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J Biotechnol. 2014 Dec 10;191:236-45. doi: 10.1016/j.jbiotec.2014.04.023. Epub 2014 May 23.

Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

Author information

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.
3
Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Department Drug Design and Optimization, Saarland University, Campus C2.3, D-66123 Saarbrücken, Germany.
4
Goethe-University Frankfurt, Faculty of Biosciences, Max-von-Laue-Str. 13, D-60438 Frankfurt am Main, Germany; University Hospital Frankfurt, Department of Anesthesiology, Intensive-Care Medicine and Pain Therapy, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
5
Clinic for Birds, Reptiles, Amphibians and Fish, Justus-Liebig University, Gießen, Frankfurter Str. 91-93, D-35392 Giessen, Germany.
6
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany. Electronic address: Kolmar@Biochemie-TUD.de.

Abstract

A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

KEYWORDS:

Semi-synthetic library; Shark vNAR antibody; Stepwise in vitro affinity maturation; Yeast surface display

PMID:
24862193
DOI:
10.1016/j.jbiotec.2014.04.023
[Indexed for MEDLINE]

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