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一种天然存在的酶，丁基胆碱酯酶（BuChE），它能够隔离或催化神经毒剂的水解。这种大分子具有提供对许多神经毒剂的保护而副作用最小的潜力，因为它可以在神经毒剂分布到毒性作用部位之前化学计量结合和/或代谢神经毒剂。开发这类用于人类的治疗药物的主要障碍是需要频繁地给药相对寿命较短的产品。我们建议创造一种新的BuChE蛋白，它可以比未经修饰的BuChE更少地施用，但具有更大的效力。我们将利用已公布的丁基胆碱酯酶的结构信息，利用半胱氨酸反应性PEG合理设计聚乙二醇(PEG)-BuChE偶联物。我们将在被认为对生物活性非必需的BuChE区域使用定向诱变引入一种新的“游离”半胱氨酸。“游离”半胱氨酸残基将作为使用巯基反应性PEG对蛋白质进行共价修饰的位点。该技术允许创建具有明确结构的新颖、完全活性的PEG- cys - buche类似物，并克服了使用标准胺反应性PEG修饰蛋白质时降低生物活性和异质性的问题。在第一阶段，我们将确定BuChE中可以在不影响蛋白质体外生物活性的情况下进行修饰的位点，并进一步证明PEGylation可以提高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，并生产了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的测试。

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.