NEW APPROACHES FOR GENERATING CATALYTIC ANTIBODIES

生成催化抗体的新方法

• 批准号: 2022835
• 负责人: THOMAS Sterling SCANLAN
• 金额: $ 9.84万
• 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/2022835
• 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.

在催化抗体领域的大部分研究已经完成。 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到， 许多反应都服从于抗体催化，标准 在这些研究中报道的第一代催化剂具有差的活性 与天然酶相比。我们对开发新方法感兴趣 产生具有更高活性的催化抗体， 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， 精心设计的我们最近制造了一种水解抗体 对映选择性氨基酸酯和酰胺水解。这是其中一 迄今为止产生的酯酶活性最好的催化抗体（kcat=4 sec-1），我们的初步机制研究表明，抗体 使用类似于丝氨酸蛋白酶的催化机制。在 此外，我们还解决了这种三维晶体结构， 具有结合半抗原的抗体，分辨率为2.5埃（与 Robert Fletterick在UCSF的团队）。活性部位结构为 与我们的动力学数据一致，并进一步支持了 类似于丝氨酸蛋白酶。有了这个系统，我们有了一个无与伦比的 描述重要结构/功能关系的机会， 水解抗体，并创造出我们第一个- 生成催化剂。该建议描述了机械研究，酰胺酶 动力学分析、诱变研究和基于酰胺酶的功能性 在杂交瘤中的选择，是围绕我们独特的催化， 抗体系统机械实验旨在证明 在催化中存在共价中间体，并确定 中间体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分析方法， 抗体催化的酰胺水解的动力学。我们的诱变研究 设计用于鉴定抗体中催化重要的残基 活性位点，并构建更多的活性抗体突变体， 在kcat和KM中。通过我们在杂交瘤中的功能选择，我们打算 来探测免疫球蛋白库中的抗体， 催化酰胺水解。这项研究的结果可以 通过定义催化抗体， 催化活性的限制，可以结合到一个 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。