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Journal of Cancer Research and Clinical Oncology

, Volume 141, Issue 6, pp 1049–1061 | Cite as

In vitro effects and ex vivo binding of an EGFR-specific immunotoxin on rhabdomyosarcoma cells

  • Judith Niesen
  • Hannes Brehm
  • Christoph Stein
  • Nina Berges
  • Alessa Pardo
  • Rainer Fischer
  • Andre ten Haaf
  • Stefan Gattenlöhner
  • Mehmet K. Tur
  • Stefan Barth
Original Article – Cancer Research

Abstract

Purpose

Rhabdomyosarcoma (RMS) is a rare and aggressive soft tissue sarcoma with limited treatment options and a high failure rate during standard therapy. New therapeutic strategies based on targeted immunotherapy are therefore much in demand. The epidermal growth factor receptor (EGFR) has all the characteristics of an ideal target. It is overexpressed in up to 80 % of embryonal RMS and up to 50 % of alveolar RMS tumors. We therefore tested the activity of the EGFR-specific recombinant immunotoxin (IT) 425(scFv)-ETA′ against EGFR+ RMS cells in vitro and ex vivo.

Methods

We tested the specific binding and internalization behavior of 425(scFv)-ETA′ in RMS cell lines in vitro by flow cytometry, compared to the corresponding imaging probe 425(scFv)-SNAP monitored by live cell imaging. The cytotoxic activity of 425(scFv)-ETA′ was tested using cell viability and apoptosis assays. Specific binding of the IT was confirmed on formalin-fixed paraffin-embedded tissue samples from two RMS patients.

Results

We confirmed the specific binding of 425(scFv)-ETA′ to RMS cells in vitro and ex vivo. Both the IT and the corresponding imaging probe were rapidly internalized. The IT killed EGFR+ RMS cells in a dose-dependent manner, while showing no effect against control cells. It showed specific apoptotic activity against one selected RMS cell line.

Conclusions

This is the first study showing the promising therapeutic potential of a recombinant, EGFR-targeting, ETA′-based IT on RMS cells. We confirmed the selective killing with IC50 values of up to 50 pM, and immunohistochemical staining confirmed the specific ex vivo binding to primary RMS material.

Keywords

Immunotoxin EGFR scFv Rhabdomyosarcoma Pseudomonas exotoxin A 

Notes

Acknowledgments

Christoph Stein was supported by the INTERREG IV A project Microbiomed. We would like to thank Radoslav Mladenov for his help with the tissue sections. We thank Dr. Agnieszka Weinandy (University Hospital Aachen, Neurosurgery Clinic, Aachen, Germany) for providing cetuximab, and we also thank Dr. Richard M. Twyman for the critical reading of the manuscript.

Conflict of interest

None.

Ethical standard

Primary tissue samples were obtained during routine clinical practice at the University Hospital Giessen approved by the appropriate ethics committee, in accordance with the principles and the ethical standards of the Declaration of Helsinki.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Judith Niesen
    • 2
  • Hannes Brehm
    • 1
  • Christoph Stein
    • 1
    • 2
  • Nina Berges
    • 1
  • Alessa Pardo
    • 1
  • Rainer Fischer
    • 2
    • 3
  • Andre ten Haaf
    • 4
  • Stefan Gattenlöhner
    • 4
  • Mehmet K. Tur
    • 4
  • Stefan Barth
    • 1
    • 2
  1. 1.Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical EngineeringUniversity Hospital RWTH AachenAachenGermany
  2. 2.Fraunhofer Institute for Molecular Biology and Applied Ecology IMEAachenGermany
  3. 3.Institute of Molecular Biotechnology (Biology VII)RWTH Aachen UniversityAachenGermany
  4. 4.Department of PathologyJustus-Liebig UniversityGiessenGermany

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