STRUCTURE/FUNCTION STUDIES OF ANTIGEN-ANTIBODY REACTIONS

抗原抗体反应的结构/功能研究

• 批准号: 6019060
• 负责人: Roy A Mariuzza
• 金额: $ 20.25万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-19 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019060
• 关键词: X ray crystallography; antigen antibody reaction; antiidiotype antibody; chemical binding; chemical stability; conformation; hydrogen bond; hydropathy; intermolecular interaction; lysozyme; microcalorimetry; monoclonal antibody; mutant; protein sequence; protein structure function; site directed mutagenesis; thermodynamics

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.

抗体是一种强大的多功能结合分子， 系统用于消除外来抗原，并因此构成了一种 阐明大分子生物学原理的极好模型 识别.抗体功能的分子基础知识是 对于深入了解免疫反应和 抗体作为治疗工具的发展（例如，通过 啮齿动物抗体的“人源化”）。 我们建议探索抗原抗体识别的分子基础 通过详细的结构-功能研究定点突变的 抗鸡卵清溶菌酶（HEL）单克隆抗体D1.3， 在我们的实验室里进行了广泛的结构研究。为 在这些实验中，蛋白质工程化的细菌表达的FvD1.3 片段将被使用，因为自由的三维结构 已知Fv和Fv-HEL复合物的分辨率为1.8埃; 这应该允许更严格地解释现场指导 诱变实验比目前可能的任何其他抗原- 抗体系统设计Fv突变体以研究 蛋白质-蛋白质（抗原-抗体）识别问题 其特征在于对抗原的亲和力和反应性。 选择的与HEL复合的FvD1.3突变体将被结晶并X射线衍射分析。 将进行晶体学分析，以精确地确定 在结构水平上特定氨基酸取代的影响。在 此外，滴定量热法将用于确定熵， 结合反应的焓变，目的是关联 这些热力学参数与X射线模型。 为了研究亲和力成熟对一个确定的蛋白抗原 决定子是由免疫系统实现的，随机诱变的 抗体基因结合最近开发的方法， 抗体在丝状噬菌体表面的展示将是 用于模拟这一过程，并分离具有改进的D1.3的变体。 对抗原的亲和力。的三维结构， 然后，将确定与HEL复合的相应Fv片段， 为了了解所观察到的增加的结构基础， 亲和力 设想这一基础工作将产生Fv突变体文库 已知的结构和亲和力，这将作为建模的基础 抗原-抗体反应的结构和热力学参数。 这将大大有助于旨在从一开始就 抗体亲和力和结合位点构象的预测，以及 从而促进用于医学的抗体分子的工程化， 化学应用。