NEW APPROACHES FOR GENERATING CATALYTIC ANTIBODIES

生成催化抗体的新方法

• 批准号: 2838611
• 负责人: THOMAS Sterling SCANLAN
• 金额: $ 10.53万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2838611
• 关键词: X ray crystallography; abzyme; active sites; chemical kinetics; chemical reaction; enzyme activity; enzyme structure; esterase; hybridomas; immunoglobulin structure; laboratory mouse; monoclonal antibody; protein engineering; protein structure function; radiotracer

The majority of research in the field of catalytic antibodies has been directed toward the discovery of new chemical reactions that can be catalyzed by antibodies. While it is now clear from this work that a large number of reactions are amenable to antibody catalysis, the standard first-generation catalysts reported in these studies have poor activity compared to natural enzymes. We are interested in developing new methods that produce catalytic antibodies with higher activity, and in understanding structure/function relationships in antibody catalysis that will allow higher activity variants of first-generation catalysts to be engineered. We recently generated a hydrolytic antibody that catalyzes enantioselective amino acid ester and amide hydrolysis. This is one of the best catalytic antibodies generated to date for esterase activity (kcat=4 sec-1), and our preliminary mechanistic studies indicate that the antibody uses a mechanism of catalysis that is similar to serine proteases. In addition, we have solved the 3-dimensional crystal structure of this antibody with bound hapten at 2.5 Angstroms resolution (in collaboration with Robert Fletterick's group at UCSF). The active site structure is consistent with our kinetic data and further supports the mechanistic analogy with serine proteases. With this system, we have an unparalleled opportunity to delineate the important structure/function relations for hydrolytic antibodies and create more active variants of our first- generation catalyst. This proposal describes mechanistic studies, amidase kinetic analyses, mutagenesis studies, and an amidase-based functional selection in hybridomas that are designed around our unique catalytic antibody system. The mechanistic experiments are aimed at proving the existence of a covalent intermediate in catalysis and identifying whether intermediate formation or breakdown is rate-limiting. We propose to develop a more sensitive radioisotope assay to accurately measure the kinetics of antibody-catalyzed amide hydrolysis. Our mutagenesis studies are designed to identify catalytically important residues in the antibody active site, and construct more active antibody mutants that are improved in both kcat and KM. With our functional selection in hybridomas we intend to probe the immunoglobulin repertoire for antibodies that are better at catalyzing amide hydrolysis. The results of this research could significantly advance the field of catalytic antibodies by defining the limits of catalytic activity that can be incorporated into an immunoglobulin combining site and providing new selection-based methods for generating catalytic antibodies with higher levels of activity.

催化抗体领域的大部分研究都是 旨在发现新的化学反应 由抗体催化。虽然现在从这项工作中可以清楚地看出， 抗体催化反应的数量，标准 这些研究报告的第一代催化剂活性较差 与天然酶相比。我们有兴趣开发新方法 产生具有更高活性的催化抗体，并且 了解抗体催化中的结构/功能关系 将允许第一代催化剂的更高活性变体 设计的。我们最近产生了一种水解抗体，可以催化 对映选择性氨基酸酯和酰胺水解。这是其中之一 迄今为止产生的酯酶活性最佳催化抗体（kcat=4 sec-1），我们的初步机制研究表明该抗体 使用类似于丝氨酸蛋白酶的催化机制。在 此外，我们还解决了这个的3维晶体结构 具有 2.5 埃分辨率的结合半抗原的抗体（合作 与加州大学旧金山分校 (UCSF) 的罗伯特·弗莱特里克 (Robert Fletrick) 小组合作)。活性位点结构为 与我们的动力学数据一致，进一步支持了该机制 与丝氨酸蛋白酶类比。有了这个系统，我们就拥有了无与伦比的 有机会描述重要的结构/功能关系 水解抗体并创建我们的第一个的更活跃的变体 生成催化剂。该提案描述了机制研究、酰胺酶 动力学分析、诱变研究和基于酰胺酶的功能 围绕我们独特的催化设计的杂交瘤的选择 抗体系统。机械实验旨在证明 催化中是否存在共价中间体并确定是否存在 中间形成或分解是限速的。我们建议 开发一种更灵敏的放射性同位素测定法来准确测量 抗体催化酰胺水解的动力学。我们的诱变研究 旨在识别抗体中具有催化作用的重要残基 活性位点，并构建更具活性的改良抗体突变体 在 kcat 和 KM 中。通过我们对杂交瘤的功能选择，我们打算 探索免疫球蛋白库，寻找更擅长的抗体 催化酰胺水解。这项研究的结果可以 通过定义显着推进催化抗体领域 可以纳入的催化活性的限制 免疫球蛋白结合位点并提供新的基于选择的方法 用于产生具有更高活性水平的催化抗体。