ENGINEERING OF ANTIDIGOXIN ANTIBODY COMBINING SITES

抗地高辛抗体结合位点的工程

• 批准号: 2223641
• 负责人: MICHAEL N MARGOLIES
• 金额: $ 26.98万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2223641
• 关键词: X ray crystallography; antibody; antibody specificity; antiidiotype antibody; chemical binding; digoxin; flow cytometry; laboratory mouse; molecular cloning; nuclear magnetic resonance spectroscopy; protein engineering; protein sequence; site directed mutagenesis; southern blotting

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.

该提案利用了蛋白质领域最新的方法学进展 工程和结构分析，以便进一步了解 抗体的结构和功能之间的关系 结合站点。 地高辛特异性抗体是一个模型系统，其中 抗体和半抗原结构经过设计和修改以影响 通过改变结合特异性而产生生物活性。 反对- 地高辛抗体是经过验证的诊断和治疗药物 逆转洋地黄糖苷引起的毒性。 单克隆 抗地高辛抗体对其位点表现出非凡的亲和力 填充疏水性配体，其结构有数百个 已知立体化学的已定义同系物。 目前已证实可行性 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 真核生物或原核生物（大肠杆菌）转染允许 突变体工程提供了“规则”的直接测试 抗体互补性。 这些研究得到了补充和完善 通过使用 X 射线分析三级结构是可行的 晶体学、核磁共振波谱学和计算机 造型。 提议的实验包括：1) 结构和结合 自发可变区体细胞突变体的分析 通过从二次反应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 Fab：同源抗体 26-10 和非结合抗体的地高辛复合物 突变体作为新可变区工程的基础 结合改变的突变体。 突变体的设计还基于 Fv 的 NMR 研究结果、预测计算机建模和研究 手中的自发突变体。 3) 体外诱变工程 抗体 40-50 的结合变体，对其进行晶体学细化 即将完成。 这些研究与 26-10 的研究类似， 不同的一级结构和结合特异性，提供了独特的 有机会比较相同半抗原的抗体。 4）确定 共享 VL 的抗体组中结合的相对链贡献 地区。 5) 单克隆的Fv、单链Fv和Fab片段 抗体和突变体的表达及其结合特征， 与核磁共振和晶体学研究相结合。 操纵 抗体基因和蛋白质用于阐明结构基础 抗原抗体互补性是更广泛的重要范式 抗体作为治疗工具的应用，无论是否直接用于 作为 Fv 或 Fab 片段的免疫疗法，或通过与效应子连接来靶向 分子。