Abstract 2732: Internalization of the dual-specific immunotoxin D2C7-(scdsFv)-PE38KDEL in malignant glioma cell lines
Authors: Hedegaard Chris J. , Pegram Charles N. , Bigner Darell D. , Poulsen Hans S. 
Published: August 2012

Abstract
Aim: The aim of this study is firstly to examine if the D2C7-immunotoxin (D2C7-IT) specifically binds to the epidermal growth factor receptor (EGFRwt) and the EGFR-mutant EGFRvIII subsequently inducing cell death. Secondly, to study the internalization of D2C7-IT after binding to EGFRwt/EGFRvIII. Background: Glioblastoma multiforme (GBM) is a malignant primary brain tumor and from diagnose median survival remains around 15 months. Tumor burden can be decreased by operation but eventually the tumor recurs. Thus, novel treatment-strategies for GBM patients are in great demand that would result prolonged median survival and increased quality of life. While EGFRwt and EGFRvIII are not found in normal brain tissue, expression of these are observed in malignant tissue of GBM, and consequently the D2C7-IT has been designed to target both EGFRwt and EGFRvIII. D2C7-IT comprises single chain disulphide stabilized fragment variables (scdsFv) of the bivalent anti-EGFR/EGFRvIII antibody D2C7, a 38 kDa fragment of the pseudomonas exotoxin A (PE38), and finally a C-terminal ‘endoplasmatic reticulum (ER) retention motif’: KDEL. In theory, the D2C7-IT is internalized after binding to EGFR/EGFRvIII and the PE38 moiety is proteolytically cleaved in the endosomal compartment, which releases the C-terminal toxic part of the PE38 into the cytosol. In the cytosol, binding of the KDEL-receptor assures transport to the ER where the toxic moiety inhibits protein synthesis and causes cell death. Methods: Western blotting was applied for examining D2C7-IT-binding to EGFRwt and EGFRvIII in different human malignant cell lines expressing varying levels of EGFRwt or EGFRvIII. MTT assays were used for assessing the impact of D2C7-IT on cell viability in these cell lines. Results: We observed that D2C7-IT binds to cells expressing EGFRwt or EGFRvIII. In addition, the binding on these cells could be inhibited by co-incubating with either EGF or the D2C7 antibody in surplus. Furthermore, D2C7-IT induced a moderate Tyr1173 phosphorylation (marker for internalization) on EGFRwt expressing cells. Concordantly, EGF induced a high level of EGFR-Tyr1173 phosphorylation on EGFRwt expressing cells, which was reduced to a moderate level when co-incubating with D2C7-IT in surplus. D2C7-IT reduced viability in cell cultures expressing EGFR or EGFRvIII while D2C7-IT had no effect on EGFRwt/EGFRvIII negative cells. We are currently trying to visualize the subcellular localizations of D2C7-IT after internalization by fluorescent microscopy. Conclusion: These preliminary results show that D2C7-IT specifically binds to EGFR and EGFRvIII, and reduces viability of EGFRwt/EGFRvIII expressing cells in vitro.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2732. doi:1538-7445.AM2012-2732