Allosteric Binding in Antibodies and Protein Antigens

描述（由申请人提供）：单克隆抗体广泛用于医学研究活动、治疗治疗和医学诊断免疫测定，构成了数十亿美元的医疗保健产业。该项目将研究最近针对选定的抗体-抗原对描述的两种新型协同结合反应：抗体的亲和力似乎随着抗原浓度的增加而增加（正协同性）；而那些抗体对蛋白质抗原上的一个表位的亲和力似乎在针对同一蛋白质抗原上不同的单独表位的第二个抗体的存在下增加。分析实验的目的是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性超离心、不对称场流分馏、等温滴定和差示扫描量热法、圆二色性和其他光谱测量。具体目标1和3是对单克隆抗体进行详细的结构和功能研究，这些单克隆抗体与各自的抗原表现出协同结合行为。实际目标包括(i)确定明显希尔系数低于或高于2.0的抗体的抗原结合化学计量，（ii）观察抗体构象变化与抗原结合的相关性。特异性目的2是对结合单克隆抗体以协同方式分离表位的蛋白抗原进行功能和结构研究。这一目的的主要目标是量化亲和效应和结合依赖的蛋白质构象变化对结合的明显协同作用的相对贡献。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。任何试图利用表现出新型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，都必须依赖于有关新型结合表达的分子机制的现有知识。该项目有望为控制这些意外活动的诊断和治疗应用提供基础知识。这也将有助于对迄今为止尚未认识到的抗体功能方面的基本了解。

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.