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Int J Mol Sci. 2018 Nov 2;19(11). pii: E3444. doi: 10.3390/ijms19113444.

A Two-Step Approach for the Design and Generation of Nanobodies.

Author information

1
Faculty of Biology, University of Freiburg, Schaenzlestraße 1, 79104 Freiburg, Germany. hanna.wagner@biologie.uni-freiburg.de.
2
Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Albertstraße 19A, 79104 Freiburg, Germany. hanna.wagner@biologie.uni-freiburg.de.
3
BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schaenzlestraße 18, 79104 Freiburg, Germany. hanna.wagner@biologie.uni-freiburg.de.
4
Faculty of Biology, University of Freiburg, Schaenzlestraße 1, 79104 Freiburg, Germany. sarah.wehrle@epfl.ch.
5
Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch, France. etienne.weiss@unistra.fr.
6
Faculty of Biology, University of Freiburg, Schaenzlestraße 1, 79104 Freiburg, Germany. marco.cavallari@bioss.uni-freiburg.de.
7
BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schaenzlestraße 18, 79104 Freiburg, Germany. marco.cavallari@bioss.uni-freiburg.de.
8
Faculty of Biology, University of Freiburg, Schaenzlestraße 1, 79104 Freiburg, Germany. wilfried.weber@biologie.uni-freiburg.de.
9
Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Albertstraße 19A, 79104 Freiburg, Germany. wilfried.weber@biologie.uni-freiburg.de.
10
BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schaenzlestraße 18, 79104 Freiburg, Germany. wilfried.weber@biologie.uni-freiburg.de.

Abstract

Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin- and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols.

KEYWORDS:

VHH; complementarity-determining region (CDR) grafting; hapten; phage display; single-domain antibody; synthetic library; toxin

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