Role of Pegylation on the Circulatory Stability and Immunogenicity of Recombinant Cholinesterases

Previous studies in rodents and non human primates demonstrated that pretreatment of animals with cholinesterases (ChEs), both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), could provide significant protection against behavioral and lethal effects of nerve agent intoxication. Currently, human (Hu) serum BChE is under development as a medical countermeasure against organophosphate chemical warfare agent toxicity. Similarly, methods for producing gram quantities of recombinant (r)Hu BChE and its development as a bioscavenger are also being pursued. In this regard, we examined the pharmacokinetic and immunologic properties of rHu ChEs and found that they displayed a relatively short half-life and enhanced immunogenicity in mice. To improve their circulatory stability, we modified recombinant (r)Hu ChEs by polyethylene glycosylation (PEGylation) and evaluated PEG modified proteins for size modification by SDS-PAGE, any changes in catalytic and inhibitory parameters, alterations in pharmacokinetic behavior, and their immunogenic activity. PEGylation of rHu ChEs with 20 kDa PEG polymer (PEG-rHu ChEs) had no deleterious effects on the catalytic properties of the enzymes. When administered into mice, a significant improvement in the circulatory stability was observed for PEG-rHu ChEs. Pharmacokinetic parameters for rHu ChEs were similar or close to those for native ChEs. However, a second injection of PEG-rHu ChEs administered 28 days later cleared rapidly from the circulation of mice as compared to the first injection. The poor circulatory stability observed for the second injection of PEG-rHu ChEs coincided with the presence of circulating anti-ChE IgG levels in mice. In conclusion, although PEGylation represents a suitable simple strategy to improve the circulatory stability of rHu ChEs, it may not be a suitable approach to eliminate the immunogenicity of these enzymes.