Cysteine variants of alpha interferon-2
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 reduce the symptoms, the occurrence or the severity of a disease or condition that can be treated by alpha interferon, comprising administering to an animal having said disease or condition a composition comprising a cysteine variant of alpha interferon-2 of SEQ ID NO: 3, wherein said cysteine variant comprises at least one cysteine residue substituted for an amino acid selected from the group consisting of: D2, L3, Q5, S8, N45, Q101, G104, V105, T106, E107, M111, R162, S163, K164, E165, or a cysteine residue added at position 166 with respect to SEQ ID NO:3.
2. The method of claim 1 , wherein said cysteine variant comprises at least one cysteine residue substituted for at least one amino acid selected from the group consisting of D2, L3, Q5, and S8.
3. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for Q5.
4. The method of claim 1 , wherein said cysteine variant comprises at least one cysteine residue substituted for at least one amino acid selected from the group consisting of Q101, G102, G104, V105C, T106,and E107.
5. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for M111.
6. 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.
7. The method of claim 1 , wherein said composition is administered by intravenous administration.
8. The method of claim 1 , wherein said composition is administered by subcutaneous administration.
9. The method of claim 1 , wherein said cysteine variant of alpha interferon-2 is modified with a cysteine-reactive moiety.
10. The method of claim 9 , wherein said cysteine reactive moiety is a polyethylene glycol.
11. The method of claim 1 , wherein said disease is cancer.
12. The method of claim 1 , wherein said disease is a viral disease.
13. The method of claim 12 , wherein said viral disease is selected from the group consisting of Hepatitis B and Hepatitis C.
14. The method of claim 11 , wherein said cancer is selected from the group consisting of leukemia, melanoma and Kaposi's sarcoma.
15. The method of claim 11 , wherein administration of the composition inhibits growth of tumor cells.
16. The method of claim 12 , wherein administration of the composition inhibits viral growth.
17. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for Q101.
18. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for T106.
19. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for E107.
20. The method of 1 , wherein said cysteine variant comprises a cysteine residue substitute for S163.
21. The method of claim 1 , wherein the arginine at position 23 with respect to SEQ ID NO:3 is substituted with a lysine.
22. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue at position 166 with respect to SEQ ID NO:3.