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J. Biol. Chem., Vol. 281, Issue 46, 35167-35175, November 17, 2006

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Selection and Characterization of Her2 Binding-designed Ankyrin Repeat Proteins^*

Christian Zahnd¹, Frédéric Pecorari², Nadine Straumann, Emanuel Wyler³, and Andreas Plückthun⁴

From the Department of Biochemistry, University of Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland and the Institute of Cell Biology, Hönggerberg, ETH Zürich, CH-8093 Zürich, Switzerland

Designed ankyrin repeat proteins (DARPins) are a novel class of binding proteins that bind their target protein with high affinity and specificity and have very favorable expression and stability properties. We describe here the in vitro selection of DARPins against human epidermal growth factor receptor 2 (Her2), an important target for cancer therapy and diagnosis. Several DARPins bind to the same epitope as trastuzumab (Herceptin), but none were selected that bind to the epitope of pertuzumab (Omnitarg). Some of the selected DARPins bind with low nanomolar affinity (K_d = 7.3 nM) to the target. Further analysis revealed that all DARPins are highly specific and do not cross-react with epidermal growth factor receptor I (EGFR1) or any other investigated protein. The selected DARPins specifically bind to strongly Her2-overexpressing cell lines such as SKBR-3 but also recognize small amounts of Her2 on weakly expressing cell lines such as MCF-7. Furthermore, the DARPins also lead to a highly specific and strong staining of plasma membranes of paraffinated sections of human mamma-carcinoma tissue. Thus, the selected DARPins might be used for the development of diagnostic tests for the status of Her2 overexpression in different adenocarcinomas, and they may be further evaluated for their potential in targeted therapy since their favorable expression properties make the construction of fusion proteins very convenient.

Received for publication, March 17, 2006 , and in revised form, September 8, 2006.

^* This work was supported by a grant from the Swiss National Center of Competence in Research in Structural Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Molecular Partners AG, c/o Department of Biochemistry, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.

² Present address: Unité de Biochimie Structurale-CNRS URA 2185, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris Cedex 15, France.

³ Present address: Institute of Biochemistry, ETH Zürich, CH-8093 Zurich, Switzerland.

⁴ To whom correspondence should be addressed. Tel.: 41-44-635-5570; Fax: 41-44-635-5902; E-mail: plueckthun{at}bioc.unizh.ch.

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