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.

项目概要 广泛的神经系统适应症，包括精神分裂症、疼痛、帕金森病和阿尔茨海默病 疾病和自闭症与 G 蛋白偶联受体蛋白 (GPCR) 相关，这使得它们具有吸引力 治疗的目标。了解 GPCR 功能障碍对于有效的治疗开发至关重要。 然而，破译 GPCR 的功能仍然是一项艰巨的任务，这在很大程度上是由于其固有的结构 GPCR 的复杂性，以及缺乏阐明 GPCR 功能的工具/试剂。抗体 (Abs) 可以 作为高度特异性的分析剂，并已被证明是高效的治疗方法。然而，高 通过传统的免疫方法很难制备高质量的 GPCR 抗体。 为了解决研究 GPCR 功能的高级工具未得到满足的需求，我们建议创建 用于快速发现和表征选择性高亲和力纳米抗体的强大、集成的管道 使用体外相位显示技术针对具有神经学重要性的已定义 GPCR 结构。我们的目标是 克服产生可再生 GPCR 结合试剂的两个主要挑战：(1) 缺乏 功能性 GPCR 抗原的可用性，以及 (2) GPCR 固有的较差免疫原性和高成本 使用常规动物免疫方法产生GPCR特异性抗体。我们的方法是 利用无细胞膜蛋白合成方法轻松制备大量高纯度、 生物素化蛋白脂质体（含有 GPCR 的脂质体），可从反应中有效纯化 链霉亲和素-磁珠混合物，用于从噬菌体展示中平行选择纳米抗体 合成纳米抗体。通过将体外 GPCR 生产与同一体外抗体选择相结合 实验室，我们预计构建一个集成管道，用于常规生成正交 GPCR- 在几周内靶向纳米抗体。在第一阶段概念验证演示中，我们将开发无细胞 合成大麻素受体 CB1 和 CB2 并生产这两种亚型的纳米抗体的方法 来自噬菌体展示的纳米抗体库的受体。所得纳米抗体的功能将是 通过生化和细胞分析确定目标 GPCR 的结合亲和力和特异性 方法。选择受体 CB1 和 CB2 进行演示是因为它们具有显着的神经生物学作用 功能。该技术可应用于第二阶段的其他 GPCR，总体目标是 1) 构建 GPCR 蛋白脂质体和 GPCR 靶向纳米抗体文库，2) 营销 GPCR 靶向 纳米抗体作为研究 GPCR 的宝贵研究试剂。由于纳米抗体的独特性质， 例如极其坚固、高度抗变性/热降解、高水溶性，以及 这些纳米抗体具有优异的身体分布、组织穿透性和穿过血脑屏障的能力 为研究脑源性 GPCR 提供宝贵的新研究工具，并可能具有诊断和 治疗效用。鉴于其模块化和可扩展性，这种 GPCR-纳米抗体管道有潜力产生 在蛋白质组范围内定制亲和试剂，为 GPCR 提供高质量且易于使用的工具包 生物和生物医学实验室将广泛使用的功能分析。