Inosine Induces Stemness Features in CAR T cells and Enhances Potency.

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8 ⁺ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data 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.
Competing Interests: Conflict of interests D.D.K, S.A.F., and C.L.M. are co-inventors on a pending patent application number 63/358,996 for inosine media supplementation during cell manufacturing. D.D.K and C.L.M. are inventors on a patent application number PCT/US2022/075584 that covers the use of T cells overexpressing ADA1/2 for cancer immunotherapy. C.L.M. is a cofounder of and holds equity in Lyell Immunopharma and Link Cell Therapies. C.L.M., and L.L. are co-founders of and hold equity in CARGO Therapeutics. L.L. consults for and holds equity in Lyell Immunopharma. E.S holds equity in Lyell Immunopharma and consults for Lepton Pharmaceuticals and Galaria. S.A.F. serves on the Scientific Advisory Boards for Alaunos Therapeutics and Fresh Wind Biotech and has equity interest in both; S.A.F. receives research funding from CARGO and Tune Therapeutics. S.P. is a current employee of and holds equity in CARGO. C.L.M. consults for Lyell, CARGO, Link, Apricity, Nektar, Immatics, Mammoth, and Ensoma. A.T.S. is a cofounder of Immunai and Cartography Biosciences. A.T.S. receives research funding from Allogene Therapeutics and Merck Research Laboratories.