IFN1 Blockade Enhances Non-Activated CAR T Cell Anti-Tumor Function Via Inhibiting the Innate Anti-Viral Response

Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented success in treating blood-based cancers. However, the durability of response is often hindered by challenges related to long-term T cell persistence and engraftment. The efficacy of CAR T cell immunotherapy relies on the differentiation status and overall fitness of the CAR T cell product. Current protocols involve the activation and ex vivo expansion of patient T cells, however, activation leads to irreversible differentiation, compromising their therapeutic potency. Manufacturing protocols utilizing non-activated T cells results in superior differentiation characteristics and reduced exhaustion, with concordant benefits in long-term tumor control. Nevertheless, as quiescent T cells are highly resistant to lentiviral infection, CAR T manufacturing yield is a significant limitation with non-activated T cells. In this study, we investigated the impact of IFN1 blockade on CAR lentivirus transduction and function of non-activated T cells. Quiescent T cells were treated with an IFN1 binding protein, or vehicle, and transduced with CAR19 lentiviral vectors. Our findings demonstrate that lentiviral vector transduction triggers an innate immune response in non-activated T cells, resulting in reduced transduction efficiency, and altered T cell differentiation. Inhibiting the anti-viral response via blockade of IFN1 during transduction promotes a more naïve and central memory phenotype. We further investigate the effect of IFN1 blockade by lentiviral-mediated expression of CAR as well as a secreted IFN1-binding protein. These CAR T cells show enhanced cytolytic activity and cytokine production against target cells after repeated antigen exposure. We, therefore, tested the effect of IFN1 blockade in a preclinically validated xenograft NALM6 model of leukemia. After establishing xenografts, a suboptimal dose of CART19 (1e6 T cells), generated in the presence of IFN1 binding protein, was infused. These CAR T cells demonstrated enhanced tumor control relative to standard CAR T cells. Our studies in vivo suggest that the sustained secretion of the protein to block IFN1 may enhance the therapeutic activity of T cells in tumors reliant on IFN-mediated immune evasion. Our results identify a novel approach to enhance non-activated CAR T cell transduction and fitness by countering anti-viral defenses triggered during gene delivery. Mechanistically we show that blocking IFN1 signaling with a binding protein improves transduction and anti-tumor function of non-activated CAR T cells during repeated antigen exposure. Given the therapeutic promise of non-activated CAR T cells, our findings have immediate translational relevance for broadening their appeal against cancer.