Assessment of Cell Cytotoxicity and Comet Assay on HER2/neu Positive Cell Line Due to 111 In Auger Electrons as DNA-Targeting Radioimmunoconjugate

Abstract

Background: Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab is a high affinity-binding monoclonal antibody against HER2/neu, which is overexpressed in breast tumors and used in radiopharmaceutical development. Objective: In this work, the lethal effects of 111In3+, 111In-DTPA-trastuzumab, and 111In-trastuzumab coupled-nuclear localizing sequence peptide (111In-DTPA-NLS-trastuzumab) on malignant cells were studied in vitro. Methods: DTPA-NLS-trastuzumab was prepared using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) conjugation with NLS peptide in the first step, followed by conjugation with diethylenetriaminepentaacetic acid (DTPA). Both DTPA-trastuzumab and DTPA-NLS-trastuzumab were labeled with 111In, followed by purification and quality control techniques. Sk-Br-3 (a HER2/neu+ cell line) was used in the cell viability assessment assay for 11In, 111In-DTPA-trastuzumab, and 111In-DTPA-NLS-trastuzumab (3.7 MBq) at 37 ºC. The cytotoxicity of the three species was studied using MTT, and comet assay was utilized by DNA damage detection. Results: A significant radiochemical purity for 111In-DTPA-NLS-trastuzumab (99.36% ± 0.30%, ITLC) at the DTPA:antibody ratio of 6.90 ± 0.34:1 was obtained. Significant cell viability difference was found for 111In-DTPA-NLS-trastuzumab compared to the other treatments at two-time points. In addition, comet assay demonstrated significant DNA damage at 144 h using 111In-DTPA-NLS-trastuzumab. Conclusion: The results of cell viability and cell death using MTT assay and comet assay, respectively, demonstrate that the NLS-peptide effectively facilitates 111In-trastuzumab transport into the HER2/neu positive cancer cell nuclei to impose the radiotherapeutic effects of Auger electrons on DNA, leading to cell death.