title: Bispecific designed ankyrin repeat proteins (DARPins) targeting epidermal growth factor receptor inhibit A431 cell proliferation and receptor recycling.
abstract: The EGF receptor (EGFR) has been implicated in the development and progression of many tumors. Although monoclonal antibodies directed against EGFR have been approved for the treatment of cancer in combination with chemotherapy, there are limitations in their clinical efficacy, necessitating the search for robust targeting molecules that can be equipped with new effector functions or show a new mechanism of action. Designed ankyrin repeat proteins (DARPins) may provide the targeting component for such novel reagents. Previously, four DARPins were selected against EGFR with (sub)nanomolar affinity. As any targeting module should preferably be able to inhibit EGFR-mediated signaling, their effect on A431 cells overexpressing EGFR was examined: three of them were shown to inhibit proliferation by inducing G(1) arrest, as seen for the Food and Drug Administration-approved antibody cetuximab. To understand this inhibitory mechanism, we mapped the epitopes of the DARPins using yeast surface display. The epitopes for the biologically active DARPins overlapped with the EGF-binding site, whereas the fourth DARPin bound to a different domain, explaining the lack of a biological effect. To optimize the biological activity of the DARPins, we combined two DARPins binding to different epitopes with a flexible linker or with a leucine zipper, leading to a homodimer. The latter DARPin was able to reduce surface EGFR by inhibiting receptor recycling, leading to a dramatic decrease in cell viability. These results indicate that multispecific EGFR-specific DARPins are superior to cetuximab and may form the basis of new opportunities in tumor targeting and tumor therapy.
authors: Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
authors: Ykelien L
authors: YL
authors: Boersma
authors: Department of Chemical Engineering and Bioengineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors: Ginger
authors: G
authors: Chao
authors: Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
authors: Daniel
authors: D
authors: Steiner
authors: Department of Chemical Engineering and Bioengineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors: K Dane
authors: KD
authors: Wittrup
authors: Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland. Electronic address: plueckthun@bioc.uzh.ch.
authors: Andreas
authors: A
authors: Plückthun
mesh_terms: D017089:Ankyrin Repeat; D017487:Ankyrins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000494:pharmacology; D059447:Cell Cycle Checkpoints; D045744:Cell Line, Tumor; D000068818:Cetuximab; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016193:G1 Phase; D006801:Humans; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques
chemical_list: D017487:Ankyrins; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D000939:Epitopes; C512478:EGFR protein, human; D066246:ErbB Receptors; D000068818:Cetuximab