¹¹¹In-Labeled Trastuzumab (Herceptin) Modified with Nuclear Localization Sequences (NLS): An Auger Electron-Emitting Radiotherapeutic Agent for HER2/neu-Amplified Breast Cancer

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

^* To whom correspondence should be addressed. E-mail: raymond.reilly{at}utoronto.ca.

	Abstract

The cytotoxicity and tumor-targeting properties of the anti-HER2/neu monoclonal antibody trastuzumab modified with peptides (CGYGPKKKRKVGG) harboring the nuclear localization sequence ([NLS] italicized) of simian virus 40 large T-antigen and radiolabeled with ¹¹¹In were evaluated. Methods: Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) for reaction with NLS-peptides and labeled with ¹¹¹In using diethylenetriaminepentaacetic acid (DTPA). The immunoreactivity of ¹¹¹In-NLS-trastuzumab was determined by its ability to displace the binding of trastuzumab to SK-BR-3 human breast cancer (BC) cells. Cellular uptake and nuclear localization were evaluated in SK-BR-3, MDA-MB-361, and MDA-MB-231 BC cells, expressing high, intermediate, or very low levels of HER2/neu, respectively, by cell fractionation and confocal microscopy. Biodistribution and nuclear uptake were compared in athymic mice bearing MDA-MB-361 xenografts. The cytotoxicity of ¹¹¹In-trastuzumab and ¹¹¹In-NLS-trastuzumab was studied by clonogenic assays, and DNA damage was assessed by probing for phosphorylated histone H2AX (H2AX) foci. Results: The dissociation constant for binding of ¹¹¹In-NLS-trastuzumab to SK-BR-3 cells was reduced <3-fold compared with that of ¹¹¹In-trastuzumab, demonstrating relatively preserved receptor-binding affinity. The receptor-mediated internalization of ¹¹¹In-trastuzumab in SK-BR-3, MDA-MB-361, and MDA-MB-231 cells increased significantly from 7.2% ± 0.9%, 1.3% ± 0.1%, and 0.2% ± 0.05% to 14.4% ± 1.8%, 6.3% ± 0.2%, and 0.9% ± 0.2% for ¹¹¹In-NLS-trastuzumab harboring 6 NLS-peptides, respectively. NLS-trastuzumab localized in the nuclei of BC cells, whereas unmodified trastuzumab remained surface-bound. Conjugation of ¹¹¹In-trastuzumab to NLS-peptides did not affect its tissue biodistribution but promoted specific nuclear uptake in MDA-MB-361 xenografts (2.4-2.9 %ID/g [percentage injected dose per gram] for ¹¹¹In-NLS-trastuzumab and 1.1 %ID/g for ¹¹¹In-trastuzumab). ¹¹¹In-NLS-trastuzumab was 5- and 2-fold more potent at killing SK-BR-3 and MDA-MB-361 cells than ¹¹¹In-trastuzumab, respectively, whereas toxicity toward MDA-MB-231 cells was minimal. ¹¹¹In-NLS-trastuzumab was 6-fold more effective at killing SK-BR-3 cells than unlabeled trastuzumab. Formation of H2AX foci occurred in a greater proportion of BC cells after incubation with ¹¹¹In-NLS-trastuzumab compared with ¹¹¹In-trastuzumab or unlabeled trastuzumab. Conclusion: NLS-peptides routed ¹¹¹In-trastuzumab to the nucleus of HER2/neu-positive human BC cells, rendering the radiopharmaceutical lethal to the cells through the emission of nanometer-micrometer range Auger electrons. The greater cytotoxic potency of ¹¹¹In-NLS-trastuzumab compared with unlabeled trastuzumab in vitro and its favorable tumor-targeting properties in vivo suggest that it could be an effective targeted radiotherapeutic agent for HER2/neu-amplified BC in humans.

Key Words: breast cancer, trastuzumab (Herceptin), ¹¹¹In, nuclear translocation, Auger electrons

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