Molecular Targeting of Cytotoxic Radiosensitizing Chemotherapies

Abstract

Resistance to radiotherapy is a significant barrier to improving outcomes of patients diagnosed with locally advanced cancers. The clinical utility of more potent radiosensitizers is curtailed by dose limiting toxicity. To provide a solution to these issues, we are developing antibody drug conjugates (ADC) to selectively deliver potent radiosensitizers to tumors based on cell surface receptor expression. We are testing the hypothesis that the therapeutic window of cetuximab, trastuzumab, and pertuzumab can be widened, by re-purposing their therapeutic task. Instead of being utilized as inhibitors of ErbB signaling for which resistance emerges from bypass signaling, we propose they be used as receptor targeted delivery vehicles for potent radiosensitizers. We synthesized theranostic ADC by conjugating ErbB antibodies to the potent anti-tubulin drug monomethyl auristatin E to create cetuximab-MMAE (C-MMAE), trastuzumab-MMAE (T-MMAE) and pertuzumab-MMAE (P-MMAE). Each ADC was labeled with Cy5 to allow for non-invasive monitoring in cell culture and murine models. Experiments were done in EGFR+ and HER2+ tumor cell lines of various histologies. Binding, functionality and toxicity of each ADC was measured by flow cytometry, confocal microscopy, cell cycle analysis and alamar blue assay. Radiosensitization was tested by neutral comet assays and clonogenic survival. For in vivo studies, tumor xenografts were grown subcutaneously in nude mice to > 100 mm3. ADC (0.25 nmoles) was given intravenously followed by focal IR (3 Gy x 3). Fluorescent imaging for Cy5 labeled ADC allowed for evaluation of tumor targeting and therapeutic efficacy was determined by tumor volume response. Statistical analysis was performed by ANOVA. In cell culture, the specificity/functionality of C-MMAE, T-MMAE, and P-MMAE was first tested. C-MMAE specifically bound to EGFR+ tumor cell lines (CAL-27, SCC-61, and A-549) and resulted in MMAE mediated G2/M arrest at 0.5-5 nM. In contrast, T-MMAE and P-MMAE bound selectively to HER2+ cells lines (OE-19, CALU-3, and BT-474). Importantly, while free MMAE decreased survival of all cell lines indiscriminately, ErbB antibody conjugation restricted MMAE toxicity and radiosensitization to EGFR+ cells or HER2+ cells in the 0.5 – 5 nM range, P < 0.05. At 20 nM, antibody alone was not tumoricidal and did radiosensitize EGFR+ or HER2+ tumor cells. In vivo, Cy5 imaging demonstrated C-MMAE localized to EGFR+ tumor xenografts (CAL27, SCC-61) while T-MMAE selectively accumulated in HER2+ OE19 tumor xenografts. Therapeutically, C-MMAE or T-MMAE and fractionated IR produced significant tumor growth delay when compared to IR and antibody alone, P < 0.01. Our studies demonstrate a potent ADC radiosensitization approach that is biomarker-driven and histology agnostic. We propose an alternative paradigm for ErbB radiosensitization, using tumor cell surface receptors as beacons for drug delivery, instead of as targets for signal inhibition.