A comparison of non-biologically active truncated EGF (EGFt) and full-length hEGF for delivery of Auger electron-emitting 111In to EGFR-positive breast cancer cells and tumor xenografts in athymic mice

Abstract

Introduction: EGFt is a truncated form of human epidermal growth factor (hEGF) that is non-biologically active but retains binding and internalization into EGFR-positive cells. Our aim was to compare EGFt and hEGF for delivery of (111)In to human breast cancer (BC) cells and tumors and evaluate its cytotoxicity against EGFR-positive BC cells, mediated by the Auger electron emissions of (111)In.

Methods: The binding, internalization and nuclear localization of EGFt and hEGF in MDA-MB-468 human BC cells were first assessed by confocal fluorescence microscopy. Subcellular fractionation was then used to quantify the cellular and nuclear uptake of (111)In-EGFt and (111)In-hEGF in MDA-MB-468 cells. The effect of exposure in vitro to (111)In-EGFt or (111)In-hEGF on the clonogenic survival of MDA-MB-468 (10(6) EGFR/cell) or MCF-7 cells (10(4) EGFR/cell) was determined. The pharmacokinetics and tumor and normal tissue biodistribution of (111)In-EGFt was compared to (111)In-hEGF in CD-1 athymic mice with s.c. MDA-MB-468 and MCF-7 tumors. Nuclear importation in MDA-MB-468 tumors was determined ex vivo by subcellular fractionation.

Results: Fluorescently-labeled EGFt and hEGF were bound, internalized and localized in the nucleus of MDA-MB-468 cells. Binding of (111)In-EGFt to MDA-MB-468 cells was 8-fold lower than (111)In-hEGF, but nuclear importation as a proportion of cell-bound (111)In was 3.6-fold greater than (111)In-hEGF. Nuclear uptake of (111)In-EGFt was lower than (111)In-hEGF when differences in cell binding were taken into account. The cytotoxicity of (111)In-EGFt (1.0MBq/mL; 10 nmols/L) against MDA-MB-468 cells was 9-fold lower than (111)In-hEGF but only 2-fold lower at a higher concentration (1.85 MBq/mL; 40 nmols/L). (111)In-EGFt and (111)In-hEGF exhibited greater cytotoxicity against MDA-MB-468 cells than MCF-7 cells. (111)In-EGFt was eliminated more slowly from the blood of tumor-bearing mice and exhibited lower liver uptake but higher kidney accumulation. Uptake of (111)In-EGFt in MDA-MB-468 tumors was 2.2-fold lower than (111)In-hEGF, and was blocked by anti-EGFR monoclonal antibody, nimotuzumab. Nuclear uptake into MDA-MB-468 tumor cells was higher for (111)In-EGFt than (111)In-hEGF, but when the lower tumor uptake of (111)In-EGFt was considered, there were no overall differences.

Conclusion: We conclude that the absence of biological activity of EGFt makes it attractive for delivery of Auger electron-emitting (111)In to EGFR-overexpressing BC, but its lower cellular and tumor uptake would limit its effectiveness compared to (111)In-hEGF.

Advances in knowledge and implications for patient care: (111)In-EGFt may reduce the adverse effects previously observed in patients administered (111)In-hEGF since it is not biologically active, but its lower uptake by BC cells and tumors would limit its effectiveness for treatment of breast cancer.

Keywords: Breast cancer; EGF; EGFR; Indium-111; Radiopharmaceuticals; Theranostics.