ENGINEERING OF ANTIDIGOXIN ANTIBODY COMBINING SITES

该建议利用了蛋白质研究方法的最新进展 工程和结构分析，以进一步了解 抗体结构与功能的关系 结合位点。 地高辛特异性抗体是模型系统，其中 抗体和半抗原结构被设计和修饰以影响 生物活性通过改变结合特异性。 抗 地高辛抗体是经证实的用于诊断的治疗剂， 逆转洋地黄苷的毒性。 单克隆 抗地高辛抗体对它们的位点显示出特殊的亲和力， 填充疏水性配体，对于其有数百种结构上的 已知立体化学的确定同系物。 目前已证实的可行性 抗体可变区基因的克隆，体外诱变，和 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）中转染 突变体的工程设计提供了对“规则”的直接测试， 抗体互补性 这些研究是相辅相成的， 通过使用X射线的三级结构分析可行 晶体学、核磁共振光谱学和计算机 建模 建议的实验包括：1）结构和绑定 具有改变的自发可变区体细胞突变体的分析 通过从二次应答获得的细胞分选选择的结合 杂交瘤2)最近确定的晶体结构的 同源抗体26-10的Fab：地高辛复合物和非结合抗体 突变体作为新可变区工程化的基础 具有改变的结合的突变体。 突变体的设计也基于 Fv的NMR研究结果、预测计算机建模和研究 自发突变体的数量3)体外诱变工程 抗体40-50的结合变体， 即将完工 这些研究与26-10的研究平行， 不同的一级结构和结合特异性， 有机会比较相同半抗原的抗体。4)确定 共享VL的抗体组中对结合的相对链贡献 地区5)单克隆抗体的Fv、单链Fv和Fab片段 表达抗体和突变体并表征其结合， 结合核磁共振和晶体学研究。 操纵 用于阐明结构基础的抗体基因和蛋白质 抗原-抗体互补性是一个重要的范例，在更广泛的 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，或通过与效应物的连接而靶向 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.