Augmenting TCR signal strength and ICOS costimulation results in metabolically fit and therapeutically potent human CAR Th17 cell therapy

Adoptive cell transfer (ACT) therapy with IL-17 producing human T cells elicits potent antitumor activity in preclinical models. However, further refinement of this novel approach is needed to position it for clinical application. While activation signal strength differentially regulates IL-17 production by human CD4+ T cells, the degree to which TCR and co-stimulation signal strength impacts antitumor Th17 cell immunity remains poorly understood. We discovered that decreasing TCR/co-stimulation signal strength by incremental reduction of αCD3/co-stimulation beads in a Th17 culture progressively diminished their effector memory phenotype but enhanced their polyfunctionality. Additional investigation revealed that Th17 cells stimulated with αCD3/ICOS beads produced more IL-17A, IFNγ, IL-2 and IL-22 than those stimulated with αCD3/CD28 beads, regardless of signal strength. Th17 cells propagated with 30-fold fewer αCD3/ICOS beads (weak signal strength, 1 bead per 10 T cells) were less reliant on glucose for growth compared to those stimulated with the standard, strong signal strength (3 beads per T cell). Further metabolomic analysis revealed Th17 cells weakly simulated with αCD3/ICOS beads favored the central carbon pathway through increased gluconeogenesis for bioenergetics, marked by abundant intracellular phosphoenoylpyruvate (PEP). Importantly, Th17 cells weakly stimulated with αCD3/ICOS beads and redirected with a chimeric antigen receptor (CAR) that recognizes mesothelin were more effective at clearing large human mesothelioma tumors when infused into mice than those manufactured using the standard FDA-approved protocols. Taken together, these data indicate Th17 ACT therapy can be improved by using fewer activation beads during T cell manufacturing, a finding that is both cost effective and directly translatable to patients.