Occurence and Mechanisms of Antibody-Antigen Allosteric Binding Behavior

在医学研究活动、治疗治疗和医学诊断免疫分析中广泛使用单抗，构成了一个价值数十亿美元的医疗保健产业。本项目将研究最近为选定的抗体-抗原对描述的两种新的协同结合反应：一种是抗体的亲和力随着抗原浓度的增加而增加(正协同性)；另一种是针对蛋白质抗原上的一个表位的抗体的亲和力在另一种抗体指向同一蛋白质抗原上的不同的单独表位时似乎增加。该项目的长期目标是了解这些新型协同结合反应的分子机制。建议在当前赠款期间进行的实验将主要集中在使用完整抗体和相应的Fab片段进行功能研究 来源于亲本结构的蛋白水解性裂解。功能研究将包括KinExA流动荧光仪上的动力学排斥分析和Biacore表面等离子体共振光谱仪上的竞争分析。具体目标#1是对表现出与各自抗原协同结合行为的单抗进行详细的结合研究。这一目标的一个目的是确定某些抗体是否在每个完整的免疫球蛋白分子上具有两个以上的抗原结合位点。具体目标#2是对以协同方式将单抗与不同表位结合的蛋白质抗原进行结合研究。这一目标的一个目标是量化亲和力效应和依赖结合的蛋白质构象变化对结合的表观协同作用的相对贡献。在这两个目标中，统一 假设潜在的分子机制是抗体-抗原结合作用导致的蛋白质构象变化。任何试图开发具有新的协同结合特性的抗体以增强当前的临床诊断或治疗应用的尝试，都必须依赖于现有的关于表达新结合的分子机制的知识。 该项目有望为操纵这些意想不到的活动用于诊断和治疗应用贡献基础知识。它还将有助于对抗体功能的一个迄今未被认识的方面的基本了解。

The widespread exploitation of monoclonal antibodies for medical research activities, therapeutic treatments, and medical diagnostic immunoassays constitutes a multi-billion dollar health care industry. This project will investigate two types of novel synergistic binding reactions recently described for selected antibody-antigen pairs: those where the affinity of the antibody appeared to increase as the antigen concentration increased (positive cooperativity); and those where the affinity of an antibody directed toward one epitope on a protein antigen appeared to increase in the presence of a second antibody directed toward a different, separate epitope on the same protein antigen. The long-term goal of this project is to understand the molecular mechanisms of these novel synergistic binding reactions. Experiments proposed for the current grant period will focus primarily on functional studies using intact antibodies and corresponding Fab fragments derived from proteolytic cleavage of the parent structures. Functional studies will include kinetic exclusion assays on a KinExA flow fluorimeter and competition assays on a BIAcore surface plasmon resonance spectrometer. Specific aim #1 is to conduct detailed binding studies on monoclonal antibodies that exhibit synergistic binding behavior with their respective antigens. One goal of this aim is to determine whether certain antibodies possess more than two antigen binding sites per intact IgG molecule. Specific aim #2 is to conduct binding studies on protein antigens that bind monoclonal antibodies to separate epitopes in a synergistic manner. One goal of this aim is to quantify the relative contributions of both avidity effects and binding-dependent protein conformation changes to the apparent synergy of binding. In both aims, the unifying hypothesis is that the underlying molecular mechanisms are protein conformation changes that occur as a consequence of the antibody-antigen binding interaction. Any attempts to exploit antibodies that exhibit novel synergistic binding properties to enhance current clinical diagnostic or therapeutic applications must be dependent on existing knowledge concerning the molecular mechanisms whereby the novel binding is expressed. This project is expected to contribute fundamental knowledge toward manipulating these unexpected activities for diagnostic and therapeutic applications. It will also contribute to a basic understanding of a heretofore unrecognized aspect of antibody function.