虽然 A2A 腺苷受体 (AR) 被认为是腺苷介导的免疫抑制的主要贡献者，但与腺苷亲和力最低的 A2B 也被认为是促进肿瘤生长的潜在贡献者。因此，在富含腺苷的肿瘤微环境（TME）中，A2B可以与A2A互补和/或补偿，同时靶向A2A和A2B AR可以为癌症免疫治疗提供更高的潜力。我们开发了 M1069 - A2A 和 A2B AR 的高度选择性双重拮抗剂。在对人类和小鼠原代免疫细胞进行的检测中，M1069 在高腺苷环境下挽救了 T 细胞（A2A 依赖性）产生 IL 2，并抑制骨髓细胞（A2B 依赖性）产生 VEGF。与 A2A 选择性拮抗剂 (A2Ai) 相比，M1069 还表现出对促肿瘤细胞因子 CXCL1、CXCL5 分泌的卓越抑制作用，并拯救腺苷分化树突状细胞的 IL 12 分泌。在一项单向混合淋巴细胞反应 (MLR) 测定中，与 A2Ai 存在下分化的树突状细胞相比，用 M1069 处理的腺苷分化的人和鼠树突状细胞表现出优异的 T 细胞刺激活性。在体内，M1069 作为单一疗法可减少肿瘤生长，并在同基因腺苷 hi/CD73hi 4T1 乳腺肿瘤模型中增强 bintrafusp alfa (BA) 或顺铂的抗肿瘤活性，但在 CD73 敲除 (KO) 4T1 肿瘤模型或腺苷低/ CD73low MC38 小鼠结肠癌模型。总之，我们的双重 A2A/A2B AR 拮抗剂 M1069 可以在体外和体内抵消高浓度腺苷的免疫抑制机制，并增强其他药物（包括 BA 和顺铂）的抗肿瘤活性。

While A2A adenosine receptor (AR) was considered as a major contributor to adenosine-mediated immunosuppression, A2B, having the lowest affinity to adenosine, has also emerged as a potential contributor to tumor promotion. Therefore, in adenosine-rich tumor microenvironment (TME), where A2B could be complementary and/or compensatory to A2A, simultaneous targeting of A2A and A2B ARs can provide higher potential for cancer immunotherapy. We developed M1069 - a highly selective dual antagonist of the A2A and A2B AR. In assays with primary human and murine immune cells, M1069 rescued IL 2 production from T cells (A2A dependent) and inhibited VEGF production by myeloid cells (A2B dependent) in adenosine-high settings. M1069 also demonstrated superior suppression of secretion of pro tumorigenic cytokines CXCL1, CXCL5, and rescue of IL 12 secretion from adenosine differentiated dendritic cells compared to an A2A selective antagonist (A2Ai). In a one-way mixed lymphocyte reaction (MLR) assay, adenosine differentiated human and murine dendritic cells treated with M1069 demonstrated superior T cell stimulatory activity compared to dendritic cells differentiated in presence of A2Ai. In vivo, M1069 decreased tumor growth as a monotherapy and enhanced anti-tumor activity of bintrafusp alfa (BA) or cisplatin in syngeneic adenosinehi/CD73hi 4T1 breast tumor model, but not in the CD73 knockout (KO) 4T1 tumor model or in adenosinelow/CD73low MC38 murine colon carcinoma model. In summary, our dual A2A/A2B AR antagonist M1069 may counteract immune-suppressive mechanisms of high concentrations of adenosine in vitro and in vivo and enhance the anti-tumor activity of other agents, including BA and cisplatin.