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

Abstract

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.

Human IL-17A protein production is controlled through a PIP5K1α-dependent translational checkpoint

“…It is possible that PIP5K1α plays additional roles in driving IL-17 production by memory T H 17 cells through supporting TCR/CD28 signaling in addition to enhancing IL-17A protein production, but this remains to be tested. In this context, it is worth highlighting that CD28 inhibits T H 17 cell generation in a dose-dependent manner when using agonistic anti-CD28 in vitro, and this inhibition can be mitigated by reducing the concentration of anti-CD3 ( 14 , 39 ). In other words, cumulative signals from the TCR and CD28 determine T H 17 cell output, much like what has been described for T H 1 and T H 2 cells ( 14 , 40 ).…”

Dual T cell receptor/chimeric antigen receptor engineered NK‐92 cells targeting the HPV16 E6 oncoprotein and the tumor‐associated antigen L1CAM exhibit enhanced cytotoxicity and specificity against tumor cells

“…It has been shown that ICOS signaling is a strong costimulatory signal for T cells and is required for the proper functionality of activated T cells. 42 Further, ICOS stimulation has been shown to augment T cell 43 and NK functionality. 44,45 To test whether concomitant activation of ICOS signaling in NK-92/CD3/CD8 cells expressing a TCR could enhance TCR-mediated cytotoxicity, we first generated a T2-derived cell line constitutively expressing ICOSL (Figure 4A).…”