Post-Translational Engineering to Improve Biotherapeutic Quality from CHO Cells

目前迫切需要开发新的医学治疗方法，以治疗农药和沙林或VX等生物战剂中存在的有机磷（OP）毒素引起的神经中毒。一种有希望的治疗选择是蛋白质生物清除剂，人类丁基胆碱酯酶（huBChE），它将结合并抑制这些神经毒剂。这种酶可以通过与神经毒剂结合并隔离它们对神经毒剂的作用来防止暴露于OP化合物的人中毒。人类乙酰胆碱酯酶的主要靶点。从人血浆中提取的HuBChE在动物模型中显示出限制OP毒剂中毒的良好结果，显示出较长的循环半衰期（平均停留时间）延长数天。不幸的是，由于人血浆供应有限，迫切需要huBChE的重组蛋白来源来满足需求。不幸的是，重组人BChE （rhuBChE）并不像血浆来源的天然huBChE那样有效，主要是因为rhuBChE的循环半衰期可以降低10倍，因为从患者体内快速去除蛋白质。血流。大黄bche不有效的一个主要原因是，目前的重组表达系统不具备产生与天然huBChE具有相似物理和化学性质的重组产品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对中国仓鼠卵巢（CHO）细胞的生产平台进行改造，以增强CHO细胞的功能。翻译后加工能力和生产长寿命的大黄酚产品。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。为了增加大黄酚和其他治疗药物的循环半衰期，CHO细胞将被修饰以合成一种重组蛋白，该蛋白含有更高水平的唾液酸和更大比例的组装四聚体。为了实现这些目标，唾液基转移酶将被过度表达，负责生成唾液酸前体的生化途径将被增强，促进huBChE四聚体组装的PRAD伴侣将被设计到CHO细胞中。将所得的大黄酚进行纯化，并将其四聚体组装状态和唾液酸含量与血浆衍生的天然大黄酚进行比较，以确定其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应用最广泛的生产生物，目前近一半的生物治疗蛋白都是由仓鼠卵巢细胞生产的。该项目将改变这个重要的生产平台，使CHO能够生产具有更长的循环寿命的产品。增加的循环寿命将导致患者服用这些药物的剂量降低，并使这些生物制药产品在美国和世界各地更便宜。所附项目将证明生产临床相关生物清除剂huBChE的能力，用于治疗杀虫剂和化学武器中存在的神经毒剂中毒。此外，这个新平台将同样适用于改善市场或临床中许多其他生物制药的循环寿命。此外，该项目还将教育研究生掌握工业所需的关键生物技术技能，并通过实践学习模块，激发来自不同生物技术教育和职业背景的巴尔的摩当地高中生的兴趣。

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.