Engineering a fifth-generation CAR T cells to overcome PD-L1-mediated immunosuppression in lung cancer

Abstract

The tumor microenvironment (TME) significantly hinders chimeric antigen receptor (CAR) T cell therapy in solid tumors, despite its success in hematological malignancies. This disparity is attributable to immunosuppressive factors, such as program death ligand 1 (PD-L1) upregulation in non-small-cell-lung cancer (NSCLC). This study aims to create and assess anti-FRα-CAR5, a novel anti-folate receptor alpha (FRα) CAR T cell designed to secrete a PD-L1 blocking single chain variable fragment (scFv). Human T lymphocytes were engineered with a lentiviral vector to express anti-FRα-CAR5, which incorporates a fourth-generation CAR backbone (CD28, 4–1BB, CD27, and CD3 zeta) augmented by a secreted anti-PD-L1 scFv derived from atezolizumab. Transfected HEK293T cells were used to evaluate surface expression of anti-FRα-CAR. The secreted anti-PD-L1 scFv was tested for binding ability on lung adenocarcinoma cell lines. Furthermore, the secreted anti-PD-L1 scFv demonstrated over 80 % inhibitory activity against PD-L1 monoclonal antibody. Importantly, anti-FRα-CAR5 T cells enhanced expansion and cytotoxicity against FRα and PD-L1 expressing lung cancer cell lines in vitro compared to an anti-FRα-CAR4 lacking the secreted anti-PD-L1 scFv. This fifth-generation CAR offers a promising strategy to enhance CAR T cell therapy efficacy in PD-L1-mediated immunosuppressive TMEs. These findings suggest that anti-FRα-CAR5 T cells therapy warrants further preclinical validation as a potential treatment strategy for NSCLC patients.