Post-Translational Engineering to Improve Biotherapeutic Quality from CHO Cells

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CBET-1264802Michael BetenbaughThere is a significant need to develop new medical treatments against nerve poisoning caused by organophosphorous (OP) toxins present in pesticides and biological warfare agents such as sarin or VX. A promising treatment option is the protein bioscavenger, human butyrylcholinesterase, (huBChE), which will bind and inhibit these nerve agents. The enzyme can prevent intoxication of humans exposed to OP compounds by binding to the nerve agents and sequestering them from acting on the nerve agent?s principal target of acetylcholinesterase in humans. HuBChE derived from human plasma has shown promising results in limiting OP agent poisoning in animal models, exhibiting a long circulatory half-life (mean residence time) extended over days. Unfortunately, since the supply of human plasma is limited, recombinant protein sources of huBChE are urgently needed to meet the need. Unfortunately, recombinant human BChE (rhuBChE) has not been as effective as plasma-derived natural huBChE, predominantly because the circulatory half-life of rhuBChE can be 10 fold lower due to rapid removal of the protein from the patient?s blood stream. A major reason that rhuBChE is not effective is because current recombinant expression systems do not have the biosynthetic capabilities needed for generating a recombinant product with similar physical and chemical properties as native huBChE. Therefore, the goal of this project is to apply metabolic engineering strategies to modify the production platform of Chinese hamster ovary (CHO) cells in order to enhance these cells? post-translational processing capabilities and produce long-lived rhuBChE products. Previous studies with huBChE have shown that two of the critical factors limiting circulatory half-lives are sialic acid content and multimeric protein assembly. In order to increase circulatory half-life of rhuBChE and other therapeutics, CHO cells will be modified to synthesize a recombinant protein that contains increased levels of sialic acid and a greater fraction of assembled tetramers. In order to accomplish these goals, sialyltransferases will be overexpressed, biochemical pathways responsible for generating sialic acid precursors will be enhanced, and the PRAD chaperone facilitating huBChE tetramer assembly will be engineered into CHO cells. The resulting rhuBChE will be purified and its tetrameric assembly status and sialic acid content compared to plasma derived natural huBChE to determine if the physical and chemical properties are similar. Chinese hamster ovary cells are the most widely used production organism in biotechnology, applied for the production of nearly half of the biotherapeutic proteins available today. This project will transform this important production platform by enabling CHO to generate products with increased circulatory lifetimes. Increased circulatory lifetimes will result in lower dosages for patients taking these drugs and make these biopharma products more affordable in the US and around the world. The enclosed project will demonstrate this capability for the production of the clinically relevant bioscavenger, huBChE, used in treating poisoning by nerve agents present in pesticides and chemical weapons. Furthermore, this new platform will be equally applicable for improving the circulatory lifetimes of many other biopharmaceuticals in the market or clinic. In addition, the project will serve to educate graduate students in critical biotechnology skill sets needed for industry and excite local Baltimore high school students from diverse backgrounds in biotechnology education and careers through hands-on learning modules.