Inactivation of HNSCC Cells by ⁹⁰Y-Labeled Cetuximab Strictly Depends on the Number of Induced DNA Double-Strand Breaks

Abstract

Radioimmunotherapy is considered to have great potential for efficient and highly specific treatment of tumors. The aim of this study was to determine the efficacy of radioimmunotherapy when using 90 Y-labeled cetuximab and to determine to what degree induction and repair of DNA double-strand breaks (DSBs) are decisive for this approach. Methods: This study was performed with 9 cell lines of squamous cell carcinoma of the head and neck (HNSCC) differing strongly in epidermal growth factor receptor (EGFR) expression. The radionuclide 90 Y was coupled by the chelator trans-cyclohexyl-diethylene-triaminepentaacetic acid (CHX-A$-DTPA)/linker construct to the EGFR-directed antibody cetuximab to yield 90 Y-Y-CHX-A$-DTPA-cetuximab with a specific activity of approximately 1.2 GBq/mg. EGFR expression was determined by immunofluorescence and Western blotting, cetuximab binding by fluorescence-activated cell sorter analysis, the number of DSBs by immunofluorescence staining gH2AX/53BP1-positive repair foci, and cell survival by colony formation. Results: For the 9 HNSCC cell lines, cetuximab binding correlated with the amount of EGFR present in the cell membrane (r 2 5 0.967, P , 0.001). When cells were exposed to 90 Y-Y-CHX-A$-DTPA-cetuximab, the number of induced DSBs increased linearly with time (r 2 5 0.968, P 5 0.016). This number was found to correlate with the amount of membranous EGFR (r 2 5 0.877, P 5 0.006). Most DSBs were repaired during incubation at 37°C, but the small number of remaining DSBs still correlated with the amount of membranous EGFR (24 h: r 2 5 0.977, P , 0.001; 48 h: r 2 5 0.947, P , 0.001). Exposure to 90 Y-Y-CHX-A$-DTPA-cetuximab also resulted in efficient cell killing, whereby the extent of cell killing correlated strongly with the respective number of remaining DSBs (r 2 5 0.989, P , 0.001) and with the amount of membranous EGFR (r 2 5 0.967, P , 0.001). No cell killing was observed for UTSCC15 cells with low EGFR expression, in contrast to the strong reduction of 86% measured for UTSCC14 cells showing a strong overexpression of EGFR. Conclusion: 90 Y-Y-CHX-A$-DTPA-cetuximab affected cell survival through the induction of DSBs. This treatment was especially efficient for HNSCC cells strongly overexpressing EGFR, whereas no effect was seen for cells with low levels of EGFR expression. Therefore, EGFR-directed radioimmunotherapy using 90 Y-Y-CHX-A$-DTPA-cetuximab appears to be a powerful tool that can be used to inactivate tumors with strong EGFR overexpression, which are often characterized by a pronounced radioresistance.

Molecular Radiotherapy Using Cleavable Radioimmunoconjugates That Target EGFR and γH2AX

“…This makes their use distinctly different from previously reported EGFR-targeting radioimmunotherapeutics, which mainly incorporate alpha-or beta-emitting radionuclides. The use of 177 Lu-, 90 Y-, and 231 Bi-labeled anti-EGFR antibodies has shown great promise for radioimmunotherapy (25)(26)(27)(28). However, because these agents were investigated using different cell models and given the unique radiobiologic characteristics of Auger electron emitters (29), a direct comparison with the method reported here is not straightforward.…”

Radiolabeled Cetuximab Conjugates for EGFR Targeted Cancer Diagnostics and Therapy

“…Unlabeled cetuximab caused a radiosensitizing effect [171,172] in one out of three cell lines [119]. Recently, an EGFR expression depending number of DNA double strand breaks (DSB) caused by 90 Y-CHX-A′′-DTPA was demonstrated in vitro [173]. Furthermore, also in different breast cancer cells, which were sensitized with an inhibitor of DNA-dependent-protein-kinase, DNA DSB have been assessed after treatment of 213 Bi-labeled cetuximab [135].…”

Combined external beam radiotherapy with carbon ions and tumor targeting endoradiotherapy

“…Therefore, dual modality therapy consisting of anti-EGFR and radiotherapy was rationally designed utilizing Cetuximab, a chimeric IgG1 antibody, and this was the first combination of targeted therapy with EBRT to receive FDA-approval for the treatment of locally advanced HNSCC [ 18 ]. Based on this success different approaches for development of anti-EGFR based theragnostics was studied using different antibodies, chelators and radionuclide combinations for radioimmunotherapy of EGFR expressing tumor entities [ 19 – 26 ]. Further, preclinical rationales for combined anti-EGFR EndoRT with EBRT were developed [ 13 , 27 – 29 ].…”