Generation of off-the-shelf CAR-NKT cells for cancer immunotherapy with genetically engineered HSCs and feeder-free differentiation culture

Current CAR-T cell therapy's clinical potential is hampered by its autologous nature that poses significant challenges in manufacturing, costs, and patient selection. This spurs demand for off-the-shelf therapies. Here we introduce an ex vivo feeder-free culture to differentiate gene-engineered HSCs into allogeneic NKT cells and their CAR-armed derivatives (AlloCAR-NKT cells). The AlloCAR-NKT cells are generated at high yield, purity, and robustness. These cells exhibit potent antitumor efficacy, showing effective tumor homing, clonal expansion, and persistence In vivo. Impressively, AlloCAR-NKT cells can alter the tumor microenvironment by selectively depleting immunosuppressive TAMs and MDSCs, and can antagonize tumor immune evasion by deploying CAR/TCR/NKR triple-targeting mechanisms. AlloCAR-NKT cells also demonstrate an appealing safety profile with low GvHD and CRS risks, and exhibit a stable “hypoimmunogenic” phenotype attributed to epigenetic and signaling regulations. The reported technology presents a scalable strategy for diverse allogeneic CAR-NKT cell products, with potential for clinical translation and commercialization against various cancers. Overall design: A total of 24 cell samples were analyzed, including 3 AlloNKT (6B11+TCRaß+), 3 AlloBCAR-NKT (6B11+TCRaß+), 3 Allo15BCAR-NKT (6B11+TCRaß+), 2 Allo15CAR19-NKT (6B11+TCRaß+), 3 PBMC-NKT (6B11+TCRaß+CD4-), 8 PBMC-Tc (6B11-TCRaß+CD4-), and 2 PBMC-NK (CD56+TCRaß-) cell samples. Note that Allo/15(CAR)-NKT cells displayed a CD4-CD8+/- phenotype, therefore the CD4- populations of PBMC-NKT and PBMC-Tc cells were used in this study. Among the 24 cell samples, 11 samples were newly sequenced in this study, and the remaining 13 samples were sequenced in our previous study (GEO Accession: GSE164425). We downloaded the raw sequencing data of these 13 samples and reanalyzed them. All the samples were subject to the same data processing and analysis procedure as denoted in the protocols fields.