640. Ex Vivo AKT Inhibition Promotes the Generation of Potent CD19CAR T Cells for Adoptive Immunotherapy
Authors: Ryan Z. Urak, ChingLam W. Wong, Elizabeth Budde, Christine E. Brown, Sandra Thomas, Stephen J. Forman, Xiuli Wang
Journal: Molecular Therapy (2016)

Insufficient persistence and effector function of chimeric antigen receptor (CAR) re-directed T cells in vivo has been a challenge for adoptive T cell therapy. Generation of long-lived potent CAR T cells is an increasing demand in the field. AKT activation triggered by convergent extracellular signals evokes a transcription program that enhances effector functions. However, sustained AKT activation severely impairs T cell memory and protective immunity because AKT drives differentiation of effectors, diminishing T cell potential to survive and differentiate into memory cells. We now investigate whether inhibition of AKT signaling can prevent terminal differentiation of T cells that are genetically modified to express CD19-specific chimeric antigen receptors (CD19CAR), as well as increase the number of memory CD19CAR T cells, which would enhance the antitumor activity following adoptive therapy. CD8+ T cells from healthy donors were isolated, activated with CD3/CD28 beads, and transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain, which carries mutations at two sites (L235E; N297Q) within the CH2 region on the IgG4-Fc spacers to block diminshed potency and persistence due to Fc receptor binding. The lentiviral vector also expressed a truncated human epidermal growth factor receptor (huEGFRt) for selection and ablation purposes. IL-2 (50U/mL) and AKT inhibitor (1uM/mL) were supplemented every other day. Transduced CD19CAR T cells without AKT inhibitor treatment were used as controls. The engineered CD19CAR T cells were expanded in vivo for 21 days before in vitro and in vivo assays. We found that AKT inhibitor did not compromise the CD19CAR T cell proliferation and survival in vitro; comparable CD19CAR T cell expansion was observed after culturing in the presence or absence of AKT inhibitor. Functionally, AKT inhibitor did not dampen the effector function of CD19CAR T cells as indicated by equivalent levels of interferon gamma production and CD107a expression upon CD19 antigen stimulation. Memory-like phenotype such as CD62L and CD28 expression on CAR T cells is associated with better antitumor activity in vivo. We therefore characterized the CD19CAR T cells after ex vivo expansion. We found that 40% of AKT-inhibited CD19CAR T cells expressed CD62L and co-expressed CD28. In contrast, only 10% of control untreated CD19CAR T cells expressed CD62L and they were CD28 negative, indicating that AKT-inhibited CD19CAR T cells may have superior anti-tumor activity following adoptive transfer. To test the potency of the AKT inhibitor treated CAR T cells, 0.5×106 CD19+ acute lymphoid leukemic cells (SupB15) that were engineered to express firely lucifierase were inoculated intravenously into NOD/Scid IL-2RgammaCnull (NSG) mice. Five days post tumor engraftment, 2×106 CD8+ CD19CAR T cells were intravenously injected into tumor bearing mice. Control mice received either no T cells, non-transduced T cells (Mock), or CD19CAR T cells that were not treated with AKT inhibitor during in vitro expansion. Tumor signals post T cells infusion were monitored by biophotonic imaging. In contrast to the untreated CD19CAR T cells, which exhibited lower and transient anti-tumor activity, AKT-inhibited CD19CAR T cells completely eradicated the CD19+ tumor in all mice (Figure 1) 21 days post CD19CAR T cell infusion. In conclusion, inhibition of AKT signaling during the ex vivo priming and expansion gives rise to a CD19CAR T cell population that possesses superior antitumor activity. These findings suggest that therapeutic modulation of AKT might be a strategy to augment antitumor immunity for adoptive CAR T cell therapy, which could easily be transitioned into the clinic with the availability of pharmaceutical grade AKT inhibitor.