ENGINEERING OF ANTIDIGOXIN ANTIBODY COMBINING SITES

抗地高辛抗体结合位点的工程

• 批准号: 3366615
• 负责人: MICHAEL N MARGOLIES
• 金额: $ 25.67万
• 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/3366615
• 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.

该提议利用了蛋白质的最新方法学的进步 工程和结构分析，以进一步了解 抗体的结构和功能之间的关系 结合站点。 地高毒素特异性抗体是一个模型系统 抗体和触觉结构的设计和修饰以影响 通过改变结合特异性的生物活性。 反对- 地高辛抗体被证明是用于诊断和的治疗剂 毒性逆转由于digitalis糖苷而引起的。 单克隆 抗毒素抗体对其部位表现出非凡的亲和力 - 填充疏水配体，在结构上有数百种 已知的立体化学的固定同源物。 现在已证明的可行性 抗体可变区域基因的克隆，体外诱变和 以下抗体或抗体结合片段的表达 在真核生物或原核生物（E Coli）允许的转染中转染 突变体的工程为“规则”进行直接测试 抗体互补性。 这些研究是补充的 通过使用X射线的三级结构的分析可行 晶体学，核磁共振光谱和计算机 造型。 建议的实验包括：1）结构和结合 分析自发变化区域突变体的分析 通过从次级响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 晶圆厂：同源抗体26-10的地高辛复合物和无结合 突变体是新变量区域工程的基础 结合改变的突变体。 突变体的设计也基于 FV，预测计算机建模和研究的NMR研究结果 手中的自发突变体。 3）通过体外诱变工程师 抗体的结合变体40-50晶体学改进 即将完成。 这些研究平行于26-10的研究 不同的主要结构和绑定特异性，提供独特的 将抗体与同一触觉进行比较的机会。 4）确定 相对链对结合抗体集的贡献共享VL 地区。 5）FV，单链FV和单克隆的Fab片段 表达抗体和突变体，其结合表征 与NMR和晶体学研究的结合。 操纵 抗体基因和蛋白质阐明结构基础 抗原抗体的互补性在更广泛的范围内很重要 是否直接使用抗体作为治疗工具 免疫疗法作为FV或FAB片段，或通过与效应器联系的目标 分子。