STRUCTURE/FUNCTION STUDIES OF ANTIGEN/ANTIBODY REACTIONS

抗体是一种功能强大、用途广泛的结合分子，它可以免疫 系统用来消除外来抗原，因此构成了一个 阐明大分子原理的极佳模型 承认。了解抗体功能的分子基础是 对于深入了解免疫反应和 开发抗体作为治疗工具(例如，通过 啮齿动物抗体的“人性化”)。 我们建议探索抗原-抗体识别的分子基础。 通过详细的定点突变体的结构和功能的研究 抗鸡蛋清溶菌酶(HEL)单抗D1.3，具有 是我们实验室广泛的结构研究的主题。为 这些实验，蛋白质工程的，细菌表达的FvD1.3 将使用的碎片作为自由的三维结构 FV和FV-HEL复合体的FV分辨率为1.8埃； 这应该允许更严格地解释站点定向 比目前对任何其他抗原都可能进行的诱变实验- 抗体系统。FV突变体旨在研究FV的特定方面 蛋白质-蛋白质(抗原-抗体)识别的问题将是 以对抗原的亲和力和反应性为特征的。 选定的FvD1.3突变体与HEL络合将被结晶并进行X射线检查 将进行结晶学分析以准确确定 特定氨基酸取代在结构水平上的影响。在……里面 此外，将使用滴定量热法来确定熵和 以关联为目的的结合反应的热变 这些热力学参数与X射线模型相吻合。 研究亲和力是如何对特定的蛋白质抗原成熟的 决定因素是由免疫系统实现的，随机突变 抗体基因与新近开发的方法相结合 丝状噬菌体表面的抗体展示将是 用于模拟这一过程并分离D1.3的变体，改进后的 对抗原的亲和力。的三维结构 与HEL络合的相应Fv片段将在 为了了解观察到的增长的结构性基础 亲和力。 据设想，这项基础工作将产生一个Fv突变体库 已知的结构和亲和力，将作为建模的基础 抗原-抗体反应的结构和热力学参数。 这将大大促进旨在从头开始的努力。 抗体亲和力和结合部位构象的预测，以及 从而促进了用于医疗和医疗的抗体分子的工程 化学应用。

Antibodies are powerful and versatile binding molecules that the immune system employs to eliminate foreign antigens and as such constitute an excellent model for elucidating the principles governing macromolecular recognition. A knowledge of the molecular basis of antibody function is also essential for an in-depth understanding of immune responses and for the development of antibodies as therapeutic tools (e.g. through the "humanization" of rodent antibodies). We propose to explore the molecular basis of antigen-antibody recognition through detailed structure-function studies of site-directed mutants of the anti-hen egg-white lysozyme (HEL) monoclonal antibody D1.3, which has been the subject of extensive structural studies in our laboratory. For these experiments, the protein-engineered, bacterially-expressed FvDl.3 fragment will be used since the three-dimensional structures of the free Fv and that of the Fv-HEL complex are known to 1.8 Angstrom resolution; this should allow a more rigorous interpretation of site-directed mutagenesis experiments than is currently possible for any other antigen- antibody system. Fv mutants designed to investigate particular aspects of the problem of protein-protein (antigen-antibody) recognition will be characterized in terms of affinity and reactivity towards the antigen. Selected FvD1.3 mutants complexed with HEL will be crystallized and X-ray crystallographic analysis will be carried out to precisely ascertain the effects of particular amino acid substitutions at the structural level. In addition, titration calorimetry will be used to determine the entropy and enthalpy changes of the binding reactions with the aim of correlating these thermodynamic parameters with the X-ray models. To investigate how affinity maturation towards a defined protein antigenic determinant is achieved by the immune system, random mutagenesis of antibody genes in combination with recently developed methods for the display of antibodies on the surface of filamentous bacteriophage will be used to mimic this process and to isolate variants of D1.3 with improved affinity for antigen. The three-dimensional structures of the corresponding Fv fragments complexed with HEL will then be determined in order to understand the structural basis for the observed increases in affinity. It is envisaged that this basic work will generate a library of Fv mutants of known structure and affinity which will serve as a basis for modelling the structural and thermodynamic parameters of antigen-antibody reactions. This should significantly contribute to efforts aimed at ab initio prediction of antibody affinity and combining site conformation, and thereby facilitate the engineering of antibody molecules for medical and chemical applications.