Isolation of a pH-Sensitive IgNAR Variable Domain from a Yeast-Displayed, Histidine-Doped Master Library
Isolation of a pH-Sensitive IgNAR Variable Domain from a Yeast-Displayed, Histidine-Doped Master Library
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DOI：
10.1007/s10126-016-9690-z
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发表时间：
2016-04-01
影响因子：
3
通讯作者：
Kolmar, Harald
中科院分区：
文献类型：
--
作者：
Koenning, Doreen;Zielonka, Stefan;Kolmar, Harald
近年来，将pH敏感性工程化到抗体以及抗体衍生片段中对于生物医学和生物技术应用已经变得越来越有吸引力。在本文中，我们报告了第一个pH敏感IgNAR可变结构域（vNAR）的分离，其分离自酵母展示的半合成主文库。该策略使得能够从富含组氨酸的CDR 3文库中直接鉴定pH依赖性结合剂。显示的vNAR变体在其12残基CDR 3环中的随机位置处平均含有两个组氨酸取代。在针对概念验证靶标EpCAM筛选七轮后（选择在pH 7.4下结合和在pH 6.0下降低结合），获得单个克隆，其显示特异性和pH依赖性结合，如通过酵母表面展示和生物层干涉测量法表征的。这种pH依赖性vNAR结构域的潜在应用包括它们在定制的亲和色谱中的应用，从而实现温和的洗脱方案。此外，利用主文库分离pH敏感性vNAR变体可以是获得具有用于生物技术、诊断和治疗的规定特征的结合实体的通用策略。
In recent years, engineering of pH-sensitivity into antibodies as well as antibody-derived fragments has become more and more attractive for biomedical and biotechnological applications. Herein, we report the isolation of the first pH-sensitive IgNAR variable domain (vNAR), which was isolated from a yeast-displayed, semi-synthetic master library. This strategy enables the direct identification of pH-dependent binders from a histidine-enriched CDR3 library. Displayed vNAR variants contained two histidine substitutions on average at random positions in their 12-residue CDR3 loop. Upon screening of seven rounds against the proof-of-concept target EpCAM (selection for binding at pH 7.4 and decreased binding at pH 6.0), a single clone was obtained that showed specific and pH-dependent binding as characterized by yeast surface display and biolayer interferometry. Potential applications for such pH-dependent vNAR domains include their employment in tailored affinity chromatography, enabling mild elution protocols. Moreover, utilizing a master library for the isolation of pH-sensitive vNAR variants may be a generic strategy to obtain binding entities with prescribed characteristics for applications in biotechnology, diagnostics, and therapy.