Department of Oncology, Ludwig Institute for Cancer Research Lausanne, University of Lausanne, Lausanne, Switzerland.
Department of Oncology, Ludwig Institute for Cancer Research Lausanne, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.
Sci Rep. 2019 Sep 6;9(1):12815. doi: 10.1038/s41598-019-49233-7.
An early bottleneck in the rapid isolation of new antibody fragment binders using in vitro library approaches is the inertia encountered in acquiring and preparing soluble antigen fragments. In this report, we describe a simple, yet powerful strategy that exploits the properties of the SpyCatcher/SpyTag (SpyC/SpyT) covalent interaction to improve substantially the speed and efficiency in obtaining functional antibody clones of interest. We demonstrate that SpyC has broad utility as a protein-fusion tag partner in a eukaryotic expression/secretion context, retaining its functionality and permitting the direct, selective capture and immobilization of soluble antigen fusions using solid phase media coated with a synthetic modified SpyT peptide reagent. In addition, we show that the expressed SpyC-antigen format is highly compatible with downstream antibody phage display selection and screening procedures, requiring minimal post-expression handling with no sample modifications. To illustrate the potential of the approach, we have isolated several fully human germline scFvs that selectively recognize therapeutically relevant native cell surface tumor antigens in various in vitro cell-based assay contexts.
利用体外文库方法快速分离新抗体片段结合物时，早期的一个瓶颈是在获取和制备可溶性抗原片段时遇到的惰性。在本报告中，我们描述了一种简单但强大的策略，利用 SpyCatcher/SpyTag（SpyC/SpyT）共价相互作用的特性，大大提高了获得功能抗体克隆的速度和效率。我们证明 SpyC 作为一种真核表达/分泌背景下的蛋白融合标签伙伴具有广泛的用途，保留其功能，并允许使用涂有合成修饰 SpyT 肽试剂的固相介质直接、选择性地捕获和固定可溶性抗原融合物。此外，我们还表明，表达的 SpyC-抗原形式与下游抗体噬菌体展示选择和筛选程序高度兼容，仅需在表达后进行最小的处理，无需样品修饰。为了说明该方法的潜力，我们已经分离了几种完全人源胚系 scFv，它们在各种基于细胞的体外检测环境中选择性地识别治疗相关的天然细胞表面肿瘤抗原。
