Antibody-binding Siglec15 and uses thereof

Abstract

To provide a therapeutic approach for cancer patients unresponsive to PD-1 / PD-L1 targeting therapies.SOLUTION: Provided herein is an isolated monoclonal antibody that specifically binds human Siglec15, or an antigen-binding portion thereof. Also provided are a nucleic acid molecule encoding the antibody or antigen-binding portion thereof, an expression vector, a host cell, and a method for expressing the antibody or antigen-binding portion thereof. The present disclosure further provides an immunoconjugate, a bispecific molecule, a chimeric antigen receptor, an oncolytic virus, and a pharmaceutical composition comprising the antibody or antigen-binding portion thereof, as well as a treatment method using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications and References This application claims priority to U.S. Provisional Patent Application No. 63 / 000,566, filed March 27, 2020.

[0002] The above-referenced applications, and all documents cited therein or during the prosecution thereof ("Application Citations"), and all documents cited or referenced herein (including, but not limited to, all documents, patents, and published patent applications cited herein) ("Documents Cited Herein"), and all documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products described herein or in any document incorporated herein by reference, are hereby incorporated by reference and may be used in the practice of the present invention. More particularly, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Any Genbank sequences described in this disclosure are incorporated by reference, along with the Genbank sequence as of the earliest effective filing date of this disclosure.

[0003] The present disclosure generally relates to isolated monoclonal antibodies, such as fully human, murine, chimeric, or humanized monoclonal antibodies, or antigen-binding portions thereof, that specifically bind to Siglec15 with high affinity and functionality. Nucleic acid molecules encoding the antibodies or antigen-binding portions, expression vectors, host cells, and methods for expressing the antibodies or antigen-binding portions are also provided. The disclosure further provides immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions comprising the antibodies or antigen-binding portions thereof, as well as diagnostic and therapeutic methods using the anti-Siglec15 antibodies of the present disclosure. [Background technology]

[0004] Immunotherapy is a groundbreaking therapeutic approach that boosts the immune system to fight diseases such as cancer. It is applicable to many indications and has low high-grade toxicity compared to other standard therapies. The PD-1 / PD-L1 pathway is the most recent target in tumor immunotherapy, and several PD-1 or PD-L1 inhibitors have been clinically approved, including the anti-PD-1 antibodies Opdivo® and Keytruda® and the anti-PD-L1 antibody Tecentriq®. However, a subset of patients do not respond to such treatment. Recent studies have revealed that targeting Siglec15 may be a complementary approach for cancer patients unresponsive to PD-1 / PD-L1-targeted therapy (Jun Wang et al., (2019) Nature Medicine 25:656-666).

[0005] Siglec15 is a member of the Siglec family, which has a sialic acid-binding immunoglobulin-type lectin structure. It contains two extracellular immunoglobulin-like domains, a transmembrane domain with a lysine residue essential for interaction with the adaptor protein DAP12, and a cytoplasmic tail (Takashi Angata et al., (2007) Glycobiology 17(8):838-846).

[0006] Siglec15 is expressed on osteoclasts and plays a role in osteoclast differentiation and bone remodeling (Hiruma Y et al., (2011) Biochemical and Biophysical Research Communications 409(3):424-429; Takashi Angata (2020) Journal of Biomedical Science 27:10). Administration of anti-Siglec15 antibodies inhibited osteoclastic bone resorption and increased bone mass in rodent models (Stuible M et al., (2014) Journal of Biological Chemistry 289(10):6498-6512; Sato D et al., (2018) Bone 116:172-180).

[0007] Siglec15 is also expressed on tumor-associated macrophages and preferentially recognizes the sialyl-Tn antigen, a tumor-associated glycan structure. Sialyl-Tn / cancer cell lines and M-CSF-induced human macrophages or Siglec15 + Co-culture of myeloid cell lines induced the production of transforming growth factor-β, which promotes epithelial-mesenchymal transition and cancer cell spread (Takamiya et al., (2013) Glycobiology 23(2):178-187). Lieping Cheng et al. recently discovered that Siglec15 is also expressed in tumor cells and / or tumor-associated stromal cells in clinical specimens of non-small cell lung cancer. They also found that Siglec15 protein suppresses T cell proliferation and activation, and that anti-Siglec15 antibodies reverse T cell suppression and attenuate cancer growth in vivo. Siglec15 and PD-L1 are mutually exclusive in cancer tissues, and Siglec15 may serve as a complementary therapeutic target, providing an alternative treatment for patients refractory to PD-1 / PD-L1 blockade, as discussed above (Jun Wang et al., (2019) supra). NC318, a humanized anti-Siglec15 antibody, has been clinically tested in patients with advanced solid tumors, including non-small cell lung cancer, ovarian cancer, melanoma, colorectal cancer, and breast cancer, with long-term disease stabilization observed in 54% of patients and responses observed in 5.4% (Sun J et al., (2021) Clin Cancer Res. 27(3):680-688).

[0008] Given the involvement of Siglec15 in bone remodeling and tumorigenesis, Siglec15 is certainly an emerging promising therapeutic target. Anti-Siglec15 antibodies with improved pharmaceutical properties are needed.

[0009] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention [Means for solving the problem]

[0010] The present disclosure provides isolated murine, chimeric, human, or humanized monoclonal antibodies, or antigen-binding portions thereof, that bind to Siglec15 (e.g., human Siglec15 and monkey Siglec15) and have binding affinity / capacity for Siglec15 and blocking activity against Siglec15 binding to ligands such as LRRC4C that are comparable, if not greater, than prior art anti-Siglec15 antibodies, such as Siglec15-ch5G9 (Nextcure). The antibodies or antigen-binding portions thereof are capable of reversing Siglec15-mediated T cell suppression.

[0011] The antibodies, or antigen-binding portions thereof, of the present disclosure may be used in a variety of applications, including the detection of Siglec15 protein and the treatment and prevention of Siglec15-related diseases, such as tumors and osteoporosis.

[0012] Thus, in one aspect, the present disclosure provides a method for detecting a nucleotide sequence that binds to Siglec15, the method comprising: i) a heavy chain variable region that may comprise a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region; a heavy chain variable region, the CDR3 region of which may comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to (1) each of SEQ ID NOs: 1, 2 (X1=D, X2=Q), and 3; (2) each of SEQ ID NOs: 1, 2 (X1=E, X2=Q), and 3; (3) each of SEQ ID NOs: 1, 2 (X1=D, X2=K), and 3; (4) each of SEQ ID NOs: 9, 10, and 11; or (5) each of SEQ ID NOs: 33, 34, and 35; and / or ii) a light chain variable region, the light chain variable region comprising a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, the VL CDR1 region, a VL CDR2 region, and a VL CDR3 region. The present invention relates to an isolated monoclonal antibody (e.g., a human, murine, chimeric, or humanized antibody), or an antigen-binding portion thereof, having a light chain variable region whose CDR3 region can comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to (1) each of SEQ ID NOs: 4, 5, and 6; (2) each of SEQ ID NOs: 12, 13, and 14; or (3) each of SEQ ID NOs: 36, 37, and 38.

[0013] The antibodies, or antigen-binding portions thereof, of the disclosure may comprise a heavy chain variable region, which may include a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region; and a light chain variable region, which may include a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, and The CDR3 region may comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) each of SEQ ID NOs: 1, 2 (X1=D, X2=Q), 3, 4, 5 and 6; (2) each of SEQ ID NOs: 1, 2 (X1=E, X2=Q), 3, 4, 5 and 6; (3) each of SEQ ID NOs: 1, 2 (X1=D, X2=K), 3, 4, 5 and 6; (4) each of SEQ ID NOs: 9, 10, 11, 12, 13 and 14; or (5) each of SEQ ID NOs: 33, 34, 35, 36, 37 and 38, and the antibody or antigen-binding fragment thereof binds to Siglec15.

[0014] The heavy chain variable region of an antibody, or antigen-binding portion thereof, of the present disclosure can comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7 (X1=D, X2=Q; X1=E, X2=Q; or X1=D, X2=K), 15, 39, 40, or 41, and the antibody or antigen-binding fragment thereof binds to Siglec15. The amino acid sequence of SEQ ID NO: 7 (X1=D, X2=Q) can be encoded by the nucleotide sequence of SEQ ID NO: 27, and the amino acid sequence of SEQ ID NO: 15 can be encoded by the nucleotide sequence of SEQ ID NO: 29.

[0015] The light chain variable region of an antibody, or antigen-binding portion thereof, of the present disclosure can comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8, 16, 42, or 43, and the antibody, or antigen-binding fragment thereof, binds to Siglec15. The amino acid sequences of SEQ ID NOs: 8 and 16 can be encoded by the nucleotide sequences of SEQ ID NOs: 28 and 30.

[0016] The antibodies or antigen-binding portions thereof of the present disclosure may comprise heavy chain variable regions and light chain variable regions having amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to (1) SEQ ID NO: 7 (X1=D, X2=Q) and 8, respectively; (2) SEQ ID NO: 7 (X1=E, X2=Q) and 8, respectively; (3) SEQ ID NO: 7 (X1=D, X2=K) and 8, respectively; (4) SEQ ID NO: 15 and 16, respectively; (5) SEQ ID NO: 39 and 42, respectively; (6) SEQ ID NO: 40 and 42, respectively; or (7) SEQ ID NO: 41 and 43, respectively, and the antibodies or antigen-binding fragments thereof bind to Siglec15.

[0017] The isolated monoclonal antibody, or antigen-binding portion thereof, of the present disclosure may comprise a heavy chain and a light chain linked by a disulfide bond, the heavy chain may comprise a heavy chain variable region and a heavy chain constant region, the light chain may comprise a light chain variable region and a light chain constant region, the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region, the heavy chain variable region and the light chain variable region may comprise the amino acid sequences described above, and the antibody, or antigen-binding portion thereof, binds to Siglec15. The heavy chain constant region may be an IgG1, IgG2, or IgG4 heavy chain constant region, for example, a human IgG1, IgG2, or IgG4 heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 17. The heavy chain constant region, such as an Fc fragment, may be engineered to have reduced or enhanced FcR binding affinity. The light chain constant region may be a kappa constant region, for example, a human kappa constant region having the amino acid sequence set forth in SEQ ID NO: 18. The amino acid sequences of SEQ ID NOs: 17 and 18 can be encoded by the nucleotide sequences of SEQ ID NOs: 31 and 32, respectively.

[0018] In certain embodiments, antibodies of the present disclosure may comprise two heavy chains and two light chains, or may consist of two heavy chains and two light chains, where each heavy chain may comprise a heavy chain constant region, heavy chain variable region, or CDR sequence described above, and each light chain may comprise a light chain constant region, light chain variable region, or CDR sequence described above, and the antibody binds to Siglec15. Antibodies or antigen-binding portions thereof of the present disclosure may be full-length antibodies of, for example, the IgG1, IgG2, or IgG4 isotype. In other embodiments, antibodies or antigen-binding portions thereof may be single-chain variable fragment (scFv) antibodies, or antibody fragments such as Fab or F(ab')2 fragments.

[0019] The present disclosure also provides bispecific molecules that may include an antibody of the present disclosure or an original binding portion thereof linked to a second functional moiety (e.g., a second antibody) having a different binding specificity than the antibody or original binding portion thereof. The present disclosure also provides immunoconjugates, such as antibody-drug conjugates, that may include an antibody of the present disclosure or an antigen-binding portion thereof linked to a therapeutic agent, such as a cytotoxin. In another embodiment, the antibody of the present disclosure or an antigen-binding portion thereof may be part of a chimeric antigen receptor (CAR). Immune cells, such as T cells and NK cells, that may include an antigen-chimeric receptor are also provided. The antibody of the present disclosure or an antigen-binding portion thereof may also be encoded by or used in conjunction with an oncolytic virus.

[0020] Also included are nucleic acid molecules encoding the antibodies or antigen-binding portions thereof of the present disclosure, as well as expression vectors that may contain such nucleic acids, and host cells that may contain such expression vectors. Also provided are methods for preparing an anti-Siglec15 antibody or antigen-binding portion thereof of the present disclosure using a host cell, which may include (i) expressing the antibody in the host cell, and (ii) isolating the antibody from the host cell or a cell culture thereof.

[0021] Also provided are compositions that can include an antibody or antigen-binding portion, immunoconjugate, bispecific molecule, oncolytic virus, CAR, CAR-T cell, nucleic acid molecule, expression vector, or host cell of the present disclosure and a pharmaceutically acceptable carrier. In certain embodiments, the composition can further include a therapeutic agent, such as an anti-cancer agent.

[0022] In yet another aspect, the present disclosure provides a method of modulating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion thereof of the present disclosure, or alternatively, a nucleic acid molecule capable of expressing the antibody in the subject, such that the immune response in the subject is modulated. Preferably, the antibody or antigen-binding portion thereof of the present disclosure enhances, stimulates, or increases the immune response in the subject, e.g., by backmutating Siglec15-mediated T cell suppression. In some embodiments, the method comprises administering a bispecific molecule, immunoconjugate, CAR-T cell, or oncolytic virus encoding or carrying the antibody of the present disclosure.

[0023] In yet another aspect, the present disclosure provides a method of inhibiting bone loss or increasing bone mass in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion thereof of the present disclosure, or alternatively, a nucleic acid molecule capable of expressing the same.

[0024] In a further aspect, the present disclosure provides a method of inhibiting tumor growth in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion thereof of the present disclosure, or alternatively, a nucleic acid molecule capable of expressing the same in the subject. In some embodiments, the method comprises administering a bispecific molecule, immunoconjugate, CAR-T cell, or oncolytic virus encoding or carrying the antibody of the present disclosure. The tumor can be a solid or non-solid tumor. In certain embodiments, the tumor is a solid tumor such as, but not limited to, non-small cell lung cancer, ovarian cancer, melanoma, colorectal cancer, breast cancer (including triple-negative breast cancer), head and neck squamous cell carcinoma, endometrial cancer, and squamous cell carcinoma. In some embodiments, at least one additional anti-cancer antibody can be administered together with an antibody or antigen-binding portion thereof of the present disclosure, e.g., an anti-VISTA antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, an anti-STAT3 antibody, and / or an anti-ROR1 antibody. In yet other embodiments, antibodies, or antigen-binding portions thereof, of the present disclosure are administered with cytokines (e.g., IL-2, IL-21, GM-CSF, and / or IL-4) or costimulatory antibodies (e.g., anti-CD137 and / or anti-GITR antibodies). In another embodiment, antibodies, or antigen-binding portions thereof, of the present disclosure are administered with a chemotherapeutic agent, which may be a cytotoxic agent such as epirubicin, oxaliplatin, and / or 5-fluorouracil (5-FU). Antibodies, or antigen-binding portions thereof, of the present disclosure may be, for example, murine, human, chimeric, or humanized antibodies or antigen-binding portions thereof.

[0025] Other features and advantages of the present disclosure will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0026] Accordingly, it is not the purpose of the present invention to encompass any previously known products, methods of making the products, or methods of using the products, and therefore, the applicant reserves the right, and hereby discloses, a disclaimer of any previously known products, processes, or methods. It is further noted that the present invention does not intend to encompass within its scope any products, processes, or methods of making the products or methods of using the products that do not satisfy the USPTO's written description and enablement requirements (35 U.S.C. § 112, first paragraph) or the EPO (European Patent Convention (EPC) Article 83), and therefore, the applicant reserves the right, and hereby discloses, a disclaimer of any previously described products, methods of making the products, or methods of using the products. Compliance with Article 53(c) EPC and Rules 28(b) and (c) EPC may be advantageous in the practice of the present invention. All rights are expressly reserved to expressly disclaim any embodiment that is the subject of any issued patent of the applicant in this application line or in any other line or in any prior application of any third party. Nothing stated herein should be construed as a warranty.

[0027] It is noted that in this disclosure, and particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like may have the meaning ascribed to them by U.S. patent law; e.g., they may mean "includes," "included," "including," and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them by U.S. patent law, e.g., they may permit elements not expressly specified, but exclude elements found in the prior art or that affect a basic or novel characteristic of the invention.

[0028] The following detailed description, presented by way of example and not intended to limit the invention to only the particular embodiments described, can be best understood in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0029] [Figure 1A] 1 shows the binding ability of antibodies A1C8C6H1, A1D1B7H9, A1D5E2H1 and A1D11A7H10 to human Siglec15 in a capture ELISA. [Figure 1B] 1 shows the binding ability of antibodies A1E7G5D1, A1E10G7H9, A2A1D2F1 and A2A5G7E8 to human Siglec15 in a capture ELISA. [Figure 1C] 1 shows the binding ability of antibodies A2A6B1C2, A2G4C8G7 and A2H5F1A1 to human Siglec15 in a capture ELISA. [Figure 2A] 1 shows the binding ability of antibodies A1C8C6H1, A1D1B7H9, A1D5E2H1, and A1D11A7H10 to human-siglec15-2D3-1E1 cells expressing human Siglec15 in a cell-based binding FACS assay. [Figure 2B] 1 shows the binding ability of antibodies A1E7G5D1, A1E10G7H9, A2A1D2F1, and A2A5G7E8 to human-siglec15-2D3-1E1 cells expressing human Siglec15 in a cell-based binding FACS assay. [Figure 2C] 1 shows the binding ability of antibodies A2A6B1C2, A2G4C8G7, and A2H5F1A1 to human-siglec15-2D3-1E1 cells expressing human Siglec15 in a cell-based binding FACS assay. [Figure 3A] 1 shows the binding ability of antibodies A1C8C6H1, A1D1B7H9, A1D5E2H1, and A1D11A7H10 to cynomolgus monkey Siglec15 in indirect ELISA. [Figure 3B] 1 shows the binding ability of antibodies A1E7G5D1, A1E10G7H9, A2A1D2F1, and A2A5G7E8 to cynomolgus monkey Siglec15 in indirect ELISA. [Figure 3C]1 shows the binding ability of antibodies A2A6B1C2, A2G4C8G7, and A2H5F1A1 to cynomolgus monkey Siglec15 in indirect ELISA. [Figure 4A] 1 shows the binding ability of antibodies A1C8C6H1, A1D1B7H9, A1D5E2H1, and A1D11A7H10 to mouse Siglec15 in indirect ELISA. [Figure 4B] 1 shows the binding ability of antibodies A1E7G5D1, A1E10G7H9, A2A1D2F1, and A2A5G7E8 to mouse Siglec15 in indirect ELISA. [Figure 4C] 1 shows the binding ability of antibodies A2A6B1C2, A2G4C8G7, and A2H5F1A1 to mouse Siglec15 in indirect ELISA. [Figure 5A] 1 shows the ability of antibodies A1C8C6H1, A1D1B7H9, A1D5E2H1 and A1D11A7H10 to block human Siglec15-LRRC4C binding in a competitive ELISA. [Figure 5B] 1 shows the ability of antibodies A1E7G5D1, A1E10G7H9, A2A1D2F1 and A2A5C7E8 to block human Siglec15-LRRC4C binding in a competitive ELISA. [Figure 5C] 1 shows the ability of antibodies A2A6B1C2, A2G4C8G7, and A2H5F1A1 to block human Siglec15-LRRC4C binding in a competitive ELISA. [Figure 6A] Figure 1 shows the ability of antibodies A1C8C6H1, A1D1B7H9, A1D5E2H1 and A1D11A7H10 to block benchmark-human Siglec15 binding in a competitive ELISA. [Figure 6B] Figure 1 shows the ability of antibodies A1E7G5D1, A1E10G7H9, A2A1D2F1 and A2A5C7E8 to block benchmark-human Siglec15 binding in a competitive ELISA. [Figure 6C]Figure 1 shows the ability of antibodies A2A6B1C2, A2G4C8G7 and A2H5F1A1 to block benchmark-human Siglec15 binding in a competitive ELISA. [Figure 7A] 1 shows the ability of antibodies A1C8C6H1, A1D1B7H9, A1D5E2H1, and A1D11A7H10 to block human Siglec15 on cell surface human LRRC4C in a cell-based blocking FACS assay. [Figure 7B] 1 shows the ability of antibodies A1E7G5D1, A1E10G7H9, A2A1D2F1, and A2A5C7E8 to block human Siglec15 on cell surface human LRRC4C in a cell-based blocking FACS assay. [Figure 7C] 1 shows the ability of antibodies A2A6B1C2, A2G4C8G7, and A2H5F1A1 to block human Siglec15 on cell surface human LRRC4C in a cell-based blocking FACS assay. [Figure 8A] 1 shows that antibodies A2A5C7E8 and A1E10G7H9 restored Siglec15-induced CD8+ cell suppression in a cell-based functional assay. [Figure 8B] 1 shows that antibodies A2A5C7E8 and A1E10G7H9 restored Siglec15-induced CD4+ cell suppression in a cell-based functional assay. [Figure 9] 1 shows the binding ability of antibodies A2A5C7E8-1, A2A5C7E8-2, and A2A5C7E8-3 to human Siglec15 in a capture ELISA. [Figure 10] 1 shows the binding ability of antibodies A2A5C7E8-1, A2A5C7E8-2, and A2A5C7E8-3 to human-siglec15-2D3-1E1 cells expressing human Siglec15 in a cell-based binding FACS assay. [Figure 11] 1 shows the binding ability of antibodies A2A5C7E8-1, A2A5C7E8-2, and A2A5C7E8-3 to cynomolgus monkey Siglec15 in indirect ELISA. [Figure 12]1 shows the binding ability of antibodies A2A5C7E8-1, A2A5C7E8-2, and A2A5C7E8-3 to mouse Siglec15 in indirect ELISA. [Figure 13] 1 shows the ability of antibodies A2A5C7E8-1, A2A5C7E8-2, and A2A5C7E8-3 to block human Siglec15-LRRC4C binding in a competitive ELISA. [Figure 14] Figure 1 shows the ability of antibodies A2A5C7E8-1, A2A5C7E8-2 and A2A5C7E8-3 to block benchmark-human Siglec15 binding in a competitive ELISA. [Figure 15A] In a cell-based functional assay, antibodies A2A5C7E8-1, A2A5C7E8-2 and A2A5C7E8-3 were shown to reverse CD8+ cell suppression induced by 93.5 nM Siglec15. [Figure 15B] In a cell-based functional assay, antibodies A2A5C7E8-1, A2A5C7E8-2 and A2A5C7E8-3 were shown to reverse CD4+ cell suppression induced by 93.5 nM Siglec15. [Figure 15C] In a cell-based functional assay, antibodies A2A5C7E8-1, A2A5C7E8-2 and A2A5C7E8-3 were shown to reverse CD8+ cell suppression induced by 186.9 nM Siglec15. [Figure 15D] In a cell-based functional assay, antibodies A2A5C7E8-1, A2A5C7E8-2 and A2A5C7E8-3 were shown to reverse CD4+ cell suppression induced by 186.9 nM Siglec15. [Figure 16] Figure 1 shows the binding ability of mouse antibodies B2D7H7A3C1, B2G12H3E8 and B2H2H1H7 to human Siglec15 in a capture ELISA. [Figure 17]1 shows the binding ability of mouse antibodies B2D7H7A3C1, B2G12H3E8, and B2H2H1H7 to human-siglec15-2D3-1E1 cells expressing human Siglec15 in a cell-based binding FACS assay. [Figure 18] 1 shows the binding ability of mouse antibodies B2D7H7A3C1, B2G12H3E8 and B2H2H1H7 to cynomolgus monkey Siglec15 in an indirect ELISA. [Figure 19] 1 shows the binding ability of mouse antibodies B2D7H7A3C1, B2G12H3E8, and B2H2H1H7 to mouse Siglec15 in an indirect ELISA. [Figure 20] Figure 1 shows the ability of murine antibodies B2D7H7A3C1, B2G12H3E8 and B2H2H1H7 to block human Siglec15-LRRC4C binding in a competitive ELISA. [Figure 21] Figure 1 shows the ability of murine antibodies B2D7H7A3C1, B2G12H3E8 and B2H2H1H7 to block benchmark-human Siglec15 binding in a competitive ELISA. [Figure 22A] We show that murine antibodies B2D7H7A3C1, B2G12H3E8 and B2H2H1H7 restored Siglec15-mediated CD8+ cell suppression in cell-based functional assays. [Figure 22B] We show that murine antibodies B2D7H7A3C1, B2G12H3E8 and B2H2H1H7 restored Siglec15-mediated CD4+ cell suppression in cell-based functional assays. DETAILED DESCRIPTION OF THE INVENTION

[0030] To ensure that this disclosure may be more readily understood, some terms are first defined. Further definitions are set forth throughout the detailed description.

[0031] The term "Siglec15" includes variants, isoforms, homologs, orthologs, and paralogs. For example, an antibody specific for human Siglec15 protein may, in some cases, cross-react with Siglec15 protein from species other than humans, such as monkeys. In other embodiments, an antibody specific for human Siglec15 protein may be completely specific for human Siglec15 protein and may not exhibit other species or other types of cross-reactivity, or may cross-react with Siglec15 from certain other species but not all other species.

[0032] The term "human Siglec15" refers to a Siglec15 protein having an amino acid sequence derived from a human, such as the amino acid sequence of human Siglec15 having Genbank accession number Q6ZMC9. The terms "monkey or rhesus Siglec15" and "mouse Siglec15" refer to monkey and mouse Siglec15 sequences, respectively, such as those having the amino acid sequences with Genbank accession numbers XP_028694069.1 and NP_001094508.1, respectively.

[0033] The term "immune response" refers to the actions of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver that result in selective damage to, for example, invading pathogens, pathogen-infected cells or tissues, destruction of cancer cells, or elimination from the human body, or in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0034] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as Siglec15, through at least one antigen-binding site, which is typically located within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-chain Fv (scFv) antibodies, heavy-chain antibodies (HCAbs), light-chain antibodies (LCAbs), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, fusion proteins comprising the antigen-binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen-binding site (e.g., dual variable domain immunoglobulin molecules), so long as the antibody exhibits the desired biological activity. Antibodies include, but are not limited to, murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies can be any of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, called alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules, such as, but not limited to, toxins and radioisotopes. Unless otherwise specified, the term "antibody," as used herein, includes the "antigen-binding portion" of an intact antibody. IgG is a glycoprotein that can comprise two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V) H The heavy chain constant region can be composed of three domains: C H1 , C H2 and C H3 Each light chain can be composed of a light chain variable region (referred to herein as V L The light chain constant region can consist of one domain, C L V H and V LThe regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0035] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a Siglec15 protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) Fab fragments, V L , V H , C L and C H1 (ii) a F(ab')2 fragment, a bivalent fragment that may contain two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and C H1 (iv) a V fragment of a single arm of an antibody; L and V H Fv fragment consisting of domains, (v) V H (vi) isolated complementarity-determining regions (CDRs); and (viii) nanobodies, heavy chain variable regions containing a single variable domain and two constant domains. In addition, Fv fragments contain two domains, V and V. L and V Hare encoded by separate genes, but they are grouped together as V L and V H These regions can be joined using recombinant methods by synthetic linkers that allow them to be produced as a single protein chain (known as a single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) in which the regions pair to form a monovalent molecule. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0036] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to Siglec15 protein is substantially free of antibodies that specifically bind to antigens other than Siglec15 protein). However, an isolated antibody that specifically binds to human Siglec15 protein may have cross-reactivity to other antigens, such as Siglec15 proteins from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0037] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0038] The term "human antibody," as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences.

[0039] The term "murine antibody," as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from mouse germline immunoglobulin sequences. The murine antibodies of the invention may include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "murine antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto murine framework sequences.

[0040] The term "chimeric antibody" refers to an antibody made by combining genetic material from a non-human source with genetic material from a human, or more generally, a chimeric antibody is an antibody that has genetic material from a particular species along with genetic material from another species.

[0041] The term "humanized antibody" as used herein refers to antibodies derived from non-human species in which the protein sequences have been modified to increase their similarity to antibody forms naturally produced in humans.

[0042] The term "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0043] The phrases "antibody that recognizes an antigen" and "antibody that is specific for an antigen" are used herein synonymously with the term "antibody that specifically binds to an antigen."

[0044] As used herein, an antibody that "specifically binds to human Siglec15" is intended to refer to an antibody that binds to human Siglec15 protein (and optionally one or more non-human derived Siglec15 proteins), but does not substantially bind to non-Siglec15 proteins. Preferably, the antibody has a "high affinity," i.e., a binding affinity of 5.0×10 -9 M or less, more preferably 1.0 × 10 -9 M or less, more preferably 1.0 × 10 -10 K below M D It binds to the human Siglec15 protein.

[0045] As used herein, the term "does not substantially bind" to proteins or cells means that the antibody does not bind to proteins or cells or does not bind with high affinity, i.e., binds with less than 1.0 x 10 -6 M or more, more preferably 1.0 × 10 -5 M or more, more preferably 1.0 × 10 -4 M or more, more preferably 1.0 × 10 -3 M or more, and even more preferably 1.0 x 10 -2 K over M D This means that the molecule binds to a protein or cell.

[0046] The term "high affinity" for an IgG antibody refers to an affinity of 5.0 × 10 for the target antigen.-9 M or less, more preferably 1.0 × 10 -9 M or less, and even more preferably 5.0 x 10 -10 M or less, and even more preferably 1.0 x 10 -10 M or less, and even more preferably 5.0 x 10 -11 K below M D However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is defined as 10 -6 M or less, more preferably 10 -7 M or less, and even more preferably, 10 -8 K below M D It refers to an antibody having the following structure:

[0047] As used herein, "K" assoc " or "K a While the term "K" is intended to refer to the binding rate of a particular antibody-antigen interaction, as used herein, dis " or "K d The term "K" as used herein is intended to refer to the off-rate of a particular antibody-antigen interaction. D The term "dissociation constant" is intended to refer to the dissociation constant, which is K d Against K a The ratio of (i.e., K d / K a ) and expressed as molar concentration (M). D The K value can be determined using methods well established in the art. D A preferred method for determining is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore™ system.

[0048] The half-maximal effective concentration (EC) is also known as 50 The term "antibody concentration" refers to the concentration of antibody that induces a response halfway between baseline and maximum after a particular exposure time.

[0049] The half maximal inhibitory concentration (IC 50 The term "antibody concentration" refers to a concentration of antibody that inhibits a specific biological or biochemical function by 50% compared to the absence of the antibody.

[0050] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals such as non-human primates, sheep, dogs, cats, cows, and horses are preferred.

[0051] The term "therapeutically effective amount" refers to an amount of an antibody or antigen-binding portion thereof of the present disclosure sufficient to prevent or ameliorate symptoms associated with a disease or condition (such as cancer) and / or reduce the severity of the disease or condition. A therapeutically effective amount is understood in relation to the condition being treated, and the actual effective amount is readily understood by one of ordinary skill in the art.

[0052] Various aspects of the disclosure are described in further detail in the following subsections.

[0053] The antibodies, or antigen-binding portions thereof, of the present disclosure specifically bind to human or monkey Siglec15 with binding affinity comparable to, if not better than, the anti-Siglec15 antibodies described above, such as Siglec15-ch5G9.

[0054] Additional functional properties include the ability to block Siglec15 binding to its ligand and to restore Siglec15-mediated T cell suppression.

[0055] Exemplary antibodies, or antigen-binding portions thereof, of the present disclosure are structurally and chemically characterized as described below and in the Examples. The amino acid sequence numbers of the heavy and light chain variable regions of the antibodies are summarized in Table 1 below. The heavy chain constant region of the antibody can be, for example, a human IgG1 heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 17, and the light chain constant region of the antibody can be, for example, a human kappa constant region having the amino acid sequence set forth in SEQ ID NO: 18. The antibodies of the present disclosure can be human, murine, chimeric, or humanized antibodies.

[0056] The heavy and light chain variable region CDRs in Table 1 are defined by the Kabat numbering system, however, as is well known in the art, CDR regions can also be determined by other systems, such as Chothia, IMGT, AbM, or Contact numbering systems / methods, based on the heavy / light chain variable region sequences.

[0057] [Table 1]

[0058] V of other anti-Siglec15 antibodies that bind to human Siglec15 H and V L The sequences (or CDR sequences) of the anti-Siglec15 antibodies of the present disclosure are H and V L Preferably, V H and V L When chains (or CDRs within such chains) are mixed and matched, a particular V H / V L V from involution H The sequence is structurally similar to V H Similarly, preferably, a particular V H / V L V from involution L The sequence is structurally similar to V L It is replaced by an array.

[0059] Thus, in one embodiment, an antibody of the disclosure, or an antigen-binding portion thereof, (a) a heavy chain variable region comprising the amino acid sequence listed above in Table 1; and (b) a light chain variable region comprising an amino acid sequence listed above in Table 1, or the V of another anti-Siglec15 antibody L wherein the antibody specifically binds to human Siglec15.

[0060] In another embodiment, an antibody, or antigen-binding portion thereof, of the present disclosure: (a) the CDR1, CDR2, and CDR3 regions of the heavy chain variable region listed above in Table 1; and (b) comprises the CDR1, CDR2, and CDR3 regions of a light chain variable region listed above in Table 1 or the CDRs of another anti-Siglec15 antibody, wherein the antibody specifically binds to human Siglec15.

[0061] In yet another embodiment, the antibody, or antigen-binding portion thereof, comprises the CDRs of another antibody that binds to human Siglec15, e.g., the heavy chain variable CDR2 region of an anti-Siglec15 antibody combined with CDR1 and / or CDR3 from the heavy chain variable region, and / or CDR1, CDR2, and / or CDR3 from the light chain variable region of a different anti-Siglec15 antibody.

[0062] Furthermore, it is well known in the art that the CDR3 domain alone, independent of the CDR1 and / or CDR2 domains, can determine the binding specificity of an antibody to a cognate antigen, and that multiple antibodies can be predictably generated with the same binding specificity based on a common CDR3 sequence. For example, Klimka et al.,British J.of Cancer 83(2):252-260(2000);Beiboer et al.,J.Mol.Biol.296:833-849(2000);Rader et al.,Proc.Natl.Acad.Sci.USA95:8910-8915(1998);Barbas et al. al.,J.Am.Chem.Soc.116:2161-2162(1994);Barbas et al.,Proc.Natl.Acad.Sci.USA92:2529-2533(1995);Ditzel et al.,J.Immunol.157:739-749(1996);Berezov et al.,BIAjournal 8:Scientific Review 8(2001);Igarashi et al. See, e.g., Bourgeois et al., J. Virol 72:807-10 (1998); Levi et al., Proc. Natl. Acad. Sci. USA 90:4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994), and Xu and Davis, Immunity 13:37-45 (2000). See also U.S. Patent Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905; and 5,760,185, each of which is incorporated herein by reference in its entirety.

[0063] Thus, in another embodiment, an antibody of the present disclosure comprises CDR2 of the heavy chain variable region of an anti-Siglec15 antibody and at least CDR3 of the heavy and / or light chain variable region of an anti-Siglec15 antibody, or the CDR3 of the heavy and / or light chain variable region of another anti-Siglec15 antibody, wherein the antibody is capable of specifically binding to human Siglec15. These antibodies preferably (a) compete for binding with Siglec15; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have similar binding affinity as the anti-Siglec15 antibody of the present disclosure. In yet another embodiment, the antibody may further comprise CDR2 of the light chain variable region of an anti-Siglec15 antibody, or the CDR2 of the light chain variable region of another anti-Siglec15 antibody, wherein the antibody is capable of specifically binding to human Siglec15. In another embodiment, an antibody of the present disclosure may further comprise CDR1 of the heavy and / or light chain variable region of an anti-Siglec15 antibody, or CDR1 of the heavy and / or light chain variable region of another anti-Siglec15 antibody, wherein the antibody is capable of specifically binding to human Siglec15.

[0064] In another embodiment, an antibody of the present disclosure comprises heavy and / or light chain variable region sequences of CDR1, CDR2, and CDR3 sequences that differ from those of an anti-Siglec15 antibody of the present disclosure by one or more conservative modifications. It is understood in the art that certain conservative sequence modifications can be made without eliminating antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0065] Thus, in one embodiment, the antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and / or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: (a) the heavy chain variable region CDR1 sequence comprises a sequence listed in Table 1 above, and / or a conservative modification thereof; and / or (b) the heavy chain variable region CDR2 sequence comprises a sequence listed in Table 1 above, and / or a conservative modification thereof; and / or (c) the heavy chain variable region CDR3 sequence comprises a sequence listed in Table 1 above, and / or a conservative modification thereof; and / or (d) the light chain variable region CDR1, and / or CDR2, and / or CDR3 sequences comprise the sequences listed in Table 1 above; and / or conservative modifications thereof; (e) The antibody specifically binds to human Siglec15.

[0066] The antibodies, or antigen-binding portions thereof, of the present disclosure retain one or more of the following functional properties described above, eg, high affinity binding to human Siglec15 and the ability to restore Siglec15-mediated T cell suppression.

[0067] In various embodiments, the antibody or antigen-binding portion thereof can be, for example, a human, murine, humanized, or chimeric antibody or antigen-binding portion thereof.

[0068] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not substantially affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the present disclosure can be substituted with other amino acid residues from the same side chain family, and the altered antibodies can be tested for retained function (i.e., the above-mentioned functions) using the functional assays described herein.

[0069] The antibodies of the present disclosure may be prepared by using the V of an anti-Siglec15 antibody of the present disclosure as starting material to engineer modified antibodies. H / V LThe antibody may be prepared using antibodies having one or more of the following sequences: H and / or V L ), for example, by modifying one or more residues in one or more CDR regions and / or one or more framework regions. Additionally or alternatively, antibodies may be engineered by modifying residues in the constant region(s), for example, to alter the effector functions of the antibody.

[0070] In certain embodiments, CDR grafting techniques can be used to engineer the variable regions of antibodies. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity-determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Because CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular natural antibody by constructing expression vectors containing CDR sequences from that particular natural antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., (1989) Proc. Natl. Acad. USA 86:10029-10033; see also U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

[0071] Accordingly, another embodiment of the present disclosure relates to isolated monoclonal antibodies, or antigen-binding portions thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above, and / or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above. H and V LWhile comprising CDR sequences, they may comprise different framework sequences.

[0072] Such framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), as well as Kabat et al., (1991) (cited above); Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836 (the contents of each of which are expressly incorporated herein by reference). As another example, germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in the HCo7 HuMAb mouse are available at the attached Genbank accession numbers 1-69 (NG--0010109, NT--024637 and BC070333), 3-33 (NG--0010109 and NT--024637) and 3-7 (NG--0010109 & NT--024637). As another example, the following heavy chain germline sequences found in the HCo12 HuMAb mouse are available under the attached Genbank accession numbers: 1-69 (NG--0010109, NT--024637, and BC070333), 5-51 (NG--0010109 and NT--024637), 4-34 (NG--0010109 and NT--024637), 3-30.3 (CAJ556644), and 3-23 (AJ406678).

[0073] The antibody protein sequence is compared against compiled protein sequence databases using one of the sequence similarity search methods known to those skilled in the art, called Gapped BLAST (Altschul et al., (1997), supra).

[0074] Preferred framework sequences for use in the antibodies of this disclosure are structurally similar to the framework sequences used by the antibodies of this disclosure. H The CDR1, CDR2, and CDR3 sequences can be grafted into framework regions having the same sequence as found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted into framework regions that contain one or more mutations compared to the germline sequence. For example, in some cases, it has been found to be beneficial to mutate residues within the framework regions to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

[0075] Another type of variable region modification is the V H and / or V L Amino acid residues within the CDR1, CDR2, and / or CDR3 regions are mutated to improve one or more binding characteristics (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effect on antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays known in the art. Preferably, conservative modifications (as known in the art) are introduced. The mutations can be amino acid substitutions, additions, or deletions, but are preferably substitutions. Furthermore, typically, no more than one, two, three, four, or five residues within the CDR regions are altered.

[0076] Thus, in another embodiment, the present disclosure provides a V that comprises (a) a sequence of the present disclosure or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions. H (b) a V region comprising an amino acid sequence of the disclosure or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; H(c) a V comprising an amino acid sequence of the present disclosure or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; H (d) a V comprising an amino acid sequence of the present disclosure or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; L (e) a V comprising an amino acid sequence of the present disclosure or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; L and (f) a V comprising a sequence of the disclosure or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions. L An isolated anti-Siglec15 monoclonal antibody, or an antigen-binding portion thereof, is provided, comprising a heavy chain variable region including a CDR3 region.

[0077] The engineered antibodies of the disclosure may be modified, e.g., to improve the properties of the antibody. H and / or V L These framework modifications include those made to framework residues within the following: (a) nucleotide sequence (SEQ ID NO: 1); (b) nucleotide sequence (SEQ ID NO: 2); (c) nucleotide sequence (SEQ ID NO: 3); (d) nucleotide sequence (SEQ ID NO: 4); (e) nucleotide sequence (SEQ ID NO: 5); (f) nucleotide sequence (SEQ ID NO: 6); (g) nucleotide sequence (SEQ ID NO: 7); (h) nucleotide sequence (SEQ ID NO: 8); (i) nucleotide sequence (SEQ ID NO: 9); (j) nucleotide sequence (SEQ ID NO: 10); (j) nucleotide sequence (SEQ ID NO: 11); (j) nucleotide sequence (SEQ ID NO: 12); (j) nucleotide sequence (SEQ ID NO: 13); (j) nucleotide sequence (SEQ ID NO: 14); (j) nucleotide sequence (SEQ ID NO: 15); (j) nucleotide sequence (SEQ ID NO: 16); (j) nucleotide sequence (SEQ ID NO: 17); (j) nucleotide sequence (SEQ ID NO: 18); (j) nucleotide sequence (SEQ ID NO: 19); (k) nucleotide sequence (SEQ ID NO: 20); (k) nucleotide sequence (SEQ ID NO: 21); (k) nucleotide sequence (SEQ ID NO: 22); (k) nucleotide sequence (SEQ ID NO: 23); (k) nucleotide sequence (SEQ ID NO: 24); (k) nucleotide sequence (SEQ ID NO: 25); (k) nucleotide sequence (SEQ ID NO: 26); (k) nucleotide sequence (SEQ ID NO: 27); (k) nucleotide sequence (SEQ ID NO: 28); (k) nucleotide sequence (SEQ ID NO: 29); (k) nucleotide sequence (SEQ ID NO: 30); (k) nucleotide sequence (SEQ ID NO: 31); (k) nucleotide sequence (SEQ ID

[0078] Another type of framework modification involves mutating one or more residues within the framework regions or one or more CDR regions to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach, also known as "deimmunization," is described in further detail in U.S. Patent Application Publication No. 20030153043.

[0079] In addition to, or instead of, modifications made within the framework or CDR regions, antibodies of the disclosure can be engineered to contain modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Furthermore, antibodies of the disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) to similarly alter one or more functional properties of the antibody or to alter its glycosylation.

[0080] In one embodiment, C H1 The hinge region of is modified such that the number of cysteine ​​residues in the hinge region is changed, e.g., increased or decreased. This technique is further described in U.S. Pat. No. 5,677,425. H1 The number of cysteine ​​residues in the hinge region of the antibody can be altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0081] In another embodiment, the Fc-hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More particularly, one or more amino acid mutations are made to the C of the Fc-hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding compared to native Fc-hinge region SpA binding. H2 -C H3 This technique is described in more detail in U.S. Patent No. 6,165,745.

[0082] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such glycosylation can increase the affinity of the antibody for an antigen. See, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861.

[0083] Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present disclosure, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)-fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose in their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see U.S. Patent Application Publication No. 2004 / 0110704 and Yamane-Ohnuki et al., (2004) Biotechnol Bioeng 87:614-22). As another example, European Patent No. 1,176,195 describes cell lines in which the FUT8 gene, encoding a fucosyltransferase, has been functionally disrupted, such that antibodies expressed in such cell lines exhibit hypofucosylation by reducing or eliminating α-1,6 bond-related enzymes. Furthermore, European Patent No. 1,176,195 describes cell lines that bind to the Fc region of antibodies or have no enzymatic activity, and have low enzymatic activity for the addition of fucose to N-acetylglucosamine, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662).WO 03 / 035835 describes Lec13 cells, a mutant CHO cell line that reduces the ability to attach fucose to Asn(297)-linked carbohydrates and also results in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Antibodies with modified glycosylation profiles can also be produced in chicken eggs, as described in WO 06 / 089231. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells, such as Lemna. Methods for producing antibodies in plant systems are disclosed in U.S. patent application corresponding to Alston & Bird LLP Attorney Docket No. 040989 / 314911, filed August 11, 2006. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit a bisecting GlcNac structure, resulting in increased ADCC activity of the antibody (see also Umana et al., (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of antibodies can be cleaved using a fucosidase enzyme; for example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al., (1975) Biochem. 14:5516-23).

[0084] Another modification of the antibodies herein contemplated by the present disclosure is pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or a fragment thereof, is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono(C1-C 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present disclosure. See, e.g., EP 154 316 and EP 0 401 384.

[0085] The antibodies of the present disclosure can be characterized by their various physical properties in order to detect and / or distinguish between different classes thereof.

[0086] For example, an antibody may contain one or more glycosylation sites in either the light chain or heavy chain variable region. Such glycosylation sites may result in increased immunogenicity or altered pK of the antibody, resulting in altered antigen binding (Marshall et al., (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al., (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al., (1985) Nature 316:452-7; Mimura et al., (2000) Mol Immunol 37:697-706). Glycosylation is known to occur at motifs containing the NXS / T sequence. In some cases, it may be preferable to have an anti-Siglec15 antibody that does not contain variable region glycosylation, which can be achieved by selecting an antibody that does not contain glycosylation motifs in the variable region or by mutating residues within the glycosylated region.

[0087] In a preferred embodiment, the antibody does not contain an asparagine isomerism site. Deamidation of asparagine can occur at NG or DG sequences, resulting in the generation of isoaspartic acid residues that introduce bonds into the polypeptide chain and reduce its stability (isoaspartic acid effect).

[0088] Each antibody has a unique isoelectric point (pI), generally within the pH range of 6 to 9.5. The pI of an IgG1 antibody is typically within the pH range of 7 to 9.5, and the pI of an IgG4 antibody is typically within the pH range of 6 to 8. It has been speculated that antibodies with a pI outside the normal range may have some unfolding and instability under in vivo conditions. Therefore, it is preferable to have an anti-Siglec15 antibody with a pI value within the normal range. This can be achieved by selecting an antibody with a pI within the normal range or by mutating charged surface residues.

[0089] In another aspect, the present disclosure provides nucleic acid molecules encoding the heavy and / or light chain variable regions, or CDRs, of an antibody of the present disclosure. The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" if it has been purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. The nucleic acids of the present disclosure can be, for example, DNA or RNA, and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0090] Nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from the gene library.

[0091] A preferred nucleic acid molecule of the present disclosure is the V H and V L V includes nucleic acid molecules encoding the CDRs. H and V L Once the DNA fragments encoding the segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L or V HThe DNA fragment encoding is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0092] V H The isolated DNA encoding the region is V H The DNA encoding the heavy chain constant region (C H1 , C H2 and C H3 The heavy chain constant region can be converted into a full-length heavy chain gene by operably linking it to another DNA molecule encoding the V. The sequences of human heavy chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene, the V H The DNA encoding the heavy chain C H1 It may be operably linked to another DNA molecule encoding only the constant region.

[0093] V L The isolated DNA encoding the region is the light chain constant region, C L V on another DNA molecule that encodes L The light chain constant region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking DNA encoding the light chain constant region to the Fab light chain. The sequences of human light chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region.

[0094] To generate the scFv gene, H and V L The sequences are linked by a flexible linkerL and V H V can be expressed as a continuous single-chain protein with H and V L The DNA fragment encoding the nucleotide sequence (Gly4-Ser) is operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (see, e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0095] The monoclonal antibodies (mAbs) of the present disclosure can be expressed in transgenic mouse platforms (e.g., CAMouse HG Fully human antibodies can be produced by immunizing transgenic mice genetically engineered to produce fully human antibodies with the target antigen, namely, Siglec15, specifically human Siglec15, using a mouse model (B000.60.01T(G15), HG5042, Chongqing CamabBiotech Ltd.). Spleen cells from immunized transgenic mice were fused with myeloma cells according to the method described in Kohler G, and Milstein C, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256:495-497 (1975). The fused "hybrid cells" were then plated, and surviving hybridoma colonies were observed microscopically 7–10 days after fusion. After, for example, two weeks, supernatants from each well could be subjected to antigen binding tests. Positive hybridomas secreting the desired antibody were subcloned by limiting dilution to ensure the clonality of the cell line, followed by purification of the monoclonal antibody.

[0096] Antibodies of the present disclosure can also be generated by other methods well known in the art, such as viral or oncogenic transformation of B lymphocytes and phage display technology.

[0097] Antibodies of the present disclosure can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods, as are well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial or full-length light and heavy chains, obtained by standard molecular biology techniques, is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene.

[0098] The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include promoters and / or enhancers derived from viral elements that direct high-level protein expression in mammalian cells, such as cytomegalovirus (CMV), simian virus 40 (SV40), and adenovirus, e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or β-globin promoter, can be used. Furthermore, regulatory elements are composed of sequences derived from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). Expression vectors and expression control sequences are selected to be compatible with the expression host cell used.

[0099] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. H The segment is located at C H operatively connected to the V L The segment is located at C LThe variable regions are used to generate full-length antibody genes of any antibody isotype by inserting them operably linked to an expression vector already encoding heavy and light chain constant regions of the desired isotype. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0100] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, the selectable marker gene typically confers resistance to drugs, such as G418, hygromycin, or methotrexate, in a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0101] For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for introducing foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. While it is theoretically possible to express the antibodies of the present disclosure in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, most preferably mammalian host cells, is most preferred, because such cells are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active antibodies.

[0102] Preferred mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO) cells (including, e.g., dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, as described in R.J. Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. Another preferred expression system, particularly for use with NSO myeloma cells, is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium that the host cells are grown in. The antibody can be recovered from the culture medium using standard protein purification methods.

[0103] The antibodies of the present disclosure can be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In ADCs, the antibody and therapeutic agent are preferably conjugated via a cleavable linker, such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Pat. Nos. 7,087,600; 6,989,452; and 7,129,261; WO 02 / 096910; WO 07 / 038,658; WO 07 / 051,081; WO 07 / 059,404; WO 08 / 083,312; and WO 08 / 103,693; U.S. Patent Application Publication Nos. 2006 / 0024317; 2006 / 0004081; and 2006 / 0247295, the disclosures of which are incorporated herein by reference.

[0104] In another aspect, the present disclosure features bispecific molecules comprising one or more antibodies of this disclosure linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. Thus, as used herein, "bispecific molecule" includes molecules with three or more specificities.

[0105] Bispecific molecules can be in many different formats and sizes. At one end of the size spectrum, bispecific molecules retain the traditional antibody format, except that instead of having two binding arms of the same specificity, they have two binding arms with different specificities. At the other end are bispecific molecules consisting of two single-chain antibody fragments (scFvs) linked by a peptide chain, the so-called Bs(scFv)2 construct. Intermediate-sized bispecific molecules contain two different F(ab) fragments linked by a peptidyl linker. These and other formats of bispecific molecules can be prepared by genetic recombination, somatic cell hybridization, or chemical methods. See, for example, Kufer et al. (cited above); Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al., Immunology Today, 21(8), 391-397 (2000), and the references cited therein.

[0106] Also provided herein are oncolytic viruses that preferentially infect and kill cancer cells. The antibodies of the present disclosure can be used in conjunction with oncolytic viruses. Alternatively, oncolytic viruses encoding the antibodies of the present disclosure can be introduced into the human body.

[0107] Also provided herein is a chimeric antigen receptor (CAR) comprising an anti-Siglec15 scFv, which comprises the CDRs and heavy / light chain variable regions described herein.

[0108] An anti-Siglec15 CAR may comprise: (a) an extracellular antigen-binding domain comprising an anti-Siglec15 scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0109] CARs may contain a signal peptide at the N-terminus of the extracellular antigen-binding domain, which directs the nascent receptor to the endoplasmic reticulum, and a hinge peptide at the N-terminus of the extracellular antigen-binding domain, which makes the receptor more accessible for binding. CARs preferably contain a major intracellular signaling domain and one or more costimulatory signaling domains in the intracellular signaling domain. The most commonly used and most effective major intracellular signaling domain is the ITAM-containing CD3-zeta cytoplasmic domain, whose phosphorylation leads to T cell activation. The costimulatory signaling domain may be derived from costimulatory proteins such as CD28, CD137, and OX40.

[0110] CARs may further include factors that promote T cell proliferation, persistence, and anti-tumor activity, such as cytokines and costimulatory ligands.

[0111] Also provided are engineered immune effector cells comprising the CARs provided herein. In some embodiments, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In some embodiments, the immune effector cells are T cells.

[0112] In another aspect, the present disclosure provides pharmaceutical compositions that can include one or more antibodies or antigen-binding portions thereof, bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates, nucleic acid molecules, expression vectors, or host cells of the present disclosure formulated with a pharmaceutically acceptable carrier. The antibodies or antigen-binding portions thereof, bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates, nucleic acid molecules, expression vectors, or host cells can be administered separately when the composition includes two or more antibodies (or antigen-binding portions thereof, bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates, nucleic acid molecules, expression vectors, or host cells). The compositions can optionally contain one or more additional pharmaceutically active ingredients, e.g., another antibody or drug, e.g., an anti-tumor drug.

[0113] Pharmaceutical compositions can contain any excipient. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonicity agents, and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003) (the disclosure of which is incorporated herein by reference).

[0114] Preferably, the pharmaceutical compositions are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate it. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion. Alternatively, antibodies of the present disclosure may be administered by a non-parenteral route, for example, a topical, epidermal, or mucosal route of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical.

[0115] The pharmaceutical compositions may be in the form of sterile aqueous solutions or dispersions. They may also be formulated as microemulsions, liposomes, or other ordered structures suitable to high drug concentration.

[0116] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular method of administration, but will generally be the amount of the composition that produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient, in combination with a pharmaceutically acceptable carrier.

[0117] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect, together with the necessary pharmaceutical carrier. Alternatively, antibodies can be administered as sustained-release formulations when less frequent administration is required.

[0118] For administration of the composition, the dosage may range from about 0.0001 to 100 mg / kg.

[0119] A "therapeutically effective dosage" of an anti-Siglec15 antibody, or antigen-binding portion thereof, or bispecific, CAR-T cell, oncolytic virus, or immunoconjugate of the present disclosure preferably reduces the severity of disease symptoms and increases the frequency and length of symptom-free periods, or prevents disability or disability due to disease affliction. For example, for treating a subject with a tumor, a "therapeutically effective dosage" preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, compared to an untreated subject. A therapeutically effective amount of a therapeutic antibody can reduce tumor size or ameliorate symptoms in a subject, which may typically be a human or another mammal.

[0120] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used.See, for example, "Sustained and Controlled Release Drug Delivery Systems," JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0121] Therapeutic compositions can be administered by medical devices, such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (U.S. Pat. No. 4,487,603); (3) transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion devices (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0122] In certain embodiments, monoclonal antibodies of the present disclosure may be formulated to ensure proper distribution in vivo. For example, to ensure that therapeutic antibodies of the present disclosure cross the blood-brain barrier, they may be formulated in liposomes, which may further contain targeting molecules to facilitate selective delivery to specific cells or organs. For example, US Pat. No. 4,522,811; US ​​Pat. No. 5,374,548; US Pat. No. 5,416,016; and US Pat. No. 5,399,331; VVRanade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al. al.,(1988)Biochem.Biophys.Res.Commun.153:1038;Bloeman et al.,(1995)FEBS Lett.357:140;M.Owais et al.,(1995)Antimicrob.Agents Chemother.39:180;Briscoe et al. al.,(1995)Am.J.Physiol.1233:134;Schreier et al.,(1994)J.Biol.Chem.269:9090;Keinanen See, e.g., and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods 4:273.

[0123] The compositions of the present disclosure have many in vitro and in vivo utilities, including, for example, the treatment of cancer and osteoporosis. The antibodies can be administered to a human subject to, for example, inhibit tumor growth or inhibit bone loss in vivo.

[0124] Given the ability of the anti-Siglec15 antibodies or antigen-binding portions of the present disclosure to restore Siglec15-mediated T cell suppression and inhibit cancer cell proliferation and survival, the present disclosure provides a method of inhibiting tumor cell growth in a subject, comprising administering a composition of the present disclosure to the subject, such that tumor growth is inhibited in the subject. Non-limiting examples of tumors that can be treated with the compositions of the present disclosure include, but are not limited to, non-small cell lung cancer, ovarian cancer, melanoma, colorectal cancer, breast cancer (including triple-negative breast cancer), head and neck squamous cell carcinoma, endometrial cancer, and squamous cell carcinoma. Additionally, refractory or recurrent malignancies whose growth can be inhibited using the antibodies of the present disclosure are included.

[0125] In another aspect, the present disclosure provides a method for inhibiting bone loss or increasing bone mass, comprising administering to a subject an effective amount of an antibody, or antigen-binding portion thereof, of the present disclosure.

[0126] In another aspect, the present disclosure provides a combination treatment method in which an anti-Siglec15 antibody, or antigen-binding portion thereof, or bispecific molecule, CAR-T cell, oncolytic virus, or immunoconjugate of the present disclosure is co-administered with one or more additional antibodies effective to inhibit tumor growth in the subject. In one embodiment, the present disclosure provides a method of inhibiting tumor growth in a subject, comprising administering to the subject an anti-Siglec15 antibody (or antigen-binding portion thereof, or CAR-T cell, oncolytic virus, or immunoconjugate) and one or more additional antibodies, e.g., an anti-VISTA antibody, an anti-LAG-3 antibody, an anti-PD-L1 antibody, and an anti-PD-1 antibody and / or an anti-CTLA-4 antibody. In certain embodiments, the subject is a human.

[0127] Siglec15 signaling activation can also be further combined with standard cancer treatment. For example, Siglec15 signaling inhibition can be combined with CTLA-4 and / or LAG-3 and / or PD-1 blockade and further chemotherapy regimens. For example, a chemotherapeutic agent, which may be a cytotoxic agent, can be administered together with an anti-Siglec15 antibody. For example, epirubicin, oxaliplatin, and 5-FU are administered to patients undergoing anti-Siglec15 therapy.

[0128] Optionally, the combination of anti-Siglec15 and one or more additional antibodies (e.g., anti-CTLA-4 and / or anti-LAG-3 and / or anti-PD-1 antibodies) can be further combined with immunogens, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), and cells transfected with genes encoding immunostimulatory cytokines (He et al., (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include tumor cells transfected to express melanoma antigen peptides, such as peptides of gp100, MAGE antigens, Trp-2, MART1, and / or tyrosinase, or the cytokine GM-CSF.

[0129] Other therapies that may be combined with anti-Siglec15 therapy include, but are not limited to, interleukin-2 (IL-2) administration, radiation, surgery, or hormone deprivation.

[0130] In another aspect, the present disclosure provides a method of combination therapy in which an anti-Siglec15 antibody, or antigen-binding portion thereof, or bispecific molecule, CAR-T cell, oncolytic virus, or immunoconjugate of the present disclosure is co-administered with one or more additional agents effective in inhibiting bone loss. In one embodiment, the present disclosure provides a method of inhibiting bone loss in a subject, comprising administering to the subject an anti-Siglec15 antibody (or antigen-binding portion thereof, or CAR-T cell, oncolytic virus, or immunoconjugate) and one or more additional antibodies for treating osteoporosis, e.g., an anti-RANKL antibody and an anti-IL-11 antibody. In certain embodiments, the subject is a human.

[0131] The combination of therapeutic agents described herein can be administered simultaneously in a single composition in a pharmaceutically acceptable carrier or as separate compositions containing each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0132] Furthermore, when two or more doses of a combination therapy are administered sequentially, the order of sequential administration may be reversed or maintained in the same order at each time of administration, sequential administration may be combined with simultaneous administration, or any combination thereof.

[0133] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents and published patent applications cited throughout this application are hereby expressly incorporated by reference. [Example]

[0134] Example 1: Production of human anti-Siglec15 monoclonal antibodies using hybridoma technology immunization Transgenic Mouse Platform CAMouse HGFully human antibodies were generated using a recombinant human Siglec15 antibody (HG5042, Chongqing CAMAB Biotech Ltd.). Transgenic mice were immunized according to the method described in E. Harlow and D. Lane, *Antibody: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998. A homemade recombinant human Siglec15 protein (amino acid sequence set forth in SEQ ID NO: 19) with human IgG1 Fc at the C-terminus was used as the immunogen, and a homemade cynomolgus monkey Siglec15-his protein (amino acid sequence set forth in SEQ ID NO: 21) was used to determine antiserum titers and to screen hybridomas secreting antigen-specific antibodies. The immunization dose included 50 μg of human Siglec15-Fc protein per mouse per injection for both the primary and booster immunizations. To increase the immune response, complete Freund's adjuvant and incomplete Freund's adjuvant (Sigma, St. Louis, Mo., USA) were used for the primary and booster immunizations, respectively. Briefly, the adjuvant-antigen mixture was first prepared by gently mixing the adjuvant in a vial using a vortex mixer. The desired amount of adjuvant was transferred to an autoclaved 1.5 mL microcentrifuge tube. Antigen was prepared in PBS or saline with concentrations ranging from 0.5 to 0.67 mg / mL. The calculated amount of antigen was then added to the microcentrifuge tube along with the adjuvant, and the resulting mixture was mixed by gentle vortexing for 2 minutes to create a water-in-oil emulsion. The adjuvant-antigen emulsion was then drawn into an appropriate syringe for injection into the animals. A total of 50 μg of antigen was injected in a volume of 150 to 200 μL. Each animal was immunized and then boosted 3 to 4 times depending on the antiserum titer. Animals with good titers were given a final booster by intraperitoneal injection before fusion.

[0135] Hybridoma fusion and screening Cells of a mouse myeloma cell line (SP2 / 0-Ag14, ATCC #CRL-1581) were cultured to reach logarithmic phase just prior to fusion. Spleen cells from immunized mice were sterilely prepared and fused with myeloma cells according to the method described by Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 256:495-497 (1975). The fused "hybrid cells" were then distributed into 96-well plates in DMEM / 20% FCS / HAT medium. Viable hybridoma colonies were observed microscopically 7–10 days after fusion. Two weeks later, supernatants from each well were subjected to indirect ELISA using recombinant cynomolgus monkey Siglec15-his protein. Positive hybridomas that bound to cynomolgus monkey Siglec15-his protein were then selected and transferred to 24-well plates. These hybridoma colonies were further tested for their activity in human Siglec15-LRRC4C binding. Hybridoma clones producing antibodies that showed highly specific cynomolgus monkey Siglec15 binding and Siglec15-LRRC4C blocking activity were subcloned by limiting dilution to ensure the clonality of the cell line, and then the monoclonal antibodies were purified. Briefly, a Protein A Sepharose column (Bestchrom (Shanghai) Biosciences, catalog no. AA0273) was washed with 5–10 column volumes of PBS buffer. Cell supernatants from the hybridoma monoclones were passed through the column, which was then washed with PBS buffer until the protein absorbance reached baseline. The column was eluted with elution buffer (0.1 M glycine-HCl, pH 2.7) and immediately collected in a 1.5 ml tube containing neutralization buffer (1 M Tris-HCl, pH 9.0). Immunoglobulin-containing fractions were pooled and dialyzed overnight in PBS at 4° C. The in vitro functional activity of the purified monoclonal antibodies was then characterized as follows.

[0136] Example 2 Determination of Binding Affinity of Anti-Siglec15 Monoclonal Antibodies Using BIACORE Surface Plasmon Resonance The purified anti-Siglec15 monoclonal antibodies (mAbs) produced in Example 1 were characterized for binding affinity and binding kinetics using a Biacore T200 system (GE healthcare, Pittsburgh, PA, USA).

[0137] Briefly, goat anti-mouse IgG (GE Healthcare, Catalog No. BR100839, Human Antibody Capture Kit) was covalently coupled via primary amines to a CM5 chip (carboxymethyl dextran-coated chip, GE Healthcare, Catalog No. BR-1005-30) using a standard amine coupling kit provided by Biacore (GE Healthcare, Pittsburgh, PA, USA). Unreacted moieties on the biosensor surface were blocked with ethanolamine. Then, a 13.3 nM concentration of the purified anti-Siglec15 antibody of the present disclosure and 13.3 nM of the anti-Siglec15 benchmark (ch5G12, also known as BM; see U.S. Patent Application Publication No. 2019 / 0202912; heavy and light chain amino acid sequences set forth in SEQ ID NOs: 24 and 25, respectively) were each flowed over the chip at a flow rate of 10 μL / min. Then, serially diluted recombinant human Siglec15-his protein (homemade, amino acid sequence set forth in SEQ ID NO: 20) or cynomolgus monkey Siglec15-his protein (homemade, amino acid sequence set forth in SEQ ID NO: 21), HBS-EP starting at 80 nM + Two-fold serial dilutions in buffer were each flowed over the chip at a flow rate of 30 μL / min. Antigen-antibody binding kinetics were followed for 2 min, and dissociation kinetics were followed for 10 min. Binding and dissociation curves were fitted to a 1:1 Langmuir binding model using Biacore evaluation software. D , K. a and K. d The values ​​were determined and are summarized in Table 2 below.

[0138] [Table 2]

[0139] All anti-Siglec15 antibodies of the present disclosure specifically bound to human Siglec15 and cynomolgus monkey Siglec15 with similar or higher binding affinity compared to the benchmarks, with antibodies A2A1D2F1, A2A5C7E8, A1E10G7H9, and A1D1B7H9 exhibiting the highest binding affinity.

[0140] Example 3 Siglec15 binding activity of anti-Siglec15 antibodies The antibodies of the present disclosure were further tested for binding activity to Siglec15 by capture ELISA, flow cytometry (FACS), and indirect ELISA.

[0141] 3.1 Capture ELISA Briefly, 96-well plates were coated with 100 μl of 2 μg / ml AffiniPure goat anti-human IgG F(ab')2 fragment specific (Jackson Immuno Research, Cat#109-005-097) in PBS for 2 hours at 37° C. Plates were washed once with wash buffer (PBS + 0.05% (v / v) Tween®-20, PBST) and then blocked overnight at 4° C. with 200 μl of blocking buffer (5% (w / v) nonfat milk in PBST). Plates were washed four times. Plates containing the capture antibodies were incubated with 100 μl of serially diluted anti-Siglec15 antibodies of the present disclosure, benchmark, and hIgG (human immunoglobulin for intravenous injection, pH 4, Hualan Biological Engineering Inc.), 5-fold dilutions in 2.5% (w / v) nonfat milk in PBST starting at 66.7 nM, at 37° C. for 40 minutes, then washed again four times. Plates containing the capture antibodies were incubated with 100 μl of biotin-labeled human Siglec15-his protein (homemade with SEQ ID NO: 20, 145 ng / ml in 2.5% (w / v) nonfat milk in PBST) for 40 minutes at 37° C., washed four times, and incubated with streptavidin-conjugated HRP (1:10,000 dilution in PBST, Jackson Immuno Research, catalog number 016-030-084, 100 μl / well) for 40 minutes at 37° C. After the final wash, the plate was incubated with 100 μl / well of ELISA substrate TMB (Innoreagents, Cat. No. TMB-S-002). The reaction was stopped with 450 μl / well of 1 M H2SO4 for 4 min at room temperature. The absorbance of each well was read on a microplate reader using dual wavelength mode with 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values ​​were then plotted against antibody concentration. Data were analyzed using Graphpad Prism software, and EC 50 The values ​​were reported.

[0142] 3.2 Cell-based coupled FACS The binding activity of anti-Siglec15 antibodies to cell surface Siglec15 was tested by flow cytometry (FACS) using homemade human-siglec15-2D3-1E1 cells expressing human Siglec15 (uniprot #Q6ZMC9 amino acid residues Met1-Pro328). Human-siglec15-2D3-1E1 cells were generated by transfecting HEK-293 cells (ATCC #CRL-1573) with the pCMV-TP plasmid, which contained the human Siglec15 coding sequence inserted between the EcoRI and XbaI sites, according to the instructions for Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Human-siglec15-2D3-1E1 cells were harvested from cell culture flasks, washed twice, and resuspended in phosphate-buffered saline (PBS) containing 2% (v / v) fetal bovine serum (FACS buffer). 2 × 10 cells were plated in a 96-well plate. 5 Cells / well were incubated in 100 μL serially diluted anti-Siglec15 antibody or control (5-fold serial dilutions starting at 66.67 nM) in FACS buffer for 40 minutes on ice. Cells were washed twice with FACS buffer, and 100 μL of R-Phycoerythrin AffiniPure goat anti-human IgG, Fcγ fragment specific (1:1000 dilution in FACS buffer, Jackson Immunoresearch, catalog number 109-115-098) was added. After 40 minutes of incubation at 4°C in the dark, cells were washed three times and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism software, and EC 50 The values ​​were reported.

[0143] 3.3 Indirect ELISA Cross-reactivity of anti-Siglec15 antibodies with cynomolgus monkey or mouse Siglec15 protein was measured. Briefly, 96-well microplates were coated with 100 μl of 2 μg / ml cynomolgus monkey Siglec15-his protein (homemade with SEQ ID NO: 21) or 2 μg / ml mouse Siglec15-his protein (homemade with SEQ ID NO: 22) in carbonate / bicarbonate buffer (pH 9.6) for 2 hours at 37°C. The ELISA plates were washed once with washing buffer (PBS + 0.05% Tween-20, PBST) and then blocked with 200 μl / well of blocking buffer (5% (w / v) non-fat milk in PBST) overnight at 4°C. Plates were washed four times and incubated with 100 μl of serially diluted anti-Siglec15 antibodies of the present disclosure or controls (starting at 66.7 nM, 5-fold dilutions in PBST with 2.5% (w / v) nonfat milk) for 40 minutes at 37° C. ELISA plates were again washed four times and incubated with peroxidase AffiniPure goat anti-human IgG, F(ab′)2 fragment specific (1:5000 dilution in PBST buffer, Jackson Immunoresearch, catalog number 109-035-097, 100 μl / well) for 40 minutes at 37° C. After the final wash, plates were incubated with 100 μl / well of TMB (Innoreagents, catalog number TMB-S-002). The reaction was stopped after 4 min at room temperature using 450 μl of 1 M H2SO4. The absorbance of each well was read on a microplate reader using dual wavelength mode with 450 nm for TMB and 630 nm as the reference wavelength. OD (450-630) values ​​were plotted against antibody concentration. Data were analyzed using Graphpad Prism software, and EC 50 The values ​​were reported.

[0144] The results of the three assays are shown in Figures 1A-1C to 4A-4C.

[0145] From Figures 1A-1C, all antibodies of the present disclosure, except for A1C8C6H1, have EC 50and high Bmax (maximum binding), indicating that it specifically bound to human Siglec15.

[0146] As shown in Figures 2A to 2C, the anti-Siglec15 antibodies A1D1B7H9, A1D11A7H10, A1E10G7H9, A2A1D2F1, A2G4C8G7, and A2H5F1A1 showed higher Bmax (lower EC 50 (in ), more efficiently bound to cell surface human Siglec15.

[0147] Figures 3A-3C show that most of the antibodies of the present disclosure bound to cynomolgus monkey Siglec15 protein with similar binding activity compared to the benchmark. Only a few antibodies of the present disclosure showed similar or better binding activity to mouse Siglec15, as shown in Figures 4A-4C. For example, antibodies A1E10G7H9 and A2A1D2F1 showed higher Bmax than the benchmark.

[0148] Example 4 Blocking activity of anti-Siglec15 antibodies against Siglec15-LRRC4C or Siglec15-benchmark binding 4.1 Ligand-Blocking ELISA The activity of the anti-Siglec15 antibodies of the present disclosure in blocking Siglec15-LRRC4C binding was measured using a competitive ELISA assay. LRRC4C is a ligand for Siglec15 and can be expressed by cancer cells (WO 2018 / 057753). Briefly, 100 μl of human Siglec15-Fc protein (homemade with the amino acid sequence of SEQ ID NO: 19) was coated onto a 96-well microplate at 2 μg / mL in carbonate / bicarbonate buffer for 2 hours at 37°C. The plate was washed once with wash buffer (PBS + 0.05% Tween-20, PBST) and blocked with 5% (w / v) non-fat milk in PBST overnight at 4°C. The plate was then washed four times with wash buffer.

[0149] Serially diluted anti-Siglec15 antibodies or controls (5-fold serial dilutions starting at 66.67 nM) in PBST containing 2.5% (w / v) nonfat milk were added to the Siglec15-Fc-bound plate at 100 μl / well and incubated with human Siglec15-Fc protein at 37°C for 40 minutes. The plate was washed four times again with wash buffer, and then 290 ng / ml of biotin-labeled human LRRC4C-Fc protein (homemade with SEQ ID NO: 23) was added and incubated with it at 100 μl / well for 40 minutes at 37°C. The plate was washed again with wash buffer. Streptavidin-conjugated HRP (1:5000 dilution in PBST buffer, Jackson Immunoresearch, Cat#016-030-084) was then added to the plate at 100 μl / well and incubated at 37°C for 40 minutes. The plate was washed again with wash buffer. Finally, TMB was added, the reaction was stopped with 1 M H2SO4, and the absorbance of each well was read on a microplate reader using a dual wavelength mode with 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values ​​were then plotted against the antibody concentration. The data were analyzed using Graphpad Prism software, and the IC 50 The values ​​were reported.

[0150] 4.2 Benchmark Blocking ELISA The ability of anti-Siglec15 antibodies of the present disclosure to block Benchmark-human Siglec15 binding was measured in a competitive ELISA assay. Briefly, Benchmark was coated onto a 96-well microplate at 100 μl / well at 2 μg / mL in PBS and incubated at 37°C for 2 hours. The plate was then washed once with wash buffer and blocked with 5% (w / v) non-fat milk in PBST overnight at 4°C. The next day, an anti-Siglec15 antibody of the present disclosure or a control was diluted in 4-fold serial dilutions starting at 80 nM with biotin-labeled human Siglec15-Fc protein (homemade protein having SEQ ID NO: 19, 37 ng / ml in PBST containing 2.5% non-fat milk) and incubated at room temperature for 40 minutes. After washing the plate four times, the antibody / Siglec15-Fc mixture was added to the Benchmark-coated plate at 100 μl / well. After incubation at 37°C for 40 minutes, the plate was again washed four times with wash buffer. Next, 100 μl / well of streptavidin-conjugated HRP was added to the plate and incubated at 37°C for 40 minutes. The plate was washed again with wash buffer. Finally, TMB was added, and the reaction was stopped with 1M H2SO4. The absorbance of each well was read on a microplate reader using dual wavelength mode with 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values ​​were then plotted against antibody concentration. Data were analyzed using Graphpad Prism software, and IC was calculated. 50 The values ​​were reported.

[0151] 4.3 Cell-based ligand-blocking FACS The activity of anti-Siglec15 antibodies in blocking Siglec15 protein binding to cell surface LRRC4C was assessed by flow cytometry (FACS) using home-grown LRRC4C-3F12-1B9 cells. Briefly, HEK-293 cells (ATCC #CRL-1573) were transfected with the pCMV-TP plasmid construct containing a nucleotide sequence encoding human LRRC4C (amino acid residues Met1-Ile640 of uniprot #Q9HCJ2) inserted between the EcoRI and XbaI sites, according to the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) instructions. Stable cells designated LRRC4C-3F12-1B9 were selected for subsequent cell-based ligand-blocking assays.

[0152] Briefly, anti-Siglec15 antibodies or controls of the present disclosure were diluted in a 5-fold serial dilution starting at 66.67 nM with human Siglec15-mouse Fc protein (homemade with SEQ ID NO: 26, 8 μg / ml in FACS buffer) and incubated at room temperature for 40 minutes. LRRC4C-3F12-1B9 cells were then harvested from cell culture flasks, washed twice, and resuspended in PBS containing 2% (v / v) fetal bovine serum (FACS buffer). 1×10 cells were then plated in a 96-well plate. 5 Cells / well were incubated in 100 μL of antibody / Siglec15-mouse Fc mixture for 40 minutes at 4°C. Plates were washed twice with FACS buffer, followed by the addition of 100 μL / well of R-Phycoerythrin AffiniPure F(ab')2 fragment goat anti-mouse IgG(H+L) (1:1000 dilution in FACS buffer, Jackson Immunoresearch, Cat. No. 115-116-146) and incubation in the dark for 40 minutes at 4°C. Cells were washed twice and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism software, and IC 50 The values ​​were reported.

[0153] The results of the three assays are shown in Figures 5A-5C to 7A-7C.

[0154] It can be seen from Figures 5A to 5C that all of the anti-Siglec15 antibodies of the present disclosure can block human Siglec15-human LRRC4C binding, and that their blocking activity is similar to or lower than the benchmark.

[0155] 6A-6C show that antibodies A1E7G5D1, A2A5C7E8, A2G4C8G7, and A2H5F1A1 can block human Siglec15-benchmark binding, suggesting that they bind to the same or similar epitopes as benchmark. Antibodies A1C8C6H1, A1D1B7H9, A1D5E2H1, A1D11A7H10, A1E10G7H9, A2A1D2F1, and A2A6B1C2 cannot block human Siglec15 binding to benchmark, suggesting that A1C8C6H1, A1D1B7H9, A1D5E2H1, A1D11A7H10, A1E10G7H9, A2A1D2F1, and A2A6B1C2 may bind to different epitopes compared to benchmark.

[0156] Furthermore, as shown in Figures 7A to 7C, all anti-Siglec15 antibodies disclosed herein were able to block the binding of human Siglec15 to cell surface human LRRC4C bearing mouse Fc with blocking activity similar to or lower than that of the benchmark.

[0157] Example 5 Thermal stability of anti-Siglec15 antibodies The anti-Siglec15 antibody was also tested for its thermal stability. Briefly, a protein thermal shift assay was used to determine the Tm (melting temperature) using the GloMelt™ Thermal Shift Protein Stability Kit (Biotium, catalog number 33022-T). The GloMelt™ dye was then thawed and allowed to reach room temperature. The vial containing the dye was vortexed and centrifuged. Next, a 10x dye was prepared by adding 5 μL of 200x dye to 95 μL of PBS. 2 μL of 10x dye and 10 μg of antibody were added, and PBS was added to bring the total reaction volume to 20 μL. The tube containing the dye and antibody was briefly spun and placed in a real-time PCR thermocycler (Roche, LightCycler 480II) set using a melting curve program with the parameters in Table 3.

[0158] [Table 3]

[0159] [Table 4]

[0160] The melting temperatures of the antibodies of the present disclosure are shown in Table 4, suggesting that the antibodies of the present disclosure are likely to be stable in the human body.

[0161] Example 6 Anti-Siglec15 antibodies that restored Siglec15-mediated T cell suppression Anti-CD3 monoclonal antibody (OKT3) 50 ng / mL (eBioscience Inc., Catalog No. 16-0037-85) in DPBS was coated onto a 96-well microplate at 100 μl / well overnight at 4° C. The next day, unbound anti-CD3 monoclonal antibody was aspirated immediately before the addition of PBMCs.

[0162] Total PBMCs from healthy human donors were washed with RPMI-1640 medium (Gibco, Cat. No. A10491-01) supplemented with 10% FBS (Gibco, Cat. No. 10099-141), centrifuged at 200 g for 15 minutes, and the supernatant was removed. PBMC cells were then labeled with 0.5 μM CFSE (Invitrogen, Cat. No. C1157) for 20 minutes on ice, with a final cell density of 1 × 10 6 Four volumes of RPMI-1640 supplemented with 10% FBS were added to the cells and incubated at room temperature for 5 minutes. The plates were centrifuged at 300g for 10 minutes and plated in RPMI-1640 medium supplemented with 10% FBS at a density of 6 x 10 cells / mL. 6 The cells were resuspended at 120 μg / mL. Meanwhile, serially diluted anti-Siglec15 antibodies or controls (4-fold diluted in RPMI-1640 medium supplemented with 10% FBS, starting at 120 μg / mL) of the present disclosure were mixed with human Siglec15-Fc protein (homemade preparation of SEQ ID NO: 19, 40 μg / mL in RPMI-1640 medium supplemented with 10% FBS) at a volume ratio of 1:1 and incubated at room temperature for 30 minutes. Next, 50 μL of medium containing PMBC cells and 50 μL of the antibody / Siglec15-Fc mixture were added to the anti-CD3-bound plate and incubated at 37°C in a CO2 incubator for 3 days. Next, 100 μL of FACS buffer was added to each well, and the cells were pipetted several times and transferred to a U-shaped plate. The U-shaped plate was centrifuged, and the supernatant was removed. The cells were then incubated with 50 μl / well of HFCR (1:10 diluted in FACS buffer, Biolegend Inc., Cat. No. 422302) in the dark for 15 minutes at 4°C. The cells were stained with anti-CD4 (Biolegend Inc., Cat. No. 357410) and anti-CD8 (Biolegend Inc., Cat. No. 301066) fluorescent mAbs (1:10 diluted in FACS buffer) for 30 minutes at 4°C. The cells were washed twice in FACS buffer (200 μL / well) and resuspended in FACS buffer (200 μL / well). Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism software, and EC50 The values ​​were reported.

[0163] The results are shown in Figures 8A and 8B.

[0164] Antibodies A2A5C7E8 and A1E10G7H9 have high EC 50 Siglec15-mediated CD8 + T cells and CD4 + It can be seen that the T cell suppression was able to be reversed. Notably, the high dose of antibody A2A5C7E8 was more effective than the benchmark in reversing T cell suppression, resulting in CD4 + and CD8 + The percentage of T cell proliferation was increased.

[0165] Example 7 Sequencing of anti-Siglec15 antibodies The two antibodies, A2A5C7E8 and A1E10G7H9, were sequenced to obtain the complete heavy and light chain variable and constant region sequences. The sequence numbers of the heavy and light chain variable regions are shown in Table 1, and the heavy / light chain isotypes were determined by sequence alignment in databases.

[0166] Example 8 Genetic Engineering of Anti-Siglec15 Antibody A2A5C7E8 For example, to avoid or reduce post-translational modifications such as deamidation and isomerization of specific amino acid residues in the CDR regions, which may adversely affect antibody production, stability, safety, and / or efficacy, antibody A2A5C7E8 (hereinafter also referred to as A2A5C7E8-1) was further modified in the heavy chain CDR2 region. A total of two modified variants, A2A5C7E8-2 and A2A5C7E8-3, were obtained, and their CDR and heavy / light chain variable region SEQ ID NOs are listed in Table 1.

[0167] Vectors containing nucleotides encoding the heavy chain variable region of A2A5C7E8-2 or A2A5C7E8-3 linked to the human IgG1 heavy chain constant region (SEQ ID NO: 17), and vectors containing nucleotides encoding the light chain variable region linked to the human kappa light chain constant region (SEQ ID NO: 18), were transiently transfected into 50 ml of 293F suspension cell cultures at a light chain:heavy chain construct ratio of 1.1:1 with 1 mg / mL PEI.

[0168] Example 9 Characterization of modified A2A5C7E8 variants The modified variants A2A5C7E8-2 and A2A5C7E8-3 were purified as described above and tested in Biacore, capture ELISA, indirect ELISA, cell-based binding FACS, competitive ELISA and cell-based functional assays according to the protocols in the examples above, with or without modifications, and also according to the protocols described below.

[0169] For Biacore studies measuring the binding affinity of modified A2A5C7E8 variants to mouse Siglec15, mouse Siglec15-his protein (homemade with SEQ ID NO: 22) was used.

[0170] In a reversion test of T cell suppression, an A2A5C7E8 variant of the present disclosure or a control at a concentration of 48 μg / ml was mixed with human Siglec15-Fc protein (SEQ ID NO: 19, 20 μg / ml in RPMI-1640 medium supplemented with 10% FBS) in a 1:1 volume ratio, and an A2A5C7E8 variant of the present disclosure or a control at a concentration of 120 μg / ml was mixed with human Siglec15-Fc protein (40 μg / ml in RPMI-1640 medium supplemented with 10% FBS) in a 1:1 volume ratio.

[0171] The Biacore test results are summarized in Tables 5-1 and 5-2. The results of other assays are shown in Figures 9-14 and 15A-15D.

[0172] [Table 5]

[0173] [Table 6]

[0174] As shown in Tables 5-1 and 5-2, the modified variants A2A5C7E8-2 and A2A5C7E8-3 specifically bound to human Siglec15 and cynomolgus monkey Siglec15 with higher binding affinity than the benchmark. Antibody A2A5C7E8-3 also showed higher binding affinity for mouse Siglec15 than the benchmark. Antibody A2A5C7E8-2 showed equivalent binding affinity for mouse Siglec15 compared to the benchmark.

[0175] As shown in Figures 9 and 10, the modified variants A2A5C7E8-2 and A2A5C7E8-3 bound to human Siglec15 more efficiently or with a higher Bmax than the benchmark, and inhibited Siglec15-LRRC4C binding more efficiently than the benchmark, as shown in Figure 13. Furthermore, as shown in Figures 11 and 12, the modified variants A2A5C7E8-2 and A2A5C7E8-3 bound to cynomolgus monkey Siglec15 and mouse Siglec15 more efficiently than the benchmark.

[0176] As shown in Figure 14, the modified variants A2A5C7E8-2 and A2A5C7E8-3 were able to block benchmark-Siglec15 binding, indicating that the modified variants A2A5C7E8-2 and A2A5C7E8-3 bound to similar epitopes relative to the benchmark.

[0177] As shown in Figures 15A-15D, the modified variants A2A5C7E8-2 and A2A5C7E8-3 inhibited Siglec15-mediated CD8 + T cells and CD4 +Remarkably, when human Siglec15-Fc protein was used at a relatively low dose, antibodies A2A5C7E8-2 and A2A5C7E8-3 were able to restore T cell suppression and achieve similar or higher cell proliferation percentages compared to the benchmark. + and CD8 + It was more effective than the benchmark in reversing T cell suppression.

[0178] Example 10: Production of mouse anti-Siglec15 monoclonal antibodies using hybridoma technology Immunization, hybridoma fusion and screening Mouse immunization, hybridoma fusion, and screening were performed according to the protocol in Example 1 with the following modifications. Specifically, normal mice were used, and the immunization dose contained 50 μg of recombinant human Siglec15-Fc protein per mouse per injection for the primary immunization and 25 μg of human Siglec15-Fc protein per mouse per injection for the booster immunization. Antigen was prepared in PBS or saline at concentrations ranging from 0.25 to 0.67 mg / ml. A total of 50 or 25 μg of antigen was injected in a volume of 150 to 200 μl. Each animal was immunized and then boosted three to four times depending on the antiserum titer. The fusion cell culture supernatant was subjected to indirect ELISA using homemade human Siglec15-his protein. Positive hybridoma screening antibodies that bound to human Siglec15-his protein were selected and transferred to 24-well plates. These hybridomas were also subjected to cell-based binding FACS, capture ELISA, indirect ELISA, and ligand-blocking ELISA. Hybridoma clones producing antibodies exhibiting highly specific human Siglec15-his binding, cynomolgus monkey Siglec15-his binding, mouse Siglec15-his binding, and human Siglec15-LRRC4C blocking activity were subcloned by limiting dilution to ensure the clonality of the cell lines, and then the monoclonal antibodies were purified.

[0179] Example 11 Determination of Binding Affinity of Mouse Anti-Siglec15 Monoclonal Antibodies Using BIACORE Surface Plasmon Resonance The purified mouse anti-Siglec15 monoclonal antibody produced in Example 10 was characterized for its binding affinity and binding kinetics by a Biacore T200 system (GE healthcare, Pittsburgh, PA, USA).

[0180] Briefly, goat anti-mouse IgG (GE healthcare, catalog number BR100838, mouse antibody capture kit) was covalently coupled via primary amines to a CM5 chip (carboxymethyl dextran-coated chip commercially available from GE healthcare, catalog number BR100530) using a standard amine coupling kit provided by Biacore (GE healthcare, Pittsburgh, PA, USA). Unreacted sites on the biosensor surface were blocked with ethanolamine. Purified mouse anti-Siglec15 antibodies of the present disclosure at a concentration of 13.3 nM were each flowed over the chip at a flow rate of 10 μL / min. Serially diluted human Siglec15-his protein (homemade, amino acid sequence set forth in SEQ ID NO: 20), cynomolgus monkey Siglec15-his protein (homemade, amino acid sequence set forth in SEQ ID NO: 21), or mouse Siglec15-his protein (homemade, amino acid sequence set forth in SEQ ID NO: 22) (starting at 80 nM and continuing in HBS-EP 5000). + A 2-fold dilution in buffer (provided by Biacore) was flowed over the chip at a flow rate of 30 μL / min. Antigen-antibody binding kinetics were followed for 2 min, and dissociation kinetics were followed for 10 min. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using Biacore evaluation software. D , K. a and K. d The values ​​were determined and are summarized in Tables 6-1 and 6-2 below.

[0181] [Table 7]

[0182] [Table 8]

[0183] All of the mouse anti-Siglec15 antibodies disclosed herein specifically bound to human Siglec15, cynomolgus monkey Siglec15, and mouse Siglec15 with high binding affinity.

[0184] Example 12 Binding activity of mouse anti-Siglec15 monoclonal antibodies The binding activity of the mouse anti-Siglec15 antibody to human Siglec15, cynomolgus monkey Siglec15, and mouse Siglec15 was further determined by capture ELISA, indirect ELISA, and flow cytometry (FACS).

[0185] A capture ELISA was performed according to the protocol in Example 3, except that AffiniPure Goat Anti-Mouse IgG, F(ab')2 Fragment Specific (Jackson ImmunoResearch, Catalog No. 115-005-072) was used at 100 μl / well instead of AffiniPure Goat Anti-Human IgG, F(ab')2 Fragment Specific. The results are shown in Figure 16.

[0186] 1.5 x 10 in a 96-well plate 5 Cell-based FACS was performed according to the protocol in Example 3, except that cells / well were incubated on ice for 40 minutes in 100 μL of serially diluted anti-Siglec15 antibody or control (starting at 66.67 nM, followed by 5-fold serial dilutions) in FACS buffer, and 100 μL of R-phycoerythrin AffiniPure F(ab')2 fragment goat anti-mouse IgG(H+L) (1:1000 dilution in FACS buffer, Jackson Immunoresearch, catalog no. 115-116-146) was added to the cells. The results are shown in Figure 17.

[0187] An indirect EELISA was performed according to the protocol in Example 3, except that peroxidase AffiniPure goat anti-mouse IgG, Fcγ fragment specific (Jackson Immunoresearch, catalog number 115-035-071) was used at 100 μl / well instead of R-phycoerythrin AffiniPure goat anti-human IgG, Fcγ fragment specific, and the results are shown in Figures 18 and 19.

[0188] The murine anti-Siglec15 antibodies of the present disclosure exhibit higher Bmax (maximum binding) and lower EC compared to the benchmark (ch5G12, also known as BM; see U.S. Patent Application Publication No. 2019 / 0202912, heavy and light chain amino acid sequences set forth in SEQ ID NOs: 24 and 25, respectively). 50 16 shows that the mouse anti-Siglec15 antibody of the present disclosure specifically bound to human Siglec15 at a concentration of 1000 kJ / mL, suggesting that it bound to a larger amount of human Siglec15 protein more efficiently. Figure 17 shows that the mouse anti-Siglec15 antibody of the present disclosure specifically bound to cell surface human Siglec15 in the FACS assay, although with slightly lower activity.

[0189] Figure 18 shows that the mouse anti-Siglec15 antibody of the present disclosure specifically binds to cynomolgus monkey Siglec15 protein with a binding activity higher than that of the benchmark, and Figure 19 shows that the mouse anti-Siglec15 antibody of the present disclosure specifically binds to mouse Siglec15 protein with a binding activity slightly lower than that of the benchmark.

[0190] Example 13 Blocking activity of mouse anti-Siglec15 antibodies against Siglec15-LRRC4C or Siglec15-benchmark binding The mouse anti-Siglec15 antibodies of the present disclosure were also tested in a ligand-blocking ELISA and a benchmark-blocking ELISA according to the protocols described above, and the results are shown in Figures 20 and 21.

[0191] FIG. 20 shows that the mouse anti-Siglec15 antibody of the present disclosure can block human Siglec15-human LRRC4C binding, and that its blocking activity is slightly higher than that of the benchmark.

[0192] Figure 21 shows that the mouse anti-Siglec15 antibodies of the present disclosure can block human Siglec15-benchmark binding, suggesting that the mouse anti-Siglec15 antibodies of the present disclosure may bind to an epitope similar to that bound by the benchmark.

[0193] Example 14 Mouse anti-Siglec15 antibodies that reverse Siglec15-mediated T cell suppression Murine anti-Siglec15 antibodies of the present disclosure were further tested for biological activity in reverting Siglec15-mediated T cell suppression according to the protocols of the examples above, with the modifications and protocols described below.

[0194] In a reversion assay of T cell suppression, a mouse anti-Siglec15 antibody of the present disclosure or a control at a concentration of 400 μg / ml was mixed with human Siglec15-Fc protein (SEQ ID NO: 19, 40 μg / ml in RPMI-1640 medium supplemented with 10% FBS) at a volume ratio of 1:1. The results are shown in Figures 22A and 22B.

[0195] The murine anti-Siglec15 antibodies of the present disclosure have lower or similar EC 50 Siglec15-mediated CD8 + T cells and CD4 + It can be seen that T cell suppression can be reversed and that treatment with high doses of the antibodies of the present disclosure resulted in a significantly higher percentage of cell proliferation.

[0196] The murine antibodies of the present disclosure were then sequenced, and the SEQ ID NOs of the heavy and light chain variable regions are summarized in Table 1. Interestingly, the three antibodies B2D7H7A3C1, B2G12H3E8, and B2H2H1A7 had the same heavy and light chain CDR sequences.

[0197] While the present disclosure has been described above in conjunction with one or more embodiments, it is to be understood that the disclosure is not limited to those embodiments, and the description is intended to cover all alternatives, modifications, and equivalents as may be included within the spirit and scope of the appended claims. All references cited herein are further incorporated by reference in their entirety.

[0198] The sequences in this application are summarized below.

[0199] [Table 9]

[0200] [Table 10]

[0201] [Table 11]

[0202] [Table 12]

[0203] [Table 13]

[0204] While preferred embodiments of the invention have been described in detail, it should be understood that the invention defined by the preceding paragraphs is not limited to the specific details set forth in the foregoing specification, as many obvious variations thereof are possible without departing from the spirit or scope of the invention.

Claims

1. An antigen-binding fragment of an isolated antibody that specifically binds to Siglec15, a heavy chain variable region partially identical to the heavy chain variable region (VH) set forth in SEQ ID NO: 7, said heavy chain variable region comprising at least the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences of SEQ ID NO: 7; and a light chain variable region partially identical to the light chain variable region (VL) set forth in SEQ ID NO: 8, said light chain variable region comprising at least the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences of SEQ ID NO: 8; Equipped with An isolated antigen-binding fragment of an antibody, wherein X1 and X2 of the heavy chain variable region of SEQ ID NO: 7 are D and K.

2. 2. The antigen-binding fragment of claim 1, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

3. (a) the heavy chain variable region comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 7, and / or the light chain variable region comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 8; (b) the heavy chain variable region and the light chain variable region comprise amino acid sequences having at least 85% identity to SEQ ID NOs: 7 and 8, respectively; (c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, and / or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8, or (d) the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth in SEQ ID NOs: 7 and 8; An antigen-binding fragment of the isolated antibody of claim 1 or 2.

4. 4. The antigen-binding fragment of any one of claims 1 to 3, wherein the VH CDR2 region comprises the amino acid sequence set forth in SEQ ID NO:2, and wherein X1 is D and X2 is K.

5. An antigen-binding fragment of an isolated antibody, comprising:

5. The antigen-binding fragment of any one of claims 1 to 4, wherein the isolated antibody is of the IgG1, IgG2, or IgG4 isotype.

6. An antigen-binding fragment of an isolated antibody described in any one of claims 1 to 5, which is a Fab fragment containing a human kappa or human lambda light chain constant region.

7. The isolated antibody antigen-binding fragment comprises: (a) a CH1 domain contained in the amino acid sequence of SEQ ID NO: 17 linked to the heavy chain variable region; or (b) a light chain constant region having the amino acid sequence of SEQ ID NO: 18 linked to the light chain variable region; 7. An antigen-binding fragment of the isolated antibody of claim 1, which is a Fab fragment comprising at least one of:

8. An antigen-binding fragment of an isolated antibody described in any one of claims 1 to 7, which is an antigen-binding fragment of a human or chimeric antibody.

9. 9. An antigen-binding fragment of the isolated antibody of any one of claims 1 to 8 conjugated to a cytotoxic agent, toxin, or radioisotope.

10. A polynucleotide encoding the heavy chain variable region and light chain variable region of an antigen-binding fragment of an isolated antibody described in any one of claims 1 to 9.

11. A vector containing the polynucleotide of claim 10.

12. (a) the polynucleotide of claim 10; (b) the vector according to claim 11 ; (c) a first polynucleotide encoding a heavy chain variable region of an antigen-binding fragment of the isolated antibody of any one of claims 1 to 9, and a second polynucleotide encoding a light chain variable region of an antigen-binding fragment of the isolated antibody of any one of claims 1 to 9; or (d) a first vector comprising a first polynucleotide encoding a heavy chain variable region of an antigen-binding fragment of the isolated antibody of any one of claims 1 to 9, and a second vector comprising a second polynucleotide encoding a light chain variable region of an antigen-binding fragment of the isolated antibody of any one of claims 1 to 9; A host cell comprising:

13. 1. A method for producing an antigen-binding fragment of an antibody that specifically binds to Siglec15, said method comprising: (a) a polynucleotide encoding the heavy chain variable region and the light chain variable region of the antigen-binding fragment of the isolated antibody of any one of claims 1 to 9; (b) a first polynucleotide encoding a heavy chain variable region of the isolated antibody of any one of claims 1 to 9, and a second polynucleotide encoding a light chain variable region of an antigen-binding fragment of the isolated antibody of any one of claims 1 to 9; (c) a vector comprising polynucleotides encoding the heavy chain variable region and the light chain variable region of the antigen-binding fragment of the isolated antibody of any one of claims 1 to 9; or (d) a first vector comprising a first polynucleotide encoding a heavy chain variable region of an antigen-binding fragment of the isolated antibody of any one of claims 1 to 9, and a second vector comprising a second polynucleotide encoding a light chain variable region of an antigen-binding fragment of the isolated antibody of any one of claims 1 to 9; 20. A method comprising culturing a host cell comprising:

14. A pharmaceutical composition comprising an antigen-binding fragment of an isolated antibody described in any one of claims 1 to 9, a polynucleotide described in claim 10, a vector described in claim 11, or a host cell described in claim 12, and a pharmaceutically acceptable carrier or excipient.

15. 1. A pharmaceutical composition for use in treating a disease or disorder expressing Siglec15 in a subject, comprising:

15. A therapeutically effective amount of an antigen-binding fragment of the isolated antibody of any one of claims 1 to 9, a polynucleotide of claim 10, a vector of claim 11, or a host cell of claim 12, or a pharmaceutical composition of claim 14, wherein the disease or disorder is cancer or bone loss. A pharmaceutical composition for use in treating a disease or disorder that expresses Siglec15 in a subject.

16. 16. The pharmaceutical composition for use according to claim 15, wherein the disease or disorder is cancer and the cancer is a solid tumor.

17. 17. The pharmaceutical composition for use according to claim 16, wherein the solid tumor is non-small cell lung cancer, ovarian cancer, melanoma, colorectal cancer, breast cancer, endometrial cancer, or squamous cell carcinoma.

18. (a) treating cancer in a subject in need thereof; (b) inhibiting bone loss in a subject; or (c) increasing bone mass in a subject; 16. Use of an antigen-binding fragment of an isolated antibody described in any one of claims 1 to 9, a polynucleotide described in claim 10, a vector described in claim 11, a host cell described in claim 12, or a pharmaceutical composition described in claim 14 in the manufacture of a medicament for the treatment of a cancer.

19. (a) treating cancer in a subject in need thereof; (b) inhibiting bone loss in a subject; or (c) increasing bone mass in a subject; 15. An antigen-binding fragment of the isolated antibody of any one of claims 1 to 9, a polynucleotide of claim 10, a vector of claim 11, a host cell of claim 12, or a pharmaceutical composition of claim 14, for use in

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