Developing an integrated pipeline for routine generation of orthogonal GPCR-targeting nanobodies

开发用于常规生成正交 GPCR 靶向纳米抗体的集成管道

• 批准号: 10603669
• 负责人: XICHUN ZHOU
• 金额: $ 21.86万
• 依托单位: VITAN-BIOTECH, LLC
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-22 至 2024-09-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10603669
• 关键词: Address; Affinity; Alzheimer' s Disease; Animals; Antibodies; Antigens; Bacteriophages; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biotinylation; Brain; CNR1 gene; CNR2 gene; Cell-Free System; Cells; Chimeric Proteins; Coupling; Detection; Development; Diagnostic; Disease; Disease Progression; Drug Targeting; Enzyme-Linked Immunosorbent Assay; Fab Immunoglobulins; Functional disorder; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Generations; Goals; Heterogeneity; Immunization; In Vitro; Individual; Laboratories; Libraries; Link; Liposomes; Marketing; Membrane Proteins; Methods; Monitor; Nerve; Neurobiology; Neurodegenerative Disorders; Neurologic; Pain; Parkinson Disease; Pathogenesis; Penetration; Phage Display; Phage Receptors; Phase; Play; Preparation; Production; Property; Protein Biosynthesis; Proteomics; Protocols documentation; Reaction; Reagent; Recombinants; Research; Research Personnel; Resistance; Role; Schizophrenia; Solubility; Specificity; Streptavidin; Structure; Symptoms; System; Technology; Therapeutic; Tissues; aqueous; autism spectrum disorder; blood-brain barrier crossing; cost; experimental study; imaging study; immunogenic; immunogenicity; improved; in vitro activity; in vivo imaging; magnetic beads; mental function; nanobodies; nervous system disorder; neuropathology; novel; optical imaging; protein activation; protein function; proteoliposomes; receptor; success; targeted treatment; therapeutic development; therapeutically effective; tool; ultra high resolution

Project Summary A broad range of neurological indications, including schizophrenia, pain, Parkinson's and Alzheimer's diseases, and autism have been linked to G-protein coupled receptor proteins (GPCRs), making them attractive targets for therapy. Understanding GPCR dysfunction is essential for effective therapeutic development. However, deciphering the functions of GPCRs remains a daunting task in large part due to the inherent structural complexity of GPCRs, and the lack of tools/reagents for elucidating the GPCRs functions. Antibodies (Abs) can serve as highly specific analytical agents and have proven to be highly effective therapeutics. However, high- quality Abs against GPCRs are very difficult to make by traditional immunization-based methods. To address the unmet need of advanced tools for studying the functions of GPCRs, we propose to create a robust, integrated pipeline for the rapid discovery and characterization of selective, high-affinity nanobodies against defined GPCR structures of neurological importance using in vitro phase-display technology. We aim to overcome the two primary challenges to enable generation of renewable GPCR-binding reagents: (1) lack of functional GPCRs antigens availability, and (2) the inherent poor immunogenicity of GPCRs and high cost in producing GPCR-specific antibodies using conventional animal immunization method. Our approach is to leverage cell-free membrane protein synthesis method for facile preparation of large quantities of high-purity, biotinylated proteoliposomes (liposome-harboring GPCRs), which can be efficiently purified from reaction mixture by streptavidin-magnetic beads and used for parallel selection of nanobodies from phage-displayed synthetic nanobodies. By coupling in vitro GPCR production to in vitro antibody selection within the same laboratory, we anticipate construction of an integrated pipeline for routine generation of orthogonal GPCR- targeting nanobodies in a matter of weeks. In Phase I proof-of-concept demonstration, we will develop cell-free approach to synthesize cannabinoid receptors CB1 and CB2 and produce nanobodies to these two subtype receptors from phage-displayed nanobody libraries. The functionalities of resulting nanobodies will be characterized to determine binding affinity and specificity for the target GPCRs by biochemical and cellular methods. The receptors CB1 and CB2 are selected for demonstration due to their significant neurobiological functions. This proposed technology can be applied to other GPCRs in Phase II with an overall goal of 1) building libraries of GPCR proteoliposomes and GPCR-targeting nanobodies, 2) marketing the GPCR-targeting nanobodies as invaluable research reagents for studying GPCRs. Due to the unique properties of nanobodies, such as extremely robust, highly resistant to denaturation/thermal degradation, high aqueous solubility, and superior body distribution, tissue penetration, and the ability to cross blood-brain barrier, these nanobodies will provide invaluable new research tools for studying brain-derived GPCRs, and may have diagnostic and therapeutic utility. Given its modularity and scalability, this GPCR-nanobody pipeline has the potential to yield customized affinity reagents on a proteomic-wide scale, providing a high-quality and easy to use toolkit for GPCR functional analysis that will be broadly accessible to the biological and biomedical laboratories.

项目摘要 广泛的神经系统迹象，包括精神分裂症，疼痛，帕金森氏症和阿尔茨海默氏症 疾病和自闭症与G蛋白偶联受体蛋白（GPCR）有关，使其具有吸引力 治疗的靶标。了解GPCR功能障碍对于有效的治疗发展至关重要。 然而，解密GPCR的功能在很大程度上仍然是一项艰巨的任务 GPCR的复杂性以及缺乏用于阐明GPCR功能的工具/试剂。抗体（ABS）可以 用作高度特定的分析剂，已被证明是高效的治疗剂。但是，高 - 通过传统的基于免疫的方法，很难对GPCR进行质量ABS。 为了满足研究GPCR功能的高级工具的未满足需求，我们建议创建 适用的集成管道，用于快速发现和表征选择性高亲和力纳米体 使用体外相位播放技术的神经学重要性的确定的GPCR结构。我们的目标 克服两个主要挑战，以使能够生成可再生的GPCR结合试剂：（1）缺乏 功能性GPCR抗原可用性，以及（2）GPCR的固有的较差的免疫原性和高成本 使用常规动物免疫方法产生GPCR特异性抗体。我们的方法是 利用无细胞的膜蛋白合成方法来轻松制备大量的高纯度， 生物素化蛋白脂质体（脂质体 - 磨刀型GPCR）可以有效地从反应中纯化 通过链霉亲和素磁珠混合物，用于从噬菌体播放中平行选择纳米体 合成纳米体。通过将体外GPCR耦合到同一体外抗体选择 实验室，我们预计建造一条集成管道，以常规生成正交GPCR- 在几周内靶向纳米体。在第一阶段的概念验证演示中，我们将开发无细胞 合成大麻素受体CB1和CB2并为这两个亚型产生纳米体的方法 来自噬菌体播放的纳米库库的受体。产生的纳米构造的功能将是 特征是通过生化和细胞确定目标GPCR的结合亲和力和特异性 方法。由于其显着的神经生物学，因此选择了受体CB1和CB2进行演示 功能。该提出的技术可以应用于II阶段的其他GPCR，总体目标是1）建造 GPCR蛋白质脂质体和靶向GPCR的纳米生物的库，2）营销靶向GPCR的靶向 纳米型作为研究GPCR的宝贵研究试剂。由于纳米生物的独特特性， 例如极强的，对变性/热降解，高水溶性和高度耐药性和 这些纳米体的卓越身体分布，组织穿透以及跨血脑屏障的能力， 为研究脑衍生的GPCR提供宝贵的新研究工具，并可能具有诊断性和 治疗效用。鉴于其模块化和可扩展性，该GPCR-Nanobody管道有可能产生 定制的亲和力试剂在蛋白质组学范围内，为GPCR提供高质量且易于使用的工具包 生物学和生物医学实验室将广泛访问的功能分析。