Construction of chimeric antigen receptor-modified T cells targeting EpCAM and assessment of their anti-tumor effect on cancer cells

Abstract

Colon cancer is a common malignancy worldwide and there is an urgent requirement to develop effective treatment strategies. In recent years, tumor immunotherapy has become a new method of effectively treating tumors. chimeric antigen receptor (cAR) T cell technology combines the precise targeting specificity of monoclonal antibodies with the strong toxicity and persistence of cytotoxic T cells to specifically recognize tumor-associated antigens and promote tumor cell death efficiently and permanently, without depending on major histocompatibility complex restriction. In the present study, epithelial cell adhesion molecule (EpCAM)-targeting CAR T cells (EpCAM-CAR-T) were developed, and their ability to kill cancer cells in vitro was assessed. Firstly, an EpCAM-CAR plasmid was constructed using molecular biology techniques, and transfected into T cells to obtain EpCAM-CAR-T cells. Transfection efficiency was assessed using reverse transcription-quantitative PCR and flow cytometry. Next, the expression levels of EpCAM in five colon cancer cell lines were examined by western blotting and flow cytometry. Finally, the effect of EpCAM-CAR-T cells on cancer cell death was examined in vitro via co-culture experiments. T cells stably expressing EpCAM-CAR were successfully obtained, and the transduction efficiency according to flow cytometry was 50.4%. In vitro experiments showed that EpCAM-CAR-T cells exhibited a significantly higher apoptotic effect on cancer cells compared with untransfected T cells. Analyses also demonstrated that this effect was dependent on the ratio of EpCAM-CAR-T cells to tumor cells, and the expression of surface EpcAM. Similarly, the ELISA results showed that interleukin (IL)-2 IL-6 and interferon-γ levels were significantly elevated following exposure to EpCAM-CAR-T cells compared to exposure to untransfected T cells, and were dependent on the number of EpCAM-CAR-T cells and the amount of EpCAM expressed on the surface of tumor cells. The present study provided a basis for the clinical application of CAR-T cell therapy against solid tumors, and a provided a new strategy for the treatment of colon cancer.