variable region of the heavy chain, or a respective combination of any 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.

[00224] Siglec-15 binding molecules may include a CDR 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%, or at least 99% identical to the amino acid sequence of a CDR from the clones listed above that exhibit immunospecific binding to Siglec-15. Petition 870200066611, dated 05 / 28 / 2020, p. 83 / 280 / 217

[00225] For example, the disclosed molecules may include one or more CDR light chains with amino acid sequences from any of the SEQ ID NO: 24-45 and 146-161. The molecule may include at least one CDR1 light chain, one CDR2 light chain, and one CDR3 light chain. For example, the molecule may include a CDR1 light chain including an amino acid sequence selected from the group consisting of SEQ ID NO: 24-31 and 146-152. The molecule may include a CDR2 light chain including an amino acid sequence selected from the group consisting of SEQ ID NO: 32-38 and 153-156. The molecule may include a CDR3 light chain including an amino acid sequence selected from the group consisting of SEQ ID NO: 39-45 and 157-161.

[00226] In particular embodiments, the molecule includes a CDR1 light chain, a CDR2 light chain, and a CDR3 light chain, wherein CDR1 light chain, CDR2 light chain, and CDR3 light chain include the following amino acid sequences: LCDR1 LCDR2 LCDR3 SEQ ID NO: SEQ ID NO: SEQ ID NO: 1B2 24 32 and 39 ; 1C3 25 33 and 40; 1H3 26 34 and 41; 1C12 24 32 and 39; 3H10 27 35 and 42; 5G12 28 36 and 43; 6F8 25 33 and 40; 8C8 25 33 and 40; 8H8 29 37 and 44; 9A5 30 38 and 45; 10G9 31 37 and 44; #6 24 32 and 157 ; #28 30 153 and 45; #63 30 38 and 45; #71 24 32 and 157 ; #77 146 32 and 157 ; #80 147 154 and 158 ; #82 148 153 and 45 ; #83 149 155 and 159 ; #92 150 156 and 160 ; #93 151 154 and 161 ; #99 30 38 and 45 ; #104 152 38 and 45 ; or #105 24 32 and 157 .

[00227] The disclosed molecules may include one or more heavy chain CDRs possessing amino acid sequences of any of the Petition 870200066611, dated 05 / 28 / 2020, page 84 / 280 / 217 SEQ ID NO: 46-73 and 162-190. The molecule may include at least one CDR1 heavy chain, one CDR2 heavy chain, and one CDR3 heavy chain. The molecule may include a CDR1 heavy chain including an amino acid sequence selected from the group consisting of SEQ ID NO: 46-55 and 162-169. The molecule may include a CDR2 heavy chain including an amino acid sequence selected from the group consisting of SEQ ID NO: 56-66 and 170-181. The molecule may include a CDR3 heavy chain including an amino acid sequence selected from the group consisting of SEQ ID NO: 67-73 and 182-190.

[00228] In particular embodiments, the molecule includes 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 Heavy chains include the amino acid sequences: HCDR1 HCDR2 HCDR3 SEQ ID NO: SEQ ID NO: SEQ ID NO: 1B2 46 56 and 67 ; 1C3 47 57 and 68 ; 1H3 48 58 and 69 ; 1C12 49 59 and 67 ; 3H10 50 60 and 70 ; 5G12 51 61 and 71 ; 6F8 52 62 and 68 ; 8C8 52 63 and 68 ; 8H8 53 64 and 72 ; 9A5 54 65 and 73 ; 10G9 55 66 and 72 ; #6 162 170 and 182 ; #28 163 171 and 183; #63 164 172 and 184; #71 162 173 and 185; #77 162 174 and 185; #80 165 175 and 186; #82 166 176 and 187; #83 167 177 and 188; #92 168 178 and 189; #93 169 179 and 190; #99 166 180 and 187; #104 164 181 and 184; or #105 162 173 and 185.

[00229] Siglec-15 binding molecules may include a amino acid sequence of a variable heavy chain and / or variable light chain 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 Petition 870200066611, dated 05 / 28 / 2020, p. 85 / 280 / 217 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of the variable heavy chain and / or light chain of the antibody produced by any of the above clones, and which exhibits immunospecific binding to human Siglec-15.

[00230] For example, disclosed Siglec-15 binding molecules may include a variable light chain region with the amino acid sequence 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 with 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 that exhibits immunospecific binding to Siglec-15.

[00231] Additionally or alternatively, the disclosed Siglec-15 linker molecules may include a variable heavy chain region having 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 variant thereof comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity with 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 that exhibits immunospecific binding to Siglec-15.

[00232] The Siglec-15 binding molecule may be an immunoglobulin molecule (e.g., an antibody, diabody, fusion protein, etc.) 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 light chain CDRs include: (1) the light chain CDR1 of the murine 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 Petition 870200066611, dated 05 / 28 / 2020, page 86 / 280 / 217 105A, or a humanized variant thereof; (2) the CDR2 light chain of murine 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 CDR3 light chain of murine 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 CDR1 light chain and the CDR2 light chain of the 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 CDR1 light chain and the CDR3 light chain of the 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 CDR2 light chain and the CDR3 light chain of the 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 mouse CDR1 light chain, CDR2 light chain and CDR3 light chain of the 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 its humanized variant.

[00233] The molecule may be an immunoglobulin molecule including one, two, or three light chain CDRs and one, two, or three CDRs Petition 870200066611, dated 05 / 28 / 2020, p. 87 / 280 / 217 of heavy chain (for example, in some embodiments, three CDRs of light chain and three CDRs of heavy chain), where the heavy chain CDRs include: (1) the murine anti-human Siglec-15 antibody heavy chain CDR1 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 its humanized variant; (2) the murine anti-human Siglec-15 antibody heavy chain CDR2 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) a murine anti-human Siglec-15 antibody heavy chain CDR3 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 CDR1 heavy chain and the CDR2 heavy chain of murine 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 CDR1 heavy chain and the CDR3 heavy chain of murine 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 CDR2 heavy chain and the CDR3 heavy chain of murine 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, the heavy chain CDR2 and the Petition 870200066611, dated 05 / 28 / 2020, page 88 / 280 / 217 CDR3 heavy chain of murine 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.

[00234] The molecule may be an immunoglobulin molecule including one, two, or three light chain CDRs and one, two, or three heavy chain CDRs (for example, in some embodiments, three light chain CDRs and three heavy chain CDRs), wherein the heavy chain CDRs include: (1) the CDR1 light chain of murine 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 CDR2 light chain of murine 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 CDR3 light chain of murine 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 CDR1 light chain and the CDR2 light chain of murine 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 CDR1 light chain and the CDR3 light chain of murine 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; Petition 870200066611, dated 05 / 28 / 2020, page 89 / 280 / 217 (6) the CDR2 light chain and the CDR3 light chain of the murine Siglec-15 anti-human 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 CDR1 light chain, the CDR2 light chain and the CDR3 light chain of murine 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, wherein the heavy chain CDRs include: (1) the murine anti-human Siglec-15 antibody heavy chain CDR1 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 murine anti-human Siglec-15 antibody heavy chain CDR2 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) a murine anti-human Siglec-15 antibody heavy chain CDR3 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 CDR1 heavy chain and the CDR2 heavy chain of murine 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 CDR1 heavy chain and the CDR3 heavy chain of the murine anti-human Siglec-15 antibody 1B2, 1C3, 1H3, 1C12, 3H10, Petition 870200066611, dated 05 / 28 / 2020, pages 90 / 280 / 217 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 CDR2 heavy chain and the CDR3 heavy chain of murine 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 CDR1 heavy chain, the CDR2 heavy chain and the CDR3 heavy chain of murine 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.

[00235] For example, the antibody may have one or more CDRs of murine 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 chimeric antibody thereof, or a humanized variant possessing the CDR(s) corresponding to the CDR(s) of the murine Siglec-15 anti-human 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.

[00236] One embodiment provides a humanized monoclonal antibody with a 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 amino acid sequence selected from the group consisting of SEQ ID NO: 227, 228 and 229 and / or a variable heavy 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 ... selected amino acid sequence from the group consisting of SEQ ID NO: 227, 228 and 229 Petition 870200066611, dated 05 / 28 / 2020, page 91 / 280 / 217 less than 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 230, 231, 233 and 235. 3. Antibody Compositions

[00237] Disclosed Siglec-15 binding molecules may be antibodies or their antigen-binding fragments. Disclosed antibodies and their antigen-binding fragments include complete immunoglobulin (i.e., an intact antibody) of any class, its fragments, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. In some embodiments, the disclosed molecule contains both an antibody light chain and at least the variable domain of an antibody heavy chain. In other embodiments, such molecules may further include one or more of the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain (especially the CH1 and hinge regions, or the CH1, hinge, and CH2 regions, or the CH1, hinge, CH2, and CH3 regions). The antibody may be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4.In some embodiments, the constant domain is a complement to the constant domain fixation where it is desired that the antibody exhibit cytotoxic activity and the class is typically IgG1. In other embodiments, where such cytotoxic activity is not desired, the constant domain may be of the IgG2 or IgG4 class. The antibody may include sequences from more than one class or isotype, and selecting particular constant domains to optimize the desired effector functions is within normal expertise in the art.

[00238] The variable domains differ in sequence between antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, the variability is generally not uniformly distributed across the variable domains of antibodies. It is usually concentrated in three segments. Petition 870200066611, dated 05 / 28 / 2020, page 92 / 280 / 217, referred to as complementarity-determining regions (CDRs) or hypervariables, both in the variable domains of the light chain and in the variable domains of the heavy chain. The most highly conserved portions of the variable domains are called framework (FR). The variable domains of native light and heavy chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form, part of the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies.

[00239] Bioactive antibody fragments are also disclosed. The fragments, whether or not linked to other sequences, include insertions, deletions, substitutions, or other selected modifications of specific amino acid regions or residues, provided that the fragment's activity is not significantly altered or diminished compared to the unmodified antibody or antibody fragment.

[00240] The techniques can also be adapted for the production of single-chain antibodies specific for an antigenic protein of the present disclosure. The methods for producing single-chain antibodies are well known to those skilled in the art. A single-chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen-binding site in a single molecule. Variable single-chain antibody fragments (scFvs) in which the C-terminus of one variable domain is linked to the N-terminus of the other variable domain via a peptide or linker of 15 to 25 amino acids have been developed without significantly disrupting antigen binding. Petition 870200066611, dated 05 / 28 / 2020, page 93 / 280 / 217 antigen or binding specificity. The ligand is chosen to allow the heavy chain and the light chain to bind in their proper conformational orientation.

[00241] Divalent single-chain variable fragments (di-scFvs) can be manipulated by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, producing tandem scFvs. scFvs can also be created with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40 times lower than the corresponding scFvs, meaning they have a much higher affinity for their target. Even shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced similarly. They exhibit an even higher affinity for their targets than diabodies.

[00242] A monoclonal antibody is obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Monoclonal antibodies specifically include chimeric antibodies, in which a portion of the light and / or heavy chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is / are identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired antagonistic activity. Petition 870200066611, dated 05 / 28 / 2020, pp. 94 / 280 / 217 the. Chimeric and Humanized Antibodies

[00243] Chimeric antibodies and their antigen-binding fragments, including one or more of the revealed sequences and their functional variants, are also provided.

[00244] Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125: 191-202; and US Patents Nos. 6,311,415, 5,807,715, 4,816,567 and 4,816,397. Chimeric antibodies including one or more CDRs from a non-human species and structural regions of a human immunoglobulin molecule can be produced using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; International Publication No. WO 91 / 09967; and US Patents Nos. 5,225,539, 5,530,101 and 5,585,089), gilding 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 Patent No. 5,565,332).

[00245] The disclosed molecules may be human or humanized antibodies, or their antigen-binding fragments. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus may give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to decrease the chance that an antibody administered to a human will evoke an undesirable immune response.

[00246] Transgenic animals (e.g., mice) can be used that are capable, after immunization, of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of Petition 870200066611, dated 05 / 28 / 2020, page 95 / 280 / 217: The gene in the antibody heavy chain binding region (J(H)) in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene matrix into such germline mutant mice will result in the production of human antibodies upon exposure to antigen.

[00247] Optionally, antibodies are generated in other species and humanized for administration in humans. Humanized forms of non-human antibodies (e.g., murine) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antibody antigen-binding subsequences), which contain a minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibody) in which residues of a complementarity-determining region (CDR) of the receptor antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit, possessing the specificity, affinity and capacity. In some cases, Fv structural residues of human immunoglobulin are replaced by the corresponding non-human residues.Humanized antibodies may also contain residues that are not found in either the recipient antibody or the imported CDR or structural sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein 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 those of a human immunoglobulin consensus sequence. Ideally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant (Fc) region, typically that of a human immunoglobulin. Petition 870200066611, dated 05 / 28 / 2020, pp. 96 / 280 / 217

[00248] Methods for humanizing non-human antibodies are well known in the art, see, for example, European Patents Nos. EP 239,400, EP 592,106 and EP 519,596; International Publications Nos. WO 91 / 09967 and WO 93 / 17105; US Patents 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., 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. , 1997, J. Biol. Chem. 272: 1067810684; 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. , 1994, J. Mol. Biol. 235: 959-973; Jones et al. , 1986, Nature 321: 522-525; Reichmann et al., 1988, Nature 332: 323-329; e Presta, 1992, Curr. Op. Struct. Biol. 2:593596).

[00249] In general, 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 the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can essentially be achieved by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. Consequently, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or fragment, in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, antibodies Petition 870200066611, dated 05 / 28 / 2020, page 97 / 280 / 217 humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.

[00250] The choice of human variable domains, both light and heavy, to be used in the manufacture of humanized antibodies can be very important to reduce antigenicity. According to the best-fit method, the variable domain sequence of a rodent antibody is tested against the entire library of known human variable domain sequences. The human sequence closest to that of the rodent is then accepted as the human structure region (FR) for the humanized antibody. Another method uses a particular structure derived from the consensus sequence of all human antibodies of a particular subset of light or heavy chains. The same structure can be used for several different humanized antibodies.

[00251] It is also important that antibodies be humanized with high affinity retention for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analyzing parental sequences and various conceptual humanized and manipulated products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of immunoglobulin sequences from the selected candidate. Inspection of these displays allows analysis of the probable role of residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.In this way, FR residues can be selected and combined from the sequences. Petition 870200066611, dated 05 / 28 / 2020, pp. 98 / 280 / 217, regarding consensus and importation, so that the desired antibody characteristic, such as higher affinity for target antigens, is achieved. In general, CDR residues are directly and most substantially involved in influencing antigen binding.

[00252] A human, humanized, or chimeric antibody derivative may include substantially all at least one, and typically two, variable domains in which all or substantially all CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all structural regions are those of a human immunoglobulin consensus sequence. Such antibodies may also include at least a portion of an immunoglobulin constant (Fc) region, typically that of a human immunoglobulin. The constant domains of such antibodies may be selected in relation to the proposed function of the antibody, in particular the effector function that may be required. In some embodiments, the constant domains of such antibodies are or may include the human IgA, IgD, IgE, IgG, or IgM domains.In one specific embodiment, constant domains of human IgG, especially of the IgG1 and IgG3 isotypes, are used when the humanized antibody derivative is intended for therapeutic use and antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, are required. In other 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 including one or more amino acid modifications that alter antibody effector functions such as those described in US Patent Application Publications Nos. 2005 / 0037000 and 2005 / 0064514.

[00253] The structure and CDR regions of a humanized antibody do not need to correspond precisely to the parental sequences, because Petition 870200066611, dated 05 / 28 / 2020, page 99 / 280 / 217, for example, the donor CDR or consensus frame may be mutagenized by substitution, insertion, or deletion of at least one residue such that the CDR or frame residue at that location does not match the donor or consensus antibody. In some embodiments, these mutations are not extensive. Typically, at least 75% of the humanized antibody residues will match those of the parental frame region (FR) and CDR sequences, most frequently 90% or greater than 95%. Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and US Patents Nos. 5,225,539, 5,530,101 and 5,585,089), plating 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., 1994, Proc. Natl. Acad. Sci. 91: 969-973), embaralhamento de cadeia (Patente US N° 5,565,332), and techniques disclosed, for example, in the Patentes US Nos. 6,407,213, 5,766,886, 5,585,089, Publicação Internacional N° WO 9317105, Tan et 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., 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.

[00254] Frequently, structural residues in structural regions will be replaced with the corresponding residue of the CDR donor antibody to alter, for example, improve antigen binding. These structural substitutions are identified by well-known methods in the art, for example, by modeling CDR residue-structure interactions for Petition 870200066611, dated 05 / 28 / 2020, p. 100 / 280 / 217 to identify structural residues important for antigen binding and sequence comparison to identify unusual structural residues at particular positions. (See, for example, Queen et al., U.S. Patent No. 5,585,089; U.S. Publications Nos. 2004 / 0049014 and 2003 / 0229208; U.S. Patents Nos. 6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101 and Riechmann et al.)

[00255] Human, chimeric, or humanized derivatives of disclosed murine anti-Siglec-15 antibodies can be used for in vivo methods in humans. Murine antibodies or antibodies from other species can be advantageously used for many applications (e.g., in vitro or in situ detection assays, acute in vivo use, etc.). Such a human or humanized antibody may include substitutions, deletions, or additions of amino acid residues in one or more non-human CDRs. The humanized antibody derivative may have substantially the same, stronger, or weaker binding when compared to a non-derived humanized antibody. In specific embodiments, one, two, three, four, or five amino acid residues of the CDR have been substituted, deleted, or added (i.e., mutated). Completely human antibodies are particularly desirable for therapeutic treatment of human subjects.

[00256] Such human antibodies can be produced by a variety of methods known in the art, including phage presentation methods using antibody libraries derived from human immunoglobulin sequences (see U.S. Patents Nos. 4,444,887 and 4,716,111; and International Publications Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735 and WO 91 / 10741). Such antibodies can be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. Petition 870200066611, dated 05 / 28 / 2020, pages 101 / 280 / 217

[00257] For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. Mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then reared to produce homozygous offspring that express human antibodies.Transgenic mice are immunized using conventional methodologies with a selected antigen, for example, all or part of a polypeptide. Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology (see, for example, US Patent No. 5,916,771). The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for the production of human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13: 65-93, which are incorporated herein by reference in their entirety).For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, for example, International Publications Nos. Petition 870200066611, dated 05 / 28 / 2020, page 102 / 280 / 217 WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patents Nos. 5,413,923, 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 entirety. In addition, companies such as Abgenix, Inc. (Fremont, California) and Medarex (Princeton, NJ) may be involved in providing human antibodies directed against a selected antigen, using technology similar to that described above.

[00258] The DNA sequences encoding human acceptor structure sequences include, but are not limited to, FR segments of the VH segment of the human germline VH1-18 and JH6 and the VL segment of the human germline VK-A26 and JK4. In one specific embodiment, one or more of the CDRs are inserted within the structure regions using routine recombinant DNA techniques. The structural regions may be natural or consensus structure regions, and human structure regions (See, for example, Chothia et al., 1998, “Structural Determinants In The Sequences Of Immunoglobulin Variable Domain,” J. Mol. Biol. 278: 457-479 for a list of human structural regions). i. 5G12 humanized

[00259] One embodiment provides a humanized 5G12 antibody or its antigen-binding fragment. b. Single-chain antibodies

[00260] Siglec-15 binding molecules can be single-chain antibodies. The methods for producing single-chain antibodies are well known to those skilled in the art. A single-chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen-binding site in a single molecule. Variable single-chain antibody fragments (scFvs) in which the C-terminus of one variable domain is linked to the N-terminus of the other variable domain through Petition 870200066611, dated 05 / 28 / 2020, page 103 / 280 / 217, a peptide or ligand of 15 to 25 amino acids has been developed without significantly disrupting antigen binding or binding specificity. The ligand is chosen to allow the heavy chain and light chain to bind in their proper conformational orientation. These Fvs do not possess the constant (Fc) regions present in the heavy and light chains of the native antibody. c. Monovalent antibodies

[00261] In vitro methods are also suitable for preparing monovalent antibodies. Antibody digestion to produce fragments thereof, particularly Fab fragments, can be performed using routine techniques known in the art. For example, digestion can be performed using papain. Antibody digestion with papain 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 yields a fragment, called the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking the antigen.

[00262] The Fab fragments produced in antibody digestion also contain the constant light chain domains and the first constant heavy chain domain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy-terminal end of the heavy chain domain, including one or more cysteines from the antibody hinge region. The F(ab')2 fragment is a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge in the hinge region. Fab'-SH is the designation in this document for Fab' in which the cysteine ​​residues of the constant domains carry a free thiol group. Antibody fragments were originally produced as pairs of Fab' fragments, which have hinge cysteines between them. Other chemical linkages of antibody fragments are also known. Petition 870200066611, dated 05 / 28 / 2020, pages 104 / 280 / 217 d. Conjugates or fusions of antibody fragments

[00263] The targeting function of the antibody can be used therapeutically by binding the antibody or a fragment thereof to a therapeutic agent. This binding of the antibody or fragment (for example, at least a portion of an immunoglobulin (Fc) constant region) to the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein comprising the antibody or antibody fragment and the therapeutic agent.

[00264] This binding of the antibody or fragment to the therapeutic agent can be achieved by making an immunoconjugate or a fusion protein or by linking the antibody or fragment to a nucleic acid, such as siRNA, comprising the antibody or antibody fragment and the therapeutic agent.

[00265] 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 is present in the circulation or at the treatment site for longer periods. For example, it may be desirable to maintain antibody titers in the circulation or at the treatment site for extended periods. Antibodies can be manipulated with Fc variants that prolong the half-life, for example, using Xtend™ antibody half-life extension technology (Xencor, Monrovia, CA). In other embodiments, the half-life of the anti-DNA antibody is decreased to reduce potential side effects. Disclosed conjugates can be used to modify a particular biological response. The drug fraction should not be understood as limited to classic chemical therapeutic agents. For example, the drug fraction may be a protein or polypeptide possessing a desired biological activity.These proteins may include, for example, a toxin such as abrin, ricin A, Pseudomonas exotoxins, or diphtheria toxins. Petition 870200066611, dated 05 / 28 / 2020, pages 105 / 280 / 217 e. Mono- and Multi-Specific Antibodies

[00266] In some embodiments, the disclosed antibodies are monospecific, binding only to Siglec-15. Bispecific derivatives of such antibodies, trispecific derivatives of such antibodies, or derivatives of higher multiple specificity are also provided, which exhibit specificity for different targets of the immune system in addition to their specificity for human Siglec-15. For example, such antibodies may bind both to human Siglec-15 and to an antigen that is important for targeting the antibody to a particular cell or tissue type (e.g., to an antigen associated with a cancerous antigen of a tumor to be treated).In another embodiment, this multispecific antibody binds to molecules (receptors or ligands) involved in alternative immunomodulatory pathways, such as B7-H1, PD-1, CTLA4, TIM3, TIM4, OX40, CD40, GITR, 4-1-BB, LIGHT, or LAG3, in order to enhance immunomodulatory effects and combine multiple mechanisms of action, such as ligand blocking, immune cell activation, and direct targeting of tumors, into a single molecule. f. Derivatives

[00267] The production and use of derivatives of any of the disclosed Siglec-15 binding molecules are also disclosed. A derivative molecule, for example, an antibody or antibody fragment, may be modified by chemical modifications using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, tunicamycin synthesis, etc. The term derivative encompasses modifications of non-amino acids, for example, amino acids that may be glycosylated (e.g., have altered content of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylated, pegylated, phosphorylated, amidated, group-derived Petition 870200066611, dated 05 / 28 / 2020, pp. 106 / 280 100 / 217 known protectors / blockers, proteolytic cleavage, bound to a cellular ligand or other protein, etc. In some embodiments, the altered carbohydrate modifications modulate one or more of the following: antibody solubilization, facilitation of subcellular transport and antibody secretion, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function.

[00268] In one specific embodiment, the altered carbohydrate modifications increase antibody-mediated effector function compared to the antibody that does not have the carbohydrate modification. Carbohydrate modifications that lead to antibody-mediated effector function are well known in the art (e.g., see Shields, RL et 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; 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 of altering carbohydrate content are known to those skilled in the art, see, 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, M. H. et al. (1989) “Studies Of Aglycosylated Chimeric Mouse-Human IgG. Papel dos carboidratos na estrutura e nas funções efetoras mediadas pela região constante da IgG humana 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 AntibodyTransplantation 60(8):847-53; Elliott, S. et al. (2003) “Enhancement Of Therapeutic Protein In Vivo Activities Through Glycoengineering Nature Biotechnol. 21:414-21;. Petição 870200066611, de 28 / 05 / 2020, pág. 107 / 280 101 / 217 Shields, R. L. et al. (2002) “Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And AntibodyDependent Cellular Toxicity.,” J. Biol. Chem. 277(30): 26733-26740).

[00269] Disclosed antibodies can be modified by recombinant means to increase the antibody's effectiveness in mediating the desired function. Thus, antibodies can be modified by substitutions using recombinant means. Typically, the substitutions will be conservative substitutions. For example, at least one amino acid in the constant region of the antibody may be replaced by a different residue. See, for example, U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol. 30:105-08 (1993). Modification of amino acids includes deletions, additions, and substitutions of amino acids. In some cases, these changes are made to reduce undesirable activities, for example, complement-dependent cytotoxicity. Frequently, antibodies are labeled by covalently or non-covalently attaching a substance that provides a detectable signal.A wide variety of labels and conjugation techniques are known and extensively reported in the scientific and patent literature. These antibodies can be screened for binding to Siglec-15 polypeptides, fragments, or fusions thereof. See, for example, Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[00270] In some embodiments, an antibody derivative will have a similar or identical function to that of the precursor antibody. In another embodiment, an antibody derivative will exhibit altered activity relative to the precursor antibody. For example, a derivative antibody (or its fragment) may bind to its epitope more strongly or be more resistant to proteolysis than the precursor antibody.

[00271] Substitutions, additions, or deletions in derived antibodies may be located in the Fc region of the antibody and may thus serve to modify the Petition 870200066611, dated 05 / 28 / 2020, pp. 108 / 280 102 / 217 antibody binding affinity to one or more FcyR. Methods for modifying antibodies with modified binding to one or more FcyR are known in the art, see, for example, PCT Publications Nos. WO 04 / 029207, WO 04 / 029092, WO 04 / 028564, WO 99 / 58572, WO 99 / 51642, WO 98 / 23289, WO 89 / 07142, WO 88 / 07089 and US Patents Nos. 5,843,597 and 5,642,821.

[00272] In some embodiments, antibodies whose Fc region has been eliminated (e.g., a Fab or F(ab)2, etc.) or modified so that the molecule exhibits decreased or no Fc receptor (FcR) binding activity or exhibits enhanced complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) activities. In some embodiments, the antibodies have altered affinity for an activating FcyR, e.g., FcyRIIIA. Such modifications may also have altered Fc-mediated effector function. Modifications affecting Fc-mediated effector function are well known in the art (see U.S. Patent No. 6,194,551, and WO 00 / 42072).In one particular embodiment, modification of the Fc region results in an antibody with an altered antibody-mediated effector function, altered binding to other Fc receptors (e.g., Fc activating receptors), altered antibody-dependent cell-mediated cytotoxicity (ADCC) activity, altered C1q binding activity, altered complement-dependent cytotoxicity (CDC) activity, altered phagocytic activity, or any combination thereof.

[00273] Derived antibodies can be used to alter the half-lives (e.g., serum half-lives) of precursor antibodies in a mammal, such as a human. For example, such an alteration can result in a half-life 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. Petition 870200066611, dated 05 / 28 / 2020, pp. 109 / 280103 / 217 months. Increasing the half-life of humanized antibodies or their fragments in a mammal, such as a human, results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus reduces the frequency of administration of said antibodies or antibody fragments and / or reduces the concentration of said antibodies or antibody fragments to be administered. Antibodies or their fragments with enhanced in vivo half-lives can be generated by techniques known to those skilled in the art. For example, antibodies or their fragments with enhanced in vivo half-lives can be generated by modifying (e.g., substituting, eliminating, or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor. Humanized antibodies can be manipulated to increase biological half-lives (see, for example, U.S. Patent No. 6,277,375).For example, humanized antibodies can be manipulated in the Fc hinge domain to have enhanced in vivo or serum half-lives.

[00274] Antibodies or fragments thereof with extended in vivo half-lives can be generated by linking said antibodies or antibody fragments to polymeric molecules such as high molecular weight polyethylene glycol (PEG). PEG can be linked to said antibodies or antibody fragments with or without a multifunctional linker through site-specific conjugation of PEG to the N- or C-terminus of said antibodies or antibody fragments or via epsilon-amino groups present in lysine residues. Derivatization of linear or branched polymers resulting in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure adequate conjugation of PEG molecules to antibodies. Unreacted PEG can be separated from antibody-PEG conjugates, for example, by size exclusion or chromatography. Petition 870200066611, dated 05 / 28 / 2020, pp. 110 / 280 104 / 217 ion exchange.

[00275] Antibodies can also be modified by the methods and coupling agents described by Davis et al. (See U.S. Patent No. 4,179,337) in order to provide compositions that can be injected into the mammalian circulatory system with substantially no immunogenic response.

[00276] The structural residues of humanized antibodies can be modified. Structural residues in structural regions can be replaced with the corresponding residue of the CDR donor antibody to alter, for example, improve antigen binding. These structural substitutions can be identified by methods well known in the art, for example, by modeling the interactions of CDR residues and structure to identify structural residues important for antigen binding and by sequence comparison to identify unusual structural residues at particular positions. (See, for example, US Patent No. 5,585,089 and Riechmann, L. et al. (1988) “Reshaping Human Antibodies For Therapy,” Nature 332:323-327).

[00277] The disclosed Siglec-15 linker molecules can be recombinantly fused or chemically conjugated (including covalently and non-covalently conjugated) to a heterologous molecule (i.e., an unrelated molecule). Fusion does not necessarily have to be direct, but can occur through ligand sequences.

[00278] In some embodiments, such heterologous molecules are polypeptides possessing 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. Such heterologous molecules may alternatively be enzymes, hormones, cell surface receptors, drug fractions, such as: targeting reagents Petition 870200066611, dated 05 / 28 / 2020, p. 111 / 280 105 / 217 specific for macrophages (such as intracellular carboxylesterase, hCE1 (Needham, LA 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, RA (2010) “Chitins And Chitosans As Immunoadjuvants And Non-Allergenic Drug Carriers,” Mar Drugs 8(2):292-312), galactosity low-density lipoprotein (Wu, F. et al. (009) “Galactosylated LDL Nanoparticles: A Novel Targeting 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 (NFormyl-Methionyl-Leucyl-Phenylalanine) Nanocarrier Uptake By Macrophages,” Bioconjug. Chem. 19(1):28-38), mannosylated or maleylated protein, such as maleylated albumin (Anatelli, F. et al.(2006) “Macrophage-Targeted Photosensitizer Conjugate Delivered By Intratumoral Injection,” Mol Pharm. 3(6):654-664; Bansal, P. et al. (1999) “MHC Class IRestricted Presentation Of Maleylated Protein Binding To Scavenger Receptors,” J. Immunol. 162 (8): 4430-4437); veja também Mukhopadhyay, A. et al. (2003) “Intracellular Delivery Of Drugs To Macrophages,” Adv. Biochem. Eng. Biotechnol.84:183-209), toxins (such as abrin, ricin A, Pseudomonas exotoxin (i.e., PE-40), diphtheria toxin, ricin, gelonin, or pokeweed antiviral protein), proteins (such as tumor necrosis factor, interferon (e.g., α-interferon, β-interferon), nerve growth factor, platelet-derived apoptotic agent (e.g., tumor necrosis factor-α, tumor necrosis factor-β), tissue plasminogen activator, or growth factor), biological response modifiers (such as, for example, lymphokines (e.g., interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6)), granulocyte macrophage colony-stimulating factor (GM-CSF), factor (G-CSF). Petition 870200066611, dated 05 / 28 / 2020, pp. 112 / 280 106 / 217 granulocyte colony-stimulating agent or macrophage colony-stimulating factor (M-CSF), or growth factors (e.g., growth hormone (GH))), cytotoxins (e.g., cytostatic or cytocidal agents such as paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, coquicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and respective analogues or homologues), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, BiCNU® (carmustine;alkylating agents (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and platinum(II) cisdichlorodiamine (DDP) (cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), or antimitotic agents (e.g., vincristine and vinblastine).

[00279] In another embodiment, the molecules are conjugated to a second antibody to form a heteroconjugate antibody as described by Segal in US Patent No. 4,676,980. These heteroconjugate antibodies may additionally bind to haptens (such as fluorescein, etc.) or to cell markers (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 to cytokines (e.g., IL-4, IL-7, IL-10, IL-12, IL-15, IL-17, TGF-beta, IFNγ, Flt3, BLys) or chemokines (e.g., CCL21), etc.

[00280] The Fc portion of the fusion protein can be varied by isotype or subclass, can be chimeric or hybrid, and / or can be modified, by Petition 870200066611, dated 05 / 28 / 2020, pp. 113 / 280 107 / 217 For example, to improve effector functions, half-life control, tissue accessibility, increased biophysical characteristics such as stability, and improved production efficiency (and be less expensive). Many useful modifications to the construction of disclosed fusion proteins and methods for producing 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 Antibodies Therapeutic, Curr. Opin. Immun. 20: 460-470. In some modalities, the Fc region is the native Fc region of IgG1, IgG2, or IgG4.In some forms, the Fc region is a hybrid, for example, a chimeric consisting of constant Fc regions of IgG2 / IgG4. Modifications to the Fc region include, but are not limited to, IgG4 modified to prevent binding to Fc gamma and complement receptors, IgG1 modified to enhance binding to one or more Fc gamma receptors, IgG1 modified to minimize effector function (amino acid alterations), IgG1 with / without altered glycan (typically altering the host of expression), and IgG1 with pH-dependent alteration to FcRn. The Fc region may include the entire hinge region, or less than the entire hinge region.

[00281] The therapeutic outcome in patients treated with rituximab (chimeric mouse / human IgG1 monoclonal antibody against CD20) for non-Hodgkin lymphoma or Waldenstrom's macroglobulinemia correlated with the individual expression of allelic variants of Fcy receptors with distinct intrinsic affinities for the Fc domain of human IgG1. In particular, patients with high-affinity alleles of the low-affinity activating Fc receptor CD16A (FcyRIIIA) showed rates of Petition 870200066611, dated 05 / 28 / 2020, pp. 114 / 280 108 / 217 higher response rates and, in cases of non-Hodgkin lymphoma, improved progression-free survival. Therefore, the Fc domain may contain one or more insertions, deletions, or amino acid substitutions in the Fc domain that reduce binding to the low-affinity inhibitory Fc receptor CD32B (FcyRIIB) and retain wild-type binding levels or increase binding to the low-affinity activating Fc receptor CD16A (FcRIIIA).

[00282] Another embodiment includes IgG2-4 hybrids and IgG4 mutants that have reduced binding to FcyR, which increases their half-life. 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, 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; and US Patent No. 6,982,323. In some embodiments, the IgG1 and / or IgG2 domain is modified; For example, Angal, S. et al. (1993) describe variants of IgG1 and IgG2 in which serine 241 is replaced by proline.

[00283] In some embodiments, the Fc domain of such molecules contains amino acid insertions, deletions, or substitutions that increase CD16A binding. A large number of substitutions in the Fc domain of human IgG1 that increase CD16A binding and reduce CD32B binding 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):88828890. Exemplary variants of human IgG1 Fc domains with reduced CD32B binding and / or enhanced CD16A binding contain Petition 870200066611, dated 05 / 28 / 2020, pp. 115 / 280 109 / 217 F243L, R929P, Y300L, V305I, or P296L substitutions. These amino acid substitutions can be present in a human IgG1 Fc domain in any combination. In one embodiment, the human IgG1 Fc domain variant contains an F243L, R929P, and Y300L substitution. In another embodiment, the human IgG1 Fc domain variant contains an F243L, R929P, Y300L, V305I, and P296L substitution. In yet another embodiment, the human IgG1 Fc domain variant contains an N297Q substitution, since this mutation abolishes FcR binding.

[00284] Técnicas para conjugar frações terapêuticas com anticorpos são bem conhecidas; ver, por exemplo, Arnon et al. , “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy ”, em 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 (2a. Ed. ), Robinson et al. (eds. ), 1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review ”, em Monoclonal Antibodies ‘84: Biological And Clinical Applications, Pinchera et al. (eds. ), 1985, pp. 475-506); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, em Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds. ), 1985, pp. 303-16, Academic Press; e Thorpe et al. (1982) “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,” Immunol. Rev. 62:119-158.。

[00285] Any of the disclosed molecules can be fused to marker sequences, such as a peptide, to facilitate purification. In some embodiments, the marker amino acid sequence is a hexahistidine peptide, the hemagglutinin marker “HA”, which corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson, IA et al. (1984) “The Structure Of An Antigenic Determinant In A Protein,” Cell, 37:767-778) and the “flag” marker (Knappik, A. et al. (1994) Petition 870200066611, dated 05 / 28 / 2020, pp. 116 / 280 110 / 217 “An Improved Affinity Tag Based On The FLAG Peptide For The Detection And Purification Of Recombinant Antibody Fragments,” Biotechniques 17(4):754-761).

[00286] Disclosed Siglec-15 binding molecules can be conjugated with a diagnostic or therapeutic agent, or another molecule for which an increased serum half-life is desired. Antibodies can be used diagnostically (in vivo, in situ or in vitro) to, for example, monitor the development or progression of a disease, disorder or infection as part of a clinical trial procedure to, for example, determine the efficacy of a given treatment or to select patients most likely to respond to a specific therapy (such as those expressing high levels of Siglec-15).

[00287] Detection can be facilitated by coupling the molecule, such as an antibody or an antigen-binding fragment thereof, 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. The detectable substance can be coupled or conjugated directly to the antibody or indirectly, through an intermediate (such as, for example, a ligand 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 with antibodies for use as diagnostics.Such diagnosis and detection can be performed by coupling the antibody with detectable substances including, but not limited to, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; prosthetic group complexes such as, among others, streptavidin / biotin and avidin / biotin; fluorescent materials such as, but not limited to, umbelliferone, fluorescein, etc. Petition 870200066611, dated 05 / 28 / 2020, pp. 117 / 280 111 / 217 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 (213Bi), carbon (14C), chromium (51Cr), cobalt (57Co), fluorine (18F), gadolinium (153Gd, 159Gd), gallium (68Ga, 67Ga), germanium (68Ge), holmium (166Ho), indium (115In, 113In, 112In, 111In), iodine (131I, 125I, 123I, 121I), lanthanum (140La), lutetium (177Lu), manganese (54Mn), molybdenum (99Mo), palladium (103Pd), phosphorus (32P), praseodymium (142Pr), promethium (149Pm), rhenium (186Re, 188Re), rhodium (105Rh), ruthenium (97Ru), samarium (153Sm), scandium (47Sc), selenium (75Se), strontium (85Sr), sulfur (35S), technetium (99Tc), thallium (201Ti), tin (113Sn,117Sn), tritium (3H), xenon (133Xe), ytterbium (169Yb,175Yb), yttrium (90Y), zinc (65Zn);Positron-emitting metals using various positron emission tomography and non-radioactive paramagnetic metal ions.

[00288] The disclosed molecules can be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen or other molecules that are capable of binding to the target antigen that has been immobilized on the support via binding to an antibody or antigen-binding fragment. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[00289] Nucleic acid molecules (DNA or RNA) that encode any of these antibodies, fusion proteins or fragments, as well as vector molecules (such as plasmids) that are capable of transmitting or replicating such nucleic acid molecules are also disclosed. Nucleic acids can be single-stranded, double-stranded, or can contain both single-stranded and double-stranded portions. 3. Method of Doing Petition 870200066611, dated 05 / 28 / 2020, pp. 118 / 280 112 / 217

[00290] Siglec-15 binding molecules can be produced by any method known in the art useful for the production of polypeptides, for example, in vitro synthesis, recombinant DNA production and the like. Humanized antibodies are typically produced by recombinant DNA technology. Antibodies can be produced using recombinant immunoglobulin expression technology. Recombinant production of immunoglobulin molecules, including humanized antibodies, is described in US Patent No. 4,816,397 (Boss et al.), US Patents Nos. 6,331,415 and 4,816,567 (both to Cabilly et al.), UK Patent GB 2,188,638 (Winter et al.), and British Patent GB 2,209,757. Techniques for 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 on the generation, design, and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies, Academic Press, San Diego (1993).

[00291] An exemplary process for the production of recombinant chimeric antibodies may include the following: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody heavy chain in which the CDRs and the variable region of an anti-Siglec-15 antibody are fused to an Fc region derived from a human immunoglobulin, thereby producing a vector for the expression of a chimeric antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody light chain of the murine anti-human Siglec-15 monoclonal antibody, thus producing a vector for the expression of the chimeric antibody light chain; c) transferring the expression vectors to a host cell by conventional molecular biology methods to Petition 870200066611, dated 05 / 28 / 2020, pp. 119 / 280 113 / 217 produce a transfected host cell for the expression of chimeric antibodies; ed) culture the transfected cell by conventional cell culture techniques in order to produce chimeric antibodies.

[00292] An exemplary process for the production of recombinant humanized antibodies may include the following: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an anti-human Siglec-15 heavy chain in which the CDRs and a minimal portion of the variable structure region that are necessary to retain donor antibody binding specificity are derived from humanized variants of anti-human Siglec-15 antibody(ies), and the remainder of the antibody is derived from a human immunoglobulin, thus producing a vector for the expression of a humanized antibody heavy chain;b) construct, using conventional molecular biology methods, an expression vector that encodes and expresses an antibody light chain in which the CDRs and a minimal portion of the variable region structure necessary to retain the donor antibody binding specificity are derived from a non-human immunoglobulin, such as the disclosed murine anti-human Siglec15 antibodies, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for the expression of the humanized antibody light chain; c) transfer the expression vectors to a host cell using conventional molecular biology methods to produce a host cell transfected for the expression of humanized antibodies; d) culture the transfected cell using conventional cell culture techniques to produce humanized antibodies.

[00293] With regard to either of the two exemplary methods, host cells can be co-transfected with such expression vectors, which may contain different selectable markers, but, with the exception of the heavy and light chain coding sequences, may be identical. This procedure provides equal expression of polypeptides from Petition 870200066611, dated 05 / 28 / 2020, pp. 120 / 280 114 / 217 heavy and light chain. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used. The coding sequences for the heavy and light chains can include cDNA or genomic DNA or both. The host cell used to express the recombinant antibody can be a bacterial cell, such as Escherichia coli, or a eukaryotic cell (e.g., a Chinese hamster ovary (CHO) cell or a HEK-293 cell). 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).

[00294] Any of the disclosed antibodies can be used to generate anti-idiotype antibodies using techniques well known to those skilled in the art (see, for example, Greenspan, NS et al. (1989) “Idiotypes: Structure And Immunogenicity,” FASEB J. 7:437-444; and Nisinoff, A. (1991) “Idiotypes: Concepts And Applications,” J. Imunol. 147(8):2429-2438). C. Ligand-Binding Molecules of Siglec-15

[00295] Molecules that bind to Siglec-15 ligands, such as Siglec-15 proteins, Siglec-15 fusion proteins, and their fragments and variants, are also provided. The Siglec-15 ligand-binding molecule can bind to Siglec-15 ligands such as a sialylated glycoprotein, LRRC4C, a Siglec-15 counter-receptor, etc. In some embodiments, the Siglec-15 ligand-binding molecule can induce signal transduction through the Siglec-15 ligand. In some embodiments, the Siglec-15 ligand-binding molecule blocks or reduces the interaction between Siglec-15 and its ligand, without inducing signal transduction through Siglec-15 or its ligand. Siglec-15 ligand-binding molecules can be used to modulate Siglec-15 activity as discussed. Petition 870200066611, dated 05 / 28 / 2020, pp. 121 / 280 115 / 217 in more detail below and illustrated in the Examples, and can be used to therapeutically treat a subject in need of it. 1. Siglec-15 Polypeptides

[00296] 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 molecules include a polypeptide that is 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, such as the extracellular domain, or a subdomain thereof, such as the IgV domain, the IgC domain, or a combination thereof. In some embodiments, the Siglec-15 polypeptide is soluble or otherwise cell-free. For example, in some embodiments, Siglec-15 lacks one or more of the transmembrane domain, the cytoplasmic domain, or the leader sequence. 2. Siglec-15 Fusion Proteins

[00297] In some embodiments, the Siglec-15 ligand-binding molecule is a Siglec-15 fusion protein. Fusion proteins containing Siglec-15 polypeptides coupled to other polypeptides to form fusion proteins are provided. Siglec-15 fusion polypeptides may have a first fusion partner comprising all or part of a Siglec-15 protein fused (i) directly to a second polypeptide or, (ii) optionally, fused to a linker peptide sequence that is fused with the second polypeptide. The fusion proteins optionally contain a domain that functions to dimerize or multimerize two or more fusion proteins. In some embodiments, the fusion protein is not or does not dimerize or multimerize. The peptide / polypeptide linker domain may be a separate domain or, alternatively, may be contained within one of the other domains (first polypeptide or second Siglec-15 polypeptide) of the fusion protein.Similarly, the domain that functions to dimerize or multimerize fusion proteins may be a domain. Petition 870200066611, dated 05 / 28 / 2020, pages 122 / 280 116 / 217 separate or, alternatively, it may be contained within one of the other domains (Siglec-15 polypeptide, second polypeptide, or polypeptide / peptide linker domain) of the fusion protein. In some embodiments, the dimerization / multimerization domain and the peptide / polypeptide linker domain are the same.

[00298] The fusion proteins described in this document are of 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 a Siglec-15 polypeptide, “R2” is an optional peptide / polypeptide binding domain, and “R3” is a second polypeptide. Alternatively, R3 could be the Siglec-15 polypeptide and R1 could be the second polypeptide.

[00299] Fusion proteins can be dimerized or multimerized. Dimerization or multimerization can occur between two or more fusion proteins through dimerization or multimerization domains. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical cross-linking. The dimers or multimers that are formed can be homodimeric / homomultimeric or heterodimeric / heteromultimeric. As discussed above, in some embodiments, the fusion protein is not or does not dimerize or multimerize.

[00300] In some embodiments, the second polypeptide contains one or more domains of a constant region of the immunoglobulin heavy chain, for example, an amino acid sequence corresponding to the hinge regions, CH2 and / or CH3 of a human immunoglobulin Cy1 chain, the hinge regions, CH2 and / or CH3 of a murine immunoglobulin CY2a chain, CH2 and / or CH3 regions of a human immunoglobulin Cy1, etc.

[00301] The Fc portion of the fusion protein can be varied by isotype. Petition 870200066611, dated 05 / 28 / 2020, pp. 123 / 280 117 / 217 or subclass, may be chimeric or hybrid, and / or may be modified, for example, to improve effector functions, half-life control, tissue accessibility, increase biophysical characteristics such as stability, and improve production efficiency (and be less expensive). Many useful modifications in the construction of disclosed fusion proteins and methods for producing them are known in the art, see, for example, 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 the native Fc region of IgG1, IgG2, or IgG4. In some forms, the Fc region is a hybrid, for example, a chimeric region consisting of constant Fc regions of IgG2 / IgG4.Modifications to the Fc region include, but are not limited to, modified IgG4 to prevent binding to Fc gamma and complement receptors, modified IgG1 to enhance binding to one or more Fc gamma receptors, modified IgG1 to minimize effector function (amino acid alterations), IgG1 with / without altered glycan (typically altering the host of expression), and IgG1 with pH-dependent alteration to FcRn. The Fc region may include the entire hinge region, or less than the entire hinge region.

[00302] The therapeutic outcome in patients treated with rituximab (chimeric mouse / human IgG1 monoclonal antibody against CD20) for non-Hodgkin lymphoma or Waldenstrom's macroglobulinemia correlated with the individual expression of allelic variants of Fcy receptors with distinct intrinsic affinities for the Fc domain of human IgG1. In particular, patients with high-affinity alleles of the low-affinity activating Fc receptor CD16A (FcyRIIIA) showed higher response rates and, in cases of non-Hodgkin lymphoma, improved progression-free survival. In another modality, the Fc domain may contain one or more amino acid insertions, deletions, or substitutions that Petition 870200066611, dated 05 / 28 / 2020, pages 124 / 280 118 / 217 reduce binding to the low-affinity inhibitory Fc receptor CD32B (FcyRIIB) and retain wild-type binding levels or increase binding to the low-affinity activating Fc receptor CD16A (FcyRIIIA).

[00303] Another embodiment includes IgG2-4 hybrids and IgG4 mutants that reduce FcR binding, increasing its half-life. 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 Immunology, 34(6): 441-452 (1997); and US Patent No. 6982323 by Wang et al. In some embodiments, the IgG1 and / or IgG2 domain is deleted, for example, Angal et al. They describe IgG1 and IgG2 having serine 241 replaced by proline.

[00304] In some embodiments, the Fc domain contains amino acid insertions, deletions, or substitutions that increase CD16A binding. A large number of substitutions in the human IgG1 Fc domain that increase CD16A binding and reduce CD32B binding are known in the art and are described in Stavenhagen et al., Cancer Res., 57 (18): 888-290 (2007). Exemplary variants of human IgG1 Fc domains with reduced CD32B binding and / or enhanced CD16A binding contain F243L, R929P, Y300L, V305I, or P296L substitutions. These amino acid substitutions may be present in a human IgG1 Fc domain in any combination. In one embodiment, the variant of the human IgG1 Fc domain contains an F243L, R929P, and Y300L substitution. In another embodiment, the human IgG1 FC domain variant contains a substitution of F243L, R929P, Y300L, V305I, and P296L.In another variant, the human IgG1 Fc domain contains an N297Q substitution, since this mutation abolishes FcR binding.

[00305] The disclosed fusion proteins optionally contain a peptide or polypeptide linker domain that separates the Siglec-15 polypeptide from the second polypeptide. In some embodiments, the domain Petition 870200066611, dated 05 / 28 / 2020, pages 125 / 280 The 119 / 217 ligand contains the hinge region of an immunoglobulin. In a preferred embodiment, the hinge region is derived from a human immunoglobulin. Suitable human immunoglobulins from which the hinge can be derived include IgG, IgD, and IgA. In a preferred embodiment, the hinge region is derived from human IgG. The amino acid sequences of the immunoglobulin hinge regions and other domains are well known in the art.

[00306] An exemplary fusion protein is the Fc Siglec15 ECD-IgG1 Fusion Protein (L234F / L235E / P331S). MEWSWVFLFFLSVTTGVHSEVRTKIDTTENLLNTEVHSSPAQR WSMQVPPEVSAEAGDAAVLPCTETHPHRHYDGPLTAIWRAGEPYAG PQVERCAAARGSELCQTALSLHGRERLLGNPRRNDLSLRVERLALAD DRRYECRVEEAGDVHDRYESRHGVRLHVTAAPRIVNISVLPSPAHAE RALCTAEGEPPPALAWSGPALGNSLAAVRSPREGHGHLVTAELPALT HDGRYTCTAANSLGRSEASVYLEREHGASGDKTHTCPPPCPAPEFEGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG (SEQ ID NO:193).

[00307] A Murine Leader Sequence is illustrated in underline. An extracellular Siglec-15 (ECD) domain is in italics. A hinge region is double underlined. The remaining sequence is derived from the Fc of IgG1. L234F / L235E / P331S mutations in the Fc domain of IgG1 are in bold and dotted-underlined.

[00308] In some embodiments, the leader sequence is cleaved or absent from the fusion protein. For example, the fusion protein may have the sequence: EVRTKIDTTENLLNTEVHSSPAQRWSMQVPPEVSAEAGDAAVL Petition 870200066611, dated 05 / 28 / 2020, pages 126 / 280 120 / 217 pctfthphrhydgpltaiwragepyagpqvfrcaaargselcqtals lhgrfrllgnprrndlslrverlaladdrryfcrvefagdvhdryes rhgvrlhvtaaprivnisvlpspahafralctaegepppalawsgpal gnslaavrspreghghlvtaelpalthdgrytctaanslgrseasvy lfrfhgasgdkthtcppcpapefeggpsvflfppkpkdtlmisrtpevt CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:194).

[00309] 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.

[00310] In some embodiments, the leader sequence, the linker (e.g., the hinge region), the second fusion partner (e.g., the Fc domain of IgG1), or a combination thereof are substitutes for other hinge sequence(s), Fc domain, etc. Suitable substitutes are well known in the art. See, for example, U.S. Patent No. 9,005,616, which is specifically incorporated by reference in its entirety. 3. Nucleic Acids and Siglec-15 Cells

[00311] Vectors encoding Siglec-15 polypeptides, their fragments and fusions are also provided. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. Thus, cells containing and expressing Siglec-15 polypeptides, their fragments and fusions are also provided. As used in this document, a vector is a replicon, such as a plasmid, phage, virus, or cosmid, into which another DNA segment can be inserted in order to trigger replication of the inserted segment. Vectors can be expression vectors. An “expression vector” is a vector that includes one or more Petition 870200066611, dated 05 / 28 / 2020, pages 127 / 280 121 / 217 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.

[00312] Nucleic acids in vectors can be operationally linked to one or more expression control sequences. As used in this document, operationally linked means incorporated into a genetic construct such that the expression control sequences 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, typically 100 nucleotides upstream of the point where transcription begins (usually near the RNA polymerase II initiation site). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter.Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can act when situated at various distances from the transcription site. An enhancer can also be located downstream of the transcription initiation site. A coding sequence is operationally linked and under the control of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which can then be translated into the protein encoded by the coding sequence.

[00313] Suitable expression vectors include, without limitation, plasmids and viral vectors derived, for example, from a bacteriophage, baculovirus, tobacco mosaic virus, herpesvirus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus and virus Petition 870200066611, dated 05 / 28 / 2020, pages 128 / 280 122 / 217 adeno-associated. Numerous vectors and expression systems are commercially available from corporations such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[00314] An expression vector may include a sequence of tags. The tag sequences are typically expressed as a fusion with the encoded polypeptide. These tags can be inserted anywhere within the polypeptide, including at the carboxyl or amino termini. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tagging (Kodak, New Haven, CT), maltose-binding protein, and protein A. In one embodiment, a nucleic acid molecule encoding a disclosed Siglec15 fusion polypeptide is present in a vector containing nucleic acids encoding one or more domains of an Ig heavy chain constant region, for example, with an amino acid sequence corresponding to the hinge, CH2, and CH3 regions of a human immunoglobulin Cy1 chain.

[00315] Vectors containing nucleic acids to be expressed can be transferred into host cells. The term “host cell” is intended to include both prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used in this document, transformed and transfected encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of several techniques. While not limited to one particular technique, several of these techniques are well established in the art. Prokaryotic cells can be transformed with nucleic acids, for example, by electroporation or calcium chloride-mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques, Petition 870200066611, dated 05 / 28 / 2020, pages 129 / 280 123 / 217 including, for example, calcium phosphate co-precipitation, DEAE-dextran mediated transfection, lipofection, electroporation or microinjection. Host cells (e.g., a prokaryotic cell or a eukaryotic cell, such as a CHO cell) can be used to, for example, produce the Siglec-15 fusion polypeptides described in this document.

[00316] The vectors described can be used to express Siglec15 in cells. An exemplary vector includes, but is not limited to, an adenoviral vector. One approach includes nucleic acid transfer into primary cells in culture, followed by autologous transplantation of the transformed cells ex vivo into the host, either systemically or into a particular organ or tissue. Ex vivo methods may include, for example, the steps of collecting cells from an individual, culturing the cells, transducing them with an expression vector, and maintaining the cells under suitable conditions for expression of the encoded polypeptides. These methods are known in molecular biology techniques. The transduction step can be achieved by any conventional means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolytic gene transfer. Alternatively, liposomes and polymeric microparticles can be used.Cells that have been successfully transduced 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 implanted into the individual. In one embodiment, expression vectors containing nucleic acids encoding the fusion proteins are transfected into cells that are administered to a subject in need.

[00317] In vivo nucleic acid therapy can be performed by direct transfer of functionally active DNA into mammalian somatic tissue or organs in vivo. For example, nucleic acids that encode Petition 870200066611, dated 05 / 28 / 2020, pages 130 / 280 124 / 217 polypeptides described in this document can be administered directly to lymphoid tissues or tumors. Alternatively, specific targeting to lymphoid tissue can be achieved using lymphoid tissue-specific transcriptional regulatory elements (TREs), such as B lymphocyte, T lymphocyte, or dendritic cell-specific TREs. Lymphoid tissue-specific TREs are known in the art.

[00318] Nucleic acids can also be administered in vivo via viral means. Nucleic acid molecules encoding fusion proteins can be packaged into retroviral vectors using packaging cell lines that produce replication-defective retroviruses, as known in the art. Other viral vectors can also be used, including recombinant adenoviruses and vaccinia viruses, which can be transformed into non-replicating vectors. In addition to pure DNA or RNA, or viral vectors, engineered bacteria can be used as vectors.

[00319] Nucleic acids can also be administered via other vehicles, including liposomes, polymeric micro- and nanoparticles, and polycations such as asialoglycoprotein / polylysine.

[00320] In addition to carrier-mediated gene transfer in vivo, well-known physical means in the technique can be used for direct DNA transfer, including plasmid DNA administration and particle bombardment-mediated gene transfer. D. Pharmaceutical Compositions

[00321] Pharmaceutical compositions including the disclosed Siglec-15 binding molecules are provided. Pharmaceutical compositions containing a Siglec-15 binding molecule may be for parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous), transdermal (passively or using ionophoresis or electroporation) or transmucosal (nasal, vaginal, rectal or sublingual) administration or using bioerodible inserts and may be formulated Petition 870200066611, dated 05 / 28 / 2020, pp. 131 / 280 125 / 217 in dosage forms appropriate for each route of administration.

[00322] In some in vivo approaches, the compositions disclosed in this document are administered to a subject in a therapeutically effective amount. As used in this document, the term “effective amount” or “therapeutically effective amount” denotes a dosage sufficient to treat, inhibit, or alleviate one or more of the symptoms of the disorder being treated or otherwise provide a desired pharmacological and / or physiological effect. The precise dosage varies according to a variety of factors, such as individual-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment to be performed.

[00323] For the disclosed Siglec-15 binding molecules, as new studies are conducted, information will emerge regarding appropriate dosage levels for the treatment of various conditions in various patients, and those skilled in the art, considering the therapeutic context, age, and general health status of the recipient, will be able to determine the appropriate dosage. The selected dosage depends on the desired therapeutic effect, the route of administration, and the desired duration of treatment. In general, for intravenous injection or infusion, the dosage may be lower.

[00324] 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 may be, for example, between 0.01 mg / kg and 20 mg / kg, 0.01 mg / kg and 10 mg / kg, 0.01 mg / kg and 5 mg / kg, 0.01 and 2 mg / kg, 0.01 and 1 mg / kg, 0.01 mg / kg and 0.75 mg / kg, 0.01 mg / kg and 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. Specific exemplary 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. A dose as low as 0.01 mg / kg is believed to be adequate. Petition 870200066611, dated 05 / 28 / 2020, pp. 132 / 280 126 / 217 to have appreciable pharmacodynamic effects. Dose levels of 0.10-1 mg / kg are anticipated to be the most appropriate. Higher doses (e.g., 1-30 mg / kg) would also be anticipated to be active.

[00325] Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to foreign polypeptides. Thus, lower doses of human antibodies and less frequent administration are often possible. Furthermore, the dosage and frequency of administration of antibodies of the invention or their fragments can be reduced by increasing the absorption and tissue penetration of the antibodies through modifications such as, for example, lipidation.

[00326] In certain embodiments, the Siglec-15 binding molecule is administered locally, for example, by injection directly into a treatment site. Typically, injection causes an enhanced localized concentration of the Siglec-15 binding molecule composition that is greater than that which can be achieved by systemic administration. Siglec-15 binding molecule compositions may be combined with a matrix as described below to assist in creating an enhanced localized concentration of the polypeptide compositions by reducing passive diffusion of the polypeptides out of the treatment site. 1. Formulations for parenteral administration

[00327] In some embodiments, the compositions disclosed in this document are administered in an aqueous solution, by injection or parenteral infusion. The formulation may also be in the form of a suspension or emulsion.

[00328] In general, pharmaceutical compositions are provided including effective amounts of a Siglec-15 binding molecule and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Petition 870200066611, dated 05 / 28 / 2020, pp. 133 / 280 127 / 217 These compositions optionally include one or more of the following: diluents, sterile water, buffered saline solution of varying buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking agents (e.g., lactose and mannitol). Examples of non-aqueous solvents or vehicles include: propylene glycol, polyethylene glycol, vegetable oils such as olive and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use.The formulation can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. 2. Controlled-management polymer matrices

[00329] The Siglec-15 binding molecules disclosed in this document can also be administered in controlled-release formulations. Controlled-release polymeric devices can be made for long-term systemical release following implantation of a polymeric device (a rod, a cylinder, a film, or a disc) or injections (microparticles). The matrix can be in the form of microparticles, such as microspheres, where the agent is dispersed within a solid polymeric matrix, or microcapsules, where the core is of a different material than the polymeric shell, and the peptide is dispersed or suspended in the core, which can be liquid or solid in nature. Unless specifically defined in this document, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer can be molded as a plate or thin film, ranging from nanometers to Petition 870200066611, dated 05 / 28 / 2020, pp. 134 / 280 128 / 217 four centimeters, a powder produced by grinding or other conventional techniques or even a gel, such as a hydrogel.

[00330] Non-biodegradable or biodegradable matrices can be used for the delivery of fusion polypeptides or nucleic acids encoding fusion polypeptides. These can be natural or synthetic polymers. Synthetic polymers typically have better characterization of degradation and release profiles. The polymer is selected based on the period during which release is desired. In some cases, linear release may be more useful, while in others a pulsed release or bulk release may provide more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% by weight of water) and may optionally be crosslinked with ions or multivalent polymers.

[00331] The matrices 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 for producing microspheres for drug delivery, as described, for example, 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).

[00332] The devices can be formulated for release in order to treat the implant or injection area – which will generally administer a dosage that is much lower than the dosage for treating a whole body – or systemic administration. They can be implanted or injected subcutaneously, into the muscle, into the fat, or swallowed. III. Method of Use

[00333] Methods for using the disclosed Siglec-15 binding molecules and Siglec-15 ligand binding are also provided. Uses of Petition 870200066611, dated 05 / 28 / 2020, pages 135 / 280 129 / 217 Such molecules are used to enhance immune responses, slow or prevent tumor growth, inhibit tumor-mediated immunosuppression, eliminate tumors, and / or deplete or block the activity of tumor-associated macrophages (TAMs) in order to alter their activity, decrease TAM-mediated immunosuppression, reduce or reverse T-cell suppression, and / or are disclosed. Uses of such molecules in the diagnosis and treatment of cancer and other diseases are also provided.

[00334] TAMs provide a link between inflammation and cancer. Macrophages are immune system cells derived from activated blood monocytes. They are primarily recognized as participants in inflammatory responses induced by pathogens or tissue damage, acting to remove (i.e., phagocytosis) pathogens, dead cells, cellular debris, and various components of the extracellular matrix (ECM). Macrophages have been found to constitute an important constituent in the tumor microenvironment and represent up to 50% of the tumor mass.

[00335] In addition to mediating phagocytosis, macrophages secrete pro-angiogenic growth factors and matrix remodeling proteases and therefore play a role in the development of the vascular infrastructure (angiogenesis) necessary for tumor development and growth (Pollard, JW, (2009). Nat. Rev. Immunol. 9:259-270). The presence of macrophages within a tumor appears to aid tumor growth. Several studies provide evidence that the presence of tumor-associated macrophages within the tumor is a negative prognostic factor for survival (Farinha, P. et al. (2005), Blood 106:2169-2174; Dave, SS 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 facilitate 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. Petition 870200066611, of 28 / 05 / 2020, p. 136 / 280 130 / 217 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).

[00336] Studies show that Siglec-15 is inductively expressed in TAMs and increases TGF-β secretion through coupling tumor cell recognition with the DAP12Syk signal transduction pathway (Takamiya, et al., Glycobiology, 23 (2): 178-87 (2013). In a specific model proposed 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 increases TGF-β production from macrophages via the DAP12Syk pathway, tilting the tumor microenvironment towards immunosuppression and thus tumor progression and even metastasis.

[00337] The binding of anti-Siglec-15 antibodies to Siglec-15 can result in the reduction, blocking, antagonization, attenuation, or complete abolition of Siglec-15's ability to bind to one or more ligands and thus decrease or prevent inhibitory immune signaling, including but not limited to Siglec-15-mediated TGF-β secretion. Increased TGF-β expression is frequently correlated with the malignancy of many cancers and with a defect in the cell growth inhibition response to TGF-β, leading to immunosuppression-based oncogenesis. Reducing Siglec-15-mediated TGF-β secretion leads to an overall increase in immune responses and a direct or indirect decrease in tumor progression in the subject.

[00338] The examples below show that anti-Siglec-15 function-reducing and blocking antibodies, including those disclosed in this document, can reverse Siglec-15-mediated suppression of CD4+ and CD8+ T cell proliferation in a peripheral blood mononuclear cell (PBMC) proliferation assay. Petition 870200066611, dated 05 / 28 / 2020, pp. 137 / 280 131 / 217

[00339] Thus, methods of reducing immunosuppression and / or increasing an immune response, most typically through the administration to a subject in need thereof of an effective amount of anti-Siglec-15 function-blocking antibody.

[00340] Suitable antibodies, polypeptides, and fusion proteins are disclosed in this document and may be further selected by in vitro assays including but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and in vivo assays such as tumor growth inhibition. Exemplary assays for testing the function of the disclosed antibodies are provided in the Examples below.

[00341] The antibodies provided in this document may also be useful in diagnostic and research applications. For example, non-neutralizing antibodies can bind to the specific antigen without inhibiting the biological or receptor-binding activity of the antigen, and can be used in capture assays and other pull-downs (e.g., ELISA). Neutralizing antibodies (e.g., function blockers) may be useful in competitive binding assays.

[00342] Antibodies can also be used to quantify Siglec-15 polypeptides or their ligand. A. Molecules that Enhance the Immune Response 1. Therapeutic and Prophylactic Uses

[00343] Therapeutic and / or prophylactic use of molecules (especially antibodies or their antigen-binding fragments) that bind immunospecifically to human Siglec-15 molecules and to Siglec-15 ligands and that are capable of reducing the binding between Siglec-15 and one or more of its ligands and / or counter-receptors are provided (e.g., antagonist molecules).

[00344] In some forms, the molecules reduce or prevent Petition 870200066611, dated 05 / 28 / 2020, pp. 138 / 280 132 / 217 The binding between Siglec-15 and a sialylated glycoprotein ligand, for example sialylated glycoproteins endogenously expressed by a subject's cells, and reduces or prevents Siglec-15-mediated signal transduction. Additionally or alternatively, the molecules may reduce or prevent the binding between Siglec-15 and a counter-receptor thereof, and reduce or prevent Siglec-15-mediated signal transduction and / or signal transduction through the counter-receptor. For example, the disclosed molecules may bind to antigens 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 binding site important for binding to a Siglec-15 counter-receptor. Exemplary compositions are illustrated in the Examples below. For example, in some disciplines, 5G12, 6F8, 8C8, 1C3, 1C12, 3H10 and 1B2 are considered "strong blockers" and 10G9, 8H8 and 9A5 are considered "partial blockers".Therefore, in some embodiments, the molecules used to modulate an immune response include 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 molecules include the variable region of the light chain and / or the variable region of the heavy chain of 5G12, 6F8, 8C8, 1C3, 1C12, 3H10, 1B2, 10G9, 8H8, or 9A5. In some forms, the 5G12, 6F8, 8C8, 1C3, 1C12, 3H10, 1B2, 10G9, 8H8 or 9A5 anti-human murine genome, or a chimeric or humanized variant thereof.

[00345] As discussed above, interactions between Siglec-15 and sialylated glycoprotein ligands and / or Siglec-15 counter-receptors can inhibit T cell proliferation and increase cytokine production, such as TGFβ. Thus, in some embodiments, administration of disclosed Siglec-15 binding molecules to a subject modulates the subject's upstream immune system by blocking or otherwise antagonizing the binding / interaction of the Siglec-15 ligand and / or counter-receptor. In another embodiment, the avidity and / or affinity of the anti-Siglec-15 antibody may be such that it binds only to Petition 870200066611, dated 05 / 28 / 2020, pp. 139 / 280 133 / 217 cells that express very high levels of Siglec-15, which are, for example, tumor-associated macrophages (TAMs) or cancer cells, thus allowing specific targeting of this cell population. Thus, in some modalities, the molecules reduce the production of TGF-β, for example, in a tumor microenvironment. In some modalities, the cells expressing Siglec-15 are monocytes. In more specific modalities, the cells expressing Siglec-15 are macrophages, for example TAMs.

[00346] As indicated above, the released antibodies and antigen-binding fragments can bind to and substantially block TAMs in order to modulate their immunosuppressive activity. Furthermore, such antibodies can be used to deplete Siglec-15 TAMs within the tumor microenvironment, or deplete their concentration in peripheral blood. In one embodiment, this modulation or depletion is achieved using Siglec-15 antibodies that bind to a site so as to impair or disrupt the normal function of Siglec-15. As a consequence of such disruption, TAM activity is decreased (modulated) and / or the actual or effective (functional) concentration of macrophages in the tumor is depleted. Alternatively, such modulation or depletion is achieved using anti-Siglec-15 antibodies that are conjugated with a toxin, such that their binding to a TAM leads to macrophage death.

[00347] Additionally, the disclosed antagonist molecules can be used to induce, enhance, or improve T cell proliferation. In some modalities, the T cell response is induced by reducing or preventing Siglec-15 from binding to a counter-receptor on the T cell. Positive modulation of the immune system is particularly desirable in the treatment of cancers and chronic infections, and thus the disclosed compositions can be used in the treatment of such disorders. 2. Subjects to be Treated Petition 870200066611, dated 05 / 28 / 2020, pages 140 / 280 134 / 217 a. Cancer Treatment

[00348] The disclosed compositions and methods for reducing function can be used to treat cancer. Generally, the methods involve stimulating or enhancing an immune response to cancer, reducing or preventing tumor growth or progression, or a combination thereof in the subject, by administering to the subject a quantity of a Siglec-15 linker. The method may reduce one or more symptoms of cancer.

[00349] Cancer cells acquire a characteristic set of functional capabilities during their development, albeit through various mechanisms. These capabilities include evasion of apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion / metastasis, unlimited explanatory potential, and sustained angiogenesis. The term "cancer cell" should encompass both pre-malignant and malignant cancer cells. In some modalities, cancer refers to a benign tumor that has remained localized. In other modalities, cancer refers to a malignant tumor that has invaded and destroyed neighboring body structures and spread to distant sites. In other modalities, cancer is associated with a specific cancer antigen (e.g., pan-carcinoma antigen (KS 1 / 4), ovarian carcinoma antigen (CA125), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2 / neu, etc.).).

[00350] The methods and compositions described in this document are useful in the treatment or prevention of a variety of cancers or other abnormal proliferative diseases, including (but not limited to) the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berketts lymphoma; tumors Petition 870200066611, dated 05 / 28 / 2020, pages 141 / 280 135 / 217 hematopoietic stem cells of the myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xenoderma pegmentosum, keratoactanthoma, seminoma, follicular thyroid carcinoma, and teratocarcinoma.

[00351] Cancers caused by aberrations in apoptosis can also be treated by the disclosed methods and compositions. Such cancers may include, but are not limited to, follicular lymphomas, carcinomas with p53 mutations, hormone-dependent breast, prostate and ovarian tumors, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes. In specific modalities, malignant or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or prevented by the methods and compositions in the ovary, bladder, breast, colon, lung, skin, pancreas or uterus. In other specific modalities, sarcoma, melanoma or leukemia is treated or prevented by the methods and compositions.

[00352] The disclosed compositions and methods are particularly useful for the treatment of cancers that are associated with cells that express abnormally high levels of Siglec-15 itself or Siglec-15 glycoprotein ligands, cancers with high numbers of tumor-associated macrophages, especially if the macrophages express Siglec-15, and / or a cancer in which other cell type(s) express(s) high levels of Siglec-15 or Siglec-15 ligands.

[00353] Specific cancers and related disorders that can be treated or prevented by methods and compositions disclosed in this Petition 870200066611, dated 05 / 28 / 2020, pages 142 / 280 136 / 217 document includes, but is not limited to, leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic (granulocytic) myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, such as, but not limited to, Hodgkin's disease or non-Hodgkin's lymphomas (e.g., diffuse anaplastic lymphoma ALK-negative kinase, large B-cell lymphoma (DLBCL); diffuse anaplastic lymphoma ALK-positive kinase, large B-cell lymphoma (DLBCL); anaplastic lymphoma (ALK)-positive kinase, anaplastic large B-cell lymphoma ALK+ (ALCL), acute myeloid lymphoma (AML));Asymptomatic multiple myelomas, such as, but not limited to, asymptomatic multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary myelomatosis and extramedullary myelomatosis; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; Bone and connective tissue sarcomas, such as, but not limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, bone fibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;Brain tumors, including but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, breast cancer; Petition 870200066611, dated 05 / 28 / 2020, pages 143 / 280 137 / 217 mucinous, tubular breast cancer, papillary breast cancer, Paget's disease and inflammatory breast cancers; adrenal cancer, including but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including but not limited to, insulinoma, gastrinoma, glaucoma, vipoma, somatostatin-secreting tumor and carcinoid or islet cell tumor; pituitary cancers, including but not limited to, Cushing's disease, prolactin-secreting tumor, acromegaly and diabetes insipidus; eye cancer, including but not limited to, ocular melanoma such as choroidal melanoma, iris melanoma and ciliary body melanoma and retinoblastoma; Types of vaginal cancers, including but not limited to adenocarcinoma, squamous cell carcinoma, and melanoma;Vulvar cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancers, including but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers, including but not limited to epithelial ovarian carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat grain (small cell) carcinoma; stomach cancers, including but not limited to adenocarcinoma, fungal (polypoid), ulcerative, superficial spread, diffuse spread, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers;Liver cancers including, but not limited to, hepatocellular carcinoma and hepatoblastoma; gallbladder cancers including, but not limited to, adenocarcinoma; cholangiocarcinomas including, but not limited to, papillary, nodular, and diffuse; lung cancers including, but not limited to, non-small cell lung cancer; Petition 870200066611, dated 05 / 28 / 2020, pp. 144 / 280 138 / 217 squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer; testicular cancers including, but not limited to, germ cell tumor, seminoma, anaplastic, classic (typical), spermatocytic, non-seminoma, embryonal carcinoma, teratoma, choriocarcinoma (embryo sac tumor), prostate cancers including, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancers; oral cavity cancers including, but not limited to, squamous cell carcinoma; basal cell carcinomas; salivary gland cancers including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma and adenoid cystic carcinoma; pharyngeal cancers including, but not limited to, squamous cell carcinoma and verrucous carcinoma;Skin cancers including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma; kidney cancers including, but not limited to, renal cell carcinomas, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter); Wilms' tumor;Bladder cancers including, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliolosarcoma, lymphangioendothelisarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinomas (for a review of these disorders, see Fishman et al., 1985, Medicine, 2nd 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). b. Treatment of Infections Petition 870200066611, dated 05 / 28 / 2020, pages 145 / 280 139 / 217

[00354] The disclosed compositions and methods for reducing function can be used to treat infections and infectious diseases. Generally, the methods involve stimulating or enhancing an immune response to an infectious agent, reducing or preventing the progression of infectious disease, or a combination thereof in the subject, by administering to the subject an amount of a Siglec-15 binding molecule. The method may reduce one or more symptoms of the infection.

[00355] The infection or disease may be caused by a bacterium, virus, protozoan, helminth, or other microbial pathogen that enters intracellularly and is attacked, that is, by cytotoxic T lymphocytes.

[00356] Infection or disease can be acute or chronic. An acute infection is typically a short-lived infection. During an acute microbial infection, immune cells begin to express immunomodulatory receptors. Therefore, in some modalities, the method involves increasing an immune-stimulating response against an acute infection.

[00357] 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.

[00358] 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, causing the infection to remain with the host for an extended period of time.

[00359] Examples of infections to be treated are chronic infections caused by a hepatitis virus, a human immunodeficiency virus (HIV), a human T-lymphotropic virus (HTLV), a herpes virus, Petition 870200066611, dated 05 / 28 / 2020, pages 146 / 280 140 / 217 an Epstein-Barr virus or a human papillomavirus.

[00360] Given that viral infections are primarily cleared by T cells, an increase in T cell activity would be therapeutically useful in situations where faster or more complete elimination of an infectious viral agent would be beneficial to an animal or human subject. Thus, the disclosed compositions can be administered for the treatment of 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 A virus) and the common cold (e.g., human rhinovirus), and other viral infections caused by, for example, HTLV, hepatitis virus, respiratory syncytial virus, vaccinia virus, and rabies virus. The molecules can be administered topically to treat viral skin conditions such as herpes or shingles lesions, or genital warts.The molecules can also be administered systemically to treat systemic viral diseases, including but not limited to AIDS, influenza, the common cold, or encephalitis.

[00361] Representative treatable infections include, but are not limited to, infections caused by microorganisms including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Haemophilus influenzae type B (HIB), Hyphomicrobium, Leptospirosis, Legionella, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococci, Streptococci, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus and Petition 870200066611, dated 05 / 28 / 2020, pp. 147 / 280 141 / 217 Treponema, Vibrio, Yersinia, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis e Schistosoma mansoni.

[00362] Other microorganisms that can be treated with the disclosed compositions and methods include bacteria, such as those of the pathogens Klebsiella, Serratia, Pasteurella; associated with cholera, tetanus, botulism, anthrax, plague and Lyme disease; or fungal or parasitic pathogens, such as Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus, Aspergillus (fumigatus, Niger, etc.), Mucorales genus (absidia, mucor, rhizophus), Sporothrix (schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis) and Histoplasma (capsulatuma), Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lamblia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondii, etc.), Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba, Histolytica, Balantidium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis Pneumonia, Plasmodium, Babesia or Trypanosoma, etc. B. Immune Response Reducing Molecules 1. Therapeutic and Prophylactic Uses

[00363] Therapeutic and / or prophylactic use of molecules (especially antibodies or their antigen-binding fragments) that bind to human Siglec-15 or a ligand thereof and that are capable of increasing or enhancing the binding between Siglec-15 and one or more of its ligands and / or counter-receptors are provided (e.g., molecules Petition 870200066611, dated 05 / 28 / 2020, pp. 148 / 280 142 / 217 agonists) either directly increase or enhance signal transduction mediated by Siglec-15 or Siglec-15 counter-receptors.

[00364] As discussed above, interactions between Siglec-15 and sialylated glycoprotein ligands and / or Siglec-15 counter-receptors can inhibit T cell proliferation and increase cytokine production, such as TGFβ. Thus, in some modalities, administration of the function that activates Siglec-15 binding molecules or activation of Siglec-15 ligand binding molecules in a subject negatively modulates the subject's immune system by induction or agonization of the Siglec-15 ligand and / or counter-receptor binding / interaction, or by directly stimulating Siglec-15 or Siglec-15 counter-receptor signal transduction. In some modalities, the molecules increase TGF-β production and / or secretion from, for example, monocytes such as macrophages.

[00365] Additionally, the disclosed Siglec-15 agonist-binding molecules and Siglec-15 ligand can be used to reduce or decrease T cell proliferation. In some embodiments, the T cell response is induced by increased or enhanced Siglec-15 binding to a counter-receptor on the T cell. Negative modulation of the immune system is particularly desirable in the treatment of inflammatory and autoimmune diseases and disorders and to treat or prevent graft rejection and / or graft host disease, and thus the disclosed compositions can be used in the treatment of such disorders. a. Inflammatory responses

[00366] In some modalities, Siglec-15 agonist molecules are used to treat or alleviate one or more symptoms of inflammation, for example, acute, chronic, or persistent inflammation.

[00367] An immune response including inflammation can be inhibited or reduced in a subject, preferably in a human, by administering an effective amount of the Siglec-15 binding molecule. Petition 870200066611, dated 05 / 28 / 2020, pp. 149 / 280 143 / 217 to inhibit or reduce the biological activity of an immune cell (e.g., T cells or B cells) or to reduce the amounts of pro-inflammatory molecules at a site of inflammation. Exemplary pro-inflammatory molecules include, but are not limited to, IL-1e, TNF-α, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. b. Hyperinflammatory Response

[00368] In some embodiments, Siglec-15 binding molecules slow down the immune system. For example, a Siglec-15 binding molecule can be used to control an immune-stimulatory response to an infection that is causing damage to healthy tissues through a hyper-inflammatory response. Therefore, in some embodiments, the agents are administered to a subject with an infection who is also experiencing a hyper-inflammatory response. In such cases, controlling excessive immune responses may be beneficial to the subject. c. Inflammatory and Autoimmune Diseases / Disorders -

[00369] The disclosed compositions can also be used to treat inflammatory or autoimmune diseases and disorders.Representative inflammatory or autoimmune diseases / disorders that may be treated include, but are not limited to, 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), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac cell dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia - fibromyositis, Graves' disease, Guillain-Barré syndrome. Petition 870200066611, dated 05 / 28 / 2020, pages 150 / 280 144 / 217 Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, diabetes (Type I), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff man syndrome, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo and Wegener's granulomatosis.

[00370] In some forms, inflammation or autoimmune disease is caused by a pathogen or is the result of an infection. d. Transplants

[00371] Siglec-15 binding molecules can be used to reduce or inhibit transplant rejection in a subject, preferably a human subject. Transplant rejection can be inhibited or reduced in a subject by administering an effective amount of a Siglec-15 agonist binding molecule to inhibit or reduce the biological activity of an immune cell or to reduce the amounts of pro-inflammatory cytokines or other molecules associated with or promoting inflammation at the transplant site.

[00372] The transplanted material may be cells, tissues, organs, limbs, digits, or a portion of the body, preferably the human body. Transplants are typically allogeneic or xenogeneic. A Siglec-15 agonist molecule is typically administered to a subject in an amount effective to reduce or inhibit transplant rejection. The molecule may be administered systemically or locally by any acceptable route of administration. In some embodiments, the molecules are Petition 870200066611, dated 05 / 28 / 2020, pages 151 / 280 145 / 217 administered to a transplant site before, at the time of, or after transplantation.

[00373] The molecules can be administered directly to cells, tissues, or organs to be transplanted ex vivo. In one embodiment, the transplant material is brought into contact with a Siglec-15 binding molecule before transplantation, after transplantation, or both.

[00374] In other modalities, a Siglec-15 binding molecule is administered to immune tissues or organs, such as lymph nodes or the spleen. i. Cells

[00375] Populations of any type of cell can be transplanted into a subject. Cells can be homogeneous or heterogeneous. Heterogeneous means that the cell population contains more than one cell type. Exemplary cells include progenitor cells such as stem cells and pluripotent cells that can be collected from a donor and transplanted into a subject. Cells are optionally treated ex vivo before transplantation. Cells can be autologous or heterologous. ii. Fabrics

[00376] Any tissue can be used as a transplant. Examples of tissues include skin, adipose tissue, cardiovascular tissue such as veins, arteries, capillaries, valves; neural tissue, bone marrow, lung tissue, eye tissue such as corneas and lenses, cartilage, bone, and mucous tissue. The tissue can be modified as discussed above. iii. Organs

[00377] Examples of 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 also be modified before transplantation, such as Petition 870200066611, dated 05 / 28 / 2020, pages 152 / 280 146 / 217 discussed above.

[00378] One embodiment provides a method of inhibiting or reducing chronic transplant rejection in a subject by administering an effective amount of a Siglec-15 binding molecule to inhibit or reduce chronic transplant rejection relative to a control. e. Graft-versus-host disease (GVHD)

[00379] The molecules can also be used to treat graft-versus-host disease (GVHD) by administering an effective amount of a Siglec-15 binding molecule 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 mount an immune attack. It can also occur in a blood transfusion under certain circumstances. Symptoms of GVD include skin rash or changes in skin color or texture, diarrhea, nausea, altered liver function, yellowing of the skin, increased susceptibility to infections, dry and irritated eyes, and dry or sensitive mouth. f. Diabetes

[00380] Siglec-15 agonist-binding molecules can also be used to treat diabetes. The method involves transplanting insulin-producing cells into a subject and administering to the subject an effective amount of a molecule to reduce or inhibit transplant rejection. Preferably, the insulin-producing cells are beta or islet cells. In certain embodiments, the insulin-producing cells are recombinant cells engineered to produce insulin.

[00381] Insulin-producing cells can be encapsulated within a matrix, such as a polymeric matrix, using suitable polymers, including but not limited to alginate, agarose, hyaluronic acid, collagen, synthetic monomers, albumin, fibrinogen, Petition 870200066611, dated 05 / 28 / 2020, pages 153 / 280 147 / 217 fibronectin, vitronectin, laminin, dextran, dextran sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, chitin, chitosan, heparan, heparan sulfate or a combination thereof. C. Therapeutic Inhibition of Osteoclastic Bone Resorption

[00382] Studies show that the expression and function of Siglec-15 are important in 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 specifically incorporated by reference in its entirety). Although the Siglec-15 protein is highly upregulated during osteoclast differentiation, the protein is undetectable in undifferentiated cells. The results indicate that Siglec-15 and DAP12 form a complex at endogenous expression levels in osteoclasts. Siglec-15 knockout mice (- / -) are mildly osteoporotic.

[00383] In vivo studies also show that the antibody Siglec-15 can inhibit osteoclast activity in a physiological context and provide a therapeutic strategy to reduce bone loss. Although the activation of receptors on the cell surface and their endocytic downregulation are often coupled as a means of limiting the intensity and duration of signaling, for Siglec-15 signaling and endocytosis appear to occur exclusively from each other, depending on whether antibody binding induces receptor clustering or simply dimerization.

[00384] Siglec-15 is an intrinsic osteoclast receptor with a highly restricted expression pattern, and therefore, therapies targeting Siglec-15 are selective. Siglec-15 antibodies inhibit osteoclast differentiation at a relatively late stage, thus preserving the coupled communication between osteoclasts and osteoblasts and avoiding complications of existing osteoclast-targeted therapies that can induce osteoclast cell death (bisphosphonates) or prevent their differentiation at an early stage (denosumab) with side effects. Petition 870200066611, dated 05 / 28 / 2020, pages 154 / 280 148 / 217 undesirable side effects include osteonecrosis of the jaw and atypical fractures of the femur.

[00385] Thus, in some embodiments, disclosed molecules (especially antibodies or their antigen-binding fragments) that bind immunospecifically to human Siglec-15 and that are capable of reducing or blocking the binding between Siglec-15 and one or more of its ligands and / or counter-receptors (e.g., agonist molecules) may 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. D. Guidance and Detection

[00386] Disclosed Siglec-15 binding and Siglec-15 ligand-binding molecules, regardless of their effect on Siglec-15 function, can be used to deliver therapeutic cargo and / or detect the presence of Siglec-15 or a ligand thereof, respectively, in cells or tissues. For example, Siglec-15 binding and Siglec-15 ligand molecules can be conjugated to a biological molecule of interest to form a conjugate. Cargo including pharmacologically active molecules, such as inorganic and organic molecules, pharmaceutical agents, drugs, peptides, proteins, genetic material, etc., can be conjugated to Siglec-15 binding or Siglec-15 ligand-binding molecules, which can then direct the cargo to cells or tissues expressing Siglec-15 or a ligand thereof, respectively. Siglec-15 molecules can be chemically linked to a polypeptide by a peptide bond or by a chemical or peptide bonding molecule.Methods for linking a drug or other small pharmaceutical molecule to an antibody fragment are well known and include bifunctional chemical ligands such as NPedition 870200066611, dated 05 / 28 / 2020, page 155 / 280. 149 / 217 succinimidyl (4-iodoacetyl)-aminobenzoate; sulfosuccinimidyl(4-iodoacetyl)aminobenzoate; inverted 4-succinimidyl-oxycarbonyl-.A.-(2pyridyldithio)toluene; sulfosuccinimidyl-6-[. inverted alpha.-methyl- .A.(pyridylthiol)-toluami-do]hexanoate; N-succinimidyl-3-(-2-pyridyldithio) propionate; succinimidyl-6-[3(-(-2-pyridyldithio)propionamido]hexanoate; sulfosuccinimidyl-6-[3(-(-2-pyridyldithio)propionamido]hexanoate; 3-(2-pyridyldithio)hydrazide, Ellman's reagent, dichlorotriazine acid, S-(2-thiopyridyl)-L-cysteine ​​and the like.

[00387] Fusion proteins can be designed to attach the protein of interest to the amino or carboxyl terminus of the antibody heavy or light chain, although the entire heavy chain may not be required. Potential configurations include the use of truncated portions of the heavy and light chains, with or without spacer sequences, as needed to maintain the functional integrity of the attached protein.

[00388] Alternatively, a universal carrier system can be designed. For example, several proteins or DNA can be conjugated with a common carrier, such as protein A, poly-L-lysine, hexhididine, and the like. The conjugated carrier will then form a complex with Siglec-15 binding molecules or Siglec-15 ligand. A small portion of the carrier molecule that is responsible for immunoglobulin binding can be used as the carrier. Other similar configurations include the design of carriers that interact with manipulated proteins in the heavy or light chain of the antibody.

[00389] In some embodiments, Siglec-15 binding molecules or Siglec-15 ligand-binding molecules are conjugated or otherwise incorporated into or on a nanocarrier to target the nanocarrier to Siglec-15 positive cells or Siglec-15 ligand. The nanocarrier, for example, micro or nano-polymeric particles, liposomes, nanotubes, etc., may include an active agent for delivery to Siglec-15 positive cells. Petition 870200066611, dated 05 / 28 / 2020, p. 156 / 280 150 / 217 or Siglec-15 binder or its microenvironment.

[00390] Similarly, Siglec-15 binding molecules or Siglec-15 ligand can be conjugated with a detectable marker or can be unconjugated and detected with a secondary reagent to detect the expression of Siglec-15 or Siglec-15 ligand, respectively, in vitro or in vivo. Thus, the molecules can be used for imaging, immunohistochemistry, and other assays. IV. Combined Therapies

[00391] The disclosed Siglec-15 binding and Siglec-15 ligand-binding molecules may 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 or Siglec-15 ligand-binding molecule and the additional therapeutic agent are administered separately but simultaneously. The Siglec-15 binding or Siglec-15 ligand-binding molecule and the additional therapeutic agent may also be administered as part of the same composition. In other embodiments, the Siglec-15 binding or Siglec-15 ligand-binding molecule and the second therapeutic agent are administered separately and at different times, but as part of the same treatment regimen.

[00392] A first therapeutic agent may be administered to the subject 1, 2, 3, 4, 5, 6 or more hours, or 1, 2, 3, 4, 5, 6, 7 or more days before the administration of a second therapeutic agent. In some embodiments, the subject may 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 a first administration of the second agent. The Siglec-15 binding molecule or Siglec-15 ligand binding agent may be the first or the second therapeutic agent. In some embodiments, one or more Siglec-15 binding molecules and one or more Siglec-15 ligand binding agents are administered in combination. Petition 870200066611, dated 05 / 28 / 2020, pp. 157 / 280 151 / 217

[00393] The Siglec-15 binding molecule and / or Siglec-15 ligand and the additional therapeutic agent may be administered as part of a therapeutic regimen. For example, if a first therapeutic agent may be administered to a subject every four days, the second therapeutic agent may be administered on the first, second, third, or fourth day, or combinations thereof. The first therapeutic agent or second therapeutic agent may be administered repeatedly throughout the treatment regimen.

[00394] Exemplary molecules include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutics, enzymes, antibiotics, antivirals (especially protease inhibitors alone or in combination with nucleosides for the treatment of HIV or hepatitis B or C), antiparasitics (helminths, protozoa), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and their bioactive fragments (including humanized, single-chain and chimeric antibodies), antigens and vaccines (including adjuvants), peptide drugs, anti-inflammatories, ligands that bind to Toll-like receptors (including, but not limited to, polyinosinic:polycyticidyl acid (polyl:C) and CpG oligonucleotides) to activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, other molecules that activate or positively regulate the action of T lymphocytes. cytotoxic,natural killer cells and helper T cells and other molecules that disable or negatively regulate suppressor or regulatory T cells.

[00395] Additional therapeutic agents are selected based on the condition, disorder, or disease being treated. For example, the Siglec-15 binding molecule may 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. Petition 870200066611, dated 05 / 28 / 2020, pp. 158 / 280 152 / 217 A. Chemotherapeutic Agents

[00396] Molecules that bind to Siglec-15 and Siglec-15 ligand-binding molecules can be combined with one or more chemotherapeutic agents and pro-apoptotic agents.Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, chrysantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafururacil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof.Representative pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ (2) and combinations thereof. B. Other immunomodulators 1. PD-1 antagonists

[00397] In some embodiments, Siglec-15 binding molecules or Siglec-15 ligand binding are co-administered with a PD-1 antagonist. Programmed death 1 (PD-1) is a member of the CD28 receptor family that provides a negative immune response when induced in T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that decreases T cell multiplication and / or the strength and / or duration of a T cell response. Suitable PD-1 antagonists are described in U.S. Patents Nos. 8,114,845, 8,609,089, and 8,709,416, which are specifically incorporated herein by reference in their Petition 870200066611, dated 05 / 28 / 2020, pp. 159 / 280 153 / 217 totality, and include compounds or agents that bind to and block a PD-1 ligand to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or bind directly to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.

[00398] In some embodiments, the PD-1 receptor antagonist can bind directly to the PD-1 receptor without triggering inhibitory signal transduction and can also bind to a PD-1 receptor ligand to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and trigger inhibitory signal transduction, fewer cells are attenuated by the negative signal transmitted by PD-1 signal transduction and a more robust immune response can be achieved.

[00399] PD-1 signaling is believed to be driven by binding to a PD-1 ligand (such as B7-H1 or B7-DC) near a peptide antigen presented by the major histocompatibility complex (MHC) (see, for example, Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Therefore, proteins, antibodies, or small molecules that prevent PD-1 and TCR co-binding on the T cell membrane are useful PD-1 antagonists.

[00400] In some embodiments, PD-1 receptor antagonists are small molecule antagonists or antibodies that reduce or interfere with PD-1 receptor signal transduction by binding to PD-1 ligands or PD-1, especially where co-binding of PD-1 with TCR does not follow this binding, thus not causing inhibitory signal transduction through the PD-1 receptor.

[00401] Other PD-1 antagonists contemplated by the methods of this invention include antibodies that bind to PD-1 or PD-1 ligands and other antibodies.

[00402] Suitable anti-PD-1 antibodies include, but are not Petition 870200066611, dated 05 / 28 / 2020, pages 160 / 280 154 / 217 limited to those described in the following U.S. Patent Nos.: 7332582, 7488802, 7521051, 7524498, 7563869, 7981416, 8088905, 8287856, 8580247, 8728474, 8779105, 9067999, 9073994, 9084776, 9205148, 9358289, 9387247, 9492539, 9492540, all of which are incorporated by reference in their entirety.

[00403] See also Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[00404] Exemplary anti-B7-H1 antibodies (also referred to as anti-PD-L1) include, but are not limited to, those described in U.S. Patents Nos. 8383796, 9102725, 9273135, 9393301, and 9580507, all of which are specifically incorporated by reference herein in their entirety.

[00405] For anti-B7-DC antibodies (also referred to as anti-PDL2), see U.S. Pat. Nos.: 7,411,051, 7,052,694, 7,390,888, 8188238 and 9255147, all of which are specifically incorporated herein by reference in their entirety.

[00406] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, including homologs and variants thereof, as well as active fragments of any of the above, and fusion proteins incorporating any of these. In some embodiments, the fusion protein includes the soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.

[00407] The PD-1 antagonist can also be a fragment of a mammalian B7-H1, for example from a mouse or primate, such as a human, where the fragment binds to and blocks PD-1 but does not result in inhibitory signal transduction through PD-1. The fragments can also be part of a fusion protein, for example, an Ig fusion protein.

[00408] Other useful PD-1 polypeptide antagonists include Petition 870200066611, dated 05 / 28 / 2020, pp. 161 / 280 155 / 217 those that bind to PD-1 receptor ligands. These include the PD-1 receptor protein, or its soluble fragments, which can bind to PD-1 ligands such as B7-H1 or B7-DC, and prevent binding to the endogenous PD-1 receptor, thus preventing inhibitory signal transduction. B7-H1 has also been shown to bind to the B7.1 protein (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include the soluble ECD portion of the PD-1 protein which includes mutations, such as the A99L mutation, which increases binding to natural ligands (Molnar et al., PNAS, 105: 10483-10488 (2008)). B7-1 or soluble fragments thereof, which can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thus avoiding inhibitory signal transduction, are also useful.

[00409] The antisense nucleic acids PD-1 and B7-H1, both DNA and RNA, as well as siRNA molecules, can also be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 in T cells as well as the production of T cell ligands such as B7-H1, PD-L1 and / or PD-L2. For example, siRNA (e.g., about 21 nucleotides long, which is specific to the gene encoding PD-1, or encoding a PD-1 ligand, and whose oligonucleotides can be readily acquired commercially) complexed with carriers such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119 (8): 22312244 (2009)), are readily taken up by cells expressing PD-1 as well as PD-1 ligands and reduce the expression of these receptors and ligands to achieve a decrease in inhibitory signal transduction in T cells, thus activating T cells. 2. CTLA4 antagonists

[00410] Other molecules useful in measuring the effects of T cells in an immune response are also contemplated as additional therapeutic agents. In some modalities, the molecule is a CTLA4 antagonist, for example, an anti-CTLA4 antagonist antibody. Petition 870200066611, dated 05 / 28 / 2020, pp. 162 / 280 156 / 217 An example of an anti-CTLA4 antibody contemplated for use in the methods of the invention includes an antibody as described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[00411] Dosages for anti-PD-1, anti-B7-H1 and anti-CTLA4 antibodies are known in the art and may be in the range of, for example, 0.1 to 100 mg / kg, or with shorter ranges of 1 to 50 mg / kg, or 10 to 20 mg / kg. An appropriate dose for a human may be between 5 and 15 mg / kg, with 10 mg / kg of antibody (e.g., human anti-PD-1 antibody) being a specific modality.

[00412] Specific examples of an anti-CTLA4 antibody useful in the methods of the invention are Ipilimumab, a human anti-CTLA4 antibody, administered at a dose of, for example, about 10 mg / kg and Tremelimumab, a human anti-CTLA4 antibody 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.

[00413] In other embodiments, the antagonist is a small molecule. A number of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277: 7363-7368 (2002)). Such small organics can be administered alone or in combination with an anti-CTLA4 antibody to reduce inhibitory T cell signal transduction. 3. Enhancing Agents

[00414] In some embodiments, additional therapeutic agents include a potentiating agent. The potentiating agent acts to increase the efficiency of the supreme regulator of the immune response, possibly by more than one mechanism, although the precise mechanism of action is not essential for the general practice of the present invention.

[00415] In some modalities, the potentiating agent is the Petition 870200066611, dated 05 / 28 / 2020, pages 163 / 280 157 / 217 Cyclophosphamide. Cyclophosphamide (CTX, Cytoxan®, or Neosar®) is an oxazosphorin drug, and its analogues include ifosfamide (IFO, Ifex), perfosfamide, trofosfamide (trofosfamide; Ixoten), and pharmaceutically acceptable salts, solvates, prodrugs, and metabolites thereof (US Patent Application 20070202077, which is incorporated in its entirety). Ifosfamide (MITOXANA®) is a structural analogue of cyclophosphamide, and its mechanism of action is considered identical or substantially similar to that of cyclophosphamide. Perfosfamide (4-hydroperoxycyclophosphamide) and troposfamide are also alkylating agents that are structurally related to cyclophosphamide. For example, perfosfamide alkylates DNA, thereby inhibiting DNA replication and RNA and protein synthesis. New oxazaphosphorine derivatives have been designed and evaluated in an attempt to improve selectivity and response while reducing host toxicity (Liang J, Huang M, Duan W, Yu XQ, Zhou S.Design of new oxazacossin anticancer drugs. Curr Pharm Des. 2007;13(9):963-78. Review). These include mafosfamide (NSC 345842), glufosfamide (D19575, beta-D-glucosylisophosphoramide mustard), S-(-)-bromophosphamide (CBM11), NSC 612567 (aldofosfamide perhydrofiazide), and NSC 613060 (aldofosfamide thiazolidine). Mafosfamide is an oxazaphosphorine analogue that is a chemically stable 4-thioethanesulfonic acid salt of 4-hydroxy-CPA. Glufosfamide is an IFO derivative in which isophosphoramide mustard, the alkylating metabolite of IFO, is glycosidically linked to a beta-D-glucose molecule. Additional cyclophosphamide analogs are described in U.S. Patent 5,190,929 entitled "Cyclophosphamide analogs useful as antitumor agents," which are incorporated herein by reference in their entirety.

[00416] Although CTX itself is not toxic, some of its metabolites are cytotoxic alkylating agents that induce DNA cross-linking and, at higher doses, strand breaks. Many cells are Petition 870200066611, dated 05 / 28 / 2020, pp. 164 / 280 158 / 217 resistant to CTX because they express high levels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). CTX targets proliferating lymphocytes because lymphocytes (but not hematopoietic stem cells) express only low levels of ALDH, and cyclable cells are more sensitive to DNA alkylating agents.

[00417] Low doses of CTX (<200 mg / kg) may have immunostimulatory effects, including stimulation of antitumor immune responses in humans and mouse cancer models (Brode & Cooke Crit Rev. Immunol. 28:109-126 (2008)). These low doses are subtherapeutic and do not have direct antitumor activity. In contrast, high doses of CTX inhibit the antitumor response. Several mechanisms may explain the role of CTX in potentiating the antitumor immune response: (a) depletion of CD4+ CD25+ FoxP3+ Tregs (and specifically proliferative Treg, which may be especially suppressive), (b) depletion of B lymphocytes; (c) induction of nitric oxide (NO), resulting in suppression of tumor cell growth; (d) mobilization and expansion of CD11b+ Gr1+ MDSCs. These primary effects have numerous secondary effects; For example, after Treg depletion, macrophages produce more IFN-γ and less IL-10.CTX has also been shown to induce type I IFN expression and promote homeostatic lymphocyte proliferation.

[00418] Treg depletion is most often cited as the mechanism by which CTX potentiates the antitumor immune response. This conclusion is based in part on the results of adoptive transfer experiments. In the AB1-HA tumor model, treatment with CTX on day 9 gives a cure rate of 75%. Transfer of purified Treg on day 12 almost completely inhibited the CTX response (van der Most et al. Cancer Immunol. Immunother. 58:1219-1228 (2009)). A similar result was observed in the HHD2 tumor model: adoptive transfer of CD4+ CD25+ Treg after CTX pretreatment eliminated the therapeutic response to the vaccine. Petition 870200066611, dated 05 / 28 / 2020, pages 165 / 280 159 / 217 (Taieb, JJ Immunol. 176:2722-2729 (2006)).

[00419] Numerous clinical trials in humans have demonstrated that low-dose CTX is a safe, well-tolerated, and effective agent for promoting antitumor immune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47:1-12 (1998)).

[00420] The ideal dose for CTX to potentiate an anti-tumor immune response is one that reduces the total T cell count by reducing Treg levels below the normal range, but is subtherapeutic (see Machiels et al. Cancer Res. 61:3689-3697 (2001)).

[00421] In human clinical trials in which CTX was used as an immunopotentiating agent, a dose of 300 mg / m2 was usually used. For an average male (6 feet, 170 pounds (78 kg) with a body surface area of ​​1.98 m2), 300 mg / m2 is 8 mg / kg or 624 mg of total protein. In mouse cancer models, efficacy was observed 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)).

[00422] For larger mammals, such as a primate, such as a human patient, such mg / m2 doses may be used, but unit doses administered over a finite time interval 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, are all specifically contemplated by the invention. The same regimen may be applied to the other potentiating agents mentioned in this document.

[00423] In other modalities, the potentiating agent is an agent that reduces the activity and / or number of regulatory T lymphocytes (T-regs), such as Sunitinib (SUTENT®), anti-TGFe or Imatinib (GLEEVAC®). The regimen of Petition 870200066611, dated 05 / 28 / 2020, pp. 166 / 280 160 / 217 The treatment described may also include the administration of an adjuvant.

[00424] Useful potentiating agents also include mitosis inhibitors such as paclitaxel, aromatase inhibitors (e.g., letrozole) and angiogenesis inhibitors (VEGF inhibitors, e.g., Avastin, VEGF-Trap) (see, for example, Li et al., Clin Cancer Res. 2006 Nov 15; 12 (22): 6808-16.), anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists and IL-18 antagonists. C. Antimicrobials

[00425] In one embodiment, Siglec-15 binding molecules or Siglec-15 ligand binding can be used in a preventive or prophylactic role in the treatment and prevention of diseases as discussed above, and also in the context of severe trauma injuries such as a severe burn, open bone fracture, accidental amputation or other wounds. Therefore, Siglec-15 binding molecules or Siglec-15 ligand binding can be administered to the subject in combination with an antimicrobial such as an antibiotic, an antifungal, an antiviral, an antiparasitic or essential oil.

[00426] In some modalities, the subject is administered Siglec-15 binding molecules and / or the antimicrobial at the time of hospital admission to prevent further bacterial, fungal, or viral complications. The antibiotic may target pathogens, and the Siglec-15 binding molecule or Siglec-15 ligand may stimulate the immune system to provide an enhanced response to treat or prevent new infections or diseases. D. Immunosuppressive Agents

[00427] In some modalities, the immune response or inflammatory / autoimmune disease / disorder is treated by administering to the subject a Siglec-15 binding or ligand-binding molecule and a second agent that is an immunosuppressant. Immunosuppressive agents include, but Petition 870200066611, dated 05 / 28 / 2020, pp. 167 / 280 161 / 217 are not limited to antibodies against other lymphocyte surface markers (e.g., CD40, alpha-4 integrin) or against 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®, Neosar®, Procytox®, Revimmune™), methotrexate (MTX) (i.e., Rheumatrex®, Trexall®), belimumab (i.e., Benlysta®) or other immunosuppressive drugs (e.g., cyclosporine A, FK506-like compounds, rapamycin compounds, or steroids), antiproliferative agents, cytotoxic agents, or other compounds that may assist in immunosuppression.

[00428] The therapeutic agent may be a CTLA-4 fusion protein, such as CTLA-4-Ig (abatacept). CTLA-4-Ig fusion proteins compete with the co-stimulatory receptor, CD28, on T cells for binding to CD80 / CD86 (B7-1 / B7-2) on antigen-presenting cells and thus function to inhibit T cell activation. In another embodiment, the therapeutic agent is a CTLA-4-Ig fusion protein known as belatacept. Belatacept contains two amino acid substitutions (L104E and A29Y) that markedly increase its avidity for CD86 in vivo. In another embodiment, the therapeutic agent is Maxy-4.

[00429] 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 oxazophorin group. It is used to treat various types of cancer and some autoimmune disorders. Cyclophosphamide (CTX) is the main drug used for diffuse proliferative glomerulonephritis in patients with lupus renal failure.

[00430] The therapeutic agent can be administered in an amount effective to reduce blood or serum levels of anti-double-stranded DNA (anti-ds DNA) autoantibodies and / or to reduce proteinuria in a patient. Petition 870200066611, dated 05 / 28 / 2020, pp. 168 / 280 162 / 217 with the same need.

[00431] In another embodiment, the therapeutic agent increases the amount of adenosine in the serum, see, for example, WO 08 / 147482. For example, the second therapeutic agent may be CD73-Ig, recombinant CD73 or another agent (e.g., a cytokine or monoclonal antibody or small molecule) that increases the expression of CD73, see, for example, WO 04 / 084933. In another embodiment, the therapeutic agent is interferon beta.

[00432] The therapeutic agent may be Tysabri or another MS therapeutic. In another embodiment, the second therapeutic agent preferentially treats chronic inflammation, so the treatment regimen targets both acute and chronic inflammation. In a preferred embodiment, the second therapy is a TNF-α blocker.

[00433] The therapeutic agent may be a small molecule that inhibits or reduces the differentiation, proliferation, activity and / or production of cytokines 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-ίβ, TNF-α, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21 and MMPs. In another embodiment, the therapeutic agent is a small molecule that interacts with Tregs, increases Treg activity, promotes or enhances IL-10 secretion by Tregs, increases the number of Tregs, increases the suppressive capacity of Tregs, or combinations thereof.

[00434] In some modalities, the composition increases Treg activity or production. Exemplary Treg-enhancing agents include, but are not limited to, fluticasone glucosorticoid, salmeteroal, antibodies against IL-12, IFN-γ and IL-4; vitamin D3 and dexamethasone, and combinations thereof.

[00435] In some modalities, the therapeutic agent is an antibody, for example, a function-blocking antibody against a Petition 870200066611, dated 05 / 28 / 2020, pp. 169 / 280 163 / 217 pro-inflammatory molecule, such as IL-6, IL-23, IL-22 or IL-21.

[00436] As used in this document, the term “rapamycin compound” includes neutral tricyclic rapamycin compounds, rapamycin derivatives, rapamycin analogues, and other macrolide compounds thought to have the same mechanism of action as rapamycin (e.g., inhibition of cytokine function). The term “rapamycin compounds” includes compounds with structural similarity to rapamycin, for example, compounds with a similar macrocyclic structure that have been modified to enhance their therapeutic efficacy. Examples of rapamycin compounds are known in the art (see, for example, WO95122972, WO95116691, WO95104738, US Patent 6,015,809; 5,989,591; US ​​Patent 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).

[00437] The term “FK506-type compounds” includes derivatives and analogues of FK506 and FK506, for example, compounds with structural similarity to FK506, for example, compounds with a similar macrocyclic structure that have been modified to enhance their therapeutic efficacy. Examples of FK506-type compounds include, for example, those described in WO 00101385. Preferably, the term “rapamycin compound” as used in this document does not include FK506-type compounds. E. Anti-inflammatories

[00438] Other suitable therapeutic agents include, but are not limited to, anti-inflammatory agents. The anti-inflammatory agent may be nonsteroidal, steroidal, or a combination thereof. One embodiment provides oral compositions 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, without limitation, oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam; Petition 870200066611, dated 05 / 28 / 2020, pages 170 / 280 164 / 217 salicylates, such as aspirin, disalcid, benorilate, trilisate, safaprine, solprine, diflunisal and fendiosal; acetic acid derivatives, such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidomethacin, acematization, fentiazac, zomepirac, clindanac, oxepinac, felbinac and ketorolac; fenamates, such as mefenamic, meclofenamic, flufenamic, niflumic and tolfenamic acids; Propionic acid derivatives, such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropene, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen and tiaprofen; pyrazoles, such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone and trimethazone. Mixtures of these nonsteroidal anti-inflammatory agents may also be used.

[00439] Representative examples of steroidal anti-inflammatory drugs include, without limitation, corticosteroids such as hydrocortisone, hydroxyl-triamcinolone, alpha-methyl dexamethasone, dexamethasone phosphate, beclomethasone dipropionates, clobetasol valerate, desonide, deoxyacetone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluchlorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortin butyl esters, fluocortolone, fluprednidene (fluprednilidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, corticone, fludrocortisone, difluorosone diacetate, fluradrenolone, fludrocortisone, diflurosone diacetate, fluradrenolone acetonide, medrisone, amcinafel, amcinafide, betamethasone and the balance of their esters, chlorprednisone,chlorprednisone acetate, clocortelone, clescinolone, dichlorisone, diflurprednate, flucoronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, Petition 870200066611, dated 05 / 28 / 2020, pages 171 / 280 165 / 217 hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone and mixtures thereof. V. Diagnostic Methods

[00440] Siglec-15 binding molecules, particularly antibodies and their antigen-binding fragments, can be used for diagnostic purposes, such as detecting, diagnosing, or monitoring diseases, disorders, or infections associated with Siglec-15 expression, or to determine or assist in determining or identifying suitable patient populations or profiles. Any of these methods can be coupled with a treatment method for the subject, for example, by administering to the subject an effective amount of one or more therapeutic Siglec-15 binding molecules.

[00441] The detection or diagnosis of a disease, disorder, or infection, including but not limited to cancer, may include: (a) testing the expression of Siglec-15 or derivatives thereof in cells, serum, plasma, blood, or a tissue sample (e.g., a tumor sample) from a subject using one or more antibodies (or fragments thereof) that bind immunospecifically to such antigens; and (b) comparing the antigen level with a control level, for example, levels in normal tissue samples, where an increase in the assayed antigen level compared with the antigen control level is indicative of disease, disorder, or infection. Such antibodies and fragments may be employed in immunoassays, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell separation (FACS).

[00442] In some modalities, antibodies or fragments are used for IHC analysis in cells from a tissue sample in vitro or in situ or in vivo. Thus, antibodies and fragments can be used Petition 870200066611, dated 05 / 28 / 2020, pp. 172 / 280 166 / 217 in the detection and diagnosis of a disease, disorder, or infection in a human. In one embodiment, such diagnosis includes: a) administering to a subject (e.g., parenterally, subcutaneously, or intraperitoneally) an effective amount of such labeled antibody or antigen-binding fragment; b) waiting for a time interval after administration to allow the labeled molecule to preferentially concentrate at sites in the subject where Siglec-15 is expressed (and for unbound labeled molecule to be eliminated to the background level); c) determining the background level; d) detecting the labeled antibody in the subject, such that localized detection of labeled antibody above or below the background level indicates that the subject has the disease, disorder, or infection and / or shows the location and relative tissue expression level of Siglec-15+.According to this modality, the antibody can be labeled with an imaging portion that is detectable in vivo using an imaging system known to one skilled in the art. The background level can be determined by various methods, including comparing the amount of labeled molecule detected with a previously determined standard value for a particular system.

[00443] Other methods include, for example, monitoring the progression of a disease, disorder or infection by (a) testing the expression of Siglec-15 in cells or in a tissue sample from a subject obtained at an early and later time point using a Siglec-15 binding molecule and (b) comparing the level of Siglec-15 expression in the subject's cells or tissue sample at early and later time points, where an increase in the assayed level of Siglec-15 later, compared to the early time point, is indicative of disease, disorder or infection progression.

[00444] One method for monitoring a response to a treatment may include (a) testing the expression of Siglec-15 in cells or in a tissue sample from a subject before and after treatment using a molecule Petition 870200066611, dated 05 / 28 / 2020, pages 173 / 280 167 / 217 of Siglec-15 binding; and (b) compare the Siglec-15 level over time, whereby a decrease in the analyzed Siglec-15 level after treatment compared to the Siglec-15 level before treatment is indicative of a favorable response to treatment.

[00445] It will be understood in the technique that the size of the subject and the imaging system used will determine the amount of image portion needed to produce diagnostic images.

[00446] Depending on several variables, including the type of labeling used and the mode of administration, the time interval after administration to allow the labeled molecule to preferentially concentrate at sites in the subject and for the unbound labeled molecule to be cleared to the background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment, the time interval after administration is 5 to 20 days or 5 to 10 days.

[00447] In one embodiment, monitoring of a disease, disorder or infection is performed by repeating the method used to diagnose the disease, disorder or infection, for example, one month after the initial diagnosis, six months after the initial diagnosis, one year after the initial diagnosis, etc.

[00448] The presence of the labeled molecule can be detected in the subject using methods known in the art for in vivo scanning. These methods depend on the type of labeling used. Those skilled in the art will be able to determine the appropriate method to detect a specific labeling. Methods and devices that can be used in diagnostic methods include, but are not limited to, computed tomography (CT), whole-body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound.

[00449] In one specific modality, the molecule is labeled with a radioisotope and detected in the patient using a surgical instrument. Petition 870200066611, dated 05 / 28 / 2020, pages 174 / 280 168 / 217 responsive to radiation (Thurston et al., US Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence-responsive scanning instrument. In another embodiment, the molecule is labeled with a positron-emitting metal and is detected in the patient using positron emission tomography. Yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI). VI. Kits

[00450] The disclosed Siglec-15 binding molecules or Siglec-15 ligand binding molecules may be packaged in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity. The molecules may be supplied as a sterile dry lyophilized powder or a water-free concentrate in a hermetically sealed container and may be reconstituted, for example, with water or saline solution to the appropriate concentration for administration to a subject. For example, the molecules may be supplied as a sterile dry lyophilized powder in a hermetically 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.Lyophilized molecules can be stored between 2 and 8°C in their original container 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.

[00451] In an alternative embodiment, the molecules are supplied in liquid form in a hermetically sealed container, indicating the quantity and concentration. In some embodiments, the liquid form of the molecules supplied in a hermetically sealed container includes at least 1 mg / ml, or at least 2.5 mg / ml, at least 5 mg / ml, at least Petition 870200066611, dated 05 / 28 / 2020, pages 175 / 280 169 / 217 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, at least 200 mg / ml of the molecules.

[00452] Pharmaceutical packaging and kits including one or more containers filled with Siglec-15 binding molecules or Siglec-15 ligand-binding molecules are also provided. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease may also be included in the pharmaceutical packaging or kit. The pharmaceutical packaging or kit may also include one or more containers filled with one or more of the ingredients of the disclosed pharmaceutical compositions. Optionally associated with such container(s) may be a warning in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, which warning reflects the agency's approval for manufacture, use, or sale for administration in humans.

[00453] Kits designed for the methods described above are also provided. The embodiments typically include one or more Siglec-15 binding molecules or Siglec-15 ligand-binding molecules. In particular embodiments, a kit may also include one or more other prophylactic or therapeutic agents useful for cancer treatment, in one or more containers. Examples Example 1: SIGLEC-15 Antibodies and Their Heavy and Light Chain Sequences Materials and methods Mouse anti-human Siglec-15 monoclonal antibodies

[00454] Siglec-15 knockout mice (n = 2) were immunized with hS15.mIg (human Siglec 15 extracellular domain [ECD] fused with mouse IgG2a) emulsified with CFA (Freund's complete adjuvant). The mice also received an injection of GM-CSF and anti Petition 870200066611, dated 05 / 28 / 2020, pp. 176 / 280 170 / 217 CD40. Mice were challenged with the same immunogen two weeks later. The titer of the antisera was evaluated by testing serum collected from tail bleeding on an ELISA plate coated with hS15.hIg (human Siglec 15 ECD fused with human IgG1) at various dilutions, from 1:1000 to 1:100,000,000. Figure 1 shows that anti-hS15 antibodies were detected at >1:100,000 dilution. Mice received a 3rd dose of antigen two weeks later. Three days after the final booster, mouse splenocytes were collected and resuspended in RPMI supplemented with 10% FBS and glutamine, and then fused to form hybridomas. Electrofusion of Siglec-15 knockout splenocytes (S15 KO)

[00455] The fused cells were plated on methylcellulose gel / medium; remaining fused cells were cryopreserved and can be thawed for another cloning batch.

[00456] Individual clones were collected and placed in 10x96 well plates (960 clones).

[00457] The supernatant was collected 2 weeks later. RACE

[00458] RACE (Rapid End Amplification of cDNA) identification of heavy and light chains was performed according to the following protocol: (1) mRNA denaturation, (2) cDNA synthesis, (3) 5'RACE reaction, (4) analysis of PCR results (on an agarose gel to visualize the amplified DNA fragment - the correct antibody variable region DNA fragments should be between 500-700 base pairs in size, (5) TOPO cloned PCR positive bands, (6) PCR amplified TOPO clones, followed by gel electrophoresis and agarose gel recovery, (7) sequenced 218 clones in total, (8) performed CDR analysis using sequencing data (CDR regions were defined using VBASE2 available through vbase2.org). Petition 870200066611, dated 05 / 28 / 2020, pages 177 / 280 171 / 217 Results

[00459] Antibodies were cloned using RACE methods. After sequencing 218 cloned DNA fragments, antibody sequence analysis identified one heavy chain and one light chain for 24 antibody samples referred to in this document 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. The sequences are provided below and above. Heavy and light chain sequences and CDRs are provided above, below, and illustrated in Figure 2A-3C. SEQUENCES 1B2: Amino acid sequence 1B2 VL in FASTA format DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQK PGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPWTFGGGTKLEIK (SEQ ID NO:3) Nucleotide sequence 1B2 VL in FASTA format GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTC TTGGAGATCAAGCCTCCATCCTTGCAGATCTAGTCAGAGCATTGT ACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACC AGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT TTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTT TATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAG GCACCAAGCTGGAAATCAAG (SEQ ID NO:74) Amino Acid Sequence of 1B2 VH in FASTA format EVQLVESGGGFVKPGGSLKLSCAASGFTFSDYGMHWVRQAPE KGLEWVAYISSGSSIIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDT AMYYCARDHYHGNGSDYWGQGTTLTVSS (SEQ ID NO:13) 1B2 VH Nucleotide Sequence in FASTA format GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTCGTGAAGCC Petition 870200066611, of 28 / 05 / 2020, p. 178 / 280 172 / 217 TGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTC AGTGACTATGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGG CTGGAGTGGGTTGCATACATTAGTAGTGGCAGTAGTATCATCTACT ATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATG CCAAGAACACCCTGTTCCTGCAAATGACCAGTCTGAGGTCTGAGG ACACGGCCATGTATTACTGTGCAAGGGACCACTACCATGGTAACG GGTCCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:85) SEQUÊNCIAS 1C3: Sequência de Aminoácidos 1C3 VL no formato FASTA DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQR PGQSPQLLIYRMSNLASGVPDRFGGSGSGTAFTLRISRVEAEDVGFYY CMQHLEYPYTFGGGTRLEIK (SEQ ID NO:4) Sequência Nucleotídica 1C3 VL no formato FASTA GATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACT CCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCC TGCATAGTAATGGCAACACTTACTTATATTGGTTCCTGCAGAGGCC AGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCC TCAGGAGTCCCAGACAGGTTCGGTGGCAGTGGGTCAGGAACTGCT TTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTTTTT ATTACTGTATGCAACATCTAGAATATCCGTACACGTTCGGAGGGG GGACCAGGCTGGAAATAAAA (SEQ ID NO:75) 1C3 VH Amino Acid Sequence in FASTA format QVQLKQSGAELVKPGASVKISCKASGYIFTDYVNWVKQRPGQ GLEWIGKIGPGSVSIYYNEKFKGCATLTADKSSSTAYMQLSSLTSEDS AVYFCASYYYGFAYWGQGTLVTVSA (SEQ ID NO:14) 1C3 VH Nucleotide Sequence in FASTA format CAGGTCCAGCTGAAGCAGTCTGGAGCTGAGCTGGTGAAGCC TGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACATCTTC Petition 870200066611, of 28 / 05 / 2020, p. 179 / 280 173 / 217 ACTGACTATTTATGTAAACTGGGTGAAGCAGAGGCCTGGACAGGGC CTTGAGTGGATTGGAAAGATTGGTCCTGGAAGTGTTAGTATTTACT ACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAAT CCTCCAGCAGCCTACATGCAGCTCAGCAGCCTGACCATGGG ACTCTGCAGTCTATTTCTGTGCAAGTTATTACTACGGGTTTGCTTAC TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:86) 1H3 SEQUENCES: 1H3 VL Amino Acid Sequence in FASTA format DIQMTQASSSLSSVSLGGRVTITCKASDHINNWLAWYQQKPGNA PRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQY WSSPLTFGAGTKLELK (SEQ ID NO:5) Nucleotide sequence of 1H3 VL in FASTA format GACATCCAGATGACACAGGCTTCATCCTCCTTGTCTGTATCTC TAGGAGGCAGAGTCACCATTGCAAGGCAAGTGACCACATTA ATAATTGGTTGGCCTGGTATCAGCAGAAACCAGGAAATGCTCCTA GGCTCTTAATCTGGTGCAACCAGTTTGGAAACTGGGGTTCTCTT AAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCAT TACCAGTCTTCAGACTGAAGATGTTGCTACTTATTACTGTCAACAG TATTGGAGTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGC TGAAA (SEQ ID NO:76) 1H3 VH Amino Acid Sequence in FASTA format QVQLKESGPGLVAPSQSLSITCTVSGFSLSNYGVHWVRQPPGKG LEWLVLIWSDGSTTYNSALKSRLSISKDNSKSQVFLKMNSLQTGDTA MYYCARHPYDDYSGYYYTMDYWGQGTSVTVSS (SEQ ID NO:15) Nucleotide sequence of 1H3 VH in FASTA format CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCC CTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTA AGCAATTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGT CTGGAGTGGCTGGTACTGATATGGAGTGAGGAGCACT Petition 870200066611, of 28 / 05 / 2020, p. 180 / 280 174 / 217 AATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCC AAGAGCCAAGTTTTCTTAAAAATGAACAGTCTCCAAACTGGTGAC ACAGCCATGTACTACTGTGCCAGACATCCCTATGATGATTTCCG GCTATTACTATACTATGGACTACTGGGGTCAAGGAACCTCAGTCCACTCACTCACTGGGGGTCAAGGAACCTCAGTCCACTCACTCACTGQGGGGTCAAGGAACCTCACTCACTCACTCQ: NO: ID77) SEQUENCE 1C12: Amino Acid Sequence of 1C12 VL in FASTA format DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQK PGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPWTFGGGTKLEIK (SEQ ID NO:3) Nucleotide sequence of 1C12 VL in FASTA format GATGTTTTGATGACCCAAAACTCCACTCTCCCTGCCTGTCAGTC TTGGAGATCAAGCCTCCATCCTTGCAGATCTAGTCAGAGCATTGT ACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACC AGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT TTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTT TATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAG GCACCAAGCTGGAAATCAA (SEQ ID NO:77) Amino Acid Sequence of 1C12 VH in FASTA format EVQLVESGGGLVKPGGSLKLSCAASGFSFSDYGMHWVRQAPE KGLEWVAYISSGSSILYYADIVKGRFTISRDNAKNTLFLQMTSLRSEDT AMYYCARDHYHGNGSDYWGQGTTLTVSS (SEQ ID NO:16) Nucleotide sequence 1C12 VH in FASTA format GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCC TGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGTTTCTCTTTC AGTGACTATGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGG CTGGAGTGGGTTGCATACATTAGTAGTGGCAGTAGTATCCTCTACT ATGCAGACATAGTGAAGGGCCGATTCACCATCTCCAGAGACAATG Petition 870200066611, dated 05 / 28 / 2020, pages 181 / 280 175 / 217 CCAAGAACACCCTGTTCCTGCAAATGACCAGTCTGAGGTCTGAGG ACACGGCCATGTATTACTGTGCAAGGGACCACTACCATGGTAACG GGTCCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:88) SEQUENCES 3H10: 3H10 VL Amino Acid Sequence in FASTA Format QIILTQSPAIMSASPGEKVTMTCSASSSTSFMHWYQQKPGTSPKR WIFDTSKLASGVPGRFIGSGSGTSYSLTISTMEAEDAATYYCHQRSAY PWTFGGGTKLEIK (SEQ ID NO:6) Nucleotide sequence 3H10 VL in FASTA format CAAATTATTCTCACCCAGTCTCCAGCAATCATGTCTGCATCCTC CAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTACAA GTTTCATGCACTGGTACCAGCAGAAGCCAGGCACCTCCCCCAAAAA GATGGATTTTTGACACATCCAAACTGGCTTCTGGAGTCCCTGGTCG CTTCATTGGTAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGC ACCATGGAGGCTGAAGATGCTGCCACTTTACTGCCATCAGCGG AGTGCTTACCCATGGACGTTCGGTGGAGGCACCAAGCTGGAAATC AAA (SEQ ID NO:78) Amino Acid Sequence of 3H10 VH in FASTA format EVQLQQSGAELVRPGASVKLSCTASGFNIKDYMHWVKERPEQ GLEWIGRIDPEDGDIEYDPKFQGKATADTSNSNTAYLQFSSLTSEDT AVYYCVTDYDYDGGWFAYWGQGTLVTVSA (SEQ ID NO:17) VH 3H10 Nucleotide Sequence in FASTA format GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAGGCC AGGGGCCTCAGTCAAGTTGTCCTGCACAGCTCTGGCTTCAACATT AAAGACTACTATATGCACTGGGTGAAAGAGAGGCCTGAACAGGGC CTGGAGTGGATTGGAAGGATTGATCCTGAGGGATTGGAGGATT TATGACCCGAAGTTCCAGGGCAAGGCCACTATGACTGCAGATACA TCCTCCAACACAGCCTACCTGCAGTTCAGCAGCCTGACATCTGAGG Petition 870200066611, of 28 / 05 / 2020, p. 182 / 280 176 / 217 ACACTGCCGTCTATTATTGTGTCACGGACTATGATTACGACGGAGG CTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:89) SEQUENCES 5G12: Amino Acid Sequence of 5G12 VL in FASTA format DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSP KTLIYRANRLVDGVPSRFSGSGCGQDYSLTISSLEYEDMGIYYCLQYD EFPYTFGGGTKLEIKR (SEQ ID NO:7) Nucleotide sequence of 5G12 VL in FASTA format GACATCAAGATGACCCAGTCTCCATCTTCATGTATGCATCT CTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATT AATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCT AAGACCCTGATCTATCGTGCAAACAGATTGGTAGATGGGGTCCCA TCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCA TCAGCAGCCTGGAGTATGAAGATATGGGAATTTTATTGTCTACA GTATGATGAGTTTCCGTACGTTCGGAGGGGGACCAAGCTGGA AATAAAA (SEQ ID NO:79) Amino Acid Sequence of 5G12 VH in FASTA format QVQLQQPGAELVKPGASVKMSCKASGYTFTSYWITWVIQRPGQ GLEWIGDIYCGSDTMHYNEKFKNKATLTVDTSSSTAYMQLSSLTSED SAVYYCARWWDYGSSYDYFDYWGQGTTLTVSS (SEQ ID NO:18) 5G12 VH Nucleotide Sequence in FASTA format CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCT GGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCA CCAGCTACTGGATAACCTGGTGATACAGAGGCCGGGACAAGGCC TTGAGTGGATTGGAGATATTTATTGTGGTAGTGATACTATGCACTA CAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACACATC CTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGA CTCTGCGGTCTATTACTGTGCAAGATGGTGGGACTACGGTAGTAGC Petition 870200066611, dated 05 / 28 / 2020, pp. 183 / 280 177 / 217 TACGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCT CCTCA (SEQ ID NO:90) 6F8 SEQUENCES: Amino acid sequence 6F8 VL in FASTA format DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQR PGQSPQLLIYRMSNLASGVPDRFGGSGSGTAFTLRISRVEAEDVGVYY CMQHLEYPYTFGGGTKLEIKR (SEQ ID NO:8) Nucleotide sequence 6F8 VL in FASTA format GATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACT CCTGGAGAGTCAGTATCCATCCCTGCAGGTCTAGTAAGAGTCTCC TGCATAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCC AGGCCAGTCTCCTCAGCTCCTGATATATCGGGATCCAACCTTGCCTT TCAGGAGTCCCAGACAGGTTCGGTGGCAGTGGGTCAGGAACTGCT TTCACACTGAGAATCAGTAGAGTGGAG...

Claims

1. Monoclonal antibody or antigen-binding fragment thereof that specifically binds to SIGLEC-15, characterized in that the antibody comprises a set of three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) selected from the group consisting of: i. SYWIS (SEQ ID NO: 208; HCDR1), DIYSGSDTTHYAEKFQG (SEQ ID NO: 374; HCDR2) and WWDYGSSYDYFDY (SEQ ID NO: 71; HCDR3); ii. SYWIT (SEQ ID NO: 204; HCDR1), DIYSGSDTMHYAEKFQG (SEQ ID NO: 205; HCDR2) and WWDYGSSYDYFY (SEQ ID NO: 71; HCDR3); and iii. SYWIT (SEQ ID NO: 204; HCDR1), DIYSGSDTTHYAEKFQG (SEQ ID NO: 374; HCDR2) and WWDYGSSYDYFY (SEQ ID NO: 71; HCDR3), and a set of three lightweight CDRs (LCDR1, LCDR2 and LCDR3) selected from the group consisting of: i. KASQDINVYLS (SEQ ID NO: 198; LCDR1), RANRLTS (SEQ ID NO: 202; LCDR2) and LQYDEFPYT (SEQ ID NO: 43; LCDR3); ii. KASQDINSYLS (SEQ ID NO: 28; LCDR1), RANRLVD (SEQ ID NO: 36; LCDR2) and LQYDEFPY (SEQ ID NO: 43; LCDR3); iii.KASQDINTYLS (SEQ ID NO: 196; LCDR1), RANRLVD (SEQ ID NO: 36; LCDR2) e LQYDEFPYT (SEQ ID NO: 43; LCDR3); iv. KASQDINVYLS (SEQ ID NO: 198; LCDR1), RANRLVD (SEQ ID NO: 36; LCDR2) e LQYDEFPYT (SEQ ID NO: 43; LCDR3); e v. KASQDIQSYLS (SEQ ID NO: 200; LCDR1), RANRLVD (SEQ ID NO: 36; LCDR2) e LQYDEFPYT (SEQ ID NO: 43; LCDR3).

2. Monoclonal antibody or antigen-binding fragment thereof, according to claim 1, characterized in that the antibody comprises a variable full-length heavy chain region selected from the group consisting of i.QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWISW Petition 870260041430, dated 04 / 05 / 2026, page 10 / 18 2 / 4 VRQAPGQGLEWMGDIYSGSDTTHYAEKFQGRVTLTVDTSTSTAYME LSSLRSEDTAVYYCARWWDYGSSYDYFDYWGQGTLVTVSS (SEQ ID NO: 207); ii.QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWITW VRQAPGQGLEWMGDIYSGSDTMHYAEKFQGRVTLTVDTSTSTAYME LSSLRSEDTAVYYCARWWDYGSSYDYFDYWGQGTLVTVSS (SEQ ID NO: 203); and iii.QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIT WVRQAPGQGLEWMGDIYSGSDTTHYAEKFQGRVTLTVDTSTSTAYM ELSSLRSEDTAVYYCARWWDYGSSYDYFDYWGQGTLVTVSS (SEQ ID NO: 206), and a variable full-length light chain region selected from the group consisting of i.DIQMTQSPSSLSASVGDRVTITCKASQDINVYLSWFQQ KPGKAPKTLIYRANRLTSGVPSRFSGSGSGTDYTLTISSLQPEDFATYY CLQYDEFPYTFGGGTKVEIK (SEQ ID NO: 201); ii.DIQMTQSPSSLSASVGDRVTITCKASQDINSYLSWFQQ KPGKAPKTLIYRANRLVDGVPSRFSGSGSGTDYTLTISSLQPEDFATYY CLQYDEFPYTFGGGTKVEIK (SEQ ID NO: 219); iii.DIQMTQSPSSLSASVGDRVTITCKASQDINTYLSWFQ QKPGKAPKTLIYRANRLVDGVPSRFSGSGSGTDYTLTISSLQPEDFATY YCLQYDEFPYTFGGGTKVEIK (SEQ ID NO: 195); iv.DIQMTQSPSSLSASVGDRVTITCKASQDINVYLSWFQ QKPGKAPKTLIYRANRLVDGVPSRFSGSGSGTDYTLTISSLQPEDFATY YCLQYDEFPYTFGGGTKVEIK (SEQ ID NO: 197); e v.DIQMTQSPSSLSASVGDRVTITCKASQDIQSYLSWFQQ KPGKAPKTLIYRANRLVDGVPSRFSGSGSGTDYTLTISSLQPEDFATYY CLQYDEFPYTFGGGTKVEIK (SEQ ID NO: 199).

3. Monoclonal antibody or antigen-binding fragment thereof, according to claim 1 or 2, characterized in that the antibody comprises HCDR1 according to SYWIS (SEQ ID NO: Petition 870260041430, dated 04 / 05 / 2026, page 11 / 18 3 / 4 208), HCDR2 according to DIYSGSDTTHYAEKFQG (SEQ ID NO: 374) and HCDR3 according to WWDYGSSYDYFDY (SEQ ID NO: 71) and LCDR1 according to KASQDINVYLS (SEQ ID NO: 198), LCDR2 according to RANRLTS (SEQ ID NO: 202) and LCDR3 according to LQYDEFPYT (SEQ ID NO: 43).

4. Monoclonal antibody or antigen-binding fragment thereof, according to claim 1, characterized in that the antibody comprises a full-length variable heavy chain region (HCVR) with an amino acid sequence according to QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWISWVRQAPGQGLE WMGDIYSGSDTTHYAEKFQGRVTLTVDTSTSTAYMELSSLRSEDTAV YYCARWWDYGSSYDYFDYWGQGTLVTVSS (SEQ ID NO: 207), and a full-length variable light chain region with an amino acid sequence according to DIQMTQSPSSLSASVGDRVTITCKASQDINVYLSWFQQKPGKAPKTLI YRANRLTSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYDEFPYT FGGGTKVEIK (SEQ ID NO: 201).

5. Pharmaceutical composition, characterized in that it comprises the monoclonal antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 4 in combination with an excipient.

6. Use of a monoclonal antibody as defined in any one of claims 1 to 4, characterized in that it is for the manufacture of a medicament for the treatment of a tumor in an individual in need thereof.

7. Use according to claim 6, characterized in that the tumor is a colorectal tumor.

8. Use according to claim 6, characterized in that the tumor is a lymphoma.

9. Use according to claim 6, characterized in that the tumor is an ovarian tumor. Petition 870260041430, dated 04 / 05 / 2026, page 12 / 18 4 / 4 10. Use of a monoclonal antibody according to claim 6, characterized in that it is for promoting an immune response in an individual in need thereof.

11. Use according to claim 10, characterized in that the immune response promoted delays or prevents tumor growth, inhibits tumor-mediated immunosuppression, eliminates tumors, depletes or blocks the activity of tumor-associated macrophages (TAMs), decreases TAM-mediated immunosuppression, reduces or reverses T cell suppression, increases T cell proliferation, or a combination thereof.

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