Modified interferon-beta (IFN-beta) polypeptides

• US 20080038224A1
• Filed: 03/27/2007
• Published: 02/14/2008
• Est. Priority Date: 03/28/2006
• Status: Abandoned Application

• Current Assignee: HanAll Biopharma Co. Ltd.
• Original Assignee: HanAll Biopharma Co. Ltd.
• Inventors: Guyon, Thierry, Drittanti, Lila, Borrelly, Gilles, Vega, Manuel
• Application Number:
  US11/729,267
• Publication Number:
  US 20080038224A1
• Time in Patent Office: Days
• US Class Current: 424/85.6
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/565 IFN-beta

Abstract

Provided are modified interferon-beta polypeptides and nucleic acid molecules encoding modified interferon-beta polypeptides and formulations containing the polypeptides and/or nucleic acid molecules. The modified polypeptides exhibit increased protein stability, including increased resistance to proteases. Also provided are methods of treatment by administering modified interferon-beta polypeptides.

Claims

1. A modified IFN-β

polypeptide, comprising;

an amino acid modification in an unmodified IFN-β

polypeptide at positions corresponding to amino acid position L5 or L6 in the mature IFN-β

polypeptide set forth in SEQ ID NO;

1, wherein a modification is a replacement (substitution), addition, deletion, or a combination thereof, of amino acid residues; and

one or more additional amino acid modifications at another position.

2. The modified IFN-β

polypeptide of claim 1 that has two amino acid replacements.

3. The modified IFN-β

polypeptide of claim 1 that retains at least one in vivo activity of an IFN-β

polypeptide.

4. The modified IFN-β

polypeptide of claim 1 that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 modifications in addition to the modification at position L5 or L6.

5. A modified IFN-β

polypeptide of claim 1 that is a mature IFN-β

polypeptide.

6. A modified IFN-β

polypeptide of claim 1 that is a precursor IFN-β

polypeptide.

7. A modified IFN-β

polypeptide of claim 1, wherein the modification at positions L5 or L6 is replacement of leucine (L) by an amino acid selected from among aspartic acid (D), glutamine (Q), asparagine (N), or glutamic acid (E).

8. A modified IFN-β

polypeptide of claim 1, wherein the one or more further amino acid modification(s) is at one or more positions corresponding to amino acid positions selected from among M1, Y3, L5, L6, F8, L9, Q10, R11, S12, S13, N14, F15, Q16, C17, Q18, K19, L20, L21, W22, Q23, L24, N25, R27, L28, E29, Y30, C31, L32, K33, D34, R35, M36, F38, D39, P41, E42, E43, K45, L47, Q48, Q49, F50, Q51, K52, E53, D54, L57, Y60, E61, M62, L63, Q64, F67, F70, R71, Q72, D73, G78, W79, N80, E81, T82, I83, E85, N86, L87, L88, A89, N90, V91, Y92, Q94, I95, H97, L98, K99, V101, L102, E103, E104, K105, L106, E107, K108, E109, D110, R113, K115, L116, M117, L120, L122, K123, R124, Y125, Y126, R128, L130, Y132, L133, K134, K136, E137, Y138, W143, R147, E149, L15I, R152, F154, Y155, F156, R159, L160, Y163, L164, and R165 in the mature IFN-β

polypeptide set forth in SEQ ID NO;

1.

9. A modified IFN-β

polypeptide of claim 8, wherein the further amino acid modification(s) correspond to modifications selected from among M1V, M1I, M1T, M1A, M1Q, M1D, M1E, M1K, M1N, M1R, M1S, M1C, Y3I, Y3H, L5V, L5I, L5T, L5Q, L5H, L5A, L5D, L5E, L5K, L5R, L5N, L5S, L6D, L6E, L6K, L6N, L6Q, L6R, L6S, L6T, L6C, L61, L6V, L6H, L6A, F8I, F8V, F8D, F8E, F8K, F8R, L9V, L91, L9T, L9Q, L9H, L9A, L9D, L9E, L9K, L9N, L9R, L9S, Q10D, Q10E, Q10K, Q10N, Q10R, Q10S, Q10T, Q10C, R11H, R11Q, R11D, S12D, S12E, S12K, S12R, S13D, S13E, S13K, S13N, S13Q, S13R, S13T, S13C, N14D, N14E, N14K, N14Q, N14R, N14S, N14T, F15I, F15V, F15D, F15E, F15K, F15R, Q16D, Q16E, Q16K, Q16N, Q16R, Q16S, Q16T, Q16C, C17D, C17E, C17K, C17N, C17Q, C17R, C17S, C17T, Q18H, Q18S, Q18T, Q18N, K19N, K19Q, K19T, K19S, K19H, L20I, L20V, L20H, L20A, L20N, L20Q, L20R, L20S, L20T, L20D, L20E, L20K, L21I, L21V, L21T, L21Q, L21H, L21A, W22S, W22H, W22D, W22E, W22K, W22R, Q23D, Q23E, Q23K, Q23R, Q23H, Q23S, Q23T, Q23N, L24I, L24V, L24T, L24Q, L24H, L24A, L24D, L24E, L24K, L24R, N25H, N25S, N25Q, R27H, R27Q, L28V, L28I, L28T, L28Q, L28H, L28A, E29Q, E29H, E29N, Y30H, Y30I, L32V, L32I, L32T, L32Q, L32H, L32A, K33Q, K33T, K33S, K33H, K33N, D34N, D34Q, D34G, R35H, R35Q, M36V, M36I, M36T, M36Q, M36A, F38I, F38V, D39N, D39Q, D39H, D39G, P41A, P41S, E42N, E42Q, E42H, E43K, E43Q, E43H, E43N, K45D, K45N, K45Q, K45T, K45S, K45H, L47V, L47I, L47T, L47Q, L47H, L47A, Q48H, Q48S, Q48T, Q48N, Q49H, Q49S, Q49T, Q49N, F50I, F50V, Q51H, Q51S, Q51T, Q51N, K52Q, K52T, K52S, K52H, K52D, K52N, E53R, E53Q, E53H, E53N, D54K, D54Q, D54N, D54G, L57I, L57V, L57T, L57Q, L57H, L57A, Y60H, Y60I, E61K, E61Q, E61H, E61N, M62I, M62V, M62T, M62Q, M62A, L63I, L63V, L63T, L63Q, L63H, L63A, Q64H, Q64S, Q64T, Q64N, F67I, F67V, F70I, F70V, R71H, R71Q, Q72H, Q72S, Q72T, Q72N, D73Q, D73H, D73G, D73N, G78D, G78E, G78K, G78R, W79H, W79S, N80D, N80E, N80K, N80R, E81Q, E81H, E81K, E81N, T82D, T82E, T82K, T82R, I83D, I83E, I83K, I83R, I83N, I83Q, I83S, I83T, E85Q, E85H, E85K, E85N, N86D, N86E, N86K, N86R, N86Q, N86S, N86T, L87D, L87E, L87K, L87R, L87N, L87Q, L87S, L87T, L87I, L87V, L87H, L87A, L88I, L88V, L88T, L88Q, L88H, L88A, A89D, A89E, A89K, A89R, N90D, N90E, N90K, N90Q, N90R, N90S, N90T, N90C, V91D, V91E, V91K, V91N, V91Q, V91R, V91S, V91T, V91C, Y92H, Y921, Q94D, Q94E, Q94K, Q94N, Q94R, Q94S, Q94T, Q94C, I95D, I95E, I95K, I95N, I95Q, I95R, I95S, I95T, H97D, H97E, H97K, H97N, H97Q, H97R, H97S, H97T, H97C, L98D, L98E, L98K, L98N, L98Q, L98R, L98S, L98T, L98C, L98I, L98V, L98H, L98A, K99N, K99Q, K99T, K99S, K99H, V101D, V101E, V101K, V101N, V101Q, V101R, V101S, V101T, V101C, L102I, L102V, L102T, L102Q, L102H, L102A, E103K, E103N, E103Q, E103H, E104Q, E104H, E104R, E104N, K105Q, K105T, K105S, K105H, K105D, K105N, L106I, L106V, L106T, L106Q, L106H, L106A, E107Q, E107H, E107R, E107N, K108D, K108N, K108Q, K108T, K108S, K108H, E109H, E109Q, E109R, E109N, D110K, D110N, D110Q, D110H, D110G, F111I, F111V, R113H, R113Q, R113E, K115D, K115Q, K115N, K115S, K115H, L116V, L116I, L116T, L116Q, L116H, L116A, M117I, M117V, M117T, M117Q, M117A, L120V, L120I, L120T, L120Q, L120H, L120A, L122I, L122V, L122T, L122Q, L122H, L122A, K123Q, K123T, K123S, K123H, K123N, R124D, R124E, R124H, R124Q, Y125H, Y125I, Y126H, Y126I, R128H, R128Q, L130V, L130I, L130T, L130Q, L130H, L130A, Y132H, Y132I, L133I, L133V, L133T, L133Q, L133H, L133A, K134Q, K134T, K134S, K134H, K134N, K136N, K136Q, K136T, K136S, K136H, E137Q, E137H, E137N, Y138H, Y138I, W143H, W143S, R147H, R147Q, E149Q, E149H, E149N, L151I, L151V, L151T, L151Q, L151H, L151A, R152D, R152H, R152Q, F154I, F154V, Y155H, Y155I, F156I, F156V, R159H, R159Q, L1601, L160V, L160T, L160Q, L160H, L160A, Y163H, Y163I, L164I, L164V, L164T, L164Q, L164H, L164A, R165D, R165H, and R165Q in the mature IFN-β

polypeptide set forth in SEQ ID NO;

1.

10. A modified IFN-β

polypeptide of claim 9, wherein the modifications correspond to modifications selected from among L5D/L6E, L5E/Q10D, L5Q/M36I, L6E/L47I, L5E/K108S, L5E/L6E, L5D/Q10D, L5N/M36I, L6Q/L47I, L5D/K108S, L5N/L6E, L5Q/Q10D, L6E/M36I, L5E/N86Q, L5Q/K108S, L5Q/L6E, L5N/Q10D, L6Q/M36I, L5D/N86Q, L5N/K108S, L5D/L6Q, L6E/Q10D, L5E/L47I, L5Q/N86Q, L6E/L6Q, L6Q/Q10D, L5D/L47I, L6Q/K108S, L5N/L6Q, L6E/M36I, L5Q/L47I, L6E/N86Q, L5Q/L6Q, L5D/M36I, L5N/L47I, L6Q/N86Q, L6E/K108S, and L5N/N86Q in the mature IFN-β

polypeptide set forth in SEQ ID NO;

1.

11. The modified IFN-β

polypeptide of claim 1, wherein the unmodified IFN-β

polypeptide contains a sequence of amino acids set forth SEQ ID NO;

1 or SEQ ID NO;

3.

12. The modified IFN-β

polypeptide of claim 1, comprising a sequence of amino acids set forth in any one of SEQ ID NOS;

88-125.

13. The modified IFN-β

polypeptide of claim 1, wherein the unmodified IFN-β

polypeptide is an allelic or species variant of the polypeptide set forth in SEQ ID NO;

1.

14. The modified IFN-β

polypeptide of claim 13, wherein the allelic or species variant has 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide set forth in SEQ ID NO;

1 excluding the amino acid modification(s).

15. The modified IFN-β

polypeptide of claim 1, wherein the modification at position 5 is or corresponds to L5D and/or the modification at position 6 is or corresponds to L6Q.

16. The modified IFN-β

polypeptide of claim 15, wherein the amino acid modification is or corresponds to L5D/L47I or to L5D/L6Q.

17. The modified IFN-β

polypeptide of claim 16, comprising the sequence of amino acids set forth in SEQ ID NO;

115.

18. The modified IFN-β

polypeptide of claim 16, comprising the sequence of amino acids set forth in SEQ ID NO;

108.

19. The modified IFN-β

polypeptide of claim 15, wherein the amino acid modification is or corresponds to L6Q/K108S.

20. The modified IFN-β

polypeptide of claim 19, comprising the sequence of amino acids set forth in SEQ ID NO;

117.

21. A modified IFN-β

polypeptide of claim 1 that exhibits increased protein stability compared to the unmodified IFN-β

polypeptide and retains one or more activities of the unmodified IFN-β

polypeptide, wherein only the primary sequence is modified.

22. The modified IFN-β

polypeptide of claim 1, further comprising one or more amino acid modifications contributing to or permitting one or more of deimmunization, glycosylation and PEGylation of the polypeptide.

23. The modified IFN-β

polypeptide of claim 22, wherein the polypeptide is glycosylated and/or conjugated to a polyethylene glycol (PEG) moiety.

24. The modified IFN-β

of claim 1 that exhibits increased protease resistance.

25. The modified IFN-β

polypeptide of claim 24, wherein the protease is selected from among one or more of pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α

-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA.

26. A modified IFN-β

polypeptide of claim 25, wherein the protease is gelatinase B and the amino acid modifications correspond to modifications selected from among L6E/K108S, L5Q/K108S, L5E/K108S, L5N/Q10D, and L5N/K108S of a mature IFN-β

polypeptide.

27. The modified IFN-β

polypeptide of claim 1, wherein the one or more amino acid modifications are selected from natural amino acids, non-natural amino acids and a combination of natural and non-natural amino acids.

28. The modified IFN-β

polypeptide of claim 1 that is a naked polypeptide chain.

29. The modified IFN-β

polypeptide of claim 1 that is pegylated, albuminated and/or glycosylated.

30. A nucleic acid molecule, comprising a sequence of nucleotides encoding a modified IFN-β

polypeptide of claim 1.

31. A vector, comprising the nucleic acid molecule of claim 30.

32. A cell, comprising the vector of claim 31.

33. The cell of claim 32 that is a eukaryotic cell.

34. The cell of claim 32 that is a prokaryotic cell.

35. The cell of claim 32 that is an algal cell.

36. A method for production of a modified IFN-β

polypeptide, comprising introducing a nucleic acid molecule of claim 30 into a cell, and culturing the cell under conditions whereby the encoded modified IFN-α

polypeptide is expressed.

37. The method of claim 36, wherein the cell is a bacterial cell.

38. The method of claim 36, wherein the cell is a eukaryotic cell.

39. The method of claim 36, wherein the cell is a Chinese hamster ovary cell.

40. The method of claim 36, wherein the cell is an algal cell.

41. A method for production of a modified IFN-β

polypeptide, comprising introducing a nucleic acid molecule of claim 30 into a cell-free system, whereby the encoded modified IFN-β

polypeptide is expressed.

42. The method of claim 36, wherein the modified IFN-β

polypeptide is glycosylated.

43. A pharmaceutical composition, comprising a modified IFN-β

polypeptide of claim 1 in a pharmaceutically acceptable excipient.

44. A pharmaceutical composition, consisting essentially of a modified IFN-β

polypeptide of claim 1.

45. The pharmaceutical composition of claim 43 that is formulated for oral, nasal or pulmonary administration.

46. The pharmaceutical composition of claim 45, wherein the pharmaceutical composition is formulated for oral administration.

47. The pharmaceutical composition of claim 43, wherein modified IFN-β

polypeptide exhibits increased protease resistance or increased conformation stability compared to an IFN-β

polypeptide without the recited modifications.

48. The pharmaceutical composition of claim 43, wherein the modified IFN-β

polypeptide exhibits increased protease resistance compared to an IFN-α

polypeptide without the recited modifications.

49. The pharmaceutical composition of claim 48, wherein the modified IFN-β

polypeptides exhibits increased protease resistance to one or more proteases in the gastrointestinal tract.

50. The pharmaceutical composition of claim 49, where the one or more proteases is/are selected from among pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α

-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA.

51. The pharmaceutical composition of claim 50, wherein the protease is gelatinase B.

52. The pharmaceutical composition of claim 43, wherein the pharmaceutical composition does not contain exogenously added protease inhibitors.

53. The pharmaceutical composition of claim 43, wherein the excipient is selected from among a binding agent, a filler, a lubricant, a disintegrant and a wetting agent.

54. The pharmaceutical composition of claim 43 that is formulated as a liquid, a pill, a tablet, a lozenge or a capsule.

55. The pharmaceutical composition of claim 54, wherein the pill or tablet is chewable.

56. The pharmaceutical composition of claim 54, wherein the lozenge delivers the modified IFN-β

polypeptide to the mucosa of the mouth, throat, or gastrointestinal tract.

57. The pharmaceutical composition of claim 43, wherein the pharmaceutical composition is formulated for controlled-release of the modified IFN-β

polypeptide.

58. The pharmaceutical composition of claim 54, wherein the pill, tablet, or capsule is coated with an enteric coating.

59. A pharmaceutical composition, comprising a nucleic acid of claim 30.

60. A method, comprising treating a subject by administering the pharmaceutical composition of claim 43, wherein the subject has a disease or condition that is responsive to administration of IFN-β

.

61. The method of claim 60, wherein the disease or condition is selected from among a viral infection, a proliferative disorder, an autoimmune disease, and an inflammatory disorder.

62. The method of claim 61, wherein the autoimmune disease is multiple sclerosis, rheumatoid arthritis, chronic viral hepatitis, hepatitis A, hepatitis B, and myocardial viral infection.

63. The method of claim 61, wherein the proliferative disease is a cancer or bone disorder.

64. The method of claim 63, wherein the cancer is selected from among uveal melanoma, colon cancer, liver cancer, and a metastatic cancer.

65. The method of claim 63, wherein the bone disorder is osteoporosis or osteopenia.

66. The method of claim 61, wherein the inflammatory disorder is selected from among asthma, Guillain-Barre syndrome, and an inflammatory bowel disease.

67. The method of claim 66, wherein the inflammatory bowel disease is ulcerative colitis or Crohn'"'"'s disease.

68. The method of claim 61, wherein the viral infection is chronic viral hepatitis or myocardial viral infection.

69. A method of treating multiple sclerosis, comprising administering a modified IFN-β

polypeptide of claim 1, wherein the polypeptide exhibits increased resistance to cleavage by gelatinase B.