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 analogs with a cysteine-reactive polyethylene glycol (PEG) reagent. Thirteen cysteine analogs of GM-CSF were constructed, primarily in non-helical regions of the protein believed to lie away from the major receptor binding sites. The GM-CSF cysteine analogs 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 analogs 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 analogs and seven PEGylated cysteine analogs were comparable to that of wild type GM-CSF in an in vitro cell proliferation assay using human TF-1 cells. One cysteine analog was modified with larger 10 kDa-, 20 kDa-and 40 kDaPEGs, 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 analogs, and provide further evidence that the non-helical regions of human GM-CSF examined are largely non-essential for biological activity of the protein.

PEGylation of therapeutic proteins

“…Genetically introduced cysteines represent an opportunity to direct the PEG moiety to an exactly determined site in the molecule. In the case of IFN-α2a, several cysteine analogues with high preserved in vitro activity were identified and used for specific PEGylations [47,48]. Another very promising group of proteins for cysteine-specific…”

PEGylation of Proteins and Liposomes: a Powerful and Flexible Strategy to Improve the Drug Delivery

“…This method offers the advantages of a relative low number of cysteine residues in protein sequences, that limit the number of different PEG isomers obtained, but its main disadvantage is the hydrophobicity of cysteine that make this amino acid often buried inside the protein structure and therefore hardly accessible for conjugation with bulky PEG moiety. A number of strategies have been investigated to overcome this limitation as, for example, to perform the PEGylation under transient light denaturating conditions able to expose partially buried cysteine residues, or to genetically replace a non-essential amino acid with a cysteine residue [24][25][26]. Another simple strategy is to reduce the disulphide bridges of a protein in order to expose new thiol residues.…”

Sortase-catalyzed transformations that improve the properties of cytokines

“…Such PEGylated molecules often show decreased biological activity, likely due to attachment of a PEG chain to a residue important for interaction with the receptor (6)-this problem may be overcome by sitespecific PEGylation. Although engineering of a carefully placed unpaired cysteine residue allows site-specific PEGylation (7,8), this method must be tailored to the specific protein target.…”