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Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-54361

Stefan, N; Martin-Killias, P; Wyss-Stoeckle, S; Honegger, A; Zangemeister-Wittke, U; Plückthun, A (2011). DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency. Journal of Molecular Biology, 413(4):826-43.

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Abstract

Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K(d)=68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
DDC:570 Life sciences; biology
Language:English
Date:2011
Deposited On:13 Jan 2012 09:50
Last Modified:05 Feb 2014 07:24
Publisher:Elsevier
ISSN:0022-2836
Publisher DOI:10.1016/j.jmb.2011.09.016
PubMed ID:21963989
Citations:Web of Science®. Times Cited: 21
Google Scholar™
Scopus®. Citation Count: 19

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