ENGINEERING OF ANTIDIGOXIN ANTIBODY COMBINING SITES

抗地高辛抗体结合位点的工程

• 批准号: 2750364
• 负责人: MICHAEL N MARGOLIES
• 金额: $ 33.35万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2750364
• 关键词: X ray crystallography; antibody specificity; antibody titering; antiidiotype antibody; chemical binding; computer simulation; digoxin; enzyme linked immunosorbent assay; haptens; immunogenetics; laboratory mouse; molecular cloning; monoclonal antibody; mutant; protein engineering; protein purification; protein sequence; protein structure function; radioimmunoassay; site directed mutagenesis

DESCRIPTION: The objective of this proposal is to modify antibody combining sites in order to change affinity and specificity for structurally related analogues. The model system consists of digoxin- specific antibodies which are proven therapeutic agents for reversal of digitalis toxicity. Monoclonal anti-digoxin antibodies display exceptional affinity for their site-filling, relatively rigid hydrophobic ligand, for which there are numerous analogues of known stereochemistry for which anti-digoxin antibodies display varying specificity. Studies of structure-function relationships and antigen combining site engineering are made feasible by the existence of x-ray crystallographic structures of two different anti-digoxin Fabs utilizing entirely different variable region genes in complex with hapten, an uncomplexed Fab, and a non-binding mutant Fab. The mutagenesis strategy hinges upon the selection of antibodies of desired specificity from large libraries of mutant Fab fragments displayed on filamentous bacteriophage. The DNA from the two anti-digoxin Fabs is cloned into phage expression vectors, and mutant Fabs with unique specificities obtained through saturation mutagenesis of complementarity-determining regions (CDRs) and framework segments are enriched by successive rounds of affinity selections. Cloned mutants are sequenced, affinity and specificity for digoxin analogues determined, and the sequence and binding data interpreted in the context of crystal structure. The results are then used for reiterative rounds of mutagenesis, employing, as appropriate, random mutagenesis of entire V regions, site-directed mutagenesis, CDR loop lengthening, and combinatorial mutagenesis involving different CDRs and both chains. Systematic mutagenesis ex vivo provides opportunities to attain specificity and affinity changes not constrained by the biases of germline gene codons and the in vitro somatic mutation process. Soluble Fabs of novel variants will be crystallized and structures determined. A selective approach employing phage-displayed Fab libraries not only promises insights into the structural basis of a combining site complementarity, but represents a paradigm for constructing antibodies of unique specificity, or antibodies for which undesirable cross-reactivity is removed, for use in clinical immunotherapy.

描述：本提案的目的是修饰抗体