Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.
Ferring Darmstadt Laboratories, Darmstadt, Germany.
Methods Mol Biol. 2023;2681:291-311. doi: 10.1007/978-1-0716-3279-6_16.
While yeast surface display (YSD) has gained traction for antibody hit discovery efforts with the first therapeutic YSD-isolated antibody sintilimab approved in 2018, a major drawback that remains is the time-consuming reformatting of monoclonal antibody (mAb) candidates. By using a Golden Gate cloning (GGC)-dependent workflow, the bulk transfer of genetic information can be performed from antibody fragments displayed on yeast cells to a bidirectional mammalian expression vector. Herein, we describe in-depth protocols for the reformatting of mAbs, starting from the generation of Fab fragment libraries in YSD vectors and ending up with IgG molecules in bidirectional mammalian vectors in a consolidated two-pot, two-step procedure.
虽然酵母表面展示（YSD）在抗体命中发现方面取得了进展，2018 年首个治疗性 YSD 分离的抗体信迪利单抗获得批准，但仍然存在一个主要缺点，即单克隆抗体（mAb）候选物的重新格式化非常耗时。通过使用 Golden Gate 克隆（GGC）依赖性工作流程，可以将从酵母细胞上展示的抗体片段到双向哺乳动物表达载体的大量遗传信息进行批量转移。本文详细描述了 mAb 重新格式化的方案，从 YSD 载体中 Fab 片段文库的生成开始，最终通过两步两步的两步法，在双向哺乳动物载体中得到 IgG 分子。
