Allosteric Binding in Antibodies and Protein Antigens

描述(由申请人提供)：广泛开发用于医学研究活动、治疗治疗和医学诊断免疫分析的单抗构成了一个数十亿美元的卫生保健产业。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。分析实验的目的是为了了解这些新的协同结合反应的分子机制。方法包括但不限于分析超速离心法、不对称场流分级法、等温滴定和差示扫描量热法、圆二色谱和其他光谱测量。具体目标1和3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构和功能研究。实际目标包括(I)确定表观希尔系数低于和高于2.0的抗体的抗原结合化学计量学，以及(Ii)观察抗体构象变化与抗原结合的关系。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功能和结构研究。这个目标的主要目的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。在这两个目的中，统一的假设是潜在的分子机制是作为抗体-抗原结合作用的结果而发生的蛋白质构象变化。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。

DESCRIPTION (provided by applicant): 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. Analytical experiments are proposed with the goal of understanding the molecular mechanisms of these novel synergistic binding reactions. Methods include, but are not limited to, analytical ultracentrifugation, asymmetric field flow fractionation, isothermal titration and differential scanning calorimetry, and circular dichroism and other spectroscopic measurements. Specific aims 1 and 3 are to conduct detailed structural and functional studies on monoclonal antibodies that exhibit synergistic binding behavior with their respective antigens. Practical goals include (i) the determination of antigen binding stoichiometries for antibodies that exhibit apparent Hill coefficients below and above 2.0 and (ii) the observation and correlation of antibody conformational changes to antigen binding. Specific aim 2 is to conduct functional and structural studies on protein antigens that bind monoclonal antibodies to separate epitopes in a synergistic manner. The principal 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.