Anti-SIGLEC15 Antibody and Its Use

Abstract

The present disclosure relates to anti-SIGLEC15 antibodies, antigen-binding fragments, and their use.

Description

Technical Field

[0001] Priority Claim This application claims the benefit of PCT Application No. PCT / CN2022 / 098378, filed on June 13, 2022, and PCT Application No. PCT / CN2022 / 134289, filed on November 25, 2022. The entire above-mentioned content is incorporated herein by reference.

[0002] Technical Field The present disclosure relates to anti-SIGLEC15 (sialic acid-binding Ig-like lectin 15) antibodies and their use.

Background Art

[0003] SIGLEC15 (sialic acid-binding immunoglobulin-like lectin 15) belongs to the SIGLEC family and is a type I transmembrane protein. It is hardly expressed in most normal human tissues and subsets of immune cells, but has relatively high expression in macrophages. In 2007, Takashi Angata, a Japanese scientist, first discovered that this protein can recognize sialic acid and classified it into the SIGLEC family. By regulating innate and adaptive immune responses, SIGLEC15 plays an important role in autoimmune diseases, inflammatory responses, and tumors.

[0004] Considering that SIGLEC15 plays an important role in autoimmune diseases, inflammatory responses, and tumors, the development of therapeutic agents targeting SIGLEC15 is required.

Summary of the Invention

[0005] The present disclosure relates to anti-SIGLEC15 antibodies, antigen-binding fragments thereof, and their use.

[0006] In one aspect, the present disclosure provides A heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence, and the heavy chain variable region, A light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, and the light chain variable region, The selected VH CDR1, 2, and 3 amino acid sequences, and the selected VL CDR1, 2, and 3 amino acid sequences are related to an antibody or an antigen-binding fragment thereof that binds to SIGLEC15 (sialic acid-binding Ig-like lectin 15), which is one of the following. (1) The selected VH CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively. (2) The selected VH CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively. (3) The selected VH CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively. (4) The selected VH CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, and 24, respectively. (5) The selected VH CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, and 30, respectively, and (6) The selected VH CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, and 36, respectively.

[0007] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0008] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.

[0009] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.

[0010] In some embodiments, according to the Chothia definition, VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.

[0011] In some embodiments, according to the definition of Chothia, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.

[0012] In some embodiments, according to the definition of Chothia, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human, mouse, monkey, or canine SIGLEC15.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), a Fab antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

[0015] In some embodiments, the antibody or antigen-binding fragment thereof is a human IgG1 antibody or antigen-binding fragment thereof, or a human IgG4 antibody or antigen-binding fragment thereof.

[0016] In one aspect, the present disclosure provides an immunoglobulin heavy chain or fragment thereof comprising a variable heavy chain region (VH) comprising complementarity-determining regions (CDR) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, wherein the VH binds to SIGLEC15 when paired with a variable light chain region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 38. An immunoglobulin light chain or a fragment thereof, comprising a variable light chain (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3, each comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, wherein said VL binds to SIGLEC15 when forming a pair with a VH comprising the amino acid sequence set forth in SEQ ID NO: 37. An immunoglobulin heavy chain or a fragment thereof, comprising a variable heavy chain (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, each comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, wherein said VH binds to SIGLEC15 when forming a pair with a variable light chain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 40. An immunoglobulin light chain or a fragment thereof, comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3, each comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, wherein said VL binds to SIGLEC15 when forming a pair with a VH comprising the amino acid sequence set forth in SEQ ID NO: 39. An immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3, each comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, wherein said VH binds to SIGLEC15 when forming a pair with a variable light chain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 42, or An immunoglobulin light chain or a fragment thereof, comprising a VL comprising CDRs 1, 2, and 3, each comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, wherein said VL binds to SIGLEC15 when forming a pair with a VH comprising the amino acid sequence set forth in SEQ ID NO: 41. Relates to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising.

[0017] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the VH of which comprises CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.

[0018] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the VL of which comprises CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0019] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the VH of which comprises CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively.

[0020] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the VL of which comprises CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.

[0021] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the VH of which comprises CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively.

[0022] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the VL of which comprises CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.

[0023] In some embodiments, VH specifically binds to human, mouse, monkey, or dog SIGLEC15 when forming a pair with VL, or VL specifically binds to human, mouse, monkey, or dog SIGLEC15 when forming a pair with VH.

[0024] In some embodiments, the immunoglobulin heavy chain or a fragment thereof is a human immunoglobulin heavy chain or a fragment thereof (e.g., a human IgG1 heavy chain or a fragment thereof, or a human IgG4 heavy chain or a fragment thereof), and the immunoglobulin light chain or a fragment thereof is a human immunoglobulin light chain or a fragment thereof. In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv), a single-arm antibody, a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid is cDNA.

[0025] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0026] In one aspect, the disclosure relates to a vector comprising two of the nucleic acids described herein, the vector encoding a VL region and a VH region that together bind to SIGLEC15.

[0027] In one aspect, the disclosure relates to a pair of vectors, each vector comprising one of the nucleic acids described herein, which together encode a VL region and a VH region that bind to SIGLEC15 when paired.

[0028] In one aspect, the disclosure relates to a cell comprising the vector described herein, or a pair of vectors described herein.

[0029] In some embodiments, the cell is a CHO cell.

[0030] In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0031] In one aspect, the present disclosure relates to a cell comprising two nucleic acids described herein.

[0032] In some embodiments, the two nucleic acids encode, as a pair, a VL region and a VH region that together bind to SIGLEC15.

[0033] In one aspect, the present disclosure relates to a method for producing an antibody or an antigen-binding fragment thereof, the method comprising: (a) culturing the cells described herein under conditions sufficient for the cells to produce an antibody or an antigen-binding fragment; and (b) recovering the antibody or the antigen-binding fragment produced by the cells.

[0034] In one aspect, the present disclosure relates to an antibody or an antigen-binding fragment thereof that binds to SIGLEC15, comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: The selected VH sequence is SEQ ID NO: 37 and the selected VL sequence is SEQ ID NO: 38, the selected VH sequence is SEQ ID NO: 39 and the selected VL sequence is SEQ ID NO: 40, and the selected VH sequence is SEQ ID NO: 41 and the selected VL sequence is SEQ ID NO: 42.

[0035] In some embodiments, VH comprises the sequence of SEQ ID NO: 37 and VL comprises the sequence of SEQ ID NO: 38.

[0036] In some embodiments, VH comprises the sequence of SEQ ID NO: 39 and VL comprises the sequence of SEQ ID NO: 40. In some embodiments, VH comprises the sequence of SEQ ID NO: 41 and VL comprises the sequence of SEQ ID NO: 42.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human, mouse, monkey, or canine SIGLEC15.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), a Fab antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

[0039] In some embodiments, the antibody or antigen-binding fragment is a human IgG1 antibody or antigen-binding fragment thereof, or a human IgG4 antibody or antigen-binding fragment thereof.

[0040] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that includes VH CDR1, 2, 3, and VL CDR1, 2, 3 of the antibody or antigen-binding fragment thereof described herein.

[0041] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof described herein.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof includes a fragment crystallizable region (Fc region).

[0043] In some embodiments, the Fc region has reduced or no complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).

[0044] In one aspect, the disclosure relates to a chimeric antigen receptor (CAR) that includes the antibody or antigen-binding fragment thereof described herein.

[0045] In one aspect, the disclosure relates to an antibody-drug conjugate that includes the antibody or antigen-binding fragment thereof described herein covalently bound to a therapeutic agent.

[0046] In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent.

[0047] In one aspect, the present disclosure relates to a method for treating a subject having cancer, the method comprising administering to the subject a composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein.

[0048] In some embodiments, the subject has a solid tumor.

[0049] In some embodiments, the cancer is non-small cell lung cancer (NSCLC), ovarian cancer, melanoma, colorectal cancer, breast cancer, colon adenocarcinoma, hematologic malignancy, head and neck cancer, gastrointestinal cancer, bladder cancer, or bone cancer.

[0050] In some embodiments, the cancer is non-Hodgkin lymphoma, lymphoma, leukemia, acute myeloid leukemia, or chronic lymphocytic leukemia.

[0051] In some embodiments, the subject is further treated with an effective amount of an anti-4-1BB antibody, anti-OX40 antibody, anti-PD-1, anti-CTLA4, or anti-PD-L1 antibody.

[0052] In one aspect, the present disclosure relates to a method for reducing tumor growth rate, the method comprising contacting tumor cells with a composition comprising an effective amount of an antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein.

[0053] In one aspect, the present disclosure relates to a method for killing tumor cells, the method comprising contacting tumor cells with a composition comprising an effective amount of an antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein.

[0054] In one aspect, the present disclosure relates to a method for increasing an immune response in a subject, the method comprising administering to the subject a composition comprising an effective amount of an antibody or antigen-binding fragment thereof described herein.

[0055] In one aspect, the present disclosure relates to a method of treating a subject having a bone disease, the method comprising administering to the subject a composition comprising a therapeutically effective amount of an antibody or an antigen-binding fragment thereof described herein.

[0056] In some embodiments, the bone disease is osteoporosis.

[0057] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0058] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier.

[0059] As used herein, the term "cancer" means cells having the ability of autonomous growth. Examples of such cells include cells having abnormal states or conditions characterized by rapid proliferation of cell growth. This term means including cancerous growths, such as tumors, carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. Malignant tumors of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, blood, nervous, hepatic, gastrointestinal, and endocrine systems, as well as most colorectal cancers, renal cell cancers, prostate cancers and / or testicular tumors, non-small cell lung cancers, and small intestine cancers are also included. "Spontaneously occurring" cancers include any cancers not experimentally induced by transplanting cancer cells into a subject, such as cancers that occur naturally, cancers caused by exposing a patient to carcinogenic substances, cancers caused by insertion of a transgenic cancer gene or knockout of a tumor suppressor gene, and cancers caused by infectious diseases, such as viral infections. The term "carcinoma" is recognized in the art and means a malignant tumor of epithelial or endocrine gland tissue. This term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" means a carcinoma derived from glandular tissue or a carcinoma in which tumor cells form recognizable glandular structures. The term "sarcoma" is recognized in the art and means a malignant tumor of mesenchymal origin. The term "hematopoietic neoplastic disease" includes diseases associated with hyperplastic / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic diseases can arise from the bone marrow, lymphatic system, or erythroid lineage, or their progenitor cells.

[0060] As used herein, the term "antibody" means any antigen-binding molecule that contains at least one (e.g., 1, 2, 3, 4, 5, or 6) complementarity-determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain the Fc region of a human antibody. The term "antibody" also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0061] As used herein, the term "antigen-binding fragment" means a portion of a full-length antibody, and the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, an antigen-binding fragment contains at least one variable domain (e.g., the variable domain of a heavy chain or the variable domain of a light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab’, F(ab’)2, and Fv fragments.

[0062] As used herein, the term "human antibody" means an antibody encoded by endogenous nucleic acids present in a human (e.g., a rearranged human immunoglobulin heavy chain or light chain locus). In some embodiments, a human antibody is recovered from a human or produced in a human cell culture (e.g., within a human hybridoma cell). In some embodiments, a human antibody is produced in non-human cells (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in bacterial cells or yeast cells. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a cow) that contains an unrearranged or rearranged human immunoglobulin locus (e.g., a heavy chain or light chain human immunoglobulin locus).

[0063] As used herein, the term "chimeric antibody" means an antibody that contains sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species such as human and mouse antibodies). Non-limiting examples of chimeric antibodies are antibodies that contain the variable domain sequences (e.g., all or part of the light chain and / or heavy chain variable domain sequences) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0064] As used herein, the term "humanized antibody" means a non-human antibody that contains a minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and also contains sequences derived from a human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (recipient antibody) in which the residues of the hypervariable (e.g., CDR) regions of the recipient antibody are replaced by the residues of the hypervariable (e.g., CDR) regions of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody having the desired specificity, affinity, and capacity. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced by the corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, a humanized antibody can contain residues not found in the recipient antibody or the donor antibody. By making these modifications, the performance of the antibody can be further refined. In some embodiments, a humanized antibody contains at least one, and typically two, variable domains, and all or substantially all of the hypervariable loops (CDRs) correspond to the hypervariable loops of a non-human (e.g., mouse) immunoglobulin, and all or substantially all of the framework regions are of human immunoglobulin sequence. A humanized antibody can also contain the immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods well known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0065] As used herein, the term "single-chain antibody" means a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) that can specifically bind to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0066] As used herein, the term "multimeric antibody" means an antibody containing four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, the multimeric antibody can crosslink one target molecule (e.g., SIGLEC15) to at least one second target molecule on the surface of a mammalian cell (e.g., a human T cell).

[0067] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and refer to an animal, human, or non-human to whom treatment according to the methods of the invention is provided. Veterinary and non-veterinary uses are contemplated by the invention. A human patient can be an adult human or a juvenile human (e.g., a human under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, suids (e.g., pigs, mini-pigs), equids, canids, felids, bovids, and other domestic, livestock, and zoo animals.

[0068] As used herein, when referring to an antibody, the phrases "specifically binds" and "specifically binds to" mean that the interaction depends on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the target molecule; in other words, the reagent generally recognizes and binds to a molecule containing a particular structure rather than the entire molecule, and an antibody preferably interacts with its target molecule (e.g., SIGLEC15) more than with other molecules. An antibody that specifically binds to a target molecule can also be referred to as a target-specific antibody. For example, an antibody that specifically binds to the SIGLEC15 molecule can also be referred to as a SIGLEC15-specific antibody or an anti-SIGLEC15 antibody.

[0069] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and mean a polymer of amino acids of any length, having at least two amino acids.

[0070] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein and mean a polymer of nucleotides of any length, having at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0072] Other features and advantages of the present invention will become apparent from the following detailed description, drawings, and claims.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0074] This disclosure provides examples of antibodies that bind to SIGLEC15 and antigen-binding fragments thereof.

[0075] SIGLEC15 and the Immune System SIGLEC15 (sialic acid-binding immunoglobulin-like lectin 15) belongs to the SIGLEC family and is a type I transmembrane protein. It is hardly expressed in most normal human tissues and immune cell subsets, but shows relatively high expression in macrophages.

[0076] SIGLECs constitute a family of cell surface proteins that play important roles in regulating immune homeostasis. Dysregulation of these proteins is associated with various diseases ranging from autoimmunity to infections and cancer. They are type I transmembrane proteins with one V-set immunoglobulin (Ig) domain containing a sialic acid-binding site and one or more C2-set Ig domains in their extracellular regions. Most SIGLECs, including CD22 (Siglec-2) and most CD33 (Siglec-3)-related SIGLECs, have immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and / or ITIM-like motifs in their cytoplasmic domains and mediate inhibitory receptor signaling. Each SIGLEC preferentially recognizes different types of sialic acids, which are groups of sugars expressed on all mammalian cells as a mechanism for distinguishing self and non-self. However, some pathogens can utilize inhibitory SIGLECs to suppress the immune response and aid in their survival. Most SIGLECs function as receptors, but some SIGLECs such as Siglec-1 can function as functional ligands. SIGLEC expression is mainly found in hematopoietic cells (mainly myeloid cells and B cells) or non-hematopoietic cells such as neurons. Among the SIGLEC family, Siglec-15 has been identified as a very unique member, selectively expressed in myeloid cells and osteoclasts (bone-specific myeloid lineages) and not normally present in other immune cells and tissues.

[0077] The amino acid sequence alignment between human and mouse Siglec-15 shows 83% identity. Unlike other Siglecs that exist on chromosome 19 or 1, the Siglec-15 gene is located on chromosome 18. Initial studies have suggested that Siglec-15 contains a conserved arginine (R143) motif in the membrane-distal IgV domain, which is important for sialic acid binding. Siglec-15 preferentially binds to sialyl-Tn (Neu5Ac alpha 2-6GalNAc), a short O-glycan with sialic acid residues, and its de novo expression or overexpression is associated with various types of epithelial cancers. Importantly, in contrast to most Siglecs that contain one IgV domain and multiple IgC2 domain "tandem repeats" in the extracellular region, Siglec-15 shows only one IgV domain and one IgC2 domain, which are commonly seen in members of the B7 family. There is high structural homology between Siglec-15 and PD-L1, and the protein sequence of the extracellular domain of Siglec-15 shows 20% - 30% identity with the B7 family, which is similar to the identity between members of the B7 family. These characteristic molecular properties highlight the unique nature of Siglec-15 and suggest the possibility of binding to B7 immune regulatory molecules.

[0078] Unlike most Siglec members, Siglec-15 does not have a typical immunoreceptor tyrosine-based inhibitory motif (ITIM) or ITIM-like motif in its intracellular domain that mediates inhibitory signaling. Instead, it has been reported to associate with the 12 kDa signaling adapter DNAX-activating protein (DAP12) and DAP10, which contain immunoreceptor tyrosine-based activation motifs (ITAM), via positively charged lysine residues (K273 in mouse Siglec-15 and K274 in human Siglec-15) within the transmembrane domain. Binding to DAP12 and / or DAP10 is a typical feature of some Siglecs with activating signaling, such as Siglec-14 and Siglec-16, and this may be achieved particularly through the recruitment of spleen tyrosine kinase (SYK) and ZAP70.

[0079] Siglec-15 has been identified as an important regulator of osteoclast differentiation and function. Yoshiharu et al. discovered that Siglec-15 is upregulated on osteoclasts immediately after stimulation by receptor activator of nuclear factor-κB ligand during attempts to identify regulators of osteoclast-like giant cell tumors. Knockdown of Siglec-15 by shRNA, or treatment with a polyclonal antibody against Siglec-15, inhibits osteoclast differentiation and bone resorption. The sialic acid / Siglec-15 axis may constitute a functional loop for osteoclast differentiation, and removal of sialic acid by sialidase, or disruption of sialylated glycan binding by the R143 mutation, inhibits osteoclast development. DAP12 may be required for Siglec-15 function in osteoclasts, because the K273 mutation that disrupts the binding between DAP12 and Siglec-15 resulted in loss of Siglec-15 function in osteoclasts. However, it remains unclear whether Siglec-15 functions mainly as a receptor or a ligand, and the extent to which the binding of DAP-12 and DAP-10 is important for the osteoclast function of Siglec-15.

[0080] Considering its structural similarity to the B7 family and its dominant expression pattern on myeloid cells, the hypothesis was put forward that Siglec-15 might be involved in immune regulation. At the beginning of 2010, using the TCAA screening platform, one group observed the inhibition of NFκB reporter activity of Jurkat T cells by HEK-293T cells expressing Siglec-15. Furthermore, Siglec-15 ectodomain fusion proteins coated on plates or supplied in soluble form potently inhibited anti-CD3 (OKT3)-induced human T cell proliferation. Along this line, Siglec-15 expression on artificial APCs suppressed the proliferation, cytokine secretion, and killing ability of mouse T cells. These in vitro data suggest a ligand-like function of Siglec-15 that suppresses human or mouse T cells via unknown receptor signaling. Subsequently, using the experimental autoimmune encephalomyelitis (EAE) mouse model, the in vivo function of Siglec-15 in T cells was verified. In Siglec-15-deficient mice, or by injection of Siglec-15 ectodomain fusion proteins, EAE was significantly exacerbated and the T cell response was remarkably amplified compared to the control group. Furthermore, Siglec-15 was observed to affect antigen-specific T cell responses, and Siglec-15-deficient mice showed much higher proliferation of OT-I T cells in the blood and spleen immediately after OVA peptide stimulation compared to WT mice, which is similar to the phenotype of PD-L1 KO mice. In this process, Siglec15-deficient mice showed a decrease in serum IL10 levels compared to WT, and the difference in OT-I T cell proliferation between WT mice and Siglec-15-deficient mice was abolished by anti-IL10 mAbs, suggesting that IL10 might be an important factor.

[0081] A detailed review of SIGLEC15 and its functions can be found in Sun, Jingwei, et al. “Siglec-15 as an emerging target for next-generation cancer immunotherapy.” Clinical Cancer Research 27.3 (2021): 680-688, which is incorporated herein by reference in its entirety.

[0082] The present disclosure provides several anti-SIGLEC15 antibodies, antigen-binding fragments thereof, and methods of using these anti-SIGLEC15 antibodies and antigen-binding fragments to inhibit tumor growth, treat cancer, and treat autoimmune diseases.

[0083] Antibodies and antigen-binding fragments The present disclosure provides anti-SIGLEC15 antibodies, and antigen-binding fragments thereof. Generally, an antibody (also called an immunoglobulin) is composed of two classes of polypeptide chains, a light chain and a heavy chain. The non-limiting antibodies of the present disclosure can be intact four-immunoglobulin chain antibodies comprising two heavy chains and two light chains. The heavy chain of the antibody can be any isotype including IgM, IgG, IgE, IgA, or IgD, or a sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a κ light chain or a λ light chain. The antibody can comprise two identical copies of the light chain, and two identical copies of the heavy chain. Each contains one variable domain (or variable region, V H ), and the heavy chains, which contain a plurality of constant domains (or constant regions), are joined to each other within those constant domains via disulfide bonds to form the “stem” of the antibody. Each contains one variable domain (or variable region, V L) and the light chains each containing one constant domain (or constant region) bind to one heavy chain via a disulfide bond. The variable region of each light chain is aligned with the variable region of the heavy chain to which it binds. The variable regions of both the light and heavy chains contain three hypervariable regions, sandwiched between more conserved framework regions (FRs).

[0084] The hypervariable regions, known as complementarity-determining regions (CDRs), form loops that include the principal antigen-binding surface of the antibody. The four framework regions mostly adopt a β-sheet structure, and the CDRs form connecting loops and in some cases part of the β-sheet structure. The CDRs of each chain are held in close proximity to the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding region.

[0085] Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and several CDR definitions are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of structural loop regions. These methods and definitions are described, for example, in Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains,” Molecular immunology 45.14 (2008): 3832-3839; Wu, T.T. and Kabat, E.A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68(1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2): 269-94 (Jan. 1998); Chothia et al., Nature 342(6252): 877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7: 64 (2007), each of which is hereby incorporated by reference in its entirety.

[0086] CDRs are important for recognizing epitopes of antigens. As used herein, an "epitope" is the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but because epitopes can depend on the three-dimensional structure of the antigen based on its secondary and tertiary structure, these amino acids need not be in a continuous linear sequence of the primary structure of the antigen.

[0087] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, with differences in their constant regions, particularly in the hinge and upper CH2 domains. The sequences and differences of IgG subclasses are well known in the art and are described, for example, in Vidarsson, et al, “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5 (2014); Irani, et al. “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2 (2015): 171 - 182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated herein by reference in its entirety.

[0088] The antibodies can also be immunoglobulin molecules derived from any species (e.g., human, rodent, mouse, camel). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies comprising immunoglobulin binding domains fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to a part of an antibody that retains the specific binding activity of the intact antibody, i.e., any part of the antibody that can specifically bind to an epitope on the target molecule of the intact antibody. This includes, for example, Fab, Fab’, F(ab’)2, and variants of these fragments. Thus, in some embodiments, the antibody or its antigen-binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising a binding domain that is an antibody-binding domain or a binding domain homologous thereto. Non-limiting examples of antigen-binding domains include, for example, heavy and / or light chain CDRs of an intact antibody, heavy and / or light chain variable regions of an intact antibody, full-length heavy or light chains of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0089] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-chain variable fragment (scFv) described herein fused to a CD3ζ transmembrane and endodomain. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains derived from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, e.g., CD3z-CD28-41BB, or CD3z-CD28-OX40, to increase potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors described herein.

[0090] In some embodiments, the scFv has one heavy chain variable domain and one light chain variable domain. In some embodiments, the scFv has two heavy chain variable domains and two light chain variable domains.

[0091] In some embodiments, bispecific antibodies targeting SIGLEC15 and additional antigens (e.g., PD-1) can be generated using the sequences of the antibodies or antigen-binding fragments thereof described herein (e.g., CDR or VH / VL sequences).

[0092] Anti-SIGLEC15 antibodies and antigen-binding fragments The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to SIGLEC15 (e.g., human SIGLEC15). The antibodies and antigen-binding fragments described herein are capable of binding to SIGLEC15. These antibodies can be agonists or antagonists. In some embodiments, these antibodies can increase the immune response. In some embodiments, these antibodies can block the SIGLEC15 pathway.

[0093] The present disclosure provides, for example, anti-SIGLEC15 antibodies 10A7, 10C9, 23F4, chimeric antibodies thereof, and human or humanized antibodies thereof.

[0094] CDR sequences for 10A7 and antibodies derived from 10A7 (e.g., human, or humanized antibodies) include the CDR sequences of the heavy chain variable domain, SEQ ID NOs: 1-3, and the CDR sequences of the light chain variable domain, SEQ ID NOs: 4-6, as defined by Kabat numbering. The CDRs can also be defined by the Chothia system. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are described in SEQ ID NOs: 19-21, and the CDR sequences of the light chain variable domain are described in SEQ ID NOs: 22-24.

[0095] Similarly, as the CDR sequences for 10C9 and antibodies derived from 10C9, there are the CDR sequences of the heavy chain variable domain defined by Kabat numbering, SEQ ID NOs: 7-9, and the CDR sequences of the light chain variable domain, SEQ ID NOs: 10-12. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are described in SEQ ID NOs: 25-27, and the CDR sequences of the light chain variable domain are described in SEQ ID NOs: 28-30.

[0096] As the CDR sequences for 23F4 and antibodies derived from 23F4, there are the CDR sequences of the heavy chain variable domain defined by Kabat numbering, SEQ ID NOs: 13-15, and the CDR sequences of the light chain variable domain, SEQ ID NOs: 16-18. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are described in SEQ ID NOs: 31-33, and the CDR sequences of the light chain variable domain are described in SEQ ID NOs: 34-36.

[0097] The amino acid sequence for the heavy chain variable region of the 10A7 antibody is described in SEQ ID NO: 37. The amino acid sequence for the light chain variable region of the 10A7 antibody is described in SEQ ID NO: 38.

[0098] The amino acid sequence for the heavy chain variable region of the 10C9 antibody is described in SEQ ID NO: 39. The amino acid sequence for the light chain variable region of the 10C9 antibody is described in SEQ ID NO: 40.

[0099] The amino acid sequence for the heavy chain variable region of the 23F4 antibody is described in SEQ ID NO: 41. The amino acid sequence for the light chain variable region of the 23F4 antibody is described in SEQ ID NO: 42.

[0100] Also provided are amino acid sequences for the heavy chain variable region and the light chain variable region of the modified antibody. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37, 39, or 41. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 38, 40, or 42. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, and together they bind to SIGLEC15.

[0101] The humanization rate means the ratio of identity of the heavy or light chain variable region sequence compared to the human antibody sequence in the International Immunogenetics Information System (IMGT) database. The top hit means that the heavy or light chain variable region sequence is closer to a specific species than to other species. For example, the top hit for humans means that the sequence is closer to humans than to other species. The top hit for humans and cynomolgus monkeys means that the sequence has the same ratio of identity to the human sequence and the cynomolgus monkey sequence, and these ratios of identity are the highest compared to the sequences of other species. In some embodiments, the humanization rate exceeds 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions of the method for determining the humanization rate and the method for determining the top hit are well known in the art and are described, for example, in Jones, et al. “The INNs and outs of antibody nonproprietary names.” MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, the entire content of which is incorporated herein by reference. A high humanization rate often has various advantages. For example, it is safer, more effective in humans, more likely to be tolerated by human subjects, and / or less likely to have side effects. In some embodiments, the variable region is fully human, for example, derived from the human heavy chain immunoglobulin locus sequence (e.g., a combination of human IGHV, human IGHD, and human IGHJ genes), and / or the human κ chain immunoglobulin locus sequence (e.g., a combination of human IGKV and human IGKJ genes).

[0102] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain 1, 2, or 3 heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1-3, SEQ ID NOs: 7-9, SEQ ID NOs: 13-15, SEQ ID NOs: 19-21, SEQ ID NOs: 25-27, and SEQ ID NOs: 31-33, and / or 1, 2, or 3 light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 4-6, SEQ ID NOs: 10-12, SEQ ID NOs: 16-18, SEQ ID NOs: 22-24, SEQ ID NOs: 28-30, and SEQ ID NOs: 34-36.

[0103] In some embodiments, an antibody can have a heavy chain variable region (VH) that includes complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence. In some embodiments, an antibody can have a light chain variable region (VL) that includes CDRs 1, 2, and 3, wherein the CDR1 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. The selected VH CDR1, 2, 3 amino acid sequences, and the selected VL CDR1, 2, 3 amino acid sequences are shown in FIGS. 11 (Kabat CDRs) and 12 (Chothia CDRs).

[0104] In some embodiments, the antibodies or antigen-binding fragments described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 2 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, and SEQ ID NO: 3 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0105] In some embodiments, the antibodies or antigen-binding fragments described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 8 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, and SEQ ID NO: 9 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0106] In some embodiments, the antibodies or antigen-binding fragments described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 14 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, and SEQ ID NO: 15 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0107] In some embodiments, the antibodies or antigen-binding fragments described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 20 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, and SEQ ID NO: 21 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0108] In some embodiments, the antibodies or antigen-binding fragments described herein can contain a heavy-chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 26 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 27 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0109] In some embodiments, the antibodies or antigen-binding fragments described herein can contain a heavy-chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 31 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 32 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 33 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0110] In some embodiments, the antibodies or antigen-binding fragments described herein can contain a light-chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 5 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 6 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0111] In some embodiments, the antibodies or antigen-binding fragments described herein can contain a light-chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 11 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 12 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0112] In some embodiments, the antibody or antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 17 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, and SEQ ID NO: 18 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0113] In some embodiments, the antibody or antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 23 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, and SEQ ID NO: 24 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0114] In some embodiments, the antibody or antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 28 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 29 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, and SEQ ID NO: 30 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0115] In some embodiments, the antibody or antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 34 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 35 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, and SEQ ID NO: 36 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0116] Insertions, deletions, and substitutions can be present within the CDR sequences, or at one or both ends of the CDR sequences. In some embodiments, the CDRs are determined based on the Kabat numbering scheme. In some embodiments, the CDRs are determined based on the Chothia numbering scheme. In some embodiments, the CDRs are determined based on a combination of the Kabat and Chothia numbering schemes.

[0117] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to SIGLEC15. The antibody or antigen-binding fragment thereof contains a heavy chain variable region (VH) comprising, or consisting of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising, or consisting of, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 37 and the selected VL sequence is SEQ ID NO: 38. In some embodiments, the selected VH sequence is SEQ ID NO: 39 and the selected VL sequence is SEQ ID NO: 40. In some embodiments, the selected VH sequence is SEQ ID NO: 41 and the selected VL sequence is SEQ ID NO: 42.

[0118] The present disclosure also provides an antibody or an antigen-binding fragment thereof that is capable of competing with the antibodies described herein. In some aspects, the antibody or antigen-binding fragment can bind to the same epitope as the antibodies described herein.

[0119] The present disclosure also provides an antibody or antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment described herein. Cross-competition assays are well known in the art and are described, for example, in Moore et al., “Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein.” Journal of virology 70.3 (1996): 1863-1872, which is incorporated herein by reference in its entirety. In one aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment described herein. Epitope-binding assays are well known in the art and are described, for example, in Estep et al. “High throughput solution-based measurement of antibody-antigen affinity and epitope binning.” MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0120] To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences to optimize alignment for comparison, and non-homologous sequences may be disregarded). Subsequently, the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. For example, sequence comparison and determination of percent identity between two sequences can be performed using the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0121] The present disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises the CDRs shown in FIG. 11 or FIG. 12, or has the sequence shown in FIG. 13. When the polypeptide forms a pair with a corresponding polypeptide (e.g., a corresponding heavy chain variable region, or a corresponding light chain variable region), the polypeptide forming the pair binds to SIGLEC15 (e.g., human SIGLEC15).

[0122] Anti-SIGLEC15 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of the antibody or antibody fragment, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, antibodies produced intracellularly (i.e., intrabodies), and antigen-binding fragments thereof. The antibody or its antigen-binding fragment can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0123] Antibody fragments are suitable for use in the provided methods as long as they retain the desired affinity and specificity of the full-length antibody. Thus, fragments of antibodies that bind to SIGLEC15 retain the ability to bind to SIGLEC15. An Fv fragment is an antibody fragment that contains the complete antigen recognition and binding site. This region is composed of a dimer of one heavy-chain variable domain and one light-chain variable domain closely associated, which can be covalently linked, for example, in an scFv. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Overall, the six CDRs or subsets thereof together confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for a particular antigen) can have the ability to recognize and bind an antigen, but usually with lower affinity than the entire binding site.

[0124] A single-chain Fv (scFv) or antibody fragment contains the VH and VL domains (or regions) of an antibody, and these domains are present within a single polypeptide chain. Generally, the scFv polypeptide further contains a polypeptide linker between the VH domain and the VL domain, which enables the scFv to form a desirable structure for antigen binding.

[0125] A Fab fragment contains the variable and constant domains of the light chain, as well as the variable domain and the first constant domain (CH1) of the heavy chain. An F(ab’)2 antibody fragment generally contains a pair of Fab fragments covalently linked near the carboxy terminus by the hinge cysteines between them. Other chemical linkages of antibody fragments are well known in the art.

[0126] A diabody is a small antibody fragment containing two antigen-binding sites, and this fragment contains VH connected to VL (VH and VL) within the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains can be made to pair with the complementary domains on another chain to generate two antigen-binding sites.

[0127] A linear antibody contains a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with a complementary light chain polypeptide. A linear antibody may be bispecific or monospecific.

[0128] A single-armed antibody can have a heavy chain fragment that includes a heavy chain and a light chain, and the CH2 and CH3 domains of IgG (B in Figure 1). In some embodiments, a single-armed antibody is an antibody that has only one of the two antigen-binding arms in a typical antibody. In some embodiments, a single-armed antibody includes an antigen-binding arm (e.g., VH+CH1 and VL+CL) and an Fc.

[0129] The antibodies and antibody fragments of the present disclosure can be modified within the Fc region to confer desired effector functions or serum half-lives. In some embodiments, the Fc region can be modified to silence or reduce complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).

[0130] Multimerization of antibodies can be achieved by natural aggregation of the antibodies or by chemical or recombinant conjugation techniques well known in the art. For example, some percentage of a purified antibody preparation (e.g., purified IgG1 molecules) will spontaneously form protein aggregates that contain antibody homodimers and other higher-order antibody multimers.

[0131] Alternatively, antibody homodimers can be formed by chemical conjugation techniques well known in the art. For example, heterobifunctional crosslinking agents including, but not limited to, SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acetylthioacetate) can be used to form antibody multimers. An exemplary procedure for forming antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94:7509-7514, 1997). Antibody homodimers can be converted to Fab’2 homodimers by pepsin digestion. Another method for forming antibody homodimers is the method using autophilic T15 peptides described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0132] In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell culture. For example, the interface can comprise at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0133] Examples of bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies within the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be produced using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0134] Methods for generating bispecific antibodies from antibody fragments are also well known in the art. For example, bispecific antibodies can be prepared using chemical linkages. Brennan et al. (Science 229:81, 1985) describe procedures in which intact antibodies are cleaved by proteolysis to generate F(ab’)2 fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize the neighboring dithiols and prevent intermolecular disulfide formation. The resulting Fab’ fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’TNB derivatives is then reconverted to Fab’ thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab’TNB derivative to form a bispecific antibody.

[0135] Any of the antibodies or antigen-binding fragments described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life of an antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans), or can extend its biological activity.

[0136] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. Antibody-drug conjugates comprising an antibody or an antigen-binding fragment thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), dion, mitoxantrone, mitomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and analogs).

[0137] In some embodiments, the antibodies or antigen-binding fragments thereof described herein recognize endogenous SIGLEC15, or recombinant SIGLEC15. In some embodiments, the antibodies or antigen-binding fragments thereof described herein recognize human SIGLEC15.

[0138] In some embodiments, the half-life of the antibodies or antigen-binding fragments thereof described herein in wild-type mice (e.g., C57BL / 6 mice) is at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days. In some embodiments, the half-life of the antibodies or antigen-binding fragments thereof described herein in SIGLEC15 gene humanized mice (e.g., hSIGLEC15 mice) is at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days. In some embodiments, the maximum concentration (Cmax) of the antibodies or antigen-binding fragments thereof described herein in SIGLEC15 gene humanized mice (e.g., hSIGLEC15 mice) is at least 50 μg / mL, at least 75 μg / mL, at least 100 μg / mL, at least 125 μg / mL, at least 150 μg / mL, at least 175 μg / mL, or at least 200 μg / mL. In some embodiments, the serum clearance rate (CL) of the antibodies or antigen-binding fragments thereof described herein in SIGLEC15 gene humanized mice (e.g., hSIGLEC15 mice) is at least 7.5 mL / day / kg, at least 10 mL / day / kg, at least 12.5 mL / day / kg, at least 15 mL / day / kg, at least 17.5 mL / day / kg, at least 20 mL / day / kg, at least 25 mL / day / kg, at least 30 mL / day / kg, at least 35 mL / day / kg, or at least 40 mL / day / kg.

[0139] In some embodiments, the clearance rate (CL) of the antibodies or antigen-binding fragments thereof described herein in wild-type mice (e.g., C57BL / 6 mice) is less than 7 mL / day / kg, less than 6 mL / day / kg, less than 5 mL / day / kg, or less than 4 mL / day / kg. In some embodiments, the clearance rate (CL) of the antibodies or antigen-binding fragments thereof described herein in SIGLEC15 gene humanized mice (e.g., hSIGLEC15 mice) is less than 15 mL / day / kg, less than 14 mL / day / kg, less than 13 mL / day / kg, or less than 12 mL / day / kg.

[0140] In some embodiments, the half-life of the antibodies or antigen-binding fragments thereof described herein (e.g., in FcRn gene humanized mice) is at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. In some embodiments, the clearance rate (CL) of the antibodies or antigen-binding fragments thereof described herein (e.g., in FcRn gene humanized mice) is less than 16 L / day / kg, less than 15 mL / day / kg, less than 14 mL / day / kg, less than 13 mL / day / kg, less than 12 mL / day / kg, less than 11 mL / day / kg, less than 10 mL / day / kg, less than 9 mL / day / kg, less than 8 mL / day / kg, or less than 7 mL / day / kg.

[0141] Properties of the Antibody The antibodies or antigen-binding fragments thereof described herein can block the binding between SIGLEC15 and a SIGLEC15 ligand (e.g., sialic acid).

[0142] The antibodies or antigen-binding fragments thereof described herein can be SIGLEC15 pathway agonists or antagonists. In some embodiments, by binding to SIGLEC15, the antibody can inhibit the SIGLEC15 signaling pathway. In some embodiments, the antibody can upregulate or downregulate the immune response.

[0143] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase the immune response, the activity or number of immune cells (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen-presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can decrease the activity or number of immune cells (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen-presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0144] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce the number of mCD45+ cells, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce the amount of G-MDSC in mCD45+ cells, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase the amount of M-MDSC in mCD45+ cells, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, or 10-fold. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase the amount of CTL in mCD45+ cells, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, or 10-fold. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase the amount of Th cells in mCD45+ cells, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, or 10-fold.

[0145] In some embodiments, the antibody (or antigen-binding fragment thereof) specifically binds to SIGLEC15 (e.g., human SIGLEC15, monkey SIGLEC15 (e.g., rhesus monkey, cynomolgus monkey), canine SIGLEC15, mouse SIGLEC15, and / or chimeric SIGLEC15) with a dissociation rate (koff) of less than 0.1 s -1 less than, 0.01 s -1 less than, 0.001 s -1 less than, 0.0001 s -1 less than, 0.00001 s -1 less than, 0.000001 s -1 less than, or 0.0000001 s -1 less than. In some embodiments, the dissociation rate (koff) is 0.01 s -1Ultra, 0.001 s -1 Ultra, 0.0001 s -1 Ultra, 0.00001 s -1 Ultra, 0.000001 s -1 Ultra, 0.0000001 s -1 Ultra, or 0.00000001 s -1 Is ultra.

[0146] In some embodiments, the association rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms, or greater than 1×10 6 / Ms. In some embodiments, the association rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms, or less than 1×10 7 / Ms.

[0147] The affinity can be estimated from the quotient of the rate constants of dissociation (KD = koff / kon). In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10-8 M, less than 1×10 -9 M, less than 1×10 -10 M, less than 1×10 -11 M, less than 1×10 -12 M, less than 1×10 -13 M, or less than 1×10 -14 M. In some embodiments, KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M, greater than 1×10 -12 M, greater than 1×10 -13 M, greater than 1×10 -14 M.

[0148] As general techniques for measuring the affinity of an antibody for an antigen, for example, ELISA, RIA, and surface plasmon resonance (SPR) can be mentioned. In some embodiments, the antibody binds to human SIGLEC15 (SEQ ID NO: 45), mouse SIGLEC15 (SEQ ID NO: 46), monkey SIGLEC15 (SEQ ID NO: 47), dog SIGLEC15 (SEQ ID NO: 48), and / or chimeric SIGLEC15 (SEQ ID NO: 67). In some embodiments, the antibody does not bind to human SIGLEC15, mouse SIGLEC15, monkey SIGLEC15, dog SIGLEC15, and / or chimeric SIGLEC15.

[0149] In some embodiments, the thermal stability is measured. The antibodies or antigen-binding fragments described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, the Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

[0150] In some embodiments, the antibodies or antigen-binding fragments described herein can bind to the same epitope of SIGLEC15. In some embodiments, the antibodies or antigen-binding fragments described herein can bind to different epitopes of SIGLEC15.

[0151] In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a purity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more as determined by size exclusion chromatography (SEC). In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a hydrophobic interaction chromatography (HIC) retention time of more than 2 minutes, more than 2.5 minutes, more than 3 minutes, more than 3.5 minutes, more than 4 minutes, or more than 4.5 minutes. In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a main peak constituting more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90% as determined by capillary isoelectric focusing (cIEF). In some embodiments, the antibodies or antigen-binding fragments thereof described herein have an isoelectric point (PI) of more than 7.5, more than 7.75, more than 8, more than 8.25, more than 8.5, or more than 8.75 as determined by capillary isoelectric focusing (cIEF). In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a half maximal effective concentration (EC50) of less than 2.5, less than 2, less than 1.5, less than 1, less than 0.75, less than 0.5, less than 0.25, or less than 0.15 μg / mL.

[0152] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can relieve the cell growth inhibition caused by hSIGLEC15. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can relieve more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, or more than 70% of the hCD4+ T cell growth inhibition caused by hSIGLEC15. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can relieve more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, or more than 70% of the hCD8+ T cell growth inhibition caused by hSIGLEC15.

[0153] In some embodiments, the antibody has a tumor growth inhibition rate (TGI%) of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or more than 200%. In some embodiments, the antibody has a tumor growth inhibition rate of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% can be measured, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, the tumor growth inhibition rate (TGI%) is calculated using the following formula: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100 Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day 0. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day 0.

[0154] In some embodiments, the antibody or antigen-binding fragment thereof described herein is a SIGLEC15 antagonist. In some embodiments, the antibody or antigen-binding fragment reduces SIGLEC15 signaling in target cells expressing SIGLEC15.

[0155] In some embodiments, the antibody or antigen-binding fragment can enhance APC (e.g., DC cell) functions, such as induction of co-stimulation and surface expression of MHC molecules, induction of pro-inflammatory cytokine production, and / or improvement of T cell triggering function.

[0156] In some embodiments, the antibody or antigen-binding fragment can bind to tumor cells expressing SIGLEC15. In some embodiments, the antibody or antigen-binding fragment can induce complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) and kill tumor cells.

[0157] In some embodiments, the antibody or antigen-binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis.

[0158] In some embodiments, the antibody or antigen-binding fragment can induce complement-dependent cytotoxicity (CDC).

[0159] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is optionally a human IgG1 antibody having an SI mutation, LALA mutation, N297A mutation, YTE mutation, and / or FLAA mutation. In some embodiments, the antibody is optionally a human IgG4 antibody having an SI mutation, LALA mutation, N297A mutation, YTE mutation, and / or FLAA mutation.

[0160] In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab, Fab’, F(ab’)2, and Fv fragment. In some embodiments, the Fc region has an LALA mutation (L234A and L235A mutations according to EU numbering), or an LALA-PG mutation (L234A, L235A, P329G mutations according to EU numbering).

[0161] In some embodiments, the Fc region has the FLAA mutations (F234A and L235A according to EU numbering). In some embodiments, the Fc has the SI mutations (S239D and I332E mutations according to EU numbering). In some embodiments, the Fc has the N297A mutation according to EU numbering. In some embodiments, the Fc has the YTE mutations (M252Y, S254T, and T256E according to EU numbering).

[0162] In some embodiments, the antibody or antigen-binding fragment described herein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 70-77.

[0163] Method for producing anti-SIGLEC15 antibody An isolated fragment of human SIGLEC15 can be used as an immunogen, and antibodies can be generated using standard techniques for the preparation of polyclonal and monoclonal antibodies. Polyclonal antibodies can be raised in animals by injecting the antigenic peptide or protein multiple times (e.g., subcutaneously or intraperitoneally). In some embodiments, the antigenic peptide or protein is injected together with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. The antigenic peptide or protein can be injected into the animal two or more times (e.g., 2, 3, or 4 times).

[0164] A full-length polypeptide or protein can be used, or an antigenic peptide fragment thereof can be used as an immunogen. The antigenic peptide of the protein contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of SIGLEC15 and encompasses the epitope of the protein such that an antibody generated against the peptide forms a specific immune complex with the protein. As described above, the full-length sequence of human SIGLEC15 (SEQ ID NO: 45) is well known in the art. In some embodiments, a human SIGLEC15 protein with an Fc tag or a His tag is used as the immunogen.

[0165] An immunogen is typically used for the preparation of antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations can contain, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., fragments of human SIGLEC15). The preparation can further contain an adjuvant such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0166] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a SIGLEC15 polypeptide, or an antigenic peptide thereof (e.g., a part of SIGLEC15), as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques such as an enzyme-linked immunosorbent assay (ELISA) using an immobilized SIGLEC15 polypeptide or peptide. If desired, antibody molecules can be isolated from a mammal (e.g., from blood) and further purified by well-known techniques such as protein G chromatography or protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, for example, when the titer of specific antibodies is at its maximum, antibody-producing cells are obtained from the subject and used to prepare monoclonal antibodies by standard techniques such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or trioma technique. Techniques for producing hybridomas are well known (see generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). For example, monoclonal antibodies can be detected by screening hybridoma culture supernatants for antibodies that bind to the polypeptide or epitope of interest using a standard ELISA assay.

[0167] Appropriate nucleotide changes can be introduced into DNA encoding a human, humanized, or chimeric antibody, or an antibody or antigen-binding fragment thereof described herein, or variants of the antibodies or antigen-binding fragments described herein can be prepared by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids of the sequence that makes up the antigen-binding site or antigen-binding domain of the antibody. In such a population of variants, some antibodies or antigen-binding fragments have an increased affinity for a target protein, such as SIGLEC15. Any combination of deletions, insertions, and / or combinations can be achieved in an antibody or antigen-binding fragment thereof with an increased binding affinity for the target. Changes in the number of glycosylation sites (e.g., increase or decrease), changes in the type of glycosylation sites (e.g., changing the amino acid sequence so that different sugars are attached by enzymes present in the cell), or introduction of new glycosylation sites, etc., can change the antibody or antigen-binding fragment, or introduce new post-translational modifications into the antibody or antigen-binding fragment by changes in the amino acids introduced into the antibody or antigen-binding fragment.

[0168] The antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of native antibodies include antibodies derived from humans, primates such as monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically engineered to produce human antibodies.

[0169] Examples of human and humanized antibodies include antibodies having variable and constant regions derived from human germline immunoglobulin sequences (or having the same amino acid sequence as those derived therefrom). Examples of human antibodies may include amino acid residues within the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutations in vivo).

[0170] Humanized antibodies typically have a human framework (FR) to which non-human CDRs are grafted. Thus, humanized antibodies have one or more amino acid sequences introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues and typically are obtained from the "import" variable domain. Humanization can essentially be carried out, for example, by replacing rodent CDRs or CDR sequences with the corresponding sequences of human antibodies. These methods are described, for example, in Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988), each of which is incorporated herein by reference in its entirety. Thus, a "humanized" antibody is a chimeric antibody in which a substantially smaller portion than the intact human V domain is replaced by the corresponding sequence derived from a non-human species. In practice, a typical humanized antibody is a mouse antibody in which some CDR residues and some FR residues are replaced by residues derived from similar sites within the human antibody.

[0171] The selection of human VH and VL domains for use in the production of humanized antibodies is very important for reducing immunogenicity. According to the so-called "best fit" method, the sequences of the V domains of mouse antibodies are screened against the entire library of known human domain sequences. The human sequence closest to the mouse sequence is then recognized as the human FR for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).

[0172] Furthermore, it is important to humanize the antibody while retaining high specificity and affinity for the antigen, as well as other desirable biological properties. To achieve this goal, humanized antibodies can be prepared by an analytical process of the parental and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that can illustrate and display the possible three-dimensional conformations of the selected candidate immunoglobulin sequences. By observing these displays, it becomes possible to analyze the possible roles of the residues in the functionalization of the candidate immunoglobulin sequences, i.e., to analyze the residues of the candidate immunoglobulin that affect its binding ability to the antigen. In this way, FR residues can be selected and combined from the recipient and import sequences to achieve the desired antibody properties, such as an increase in affinity for the target antigen.

[0173] Typically, amino acid sequence variants of anti-SIGLEC15 antibodies that are human, humanized, or chimeric contain amino acid sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity percent to the sequences present in the light or heavy chain of the original antibody.

[0174] In some embodiments, a mouse having a humanized heavy chain immunoglobulin locus and a humanized κ chain immunoglobulin locus (e.g., RenMab TMUsing a mouse, antibodies are generated. A heavy-chain immunoglobulin locus is a region on a chromosome that contains genes for the heavy chain of an antibody. The locus can include, for example, human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy-chain constant domain genes. A κ-chain immunoglobulin locus is a region on a chromosome that contains genes encoding the light chain (κ-chain) of an antibody. Examples of κ-chain immunoglobulin loci can include human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light-chain constant domain genes. RenMab TM Detailed descriptions regarding the mouse can be found in PCT / CN2020 / 075698 or US20200390073A1, the entire contents of which are incorporated herein by reference.

[0175] In some embodiments, a mouse having a humanized heavy-chain immunoglobulin locus and a humanized κ-chain immunoglobulin locus (e.g., RenLite TM mouse) is used to generate antibodies. A heavy-chain immunoglobulin locus is a region on a chromosome that contains genes for the heavy chain of an antibody. The locus can include, for example, human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy-chain constant domain genes. A κ-chain immunoglobulin locus is a region on a chromosome that contains genes encoding a common light chain. Examples of κ-chain immunoglobulin loci can include human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light-chain constant domain genes. RenLite TM Detailed descriptions regarding the mouse can be found in PCT / CN2021 / 097652, the entire contents of which are incorporated herein by reference.

[0176] Antibodies generated by mice have a fully human VH, a fully human VL, and a murine constant region. In some embodiments, the human VH and human VL bind to a human IgG constant region (e.g., IgG1, IgG2, IgG3, and IgG4). In some embodiments, the constant region has a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs: 70-77.

[0177] Identity or homology to the original sequence is typically the percentage of amino acid residues present in a candidate sequence that is identical to the sequence present within a human, humanized, or chimeric anti-SIGLEC15 antibody or fragment, where the sequences are aligned, gaps are introduced if necessary to achieve maximum percent sequence identity, and conservative substitutions as part of sequence identity are not considered.

[0178] Further modifications can be made to the anti-SIGLEC15 antibody or antigen-binding fragment. For example, cysteine residues can be introduced into the Fc region to enable interchain disulfide bond formation within this region. The homodimeric antibodies thus generated may have some increased in vitro and / or in vivo half-life. For example, homodimeric antibodies with increased in vitro and / or in vivo half-life can also be prepared using heterobifunctional crosslinking agents as described in Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, antibodies having a double Fc region can be engineered (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0179] In some embodiments, a covalent modification can be added to the anti-SIGLEC15 antibody or its antigen-binding fragment. These covalent modifications can be added by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting the target amino acid residue of the antibody or fragment with an organic derivatizing agent capable of reacting with the selected side chain, or the N- or C-terminal residue.

[0180] In some embodiments, provided are antibody variants having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined, for example, by calculating the average amount of fucose in the sugar chain located at Asn297 relative to the total of all sugar structures (e.g., complex, hybrid, and high-mannose structures) bound to Asn297, as measured by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 in the Fc region (numbering according to Eu of the Fc region residues, or position 314 in the Kabat numbering), however, Asn297 may also be located between about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody is further recombinantly engineered to replace the asparagine at position 297 with alanine (N297A).

[0181] In some embodiments, to promote production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further engineered to replace serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is provided, for example, in Silva et al. “The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.” Journal of Biological Chemistry 290.9 (2015): 5462-5469, which is incorporated herein by reference in its entirety.

[0182] Recombinant vector The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cells contain the polynucleotide and / or the vector containing the polynucleotide), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

[0183] As used herein, a "vector" is any construct that can deliver one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" can deliver one or more polynucleotides of interest as an expressed polypeptide within a host cell into which the expression vector has been introduced. Thus, within the expression vector, the polynucleotide of interest is placed at, near, or adjacent to the integration site of the polynucleotide of interest such that it is operably linked to control elements such as a promoter, enhancer, and / or polyA tail either within the vector or in the genome of the host cell, so that the polynucleotide of interest is translated within the host cell into which the expression vector has been introduced for expression within the vector.

[0184] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant virus). Thus, non-limiting examples of vectors include viral vectors (those that can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0185] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses), which may involve the use of non-pathogenic (defective) replicable viruses or non-replicable viruses. In the latter case, viral propagation generally occurs only in complementary virus packaging cells. For example, Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berchner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207 disclose suitable systems. Techniques for incorporating DNA into such expression systems are well known to those of skill in the art. DNA may also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. Incorporation of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0186] For expression, the DNA insert containing a polynucleotide encoding an antibody or a polypeptide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage λPL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters, and the promoter of the retroviral LTR. Other suitable promoters are known to those skilled in the art. The expression construct can further contain sites for transcription initiation and termination, and, within the transcription region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct can include a translation start at the beginning and a stop codon (UAA, UGA or UAG) approximately located at the end of the polypeptide to be translated.

[0187] As shown, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase for eukaryotic cell culture, or neomycin resistance, and the tetracycline or ampicillin resistance genes for E. coli and other bacterial cultures. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Suitable culture media and conditions for the host cells described herein are well known in the art.

[0188] Non-limiting vectors for use in bacteria include pQE70, pQE60, and pQE-9 available from Qiagen, pBS vectors available from Stratagene, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic cell vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0189] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters, and trp promoter. Suitable eukaryotic cell promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTR promoters such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0190] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters such as the alpha factor, alcohol oxidase, and PGH may be used. See Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y, and Grant et al., Methods Enzymol., 153:516-544 (1997) for a review.

[0191] Introduction of the construct into host cells can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), the entirety of which is incorporated herein by reference.

[0192] Transcription of the DNA encoding the antibodies of the present disclosure by more eukaryotes can be increased by inserting enhancer sequences into the vector. Enhancers are typically cis-acting elements of DNA, about 10 - 300 bp, that serve to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located behind the origin of replication at base pairs 100 - 270, the cytomegalovirus immediate early promoter enhancer, the polyoma enhancer behind the origin of replication, and the adenovirus enhancer.

[0193] To secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. The signal can be endogenous to the polypeptide or the signal can be a heterologous signal.

[0194] A polypeptide (e.g., an antibody) can be expressed in a modified form such as a fusion protein (e.g., a GST fusion), or with a histidine tag, and can contain not only a secretion signal but also additional heterologous functional regions. For example, regions of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and durability in the host cell during purification or subsequent handling and storage. Also, a peptide moiety can be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. Adding a peptide moiety to a polypeptide to, inter alia, effect secretion or excretion, improve stability, and facilitate purification is well-known and routine in the art.

[0195] Treatment method The antibodies or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.

[0196] In one aspect, the present disclosure provides a method for treating cancer in a subject, a method for decreasing over time the rate of increase in tumor volume in a subject, a method for reducing the risk of metastasis occurring, or a method for reducing the risk of further metastasis occurring in a subject. In some embodiments, the treatment can interrupt, slow, arrest, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in the subject.

[0197] In one aspect, the present disclosure relates to a method comprising administering to a subject in need thereof (e.g., a subject having or identified or diagnosed as having cancer, e.g., breast cancer (e.g., triple negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or a hematologic malignancy) a therapeutically effective amount of an antibody or an antigen-binding fragment thereof disclosed herein. In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the cancer is NSCLC, ovarian cancer, melanoma, colorectal cancer, breast cancer, hematologic malignancy, head and neck cancer, gastrointestinal cancer, bladder cancer, or bone cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple negative breast cancer (TNBC), or colon cancer. In some embodiments, the subject has Hodgkin lymphoma. In some embodiments, the subject has triple negative breast cancer (TNBC), gastric cancer, urothelial cancer, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, hematologic malignancy, particularly non-Hodgkin lymphoma, lymphoma, chronic lymphocytic leukemia, or progressive solid tumor.

[0198] In some embodiments, the compositions and methods disclosed herein can be used for the treatment of patients at risk of cancer. Patients having cancer can be identified by various methods well known in the art.

[0199] In one aspect, the present disclosure provides a method for treating, preventing, or reducing the risk of developing a bone disease such as fibrous dysplasia, renal disease (renal osteodystrophy, Fanconi syndrome), tumor-induced osteomalacia, hypophosphatasia, McCune-Albright syndrome, or osteogenesis imperfecta with calcification defects. In some embodiments, the bone calcification disorder is osteoporosis.

[0200] In one aspect, the present disclosure provides a method for treating, preventing, or reducing the risk of developing an abnormal or undesirable immune response, such as a disorder associated with an autoimmune disease, for example, by affecting the functional properties of APC cells (e.g., by blocking the interaction between SIGLEC15 and its ligand). These autoimmune diseases include, but are not limited to, alopecia areata, lupus, ankylosing spondylitis, Meniere's disease, antiphospholipid syndrome, mixed connective tissue disease, autoimmune Addison's disease, multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, autoimmune hepatitis, pemphigus vulgaris, Behcet's disease, pernicious anemia, bullous pemphigoid, polyarthritis nodosa, myocarditis, polychondritis, dermatitis herpetiformis, polyglandular syndrome, chronic fatigue immune dysfunction syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelination, polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary agammaglobulinemia, Churg-Strauss syndrome, primary biliary cirrhosis, cicatricial pemphigoid, psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, rheumatic fever, discoid lupus, rheumatoid arthritis, cold globulinemia sarcoidosis, fibromyalgia, scleroderma, Graves' disease, Sjogren's syndrome, Guillain-Barre, stiff man syndrome, Hashimoto's thyroiditis, Takayasu arteritis, idiopathic pulmonary fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenic purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes (e.g., type I), vasculitis, lichen planus, and vitiligo. An anti-SIGLEC15 antibody or an antigen-binding fragment thereof can also be administered to a subject to treat, prevent, or reduce the risk of progression of a disorder associated with an abnormal or undesirable immune response related to cell, tissue, or organ transplantation, such as kidney, liver, and heart transplantation, for example, graft-versus-host disease (GVHD), or to prevent allograft rejection. In some embodiments, the subject suffers from a skin disease, a liver disease (e.g., cirrhosis), hidradenitis suppurativa, or experimental autoimmune encephalomyelitis. In some embodiments, the subject has a kidney disease, lupus, Sjogren's syndrome, ulcerative colitis, psoriasis, hidradenitis suppurativa, immune thrombocytopenia (ITP), or other inflammatory arthritis. In some embodiments, the subject suffers from multiple sclerosis or myasthenia gravis.In some embodiments, the subject has Crohn's disease, ulcerative colitis, or type 1 diabetes. In some embodiments, the subject has an autoimmune thyroid disease, Graves' disease, multiple sclerosis, psoriasis, an inflammatory bowel disease (e.g., Crohn's disease (CD) and ulcerative colitis), rheumatoid arthritis, Sjogren's syndrome, autoimmune nephritis, or systemic lupus erythematosus. In some embodiments, the method comprises administering to the subject a composition comprising an effective amount of an antibody or antigen-binding fragment thereof described herein.

[0201] As used herein, "effective amount" means an amount or dose sufficient to produce a beneficial or desired result, including interrupting, slowing, preventing, or inhibiting the progression of a disease, e.g., an autoimmune disease or cancer. The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition thereof is administered, the severity of the symptoms, and the route of administration, and thus, administration can be determined on an individual basis.

[0202] The effective amount can be administered in one or more administrations. For example, an effective amount of an antibody or antigen-binding fragment is an amount sufficient to alleviate, arrest, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or, in vitro, an amount sufficient to alleviate, arrest, stabilize, reverse, slow, and / or delay the growth of a cell (e.g., a biopsy cell, any of the cancer cells described herein, or a cell line (e.g., a cancer cell line)). As will be understood in the art, the effective amount of an antibody or antigen-binding fragment can vary depending, inter alia, on the patient's medical history, as well as other factors such as the type (and / or dose) of antibody used.

[0203] The effective amount and schedule for administering the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein can be determined experimentally, and making such determinations is within the scope of those skilled in the art. Those skilled in the art will understand that the dosage that needs to be administered will vary depending on, for example, the mammal receiving the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein, the route of administration, the specific type of antibody, the polynucleotide encoding the antibody, the antigen-binding fragment, and / or the composition disclosed herein that is being used, and other agents being administered to the mammal. Guidance for selecting an appropriate dosage for an antibody or antigen-binding fragment can be found in the literature regarding the therapeutic use of antibodies and antigen-binding fragments, such as Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

[0204] The typical daily dose of the effective amount of the antibody is from 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose can be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose can be more than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dose is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0205] In any of the methods described herein, at least one antibody, an antigen-binding fragment thereof, or a pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein), and optionally, at least one additional therapeutic agent, can be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment, and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment, and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment, and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered as a sustained-release oral formulation.

[0206] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administering at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents, and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that there is an overlap between the biological activity period of the one or more additional therapeutic agents and the biological activity period of at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) within the subject.

[0207] In some embodiments, at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) can be administered to a subject over a long period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of the treatment period using any of the methods described herein to diagnose or monitor the effectiveness of the treatment (e.g., observe at least one symptom of cancer). As described herein, a skilled medical professional can also vary (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to the subject, and can adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and methods well known in the art).

[0208] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, inhibitors of anaplastic lymphoma kinase (ALK), inhibitors of phosphatidylinositol 3-kinase (PI3K), Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, inhibitors of Bruton's tyrosine kinase (BTK), and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) (e.g., epacadostat).

[0209] In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of the activated hedgehog signaling pathway, and an agent that selectively degrades estrogen receptor.

[0210] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, reolysin, alimta, zykadia, sutent, temsirolimus, axitinib, everolimus, sorafenib, votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitor, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0211] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF)α, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0212] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0213] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, an anti-ICOS antibody, an anti-CD27 antibody, an anti-OX40 antibody, an anti-4-1BB antibody, and / or an anti-GITR antibody.

[0214] In one aspect, the present disclosure provides a combination therapy. In some embodiments, the anti-SIGLEC15 antibody or an antigen-binding fragment thereof (e.g., any antibody described herein) can be administered together with an anti-PD-L1 antibody. In some embodiments, the anti-SIGLEC15 antibody or an antigen-binding fragment thereof (e.g., any antibody described herein) can be administered together with an anti-OX40 antibody. In some embodiments, the anti-SIGLEC15 antibody or an antigen-binding fragment thereof (e.g., any antibody described herein) can be administered together with an anti-4-1-BB antibody.

[0215] Pharmaceutical Compositions and Routes of Administration Also provided herein are pharmaceutical compositions containing at least one (e.g., 1, 2, 3, or 4) of the antibodies or antigen-binding fragments described herein. Any two or more (e.g., 2, 3, or 4) of the antibodies or antigen-binding fragments described herein can be present in the pharmaceutical composition in any combination. The pharmaceutical composition can be formulated in any manner well known in the art.

[0216] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition can include a sterile diluent (e.g., sterile water or saline), a nonvolatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, an antibacterial or antifungal agent (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), an antioxidant (such as ascorbic acid or sodium bisulfite), a chelating agent (such as ethylenediaminetetraacetic acid), a buffer (such as acetate, citrate, or phosphate), and an isotonic agent (e.g., a sugar (e.g., dextrose), a polyalcohol (e.g., mannitol or sorbitol), or a salt (e.g., sodium chloride)), or any combination thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The preparation of the composition can be formulated and enclosed in ampoules, disposable syringes, or multi-dose vials. If necessary (e.g., as in injectable formulations), appropriate fluidity can be maintained, for example, by using a coating such as lecithin or a surfactant. The absorption of the antibody or its antigen-binding fragment can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implant and microencapsulation delivery systems, which can include biodegradable and biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0217] A composition containing any one or more of the antibodies or antigen-binding fragments described herein can be formulated in unit dosage forms (i.e., physically discrete units containing a predetermined quantity of the active compound, which facilitate administration and provide a uniform dosage) for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration.

[0218] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. The pharmaceutical composition can be provided in unit dosage forms (i.e., dosage for a single administration). The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the selected route of administration. For injection, the antibody is formulated in an aqueous solution, preferably in a physiologically compatible buffer, which can reduce discomfort at the injection site. The solution can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. Alternatively, the antibody can be in lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0219] The toxicity and therapeutic effect of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Agents with a high therapeutic index are preferred. If the agent exhibits undesirable side effects, care must be taken to minimize the potential for harm (i.e., reduce the undesirable side effects). The toxicity and therapeutic effect can be determined by other standard pharmaceutical procedures.

[0220] Data obtained from cell culture assays and animal studies can be used in formulating appropriate dosages of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., 1, 2, 3, or 4) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) is an amount that treats (e.g., kills cancer cells) the subject's disease in a subject (e.g., a human subject identified as having cancer), or a subject identified as being at risk of developing a disease (e.g., a subject who previously had cancer but is currently in remission), and reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human). The effectiveness and administration of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or a veterinary professional using methods well known in the art, in addition to observing one or more symptoms of the disease in the subject (e.g., a human). Certain factors can affect the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and the presence of other diseases).

[0221] Exemplary dosages include any amount (milligrams or micrograms) of any of the antibodies or antigen-binding fragments described herein per kilogram of the subject's body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 1 μg / kg to about 50 μg / kg). Although these dosages cover a wide range, those skilled in the art will understand that therapeutic agents containing antibodies and their antigen-binding fragments can have their efficacy and effective amounts determined by methods well known in the art. Typically, a relatively low dosage is first administered, and a responsible health care professional or veterinary professional (in the case of therapeutic use), or a researcher (if still working in the development stage), can subsequently and gradually increase the dosage until an appropriate response is obtained. In addition, the specific dosage level for any particular subject is understood to depend on various factors including the activity of the specific compound used, the age, weight, general health, sex, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in vivo.

[0222] The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for use in administration. The present disclosure also provides methods for manufacturing antibodies or their antigen-binding fragments for various uses described herein.

Examples

[0223] The present invention will be further illustrated by the following examples, which do not limit the scope of the present invention described in the claims.

[0224] Example 1. Generation of a Human Anti-SIGLEC15 Antibody To generate an antibody against human SIGLEC15, RenMab TM mice were immunized with human SIGLEC15. Anti-SIGLEC15 antibodies were produced by the method described below.

[0225] RenMab TM The mouse has both a humanized heavy-chain immunoglobulin locus and a humanized κ-chain immunoglobulin locus. The heavy-chain immunoglobulin locus is a region on a chromosome that contains genes for the heavy chains of antibodies. The locus includes IGHV (variable), IGHD (diversity), IGHJ (joining), and heavy-chain constant domain genes. The κ-chain immunoglobulin locus is a region on a chromosome that contains genes encoding the light chains (κ-chains) of antibodies. The κ-chain immunoglobulin locus includes IGKV (variable), IGKJ (joining), and light-chain constant domain genes. RenMab TM A detailed description of the mouse can be found in PCT / CN2020 / 075698, which is hereby incorporated by reference in its entirety.

[0226] Immunization of the mouse Human SIGLEC15 protein (hSIGLEC15-Fc, ACRO Biosystems Inc., catalog number: SG5-H5253, including positions 20 to 263 of SEQ ID NO: 45) and mouse SIGLEC15 protein (mSIGLEC15-His, ACRO Biosystems Inc., catalog number: SG5-M52H7, including positions 24 to 262 of SEQ ID NO: 46) were emulsified with an adjuvant and used for immunizing RenMab TM The mouse. Retro-orbital blood was collected as a negative control before immunization.

[0227] Complete Freund's adjuvant CFA was used for the first immunization, and incomplete Freund's adjuvant IFA was used for the second, third, and fourth immunizations. A total of 4 immunizations were performed. The first and second immunizations were spaced 2 weeks apart, and the remaining immunizations were spaced 1 week apart. One week after the fourth immunization, retro-orbital blood was collected, and the antibody titer in the serum was detected by FACS.

[0228] In another experiment, several mice (SIGLEC15 KO mice) are immunized by injecting an expression plasmid encoding human SIGLEC15 or mouse SIGLEC15 into the mice. The plasmid encoding the antigen is injected into the anterior tibialis muscle (intramuscular injection, i.m. injection). At least 4 injections are performed at intervals of at least 14 days between two injections. Blood (serum) is collected 7 days after the last immunization, and the antibody titer of the serum is tested by FACS.

[0229] At least 14 days after the previous immunization, a procedure to enhance immunization (either by injecting protein or injecting plasmid) was performed. SIGLEC15 protein was injected intraperitoneally, and CHO-S cells expressing human SIGLEC15 antigen were injected via the tail vein.

[0230] Antigen-specific immune cells were isolated from the immunized mice, and further, anti-SIGLEC15 antibodies were obtained, or the variable region sequences of the light and heavy chains of anti-SIGLEC15 antibodies were obtained. For example, single-cell technology (e.g., Beacon® Optofluidic System, Berkeley Lights Inc.) was used to screen and discover plasma cells secreting antigen-specific monoclonal antibodies, and reverse transcription and PCR sequencing were used to obtain antibody variable region sequences. The obtained variable region sequences were used for antibody expression, and FACS was used to verify the binding affinity for SIGLEC15. Specifically, the obtained VH sequence and VL sequence were each connected to the human IgG1 constant region containing the N297A mutation. Exemplary antibodies obtained by this method included 10A7, 10C9, and 23F4.

[0231] The amino acid sequences of the heavy chain CDR1, 2, 3 and the light chain CDR1, 2, 3 for 10A7 are shown in SEQ ID NOs: 1-6 (Kabat numbering) or SEQ ID NOs: 19-24 (Chothia numbering), respectively. The human heavy chain variable region and human light chain variable region for 10A7 are shown in SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

[0232] The amino acid sequences of the heavy chain CDR1, 2, 3 and the light chain CDR1, 2, 3 for 10C9 are shown in SEQ ID NOs: 7-12 (Kabat numbering) or SEQ ID NOs: 25-30 (Chothia numbering), respectively. The human heavy chain variable region and the human light chain variable region for 10C9 are shown in SEQ ID NO: 39 and SEQ ID NO: 40, respectively.

[0233] The amino acid sequences of the heavy chain CDR1, 2, 3 and the light chain CDR1, 2, 3 for 23F4 are shown in SEQ ID NOs: 13-18 (Kabat numbering) or SEQ ID NOs: 31-36 (Chothia numbering), respectively. The human heavy chain variable region and the human light chain variable region for 23F4 are shown in SEQ ID NO: 41 and SEQ ID NO: 42, respectively.

[0234] Construction of single-armed antibody Furthermore, a single-armed antibody was constructed, and the antibody has an anti-SIGLEC15 arm containing a heavy chain and a light chain, and a heavy chain fragment containing the CH2 and CH3 domains of IgG (B in FIG. 1).

[0235] Vectors expressing each polypeptide chain of the single-armed antibody were co-transfected into CHO cells. After culturing for 14 days, the cell supernatant was collected and purified by protein A affinity chromatography. The constant domain of the antibody was selected from either human IgG1 or IgG4. In particular, mutations within IgG1, such as the N297A mutation, were also introduced to reduce the binding affinity of the Fc receptor. These Fc mutations can improve the safety of the antibody by minimizing the effector function of the antibody. Exemplary antibodies obtained by this method included 10C9-S and 23F4-S.

[0236] In the single-armed antibody, a knob-into-hole (KIH) mutation was also introduced into the constant region. Using the knob-into-hole technology, a knob mutation (T366W) was introduced into the Fc of the full-length heavy chain, and a hole mutation (T366S, L368A, and Y407V) was introduced into a single Fc fragment to construct a single-armed anti-SIGLEC15 antibody (Figure 1). The single-armed anti-SIGLEC15 antibody 10C9-S and human IgG1-N297A were prepared (knob-containing full-length heavy chain SEQ ID NO: 43, single Fc fragment SEQ ID NO: 44, full-length light chain SEQ ID NO: 78).

[0237] Example 2. Inter-species binding of anti-SIGLEC15 antibody CHO-S-hSIGLEC15 cells, CHO-S-mSIGLEC15 cells, CHO-S-fasSIGLEC15 cells, or CHO-S-dSIGLEC15 cells were each transferred to a 96-well plate at a density of 5×10 4 cells / well. Serial diluted sample anti-SIGLEC15 antibodies were added to the 96-well plate and incubated at 4°C for 30 minutes. Next, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alex Flour 647 (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., 109-606-170) in the dark at 4°C for 15 minutes, followed by flow cytometry analysis.

[0238] CHO-S-hSIGLEC15 cells, CHO-S-mSIGLEC15 cells, CHO-S-fasSIGLEC15 cells, or CHO-S-dSIGLEC15 cells were obtained by transfecting CHO-S cells with the human SIGLEC15 amino acid sequence (hSIGLEC15, SEQ ID NO: 45), mouse SIGLEC15 amino acid sequence (mSIGLEC15, SEQ ID NO: 46), positions 60-387 of the Macaca fascicularis (cynomolgus monkey) SIGLEC15 amino acid sequence (fasSIGLEC15, SEQ ID NO: 47), and dog SIGLEC15 amino acid sequence (dSIGLEC15, SEQ ID NO: 48), respectively.

[0239] The test results are shown in the following table. All anti-SIGLEC15 antibodies 10A7, 10C9, and 23F4 can bind to hSIGLEC15, mSIGLEC15, fasSIGLEC15, and dSIGLEC15.

[0240]

Table 1

[0241] To determine whether the anti-SIGLEC15 antibodies can bind to other SIGLEC family proteins, another similar experiment was conducted. CHO-S-hSIGLEC1 cells (expressing hSIGLEC1, SEQ ID NO: 49), CHO-S-hSIGLEC2 cells (expressing hSIGLEC2, SEQ ID NO: 50), CHO-S-hSIGLEC4 cells (expressing hSIGLEC4, SEQ ID NO: 51), CHO-S-hSIGLEC5 cells (expressing hSIGLEC5, SEQ ID NO: 52), CHO-S-hSIGLEC6 cells (expressing hSIGLEC6, SEQ ID NO: 53), CHO-S-hSIGLEC7 cells (expressing hSIGLEC7, SEQ ID NO: 54), CHO-S-hSIGLEC8 cells (expressing hSIGLEC8, SEQ ID NO: 55), CHO-S-hSIGLEC9 cells (expressing hSIGLEC9, SEQ ID NO: 56), CHO-S-hSIGLEC10 cells (expressing hSIGLEC10, SEQ ID NO: 57), CHO-S-hSIGLEC11 cells (expressing hSIGLEC11, SEQ ID NO: 58), CHO-S-hSIGLEC12 cells (expressing hSIGLEC12, SEQ ID NO: 59), CHO-S-hSIGLEC14 cells (expressing hSIGLEC14, SEQ ID NO: 60), or CHO-S-hSIGLEC16 cells (expressing hSIGLEC16, SEQ ID NO: 61) were used.

[0242] The results are shown in the following table. The anti-SIGLEC15 antibodies 10A7, 10C9, and 23F4 do not bind to other SIGLEC family proteins.

[0243]

Table 2

[0244] Example 3. Binding Affinity of Anti-SIGLEC15 Antibody The binding affinity of the anti-SIGLEC15 antibody to His-tagged SIGLEC15 proteins of human (hSIGLEC15-His, Novoprotein Scientific Inc., catalog number: CW37) (positions 20 to 263 of SEQ ID NO: 45), mouse (mSIGLEC15-His, ACROBiosystems Inc., catalog number: SG5-M52H7) (positions 24 to 262 of SEQ ID NO: 46), or monkey (fasSIGLEC15-His, ACROBiosystems Inc., catalog number: SG5-C52H6) (SEQ ID NO: 62) was verified by surface plasmon resonance (SPR) using a Biacore 8K biosensor equipped with a pre-immobilized Protein A sensor chip. TM (Biacore, Inc., Piscataway N.J.)

[0245] The purified anti-SIGLEC15 antibody was captured on a Protein A chip (Series S Sensor Chip Protein A) for detection. The purified anti-SIGLEC15 antibody at 1 μg / mL was loaded at 10 μL / min and bound to gradient concentrations (50, 25, 12.5, 6.25, 3.125, 1.5626, 0.78125, and 0 nM) of recombinant hSIGLEC15, fasSIGLEC15, and mSIGELC15. The flow rate was 30 μL / min, and the binding time and dissociation time were set to 180 s and 600 s, respectively. After the last injection of each titrant, the chip was regenerated with a glycine solution (pH 2.0) at 30 μL / min for 30 s.

[0246] Biacore TMUsing 8K Evaluation software 3.0, the overall data was fitted to a 1:1 Langmuir binding model (Karlsson, R., Roos, H., Fagerstam, L., Petersson, B., 1994. Methods Enzymology 6.99 - 110) to simultaneously obtain the association rate (kon) and dissociation rate (koff). Affinity was estimated from the quotient of the rate constants (KD = koff / kon).

[0247] As will be understood by those skilled in the art, the same method with appropriate adjustment of parameters (e.g., antibody concentration) was performed for each test antibody. The results for the test antibodies are summarized in the table below.

[0248]

Table 3

[0249] 5G12 is a humanized IgG1 monoclonal antibody targeting human SIGLEC15 in the Phase I / II clinical development at NextCure for the treatment of patients with locally advanced or metastatic solid tumors. The heavy chain sequence of the 5G12 antibody containing the N297A mutation is shown in SEQ ID NO: 63. The light chain sequence of the 5G12 antibody is shown in SEQ ID NO: 64.

[0250] The results show that all of the anti - SIGLEC15 antibodies 10C9 and 23F4 can bind to human and monkey SIGLEC15 with high affinity.

[0251] Example 4. Epitope analysis of anti - SIGLEC15 antibodies The relative positions of the target protein epitopes between pairs of purified anti - SIGLEC15 antibodies were analyzed by biolayer interferometry (BLI) using a ForteBio Octet system at 30°C. A total of four purified antibodies, 5G12, 10A7, 10C9, and 23F4, were used.

[0252] 1× HBS-EP+ buffer (diluted from HBS-EP+ buffer (10×), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P20, pH 7.4)) was used as the running buffer throughout the experiment. Approximately 100 RU of hSIGLEC15 protein was captured at a flow rate of 10 μL / min, and 200 nM antibody was injected at a flow rate of 30 μL / min to bind to the ligand. To determine whether the binding of different antibodies interfered with each other, another antibody was injected under the same conditions. The binding time was 300 s for each antibody.

[0253] The binding values of each antibody were obtained using Data Analysis HT 12.0. To quantify the interference of the binding of one antibody to another, the binding ratio was calculated and each pair of antibodies was compared. The binding ratio was defined as the binding value of the second antibody (sample 2) divided by the binding value of the first antibody (sample 1). The binding ratios of each antibody pair were summarized in a matrix table as shown in Table 4. More specifically, when sample 1 showed an inhibitory effect on sample 2, the binding ratio was -0.1 to 0.5. When sample 1 did not show an inhibitory effect on sample 2, the binding ratio was 0.5 to 1.2. Generally, antibody pairs that interfere with each other have identical or overlapping epitopes.

[0254] The results of the epitope binding analysis showed that 23F4 and 10C9 recognized different epitopes, and 10A7, 10C9, and 5G12 showed a strong correlation with each other.

[0255]

Table 4

[0256] Example 5. Stability Analysis of Anti-SIGLEC15 Antibodies The anti-SIGLEC15 antibodies 10C9 and 23F4 were diluted to 2 mg / mL using a buffer at pH 6.0 (3 mg / mL histidine, 80 mg / mL sucrose, and 0.2 mg / mL Tween 80). The diluted antibodies were kept in sealed Eppendorf tubes at 5 ± 3°C (hereinafter referred to as 4°C) for 7 days, at 25 ± 2°C (hereinafter referred to as 25°C) for 7 days, or at 40 ± 2°C (hereinafter referred to as 40°C) for 7 days to evaluate their thermal stability.

[0257] The freeze-thaw stability was determined in the following experiments: "Freeze 1": frozen at -80°C for 5 days and then thawed at 4°C for detection; "Freeze 10": frozen at -80°C and the freeze-thaw cycle was repeated 10 times within 5 days (frozen at -80°C and thawed at 4°C).

[0258] Low pH stability: The stability index of the antibody was determined before and after incubation for 6 hours in 1 mol / L acetic acid (pH 3.5).

[0259] Specifically, the following tests were conducted: (1) Observation of the appearance of the solution and the presence of visible insoluble objects; (2) Detection of changes in the purity of the antibody by size exclusion ultra-high performance liquid chromatography (SEC-UPLC) (shown as the percentage of the main peak area to the total area of all peaks (purity, %)); (3) Detection of changes in the apparent hydrophobicity of the antibody using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) method (shown as the retention time of the main peak (HIC, minutes)); (4) Detection of charge variants in the antibody by capillary isoelectric focusing electrophoresis (cIEF) (shown as the percentage of the main component, acidic component, and alkaline component); (5) Detection of antibody activity (EC50) using flow cytometry.

[0260] In the SEC-UPLC experiment, an Agilent 1290 chromatograph system (XBridge TMA Protein BEH SEC column (200 Å, Waters Corporation) was used. The antibody sample was diluted to 1 mg / mL with purified water. The following parameters were used: mobile phase: 100 mmol / L phosphate buffer (PB) (pH 7.4) + 0.2 mol / L NaCl + 10% acetonitrile; flow rate: 1.8 mL / min; column temperature: 25 °C; detection wavelength: 280 nm; injection volume: 10 μg; sample tray temperature: 6 °C; and run time: 7 min.

[0261] For the HIC-HPLC experiment, an Agilent 1260 chromatograph system (ProPac TM HIC-10 column (4.6 × 250 mm, Thermo Scientific) was used and the sample was diluted to 0.5 mg / mL with mobile phase A. The following parameters were used: mobile phase A: 0.9 M ammonium sulfate, 0.1 M PB, 10% acetonitrile (pH 6.5); mobile phase B: 0.1 M PB, 10% acetonitrile (pH 6.5); flow rate: 0.8 mL / min; gradient: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A; column temperature: 30 °C; detection wavelength: 280 nm; injection volume: 10 μg; sample tray temperature: approximately 6 °C; and run time: 45 min.

[0262] In the cIEF experiment, for sample preparation, the Maurice cIEF Method Development Kit (Protein Simple, Catalog Number: PS-MDK01-C) was used. Specifically, 40 μg of protein sample was mixed in the kit with the following reagents: 1 μL of Maurice cIEF pI Marker-4.05, 1 μL of Maurice cIEF pI Marker-9.99, 35 μL of 1% methylcellulose solution, 2 μL of Maurice cIEF 500 mM arginine, 4 μL of ampholytes (Pharmalyte pH range 3-10), and water (added to a final volume of 100 μL). Using a Maurice analyzer (Protein Simple, Santa Clara, CA) and a Maurice cIEF cartridge (PS-MC02-C), an imaging capillary isoelectric focusing electrophoresis spectrum was generated. The sample was focused for a total of 10 minutes. The absorbance of the protein focused at 280 nm was analyzed using the analysis software installed on the instrument.

[0263] In the activity detection experiment, 30 μL of CHO-S-hSIGLEC15 cells were added to a culture plate, and the test antibody was added at gradient dilutions (90, 30, 10-0.000152, 0.00051 μg / mL) and incubated at 4 °C for 30 minutes. The plate was washed with PBS, and the diluted secondary antibody Anti-hIgG-Fc-Alex Flour 647 (RL1-H) (1:2000) was added and incubated at 4 °C for 30 minutes. The plate was washed once with PBS, and the cells were resuspended for flow cytometry detection. The MFI value was derived by FlowJo 7.6.1 analysis to calculate the EC50.

[0264] The detailed results of 10C9, 10C9-S, and 23F4 are shown in the following table. The results indicated that these anti-SIGLEC15 antibodies have excellent stability and physical and chemical properties.

[0265]

Table 5

[0266] Example 6. Human PBMC Proliferation Assay The proliferation assay was performed using human peripheral blood mononuclear cells (PBMC). Human PBMC (AllCells, catalog number: FPB003F-C) was stained with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE, Thermo Fisher, catalog number: C34554) according to the manufacturer's instructions. A 96-well plate was coated with 0.01 μg / mL anti-CD3 monoclonal antibody (ACROBiosystems Inc., catalog number: CDE-M120a). The coating was performed overnight at 4°C. The plate was washed three times with PBS. 5.00 μg / mL (final concentration) of human SIGLEC15 protein (hSIGLEC15, ACROBiosystems Inc., catalog number: SG5-H5253) was added together with different anti-SIGLEC15 antibodies at various concentrations. The plate was incubated at 37°C for 3 - 4 hours. 2×10 5 individual PBMC cells labeled with CFSE were added to each well and incubated at 37°C with 5% CO2 for 72 hours. The PBMC cells were stained with anti-CD4 antibody (Biolegend, catalog number: 300514) and anti-CD8a antibody (Biolegend, catalog number: 301036), and flow cytometry analysis was performed. The details and results of the incubation scheme are shown in Table 6. The results showed that 23F4, 10C9, and 10C9-S reversed the inhibition of T cell proliferation induced by human SIGLEC15 protein, and the effect of reversing the inhibition of T cell proliferation became stronger as the dose increased.

[0267]

Table 6

[0268] Example 7. Pharmacokinetic (PK) Analysis A humanized SIGLEC15 mouse model (hSIGLEC15 mouse) was recombinantly expressed with a chimeric SIGLEC15 protein (SEQ ID NO: 67), wherein a part of the extracellular region of the mouse SIGLEC15 protein was replaced with the corresponding human SIGLEC15 extracellular region. A detailed description of the humanized SIGLEC15 mouse model can be found in PCT / CN2022 / 080427, which is hereby incorporated by reference in its entirety.

[0269] The pharmacokinetic clearance rate of the anti-SIGLEC15 antibody was determined in hSIGLEC15 mice. Specifically, the mice were divided into two groups (3 mice per group), and 23F4 or 5G12 was administered at 10 mg / kg by intravenous injection. Blood samples were collected 4 days before administration and at 15 minutes, 4 hours, 1 day, 3 days, 7 days, 10 days, 14 days, and 21 days after administration.

[0270] The serum levels of human antibodies were measured by sandwich ELISA. Briefly, goat anti-human IgG (H+L) (Jackson ImmunoResearch Inc., catalog number: 109-005-088) was diluted to a final concentration of 2000 ng / mL and added to a 96-well plate (ELISA plate) at 100 μL / well, and then incubated overnight at 4°C. After incubation, the plate was washed with PBS-T buffer (Tween TMIt was washed 4 times with PBS supplemented with 20. The region where the antibody was not bound was blocked with 2% BSA (bovine serum albumin) at 37 °C for 2 hours. Then, the plate was washed 4 times with PBS-T buffer. After washing, 100 μL of blocking buffer (2% BSA) was added to each well. The wells were sealed and incubated at 37 °C for 1 hour. After the plate was washed with a plate washer, Peroxidase AffiniPure F(ab’)2 fragment Goat anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch Inc., catalog number: 109-036-098) was added to each well of the plate at 100 μL / well and incubated at 37 °C for 1 hour. After the plate was washed, tetramethylbenzidine (TMB) solution was added as a substrate to the 96-well plate at 100 μL / well. After incubation at room temperature in the dark, 100 μL of stop solution (Beyotime, catalog number: P0215) was added to each well. Using a microplate reader, the absorbance values of each well were read at wavelengths of 450 nm and 630 nm. Gen5 of the analysis software TM was used to analyze the data. Using the absorbance values and the corresponding concentrations of the calibration samples prepared with each test product, a standard curve with four parameters (i.e., T 1 / 2 , C max , AUC 0~21日 , and CL) was produced. Using the standard curve, the antibody concentration of each serum sample was calculated. A drug concentration-time curve was produced using the calculated sample concentrations at each time point. Phoenix TM WinNolin 8.3 was used to calculate the pharmacokinetic parameters. The results are shown in the following table.

[0271]

Table 7

[0272] Four days before antibody administration, the antibody concentration was detected as 0 μg / mL (results not shown). As shown in FIGS. 2A-2C and Table 7, the results were consistent with typical pharmacokinetic properties, indicating that after injection of the anti-SIGLEC15 antibody, the concentration of the antibody in the serum of hSIGLEC15 mice decreased over time. The half-lives of 10A7, 23F4, and 5G12 in hSIGLEC15 mice were 11.51 days, 9.79 days, and 4.19 days, respectively.

[0273] In a similar experiment, the PK analysis results of 23F4-S and 10C9-S are shown in Table 8.

[0274]

Table 8

[0275] Example 8. Anti-tumor Activity in hSIGLEC15 Mice Anti-tumor Activity of 23F4 in Combination with Anti-PD-L1 Antibody hSIGLEC15 mice were used to determine the anti-tumor activity of the anti-SIGLEC15 antibody. Approximately 5×10 5 MC38 cells were subcutaneously injected into hSIGLEC15 mice, and when the tumor volume grew to approximately 100-150 mm 3 , the mice were divided into different groups based on tumor size (5 mice per group). The treatment groups were randomly selected for 23F4 treatment, atezolizumab analog treatment, or a combination of atezolizumab analog and 23F4 treatment. Mice in the control group were injected with phosphate-buffered saline (PBS). The dosing frequency was twice a week (for a total of 6 doses). The tumor volume was measured twice a week, and the body weight of the mice was also measured. When the tumor volume of the mice reached 3000 mm 3 , euthanasia was performed. The details of the dosing scheme are shown in the table below.

[0276]

Table 9

[0277] The lengths of the long and short axes of the tumor were measured, and the tumor volume was calculated as 0.5×(long axis)×(short axis). 2 The body weight of the mice was also measured twice a week.

[0278] The tumor growth inhibition rate (TGI%) was calculated using the following formula: [1 - (Ti - T0) / (Vi - V0)]×100. Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day 0. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day 0.

[0279] A t-test was performed for statistical analysis. P < 0.05 is the threshold for indicating a significant difference.

[0280] The body weights of the mice in all different groups increased. On the day of group assignment (day 0), the average body weight of each group was in the range of 19.3 g to 19.7 g. At the end of the experiment (day 25), the average body weight of each group was in the range of 22.8 g to 24.9 g. The average body weight of each group was in the range of 118.0% to 126.7%. The results showed that the tested antibody had good tolerance and was clearly non-toxic to mice.

[0281] The tumor size data of the group treated with the antibody are shown in Figure 3. Table 10 below summarizes the results of this experiment, including the tumor volume at the day of group division (day 0), 14 days after group division (day 14), and at the end of the experiment (day 25); the survival rate of the mice; the tumor growth inhibition value (TGI); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0282]

Table 10

[0283] As shown in Fig. 3 and Table 10, compared with the control group (G1), tumor growth in the treatment groups (G2 and G6) was suppressed to varying degrees. Among the treatment groups G3 - G5 treated only with 23F4, G4 showed the best tumor suppression effect. Furthermore, 23F4 combined with the atezolizumab analog (G6) achieved a better tumor suppression effect compared with the 23F4 treatment group (G5) or the anti-PD-L1 antibody treatment group (G2), indicating that the anti-SIGLEC15 antibody enhanced the antitumor effect of the anti-PD-L1 antibody in the MC38 model.

[0284] Antitumor activity of 23F4 at a dose of 3 mg / kg In another experiment, to determine the antitumor activity of 23F4, approximately 5×10 5 MC38 cells were subcutaneously injected into hSIGLEC15 mice, and when the tumor volume grew to approximately 100 mm 3 the mice were divided into one control group and two treatment groups based on tumor size (7 mice per group). The treatment groups were randomly selected for 23F4 treatment (G2, 3 mg / kg) or 5G12 treatment (G3, 10 mg / kg). The mice in the control group were injected with PBS (G1). The dosing frequency was twice a week (for a total of 6 doses). The tumor volume was measured twice a week, and the body weight of the mice was also measured.

[0285] The body weights of the mice in all groups increased. On day 0, the average body weight of each group ranged from 19.9 g to 20.1 g. At the end of the experiment (day 21), the average body weight of each group ranged from 23.1 g to 23.6 g. The average body weight of each group ranged from 115.6% to 118.1%. The results showed that the tested antibodies had good tolerance and were clearly non-toxic to mice.

[0286] The tumor sizes of the groups treated with the antibody are shown in Fig. 4 and Table 11, indicating that compared with the control group (G1), tumor growth in the treatment groups (G2 and G3) was suppressed to varying degrees, and 23F4 at a dose of 3 mg / kg achieved a better tumor suppression effect compared with 5G12.

[0287]

Table 11

[0288] TIL Analysis in the MC38 Tumor Model In another experiment, to determine the antitumor activities of 23F4 and 23F4-S, approximately 5×10 5 MC38 cells were subcutaneously injected into hSIGLEC15 mice, and when the tumor volume grew to approximately 100 mm 3 The mice were divided into one control group and four treatment groups (7 mice per group) based on tumor size. The treatment groups were randomly selected for 23F4 treatment (G2 (1 mg / kg) and G3 (3 mg / kg)) or 23F4-S treatment (G4 (1 mg / kg)). The mice in the control group were injected with PBS (G1). The dosing frequency was twice a week (a total of 6 doses). The tumor volume was measured twice a week, and the body weight of the mice was also measured.

[0289] The body weights of the mice in all different groups increased. On day 0, the average body weight of each group was in the range of 20.8 g to 21.3 g. At the end of the experiment (day 20), the average body weight of each group was in the range of 22.9 g to 23.9 g. The average body weight of each group was in the range of 109.7% to 114.0%. The results showed that the tested antibodies had good tolerance and were clearly non-toxic to the mice.

[0290] The tumor size data of the groups treated with the antibody are shown in Table 12, indicating that the growth of tumors in the treatment groups (G2 - G4) was suppressed to different degrees compared with the control group (G1).

[0291] [Table 12]

[0292] At the end of the experiment, tumor tissues were collected, digested, and then resuspended as a single-cell suspension for flow cytometry analysis. Anti-mouse CD45 antibody APC / Cyanine7 anti-mouse CD45 antibody (BioLegend, catalog number: 103116), anti-mouse CD16 / 32 antibody purified anti-mouse CD16 / 32 antibody (BioLegend, catalog number: 101302), anti-mouse CD3 antibody PerCP / Cyanine5.5 anti-mouse CD3ε antibody (BioLegend, catalog number: 100328), anti-mouse CD4 antibody FITC anti-mouse CD4 antibody (BioLegend, catalog number: 100406), anti-mouse CD8a antibody Brilliant Violet 605 TM anti-mouse CD8a antibody (BioLegend, catalog number: 100744), anti-mouse NK1.1 antibody Brilliant Violet421 TM anti-mouse NK1.1 antibody (BioLegend, catalog number: 108732), anti-mouse CD11b antibody PE anti-mouse / human CD11b antibody (BioLegend, catalog number: 101208), anti-mouse ly-6G antibody Alexa Fluor® 700 anti-mouse ly-6G antibody (BioLegend, catalog number: 127622), anti-mouse mLy-6C antibody APC anti-mouse mLy-6C antibody (BioLegend, catalog number: 128016), anti-mouse Foxp3 antibody (eBioscience, catalog number: 25-5773-82) were used for cell staining before flow cytometry. The cell sorting method for TIL (tumor-infiltrating lymphocyte) analysis is shown in Figure 5.

[0293] As shown in A-E of Figure 6, the anti-SIGLEC15 antibody can significantly reduce the number of G-MDSC (granulocytic myeloid-derived suppressor cells) in the tumor microenvironment, and then reduce the immunosuppression caused by G-MDSC, which appears as a slight increase in the number of T helper (Th) cells and CTL (cytotoxic T lymphocytes). Since G-MDSC occupies a higher proportion in the tumor microenvironment compared to M-MDSC (monocytic myeloid-derived suppressor cells), the effect of G-MDSC on the immunosuppressive effect may be greater than that of M-MDSC.

[0294] Antitumor activity of 10C9 A total of 15 hSIGLEC15 mice were subcutaneously injected with MC38 cells (5×10 5 / mouse). When the tumors reached a volume of 100 mm 3 the mice were randomly divided into three groups of 5 mice each. The treatment groups were intraperitoneally injected with 10C9 or the positive control antibody 5G12 at a dose of 3 mg / kg, and the control group was injected with PBS twice a week. The body weight and tumor volume of the mice were measured twice a week until the experiment ended after 3 weeks.

[0295] Throughout the treatment period, the average body weight of the mice in the control and treatment groups increased steadily, and no significant difference was found between the groups, indicating that these 10C9 were clearly non-toxic to mice. Table 13 below shows the results of TGI% (21 days after grouping) for each group. Compared with the control group, tumor growth in the treatment groups was suppressed to varying degrees, and the 10C9 treatment group obtained a better tumor suppression effect compared with 5G12.

[0296]

Table 13

[0297] Antitumor activity of 10A7 in combination with anti-PD-L1 antibody A total of 20 hSIGLEC15 mice were subcutaneously injected with MC38 cells (5×10 5 / mouse). When the tumors reached a volume of 100 mm 3 the mice were divided into one control group and three treatment groups (5 mice per group) based on tumor size. The treatment groups were randomly selected for anti-PD-L1 antibody atezolizumab analog treatment, 10A7 treatment, or a combination of atezolizumab analog and 10A7 treatment. The control group mice were injected with PBS. Tumor volume was measured twice a week, and the body weight of the mice was also measured. Euthanasia was performed when the tumor volume of the mice reached 3000 mm 3 The details of the dosing scheme are shown in the table below.

[0298]

Table 14

[0299] Atezolizumab is a humanized anti-PD-L1 monoclonal antibody. The heavy chain sequence and the light chain sequence are shown in SEQ ID NOs: 65-66.

[0300] Throughout the treatment period, the average body weights of the mice in the control group and the treatment group increased steadily, and no significant difference was found between the groups, indicating that these antibodies were clearly non-toxic to the animals.

[0301] The tumor sizes in the group treated with the antibody are shown in Figure 7. The following Table 15 shows the results of TGI% (24 days after grouping) for each group. Compared with the control group, the tumor growth in the treatment group was suppressed to varying degrees, and anti-SIGLEC15 antibody 10A7 combined with the anti-PD-L1 antibody treatment group obtained a better tumor suppression effect compared with the 10A7 treatment group or the anti-PD-L1 antibody treatment group.

[0302]

Table 15

[0303] Example 9. Anti-tumor activity in C57BL / 6 mice C57BL / 6 mice were subcutaneously injected with MC38 cells (5×10 5 / mouse). When the tumors reached a volume of 100 mm 3 , the mice were randomly divided into 3 groups of 7 mice each. The treatment group was intraperitoneally injected with 10C9-S, and the control group was injected with PBS twice a week. The body weights and tumor volumes of the mice were measured twice a week until the experiment ended after 24 days. The details of the dosing scheme are shown in the following table.

[0304]

Table 16

[0305] Throughout the entire treatment period, the average body weights of the mice in the control group and the treatment group increased steadily, and no significant difference was found between the groups, indicating that 10C9-S was clearly non-toxic to animals. The results of TGI% (24 days after grouping) for each group are shown in Table 17 below. Compared with the control group, tumor growth in the 10C9-S treatment group was suppressed to varying degrees.

[0306]

Table 17

[0307] Anti-tumor activity in Example 10.h4-1BB mice A humanized 4-1BB mouse model (h4-1BB mouse) was recombinantly engineered to express a chimeric 4-1BB protein (SEQ ID NO: 68), where a portion of the extracellular region of the mouse 4-1BB protein was replaced with the corresponding human 4-1BB extracellular region. A detailed description of the humanized 4-1BB mouse model can be found in PCT / CN2021 / 087867, which is hereby incorporated by reference in its entirety.

[0308] Approximately 5×10 5 individual mouse colon cancer cells MC38 were subcutaneously injected into h4-1BB mice. When the tumor volume grew to approximately 100 mm 3 the mice were divided into one control group and three treatment groups (5 mice per group) based on tumor size. The treatment groups were randomly selected for anti-4-1BB antibody YH004 treatment (G2), 23F4 treatment (G3), or a combination of YH004 and 23F4 treatment (G4). A detailed description of YH004 can be found in PCT / CN2019 / 105315, which is hereby incorporated by reference in its entirety. The mice in the control group were injected with phosphate-buffered saline (PBS) (G1). The tumor volume was measured twice a week, and the body weight of the mice was also measured. When the tumor volume of the mice reached 3000 mm 3 euthanasia was performed. The details of the dosing scheme are shown in the table below.

[0309]

Table 18

[0310] The body weights of the mice in all different groups increased. On the day of group assignment (day 0), the average body weight of each group was in the range of 19.2 g to 19.4 g. At the end of the experiment (day 21), the average body weight of each group was in the range of 20.9 g to 22.0 g. The average body weight of each group was in the range of 109.0% to 114.4%. The results showed that the tested antibody had good tolerance and was clearly non-toxic to mice.

[0311] As shown in Figure 8 and Table 19, compared with the control group, the tumor growth in the treatment group was suppressed to different degrees. Among the treatment groups, the anti-SIGLEC15 antibody combined with the anti-4-1BB antibody was able to significantly inhibit tumor growth with excellent efficacy.

[0312]

Table 19

[0313] Antitumor Activity of Example 11. hOX40 Mice A humanized OX40 mouse model (hOX40 mouse) was recombinantly expressed to express a chimeric OX40 protein (SEQ ID NO: 69), where a part of the extracellular region of the mouse OX40 protein was replaced with the corresponding human OX40 extracellular region. A detailed description of the humanized OX40 mouse model can be found in PCT / CN2017 / 099575, which is hereby incorporated by reference in its entirety.

[0314] Approximately 5×10 5 individual mouse colon cancer cells MC38 were subcutaneously injected into hOX40 mice, and the tumor volume was approximately 100 mm 3Once it had grown to this size, the mice were divided into one control group and four treatment groups (five mice per group) based on tumor size. The treatment groups were randomly selected for anti-OX40 antibody YH002 treatment (G2), 10A7 treatment (G3), combination treatment with YH002 and 10A7 (G4), or combination treatment with YH002 and 10C9 (G5). A detailed description of YH002 can be found in PCT / CN2017 / 112832, which is hereby incorporated by reference in its entirety. Mice in the control group were injected with an equal volume of PBS (G1). The details of the dosing schedule are shown in the table below.

[0315]

Table 20

[0316] The body weights of the mice in all groups increased. On the day of group allocation (day 0), the average body weight of each group ranged from 19.7 g to 20.1 g. At the end of the experiment (day 24), the average body weight of each group ranged from 22.2 g to 25.8 g. The average body weight of each group ranged from 111.8% to 129.3%. The results showed that the tested antibodies had good tolerability and were clearly non-toxic to the mice.

[0317] The following Table 21 summarizes the results of this experiment, including the tumor volume at the day of group allocation (day 0), 14 days after group allocation (day 14), and at the end of the experiment (day 24); TGI and the P value of the tumor volume between the treatment groups and the control group. The results showed that the anti-OX40 antibody in combination with anti-SIGLEC15 antibody 10A7 or 10C9 had excellent efficacy and could significantly inhibit tumor growth.

[0318]

Table 21

[0319] Example 12. In vivo toxicity of anti-SIGLEC15 antibody The in vivo toxicity of anti-SIGLEC15 antibodies in hSIGLEC15 mice was evaluated. hSIGLEC15 mice were divided into different groups according to body weight (3 mice per group), and the treatment groups were intraperitoneally injected with 10A7, 23F4, or the positive control antibody 5G12 at a dose of 30 mg / kg for treatment, and the control group was injected with PBS. The treatment was carried out on days 0, 3, 7, and 10. The body weight of the mice was measured twice a week until the end of the experiment. On days 11, 16, and 21, the peripheral blood and serum of hSIGLEC15 mice were collected and analyzed using biochemical tests (alanine transaminase (ALT) and aspartate (AST)) and hematological tests (white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit or red blood cell volume (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), percentage of lymphocytes (LYMPH%), percentage of monocytes (MONO%), percentage of neutrophils (NEUT%) including complete blood count).

[0320] As shown in FIGS. 9A-9B, the average body weight of the mice in the control group and the treatment groups increased steadily throughout the treatment period. FIGS. 10A-M show the results of the hematological and biochemical tests, and there were no significant differences between the groups. These results indicated that the anti-SIGLEC15 antibodies 23F4 and 10A7 were not toxic to mice.

[0321] Similar to the verification of the in vivo toxicity of 23F4, the toxicity analysis of 10C9 and 10C9-S was also carried out. The results showed that the average body weight of the mice in the control group and the treatment groups increased steadily throughout the treatment period, and there were no significant differences between the groups in the results of the hematological and biochemical tests. These results indicated that the anti-SIGLEC15 antibodies were clearly not toxic to mice.

[0322] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the above description is for illustrative purposes only and does not limit the scope of the present invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to SIGLEC15 (sialic acid-conjugated IgG-like lectin 15), A heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region of the VH includes an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the CDR2 region of the VH includes an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the CDR3 region of the VH includes an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence, and A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the CDR1 region of the VL comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the CDR2 region of the VL comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the CDR3 region of the VL comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence, and the light chain variable region comprises the light chain variable region, The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of the following antibodies or antigen-binding fragments. (1) (1-a) The selected VH CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 13, 14, and 15, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 16, 17, and 18, respectively, or (1-b) The selected VH CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 31, 32, and 33, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 34, 35, and 36, respectively. (2) (2-a) The selected VH CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 4, 5, 6, respectively, or (2-b) The selected VH CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 19, 20, 21, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 22, 23, 24, respectively, and (3) (3-a) The selected VH CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 7, 8, and 9, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 10, 11, and 12, respectively, or (3-b) The selected VH CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 25, 26, and 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are described in SEQ ID NOs: 28, 29, and 30, respectively.

2. (1) (1-a) According to Kabat's definition, the VH includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs: 13, 14, and 15, respectively, and the VL includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs: 16, 17, and 18, respectively, or (1-b) According to Chothia's definition, the VH includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs: 31, 32, and 33, respectively, and the VL includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs: 34, 35, and 36, respectively. (2) (2-a) According to Kabat's definition, the VH includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs: 1, 2, and 3, respectively, and the VL includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs: 4, 5, and 6, respectively, or (2-b) According to Chothia's definition, the VH includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs: 19, 20, and 21, respectively, and the VL includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs: 22, 23, and 24, respectively, or (3) (3-a) According to Kabat's definition, the VH includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs. 7, 8, and 9, respectively, and the VL includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs. 10, 11, and 12, respectively, or (3-b) According to Chothia's definition, the VH includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs. 25, 26, and 27, respectively, and the VL includes CDR1, 2, 3 having the amino acid sequences described in SEQ ID NOs. 28, 29, and 30, respectively. The antibody or antigen-binding fragment thereof according to claim 1.

3. The antibody or antigen-binding fragment thereof according to Claim 1, having one or more of the following characteristics selected from the following. (i) The antibody or its antigen-binding fragment specifically binds to SIGLEC15 in humans, mice, monkeys, or dogs; (ii) The antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment, a single-chain variable fragment (scFv), a single-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody); (iii) The antibody or its antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment, or a human IgG4 antibody or its antigen-binding fragment.

4. A nucleic acid comprising a polynucleotide encoding a polypeptide, comprising any of the following: (1) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising complementarity-determining regions (CDR) 1, 2, and 3, respectively, which include the amino acid sequences described in SEQ ID NOs. 13, 14, and 15 or SEQ ID NOs. 31, 32, and 33, wherein the VH binds to SIGLEC15 when paired with a light chain variable region (VL) containing the amino acid sequence described in SEQ ID NO.

42. (2) An immunoglobulin light chain or fragment thereof comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3, each comprising the amino acid sequences described in SEQ ID NOs. 16, 17, and 18 or SEQ ID NOs. 34, 35, and 36, wherein the VL binds to SIGLEC15 when paired with a VH comprising the amino acid sequence described in SEQ ID NO.

41. (3) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences described in SEQ ID NOs: 1, 2, and 3, or SEQ ID NOs: 19, 20, and 21, wherein the VH binds to SIGLEC15 when paired with a light chain variable region (VL) containing the amino acid sequence described in SEQ ID NO: 38, (4) An immunoglobulin light chain or fragment thereof comprising a VL containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences described in SEQ ID NOs: 4, 5, and 6 or SEQ ID NOs: 22, 23, and 24, wherein the VL binds to SIGLEC15 when paired with a VH containing the amino acid sequence described in SEQ ID NO:

37. (5) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences described in SEQ ID NOs. 7, 8, and 9 or SEQ ID NOs. 25, 26, and 27, wherein the VH binds to SIGLEC15 when paired with a light chain variable region (VL) containing the amino acid sequence described in SEQ ID NO.

40. (6) An immunoglobulin light chain or fragment thereof comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3, each comprising the amino acid sequences described in SEQ ID NOs. 10, 11, and 12 or SEQ ID NOs. 28, 29, and 30, wherein the VL binds to SIGLEC15 when paired with a VH comprising the amino acid sequence described in SEQ ID NOs.

39.

5. A vector comprising one or more nucleic acids as described in Claim 4, wherein the vector optionally comprises two nucleic acids, and the vector encodes the VL region and the VH region that bind together to SIGLEC15.

6. A cell comprising (i) one or more nucleic acids according to Claim 4, (ii) a vector comprising one or more of the nucleic acids, or (iii) a pair of vectors, each vector comprising one of the nucleic acids and, when paired, encoding a VL region and a VH region that bind together to SIGLEC15, Optionally, the cell contains two nucleic acids, and optionally, the two nucleic acids encode a VL region and a VH region that bind together to SIGLEC15. The cells are, by choice, CHO cells.

7. A method for producing an antibody or its antigen-binding fragment, (a) Culturing the cells described in claim 6 under conditions sufficient to produce the antibody or the antigen-binding fragment, (b) Recovering the antibody or antigen-binding fragment produced by the cells, Methods that include...

8. An antibody or antigen-binding fragment thereof that binds to SIGLEC15, An antibody or antigen-binding fragment comprising a heavy chain variable region (VH) containing an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) containing an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following. (1) The selected VH sequence is sequence number 41, and the selected VL sequence is sequence number 42. (2) The selected VH sequence is sequence number 37, and the selected VL sequence is sequence number 38. (3) The selected VH sequence is sequence number 39, and the selected VL sequence is sequence number 40. 。

9. (i) The VH includes the sequence of sequence number 41, and the VL includes the sequence of sequence number 42, (ii) The VH includes the sequence of sequence number 37, and the VL includes the sequence of sequence number 38, or (iii) The VH includes the sequence of sequence number 39, and the VL includes the sequence of sequence number 40, The antibody or antigen-binding fragment thereof according to claim 8.

10. An antibody or antigen-binding fragment thereof, comprising VH CDR1, 2, 3 and VL CDR1, 2, 3 of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and 8 to 9.

11. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and 8 to 9.

12. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and 8 to 9, covalently bound to a therapeutic agent, wherein the therapeutic agent is optionally a cytotoxic agent or a cell proliferation inhibitor.

13. A pharmaceutical composition for treating a subject having cancer, for reducing the rate of tumor growth, and / or killing tumor cells, comprising an antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, according to any one of claims 1 to 3 and 8 to 9, wherein the antibody-drug conjugate comprises an antibody or antigen-binding fragment thereof covalently bound to a therapeutic agent.

14. The pharmaceutical composition according to claim 13, having one or more selected from the following characteristics. (i) The subject suffers from a solid tumor, (ii) The cancer is non-small cell lung cancer (NSCLC), ovarian cancer, melanoma, colorectal cancer, breast cancer, colon adenocarcinoma, hematological malignancies, head and neck cancer, gastrointestinal cancer, bladder cancer, or bone cancer, or The aforementioned cancers are non-Hodgkin lymphoma, lymphoma, leukemia, acute myeloid leukemia, or chronic lymphocytic leukemia. (iii) The subjects are further treated with an effective dose of anti-4-1BB antibody, anti-OX40 antibody, anti-PD-1, anti-CTLA4, or anti-PD-L1 antibody.

15. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3 and 8 to 9, for increasing the immune response in a subject and / or treating a subject having a bone disease, wherein the bone disease is optionally osteoporosis.

16. A pharmaceutical composition comprising an antibody or its antigen-binding fragment or antibody-drug conjugate according to any one of claims 1 to 3 and 8 to 9, and a pharmaceutically acceptable carrier, wherein the antibody-drug conjugate comprises an antibody or its antigen-binding fragment covalently bound to a therapeutic agent.

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