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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23020 43409 2014 10 PDF Available
Title
Shark Attack: High affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation
Abstract

•A novel method for stepwise affinity maturation of antigen-specific vNAR antibody domains was established.•Antigen-specific molecules were selected against three different targets (EpCAM, EphA2, HTRA1) using yeast surface display.•Affinity maturation was achieved by subsequent CDR1 diversification of target-binding populations.

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
Shark vNAR antibody; Semi-synthetic library; Stepwise in vitro affinity maturation; Yeast surface display
First Page Preview
Shark Attack: High affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 191, 10 December 2014, Pages 236–245
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering