Generation and Evaluation of PEGylated Recombinant Human Acetylcholinesterase as an Optimal OP-Bioscavenger

摘要

We have shown in the past that recombinant human AChE (rHuAChE) can be converted into circulatory long-lived molecular forms by conjugation of polyethylene glycol (PEG) chains to lysine residues. In the present study, we determined the factors that govern circulatory residence of PEGylated AChE, and established the lysine mutant configurations that allow maximal circulatory longevity. We further demonstrated that PEGylated rHuAChEs are less immunogenic than non-modified enzyme. In addition, we incorporated favorable kinetic characteristics for OP-scavenging into the rHuAChE enzyme, utilizing genetic engineering methods. Thus, incorporation of the F338A mutation into circulatory long-lived PEG-hypolysine AChE resulted in an enzyme displaying reduced aging rates that could protect mice against repeated OP-compound exposures. Finally, we established a method for production of enzymatically active rHAChE at appreciable amounts and relatively low cost utilizing the Pichia pastoris yeast cell system. Taken together, these different lines of study pave the way for large-scale production of PEGylated recombinant human AChE derivatives displaying catalytic and pharmacokinetic properties optimized for effective in- vivo bioscavenging of OP-compounds.