Abstract
Abstract Objective: Radioimmunotherapy has an advantage in treating tumors scattered in the body. Auger electrons have high cell killing effect because of its high LET, and the short range can prevent damages to normal tissue. However, to achieve higher therapeutic effect, it is desirable to deliver Auger electrons into nucleus and locate close to DNA. The use of nuclear localizing signal (NLS) has been proposed to transport Auger electron emitters into nucleus. To evaluate the cytotoxicity of nuclear localized Auger electron emitter, we performed in-vitro assay using 111In-labeled anti-HER2 antibody attached with NLS peptides to human breast cancer cell lines overexpressing HER2. Material and methods: Trastuzumab, a monoclonal antibody to HER2, was conjugated with CHX-A”-DTPA (DTPA) and derivatized with sulfosuccinimdyl-4-(N-maleimidomethyl) cyclohexane-1carboxylate (sulfo-SMCC) for reaction with synthetic 13-mer NLS peptides (CGYGPKKKRKVGG). 111In was labeled with either trastuzumab-DTPA or NLS-trastuzumab-DTPA, NLS-trastuzumab-DTPA-S carried approximately 4 NLS peptides per antibody and NLS-trastuzumab-DTPA-L carried 10. The radiochemical labeling of 111In-NLS-trastuzumab and 111In-trastuzumab was determined by HPLC. Human mammary epithelial cells HMEpC and two types of human breast cancer cells with low and high HER2 expression, MCF-7 and SKBR3, respectively, were employed for in-vitro study. Nuclear accumulation of 111In was measured in SKBR3 cells by subcellular fraction. Cell viability was evaluated with dye-based cell viability assays in all cell lines. DNA damages were examined with γ-H2AX immunohistochemistry in SKBR3 cells. Results: Nuclear uptake of 111In was 1.3-1.5-fold higher in SKBR3 cells treated with 111In-NLS-trastuzumab than 111In-trastuzumab and increased depending on the number of NLS peptides. Cell viability was significantly reduced with 111In-NLS-trastuzumab comparing 111In-trastuzumab in SKBR3 cells. Both of 111In-NLS-trastuzumab had little or no cytotoxicity on MCF-7 cells and HMEpC cells. SKBR3 cells treated with 111In-NLS-trastuzumab-L showed more γ-H2AX foci than 111In-trastuzumab. Conclusions: The cytotoxicity of 111In-NLS-trastuzumab was higher than that of 111In-trastuzumab by inducing more DNA damages in HER2-overexpressing human breast cancer cells. These data demonstrate that delivery of 111In into cell nucleus by anti-HER2 antibody harboring NLS is an effective strategy for targeted cell killing of the human cancer cells overexpressing HER2. Citation Format: Huizi Li, Sumitaka Hasegawa, Tadashi Kamada, Tsuneo Saga. Effective auger electron radioimmunotherapy using modified anti-HER2 antibody with nuclear localizing signal. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1814. doi:10.1158/1538-7445.AM2015-1814
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