SEQUENCE, SHAPE AND SPECIFICITY OF ANTIBODIES

抗体的序列、形状和特异性

• 批准号: 2894503
• 负责人: MICHAEL N MARGOLIES
• 金额: $ 37.75万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2894503
• 关键词: X ray crystallography; antibody specificity; chemical binding; gene mutation; gene rearrangement; haptens; immunoglobulin structure; laboratory mouse; protein engineering; protein sequence; site directed mutagenesis

DESCRIPTION: (Adapted from the applicant's abstract) A majority of antibodies raised against p-azophenylarsonate (Ars)-protein conjugates in A/J mice share a heritable cross-reactive idiotype, as they are encoded by a single combination of variable region germline gene segments termed "canonical." The dominance of the canonically-encoded structure is due to the favorable intrinsic Ars affinity of the V region germline gene combination and its ability to sustain somatic mutations conferring higher affinity leading to preferred antigen-driven selection of B cells expressing canonical V regions. The Ars systems is an important model for defining combining site structural changes occurring temporally in immune responses (affinity maturation), as functional differences among these antibodies can be related structurally by comparison to unmutated precursors. The goals of this project include: 1) To access the differentiative capacity of the germline canonical structure to generate increased affinity and to change specificity for structurally-related analogues as compared to that of a higher-affinity somatically mutated canonical antibody of known crystal structure. 2) To engineer changes in specificity and affinity ex vivo that may not be possible in vivo owing to biases inherent in the germline sequence and the somatic mutation process. A "selective" approach will be used primarily, in which libraries of mutant Fabs derived from germline (unmutated) canonical anti-Ars antibodies and high-affinity (somatically mutated) canonical anti-Ars antibodies are displayed on filamentous bacteriophage and sorted by affinity, and specificity of these mutants for Ars analogues are determined and the results interpreted in the context of the crystal structures of anti-Ars Fabs. In addition to saturation mutagenesis of different CDRs, "random" mutagenesis of entire V regions, combinatorial mutagenesis of CDRs and regions from both chains, as well as site-directed mutagenesis, will be carried out. Novel mutants will be crystallized and their structures determined. The feasibility of antibody combining site engineering to design antibodies with altered function, and in particular to increase specificity, is critical for the success of clinical immunotherapy.

描述：（改编自申请人的摘要）大多数 对-偶氮苯胂酸盐（Ars）-蛋白质缀合物产生的抗体， A/J小鼠共有一种可遗传的交叉反应独特型，因为它们是由一种 可变区种系基因区段的单一组合称为 “canonical.“规范编码结构的主导地位是由于 V区生殖系基因的有利的内在Ars亲和力 组合及其维持体细胞突变的能力，赋予更高的 亲和力导致优选的抗原驱动选择表达的B细胞 典型V区 Ars系统是一个重要的模型， 免疫反应中暂时发生的结合位点结构变化 （亲和力成熟），因为这些抗体之间的功能差异可以 与未突变的前体相比，在结构上相关。 的目标 该项目包括：1）评估的差异化能力， 生殖系典型结构，以产生增加的亲和力， 结构相关类似物的特异性相比， 已知晶体高亲和力体细胞突变的典型抗体 结构 2)为了在体外设计特异性和亲和力的变化， 由于生殖系固有的偏差， 序列和体细胞突变过程。 一个“选择性”的方法将是 主要使用，其中源自种系的突变Fab文库 （未突变的）典型抗Ars抗体和高亲和力（体细胞）抗Ars抗体 突变的）典型抗Ars抗体展示在丝状 噬菌体和排序的亲和力，和特异性，这些突变体， Ars类似物的测定和结果解释的背景下， 反Ars Fab的晶体结构 除了饱和 不同CDR的诱变，整个V区的“随机”诱变， 来自两条链的CDR和区域的组合诱变，以及 将进行定点诱变。 新的突变体将是 结晶并确定其结构。 抗体的可行性 结合位点工程设计功能改变的抗体， 特别是增加特异性，对于 临床免疫治疗