DNA damage responses (DDR) occur during oncogenesis and therapeutic responses to DNA damaging cytotoxic drugs. Thus, a real-time method to image DNA damage in vivo would be useful to diagnose cancer and monitor its treatment. Toward this end, we have developed fluorophore-and radioisotope-labeled immunoconjugates to target a DDR signaling protein, phosphorylated histone H2A variant H2AX (gH2AX), which forms foci at sites of DNA double-strand breaks. Anti-gH2AX antibodies were modified by the addition of diethylenetriaminepentaacetic acid (DTPA) to allow 111 In labeling or the fluorophore Cy3. The cell-penetrating peptide Tat (GRKKRRQRRRPPQGYG) was also added to the immunoconjugate to aid nuclear translocation. In irradiated breast cancer cells, confocal microscopy confirmed the expected colocalization of anti-gH2AX-Tat with gH2AX foci. In comparison with nonspecific antibody conjugates, 111 In-anti-gH2AX-Tat was retained longer in cells.Anti-gH2AX-Tat probes were also used to track in vivo DNA damage, using a mouse xenograft model of human breast cancer. After local X-ray irradiation or bleomycin treatment, the anti-gH2AX-Tat probes produced fluorescent and single photon emission computed tomography signals in the tumors that were proportionate to the delivered radiation dose and the amount of gH2AX present. Taken together, our findings establish the use of radioimmunoconjugates that target gH2AX as a noninvasive imaging method to monitor DNA damage, with many potential applications in preclinical and clinical settings. Cancer Res; 71(13); 4539-49. Ó2011 AACR.

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The intracellular distribution of Auger electron-emitting radiopharmaceuticals is a determinant of cytotoxicity. However, the mechanisms by which these agents are routed through the cell are ill understood. The aim of this study was to investigate how trafficking of 111 In-labeled human epidermal growth factor ( 111 In-DTPA-hEGF) relates to that of the EGF receptor (EGFR) and whether coadministration of agents that modulate EGFR signaling alters the efficacy of 111 In-DTPA-hEGF. Methods: The spatiotemporal interaction between AlexaFluor488-EGF (AF488-EGF) and Cy3-conjugated anti-EGFR antibody (Cy3-anti-EGFR) was studied in the breast cancer cell line MDA-MB-468 using fluorescence resonance energy transfer and 2-photon fluorescence lifetime imaging. 111 In internalization and nuclear fractionation assays were performed to investigate the effect of the ErbB-2-blocking antibody trastuzumab and a prenyltransferase inhibitor, L-778,123, on the subcellular localization of 111 In-DTPA-hEGF in MDA-MB-468 (1.3 · 10 6 EGFR per cell; ErbB-2 negative) and 231-H2N (0.2 · 10 6 EGFR per cell; 0.4 · 10 5 ErbB-2 per cell) cell lines. The cytotoxicity of 111 In-DTPA-hEGF (0-64 nM) plus trastuzumab (0-50 mg/mL) or L-778,123 (0-22.5 mM) was measured using clonogenic assays in a panel of breast cancer cell lines that express different levels of EGFR and ErB-2. Clonogenic survival data were used to calculate combination indices. Tumor growth inhibition was measured in vivo in 231-H2N xenograft-bearing mice treated with 111 In-DTPA-hEGF plus trastuzumab or L-788,123. Results: Using fluorescence resonance energy transfer, we showed that EGF interacts with EGFR in the cytoplasm and nucleus after internalization of the ligand-receptor complex in MDA-MB-468 cells. Nuclear localization of 111 In-DTPA-hEGF is enhanced by trastuzumab and L-788,123. Trastuzumab and L-788,123 sensitized 231-H2N cells to 111 In-DTPA-hEGF. Nuclear localization and cytotoxicity of 111 In-DTPA-hEGF were significantly increased in 231-H2N xenografts by cotreatment with L-788,123 (P , 0.0001). Conclusion: The therapeutic efficacy of 111 In-DTPA-hEGF is increased through the coadministration of selected molecularly targeted drugs that modulate EGFR signaling and trafficking.