ENGINEERING OF ANTIDIGOXIN ANTIBODY COMBINING SITES

抗地高辛抗体结合位点的工程

基本信息

批准号：
6043778
负责人：
MICHAEL N MARGOLIES
金额：
$ 34.68万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-08-01 至 2001-07-31
项目状态：
已结题

项目摘要

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：本提案的目的是修改抗体组合位点以改变亲和力和特异性结构相关的类似物。该模型系统由地高辛- 特异性抗体，已被证明是逆转疾病的治疗剂洋地黄毒性。单克隆抗地高辛抗体展示对其位点填充具有特殊的亲和力，相对刚性疏水性配体，有许多已知的类似物抗地高辛抗体表现出不同的立体化学特异性。结构-功能关系和抗原的研究 X 射线的存在使得结合点工程变得可行利用两种不同的抗地高辛 Fab 的晶体结构完全不同的可变区基因与半抗原复合，未复合的 Fab 和非结合突变体 Fab。诱变策略取决于从大量抗体中选择具有所需特异性的抗体丝状噬菌体上展示的突变 Fab 片段文库。来自两个抗地高辛 Fab 的 DNA 被克隆到噬菌体表达中载体，以及通过获得的具有独特特异性的突变Fab 互补决定区（CDR）的饱和诱变和框架片段通过连续几轮的亲和力而丰富选择。对克隆突变体进行测序、亲和力和特异性确定的地高辛类似物，以及序列和结合数据在晶体结构的背景下解释。那么结果就是用于重复轮次诱变，酌情采用，整个 V 区的随机诱变、定点诱变、CDR 环延长和涉及不同 CDR 的组合诱变和两条链。离体系统诱变提供了机会获得不受偏见限制的特异性和亲和力变化种系基因密码子和体外体细胞突变过程。新变体的可溶性工厂将被结晶和结构决定。采用噬菌体展示 Fab 的选择性方法图书馆不仅能够洞察事物的结构基础结合了位点互补性，但代表了一个范例构建具有独特特异性的抗体，或针对其消除不良交叉反应，用于临床免疫疗法。