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Journal of Cancer Research and Clinical Oncology

, Volume 141, Issue 12, pp 2079–2095 | Cite as

Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities

  • Judith Niesen
  • Christoph Stein
  • Hannes Brehm
  • Grit Hehmann-Titt
  • Rolf Fendel
  • Georg Melmer
  • Rainer Fischer
  • Stefan Barth
Original Article – Cancer Research

Abstract

Purpose

The epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors. EGFR-specific monoclonal antibodies (mAbs), such as cetuximab and panitumumab, have been approved for the treatment of colorectal and head and neck cancer. To increase tissue penetration, we constructed single-chain fragment variable (scFv) antibodies derived from these mAbs and evaluated their potential for targeted cancer therapy. The resulting scFv-based EGFR-specific immunotoxins (ITs) combine target specificity of the full-size mAb with the cell-killing activity of a toxic effector domain, a truncated version of Pseudomonas exotoxin A (ETA′).

Methods

The ITs and corresponding imaging probes were tested in vitro against four solid tumor entities (rhabdomyosarcoma, breast, prostate and pancreatic cancer). Specific binding and internalization of the ITs scFv2112-ETA′ (from cetuximab) and scFv1711-ETA′ (from panitumumab) were demonstrated by flow cytometry and for the scFv-SNAP-tag imaging probes by live cell imaging. Cytotoxic potential of the ITs was analyzed in cell viability and apoptosis assays. Binding of the ITs was proofed ex vivo on rhabdomyosarcoma, prostate and breast cancer formalin-fixed paraffin-embedded biopsies.

Results

Both novel ITs showed significant pro-apoptotic and anti-proliferative effects toward the target cells, achieving IC50 values of 4 pM (high EGFR expression) to 460 pM (moderate EGFR expression). Additionally, rapid internalization and specific in vitro and ex vivo binding on patient tissue were confirmed.

Conclusions

These data demonstrate the potent therapeutic activity of two novel EGFR-specific ETA′-based ITs. Both molecules are promising candidates for further development toward clinical use in the treatment of various solid tumors to supplement the existing therapeutic regimes.

Keywords

Epidermal growth factor receptor (EGFR) Immunotoxin (IT) Single-chain fragment variable (scFv) Pseudomonas exotoxin A (ETA′) SNAP-tag Cancer therapy 

Notes

Acknowledgments

Christoph Stein was supported by the INTERREG IV A project Microbiomed. This work was funded in part by a grant from the German province NRW from EFRE “European Fund for Regional Development” under the theme “Europe—Investment in our Future.” We would like to thank Radoslav Mladenov and Nina Berges (Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical Engineering, RWTH Aachen University Clinic, Aachen, Germany) for their help with immunohistochemistry and confocal microscopy. For obtaining the tissue sections, we want to thank Dr. Mehmet Kemal Tur (Department of Pathology, Justus-Liebig University, Giessen, Germany). We also thank Dr. Richard M. Twyman for critical reading of the manuscript.

Conflict of interest

Georg Melmer is a stakeholder of Pharmedartis GmbH and Grit Hehmann-Titt is employed by Pharmedartis. The other authors declare no conflicts of interest.

Ethical standard

In accordance with the Helsinki Declaration of 1975, primary tissue samples were obtained during routine clinical practice at the University Hospital Giessen approved by the appropriate ethics committee.

Supplementary material

432_2015_1975_MOESM1_ESM.tif (278 kb)
Fig. S1 The scFv2112-SNAP construct labeled with BG-Alexa Fluor® 488 incubated with the EGFR- cell line U937. Hoechst-stained nucleoli are shown in the left image. No unspecific binding of the ITs to U937 cells was detected (middle image). The right picture shows the white light channel. (TIFF 278 kb)
432_2015_1975_MOESM2_ESM.tif (272 kb)
Fig. S2 The scFv-SNAP constructs, scFv2112-SNAP and scFv1711-SNAP, the parental mAbs cetuximab and panitumumab and a non-binding mock-ETA’ construct were used as controls in XTT assays. Unspecific effects were not observed up to the highest starting concentration used for the ITs (80 nM). As before the experiments were carried out at least four times in triplicate or quadruplicate, GraphPad Prism software was used for calculation of potential reduction in protein synthesis. (TIFF 271 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Judith Niesen
    • 1
  • Christoph Stein
    • 1
    • 2
  • Hannes Brehm
    • 2
  • Grit Hehmann-Titt
    • 4
  • Rolf Fendel
    • 1
    • 2
  • Georg Melmer
    • 4
  • Rainer Fischer
    • 1
    • 3
  • Stefan Barth
    • 1
    • 2
  1. 1.Fraunhofer Institute for Molecular Biology and Applied Ecology IMEAachenGermany
  2. 2.Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical EngineeringRWTH Aachen University ClinicAachenGermany
  3. 3.Institute of Molecular Biotechnology (Biology VII)RWTH Aachen UniversityAachenGermany
  4. 4.Pharmedartis GmbHAachenGermany

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