Sie befinden Sich nicht im Netzwerk der Universität Paderborn. Der Zugriff auf elektronische Ressourcen ist gegebenenfalls nur via VPN oder Shibboleth (DFN-AAI) möglich. mehr Informationen...
Shark Attack: High affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation
Ist Teil von
Journal of biotechnology, 2014-12, Vol.191, p.236-245
Ort / Verlag
Netherlands: Elsevier B.V
Erscheinungsjahr
2014
Link zum Volltext
Quelle
MEDLINE
Beschreibungen/Notizen
•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.