Laboratory of Regeneromics, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
Department of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, WA 99210, USA.
Int J Mol Sci. 2021 Apr 29;22(9):4711. doi: 10.3390/ijms22094711.
G-protein-coupled receptors (GPCRs), especially chemokine receptors, are ideal targets for monoclonal antibody drugs. Considering the special multi-pass transmembrane structure of GPCR, it is often a laborious job to obtain antibody information about off-targets and epitopes on antigens. To accelerate the process, a rapid and simple method needs to be developed. The split-ubiquitin-based yeast two hybrid system (YTH) was used as a blue script for a new method. By fusing with transmembrane peptides, scFv antibodies were designed to be anchored on the cytomembrane, where the GPCR was co-displayed as well. The coupled split-ubiquitin system transformed the scFv-GPCR interaction signal into the expression of reporter genes. By optimizing the topological structure of scFv fusion protein and key elements, including signal peptides, transmembrane peptides, and flexible linkers, a system named Antigen-Antibody Co-Display (AACD) was established, which rapidly detected the interactions between antibodies and their target GPCRs, CXCR4 and CXCR5, while also determining the off-target antibodies and antibody-associated epitopes. The AACD system can rapidly determine the association between GPCRs and their candidate antibodies and shorten the research period for off-target detection and epitope identification. This system should improve the process of GPCR antibody development and provide a new strategy for GPCRs antibody screening.
G 蛋白偶联受体（GPCRs），特别是趋化因子受体，是单克隆抗体药物的理想靶点。考虑到 GPCR 的特殊多跨膜结构，获得针对非靶点和抗原表位的抗体信息通常是一项艰巨的工作。为了加快这一进程，需要开发一种快速而简单的方法。基于分离泛素的酵母双杂交系统（YTH）被用作一种新方法的蓝本。通过与跨膜肽融合，设计 scFv 抗体锚定在细胞质膜上，同时也展示了 GPCR。偶联的分离泛素系统将 scFv-GPCR 相互作用信号转化为报告基因的表达。通过优化 scFv 融合蛋白的拓扑结构和关键元件，包括信号肽、跨膜肽和柔性接头，建立了一个名为抗原-抗体共展示（AACD）的系统，该系统可快速检测抗体与其靶标 GPCR（CXCR4 和 CXCR5）之间的相互作用，同时确定非靶标抗体和抗体相关表位。AACD 系统可以快速确定 GPCR 与其候选抗体之间的关联，并缩短非靶标检测和表位鉴定的研究周期。该系统应改进 GPCR 抗体开发的过程，并为 GPCR 抗体筛选提供新的策略。
