eVLP-mediated CREM editing enhances cytotoxicity of bicistronic IL-15-armored CD19/BCMA dual-target CAR-NK cells

Abstract

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells represent a promising platform for cancer immunotherapy; however, optimization of CAR architecture and additional engineering strategies remain necessary to improve their functional activity. In this study, we generated CD19/BCMA dual-target CAR constructs based on tandem and bicistronic configurations with or without membrane-bound interleukin-15 (mbIL-15), and further evaluated engineered virus-like particle (eVLP)-mediated CREM editing as an additional strategy for CAR-NK cell engineering. Four CAR configurations were evaluated in primary human T and NK cells for CAR expression, cytotoxicity, cytokine production, and proliferative activity. Bicistronic CD19/BCMA CAR-mbIL-15 cells exhibited enhanced functional activity compared with corresponding tandem CAR-mbIL-15 cells, including increased tumor-cell killing and activation-associated functional responses in vitro . In a CD19/BCMA-positive leukemia xenograft model, bicistronic CD19/BCMA CAR-mbIL-15 T and NK cells reduced tumor burden, delayed disease progression, and prolonged survival. Furthermore, eVLP-mediated CREM editing achieved efficient genome modification in primary NK cells and remained compatible with subsequent CAR engineering. CREM-edited CAR-NK cells exhibited increased cytotoxic activity compared with unedited CAR-NK cells in vitro . Collectively, these findings support the development of modular CAR-NK engineering strategies integrating CAR architecture optimization, membrane-bound cytokine armoring, and transient genome engineering.