Polymer conjugates of mutated neublastin
View Patent ↗A dimer comprising a mutated neublastin polypeptide coupled to a polymer is disclosed. Such dimers exhibit prolonged bioavailability and, in preferred embodiments, prolonged biological activity relative to wild-type forms of neublastin.
1. An isolated polypeptide comprising an amino acid sequence at least 90% identical to amino acids 8-113 of SEQ ID NO:1, wherein the polypeptide includes at least one amino acid substitution selected from the group consisting of:
(a) an amino acid other than arginine at the position corresponding to position 14 in SEQ ID NO:1;
(b) an amino acid other than arginine at the position corresponding to position 39 in SEQ ID NO:1;
(c) an amino acid other than arginine at the position corresponding to position 68 in SEQ ID NO:1; and
(d) an amino acid other than asparagine at the position corresponding to position 95 in SEQ ID NO:1,
wherein the polypeptide, when dimerized, binds to GFRα3.
2. The polypeptide of claim 1 , wherein the amino acid at the position corresponding to position 95 in SEQ ID NO:1 is an amino acid other than asparagine.
3. The polypeptide of claim 1 , wherein the amino acid at the position corresponding to position 95 in SEQ ID NO:1 is lysine.
4. The polypeptide of claim 1 , wherein the polypeptide comprises amino acids 8-113 of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, wherein an amino acid other than asparagine is substituted for the asparagine at position 95.
5. The polypeptide of claim 1 , wherein the polypeptide comprises amino acids 1-113 of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, wherein lysine is substituted for asparagine at position 95.
6. The polypeptide of claim 1 , wherein the amino acid sequence is at least 95% identical to amino acids 8-113 of SEQ ID NO:1.
7. The polypeptide of claim 1 , wherein the amino acid sequence is at least 95% identical to amino acids 8-113 of SEQ ID NO:2.
8. The polypeptide of claim 2 , wherein the amino acid sequence is at least 95% identical to amino acids 8-113 of SEQ ID NO:2.
9. The polypeptide of claim 3 , wherein the amino acid sequence is at least 95% identical to amino acids 8-113 of SEQ ID NO:2.
10. A fusion protein comprising the polypeptide of claim 1 fused to a second moiety.
11. The fusion protein of claim 10 , wherein the second moiety is a human serum albumin sequence.
12. A dimer comprising two polypeptides according to claim 1 .
13. A dimer comprising two fusion proteins according to claim 10 .
14. A conjugate comprising the polypeptide of claim 1 conjugated to a non-naturally occurring polymer.
15. A conjugate comprising the polypeptide of claim 7 conjugated to a non-naturally occurring polymer.
16. A conjugate comprising the polypeptide of claim 8 conjugated to a non-naturally occurring polymer.
17. A conjugate comprising the polypeptide of claim 9 conjugated to a non-naturally occurring polymer.
18. A conjugate comprising the fusion protein of claim 10 conjugated to a non-naturally occurring polymer.
19. The conjugate of claim 14 , wherein the polymer is a polyalkylene glycol.
20. The conjugate of claim 19 , wherein the polyalkylene glycol is polyethylene glycol.
21. The conjugate of claim 15 , wherein the polymer is a polyalkylene glycol.
22. The conjugate of claim 21 , wherein the polyalkylene glycol is polyethylene glycol.
23. The conjugate of claim 16 , wherein the polymer is a polyalkylene glycol.
24. The conjugate of claim 23 , wherein the polyalkylene glycol is polyethylene glycol.
25. The conjugate of claim 17 , wherein the polymer is a polyalkylene glycol.
26. The conjugate of claim 25 , wherein the polyalkylene glycol is polyethylene glycol.
27. The conjugate of claim 19 , wherein the polyalkylene glycol is conjugated to a lysine residue substituted at a position corresponding to position 14, 39, 68, or 95 in SEQ ID NO:1.
28. The conjugate of claim 19 , wherein the polyalkylene glycol is conjugated to the N terminus of the polypeptide.
29. The conjugate of claim 19 , wherein the polypeptide is glycosylated.
30. A pharmaceutical composition comprising the polypeptide of claim 1 and physiologically acceptable vehicle.
31. A pharmaceutical composition comprising the fusion protein of claim 10 and physiologically acceptable vehicle.
32. A pharmaceutical composition comprising the conjugate of claim 19 and physiologically acceptable vehicle.
33. A polypeptide comprising an amino acid sequence at least 95% identical to amino acids 15-113 of SEQ ID NO:2 (NBN99), wherein the polypeptide includes at least one amino acid substitution selected from the group consisting of:
(a) an amino acid other than arginine at the position corresponding to position 39 in SEQ ID NO:2;
(b) an amino acid other than arginine at the position corresponding to position 68 in SEQ ID NO:2; and
(c) an amino acid other than asparagine at the position corresponding to position 95 in SEQ ID NO:2,
wherein the polypeptide, when dimerized, binds to GFRα3.
34. The polypeptide of claim 33 , wherein the amino acid at the position corresponding to position 39 in SEQ ID NO:2 is an amino acid other than arginine.
35. The polypeptide of claim 33 , wherein the amino acid at the position corresponding to position 39 in SEQ ID NO:2 is lysine.
36. The polypeptide of claim 33 , wherein the amino acid at the position corresponding to position 68 in SEQ ID NO:2 is an amino acid other than arginine.
37. The polypeptide of claim 33 , wherein the amino acid at the position corresponding to position 68 in SEQ ID NO:2 is lysine.
38. The polypeptide of claim 33 , wherein the amino acid at the position corresponding to position 95 in SEQ ID NO:2 is an amino acid other than asparagine.
39. The polypeptide of claim 33 , wherein the amino acid at the position corresponding to position 95 in SEQ ID NO:2 is lysine.
40. The polypeptide of claim 33 , wherein the polypeptide comprises amino acids 15-113 of SEQ ID NO:2 (NBN99), wherein an amino acid other than asparagine is substituted for the asparagine at position 95.
41. The polypeptide of claim 33 , wherein the polypeptide comprises amino acids 15-113 of SEQ ID NO:2 (NBN99), wherein lysine is substituted for asparagine at position 95.
42. A conjugate comprising the polypeptide of claim 33 conjugated to a non-naturally occurring polymer.
43. A conjugate comprising the polypeptide of claim 34 conjugated to a non-naturally occurring polymer.
44. A conjugate comprising the polypeptide of claim 35 conjugated to a non-naturally occurring polymer.
45. A conjugate comprising the polypeptide of claim 36 conjugated to a non-naturally occurring polymer.
46. A conjugate comprising the polypeptide of claim 37 conjugated to a non-naturally occurring polymer.
47. A conjugate comprising the polypeptide of claim 38 conjugated to a non-naturally occurring polymer.
48. A conjugate comprising the polypeptide of claim 39 conjugated to a non-naturally occurring polymer.
49. A conjugate comprising the polypeptide of claim 40 conjugated to a non-naturally occurring polymer.
50. A conjugate comprising the polypeptide of claim 41 conjugated to a non-naturally occurring polymer.
51. The conjugate of claim 42 , wherein the polymer is a polyalkylene glycol.
52. The conjugate of claim 51 , wherein the polyalkylene glycol is polyethylene glycol.
53. The conjugate of claim 43 , wherein the polymer is a polyalkylene glycol.
54. The conjugate of claim 53 , wherein the polyalkylene glycol is polyethylene glycol.
55. The conjugate of claim 44 , wherein the polymer is a polyalkylene glycol.
56. The conjugate of claim 55 , wherein the polyalkylene glycol is polyethylene glycol.
57. The conjugate of claim 45 , wherein the polymer is a polyalkylene glycol.
58. The conjugate of claim 57 , wherein the polyalkylene glycol is polyethylene glycol.
59. The conjugate of claim 46 , wherein the polymer is a polyalkylene glycol.
60. The conjugate of claim 9 , wherein the polyalkylene glycol is polyethylene glycol.
61. The conjugate of claim 47 , wherein the polymer is a polyalkylene glycol.
62. The conjugate of claim 61 , wherein the polyalkylene glycol is polyethylene glycol.
63. The conjugate of claim 48 , wherein the polymer is a polyalkylene glycol.
64. The conjugate of claim 63 , wherein the polyalkylene glycol is polyethylene glycol.
65. The conjugate of claim 49 , wherein the polymer is a polyalkylene glycol.
66. The conjugate of claim 65 , wherein the polyalkylene glycol is polyethylene glycol.
67. The conjugate of claim 50 , wherein the polymer is a polyalkylene glycol.
68. The conjugate of claim 67 , wherein the polyalkylene glycol is polyethylene glycol.