Simultaneous Targeting of Ovarian Cancer and Its Microenvironment Using Stem Cell-Engineered Off-The-Shelf CAR-NKT Cells

Ovarian cancer (OC) poses a significant challenge due to frequent recurrence linked to tumor heterogeneity and an immunosuppressive tumor microenvironment (TME). Targeting both OC cells and TME simultaneously holds promise for effective treatment. This study comprehensively analyzed 17 chemonaive and 18 refractory/recurrent OC samples, identifying mesothelin (MSLN) and natural killer receptor (NKR) ligands as tumor targets and CD1d as an OC TME target. Leveraging hematopoietic stem cell (HSC) gene engineering and feeder-free differentiation culture, we generated allogeneic MSLN-targeting CAR (MCAR)-engineered invariant natural killer T (MCAR-NKT) cells, demonstrating high-yield and high-purity production, potent antitumor efficacy, multiple tumor targeting mechanism, and significant in vivo expansion and persistence. Notably, these cells selectively deplete immunosuppressive TAMs and MDSCs, counteract tumor immune evasion, dispaly a favorable safety profile with low risks of GvHD and CRS, and exhibit a stable “hypoimmunogenic” phenotype attributed to epigenetic and signaling regulations. These findings underscore the preclinical feasibility and therapeutic potential of allogeneic MCAR-NKT cell products for OC, laying a foundation for translational and clinical development. 6 samples, including 3 allogeneic IL15-enhanced HSC-derived mesothelin-targeting CAR-engineered NKT (Allo15MCAR-NKT) cells, and 3 PBMC-derived MCAR-engineered T (MCAR-T) cells were included in this study. Genomic DNA was isolated from experimental samples using a QIAGEN DNeasy Blood & Tissue kit, then sonicated using a Covaris M220 Focused-ultrasonicator, following the manufacturers’ instructions. Then the genomic DNA was sent to UCLA TCGB core to conduct the WGBS library construction and sequencing.