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.

针对不同蛋白聚集体的免疫治疗，如淀粉样蛋白-β(A-β)、tau和α-突触核蛋白 (αSYN)处于不同的临床开发阶段，总体上是 制药业应对以此类证词为特征的疾病[1-3]。其中大部分都是 潜在的治疗方法是全抗体。对抗体片段的关注要少得多，抗体片段具有 某些优势以及它们的治疗和诊断潜力应该进一步挖掘。 具体地说，单域抗体(SdAbbs)特别令人感兴趣，因为它们体积小(13 KDA)改善了组织渗透，包括通过血脑屏障，从而更好地进入 针对目标分子及其隐藏表位的抗体(150 KDa)。同样重要的是，它们具有很高的亲和力， 而且易于工程设计和大量种植。 具体目的是确定骆驼单域重链的治疗潜力。 抗αSYN蛋白的抗体片段(SdAb)。据推测，它们的小尺寸将提供 相对于整个抗体的治疗益处，主要是因为更容易接触到靶点，以及一些 这是由于它们与较大的抗体无法获得的新表位结合所致。 我们已经产生了51个sdAbs克隆，具有独特的互补决定区域(CDR)， 识别各种形式的αSYN，并建议在不同的模型中确定它们的治疗潜力， 以及澄清他们的行动机制。对于初始屏幕，最多将有30个单独的sdAb 在果蝇共核病模型的神经元中全局表达，以及它们预防/减弱 α同步病理监测。随后，5个最有效的sdAb将在苍蝇身上进一步检测，方法是 在星形胶质细胞或外周表达，在机械性突触核病的细胞培养模型中表达 洞察力，这是在动物模型中进行的最有效的研究。我们的初步数据支持 筛选针对tau蛋白的抗体片段在苍蝇模型中治疗效果的可行性。 具体地说，我们展示了抗tau单链可变抗体片段(ScFv)神经元表达 防止过表达人tau的发育毒性，无论是否存在tau突变，以及 显著延长了变态苍蝇的寿命。我们预计表达抗αSYN的sdAbs 并核症苍蝇也将提供治疗益处。 拟议的研究可能确定路易体痴呆的一类新的候选治疗方法， 帕金森氏病、阿尔茨海默病合并路易体、多系统萎缩等 联核病，与其他各种蛋白质错误折叠障碍直接相关。