IL-1ra variants

• US 7,619,066 B2
• Filed: 04/01/2005
• Issued: 11/17/2009
• Est. Priority Date: 04/02/2004
• Status: Active Grant

• Current Assignee: Swedish Orphan Biovitrum AB (publ)
• Original Assignee: Amgen Inc.
• Inventors: Raibekas, Andrei, Kerwin, Bruce
• Primary Examiner(s): Nickol; Gary B
• Assistant Examiner(s): Xie; Xiaozhen
• Application Number:
  US11/097,453
• Publication Number:
  US 20050282752A1
• Time in Patent Office: 1,691 Days
• Field of Search: None
• 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 making a variant of an IL-1ra comprising the amino acid sequence set forth in SEQ ID NO:

3,

wherein the variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO;

3;

wherein;

a) the variant has an amino acid that does not have a charge at the position corresponding to Lysine-93 of SEQ ID NO;

3;

b) the variant has an amino acid that does not have a charge at the position corresponding to Arginine-97 of SEQ ID NO;

3;

or

c) the variant has an amino acid that does not have a charge at the position corresponding to Lysine-93 of SEQ ID NO;

3 and an amino acid that does not have a charge at the position corresponding to Arginine-97 of SEQ ID NO;

3; and

wherein if the variant has an arginine at the position corresponding to Arginine-97 of SEQ ID NO;

3, the variant does not have a threonine or a histidine at the position corresponding to Lysine-93 of SEQ ID NO;

3;

thereby producing an IL-1ra having reduced aggregation.

2. The method of claim 1, wherein the variant has an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine at the position corresponding to Lysine-93 of SEQ ID NO:

3.

3. The method of claim 2, wherein the variant has an amino acid selected from alanine and glycine at the position corresponding to Lysine-93 of SEQ ID NO:

3.

4. The method of claim 1, comprising making a variant of an IL-1ra comprising SEQ ID NO:

3 with a methionine at the N-terminus, wherein the variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO;

3 with a methionine at the N-terminus.

5. The method of claim 1, wherein the variant comprises SEQ ID NO:

3 with a methionine at the N-terminus and has an alanine at the position corresponding to Lysine-93 of SEQ ID NO;

3.

6. The method of claim 1, wherein the variant has an amino acid that does not have a charge at the position corresponding to Arginine-97 of SEQ ID NO:

3.

7. The method of claim 1, wherein the variant has an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine at the position corresponding to Arginine-97 of SEQ ID NO:

3.

8. The method of claim 7, wherein the variant has an amino acid selected from alanine and glycine at the position corresponding to Arginine-97 of SEQ ID NO:

3.

9. The method of claim 1, further comprising incubating the IL-1ra having reduced aggregation with at least one accessory molecule, wherein at least one accessory molecule is selected from a sugar and a multiple-charge anion.

10. The method of claim 9, wherein at least one accessory molecule is a multiple-charge anion.

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

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

13. The method of claim 9, wherein at least one accessory molecule is a sugar.

14. The method of claim 13, wherein said sugar is glycerol, sucrose, or sorbitol.

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

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

17. A method of preparing an interleukin-1 receptor antagonist (IL-1ra) drug formulation comprising making a variant of an IL-1ra comprising the amino acid seguence set forth in SEQ ID NO:

3,

wherein the variant comprises an amino acid seguence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO;

3;

wherein;

a) the variant has an amino acid that does not have a charge at the position corresponding to Lysine-93 of SEQ ID NO;

3;

b) the variant has an amino acid that does not have a charge at the position corresponding to Arginine-97 of SEQ ID NO;

3;

or

c) the variant has an amino acid that does not have a charge at the position corresponding to Lysine-93 of SEQ ID NO;

3 and an amino acid that does not have a charge at the position corresponding to Arginine-97 of SEQ ID NO;

3 and

wherein if the variant has an arginine at the position corresponding to Arginine-97 of SEQ ID NO;

3, the variant does not have a threonine or a histidine at the position corresponding to Lysine-93 of SEQ ID NO;

3;

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

18. The method of claim 17, wherein the variant has an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine at the position corresponding to Lysine-93 of SEQ ID NO:

3.

19. The method of claim 18, wherein the variant has an amino acid selected from alanine and glycine at the position corresponding to Lysine-93 of SEQ ID NO:

3.

20. The method of claim 19, comprising making a variant of an IL-1ra comprising SEQ ID NO:

3 with a methionine at the N-terminus, wherein the variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO;

3 with a methionine at the N-terminus.

21. The method of claim 17, wherein the variant comprises SEQ ID NO:

3 with a methionine at the N-terminus and has an alanine at the position corresponding to Lysine-93 of SEQ ID NO;

3.

22. The method of claim 17, wherein the variant has an amino acid that does not have a charge at the position corresponding to Arginine-97 of SEQ ID NO:

3.

23. The method of claim 17, wherein the variant has an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine at the position corresponding to Arginine-97 of SEQ ID NO:

3.

24. The method of claim 23, wherein the variant has an amino acid selected from alanine and glycine at the position corresponding to Arginine-97 of SEQ ID NO:

3.

25. The method of claim 17, further comprising incubating the IL-1ra having reduced aggregation with at least one accessory molecule, wherein at least one accessory molecule is selected from a sugar and a multiple-charge anion.

26. The method of claim 25, wherein at least one accessory molecule is a multiple-charge anion.

27. The method of claim 26, wherein said multiple-charge anion is 1 to 20 mM pyrophosphate.

28. The method of claim 26, wherein said multiple-charge anion is 1 to 20 mM citrate.

29. The method of claim 25, wherein at least one accessory molecule is a sugar.

30. The method of claim 29, wherein said sugar is glycerol, sucrose, or sorbitol.

31. The method of claim 29, wherein said sugar is at a concentration of from 1 to 3 percent.

32. The method of claim 17, further comprising incubating the IL-1ra having reduced aggregation with at least one accessory molecule, 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.

33. An isolated protein having the amino acid sequence shown in SEQ ID NO:

12, wherein X_aa at position 94 is an amino acid that does not have a charge, and wherein X_aa at position 94 is not a threonine or a histidine.

34. The protein of claim 33, wherein X_aa at position 94 is an amino acid selected from alanine, glycine, glutamine, asparagine, serine, and tyrosine.

35. The protein of claim 34, wherein X_aa at position 94 is an amino acid selected from alanine and glycine.

36. The protein of claim 33 produced in E. coli.

37. An isolated protein having the amino acid sequence shown in SEQ ID NO:

12, wherein X_aa at position 98 is an amino acid that does not have a charge.

38. The protein of claim 37, wherein X_aa at position 98 is an amino acid selected from alanine, glycine, glutamine, asparagine, serine, threonine, and tyrosine.

39. The protein of claim 38, wherein X_aa at position 98 is an amino acid selected from alanine and glycine.

40. The protein of claim 37 produced in E. coli.