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,potentantitumor 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, display 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. To comprehensively study the gene expression profile across the allogeneic MCAR-NKT cell populations, we performed and analyzed bulk RNA-Seq of allogeneic MCAR-NKT cells and other immune cells, including 4 AlloMCAR-NKT, 4 Allo15MCAR-NKT, 3 PBMC-NKT, 7 PBMC-Tc, and 2 PBMC-NK. This study sequenced 4 AlloMCAR-NKT, 4 Allo15MCAR-NKT, and 2 PBMC-Tc cell samples. The remaining samples were sequenced in our previous study (GEO Accession: GSE164425), thus we downloaded the raw sequencing data and reanalyzed them. All 20 samples were subject to the same processing and analysis procedure as denoted in the PROTOCOLS section. Re-analysed Samples PRJNA690603/GSE164425: GSM5009508 SRR13381422/SRX9804363 SAMN17255538 GSM5009509 SRR13381423/SRX9804364 SAMN17255537 GSM5009510 SRR13381424/SRX9804365 SAMN17255536 GSM5009511 SRR13381425/SRX9804366 SAMN17255535 GSM5009512 SRR13381426/SRX9804367 SAMN17255534 GSM5009496 SRR13381435/SRX9804351 SAMN17255123 GSM5009497 SRR13381436/SRX9804352 SAMN17255122 GSM5009498 SRR13381437/SRX9804353 SAMN17255121 GSM5009499 SRR13381438/SRX9804354 SAMN17255120 GSM5009500 SRR13381439/SRX9804355 SAMN17255119