Anti-SIGLEC15 antibodies and uses thereof

Abstract

The present disclosure relates to anti-SIGLEC15 antibodies, antigen binding fragments, and uses thereof.

Description

[0001] Priority declaration

[0002] 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 contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to anti-SIGLEC15 (sialic acid-binding Ig-like lectin 15) antibodies and uses thereof. Background Art

[0004] SIGLEC15 (sialic acid-binding immunoglobulin-like lectin 15) belongs to the SIGLEC family and is a type I transmembrane protein. It is rarely expressed in most normal human tissues and immune cell subsets, but has relatively high expression in macrophages. In 2007, Japanese scientist Takashi Angata first discovered that this protein can recognize sialic acid, so it was classified as a SIGLEC family. By regulating innate and adaptive immune responses, SIGLEC15 plays an important role in autoimmune diseases, inflammatory responses, and tumors.

[0005] Considering the important role of SIGLEC15 in autoimmune diseases, inflammatory responses, and tumors, there is a need to develop therapeutic agents targeting SIGLEC15. Summary of the invention

[0006] The present disclosure relates to anti-SIGLEC15 antibodies, antigen-binding fragments thereof, and uses thereof.

[0007] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to SIGLEC15 (sialic acid-binding Ig-like lectin 15), the antibody or antigen-binding fragment thereof comprising:

[0008] a heavy chain variable region (VH) comprising complementarity determining regions (CDR) 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

[0009] a light chain variable region (VL) comprising CDR1, 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,

[0010] wherein the selected VH CDR1, 2 and 3 amino acid sequence and the selected VL CDR1, 2 and 3 amino acid sequence are one of the following:

[0011] (1) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1, 2, 3, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4, 5, 6, respectively;

[0012] (2) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 7, 8, 9, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 10, 11, 12, respectively;

[0013] (3) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13, 14, 15, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 16, 17, 18, respectively;

[0014] (4) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 19, 20, 21, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22, 23, 24, respectively;

[0015] (5) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 25, 26, 27, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 28, 29, 30, respectively; and

[0016] (6) The selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 31, 32, 33, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 34, 35, 36, respectively.

[0017] In some embodiments, according to the Kabat definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively.

[0018] In some embodiments, according to the Kabat definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12, respectively.

[0019] In some embodiments, according to the Kabat definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 13, 14 and 15, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 16, 17 and 18, respectively.

[0020] In some embodiments, according to the Chothia definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 19, 20 and 21, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 22, 23 and 24, respectively.

[0021] In some embodiments, according to the Chothia definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 25, 26 and 27, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30, respectively.

[0022] In some embodiments, according to the Chothia definition, VH includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 31, 32 and 33, respectively, and VL includes CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 34, 35 and 36, respectively.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds human, mouse, monkey or dog SIGLEC15.

[0024] 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 one-armed antibody, and / or a multispecific antibody (eg, a bispecific antibody).

[0025] 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.

[0026] In one aspect, the present disclosure relates to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

[0027] An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity determining regions (CDRs) 1, 2, and 3 having amino acid sequences as shown in SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH binds to SIGLEC15 when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 38;

[0028] An immunoglobulin light chain or fragment thereof comprising a VL comprising complementarity determining regions (CDRs) 1, 2, and 3 having amino acid sequences as shown in SEQ ID NOs: 4, 5, and 6, respectively, and wherein the VL binds to SIGLEC15 when paired with a VH comprising the amino acid sequence as shown in SEQ ID NO: 37;

[0029] An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity determining regions (CDRs) 1, 2, and 3 having amino acid sequences as shown in SEQ ID NOs: 7, 8, and 9, respectively, and wherein the VH binds to SIGLEC15 when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 40;

[0030] An immunoglobulin light chain or fragment thereof comprising a VL comprising complementarity determining regions (CDRs) 1, 2, and 3 having amino acid sequences as shown in SEQ ID NOs: 10, 11, and 12, respectively, and wherein the VL binds to SIGLEC15 when paired with a VH comprising the amino acid sequence as shown in SEQ ID NO: 39;

[0031] An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity determining regions (CDRs) 1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 13, 14 and 15, respectively, and wherein the VH binds to SIGLEC15 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 42; or

[0032] An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 16, 17 and 18, respectively, and wherein the VL binds to SIGLEC15 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO: 41.

[0033] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 1, 2 and 3, respectively.

[0034] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively.

[0035] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively.

[0036] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NOs: 10, 11 and 12, respectively.

[0037] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 13, 14 and 15, respectively.

[0038] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NOs: 16, 17 and 18, respectively.

[0039] In some embodiments, the VH specifically binds to human, mouse, monkey, or dog SIGLEC15 when paired with the VL, or the VL specifically binds to human, mouse, monkey, or dog SIGLEC15 when paired with the VH.

[0040] In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof (e.g., a human IgG1 heavy chain or fragment thereof; or a human IgG4 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof.

[0041] In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv), a one-armed antibody, a multispecific antibody (e.g., a bispecific antibody) or a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid is a cDNA.

[0042] In one aspect, the disclosure relates to a vector comprising one or more nucleic acids described herein.

[0043] In one aspect, the disclosure relates to a vector comprising two nucleic acids described herein, wherein the vector encodes a VL region and a VH region that together bind to SIGLEC15.

[0044] In one aspect, the disclosure relates to a pair of vectors, wherein each vector comprises a nucleic acid described herein, wherein the pair of vectors together encode a VL region and a VH region that together bind to SIGLEC15.

[0045] In one aspect, the disclosure relates to a cell comprising a vector described herein or a vector pair described herein.

[0046] In some embodiments, the cell is a CHO cell.

[0047] In one aspect, the disclosure relates to a cell comprising one or more nucleic acids described herein.

[0048] In one aspect, the disclosure relates to a cell comprising two nucleic acids described herein.

[0049] In some embodiments, the two nucleic acids together encode a VL region and a VH region that together bind to SIGLEC15.

[0050] In one aspect, the present disclosure relates to a method of producing an antibody or an antigen-binding fragment thereof, the method comprising

[0051] (a) culturing the cell described herein under conditions sufficient for the cell to produce the antibody or antigen-binding fragment;

[0052] as well as

[0053] (b) collecting the antibody or antigen-binding fragment produced by the cell.

[0054] In one aspect, the present disclosure relates to an antibody or an antigen-binding fragment thereof that binds to SIGLEC15, the antibody or the antigen-binding fragment thereof comprising: a heavy chain variable region (VH), the VH comprising an amino acid sequence having at least 90% identity with a selected VH sequence; and a light chain variable region (VL), the VL comprising an amino acid sequence having at least 90% identity with a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0055] The selected VH sequence is SEQ ID NO:37, and the selected VL sequence is SEQ ID NO:38;

[0056] The selected VH sequence is SEQ ID NO:39, and the selected VL sequence is SEQ ID NO:40; and

[0057] The selected VH sequence is SEQ ID NO:41, and the selected VL sequence is SEQ ID NO:42.

[0058] In some embodiments, VH comprises the sequence of SEQ ID NO:37, and VL comprises the sequence of SEQ ID NO:38.

[0059] In some embodiments, VH comprises the sequence of SEQ ID NO:39, and VL comprises the sequence of SEQ ID NO:40.

[0060] In some embodiments, VH comprises the sequence of SEQ ID NO:41, and VL comprises the sequence of SEQ ID NO:42.

[0061] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds human, mouse, monkey or dog SIGLEC15.

[0062] 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 one-armed antibody, and / or a multispecific antibody (eg, a bispecific antibody).

[0063] 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.

[0064] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof comprising the VH CDR1, 2, 3 and VL CDR1, 2, 3 of the antibody or antigen-binding fragment thereof described herein.

[0065] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that cross-competes with an antibody or antigen-binding fragment thereof described herein.

[0066] In some embodiments, the antibody or antigen-binding fragment thereof includes a fragment crystallizable region (Fc region).

[0067] In some embodiments, the Fc region has reduced or no complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).

[0068] In one aspect, the present disclosure relates to a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof described herein.

[0069] In one aspect, the present disclosure relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof described herein covalently bound to a therapeutic agent.

[0070] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0071] In one aspect, the disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein or an antibody drug conjugate described herein.

[0072] In some embodiments, the subject has a solid tumor.

[0073] In some embodiments, 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.

[0074] In some embodiments, the cancer is non-Hodgkin lymphoma, lymphoma, leukemia, acute myeloid leukemia, or chronic lymphocytic leukemia.

[0075] In some embodiments, the subject is further treated with an effective amount of an anti-4-1BB antibody, an anti-OX40 antibody, an anti-PD-1, an anti-CTLA4, or an anti-PD-L1 antibody.

[0076] In one aspect, the disclosure relates to a method of reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein or an antibody drug conjugate described herein.

[0077] In one aspect, the disclosure relates to a method of killing a tumor cell, the method comprising contacting the tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein or an antibody drug conjugate described herein.

[0078] In one aspect, the disclosure relates to a method of increasing an immune response in a subject, the method comprising: administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0079] In one aspect, the disclosure relates to a method of treating a subject having a bone disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0080] In some embodiments, the bone disease is osteoporosis.

[0081] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0082] In one aspect, the present disclosure relates to a pharmaceutical composition comprising the antibody drug conjugate described herein and a pharmaceutically acceptable carrier.

[0083] As used herein, the term "cancer" refers to a cell with autonomous growth ability. Examples of such cells include cells with abnormal states or conditions characterized by rapid proliferation of cell growth. The term is intended to include cancerous growth (e.g., tumors), carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of their histopathological type or invasion stage. Malignant tumors of various organ systems are also included, such as the respiratory system, cardiovascular system, renal system, reproductive system, blood system, nervous system, liver system, gastrointestinal system, and endocrine system; and adenocarcinomas, which include malignant tumors, such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancers, and small intestinal cancers. "Naturally occurring" cancers include any cancer that is not experimentally induced by implanting cancer cells into a subject, and include, for example, spontaneously occurring cancers, cancers caused by exposure of patients to carcinogens, cancers caused by insertion of transgenic oncogenes or knockout of tumor suppressor genes, and cancers caused by infection (e.g., viral infection). The term "cancer" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcoma, which includes malignant tumors composed of carcinoma and sarcoma tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or in which the tumor cells form a recognizable glandular structure. The term "sarcoma" is recognized in the art and refers to a malignant tumor derived from mesenchyme. The term "hematopoietic neoplastic disease" includes diseases involving hyperplastic / tumor cells of hematopoietic origin. Hematopoietic neoplastic diseases can be caused by myeloid cells, lymphocytes or erythroid cells or their precursor cells.

[0084] As used herein, the term "antibody" refers to any antigen binding molecule that contains at least one (e.g., one, two, three, four, five or six) complementary determining regions (CDRs) (e.g., any one of the three CDRs from an immunoglobulin light chain or any one 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 (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives such as bispecific antibodies, single-chain antibodies, double antibodies, linear antibodies, and multispecific antibodies formed by antibody fragments.

[0085] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0086] As used herein, the term "human antibody" refers to an antibody encoded by an endogenous nucleic acid present in a human (e.g., a rearranged human immunoglobulin heavy chain or light chain locus). In some embodiments, human antibodies are collected from humans or produced in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacteria or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., cattle) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy chain or light chain human immunoglobulin loci).

[0087] As used herein, the term "chimeric antibody" refers to an antibody containing sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody containing a variable domain sequence (e.g., all or part of a light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and a constant domain of a human antibody. Other examples of chimeric antibodies are described herein and are known in the art.

[0088] As used herein, the term "humanized antibody" refers to a non-human antibody, which contains a minimum sequence derived from a non-human (e.g., mouse) immunoglobulin, and contains a sequence derived from a human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (receptor antibody), wherein the hypervariable region (e.g., CDR) residues of the receptor antibody are replaced by hypervariable regions (e.g., CDR) residues from a non-human antibody (e.g., donor antibody) with desired specificity, affinity and ability, such as mouse, rat or rabbit antibody. In some embodiments, the Fv framework residues of human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, a humanized antibody may contain residues not found in a receptor antibody or a donor antibody. These modifications may be performed to further improve antibody performance. In some embodiments, humanized antibodies include at least one and generally substantially all of two variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to the hypervariable loops of non-human (e.g., mouse) immunoglobulins, and all or substantially all of the framework regions are framework regions of human immunoglobulins. Humanized antibodies may also include at least a portion of an immunoglobulin constant region (Fc), generally a constant region of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for producing humanized antibodies are described herein.

[0089] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) that are capable of specific binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0090] As used herein, the term "multimeric antibody" refers to an antibody containing four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, the multimeric antibody is capable of cross-linking a target molecule (e.g., SIGLEC15) on the surface of a mammalian cell (e.g., a human T cell) to at least one second target molecule.

[0091] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification, and describe an animal (human or non-human) for which treatment is provided according to the method of the present invention. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adults or adolescents (e.g., people 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. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domestic animals, farm animals, and zoo animals.

[0092] As used herein, when referring to an antibody, the phrase "specifically binds" means that the antibody preferentially interacts with its target molecule (e.g., SIGLEC15) over other molecules because the interaction depends on the presence of a specific structure (i.e., antigenic determinant or epitope) on the target molecule; in other words, the agent recognizes and binds to molecules including a specific structure, rather than all molecules as is usually the case. An antibody that specifically binds to a target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a SIGLEC15 molecule may be referred to as a SIGLEC15-specific antibody or an anti-SIGLEC15 antibody.

[0093] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to amino acid polymers of any length of at least two amino acids.

[0094] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to nucleotide polymers of any length of at least two nucleotides, and include but are not limited to DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0095] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may 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 herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0096] From the following specific implementation and attached picture Other features and advantages of the invention will be apparent from the accompanying claims. Attached picture illustrate

[0097] Figure 1A to Figure 1B Exemplary structures of antibodies described herein are shown.

[0098] Figure 2A to Figure 2C Shown are changes in human antibody serum levels as measured by sandwich ELISA over time.

[0099] Figure 3 Shown are tumor size data for groups treated with PBS, 3 mg / kg of the atezolizumab analog, 1 mg / kg, 3 mg / kg, and 10 mg / kg of 23F4, or a combination of 3 mg / kg of the atezolizumab analog and 10 mg / kg of 23F4.

[0100] Figure 4 Shown are tumor size data for groups treated with PBS, 3 mg / kg of 23F4, or 10 mg / kg of 5G12.

[0101] Figure 5 Shown is the cell sorting strategy for TIL (tumor infiltrating lymphocyte) analysis.

[0102] Figure 6A to Fig. 6E Shown are the results of TIL analysis in the MC38 tumor model as determined by flow cytometry.

[0103] Figure 7 Shown are tumor size data for groups treated with PBS, 1 mg / kg of atezolizumab analog, 10 mg / kg of 10A7, or a combination of 1 mg / kg of atezolizumab analog and 10 mg / kg of 10A7.

[0104] Figure 8Shown are tumor size data for groups treated with PBS, 0.3 mg / kg of YH004, 10 mg / kg of 23F4, or a combination of 0.3 mg / kg of YH004 and 10 mg / kg of 23F4.

[0105] Fig. 9A The mean body weights of mice in the control and treatment groups are shown.

[0106] Fig. 9B Shown are the changes in body weight of mice in the control and treatment groups.

[0107] Figure 10A to Figure 10M Test results from biochemical tests (alanine aminotransferase (ALT) and aspartate (AST)) and hematological tests (complete blood count, including white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit or packed cell volume (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), lymphocyte percentage (LYMPH%), monocyte percentage (MONO%), neutrophil percentage (NEUT%)) are shown.

[0108] Fig.11 The CDR sequences of anti-SIGLEC15 antibodies (10A7, 10C9, and 23F4) and the CDR sequences of their related anti-SIGLEC15 antibodies defined by Kabat numbering are listed.

[0109] Fig.12 The CDR sequences of anti-SIGLEC15 antibodies (10A7, 10C9, and 23F4) and the CDR sequences of their related anti-SIGLEC15 antibodies defined by Chothia numbering are listed.

[0110] Fig.13 The amino acid sequences of the heavy chain variable regions and light chain variable regions of the anti-SIGLEC15 antibodies (10A7, 10C9, and 23F4) are listed.

[0111] Fig.14 Certain amino acid sequences discussed in this disclosure are listed. DETAILED DESCRIPTION

[0112] The present disclosure provides examples of antibodies, antigen-binding fragments thereof, that bind SIGLEC15.

[0113] SIGLEC15 and the immune system

[0114] SIGLEC15 (sialic acid-binding immunoglobulin-like lectin 15) belongs to the SIGLEC family and is a type I transmembrane protein. It is rarely expressed in most normal human tissues and immune cell subsets, but has a relatively high expression in macrophages.

[0115] SIGLECs constitute a family of cell surface proteins that play an important role in the regulation of immune homeostasis. Disorders of these proteins are associated with a variety of diseases ranging from autoimmunity to infection and cancer. SIGLECs are type I transmembrane proteins that have a V group immunoglobulin (Ig) domain containing a sialic acid binding site and one or more C2 group Ig domains in their extracellular regions. Most Siglecs (including CD22 (Siglec-2) related Siglecs 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 acid (a group of sugars expressed on all mammalian cells) as a mechanism to distinguish between self and non-self. However, some pathogens can use inhibitory Siglecs to suppress immune responses and facilitate their survival. Although most Siglecs act as receptors, some Siglecs can act as functional ligands, such as Siglec-1. Siglec expression has been found to be mainly on hematopoietic cells (mainly on myeloid cells and B cells) or on non-hematopoietic cells such as neurons. In the Siglec family, Siglec-15 has been identified as a very unique member, selectively expressed on myeloid cells and osteoclasts (bone-specific myeloid lineage) and generally absent from other immune cells and tissues.

[0116] Amino acid sequence alignment between human Siglec-15 and mouse Siglec-15 showed 83% identity, and unlike other Siglecs located within chromosomes 19 or 1, the Siglec-15 gene is located within chromosome 18. Early studies have shown that Siglec-15 contains a conserved arginine (R143) motif in the membrane distal IgV domain, which is critical for sialic acid binding. Siglec-15 preferentially binds Sialyl-Tn (Neu5Ac alpha 2–6GalNAc), a short O-glycan with sialic acid residues, whose neo-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 domains “tandem repeats” in the extracellular region, Siglec-15 displays only one IgV and one IgC2 domain, a structure that is common among B7 family members. There is a high degree of structural homology between Siglec-15 and PD-L1, and the protein sequence of the extracellular domain of Siglec-15 exhibits 20%–30% identity with the B7 family, which is similar to the identity between B7 family members. These unique molecular features highlight the unique properties of Siglec-15 and suggest that Siglec-15 may be related to B7 immunomodulatory molecules.

[0117] Unlike most Siglec members, Siglec-15 does not have a typical ITIM or ITIM-like motif in its intracellular domain that mediates inhibitory signaling. Instead, it is reported to bind to the 12kDa signaling adaptor DNAX-activated protein (DAP12) and DAP10 (which contains an immunoreceptor tyrosine-based activation motif (ITAM)) through positively charged lysine residues in its transmembrane domain (K273 in mouse Siglec-15; K274 in human Siglec-15). Binding to DAP12 and / or DAP10 is a typical feature of some Siglecs with activated signaling (such as Siglec-14 and Siglec-16), which may be achieved by recruiting spleen tyrosine kinase (SYK) and ZAP70, etc.

[0118] Siglec-15 has been identified as an important regulator in osteoclast differentiation and function. In an attempt to identify regulators of osteoclast-like giant cell tumor, Yoshiharu and colleagues found that Siglec-15 is upregulated in osteoclasts upon stimulation with receptor activator of nuclear factor-κB ligand. Knockdown of Siglec-15 with 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—removal of sialic acid by sialidases or disruption of sialylated glycan binding by R143 mutations impairs osteoclast development. DAP12 may be required for Siglec-15 function in osteoclasts, as the K273 mutation that disrupts DAP12 binding to Siglec-15 results in loss of Siglec-15 function in osteoclasts. However, it is unclear whether Siglec-15 functions primarily as a receptor or a ligand, or how important the binding of DAP-12 and DAP-10 is for the osteoclast function of Siglec-15.

[0119] Considering that Siglec-15 is structurally similar to the B7 family and has a dominant expression pattern in myeloid cells, it has been speculated that it may be involved in immune regulation. In early 2010, a research group used the TCAA screening platform to observe the inhibition of NFκB reporter gene activity in Jurkat T cells by HEK-293T cells expressing Siglec-15. In addition, Siglec-15 extracellular domain fusion protein, whether coated on the plate or provided in soluble form, can effectively inhibit anti-CD3 (OKT3)-induced human T cell proliferation. Consistent with this, the expression of Siglec-15 on artificial APCs inhibited mouse T cell proliferation, cytokine secretion, and killing ability. These in vitro data suggest a ligand-like function of Siglec-15, which inhibits human or mouse T cells through unknown receptor signaling. The in vivo function of Siglec-15 on T cells was subsequently verified using the experimental autoimmune encephalomyelitis (EAE) mouse model. The study found that EAE was significantly worsened and T cell responses were significantly enhanced in Siglec-15-deficient mice or mice injected with Siglec-15 extracellular domain fusion protein compared with the control group. In addition, it was observed that Siglec-15 affects antigen-specific T cell responses—the expansion of OT-I T cells in the blood and spleen of Siglec-15-deficient mice was much higher under OVA peptide stimulation compared with WT mice, which is similar to the phenotype of PD-L1 KO mice. IL10 may be an important factor in this process, because the IL10 level in the serum of Siglec15-deficient mice showed a decrease compared with WT mice, and anti-IL10 mAb eliminated the difference in OT-I T cell expansion between WT mice and Siglec-15-deficient mice.

[0120] For a detailed review of SIGLEC15 and its functions, see Sun, Jingwei et al., “Siglec-15 as an emerging target for next-generation cancer immunotherapy.” Clinical Cancer Research 27.3 (2021): pp. 680-688, the entire text of which is incorporated herein by reference.

[0121] 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.

[0122] Antibodies and antigen-binding fragments

[0123] The present disclosure provides anti-SIGLEC15 antibodies and antigen-binding fragments thereof. Generally, antibodies (also known as immunoglobulins) are composed of two types of polypeptide chains, namely light chains and heavy chains. The non-limiting antibodies of the present disclosure can be complete four-immunoglobulin chain antibodies including 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 subisotype (including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.). The light chain can be a kappa light chain or a lambda light chain. The antibody may include two identical copies of a light chain and two identical copies of a heavy chain. Each contains a variable domain (or variable region, V H ) and multiple constant domains (or constant regions) are bound to each other through disulfide bonds within their constant domains to form the "backbone" of the antibody. L ) and a light chain of a constant domain (or constant region) each bind to a heavy chain through 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, which are sandwiched between more conserved framework regions (FR).

[0124] These hypervariable regions, called complementarity determining regions (CDRs), form loops that comprise the major antigen-binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation, while the CDRs form loops that connect and in some cases form part of the beta-sheet structure. The CDRs in each chain are held tightly together by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding region.

[0125] Methods for identifying the CDR regions of an antibody by analyzing its amino acid sequence are well known, and a number of definitions of CDRs 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, pp. 422-439; Abhinandan et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains", Molecular immunology 45.14 (2008): pp. 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med., Vol. 132: pp. 211-250; Martin et al., Methods Enzymol. Vol. 203: pp. 121-153 (1991); Morea et al., Biophys Chem. Vol. 68, No. 1-3: pp. 9-16 (October 1997); Morea et al., J. Chem. Vol. 68, No. 1-3: pp. 9-16 (October 1997); Morea et al., J. Mol Biol. Vol. 275 No. 2: pp. 269-294 (January 1998); Chothia et al., Nature Vol. 342 No. 6252: pp. 877-883 (December 1989); Ponomarenko and Bourne, BMC Structural Biology: Vol. 7: p. 64 (2007), all of which are incorporated herein by reference in their entirety.

[0126] CDR is very important for recognizing the epitope of an antigen. As used herein, an "epitope" is the smallest part 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 three, four, five, six or seven amino acids, but these amino acids do not have to be in a continuous linear sequence of the primary structure of the antigen, because the epitope can depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structures of the antigen.

[0127] In some embodiments, the antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, but differ in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and are described in, for example, 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, eds., "The human IgG subclasses: molecular analysis of structure, function and regulation." Elsevier, 2016, each of which is incorporated herein by reference in its entirety.

[0128] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camelids). Antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies comprising immunoglobulin binding domains fused to another polypeptide. The term "antigen binding domain" or "antigen binding fragment" is a part of an antibody that retains the specific binding activity of a complete antibody, that is, any part of an antibody that can specifically bind to an epitope on a target molecule of a complete antibody. It includes, for example, Fab, Fab', F(ab')2 and variants of these fragments. Therefore, in some embodiments, an antibody or its antigen binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, double antibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed by antibody fragments, and any polypeptide comprising an antibody binding domain or a binding domain homologous thereto. Non-limiting examples of antigen binding domains include, e.g., heavy and / or light chain CDRs of an intact antibody, heavy and / or light chain variable regions of an intact antibody, the full length heavy or light chain of an intact antibody, or a single CDR from a heavy or light chain of an intact antibody.

[0129] In some embodiments, the antigen binding fragment may form a part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-chain variable fragment (scFv) fused to a CD3-ζ transmembrane domain and an intracellular domain as described herein. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to increase effectiveness. Therefore, on the one hand, the present disclosure also provides cells (e.g., T cells) expressing chimeric antigen receptors as described herein.

[0130] 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.

[0131] In some embodiments, the sequences of the antibodies or antigen-binding fragments thereof described herein (eg, CDR or VH / VL sequences) can be used to generate bispecific antibodies targeting SIGLEC15 and an additional antigen (eg, PD-1).

[0132] Anti-SIGLEC15 antibodies and antigen-binding fragments

[0133] 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 an immune response. In some embodiments, these antibodies can block the SIGLEC15 pathway.

[0134] The present disclosure provides, for example, anti-SIGLEC15 antibodies 10A7, 10C9, 23F4, chimeric antibodies thereof, and human or humanized antibodies thereof.

[0135] The CDR sequences of 10A7 and 10A7-derived antibodies (e.g., human or humanized antibodies) include CDRs of the heavy chain variable domain (SEQ ID NOs: 1-3) and CDRs of the light chain variable domain (SEQ ID NOs: 4-6) as defined by Kabat numbering. CDRs may also be defined by the Chothia system. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 19-21, and the CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 22-24.

[0136] Similarly, the CDR sequences of 10C9 and 10C9-derived antibodies include the CDRs of the heavy chain variable domain (SEQ ID NOs: 7-9) and the CDRs of the light chain variable domain (SEQ ID NOs: 10-12) as defined by Kabat numbering. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 25-27, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 28-30.

[0137] The CDR sequences of 23F4 and 23F4-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NOs: 13-15) and CDRs of the light chain variable domain (SEQ ID NOs: 16-18) as defined by Kabat numbering. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 31-33, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 34-36.

[0138] The amino acid sequence of the heavy chain variable region of the 10A7 antibody is shown in SEQ ID NO: 37. The amino acid sequence of the light chain variable region of the 10A7 antibody is shown in SEQ ID NO: 38.

[0139] The amino acid sequence of the heavy chain variable region of the 10C9 antibody is shown in SEQ ID NO: 39. The amino acid sequence of the light chain variable region of the 10C9 antibody is shown in SEQ ID NO: 40.

[0140] The amino acid sequence of the heavy chain variable region of the 23F4 antibody is shown in SEQ ID NO: 41. The amino acid sequence of the light chain variable region of the 23F4 antibody is shown in SEQ ID NO: 42.

[0141] Also provided are amino acid sequences of 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 they bind SIGLEC15 together.

[0142] Humanization percentage means the percentage of identity of the heavy chain or light chain variable region sequence compared to the human antibody sequence in the International Immunogenetics Information System (IMGT) database. The best comparison (top hit) means that the heavy chain or light chain variable region sequence is closer to a specific species than to other species. For example, the best comparison with man means that the sequence is closer to people than to other species. The best comparison with humans and cynomolgus monkeys (Macaca fascicularis) means that the sequence has the same percentage of identity with humans and cynomolgus monkey sequences, and compared with other species sequences, these percentages of identity are the highest. In some embodiments, the humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. Detailed descriptions of how to determine the percentage of humanization and how to determine the optimal alignment are 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, which is incorporated herein by reference in its entirety. A high percentage of humanization generally has a variety of advantages, for example, being safer and more effective in humans, being more likely to be tolerated by human subjects, and / or having less likelihood of side effects. In some embodiments, the variable region is fully human, for example, derived from a human heavy chain immunoglobulin locus sequence (e.g., recombination of human IGHV, human IGHD, and human IGHJ genes), and / or a human kappa chain immunoglobulin locus sequence (e.g., recombination of human IGKV and human IGKJ genes).

[0143] In addition, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may also contain one, two or three heavy chain variable region CDRs selected from the following items: SEQ ID NO: 1-3, SEQ ID NO: 7-9, SEQ ID NO: 13-15, SEQ ID NO: 19-21, SEQ ID NO: 25-27 and SEQ ID NO: 31-33; and / or one, two or three light chain variable region CDRs selected from the following items: SEQ ID NO: 4-6, SEQ ID NO: 10-12, SEQ ID NO: 16-18, SEQ ID NO: 22-24, SEQ ID NO: 28-30 and SEQ ID NO: 34-36.

[0144] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity determining regions (CDR) 1, 2, 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, the antibody may have a light chain variable region (VL) comprising CDR1, 2, 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 Fig.11 (Kabat CDR) and Fig.12 (Chothia CDR).

[0145] In some embodiments, the antibodies or antigen-binding fragments described herein may include a heavy chain variable domain containing one, two or three of the following CDRs: a CDR of SEQ ID NO: 1 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 2 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 3 having zero, one or two amino acid insertions, deletions or substitutions.

[0146] In some embodiments, the antibodies or antigen-binding fragments described herein may include a heavy chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO:7 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO:8 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO:9 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0147] In some embodiments, the antibodies or antigen-binding fragments described herein may include a heavy chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO: 13 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 14 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 15 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0148] In some embodiments, the antibodies or antigen-binding fragments described herein may include a heavy chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO: 19 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 20 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO: 21 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0149] In some embodiments, the antibodies or antigen-binding fragments described herein may include a heavy chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO:25 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO:26 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO:27 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0150] In some embodiments, the antibodies or antigen-binding fragments described herein may include a heavy chain variable domain containing one, two or three of the following CDRs: a CDR of SEQ ID NO:31 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:32 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:33 having zero, one or two amino acid insertions, deletions or substitutions.

[0151] In some embodiments, the antibodies or antigen-binding fragments described herein may include a light chain variable domain containing one, two or three of the following CDRs: a CDR of SEQ ID NO:4 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:5 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:6 having zero, one or two amino acid insertions, deletions or substitutions.

[0152] In some embodiments, the antibodies or antigen-binding fragments described herein may include a light chain variable domain containing one, two or three of the following CDRs: a CDR of SEQ ID NO: 10 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 11 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 12 having zero, one or two amino acid insertions, deletions or substitutions.

[0153] In some embodiments, the antibodies or antigen-binding fragments described herein may include a light chain variable domain containing one, two or three of the following CDRs: a CDR of SEQ ID NO: 16 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 17 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 18 having zero, one or two amino acid insertions, deletions or substitutions.

[0154] In some embodiments, the antibodies or antigen-binding fragments described herein may include a light chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO:22 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO:23 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO:24 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0155] In some embodiments, the antibodies or antigen-binding fragments described herein may include a light chain variable domain containing one, two or three of the following CDRs: a CDR of SEQ ID NO:28 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:29 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:30 having zero, one or two amino acid insertions, deletions or substitutions.

[0156] In some embodiments, the antibodies or antigen-binding fragments described herein may include a light chain variable domain containing one, two, or three of the following CDRs: a CDR of SEQ ID NO:34 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO:35 having zero, one, or two amino acid insertions, deletions, or substitutions; a CDR of SEQ ID NO:36 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0157] Insertions, deletions and substitutions may be within the CDR sequence or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on the Kabat numbering convention. In some embodiments, the CDR is determined based on the Chothia numbering convention. In some embodiments, the CDR is determined based on a combination of the Kabat and Chothia numbering conventions.

[0158] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to SIGLEC15. The antibody or antigen-binding fragment thereof contains: a heavy chain variable region (VH), the VH comprising or consisting of an amino acid sequence having at least 80%, 85%, 90% or 95% identity with a selected VH sequence; and a light chain variable region (VL), the VL comprising or consisting of an amino acid sequence having at least 80%, 85%, 90% or 95% identity with 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.

[0159] The present disclosure also provides antibodies or antigen-binding fragments thereof that can compete with the antibodies described herein.In some aspects, the antibodies or antigen-binding fragments can bind to the same epitope as the antibodies described herein.

[0160] The present disclosure also provides antibodies or antigen-binding fragments thereof that cross-compete with any antibody or antigen-binding fragment as described herein. Cross-competition assays are known in the art and are described in, for example, Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1gp120exterior envelope glycoprotein.", Journal of virology, 70.3 (1996): pp. 1863-1872, which is incorporated herein by reference in its entirety. On the one hand, the present disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any antibody or antigen-binding fragment as described herein. Epitope grouping assays are known in the art and are described in, for example, Estep et al., "High throughput solution-based measurement ofantibody-antigen affinity and epitope binning.", MAbs. Vol. 5, No. 2, Taylor & Francis, 2013, which are incorporated herein by reference in their entirety.

[0161] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, these sequences are compared for the best comparison purpose (for example, a gap can be introduced in one or both of the first and second amino acid or nucleic acid sequences for the best comparison, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, these molecules are identical at that position. Considering the number of gaps and the length of each gap that need to be introduced to achieve the best comparison of the two sequences, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences. For example, the comparison of the sequence and the determination of the percent identity between the two sequences can be completed using the Blossum 62 scoring matrix, wherein the gap penalty is 12, the gap extension penalty is 4, and the frameshift gap penalty is 5.

[0162] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising an immunoglobulin heavy chain or an immunoglobulin light chain. An immunoglobulin heavy chain or an immunoglobulin light chain comprises Fig.11 or Fig.12 The CDRs shown, or having Fig.13When the polypeptide is paired with a corresponding polypeptide (eg, a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to SIGLEC15 (eg, human SIGLEC15).

[0163] Anti-SIGLEC15 antibodies and antigen-binding fragments may also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multispecific (e.g., bispecific) antibodies or antibody fragments. Other antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific (multimers, e.g., bispecific) antibodies, human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, antibodies prepared intracellularly (i.e., intrabodies), and antigen-binding fragments thereof. The antibody or its antigen-binding fragment may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), category (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.

[0164] As long as the antibody fragment retains the desired affinity and specificity of the full-length antibody, it is suitable for the provided method. Therefore, the antibody fragment that binds to SIGLEC15 will retain the ability to bind to SIGLEC15. The Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain that are tightly bound, and the tight binding can be covalent in nature, such as scFv. In this configuration, it is the three CDRs of each variable domain that interact to define the antigen binding site on the surface of the VH-VL dimer. In general, the six CDRs or a subset thereof together confer antigen binding specificity to the antibody. However, even a single variable domain (or half an Fv that includes only three CDRs specific to a certain antigen) has the ability to recognize and bind to an antigen, but the affinity is usually lower than that of the entire binding site.

[0165] Single-chain Fv (scFv) antibody fragments comprise the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide also includes a polypeptide linker between the VH and VL domains so that the scFv forms a desired structure for antigen binding.

[0166] The Fab fragment contains the variable domain and constant domain of the light chain and the variable domain and the first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment comprises a pair of Fab fragments, which are usually covalently linked near their carboxyl termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0167] Diabodies are small antibody fragments with two antigen-binding sites that contain VH and VL linked on 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 are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0168] Linear antibodies include a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0169] The one-armed antibody may have a heavy chain and a light chain, as well as a heavy chain fragment including the CH2 and CH3 domains of IgG ( Figure 1B ). In some embodiments, a one-armed antibody is an antibody that has only one of the two antigen-binding arms in a typical antibody. In some embodiments, a one-armed antibody includes one antigen-binding arm (e.g., VH+CH1 and VL+CL) and an Fc region.

[0170] The antibodies and antibody fragments of the present disclosure may be modified in the Fc region to provide desired effector functions or serum half-life. In some embodiments, the Fc region may be modified to silence or reduce complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).

[0171] The multimerization of antibodies can be achieved by natural aggregation of antibodies or by chemical or recombinant connection techniques known in the art. For example, a certain percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0172] Alternatively, antibody homodimers can be formed by chemical connection techniques known in the art. For example, heterologous bifunctional cross-linking agents include, but are not limited to, SMCC (succinimidyl 4-(maleimidomethyl) cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acetylthioacetate) can be used to form antibody polymers. An exemplary scheme for forming antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. USA, Vol. 94: pp. 7509-7514, 1997). By pepsin digestion, antibody homodimers can be converted into Fab'2 homodimers. Another method for forming antibody homodimers is by using an autophagic T15 peptide, which is described in Zhao et al. (J. Immunol. Vol. 25: pp. 396-404, 2002).

[0173] In some embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be prepared by engineering the interface between a pair of antibody molecules so that the percentage of the heterodimer recovered from the recombinant cell culture is maximized. For example, the interface can include 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 by larger side chains (for example, tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (for example, alanine or threonine), a compensation "cavity" identical or similar to the large side chain size is produced on the interface of the second antibody molecule. This provides a mechanism that can make the yield of heterodimers higher than other unwanted end products (such as homodimers). The method is described in, for example, WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0174] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other antibody can be coupled to biotin. Heteroconjugate antibodies can also be prepared using any convenient cross-linking method. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

[0175] Methods for producing bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. (Science, Vol. 229: p. 81, 1985) described a method in which an intact antibody is proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, sodium arsenite, to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'TNB derivatives is then converted into a Fab'thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'TNB derivative to form a bispecific antibody.

[0176] Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or 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 stabilizing molecules can increase the half-life of the antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored in a pharmaceutical composition) or in vivo (e.g., in the human body) or extend its biological activity.

[0177] In some embodiments, the antibodies or antigen binding fragments described herein may be conjugated to a therapeutic agent. Antibody drug conjugates including antibodies or antigen binding fragments thereof may be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoid alkaloids (such as DM-1 and DM-4), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and the like).

[0178] In some embodiments, the antibodies or antigen-binding fragments thereof described herein recognize endogenous SIGLEC15 or recombinant SIGLEC 15. In some embodiments, the antibodies or antigen-binding fragments thereof described herein recognize human SIGLEC15.

[0179] 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 antibody or antigen-binding fragment thereof described herein in a SIGLEC15 gene humanized mouse (e.g., hSIGLEC15 mouse) 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 (CL) of the antibody or antigen-binding fragment thereof described herein in a SIGLEC15 gene humanized mouse (e.g., hSIGLEC15 mouse) 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.

[0180] In some embodiments, the clearance rate (CL) of the antibody or antigen-binding fragment 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 antibody or antigen-binding fragment 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.

[0181] In some embodiments, the half-life of the antibodies or antigen-binding fragments thereof described herein (e.g., in humanized FcRn 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 humanized FcRn mice) is less than 16 ml / 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.

[0182] Antibody characteristics

[0183] The antibodies or antigen-binding fragments thereof described herein can block the binding between SIGLEC15 and a SIGLEC15 ligand (eg, sialic acid).

[0184] The antibodies or antigen-binding fragments thereof described herein may be agonists or antagonists of the SIGLEC15 pathway. In some embodiments, by binding to SIGLEC15, the antibodies may inhibit the SIGLEC15 signaling pathway. In some embodiments, the antibodies may upregulate an immune response or downregulate an immune response.

[0185] In some embodiments, antibodies or antigen-binding fragments thereof as described herein can increase the activity or number of immune responses, 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, antibodies or antigen-binding fragments thereof as described herein can reduce 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.

[0186] In some embodiments, the antibodies or antigen-binding fragments thereof as 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 as 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 as 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, 10-fold. In some embodiments, the antibodies or antigen-binding fragments thereof as described herein can increase the amount of CTLs in mCD45+ cells, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold. In some embodiments, the antibodies or antigen-binding fragments thereof as 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, 10-fold.

[0187] In some embodiments, the antibody (or antigen-binding fragment thereof) is activated at a rate of less than 0.1 s -1 Less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 , less than 0.00001s -1 , less than 0.000001s -1 or less than 0.0000001s -1 The dissociation rate (koff) of the antibody specifically binds to SIGLEC15 (e.g., human SIGLEC15, monkey SIGLEC15 (e.g., macaque, cynomolgus monkey (Macaca fascicularis)), dog SIGLEC15, mouse SIGLEC15 and / or chimeric SIGLEC15). In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 , greater than 0.000001s -1 , greater than 0.0000001s -1 or greater than 0.00000001s -1 .

[0188] In some embodiments, the kinetic binding 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 kinetic binding rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0189] Affinity can be derived from the quotient of the kinetic rate constants (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 In some embodiments, the 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, the 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.

[0190] General techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). 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.

[0191] In some embodiments, thermal stability is determined.Antibodies or antigen-binding fragments as described herein can have a Tm greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C. In some embodiments, the Tm is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C.

[0192] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein may bind to the same epitope of SIGLEC15. In some embodiments, the antibodies or antigen-binding fragments thereof as described herein may bind to different epitopes of SIGLEC15.

[0193] In some embodiments, the antibody or antigen-binding fragment thereof as described herein has a purity of more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, or more than 98% as determined by size exclusion chromatography (SEC). In some embodiments, the hydrophobic interaction chromatography (HIC) retention time of the antibody or antigen-binding fragment thereof as described herein is higher than 2 minutes, higher than 2.5 minutes, higher than 3 minutes, higher than 3.5 minutes, higher than 4 minutes, or higher than 4.5 minutes. In some embodiments, the antibody or antigen-binding fragment thereof as described herein has a main peak of 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 as described herein have an isoelectric point (PI) of greater than 7.5, greater than 7.75, greater than 8, greater than 8.25, greater than 8.5, or greater than 8.75 as determined by capillary isoelectric focusing (cIEF). In some embodiments, the antibodies or antigen-binding fragments thereof as described herein have a half-maximal effective concentration (EC50) of less than 2.5 μg / mL, less than 2 μg / mL, less than 1.5 μg / mL, less than 1 μg / mL, less than 0.75 μg / mL, less than 0.5 μg / mL, less than 0.25 μg / mL, less than 0.15 μg / mL.

[0194] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein can relieve the inhibition of cell proliferation caused by hSIGLEC15. In some embodiments, the antibodies or antigen-binding fragments thereof as described herein can relieve the inhibition of hCD4+T cell proliferation caused by hSIGLEC15 by greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. In some embodiments, the antibodies or antigen-binding fragments thereof as described herein can relieve the inhibition of hCD8+T cell proliferation caused by hSIGLEC15 by greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%.

[0195] In some embodiments, the antibody has a tumor growth inhibition percentage (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. In some embodiments, the antibody has a tumor growth inhibition percentage less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. TGI% can be determined, for example, at 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days or 30 days after the start of treatment, or at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months after the start of treatment. As used herein, tumor growth inhibition percentage (TGI%) is calculated using the formula:

[0196] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100

[0197] Ti is the mean tumor volume in the treatment group at day i. T0 is the mean tumor volume in the treatment group at day zero. Vi is the mean tumor volume in the control group at day i. V0 is the mean tumor volume in the control group at day zero.

[0198] In some embodiments, an antibody or antigen-binding fragment thereof as described herein is a SIGLEC15 antagonist. In some embodiments, the antibody or antigen-binding fragment reduces SIGLEC15 signaling in a target cell expressing SIGLEC15.

[0199] In some embodiments, the antibody or antigen-binding fragment can enhance APC (eg, DC cell) function, for example, inducing surface expression of co-stimulatory and MHC molecules, inducing the production of pro-inflammatory cytokines, and / or enhancing T cell triggering function.

[0200] 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 cellular cytotoxicity (ADCC), and kill tumor cells.

[0201] 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.

[0202] In some embodiments, the antibody or antigen-binding fragment can induce complement dependent cytotoxicity (CDC).

[0203] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3 or human IgG4. In some embodiments, the antibody is a human IgG1 antibody, optionally with an SI mutation, a LALA mutation, a N297A mutation, a YTE mutation and / or a FLAA mutation. In some embodiments, the antibody is a human IgG4 antibody, optionally with an SI mutation, a LALA mutation, a N297A mutation, a YTE mutation and / or a FLAA mutation.

[0204] 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 Fab, Fab', F(ab')2 and Fv fragment. In some embodiments, the Fc region has LALA mutation (according to the L234A and L235A mutations of EU numbering), or LALA-PG mutation (according to the L234A, L235A, P329G mutations of EU numbering). In some embodiments, the Fc region has FLAA mutation (according to the F234A and L235A of EU numbering). In some embodiments, Fc has SI mutation (according to the S239D and I332E mutations of EU numbering). In some embodiments, Fc has N297A mutation according to EU numbering. In some embodiments, Fc has YTE mutation (according to the M252Y, S254T and T256E of EU numbering).

[0205] In some embodiments, the antibodies or antigen-binding fragments described herein include an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any of SEQ ID NOs: 70-77. Methods of Making Anti-SIGLEC15 Antibodies

[0206] Separated human SIGLEC15 fragments can be used as immunogens to produce antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of antigenic peptides or proteins. 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. Animals can be injected with more than one (e.g., two, three, or four) antigenic peptides or proteins.

[0207] The full-length polypeptide or protein, or an antigenic peptide fragment thereof, can be used as an immunogen. The antigenic peptide of the protein includes at least 8 (e.g., at least 10, 15, 20 or 30) amino acid residues of the amino acid sequence of SIGLEC15, and covers the epitope of the protein, so that the antibody produced 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 known in the art. In some embodiments, a human SIGLEC15 protein with an Fc tag or a His tag is used as an immunogen.

[0208] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may contain, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., fragments of human SIGLEC15). The preparation may also include an adjuvant (such as Freund's complete or incomplete adjuvant) or a similar immunostimulant.

[0209] 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 portion of SIGLEC15) as an immunogen. Antibody titers in immunized subjects can be monitored over time using immobilized SIGLEC15 polypeptides or peptides by standard techniques, such as enzyme-linked immunosorbent assay (ELISA). If necessary, antibody molecules can be isolated from mammals (e.g., from blood) and further purified by well-known techniques (such as protein A or protein G chromatography) to obtain IgG fractions. At an appropriate time after immunization, for example, when specific antibody titers are highest, antibody-producing cells can be 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, Vol. 256: pp. 495-497, 1975), human B cell hybridoma technology (Kozbor et al., Immunol. Today, Vol. 4: p. 72, 1983), EBV-hybridoma technology (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., ed., John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing monoclonal antibodies are detected by screening hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, for example, using a standard ELISA assay.

[0210] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding the human, humanized or chimeric antibodies or antigen-binding fragments thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence constituting the antigen-binding site of the antibody or antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein (e.g., SIGLEC15). Any combination of deletions, insertions and / or combinations may be performed to obtain antibodies or antigen-binding fragments thereof with increased binding affinity to the target. Amino acid changes introduced into antibodies or antigen-binding fragments may also change or introduce new post-translational modifications into antibodies or antigen-binding fragments, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation sites (e.g., changing the amino acid sequence so that different sugars are connected by enzymes present in the cell), or introducing new glycosylation sites.

[0211] The antibodies disclosed herein can be derived from animals of any species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, 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).

[0212] 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 antibodies derived from human germline immunoglobulin sequences). Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutations in vivo), such as in CDRs.

[0213] Humanized antibodies are usually non-human CDRs transplanted onto human frameworks (FRs). Therefore, humanized antibodies have one or more amino acid sequences introduced therein from non-human sources. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domains. Humanization can be performed essentially by, for example, replacing the corresponding sequences of human antibodies with rodent CDRs or CDR sequences. These methods are described in, for example, Jones et al., Nature, Vol. 321: pp. 522-525 (1986); Riechmann et al., Nature, Vol. 332: pp. 323-327 (1988); Verhoeyen et al., Science, Vol. 239: pp. 1534-1536 (1988); each of these documents is incorporated herein by reference in its entirety. Therefore, "humanized" antibodies are chimeric antibodies in which a small portion of the complete human V domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.

[0214] The selection of human VH and VL domains for preparing humanized antibodies is very important for reducing immunogenicity. According to the so-called "best fit" method, the V domain sequence of mouse antibodies is screened for the entire library of known human domain sequences. Then, the human sequence closest to the mouse sequence is accepted as the human FR of humanized antibodies (Sims et al., J.Immunol., Vol. 151: p. 2296 (1993); Chothia et al., J.Mol.Biol., Vol. 196: p. 901 (1987)).

[0215] More importantly, the antibody humanization retains high specificity and affinity to antigen and other favorable biological properties.In order to achieve this goal, humanized antibodies can be prepared by using the three-dimensional model analysis of parental sequence and various conceptual humanized products of parental and humanized sequences.Three-dimensional immunoglobulin model is usually available and familiar to those skilled in the art.The computer program of the possible three-dimensional conformational structure of the candidate immunoglobulin sequence of explanation and display selection can be obtained.The inspection of these displays allows the analysis of the possible effect of residues in the function of the candidate immunoglobulin sequence, i.e., analyzes the residues that affect the ability of the candidate immunoglobulin to bind its antigen.By this method, can select and combine FR residues from receptor and imported sequence, so as to obtain desired antibody characteristics, such as the affinity to the target antigen increased.

[0216] Typically, the amino acid sequence variants of a human, humanized or chimeric anti-SIGLEC15 antibody will contain an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the sequence present in the light or heavy chain of the original antibody.

[0217] In some embodiments, a mouse (e.g., RenMab) having a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus is used. TM Mouse) produces antibodies. The heavy chain immunoglobulin locus is a region on the chromosome that contains antibody heavy chain genes. 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. The κ chain immunoglobulin locus is a region on the chromosome that contains genes encoding antibody light chains (κ chains). The κ chain immunoglobulin locus can include, for example, human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light chain constant domain genes. About RenMab TM A detailed description of mice can be found in PCT / CN2020 / 075698 or US20200390073A1, the entire contents of which are incorporated herein by reference.

[0218] In some embodiments, mice (e.g., RenLite TMMouse) produces antibodies. The heavy chain immunoglobulin locus is a region on the chromosome that contains antibody heavy chain genes. 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. The κ chain immunoglobulin locus is a region on the chromosome that contains genes encoding common light chains. The κ chain immunoglobulin locus can include, for example, human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light chain constant domain genes. About RenLite TM A detailed description of mice can be found in PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety.

[0219] The antibody produced by the mouse has a complete human VH, a complete human VL, and a mouse constant region. In some embodiments, the human VH and human VL are connected to a human IgG constant region (e.g., IgG1, IgG2, IgG3, and IgG4). In some embodiments, the constant region has a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to one of SEQ ID NOs: 70-77.

[0220] Identity or homology relative to a native sequence is typically the percentage of amino acid residues present in a candidate sequence that are identical to the sequence present in a human, humanized or chimeric anti-SIGLEC15 antibody or fragment, after aligning the candidate sequence with the native sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

[0221] The anti-SIGLEC15 antibody or antigen-binding fragment may be further modified. For example, cysteine ​​residues may be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus produced may have any increased in vitro and / or in vivo half-life. Homodimeric antibodies with increased in vitro and / or in vivo half-life may also be prepared using heterologous bifunctional cross-linkers, as described, for example, in Wolff et al. (Cancer Res., Vol. 53: pp. 2560-2565, 1993). Alternatively, the antibody may be engineered to have dual Fc regions (see, e.g., Stevenson et al., Anti-Cancer Drug Design, Vol. 3: pp. 219-230, 1989).

[0222] In some embodiments, the anti-SIGLEC15 antibody or antigen-binding fragment thereof may be covalently modified. These covalent modifications may be performed 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 targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that is capable of reacting with selected side chains or N-terminal or C-terminal residues.

[0223] In some embodiments, antibody variants are provided, which have a carbohydrate structure lacking fucose (directly or indirectly) connected to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) connected to Asn297, as measured by MALDI-TOF mass spectrometry (e.g., as described in WO 2008 / 077546). Asn297 refers to an asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, due to minor sequence variations in antibodies, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function.In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0224] In some embodiments, to promote production efficiency by avoiding Fab arm exchange, the Fc region of the antibody is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). For a detailed description of the S228 mutation, see, for example, 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.

[0225] Recombinant vector

[0226] 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 these recombinant vectors are introduced (i.e., such that these host cells contain the polynucleotides and / or vectors comprising the polynucleotides), and recombinant antibody polypeptides or fragments thereof produced by recombinant technology.

[0227] As used herein, "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. "Expression vector" can deliver one or more polynucleotides of interest in a host cell into which the expression vector has been introduced and express them as encoded polypeptides. Therefore, in an expression vector, by being operably connected to a regulatory element (such as a promoter, enhancer and / or poly-A tail) at or near or on both sides of the integration site of the polynucleotide of interest in the vector or in the genome of the host cell, the polynucleotide of interest is positioned to be expressed in the vector so that the polynucleotide of interest will be translated in the host cell into which the expression vector is introduced.

[0228] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection and infection and / or transduction (e.g., with a recombinant virus). Therefore, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0229] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of non-pathogenic (defective), replication-competent viruses, or replication-defective viruses may be used. In the latter case, viral propagation typically occurs only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA, Vol. 86: 317-321; Flexner et al., 1989, Ann. NY Acad Sci., Vol. 569: 86-103; Flexner et al., 1990, Vaccine, Vol. 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; Berkner-Biotechniques, Vol. 6: pp. 616-627, 1988; Rosenfeld et al., 1991, Science, Vol. 252: pp. 431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, Vol. 91: pp. 215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 11498-11502; Guzman et al., 1993, Circulation, Vol. 88: pp. 2838-2848; and Guzman et al., 1993, Cir. Res., Vol. 73: pp. 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., 1993, Science, Vol. 259: pp. 1745-1749, and Cohen, 1993, Science, Vol. 259: pp. 1691-1692. The uptake of naked DNA may be increased by coating the DNA on biodegradable beads that are efficiently transported into cells.

[0230] For expression, the DNA insert comprising the polynucleotide encoding the antibody or polypeptide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the bacteriophage λPL promoter, E. coli (E. coli) lac, trp and tac promoters, SV40 early and late promoters, and promoters of retroviral LTRs, etc. Other suitable promoters are known to those skilled in the art. The expression construct may also contain transcription initiation and termination sites, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct may include a translation initiation codon at the start of the polypeptide to be translated and a termination codon (UAA, UGA or UAG) at an appropriate position at the end of the polypeptide to be translated.

[0231] As described above, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as Escherichia coli (E.coli), Streptomyces (Streptomyces) and Salmonella typhimurium (Salmonella typhimurium) cells; fungal cells, such as yeast cells; insect cells, such as fruit fly S2 and fall armyworm Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma and HK 293 cells; and plant cells. Suitable culture media and conditions for host cells described herein are known in the art.

[0232] Non-limiting vectors for bacteria include pQE70, pQE60, and pQE-9 from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene; and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0233] Suitable non-limiting bacterial promoters include E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, lambda PR and PL promoters and trp promoter. Suitable eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, promoters of retroviral LTR (such as the promoter of Rous sarcoma virus (RSV)) and metallothionein promoters (such as mouse metallothionein-I promoter).

[0234] In Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters are available, such as alpha factor, alcohol oxidase, and PGH. For review, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., Vol. 153: 516-544 (1997).

[0235] The construct is introduced into the host cell by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. These methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0236] The transcription of the DNA encoding the antibodies of the present disclosure by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, usually about 10 bp-300 bp, that acts to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located on the late side of the replication origin at base pairs 100-270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

[0237] In order to secrete the translated protein into 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 an endogenous signal of the polypeptide or a heterologous signal.

[0238] Polypeptides (e.g., antibodies) can be expressed in a modified form, such as a fusion protein (e.g., a GST fusion protein) or with a histidine tag, and can include not only a secretion signal, but also additional heterologous functional regions. For example, a region of additional amino acids (particularly charged amino acids) can be added to the N-terminus of the polypeptide to improve stability and durability in a host cell, during purification, or during subsequent processing and storage. In addition, peptide moieties can be added to the polypeptide to promote purification. These regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to a polypeptide to cause secretion or excretion, improve stability, and promote purification, etc., is a conventional technique well known in the art.

[0239] Treatment

[0240] The antibodies or antigen-binding fragments thereof disclosed herein can be used for a variety of therapeutic purposes.

[0241] In one aspect, the present disclosure provides methods for treating cancer in a subject, methods for reducing the rate of increase of tumor volume over time in a subject, methods for reducing the risk of forming metastases, or methods for reducing the risk of forming additional metastases in a subject. In some embodiments, treatment may terminate, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment may reduce the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0242] In one aspect, the disclosure features a method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject suffering from or identified or diagnosed as suffering from cancer), such as breast cancer (e.g., triple-negative breast cancer), carcinoid, 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, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematological malignancies. In some embodiments, the cancer is unresectable melanoma 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, hematological malignancies, head and neck cancer, gastrointestinal cancer, bladder cancer, or bone cancer. In some embodiments, the subject suffers from 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 colorectal cancer. In some embodiments, the subject suffers from Hodgkin's lymphoma. In some embodiments, the subject suffers from triple negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, hematological malignancies, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

[0243] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer.Cancer patients can be identified using various methods known in the art.

[0244] In one aspect, the disclosure provides methods for treating, preventing, or reducing the risk of developing a bone disease, such as rickets, renal disease (renal osteodystrophy, Fanconi syndrome), tumor-induced osteomalacia, hypophosphatasia, McCune-Albright syndrome, or osteogenesis imperfecta with a mineralization defect (Syndrome Similar to Osteogenesis Imperfecta (SROI)). In some embodiments, the bone mineralization disorder is osteoporosis.

[0245] In one aspect, the present disclosure provides methods for treating, preventing, or reducing the risk of developing diseases associated with abnormal or unwanted immune responses (e.g., autoimmune diseases), such as by affecting the functional properties of APC cells (e.g., by blocking the interaction between SIGLEC15 and SIGLEC15 ligands). 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, disease), multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, autoimmune hepatitis, pemphigus vulgaris, Behcet's disease, pernicious anemia, bullous pemphigoid, polyarthritis nodularis, cardiomyopathy, polychondritis, celiac dermatitis, polyglandular syndrome, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelinating, 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, cryoglobulinemia, sarcoidosis, fibromyalgia, scleroderma, Graves' disease, Sjögren's syndrome, Guillain-Barre syndrome, 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 diabetes), vasculitis, lichen planus, and vitiligo. Anti-SIGLEC15 antibodies or antigen-binding fragments thereof can also be administered to a subject to treat, prevent, or reduce the risk of development of a disease associated with an abnormal or unwanted immune response associated with cell, tissue, or organ transplantation (e.g., kidney, liver, and heart transplantation (e.g., graft-versus-host disease (GVHD)), or to prevent allogeneic transplant rejection. In some embodiments, the subject suffers from skin disease, liver disease (e.g., cirrhosis), hidradenitis, experimental autoimmune encephalomyelitis. In some embodiments, the subject suffers from nephropathy, lupus, Sjögren'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 suffers from Crohn's disease, ulcerative colitis, or type 1 diabetes. In some embodiments, the subject suffers from autoimmune thyroid disease, Graves' disease, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., Crohn's disease (CD) and ulcerative colitis), rheumatoid arthritis, Sjögren's syndrome, autoimmune nephritis, or systemic lupus erythematosus.In some embodiments, the method involves administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein.

[0246] As used herein, "effective amount" refers to an amount or dosage sufficient to achieve beneficial or desired results, including terminating, slowing, delaying 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, antibody encoding polynucleotide, vector comprising the polynucleotide and / or composition thereof is to be administered, the severity of the symptoms and the route of administration, and therefore can be determined on an individual basis.

[0247] The effective amount can be applied in one or more administrations. For example, the effective amount of an antibody or antigen binding fragment is enough to improve, terminate, stabilize, reverse, inhibit, slow down and / or delay the amount of the progress of an autoimmune disease or cancer in a patient, or is enough to improve, terminate, stabilize, reverse, slow down and / or delay the amount of a cell (e.g., a biopsy cell, any one of the cancer cells described herein or a cell line (e.g., a cancer cell line)) in vitro propagation. As understood in the art, the effective amount of an antibody or antigen binding fragment can be based on, especially the patient's medical history and other factors (such as the type (and / or dosage) of the antibody used) change.

[0248] The effective amount and dosage regimen of the antibodies, polynucleotides encoding antibodies and / or compositions disclosed herein can be determined empirically, and making such determinations is within the technical scope of the art. It will be understood by those skilled in the art that the dosage that must be applied will vary according to, for example, the mammal to which the antibodies, polynucleotides encoding antibodies and / or compositions disclosed herein will be received, the route of administration, the specific type of the antibodies, polynucleotides encoding antibodies, antigen-binding fragments and / or compositions disclosed herein used, and other drugs administered to mammals. Guidance on selecting the appropriate dosage of an antibody or antigen-binding fragment can be found in the literature on the therapeutic use of antibodies and antigen-binding fragments, such as Handbook of Monoclonal Antibodies, Ferrone et al., Noges Publications, Park Ridge, NJ, 1985, Chapter 22, pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., Raven Press, New York, 1977, pp. 365-389.

[0249] The typical daily dose of an effective amount of an antibody is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dosage may 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 dosage may be greater 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 dosage 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.

[0250] In any of the methods described herein, at least one antibody, its antigen binding fragment or pharmaceutical composition (e.g., any of the antibodies, antigen binding fragments or pharmaceutical compositions described herein) and optionally at least one additional therapeutic agent may 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 with 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 with the same composition (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 in the form of a pill, tablet or capsule. In some embodiments, at least one additional therapeutic agent is administered with a sustained release oral formulation.

[0251] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of 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 overlap in the biologically active periods of the one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

[0252] 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) may be administered to a subject over an extended 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 may determine the length of the treatment period using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., to observe at least one symptom of cancer). As described herein, a skilled medical professional may also change the type and amount (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject, and may also adjust (e.g., increase or decrease) the dose or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0253] In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agent may include one or more inhibitors, and the one or more inhibitors are selected from the group consisting of: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0254] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of: a HER3 inhibitor, a LSD1 inhibitor, a MDM2 inhibitor, a BCL2 inhibitor, a CHK1 inhibitor, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

[0255] In some embodiments, the additional therapeutic agent may include 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, sorafenib, enib), Votrient, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0256] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a 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 CX3CL1-targeted therapy, a CXCL9-targeted therapy, a CXCL10-targeted therapy, a CCL5-targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0257] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0258] 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-4-1BB antibody, and / or an anti-GITR antibody.

[0259] On the one hand, the present disclosure provides a combination therapy. In some embodiments, an anti-SIGLEC15 antibody or an antigen-binding fragment thereof (e.g., any antibody described herein) may be administered together with an anti-PD-L1 antibody. In some embodiments, an anti-SIGLEC15 antibody or an antigen-binding fragment thereof (e.g., any antibody described herein) may be administered together with an anti-OX40 antibody. In some embodiments, an anti-SIGLEC15 antibody or an antigen-binding fragment thereof (e.g., any antibody described herein) may be administered together with an anti-4-1-BB antibody.

[0260] Pharmaceutical compositions and routes of administration

[0261] Also provided herein are pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) antibody or antigen-binding fragment described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein may be present in any combination in a pharmaceutical composition. The pharmaceutical composition may be formulated in any manner known in the art.

[0262] Pharmaceutical compositions are formulated to be compatible with their intended routes of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous or intraperitoneal). Compositions may include sterile diluents (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (such as benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (such as ascorbic acid or sodium bisulfite), chelating agents (such as ethylenediaminetetraacetic acid), buffers (such as acetate, citrate or phosphate) and isotonic agents (such as sugars (e.g., glucose), polyols (e.g., mannitol or sorbitol) or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The preparation of the composition may be formulated and encapsulated in ampoules, disposable syringes or multi-dose vials. When needed (as, for example, in injectable formulations), suitable fluidity can be maintained by, for example, using a coating (such as lecithin) or a surfactant. Absorption of the antibody or its Fab can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza and Nova Pharmaceuticals).

[0263] Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing predetermined quantities of active compound to facilitate administration and uniformity of dosage).

[0264] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and produced under good manufacturing practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a single dose). Pharmaceutical compositions 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 can be formulated in an aqueous solution, preferably in a physiologically compatible buffer to reduce discomfort at the injection site. The solution may contain a preparatant, such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the antibody can be in a lyophilized form so that it can be formulated with a suitable solvent (e.g., sterile pyrogen-free water) before use.

[0265] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical methods in cell culture or experimental animals (e.g., monkeys). For example, the LD50 (the dose that is lethal to 50% of the population) and the ED50 (the dose that is therapeutically effective to 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. When an agent exhibits adverse side effects, care should be taken to minimize potential damage (i.e., reduce adverse side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical methods.

[0266] The data obtained from cell culture assays and animal studies can be used to formulate any given agent for a subject (e.g., a human). The therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or their antigen-binding fragments (e.g., any one of the antibodies or antibody fragments described herein) will be a subject (e.g., a human subject identified as having cancer) or a subject identified as having a risk of illness (e.g., a subject previously suffering from cancer but now cured) to treat the disease (e.g., kill cancer cells), reduce the severity, frequency, and / or duration of one or more symptoms of the disease of the subject (e.g., a human). The effectiveness and dosage of any one of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or a veterinary professional using methods known in the art and by observing one or more symptoms of the disease in the subject (e.g., a human). Certain factors may affect the dosage and timing required for the effective treatment of a subject (e.g., the severity of the disease or condition, previous treatment, the general health and / or age of the subject, and the presence of other diseases).

[0267] Exemplary dosages include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of subject 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, it will be appreciated by those of ordinary skill in the art that the efficacy of therapeutic agents (including antibodies and antigen-binding fragments thereof) varies, and that effective amounts can be determined by methods known in the art. Typically, a relatively low dose is first administered, after which the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (when still working in the development phase) can gradually increase the dose until an appropriate response is obtained. Furthermore, it should be understood that the specific dosage level for any particular subject will depend on a variety of 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.

[0268] The pharmaceutical composition may be included in a container, pack, or dispenser together with instructions for administration. The present disclosure also provides methods for preparing antibodies or antigen-binding fragments thereof for various uses as described herein.

[0269] Example

[0270] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0271] Example 1. Generation of human anti-SIGLEC15 antibodies

[0272] To generate antibodies against human SIGLEC15, RenMab was immunized with human SIGLEC15. TM Mouse. Anti-SIGLEC15 antibody was prepared by the method described below.

[0273] RenMab TM The mouse has a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus. The heavy chain immunoglobulin locus is the region on the chromosome that contains the antibody heavy chain genes. The locus includes IGHV (variable), IGHD (diversity), IGHJ (joining), and heavy chain constant domain genes. The kappa chain immunoglobulin locus is the region on the chromosome that contains the genes encoding the antibody light chain (kappa chain). The kappa chain immunoglobulin locus includes IGKV (variable), IGKJ (joining), and light chain constant domain genes. About RenMab TMA detailed description of mice can be found in PCT / CN2020 / 075698, the entire text of which is incorporated herein by reference.

[0274] Mouse immunization

[0275] Human SIGLEC15 protein (hSIGLEC15-Fc, Beijing Biopsies Biotechnology Co., Ltd., catalog number: SG5-H5253, including positions 20-263 of SEQ ID NO: 45) and mouse SIGLEC15 protein (mSIGLEC15-His, Beijing Biopsies Biotechnology Co., Ltd., catalog number: SG5-M52H7, including positions 24-262 of SEQ ID NO: 46) were emulsified with adjuvant and used to immunize RenMab TM Mice. Before immunization, retro-orbital blood was collected as a negative control.

[0276] The first immunization used Freund's complete adjuvant CFA, and the second, third and fourth immunizations used Freund's incomplete adjuvant IFA. A total of four immunizations were performed. The first and second immunizations were separated by two weeks, and the remaining immunizations were separated by one week. Retro-orbital blood was collected one week after the fourth immunization, and the antibody titer of the serum was detected by FACS.

[0277] In another experiment, several mice were immunized by injecting expression plasmids encoding human SIGLEC15 or mouse SIGLEC15 into mice (SIGLEC15 KO mice). Plasmids encoding antigens were injected into the tibialis anterior muscle (intramuscular (im) injection). At least four injections were performed, with at least 14 days between injections. Blood (serum) was collected seven days after the last immunization, and the serum was tested for antibody titers by FACS.

[0278] A booster immunization step (by protein injection or by plasmid injection) was also performed at least fourteen days after the previous immunization. SIGLEC15 protein was injected intraperitoneally and CHO-S cells expressing human SIGLEC15 antigen were injected via the tail vein.

[0279] Antigen-specific immune cells are isolated from immunized mice to further obtain anti-SIGLEC15 antibodies or obtain the light chain and heavy chain variable region sequences of anti-SIGLEC15 antibodies. For example, using single cell technology (e.g., using Optical fluid system, Berkeley Lights Company) to screen and find plasma cells secreting antigen-specific monoclonal antibodies, and reverse transcription and PCR sequencing are used to obtain antibody variable region sequences. The obtained variable region sequences are used for antibody expression to verify the binding affinity to SIGLEC15 using FACS. Specifically, the obtained VH sequence and VL sequence are respectively connected to the human IgG1 constant region including the N297A mutation. Exemplary antibodies obtained by this method include: 10A7, 10C9 and 23F4.

[0280] The amino acid sequences of the heavy chain CDR1, 2, 3 and light chain CDR1, 2, 3 of 10A7 are shown in SEQ ID NO: 1-6 (Kabat numbering) or SEQ ID NO: 19-24 (Chothia numbering), respectively. The human heavy chain variable region and human light chain variable region of 10A7 are shown in SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

[0281] The amino acid sequences of the heavy chain CDR1, 2, 3 and light chain CDR1, 2, 3 of 10C9 are shown in SEQ ID NO: 7-12 (Kabat numbering) or SEQ ID NO: 25-30 (Chothia numbering), respectively. The human heavy chain variable region and human light chain variable region of 10C9 are shown in SEQ ID NO: 39 and SEQ ID NO: 40, respectively.

[0282] The amino acid sequences of the heavy chain CDR1, 2, 3 and light chain CDR1, 2, 3 of 23F4 are shown in SEQ ID NO: 13-18 (Kabat numbering) or SEQ ID NO: 31-36 (Chothia numbering), respectively. The human heavy chain variable region and human light chain variable region of 23F4 are shown in SEQ ID NO: 41 and SEQ ID NO: 42, respectively.

[0283] Construction of one-armed antibodies

[0284] In addition, a one-armed antibody was constructed that had an anti-SIGLEC15 arm including a heavy chain and a light chain, and a heavy chain fragment including the CH2 and CH3 domains of IgG ( Figure 1B ).

[0285] The vectors expressing each polypeptide chain of the one-armed antibody are co-transfected into CHO cells. After 14 days of culture, the cell supernatant is collected and purified by protein A affinity chromatography. The constant domain of the antibody is selected from human IgG1 or IgG4. In particular, IgG1 also introduces mutations, such as N297A mutations, to reduce Fc receptor binding affinity. These Fc mutations can improve antibody safety by minimizing antibody effector functions. Exemplary antibodies obtained by this method include: 10C9-S and 23F4-S.

[0286] In the single-armed antibody, a knob-hole (KIH) mutation was also introduced into the constant region. The knob-hole technique was used to construct a single-armed anti-SIGLEC15 antibody ( FIG. 1 ) by introducing a knob mutation (T366W) in the Fc of the full-length heavy chain and a hole mutation (T366S, L368A and Y407V) in a single Fc fragment. The single-armed anti-SIGLEC15 antibody 10C9-S, human IgG1-N297A (full-length heavy chain with knob SEQ ID NO: 43, single Fc fragment SEQ ID NO: 44, full-length light chain SEQ ID NO: 78) was generated.

[0287] Example 2. Cross-species binding of anti-SIGLEC15 antibodies

[0288] CHO-S-hSIGLEC15 cells, CHO-S-mSIGLEC15 cells, CHO-S-fasSIGLEC15 cells or CHO-S-dSIGLEC15 cells were respectively cultured at 5×10 4 The density of cells / well was transferred to a 96-well plate. Serially diluted samples of anti-SIGLEC15 antibody were added to the 96-well plate and incubated at 4°C for 30 minutes. Then, the cells were incubated with secondary antibody anti-hIgG-Fc-Alex Flour 647 (RL1-H) (Jackson ImmunoResearch Laboratories, 109-606-170) at 4°C and dark conditions for 15 minutes before flow cytometry analysis.

[0289] CHO-S-hSIGLEC15 cells, CHO-S-mSIGLEC15 cells, CHO-S-fasSIGLEC15 cells or CHO-S-dSIGLEC15 cells are obtained by transfecting CHO-S cells with human SIGLEC15 amino acid sequence (hSIGLEC15, SEQ ID NO: 45), mouse SIGLEC15 amino acid sequence (mSIGLEC15, SEQ ID NO: 46), cynomolgus monkey (Macaca fascicularis) SIGLEC15 amino acid sequence positions 60-387 (fasSIGLEC15, SEQ ID NO: 47) and dog SIGLEC15 amino acid sequence (dSIGLEC15, SEQ ID NO: 48), respectively.

[0290] The test results are shown in the table below. All anti-SIGLEC15 antibodies 10A7, 10C9 and 23F4 can bind to hSIGLEC15, mSIGLEC15, fasSIGLEC15 and dSIGLEC15.

[0291] Table 1

[0292]

[0293]

[0294] Another similar experiment was performed to determine whether the anti-SIGLEC15 antibody could bind to other SIGLEC family proteins. 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), and CHO-S-hSIGLEC11 cells (expressing hSIGLEC12, SEQ ID NO:58). NO:57), CHO-hSIGLEC11 cells (expressing hSIGLEC11, SEQ ID NO:58), CHO-hSIGLEC12 cells (expressing hSIGLEC12, SEQ ID NO:59), CHO-hSIGLEC14 cells (expressing hSIGLEC14, SEQ ID NO:60) or CHO-hSIGLEC16 cells (expressing hSIGLEC16, SEQ ID NO:61).

[0295] The results are shown in the table below. Anti-SIGLEC15 antibodies 10A7, 10C9 and 23F4 did not bind to other SIGLEC family proteins.

[0296] Table 2

[0297]

[0298]

[0299]

[0300] Example 3. Binding affinity of anti-SIGLEC15 antibodies

[0301] A Biacore was used with a pre-immobilized protein A sensor chip. TM(Biacore, Inc., Piscataway, NJ, USA) 8K biosensor, using surface plasmon resonance (SPR) to verify the binding affinity of anti-SIGLEC15 antibodies to human His-tagged SIGLEC15 protein (hSIGLEC15-His, Novoprotein Scientific, catalog number: CW37) (positions 20-263 of SEQ ID NO:45), mouse His-tagged SIGLEC15 protein (mSIGLEC15-His, Beijing Biopsies Biotechnology Co., Ltd., catalog number: SG5-M52H7) (positions 24-262 of SEQ ID NO:46) or monkey His-tagged SIGLEC15 protein (fasSIGLEC15-His, Beijing Biopsies Biotechnology Co., Ltd., catalog number: SG5-C52H6) (SEQ ID NO:62).

[0302] Purified anti-SIGLEC15 antibodies were captured on a protein A chip (Series S Sensor Chip Protein A) for detection. 1 μg / mL purified anti-SIGLEC15 antibodies were loaded at 10 μL / min to bind gradient concentrations of recombinant hSIGLEC15, fasSIGLEC15, and mSIGELC15 (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5626 nM, 0.78125 nM, and 0 nM). The flow rate was 30 μL / min, and the association and dissociation times were set to 180 s and 600 s, respectively. After the last injection of each round of titration, the chip was regenerated with a glycine solution (pH 2.0) at a rate of 30 μL / min for 30 seconds.

[0303] By using Biacore TM 8K Evaluation Software 3.0 fits the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994, Methods Enzymology, Vol. 6, pp. 99-110), thereby simultaneously obtaining the kinetic association rate (kon) and dissociation rate (koff). Affinity is derived from the quotient of the kinetic rate constant (KD = koff / kon).

[0304] As will be appreciated by those of ordinary skill in the art, the same method was used for each antibody tested, with appropriate adjustments to parameters (eg, antibody concentration). The results for the tested antibodies are summarized in the table below.

[0305] Table 3

[0306]

[0307] 5G12 is a humanized IgG1 monoclonal antibody targeting human SIGLEC15 in 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.

[0308] The results showed that both anti-SIGLEC15 antibodies 10C9 and 23F4 could bind to human SIGLEC15 and monkey SIGLEC15 with high affinity.

[0309] Example 4. Epitope analysis of anti-SIGLEC15 antibodies

[0310] The relative position of the target protein epitope between a pair of purified anti-SIGLEC15 antibodies was analyzed by biolayer interferometry (BLI) using the ForteBio Octet system at 30° C. A total of 4 purified antibodies were used: 5G12, 10A7, 10C9, and 23F4.

[0311] Throughout the experiment, 1×HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P20, pH 7.4) diluted from HBS-EP+ buffer (10×) was used as the running buffer. 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 the ligand. Another antibody was injected under the same conditions to determine whether the binding of different antibodies interfered with each other. The binding time for each antibody was 300 s.

[0312] Data Analysis HT 12.0 was used to obtain the binding values ​​of each antibody. In order to quantify the interference of one antibody on the binding of another antibody, the binding rate was calculated to compare each pair of antibodies. The binding rate is defined as the binding value of the second antibody (analyte 2) divided by the binding value of the first antibody (analyte 1). The matrix table shown in Table 4 summarizes the binding rates of each antibody pair. More specifically, when analyte 1 shows a blocking effect on analyte 2, the binding rate is -0.1 to 0.5. When analyte 1 has no blocking effect on analyte 2, the binding rate is 0.5 to 1.2. Typically, the antibody pairs that interfere with each other have the same or overlapping epitopes.

[0313] The results of epitope binding assays showed that 23F4 and 10C9 recognized different epitopes, and 10A7, 10C9, and 5G12 showed strong correlation with each other.

[0314] Table 4

[0315] Antibody 5G12 10A7 10C9 23F4 5G12 0.08 -0.08 -0.05 0.51 10A7 0.20 0.14 0.16 0.43 10C9 0.23 0.11 0.11 0.72 23F4 0.48 0.18 0.62 0.07

[0316] Example 5. Stability analysis of anti-SIGLEC15 antibodies

[0317] Anti-SIGLEC15 antibodies 10C9 and 23F4 were diluted to 2 mg / mL using a pH 6.0 buffer (3 mg / mL histidine, 80 mg / mL sucrose, and 0.2 mg / mL Tween 80). The diluted antibodies were placed in sealed Eppendorf tubes and stored at 5°C ± 3°C (hereinafter referred to as 4°C) for 7 days; at 25°C ± 2°C (hereinafter referred to as 25°C) for 7 days; or at 40°C ± 2°C (hereinafter referred to as 40°C) for 7 days, and their thermal stability was evaluated.

[0318] The freeze-thaw stability was determined using the following assay: "Freeze 1": frozen at -80°C for 5 days, then thawed at 4°C for testing; "Freeze 10": frozen at -80°C, repeated freeze-thaw 10 times within 5 days (frozen at -80°C, thawed at 4°C);

[0319] Low pH stability: The stability index of the antibodies was determined before and after incubation in 1 mol / L acetic acid, pH 3.5 for six hours.

[0320] Specifically, the following tests were performed: (1) observing the appearance of the solution and the presence of visible insoluble objects; (2) detecting changes in antibody purity by size exclusion ultra-high performance liquid chromatography (SEC-UPLC) (expressed as the percentage of the main peak area to the sum of all peak areas (purity, %)); (3) detecting changes in the apparent hydrophobicity of the antibody by the hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) method (expressed as the retention time of the main peak (HIC, min); (4) detecting charge variations in the antibody by capillary isoelectric focusing (cIEF) (expressed as the percentage of the main component, acidic component and basic component); (5) detecting antibody activity (EC50) by flow cytometry.

[0321] In the SEC-UPLC experiment, an Agilent 1290 chromatography system (with XBridge TM Protein BEH SEC column ( Waters Corporation). Antibody samples were 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 plate temperature: 6°C; run time: 7 minutes.

[0322] In the HIC-HPLC experiments, an Agilent 1260 chromatography system (with ProPac TM HIC-10 column (4.6×250 mm, Thermo Scientific) was connected), and the sample was diluted to 0.5 mg / mL using 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 plate temperature: about 6° C.; run time: 45 min.

[0323] In the cIEF experiment, the Maurice cIEF method development kit (Protein Simple, catalog number: PS-MDK01-C) was used for sample preparation. Specifically, 40 μg of protein sample was mixed with the following reagents in the kit: 1 μL Maurice cIEF pI marker-4.05, 1 μL Maurice cIEF pI marker-9.99, 35 μL 1% methylcellulose solution, 2 μL Maurice cIEF 500mM arginine, 4 μL ampholytes (Pharmalyte pH range 3-10) and water (added to make the final volume 100 μL). On the Maurice analyzer (Protein Simple, Santa Clara, CA), the imaging capillary isoelectric focusing spectrum was generated using the Maurice cIEF box (PS-MC02-C). The sample was focused for a total of 10 minutes. The analysis software installed on the instrument was used to analyze the absorbance of the focused protein at 280nm.

[0324] In the activity detection experiment, 30 μL of CHO-S-hSIGLEC15 cells were added to the culture plate, and the antibody to be tested was added in a gradient dilution (90 μg / mL, 30 μg / mL, 10 μg / mL~0.000152 μg / mL, 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. MFI values ​​were derived by FlowJo 7.6.1 analysis to calculate EC50.

[0325] The detailed results of 10C9, 10C9-S and 23F4 are shown in the table below. The results show that these anti-SIGLEC15 antibodies have good stability and physicochemical properties.

[0326] Table 5

[0327]

[0328]

[0329] Note: -: Not detected.

[0330] Example 6. Human PBMC proliferation assay

[0331] Proliferation assays were performed using human peripheral blood mononuclear cells (PBMCs). Human PBMCs (AllCells, catalog number: FPB003F-C) were stained with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE, Thermo Fisher, catalog number: C34554) according to the manufacturer's instructions. 96-well plates were coated with 0.01 μg / mL anti-CD3 monoclonal antibody (Beijing Biopsies Biotechnology Co., Ltd., catalog number: CDE-M120a). Coating was performed overnight at 4°C. The plates were washed three times with PBS. 5.00 μg / mL (final concentration) of human SIGLEC15 protein (hSIGLEC15, Beijing Biopsies Biotechnology Co., Ltd., catalog number: SG5-H5253) was added together with various concentrations of different anti-SIGLEC15 antibodies. The plates were incubated at 37°C for 3 hours to 4 hours. 2×10 5 PBMC cells were added to each well and incubated at 37°C with 5% CO2 for 72 hours. PBMC cells were stained with anti-CD4 antibody (Biolegend, catalog number: 300514) and anti-CD8a antibody (Biolegend, catalog number: 301036) for flow cytometry analysis. The details of the culture protocol and the results are shown in Table 6. The results showed that 23F4, 10C9 and 10C9-S reversed the inhibition of T cell proliferation by human SIGLEC15 protein, and the inhibitory effect of reversing T cell proliferation increased with increasing dose.

[0332] Table 6

[0333]

[0334] Example 7. Pharmacokinetic (PK) analysis

[0335] The humanized SIGLEC15 mouse model (hSIGLEC15 mouse) is engineered to express a chimeric SIGLEC15 protein (SEQ ID NO: 67), in which part of the extracellular region of the mouse SIGLEC15 protein is replaced by the corresponding human SIGLEC15 extracellular region. A detailed description of the humanized SIGLEC15 mouse model can be found in PCT / CN2022 / 080427, which is incorporated herein by reference in its entirety.

[0336] The pharmacokinetic clearance rate of anti-SIGLEC15 antibodies in hSIGLEC15 mice was determined. Specifically, mice were divided into two groups (3 mice per group) and administered 10 mg / kg of 23F4 or 5G12 by intravenous injection. Blood samples were collected 4 days before administration and 15 minutes, 4 hours, 1 day, 3 days, 7 days, 10 days, 14 days and 21 days after administration.

[0337] Serum levels of human antibodies were determined by sandwich ELISA. Briefly, goat anti-human IgG (H+L) (Jackson ImmunoResearch, catalog number: 109-005-088) was diluted to a final concentration of 2000 ng / mL, added to a 96-well plate (ELISA plate) at 100 μL / well, and then incubated overnight at 4°C. After the incubation, the cells were washed with PBS-T buffer (supplemented with Tween TM The plate was washed 4 times with PBS (20% PBS). The unbound area of ​​the antibody was blocked with 2% BSA (bovine serum albumin) at 37°C for 2 hours. The plate was then 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 washing the plate using a plate washer, 100 μL / well of peroxidase AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ specific fragment (Jackson ImmunoResearch, catalog number: 109-036-098) was added to each well of the plate and incubated at 37°C for 1 hour. After washing the plate, tetramethylbenzidine (TMB) solution was added to a 96-well plate as a substrate in an amount of 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. The absorbance of each well was read at 450 nm and 630 nm using a microplate reader. TM The data were analyzed using the data analysis software. The absorbance values ​​and corresponding concentrations of the calibration samples prepared for each test product were used to establish a calibration curve with four parameters (i.e., T 1 / 2 , C max , AUC 0天-21天The standard curve was used to calculate the antibody concentration of each serum sample. The drug concentration-time curve was established using the sample concentration calculated at each time point. Phoenix TM WinNolin 8.3 was used to calculate the pharmacokinetic parameters. The results are shown in the table below.

[0338] Table 7

[0339]

[0340] Four days before antibody administration, the antibody concentration tested was 0 μg / mL (results not shown). Figure 2A to Figure 2C As shown in Table 7, the results are consistent with typical pharmacokinetic characteristics, indicating that the concentration of antibodies in the serum of hSIGLEC15 mice decreases over time after injection of anti-SIGLEC15 antibodies. The half-lives of 10A7, 23F4, and 5G12 in hSIGLEC15 mice were 11.51 days, 9.79 days, and 4.19 days, respectively.

[0341] In similar experiments, the results of PK analysis of 23F4-S and 10C9-S are shown in Table 8.

[0342] Table 8

[0343]

[0344] Example 8. Antitumor activity in hSIGLEC15 mice

[0345] Antitumor activity of 23F4 in combination with anti-PD-L1 antibody

[0346] hSIGLEC15 mice were used to determine the anti-tumor activity of anti-SIGLEC15 antibodies. 5 MC38 cells were injected subcutaneously into hSIGLEC15 mice, and when the tumor volume grew to approximately 100 mm 3 -150mm 3 The mice were divided into different groups (5 mice per group) according to the tumor size. The treatment groups were randomly selected for 23F4 treatment, atezolizumab analog treatment, or the combination of atezolizumab analog and 23F4. The control group mice were injected with phosphate buffered saline (PBS). The administration frequency was twice a week (6 times in total). The tumor volume was measured twice a week and the weight of the mice was measured. When the tumor volume of the mice reached 3000mm 3 The detailed dosing schedule is shown in the table below.

[0347] Table 9

[0348]

[0349] The lengths of the long and short axes of the tumor were measured and the value was 0.5 × (long axis) × (short axis). 2 The tumor volume was calculated and the body weight of the mice was measured twice a week.

[0350] The tumor growth inhibition percentage (TGI%) was 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 zero. 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 zero.

[0351] Statistical analysis was performed using the t-test, with P < 0.05 being the threshold for significant differences.

[0352] The weight of mice in different groups increased. On the day of grouping (day 0), the average body weight of each group was in the range of 19.3g-19.7g. At the end of the experiment (day 25), the average body weight of each group was in the range of 22.8g-24.9g. The average body weight of each group was in the range of 118.0%-126.7%. The results show that the antibodies tested are well tolerated and have no obvious toxicity to mice.

[0353] The tumor size data in the antibody-treated group are shown in Figure 3 Table 10 below summarizes the results of the experiment, including the tumor volumes on the day of grouping (day 0), 14 days after grouping (day 14), and at the end of the experiment (day 25); the survival rate of mice; the tumor growth inhibition value (TGI%); and the statistical difference (P value) in tumor volume between the treatment group and the control group.

[0354] Table 10

[0355]

[0356] like Figure 3 As shown in Table 10, tumor growth in the treatment groups (G2 and G6) was inhibited to varying degrees compared with the control group (G1). Among the treatment groups G3-G5 treated with 23F4 alone, G4 showed the best tumor inhibition. In addition, the combination of 23F4 and atezolizumab analogs (G6) achieved better tumor inhibition than the 23F4 treatment group (G5) or the anti-PD-L1 antibody treatment group (G2), indicating that anti-SIGLEC15 antibodies enhance the anti-tumor efficacy of anti-PD-L1 antibodies in the MC38 model.

[0357] Antitumor activity of 23F4 at a dose of 3 mg / kg

[0358] In another experiment, about 5 × 10 5 MC38 cells were injected subcutaneously into hSIGLEC15 mice to determine the antitumor activity of 23F4. When the tumor volume grew to approximately 100 mm 3 At the time of the experiment, mice were divided into a control group and two treatment groups (7 mice in each group) according to the tumor size. The treatment groups were randomly selected for 23F4 treatment (G2, 3 mg / kg) or 5G12 treatment (G3, 10 mg / kg). The control group mice were injected with PBS (G1). The frequency of administration was twice a week (6 times in total). The tumor volume was measured twice a week, and the weight of the mice was measured.

[0359] The weight of mice in different groups increased. On day 0, the average weight of each group was in the range of 19.9g-20.1g. At the end of the experiment (day 21), the average weight of each group was in the range of 23.1g-23.6g. The average weight of each group was in the range of 115.6%-118.1%. The results show that the antibodies tested are well tolerated and have no obvious toxicity to mice.

[0360] The tumor sizes in the antibody-treated groups are shown in Figure 4 and Table 11, which show that compared with the control group (G1), the tumor growth of the treatment groups (G2 and G3) was inhibited to varying degrees, and compared with 5G12, 23F4 at a dose of 3 mg / kg achieved better tumor inhibition.

[0361] Table 11

[0362]

[0363] TIL analysis in the MC38 tumor model

[0364] In another experiment, about 5 × 10 5 MC38 cells were injected subcutaneously into hSIGLEC15 mice to determine the antitumor activity of 23F4 and 23F4-S. When the tumor volume grew to approximately 100 mm 3 At the time of the experiment, mice were divided into a control group and four treatment groups (7 mice in each group) according to the 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 control group mice were injected with PBS (G1). The frequency of administration was twice a week (6 times in total). The tumor volume was measured twice a week, and the weight of the mice was measured.

[0365] The weight of mice in different groups increased. On day 0, the average weight of each group was in the range of 20.8g-21.3g. At the end of the experiment (day 20), the average weight of each group was in the range of 22.9g-23.9g. The average weight of each group was in the range of 109.7%-114.0%. The results show that the antibodies tested are well tolerated and have no obvious toxicity to mice.

[0366] The tumor size data in the antibody-treated groups are shown in Table 12, which shows that tumor growth in the treatment groups (G2-G4) was inhibited to varying degrees compared to the control group (G1).

[0367] Table 12

[0368]

[0369] At the end of the experiment, tumor tissues were collected, digested, and then resuspended into single-cell suspensions for flow cytometry analysis. Before flow cytometry, 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 were used. TM Anti-mouse CD8a antibody (BioLegend, catalog number: 100744), anti-mouse NK1.1 antibody Brilliant Violet 421 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 Cell staining was performed with 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), and anti-mouse Foxp3 antibody (eBioscience, catalog number: 25-5773-82). Cell sorting strategy for TIL (tumor infiltrating lymphocyte) analysis is as follows Figure 5 shown.

[0370] like Figure 6A to Fig. 6E As shown, anti-SIGLEC15 antibodies can significantly reduce the number of G-MDSC (granulocytoid myeloid-derived suppressor cells) in the tumor microenvironment, thereby alleviating the immunosuppression caused by G-MDSC, which is manifested as a slight increase in the number of T helper cells (Th) and CTL (cytotoxic T lymphocytes). Compared with M-MDSC (monocyte-like myeloid-derived suppressor cells), G-MDSC occupies a higher proportion in the tumor microenvironment, so the effect of G-MDSC on immunosuppression may be greater than that of M-MDSC.

[0371] Antitumor activity of 10C9

[0372] A total of fifteen hSIGLEC15 mice were injected subcutaneously with MC38 cells (5×10 5 When the tumor volume reached 100 mm 3 At 14:00, mice were randomly divided into 3 groups, with 5 mice in each group. The treatment group was treated with 10C9 or positive control antibody 5G12 at a dose of 3 mg / kg by intraperitoneal injection, 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 end of the experiment after 3 weeks.

[0373] During the entire treatment period, the average body weight of mice in the control group and the treatment group increased steadily, with no significant difference between the groups, indicating that 10C9 had no significant toxicity to mice. Table 13 below shows the TGI% (21 days after grouping) results of each group. Compared with the control group, the tumor growth in the treatment group was inhibited to varying degrees, among which the 10C9 treatment group achieved better tumor inhibition compared with 5G12.

[0374] Table 13

[0375] Group Antibody <![CDATA[Average tumor volume (mm 3 )]]> TGI % P-value G1 PBS 1632±238 NA NA G2 5G12 (3 mg / kg) 1293±163 21.9％ 0.274 G3 10C9 (3 mg / kg) 1157±225 30.8％ 0.185

[0376] Antitumor activity of 10A7 in combination with anti-PD-L1 antibody

[0377] A total of twenty hSIGLEC15 mice were injected subcutaneously with MC38 cells (5×10 5 When the tumor volume reached 100 mm 3 At the time of the study, mice were divided into 1 control group and 3 treatment groups (5 mice in each group) according to tumor size. Treatment groups were randomly selected for treatment with the anti-PD-L1 antibody atezolizumab analog, 10A7, or a combination of atezolizumab analog and 10A7. The control group mice were injected with PBS. Tumor volume was measured twice a week, and the weight of the mice was measured. When the tumor volume of the mice reached 3000mm 3 The detailed dosing schedule is shown in the table below.

[0378] Table 14

[0379]

[0380] Atezolizumab is a humanized anti-PD-L1 monoclonal antibody. The heavy chain sequence and light chain sequence are shown in SEQ ID NO: 65-66.

[0381] Throughout the treatment period, the average body weight of mice in the control and treatment groups increased steadily, with no significant differences between the groups, indicating that these antibodies had no obvious toxicity to the animals.

[0382] The tumor sizes in the antibody-treated groups are shown in Figure 7 Table 15 below shows the TGI% (24 days after grouping) results of each group. Compared with the control group, the tumor growth in the treatment group was inhibited to varying degrees, among which the combination treatment group of anti-SIGLEC15 antibody 10A7 and anti-PD-L1 antibody achieved better tumor inhibition effect compared with the 10A7 treatment group or the anti-PD-L1 antibody treatment group.

[0383] Table 15

[0384]

[0385] Example 9. Antitumor activity in C57BL / 6 mice

[0386] C57BL / 6 mice were injected subcutaneously with MC38 cells (5×10 5 When the tumor volume reached 100 mm 3 At 14:00, mice were randomly divided into 3 groups, 7 mice in each group. The treatment group was treated by intraperitoneal injection of 10C9-S, and the control group was injected with PBS, and the administration was done twice a week. The body weight and tumor volume of the mice were measured twice a week until the end of the experiment after 24 days. The detailed dosing schedule is shown in the table below.

[0387] Table 16

[0388]

[0389] During the entire treatment period, the average body weight of mice in the control group and the treatment group increased steadily, with no significant difference between the groups, indicating that 10C9-S had no significant toxicity to the animals. Table 17 below shows the TGI% (24 days after grouping) results of each group. Compared with the control group, the tumor growth in the 10C9-S treatment group was inhibited to varying degrees.

[0390] Table 17

[0391]

[0392] Example 10. Antitumor activity in h4-1BB mice

[0393] Humanized 4-1BB mouse model (h4-1BB mouse) is engineered to express chimeric 4-1BB protein (SEQ ID NO: 68), wherein part of the extracellular region of mouse 4-1BB protein is replaced by 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 incorporated herein by reference in its entirety.

[0394] About 5×10 5 Mouse colon cancer cells MC38 were injected subcutaneously into h4-1BB mice. When the tumor volume grew to about 100 mm 3 When, mice were divided into 1 control group and 3 treatment groups (5 mice per group) according to tumor size. The treatment groups were randomly selected for anti-4-1BB antibody YH004 treatment (G2), 23F4 treatment (G3) or combination treatment of YH004 and 23F4 (G4). A detailed description of YH004 can be found in PCT / CN2019 / 105315, which is incorporated herein by reference in its entirety. Control group mice were injected with phosphate buffered saline (PBS) (G1). Tumor volume was measured twice a week, and the weight of mice was measured. When the tumor volume of mice reached 3000mm 3 The detailed dosing schedule is shown in the table below.

[0395] Table 18

[0396]

[0397] The weight of mice in different groups increased. On the day of grouping (day 0), the average body weight of each group was in the range of 19.2g-19.4g. At the end of the experiment (day 21), the average body weight of each group was in the range of 20.9g-22.0g. The average body weight of each group was in the range of 109.0%-114.4%. The results show that the antibodies tested are well tolerated and have no obvious toxicity to mice.

[0398] like Figure 8 As shown in Table 19, the tumor growth in the treatment group was inhibited to varying degrees compared with the control group. In the treatment group, the combination of anti-SIGLEC15 antibody and anti-4-1BB antibody can significantly inhibit tumor growth and has excellent efficacy.

[0399] Table 19

[0400]

[0401] Example 11. Antitumor activity in hOX40 mice

[0402] The humanized OX40 mouse model (hOX40 mouse) is engineered to express a chimeric OX40 protein (SEQ ID NO: 69), in which part of the extracellular region of the mouse OX40 protein is replaced by the corresponding human OX40 extracellular region. A detailed description of the humanized OX40 mouse model can be found in PCT / CN2017 / 099575, which is incorporated herein by reference in its entirety.

[0403] About 5×10 5 Mouse colon cancer cells MC38 were injected subcutaneously into hOX40 mice. When the tumor volume grew to about 100 mm 3 When, mice were divided into 1 control group and 4 treatment groups (5 mice in each group) according to tumor size. The treatment groups were randomly selected for anti-OX40 antibody YH002 treatment (G2), 10A7 treatment (G3), combination treatment of YH002 and 10A7 (G4) or combination treatment of YH002 and 10C9 (G5). A detailed description of YH002 can be found in PCT / CN2017 / 112832, which is incorporated herein by reference in its entirety. The control group mice were injected with an equal volume of PBS (G1). The detailed dosing regimen is shown in the table below.

[0404] Table 20

[0405]

[0406]

[0407] The weight of mice in different groups increased. On the day of grouping (day 0), the average body weight of each group was in the range of 19.7g-20.1g. At the end of the experiment (day 24), the average body weight of each group was in the range of 22.2g-25.8g. The average body weight of each group was in the range of 111.8%-129.3%. The results show that the antibodies tested are well tolerated and have no obvious toxicity to mice.

[0408] Table 21 below summarizes the results of the experiment, including the tumor volume on the day of grouping (day 0), 14 days after grouping (day 14), and at the end of the experiment (day 24); TGI between the treatment group and the control group and the P value of the tumor volume. The results showed that the combination of anti-OX40 antibody and anti-SIGLEC15 antibody 10A7 or 10C9 can significantly inhibit tumor growth and has excellent efficacy.

[0409] Table 21

[0410]

[0411] Example 12. In vivo toxicity of anti-SIGLEC15 antibodies

[0412] The in vivo toxicity of anti-SIGLEC15 antibodies in hSIGLEC15 mice was evaluated. hSIGLEC15 mice were divided into different groups (3 mice per group) according to weight, and the treatment groups were treated with 10A7, 23F4 or positive control antibody 5G12 at a dose of 30 mg / kg by intraperitoneal injection, and the control group was injected with PBS. Treatment was implemented 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, peripheral blood and serum were collected from hSIGLEC15 mice and analyzed using biochemical tests (alanine aminotransferase (ALT) and aspartate (AST)) and hematological tests (complete blood cell count, including white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit or packed cell volume (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), lymphocyte percentage (LYMPH%), monocyte percentage (MONO%), neutrophil percentage (NEUT%)).

[0413] like FIG. 9A to FIG. 9B As shown, the average body weight of mice in both the control and treatment groups increased steadily throughout the treatment period. Figure 10A to Figure 10M Hematological and biochemical test results showed no significant differences between the groups. These results suggest that the anti-SIGLEC15 antibodies 23F4 and 10A7 are not toxic to mice.

[0414] Similar to the in vivo toxicity validation of 23F4, toxicity analysis of 10C9 and 10C9-S was also performed. The results showed that the average body weight of mice in the control and treatment groups increased steadily throughout the treatment period, and there were no significant differences in the results of hematological and biochemical tests between the groups. These results indicate that anti-SIGLEC15 antibodies have no obvious toxicity to mice.

[0415] Other Implementations

[0416] It should be understood that although the invention has been described in conjunction with the specific embodiments of the invention, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of 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-binding Ig-like lectin 15), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDR) 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 a light chain variable region (VL) comprising CDR1, 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, wherein the selected VH CDR1, 2 and 3 amino acid sequence and the selected VL CDR1, 2 and 3 amino acid sequence are one of the following: (1) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1, 2, 3, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4, 5, 6, respectively; (2) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 7, 8, 9, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 10, 11, 12, respectively; (3) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13, 14, 15, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 16, 17, 18, respectively; (4) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 19, 20, 21, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22, 23, 24, respectively; (5) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 25, 26, 27, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 28, 29, 30, respectively; and (6) The selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 31, 32, 33, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 34, 35, 36, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12, respectively.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 13, 14 and 15, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 16, 17 and 18, respectively.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein according to the Chothia definition, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 19, 20 and 21, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 22, 23 and 24, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein according to the Chothia definition, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 25, 26 and 27, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30, respectively.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein according to the Chothia definition, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 31, 32 and 33, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 34, 35 and 36, respectively.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof specifically binds to human, mouse, monkey or dog SIGLEC15.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), a one-armed antibody and / or a multispecific antibody (e.g., a bispecific antibody).

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein 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.

11. A nucleic acid comprising a polynucleotide encoding a polypeptide comprising: (1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, and wherein the VH binds to SIGLEC15 when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 38; (2) an immunoglobulin light chain or a fragment thereof comprising a VL, wherein the VL comprises complementarity determining regions (CDRs) 1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively, and wherein the VL binds to SIGLEC15 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO: 37; (3) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity determining regions (CDRs) 1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively, and wherein the VH binds to SIGLEC15 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 40; (4) an immunoglobulin light chain or a fragment thereof comprising a VL comprising complementarity determining regions (CDRs) 1, 2 and 3 having the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12, respectively, and wherein the VL binds to SIGLEC15 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO: 39; (5) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NOs: 13, 14 and 15, respectively, and wherein the VH binds to SIGLEC15 when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 42; or (6) An immunoglobulin light chain or a fragment thereof comprising a VL, wherein the VL comprises CDR1, 2 and 3 having the amino acid sequences shown in SEQ ID NO:16, 17 and 18, respectively, and wherein the VL binds to SIGLEC15 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO:

41.

12. The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing a VH, wherein the VH comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 1, 2, and 3, respectively.

13. The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof containing a VL, wherein the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 4, 5, and 6, respectively.

14. The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing a VH, wherein the VH comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NOs: 7, 8, and 9, respectively.

15. The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof containing a VL, wherein the VL comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NOs: 10, 11, and 12, respectively.

16. The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing a VH, wherein the VH comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NOs: 13, 14, and 15, respectively.

17. The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof containing a VL, wherein the VL comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NOs: 16, 17, and 18, respectively.

18. The nucleic acid of any one of claims 11-17, wherein the VH specifically binds to human, mouse, monkey or dog SIGLEC15 when paired with the VL, or the VL specifically binds to human, mouse, monkey or dog SIGLEC15 when paired with the VH.

19. The nucleic acid of any one of claims 11-18, wherein the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof (e.g., a human IgG1 heavy chain or fragment thereof; or a human IgG4 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof.

20. The nucleic acid of any one of claims 11-19, wherein the nucleic acid encodes a single-chain variable fragment (scFv), a one-armed antibody, a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR).

21. The nucleic acid according to any one of claims 11-20, wherein the nucleic acid is cDNA.

22. A vector comprising one or more nucleic acids according to any one of claims 11-21.

23. A vector comprising two nucleic acids according to any one of claims 11 to 21, wherein the vector encodes a VL region and a VH region, and the VL region and the VH region together bind to SIGLEC15.

24. A pair of vectors, wherein each vector comprises a nucleic acid as described in any one of claims 11-21, wherein the pair of vectors jointly encode a VL region and a VH region, and the VL region and the VH region together bind to SIGLEC15.

25. A cell comprising the vector of claim 22 or 23, or the vector pair of claim 24.

26. The cell according to claim 25, wherein the cell is a CHO cell.

27. A cell comprising one or more nucleic acids of any one of claims 11-21.

28. A cell comprising two nucleic acids as described in any one of claims 11-21.

29. The cell of claim 28, wherein the two nucleic acids together encode a VL region and a VH region that together bind SIGLEC15.

30. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising (c) culturing the cell under conditions sufficient for the cell to produce the antibody or antigen-binding fragment as described in any one of claims 25 to 29; and (d) collecting the antibodies or antigen-binding fragments produced by the cells.

31. An antibody or antigen-binding fragment thereof that binds to SIGLEC15, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH), the VH comprising an amino acid sequence that is at least 90% identical to a selected VH sequence; and a light chain variable region (VL), the 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: (1) the selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is SEQ ID NO: 38; (2) the selected VH sequence is SEQ ID NO:39, and the selected VL sequence is SEQ ID NO:40; and (3) The selected VH sequence is SEQ ID NO:41, and the selected VL sequence is SEQ ID NO:

42.

32. The antibody or antigen-binding fragment thereof of claim 31, wherein the VH comprises the sequence of SEQ ID NO: 37 and the VL comprises the sequence of SEQ ID NO:

38.

33. The antibody or antigen-binding fragment thereof of claim 31, wherein the VH comprises the sequence of SEQ ID NO: 39, and the VL comprises the sequence of SEQ ID NO:

40.

34. The antibody or antigen-binding fragment thereof of claim 31, wherein the VH comprises the sequence of SEQ ID NO:41, and the VL comprises the sequence of SEQ ID NO:

42.

35. The antibody or antigen-binding fragment thereof according to any one of claims 31-34, wherein the antibody or antigen-binding fragment thereof specifically binds to human, mouse, monkey or dog SIGLEC15.

36. The antibody or antigen-binding fragment thereof according to any one of claims 31-35, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), a one-armed antibody and / or a multispecific antibody (e.g., a bispecific antibody).

37. The antibody or antigen-binding fragment thereof according to any one of claims 31 to 36, wherein 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.

38. 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 1-10 and 31-37.

39. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 31-38.

40. The antibody or antigen-binding fragment thereof according to any one of claims 1-10 and 31-39, wherein the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region).

41. The antibody or antigen-binding fragment thereof of claim 40, wherein the Fc region has reduced complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).

42. A chimeric antigen receptor (CAR), comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-10 and 31-39.

43. An antibody drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 31-41 covalently bound to a therapeutic agent.

44. The antibody drug conjugate of claim 43, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.

45. A method of treating a subject having cancer, the method comprising: A therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 31-41, or the antibody drug conjugate of claim 43 or 44 is administered to the subject.

46. ​​The method of claim 45, wherein the subject has a solid tumor.

47. The method of claim 45, wherein 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.

48. The method of claim 45, wherein the cancer is non-Hodgkin lymphoma, lymphoma, leukemia, acute myeloid leukemia, or chronic lymphocytic leukemia.

49. The method of claim 45, wherein the subject is further treated with an effective amount of an anti-4-1BB antibody, an anti-OX40 antibody, an anti-PD-1, an anti-CTLA4, or an anti-PD-L1 antibody.

50. A method for reducing tumor growth rate, the method comprising The tumor cells are contacted with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 31-41, or the antibody drug conjugate of claim 43 or 44.

51. A method for killing tumor cells, the method comprising The tumor cells are contacted with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 31-41, or the antibody drug conjugate of claim 43 or 44.

52. A method of increasing an immune response in a subject, the method comprising An effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 31-41 is administered to the subject.

53. A method of treating a subject having a bone disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 31-41.

54. The method of claim 53, wherein the bone disease is osteoporosis.

55. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-10 and 31-41 and a pharmaceutically acceptable carrier.

56. A pharmaceutical composition comprising the antibody drug conjugate of claim 43 or 44 and a pharmaceutically acceptable carrier.

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