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Trastuzumab-Resistant Breast Cancer Cells Remain Sensitive to the Auger Electron–Emitting Radiotherapeutic Agent ¹¹¹In-NLS-Trastuzumab and Are Radiosensitized by Methotrexate

¹ Leslie Dan Faculty of Pharmacy, Department of Pharmaceutical Sciences, University of Toronto, Ontario, Canada; ² Department of Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom; ³ Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada; and ⁴ Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada

Correspondence: For correspondence or reprints contact: Raymond M. Reilly, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, Ontario M5S 3M2, Canada. E-mail: raymond.reilly{at}utoronto.ca

Our goals in this study were to determine whether ¹¹¹In-trastuzumab coupled to peptides harboring nuclear localizing sequences (NLSs) could kill trastuzumab-resistant breast cancer cell lines through the emission of Auger electrons and whether the combination of radiosensitization with methotrexate (MTX) would augment the cytotoxicity of this radiopharmaceutical. Methods: Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate for reaction with NLS peptides and then conjugated with diethylenetriaminepentaacetic acid for labeling with ¹¹¹In. HER2 expression was determined by Western blot and by radioligand binding assay using ¹¹¹In-trastuzumab in a panel of breast cancer cell lines, including SK-BR-3, MDA-MB-231 and its HER2-transfected subclone (231-H2N), and 2 trastuzumab-resistant variants (TrR1 and TrR2). Nuclear importation of ¹¹¹In-NLS-trastuzumab and ¹¹¹In-trastuzumab in breast cancer cells was measured by subcellular fractionation, and the clonogenic survival of these cells was determined after incubation with ¹¹¹In-NLS-trastuzumab, ¹¹¹In-trastuzumab, or trastuzumab (combined with or without MTX). Survival curves were analyzed according to the dose-response model, and the radiation-enhancement ratio was calculated from the survival curve parameters. Results: The expression of HER2 was highest in SK-BR-3 cells (12.6 x 10⁵ receptors/cell), compared with 231-H2N and TrR1 cells (6.1 x 10⁵ and 5.1 x 10⁵ receptors/cell, respectively), and lowest in MDA-MB-231 and TrR2 cells (0.4 x 10⁵ and 0.6 x 10⁵ receptors/cell, respectively). NLS peptides increased the nuclear uptake of ¹¹¹In-trastuzumab in MDA-MB-231, 231-H2N, TrR1, and TrR2 cells from 0.1% ± 0.01%, 2.5% ± 0.2%, 2.8% ± 0.7%, and 0.5% ± 0.1% to 0.5% ± 0.1%, 4.6% ± 0.1%, 5.2% ± 0.6%, and 1.5% ± 0.2%, respectively. The cytotoxicity of ¹¹¹In-NLS-trastuzumab on breast cancer cells was directly correlated with the HER2 expression densities of the cells. On a molar concentration basis, the effective concentration required to kill 50% of 231-H2N and TrR1 cells for ¹¹¹In-NLS-trastuzumab was 9- to 12-fold lower than for ¹¹¹In-trastuzumab and 16- to 77-fold lower than for trastuzumab. MDA-MB-231 and TrR2 cells were less sensitive to ¹¹¹In-NLS-trastuzumab or ¹¹¹In-trastuzumab, and both cell lines were completely insensitive to trastuzumab. The radiation-enhancement ratio induced by MTX for 231-H2N and TrR1 cells after exposure to ¹¹¹In-NLS-trastuzumab was 1.42 and 1.68, respectively. Conclusion: Targeted Auger electron radioimmunotherapy with ¹¹¹In-NLS-trastuzumab can overcome resistance to trastuzumab, and MTX can potently enhance the sensitivity of HER2-overexpressing breast cancer cells to the lethal Auger electrons emitted by this radiopharmaceutical.

Key Words: breast cancer • trastuzumab (Herceptin) • radiosensitization • methotrexate • Auger electrons

COPYRIGHT © 2008 by the Society of Nuclear Medicine, Inc.

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