Neublastin Variants

• US 20080039385A1
• Filed: 08/18/2005
• Published: 02/14/2008
• Est. Priority Date: 08/19/2004
• Status: Active Grant

• Current Assignee: Sinfonia Biotherapeutics AB
• Original Assignee: Biogen Idec MA Inc. (Biogen, Inc.)
• Inventors: Pepinsky, R., Rossomando, Anthony, Silvian, Laura
• Granted Patent: US 8,263,553 B2
• Time in Patent Office: Days
• US Class Current: 514/12
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/185 Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/4756 Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
• CPC Class Codes: C07K 14/495 Transforming growth factor [TGF]
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• 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

Compositions and methods for folding proteins belonging to the transforming growth factor beta superfamily are disclosed. The compositions and methods allow for the folding of such proteins when produced in an expression system that does not yield a properly folded, biologically active product.

Claims

1. A polypeptide comprising an amino acid sequence at least 80% identical to amino acids 15-113 of SEQ ID NO:

1, wherein the amino acid sequence comprises at least one amino acid substitution, relative to SEQ ID NO;

1, selected from the group consisting of;

an amino acid other than arginine at the position corresponding to position 48 of SEQ ID NO;

1;

an amino acid other than arginine at the position corresponding to position 49 of SEQ ID NO;

1; and

an amino acid other than arginine at the position corresponding to position 51 of SEQ ID NO;

1,

wherein the polypeptide, when dimerized, binds to a complex containing GFRalpha3 and RET.

2. The polypeptide of claim 1, wherein the amino acid sequence comprises an amino acid other than arginine at the position corresponding to position 48 of SEQ ID NO:

1.

3. The polypeptide of claim 2, wherein the arginine residue at position 48 of SEQ ID NO:

1 is substituted with a non-conservative amino acid residue.

4. The polypeptide of claim 2, wherein the arginine residue at position 48 of SEQ ID NO:

1 is substituted with glutamic acid.

5. The polypeptide of claim 1, wherein the amino acid sequence comprises an amino acid other than arginine at the position corresponding to position 49 of SEQ ID NO:

1.

6. The polypeptide of claim 5, wherein the arginine residue at position 49 of SEQ ID NO:

1 is substituted with a non-conservative amino acid residue.

7. The polypeptide of claim 5, wherein the arginine residue at position 49 of SEQ ID NO:

1 is substituted with glutamic acid.

8. The polypeptide of claim 1, wherein the amino acid sequence comprises an amino acid other than arginine at the position corresponding to position 51 of SEQ ID NO:

1.

9. The polypeptide of claim 8, wherein the arginine residue at position 51 of SEQ ID NO:

1 is substituted with a non-conservative amino acid residue.

10. The polypeptide of claim 8, wherein the arginine residue at position 51 of SEQ ID NO:

1 is substituted with glutamic acid.

11. The polypeptide of claim 1, wherein the amino acid sequence comprises amino acids other than arginine at the positions corresponding to position 48 and position 49 of SEQ ID NO:

1.

12. The polypeptide of claim 11, wherein the arginine residue at position 48 and the arginine reside at position 49 of SEQ ID NO:

1 are substituted with non-conservative amino acid residues.

13. The polypeptide of claim 11, wherein the arginine residue at position 48 and the arginine reside at position 49 of SEQ ID NO:

1 are each substituted with glutamic acid.

14. The polypeptide of claim 1, wherein the amino acid sequence is at least 90% identical to amino acids 15-113 of SEQ ID NO:

1.

15. The polypeptide of claim 1, wherein the amino acid sequence is at least 95% identical to amino acids 15-113 of SEQ ID NO:

1.

16. The polypeptide of claim 1, wherein the amino acid sequence is at least 98% identical to amino acids 15-113 of SEQ ID NO:

1.

20. A dimer comprising two polypeptides according to claim 1.

21. A conjugate comprising the polypeptide of claim 1 conjugated to a non-naturally occurring polymer.

22. A fusion protein comprising the polypeptide of claim 1 and a heterologous amino acid sequence.

23. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier or excipient.

24. A nucleic acid comprising a sequence that encodes the polypeptide of claim 1.

25. An expression vector comprising the nucleic acid of claim 24.

26. A cell comprising the expression vector of claim 25.

27. A method of making a polypeptide, the method comprising:

providing the cell of claim 26, and

culturing the cell under conditions that permit expression of the nucleic acid.

28. A method of treating or preventing a nervous system disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition of claim 23.

29. A method of treating neuropathic pain in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition of claim 23.

30. A method of activating the RET receptor in a mammal, the method comprising administering to the mammal an effective amount of the polypeptide of claim 1.

17. A polypeptide comprising amino acids 15-113 of SEQ ID NO:

2, amino acids 15-113 of SEQ ID NO;

3, amino acids 15-113 of SEQ ID NO;

4, amino acids 15-113 of SEQ ID NO;

5, amino acids 15-113 of SEQ ID NO;

8, or amino acids 15-113 of SEQ ID NO;

9.

18. The polypeptide of claim 17, wherein the polypeptide comprises amino acids 10-113 of SEQ ID NO:

2, amino acids 10-113 of SEQ ID NO;

3, amino acids 10-113 of SEQ ID NO;

4, amino acids 10-113 of SEQ ID NO;

5, amino acids 10-113 of SEQ ID NO;

8, or amino acids 10-113 of SEQ ID NO;

9.

19. The polypeptide of claim 17, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

2, the amino acid sequence of SEQ ID NO;

3, the amino acid sequence of SEQ ID NO;

4, the amino acid sequence of SEQ ID NO;

5, the amino acid sequence of SEQ ID NO;

8, or the amino acid sequence of SEQ ID NO;

9.

31. A polypeptide comprising an amino acid sequence at least 80% identical to amino acids 15-113 of SEQ ID NO:

1, wherein the amino acid sequence comprises at least one amino acid substitution, relative to SEQ ID NO;

1, selected from the group consisting of;

an amino acid other than serine at the position corresponding to position 20 of SEQ ID NO;

1;

an amino acid other than glutamine at the position corresponding to position 21 of SEQ ID NO;

1;

an amino acid other than histidine at the position corresponding to position 32 of SEQ ID NO;

1;

an amino acid other than arginine at the position corresponding to position 33 of SEQ ID NO;

1;

an amino acid other than arginine at the position corresponding to position 39 of SEQ ID NO;

1;

an amino acid other than serine at the position corresponding to position 46 of SEQ ID NO;

1;

an amino acid other than arginine at the position corresponding to position 68 of SEQ ID NO;

1;

an amino acid other than glycine at the position corresponding to position 72 of SEQ ID NO;

1;

an amino acid other than serine at the position corresponding to position 73 of SEQ ID NO;

1; and

an amino acid other than valine at the position corresponding to position 94 of SEQ ID NO;

1,

wherein the polypeptide, when dimerized, binds to a complex containing GFRalpha3 and RET.

32. The polypeptide of claim 31, wherein the amino acid sequence is at least 90% identical to amino acids 15-113 of SEQ ID NO:

1.

33. The polypeptide of claim 31, wherein the amino acid sequence is at least 95% identical to amino acids 15-113 of SEQ ID NO:

1.

34. The polypeptide of claim 31, wherein the amino acid sequence is at least 98% identical to amino acids 15-113 of SEQ ID NO:

1.

48. A dimer comprising two polypeptides according to claim 31.

49. A conjugate comprising the polypeptide of claim 31 conjugated to a non-naturally occurring polymer.

50. A fusion protein comprising the polypeptide of claim 31 and a heterologous amino acid sequence.

51. A pharmaceutical composition comprising the polypeptide of claim 31 and a pharmaceutically acceptable carrier or excipient.

52. A nucleic acid comprising a sequence that encodes the polypeptide of claim 31.

53. An expression vector comprising the nucleic acid of claim 52.

54. A cell comprising the expression vector of claim 53.

55. A method of making a polypeptide, the method comprising:

providing the cell of claim 54, and

culturing the cell under conditions that permit expression of the nucleic acid.

56. A method of treating or preventing a nervous system disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition of claim 51.

57. A method of treating neuropathic pain in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition of claim 51.

58. A method of activating the RET receptor in a mammal, the method comprising administering to the mammal an effective amount of the polypeptide of claim 31.

35. A polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:

1, wherein the amino acid sequence comprises at least one amino acid substitution, relative to SEQ ID NO;

1, selected from the group consisting of;

an amino acid other than arginine at the position corresponding to position 7 of SEQ ID NO;

1;

an amino acid other than arginine at the position corresponding to position 9 of SEQ ID NO;

1; and

an amino acid other than arginine at the position corresponding to position 14 of SEQ ID NO;

1,

wherein the polypeptide, when dimerized, binds to a complex containing GFRalpha3 and RET.

36. The polypeptide of claim 35, wherein the amino acid sequence comprises an amino acid other than arginine at the position corresponding to position 7 of SEQ ID NO:

1.

37. The polypeptide of claim 36, wherein the arginine residue at position 7 of SEQ ID NO:

1 is substituted with a non-conservative amino acid residue.

38. The polypeptide of claim 36, wherein the arginine residue at position 7 of SEQ ID NO:

1 is substituted with glutamic acid.

39. The polypeptide of claim 35, wherein the amino acid sequence comprises an amino acid other than arginine at the position corresponding to position 9 of SEQ ID NO:

1.

40. The polypeptide of claim 39, wherein the arginine residue at position 9 of SEQ ID NO:

1 is substituted with a non-conservative amino acid residue.

41. The polypeptide of claim 39, wherein the arginine residue at position 9 of SEQ ID NO:

1 is substituted with glutamic acid.

42. The polypeptide of claim 35, wherein the amino acid sequence comprises an amino acid other than arginine at the position corresponding to position 14 of SEQ ID NO:

1.

43. The polypeptide of claim 42, wherein the arginine residue at position 14 of SEQ ID NO:

1 is substituted with a non-conservative amino acid residue.

44. The polypeptide of claim 42, wherein the arginine residue at position 14 of SEQ ID NO:

1 is substituted with glutamic acid.

45. The polypeptide of claim 35, wherein the amino acid sequence is at least 90% identical to SEQ ID NO:

1.

46. The polypeptide of claim 35, wherein the amino acid sequence is at least 95% identical to SEQ ID NO:

1.

47. The polypeptide of claim 35, wherein the amino acid sequence is at least 98% identical to amino acids 15-113 of SEQ ID NO:

1.