Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin.

BACKGROUND: Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein. RESULTS: Secretion level of the fusion protein produced in vitro in BHK cells was approximately 30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04-1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of approximately 150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (approximately 32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (approximately 3 h). In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested. CONCLUSION: Both the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.

Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning.

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat beta-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.