CRISPR-Cas9 knockout of DGKα/ζ improves the anti-tumor activities of TAG-72 CAR-T cells in ovarian cancer

High recurrence and chemoresistance in solid tumors, like ovarian cancer, stress the need for new therapies. Chimeric antigen receptor (CAR)-T cells show promise but face challenges due to tumor heterogeneity and immune suppression in the tumor microenvironment (TME). Thus, novel approaches are needed to further enhance the efficacy of CAR-T cell therapies. In T cell therapies, inhibiting checkpoint molecules is crucial for overcoming exhaustion and boosting anti-tumor activity. Additionally, prioritizing safety by engineering cells to target markers absent on normal healthy cells reduces off-target risks. We targeted tumor-associated glycoprotein 72 (TAG-72), an oncofetal antigen highly expressed in adenocarcinomas like ovarian cancer, by engineering TAG-72 CAR-T cells and used CRISPR-Cas9 to knock out the T cell-inhibitory enzymes diacylglycerol kinase (DGK) α and ζ. DGKα/ζ knockout (KO) did not impact CAR-T cell viability or phenotype. These cells selectively killed TAG-72-expressing cancer cells <i>in vitro</i> and ablated established tumors <i>in vivo</i> for up to 100 days, whereas non-deleted control TAG-72 CAR-T cells showed tumor relapse around 40 days. These findings highlight the potential of CRISPR-induced DGKα/ζ KO to enhance CAR-T cell efficacy against solid tumors such as ovarian cancer, offering a promising avenue for improved cancer therapies.