Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression
(1) Ma L (2) Zhang K (3) Xu J (4) Wang J (5) Jiang T (6) Du X (7) Zhang J (8) Huang J (9) Ren F (10) Liu D (11) Xue W (12) Kan D (13) Yao M (14) Liang Y (15) Jason-Sun H
To improve safety and efficacy of CAR T cell therapy for solid tumors, Ma, Zhang, and Xu et al. utilized the endogenous IFNγ promoter to regulate the expression of IL-15, which was inserted into the 3-UTR of the IFNγ gene. The IL-15 transgene did not impact IFNγ expression and was co-expressed with IFNγ in an antigen-dependent manner. In murine proof-of-concept studies, IL-15 expression, under control of the IFNγ promoter, dramatically increased the antitumor activity of claudin-targeted CAR T cells. A two-gene (CAR and IL-15) knock-in approach using a single AAV vector to streamline the process was developed, which was also effective in tumor control.
Contributed by Katherine Turner
(1) Ma L (2) Zhang K (3) Xu J (4) Wang J (5) Jiang T (6) Du X (7) Zhang J (8) Huang J (9) Ren F (10) Liu D (11) Xue W (12) Kan D (13) Yao M (14) Liang Y (15) Jason-Sun H
To improve safety and efficacy of CAR T cell therapy for solid tumors, Ma, Zhang, and Xu et al. utilized the endogenous IFNγ promoter to regulate the expression of IL-15, which was inserted into the 3-UTR of the IFNγ gene. The IL-15 transgene did not impact IFNγ expression and was co-expressed with IFNγ in an antigen-dependent manner. In murine proof-of-concept studies, IL-15 expression, under control of the IFNγ promoter, dramatically increased the antitumor activity of claudin-targeted CAR T cells. A two-gene (CAR and IL-15) knock-in approach using a single AAV vector to streamline the process was developed, which was also effective in tumor control.
Contributed by Katherine Turner
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.
