Background CAR T cells have been highly effective against refractory B cell malignancies but have not demonstrated sustained antitumor effects against solid tumors. Intense effort is underway to augment the potency of CAR T cells in order to overcome the suppressive tumor microenvironment, which is associated with T cell exhaustion. Adenosine is a major mediator of immune suppression. CD39 (ecto-ATP diphosphohydrolase-1) plays a central role in the generation of adenosine by catalyzing the metabolism of ATP into ADP/AMP. CD73 (5'-ectonucleotidase) subsequently metabolizes ADP/AMP into adenosine which mediates immune suppression through adenosine associated receptor signaling. CD39 is also expressed by exhausted CD8+ and tumor reactive T cells within the tumor microenvironment, where it is associated with tumor progression, but it remains unclear whether exhausted and/or tumor reactive CD39+CD8+ T cells mediate immune suppression via adenosine.

Methods We developed a high affinity version of the disialoganglioside (GD2)-targeting CAR (HA-GD2) that spontaneously clusters on the surface of human T cells in the absence of antigen and mimics chronic antigen exposure leading to T cell exhaustion. Using this model, we demonstrate that exhausted CD39+CD8+ CAR T cells actively produce adenosine and mediate immune suppression through surface upregulation of CD39/73. In an attempt to generate adenosine resistance and enhance the function of exhausted CAR T cells, we knocked out CD39, CD73, or A2aR and overexpressed transmembrane-bound adenosine deaminase (ADA-TM).

Results Only overexpression of ADA-TM, which metabolizes adenosine to inosine, induced significant transcriptomic changes, higher frequency of stem- and central- like memory T cells, and a simultaneous decrease of exhausted T cell subpopulations. Direct exposure of HA-GD2 CAR T cells to high inosine concentration during cell manufacturing process, lead to a higher frequency of central-like memory cells and significant fitness enhancement associated with broad changes at the metabolic level. RNAseq and cyTOF analysis indicated decreased glycolytic flux, increased mitochondrial activity and glutamine and polyamine metabolism. Further, inosine altered the epigenetic state of HA-GD2 CAR T cells. We observed significant enrichment of IRF and NF-κB transcription factor motifs and motifs associated with memory differentiation in T cells grown in the presence of inosine. Finally, we showed that production of exhausted HA-GD2 and clinically-relevant version of GD2 CAR T cells in inosine-containing culture media enhances there in vivo efficacy, leading to improved survival of mice.

Conclusions We propose introducing inosine during cell manufacturing process as a novel strategy for improving clinical outcomes of CAR T cell therapy.

Ethics Approval Immunocompromised NOD-SCID-Il2rg−/− (NSG) mice were purchased from JAX and bred inhouse. All mice were bred, housed, and treated in ethical compliance with Stanford University

ACUC (APLAC) approved protocols Protocol ID 31287