Cocaine degradation: Improving antibody efficacy

描述（由申请人提供）：该提案提出了一种用于治疗可卡因成瘾的免疫治疗策略，其中催化抗体进化为能够将可卡因水解为其非精神活性产物，ecognine甲酯和苯甲酸。以先前鉴定的可卡因催化剂（称为GNL）为起点，将应用两种不同的定向进化技术构建新的scFv文库。第一种方法涉及基于Wilson和Janda实验室未发表的晶体结构结果的互补决定区（CDR）行走的亲和成熟，而第二种方法依赖于随机重组或脱氧核糖核酸（DMA）改组。将使用高通量方法筛选这些GNLM （gnl突变体）文库，并评估其水解可卡因的能力；前两名抗体将通过比较它们的动力学参数来确定。选择的GNLM催化剂将被检查其影响大鼠运动活动的能力，因为运动行为提供了标准化条件下药物作用的敏感和可重复的测量。

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 The first involves affinity maturation by complementarity determining region (CDR) walking based on unpublished crystal structure results from the Wilson and Janda laboratories, while the second relies on random recombination, or deoxyribose nucleic acid (DMA) shuffling.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.