title: In vitro effects and ex vivo binding of an EGFR-specific immunotoxin on rhabdomyosarcoma cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, 52074, Aachen, Germany.
authors.forename: Judith
authors.initials: J
authors.lastname: Niesen
authors.forename: Hannes
authors.initials: H
authors.lastname: Brehm
authors.forename: Christoph
authors.initials: C
authors.lastname: Stein
authors.forename: Nina
authors.initials: N
authors.lastname: Berges
authors.forename: Alessa
authors.initials: A
authors.lastname: Pardo
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Andre
authors.initials: A
authors.lastname: Ten Haaf
authors.forename: Stefan
authors.initials: S
authors.lastname: Gattenlöhner
authors.forename: Mehmet K
authors.initials: MK
authors.lastname: Tur
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D066246:ErbB Receptors / Q000378:metabolism*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D007167:Immunotherapy / Q000379:methods*; D000922:Immunotoxins / Q000494:pharmacology*; D066298:In Vitro Techniques; D018613:Microscopy, Confocal; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012208:Rhabdomyosarcoma / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D018232:Rhabdomyosarcoma, Alveolar / Q000188:drug therapy / Q000473:pathology; D018233:Rhabdomyosarcoma, Embryonal / Q000188:drug therapy / Q000473:pathology; D016896:Treatment Outcome; D015854:Up-Regulation
chemical_list: D000922:Immunotoxins; D011994:Recombinant Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors