Introduction: The adenosine pathways via type A2a and A2b receptors are known to suppress immunity by regulating the functions of immune cells, such as macrophages, dendritic cells, MDSC, T cells and NK cells in tumor microenvironment. Also, the adenosine pathway through an A1 receptor induces tumor growth by promoting cancer cell proliferation. On the other hand, it has been recently reported that the downregulation of A1R induces expression of tumor PD-L1. Herein, we introduce a novel antagonist of adenosine receptor, HM87277, which has potent antagonistic activity against three types of adenosine receptors A1R, A2aR and A2bR, and present preclinical results that the triple antagonist HM87277 may be more effective in treating cancer patients through appropriate combination with immune checkpoint blockers (ICBs).

Materials and Methods: Receptor binding affinities were assessed using human adenosine receptors of type A1, A2a, A2b and A3. Cellular functional activity tests such as cAMP production. To identify adenosine receptor downstream signaling in cancer cells, p-ERK and p-JNK were measured by western blotting in SK-BR3 cells. PD-L1 induction by adenosine receptor antagonist was determined by flow cytometry using Canto II (BD) in PD-L1 expressing SK-MEL-28 cells. For cytotoxicity assay of T cell, isolated human CD8+ T cells were co-cultured with A1R expressing cancer cells in the presence of a high concentration of adenosine. Antitumor activity of HM87277 alone and in combination with ICBs was assessed in syngeneic mouse models using murine cancer cell lines.

Results: HM87277 showed strong binding affinity toward human adenosine receptors of type A1, A2a and A2b, and potently inhibited the NECA-mediated production of intracellular cAMP in HEK293-A2aR cells. HM87277 significantly increased the proliferation of effector T cells. HM87277 potently inhibited the p-ERK and p-JNK, and induced the expression of PD-L1 in A1R-expressing cancer cells. As well, the combination of HM87277 and anti-PD-L1 mAb was observed to have synergistic inhibitory effect on tumor cell growth in co-culture with SK-MEL-28 and CD8 + T cells. Furthermore, in the mouse syngeneic model using murine cancer cell lines, HM87277 effectively inhibited tumor growth alone or in combination with anti-PD-L1 mAb.

Conclusion: These results indicate that a triple antagonist HM87277, which simultaneously antagonizes type A1, A2a and A2b adenosine receptors, may not only stimulate immune cells such as cytotoxic CD8+ T cell but also inhibit the growth of cancer cells themselves through inhibition of ERK and JNK pathway. In addition, it showed a strong combination effect by enhancing the sensitivity to ICBs through the increase of PD-L1 in tumor cells. Therefore, it is considered that the triple antagonist HM87277 is worth developing as a potential anticancer agent.

Citation Format: Sunyoung Jang, Seunah Jun, Hosun Lee, Yongtaek Lee, Joo-Yun Byun, Junghwa Park, Yu-Yon Kim, Young Gil Ahn, YoungHoon Kim, Kwee Hyun Suh. Discovery and characterization of a novel triple A1/A2a/A2b adenosine receptor antagonist for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1704.