Rational engineering of an improved adenosine deaminase 2 enzyme for weaponizing T-cell therapies.

Adenosine is a potent immunosuppressive metabolite that accumulates in the extracellular space within solid tumors and inhibits the antitumor function of native immune cell responses as well as chimeric antigen receptor (CAR) T-cell therapies. Here, we show that engineered human cells can degrade extracellular adenosine through secretion of adenosine deaminase (ADA) enzymes-a possible therapeutic enhancement for CAR T cells. We first determine that the high-activity ADA1 isoform is naturally intracellularly restricted and show that the addition of canonical or computationally predicted secretory peptides did not allow for improved secretion. We did, however, determine that the lower-activity ADA2 isoform is naturally secreted. Thus, we utilized phylogenetic-based structural comparisons to guide a mutational survey of ADA2 active site residues, which when coupled with a high-throughput screen for enhanced ADA2-mediated extracellular adenosine rate allowed isolation of the most catalytically efficient ADA2 variant reported to date. When expressed by human cells, this variant exhibits 30× higher extracellular adenosine degradation activity than the wild-type enzyme. Finally, we demonstrate that Jurkat and CAR T cells engineered to express this secreted, high-activity ADA2 variant can degrade significant amounts of extracellular adenosine