As one of the most promising adoptive cell therapies, CAR-T cell therapy has achieved notable clinical effects in patients with hematological tumors. However, several treatment-related obstacles remain in CAR-T therapy, such as cytokine release syndrome, neurotoxicity, and high-frequency recurrence, which severely limit the long-term effects and can potentially be fatal. Therefore, strategies to increase the controllability and safety of CAR-T therapy are urgently needed. In this study, we engineered a genetic code expansion-based therapeutic system to achieve rapid CAR protein expression and regulation in response to cognate unnatural amino acids at the translational level. When the unnatural amino acid N-ε-((tert-butoxy) carbonyl)-l-lysine (BOCK) is absent, the CAR protein cannot be completely translated, and CAR-T is “closed”. When BOCK is present, complete translation of the CAR protein is induced, and CAR-T is “open”. Therefore, we investigated whether the BOCK-induced device can control CAR protein expression and regulate CAR-T cell function using a series of in vitro and in vivo experiments. First, we verified that the BOCK-induced genetic code expansion system enables the regulation of protein expression as a controllable switch. We subsequently demonstrated that when the system was combined with CAR-T cells, BOCK could effectively and precisely control CAR protein expression and induce CAR signaling activation. When incubated with tumor cells, BOCK regulated CAR-T cells cytotoxicity in a dose-dependent manner. Our results revealed that the presence of BOCK enables the activation of CAR-T cells with strong anti-tumor cytotoxicity in a NOG mouse model. Furthermore, we verified that the BOCK-induced CAR device provided NK cells with controllable anti-tumor activity, which confirmed the universality of this device. Our study systematically demonstrated that the BOCK-induced genetic code expansion system effectively and precisely regulates CAR protein expression and controls CAR-T cell anti-tumor effects in vitro and in vivo. We conclude that this controllable and reversible switch has the potential for more effective, secure, and clinically available CAR-based cellular immunotherapies.

中文翻译：

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通过遗传密码扩展系统为基于 CAR 的细胞免疫疗法设计可控且可逆的开关


CAR-T 细胞疗法作为最具前景的过继细胞疗法之一，在血液肿瘤患者中取得了显著的临床效果。然而，CAR-T 疗法仍然存在一些与治疗相关的障碍，例如细胞因子释放综合征、神经毒性和高频复发，这些障碍严重限制了长期影响，并且可能是致命的。因此，迫切需要提高 CAR-T 疗法可控性和安全性的策略。在这项研究中，我们设计了一个基于遗传密码扩展的治疗系统，以实现 CAR 蛋白的快速表达和调节，以响应翻译水平的同源非天然氨基酸。当非天然氨基酸 N-ε-（叔丁氧基）羰基）-l-赖氨酸 （BOCK） 不存在时，CAR 蛋白就不能完全翻译，CAR-T 就“闭合”了。当 BOCK 存在时，会诱导 CAR 蛋白的完全翻译，并且 CAR-T 是“开放的”。因此，我们通过一系列体外和体内实验研究了 BOCK 诱导的装置是否可以控制 CAR 蛋白表达和调节 CAR-T 细胞功能。首先，我们验证了 BOCK 诱导的遗传密码扩展系统能够将蛋白质表达作为可控开关进行调节。我们随后证明，当该系统与 CAR-T 细胞结合时，BOCK 可以有效、精确地控制 CAR 蛋白表达并诱导 CAR 信号激活。当与肿瘤细胞一起孵育时，BOCK 以剂量依赖性方式调节 CAR-T 细胞的细胞毒性。我们的结果表明，BOCK 的存在能够在 NOG 小鼠模型中激活具有强抗肿瘤细胞毒性的 CAR-T 细胞。 此外，我们验证了 BOCK 诱导的 CAR 装置为 NK 细胞提供了可控的抗肿瘤活性，这证实了该装置的普遍性。我们的研究系统地证明了 BOCK 诱导的遗传密码扩增系统在体外和体内有效、精确地调节 CAR 蛋白表达并控制 CAR-T 细胞的抗肿瘤作用。我们得出结论，这种可控和可逆的转换有可能实现更有效、更安全和临床可用的基于 CAR 的细胞免疫疗法。