Simultaneous Inhibition of Epidermal Growth Factor Receptor (EGFR) Signaling and Enhanced Activation of Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Receptor-mediated Apoptosis Induction by an scFv:sTRAIL Fusion Protein with Specificity for Human EGFR

Wijnand Helfrich,Lou F.M.H. de Leij,Edwin Bremer,Marike H. Dijkstra,Linda van Genne,Douwe F. Samplonius,Bart Jan Kroesen

doi:10.1074/jbc.m413673200

Abstract

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.

Highlights

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction
Strong binding of scFv425:soluble TRAIL (sTRAIL) was detected on the cell surface (Fig. 1A, solid line) that could be inhibited by preincubation with parental EGFR-blocking mAb 425 (Fig. 1A, dashed line)
Binding of scFv425:sTRAIL to cell surface-expressed EGFR results in the reciprocal activation of agonistic Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors in a paracrine fashion, whereby apoptotic activity can be directed toward neighboring tumor cells that are devoid of EGFR expression

Summary

Cell Lines

The following cell lines were purchased from the ATTC (Manassas, VA): Jurkat (ALL T-cell line), A431 (epidermoid vulva carcinoma), A172, Hs683 (glioblastoma), SW948, and WiDr (colon carcinoma). Cell line Jurkat.EGFRvIII was generated by electroporation of Jurkat cells with plasmid pH␤Apr-1-neo/EGFRvIII Duke University Medical Center, NC), after which transfectants were selected by G418 selection (500 ␮g/ml, Invitrogen). Cell lines were cultured at 37 °C in a humidified 5% CO2 atmosphere. SW948, and WiDr cells were cultured in RPMI 1640 (Cambrex Bio Science, Verviers, France) supplemented with 15% fetal calf serum. A431, HS683, and A172 cells were cultured in Dulbecco’s modified Eagle’s medium, 10% fetal calf serum, and 4 mM L-glutamine (Cambrex Bio Science)

Monoclonal Antibodies and Inhibitors

Apoptosis Assessed by Viability Assay

Immunoblot Analysis

RESULTS

DISCUSSION