Site-specific PEGylation of engineered cysteine analogues of recombinant human granulocyte-macrophage colony-stimulating factor

Abstract

Granulocyte macrophage colony-stimulating factor (GM-CSF) stimulates proliferation of hematopoietic cells of the macrophage and granulocyte lineages and is used clinically to treat neutropenia and other myeloid disorders. Because of its short circulating half-life, GM-CSF is administered to patients by daily injection. We describe here the engineering of highly potent, long-acting human GM-CSF proteins through site-specific modification of GM-CSF cysteine analogues with a cysteine-reactive poly(ethylene glycol) (PEG) reagent. Thirteen cysteine analogues of GM-CSF were constructed, primarily in nonhelical regions of the protein believed to lie away from the major receptor binding sites. The GM-CSF cysteine analogues were properly processed but insoluble following secretion into the Escherichia coli periplasm. The proteins were refolded and purified by column chromatography. Ten of the cysteine analogues could be modified with a 5-kDa maleimide PEG, and seven of the mono-PEGylated proteins were purified by ion-exchange column chromatography. Biological activities of the 13 cysteine analogues and 7 PEGylated cysteine analogues were comparable to that of wild-type GM-CSF in an in vitro cell proliferation assay using human TF-1 cells. One cysteine analogue was modified with larger 10-, 20-, and 40-kDa PEGs, with only minimal loss of in vitro bioactivity. Pharmacokinetic experiments in rats demonstrated that the PEGylated proteins had up to 47-fold longer circulating half-lives than wild-type GM-CSF. These data demonstrate the utility of site-specific PEGylation for creating highly potent, long-acting GM-CSF analogues and provide further evidence that the nonhelical regions of human GM-CSF examined are largely nonessential for biological activity of the protein.

Figure 1

SDS-PAGE analysis of purified wild type GM-CSF. Western blot of a wild type GM-CSF standard (lanes 1 and 3; R & D Systems, Inc.) and our purified wild type GM-CSF (lanes 2 and 4) following non-reducing (lanes 1 and 2) and reducing (lanes 3 and 4) SDS-PAGE. Positions of molecular weight standards are shown to the left.

Figure 2

SDS-PAGE analysis and in vitro bioactivities of purified wild type GM-CSF and GM-CSF cysteine analogs. Panel A shows non-reducing SDS-PAGE analysis of column pools of the purified GM-CSF cysteine analogs. Lane 1, molecular weight standards; Lane 2, wild type GM-CSF; Lane 3, *-1C; Lane 4, A1C; Lane 5, A3C; Lane 6, S5C, Lane 7, S7C; Lane 8, N27C; Lane 9, S69C; Lane 10, E93C; Lane 11, T94C; Lane 12, T102C; Lane 13, V125C; Lane 14, Q126C; and Lane 15, *128C. Proteins were stained with Coomassie Blue. Panel B shows dose response curves for our wild type GM-CSF and representative GM-CSF cysteine analogs for stimulating proliferation of TF-1 cells. Data are means of triplicate wells ± SD from representative experiments. SD were generally less than 5% of the means.

Figure 3

Purification of the PEGylated GM-CSF S7C protein and inability of wild type GM-CSF to react with maleimide-PEG. Panel A shows Q-Sepharose column chromatography of the S7C PEGylation reaction products. Panel B shows non-reducing SDS-PAGE analysis of the Q-sepharose column fractions. Lane 1, molecular weight standards; Lane 2, S7C; Lane 3, S7C PEGylation reaction products; Lanes 4–12 represent Q-Sepharose column fractions 30–38. Fractions 30–34 (Lanes 4–8) contain predominantly PEGylated S7C. Fractions 36–38 (Lanes 10–12) contain unreacted S7C. Panel C is non-reducing SDS-PAGE analysis of wild type GM-CSF PEGylation reaction products. Lane 1, molecular weight standards; Lane 2, wild type GM-CSF; Lane 3, GM-CSF +5 kDa-maleimide-PEG; Lane 4, GM-CSF + 5 kDa-maleimide-PEG + TCEP.

Figure 4

SDS-PAGE analysis and in vitro bioactivities of purified 5 kDa-maleimide PEG-GM-CSF cysteine analogs. Panel A shows non-reducing SDS-PAGE analysis of column pools of purified PEG-GM-CSF cysteine analogs. Lane 1, molecular weight standards; Lane 2, wild type GM-CSF; Lane 3, 5 kDa-PEG-*-1C; Lane 4, 5 kDa -PEG-A1C; Lane 5, 5 kDa -PEG-A3C; Lane 6, 5 kDa -PEG-S5C; Lane 7, 5 kDa -PEG-S7C; Lane 8, 5 kDa -PEG-N27C; Lane 9, 5 kDa -PEG-S69C; Lane 10, 5 kDa -PEG-E93C; Lane 11, 5 kDa -PEG-T94C; Lane 12, 5 kDa -PEG-T102C. Proteins were stained with Coomassie Blue. Panel B shows dose-response curves for 5 kDA-PEG GM-CSF cysteine analogs and our wild type GM-CSF for stimulating proliferation of TF-1 cells. Data are means of triplicate wells ± SD from representative experiments. SD were generally less than 5% of the means.

Figure 5

SDS-PAGE analysis and in vitro bioactivities of the GM-CSF A3C protein modified with different size PEGs. Panel A shows non-reducing SDS-PAGE analysis of the purified proteins. Lane 1, molecular weight standards; Lane 2, wild type GM-CSF; Lanes 3–6 are column pools of the A3C protein modified with 5 kDa-, 10 kDa-, 20 kDa- and 40 kDa-maleimide PEGs, respectively. Panel B shows dose response curves for the 10 kDa-, 20 kDa-, and 40 kDa-PEG A3C proteins for stimulating proliferation of TF-1 cells. SD were generally less than 5% of the means.

Figure 6

Circulating plasma levels of Leukine^®, E. coli-derived wild type GM-CSF and the GM-CSF A3C protein modified with different size PEGs following (A) intravenous or (B) subcutaneous administration in rats. Data are means ± SD for three rats per group and were measured using human GM-CSF ELISAs. SD were generally less than 25% of the means. The 120 h time point samples for the 10 kDa-PEG-A3C protein in the subcutaneous study shown in (B) were lost and could not be analyzed.