Juno Therapeutics GmbH, A Bristol-Myers Squibb Company, Grillparzerstr. 10, Munich 81675, Germany.
Juno Therapeutics GmbH, A Bristol-Myers Squibb Company, Grillparzerstr. 10, Munich 81675, Germany.
J Immunol Methods. 2024 Mar;526:113617. doi: 10.1016/j.jim.2024.113617. Epub 2024 Jan 11.
Immunotherapy using TCR and especially CAR transgenic T cells is a rapidly advancing field with the potential to become standard of care for the treatment of multiple diseases. While all current FDA approved CAR T cell products are generated using lentiviral gene transfer, extensive work is put into CRISPR/Cas mediated gene delivery to develop the next generation of safer and more potent cell products. One limitation of all editing systems is the size restriction of the knock-in cargo. Targeted integration under control of an endogenous promotor and/or signaling cascades opens the possibility to reduce CAR gene size to absolute minimum. Here we demonstrate that a first-generation CAR payload can be reduced to its minimum component - the antigen-binding domain - by targeted integration under control of the CD3ε promoter generating a CAR-CD3ε fusion protein that exploits the endogenous TCR signaling cascade. Miniaturizing CAR payload in this way results in potent CAR activity while simultaneously retaining the primary antigen recognition function of the TCR. Introducing CAR-specificity using a CAR binder only while maintaining endogenous TCR function may be an appealing design for future autologous CAR T cell therapies.
免疫疗法使用 TCR 和特别是 CAR 转基因 T 细胞是一个快速发展的领域，有可能成为治疗多种疾病的标准治疗方法。虽然所有目前获得 FDA 批准的 CAR T 细胞产品都是使用慢病毒基因转移生成的，但大量工作正在投入到 CRISPR/Cas 介导的基因传递中，以开发下一代更安全、更有效的细胞产品。所有编辑系统的一个限制是敲入 cargo 的大小限制。在内源性启动子和/或信号级联的控制下进行靶向整合，为将 CAR 基因大小减小到绝对最小值开辟了可能性。在这里，我们证明通过靶向整合在 CD3ε 启动子的控制下，可以将第一代 CAR 有效载荷减小到其最小组成部分 - 抗原结合结构域 - 生成利用内源性 TCR 信号级联的 CAR-CD3ε 融合蛋白。通过这种方式使 CAR 有效载荷小型化可产生有效的 CAR 活性，同时保留 TCR 的主要抗原识别功能。仅使用 CAR 结合物引入 CAR 特异性，同时保持内源性 TCR 功能，可能是未来自体 CAR T 细胞疗法的一种有吸引力的设计。
