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

Background: Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab, a high affinity-binding monoclonal antibody against HER2/neu is which is over-expressed in breast tumors, is used in radiopharmaceutical development.

Objectives: 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 ¹¹¹In, 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 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 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.