Isolation of a pH-Sensitive IgNAR Variable Domain from a Yeast-Displayed, Histidine-Doped Master Library

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• 作者：Doreen Könning ; Stefan Zielonka ; Carolin Sellmann…
• 关键词：Shark antibody ; IgNAR ; vNAR ; Single domain antibody ; pH ; dependent binding ; Yeast surface display
• 刊名：Marine Biotechnology
• 出版年：2016
• 出版时间：April 2016
• 年：2016
• 卷：18
• 期：2
• 页码：161-167
• 全文大小：742 KB
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• 作者单位：Doreen Könning (1)
  Stefan Zielonka (1)
  Carolin Sellmann (1)
  Christian Schröter (1)
  Julius Grzeschik (1)
  Stefan Becker (2)
  Harald Kolmar (1)
  
  1. Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Straße 4, D-64287, Darmstadt, Germany
  2. Protein Engineering and Antibody Technologies, Merck-Serono, Merck KGaA, Frankfurter Straße 250, D-64293, Darmstadt, Germany
  
• 刊物类别：Earth and Environmental Science
• 刊物主题：Earth sciences
  Oceanography
  
• 出版者：Springer New York
• ISSN：1436-2236

文摘

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.