Antibody engineering in mammalian cells offers the important advantage of expression and screening of libraries in their native conformation, increasing the likelihood of generating candidates with more favorable molecular properties. Major advances in cellular engineering enabled by CRISPR-Cas9 genome editing have made it possible to expand the use of mammalian cells in biotechnological applications. Here, we describe an antibody engineering and screening approach where complete variable light (V_L) and heavy (V_H) chain cassette libraries are stably integrated into the genome of hybridoma cells by enhanced Cas9-driven homology-directed repair (HDR), resulting in their surface display and secretion. By developing an improved HDR donor format that utilizes in situ linearization, we are able to achieve >15-fold improvement of genomic integration, resulting in a screening workflow that only requires a simple plasmid electroporation. This proved suitable for different applications in antibody discovery and engineering. By integrating and screening an immune library obtained from the variable gene repertoire of an immunized mouse, we could isolate a diverse panel of >40 unique antigen-binding variants. Additionally, we successfully performed affinity maturation by directed evolution screening of an antibody library based on random mutagenesis, leading to the isolation of several clones with affinities in the picomolar range.

哺乳动物细胞中的抗体工程化提供了以其天然构象表达和筛选文库的重要优势，从而增加了产生具有更有利分子特性的候选基因的可能性。通过CRISPR-Cas9基因组编辑实现的细胞工程学的重大进步，使得在生物技术应用中扩展哺乳动物细胞的应用成为可能。在这里，我们描述了一种抗体工程和筛选方法，其中完整的可变轻链（V _L）和重链（V _H）盒式磁带库通过增强的Cas9驱动的同源导向修复（HDR）稳定地整合到杂交瘤细胞的基因组中，在他们的表面展示和分泌。通过开发一种改进的HDR供体格式，该格式可以在原地使用线性化，我们能够实现> 15倍的基因组整合改善，从而实现仅需简单质粒电穿孔的筛选工作流程。事实证明，此方法适用于抗体发现和工程设计中的不同应用。通过整合和筛选从免疫小鼠可变基因库获得的免疫文库，我们可以分离出包含> 40种独特抗原结合变体的多样化面板。此外，我们通过基于随机诱变的抗体文库的定向进化筛选成功完成了亲和力成熟，从而分离出了几个亲和力在皮摩尔范围内的克隆。