Claims
- 1. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one amino acid located in at least one region of granulocyte colony-stimulating factor selected from the group consisting of: the A-B loop, the B-C loop, the first three or last three amino acids in helix B, the first three or last three amino acids in helix C, the first three or last three amino acids in helix D, and the amino acids following helix D; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 2. The cysteine variant according to claim 1, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of: K40, S53, G55, W58, A59, P60, S62, S63, P65, S66, Q67, A68, Q70, A72, Q90, A91, E93, G94, S96, E98, G100, A143, Q145, Q173 and P174.
- 3. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein said variant contains T133 and wherein a cysteine residue is substituted for at least one amino acid located in at least one region of granulocyte colony-stimulating factor selected from the group consisting of: the A-B loop, the B-C loop, the C-D loop, the first three or last three amino acids in helix B, the first three or last three amino acids in helix C, the first three or last three amino acids in helix D, and the amino acids following helix D; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 4. The cysteine variant according to claim 3, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of: K40, S53, G55, W58, A59, P60, S62, S63, P65, S66, Q67, A68, Q70, A72, Q90, A91, E93, G94, S96, E98, G100, G125, M126, A127, A129, Q131, Q134, G135, A136, A139, A141, S142, A143, Q145, Q173 and P174.
- 5. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one amino acid located in the region of granulocyte colony-stimulating factor following helix D; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 6. The cysteine variant according to claim 5, wherein a cysteine residue is substituted for Q173.
- 7. The cysteine variant according to claim 5, wherein a cysteine residue is substituted for P174.
- 8. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one amino acid located in the A-B loop of granulocyte colony-stimulating factor; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 9. The cysteine variant according to claim 8, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of K40, S53, G55, W58, A59, P60, S62, S63, P65, S66, Q67, A68, and Q70.
- 10. The cysteine variant according to claim 8, wherein a cysteine residue is substituted for W58.
- 11. The cysteine variant according to claim 8, wherein a cysteine residue is substituted for A68.
- 12. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one amino acid located in the B-C loop of granulocyte colony-stimulating factor; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 13. The cysteine variant according to claim 12, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of E93, G94, S96, and E98.
- 14. The cysteine variant according to claim 12, wherein a cysteine residue is substituted for E93.
- 15. The cysteine variant according to claim 12, wherein a cysteine residue is substituted for E98.
- 16. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one amino acid located in the C-D loop of granulocyte colony-stimulating factor; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 17. The cysteine variant according to claim 16, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of G125, M126, A127, A129, Q131, Q134, G135, A136, A139, A141, and S142.
- 18. The cysteine variant according to claim 16, wherein a cysteine residue is substituted for A129.
- 19. The cysteine variant according to claim 16, wherein a cysteine residue is substituted for Q131.
- 20. The cysteine variant according to claim 16, wherein a cysteine residue is substituted for T133.
- 21. The cysteine variant according to claim 16, wherein a cysteine residue is substituted for Q134.
- 22. The cysteine variant according to claim 16, wherein a cysteine residue is substituted for A136.
- 23. The cysteine variant according to claim 16, wherein a cysteine residue is substituted for A139.
- 24. The cysteine variant according to claim 16, wherein a cysteine residue is substituted for A141.
- 25. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein said variant contains T133 and wherein a cysteine residue is substituted for at least one amino acid located in the C-D loop of granulocyte colony-stimulating factor; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 26. The cysteine variant according to claim 25, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of G125, M126, A127, A129, Q131, Q134, G135, A136, A139, A141, and S142.
- 27. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one of the first three amino acids in helix A of granulocyte colony-stimulating factor; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 28. The cysteine variant according to claim 27, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of Q11 and S12.
- 29. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for T38; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 30. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one amino acid located in at least one region of granulocyte colony-stimulating factor selected from the group consisting of the first three amino acids in helix B and the last three amino acids in helix B; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 31. The cysteine variant according to claim 30, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of A72, Q90 and A91.
- 32. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one amino acid located in at least one region of granulocyte colony-stimulating factor selected from the group consisting of the first three amino acids in helix C and the last three amino acids in helix C; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 33. The cysteine variant according to claim 33, wherein a cysteine residue is substituted for G100.
- 34. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one amino acid located in at least one region of granulocyte colony-stimulating factor selected from the group consisting of the first three amino acids in helix D and the last three amino acids in helix D; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 35. The cysteine variant according to claim 34, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of A143 and Q145.
- 36. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one amino acid of granulocyte colony-stimulating factor selected from the group consisting of P2, S7, S8, L9 and P10; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 37. The cysteine variant according to claim 36, wherein a cysteine residue is substituted for P2.
- 38. The cysteine variant according to claim 36, wherein a cysteine residue is substituted for S7.
- 39. The cysteine variant according to any one of claims 1, 3, 5, 8, 12, 16, 25, 27, 29, 30, 32, 34 or 36, wherein a non-cysteine amino acid is substituted for C17.
- 40. The cysteine variant according to claim 39, wherein the non-cysteine amino acid substituted for C17 is selected from the group consisting of serine and alanine.
- 41. The cysteine variant according to any one of claims 1, 3, 5, 8, 12, 16, 25, 27, 29, 30, 32, 34 or 36, wherein the substituted cysteine residue is modified with a cysteine-reactive moiety.
- 42. The cysteine variant according to any one of claims 1, 3, 5, 8, 12, 16, 25, 27, 29, 30, 32, 34 or 36, wherein the substituted cysteine residue is modified with a cysteine-reactive moiety, and wherein a non-cysteine amino acid is substituted for C17.
- 43. The cysteine variant according to claim 42, wherein the non-cysteine amino acid substituted for C17 is selected from the group consisting of serine and alanine.
- 44. The cysteine variant according to any one of claims 1, 3, 5, 8, 12, 16, 25, 27, 29, 30, 32, 34 or 36, wherein the substituted cysteine residue is modified with polyethylene glycol.
- 45. The cysteine variant according to any one of claims 1, 3, 5, 8, 12, 16, 25, 27, 29, 30, 32, 34 or 36, wherein the substituted cysteine residue is modified with polyethylene glycol, and wherein a non-cysteine amino acid is substituted for C17.
- 46. The cysteine variant according to claim 45, wherein the non-cysteine amino acid substituted for C17 is selected from the group consisting of serine and alanine.
- 47. The cysteine variant according to any one of claims 1, 3, 5, 8, 12, 16, 25, 27, 29, 30, 32, 34 or 36, wherein said cysteine variant is modified with at least one polyethylene glycol.
- 48. The cysteine variant according to any one of claims 1, 3, 5, 8, 12, 16, 25, 27, 29, 30, 32, 34 or 36, wherein said cysteine variant is modified with at least one polyethylene glycol, and wherein a non-cysteine amino acid is substituted for C17.
- 49. The cysteine variant according to claim 48, wherein the non-cysteine amino acid substituted for C17 is selected from the group consisting of serine and alanine.
- 50. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of T1, L3, G4, P5, and A6, and wherein the substituted cysteine residue is modified with a cysteine-reactive moiety; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 51. The cysteine variant according to claim 50, wherein a cysteine residue is substituted for T1.
- 52. The cysteine variant according to claim 50, wherein a cysteine residue is substituted for L3.
- 53. The cysteine variant according to claim 50, wherein a cysteine residue is substituted for A6.
- 54. A cysteine variant of granulocyte colony-stimulating factor of SEQ ID NO:6, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of T1, L3, G4, P5, and A6, and wherein the substituted cysteine residue is modified with a polyethylene glycol; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to granulocyte colony-stimulating factor.
- 55. The cysteine variant according to claim 54, wherein a cysteine residue is substituted for T1.
- 56. The cysteine variant according to claim 54, wherein a cysteine residue is substituted for L3.
- 57. The cysteine variant according to claim 54, wherein a cysteine residue is substituted for A6.
- 58. The cysteine variant according to any one of claims 50 or 53, wherein a non-cysteine amino acid is substituted for C17.
- 59. The cysteine variant according to claim 58, wherein the non-cysteine amino acid substituted for C17 is selected from the group consisting of serine and alanine.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser. No. 10/400,377, filed Mar. 26, 2003, which is a divisional of U.S. application Ser. No. 09/462,941, filed Jan. 14, 2000, now U.S. Pat. No. 6,608,183, which is a national stage application under 35 U.S.C. § 371 of PCT Application Serial No. PCT/US98/14497, filed Jul. 13, 1998, which claims the benefit of priority from U.S. Provisional Application Ser. No. 60/052,516, filed Jul. 14, 1997, each of which is incorporated herein by reference in its entirety.
GOVERNMENT SUPPORT
[0002] This invention was made in part with government support under Grant Nos. 1R43 CA78094-01 and 2R44 CA78094-02, each awarded by the National Institutes of Health. The government has certain rights in the invention.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60052516 |
Jul 1997 |
US |
Divisions (1)
|
Number |
Date |
Country |
Parent |
09462941 |
Jan 2000 |
US |
Child |
10400377 |
Mar 2003 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
10400377 |
Mar 2003 |
US |
Child |
10856219 |
May 2004 |
US |