Novel LILRB2 antibodies and uses thereof

Abstract

Provided herein are antibodies that bind to LILRB2 and uses of such antibodies in the detection and treatment of cancer and autoimmune diseases. TIFF2023513158000023.tif95139

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of priority to U.S. Provisional Application No. 62 / 970,496, filed February 5, 2020, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing The sequence listing contained in the file entitled "UTFHP0359WO_ST25", 213 KB (measured in Microsoft Windows) created on January 27, 2021, has been submitted herewith by electronic submission and is incorporated herein by reference. [Background technology]

[0003] background 1. Field The present disclosure relates generally to the fields of medicine, oncology, immunology, and immuno-oncology. More specifically, the present disclosure relates to antibodies that bind to LILRB and can treat cancers, including leukemia and solid tumors.

[0004] 2. Description of Related Art Current immune checkpoint blockade strategies have been successful in treating certain types of solid cancer. However, most cancer patients do not respond to current checkpoint blockade or relapse after treatment. Additionally, checkpoint blockade monotherapy has not been successful against most hematological malignancies, including multiple myeloma and leukemia.

[0005] The tumor microenvironment (TME) is thought to play an essential role in regulating the immune response against tumors. Among the complex factors and components that make up the tumor microenvironment, myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and the extracellular matrix all play essential roles in suppressing the immune response against tumors.

[0006] Recently, the inhibitory leukocyte immunoglobulin-like receptor (LILRB) and the related immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor LAIR1 have been shown to have tumor-promoting functions in various hematopoietic and solid cancer cells and in the immunosuppressive tumor microenvironment. ITIM-containing receptors are expressed on a wide range of immune cells and signal through recruitment of the phosphatases SHP-1, SHP-2, or SHIP, leading to negative regulation of immune cell activation. Similar to CTLA-4 and PD-1, LILRB is considered an immune checkpoint factor.

[0007] LILRB2 has been identified as a key regulator of myeloid cell phenotype in vitro and in vivo, and its activation by various ligands suppresses the pro-inflammatory activity of myeloid cells. Because myeloid cells with an inhibitory / anti-inflammatory phenotype can downregulate T cell activation, proliferation, and cytotoxic activity in the solid tumor microenvironment, therapeutic blockade of LILRB2 in myeloid-rich solid tumors has the potential to reactivate or enhance antitumor immune responses in patients with disease refractory / relapsed to T cell checkpoint inhibitors.

[0008] LILRB2 can inhibit the activity of multiple immune cell types that promote tumor immune evasion. LILRB2 belongs to the subfamily B class of LIR receptors, which contain two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The receptor is expressed on myeloid cells and binds to multiple types of ligands, including HLA class I molecules, ANGPTL, myelin inhibitors (including Nogo66, MAG, and OMgp), and β-amyloid, transducing negative signals that inhibit the stimulation of immune responses. It is thought to regulate inflammatory responses and cytotoxicity, helping to focus the immune response and limit autoreactivity. Multiple transcript variants encoding different isoforms have been found for this gene.

[0009] Conversely, agonizing LILRB family receptors may suppress immune responses or inflammation found in autoimmune or inflammatory diseases. Summary of the Invention

[0010] overview Thus, in one aspect, the present disclosure provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to LILRB2. In certain embodiments, the antibody or antigen-binding fragment modulates the activation of LILRB2 when bound to LILRB2. In certain embodiments, the antibody or antigen-binding fragment activates LILRB2 when bound to LILRB2. In certain embodiments, the antibody or antigen-binding fragment inhibits the activation of LILRB2 when bound to LILRB2. In certain embodiments, the antibody or antigen-binding fragment specifically blocks the binding of MHC and other ligands to LILRB2 when bound to LILRB2.

[0011] In one aspect, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain (HC) variable region (VH) and a light chain (LC) variable region (VL) comprising the clone CDR sequence pair set forth in Table 2; and variants thereof in which one or more of the LC-CDRs have one, two, or three amino acid substitutions, additions, deletions, or a combination thereof. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the isolated monoclonal antibody is a murine antibody, a rodent antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a human antibody. The isolated monoclonal antibody or antigen-binding fragment thereof may have VH and VL chains with amino acid sequences at least 90% or 95% identical to the clone sequence pair in Appendix II and IV, respectively. The isolated monoclonal antibody or antigen-binding fragment thereof may have VH and VL chains encoded by nucleic acid sequences at least 80% or 90% identical to the clone sequence pair in Appendix I and III, respectively. The isolated monoclonal antibody or antigen-binding fragment thereof may have VH and VL chains that have amino acid sequences identical to the clone sequence pair in Appendices II and IV, respectively. The isolated monoclonal antibody or antigen-binding fragment thereof may have VH and VL chains that are encoded by nucleic acid sequences identical to the clone sequence pair in Appendices I and III, respectively.

[0012] A variant may have one, two, or three amino acid substitutions, additions, deletions, or a combination thereof, in one or more of the HC-CDRs or LC-CDRs. In certain embodiments, each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat and Chothia definitions, the AbM definition, or the contact definition of a CDR.

[0013] In another aspect, the present disclosure provides an isolated monoclonal antibody or antigen-binding fragment thereof that competes for the same epitope with an antibody having a clone heavy chain-light chain CDR sequence pair from Table 2. In certain embodiments, the epitope to which the antibody or antigen-binding fragment binds is located within the linker region between the D1 and D2 domains of human LILRB2.

[0014] In certain embodiments, the isolated monoclonal antibodies described herein are chimeric, humanized, or human antibodies. In certain embodiments, the isolated monoclonal antibodies described herein are IgG1, IgG2, IgG3, or IgG4 types. In certain embodiments, the antigen-binding fragments described herein are recombinant ScFv (single-chain variable fragment) antibodies, Fab fragments, F(ab')2 fragments, or Fv fragments.

[0015] In another aspect, there is provided a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof provided herein and at least one pharmaceutically acceptable carrier.

[0016] In another aspect, there is provided an isolated nucleic acid encoding the isolated monoclonal antibody or antigen-binding fragment thereof provided herein.

[0017] In another aspect, there is provided a vector comprising the isolated nucleic acid provided herein.

[0018] In another aspect, a host cell is provided that contains the vector provided herein. The host cell may be a mammalian cell. The host cell may be a CHO cell.

[0019] In another aspect, a hybridoma is provided that encodes or produces the isolated monoclonal antibody provided herein.

[0020] In another aspect, methods of producing an antibody are provided, which may include culturing a host cell provided herein under conditions suitable for expression of the antibody, and recovering the antibody.

[0021] In another aspect, provided is a chimeric antigen receptor (CAR) protein comprising the antigen-binding fragment provided herein.

[0022] In another aspect, provided is an isolated nucleic acid encoding a CAR protein provided herein.

[0023] In another aspect, an engineered cell is provided that contains the isolated nucleic acid provided herein. In certain embodiments, the cell is a T cell, an NK cell, or a bone marrow cell.

[0024] In another aspect, provided is a method of treating or ameliorating the effects of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof defined herein.

[0025] The method may reduce or eradicate tumor burden in a subject and / or may slow tumor growth rate, reduce the number of tumor cells, shrink tumor size, reduce tumor invasion, reduce tumor metastasis, or eradicate tumor in a subject. The cancer may be a solid tumor or a hematological malignancy.

[0026] In certain embodiments, the cancer is a solid tumor, including adrenal gland cancer, bile duct cancer, bone cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, mesothelioma, melanoma, Merkel cell carcinoma, nasopharyngeal carcinoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, penile cancer, pinealoma, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, skin cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, and vaginal cancer.

[0027] In some embodiments, the cancer is metastatic, recurrent, or drug-resistant cancer.

[0028] In some embodiments, the cancer is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic lymphoblastic leukemia (CLL), chronic myelomonocytic leukemia (CMML), chronic myelogenous leukemia (CML), pre-B cell acute lymphocytic leukemia (pre ... Hematological malignancies including ALL), diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, hairy cell leukemia, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocytocyte-rich B-cell lymphoma, heavy chain disease, Hodgkin lymphoma, non-Hodgkin lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma (MM), myelodysplastic syndromes (MDS), myeloproliferative neoplasms, and polycythemia vera.

[0029] The antibody or antigen-binding fragment thereof may be administered intravenously, intraarterially, intratumorally, or subcutaneously.

[0030] In certain embodiments, the method includes administering topoisomerase inhibitors, anthracycline topoisomerase inhibitors, anthracyclines, daunorubicin, nucleoside metabolic inhibitors, cytarabine, hypomethylating agents, low-dose cytarabine (LDAC), a combination of daunorubicin and cytarabine, injectable daunorubicin and cytarabine liposomes, Vixeos®, azacitidine, Vidaza®, decitabine, all-trans-retinoic acid (ATRA), arsenic, arsenic trioxide, histamine dihydrochloride, Sepren®. , interleukin-2, aldesleukin, Proleukin®, gemtuzumab ozogamicin, Mylotarg®, FLT-3 inhibitors, midostaurin, Rydapt®, clofarabine, farnesyltransferase inhibitors, decitabine, IDH1 inhibitors, ivosidenib, tibsovo®, IDH2 inhibitors, enasidenib, Idifa®, Smoothened (SMO) inhibitors, glasdegib, arginase inhibitors, IDO inhibitors, epacadostat, BCL-2 inhibitors (BCL-2 inihbitor), venetoclax, Venclexta®, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitors, ibrutinib, Imbruvica®, acalabrutinib, Calquence®, zanubrutinib, PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, TIGIT antibodies, TIM3 antibodies, CD40 antibodies, 4-1BB antibodies, CD47 antibodies, SIRP1α antibodies or fusion proteins, CD70 antibodies, and CLL1 The method may further comprise administering to the subject one or more drugs selected from the group consisting of: an antibody, a CD123 antibody, an E-selectin antagonist, an antibody that binds to a tumor antigen, an antibody that binds to a T cell surface marker, an antibody that binds to a myeloid cell or NK cell surface marker, an alkylating agent, a nitrosourea agent, an antimetabolite, an antitumor antibiotic, an alkaloid derived from a plant, a hormone therapy agent, a hormone antagonist, an aromatase inhibitor, and a P-glycoprotein inhibitor.

[0031] The isolated monoclonal antibody or antigen-binding fragment thereof may comprise an anti-tumor drug linked thereto. The anti-tumor drug may be linked to the antibody via a photodegradable linker. The anti-tumor drug may be linked to the antibody via an enzyme-cleavable linker. The anti-tumor drug may be a toxin, a radioisotope, a cytokine, or an enzyme.

[0032] In another embodiment, a method for detecting cancer cells or cancer stem cells in a sample or subject is provided, comprising: (a) contacting the subject or a sample derived from the subject with an antibody or antigen-binding fragment thereof defined herein; and (b) detecting binding of the antibody to the cancer cells or cancer stem cells in the subject or sample. The sample may be a body fluid or biopsy, or blood, bone marrow, sputum, tears, saliva, mucus, serum, urine, or feces. Detection may involve immunohistochemistry, flow cytometry, immunoassays (including ELISA, RIA, etc.), or Western blotting. The method may further include performing steps (a) and (b) a second time and determining a change in the level of detection compared to the first time. The isolated monoclonal antibody or antigen-binding fragment thereof may further comprise a label (e.g., a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye). The isolated monoclonal antibody or antigen-binding fragment thereof may be conjugated to a liposome or nanoparticle.

[0033] In yet another aspect, a method for treating or ameliorating the effects of autoimmune disease in a subject is provided, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as defined herein.The antibody or antigen-binding fragment thereof may be administered intravenously, intraarterially, intratumorally, or subcutaneously.The method may further comprise administering to the subject one or more drugs selected from the group consisting of steroids or NSAIDs.Autoimmune diseases include Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, undifferentiated spondyloarthropathy, juvenile spondyloarthropathy, Behcet's disease, enthesitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, fibromyalgia, chronic fatigue syndrome, pain conditions associated with systemic inflammatory diseases, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile-onset diabetes mellitus (also known as type 1 diabetes), Wegener's granulomatosis, polymyositis, Dermatomyositis, inclusion body myositis, multiple endocrinopathy, Schmidt's syndrome, autoimmune uveitis, Addison's disease, Graves' disease, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, multiple sclerosis, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler's syndrome, myasthenia gravis, Eaton-Lambert syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia, scleroderma, progressive systemic sclerosis, CR EST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), adult-onset diabetes mellitus (also known as type II diabetes), mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, antiphospholipid syndrome, erythema multiforme, Cushing's syndrome, autoimmune chronic active hepatitis, allergic diseases, allergic encephalomyelitis, transfusion reactions, leprosy, malaria, leishmaniasis The condition may be rheumatic fever, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, eczema, lymphomatoid granulomatosis, Kawasaki disease, endophthalmitis, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman syndrome, Felty syndrome, Fuchs's cycle disease, IgA nephropathy, Henoch-Schönlein purpura, graft-versus-host disease, transplant rejection, tularemia, periodic fever syndrome, septic arthritis, familial Mediterranean fever, TNF receptor-associated periodic syndrome (TRAPS), Muckle-Wells syndrome, or hyper-IgD syndrome.

[0034] Also provided are monoclonal antibodies that bind to LILRB2 and (a) do not bind to LILRA or another LILRB; (b) bind to LILRB2 domain 1 or 4; (c) activate or antagonize LILRB2; (d) enhance the inflammatory potential of monocytes; (e) prevent myeloid-derived suppressor cell function; and / or (f) inhibit leukemia cell migration and / or infiltration in vivo.

[0035] The use of the words "a" or "an" when used in conjunction with the word "comprising" in the claims and / or this specification can mean "one," but it is also consistent with the meanings of "one or more," "at least one," and "one or more than one." The word "about" means plus or minus 5% of the stated number.

[0036] [The present invention 1001] An isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain (HC) variable region (VH) and a light chain (LC) variable region (VL) comprising the clone CDR sequence pairs set forth in Table 2; and variants thereof in which one or more of the HC-CDRs and / or LC-CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. [The present invention 1002] 1001. The isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, wherein said isolated monoclonal antibody is a murine antibody, a rodent antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a human antibody. [The present invention 1003] 1001. The isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, wherein said antigen-binding fragment is a recombinant ScFv (single-chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. [The present invention 1004] 1001. The isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, wherein said isolated monoclonal antibody is a human antibody. [The present invention 1005] 1001. An isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, wherein said VH and VL chains have amino acid sequences that are at least 90% or 95% identical to the clone sequence pairs of Appendix II and IV, respectively. [The present invention 1006] 1001. An isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, wherein said VH and VL chains are encoded by nucleic acid sequences that are at least 80% or 90% identical to the clone sequence pairs of Appendices I and III, respectively. [The present invention 1007] 1005. An isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, wherein said VH and VL chains have amino acid sequences identical to the clone sequence pair in Appendix II and IV, respectively. [The present invention 1008] 1005. An isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, wherein said VH and VL chains are encoded by nucleic acid sequences identical to the clone sequence pair in Appendix I and III, respectively. [The present invention 1009] 1009. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1001 to 1008, wherein the isolated monoclonal antibody is a humanized antibody. [The present invention 1010] 1001. The isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, wherein said antibody is a chimeric antibody. [The present invention 1011] 1001. An isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, which induces activation of LILRB2. [The present invention 1012] 1001. An isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, which inhibits the activation of LILRB2. [The present invention 1013] An isolated monoclonal antibody or antigen-binding fragment thereof that competes with any of the isolated monoclonal antibodies or antigen-binding fragments thereof of the present inventions 1001 to 1012 for the same epitope. [The present invention 1014] A pharmaceutical composition comprising any one of the isolated monoclonal antibodies or antigen-binding fragments thereof of the present inventions 1001 to 1013 and a pharmaceutically acceptable carrier. [The present invention 1015] An isolated nucleic acid encoding any one of the isolated monoclonal antibodies of the present inventions 1001 to 1013. [The present invention 1016] A vector comprising the isolated nucleic acid of the present invention. [The present invention 1017] A host cell comprising a vector of the present invention. [The present invention 1018] The host cell of the present invention 1017, which is a mammalian cell. [The present invention 1019] The host cell of the present invention 1017, which is a CHO cell. [The present invention 1020] A hybridoma or engineered cell that encodes and / or produces any of the isolated monoclonal antibodies of the present invention 1001 to 1013. [The present invention 1021] A method for producing an antibody, comprising culturing a host cell of the invention 1017 under conditions suitable for expression of said antibody, and recovering said antibody. [The present invention 1022] A chimeric antigen receptor (CAR) protein comprising an antigen-binding fragment of any one of 1001 to 1013 of the present invention. [The present invention 1023] An isolated nucleic acid encoding a CAR protein of the present invention. [The present invention 1024] A vector comprising the isolated nucleic acid of the present invention. [The present invention 1025] An engineered cell comprising an isolated nucleic acid of the invention 1023. [The present invention 1026] The engineered cell of the present invention 1025, which is a T cell, a NK cell, or a macrophage. [The present invention 1027] A method for treating cancer or ameliorating the effects of cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of any of 1001 to 1013 of the present invention, or an engineered cell of 1025 or 1026 of the present invention. [The present invention 1028] The method of claim 1027, wherein the method reduces or eradicates tumor burden in said subject. [The present invention 1029] The method of claim 1027, wherein the number of tumor cells is reduced and / or the rate of tumor growth is slowed. [The present invention 1030] The method of claim 1027, wherein the tumor size is reduced. [The present invention 1031] The method of claim 1027, wherein the method reduces or prevents tumor metastasis. [The present invention 1032] The method of claim 1027, wherein the tumor is eradicated in the subject. [The present invention 1033] The method of claim 1027, wherein the cancer is a solid cancer. [The present invention 1034] 1033. The method of claim 1033, wherein the solid cancer is selected from the group consisting of adrenal gland cancer, bile duct cancer, bone cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, mesothelioma, melanoma, Merkel cell carcinoma, nasopharyngeal carcinoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, penile cancer, pinealoma, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, skin cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, and vaginal cancer. [This invention 1035] The method of the present invention 1027, which targets monocytes, macrophages, dendritic cells, neutrophils, and other myeloid cells, myeloid-derived suppressor cells, and tumor-associated macrophages. [The present invention 1036] 1027. The method of claim 1027, wherein said cancer is a hematological malignancy. [This invention 1037] The hematological malignancies are acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia, chronic lymphoblastic leukemia (CLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myelomonocytic leukemia (CMML), chronic myelogenous leukemia (CML), pre-B-cell acute lymphocytic leukemia (pre-B ALL), diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, hairy cell leukemia, heavy chain disease, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocytocyte-rich B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasm, and polycythemia vera. [The present invention 1038] 1027. The method of claim 1027, wherein said antibody or antigen-binding fragment thereof is administered intravenously, intraarterially, intratumorally, or subcutaneously. [This invention 1039] Topoisomerase inhibitors, anthracyclines, topoisomerase inhibitors, anthracyclines, daunorubicin, nucleoside metabolic inhibitors, cytarabine, hypomethylating agents, low-dose cytarabine (LDAC), daunorubicin and cytarabine combinations, daunorubicin and cytarabine liposomes for injection, Vixeos®, azacitidine, Vidaza®, decitabine, all-trans-retinoic acid (ATRA), arsenic, arsenic trioxide, histamine dihydrochloride, Sepren®, interlo Ikin-2, aldesleukin, Proleukin (registered trademark), gemtuzumab ozogamicin, Mylotarg (registered trademark), FLT-3 inhibitors, midostaurin, Rydapt (registered trademark), clofarabine, farnesyltransferase inhibitors, decitabine, IDH1 inhibitors, ivosidenib, tibsovo (registered trademark), IDH2 inhibitors, enasidenib, Idifa (registered trademark), Smoothened (SMO) inhibitors, glasdegib, arginase inhibitors, IDO inhibitors, epacadostat, BCL-2 inhibitors, bevacizumab Netoclax, Venclexta®, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitors, ibrutinib, Imbruvica®, acalabrutinib, Calquence®, zanubrutinib, PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, TIGIT antibodies, TIM3 antibodies, CD40 antibodies, 4-1BB antibodies, CD47 antibodies, SIRP1α antibodies or fusion proteins, CD70 antibodies, and and administering to the subject one or more drugs selected from the group consisting of a CLL1 antibody, a CD123 antibody, an E-selectin antagonist, an antibody that binds to a tumor antigen, an antibody that binds to a T cell surface marker, an antibody that binds to a myeloid cell or NK cell surface marker, an alkylating agent, a nitrosourea agent, an antimetabolite, an antitumor antibiotic, an alkaloid derived from a plant, a hormone therapy agent, a hormone antagonist, an aromatase inhibitor, and a P-glycoprotein inhibitor. [The present invention 1040] 1039. The method of any of claims 1027 to 1039, wherein said isolated monoclonal antibody or antigen-binding fragment thereof further comprises an anti-tumor drug linked thereto. [The present invention 1041] 1040. The method of claim 1040, wherein said anti-tumor drug is linked to said antibody via a photolabile linker. [The present invention 1042] 1040. The method of claim 1040, wherein said anti-tumor drug is linked to said antibody via an enzyme-cleavable linker. [This invention 1043] The method of claim 1040, wherein said anti-tumor drug is a toxin, a radioisotope, a cytokine, or an enzyme. [This invention 1044] 1. A method for detecting cancer cells or cancer stem cells in a sample or a subject, comprising: (a) contacting a subject or a sample derived from a subject with any one of the antibodies or antigen-binding fragments thereof of the present invention 1001 to 1011; and (b) detecting binding of the antibody to cancer cells or cancer stem cells in the subject or the sample; A method comprising: [This invention 1045] The method of claim 1044, wherein said sample is a body fluid or a biopsy. [The present invention 1046] 104. The method of claim 1044, wherein said sample is blood, bone marrow, sputum, tears, saliva, mucus, serum, urine or feces. [This invention 1047] The method of claim 1044, wherein detecting comprises immunohistochemistry, flow cytometry, immunoassay (including ELISA, RIA, etc.) or Western blot. [This invention 1048] The method of claim 1044, further comprising performing steps (a) and (b) a second time and determining a change in the level of detection compared to the first time. [This invention 1049] 104. The method of claim 1044, wherein said isolated monoclonal antibody or antigen-binding fragment thereof further comprises a label. [The present invention 1050] 1049. The method of claim 1049, wherein said label is a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. [This invention 1051] 1050. The method of any of claims 1027 to 1050, wherein said isolated monoclonal antibody or antigen-binding fragment thereof is conjugated to a liposome or nanoparticle. [This invention 1052] A method for treating an autoimmune disease or ameliorating the effects of an autoimmune disease in a subject, comprising the step of administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of any of 1001 to 1013 of the present invention, or an engineered cell of 1025 or 1026 of the present invention. [This invention 1053] The method of the present invention 1052 targets monocytes, macrophages, dendritic cells, and neutrophils, as well as other myeloid cells. [This invention 1054] 1052. The method of claim 1052, wherein the antibody or antigen-binding fragment thereof is administered intravenously, intraarterially, intratumorally, or subcutaneously. [This invention 1055] The method of claim 1052, further comprising the step of administering to said subject one or more drugs selected from the group consisting of steroids or NSAIDs. [This invention 1056] The autoimmune disease is selected from the group consisting of Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, undifferentiated spondyloarthropathy, juvenile spondyloarthropathy, Behcet's disease, enthesitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, fibromyalgia, chronic fatigue syndrome, pain conditions associated with systemic inflammatory diseases, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile-onset diabetes mellitus (also known as type 1 diabetes), Wegener's granulomatosis, polymyositis, Dermatomyositis, inclusion body myositis, multiple endocrinopathy, Schmidt syndrome, autoimmune uveitis, Addison's disease, Graves' disease, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, multiple sclerosis, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler syndrome, myasthenia gravis, Eaton-Lambert syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), adult-onset diabetes mellitus (also known as type II diabetes), mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, antiphospholipid syndrome, erythema multiforme, Cushing's syndrome, autoimmune chronic active hepatitis, allergic diseases, allergic encephalomyelitis, transfusion reactions, leprosy, malaria, leishmaniasis, avian The method of claim 1052, wherein the disease is panosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, eczema, lymphomatoid granulomatosis, Kawasaki disease, endophthalmitis, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman syndrome, Felty syndrome, Fuchs's cyclitis, IgA nephropathy, Henoch-Schönlein purpura, graft-versus-host disease, transplant rejection, tularemia, periodic fever syndrome, septic arthritis, familial Mediterranean fever, TNF receptor-associated periodic syndrome (TRAPS), Muckle-Wells syndrome, or hyper-IgD syndrome. [This invention 1057] A method for enhancing T cell activation in a subject, comprising the step of administering to the subject an antibody or antigen-binding fragment thereof of any of 1001 to 1013 of the present invention or an engineered cell of 1025 or 1026 of the present invention. [This invention 1058] A method for modulating the M2a macrophage phenotype in a subject, comprising the step of administering to the subject an antibody or antigen-binding fragment thereof of any of 1001 to 1013 of the present invention or an engineered cell of 1025 or 1026 of the present invention. [This invention 1059] binds to LILRB2, and (a) does not bind to LILRA or another LILRB; or (b) binds to LILRB2 domain 1 or 4; or (c) activating or antagonizing LILRB2; or (d) enhance the inflammatory potential of monocytes; or (e) enhances T cell activation; or (f) modulates the M2a macrophage phenotype; or (g) preventing myeloid-derived suppressor cell function; or (h) inhibiting leukemia cell migration and / or invasion in vivo; Monoclonal antibodies. It is contemplated that any method or composition described herein may be implemented with respect to any other method or composition described herein. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, and that various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0037] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0038] [Figure 1A] Figure 1A-C. Screening of monoclonal antibodies specific to LILRB2. (Figure 1A) Representative flow cytometry profile showing that monoclonal antibodies bind to LILRB2 reporter cells. Monoclonal antibody binding was screened using a flow cytometer on LILRB2 reporter cells. Bound antibodies were detected with an allophycocyanin (APC)-conjugated goat anti-human IgG secondary antibody. [Figure 1B] (Figure 1B) Quantification of the binding ability of monoclonal antibodies to LILRB2 reporter cells. B2-7, B2-15, B2-16, B2-17, B2-19, B2-8, B2-24, B2-25, B2-10, B2-12, and B2-18 highly bind to LILRB2 reporter cells. [Figure 1C-1] (Figure 1C) Quantification of the binding ability of monoclonal antibodies B2-7, B2-15, B2-16, B2-17, B2-19, B2-8, B2-24, B2-25, B2-10, B2-12, and B2-18 to LILRA, LILRB, and LAIR1 reporter cells. [Figure 1C-2]A continuation of Figure 1C-1 is shown. [Figure 2] Figure 2A-B. Antibodies B2-8, B2-24, B2-10, B2-10, and B2-15 increase GFP signaling in LILRB2 reporter cells. (Figure 2A) GFP signaling in LILRB2 reporter cells incubated with soluble antibodies. (Figure 2B) GFP signaling in LILRB2 reporter cells incubated with soluble antibodies in combination with K562. [Figure 3A] Figure 3A-C. Antibodies B2-7, B-15, B2-16, B2-17, and B2-19 block GFP signaling in LILRB2 reporter cells activated by coated ANGPTL2. (Figure 3A) Representative flow cytometry profiles show that coated ANGPTL2 stimulates GFP expression in LILRB2 reporter cells. [Figure 3B] (Figure 3B) Representative flow cytometry profiles showed that B2-7, B-15, B2-16, B2-17, and B2-19 effectively blocked GFP expression in coated ANGPTL2-activated LILRB2 reporter cells. [Figure 3C] (Figure 3C) Dose-dependent inhibitory activity of B2-7, B-15, B2-16, B2-17, and B2-19 on GFP expression induced by coated ANGPTL2. The blocking potencies (IC50) of B2-19, B2-16, B2-7, B2-15, and B2-17 were 48.54 ng / ml, 131.4 ng / ml, 221.1 ng / ml, 341.3 ng / ml, and 405.1 ng / ml, respectively. [Figure 4A] Figure 4A-C. Antibodies B2-7, B-15, B2-16, B2-17, and B2-19 block GFP signaling in LILRB2 reporter cells activated by coated SEMA4A. (Figure 4A) Representative flow cytometry profiles show that coated SEMA4A stimulates GFP expression in LILRB2 reporter cells. [Figure 4B](Figure 4B) Representative flow cytometry profiles showed that B2-7, B-15, B2-16, B2-17, and B2-19 effectively blocked GFP expression in coated SEMA4A-activated LILRB2 reporter cells. [Figure 4C] (Figure 4C) Dose-dependent inhibition of GFP expression induced by coated SEMA4A by B2-7, B-15, B2-16, B2-17, and B2-19. The blocking potencies (IC50s) of B2-19, B2-16, B2-7, B2-15, and B2-17 were 167.1 ng / ml, 449 ng / ml, 701 ng / ml, 1001 ng / ml, and 1034 ng / ml, respectively. [Figure 5] Figures 5A-C. Antibodies B2-7, B-15, B2-16, B2-17, and B2-19 block GFP signaling in LILRB2 reporter cells activated by HLA-G overexpressed on K562 cells. (Figure 5A) Representative flow cytometry profiles show that HLA-G overexpressed on K562 cells stimulates GFP expression in LILRB2 reporter cells. (Figure 5B) Representative flow cytometry profiles show that B2-7, B-15, B2-16, B2-17, and B2-19 effectively block GFP expression in LILRB2 reporter cells activated by HLA-G overexpressed on K562 cells. (Figure 5C) Quantification of the GFP percentage shown in Figure 5B. [Figure 6A] Figure 6A-C. Effect of LILRB2 antibody on LPS responses in primary human monocytes. (Figure 6A-B) Representative flow cytometry profiles showed surface CD86 and intracellular TNFα staining in cells gated on CD33+ monocytes. PBMCs were cultured with anti-LILRB2 antibody (10 ng / ml) for 48 hours, followed by 6 hours of LPS stimulation (50 ng / ml) in the presence of brefeldin A. [Figure 6B] See legend to Figure 6A. [Figure 6C](Figure 6C) Quantification of fold change was defined as the ratio of the mean fluorescence intensity (MFI) of CD86 and TNFα in anti-LILRB2-treated samples to their respective MFIs in the IgG-treated samples shown in Figure 6A-B. MFI values ​​represent cells gated on CD33+ monocytes. [Figure 7A] Figure 7A-J. Antagonist LILRB2 mAb inhibits leukemia cell development in the C1498-LILRB2 tumor-bearing model. (Figure 7A) Human LILRB2 expression on murine C1498 parental cells and human LILRB2 retrovirally transduced C1498 cells. [Figure 7B] (FIG. 7B) LILRB2 promotes death in leukemia-bearing mice. Kaplan-Meier survival curves of humanized NSG mice injected iv with LILRB2-overexpressing or control C1498 cells (1×10 cells / mouse). [Figure 7C] (Figure 7C-D) Representative flow cytometry plots and summary of leukemic cell infiltration in bone marrow (BM), peripheral blood (PB), liver (LV), and spleen (SP) from C57BL / 6 mice intravenously injected with LILRB2-overexpressing or control C1498 cells (1 × 106 cells / mouse). [Figure 7D] See legend to Figure 7C. [Figure 7E] (FIG. 7E) Kaplan-Meier survival curves of C57BL / 6 mice iv injected with LILRB2-overexpressing or control C1498 cells (1×10 6 cells / mouse). [Figure 7F] (Figure 7F-G) Representative flow cytometry plots and summary of myeloid cell infiltration in peripheral blood (PB) from C57BL / 6 mice iv injected with LILRB2-overexpressing or control C1498 cells (1 × 106 cells / mouse). [Figure 7G] See legend to Figure 7F. [Figure 7H](Figure 7H-I) Representative flow cytometry plots and summary of leukemic cell infiltration in peripheral blood (PB) from C56BL / 6 mice treated with LALAPG-mutant anti-LILRB2 antibody or IgG control after leukemic cell transplantation. LILRB2-overexpressing C1498 cells (1 x 10 cells / mouse) were injected into C57BL / 6 mice, followed by treatment with LALAPG-Fc mutant anti-LILRB2 antibody or LALAPG-Fc mutant IgG. The percentage of leukemic cells (GFP+) from peripheral blood (PB) was determined by flow cytometry 20 days after transplantation. [Figure 7I] See legend to Figure 7H. [Figure 7J] (FIG. 7J) Summary of myeloid cell infiltration in peripheral blood (PB) from C56BL / 6 mice treated with LALAPG mutant anti-LILRB2 antibodies or LALAPG Fc mutant IgG control at the indicated time points after leukemia cell transplantation. [Figure 8A] Figure 8A-F. Antagonist LILRB2 mAb inhibits leukemia cell development in the MLL-AF9 leukemia model. (Figure 8A) Expression of LILRB2 on PIRB-KO MLL-AF9 leukemia cells transduced with LILRB2 (LILRB2) or control (control) leukemia cells. [Figure 8B] (Figure 8B-C) Representative flow cytometry plots and summary of leukemic cell infiltration in peripheral blood (PB) from C56BL / 6 mice transplanted with PirB-KO MLL-AF9 leukemic cells transduced with LILRB2 (LILRB2) or control PirB-KO MLL-AF9 (control) leukemic cells. [Figure 8C] See legend to Figure 8B. [Figure 8D] (Figure 8D) Kaplan-Meier survival curves of leukemia mice transplanted with LILRB2-overexpressing or control PirB-KO MLL-AF9 cells. [Figure 8E](Figure 8E-F) Representative flow cytometry plots and summary of leukemic cell infiltration in peripheral blood (PB) from C56BL / 6 mice transplanted with PirB-KO LILRB2 leukemic cells and subsequently treated with LALAPG mutant anti-LILRB2 antibody or IgG control. [Figure 8F] See legend to Figure 8E. [Figure 9] Figures 9A-G. Anti-LILRB2 antibody inhibits AML cell migration and invasion. (Figure 9A) LILRB2 expression on THP-1 cells was confirmed using a commercial phycoerythrin (PE)-anti-LILRB2 antibody. (Figure 9B) Short-term (20 h) infiltration of leukemia cells in NSG mice treated with LALAPG Fc mutant anti-LILRB2 antibody or LALAPG Fc mutant IgG control after leukemia transplantation. THP-1 cells (1 x 107 cells / mouse) were injected into NSG mice, followed immediately by treatment with LALAPG mutant IgG control or anti-LILRB2 antibody. 20 h after transplantation, the number of leukemia cells (GFP+) from the bone marrow (BM), liver (LV), and spleen (SP) was determined by flow cytometry and normalized to the number in peripheral blood (PB). (Figure 9C) Percentage of leukemia cells (GFP+) in the indicated organs, e.g., liver (LV), bone marrow (BM), spleen (SP), and peripheral blood (PB), at 21 days post-transplant in NSG mice treated with LALAPG mutant anti-LILRB2 antibodies or IgG control after THP-1 injection. (Figure 9D) The body weight of each mouse shown in Figure 9B was measured on the indicated days after THP-1 cell injection. (Figure 9E) Comparison of liver size from NSG mice treated with IgG or anti-LILRB2 antibodies containing the LALAPG Fc mutation 28 days after THP-1 transplantation. (Figure 9F) Quantification of liver weight shown in Figure 9E normalized to the body weight of each individual mouse. (Figure 9G) Survival curve of NSG mice treated with LALAPG Fc mutant IgG control or anti-LILRB2 antibodies after THP-1 transplantation. [Figure 10]Figures 10A-E. Antagonist LILRB2 mAb inhibits leukemia cell development in patient-derived xenograft (PDX) models. (Figure 10A) LILRB2 expression pattern on primary leukemia cells from AML M5 patients. (Figure 10B) Analysis of the correlation between LILRB2 mRNA levels and overall survival of patients with AML-M5 (n=132, divided into two groups based on gene expression) in the TCGA database (https: / / xena.ucsc.edu) by Kaplan-Meier log-rank test. (Figure 10C) Infiltration of human CD45+CD33+ leukemia cells in the peripheral blood (PB), bone marrow (BM), spleen (SP), and liver (LV) of NSG mice transplanted with primary AML-M5 leukemia samples and treated with anti-LILRB2 antibody or control IgG. (Figure 10D) Representative brightfield microscopy images of primary AML-M5 leukemia cells cultured in the presence of LALAPG mutant anti-LILRB2 antibody or IgG control. Cells treated with anti-LILRB2 showed a more adherent, differentiated morphology. (Figure 10E) Intracellular expression of CD68 on primary AML-M5 leukemia cells cultured in the presence of LALAPG mutant anti-LILRB2 antibody or IgG control. [Figure 11]Figures 11A-G. Antagonist LILRB2 mAb can prevent the T cell suppressive function of myeloid-derived suppressor cells (MDSCs) in vitro. (Figure 11A) One representative histogram shows that antagonist LILRB2 mAb attenuates the suppressive function of MDSCs on CD8+ T cells. MDSCs were isolated from PBMCs of patients with solid tumors by depleting HLD-DRbright cells and then enriching for CD14+ cells using autoMACS. To monitor cell proliferation, MDSCs were cocultured with T cells from the same donor that had been pre-stained with CSFE (E:T=1). 10 μg / mL of LALAPG mutant IgG, B2-7, or B2-19 was added to the cell culture. The percentage of proliferating T cells, indicated by reduced intensity of the CFSE signal, was determined by flow cytometry 5 days after treatment. (Figure 11B) Quantification of the effect of anti-LILRB2 mAb on the inhibitory function of MDSCs against T cells. The percentage of proliferating T cells (left panel: CD8+ T cells, right panel: CD4+ T cells), indicated by reduced intensity of the CFSE signal, was determined by flow cytometry 5–7 days after treatment. (Figure 11C) Anti-LILRB2 mAb attenuated the inhibitory function of MDSCs against T cells, as assessed by measuring IFN-γ secretion in the supernatant of T cell cultures. (Figure 11D) Anti-LILRB2 mAb reduced the expression of M2 macrophage markers on MDSCs while simultaneously increasing the expression of M1 macrophage markers. MDSCs isolated from the peripheral blood of patients with solid tumors were cultured with 10 μg / mL of anti-LILRB2 antibody for 7 days. The expression of CD163, CD206, and CD86 was analyzed by flow cytometry. (Figure 11E) Anti-LILRB2 mAb reduced the expression of M2 markers on monocyte-derived macrophages from healthy donors, while simultaneously increasing the expression of M1 markers. Monocytes isolated from peripheral blood of healthy donors were cultured and incubated with 10 μg / mL of anti-LILRB2 antibody for 7 days. Expression of CD163, CD206, and CD86 was analyzed by flow cytometry.(Figure 11F) Anti-LILRB2 mAb reduced the expression of M2 markers on the cell surface of tumor-associated macrophages / monocytes in ascites from patients with ovarian cancer, while simultaneously increasing the expression of M1 markers. CD14+ cells were isolated from ascites by autoMACS and cultured with anti-LILRB2 mAb with IgG4 Fc for 7 days. CD163, CD206, and CD86 were analyzed by flow cytometry. (Figure 11G) Anti-LILRB2 mAb increased M1 macrophage cytokines and chemokines secreted by MDSCs from 4–5 patients with solid tumors. [Figure 12A] Figures 12A-B. Antibody sequence analysis of positive phages. (Figure 12A) Phylogenetic tree of heavy chain variable region (VH) and light chain variable region (VL). [Figure 12B] (Figure 12B) ELISA binding of 24 positive phages to LILRB2. [Figure 13] Figures 13A-B. ELISA binding EC50 for LILRB2. (Figure 13A) ELISA binding curve of LILRB2 antibody. (Figure 13B) Calculated EC50 value of LILRB2 antibody. [Figure 14A-1] Figure 14A-D. Binding specificity of LILRB2 antibodies. (Figure 14A) ELISA binding of LILRB2 antibodies to antigens of other members of the LILR family. [Figure 14A-2] This shows a continuation of Figure 14A-1. [Figure 14B] (Figures 14B-D) Comparison of ELISA binding curves of antibodies (Figure 14B) B2-10, (Figure 14C) B2-12, and (Figure 14D) B2-18 to LILRB2 and LILRA1. [Figure 14C] See legend to Figure 14B. [Figure 14D] See legend to Figure 14B. [Figure 15]Epitope binning of LILRB2 antibodies. Epitope binning was performed in a sandwich format on the Octet RED 96 System. Each antibody was loaded as the first antibody onto a Protein A biosensor. After blocking the biosensor with an unrelated IgG, the LILRB2 antigen was then captured, and the biosensor was further incubated with the remaining other antibodies (secondary antibodies). A "+" indicates that the first antibody blocked the signal of the secondary antibody. Antibodies belonging to the same bin were highlighted in different colors. [Figure 16] Figures 16A-D. Binding domains on LILRB2 by antibodies. (Figure 16A) Schematic diagram showing different truncated ECD proteins with Fc fusion. (Figure 16B) SDS-PAGE of purified fusion proteins. (Figure 16C) ELISA binding of antibodies to different truncated proteins. (Figure 16D) Summary of antibody binding domains. [Figure 17-1] Figures 17A-F. Mapping of key residues on D1 and D4. (Figures 17A-B) Alignment of the (Figure 17A (SEQ ID NOS:593-595)) D1 and (Figure 17B (SEQ ID NOS:596-597)) D4 domains of LILRB2 and LILRB1. Regions that differ between LILRB2 and LILRB1, are exposed, and are located in the loop regions are boxed. (Figures 17C-D) Sequences of mutations on the (Figure 17C (SEQ ID NOS:598-605)) D1 and (Figure 17D (SEQ ID NOS:606-613)) D4 domains. [Figure 17-2] (Figures 17E-F) Loss of antibody binding to mutants of the (Figure 17E) D1 and (Figure 17F) D4 domains based on ELISA. Percent binding to each mutant relative to wild-type B2-ECD is plotted as a stacked bar graph. [Figure 18] Blocking antibody affinity. The antibody was captured on a Protein A biosensor. The sensor was immersed in serially diluted LILRB2 solutions for 300 seconds to allow association, and then immersed in kinetic buffer for 600 seconds to allow dissociation. The association and dissociation curves are shown as solid blue lines, and the two phases are separated by a dotted red line. [Figure 19]Figures 19A-B. ELISA binding measurements of antibodies against human and non-human primate (cynomolgus monkey, cyno) LILRB2 recombinantly produced in HEK293 cells. (Figure 19A) ELISA binding against human LILRB2. (Figure 19B) ELISA binding against cynomolgus monkey LILRB2. Fusion proteins of the extracellular domain (ECD) of human LILRB2 or NHP-LILRB2 with the Fc of mouse IgG2a were used to coat ELISA plates, and the antibodies were titrated at different concentrations as shown on the graph. [Figure 20] Figures 20A-B. Measurement of cell surface LILRB2 binding by monoclonal antibodies by flow cytometry. (Figure 20A) Quantification of the binding ability of monoclonal antibodies to LILRB2 reporter cells. Monoclonal antibodies were screened using a flow cytometer on LILRB2 reporter cells labeled with allophycocyanin (APC) goat anti-human IgG secondary antibody. (Figure 20B) Representative flow cytometry profiles showing that HCB2-5 and HCB2-10 monoclonal antibodies highly bind to LILRB2 reporter cells, while HCB2-2 binds weakly to LILRB2 reporter cells. NC: unlabeled reporter cells. ISO: incubation of reporter cells with only secondary Ab (anti-human Fc specific) conjugated to APC (ISO). Numbers indicate the mean fluorescence intensity (MFI) of APC or AF647. [Figure 21-1] Detection of LILRB2 antibody binding to cynomolgus monkey LILRB2 expressed on the cell surface. Antibody binding was detected by flow cytometry using HEK293 cells stably expressing full-length cynomolgus monkey LILRB2 with an N-terminal FLAG tag. Isotype human IgG1 (shown as a black line) was used as a control, and the solid pink peak indicates binding of the LILRB2 antibody to cynomolgus monkey LILRB2 on the cell surface. A mouse anti-Flag monoclonal antibody was used for flow cytometric detection. [Figure 21-2] A continuation of Figure 21-1 is shown. [Figure 22]Determination of the involvement of GFP reporter signaling with immobilized LILRB2 antibodies. NC, negative control and isotype control IgG. [Figure 23] Figures 23A-B. Determination of the contribution of soluble antibodies to GFP signaling in LILRB2 reporter cells in the presence or absence of K562 cells. (Figure 23A) GFP signaling in LILRB2 reporter cells incubated with soluble antibodies. (Figure 23B) GFP signaling in LILRB2 reporter cells incubated with soluble antibodies in combination with K562. [Figure 24] Figures 24A-B. Specific binding of LILRB2 by LILRB2 monoclonal antibody. (Figure 24A) Representative flow cytometry profile showing that only the positive control mAb (POS) binds to LILRA reporter cells (RC), but not the LILRB2 mAb. (Figure 24B) Representative flow cytometry profile showing that the LILRB2 mAb binds to LILRB2-expressing reporter cells, but not to reporter cells expressing LILRB and LAIR1. Binding of the positive control mAb (POS) demonstrates the expression level of each receptor. Binding of the LILRB2 monoclonal antibody was detected using flow cytometry on LILRA-, LILRB-, or LAIR1-expressing reporter cells using an allophycocyanin (APC)-labeled goat anti-human IgG Fc-specific secondary antibody. Non: unstained reporter cells. NEG: cells incubated with secondary Ab only. POS: cells incubated with commercial antibodies conjugated to APC or AF647 against the respective LIL or LAIR-1 receptors. Numbers indicate the mean fluorescence intensity (MFI) of APC or AF647. [Figure 25]Figures 25A-B. Blocking activity of LILRB2 antibodies assayed on GFP signaling of LILRB2 reporter cells activated by HLA-G overexpressed on K562 cells. (Figure 25A) Representative flow cytometry profiles showed that HLA-G overexpressed on K562 cells stimulated GFP induction in LILRB2 reporter cells. (Figure 25B) Representative flow cytometry profiles showed that HCB2-5 and HCB2-10 effectively blocked GFP signaling of LILRB2 reporter cells activated by HLA-G-overexpressing K562 cells. IgG, isotype control. [Figure 26] Figures 26A-C. Blocking activity of LILRB2 antibodies assayed on GFP signaling in LILRB2 reporter cells activated by coated ANGPTL2. (Figure 26A) Representative flow cytometry profiles showed that HCB2-5 and HCB2-10 effectively blocked GFP signaling in LILRB2 reporter cells activated by coated ANGPTL2. (Figure 26B) Quantification of the GFP percentage shown in Figure 26A. (Figure 26C) Dose-dependent inhibitory ability of HCB2-5 and HCB2-10 on GFP signaling induced by coated ANGPTL2. [Figure 27] Figures 27A-C. Blocking activity of HCB2-5 and HCB2-10 assayed on GFP signaling in LILRB2 reporter cells activated by coated SEMA4A. (Figure 27A) Representative flow cytometry profiles showed that HCB2-5 and HCB2-10 effectively blocked GFP signaling in LILRB2 reporter cells activated by coated SEMA4A. (Figure 27B) Quantification of the GFP percentage shown in Figure 27A. (Figure 27C) Dose-dependent inhibitory ability of HCB2-5 and HCB2-10 on GFP signaling induced by coated SEMA4A. [Figure 28] Figures 28A-B. Phylogenetic trees of antibody VH and VL. [Figure 29] Binding of LILRB2 antibodies on a reporter cell line expressing the ectodomain of LILR family proteins on the cell surface. Bound antibodies were detected with an anti-human Fc-specific secondary antibody (2nd Ab) conjugated to AF647. Expression of each LILR was confirmed using commercially available antibodies (Ctl+Ab) directly conjugated to AF647 or APC. All incubations were performed at 4°C for 30 minutes. Data shown are the mean fluorescence intensity after sample acquisition on a flow cytometer. [Figure 30] Binding of LILRB2 antibody on leukocytes from human whole blood harvested from healthy donors. LILRB2 antibody was directly conjugated with AF647. One hundred microliters of whole blood was incubated with antibodies for cell surface markers and LILRB2 antibody according to the protocol available in the literature (Hensley et al., J Vis Exp 2012;67:4302). Data shown are averaged geometric mean fluorescence intensity ± standard error of the mean (sem, N = 2 donors) after sample acquisition on a flow cytometer (BD FACS Celesta), subtracting background fluorescence from stained samples in which the LILRB2 antibody was omitted. [Figure 31] Binding of LILRB2 antibodies on HEK293 cells stably expressing full-length human LILRB2. Fifty thousand cells were incubated with a dilution series of LILRB2 antibodies (40–0.0006 μg / mL) in a final volume of 100 μL. Bound antibodies were detected with an anti-human Fc-specific secondary antibody conjugated to AF647. All incubations were performed at 4°C for 30 minutes. Data shown are the averaged geometric mean fluorescence intensity ± standard error of the mean (sem) minus background fluorescence for samples incubated with secondary antibody alone after sample acquisition (in duplicate) on a flow cytometer (BD FACS Celesta). [Figure 32]Binding of LILRB2 antibody on CD14+CD16- monocytes isolated from human PBMCs from healthy donors. Fifty thousand cells were incubated with a dilution series of LILRB2 antibody directly conjugated to AF647 (40-0.0006 μg / mL) in a final volume of 100 μL for 30 minutes at 4°C. Data shown are averaged geometric means of fluorescence intensity from duplicate samples acquired on a flow cytometer (BD FACS Celesta) from one donor and are representative of two experiments using cells isolated from different donors. [Figure 33] Inhibition of HLA-G-His (5 μg / mL) binding on HEK293 cells stably expressing full-length LILRB2 in the presence of a dilution series of competing LILRB2 antibodies (40–0.0098 μg / mL). Bound HLA-G was detected by flow cytometry using an anti-His antibody directly conjugated to APC. All incubations were performed at 4°C for 30 minutes. Data shown are the averaged geometric mean ± standard error of the mean (sem) of fluorescence intensity from duplicate samples acquired on a flow cytometer (BD FACS Celesta). [Figure 34] Inhibition of SEMA4A-hFc-AF647 (5 μg / mL) binding on HEK293_LILRB2 cells in the presence of a dilution series of competing LILRB2 antibodies (40–0.0098 μg / mL). Incubation was performed at 4°C for 30 min. Data shown are the averaged geometric mean ± standard error of the mean (sem) of fluorescence intensity from duplicate samples acquired on a flow cytometer (BD FACS Celesta). [Figure 35]Effect of LILRB2-blocking antibodies on the levels of TNF-α secreted by PBMCs stimulated with 50 ng / mL LPS. Data shown are from two donors and are representative of six donors (total N = 6 donors). PBMCs isolated from healthy donors were incubated (in duplicate) with LPS (Sigma-Aldrich) and various concentrations of antibodies for three days. Cytokines were measured in the culture medium supernatant using a Human Cytokine Premixed Magnetic Luminex Performance Assay. [Figure 36] Effect of LILRB2 blocking antibodies on the levels of IFN-γ secreted by PBMCs stimulated with 50 ng / mL LPS. Data shown are from two donors and are representative of five donors (total N = 6 donors). PBMCs isolated from healthy donors were incubated (in duplicate) with LPS (Sigma-Aldrich) and various concentrations of antibodies for 3 days. Cytokines were measured in the culture medium supernatant using a Human Cytokine Premixed Magnetic Luminex Performance Assay. [Figure 37] Effect of LILRB2 blocking antibody (40 μg / mL) on the levels of IL-12p40 secreted by PBMCs stimulated with 50 ng / mL LPS (total N = 3 donors). PBMCs isolated from healthy donors were incubated (in duplicate) with LPS (Sigma-Aldrich) and 40 μg / mL B2-19 antibody for 3 days. IL-12p40 concentrations were measured in the culture medium supernatants using a human IL-12 ELISA assay (BD Biosciences). [Figure 38]Effect of LILRB2 blocking antibodies on the levels of IFN-γ secreted by PBMCs stimulated with 10 ng / mL of the anti-CD3 activating antibody HIT3a. Data shown are from two donors and are representative of six donors (total N = 6 donors). PBMCs isolated from healthy donors were incubated (in duplicate) with HIT3a (Biolegend) and various concentrations of antibody for three days. Cytokines were measured in the culture medium supernatant using a Human Cytokine Premixed Magnetic Luminex Performance Assay. [Figure 39] Effect of LILRB2 blocking antibodies on the levels of TNF-α secreted by PBMCs stimulated with 10 ng / mL of the anti-CD3 activating antibody HIT3a. Data shown are from two donors and are representative of six donors (total N = 6 donors). PBMCs isolated from healthy donors were incubated (in duplicate) with HIT3a (Biolegend) and various concentrations of antibody for three days. Cytokines were measured in the culture medium supernatant using a Human Cytokine Premixed Magnetic Luminex Performance Assay. [Figure 40] Effect of LILRB2 blocking antibodies on the levels of GM-CSF secreted by PBMCs stimulated with 10 ng / mL of the anti-CD3 activating antibody HIT3a. Data shown are from two donors and are representative of six donors (total N = 6 donors). PBMCs isolated from healthy donors were incubated (in duplicate) with HIT3a (Biolegend) and various concentrations of antibody for three days. Cytokines were measured in the culture medium supernatant using a Human Cytokine Premixed Magnetic Luminex Performance Assay. [Figure 41]Effect of LILRB2 blocking antibodies on the levels of IL-1α secreted by PBMCs stimulated with 10 ng / mL of the anti-CD3 activating antibody HIT3a. Data shown are from two donors and are representative of six donors (total N = 6 donors). PBMCs isolated from healthy donors were incubated (in duplicate) with HIT3a (Biolegend) and various concentrations of antibody for three days. Cytokines were measured in the culture medium supernatant using a Human Cytokine Premixed Magnetic Luminex Performance Assay. [Figure 42] Effect of LILRB2 blocking antibodies on the levels of IL-1β secreted by PBMCs stimulated with 10 ng / mL of the anti-CD3 activating antibody HIT3a. Data shown are from two donors and are representative of six donors (total N = 6 donors). PBMCs isolated from healthy donors were incubated (in duplicate) with HIT3a (Biolegend) and various concentrations of antibody for 3 days. Cytokines were measured in the culture medium supernatant using a Human Cytokine Premixed Magnetic Luminex Performance Assay. [Figure 43] Effect of LILRB2 blocking antibodies on the levels of IL-6 secreted by PBMCs stimulated with 10 ng / mL of the anti-CD3 activating antibody HIT3a. Data shown are from two donors and are representative of five donors (total N = 6 donors). PBMCs isolated from healthy donors were incubated (in duplicate) with HIT3a (Biolegend) and various concentrations of antibody for 3 days. Cytokines were measured in the culture medium supernatant using a Human Cytokine Premixed Magnetic Luminex Performance Assay. [Figure 44]Effect of LILRB2 blocking antibodies on the levels of CXCL2 secreted by PBMCs stimulated with 10 ng / mL of the anti-CD3 activating antibody HIT3a. Data shown are from two donors and are representative of six donors (total N = 6 donors). PBMCs isolated from healthy donors were incubated (in duplicate) with HIT3a (Biolegend) and various concentrations of antibody for three days. Cytokines were measured in the culture medium supernatant using a Human Cytokine Premixed Magnetic Luminex Performance Assay. [Figure 45] Effect of 10 μg / mL B2-19 antibody on monocyte-derived macrophage cell surface markers. CD14+CD16- monocytes isolated from human PBMCs from healthy donors were differentiated into macrophages in the presence of 100 ng / mL human CSF-1 for 6 days, followed by incubation with 100 ng / mL human CSF-1, 20 ng / mL human IL-4, and B2-19 antibody or isotype control for 24 hours. Cells were detached and stained for flow cytometry analysis (FACS Celesta) using standard protocols. Data shown are the fold change in geometric mean fluorescence intensity (MFI) for B2-19-treated cells relative to cells treated with isotype control. [Figure 46] Effect of 40 μg / mL of B2-19 antibody on cell surface expression of CD25 on CD8+ T cells. PBMCs isolated from healthy donors were incubated with 10 ng / mL of HIT3a (Biolegend) and 40 μg / mL of B2-19 antibody for 3 days. CD8+ T cells were analyzed for cell surface CD25 expression by flow cytometry analysis (FACS Celesta) using standard protocols. Data shown are the percent change in geometric mean fluorescence intensity (MFI) for cells treated with B2-19 relative to cells treated with an isotype control. [Figure 47] B2-19 antibody enhances cytokine and chemokine production in immature DCs treated with IL-10 for 2 days (to induce tolerogenic DCs). Each line represents results from a different donor. [Figure 48] B2-19 antibody enhanced the pro-inflammatory phenotype of LPS-treated DCs, as evidenced by changes in the expression of several cell surface markers. Each line represents results from a different donor. [Figure 49] The B2-19 antibody exhibits the expected pharmacokinetic profile (CL and half-life) of a human IgG4 dosed at 5 mg / kg in C57BL / 6J wild-type mice. [Figure 50] Figures 50A-B. (Figure 50A) B2-19 antibody monotherapy reduces tumor growth rate in humanized NSG-SGM3 mice xenografted with the SK-MEL-5 melanoma cell line. (Figure 50B) B2-19 antibody monotherapy causes tumor growth inhibition in humanized NSG-SGM3 mice xenografted with the SK-MEL-5 melanoma cell line. [Figure 51] Representative flow cytometry profiles showed that coated human ANGPTL2 (hANGPLT2) and mouse Angptl2 (mAngptl2) stimulated GFP expression in LILRB2 reporter cells, and B2-19 effectively blocked GFP expression in LILRB2 reporter cells activated by coated hANGPLT2 and mAngptl2. [Figure 52] Representative flow cytometry profiles showed that coated human CD1d (hCD1d) and mouse CD1d (mCD1d) stimulated GFP expression in LILRB2 reporter cells, and B2-19 effectively blocked GFP expression in LILRB2 reporter cells activated by coated hCD1d and mCD1d. DETAILED DESCRIPTION OF THE INVENTION

[0039] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The present inventors have determined that LILRB2 plays an essential role in regulating both innate and adaptive immunity. LILRB2 is expressed on several types of immune cells, such as normal monocytes, dendritic cells, granulocytes, and myeloid-derived suppressor cells (MDSCs). The present inventors have isolated a group of novel monoclonal antibodies that recognize the LILRB2 protein and can be used to treat cancer and autoimmune diseases. Within this group of anti-LILRB2 antibodies, there are examples of antagonists and agonists of LILRB2 signaling.

[0040] The following description of the present disclosure is intended to merely illustrate various aspects of the present disclosure. Therefore, the specific improvements discussed should not be construed as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, modifications, and improvements may be made without departing from the scope of the present disclosure, and it is understood that such equivalents are encompassed by the present invention. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0041] I. Definition It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of other forms of terms, such as "including," "includes," and "included," is not limiting. Furthermore, terms such as "component" or "ingredients" include both components and ingredients containing one unit and components and ingredients containing more than one subunit, unless specifically stated otherwise. Furthermore, the use of the term "portion" can include a portion of a moiety or the entire moiety.

[0042] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0043] As used herein, the term "about" when referring to a measurable value, such as an amount, time period, and the like, is intended to encompass a variation of up to ±10% from the specified value. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the disclosed subject matter. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed, at the very least, in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0044] The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to a target antigen, including, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. An "antibody" is a type of antigen-binding protein. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, although in some instances they may contain fewer chains, such as naturally occurring antibodies in camelids that contain only heavy chains. An antibody may be derived from only a single source or may be "chimeric," i.e., different portions of the antibody may be derived from two different antibodies, as described further below. Antigen-binding proteins, antibodies, or binding fragments may be produced in hybridomas, by recombinant DNA technology, or by enzymatic or chemical degradation of intact antibodies. Unless otherwise indicated, the term "antibody" includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are described below. Furthermore, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

[0045] Natural antibody structural units typically comprise a tetramer. Each such tetramer typically consists of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (in certain embodiments, approximately 25 kDa) and one full-length "heavy" chain (in certain embodiments, approximately 50-70 kDa). The amino-terminal portion of each chain typically contains a variable region of approximately 100-110 amino acids, which is typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that may be involved in effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. IgA is similarly further divided into subclasses, including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant regions are typically connected by a "J" region of about 12 or more amino acids, with the heavy chain also containing a "D" region of about 10 additional amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (incorporated by reference in its entirety for all purposes). Each light chain / heavy chain variable region pair typically forms an antigen-binding site.

[0046] The term "variable region" or "variable domain" refers to a portion of an antibody's light and / or heavy chain, typically comprising approximately the amino-terminal 120-130 amino acids in the heavy chain and the amino-terminal 100-110 amino acids in the light chain. In certain embodiments, the variable regions of different antibodies vary significantly in amino acid sequence, even among antibodies of the same species. The variable region of an antibody typically determines the specificity of a particular antibody for its target.

[0047] Variable regions typically share the same general structure: relatively conserved framework regions (FRs) linked by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs of the two chains of each pair are typically aligned by the framework regions, enabling binding to a specific epitope. Both light and heavy chain variable regions typically contain the following domains, from N- to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is typically made according to the definitions of the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987), or Chothia et al., Nature, 342:878-883 (1989).

[0048] In certain embodiments, the antibody heavy chain binds to the antigen in the absence of the antibody light chain. In certain embodiments, the antibody light chain binds to the antigen in the absence of the antibody heavy chain. In certain embodiments, the binding region of the antibody binds to the antigen in the absence of the antibody light chain. In certain embodiments, the binding region of the antibody binds to the antigen in the absence of the antibody heavy chain. In certain embodiments, each variable region specifically binds to the antigen in the absence of other variable regions.

[0049] In certain embodiments, the definitive description of CDR and the identification of the residues that constitute antibody binding site are achieved by elucidating the structure of antibody and / or the structure of antibody-ligand complex.In certain embodiments, this can be achieved by any of various techniques known to those skilled in the art, such as X-ray crystallography.In certain embodiments, various analytical methods can be used to identify or roughly determine CDR regions.Examples of such methods include, but are not limited to, Kabat's definition, Chothia's definition, AbM's definition, and contact definition.

[0050] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, for example, Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but takes into account the location of certain structural loop regions. See, for example, Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses a comprehensive set of computer programs produced by the Oxford Molecular Group to model antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition uses a combination of knowledge databases and ab initio methods to model the tertiary structure of an antibody from its primary sequence, as described in Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl, 3:194-198 (1999). The contact definition is based on an analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol, 5:732-45 (1996).

[0051] By convention, the CDR regions in the heavy chain are typically referred to as H1, H2, and H3, numbered sequentially from the amino terminus to the carboxy terminus, and the CDR regions in the light chain are typically referred to as L1, L2, and L3, numbered sequentially from the amino terminus to the carboxy terminus.

[0052] The term "light chain" includes full-length light chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length light chain contains a variable region domain, VL, and a constant region domain, CL. The variable region domain of a light chain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0053] The term "heavy chain" includes full-length heavy chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length heavy chain contains a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino terminus of the polypeptide, the CH domain is at the carboxyl terminus, and CH3 is closest to the carboxy terminus of the polypeptide. The heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0054] Bispecific or bifunctional antibodies are typically artificial hybrid antibodies with two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by various methods, including, but not limited to, hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai et al., Clin. Exp. Immunol, 79:315-321(1990); Kostelny et al., J. Immunol, 148:1547-1553(1992).

[0055] The term "antigen" refers to a substance that can induce an adaptive immune response. Specifically, an antigen is a substance that serves as a target for a receptor of the adaptive immune response. Typically, an antigen is a molecule that binds to an antigen-specific receptor that is not itself capable of inducing an immune response in the body. Antigens are usually proteins and polysaccharides, and less frequently lipids. Suitable antigens include, but are not limited to, bacteria (external membrane, capsule, cell wall, flagella, fimbria, and toxins), viruses, and parts of other microorganisms. Antigens also include tumor antigens, e.g., antigens generated by mutations in tumors. As used herein, antigens also include immunogens and haptens.

[0056] As used herein, "antigen binding protein" ("ABP") refers to any protein that binds to a specific target antigen. In this application, the specific target is the LILRB protein or a fragment thereof. "Antigen binding proteins" include, but are not limited to, antibodies and antigen-binding fragments thereof. Peptibodies are another example of antigen binding proteins.

[0057] As used herein, the term "antigen-binding fragment" refers to a portion of a protein that can specifically bind to an antigen. In certain embodiments, the antigen-binding fragment is derived from an antibody or any other antibody fragment that contains one or more CDRs that bind to the antigen but do not comprise the intact native antibody structure. In certain embodiments, the antigen-binding fragment is not derived from an antibody but from a receptor. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, single-domain antibodies (sdAbs), camelid antibodies or nanobodies, domain antibodies, and bivalent domain antibodies. In certain embodiments, the antigen-binding fragment can bind to the same antigen as the parent antibody. In certain embodiments, the antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies. In certain embodiments, the antigen-binding fragment is derived from a receptor and contains one or more mutations. In certain embodiments, the antigen-binding fragment does not bind to the natural ligand of the receptor from which the antigen-binding fragment is derived.

[0058] A "Fab fragment" contains one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0059] A "Fab' fragment" contains one light chain and a portion of one heavy chain containing the VH domain and the CH1 domain, and also containing the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bond formation between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0060] An "F(ab')2 fragment" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. An F(ab')2 fragment is thus composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0061] The "Fc" region comprises two heavy chain fragments comprising the CH1 and CH2 domains of an antibody, held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0062] The "Fv region" comprises the variable regions of both the heavy and light chains, but lacks the constant regions.

[0063] A "single-chain antibody" is an Fv molecule in which the heavy and light chain variable regions are connected by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

[0064] A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently linked by a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0065] A "bivalent antigen-binding protein" or "bivalent antibody" comprises two antigen-binding sites. In some instances, the two binding sites have the same antigen specificity. Bivalent antigen-binding proteins and bivalent antibodies can be bispecific. See below. Bivalent antibodies other than "multispecific" or "multifunctional" antibodies are understood to, in certain embodiments, typically have each of their binding sites identical.

[0066] A "multispecific antigen-binding protein" or "multispecific antibody" is one that targets more than one antigen or epitope.

[0067] "Bispecific," "dual-specific," or "bifunctional" antigen-binding proteins or antibodies are hybrid antigen-binding proteins or antibodies, each having two different antigen-binding sites. Bispecific antigen-binding proteins and antibodies are a type of multispecific antigen-binding protein antibody and can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes, which can be present on the same or different protein targets.

[0068] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, such as those described herein. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0069] An antibody that "specifically binds" or is "specific" for a particular polypeptide or an epitope on a particular polypeptide is an antibody that binds to that particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes. For example, a LILRB2-specific antibody of the present invention is specific for LILRB2. In some embodiments, an antibody that binds to LILRB2 has a specificity of ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 As used herein, the dissociation constant Kd is the ratio of the dissociation rate to the association rate (k M), which can be determined using any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (e.g., FACS). off / k onIn certain embodiments, the Kd value may be suitably determined by using flow cytometry.

[0070] The term "compete" when used in the context of antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof) that compete for the same epitope refers to competition between the antigen-binding proteins as determined by an assay in which the antigen-binding protein (e.g., antibody or antigen-binding fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., LILRB or a fragment thereof). Many types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, including, for example, solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIAs (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid-phase direct label assays; solid-phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); and solid-phase direct label RIAs using 1 to 125 labels (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15). These include solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either an unlabeled test antigen-binding protein or a labeled reference antigen-binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. The test antigen-binding protein is usually present in excess.Antigen-binding proteins identified by competitive assays (competitor antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein and antigen-binding proteins that bind to an adjacent epitope that is sufficiently proximal to the epitope bound by the reference antigen-binding protein so that steric hindrance occurs. Additional details regarding methods for determining competitive binding are provided in the Examples herein. Typically, when a competitor antigen-binding protein is present in excess, it inhibits (e.g., reduces) specific binding of the reference antigen-binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some examples, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0071] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. An epitope can be either a linear epitope or a conformational epitope. A linear epitope is formed by a continuous sequence of amino acids in an antigen and interacts with an antibody based on its primary structure. On the other hand, a conformational epitope is composed of discontinuous portions of the antigen's amino acid sequence and interacts with an antibody based on the antigen's 3D structure. Generally, an epitope is about 5 or 6 amino acids in length. Two antibodies can bind to the same epitope within an antigen if they exhibit competitive binding for the antigen.

[0072] A "cell," as used herein, can be prokaryotic or eukaryotic. Prokaryotic cells include, for example, bacteria. Eukaryotic cells include, for example, fungi, plant cells, and animal cells. Types of animal cells (e.g., mammalian cells or human cells) include, for example, cells from the circulatory / immune system or organs, such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, granulocytes (e.g., basophilic granulocytes, eosinophilic granulocytes, neutrophilic granulocytes, and hypersegmented neutrophils), monocytes or macrophages, erythroid cells (e.g., reticulocytes), mast cells, thrombocytes or megakaryocytes, and dendritic cells; cells from the endocrine system or organs, such as thyroid cells (e.g., thyroid epithelial cells, parafollicular cells), parathyroid cells (e.g., parathyroid chief cells, eosinophilic cells), adrenal cells (e.g., chromaffin cells), and pineal cells (e.g., pinealocytes); Cells from the nervous system or organs, such as glioblasts (e.g., astrocytes and oligodendrocytes), microglia, magnocellular neurosecretory cells, astrocytes, Boettcher cells, and pituitary cells (e.g., gonadotropes, adrenocorticotropes, thyrotropes, somatotropes, and prolactinocytes); cells from the respiratory system or organs, such as alveolar epithelial cells (type I pneumocytes and type II pneumocytes), Clara cells, goblet cells, and alveolar macrophages; cells from the circulatory system or organs (e.g., cardiac myocytes and pericytes); cells from the digestive system or organs, such as gastric chief cells, parietal cells, goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells, enteroendocrine cells, enterochromaffin cells, APUD cells, and liver cells (e.g., hepatocytes and Kupffer cells);Cells from the integumentary system or organs, such as bone cells (e.g., osteoblasts, osteocytes, and osteoclasts), dental cells (e.g., cementoblasts and ameloblasts), cartilage cells (e.g., chondrocytes and chondrocytes), skin / hair cells (e.g., trichocytes, keratinocytes, and melanocytes (nevus cells), muscle cells (e.g., myocytes), adipocytes, fibroblasts, and tenocytes; cells from the urinary system or organs (e.g., podocytes, juxtaglomerular cells, intraglomerular mesangial cells, extraglomerular mesangial cells, renal proximal tubule brush border cells, and macula densa cells); and cells from the reproductive system or organs (e.g., sperm, Sertoli cells, Leydig cells, eggs, oocytes). Cells can be normal, healthy cells, or diseased or unhealthy cells (e.g., cancer cells). Cells further include mammalian zygotes or stem cells, including embryonic stem cells, fetal stem cells, induced pluripotent stem cells, and adult stem cells. Stem cells are cells that can undergo cycles of cell division while maintaining an undifferentiated state and differentiate into specialized cell types. Stem cells can be pluripotent stem cells, multipotent stem cells, oligopotent stem cells, and unipotent stem cells, any of which can be derived from somatic cells. Stem cells can also include cancer stem cells. Mammalian cells can be rodent cells, e.g., mouse, rat, or hamster cells. Mammalian cells can be lagomorph cells, e.g., rabbit cells. Mammalian cells can also be primate cells, e.g., human cells.

[0073] The term "chimeric antigen receptor" or "CAR," as used herein, refers to an artificially constructed hybrid protein or polypeptide containing an antibody antigen-binding domain (e.g., a single-chain variable fragment (scFv)) linked to a domain or signaling domain that activates immune cells, e.g., T cells or NK cells, e.g., a T cell signaling or T cell activation domain (see, e.g., Kershaw et al., supra; Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724(1993); and Sadelain et al., Curr. Opin. Immunol. 21(2):215-223(2009)). CARs take advantage of the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of immune cells to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition endows CAR-expressing immune cells with the ability to recognize antigens independently of antigen processing, thereby bypassing a major mechanism of tumor evasion. Additionally, when expressed in T cells, CARs advantageously do not dimerize with the endogenous T cell receptor (TCR) alpha and beta chains.

[0074] As used herein, "essentially free" with respect to a specified component means that none of the specified components are intentionally incorporated into the composition and / or are present only as contaminants or in trace amounts. The total amount of the specified component resulting from any unintentional contamination of the composition is therefore well below 0.05%, preferably less than 0.01%. Most preferred are compositions in which no amount of the specified component can be detected using standard analytical methods.

[0075] The term "host cell" means a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not they are identical in morphology or genetic make-up to the original parent cell, so long as the gene of interest is present.

[0076] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percent of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G, 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0077] In calculating percent identity, the sequences to be compared are typically aligned to maximize the match between the sequences. An example of a computer program that can be used to determine percent identity is the GCG program package, including GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or polynucleotides whose percent sequence identity is to be determined. The sequences are aligned for the optimal match of their respective amino acids or nucleotides (the "matched span" determined by the algorithm). A comparison matrix, such as PAM 250 or BLOSUM 62, is used in conjunction with the algorithm, along with a gap opening penalty (calculated as 3 times the average diagonal, where "average diagonal" is the average of the diagonals of the comparison matrix used, and "diagonal" is the score or number assigned to each perfect amino acid match by a specific comparison matrix) and a gap extension penalty (usually 1 / 10 times the gap opening penalty). In certain embodiments, standard comparison matrices (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919 for the BLOSUM 62 comparison matrix) are also used by the algorithm.

[0078] Examples of parameters that can be used to determine percent identity of polypeptide or nucleotide sequences using the GAP program can be found in Needleman et al., 1970, J. Mol. Biol. 48:443-453.

[0079] Certain alignment schemes for aligning two amino acid sequences may result in matching only short regions of the two sequences, and this small aligned region may have very high sequence identity even if there is no significant relationship between the two full-length sequences. Thus, the selected alignment method (GAP program) can be adjusted, if so desired, to result in alignment over at least 50 or other number of contiguous amino acids of the target polypeptide.

[0080] The term "linked," as used herein, refers to association via intramolecular interactions, such as covalent, metallic, and / or ionic bonds, or intermolecular interactions, such as hydrogen bonds or non-covalent bonds.

[0081] Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) is a protein encoded by the LILRB2 gene in humans. This gene is a member of the leukocyte immunoglobulin-like receptor (LIR) family found in a gene cluster on chromosome 19q13.4. The encoded protein belongs to the subfamily B class of LIR receptors, which contain two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). This receptor is expressed on myeloid cells; it binds to multiple types of ligands, including HLA class I molecules, ANGPTL, myelin inhibitors (e.g., Nogo66, MAG, OMgp), and beta-amyloid; and it transmits negative signals that inhibit stimulation of the immune response. It is thought to regulate inflammatory responses and cytotoxicity, helping to focus the immune response and limit autoreactivity.

[0082] The term "operably linked" refers to an arrangement of components configured so that the components so described perform their normal function. Thus, a given signal peptide operably linked to a polypeptide directs the secretion of the polypeptide from a cell. In the case of a promoter, a promoter operably linked to a coding sequence directs the expression of the coding sequence. A promoter or other control elements need not be contiguous with a coding sequence, so long as they function to direct its expression. For example, there can be intervening untranslated but transcribed sequences between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0083] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive, provided that this disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second, or more.

[0084] The terms "polynucleotide" or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. The nucleotides that make up a polynucleotide may be ribonucleotides or deoxyribonucleosides or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates, and phosphoramidates.

[0085] The term "polypeptide" or "protein" refers to a polymer having the amino acid sequence of a native protein, i.e., a protein produced by a naturally occurring non-recombinant cell, or it includes molecules produced by genetically engineered or recombinant cells and having the amino acid sequence of a native protein or molecules having one or more amino acid deletions, additions, and / or substitutions of the native sequence. The terms also include amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. The terms "polypeptide" and "protein" specifically encompass LILRB antigen-binding proteins, antibodies, or sequences having one or more amino acid deletions, additions, and / or substitutions of antigen-binding proteins. The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to the full-length native protein. Such fragments can also contain modified amino acids compared to the native protein. In certain embodiments, the fragments are about 5-500 amino acids in length. For example, a fragment may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies, such as binding domains. In the case of LILRB-binding antibodies, useful fragments include, but are not limited to, CDR regions, heavy and / or light chain variable domains, portions of antibody chains containing two CDRs, or only the variable regions thereof.

[0086] Pharmaceutically acceptable carriers useful in the present invention are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually comprise injectable fluids containing pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, or glycerol, as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the administered pharmaceutical composition may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate.

[0087] As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, a subject is a human being. A subject may be a patient, which refers to a human who presents to a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0088] The term "therapeutically effective amount" or "effective dosage" as used herein refers to a dosage or concentration of a drug that is effective for treating a disease or condition. For example, with respect to the use of the monoclonal antibody or antigen-binding fragment thereof disclosed herein to treat cancer, a therapeutically effective amount is a dosage or concentration of the monoclonal antibody or antigen-binding fragment thereof that can reduce tumor volume, eradicate all or part of a tumor, inhibit or delay tumor growth or cancer cell infiltration into other organs, inhibit the growth or proliferation of cells that mediate a cancerous condition, inhibit or delay metastasis of tumor cells, ameliorate any symptoms or markers associated with a tumor or cancerous condition, prevent or delay the onset of a tumor or cancerous condition, or some combination thereof.

[0089] As used herein, "treating" a condition or "treatment" includes preventing or alleviating the condition, slowing the onset or rate of onset of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0090] As used herein, a "vector" refers to a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may contain a nucleic acid sequence that enables its replication in the host cell, such as an origin of replication. A vector may also contain one or more therapeutic genes and / or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform, or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell. A vector may also contain materials that assist in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating, etc.

[0091] II. LILRB2-related diseases LILRB2 has been identified as a key regulator of myeloid cell phenotype. Activation of LILRB2 suppresses the pro-inflammatory activity of myeloid cells. Myeloid cells with an inhibitory / anti-inflammatory phenotype can downregulate T cell activation, proliferation, and cytotoxic activity, and modulation of LILRB2 has potential therapeutic uses in conditions and disorders including cancer, autoimmune diseases, and inflammatory diseases.

[0092] Hyperproliferative diseases can be associated with any disease that causes cells to begin to multiply uncontrollably, the prototypical example being cancer.

[0093] Examples of cancers can be generally categorized into solid tumors and hematological malignancies. Solid tumors include adrenal gland cancer, bile duct cancer, bone cancer, brain cancer (e.g., astrocytoma, brain stem glioma, craniopharyngioma, ependymoma, hemangioblastoma, medulloblastoma, meningioma, oligodendroglioma, spinal axis tumor), breast cancer (including acoustic neuroma, basal breast cancer, ductal carcinoma, and lobular breast cancer), cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer (including Wilms' tumor), liver cancer (including hepatocellular carcinoma (HCC)), lung cancer (bronchogenic carcinoma, non-small cell lung cancer (squamous / non-squamous), bronchioloalveolar cell lung cancer, papillary gland cancer, and thyroid cancer). cancer), mesothelioma, melanoma, Merkel cell carcinoma, nasopharyngeal carcinoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, penile cancer, pinealoma, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma (including chondrosarcoma, Ewing's sarcoma, fibrosarcoma, leiomyosarcoma, liposarcoma, myxosarcoma, osteogenic sarcoma, rhabdomyosarcoma, synovial sarcoma), skin cancer (including basal cell carcinoma, sebaceous carcinoma, and squamous cell carcinoma), testicular cancer (including seminoma), thymic carcinoma, thyroid cancer (e.g., medullary thyroid carcinoma, papillary thyroid carcinoma), uterine cancer, and vaginal cancer.

[0094] Hematological malignancies include blastic plasmacytoid dendritic cell neoplasm (BPDCN), heavy chain disease, leukemia (acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML) (including but not limited to acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic leukemia or M5 AML), B-cell leukemia, chronic lymphoblastic leukemia (CLL), chronic myelomonocytic leukemia (CMML), chronic myeloid leukemia (CML), pre-B-cell acute lymphocytic leukemia (pre-B-cell acute lymphocytic leukemia), and leukemia (pre-B-cell acute lymphocytic leukemia). ALL), diffuse large B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, hairy cell leukemia, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell / histiocytocyte-rich B-cell lymphoma), lymphomas (including but not limited to Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia), multiple myeloma (MM), myelodysplastic syndromes (MDS), myeloproliferative neoplasms, and polycythemia vera.

[0095] Immunotherapy holds great promise for achieving long-lasting antitumor effects. Blockade of immune checkpoints PD-1 and CTLA-4 has been successful in treating some types of cancer but not others. These immunotherapies target inhibitory molecules on T cells to reactivate dysfunctional T cells within the tumor microenvironment (TME). Other populations of immune cells, including monocytic cells, are present in the TME in even greater numbers than T cells. In fact, monocyte-derived macrophages are the most abundant immune cell population in tumor tissue. These innate cells have the ability to kill tumor cells and prime or reactivate T cells, but they become dysfunctional in the TME and become MDSCs and tumor-associated macrophages (TAMs), which support tumor development and suppress immune surveillance and attack. MDSCs, including monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs), are heterogeneous populations of immature myeloid cells that do not terminally differentiate. TAMs are a mixed macrophage population in the TME. They are anti-inflammatory and correlate with poor prognosis. Despite phenotypic plasticity, MDSCs and TAMs are defined by their immunosuppressive functions. Removing, reprogramming, or blocking the trafficking of these immunosuppressive monocytic cells is becoming an attractive anticancer therapeutic strategy.

[0096] LILRB2 is expressed on MDSCs and TAMs in the TME. Therapeutic blockade of LILRB2 in myeloid-rich solid tumors has the potential to reactivate or enhance antitumor immune responses in patients with non-responsive / relapsed disease to T cell checkpoint inhibitors.

[0097] LILRB2 expression on myeloid cells may regulate systems involved in autoimmune and inflammatory diseases. Therapeutic activation or agonization of LILRB2 has the potential to treat autoimmune or inflammatory diseases.

[0098] Autoimmune or inflammatory diseases include acquired immune deficiency syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac sprue-dermatitis herpetiformis; chronic fatigue immune deficiency syndrome (CFIDS), and chronic inflammatory demyelinating polyneuropathy. CIPD, cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia anemia), polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, systemic sclerosis, progressive systemic sclerosis (PSS), systemic sclerosis (SS), Sjögren's syndrome, stiff-man syndrome, systemic lupus erythematosus (SLE), Takayasu's arteritis, temporal arteritis / giant cell arteritis, Inflammatory bowel disease (IBD), ulcerative colitis, Conn's disease, intestinal mucosal inflammation, colitis-associated wasting disease, uveitis, vitiligo and Wegener's granulomatosis, Alzheimer's disease, asthma, atopic allergy, allergies, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemia, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy, ventilator-induced lung injury, viral infections, and autoimmune diabetes, etc. Inflammatory disorders include, for example, chronic and acute inflammatory disorders.

[0099] III. Monoclonal Antibodies and Their Production The monoclonal antibodies described herein can be prepared using standard methods, followed by screening, characterization, and functional evaluation. After sequencing the variable regions, they can be subcloned into human expression vectors to produce chimeric antibody genes, which are then expressed and purified. These chimeric antibodies can be tested in antigen binding, signal transduction blockade, and xenotransplantation experiments. The monoclonal antibodies described herein can also be prepared using phage display methods, in which a large library of phage-displayed human scFvs is panned against the target protein. The human scFvs selected to specifically bind to the target protein can be sequenced and then subcloned into human expression vectors to produce the desired human antibodies.

[0100] A. General method It will be understood that monoclonal antibodies that bind to LILRB2 have several uses. Uses include the manufacture of diagnostic kits for use in cancer detection and diagnosis, as well as cancer therapy. In these contexts, such antibodies can be linked to diagnostic or therapeutic agents, and can be used as capture agents or competitors in competitive assays, or can be used individually without binding additional agents. Antibodies can be mutated or modified, as further discussed below. Methods for preparing and characterizing antibodies are well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; US Patent 4,196,265).

[0101] Classical methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as methods for preparing polyclonal antibodies. The first step in both of these methods is immunization of a suitable host. As is well known in the art, a given composition for immunization can vary in its immunogenicity. Therefore, it is often necessary to enhance the host immune system, which can be achieved by linking a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins, such as ovalbumin, mouse serum albumin, or rabbit serum albumin, can also be used as carriers. Means for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bisdiazotized benzidine. As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of nonspecific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0102] The amount of immunogen composition used in the production of polyclonal antibodies varies depending on the nature of the immunogen as well as the animal used for immunization. Various routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal). The production of polyclonal antibodies can be monitored by sampling the blood of the immunized animal at various times after immunization. A second booster injection may be given. The process of boosting and titration is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled to isolate and store serum, and / or the animal can be used to generate MAbs.

[0103] After immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in MAb generation protocols. These cells can be obtained from biopsied spleens or lymph nodes, or from circulating blood. Antibody-producing B lymphocytes from the immunized animal are then fused with immortal myeloma cells, generally cells of the same species as the immunized animal, or human or human / mouse chimeric cells. Myeloma cell lines suitable for use in hybridoma production fusion procedures are preferably non-antibody-producing, have high fusion efficiency, and possess enzyme deficiencies that render them unable to grow in certain selective media that support the growth of only the desired fused cells (hybridomas). As known to those skilled in the art, any of a number of myeloma cell types can be used (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984).

[0104] Methods for generating hybrids between antibody-producing spleen or lymph node cells and myeloma cells typically involve mixing somatic cells with myeloma cells in a 2:1 ratio, although the ratio can vary from about 20:1 to about 1:1, respectively, in the presence of one or more agents (chemical or electrical) that promote cell membrane fusion. A fusion method using Sendai virus was described by Kohler and Milstein (1975; 1976), and a fusion method using polyethylene glycol (PEG), e.g., 37% (v / v) PEG, was described by Gefter et al. (1977). Electrically induced fusion methods are also suitable (Goding, pp. 71-74, 1986). The fusion procedure typically involves mixing approximately 1 × 10 -6 ~1×10 -8 This produces viable hybrids at a low frequency. However, this is not a problem because viable fused hybrids differentiate from parental unfused cells (especially unfused myeloma cells, which usually continue to divide indefinitely) by culturing in a selective medium. Selective media are generally media containing agents that block the de novo synthesis of nucleotides in tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block the de novo synthesis of both purines and pyrimidines, while azaserine blocks only the synthesis of purines. When aminopterin or methotrexate is used, hypoxanthine and thymidine are added to the medium as a source of nucleotides (HAT medium). When azaserine is used, hypoxanthine is added to the medium. If the B cell source is an Epstein-Barr virus (EBV) transformed human B cell line, ouabain is added to remove EBV transformed cells that have not fused to myeloma.

[0105] The preferred selective medium is HAT or HAT containing ouabain. Only cells capable of operating the nucleotide salvage pathway can survive in HAT medium. Myeloma cells lack key enzymes in the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), and cannot survive. B cells can operate this pathway, but they have a limited lifespan in culture and generally die within about two weeks. Therefore, the only cells that can survive in selective medium are hybrids formed from myeloma and B cells. When the source of B cells used for fusion is an EBV-transformed B cell line, as in this case, ouabain is also used for drug selection of the hybrid, since EBV-transformed B cells are sensitive to drug killing, while the myeloma partner used is selected to be ouabain-resistant.

[0106] Culture yields a population of hybridomas from which specific hybridomas are selected. Hybridoma selection is typically performed by culturing diluted cells to single clones in microtiter plates and then testing the supernatants of individual clones (after approximately 2–3 weeks) for the desired reactivity. Assays should be sensitive, simple, and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, or plaque or dot immunobinding assays. Selected hybridomas are then serially diluted or single-cell sorted by flow cytometry sorting, cloned into individual antibody-producing cell lines, and the clones can then be propagated indefinitely to provide mAbs. Cell lines can be exploited for MAb production in two basic ways: A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse). Optionally, the animal can be primed with a hydrocarbon, particularly an oil such as pristane (tetramethylpentadecane), prior to injection. When human hybridomas are used in this method, they are best injected into immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animals develop tumors secreting the specific monoclonal antibodies produced by the fused cell hybrids. The animal's body fluids, such as serum or ascites fluid, can then be harvested to provide high concentrations of MAbs. Individual cell lines can also be cultured in vitro, where MAbs are naturally secreted into the culture medium, from which they can be easily obtained in high concentrations. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in the cell supernatant. To optimize the ability to recover highly pure human monoclonal immunoglobulins, cell lines can be adapted for growth in serum-free medium.

[0107] MAbs produced by either means may be further purified, if necessary, using filtration, centrifugation, and various chromatographic methods, such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the present disclosure can be obtained from the purified monoclonal antibodies by methods including digestion with enzymes, such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0108] It is also contemplated that a molecular cloning approach may be used to generate monoclonal antibodies. For this purpose, RNA is isolated from hybridoma strains, antibody genes are obtained by RT-PCR, and cloned into immunoglobulin expression vectors. Alternatively, a combinatorial immunoglobulin phagemid library is prepared from RNA isolated from cell strains, and phagemids expressing suitable antibodies are selected by panning using viral antigens. The advantage of this approach over traditional hybridoma technology is that it allows approximately 10 4 The advantages of this approach are that twice as many antibodies can be produced and screened, and that new specificities can be generated by combining heavy and light chains, further increasing the chances of finding a suitable antibody.

[0109] Recently, additional methods for generating mAbs have been developed, such as scFv phage display (see C. M. Hammers and J. R. Stanley, Antibody phage display: technique and applications, J. Invest. Dermatol (2014) 134:e17). Generally, a panel of human mAbs that bind to a target protein, such as human LILRB2, is generated by panning a large diversity human scFv phage-displayed antibody library.

[0110] To generate a human scFv phage-displayed antibody library, RNA is extracted from a selected cell source, such as peripheral blood mononuclear cells. The RNA is then reverse transcribed into cDNA, and the cDNA is used for PCR of the encoded antibody VH and VL chains. A defined set of primers specific for different VH and VL chain gene families allows for the amplification of all transcribed rearranged variable regions within a given immunoglobulin repertoire, reflecting all antibody specificities in a particular individual.

[0111] The VH and VL PCR products representing the antibody repertoire are ligated into a phage display vector engineered to express the VH and VL as scFvs fused to the pIII minor capsid protein of E. coli filamentous bacteriophage, originally derived from M13 bacteriophage, generating a library of phage, each of which expresses an scFv on its surface and harbors a vector carrying each nucleotide sequence.

[0112] The library is then screened for phages that bind to the target antigen through their expressed surface scFvs by a technique called biopanning. Briefly, the target protein is coated onto a solid phase for incubation with the phage library. After washing and elution, the antigen-enriched phages are recovered and used for the next round of phage panning. After at least three rounds of phage panning, single bacterial colonies are picked for phage ELISA and other functional / genetic analyses.

[0113] Positive hits are sequenced for the scFv region and converted into fully human IgG heavy and light chain constructs, which are then used to generate the mAb of interest using the methods described above. For example, the IgG expression plasmids are co-transfected into Expi293 cells using the transfection reagent PEI. After 7 days of expression, the supernatant is harvested and the antibody is purified by affinity chromatography using Protein A resin.

[0114] Other U.S. patents that teach the production of antibodies useful in the present disclosure, each of which is incorporated herein by reference, include U.S. Pat. No. 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Pat. No. 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Pat. No. 4,867,973, which describes antibody-therapeutic agent conjugates.

[0115] B. Antibodies of the Present Disclosure 1. Antibody against LILRB2 Antibodies or antigen-binding fragments thereof according to the present disclosure can be defined in a first instance by their binding specificity, in this case for LILRB2. One skilled in the art can determine whether an antibody falls within the scope of the present claims by assessing the binding specificity / affinity of a given antibody using techniques well known to those skilled in the art.

[0116] In one aspect, antibodies and antigen-binding fragments that specifically bind to LILRB2 are provided. In some embodiments, such antibodies modulate the activation of LILRB2 when bound to LILRB2. In certain embodiments, the antibodies or antigen-binding fragments activate LILRB2 when bound to LILRB2. In certain embodiments, the antibodies or antigen-binding fragments suppress the activation of LILRB2 when bound to LILRB2. In certain embodiments, the antibodies or antigen-binding fragments can specifically interfere with, specifically block, or reduce the interaction of LILRB2 with its binding partner when bound to LILRB2. In certain embodiments, the antibodies or antigen-binding fragments provided herein have the ability to inhibit the immunosuppressive activity of MDSCs and other solid tumor-infiltrating myeloid cells, such as tumor-associated macrophages (TAMs) and tolerogenic dendritic cells (DCs). In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically or selectively bind to human LILRB2.

[0117] In some embodiments, the antibody or antigen-binding fragment specifically binds to human LILRB2 and / or substantially inhibits binding of human LILRB2 to HLA-G, ANGPTL, SEMA4A by at least about 20%-40%, 40-60%, 60-80%, 80-85%, or more (e.g., by assays such as those disclosed in the Examples). -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13In some embodiments, the antibody or antigen-binding fragment has an IC50 for blocking HLA-G, ANGPTL, SEMA4A binding to LILRB2 of less than 10 uM, 10 uM to 1 uM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, or 10 pM to 1 pM.

[0118] In some embodiments, the antibodies or antigen-binding fragments provided herein have the clonal CDR pairs shown in Table 2.

[0119] In certain embodiments, antibodies may be defined by their variable sequences, including additional "framework" regions. Antibodies are characterized by cloned heavy and light chain amino acid sequence pairs from Appendices I and III. Furthermore, antibody sequences may vary from these sequences, particularly in regions outside the CDRs. For example, amino acids may be varied from those listed above at a given percentage of homology, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or amino acids may be varied from those listed above by allowing for conservative substitutions (discussed below). Each of the above applies to the amino acid sequences in Appendices I and III. In another embodiment, an antibody derivative of the present disclosure comprises VL and VH domains with up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more conservative or non-conservative amino acid substitutions, while still exhibiting desired binding and functional properties.

[0120] Although the antibodies of the present disclosure were generated as IgGs, it may be useful to modify the constant regions to alter their function. The constant region of an antibody typically mediates the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Thus, the term "antibody" includes intact immunoglobulins of IgA, IgG, IgE, IgD, and IgM types (and their subtypes), and the immunoglobulin light chains may be of the kappa or lambda type. Within the light and heavy chains, the variable and constant regions are connected by a 35-member "J" region of about 12 or more amino acids, with the heavy chain also containing a "D" region of about 10 additional amino acids. Generally, the constant regions of an antibody are described in Fundamental Immunology Ch. 7 (Paul, W., ed., 2002). nd ed. Raven Press, NY (1989).

[0121] The present disclosure further includes nucleic acids that hybridize to nucleic acids encoding the antibodies disclosed herein. Generally, nucleic acids hybridize under medium or high stringency conditions to nucleic acids encoding the antibodies disclosed herein, and also to nucleic acids encoding antibodies that maintain the ability to specifically bind to LILRB2. A first nucleic acid molecule is "hybridizable" to a second nucleic acid molecule when the first nucleic acid molecule in single-stranded form can anneal to the second nucleic acid molecule under appropriate conditions of temperature and solution ionic strength (Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3 rd(See, e.g., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001). Temperature and ionic strength conditions determine the "stringency" of hybridization. Typical medium-stringency hybridization conditions are 40% formamide, 5x or 6x SSC, and 0.1% SDS at 42°C. High-stringency hybridization conditions are 50% formamide, 5x or 6x SSC (0.15 M NaCl and 0.015 M Na citrate), 42°C, or optionally higher temperatures (e.g., 57°C, 59°C, 60°C, 62°C, 63°C, 65°C, or 68°C). Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for nucleic acid hybridization depends on the length of nucleic acid and the degree of complementarity, variables well known in the art.The greater the degree of similarity or homology between two nucleotide sequences, the higher the stringency that nucleic acid can hybridize.For hybrids longer than 100 nucleotides, a formula for calculating melting temperature has been derived (see Sambrook et al., supra).For hybridization with shorter nucleic acids, such as oligonucleotides, the position of mismatch becomes more important, and the length of oligonucleotide determines its specificity (see Sambrook et al., supra).

[0122] 2. Exemplary Epitopes and Competing Antigen-Binding Proteins In another aspect, the present disclosure provides epitopes to which anti-LILRB2 antibodies bind. In some embodiments, the epitopes bound by the antibodies described herein are useful. In certain embodiments, the epitopes provided herein can be used to isolate antibodies or antigen-binding proteins that bind to LILRB2. In certain embodiments, the epitopes provided herein can be used to generate antibodies or antigen-binding proteins that bind to LILRB2. In certain embodiments, the epitopes or sequences comprising the epitopes provided herein can be used as immunogens to generate antibodies or antigen-binding proteins that bind to LILRB2. In certain embodiments, the epitopes described herein or sequences comprising the epitopes described herein can be used to interfere with the biological activity of LILRB2.

[0123] In some embodiments, antibodies or antigen-binding fragments thereof that bind to any of the epitopes are particularly useful. In some embodiments, the epitopes provided herein modulate the biological activity of LILRB2 when bound by the antibody. In some embodiments, the epitopes provided herein activate LILRB2 when bound by the antibody. In some embodiments, the epitopes provided herein inhibit the activation of LILRB2 when bound by the antibody. In some embodiments, the epitopes provided herein block the interaction of LILRB2 with its binding partner when bound by the antibody.

[0124] In some embodiments, domains / regions containing residues that contact or are covered by antibodies can be identified by mutating specific residues in LILRB2 and determining whether the antibody can bind to the mutated LILRB2 protein. By making a large number of individual mutations, it is possible to identify residues that play a direct role in binding or residues that are sufficiently close to the antibody so that mutations can affect the binding of the antibody to the antigen. From knowledge of these amino acids, it is possible to elucidate the domains or regions of the antigen that contain residues that contact or are covered by the antigen-binding protein. Such domains can comprise the binding epitope of the antigen-binding protein.

[0125] In another aspect, the present disclosure provides antigen-binding proteins that compete with one of the exemplified antibodies or antigen-binding fragments that bind to the epitope described herein for specific binding to LILRB2. Such antigen-binding proteins may also bind to the same epitope as one of the exemplified antibodies or antigen-binding fragments herein, or to an overlapping epitope. Antigen-binding proteins that compete with or bind to the same epitope as the exemplified antibodies are expected to exhibit similar functional properties. Exemplary antibodies include those described above, such as those having the heavy and light chain variable regions and CDRs contained in Table 1, the heavy and light chains set forth in Appendices I and II, and the heavy and light chain coding regions set forth in Appendices II and IV.

[0126] C. Manipulation of antibody sequences In various embodiments, one may choose to engineer the sequence of an identified antibody for various reasons, such as improved expression, improved cross-reactivity, or reduced off-target binding. Below is a general discussion of relevant techniques for antibody engineering.

[0127] After culturing the hybridomas, cells can be lysed and total RNA extracted. After generating cDNA copies of the RNA using random hexamers in RT, PCR can be performed using a multiplexed mixture of PCR primers expected to amplify all human variable gene sequences. The PCR products can be cloned into the pGEM-T Easy vector and sequenced by automated DNA sequencing using standard vector primers. Antibodies recovered from hybridoma supernatants and purified by FPLC using a Protein G column can be used for binding and neutralization assays. Recombinant full-length IgG antibodies can be generated by subcloning the heavy and light chain Fv DNA from the cloning vector into an IgG plasmid vector, transfecting it into 293 Freestyle or CHO cells, and the antibodies can be recovered and purified from the supernatants of 293 or CHO cells.

[0128] The rapid availability of antibodies produced in the same host cell and cell culture method as the final cGMP manufacturing process has the potential to reduce the duration of method development programs. Lonza has developed a general method for the rapid production of small amounts (up to 50 g) of antibodies in CHO cells using pooled transfectants grown in CDACF medium. While somewhat slower than true transient systems, advantages include higher product concentrations and the use of the same host and process as the production cell line. Single-use bioreactors operated in fed-batch mode: In an example of growth and productivity of a GS-CHO pool expressing a model antibody in a single-use bag bioreactor culture (5 L working volume), a harvested antibody concentration of 2 g / L was achieved within 9 weeks of transfection.

[0129] Antibody molecules include, for example, fragments produced by proteolytic cleavage of mAbs (F(ab'), F(ab')2, etc.), or single-chain immunoglobulins that can be produced, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with each other, or with other antibody fragments or receptor ligands, to form "chimeric" binding molecules. Notably, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.

[0130] 1. Alteration of antigen binding In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody containing the same CDR sequences as those in the disclosed antibodies (e.g., a chimeric antibody or a CDR-grafted antibody). Alternatively, modifications, such as the introduction of conservative changes, into the antibody molecule may be desired. In making such modifications, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydropathic characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

[0131] It is also understood in the art that substitutions of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with the biological properties of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartic acid (+3.0±1), glutamic acid (+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur-containing amino acids: cysteine ​​(-1.0) and methionine (-1.3); hydrophobic nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5±1), alanine (-0.5), and glycine (0); Hydrophobic aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0132] It is understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity to produce a biologically or immunologically modified protein, with substitution of amino acids whose hydrophilicity values ​​are within ±2 being preferred, those within ±1 being particularly preferred, and those within ±0.5 being even more particularly preferred.

[0133] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration various of the above characteristics are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0134] The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functions can be achieved. For example, changing to IgG1 can enhance antibody-dependent cellular cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve binding valency.

[0135] The modified antibodies can be prepared by any technique known to those skilled in the art, including expression through standard molecular biology techniques or chemical synthesis of the polypeptide. Methods of recombinant expression are covered elsewhere in this document.

[0136] 2. Fc region modification The antibodies disclosed herein can also be engineered to contain modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or effector function (e.g., antigen-dependent cellular cytotoxicity). Furthermore, the antibodies disclosed herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, also to alter one or more functional properties of the antibody. Each of these aspects is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat. The antibodies disclosed herein also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Pat. No. 5,624,821; WO2003 / 086310; WO2005 / 120571; WO2006 / 0057702. Such modifications can be used to enhance or suppress various immune system responses, which can have beneficial effects in diagnosis and therapy. Modifications of the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and the addition of multiple Fc domains. Fc modifications can also alter the half-life of therapeutic antibodies, which allows for less frequent dosing, thus increasing convenience and reducing material usage. This mutation has been reported to eliminate heterogeneity between heavy chain disulfide bridges in the hinge region.

[0137] In one embodiment, the hinge region of CH1 is modified to increase or decrease the number of cysteine ​​residues in the hinge region. This approach is further described in U.S. Patent No. 5,677,425. Altering the number of cysteine ​​residues in the hinge region of CH1 can improve or decrease the stability of the antibody, for example, by facilitating assembly of the light and heavy chains. In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, as described in U.S. Patent No. 6,277,375, one or more of the following mutations can be introduced: T252L, T254S, T256F. Alternatively, to increase biological half-life, the antibody can be altered within the CH1 or CL region to contain salvage receptor binding epitopes from two loops of the CH2 domain of the IgG Fc region, as described in U.S. Patent Nos. 5,869,046 and 6,121,022. In yet another embodiment, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be substituted with a different amino acid residue so that the antibody has altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260.

[0138] In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the antibody's ability to fix complement. This approach is further described in PCT Publication WO 94 / 29351. In yet another example, the Fc region can be modified to include one or more of the following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 270, 272, 276 The antibody may be modified to enhance or reduce its ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to improve or reduce its affinity for Fcγ receptors by modifying one or more amino acids at positions 1, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is further described in PCT Publication WO 00 / 42072. Additionally, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described. Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. Additionally, the following combinations of mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A.

[0139] In one embodiment, the Fc region is modified to reduce the ability of the antibody to mediate effector function and / or increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of an antibody is modified by changing the residues at positions 243 and 264 to alanine. In one embodiment, the Fc region is modified to reduce the ability of the antibody to mediate effector function and / or increase anti-inflammatory properties by modifying residues 243, 264, 267, and 328.

[0140] In one embodiment, the Fc region is altered to abolish the ability of the antibody to mediate effector function by altering residues 243, 235, and 329 to alanine or glycine (L243A-L235A-P329G).

[0141] In yet another embodiment, the antibody comprises a specific glycosylation pattern. For example, an aglycosylated antibody can be prepared (i.e., the antibody lacks glycosylation). The glycosylation pattern of the antibody can be altered to, for example, increase the affinity or avidity of the antibody for an antigen. Such modification can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such glycosylation can increase the affinity or avidity of the antibody for an antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0142] Antibodies can also be prepared with glycosylation patterns comprising hypofucosylated or nonfucosylated glycans; hypofucosylated or nonfucosylated antibodies have a reduced amount of fucosyl residues on the glycans. Antibodies may also contain increased amounts of glycans with biantennary GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such modifications can be achieved, for example, by expressing the antibody in a host cell whose glycosylation pathway has been genetically engineered to produce a glycoprotein with a particular glycosylation pattern. These cells have been described in the art and can be used as host cells to express the recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)-fucosyltransferase), and therefore antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrate chains. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see U.S. Patent Application Publication No. 20040110704). As another example, EP1176195 describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation due to reduced or eliminated α-1,6 bond-related enzymes. EP1176195 also describes cell lines with low or no enzymatic activity that adds fucose to N-acetylglucosamine attached to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL PCT Publication WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked glycans, and also results in hypofucosylation of antibodies expressed in the host cells.Antibodies with modified glycosylation profiles can also be produced in chicken eggs, as described in PCT Publication WO 06 / 089231. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells, such as Lemna plant cells (U.S. Patent No. 7,632,983). Methods for producing antibodies in plant systems are disclosed in U.S. Patent Nos. 6,998,267 and 7,388,081. PCT Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), where antibodies expressed in the engineered cell lines exhibit increased biantennary GlcNac structures, resulting in increased ADCC activity of the antibodies.

[0143] Alternatively, fucosidase enzymes can be used to cleave the fucose residues from antibodies; for example, fucosidase α-L-fucosidase removes fucosyl residues from antibodies. The antibodies disclosed herein further include antibodies produced in lower eukaryotic host cells, particularly fungal host cells, such as yeast and filamentous fungi, that are genetically engineered to produce glycoproteins with mammalian- or human-like glycosylation patterns. A particular advantage of these genetically engineered host cells over currently used mammalian cell lines is the ability to control the glycosylation profile of glycoproteins produced in the cells, thereby producing glycoprotein compositions in which specific N-glycan structures predominate (see, for example, U.S. Patent Nos. 7,029,872 and 7,449,308). These genetically engineered host cells have been used to produce antibodies with predominantly specific N-glycan structures.

[0144] In addition, fungi, such as yeast or filamentous fungi, lack the ability to produce fucosylated glycoproteins, and therefore antibodies produced in such cells lack fucose unless the cells are further modified to contain an enzymatic pathway for producing fucosylated glycoproteins (see, e.g., PCT Publication WO2008112092). In certain embodiments, the antibodies disclosed herein further include antibodies produced in lower eukaryotic host cells and comprising fucosylated and non-fucosylated hybrid and complex N-glycans, including biantennary and multiantennary species, including, but not limited to, N-glycans such as GlcNAc(1-4)Man3GlcNAc2; Gal(1-4)GlcNAc(1-4)Man3GlcNAc2; NANA(1-4)Gal(1-4)GlcNAc(1-4)Man3GlcNAc2. In certain embodiments, the antibody compositions provided herein may comprise an antibody having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2; GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2. In certain aspects, the hybrid N-glycan is the predominant N-glycan species in the composition. In further aspects, the hybrid N-glycan is a particular N-glycan species that constitutes about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the hybrid N-glycans in the composition.

[0145] In certain embodiments, the antibody compositions provided herein comprise antibodies having at least one complex N-glycan selected from the group consisting of GlcNAcMan3GlcNAc2; GalGlcNAcMan3GlcNAc2; NANAGalGlcNAcMan3GlcNAc2; GlcNAc2Man3GlcNAc2; GalGlcNAc2Man3GlcNAc2; Gal2GlcNAc2Man3GlcNAc2; NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2. In certain aspects, the complex N-glycan is the predominant N-glycan species in the composition. In a further aspect, the complex N-glycan is a specific N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycans in the composition. In certain embodiments, the N-glycan is fucosylated. Generally, fucose is linked to GlcNAc at the reducing end of the N-glycan via α1,3-linkage, GlcNAc at the reducing end of the N-glycan via α1,6-linkage, Gal at the non-reducing end of the N-glycan via α1,2-linkage, GlcNAc at the non-reducing end of the N-glycan via α1,3-linkage, or GlcNAc at the non-reducing end of the N-glycan via α1,4-linkage.

[0146] Thus, in certain aspects of the above glycoprotein compositions, the glycoform is an α1,3-linked or α1,6-linked fucose, resulting in a glycoform selected from the group consisting of Man5GlcNAc2(Fuc), GlcNAcMan5GlcNAc2(Fuc), Man3GlcNAc2(Fuc), GlcNAcMan3GlcNAc2(Fuc), GlcNAc2Man3GlcNAc2(Fuc), GalGlcNAc2Man3GlcNAc2(Fuc), Gal2GlcNAc2Man3GlcNAc2(Fuc), NANAGal2GlcNAc2Man3GlcNAc2(Fuc), and NANA2Gal2GlcNAc2Man3GlcNAc2(Fuc); an α1,3-linked or α1,4-linked fucose, giving rise to a glycoform selected from the group consisting of GlcNAc(Fuc)Man5GlcNAc2, GlcNAc(Fuc)Man3GlcNAc2, GlcNAc2(Fuc1-2)Man3GlcNAc2, GalGlcNAc2(Fuc1-2)Man3GlcNAc2, Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2, NANAGal2GlcNAc2(Fuc1-2)Man3GlcNAc2, and NANA2Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2; or an α1,2-linked fucose, giving rise to a glycoform selected from the group consisting of Gal(Fuc)GlcNAc2Man3GlcNAc2, Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2, NANAGal2(Fuc1-2)GlcNAc2Man3GlcNAc2, and NANA2Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2.

[0147] In a further aspect, the antibody comprises high-mannose N-glycans, including, but not limited to, N-glycans consisting of the Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or Man3GlcNAc2 N-glycan structure. In a further aspect of the above, the complex N-glycans further include fucosylated and non-fucosylated biantennary and multiantennary species. As used herein, the terms "N-glycan" and "glycoform" are used interchangeably and refer to N-linked oligosaccharides, such as those attached via an asparagine-N-acetylglucosamine linkage to an asparagine residue in a polypeptide. N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in a protein.

[0148] D. Single chain antibody Single-chain variable fragments (scFvs) are fusions of the variable regions of immunoglobulin heavy and light chains linked together with a short (usually serine or glycine) linker. These chimeric molecules retain the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide. This modification typically leaves the specificity unchanged. These molecules were historically generated to facilitate phage display, where it is highly convenient to express the antigen-binding domain as a single peptide. Alternatively, scFvs can be generated directly from subcloned heavy and light chains derived from hybridomas. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules and therefore the common binding site (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using protein L, as protein L interacts with the variable region of the kappa light chain.

[0149] Flexible linkers are generally composed of helix- and turn-promoting amino acid residues, such as alanine, serine, and glycine; however, other residues may also function. Tang et al. (1996) used phage display as a means to rapidly select specialized linkers for single-chain antibodies (scFv) from protein linker libraries. A random linker library was constructed in which genes for heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approximately 5×10 6 The tethers (10 distinct members) were displayed on filamentous phage and subjected to affinity selection with the hapten. The population of selected variants exhibited significantly increased binding activity while retaining considerable sequence diversity. Screening of 1054 individual variants subsequently yielded catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed the V as the only common feature of the selected tethers. H A conserved proline in two residues of the linker after the C-terminus of and numerous arginines and prolines in other positions were revealed.

[0150] The recombinant antibodies of the present disclosure may also include sequences or moieties that allow receptor dimerization or multimerization. Such sequences include sequences derived from IgA that allow for the formation of multimers in combination with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents that allow the combination of two antibodies, such as biotin / avidin.

[0151] In another embodiment, single-chain antibodies can be produced by linking the light and heavy chains of the receptor using a non-peptide linker or chemical unit. Generally, the light and heavy chains are produced in separate cells, purified, and then linked together in a suitable manner (i.e., the N-terminus of the heavy chain is attached to the C-terminus of the light chain via a suitable chemical crosslinker).

[0152] Cross-linking reagents, such as stabilizers and coagulants, are used to form molecular bridges that connect the functional groups of two different molecules.However, it is contemplated that the heteromeric complexes that are composed of the dimer or multimer of the same analog or different analogs can be produced.To link two different compounds in a step-by-step manner, heterobifunctional cross-linking agents can be used, which eliminates the formation of undesired homopolymers.

[0153] Exemplary heterobifunctional crosslinkers contain two reactive groups, one that reacts with primary amine groups (e.g., N-hydroxysuccinimide) and the other that reacts with thiol groups (e.g., pyridyl disulfide, maleimide, halogen, etc.). Through the primary amine reactive group, the crosslinker may react with a lysine residue of one protein (e.g., a selected antibody or fragment), and through the thiol reactive group, the crosslinker already attached to the first protein reacts with a cysteine ​​residue (free sulfhydryl group) of another protein (e.g., a selective agent).

[0154] It is preferable to use a crosslinker that has reasonable stability in blood.Many types of disulfide bond-containing linkers are known that can be successfully used to conjugate targeting agents and therapeutic / prophylactic agents.Linkers that contain sterically hindered disulfide bonds may provide greater stability in vivo and prevent the release of targeting peptides before reaching the site of action.Therefore, these linkers are a group of linking agents.

[0155] Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by adjacent benzene rings and methyl groups. The steric hindrance of the disulfide bond is thought to function to protect the bond from attack by thiolate anions, such as glutathione, that may be present in tissues and blood, thereby helping to prevent decoupling of the conjugate prior to delivery of the bound agent to the target site.

[0156] Like many other known cross-linking reagents, the SMPT cross-linking reagent offers the ability to cross-link functional groups such as the SH of cysteine ​​or primary amines (e.g., the epsilon-amino group of lysine). Another possible class of cross-linking reagents includes heterobifunctional photoreactive phenyl azides containing a cleavable disulfide bond, such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups, and the phenyl azide reacts nonselectively (by photolysis) with any amino acid residue.

[0157] In addition to hindered cross-linkers, unhindered linkers can also be used in accordance with the present invention. Other useful cross-linkers that are not thought to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[0158] U.S. Patent No. 4,680,338 describes bifunctional linkers useful for preparing conjugates of ligands with amine-containing polymers and / or proteins, particularly for forming antibody conjugates with chelators, drugs, enzymes, detectable labels, and the like. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that can be cleaved under a variety of mild conditions. This linker is particularly useful in that the agent of interest can be directly attached to the linker, and cleavage can result in release of the active agent. Specific uses include adding free amino or free sulfhydryl groups to proteins, such as antibodies, or drugs.

[0159] U.S. Patent No. 5,856,456 provides peptide linkers for use in connecting polypeptide components to prepare fusion proteins, such as single-chain antibodies. The linkers are up to about 50 amino acids in length, contain at least one proline residue after a charged amino acid (preferably arginine or lysine), and are characterized by greater stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers useful in various immunodiagnostic and separation techniques.

[0160] E. Purification In certain embodiments, the antibodies of the present disclosure may be purified. As used herein, the term "purified" is intended to refer to a composition that can be isolated from other components and that is purified to any degree compared to the state in which the protein can be obtained in nature. Thus, a purified protein also refers to a protein that has been released from the environment in which it can naturally occur. When the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms the majority of the composition, for example, constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0161] Protein purification techniques are well known to those skilled in the art. At one level, these techniques involve crude fractionation of the cellular milieu into polypeptide and non-polypeptide fractions. Once the polypeptide has been separated from other proteins, chromatographic and electrophoretic techniques can be used to further purify the polypeptide of interest to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of pure peptides include ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, or heat denaturation followed by centrifugation; gel filtration, reverse-phase, hydroxylapatite, and affinity chromatography; and combinations of such and other techniques.

[0162] In purifying the antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions.The polypeptide can be purified from other cellular components using an affinity column that binds to the tagged portion of the polypeptide.As is generally known in the art, the order in which various purification steps are performed may be changed, or certain steps may be omitted, and still result in a suitable method for preparing a substantially purified protein or peptide.

[0163] Typically, whole antibodies are fractionated using an agent that binds to the Fc portion of the antibody (i.e., protein A). Alternatively, the antigen may be used to simultaneously purify and select the appropriate antibodies. Such methods often utilize a selection agent bound to a support, such as a column, filter, or beads. The antibody is bound to the support, contaminants are removed (e.g., by washing), and the antibody is released by applying conditions (salt, heat, etc.).

[0164] Various methods for quantifying the degree of purification of a protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction or assessing the amount of polypeptide within a fraction by SDS / PAGE analysis. Another way to assess the purity of a fraction is to calculate the specific activity of the fraction and compare it to the specific activity of the initial extract, thereby calculating the degree of purity. The actual units used to express the amount of activity will, of course, depend on the particular assay technique chosen for purification and whether the expressed protein or peptide exhibits detectable activity.

[0165] It is known that the migration of polypeptides can vary, sometimes significantly, using different conditions of SDS / PAGE (Capaldi et al., 1977). Therefore, it will be understood that the apparent molecular weight of purified or partially purified expression products may vary under different electrophoretic conditions.

[0166] V. Cancer Treatment A. Formulation and Administration The present disclosure provides pharmaceutical compositions containing anti-LILRB2 antibodies and antigens for generating them. The compositions comprise a prophylactically or therapeutically effective amount of the antibody or fragment thereof and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, more specifically humans, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia. The term "carrier" refers to a diluent, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0167] If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations can contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences." Such compositions contain a prophylactically or therapeutically effective amount of an antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which may be oral, intravenous, intraarterial, buccal, intranasal, aerosol, bronchial inhalation, or delivery via mechanical ventilation.

[0168] The antibodies of the present disclosure described herein can be formulated for parenteral administration, e.g., for injection via intradermal, intravenous, intraarterial, intramuscular, subcutaneous, intratumoral, or intraperitoneal routes. Alternatively, antibodies may be administered topically directly to a mucous membrane, e.g., by nasal drops, inhalation, or nebulizer. Pharmaceutically acceptable salts include acid salts and salts formed with inorganic acids, e.g., hydrochloric acid or phosphoric acid, or organic acids, e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases, e.g., isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0169] Passive transfer of antibodies, known as artificially acquired passive immunity, generally involves the use of intravenous injections. Antibodies can be in the form of pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, high-titer human IVIG or IG from immunized or disease-recovering donors, and human or animal plasma or serum as monoclonal antibodies (MAbs). Such immunity is generally short-lived, and there are potential risks of hypersensitivity reactions and serum sickness, particularly from gamma globulins of non-human origin. However, passive immunization provides immediate protection. The antibody is formulated in a carrier suitable for injection, i.e., passable through a sterile needle.

[0170] Generally, the components of the compositions of the present disclosure are supplied separately or mixed together in unit dosage form, for example, as lyophilized powder or water-free concentrate, for example, in a hermetically sealed container, for example, an ampoule or sachet indicating the amount of active ingredient.When the composition is administered by injection, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0171] The compositions of the present disclosure can be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0172] B. Cell therapy In another aspect, the present disclosure provides an immune cell expressing a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen-binding fragment provided herein. In one embodiment, the CAR protein comprises, from N-terminus to C-terminus, a leader peptide, an anti-LILRB2 heavy chain variable domain, a linker domain, an anti-LILRB2 light chain variable domain, a human IgG1-CH2-CH3 domain, a spacer region, a CD28 transmembrane domain, a 4-1BB intracellular costimulatory signaling domain, and a CD3ζ intracellular T cell signaling domain.

[0173] Also provided is a method of immunotherapy comprising administering an effective amount of the immune cells of the present disclosure. In one embodiment, a medical disease or disorder is treated by transplantation of an immune cell population that induces an immune response. In certain embodiments of the present disclosure, cancer or an infectious disease is treated by transplantation of an immune cell population that induces an immune response. Provided herein is a method of treating or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of an antigen-specific cell therapy.

[0174] Immune cells can be T cells (for example, regulatory T cells, CD4+ T cells, CD8+ T cells, or γδ T cells), NK cells, invariant NK cells, NKT cells, or macrophages. In addition to the methods for producing and manipulating immune cells, the methods for using and administering the cells for adoptive cell therapy are also provided herein, and in adoptive cell therapy, the cells can be autologous or allogeneic. Therefore, immune cells can be used as immunotherapy, such as targeting cancer cells.

[0175] Immune cells can be isolated from subjects, particularly human subjects.Immune cells can be obtained from healthy human subjects, healthy volunteers, or healthy donors.Immune cells can be obtained from the subject of interest, for example, the subject suspected of having a specific disease or pathological condition, the subject suspected of having a predisposition to a specific disease or pathological condition, or the subject undergoing therapy for a specific disease or pathological condition.Immune cells can be collected from any location where they exist in subjects, including but not limited to blood, umbilical cord blood, spleen, thymus, lymph node, and bone marrow.Immune cells isolated can be used directly, or can be stored for a certain period of time, such as by freezing.

[0176] Immune cells may be enriched / purified from any tissue in which they exist, including, but not limited to, blood (including blood collected by blood banks or umbilical cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained through biopsy procedures. The tissues / organs from which immune cells are enriched, isolated, and / or purified may be isolated from both living and non-living subjects, and non-living subjects are organ donors. In certain embodiments, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood. In some aspects, immune cells isolated from umbilical cord blood have enhanced immunoregulatory capabilities, such as those measured by CD4 or CD8 positive T cell suppression. In certain aspects, immune cells are isolated from pooled blood, particularly pooled umbilical cord blood, for enhanced immunoregulatory capabilities. Pooled blood may be from two or more sources, for example, 3, 4, 5, 6, 7, 8, 9, 10, or more sources (e.g., donor subjects).

[0177] The population of immune cells can be obtained from the subject who needs therapy or suffers from a disease associated with reduced immune cell activity.Therefore, the cells are autologous to the subject who needs therapy.Alternatively, the population of immune cells can be obtained from a donor, preferably a histocompatible matched donor.The population of immune cells can be collected from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue where immune cells exist in the subject or donor.The immune cells can be isolated from a pool of subjects and / or donors, for example, pooled umbilical cord blood.

[0178] When the population of immune cells is obtained from a donor other than the subject, the donor is preferably allogeneic, but the obtained cells are subject-compatible in that they can be introduced into the subject.Allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible.To be subject-compatible, allogeneic cells can be treated to reduce immunogenicity.

[0179] Immune cells can be genetically engineered to express antigen receptors, such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, host cells (e.g., autologous or allogeneic T cells) are modified to express T cell receptors (TCRs) with antigen specificity for cancer antigens. In certain embodiments, NK cells are engineered to express TCRs. NK cells can also be engineered to express CARs. Multiple CARs and / or TCRs, such as those for different antigens, can be added to a single cell type, such as T cells or NK cells.

[0180] Suitable methods of modification are known in the art. See, for example, Sambrook et al., supra; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. For example, cells can be transduced to express T cell receptors (TCRs) with antigen specificity for cancer antigens using the transduction techniques described in Heemskerk et al. (2008) and Johnson et al. (2009).

[0181] In some embodiments, the cells contain one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors, and the genetically engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or sample obtained from the cell, e.g., obtained from another organism or cell that is not normally found, e.g., in the cell being engineered and / or in the organism from which such cell is derived. In some embodiments, the nucleic acid is non-naturally occurring, such as a nucleic acid not found in nature (e.g., chimeric).

[0182] C. Combination Therapy It may also be desirable to provide a combination therapy using the antibody of the present disclosure in combination with an additional anti-cancer therapy. These therapies are provided in a combined amount effective to achieve a reduction in one or more disease parameters. This method may involve contacting cells / subjects with both agents / therapies simultaneously, for example, using a single composition or pharmacological formulation containing both agents, or by simultaneously contacting cells / subjects with two separate compositions or formulations, one composition containing the antibody and the other composition containing the other agent.

[0183] Alternatively, the antibody may precede or follow the other treatment by intervals ranging from minutes to weeks. Generally, a long period of time should not pass between each delivery time point to ensure that the therapies can still exert a beneficial combined effect on the cell / subject. In such instances, it is contemplated to contact the cell with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay of only about 12 hours. However, in some situations, it may be desirable to significantly extend the duration of treatment, allowing tens of days (e.g., 2, 3, 4, 5, 6, or 7) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) to pass between each administration.

[0184] It may be desirable to administer multiple doses of either the anti-LILRB2 antibody or other therapy. Various combinations, where the antibody is "A" and the other therapy is "B," can be used, as exemplified below. TIFF0007767291000001.tif17128

[0185] Other combinations are contemplated. The methods and compositions of the present invention can be used to contact target cells or sites with an antibody and at least one other therapy to kill cells, inhibit cell proliferation, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells. These therapies are provided in a combined amount effective to kill or inhibit the growth of cancer cells. The method may involve contacting the cells / sites / subjects with the agents / therapies simultaneously.

[0186] Specific agents contemplated for combination therapy with the antibodies of the present disclosure include chemotherapy and hematopoietic stem cell transplantation. Chemotherapeutic agents include cytarabine (ara-C) and an anthracycline (most often daunorubicin), high-dose cytarabine alone, induction chemotherapy, usually an anthracycline plus all-trans retinoic acid (ATRA), histamine dihydrochloride (Cepren) and interleukin 2 (Proleukin) after completion of consolidation therapy, gemtuzumab ozogamicin (Mylotarg) for patients over 60 years old with relapsed AML who are not candidates for high-dose chemotherapy, clofarabine, as well as targeted therapy such as kinase inhibitors, farnesyltransferase inhibitors, decitabine, and inhibitors of MDR1 (multidrug resistance protein), or arsenic trioxide for relapsed acute promyelocytic leukemia (APL).

[0187] In certain embodiments, the agents for combination therapy are topoisomerase inhibitors, anthracycline topoisomerase inhibitors, anthracyclines, daunorubicin, nucleoside metabolic inhibitors, cytarabine, hypomethylating agents, low-dose cytarabine (LDAC), a combination of daunorubicin and cytarabine, injectable daunorubicin and cytarabine liposomes, Vixeos®, azacitidine, Vidaza®, decitabine, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine dihydrochloride, Sepren®, Interleukin-2, aldesleukin, Proleukin®, gemtuzumab ozogamicin, Mylotarg®, FLT-3 inhibitors, midostaurin, Rydapt®, clofarabine, farnesyltransferase inhibitors, decitabine, IDH1 inhibitors, ivosidenib, tibsovo®, IDH2 inhibitors, enasidenib, Idhifa®, Smoothened (SMO) inhibitors, glasdegib, arginase inhibitors, IDO inhibitors, epacadostat, BCL-2 inhibitors (BCL-2 inihbitor), venetoclax, Venclexta (registered trademark), platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitors, ibrutinib, Imbruvica (registered trademark), acalabrutinib, Calquence (registered trademark), zanubrutinib, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG3 antibody, ICOS antibody, TIGIT antibody, TIM3 antibody, CD40 antibody, 4-1BB antibody, CD47 antibody, SIRP1α antibody The therapeutic agent is one or more drugs selected from the group consisting of antibodies or fusion proteins, CD70 antibodies, CLL1 antibodies, CD123 antibodies, E-selectin antagonists, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to myeloid cell or NK cell surface markers, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant-derived alkaloids, hormone therapy agents, hormone antagonists, aromatase inhibitors, and P-glycoprotein inhibitors.

[0188] VI. Antibody Conjugates The antibodies of the present disclosure can be linked to at least one agent to form an antibody conjugate. To increase the effectiveness of antibody molecules as diagnostic or therapeutic agents, it is common practice to link, covalently bond, or complex at least one desired molecule or moiety to the antibody molecule. Such molecules or moieties may be, but are not limited to, at least one effector or reporter molecule. Effector molecules include molecules with desired activity, such as cytotoxic activity. Non-limiting examples of effector molecules attached to antibodies include toxins, antitumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelators, cytokines, growth factors, and oligo- or polynucleotides. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or ligands, such as biotin.

[0189] Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapeutics. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cell-killing drug. This approach combines the high specificity of MAbs for antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver their payload (drug) to tumor cells bearing concentrated levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in reduced toxicity and an improved therapeutic index. This approach was validated by the FDA's approval of two ADC drugs, Adcetris® (brentuximab vedotin) in 2011 and Kadcyla® (trastuzumab emtansine or T-DM1) in 2013. There are currently more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become more mature, the discovery and development of new ADCs increasingly depends on the identification and validation of new targets suitable for this approach and the generation of targeting MAbs. Two criteria for ADC targets are upregulated / high levels of expression in tumor cells and robust internalization.

[0190] Antibody conjugates are also preferred for use as diagnostic agents. Antibody diagnostics generally fall into two categories: those for use in in vitro diagnostics, such as in various immunoassays, and those for use in in vivo diagnostic protocols, commonly known as "antibody-directed imaging." Many suitable imaging agents are known in the art, as are methods for their attachment to antibodies (see, for example, U.S. Patent Nos. 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorescent dyes, NMR-detectable substances, and X-ray imaging agents.

[0191] In the case of paramagnetic ions, examples include ions such as chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III) and / or erbium(III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum(III), gold(III), lead(II), and especially bismuth(III).

[0192] For radioactive isotopes for therapeutic and / or diagnostic applications, astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 Cobalt, Copper 67 , 152 Eu, gallium 67 , 3 Hydrogen, iodine 123 , iodine 125 , iodine 131 ,indium 111 , 59 iron, 32 Phosphorus, rhenium 186 ,rhenium 188 , 75 Selenium, 35 Sulfur, technicium 99m and / or yttrium 90 Examples include: 125 I is often preferred for use in certain embodiments, and technicium 99m and / or indium 111are also often preferred due to their low energy and suitability for long-distance detection. Radiolabeled monoclonal antibodies of the present disclosure can be produced according to methods well known in the art. For example, monoclonal antibodies can be iodinated by contacting them with sodium and / or potassium iodide and a chemical oxidizing agent, such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the present disclosure can also be iodinated with technetium by a ligand exchange method, for example, by reducing pertechnetate with a stannous solution, chelating the reduced technetium to a Sephadex column, and applying the antibody to the column. 99m Alternatively, direct labeling techniques may be used, for example, by incubating pertechnetate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediate functional groups often used to attach radioisotopes that exist as metal ions to antibodies are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0193] Fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine, and / or Texas Red.

[0194] Another type of antibody conjugate contemplated in the present disclosure is primarily intended for in vitro use, in which the antibody is linked to an enzyme (enzyme tag) that produces a colored product upon contact with a secondary binding ligand and / or a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

[0195] Yet another known method for site-specific attachment of molecules to antibodies involves reacting the antibody with a hapten-based affinity tag. Essentially, the hapten-based affinity tag reacts with amino acids in the antigen-binding site, thereby disrupting this site and blocking specific antigen reaction. However, this can be disadvantageous because it results in loss of antigen binding by the antibody conjugate.

[0196] Molecules containing azide groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogs of purine nucleotides have been used as site-directed photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). 2- and 8-azido nucleotides have also been used to map nucleotide-binding domains in purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can be used as antibody binders.

[0197] Several methods for attaching or conjugating antibodies to their conjugate moieties are known in the art. Some attachment methods involve, for example, the use of organic chelating agents attached to antibodies, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or metal chelate complexes using tetrachloro-3α-6α-diphenylglycouril-3 (U.S. Patent Nos. 4,472,509 and 4,938,948). Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents, such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies, with a detectable imaging moiety attached to the antibody using a linker, such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0198] In another embodiment, immunoglobulins are derivatized by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin using reaction conditions that do not alter the antibody's binding site. Antibody conjugates produced according to this methodology have been disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Patent No. 5,196,066; incorporated herein by reference). Site-specific attachment of effector or reporter molecules, in which the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region, has also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce antibodies with diagnostic and therapeutic potential that are currently undergoing clinical evaluation.

[0199] VII. Immunodetection Methods In yet a further aspect, the present disclosure relates to immunodetection methods for binding, purifying, removing, quantifying, and generally detecting LILRB-associated cancers. While such methods can be applied in a traditional sense, another use is in the quality control and monitoring of vaccines and other virus stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e., long-term stability) of H1 antigen in the virus. Alternatively, the methods can be used to screen various antibodies for appropriate / desired reactivity profiles.

[0200] Some immunodetection methods include, to name a few, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescence assay, bioluminescence assay, and Western blot. In particular, competitive assays for detecting and quantifying LILRB are also provided. The steps of various useful immunodetection methods are described in scientific literature, for example, Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). Generally, immunobinding methods include obtaining a sample suspected of containing LILRB-associated cancer, and optionally contacting the sample with a first antibody according to the present disclosure under conditions effective to allow the formation of an immune complex.

[0201] These methods include methods for detecting or purifying LILRB or LILRB-associated cancer cells from a sample. The antibody is preferably linked to a solid support, e.g., in the form of a column matrix, and a sample suspected of containing LILRB-associated cancer cells is applied to the immobilized antibody. Undesired components are washed from the column, leaving LILRB-expressing cells immunocomplexed to the immobilized antibody, and the complex is then recovered by removing the organism or antigen from the column.

[0202] Immunobinding methods also include methods for detecting and quantifying the amount of LILRB-associated cancer cells or related components in a sample, as well as for detecting and quantifying any immune complexes formed during the binding process. Here, a sample suspected of containing LILRB-associated cancer cells is obtained and contacted with an antibody that binds to LILRB or its components, and then the immune complexes formed under specific conditions are detected and the amount thereof is quantified. With regard to antigen detection, the biological sample analyzed may be any sample suspected of containing LILRB-associated cancer, such as a tissue section or specimen, a homogenized tissue extract, a body fluid such as blood and serum, or a secretion such as feces or urine.

[0203] Contacting a selected biological sample with an antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) generally involves simply adding the antibody composition to the sample and incubating the mixture for a period of time sufficient for the antibody to form immune complexes, i.e., bind to the LILRB. After this time, the sample-antibody composition, e.g., tissue section, ELISA plate, dot blot, or Western blot, is generally washed to remove any non-specifically bound antibody species, allowing only specifically bound antibodies within the primary immune complexes to be detected.

[0204] Generally, the detection of immune complex formation is well known in the art and can be achieved through the application of many approaches.These methods are generally based on the detection of label or marker, for example, radioactive tag, fluorescent tag, biological tag and enzyme tag.The patents relating to the use of such labels include U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.Of course, as known in the art, additional advantages can be found through the use of secondary binding ligand, for example, secondary antibody and / or biotin / avidin ligand binding configuration.

[0205] The antibody used for detection may itself be linked to a detectable label, in which case the amount of primary immune complexes in the composition can be determined simply by detecting the label. Alternatively, the first antibody bound within the primary immune complex may be detected by a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is often itself an antibody, in which case it may be referred to as a "secondary" antibody. The primary immune complexes are contacted with a labeled secondary binding ligand or antibody under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, after which the label remaining in the secondary immune complexes is detected.

[0206] Another method involves detecting primary immune complexes using a two-step approach. As described above, a second binding ligand, such as an antibody, having binding affinity for the antibody is used to form secondary immune complexes. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing the detection of the tertiary immune complexes thus formed. This system can provide signal amplification, if desired.

[0207] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and then a second antibody is used to detect the biotin bound to the complexed biotin. In this method, the sample to be tested is first incubated in a solution containing the first-step antibody. If the target antigen is present, a portion of the antibody binds to the antigen, forming a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding an additional biotin moiety to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing a second-step antibody directed against biotin. This second-step antibody is labeled with an enzyme that can be used to detect the presence of the antibody / antigen complex, for example, by histoenzymology using a chromogenic substrate. Once suitably amplified, a macroscopically visible conjugate can be produced.

[0208] Another known method of immunodetection utilizes immuno-PCR (polymerase chain reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, but instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody complex is washed with a low pH or high salt buffer, which releases the antibody. The resulting wash solution is then used to perform a PCR reaction using suitable primers along with appropriate controls. At least in theory, the enormous amplification power and specificity of PCR can be utilized to detect single antigen molecules.

[0209] 1. ELISA In the simplest and most straightforward sense, immunoassay is binding assay.Some preferred immunoassays are various types of enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) known in the art.Immunohistochemical detection using tissue section is also particularly useful.However, it is easy to understand that detection is not limited to such technology, and Western blotting, dot blotting, FACS analysis, etc. can also be used.

[0210] In one exemplary ELISA, the antibodies of the present disclosure are immobilized on a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. A test composition suspected of containing LILRB-associated cancer cells is then added to the well. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding another anti-LILRB antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection can also be achieved by adding a second anti-LILRB2 antibody followed by a third antibody linked to a detectable label that has binding affinity to the second antibody.

[0211] In another exemplary ELISA, a sample suspected of containing LILRB2-associated cancer cells is immobilized on a well surface and then contacted with the anti-LILRB2 antibody of the present disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-LILRB2 antibody is detected. If the first anti-LILRB2 antibody is linked to a detectable label, the immune complex may be detected directly. Again, the immune complex may be detected using a second antibody (the second antibody is linked to a detectable label) that has binding affinity to the first anti-LILRB2 antibody.

[0212] Regardless of the format used, ELISAs have certain features in common, such as coating, incubation and binding, washing to remove non-specifically bound species, and detection of bound immune complexes, which are described below.

[0213] Coating a plate with either an antigen or an antibody generally involves incubating the wells of the plate with a solution of the antigen or antibody overnight or for a specified period of time. The wells are then washed to remove any incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with respect to the test antiserum. These include bovine serum albumin (BSA), casein, or a solution of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, thus reducing the background caused by nonspecific binding of the antiserum to the surface.

[0214] In ELISA, rather than a direct procedure, it is probably more conventional to use secondary or tertiary detection. Thus, after binding a protein or antibody to a well, coating it with a non-reactive material to reduce background, and washing to remove unbound material, the biological sample to be tested is contacted with the immobilized surface under conditions effective to allow immune complex (antigen / antibody) formation. Next, detection of the immune complex requires a labeled secondary binding ligand or antibody, and the secondary binding ligand or antibody in combination with a labeled tertiary antibody or third binding ligand.

[0215] "Under conditions effective to allow immune complex (antigen / antibody) formation" means that the conditions preferably include diluting the antigen and / or antibody in a solution such as BSA, bovine gamma globulin (BGG), or phosphate buffered saline (PBS) / Tween. These added agents also tend to aid in the reduction of nonspecific background.

[0216] "Suitable" conditions also mean that the incubation is carried out at a temperature or for a period of time sufficient to allow effective binding. The incubation step is typically carried out for about 1 to 2 to 4 hours, preferably at a temperature of about 25°C to 27°C, or may be carried out overnight at about 4°C.

[0217] After all incubation steps in ELISA, the contacted surface is washed to remove uncomplexed material. A preferred washing procedure involves washing with a solution such as PBS / Tween or borate buffer. After the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the presence of even minute amounts of immune complexes can be determined.

[0218] To provide a means of detection, the second or third antibody has an associated label to allow detection. Preferably, this is an enzyme that generates color upon incubation with an appropriate chromogenic substrate. Thus, for example, it may be desirable to contact or incubate the first and second immune complexes with urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase-conjugated antibodies for a period and under conditions that favor the formation of additional immune complexes (e.g., incubation in a PBS-containing solution, such as PBS-Tween, at room temperature for 2 hours).

[0219] After incubation with the labeled antibody, followed by washing to remove unbound material, the amount of label is quantified, for example, by incubation with a chromogenic substrate, e.g., urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or HO in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the extent of color development, e.g., using a visible spectrum spectrophotometer.

[0220] 2. Western Blot Western blot (or protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by polypeptide length (denaturing conditions) or by the protein's 3D structure (native / non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF) and probed (detected) using an antibody specific for the target protein.

[0221] Samples can be taken from whole tissues or cell cultures. In most cases, solid tissues are first mechanically disrupted using a blender (for large sample volumes), a homogenizer (for small volumes), or sonication. Cells may also be disrupted by one of the mechanical methods mentioned above. However, it should be noted that bacterial, viral, or environmental samples can be sources of proteins, and Western blotting is not limited to cellular studies alone. A combination of detergents, salts, and detergents may be used to promote cell lysis and solubilize proteins. Protease and phosphatase inhibitors are often added to prevent digestion of the sample by its own enzymes. Tissue preparation is often performed at low temperatures to avoid protein denaturation.

[0222] Gel electrophoresis is used to separate proteins from a sample. Proteins can be separated by isoelectric point (pi), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the sample treatment and the properties of the gel. This is a very useful method for determining proteins. Two-dimensional (2-D) gels can also be used, which spread proteins from a single sample across two dimensions. Proteins are separated according to isoelectric point (pH at which they have a neutral net charge) in the first dimension and according to molecular weight in the second dimension.

[0223] To make proteins accessible for antibody detection, they are transferred from the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter paper is placed on top of it. The entire stack is then placed in a buffer solution, which wicks upward toward the paper by capillary action, carrying the proteins with it. Another method of protein transfer, called electroblotting, uses an electric current to draw proteins from the gel into a PVDF or nitrocellulose membrane. The proteins migrate from the gel onto the membrane while maintaining their organization within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both types of membrane are chosen for their nonspecific protein-binding properties (i.e., they bind all proteins equally). Protein binding is based on charge interactions between the membrane and the protein, as well as hydrophobic interactions. Nitrocellulose membranes are less expensive than PVDF but are much more fragile and do not withstand repeated probing as well. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once the proteins have been transferred, they are detected using a labeled primary antibody, or an unlabeled primary antibody followed by indirect detection using labeled Protein A or a secondary labeled antibody that binds to the Fc region of the primary antibody.

[0224] 3. Immunohistochemistry The antibodies of the present disclosure can also be used in combination with either fresh-frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for immunohistochemical (IHC) studies. Methods for preparing tissue blocks from these particulate specimens have been used successfully in prior IHC studies of various prognostic factors and are well known to those skilled in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0225] Briefly, frozen sections can be prepared by rehydrating 50 ng of frozen "ground" tissue in a small plastic capsule in phosphate-buffered saline (PBS) at room temperature, pelleting the particles by centrifugation, resuspending them in a viscous embedding medium (OCT), inverting the capsule and / or pelleting again by centrifugation, snap-cooling in -70°C isopentane, cutting the plastic capsule and / or removing the frozen tissue cylinder, mounting the tissue cylinder on a cryostat microtome chuck, and / or cutting 25-50 serial sections from the capsule. Alternatively, the entire frozen tissue sample can be used for serial sectioning.

[0226] Permanent sections can be prepared by a similar method involving rehydrating a 50 mg sample in a plastic microcentrifuge tube, pelleting, resuspending in 10% formalin and fixing for 4 hours, washing / pelleting, resuspending in warm 2.5% agar, pelleting, chilling in ice-cold water to solidify the agar, removing the tissue / agar block from the tube, infiltrating and / or embedding the block in paraffin, and / or cutting up to 50 serial permanent sections. Again, the entire tissue sample may be substituted.

[0227] 4. Immunodetection Kit In yet a further aspect, the present disclosure relates to an immunodetection kit for use with the immunodetection method described above. The antibody can be used to detect LILRB-associated cancer cells, and therefore the antibody can be included in the kit. Thus, the immunodetection kit includes, in a suitable container means, a first antibody that binds to LILRB, and optionally an immunodetection reagent.

[0228] In certain embodiments, the antibody may be pre-bound to a solid support, for example, a column matrix and / or the well of a microtiter plate.The immunodetection reagent of the kit can be any one of various forms, such as a detectable label associated or linked to a given antibody.Detectable labels associated or linked to secondary binding ligands are also contemplated.An exemplary secondary ligand is a secondary antibody that has binding affinity to the first antibody.

[0229] Further suitable immunodetection reagents for use in the kits of the present invention include two-component reagents that include a second antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, where the third antibody is linked to a detectable label. As noted above, numerous exemplary labels are known in the art, and all such labels can be used in connection with the present disclosure.

[0230] The kit may further comprise a suitably dispensed composition of LILRB, which may be labeled or unlabeled, so that it can be used to generate a standard curve for the detection assay. The kit may contain the antibody-label conjugate in a fully conjugated form, in the form of an intermediate, or as separate moieties that are conjugated by the user of the kit. The components of the kit may be packaged either in aqueous medium or in lyophilized form.

[0231] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody may be placed, or preferably suitably dispensed. The kits of the present disclosure will also typically include means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection- or blow-molded plastic containers for retaining the desired vials therein.

[0232] 5. Flow cytometry and FACS The antibody of the present disclosure can also be used in flow cytometry or FACS.Flow cytometry is a laser or impedance-based technology used in many detection assays, including cell counting, cell sorting, biomarker detection and protein manipulation.In this technology, cells are suspended in a fluid stream and passed through an electronic detection device, allowing simultaneous multiparameter analysis of the physical and chemical characteristics of up to thousands of particles per second.Flow cytometry is routinely used in diagnosis disorders, especially blood cancer, but has many other applications in basic research, clinical practice and clinical trials.

[0233] Fluorescence-activated cell sorting (FACS) is a specialized type of cytometry. It provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence characteristics of each cell. Generally, the technique involves a cell suspension entrained in the center of a narrow, rapidly flowing stream of liquid. The stream is configured so that there is a large separation between cells compared to their diameter. A vibrating mechanism breaks the stream of cells into individual droplets. Just before the stream breaks into droplets, it passes through a fluorescence measurement station, where the fluorescence of each cell is measured. An electrically charged ring is placed exactly at the point where the stream breaks into droplets. A charge is placed on the ring base just before the fluorescence intensity is measured, and an opposite charge is trapped on the droplets as they break from the stream. The charged droplets then fall through an electrostatic deflection system, which diverts the droplets into containers based on their charge.

[0234] In certain embodiments used in flow cytometry or FACS, antibodies of the present disclosure are labeled with a fluorophore and then allowed to bind to cells of interest, which are analyzed in a flow cytometer or sorted by a FACS machine. [Example]

[0235] VIII. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention and, as such, can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0236] Example 1 Screening for specific monoclonal antibodies against LILRB2. We used a stable reporter cell system to detect the binding ability of selected monoclonal antibodies against LILRB2. In this chimeric receptor reporter system, the extracellular domain (ECD) of LILRB2 is fused to the transmembrane / intracellular domain of PILRb, which associates with the adaptor protein DAP12, which contains an immunoreceptor tyrosine-based activation motif. Twenty-seven monoclonal antibodies were screened. LILRB2 reporter cells were incubated with the selected monoclonal antibodies and then labeled with a goat anti-human IgG secondary antibody (APC). Flow cytometry analysis demonstrated that more than 95% of the LILRB2 reporter cells were labeled with clones B2-7, B2-15, B2-16, B2-17, B2-18, and B2-19. A lower percentage of LILRB2 reporter cells were labeled with clones B2-8, B2-10, B2-12, B2-24, and B2-25. Other clones did not have the ability to bind to LILRB2 reporter cells (Figure 1A-B). Because members of the LILRA and LILRB families share high sequence homology, we detected potential cross-reactivity of B2-7, B2-15, B2-16, B2-17, B2-18, B2-19, B2-8, B2-10, B2-12, B2-24, and B2-25 with LILRB and LILRB. We generated LILRB and LILRB reporters stably transfected with the extracellular domains (ECDs) of each receptor. Flow cytometry analysis showed that B2-7, B2-15, B2-16, B2-17, B2-18, B2-19, B2-8, B2-24, and B2-25 did not cross-react with other LILRBs except for LILRA and LILRB2. B2-10, B2-12, and B2-18 bound nonspecifically to LILRA1 (Fig. 1C and Fig. S29). Thus, these eight antibodies were identified as specific monoclonal antibodies against LILRB2.The present inventors additionally demonstrated that B2-7, B2-15, B2-16, B2-17, and B2-19 bound only to bone marrow cells in human peripheral blood samples (Figure 30), further confirming binding in cells expressing endogenous LILRB2. The present inventors also confirmed that B2-7, B2-15, B2-16, B2-17, and B2-19 bound in a dose-dependent manner to HEK293 cells stably expressing full-length LILRB2 (HEK293_LILRB2) (Figure 31) and primary monocytes (Figure 32). Binding EC 50 Values ​​range from 0.101 to 0.277 μg / mL in HEK293_LILRB2 (Figure 31) and from 0.056 to >10 μg / mL in primary monocytes (Figure 32).

[0237] Screening for candidate antagonistic and agonistic antibodies against LILRB2. Typically, receptor-blocking antibodies target the ligand-binding site, compete with the ligand, and / or block the receptor's interaction with its ligand. LILRB2 ligands include angiopoietin-like proteins (ANGPTL), semaphorin 4A (SEMA4A), TIM-3, CD-1 molecules, amyloid-β (Aβ) oligomers, classical human leukocyte antigen (HLA) class I molecules (HLA-A, HLA-B, HLA-C), and non-classical HLA-class I molecules (HLA-E, HLA-F, HLA-G, and HLA-H). The LILRB2 reporter system provides a powerful tool for screening candidate antagonistic antibodies against LILRB2. Upon activation of the chimeric receptor by ligand binding to the ECD of LILRB2, ZAP70 or Syk kinase is recruited to the immunoreceptor tyrosine-based activation motif of the adaptor DAP12, activating NFAT and promoting GFP expression driven by an NFAT-responsive promoter. Antibodies that significantly reduced GFP signaling were considered candidate antagonist antibodies.

[0238] We tested the ability of LILRB2 mAbs to block the activation of LILRB2 reporter cells stimulated by coated ANGPTL2 (Figures 3A-C; Figure 5I) and SEMA4A (Figures 4A-C). The results showed that B2-7, B2-15, B2-16, B2-17, and B2-19 could dose-dependently prevent the activation of LILRB2 reporter cells by the coated ligands. The B2-19 antibody also blocked the activation of LILRB2 reporter cells stimulated by coated CD1d (Figure 52). LILRB2 reporter cells were also cocultured with K562 cells overexpressing HLA-G and then treated with LILRB2 mAbs. The results also showed that these LILRB2 mAbs blocked the activation of LILRB2 reporter cells stimulated by HLA-G on K562 cells (Figures 5A-C). The LILRB2 antibodies were also shown to block HLA-G and SEMA4A binding to HEK293 cells expressing full-length LILRB2 in a dose-dependent manner (Figures 33 and 34, respectively). These data demonstrated that B2-7, B2-15, B2-16, B2-17, and B2-19 are antagonistic LILRB2 mAbs.

[0239] In contrast, several LILRB2 mAbs (B2-8, B2-24, B2-25, B2-10, B2-12, and B2-18) were able to activate LILRB2 reporter cells when added to the cell culture medium (Figure 2A) or when co-cultured with K562 (Figure 2B), demonstrating that these LILRB2 mAbs are agonistic antibodies.

[0240] Effect of anti-LILRB2 antibodies on LPS responses in primary human monocytes. LILRB2 is expressed on hematopoietic stem cells, monocytes, macrophages, dendritic cells, and in some individuals, basophils, decidual macrophages, mast cell precursors, endothelial cells, and osteoclasts, but not on lymphoid cells. LILRB2 is classified as an immune inhibitory receptor and is associated with downregulation of immune responses. To determine the functionality of the screened anti-LILRB2 antibodies, peripheral blood mononuclear cell (PBMC)-based functional assays were performed to assess whether the screened antibodies could amplify or inhibit monocyte activation. B2-7, B2-15, B2-16, B2-17, and B2-19 were able to enhance CD86 and TNFα levels in the presence of LPS. B2-8, B2-24, and B2-25 showed inhibition of CD86 levels upon LPS stimulation but did not appear to have an effect on TNFα levels (Figure 6A-C). B2-7, B2-16, and B2-19 were able to enhance IFN-γ (Figure 35) and TNF-α (Figure 36) secretion from PBMCs stimulated with LPS. B2-19 was also able to enhance IL-12p40 secretion from PBMCs stimulated with LPS (Figure 37). We identified B2-7, B2-15, B2-16, B2-17, and B2-19 LILRB2-specific antibodies that enhance the inflammatory potential of monocytes in response to LPS stimulation. All of these antibodies are functional antagonist antibodies. B2-8, B2-24, and B2-25 are candidates for effective antibodies against LILRB2.

[0241] Effect of anti-LILRB2 antibodies on PBMCs stimulated with anti-CD3 activating antibodies. We evaluated the indirect effect of LILRB2 blockade on T cell activation by activating anti-CD3 antibodies. B2-7 and B2-19 antibodies were able to enhance the secretion of IFN-γ (Figure 38), TNF-α (Figure 39), GM-CSF (Figure 40), IL-1α (Figure 41), IL-1β (Figure 42), IL-6 (Figure 43), and CXCL2 (Figure 44). B2-19 also enhanced the secretion of CD8 +It was possible to enhance the cell surface expression of CD25 on T cells (Figure 46). We identified B2-7 and B2-19 LILRB2-specific antibodies that enhance T cell activation.

[0242] Effect of anti-LILRB2 antibody on monocyte-derived M2a macrophages. We evaluated the effect of B2-19 on the phenotype of M2a macrophages in vitro. In M2a macrophages differentiated from monocytes from several donors, B2-19 reduced the expression of CD64, CD163, and CD14 (Figure 45). We identified B2-19 as a LILRB2-specific antibody capable of modulating the phenotype of M2a macrophages.

[0243] Anti-LILRB2 antibodies inhibit leukemia cell development in the C1498-LILRB2 tumor-bearing model. We evaluated whether LILRB2 blockade could inhibit tumor progression in the C1498 leukemia model. A C1498 subline (C1498-LILRB2) overexpressing human LILRB2 was generated by retroviral transduction. LILRB2 was stably expressed on C1498-LILRB2 (Figure 7A). LILRB2 could promote leukemia development. Overexpressed LILRB2 promoted leukemia development in NSG mice (Figure 7B). LILRB2 overexpression resulted in poorer leukemia infiltration (Figures 7C-D), survival curve (Figure 7E), and myoid cell infiltration (Figure 7G). Furthermore, LALAPG variants of B2-7 and B2-19 containing Fc mutations were generated to abolish complement binding and fixation as well as Fc-γ-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) in both mouse IgG2a and human IgG1. Treatment with LALAPG mutant antagonist antibodies significantly suppressed myeloid leukemia growth (Figure 7H-J).

[0244] Anti-LILRB2 antibody inhibits leukemia cell development in the MLL-AF9 model. We evaluated whether LILRB2 blockade could inhibit tumor progression in the MLL-AF9 leukemia model. A PirB-KO MLL-AF9 leukemia subline (LILRB2) overexpressing human LILRB2 was generated by retroviral transduction and mouse transplantation. LILRB2 expression could be detected on PirB-KO MLL-AF9 LILRB2 or control leukemia cells (Figure 8A). In this MLL-AF9 model, LILRB2 overexpression resulted in poorer leukemia infiltration (Figures 8B-C) and survival curve (Figure 8D). Furthermore, treatment with a LALAPG mutant antagonist antibody significantly suppressed leukemia infiltration (Figures 8E-F).

[0245] LILRB2 blockade inhibits AML cell migration and invasion. LILRB2 also plays various roles in cancer biology. LILRB2 expression could be detected on THP-1, a human leukemia cell line (Figure 9A). We used a humanized mouse xenograft model to examine the function of LILRB2 in leukemia. LILRB2 blockade reduced homing and engraftment of leukemia cells to hematopoietic organs (Figure 9B-C), resulting in delayed weight loss (Figure 9D), less severe infiltration of myeloid leukemia cells into the liver (Figure 9E-F), and prolonged survival of xenografted mice. Taken together, LILRB2 blockade reduced leukemia migration and invasion.

[0246] Antagonist LILRB2 mAb inhibits leukemia cell development in patient-derived xenograft (PDX) models. Flow cytometry analysis showed that LILRB2 was expressed in some cases of primary monocytic AML (M5) (Figure 10A). In the TCGA database, LILRB2 mRNA levels negatively correlated with AML patient survival (Figure 10B). In PDX models, LALAPG mutant antagonist antibodies B2-7 and B2-19 significantly suppressed leukemia infiltration (Figure 10C). In in vitro culture systems, LALAPG mutant antagonist antibodies promoted differentiation of AML-M5 leukemia cells (Figures 10D-E).

[0247] Antagonist LILRB2 mAb prevents the T cell suppressive function of myeloid-derived suppressor cells (MDSCs) in vitro and inhibits CD14 T cells from cancer-derived ascites. + The function of antagonistic LILRB2 antibodies on the T cell suppressive function of MDSCs was tested in vitro. Pairs of MDSCs and T cells from the same tumor patient were co-cultured and treated with antagonistic LILRB2 antibodies. Treatment with B2-7 and B2-19 antibodies significantly attenuated the inhibition of T cell proliferation induced by MDSCs, likely by converting immunosuppressive myeloid cells into proinflammatory myeloid cells (Figures 11A-E and 11G). B2-7, B2-15, B2-16, B2-17, and B2-19 also inhibited primary CD14 cells isolated from cancer-derived ascites. + We were able to decrease the anti-inflammatory markers CD163 and CD206 and increase the pro-inflammatory marker CD86 in the cells (Figure 11F).

[0248] Example 2 Generation and Characterization of LILRB2 Antibodies. LILRB2 antibodies were generated by panning a phage-displayed human antibody scFv library. LILRB2-His antigen was coated onto a solid phase for three rounds of panning. In each round of phage panning, LILRB1-His antigen was used for exclusion selection. After phage panning, single bacterial colonies were picked for phage ELISA testing. Positive phage ELSIA results were sequenced and analyzed for their scFv regions. As shown in Figure 12A, 24 unique antibody sequences were obtained. Phages displaying the scFvs showed binding to the LILRB2 antigen in phage ELISA (Figure 12B). These 24 scFvs were then converted to whole human IgG1 for further analysis.

[0249] ELISA binding EC of LILRB2 antibody 50 ELISA binding EC of these 24 antibodies 50was measured by indirect ELISA. Briefly, ELISA plates were coated with LILRB2-His antigen. Serially diluted antibodies were incubated with the antigen-coated plates. After incubation, a goat anti-human Fc secondary antibody conjugated with HRP was incubated with the plates. TMB substrate was added for color development and measurement of absorbance at 450 nm. As shown in Figures 13A-B, these antibodies bound to LILRB2 in a dose-dependent manner (Figure 13A), and their EC 50 The values ​​ranged from 0.355 to 46.49 nM (Figure 13B).

[0250] Binding specificity of LILRB2 antibodies. The binding specificity of LILRB2 antibodies was evaluated by determining their ELISA binding activity with antigens for other members of the LILRA family. As shown in Figure 14A, of the 24 antibodies, 21 showed highly specific binding to LILRB2, and three antibodies (B2-10, -12, and -18) showed weak cross-binding to LILRA1. Further titration of these three antibodies with LILRB2 and LILRA1 antigens showed that the binding ability of these three antibodies to LILRB2 was much stronger (>100-fold) than that to LILRA1 (Figure 14B).

[0251] Epitope binning of LILRB2 antibodies. Epitope binning was performed on 22 antibodies in a sandwich format on the OctetRED96 system. Each antibody was cross-binned with the remaining antibodies to determine whether they could compete with each other. As shown in Figure 15, these antibodies can be divided into three bins. In each bin, antibodies compete with each other for LILRB2 binding. Antibodies in bin 1 and bin 2 showed overlap in their blocking profiles.

[0252] Domain mapping of LILRB2 antibodies. LILRB2 has four Ig-like domains (D1, D2, D3, and D4) and a juxtamembrane domain (JM). To determine the binding domain of LILRB2 antibodies, the full-length extracellular domain (ECD) or truncated ECD of LILRB2 was fused to the Fc fragment of mouse IgG2a for recombinant protein expression (Figures 16A-B). The binding ability of LILRB2 antibodies to these domain proteins was then determined. Deletion of the D1 domain resulted in loss of binding to antibodies B2-7, -15, -16, -17, and -19, but not to other antibodies, indicating that these antibodies bind to the D1 domain of LILRB2 (Figure 16C). Similarly, the binding domains of other antibodies were determined and summarized (Figure 16D).

[0253] Key Residues for Antibody Binding. To finely map the key residues for antibody binding, we generated a series of mutant B2-ECD proteins and assessed antibody binding. Because most antibodies bind to the D1 or D4 domain, we designed mutations only in the D1 or D4 domain. The mutations were based on two criteria: 1) residues that differ between LILRB2 and LILRB1; and (2) residues located in exposed, flexible loop regions. As shown in Figures 17A-C, four regions in the D1 domain of LILRB2 were mutated to their corresponding sequences in LILRB1. Similarly, four regions in the D4 domain of LILRB2 were mutated (Figures 17B-D). The four D1 mutants were used to test binding with five antibodies (B2-7, -15, -16, -17, and -19) that bind to the D1 domain, and antibody B2-18, which binds to the D4 domain, was used as a control. As shown in Figure 17E, the D1M4 and D1M2 mutants completely abolished binding by antibodies B2-7, -15, and -17, indicating that these two regions are key to the binding activity of these antibodies; D1M4 completely, and D1M2 partially, abolished binding by antibodies B2-16 and -19, indicating that the M4 region is key to the binding activity of the two antibodies. As a control, none of these mutants abolished binding by antibody B2-18. Similar methods were used to determine the regions on the D4 domain that are key to the binding activity of other antibodies (Figure 17F).

[0254] Affinity measurements of blocking antibodies. The affinities of five blocking antibodies were measured on the Octet RED 96 system. As shown in Figure 18 and Table 1, the affinities range from 1.87 to 35.9 nM.

[0255] Example 3 Classical monocytes isolated from PBMCs of apparently healthy donors were differentiated into immature DCs using 50 ng / mL GM-CSF and 35 ng / mL IL-4 for 6 days. On day 6, DCs were treated with 40 ng / mL IL-10 (as a tolerogenic stimulus) and 10 μg / mL B2-19 or its isotype control. After 48 hours, cytokine and chemokine levels in the culture medium supernatant were measured using a Luminex assay (R&D Systems). B2-19 triggered enhanced production of the pro-inflammatory cytokine IL-6 and chemokines known to recruit monocytes (CCL2) and neutrophils (CXCL8) (Figure 47).

[0256] Classical monocytes isolated from PBMCs prepared from apparently healthy donors were differentiated into immature DCs using 50 ng / mL GM-CSF and 35 ng / mL IL-4 for 6 days. On day 6, DCs were treated with 100 ng / mL LPS (as a maturation stimulus) and 10 μg / mL B2-19 or its isotype control. Forty-eight hours later, the expression levels of several cell surface markers were measured by flow cytometry. B2-19 increased the expression of CD83 (a DC maturation marker), CD86 (a costimulatory molecule), and HLA-DR (antigen presentation). On the other hand, B2-19 reduced the expression levels of the inhibitory receptors LILRB4 and CD209. Taken together, these data indicate that B2-19 further promotes a pro-inflammatory phenotype in LPS-stimulated DCs (Figure 48).

[0257] The pharmacokinetics of B2-19 was evaluated in wild-type mice (without LILRB2 expression). Nine C57BL / 6J mice received a single 5 mg / kg intravenous dose of B2-19 and were randomized into three groups for blood collection at alternating time points. Serum was prepared from the blood samples, and B2-19 concentrations were determined using ELISA. Antibodies in the serum were captured with plate-coated antigen (LILRB2-ECD, R&D Systems) and detected using a goat anti-human IgG Fc antibody conjugated with HRP (Jackson ImmunoResearch) and TMB substrate. The results show that the B2-19 antibody conferred the expected pharmacokinetic profile (CL and half-life) of human IgG4 in C57BL / 6J wild-type mice dosed at 5 mg / kg. These results demonstrate that in vivo exposure to B2-19 (in mice) does not reduce its binding activity, as the ELISA format measures active antibody.

[0258] The antitumor efficacy of the B2-19 antibody was evaluated in humanized NSG-SGM3 mice xenografted with the SK-MEL-5 melanoma cell line. 1 × 10 cells isolated from the umbilical cord blood of individual donors were used. 5 CD34 + NSG-SGM3 mice (The Jackson Laboratory, stock number 013062) were humanized using hematopoietic cells. Six weeks after humanization, mice were injected subcutaneously with 1 × 10 6 SK-MEL-5 cells were transplanted. The average tumor size was 70-80 mm. 3 When the following criteria were met (29 days after tumor implantation): human CD45 in the blood; + CD14 + % of cells, tumor volume and donor (each CD34 per treatment cohort) +Mice were randomized into two treatment groups with six mice per group based on the number of donors (two mice per donor). Mice were treated intravenously with 20 mg / kg of B2-19 or its isotype control every three days for three doses. Compared to its isotype control, B2-19 monotherapy reduced tumor growth rate and caused a 29% reduction in tumor size (Figures 50A-B). These data demonstrate that B2-19 antibody monotherapy exhibits antitumor efficacy in mouse models of solid tumors.

[0259] Table 1. Affinity of LILRB2 blocking antibodies TIFF0007767291000002.tif42128

[0260] Table 2: Amino acid sequences of CDRs of LILRB2 antibodies TIFF0007767291000003.tif234134TIFF0007767291000004.tif234136

[0261] Table 3: DNA sequences of CDRs of LILRB2 antibodies TIFF0007767291000005.tif232141TIFF0007767291000006.tif232159TIFF0007767291000007.tif232151TIFF0007767291000008.tif23228

[0262] Table 4: DNA sequence of antibody heavy chain (HC) TIFF0007767291000009.tif221150TIFF0007767291000010.tif228150TIFF0007767291000011.tif224150TIFF0007767291000012.tif164150

[0263] Table 5: Amino acid sequence of antibody heavy chain (HC) TIFF0007767291000013.tif213149TIFF0007767291000014.tif202150

[0264] Table 6: DNA sequence of the LILRB2 antibody light chain (LC) TIFF0007767291000015.tif217150TIFF0007767291000016.tif224150TIFF0007767291000017.tif224150TIFF0007767291000018.tif164150

[0265] Table 7. Amino acid sequence of the LILRB2 antibody light chain (LC) TIFF0007767291000019.tif213150TIFF0007767291000020.tif202150

[0266] All of the methods disclosed and claimed in this application can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods described herein and in the steps or sequence of steps of the methods without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0267] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007767291000021.tif45150

Claims

1. i) a heavy chain (HC) variable region (VH) comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:97, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:98, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:99; and ii) a light chain (LC) variable region (VL) comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 100, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 101, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 102; An isolated monoclonal antibody or antigen-binding fragment thereof that binds to LILRB2.

2. (a) the isolated monoclonal antibody is a murine antibody, a rodent antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a human antibody; or (b) the antigen-binding fragment is a recombinant ScFv (single-chain variable fragment) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment; 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1.

3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence set forth in SEQ ID NO:498, and the VL comprises an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence set forth in SEQ ID NO:

572.

4. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. An isolated nucleic acid encoding the isolated monoclonal antibody of any one of claims 1 to 3.

6. A vector comprising the isolated nucleic acid of claim 5.

7. A host cell comprising the vector of claim 6.

8. The host cell of claim 7, which is a mammalian cell or a CHO cell.

9. A hybridoma or engineered cell that encodes and / or produces the isolated monoclonal antibody of any one of claims 1 to 3.

10. 9. A method for producing an antibody, comprising culturing a host cell according to claim 7 or 8 under conditions suitable for expressing said antibody, and recovering said antibody.

11. A chimeric antigen receptor (CAR) protein comprising the antigen-binding fragment of any one of claims 1 to 3.

12. 12. An isolated nucleic acid encoding the CAR protein of claim 11.

13. 13. An engineered cell comprising the isolated nucleic acid of claim 12.

14. 14. The engineered cell of claim 13, which is a T cell, an NK cell, or a macrophage.

15. 14. A medicament comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 3 or the engineered cell of claim 13 for use in a method of treating cancer or ameliorating the effects of cancer in a subject, comprising: the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof or the engineered cell; Medicine.

16. (a) the method comprises: (i) reducing or eradicating the tumor burden in said subject; (ii) reducing the number of tumor cells and / or slowing the rate of tumor growth; (iii) reducing tumor size; (iv) reducing or preventing tumor metastasis; (v) eradicating the tumor in said subject; or (vi) topoisomerase inhibitors, anthracyclines, topoisomerase inhibitors, anthracyclines, daunorubicin, nucleoside metabolic inhibitors, cytarabine, hypomethylating agents, low-dose cytarabine (LDAC), daunorubicin and cytarabine combinations, daunorubicin and cytarabine liposomes for injection, Vixeos®, azacitidine, Vidaza®, decitabine, all-trans-retinoic acid (ATRA), arsenic, arsenic trioxide, histamine dihydrochloride, Sepren®, Interleukin-2, aldesleukin, Proleukin®, gemtuzumab ozogamicin, Mylotarg®, FLT-3 inhibitors, midostaurin, Rydapt®, clofarabine, farnesyltransferase inhibitors, decitabine, IDH1 inhibitors, ivosidenib, tibsovo®, IDH2 inhibitors, enasidenib, Idifa®, Smoothened (SMO) inhibitors, glasdegib, arginase inhibitors, IDO inhibitors, epacadostat, BCL- 2 inhibitors, venetoclax, Venclexta®, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitors, ibrutinib, Imbruvica®, acalabrutinib, Calquence®, zanubrutinib, PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, TIGIT antibodies, TIM3 antibodies, CD40 antibodies, 4-1BB antibodies, CD47 antibodies, SIRP1α antibodies or fusion proteins, C or further comprising administering to the subject a D70 antibody and one or more drugs selected from the group consisting of a CLL1 antibody, a CD123 antibody, an antagonist of E-selectin, an antibody that binds to a tumor antigen, an antibody that binds to a T cell surface marker, an antibody that binds to a myeloid cell or NK cell surface marker, an alkylating agent, a nitrosourea agent, an antimetabolite, an antitumor antibiotic, an alkaloid derived from a plant, a hormone therapy agent, a hormone antagonist, an aromatase inhibitor, and a P-glycoprotein inhibitor; or (b) the cancer is a solid cancer or a hematological malignancy; (c) the antibody or antigen-binding fragment thereof or the engineered cell targets monocytes, macrophages, dendritic cells, neutrophils, other myeloid cells, myeloid-derived suppressor cells, or tumor-associated macrophages; or (d) the antibody or antigen-binding fragment thereof is administered intravenously, intraarterially, intratumorally, or subcutaneously; The pharmaceutical composition according to claim 15.

17. 17. The pharmaceutical composition of claim 16, wherein the solid cancer is selected from the group consisting of adrenal gland cancer, bile duct cancer, bone cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, mesothelioma, melanoma, Merkel cell carcinoma, nasopharyngeal cancer, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, penile cancer, pinealoma, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, skin cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, and vaginal cancer.

18. The pharmaceutical of claim 16 or 17, wherein the antibody or antigen-binding fragment thereof further comprises an anti-tumor drug linked thereto.

19. The antitumor drug is (i) linked to the antibody via a photodegradable linker; (ii) linked to the antibody via an enzyme-cleavable linker, or (iii) is a toxin, radioisotope, cytokine, or enzyme; The pharmaceutical composition according to claim 18.

20. 1. An in vitro method for detecting LILRB2-expressing cells in a sample, comprising: (a) contacting a sample from a subject with the antibody or antigen-binding fragment thereof of any one of claims 1 to 3; and (b) detecting binding of the antibody to LILRB2-expressing cells in the sample A method comprising:

21. (a) the sample is a body fluid or a biopsy; (b) the sample is blood, bone marrow, sputum, tears, saliva, mucus, serum, urine or feces; and / or (c) detection includes immunohistochemistry, flow cytometry, immunoassay or Western blot; 21. The method of claim 20.

22. 22. The method of claim 21, wherein the immunoassay is an ELISA or RIA.

23. (a) the method further comprises performing steps (a) and (b) a second time and determining a change in the level of detection compared to the first time; or (b) the isolated monoclonal antibody or antigen-binding fragment thereof further comprises a label; 21. The method of claim 20.

24. 24. The method of claim 23, wherein the label is a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye.

25. 25. The method of claim 23 or 24, wherein the isolated monoclonal antibody or antigen-binding fragment thereof is conjugated to a liposome or nanoparticle.

26. 14. A medicament comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 3 or the engineered cell of claim 13 for use in a method of treating or ameliorating the effects of an autoimmune disease in a subject, comprising: the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof or the engineered cell; The medicine.

27. (a) the antibody or antigen-binding fragment thereof or the engineered cell targets monocytes, macrophages, dendritic cells, neutrophils, or other myeloid cells; (b) the antibody or antigen-binding fragment thereof is administered intravenously, intraarterially, intratumorally, or subcutaneously; (c) the method further comprises administering to the subject one or more drugs selected from the group consisting of steroids or NSAIDs; or (d) the autoimmune disease is selected from the group consisting of Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, undifferentiated spondyloarthropathy, juvenile spondyloarthropathy, Behcet's disease, enthesitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, fibromyalgia, chronic fatigue syndrome, pain conditions associated with systemic inflammatory diseases, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile-onset diabetes mellitus (also known as type 1 diabetes), Wegener's granulomatosis, multiple myeloma, and the like. Myositis, dermatomyositis, inclusion body myositis, multiple endocrinopathy, Schmidt's syndrome, autoimmune uveitis, Addison's disease, Graves' disease, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, multiple sclerosis, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler's syndrome, myasthenia gravis, Eaton-Lambert syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia, scleroderma, progressive systemic sclerosis CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), adult-onset diabetes mellitus (also known as type II diabetes), mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, antiphospholipid syndrome, erythema multiforme, Cushing's syndrome, autoimmune chronic active hepatitis, allergic diseases, allergic encephalomyelitis, transfusion reactions, leprosy, malaria, Leprosy Schistosomiasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, eczema, lymphomatoid granulomatosis, Kawasaki disease, endophthalmitis, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman syndrome, Felty syndrome, Fuchs's cyclitis, IgA nephropathy, Henoch-Schönlein purpura, graft-versus-host disease, transplant rejection, tularemia, periodic fever syndrome, septic arthritis, familial Mediterranean fever, TNF receptor-associated periodic syndrome (TRAPS), Muckle-Wells syndrome, or hyper-IgD syndrome. The pharmaceutical composition of claim 26.

Images