Abstract
BackgroundCAR 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).MethodsIn 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.ResultsOur 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.ConclusionAltogether, 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.
Highlights
CAR T cell-based therapies have shown promising results in hematological malignancies
Generation of MSLN-CAR T cell and adenosine 2a receptor (A2aR) targeted MSLNCAR T cell using lentiviral gene transfer As alluded to earlier, in this study we aimed to combine the effects of mesothelin-targeting with diminished expression of A2aR adenosine receptors in CAR T cells
As described in materials and methods, four types of MSLN- CAR T cells were generated and analyzed: MSLN-CAR T cell; i.e. primary human T cells that were transduced with third generation lentiviral particles containing fully human anti mesothelin Single-chain variable fragments (scFv), a CD8a hinge domain, human 4-1BB TM region, and an intracellular signaling domain containing 4-1BB and CD3ζ domains (Fig. 1a); and three groups of anti-A2aR shRNAcontaining CAR T cells were referred as A2aR.KD1MSLN CAR T cells, A2aR.KD2-MSLN CAR T cells, and A2aR.KD3-MSLN CAR T cells (Fig. 1a)
Summary
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. CAR T cell-based therapies have shown promising results in the treatment of hematological malignancies including CD19 or CD22 positive B-cell acute lymphoblastic leukemia (ALL). Various factors have been suggested to contribute to such a lack of efficacy, including the scarcity of targetable tumor associated antigens and hostile TME, which might act as a key impediment for T cell function [1]. A2aR which is expressed at the surface of activated T cells, binds to adenosine with high affinity and leads to enhanced production of intracellular cyclic AMP (cAMP). Elevated levels of cAMP can attenuate the anti-tumor T cell responses [4, 7, 8]
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