Methods of use for cysteine variants of human G-CSF
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. Also disclosed are therapeutic methods for using the cysteine variants of the invention.
1. A method to stimulate proliferation and differentiation of hematopoietic cells in an animal, comprising administering to said animal a composition comprising a cysteine variant of G-CSF of SEQ ID NO: 6, wherein said G-CSF cysteine variant comprises a cysteine residue substituted for one amino acid selected from the group consisting of P2, S7, W58, A68, E93, A129, Q131, T133, Q134, A136, A139, and A141 or wherein a cysteine residue is added preceding the first amino acid of the mature protein or following the last amino acid of the mature protein, 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 method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for A141.
3. The method of claim 1 , wherein said cysteine variant comprises a non-cysteine amino acid substituted for C17.
4. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for A141 and a non-cysteine amino acid substituted for C17.
5. The method of claim 1 , wherein said cysteine variant is modified with a cysteine-reactive moiety.
6. The method of claim 1 , wherein said cysteine variant is modified with a cysteine-reactive moiety and wherein said cysteine variant comprises a non-cysteine amino acid substituted for C17.
7. The method of claim 5 , wherein said cysteine-reactive moiety is a polyethylene glycol.
8. The method of claim 6 , wherein said cysteine-reactive moiety is a polyethylene glycol.
9. The method of claim 1 , wherein said composition is administered by a route selected from the group consisting of intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration, subcutaneous administration, transdermal delivery, intratracheal administration, intraarticular administration, intraventricular administration, inhalation, intranasal, intracranial, intraspinal, intraocular, aural, intranasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue.
10. The method of claim 1 , wherein said composition is administered by intravenous administration.
11. The method of claim 1 , wherein said composition is administered by subcutaneous administration.
12. The method of claim 1 , wherein said animal has a deficiency of hematopoietic cells.
13. The method of claim 12 , wherein said deficiency of hematopoietic cells is associated with myelosuppressive chemotherapy.
14. The method of claim 12 , wherein said deficiency of hematopoietic cells is associated with bone marrow transplantation.
15. The method of claim 12 , wherein said deficiency of hematopoietic cells is associated with infection with the human immunodeficiency virus.
16. The method of claim 12 , wherein said deficiency of hematopoietic cells is associated with chronic neutropenia.
17. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for P2.
18. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for S7.
19. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for W58.
20. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for A68.
21. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for E93.
22. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for A129.
23. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for Q131.
24. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for T133.
25. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for Q134.
26. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for A136.
27. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for A139.
28. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue added preceding the first amino acid of the mature protein.
29. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue added following the last amino acid of the mature protein.
30. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for P2 and a second cysteine substituted for A141.
31. The method of claim 1 , wherein said hematopoietic cell is selected from the group consisting of a granulocyte, a neutrophil, an eosinophil, a monocyte, a macrophage, a white blood cell, a lymphocyte, an erythroid cell, an erythrocyte, a red blood cell, a thrombocyte cell, a megakaryocyte cell, and a platelet cell.
32. The method of claim 31 , wherein said hematopoietic cell is a neutrophil.
33. The method of claim 32 , wherein said hematopoietic cell is a white blood cell.