Genetic and pharmacological targeting of A2a receptor improves function of anti-mesothelin CAR T cells

Abstract

Background: CAR T cell-based therapies have shown promising results in hematological malignancies. Results of CAR T cell projects in solid tumors have been less impressive, and factors including lack of targetable antigens and immunosuppressive tumor microenvironment (TME) have been suggested as culprits. Adenosine, a metabolite which is highly produced in TME, is known to mediate the suppression of anti-tumor T cell responses via binding and signaling through adenosine 2a receptor (A2aR). Methods: In this study, the expression of A2aR and the effects of its activation on the function of fully human anti-mesothelin CAR T cells (MSLN-CAR T), were analyzed. Afterwards, the molecular and pharmacological means to overcome the inhibitory effects of A2aR signaling on CAR T cell function were used. This was performed by targeting A2aR expression in MSLN-CAR T cells using various anti-A2aR shRNA sequences embedded in the CAR vector and an A2aR pharmacological antagonist, SCH-58261. Statistical analyses were performed Prism 7 software. Results: Our experiments showed significant A2aR upregulation on T cells during the CAR T cell production procedure (cell activation and transduction). Activation of adenosine signaling using adenosine analog led to the suppression of all major anti-tumor functions in MSLN-CAR T cells. Interestingly, CAR T cells that carried the anti-A2aR shRNA sequences were resistant to the inhibitory effects of adenosine signaling. Pharmacological inhibition of A2aR reversed the reduction in CAR T cell proliferation and cytokine response caused by the adenosine analog; however, it failed to rescue the cytotoxic function of the cells. Conclusion: Altogether, our results indicate that mitigating A2aR signaling by genetic targeting of the receptor might be a promising approach in improving CAR T cell function in an unreceptive microenvironment and could potentially improve the outcome of treatment in clinical settings.

The role of dysregulated immune responses in COVID-19 pathogenesis

“…Similar to some chronic infections, persistent virus exposure and/or inflammation along with the high levels of IL-10, as an inhibitory cytokine, lead to exhaustion T cells. Cytotoxic T cells, which play an important role against viral infections and cancers, increase the expression of some checkpoint inhibitor including PD-1 and Tim-3 and A2aR, leading to a loss of cytokine production capability, reduced cytotoxic function and T cell proliferation ( Diao et al, 2020 ; Masoumi et al, 2020 ).. Interestingly, the exhausted CD94/NK group 2 member A (NKG2A)+ cytotoxic lymphocytes increase in COVID-19 patients. NK cells also get exhausted of increasing the expression of NKG2A as an inhibitory receptor.…”

Mesothelin-targeted CAR-T cell therapy for solid tumors

“…Adenosine is an important immunosuppressive metabolite of the tumor microenvironment that through binding to its cognate receptor A2aR expressed by activated T cells leads to blockade of T cell anti-tumor activity [105]. For this reason, a recent study tested the activity of a fully human CD3ζ/4-1ΒΒ anti-MSLN CAR-T cell in combination with an A2aR knockdown approach via addition of anti-A2aR shRNA sequences in the CAR vector or in combination with an A2aR pharmacological antagonist [105].…”

“…Adenosine is an important immunosuppressive metabolite of the tumor microenvironment that through binding to its cognate receptor A2aR expressed by activated T cells leads to blockade of T cell anti-tumor activity [105]. For this reason, a recent study tested the activity of a fully human CD3ζ/4-1ΒΒ anti-MSLN CAR-T cell in combination with an A2aR knockdown approach via addition of anti-A2aR shRNA sequences in the CAR vector or in combination with an A2aR pharmacological antagonist [105]. The anti-MSLN CAR-T cells containing the anti-A2aR shRNA sequences were protected from the immunosuppressive and inhibitory effects of adenosine signaling and were able to exert their proliferative, cytokineproducing, and cytotoxic functions but the pharmacological inhibition of A2aR, although able to protect the proliferative and cytokine-producing functions of the CAR-T cells, failed to rescue their cytotoxic ability [105].…”

The Great War of Today: Modifications of CAR-T Cells to Effectively Combat Malignancies

“…The activation of CAR-T cells through either the TCR or CAR leads to a further increase of A2aR expression [ 191 ]. Targeting A2aR with either genetic or pharmacological blockade [ 192 ], which is now thoroughly investigated as an anticancer drug [ 193 ], was shown to enhance CAR-T cell efficacy, especially in combination with PD-1 inhibitors [ 191 ].…”