Amplification of IR-induced DNA damage by Auger electron treatment with TAT-radioimmunoconjugates

Objectives The most deleterious DNA lesions caused by ionizing radiation are double strand breaks (DSB). We hypothesise that it is possible to amplify DSB damage using a novel Auger electron-emitting radiopharmaceutical, ¹¹¹In-DTPA-anti-γH2AX-TAT, which targets γH2AX foci at sites of DSB. TAT-peptide conjugation confers cell-penetrating and nuclear localisation properties to the radioimmunoconjugate.

Methods ¹¹¹In-DTPA-anti-γH2AX-TAT was synthesised using EDC/NHS chemistry for TAT-peptide linkage and cDTPA coupling for ¹¹¹In chelation. Internalisation and retention assays were performed on a panel of irradiated (up to 10Gy) and non-irradiated breast cancer cells, using ¹¹¹In-DTPA-mouseIgG-TAT as a non-specific control. Clonogenic survival and neutral comet assays were performed to study amplification of cell kill.

Results TAT:IgG ratio was 5:1, DTPA:IgG ratio was 1.5:1. Radiochemical yield was 89% and radiochemical purity was >97%. The retention of ¹¹¹In-DTPA-anti-γH2AX-TAT in MDA-MB-468 cells was increased following IR (4 Gy) compared to non-irradiated controls (80 vs. 20 % retention at 4 h, respectively). The retention of ¹¹¹In-DTPA-mouseIgG-TAT was similar to that of ¹¹¹In-DTPA-anti-γH2AX-TAT in control cells but was unaffected by IR. ¹¹¹In-DTPA-anti-γH2AX-TAT but not ¹¹¹In-DTPA-mouseIgG-TAT caused decreased clonogenic survival of cells exposed to IR. Both ¹¹¹In-DTPA-anti-γH2AX-TAT and ¹¹¹In-DTPA-mouseIgG-TAT alone caused minimal cell kill. Comet assays indicated augmentation of DNA damage in cells exposed to IR plus ¹¹¹In-DTPA-anti-γH2AX-TAT.

Conclusions ¹¹¹In-DTPA-anti-γH2AX-TAT results in amplification of DNA DSB damage in cancer cells.