Polyethylene glycol (PEG) modification of granulocyte-macrophage colony stimulating factor (GM-CSF) enhances neutrophil priming activity but not colony stimulating activity

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Abstract
PEG-modified proteins have numerous advantages over their unmodified counterparts (increased half life, reduced antigenicity, improved solubility), but almost without exception, they show a modest to marked reduction in biological or enzymatic activity. However, while investigating a new protocol for the preparation of PEG-proteins, we compared PEG-modified and unmodified GM-CSF with respect to their polymorphonuclear neutrophil granulocyte (PMN) priming activities. PEG-GM-CSF was unexpectedly more active than GM-CSF in its ability to prime neutrophils to respond to the synthetic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) with an oxidative burst (assessed both by nitroblue tetrazolium reduction and ferricytochrome c reduction). These results were in contrast to the findings for colony stimulating activity and with GM-CSF induced thymidine uptake, where the biological activity was unchanged or reduced. The enhanced neutrophil priming activity of PEG-GM-CSF was confirmed using FPLC fractionated PEG-modified GM-CSF. This showed changes in the bioactivity profile consistent with both the shift in protein elution profile and enhanced activity of the PEG-modified material (reflected in the increased area under the bioactivity curve). We also excluded a neutrophil priming action for PEG-modified fetal calf serum proteins, carrier proteins and 'irrelevant' cytokine, erythropoietin. The dissociation of the two bioactivities was confirmed using individual FPLC fractions. These results suggest the presence of differences in either binding, receptor/ligand processing or signal transduction for neutrophils versus progenitors, that are differentially affected by PEG-modification of GM-CSF. The demonstration that PEG-modification can partially dissociate two biological activities suggests the feasibility of using PEG-modification to produce proteins with subtly altered spectra of biological activity and hence new ranges of clinical applications.