EphA2 chimeric antigen receptor-modified T cells for the immunotherapy of esophageal squamous cell carcinoma

Hui Shi, Feng Yu, Yinting Mao, Qianqian Ju, Yingcheng Wu, Wen Bai, Peiwen Wang, Ran Xu, Maorong Jiang, Jiahai Shi


Background: It is urgent to explore an effective potential therapeutic strategy for ESCC. In recent years, cell-based cancer immunotherapy has become a potentially close for carcinoma therapy. Chimeric antigen receptor (CAR) T cell technology is a kind of adoptive cell therapy technique which has been developed rapidly. We sought to obtain EphA2.CAR-T cell and revealed the ability of EphA2.CAR-T cells to kill esophageal squamous cell carcinoma (ESCC) cells in vitro.
Methods: Firstly, the expression and location of EphA2 in ESCC tissues and cells was tested by immunohistochemistry staining and Western blot. Secondly, the second generation of EphA2.CAR was constructed via molecular biology technology, and transduced into T cells to obtain the EphA2.CAR-T cell. The transduction efficacies were assessed using flow cytometry (FCM). Thirdly, the effect of cell killing of EphA2.CAR-T cell on ESCC cells in vitro was detected by co-culture experiments. The productions of cytokines (TNF-α and IFN-γ) by EphA2.CAR-T cell after co-culture with ESCC cells were analyzed by ELISA assay.
Results: The expression of EphA2 was significantly upregulated in ESCC tissues and cells (P<0.05). EphA2 was expressed on the membrane of ESCC cells, so it could be served as tumor-associated surface antigens (TAA) of CAR for ESCC treatment. The EphA2.CAR-T cell was obtained successfully, and its’ transduction efficacies was 61.4% by FCM. The ability of cell killing of EphA2.CAR-T cell was better than that of T cells (P<0.01), and demonstrated a dose-dependent cell killing. The results of ELISA assay showed that the levels of TNF-α and IFN-γ in EphA2.CAR-T cells were notably raised compared with T cells (P<0.05).
Conclusions: We firstly constructed the second generation of EphA2.CAR and established EphA2.CAR-T cells. The EphA2.CAR-T cells showed a dose-dependent cell killing of ESCC cells, and promoted the production of cytokines in vitro. These findings open a new way for treatment of ESCC by immunotherapy in the future.