Cocaine degradation: Improving antibody efficacy

描述(由申请人提供)：本提案提出了一种治疗可卡因成瘾的免疫治疗策略，在该策略中，催化抗体的进化是因为它们能够将可卡因水解为其非精神活性产物、生态甲酯和苯甲酸。使用以前发现的可卡因催化剂，称为GNL，作为起点，两种不同的定向进化技术将被应用于构建新型单链抗体文库。第一种是基于Wilson和Janda实验室未发表的晶体结构的互补决定区(CDR)行走的亲和力成熟，第二种是依赖随机重组或脱氧核糖核酸(DMA)改组。这些GNLM(GNL突变)文库将使用高通量方法进行筛选，并评估它们水解可卡因的能力；排名前两位的抗体将通过比较它们的动力学参数来确定。选定的GNLM催化剂将被测试它们影响大鼠运动活动的能力，因为在标准化条件下，运动行为提供了一种灵敏和可重复的药物作用衡量标准。

DESCRIPTION (provided by applicant): This proposal presents an immunotherapeutic strategy for the treatment of cocaine addiction in which catalytic antibodies are evolved for their ability to hydrolyze cocaine to its non-psychoactive products, ecognine methyl ester and benzoic acid. Using previously identified cocaine catalysts, termed GNL, as a starting point, two different directed evolution techniques will be applied for the construction of novel scFv libraries. The first involves affinity maturation by complementarity determining region (CDR) walking based on unpublished crystal structure results from the Wilson and Janda laboratories, while the second relies on random recombination, or deoxyribose nucleic acid (DMA) shuffling. These GNLM (GNL-mutant) libraries will be screened using high throughput methodology and assessed for their ability to hydrolyze cocaine; the top two antibodies will be determined by a comparison their kinetic parameters. Selected GNLM catalysts will be examined for their ability to affect locomotor activity in rats, since motor behavior provides a sensitive and reproducible measure of drug action under standardized conditions.