Inosine Induces Stemness Features in HA-CAR-T cells and Enhances Potency [ATAC-seq]

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ HA-CAR-T cells commonly express CD39 and CD73, which mediate proximal steps in Ado generation. Here we sought to enhance HA-CAR-T cell potency by knocking out CD39, CD73 or adenosine receptor 2a (A2aR), but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced HA-CAR-T functionality. Similarly, and to a greater extent, exposure of HA-CAR-T cells to INO augmented HA-CAR-T cell function and induced hallmark features of T cell stemness. INO induced profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency HA-CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing. On day 14, CD39+ and CD39- CD4+ or CD8+ subsets were isolated using a BD FACSAria cell sorter (Stem Cell FACS Core, Stanford University School of Medicine). ADA-OE, CD39KO, CD73KO, A2aRKO and AAVS1KO HA-CAR-T cells were collected at day 15 post-activation. HA-CAR-T cells cultured in regular media or in the media containing INO but not GLC were collected at day 14.