ENGINEERING OF ANTIDIGOXIN ANTIBODY COMBINING SITES

这一建议充分利用了蛋白质方法学的最新进展。 为了加深对工程和结构分析的了解 抗体结构与功能的关系 结合点。地高辛特异性抗体是一个模型系统，在其中 抗体和半抗原结构被设计和修改以影响 通过结合专一性的改变而产生的生物活性。反- 地高辛抗体是公认的诊断和治疗药物 洋地黄苷的毒性逆转作用。单克隆 抗地高辛抗体对其部位表现出非凡的亲和力- 填充疏水配体，其结构上有数百个 已知立体化学的已定义的同系物。现已证实的可行性 抗体可变区基因的克隆，体外诱变，以及 下列抗体或抗体结合片段的表达 允许在真核生物或原核生物(大肠杆菌)中进行转基因 突变体工程学，提供对 抗体互补性。这些研究是相辅相成的 用X射线分析三级结构的可行性 结晶学、核磁共振光谱学和计算机 模特儿。建议的实验包括：1)结构和结合 突变体细胞自发可变区的分析 通过从二次反应中获得的细胞分类选择的结合 杂交瘤。2)最新测定的晶体结构 Fab：同源抗体26-10的地高辛复合体和一个非结合 突变体是构建新的可变区的基础 结合方式改变的突变体。突变体的设计也是基于 FV的核磁共振研究结果、预测性计算机建模和研究 自发变种人的手中。3)体外诱变工程 抗体40-50的结合变异体的结晶学提纯 已经接近尾声。这些研究与26-10年度的研究平行，后者有 不同的一级结构和结合特异性，提供了独特的 有机会比较同一半抗原的抗体。4)确定 共享VL的抗体集对结合的相对链贡献 地区。5)单抗Fv、单链Fv和Fab片段 表达抗体和突变体，并表征它们的结合。 结合核磁共振和结晶学研究。操纵 抗体基因和蛋白质用于阐明其结构基础 抗原-抗体互补性是一个更广泛的重要范例 抗体作为治疗工具的应用，无论是直接用于 作为Fv或Fab片段的免疫治疗，或通过与效应器的连锁靶向治疗 分子。

This proposal capitalizes on recent methodological advances in protein engineering and structural analysis in order to further understanding of the relationship between the structure and function of the antibody combining site. Digoxin specific antibodies are a model system in which antibody and hapten structures are designed and modified to affect biologic activity through alterations in binding specificity. Anti- digoxin antibodies are proven therapeutic agents for diagnosis and reversal of toxicity due to digitalis glycosides. Monoclonal antidigoxin antibodies display exceptional affinity for their site- filling hydrophobic ligand, for which there are hundreds of structurally defined congeners of known stereochemistry. The now proven feasibility of cloning of antibody variable region genes, in vitro mutagenesis, and expression of antibodies or antibody binding fragments following transfection in eukaryotes, or in prokaryotes (E coli) permits engineering of mutants providing direct tests of the "rules" for antibody complementarity. These studies are complemented and made feasible by analyses of tertiary structure employing X-ray crystallography, nuclear magnetic resonance spectroscopy, and computer modeling. Proposed experiments include: 1) structural and binding analysis of spontaneous variable region somatic mutants with altered binding selected by cell sorting obtained from secondary response hybridomas. 2) The recently determined crystal structures of the Fab:digoxin complex of the cognate antibody 26-10 and a nonbinding mutant serve as the basis for engineering of new variable region mutants with altered binding. The design of mutants is based also on results of NMR studies of Fv, predictive computer modeling, and studies of spontaneous mutants in hand. 3) Engineer by in vitro mutagenesis binding variants of antibody 40-50 for which crystallographic refinement is near completion. These studies parallel those for 26-10, which has different primary structure and binding specificity, providing a unique opportunity to compare antibodies to the same hapten. 4) Determine relative chain contribution to binding in antibody sets sharing VL regions. 5) Fv, single chain Fv and Fab fragments of monoclonal antibodies and mutants are expressed and their binding characterized, in conjunction with NMR and crystallographic studies. The manipulation of antibody genes and proteins for the elucidation of the structural basis of antigen-antibody complementarity is a paradigm important in the wider application of antibodies as therapeutic tools, whether used directly in immunotherapy as Fv or Fab fragments, or targeted by linkage to effector molecules.