The activity of EGFR CAR-NK and CAR-T cells against EGFR inhibitor-resistant NSCLC and drug-tolerant persister cells

Patients with NSCLC harboring EGFR mutations typically have significant clinical benefits from EGFR tyrosine kinase inhibitors (TKIs) such as osimertinib. However, a residual population of drug-tolerant persister cells (DTPCs) inevitably remains, which ultimately gives rise to fully drug-resistant cells (DRCs). This study evaluates the activity of EGFR chimeric antigen receptor (CAR)-based therapies in this context. We developed EGFR CAR-T and CAR-NK cells and evaluated their antitumor activity against parental cells, DTPCs, and DRCs in vitro and in vivo. We investigated the mechanisms regulating the sensitivity of DTPCs and DRCs to CAR-T or CAR-NK cells, including NK-activating ligands, TGF-β signaling, and EGFR surface levels. Additionally, we developed strategies that included galunisertib treatment and the expression of dominant-negative TGF-β receptor II (DNR) in CAR-NK cells. DTPCs demonstrated increased sensitivity to both EGFR CAR-T and CAR-NK. DRCs were relatively resistant to CAR-T cells but more sensitive to CAR-NK cells. DRCs and DTPCs had higher levels of NKp30 or NKG2D ligands, which enhance the effectiveness of CAR-NK. Elevated TGF-β levels in DRCs impaired CAR function, but this was reversed by co-expression of galunisertib or DNR in CAR-NK cells. Continued TKI treatment increased EGFR expression on DRCs, possibly contributing to the improved killing activity seen with TKI/CAR combinations compared to CARs alone in TKI-resistant cells. EGFR-directed cellular therapies, particularly EGFR CAR-NK cells, demonstrate activity against EGFR-mutant DTPCs and DRCs in vitro and in vivo, with enhanced activity observed when combined with EGFR TKIs or TGF-β pathway blockade.