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Construction of Histidine-Enriched Shark IgNAR Variable Domain Antibody Libraries for the Isolation of pH-Sensitive vNAR Fragments

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Antibody Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1827))

Abstract

The adaptive immune system of sharks comprises a heavy chain-only antibody isotype, referred to as immunoglobulin new antigen receptor (IgNAR). Antigen binding in case of IgNAR antibodies is mediated by a single variable domain (vNAR). Due to their inherent beneficial biophysical properties, such as small size and high thermal stability combined with a high specificity and affinity to their target antigens, vNAR domains emerged as promising tools for biotechnological and biomedical applications. Herein, we present detailed protocols for the engineering of pH-sensitivity into IgNAR V domains by constructing histidine-enriched and CDR3-diversified semisynthetic antibody libraries which can then be screened upon using yeast surface display. Protonation or deprotonation of incorporated histidine residues at different pH values results in structural transitions caused by altered electrostatic interactions. These interactions account for an altered binding behavior toward the target antigen. In the following protocol, we describe the generation of a semisynthetic vNAR master library that comprises two histidine residues on average in the 12-residue CDR3 loop. Moreover, once a pH-dependent vNAR population toward the target antigen is identified, this population can further be optimized in terms of affinity and pH sensitivity upon conducting a CDR1-mediated affinity maturation.

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Acknowledgments

This work was in part funded by the Merck Lab at Technische Universität Darmstadt.

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Authors and Affiliations

  1. Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany

    Doreen Könning, Steffen Hinz, Julius Grzeschik, Simon Krah, Stefan Zielonka & Harald Kolmar

  2. Antibody-Drug Conjugates and Targeted NBE Therapeutics, Merck KGaA, Darmstadt, Germany

    Doreen Könning & Christian Schröter

  3. Protein Engineering and Antibody Technologies, Merck KGaA, Darmstadt, Germany

    Simon Krah & Stefan Zielonka

Authors
  1. Doreen Könning
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  2. Steffen Hinz
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  3. Julius Grzeschik
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  4. Christian Schröter
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  5. Simon Krah
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  6. Stefan Zielonka
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  7. Harald Kolmar
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Corresponding author

Correspondence to Harald Kolmar .

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Editors and Affiliations

  1. Immunology Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia

    Damien Nevoltris

  2. Institut Paoli-Calmettes, CRCM, Aix Marseille University, CNRS, INSERM, Marseille, France

    Patrick Chames

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Könning, D. et al. (2018). Construction of Histidine-Enriched Shark IgNAR Variable Domain Antibody Libraries for the Isolation of pH-Sensitive vNAR Fragments. In: Nevoltris, D., Chames, P. (eds) Antibody Engineering. Methods in Molecular Biology, vol 1827. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8648-4_6

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  • DOI: https://doi.org/10.1007/978-1-4939-8648-4_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8647-7

  • Online ISBN: 978-1-4939-8648-4

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