EARLY DETECTION OF TAU ACETYLATION USING ULTRA-HIGH AFFINITY ANTIBODIES

使用超高亲和力抗体早期检测 TAU 乙酰化

• 批准号: 9227696
• 负责人: Yongku Peter Cho
• 金额: $ 7.47万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9227696
• 关键词: Acetylation; Address; Affinity; Alzheimer' s Disease; Antibodies; Antibody Affinity; Antibody Dissociations; Antibody Specificity; Antigens; Binding; Binding Sites; Biological; Brain; Cells; Chemicals; Chickens; Color; Communities; Deposition; Detection; Development; Directed Molecular Evolution; Disease Progression; Early Diagnosis; Engineering; Enhancing Antibodies; Ensure; Future; Generations; Genes; Homeostasis; Human; Hybridomas; Immune Sera; Immunoglobulin G; In Vitro; Investigation; Iowa; Label; Measurement; Measures; Mediating; Methods; Modification; Monitor; Monoclonal Antibodies; Nerve Degeneration; Neurodegenerative Disorders; Outcome; Passive Immunotherapy; Pathology; Patients; Post-Translational Protein Processing; Property; Reagent; Recombinant Antibody; Reproducibility; Resolution; Route; Scheme; Sensitivity and Specificity; Site; Specificity; Staging; Tauopathies; Testing; Tissue Sample; Transgenic Mice; Universities; Validation; antibody engineering; antibody libraries; base; clinically relevant; cross reactivity; high throughput screening; improved; insight; mouse model; mutant; neurotoxicity; novel; novel therapeutics; polyclonal antibody; prevent; process optimization; protein aminoacid sequence; research study; screening; tau Proteins; tau phosphorylation; tau-1; therapeutic development; tool

Project Summary Acetylation of tau is a recently identified mechanism critical in the initial stages of pathology mediated by tau. Initial results show that tau acetylation occurs early on in disease progression, reduces tau turnover, and promotes aggregation of phosphorylated tau. Therefore, early detection of tau acetylation and monitoring its synergy with tau phosphorylation is a promising route to identifying the mechanism underlying tauopathies. However, existing methods to detect this critical modification have relied on polyclonal antibodies, which have poorly characterized binding properties. In this proposal, we hypothesize that enhancing the sensitivity of detecting acetylated tau would dramatically improve our ability to study the initial stages of tau mediated pathology. However, yielding high affinity and specificity antibodies against specific protein modification sites is challenging. This requires enhancing antibody affinity while preventing unwanted cross reactivity towards similar binding sites. Even though numerous methods have been developed to screen antibody sequences to quantify and engineer affinity, methods to optimize antibody specificity based on measurement of cross reactivity are virtually non-existent. To address this problem, a novel antibody screening approach based on quantitative measure of antibody specificity will be developed. The proposed high-throughput screening approach will enable simultaneous optimization of antibody specificity and affinity. Using this approach, ultra- high affinity monoclonal antibodies against key acetylation sites specific to AD patients will be generated. The specific aims during this project period are 1) Identifying acetylated tau binding antibodies by screening recombinant antibody libraries, 2) optimizing specificity and affinity of the antibodies through directed evolution, and 3) validation of the antibodies using tissue samples from mouse models and Alzheimer's disease patients. The outcomes of this project will be high quality monoclonal antibodies targeting acetylated tau. If successful, the project will enable early detection of acetylated tau, which has enormous impact on basic biological investigations and novel therapeutic development in tau mediated neurodegeneration.

项目概要 tau 的乙酰化是最近发现的一种机制，在 tau 介导的病理学初始阶段至关重要。 初步结果表明，tau 乙酰化发生在疾病进展的早期，减少了 tau 更新，并且 促进磷酸化 tau 蛋白的聚集。因此，早期检测 tau 乙酰化并监测其 与 tau 磷酸化的协同作用是识别 tau 病潜在机制的一条有前途的途径。 然而，检测这种关键修饰的现有方法依赖于多克隆抗体，该抗体已 结合特性的表征不佳。在这个提案中，我们假设增强 检测乙酰化 tau 将极大地提高我们研究 tau 介导的初始阶段的能力 病理。然而，产生针对特定蛋白质修饰位点的高亲和力和特异性抗体是 具有挑战性的。这需要增强抗体亲和力，同时防止不必要的交叉反应 相似的结合位点。尽管已经开发了多种方法来筛选抗体序列 量化和设计亲和力，基于交叉测量优化抗体特异性的方法 反应活性几乎不存在。为了解决这个问题，一种基于 将开发抗体特异性的定量测量方法。拟议的高通量筛选 该方法将能够同时优化抗体特异性和亲和力。使用这种方法，超 将产生针对 AD 患者特有的关键乙酰化位点的高亲和力单克隆抗体。这 该项目期间的具体目标是 1) 通过筛选鉴定乙酰化 tau 结合抗体 重组抗体文库，2)通过定向进化优化抗体的特异性和亲和力， 3) 使用小鼠模型和阿尔茨海默病患者的组织样本验证抗体。 该项目的成果将是针对乙酰化 tau 的高质量单克隆抗体。如果成功的话， 该项目将实现乙酰化 tau 的早期检测，这对基础生物学产生巨大影响 tau 介导的神经变性的研究和新疗法的开发。