Novel Anti- lilrb4 antibodies and derived products

Abstract

To provide new anti- LILRB4 antibodies.SOLUTION: A heavy chain variable region comprising a heavy chain complementary determining region (HC-CDR) 1 having a particular amino acid sequence, a HC - CDR2 having a particular amino acid sequence, and a HC - CDR3 having a particular amino acid sequence; and b) a light chain variable region comprising a light chain complementary determining region (LC-CDR) 1 having a particular amino acid sequence comprising a mutation at amino acid residue NS, a LC - CDR2 having a particular amino acid sequence, and a LC - CDR3 having a particular amino acid sequence. LILRB4. Further provided are anti- LILRB4 chimeric antigen-receptor proteins, isolated polynucleotides encoding them, pharmaceutical compositions comprising them, and uses thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 988,892, filed March 12, 2020, the disclosure of which is incorporated herein by reference. Sequence Listing

[0002] The Sequence Listing contained in the file designated "066564-8013WO01_ST25", which is 151 KB (as measured by Microsoft Windows) and created on March 12, 2021, has been submitted herewith by electronic submission and is incorporated herein by reference. FIELD OF THE INVENTION

[0003] The present disclosure relates generally to the fields of medicine, oncology, and immunology. More specifically, the present disclosure relates to antibodies that bind to LILRB4. [Background technology]

[0002]

[0004] Human leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4), also known as immunoglobulin-like transcript 3 (ILT3 or ILT-3), leukocyte immunoglobulin-like receptor 5 (LIR5 or LIR-5), and CD85k or CD85K, is a type 1 membrane protein that contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and is involved in the negative regulation of immune cell activation. LILRB4 is expressed on monocytes, macrophages, and dendritic cells and can inhibit innate immunity in a cell-autonomous manner and suppress T cell activation through an indirect mechanism. LILRB4 is a specific marker for monocytic acute myeloid leukemia (AML), including refractory and relapsed disease. It has been shown that LILRB4 supports tumor cell infiltration into tissues and suppresses T cell activity in AML cells through a signaling pathway involving APOE, LILRB4, SHP-2, uPAR, and ARG1 (Deng M. et al., Nature (2018) 562:605-09). There is a significant need for novel anti-LILRB4 antibodies. Summary of the Invention

[0003]

[0005] The present disclosure provides anti-LILRB4 antibodies and antigen-binding fragments thereof, their amino acid and nucleotide sequences, anti-LILRB4 chimeric antigen receptors, and uses thereof.

[0006] In one aspect, the disclosure provides an isolated anti-LILRB4 antibody or antigen-binding fragment thereof. In some embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain complementarity-determining region (HC-CDR)1 having the amino acid sequence of SEQ ID NO:5, an HC-CDR2 having the amino acid sequence of SEQ ID NO:6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7; and (b) a light chain variable region comprising a light chain complementarity-determining region (LC-CDR)1 having the amino acid sequence of SEQ ID NO:8 containing a mutation at amino acid residue NS, an LC-CDR2 having the amino acid sequence of SEQ ID NO:9, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:10.

[0004]

[0007] In a particular embodiment, LC-CDR1 has the amino acid sequence of SEQ ID NO:28.

[0008] In certain embodiments, the heavy chain variable region has an amino acid sequence at least about 90% identical to SEQ ID NO:1; and the light chain variable region has an amino acid sequence at least about 90% identical to SEQ ID NO:27.

[0005]

[0009] In certain embodiments, the heavy chain variable region has the amino acid sequence of SEQ ID NO: 1, and the light chain variable region has the amino acid sequence of SEQ ID NO:27.

[0010] In certain embodiments, the antibody or antigen-binding fragment further comprises an immunoglobulin constant region, optionally the constant region of an IgG, or optionally the constant region of a human IgG.

[0006]

[0011] In certain embodiments, the antibodies described herein are of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0012] In certain embodiments, the antibody or antigen-binding fragment is humanized.

[0007]

[0013] In certain embodiments, the antigen-binding fragment is a camelized single domain antibody, a diabody, a ds (disulfide stabilized) diabody or a ds diabody, an scFv, an scFv dimer, a BsFv, a dsFv, a (dsFv)2, a ds Fv-dsFv', Fv fragment, Fab, Fab', F(ab')2, bispecific antibody, The antibody may be a monobody, a domain antibody, or a bivalent antibody.

[0008]

[0014] In certain embodiments, the anti-LILRB4 antibodies described herein are bispecific antibodies. In some embodiments, the anti-LILRB4 bispecific antibodies are directed against a T cell receptor, such as CD3. In some embodiments, the anti-LILRB4 bispecific antibodies are directed against a NK cell receptor, such as CD16A.

[0009]

[0015] Thus, in another aspect, the present disclosure provides a bispecific antibody or antigen-binding fragment capable of binding to LILRB4 and CD3.

[0016] In certain embodiments, the bispecific antibody or antigen-binding fragment provided herein comprises: (a) a first light chain variable (V L ) domain and the first heavy chain variable (V H (b) a first antigen-binding region comprising a V L Domain and Second V H and a second antigen-binding region comprising a domain, wherein the first antigen-binding region is capable of binding to LILRB4 and the second antigen-binding region is capable of binding to CD3, or vice versa.

[0010]

[0017] In certain embodiments, the first V Ldomain and first heavy chain variable domain are linked to a first pair of constant domains, and a second V L Domain and Second V H The domains are each linked to a second pair of constant domains.

[0011]

[0018] In certain embodiments, the first V L The first light chain constant (C L ) domain and the first V H The domain is the first heavy chain constant domain 1 (C H 1). In certain embodiments, the first V L Domain is the first C H 1 domain and V H Domain is the second C L It is linked to the domain.

[0012]

[0019] In certain embodiments, the second V L Domain is the second C L domain, and a second V H Domain is the second C H In certain embodiments, the second V L Domain is the second C H linked to one domain, and a second V H Domain is second C L In certain embodiments, the second V L The domain is linked to a T cell receptor (TCR) α chain constant domain and a second V H The domain is the TCR β chain constant In certain embodiments, the second V L The domain is the TCR β chain constant domain. Connected to the main and second V H The domain is linked to the TCR alpha chain constant domain .

[0013]

[0020] In certain embodiments, the first antigen-binding region and / or the second antigen-binding region is a single-chain variable fragment (scFv).

[0021] In certain embodiments, the antibodies or antigen-binding fragments provided herein: The Third V L Domains and Third V H The antibody further comprises a third antigen-binding region comprising a domain, wherein the third antigen-binding region is capable of binding to LILRB4 or CD3.

[0014]

[0022] In certain embodiments, a third V L domain and third heavy chain variable domain are each linked to a first pair of constant domains. In certain embodiments, a third V L Domain is the third C L domain, and a third V H Domain is the third C H 1 Domestic In certain embodiments, a third V L Domain is the third C H 1 domain, and the third V H Domain is the third C L In certain embodiments, the third V L The domain is linked to a second TCR α chain constant domain and a third V H In certain embodiments, the third V is linked to a second TCR β chain constant domain. L The domain is linked to a second TCR β chain constant domain and a third V H The domain is linked to a second TCR alpha chain constant domain.

[0015]

[0023] In certain embodiments, the TCR alpha chain constant domain has the amino acid sequence of SEQ ID NO: 89. In certain embodiments, the TCR alpha chain constant domain has the S91A mutation of SEQ ID NO:89.

[0016]

[0024] In certain embodiments, the antibody or antigen-binding fragment is linked to one or more conjugate moieties, hi certain embodiments, the conjugate moiety comprises a clearance modifier, a toxin, a detectable label, a chemotherapeutic agent, or a purification moiety.

[0017]

[0025] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0026] In another aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof described herein.

[0018]

[0027] In another aspect, the present disclosure provides a vector comprising an isolated polynucleotide described herein.

[0028] In another aspect, the present disclosure provides a host cell comprising a vector described herein. In certain embodiments, the host cell is a mammalian cell, such as a CHO cell.

[0019]

[0029] In another aspect, the present disclosure provides hybridomas encoding or producing anti-LILRB4 antibodies as provided herein.

[0030] In another aspect, the present disclosure provides methods of expressing an antibody or antigen-binding fragment thereof described herein. In some embodiments, the method comprises culturing a host cell described herein under conditions in which a vector described herein is expressed.

[0020]

[0031] In another aspect, the present disclosure provides methods of treating or ameliorating the effects of cancer in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition described herein.

[0021]

[0032] The method may reduce or eradicate tumor burden in a subject, may reduce tumor cell number, may reduce tumor size, may reduce tumor invasion, may reduce tumor metastasis, or may eradicate tumor in a subject. The cancer may be a solid tumor or a hematological malignancy.

[0022]

[0033] In certain embodiments, the 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, stomach cancer, gastroesophageal cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, mesothelioma, squamous cell carcinoma, Cancer, melanoma, Merkel cell carcinoma, nasopharyngeal carcinoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, skin cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, and vaginal cancer.

[0023]

[0034] In some embodiments, the cancer is metastatic, recurrent, or drug-resistant cancer.

[0035] In some embodiments, the cancer is acute lymphocytic / lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic lymphoblastic leukemia (CLL), chronic myelomonocytic leukemia (CMML), chronic myeloid leukemia (CML), diffuse giant B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, hairy cell leukemia, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, pre-B acute lymphocytic leukemia (pre-B Hematological malignancies including ALL), primary CNS lymphoma, primary mediastinal giant cell B-cell lymphoma, T-cell / histiocytic-rich B-cell lymphoma, heavy chain disease, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma (MM), myelodysplastic syndromes (MDS), myeloproliferative neoplasms, and polycythemia vera.

[0024]

[0036] In certain embodiments, said hematological malignancies include subsets or subtypes of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic / monoblastic leukemia or M5 AML, and acute myeloblastic leukemia.

[0025]

[0037] In certain embodiments, the hematological malignancy includes acute myeloid leukemia (AML) that is resistant to venetoclax, or venetoclax in combination with azacitidine (azacytidine / azacitidine), or that has relapsed after treatment with azacitidine and / or venetoclax, or that is resistant to venetoclax in combination with decitabine, or that has relapsed after treatment with azacitidine and decitabine.

[0026]

[0038] In certain embodiments, the antibody or antigen-binding fragment thereof is administered intravenously, intraarterially, intratumorally, or subcutaneously.

[0039] In certain embodiments, the method comprises administering to the subject one or more of a topoisomerase inhibitor, an anthracycline topoisomerase inhibitor, an anthracycline, daunorubicin, a nucleoside metabolic inhibitor, cytarabine, a hypomethylating agent, low-dose cytarabine (LDAC), a combination of daunorubicin and cytarabine, injectable daunorubicin and cytarabine liposomal, Vyxeos®, azacitidine, Vidaza®, decitabine, all-trans retinoic acid (ATRA), arsenic, arsenous acid, histamine dihydrochloride, Ceplene®, interleukin-2, aldesleukin, Proleukin®, gemtuzumab ozogamicin, Mylotarg®, an FLT-3 inhibitor, midostaurin, Rydapt®, clofarabine, a farnesyltransferase inhibitor, decitabine , IDH1 inhibitors, ivosidenib, Tibsovo®, IDH2 inhibitors, enasidenib, Idhifa®, 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, CD70 antibodies, CLL1 antibodies, CD123 antibodies, E-selectin antibodies The method further comprises administering one or more agents selected from the group consisting of 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, a plant-derived alkaloid, a hormone therapy agent, a hormone antagonist, an aromatase inhibitor, and a P-glycoprotein inhibitor.

[0027]

[0040] In certain embodiments, the method further comprises first administering to the subject anti-LILRB4 antibody monotherapy for a period of time, followed by the addition of one or more agents selected from the group consisting of azacitidine, Vidaza®, a BCL-2 inhibitor, venetoclax, and Venclexta®.

[0028]

[0041] In yet another aspect, the present disclosure provides a method for detecting cancer cells or cancer stem cells in a sample or a subject. In certain embodiments, the method comprises: (a) contacting a subject or a sample from a subject with an antibody or antigen-binding fragment thereof described herein; and (b) detecting binding of the antibody to cancer cells or cancer stem cells in the subject or sample.

[0029]

[0042] In some embodiments, the sample is a bodily fluid or biopsy. In some embodiments, the sample is blood, sputum, tears, saliva, mucus, serum, urine, or feces.

[0043] In some embodiments, detection comprises immunohistochemistry, flow cytometry, immunoassays (including ELISA, RIA, etc.) or Western blot.

[0030]

[0044] In some embodiments, the method further comprises performing steps (a) and (b) a second or further time and determining a change in the level of detection as compared to the first time.

[0031]

[0045] The anti-LILRB4 antibody or antigen-binding fragment thereof may further comprise a label, such as 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.

[0032]

[0046] In another aspect, the disclosure provides the use of an antibody or antigen-binding fragment thereof described herein in the manufacture of a medicament for treating cancer in a subject.

[0047] In another aspect, the present disclosure provides kits comprising the antibodies or antigen-binding fragments thereof described herein, useful in detecting LILRB4.

[0033]

[0048] In another aspect, the present disclosure provides an anti-LILRB4 chimeric antigen receptor (CAR) protein. In some embodiments, the CAR protein comprises: (a) a heavy chain variable region comprising an HC-CDR1 having the amino acid sequence of SEQ ID NO: 5, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 9, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 10, each of which includes a mutation at amino acid residue NS. In some embodiments, the LC-CDR1 has the amino acid sequence of SEQ ID NO: 28.

[0034]

[0049] In some embodiments, the heavy chain variable region of the LILRB4 CAR protein has an amino acid sequence at least about 90% identical to SEQ ID NO: 1; and the light chain variable region of the CAR protein has an amino acid sequence at least about 90% identical to SEQ ID NO: 27. In some embodiments, the heavy chain variable region has the amino acid sequence of SEQ ID NO: 1; and the light chain variable region has the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CAR protein The R protein comprises a single chain variable fragment (scFv) having an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 66 or SEQ ID NO: 68. In some embodiments, the CAR protein has an scFv having an amino acid sequence that is identical to SEQ ID NO: 66 or SEQ ID NO: 68.

[0035]

[0050] In another aspect, the present disclosure provides a polynucleotide molecule encoding a CAR protein described herein. In some embodiments, the polynucleotide molecule further comprises a promoter active in eukaryotic cells. In some embodiments, the polynucleotide molecule is an expression vector.

[0036]

[0051] In another aspect, the present disclosure provides an engineered cell comprising a polynucleotide molecule encoding a CAR protein described herein. In some embodiments, the cell is a T cell, an NK cell, or a macrophage.

[0037]

[0052] In another aspect, the present disclosure provides a method of treating or ameliorating cancer in a subject in need of treatment, comprising administering to the subject an effective amount of a cell therapy comprising one or more cells comprising a polynucleotide molecule encoding a CAR protein described herein. In some embodiments, the method further comprises administering to the human subject a second cancer therapy. In some embodiments, the second cancer therapy is chemotherapy, immunotherapy, radiation therapy, hormonal therapy, or surgery. In some embodiments, the second cancer therapy is administered simultaneously with the cell therapy. In some embodiments, the second cancer therapy is administered before or after the cell therapy. In some embodiments, the method further comprises administering to the human subject a second administration of an effective amount of one or more cells comprising a polynucleotide molecule encoding a CAR protein described herein.

[0038]

[0053] In some embodiments, the cell therapy is administered locally to the cancer site, regionally to the cancer site, or systemically.

[0054] In some embodiments, the cancer is acute lymphocytic / lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic lymphoblastic leukemia (CLL), chronic myelomonocytic leukemia (CMML), chronic myeloid leukemia (CML), diffuse giant B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, follicular lymphoma, hairy cell leukemia, HHV8-associated primary effusion lymphoma, plasmablastic lymphoma, pre-B acute lymphocytic leukemia (pre-B Hematological malignancies including ALL), primary CNS lymphoma, primary mediastinal giant cell B-cell lymphoma, T-cell / histiocytic-rich B-cell lymphoma, heavy chain disease, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma (MM), myelodysplastic syndromes (MDS), myeloproliferative neoplasms, and polycythemia vera.

[0039]

[0055] In certain embodiments, said hematological malignancies include subsets or subtypes of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic / monoblastic leukemia or M5 AML, and acute myeloblastic leukemia.

[0040]

[0056] In yet a further aspect, there is provided a method of treating or ameliorating the effects of an autoimmune disease in a subject, 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, intraperitoneally, or subcutaneously. The method may further comprise administering to the subject one or more agents selected from the group consisting of steroids or NSAIDs. The autoimmune disease may be Guillain-Barré syndrome. group, chronic inflammatory demyelinating polyneuropathy, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, undifferentiated spondyloarthropathy, juvenile spondyloarthropathy, Behçet's disease, enthesitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, fibromyalgia, chronic fatigue syndrome, pain states associated with systemic inflammatory diseases, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, early-onset diabetes mellitus (also known as type 1 diabetes), Wegener's granulomatosis, polymyositis, dermatomyositis, inclusion body myositis, polymyositis, Endocrine deficiency, 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, Dresser'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, leukocytosis) Inno's phenomenon, esophageal hypoperistalsis, 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 maniasis, 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, Hook'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. [Brief explanation of the drawings]

[0041] [Figure 1]

[0057] FIG. 1 shows a schematic diagram of the mechanism of action of anti-LILRB4 antibodies and derived products. [Figure 2]

[0058] FIG. 2 shows a comparison of icIEF results for H7K3 (molecule A) and H7K3m5 (molecule B) at 40° C. for 2 or 4 weeks. [Figure 3]

[0059] FIG. 3 shows the recognition of human endogenous LILRB4 on THP-1 cells by the anti-LILRB4 antibody H7K3m5. [Figure 4]

[0060] FIG. 4 shows ADCC of THP-1-GFP cells by wild-type (WT) or afucosylated (afu) H7K3m5. [Figure 5]

[0061] FIG. 5 shows LILRB4 expression on human monocytes and plasmacytoid dendritic cells (pDCs). [Figure 6]

[0062] FIG. 6 shows upregulation of LILRB4 expression by IL-10 and IFNα treatment in human monocytes. [Figure 7]

[0063] FIG. 7 shows downregulated LILRB4 levels and upregulated uPAR levels on human monocytes stimulated by LPS. [Figure 8-1]

[0064] FIG. 8A shows LIRB4 expression on human monocyte-differentiated macrophages in vitro. [Figure 8-2] FIG. 8B shows the copy number of LILRB4 on monocyte-derived human macrophages in vitro. [Figure 9]

[0065] FIG. 9 shows the copy number of LILRB4 on in vitro differentiated MDSCs. [Figure 10-1]

[0066] Figure 10A shows the copy number of LILRB4 on human monocyte-derived dendritic cells (DCs). LILRB4 levels are found in the following order, from highest to lowest: tolerogenic DCs > activated DCs > immature DCs. [Figure 10-2]Figure 10B shows the copy number of LILRB4 on human monocyte-derived dendritic cells (DCs). LILRB4 levels are found in the following order, from highest to lowest: tolerogenic DCs > activated DCs > immature DCs. [Figure 11]

[0067] Figure 11 shows a comparison of LILRB4 mRNA expression levels between solid tumor samples from the TCGA RNA sequencing database, including high and low signals for macrophage infiltration, based on macrophage gene expression "signatures" generated through a computational biology approach. The total number of samples for each tumor type included in the analysis is shown in parentheses. The results shown here are based in part on RNA sequencing data generated by the TCGA Research Network. [Figure 12]

[0068] Figures 12A-12B illustrate representative flow cytometry data showing that H7K3m5 specifically binds to monocytic myeloid cells infiltrated within the solid tumor microenvironment (TME) as well as peripheral blood monocytic myeloid cells from solid tumor patients. Histograms filled in dark gray: samples incubated with H7K3m5; histograms filled in light gray: samples incubated with human IgG1 isotype control. Figure 12A. Binding signals of H7K3m5 or its isotype control on various myeloid cell subsets infiltrated into the TME. Figure 12B. Binding signals of H7K3m5 or its isotype control on various myeloid cell subsets derived from peripheral blood. [Figure 13-1]

[0069] Figures 13A-13D show the absence of H7K3m5-mediated monocyte killing in autologous ADCC using fresh PBMCs. Freshly isolated PBMCs from healthy donors were incubated overnight in the presence of serially titrated H7K3m5, isotype control human IgG1, or rituximab as a positive control. Monocytes and B cells were identified and enumerated by flow cytometry as CD14+CD19- and CD19+CD14-. Figures 13A and 13C show the absence of monocyte killing by PBMCs from two different donors, and Figures 13B and 13D show the corresponding B cell killing as a positive control. [Figure 13-2] Figures 13A-13D show the absence of H7K3m5-mediated monocyte killing in autologous ADCC using fresh PBMCs. Freshly isolated PBMCs from healthy donors were incubated overnight in the presence of serially titrated H7K3m5, isotype control human IgG1, or rituximab as a positive control. Monocytes and B cells were identified and enumerated by flow cytometry as CD14+CD19- and CD19+CD14-. Figures 13A and 13C show the absence of monocyte killing by PBMCs from two different donors, and Figures 13B and 13D show the corresponding B cell killing as a positive control. [Figure 14-1]

[0070] Figures 14A-14D show autologous ADCC of normal monocytes by wild-type (WT) or afucosylated (afu) H7K3m5. Figures 14A and 14C show representative monocyte killing via ADCC by PBMCs from two different donors. ADCC against monocytes by afucosylated H7K3m5 was observed in donor 024, while activity was minimal in donor 13. No ADCC was observed with wild-type H7K3m5. Figures 14B and 14D show the corresponding B cell killing as a positive control. [Figure 14-2]Figures 14A-14D show autologous ADCC of normal monocytes by wild-type (WT) or afucosylated (afu) H7K3m5. Figures 14A and 14C show representative monocyte killing via ADCC by PBMCs from two different donors. ADCC against monocytes by afucosylated H7K3m5 was observed in donor 024, while activity was minimal in donor 13. No ADCC was observed with wild-type H7K3m5. Figures 14B and 14D show the corresponding B cell killing as a positive control. [Figure 15-1]

[0071] Figures 15A-15D show autologous ADCC of pDCs or monocytes mediated by wild-type (WT) or afucosylated (afu) H7K3m5. ADCC against pDCs was observed with both wild-type and afucosylated H7K3m5 in two donors. Meanwhile, monocytes could only be killed by afucosylated H7K3m5, depending on the donor. Furthermore, afucosylated H7K3m5 showed much stronger ADCC activity against pDCs or monocytes than wild-type H7K3m5 (Figures 15A-15B). [Figure 15-2] Figures 15A-15D show autologous ADCC of pDCs or monocytes by wild-type (WT) or afucosylated (afu) H7K3m5-mediated autologous ADCC. ADCC of pDCs was observed with both wild-type and afucosylated H7K3m5 in two donors. Meanwhile, monocytes could only be killed by afucosylated H7K3m5, depending on the donor. [Figure 16]

[0072] Figures 16A-16B show dose-dependent ADCC of CD33+ MDSC cells with purified NK cells in the presence of H7K3m5, which has no ADCC effect on monocytes at the same dose level. H7K3m5 also showed ADCC activity against the AML cell line THP-1 in the same experiment (Figure 16B). [Figure 17-1]

[0073] Figure 17A shows ADCP of THP-1-GFP with anti-LILRB4. THP-1-GFP cells were cocultured with in vitro-differentiated macrophages for 24 hours in the presence of serially titrated wild-type H7K3m5 or isotype control human IgG1. THP-1-GFP cells and macrophages were identified and quantified as GFP+ and CD163+CD206+, respectively. The percentage of killed THP-1 cells was calculated from the absolute number of GFP+ cells or from the GFP+% cells. Figures 17A and 17B show ADCP of THP-1-GFP cells with wild-type H7K3m5 derived from macrophages differentiated from two separate healthy donors. [Figure 17-2] Figure 17B shows ADCP of THP-1-GFP with anti-LILRB4. THP-1-GFP cells were cocultured with in vitro-differentiated macrophages for 24 hours in the presence of serially titrated wild-type H7K3m5 or isotype control human IgG1. THP-1-GFP cells and macrophages were identified and quantified as GFP+ and CD163+CD206+, respectively. The percentage of killed THP-1 cells was calculated from the absolute number of GFP+ cells or from the GFP+% cells. Figures 17A and 17B show ADCP of THP-1-GFP cells with wild-type H7K3m5 derived from macrophages differentiated from two separate healthy donors. [Figure 18]

[0074] Figure 18 shows in vitro T cell cytotoxicity against THP-1-GFP cells with anti-LILRB4. THP-1-GFP cells were co-cultured with purified naive T cells. Anti-LILRB4 H7K3m5 can induce T cell cytotoxicity against AML cells. Effector pan-T cells were derived from three different healthy donors. Curves are plotted as mean ± SD. EC50 values ​​are in nanomolar units. [Figure 19]

[0075] Figure 19 shows representative cytokine production profiles in supernatants from in vitro T cell cytotoxicity assay samples. Anti-LILRB4-induced T cell cytotoxicity against THP-1 cells is reflected by elevated cytokines during co-culture. Curves are plotted as mean ± SD. EC50 values ​​are in nanomolar. EC50 values ​​are not available for IL-6. [Figure 20]

[0076] Figure 20 shows the assessment of T cell activation by flow cytometry. A-B: Surface staining of T cell activation markers CD69 (A) and CD25 (B). C-E: Intracellular cytokine staining of co-cultured T cells and THP-1 cells by flow cytometry. C: Cells producing both IFNγ and TNFα; D: Cells producing IFNγ but not TNFα; E: Cells producing TNFα but not IFNγ. [Figure 21]

[0077] Figure 21 shows the increase in T cell activation markers and MHC expression on THP-1 cells upon H7K3m5 treatment. D428 = donor 428. MFI = geometric mean fluorescence intensity. [Figure 22]

[0078] Figure 22 shows surface expression of activation markers on THP-1 AML cells by flow cytometry. MFI = geometric mean fluorescence intensity. [Figure 23]

[0079] Figure 23 shows that H7K3m5 is effective in an AML xenograft model. This experiment evaluated the growth kinetics of THP-1.luc cells and used bioimaging to determine the efficacy of H7K3m5 in a THP-1.luc human AML xenograft model in female NSG mice. 1x10 THP-1.luc cells were intravenously implanted into JAX female NSG mice via the tail vein. After whole-body bioluminescence imaging on day 1 (4-6 hours after cell injection), animals were randomized and received a single intravenous dose of vehicle control or H7K3m5 (1 mg / kg). Whole-body bioluminescence imaging data were collected for control and treated animals on days 7, 14, 17, and 21. [Figure 24]

[0080] Figure 24 illustrates flow cytometry data showing that H7K3m5 enhances the maturation / activation of monocyte-derived dendritic cells (Mo-DCs) in response to Toll-like receptor (TLR) signaling. H7K3m5 increased the expression of activation markers (CD86, HLA-DR) while decreasing the expression of the tolerogenic marker CD209. Each line represents results from a different healthy donor. The percentage of donors in which H7K3m5 produced the desired pro-inflammatory effect is indicated in parentheses. *p<0.05 (paired t-test). [Figure 25]

[0081] Figure 25 illustrates flow cytometry data showing that H7K3m5 enhances the expression of activation markers (CD86 and HLA-DR) on the surface of mature monocyte-derived DCs (Mo-DCs) during mixed leukocyte reactions with allogeneic T cells. To study the effect of H7K3m5 on immature and mature Mo-DCs, respectively, the effect of H7K3m5 was assessed in the absence (-CD40L) or presence (+CD40L) of CD40 ligand. Each line represents results from a different healthy donor (n=3 donors). [Figure 26]

[0082] Figure 26 illustrates ELISA data showing that H7K3m5 enhances IL-12 production in allogeneic mixed leukocyte reactions of T cells and mature monocyte-derived DCs (Mo-DCs). To study the effect of H7K3m5 on immature and mature Mo-DCs, respectively, the effect of H7K3m5 was assessed in the absence (-CD40L) or presence (+CD40L) of CD40 ligand. Data are presented as mean ± SEM, and data for each donor are also shown as individual data points (n = 2-3 donors). [Figure 27]

[0083] Figure 27 illustrates ELISA data showing that H7K3m5 enhances IFN-γ production in allogeneic mixed leukocyte reactions of T cells and monocyte-derived DCs (Mo-DCs). To study the effects of H7K3m5 on immature and mature Mo-DCs, respectively, the effect of H7K3m5 was evaluated in the absence (-CD40L) or presence (+CD40L) of CD40 ligand. Data are presented as mean ± SEM, and data for each donor are also shown as individual data points (n = 3 donors). [Figure 28]

[0084] Figures 28A and 28B show schematic diagrams of the configuration of LILRB4 / CD3 bispecific antibodies. [Figure 29]

[0085] Figures 29A and 29B show binding of CD3 and LILRB4 bispecific antibodies to normal monocytes and the AML cell line THP-1 as measured by FACS. Similar binding affinity trends were observed between monocytes and THP-1 across different anti-LILRB4 monospecific and bispecific antibodies. [Figure 30]

[0086] Figures 30A and 30B show T cell-mediated cytotoxicity of bispecific CD3 / LILRB4 antibodies against monocytes (Figure 22A) and THP-1-luc-GFP cells (Figure 22B). [Figure 31-1]

[0087] Figures 31A and 31B show auto-killing of monocytes by LILRB4xCD3 bispecific antibody (Figure 31A) and auto-killing of B cells by Rituxan as a control (Figure 31B). [Figure 31-2] Figures 31A and 31B show auto-killing of monocytes by LILRB4xCD3 bispecific antibody (Figure 31A) and auto-killing of B cells by Rituxan as a control (Figure 31B). [Figure 32]

[0088] FIG. 32 shows a comparison of binding affinities using H7K3m5 full-length IgG and ScFv proteins to human primary monocytes and the human leukemia cell line THP-1 in a flow cytometry assay. [Figure 33]

[0089] Figure 33 shows a schematic diagram of the DNA construct for expressing the anti-LILRB4 CAR protein. The DNA construct was based on a second-generation CAR construct containing a CD3 zeta activation domain and a CD28 or 4-1BB costimulatory domain. The scFv was derived from the anti-LILRB4 monoclonal antibody H7K3m5. The 5' and 3' homology arms are homologous sequences upstream and downstream of the Cas9 DNA cleavage site in the TRAC gene (based on the gRNA design). The promoter and leader peptide are elements for gene expression and cell export location. An SV40 polyA tail was included to improve transcript stability and translation. [Figure 34]

[0090] Figure 34 shows the efficient generation of LILRB4 CAR-T cells using the CRISPR knockout and knockin method. Human primary T cells were transfected with CRISPR-Cas9 RNP complexes designed to inactivate the TCR alpha (TRAC) locus, with or without a DNA template for homologous recombination-based knockin. After transfection, cells were grown in culture for two weeks. Anti-LILRB4 CAR-T cells were identified by binding to an LILRB4-Fc fusion protein (ACRObiosystems CDK-H5259) and an anti-Fc antibody (Biolegend B278652, negative control). Successful TCR alpha (TRAC) inactivation (knockout or KO) was measured by anti-CD3 staining (anti-CD3 PE, BD555333). ATC, activated T cells; KO, TCR alpha (TRAC) inactivated T cells; RB4_CD28, T cells expressing anti-LILRB4 CAR containing a CD28 costimulatory domain; RB4_41BB, T cells expressing anti-LILRB4 CAR containing a 4-1BB costimulatory domain. [Figure 35]

[0091] Figure 35 shows the proliferation of TCR alpha (TRAC) inactivated T cells (KO) and anti-LILRB4 CAR (or control CAR) knock-in T cells. After knockout of TCR alpha, cells were grown in complete Optimizer medium containing IL-2 300 IU / ml and no anti-CD3 / 28 added. Fold expansion was plotted by dividing the total T cell number on that day (as indicated) by the initial culture number. Anti-LILRB4 CAR-T cells showed significantly higher fold expansion compared to control CAR-T cells. ATC, activated T cells; KO, TCR alpha (TRAC) inactivated T cells; ctrl_CD28, T cells expressing a control CAR containing a CD28 costimulatory domain; ctrl_41BB, T cells expressing a control CAR containing a 4-1BB costimulatory domain; RB4_CD28, T cells expressing an anti-LILRB4 CAR containing a CD28 costimulatory domain; RB4_41BB, T cells expressing an anti-LILRB4 CAR containing a 4-1BB costimulatory domain. [Figure 36-1]

[0092] Figures 36A-36H show antigen-dependent activation of CAR-T cultures. 1 μg / ml of recombinant control antigen or LILRB4 antigen was coated onto a 96-well plate overnight in PBS buffer. The plate was washed twice with PBS buffer. 1×10 CAR-T cells in medium (without any added cytokines) were added to each well and incubated for 72 hours. Cell culture supernatants were collected for cytokine release measurement by Luminex assay. ATC, activated T cells; KO, TCR alpha (TRAC) inactivated T cells; anti-RB4_CD28CART, T cells expressing an anti-LILRB4 CAR containing a CD28 costimulatory domain; anti-RB4-41BBCART, T cells expressing an anti-LILRB4 CAR containing a 4-1BB costimulatory domain; control_CD28CART, T cells expressing a control CAR containing a CD28 costimulatory domain; control_41BBCART, T cells expressing a control CAR containing a 4-1BB costimulatory domain. [Figure 36-2]Figures 36A-36H show antigen-dependent activation of CAR-T cultures. 1 μg / ml of recombinant control antigen or LILRB4 antigen was coated onto a 96-well plate overnight in PBS buffer. The plate was washed twice with PBS buffer. 1×10 CAR-T cells in medium (without any added cytokines) were added to each well and incubated for 72 hours. Cell culture supernatants were collected for cytokine release measurement by Luminex assay. ATC, activated T cells; KO, TCR alpha (TRAC) inactivated T cells; anti-RB4_CD28CART, T cells expressing an anti-LILRB4 CAR containing a CD28 costimulatory domain; anti-RB4-41BB CART, T cells expressing an anti-LILRB4 CAR containing a 4-1BB costimulatory domain; control_CD28CART, T cells expressing a control CAR containing a CD28 costimulatory domain; control_41BBCART, T cells expressing a control CAR containing a 4-1BB costimulatory domain. [Figure 36-3] Figures 36A-36H show antigen-dependent activation of CAR-T cultures. 1 μg / ml of recombinant control antigen or LILRB4 antigen was coated onto a 96-well plate overnight in PBS buffer. The plate was washed twice with PBS buffer. 1×10 CAR-T cells in medium (without any added cytokines) were added to each well and incubated for 72 hours. Cell culture supernatants were collected for cytokine release measurement by Luminex assay. ATC, activated T cells; KO, TCR alpha (TRAC) inactivated T cells; anti-RB4_CD28CART, T cells expressing an anti-LILRB4 CAR containing a CD28 costimulatory domain; anti-RB4-41BB CART, T cells expressing an anti-LILRB4 CAR containing a 4-1BB costimulatory domain; control_CD28CART, T cells expressing a control CAR containing a CD28 costimulatory domain; control_41BBCART, T cells expressing a control CAR containing a 4-1BB costimulatory domain. [Figure 37-1]

[0093] Figures 37A-37C show the characterization of CAR-T cells after 2 weeks of expansion. Frozen CAR-T cells stored in liquid nitrogen were thawed and maintained in culture for 2-3 days before flow cytometry analysis. The antibodies used were anti-CD8 APC Cy7 (BD561945), anti-PD1 PE (BD560908), and anti-TIM3 BV421 (BD565562). Anti-LILRB4 CAR-T cells were identified by binding to LILRB4-Fc fusion protein (ACRObiosystems CDK-H5259) and anti-Fc antibody (Biolegend B278652). [Figure 37-2] Figures 37A-37C show the characterization of CAR-T cells after 2 weeks of expansion. Frozen CAR-T cells stored in liquid nitrogen were thawed and maintained in culture for 2-3 days before flow cytometry analysis. The antibodies used were anti-CD8 APC Cy7 (BD561945), anti-PD1 PE (BD560908), and anti-TIM3 BV421 (BD565562). Anti-LILRB4 CAR-T cells were identified by binding to LILRB4-Fc fusion protein (ACRObiosystems CDK-H5259) and anti-Fc antibody (Biolegend B278652). [Figure 37-3] Figures 37A-37C show the characterization of CAR-T cells after 2 weeks of expansion. Frozen CAR-T cells stored in liquid nitrogen were thawed and maintained in culture for 2-3 days before flow cytometry analysis. The antibodies used were anti-CD8 APC Cy7 (BD561945), anti-PD1 PE (BD560908), and anti-TIM3 BV421 (BD565562). Anti-LILRB4 CAR-T cells were identified by binding to LILRB4-Fc fusion protein (ACRObiosystems CDK-H5259) and anti-Fc antibody (Biolegend B278652). [Figure 38]

[0094] Figure 38 shows the cytotoxicity of anti-LILRB4 CAR-T cells. CHO K1 RB4 cells were seeded at different densities (6x104, 2x104, or 7x104) for 12 hours, 1x105 CAR-T cells were added, and cytotoxicity was measured by removing the supernatant CAR-T cells and washing the plate twice with PBS. Total viable and adherent CHO K1 RB4 cells were measured using a Promega CTG2.0 luminescence kit. The % cytotoxicity was calculated by dividing the luminescence signal of each condition by that of a T cell control activated at the same E:T ratio. [Figure 39-1]

[0095] Figures 39A-39B show schematics of a Phase 1, first-in-human clinical trial. Figure 39A is a schematic of the "window" design for dose escalation. Figure 39B is a schematic of anti-LILRB4 monotherapy. Figures 39C-39D are schematics of potential combination studies of anti-LILRB4 antibodies with azacitidine and / or venetoclax. Other potential combinations with anti-LILRB4 follow the same or similar schema. AZA, azacitidine; VEN, venetoclax; C1D1, cycle 1 day 1; cycle 2 day 1; DLT, dose-limiting toxicity; MTD1, maximum tolerated dose of anti-LILRB4 monotherapy; MTD2, maximum tolerated dose of anti-LILRB4 in combination with azacitidine. Anti-LILRB4 will be administered as monotherapy or in combination with other agents every 14 days until disease progression or death. [Figure 39-2] Figures 39C-39D are schematic diagrams of potential combination studies of anti-LILRB4 antibodies with azacitidine and / or venetoclax. Other potential combinations with anti-LILRB4 follow the same or similar schema. AZA, azacitidine; VEN, venetoclax; C1D1, cycle 1 day 1; cycle 2 day 1; DLT, dose-limiting toxicity; MTD1, maximum tolerated dose of anti-LILRB4 monotherapy; MTD2, maximum tolerated dose of anti-LILRB4 in combination with azacitidine. Anti-LILRB4 is administered as monotherapy or in combination with other agents every 14 days until disease progression or death. DETAILED DESCRIPTION OF THE INVENTION

[0042]

[0096] The following description of the present disclosure is intended merely to illustrate various aspects of the disclosure. As such, the specific modifications discussed are not to be considered limitations on the scope of the disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the disclosure, and it is understood that such equivalent aspects are intended to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein in their entirety.

[0043]

[0097] I. Definition

[0098] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and do not limit the invention as claimed. In this application, the use of the singular includes the plural unless expressly stated otherwise. In this disclosure, the term "or" is used to mean "and / or," unless expressly indicated to refer to alternatives only or where the alternatives are mutually exclusive. As used herein, "another" may mean at least a second or more. Furthermore, the use of the term "including" and other forms, such as "includes" and "included," is not limiting. Also, terms such as "element" or "component" include both elements or components that contain one unit or element, and elements or components that contain more than one subunit, unless otherwise stated. Also, the use of the term "portion" can include a part of a moiety or an entire portion.

[0044]

[0099] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0100] The term "antibody," as used herein, includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A natural, intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, and each heavy chain contains a variable domain (V H ), and the first, second, and third constant domains (C H1 , C H2 , C H3 Mammalian light chains are classified as λ or κ, while each light chain consists of a constant region containing a variable domain (V L ) and constant domains (C L ). A typical IgG antibody has a "Y" shape, with the stem of the Y typically consisting of the second and third constant domains of two heavy chains linked together through disulfide bonds. Each arm of the Y contains the variable domain and first constant domain of a single heavy chain linked to the variable and constant domains of a single light chain. The variable domains of the light and heavy chains are involved in antigen binding. Both chains The variable domains therein generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs comprising LCDR1, LCDR2, and LCDR3; heavy chain CDRs comprising HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the Kabat, IMGT, Chothia, or Al-Lazikani conventions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. (1985) 186(3):651-63; Chothia, C. and Lesk, AM, J. Mol. Biol. (1987) 196:901; Chothia, C. et al., Nature (1989) 342(6252):877-83; Marie-Paule Lefranc et al., Developmental and Comparative Immunology (2003) 27: 55-77; Marie-Paule Lefranc et al. Immunome Research (2005) 1(3); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), chapter 26, 481-514, (2015)). The three CDRs are interposed between adjacent stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant domains of the heavy and light chains are not involved in antigen binding but exhibit diverse effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are divided into subclasses, e.g., IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain).

[0045]

[0101] The term "antigen" refers to a substance capable of inducing an adaptive immune response. In particular, an antigen is a substance that is specifically bound by an antibody or a T lymphocyte antigen receptor. Antigens are usually proteins and polysaccharides, and less frequently lipids. Suitable antigens include, without limitation, parts of bacteria (coats, capsules, cell walls, flagella, fimbriae, and toxins), viruses, and other microorganisms. Antigens also include tumor antigens, such as antigens generated by mutations in tumors. As used herein, antigens also include immunogens and haptens.

[0046]

[0102] The term "antigen-binding fragment," as used herein, refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain an intact native antibody structure. Examples of antigen-binding fragments include, without limitation, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide bridges, and the like. Stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsF Antigen-binding fragments include single-domain antibodies (single-domain antibodies), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), diabodies, multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments are capable of binding to the same antigen as that bound by the parent antibody.

[0047]

[0103] A "Fab fragment" is a fragment of one light chain and one heavy chain. H 1 and variable The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0048]

[0104] A "Fab' fragment" is a fragment containing one light chain and one V H Domain and C H 1 dollar A portion of one heavy chain containing a C H 1 and C H It includes the region between the two domains such that an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0049]

[0105] A "F(ab')2 fragment" is a fragment that contains two light chains and a C H 1 and C H 2D A F(ab')2 fragment therefore contains two heavy chains containing a portion of the constant region between the heavy chains, such that an interchain disulfide bond is formed between the two heavy chains. It consists of two Fab' fragments held together by a disulfide bond between the heavy chains.

[0050]

[0106] "Fv," with respect to an antibody, refers to the minimum fragment of an antibody that retains a complete antigen-binding site. The Fv fragment consists of the variable domain of a single light chain linked to the variable domain of a single heavy chain.

[0051]

[0107] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable domain and a heavy chain variable domain linked to each other directly or through a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA (1988) 85:5879).

[0052]

[0108] The "Fc" region is the C H 2 and C H The two heavy chain fragments contain two heavy chain fragments each containing a C3 domain. The two heavy chain fragments are connected by two or more disulfide bonds and by C H The Fc region of an antibody is held together by hydrophobic interactions between the three domains. They are involved in important effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but do not function in antigen binding.

[0053]

[0109] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0110] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable domain of a single light chain and the variable domain of a single heavy chain is a disulfide bond. In some embodiments, a "(dsFv)2" or "(dsFv-dsFv')" comprises three peptides. V chain: linked by a peptide linker (e.g., a long, flexible linker) and each connected by a disulfide bridge to two V L Two Vs joined to a domain H In some embodiments, the dsFv-dsFv' is bispecific, with each disulfide-paired heavy and light chain having a different antigen specificity.

[0054]

[0111] "Camelized single domain antibodies," "heavy chain antibodies," or "HCAbs" are antibodies that contain two V H refers to an antibody that contains a light chain domain and does not contain a light chain (Riechman (N L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from the Camelidae family (camels, dromedaries, and llamas). Although they lack light chains, camelized antibodies have authentic antigen-binding repertoires (Hamers-Casterman C. et al., Nature (1993) 363:446-8; Nguyen VK. et al., Immunogenetics (2002) 54:39-47; Nguyen VK. et al., Immunology (2003) 109:93-101). The variable domains of heavy chain antibodies (VHH domains) represent the smallest known antigen-binding units generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. (2007) 21:3490-8).

[0055]

[0112] "Nanobody" refers to an antibody fragment consisting of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.

[0113] "Diabodies" or "dAbs" are fragments of V L V linked to domain H Domain Included (V H -V L or V L -V H), including small antibody fragments with two antigen-binding sites (see, e.g., Holliger P. et al., Proc Natl Acad Sci US A. Jul 15;90(14):6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, thereby generating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0056]

[0114] In certain embodiments, an "scFv dimer" is a divalent or bivalent single-chain variable fragment (di-scFv, bi-scFv) that can be engineered by linking two scFvs. H -V L V dimerized with the moiety H -V L Bivalent diabodies or bivalent scFvs (BsFv, di-scFv, bi-scFv) comprise a V of one part (linked by a peptide linker). H is the other part V L and in concert with the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a dimer of V H1 and V L1 cooperate, and V H2 and V L2 V cooperates, and each cooperated pair has a different antigenic property. H1 -V L2 (linked by a peptide linker) and V L1 -V H2 (also linked by a peptide linker) associated therewith.

[0057]

[0115] A "domain antibody" refers to an antibody fragment containing only the variable domain of a heavy chain or the variable domain of a light chain. In particular examples, two or more V H Domain is The two Vs of a bivalent domain antibody are linked by a peptide linker to form a bivalent or multivalent domain antibody. H The domains may target the same or different antigens.

[0058]

[0116] A "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes, which may be on the same antigen or on two different antigens.

[0059]

[0117] "Cancer," as used herein, refers to any medical condition characterized by malignant cell growth or neoplasia, abnormal growth, invasion, or metastasis, and includes both solid tumors and non-solid tumors (hematological malignancies), such as leukemia. As used herein, "solid tumor" refers to a solid mass of neoplastic and / or malignant cells. Examples of cancers or tumors include hematological malignancies, oral cancer (e.g., cancer of the lip, tongue, or pharynx), digestive tract (e.g., esophagus, stomach, small intestine, colon, large intestine, or rectum), peritoneum, liver and biliary tract, pancreas, respiratory system (e.g., larynx or lung (small cell and non-small cell), bone, connective tissue, skin (e.g., melanoma), breast, reproductive organs (fallopian tubes, uterus, cervix, testes, ovaries, or prostate), urinary tract (e.g., bladder or kidney), brain, and endocrine glands, such as the thyroid gland. In certain embodiments, the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, renal cancer, bladder cancer, hepatocellular carcinoma, and colorectal cancer. In certain embodiments, the cancer is selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and B-cell lymphoma.

[0060]

[0118] The term "chimeric" is used herein to refer to a chimeric antibody that contains heavy chains and / or antibodies derived from one species. "Chimeric" refers to an antibody or antigen-binding fragment having a portion of a heavy or light chain and the remaining portions of a heavy and / or light chain from a different species. In an illustrative example, a chimeric antibody may contain a constant region from a human and a variable region from a non-human animal, such as a mouse or rabbit. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0061]

[0119] The terms "specific binding" or "specifically bind" as used herein refer to a non-random binding reaction between two molecules, such as an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein have a binding affinity of ≦10 -6 M (e.g., ≦5x10 -7 M, ≤2x10 -7 M, ≤10 -7 M, ≤5x10 -8 M, ≤2x10 -8 M, ≤10 -8 M, ≤5x10 -9 M, ≤4x10 -9 M, ≤3x10 -9 M, ≤2x10 -9 M, or ≤ 10 -9 M) binding affinity (K D ) and specifically binds to human LILRB4. Te, K D is the ratio of the dissociation rate to the association rate (k off / k on ), which can be determined by 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). In certain embodiments, K D The value may be suitably determined by using flow cytometry.

[0062]

[0120] The ability to "block binding" or "compete for the same epitope" herein refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human LILRB4 and an anti-LILRB4 antibody) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 85%, or greater than 90%.

[0063]

[0121] Those skilled in the art will recognize that, without undue experimentation, it is possible to determine whether a given antibody binds to the same epitope as an antibody of the present disclosure by determining whether the former prevents the latter from binding to the LILRB4 antigen polypeptide. If a given antibody competes with an antibody of the present disclosure, as indicated by reduced binding to the LILRB4 antigen polypeptide by the antibody of the present disclosure, then the two antibodies bind to the same or closely related epitopes. Alternatively, if the binding of a given antibody to the LILRB4 antigen polypeptide is inhibited by an antibody of the present disclosure, then the two antibodies bind to the same or closely related epitopes.

[0064]

[0122] The term "chimeric antigen receptor" or "CAR," as used herein, refers to an engineered receptor that can transfer a desired specificity for an antigen into immune effector cells, such as T cells, NK cells, and macrophages. Typically, a CAR protein comprises an extracellular domain that conveys the desired specificity, a transmembrane domain, and an intracellular domain that transmits a signal to the immune effector cell when the immune effector cell binds to the antigen. In certain embodiments, the extracellular domain comprises a leader peptide, an antigen recognition region, and a spacer region. In certain embodiments, the antigen recognition region is derived from an antibody that specifically binds to the antigen. In certain embodiments, the antigen recognition region is a single-chain variable fragment (scFv) derived from an antibody. In certain embodiments, the single-chain variable fragment (scFv) is derived from a humanized antibody. In certain embodiments, the single-chain variable fragment comprises a heavy chain variable region fused to a light chain variable region via a flexible linker.

[0065]

[0123] A "conservative substitution" with respect to an amino acid sequence refers to the replacement of an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, a conservative substitution may be made with an amino acid residue having a hydrophobic side chain (e.g., Met, Ala, Val, Leu, and Conservative substitutions may be made between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions usually do not cause significant changes in the conformational structure of the protein and, therefore, may retain the biological activity of the protein.

[0066]

[0124] "Effector function," as used herein, refers to a biological activity that can contribute to the binding of the Fc region of an antibody to effectors such as the C1 complex and Fc receptors. Exemplary effector functions include complement-dependent cytotoxicity (CDC), which is induced by the interaction of an antibody and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC), which is induced by the binding of the Fc region of an antibody to an Fc receptor on an effector cell; and phagocytosis.

[0067]

[0125] The term "epitope" as used herein refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitopes within the antigen. For example, if an antibody or antigen-binding fragment blocks the binding of a reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%, the antibody or antigen-binding fragment can be considered to bind to the same / closely related epitope as the reference antibody.

[0068]

[0126] The terms "homologue" and "homologous" are used interchangeably herein and refer to a nucleic acid sequence (or its complementary strand) or amino acid sequence that, when optimally aligned, has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence.

[0069]

[0127] The phrase "host cell," as used herein, refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0128] The term "humanized" as used herein means that the antibody or antigen-binding fragment contains CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, constant regions derived from a human.

[0070]

[0129] An "isolated" material is one that has been altered by the hand of man from its natural state. When an "isolated" composition or material exists in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated," but the same polynucleotide or polypeptide is "isolated" if it is sufficiently separated from the materials with which it naturally occurs so that it exists in a substantially pure state. An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment that has a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (e.g., ion exchange chromatography or reverse-phase HPLC).

[0071]

[0130] "Leader peptide" refers to a peptide having a length of approximately 5-30 amino acids present at the N-terminus of newly synthesized proteins that form part of the secretory pathway. Proteins of the secretory pathway include, but are not limited to, those that are secreted into specific intracellular organelles (e.g., the endoplasmic reticulum, the Golgi, and the cytoplasm). In some embodiments, the leader peptide forms part of the transmembrane domain of the protein.

[0072]

[0131] "LILRB4," as used herein, refers to LILRB4 from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). Exemplary sequences of human LILRB4 include GenBank sequence reference numbers NP_001265355, AAH26309, ABM83015, ABM86208, and AIC55892. The term "LILRB4," as used herein, is intended to include any form of human LILRB4, for example: 1) a native, unprocessed LILRB4 molecule, a "full-length" LILRB4 chain, or a naturally occurring variant of LILRB4, including a splice variant or allelic variant; 2) any form of LILRB4 that results from processing in a cell; or 3) a full-length, fragment (e.g., truncated, extracellular / transmembrane domain), or modified (e.g., mutated, glycosylated / PEGylated, His-tag / immunofluorescence fusion) LILRB4 subunit produced through recombinant methods.

[0073]

[0132] The term "anti-LILRB4 antibody" refers to an antibody capable of specifically binding to LILRB4 (e.g., human or monkey LILRB4).

[0133] As used herein, a "LILRB4-associated" disease or condition refers to any disease or condition caused by, exacerbated by, or otherwise associated with increased or decreased expression or activity of LILRB4. In some embodiments, the LILRB4-associated condition is an immune-related disorder, such as cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0074]

[0134] The term "linked" as used herein refers to association through intramolecular interactions, such as covalent, metallic, and / or ionic bonds, or intermolecular interactions, such as hydrogen bonds or non-covalent bonds.

[0075]

[0135] The term "operably linked" refers to an arrangement of elements in which the described components are configured so as to perform their normal function. Thus, a given signal peptide operably linked to a polypeptide will direct the secretion of the polypeptide from a cell. In the case of a promoter, a promoter operably linked to a coding sequence will direct 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, intervening untranslated but still transcribed sequences can be present between the promoter and coding sequence and the promoter sequence would still be considered "operably linked" to the coding sequence.

[0076]

[0136] "Percent (%) sequence identity" is defined, with respect to an amino acid sequence (or nucleic acid sequence), as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be performed using, for example, publicly available tools, such as BLASTN, BLASTp (US National Center for Available on the National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al., J. Mol. Biol. (1990) 215:403-410; Stephen F. et al., Nucleic Acids Res. (1997) 25:3389-3402. This can be achieved using the following software: ClustalW2 (available on the European Bioinformatics Institute website; see also Higgins DG et al., Methods in Enzymology (1996) 266:383-402; Larkin MA et al., Bioinformatics (2007) 23:2947-8), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool or may customize the parameters as appropriate for the alignment, for example, by selecting an appropriate algorithm.

[0077]

[0137] 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 deoxyribonucleotides 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, phosphoroanilothiones, phosphoraniladates, and phosphoramidates.

[0078]

[0138] The term "polypeptide" or "protein" refers to a string of at least two amino acids linked together by peptide bonds. Polypeptides and proteins may contain moieties in addition to amino acids (e.g., may be glycosylated) and / or may be otherwise processed or modified. One of ordinary skill in the art will recognize that a "polypeptide" or "protein" may be the entire polypeptide chain (with or without a signal sequence) as produced by a cell, or a functional portion thereof. One of ordinary skill in the art will further recognize that a polypeptide or protein may sometimes comprise more than one polypeptide chain, linked, for example, by one or more disulfide bonds or associated by other means. 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.

[0079]

[0139] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient(s), and / or salt is generally chemically and / or physically compatible with other ingredients contained in the formulation, and physiologically compatible with the recipient thereof.

[0080]

[0140] 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). Humans include prenatal and postnatal. In many embodiments, a subject is a human. A subject may be a patient, which refers to a person who visits a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be affected by or susceptible to a disease or injury, but may or may not exhibit symptoms of the disease or disorder.

[0081]

[0141] 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 antibodies or antigen-binding fragments thereof disclosed herein, a therapeutically effective amount may be one that reduces tumor volume, eradicates all or part of a tumor, inhibits or delays tumor growth or cancer cell invasion into other organs, inhibits the growth or proliferation of cells that mediate a cancerous condition, inhibits or delays tumor cell metastasis, or inhibits or delays tumor or The dosage or concentration of a monoclonal antibody or antigen-binding fragment thereof that can ameliorate any symptoms or markers associated with a tumor or cancerous condition, prevent or slow the development of a tumor or cancerous condition, or some combination thereof.

[0082]

[0142] "Treating" a condition or "treatment," as used herein, includes preventing or alleviating the condition, delaying the onset of the condition or the rate at which the condition develops, reducing the risk of developing the condition, preventing or delaying the development 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.

[0083]

[0143] The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to cause expression of the protein. A vector can be used to transform, transduce, or transfect a host cell to cause expression of the genetic elements carried by the vector within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). A vector may contain various elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selectable element, and a reporter gene. Additionally, a vector may contain an origin of replication. A vector may also contain a substance that assists entry into a cell, including, but not limited to, a viral particle, a liposome, or a protein coating. A vector may be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda phage, and M13 phage, and the plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, and pALTER. , pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBA BE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0084]

[0144] II. Anti-LILRB4 Antibodies and Antigen-Binding Fragments

[0145] In one aspect, the present disclosure provides anti-LILRB4 antibodies and antigen-binding fragments thereof that have high binding affinity to LILRB4. In some embodiments, when bound to LILRB4, such antibodies regulate the activation of LILRB4. In certain embodiments, the antibodies or antigen-binding fragments, when bound to LILRB4, regulate the activation of LILRB4. In certain embodiments, the antibody or antigen-binding fragment provided herein can specifically interfere with, block, or reduce the interaction between ApoE and LILRB4 when bound to LILRB4. In certain embodiments, the antibody or antigen-binding fragment provided herein can inhibit the ApoE-mediated activity of LILRB4. In certain embodiments, the antibody or antigen-binding fragment provided herein specifically or selectively binds to human LILRB4.

[0085]

[0146] The binding affinity of the antibodies or antigen-binding fragments provided herein is determined by the K D The value may be expressed by the value at which binding between the antigen and the antigen-binding molecule reaches equilibrium. The ratio of dissociation rate to association rate (k off / k on ) indicates antigen binding affinity (e.g., BaK D ) can be used in a variety of ways known in the art, including, for example, biolayer interferometry. The amount of the ion exchange reaction may be suitably determined using any suitable method available.

[0086]

[0147] The binding of the antibody to human LILRB4 was also determined by the "half maximal effective concentration" (EC 50 ) value, which refers to the antibody concentration at which 50% of its maximal effect (e.g., binding or inhibition) is observed. 50 Levels can be measured by methods known in the art, such as sandwich assays, eg, ELISA, Western blots, flow cytometry assays, and other binding assays.

[0087]

[0148] Specific anti-LILRB4 antibody

[0149] In one aspect, the present disclosure provides anti-LILRB4 antibodies and antigen-binding fragments thereof comprising one or more (e.g., one, two, three, four, five, or six) CDR sequences of the anti-LILRB4 antibodies disclosed herein. Although CDRs are known to be involved in antigen binding, it has been found that not all six CDRs are essential or invariable. In other words, it is possible to replace, change, or modify one or more CDRs in the anti-LILRB4 antibodies disclosed herein while still substantially retaining specific binding affinity to LILRB4.

[0088]

[0150] In certain embodiments, the LILRB4 antibody is derived from the antibody H7K3, which has the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 3. In certain embodiments, the anti-LILRB4 antibody has enhanced stability compared to H7K3, yet is able to substantially retain specific binding affinity to LILRB4.

[0089]

[0151] In certain embodiments, the LILRB4 antibody has a heavy chain variable region comprising heavy chain complementarity determining region (HC-CDR) 1 having the amino acid sequence of SEQ ID NO: 5, HC CDR2 having the amino acid sequence of SEQ ID NO: 6, and HC CDR3 having the amino acid sequence of SEQ ID NO: 7. In certain embodiments, HC-CDR3 has the amino acid sequence of SEQ ID NO: 7, including a mutation at amino acid residue W.

[0090]

[0152] In certain embodiments, the LILRB4 antibody has a light chain variable region comprising a light chain complementarity determining region (LC-CDR)1 having the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 9, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the light chain variable region comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 9, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 10, each of which comprises a mutation at amino acid residue NS. In certain embodiments, LC-CDR1 has an amino acid sequence selected from SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34.

[0091]

[0153] In certain embodiments, the LILRB4 antibody is selected from the group consisting of antibodies to LILRB4, ... It has a CDR sequence.

[0154] Table 1. CDR sequences of anti-LILRB4 antibodies

[0092] [Table 1]

[0093]

[0155] The heavy and light chain variable region amino acid sequences of the above anti-LILRB4 antibodies are provided below.

[0094] [ka]

[0095] [ka]

[0096]

[0156] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof are capable of specifically binding to LILRB4. The CDR sequences provided in Table 1 can be grafted onto any suitable FR sequence of any suitable species, such as mouse, human, rat, rabbit, etc., using suitable methods known in the art, such as recombinant techniques.

[0097]

[0157] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are humanized. Humanized antibodies or antigen-binding fragments are desirable because they have reduced immunogenicity in humans. Humanized antibodies are chimeric in the variable region, with non-human CDR sequences grafted into humans or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can be essentially carried out by substituting non-human (e.g., murine) CDR genes for the corresponding human CDR genes in human immunoglobulin genes (see, e.g., Jones et al., Nature (1986) 321:522-525; Riechmann et al., Nature (1988) 332:323-327; Verhoeyen et al., Science (1988) 239:1534-1536).

[0098]

[0158] Suitable human heavy and light chain variable domains can be selected to achieve this goal using methods known in the art. In an illustrative example, a "best-fit" approach can be used, in which a non-human (e.g., rodent) antibody variable domain sequence is screened, or BLASTed, against a database of known human variable domain sequences, and the human sequence that is closest to the non-human query sequence is identified and used as a human scaffold for grafting the non-human CDR sequences (e.g., Sims et al., J. Immunol. (1993) 151:2296; Chothia et al., J. Mot. Biol. (1987) 196:901. Alternatively, frameworks derived from the consensus sequence of all human antibodies may be used for grafting non-human CDRs (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA (1992) 89:4285; Presta et al., J. Immunol. (1993) 151:2623).

[0099]

[0159] In certain embodiments, a humanized antibody or antigen-binding fragment as provided herein is composed substantially entirely of human sequences, except for CDR sequences, which are non-human. In some embodiments, the variable region FRs, and, if present, the constant region, are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences may be derived from different human immunoglobulin genes, e.g., the FR sequences may be derived from one human antibody and the constant region from another human antibody.

[0100]

[0160] In certain embodiments, the humanized antibodies and antigen-binding fragments thereof provided herein comprise the heavy chain FR sequence of H7 and / or the light chain FR sequence of K3. In some embodiments, the human-derived FR region may comprise the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues in the human FR are replaced with the corresponding residue from the parent non-human antibody. This may be desirable in certain embodiments to more closely resemble the non-human parent antibody structure. In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein comprise no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each human FR sequence, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all FRs of the heavy or light chain variable domain. In some embodiments, such amino acid residue changes may occur only in the heavy chain FR region, only in the light chain FR region, or in both chains.

[0101]

[0161] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain variable domain sequence selected from the group consisting of SEQ ID NOs: 1, 11, 13, 15, and 17. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a light chain variable domain selected from the group consisting of SEQ ID NOs: 3, 19, 21, 23, 25, 27, 29, 31, and 33.

[0102]

[0162] In some embodiments, the anti-LILRB4 antibodies and antigen-binding fragments provided herein comprise all or a portion of the heavy chain variable domain and / or all or a portion of the light chain variable domain. In one embodiment, the anti-LILRB4 antibodies and antigen-binding fragments provided herein are single-domain antibodies consisting of all or a portion of the heavy chain variable domain provided herein. Further information on such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0103]

[0163] In certain embodiments, the anti-LILRB4 antibodies and fragments thereof provided herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and / or a light chain constant region. The heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises a Cκ or Cλ region.

[0104]

[0164] The antibodies or antigen-binding fragments thereof provided herein may be monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, labeled antibodies, bivalent antibodies, or anti-idiotypic antibodies. These antibodies are produced in vitro using recombinant methods rather than by immunohistochemistry.

[0105]

[0165] Antibody variants

[0166] The antibodies and antigen-binding fragments thereof provided herein also include various variants thereof. In certain embodiments, the antibodies and antigen-binding fragments thereof include various types of variants of the exemplary antibodies provided herein.

[0106]

[0167] In certain embodiments, the antibody variants comprise one or more CDR sequences as provided in Table 1, one or more variable region sequences as provided herein (but not any of the CDR sequences), and / or one or more modifications or substitutions in the constant region (e.g., Fc region). Such variants retain the specific binding affinity of the parent antibody to LILRB4 but have one or more desirable properties conferred by the modification(s) or substitution(s). For example, the antibody variants may have improved antigen binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamidation or deamination, improved or increased effector function(s), reduced or eliminated effector function(s), improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine ​​residues).

[0107]

[0168] The parent antibody sequence may be screened to identify suitable or preferred residues to modify or replace using methods known in the art, such as "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) may be identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies may be produced and screened for properties of interest. If substitution at a particular amino acid position exhibits a functional change of interest, that position may be identified as a potential residue for modification or replacement. Potential residues may be further evaluated by substituting with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0108]

[0169] Affinity mutants

[0170] The affinity variants may contain modifications or substitutions in one or more CDR sequences, one or more FR sequences, or heavy or light chain variable regions provided herein. The affinity variants retain the specific binding affinity for LILRB4 of the parent antibody, or even have improved specific binding affinity for LILRB4 over that of the parent antibody.

[0109]

[0171] Various methods known in the art can be used to achieve this goal. For example, a library of antibody variants (e.g., Fab or scFv variants) can be generated and expressed using phage display technology, and then screened for binding affinity to human LILRB4. For another example, computer software can be used to virtually simulate the binding of an antibody to human LILRB4 and identify amino acid residues on the antibody that form the binding interface. Such residues can be avoided in substitution to prevent a decrease in binding affinity, or targeted for substitution to provide stronger binding.

[0110]

[0172] In certain embodiments, the humanized antibodies or antigen-binding The fragments contain one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences, hi certain embodiments, the affinity variants do not contain more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in total in the CDR and / or FR sequences.

[0111]

[0173] In certain embodiments, the anti-LILRB4 antibodies and antigen-binding fragments thereof comprise one, two, or three CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to those(s) listed in Table 1, while retaining a similar, or even higher, level of binding affinity to LILRB4 than the parent antibody.

[0112]

[0174] In certain embodiments, anti-LILRB4 antibodies and antigen-binding fragments thereof comprise one or more variable regions having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to those provided herein(s), while retaining a similar, or even higher, level of binding affinity to LILRB4 as the parent antibody. In some embodiments, substitutions, insertions, or deletions occur in regions outside of the CDRs (e.g., in the FRs).

[0113]

[0175] Glycosylation variants

[0176] In yet another embodiment, the antibody comprises a particular glycosylation pattern. For example, an aglycosylated antibody may be generated (i.e., the antibody lacks glycosylation). The glycosylation pattern of an antibody may be altered to, for example, increase the affinity or avidity of the antibody for an antigen. Such modifications may be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions may be made that result in the removal of one or more of the variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation may increase the affinity or avidity of the antibody for an antigen. See, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861.

[0114]

[0177] Antibodies can also be generated whose glycosylation pattern includes hypofucosylated or afucosylated glycans; for example, hypofucosylated or afucosylated antibodies have a reduced amount of fucosyl residues on the glycans. Antibodies can also include increased amounts of glycans with biantennary GlcNac structures. Such glycosylation pattern alterations 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 glycoproteins 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, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)-fucosyltransferase), and therefore antibodies expressed in Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. 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 Publication No. 20040110704). As another example, EP 1 176 195 describes cell lines with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, and therefore antibodies expressed in such cell lines lack fucose on their carbohydrates by reducing or eliminating the α-1,6 bond-related enzyme. Thus, they exhibit hypofucosylation. EP 1 176 195 also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine attached to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO 2003 / 035835 describes a mutant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting 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 (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 1999 / 054342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), and antibodies expressed in the engineered cell lines exhibit increased biantennary GlcNac structures, resulting in increased ADCC activity of the antibodies. Hypofucosylation is also referred to as afucosylation when fucosylation is minimal on the antibody.

[0115]

[0178] Alternatively, the fucose residues of antibodies can be cleaved using a fucosidase enzyme; for example, fucosidase, α-L-fucosidase, removes fucosyl residues from antibodies. The antibodies disclosed herein also include those produced in lower eukaryotic host cells, and fungal host cells, such as yeast and filamentous fungi, have been genetically engineered to produce glycoproteins with mammalian or human-like glycosylation patterns. A particular advantage of these genetically modified host cells over currently used mammalian cell lines is the ability to adjust the glycosylation profile of glycoproteins produced in the cells, allowing for the production of glycoprotein compositions dominated by specific N-glycan structures (see, e.g., U.S. Patent Nos. 7,029,872 and 7,449,308). These genetically modified host cells have been used to produce antibodies primarily with specific N-glycan structures.

[0116]

[0179] Furthermore, fungi, such as yeast or filamentous fungi, lack the ability to produce fucosylated glycoproteins, and therefore antibodies produced in such cells will 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 those produced in lower eukaryotic host cells and comprising fucosylated and nonfucosylated hybrid and complex N-glycans, including N-glycans such as, but not limited to, GlcNAc(1-4)Man3GlcNAc2; Gal(1-4)GlcNAc(1-4)Man3GlcNAc2; NANA(1-4)Gal(1-4)GlcNAc(1-4)Man3GlcNAc2, including biantennary and multiantennary species. 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.

[0117]

[0180] In certain embodiments, the antibody compositions provided herein comprise GlcNAcMan3GlcNAc2; GalGlcNAcMan3GlcNAc2; NANA The present invention also includes antibodies having at least one complex N-glycan selected from the group consisting of GalGlcNAcMan3GlcNAc2; GlcNAc2Man3GlcNAc2; GalGlcNAc2Man3GlcNAc2; Gal2GlcNAc2Man3GlcNAc2; NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2. In certain aspects, the complex N-glycan is the predominant N-glycan species in the composition. In further aspects, the complex N-glycan is a specific N-glycan species that constitutes 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-glycans are fucosylated. Generally, fucose is α1,3-linked to GlcNAc at the reducing end of the N-glycan, α1,6-linked to GlcNAc at the reducing end of the N-glycan, α1,2-linked to Gal at the non-reducing end of the N-glycan, α1,3-linked to GlcNAc at the non-reducing end of the N-glycan, or α1,4-linked to GlcNAc at the non-reducing end of the N-glycan.

[0118]

[0181] Thus, in certain aspects of the above glycoprotein compositions, the glycoforms are at α1,3-linked or α1,6-linked fucose, producing glycoforms 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); GlcNAc(Fuc)Man5GlcNAc2, GlcNAc(Fuc)Man3GlcNAc2, GlcNAc2(Fuc) 2) α1,3-linked or α1,4-linked glycoforms that produce glycoforms selected from the group consisting of Man3GlcNAc2, GalGlcNAc2(Fuc1-2)Man3GlcNAc2, Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2, NANAGal2GlcNAc2(Fuc1-2)Man3GlcNAc2, and NANA2Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2 -linked fucose; or α1,2-linked fucose that produces a glycoform selected from the group consisting of Gal(Fuc)GlcNAc2Man3GlcNAc2, Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2, NANAGal2(Fuc1-2)GlcNAc2Man3GlcNAc2, and NANA2Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2.

[0119]

[0182] 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 the above further aspect, the complex N-glycans further include fucosylated and nonfucosylated (or afucosylated) biantennary and multiantennary species. As used herein, the terms "N-glycan" and "glycoform" are used interchangeably and refer to N-linked oligosaccharides, e.g., 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.

[0120]

[0183] The anti-LILRB4 antibodies and antigen-binding fragments provided herein also include glycosylation variants, which can be obtained to either increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment.

[0121]

[0184] An antibody or antigen-binding fragment thereof may contain one or more amino acid residues having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of a sugar, N-acetylgalactosamine, galactose, or xylose, to one hydroxyamino acid, most commonly serine or threonine. Removal of native glycosylation sites can be suitably accomplished, for example, by modifying the amino acid sequence so that one of the above-mentioned triplets (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence is substituted. New glycosylation sites can be generated in a similar manner by introducing such triplet peptide sequences or serine or threonine residues.

[0122]

[0185] One type of glycosylation modification is performed using antibody-producing cells deficient in specific enzymatic pathways involved in site-specific glycosylation, including fucosylation. For example, antibodies lacking fucosylation (referred to as afucosylated antibodies) generally have enhanced ADCC activity. Using afucosylated H7K3m5, killing of normal monocytes through ADCC was observed in 25–50% of PBMC donors tested (Figures 14A–14D and 15C–15D). Furthermore, as shown in Figures 15A–15B, both afucosylated and wild-type H7K3m5 resulted in killing of pDCs through autologous ADCC. On the other hand, depending on the donor, monocytes may be killed only by afucosylated H7K3m5 and not by wild-type H7K3m5 (Figures 15C–15D).

[0123]

[0186] Cysteine-engineered mutants

[0187] The anti-LILRB4 antibodies and antigen-binding fragments provided herein also include cysteine ​​engineered variants, which contain one or more introduced free cysteine ​​amino acid residues.

[0124]

[0188] Free cysteine ​​residue is not part of disulfide bridge.Cysteine ​​engineered variants are useful for example, inter alia, cytotoxic and / or imaging compounds, labels, or radioisotopes, for example, via maleimide or haloacetyl, to conjugate at engineered cysteine ​​site.Methods for engineering antibody or antigen-binding fragment to introduce free cysteine ​​residue are known in the art, for example, see WO2006 / 034488.

[0125]

[0189] Fc variants

[0190] The antibodies disclosed herein may 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 may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) also to alter one or more functional properties of the antibody, or to alter its glycosylation. 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 containing modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Patent Nos. 5,624,821; WO2003 / 086310; US2004 / 0002587; US2005 / 0152894; US2005 / 0249723; WO2006 / 019447. Such modifications can be used to identify potential beneficial compounds in diagnostics and therapeutics. The Fc region may enhance or suppress various responses of the immune system, with beneficial effects. Modifications of the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and the addition of multiple Fc fragments. Changes to the Fc region may also alter the half-life of antibodies in therapeutic antibodies, allowing for less frequent dosing and thus increasing convenience and reducing substance use. This mutation has been reported to eliminate heterogeneity in inter-heavy chain disulfide bridges in the hinge region.

[0126]

[0191] 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. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In another embodiment, the antibody is modified to increase biological half-life. Various approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375. Alternatively, to increase biological half-life, the antibody may be modified within the CH1 or CL region to contain salvage receptor binding epitopes recruited 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 other embodiments, the Fc region is modified by substituting at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 may be substituted with a different amino acid residue so that the antibody has altered affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered may 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.

[0127]

[0192] In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in PCT Publication WO1994 / 029351. In yet another example, the following digits: 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, 333, 334, The Fc region is modified to increase or decrease the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or increase or decrease the affinity of the antibody for the Fcγ receptor by modifying one or more amino acids at 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 2000 / 042072. Additionally, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and mutants 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 mutations have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A.

[0128]

[0193] In one embodiment, the Fc region is modified to decrease 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 the antibody is modified by changing residues 243 and 264 to alanine. In one embodiment, the Fc region is modified to decrease the ability of the antibody to mediate effector function and / or increase anti-inflammatory properties by modifying residues 243, 264, 267, and 328.

[0129]

[0194] In one embodiment, the Fc region is modified to abolish the ability of the antibody to mediate effector function by modifying residues 234, 235 and 329 to alanine or glycine (L234A-L235A-P329G).

[0130]

[0195] The anti-LILRB4 antibodies and antigen-binding fragments provided herein also include Fc variants, which comprise one or more amino acid residue modifications or substitutions in the Fc region and / or hinge region.

[0131]

[0196] In certain embodiments, anti-LILRB4 antibodies and antigen-binding fragments contain one or more amino acid substitution(s) that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants may have extended pharmacokinetic half-lives because they bind to FcRn at acidic pH, allowing them to escape degradation in lysosomes and then be translocated and released outside the cell. Methods for engineering antibodies and antigen-binding fragments thereof to improve their binding affinity to FcRn are well known in the art, see, for example, Vaughn, D. et al., Structure, 6(1): 63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research (2010) 70: 3269-3277; and Hinton, P. et al., J. Immunology (2006) 176:346-356.

[0132]

[0197] In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment contains one or more amino acid substitutions that alter antibody-dependent cellular cytotoxicity (ADCC). Specific amino acid residues in the CH2 domain of the Fc region may be substituted to provide enhanced ADCC activity. Alternatively, or in addition, the carbohydrate structure on the antibody may be altered to enhance ADCC activity. Methods for modifying ADCC activity by antibody engineering have been described in the art, for example, Shields R L. et al., J Biol Chem. (2001) 276(9): 6591-604; Idusogie E E. et al., J Immunol. (2000) 164(8):4178-84; Steurer W. et al., J Immunol. (1995) 155(3): 1165-74; Idusogie E E. et al., J Immunol. (2001) 166(4): 2571-5; Lazar G A. et al., PNAS (2006) 103(11): 4005-4010; Ryan M C. et al., Mol. Cancer Ther. (2007) 6: 3009-3018; Richards See JO. et al., Mol Cancer Ther. (2008) 7(8): 2517-27; Shields RL et al., J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. et al., J. Biol. Chem (2003) 278: 3466-3473.

[0133]

[0198] In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment is and WO1994 / 029351 for other examples of Fc region variants. See the reference.

[0134]

[0199] In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment contains one or more amino acid substitutions at the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include the introduction of a knot into a first Fc polypeptide and a cavity into a second polypeptide, where the knot can be positioned within the cavity to promote interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies containing these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0135]

[0200] antigen binding fragment

[0201] Anti-LILRB4 antigen-binding fragments are also provided herein. Various types of antigen-binding fragments are known in the art and may be developed based on the anti-LILRB4 antibodies provided herein, including, for example, the exemplary antibodies whose CDRs and variable sequences are provided herein, and different variants thereof (e.g., affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered variants, etc.).

[0136]

[0202] In certain embodiments, the anti-LILRB4 antigen-binding fragments provided herein are camelized single domain antibodies, diabodies, single chain Fv fragments (scFv), scFv dimers, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragments, F ab, Fab', F(ab')2, bispecific antibody, ds diabody, nanobody, do It may be a main antibody, a single domain antibody, or a bivalent domain antibody.

[0137]

[0203] Single-chain variable fragments (scFvs) are fusions of the variable regions of immunoglobulin heavy and light chains linked together by a short (usually serine or glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were historically generated to facilitate phage display, which is highly suitable for expressing antigen-binding domains as single peptides. Alternatively, scFvs can be generated directly from subcloned heavy and light chains from hybridomas. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules and therefore lack the consensus binding site (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using protein L because protein L interacts with the variable region of the kappa light chain.

[0138]

[0204] Flexible linkers are generally composed of helical and rotation-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 tailored linkers for single-chain antibodies (scFv) from protein linker libraries. They constructed a random linker library 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 5x10 6 different members) The mutants were displayed on filamentous phage and subjected to affinity selection with the hapten. The selected mutant population showed a significant increase in binding activity while retaining considerable sequence diversity. 1054 individual mutants were subsequently screened to identify those that could be efficiently produced in a soluble form. The resulting catalytically active scFvs were obtained. Sequence analysis revealed that the only common features of the selected tethers were a conserved proline in the linker two residues after the VH C-terminus, and an abundance of arginine and proline at other positions.

[0139]

[0205] The recombinant antibodies of the present disclosure may also contain sequences or moieties that allow receptor dimerization or multimerization. Such sequences include those derived from IgA, which allow the formation of multimers in conjunction 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.

[0140]

[0206] In a separate embodiment, single-chain antibodies may be produced by linking the receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in separate cells, purified, and then linked together in an appropriate manner (i.e., the N-terminus of the heavy chain will be attached to the C-terminus of the light chain through a suitable chemical bridge).

[0141]

[0207] Cross-linking reagents are used to form molecular bridges connecting the functional groups of two different molecules, such as a stabilizer and a coagulant. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes composed of different analogs may be produced. To link two different compounds in a stepwise manner, heterobifunctional cross-linkers may be used, which eliminates the formation of undesired homopolymers.

[0142]

[0208] Exemplary heterobifunctional crosslinkers contain two reactive groups: one reactive group that reacts with primary amine groups (e.g., N-hydroxysuccinimide), and another reactive group that reacts with thiol groups (e.g., pyridyl disulfide, maleimide, halogen, etc.). Through the primary amine reactive group, the crosslinker reacts with one protein (e.g., an antibody or fragment of choice), and through the thiol reactive group, the crosslinker already attached to the first protein reacts with a cysteine ​​residue (free sulfhydryl group) on another protein (e.g., an agent of choice).

[0143]

[0209] Preferably, a crosslinker with reasonable stability in blood is used.Many types of disulfide bond-containing linkers are known, which can be successfully used to conjugate targeting agent and therapeutic / prophylactic agent.Containing sterically hindered disulfide bond linkers may prove to produce greater stability in vivo and prevent targeting peptide from being released before reaching the site of action.These linkers are therefore one group of linking agents.

[0144]

[0210] Another cross-linking reagent is SMPT, a bifunctional cross-linker containing a "sterically hindered" disulfide bond via adjacent benzene rings and methyl groups. The steric hindrance of the disulfide bond serves to protect the bond from attack by thiolate anions, such as glutathione, that may be present in tissues and blood, thereby helping to prevent unconjugation of the conjugate before the attached agent is delivered to the target site.

[0145]

[0211] The SMPT cross-linking reagent, like many other known cross-linking reagents, 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 type of cross-linker 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 (upon photolysis) nonselectively with any amino acid residue.

[0146]

[0212] In addition to hindered crosslinkers, non-hindered linkers may also be used in accordance with the present specification. Other useful crosslinkers that are not considered to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such crosslinkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[0147]

[0213] U.S. Patent No. 4,680,338 describes bifunctional linkers useful for producing conjugates of amine-containing polymers and / or proteins with ligands, particularly to form antibody conjugates containing chelators, drugs, enzymes, detectable labels, etc. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that are cleavable under a variety of mild conditions. This linker is particularly useful when an agent of interest can be directly attached to the linker, resulting in release of the active agent upon cleavage. Specific uses include the addition of free amino or free sulfhydryl groups to proteins, such as antibodies or drugs.

[0148]

[0214] U.S. Patent No. 5,856,456 provides peptide linkers for use in linking polypeptide components to create fusion proteins, such as single-chain antibodies. The linkers are up to about 50 amino acids in length, contain at least one charged amino acid (preferably arginine or lysine) followed by proline, and are characterized by greater stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separation techniques.

[0149]

[0215] A variety of techniques may be used to produce such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods (1992) 24:107-117; and Brennan et al., Science (1985) 229:81), recombinant expression in host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., for ScFv), and chemical coupling of two Fab'-SH fragments to form an F(ab')2 fragment. (Carter et al., Bio / Technology (1992) 10:163-167). Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0150]

[0216] In certain embodiments, the antigen-binding fragment is an scFv. The production of scFv is described, for example, in WO 93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. The scFv can be fused to an effector protein at either the amino or carboxyl terminus to provide a fusion protein (see, for example, Antibody Engineering, edited by Borrebaeck).

[0151]

[0217] bispecific antibody

[0218] In certain embodiments, the LILRB4 antibody disclosed herein is a bispecific antibody. In certain embodiments, the LILRB4 bispecific antibody can be used to treat hematological and solid malignancies by binding to both an antigen on T cells or NK cells and LILRB4 on cancer cells, thereby redirecting cytotoxic T cells or NK cells to cancer cells. In some embodiments, the anti-LILRB4 bispecific antibody is directed against a T cell receptor, such as CD3. In some embodiments, the anti-LILRB4 bispecific antibody is directed against an NK cell receptor, such as CD16A.

[0152]

[0219] The anti-LILRB4 bispecific antibodies of the present disclosure may have a variety of types and structures, which can be seen by an exemplary embodiment of a bispecific antibody that specifically binds to LILRB4 and CD3 (LILRB4 / CD3 bispecific) as illustrated in Figures 28A and 28B.

[0153]

[0220] As illustrated in Figure 28A, in an exemplary embodiment of the invention, the LILRB4 / CD3 bispecific antibody is Y-shaped and comprises two arms. One arm of the antibody formed by a portion of the first heavy chain polypeptide and the first light chain polypeptide comprises a heavy chain variable domain (V H 1) and the light chain variable domain (V L The other arm of the antibody formed by the second heavy chain polypeptide and the second light chain polypeptide comprises a second pair of heavy chain variable domains (V H 2) and the light chain variable domain (V L 2) V H 2 and V L 2 forms a second antigen-binding site capable of specifically binding to CD3. In the configuration, each bispecific antibody contains a single copy of the antigen-binding site for LILRB4 and a single copy of the antigen-binding site for CD3, which is referred to as 1+1.

[0154]

[0221] Referring to embodiment 4-3ab in Figure 28A, the first heavy chain polypeptide comprises, from N-terminus to C-terminus, V H 1-C H 1-C H 2-C H 3, wherein C H 1. C H 2, and C H 3 refers to heavy chain constant domains 1, 2 and 3; the first light chain polypeptide is the N-terminal From the C-terminus, V L 1-C L wherein C L refers to the light chain constant domain; the second heavy chain The polypeptide is composed of, from the N-terminus to the C-terminus, H 2-TCRβ-C H 2-C H 3 includes; and The second light chain polypeptide comprises, from N-terminus to C-terminus, V L 2-Containing TCRα. Bispecific The use of TCR alpha and TCR beta constant domains in antibodies allows for the correct association of the light chain and its cognate heavy chain, resulting in higher yields of the desired bispecific antibody against LILRB4 and CD3 (see, e.g., WO2019057122A1). In a specific embodiment, the TCR alpha domain has the amino acid sequence of SEQ ID NO: 89, and the TCR beta domain has the amino acid sequence of SEQ ID NO: 90.

[0155]

[0222] Alternatively, as exemplified in the 4ab-3 embodiment in Figure 28A, the first heavy chain polypeptide can comprise, from N-terminus to C-terminus, H 1-TCRβ-C H 2-C H 3 including; 1st The light chain polypeptide of L 1 - Contains TCRα; The peptide is composed of, from the N-terminus to the C-terminus, H 2-C H 1-C H 2-C H3; and the second light chain polypeptide comprises, from N-terminus to C-terminus, V L 2-C L Includes.

[0156]

[0223] As illustrated in Figure 28A, the stem of a Y-shaped antibody comprises the second and third constant domains (C) of the first and second heavy chain polypeptides joined together through disulfide bonds. H 2 and C H 3). In certain embodiments, the Fc region is engineered using knobs-in-holes (KiH) technology (Ridgway JB et al., Protein Eng (1996) 9:617-21; Atwell S et al., J Mol Biol (1997) 270:26-35; Merchant et al., Nature Biotech (1998) 16, 677-681), which prevents homodimerization of heavy chain polypeptides. Briefly, the constant regions of the two heavy chain polypeptides are each mutated to generate either knobs or holes, which pair to promote heterodimerization. Designing LILRB4 / CD3 bispecific antibodies using KiH technology allows for heterodimerization of the heavy chains and the correct association of the light chain and its cognate heavy chain, resulting in higher yields of the desired bispecific antibodies against LILRB4 and CD3.

[0157]

[0224] In certain embodiments, the configuration of the bispecific antibody comprises two copies of the antigen-binding site for LILRB4 and one copy of the antigen-binding site for CD3. This configuration is referred to as 2+1 and is illustrated in Figure 28A. In such a configuration, the LILRB4 / CD3 bispecific antibody comprises two pairs of heavy / light chain polypeptides, which form a first antigen-binding region and a second antigen-binding region that bind to LILRB4 and CD3, respectively. Unlike the 1+1 configuration, in the 2+1 configuration, one heavy chain polypeptide binds a third heavy chain variable domain (V) to a third light chain variable domain in a third light chain polypeptide. H 3) A third antigen that contains a homologue and binds to LILRB4 Forming a bonding region.

[0158]

[0225] Referring to embodiment 44-3ab in Figure 28A, in an exemplary embodiment of the invention, a LILRB4 / CD3 bispecific antibody is Y-shaped and comprises two arms, comprising a first heavy chain polypeptide, a first light chain polypeptide, a second heavy chain polypeptide, a second light chain polypeptide, and a third light chain polypeptide. The first heavy chain polypeptide comprises, from N-terminus to C-terminus, H 3-C H 1-LV H 1-C H 1-C H 2-C H 3, wherein L is a linker (e.g., (G4S)2); the first light chain polypeptide comprises, from N-terminus to C-terminus, V L 1-C L the second heavy chain polypeptide comprises, from N-terminus to C-terminus, V H 2-TC Rβ-C H 2-C H 3; and the second light chain polypeptide comprises, from N-terminus to C-terminus, V L 2—TCRα; the third light chain polypeptide comprises, from N-terminus to C-terminus, V L 3-C L of On one arm of the antibody, V H 1 and V L 1 forms the primary antigen-binding site for LILRB4, while V H 3 and V L 3 forms the second antigen-binding site for LILRB4. On the other arm of the antibody, V H 2 and V L In certain embodiments, the third light chain polypeptide V forms an antigen-binding site capable of specifically binding to CD3. L 3 -C L is the first light chain polypeptide V L 1-C L may be the same as

[0159]

[0226] Referring to embodiment 4ab4ab-3 in Figure 28A, in an exemplary embodiment of the invention, a LILRB4 / CD3 bispecific antibody comprises a first heavy chain polypeptide, a first light chain polypeptide, a second heavy chain polypeptide, a second light chain polypeptide, and a third light chain polypeptide. The first heavy chain polypeptide comprises, from N-terminus to C-terminus, H 3-TCR β-LV H 1-TCRβ-C H 2-C H 3, wherein L is a linker (e.g., (G4S)2); the first light chain polypeptide comprises, from N-terminus to C-terminus, V L the second heavy chain polypeptide comprises, from N-terminus to C-terminus, V H 2-C H 1-C H 2-C H 3; and the second light chain polypeptide comprises, from N-terminus to C-terminus, V L 2-C L the third light chain polypeptide comprises, from N-terminus to C-terminus, V L 3 - Contains TCRα. On one arm of the antibody So, V H 1 and V L 1 forms the primary antigen-binding site for LILRB4, while V H 3 and V L 3 forms the second antigen-binding site for LILRB4. On the arm of V H 2 and V L In certain embodiments, the third light chain polypeptide V forms an antigen-binding site capable of specifically binding to CD3. L 3-TCRα is the first light chain polynucleotide Peptide V L 1-TCRα.

[0160]

[0227] Referring to embodiment 43ab-4 in Figure 28, in an exemplary embodiment of the invention, a LILRB4 / CD3 bispecific antibody comprises a first heavy chain polypeptide, a first light chain polypeptide, a second heavy chain polypeptide, a second light chain polypeptide, and a third light chain polypeptide. The first heavy chain polypeptide comprises, from N-terminus to C-terminus, H 3-C H 1-LV H 2-TCRβ-C H 2-C H 3, where L is a linker (e.g., (G4S)2) the first light chain polypeptide comprises, from N-terminus to C-terminus, V L 2 - Contains TCRα; second helical The chain polypeptide is composed of, from the N-terminus to the C-terminus, H 1-C H 1-C H 2-C H 3; and the second light chain polypeptide comprises, from N-terminus to C-terminus, V L 1-C L the third light chain polypeptide comprises, from N-terminus to C-terminus, V L 3-C L On one arm of the antibody, V H 1 and BiV L 1 forms the first antigen-binding site for LILRB4. On the platform, V H 3 and V L 3 forms the second antigen-binding site for LILRB4, while V H 2 and V L In certain embodiments, the third light chain polypeptide V forms an antigen-binding site capable of specifically binding to CD3. L 3-C L is the first light chain polypeptide V L 1- C L may be the same as

[0161]

[0228] Referring to embodiment 4ab3-4ab in Figure 28A, in an exemplary embodiment of the invention, a LILRB4 / CD3 bispecific antibody comprises a first heavy chain polypeptide, a first light chain polypeptide, a second heavy chain polypeptide, a second light chain polypeptide, and a third light chain polypeptide. The first heavy chain polypeptide comprises, from N-terminus to C-terminus, H 3-TCR β-LV H 2-C H 1-C H 2-C H 3, wherein L is a linker (e.g., (G4S)2); the first light chain polypeptide chain comprises, from N-terminus to C-terminus, V L 2-C L the second heavy chain polypeptide comprises, from N-terminus to C-terminus, V H 1-TCRβ-C H 2-C H Includes 3; and the second light chain polypeptide is, from N-terminus to C-terminus, V L 1 - contains TCRα; 3 The light chain polypeptide of L 3 - Contains TCRα. On the platform, V H 1 and V L 1 forms the first antigen-binding site for LILRB4. On the other arm of the antibody, V H 3 and V L 3 forms the second antigen-binding site for LILRB4, while V H 2 and V L In certain embodiments, the third light chain polypeptide V forms an antigen-binding site for CD3. L 3-TCRα is the first light chain polypeptide DoV L 1-TCRα.

[0162]

[0229] In a particular embodiment, the antigen binding site directed against CD3 is selected from the group consisting of anti-CD3 antibodies known in the art, such as the anti-CD3 antibodies described in WO2019057099, SP34 (Pessano et al. EMBO J (1985) 4, 337-334), OKT3 (Ortho, Raritan, NJ; Van Wauwe et al., J Immunol (1984) 133, 129-32), M291 (Protein Design Laboratories, Fremont, CA), BC3 (Fred Hutchinson Cancer Research Center, Seattle, WA), TR66 (Novus Biologicals, Centennial, CO), and BMA030 (Walker C et al., Eur J Immunol. (1907) 17:1611-8).

[0163]

[0230] In certain embodiments, LILRB4 / CD3 bispecific antibodies may be engineered to improve uniformity and manufacturability. In some embodiments, the TCRα may be mutated at S91A of SEQ ID NO: 89 to remove the O-glycan modification site, as illustrated in Figure 28B. In some embodiments, a Q1E mutation may be made at the N-terminus of the heavy or light chain polypeptide to prevent N-terminal pyro-Q formation.

[0164]

[0231] In certain embodiments, anti-LILRB4 bispecific antibodies may be constructed in many other ways, as reviewed in Konterman et al. 2017, 9 182-212. In particular, anti-LILRB4 bispecific antibodies include covalent antibody conjugates, asymmetric F(ab')2, CovX bodies, mouse / rat chimeric IgG, kappa / lambda bodies containing a common heavy chain, tandem single variable domains (scFv), BiTEs, triple bodies, diabodies, tandem domain antibodies, scFv fusions with CH1 / CL domains, Fab-scFv bibodies or tribodies, Fab-Fv fusions, Fab-single domain antibody (sdAb) / VHH fusions, orthogonal Fab-Fab, scFv2-albumin / toxin fusions, single chain diabody-albumin / toxin fusions, tandem scFv albumin / toxin fusions, dock-and-lock (DNL) Fab3, DNL-Fab2-scFv, DNL-Fab-IgG fusions, ImmTAC TCR-scFv fusions, IgGs containing different heavy chains and different or common light chains, IgG-scFvs attached to the heavy or light chain N- or C-terminus, v fusions, IgG single chain Fab (scFab) fusions to the N- or C-terminus of the heavy or light chain, single chain IgG (scIgG) including scFv fusions, dual variable domain (DVD) diabodies, asymmetric scFv-Fc, tandem-scFv-Fc fusions, dual affinity re-targeting antibodies (DART) with or without Fc fusions, asymmetric Fab-scFv-Fc fusions, scFv-CH3 fusions, TriFab, IgG tandem scFv fusions, IgG-cross Fab fusions, tandem Fab-IgG fusions including orthogonal Fabs, didiabody-Fc fusions, single chain diabody-Fc fusions, Fab-scFv-Fc fusions, scFv-Fc fusions, scFv2-Fcab, di-diabody, single chain diabody CH3 fusions, IgE / M CH2 fusions, F(ab')2 fusions, CH1 / CK fusions, 2-in-1 de Dual-acting Fab (DAF), or DutaMab, a DNL-Fab2-IgG fusion It may be constructed as:

[0165]

[0232] Examples include, but are not limited to, knobs-in-holes (Ridgway et al. PEDS 1996; Atwell et al. J Mol Biol 1997; Merchant et al. Nat Biotechnol 1998), HA-TF mutations (Moore et al. Mabs 2011), ZW1 (Von Kreudenstein et al. MAbs 2013), CH3 charge pair (Gunasekaran et al. J Biol Chem 2010), IgG1 hinge / CH3 charge pair (Strop et al. J Mol Biol 2012), IgG2 hinge / CH3 charge pair (Strop et al. J Mol Biol 2012), EW-RVT mutation with or without engineered disulfides (Choi et al. Mol Cancer Ther 2013; Choi et al. Mol Immunol 2015), biclonic (Geuijen et al. J Clinical Onc 2014), DuoBody (Labrijn et al. Proc Natl Acad Heterodimerization of heavy chains for bispecific antibodies containing Fc domains may be achieved by several means, including (but not limited to) CrossMab (Schaefer et al. Cancer Cell 2011), orthogonal Fab (Lewis et al. Nat Biotechnol 2014), T cell receptor fusions (Wu et al. MAbs 2015), CR3 (Golay et al. J Immunol 2016), MUT4 (Golay et al. J Immunol 2016), DuetMab (Mazor et al. MAbs 2015, 7, 377-89; Mazor et al. MAbs 2015, 7, 461-669).

[0166]

[0233] In certain embodiments, the bispecific antibody may target LILRB4 and additional targets, including but not limited to, CD3, CD2, CD16a, NKp46, CD137, OX40, PD-1, PD-L1, CD40, CTLA4, LAG3, TIM3, CD47.

[0167]

[0234] Conjugates

[0235] In some embodiments, the anti-LILRB4 antibodies and antigen-binding fragments thereof further comprise a conjugate moiety. The conjugate moiety may be linked to the antibody and antigen-binding fragment thereof. The conjugate moiety is a proteinaceous or non-proteinaceous moiety that can be attached to the antibody or antigen-binding fragment thereof. A variety of conjugate moieties may be linked to the antibodies or antigen-binding fragments provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (ed.), Carger Press, New York, (1989). These conjugate moieties may be linked to the antibody or antigen-binding fragment by covalent bonding, affinity binding, intercalation, coordinate bonding, complex formation, association, blending, or addition, among other methods.

[0168]

[0236] In certain embodiments, the antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope-binding portion that can be utilized for binding to one or more conjugate moieties. For example, such sites may include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, that facilitate covalent linkage to a conjugate moiety.

[0169]

[0237] In certain embodiments, the antibody may be indirectly linked to the conjugate moiety or through another conjugate moiety. For example, the antibody or antigen-binding fragment may be conjugated to biotin and then indirectly conjugated to a second conjugate that is conjugated to avidin. The conjugate may be a clearance modifier, a toxin (e.g., a chemotherapeutic agent), a detectable label (e.g., a radioisotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label), or a purification moiety.

[0170]

[0238] A "toxin" may be any agent that is harmful to cells or capable of damaging or killing cells. Examples of toxins include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), mertansine, emtansine, DM1, maytansinoid DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil ... These include uracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin-binding agents.

[0171]

[0239] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luceriferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I,125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y, 88 Y , 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 P, other lanthanides), luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules or gold for detection.

[0172]

[0240] In certain embodiments, the conjugated moiety may be a clearance modifier, which helps increase the half-life of the antibody. Illustrative examples include water-soluble polymers, e.g., These include PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, etc. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules.

[0173]

[0241] In certain embodiments, the conjugated moiety may be a purification moiety such as a magnetic bead.

[0242] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are used for the base of a conjugate.

[0174]

[0243] Polynucleotides and Recombinant Methods

[0244] The present disclosure provides isolated polynucleotides encoding anti-LILRB4 antibodies and antigen-binding fragments thereof. In certain embodiments, the isolated polynucleotides comprise one or more nucleotide sequences, such as those set forth in SEQ ID NOS: 2, 4, and 35, which encode the variable regions of exemplary antibodies provided herein. DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). The encoding DNA can also be obtained by synthetic methods.

[0175]

[0245] Isolated polynucleotides encoding anti-LILRB4 antibodies and antigen-binding fragments may be inserted into vectors for further cloning (amplification of the DNA) or expression using recombinant techniques known in the art. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0176]

[0246] The present disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, and M13 phage, as well as the plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, and pALTER. , pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBA BE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0177]

[0247] A vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment may be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotes, yeast, or the higher eukaryotic cells described above. Prokaryotes suitable for this purpose include eubacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae. Bacteria include bacteria of the genus Oobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli, e.g., B. subtilis and B. licheniformis, Pseudomonas, e.g., P. aeruginosa, and Streptomyces.

[0178]

[0248] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for anti-LILRB4 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used lower eukaryotic host microorganism. However, several other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus (ATCC 56,500). marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentali; and filamentous fungal hosts, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus, such as A. nidulans and A. niger.

[0179]

[0249] Suitable host cells for expressing the glycosylated antibodies or antigen-binding fragments provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and mutants have been identified, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Various virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as viruses herein in accordance with the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of corn, potato, soybean, petunia, tomato, and tobacco may also be utilized as hosts.

[0180]

[0250] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cells (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. (1977) 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (CHO), dihydrofolate reductase (DHFR)-deficient CHO cells, CHO-DHFR (Urlaub et al., Proc. Natl. Acad. Sci. USA (1980) 77:4216); mouse Sertoli cells (TM4, Mather, Biol. Reprod. (1980) 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. (1982) 383:44-68); MRC 5 cells; FS4 cells; and human hepatoma cells (Hep G2). In some preferred embodiments, the host cells are 293F cells.

[0181]

[0251] For production of anti-LILRB4 antibodies, host cells are transformed with the above-described expression or cloning vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, antibodies can be produced by homologous recombination, as known in the art.

[0182]

[0252] The host cells used to produce the antibodies or antigen-binding fragments provided herein may be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), minimal essential medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM) (Sigma), are suitable for culturing host cells. Further, see Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. (1980) 102:255, U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; Any of the media described in WO 90 / 03430; WO 87 / 00195; or U.S. Patent No. Re. 30,985 may be used as the culture medium for the host cells. Any of these media may optionally contain hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin), and the like. TM The culture medium may be supplemented with nutrients (e.g., soluble or non-soluble drugs), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions, such as temperature, pH, etc., will be those previously employed with the host cell selected for expression and will be apparent to one of ordinary skill in the art.

[0183]

[0253] When using recombinant techniques, antibodies can be produced intracellularly in the periplasmic space or directly secreted into the culture medium. If antibodies are produced intracellularly, the first step is to remove particulate debris, either host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology (1992) 10:163-167, describe a method for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein filtration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. A protease inhibitor, such as PMSF, may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0184]

[0254] The anti-LILRB4 antibodies and antigen-binding fragments thereof prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0185]

[0255] In certain embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A may be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. (1983) 62:1-13). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. (1986) 5:1567-75). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices, such as controlled-pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX TM Resins (JT Baker, Phillipsburg, NJ) are useful for purification. Other techniques for protein purification include fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, heparin SEPHAROSE on anion or cation exchange resins (e.g., polyaspartic acid columns). TM Chromatography on chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available, depending on the antibody to be recovered.

[0186]

[0256] After any preliminary purification step(s), the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, preferably performed at a low salt concentration (e.g., about 0-0.25 M salt), using an elution buffer at a pH between about 2.5 and 4.5.

[0187]

[0257] purification

[0258] In certain embodiments, the antibodies of the present disclosure may be purified. The term "purified" is intended herein to refer to a composition isolatable from other components, in which the protein has been purified to any degree relative to its naturally obtainable state. Thus, a purified protein also refers to a protein that has been freed from the environment in which it may naturally occur. When the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms a major component of the composition, e.g., comprises about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0188]

[0259] Protein purification techniques are well known to those skilled in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Once the polypeptide has been separated from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of pure peptides are ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc., or heat denaturation followed by centrifugation; gel filtration, reverse phase, hydroxylapatite, and affinity chromatography; and combinations of these and other techniques.

[0189]

[0260] When purifying the antibodies 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 may be purified from other cellular components using an affinity column that binds to a tagged portion of the polypeptide. As is generally known in the art, the order in which the various purification steps are performed may be varied, or certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

[0190]

[0261] Typically, whole antibodies are fractionated using agents that bind to the Fc portion of antibodies (i.e., protein A). Alternatively, antigens may be used to simultaneously purify and select suitable antibodies. These 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., washed away), and the antibody is released by applying conditions (salt, heat, etc.).

[0191]

[0262] In light of the present disclosure, those skilled in the art will know a variety of methods for quantifying the degree of purification of a protein or peptide. These include, for example, determining the specific activity of an active fraction or assessing the number of polypeptides within a fraction by SDS / PAGE analysis. Another method for assessing 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, and thus calculate the purity. The actual units used to express the amount of activity will, of course, depend on the particular assay technique chosen to track purification and whether the expressed protein or peptide exhibits detectable activity.

[0192]

[0263] It is known that the migration of polypeptides can vary, sometimes significantly, under different conditions of SDS / PAGE (Capaldi et al., 1977). It will therefore be appreciated that the apparent molecular weight of purified or partially purified expression products may vary under different electrophoretic conditions.

[0193]

[0264] III. Pharmaceutical Compositions

[0265] The present disclosure further provides pharmaceutical compositions comprising an anti-LILRB4 antibody or antigen-binding fragment thereof and one or more pharmaceutically acceptable carriers.

[0194]

[0266] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein include, for example, pharmaceutically acceptable liquid, gel, or solid carriers. The composition may include, for example, an aqueous vehicle, a non-aqueous vehicle, an antimicrobial agent, an isotonic agent, a buffer, an antioxidant, an anesthetic, a suspending / dispersing agent, a sequestrant or chelating agent, a diluent, an adjuvant, an excipient, or other non-toxic auxiliary substance, or other component known in the art, or various combinations thereof.

[0195]

[0267] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, coloring agents, emulsifiers, or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisol, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, inclusion of one or more antioxidants, such as methionine, in compositions comprising antibodies or antigen-binding fragments and conjugates as provided herein reduces oxidation of the antibodies or antigen-binding fragments. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Thus, in certain embodiments, compositions are provided that include one or more antibodies or antigen-binding fragments as disclosed herein and one or more antioxidants, such as methionine. Also provided are methods for preventing oxidation, extending the shelf life, and / or improving the effectiveness of antibodies or antigen-binding fragments as provided herein by combining the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0196]

[0268] To further illustrate, pharmaceutically acceptable carriers include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents in bactericidal or fungicidal concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; Other suitable excipients may include acetaminophen, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multidose containers and include phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0197]

[0269] The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained-release formulation, or powder. Oral formulations may include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0198]

[0270] In certain embodiments, pharmaceutical compositions are formulated into injectable compositions.Injectable pharmaceutical compositions can be prepared in any conventional form, for example, in liquid solution, suspension, emulsion, or in solid form suitable for forming liquid solution, suspension, or emulsion.Preparations for injection can include sterile and / or non-pyrogenic solution ready for injection, sterile dry soluble product ready to be combined with solvent immediately before use, such as lyophilized powder, and include hypodermic tablets, sterile suspension ready for injection, sterile dry insoluble product ready to be combined with vehicle immediately before use, and sterile and / or non-pyrogenic emulsion.Solution can be either aqueous or non-aqueous.

[0199]

[0271] In certain embodiments, the unit dose parenteral preparation is packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile and non-pyrogenic, as known and practiced in the art.

[0200]

[0272] In certain embodiments, sterile lyophilized powders are prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent. The solvent may contain excipients to improve stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Possible excipients include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. In one embodiment, the solvent may contain a buffer, such as citric acid, sodium or potassium phosphate, or other such buffers known to those skilled in the art, at approximately neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of an anti-LILRB4 antibody or antigen-binding fragment thereof, or a composition thereof. To facilitate accurate sample removal and accurate dosing, overfilled vials containing a small amount (e.g., about 10%) in excess of that needed for a dose or dose set are acceptable. The lyophilized powder may be stored under appropriate conditions, for example, at about 4°C to room temperature.

[0201]

[0273] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, sterile and / or pyrogen-free water or other liquid suitable for the carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the selected therapy given and can be determined empirically.

[0202]

[0274] IV. Uses of Anti-LILRB4 Antibodies

[0275] The present disclosure also provides a therapy comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment as provided herein to a subject in need thereof, thereby treating or preventing a LILRB4-associated condition or disorder. In some embodiments, the LILRB4-associated condition or disorder is cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0203]

[0276] Examples of cancers can be generally categorized into solid tumors and hematological malignancies. Solid tumors include, but are not limited to, non-small cell lung cancer (squamous / non-squamous), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal, ductal, and lobular breast cancer), pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, multiple myeloma, mycoses fungoides, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovium / synovial sarcoma, mesothelioma, uterine leukemia, thyroid cancer ... These include: Lee's sarcoma, leiomyosarcoma, rhabdomyosarcoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical carcinoma, testicular tumor, seminoma, mast cell-derived tumors, EBV-positive and -negative PTLD, nasopharyngeal carcinoma, spinal axis tumor, brain stem glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0204]

[0277] Solid tumors are characterized by numerous biological features, including the maintenance of proliferative signaling, evasion of growth inhibitors, resistance to cell death, replicative immortality, angiogenesis, activation of invasion and metastasis, tumor-promoting inflammation, evasion of immune destruction, genomic instability and mutations, and dysregulated cellular energetics. Therapeutic efforts range from cytotoxic chemotherapy targeting rapidly dividing cells to small molecules that inhibit selected signaling pathways and monoclonal antibodies that target surface proteins. More recently, the concept of cancer immunotherapy, which reactivates endogenous immunity, or cell therapy utilizing synthetic immune responses, has shown promise. Despite these advances, the majority of patients with advanced solid tumors still do not survive long-term. The use of immune checkpoint inhibitors, such as anti-CTLA-4 or anti-PD-1 / PD-L1, has led to long-term progression-free and overall survival in a minority of patients.

[0205]

[0278] To improve outcomes, newer immunotherapeutic approaches that target different aspects of immunobiology and different tumor-infiltrating cells are needed, such as those targeting LILRB4 as an inhibitory receptor expressed on myeloid cell subsets, including myeloid-derived suppressor cells (MDSCs). These myeloid cells are functionally described as myeloid-derived suppressor cells because their immunosuppressive / anti-inflammatory phenotype can inhibit the activation, proliferation, and cytotoxic activity of tumor antigen-specific T cells.

[0206]

[0279] In some embodiments, depletion of MDSCs can reverse the suppressive effects on tumor antigen-specific T cells for solid tumor treatment.

[0280] In some embodiments, blocking LILRB4 on bone marrow cells can also relieve its inhibitory effect on antigen-presenting cells (APCs), including dendritic cells or myeloid leukemia cells that express LILRB4. Increased antigen-presenting activity by certain cytokine-producing APCs can be observed, which can lead to T cell activation, cytotoxicity, and T cell cytokine production.

[0207]

[0281] Hematological malignancies include, but are not limited to, acute lymphocytic / lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic lymphoblastic leukemia (CLL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), classical Hodgkin's lymphoma (CHL), diffuse giant B-cell lymphoma (DLBCL), extranodal NK / T-cell lymphoma, hairy cell leukemia, heavy chain disease, HHV8-associated primary effusion lymphoma, lymphoid malignancies, multiple myeloma (MM), myelodysplasia, myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma, plasmablastic lymphoma, pre-B acute lymphocytic leukemia (pre-B ALL), primary CNS lymphoma, primary mediastinal giant B-cell lymphoma, T-cell / histiocytic-rich B-cell lymphoma, myeloproliferative neoplasms, and Waldenström's macroglobulinemia.

[0208]

[0282] Autoimmune or inflammatory diseases include, but are not limited to, acquired immune deficiency syndrome (AIDS, a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, and autoimmune hepatitis. , autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac disease-dermatitis herpetiformis, chronic fatigue and immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos disease, juvenile dermatomyositis, discoid lupus erythematosus, essential mixed cryog Globulinemia, 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, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis, and and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, systemic sclerosis, progressive systemic sclerosis (PSS), systemic sclerosis (SS), Sjogren's syndrome, stiff-body syndrome, systemic lupus erythematosus (SLE), Takayasu's arteritis, temporal arteritis / giant cell arteritis, inflammatory bowel disease (IBD), ulcerative colitis These include enteritis, Crohn'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, autoimmune diabetes, etc. Inflammatory disorders include, for example, chronic and acute inflammatory disorders.

[0209]

[0283] Infectious diseases include, but are not limited to, fungal infections, parasitic / protozoan infections, or chronic viral infections, such as malaria, coccidioidmycosis immitis, histoplasmosis, onychomycosis, aspergillosis, blastomycosis, candida albicans, albicans), paracoccidiodomycosis, microsporidiosis, acanthamoeba keratopathy, amebiasis, ascariasis, babesiosis, balantidiosis, Baylisascariasis, Chagas disease, clonorchiasis, cochliomyiasis, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, filariasis, giardiasis, gnathostomiasis, hymenococcosis, isosporiasis, Katayama fever, leishmaniasis, Lyme disease, yokogawa fluke disease, myiasis, onchocerciasis, lice infestation These include infections with: scabies, schistosomiasis, sleeping sickness, strongyloidiasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, trichuriasis, trypanosomiasis, helminth infections, hepatitis B (HBV), hepatitis C (HCV), herpesvirus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human papillomavirus, adenovirus, human immunodeficiency virus I, human immunodeficiency virus II, Kaposi's sarcoma-associated herpesvirus infection, Schingling virus (Turketeno virus), human T-lymphotropic virus I, human T-lymphotropic virus II, varicella-zoster, JC virus, or BK virus.

[0210]

[0284] A therapeutically effective amount of an antibody or antigen-binding fragment as provided herein will depend on a variety of factors known in the art, such as body weight, age, past medical history, current medications, the subject's health and potential for cross-reactivity, allergies, sensitivities and adverse side effects, as well as the route of administration and the stage of disease progression. Dosages can be proportionally reduced or increased by one of ordinary skill in the art (e.g., a physician or veterinarian) as indicated by these and other circumstances or requirements.

[0211]

[0285] In certain embodiments, the antibodies or antigen-binding fragments as provided herein are administered at a therapeutically effective dosage of about 0.0001 mg / kg to about 100 mg / kg. In certain of these embodiments, the antibody or antigen-binding fragment is administered at a dosage of about 50 mg / kg or less, and in certain of these embodiments, the dosage is 10 mg / kg or less, 5 mg / kg or less, 3 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the administered dosage may vary over the course of treatment. For example, in certain embodiments, the initial administered dosage may be higher than subsequent administered dosages. In certain embodiments, the administered dosage may vary over the course of treatment depending on the subject's response.

[0212]

[0286] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered or several divided doses may be administered over time.

[0213]

[0287] The antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, for example, parenterally (e.g., subcutaneously, intraperitoneally, intravenously, including intravenous infusion, intramuscularly, or intradermal injection) or non-parenterally (e.g., orally, intranasally, intraocularly, sublingually, rectally, or topically).

[0214]

[0288] In some embodiments, the antibodies or antigen-binding fragments disclosed herein may be administered alone or in combination with one or more additional therapeutic procedures or agents. For example, the antibodies or antigen-binding fragments disclosed herein may be administered in combination with another therapeutic agent, such as a chemotherapeutic or anti-cancer agent.

[0215]

[0289] In certain of these embodiments, an antibody or antigen-binding fragment as disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the antibody or antigen-binding fragment and the additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment administered "in combination with" another therapeutic agent need not be administered simultaneously with or in the same composition as such agent. An antibody or antigen-binding fragment that is administered before or after another agent is considered to be administered "in combination with" such agent, as the phrase "in combination" is used herein, even if the antibody or antigen-binding fragment and the second agent are administered via different routes. Where possible, additional therapeutic agents administered in combination with an antibody or antigen-binding fragment disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Prescriber's Digital Reference (available online only at pdr.net) or protocols well known in the art.

[0216]

[0290] Specific agents contemplated for combination therapy with the antibodies of the present disclosure include chemotherapy, which may include cytarabine (ara-C) and an anthracycline (most often daunorubicin), high-dose cytarabine alone, induction chemotherapy, usually all-trans retinoic acid (ATRA) added to an anthracycline, histamine dihydrochloride (Ceplene) and interleukin-2 (Proleukin) after completion of consolidation therapy, gemtuzumab ozogamicin (Mylotarg) for patients over 60 years of age with relapsed AML who are not candidates for high-dose chemotherapy, clofarabine, and targeted therapies such as kinase inhibitors, farnesyltransferase inhibitors, decitabine, and inhibitors of MDR1 (multidrug resistance protein), or arsenic trioxide, or relapsed acute promyelocytic leukemia (APL).

[0217]

[0291] 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), daunorubicin and cytarabine combinations, daunorubicin and cytarabine liposomes for injection, Vyxeos®, azacitidine, Vedaza®, decitabine, all-trans retinoic acid (ATRA), arsenic, arsenous acid, histamine dihydrochloride, Ceplene® ), 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, I DO inhibitors, epacadostat, BCL-2 inhibitors, 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 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, TIGIT antibodies, TIM3 antibodies, CD40 antibodies, The agent is one or more agents selected from the group consisting of a 4-1BB antibody, a CD47 antibody, a SIRP1α antibody or fusion protein, 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, a plant-derived alkaloid, a hormone therapy agent, a hormone antagonist, an aromatase inhibitor, and a P-glycoprotein inhibitor.

[0218]

[0292] In certain embodiments, the LILRB4-associated condition or disorder is acute myeloid leukemia (AML), acute myelomonocytic leukemia (FAB M4) subtype, and acute monoblastic / monocytic leukemia (FAB M5) subtype.

[0219]

[0293] In certain embodiments, AML that is resistant or refractory to standard therapies, such as venetoclax and azacitidine.

[0294] AML is an aggressive malignancy with a poor prognosis. In the World Health Organization (WHO) classification, acute myelomonocytic leukemia (M4 in the French-American-British [FAB] classification) and acute monoblastic / monocytic leukemia (M5 in the FAB classification) are subtypes of AML, not otherwise specified (AML, NOS).

[0220]

[0295] WHO-series acute myelomonocytic leukemia (FAB M4) has blasts (≥20% total) consisting of myeloblasts, monoblasts, and promonocytes, and ≥20%–79% monocytic cell lineage. Acute monoblastic / monocytic leukemia (FAB M5) has ≥20% monoblasts / promonocytes and ≥80% myeloid cells with monocytic characteristics. Acute myelomonocytic leukemia (M4) and acute monocytic leukemia (M5) account for approximately 20% and 10% of all AML cases, respectively (Ganzel et al., 2016). AML patients with a significant monocytic component often present with extramedullary disease (Ganzel et al., 2016) and hyperleukocytosis (≥100x10 in peripheral blood). 3 / μL leukocytes) (Rol lig and Ehninger, 2015) have evidence.

[0221]

[0296] LILRB4 is expressed on AML cells with monocytic differentiation (Deng et al., 2018; Dobrowolska et al., 2013) and CMML (Chien et al., 2019). LILRB4 expression levels on AML blasts with monocytic differentiation are comparable to or may be up to 10-fold higher than normal monocytes (Deng et al., 2019). 8). Furthermore, both functional and immunophenotypic studies suggest that LILRB4 is expressed by leukemia stem cells derived from monocytic AML (Deng et al., 2018).

[0222]

[0297] In 2008, AZA was approved by the European Medicines Agency (EMA) for the treatment of AML patients who have 20% to 30% bone marrow (BM) blasts, are older than 64 years, and are ineligible for hematopoietic stem cell transplantation (HSCT). Hematologists at specialized centers began treating AML patients with >30% BM blasts with AZA as early as 2007, indicating that physicians were confident they were doing what was best for their patients. This assumption is based on the significant improvement in overall survival (OS) achieved in the AZA-MDS-001 trial and in the Cancer and Leukemia Group B protocols, where 32% and 38% of the study populations, respectively, had AML with 20% to 30% BM blasts.

[0223]

[0298] In 2010, patients with newly diagnosed AML had >30% BM blasts and ≤15x10 9 In AML patients older than 65 years with a white blood cell (WBC) count of 1 / L, The international phase 3 randomized AZA-AML-001 clinical trial was initiated, testing AZA against a controlled treatment regimen (CCR) (preselected by the treating physician) consisting of intensive chemotherapy, low-dose cytarabine, or best supportive care. This trial reported a clinically meaningful improvement in OS (10.4 vs. 6.5 months; p = 0.1009) for AZA versus CCR. Furthermore, overall response (complete remission [CR] + complete remission with incomplete blood cell recovery [CRi]) rates were comparable in the AZA (27.8%) and CCR (25.1%) arms (p = 0.5384), and EMA approval of AZA was expanded to include AML patients with >30% BM blasts on October 30, 2015.

[0224]

[0299] In certain embodiments, the LILRB4-associated condition or disorder is chronic myelomonocytic leukemia (CMML).

[0300] The CMML diagnostic classification is based on clinical examination, morphology, cytogenetics, and, whenever possible, flow cytometry, and incorporates molecular biology, as defined by the WHO Patients should be classified according to the 2016 WHO classification (Arber et al., 2016), which includes CMML-0 for cases with <2% blasts in peripheral blood and <5% blasts in BM; CMML-1 for cases with 2%-4% blasts in peripheral blood and 5%-9% blasts in BM; and CMML-2 for cases with 5%-19% blasts in peripheral blood, 10%-19% blasts in BM, and / or the presence of Auer rods.

[0225]

[0301] 13x10 9 Based on a WBC cutoff of / L, the FAB classification The distinction initially proposed between "dysplastic" and "proliferative" CMML remains useful because their clinical features differ (cytopenias versus organomegaly, elevated WBC, and constitutional symptoms) and, consequently, clinical management differs.

[0226]

[0302] Extramedullary leukemia includes primarily specific serous effusions (pleural effusion and less frequently pericardial or ascites) and specific skin infiltrates, apart from splenomegaly and hepatomegaly, all of which are associated with a poorer prognosis.

[0227]

[0303] VIDAZA® (azacitidine) is a nucleoside metabolic inhibitor indicated for the treatment of patients with the following FAB myelodysplastic syndromes (MDS) subtypes: refractory anemia or refractory anemia with ringed sideroblasts (if associated with neutropenia or thrombocytopenia or requiring transfusions), refractory anemia with excess blasts, refractory anemia with excess transformed blasts, and chronic myelomonocytic leukemia (CMML).

[0228]

[0304] The present disclosure provides an antibody to detect the presence or amount of LILRB4 in a sample. Further provided is a method of using a LILRB4 antibody or antigen-binding fragment thereof, comprising contacting a sample with the antibody or antigen-binding fragment thereof and determining the presence or amount of LILRB4 in the sample. Methods for detecting LILRB4 using an anti-LILRB4 antibody include, but are not limited to, ELISA, Western blot, flow cytometry, and FACS.

[0229]

[0305] In some embodiments, the present disclosure provides methods for diagnosing a LILRB4-associated disease or condition in a subject, the method comprising the steps of: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; b) determining the presence or amount of LILRB4 in the sample; and c) correlating the presence of LILRB4 with a LILRB4-associated disease or condition in the subject.

[0230]

[0306] In some embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein, optionally conjugated to a detectable moiety, which may be useful for detecting LILRB4 or diagnosing a LILRB4-related disease.

[0231]

[0307] In some embodiments, the present disclosure also provides the use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a LILRB4-related disease or condition in a subject, or in the manufacture of a diagnostic reagent for diagnosing a LILRB4-related disease or condition.

[0232]

[0308] V. Chimeric Antigen Receptor

[0309] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) protein (LILRB4 CAR protein) that binds to LILRB4. In certain embodiments, the CAR protein comprises an antigen-recognition region, i.e., an antibody or antigen-binding fragment that recognizes LILRB4 as described herein, and other membrane and intracellular components. In some embodiments, the LILRB4 CAR protein comprises a LILRB4 antigen-recognition region, a transmembrane domain, and an intracellular costimulatory signal domain. In certain embodiments, the single-chain LILRB4 CAR protein also comprises a leader peptide, a spacer region, and an intracellular T cell signaling domain.

[0233]

[0310] In certain embodiments, the antigen recognition region comprises multiple polypeptide chains.

[0311] In some embodiments, the CAR protein comprises a first polypeptide comprising an antibody heavy chain variable domain, and a polypeptide comprising an antibody light chain variable domain, wherein the first or second polypeptide further comprises a transmembrane domain, and wherein the antibody heavy chain variable domain and the antibody light chain variable domain together form an antigen recognition region.

[0234]

[0312] In some embodiments, the CAR protein comprises a first polypeptide comprising an antibody heavy chain variable domain and a second polypeptide comprising an antibody light chain variable domain and an antibody light chain constant domain, wherein the first polypeptide further comprises a transmembrane domain, and the antibody heavy chain variable domain, antibody light chain variable domain, and antibody light chain constant domain together form an antigen recognition region. In some embodiments, the first portion further comprises an intracellular costimulatory signaling domain and a CD3ζ intracellular T cell signaling domain.

[0235]

[0313] In some embodiments, the CAR protein comprises a first polypeptide comprising an antibody heavy chain variable domain and an antibody heavy chain constant domain, and a second polypeptide comprising an antibody light chain variable domain, wherein the first polypeptide further comprises a transmembrane domain, and the antibody heavy chain variable domain, antibody heavy chain constant domain, and antibody light chain variable domain. The domains together form an antigen recognition region. In some embodiments, the first portion further comprises an intracellular costimulatory signaling domain and a CD3ζ intracellular T cell signaling domain.

[0236]

[0314] In some embodiments, the CAR protein comprises a first polypeptide comprising an antibody heavy chain variable domain and a second polypeptide comprising an antibody light chain variable domain, wherein the second polypeptide further comprises a transmembrane domain, and the antibody heavy chain variable domain, antibody light chain variable domain, and antibody light chain constant domain together form an antigen recognition region. In some embodiments, the second portion further comprises an intracellular costimulatory signaling domain and a CD3ζ intracellular T cell signaling domain.

[0237]

[0315] In some embodiments, the CAR protein comprises a first polypeptide comprising an antibody heavy chain variable domain and an antibody heavy chain constant domain, and a second polypeptide comprising an antibody light chain variable domain, wherein the second polypeptide further comprises a transmembrane domain, and the antibody heavy chain variable domain, antibody heavy chain constant domain, and antibody light chain variable domain together form an antigen recognition region. In some embodiments, the second portion further comprises an intracellular costimulatory signaling domain and a CD3ζ intracellular T cell signaling domain.

[0238]

[0316] In certain embodiments, the CAR protein is an anti-LILRB4 scFv as described herein, i.e., a single-chain polypeptide comprising an anti-LILRB4 heavy chain variable domain and an anti-LILRB4 light chain variable domain linked by a linker domain. In one embodiment, the CAR protein comprises, from N-terminus to C-terminus: a leader peptide, an anti-LILRB4 heavy chain variable domain, a linker domain, an anti-LILRB4 light chain variable domain, a hinge region, a transmembrane domain, and an intracellular costimulatory signal domain. In one embodiment, the CAR protein comprises, from N-terminus to C-terminus: a leader peptide, an anti-LILRB4 light chain variable domain, a linker domain, an anti-LILRB4 heavy chain variable domain, a hinge region, a transmembrane domain, and an intracellular costimulatory signal domain. In some embodiments, the CAR protein further comprises a CD3ζ intracellular T cell signaling domain.

[0239]

[0317] In certain embodiments, the linker domain is generally composed of helical and rotation-promoting amino acid residues, such as alanine, serine, and glycine. However, other residues may also function. In some embodiments, the linker domain is inserted between the VH and VL of an scFv. In some embodiments, the linker domain is between the transmembrane domain and the intracellular costimulatory signaling domain. In some embodiments, the linker domain is between the intracellular T cell signaling domain and the intracellular costimulatory signaling domain. In some embodiments, the linker domain comprises the sequence GGGGSGGGSGGGGS (SEQ ID NO: 70).

[0240]

[0318] In some embodiments, the transmembrane domain is a CD8α transmembrane domain having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity compared to a naturally occurring CD8α transmembrane domain polypeptide (SEQ ID NO: 71). In some embodiments, the CD8α transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 72.

[0241]

[0319] In some embodiments, the transmembrane domain is a CD28 transmembrane domain having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity compared to a naturally occurring CD28 transmembrane domain polypeptide (SEQ ID NO: 73). is encoded by the nucleic acid sequence

[0242]

[0320] The intracellular costimulatory signaling domain comprises an amino acid sequence capable of providing costimulatory signaling in response to binding of an antigen to the CAR. In some embodiments, signaling of the costimulatory signaling domain results in cytokine production and proliferation of T cells or NK cells expressing such cytokines. In some embodiments, the intracellular costimulatory signaling domain is a CD28 intracellular costimulatory signaling domain, a 4-1BB intracellular costimulatory signaling domain, an ICOS intracellular costimulatory signaling domain, an OX-40 intracellular costimulatory signaling domain, or any combination thereof. In some embodiments, the CD28 costimulatory signaling domain has the polypeptide sequence of SEQ ID NO: 75. In some embodiments, the CD28 intracellular costimulatory signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 76. In some embodiments, the 4-1BB intracellular costimulatory signaling domain has the polypeptide sequence of SEQ ID NO: 77. In some embodiments, the 4-1BB intracellular costimulatory signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 78.

[0243]

[0321] As provided herein, a "hinge region" is a polypeptide that links an antigen-binding region to a transmembrane domain. In some embodiments, the hinge region links a heavy chain variable region to a transmembrane domain. In some embodiments, the hinge region links a heavy chain constant region to a transmembrane domain. In some embodiments, the hinge region links a light chain variable region to a transmembrane domain. In some embodiments, the hinge region links a light chain constant region to a transmembrane domain. In some embodiments, the binding affinity of the antigen-binding region to an antigen is increased compared to the absence of the hinge region. In some embodiments, steric hindrance between the antigen-binding region and the antigen is reduced in the presence of the hinge region. In some embodiments, the hinge region is a CD8α hinge region. In some embodiments, the hinge region is a CD28 hinge region.

[0244]

[0322] In some embodiments, the intracellular T cell signaling domain includes the signaling domain of the zeta (ζ) chain of the human CD3 complex, i.e., the CD3ζ intracellular T cell signaling domain. In some embodiments, the intracellular T cell signaling domain is the protein CD3zIso1 comprising the amino acid sequence of SEQ ID NO: 79. In some embodiments, the intracellular T cell signaling domain is the protein CD3zIso3 comprising the amino acid sequence of SEQ ID NO: 80, which is encoded by the nucleic acid sequence of SEQ ID NO: 81.

[0245]

[0323] In one example, the CAR protein is a single-chain polypeptide comprising, from N-terminus to C-terminus: a CD8α leader peptide, an anti-LILRB4 scFv, a CD8α hinge region, a CD8α transmembrane domain (or a CD28 transmembrane domain), a 4-1BB intracellular costimulatory signaling domain (or a CD28 intracellular costimulatory signaling domain, or a CD28 intracellular costimulatory signaling domain followed by a 4-1BB intracellular costimulatory signaling domain), and a CD3ζ intracellular T cell signaling domain of one of two isoforms (CD3zIso1 or CD3zIso3).

[0246]

[0324] In certain embodiments, the LILRB4 CAR proteins provided herein exhibit high affinity for LILRB4. In certain embodiments, the CAR proteins provided herein exhibit a binding affinity (EC 2 as measured by ELISA) for LILRB4 of less than 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, or 0.05 nM. 50 For the purposes of this application, ELISA EC 50 The value can be determined as follows: LILRB- The 4 extracellular domain protein (containing a 6HIS tag at the C-terminus) was recombinantly produced in HEK293 cells and coated onto high-binding 96-well clear plates (Corning-Costar, Fisher Scientific) at a concentration of 1 μg / ml (100 μl / well) for 14–16 hours at 4°C. The coated plates were washed briefly with PBS, pH 7.4, and blocked with 200 μl / well of 5% nonfat milk in PBS for 2 hours at 37°C. Serial dilutions of test monoclonal antibodies (IgG or scFv fragments), starting at 10 μg / ml and titrated downwards three-fold in 12 steps, were added to the 96-well plates for binding by incubating the assay plate with the lid on for 45 minutes at 37°C. The plates were then washed three times with PBS containing Tween 20 (0.05% concentration) and once with PBS. Secondary antibodies (Jackson ImmunoResearch), anti-human or anti-rabbit, or other species IgG-specific antibodies containing HRP conjugates, were added according to the manufacturer's suggested dilution for 1 hour at room temperature for incubation. Detection was performed by adding HRP substrate, TMB (ThermoFisher), for 10 minutes and stopped by adding 50 μl / well of 2N H2SO4. Plates were read for absorbance at 450 nm using a plate reader (SpectraMax M4, Molecular Devices). Data were collected and analyzed using EC50 For calculations, graphs were generated using a four-parameter curve fit in GrapPad Prism 7 software.

[0247]

[0325] In another aspect, the present disclosure provides a polynucleotide molecule encoding a CAR protein described herein. In some embodiments, the polynucleotide molecule further comprises a promoter active in eukaryotic cells. In some embodiments, the promoter is a JeT promoter. The JeT promoter is a recombinant promoter with transcriptional activity comparable to many strong mammalian promoters. The JeT promoter consists of five main elements: (1) a TATA box; (2) a transcription initiation site (Inr); (3) a CAT consensus sequence combined with a CArG element; and finally (5) two tandemly arranged four Sp1 transcription binding sites (GGGCGG) (US2002 / 0098547 A1). In some embodiments, the polynucleotide molecule is an expression vector. In some embodiments, the vector is based on Clontech's pLVX-EF1alpha-IRES-ZsGreen, or pSIN-EF1alpha-IRES-Puromycin or pSIN-EF1alpha. In one example, a polynucleotide molecule of the present disclosure comprises the following elements in succession: (1) a JeT promoter; (2) a sequence encoding a CD8-alpha leader; (3) a sequence encoding a heavy chain variable region; (4) a sequence encoding a linker; (5) a sequence encoding a light chain variable region; (6) a sequence encoding a CD8 hinge and TM domain; (7) a sequence encoding a 4-1BB costimulatory domain; and (8) a sequence encoding a CD3-zeta activation domain. In one example, the above elements are flanked by 5' and 3' homology arms that facilitate insertion of the polynucleotide molecule into a target locus, such as the T cell receptor alpha constant (TRAC) locus.

[0248]

[0326] VI. Engineered cells expressing anti-LILRB4 CAR protein

[0327] In another aspect, the present disclosure provides engineered immune cells that express the CAR proteins described herein. The immune cells may be T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), natural killer (NK) cells, invariant NK cells, NKT cells, or macrophages. Also provided herein are methods for producing and engineering immune cells, as well as methods for using and administering cells for adoptive cell therapy, in which the cells may be autologous or allogeneic. Thus, engineered immune cells may be used as immunotherapies, for example, to target cancer cells.

[0249]

[0328] Expression of CAR proteins allows engineered immune cells to bind to target cells, such as cancer cells, by recognizing antigens present on the target cells. Upon binding to the target cells, the engineered immune cells are activated, then begin to proliferate, become cytotoxic, and ultimately destroy the target cells. CAR-T cell immunotherapy has shown success in clinical trials and has been approved by the US FDA for the treatment of refractory B-cell acute lymphoblastic leukemia and B-cell non-Hodgkin's lymphoma (Hartmann J et al., EMBO Mol Med (2017) 9:1183-97). In addition to CAR-T cells, CAR NK cells and CAR macrophages have recently been developed as immunotherapy options (Kloess S et al., Transfusion Medicine and Hemotherapy (2019) 46:4-13; Klichinsky M et al., AACR Annual Meeting 2017, Abstract 4575). Thus, in certain embodiments of the present disclosure, the immune cell expressing a CAR protein described herein is a T cell, an NK cell, or a macrophage.

[0250]

[0329] Immune cells can be isolated from a subject, particularly a human subject.Immune cells can be obtained from a subject of interest, such as a subject suspected of having a specific disease or condition, a subject suspected of having a predisposition to a specific disease or condition, a subject undergoing therapy for a specific disease or condition, a healthy volunteer or healthy donor, or from a blood bank.Immune cells can be collected from any location present in a subject, including but not limited to blood, umbilical cord blood, spleen, thymus, lymph node and bone marrow.Immune cells can be directly used or can be stored for a certain period, for example, by freezing.

[0251]

[0330] Immune cells may be enriched / purified from any tissue in which they are present, including, but not limited to, blood (including blood collected by a blood bank or umbilical cord blood bank), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained by biopsy. The tissues / organs from which immune cells are enriched, isolated, and / or purified may be isolated from both living and non-living subjects, with the non-living subject being an organ donor. 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 capacity, as measured, for example, 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 capacity. Pooled blood may be derived from two or more sources, for example, three, four, five, six, seven, eight, nine, ten or more sources (eg, donor subjects).

[0252]

[0331] The immune cell population may be obtained from a subject needing therapy or suffering from a disease associated with decreased immune cell activity. Thus, the cells will be autologous to the subject needing therapy. Alternatively, the immune cell population may be obtained from a donor, preferably a histocompatibility-matched donor. The immune cell population may be collected from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells are present in the subject or donor. The immune cells may be isolated from a pool of subjects and / or donors, such as pooled umbilical cord blood.

[0253]

[0332] If the immune cell population is obtained from a donor different from the subject, the donor is preferably allogeneic, provided that the resulting 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 may be treated to reduce immunogenicity. stomach.

[0254]

[0333] Immune cells can be genetically engineered to express CAR using appropriate modification methods known in the art. For example, see Sambrook and Ausubel, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In some embodiments, immune cells contain one or more nucleic acids encoding one or more CAR proteins, which are introduced through genetic engineering. In certain embodiments, the nucleic acid encoding CAR protein is inserted into the immune cell genome using gene editing methods, such as CRISPR / Cas technology. In one example, the nucleic acid encoding CAR protein is inserted at the T cell receptor alpha constant (TRAC) locus (for example, see Eyquem J et al., Nature (2017) 543:113-117).

[0255]

[0334] Also provided is a method for immunotherapy, comprising administering an effective amount of the immune cells of the present disclosure. In some embodiments, a medical disease or disorder is treated by transferring the immune cell population described herein, which induces an immune response. In certain embodiments, the medical disease or disorder is cancer. In certain embodiments, the medical disease or disorder is an autoimmune or inflammatory disease. [Example]

[0256]

[0335] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate particular embodiments falling within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the methods described herein while still remaining within the scope of the invention. It is the inventors' intention that such variations be included within the scope of the invention.

[0257] Example 1

[0336] material and method

[0337] SEC The size exclusion chromatography (SEC) method was as follows: If the sample concentration exceeded 10.0 mg / mL, the sample was diluted to 10.0 mg / mL with the mobile phase before SEC analysis. 100 μg of sample was injected into the column. The equipment used was an Agilent 1260 HPLC system equipped with a TSKgel G3000SWXL column (7.8 × 300 mm, 5 μm particle size) and a UV detector (detection wavelength: 280 nm). The mobile phase was 50 mM phosphate buffer (pH 6.8 ± 0.1) containing 300 mM sodium chloride. An isocratic gradient was applied at a flow rate of 1.0 mL / min for 20 min.

[0258]

[0338] Imaging capillary isoelectric focusing (icIEF) 20 μL of reference standard or sample (diluted to 1.0 mg / mL) was individually mixed with ~80 μL of a master mix consisting of 0.5 μL of pI 7.40 marker, 0.5 μL of pI 9.77 marker, 1.0 μL of Pharmalyte 3-10, 3.0 μL of Pharmalyte 8-10.5, 35 μL of 1% methylcellulose (MC), 37.5 μL of 8 M urea solution, 0.07 μL of acetic acid, and 2.5 μL of ultrapure water. The loading mixture was then analyzed by iCE3 capillary isoelectric focusing equipped with an FC-coated whole-column detection capillary. The analysis was performed on an instrument. Electrophoresis was performed in two steps: (1) 1.5 kV for 1 minute; (2) 3 kV for 8 minutes, and the autosampler tray was maintained at 15°C. Absorbance detection was performed at 280 nm. After analysis, the raw data were processed with Empower 3.

[0259]

[0339] EC50 FACS The binding ability of stable samples of the H7K3 antibody was determined using stable CHO-K1 cells expressing either human LILRB4 or flag-tagged cynomolgus monkey LILRB4. CHO-K1 cells (1x10 5 ) were stained with serially titrated stability samples, followed by fluorescently conjugated anti-human Secondary staining was performed with IgG (Biolegend). Geometric mean fluorescence (MFI) was measured and EC was analyzed by Prism. 50 was calculated.

[0260]

[0340] EC50 ELISAHuman LILRB4 ECD-his recombinant protein (Sino Biological) or cynomolgus monkey LILRB4 ECD-his recombinant protein (ACRObiosystems) was coated onto EIA / RIA plates (Corning) overnight at 4°C. After blocking with 5% nonfat milk for 2 hours at 37°C, 100 μL of serially diluted anti-LILRB4 antibody was added to the wells and incubated for 45 minutes at 37°C. The plate was then washed three times with PBS-Tween 20 (0.05%) and once with PBS, followed by incubation with HRP-conjugated anti-hFc antibody (Jackson ImmunoResearch Laboratories) for 35 minutes at room temperature. Signals were developed with TMB substrate (Sigma) and stopped by adding 2M sulfuric acid. The OD was then read at 450 nm on a plate reader (Molecular Devices) using Prism (GraphPad). 450 Based on the measurements, EC 50 was calculated.

[0261]

[0341] ApoE inhibition (IC50) The ligand-blocking ability of H7K3 mutants was screened using a mouse T hybridoma cell line expressing LILRB4 extracellularly coupled to a nuclear factor of activated T cells (NFAT)-GFP reporter system. LILRB4 reporter cells (2 x 10 per well) were cultured. 4 ) and fixed recombinant APOE3 protein Cells were co-cultured with 10 μg / mL of protein (Novoprotein Catalog No. C102) to induce GFP expression by binding of APOE to its receptor LILRB4, and the GFP signal was quantified using flow cytometry. In the presence of serially titrated LILRB4 blocking antibody H7K3, GFP expression decreased in a dose-dependent manner, and the flow cytometry signal indicated IC 50 was calculated.

[0262]

[0342] Self ADCCPBMCs were freshly isolated from healthy donors and cultured overnight in the presence of 50 ng / mL rhIL-2 and serially titrated antibodies. Cells were stained with fluorescently conjugated anti-CD14, anti-CD19, anti-CD303, and anti-CD123 antibodies and acquired by FACS Celesta to count viable monocytes, pDCs, and B cells. Dead cells were excluded by adding 7-AAD. Percent cell killing was calculated as 100 minus [(number of antibody-treated cells) / (number of non-antibody-treated cells)].

[0263]

[0343] ADCC of THP-1-GFP cells THP-1-GFP cells and freshly isolated PBMCs were co-cultured overnight in the presence of 50 ng / mL rhIL-2 and serial titration of antibodies (E:T ratio = 50:1). + and 7-AAD - The number of viable THP-1-GFP cells was obtained by gating on the cells. Percent cell killing was calculated using the same formula as for autologous ADCC.

[0264]

[0344] ADCC in MDSCs MDSCs and NK cells were prepared from two different healthy donors. PBMCs were cultured in the presence of 40 ng / mL GM-CSF. MDSCs were generated by co-culture with L5 cells for 8 days and purified using CD33+ microbeads. IL-2 (100 ng / mL) was added for NK cell activation. MDSC:NK = 1:2.5 (duplicate) containing 50,000 MDSCs and 125,000 NK cells were co-cultured for 21 hours. After 21 hours of incubation, cells were stained with CD14-FITC. THP-1-luc-GFP cells were used as a positive control under the same settings. The percentage of cell killing was calculated using the same formula as for autologous ADCC.

[0265]

[0345] ADCPHuman monocytes were isolated from PBMCs from healthy donors using negative selection (Miltenyi Biotec) and cultured in X-vivo 10 + 10% FBS medium for 7 days in the presence of 50 ng / mL M-CSF (R&D system). For the final 24 hours, 50 ng / mL interferon-gamma was added to prime the macrophages. THP-1-GFP cells (2.5 x 10 4 ) continuously They were co-cultured with macrophages in the presence of titrated anti-LILRB4 antibodies (E:T=5:1) for 24 hours and stained with RPE-conjugated anti-CD163 and anti-CD206. + Viable THP-1 cells were obtained by flow cytometry by gating on cells. Absolute numbers of THP-1 cells and GFP + % cell killing Cents were calculated.

[0266]

[0346] T cell-mediated cytotoxicity A FACS-based approach was used to determine the ability of anti-LILRB4 to mediate tumor cell killing by naive T cells. Human buffy coats were obtained from healthy donors, and peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats by Ficoll Paque Plus (GE Healthcare catalog number 17-1440-03) density gradient cell separation. Pan T cells were further isolated from PBMCs using a human pan T cell isolation kit (Miltenyi Biotec catalog number 130-096-535). 4 x 10 cells were cultured at a 4:1 ratio. 5 Freshly isolated human pan-T thin Cells were used as effector cells, and 1x10 5 Targeting THP-1-GFP Human pan-T cells, THP-1-GFP cells, and increasing concentrations of anti-LILRB4 antibody or isotype control human IgG1 (BioXcell catalog no. BE0297) were mixed in a total volume of 200 μL in RPMI 1640 (Gibco catalog no. 61870-036) + 10% heat-inactivated FBS (Gibco catalog no. 10082-147) in a U-shaped 96-well plate and incubated at 37°C for 48 hours. At the end of the incubation, 40 μL of supernatant was collected for cytokine Luminex assay. 7-AAD (BD Pharmingen catalog no. 559925) was added to the cells, and 100 μL of cells were acquired by FACS Celesta, and the percentage of GFP-positive cells was measured. Flowjo software (Flowjo Flow cytometry data were analyzed using LLC (Liquid Crystal Life Sciences) and cytotoxicity was calculated as follows: Percent cytotoxicity = 100 - ([T / NT] x 100), where T and NT are the percentages of GFP treated with or without test antibody, respectively. + The percentage of cells was calculated as:

[0267]

[0347] Luminex cytokine assay Cell supernatants were tested using a custom 15-plex panel kit (R&D Systems). To perform the DA Bead assay using the wallless plate and reagents from the 15-plex kit, the protocol for the R&D Systems kit was slightly modified. First, the DA Bead wallless plate was blocked with 10 μL of 1% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature. Subsequently, the DA Bead plate was washed once with 0.1% BSA 0.05% Tween 20 in PBS (wash buffer) using an automated washing station LT MX (Curiox Biosystems). 7.5 μL of premixed magnetic beads were added to each well. Then, 7.5 μL of diluted sample, standard, or blank was added to the appropriate well. The DA Bead plate was then analyzed using an analog microprobe at an intensity level of 4. The DA Bead plate was vortexed for approximately 10 seconds on a Rate Genie Shaker (Scientific Industries Inc., Bohemia, NY). The DA Bead plate was placed on a 3 mm span orbital shaker (Orbit 300, Labnet, Edison, NJ) and shaken at 350 revolutions per minute (rpm) (0.2 x g) for 120 minutes at room temperature. The DA Bead plate was then washed three times in an LT MX wash station. 10 μL of detection antibody dilution was added to each used well. The DA Beads were then placed on an analog microplate Genie shaker as described above for approximately 10 seconds and incubated on an orbital shaker at 350 rpm for 60 minutes at room temperature. The DA Bead plate was then washed three times using the LT MX station. 10 μL of streptavidin-phycoerythrin dilution was added to each well. The DA Bead plate was placed on a Genie shaker as described above for approximately 10 seconds and incubated on an orbital shaker at 350 rpm at room temperature for 30 minutes. The DA Bead plate was washed three times in the LT MX station. The beads were resuspended in a total volume of 65 μL of wash buffer and transferred to a skirted PCR plate and read in a Luminex reader for data acquisition (MAGPIX, a dual-laser flow-based detection device, Luminex).

[0268]

[0348] FACS analysis of co-cultured T cells and THP-1 cells Human buffy coats were obtained from healthy donors, and peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats by Ficoll Paque Plus (GE Healthcare catalog number 17-1440-03) density gradient cell separation. Pan T cells were further isolated from frozen PBMCs by negative depletion using a human pan T cell isolation kit (Miltenyi Biotec catalog number 130-096-535). 8 x 10 cells were cultured at an 8:1 ratio. 5 Refined Hi Pan T cells were used as effector cells, and 1 x 10 5 THP-1-GFP Human pan-T cells, THP-1-GFP cells, and anti-LILRB4 antibody or isotype control human IgG1 (BioXcell catalog no. BE0297) were mixed in a total volume of 200 μL in X-vivo 10 (Lonza catalog no. 04-380Q) + 10% heat-inactivated fetal bovine serum (FBS; Gibco catalog no. 10082-147) in a U-shaped 96-well plate and incubated at 37°C for 48 hours. The final concentration of anti-LILRB4 antibody or human IgG1 was 3 μg / mL (20 nM). To measure intracellular TNFα and IFNγ production, a protein transport inhibitor (BD Biosciences catalog no. 555029) was added for the last 11 hours of incubation. At the end of the incubation, cells were spun off from the medium containing protein transport inhibitors and incubated with human IgG (Sigma Aldrich Catalog No. I4506) for 10 minutes at room temperature to block Fc receptors. Cells were then stained for surface antigens with directly conjugated anti-CD4 (Cat. No. 564975) and anti-CD8 (Cat. No. 563256), fixed and permeabilized using a fixation / permeabilization kit (Cat. No. 555028), and stained with anti-IFNγ (Cat. No. 554552) and anti-TNFα (Cat. No. 554514) or isotype control antibodies (Cat. Nos. 554681, 555749). Antibodies against surface and intracellular antigens were from Becton, Dickinson, and Company.

[0269]

[0349] FACS analysis of T cell and THP-1 cell activationSurface expression of T cell activation markers was assessed with directly conjugated anti-CD4 (Cat. No. 564975), anti-CD8 (Cat. No. 563256), anti-CD69 (Cat. No. 555533), and anti-CD25 (Cat. No. 555432) antibodies from BD Biosciences. Surface expression of THP-1 cell markers was assessed with directly conjugated anti-HLA-DR (Cat. No. 559866), anti-CD80 (Cat. No. 563084), anti-CD83 (Cat. No. 565336), and anti-CD86 (Cat. No. 555432) antibodies from BD Biosciences. Antibodies were assessed using antibodies against BD Pharmingen's anti-CD40 (catalog no. 334310), anti-HLA-A, B, C (catalog no. 311406), and anti-LILRB4 (catalog no. 333008). Cells were acquired by FACS Celesta after adding 7-aminoactinomycin D (7AAD; BD Pharmingen catalog no. 559925). Flow cytometry data were analyzed using Flowjo software (Flowjo LLC) and graphed using Prism GraphPad software.

[0270]

[0350] PCR fragment for CAR coding fragment PCR was performed with PRIMESTAR DNA polymerase (Takara Bio R010B) using a Venti thermocycler under the following conditions: 98°C for 30 seconds, (98°C for 10 seconds, 64°C for 5 seconds, 72°C for 30 seconds) x 35 cycles, 72°C for 7 minutes. The PCR product was further purified using a PCR clean-up kit (Macherey-Nagel 740609.250), and the eluted DNA was further cleaned / concentrated by ethanol precipitation.

[0271]

[0351] gene targetingWe first activated T cells with TransAct and then washed out the TransAct before electroporation. 72 hours after TransAct activation of PBMCs, CD3 / CD28 beads were magnetically removed, and T cells were transfected with 5 μg and 100 pM / L RNA duplex using the Neon transfection system (Thermo Fisher, 10 μL tip). 4 × 10 5 The cells are then treated with the RNP complex and the CAR-encoding The cells were mixed with 2.5 μg of PCR fragment. After electroporation, the cells were diluted into culture medium and incubated at 37°C and 7% CO2. The edited cells were then cultured under standard conditions (37°C and ∼1 × 10 per ml). 6 Necessary for maintaining cell density The cells were cultured in T cell expansion medium (expanded in T cell expansion medium, replenishing the medium every 2-3 days to allow for cell proliferation).

[0272]

[0352] TCR alpha KO T cells Human primary T cells were transfected with CRISPR-Cas9 RNP complexes containing guide RNA targeting the 5' end of the first exon of TRAC and provided a DNA template for homologous recombination-based knock-in. After knockout of TCR alpha, cells were grown in complete Optimizer medium containing 300 IU / ml IL-2 and no anti-CD3 / 28. After transfection, cells were grown in culture for 2 weeks.

[0273]

[0353] Flow cytometry assay of CAR-T transduction LILRB4 was detected by binding to the LILRB4-Fc fusion protein (ACRObiosystems CDK-H5259) and anti-Fc antibody (Biolegend B278652). CAR-T were identified. Successful knockout (KO) of the endogenous TCR alpha (TRAC) locus was measured by anti-CD3 staining (anti-CD3 PE, BD 555333).

[0274]

[0354] Antigen stimulation assay : 1 μg / ml recombinant control antigen or LILRB4 antigen in PBS buffer was coated onto a 96-well plate overnight. The plate was washed twice with PBS buffer. 1×10 5 CAR-T cells were added to each well and incubated for 72 hours. Cell culture supernatants were collected for cytokine release measurement by minex assay (R&D Systems FCSTM-18).

[0275]

[0355] CAR-T cell-mediated cytotoxicity assay :CHO K1 RB4 cells With different densities (6X10 4 , 2X10 4 or 7X10 3 ) Planted for 12 hours. 1X10 5 Cytotoxicity was measured by adding CAR-T cells, removing the supernatant, and washing the plate twice with PBS. The total viable adherent CHO K1 RB4 cells were measured using a Promega CTG2.0 luminescence kit, and the % cytotoxicity was calculated by dividing the luminescence signal of each condition by that of a T cell control activated at the same E:T ratio.

[0276]

[0356] Phase 1 designDuring Part 1A, a monotherapy expansion phase (single dose on day 1), patients will be enrolled in sequential cohorts of increasing doses of anti-LILRB4 monotherapy. The objective of Part 1A is to determine the maximum tolerated dose (MTD) of anti-LILRB4 monotherapy (MTD1). DLTs at MTD1 will be assessed during the first 14 days of treatment (before the first dose of anti-LILRB4 in combination with azacitidine), i.e., during the first dose interval of anti-LILRB4. The initial dose expansion will begin with an accelerated titration setting, followed by a standard expansion phase using a 3+3 design. Part 1 will include both relapsed and / or refractory myelomonocytic (M4) and monocytic / monoblastic (M5) AML patients and chronic myelomonocytic leukemia (CMML) patients, as described above. This 2-week monotherapy lead-in ("window") of anti-LILRB4 will allow for the study of the impact of a monoclonal antibody specifically targeting LILRB4 as monotherapy. In Part 1B (starting on day 15), patients without DLT in Part 1A received standard doses of azacitidine (75 mg / m2 subcutaneously, 2 The same dose of anti-LILRB4 as administered in Part 1A will be administered in combination with azacitidine (7 days every 8 days). The MTD of anti-LILRB4 in combination with azacitidine (MTD2) will be determined with a 28-day DLT window, consisting of 14 days of monotherapy and 14 days of combination treatment. The overall DLT period for Part 1 (Parts 1A and 1B combined) will be 28 days. Subsequent cycles will be anti-LILRB4 in combination with azacitidine.

[0277] Example 2

[0357] The present inventors previously identified a rabbit anti-LILRB4 antibody, designated B4-193, that has high binding affinity for human LILRB4 and is capable of inhibiting cancer development in a xenograft AML mouse model (see U.S. Provisional Application No. 62 / 730,715, the disclosure of which is incorporated herein by reference in its entirety).

[0278]

[0358] The present inventors have generated a humanized anti-LILRB4 antibody containing the same CDRs as B4-193. The humanized anti-LILRB4 antibody, designated H7K3, has the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 3.

[0279]

[0359] Computational analysis of H7K3 indicated that the antibody has a potential oxidation site at amino acid residue W in heavy chain CDR3 and a potential deamidation site at amino acid residue NS in light chain CDR1, which could potentially reduce the stability of the antibody.

[0280]

[0360] We then evaluated the stability of anti-H7K3 in PBS or formulation buffer at 40° C., the results of which are summarized in Tables 2 and 3. The results show that anti-H7K3 is unstable.

[0281]

[0361] Table 2. H7K3 stability in PBS

[0282] [Table 2]

[0283]

[0362] Table 3. Stability of H7K3 in formulation buffer

[0284] [Table 3]

[0285] Example 3

[0363] To re-engineer H7K3 to correct its deamination and oxidation propensity, we generated a series of mutants with mutations at potential oxidation and deamination sites. The sequences of the H7K3 mutants are summarized in Table 4.

[0286]

[0364] Table 4. H7K3 mutants

[0287] [Table 4]

[0288]

[0365] We tested the binding affinity of H7K3 mutants using CHO stable cells expressing human and cynomolgus monkey (cyno) LILRB4 through FACS. The results are summarized in Table 5 below.

[0289]

[0366] Table 5. Binding affinities of H7K3 mutants

[0290] [Table 5]

[0291]

[0367] We further tested the stability of H7K3 mutants. As summarized in Table 6 below, oxidized species were observed in H7K3 after 4 weeks of incubation at 40°C, compared to the control sample without incubation at 40°C (confirmed by mapping results). No obvious oxidation was observed in H7K3m5 after 4 weeks of incubation at 40°C. It can be concluded that H7K3m5 is more stable than H7K3.

[0292]

[0368] Table 6. Comparison of deglycosylated reduced mass (DRM) results for H7K3 and H7K3m5

[0293] [Table 6]

[0294]

[0369] We further tested the stability of the H7K3 mutants. Compared to the control sample not incubated at 40°C, we observed a decrease of approximately 50% in the main peak of H7K3 after 4 weeks of incubation at 40°C. Compared to the control, we observed a decrease of approximately 16% in the main peak of H7K3m5 after 4 weeks of incubation at 40°C. We can conclude that H7K3m5 is more stable than H7K3.

[0295]

[0370] Table 7. Comparison of icIEF results for H7K3 and H7K3m5

[0296] [Table 7]

[0297]

[0371] No significant oxidation of H7K3m5 was detected under light exposure. Consistency between DRM and peptide mapping results was obtained for light-exposed H7K3m5. The results indicated that H7K3m5 exhibits acceptable stability under oxidative conditions (light).

[0298]

[0372] For H7K3m5 after 4 weeks of incubation at 40°C, the DRM results showed no significant oxidation. H7K3m5 appears to be more stable than H7K3 in terms of the DRM and icIEF results. icIEF can monitor oxidation and deamidation even at low ratios.

[0299] Example 4

[0373] This example illustrates the biological function of H7K3m5.

[0374] We first evaluated whether H7K3m5 binds to LILRB4 expressed on the cell surface. As shown in Figure 3, H7K3m5 recognized LILRB4 expressed on THP-1-GFP cells. As shown in Figure 4, H7K3m5 can induce ADCC activity against THP-1 cells in vitro in the presence of PBMCs. Notably, afucosylated H7K3m5 exhibited enhanced cell killing compared to wild-type H7K3m5, with an EC50 increase of more than 100-fold. As shown in Figure 5, H7K3m5 also binds to LILRB4 expressed on human monocytes and plasmacytoid dendritic cells (pDCs).

[0300]

[0375] As shown in Figure 6, LILRB4 expression in human monocytes can be upregulated by IL-10 and IFNα treatment. Human monocytes were isolated from human PBMCs by negative selection and stimulated with 50 ng / ml IL-10 plus 1500 U IFNα for 24 hours in RPMI medium containing 10% FBS and 1x L-glutamine. Cells were stained with CD14-PE and 2 μg / ml anti-LILRB4-APC.

[0301]

[0376] On the other hand, human monocytes treated with LPS downregulated LILRB4 expression and increased uPAR expression levels (FIG. 7).

[0377] As shown in Figures 8A and 8B, LILRB4 is expressed on in vitro differentiated human macrophages. The copy number of LILRB4 on in vitro monocyte-derived human macrophages is very high, approaching 150,000 copies / cell.

[0302]

[0378] LILRB4 expression is greatly increased in myeloid-derived suppressor cells (MDSCs), as shown in Figure 9. The copy number of LILRB4 on in vitro differentiated MDSCs can be as high as 200,000 copies / cell.

[0303]

[0379] As shown in Figures 10A-10B, LILRB4 is also highly expressed on human monocyte-derived dendritic cells (DCs). LILRB4 levels are found in the following order, from highest to lowest: tolerogenic DCs > activated DCs > immature DCs.

[0304]

[0380] Table 8. Comparison of LILRB4 copy numbers on different types of human primary cells and AML cell lines

[0305] [Table 8]

[0306]

[0381] We then compared LILRB4 mRNA expression levels between solid tumor samples with high and low signals for macrophage infiltration. Using computational biology and statistical approaches, we analyzed RNA sequencing from the TCGA database to identify samples within each tumor type that exhibited coordinated upregulated expression of multiple transcripts primarily expressed by tumor-associated macrophages (collectively defining a macrophage gene expression "signature"). Samples exhibiting a high signal for the macrophage gene expression "signature" were classified as highly infiltrated by tumor-associated macrophages. The expression levels of LILRB4 transcripts were then compared between these tumor samples and the remaining samples lacking the macrophage gene expression "signature" (grouped as low-macrophage infiltration samples). As shown in Figure 11, higher LILRB4 mRNA expression levels correlate with macrophage infiltration.

[0307]

[0382] We next determined the cell types bound by the LILRB4 antibody (H7K3m5) in solid tumors. Tissue samples from solid tumors were dissociated into single cells using mechanical methods and PBS-10 mM EDTA. In some cases, peripheral blood samples were also obtained from the same donors as the tumor tissue samples and processed for flow cytometry analysis using standard methods. The resulting cells were incubated with a cocktail of antibodies against H7K3m5 and myeloid cell markers at 4°C using standard methods, and the stained samples were analyzed by flow cytometry. Gating in tumor samples was performed as follows: Myeloid dendritic cells (DCs, CD11b + CD15 - CD14 - HLA-DR + CD11c + ), HLA-DR high tumor-associated macrophages (TAM) / monocytes (CD11b + CD15 - CD14 + HLA-DR + ), HLA-DR low TAM / monocytic myeloid-derived suppressor cells (M-MDSC, CD11b + CD15 - CD14 + HLA-DR - ), PMN-MDSC (CD11b + CD15 + CD14 - The gating procedure for peripheral blood was as follows: Bone marrow DC (CD11b + CD14 - CD11c + ), monocytes (CD11b + CD14 + HL A-DR high), M-MDSC (CD11b + CD14 + HLA-DR low), PMN-MDSC (CD11b + CD15 + CD14 - HLA-DR - Lox-1 + ) As shown in Figures 12A-12B As shown, the results of these experiments demonstrate that H7K3m5 binds to monocytic (but not granulocytic) myeloid cells in the tumor microenvironment and in the periphery, a finding consistent with the expression pattern of LILRB4, which is restricted to myeloid cells of the monocytic lineage.

[0308]

[0383] Although LILRB4 is expressed on primary normal monocytes, binding of H7K3m5 did not result in monocyte killing via ADCC (Figures 13A-13D). With afucosylated H7K3m5, normal monocyte killing via ADCC was observed in 25-50% of PBMC donors tested (Figures 14A-14D and Figures 15C-15D). Furthermore, as shown in Figures 15A-15B, both afucosylated and wild-type H7K3m5 resulted in the killing of pDCs via autologous ADCC. On the other hand, monocytes could only be killed by afucosylated H7K3m5, depending on the donor (Figures 15C-15D).

[0309]

[0384] As shown in Figures 16A and 16B, anti-LILRB4 antibodies, such as H7K3m5, depleted in vitro-derived (tumor cell-conditioned) myeloid-derived suppressor cells (MDSCs) through ADCC, which is a potential mechanism by which anti-LILRB4 may act in the treatment of solid tumors.

[0310]

[0385] In addition to ADCC, wild-type H7K3m5 also exhibited cell killing through ADCP against THP-1 cells (FIGS. 17A-17B).

[0386] Anti-LILRB4 antibodies also enhance T cell-mediated cytotoxicity against tumor cells. As shown in Figure 18, anti-LILRB4 can induce T cell cytotoxicity against the AML cell line THP-1-GFP, whereas no T cell cytotoxicity was observed with an isotype control antibody. H7K3m5 treatment induced naive T cells to kill THP-1 AML cells in a dose-dependent manner. The average EC50 was 0.208 ± 0.125 nM (31.2 ± 18.8 ng / mL) (n = 3).

[0311]

[0387] Cytokine measurements from the supernatants of cytotoxicity assay samples using a multiplex Luminex assay showed a dose-dependent increase in IFNγ and TNFα in H7K3m5-treated samples compared to control samples (Figure 19). The cytokine profile changes were consistent with the increased cell-killing activity measured by THP-1 cell quantification (Figure 18). In addition to increased TNFα and IFNγ levels, which are likely produced by activated cytotoxic T cells, a dose-dependent increase in MCP-1 and IL-1Rα, known to be produced by monocytes and macrophages, was also observed, likely from activated THP-1 cells. IL-6, IL-8, and IL-10 levels also increased in response to H7K3m5 treatment compared to controls. These cytokines likely originate from both activated T cells and THP-1 cells.

[0312]

[0388] T cell activation markers were assessed by flow cytometry (see Figure 20). A two-fold higher percentage of CD69-expressing T cells was observed with H7K3m5 treatment (Figure 20A). Here, 4.7% of CD4+ T cells and 23.6% of CD8+ T cells treated with H7K3m5 expressed CD69, whereas only 2.6% of CD4+ T cells and 12.2% of CD8+ T cells treated with isotype control expressed CD69. When T cells were cultured alone, H7K3m5 treatment did not increase the number of CD69+ T cells. H7K3m5 had only a modest effect on the expansion of CD25+ T cells (Figure 20B).

[0313]

[0389] Cytokine production in cocultured T cells and THP-1 AML cells was also assessed by intracellular staining for flow cytometry. Compared to the isotype control, H7K3m5 treatment increased IFNγ- and TNFα-producing CD4+ and CD8+ T cells by 2.7- and 34-fold, respectively (Figure 20C). IFNγ-producing CD4+ and CD8+ T cells increased 2- and 4-fold with H7K3m5 treatment, and IFNγ was not produced by THP-1 cells (Figure 20D). In contrast, TNFα-producing THP-1 cells increased 3-fold with H7K3m5 treatment when cocultured with T cells (Figure 18E). H7K3m5 treatment of THP-1 cells alone, without T cells, did not result in an increase in TNFα-producing THP-1 cells. These data showed that IFNγ was produced exclusively by activated T cells, and TNFα was produced by both activated T cells and THP-1 AML cells.

[0314]

[0390] Taken together, H7K3m5 enhanced antigen presentation and IFNγ and TNFα production in THP-1 cells and activated T cells.

[0391] The proposed mechanism for enhanced T cell-mediated cytotoxicity involves blocking LILRB4 inhibitory receptor signaling with anti-LILRB4 blocking antibodies, reducing arginase production by THP-1 cells, cytokine production by THP-1 cells (Figures 19 and 20), and enhancing THP-1 antigen-presenting ability (Figure 21). As shown in Figure 21, THP-1 AML cells and naive T cells were cocultured at a 4:1 E:T ratio in the presence of 1.5 μg / mL (10 nM) H7K3m5 or human IgG1 at 37°C for 48 hours. T cell and THP-1 cell activation markers were assessed by flow cytometry (Figure 21). A mild upregulation of the costimulatory molecule CD83 was detected on THP-1 AML cells in all experiments. Each experiment used pan-T cells from a different donor (n = 4). Moderate upregulation of the MHC class II molecule HLADR (n = 3), MHC class I molecule (n = 1), and another costimulatory molecule, CD86 (n = 2), was also detected on THP-1 cells. These data indicate that H7K3m5 treatment enhances the antigen-presenting activity of THP-1 cells. The T cell activation marker CD69 was moderately induced by H7K3m5 treatment (n = 1), which is consistent with increased cytotoxic activity. Taken together, enhanced antigen presentation of THP-1 cells may induce T cell activation and lead to enhanced cytotoxic activity.

[0315]

[0392] When THP-1 cells were cocultured with naive T cells in the presence of H7K3m5, H7K3m5 activated T cell cytotoxicity against THP-1 cells (Figure 20). To further understand the changes in this activity for both cell types, naive T cells and THP-1 AML cells were cocultured at an E:T ratio of 8:1 in the presence of 3 μg / ml (20 nM) H7K3m5 or human IgG1 for 48 hours at 37°C. THP-1 activation was assessed by flow cytometry (Figure 22). Expression of HLA class I (A, B, C) and class II (HLA-DR) and costimulatory molecules CD40, CD86, CD80, and CD83 was induced by H7K3m5 treatment, indicating that H7K3m5 enhances the antigen-presenting function of THP-1 cells. In contrast, expression of the inhibitory molecules CD205 and LILRB4 was not reduced by H7K3m5. Interestingly and unexpectedly, H7K3m5 treatment also increased the expression of uPAR, a downstream NF-κB target of LILRB4 that is highly expressed in monocytic AML cells and is known to promote cancer invasion, metastasis, survival, and angiogenesis ( Deng et al., 2018 ).

[0316]

[0393] When THP-1 AML cells were co-cultured with naive T cells, T cell activity was significantly increased. No activation or cytotoxicity was observed. This is likely due to impaired antigen-presenting function of THP-1 AML cells with high LILRB4 expression. When H7K3m5 was added to cocultured THP-1 and naive T cells, enhanced antigen-presenting function of THP-1 cells was experimentally observed, as evidenced by increased HLA-DR and CD38 expression. Corresponding activation of naive T cells was observed, particularly cytotoxic activation against THP-1 cells. This was demonstrated by increased secretion of cytokines (i.e., TNFα and IFNγ) in the tissue culture medium. These data indicate that H7K3m5 can activate T cell cytotoxicity against THP-1 AML cells.

[0317]

[0394] H7K3m5-mediated cytotoxicity of normal monocytes by naive T cells was also evaluated in this in vitro system, but no killing of normal monocytes was observed (data not shown). The LILRB4 density on normal monocytes was 10-fold lower than that on THP-1 AML cells. More importantly, the antigen-presenting capacity of normal monocytes is expected to be significantly lower than that of tumor AML cells due to the lack of tumor-associated antigens. Although we found that patient AML cells could express similar or higher levels of LILRB4 than those on normal monocytes, it is plausible that AML blasts are phenotypically more THP-1 cell-like and can be killed by activated T cells when treated with H7K3m5.

[0318]

[0395] We then tested the effect of H7K3m5 on monocyte-derived dendritic cells (Mo-DCs). Classical monocytes were isolated from healthy donor PBMCs and differentiated into immature dendritic cells (Mo-DCs) in DC medium (StemXVivo, 50 μg / mL gentamicin, 50 ng / mL GM-CSF, and 35 ng / mL IL-4) for 6 days. The immature monocyte-derived dendritic cells (Mo-DCs) were then incubated with antibodies (100 nM) in the presence of 100 ng / mL LPS (a TLR4 agonist) to induce dendritic cell maturation and activation. After 2 days, the cells were analyzed by flow cytometry for the expression of cell surface markers. As shown in Figure 24, increased expression of HLA-DR and the costimulatory molecule CD86, and decreased expression of the tolerogenic marker CD209, indicate that H7K3m5 enhances the antigen-presenting and pro-inflammatory capacity of Mo-DCs in response to TLR signaling.

[0319]

[0396] Immature dendritic cells (Mo-DCs) were differentiated from monocytes isolated from PBMCs of healthy donors in DC medium (StemXVivo, 50 μg / mL gentamicin, 100 ng / mL GM-CSF, and 35 ng / mL IL-4) for 5 days. On day 5, Mo-DCs were supplemented with fresh DC medium and treated with 30 μg / mL H7K3m5 or its isotype control in the absence or presence of 5 μg / mL CD40 ligand for an additional 2 days. On day 7, T cells were isolated from PBMCs of a healthy, unrelated donor and suspended in fresh medium containing a cytokine cocktail and 30 μg / mL H7K3m5. Cultures of T cells alone and Mo-DCs alone were included as controls. At the end of the 4-day period, IFN-γ and IL-12 levels in the culture supernatant were measured by ELISA, while the cell surface phenotype of Mo-DCs was analyzed by flow cytometry. The experimental results illustrated in Figures 24-27 show that the pro-inflammatory effect of H7K3m5 on Mo-DCs was more pronounced if the Mo-DCs had been matured with CD40 ligand.

[0320] Example 5

[0397] This example illustrates the generation of a LILRB4 / CD3 bispecific antibody based on the heavy and light chain variable domain sequences of H7K3m5, as well as the antibody disclosed in WO2019057099. Heavy chain heterodimerization was controlled by knob and hole mutations (Merchant et al. Nature Biotech 1998, 16, 677-681) stabilized by engineered disulfide bonds (Carter J Immunol Methods 2001, 248, 7-15). Light chain C kappa or heavy chain Correct light chain pairing was controlled by replacing CH1 with the human T cell reporter alpha (TRAC) and beta (TRBC) constant domains as previously described (WO2019057122A1). Mutations were also introduced into the human IgG1 constant domain to reduce effector function, improve stability, and increase productivity in CHO (Alegre et al. Transplantation 1994, 57 1537-43 and Hu et al. Biotechnol Prog 2017, 33 786-794).

[0321]

[0398] Expression and purification of LILRB4 / CD3 bispecific antibodies

[0399] Figure 28A shows a schematic diagram of six first-generation LILRB4 / CD3 bispecific antibodies in 1+1 or 2+1 configurations. Bispecific antibodies in the 1+1 configuration (4-3ab and 4ab-3) were engineered to bind to a single copy of CD3 epsilon and LILRB4. Bispecific antibodies in the 2+1 configuration (44-4ab, 4ab4ab-3, 43ab-4, and 4ab3-4ab) were engineered to bind to a single copy of CD3 epsilon and two copies of LILRB4. The 44-3ab and 4ab4ab-3 configurations have both LILRB4 binding moieties in tandem on one arm of the bispecific. The 43ab-4 and 4ab3-4ab configurations have LILRB4 binding moieties on both arms of the bispecific. The polypeptide chains of each first-generation bispecific antibody and their amino acid sequences are listed in Table 9.

[0322]

[0400] Table 9. Sequences of first generation LILRB4 / CD3 bispecific antibodies

[0323] [Table 9]

[0324]

[0401] To generate first-generation bispecific antibodies, DNA encoding the first-generation bispecific antibodies was cloned into a mammalian expression vector after gene synthesis. The bispecific antibodies were then expressed by transiently transfecting the appropriate mixture of vectors into Expi293 cells and expressing at a 100 mL scale. All samples were first purified from the supernatant by Protein A affinity chromatography. Bispecific antibodies 1-3 were further purified by size exclusion chromatography (SEC), while bispecific antibodies 4-6 were purified by anion exchange chromatography (AEX). All samples were analyzed for purity by SDS-PAGE and analytical SEC. The results are shown in Table 10. Aliquots were deglycosylated with PNGase F (MEDNA Bio M3103) and purified using an Acquity UPLC Protein BEH SEC column coupled to a Xevo G2-XS QTOF. The compounds were characterized by mass spectrometry using a utility UPLC. The results are shown in Table 11.

[0325]

[0402] Table 10. Yield and purity of first-generation bispecific antibodies

[0326] [Table 10]

[0327]

[0403] Table 11. Mass spectrometry data for first generation bispecific antibodies

[0328] [Table 11]

[0329]

[0404] To improve homogeneity and manufacturability, second-generation bispecific antibodies were designed. Specifically, this was done by mutating S91A in the TCRa domain to remove the O-glycan modification or by creating a Q1E mutation to prevent N-terminal pyroQ formation. The polypeptide chains and their amino acid sequences of each first-generation bispecific antibody are listed in Table 12. DNA encoding the second-generation bispecific antibodies and controls was cloned into mammalian expression vectors using gene synthesis or standard molecular biology protocols starting from the first-generation bispecific antibody. The bispecific antibodies were then expressed using the appropriate vectors transiently transfected into CHO-K1 cells and expressed at a 1 L scale for 14 days using a fed-batch protocol. The bispecific antibodies were purified from the supernatants from all samples using a Protein A column and polished by SEC. All samples were analyzed for purity by SDS-PAGE and analytical SEC. The results are shown in Table 13. An aliquot of the sample was deglycosylated and characterized by mass spectrometry, which showed that the sample was devoid of first generation bispecific antibody impurities (see Table 14).

[0330]

[0405] Table 12. Sequences of second-generation LILRB4 / CD3 bispecific antibodies

[0331] [Table 12]

[0332]

[0406] Table 13. Yield and purity of second-generation bispecific antibodies

[0333] [Table 13]

[0334]

[0407] Table 14. Mass spectrometry data of second-generation bispecific antibodies

[0335] [Table 14]

[0336]

[0408] In vitro cytotoxicity assay

[0409] A FACS-based approach was used to determine the ability of LILRB4 / CD3 bispecific antibodies to mediate target cell killing by T cells. In vitro cytotoxicity of human AMP cell lines THP-1 and MV-4-11 cells: Human AMP cell lines THP-1 and MV-4-11 cells were engineered to express green fluorescent protein (GFP). Human buffy coats were obtained from healthy donors collected by the Stanford Blood Center. Human peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats by Ficoll Paque Plus (GE Healthcare catalog number 17-1440-03) density gradient cell separation. Pan T cells were further isolated from the PBMCs using a human pan T cell isolation kit (Miltenyi Biotec catalog number 130-096-535). 5x10 cells were cultured at a 5:1 ratio. 5 Freshly isolated human pan-T cells were used as effector cells. and 1x10 5 THP-1-GFP cells were used as target cells. MV4 In the -11 cell killing assay, 9x10 5 Freshly isolated human pan-T cells used as vector cells, and 1x10 5 MV4-11-GFP as target cells Human pan T cells, THP-1-GFP or MV4-11-GFP cells, and increasing concentrations of IO-202 or isotype control human IgG1 (BioXcell catalog no. BE0297) were mixed in a total volume of 200 μL in RPMI 1640 (Gibco catalog no. 61870-036) + 10% heat-inactivated fetal bovine serum (FBS; Gibco catalog no. 10082-147) in a U-shaped 96-well plate and incubated at 37°C for 48 hours. At the end of the incubation, 7-aminoactinomycin D (7-AAD; BD Pharmingen catalog no. 559925) was added to the cells, and 100 μL of cells were acquired by FACS Celesta, and the percentage of GFP-positive cells was measured. Flow cytometry data were analyzed using Flowjo software (Flowjo LLC), and cytotoxicity was calculated as follows: Percent cytotoxicity = 100 - ([T / NT] x 100), where T and NT are the percentages of GFP treated with or without test antibody, respectively. + The percentage of cells was calculated as:

[0337]

[0410] Autologous killing of normal human monocytes: 1x10 6 PBMCs and increasing concentrations of The LILRB4 / CD3 bispecific antibody or control was mixed in RPMI + 10% heat-inactivated FBS and 50 ng / ml IL-2 (R&D systems catalog no. 202-IL / CF) in a total volume of 200 μL in a U-shaped 96-well plate and incubated at 37°C for 48 hours. PBMCs incubated with increasing concentrations of rituximab (Biogen / Genentech) served as assay controls. After incubation, cells were washed and incubated with 5 μL of Fc receptor blocker (10 mg / mL human immunoglobulin G [IgG], Sigma Aldrich catalog no. I4506) for 10 minutes at room temperature, followed by incubation with 100 μL of fluorescently conjugated CD14 (clone M5E2, catalog no. 555397) and CD19 (clone HIB19, catalog no. 555415) from BD Biosciences for 30 minutes on ice. Monocytes were identified as CD14 positive cells, while B cells were identified as CD19 positive cells.

[0338]

[0411] As shown in Figures 29A-29B, similar binding affinity trends were observed between monocytes and THP-1 cells across different anti-LILRB4 monospecific and bispecific antibodies. As shown in Figures 30A-30B, T cell-mediated cytotoxicity of bispecific LILRB4 / CD3 antibodies against monocytes and THP-1-luc-GFP cells was observed. As shown in Figures 31A-31B, autocytic killing of monocytes by LILRB4 / CD3 bispecific antibodies (Figure 31A) and autocytic killing of B cells by Rituxan (Figure 31B) as a control was observed.

[0339]

[0412] Cell kill curve generation and EC2 were performed using Prism GraphPad software using nonlinear sigmoidal dose-response curve fitting. 50 Calculations were performed. Mean ± SD was calculated by Excel. Results are reported in Tables 15 and 16.

[0340]

[0413] Table 15. T cell cytotoxicity of first generation bispecific antibodies.

[0341] [Table 15]

[0342] * Approximate EC 50 ** The EC50 cannot be determined precisely; 4-3ab, 4ab-3, and 44-3ab have weaker potency than 4ab4ab-3, 43ab-4, and 4ab3-4ab.

[0343]

[0414] Table 16. T cell cytotoxicity of second generation bispecific antibodies.

[0344] [Table 16]

[0345]

[0415] Surface plasmon resonance (Biacore)

[0416] The binding affinity of first-generation bispecific antibodies to recombinant CD3e-CD3d heterodimer protein (Acro Biosystems) or LILRB4 recombinant protein (Sino Biological) was measured by surface plasmon resonance using a Biacore 8K instrument. Briefly, CD3e-CD3d or LILRB4 proteins were immobilized on a CM5 chip with EDC and NHS according to standard protocols. The bispecific antibody analytes were then applied to six concentrations (1.25, 2.5, 5, 10, 20, and 40 nM) or seven concentrations (20, 20 nM) for LILRB4 and CD3e-CD3d, respectively, in 1xHBX-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4). The chip was regenerated using 10 mM glycine pH 1.5 as the regeneration buffer. Sensograms of the reference channel Fc1 and the buffer channel were subtracted from the test sensograms, and the experimental data were fitted by a 1:1 binding model. For affinities, see Table 17.

[0346]

[0417] Table 17. Affinity of first-generation bispecific antibodies to recombinant proteins and cells

[0347] [Table 17]

[0348]

[0418] Bio-Layer Interferometry (Gator Bio)

[0419] The binding affinities of second-generation bispecific antibodies were measured by Bio-Layer Interferometry (BLI) using a Gator Bio instrument against recombinant human CD3e (Acro, Cat. No. CDE-H5223), cyno CD3e (Acro, Cat. No. CDE-C5226), human LILRB4 (Sino Biological, Cat. No. 16742-H08H), and cyno LILRB4 (Acro, Cat. No. CDK-C5227). Bispecific antibodies were captured using a 5 μg / mL immobilized anti-human Fc antibody (HFC) probe (Gator Bio, Cat. No. PL168-160003). Human CD3e and cyno LILRB4 were captured, respectively. Binding affinities were measured for CD3e, human LILRB4, and cyno LILRB4 using six concentrations (0.6-159 nM, 0.7-164 nM, 0.8-200 nM, and 2.5-595 nM). Binding affinity constants were determined using a 1:1 fit model (Global Fit) in Gator data analysis software 1.6.1.1203, and KD was calculated using the ratio Kdis / Kon. See Table 18 for affinities.

[0349]

[0420] Table 18. Affinity of second-generation bispecific antibodies to recombinant proteins and cells

[0350] [Table 18]

[0351]

[0421] Flow cytometry

[0422] Binding of first- and second-generation bispecific antibodies to CD3 or LILRB4 was measured by FACS on Jurkat or THP-1 cells. To measure bispecific antibody binding to THP-1 cells, THP-1 cells were first incubated with 10 μg of human Fc blocker (BD Pharmingen Catalog No. 564220) per million cells for 10 minutes at room temperature, followed by incubation with serially diluted bispecific antibodies on ice for 30 minutes. Cells were washed twice with BSA staining buffer (BD Pharmingen Catalog No. 554657) and then incubated with 5 μg / mL of secondary Alexa647-conjugated anti-human IgG Fc monoclonal antibody (Biolegend Catalog No. 409306) for 30 minutes on ice. After the final wash, 7-AAD was applied to exclude dead cells. To measure binding of first-generation bispecific antibodies to Jurkat cells, each bispecific antibody was diluted in buffer containing 1% BSA (5-fold dilution series, 400 nM being the highest concentration) and incubated with the cells at 4°C for 30 minutes. The bispecific antibodies were then stained with 5 μg / mL Alexa647 anti-human IgG Fc for 0.5 hours at 4°C and then analyzed. Although binding to CD3 was clearly observed on the cells compared to the isotype control, the EC50 could not be calculated or could only be approximated because the binding curve did not reach a plateau. See Tables 17 and 18 for EC50 values.

[0352] Example 6

[0423] This example illustrates the generation of CAR-T cells expressing CAR proteins based on the heavy and light chain variable region sequences of H7K3m5.

[0353]

[0424] Using flow cytometry, two different configurations of single-chain Fv (scFv) from anti-LILRB4 monoclonal antibody H7K3m5 were tested for binding to human primary monocytes and human leukemia cells THP-1. As shown in Figure 32, the VlVh configuration maintains the binding affinity of H7K3m5, while the VhVl configuration loses some binding affinity. Therefore, VlVh is selected for CAR construction.

[0354]

[0425] As illustrated in Figure 33, the DNA construct for expressing the anti-LILRB4 CAR protein is a second-generation CAR construct containing a CD28 or 4-1BB costimulatory domain, including a CD3 zeta activation domain. The scFv is derived from the anti-LILRB4 monoclonal antibody H7K3m5. The 5' and 3' homology arms are located upstream and downstream of the Cas9 DNA cleavage site in the TRAC gene (based on the gRNA design). The promoter and leader peptide are essential elements for gene expression and cell extravasation. Here, we used the JeT promoter to control scFv expression (Eyquem J et al. Nature (2017) 543:113-117). The SV40 poly(A) tail was included to improve transcript stability and translation.

[0355]

[0426] Human primary T cells were transfected with CRISPR-Cas9 RNP complexes containing guide RNA targeting the 5' end of the first exon of TRAC and supplied with a DNA construct for homologous recombination-based knock-in. After knockout of TCR alpha, cells were grown in complete Optimizer medium containing 300 IU / ml of IL-2 and no anti-CD3 / 28. After transfection, cells were grown in culture for 2 weeks. Anti-LILRB4 CAR-T cells were identified by binding to an LILRB4-Fc fusion protein (ACRObiosystems CDK-H5259) and an anti-Fc antibody (Biolegend B278652). Successful knockout (KO) of the endogenous TCR alpha (TRAC) locus was measured by anti-CD3 staining (anti-CD3 PE, BD 555333). Efficient generation of anti-LILRB4 CAR-T cells was confirmed, as shown in Figure 34.

[0356]

[0427] As shown in Figure 35, TCR alpha (TRAC) inactivated T cells (KO) and anti-LILRB4 CAR (or control CAR) knock-in T cells undergo cell proliferation and expansion in vitro. Anti-LILRB4 CAR-T cells had a significantly higher fold expansion compared to control CAR-T cells.

[0357]

[0428] To test the antigen-dependent activation of CAR-T cultures, 1 μg / ml recombinant control antigen or LILRB4 antigen was coated onto a 96-well plate in PBS buffer overnight. The plate was washed twice with PBS buffer. 1×10 5 CAR-T cells were added to each well and incubated for 72 hours. The cells were incubated for 1 h. Cell culture supernatants were collected for cytokine release measurement by Luminex assay. As shown in Figures 36A-36H, release of cytokines IL-8, IFNγ, IL-10, IL-1ra, IL-2, IL-6, TNFα, and IL1 alpha by anti-LILRB4 CAR-T cells is dependent on the presence of LILRB4.

[0358]

[0429] Figures 37A-37C show the characterization of CAR-T cells after 2 weeks of expansion. Frozen CAR-T cells stored in liquid nitrogen were thawed and maintained in culture for 2-3 days before flow cytometry analysis. The antibodies used were anti-CD8 APC Cy7 (BD561945), anti-PD1 PE (BD560908), and anti-TIM3 BV421 (BD565562). LILRB4-Fc fusion protein (ACRObiosystems) was used. LILRB4 CAR-T cells were identified by binding to anti-LILRB4_CD28 (CDK-H5259) and anti-Fc antibody (Biolegend B278652). As shown in Figure 37A, expression of anti-LILRB4_CD28 slightly reduced the percentage of CD8+ T cells (from 61.8% to 41.8%) and did not substantially change the expression of PD-1 and TIM3 on the cells. As shown in Figure 37B, expression of anti-LILRB4_4-1BB reduced the percentage of CD8+ T cells from 48.7% to 3.61% and increased PD-1 expression. As shown in Figure 37C, expression of knockout (KO) TCR alpha at the TRAC locus, where the CAR construct is inserted, did not substantially change the percentage of CD8+ T cells or the expression of PD-1 or TIM3.

[0359]

[0430] To test the cytotoxicity of anti-LILRB4 CAR-T cells, CHO K1 RB4 cells at different densities (6x10 4 , 2X10 4 or 7X10 3 )12 hours Planting, 1X10 5 Adding CAR-T cells and removing the supernatant CAR-T cells Cytotoxicity was then measured by washing the plates twice with PBS. The total viable adherent CHO K1 RB4 cells were measured by ega CTG2.0 luminescence kit, and the % cytotoxicity was calculated by dividing the luminescence signal of each condition by that of the T cell control activated at the same E:T ratio. As shown in Figure 38, anti-LILRB4 CAR-T cells (44) killed CHO K1 RB4 cells, but anti-CD19 CAR-T cells (94) did not.

[0360] Example 7

[0431] This example presents the design of a Phase 1, first in human clinical trial.

[0432] As shown in Figure 39A, a "window" design involving a 2-week monotherapy introduction ("window") of anti-LILRB4 allows for the study of the impact of a monoclonal antibody that specifically targets LILRB4 as a monotherapy.

[0361]

[0433] During Part 1A, a monotherapy expansion phase (single dose on day 1), patients will be enrolled in sequential cohorts of increasing doses of anti-LILRB4 monotherapy. The objective of Part 1A is to determine the MTD of anti-LILRB4 monotherapy (MTD1). DLTs at MTD1 will be assessed during the first 14 days of treatment (prior to the first dose of anti-LILRB4 in combination with azacitidine), i.e., the first dose interval of anti-LILRB4. The first dose expansion will begin with an accelerated titration setting, followed by a standard expansion phase using a 3+3 design. Part 1 will include both relapsed and / or refractory myelomonocytic (M4) and monocytic / monoblastic (M5) AML patients and chronic myelomonocytic leukemia (CMML) patients.

[0362]

[0434] In Part 1B (starting on Day 15), patients without DLTs in Part 1A will receive azacitidine in combination with standard doses of azacitidine (75 mg / m2 subcutaneously for 7 days every 28 days). Part 1A will receive the same dose of anti-LILRB4 as administered in Part 1A. The MTD of anti-LILRB4 in combination with azacitidine (MTD2) will be determined over a 28-day DLT window, consisting of 14 days of monotherapy and 14 days of combination treatment.

[0363]

[0435] The overall DLT period in Part 1 (Parts 1A and 1B combined) was 28 days, which was easily translatable to 42 days, including the first 14 days for monotherapy DLT and the last 28 days for combination therapy DLT (with the addition of azacitidine).

[0364]

[0436] Subsequent cycles will be anti-LILRB4 in combination with azacytidine.

[0437] As shown in Figures 39B and 39C, once the MTD and / or RP2D for the anti-LILRB4 plus azacitidine combination is identified and approval is granted by the Safety Review Committee (SRC), enrollment into one of two expansion arms (Figures 39B and 39C) will begin in patients with relapsed and / or refractory monoblastic / monocytic leukemia.

[0365]

[0438] As shown in Figure 39B, this study will enroll a monotherapy cohort of anti-LILRB4 in patients with relapsed / refractory AML with monocytic differentiation. Figure 39B also represents a possible design for a first-in-human Phase 1 clinical trial if the designed "window" is not approved.

[0366]

[0439] As shown in Figure 39C, this study will enroll a combination cohort of anti-LILRB4 plus azacitidine in patients with relapsed / refractory AML with monocytic differentiation.

[0367]

[0440] As shown in Figure 39D, this study will enroll combination cohorts of anti-LILRB4 + azacitidine + venetoclax in patients with relapsed / refractory AML with monocytic differentiation and in newly diagnosed AML patients with monocytic differentiation.

[0368]

[0441] All of the compositions and methods disclosed and claimed herein 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 and steps or the order of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. Such similar substitutions 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.

Claims

1. a) a heavy chain variable region comprising a heavy chain complementarity determining region (HC-CDR) 1 having the amino acid sequence of SEQ ID NO: 5, a HC-CDR2 having the amino acid sequence of SEQ ID NO: 6, and a HC-CDR3 having the amino acid sequence of SEQ ID NO: 7; and b) a light chain variable region comprising a light chain complementarity determining region (LC-CDR) 1 having the amino acid sequence of SEQ ID NO: 8 containing a mutation at amino acid residue NS, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 9, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:

10. An isolated anti-LILRB4 antibody or antigen-binding fragment thereof, comprising:

2. The antibody or antigen-binding fragment thereof of claim 1, wherein LC-CDR1 has the amino acid sequence of SEQ ID NO:

28.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region has an amino acid sequence at least about 90% identical to SEQ ID NO: 1; and the light chain variable region has an amino acid sequence at least about 90% identical to SEQ ID NO:

27.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 1 and the light chain variable region has the amino acid sequence of SEQ ID NO:

27.

5. 10. The antibody or antigen-binding fragment thereof of any of the preceding claims, further comprising an immunoglobulin constant region, optionally the constant region of an IgG, or optionally the constant region of a human IgG.

6. 10. The antibody or antigen-binding fragment thereof of any preceding claim, which is humanized.

7. Camelized single domain antibodies, diabodies, scFv, scFv dimers, BsFv, dsFv, (dsFv) 2 , dsFv-dsFv', Fv fragment, F ab, Fab', F(ab') 2 , bispecific antibodies, ds diabodies, nanobodies, do 10. The antibody or antigen-binding fragment thereof of any preceding claim, which is a main antibody or a bivalent antibody.

8. The antibody or antigen-binding fragment thereof of claim 7, which is a bispecific antibody against LILRB4 and CD3.

9. 10. The antibody or antigen-binding fragment thereof of any preceding claim, linked to one or more conjugate moieties.

10. 10. The antibody or antigen-binding fragment thereof of claim 9, wherein the conjugate moiety comprises a clearance modifier, a toxin, a detectable label, a chemotherapeutic agent, a cytokine, or a purification moiety.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any of the preceding claims and a pharmaceutically acceptable carrier.

12. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of claims 1 to 10.

13. A vector comprising the isolated polynucleotide of claim 12.

14. A host cell comprising the vector of claim 13.

15. A method for expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 10, comprising:

15. The method comprising culturing the host cell of claim 14 under conditions in which the vector of claim 13 is expressed.

16. A method for treating or ameliorating the effects of cancer in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any of claims 1 to 10 or the pharmaceutical composition of claim 11.

17. 17. The method of claim 16, wherein the cancer is selected from the group consisting of adrenal gland cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.

18. 17. The method of claim 16, wherein the cancer has an immunosuppressive microenvironment and the antibody or antigen-binding fragment thereof kills at least one myeloid-derived suppressor cell (MDSC).

19. 17. The method of claim 16, wherein the cancer is selected from the group consisting of lymphoma, lymphocytic leukemia, Hodgkin's disease, acute myeloid leukemia (AML), acute lymphocytic / lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia (CML), myelodysplastic syndrome (MDS), myeloproliferative neoplasms, and chronic myelomonocytic leukemia (CMML).

20. The method of any of claims 16 to 19, wherein the subject is a human.

21. The method of any of claims 16 to 20, wherein the antibody or antigen-binding fragment thereof is administered intravenously, intraarterially, intratumorally, or subcutaneously.

22. Topoisomerase inhibitors, anthracycline topoisomerase inhibitors, anthracyclines, daunorubicin, nucleoside metabolic inhibitors, cytarabine, hypomethylating agents, low-dose cytarabine (LDAC), daunorubicin and cytarabine combination, injectable daunorubicin and cytarabine liposome, Vyxeos®, azacitidine, Vidaza®, decitabine, all-trans retinoic acid (ATRA), arsenic, arsenous acid, histamine dihydrochloride, Ceplene®, 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, epacad Stats, BCL-2 inhibitors, venetoclax, Venclexta®, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitors, ibrutinib, IMBRUVICA®, acalabrutinib, CALQUEENCE®, 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, SIR 22. The method of any of claims 16-21, further comprising administering one or more agents selected from the group consisting of a P1α antibody or fusion protein, 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, a plant-derived alkaloid, a hormone therapy agent, a hormone antagonist, an aromatase inhibitor, and a P-glycoprotein inhibitor.

23. A method for treating or ameliorating an autoimmune or inflammatory disease in a subject. The method comprises the step of administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 10, or the pharmaceutical composition of claim 11.

24. Autoimmune or inflammatory diseases include acquired immune deficiency syndrome (AIDS), 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 disease-dermatitis herpetiformis, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold sores, and flu-like illnesses. Cold agglutinin disease, CREST syndrome, Crohn's disease, Degos disease, juvenile dermatomyositis, discoid lupus erythematosus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre 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, 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-body syndrome, systemic lupus erythematosus (SLE), Takayasu's arteritis, temporal arteritis / giant cell arteritis, inflammatory bowel disease (IB) D) The method of claim 23, wherein the agent is selected from the group consisting of ulcerative colitis, Crohn's disease, intestinal mucosal inflammation, colitis-associated wasting disease, uveitis, vitiligo and Wegener's granulomatosis, Alzheimer's disease, asthma, atopic allergy, allergy, 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 infection, and autoimmune diabetes.

25. A method for killing cells in a subject, the method comprising administering to the subject the antibody or antigen-binding fragment thereof of any of claims 1 to 10.

26. 26. The method of claim 25, wherein the cell is a monocyte, a plasmacytoid dendritic cell (pDC), a macrophage, a myeloid-derived suppressor cell (MDSC), a monocyte-derived macrophage, a monocyte-derived dendritic cell, or a cancerous cell.

27. The method of claim 25, wherein the cells have antigen-presenting function.

28. 26. The method of claim 25, wherein the cell has at least 2,000, at least 50,000, at least 100,000, or at least 150,000 copies of the LILRB4 protein expressed on the cell surface.

29. 26. The method of claim 25, wherein the cells are killed through antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP).

30. 26. The method of claim 25, wherein the cells are killed through T cell cytotoxicity.

31. A method for activating T cells, comprising the step of contacting T cells with the antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

32. A method for activating T cells, comprising the step of contacting T cells with the antibody or antigen-binding fragment thereof of any one of claims 1 to 10 in the presence of cancerous cells.

33. 33. The method of claim 32, wherein the cancerous cells have increased antigen-presenting activity.

34. 33. The method of claims 31-32, wherein the T cells are cultured in vitro.

35. 1. A method for detecting cancer cells or cancer stem cells in a sample or subject, comprising: (a) contacting a subject or a sample derived from a subject with the antibody or antigen-binding fragment thereof of any one of claims 1 to 10; and (b) detecting binding of said antibody to cancer cells or cancer stem cells in said subject or sample. The method comprises the steps of:

36. 36. The method of claim 35, wherein the sample is a body fluid or a biopsy.

37. 36. The method of claim 35, wherein the sample is blood, sputum, tears, saliva, mucus, serum, urine, or feces.

38. 36. The method of claim 35, wherein detection comprises immunohistochemistry, flow cytometry or FACS, immunoassay (including ELISA, RIA, etc.) or Western blot.

39. 36. The method of claim 35, further comprising performing steps (a) and (b) a second or further time and determining a change in the level of detection as compared to the first time.

40. 11. Use of the antibody or antigen-binding fragment thereof of any of claims 1 to 10 in the manufacture of a medicament for treating cancer in a subject.

41. A kit useful for detecting LILRB4, comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

42. 1. A chimeric antigen receptor (CAR) protein comprising: a) a heavy chain variable region comprising an HC-CDR1 having the amino acid sequence of SEQ ID NO:5, an HC-CDR2 having the amino acid sequence of SEQ ID NO:6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:7; and b) a light chain variable region comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO: 8 containing a mutation at amino acid residue NS, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 9, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:

10. The CAR protein comprising:

43. The CAR protein of claim 42, wherein LC-CDR1 has the amino acid sequence of SEQ ID NO:

28.

44. 43. The CAR protein of claim 42, wherein the heavy chain variable region has an amino acid sequence at least about 90% identical to SEQ ID NO: 1; and the light chain variable region has an amino acid sequence at least about 90% identical to SEQ ID NO:

27.

45. 43. The CAR protein of claim 42, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 1; and the light chain variable region has the amino acid sequence of SEQ ID NO:

27.

46. 43. The CAR protein of claim 42, comprising a single chain variable fragment (scFv) having an amino acid sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 66 or 68.

47. 5. The method of claim 4, comprising administering to a subject an scFv having an amino acid sequence identical to SEQ ID NO: 66 or 68. 2 CAR protein.

48. 43. The CAR protein of claim 42, further comprising a CD8α transmembrane domain or a CD28 transmembrane domain.

49. The CAR protein of claim 42, further comprising a 4-1BB intracellular costimulatory signaling domain or a CD28 intracellular costimulatory signaling domain.

50. 43. The CAR protein of claim 42, further comprising a CD3ζ intracellular T cell signaling domain.

51. A polynucleotide molecule encoding the CAR protein of any one of claims 42 to 50.

52. 52. The polynucleotide molecule of claim 51, further comprising a promoter that is active in a eukaryotic cell.

53. 52. The polynucleotide molecule of claim 51 further defined as an expression vector.

54. 52. An engineered cell comprising the polynucleotide molecule of claim 51.

55. 55. The cell of claim 54, wherein the cell is a T cell, a NK cell, or a macrophage.

56. 56. A method of treating or ameliorating cancer in a subject in need of treatment, comprising administering to the subject a cell therapy comprising a therapeutically effective amount of cells according to claim 54 or 55.

57. 57. The method of claim 56, wherein the cell therapy is administered locally to the cancer site, regionally to the cancer site, or systemically.

58. 58. The method of claim 57, further comprising administering to the human subject a second cancer therapy.

59. 59. The method of claim 58, wherein said second cancer therapy is chemotherapy, immunotherapy, radiation therapy, hormone therapy, or surgery.

60. 59. The method of claim 58, wherein said second cancer therapy is administered simultaneously with the cell therapy.

61. 59. The method of claim 58, wherein said second cancer therapy is administered before or after the cell therapy.

62. 58. The method of claim 57, further comprising administering to the human subject a second administration of an effective amount of one or more cells of claim 54 or 55.

63. 57. The method of claim 56, wherein the cancer is metastatic, recurrent, or drug-resistant cancer.

64. 57. The method of claim 56, wherein the cancer is AML.

65. 57. The method of claim 56, wherein the cancer is selected from the group consisting of pre-B acute lymphocytic leukemia (pre-B ALL), B-cell leukemia, chronic lymphoblastic leukemia (CLL), multiple myeloma (MM), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), myeloproliferative neoplasm, and blastic plasmacytoid dendritic cell neoplasm (BPDCN).

66. 57. The method of claim 56, wherein the cancer is breast cancer, lung cancer, pancreatic cancer, or prostate cancer.

67. 56. A method for treating or ameliorating an autoimmune or inflammatory disease in a subject, comprising administering to the subject a cell therapy comprising a therapeutically effective amount of the cells of claim 54 or 55.

68. Autoimmune or inflammatory diseases include acquired immune deficiency syndrome (AIDS), 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 disease-dermatitis herpetiformis, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold sores, and flu-like illnesses. Cold agglutinin disease, CREST syndrome, Crohn's disease, Degos disease, juvenile dermatomyositis, discoid lupus erythematosus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre 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, 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-body syndrome, systemic lupus erythematosus (SLE), Takayasu's arteritis, temporal arteritis / giant cell arteritis, inflammatory bowel disease (IB) D) The method of claim 67, wherein the agent is selected from the group consisting of ulcerative colitis, Crohn's disease, intestinal mucosal inflammation, colitis-associated wasting disease, uveitis, vitiligo and Wegener's granulomatosis, Alzheimer's disease, asthma, atopic allergy, allergy, 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 infection, and autoimmune diabetes.

69. A bispecific antibody or antigen-binding fragment capable of binding to LILRB4 and CD3.

70. (a) a first light chain variable (V L ) domain and the first heavy chain variable (V H a first antigen-binding region comprising a nucleotide sequence (SEQ ID NO: 1) domain; and (b) Second V L Domain and second V H and a second antigen-binding region comprising a domain, wherein the first antigen-binding region is capable of binding to LILRB4 and the second antigen-binding region is capable of binding to CD3, or vice versa.

70. The bispecific antibody or antigen-binding fragment of claim 69.

71. First V L The first heavy chain variable domain and the first heavy chain variable domain are, respectively, the first heavy chain variable domain and the first heavy chain variable domain. and a second V L Domain and second V H 71. The bispecific antibody or antigen-binding fragment of claim 70, wherein each of the domains is linked to a second pair of constant domains.

72. (a) First V L The first light chain constant (C L ) domain and a first V H The domain is the first heavy chain constant domain 1 (C H 1) or (b) First V L Domain is the first C H 1 domain and V H The domain is Second C L Linked to a domain 72. The bispecific antibody or antigen-binding fragment thereof of claim 71.

73. (a) Second V L Domain is the second C L domain and a second V H domain is the second C H linked to one domain, or (b) Second V L Domain is the second C H linked to one domain and a second V H Domain N is the second C L linked to a domain, or (c) Second V L The domain is linked to a T cell receptor (TCR) α chain constant domain, And the second V H the domain is linked to a TCR β chain constant domain, or (d) Second V L domain is linked to the TCR β chain constant domain, and a second V H The domain is linked to the TCR alpha chain constant domain 73. The bispecific antibody or antigen-binding fragment of claim 72.

74. 74. The bispecific antibody or antigen-binding fragment of claim 73, wherein the TCR alpha chain constant domain has an S91A mutation.

75. 71. The bispecific antibody or antigen-binding fragment of claim 70, wherein the first antigen-binding region and / or the second antigen-binding region is a single-chain variable fragment (scFv).

76. The Third V L Domain and Third V H 71. The bispecific antibody or antigen-binding fragment of claim 70, further comprising a third antigen-binding region comprising a domain, wherein the third antigen-binding region is capable of binding to LILRB4 or CD3.

77. The Third V L The heavy chain variable domain and the third heavy chain variable domain are, respectively, the first constant domain and the second constant domain.

77. The bispecific antibody or antigen-binding fragment of claim 76, wherein the bispecific antibody or antigen-binding fragment is linked to a pair of:

78. (a) Third V L Domain is the third C L domain and a third V H domain is the third C H linked to one domain, or (b) Third V L Domain is the third C H linked to one domain and a third V H Domain N is the third C L linked to a domain, or (c) Third V L domain is linked to a second TCR α chain constant domain, and a third V H the domain is linked to a second TCR β chain constant domain, or (d) Third V L domain is linked to a second TCR β chain constant domain, and a third V H The domain is linked to a second TCR alpha chain constant domain.

78. The bispecific antibody or antigen-binding fragment of claim 77.

79. (a) a first VHC-CDR1 having the amino acid sequence of SEQ ID NO:5, a first HC-CDR2 having the amino acid sequence of SEQ ID NO:6, and a first HC-CDR3 having the amino acid sequence of SEQ ID NO:7; H domain; and (b) a first V LC-CDR1 having the amino acid sequence of SEQ ID NO: 8, including a mutation at amino acid residue NS, a first LC-CDR2 having the amino acid sequence of SEQ ID NO: 9, and a first LC-CDR3 having the amino acid sequence of SEQ ID NO: 10; L domain 71. The bispecific antibody or antigen-binding fragment of claim 70, comprising:

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