A_2B adenosine receptor antagonists rescue lymphocyte activity in adenosine-producing patient-derived cancer models

Adenosine is a metabolite that suppresses antitumor immune response of T and NK cells via extracellular binding to the two subtypes of adenosine-2 receptors, A₂ARs. While blockade of the A_2AARs subtype effectively rescues lymphocyte activity, with four A_2AAR antagonists currently in anticancer clinical trials, less is known for the therapeutic potential of the other A_2BAR blockade within cancer immunotherapy. Recent studies suggest the formation of A_2AAR/A_2BAR dimers in tissues that coexpress the two receptor subtypes, where the A_2BAR plays a dominant role, suggesting it as a promising target for cancer immunotherapy.

We report the synthesis and functional evaluation of five potent A_2BAR antagonists and a dual A_2AAR/A_2BAR antagonist. The compounds were designed using previous pharmacological data assisted by modeling studies. Synthesis was developed using multicomponent approaches. Flow cytometry was used to evaluate the phenotype of T and NK cells on A_2BAR antagonist treatment. Functional activity of T and NK cells was tested in patient-derived tumor spheroid models.

We provide data for six novel small molecules: five A_2BAR selective antagonists and a dual A_2AAR/A_2BAR antagonist. The growth of patient-derived breast cancer spheroids is prevented when treated with A_2BAR antagonists. To elucidate if this depends on increased lymphocyte activity, immune cells proliferation, and cytokine production, lymphocyte infiltration was evaluated and compared with the potent A_2AAR antagonist AZD-4635. We find that A_2BAR antagonists rescue T and NK cell proliferation, IFNγ and perforin production, and increase tumor infiltrating lymphocytes infiltration into tumor spheroids without altering the expression of adhesion molecules.

Our results demonstrate that A_2BAR is a promising target in immunotherapy, identifying ISAM-R56A as the most potent candidate for A_2BAR blockade. Inhibition of A_2BAR signaling restores T cell function and proliferation. Furthermore, A_2BAR and dual A_2AAR/A_2BAR antagonists showed similar or better results than A_2AAR antagonist AZD-4635 reinforcing the idea of dominant role of the A_2BAR in the regulation of the immune system.