Glypican-3–Specific CAR T Cells Coexpressing IL15 and IL21 Have Superior Expansion and Antitumor Activity against Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death in the world, and curative systemic therapies are lacking. Chimeric antigen receptor (CAR)-expressing T cells induce robust antitumor responses in patients with hematologic malignancies but have limited efficacy in patients with solid tumors, including HCC. IL15 and IL21 promote T-cell expansion, survival, and function and can improve the antitumor properties of T cells. We explored whether transgenic expression of IL15 and/or IL21 enhanced glypican-3-CAR (GPC3-CAR) T cells' antitumor properties against HCC. We previously optimized the costimulation in GPC3-CARs and selected a second-generation GPC3-CAR incorporating a 4-1BB costimulatory endodomain (GBBz) for development. Here, we generated constructs encoding IL15, IL21, or both with GBBz (15.GBBz, 21.GBBz, and 21.15.GBBz, respectively) and examined the ability of transduced T cells to kill, produce effector cytokines, and expand in an antigen-dependent manner. We performed gene-expression and phenotypic analyses of GPC3-CAR T cells and CRISPR-Cas9 knockout of the