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

Lauren Giuffrida,Christina Mølck,Benjamin J Solomon,Kirsten L Todd,Amanda X Y Chen,Junyun Lai,Phillip K Darcy,Simon J Harrison,Ian A Parish,Gregory D Stewart,Melissa A Henderson,Sherly Mardiana,Emma V Petley,Imran G House,Lev M Kats,Kevin Sek,Paul J Neeson,Paul A Beavis,Deborah Meyran

doi:10.1038/s41467-021-23331-5

Abstract

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 A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR 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 A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-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. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.

Highlights

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 A2A receptor (A2AR)
chimeric antigen receptor (CAR) T cells were stimulated with an anti-CAR antibody in the presence or absence of NECA, an adenosine mimetic, as well as SCH58261, a selective A2AR antagonist
In the context of cells activated through the CAR, transcriptional changes induced by NECA were fully reversible with addition of the A2AR antagonist SCH58261, demonstrating that adenosine acts on CAR T-cell effector function principally through A2AR activation (Fig. 1B)

Summary

Introduction

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 A2A receptor (A2AR) Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas[9] strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Approved for the treatment of relapsed B-cell acute lymphoblastic leukemia and aggressive lymphoma following remarkable clinical response rates and defined curative potential[3] These effects have not been recapitulated in the solid tumor setting, where CAR T cells are faced with additional barriers such as tumor antigen heterogeneity, the requirement to traffic to the tumor site, and an immunosuppressive, hypoxic tumor microenvironment[1,2]. This work has set the scene for alternative strategies to target this pathway, including therapeutics directed toward the upstream ectoenzymes CD73, CD39, and CD38, or the upstream Hypoxia-HIF-1α axis itself[8,10] signifying the clinical interest in targeting this pathway[13]

Methods

Results

Conclusion