Tnf-alpha binding proteins

Abstract

TNF-α binding proteins, including chimeric, CDR-grafted, and humanized antibodies that bind TNF-α are provided. Binding proteins have high affinity for TNF-α and neutralize TNF-α activity. A binding protein can be a full-length antibody or a TNF-α-binding portion thereof. Methods of making and methods of using the binding proteins are also described. The TNF-α binding proteins are useful for detecting TNF-α and for inhibiting TNF-α activity, including in a human subject suffering from a disease or disorder in which TNF-α activity is detrimental.

1 - 53 . (canceled) 
     
     
         54 . A humanized affinity matured binding protein that binds human tumor necrosis factor alpha (TNF-α), or an or antigen binding portion thereof, wherein the binding protein or antigen binding portion thereof comprises a heavy chain variable region (VH) having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 37, 38, 39, 40, 41, and 42, and a light chain variable region (VL) having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: SEQ ID NOS: 35, 43, 44, 45, and 46, and wherein the binding protein comprises at least one amino acid substitution in one or more complementarity determining regions (CDRs). 
     
     
         55 . The binding protein or antigen binding portion thereof of  claim 54 , wherein the binding protein or antigen binding portion thereof comprises a VH and a VL comprising the respective amino acid sequences:
 SEQ ID NO: 37 and SEQ ID NO: 35;   SEQ ID NO: 37 and SEQ ID NO: 43;   SEQ ID NO: 37 and SEQ ID NO: 44;   SEQ ID NO: 37 and SEQ ID NO: 45;   SEQ ID NO: 37 and SEQ ID NO: 46;   SEQ ID NO: 38 and SEQ ID NO: 35;   SEQ ID NO: 38 and SEQ ID NO: 43;   SEQ ID NO: 38 and SEQ ID NO: 44;   SEQ ID NO: 38 and SEQ ID NO: 45;   SEQ ID NO: 38 and SEQ ID NO: 46;   SEQ ID NO: 39 and SEQ ID NO: 35;   SEQ ID NO: 39 and SEQ ID NO: 43;   SEQ ID NO: 39 and SEQ ID NO: 44;   SEQ ID NO: 39 and SEQ ID NO: 45;   SEQ ID NO: 39 and SEQ ID NO: 46;   SEQ ID NO: 40 and SEQ ID NO: 35;   SEQ ID NO: 40 and SEQ ID NO: 43;   SEQ ID NO: 40 and SEQ ID NO: 44;   SEQ ID NO: 40 and SEQ ID NO: 45;   SEQ ID NO: 40 and SEQ ID NO: 46;   SEQ ID NO: 41 and SEQ ID NO: 35;   SEQ ID NO: 41 and SEQ ID NO: 43;   SEQ ID NO: 41 and SEQ ID NO: 44;   SEQ ID NO: 41 and SEQ ID NO: 45;   SEQ ID NO: 41 and SEQ ID NO: 46;   SEQ ID NO: 42 and SEQ ID NO: 35;   SEQ ID NO: 42 and SEQ ID NO: 43;   SEQ ID NO: 42 and SEQ ID NO: 44;   SEQ ID NO: 42 and SEQ ID NO: 45; or   SEQ ID NO: 42 and SEQ ID NO: 46.   
     
     
         56 . The binding protein of  claim 54 , wherein the one or more CDRs are selected from the group consisting of:
 CDR-H1 comprising the amino acid sequence NYGMN (residues 31-35 of SEQ ID NOS: 37, 38, 39, 40, 41, and 42);   CDR-H2 comprising the amino acid sequence WINTYTGEPTYADDFKG (residues 50-66 of SEQ ID NOS: 37, 38, 39, 40, 41, and 42);   CDR-H3 comprising the amino acid sequence KFLTTVVVTDYAMDY (residues 99-113 of SEQ ID NOS: 37, 38, 39, 40, 41, and 42);   CDR-L1 comprising the amino acid sequence RASQDISNYLN (residues 24-34 of SEQ ID NOS: 35, 43, 44, 45, and 46);   CDR-L2 comprising the amino acid sequence YTSRLQS (residues 50-56 of SEQ ID NOS: 35, 43, 44, 45, and 46); and   CDR-L3 comprising the amino acid sequence QQGNTLPPT (residues 89-97 of SEQ ID NOS: 35, 43, 44, 45, and 46)   
     
     
         57 . The binding protein of  claim 54 , wherein the binding protein is an immunoglobulin molecule, a disulfide linked Fv, a monoclonal antibody, an scFv, a chimeric antibody, a single domain antibody, a CDR-grafted antibody, a diabody, a humanized antibody, a multispecific antibody, an Fab, an Fab′, a bispecific antibody, an F(ab′)2, or an Fv. 
     
     
         58 . The binding protein of  claim 56 , wherein the binding protein comprises a human IgM constant domain, a human IgG4 constant domain, a human IgG1 constant domain, a human IgE constant domain, a human IgG2 constant domain, a human IgG3 constant domain, or a human IgA constant domain. 
     
     
         59 . The binding protein of  claim 56 , further comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. 
     
     
         60 . The binding protein of  claim 56 , further comprising a light chain constant region having an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. 
     
     
         61 . The binding protein of  claim 54 , wherein the binding protein is capable of neutralizing human TNF-α. 
     
     
         62 . The binding protein of  claim 61 , wherein the binding protein has a dissociation constant (KD) to TNF-α of at most about 10 −9  M, and wherein the binding protein neutralizes TNF-α with an IC50 of at most about 10 −9  M. 
     
     
         63 . The binding protein of  claim 54 , wherein the binding protein further comprises an immunoadhesion molecule, an imaging agent, a therapeutic agent, or a cytotoxic agent. 
     
     
         64 . The binding protein of  claim 63 , wherein the imaging agent is a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin. 
     
     
         65 . The binding protein of  claim 64 , wherein the radiolabel is  3 H,  14 C,  35 S,  90 Y,  99 Tc,  111 In,  125 I,  131 I,  177 Lu,  166 Ho, or  153 Sm. 
     
     
         66 . The binding protein of  claim 63 , wherein the therapeutic or cytotoxic agent is an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, and an apoptotic agent. 
     
     
         67 . The binding protein of  claim 54 , wherein the binding protein comprises a human glycosylation pattern. 
     
     
         68 . The binding protein of  claim 54 , wherein the binding protein is a crystallized binding protein. 
     
     
         69 . An isolated nucleic acid encoding a binding protein amino acid sequence of  claim 54 . 
     
     
         70 . A vector comprising the isolated nucleic acid of  claim 69 . 
     
     
         71 . The vector of  claim 70 , wherein the vector is pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, pHybE, or pBJ. 
     
     
         72 . A host cell comprising the vector of  claim 70 . 
     
     
         73 . The host cell of  claim 72 , wherein the host cell is a prokaryotic cell. 
     
     
         74 . The host cell of  72 , wherein the host cell is a eukaryotic cell. 
     
     
         75 . The host cell of  claim 74 , wherein the eukaryotic cell is a protist cell, an animal cell, a plant cell, a fungal cell, a mammalian cell, an avian cell, or an insect cell. 
     
     
         76 . The host cell of  claim 74 , wherein the eukaryotic cell is  S. cerevisiae , a CHO cell, a COS cell, or an SF9 cell. 
     
     
         77 . A method of producing a protein capable of binding TNF-α, the method comprising the steps of culturing the host cell described of in  claim 72  in culture medium under conditions sufficient to produce a binding protein capable of binding TNF-α. 
     
     
         78 . A protein produced according to the method of  claim 77 . 
     
     
         79 . A pharmaceutical composition comprising the binding protein of  claim 54 , and a pharmaceutically acceptable carrier. 
     
     
         80 . The pharmaceutical composition of  claim 79 , further comprising at least one additional agent for treating a disorder in which TNF-α activity is detrimental. 
     
     
         81 . The pharmaceutical composition of  claim 80 , wherein the additional agent is a therapeutic agent; an imaging agent; a cytotoxic agent; an angiogenesis inhibitor; a kinase inhibitor; a co-stimulation molecule blocker; an adhesion molecule blocker; an anti-cytokine antibody or functional fragment thereof; methotrexate; cyclosporin; rapamycin; FK506; a detectable label or reporter; a TNF antagonist; an anti-rheumatic; a muscle relaxant; a narcotic; a non-steroid anti-inflammatory drug (NSAID); an analgesic; an anesthetic; a sedative; a local anesthetic; a neuromuscular blocker; an antimicrobial; an antipsoriatic; a corticosteroid; an anabolic steroid; an erythropoietin; an immunization; an immunoglobulin; an immunosuppressive; a growth hormone; a hormone replacement drug; a radiopharmaceutical; an antidepressant; an antipsychotic; a stimulant; an asthma medication; a beta agonist; an inhaled steroid; an oral steroid; an epinephrine or analog thereof; a cytokine; or a cytokine antagonist. 
     
     
         82 . A method for treating a mammal comprising the step of administering to the mammal an effective amount of the composition of  claim 79 . 
     
     
         83 . A method for reducing human TNF-α activity comprising contacting human TNF-α with the binding protein of  claim 54  such that human TNF-α activity is reduced. 
     
     
         84 . A method for reducing human TNF-α activity in a human subject suffering from a disorder in which TNF-α activity is detrimental, comprising administering to the human subject the binding protein of  claim 54  such that human TNF-α activity in the human subject is reduced. 
     
     
         85 . A method for treating a subject for a disease or a disorder in which TNF-α activity is detrimental by administering to the subject the binding protein of  claim 54  such that treatment is achieved. 
     
     
         86 . The method of  claim 85 , wherein the disorder is an autoimmune and/or inflammatory disorder. 
     
     
         87 . The method of  claim 86 , wherein the disorder is Crohn's disease, plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus, ankylosing spondylitis, insulin-dependent diabetes mellitus, autoimmune diabetes, allergy, and autoimmune uveitis. 
     
     
         88 . The method of  claim 86 , wherein the disorder is a respiratory disorder; asthma; allergic and nonallergic asthma; asthma due to infection; asthma due to infection with respiratory syncytial virus (RSV); chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation; eosinophilia; fibrosis and excess mucus production; cystic fibrosis; pulmonary fibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema; allergic rhinitis; allergic enterogastritis; an inflammatory and/or autoimmune condition of the skin; an inflammatory and/or autoimmune condition of gastrointestinal organs; inflammatory bowel diseases (IBD); ulcerative colitis; Crohn's disease; an inflammatory and/or autoimmune condition of the liver; liver cirrhosis; liver fibrosis; liver fibrosis caused by hepatitis B and/or C virus; scleroderma; tumors or cancers; hepatocellular carcinoma; glioblastoma; lymphoma; Hodgkin's lymphoma; a viral infection; a bacterial infection; a parasitic infection; HTLV-1 infection; suppression of expression of protective type 1 immune responses, and suppression of expression of a protective type 1 immune response during vaccination. 
     
     
         89 . A method of treating a patient suffering from a disorder in which TNF-α is detrimental comprising administering the binding protein of  claim 54  before, concurrent, or after the administration of a second agent, wherein the second agent is an antibody, or fragment thereof, capable of binding human IL-12; PGE2; LPA; NGF; CGRP; SubP; RAGE; histamine; a histamine receptor blocker; bradykinin; IL-1alpha; IL-1beta; VEGF; PLGF; methotrexate; a corticosteroid, a glucocorticoid receptor modulator; cyclosporin, rapamycin, FK506, or a non-steroidal anti-inflammatory agent. 
     
     
         90 . A method of treating a patient suffering from a disorder in which TNF-α is detrimental, the method comprising the step of administering the binding protein of  claim 54  before, concurrently, or after the administration of a second agent, wherein the second agent is chosen from TNF antagonists; a soluble fragment of a TNF receptor; ENBREL®; TNF enzyme antagonists; TNF converting enzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-beta antagonists; interferon gamma; perfenidone; chemotherapeutic agents, methotrexate; leflunomide; sirolimus (rapamycin) or an analog thereof, CCI-779; COX2 or cPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors; TPL-2, MK-2 and NFkB inhibitors; budenoside; epidermal growth factor; corticosteroids; cyclosporine; sulfasalazine; amino salicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1b antibodies; anti-IL-6 antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies or agonists of TNF, LT, IL-1β, IL-2, IL-3, IL-4, IL5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II, GM-CSF, FGF, or PDGF; antibodies of CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands; FK506; rapamycin; mycophenolate mofetil; ibuprofen; prednisolone; phosphodiesterase inhibitors; adensosine agonists; antithrombotic agents; complement inhibitors; adrenergic agents; IRAK, NIK, IKK, p38, or MAP kinase inhibitors; IL-1β converting enzyme inhibitors; TNFa converting enzyme inhibitors; T-cell signaling inhibitors; metalloproteinase inhibitors; 6-mercaptopurines; angiotensin converting enzyme inhibitors; soluble cytokine receptors; soluble p55 TNF receptor; soluble p75 TNF receptor; sIL-1RI; sIL-1RII; sIL-6R; anti-inflammatory cytokines; and TGFb. 
     
     
         91 . The method of  claim 82 , wherein the administering to the subject is by at least parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal. 
     
     
         92 . A method of detecting human TNF-α in a sample comprising:
 (i) contacting the sample with an TNF-α binding protein as described in  claim 54  or an TNF-α binding portion thereof;
 and 
 
 (ii) detecting formation of a complex between the TNF-α binding protein or binding portion thereof and TNF-α in the sample, wherein a statistically significant change in the formation of the complex in the sample relative to that in a control sample or relative TNF-α in the sample. 
 
     
     
         93 . The method according to  claim 92 , wherein the sample is whole blood, plasma, serum, urine, saliva, or tissue biopsy. 
     
     
         94 . A method of detecting human TNF-α in a human subject comprising:
 (i) administering an TNF-α binding protein as described in  claim 54 , or an TNF-α binding portion thereof, to a test subject or a control subject under conditions that allow binding of the TNF-α binding protein, or TNF-α binding portion thereof, to human TNF-α;
 and 
 
 (ii) detecting formation of a complex between the binding protein or binding portion thereof and TNF-α, wherein a statistically significant change in the formation of the complex in the test subject relative to the control subject or relative to formation of the complex in the test subject at an earlier time point is indicative of the presence of TNF-α.