Ex vivo AKT-inhibition facilitates generation of polyfunctional stem cell memory-like CD8⁺ T cells for adoptive...

Adoptive T cell therapy has shown clinical potential for patients with cancer, though effective treatment is dependent on longevity and potency of the exploited tumor-reactive T cells. Previously, we showed that ex vivo inhibition of AKT using the research compound Akt-inhibitor VIII retained differentiation and improved functionality of minor histocompatibility antigen (MiHA)-specific CD8⁺ T cells. Here, we compared a panel of clinically applicable AKT-inhibitors with an allosteric or adenosine triphosphate-competitive mode of action. We analyzed phenotype, functionality, metabolism and transcriptome of AKT-inhibited CD8⁺ T cells using different T cell activation models. Most inhibitors facilitated T cell expansion while preserving an early memory phenotype, reflected by maintenance of CD62L, CCR7 and CXCR4 expression. Moreover, transcriptome profiling revealed that AKT-inhibited CD8⁺ T cells clustered closely to naturally occurring stem cell-memory CD8⁺ T cells, while control T cells resembled effector-memory T cells. Interestingly, AKT-inhibited CD8⁺ T cells showed enrichment of hypoxia-associated genes, which was consistent with enhanced glycolytic function. Notably, AKT-inhibition during MiHA-specific CD8⁺ T cell priming uncoupled preservation of early memory differentiation from ex vivo expansion. Furthermore, AKT-inhibited MiHA-specific CD8⁺ T cells showed increased polyfunctionality with co-secretion of IFN-γ and IL-2 upon antigen recall. Together, these data demonstrate that AKT-inhibitors with different modality of action promote the ex vivo generation of stem cell memory-like CD8⁺ T cells with a unique metabolic profile and retained polyfunctionality. Akt-inhibitor VIII and GDC-0068 outperformed other inhibitors, and are therefore promising candidates for ex vivo generation of superior tumor-reactive T cells for adoptive immunotherapy in cancer patients.