Inhibition of AKT-signaling uncouples cellular differentiation from expansion for receptor-engineered T cells

Adoptive immunotherapies using genetically-redirected T cells expressing a chimeric antigen receptor (CAR) or T cell receptor (TCR) are poised to enter mainstream clinical practice. Despite encouraging results, some patients fail to respond to current therapies. In part, this phenomenon has been associated with infusion of a reduced number of early memory T cells. Herein, we report that pharmacologic disruption of AKT-signaling (AKTi) is compatible with the transduction of both CARs and TCRs into human T cells and promotes a minimally differentiated CD62L-expressing phenotype. Critically, this intervention did not compromise cell yield. Mechanistically, disruption of AKT-signaling preserved MAPK activation and promoted the intra-nuclear accumulation of FOXO1, a key transcriptional regulator of T-cell memory. Consequently, AKTi synchronized the T-cell transcriptional profile for FOXO1-dependent target genes across multiple donors. Expression of an AKT-resistant FOXO1 mutant phenocopied the influence of AKTi while addition of AKTi to T cells expressing mutant FOXO1 failed to further augment the frequency of CD62L-expressing cells. Finally, CD19 CAR-modified T cells transduced and expanded in AKTi treated established B-cell acute lymphoblastic leukemia superiorly to conventionally grown T cells in a murine xenograft model. Thus, inhibition of AKT-signaling represents a generalizable strategy to generate large numbers of receptor-modified T cells with an early memory phenotype. PBMCs from 3 patients (Patients C, M, and P) were enriched for CD8+ T cells using Miltenyi kit. 1e6 cells per sample. Time 0 represents start of culture. Whole PBMCs (not CD8-enriched) were activated with anti-CD3 and retrovirally-transduced with CD19 CAR and expanded in presence or absence of pharmacologic inhibitor of Akt. Veh denotes cells expanded in conventional conditions. Akt denotes conditions expanded in 1uM Akt inhibitor. At day 10, were enriched for CD8+ T cells using Miltenyi kit and 1e6 cells per sample were preserved in 40ul RNA later and stored in -80C. N=33 samples.