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