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)大部分 抗对偶氮苯基砷酸盐(Ars)-蛋白质结合物的抗体 A/J小鼠共有一种可遗传的交叉反应独特型，因为它们由一种 称为可变区生殖系基因片段的单一组合 “经典的。”规范编码结构的优势是由于 V区生殖系基因良好的内在ARS亲和力 组合及其维持较高体细胞突变的能力 亲和力导致优先抗原驱动的B细胞表达 典型的V区。ARS系统是定义 结合免疫反应中暂时发生的部位结构变化 (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，在结构上是相关的。的目标 该项目包括：1)获取客户的差异化能力 生殖系规范结构产生更高的亲和力并改变 结构相关类似物的专一性与 已知晶体的高亲和力体细胞突变正则抗体 结构。2)在体外改变特异性和亲和力， 由于生殖系固有的偏见，在体内可能是不可能的 序列和体细胞突变过程。“有选择性的”方法将是 主要用于其中从生殖系衍生的突变Fabs文库 (未突变的)典范抗ARs抗体和高亲和力(躯体 突变)典型的抗Ars抗体显示在丝状细胞上 噬菌体，并根据亲和力进行分类，以及这些突变体对 ARS类似物的确定和结果在以下上下文中解释 抗Ars纤维的晶体结构。除了饱和之外 不同CDR的突变，整个V区的随机突变， CDR和来自两个链的区域的组合突变 将进行定点突变。新的突变体将是 结晶并确定了它们的结构。抗体的可行性 结合现场工程设计具有改变功能的抗体，以及 尤其是增加特异性，对成功的 临床免疫治疗。