Despite the remarkable success of chimeric antigen receptor (CAR) T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous interferon (IFN)-γ promoter for transgenic interleukin (IL)-15 expression. We demonstrate that IFN-γ expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3′ UTR of the IFN-γ gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. and data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Last, as a part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.

中文翻译：

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利用内源性 IFN-γ 启动子进行细胞因子表达，构建新型 TRUCK


尽管嵌合抗原受体（CAR）T疗法在血液恶性肿瘤中取得了显着成功，但其在实体瘤中的疗效仍然有限。细胞因子工程化的 CAR T 细胞提供了一条有前途的途径，但其临床转化受到与组成型细胞因子表达相关的风险的阻碍。在这项概念验证研究中，我们利用内源干扰素 (IFN)-γ 启动子进行转基因白细胞介素 (IL)-15 表达。我们证明 IFN-γ 表达受到 T 细胞受体信号传导的严格调控。通过同源定向修复介导的敲入，将内部核糖体进入位点 IL15 引入 IFN-γ 基因的 3'UTR，我们证实 IL-15 表达可以与 IFN-γ 以抗原刺激依赖性方式共表达方式。重要的是，转基因的插入不会损害内源性 IFN-γ 的表达。数据表明，IFN-γ启动子驱动的IL-15可显着提高CAR T细胞的抗肿瘤活性，表明IL-15表达的有效性。最后，作为我们临床转化努力的一部分，我们开发了一种创新的双基因敲入方法。这种方法能够使用单个 AAV 载体将 CAR 和 IL-15 基因同时整合到 TRAC 和 IFN-γ 基因位点中。使用这种方法设计表达 IL-15 的 CAR T 细胞表现出增强的抗肿瘤功效。总体而言，我们的研究强调了利用内源启动子在 CAR T 细胞中表达转基因细胞因子的可行性。