Effect of iPSC-derived NK cells with site-specific integration of CAR19 and IL24 at the rDNA locus on anti-tumor activity and proliferation.

175 Background: The generation of CAR-NK cells using induced pluripotent stem cells (iPSCs) has emerged as a paradigm for manufacturing off-the-shelf cell products for universal immunotherapy. However, enhancing the potency, safety and multi-actions of CAR-NK cells is still full of challenges. Methods: Interleukin 24 (IL24) and CD19-specific chimeric antigen receptor (CAR19) were site-specifically integrated at the ribosomal DNA (rDNA) locus in human iPSCs by using TALENickases. The engineered iPSCs were differentiated into NK (CAR-iNK) cells by adopting a 38-day differentiation protocol followed by expansion using magnetic beads in vitro. Results: Compared with the CAR-iNK cells, IL24 armored CAR-iNK (CAR19-IL24-iNK) cells showed higher cytotoxic capacity and amplification ability in vitro. Meanwhile, CAR19-IL24-iNK cells inhibited tumor progression more effectively and exhibited better survival without significant side effects in the B-ALL (Nalm-6(Luc1))-bearing mouse model. Interestingly, RNA-sequencing analysis suggested that IL24 may enhance iNK cell function by attracting neutrophils and upregulating FAS and TNFSF10-related genes while exerting a direct effect on tumor cells. Conclusions: This study encourages the exploration of IL24 and other molecules to enhance antitumor properties of CAR-NK cells, suggesting a novel strategy to modulate tumor microenvironment while attacking tumor cells with the potential of promising off-the-shelf immunotherapy.