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

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。有趣的是，淀粉样蛋白 不是形成具有单个3D结构的聚集体，而是具有多个相关但不同的3D结构， 与独特的生物活性有关(类似于普恩病毒株)。这些结构独特的集合体是如何 调节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。 菲尔德。针对淀粉样变性聚集体的构象抗体对于研究这一点是非常有价值的。 耐人寻味的问题，更广泛地说，因为它们可能被用作特定疾病的诊断和治疗 探员们。这种抗体的用途源于它们能够灵敏地检测特定类型的蛋白质。 并选择性地干扰它们的生物活性。然而，这些类型的抗体是 很难产生，因为许多抗原(免疫优势表位)和 特定于蛋白质聚集体(多价，高度疏水，在某些情况下，动力学不稳定)。这个 这项提议的目标是开发结构导向的方法来体外产生抗体。 在淀粉样变性聚集体中预测的六类构象表位，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。被预测的 表位-包括寡聚体和纤维中通用抗体表位的主要候选者-有 之前没有使用单抗进行过特定的靶向或验证。我们将主要针对- 突触核蛋白(与帕金森氏病有关)，形成多种类型的寡聚体和纤维。这些 研究将解决四个关键的基本问题(第一季度、第二季度)和方法问题(第三季度、第四季度)。1)哪种通用 预测的构象抗体表位存在于-突触核蛋白等的寡聚体和纤维中 淀粉样蛋白？2)预测的构象表位存在于哪些蛋白质特定类别 -突触核蛋白聚集体的不同结构(株)变体？3)什么是最佳设计参数 生成体外抗体库，最大限度地分离具有高特异性的抗体 除了高亲和力之外？4)体外靶向抗体库的最有效方法是什么？ 蛋白质聚集体中的选定表位，以克服与以下方面相关的常见问题 免疫优势表位和较差的抗原质量？这些建议的研究基于多个 特西尔实验室的发现：I)用于分离展示的酵母的新的体外文库设计和分类方法 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；以及iii) A-纤维中多类构象表位的鉴定这些发现为我们提供了一个强有力的 合理地产生针对不同类型的淀粉样变性聚集体的抗体并提供 对与独特的人类疾病有关的蛋白质聚集体之间的构象差异的关键见解。

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 - 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 -synuclein and other amyloidogenic proteins? 2) Which protein-specific classes of predicted conformational epitopes exist in different structural (strain) variants of -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 A 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.