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

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 NGFR control, A1R and A3R constitutively expressing mouse anti-HER2 CAR T cells were stimulated with E0771-HER2 breast cancer cells for 16 hours. CAR-T cells were FACS sorted and RNA extracted using RNAeasy (Qiagen) kit as per manufacturer's instructions.