Inactivation of HNSCC cells by 90Y-labeled cetuximab strictly depends on the number of induced DNA double-strand breaks.
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-triamine-pentaacetic 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 γH2AX/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) = 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) = 0.968, P = 0.016). This number was found to correlate with the amount of membranous EGFR (r(2) = 0.877, P = 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) = 0.977, P < 0.001; 48 h: r(2) = 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) = 0.989, P < 0.001) and with the amount of membranous EGFR (r(2) = 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.


CONCLUSIONS
(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.
Laboratory of Radiobiology and Experimental Radiooncology, University Medical Centre Hamburg-Eppendorf/Hubertus Wald Cancer Centre, Hamburg, Germany.
Jarob
J
Saker
Malte
M
Kriegs
Martin
M
Zenker
Jan-Martin
JM
Heldt
Iris
I
Eke
Hans-Jürgen
HJ
Pietzsch
Reidar
R
Grénman
Nils
N
Cordes
Cordula
C
Petersen
Michael
M
Baumann
Jörg
J
Steinbach
Ekkehard
E
Dikomey
Ulla
U
Kasten-Pisula
D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology / Q000532:radiotherapy*; D016923:Cell Death / Q000528:radiation effects; D045744:Cell Line, Tumor; D000068818:Cetuximab; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006258:Head and Neck Neoplasms / Q000378:metabolism / Q000473:pathology / Q000532:radiotherapy*; D006801:Humans; D018796:Immunoconjugates / Q000627:therapeutic use*; D034741:RNA, Small Interfering / Q000235:genetics; D011836:Radiation Tolerance; D016499:Radioimmunotherapy; D015021:Yttrium Radioisotopes / Q000627:therapeutic use*
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D018796:Immunoconjugates; D034741:RNA, Small Interfering; D015021:Yttrium Radioisotopes; D066246:ErbB Receptors; D000068818:Cetuximab