TNF alpha INHIBITOR POLYPEPTIDES, POLYNUCLEOTIDES ENCODING SAME, CELLS EXPRESSING SAME AND METHODS OF PRODUCING SAME

Abstract

A plant produced TNF alpha polypeptide inhibitor is provided. Also provided are methods of generating and using same.

1 - 5 . (canceled) 
     
     
         6 . A plant produced TNFα polypeptide inhibitor comprising a soluble TNFR fragment which specifically binds TNFα, wherein said soluble TNFR fragment is part of a chimeric polypeptide comprising:
 (i) a first domain which comprises a TNFα binding domain of a TNF receptor; and 
 (ii) a second domain which comprises an Fc domain of an immunoglobulin; wherein said first domain and said second domain are N-terminally to C-terminally respectively sequentially translationally fused and wherein the chimeric polypeptide specifically binds TNFα. 
 
     
     
         7 . The TNFα polypeptide inhibitor of  claim 6 , wherein said chimeric polypeptide further comprises a third domain which comprises an endoplasmic reticulum retention signal, wherein said first domain, second domain and third domain are N-terminally to C-terminally respectively sequentially translationally fused. 
     
     
         8 . The TNFα polypeptide inhibitor of  claim 6 , comprising an additional domain encoding an endoplasmic reticulum signal peptide translationally fused N-terminally to said first domain. 
     
     
         9 . The TNFα polypeptide inhibitor of  claim 8 , wherein said signal peptide is a plant signal peptide. 
     
     
         10 . The TNFα polypeptide inhibitor of  claim 9 , wherein said plant signal peptide is as set forth in SEQ ID NO: 4. 
     
     
         11 . The TNFα polypeptide inhibitor of  claim 6 , wherein said first domain is 200-250 amino acids long. 
     
     
         12 . The TNFα polypeptide inhibitor of  claim 11 , wherein said first domain comprises the amino acid sequence LCAP (SEQ ID NO: 11) and VFCT (SEQ ID NO: 12). 
     
     
         13 . The TNFα polypeptide inhibitor of  claim 12 , wherein said first domain further comprises the amino acid sequence LPAQVAFXPYAPEPGSTC (SEQ ID NO: 13). 
     
     
         14 . The TNFα polypeptide inhibitor of  claim 13 , wherein said first domain is as set forth in SEQ ID NO: 2. 
     
     
         15 . The TNFα polypeptide inhibitor of  claim 6 , wherein said immunoglobulin is IgG 1 . 
     
     
         16 . The TNFα polypeptide inhibitor of  claim 6 , wherein said second domain is as set forth in SEQ ID NO: 9. 
     
     
         17 . The TNFα polypeptide inhibitor of  claim 6 , wherein said chimeric polypeptide is as set forth in SEQ ID NO:  6 . 
     
     
         18 . The TNFα polypeptide inhibitor of  claim 7 , wherein said chimeric polypeptide is as set forth in SEQ ID NO: 7, 204 OR 205 or 214. 
     
     
         19 . The TNFα polypeptide inhibitor of  claim 6 , wherein said chimeric polypeptide is capable of inhibiting TNFα-induced apoptosis. 
     
     
         20 . The TNFα polypeptide inhibitor of  claim 6 , wherein said TNFα polypeptide inhibitor comprises a plant-specific glycan. 
     
     
         21 . The TNFα polypeptide inhibitor of  claim 20 , wherein said plant-specific glycan is selected from the group consisting of a core xylose and a core α-(1,3) fucose. 
     
     
         22 . The TNFα polypeptide inhibitor of  claim 6 , wherein the polypeptide is purified to at least 98% homogeneity. 
     
     
         23 . The TNFα polypeptide inhibitor of  claim 6 , capable of inhibiting TNFα-induced apoptosis. 
     
     
         24 . An isolated polynucleotide comprising a nucleic acid sequence encoding the TNFα polypeptide inhibitor of  claim 6 . 
     
     
         25 - 26 . (canceled) 
     
     
         27 . A nucleic acid expression construct comprising a nucleic acid sequence encoding the polynucleotide of  claim 24  and a cis-acting regulatory element active in a plant cell. 
     
     
         28 . (canceled) 
     
     
         29 . A plant cell comprising the nucleic acid construct of  claim 27 . 
     
     
         30 . The plant cell of  claim 29  being a  Nicotiana tabacum  plant cell. 
     
     
         31 . The plant cell of  claim 30 , wherein said  Nicotiana tabacum  L. cv plant cell is a Bright Yellow (BY-2) cell. 
     
     
         32 . The plant cell of  claim 29 , being lyophilized. 
     
     
         33 . A plant cell suspension culture comprising the plant cell of  claim 29 . 
     
     
         34 - 35 . (canceled) 
     
     
         36 . A method of treating a TNFα-associated medical condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of  claim 6 , thereby treating the TNFα-associated medical condition in the subject. 
     
     
         37 . A method of treating a TNFα-associated medical condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the plant cells of  claim 29 , thereby treating the TNFα-associated medical condition in the subject. 
     
     
         38 - 41 . (canceled) 
     
     
         42 . The method of  claim 36 , wherein said medical condition is an inflammatory disease. 
     
     
         43 . The method of  claim 36 , wherein said medical condition is an autoimmune disease. 
     
     
         44 . (canceled) 
     
     
         45 . The method of  claim 36 , wherein said medical condition is an inflammatory bowel disease. 
     
     
         46 . The method of  claim 45 , wherein said inflammatory bowel disease is selected from the group consisting of colitis and Crohn's disease. 
     
     
         47 - 50 . (canceled) 
     
     
         51 . The method of  claim 37 , wherein said plant cells are formulated for enteral administration and wherein said medical condition is not a obesity, metabolic syndrome, diabetes and a liver disease or disorder. 
     
     
         52 . A method of polypeptide production, comprising:
 providing a cell according to  claim 29 ; and   culturing said cell so as to produce said polypeptide.   
     
     
         53 - 55 . (canceled) 
     
     
         56 . The method of  claim 37 , wherein said medical condition is an inflammatory disease. 
     
     
         57 . The method of  claim 37 , wherein said medical condition is an autoimmune disease. 
     
     
         58 . The method of  claim 37 , wherein said medical condition is an inflammatory bowel disease. 
     
     
         59 . The method of  claim 58 , wherein said inflammatory bowel disease is selected from the group consisting of colitis and Crohn's disease.