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从小低聚物到大淀粉样蛋白的有毒物种 从病理上讲，纤维与阿尔茨海默病等神经退行性疾病有关。构象 具有蛋白质聚集性的抗体对于研究不同类型的蛋白质聚集的作用是重要的 神经退行性疾病中的聚集体，以及潜在地治疗这些衰弱的疾病。它 然而，产生针对蛋白质聚集体的构象抗体是极其困难的，因为 有几个因素：一)免疫的局限性(缺乏对抗原提呈的控制)；二)固定的 每个抗体的结合位点数，这限制了多价体用于靶向多聚体蛋白的使用 凝聚体；以及iii)使用朴素抗体库通过在体内获得构象抗体的困难 体外筛选方法。为了应对这些挑战，我们最近开发了一种系统的方法来 产生对蛋白质聚集体具有特异性的区域抗体。这项提议的目标是利用这一点 产生针对tau寡聚体和纤维的构象结构域抗体的方法，并使用这些抗体 用抗体评价不同类型的tau聚集体在介导tau病理中的相对重要性 在动物模型中。我们建议的方法建立在我们在以下方面的集体经验之上：i)设计领域 基于相同多肽之间同型相互作用的具有蛋白质聚集体特异性的抗体 基序；ii)利用定向进化提高结构域抗体的亲和力和特异性；iii)设计 与低聚蛋白质高亲和力和高特异性结合的多价分子；iv)组装和 分离tau寡聚体和纤维；以及v)评估抗体预防和逆转病理的能力 在tau动物模型中。在目标1中，我们将测试我们的假设，即具有增强的结构域抗体 可以很容易地从抗体中选择tau寡聚体和纤维的构象特异性和亲和力 将含有tau淀粉样多肽的文库嫁接到主结合环(CDR3)中。接下来，在Aim 2a中，我们将 评估我们的假设，即多价态可以用来增加构象特异性和亲和力 通过以一种能够控制数量的方式生成多价版本来获得tau结构域抗体 和区域抗体的间距。在目标2B中，我们将产生结合tau的双特异性结构域抗体 和血脑屏障(BBB)靶向结构域抗体，并评价其药代动力学和靶向性 Tau转基因(PS19)小鼠的参与。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，最适合在小鼠体内结合tau 脑内，抑制tau的种植、自发聚集和体内病理利用tau小鼠模型。 我们研究的重要成果将是产生双特异性抗体的系统化方法， 识别不同类型的蛋白质聚集体，并有效地进入大脑，并评估 Tau寡聚体和纤维在介导病理过程中的相对重要性。

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.