title: Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities.
abstract: OBJECTIVE
  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.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074, Aachen, Germany. judith.niesen@ime.fraunhofer.de.
authors.forename: Judith
authors.initials: J
authors.lastname: Niesen
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074, Aachen, Germany.|Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical Engineering, RWTH Aachen University Clinic, Aachen, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Stein
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical Engineering, RWTH Aachen University Clinic, Aachen, Germany.
authors.forename: Hannes
authors.initials: H
authors.lastname: Brehm
authors.affiliation: Pharmedartis GmbH, Aachen, Germany.
authors.forename: Grit
authors.initials: G
authors.lastname: Hehmann-Titt
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074, Aachen, Germany.|Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical Engineering, RWTH Aachen University Clinic, Aachen, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Fendel
authors.affiliation: Pharmedartis GmbH, Aachen, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Melmer
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074, Aachen, Germany.|Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074, Aachen, Germany.|Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical Engineering, RWTH Aachen University Clinic, Aachen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001427:Bacterial Toxins / Q000378:metabolism; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D000068818:Cetuximab / Q000494:pharmacology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D005098:Exotoxins / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology; D000922:Immunotoxins / Q000494:pharmacology*; D009369:Neoplasms / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D000077544:Panitumumab; D057127:Single-Chain Antibodies / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D037521:Virulence Factors / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D000068818:Cetuximab; D066246:ErbB Receptors; D005098:Exotoxins; D007155:Immunologic Factors; D000922:Immunotoxins; D000077544:Panitumumab; D057127:Single-Chain Antibodies; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C512478:EGFR protein, human
keywords: Cancer therapy; Epidermal growth factor receptor (EGFR); Immunotoxin (IT); Pseudomonas exotoxin A (ETA′); SNAP-tag; Single-chain fragment variable (scFv)