In vitro effects and ex vivo binding of an EGFR-specific immunotoxin on rhabdomyosarcoma cells
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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.
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.
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.
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.
KeywordsImmunotoxin EGFR scFv Rhabdomyosarcoma Pseudomonas exotoxin A
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
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.
- Barth S (2002) Technology evaluation: BL22. NCI current opinion in molecular therapeutics 4:72–75Google Scholar
- Brehm H et al (2014) A CSPG4-specific immunotoxin kills rhabdomyosarcoma cells and binds to primary tumor tissues Cancer Lett. doi: 10.1016/j.canlet.2014.07.006
- Cizeau J, Grenkow DM, Brown JG, Entwistle J, MacDonald GC (2009) Engineering and biological characterization of VB6-845, an anti-EpCAM immunotoxin containing a T-cell epitope-depleted variant of the plant toxin bouganin. J Immunother 32:574–584. doi: 10.1097/CJI.0b013e3181a6981c CrossRefPubMedGoogle Scholar
- Gattenlohner S et al (2010) A human recombinant autoantibody-based immunotoxin specific for the fetal acetylcholine receptor inhibits rhabdomyosarcoma growth in vitro and in a murine transplantation model. J Biomed Biotechnol 2010:187621. doi: 10.1155/2010/187621 CrossRefPubMedCentralPubMedGoogle Scholar
- Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- Jain S, Xu R, Prieto VG, Lee P (2010) Molecular classification of soft tissue sarcomas and its clinical applications Int J. Clin Exp Pathol 3:416–428Google Scholar
- Kampmeier F, Ribbert M, Nachreiner T, Dembski S, Beaufils F, Brecht A, Barth S (2009) Site-specific, covalent labeling of recombinant antibody fragments via fusion to an engineered version of 6-O-alkylguanine DNA alkyltransferase. Bioconjug Chem 20:1010–1015. doi: 10.1021/bc9000257 CrossRefPubMedGoogle Scholar
- Sumegi J, Streblow R, Frayer RW, Dal Cin P, Rosenberg A, Meloni-Ehrig A, Bridge JA (2010) Recurrent t(2;2) and t(2;8) translocations in rhabdomyosarcoma without the canonical PAX-FOXO1 fuse PAX3 to members of the nuclear receptor transcriptional coactivator family. Genes Chromosom Cancer 49:224–236. doi: 10.1002/gcc.20731 PubMedCentralPubMedGoogle Scholar