Efficacy and Mechanism of Action of Heavy-Chain α-Synuclein Antibody Fragments Derived from Llama

美洲驼重链α-突触核蛋白抗体片段的功效和作用机制

• 批准号: 9762785
• 负责人: Einar M Sigurdsson
• 金额: $ 21.19万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762785
• 关键词: Affinity; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Animal Model; Antibodies; Antibody Therapy; Astrocytes; Attention; Attenuated; B-Lymphocytes; Binding; Biological Assay; Blood - brain barrier anatomy; Brain; Cell Culture Techniques; Cell membrane; Clinical Trials; Data; Deposition; Diagnostic; Disease; Drosophila genus; Drug Industry; Endocytosis; Engineering; Epitopes; Half-Life; Human; Image; Immunize; Immunoglobulin Fragments; Immunoglobulin G; Immunotherapy; In Vitro; Individual; Intravenous; Laboratories; Length; Lesion; Lewy Bodies; Lewy Body Dementia; Libraries; Llama; Longevity; Mediating; Modeling; Monitor; Multiple System Atrophy; Mus; Mutation; Neurons; Parkinson Disease; Pathogenesis; Pathologic; Pathology; Penetration; Peripheral; Phage Display; Phase II Clinical Trials; Prions; Proteins; Public Health; Publishing; Reporting; Research; Signal Transduction; Tauopathies; Therapeutic; Time; Tissues; Treatment Efficacy; alpha synuclein; base; clinical development; cost; developmental toxicity; fly; improved; in vivo; insight; interest; intravenous injection; mouse model; nanobodies; novel; overexpression; prevent; protein aggregate; protein aggregation; protein misfolding; receptor; screening; synucleinopathy; tau Proteins; tau aggregation; uptake

Immunotherapies targeting various protein aggregates such as amyloid-β (Aβ), tau and α-synuclein (αsyn) are in different stages of clinical development, and collectively are the most common approach by the pharmaceutical industry to tackle diseases characterized by such depositions [1-3]. The majority of these potential therapies are whole antibodies. Much less attention has been paid to antibody fragments, which have certain advantages and their therapeutic and diagnostic potential should be explored further. Specifically, single domain antibodies (sdAbs) are of particular interest because their small size (13 kDa) improves tissue penetration, including through the blood-brain-barrier, allowing better access over antibodies (150 kDa) to the target molecule and its hidden epitopes. Importantly as well, they are high affinity, and easy to engineer and grow in large quantities. The Specific Aim is to determine the therapeutic potential of camelid single-domain heavy chain antibody fragments (sdAbs) against the αsyn protein. It is hypothesized that their small size will provide therapeutic benefits over whole antibodies, primarily because of greater access to the target, and to some extent due to their binding to novel epitopes that the larger antibodies cannot access. We have generated 51 clones of sdAbs, with unique complementary determining regions (CDRs), that recognize various forms of αsyn and propose to determine their therapeutic potential in different models, as well as to clarify their mechanism of action. For the initial screen, up to 30 of the individual sdAbs will be expressed globally in neurons of fruit fly models of synucleinopathy, and their ability to prevent/attenuate the αsyn pathology monitored. Subsequently, the 5 most efficacious sdAbs will be examined further in flies by expressing them in astrocytes or peripherally, and in cell culture models of synucleinopathy for mechanistic insight, and the most effective one from that study in an animal model. Our preliminary data support the feasibility of screening for therapeutic efficacy of antibody fragments targeting the tau protein in fly models. Specifically, we show that neuronal expression of an anti-tau single chain variable antibody fragment (scFv) prevents developmental toxicity of overexpression of human tau with or without a tauopathy mutation, and significantly extends the life span of the tauopathy flies. We expect that expressing sdAbs against αsyn in synucleinopathy flies will also provide therapeutic benefits. The proposed studies may identify a novel class of therapy candidates for Lewy Body Dementia, Parkinson's disease, Alzheimer's disease with Lewy Bodies, Multiple System Atrophy and for other synucleinopathies, with direct relevance to various other protein misfolding disorders.

靶向各种蛋白质聚集体（如淀粉样蛋白-β（A β）、tau和α-突触核蛋白）的免疫疗法 （α syn）处于临床开发的不同阶段，并且总体上是最常见的方法。 制药行业，以解决疾病的特点，这种沉积[1 - 3]。大多数这些 潜在的治疗方法是全抗体。对抗体片段的关注要少得多， 应进一步探索其某些优点及其治疗和诊断潜力。 具体地，单结构域抗体（sdAb）是特别感兴趣的，因为它们的小尺寸（13 kDa）改善组织渗透，包括通过血脑屏障， 靶分子及其隐藏表位的抗体（150 kDa）。同样重要的是，它们具有高亲和力， 并且易于工程化和大量种植。 具体目的是确定骆驼科动物单结构域重链的治疗潜力 针对α syn蛋白的抗体片段（sdAb）。据推测，它们的小尺寸将提供 治疗效益超过整个抗体，主要是因为更大的访问目标，和一些 这是由于它们与较大抗体不能接近的新表位结合。 我们已经产生了51个sdAb克隆，具有独特的互补决定区（CDR）， 该研究识别各种形式的α-syn，并提出确定它们在不同模型中的治疗潜力， 并阐明其作用机制。对于初始筛选，将筛选多达30个单独的sdAb。 在突触核蛋白病的果蝇模型的神经元中全面表达，以及它们预防/减弱突触核蛋白病的能力。 监测α syn病理学。随后，将通过以下方法在果蝇中进一步检查5种最有效的sdAb： 在星形胶质细胞或外周表达，以及在突触核蛋白病的细胞培养模型中表达， 洞察力，也是动物模型研究中最有效的一种。我们的初步数据支持 在果蝇模型中筛选靶向tau蛋白的抗体片段的治疗功效的可行性。 具体地说，我们发现抗tau蛋白单链可变抗体片段（scFv）的神经元表达， 防止具有或不具有tau蛋白病突变的人tau的过表达的发育毒性，和 显著延长了tau蛋白病果蝇的寿命。我们预期，在大肠杆菌中表达抗α syn的sdAb， 共核蛋白病果蝇也将提供治疗益处。 拟议的研究可能会确定一类新的治疗路易体痴呆症的候选人， 帕金森病、阿尔茨海默病伴路易体、多系统萎缩和其他 突触核蛋白病，与各种其他蛋白质错误折叠疾病直接相关。