利用内源性 IFN-γ 启动子驱动细胞因子表达构建新型 TRUCK

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)- promoter for transgenic interleukin (IL)-15 expression.

We demonstrate that IFN- expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3' UTR of the IFN- gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN- in an antigen stimulation-dependent manner.

Importantly, the insertion of transgenes does not compromise endogenous IFN- expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN- 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- 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.