A Homologous Non-Human Primate Model System for Producing and Testing Recombinant Human Compatible Serum Butyrylcholinesterase

Nerve gas and insecticides pose ever increasing military and civilian threats. In recent years exogenous administration of cholinesterases have been successfully used as safe and efficacious prophylactic treatments due to their capacity to scavenge organo-phosphate (OP) toxins before inhibition of the targeted endogenous acetylcholinesterase (AChE) can occur. Based on availability, broad spectrum efficacy, stability and safety, homologous native BChE represents to date the best candidate in terms of developing a human treatment. However, while the currently used methods of native enzyme isolation from frozen human plasma are effective on a laboratory scale, they are time consuming and expensive. These factors, coupled with potential safety issues associated with the purification of proteins from human blood (i.e. hepatitis, HIV, prion and West Nile virus contamination), highlight the importance of developing recombinant BChE technology as an alternate and parallel strategy for commercial production. The overall objective of this Phase 1 proposal is to develop an homologous non-human primate model in which recombinant(r)macaque(Ma)BChE, can be produced and tested in macaques as an alternate pharmaceutical bioscavenger to the native enzyme. The recombinant product/s should exhibit chemical and functional properties similar to native molecules i.e. long circulatory stability and no immunogenicity following injection into homologous hosts. This study aims to (i) Express both recombinant tetrameric and monomeric forms of MaBChE by stable trasnsfection of CHO cells.(ii) Optimize in vivo stability of both MaBChE forms by chemical modification using polyethylene glycol, in order to overcome the deleterious effects of incomplete sialyation of recombinant molecules (iii) Monitor the pharmacokinetics of clearance in mice injected with native MaBChE compared to those receiving the modified and unmodified tetrameric and monomeric rMaBChE. (iv) Produce 40mg (28,000U) of eaqch of the most stable monomers and tetramers and assess their rates of clearance and ability to induce anti-BChE antibody. In phase II, through collaborative efforts or partnerships, scaled-up production of human rBChE will be performed under GMP conditions to conduct human trials.