ENGINEERING OF ANTIDIGOXIN ANTIBODY COMBINING SITES

描述：这项提议的目的是修饰抗体 结合位点以改变亲和力和特异性 结构上相关的类似物。模型系统由地高辛- 已被证明是逆转白血病的治疗药物的特异性抗体 洋地黄毒性。抗地高辛单抗展示 对他们的站点填充具有特殊的亲和力，相对僵硬 疏水配体，有许多已知的类似物 抗地高辛抗体表现不同的立体化学 专一性。结构-功能关系与抗原的研究 由于x射线的存在，联合场地工程成为可能。 两种不同抗地高辛纤维的晶体结构 与半抗原复合体中完全不同的可变区基因，以及 未复合的Fab和非结合突变体Fab。诱变策略 关键在于从大鼠体内选择具有所需特异性的抗体 展示在丝状噬菌体上的突变Fab片段文库。 将两株抗地高辛抗体的DNA克隆到噬菌体表达载体中 载体和具有独特特异性的突变FB，通过 互补决定区(CDR)的饱和突变和 框架片段通过连续几轮的亲和力得到丰富 选择。克隆的突变体进行了测序，亲和力和特异性 测定地高辛类似物，测序和结合数据 在晶体结构的背景下进行解释。然后，结果是 用于反复的几轮诱变，酌情采用 全V区的随机突变、定点突变、CDR 环延长和涉及不同CDR的组合突变 和两条锁链。体外系统诱变提供了机会 为了获得不受偏见限制的特异性和亲和力变化 生殖系基因密码子和体外体细胞突变过程。 新型变种的可溶性纤维将被晶化和结构 下定决心。一种利用噬菌体展示Fab的选择性方法 库不仅承诺深入了解 结合站点互补性，但代表了 构建具有独特特异性的抗体，或针对其 消除了不需要的交叉反应，用于临床 免疫疗法。

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.