Abstract 2549: Development of CAR T-cell therapy targeting glypican-3 in liver cancer

Abstract Background: Glypican-3 (GPC3) is a candidate therapeutic target in hepatocellular carcinoma (HCC). We have generated the HN3 and hYP7 antibodies that recognize the N-terminus and C-terminus of GPC3, respectively. Here, we engineered human T cells that express GPC3-specific chimeric antigen receptors (CARs) and evaluated their potential for the treatment of HCC. Methods: The GPC3-specific CARs (CAR.HN3 and CAR.hYP7) contain a CD3ζ chain and the 4-1BB co-stimulatory endodomain along with a truncated human EGFR polypeptide (huEGFRt) that retains the cetuximab epitope. A set of GPC3 positive-liver cancer cell lines including Hep3B, HepG2 and Huh7 were engineered to express the firefly luciferase gene. These cells were co-cultured with GPC3 CAR T cells at various ratios for 24 hours. A bioluminescent luciferase reporter assay was then used to evaluate the cytolytic activity of effector CAR T cells. We also evaluated the anti-tumor activity of CAR T cells in xenograft models with intraperitoneal injection of luciferase-expressing HCC cells into NOD/SCID/IL2gnull (NSG) mice. The tumor growth was measured via bioluminescence tumor imaging. Results: We found that the CAR.hYP7 cells targeting the C-terminus of GPC3 showed the highest cytolytic activity against GPC3-positive HCC cells. In two HCC intraperitoneal xenograft models (Hep3B and HepG2), luciferase-expressing HCC cells preferentially grew on the mouse liver. More importantly, we found that a single intraperitoneal treatment with a high dose of CAR.hYP7 cells (40 million per mouse) exhibited sustainable antitumor efficacy in the Hep3B model and all of the mice survived after 9 weeks post CAR T-cell treatment without recurrence. The serum levels of alpha fetoprotein (AFP) in mice treated with a high dose of CAR.hYP7 cells were less than 50 ng/ml, the threshold value for HCC diagnosis. Additionally, a single intraperitoneal treatment with a low dose of CAR.hYP7 cells (10 million per mouse) caused regression of liver tumor xenografts. In the low dose CAR.hYP7 group, Hep3B tumors in the mice grew locally and restricted to the fat tissues far from the mouse liver, indicating that CAR T cells prevented tumors from seeding and growing in the liver. Although CAR.HN3 cells targeting the N- terminus of GPC3 also exhibited high cytotoxicity to HCC cells in vitro, no significant Hep3B tumor growth inhibition was seen in the mice treated with CAR.HN3 cells. Conclusion: Our work suggests that GPC3-targeting CAR T cells, in particular CAR.hYP7, is a promising therapeutic intervention for liver cancer that can be translated to human use. Citation Format: Dan Li, Nan Li, Yifan Zhang, Haiying Fu, Madeline B. Torres, Qun Wang, Tim F. Greten, Mitchell Ho. Development of CAR T-cell therapy targeting glypican-3 in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2549.