Abstract 4551: 111In-NLS-Nimotuzumab: A potent auger electron-emitting radiotherapeutic agent for EGFR-overexpressing breast cancer

Abstract

Abstract Objective: To evaluate the cytotoxicity of nimotuzumab (Nmab), a monoclonal antibody specific for EGFR labeled with 111In and modified with nuclear localization sequence (NLS) peptides for importation into the nucleus of human breast cancer (BC) cells, where the emitted Auger electrons are most damaging to DNA and lethal. Methods: NMab was derivatized with sulfo-SMCC to introduce maleimide groups for reaction with NLS-peptides (CGYGPKKKRKVGG) and benzyl-DTPA (bn-DTPA) for complexing 111In. NLS substitution was quantified by a band shift on SDS-PAGE and bnDTPA substitution by an 111In-binding assay. The immunoreactivity (Kd) of NLS-Nmab was determined by the displacement of binding of 111In-Nmab to MDA-MB-468 BC cells (106 EGFR/cell). Internalization and nuclear importation were determined in MDA-MB-468 and MCF-7 (104 EGFR/cell) BC cells by subcellular fractionation at 37 oC and 4 oC up to 3 h. Clonogenic survival (CS) was measured in MDA-MB-468 and MCF-7 cells exposed to 2 to 40 nM (450-900 KBq/mL) of 111In-NLS-Nmab for 3 days. Results: Nmab was modified with 8.5±0.2 NLS and 2.1±2.1 bn-DTPA groups. The Kd for binding of 111In-NLS-Mab to MDA-MB-468 cells was 9.8±0.9 nM. NLS-Nmab was labeled with 111In to a radiochemical purity of 99.2±0.1% (specific activity 350±9 MBq/mg). The proportion of cell-bound 111In internalized into MDA-MB-468 cells at 3 h was 27.8±0.8% at 37 oC and 4.4±0.2 at 4 oC. Nuclear uptake was 31.2±2.6% for 111In-NLS-Nmab and 16.0±0.6% for 111In-Nmab (P<0.001). Binding and nuclear uptake were 20-fold and 6-fold lower, respectively for MCF-7 cells. The CS of MDA-MB-468 cells exposed to 111In-NLS-Nmab (20 nM) was 2.8±0.2% compared to 21.0±5.3% for 111In-Nmab (P=0.004) and 60.0±7.0% for Nmab (P=0.001). The CS of MCF-7 cells exposed to 111In-NLS-Nmab (40 nM) was 82.6±6.4% vs. 80.0±1.6% for 111In-Nmab (p>0.05) and 95±19.4% for Nmab (p>0.05). Conclusion: 111In-NLS-Nmab is a promising targeted radiotherapeutic agent for EGFR-overexpressing BC as evidenced by a 20-fold increased toxicity towards MDA-MB-468 cells compared to Nmab but lack of toxicity towards MCF-7 cells with 10-fold lower EGFR expression similar to that on most normal epithelial cells. Supported by the Canadian Breast Cancer Research Alliance (Grant 019513). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4551.