Engineering Antibodies on the Surface of CHO Cells.
Engineering Antibodies on the Surface of CHO Cells.
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DOI：
10.1007/978-1-4939-9853-1_22
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发表时间：
2020
影响因子：
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通讯作者：
Annalee W. Nguyen;Kevin C. Le;J. Maynard
中科院分区：
文献类型：
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作者：
Annalee W. Nguyen;Kevin C. Le;J. Maynard
虽然抗体库传统上是以噬菌体、细菌或酵母展示的形式进行筛选，但它们是大规模生产的，用于哺乳动物细胞系的制药和商业用途。用于筛选的更简单的生物具有与哺乳动物细胞显著不同的折叠和糖基化机制；因此，从这些文库产生的克隆可能需要进一步优化哺乳动物细胞的表达。为了简化抗体发现过程，我们开发了一个基于中国仓鼠卵巢(CHO)细胞的选择系统，允许长期展示抗体Fab片段。该系统由半稳定的Epi-CHO表型平台促进，可在长达2个月的时间内保持抗体表达，并与标准的基于聚合酶链式反应的突变策略兼容。该方案以HER2结合抗体为例，描述了CHO显示结合流式细胞术的简单和易行的使用，以丰富具有更高配基结合亲和力的抗体变体。
While antibody libraries are traditionally screened in phage, bacterial, or yeast display formats, they are produced in large scale for pharmaceutical and commercial use in mammalian cell lines. The simpler organisms used for screening have significantly different folding and glycosylation machinery than mammalian cells; consequently, clones resulting from these libraries may require further optimization for mammalian cell expression. To streamline the antibody discovery process, we developed a Chinese hamster ovary (CHO) cell-based selection system that allows for long-term display of antibody Fab fragments. This system is facilitated by a semi-stable Epi-CHO episomal platform to maintain antibody expression for up to 2 months and is compatible with standard PCR-based mutagenesis strategies. This protocol describes the simple and accessible use of CHO display coupled with flow cytometry to enrich for antibody variants with increased ligand-binding affinity from large libraries of ~106variants, using HER2-binding antibodies as an example.