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

IntroductionThe 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).MethodsThe 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.Results99mTc-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.ConclusionsWe 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.