title: Effect of polyethylene glycol modification on the circulatory stability and immunogenicity of recombinant human butyrylcholinesterase.
abstract: The therapeutic value of human serum butyrylcholinesterase (Hu BChE) as a bioscavenger of chemical warfare agents is due to its high reactivity with organophosphorus compounds and prolonged circulatory stability. Native Hu BChE is mostly tetrameric in form while the enzyme produced using molecular cloning technology is a mixture of tetramers, dimers, and monomers. Previous studies revealed that monomers and dimers of recombinant human (rHu) BChE cleared rapidly from the circulation of mice compared to tetrameric rHu BChE and native Hu BChE, which have mean residence times (MRTs) of 18h and 45h, respectively. It was also shown that polyethylene glycol-20K (PEG) modification of tetrameric rHu BChE prolonged its circulatory stability and bioavailability in vivo. The goal of this study was to determine if modification with PEG could prolong the circulatory stability and eliminate the immunogenicity of monomeric rHu BChE. Monomeric rHu BChE was expressed in human 293A cells using a cDNA lacking the 45 amino acid tetramerization domain from the carboxyl terminus and the adenovirus expression system. The catalytic and inhibitory properties of purified monomeric rHu BChE were similar to those for native Hu BChE and were not affected by PEG modification. As expected, monomeric rHu BChE rapidly cleared from the circulation of mice (MRT=3.2+/-0.3h) while monomeric PEG-rHu BChE demonstrated significant improvement in its bioavailability and circulatory stability in blood (MRT=31.4+/-5.4h). However, a second injection of monomeric PEG-rHu BChE, 28 days after the first, displayed a much shorter MRT=11.6+/-0.4h, and circulating anti-monomeric PEG-rHu BChE antibodies were detected in the blood of mice. These results suggest that PEG modification increased the circulatory stability of monomeric rHu BChE but failed to reduce or eliminate its immunogenicity.
authors.affiliation: Division of Biochemistry, Walter Reed Army Institute of Research, Silver Spring, MD 20910-7500, USA. nageswararao.chilukuri@amedd.army.mil
authors.forename: Nageswararao
authors.initials: N
authors.lastname: Chilukuri
authors.forename: Wei
authors.initials: W
authors.lastname: Sun
authors.forename: Ramachandra S
authors.initials: RS
authors.lastname: Naik
authors.forename: Kalpana
authors.initials: K
authors.lastname: Parikh
authors.forename: Lin
authors.initials: L
authors.lastname: Tang
authors.forename: Bhupendra P
authors.initials: BP
authors.lastname: Doctor
authors.forename: Ashima
authors.initials: A
authors.lastname: Saxena
mesh_terms: D002091:Butyrylcholinesterase / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D002091:Butyrylcholinesterase