iPSC-derived NK cells with site-specific integration of CAR19 and IL24 at the multi-copy rDNA locus enhanced antitumor activity and proliferation.

The generation of chimeric antigen receptor-modified natural killer (CAR-NK) cells using induced pluripotent stem cells (iPSCs) has emerged as one of the paradigms for manufacturing off-the-shelf universal immunotherapy. However, there are still some challenges in enhancing the potency, safety, and multiple actions of CAR-NK cells. Here, iPSCs were site-specifically integrated at the ribosomal DNA (rDNA) locus with interleukin 24 (IL24) and CD19-specific chimeric antigen receptor (CAR19), and successfully differentiated into iPSC-derived NK (iNK) cells, followed by expansion using magnetic beads in vitro. Compared with the CAR19-iNK cells, IL24 armored CAR19-iNK (CAR19-IL24-iNK) cells showed higher cytotoxic capacity and amplification ability in vitro and inhibited tumor progression more effectively with better survival in a B-cell acute lymphoblastic leukaemia (B-ALL) (Nalm-6 (Luc1))-bearing mouse model. Interestingly, RNA-sequencing analysis showed that IL24 may enhance iNK cell function through nuclear factor kappa B (NFκB) pathway-related genes while exerting a direct effect on tumor cells. This study proved the feasibility and potential of combining IL24 with CAR-iNK cell therapy, suggesting a novel and promising off-the-shelf immunotherapy strategy.