Method of treatment using cysteine mutants of beta interferon
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 treat an animal with a disease or condition that can be treated by wild-type beta interferon, comprising administering to said animal a composition comprising a beta interferon cysteine mutein of SEQ ID NO:5, wherein a cysteine residue is substituted for an amino acid selected from the group consisting of: M1, S2, Q23, N25, G26, N80, E109, D110, F111, T112, R113, G114, K115 and N166, or wherein a cysteine residue is added following the last amino acid of the protein, and wherein said variant has biological activity in vitro, as measured by inhibition of proliferation of a cell line whose proliferation is inhibited in response to beta interferon.
2. The method of claim 1 , wherein said cysteine variant comprises a non-cysteine amino acid substituted for C17.
3. The method of claim 1 , wherein a cysteine residue is substituted for F111.
4. The method of claim 1 , wherein said cysteine variant comprises a cysteine residue substituted for F111 and a non-cysteine amino acid substituted for C17.
5. The method of claim 4 , wherein said beta interferon cysteine mutein is modified with at least one polyethylene glycol.
6. The method of claim 1 , wherein a cysteine residue is substituted for an amino acid selected from the group consisting of M1 and S2.
7. The method of claim 1 , wherein a cysteine residue is substituted for an amino acid selected from the group consisting of Q23, N25 and G26.
8. The method of claim 1 , wherein a cysteine residue is substituted for an amino acid selected from the group consisting of N80 and N166.
9. The method of claim 1 , wherein a cysteine residue is substituted for an amino acid selected from the group consisting of D110, T112, R113, G114 and K115.
10. The method of claim 1 , wherein a cysteine residue is added following the last amino acid of the protein.
11. The method of claim 2 , wherein said non-cysteine residue is selected from the group consisting of alanine and serine.
12. 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 administration, intracranial administration, intraspinal administration, intraocular administration, aural administration, oral administration, pulmonary administration, impregnation of a catheter, and direct injection into a tissue.
13. The method of claim 1 , wherein said composition is administered by intravenous administration.
14. The method of claim 1 , wherein said composition is administered by subcutaneous administration.
15. The method of claim 1 , wherein said animal has a viral disease or condition.
16. The method of claim 1 , wherein said animal has a tumor.
17. The method of claim 1 , wherein said animal has an immunological disease or condition.
18. The method of claim 1 , wherein said beta interferon cysteine mutein is modified with at least one polyethylene glycol.
19. The method of claim 1 , wherein said beta interferon cysteine mutein is modified with a cysteine-reactive moiety.
20. The method of claim 19 , wherein said cysteine-reactive moiety is a polyethylene glycol.