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：该提议的目的是修改抗体结合站点以改变亲和力和特异性与结构相关的类似物。模型系统由高辛 - 特定的抗体，这些抗体被证明是逆转的治疗剂 Digitalis毒性。单克隆抗二氧抗体抗体显示对其现场填充，相对僵化的特殊亲和力疏水配体，有许多已知的类似物立体化学的抗二高氧蛋白抗体显示不同特异性。结构功能关系和抗原的研究 X射线的存在使场地工程相结合是可行的利用两种不同抗二高氧蛋白的晶体结构与Hapten复合物中完全不同的变量区域基因，未复杂的Fab和非结合突变型晶圆厂。诱变策略取决于从大的选择所需特异性的抗体在丝状噬菌体上显示的突变型晶圆厂碎片的库。将两个抗二氧蛋白晶圆厂的DNA克隆到噬菌体表达中向量和具有独特特异性的突变型晶圆厂互补性区域（CDR）和框架段通过连续的亲和力丰富选择。克隆突变体是测序的，亲和力和特异性地高辛类似物确定，序列和结合数据在晶体结构的背景下进行解释。结果是用于诱变的重复性回合，适当地采用整个V区域的随机诱变，位置定向诱变，CDR 循环延长和组合诱变涉及不同的CDR 和两个连锁店。系统诱变后体内提供机会达到特异性和亲和力变化不受偏见的约束种系基因密码子和体外体细胞突变过程。新型变体的可溶性晶圆厂将结晶并结构决定。采用噬菌体播放的晶圆厂的选择性方法图书馆不仅承诺对一个结构基础的见解结合场地互补性，但代表了构建具有独特特异性或抗体的抗体除去不良的交叉反应性，用于临床免疫疗法。