PEGylated Butyrylcholinesterase used as a Bioscavenger

描述（由申请人提供）：处理神经毒剂毒性的新概念涉及利用天然存在的酶丁酰胆碱酯酶（BuChE），其能够隔离或催化神经毒剂的水解。这种大分子有可能以最小的副作用提供针对多种神经毒剂的保护，因为它可以在神经毒剂分布到毒性作用部位之前以化学计量结合和/或代谢神经毒剂。开发这种用于人类的治疗方法的主要障碍是需要频繁施用相对短期的产品。我们建议创建一种新型 BuChE 蛋白，与未修饰的 BuChE 相比，其给药频率较低，但效力更强。我们将利用已发表的丁基胆碱酯酶结构信息来实现这一目标，以使用半胱氨酸反应性 PEG 合理设计聚乙二醇 (PEG)-BuChE 缀合物。我们将在 BuChE 区域中使用 she 定向诱变引入一种新的“游离”半胱氨酸，这些区域被认为对生物活性不是必需的。 “游离”半胱氨酸残基将作为使用硫醇反应性 PEG 对蛋白质进行共价修饰的位点。该技术可以创建具有明确结构的新型、完全活性的 PEG-Cys-BuChE 类似物，并克服了使用标准胺反应性 PEG 修饰蛋白质时生物活性降低和异质性降低的问题。在第一阶段，我们将确定 BuChE 中可以进行修饰而不影响蛋白质体外生物活性的位点，并进一步证明聚乙二醇化可增强 BuChE 的循环半衰期和体内功效。在第二阶段，我们开发了一种具有成本效益的制造工艺，并生产足够数量的聚乙二醇化重组 BuChE，用于动物毒性模型和生化表征测试。

DESCRIPTION (provided by applicant): A novel concept for dealing with nerve agent toxicity involves taking advantage of a naturally occurring enzyme, butyrylcholinesterase (BuChE) that is able to sequester or catalyze the hydrolysis of nerve agents. This macromolecule has the potential of providing protection against a number of nerve agents with minimal side effects, since it can stoichiometrically bind and/or metabolize a nerve agent before its distribution to the site of toxic effect. The main obstacle to development of this type of therapeutic for use in humans is the need for frequent administration of a relatively short-lived product. We propose to create a novel BuChE protein that can be administered less frequently, but with greater potency, than the unmodified BuChE. We will achieve this goal using the published structural information for butylcholinesterase to rationally design polyethylene glycol (PEG)-BuChE conjugates using cysteine-reactive PEG. We will introduce a new "free" cysteine using she-directed mutagenesis in regions of BuChE that are believed to be non-essential for biological activity. The "free" cysteine residue will serve as the site for the covalent modification of the protein using a thiol-reactive PEG. This technology allows for the creation of novel, fully active PEG-Cys-BuChE analogues of defined structure and overcomes the problems of reduced bioactivity and heterogeneity when a protein is modified using a standard amine-reactive PEG. During Phase I we will identify sites in BuChE that can be modified without affecting the protein's in vitro bioactivity and further, demonstrate that PEGylation enhances BuChE's circulating half-life and efficacy in vivo. During Phase II, we develop a cost effective manufacturing process and produce sufficient quantities of the PEGylated recombinant BuChE for testing in animal toxicity models and biochemical characterization.