Single domain antibodies for diagnosis and treatment of synucleinopathies

摘要 针对淀粉样蛋白-β（Aβ）、tau和α-突触核蛋白（αsyn）聚集体的免疫疗法是目前最常见的免疫疗法。 常见的实验方法，以这种沉积物为特征的疾病。这些潜力中的大多数 治疗方法是全抗体。抗体片段相对于抗体具有某些优点，但其 诊断和治疗潜力尚未得到充分开发。5个抗αsyn IgG抗体和1个αsyn 疫苗处于1-2期临床试验中，一种Aβ抗体最近在3期试验中显示出临床益处， 这有力地支持了这种治疗方法。关于抗体片段，几种抗αsyn单克隆抗体， 链可变区片段（scFvs）在培养和体内是有效的，并且两个抗αsyn单结构域 抗体（sdAb）已经在结合测定中表征，但是它们的治疗功效或诊断功效 成像潜力尚未报道。SdAb与scFv相比具有几个优点，包括更高的免疫原性。 亲和性、与隐蔽表位的结合和更简单的工程化。它们也更适合基因治疗 由于简单的表达和可预测的蛋白质折叠，鉴于其单一结构域， 高溶解度。 我们从噬菌体筛选中获得了58个具有独特结合区的抗αsyn sdAb克隆， 展示用αsyn免疫的美洲驼的B细胞产生的文库。提出了三个目标，以澄清 它们在体内检测和清除αsyn的潜力。目的1将确定sdAb的体内成像潜力 瞄准α合成体目的2阐明靶向α syn的sdAb清除α syn的潜力和作用机制， 目的3探讨以αsyn为靶点的sdAb基因治疗的可行性。 初步研究结果表明，sdAb：1）以高亲和力识别各种形式的αsyn; 2） 在培养模型中清除αsyn并防止其毒性; 3）从αsyn小鼠脑间质液中清除αsyn; 4） 在静脉注射后与αsyn小鼠脑中的αsyn结合，与体内脑信号相关性良好 5）静脉注射sdAb后，用改良的αsyn脑负荷清除脑中的αsyn 通过其PROTAC衍生物清除，以及; 6）在静脉内sdAb基因治疗后从脑中清除αsyn。它 假设sdAb的小尺寸将提供相对于整体的诊断和治疗益处。 抗体，主要是因为更容易进入大脑和靶点，基因治疗的正确折叠， 并且在某种程度上是由于它们与较大抗体不能接近的新表位结合。 总体而言，拟议的研究可能有助于阐明αsyn的发病机制， sdAb的作用，并确定有前途的诊断和治疗sdAb的临床试验， 突触核蛋白病如路易体痴呆、帕金森病、路易体阿尔茨海默病， 和多系统萎缩

Abstract Immunotherapies targeting aggregates of amyloid-β (Aβ), tau and α-synuclein (αsyn) are the most common experimental approach for diseases characterized by such depositions. Most of these potential therapies are whole antibodies. Antibody fragments have certain advantages over antibodies but their diagnostic and therapeutic potential has not been well explored. Five anti-αsyn IgG antibodies and one αsyn vaccine are in Phase 1-2 clinical trials, and one Aβ antibody recently showed clinical benefit in a Phase 3 trial, which strongly supports this therapeutic approach. Regarding antibody fragments, several anti-αsyn single chain variable fragments (scFvs) are effective in culture and in vivo, and two anti-αsyn single domain antibodies (sdAbs) have been characterized in a binding assay but their therapeutic efficacy or diagnostic imaging potential have not been reported. SdAbs have several advantages over scFvs, including higher affinity, binding to cryptic epitopes and simpler engineering. They are also more suitable for gene therapy because of uncomplicated expression and predictable protein folding, given their single domain, resulting in high solubility. We have developed 58 anti-αsyn sdAb clones with unique binding regions from a screen of phage display libraries generated from B-cells of a llama immunized with αsyn. Three aims are proposed to clarify their potential to detect and clear αsyn in vivo. Aim 1 will determine the in vivo imaging potential of sdAbs targeting αsyn. Aim 2 will clarify αsyn clearance potential and mechanism of action of sdAbs targeting αsyn, and Aim 3 will explore the feasibility of sdAb gene therapy targeting αsyn. Preliminary findings indicate that the sdAbs: 1) recognize various forms of αsyn with high affinity; 2) clear αsyn and prevent its toxicity in culture models; 3) clear αsyn from brain interstitial fluid in αsyn mice; 4) bind to αsyn in the brain of αsyn mice after intravenous injection with in vivo brain signal that correlates well with αsyn brain burden; 5) clear αsyn from the brain after intravenous injection of sdAb with improved clearance by its PROTAC derivative, and; 6) clear αsyn from the brain after intravenous sdAb gene therapy. It is hypothesized that the small size of the sdAbs will provide diagnostic and therapeutic benefits over whole antibodies, primarily because of greater access into the brain and to the target, proper folding for gene therapy, and to some extent due to their binding to novel epitopes that the larger antibodies cannot access. Overall, the proposed studies are likely to aid in clarifying αsyn pathogenesis, decipher the mechanism of action of the sdAbs, and identify promising diagnostic and therapeutic sdAbs for clinical trials on synucleinopathies such as Lewy Body Dementia, Parkinson's disease, Alzheimer's disease with Lewy Bodies, and Multiple System Atrophy.