Glypican-3-specific CAR T cells co-expressing 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 co-stimulation 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 knock-out of the TCF7 gene . Finally, we measured GPC3-CAR T cell antitumor activity in murine xenograft models of GPC3+ tumors.The increased proliferation of 21.15.GBBz T cells was at least in part dependent on upregulation and maintenance of TCF-1 (encoded by TCF7 ) and associated with a higher percentage of stem cell memory and central memory populations after manufacturing. T cells expressing 21.15.GBBz had superior in vitro and in vivo expansion and persistence, and the most robust antitumor activity in vivo . These results provided preclinical evidence to support the clinical evaluation of 21.15.GPC3-CAR T cells in patients with HCC.