Method of treatment of an infection in an animal with GM-CSF cysteine muteins in the proximal region to helix A
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 having a disease or condition that can be treated by wild-type granulocyte-macrophage colony stimulating factor (GM-CSF), comprising administering to an animal having said disease or condition a composition comprising a human granulocyte-macrophage colony stimulating factor (GM-CSF) cysteine mutein protein of SEQ ID NO:8, wherein a cysteine residue is substituted for an amino acid selected from the group consisting of: A1, A3, S5, and S7.
2. The method of claim 1 , wherein said cysteine mutein comprises a cysteine residue substituted for the alanine at position 3 of human GM-CSF (SEQ ID NO:8).
3. The method of claim 1 , wherein said cysteine mutein comprises a cysteine residue substituted for the alanine at position 1 of human GM-CSF (SEQ ID NO:8).
4. The method of claim 1 , wherein said cysteine mutein comprises a cysteine residue substituted for the serine at position 5 of human GM-CSF (SEQ ID NO:8).
5. The method of claim 1 , wherein said cysteine mutein comprises a cysteine residue substituted for the serine at position 7 of human GM-CSF (SEQ ID NO:8).
6. The method of claim 1 , wherein the disease or condition is selected from the group consisting of: neutropenia, a refractory wound, a cancer and an infection.
7. The method of claim 6 , wherein said disease is an infection.
8. The method of claim 7 , wherein said GM-CSF cysteine mutein is used as an adjuvant for a vaccine.
9. The method of claim 7 , wherein said infection is selected from the group consisting of a bacterial infection, a fungal infection and a viral infection.
10. 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.
11. The method of claim 1 , wherein said composition is administered by intravenous administration.
12. The method of claim 1 , wherein said composition is administered by subcutaneous administration.
13. The method of claim 1 , wherein said GM-CSF cysteine mutein is modified with at least one polyethylene glycol.
14. The method of claim 1 , wherein said GM-CSF cysteine mutein is modified with a cysteine-reactive moiety.
15. The method of claim 1 , wherein said cysteine-reactive moiety is a polyethylene glycol.