Generation of Recombinant Human AChE Op-Scavengers With Extended Circulatory Longevity

We demonstrated in the past that chemical conjugation of polyethylene glycol (PEG) moieties to recombinant human acetylcholinesterase (rHuAChE) gives rise to OP bioscavenger species which reside for very long period of time in the circulation of mice, regardless of their post-translation- modification state. These findings allow for production of rHuAChE not only in eukaryotic cells but also in bacterial cost-effective systems. A synthetic human AChE gene was constructed and its expression was analyzed under control of various potent transcription promoters in Bacillus brevis cells. The potential use of this system for large-scale production of AChE is presently being assessed. We have begun a series of studies to determine the effect of removal of human AChE lysine residues, which serve as tar et sites for PEG-conjugation, on the enzymatic and pharmacokinetic performance and on the degree of homogeneity of the enzyme product. In other studies we demonstrated that AChE PEGylation results in a major reduction of the immunogenicity of the enzyme. In structure-function studies of AChE, we compared the reactivities of enantiomers of VX and their noncharged isosters, as well as ecothiophate, in conjunction with a battery of AChE mutants. These studies allowed us to define two subsites, located in the active site and peripheral anionic subsites, which confer enzyme stereoselectivity to CW agents such as VX.