Gene expression profiles of stimulated CAR-T cells targeting TNF-NTF

Chimeric antigen receptor (CAR)-T cell therapy is a promising therapeutic approach for relapsed or refractory acute myeloid leukemia (AML). However, optimal target antigens have yet to be established. A subset of AML cells express TNF, which is initially expressed on the cell surface and subsequently secreted by proteolytic cleavage of the extracellular portion. We generated CAR-T cells targeting the N-terminal fragment of TNF (TNF-NTF) that remains on the cell surface after shedding. We ablated TNF gene to avoid fratricide and coexpressed chimeric cytokine receptor (G6/7R) that we had previously developed in CAR-T cells to provide constitutive IL-7 signaling. The G6/7R-expressing TNF-TNF CAR-T cells exhibited potent and durable therapeutic efficacy in vivo. Our data suggest that TNF-TNF may be a promising target antigen for CAR-T cells applicable to AML. Overall design: Human CD8+ T cells derived from three different healthy donors with TNF gene knockout were retrovirally transduced with an anti-TNF-NTF CAR gene or the CAR gene + G6/7R. The CAR-T cells were then stimulated with NALM-6 transduced with TNF NTF (NALM-6 TNF-NTF, high antigen density) or THP1, which endogenously expresses TNF (low antigen density) for 24h. Total RNA was collected, and gene expression profiles were analyzed by RNA sequencing.