G-CSF conjugates
Polypeptide conjugates with G-CSF activity comprising a polypeptide having at least one introduced lysine residue and at least one removed lysine residue compared to the sequence of human G-CSF, and which are conjugated to 2-6 polyethylene glycol moieties. The conjugates have a low in vitro bioactivity, a long in vivo half-life, a reduced receptor-mediated clearance, and provide a more rapid stimulation of production of white blood cells and neutrophils than non-conjugated recombinant human G-CSF.
1. A method for preparing a G-CSF conjugate, the method comprising
(a) providing a polypeptide comprising an amino acid sequence consisting of SEQ ID NO:1 with the substitutions K16R, K34R, K40R, T105K and S159K, and
(b) reacting the polypeptide with amine-reactive polyethylene glycol moieties under conditions sufficient to produce a G-CSF conjugate having a polyethylene glycol moiety covalently bound to each of at least two attachment groups of the polypeptide,
wherein the G-CSF conjugate exhibits G-CSF cell proliferation activity.
2. The method of claim 1 , wherein the polyethylene glycol moieties each have a molecular weight of about 5 kilodaltons.
3. The method of claim 1 , wherein the amine-Reactive polyethylene glycol moieties are mPEG-succinimidyl propionate (mPEG-SPA).
4. The method of claim 3 , wherein the amine-reactive polyethylene glycol moieties are mPEG-SPA 5000.
5. The method of claim 1 , wherein the amine-reactive polyethylene glycol moieties are attached to the N-terminal amino group and to one or more lysine residues.
6. The method of claim 5 , wherein the one or more lysine residues are selected from Lys23, Lys105, and Lys159.
7. The method of claim 1 , wherein the step of providing the polypeptide comprises:
providing an isolated host cell comprising a nucleic acid that comprises a promoter operably linked to a nucleotide sequence encoding the polypeptide,
culturing the host cell in a nutrient medium under conditions which permit expression of the polypeptide, and
recovering the polypeptide from the nutrient medium or from the cultured host cells.
8. The method of claim 7 , wherein the host cell is a bacterial host cell.
9. The method of claim 7 , wherein the host cell is a glycosylating host cell.
10. The method of claim 9 , wherein the host cell is selected from an S. cerevisiae cell, a Pichia pastoris cell, a CHO cell, a BHK cell, an HEK 293 cell, and an SF9 cell.
11. The method of claim 8 , wherein the host cell is E. coli.
12. The method of claim 10 , wherein the host cell is an S. cerevisiae cell.
13. The method of claim 10 , wherein the host cell is a CHO cell.
14. The method of claim 7 , wherein the polypeptide is recovered from the nutrient medium.
15. The method of claim 7 , wherein the polypeptide is recovered from the cultured host cells.
16. The method of claim 7 , wherein the method further comprises a purification step to purify the recovered polypeptide.
17. The method of claim 16 , wherein the purification step comprises a chromatography method.
18. The method of claim 17 , wherein the chromatography method is ion exchange chromatography.
19. The method of claim 1 , wherein the amine-reactive polyethylene glycol moieties comprise a succinimidyl group.
20. The method of claim 1 , wherein the method further comprises a purification step.
21. The method of claim 20 , wherein the purification step comprises cation exchange chromatography.
22. The method of claim 1 , wherein the conditions are a molar excess of the amine-reactive polyethylene glycol moieties relative to the number of polypeptide attachment sites.
23. The method of claim 1 , wherein the conditions are a molar ratio of amine-reactive polyethylene glycol moieties to polypeptide that is greater than 10:1 and up to about 100:1.
24. The method of claim 22 , wherein the conditions are about a 5:1 molar excess of the amine-reactive polyethylene glycol moieties relative to the number of polypeptide attachment sites.
25. The method of claim 22 , wherein the conditions are about a 10:1 molar excess of the amine-reactive polyethylene glycol moieties relative to the number of polypeptide attachment sites.
26. The method of claim 22 , wherein the conditions are about a 1000:1 molar excess of the amine-reactive polyethylene glycol moieties relative to the number of polypeptide attachment sites.
27. The method of claim 1 , wherein the G-CSF conjugate comprises 2-8 polyethylene glycol moieties.
28. The method of claim 1 , wherein the G-CSF conjugate comprises 3-6 polyethylene glycol moieties.
29. The method of claim 19 , wherein the amine-reactive polyethylene glycol moiety is a methoxypolyethylene glycol (mPEG) moiety.
30. The method of claim 29 , wherein the amine-reactive polyethylene glycol moiety is a linear molecule.