Laboratory of Radiobiology and Experimental Radiooncology, University Medical Centre Hamburg-Eppendorf/Hubertus Wald Cancer Centre, Hamburg, Germany.
J Nucl Med. 2013 Mar;54(3):416-23. doi: 10.2967/jnumed.111.101857. Epub 2013 Jan 23.
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.
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.
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.
(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.
本研究旨在确定使用放射性核素（90）Y 标记的西妥昔单抗进行放射免疫治疗的疗效，并确定诱导和修复 DNA 双链断裂（DSB）在多大程度上对此方法起决定性作用。
本研究使用 9 株头颈鳞状细胞癌（HNSCC）细胞系进行，这些细胞系在表皮生长因子受体（EGFR）表达方面差异很大。放射性核素（90）Y 通过螯合剂反式环已基二乙三胺五乙酸（CHX-A″-DTPA）/连接物构建体与 EGFR 导向抗体西妥昔单抗偶联，得到特定活性约为 1.2GBq/mg 的（90）Y-Y-CHX-A″-DTPA-西妥昔单抗。EGFR 表达通过免疫荧光和 Western blot 进行测定，西妥昔单抗结合通过荧光激活细胞分选分析进行测定，DSB 的数量通过免疫荧光染色γH2AX/53BP1 阳性修复焦点进行测定，细胞存活通过集落形成进行测定。
对于 9 株 HNSCC 细胞系，西妥昔单抗结合与细胞膜上存在的 EGFR 量相关（r²=0.967，P<0.001）。当细胞暴露于（90）Y-Y-CHX-A″-DTPA-西妥昔单抗时，诱导的 DSB 数量随时间呈线性增加（r²=0.968，P=0.016）。发现该数量与膜 EGFR 的量相关（r²=0.877，P=0.006）。大多数 DSB 在 37°C 孵育期间得到修复，但仍有少量残留的 DSB 与膜 EGFR 的量相关（24 小时：r²=0.977，P<0.001；48 小时：r²=0.947，P<0.001）。暴露于（90）Y-Y-CHX-A″-DTPA-西妥昔单抗也导致有效的细胞杀伤，其中细胞杀伤的程度与残留的 DSB 数量密切相关（r²=0.989，P<0.001）和膜 EGFR 的量（r²=0.967，P<0.001）。与强烈表达 EGFR 的 UTSCC14 细胞相比，EGFR 表达低的 UTSCC15 细胞观察到强烈的 86%的减少，而没有观察到细胞杀伤。
（90）Y-Y-CHX-A″-DTPA-西妥昔单抗通过诱导 DSB 影响细胞存活。对于强烈过表达 EGFR 的 HNSCC 细胞，这种治疗方法特别有效，而对于 EGFR 表达水平低的细胞则没有效果。因此，使用（90）Y-Y-CHX-A″-DTPA-西妥昔单抗进行 EGFR 导向放射免疫治疗似乎是一种强大的工具，可以用于灭活具有强烈 EGFR 过表达的肿瘤，这些肿瘤通常表现出明显的放射抗性。
