Clearance and In Vivo Detection of Tau Pathology.

摘要： Tau和淀粉样蛋白-β免疫疗法是制药行业最常见的方法 来治疗阿尔茨海默病，这种疾病的特征是这样的证词。大多数有希望的 治疗是全单克隆抗体（mAb）。对抗体片段的关注要少得多， 如单链可变区片段（scFv）和单结构域抗体（sdAb），其具有某些 这些优势证明了探索其治疗和诊断潜力的合理性。在这两个目标中，tau可能更重要。 因为它的病理学与痴呆的相关性比Aβ病变更好。该提案的目的是： 1)为了阐明体内双光子成像方法的动力学、机制和功能 tau抗体疗法的后果，包括其片段，和; 2）为了确定tau抗体疗法的诊断结果， 最小的生物结合剂sdAb对tau蛋白的成像潜力。 根据我们的初步数据，假设在体双光子成像是最敏感的 和全面的体内方法，在很短的时间内，以澄清动力学和治疗潜力， tau mAb/scFv/sdAb。此外，这种技术应该提供一个机械的洞察力， 治疗起作用，有益于神经功能。同样，假设sdAb的小尺寸将 与较大的单克隆抗体和单链抗体相比， 以及tau靶点，并且在某种程度上是由于它们与新表位的结合。 我们已经应用并进一步开发了各种双光子成像方法，以澄清 tau抗体疗法的机制。具体地，共注射脑可渗透的tau β-折叠染料，FSB， 与荧光标记的tau mAb/scFv/sdAb，允许检查它们与 tau聚集体和随后的tau损伤的清除。此外，病毒神经元表达的钙 和谷氨酸指示剂提供了与tau病理学相关的功能缺陷的窗口， 研究tau mAb/scFv/sdAb减弱和/或逆转这些信号传导异常的能力。 对于这些研究，我们有45个tau mAb的克隆、许多scFv和50个sdAb的克隆， 识别各种形式的tau蛋白的独特结合区域。对于治疗和机械 研究，我们将研究其中的一些，根据我们公布的和初步的数据已经显示出潜力 以清除病理性tau蛋白并防止其毒性。对于诊断研究，将选择最多20个sdAb 基于使用体内成像系统（IVIS）的用于体内成像研究的各种体外结合测定。 随后，最有希望的将被进一步检查作为潜在的诊断正电子发射 在一个实施方案中，通过在microPET中对tau蛋白病小鼠进行成像，来对tau蛋白断层扫描（PET）配体进行成像。 这一建议与其他蛋白质聚集疾病密切相关，应阐明 研究人员发现了tau免疫疗法的机制，并且可以为临床试验提供治疗和诊断候选物。

Abstract: Tau and amyloid-β immunotherapies are the most common approaches by the pharmaceutical industry to tackle Alzheimer's disease, which is characterized by such depositions. The majority of these promising therapies are whole monoclonal antibodies (mAbs). Much less attention has been paid to antibody fragments, like single-chain variable fragments (scFvs) and single domain antibodies (sdAbs), which have certain advantages that justify exploring their therapeutic and diagnostic potential. Of the two targets, tau may be more promising because its pathology correlates better with dementia than Aβ lesions. The aims of the proposal are: 1) To clarify with in vivo two-photon imaging approaches the kinetics, mechanisms, and functional consequences of tau antibody therapies, including their fragments, and; 2) To determine the diagnostic imaging potential of the smallest biological binders, sdAbs, to the tau protein. Based on our preliminary data, it is hypothesized that in vivo two-photon imaging is the most sensitive and comprehensive in vivo approach, within a short timeframe, to clarify kinetics and therapeutic potential of tau mAbs/scFvs/sdAbs. Furthermore, this technique should provide a mechanistic insight into how the therapies work, and benefit neuronal function. Likewise, it is hypothesized that the small size of the sdAbs will provide diagnostic benefits over the larger mAbs and scFvs, primarily because of greater access into the brain and to the tau target, and to some extent due to their binding to novel epitopes. We have applied and further developed various two-photon imaging approaches to clarify the mechanisms of tau antibody therapies. Specifically, co-injection of a brain permeable tau β-sheet dye, FSB, with a fluorescently labeled tau mAb/scFv/sdAb, allows for examining the kinetics of their colocalization with tau aggregates and subsequent clearance of tau lesions. Furthermore, viral neuronal expression of calcium and glutamate indicators provides a window into functional deficits associated with tau pathology, and allow study of the ability of tau mAbs/scFvs/sdAbs to attenuate and/or reverse these signaling abnormalities. For these studies, we have 45 clones of tau mAbs, numerous scFvs, and 50 clones of sdAbs with unique binding regions that recognize various forms of the tau protein. For the therapeutic and mechanistic studies, we will examine a few of these that based on our published and preliminary data have shown potential to clear pathological tau and prevent its toxicity. For the diagnostic studies, up to 20 sdAbs will be selected based on various in vitro binding assays for in vivo imaging studies using the In Vivo Imaging System (IVIS). Subsequently, the most promising ones will be examined further as potential diagnostic positron emission tomography (PET) ligands by imaging tauopathy mice in a microPET. This proposal, with strong relevance to other protein aggregation diseases, should clarify the mechanisms of tau immunotherapies and may provide therapeutic and diagnostic candidates for clinical trials.