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：（根据申请人的摘要进行了改编）大多数 针对P-琼斯苯二酚（ARS） - 蛋白偶联物的抗体中添加的抗体 A/J鼠子共享一种可遗传的交叉反应性愚蠢，因为它们是由 可变区种系基因段的单一组合称为 “典范。” 规范编码结构的主导地位是由于 V区种系基因的有利的内在ARS亲和力 结合及其维持躯体突变的能力赋予更高 亲和力导致表达B细胞的首选抗原驱动选择 规范V区域。 ARS系统是定义的重要模型 结合位点结构变化在免疫反应中发生的时间变化 （亲和力成熟），因为这些抗体之间的功能差异可以 通过与未成形的前体进行比较，在结构上与之相关。 目标 该项目包括：1）访问 种系规范结构，产生增强的亲和力并改变 与A的特异性相比 较高亲和力的已知晶体的体积突变典型抗体 结构。 2）设计特异性和亲和力的变化 由于种系中固有的偏见，可能无法在体内 序列和体细胞突变过程。 “选择性”方法将是 主要使用的是，突变型晶圆厂的库是从种系衍生的 （未分解的）规范抗ARS抗体和高亲和力 突变的）规范抗ARS抗体在丝状上显示 噬菌体并按亲和力排序，这些突变体的特异性 确定ARS类似物，并在 抗ARS Fabs的晶体结构。 除了饱和 不同CDR的诱变，整个V区域的“随机”诱变， CDR和区域的组合诱变，以及 将进行定向的诱变。 新型突变体将是 结晶及其结构确定。 抗体的可行性 将现场工程结合到设计抗体与功能变化的设计，并且 特别是提高特异性，对于成功的成功至关重要 临床免疫疗法。