Chimeric antigen receptors targeting siglec-15 and uses thereof

Abstract

The present application relates to antibodies and chimeric antigen receptors targeting siglec-15 and applications thereof. Specifically, the present application relates to an scFv specifically targeting siglec-15, antibodies and CAR-T cells specific thereto. The present application also provides a chimeric antigen receptor immune cell simultaneously targeting siglec-15, which can effectively treat cancer or tumor, and in addition, the present application also provides a marker target siglec-15 for treating AML hematological tumor.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy, and more specifically to a chimeric antigen receptor targeting siglec-15 and its applications. Background Technology

[0002] Chimeric antigen receptors (CARs) are artificial receptors that mimic the function of T-cell receptors, combining the specificity of antigen-antibody or ligand-receptor recognition and binding with the killing ability of effector T cells against recognized tumor cells. A CAR consists of a CD8a guide peptide, an antigen recognition region (ligand, single-chain antibody, or Fab fragment), a transmembrane region, and a series of signal transduction domains of the T cell (CD28, CD3, CD137 intracellular signal transduction domains). After modification, the CAR expressed on the surface of T cells first binds to tumor cell surface antigens through the antigen recognition region, and then transmits activation signals into the cell through its signal transduction domain, targeting and activating the T cell's tumor-killing activity. The DNA sequence expressing the CAR is cloned into a lentiviral expression vector and used to infect T cells isolated from the patient's blood, causing the T cells to express the corresponding CAR. These modified T cells are then reinfused into the patient, allowing them to target and kill tumor cells expressing the relevant antigens, thus achieving tumor cell elimination.

[0003] Acute myeloid leukemia (AML) is a highly heterogeneous malignant tumor characterized by the malignant proliferation of immature bone marrow stem cells in the bone marrow and peripheral blood. The classic AML classifications include the FAB and MIC classifications. The FAB classification (divided into M0 to M7 types) is particularly important. Although research into AML treatment approaches has deepened in recent years, and the development of new therapeutic drugs and hematopoietic stem cell transplantation technology has greatly improved the prognosis of AML, the main treatment for AML remains the classic "3+7 regimen," namely chemotherapy and hematopoietic stem cell transplantation. Moreover, the 5-year survival rate remains only around 30%. Given the current characteristics of AML—low cure and survival rates, and high relapse rates—the search for new biomarkers and targeted therapeutic molecules is particularly important.

[0004] Therefore, there is an urgent need in this field to develop an effective and safe treatment for cancers or tumors, such as acute myeloid leukemia. Summary of the Invention

[0005] The purpose of this invention is to provide an effective and safe treatment for cancer or tumors, such as acute myeloid leukemia.

[0006] In a first aspect, the present invention provides an antibody heavy chain variable region comprising three complementarity-determining regions (CDRs): CDR1 as shown in SEQ ID NO: 1, 2, 3 or 4 CDR2 as shown in SEQ ID NO:5, 6, 7 or 8, and CDR3 as shown in SEQ ID NO:9, 10, 11 or 12.

[0007] In another preferred embodiment, the CDR of the heavy chain variable region includes SEQ ID NO:N H N H +4, and N H The three CDRs shown in +8 include N. H The numbers are 1, 2, 3, or 4.

[0008] In another preferred embodiment, any of the amino acid sequences described above further includes a derived sequence that has optionally been added, deleted, modified, and / or substituted at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and is capable of retaining siglec-15 binding affinity.

[0009] In another preferred embodiment, the heavy chain variable region further includes a human-derived FR region or a mouse-derived FR region.

[0010] In another preferred embodiment, the heavy chain variable region has an amino acid sequence shown in any of SEQ ID NO:25-28.

[0011] A second aspect of the present invention provides a heavy chain of an antibody having a heavy chain variable region as described in the first aspect of the present invention.

[0012] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0013] In another preferred embodiment, the heavy chain constant region is of human, mouse, or rabbit origin.

[0014] A third aspect of the present invention provides a light chain variable region of an antibody, wherein the light chain variable region includes the following three complementarity-determining regions (CDRs): CDR1' as shown in SEQ ID NO: 13, 14, 15 or 16, CDR2' shown in SEQ ID NO:17, 18, 19 or 20, and CDR3' as shown in SEQ ID NO:21, 22, 23 or 24.

[0015] In another preferred embodiment, the CDR of the light chain variable region includes SEQ ID NO:N L N L +4, and N L The three CDRs shown in +8 include N.L The numbers are 13, 14, 15, or 16 respectively.

[0016] In another preferred embodiment, any of the amino acid sequences described above further includes a derived sequence that has optionally been added, deleted, modified, and / or substituted at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and is capable of retaining siglec-15 binding affinity.

[0017] In another preferred embodiment, the light chain variable region further includes a human-derived FR region or a mouse-derived FR region.

[0018] In another preferred embodiment, the light chain variable region has any of the amino acid sequences shown in SEQ ID NO:29-32.

[0019] A fourth aspect of the present invention provides a light chain of an antibody having a light chain variable region as described in a third aspect of the present invention.

[0020] In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

[0021] In another preferred embodiment, the light chain constant region is of human, mouse, or rabbit origin.

[0022] The fifth invention provides an antibody having: (1) The heavy chain variable region as described in the first aspect of the present invention; and / or (2) The light chain variable region as described in the third aspect of the present invention.

[0023] In another preferred embodiment, the antibody has: a heavy chain as described in the second aspect of the invention; and / or a light chain as described in the fourth aspect of the invention.

[0024] In another preferred embodiment, the KD(M) of the antibody’s affinity for human siglec-15 (wild type) is ≤1×10⁻⁶. -7 Preferably ≤1×10 -8 Better ≤8×10 -9 .

[0025] In another preferred embodiment, the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, or combinations thereof.

[0026] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0027] In another preferred embodiment, the antibody is a monoclonal antibody.

[0028] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0029] In another preferred embodiment, the heavy chain variable region sequence of the antibody is as shown in any of SEQ ID NO: 25-28; and / or The light chain variable region sequence of the antibody is shown in any one of SEQ ID NO: 29-32.

[0030] In another preferred embodiment, the antibody is of the IgG type.

[0031] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0032] A sixth aspect of the present invention provides an scFv, the scFv comprising an antibody heavy chain variable region as described in the first aspect of the present invention and an antibody light chain variable region as described in the third aspect of the present invention.

[0033] In another preferred embodiment, the scFv further comprises a linker peptide located between the heavy chain variable region and the light chain variable region.

[0034] In another preferred embodiment, the scFv is shown in equation A or equation B as follows: V H -V L , (A); or V L -V H (B); In the formula, V H V is the variable region of the antibody heavy chain; L "-" represents the variable region of the antibody light chain; "-" represents a linking peptide (or flexible linker) or peptide bond.

[0035] In another preferred embodiment, the structures A and B are arranged from the N end to the C end.

[0036] In another preferred embodiment, the V H and V L The linking peptides are 1-4 consecutive GGGGS sequences, preferably 1-4, more preferably 3-4.

[0037] In another preferred embodiment, V H The amino acid sequence includes V selected from any of SEQ ID No.: 25-28. H or its derivative V H (or its active fragment).

[0038] In another preferred embodiment, V L The amino acid sequence includes V selected from any of SEQ ID No.:29-32. L or its derivative V L (or its active fragment).

[0039] In another preferred embodiment, the scFv comprises an antibody heavy chain variable region as shown in SEQ ID NO.: 25 and an antibody light chain variable region as shown in SEQ ID NO.: 29.

[0040] In another preferred embodiment, the scFv comprises an antibody heavy chain variable region as shown in SEQ ID NO.: 26 and an antibody light chain variable region as shown in SEQ ID NO.: 30.

[0041] In another preferred embodiment, the scFv includes an antibody heavy chain variable region as shown in SEQ ID NO.: 27 and an antibody light chain variable region as shown in SEQ ID NO.: 31.

[0042] In another preferred embodiment, the scFv includes an antibody heavy chain variable region as shown in SEQ ID NO.: 28 and an antibody light chain variable region as shown in SEQ ID NO.: 32.

[0043] In another preferred embodiment, the scFv comprises an amino acid sequence as shown in SEQ ID NO.: 33-36.

[0044] In another preferred embodiment, the amino acid sequence of the scFv is shown in SEQ ID NO.: 33-36.

[0045] In another preferred embodiment, the amino acid sequence of the scFv has at least 70%, preferably at least 75%, 80%, 85%, 90%, and more preferably at least 95%, 96%, 97%, 98%, 99% sequence identity with the sequence shown in SEQ ID NO.: 33-36.

[0046] In another preferred embodiment, the scFv targets or binds to human siglec-15.

[0047] In another preferred embodiment, the scFv is a murine, human, human-mice chimeric, or fully humanized single-chain antibody variable region fragment.

[0048] A seventh aspect of the present invention provides a recombinant protein, said recombinant protein having: (i) the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, the antibody as described in the fifth aspect of the present invention, or the scFv as described in the sixth aspect of the present invention; and (ii) Optional tag sequences to assist in expression and / or purification.

[0049] In another preferred embodiment, the tag sequence includes a 6His tag.

[0050] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0051] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0052] The eighth aspect of the present invention provides a chimeric antigen receptor (CAR) comprising scFv as described in the sixth aspect of the present invention.

[0053] In another preferred embodiment, the structure of the CAR is shown in Equation I: Z1-TH-TM-C-Z2 (I) In the formula, Each "-" independently represents a linking peptide or peptide bond; Z1 is either absent or a signal peptide sequence; T represents scFv as described in the sixth aspect of this invention; H represents the area with no hinge or no connection. TM represents a transmembrane domain; C is a co-stimulatory signaling molecule; Z2 is a cytoplasmic signal transduction sequence derived from CD3ζ.

[0054] In another preferred embodiment, Z1 is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0055] In another preferred embodiment, Z1 is a signal peptide selected from the group consisting of: CD8.

[0056] In another preferred embodiment, the amino acid sequence of Z1 is shown in SEQ ID NO.:37.

[0057] In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

[0058] In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of: CD8.

[0059] In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID NO.:38.

[0060] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD3 epsilon, CD4, CD8, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1, LAG-3, or a combination thereof.

[0061] In another preferred embodiment, the TM includes a transmembrane region derived from CD8.

[0062] In another preferred embodiment, the amino acid sequence of the TM is shown in SEQ ID NO.:39.

[0063] In another preferred embodiment, C is a co-stimulatory signaling molecule selected from the group consisting of: OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, or a combination thereof.

[0064] In another preferred embodiment, C comprises a co-stimulatory signaling molecule derived from 4-1BB.

[0065] In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO.:40.

[0066] In another preferred embodiment, the CD3ζ has an amino acid sequence as shown in SEQ ID NO.: 41.

[0067] In another preferred embodiment, the amino acid sequence of the CAR is as shown in any of SEQ ID No.:43-46.

[0068] A ninth aspect of the present invention provides an engineered immune cell, said immune cell comprising: Expression cassettes for expressing exogenous chimeric antigen receptors as described in the eighth aspect of the present invention.

[0069] In another preferred embodiment, the expression cassette contains a nucleic acid sequence encoding the chimeric antigen receptor described in the eighth aspect of the invention.

[0070] In another preferred embodiment, the expression box further includes a promoter and / or a terminator.

[0071] In another preferred embodiment, the promoter is a mammalian promoter, preferably the EF1 promoter.

[0072] In another preferred embodiment, the sequence of the promoter is shown in SEQ ID NO:42.

[0073] In another preferred embodiment, the expression cassette is located on a vector or integrated into the chromosome of the engineered immune cell.

[0074] In another preferred embodiment, the cell is a mammalian cell.

[0075] In another preferred embodiment, the immune cells are ex vivo.

[0076] In another preferred embodiment, the immune cells are autologous.

[0077] In another preferred embodiment, the immune cells are non-autologous.

[0078] In another preferred embodiment, the immune cells are derived from humans or non-human mammals (such as mice).

[0079] In another preferred embodiment, the immune cells are derived from primates (preferably humans).

[0080] In another preferred embodiment, the immune cells are selected from the group consisting of: (i) Chimeric antigen receptor T cells (CAR-T cells); (ii) Chimeric antigen receptor NK cells (CAR-NK cells); or (iii) Exogenous T cell receptor (TCR) T cells (TCR-T cells) In another preferred embodiment, the immune cells include: NK cells, T cells, NKT cells, (γδ) T cells, monocytes, or macrophages.

[0081] The tenth aspect of this invention provides an antibody-drug conjugate, the antibody-drug conjugate comprising: (a) the heavy chain variable region as described in the first aspect of the invention, the heavy chain as described in the second aspect of the invention, the light chain variable region as described in the third aspect of the invention, the light chain as described in the fourth aspect of the invention, the antibody as described in the fifth aspect of the invention, or the scFv as described in the sixth aspect of the invention; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0082] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0083] The eleventh aspect of the present invention provides a nucleic acid molecule that encodes the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, the antibody of the fifth aspect of the present invention, the scFv of the sixth aspect of the present invention, the recombinant protein of the seventh aspect of the present invention, the chimeric antigen receptor of the eighth aspect of the present invention, and the antibody-drug conjugate of the tenth aspect of the present invention.

[0084] The twelfth aspect of the present invention provides a carrier containing the nucleic acid molecule described in the eleventh aspect of the present invention.

[0085] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0086] In another preferred embodiment, the vector is a lentiviral vector.

[0087] The thirteenth aspect of the present invention provides a host cell containing a vector or chromosome according to the twelfth aspect of the present invention, or having an exogenous nucleic acid molecule according to the eleventh aspect of the present invention integrated into it, or expressing a chimeric antigen receptor according to the eighth aspect of the present invention.

[0088] In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0089] In another preferred embodiment, the cell is a mammalian cell, preferably a human cell.

[0090] In another preferred embodiment, the host cell comprises engineered immune cells.

[0091] In another preferred embodiment, the engineered immune cells include T cells, NK cells, or macrophages.

[0092] In another preferred embodiment, the cell is a T cell.

[0093] In another preferred embodiment, the engineered immune cells are selected from the group consisting of: (i) Chimeric antigen receptor T cells (CAR-T cells); (ii) Chimeric antigen receptor NK cells (CAR-NK cells); or (iii) Exogenous T cell receptor (TCR) T cells (TCR-T cells).

[0094] In another preferred embodiment, the immune cells are autologous.

[0095] In another preferred embodiment, the immune cells are allogeneic.

[0096] In another preferred embodiment, the cell is a CAR-T cell that expresses the chimeric antigen receptor described in the third aspect of the present invention.

[0097] The fourteenth aspect of the present invention provides a method for preparing engineered immune cells, wherein the engineered immune cells express the chimeric antigen receptor described in the eighth aspect of the present invention, comprising the following steps: transducing the nucleic acid molecule described in the eleventh aspect of the present invention or the vector described in the twelfth aspect of the present invention into the immune cells, thereby obtaining the engineered immune cells.

[0098] In another preferred embodiment, the import includes simultaneous, sequential, or sequential import.

[0099] In another preferred embodiment, the immune cells are T cells or NK cells.

[0100] In another preferred embodiment, the method further includes a step of testing the function and effectiveness of the obtained engineered immune cells.

[0101] The fifteenth aspect of the present invention provides a pharmaceutical composition comprising the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, the antibody of the fifth aspect of the present invention, the scFv of the sixth aspect of the present invention, the recombinant protein of the seventh aspect of the present invention, the chimeric antigen receptor of the eighth aspect of the present invention, the engineered immune cell of the ninth aspect of the present invention, or the antibody-drug conjugate of the tenth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0102] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0103] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0104] In another preferred embodiment, the host cell comprises engineered immune cells.

[0105] In another preferred embodiment, the engineered immune cells are (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) chimeric antigen receptor NK cells (CAR-NK cells).

[0106] In another preferred embodiment, the concentration of the cells in the pharmaceutical composition is 1 × 10⁻⁶. 3 -1×10 10 Cells / ml, preferably 1×10⁻⁶ 4 -1×10 7 Cells / ml

[0107] In another preferred embodiment, the pharmaceutical composition also contains other drugs for the prevention and / or treatment of cancer or tumors (such as antibody drugs, chemotherapy drugs, or other CAR-T drugs).

[0108] The sixteenth aspect of the present invention provides the use of the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, the antibody of the fifth aspect of the present invention, the scFv of the sixth aspect of the present invention, the recombinant protein of the seventh aspect of the present invention, the chimeric antigen receptor of the eighth aspect of the present invention, the engineered immune cell of the ninth aspect of the present invention, the antibody-drug conjugate of the tenth aspect of the present invention, the nucleic acid molecule of the eleventh aspect of the present invention, the carrier of the twelfth aspect of the present invention, the host cell of the thirteenth aspect of the present invention, or the pharmaceutical composition of the fifteenth aspect of the present invention for (i) preparing a medicament or preparation for the prevention and / or treatment of cancer or tumor; and / or (ii) preparing a diagnostic reagent or kit.

[0109] In another preferred embodiment, the antibody comprises an antibody in the form of a drug conjugate (ADC).

[0110] In another preferred embodiment, the cancer or tumor includes solid tumors and hematologic malignancies.

[0111] In another preferred embodiment, the cancer or tumor includes tumors that are highly expressed or positive for siglec-15.

[0112] In another preferred embodiment, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myelodysplastic / myeloproliferative disorder (MDS / MPD), chronic myeloproliferative disorder (SMPD), pre-B lymphoblastic leukemia / lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, B lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma / plasmacytoma, MALT-type marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma / leukemia, T / NK cell tumors, pre-lymphoblastic lymphoma / leukemia, and T-cell leukemia. T-cell lymphoblastic leukemia, aggressive NK-cell leukemia, mature T-cell lymphoma / leukemia, extranodal nasal NK / T-cell lymphoma, enteropathic T-cell lymphoma, mycosis fungoides / sezary syndrome (MF / SS), peripheral T-cell lymphoma, anaplastic large cell lymphoma, multiple myeloma (MM), acute lymphoblastic leukemia (ALL); pro-T-cell lymphoma / leukemia; Hodgkin's lymphoma; mastocytosis, mast cell leukemia (MCL), mast cell sarcoma (MCS); macrophage / histiocytic tumors, dendritic cell tumors, Langerhans cell histiocytosis (LCH), Langerhans cell sarcoma (LCS), follicular dendritic cell sarcoma / tumor, dendritic cell sarcoma, or combinations thereof.

[0113] In another preferred embodiment, the solid tumor is selected from the group consisting of: head and neck tumors, pharyngeal cancer, lung cancer, non-small cell lung cancer, bronchial cancer, gastric cancer, peritoneal metastases of gastric cancer, esophageal cancer, bile duct cancer, pancreatic cancer, colorectal cancer, peritoneal metastases of colorectal cancer, small bowel cancer, kidney tumors, renal cancer, bladder tumors, transitional epithelial malignant tumors, endocrine tumors, thyroid cancer, adrenal tumors, breast cancer, cervical cancer, ovarian cancer, peritoneal metastases of ovarian cancer, endometrial cancer, choriocarcinoma, prostate cancer, testicular tumors, germ cell tumors, seminoma, embryonal tumors, nervous system tumors, glioma, neuroblastoma, skin tumors, malignant melanoma, lymphoma, thymic tumors, nasopharyngeal carcinoma, bone cancer, sarcoma, rhabdomyosarcoma, liposarcoma, angiosarcoma, leiomyosarcoma, fibrosarcoma, osteosarcoma, Ewing's sarcoma, metastatic solid tumors such as metastases in the abdominal cavity, thoracic cavity, pelvis, solid organs, etc., or combinations thereof.

[0114] In another preferred embodiment, the detection reagent or kit is used to diagnose tumors that express or highly express siglec-15.

[0115] In another preferred embodiment, the detection reagent or kit is used to detect the siglec-15 protein in a sample.

[0116] In another preferred embodiment, the detection reagent is a detection strip.

[0117] The seventeenth aspect of the present invention provides a kit for the prevention and / or treatment of cancer or tumors, the kit comprising a container and, within the container, a heavy chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, a light chain variable region as described in the third aspect of the present invention, a light chain as described in the fourth aspect of the present invention, an antibody as described in the fifth aspect of the present invention, an scFv as described in the sixth aspect of the present invention, a recombinant protein as described in the seventh aspect of the present invention, a chimeric antigen receptor as described in the eighth aspect of the present invention, engineered immune cells as described in the ninth aspect of the present invention, an antibody-drug conjugate as described in the tenth aspect of the present invention, a nucleic acid molecule as described in the eleventh aspect of the present invention, a vector as described in the twelfth aspect of the present invention, or a host cell as described in the thirteenth aspect of the present invention, or a pharmaceutical composition as described in the fifteenth aspect of the present invention.

[0118] In another preferred embodiment, the kit also includes a label or instructions for use.

[0119] The eighteenth aspect of the present invention provides a method for treating a disease, comprising: administering to a subject requiring treatment a safe and effective amount of the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, an antibody of the fifth aspect of the present invention, the scFv of the sixth aspect of the present invention, a recombinant protein of the seventh aspect of the present invention, a chimeric antigen receptor of the eighth aspect of the present invention, engineered immune cells of the ninth aspect of the present invention, an antibody-drug conjugate of the tenth aspect of the present invention, a nucleic acid molecule of the eleventh aspect of the present invention, a vector of the twelfth aspect of the present invention, or a host cell of the thirteenth aspect of the present invention, or a pharmaceutical composition of the fifteenth aspect of the present invention.

[0120] In another preferred embodiment, the disease includes cancer or tumor.

[0121] In another preferred embodiment, the cancer or tumor includes solid tumors and hematologic malignancies.

[0122] In another preferred embodiment, the cancer or tumor includes tumors that are highly expressed or positive for siglec-15.

[0123] In another preferred embodiment, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myelodysplastic / myeloproliferative disorder (MDS / MPD), chronic myeloproliferative disorder (SMPD), pre-B lymphoblastic leukemia / lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, B lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma / plasmacytoma, MALT-type marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma / leukemia, T / NK cell tumors, pre-lymphoblastic lymphoma / leukemia, and T-cell leukemia. T-cell lymphoblastic leukemia, aggressive NK-cell leukemia, mature T-cell lymphoma / leukemia, extranodal nasal NK / T-cell lymphoma, enteropathic T-cell lymphoma, mycosis fungoides / sezary syndrome (MF / SS), peripheral T-cell lymphoma, anaplastic large cell lymphoma, multiple myeloma (MM), acute lymphoblastic leukemia (ALL); pro-T-cell lymphoma / leukemia; Hodgkin's lymphoma; mastocytosis, mast cell leukemia (MCL), mast cell sarcoma (MCS); macrophage / histiocytic tumors, dendritic cell tumors, Langerhans cell histiocytosis (LCH), Langerhans cell sarcoma (LCS), follicular dendritic cell sarcoma / tumor, dendritic cell sarcoma, or combinations thereof.

[0124] In another preferred embodiment, the solid tumor is selected from the group consisting of: head and neck tumors, pharyngeal cancer, lung cancer, non-small cell lung cancer, bronchial cancer, gastric cancer, peritoneal metastases of gastric cancer, esophageal cancer, liver cancer, bile duct cancer, pancreatic cancer, colorectal cancer, peritoneal metastases of colorectal cancer, small bowel cancer, kidney tumors, renal cancer, bladder tumors, transitional epithelial malignancies, endocrine tumors, thyroid cancer, adrenal tumors, breast cancer, cervical cancer, ovarian cancer, peritoneal metastases of ovarian cancer, and endometrium. Cancer, choriocarcinoma, prostate cancer, testicular tumor, germ cell tumor, seminoma, embryonal tumor, nervous system tumor, glioma, neuroblastoma, skin tumor, malignant melanoma, lymphoma, thymic tumor, nasopharyngeal carcinoma, bone cancer, sarcoma, rhabdomyosarcoma, liposarcoma, angiosarcoma, leiomyosarcoma, fibrosarcoma, osteosarcoma, Ewing's sarcoma, metastatic solid tumors such as metastases in the abdominal cavity, thoracic cavity, pelvis, solid organs, etc., or combinations thereof.

[0125] The nineteenth aspect of the present invention provides a method for in vitro detection of siglec-15 protein in a sample, the method comprising the steps of: (1) In vitro, the sample is contacted with the antibody as described in the fifth aspect of the present invention; (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of siglec-15 protein in the sample.

[0126] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0127] The twentieth aspect of the present invention provides a detection plate, the detection plate comprising: a substrate (support plate) and a test strip, the test strip containing an antibody as described in the fifth aspect of the present invention or an antibody-drug conjugate as described in the ninth aspect of the present invention.

[0128] The twenty-first aspect of the present invention provides a diagnostic kit comprising: (1) A first container containing the antibody as described in the fifth aspect of the present invention; and / or (2) A second container containing a secondary antibody against the antibody described in the fifth aspect of the present invention.

[0129] In another preferred embodiment, the kit contains the detection plate described in the twentieth aspect of the present invention.

[0130] The twenty-second aspect of the present invention provides the use of the Siglec-15 gene, mRNA, cDNA, or protein, or a detection reagent thereof, (i) as a biomarker for detecting hematologic malignancies; and / or (ii) for the preparation of diagnostic reagents or kits for detecting hematologic malignancies.

[0131] In another preferred embodiment, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myelodysplastic / myeloproliferative disorder (MDS / MPD), chronic myeloproliferative disorder (SMPD), pre-B lymphoblastic leukemia / lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, B lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma / plasmacytoma, MALT-type marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma / leukemia, T / NK cell tumors, pre-lymphoblastic lymphoma / leukemia, and T-cell leukemia. T-cell lymphoblastic leukemia, aggressive NK-cell leukemia, mature T-cell lymphoma / leukemia, extranodal nasal NK / T-cell lymphoma, enteropathic T-cell lymphoma, mycosis fungoides / sezary syndrome (MF / SS), peripheral T-cell lymphoma, anaplastic large cell lymphoma, multiple myeloma (MM), acute lymphoblastic leukemia (ALL); pro-T-cell lymphoma / leukemia; Hodgkin's lymphoma; mastocytosis, mast cell leukemia (MCL), mast cell sarcoma (MCS); macrophage / histiocytic tumors, dendritic cell tumors, Langerhans cell histiocytosis (LCH), Langerhans cell sarcoma (LCS), follicular dendritic cell sarcoma / tumor, dendritic cell sarcoma, or combinations thereof.

[0132] In another preferred embodiment, the detection reagent further includes detection reagents for HLA-DR, CD38, CD117, MPO, TIM3, CD70, CD371, and CD33.

[0133] In another preferred embodiment, the diagnostic reagent includes antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips), or protein chips.

[0134] In another preferred embodiment, the diagnostic reagents include diagnostic reagents of HLA-DR, CD38, CD117, MPO, TIM3, CD70, CD371, and CD33.

[0135] In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

[0136] In another preferred embodiment, the Siglec-15 gene, mRNA, cDNA, or protein is derived from mammals, preferably rodents (such as mice and rats), primates, and humans, and more preferably from patients diagnosed with hematologic malignancies.

[0137] In another preferred embodiment, the Siglec-15 gene, mRNA, cDNA, or protein is derived from a hematologic malignancy patient.

[0138] In another preferred embodiment, the accession number of the Siglec-15 gene is 284266.

[0139] In another preferred embodiment, the accession number of the Siglec-15 mRNA is NM_213602.3.

[0140] In another preferred embodiment, the accession number of the Siglec-15 protein is NP_998767.1.

[0141] In another preferred embodiment, the detection is a cell or tissue sample detection.

[0142] In another preferred embodiment, the detection includes immunohistochemistry, Western blotting, and quantitative real-time PCR.

[0143] In another preferred embodiment, the detection is to measure blood tumor tissue or general tissue samples.

[0144] In another preferred embodiment, the general tissue includes adjacent normal tissue.

[0145] In another preferred embodiment, the detection reagent includes a specific antibody against Siglec-15, a specific binding molecule against Siglec-15, specific amplification primers, a probe, or a chip.

[0146] In another preferred embodiment, the Siglec-15 protein or its specific antibody or specific binding molecule is conjugated with or carries a detectable label.

[0147] In another preferred embodiment, the detectable marker is selected from the group consisting of chromophores, chemiluminescent groups, fluorophores, isotopes, or enzymes.

[0148] In another preferred embodiment, the specific antibody for Siglec-15 is a monoclonal antibody or a polyclonal antibody.

[0149] The twenty-third aspect of the present invention provides a diagnostic kit for detecting hematologic malignancies, the kit comprising a container containing a detection reagent for detecting the Siglec-15 gene, mRNA, cDNA, or protein; and a label or instruction manual indicating that the kit is for detecting hematologic malignancies.

[0150] In another preferred embodiment, the container further contains one or more detection reagents selected from the group consisting of HLA-DR, CD38, CD117, MPO, TIM3, CD70, CD371, CD33 genes, mRNA, cDNA, or proteins.

[0151] In another preferred embodiment, the detection reagent for detecting the Siglec-15 gene, mRNA, cDNA, or protein includes: (a) Specific antibodies against Siglec-15 protein; and / or (b) Specific primers for specific amplification of Siglec-15 mRNA or cDNA.

[0152] In another preferred embodiment, the detection is a cell or tissue sample detection.

[0153] In another preferred embodiment, the label or instruction manual includes the following information: (i) When the ratio of Siglec-15 expression level E1 in cells or tissues of hematologic malignancies to Siglec-15 expression level E2 in normal cells or tissues is ≥2, it suggests that the probability of the subject having hematologic malignancies is higher than that of the general population. Wherein, E2 refers to the expression level of Siglec-15 in general cells or tissues of the general population; (ii) If the co-expression of Siglec-15 with one or more of HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371 in the cells or tissues (such as bone marrow samples) of the subject being tested is greater than 30%, it suggests that the subject being tested has a higher probability of having a hematologic malignancy than the general population.

[0154] In another preferred embodiment, the general cells or tissues include adjacent cells or tissues.

[0155] The twenty-fourth aspect of the present invention provides a method for detecting hematologic malignancies, the method comprising: a) Provide test samples from the subjects; b) Detect the expression level E1 of Siglec-15 protein in the test sample; and / or the expression level E2 containing, but not limited to, one or more of HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371; and c) Compare the expression levels of Siglec-15 protein determined in step b) with those of a control, including but not limited to the expression levels of one or more of HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371. Compared with the control group, the expression level of Siglec-15 protein in the sample was higher than the reference value, indicating that the subject had a higher risk of hematologic malignancies than the general population (control group); and / or Compared to the control group, the expression level of Siglec-15 protein in the sample was lower than the reference value, indicating that the risk of hematologic malignancies in the subjects was lower than that in the general population (control group); and / or Compared with the control group, if the co-expression level of Siglec-15 in the sample is greater than 30% with one or more of HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371, it indicates that the subject has a higher risk of hematologic malignancies than the general population (control group).

[0156] In another preferred embodiment, the subject is a human or a non-human mammal.

[0157] In another preferred embodiment, the test sample is cells or tissue of a hematologic malignancy.

[0158] In another preferred embodiment, the reference value is a cut-off value.

[0159] In another preferred embodiment, the reference value is the relative expression level of Siglec-15 in the sample.

[0160] In another preferred embodiment, the reference value is 2.

[0161] In another preferred embodiment, the detection step (b) includes detecting the amount of Siglec-15 mRNA or Siglec-15 cDNA; and / or detecting the amount of Siglec-15 protein; and / or detecting the amount of mRNA or cDNA containing, but not limited to, one or more of HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371; and / or detecting the amount of its protein.

[0162] In another preferred embodiment, the expression level of Siglec-15 protein in the sample is detected by quantitative real-time PCR or immunohistochemistry; and / or includes, but is not limited to, the expression level of one or more of HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371.

[0163] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0164] The twenty-fifth aspect of the present invention provides a method for determining a treatment plan, comprising: a) Provide test samples from the subjects; b) Detect the expression level of Siglec-15 protein in the test sample; and / or the expression level of proteins including, but not limited to, one or more of HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371; and c) Determine the treatment regimen based on the expression level of the Siglec-15 protein in the sample; and / or the expression level of proteins including, but not limited to, one or more of HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371.

[0165] In another preferred embodiment, the subject is a human or a non-human mammal.

[0166] In another preferred embodiment, when the expression level of Siglec-15 protein in the sample is higher than that of a reference value, indicating that the subject has a higher risk of developing hematologic malignancies than the general population (control group), the treatment regimen includes Siglec-15 inhibitor therapy.

[0167] In another preferred embodiment, when the co-expression of Siglec-15 in the sample with one or more of, but not limited to, HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371 is greater than 30%, it indicates that the subject has a higher risk of hematologic malignancy than the general population (control group), and the treatment regimen includes Siglec-15 inhibitor therapy.

[0168] In another preferred embodiment, the Siglec-15 inhibitor therapy is selected from the group consisting of: Siglec-15 inhibitor therapy: antibodies, small molecule compounds, microRNA, siRNA, shRNA, or combinations thereof; In another preferred embodiment, when the subject has a higher risk of developing hematologic malignancies than the general population (control group), the treatment regimen may also include Siglec-15 inhibitor therapy or a combination of Siglec-15 inhibitors with other hematologic malignancies treatments.

[0169] In another preferred embodiment, the other drugs for treating hematologic malignancies are selected from the group consisting of chemotherapy drugs, monoclonal antibody drugs, immune cell drugs, or combinations thereof.

[0170] In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of: methotrexate, cytarabine, hydroxyurea, fludarabine, 6-mercaptopurine, gemcitabine, deoxymyopicin, 2-chlorodeoxyadenosine, paclitaxel, vincristine, vinblastine, homoharringtonine, camptothecin and its derivatives, daunorubicin, doxorubicin, mitoxantrone, etoposide, teniposide, acridine, cyclophosphamide, nitrogen mustard, asparaginase, busulfan, carboplatin, cisplatin, fluorouracil, or combinations thereof.

[0171] In another preferred embodiment, the monoclonal antibody drug includes rituximab (Romeovart).

[0172] The twenty-sixth aspect of the present invention provides the use of an inhibitor of the Siglec-15 gene or its protein for the preparation of a composition or formulation for (a) inhibiting the growth or proliferation of hematologic malignancies; and / or (d) diagnosing and / or preventing and / or treating hematologic malignancies.

[0173] In another preferred embodiment, the Siglec-15 gene or its protein inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, or combinations thereof.

[0174] In another preferred embodiment, the inhibitor includes an inhibitor that inhibits the expression of the Siglec-15 gene or its protein.

[0175] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0176] In another preferred embodiment, the composition comprises a therapeutically effective amount of an inhibitor of the Siglec-15 gene or its protein, and a pharmaceutically acceptable carrier.

[0177] In another preferred embodiment, the drug is administered via a method selected from the group consisting of: oral, intravenous, intramuscular, subcutaneous, sublingual, rectal, nasal spray, oral spray, local or systemic transdermal administration.

[0178] In another preferred embodiment, the formulation is selected from the group consisting of tablets, capsules, injections, granules, and sprays.

[0179] The twenty-seventh aspect of the present invention provides a pharmaceutical composition comprising: (a1) Inhibitors of the Siglec-15 gene or its protein; and (b) Pharmaceutically acceptable carriers.

[0180] In another preferred embodiment, the pharmaceutical composition further includes: (c) Other medications for the prevention and / or treatment of hematologic malignancies.

[0181] In another preferred embodiment, the content of component (a1) in the pharmaceutical composition is 1%-99%, more preferably 10%-90%, and even more preferably 30%-70%.

[0182] In another preferred embodiment, the content of component (c) in the pharmaceutical composition is 1%-99%, more preferably 10%-90%, and even more preferably 30%-70%.

[0183] In another preferred embodiment, the component (a1) and optional component (c) constitute 0.01-99.99 wt% of the total weight of the pharmaceutical composition, more preferably 0.1-90 wt%, and more preferably 1-80 wt%.

[0184] In another preferred embodiment, the dosage form of the pharmaceutical composition includes injectable and oral dosage forms.

[0185] In another preferred embodiment, the oral dosage form includes tablets, capsules, films, and granules.

[0186] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a sustained-release dosage form and a non-sustained-release dosage form.

[0187] The twenty-eighth aspect of the present invention provides a medicine box, comprising: (a1) A first container, and an inhibitor of the Siglec-15 gene or its protein located in the first container, or a drug containing an inhibitor of the Siglec-15 gene or its protein.

[0188] In another preferred embodiment, the pillbox further includes: (b1) A second container, and other drugs for the prevention and / or treatment of hematologic malignancies located in the second container, or drugs containing other drugs for the prevention and / or treatment of hematologic malignancies.

[0189] In another preferred embodiment, the kit further includes (c1) a third container and a Siglec-15 test reagent located in the third container.

[0190] In another preferred embodiment, the first container, the second container, and the third container may be the same or different containers.

[0191] In another preferred embodiment, the drug in the first container is a single-ingredient preparation containing an inhibitor of the Siglec-15 gene or its protein.

[0192] In another preferred embodiment, the drug in the second container is a single-ingredient preparation containing other drugs for the prevention and / or treatment of hematologic malignancies.

[0193] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injectable dosage form.

[0194] In another preferred embodiment, the kit also includes instructions for use.

[0195] In another preferred embodiment, the specification includes one or more descriptions selected from the group consisting of: (a) Using inhibitors of the Siglec-15 gene or its protein for (i) inhibiting the growth or proliferation of hematologic malignancies; and / or (ii) preventing and / or treating hematologic malignancies; (b) Using an inhibitor of the Siglec-15 gene or its protein in combination with other drugs for the prevention and / or treatment of hematologic malignancies to (i) inhibit the growth or proliferation of hematologic malignancies; and / or (ii) prevent and / or treat hematologic malignancies; (c) Detecting the expression level of the Siglec-15 gene or its protein in patients with hematologic malignancies, while administering an inhibitor of the Siglec-15 gene or its protein to (i) inhibit the growth or proliferation of hematologic malignancies; and / or (ii) methods for the prevention and / or treatment of hematologic malignancies; (d) Methods for detecting the expression level of the Siglec-15 gene or its protein in patients with hematologic malignancies, in combination with an inhibitor of the Siglec-15 gene or its protein; and other drugs for the prevention and / or treatment of hematologic malignancies to (i) inhibit the growth or proliferation of hematologic malignancies; and / or (ii) prevent and / or treat hematologic malignancies.

[0196] The twenty-ninth aspect of the present invention provides the use of the pharmaceutical composition of the twenty-seventh aspect of the present invention or the medicament of the twenty-eighth aspect of the present invention for (i) inhibiting the growth or proliferation of hematologic tumor cells; and / or (ii) preventing and / or treating hematologic tumors.

[0197] In another preferred embodiment, the concentration of the inhibitor of the Siglec-15 gene or its protein in the pharmaceutical composition is 100-2000 ng / ml, more preferably 500-1500 ng / ml, and even more preferably 800-1000 ng / ml.

[0198] In another preferred embodiment, the concentration of the other drug for preventing and / or treating hematologic malignancies in the pharmaceutical composition is 500-4000 ng / ml, more preferably 1500-3500 ng / ml, and even more preferably 2000-3000 ng / ml.

[0199] In another preferred embodiment, the pharmaceutical composition or kit comprises (a) an inhibitor of the Siglec-15 gene or its protein; and (b) optionally other drugs for the prevention and / or treatment of hematologic malignancies; and (d) a pharmaceutically acceptable carrier.

[0200] In another preferred embodiment, the pharmaceutical composition or kit contains (b) an inhibitor of the Siglec-15 gene or its protein; and (c) optional other drugs for the prevention and / or treatment of hematologic malignancies, comprising 0.01-99.99 wt%, more preferably 0.1-90 wt%, and more preferably 1-80 wt% of the total weight of the pharmaceutical composition or kit.

[0201] The thirtieth aspect of this invention provides a method for preventing and / or treating hematologic malignancies, comprising: Administer an inhibitor of the Siglec-15 gene or its protein to the desired recipient; or the pharmaceutical composition described in aspect twenty-seven of the present invention or the medicament described in aspect twenty-eight of the present invention.

[0202] In another preferred embodiment, the object includes a person or non-human mammal suffering from a blood cancer.

[0203] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0204] In another preferred embodiment, the dose of the inhibitor of the Siglec-15 gene or its protein is 0.5-5 mg / kg body weight, more preferably 1-4 mg / kg body weight, and most preferably 2-3 mg / kg body weight.

[0205] In another preferred embodiment, the dosage of the other drug for the prevention and / or treatment of hematologic malignancies is 5-70 mg / kg body weight, more preferably 10-50 mg / kg body weight, and most preferably 20-40 mg / kg body weight.

[0206] In another preferred embodiment, the inhibitor of the Siglec-15 gene or its protein is administered 1-4 times per week, more preferably 2-3 times per week.

[0207] In another preferred embodiment, the administration time of the inhibitor of the Siglec-15 gene or its protein is 20-90 days, more preferably 20-60 days, and most preferably 30-40 days.

[0208] In another preferred embodiment, the administration period of the other drugs for the prevention and / or treatment of hematologic malignancies is 20-90 days, more preferably 20-60 days, and most preferably 30-40 days.

[0209] In another preferred embodiment, an inhibitor of the Siglec-15 gene or its protein is administered simultaneously or sequentially with optional other drugs for the prevention and / or treatment of hematologic malignancies.

[0210] The thirty-first aspect of the present invention provides a method for inhibiting the growth or proliferation of hematologic tumor cells in vitro without therapeutic effect, comprising the steps of: culturing hematologic tumor cells in the presence of the Siglec-15 gene or an inhibitor of its protein, thereby inhibiting the growth or proliferation of hematologic tumor cells.

[0211] In another preferred embodiment, the Siglec-15 gene or its protein inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, or combinations thereof.

[0212] In another preferred embodiment, the hematologic tumor cells highly express the Siglec-15 protein.

[0213] In another preferred embodiment, the method further includes adding other drugs for the prevention and / or treatment of hematologic malignancies to the culture system of hematologic malignancies, thereby inhibiting the growth or proliferation of hematologic malignancies.

[0214] In another preferred embodiment, the blood tumor cells are cells cultured in vitro.

[0215] The thirty-second aspect of this invention provides a method for screening candidate compounds for the prevention and / or treatment of hematological malignancies, the method comprising the steps of: (a) In the test group, the test compound was added to the cell culture system, and the expression level (E1) and / or activity (A1) of Siglec-15 in the cells of the test group were observed; in the control group, the test compound was not added to the same cell culture system, and the expression level (E0) and / or activity (A0) of Siglec-15 in the cells of the control group were observed. If the expression level (E1) and / or activity (A1) of Siglec-15 in the test group is significantly lower than that in the control group, it indicates that the test compound is a candidate compound for the prevention and / or treatment of hematological malignancies that inhibits the expression and / or activity of Siglec-15.

[0216] In another preferred embodiment, the expression level of Siglec-15 is determined by quantitative real-time PCR or immunohistochemistry.

[0217] In another preferred embodiment, the method further includes the step of: (b) For the candidate compounds obtained in step (a), further test their inhibitory effect on the growth or proliferation of hematologic tumor cells; and / or further test whether they have a downregulatory effect on the Siglec-15 gene.

[0218] In another preferred embodiment, step (b) includes the steps of: in the test group, adding the test compound to the culture system of hematologic malignancies and observing the number and / or growth of the hematologic malignancies; in the control group, not adding the test compound to the culture system of hematologic malignancies and observing the number and / or growth of the hematologic malignancies; wherein, if the number or growth rate of hematologic malignancies in the test group is less than that in the control group, it indicates that the test compound is a candidate compound for the prevention and / or treatment of hematologic malignancies that has an inhibitory effect on the growth or proliferation of hematologic malignancies.

[0219] In another preferred embodiment, the method includes step (c): applying the candidate compound identified in step (a) to a mammalian model and determining its effect on the mammal.

[0220] In another preferred embodiment, the mammal is a mammal suffering from a blood tumor.

[0221] In another preferred embodiment, “significantly lower than” means E1 / E0 ≤ 1 / 2, more preferably ≤ 1 / 3, and even more preferably ≤ 1 / 4.

[0222] In another preferred embodiment, “significantly lower than” means A1 / A0 ≤ 1 / 2, more preferably ≤ 1 / 3, and even more preferably ≤ 1 / 4.

[0223] In another preferred embodiment, the cells include hematologic tumor cells.

[0224] In another preferred embodiment, the cells are cells cultured in vitro.

[0225] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0226] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0227] Figure 1 The binding ability of three Siglec-15 (S15) antibodies to human and mouse proteins was demonstrated; The binding of three antibodies (clone 128, 413, and 304) to human and mouse S15 was monitored using CHO cell lines that highly expressed human S15 and CHO cell lines that highly expressed mouse S15 constructed in the laboratory. The antibody with clone number 128 only bound to human S15, while 413 and 304 could bind to both human and mouse S15.

[0228] Figure 2 The results showed that antibodies 304 and 216 specifically bound to the high-expressing cell line and the AML cell line. Antibodies 128, 413, 304, and 216 stained the high-expressing cell line and the AML cell line U937, respectively, and antibodies 304 and 216 bound to U937 cells.

[0229] Figure 3 The differential expression of Siglec-15 in different tumor cell lines was observed. Jurkat, Nalm6, and K562 cells showed no S15 expression, while U937 cells derived from malignant histiocytic lymphoma and THP1, Kasumi-1, and HL60 cells from acute leukemia cell lines all expressed Siglec-15. This indicates that the 304 antibody can specifically bind to AML cells.

[0230] Figure 4 The results showed that the 304 antibody can specifically bind to tumor cells from AML patients; the 304 antibody was used to stain tumor cell lines from AML patients, and the 304 antibody can bind to tumor cells from AML patients.

[0231] Figure 5 The results showed that Siglec-15 is not expressed in normal bone marrow stem cells. Both bone marrow samples from healthy individuals contained stem cells (CD34+), and Siglec-15 expression was less than 10% in both cases.

[0232] Figure 6 The in vitro killing ability of 304-CAR-T cells was demonstrated. In the in vitro killing assay, 304-CAR-T cells were constructed using the 304 antibody sequence, and CAR-T cells were used to kill U937 cells and the laboratory-constructed Siglec-15 high-expression strain Nalm6-S15 cells, respectively. Both cells showed specific killing ability and high IFN-R secretion.

[0233] Figure 7 The in vivo killing ability of 304-CAR-T was demonstrated. An NCG mouse model was constructed using the U937 cell line, and the mice were treated with 304-CAR-T and TX103 CAR-T targeting unrelated targets, respectively. It was found that 304-CAR-T could significantly inhibit tumor growth. Detailed Implementation

[0234] Through extensive and in-depth research, the inventors have unexpectedly discovered for the first time a specific scFv targeting siglec-15, an antibody, and specific CAR-T cells. Specifically, this invention unexpectedly yielded an anti-siglec-15 monoclonal antibody with extremely excellent affinity and specificity, and based on this antibody, a humanized antibody was obtained. The antibody of this invention can bind to the siglec-15 antigen with high specificity and exhibits high affinity. Furthermore, the scFv of this invention has high affinity and good specificity for siglec-15, specifically targeting siglec-15, especially the full-length siglec-15 antigen and its extracellular region. In addition, this invention also provides a chimeric antigen receptor immune cell targeting siglec-15, which can effectively treat cancer or tumors.

[0235] Furthermore, this invention has unexpectedly discovered that in hematologic tumor cells or tissues... siglec-15 The expression of the gene or its protein was significantly higher than that of the siglec-15 gene or its protein in normal tissues, and the applicant also found that, therefore, siglec-15 The gene or its protein can be used as a biomarker for detecting hematologic malignancies, and the siglec-15 gene or its protein can be combined with HLA-DR, CD38, CD117, MPO, TIM3, CD70, CD371, and CD33 genes or their proteins for the detection of hematologic malignancies. Furthermore, the applicant unexpectedly discovered that... siglec-15 Inhibitors of genes or their proteins can effectively (a) inhibit the growth or proliferation of hematologic malignancies; and / or (b) prevent and / or treat hematologic malignancies, and, siglec-15 Inhibitors of genes or their proteins can be used in combination with other optional drugs for the prevention and / or treatment of hematological malignancies, and have significant therapeutic effects on hematological malignancies. Based on this, the present invention was completed.

[0236] This invention uses CAR-T cells as an example to provide a detailed description of the engineered immune cells of this invention. The engineered immune cells of this invention are not limited to the CAR-T cells described in the context; the engineered immune cells of this invention have the same or similar technical features and beneficial effects as the CAR-T cells described in the context. Specifically, when immune cells express chimeric antigen receptor (CAR), NK cells are equivalent to T cells (or T cells can replace NK cells); when immune cells are T cells, TCR is equivalent to CAR (or CAR can be replaced by TCR).

[0237] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0238] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0239] As used herein, a "chimeric antigen receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a different polypeptide, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is also known as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)." The "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding a particular antigen. The "intracellular domain" refers to any oligopeptide or polypeptide known to act as a signaling domain for activating or inhibiting intracellular biological processes.

[0240] As used in this article, a "domain" refers to a region in a polypeptide that is independent of other regions and folds into a specific structure.

[0241] As used in this article, "tumor antigen" refers to a biomolecule that has antigenic properties and whose expression leads to cancer.

[0242] As used herein, the terms “administer” and “treatment” refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. “Administer” and “treatment” can refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and a cell, as well as contact between a reagent and a fluid, and contact between a fluid and a cell. “Administer” and “treatment” also mean treatment by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to a human, animal, or research subject, “treatment” refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic procedures; including contact between anti-human Siglec-15 antibody and a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

[0243] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent, comprising any of the anti-human Siglec-15 antibodies of the present invention and combinations thereof, to a patient having one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount that effectively relieves one or more disease symptoms (therapeutic effective amount).

[0244] As used herein, the terms “optional” or “optionally” mean that the events or conditions described below may occur but are not required to occur. For example, “optionally containing 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a particular sequence may be present but are not required to be present, and may be 1, 2 or 3.

[0245] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0246] scFv As used herein, scFv is a single-chain antibody variable region fragment that specifically recognizes antigens. Alternatively, in this invention, it can also be an antigen-binding domain.

[0247] In this invention, the scFv of this invention includes the heavy chain variable region described in the first aspect of this invention and the light chain variable region described in the third aspect of this invention.

[0248] In another preferred embodiment, the scFv further comprises a linker peptide between the heavy chain variable region and the light chain variable region.

[0249] In another preferred embodiment, the scFv is shown in equation A or equation B as follows: V H -V L , (A); or V L -V H (B) In the formula, V H V is the variable region of the antibody heavy chain; L "-" represents the variable region of the antibody light chain; "-" represents a linking peptide or peptide bond.

[0250] In another preferred embodiment, the V H and V L The linking peptides are 1-4 consecutive GGGGS sequences, preferably 2-4, and more preferably 3-4.

[0251] In another preferred embodiment, the amino acid sequence of the scFv is shown in SEQ ID NO.: 33-36.

[0252] In another preferred embodiment, the scFv targets or binds to human siglec-15.

[0253] Antibody As used herein, the term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its heavy chain region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in the constant region. Each of the five classes of Ig can have either a κ or λ chain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

[0254] The antibody light chain of the present invention may further include a light chain constant region, wherein the light chain constant region comprises a human or mouse κ, λ chain or a variant thereof.

[0255] In this invention, the antibody heavy chain may further include a heavy chain constant region, which contains human or mouse IgG1, IgG2, IgG3, IgG4, or variants thereof. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably and is called the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the terminal terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0256] The antibodies of this invention include murine antibodies, chimeric antibodies, and humanized antibodies, with humanized antibodies being preferred. The term "murine antibody" in this invention refers to a monoclonal antibody against human siglec-15 prepared according to the knowledge and skills in the art. Preparation involves injecting the test subject with the siglec-15 antigen, followed by isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. In a preferred embodiment of this invention, the murine siglec-15 antibody or its antigen-binding fragment may further include a light chain constant region of a murine κ, λ chain, or a variant thereof, or further include a heavy chain constant region of murine IgG1, IgG2, IgG3, or a variant thereof.

[0257] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by murine antibodies.

[0258] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into the variable region framework of a human antibody, i.e., a different type of human germline antibody framework sequence. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies, which carry a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. To avoid a decrease in immunogenicity along with a decrease in activity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity.

[0259] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen (e.g., siglec-15). It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include... (i) Fab fragment, composed of V L V H A monovalent fragment composed of the CL and CH1 domains; (ii) F(ab')2 fragment, which is a divalent fragment containing two Fab fragments connected by a disulfide bridge on the chain region; (iii) By V H The Fd fragment is composed of the CH1 domain; (iv) V from the single arm of the antibody H and V L Fv segments composed of structural domains.

[0260] Fv antibodies contain variable regions of the antibody heavy chain and light chain, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites. Generally, Fv antibodies also contain V... H and V L It forms a polypeptide linker between domains and can form the structure required for antigen binding.

[0261] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of these six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242.

[0262] The term "epitope" or "antigenic determinant" refers to the site on an antigen where an immunoglobulin or antibody specifically binds (e.g., a specific site on the siglec-15 molecule). Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation.

[0263] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example, approximately less than 10 -8 M, 1O -9 M or lO -10 M or lower affinity (KD) binding.

[0264] The term "competitive binding" refers to an antibody that recognizes or binds to the same epitope (also called an antigenic determinant) or a portion of the same epitope on the extracellular region of human siglec-15 as the monoclonal antibody of the present invention. An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of human siglec-15 recognized by the monoclonal antibody of the present invention.

[0265] The term "KD" or "Kd" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies of this invention have a dissociation equilibrium constant of less than approximately 10. -7 M, for example, less than approximately 10 -8 M, 1O -9 M or lO -10 The dissociation equilibrium constant (KD) of M or smaller, combined with siglec-15, was determined in a BIACORE instrument using surface plasmon resonance (SPR) technology.

[0266] As used herein, the term "antigen determinant" refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by the antibody or antigen-binding fragment of the present invention.

[0267] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0268] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be prepared using DNA recombination techniques well known in the art.

[0269] As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone derived from a single cell. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. The cell may be a eukaryotic, prokaryotic, or phage clone.

[0270] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0271] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0272] Table A Human siglec-15 specific antibody This invention provides an anti-human siglec-15 antibody (hereinafter referred to as siglec-15 antibody). Specifically, this invention provides an antibody with high specificity and high affinity against siglec-15, comprising a heavy chain and a light chain, wherein the heavy chain contains a heavy chain variable region (V). H The amino acid sequence, wherein the light chain contains a light chain variable region (V L The amino acid sequence is not specified. The siglec-15 antibody of this invention enhances the anti-tumor effect of T cells by stimulating antigen-specific T cell responses, thereby maximizing the patient's own immune system response to the tumor and achieving the goal of killing tumor cells.

[0273] Preferably, the heavy chain variable region (V H Amino acid sequence and light chain variable region (V L The CDRs of the amino acid sequences are selected from the following groups: (1) SEQ ID NO:1 (2) SEQ ID NO:2 (3) SEQ ID NO:3 (4) SEQ ID NO:4 (5) SEQ ID NO:5 (6) SEQ ID NO:6 (7) SEQ ID NO:7 (8) SEQ ID NO:9 (9) SEQ ID NO:10 (10) SEQ ID NO:11 (11) SEQ ID NO:12 (12) SEQ ID NO:13 (13) SEQ ID NO:14 (14) SEQ ID NO:15 (15) SEQ ID NO:16 (16) SEQ ID NO:17 (17) SEQ ID NO:18 (18) SEQ ID NO:19 (19) SEQ ID NO:20 (20) SEQ ID NO:21 (21) SEQ ID NO:22 (22) SEQ ID NO:23 (23) SEQ ID NO:24; The above-mentioned amino acid sequences are sequences that have been modified by adding, deleting, modifying and / or substituting at least one (e.g., 1-5, 1-3, preferably 1-2, more preferably 1) amino acid to form a sequence with siglec-15 binding affinity.

[0274] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence preferably has a homology of at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the amino acid sequence.

[0275] The antibody of the present invention can be a double-chain or single-chain antibody, and can be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0276] The antibody derivatives described in this invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2, or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes of antibodies.

[0277] The animal is preferably a mammal, such as a mouse.

[0278] The antibodies of this invention can be murine antibodies, chimeric antibodies, humanized antibodies, CDR-grafted and / or modified antibodies targeting human siglec-15.

[0279] In a preferred embodiment of the present invention, any one or more sequences in SEQ ID NO:1-12 above, or sequences having siglec-15 binding affinity after addition, deletion, modification and / or substitution of at least one amino acid, are located in the heavy chain variable region (V). H ) of the CDR area.

[0280] In a preferred embodiment of the present invention, any one or more sequences of SEQ ID NO: 13-24 above, or sequences having siglec-15 binding affinity after addition, deletion, modification and / or substitution of at least one amino acid, are located in the light chain variable region (V L ) of the CDR area.

[0281] In a more preferred embodiment of the present invention, V H CDR1, CDR2, and CDR3 are each independently selected from any one or more sequences in SEQ ID NO: 1-12, or sequences with siglec-15 binding affinity that have been added, deleted, modified, and / or substituted with at least one amino acid; V L CDR1, CDR2, and CDR3 are each independently selected from any one or more sequences in SEQ ID NO: 13-24, or sequences with siglec-15 binding affinity that have been added, deleted, modified, and / or substituted with at least one amino acid.

[0282] In the above-described content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0283] In this invention, the number of added, deleted, modified and / or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, and most preferably 1.

[0284] Antibody preparation Any method suitable for producing monoclonal antibodies can be used to produce the siglec-15 antibody of the present invention. For example, animals can be immunized with the linked or naturally occurring siglec-15 protein or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, or one or more routes.

[0285] Any suitable form of siglec-15 can serve as an immunogen (antigen) for generating non-human antibodies specific to siglec-15 and screening for the biological activity of said antibodies. The immunogen can be used alone or in combination with one or more immunogenic enhancers known in the art. The immunogen can be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA). The DNA encoding the immunogen can be expressed using suitable genetic vectors, including but not limited to adenovirus vectors, baculovirus vectors, plasmids, and nonviral vectors.

[0286] Exemplary methods for producing the siglec-15 antibody of the present invention are described in Examples 1 and 2.

[0287] Humanized antibodies can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. In this invention, the antibody is an IgG antibody, using either the IgG1 or IgG4 subtype.

[0288] Similarly, any type of light chain can be used in the compounds and methods described herein. Specifically, κ, λ chains, or variations thereof, can be used in the compounds and methods of this invention.

[0289] An exemplary method for humanizing the siglec-15 antibody of the present invention is described in Example 6.

[0290] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, coding sequences of different light and heavy chains can be fused together in different combinations to form single-chain antibodies. Optimized single-chain antibodies can be obtained by detecting and analyzing the functions of single-chain antibodies with different combinations or linkage modifications.

[0291] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0292] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences are obtained by first synthesizing multiple small fragments and then ligating them. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0293] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0294] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one implementation, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked.

[0295] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0296] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant or animal cells (such as mammalian cells).

[0297] The steps of transforming host cells with recombinant DNA as described in this invention can be performed using techniques well known in the art. The obtained transformants can be cultured using conventional methods, and the transformants express the polypeptide encoded by the gene of this invention. Depending on the host cell used, the cells are cultured in a conventional culture medium under suitable conditions.

[0298] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0299] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0300] siglec-15 The sialic acid-binding Ig-like lectins family, or Siglecs family, is a classic family of immunoglobulin-like lectin proteins. Under physiological conditions, only 15 Siglec molecules are expressed on the surface of myeloid cells and immune cells that mediate immunosuppression, thus possessing immunosuppressive properties. When Siglecs recognize sialic acid-containing glycans expressed by all mammalian cells, they can help immune cells distinguish between self and non-self. Sialidized pathogens can also disrupt or promote immune responses, or downregulate immune cell responses and evade immune surveillance, through Siglec-dependent interactions. Siglecs play a crucial role in the regulation of immune cell activation and inhibition receptors, influencing host-pathogen interactions, neurodegeneration, osteoclast differentiation in autoimmune diseases, and cancer. Due to their restricted expression on immune cells, endocytic properties, and ability to regulate receptor signaling, they have become important targets for cell-directed therapy.

[0301] Siglec-15 (S15 for short) is a cell surface transmembrane receptor composed of an immunoglobulin (Ig)-like domain, a transmembrane domain, and a short cytoplasmic tail. The immunoglobulin-like domain consists of two extracellular Ig domains, including an N-terminal V-set domain containing a sialic acid binding site and a type II constant region (IgC2). It associates with the tyrosine-containing activation motif (ITAM) adapter molecule DAP12 via positively charged amino acids in its transmembrane domain and activates the receptor by recruiting SYK kinases.

[0302] Siglec-15 differs from other family members. It exhibits similar domain components and high homology to B7-H1 and many other B7 family members. More importantly, the expression of Siglec-15 and B7-H1 in human lung cancer tissues is mutually exclusive. Therefore, in addition to its immunomodulatory functions, Siglec-15 may possess a unique regulatory mechanism: besides binding to TCR and CD44, Siglec-15 has not been found to bind to other receptors and does not interact with B7-H1, PD-1, B7-1, or any other known B7 family ligands or receptors.

[0303] Siglec-15 is expressed in various human tumor tissues on tumor cells, tumor-associated stromal cells, and myeloid cells such as tumor-infiltrating macrophages or monocytes. Siglec-15 gene knockout does not cause obvious autoimmune diseases or other symptoms in mice. Myeloid-derived Siglec-15 from macrophages or monocytes can suppress antigen-specific T cell immune responses by regulating cell growth. In various mouse tumor models, the anti-Siglec-15 monoclonal antibody α-S15 can block the immunosuppressive effect of myeloid-derived Siglec-15 from macrophages or monocytes, thereby enhancing the in vivo anti-tumor immune response.

[0304] In this invention, samples of tumor cells, such as acute myeloid leukemia (AML) tumor cells, were tested and found that siglec-15 was highly expressed in some tumor cells. The chimeric antigen receptor targeting siglec-15 prepared in this invention can effectively kill tumor cells in vitro and in vivo, and showed a good anti-tumor effect on AML tumor cells.

[0305] Exogenous T-cell antigen receptor As used in this article, the exogenous T cell antigen receptor (TCR) is a TCR whose α and β chains are cloned from tumor reactive T cells through gene transfer technology and then exogenously transferred into T cells using lentiviruses or retroviruses as vectors through genetic engineering.

[0306] T cells modified with exogenous TCRs can specifically recognize and kill tumor cells. By optimizing the affinity of TCRs for tumor-specific antigens, the affinity of T cells for tumors can be improved, thereby enhancing the anti-tumor effect.

[0307] Chimeric antigen receptor (CAR) Chimeric antigen receptors (CARs) consist of an extracellular antigen recognition region, typically a single-chain variable fragment (scFv), a transmembrane region, and an intracellular co-stimulatory signaling region. CAR design has evolved as follows: First-generation CARs contain only one intracellular signaling component, CD3ζ or FcγRI. Because they have only one activation domain, they can only induce transient T cell proliferation and limited cytokine secretion, failing to provide long-term T cell proliferation signals and sustained in vivo anti-tumor effects, thus failing to achieve satisfactory clinical efficacy. Second-generation CARs introduce a co-stimulatory molecule, such as CD28, 4-1BB, OX40, or ICOS, into the existing structure, significantly improving function compared to first-generation CARs and further enhancing the persistence of CAR-T cells and their ability to kill tumor cells. Third- and fourth-generation CARs are developed by tandem with new immune co-stimulatory molecules such as CD27 and CD134.

[0308] The extracellular domain of CARs recognizes a specific antigen, which is then transduced through intracellular domains, leading to cell activation and proliferation, cytotoxicity, and cytokine secretion, ultimately eliminating target cells. The process involves first isolating autologous cells (or xenograft cells), activating and genetically modifying them to produce CAR-containing immune cells, and then injecting these cells into the same patient. This method results in an extremely low probability of graft-versus-host disease, as the antigen is recognized by immune cells in a non-MHC-restricted manner.

[0309] CAR-immunotherapy has achieved a very high clinical response rate in the treatment of hematologic malignancies, a rate that no previous treatment method could achieve, sparking a surge of clinical research worldwide.

[0310] Specifically, the chimeric antigen receptor (CAR) of the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain containing a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen, rather than an antigen receptor or its ligands.

[0311] A linker may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of the polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2-100 amino acids, and most preferably 3-50 amino acids. The CAR of the present invention, when expressed in T cells, is capable of antigen recognition based on antigen-binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to stop growing, be induced to die, or otherwise be affected, resulting in a reduction or elimination of the patient's tumor burden. The antigen-binding domain is preferably fused with an intracellular domain derived from one or more of the co-stimulatory molecules and the ζ chain. Preferably, the antigen-binding domain is fused with an intracellular domain combining a CD28 signaling domain and a CD3ζ signaling domain.

[0312] As used herein, "antigen-binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen-binding activity. Fv antibodies contain variable regions of the antibody heavy chain and light chain, but no constant regions, and are the smallest antibody fragments possessing all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The antigen-binding domain is typically scFv (single-chain variable fragment). The size of an scFv is generally 1 / 6 of a complete antibody. Single-chain antibodies are preferably a single amino acid chain sequence encoded by a single nucleotide chain. As a preferred embodiment of the invention, the scFv contains an antibody that specifically recognizes tumor-overexpressed antigens CD19 and / or BCMA, preferably a single-chain antibody.

[0313] In this invention, the scFv of this invention also includes its conserved variants, which refer to the amino acid sequence of the scFv of this invention having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids replaced by amino acids with similar or analogous properties to form a polypeptide.

[0314] In this invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0315] In this invention, the number of added, deleted, modified and / or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, and most preferably 1.

[0316] For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one implementation, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected, or modified by amino acid substitution, to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0317] The intracellular domains in the CAR of the present invention include the CD28 and / or 4-1BB signal transduction domains and the CD3ζ signal transduction domain.

[0318] In a preferred embodiment of the present invention, the amino acid sequence of the CAR is shown in any of SEQ ID NO.:43-46.

[0319] Chimeric antigen receptor T cells (CAR-T cells) As used herein, the terms "CAR-T cell", "CAR-T", and "CAR-T cell of the present invention" all refer to the CAR-T cell described in the first aspect of the present invention.

[0320] CAR-T cells have the following advantages over other T-cell-based therapies: (1) The action of CAR-T cells is not restricted by MHC; (2) Given that many tumor cells express the same tumor antigens, once the CAR gene targeting a certain tumor antigen is constructed, it can be widely used; (3) CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, thus expanding the target range of tumor antigens; (4) Using the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0321] Chimeric antigen receptor NK cells (CAR-NK cells) As used herein, the terms "CAR-NK cell," "CAR-NK," and "CAR-NK cell of the present invention" all refer to the CAR-NK cell described in the first aspect of the present invention. The CAR-NK cell of the present invention can be used to treat tumors with high expression of CD19 and BCMA.

[0322] Natural killer (NK) cells are a major type of immune effector cells that protect the body from viral infections and tumor cell invasion through non-antigen-specific pathways. Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.

[0323] Compared with autologous CAR-T cells, CAR-NK cells have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzymes, without killing normal cells in the body; (2) they release very little cytokine, thus reducing the risk of cytokine storm; and (3) they are very easy to expand in vitro and develop into "ready-made" products. In addition, they are similar to CAR-T cell therapy.

[0324] carrier The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a vector known to contain the gene, or by directly isolating the gene from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be synthesized.

[0325] This invention also provides vectors in which the expression cassette of this invention is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0326] In short, the expression cassette or nucleic acid sequence of this invention is typically operatively linked to a promoter and incorporated into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, an initial sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0327] The expression constructs of the present invention can also be used with standard gene delivery protocols for nucleic acid immunotherapy and gene therapy. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0328] This nucleic acid can be cloned into many types of vectors. For example, it can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0329] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0330] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0331] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. These are typically located in a 30–110 bp region upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible to maintain promoter function when an element is inverted or moved relative to another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to initiate transcription.

[0332] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered as part of the invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operatively linked to the inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0333] To assess the expression of CAR peptides or portions thereof, the expression vector introduced into cells may also contain either or both of an optional marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector. Alternatively, the optional marker may be carried on a separate DNA segment and used in co-transfection procedures. Both the optional marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful optional markers include, for example, antibiotic resistance genes such as neo.

[0334] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is either absent from or expressed by the recipient organism or tissue, and that encodes a polypeptide whose expression is clearly indicated by readily detectable properties such as enzyme activity. After DNA has been introduced into the recipient cells, reporter gene expression is measured at an appropriate time. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available. Typically, a construct with at least five flanking regions exhibiting the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be ligated into reporter genes and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0335] Methods for introducing genes into cells and expressing genes into cells are known in the art. Within the scope of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0336] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and so on. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0337] Biological approaches to introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0338] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in both in vitro and in vivo is the liposome (e.g., an artificial membrane capsule).

[0339] In the case of using a non-viral delivery system, an exemplary delivery tool is a liposome. Consider using a lipid formulation to introduce nucleic acid into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid may be associated with a lipid. Lipid-associated nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, conjugated with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA, or lipid / expression vector associated with the composition are not limited to any specific structure in solution. For example, they may be present in a bilayer structure, as micelles, or have a “collapsed” structure. They may also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm and in compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

[0340] In a preferred embodiment of the present invention, the carrier is a lentivirus carrier.

[0341] preparation This invention provides a formulation comprising the chimeric antigen receptor described in the third aspect of this invention, the nucleic acid molecule described in the sixth aspect of this invention, the carrier described in the seventh aspect of this invention, or the engineered immune cells described in the eighth aspect of this invention, as well as a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation. Preferably, the concentration of the CAR-T cells in the formulation is 1 × 10⁻⁶. 3 -1×10 10 Cells / ml, more optimal 1×10 4 -1×10 9Cells / ml. In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

[0342] Therapeutic applications This invention includes therapeutic applications using cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of this invention. The transduced T cells can target the tumor cell marker Siglec-15, synergistically activating T cells and evoking an immune response, thereby significantly enhancing their efficiency in killing tumor cells.

[0343] Therefore, the present invention also provides a method for stimulating a T-cell-mediated immune response to a target cell population or tissue in a mammal, comprising the step of administering the CAR-T cells of the present invention to a mammal.

[0344] In one embodiment, the present invention includes a type of cell therapy in which the patient's own T cells (or those from a heterologous donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then injected into the same patient. This method results in an extremely low probability of graft-versus-host disease, and the antigen is recognized by the T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-lasting, durable antibodies that lead to sustained tumor control.

[0345] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and sustain for an extended period of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein CAR-modified T cells induce an immune response specific to the antigen-binding domain in the CAR. For example, anti-Siglec-15 CAR-T cells elicit a specific immune response against cells expressing Siglec-15.

[0346] Although the data disclosed herein specifically disclose lentiviral vectors including anti-Siglec-15 scFv, hinge and transmembrane regions, and CD28 and / or 4-1BB; and CD3ζ signaling domains, the invention should be construed as including any number of variations in each of the construct components.

[0347] Treatable cancers include tumors that are not vascularized or are substantially not vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematologic malignancies, such as leukemia and lymphoma) or may include solid tumors. Types of cancers treatable with the CAR of this invention include, but are not limited to, carcinomas, germ cell tumors, and sarcomas, and certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included.

[0348] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or blood-borne) cancers include leukemia, including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and spinal dysplasia.

[0349] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcoma and carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoma, pancreatic cancer, and ovarian cancer.

[0350] The CAR-modified T cells of the present invention can also be used as a type of vaccine for in vitro immunization and / or in vivo therapy in mammals. Preferably, the mammal is human.

[0351] For in vitro immunization, at least one of the following occurs in vitro before the cells are administered into a mammal: i) cell expansion, ii) introduction of nucleic acid encoding CAR into the cells, and / or iii) cryopreservation of the cells.

[0352] In vitro procedures are well known in the art and are discussed more fully below. Simply put, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) using a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefit. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0353] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against antigens in a patient.

[0354] The present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need of the treatment.

[0355] The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with diluents and / or other components such as IL-2, IL-17, or other cytokines or other genetically engineered or cell populations. In short, the pharmaceutical compositions of the present invention may comprise target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0356] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the treatment (or prevention) of a disease. The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease—although the appropriate dosage can be determined by clinical trials.

[0357] When referring to "immunologically effective amount," "antitumor effective amount," "tumor-suppressive effective amount," or "therapeutic amount," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. It can generally be indicated that a pharmaceutical composition including T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6 The T-cell composition can be administered at a dose of cells per kg of body weight (including all integer values ​​within those ranges). These doses can also be administered multiple times. The cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a physician skilled in the medical field by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

[0358] The application of the target composition can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (iv), or intracavitarily (e.g., intraperitoneally). In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or metastatic site.

[0359] In some embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with any number of relevant treatment modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or nastatinumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an injection of the expanded immune cells of the present invention after transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0360] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 6 One to 1×10 10 The modified T cells of this invention (e.g., CAR-T cells) are administered to a patient via, for example, intravenous infusion.

[0361] Hematologic malignancies Hematologic malignancies mainly refer to tumors of hematopoietic and lymphoid tissues. Based on cell lineage, they can be classified into four types: myeloid tumors, lymphoid tumors, histiocytic tumors, and mast cell tumors. Myeloid tumors include acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloproliferative disorder (CMPD), and myelodysplastic / chronic myeloproliferative disorder (MD / CMPD). Lymphoid tumors include acute lymphoblastic leukemia, Burkitt lymphoma / burkitt lymphocytic leukemia, pre-ALL, chronic lymphocytic leukemia / small cell lymphoma (CLL / SLL), hairy cell leukemia (HCL), plasma cell myeloma (multiple myeloma / plasmacytoma MM), large granular lymphocytic leukemia (GLL), Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), and lymphoma cell leukemia.

[0362] sample As used herein, the term "sample" or "sample" refers to material specifically associated with a subject from which specific information relating to the subject can be determined, calculated, or inferred. A sample may consist wholly or partially of biological material from the subject. A sample may also be material that has been in contact with the subject in a manner that allows testing of the sample to provide information relating to the subject. A sample may also be material that has been in contact with other materials, not belonging to the subject, but which enable subsequent testing of the first material to determine information relating to the subject; for example, a sample may be a cleaning solution for a probe or scalpel. A sample may be a source of biological material other than that in contact with the subject, as long as those skilled in the art can still determine information relating to the subject from the sample.

[0363] Express As used herein, the term "expression" includes the production of mRNA from a gene or gene segment, and includes the production of proteins encoded by RNA or a gene or gene segment, as well as the appearance of detection substances associated with expression. For example, the binding of cDNA, ligand-binding ligands (such as antibodies) to genes or other oligonucleotides, proteins, or protein fragments, and the chromogenic portion of the ligand-binding ligand are all included within the scope of the term "expression." Therefore, an increase in the density of the upper half-spot in immunoblotting such as Western blotting also falls within the scope of the biologically molecular-based term "expression."

[0364] Reference value As used herein, the term "reference value" refers to a value that is statistically relevant to a particular outcome when compared with the results of an analysis. In a preferred embodiment, the reference value is determined based on a statistical analysis of studies comparing siglec15 expression with known clinical outcomes. Some such studies are shown in the Examples section of this document. However, studies from the literature and user experience with the methods disclosed herein can also be used to produce or adjust reference values. Reference values ​​can also be determined by considering circumstances and outcomes that are particularly relevant to the patient's medical history, genetics, age, and other factors.

[0365] In this invention, the reference value refers to the cut-off value, which refers to the relative expression level of siglec15 in hematologic tumor cells or tissues, preferably a relative expression level of 2 (quantitative real-time PCR).

[0366] Non-hematologic malignancies As used herein, the term “non-hematologic malignancy sample” includes, but is not limited to, individuals who do not have hematologic malignancies and non-hematologic malignancy tissue from patients with hematologic malignancies.

[0367] siglec15 protein and polynucleotides In this invention, the terms "inventive protein," "siglec15 protein," and "siglec15 polypeptide" are used interchangeably and all refer to proteins or polypeptides having the siglec15 amino acid sequence. These include siglec15 proteins with or without a starting methionine. Furthermore, the term also includes the full-length siglec15 and fragments thereof. The siglec15 protein referred to in this invention includes its complete amino acid sequence, its secreted protein, its mutants, and its functionally active fragments.

[0368] The sialic acid-binding Ig-like lectins family, or Siglecs family, is a classic family of immunoglobulin-like lectin proteins. Under physiological conditions, only 15 Siglec molecules are expressed on the surface of myeloid cells and immune cells that mediate immunosuppression, thus possessing immunosuppressive properties. When Siglecs recognize sialic acid-containing glycans expressed by all mammalian cells, they can help immune cells distinguish between self and non-self. Sialidized pathogens can also disrupt or promote immune responses, or downregulate immune cell responses and evade immune surveillance, through Siglec-dependent interactions. Siglecs play a crucial role in the regulation of immune cell activation and inhibition receptors, influencing host-pathogen interactions, neurodegeneration, osteoclast differentiation in autoimmune diseases, and cancer. Due to their restricted expression on immune cells, endocytic properties, and ability to regulate receptor signaling, they have become important targets for cell-directed therapy.

[0369] Siglec-15, a cell surface transmembrane receptor, consists of an immunoglobulin (Ig)-like domain, a transmembrane domain, and a short cytoplasmic tail. The immunoglobulin-like domain comprises two extracellular Ig domains: an N-terminal V-set domain containing a sialic acid binding site and a type II constant region (IgC2). It associates with the tyrosine-containing activation motif (ITAM) adapter molecule DAP12 via positively charged amino acids in its transmembrane domain and activates the receptor by recruiting SYK kinases.

[0370] Siglec-15 differs from other family members. It exhibits similar domain components and high homology to B7-H1 and many other B7 family members. More importantly, the expression of Siglec-15 and B7-H1 in human lung cancer tissues is mutually exclusive. Therefore, in addition to its immunomodulatory functions, Siglec-15 may possess a unique regulatory mechanism: besides binding to TCR and CD44, Siglec-15 has not been found to bind to other receptors and does not interact with B7-H1, PD-1, B7-1, or any other known B7 family ligands or receptors.

[0371] Siglec-15 is expressed in various human tumor tissues on tumor-associated stromal cells or tumor cells, as well as tumor-infiltrating macrophages / myeloid cells. Siglec-15 gene knockout does not induce autoimmune diseases or other symptoms in mice. Macrophage / myeloid-derived Siglec-15 can suppress antigen-specific T cell immune responses by regulating cell growth. In various mouse tumor models, the anti-Siglec-15 monoclonal antibody α-S15 can block the immunosuppressive effect of macrophage / myeloid-derived Siglec-15, thereby enhancing in vivo anti-tumor immune responses.

[0372] The human Siglec-15 protein is 328 amino acids long (accession number NP_998767.1). The mouse Siglec-15 protein has accession number NP_001094508.1.

[0373] In this invention, the terms "Siglec-15 gene" and "Siglec-15 polynucleotide" are used interchangeably and both refer to nucleic acid sequences having the Siglec-15 nucleotide sequence.

[0374] The full-length genome of the human Siglec-15 gene is 18420 bp (NCBI GenBank accession number, Gene ID: NC_000018.10), and the full-length mRNA sequence of its transcription product is 2948 bp (CDS 987 bp) (NCBI GenBank accession number: NM_213602.3).

[0375] The full-length genome of the mouse Siglec-15 gene is 14949 bp (NCBI GenBank accession number, Gene ID: NC_000084.7), and the full-length mRNA sequence of its transcription product is 2323 bp (CDS 1026 bp) (NCBI GenBank accession number: NM_001101038.2).

[0376] The human and mouse Siglec-15 protein sequence shares 82% similarity.

[0377] It should be understood that nucleotide substitutions in a codon are acceptable when encoding the same amino acid. Furthermore, it should be understood that nucleotide substitutions are also acceptable when they result in conserved amino acid substitutions.

[0378] Once the amino acid fragment of Siglec-15 is obtained, the nucleic acid sequence encoding it can be constructed, and specific probes can be designed based on the nucleotide sequence. The full-length nucleotide sequence or its fragments can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the Siglec-15 nucleotide sequence disclosed in this invention, especially the open reading frame sequence, and the relevant sequence can be amplified using a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0379] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0380] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0381] Currently, the DNA sequence encoding the protein of this invention (or its fragments, derivatives) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0382] Using conventional recombinant DNA techniques, the polynucleotide sequence of this invention can be used to express or produce recombinant Siglec-15 peptides. Generally, the following steps are involved: (1). Transform or transduce suitable host cells with the polynucleotide (or variant) encoding the human Siglec-15 polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide; (2) Host cells cultured in a suitable culture medium; (3) Isolate and purify proteins from culture media or cells.

[0383] In this invention, the Siglec-15 polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0384] Methods well known to those skilled in the art can be used to construct expression vectors containing a Siglec-15 coding DNA sequence and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0385] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0386] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0387] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces bacteria; fungal cells such as yeast; plant cells; insect cells; and animal cells.

[0388] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0389] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0390] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0391] Specific antibodies In this invention, the terms "antibody of the present invention" and "specific antibody against Siglec-15" are used interchangeably.

[0392] This invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, that are specific to the human Siglec-15 polypeptide. Here, "specificity" means that the antibody can bind to the human Siglec-15 gene product or fragment. Preferably, it refers to antibodies that can bind to the human Siglec-15 gene product or fragment but do not recognize or bind to other unrelated antigen molecules. Antibodies in this invention include molecules that can bind to and inhibit the human Siglec-15 protein, as well as antibodies that do not affect the function of the human Siglec-15 protein. This invention also includes antibodies that can bind to modified or unmodified forms of the human Siglec-15 gene product.

[0393] This invention includes not only complete monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab)2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules (Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have mouse antibody binding specificity but still retain the antibody portion derived from humans.

[0394] The antibodies of this invention can be prepared using various techniques known to those skilled in the art. For example, purified human Siglec-15 gene product or its antigenic fragment can be administered to animals to induce the production of polyclonal antibodies. Similarly, cells expressing human Siglec-15 protein or its antigenic fragment can be used to immunize animals to produce antibodies. The antibodies of this invention can also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al.). Nature 256;495, 1975; Kohler et al., Eur.J.Immunol. 6:511, 1976; Kohler et al., Eur.J.Immunol 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas (Elsevier, NY, 1981). The antibodies of this invention include antibodies that block the function of human Siglec-15 protein and antibodies that do not affect the function of human Siglec-15 protein. The various antibodies of this invention can be obtained using fragments or functional regions of the human Siglec-15 gene product through conventional immunoassay techniques. These fragments or functional regions can be prepared using recombinant methods or synthesized using a peptide synthesizer. Antibodies that bind to the unmodified form of the human Siglec-15 gene product can be used with prokaryotic cells (e.g., Elsevier, NY, 1981). E. Coli Gene products produced in eukaryotic cells (e.g., yeast or insect cells) are used to immunize animals; antibodies that bind to post-translational modifications (such as glycosylated or phosphorylated proteins or peptides) can be obtained by immunizing animals with gene products produced in eukaryotic cells (e.g., yeast or insect cells).

[0395] Antibodies against human Siglec-15 protein can be used in immunohistochemistry to detect human Siglec-15 protein in specimens (especially tissue or cell samples).

[0396] Detection methods Utilizing the presence of Siglec-15 in the cells or tissues of hematologic malignancies, this invention also provides a method for detecting hematologic malignancies.

[0397] In a preferred embodiment of the present invention, the present invention provides a high-throughput next-generation sequencing method for detecting Siglec-15, as well as Sanger sequencing, quantitative PCR (qPCR), in situ immunofluorescence (FISH), and immunohistochemistry.

[0398] Test kit Based on the correlation between Siglec-15 and hematologic malignancies, namely that Siglec-15 is present in the cells or tissues of hematologic malignancies, Siglec-15 can serve as a diagnostic biomarker for hematologic malignancies.

[0399] The present invention also provides a kit for detecting hematologic malignancies, comprising a detection reagent for detecting the Siglec-15 gene, mRNA, cDNA, or protein; and a label or instruction manual indicating that the kit is used for detecting hematologic malignancies. Preferably, the container further comprises one or more detection reagents selected from the group consisting of: HLA-DR, CD38, CD117, MPO, TIM3, CD70, CD371, CD33 gene, mRNA, cDNA, or protein detection reagents.

[0400] The label or instruction manual shall specify the following: (i) When the ratio of Siglec-15 expression level E1 in cells or tissues of hematologic malignancies to Siglec-15 expression level E2 in normal cells or tissues is ≥2, it suggests that the probability of the subject having hematologic malignancies is higher than that of the general population. Wherein, E2 refers to the expression level of Siglec-15 in general cells or tissues of the general population; (ii) If the co-expression of Siglec-15 with one or more of HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371 in the cells or tissues (such as bone marrow samples) of the subject being tested is greater than 30%, it suggests that the subject being tested has a higher probability of having a hematologic malignancy than the general population.

[0401] Detection methods and kits This invention relates to diagnostic assays for the quantitative and localization detection of protein or mRNA levels of human Siglec-15 and / or one or more of HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371. These assays are well known in the art. The detection of protein levels of human Siglec-15 and / or one or more of HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371 in these assays can be used for the diagnosis (including as an adjunct to the diagnosis) of hematologic malignancies.

[0402] One method for detecting the presence of Siglec-15 protein in a sample is to use a specific antibody against Siglec-15 protein. The method includes: contacting the sample with the specific antibody against Siglec-15 protein; observing whether an antibody complex is formed. The formation of an antibody complex indicates the presence of Siglec-15 protein in the sample.

[0403] Siglec-15s protein or its polynucleotides can be used for the diagnosis and treatment of Siglec-15 protein-related diseases. A portion or all of the polynucleotides of this invention can be immobilized as probes on microarrays or DNA chips for differential gene expression analysis and gene diagnosis in tissues. Anti-Siglec-15 antibodies can be immobilized on protein chips for detecting Siglec-15 protein in samples. In a preferred embodiment, Siglec-15 protein or its polynucleotides can be combined with one or more proteins or their polynucleotides comprising, but not limited to, HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371 for the diagnosis and treatment of Siglec-15 protein-related diseases. A portion or all of the polynucleotides of this invention can be immobilized as probes on microarrays or DNA chips for differential gene expression analysis and gene diagnosis in tissues. Antibodies against Siglec-15 or containing, but not limited to, one or more of the following proteins: HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371 can be immobilized on a protein chip for the detection of Siglec-15 protein or containing, but not limited to, one or more of the following proteins: HLA-DR, CD38, CD117, MPO, TIM3, CD70, and CD371 in a sample.

[0404] The main advantages of this invention include: (1) The antibody of the present invention has the characteristics of high affinity and high specificity.

[0405] (2) The humanized antibody or scFv of the present invention still has high affinity and high specificity for Siglec-15.

[0406] (3) The scFv of the present invention has high affinity and good specificity for Siglec-15 and can specifically target Siglec-15, especially the full-length antigen and extracellular region of Siglec-15.

[0407] (4) The chimeric antigen receptor targeting siglec-15 prepared in this invention can effectively kill tumor cells, such as AML tumor cells, in vitro and in vivo, showing a good anti-tumor effect.

[0408] (5) This invention is the first to discover that the Siglec-15 (S15) antigen constructed in vitro and the S15 antigen expressed on AML cells have structural differences: the antibodies generated by immunization with the constructed S15 antigen can bind to the constructed high-expression strain in part, bind to the AML cell strain in part, and bind to both in part. This phenomenon has rarely occurred in previous antibody production.

[0409] (6) This invention is the first to discover that the expression of the Siglec-15 gene or its protein in cells or tissues of hematologic malignancies is significantly higher than that in normal tissues. Therefore, the Siglec-15 gene or its protein can be used to detect hematologic malignancies.

[0410] (7) This invention is the first to discover that inhibitors of the Siglec-15 gene or its protein can effectively (a) inhibit the growth or proliferation of hematologic malignancies; and / or (b) prevent and / or treat hematologic malignancies.

[0411] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0412] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available products.

[0413] The sequence of elements in the CDR of the heavy chain variable region, the CDR of the light chain variable region, the heavy chain variable region, the light chain variable region, and the CAR structure is shown in Tables 1-5.

[0414] Table 1. Complementarity-determining region (CDR) sequences in the variable region of heavy chains Example 1: Immunization of mice Female Balb / c mice aged 8-10 weeks were subcutaneously immunized (sc) at multiple sites with a mixture of 100 μL of a fusion protein containing 50 μg of human siglec15 and mouse Fc (hSiglec15-mFc) and complete Freund's adjuvant (CFA) (Sigma-Aldrich). Two weeks later, the mice were reimmunized with 50-100 μg of the fusion protein and incomplete Freund's adjuvant (IFA) (Sigma-Aldrich), and this process was repeated every two weeks for a total of three immunizations. Serum titers were measured two weeks after each immunization. When the titer was sufficient for fusion, the mice were further immunized via intraperitoneal injection (ip) of PBS containing 60 μg of the fusion protein.

[0415] Example 2 Antibody Screening Hybridoma cells were obtained by fusing immunized mouse spleen cells and SP2 / 0-Ag14 myeloma cells (from ATCC): The immunized mice were euthanized with carbon dioxide, and the spleens were aseptically harvested. The entire spleen was dissociated into a single-cell suspension and erythrocytes were lysed using ACK buffer (from LONZA). SP2 / 0-Ag14 myeloma cells and spleen cells were mixed in a 1:1 ratio in 50 ml conical centrifuge tubes. After centrifugation, the supernatant was discarded, and 50% polyethylene glycol (PEG, Roche) was added for cell fusion. The fused cells were cultured in HAT selective medium for 8–10 days. Hybridoma cells that produced antibodies binding to hSiglec15-expressing cells were screened using enzyme-linked immunosorbent assay (ELISA), and the results were verified by flow cytometry. Four positive clones were selected, numbered 128, 413, 216, and 304, which could bind to the hSiglec15 protein expressed on CHO cells. Then, limiting dilution techniques were used to subclone positive hybridomas to obtain pure monoclonal strains.

[0416] Example 3: Specificity of anti-siglec15 antibody binding to human and mouse S15 proteins DNA fragments of human and mouse Siglec15 were obtained using PCR to construct a plasmid for high expression of Siglec15 (S15). The constructed plasmid was used for lentiviral vector packaging, and the lentiviral vector was harvested and concentrated by ultrafiltration. The obtained lentiviral vector was used to transduce CHO cells, obtaining CHO engineered cells stably expressing human Siglec15 (CHO / hS15) and mouse Siglec15 (CHO / mS15). Flow cytometry staining was performed on both cell types using 200 ng of the aforementioned 128, 413, and 304 antibodies, respectively. The cells were incubated at 4°C in the dark for 30 min, washed twice with PBS, and then 1 μL of APC anti-mouse IgG (Biolegend) was added. The cells were incubated at 4°C in the dark for 30 min, washed twice with PBS, and the binding ability of the three antibodies to human and mouse Siglec15 protein was detected by flow cytometry.

[0417] The results are as follows Figure 1 As shown in the figure. The results indicate that antibody 128 binds only to human siglec15; antibodies 413 and 304 can bind to both human and mouse siglec15 proteins.

[0418] Example 4: Verification of the binding ability of anti-siglec15 antibody to acute myeloid leukemia (AML) tumor cells. The DNA fragment of human Siglec15 was obtained by PCR and used to construct a plasmid for high expression of S15. The constructed plasmid was used for packaging a lentiviral vector, which was then harvested and concentrated by ultrafiltration. The obtained lentiviral vector was used to transduce Nalm6 cells to obtain Nalm6 cells stably expressing human Siglec15 (Nalm6-S15). Nalm6-S15 cell lines and acute myeloid leukemia (AML) tumor cell line U937 were stained with antibodies 128, 304, 413, and 216, incubated at 4°C in the dark for 30 min, and washed twice with PBS. 1 μL of PE anti-mouse IgG (Biolegend) was added, and the cells were incubated at 4°C in the dark for 30 min. After washing twice with PBS, the binding ability of the above antibodies to the AML cell line was analyzed by flow cytometry.

[0419] The results are as follows Figure 2 As shown, antibodies 128 and 413 can only bind to the Nalm6-S15 cell line; antibodies 304 and 216 can bind to both the Nalm6-S15 cell line and the U937 AML tumor cell line. This indicates that antibodies 216 and 304 can specifically recognize and bind to the Siglec15 protein antigen on tumor cells, with 304 showing a relatively stronger binding ability. Therefore, 304 was chosen for later experiments.

[0420] Example 5: Expression of siglec15 in different tumor cells Flow cytometry was used to stain hematologic malignancy-nonAML cell lines Jurkat, Nalm6, and K562, and AML cell lines Kasumi-1, THP1, U937, and HL60 with 304 antibody. The staining method was as follows: 1-5 × 10⁻⁶ cells were used. 5 Cultured tumor cells were washed twice with 2 ml of PBS, then incubated with 200 ng of 304 antibody at 4°C in the dark for 30 min. After washing twice with 2 ml of PBS, 1 μL of anti-mouse IgG (PE-labeled, Biolegend) was added and incubated at 4°C in the dark for 30 min. After washing twice with PBS, the cells were analyzed by flow cytometry to verify the binding ability of the 304 antibody to different tumor cells.

[0421] The results are as follows Figure 3 As shown, antibody 304 did not bind to Jurkat, Nalm6, and K562 cells, suggesting that these three non-AML cell lines do not express Siglec15; while antibody 304 could bind to AML cells Kasumi-1, THP1, U937, and HL60, indicating that there is extensive expression of Siglec15 protein in AML tumor cell lines, and antibody 304 has an effective binding ability to this protein.

[0422] Example 6: Expression of siglec15 in tumor cells of AML patients Flow cytometry staining of AML patient tumor cells with 304 antibody: Clinically obtained AML bone marrow samples were centrifuged at 500g for 5 min, the supernatant was discarded, and 3-5 volumes of ACK lysing buffer (Lonza) were added for erythrocyte lysis. The samples were incubated at room temperature for 3-5 min. After washing with PBS containing 2% FBS, the samples were centrifuged at 500g for 5 min and washed twice. 200 ng of 304 antibody was added and incubated at 4℃ in the dark for 30 min, followed by washing twice with PBS. 1 μL of anti-mouse IgG (APC-labeled, Biolegend) was added and incubated at 4℃ in the dark for 30 min, followed by washing twice with PBS. CD45 (V500-labeled, Biolegend) and CD371 antibody (PE-labeled, Biolegend) were added and incubated at 4℃ in the dark for 30 min, followed by washing twice with PBS. Flow cytometry was used to detect and analyze the samples to verify the binding ability of 304 antibody to tumor cells in bone marrow samples from clinical AML patients.

[0423] Figure 4The results show the flow cytometry results of tumor cells from three patients with acute myeloid leukemia (AML). The results show that the 304 antibody can bind to most of the tumor cells in the AML patient samples, indicating that AML tumor cells have high expression of Siglec15 antigen, and that the 304 antibody can specifically bind to Siglec15 on AML tumor cells.

[0424] Example 7: Expression of siglec15 in normal human bone marrow samples Two randomly selected discarded bone marrow samples from healthy individuals were collected, 1 ml each. Sample processing was as follows: centrifugation at 500g for 5 min, discarding the supernatant, adding 3-5 volumes of ACK lysing buffer (Lonza) for erythrocyte lysis, and incubating at room temperature for 3-5 min; washing twice with PBS containing 2% FBS; adding 200 ng of 304 antibody and incubating at 4°C in the dark for 30 min; washing twice with PBS, adding 1 μL of anti-mouse IgG (APC-labeled, Biolegend), and incubating at 4°C in the dark for 30 min; washing twice with PBS, adding 1 μL of CD34 antibody (Percp-cy5.5-labeled, Biolegend), and incubating at 4°C in the dark for 30 min; washing twice with PBS, and analyzing by flow cytometry. The expression of siglec15 in stem cells in the bone marrow samples was verified.

[0425] like Figure 5 The results of the test are shown in two bone marrow samples from healthy individuals. Both samples contained CD34+ stem cells. Analysis of Siglec15 expression in the cell population showed that Siglec15 positive cells accounted for no more than 10% of the stem cell population, indicating that healthy bone marrow stem cells only express a small amount of Siglec15 protein, suggesting the safety of Siglec15 as a target.

[0426] Example 8: In vitro killing and cytokine secretion The single-chain antibody fragment (scFv) of the 304 antibody, the human CD8α hinge region and transmembrane region, the human 41BB intracellular region or CD28 intracellular region gene, and the human CD3ζ intracellular region gene sequence were ligated using overlap PCR to form a complete S15-CAR gene vector. A control CAR-T cell gene vector (control CAR-T, TX103 CAR-T) was constructed using the same method. CAR lentiviral vectors were prepared using a third-generation 4-plasmid packaging system, and the prepared lentiviruses were then ultra-concentrated.

[0427] Healthy human PBMCs were activated with CD3 / CD28 activator. After 18-24 hours of activation, lentiviral vectors were added for transduction. Transduced cells were cultured until day 9, and Control CAR-T and S15 CAR-T cells were harvested. These two types of CAR-T cells were co-incubated with Nalm6-S15 cells overexpressing S15 and U937 AML cell line naturally expressing S15 for in vitro killing and cytokine secretion assays. Tumor cells were washed twice with calcium- and magnesium-free PBS; 2×10⁻⁶ cells were used for transduction. 6 Resuspend cells at a density of 1 / ml; add 5mM CFSE (Invitrogen) to a final concentration of 5uM, incubate at 37℃ for 7 min; terminate incubation by adding basal culture medium containing 10% FBS; after centrifugation, adjust the tumor cell concentration to 5×10⁻⁶ cells / ml with complete culture medium. 5 / ml; After CAR-T cell counting, adjust the cell count to 5×10⁶. 6 CAR + Cells were added to 96-well plates at CAR-T cell:tumor cell (E:T) ratios of 10:1, 5:1, 2.5:1, 1.25:1, and 0.625:1, with three replicates for each ratio and a final volume of 200 μL per well. After incubation at 37°C in the dark for 12 hours, 15-20 μL was aspirated from each well for cytokine detection (BD, CBA Kit). The incubated cells were then collected into flow cytometry tubes, and 10 μL of 5 μg / ml DAPI was added to each tube before analysis to label dead cells. The cells were then analyzed using flow cytometry.

[0428] The results are as follows Figure 6 As shown: 304 S15-CAR-T showed no killing ability against S15-negative Nalm6 cell lines and low IFN-γ secretion levels; 304 S15-CAR-T showed higher in vitro cell-killing levels and IFN-γ secretion against Nalm6-S15 cells overexpressing S15; compared with control CAR-T, 304 S15-CAR-T showed stronger killing levels and higher IFN-γ secretion against U937 cell lines. This indicates that 304 S15-CAR-T has specific S15-targeting killing ability in in vitro experiments and also has a killing effect on AML cell lines naturally expressing S15.

[0429] Example 9: S15 CAR-T in vivo efficacy test Animal experiments were conducted using the above-mentioned S15 CAR-T cells in a mouse tumor model: 6-8 week old NCG immunodeficient mice (Jiangsu Jicui Biotechnology Co., Ltd.) were purchased, and after 1 week of quarantine, the mice were injected via tail vein with 5×10⁻⁶ CAR-T cells. 4Nalm6-S15-Luc (firefly luciferase) tumor cells; 4 days after tumor inoculation, mice were weighed and intraperitoneally injected with D-Luciferase substrate at a dose of 10ul / 10g. Ten minutes later, mice were anesthetized with isoflurane and placed in a small animal in vivo imaging system to monitor tumor size. Mice were ranked according to their Total Flux values ​​from the imaging results and then randomly divided into 3 groups of 5 mice each. On the same day, each group received PBS, 1×10⁻⁶ mmol / L, or 1000 mg / L of D-Luciferase substrate. 7 CAR-TS15 CAR-T or Control CAR-T were administered via tail vein injection, with a treatment volume of 200 μL per animal. The day of treatment was marked as D0. Tumor size was continuously monitored by in vivo imaging of the small animals on days 6 and 9 post-treatment, following the same method.

[0430] The results are as follows Figure 7 As shown, tumors in mice treated with Control CAR-T cells grew rapidly, spreading throughout the body by day 6, and showing no significant difference from the PBS group by day 9. In the S15 CAR-T group, tumor cell growth was significantly inhibited compared to the Control CAR-T group on days 6 and 9 after tumor injection. This indicates that S15 CAR-T cells can significantly inhibit the growth of S15-positive tumor cells in mice.

[0431] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Fuzhou Tuoxin Tiancheng Biotechnology Co., Ltd. <120> Chimeric antigen receptors targeting siglec-15 and their applications <130> P2020-0703 <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <400> 1 Gly Tyr Thr Phe Thr Ala Tyr Asn 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <400> 2 Gly Tyr Thr Phe Thr Asn Tyr Val 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial sequence <400> 3 Gly Tyr Ser Phe Thr Asp Tyr Thr 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial sequence <400> 4 Gly Tyr Thr Phe Thr Asn His His 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial sequence <400> 5 Ile Tyr Pro Tyr Ser Gly Gly Thr 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <400> 6 Ile Asn Pro Tyr Asn Asp Val Ile 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial sequence <400> 7 Ile Asn Pro Tyr Asn Gly Gly Thr 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial sequence <400> 8 Ile Asn Pro Tyr Asn Asp Tyr Thr 1 5 <210> 9 <211> 11 <212> PRT <213> Artificial sequence <400> 9 Ala Arg Tyr Phe Asp Tyr Gly Gly Phe Ala Phe 1 5 10 <210> 10 <211> 12 <212> PRT <213> Artificial sequence <400> 10 Ala Lys Glu Gly Tyr Asp Asn Asp Pro Leu Asp Val 1 5 10 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <400> 11 Ala Arg Phe Asp Tyr Phe Asp Tyr 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial sequence <400> 12 Ala Arg Arg Tyr Ala Asn Tyr Leu Asp Tyr 1 5 10 <210> 13 <211> 5 <212> PRT <213> Artificial sequence <400> 13 Ser Ser Val Asn Tyr 1 5 <210> 14 <211> 12 <212> PRT <213> Artificial sequence <400> 14 Gln Ser Leu Leu Trp Ser Val Asn Gln Asn Asn Tyr 1 5 10 <210> 15 <211> 6 <212> PRT <213> Artificial sequence <400> 15 Gln Asp Ile Val Lys Asn 1 5 <210> 16 <211> 5 <212> PRT <213> Artificial sequence <400> 16 Ser Ser Val Asn Tyr 1 5 <210> 17 <211> 3 <212> PRT <213> Artificial sequence <400> 17 Tyr Thr Ser 1 <210> 18 <211> 3 <212> PRT <213> Artificial sequence <400> 18 Gly Ala Ser 1 <210> 19 <211> 3 <212> PRT <213> Artificial sequence <400> 19 Tyr Ala Thr 1 <210> 20 <211> 3 <212> PRT <213> Artificial sequence <400> 20 Tyr Thr Ser 1 <210> twenty one <211> 9 <212> PRT <213> Artificial sequence <400> twenty one Gln Gln Phe Thr Ser Ser Pro Trp Thr 1 5 <210> twenty two <211> 11 <212> PRT <213> Artificial sequence <400> twenty two Gln His Asn His Gly Ser Phe Leu Pro Tyr Thr 1 5 10 <210> twenty three <211> 8 <212> PRT <213> Artificial Sequence <400> 23 Leu Gln Phe Tyr Glu Phe Pro Thr 1 5 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <400> 24 Gln Gln Phe Thr Ser Ser Pro Phe Thr 1 5 <210> 25 <211> 118 <212> PRT <213> Artificial Sequence <400> 25 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30 Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Ser Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Ser Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Asp Tyr Gly Gly Phe Ala Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 26 <211> 119 <212> PRT <213> Artificial sequence <400> 26 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Thr Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Tyr Asp Asn Asp Pro Leu Asp Val Trp Gly Ala Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 27 <211> 115 <212> PRT <213> Artificial Sequence <400> 27 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Ile Leu Thr 100 105 110 Val Ser Ser 115 <210> 28 <211> 117 <212> PRT <213> Artificial sequence <400> 28 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Phe Gly Tyr Thr Phe Thr Asn His 20 25 30 Gly Tyr Ile Asn Pro Tyr Asn Asp Tyr Thr Ser Tyr Asn Gln Lys Phe 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Tyr Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys 85 90 95 Ala Arg Arg Tyr Ala Asn Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 29 <211> 106 <212> PRT <213> Artificial sequence <400> 29 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Ser Asp Ala Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Tyr Thr Ser Asn Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Gly Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 30 <211> 116 <212> PRT <213> artificial sequence <400> 30 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Arg Cys Lys Ser Ser Gln Ser Leu Leu Trp Ser 20 25 30 Val Asn Gln Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Gln Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Ile Arg Glu Ser Trp Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Asn Val His Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln His 85 90 95 Asn His Gly Ser Phe Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu 100 105 110 Glu Ile Lys Arg 115 <210> 31 <211> 107 <212> PRT <213> Artificial sequence <400> 31 Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val Lys Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Ser Phe Leu Ile 35 40 45 Tyr Tyr Ala Thr Glu Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe Pro Thr 85 90 95 Phe Gly Gly Gly Thr Thr Leu Glu Ile Lys Arg 100 105 <210> 32 <211> 106 <212> PRT <213> Artificial sequence <400> 32 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Arg Ala Ile Ser Ser Val Asn Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Ser Asp Ala Ser Pro Lys Leu Trp Ile His 35 40 45 Tyr Thr Ser Asn Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Gly Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 33 <211> 239 <212> PRT <213> Artificial Sequence <400> 33 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30 Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Ser Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Ser Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Asp Tyr Gly Gly Phe Ala Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met 130 135 140 Ser Ala Ser Leu Gly Glu Lys Val Thr Met Ser Cys Arg Ala Ser Ser 145 150 155 160 Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln Lys Ser Asp Ala Ser Pro 165 170 175 Lys Leu Trp Ile Tyr Tyr Thr Ser Asn Leu Ala Pro Gly Val Pro Ala 180 185 190 Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser 195 200 205 Ser Met Glu Gly Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr 210 215 220 Ser Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 225 230 235 <210> 34 <211> 250 <212> PRT <213> Artificial sequence <400> 34 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Thr Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Tyr Asp Asn Asp Pro Leu Asp Val Trp Gly Ala Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ala Val Thr Ala Gly Glu Lys Val Thr Met Arg Cys Lys Ser Ser 145 150 155 160 Gln Ser Leu Leu Trp Ser Val Asn Gln Asn Asn Tyr Leu Ser Trp Tyr 165 170 175 Gln Gln Lys Gln Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser 180 185 190 Ile Arg Glu Ser Trp Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Asn Val His Ala Glu Asp Leu Ala 210 215 220 Val Tyr Tyr Cys Gln His Asn His Gly Ser Phe Leu Pro Tyr Thr Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 245 250 <210> 35 <211> 237 <212> PRT <213> Artificial Sequence <400> 35 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Ile Leu Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser 130 135 140 Leu Gly Asp Arg Ile Thr Ile Thr Cys Gln Ala Thr Gln Asp Ile Val 145 150 155 160 Lys Asn Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Ser Phe 165 170 175 Leu Ile Tyr Tyr Ala Thr Glu Leu Ala Glu Gly Val Pro Ser Arg Phe 180 185 190 Ser Gly Ser Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Asn Leu 195 200 205 Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Phe Tyr Glu Phe 210 215 220 Pro Thr Phe Gly Gly Gly Thr Thr Leu Glu Ile Lys Arg 225 230 235 <210> 36 <211> 238 <212> PRT <213> Artificial sequence <400> 36 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Phe Gly Tyr Thr Phe Thr Asn His 20 25 30 His Ile Asp Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Tyr Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys 85 90 95 Ala Arg Arg Tyr Ala Asn Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser 130 135 140 Ala Ser Leu Gly Glu Lys Val Thr Met Ser Cys Arg Ala Ile Ser Ser 145 150 155 160 Val Asn Tyr Met Tyr Trp Tyr Gln Gln Lys Ser Asp Ala Ser Pro Lys 165 170 175 Leu Trp Ile His Tyr Thr Ser Asn Leu Ala Pro Gly Val Pro Ala Arg 180 185 190 Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser 195 200 205 Met Glu Gly Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser 210 215 220 Ser Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 225 230 235 <210> 37 <211> 21 <212> PRT <213> Artificial sequence <400> 37 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 38 <211> 45 <212> PRT <213> Artificial sequence <400> 38 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 39 <211> 24 <212> PRT <213> Artificial sequence <400> 39 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 40 <211> 42 <212> PRT <213> Artificial sequence <400> 40 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 41 <211> 112 <212> PRT <213> Artificial Sequence <400> 41 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 42 <211> 544 <212> DNA <213> singular sequence (artificial sequence) <400> 42 ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg 60 agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gccttttttcc cgagggtggg ggagaaccgt 180 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac 240 agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300 gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg 360 cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc 420 cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac 480 tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg tgaccggcgc 540 ctac 544 <210> 43 <211> 483 <212> PRT <213> singular sequence (artificial sequence) <400> 43 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu 20 25 30 Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Ala Tyr Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys 50 55 60 Ser Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Ser Gly Gly Thr Ala 65 70 75 80 Tyr Asn Gln Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Ser Ser 85 90 95 Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Tyr Phe Asp Tyr Gly Gly Phe Ala Phe 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asn Val Leu Thr Gln 145 150 155 160 Ser Pro Ala Ile Met Ser Ala Ser Leu Gly Glu Lys Val Thr Met Ser 165 170 175 Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln Lys 180 185 190 Ser Asp Ala Ser Pro Lys Leu Trp Ile Tyr Tyr Thr Ser Asn Leu Ala 195 200 205 Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr 210 215 220 Ser Leu Thr Ile Ser Ser Met Glu Gly Glu Asp Ala Ala Thr Tyr Tyr 225 230 235 240 Cys Gln Gln Phe Thr Ser Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys 245 250 255 Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 260 265 270 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg 275 280 285 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 290 295 300 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 305 310 315 320 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 325 330 335 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 340 345 350 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 355 360 365 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 370 375 380 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 385 390 395 400 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 405 410 415 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 420 425 430 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 435 440 445 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 450 455 460 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 465 470 475 480 Pro Pro Arg <210> 44 <211> 494 <212> PRT <213> artificial sequence <400> 44 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu 20 25 30 Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Asn Tyr Val Ile His Trp Val Lys Gln Lys Pro Gly Gln 50 55 60 Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Val Ile Lys 65 70 75 80 Tyr Asn Glu Lys Phe Thr Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser 85 90 95 Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Tyr Cys Ala Lys Glu Gly Tyr Asp Asn Asp Pro Leu Asp 115 120 125 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr 145 150 155 160 Gln Ser Pro Ser Ser Leu Ala Val Thr Ala Gly Glu Lys Val Thr Met 165 170 175 Arg Cys Lys Ser Ser Gln Ser Leu Leu Trp Ser Val Asn Gln Asn Asn 180 185 190 Tyr Leu Ser Trp Tyr Gln Gln Lys Gln Gly Gln Pro Pro Lys Leu Leu 195 200 205 Ile Tyr Gly Ala Ser Ile Arg Glu Ser Trp Val Pro Asp Arg Phe Thr 210 215 220 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val His 225 230 235 240 Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln His Asn His Gly Ser Phe 245 250 255 Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr 260 265 270 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 275 280 285 Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly 290 295 300 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 305 310 315 320 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 325 330 335 Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 340 345 350 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 355 360 365 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 370 375 380 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 385 390 395 400 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 405 410 415 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 420 425 430 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 435 440 445 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 450 455 460 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 465 470 475 480 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 45 <211> 481 <212> PRT <213> Artificial Sequence <400> 45 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu 20 25 30 Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Ser Phe Thr Asp Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys 50 55 60 Asn Leu Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Thr 65 70 75 80 Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser 85 90 95 Ser Asn Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Asp Asp Ser 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Phe Asp Tyr Phe Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Ile Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Glu Ile Gln Met Thr Gln Ser Pro Ser 145 150 155 160 Ser Met Ser Ala Ser Leu Gly Asp Arg Ile Thr Ile Thr Cys Gln Ala 165 170 175 Thr Gln Asp Ile Val Lys Asn Leu Asn Trp Tyr Gln Gln Lys Pro Gly 180 185 190 Lys Pro Pro Ser Phe Leu Ile Tyr Tyr Ala Thr Glu Leu Ala Glu Gly 195 200 205 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Tyr Ser Leu 210 215 220 Thr Ile Ser Asn Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu 225 230 235 240 Gln Phe Tyr Glu Phe Pro Thr Phe Gly Gly Gly Thr Thr Leu Glu Ile 245 250 255 Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 260 265 270 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala 275 280 285 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile 290 295 300 Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser 305 310 315 320 Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr 325 330 335 Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu 340 345 350 Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu 355 360 365 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 370 375 380 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 385 390 395 400 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 405 410 415 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 420 425 430 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 435 440 445 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 450 455 460 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 465 470 475 480 Arg <210> 46 <211> 482 <212> PRT <213> Artificial sequence <400> 46 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu 20 25 30 Val Arg Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Phe Gly Tyr 35 40 45 Thr Phe Thr Asn His His Ile Asp Trp Val Lys Gln Arg Pro Gly Gln 50 55 60 Gly Leu Asp Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Tyr Thr Ser 65 70 75 80 Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser 85 90 95 Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Phe Tyr Cys Ala Arg Arg Tyr Ala Asn Tyr Leu Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asn Val Leu Thr Gln Ser 145 150 155 160 Pro Ala Ile Met Ser Ala Ser Leu Gly Glu Lys Val Thr Met Ser Cys 165 170 175 Arg Ala Ile Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln Lys Ser 180 185 190 Asp Ala Ser Pro Lys Leu Trp Ile His Tyr Thr Ser Asn Leu Ala Pro 195 200 205 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser 210 215 220 Leu Thr Ile Ser Ser Met Glu Gly Glu Asp Ala Ala Thr Tyr Tyr Cys 225 230 235 240 Gln Gln Phe Thr Ser Ser Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu 245 250 255 Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 260 265 270 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro 275 280 285 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 290 295 300 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 305 310 315 320 Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu 325 330 335 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 340 345 350 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 355 360 365 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 370 375 380 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 385 390 395 400 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 405 410 415 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 420 425 430 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 435 440 445 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 450 455 460 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 465 470 475 480 Pro Arg

Claims

1. An antibody that binds to siglec-15, characterized in that, The antibody has a heavy chain variable region and a light chain variable region; The antibody heavy chain variable region includes the following three complementarity-determining regions (CDRs): CDR1 shown in SEQ ID NO:1 CDR2 shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:9; The light chain variable region includes the following three complementary determinant regions (CDRs): CDR1' shown in SEQ ID NO:13 CDR2' shown in SEQ ID NO:17, and CDR3' as shown in SEQ ID NO:

21.

2. The antibody as described in claim 1, characterized in that, The heavy chain variable region sequence of the antibody is shown in SEQ ID NO:25; and the light chain variable region sequence of the antibody is shown in SEQ ID NO:

29.

3. The antibody as described in claim 1, characterized in that, The antibody mentioned is of the IgG type.

4. An SCFV incorporating Siglec-15, characterized in that, The scFv contains a heavy chain variable region and an antibody light chain variable region; The antibody heavy chain variable region includes the following three complementarity-determining regions (CDRs): CDR1 shown in SEQ ID NO:1 CDR2 shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:9; The light chain variable region includes the following three complementary determinant regions (CDRs): CDR1' shown in SEQ ID NO:13 CDR2' shown in SEQ ID NO:17, and CDR3' as shown in SEQ ID NO:

21.

5. The scFv as described in claim 4, characterized in that, The scFv is shown in either formula A or formula B: V H -V L (A); or V L -V H (B) In the formula, V H V is the variable region of the antibody heavy chain; L " " represents the variable region of the antibody light chain; "-" represents a linking peptide or peptide bond.

6. The scFv as described in claim 4, characterized in that, The amino acid sequence of the scFv is shown in SEQ ID NO:

33.

7. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the antibody as claimed in claim 1 or the scFv as claimed in claim 4; and (ii) Tag sequences that assist in expression and / or purification.

8. A chimeric antigen receptor (CAR), characterized in that, The CAR includes the scFv as described in claim 4.

9. An engineered immune cell, characterized in that, The immune cells mentioned include: Expression cassette for expressing exogenous chimeric antigen receptors as described in claim 8.

10. An antibody conjugate, characterized in that, The antibody conjugate contains: (a) the antibody as claimed in claim 1 or the scFv as claimed in claim 4; and (b) A conjugation portion coupled to the antibody portion, wherein the conjugation portion is a detectable marker.

11. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody as described in claim 1, the scFv as described in claim 4, the recombinant protein as described in claim 7, the chimeric antigen receptor as described in claim 8, and the antibody conjugate as described in claim 10.

12. A carrier, characterized in that, The carrier contains the nucleic acid molecule as described in claim 11.

13. A host cell, characterized in that, The host cell contains the vector of claim 12 or the chromosome in which the exogenous nucleic acid molecule of claim 11 is integrated or expresses the chimeric antigen receptor of claim 8.

14. A method for preparing engineered immune cells, characterized in that, The engineered immune cells expressing the chimeric antigen receptor of claim 8 include the following steps: transducing the nucleic acid molecule of claim 11 or the vector of claim 12 into immune cells to obtain the engineered immune cells.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the engineered immune cells of claim 9, wherein the engineered immune cells are T cells or NK cells, and a pharmaceutically acceptable carrier, diluent or excipient.

16. Use of the engineered immune cells of claim 9, wherein the engineered immune cells are T cells or NK cells, for (i) the preparation of drugs or preparations for the prevention and / or treatment of cancer or tumors, wherein the cancer or tumor is a cancer with high expression of siglec-15, namely: acute myeloid leukemia, lung cancer, ovarian cancer, head and neck tumors, breast cancer, colorectal cancer, endometrial cancer, or a combination thereof.

17. The use as described in claim 16, characterized in that, The cancer or tumor mentioned is acute myeloid leukemia.

18. Use of the antibody of claim 1, the scFv of claim 6, the recombinant protein of claim 7, or the antibody conjugate of claim 10 for (ii) the preparation of a detection reagent or kit for detecting siglec-15.

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