NEW APPROACHES FOR GENERATING CATALYTIC ANTIBODIES

催化抗体领域的大多数研究都是 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到，一个大的 反应的数量是服从抗体催化的标准 这些研究报告的第一代催化剂的活性很差。 与天然酶相比。我们对开发新方法很感兴趣 产生具有更高活性的催化抗体，并且在 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。我们最近产生了一种水解性抗体，可以催化 对映体选择性氨基酸酯和酰胺的水解。这是世界上 迄今为止产生的最好的酯酶活性催化抗体(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……里面 此外，我们还解决了这一问题的三维晶体结构 结合半抗原分辨率为2.5埃的抗体(合作中 与罗伯特·弗莱特里克在加州大学旧金山分校的团队合作)。活动站点结构为 与我们的动力学数据一致，并进一步支持 类似于丝氨酸蛋白酶。有了这个系统，我们拥有了无与伦比的 有机会确定重要的结构/职能关系 并创造出我们第一个更活跃的变种- 一代催化剂。这项建议描述了机械学研究。 动力学分析、诱变研究和基于酰胺酶的功能 在杂交瘤中的选择是围绕我们独特的催化设计的 抗体系统。机械实验的目的是证明 催化中共价中间体的存在及鉴定 中间地层或分解是限速的。我们建议 开发一种更灵敏的放射性同位素分析方法，以准确地测量 抗体催化的酰胺水解动力学。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 活性部位，构建更多的活性抗体突变体，并进行改进 在KCAT和KM中都有。我们打算在杂交瘤中进行功能选择 探索免疫球蛋白谱系中更好的抗体 催化酰胺水解物。这项研究的结果可能 通过定义催化抗体，显著推进了催化抗体领域 可并入到 免疫球蛋白结合部位及提供新的选择方法 以产生活性更高的催化抗体。

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.