Tumor-site directed A1R expression enhances CAR T cell function and improves efficacy against solid tumors. [Human RNAseq inducible]

The efficacy of Chimeric Antigen Receptor (CAR) T cells against solid tumors is limited by immunosuppressive factors in the tumor microenvironment (TME) including adenosine, which suppresses CAR T cells through activation of the A2A receptor (A2AR). To overcome this, CAR T cells were engineered to express A1 receptor (A1R), a receptor that signals inversely to A2AR. Using murine and human CAR T cells, constitutive A1R overexpression was demonstrated to significantly enhance CAR T cell effector function albeit at the expense of CAR T cell persistence. Through a novel CRISPR/Cas9 “knock-in” approach we demonstrated that CAR T cells engineered to express A1R in a tumor-localized manner, led to enhanced anti-tumor efficacy dependent on the transcription factor IRF8 and was transcriptionally unique when compared to A2AR deletion. This data provides a novel approach for enhancing CAR T cell efficacy in solid tumors and provides proof of principle for site-directed expression of factors that promote effector T cell differentiation. Overall design: RNA-seq profiling of NR4A2 promoter driven inducible A1R CD8+ human anti-LewisY CAR T cells chronically stimulated with up to 3 rounds of OVCAR ovarian tumors over 72 hours in the presence or absence of A2AR agonist NECA (10uM). CAR-T cells were FACS sorted and RNA extracted using RNAeasy (Qiagen) kit as per manufacturer's instructions.