Allosteric Binding in Antibodies and Protein Antigens

描述（由申请人提供）：单克隆抗体用于医学研究活动、治疗性治疗和医学诊断免疫测定的广泛开发构成了数十亿美元的医疗保健行业。本计画将研究两种最近被描述的针对特定抗体-抗原对的新型协同结合反应：抗体的亲和力随抗原浓度增加而增加的反应。（积极协同性）;以及其中针对蛋白质抗原上的一个表位的抗体的亲和力在针对不同表位的第二抗体存在下似乎增加的那些，在相同的蛋白质抗原上有不同的表位。分析实验提出了理解这些新的协同结合反应的分子机制的目标。方法包括但不限于分析超离心、不对称场流分级、等温滴定和差示扫描量热法以及圆二色性和其它光谱测量。具体目标1和3是对表现出与其各自抗原的协同结合行为的单克隆抗体进行详细的结构和功能研究。实际目标包括（i）确定表现出低于和高于2.0的表观希尔系数的抗体的抗原结合化学计量，以及（ii）观察抗体构象变化与抗原结合的相关性。具体目标2是对以协同方式将单克隆抗体结合到单独表位的蛋白质抗原进行功能和结构研究。这个目标的主要目标是量化的相对贡献的亲合力效应和结合依赖性蛋白质的构象变化的结合的表观协同作用。在这两个目标中，统一的假设是，潜在的分子机制是由于抗体-抗原结合相互作用而发生的蛋白质构象变化。任何利用表现出新的协同结合特性的抗体来增强当前临床诊断或治疗应用的尝试必须依赖于关于表达新结合的分子机制的现有知识。该项目预计将有助于操纵这些意想不到的活动的诊断和治疗应用的基础知识。它也将有助于对抗体功能的一个迄今未被认识的方面的基本理解。

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.