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Background Despite remarkable response rates mediated by anti-CD19 chimeric antigen receptor (CAR) T cells in selected B cell malignancies, CAR T cell therapy still lacks efficacy in the vast majority of tumors. A substantial limiting factor of CAR T cell function is the immunosuppressive tumor microenvironment. Among other mechanisms, the accumulation of adenosine within the tumor can contribute to disease progression by suppressing anti-tumor immune responses. Adenosine 2a- and 2b-receptor (A2A and A2B)-mediated cAMP build-up suppresses T cell effector functions. In the present study we hypothesize, that combination therapy with the selective A2A/A2B dual antagonist AB928 (etrumadenant) enhances CAR T cell efficacy. Materials and Methods Second generation murine (anti-EPCAM) and human (anti-MSLN) CAR constructs, containing intracellular CD28 and CD3ζ domains, were fused via overlap extension PCR cloning. Murine or human T cells were retrovirally transduced to stably express the CAR constructs. A2A/A2B signaling in CAR T cells was analyzed by phospho-specific flow cytometry of CREB (pS133)/ATF-1 (pS63). CAR T cell activation was quantified by flow cytometry and enzyme-linked immunosorbent assay (ELISA) of IFN-γ, IL-2 and TNF-α. CAR T cell proliferation was assessed by flow cytometry. CAR T cell cytotoxicity was assessed by impedance based real-time cell analysis. Results AB928 protected murine CAR T cells from cAMP response element-binding protein (CREB) phosphorylation in the presence of stable adenosine analogue 5′-N-ethylcarboxamidoadenosine (NECA). NECA inhibited antigen-dependent CAR T cell cytokine secretion in response to four murine tumor cell lines. CAR T cell-mediated tumor cell lysis as well as proliferation were decreased in the presence of NECA or adenosine. Importantly, AB928 fully restored CAR T cell cytotoxicity, proliferation, and cytokine secretion in a dose dependent manner. Further, AB928 also restored antigen dependent cytokine secretion of human CAR T cells in the presence of NECA. Conclusions Here we used the A2A/A2B dual antagonist AB928 to overcome adenosine-mediated suppression of CAR T cells. We found that AB928 enhanced important CAR T cell effector functions in the presence of the adenosine analogue, suggesting that combination therapy with AB928 may improve CAR T cell efficacy. This study was limited to in vitro experiments. To confirm the relevance of our findings, this combination therapy must be further investigated in an in vivo setting. Disclosure Information M. Seifert: None. M. Benmebarek : None. B. Cadilha : None. J. Jobst: None. J. Dorr: None. T. Lorenzini: None. D. Dhoqina: None. J. Zhang: None. J. Zhang: None. U. Schindler: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Amgen Inc., Arcus Biosciences. Other; Significant; Arcus Biosciences. S. Endres: None. S. Kobold: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Arcus Biosciences. |
