Clearance and In Vivo Detection of Tau Pathology.

摘要： Tau和淀粉样蛋白-β免疫疗法是制药业最常用的方法 来对付阿尔茨海默氏症，这种疾病的特点是这样的证词。其中大部分都是有希望的 治疗方法是全单抗(MAbbs)。对抗体片段的关注要少得多， 如单链可变区(ScFv)和单域抗体(SdAbs)，它们有一定的 值得探索其治疗和诊断潜力的优势。在这两个目标中，tau可能更多 很有希望，因为它的病理与痴呆症的相关性比Aβ病变更好。这项建议的目的是： 1)用活体双光子成像方法阐明其动力学、机制和功能。 Tau抗体疗法的后果，包括它们的片段，以及；2)确定诊断 最小的生物结合剂sdAbs对tau蛋白的成像潜力。 根据我们的初步数据，假设体内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单抗/单链抗体/sdAbs。此外，这项技术应该提供一种机械性的洞察，以了解 治疗有效，并有益于神经元功能。同样，假设sdAbs的小尺寸将 提供比更大的单抗和单链抗体更好的诊断优势，主要是因为更容易进入大脑 以及tau靶标，在某种程度上是由于它们与新的表位结合。 我们已经应用并进一步开发了各种双光子成像方法来澄清 Tau抗体治疗的机制。具体地说，共注射脑透性tauβ片状染料FSB， 用荧光标记的tau mAb/scFv/sdAb，可以检测它们与 Tau聚集体和随后tau损伤的清除。此外，病毒神经元钙离子的表达 谷氨酸指示物提供了了解与tau病理相关的功能缺陷的窗口，并允许 研究tau单抗/单链抗体/sdAbs减弱和/或逆转这些信号异常的能力。 在这些研究中，我们有45个tau单抗的克隆，大量的scFv，以及50个sdAbbs的克隆 识别各种形式的tau蛋白的独特结合区。对于治疗性和机械性 ，我们将根据已发表的和初步的数据来检验其中一些具有潜力的研究 清除病理性tau，预防其毒性。对于诊断性研究，将选择多达20个sdAbbs 基于使用活体成像系统(IVIS)进行体内成像研究的各种体外结合分析。 随后，最有希望的将作为潜在的诊断正电子发射进行进一步检查。 断层扫描(PET)通过在微型PET中对直立性病变小鼠进行成像。 这项建议，与其他蛋白质聚集性疾病密切相关，应该澄清 研究tau免疫疗法的机制，并可能为临床试验提供治疗和诊断候选方案。

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.