Developing oxidative stress-resistant CAR-T for solid tumors Allison J Nipper1, Emilie AK Warren1, Caroline E Porter2,3, Tim Sauer3, Mariana Villanueva1, Hugo Villanueva1, Masataka Suzuki2,3, Andrew G Sikora1,4 1Department of Otolaryngology, Baylor College of Medicine, Houston, TX 2Department of Medicine; BCM 3Center for Cell and Gene Therapy, BCM; 4Department of Head and Neck Surgery, MD Anderson, Houston TX High levels of oxidative stress in the solid tumor microenvironment inflict cellular damage and alter functionality of tumor infiltrating lymphocytes. The hostile environment of solid tumors therefore poses a challenge to immunotherapy, which moderates the action of immune cells to combat tumor growth. As a result, T cells transduced with Chimeric Antigen Receptors (CAR-T) for solid tumors have not shown the success observed for leukemia and lymphoma. To combat the effect of oxidative stress on tumor-specific T cells, we have developed a novel approach to CAR-T protection by incorporating the damage suppressor (Dsup) gene from the “extremotolerant” organism the tardigrade. Tardigrades show extreme oxidative stress resistance to alleviate cellular damage during desiccation. Therefore, we hypothesize that transgenic expression of tardigrade oxidative stress response proteins in CAR-T will enhance their survival and function in solid tumors and will mitigate effects of stress on anti-tumor response. To this end, we have generated CAR-T expressing tardigrade oxidative stress response gene Dsup to explore their functionality in solid tumor environments. To determine if Dsup enhanced T cell survival under metabolic stress, we transduced primary T cells with tardigrade stress response gene Dsup or a vector only control. When cultured with H2O2 to generate oxidative stress, T cells transduced with Dsup have greater viability than T cells receiving only the control vector. To determine if the expression of Dsup affected the anti-tumor efficacy of CAR T we cocultured HER2 CAR-T with and without Dsup for 48 hours with HER2-expressing FaDu tumor cells, then quantified tumor cell death by flow cytometry. In preliminary data, we observed HER2-specific CAR-T with Dsup to kill tumor cells in culture. To determine if Dsup improves CAR-T ability to target or kill cells in vivo, CAR-T transduced with antioxidant gene Dsup were used to treat FaDu tumors grown in the chick chorioallantoic membrane model. After treatment with Dsup-HER2.CAR-T, tumors were harvested for analysis, at which time T cell infiltrates were seen throughout the tumor in 3D reconstructions generated by confocal microscopy. Together, this work suggests antioxidant genes have potential to enhance CAR-T efficacy in solid tumors through oxidative stress protection.