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: 10599101
• 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/10599101
• 关键词: Affinity; Antibodies; Antibody Specificity; Antigens; Bioinformatics; Biological; Biological Phenomena; Diagnosis; Diagnostic; Disease; Disparate; Epitopes; Evaluation; 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; Specificity; Structure; Therapeutic Agents; Variant; Work; Yeasts; alpha synuclein; antibody libraries; design; 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.

淀粉样蛋白聚集是看似不同的生物现象中的一个关键事件，包括 从细菌生物膜到神经退行性疾病。 不是用单个 3D 结构形成聚合体，而是用几个相关但不同的 3D 结构形成聚合体 这些结构独特的聚集体如何与独特的生物活性相关（类似于朊病毒株）。 介导不同的毒性或有益活性是蛋白质聚集中最重要的问题之一 淀粉样蛋白形成聚集物特异性的构象抗体对于研究这一领域非常有价值。 有趣的问题，更普遍的是，它们作为疾病特异性诊断和治疗的潜在用途 此类抗体的实用性源于它们能够灵敏地检测特定类型的蛋白质。 然而，这些类型的抗体会聚集并选择性地干扰其生物活性。 由于许多抗原（免疫显性表位）和那些抗原所共有的问题，很难生成 特定于蛋白质聚集体（多价、高度疏水性，在某些情况下，动力学不稳定）。 该提案的目标是开发结构引导的方法来体外生成针对 淀粉样蛋白形成聚集物中六类预测的构象表位，并使用这些抗体 评估体内形成的疾病相关聚集体之间的构象差异。 表位（包括寡聚物和原纤维中通用抗体表位的主要候选者） 之前没有使用单克隆抗体进行专门针对或验证，我们将主要针对 a-。 突触核蛋白（与帕金森病相关），形成多种类型的寡聚体和原纤维。 研究将解决四个关键的基本问题（Q1、Q2）和方法论问题（Q3、Q4）： 1) 哪些是通用的。 预测的构象抗体表位类别存在于α-突触核蛋白和其他物质的寡聚体和原纤维中 2) 哪些蛋白质特异性的预测构象表位存在于 α-突触核蛋白聚集体的不同结构（应变）变体 3) 最佳设计参数是什么？ 生成体外抗体库，最大限度地提高分离高特异性抗体的可能性 除了高亲和力之外，还有哪些最有效的体外方法来靶向抗体库？ 选择蛋白质聚集体中的表位以克服与 免疫显性表位和抗原质量差？这些拟议的研究是基于多个 Tessier 实验室的发现：i) 用于分离酵母展示的新型体外文库设计和分选方法 具有极高特异性的抗体；ii) 预测抗体特异性的生物信息学方法； 抗体原纤维中多种构象表位的鉴定提供了强有力的证据。 合理产生针对不同类型淀粉样蛋白聚集物的抗体并提供的基础 对与独特的人类疾病相关的蛋白质聚集体之间的构象差异的关键见解。