Abstract A10: The dual A2aR/A2bR antagonist AB928 reverses adenosine-mediated immune suppression and inhibits tumor growth in vivo

Abstract

Introduction: High levels of immunosuppressive adenosine are found in the tumor microenvironment, reaching 50-100 μM in experimental models. Adenosine exerts its effects on immune cells primarily through the adenosine receptors A2aR/A2bR, which increase intracellular levels of cyclic AMP, leading to CREB phosphorylation (pCREB). We have previously shown that the dual A2aR/A2bR antagonist AB928 is capable of inhibiting adenosine-induced pCREB in healthy human volunteer (HV) blood lymphocytes. AB928 has also been shown to relieve adenosine-mediated T-cell suppression in vitro and exhibit combinatorial effects with standard-of-care chemotherapeutics in mouse syngeneic tumor models. Herein, we show that AB928 is capable of inhibiting NECA-induced gene expression changes and CREB phosphorylation in non-small cell lung carcinoma (NSCLC) patient whole blood (WB). Additionally, observations from our in vitro human studies showing the combinatorial effect of AB928 and α-PD-1 were reproduced in B16F10 syngeneic tumors. Methods: Human WB was stimulated with 5 μM of NECA and flow cytometry was used to quantify AB928-mediated inhibition of pCREB and CD3ζ phosphorylation. B16F10 tumors were treated with α-PD-1 +/- AB928 and gene expression was determined from excised mouse tumors using the nCounter PanCancer panel. Results: To ensure AB928 can successfully inhibit the high levels of intratumoral adenosine, we found that 5 μM NECA provides maximal stimulation and is significantly more potent (>20 fold) than adenosine in the pCREB assay. Additional experiments demonstrated that AB928 has comparable potency in NECA-stimulated WB from both HV and NSCLC patients. In addition to blocking downstream signaling, NanoString analysis showed that AB928 could prevent NECA-stimulated gene expression changes in NSCLC WB. We also found that NECA stimulation, alone or in combination with PD-1 inhibition, significantly reduced proximal TCR signaling, leading to reduced levels of CD3ζ phosphorylation at TYR142 (pTYR142). These reduced pTYR142 levels, with and without α-PD-1, could be significantly rescued by AB928, suggesting that blocking adenosine immunosuppression may provide additional benefit to PD-1 inhibition in tumors. Consistent with these results, AB928 was capable of suppressing growth (volume in mm3) of B16-F10 tumors both as a single agent (vehicle: 462 +/- 58; AB928: 292 +/- 55; p<0.05) or in combination with α-PD-1 therapy (α-PD-1: 341 +/- 60; AB928+α-PD-1: 123 +/- 30; p<0.05). Further, NanoString analysis of the tumors showed an increased “T-cell Functions” score with both α-PD-1 and AB928 monotherapy, which was further enhanced in combination treatment. Conclusions: Collectively, these results support a role for AB928 in relieving adenosine-mediated immunosuppression by blocking A2aR/A2bR-induced signaling events, gene expression changes, and suppressing tumor growth in vivo. Citation Format: Daniel M. DiRenzo, Nell Narasappa, Dana Piovesan, Devika Ashok, Park Adam, Jenna L. Jeffrey, Manmohan R. Leleti, Joanne B.L. Tan, Lisa Seitz, Steve W. Young, Jay P. Powers, Matthew J. Walters. The dual A2aR/A2bR antagonist AB928 reverses adenosine-mediated immune suppression and inhibits tumor growth in vivo [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A10.