gH2AX: a target for imaging and systemic radiation therapy

Bart Cornelissen, Kate Sleeth, Jody Mitchell, Sonali Darbar, Veerle Kersemans, Sean Smart, Katherine Vallis

Gray Institute for Radiation Oncology and Biology, University of Oxford, UK

Proffered paper presentation

Objectives
The efficacy of most anti-cancer treatments, including ionizing radiation (IR) and many cytotoxic drugs, depends on an ability to cause DNA damage. It follows that quantification of intra-tumoural DNA damage could be used to predict early response to treatment. Also, radiopharmaceuticals that target components of the DNA repair machinery could amplify damage and enhance the cytotoxicity of conventional anti-cancer agents. The goal in this study was to design, synthesise and test Auger electron-emitting radiopharmaceuticals that target the DNA repair protein, γH2AX, which accumulates at DNA double-strand breaks (dsb).

Methods
¹¹¹In-DTPA- and Cy3- anti-γH2AX-TAT were synthesized using EDC/NHS chemistry for TAT-peptide linkage. TAT-peptide confers cell-penetrating and nuclear localisation properties. Nuclear localization of Cy3-anti-γH2AX-TAT was studied in irradiated breast cancer cells using confocal microscopy. Internalization and retention assays were performed on a panel of irradiated 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 and the formation of dsb. SPECT and optical imaging were performed on mice bearing MDA-MB-468 breast cancer xenografts following administration of γH2AX-targeted radiopharmaceutical and delivery of X-irradiation (4Gy) to tumour.

Results
Cy3-anti-γH2AX-TAT co-localized with γH2AX foci in irradiated cells. Retention of ¹¹¹In-DTPA-anti-γH2AX-TAT in cells increased following IR (4Gy) compared to non-irradiated controls (80 vs. 20 % retention at 4 h, respectively). Retention of ¹¹¹In-DTPA-mouseIgG-TAT was similar to that of ¹¹¹In-DTPA-anti-γH2AX-TAT in control cells but did not increase following IR. Optical imaging demonstrated retention of Cy3-anti-γH2AX-TAT in irradiated bur not in control non-irradiated tumour. Cy3-mouseIgG-TAT did not accumulate in tumour. Similar results were obtained using ¹¹¹In-DTPA-anti-γH2AX-TAT and SPECT imaging. ¹¹¹In-DTPA-anti-γH2AX-TAT (but not ¹¹¹In-DTPA-mouseIgG-TAT) caused decreased clonogenic survival of cells exposed to IR. In comet assays, ¹¹¹In-DTPA-anti-γH2AX-TAT resulted in the conversion of dsb to complex lesions in cells that were exposed to IR.

Conclusions
¹¹¹In-DTPA-anti-γH2AX-TAT can be used to image and amplify dsb in cancer cells.