Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression
Liya Ma, Kaiwen Zhang, Jian Xu, Jian Wang, Ting Jiang, Xiaolong Du, Jiaxin Zhang, Jing Huang, Fengyi Ren, Dong Liu, Weiwei Xue, Dongxu Kan, Mengjiao Yao, Yutian Liang, Hongxing Jason-Sun
Abstract
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. In vitro and in vivo 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.