A Hybrid Adherent/Suspension Cell-Based Selection Strategy for Discovery of Antibodies Targeting Membrane Proteins.
A Hybrid Adherent/Suspension Cell-Based Selection Strategy for Discovery of Antibodies Targeting Membrane Proteins.
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DOI：
10.1007/978-1-0716-2285-8_11
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发表时间：
2022
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膜蛋白是受欢迎的药物靶点，抗体治疗代表了增长最快的药物类别。然而，仍然需要快速有效的方法来发现识别膜蛋白的抗体，以开发用于靶向治疗的稳健的临床管道。与膜蛋白的重组表达相关的挑战使得全细胞筛选技术是可取的，因为这些策略允许以其天然构象呈递靶膜蛋白。在这里，我们描述了一个工作流程，采用贴壁细胞为基础的和悬浮细胞为基础的全细胞淘选方法，以丰富酵母展示的抗体库内的特异性结合剂。第一轮选择由基于贴壁细胞的方法组成，其中在表达靶的哺乳动物细胞单层上淘选多样性文库以使幼稚文库减容。随后的轮次涉及使用基于悬浮细胞的方法，用磁激活细胞分选（MACS）或荧光激活细胞分选（FACS）促进，以实现进一步的文库富集。最后，我们描述了一种高通量的方法来筛选目标结合和特异性的单个克隆分离的选择活动。
Membrane proteins are favored drug targets and antibody therapeutics represent the fastest-growing category of pharmaceuticals. However, there remains a need for rapid and effective approaches for the discovery of antibodies that recognize membrane proteins to develop a robust clinical pipeline for targeted therapeutics. The challenges associated with recombinant expression of membrane proteins make whole cell screening techniques desirable, as these strategies allow presentation of the target membrane proteins in their native conformations. Here, we describe a workflow that employs both adherent cell-based and suspension cell-based whole cell panning methodologies to enrich for specific binders within a yeast-displayed antibody library. The first round of selection consists of an adherent cell-based approach, wherein a diverse library is panned over target-expressing mammalian cell monolayers in order to debulk the naïve library. Subsequent rounds involve the use of suspension cell-based approaches, facilitated with magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS), to achieve further library enrichment. Finally, we describe a high-throughput approach to screen target binding and specificity of individual clones isolated from selection campaigns.