Design and Evolution of Polyvalent Domain Antibodies Specific for Tau Aggregates

Tau 聚集体特异性多价域抗体的设计和进化

• 批准号: 10585480
• 负责人: Ravi S. Kane
• 金额: $ 42.19万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10585480
• 关键词: Address; Affinity; Alzheimer' s Disease; Amyloid Fibrils; Animal Model; Antibodies; Antigen Presentation; Binding; Binding Sites; Bispecific Antibodies; Blood - brain barrier anatomy; Brain; Directed Molecular Evolution; Disease; Drug Kinetics; Evaluation; Evolution; Fibrinogen; Goals; Immunization; In Vitro; Link; Mediating; Methods; Molecular Conformation; Mus; Neurodegenerative Disorders; Outcome; Pathologic; Pathology; Peptides; Polyvalence; Proteins; Role; Specificity; Tauopathies; Testing; Transgenic Organisms; Work; antibody libraries; base; design; experience; in vivo; mouse model; prevent; protein aggregation; protein misfolding; self assembly; tau Proteins; tau aggregation

Protein misfolding and aberrant self-assembly into toxic species ranging from small oligomers to large amyloid fibrils are pathologically linked to neurodegenerative disorders such as Alzheimer’s disease. Conformational antibodies with specificity for protein aggregates are important for investigating the role of different types of aggregates in neurodegenerative diseases as well as for potentially treating these debilitating diseases. It has, however, been extremely difficult to generate conformational antibodies against protein aggregates due to several factors: i) the limitations of immunization (lack of control over antigen presentation); ii) the fixed number of binding sites per antibody, which limits the use of polyvalency for targeting multimeric protein aggregates; and iii) the difficulty in using naïve antibody libraries to obtain conformational antibodies via in vitro selection methods. To address these challenges, we have recently developed a systematic approach for generating domain antibodies with specificity for protein aggregates. The goal of this proposal is to use this approach to generate conformational domain antibodies specific for tau oligomers and fibrils, and to use these antibodies to evaluate the relative importance of different types of tau aggregates in mediating tau pathology in animal models. Our proposed approach builds on our collective experience in: i) designing domain antibodies with specificity for protein aggregates based on homotypic interactions between identical peptide motifs; ii) enhancing the affinity and specificity of domain antibodies using directed evolution; iii) designing polyvalent molecules that bind to oligomeric proteins with high affinity and specificity; iv) assembling and isolating tau oligomers and fibrils; and v) evaluating the ability of antibodies to prevent and reverse pathology in tau animal models. In Aim 1, we will test our hypothesis that domain antibodies with enhanced conformational specificity and affinity for tau oligomers and fibrils can be readily selected from antibody libraries with tau amyloidogenic peptides grafted into the main binding loop (CDR3). Next, in Aim 2A, we will evaluate our hypothesis that polyvalency can be used to increase the conformational specificity and affinity of tau domain antibodies by generating polyvalent versions in a manner that affords control over the number and spacing of domain antibodies. In Aim 2B, we will generate bispecific domain antibodies that combine tau and blood-brain barrier (BBB) targeting domain antibodies, and evaluate their pharmacokinetics and target engagement in tau transgenic (PS19) mice. Finally, in Aim 3, we will test the ability of the most specific and inhibitory tau/BBB bispecific antibodies generated in Aim 2, which are best at engaging tau in the mouse brain, to inhibit tau seeding, spontaneous aggregation and pathology in vivo using tau mouse models. Significant outcomes of our studies will be systematic methods for generating bispecific antibodies that recognize different types of protein aggregates and which efficiently enter the brain, and evaluation of the relative importance of tau oligomers and fibrils in mediating pathology.

蛋白质错误折叠和异常自组装成有毒物质，从小寡聚体到大淀粉样蛋白 原纤维在病理上与阿尔茨海默病等神经退行性疾病有关。 对蛋白质聚集体具有特异性的抗体对于研究不同类型的蛋白质的作用非常重要 聚集在神经退行性疾病中，并有可能治疗这些使人衰弱的疾病。 然而，由于蛋白质聚集体的构象抗体的产生极其困难 几个因素：i) 免疫接种的局限性（缺乏对抗原呈递的控制）；ii) 固定的 每个抗体的结合位点数量，这限制了多价靶向多聚蛋白的使用 聚集体；以及 iii) 使用天然抗体库通过 in 获得构象抗体的困难 为了应对这些挑战，我们最近开发了一种系统方法。 该提案的目标是利用这一点生成对蛋白质聚集体具有特异性的域抗体。 生成特异于 tau 寡聚体和原纤维的构象域抗体的方法，并使用这些抗体 评估不同类型 tau 聚集体在介导 tau 病理学中的相对重要性的抗体 我们提出的方法建立在我们以下方面的集体经验之上：i) 设计领域。 基于相同肽之间的同型相互作用，对蛋白质聚集体具有特异性的抗体 基序；ii) 使用定向进化增强域抗体的亲和力和特异性； iv) 以高亲和力和特异性结合寡聚蛋白的多价分子； 分离 tau 寡聚体和原纤维；和 v) 评估抗体预防和逆转病理的能力 在 tau 动物模型中，我们将测试我们的假设，即域抗体具有增强功能。 可以很容易地从抗体中选择对 tau 寡聚体和原纤维的构象特异性和亲和力 接下来，在目标 2A 中，我们将构建具有 tau 淀粉样蛋白生成肽的文库。 评估我们的假设，即多价可用于增加构象特异性和亲和力 通过以控制数量的方式生成多价版本来构建 tau 结构域抗体 在 Aim 2B 中，我们将生成结合 tau 的双特异性域抗体。 和血脑屏障（BBB）靶向域抗体，并评估其药代动力学和靶点 最后，在目标 3 中，我们将测试最具体和最有效的能力。 Aim 2 中生成的抑制性 tau/BBB 双特异性抗体，最能在小鼠体内接合 tau 使用 tau 小鼠模型抑制体内 tau 播种、自发聚集和病理学。 我们研究的重要成果将是产生双特异性抗体的系统方法 识别不同类型的蛋白质聚集体并有效进入大脑，并评估 tau 寡聚体和原纤维在介导病理学中的相对重要性。