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Generation and functional characterization of intracellular antibodies interacting with the kinase domain of human EGF receptor

Oncogene volume 22, pages 1557–1567 (2003)Cite this article

Abstract

Intracellular expression of single-chain antibodies (scFvs) represents a promising approach for selective interference with cellular proto-oncogenes such as the epidermal growth factor receptor (EGFR). Previously, we have shown that intrabodies targeted to the lumen of the endoplasmic reticulum prevent the transit of EGFR or the related ErbB2 molecule to the cell surface, thereby inactivating their transforming potential. While intramolecular disulfide bridges important for antibody stability are correctly formed during expression in the secretory pathway, scFvs expressed in the reducing environment of the cytosol are often inactive. To overcome this problem and to generate antibody fragments that interact with the intracellular domain of human EGFR in the cytoplasm, here we have chosen a two-step approach combining classical selection of scFvs by phage display with subsequent expression in yeast. After enrichment of EGFR-specific antibody fragments from a combinatorial library by biopanning, a yeast two-hybrid screen was performed using the intracellular domain of EGFR as bait. Screening of 1.5 × 105 preselected scFv plasmids under highly stringent conditions yielded 223 colonies that represented at least five independent scFv clones functional in the intracellular milieu of eukaryotic cells. Interaction of selected antibody fragments with the intracellular domain of EGFR was confirmed in GST pull-down and coimmunoprecipitation experiments. Upon cytoplasmic expression in human tumor cells, scFvs colocalized with EGFR at the plasma membrane demonstrating their functionality in vivo.

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Acknowledgements

We thank Gesa Brochmann-Santos for excellent technical assistance during the initial phase of the project. This work was supported in part by grants to WW from the National Genome Research Network (NGFN) program of the German ‘Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie’ (BMBF), and the German–Israeli Foundation for Scientific Research and Development. The laboratory of NEH is supported by the Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute for Biomedical Research.

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  1. Roger R Beerli

    Present address: Cytos Biotechnology AG, CH-8952, Zürich-Schlieren, Switzerland

Authors and Affiliations

  1. Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt am Main, D-60596, Germany

    Stephen Hyland & Winfried Wels

  2. The Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA

    Roger R Beerli & Carlos F Barbas III

  3. Friedrich Miescher Institute, Basel, CH-4002, Switzerland

    Roger R Beerli & Nancy E Hynes

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Correspondence to Winfried Wels.

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Hyland, S., Beerli, R., Barbas, C. et al. Generation and functional characterization of intracellular antibodies interacting with the kinase domain of human EGF receptor. Oncogene 22, 1557–1567 (2003). https://doi.org/10.1038/sj.onc.1206299

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