Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8 CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.

中文翻译：

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肌苷诱导 CAR-T 细胞干性特征并增强效力

腺苷 (Ado) 介导肿瘤微环境中的免疫抑制，并且耗尽的 CD8 CAR-T 细胞表达 CD39 和 CD73，从而介导 Ado 生成的近端步骤。在这里，我们试图通过敲除 CD39、CD73 或腺苷受体 2a (A2aR) 来增强 CAR-T 细胞效力，但仅观察到适度的效果。相比之下，Ado 脱氨酶 (ADA-OE) 的过度表达（将 Ado 代谢为肌苷 (INO)）会诱导干性并增强 CAR-T 功能。同样，CAR-T 细胞暴露于 INO 会增强功能并诱导干性特征。INO 诱导深刻的代谢重编程，减少糖酵解，增加线粒体和糖酵解能力，谷氨酰胺分解和多胺合成，并将表观基因组重新编程为更大的干性。使用 INO 进行临床规模生产，产生符合临床剂量标准的增强效力 CAR-T 细胞产品。这些结果表明 INO 是 CAR-T 细胞代谢和表观遗传干性编程的有效调节剂，并为细胞制造提供增强的效力平台。