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

项目摘要 广泛的神经症状，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(GPCRs)有关，这使它们具有吸引力 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GPCRs的职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 GPCRs的复杂性，以及缺乏工具/试剂来阐明GPCRs的功能。抗体(Abs)可以 作为高度特异的分析试剂，已被证明是高度有效的疗法。然而，高- 用传统的免疫方法很难制备高质量的抗GPCRs抗体。 为了满足研究GPCRs功能的先进工具的未得到满足的需求，我们建议创建 用于快速发现和表征选择性高亲和力纳米体的强大、集成的流水线 使用体外相显示技术对确定的具有神经重要性的gpr结构进行对照。我们的目标是 克服两个主要挑战，以产生可再生的GPCR结合试剂：(1)缺乏 功能性GPCRs抗原的可用性，以及(2)GPCRs固有的低免疫原性和较高的成本 使用传统的动物免疫方法产生gpr特异性抗体。我们的方法是 利用无细胞膜蛋白合成法简便制备大量高纯度， 生物素化蛋白脂质体(脂质体-GPCRs)，可从反应中高效纯化 链霉亲和素-磁珠混合物并用于从噬菌体展示中平行选择纳米体 人工合成的纳米物体。通过将体外GPCR产物与体外抗体选择在同一 实验室，我们预计将建造一条综合管道，用于常规产生正交gpr- 在几周内锁定纳米体靶向。在第一阶段概念验证演示中，我们将开发无单元格 大麻素受体CB1和CB2的合成及针对这两个亚型的纳米抗体的制备方法 噬菌体展示纳米体库中的受体。由此产生的纳米体的功能将是 其特征是通过生物化学和细胞学方法确定靶GPCRs的结合亲和力和特异性 方法：研究方法。由于受体cb1和cb2具有重要的神经生物学意义，因此选择它们作为实验对象。 功能。这项拟议的技术可以应用于第二阶段的其他GPCR，总体目标是1)建立 GPCR靶向纳米体库和GPCR靶向纳米体库，2)GPCR靶向市场 纳米体作为研究GPCRs的宝贵研究试剂。由于纳米体的独特性质， 例如极其坚固、高度抗变性/热降解、高水溶解度以及 优越的身体分布、组织穿透性和跨越血脑屏障的能力，这些纳米体将 为研究脑源性GPCRs提供宝贵的新研究工具，并可能具有诊断和 治疗效用。鉴于其模块化和可伸缩性，这种GPCR-纳米体管道有可能产生 在蛋白质组范围内定制亲和试剂，为GPCR提供高质量和易于使用的工具包 生物和生物医学实验室将广泛使用的功能分析。

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.