111In- or 99mTc-labeled recombinant VEGF bioconjugates: in vitro evaluation of their cytotoxicity on porcine aortic endothelial cells overexpressing Flt-1 receptors

Introduction: The aims of this study were to (a) synthesize and characterize a novel vascular endothelial growth factor (VEGF-2K) recombinant protein expressed in Pichia pastoris and (b) compare its cytotoxicity when labeled with the Auger electron emitter 111In or 99mTc, both of which are in the nanometer-micrometer range, toward porcine aortic endothelial (PAE) cells transfected with the flt-1 gene to overexpress Flt-1 receptors (PAE-Flt-1). Methods: The gene for the VEGF165 isoform was fused to a sequence encoding an extended flexible peptide (KGGGGSK) with two accessible lysines for preferential derivatization with diethylenetriaminepentaacetic acid (DTPA) for complexing 111In and a sequence for a His6 affinity tag that bound the [99mTc(CO)3(H2O)3]+ tricarbonyl complex. P. pastoris strain KM71H was transfected with the recombinant gene, the VEGF-2K protein expressed with methanol induction, and then purified by metal-affinity chromatography. VEGF-2K was modified with 13-mer peptides [CGYGPKKKRKVGG] containing the nuclear localization sequence (NLS) of SV-40 large T-antigen (underlined) to promote nuclear uptake following its receptor-mediated internalization. Results: 99mTc-DTPA-VEGF-2K bound strongly and preferentially to PAE-Flt-1 cells compared with non-transfected PAE cells, but NLS modification diminished the ratio of PAE-Flt-1 to PAE binding to 2.3-fold. Nuclear accumulation of 99mTc-labeled DTPA-VEGF-2K was not enhanced by NLS modification but was enhanced by 1.5-fold for 111In-DTPA-VEGF-2K-NLS. However, confocal microscopy revealed intranuclear distribution of DTPA-VEGF-2K-NLS, whereas DTPA-VEGF-2K distribution was mainly perinuclear. 111In-DTPA-VEGF-2K-NLS was the most cytotoxic to PAE-Flt-1 cells, reducing their clonogenic survival by 4-fold. 111In-DTPA-VEGF-2K, 99mTc-DTPA-VEGF-2K or 99mTc-DTPA-VEGF-2K-NLS had less effect on the clonogenic survival of PAE-Flt-1 or PAE cells. The strong cytotoxicity of 111In-DTPA-VEGF-2K-NLS toward PAE-Flt-1 cells was associated with a 27-fold increase in nuclear foci of immunofluorescence for phosphorylated histone-2AX corresponding to sites of unrepaired DNA double-strand breaks. Monte Carlo modeling revealed that radionuclide decay in the nucleus would provide a 5-fold higher radiation absorbed dose for 111In than for 99mTc, explaining their differential cytotoxicity, and intranuclear localization would amplify the radiation dose delivered by 111In by 3-fold, explaining the greater potency of 111In-DTPA-VEGF-2K-NLS compared with 111In-DTPA-VEGF-2K. Conclusions: We conclude that targeted Auger electron radiotherapy aimed at Flt-1 receptors is a promising strategy that should be explored further for treatment of tumors in which this angiogenic pathway is up-regulated. 111In is a more cytotoxic radionuclide than 99mTc, unless DNA delivery can be achieved, due to the short range of the electrons emitted.