Anti-SIGLEC15 antibodies and uses thereof

Abstract

The present disclosure relates to antibodies, or antigen-binding fragments thereof, that bind to SIGLEC15, isolated polynucleotides, isolated vectors, host cells, pharmaceutical compositions, or kits associated with antibodies, or antigen-binding fragments thereof, that bind to SIGLEC15. The present disclosure also provides the use of an antibody or an antigen-binding fragment thereof in the preparation of a therapeutic agent for the prevention or treatment of a disease.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to PCT application number PCT / CN2022 / 107531 filed on July 22, 2022. The contents of the prior application are considered part of this disclosure and are incorporated herein in their entirety.

[0003] References to sequence listings

[0004] The sequence listing entitled DCF230623WO-Seqlisting.xml was created on July 18, 2023, is 68,117 bytes in size, and is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates to antibodies or antigen-binding fragments thereof that bind to SIGLEC15 and uses thereof. Background Art

[0006] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0007] Sialic acid-binding Ig-like lectins ("SIGLECs") are members of the Ig superfamily. These type 1 transmembrane proteins include a sialic acid-binding N-terminal V-set domain, a variable region of the C2-set domain, a transmembrane region, and a cytoplasmic tail, and specifically bind to sialic acid attached to the terminal region of cell surface glycoconjugates. Two major subsets of SIGLECs have been identified: one subset includes CD33-related sequence-variable SIGLECs, such as SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9, SIGLEC10, SIGLEC11, SIGLEC14, and SIGLEC16. The second subset includes highly conserved SIGLECs, such as Sn (sialoadhesin) (SIGLEC1), CD22 (SIGLEC2), MAG (myelin-associated glycoprotein) (SIGLEC4), and SIGLEC15.

[0008] SIGLEC15 is an inhibitory molecule with immunosuppressive function. Recent studies have shown that SIGLEC15 can continuously inhibit the activity of T cells, suggesting that SIGLEC15 can be used as a potential target for tumor immunotherapy (Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy, Jun Wang et al., Nature Medicine 4(25):656-666 (2019)).

[0009] Therefore, there is still a need for novel antibodies or antigen-binding fragments thereof that bind to SIGLEC15 in cancer immunotherapy. Summary of the invention

[0010] For the above purposes, provided herein are novel antibodies or antigen-binding fragments thereof that bind to SIGLEC15 and uses thereof.

[0011] In one aspect, provided herein is an antibody or antigen-binding fragment thereof that binds to SIGLEC15, the antibody or antigen-binding fragment thereof comprising at least one heavy chain variable region (VH) and at least one light chain variable region (VL), wherein VH comprises CDRs 1, 2, and 3, and the CDR1 of VH differs from the amino acid sequence shown in SEQ ID NO: 55, 61, or 67 by no more than 5, 4, 3, 2, or 1 amino acid; the CDR2 of VH differs from the amino acid sequence shown in SEQ ID NO: 56, 62, or 68 by no more than 5, 4, 3, 2, or 1 amino acid; the CDR3 of VH differs from the amino acid sequence shown in SEQ ID NO: 57, 63, or 69 by no more than 5, 4, 3, 2, or 1 amino acid; the VL comprises CDRs 1, 2, and 3, and the CDR1 of VL differs from the amino acid sequence shown in SEQ ID NO: 58, 64, or 70 by no more than 5, 4, 3, 2, or 1 amino acid; the CDR2 of VL differs from the amino acid sequence shown in SEQ ID NO: 5 The difference between the amino acid sequence shown in NO:59, 65 or 71 does not exceed 5, 4, 3, 2 or 1 amino acids; the difference between the CDR3 of VL and the amino acid sequence shown in SEQ ID NO:60, 66 or 72 does not exceed 5, 4, 3, 2 or 1 amino acids.

[0012] In some embodiments, the VH of the antibody or its antigen-binding fragment that binds to SIGLEC15 comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 55, 56, and 57, respectively; and the VL of the antibody or its antigen-binding fragment that binds to SIGLEC15 comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 58, 59, and 60, respectively.

[0013] In some embodiments, the VH of the antibody or its antigen-binding fragment that binds to SIGLEC15 comprises a CDR1 having the amino acid sequence shown in SEQ ID NO:61, a CDR2 having an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:62 by no more than 5, 4, 3, 2 or 1 amino acids, and a CDR3 having the amino acid sequence shown in SEQ ID NO:63; and the VL of the antibody or its antigen-binding fragment that binds to SIGLEC15 comprises CDRs 1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs:64, 65 and 66, respectively.

[0014] In some embodiments, the VH of the antibody or its antigen-binding fragment that binds to SIGLEC15 comprises a CDR1 having the amino acid sequence shown in SEQ ID NO:67, a CDR2 having an amino acid sequence that differs by no more than 5, 4, 3, 2 or 1 amino acids from the amino acid sequence shown in SEQ ID NO:68, and a CDR3 having the amino acid sequence shown in SEQ ID NO:69; and the VL of the antibody or its antigen-binding fragment that binds to SIGLEC15 comprises a CDR1 having an amino acid sequence that differs by no more than 5, 4, 3, 2 or 1 amino acids from the amino acid sequence shown in SEQ ID NO:70, a CDR2 having the amino acid sequence shown in SEQ ID NO:71, and a CDR3 having the amino acid sequence shown in SEQ ID NO:72.

[0015] In some embodiments, CDR2 of VH comprises a mutation at a protein post-translational modification site, and / or CDR1 of VL comprises a mutation at a protein post-translational modification site.

[0016] In some embodiments, CDR2 of VH comprises mutations N55Q and / or G56A.

[0017] In some embodiments, CDR1 of VL comprises mutations G33A and / or D32E.

[0018] In some embodiments, VH is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and SEQ ID NOs: 7-25; VL is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and SEQ ID NOs: 26-40.

[0019] In some embodiments, VH comprises mutation N55Q and / or mutation G56A. In some embodiments, VL comprises mutation G33A and / or D32E.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof that binds to SIGLEC15 comprises a mutation N55Q in VH and a mutation G33A in VL.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof that binds to SIGLEC15 comprises a mutation N55Q in VH and a mutation D32E in VL.

[0022] In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and SEQ ID NOs: 7-25; and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and SEQ ID NOs: 26-40.

[0023] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:1, 2 or 3; and VL comprises the amino acid sequence shown in SEQ ID NO:4, 5 or 6.

[0024] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:7, 8 or 9, and VL comprises the amino acid sequence shown in SEQ ID NO:26.

[0025] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and VL comprises the amino acid sequence shown in SEQ ID NO:27 or 28.

[0026] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:20, 21, 22, 23, 24 or 25, and VL comprises the amino acid sequence shown in SEQ ID NO:29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40.

[0027] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:1, and VL comprises the amino acid sequence shown in SEQ ID NO:4; VH comprises the amino acid sequence shown in SEQ ID NO:2, and VL comprises the amino acid sequence shown in SEQ ID NO:5; VH comprises the amino acid sequence shown in SEQ ID NO:3, and VL comprises the amino acid sequence shown in SEQ ID NO:6; VH comprises the amino acid sequence shown in SEQ ID NO:7, 8 or 9, and VL comprises the amino acid sequence shown in SEQ ID NO:26; VH comprises the amino acid sequence shown in SEQ ID NO:10, 11 or 12, and VL comprises the amino acid sequence shown in SEQ ID NO:27; VH comprises the amino acid sequence shown in SEQ ID NO:13 or 14, and VL comprises the amino acid sequence shown in SEQ ID NO:28; VH comprises the amino acid sequence shown in SEQ ID NO:15, 16 or 17, and VL comprises the amino acid sequence shown in SEQ ID NO:27; VH comprises the amino acid sequence shown in SEQ ID NO:17, 18 or 19, and VL comprises the amino acid sequence shown in SEQ ID NO:28; VH comprises SEQ ID NO:20, and VL comprises the amino acid sequence shown in SEQ ID NO:29; VH comprises the amino acid sequence shown in SEQ ID NO:21, and VL comprises the amino acid sequence shown in SEQ ID NO:30, 31, 32, 33, 34 or 35; VH comprises the amino acid sequence shown in SEQ ID NO:22, and VL comprises the amino acid sequence shown in SEQ ID NO:36 or 37; VH comprises the amino acid sequence shown in SEQ ID NO:23, and VL comprises the amino acid sequence shown in SEQ ID NO:36 or 37; VH comprises the amino acid sequence shown in SEQ ID NO:24, and VL comprises the amino acid sequence shown in SEQ ID NO:38 or 39; or VH comprises the amino acid sequence shown in SEQ ID NO:25, and VL comprises the amino acid sequence shown in SEQ ID NO:40.

[0028] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein block the interaction between human SIGLEC15 and sTn. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein block the interaction between human SIGLEC15 and LRRC4C.

[0029] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of rescuing monocyte apoptosis, for example, apoptosis of monocytes in PBMCs mediated by soluble SIGLEC15 protein.

[0030] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of down-regulating CD14 expression, or reversing soluble SIGLEC15 protein-mediated inhibition of human monocytes, the reversal being indicated by down-regulation of CD14 expression.

[0031] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of inhibiting or increasing TNFα secretion, for example, TNFα secretion stimulated by a soluble SIGLEC15 protein.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof can increase CD4 and CD8 T cell proliferation and / or increase INF-γ secretion. In some embodiments, the antibody or antigen-binding fragment thereof provided herein can reverse the inhibition of PBMC T cells mediated by soluble SIGLEC15 protein by increasing CD4 and CD8 T cell proliferation and / or increasing INF-γ secretion.

[0033] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of eliciting immunological memory.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof binds to SIGLEC15 with a KD of no more than 9.9E-8M, or no more than 9.9E-9M.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and sTn, wherein the IC50 is no more than 10, 8, 6, 5, 4 or 3 μg / mL; or the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and sTn, wherein the IC50 is no more than 50, 40, 35 or 30 nM.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and LRRC4C, wherein the IC50 is no more than 8, 6, 5, 4, 3 or 1 μg / mL; or the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and LRRC4C, wherein the IC50 is no more than 10, 8, 6, 5 or 3 nM.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof can mediate ADCC (antibody-dependent cell-mediated cytotoxicity) function and / or ADCP (antibody-dependent cellular phagocytosis) function. In some embodiments, the antibody or antigen-binding fragment thereof can rescue the downregulation of APC function and / or immune response of monocytes and monocyte-derived myeloid cells mediated by SIGLEC15.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof can enhance the immune response, optionally by rescuing SIGLEC15-mediated down-regulation of APC (antigen presenting cell) function and / or monocyte immune response.

[0039] In one aspect, provided herein is an antibody or antigen-binding fragment thereof that binds to SIGLEC15, wherein the antibody or antigen-binding fragment thereof binds to an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, 45, 46, and 47, and combinations thereof. In one aspect, the antibody or antigen-binding fragment thereof binds to the amino acid sequence shown in SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, or SEQ ID NOs: 45, 46, and 47.

[0040] In one aspect, provided herein is an isolated polynucleotide encoding the above-described antibody or antigen-binding fragment thereof.

[0041] In one aspect, provided herein is an isolated vector comprising the above-described polynucleotide.

[0042] In one aspect, provided herein is a host cell comprising the above-described isolated polynucleotide or the above-described isolated vector.

[0043] In one aspect, provided herein is a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated vector or host cell, and a pharmaceutically acceptable carrier.

[0044] In one aspect, provided herein is a kit comprising the above-mentioned antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated vector, host cell or pharmaceutical composition.

[0045] In one aspect, provided herein is a use of the above-mentioned antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated vector, host cell, pharmaceutical composition or kit for preparing a therapeutic agent for binding to SIGLEC15.

[0046] In some embodiments, the above-mentioned antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated vector, host cell, pharmaceutical composition or the above-mentioned kit is used to prepare a therapeutic agent for blocking the interaction between human SIGLEC15 and sTn or LRRC4C.

[0047] In one aspect, provided herein is the use of the above-mentioned antibodies or antigen-binding fragments thereof, isolated polynucleotides, isolated vectors, host cells, pharmaceutical compositions or kits for the preparation of a therapeutic agent for enhancing the immune response to a condition, disorder or disease (optionally a condition, disorder or disease associated with SIGLEC15).

[0048] In one aspect, provided herein is the use of the above-mentioned antibodies or antigen-binding fragments thereof, isolated polynucleotides, isolated vectors or host cells, pharmaceutical compositions or kits in the preparation of therapeutic agents for diagnosing, preventing or treating conditions, disorders or diseases.

[0049] In some embodiments, the condition, disorder or disease comprises a tumor.

[0050] In one aspect, provided herein is a method for enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the above-mentioned antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated vector, host cell, pharmaceutical composition, or kit.

[0051] In some embodiments, the above antibodies or antigen-binding fragments thereof can optionally enhance the immune response by rescuing SIGLEC15-mediated downregulation of monocyte APC function and immune response.

[0052] In some embodiments, the above-mentioned antibodies or antigen-binding fragments thereof are capable of reversing soluble SIGLEC15 protein-mediated human monocyte inhibition, increasing or inhibiting TNFα secretion, increasing CD4 and CD8 T cell proliferation and / or increasing INF-γ secretion.

[0053] In one aspect, provided herein is a method for diagnosing, preventing or treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the above-described antibody or antigen-binding fragment, isolated polynucleotide, isolated vector, host cell, pharmaceutical composition or kit.

[0054] In some embodiments, the subject comprises a mammal.

[0055] In some embodiments, the subject includes humans, mice, and cynomolgus monkeys.

[0056] In one aspect, provided herein is a binding site on SIGLEC15. The binding site comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 41, 42, 43, 44, 45, 46 and 47, and combinations thereof.

[0057] In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NO: 41 and / or SEQ ID NO: 42. In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NO: 43 and / or SEQ ID NO: 44. In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NO: 45, SEQ ID NO: 46, and / or SEQ ID NO: 47. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The following is a brief description of the drawings, which are presented for the purpose of illustrating exemplary embodiments disclosed herein and not for the purpose of limiting the same.

[0059] Figure 1 The expression of SIGLEC15 on human myeloid cells measured by fluorescence activated cell sorting (FACS) detection is shown. MFI is the abbreviation of mean fluorescence intensity, M1 is the abbreviation of M1 macrophages, M2 is the abbreviation of M2 macrophages, DC is the abbreviation of dendritic cells, IL-10 is the abbreviation of interleukin 10, iso(PGLALA) represents the isotype control with PGLALA mutation, and Blank represents the blank control.

[0060] Figure 2 The SIGLEC15 protein was shown to be associated with CD4+T cells, CD8+T cells, CD14+monocytes, and CD19+B cells (S15-Fc-biotin protein, Figure 2 A; S15-His-biotin, Figure 2 B) and monocytes, M1 macrophages, M2 macrophages, THP-1 cell line (S15-Fc-biotin, Figure 2 C), S15 is the abbreviation of SIGLEC15, PBS stands for phosphate buffered saline, S15-Fc-biotin and S15-His-biotin represent biotinylated S15-Fc (silenced Fc) or S15-His protein treatment.

[0061] Figure 3 Shown by OKT3 ( Figure 3 A and Figure 3 B), PMA and ionomycin ( Figure 3 C and Figure 3 D) PHA Figure 3 E. Figure 3 F and Figure 3 G) and CD3 / CD28 beads ( Figure 3 H. Figure 3 I. Figure 3 J) Inhibitory effect of SIGLEC15 on T cells after stimulation. S15-Fc represents treatment with S15-Fc protein, iono is the abbreviation of ionomycin, no stimulation and medium represent negative controls, and hIgG1 represents isotype control.

[0062] Figure 4 SIGLEC15-induced monocyte apoptosis and monocyte suppressive activity are shown. Figure 4 A- Figure 4 C shows that S-15 dose-dependently induces monocyte apoptosis (induced apoptosis can be blocked by caspase inhibitors) and inhibits monocyte activity, respectively. CD86, HLA-DR and CD14 are monocyte surface markers, vehicle represents a negative control, and hIgG represents an isotype control.

[0063] Figure 5 The chimeric antibody was shown to be specific for CHOK1-human SIGLEC15 ( Figure 5 A), CHOK1-cynomolgus monkey SIGLEC15 ( Figure 5 B), CHOK1-mouse SIGLEC15 ( Figure 5 C) and human malignant glioma cell line U87 MG ( Figure 5 CHOK1-hS15 is the abbreviation of CHOK1-human SIGLEC15, CHOK1-cynoS15 is the abbreviation of CHOK1-cynomolgus monkey SIGLEC15, CHOK1-mS15 is the abbreviation of CHOK1-mouse SIGLEC15, and Ab Conc. is the abbreviation of antibody concentration.

[0064] Figure 6 Affinity tests of chimeric antibodies (C07, C70, C22, 5G12) by biolayer interferometry (Octet) are shown, Sec is the abbreviation for mean seconds.

[0065] Figure 7 A- Figure 7 K shows the binding specificity of the chimeric antibody to the human SIGLEC family SIGLEC15, SIGLEC1, SIGLEC2, SIGLEC3, SIGLEC4, SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9 and SIGLEC10 analyzed by ELISA. OD450 is the abbreviation for the optical density at 450 nm, OD570 is the abbreviation for the optical density at 570 nm, and hIgG1 LALA represents the isotype control.

[0066] Figure 8 The chimeric antibodies (C07, C22, C70, 5G12) analyzed by ELISA for SIGLEC15 and potential ligand sTn ( Figure 8 A) and LRRC4C ( Figure 8 B) Blocking activity of binding, Ab is the abbreviation for antibody, mAb is the abbreviation for mouse antibody, hS15 is the abbreviation for human SIGLEC15, and hIgG represents the isotype control.

[0067] Fig. 9 The chimeric antibodies (C07, C22, 5G12) rescued monocyte apoptosis induced by SIGLEC15 protein. S-15 represents SIGLEC15 treatment only, no S-15 represents negative control, hIgG1 represents isotype control, and other groups were treated with 10 μg / ml SIGLEC15 and different doses of isotype hIgG1, 5G12, C07 and C22.

[0068] Fig.10 The rescue of monocyte CD14 surface expression inhibited by SIGLEC15 protein by chimeric antibodies (C07, C22, 5G12) is shown, hIgG1 represents the isotype control, hIgG1 LALA P329G represents the isotype control with a mutant Fc fragment, and 5G12 LALA P329G represents the 5G12 antibody with a mutant Fc fragment.

[0069] Fig.11 Chimeric antibodies (C07, C22, 5G12) are shown for SIGLEC15-stimulated monocyte TNF-α secretion. Med. represents treatment with cell culture medium, hIgG1 represents isotype treatment only, S-15 represents SIGLEC15 treatment only, and other groups were treated with 5 μg / ml SIGLEC15 and different doses of isotypes 5G12, C07, and C22.

[0070] Fig.12 PBMC T cell activation assay for evaluation of chimeric antibodies (C07, C22, C70, 5G12) is shown. Fig.12 A and Fig.12 D shows the proliferation analysis of CD4, Fig.12 B and Fig.12 E shows the proliferation analysis of CD8, Fig.12 C and Fig.12 F shows the analysis of INF-γ production. No stimulation and no OKT3 represent negative controls, S15-Fc or S15-hFc represent treatment with S15-Fc protein, and NC-FC represents anti-HEL (hen egg lysozyme) antibody with the same isotype as S15-Fc as a negative control.

[0071] Fig.13 Evaluation of ADCC function of chimeric antibodies (C07, C22, C70, 5G12) is shown.

[0072] Fig.14 Evaluation of ADCP function of chimeric antibodies (C07, C22, C70, 5G12) is shown. hIgG1 represents isotype control and medium represents negative control.

[0073] Fig.15 Antibody epitopes identified by HDX / MS (hydrogen deuterium exchange mass spectrometry) are shown.

[0074] Fig.16 Single-dose PK of chimeric antibodies (C07, C22, 5G12) in hS15 KI mice bearing MC38-hS15 is shown ( Fig.16 A) and multiple dose PK ( Fig.16 B) curve.

[0075] Fig.17 The antitumor efficacy of chimeric antibodies (C07, C22, C70, 5G12) in MC38-hS15 tumors inoculated in the hS15 KI C57 mouse model was shown, where Fig.17 A- Fig.17 C shows the mean tumor growth curves, tumor weights and body weights of all treatment groups at the endpoint, respectively. Vehicle represents the negative control.

[0076] Fig.18 Individual antitumor efficacy of chimeric antibodies (C07, C22, C70, 5G12) in MC38-hS15 tumors inoculated in the hS15 KI C57 mouse model is shown. Vehicle represents a negative control.

[0077] Fig.19 Shown are tumor growth curves of MC38-hS15 in the hS15 KI C57 mouse rechallenge study. Fig.19 A shows the plasma concentration curve, and Fig.19 B shows the tumor growth curve.

[0078] Fig. 20 Individual tumor growth curves of MC38-hS15 in hS15 KI C57 mouse rechallenge study are shown. Mice were previously treated with antibodies C07, C22, C70 or 5G12, and naive mice without antibody treatment represent blank controls.

[0079] Fig.21The results show that humanized C22 antibodies (H22.015 (G56A), H22.016 (G56A) and H22.030 (G56A)) can inhibit the expression of SIGLEC15 and sTn ( Fig.21 A) and LRRC4C ( Fig.21 B) Blocking activity of binding, hIgG represents isotype control.

[0080] Fig. 22 Evaluation of C22 humanized antibody by PBMC (peripheral blood mononuclear cell) T cell assay is shown. Fig. 22 A and Fig. 22 B shows proliferation analysis of CD8 and CD4, respectively. Fig. 22 C and Fig. 22 D shows the analysis of INF-γ and TNF-α concentrations, respectively. S15-Fc represents the treatment of biotinylated S15-Fc protein, NC-FC represents anti-HEL antibody with the same isotype as S15-Fc as a negative control, hIgG1 represents an isotype control, and no OKT3 represents a negative control.

[0081] Fig.23 Evaluation of the C70 humanized antibody by PBMC T cell assay is shown. Fig.23 A and Fig.23 B shows proliferation analysis of CD8 and CD4, respectively. Fig.23 C and Fig.23 D shows the analysis of INF-γ and TNF-α concentrations, respectively.

[0082] Fig.24 Shown is a single dose PK profile of a humanized antibody (H22.015(G56A)) in cynomolgus monkeys. DETAILED DESCRIPTION

[0083] The disclosure will be explained in more detail below. This specification is not intended to be a list of all the different ways in which the invention can be implemented or all the features that can be added to the invention. For example, features shown in relation to one embodiment may be incorporated into other embodiments, and features shown in relation to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the disclosure without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention, rather than to exhaustively specify all permutations, combinations, and variations thereof.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can be used in the testing practice of the present disclosure, preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terms will be used.

[0085] SIGLEC15 (S15) is a member of the SIGLEC family (sialic acid-binding Ig-like lectin), which plays an important regulatory role in innate immunity and adaptive immunity by recognizing sialic acid-containing sugar chain structures and mediating cell-to-cell or cell-to-pathogen interactions. TCGA has shown that SIGLEC15 mRNA is upregulated in many cancers including lung cancer, kidney cancer, liver cancer, colorectal cancer, bladder cancer, endometrial cancer, etc. SIGLEC15 has been detected in tumor cells and tumor-associated macrophages, and its interaction with T cell surface receptors can inhibit the proliferation activity of T cells, ultimately leading to the "immune escape" of tumor cells (Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy, Jun Wang et al., Nature Medicine 4 (25): 656-666 (2019)).

[0086] SIGLEC15 is a potential target for tumor immunotherapy. Blockade of SIGLEC15 can promote anti-tumor immunity.

[0087] The present disclosure provides embodiments of antibodies or antigen-binding fragments thereof that bind to SIGLEC15, and uses of these antibodies and antigen-binding fragments thereof in the preparation of therapeutic agents (eg, drugs) for binding to SIGLEC15.

[0088] definition

[0089] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as these structures exhibit the desired antigen-binding activity.

[0090] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, an antigen binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain).

[0091] As used herein, the term "humanized antibody" refers to a non-human antibody that contains minimal sequence derived from non-human (eg, mouse) immunoglobulin and contains sequence derived from human immunoglobulin.

[0092] As used herein, the term "subject" refers to humans and non-human animals. Non-human animals include all mammals such as mice, rats, rabbits, cats, dogs, pigs, monkeys, chimpanzees, gorillas, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

[0093] As used herein, the term "polynucleotide" refers to a nucleotide polymer of any length of at least two nucleotides, and includes, but is not limited to, DNA, RNA, DNA / RNA hybrids, and modified forms thereof.

[0094] As used herein, the term "vector" refers to a polynucleotide that can be engineered to contain one or more clones that can be propagated in a host cell. A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences (e.g., promoters and / or enhancers) that regulate the expression of a polypeptide of interest, and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used for colorimetric assays, such as β-galactosidase).

[0095] As used herein, the term "host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate cells.

[0096] As used herein, the term "PK" is an acronym for "pharmacokinetics" and encompasses the properties of a compound including, for example, absorption, distribution, metabolism, and elimination by a subject.

[0097] As used herein, the term “EC 50 ” refers to the 50% effective concentration. The term “IC 50 ” refers to 50% inhibition concentration.

[0098] As used herein, the term "PBMC" refers to peripheral blood mononuclear cells.

[0099] As used herein, the term "ADCP" refers to antibody-dependent cellular phagocytosis.

[0100] As used herein, the term "ADCC" refers to antibody-dependent cell-mediated cytotoxicity.

[0101] As used herein, the term "INF-γ" refers to interferon gamma.

[0102] As used herein, the term "TNFα" refers to tumor necrosis factor alpha.

[0103] As used herein, the term "APC" refers to antigen presenting cell.

[0104] As used herein, the term "DC" refers to dendritic cells.

[0105] As used herein, the term "MCSF" refers to macrophage colony stimulating factor, and the terms "MCSF" and "M-CSF" are used interchangeably herein.

[0106] As used herein, the term "GMCSF" refers to granulocyte-macrophage colony stimulating factor, and the terms "GMCSF" and "GM-CSF" are used interchangeably herein.

[0107] As used herein, the term "IL10" refers to interleukin 10, the term "IL4" refers to interleukin 4, the terms "IL10" and "IL-10" are used interchangeably herein, and the terms "IL4" and "IL-4" are used interchangeably herein.

[0108] As used herein, the term "sTn" refers to sialyl-Tn.

[0109] As used herein, the term "LRRC4C" refers to leucine-rich repeat 4C.

[0110] As used herein, the term "AST" refers to aspartate aminotransferase.

[0111] As used herein, the term "ALP" refers to alkaline phosphatase.

[0112] As used herein, the term "ALT" refers to alanine aminotransferase.

[0113] As used herein, the term "TBIL" refers to total bilirubin.

[0114] As used herein, the term "RBC" refers to red blood cells.

[0115] As used herein, the term "HGB" refers to hemoglobin.

[0116] As used herein, the term "HCT" refers to the specific volume of red blood cells.

[0117] As used herein, the term "HLA-DR" refers to human leukocyte antigen-DR. The term "CD14" refers to the human CD14 molecule, and the term "CD86" refers to the human CD86 molecule.

[0118] Antibodies and their antigen-binding fragments

[0119] Provided herein are antibodies or antigen-binding fragments thereof that bind to SIGLEC15 or anti-SIGLEC15 antibodies or antigen-binding fragments thereof.

[0120] Typically, antibodies are composed of two types of polypeptide chains, light chains and heavy chains. Antibodies can contain two identical copies of light chains and two identical copies of heavy chains. The heavy chains, each containing a variable region (or variable domain, VH) and multiple constant regions (or constant domains), are bound to each other via disulfide bonds in their constant domains. The light chains, each containing a variable region (or variable domain, VL) and a constant region (or constant domain), are each bound to a heavy chain via a disulfide bond. The variable regions of both light and heavy chains contain three highly variable regions sandwiched between more conservative framework regions (FR).

[0121] The highly variable regions, called complementarity determining regions (CDRs), form a loop that encompasses the primary antigen binding surface of an antibody. CDRs are important for identifying antigenic epitopes. As used herein, "epitope" refers to the smallest portion of a target molecule that can be specifically bound by an antibody's antigen binding domain. In some embodiments, a binding site comprises an epitope.

[0122] The present disclosure provides an antibody or antigen-binding fragment thereof that binds to SIGLEC15, the antibody or antigen-binding fragment thereof comprising at least one heavy chain variable region (VH) and at least one light chain variable region (VL), VH comprising CDRs 1, 2, and 3, CDR1 of VH differing from the amino acid sequence shown in SEQ ID NO:55, 61, or 67 by no more than 5, 4, 3, 2, or 1 amino acid; CDR2 of VH differing from the amino acid sequence of SEQ ID NO:56, 62, or 68 by no more than 5, 4, 3, 2, or 1 amino acid; CDR3 of VH differing from the amino acid sequence of SEQ ID NO:57, 63, or 69 by no more than 5, 4, 3, 2, or 1 amino acid; and VL comprising CDRs 1, 2, and 3, CDR1 of VL differing from the amino acid sequence of SEQ ID NO:58, 64, or SEQ ID NO:70 by no more than 5, 4, 3, 2, or 1 amino acid; CDR2 of VL differing from the amino acid sequence of SEQ ID NO:5 The amino acid sequence shown in NO:59, 65 or 71 differs by no more than 5, 4, 3, 2 or 1 amino acids; the CDR3 of VL differs by no more than 5, 4, 3, 2 or 1 amino acids from the amino acid sequence shown in SEQ ID NO:60, 66 or 72. The CDR sequences in the present disclosure are annotated using the Kabat numbering system (e.g., MOE software).

[0123] In some embodiments, the VH of the antibody or its antigen-binding fragment comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 55, 56, and 57, respectively; and the VL of the antibody or its antigen-binding fragment comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 58, 59, and 60, respectively.

[0124] In some embodiments, the VH of the antibody or its antigen-binding fragment comprises a CDR1 having the amino acid sequence shown in SEQ ID NO:61, a CDR2 having an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:62 by no more than 5, 4, 3, 2 or 1 amino acids, and a CDR3 having the amino acid sequence shown in SEQ ID NO:63; and the VL of the antibody or its antigen-binding fragment comprises CDRs 1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs:64, 65 and 66, respectively.

[0125] In some embodiments, the VH of the antibody or its antigen-binding fragment comprises a CDR1 having the amino acid sequence shown in SEQ ID NO:67, a CDR2 having an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:68 by no more than 5, 4, 3, 2 or 1 amino acids, and a CDR3 having the amino acid sequence shown in SEQ ID NO:69; and the VL of the antibody or its antigen-binding fragment comprises a CDR1 having an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:70 by no more than 5, 4, 3, 2 or 1 amino acids, a CDR2 having the amino acid sequence shown in SEQ ID NO:71, and a CDR3 having the amino acid sequence shown in SEQ ID NO:72.

[0126] In some embodiments, the CDR2 of VH contains a mutation at a protein post-translational modification site. In some embodiments, the CDR1 of VL contains a mutation at a protein post-translational modification site.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof comprises a mutation at position 55, 56, 32, 33, or a combination thereof. In some embodiments, the CDR2 of VH comprises a mutation N55Q, G56A, or both. In some embodiments, the CDR1 of VL comprises a mutation G33A, D32E, or both.

[0128] The term "position" refers to the amino acid residue numbering counted from the N-terminus of a mutant protein, polypeptide, protein or antibody. When there is a signal peptide, the amino acid residue numbering can start from the first amino acid after the signal peptide, or from the first amino acid of a mature protein, or the term "position" is defined by the Kabat numbering and / or Chothia system. Taking mutation G56A as an example, at position 56, i.e. the 56th amino acid at the N-terminus of a protein or antibody, amino acid "G" is replaced by amino acid "A". Amino acid abbreviations, such as "G" and "A" are standard single-letter codes, and should be understood in a common manner in the art.

[0129] In some embodiments, the CDR2 of VH comprises the mutation N55Q. In some embodiments, the CDR2 of VH comprises the mutation G56A. In some embodiments, the CDR1 of VL comprises the mutation D32E. In some embodiments, the CDR1 of VL comprises the mutation G33A. In some embodiments, the CDR2 of VH comprises the mutation N55Q and the CDR1 of VL comprises the mutation D32E. In some embodiments, the CDR2 of VH comprises the mutation N55Q and the CDR1 of VL comprises the mutation G33A.

[0130] In some embodiments, the VH of the antibody or its antigen-binding fragment that binds to SIGLEC15 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and SEQ ID NOs: 7-25; the VL of the antibody or its antigen-binding fragment is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and SEQ ID NOs: 26-40.

[0131] Methods on how to determine the percent identity of two amino acid sequences are known in the art, including but not limited to BLAST (Basic Local Alignment Search Tool) at NCBI.

[0132] In some embodiments, the antibody or antigen-binding fragment thereof comprises mutation N55Q and / or mutation G56A in the heavy chain variable region.

[0133] In some embodiments, the antibody or antigen-binding fragment thereof comprises mutations G33A and / or D32E in the light chain variable region.

[0134] In some embodiments, the antibody or its antigen-binding fragment comprises a mutation G56A in VH and a mutation G33A in VL. In some embodiments, the antibody or its antigen-binding fragment comprises a mutation G56A in VH and a mutation D32E in VL. In some embodiments, the antibody or its antigen-binding fragment comprises a mutation N55Q in VH and a mutation G33A in VL. In some embodiments, the antibody or its antigen-binding fragment comprises a mutation N55Q in VH and a mutation D32E in VL.

[0135] In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and SEQ ID NOs: 7-25; and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and SEQ ID NOs: 26-40.

[0136] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:1, 2 or 3; and VL comprises the amino acid sequence shown in SEQ ID NO:4, 5 or 6.

[0137] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:7, 8 or 9, and VL comprises the amino acid sequence shown in SEQ ID NO:26.

[0138] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and VL comprises the amino acid sequence shown in SEQ ID NO:27 or 28.

[0139] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:20, 21, 22, 23, 24 or 25, and VL comprises the amino acid sequence shown in SEQ ID NO:29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40.

[0140] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:1, and VL comprises the amino acid sequence shown in SEQ ID NO:4; VH comprises the amino acid sequence shown in SEQ ID NO:2, and VL comprises the amino acid sequence shown in SEQ ID NO:5; VH comprises the amino acid sequence shown in SEQ ID NO:3, and VL comprises the amino acid sequence shown in SEQ ID NO:6.

[0141] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:7, 8 or 9, and VL comprises the amino acid sequence shown in SEQ ID NO:26.

[0142] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:10, 11 or 12, and VL comprises the amino acid sequence shown in SEQ ID NO:27.

[0143] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:13 or 14, and VL comprises the amino acid sequence shown in SEQ ID NO:28.

[0144] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:15, 16 or 17, and VL comprises the amino acid sequence shown in SEQ ID NO:27.

[0145] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:17, 18 or 19, and VL comprises the amino acid sequence shown in SEQ ID NO:28.

[0146] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:20, and VL comprises the amino acid sequence shown in SEQ ID NO:29.

[0147] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:21, and VL comprises the amino acid sequence shown in SEQ ID NO:30, 31, 32, 33, 34 or 35.

[0148] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:22, and VL comprises the amino acid sequence shown in SEQ ID NO:36 or 37.

[0149] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:23, and VL comprises the amino acid sequence shown in SEQ ID NO:36 or 37.

[0150] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:24, and VL comprises the amino acid sequence shown in SEQ ID NO:38 or 39.

[0151] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:25, and VL comprises the amino acid sequence shown in SEQ ID NO:40.

[0152] The antibodies or their antigen-binding fragments provided herein are murine, chimeric or humanized. In non-limiting embodiments, humanized antibodies are human antibodies, wherein the hypervariable region residues are replaced by the hypervariable region residues from non-human antibodies (e.g., mouse, rat or rabbit antibodies). In some embodiments, humanized antibodies are human antibodies, wherein the framework region residues are replaced by the corresponding non-human (e.g., mouse) framework region residues. In some embodiments, humanized antibodies can also contain at least a portion of human immunoglobulin constant region (Fc). Molecular biological methods known in the art can be used to produce humanized antibodies.

[0153] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be produced by hybridoma cells, optionally hybridoma cells that stably express SIGLEC15, such as modified CHOK1 cells that express human, cynomolgus monkey or mouse SIGLEC15. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be produced by hybridoma cells that stably express human SIGLEC15.

[0154] Provided herein are isolated hybridoma cell lines, wherein the cells produce an antibody or antigen-binding fragment thereof provided herein.

[0155] The above-mentioned antibody or antigen-binding fragment is a modified antibody or antigen-binding fragment thereof, which can bind to human SIGLEC15. In some embodiments, the antibody or antigen-binding fragment thereof is modified to maintain or enhance or reduce immunogenicity, binding to SIGLEC15, or reduce function.

[0156] In one aspect, provided herein is an antibody or antigen-binding fragment thereof that binds to an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-47 and combinations thereof.

[0157] In some embodiments, the antibody or antigen-binding fragment thereof binds to the amino acid sequence shown in SEQ ID NO:41. In some embodiments, the antibody or antigen-binding fragment thereof binds to the amino acid sequence shown in SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment thereof binds to the amino acid sequence shown in SEQ ID NO:43. In some embodiments, the antibody or antigen-binding fragment thereof binds to the amino acid sequence shown in SEQ ID NO:44. In some embodiments, the antibody or antigen-binding fragment thereof binds to the amino acid sequence shown in SEQ ID NO:45. In some embodiments, the antibody or antigen-binding fragment thereof binds to the amino acid sequence shown in SEQ ID NO:46. In some embodiments, the antibody or antigen-binding fragment thereof binds to the amino acid sequence shown in SEQ ID NO:47.

[0158] In some embodiments, the antibody or antigen-binding fragment thereof binds to the amino acid sequences shown in SEQ ID NOs: 41 and 42. In some embodiments, the antibody or antigen-binding fragment thereof binds to the amino acid sequences shown in SEQ ID NOs: 43 and 44. In some embodiments, the antibody or antigen-binding fragment thereof binds to the amino acid sequences shown in SEQ ID NOs: 45, 46 and 47.

[0159] In one aspect, provided herein is a binding site or binding epitope on SIGLEC15, wherein the binding site comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 41-47 and combinations thereof. In some embodiments, SIGLEC15 is human SIGLEC15.

[0160] In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NO:41. In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NO:42. In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NO:43. In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NO:44. In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NO:45. In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NO:46. In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NO:47.

[0161] In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NOs: 41 and 42. In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NOs: 43 and 44. In some embodiments, the binding site comprises the amino acid sequence shown in SEQ ID NOs: 45, 46, and 47.

[0162] In this disclosure, the term "binding epitope" refers to a molecular region on the surface of an antigen (or therapeutic target) that is capable of eliciting an immune response and combining with specific antibodies generated by such a response.

[0163] Antibody characteristics

[0164] The antibody or antigen-binding fragment thereof produced by the hybridoma cell specifically binds to SIGLEC15, and exemplary SIGLEC15 includes human SIGLEC15.

[0165] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between SIGLEC15 (eg, human SIGLEC15) and its potential ligands (eg, LRRC4C and sTn).

[0166] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and sTn.

[0167] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and sTn, wherein the IC 50 No more than 50, 40, 35 or 30 nM, as assessed by competitive ELISA as appropriate. In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and sTn, wherein the IC 50 No more than 10, 8, 6, 5, 4 or 3 μg / mL, an exemplary measurement method may be competitive FACS.

[0168] Typically, IC 50 The value represents the concentration at which the substance exerts half of its maximal inhibitory effect. This value is typically used to characterize antagonists of biological processes.

[0169] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and LRRC4C.

[0170] In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and LRRC4C, wherein the IC 50 No more than 10, 8, 6, 5 or 3 nM, optionally assessed by competitive ELISA. In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and LRRC4C with an IC50 of no more than 8, 6, 5, 4, 3 or 1 μg / mL, and an exemplary measurement method may be competitive FACS.

[0171] In some embodiments, the antibody or antigen-binding fragment thereof is capable of rescuing monocyte apoptosis, for example, soluble SIGLEC15 protein-mediated monocyte apoptosis in PBMCs (peripheral blood mononuclear cells), exemplary PBMCs include human PBMCs.

[0172] In some embodiments, the antibody or antigen-binding fragment thereof is capable of dose-dependently rescuing soluble SIGLEC15 protein-mediated apoptosis of monocytes in PBMCs.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof is capable of reversing monocyte inhibition, for example, soluble SIGLEC15 protein-mediated inhibition of human monocytes.

[0174] In some embodiments, the antibody or antigen binding fragment thereof is capable of downregulating CD14 expression. In some embodiments, the antibody or antigen binding fragment thereof is capable of reversing human monocyte inhibition. In some embodiments, the antibody or antigen binding fragment thereof is capable of reversing soluble SIGLEC15 protein-mediated human monocyte inhibition, the reversal being indicated by downregulation of CD14 expression.

[0175] In some embodiments, the antibody or antigen-binding fragment thereof is capable of reversing soluble SIGLEC15 protein-mediated suppression of human monocytes in a dose-dependent manner, with the reversal being indicated by downregulation of CD14 expression.

[0176] In some embodiments, the antibody or antigen-binding fragment thereof is capable of inhibiting or increasing TNFα (tumor necrosis factor α) secretion, such as TNFα secretion stimulated by soluble SIGLEC15 protein.

[0177] In some embodiments, the antibody or antigen-binding fragment thereof is capable of increasing CD4 and CD8 T cell proliferation and / or increasing INF-γ (interferon-γ) secretion.

[0178] In some embodiments, the antibody or antigen-binding fragment thereof is capable of reversing soluble SIGLEC15 protein-mediated suppression of PBMC T cells by increasing CD4 and CD8 T cell proliferation and / or increasing INF-γ (interferon-γ) secretion.

[0179] In some embodiments, the antibody or antigen-binding fragment thereof is capable of eliciting immunological memory.

[0180] In some embodiments, the antibody or antigen-binding fragment thereof binds to SIGLEC15, particularly wherein the KD is no more than 9.9E-8M or no more than 9.9E-9M, an exemplary measurement method can be the octet rule.

[0181] Typically, the KD value or affinity constant is the equilibrium dissociation constant or Kd / Ka ratio between an antibody and its antigen. Kd refers to the dissociation constant and Ka refers to the association constant.

[0182] In some embodiments, the antibody or antigen-binding fragment thereof is capable of mediating ADCC function and / or ADCP function.

[0183] In some embodiments, the antibody or antigen-binding fragment thereof (wherein VH comprises the amino acid sequence shown in SEQ ID NO: 7, 8 and 9, and VL comprises the amino acid sequence shown in SEQ ID NO: 26) binds to SIGLEC15, wherein EC 50 Not exceeding 5, 4, 3, 2 or 1 nM.

[0184] In some embodiments, the antibody or antigen-binding fragment thereof (wherein VH comprises the amino acid sequence shown in SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and VL comprises the amino acid sequence shown in SEQ ID NO: 27 or 28) binds to SIGLEC15, wherein EC 50 Not exceeding 5, 4, 3 or 2 nM.

[0185] In some embodiments, the antibody or antigen-binding fragment thereof (wherein VH comprises the amino acid sequence shown in SEQ ID NO: 20, 21, 22, 23, 24 or 25, and VL comprises the amino acid sequence shown in SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40) binds to SIGLEC15, wherein EC 50 Not exceeding 15, 14, 13, 12, 11 or 10 nM.

[0186] Without being bound by any theory, in some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of enhancing an immune response.

[0187] Without being bound by any theory, in some embodiments, the antibody or antigen-binding fragment thereof can enhance the immune response by rescuing SIGLEC15-mediated down-regulation of APC function and immune response of monocytes.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof is capable of rescuing SIGLEC15-mediated downregulation of APC function and immune responses in monocytes and monocyte-derived myeloid cells.

[0189] In some embodiments, the antibody or antigen-binding fragment thereof is safe and has no or low toxicity to mammals, such as cynomolgus monkeys.

[0190] Polynucleotides, vectors and host cells

[0191] The present disclosure provides isolated polynucleotides encoding the above-mentioned antibodies or antigen-binding fragments thereof.

[0192] A polynucleotide is a polymer of DNA, RNA, a DNA / RNA hybrid, or a modified form thereof. In some embodiments, the polynucleotide is a polymer of DNA. The polynucleotide is a polymer of RNA.

[0193] DNA or RNA encoding an antibody or antigen-binding fragment thereof that binds to SIGLEC15 can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody). Encoding DNA or RNA can also be obtained by synthetic methods.

[0194] Using recombinant techniques known in the art, the isolated polynucleotide encoding the antibody or its antigen-binding fragment can be inserted into a vector for further cloning (amplification of the DNA) or expression. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer component, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0195] In one aspect, provided herein is an isolated vector comprising a polynucleotide encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0196] In one aspect, provided herein is a host cell comprising the above-described isolated polynucleotide or the above-described isolated vector.

[0197] The vector comprising the polynucleotide sequence encoding the above-mentioned antibody or its antigen-binding fragment can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA or RNA in the vector herein are the above-mentioned prokaryotes, yeast or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), and Shigella, as well as Bacilli (e.g., B. subtilis and B. licheniformis), Pseudomonas (e.g., P. aeruginosa), and Streptomyces.

[0198] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for anti-SIGLEC15 antibody encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are commonly available and can be used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts such as A. nidulans and A. niger.

[0199] Host cells are transfected with the above-mentioned expression or cloning vectors for producing antibodies or antigen-binding fragments thereof or anti-SIGLEC15 antibodies or antigen-binding fragments thereof, and cultured in a conventional nutrient medium, the medium being modified to be suitable for inducing promoters, selecting transformants or amplifying genes encoding the desired sequences. In another embodiment, antibodies can be produced by homologous recombination as known in the art. In certain embodiments, host cells are capable of producing antibodies or antigen-binding fragments thereof provided herein.

[0200] Pharmaceutical composition

[0201] In one aspect, the present disclosure further provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers.

[0202] In some embodiments, the present disclosure further provides pharmaceutical compositions comprising an isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers.

[0203] In some embodiments, the present disclosure further provides a pharmaceutical composition comprising an isolated vector comprising a polynucleotide encoding an antibody or an antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers.

[0204] In some embodiments, the present disclosure further provides a pharmaceutical composition comprising a host cell containing an isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof, or an isolated vector comprising the above polynucleotide.

[0205] The pharmaceutical compositions provided herein can be formulated in any manner known in the art, such as pharmaceutical compositions provided herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound to facilitate administration and uniformity of dosage).

[0206] The pharmaceutical composition is formulated to be compatible with its intended route of administration (eg, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0207] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering agents or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0208] Suitable components may include, for example, antioxidants, fillers, adhesives, disintegrating agents, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers (such as sugars and cyclodextrins). Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylhydroxymethoxybenzene, butylhydroxytoluene and / or propyl gallate. As disclosed herein, one or more antioxidants (such as methionine) are included in the composition comprising the antibody or its antigen-binding fragment and conjugate provided herein to reduce the oxidation of the antibody or its antigen-binding fragment. This reduction in oxidation can prevent or reduce the loss of binding affinity, thereby improving antibody stability and maximizing the shelf life. Therefore, in certain embodiments, a pharmaceutical composition comprising one or more antibodies or their antigen-binding fragments as disclosed herein and one or more antioxidants (such as methionine) is provided.

[0209] To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous vehicles (such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection), non-aqueous vehicles (such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents (such as sodium chloride or dextrose), buffers (such as phosphate or citrate buffers), antioxidants (such as sodium bisulfate), local anesthetics (such as procaine hydrochloride), suspending and dispersing agents (such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone), emulsifiers (such as polysorbate 80 (Tween-80)), sequestrants or chelating agents (such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multiple dose containers, which contain phenol or cresol, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, normal saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or medicaments (such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin).

[0210] In some embodiments, the pharmaceutical composition may be a liquid solution, suspension, or emulsion.

[0211] In some embodiments, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solutions, suspensions, emulsions, or solid forms suitable for producing liquid solutions, suspensions, or emulsions. Injectable preparations can include sterile and / or pyrogen-free solutions for injection, sterile dry soluble products (such as lyophilized powders) (including subcutaneous injection tablets) prepared for combination with solvents before use, sterile suspensions for injection, sterile dry insoluble products prepared for combination with media before use, and sterile and / or pyrogen-free emulsions. The solution can be aqueous or non-aqueous.

[0212] In some embodiments, the unit dose of parenteral formulations is packaged in an ampoule, a vial, or a syringe with a needle. As known and practiced in the art, all preparations for parenteral administration should be sterile and pyrogen-free.

[0213] In some embodiments, a sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability of the powder or the reconstituted solution prepared from the powder or other pharmacological components. Excipients that can be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may contain a buffer (such as citrate, sodium phosphate or potassium phosphate) of about neutral pH in one embodiment, or other such buffers known to those skilled in the art. The solution is then sterile filtered and then freeze-dried under standard conditions known to those familiar with the technology to obtain the desired formulation. In one embodiment, the resulting solution will be dispensed into vials for freeze-drying. Each vial may contain a single dose or multiple doses of an antibody or antigen-binding fragment thereof that binds to SIGLEC15, or a composition thereof. It is acceptable to overfill vials with a small amount (e.g., about 5%, 10%, or 15%) higher than that required for one dose or a group of doses to facilitate accurate sample extraction and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0214] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. In one embodiment, sterile and / or pyrogen-free water or other liquid suitable carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the selected therapy to be administered and can be determined empirically.

[0215] Reagent test kit

[0216] In one aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof, an isolated polynucleotide, an isolated vector, and / or a pharmaceutical composition provided herein.

[0217] In some embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of: a chemotherapeutic agent, an anticancer agent, a radiotherapy, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapy, a cell therapy, a gene therapy, a hormone therapy, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, and a cytokine.

[0218] If desired, such a kit may further include one or more of the various conventional pharmaceutical kit components, such as containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be apparent to those skilled in the art. Instructions such as inserts or labels indicating the quantities of the components to be administered, directions for administration, and / or directions for mixing the components may also be included in the kit.

[0219] In some embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein (optionally conjugated with a detectable moiety), which can be used to detect a SIGLEC15-related disease, disorder or condition. The kit may further comprise instructions for use.

[0220] Use in the preparation of therapeutic agents

[0221] The present disclosure provides uses of the above-mentioned antibodies or antigen-binding fragments thereof, isolated polynucleotides, isolated vectors, host cells, pharmaceutical compositions, and / or kits in the preparation of therapeutic agents for diagnosing, preventing or treating conditions, disorders or diseases.

[0222] In some embodiments, the present disclosure provides use of the above-mentioned antibodies or antigen-binding fragments thereof, isolated polynucleotides, isolated vectors, host cells, pharmaceutical compositions, and / or kits in the preparation of therapeutic agents for binding to human SIGLEC15.

[0223] In some embodiments, provided herein is a use of the above-mentioned antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated vector, host cell, pharmaceutical composition, and / or kit for preparing a therapeutic agent for blocking the interaction between human SIGLEC15 and sTn or LRRC4C.

[0224] In some embodiments, provided herein is the use of the above-mentioned antibodies or antigen-binding fragments thereof, isolated polynucleotides, isolated vectors, host cells, pharmaceutical compositions, and / or kits in the preparation of therapeutic agents for diagnosing, preventing or treating conditions, disorders or diseases associated with SIGLEC15.

[0225] In some embodiments, provided herein is the use of the above-mentioned antibodies or antigen-binding fragments thereof, isolated polynucleotides, isolated vectors, host cells, pharmaceutical compositions, and / or kits for the preparation of therapeutic agents for enhancing the immune response to a condition, disorder or disease (preferably a condition, disorder or disease associated with SIGLEC15).

[0226] In some embodiments, the condition, disorder or disease comprises a tumor, which can be, without limitation, a blood tumor or a solid tumor. In some embodiments, the tumor can be malignant or benign. In some embodiments, the disease is cancer.

[0227] Treatment

[0228] The present disclosure further provides a method of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated vector, host cell, pharmaceutical composition, and / or kit provided herein.

[0229] Without being bound by any theory, in some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of enhancing immune responses by rescuing SIGLEC15-mediated down-regulation of monocyte APC function and immune responses.

[0230] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of increasing or inhibiting TNFα secretion, increasing CD4 and CD8 T cell proliferation, and / or increasing INF-γ secretion.

[0231] Without being bound by any theory, in some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of reversing soluble SIGLEC15 protein-mediated human monocyte inhibition, increasing or inhibiting TNFα secretion, increasing CD4 and CD8 T cell proliferation and / or increasing INF-γ secretion.

[0232] The present disclosure further provides a method for diagnosing, preventing or treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated vector, host cell, pharmaceutical composition and / or kit provided herein.

[0233] In some embodiments, the disease, disorder or condition comprises a tumor. In some embodiments, the cancer is a cancer expressing SIGLEC15. The term "cancer expressing SIGLEC15" refers to a cancer characterized by expression of SIGLEC15 protein in cancer cells, tumor-infiltrating immune cells, or immunosuppressive cells, or a cancer expressing SIGLEC15 in cancer cells, tumor-infiltrating immune cells, or immunosuppressive cells at a significantly higher level than expected in normal cells.

[0234] In some embodiments, the subject has been identified as having cancer cells or tumor-infiltrating immune cells or immunosuppressive cells that express SIGLEC15, optionally at levels significantly higher than those typically found on non-cancerous cells or non-immunosuppressive cells.

[0235] In some embodiments, the subject comprises a mammal. In some embodiments, the subject comprises a human, a mouse or a cynomolgus monkey. In some embodiments, the subject is a human (or a human patient) and may be an adult or a minor (e.g., a person under the age of 18). In some embodiments, the subject is a non-human animal, including but not limited to non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, rabbits), pigs (e.g., pigs), equines, canines, felines, bovines and other domestic, farm and zoo animals.

[0236] The therapeutically effective amount of the antibodies or antigen-binding fragments provided herein will depend on various factors known in the art, such as body weight, age, past medical history, current medication, the health status of the subject and the possibility of cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease development. According to these and other circumstances or required indications, a person skilled in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage.

[0237] In certain embodiments, the antibodies or antigen binding fragments provided herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the dosage can be changed during the course of treatment. For example, in certain embodiments, the initial dosage may be higher than the subsequent dosage. In certain embodiments, the dosage can be changed during the course of treatment, depending on the subject's response.

[0238] Dosage regimens may be adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0239] In some embodiments, the dosage may be no less than 500, 400, 300, 200, 100, 80, 60, 40, 30, 20, 10 mg / kg in a single dose.

[0240] In some embodiments, the method of enhancing the immune response of a subject in need thereof or the method of diagnosing, preventing or treating a disease, disorder or condition of a subject in need thereof rarely causes acute toxicity to the subject. Acute toxicity to the subject can be assessed by common physiological indicators in the art, exemplary physiological indicators include ALT, AST, CK, ALP, TBIL, HGB and HCT.

[0241] The antibodies or antigen-binding fragments thereof provided herein can be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal or topical) routes.

[0242] Example

[0243] Example 1. Generation of reagents

[0244] 1.1 Reference Antibodies

[0245] Reference antibody 5G12 and humanized 5G12 were produced according to patent WO 2018057735 A1 (SEQ ID NO: 7 and 18) and (SEQ ID NO: 203 and 209), respectively. The variable region sequences of 5G12 and humanized 5G12 are shown in Table 1.

[0246] Table 1. Variable region sequences of 5G12 and humanized 5G12

[0247]

[0248] 1.2 Stable cell lines

[0249] Cell lines CHOK1-hS15, CHOK1-cynoS15 and CHOK1-mS15 stably expressing human, cynomolgus monkey and mouse SIGLEC15 have been generated for hybridoma screening and in vitro assays. The prefix "h" stands for human, "c" stands for cynomolgus monkey, "m" stands for mouse, and the word "S15" stands for SIGLEC15. To generate cell lines, CHOK1 cells were transfected with human, cynomolgus monkey and mouse SIGLEC15 expression lentiviruses and selectively cultured in medium containing 10 μg / mL puromycin for 2 weeks. Single cell clones were then isolated by limiting dilution and screened by FACS to obtain single cell lines stably expressing SIGLEC15.

[0250] 1.3 Recombinant proteins

[0251] Human SIGLEC15 extracellular domain (ECD, Uniprot accession number Q6ZMC9-1, AA Phe20-Thr 263) recombinant protein with human Fc tag or 6xHis tag was produced and purified for immunization and hybridoma screening.

[0252] A series of recombinant proteins of SIGLEC family members and potential ligands of SIGLEC15 were also purchased for in vitro assays. Human SIGLEC1, SIGLEC2, SIGLEC3, SIGLEC4, SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9 and SIGLEC10 recombinant proteins with 6xHis tag or human Fc tag were purchased from Acrobiosystems. Recombinant protein of human LRRC4C with 6xHis tag was purchased from Sinobiological. Synthetic SiaTn (Neu5Aca6Tn-C3) was purchased from Lectinity.

[0253] Example 2. Mechanism of SIGLEC15 in Immuno-Oncology

[0254] 2.1 Expression of SIGLEC15 on human myeloid cells

[0255] Human myeloid cells were induced in vitro by culture-purified monocytes from healthy donor PBMCs with different cytokines. M2 and M1 macrophages were induced by adding 20ng / mL recombinant human MCSF and GMCSF to the culture medium for 5 days, respectively. M2c macrophages were induced by adding 50ng / ml IL10 for 2 days on day 5 and adding 20ng / mL MCSF during the culture period. DC cells were induced by adding 50ng / ml IL4 for 1 day on day 7 and adding 20ng / mL MCSF during the culture period. On day 8, the cells were collected, washed, and inoculated with Fc blocker for 30 minutes at 4°C. Then the SIGLEC15 antibody 5G12-LALAPG or isotype-LALAPG (human IgG1 with L234A, L235A and P329G mutations) at a final concentration of 10μg / ml was added to the wells, respectively, and inoculated at 4°C for 30 minutes. Secondary antibodies and surface markers used to distinguish cell types were added before FACS detection. As Figure 1 As shown in , S15 is expressed on human monocytes, induced M2 macrophages, and DC cells, but not in induced M1 macrophages.

[0256] 2.2 SIGLEC15 mainly binds to myeloid cells

[0257] PBMCs from healthy donors were inoculated with 10 μg / ml biotinylated S-15 / Fc protein (silenced Fc, S15-Fc) or S-15 / His biotin (His tag biotin) and detected with SA-FITC (streptavidin-FITC). Antibodies against different types of immune cells were also added to PBMCs to detect surface markers. CD4 T cells, CD8 T cells, CD14-positive monocytes, and CD19-positive B cells were gated for S15 binding. Figure 2 A and Figure 2 As shown in B, S15 mainly binds to monocytes. No significant binding to CD4 T cells, CD8 T cells, and B cells was observed. Figure 2 S15 also binds to M1 and M2 macrophages and THP-1 cell lines as shown in C. Anti-S15 antibody 5G12 dose-dependently inhibited binding to THP-1.

[0258] 2.3 SIGLEC15 does not directly inhibit T cell activation under different stimuli

[0259] To detect the inhibitory effect of S15 on T cells under different stimuli, four commonly used T cell stimuli were tested: OKT3 (purchased from Invitrogen) (0.3μg / ml coating), PMA (phorbol myristate acetate) (2ng / ml) and (1μg / ml), PHA (phytohemagglutinin) (10μg / ml) and CD3 / CD28 beads (1:50 and 1:200 dilutions). PMA, ionomycin and PHA were purchased from Sigma. CD3 / CD28 beads were purchased from Miltenyi. PBMCs from healthy donors were treated with four stimuli and serially diluted S15-Fc or isotypes for 72 hours. The proliferation of CD4 and CD8 T cells was evaluated using CFSE (carboxyfluorescein diacetate succinimidyl ester) dilutions. INFγ in the end point supernatant was detected by HTRF (homogeneous time-resolved fluorescence). Figure 3 A and Figure 3 As shown in B, under OKT3 stimulation, S15-Fc can inhibit T cell proliferation in a dose-dependent manner. Figure 3 C and Figure 3 D) PHA Figure 3 E. Figure 3 F and Figure 3 G) and CD3 / CD28 beads ( Figure 3 H. Figure 3 I and Figure 3J) Under stimulation conditions, S15-Fc cannot inhibit T cell activity, which means that S15-Fc cannot directly act on T cells and inhibit T cell activity, but mainly acts on monocytes and deteriorates the APC function of monocytes.

[0260] SIGLEC15 induces monocyte apoptosis and inhibits monocyte activity

[0261] To detect the function of S15 on monocytes, apoptotic monocytes were stained with PI (propidium iodide), annexin V and surface marker CD14. Figure 4 As shown in A, S15 dose-dependently induced monocyte cell death after 6 h of incubation with PBMCs in the presence of Fc blocker. To explore the cause of monocyte cell death, inhibitors of caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), caspase-3 (Z-DEVD-FMK), caspase-1 (Belnacasan), and pan-caspase (Z-VAD-FMK) were added to the culture medium in the same assay. Figure 4 As shown in B, caspase-3 and pan-caspase inhibitors inhibited monocyte death. Caspase-9, caspase-8 inhibitors, and caspase-1 inhibitors were unable to inhibit monocyte death, indicating that SIGLEC15 induced monocyte death but not through the caspase-9, caspase-8, and caspase-1 activation pathways. The remaining live monocytes were tested for cell surface markers associated with APC function and monocyte activation. CD86, HLA-DR, and CD14 surface expression were detected in the remaining live monocytes. Figure 4 As shown in C, S15 down-regulates the expression of CD86, HLA-DR and CD14 on the surface of monocytes, suggesting that S15 will down-regulate the APC function and immune response of monocytes.

[0262] Based on the newly discovered mechanism of action of SIGLEC15 on monocytes and monocyte-derived myeloid cells, in vitro and ex vivo assays were designed to screen antibodies.

[0263] Example 3. Hybridoma Development and Screening

[0264] 3.1 Immunity and Fusion

[0265] A rapid immunization strategy was used to immunize 20 mice from two strains (Balb / c, SJL) with Fc-tagged human SIGLEC15 ECD recombinant protein and SIGLEC15 overexpressing cell lines. The serum titers of immunized mice were detected by ELISA analysis using 6xHis-tagged human SIGLEC15 ECD recombinant protein as antigen. When the serum titer reached a high level, the final booster immunization was performed. Three days after the final booster immunization, the combined spleen cells were harvested and fused with SP2 / 0 mouse myeloma cells. The fused cells were then inoculated into 384-well plates for screening.

[0266] 3.2 Primary screening and secondary screening

[0267] 10-12 days after fusion, supernatants harvested from each well of hybridoma cells were screened by ELISA analysis using 6xHis-tagged human SIGLEC15 ECD recombinant protein as antigen. Hybridomas in positive wells were expanded and confirmed by secondary screening using the same ELISA analysis as the primary screen. FACS binding using CHOK1-hS15, CHOK1-cynoS15, and CHOK1-mS15 was also included in the secondary screening. Hybridomas secreting antibodies with the highest human SIGLEC15 binding or cross-reactive binding with cynomolgus monkey and mouse SIGLEC15 activity were then subcloned.

[0268] 3.3 Hybridoma subcloning and screening

[0269] The selected hybridoma cells were limitedly diluted into 96-well plates at a density of 1 cell / well to obtain monoclonal hybridoma cells. The supernatant harvested from these monoclonal cells was screened by the same ELISA and FACS combined assay as in Example 2.2. The antibodies secreted by the positive clones were quantified by biolayer interferometry, and then the human SIGLEC15 binding affinity and the activity of blocking the interaction between SIGLEC15 and its ligand (see the method described in 4.2.4) and functional assays were determined. As shown in Table 2, the three monoclonal antibodies secreted by clones 25E6E3, 120C8D8 and 202B8F8 showed nanomolar human SIGLEC15 binding affinity and good blocking activity.

[0270] Table 2. Summary of mouse antibody characteristics

[0271]

[0272] Example 4. Chimeric Antibody Production and Characterization

[0273] 4.1 Chimeric Antibody Production

[0274] The heavy and light chain variable regions of the monoclonal antibodies secreted by clones 25E6E3, 120C8D8 and 202B8F8 were sequenced. Based on the sequencing results, three human IgG1 chimeric antibodies were generated and named C07, C22 and C70, respectively, where the prefix "C" stands for chimeric.

[0275] The amino acid sequences of C07, C22 and C70 are listed in Table 2-1. The CDRs are underlined.

[0276] Table 2-1. Amino acid sequences of C07, C22 and C70

[0277]

[0278] 4.2 Chimeric Antibody Characterization

[0279] 4.2.1 Binding activity

[0280] The binding activity of C07, C22, C70 and the benchmark antibody 5G12 to cell membrane human, cynomolgus monkey and mouse SIGLEC15 was tested by FACS assay using CHOK1-hS15, CHOK1-cynoS15, CHOK1-mS15 and U87 MG cell lines. All tested mAbs (mouse antibodies) were bound to CHOK1-hS15, CHOK1-cynoS15 ( Figure 5 A and Figure 5 B) and U87 MG( Figure 5 D) strongly binds with similar affinities. Except for C07 which shows weak binding, other mAbs also show cross-binding to CHOK1-mS15 ( Figure 5 C). Table 3 summarizes the EC values ​​analyzed using a four-parameter nonlinear fit using GraphPad Prism 9.0. 50 and the highest MFI value.

[0281] Table 3. Binding of C07, C22 and C70 to membrane SIGLEC15

[0282]

[0283] 4.2.2 Affinity detection

[0284] The binding affinity of C07, C22, C70 and 5G12 to human SIGLEC15 ECD recombinant protein was determined using biolayer interferometry (Octet). The association and dissociation curves were fitted with a 1:1 binding model, and the Ka / Kd / KD values ​​of each antibody were calculated and summarized in Table 4. The binding curves of each antibody are shown in Figure 6 A. Figure 6 B. Figure 6 C and Figure 6D. C07 showed similar affinity to 5G12, while C22 and C70 showed higher affinity to the SIGLEC15 protein, with C22 showing the highest affinity.

[0285] Table 4. Human SIGLEC15 binding affinities of C07, C22, C70 and 5G12

[0286] Ab Code ka(1 / Ms) kd(1 / s) KD(M) C07 1.21E+06 1.74E-04 1.44E-10 C22 6.74E+05 <1.0E-07 <1.0E-12 C70 1.21E+06 9.14E-05 7.57E-11 5G12 2.13E+06 3.85E-04 1.80E-10

[0287] 4.2.3 Binding specificity

[0288] Using human SIGLEC1, SIGLEC2, SIGLEC3, SIGLEC4, SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9, SIGLEC10 and SIGLEC15 ECD recombinant proteins as antigens, the binding of C07, C22, C70 and 5G12 to human SIGLEC family members was detected by ELISA analysis. Briefly, human SIGLEC1 to SIGLEC10 and SIGLEC15 were coated at 1.0 μg / mL overnight in 100 μl PBS at 4°C. The test Ab was diluted to 10 μg / mL. Anti-human Fab HRP (rabbit anti-human IgG) was diluted 1:2500 as the detection antibody.

[0289] TMB (3,3',5,5'-tetramethylbenzidine) was added as substrate to each well and the color reaction was developed for 10 minutes and then detected at 450 and 570 nm in a microplate reader. Figure 7 A- Figure 7 As shown in K, C07, C22, C70, and 5G12 did not bind to other SIGLEC family members except SIGLEC15, indicating good binding specificity.

[0290] 4.2.4 Blocking activity

[0291] The activity of C07, C22, C70 and 5G12 in blocking the interaction between SIGLEC15 and its potential ligands sTn and LRRC4C was evaluated by competitive ELISA. Briefly, human sTn or LRRC4C-his ECD protein was coated at 5 μg / mL and 1 μg / mL, respectively, in 100 μl PBS at 4°C overnight. Diluted Ab (30 μg / ml, 1 / 5 dilution, 8 points) in blocking buffer containing 1.043 μg / ml hS15-mFC protein was added and incubated at 37°C for 30 minutes. Anti-mouse IgG (Fc specific, 1:5000 dilution) was added at 100 μl / well and incubated at 37°C for 0.5 hours after blocking and washing. TMB was added to each well as a substrate and the color reaction was developed for 10 minutes and then detected at 450 nm in a microplate reader. As Figure 8 As shown in A, C07, C22, C70, and 5G12 all exhibited strong SIGLEC15 / sTn interaction blocking activity. C22, C70, and 5G12 also exhibited strong SIGLEC15 / LRRC4C interaction blocking activity, while C07 strongly enhanced this interaction ( Figure 8 B) The blocking parameters are listed in Table 5.

[0292] Table 5. Blocking activity of C07, C22, C70 and 5G12

[0293]

[0294] 4.2.5 Chimeric Antibodies Targeting Soluble S15 Protein-Mediated Monocyte Apoptosis

[0295] The effects of C07, C22 and 5G12 on reversing soluble S15 protein-mediated monocyte apoptosis were investigated. Briefly, 1E5 human PBMCs from healthy donors were seeded into 96-well plates containing soluble SIGLEC15 fusion protein at a concentration of 10 μg / ml in culture medium supplemented with serial 2-fold dilutions of mAb starting from 30 μg / ml and incubated for 14 h at 37°C, 5% CO2. Cells were harvested and stained with CD14, Annexin-V FITC and PI for FACS analysis. Annexin-V FITC and PI double-negative cells were gated as live cells. As Fig. 9 As shown in , C07, C22, and 5G12 dose-dependently rescued monocyte apoptosis mediated by soluble S15 protein in PBMCs.

[0296] 4.2.6 Chimeric Antibodies Targeting Soluble S15 Protein-Mediated Monocyte Inhibition

[0297] The effects of C07, C22 and 5G12 on reversing soluble S15 protein-mediated monocyte inhibition (indicated by CD14 surface expression) were also studied. In brief, human PBMCs from healthy donors were seeded into 96-well plates containing soluble SIGLEC15 fusion protein at a concentration of 10 μg / ml added in culture medium and three-fold dilutions of antibodies starting from 30 μg / ml and incubated for 4 hours at 37°C and 5% CO2. Then, the cells were harvested and FACS analyzed. CD14-positive monocytes in live cells were gated. Antibody-rescued CD14 expression was calculated by the following formula:

[0298]

[0299] like Fig.10 As shown in Table 6, C07, C22 and 5G12 reversed soluble S15 protein-mediated suppression of human monocytes in a dose-dependent manner, as indicated by downregulation of CD14 expression.

[0300] Table 6. Rescue of CD14 expression by C07, C22 and 5G12

[0301]

[0302] 4.2.7 Chimeric Antibodies Targeting Soluble S15 Protein-Stimulated Monocyte TNF-α Secretion

[0303] The effects of C07, C22 and 5G12 on TNFα secretion from monocytes stimulated by soluble S15 protein after LPS (lipopolysaccharide) stimulation were also studied. Briefly, human CD14+ monocytes were isolated from human (donor: A19k287098) PBMCs from healthy donors by a magnetic bead positive selection kit (Miltenyi Biotec) and seeded into 96-well plates containing soluble SIGLEC15 fusion protein at a concentration of 5 μg / ml added to the culture medium and anti-S15 mAb added at 3.3, 10 and 30 μg / ml, and inoculated overnight at 37°C, 5% CO2. 100 ng / ml LPS was added to the culture medium, and the supernatant was harvested after 6 h for TNFα concentration determination. As Fig.11 As shown in , after LPS stimulation, C07 and 5G12 inhibited soluble S15 protein-stimulated TNFα secretion, while C22 increased TNFα secretion.

[0304] 4.2.8 Chimeric antibodies reverse soluble S15 protein-mediated inhibition of PBMC T cells

[0305] The effects of C07, C22, C70 and 5G12 on reversing soluble S15-mediated PBMC T activation were investigated. Briefly, peripheral blood mononuclear cells collected from healthy human donors previously stained with CFSE were cultured overnight at 4°C in 96-well plates pre-coated with 0.1 μg / ml OKT3. 3 μg / ml S15-Fc and serial concentrations of anti-S15 mAb were added to the wells and inoculated for 72 h at 37°C, 5% CO2. Supernatants were collected at 72 h for INF-γ detection. CD4 and CD8 T cells were gated for proliferation analysis. Fig.12 A- Fig.12 As shown in F, C07, C22, and C70 rescued S15-Fc-mediated T cell suppression by increasing CD4 and CD8 T cell proliferation and increasing INF-γ secretion, which was comparable to the benchmark mAb 5G12.

[0306] 4.2.9 ADCC assay

[0307] The ADCC function of C07, C22, C70 and 5G12 on the human SIGLEC15 overexpressing cell line 293F-S15 was tested. The 293F-S15 cell line is a monoclonal cell line with SIGLEC15 overexpressed on the cell surface, generated by infecting the human fetal kidney (HEK) cell line 293F with a SIGLEC15 overexpressing lentivirus. Briefly, 1E6 / ml 293F-S15 cells in assay buffer containing a fluorescence enhancing ligand (cytotoxicity assay reagent from PerkinElmer) were incubated in a cell culture incubator for 20 min at 37°C (gently mixed at 10 min). Suspended NK92-CD16a cell line in RPMI 1640 complete medium plus 10% FBS (fetal bovine serum) was added to the assay plate at 50 μl per well as effector cells. 100 μl of labeled target cells were added to the wells. The E / T ratio was 1:2. Antibodies were diluted 10-fold from 10 μg / ml and added to the wells. After 4 h of inoculation at 37°C, 5% CO2, 25 μL / well of supernatant was transferred to a flat-bottom assay plate and 200 μL / well of europium solution was added. Fluorescence was measured in a time-resolved fluorimeter over 5 h. A maximum LDH release control was prepared by adding 10 μL of lysis buffer to the target cell wells and lysing for 10 min. Killing index was calculated and reported in Fig.13 As shown in Fig.13 As shown in , C07, C22, C70 and 5G12 mediated similar ADCC functions.

[0308] C07 and C70 showed slightly superior ADCC function.

[0309] 4.2.10 ADCP assay

[0310] C07, C22, C70 and 5G12 were tested for the ADCP function of human SIGLEC15 overexpression cell line 293F-S15. Monocytes were separated from PBMC with CD14 beads and resuspended with RPMI 1640 complete medium supplemented with 50ng / ml M-CSF, and then cultured in an incubator (37°C, 5% CO2) for 4 days. The culture medium was half-renewed with fresh RPMI 1640 complete medium supplemented with 50ng / mL M-CSF, and cultured in an incubator (37°C, 5% CO2) for another 3 days. Macrophages were digested and inoculated into 96-well flat-bottom plates at 10,000 macrophages / 100 μL / well, and then incubated overnight in an incubator as effector cells. 293F-S15 was labeled with Celltrace violet as a target cell, and then added to the well at 20,000 cells / 50 μL / well. Antibodies were added at the indicated concentrations. After incubation for 2 hours at 37°C in a 5% CO2 incubator, cells were harvested, washed and stained with APC-anti-human CD11b, followed by FACS analysis to determine the phagocytic index %. Fig.14 As shown in , C07, C22, C70, and 5G12 exhibited similar phagocytic indices at the same dose levels.

[0311] 4.2.11 Epitope analysis

[0312] Epitope identification of C07, C22, C70 and 5G12 was performed by Octet. In brief, the HIS1K sensor was hydrolyzed for 10 minutes, followed by capture of the 6xHis-tagged human SIGLEC15 ECD recombinant protein to 0.3 nm. The SIGLEC15-captured sensor was then exposed to Ab1 and Ab2 in series or directly to Ab2. The binding signals of Ab2 and SIGLEC15 under different conditions were detected respectively. Finally, the relative binding signal of Ab2 in series was calculated by normalization. A high relative binding signal indicates low competition between Ab2 and Ab1 for binding to the antigen. Similarly, a low relative association signal indicates high competition between Ab2 and Ab1. As shown in Table 7, C70 and C22 completely compete for the binding of 5G12 to SIGLEC15, while C07 shows partial competition with 5G12.

[0313] Table 7. Epitope identification of C07, C22, C70 and 5G12

[0314] AB# 5G12 C70 C22 C07 5G12 5％ 5％ 3％ 60％ C70 18％ 13％ 11％ 79％ C22 39％ 10％ 6％ 92％ C07 30％ 24％ 97％ 8％

[0315] The binding epitopes of C07, C22, C70, and 5G12 on SIGLEC15 were further mapped using hydrogen deuterium exchange mass spectrometry (HDX-MS). Fig.15As shown in, the binding of C07, C22, C70 and 5G12 results in a decrease in hydrogen-deuterium exchange in the labeled peptides located in the IgV and / or IgC2 domains of the SIGLEC15 protein. 5G12 results in a decrease in hydrogen-deuterium exchange in the peptides "HSSPAQRWSMQVPPEVSAE (SEQ ID NO: 52)" and "RAGEPYAGPQVF (SEQ ID NO: 53)" in the IgV domain and in the peptide "ALTHDGRYTC (SEQ ID NO: 54)" in the IgC2 domain. C07 results in a decrease in hydrogen-deuterium exchange in the peptides "RAGEPYAGPQVFRC (SEQ ID NO: 41)" in the IgV domain and in the peptides "PALTHPGRYTCT (SEQ ID NO: 42)" in the IgC2 domain. C22 leads to a decrease in hydrogen-deuterium exchange in the peptides "QVPPEVSA (SEQ ID NO: 43)" and "RAGEPYAGPQVF (SEQ ID NO: 44)" in the IgV domain only. C70 leads to a decrease in hydrogen-deuterium exchange in the peptides "QVPPEVSAE (SEQ ID NO: 45)" and the long peptide "RAGEPYAGPQVFRCAAARGSE (SEQ ID NO: 46)" in the IgV domain, as well as in the unique peptide "ALCTAEGEPPPAL (SEQ ID NO: 47)" in the IgC2 domain.

[0316] 4.2.12 PK curve of chimeric antibody in hS-15KI mice carrying MC38-hS15

[0317] Single dose PK curves of C07, C22 and 5G12 were tested in hS15 KI (knock-in) mice carrying MC38-hS15. On day 0, mice were intraperitoneally administered 10 mg / kg of the test article, and 60-80 μl of blood was collected from half of the mice in the group at each time point of 15 min, 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, 120 h, 168 h and 240 h, and the other half of the mice were replaced at the next time point. The blood concentration of the test article was measured by a pre-qualified ELISA assay. PK parameters were calculated by WinNonlin software. Fig.16 As shown in Figure A and Table 8, C22 and 5G12 showed similar PK profiles, while C07 showed low Cmax and AUC, indicating lower blood exposure.

[0318] Multiple dose PK profiles of C07, C22 and 5G12 were tested in hS15 KI mice carrying MC38-hS15. Mice were given 4 doses of 5 mg / kg test article QW intraperitoneally, and 60-80 μl of blood was collected at 15 min, 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, 168 h after the first dose and before administration, and 15 min, 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, 168 h, 240 h, 336 h, 408 h, 504 h, 672 h, 840 h after the last dose. Blood concentrations of the test article were measured by a pre-qualified ELISA assay. PK parameters were calculated by WinNonlin software. Fig.16 B and shown in Table 9, C22 showed the longest blood retention after 4 doses, while 5G12 and C07 showed reduced blood exposure at the last dose. The AUC ratio (AR) of the first dose to the last dose of C22 was 1.2, while the AUC ratios of 5G12 and C07 were 0.3 and 0.7, respectively.

[0319] Table 8. PK parameters of single doses of C07 and C22 in hS-15KI mice bearing MC38-hS15

[0320]

[0321] Table 9. PK parameters of multiple doses of C07 and C22 in hS-15KI mice bearing MC38-hS15

[0322]

[0323] *:AUC 0-168h / AUC 0-inf Less than 80%.T 1 / 2 For reference only.

[0324] 4.2.13 In vivo efficacy studies

[0325] Human SIGLEC15 knock-in C57 mice were inoculated with monoclonal cells overexpressing MC38-hS15. Treatment groups included vehicle (PBS), isotype control, 5 mg / kg C07, C22, C70, and 5G12. When tumors reached an average volume of 65-70 mm 3 Treatment was started at 1:10 p.m. mAb was administered intraperitoneally (ip) twice weekly for up to 6 doses. Tumor volume was measured three times weekly. Two-way ANOVA was performed to compare the mean tumor volume of the different treatment groups with that of the isotype control group. The mean tumor growth curves of the different treatment groups ( Fig.17 A) shows that C07, C22, C70 and 5G12 significantly inhibited MC38 / hS15 tumor growth. The tumor weight of each group at the end point is shown in Fig.17 In B. The C22-treated group achieved 6 / 6 CR (complete response) without tumor growth in all treated mice at the endpoint, indicating an effective anti-tumor effect. Fig.17 C shows that there were no differences in body weight among all treatment groups. Individual tumor growth curves are shown in Fig.18 In. Fig.18 As shown in , the C22 treated group achieved 6 / 6 CR and maintained CR during the study, the C07 treated group achieved 6 / 6 CR before day 35 but was 5 / 6 CR at the endpoint, the C70 treated group achieved 3 / 6 CR, and the 5G12 treated group achieved 4 / 6 CR at the endpoint. Among the mAbs tested, C22 showed the best in vivo efficacy. Tumor-free mice were re-challenged with the same tumor cell line MC38 / hS15 until all dosed animals showed plasma Ab concentrations below the BQL (below the limit of quantification) (10 ng / ml). As shown in Fig.19 As shown in A, C22 and C70 have the longest blood concentration duration compared to C07 and 5G12. 5G12 shows the shortest blood retention duration, which indicates that C22, C70, and C07 can show better PK profiles after multiple administrations compared to 5G12. Fig.19 As shown in B, no tumors grew in any tumor-free mice previously treated with C07, C22, C70 or 5G12, indicating that the antibody treatment triggered immune memory. The tumor growth curves for each group are shown in Fig. 20 middle.

[0326] Example 5. Antibody humanization

[0327] 5.1 Humanized Design

[0328] C07, C22, and C70 were humanized using complementarity determining region (CDR) grafting.

[0329] C07 humanization

[0330] Briefly, IGHV3-33*01 (IMGT allele name) and IGKV1-12*01 (IMGT allele name) were first selected as humanized templates for heavy and light chains, respectively, based on their homology to the original mouse antibody sequences. CDRs were defined using the Kabat definition, except for the heavy chain CDR1, which was defined using a combination of the Kabat and Chothia systems. For transplantation, different combinations of CDRs and typical residues from C07 were transplanted onto the template. The resulting variants were generated, and the highest forms of mAbs were designated H07.001 to H07.088, where the prefix "H" means "humanized" and the numbers in the suffix represent the serial numbers. All variants were tested in multiple in vitro assays to select the best variants that retain the properties of the parent antibody.

[0331] C22 humanization

[0332] Briefly, IGHV1-69-2*01 (IMGT allele name) and IGKV4-1*01 (IMGT allele name) were first selected as humanized templates for heavy and light chains, respectively, based on their homology to the original mouse antibody sequences. CDRs were defined using the Kabat definition, except for the heavy chain CDR1, which was defined using a combination of the Kabat and Chothia systems. For transplantation, different combinations of CDRs and typical residues from C22 were transplanted onto the template. Potential PTM (protein post-translational modification) sites in the CDR2 of VH in the parent C22 were mutated, tested and used for the highest form of mAb. The resulting variants were generated and designated as hu22.001 to hu22.080, where the prefix "H" means "humanized" and the numbers in the suffix represent the serial number. All variants were tested in multiple in vitro assays to select the best variants that retain the properties of the parent antibody.

[0333] C70 humanization

[0334] Briefly, IGHV1-18*01 (IMGT allele name) and IGKV7-3*01 (IMGT allele name) were first selected as humanized templates for heavy and light chains, respectively, based on their homology to the original mouse antibody sequences. CDRs were defined using the Kabat definition, except for the heavy chain CDR1, which was defined using a combination of the Kabat and Chothia systems. For transplantation, different combinations of CDRs and typical residues from C70 were transplanted onto the template. The potential PTM site NG in VH of the parent C70 was mutated to NA, and the DG in VL was mutated to DA, which was then tested and used for the highest form of mAb. The resulting variants were generated and designated as hu70.001 to hu70.050, where the prefix "H" means "humanized" and the numbers in the suffix represent the serial number. All variants were tested in multiple in vitro assays to select the best variants that retain the properties of the parent antibody.

[0335] 5.2 Characterization of humanized variants

[0336] 5.2.1 Affinity and Binding Activity

[0337] C07 humanized form

[0338] The binding affinity of the humanized variants derived from C07 to the human SIGLEC15 ECD protein was determined using biolayer interferometry (Octet). The association and dissociation curves were fitted with a 1:1 binding model, and the Ka / Kd / KD values ​​of each antibody were calculated and summarized in Table 10. The binding of C07 and C07-derived humanized variants to the human SIGLEC15 ECD protein was detected by ELISA. The binding of C07 and C07-derived humanized variants to cell membrane human SIGLEC15 was detected by FACS using CHOK1 / human SIGLEC15 cells. Table 10 summarizes the EC50 and highest MFI values ​​analyzed using a four-parameter nonlinear fit of GraphPad Prism 9.0. As shown in Table 10, H07.027, H07.028, and H07.029 retain the human SIGLEC15 binding activity of the parent antibody C07.

[0339] Table 10. Affinity and binding of C07 and C07-derived humanized variants to human SIGLEC15 protein and CHOK1 / SIGLEC15

[0340]

[0341] The sequences of H07.027, H07.028 and H07.029 are shown in Table 10-1. The CDRs are underlined.

[0342] Table 10-1. Sequences of H07.027, H07.028 and H07.029

[0343]

[0344] C22 humanized form

[0345] The binding affinity of C22-derived humanized variants to human SIGLEC15 ECD protein was determined using biolayer interferometry (Octet). The association and dissociation curves were fitted with a 1:1 binding model, and the Ka / Kd / KD values ​​of each antibody were calculated and summarized in Table 11. The binding of C22 and C22-derived humanized variants to human SIGLEC15 ECD protein was detected by ELISA. CHOK1 / hS15 cells were used to detect the binding of C22 and C22-derived humanized variants to cell membrane human SIGLEC15 by FACS determination. Table 11 summarizes the EC50 and maximum MFI values ​​analyzed using a four-parameter nonlinear fit of GraphPad Prism 9.0. As shown in Table 11, H22.014, H22.015, H22.016, H22.026, H22.029, H22.030 and de-risked versions thereof (G56A, the 56th amino acid glycine in VH was mutated to alanine) retained the human SIGLEC15 binding activity of the parent antibody C22.

[0346] Table 11. Affinity and binding of C22 and C22-derived humanized variants to human SIGLEC15 protein and CHOK1 / SIGLEC15

[0347]

[0348] The sequences of H22.014, H22.015, H22.016, H22.026, H22.029, H22.030 and their de-risked forms are shown in Table 11-1. The CDRs are underlined.

[0349] Table 11-1. Sequences of H22.014, H22.015, H22.016, H22.026, H22.029, H22.030 and their de-risked forms

[0350]

[0351]

[0352] The mutated amino acids are shown in bold.

[0353] C70 humanized form

[0354] The binding affinity of the humanized variants derived from C70 to the human SIGLEC15 ECD protein was determined using biolayer interferometry (Octet). The association and dissociation curves were fitted with a 1:1 binding model, and the Ka / Kd / KD values ​​of each antibody were calculated and summarized in Table 12. The binding of C70 and C70-derived humanized variants to cell membrane human SIGLEC15 was detected by FACS assay using CHOK1 / human SIGLEC15 cells. Table 12 summarizes the EC50 and maximum MFI values ​​analyzed using a four-parameter nonlinear fit of GraphPad Prism 9.0. As shown in Table 12, H70.012, H70.013, H70.014, H70.029, H70.030, H70.031, H70.032 and four de-risked forms C70 (VH-NG / QG+VL-DG / DA), C70 (VH-NG / QG+VL-DG / EG), C70 (VH-NG / NA+VL-DG / DA), C70 (VH-NG / NA+VL-DG / EG) retained the human SIGLEC15 binding activity of the parent antibody C70.

[0355] Table 12. Affinity and binding of C70 and C70-derived humanized variants to human SIGLEC15 protein and CHOK1 / SIGLEC15

[0356]

[0357]

[0358] The sequences of H70.012, H70.013, H70.014, H70.029, H70.030, H70.031, H70.032 and four de-risked versions are shown in Table 11-1. The CDRs are underlined.

[0359] Table 11-1. H70.012, H70.013, H70.014, H70.029, H70.030, H70.031, H70.032 and the sequences of the four de-risked forms

[0360]

[0361]

[0362] The mutated amino acids are shown in bold.

[0363] 5.2.2 Blocking activity

[0364] The activity of C22 and selected C22-derived human variants in blocking the interaction between SIGLEC15 and sTn and LRRC4C was assessed by competitive FACS ( Fig.21 A and Fig.21 B, refer to the method described in Example 4.2.4). Table 13 summarizes the IC50 values ​​and the highest blocking rates analyzed using a four-parameter nonlinear fitting analysis of GraphPad Prism 9.0. H22.015 (G56A), H22.016 (G56A) and H22.030 (G56A) retained the SIGLEC15 / sTn and SIGLEC15 / LRRC4C interaction blocking activity of the parent antibody C22.

[0365] Table 13. Blocking activity of C22 and C22-derived humanized variants

[0366]

[0367] 5.2.3 Functional Verification

[0368] The functionality of the selected humanized C22 and C70 variants was verified using a PBMC T cell activation assay (see methods described in Example 4.2.8). Fig. 22 A- Fig. 22 As shown in D, H22.015(G56A), H22.016(G56A) and H22.030(G56A) showed similar potency to 5G12 and the parent antibody C22. Fig.23 A- Fig. 22 As shown in D, H70.011 (NG / NA+DG / EG), H70.012 (NG / NA+DG / EG), and H70.014 (NG / NA+DG / EG) exhibited similar potency to 5G12 and the parental antibody C70.

[0369] 5.2.4 Single-dose PK and acute toxicity evaluation in cynomolgus monkeys

[0370] The purpose of this study was to determine the potential toxicity of humanized 5G12 (5G12.H1) and H22.015 (G56A) after a single intravenous (IV) infusion into cynomolgus monkeys, with an observation period of 21 days. In addition, the pharmacokinetics (PK) of humanized 5G12 and H22.015 (G56A) were determined. A male monkey was assigned to Group 1 to determine the toxicity of H22.015 (G56A), and a female monkey was assigned to Group 2 to determine the toxicity of humanized 5G12. The animals were approximately 3 to 3.5 years old, and the weight of the male monkey was 4.0 kg and the weight of the female monkey was 2.7 kg at the beginning of dosing. The first day of dosing was designated as Day 1. All animals were autopsied on Day 22. As shown in Table 14, the changes in AST, CK, and ALP were within or close to the reference range. A transient and slightly increased ALT (about 2.3 times, compared to before dosing) and TBIL (about 1.8 times, compared to before dosing) were observed in H22.015(G56A) treated animals on day 4, which was completely recovered on day 8. As shown in Table 15, a slight decrease in RBC, HGB, and HCT (compared to before dosing) was observed in both animals after dosing, which may be caused by multiple blood draws.

[0371] like Fig.24 As shown in Table 16, the PK profiles of H22.015 (G56A) and 5G12.H1 were comparable. There was a trend that H22.015 (G56A) maintained a higher blood concentration than 5G12.H1 at the same time point, indicating that H22.015 (G56A) was able to show a better PK profile.

[0372] Table 14. Serum Chemistry of Treated Animals

[0373]

[0374] Table 15. Hematology of treated animals

[0375]

[0376] Table 16. PK parameters of H22.015 (G56A) and 5G12.H1 at 3 mg / kg dose level *: AUC0-last / AUC0-inf is less than 80%. T1 / 2 is for reference only.

[0377] Although particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents may occur that are not or may not currently be foreseen by the applicant or others skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that binds to SIGLEC15, the antibody or antigen-binding fragment thereof comprising at least one heavy chain variable region (VH) and at least one light chain variable region (VL), wherein the VH comprises CDRs 1, 2, and 3, the CDR1 of the VH differs from the amino acid sequence shown in SEQ ID NO: 55, 61, or 67 by no more than 5, 4, 3, 2, or 1 amino acid; the CDR2 of the VH differs from the amino acid sequence shown in SEQ ID NO: 56, 62, or 68 by no more than 5, 4, 3, 2, or 1 amino acid; the CDR3 of the VH differs from the amino acid sequence shown in SEQ ID NO: 57, 63, or 69 by no more than 5, 4, 3, 2, or 1 amino acid; the VL comprises CDRs 1, 2, and 3, the CDR1 of the VL differs from the amino acid sequence shown in SEQ ID NO: 58, 64, or 70 by no more than 5, 4, 3, 2, or 1 amino acid; the CDR2 of the VL differs from the amino acid sequence shown in SEQ ID NO: 59, The difference between the amino acid sequence shown in NO:59, 65 or 71 does not exceed 5, 4, 3, 2 or 1 amino acids; the difference between the CDR3 of the VL and the amino acid sequence shown in SEQ ID NO:60, 66 or 72 does not exceed 5, 4, 3, 2 or 1 amino acids.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 55, 56 and 57, respectively; and the VL comprises CDRs 1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 58, 59 and 60, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises a CDR1 having the amino acid sequence shown in SEQ ID NO:61, a CDR2 having an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:62 by no more than 5, 4, 3, 2 or 1 amino acids, and a CDR3 having the amino acid sequence shown in SEQ ID NO:63; and the VL comprises CDRs 1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs:64, 65, and 66, respectively.

4. The antibody or antigen-binding fragment thereof as claimed in claim 1 or 3, wherein the CDR2 of the VH comprises a mutation at a protein post-translational modification site.

5. The antibody or antigen-binding fragment thereof of claim 1 or 3, wherein the CDR2 of the VH comprises the mutation G56A.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises a CDR1 having the amino acid sequence shown in SEQ ID NO:67, a CDR2 having an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:68 by no more than 5, 4, 3, 2 or 1 amino acids, and a CDR3 having the amino acid sequence shown in SEQ ID NO:69; and the VL comprises a CDR1 having an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:70 by no more than 5, 4, 3, 2 or 1 amino acids, a CDR2 having the amino acid sequence shown in SEQ ID NO:71, and a CDR3 having the amino acid sequence shown in SEQ ID NO:

72.

7. The antibody or antigen-binding fragment thereof of claim 1 or 6, wherein the CDR2 of the VH comprises a mutation at a protein post-translational modification site, and / or the CDR1 of the VL comprises a mutation at a protein post-translational modification site.

8. The antibody or antigen-binding fragment thereof of claim 1 or 6, wherein the CDR2 of the VH comprises the mutations N55Q and / or G56A; and / or the CDR1 of the VL comprises the mutations G33A and / or D32E.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the CDR2 of the VH comprises the mutation N55Q; and the CDR1 of the VL comprises the mutation G33A or D32E.

10. The antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein the VH is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and SEQ ID NOs: 7-25; and the VL is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and SEQ ID NOs: 26-40.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the VH comprises mutation N55Q and / or mutation G56A.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the VL comprises mutation G33A and / or mutation D32E.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof comprises the mutation G56A in VH; the antibody or antigen-binding fragment thereof comprises the mutation N55Q in VH and the mutation G33A in VL; or the antibody or antigen-binding fragment thereof comprises the mutation N55Q in VH and the mutation D32E in VL.

14. The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and SEQ ID NOs: 7-25; and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and SEQ ID NOs: 26-40.

15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 1, 2 or 3; and the VL comprises the amino acid sequence shown in SEQ ID NO: 4, 5 or 6; The VH comprises the amino acid sequence shown in SEQ ID NO: 7, 8 or 9, and the VL comprises the amino acid sequence shown in SEQ ID NO: 26; The VH comprises the amino acid sequence shown in SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and the VL comprises the amino acid sequence shown in SEQ ID NO: 27 or 28; or The VH comprises the amino acid sequence shown in SEQ ID NO:20, 21, 22, 23, 24 or 25, and the VL comprises the amino acid sequence shown in SEQ ID NO:29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 1, and the VL comprises the amino acid sequence shown in SEQ ID NO: 4; The VH comprises the amino acid sequence shown in SEQ ID NO: 2, and the VL comprises the amino acid sequence shown in SEQ ID NO: 5; The VH comprises the amino acid sequence shown in SEQ ID NO:3, and the VL comprises the amino acid sequence shown in SEQ ID NO:6; The VH comprises the amino acid sequence shown in SEQ ID NO: 7, 8 or 9, and the VL comprises the amino acid sequence shown in SEQ ID NO: 26; The VH comprises the amino acid sequence shown in SEQ ID NO: 10, 11 or 12, and the VL comprises the amino acid sequence shown in SEQ ID NO: 27; The VH comprises the amino acid sequence shown in SEQ ID NO: 13 or 14, and the VL comprises the amino acid sequence shown in SEQ ID NO: 28; The VH comprises the amino acid sequence shown in SEQ ID NO: 15, 16 or 17, and the VL comprises the amino acid sequence shown in SEQ ID NO: 27; The VH comprises the amino acid sequence shown in SEQ ID NO: 17, 18 or 19, and the VL comprises the amino acid sequence shown in SEQ ID NO: 28; The VH comprises the amino acid sequence shown in SEQ ID NO: 20, and the VL comprises the amino acid sequence shown in SEQ ID NO: 29; The VH comprises the amino acid sequence shown in SEQ ID NO: 21, and the VL comprises the amino acid sequence shown in SEQ ID NO: 30, 31, 32, 33, 34 or 35; The VH comprises the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises the amino acid sequence shown in SEQ ID NO: 36 or 37; The VH comprises the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises the amino acid sequence shown in SEQ ID NO: 36 or 37; The VH comprises the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises the amino acid sequence shown in SEQ ID NO: 38 or 39; or The VH comprises the amino acid sequence shown in SEQ ID NO:25, and the VL comprises the amino acid sequence shown in SEQ ID NO:

40.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and sTn.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and LRRC4C.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof is capable of rescuing monocyte apoptosis, optionally, soluble SIGLEC15 protein-mediated monocyte apoptosis in PBMCs.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, wherein the antibody or antigen-binding fragment thereof is capable of down-regulating CD14 expression.

21. The antibody or antigen-binding fragment thereof of any one of claims 1-20, wherein the antibody or antigen-binding fragment thereof is capable of reversing human monocyte inhibition, optionally, soluble SIGLEC15 protein-mediated human monocyte inhibition, the reversal being indicated by downregulation of CD14 expression.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment thereof is capable of inhibiting or increasing TNFα secretion, optionally, TNFα secretion stimulated by a soluble SIGLEC15 protein.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein the antibody or antigen-binding fragment thereof is capable of increasing CD4 and CD8 T cell proliferation and / or increasing INF-γ secretion.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment thereof is capable of reversing the inhibition of PBMC T cells mediated by soluble SIGLEC15 protein by increasing CD4 and CD8 T cell proliferation and / or increasing INF-γ secretion.

25. The antibody or antigen-binding fragment thereof of any one of claims 1-24, wherein the antibody or antigen-binding fragment thereof is capable of eliciting immunological memory.

26. The antibody or antigen-binding fragment thereof of any one of claims 1-25, wherein the antibody or antigen-binding fragment thereof binds to SIGLEC15 with a KD of no more than 9.9E-8M or no more than 9.9E-9M.

27. The antibody or antigen-binding fragment thereof of any one of claims 1 to 26, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and sTn, wherein IC 50 No more than 10, 8, 6, 5, 4 or 3 μg / mL; or the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and sTn, wherein IC 50 Not exceeding 50, 40, 35 or 30 nM.

28. The antibody or antigen-binding fragment thereof of any one of claims 1 to 27, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and LRRC4C, wherein IC 50 No more than 8, 6, 5, 4, 3 or 1 μg / mL; or the antibody or antigen-binding fragment thereof blocks the interaction between human SIGLEC15 and LRRC4C, wherein the IC 50 Not exceeding 10, 8, 6, 5 or 3 nM.

29. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 28, wherein the antibody or antigen-binding fragment thereof is capable of mediating ADCC function and / or ADCP function.

30. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, wherein the antibody or antigen-binding fragment thereof is capable of rescuing SIGLEC15-mediated downregulation of APC function and / or immune response of monocytes and monocyte-derived myeloid cells.

31. The antibody or antigen-binding fragment thereof of any one of claims 1-30, wherein the antibody or antigen-binding fragment thereof is capable of enhancing the immune response by optionally rescuing SIGLEC15-mediated down-regulation of APC function of monocytes and / or immune response.

32. An antibody or an antigen-binding fragment thereof that binds to SIGLEC15, wherein the antibody or the antigen-binding fragment thereof binds to an amino acid sequence selected from the group consisting of: SEQ ID NO: 41, 42, 43, 44, 45, 46 and 47, and combinations thereof.

33. The antibody or antigen-binding fragment thereof of claim 32, wherein the antibody or antigen-binding fragment thereof binds to the amino acid sequences shown in SEQ ID NOs: 41 and 42; The antibody or antigen-binding fragment thereof binds to the amino acid sequences shown in SEQ ID NOs: 43 and 44; or The antibody or antigen-binding fragment thereof binds to the amino acid sequences shown in SEQ ID NOs: 45, 46 and 47.

34. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-33.

35. An isolated vector comprising the isolated polynucleotide of claim 34.

36. A host cell comprising the isolated polynucleotide of claim 34 or the isolated vector of claim 35.

37. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 33, or the isolated polynucleotide as described in claim 34, or the isolated vector as described in claim 35, or the host cell as described in claim 36, and a pharmaceutically acceptable carrier.

38. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33, or the isolated polynucleotide according to claim 34, or the isolated vector according to claim 35, or the host cell according to claim 36, or the pharmaceutical composition according to claim 37.

39. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33, or the isolated polynucleotide according to claim 34, or the isolated vector according to claim 35, or the host cell according to claim 36, or the pharmaceutical composition according to claim 37, or the kit according to claim 38 in the preparation of a therapeutic agent for binding to SIGLEC15.

40. The use according to claim 39, wherein the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33, or the isolated polynucleotide according to claim 34, or the isolated vector according to claim 35, or the host cell according to claim 36, or the pharmaceutical composition according to claim 37, or the kit according to claim 38 is used for the preparation of a therapeutic agent for blocking the interaction between human SIGLEC15 and sTn or LRRC4C.

41. Use of an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 33, or an isolated polynucleotide as described in claim 34, or an isolated vector as described in claim 35, or a host cell as described in claim 36, or a pharmaceutical composition as described in claim 37, or a kit as described in claim 38 in the preparation of a therapeutic agent for enhancing an immune response to a condition, disorder or disease.

42. Use of an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 33, or an isolated polynucleotide as described in claim 34, or an isolated vector as described in claim 35, or a host cell as described in claim 36, or a pharmaceutical composition as described in claim 37, or a kit as described in claim 38 in the preparation of a therapeutic agent for diagnosing, preventing or treating a condition, disorder or disease.

43. A method of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-33, or an isolated polynucleotide as described in claim 34, or an isolated vector as described in claim 35, or a host cell as described in claim 36, or a pharmaceutical composition as described in claim 37, or a kit as described in claim 38.

44. The method of claim 43, wherein the antibody or antigen-binding fragment thereof of any one of claims 1-33 is capable of enhancing the immune response by optionally rescuing SIGLEC15-mediated down-regulation of monocyte APC function and immune response.

45. The method of claim 43 or 44, wherein the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33 is capable of increasing or inhibiting TNFα secretion, increasing CD4 and CD8 T cell proliferation and / or increasing INF-γ secretion.

46. ​​A method of diagnosing, preventing or treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-33, or an isolated polynucleotide as described in claim 34, or an isolated vector as described in claim 35, or a host cell as described in claim 36, or a pharmaceutical composition as described in claim 37, or a kit as described in claim 38.

47. The method of claim 42 or 46, wherein the disease, disorder or condition comprises a tumor.

48. The method of any one of claims 43-46, wherein the subject comprises a mammal.

49. The method of claim 48, wherein the subject comprises humans, mice, and cynomolgus monkeys.

50. A binding site on human SIGLEC15, wherein the binding site comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 41, 42, 43, 44, 45, 46 and 47 and combinations thereof.

51. The binding site on human SIGLEC15 as described in claim 50, wherein the binding site comprises the amino acid sequence shown in SEQ ID NO:41 and / or SEQ ID NO:42; The binding site comprises the amino acid sequence shown in SEQ ID NO:43 and / or SEQ ID NO:44; or The binding site comprises the amino acid sequence shown in SEQ ID NO:45, SEQ ID NO:46 and / or SEQ ID NO:47.

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