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Selection of High-Affinity Heterodimeric Antigen-Binding Fc Fragments from a Large Yeast Display Library

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Genotype Phenotype Coupling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2681))

Abstract

Antigen-binding Fc (Fcab™) fragments, where a novel antigen binding site is introduced by the mutagenesis of the C-terminal loops of the CH3 domain, function as parts of bispecific IgG-like symmetrical antibodies when they replace their wild-type Fc. Their homodimeric structure typically leads to bivalent antigen binding. In particular, biological situations monovalent engagement, however, would be preferred, either for avoiding agonistic effects leading to safety issues, or the attractive option of combining a single chain (i.e., one half) of an Fcab fragment reactive with different antigens in one antibody. We present the strategies for construction and selection of yeast libraries displaying heterodimeric Fcab fragments and discuss the effects of altered thermostability of the basic Fc scaffold and novel library designs that lead to isolation of highly affine antigen binding clones.

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Acknowledgments

The financial support by the company F-star Therapeutics, Christian Doppler Society, Austrian Federal Ministry for Digital and Economic Affairs and the National Foundation for Research, Technology and Development is gratefully acknowledged (CD Laboratory for innovative Immunotherapeutics, grant to GWK). FB was a fellow of the international PhD program “BioToP-Biomolecular Technology of Proteins,” funded by the Austrian Science Fund (FWF) (W1224). This project was also supported by EQ-BOKU VIBT GmbH and BOKU Core Facility for Biomolecular and Cellular Analysis. ™ Fcab and mAb2 are the trademarks of F-star Therapeutics Limited (Cambridge, United Kingdom).

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Authors and Affiliations

  1. Christian Doppler Laboratory for Innovative Immunotherapeutics, Institute of Molecular Biology, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

    Filippo Benedetti, Gerhard Stadlmayr, Katharina Stadlbauer, Florian Rüker & Gordana Wozniak-Knopp

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  1. Filippo Benedetti
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  2. Gerhard Stadlmayr
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  3. Katharina Stadlbauer
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  4. Florian Rüker
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  5. Gordana Wozniak-Knopp
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Correspondence to Gordana Wozniak-Knopp .

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Editors and Affiliations

  1. Protein Engineering and Antibody Technologies (PEAT), Merck Healthcare KGaA, Darmstadt, Germany

    Stefan Zielonka

  2. Protein Engineering and Antibody Technologies (PEAT), Merck Healthcare KGaA, Darmstadt, Germany

    Simon Krah

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Benedetti, F., Stadlmayr, G., Stadlbauer, K., Rüker, F., Wozniak-Knopp, G. (2023). Selection of High-Affinity Heterodimeric Antigen-Binding Fc Fragments from a Large Yeast Display Library. In: Zielonka, S., Krah, S. (eds) Genotype Phenotype Coupling. Methods in Molecular Biology, vol 2681. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3279-6_9

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  • DOI: https://doi.org/10.1007/978-1-0716-3279-6_9

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