Ex Vivo AKT Inhibition Promotes the Generation of Potent CD19CAR T Cells for Adoptive Immunotherapy
Authors: Urak Ryan , Wong ChingLam , Chang Wen-Chung , Budde Elizabeth E. , Brown Christine , Thomas Sandra H. , Forman Stephen J. , Wang Xiuli 
Published: October 2019

Abstract
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, therefore diminishing T cell potential to survive and differentiate into memory cells. We now investigate whether inhibition of AKT signaling during ex vivo expansion can prevent terminal differentiation of CD19- chimeric antigen receptor (CD19 CAR) engineered T cells and increase the number of memory CD19 CAR 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 then transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain and two mutations (L235E; N297Q) within the CH2 region on the IgG4-Fc spacers which enhances potency and persistence by blocking Fc receptor binding. In addition, the lentiviral construct also expresses a truncated human epidermal growth factor receptor (huEGFRt) which allows us to use as a selectable marker and a mechanism to ablate the CAR T cells if necessary. 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 vitro for 21 days before in vitro and in vivo analyses. We found that AKT inhibitor did not compromise the CD19CAR T cell proliferation and survival in vitro. There was a comparable CD19CAR T cell expansion after culturing in the presence or absence 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. More importantly, no exhaustion markers such as KRLG and PD-1 were induced on the AKT inhibitor treated cells. In contrast, only 10% of control untreated CD19CAR T cells expressed CD62L and they were CD28 negative, indicating that AKT-inhibited CD19CAR T cells with higher levels of CD62L and CD28 expression may have superior anti-tumor activity following adoptive transfer. To test the potency of the AKT inhibitor treated CAR T cells, 0.5x106 CD19+ acute lymphoid leukemic cells (SupB15) engineered to express firefly luciferase were inoculated intravenously into NOD/Scid IL-2RgammaCnull (NSG) mice. Five days post tumor engraftment, 2x106 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 cell infusion were monitored by biophotonic imaging. Compared to the untreated CD19CAR T cells, which exhibited lower and transient anti-tumor activity, AKT inhibitor treated CD19CAR T cells completely eradicated the CD19+ tumor in all mice (Figure 1) 21 days post CD19CAR T cell infusion. In conclusion, our results demonstrate that 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 ex vivo 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.

Disclosures
Forman: Amgen: Consultancy; Mustang: Research Funding.