title: DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency.
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.
authors.affiliation: Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Nikolas
authors.initials: N
authors.lastname: Stefan
authors.forename: Patricia
authors.initials: P
authors.lastname: Martin-Killias
authors.forename: Sascha
authors.initials: S
authors.lastname: Wyss-Stoeckle
authors.forename: Annemarie
authors.initials: A
authors.lastname: Honegger
authors.forename: Uwe
authors.initials: U
authors.lastname: Zangemeister-Wittke
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000951:Antigens, Neoplasm / Q000302:isolation & purification* / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D003090:Coliphages / Q000378:metabolism; D019020:Directed Molecular Evolution; D000071858:Epithelial Cell Adhesion Molecule; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008967:Molecular Biology / Q000379:methods; D009124:Muscle Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016296:Mutagenesis; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D019151:Peptide Library; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012270:Ribosomes / Q000378:metabolism
chemical_list: C472289:ANKRD23 protein, human; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D009124:Muscle Proteins; D050505:Mutant Proteins; D009687:Nuclear Proteins; D019151:Peptide Library