Structure-guided antibody targeting of pre-selected epitopes in amyloidogenic aggregates

结构引导抗体靶向淀粉样蛋白聚集体中预先选择的表位

• 批准号: 10372055
• 负责人: Peter M Tessier
• 金额: $ 26.97万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10372055
• 关键词: Address; Affinity; Antibodies; Antibody Specificity; Antigens; Bioinformatics; Biological; Biological Phenomena; Diagnostic; Disease; Epitopes; Event; Generations; Goals; Human; Hydrophobicity; Immunodominant Epitopes; In Vitro; Kinetics; Libraries; Link; Mediating; Methodology; Methods; Microbial Biofilms; Molecular Conformation; Monoclonal Antibodies; Neurodegenerative Disorders; Parkinson Disease; Prions; Proteins; Research; Sorting - Cell Movement; Specificity; Structure; Therapeutic Agents; Variant; Work; Yeasts; alpha synuclein; antibody libraries; base; design; diagnosis evaluation; improved; in vivo; insight; novel; particle; protein aggregation; protein complex; stem; three dimensional structure

Amyloidogenic protein aggregation is a key event in seemingly disparate biological phenomena, including those ranging from bacterial biofilms to neurodegenerative diseases. Intriguingly, amyloidogenic proteins do not form aggregates with a single 3D structure, but rather with several related yet distinct 3D structures that are linked to unique biological activities (akin to prion strains). How these structurally-unique aggregates mediate different toxic or beneficial activities is one of the most important questions in the protein aggregation field. Conformational antibodies specific for amyloidogenic aggregates are invaluable for investigating this intriguing problem and, more generally, for their potential use as disease-specific diagnostic and therapeutic agents. The utility of such antibodies stems from their ability to sensitively detect specific types of protein aggregates and selectively interfere with their biological activities. However, these types of antibodies are difficult to generate because of issues common to many antigens (immunodominant epitopes) and those specific to protein aggregates (multivalent, highly hydrophobic and, in some cases, kinetically unstable). The goals of this proposal are to develop structure-guided approaches for in vitro generation of antibodies against six classes of predicted conformational epitopes in amyloidogenic aggregates, and to use these antibodies to evaluate conformational differences between disease-associated aggregates formed in vivo. The predicted epitopes – which include the leading candidates for generic antibody epitopes in oligomers and fibrils – have not been specifically targeted or verified previously using monoclonal antibodies. We will primarily target a- synuclein (associated with Parkinson’s disease), which forms multiple types of oligomers and fibrils. These studies will address four key fundamental (Q1, Q2) and methodological (Q3, Q4) questions. 1) Which generic classes of predicted conformational antibody epitopes exist in oligomers and fibrils of a-synuclein and other amyloidogenic proteins? 2) Which protein-specific classes of predicted conformational epitopes exist in different structural (strain) variants of a-synuclein aggregates? 3) What are optimal design parameters for generating in vitro antibody libraries that maximize the likelihood of isolating antibodies with high specificity in addition to high affinity? 4) What are the most effective in vitro methods for targeting antibody libraries to pre- selected epitopes in protein aggregates in order to overcome common problems associated with immunodominant epitopes and poor antigen quality? These proposed studies are based on multiple discoveries in the Tessier lab: i) novel in vitro library design and sorting methods for isolating yeast-displayed antibodies with extremely high specificity; ii) bioinformatics methods for predicting antibody specificity; and iii) identification of multiple classes of conformational epitopes in Ab fibrils. These discoveries provide a strong basis for rationally generating antibodies against diverse types of amyloidogenic aggregates and for providing key insights into conformational differences between protein aggregates linked to unique human disorders.

淀粉样蛋白蛋白聚集是看似不同的生物学现象的关键事件，包括 从细菌生物膜到神经退行性疾病的人。有趣的是，淀粉样蛋白DO 不是具有单个3D结构的聚集体，而是具有几种相关但不同的3D结构 与独特的生物学活动有关（类似于主要菌株）。这些结构唯一的聚合如何 调解不同的有毒或有益活动是蛋白质聚集中最重要的问题之一 field。针对淀粉样蛋白生成骨特异性抗体对于研究这一点很有吸引力 有趣的问题，更普遍地是因为它们可能用作特定疾病的诊断和治疗 代理商。此类抗体植物的效用是从敏感地检测特定类型蛋白质的能力 聚集并有选择地干扰其生物学活动。但是，这些类型的抗体是 由于许多抗原常见的问题（免疫主导表现），很难产生 特定于蛋白质聚集体（多价，高度疏水，在某些情况下，在动力学上不稳定）。 该建议的目标是开发用于体外产生抗体的结构引导方法 淀粉样蛋白酶骨料中的六类预测会议表位，并使用这些抗体 评估体内形成的疾病相关骨料之间的会议差异。预测 表位 - 包括低聚物和原纤维中通用抗体表位的主要候选者 - 没有使用单克隆抗体对先前的靶向或验证。我们将主要目标a- 综合蛋白（与帕金森氏病有关），形成多种类型的低聚物和原纤维。这些 研究将解决四个关键基本（Q1，Q2）和方法论学（Q3，Q4）问题。 1）哪个通用 A-突触核蛋白和其他的原纤维中存在预测会议抗体表位的类别 淀粉样蛋白？ 2）在哪些蛋白质特异性类别的预测会议表位中存在 A核蛋白聚集体的不同结构（应变）变体？ 3）什么是最佳设计参数 生成体外抗体文库，以最大程度地隔离具有高特异性的抗体的可能性 除了高亲和力吗？ 4）靶向抗体文库以预先靶向抗体的最有效的体外方法是什么 蛋白质聚集体中选定的表位，以克服与之相关的常见问题 免疫主流表位和抗原质量差？这些提出的研究是基于多重的 Tessier实验室中的发现：i）新颖的体外图书馆设计和分类方法用于隔离酵母菌播放 具有极高特异性的抗体； ii）用于预测抗体特异性的生物信息学方法；和iii） 鉴定AB原纤维中多种构象表位。这些发现提供了强大的 基于对潜水员类型的淀粉样蛋白生成骨料类型的抗体的基础，并提供 对与独特人类疾病相关的蛋白质聚集体之间构象差异的关键见解。