Overexpression of SMAD7 improves the function of EGFR‐targeted human CAR‐T cells against non‐small‐cell lung cancer

Background and Objective Recent advancements in immunotherapy led to the development of Chimeric antigen receptor (CAR) T‐cell therapy. CAR‐T cell therapy in non‐small cell lung cancer (NSCLC) is hindered by overexpression of transforming growth factor (TGFβ) in the cancer cells that have a negative regulatory role on T‐cells activity. This study characterized CAR‐T with overexpression of mothers against decapentaplegic homologue 7 (SMAD), a negative regulator of TGFβ downstream signalling. Methods We have generated three types of CAR‐T: epidermal growth factor receptor (EGFR)‐CAR‐T, EGFR‐dominant‐negative TGFbeta receptor 2 (DNR)‐CAR‐T, and EGFR‐SMAD7‐CAR‐T by transducing human T‐cells with the lentivirus constructs. We characterized the proliferation, expression of proinflammatory cytokines, activation profile, and lysis capacity in co‐cultures with A549 lung carcinoma cells with and without TGFβ neutralizing antibodies. We also tested the therapeutic potential of EGFR‐SMAD7‐CAR‐T in the A549 cells tumour‐bearing mice model. Results Both EGFR‐DNR‐CAR‐T and EGFR‐SMAD7‐CAR‐T demonstrated a higher proliferation rate and lysis capacity to A549 than traditional EGFR‐CAR‐T. Neutralization of TGFβ by the antibodies resulted in increased performance of EGFR‐CAR‐T. In vivo, both EGFR‐DNR‐CAR‐T and EGFR‐SMAD7‐CAR‐T resulted in complete tumour resorption by day 20, whereas conventional CAR‐T only has a partial effect. Conclusion We demonstrated the high efficacy and resistance to negative TGFβ regulation of EGFR‐SMAD7‐CAR‐T comparable with EGFR‐DNR‐CAR‐T and without the systemic effect of TGFβ inhibition. The efficacy of TGF‐β signalling‐resistant CAR‐T based on SMAD7 overexpression is restricted to CAR‐T cells themselves and potentially reduces systemic effects on TGF‐β signalling. SMAD7‐CAR‐T demonstrates complete elimination of cancer in vivo, high levels of SMAD7‐CAR‐T proliferation, and activation that are not significantly different from the DNR‐CAR‐T design.