Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities
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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′).
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.
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.
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.
KeywordsEpidermal growth factor receptor (EGFR) Immunotoxin (IT) Single-chain fragment variable (scFv) Pseudomonas exotoxin A (ETA′) SNAP-tag Cancer therapy
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.
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.
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