Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells for adoptive immunotherapy

Abstract

BackgroundInsufficient persistence and effector function of chimeric antigen receptor (CAR)-redirected T cells have been challenging issues for adoptive T cell therapy. Generating potent CAR T cells is of increasing importance in the field. Studies have demonstrated the importance of the Akt pathway in the regulation of T cell differentiation and memory formation. We now investigate whether inhibition of Akt signaling during ex vivo expansion of CAR T cells can promote the generation of CAR T cells with enhanced antitumor activity following adoptive therapy in a murine leukemia xenograft model.MethodsVarious T cell subsets including CD8+ T cells, bulk T cells, central memory T cells and naïve/memory T cells were isolated from PBMC of healthy donors, activated with CD3/CD28 beads, and transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain. The transduced CD19CAR T cells were expanded in the presence of IL-2 (50U/mL) and Akt inhibitor (Akti) (1 μM) that were supplemented every other day. Proliferative/expansion potential, phenotypical characteristics and functionality of the propagated CD19CAR T cells were analyzed in vitro and in vivo after 17-21 day ex vivo expansion. Anti-tumor activity was evaluated after adoptive transfer of the CD19CAR T cells into CD19+ tumor-bearing immunodeficient mice. Tumor signals were monitored with biophotonic imaging, and survival rates were analyzed by the end of the experiments.ResultsWe found that Akt inhibition did not compromise CD19CAR T cell proliferation and expansion in vitro, independent of the T cell subsets, as comparable CD19CAR T cell expansion was observed after culturing in the presence or absence of Akt inhibitor. Functionally, Akt inhibition did not dampen cell-mediated effector function, while Th1 cytokine production increased. With respect to phenotype, Akti-treated CD19CAR T cells expressed higher levels of CD62L and CD28 as compared to untreated CD19CAR T cells. Once adoptively transferred into CD19+ tumor-bearing mice, Akti treated CD19CAR T cells exhibited more antitumor activity than did untreated CD19CAR T cells.ConclusionsInhibition of Akt signaling during ex vivo priming and expansion gives rise to CD19CAR T cell populations that display comparatively higher antitumor activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-017-0227-4) contains supplementary material, which is available to authorized users.

Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy

“…For example, the efficiency of in vitro sensitization of allogenic minor histocompatibility-specific T cells (28) and the ex vivo expansion of melanoma TILs (29) were both improved following exposure to AKTi. More recently and consistent with our findings, Urak et al reported that blockade of AKT activation prevented the terminal differentiation of CAR-modified T cells expanded to therapeutic doses (83).…”

Transcriptional Profiling of CD8+ CMV-Specific T Cell Functional Subsets Obtained Using a Modified Method for Isolating High-Quality RNA From Fixed and Permeabilized Cells

“…As STAT5 activity is primarily dependent upon cytokine receptor signaling, it is possible that unchecked mTORC1 activity may lead to reductions in cytokine signaling through STAT5, and that modulation of mTORC1 activity without complete inhibition is necessary to allow STAT5 activity without compromising effector function and proliferation. This is further supported by evidence that persistent activation of Akt, a primary upstream activator of mTORC1 activity, has been shown to impair LCMV memory cell formation and suppress cytokine expression and STAT5 activity, and that incomplete mTORC1 inhibition can partially rescue this phenomenon ( 73 , 95 , 96 ). Moreover, inhibition of Akt during adoptive T cell expansion has been associated with improved antigen-specific T cell functionality ( 95 – 98 ).…”

Prolonged Persistence of Chimeric Antigen Receptor (CAR) T Cell in Adoptive Cancer Immunotherapy: Challenges and Ways Forward

“…Protein Kinase B (PKB or AKT) pathway, as a key cell survival signal, has prominent role in the multiple cellular functions such as T cells differentiation, survival, and memory formation. Various studies have demonstrated that blocking of AKT signaling pathway in CAR T cells is associated with generation of less differentiated memory cells, thereby conferring survival advantage compared to untreated T cells (50,55). These data showed that inhibition of Akt signaling during ex vivo expansion and priming leads to generation of large numbers of memory CAR T cells with superior antitumor activity (55).…”

“…Despite the great strides that have been made by CAR T cell therapy so far, some patients still do not respond to this method of therapy due to, for example, paucity or deficiency of long-lived T cells (48,49). It is well-known that expansion, differentiation, and survival of T cells depend on the integrated signals coming from TCR engagement, cytokine receptors, and costimulatory molecules (50,51). These signals lead to activation of two main signal transduction networks in T cells, MAPK and PI3K/AKT/mTOR pathways.…”