Humanized Anti-HLA-DR Antibodies

Abstract

The present invention concerns compositions and methods of use of humanized anti-HLA-DR antibodies. In preferred embodiments, the antibodies induce apoptosis and inhibit proliferation of lymphoma cells without inducing CDC or ADCC. In more preferred embodiments, the humanized anti-HLA-DR antibodies bind to the same epitope of HLA-DR as, or compete for binding to HLA-DR with, a murine L243 antibody. Most preferably, the humanized anti-HLA-DR antibody exhibits a higher affinity for HLA-DR than the parental murine antibody. The humanized HLA-DR antibody is of use for therapy of various diseases such as cancer, autoimmune disease or immune dysregulatory function, and is of particular use for therapy of B cell lymphomas and leukemias. In most preferred embodiments, the humanized anti-HLA-DR antibody is capable of inducing at least partial remission of lymphomas that are resistant to other B cell antibodies, such as rituximab.

What is claimed is: 
     
         1 . A method of treating autoimmune disease comprising administering to a subject with autoimmune disease a humanized anti-HLA-DR antibody or antigen-binding fragment thereof comprising the heavy chain complementarity determining region (CDR) sequences CDR1 (NYGMN (SEQ ID NO: 39)), CDR2 (WINTYTREPTYADDFKG (SEQ ID NO: 40)), and CDR3 (DITAVVPTGFDY (SEQ ID NO: 41)) and the light chain CDR sequences CDR1 (RASENIYSNLA (SEQ ID NO: 42)), CDR2 (AASNLAD (SEQ ID NO: 43)), and CDR3 (QHFWTTPWA (SEQ ID NO: 44));
 wherein the humanized antibody is an IgG4 antibody comprising a Ser24lPro mutation, further comprising light chain murine L243 FR residues R37, K39, V48, F49, and G100 and heavy chain murine L243 FR residues F27, K38, K46, A68, and F91.   
     
     
         2 . The method of  claim 1 , wherein the autoimmune disease is selected from the group consisting of acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitis obliterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis, dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis, polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis and fibrosing alveolitis. 
     
     
         3 . The method of  claim 1 , wherein said humanized anti-HLA-DR antibody has a higher affinity for HLA-DR than a murine L243 antibody that comprises the heavy chain CDR sequences CDR1 (NYGMN (SEQ ID NO: 39)), CDR2 (WINTYTREPTYADDFKG (SEQ ID NO: 40)), and CDR3 (DITAVVPTGFDY (SEQ ID NO: 41)) and the light chain CDR sequences CDR1 (RASENIYSNLA (SEQ ID NO: 42)), CDR2 (AASNLAD (SEQ ID NO: 43)), and CDR3 (QHFWTTPWA (SEQ ID NO: 44)). 
     
     
         4 . The method of  claim 1 , wherein said humanized anti-HLA-DR antibody is a naked antibody. 
     
     
         5 . The method of  claim 4 , further comprising administering to said subject at least one therapeutic agent selected from the group consisting of an antibody, an antibody fragment, an immunoconjugate, an immunomodulator, a hormone, a cytotoxic agent, an enzyme, a hormone, a cytokine, an anti-angiogenic agent, a pro-apoptosis agent, a photodynamic agent, a drug and a toxin. 
     
     
         6 . The method of  claim 5 , wherein the antibody, antibody fragment, or immunoconjugate binds to an antigen selected from the group consisting of A3, BrE3-antigen, CD1, CD1a, CD3, CD5, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80, HLA-DR, NCA95, NCA90, HCG, CEACAM-5, CEACAM-6, CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu, hypoxia inducible factor (HIF), KC4-antigen, KS-1 antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, MUC16, PAM-4-antigen, PSA, PSMA, RS5, 5100, TAG-72, p53, tenascin, IL-6, IL-8, insulin growth factor-1 (IGF-1), Tn antigen, VEGF, 17-1A-antigen, ED-B fibronectin, HM1.24, VEGF, ILGF, placental growth factor and carbonic anhydrase IX. 
     
     
         7 . The method of  claim 5 , wherein the drug is selected from the group consisting of 5-fluorouracil, aplidin, azaribine, anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatinum, Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, estramustine, epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenalidomide, leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, nitrosourea, plicamycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, SN-38, streptozocin, tamoxifen, temazolomide (an aqueous form of DTIC), transplatinum, thalidomide, thioguanine, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine and vinca alkaloids. 
     
     
         8 . The method of  claim 5 , wherein the toxin is selected from the group consisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), onconase, DNase I,  Staphylococcal  enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,  Pseudomonas  exotoxin and  Pseudomonas  endotoxin. 
     
     
         9 . The method of  claim 5 , wherein the anti-angiogenic agent is selected from the group consisting of angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-P1GF peptides and antibodies, anti-vascular growth factor antibodies, anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF (macrophage migration-inhibitory factor) antibodies, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin-12, IP-10, Gro-β, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxyamidotriazole, CM101, pentosan polysulphate, angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, roquinimex, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minocycline. 
     
     
         10 . The method of  claim 5 , wherein the immunomodulator is selected from the group consisting of erythropoietin, thrombopoietin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α, interferon-β, interferon-γ, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-a, tumor necrosis factor-β, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, NGF-β, platelet-growth factor, TGF-α, TGF-β, insulin-like growth factor-I, insulin-like growth factor-II, macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand, angiostatin, thrombospondin, endostatin, tumor necrosis factor and lymphotoxin (LT). 
     
     
         11 . The method of  claim 1 , wherein said humanized anti-HLA-DR antibody is attached to at least one therapeutic agent. 
     
     
         12 . The method of  claim 11 , wherein the therapeutic agent is selected from the group consisting of a drug, a toxin, an enzyme, an immunomodulator, a cytokine, a hormone, an antibody or fragment thereof, an anti-angiogenic agent, a cytotoxic agent, a pro-apoptosis agent, and a photodynamic agent. 
     
     
         13 . The method of  claim 12 , wherein the drug is selected from the group consisting of 5-fluorouracil, aplidin, azaribine, anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatinum, Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, estramustine, epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenalidomide, leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, nitrosourea, plicamycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, SN-38, streptozocin, tamoxifen, temazolomide (an aqueous form of DTIC), transplatinum, thalidomide, thioguanine, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine and vinca alkaloids. 
     
     
         14 . The method of  claim 12 , wherein the drug is SN-38. 
     
     
         15 . The method of  claim 12 , wherein the toxin is selected from the group consisting of ricin, abrin, saporin, ribonuclease (RNase), onconase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,  Pseudomonas  exotoxin and  Pseudomonas  endotoxin. 
     
     
         16 . The method of  claim 12 , wherein the anti-angiogenic agent is selected from the group consisting of angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-P1GF peptides and antibodies, anti-vascular growth factor antibodies, anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF (macrophage migration-inhibitory factor) antibodies, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin-12, IP-10, Gro-β, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxyamidotriazole, CM101, pentosan polysulphate, angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, roquinimex, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minocycline. 
     
     
         17 . The method of  claim 12 , wherein the immunomodulator is selected from the group consisting of erythropoietin, thrombopoietin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α, interferon-β, interferon-γ, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, NGF-β, platelet-growth factor, TGF-α, TGF-β, insulin-like growth factor-I, insulin-like growth factor-II, macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand, angiostatin, thrombospondin, endostatin, tumor necrosis factor and lymphotoxin (LT). 
     
     
         18 . The method of  claim 6 , wherein the administration of a combination of said humanized anti-HLA-DR antibody with an anti-CD20 antibody inhibits proliferation of lymphoma cells more than either anti-HLA-DR antibody or anti-CD20 antibody alone. 
     
     
         19 . The method of  claim 1 , wherein the subject is a human subject. 
     
     
         20 . The method of  claim 1  wherein the humanized anti-HLA-DR antibody or antigen-binding fragment thereof is administered intravenously, subcutaneously, or intramuscularly at a dose of between 20 and 2000 mg.