| Summary: | Monoclonal antibodies (mAbs) conjugated to nuclear localization sequences (NLS) and labeled with Auger electron-emitters have great potential for targeted radiotherapy of cancer. This approach may be especially appropriate for the 25-30% of patients with breast cancer whose tumors display overexpression of HER2. Trastuzumab (Herceptin) is a humanized anti-HER2 mAb approved for immunotherapy of HER2-amplified breast cancer. The goal of this research was to radiolabel trastuzumab with [111]In, and to modify it with peptides harboring the NLS (CGYGPKKKRKVGG) of the simian virus 40 large-T antigen for targeted radiotherapy of breast cancer. It was hypothesized that the NLS-peptides would mediate the translocation of covalently linked [111]In-trastuzumab molecules into the nuclei of HER2-overexpressing breast cancer cells where subcellular-range Auger electrons are most damaging to DNA and lethal to cells.
Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate for reaction with NLS-peptides and labeled with [111]In using diethylenetriaminepentaacetic acid. The dissociation constant for binding of [111]In-NLS-trastuzumab to HER2-overexpressing SK-BR-3 human breast cancer cells was reduced < 3-fold compared to [111]In-trastuzumab, demonstrating relatively preserved receptor-binding affinity. The NLS-peptides did not affect the biodistribution of [111]In-trastuzumab, but promoted its nuclear uptake in HER2-overexpressing MDA-MB-361 xenografts. The cytotoxicity of [111]In-NLS-trastuzumab on breast cancer cells correlated with their HER2 expression. Moreover, [111]In-NLS-trastuzumab was 2-fold and 5-fold more potent at killing MDA-MB-361 and SK-BR-3 cells compared to [111]In-trastuzumab, and nearly 3-fold and 6-fold more effective than unlabeled trastuzumab, respectively. Methotrexate is a known radiosensitizer that can amplify the lethal effects of ionizing radiation on tumor cells. Non-cytotoxic, but radiosensitizing doses of methotrexate were therefore combined with [111]In-NLS-trastuzumab; this enhanced the sensitivity of HER2-overexpressing breast cancer cells to [111]In-NLS-trastuzumab. The blood t1/2 of [111]In-NLS-trastuzumab in non-tumor bearing BALB/c mice was 23-34 h when administered intravenously or intraperitoneally. The maximum tolerated dose was 9.2-18.5 MBq; doses >18.5 MBq caused decreased leukocyte and platelet counts. [111]In-NLS-trastuzumab exhibited strong anti-tumor effects against HER2-overexpressing MDA-MB-361 xenografts, reducing their growth rate 2-fold and 3-fold compared to mice administered [111]In-trastuzumab or unlabeled trastuzumab, respectively.
These promising results suggest that [111]In-NLS-trastuzumab may be a useful Auger electron radioimmunotherapeutic agent for HER2-positive breast cancer in humans.
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