Bispecific Ligand‐Based EphB4 CAR‐T Cells Generated Using Ephrin‐B2 Show Potent Antitumor Activity Against Lung Adenocarcinoma

Chimeric antigen receptor (CAR)-T cells generated using ephrin-B2, the natural ligand for ephrin type-B receptor 4 (EphB4), have demonstrated antitumor activity; however, their efficacy remains unvalidated. Therefore, we evaluated the dual-targeting antitumor ability of these cells and confirmed their efficacy against lung adenocarcinoma. First, we evaluated EphB4 and EphA2 expression in samples from 74 patients with lung adenocarcinoma using immunohistochemistry. Next, EphB4 CAR-T cells were generated via piggyBac-mediated gene transfer, and their phenotype was evaluated. Subsequently, we conducted an antigen stimulation assay to assess the bispecificity of the EphB4 CAR-T cells. Finally, the antitumor effects of EphB4 CAR-T cells on lung adenocarcinoma cell lines were assessed. EphB4 and EphA2 positivity in lung adenocarcinoma samples was 93% and 100%, respectively. EphB4 CAR-T cells were successfully generated with high CAR positivity and a high proportion of naïve/stem cell memory-like T cells. In the antigen stimulation assay, the cells responded in an antigen-specific manner after stimulation with both EphB4 and EphA2 proteins. In the co-culture experiment, EphB4 CAR-T cells significantly suppressed the growth of all four lung adenocarcinoma cell lines compared to mock-T cells. In vivo, mice treated with EphB4 CAR-T cells displayed a significantly lower tumor burden than those treated with either CD19 CAR-T cells or phosphate-buffered saline. Additionally, mice treated with EphB4 CAR-T cells survived significantly longer than those in the other groups. In conclusion, ligand-based EphB4 CAR-T cells are bispecific, targeting both EphB4 and EphA2. Furthermore, these cells exert significant antitumor effects against lung adenocarcinoma.