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

项目摘要 广泛的神经系统适应症，包括精神分裂症，疼痛，帕金森氏症和阿尔茨海默氏症 自闭症与G蛋白偶联受体蛋白（GPCRs）有关，使其具有吸引力 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固有的结构性缺陷。 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 用作高度特异性的分析试剂，并已被证明是高度有效的治疗剂。然而，高- 通过传统的基于免疫的方法很难制备针对GPCR的高质量Ab。 为了满足研究GPCR功能的先进工具的未满足需求，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管道 使用体外相位显示技术，针对神经学重要性的定义的GPCR结构。我们的目标是 克服了两个主要挑战以能够产生可再生的GPCR结合试剂：（1）缺乏 功能性GPCR抗原的可用性，和（2）GPCR固有的免疫原性差和免疫治疗的高成本。 使用常规动物免疫方法产生GPCR特异性抗体。我们的做法是 利用无细胞膜蛋白合成方法简便制备大量高纯度， 生物素化的蛋白脂质体（脂质体携带GPCR），可从反应中有效纯化 链霉亲和素-磁珠混合物，用于从噬菌体展示的 合成纳米抗体。通过将体外GPCR产生与体外抗体选择在相同条件下偶联， 实验室，我们预计建设一个综合管道的常规产生的正交GPCR- 针对纳米抗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CB 1和CB 2并产生针对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 其特征在于通过生物化学和细胞生物学方法测定对靶GPCR的结合亲和力和特异性， 方法.受体CB 1和CB 2由于其显著的神经生物学特性而被选择用于证明。 功能协调发展的该提议的技术可以应用于第二阶段中的其他GPCR，总体目标是：1）建立 GPCR蛋白脂质体和GPCR靶向纳米抗体的文库，2）销售GPCR靶向纳米抗体， 纳米抗体是研究GPCR的宝贵研究试剂。由于纳米抗体的独特性质， 例如非常坚固、高度抗变性/热降解、高水溶性， 这些纳米抗体具有上级的体内分布、组织渗透性和穿过血脑屏障的能力， 为研究脑源性GPCR提供了宝贵的新研究工具，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-纳米抗体管道具有产生 在蛋白质组学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可广泛使用的功能分析。

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.