Agonistic anti-tumor necrosis factor receptor 2 antibodies

Abstract

The invention provides agonistic TNFR2 antibodies and antigen-binding fragments thereof and encompasses the use of these antibodies as therapeutics to promote the proliferation of regulatory T cells (T-reg) for the treatment of immunological diseases. Antibodies of the invention can be used to potentiate the T-reg-mediated deactivation of self- and allergen-reactive T- and B-eases. Antibodies and can thus be used to treat a wide variety of indications, including autoimmune diseases, allergic reactions, asthma, graft-versus-host disease, and allograft rejection, among others.

1 . An antibody or antigen-binding fragment thereof that specifically binds human TNFR2, wherein said antibody or antigen-binding fragment thereof specifically binds:
 (a) an epitope within human TNFR2 comprising amino acids 56-60 of SEQ ID NO: 366; and/or   (b) a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-341, 346, and 367;   and wherein said antibody or antigen-binding fragment thereof does not specifically bind an epitope within human TNFR2 comprising amino acids 142-146 of SEQ ID NO: 366.   
     
     
         2 - 5 . (canceled) 
     
     
         6 . The antibody or antigen-binding fragment thereof of  claim 1 , wherein said antibody or antigen-binding fragment thereof:
 (a) promotes proliferation of a population of T-regulatory (T-reg) cells, optionally in the presence of TNFα;   (b) promotes cell death in a population of CD8+ T-cells;   (c) promotes an increase in the level of one or more mRNA molecules encoding a protein selected from the group consisting of cIAP2, TRAF2, Etk, VEGFR2, PI3K, Akt, a protein involved in the angiogenic pathway, an IKK complex, RIP, NIK, MAP3K, a protein involved in the NFkB pathway, NIK, JNK, AP-1, a MEK, MKK3, NEMO, IL2R, Foxp3, IL2, TNF, and lymphotoxin;   (d) promotes an increase in the level of one or more proteins selected from the group consisting of cIAP2, TRAF2, Etk, VEGFR2, PI3K, Akt, a protein involved in the angiogenic pathway, an IKK complex, RIP, NIK, MAP3K, a protein involved in the NFkB pathway, NIK, JNK, AP-1, a MEK, MKK3, NEMO, IL2R, Foxp3, IL2, TNF, and lymphotoxin;   (e) activates TNFR2 signaling;   (f) specifically binds TNFR2 with a K D  of no greater than about 10 nM;   (g) specifically binds TNFR2 to form an antibody-antigen complex with a k on  of at least about 10 4  M −1 s −1 ;   (h) specifically binds TNFR2 to form an antibody-antigen complex, and wherein said complex dissociates with a k off  of no greater than about 10 −3 s −1 ; and/or   (i) does not specifically bind another tumor necrosis factor receptor (TNFR) superfamily member.   
     
     
         7 - 9 . (canceled) 
     
     
         10 . The antibody or antigen-binding fragment thereof of  claim 1 , wherein said antibody or antigen-binding fragment thereof specifically binds:
 (a) an epitope within human TNFR2 comprising at least five discontinuous or continuous residues within amino acids 96-154 of SEQ ID NO: 366   (b) an epitope within amino acids 111-150 of SEQ ID NO: 366;   (c) an epitope within amino acids 115-142 of SEQ ID NO: 366;   (d) an epitope within amino acids 122-136 of SEQ ID NO: 366;   (e) an epitope within amino acids 101-107 of SEQ ID NO: 366;   (f) an epitope within amino acids 48-67 of SEQ ID NO: 366; and/or   (g) said epitope comprising amino acids 56-60 of SEQ ID NO: 366 with a K D  of less than about 10 nM.   
     
     
         11 - 26 . (canceled) 
     
     
         27 . A method of identifying a TNFR2 agonist antibody or antigen-binding fragment thereof comprising:
 (a) contacting a mixture of antibodies or fragments thereof with at least one peptide having the amino acid sequence of any one of SEQ ID NOs: 1-341, 346, and 367; and   (b) separating antibodies or fragments thereof that specifically bind said peptide from said mixture, thereby producing an enriched antibody mixture comprising at least one said TNFR2 agonist antibody or antigen-binding fragment thereof.   
     
     
         28 . The method of  claim 27 , wherein:
 (a) said method comprises determining the amino acid sequence of one or more of the antibodies or antigen-binding fragments thereof in said enriched antibody mixture;   (b) said peptide is bound to a surface;   (c) said antibody or antigen-binding fragment thereof is expressed on the surface of a phage, bacterial cell, or yeast cell or said antibody or antigen-binding fragment thereof is expressed on the surface of a phage, bacterial cell, or yeast cell;   (d) said peptide is conjugated to a detectable label;   (e) steps (a) and (b) are sequentially repeated one or more times; and/or   (f) the method further comprises:
 i) exposing said enriched antibody mixture to at least one peptide comprising the amino acid sequence of a TNFR superfamily member other than TNFR2, and retaining antibodies or fragments thereof that do not specifically bind said peptide, thereby producing a TNFR2-specific antibody mixture comprising at least one TNFR2 agonist antibody or antigen-binding fragment thereof that does not specifically bind a TNFR superfamily member other than TNFR2; and/or 
 ii) exposing said enriched antibody mixture to at least one peptide comprising amino acids 142-146 of SEQ ID NO: 366, and retaining antibodies or fragments thereof that do not specifically bind said peptide, thereby producing an antibody mixture comprising at least one TNFR2 agonist antibody or antigen-binding fragment thereof that does not specifically bind a peptide comprising amino acids 142-146 of SEQ ID NO: 366. 
   
     
     
         29 - 38 . (canceled) 
     
     
         39 . A method of producing a TNFR2 agonist antibody or antigen-binding fragment thereof comprising immunizing a non-human mammal with a peptide comprising the sequence of any one of SEQ ID NOs: 1-341, 346, and 367 and collecting serum comprising said TNFR2 agonist antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is capable of specifically binding an epitope comprising amino acids 56-60 of SEQ ID NO: 366. 
     
     
         40 . The method of  claim 39 , wherein:
 (a) said non-human mammal is selected from the group consisting of a rabbit, mouse, rat, goat, guinea pig, hamster, horse, and sheep; and/or   (b) said peptide comprises the amino acid sequence of SEQ ID NO: 11.   
     
     
         41 . (canceled) 
     
     
         42 . An antibody or antigen-binding fragment thereof that is produced by the method of  claim 39 . 
     
     
         43 - 53 . (canceled) 
     
     
         54 . The antibody or antigen-binding fragment thereof of  claim 1 , wherein said antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multi-specific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab′) 2  molecule, or a tandem scFv (taFv). 
     
     
         55 . (canceled) 
     
     
         56 . The antibody of  claim 1 , wherein said antibody comprises an immunoglobulin subtype selected from the group consisting of IgG, IgM, IgA, IgD, and IgE. 
     
     
         57 . A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of  claim 1  and a pharmaceutically acceptable carrier or excipient. 
     
     
         58 . The pharmaceutical composition of  claim 57 , wherein said pharmaceutical composition further comprises an additional therapeutic agent, optionally wherein said additional therapeutic agent is selected from the group consisting of TNFα and BCG. 
     
     
         59 - 61 . (canceled) 
     
     
         62 . A polynucleotide encoding the antibody or antigen-binding fragment thereof of  claim 1 . 
     
     
         63 . A vector comprising the polynucleotide of  claim 62 . 
     
     
         64 - 70 . (canceled) 
     
     
         71 . An isolated host cell comprising the vector of  claim 63 . 
     
     
         72 . The host cell of  claim 71 , wherein said host cell is:
 (a) a prokaryotic cell; or   (b) a eukaryotic cell; optionally wherein said eukaryotic cell is a CHO cell, a DHFR CHO cell, a NSO myeloma cell, a COS cell, a 293 cell, or a SP2/0 cell.   
     
     
         73 - 75 . (canceled) 
     
     
         76 . A method of producing the antibody or antigen-binding fragment thereof of  claim 1 , said method comprising expressing a polynucleotide encoding said antibody or antigen-binding fragment thereof in a host cell and recovering the antibody or antigen-binding fragment thereof from host cell medium. 
     
     
         77 . A method of inhibiting an immune response mediated by a B cell or CD8+ T cell in a subject, said method comprising administering to the subject the antibody or antigen-binding fragment thereof of  claim 1 . 
     
     
         78 . A method of treating an immunological disease in a subject, said method comprising administering to the subject the antibody or antigen-binding fragment thereof of  claim 1 . 
     
     
         79 . The method of  claim 78 , wherein said subject is in need of a tissue or organ regeneration. 
     
     
         80 . The method of  claim 79 , wherein said tissue or organ is selected from the group consisting of a pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerves, structures of the head, eye, thymus, tongue, bone, liver, small intestine, large intestine, gut, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structures, embryos, and testes. 
     
     
         81 . The method of  claim 78 , wherein said immunological disease is selected from the group consisting of an autoimmune disease, a neurological condition, an allergy, asthma, macular degeneration, muscular atrophy, a disease related to miscarriage, atherosclerosis, bone loss, a musculoskeletal disease, obesity, a graft-versus-host disease, and an allograft rejection. 
     
     
         82 . The method of  claim 81 , wherein:
 (a) said autoimmune disease is selected from the group consisting of type I diabetes, Alopecia Areata, Ankylosing Spondylitis, Antiphospholipid Syndrome, Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Behcet's Disease, Bullous Pemphigoid, Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic Inflammatory Demyelinating Polyneuropathy, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CREST Syndrome, Cold Agglutinin Disease, Crohn's Disease, Essential Mixed Cryoglobulinemia, Fibromyalgia-Fibromyositis, Graves' Disease, Guillain-Barré, Hashimoto's Thyroiditis, Hypothyroidism, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, Juvenile Arthritis, Lichen Planus, Lupus, Meniere's Disease, Mixed Connective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, Polyglandular Syndromes, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma, Sjögren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis, Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo, and Wegener's Granulomatosis;   (b) said neurological condition is selected from the group consisting of a brain tumor, a brain metastasis, a spinal cord injury, schizophrenia, epilepsy, Amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, and stroke;   (c) said allergy is selected from the group consisting of food allergy, seasonal allergy, pet allergy, hives, hay fever, allergic conjunctivitis, poison ivy allergy oak allergy, mold allergy, drug allergy, dust allergy, cosmetic allergy, and chemical allergy;   (d) said graft-versus-host disease arises from a bone marrow transplant or one or more blood cells selected from the group consisting of hematopoietic stem cells, common myeloid progenitor cells, common lymphoid progenitor cells, megakaryocytes, monocytes, basophils, eosinophils, neutrophils, macrophages, T-cells, B-cells, natural killer cells, and dendritic cells; and/or   (e) said allograft rejection is selected from the group consisting of skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection, and organ graft rejection, optionally wherein:
 (i) said ligament graft rejection is selected from the group consisting of cricothyroid ligament graft rejection, periodontal ligament graft rejection, suspensory ligament of the lens graft rejection, palmar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, ulnar collateral ligament graft rejection, radial collateral ligament graft rejection, suspensory ligament of the breast graft rejection, anterior sacroiliac ligament graft rejection, posterior sacroiliac ligament graft rejection, sacrotuberous ligament graft rejection, sacrospinous ligament graft rejection, inferior pubic ligament graft rejection, superior pubic ligament graft rejection, anterior cruciate ligament graft rejection, lateral collateral ligament graft rejection, posterior cruciate ligament graft rejection, medial collateral ligament graft rejection, cranial cruciate ligament graft rejection, caudal cruciate ligament graft rejection, and patellar ligament graft rejection; and/or 
 (ii) said organ graft rejection is selected from the group consisting of heart graft rejection, lung graft rejection, kidney graft rejection, liver graft rejection, pancreas graft rejection, intestine graft rejection, and thymus graft rejection. 
   
     
     
         83 - 88 . (canceled) 
     
     
         89 . The method of  claim 78 , wherein said method further comprises administering to said subject an additional therapeutic agent. 
     
     
         90 . The method of  claim 89 , wherein said additional therapeutic agent is selected from the group consisting of TNFα and BCG. 
     
     
         91 - 92 . (canceled) 
     
     
         93 . The method of  claim 78 , wherein said subject is a mammal. 
     
     
         94 . The method of  claim 93 , wherein said mammal is a human. 
     
     
         95 . The method of  claim 78 , wherein said antibody is 8E6.D1 or a humanized antibody or antigen-binding fragment thereof comprising one or more heavy chain or light chain CDRs of 8E6.D1. 
     
     
         96 . (canceled) 
     
     
         97 . A kit comprising the antibody or antigen-binding fragment thereof of  claim 1 , a polynucleotide encoding the antibody or antigen-binding fragment thereof, a vector comprising the polynucleotide, or a host cell comprising the vector or polynucleotide, wherein the kit optionally comprises a package insert that instructs a user of said kit to administer said antibody or antigen-binding fragment thereof, polynucleotide, vector, or host cell to a human patient suffering from an immunological disease. 
     
     
         98 - 129 . (canceled) 
     
     
         130 . The antibody or antigen-binding fragment thereof of  claim 1 , wherein said antibody or antigen-binding fragment thereof comprises a non-native constant region. 
     
     
         131 . The antibody or antigen-binding fragment thereof of  claim 1 , wherein said antibody or antigen-binding fragment thereof is an isolated, non-murine antibody. 
     
     
         132 - 133 . (canceled)