IL-1ra variants

• US 20050282752A1
• Filed: 04/01/2005
• Published: 12/22/2005
• Est. Priority Date: 04/02/2004
• Status: Active Grant

• Current Assignee: Swedish Orphan Biovitrum AB (publ)
• Original Assignee: Amgen Inc.
• Inventors: Kerwin, Bruce, Raibekas, Andrei
• Granted Patent: US 7,619,066 B2
• Time in Patent Office: Days
• US Class Current: 530/351
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/7012 Compounds having a free or esterified carboxyl group attached, directly or through a carbon chain, to a carbon atom of the saccharide radical, e.g. glucuronic acid, neuraminic acid gluconic acid A61K31/191; ascorbic acid A61K31/375
• CPC Class Codes: A61K 33/42 Phosphorus; Compounds thereof
• CPC Class Codes: A61K 38/20 Interleukins [IL]
• CPC Class Codes: A61K 47/02 Inorganic compounds
• CPC Class Codes: A61K 47/12 Carboxylic acids; Salts or anhydrides thereof
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 13/12 of the kidneys
• 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 35/02 specific for leukemia
• CPC Class Codes: C07K 14/54 Interleukins [IL]
• CPC Class Codes: C07K 14/545 IL-1
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention provides for an IL-1ra having reduced aggregation, methods of reducing aggregation of IL-1ra, and kits comprising an IL-1ra having reduced aggregation.

Claims

1. A method of reducing aggregation of an aggregating interleukin-1 receptor antagonist (IL-1ra), comprising:

a) replacing Lysine-93 of IL-1ra with an amino acid that does not have a charge;

b) replacing Arginine-97 of IL-1ra with an amino acid that does not have a charge;

or

c) replacing Lysine-93 of IL-1ra with an amino that does not have a charge and replacing Arginine-97 with an amino acid that does not have a charge;

thereby producing an IL-1ra having reduced aggregation.

2. The method of claim 1, wherein Lysine-93 is replaced with an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

3. The method of claim 2, wherein Lysine-93 is replaced with an amino acid selected from alanine and glycine.

4. The method of claim 1, wherein Arginine-97 is replaced with an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

5. The method of claim 4, wherein Arginine-97 is replaced with an amino acid selected from alanine and glycine.

6. The method of claim 1, further comprising incubating the IL-1ra having reduced aggregation with at least one accessory molecule.

7. The method of claim 6, wherein at least one accessory molecule is selected from a sugar and a multiple-charge anion.

8. The method of claim 7, wherein at least one accessory molecule is a multiple-charge anion.

9. The method of claim 8, wherein said multiple-charge anion is 1 to 20 mM pyrophosphate.

10. The method of claim 8, wherein said multiple-charge anion is 1 to 20 mM citrate.

11. The method of claim 7, wherein at least one accessory molecule is a sugar.

12. The method of claim 11, wherein said sugar is glycerol, sucrose, or sorbitol.

13. The method of claim 11, wherein said sugar is at a concentration of from 1 to 3 percent.

14. The method of claim 6, wherein at least one accessory molecule is selected from a lysine-reactive accessory molecule and an arginine-reactive accessory molecule.

15. The method of claim 6, wherein at least one accessory molecule is selected from 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD-X), methyl acetyl phosphate (MAP), and citraconic anhydride.

16. A method of preparing an interleukin-1 receptor antagonist (IL-1ra) drug formulation comprising:

a) replacing Lysine-93 of an aggregating IL-1ra with an amino acid that does not have a charge;

b) replacing Arginine-97 of an aggregating IL-1ra with an amino acid that does not have a charge;

or

c) replacing Lysine-93 of an aggregating IL-1ra with an amino that does not have a charge and replacing Arginine-97 of the aggregating IL-1ra with an amino acid that does not have a charge;

thereby producing a drug formulation comprising an IL-1ra having reduced aggregation.

17. The method of claim 16, wherein Lysine-93 is replaced with an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

18. The method of claim 17, wherein Lysine-93 is replaced with an amino acid selected from alanine and glycine.

19. The method of claim 16, wherein Arginine-97 is replaced with an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

20. The method of claim 19, wherein Arginine-97 is replaced with an amino acid selected from alanine and glycine.

21. The method of claim 16, further comprising incubating the IL-1ra having reduced aggregation with at least one accessory molecule.

22. The method of claim 21, wherein at least one accessory molecule is selected from a sugar and a multiple-charge anion.

23. The method of claim 22, wherein at least one accessory molecule is a multiple-charge anion.

24. The method of claim 23, wherein said multiple-charge anion is 1 to 20 mM pyrophosphate.

25. The method of claim 23, wherein said multiple-charge anion is 1 to 20 mM citrate.

26. The method of claim 22, wherein at least one accessory molecule is a sugar.

27. The method of claim 26, wherein said sugar is glycerol, sucrose, or sorbitol.

28. The method of claim 26, wherein said is at a concentration of from 1 to 3 percent.

29. The method of claim 16, wherein at least one accessory molecule is selected from a lysine-reactive accessory molecule and an arginine-reactive accessory molecule.

30. The method of claim 16, wherein at least one accessory molecule is selected from 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD-X), methyl acetyl phosphate (MAP), and citraconic anhydride.

31. A method of treating a patient comprising administering to the patient a therapeutically effective amount of an IL-1ra having reduced aggregation, wherein the IL-1ra having reduced aggregation is selected from:

a) an IL-1ra wherein Lysine-93 is replaced with an amino acid that does not have a charge;

b) an IL-1ra wherein Arginine-97 is replaced with an amino acid that does not have a charge;

c) an IL-1ra wherein Lysine-93 is replaced with a first amino acid that does not have a charge and Arginine-97 is replaced with a second amino acid that does not have a positive charge, wherein the first amino acid that does not have a positive charge and the second amino acid that does not have a positive charge are the same or different.

32. The method of claim 31, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Lysine-93 is replaced with an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

33. The method of claim 32, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Lysine-93 is replaced with an amino acid selected from alanine and glycine.

34. The method of claim 31, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Arginine-97 is replaced with an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

35. The method of claim 34, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Arginine-97 is replaced with an amino acid selected from alanine and glycine.

36. The method of claim 31, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Lysine-93 is replaced with an amino acid selected from alanine and glycine and Arginine-97 is replaced with an amino acid selected from alanine and glycine.

37. A method of treating a patient having rheumatoid arthritis comprising administering to the patient a therapeutically effective amount of an IL-1ra having reduced aggregation, wherein the IL-1ra having reduced aggregation is selected from:

a) an IL-1ra wherein Lysine-93 is replaced with an amino acid that does not have a positive charge;

b) an IL-1ra wherein Arginine-97 is replaced with an amino acid that does not have a positive charge;

c) an IL-1ra wherein Lysine-93 is replaced with a first amino acid that does not have a positive charge and Arginine-97 is replaced with a second amino acid that does not have a positive charge, wherein the first amino acid that does not have a positive charge and the second amino acid that does not have a positive charge are the same or different.

38. The method of claim 37, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Lysine-93 is replaced with an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

39. The method of claim 38, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Lysine-93 is replaced with an amino acid selected from alanine and glycine.

40. The method of claim 37, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Arginine-97 is replaced with an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

41. The method of claim 40, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Arginine-97 is replaced with an amino acid selected from alanine and glycine.

42. The method of claim 37, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Lysine-93 is replaced with an amino acid selected from alanine and glycine and Arginine-97 is replaced with an amino acid selected from alanine and glycine.

43. A method of treating a patient having osteoarthritis comprising administering to the patient a therapeutically effective amount of an IL-1ra having reduced aggregation, wherein the IL-1ra having reduced aggregation is selected from:

a) an IL-1ra wherein Lysine-93 is replaced with an amino acid that does not have a positive charge;

b) an IL-1ra wherein Arginine-97 is replaced with an amino acid that does not have a positive charge;

c) an IL-1ra wherein Lysine-93 is replaced with a first amino acid that does not have a positive charge and Arginine-97 is replaced with a second amino acid that does not have a positive charge, wherein the first amino acid that does not have a positive charge and the second amino acid that does not have a positive charge are the same or different.

44. The method of claim 43, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Lysine-93 is replaced with an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

45. The method of claim 44, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Lysine-93 is replaced with an amino acid selected from alanine and glycine.

46. The method of claim 43, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Arginine-97 is replaced with an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

47. The method of claim 46, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Arginine-97 is replaced with an amino acid selected from alanine and glycine.

48. The method of claim 43, wherein the IL-1ra having reduced aggregation is an IL-1ra wherein Lysine-93 is replaced with an amino acid selected from alanine and glycine and Arginine-97 is replaced with an amino acid selected from alanine and glycine.

49. A protein having the amino acid sequence shown in FIG. 11 (SEQ ID NO:

12) wherein X₃ is a first amino acid that does not have a positive charge and X₄ is a second amino acid that does not have a positive charge, wherein the first amino acid that does not have a positive charge and the second amino acid that does not have a positive charge are the same or different.

50. The protein of claim 49, wherein X₃ is an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

51. The protein of claim 50, wherein X₃ is an amino acid selected from alanine and glycine.

52. The protein of claim 49, wherein X₄ is an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

53. The protein of claim 52, wherein X₄ is an amino acid selected from alanine and glycine.

54. The protein of claim 49, wherein X₃ is an amino acid selected from alanine and glycine and X₄ is an amino acid selected from alanine and glycine.

55. The protein of claim 49 produced in E. coli.

56. The protein of claim 54 produced in E. coli.

57. A protein having the amino acid sequence shown in FIG. 11 (SEQ ID NO:

12) wherein X₃ is an amino acid that does not have a positive charge.

58. The protein of claim 57, wherein X₃ is an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

59. The protein of claim 58, wherein X₃ is an amino acid selected from alanine and glycine.

60. The protein of claim 57 produced in E. coli.

61. A protein having the amino acid sequence shown in FIG. 11 (SEQ ID NO:

12) wherein X₄ is an amino acid that does not have a positive charge.

62. The protein of claim 61, wherein X₄ is an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

63. The protein of claim 62, wherein X₄ is an amino acid selected from alanine and glycine.

64. The protein of claim 61 produced in E. coli.