Antibodies specific for siglec-15 and uses thereof

Abstract

The present invention relates to antibodies specifically binding to Siglec-15, an antigen-binding fragment thereof, and uses thereof. If the antibody or antigen-binding fragment thereof of the present invention is used, it is possible to obtain an excellent anticancer effect by effectively restoring the activity of inhibited T cells and inducing the death of cancer cells.

Description

SIGLEC-15 Specific Antibody and Uses

[0001] The present invention was carried out under Project No. 1465039676 with the support of the Ministry of Health and Welfare of the Republic of Korea, the research management agency for the said project is the Korea Health Industry Development Institute, the research project name is "Infectious Disease Prevention and Treatment Technology Development Project," the research task name is "Development of Source Technology for Peptide-Fusion Antibodies for Preemptive Response and Effective Treatment of Coronavirus," the lead institution is Kookmin University, and the research period is 2023.01.01 - 2023.12.31.

[0002] This patent application claims priority to Korean Patent Application No. 10-2024-0202843 filed with the Korean Intellectual Property Office on December 31, 2024, the disclosures of said patent application are incorporated herein by reference.

[0003] The present invention relates to an antibody that specifically binds to Siglec-15, its antigen-binding fragment, and its uses.

[0004]

[0005] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0006] Recently, in the field of cancer treatment, immunotherapy, which activates the patient's immune system to attack cancer cells, has established itself as an innovative therapeutic strategy. In particular, immune checkpoint inhibitors (e.g., Keytruda, Opdivo, Tecentriq, etc.) targeting immune checkpoint proteins such as PD-1 or PD-L1 have demonstrated remarkable therapeutic effects across various cancer types. However, the objective response rate (ORR) of these existing immune checkpoint inhibitors in the overall solid tumor patient population remains at the 20–40% level depending on the cancer type, and problems persist where a significant number of patients do not respond (primary resistance) or relapse after acquiring resistance during treatment. Therefore, there is an urgent need to discover new immune checkpoint targets with mechanisms independent of or complementary to the PD-1 / PD-L1 pathway and to develop novel therapies capable of controlling them.

[0007] Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15) is attracting attention as a promising target to address these unmet medical needs. Siglec-15 is a member of the sialic acid-binding immunoglobulin-like lectin family and is known to be overexpressed primarily on tumor-associated macrophages (TAMs) and the surfaces of various cancer cells. Studies suggest that Siglec-15 helps cancer cells evade immune surveillance by interacting with inhibitory receptors (e.g., CD11b) present on T cell surfaces within the tumor microenvironment (TME), thereby inhibiting T cell proliferation and activation. Interestingly, Siglec-15 expression often tends to be mutually exclusive with PD-L1 expression, leading to expectations that it could serve as a novel therapeutic alternative for patient groups that do not respond to existing PD-1 / PD-L1 therapies.

[0008] Efforts are underway to develop antibody therapies targeting Siglec-15; however, there is still a shortage of high-efficiency antibody technologies capable of going beyond simple binding to effectively block Siglec-15’s immunosuppressive signals while simultaneously reducing Siglec-15 expression on the surface of cancer cells. In particular, antibodies with a dual mechanism—which not only potently inhibit the binding between Siglec-15 and its ligand (CD11b) but also induce the internalization of the target protein within cells after binding to reduce its density on the cell surface—are considered an ideal strategy for maximizing therapeutic efficiency. Furthermore, for efficacy evaluation prior to clinical trials, it is crucial to secure antibodies that cross-react with Siglec-15 in both humans and experimental animals (such as mice), facilitating animal model experiments.

[0009]

[0010] The inventors have made diligent research efforts to develop an antibody that specifically binds to Siglec-15. As a result, the inventors selected a novel human antibody that specifically binds to both human and mouse Siglec-15, blocks T cell inhibitory signals, and reduces Siglec-15 on the surface of cancer cells through antigen internalization, and completed the present invention by identifying its excellent anticancer efficacy through in vitro and in vivo experiments.

[0011] Accordingly, the object of the present invention is to provide an antibody that specifically binds to Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15) or an antigen-binding fragment thereof.

[0012] Another object of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or its antigen-binding fragment.

[0013] Another objective of the present invention is to provide a recombinant vector comprising the nucleic acid molecule.

[0014] Another objective of the present invention is to provide a host cell comprising the recombinant vector.

[0015] Another objective of the present invention is to provide a pharmaceutical composition for the treatment or prevention of cancer comprising the antibody or an antigen-binding fragment thereof.

[0016] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0017]

[0018] The present invention provides the inventions of 1 to 21 below.

[0019] 1. An antibody or antigen-binding fragment thereof that specifically binds to Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15), comprising the following:

[0020] (a) a heavy chain CDR1 (HCDR1) having the amino acid sequence of SEQ ID NO. 1, a heavy chain CDR2 (HCDR2) having the amino acid sequence of SEQ ID NO. 2, a heavy chain CDR3 (HCDR3) having the amino acid sequence of SEQ ID NO. 3, a light chain CDR1 (LCDR1) having the amino acid sequence of SEQ ID NO. 4, a light chain CDR2 (LCDR2) having the amino acid sequence of SEQ ID NO. 5, and a light chain CDR3 (LCDR3) having the amino acid sequence of SEQ ID NO. 6;

[0021] (b) HCDR1 having the amino acid sequence of SEQ ID NO. 9, HCDR2 having the amino acid sequence of SEQ ID NO. 10, HCDR3 having the amino acid sequence of SEQ ID NO. 11, LCDR1 having the amino acid sequence of SEQ ID NO. 12, LCDR2 having the amino acid sequence of SEQ ID NO. 13, and LCDR3 having the amino acid sequence of SEQ ID NO. 14;

[0022] (c) HCDR1 having the amino acid sequence of SEQ ID NO. 17, HCDR2 having the amino acid sequence of SEQ ID NO. 18, HCDR3 having the amino acid sequence of SEQ ID NO. 19, LCDR1 having the amino acid sequence of SEQ ID NO. 20, LCDR2 having the amino acid sequence of SEQ ID NO. 21, and LCDR3 having the amino acid sequence of SEQ ID NO. 22;

[0023] (d) HCDR1 having the amino acid sequence of SEQ ID NO. 25, HCDR2 having the amino acid sequence of SEQ ID NO. 26, HCDR3 having the amino acid sequence of SEQ ID NO. 27, LCDR1 having the amino acid sequence of SEQ ID NO. 28, LCDR2 having the amino acid sequence of SEQ ID NO. 29, and LCDR3 having the amino acid sequence of SEQ ID NO. 30;

[0024] (e) HCDR1 having the amino acid sequence of SEQ ID NO. 33, HCDR2 having the amino acid sequence of SEQ ID NO. 34, HCDR3 having the amino acid sequence of SEQ ID NO. 35, LCDR1 having the amino acid sequence of SEQ ID NO. 36, LCDR2 having the amino acid sequence of SEQ ID NO. 37, and LCDR3 having the amino acid sequence of SEQ ID NO. 38;

[0025] (f) HCDR1 having the amino acid sequence of SEQ ID NO. 41, HCDR2 having the amino acid sequence of SEQ ID NO. 42, HCDR3 having the amino acid sequence of SEQ ID NO. 43, LCDR1 having the amino acid sequence of SEQ ID NO. 44, LCDR2 having the amino acid sequence of SEQ ID NO. 45, and LCDR3 having the amino acid sequence of SEQ ID NO. 46;

[0026] (g) HCDR1 having the amino acid sequence of SEQ ID NO. 49, HCDR2 having the amino acid sequence of SEQ ID NO. 50, HCDR3 having the amino acid sequence of SEQ ID NO. 51, LCDR1 having the amino acid sequence of SEQ ID NO. 52, LCDR2 having the amino acid sequence of SEQ ID NO. 53, and LCDR3 having the amino acid sequence of SEQ ID NO. 54;

[0027] (h) HCDR1 having the amino acid sequence of SEQ ID NO. 57, HCDR2 having the amino acid sequence of SEQ ID NO. 58, HCDR3 having the amino acid sequence of SEQ ID NO. 59, LCDR1 having the amino acid sequence of SEQ ID NO. 60, LCDR2 having the amino acid sequence of SEQ ID NO. 61, and LCDR3 having the amino acid sequence of SEQ ID NO. 62; or

[0028] (i) HCDR1 having the amino acid sequence of SEQ ID NO. 65, HCDR2 having the amino acid sequence of SEQ ID NO. 66, HCDR3 having the amino acid sequence of SEQ ID NO. 67, LCDR1 having the amino acid sequence of SEQ ID NO. 68, LCDR2 having the amino acid sequence of SEQ ID NO. 69, and LCDR3 having the amino acid sequence of SEQ ID NO. 70.

[0029] 2. The antibody or its antigen-binding fragment according to 1, wherein the antibody or its antigen-binding fragment comprises the following heavy chain variable region (VH) and light chain variable region (VL):

[0030] (a) VH comprising the amino acid sequence of SEQ ID NO. 7 and VL comprising the amino acid sequence of SEQ ID NO. 8;

[0031] (b) VH containing the amino acid sequence of SEQ ID NO. 15 and VL containing the amino acid sequence of SEQ ID NO. 16;

[0032] (c) VH containing the amino acid sequence of SEQ ID NO. 23 and VL containing the amino acid sequence of SEQ ID NO. 24;

[0033] (d) VH containing the amino acid sequence of SEQ ID NO. 31 and VL containing the amino acid sequence of SEQ ID NO. 32;

[0034] (e) VH containing the amino acid sequence of SEQ ID NO. 39 and VL containing the amino acid sequence of SEQ ID NO. 40;

[0035] (f) VH containing the amino acid sequence of SEQ ID NO. 47 and VL containing the amino acid sequence of SEQ ID NO. 48;

[0036] (g) VH containing the amino acid sequence of SEQ ID NO. 55 and VL containing the amino acid sequence of SEQ ID NO. 56;

[0037] (h) VH comprising the amino acid sequence of SEQ ID NO. 63 and VL comprising the amino acid sequence of SEQ ID NO. 64; or

[0038] (i) VH containing the amino acid sequence of SEQ ID NO. 71 and VL containing the amino acid sequence of SEQ ID NO. 72.

[0039] 3. In 1 or 2, the antibody has an equilibrium dissociation constant (K) of 10 nM or less with respect to human Siglec-15 protein. D An antibody or its antigen-binding fragment having a ) value.

[0040] 4. An antibody or an antigen-binding fragment thereof, wherein, in any one of 1 to 3, the antibody or the antigen-binding fragment thereof specifically binds to an epitope consisting of the amino acid sequence represented by SEQ ID NO. 146 within the extracellular domain (ECD) of human Siglec-15.

[0041] 5. A nucleic acid molecule comprising a nucleotide sequence encoding any one of 1 to 4 antibodies or an antigen-binding fragment thereof.

[0042] 6. In 5, the nucleic acid molecule is a nucleic acid molecule that is as follows:

[0043] (a) comprising the nucleotide sequences of SEQ ID NOs 73 to 78;

[0044] (b) comprising the nucleotide sequences of SEQ ID NOs 81 to 86;

[0045] (c) comprising the nucleotide sequences of SEQ ID NOs 89 to 94;

[0046] (d) comprising the nucleotide sequences of SEQ ID NOs 97 to 102;

[0047] (e) comprising the nucleotide sequences of SEQ ID NOs 105 to 110;

[0048] (f) comprising the nucleotide sequences of SEQ ID NOs 113 to 118;

[0049] (g) comprising the nucleotide sequences of SEQ ID NOs 121 to 126;

[0050] (h) comprising the nucleotide sequences of SEQ ID NOs 129 to 134; or

[0051] (i) comprising the nucleotide sequences of SEQ ID NOs 137 to 142.

[0052] 7. In 5 or 6, the nucleic acid molecule is a nucleic acid molecule such that it is as follows:

[0053] (a) comprising the nucleotide sequences of SEQ ID NOs 79 and 80;

[0054] (b) comprising the nucleotide sequences of SEQ ID NOs 87 and 88;

[0055] (c) comprising the nucleotide sequences of SEQ ID NOs 95 and 96;

[0056] (d) comprising the nucleotide sequences of SEQ ID NOs 103 and 104;

[0057] (e) comprising the nucleotide sequences of SEQ ID NOs 111 and 112;

[0058] (f) comprising the nucleotide sequences of SEQ ID NOs 119 and 120;

[0059] (g) comprising the nucleotide sequences of SEQ ID NOs 127 and 128;

[0060] (h) comprising the nucleotide sequences of SEQ ID NOs 135 and 136; or

[0061] (i) comprising the nucleotide sequences of SEQ ID NOs 143 and 144.

[0062] 8. A recombinant vector comprising any one of the nucleic acid molecules of 5 to 7.

[0063] 9. Host cell containing the recombinant vector of 8.

[0064] 10. A pharmaceutical composition for the prevention or treatment of cancer comprising any one of 1 to 4 antibodies or an antigen-binding fragment thereof.

[0065] 11. A pharmaceutical composition for preventing or treating cancer, wherein, in 10, the cancer is a cancer expressing Siglec-15.

[0066] 12. A pharmaceutical composition for preventing or treating cancer, wherein, in 10 or 11, the cancer is selected from the group consisting of gastric cancer, rectal cancer, colon cancer, colorectal cancer, liver cancer, lung cancer, ovarian cancer, melanoma, pancreatic cancer, uterine cancer, testicular cancer, breast cancer, and combinations thereof.

[0067] 13. A pharmaceutical composition for the prevention or treatment of cancer, wherein, in any one of 10 to 12, the pharmaceutical composition additionally comprises an immune checkpoint inhibitor.

[0068] 14. A method for preventing or treating cancer, comprising the step of administering to a subject requiring treatment an antibody or an antigen-binding fragment thereof of any one of 1 to 4; or a pharmaceutical composition of any one of 10 to 13.

[0069] 15. A method for preventing or treating cancer, wherein the cancer in 14 is a cancer expressing Siglec-15.

[0070] 16. A method for preventing or treating cancer, wherein, in 14 or 15, the cancer is selected from the group consisting of gastric cancer, rectal cancer, colon cancer, colorectal cancer, liver cancer, lung cancer, ovarian cancer, melanoma, pancreatic cancer, uterine cancer, testicular cancer, breast cancer, and combinations thereof.

[0071] 17. A method for preventing or treating cancer, wherein, in any one of 14 to 16, the pharmaceutical composition additionally comprises an immune checkpoint inhibitor.

[0072] 18. Use in the manufacture of a preparation for the prevention or treatment of cancer of any one of 1 to 4 antibodies or antigen-binding fragments thereof.

[0073] 19. In 18, the above cancer is a cancer expressing Siglec-15.

[0074] 20. Use in which, in 18 or 19, the cancer is selected from the group consisting of gastric cancer, rectal cancer, colon cancer, colorectal cancer, liver cancer, lung cancer, ovarian cancer, melanoma, pancreatic cancer, uterine cancer, testicular cancer, breast cancer and combinations thereof.

[0075] 21. Use in any one of 18 to 20, wherein the pharmaceutical composition additionally comprises an immune checkpoint inhibitor.

[0076]

[0077] According to one aspect of the present invention, the present invention provides an antibody that specifically binds to Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15) or an antigen-binding fragment thereof comprising:

[0078] (a) a heavy chain CDR1 (HCDR1) having the amino acid sequence of SEQ ID NO. 1, a heavy chain CDR2 (HCDR2) having the amino acid sequence of SEQ ID NO. 2, a heavy chain CDR3 (HCDR3) having the amino acid sequence of SEQ ID NO. 3, a light chain CDR1 (LCDR1) having the amino acid sequence of SEQ ID NO. 4, a light chain CDR2 (LCDR2) having the amino acid sequence of SEQ ID NO. 5, and a light chain CDR3 (LCDR3) having the amino acid sequence of SEQ ID NO. 6;

[0079] (b) HCDR1 having the amino acid sequence of SEQ ID NO. 9, HCDR2 having the amino acid sequence of SEQ ID NO. 10, HCDR3 having the amino acid sequence of SEQ ID NO. 11, LCDR1 having the amino acid sequence of SEQ ID NO. 12, LCDR2 having the amino acid sequence of SEQ ID NO. 13, and LCDR3 having the amino acid sequence of SEQ ID NO. 14;

[0080] (c) HCDR1 having the amino acid sequence of SEQ ID NO. 17, HCDR2 having the amino acid sequence of SEQ ID NO. 18, HCDR3 having the amino acid sequence of SEQ ID NO. 19, LCDR1 having the amino acid sequence of SEQ ID NO. 20, LCDR2 having the amino acid sequence of SEQ ID NO. 21, and LCDR3 having the amino acid sequence of SEQ ID NO. 22;

[0081] (d) HCDR1 having the amino acid sequence of SEQ ID NO. 25, HCDR2 having the amino acid sequence of SEQ ID NO. 26, HCDR3 having the amino acid sequence of SEQ ID NO. 27, LCDR1 having the amino acid sequence of SEQ ID NO. 28, LCDR2 having the amino acid sequence of SEQ ID NO. 29, and LCDR3 having the amino acid sequence of SEQ ID NO. 30;

[0082] (e) HCDR1 having the amino acid sequence of SEQ ID NO. 33, HCDR2 having the amino acid sequence of SEQ ID NO. 34, HCDR3 having the amino acid sequence of SEQ ID NO. 35, LCDR1 having the amino acid sequence of SEQ ID NO. 36, LCDR2 having the amino acid sequence of SEQ ID NO. 37, and LCDR3 having the amino acid sequence of SEQ ID NO. 38;

[0083] (f) HCDR1 having the amino acid sequence of SEQ ID NO. 41, HCDR2 having the amino acid sequence of SEQ ID NO. 42, HCDR3 having the amino acid sequence of SEQ ID NO. 43, LCDR1 having the amino acid sequence of SEQ ID NO. 44, LCDR2 having the amino acid sequence of SEQ ID NO. 45, and LCDR3 having the amino acid sequence of SEQ ID NO. 46;

[0084] (g) HCDR1 having the amino acid sequence of SEQ ID NO. 49, HCDR2 having the amino acid sequence of SEQ ID NO. 50, HCDR3 having the amino acid sequence of SEQ ID NO. 51, LCDR1 having the amino acid sequence of SEQ ID NO. 52, LCDR2 having the amino acid sequence of SEQ ID NO. 53, and LCDR3 having the amino acid sequence of SEQ ID NO. 54;

[0085] (h) HCDR1 having the amino acid sequence of SEQ ID NO. 57, HCDR2 having the amino acid sequence of SEQ ID NO. 58, HCDR3 having the amino acid sequence of SEQ ID NO. 59, LCDR1 having the amino acid sequence of SEQ ID NO. 60, LCDR2 having the amino acid sequence of SEQ ID NO. 61, and LCDR3 having the amino acid sequence of SEQ ID NO. 62; or

[0086] (i) HCDR1 having the amino acid sequence of SEQ ID NO. 65, HCDR2 having the amino acid sequence of SEQ ID NO. 66, HCDR3 having the amino acid sequence of SEQ ID NO. 67, LCDR1 having the amino acid sequence of SEQ ID NO. 68, LCDR2 having the amino acid sequence of SEQ ID NO. 69, and LCDR3 having the amino acid sequence of SEQ ID NO. 70.

[0087] The inventors have made diligent research efforts to develop an antibody that specifically binds to Siglec-15. As a result, the inventors selected a novel human antibody that specifically binds to both human and mouse Siglec-15, blocks T cell inhibitory signals, and reduces Siglec-15 on the surface of cancer cells through antigen internalization, and elucidated its excellent anticancer efficacy through in vitro and in vivo experiments.

[0088] Where an embodiment is provided in which the term "comprising" is used in this specification, an embodiment "consisting essentially of" or "consisting of" is also provided.

[0089] In this specification, the term "Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15)" refers to a type I transmembrane protein belonging to the immunoglobulin-like lectin family that binds to sialic acid, characterized by being overexpressed primarily on the surface of tumor-associated macrophages (TAMs) and various cancer cells. Siglec-15 acts as an important immunosuppressive regulator within the tumor microenvironment (TME). Specifically, it plays a role in inducing cancer cells to evade the immune surveillance system by interacting with inhibitory receptors such as CD11b present on the surface of T cells to inhibit T cell proliferation and cytokine production. This immunosuppressive mechanism of Siglec-15 is known to act independently or complementarily with the existing PD-1 / PD-L1 pathway, and is therefore considered a novel target protein for immune checkpoint inhibitors.

[0090] In this specification, the term "antibody" includes not only the complete antibody form but also the antigen-binding fragment of the antibody molecule.

[0091] A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, each light chain being connected to a heavy chain by a disulfide bond.

[0092] The heavy chain invariant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain invariant region has kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

[0093] In this specification, the term "antigen-binding fragment" refers to a fragment possessing an antigen-binding function and includes Fab, F(ab'), F(ab')2, chemically linked F(ab')2 and Fv, etc. Among the antibody fragments, Fab has a structure having a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Recombinant technology for generating Fv fragments as minimal antibody fragments having only a heavy chain variable region and a light chain variable region is disclosed in PCT international published patent applications WO 88 / 10649, WO 88 / 106630, WO 88 / 07085, WO 88 / 07086 and WO 88 / 09344. Two-chain Fv has a heavy chain variable region and a light chain variable region connected by non-covalent bonds, while single-chain Fv generally has a heavy chain variable region and a single chain variable region connected through a peptide linker or directly connected at the C-terminus, so they can form a dimer-like structure similar to two-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, limiting the whole antibody with papain yields Fab, and limiting it with pepsin yields F(ab')2 fragment), or they can be produced through genetic recombination technology.

[0094] In the present invention, the antibody is specifically in the scFv form or in the complete antibody form. Additionally, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). The light chain constant region may be of the kappa or lambda form.

[0095] In this specification, the term “heavy chain” refers to both the full-length heavy chain and fragments thereof, comprising a variable region domain VH and three constant region domains CH1, CH2, and CH3, which have an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. Additionally, in this specification, the term “light chain” refers to both the full-length light chain and fragments thereof, comprising a variable region domain VL and a constant region domain CL, which have an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen.

[0096] In this specification, the term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chains (CDR-H1, CDR-H2, and CDR-H3) and light chains (CDR-L1, CDR-L2, and CDR-L3) each contain three CDRs. The CDRs provide major contact residues for the antibody to bind to an antigen or epitope.

[0097] In this specification, the antibody or its antigen-binding fragment includes not only full-length or original polyclonal or monoclonal antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fab3, Fv and variants thereof), fusion proteins comprising one or more antibody portions, human antibodies, humanized antibodies, chimeric antibodies, minibodies, diabodies, tribodies, tetrabodies, linear antibodies, monochain antibodies (scFv), scFv-Fc, bispecific antibodies, multispecific antibodies, antibody-drug conjugates, CAR-T, CAR-NK, antibody-targeting viral gene delivery vehicles (e.g., adenoviruses, adeno-associated viruses, lentiviruses, retroviruses), and other modified arrangements of immunoglobulin molecules comprising an antigen recognition site of the required specificity, such as glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Specific examples of modified antibodies and their antigen-binding fragments include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engagers), TandAbs (tandem diabodies), DAFs (dual acting Fabs), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), duobodies, and triomAbs. The list of such antibodies and their antigen-binding fragments is not limited to the above.

[0098] In this specification, the terms "Framework" or "FR" refer to variable domain residues other than hypervariable region (HVR) residues. The FR of the variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4.

[0099] Therefore, HVR and FR sequences generally appear in the following order in VH (or VL / Vk):

[0100] (a) FRH1 (Framework region 1 of Heavy chain)-CDRH1 (complementarity determining region 1 of Heavy chain)-FRH2-CDRH2-FRH3-CDRH3-FRH4; and

[0101] (b) FRL1 (Framework region 1 of Light chain)-CDRL1 (complementarity determining region 1 of Light chain)-FRL2-CDRL2-FRL3-CDRL3-FRL4.

[0102] In this specification, the terms “variable region” or “variable domain” refer to a domain of the antibody heavy chain or light chain associated with binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain includes four conserved framework regions (FR) and three hypervariable regions (HVR) (Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Additionally, an antibody binding to a specific antigen may be isolated using the VH or VL domain from an antibody that screens a library of VL or VH domains that are complementary to the antigen.

[0103] In this specification, the term "specifically binds" or similar means that an antibody or its antigen-binding fragment, or other components such as scFvs, form a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 x 10⁻⁶ -6 M or less (e.g., 9 x 10 -7 M, 8 x 10 -7 M, 7 x 10 -7 M, 6 x 10 -7 M, 5 x 10 -7 M, 4 x 10 -7 M, 3 x 10 -7 M, 2 x 10 -7 M, or 1 x 10 -7 M), preferably 1 x 10 -7 M or less (e.g., 9 x 10 -8 M, 8 x 10 -8 M, 7 x 10 -8 M, 6 x 10-8 M, 5 x 10 -8 M, 4 x 10 -8 M, 3 x 10 -8 M, 2 x 10 -8 M, or 1 x 10 -8 M), more preferably 1 x 10 -8 M or less (e.g., 9 x 10 -9 M, 8 x 10 -9 M, 7 x 10 -9 M, 6 x 10 -9 M, 5 x 10 -9 M, 4 x 10 -9 M, 3 x 10 -9 M, 2 x 10 -9 M, or 1 x 10 -9 The equilibrium dissociation constant of M) (e.g., K smaller than this). D It can be characterized as (indicating a tighter bond). Methods for determining whether two molecules specifically bind are well known in the industry, including, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0104] In this specification, the term “affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., antibody) and its binding partner (e.g., antigen). Unless otherwise specified, as used herein, “binding affinity” refers to the intrinsic binding affinity reflecting the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule Y and its partner Y can generally be expressed by a dissociation constant (Kd). Affinity may be measured by conventional methods known in the art, including those described herein.

[0105] Additionally, as specified herein, the term “human antibody” has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody excludes humanized antibodies containing non-human antigen-binding residues.

[0106] In this specification, the term “chimeric” antibody means an antibody in which part of the heavy chain and / or light chain is derived from a specific source or species, and the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0107] In this specification, the term “humanized antibody” refers to a chimeric immunoglobulin containing a minimal sequence derived from a non-human (e.g., mouse) antibody, its immunoglobulin chain, or fragment (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody). In most cases, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the recipient’s complementarity-determining region (CDR) are replaced by residues of the CDR of a non-human species (donor antibody), e.g., mouse, rat, or rabbit, having the desired specificity, affinity, and ability. In some cases, residues of the Fv framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. Additionally, the humanized antibody may contain residues not found in the recipient antibody or in the imported CDR or framework sequence. Such modifications are made to further improve and optimize antibody performance. Generally, the humanized antibody will comprise at least one, and typically two, substantially all variable domains, wherein all or substantially all of the CDR region in the domain corresponds to the CDR region of a non-human immunoglobulin, and all or substantially all of the FR region has the sequence of the FR region of a human immunoglobulin. The humanized antibody comprises at least a portion of the immunoglobulin constant region (Fc region) or a substantial sequence of the human immunoglobulin constant region (Fc region).

[0108] The above variants are said to have "substantial similarity," meaning that when two peptide sequences are optimally aligned, such as by a program GAP or BESTFIT using default gap weights, they share at least about 90% sequence identity, more preferably at least about 95%, 98%, or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. "Conservative amino acid substitution" is when an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functionality of the protein. Where two or more amino acid sequences differ from each other due to conservative substitutions, the percentage or degree of similarity may be upregulated to compensate for the conservative nature of the substitution.

[0109] These amino acid variations are based on the relative similarities of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0110] In introducing mutations, the hydropathic index of the amino acid may be considered. Each amino acid is assigned a hydropathic index based on hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0111] The hydrophobic amino acid index is very important in conferring interactive biological functions of proteins. It is a known fact that similar biological activity can be achieved by substituting with amino acids having similar hydrophobic indices. When introducing a variation based on the hydrophobic index, the substitution is preferably made between amino acids exhibiting a difference in hydrophobic index within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0112] Meanwhile, it is also well known that substitution between amino acids having similar hydrophilicity values ​​results in proteins having uniform biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspalate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); Phenylalanine (-2.5); tryptophan (-3.4). When introducing a variation based on hydrophilicity values, substitution is made between amino acids that exhibit a difference in hydrophilicity values ​​within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0113] Amino acid exchanges in proteins that do not change the overall activity of the molecule are known in the art (H. Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0114] The antibody or its antigen-binding fragment of the present invention comprises an antibody or its antigen-binding fragment comprising a slight change with respect to the amino acid sequence described above, that is, a modification that has little effect on the tertiary structure and the function of the antibody. Accordingly, in some embodiments, even if it does not match the sequence described above, it may have at least 100%, 93%, 95%, 96%, 97%, or 98% or more similarity.

[0115] In one embodiment of the present invention, the antibody or its antigen-binding fragment comprises the following heavy chain variable region (VH) and light chain variable region (VL):

[0116] (a) VH comprising the amino acid sequence of SEQ ID NO. 7 and VL comprising the amino acid sequence of SEQ ID NO. 8;

[0117] (b) VH containing the amino acid sequence of SEQ ID NO. 15 and VL containing the amino acid sequence of SEQ ID NO. 16;

[0118] (c) VH containing the amino acid sequence of SEQ ID NO. 23 and VL containing the amino acid sequence of SEQ ID NO. 24;

[0119] (d) VH containing the amino acid sequence of SEQ ID NO. 31 and VL containing the amino acid sequence of SEQ ID NO. 32;

[0120] (e) VH containing the amino acid sequence of SEQ ID NO. 39 and VL containing the amino acid sequence of SEQ ID NO. 40;

[0121] (f) VH containing the amino acid sequence of SEQ ID NO. 47 and VL containing the amino acid sequence of SEQ ID NO. 48;

[0122] (g) VH containing the amino acid sequence of SEQ ID NO. 55 and VL containing the amino acid sequence of SEQ ID NO. 56;

[0123] (h) VH comprising the amino acid sequence of SEQ ID NO. 63 and VL comprising the amino acid sequence of SEQ ID NO. 64; or

[0124] (i) VH containing the amino acid sequence of SEQ ID NO. 71 and VL containing the amino acid sequence of SEQ ID NO. 72.

[0125] In one embodiment of the present invention, the antibody or its antigen-binding fragment is cross-reactive with human Siglec-15 and mouse Siglec-15.

[0126] In one embodiment of the present invention, the antibody has an equilibrium dissociation constant (K) of 10 nM or less with respect to human Siglec-15 protein. D It has a value.

[0127] In the present invention, the "binding affinity" of an antibody is a very important indicator that determines the efficacy of the antibody. Binding affinity is the equilibrium dissociation constant (K D It can be quantified as a value, which is a numerical value indicating how easily the antibody and antigen are separated (dissociated) from the bound state (antibody-antigen complex).

[0128] K D The value is the association rate constant (K on ) and dissociation rate constant (K off The ratio of )(K off / K on It is calculated as ). Therefore, K D A low value indicates that the antibody detaches slowly from the antigen (low K off ) or that it binds rapidly to antigens (high K on It means that the antibody binds to the antigen more strongly and stably as a result.

[0129] In the present invention, K is used with Surface Plasmon Resonance (SPR).D The values ​​were measured. Specifically, after immobilizing antigens (rhSiglec-15 or rmSiglec-15) on the surface of the CM5 sensor chip, antibodies were flowed at various concentrations, and the binding and dissociation processes were monitored in real time. Through this, the binding rate constant (K on ) and dissociation rate constant (K off Directly measure ), and based on this, the equilibrium dissociation constant (K D The value of ) was calculated precisely. However, K D The method of measuring the value is not limited to this, and K may be measured using any technique available to a person skilled in the art, such as bio-layer interferometry (BLI) or enzyme-linked immunosorbent assay (ELISA). D The value can be measured.

[0130] In therapeutic antibodies, a low K of 10 nM or less, preferably 1 nM or less D The value holds very significant meaning. K D A value of 10 nM or less means that the antibody binds to the target antigen (Siglec-15 in the present invention) with very high affinity, so that it does not easily detach in the body and can remain in the target cells for a long time. This results in effects such as improved therapeutic efficacy and a low dosage.

[0131] Therefore, the present invention provides an antibody having a strong binding affinity of 10 nM or less for Siglec-15 protein, thereby allowing for a superior therapeutic effect compared to existing antibodies.

[0132] In one embodiment of the present invention, the equilibrium dissociation constant (K D The lower bound of the ) value is not specifically limited. K DThis is because the value is an indicator that the lower the value, the stronger the binding affinity between the antibody and the antigen; therefore, any low value below the claimed upper limit (10 nM) is more advantageous for achieving the superior therapeutic efficacy intended by the present invention. A person with ordinary knowledge in the relevant technical field (a person skilled in the art) would clearly understand this technical significance.

[0133] The equilibrium dissociation constant of the antibody or its antigen-binding fragment for the Siglec-15 protein may be, for example, 0.1 nM to 10 nM, 0.1 nM to 8 nM, 0.1 nM to 6 nM, 0.1 nM to 5 nM, 0.1 nM to 4 nM, 0.1 nM to 3 nM, 0.1 nM to 2 nM, 0.1 nM to 1 nM, 0.5 nM to 10 nM, 1 nM to 10 nM, 3 nM to 10 nM, 5 nM to 10 nM, 8 nM to 10 nM, 0.1 nM to 5 nM, or 1 nM to 5 nM, but is not limited thereto.

[0134] In one embodiment of the present invention, the antibody or its antigen-binding fragment specifically binds to an epitope consisting of an amino acid sequence represented by SEQ ID NO. 146 within the extracellular domain (ECD) of human Siglec-15.

[0135] Specifically, the epitope is an amino acid sequence represented by SEQ ID NO. 146, that is, a region consisting of residues from the 70th histidine (His70) to the 79th alanine (Ala79) of human Siglec-15. The inventors confirmed through epitope mapping using an overlapping peptide library that the antibody (K110.2) of the present invention binds most strongly to a fragment containing the corresponding sequence, and demonstrated through competitive ELISA analysis that the peptide effectively inhibits the binding between the antibody and full-length Siglec-15.

[0136] The epitope region of SEQ ID NO. 146 is a site that performs a critical function in the interaction of Siglec-15 with CD11b, an immunosuppressive receptor. Protein-protein inhibition analysis revealed that when the antibody of the present invention is pre-incubated with the corresponding epitope peptide to saturate the binding site, the antibody's ability to block the Siglec-15-CD11b interaction is significantly reduced. This suggests that the antibody of the present invention possesses a mechanism of action in which it binds to the corresponding epitope, thereby inducing steric hindrance and directly interfering with the ligand binding of Siglec-15, and consequently blocking T cell inhibitory signals. Furthermore, molecular docking simulation results revealed that the complementarity determining site (CDR) of the antibody of the present invention, particularly the CDR-L1, CDR-L3, and CDR-H3 loops, forms a structurally stable complex by forming extensive interactions with the epitope residues.

[0137] In one aspect of the present invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antigen-binding fragment thereof.

[0138] In this specification, the term “nucleic acid molecule” has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0139] It is obvious to those skilled in the art that the nucleotide sequence encoding the antibody or its antigen-binding fragment of the present invention is sufficient to be a nucleotide sequence encoding an amino acid sequence constituting the antibody or its antigen-binding fragment, and is not limited to any specific nucleotide sequence.

[0140] This is because even if a mutation occurs in the nucleotide sequence, there are cases where expressing the mutated nucleotide sequence as a protein does not lead to a change in the protein sequence. This is called codon degeneracy. Therefore, the above nucleotide sequence includes a nucleotide sequence containing functionally equivalent codons or codons coding for the same amino acid (for example, due to codon degeneracy, there are six codons for arginine or serine), or codons coding for biologically equivalent amino acids.

[0141] Considering the variant having the aforementioned biological equivalence activity, the nucleic acid molecule of the present invention encoding the amino acid sequence constituting the antibody or its antigen-binding fragment is interpreted to also include a sequence exhibiting substantial identity therewith. The above substantial identity is, when the sequence of the present invention described above is aligned with any other sequence to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art, at least 60% homology (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), more specifically 70% homology (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%), even more specifically 80% homology (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), and even more specifically 90% homology (e.g. It means a sequence exhibiting 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), most specifically, 95% or more homology (e.g., 95%, 96%, 97%, 98%, or 99%). All integers between 60% and 100% and decimals existing in between are included within the scope of the present invention with respect to % homology.

[0142] Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are cited in Smith and Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. Mol. Bio. 48:443(1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins and Sharp, CABIOS 5:151-3(1989); Corpet et al., Nuc. Acids Res. 16:10881-90(1988); Huang et al., Comp. Appl. BioSci. 8:155-65(1992) and Pearson et al., Meth. Mol. Biol. It is disclosed in 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) is accessible from sources such as the National Center for Biological Information (NBCI) and can be used online in conjunction with sequencing analysis programs such as blastp, blastn, blastx, tblastn, and tblastx. BLAST can be accessed through the BLAST page on the NCBI website. Methods for comparing sequence homology using this program can be found on the BLAST help page of the NCBI website.

[0143] In one embodiment of the present invention, the nucleic acid molecule is as follows:

[0144] (a) comprising the nucleotide sequences of SEQ ID NOs 73 to 78;

[0145] (b) comprising the nucleotide sequences of SEQ ID NOs 81 to 86;

[0146] (c) comprising the nucleotide sequences of SEQ ID NOs 89 to 94;

[0147] (d) comprising the nucleotide sequences of SEQ ID NOs 97 to 102;

[0148] (e) comprising the nucleotide sequences of SEQ ID NOs 105 to 110;

[0149] (f) comprising the nucleotide sequences of SEQ ID NOs 113 to 118;

[0150] (g) comprising the nucleotide sequences of SEQ ID NOs 121 to 126;

[0151] (h) comprising the nucleotide sequences of SEQ ID NOs 129 to 134; or

[0152] (i) comprising the nucleotide sequences of SEQ ID NOs 137 to 142.

[0153] The nucleotide sequences included in (a) to (i) above each represent sequences encoding the CDR of antibodies K110.2, K110.1, and K110.3 to K110.9.

[0154] In one embodiment of the present invention, the nucleic acid molecule is as follows:

[0155] (a) comprising the nucleotide sequences of SEQ ID NOs 79 and 80;

[0156] (b) comprising the nucleotide sequences of SEQ ID NOs 87 and 88;

[0157] (c) comprising the nucleotide sequences of SEQ ID NOs 95 and 96;

[0158] (d) comprising the nucleotide sequences of SEQ ID NOs 103 and 104;

[0159] (e) comprising the nucleotide sequences of SEQ ID NOs 111 and 112;

[0160] (f) comprising the nucleotide sequences of SEQ ID NOs 119 and 120;

[0161] (g) comprising the nucleotide sequences of SEQ ID NOs 127 and 128;

[0162] (h) comprising the nucleotide sequences of SEQ ID NOs 135 and 136; or

[0163] (i) comprising the nucleotide sequences of SEQ ID NOs 143 and 144.

[0164] The nucleotide sequences included in (a) to (i) above each represent sequences encoding the heavy chain variable region and light chain variable region of antibodies K110.2, K110.1, and K110.3 to K110.9.

[0165] In one aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule.

[0166] In this specification, the term "vector" includes, but is not limited to, plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors as means for expressing a target gene in a host cell.

[0167] According to one embodiment of the present invention, in the vector of the present invention, the nucleic acid molecule is operatively linked with a promoter.

[0168] In this specification, the term “operatively linked” means a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, thereby allowing the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0169] The recombinant vector system of the present invention can be constructed through various methods known in the art, specific methods for which are disclosed in Sambrook et al., *Molecular Cloning*, *A Laboratory Manual*, *Cold Spring Harbor Laboratory Press* (2001), which is incorporated herein by reference.

[0170] The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. Additionally, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0171] For example, when the vector of the present invention is an expression vector and the host is a eukaryotic cell, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, beta-actin promoter, human hemoglobin promoter and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter and Rhoese's sarcoma virus (RSV) promoter) may be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0172] The vector of the present invention may be fused with other sequences to facilitate the purification of antibodies expressed therefrom. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His(hexahistidine; Quiagen, USA).

[0173] Meanwhile, the expression vector of the present invention includes antibiotic resistance genes commonly used in the art as selection markers, such as resistance genes for ampicillin, gentamicin, cabbageillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0174] Optionally, the vector may additionally carry a gene encoding a reporter molecule (e.g., luciferase and γ-glucuronidase).

[0175] According to one embodiment of the present invention, the expression vector is a recombinant vector for host cell expression that includes a nucleic acid molecule encoded in an antibody or an antigen-binding fragment thereof, a promoter that is operatively linked to the nucleotide sequence of the nucleic acid molecule and forms an RNA molecule in a host cell, and a poly A signal sequence that acts in a host cell to cause polyadenylation of the 3'-terminus of the RNA molecule.

[0176]

[0177] In one aspect of the present invention, the present invention provides a host cell comprising the recombinant vector.

[0178] The above host cell is transformed by the recombinant vector described above.

[0179] Any host cell known in the art that can stably and continuously clone and express the vector of the present invention may be used, for example, suitable eukaryotic host cells for the vector include, but are not limited to, yeast (Saccharomyce cerevisiae), insect cells, monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line, HuT 78 cells, and HEK-293 cells.

[0180] In this specification, the terms "transformed," "transduced," or "transfected" refer to the process in which exogenous nucleic acids are delivered or introduced into a host cell. "Transformed," "transduced," or "transfected" cells are cells that have been transformed, introduced, or transfected with exogenous nucleic acids, and said cells include said cells and progeny cells resulting from their passage.

[0181] A method for delivering the vector of the present invention into a host cell, when the host cell is a eukaryotic cell, involves delivering the vector into the host cell by microinjection (Capecchi, MR, Cell, 22:479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology, 52:456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)), etc. It can be injected.

[0182] In the present invention, the recombinant vector injected into a host cell can express the above-mentioned recombinant protein complex within the host cell, and in such cases, a large amount of protein complex is obtained. For example, if the expression vector includes a lac promoter, gene expression can be induced by treating the host cell with IPTG.

[0183] The above culture is typically carried out under aerobic conditions, such as by shaking culture or rotation using a rotary machine. The culture temperature is preferably carried out in the range of 10 to 40°C, and the culture time is generally carried out for 5 hours to 7 days. The pH of the medium is preferably maintained in the range of 3.0 to 9.0 during culture. The pH of the medium can be adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc. During culture, if necessary, antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline may be added for the maintenance and expression of the recombinant vector. When culturing host cells transformed with a recombinant expression vector having an induction-capable promoter, an inducer suitable for the medium may be added if necessary. For example, if the expression vector contains a lac promoter, IPTG (isopropyl-beta-D-thiogalactopyranoside) can be added, and if it contains a trp promoter, indoleacrylic acid can be added to the medium.

[0184]

[0185] In one aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the antibody or an antigen-binding fragment thereof.

[0186] Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0187] The pharmaceutical composition of the present invention may be administered orally or parenterally, for example by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, local administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0188] Suitable dosages of the pharmaceutical composition of the present invention vary depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, diet, time of administration, route of administration, rate of excretion, and responsiveness, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg. As specified herein, the term "pharmaceutical effective dose" means an amount sufficient to prevent or treat the aforementioned disease.

[0189] In this specification, the term "prevention" means the prevention or protective treatment of a disease or diseased state. In this specification, the term "treatment" means the reduction, suppression, soothing, or eradication of a diseased state.

[0190] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0191] In one embodiment of the present invention, the cancer is a cancer that expresses Siglec-15.

[0192] In this specification, the term “cancer” is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread locally or to other parts of the body through the bloodstream and lymphatic system. The cancer includes solid tumors and non-solid tumors (e.g., hematogenous tumors).

[0193] In this specification, the term "tumor" refers to an abnormal growth of benign, pre-cancerous, malignant, or metastatic tissue.

[0194] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or fluid regions. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells forming the tumor (e.g., sarcoma, carcinoma, and lymphoma). Solid tumors, for example, examples of sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, and sweat gland carcinoma. carcinoma), medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma,Bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (e.g., glioma (e.g., brainstem glioma and mixed gliomas)), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma, craniopharyngoma, ependymoma, pinealoma, Includes hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases.

[0195] A hematological tumor or blood cancer is cancer of the blood or bone marrow. Examples of blood (or hematopoietic) cancers include leukemias, e.g., acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic myelocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, and non-Hodgkin's disease. Includes lymphoma (non-Hodgkin's lymphoma) (delayed and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0196] Since the pharmaceutical composition of the present invention utilizes the antibody or antigen-binding fragment thereof described above as an active ingredient, the details common to both are omitted to avoid excessive complexity in this specification.

[0197] In one embodiment of the present invention, the cancer is selected from the group consisting of gastric cancer, rectal cancer, colon cancer, colorectal cancer, liver cancer, lung cancer, ovarian cancer, melanoma, pancreatic cancer, uterine cancer, testicular cancer, breast cancer, and combinations thereof.

[0198] In one embodiment of the present invention, the pharmaceutical composition additionally comprises an immune checkpoint inhibitor.

[0199] In this specification, the term "immune checkpoint inhibitor" collectively refers to a substance that restores the activity of immune cells, such as T cells, and enhances the anti-cancer immune response by antagonistically blocking inhibitory signaling pathways (immune checkpoints) that cancer cells utilize to evade attacks by the immune system. Since the antibody of the present invention blocks the Siglec-15 pathway, complementary or synergistic effects can be expected when administered in combination with immune checkpoint inhibitors of other mechanisms.

[0200] Immun checkpoint inhibitors that can be used in combination may be classified according to the target receptor or ligand, and include, but are not limited to, the following examples:

[0201] (1) PD-1 (Programmed cell death protein 1) inhibitors: Pembrolizumab, Nivolumab, Cemiplimab, Camrelizumab, Sintilimab, etc.;

[0202] (2) PD-L1 (Programmed death-ligand 1) inhibitors: Atezolizumab, Avelumab, Durvalumab, Envafolimab, etc.;

[0203] (3) CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4) inhibitors: Ipilimumab, Tremelimumab, etc.;

[0204] (4) Other novel immune checkpoint target inhibitors: including Relatlimab, a lymphocyte activation gene-3 (LAG-3) inhibitor, TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) inhibitor, TIGIT (T cell immunoreceptor with Ig and ITIM domains) inhibitor, VISTA (V-domain Ig suppressor of T cell activation) inhibitor, BTLA (B- and T-lymphocyte attenuator) inhibitor, etc. These immune checkpoint inhibitors may be in the form of antibodies, as well as peptides, small molecules, or nucleic acid molecules (e.g., siRNA, mRNA), and may be administered simultaneously, sequentially, or as separate formulations with the antibody of the present invention.

[0205]

[0206] In one aspect of the present invention, the present invention provides a method for preventing or treating cancer comprising the step of administering to a subject requiring treatment an antibody or antigen-binding fragment thereof that specifically binds to Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15); or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that specifically binds to Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15).

[0207] The above-described preventive or therapeutic method has commonalities with the aforementioned antibody or antigen-binding fragment thereof; or pharmaceutical composition in that it comprises an antibody that specifically binds to Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15) or an antigen-binding fragment thereof, and to prevent excessive duplication of descriptions in the specification, the description of duplicated matters is omitted.

[0208] In one aspect of the present invention, the present invention provides a use in the manufacture of a cancer prevention or treatment agent of an antibody or an antigen-binding fragment thereof that specifically binds to Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15).

[0209] The above-mentioned preventive or therapeutic use has commonalities with the aforementioned antibody or antigen-binding fragment thereof; or pharmaceutical composition in that it includes an antibody that specifically binds to Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15) or an antigen-binding fragment thereof; and to prevent excessive duplication of descriptions in the specification, the description of duplicated matters is omitted.

[0210]

[0211] The features and advantages of the present invention are summarized as follows:

[0212] (a) The present invention provides an antibody that specifically binds to Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15) or an antigen-binding fragment thereof.

[0213] (b) The present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or the antigen-binding fragment thereof.

[0214] (c) The present invention provides a recombinant vector comprising the nucleic acid molecule.

[0215] (d) The present invention provides a host cell comprising the recombinant vector.

[0216] (e) The present invention provides a pharmaceutical composition for the treatment or prevention of cancer comprising the antibody or an antigen-binding fragment thereof.

[0217] (f) When using the antibody of the present invention or its antigen-binding fragment, the activity of suppressed T cells can be effectively restored and the death of cancer cells can be induced, thereby obtaining an excellent anticancer effect.

[0218]

[0219] Figure 1 illustrates the process of selecting antibodies specific to human and mouse Siglec-15 from a human synthetic scFv library. Figure 1a is a schematic diagram of the biopanning process, and Figure 1b is the ELISA result confirming the antigen binding ability of the selected phage clones.

[0220] Figure 2 shows the results of analyzing the biophysical characteristics of the selected antibodies. Figure 2a is a graph showing the antibody production yield, Figure 2b is the result of analyzing the purity of the antibodies using SDS-PAGE, and Figure 2c is the ELISA result confirming the binding specificity to recombinant human (rhSiglec-15) and mouse (rmSiglec-15) Siglec-15. Figure 2d shows the result of evaluating the thermal stability of the antibodies through a protein thermal shift assay.

[0221] Figure 3 is a sensorgram analyzing the binding affinity between selected antibodies (K110.1, K110.2, K110.3, K110.4) and rhSiglec-15 and rmSiglec-15 using surface plasmon resonance (SPR).

[0222] Figure 4 shows the results of flow cytometry confirming the binding specificity of selected antibodies (K110.1, K110.2, K110.3) against Siglec-15 expressed on the surface of colorectal cancer cell lines (HCT116). It shows that the binding of the antibodies to the cell surface decreases upon antigen (rhSiglec-15) pretreatment.

[0223] Figure 5 shows the results of confirming the effect of T-cell activation recovery by selected antibodies (K110.1, K110.2, K110.3). Figure 5a is a graph showing the recovery of T-GFP cell activation suppressed by rhSiglec-15 protein, and Figure 5b is a graph showing the degree of recovery of suppressed T-cell activation when co-cultured with Siglec-15-expressing cancer cells (HCT116) compared with atezolizumab.

[0224] Figure 6 shows the results of analyzing the effect of restoring T-cell activation by K110.2 on cancer cell death. Figure 6a shows the cancer cell (HCT116-GFP) death effect according to the concentration of K110.2 when co-cultured with human PBMC, and Figure 6b is a graph showing that the cancer cell death effect is inhibited when treated with the T-cell inhibitor Cyclosporin A.

[0225] Figure 7 is a graph of ELISA results confirming that K110.2 inhibits the interaction between Siglec-15 and CD11b in a concentration-dependent manner.

[0226] Figure 8 shows the results of confirming the antibody internalization and the effect of reducing Siglec-15 expression on the surface of cancer cells by K110.2. Figures 8a and 8b show the intracellular internalization of K110.2 as fluorescence images and quantitative graphs, and Figure 8c is a histogram confirming the downregulation of Siglec-15 expression on the surface of HCT116 cells by K110.2 treatment through flow cytometry.

[0227] Figure 9 shows the results of identifying the epitope of hSiglec-15 recognized by K110.2. Figure 9a is a schematic diagram of the various fragments used for epitope mapping, Figure 9b shows the results of binding affinity analysis for primary fragments (A1-A7), and Figure 9c shows the results of binding affinity analysis for secondary fragments (B1-B3). Figure 9d shows the results of a competitive ELISA using the B3 fragment, and Figure 9e is a graph confirming that the inhibitory ability of K110.2 to bind to Siglec-15-CD11b decreases upon pretreatment with the identified epitope peptide.

[0228] Figure 10 is a visualization of the three-dimensional structure and antigen-antibody binding interface of the complex of the Fv region of K110.2 and the extracellular domain (ECD) of hSiglec-15 predicted through molecular docking simulations.

[0229] Figure 11 shows the results of evaluating the anticancer efficacy of K110.2 and K110.3 in a colorectal cancer synaptic transplant mouse model (CT26 / BALB / c or CT26 / mSiglec-15 / BALB / c). Figure 11a is a schematic diagram showing the experimental schedule and administration groups, and Figures 11b and 11c are graphs showing the change in tumor volume following the administration of K110.2 and K110.3.

[0230]

[0231] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0232]

[0233] Examples

[0234] Example 1: Materials and Method

[0235] 1-1. Cell Culture

[0236] HCT116 human colorectal cancer (CRC) cells (Korean Cell Bank, Seoul, Korea), GFP-stable HCT116 cell line (HCT116-GFP; GeneCopeia, Rockville, MD, USA), Jurkat clone E6-1 cell line (Jurkat T; Korean Cell Line Bank), NF-κB / Jurkat / GFP TM Transcription reporter cell lines (T-GFP; System Bioscience, Palo Alto, CA, USA) and human peripheral blood monocytes (hPBMC; ZenBio, Durham, NC, USA) were cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Gibco, Waltham, MA, USA) supplemented with 10% (v / v) fetal bovine serum (FBS; Gibco) and 1% (v / v) penicillin-streptomycin (Gibco). The cells were maintained at 37°C and 5% carbon dioxide (CO2). Expi293F cells (Gibco) were cultured in Expi293 expression medium (Gibco) in a humidified shaking incubator at 37°C and 8% carbon dioxide (CO2).

[0237]

[0238] 1-2. Selection of Siglec-15 Specific Fully Human Antibodies

[0239] Biopanning was performed to screen for fully human single-strand variable fragment (scFv) antibodies specific to recombinant human and mouse Siglec-15 (rhSiglec-15 and rmSiglec-15; Acrobiosystems, Newark, DE, USA) from a human synthetic scFv library. Specifically, to screen for scFv clones that cross-react with the two proteins, magnetic beads (M-270 epoxy Dynabeads) coated with 4 μg of rhSiglec-15 or rmSiglec-15 were used. TMBiopanning was performed using alternating Invitrogen (Invitrogen, Waltham, MA, USA) for a total of 6 rounds. Subsequently, 96 phage clones were randomly selected from the resulting colonies, and their reactivity to rhSiglec-15 and rmSiglec-15 was evaluated using phage enzyme-linked immunosorbent assay (ELISA). Based on deoxyribonucleic acid (DNA) sequencing, 9 distinct scFv clones with unique complementarity determining site (CDR) sequences were identified.

[0240]

[0241] 1-3. Phage ELISA

[0242] Phage enzyme-linked immunosorbent assay (ELISA) was performed to select clones specific to both rhSiglec-15 and rmSiglec-15. Ninety-six single colonies randomly selected from the 6th round of biopanning were cultured in 96-deep-well plates (Axygen, Union City, CA, USA) at 37°C for 6 hours in Super Broth (SB) medium (3% (w / v) tryptone, 2% (w / v) yeast extract, 1% (w / v) MOPS; pH 7.0) supplemented with 50 μg / mL carbenicillin. After culture, 10 9VCSM13 helper phages of plaque-forming units and 70 μg / mL kanamycin were added to the plate, and the mixture was incubated overnight at 37°C. Subsequently, the plate was centrifuged at 3,000 µg for 30 minutes to separate the phage supernatant. 0.1 μg of rhSiglec-15 or rmSiglec-15 dissolved in phosphate-buffered saline (PBS) was incubated overnight at 4°C on 96-well high-binding plates (Corning, Corning, NY, USA), either coated or uncoated. Afterward, the plates were blocked for 2 hours at 37°C with 3% (w / v) bovine serum albumin (BSA) dissolved in PBS (PBS-T) containing 0.05% (v / v) Tween 20. After blocking, the phage supernatant was added to the plate and incubated at 37°C for 2 hours. After washing the plate three times with PBS-T, HRP (horseradish peroxidase)-conjugated anti-hemagglutinin antibody (1:3,000; Bethyl Laboratories, Montgomery, TX, USA) was added and incubated at 37°C for 1 hour. After washing three times with PBS-T, a 3,3',5,5'-tetramethylbenzidine (TMB; Thermo Fisher Scientific, Waltham, MA, USA) substrate solution was added to induce a color reaction, and the reaction was stopped with a 1 M sulfuric acid (H2SO4) solution. The absorbance (optical density) was measured at 450 nm using a microplate reader (Synergy H1, BioTek, Winooski, VT, USA).

[0243]

[0244] 1-4. Production, Expression, and Purification of Antibodies

[0245] To convert nine selected rhSiglec-15 and rmSiglec-15 specific scFv clones into immunoglobulin G1 (IgG1) monoclonal antibodies (mAbs), their variable heavy and light chain genes were cloned into a bicistronic mammalian expression vector based on pcDNA3.1 (Invitrogen). This vector encodes the IgG1 backbone, and the resulting Siglec-15 specific antibodies (α-Siglec-15 Abs) were named K110.1 to K110.9. Additionally, to construct K110.2 (K110.2-HA) with an HA tag, an HA-tag sequence (YPYDVPDYA) was inserted into the C-terminus of the crystallizable fragment (Fc) region of human IgG1. To construct anti-CD3ε scFv-Fc (α-CD3ε Ab), scFv fragments derived from OKT3 were subcloned into a pCEP4 mammalian expression vector (Invitrogen) containing the Fc region of IgG1. Each antibody construct was transiently expressed in Expi293F cells using the Expi293 expression system (Thermo Fisher Scientific) according to the manufacturer's instructions. The antibodies were purified from the culture medium by affinity column chromatography using Protein A-Sepharose (Repligen, Waltham, MA, USA) and dialyzed with PBS using a Slide-A-Lyzer dialysis cassette (3,500 Da MWCO, Thermo Fisher Scientific).

[0246]

[0247] 1-5. Analysis of IgG Antibody Size and Purity

[0248] After expression and purification, 2 μg of each antibody sample was developed on a 12% polyacrylamide gel using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For reduction conditions, the samples were prepared in an SDS sample buffer containing dithiothreitol (DTT) and heated to reduce disulfide bonds. After electrophoretic separation, protein bands were visualized by staining with Coomassie Brilliant Blue (CBB) EZ-Gel staining solution (DoGenBio, Seoul, Korea).

[0249]

[0250] 1-6. Enzyme-linked immunosorbent assay (ELISA)

[0251] To evaluate the binding specificity of IgG antibodies, 0.1 μg of rhSiglec-15 or rmSiglec-15 was incubated overnight at 4°C in 96-well Corning plates, either coated or uncoated. Subsequently, the plates were blocked with 3% (w / v) BSA dissolved in 0.05% PBS-T at 37°C for 2 hours, and then 20 μg / mL of each antibody was added and incubated at 37°C for 2 hours. The plates were washed three times with 0.05% PBS-T, followed by the addition of HRP-conjugated anti-human Fc secondary antibody (1:5,000; Invitrogen) and incubated at 37°C for 1 hour. After washing three times with 0.05% PBS-T, TMB (Thermo Fisher Scientific) substrate solution was added to initiate the colorimetric reaction, and the reaction was stopped with 1 M sulfuric acid (H2SO4) solution. Absorbance was measured at 450 nm using a microplate reader (Synergy H1, BioTek).

[0252]

[0253] 1-7. Protein Thermal Shift Assay

[0254] The thermal stability of the selected antibodies was evaluated using the protein thermal change assay. Briefly, MicroAmp TM 5 μg of each antibody or negative control PBS and 2.5 μL of 8X Protein Thermal Shift Dye (Applied Biosystems, Waltham, MA, USA) were mixed in each well of the Optical 8-Tube Strip (Applied Biosystems). Thermal change analysis was performed using QuantStudio according to the manufacturer's instructions. TM 3 Real-Time PCR was performed using an Applied Biosystems instrument. All experiments were performed in duplicate to ensure accuracy and reproducibility.

[0255]

[0256] 1-8. Surface Plasmon Resonance (SPR)

[0257] The binding kinetics between antibodies and antigens were analyzed using SPR with a Biacore T200 instrument (Cytiva, Marlborough, MA, USA). Specifically, rhSiglec-15 or rmSiglec-15 were covalently immobilized on a CM5 sensor chip (Cytiva). The binding interactions between the chip immobilized with rhSiglec-15 or rmSiglec-15 and antibodies K110.1, K110.2, K110.3, and K110.4 were evaluated by sequentially increasing the antibody concentration (8, 16, 32, 64, and 128 nM) in HBS-T buffer (10 mM HEPES, 150 mM NaCl, and 0.005% (v / v) Tween-20; pH 7.4; Cytiva). After each binding cycle, the sensor chip was regenerated with 10 mM glycine-HCl (pH 2.4) to remove the bound antibodies. Equilibrium dissociation constant (K DThe ) value was calculated using Biacore T200 Control Software Version 3.2 (Cytiva).

[0258]

[0259] 1-9. Flow cytometry

[0260] To evaluate the specificity of the selected antibody against Siglec-15 on colorectal cancer (CRC) cells, 2 μg / mL of the antibody was pre-incubated at room temperature (RT) for 1 hour with or without 20 μg / mL of rhSiglec-15. Subsequently, 1 x 10⁶ 6 Canine HCT116 cells were fixed with 4% (w / v) paraformaldehyde (PFA) and incubated with the mixture at room temperature for 1 hour. After washing several times with PBS containing 1% (w / v) BSA, the cells were incubated with Alexa Fluor 488-labeled anti-human Fc IgG (1:200; Invitrogen) at room temperature under dark conditions for 1 hour. Fluorescence intensity was measured using a flow cytometer (Millipore, Burlington, MA, USA).

[0261] To evaluate the effect of K110.2 on the downregulation of Siglec-15 expression on the surface of colorectal cancer cell lines, 2 X 10 5Canine HCT116 cells were cultured in PBS containing 1% (w / v) BSA at 37°C for 4 hours under conditions of 20 μg / ml K110.2 treatment or no treatment, and then fixed with 4% (w / v) PFA. The fixed cells were washed twice with PBS containing 1% (w / v) BSA and stained with anti-Siglec-15 polyclonal antibody (Invitrogen) at 4°C for 2 hours. Afterward, the cells were washed three times with PBS containing % (w / v) BSA, reacted with Alexa Fluor 647-labeled anti-rabbit IgG (1:1,000; Invitrogen), and then analyzed using a flow cytometer (Millipore).

[0262]

[0263] 1-10. T cell activation assay

[0264] To evaluate the effect of selected antibodies restoring T-cell activity inhibited by Siglec-15 at the molecular level, 3 X 10 4 T-GFP cells were seeded into each well of a 96-well plate coated or uncoated with 4 μg / mL of rhSiglec-15. Then, the T-GFP cells were treated with 1 μg / mL of anti-CD3ε antibody (α-CD3ε Ab) in the presence or absence of 20 μg / mL of selected antibodies (K110.1, K110.2, and K110.3).

[0265] To further evaluate the effect of selected antibodies in restoring T-cell activity suppressed by Siglec-15-expressing colorectal cancer cell lines, 9 X 10 4 3 x 10⁶ HCT116 cells in each well of a 96-well plate, seeded or unseeded 4T-GFP cells were seeded. Then, T-GFP cells were treated with 1 μg / mL of anti-CD3ε antibody in the presence or absence of 20 μg / mL of selected antibodies (K110.1, K110.2, and K110.3) or atezolizumab, and cultured at 37°C for 18 hours. Fluorescence images were acquired using an Incucyte SX1 live cell analysis system (Sartorius, Gottingen, Germany) equipped with a 10x objective lens, and GFP expression levels were measured to quantify immune activation.

[0266]

[0267] 1-11. PBMC-mediated cancer cell death assay

[0268] To evaluate whether K110.2 enhances PBMC-mediated Siglec-15-expressing colorectal cancer cell death, HCT116-derived cell lines stably expressing GFP (HCT116-GFP) were used. A total of 5 x 10 3 10 HCT116-GFP cells were seeded into each well of a 96-well plate and cultured overnight at 37°C. Afterwards, 5 x 10 4 Canine human PBMCs were cultured with HCT116-GFP cells in the presence or absence of specified concentrations of K110.2 or 1 μg / mL of cyclosporin A. After 48 hours of culture, fluorescence intensity was measured using the Incucyte SX1 live cell assay system (Sartorius).

[0269]

[0270] 1-12. Protein-protein interaction inhibition assay

[0271] An ELISA was performed to evaluate the ability of K110.2 to inhibit the interaction between Siglec-15 and CD11b. Briefly, 96-well Corning plates were coated with 2 μg / mL of recombinant CD11b / CD18 heterodimer (R&D Systems, Minneapolis, MN, USA) and incubated overnight at 4°C. Subsequently, the plates were blocked with 3% (w / v) BSA dissolved in 0.05% PBS-T at 37°C for 2 hours. Simultaneously, His tagged-rhSiglec-15 was pre-incubated at room temperature (RT) for 1 hour with or without K110.2 at various concentrations. The mixtures were added to the prepared wells and incubated at room temperature for 1 hour. After washing the plate three times with 0.05% PBS-T, HRP-conjugated anti-His secondary antibody (1:5,000; R&D Systems, Minneapolis, MN, USA) was added to each well and incubated at 37°C for 1 hour. After washing three times with 0.05% PBS-T, TMB (Thermo Fisher Scientific) substrate solution was added to initiate the colorimetric reaction, and the reaction was stopped with 1 M sulfuric acid (H2SO4) solution. Absorbance was measured at 450 nm using a microplate reader (Synergy H1, BioTek).

[0272]

[0273] 1-13. Measurement of antibody internalization

[0274] Antibody internalization within HCT116 colorectal cancer cells was measured using FabFluor-pH Red Antibody Labeling Reagent (Sartorius) according to the manufacturer's instructions. It is known that the red fluorescence intensity of the FabFluor-pH reagent is low at neutral or basic pH outside the cell, but significantly increases at the acidic pH of endosomes and lysosomes after antibody internalization. Briefly, 5 x 10⁶ cells per well in a 96-well culture plate 3 HCT116 cells were seeded and allowed to attach overnight. Control IgG and K110.2 were each mixed with FabFluor-pH red antibody labeling reagent in PBS at a molar ratio of 1:3 and reacted for 15 minutes. The labeled antibodies were added to the cell medium to achieve a final concentration of 2 μg / mL. The plates were transferred to an Incucyte SX1 live cell analyzer (Sartorius), images were acquired for 24 hours using a 10x objective lens, and fluorescence intensity was measured.

[0275]

[0276] 1-14. Preparation of Fc-tagged hSiglec-15 fragments

[0277] To identify the specific binding epitope of K110.2, a set of overlapping peptides across the entire extracellular domain (ECD) of hSiglec-15 was designed. Fragments 1 through 6 (A1-A6) consist of 60 amino acids, and fragment 7 (A7) consists of 64 amino acids, with each fragment overlapping by 30 amino acids. Additionally, the overlapping region between fragments A1 and A2 was further subdivided into three non-overlapping fragments (B1-B3). The DNA sequences encoding these fragments were amplified by PCR. The PCR products were subcloned into a pCEP4 mammalian expression vector containing the Fc region of human IgG1, which includes a hinge mutation designed for monomeric protein expression. The recombinant plasmid was transfected into Expi293 cells using the Expi293 Transfection Kit (Thermo Fisher Scientific) according to the manufacturer's instructions, and cultured under conditions of 37°C, 125 rpm shaking, and 8% carbon dioxide (CO2). Five days after transfection, the supernatant was collected, and Fc-tagged fragments were purified by affinity chromatography using protein A-cephalosporose (GE Healthcare, Chicago, IL, USA).

[0278]

[0279] 1-15. Epitope Mapping

[0280] Epitope mapping using ELISA was performed to determine the binding sites of K110.2 on hSiglec-15. 0.1 μg of rhSiglec-15 and each purified Fc-tagged fragment (A1-A7 and B1-B3) were coated onto 96-well Corning plates and incubated overnight at 4°C. After blocking with 3% (w / v) BSA dissolved in 0.05% PBS-T at 37°C for 2 hours, 100 nM HA-tagged K110.2 was added to each well and incubated at 37°C for 2 hours. After washing three times with 0.05% PBS-T, HRP-conjugated anti-HA antibody (1:5,000; Bethyl Laboratories) was added and incubated at 37°C for 1 hour. After washing three additional times, TMB substrate solution (Thermo Fisher Scientific) was added to initiate the color reaction, and the reaction was stopped with 1 M sulfuric acid (H2SO4). Absorbance was measured at 450 nm using a microplate reader (Synergy H1, BioTek).

[0281]

[0282] 1-16. Competition ELISA

[0283] 0.1 μg of rhSiglec-15 (ACROBiosystems) was coated onto a 96-well high-binding microplate and incubated overnight at 4°C. HA-tagged K110.2 was pre-incubated for 2 hours at room temperature (RT) with or without a 50-fold molar excess of B3 fragment. Subsequently, the mixture was added to the coated wells and incubated for an additional 2 hours at room temperature. After washing several times with 0.05% PBS-T, HRP-conjugated anti-HA antibody (1:5,000; Bethyl Laboratories) was added and incubated for 1 hour at 37°C. Finally, TMB substrate solution (Thermo Fisher Scientific) was added to initiate the colorimetric reaction, and the reaction was stopped with 1 M sulfuric acid (H2SO4). Absorbance was measured at 450 nm using a microplate reader (Synergy H1, BioTek).

[0284]

[0285] 1-17. Molecular modeling and docking

[0286] The three-dimensional (3D) structures of the variable fragment (Fv) of K110.2 and the extracellular domain (ECD) of hSiglec-15 were predicted using the AlphaFold Server based on AlphaFold3 (PMID: 38718835), one of the latest tools for protein 3D structure prediction. For the antigen model, the crystal structure of hSiglec-15 (PDB ID: 7ZOZ) was used as the true conformation of hSiglec-15. Among the five models generated by the AlphaFold Server, the top model was selected for K110.2 Fv and hSiglec-15 ECD, respectively, and docking simulations were performed using Haddock3 (PMID: 40526044). Ambiguous interaction restrictions (AIRs), representing residues potentially interacting with other molecules, were defined as CDRs for K110.2 Fv and epitope regions identified by epitope mapping for hSiglec-15 ECD. The optimal docking model was selected based on scores generated by the default scoring settings of Haddock3, and interactions between the two molecules were elucidated using PDBe Arpeggio (PMID: 27964945). The docking model was evaluated by comparing the residues within hSiglec-15 ECD that interact with K110.2 with the binding regions discovered by epitope mapping.

[0287]

[0288] 1-18. In vivo efficacy testing

[0289] Animals were maintained in a Specified Pathogen-Free (SPF) environment and acclimatized to laboratory conditions for at least one week prior to the experiment. The rearing and management of mice were carried out at the animal facility of Kookmin University in accordance with the animal care policies of the Association for Accreditation of Laboratory Animal Care (AAALAC). 2.5 x 10⁶ lesions were inscribed on both flanks of 5-week-old female BALB / c mice. 5Canine CT26 cells or mSiglec-15 overexpressing CT26 cells (CT26 / mSiglec-15) were injected subcutaneously. The tumor volume was approximately 100 mm³ 3 When [the threshold] was reached, mice were randomly divided into groups (n=4) and administered PBS (negative control), 10 mg / kg K110.2, and 10 mg / kg K110.3 intraperitoneally twice weekly for 3 weeks. Tumor volume was measured every 3 or 4 days using a digital caliper (Mitutoyo, Utsunomiya, Japan), and the total volume was calculated using the formula (long axis × short axis). 2 cubic millimeters (mm) using ) / 2 3 It was calculated as ). Here, the major axis refers to the longest axis, and the minor axis refers to the distance perpendicular to the major axis.

[0290]

[0291] 1-19. Statistical Analysis

[0292] All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA). Two-tailed Student's t-tests were used for comparisons between two groups, while one-way ANOVA with Dunnett's correction was used for multiple group comparisons. Data were expressed as mean ± standard deviation (SD). Statistical significance was defined as a P-value less than 0.05, and the significance level was indicated on the graph as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

[0293]

[0294] Example 2: Selection of Siglec-15 Specific Human Antibodies

[0295] To isolate human antibodies specific to both human and mouse Siglec-15, biopanning was performed using magnetic beads immobilized with rhSiglec-15 or rmSiglec-15 from a human synthetic scFv library and phage display technology (Fig. 1a). After six rounds of biopanning, 96 phage clones were randomly selected from the resulting colonies, and their binding affinity to rhSiglec-15 and rmSiglec-15 was evaluated via phage ELISA.

[0296] The results are shown in Fig. 1b.

[0297] As shown in Figure 1b, clones that exhibited strong reactivity to both antigens and did not bind to the negative control BSA were selected. Finally, nine scFv clones with different complementarity determining region (CDR) sequences were identified through DNA sequencing analysis and were named K110.1, K110.2, K110.3, K110.4, K110.5, K110.6, K110.7, K110.8, and K110.9, respectively.

[0298]

[0299] Example 3: Determination of Biophysical Characteristics of Selected Antibodies

[0300] 3-1. Antibody Production and Purity Analysis

[0301] Selected scFv clones were converted into human IgG antibody forms and transiently expressed in Expi293F cells, after which they were purified using protein A-cephalosporose affinity column chromatography. The production yields of the nine purified antibodies were all over 50 mg / L, and K110.2, in particular, was found to have a very excellent production yield of 223 mg / L (Fig. 2a).

[0302] To confirm the molecular weight and purity of the selected IgG antibodies, SDS-PAGE was performed under reducing conditions containing dithiothreitol (DTT), and the samples were stained with Coomassie Brilliant Blue (CBB) for analysis. As a result, all purified IgG antibodies showed a purity of over 90%, and clear bands corresponding to the expected molecular weights of the heavy chain and light chain were observed (Fig. 2b).

[0303]

[0304] 3-2. Evaluation of Bond Specificity and Thermal Stability

[0305] To evaluate the binding specificity of the selected antibodies to human and mouse Siglec-15, an ELISA was performed using plates coated with rhSiglec-15 or rmSiglec-15. Wells coated with BSA were used as negative controls. Analysis revealed that all three antibodies exhibited specific binding to rhSiglec-15 and rmSiglec-15, while not binding to BSA (Fig. 2c).

[0306] In addition, the thermal stability of the selected antibodies was evaluated through a protein thermal shift assay. The measured melting points (Tm) were 74.9°C for K110.1, 76.9°C for K110.2, 66.6°C for K110.3, and 68.4°C for K110.4, with K110.1 and K110.2 showing particularly excellent thermal stability (Fig. 2d).

[0307]

[0308] 3-3. Affinity Analysis

[0309] Real-time reaction kinetics analysis using Surface Plasmon Resonance (SPR) was performed to evaluate the binding affinity of the selected antibodies for rhSiglec-15 and rmSiglec-15. After immobilizing the antigen on the sensor chip and analyzing the results while increasing the antibody concentration, the equilibrium dissociation constant (K) for rhSiglec-15 D ) was measured to be 3.29 nM, 4.36 nM, 7.18 nM, and 56.75 nM for K110.1, K110.2, K110.3, and K110.4, respectively. K in rmSiglec-15 D The values ​​were measured as 2.23 nM, 3.29 nM, and 1.52 nM for K110.1, K110.2, and K110.3, respectively, while K110.4 was not measured. These results demonstrate that all three antibodies (K110.1, K110.2, K110.3) exhibit cross-species reactivity and possess high binding affinities at the nanomolar (nM) level (Fig. 3, Table 1).

[0310]

[0311] Evaluation of binding affinity for human and mouse Siglec-15 Antibody name rhSiglec-15rmSiglec-15K D (nM)k a (1 / Ms)k d (1 / s)K D (nM)k a (1 / Ms)k d (1 / s)K110.13.291.10Х10 5 3.63×10 -4 2.231.24×10 5 2.77×10 -4 K110.24.366.74×10 5 2.94×10 -3 3.291.07×10 6 3.51×10 -3 K110.37.184.23×10 5 3.04×10 -3 1.521.00×105 1.52×10 -4 K110.456.753.49Х10 4 2.07×10 -3 NDNDND

[0312] K D , equilibrium dissociation constant; K a , association constant; K d , dissociation constant; ND, not determined

[0313]

[0314] 3-4. Verification of binding specificity to cell surface Siglec-15

[0315] To confirm whether the selected antibody specifically binds to Siglec-15 expressed on the surface of colorectal cancer cells, flow cytometry was performed using HCT116 cells. When the cells were treated with the antibody either pre-incubated with rhSiglec-15 or without pre-incubation, strong binding to HCT116 cells was observed when the antibody was treated alone. In contrast, cell surface binding was significantly reduced when the antibody was pre-incubated with rhSiglec-15. This suggests that the selected antibody specifically recognizes and binds to Siglec-15 present on the cell surface (Fig. 4).

[0316]

[0317] Example 4: T-cell activating effect of selected Siglec-15 specific antibody

[0318] 4-1. Reversal Effect of Siglec-15-Mediated T-Cell Inhibition

[0319] To investigate whether the selected antibody could reverse T-cell suppression induced by Siglec-15, T-GFP cells were cultured with rhSiglec-15 or Siglec-15-expressing colorectal cancer (CRC) cell lines and compared under conditions with or without the selected antibody. In this case, GFP expression was used as an indicator of T-cell activation.

[0320] First, the reversal effect of antibodies on Siglec-15-mediated T-cell inhibition was evaluated at the molecular level. The results are shown in Figure 5a. As shown in Figure 5a, T-GFP cells treated with rhSiglec-15 exhibited a GFP signal inhibited to approximately 52% of the level of full activation induced by the anti-CD3ε antibody (α-CD3ε Ab). After treatment with each Siglec-15 antibody, K110.1 showed no significant effect, whereas K110.2 and K110.3 effectively reactivated T cells by restoring GFP intensity to 90.2% and 87.3%, respectively.

[0321] Next, co-culture assays were performed to evaluate the effects at the cellular level. T-GFP cells were cultured with HCT116 cells expressing Siglec-15 in the presence or absence of antibodies. The results are shown in Figure 5b. As shown in Figure 5b, without antibody treatment, the GFP intensity of T-GFP cells was suppressed to approximately 45% of the fully activated level due to the immunosuppressive effect of HCT116 cells. In contrast, when treated with K110.1, K110.2, and K110.3, the GFP intensity was significantly restored to 72%, 92.2%, and 80.2%, respectively. Notably, K110.2 restored GFP intensity to levels equivalent to or higher than those of atezolizumab, a PD-L1 targeting antibody. These results highlight the superior efficacy of K110.2 in reversing Siglec-15-mediated immunosuppression and served as the basis for selecting K110.2 as a key candidate for further investigation in this study.

[0322]

[0323] 4-2. Effect of Promoting T-cell-Mediated Cancer Cell Apoptosis

[0324] A co-culture assay was performed to determine whether the restoration of T-cell activation by K110.2 promotes T-cell-mediated cancer cell death. HCT116-GFP cells were co-cultured with human PBMCs (hPBMCs) in the presence or absence of K110.2, and GFP intensity was measured as a viability indicator for HCT116-GFP cells to monitor cancer cell death.

[0325] The results are shown in Figs. 6a and 6b.

[0326] As shown in Figure 6a, in the absence of antibodies, co-culture of hPBMC and HCT116-GFP cells resulted in only a slight decrease in GFP intensity, suggesting that T-cell-mediated cancer cell death is limited due to T-cell inhibition. On the other hand, when K110.2 was treated, GFP intensity decreased, and 65.8% of cancer cells were killed upon treatment with 50 μg / mL of K110.2, confirming that T-cell activity restored by K110.2 effectively promoted cancer cell death.

[0327] In addition, as shown in Figure 6b, when K110.2 was treated with cyclosporin A, a T-cell activation inhibitor, the survival rate of cancer cells, which was 40% when K110.2 was treated alone, increased to 82% when treated with cyclosporin A, confirming that 63.2% of cancer cell death caused by K110.2 was involved in T-cell activation.

[0328]

[0329] Example 5: Elucidation of the Mechanism of Action (MoA) of K110.2

[0330] 5-1. Blocking effect of the interaction between Siglec-15 and CD11b

[0331] Siglec-15 contributes to T-cell suppression through interactions with inhibitory receptors such as CD11b within the tumor microenvironment (TME). To evaluate whether K110.2 blocks these immunosuppressive interactions between Siglec-15 and CD11b, an ELISA-based receptor binding inhibition assay was performed.

[0332] The results are shown in Figure 7 and Table 2.

[0333] As shown in Figure 7 and Table 2, K110.2 effectively inhibited the interaction between Siglec-15 and CD11b in a concentration-dependent manner, and the half-inhibitory concentration (IC10.2) of K110.2 50 The value was determined to be 0.48 nM (Fig. 7, Table 2).

[0334]

[0335] Evaluation of binding affinity for human and mouse Siglec-15 Antibody name IC 50 values ​​(nM)CD11bK110.20.48 ± 0.181

[0336] IC 50 , Half-maximal inhibition concentration

[0337]

[0338] 5-2. Induction of Antibody Internalization and Reduction of Siglec-15 Expression

[0339] To elucidate the mechanism of Siglec-15 function regulation by K110.2, the effects on antibody internalization and surface expression were investigated. K110.2 or control IgG were conjugated with FabFluor-pH reagent and treated to HCT116 cells; internalization efficiency was monitored by quantitatively measuring fluorescence intensity for 24 hours. The results are shown in Figures 8a and 8b. As shown in Figures 8a and 8b, while the control IgG showed almost no internalization, it was confirmed that K110.2 was effectively internalized into HCT116 cells.

[0340] Next, flow cytometry was performed to evaluate the effect of K110.2 on the downregulation of Siglec-15 expression on the surface of colorectal cancer cells. Siglec-15 levels on the cell surface were measured by comparing conditions in which HCT116 cells were treated with K110.2 or control IgG, or were not treated. The results are shown in Figure 8c. As shown in Figure 8c, no change was observed when treated with control IgG, whereas treatment with K110.2 significantly reduced Siglec-15 expression on the surface of HCT116 cells.

[0341] Taken together, these results suggest that K110.2 not only blocks the interaction between Siglec-15 and CD11b but also promotes the internalization of Siglec-15, thereby inducing the downregulation of Siglec-15 expression on the surface of colorectal cancer cells. This dual mechanism of action by K110.2 may reduce the immunosuppressive effect on T cells by limiting the availability of Siglec-15 on the cancer cell surface.

[0342]

[0343] Example 6: K110.2 Epitope Mapping and Structural Analysis on hSiglec-15

[0344] 6-1. Identification of binding epitopes of K110.2

[0345] ELISA-based epitope mapping was performed to identify the binding sites (epitopes) of K110.2 to hSiglec-15. For this purpose, partially overlapping Fc-tag fragments (A1-A7) were designed to encompass the entire extracellular domain (ECD) of hSiglec-15. Fragments A1 through A6 consisted of 60 amino acids, and A7 consisted of 64 amino acids; each fragment was designed to overlap by 30 amino acids and fused to the Fc domain to aid in purification (Fig. 9a).

[0346] The reactivity of K110.2 toward these fragments was evaluated by ELISA, and K110.2 showed strong binding to fragments A1 and A2, while showing no significant binding to the remaining fragments (Fig. 9b). To identify the epitope more precisely, the 30-amino acid overlap region between A1 and A2 was subdivided into three non-overlapping fragments (B1-B3), each of which was expressed as an Fc-fusion protein consisting of 10 amino acids. ELISA analysis showed that K110.2 exhibited the strongest reactivity toward fragment B3, indicating that the epitope of K110.2 is located within the amino acid sequence corresponding to B3 (Fig. 9c).

[0347]

[0348] 6-2. Verification of Epitope Specificity and Functional Importance

[0349] To further confirm the specificity of the identified epitope, a competition ELISA using the B3 fragment was performed. Before reacting with immobilized full-length rhSiglec-15, K110.2 was pretreated with a 50-fold molar excess of B3. As a result, the presence of the B3 fragment significantly inhibited the binding of K110.2 to full-length rhSiglec-15, which supports the fact that K110.2 specifically recognizes the epitope within that region (Fig. 9d).

[0350] In addition, to confirm the functional importance of the epitope region recognized by K110.2, a protein-protein interaction inhibition assay was performed using a synthetic peptide corresponding to the mapped epitope (his70-ala79 residue of hSiglec-15). Prior to evaluating the ability of K110.2 to inhibit the interaction between Siglec-15 and CD11b, K110.2 was pretreated with the epitope peptide. As a result, pretreatment with the peptide significantly attenuated the inhibitory activity of K110.2 on the Siglec-15-CD11b interaction (Fig. 9e). These results demonstrate that the receptor blocking function of K110.2 depends on the specific recognition of the his70-ala79 epitope, confirming that this region is a functionally important site for K110.2-mediated immune regulation.

[0351]

[0352] 6-3. Molecular Modeling and Docking Analysis

[0353] To gain structural insights into antibody-antigen interactions, molecular docking simulations were performed using the modeled structures of K110.2 Fv and hSiglec-15 ECD. From over 200 predicted docking models, four consistent models were selected based on structural similarity. Among these, a single model was selected that matched the binding interface with the experimentally mapped epitope (Residue His70-Ala79 of Siglec-15). Docking analysis revealed that K110.2 binds to hSiglec-15 primarily through the CDR-L1, CDR-L3, and CDR-H3 loops, forming extensive interactions within the mapped epitope region (Fig. 10). Taken together, these findings demonstrate consistency between the structural model and ELISA-based mapping results, providing a molecular basis for the receptor blocking activity of K110.2.

[0354]

[0355] Example 7: Evaluation of In vivo efficacy of K110.2 in a colorectal cancer synaptic transplant mouse model

[0356] To evaluate the in vivo efficacy of K110.2 on colorectal cancer growth, a syngeneic mouse model was established by subcutaneously injecting mouse colorectal cancer cells, CT26 and mSiglec-15 overexpressing CT26 cells, into both flanks of BALB / c mice.

[0357] Tumor volume 100 mm² 14 days after tumor transplantation 3 When the time was reached, mice were randomly divided into three groups (n=4). They were then classified into negative control groups (PBS) and groups administered K110.2 and K110.3, and received intraperitoneal injections twice a week for three weeks (Fig. 11a).

[0358] It was confirmed that the CT26 colorectal cancer tumor growth inhibitory effect was observed in the K110.2 and K110.3 administration groups (Fig. 11b), and it was confirmed that the tumor growth inhibitory effect of K110.2 was further increased when mSiglec-15 overexpressing CT26 cells were used, confirming that the anticancer effect by K110.2 occurs in a Siglec-15-dependent manner (Fig. 11c).

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15), comprising the following: (a) a heavy chain CDR1 (HCDR1) having the amino acid sequence of SEQ ID NO. 1, a heavy chain CDR2 (HCDR2) having the amino acid sequence of SEQ ID NO. 2, a heavy chain CDR3 (HCDR3) having the amino acid sequence of SEQ ID NO. 3, a light chain CDR1 (LCDR1) having the amino acid sequence of SEQ ID NO. 4, a light chain CDR2 (LCDR2) having the amino acid sequence of SEQ ID NO. 5, and a light chain CDR3 (LCDR3) having the amino acid sequence of SEQ ID NO. 6; (b) HCDR1 having the amino acid sequence of SEQ ID NO. 9, HCDR2 having the amino acid sequence of SEQ ID NO. 10, HCDR3 having the amino acid sequence of SEQ ID NO. 11, LCDR1 having the amino acid sequence of SEQ ID NO. 12, LCDR2 having the amino acid sequence of SEQ ID NO. 13, and LCDR3 having the amino acid sequence of SEQ ID NO. 14; (c) HCDR1 having the amino acid sequence of SEQ ID NO. 17, HCDR2 having the amino acid sequence of SEQ ID NO. 18, HCDR3 having the amino acid sequence of SEQ ID NO. 19, LCDR1 having the amino acid sequence of SEQ ID NO. 20, LCDR2 having the amino acid sequence of SEQ ID NO. 21, and LCDR3 having the amino acid sequence of SEQ ID NO. 22; (d) HCDR1 having the amino acid sequence of SEQ ID NO. 25, HCDR2 having the amino acid sequence of SEQ ID NO. 26, HCDR3 having the amino acid sequence of SEQ ID NO. 27, LCDR1 having the amino acid sequence of SEQ ID NO. 28, LCDR2 having the amino acid sequence of SEQ ID NO. 29, and LCDR3 having the amino acid sequence of SEQ ID NO. 30; (e) HCDR1 having the amino acid sequence of SEQ ID NO. 33, HCDR2 having the amino acid sequence of SEQ ID NO. 34, HCDR3 having the amino acid sequence of SEQ ID NO. 35, LCDR1 having the amino acid sequence of SEQ ID NO. 36, LCDR2 having the amino acid sequence of SEQ ID NO. 37, and LCDR3 having the amino acid sequence of SEQ ID NO. 38; (f) HCDR1 having the amino acid sequence of SEQ ID NO. 41, HCDR2 having the amino acid sequence of SEQ ID NO. 42, HCDR3 having the amino acid sequence of SEQ ID NO. 43, LCDR1 having the amino acid sequence of SEQ ID NO. 44, LCDR2 having the amino acid sequence of SEQ ID NO. 45, and LCDR3 having the amino acid sequence of SEQ ID NO. 46; (g) HCDR1 having the amino acid sequence of SEQ ID NO. 49, HCDR2 having the amino acid sequence of SEQ ID NO. 50, HCDR3 having the amino acid sequence of SEQ ID NO. 51, LCDR1 having the amino acid sequence of SEQ ID NO. 52, LCDR2 having the amino acid sequence of SEQ ID NO. 53, and LCDR3 having the amino acid sequence of SEQ ID NO. 54; (h) HCDR1 having the amino acid sequence of SEQ ID NO. 57, HCDR2 having the amino acid sequence of SEQ ID NO. 58, HCDR3 having the amino acid sequence of SEQ ID NO. 59, LCDR1 having the amino acid sequence of SEQ ID NO. 60, LCDR2 having the amino acid sequence of SEQ ID NO. 61, and LCDR3 having the amino acid sequence of SEQ ID NO. 62; or (i) HCDR1 having the amino acid sequence of SEQ ID NO. 65, HCDR2 having the amino acid sequence of SEQ ID NO. 66, HCDR3 having the amino acid sequence of SEQ ID NO. 67, LCDR1 having the amino acid sequence of SEQ ID NO. 68, LCDR2 having the amino acid sequence of SEQ ID NO. 69, and LCDR3 having the amino acid sequence of SEQ ID NO.

70.

2. The antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment comprises the following heavy chain variable region (VH) and light chain variable region (VL): (a) VH comprising the amino acid sequence of SEQ ID NO. 7 and VL comprising the amino acid sequence of SEQ ID NO. 8; (b) VH containing the amino acid sequence of SEQ ID NO. 15 and VL containing the amino acid sequence of SEQ ID NO. 16; (c) VH containing the amino acid sequence of SEQ ID NO. 23 and VL containing the amino acid sequence of SEQ ID NO. 24; (d) VH containing the amino acid sequence of SEQ ID NO. 31 and VL containing the amino acid sequence of SEQ ID NO. 32; (e) VH containing the amino acid sequence of SEQ ID NO. 39 and VL containing the amino acid sequence of SEQ ID NO. 40; (f) VH containing the amino acid sequence of SEQ ID NO. 47 and VL containing the amino acid sequence of SEQ ID NO. 48; (g) VH containing the amino acid sequence of SEQ ID NO. 55 and VL containing the amino acid sequence of SEQ ID NO. 56; (h) VH comprising the amino acid sequence of SEQ ID NO. 63 and VL comprising the amino acid sequence of SEQ ID NO. 64; or (i) VH containing the amino acid sequence of SEQ ID NO. 71 and VL containing the amino acid sequence of SEQ ID NO.

72.

3. In paragraph 1, the antibody has an equilibrium dissociation constant (K) of 10 nM or less with respect to human Siglec-15 protein. D An antibody or its antigen-binding fragment having a ) value.

4. The antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment specifically binds to an epitope consisting of the amino acid sequence represented by SEQ ID NO. 146 within the extracellular domain (ECD) of human Siglec-15.

5. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody of any one of claims 1 to 4 or the antigen-binding fragment thereof.

6. In paragraph 5, the nucleic acid molecule is a nucleic acid molecule that is as follows: (a) comprising the nucleotide sequences of SEQ ID NOs 73 to 78; (b) comprising the nucleotide sequences of SEQ ID NOs 81 to 86; (c) comprising the nucleotide sequences of SEQ ID NOs 89 to 94; (d) comprising the nucleotide sequences of SEQ ID NOs 97 to 102; (e) comprising the nucleotide sequences of SEQ ID NOs 105 to 110; (f) comprising the nucleotide sequences of SEQ ID NOs 113 to 118; (g) comprising the nucleotide sequences of SEQ ID NOs 121 to 126; (h) comprising the nucleotide sequences of SEQ ID NOs 129 to 134; or (i) comprising the nucleotide sequences of SEQ ID NOs 137 to 142.

7. In paragraph 5, the nucleic acid molecule is a nucleic acid molecule that is as follows: (a) comprising the nucleotide sequences of SEQ ID NOs 79 and 80; (b) comprising the nucleotide sequences of SEQ ID NOs 87 and 88; (c) comprising the nucleotide sequences of SEQ ID NOs 95 and 96; (d) comprising the nucleotide sequences of SEQ ID NOs 103 and 104; (e) comprising the nucleotide sequences of SEQ ID NOs 111 and 112; (f) comprising the nucleotide sequences of SEQ ID NOs 119 and 120; (g) comprising the nucleotide sequences of SEQ ID NOs 127 and 128; (h) comprising the nucleotide sequences of SEQ ID NOs 135 and 136; or (i) comprising the nucleotide sequences of SEQ ID NOs 143 and 144.

8. A recombinant vector comprising the nucleic acid molecule of claim 5.

9. A host cell containing the recombinant vector of claim 8.

10. A pharmaceutical composition for the prevention or treatment of cancer comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

11. A pharmaceutical composition for the prevention or treatment of cancer, wherein, in the case of claim 10, the cancer is a cancer expressing Siglec-15.

12. A pharmaceutical composition for the prevention or treatment of cancer according to claim 10, wherein the cancer is selected from the group consisting of gastric cancer, rectal cancer, colon cancer, colorectal cancer, liver cancer, lung cancer, ovarian cancer, melanoma, pancreatic cancer, uterine cancer, testicular cancer, breast cancer, and combinations thereof.

13. A pharmaceutical composition for the prevention or treatment of cancer, wherein, in paragraph 10, the pharmaceutical composition additionally comprises an immune checkpoint inhibitor.

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