GPC2-Targeted CAR T Cells Engineered with NFAT-Inducible Membrane-Tethered IL15/IL21 Exhibit Enhanced Activity against Neuroblastoma

T-cells redirected for antigen-unrestricted cytokine-initiated killing (TRUCKs) are engineered chimeric antigen receptor (CAR) T-cells that constitutively or inducibly release cytokines upon CAR engagement. Their purpose is to enhance the function of effector cells in the immune-suppressive tumor microenvironment (TME) of particularly solid tumors that is often devoid of pro-inflammatory cytokines. We capitalize on the pleiotropic effects of two γc family cytokines, interleukin (IL)-15 and IL-21, and use them synergistically for TRUCK engineering. We demonstrate that TRUCKs with soluble IL-15/IL-21 eradicate CAR T-cell–resistant neuroblastoma, the most common extra-cranial solid tumor of childhood, but are associated with significant toxicities in mice. These toxicities can be delayed when we constitutively tether the cytokines to the surface of T-cells but are abrogated when we engineer CAR T-cells with NFAT-induced membrane-tethered IL-15/IL-21 expression. To investigate the difference in splenocyte population and tumor microenvironment status between GPC2-CAR T cells expressing membrane tethered cytokines constitutively or conditionally (by NFAT system), we inoculated mice orthotopically with neuroblastoma cell line IMR-5 or a PDX; SJNBL013762. Around 3 weeks after the tumor injection, we injected the following GPC2-CAR T cells via tail vein. 1) Untransduced control T cells, 2) CAR T cells, 3) CAR T cells with mteth.IL15.IL21, 4) CAR T cells with NFAT.mteth.IL15.IL21. After 5-6 weeks after the CAR T injection, we harvested spleens and tumors from mice treated with 3) or 4). As for 4), we included samples from good responders and poor responders. After enzymatic dissociation, the cells were analyzed using scRNA seq.