Humanized Organophosphorus Hydrolase Expressed in Human Embryonic Kidney Cells: Pharmacokinetics in the Guinea Pig Model, 01-R8280
Tony E. Reeves
Inclusive Dates: 01/02/12 – Current
Background — Current proteins under consideration for use as prophylactic drugs in the treatment of organophosphorus nerve agent intoxication lack sufficient circulatory half life to afford reasonable in vivo protective efficacy for extended periods of time. Most are cleared from circulation rapidly and possess half lives on the order of hours. The vast majority of proteins found in the blood/plasma are glycosylated, meaning they have carbohydrates (sugar molecules) covalently linked to their surface. Using exogenous or "non-self" proteins with different properties, or entirely lacking this glycan surface modification, makes them susceptible to clearance from the blood stream by multiple mechanisms and it is generally accepted that this rapid clearance results from the carbohydrate decoration on the surfaces of these proteins and not the protein sequence itself. Of those proteins under investigation, the most promising are of nonhuman origin (bacterial) or utilize nonhuman expression systems for their production (bacterial, plant, transgenic animals). To extend the circulatory half lives of these molecules to days, rather than hours, it is necessary to modify their surface decorations. Using an expression system that can accomplish this during expression is a significant cost-saving measure over post-purification modification by chemical means.
Approach — To address the issue of "non-self" carbohydrates, this project is using human embryonic kidney cells (HEK 293) to express a protein encoded by a gene of bacterial origin. This protein has been successfully expressed and glycosylated in transgenic corn.
Accomplishments — The genetic information encoded by the bacterial organophosphorus hydrolase (OPH) gene has been optimized for expression in human cells. To achieve this, the codons for each amino acid of OPH were modified to maximize translation efficiency for high levels of protein expression. The humanized OPH gene was used to generate transient and stably transfected HEK cell lines, and these transgenic cell lines express functional humanized OPH at a constant level over time. Preliminary data indicates the protein hydrolyses the pesticides paraoxon and demeton-S, as well as the nerve agent VX, but is not glycosylated as expected. It has not been determined if the protein is never glycosylated or if it is but then is processed in the cell removing the carbohydrates. Detailed mass analysis is planned for peptide fragments to determine if there is carbohydrate remains on the amino acid attachment sites to answer this. New gene constructs are being developed to have the expressed protein secreted from the cell into the media as it is being expressed to alleviate intracellular processing.