CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A(2A) receptor (A(2A)R). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A(2A)R with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A(2A)R are superior to shRNA mediated knockdown or pharmacological blockade of A(2A)R. Mechanistically, human A(2A)R-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A(2A)R deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A(2A)R for the improvement of CAR T cell function in the clinic.