Post-Translational Engineering to Improve Biotherapeutic Quality from CHO Cells

CBET-1264802Michael BetenbaughThere is a significant need to develop new medical treatment against nervous poisoning caused by organophosphorous（OP）toxins present in pesticides and biological warfare agents such as sarin or VX. 一个有前途的治疗选择是蛋白质生物清除剂，人丁酰胆碱酯酶（huBChE），它将结合和抑制这些神经毒剂。 这种酶可以通过与神经毒剂结合并隔离它们而不作用于神经毒剂来防止暴露于OP化合物的人中毒？乙酰胆碱酯酶的主要靶点。 来源于人血浆的HuBChE在动物模型中显示出限制OP剂中毒的有希望的结果，表现出延长数天的长循环半衰期（平均停留时间）。不幸的是，由于人血浆的供应有限，迫切需要huBChE的重组蛋白源来满足需求。不幸的是，重组人BChE（rhuBChE）并没有血浆来源的天然huBChE那么有效，主要是因为rhuBChE的循环半衰期可以低10倍，由于蛋白质从患者体内快速去除？的血流。rhuBChE无效的主要原因是因为目前的重组表达系统不具有产生具有与天然huBChE相似的物理和化学性质的重组产物所需的生物合成能力。 因此，本项目的目标是应用代谢工程策略来修改中国仓鼠卵巢（CHO）细胞的生产平台，以提高这些细胞？翻译后加工能力，并产生长寿命的rhuBChE产物。 先前对huBChE的研究表明，限制循环半衰期的两个关键因素是唾液酸含量和多聚体蛋白组装。 为了增加rhuBChE和其他治疗剂的循环半衰期，将修饰CHO细胞以合成含有增加水平的唾液酸和更大比例的组装四聚体的重组蛋白。 为了实现这些目标，唾液酸转移酶将被过表达，负责产生唾液酸前体的生化途径将被增强，并且促进huBChE四聚体组装的PRAD分子伴侣将被工程化到CHO细胞中。 将纯化所得rhuBChE，并将其四聚体组装状态和唾液酸含量与血浆衍生的天然huBChE进行比较，以确定物理和化学性质是否相似。 中国仓鼠卵巢细胞是生物技术中使用最广泛的生产生物体，用于生产当今可用的近一半的生物蛋白。该项目将通过使CHO能够生产具有更长循环寿命的产品来改变这一重要的生产平台。 循环寿命的延长将导致服用这些药物的患者剂量降低，并使这些生物制药产品在美国和世界各地更便宜。 所附项目将展示生产临床相关生物清除剂huBChE的能力，用于治疗农药和化学武器中存在的神经毒剂中毒。此外，这个新平台同样适用于改善市场或临床上许多其他生物药物的循环寿命。 此外，该项目将有助于教育研究生掌握工业所需的关键生物技术技能，并通过动手学习模块激发来自生物技术教育和职业背景不同的当地巴尔的摩高中学生的兴趣。

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.