Cysteine variants of granulocyte colony-stimulating factor
The growth hormone supergene family comprises greater than 20 structurally related cytokines and growth factors. A general method is provided for creating site-specific, biologically active conjugates of these proteins. The method involves adding cysteine residues to non-essential regions of the proteins or substituting cysteine residues for non-essential amino acids in the proteins using site-directed mutagenesis and then covalently coupling a cysteine-reactive polymer or other type of cysteine-reactive moiety to the proteins via the added cysteine residue. Disclosed herein are preferred sites for adding cysteine residues or introducing cysteine substitutions into the proteins, and the proteins and protein derivatives produced thereby.
1. A cysteine variant of granulocyte colony-stimulating factor (G-CSF) of SEQ ID NO:6, wherein a cysteine residue is substituted for an amino acid selected from the group consisting of: P2 and S7; and 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 P2.
3. The cysteine variant according to claim 1 , wherein a cysteine residue is substituted for S7.
4. A cysteine variant of granulocyte colony-stimulating factor (G-CSF) of SEQ ID NO:6, wherein a cysteine residue is substituted for an amino acid selected from the group consisting of: W58 and A68; and 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.
5. The cysteine variant according to claim 4 , wherein a cysteine residue is substituted for W58.
6. The cysteine variant according to claim 4 , wherein a cysteine residue is substituted for A68.
7. A cysteine. variant of granulocyte colony-stimulating factor (G-CSF) of SEQ ID NO:6, wherein a cysteine residue is substituted for the amino acid E93; and 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.
8. A cysteine variant of granulocyte colony-stimulating factor (G-CSF) of SEQ ID NO:6, wherein a cysteine residue is substituted for an amino acid selected from the group consisting of: A129, Q131, T133, Q134, A136, A139, and A141; and 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 A129.
10. The cysteine variant according to claim 8 , wherein a cysteine residue is substituted for Q131.
11. The cysteine variant according to claim 8 , wherein a cysteine residue is substituted for T133.
12. The cysteine variant according to claim 8 , wherein a cysteine residue is substituted for Q134.
13. The cysteine variant according to claim 8 , wherein a cysteine residue is substituted for A136.
14. The cysteine variant according to claim 8 , wherein a cysteine residue is substituted for A139.
15. The cysteine variant according to claim 8 , wherein a cysteine residue is substifuted for A141.
16. The cysteine variant according to claim 15 , wherein a cysteine residue is substituted for P2.
17. A cysteine variant of granulocyte colony-stimulating factor (G-CSF) of SEQ ID NO:6, wherein a cysteine residue is substituted for the amino acid Q173; and 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.
18. A cysteine variant of granulocyte colony-stimulating factor (G-CSF) of SEQ ID NO:6, wherein a cysteine residue is substituted for an amino acid selected from the group consisting of: P2, S7, W58, A68, E93, A129, Q131, T133, Q134, A136, A139, and A141; wherein a non-cysteine amino acid residue is substituted for C17; and 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.
19. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for P2.
20. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for S7.
21. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for W58.
22. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for A68.
23. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for E93.
24. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for A129.
25. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for Q131.
26. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for T133.
27. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for Q134.
28. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for A136.
29. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for A139.
30. The cysteine variant according to claim 18 , wherein a cysteine residue is substituted for A141.
31. The cysteine variant according to claim 18 , wherein the non-cysteine amino acid substituted for C17 is serine.
32. The cysteine variant according to claim 18 , wherein the non-cysteine amino acid substituted for C17 is alanine.
33. The cysteine variant according to any one of claims 1 , 2 , 3 - 16 , 18 , 19 , 20 - 30 , 31 or 32 , wherein the substituted cysteine residue is modified with a cysteine-reactive moiety.
34. The cysteine variant according to any one of claims 1 , 2 , 3 - 16 , 18 , 19 , 20 - 30 , 31 or 32 , wherein the substituted cysteine residue is modified with polyethylene glycol.
35. The cysteine variant according to any one of claims 1 , 2 , 3 - 16 , 18 , 19 , 20 - 30 , 31 or 32 , wherein the cysteine variant is modified with at least one polyethylene glycol.
36. A cysteine variant of granulocyte colony-stimulating factor (G-CSF) of SEQ ID NO:6, wherein a cysteine residue is substituted for the amino acid A6; and 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. A cysteine variant of granulocyte colony-stimulating factor (G-CSF) of SEQ ID NO:6, wherein a cysteine residue is substituted for the amino acid A6; wherein a non-cysteine amino acid residue is substituted for C17; and 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.
38. The cysteine variant according to claim 37 , wherein the non-cysteine amino acid substituted for C17 is serine.
39. The cysteine variant according to claim 37 , wherein the non-cysteine amino acid substituted for C17 is alanine.
40. The cysteine variant according to any one of claims 36 , 37 , wherein the substituted cysteine residue is modified with a cysteine-reactive moiety.
41. The cysteine variant according to any one of claims 36 , 37 , wherein the substituted cysteine residue is modified with polyethylene glycol.
42. The cysteine variant according to any one of claims 36 - 39 , wherein the cysteine variant is modified with at least one polyethylene glycol.
43. A cysteine variant of granulocyte colony-stimulating factor (G-CSF) of SEQ ID NO:6, wherein a cysteine residue is substituted for the amino acid Q173; wherein a non-cysteine amino acid residue is substituted for C17; and 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.
44. The cysteine variant according to claim 43 , wherein the non-cysteine amino acid substituted for C17 is serine.
45. The cysteine variant according to claim 43 , wherein the non-cysteine amino acid substituted for C17 is alanine.
46. The cysteine variant according to any one of claims 17 or 43 - 45 , wherein the substituted cysteine residue is modified with a cysteine-reactive moiety.
47. The cysteine variant according to any one of claims 17 or 43 - 45 , wherein the substituted cysteine residue is modified with polyethylene glycol.
48. The cysteine variant according to any one of claims 17 or 43 - 45 , wherein the cysteine variant is modified with at least one polyethylene glycol.