Liu Yue, An Lingna, Wang Xiaoqi, Dai Yueyu, Zhang Cheng, Wen Qin, Zhang Xi
Medical Center of Hematology, Xinqiao Hospital of Army Medical University, Chongqing, 400037, China.
State Key Laboratory of Trauma and Chemical Poisoning, Chongqing Key Laboratory of Hematology and Microenvironment, Chongqing, 400037, China.
J Hematol Oncol. 2024 Dec 18;17(1):122. doi: 10.1186/s13045-024-01648-0.
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
背景：作为最有前景的过继性细胞疗法之一，嵌合抗原受体T细胞（CAR-T）疗法在血液系统肿瘤患者中取得了显著的临床疗效。然而，CAR-T疗法仍存在一些与治疗相关的障碍，如细胞因子释放综合征、神经毒性和高频复发等，这些严重限制了其长期疗效，甚至可能是致命的。因此，迫切需要提高CAR-T疗法可控性和安全性的策略。 方法：在本研究中，我们构建了一种基于遗传密码扩展的治疗系统，以在翻译水平上实现对同源非天然氨基酸响应的CAR蛋白快速表达和调控。当不存在非天然氨基酸N-ε-（（叔丁氧基）羰基）-L-赖氨酸（BOCK）时，CAR蛋白无法完全翻译，CAR-T处于“关闭”状态。当存在BOCK时，可诱导CAR蛋白的完全翻译，CAR-T处于“开启”状态。因此，我们通过一系列体外和体内实验研究了BOCK诱导装置是否能够控制CAR蛋白表达并调节CAR-T细胞功能。 结果：首先，我们验证了BOCK诱导的遗传密码扩展系统能够作为一个可控开关调节蛋白表达。随后我们证明，当该系统与CAR-T细胞结合时，BOCK能够有效且精确地控制CAR蛋白表达并诱导CAR信号激活。与肿瘤细胞共孵育时，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，在NOG小鼠模型中，BOCK的存在能够激活具有强大抗肿瘤细胞毒性的CAR-T细胞。此外，我们验证了BOCK诱导的CAR装置赋予自然杀伤细胞（NK细胞）可控的抗肿瘤活性，这证实了该装置的通用性。 结论：我们的研究系统地证明了BOCK诱导的遗传密码扩展系统在体外和体内均能有效且精确地调节CAR蛋白表达并控制CAR-T细胞的抗肿瘤作用。我们得出结论，这种可控且可逆的开关具有实现更有效、安全且临床可用的基于CAR的细胞免疫疗法的潜力。
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