Design and Evolution of Polyvalent Domain Antibodies Specific for Tau Aggregates

蛋白质错误折叠和异常自组装成毒性物质，从小的寡聚体到大的淀粉样蛋白 纤维在病理学上与神经退行性疾病如阿尔茨海默病有关。构象 对蛋白质聚集体具有特异性的抗体对于研究不同类型蛋白质聚集体的作用非常重要。 聚集体在神经变性疾病中的应用以及潜在地治疗这些使人衰弱的疾病。它 然而，由于蛋白质聚集体的存在，产生针对蛋白质聚集体的构象抗体极其困难， 几个因素：i）免疫的局限性（缺乏对抗原呈递的控制）; ii）固定的 每个抗体的结合位点数，这限制了多价用于靶向多聚体蛋白的使用 聚集体;和iii）使用幼稚抗体文库通过免疫抑制获得构象抗体的困难。 离体筛选方法为了应对这些挑战，我们最近制定了一个系统的方法， 产生对蛋白质聚集体具有特异性的结构域抗体。该提案的目的是利用这一点 产生对tau寡聚体和原纤维具有特异性的构象结构域抗体的方法，以及使用这些方法 评价不同类型的tau聚集体在介导tau病理中的相对重要性的抗体 在动物模型中。我们提出的方法建立在我们的集体经验，在：i）设计域 基于相同肽间同型相互作用的对蛋白质聚集体具有特异性的抗体 基序; ii）使用定向进化增强结构域抗体的亲和力和特异性; iii）设计 以高亲和力和特异性结合寡聚蛋白的多价分子; iv）组装和 分离tau寡聚体和原纤维;和v）评价抗体预防和逆转病理学的能力 在tau动物模型中。在目标1中，我们将测试我们的假设，即具有增强的 对tau寡聚体和原纤维的构象特异性和亲和力可以容易地从抗体 将tau淀粉样蛋白生成肽移植到主要结合环（CDR 3）中的文库。在目标2A中，我们将 评估我们的假设，即多价可以用来增加构象特异性和亲和力 通过以提供对tau结构域抗体的数量的控制的方式产生多价形式， 和结构域抗体的间隔。在Aim 2B中，我们将产生双特异性结构域抗体，其联合收割机tau 和血脑屏障（BBB）靶向结构域抗体，并评估其药代动力学和靶向 在tau转基因（PS19）小鼠中的接合。最后，在目标3中，我们将测试最具体和 在Aim 2中产生的抑制性tau/BBB双特异性抗体，其在小鼠中最好地接合tau 脑，以使用tau小鼠模型在体内抑制tau播种、自发聚集和病理学。 我们研究的重要结果将是用于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，并有效地进入大脑， tau寡聚体和原纤维在介导病理学中的相对重要性。

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.