202. Development of T-Cells Carrying Two Complementary Chimeric Antigen Receptors Against GPC3 and ASGR1 for the Treatment of Hepatocellular Carcinoma

Abstract

Recently, we reported that GPC3-targeted CAR-T cells could eradicate hepatocellular carcinoma xenografts in mice. However, we do not know whether on-target off-tumor toxicity, a major toxicity associated with CAR-T cells will happen when using these GPC3-CAR T cells in human being. We propose that dual-targeted CAR-T cells co-expressing a GPC3- and a ASGR1 (a liver tissue-specific protein)-targeted CAR that signal using CD3ζ and 28BB (containing both CD28 and 4-1BB signaling domain) respectively might be used to reduce on-target off-tumor toxicity. We first demonstrated that GPC3 and ASGR1 were co-expressed in 54.7% of the tested HCC samples. We further demonstrated that dual-targeted CAR-T cells could exhibit no cytotoxic activities on ASGR1+GPC3− tumor cells. However, the cytotoxic activities of GPC3-targeted CAR-T cells and dual-targeted CAR-T cells have similar anti-tumor activities in vitro on ASGR1-GPC3+ or GPC3+ASGR1+HCC cells. Interestingly, dual-targeted CAR-T cells showed significantly higher cytokine secretion, proliferation and anti-apoptosis ability against tumor cells bearing both antigens in vitro than single-targeted CAR-T cells. Furthermore the dual-targeted CAR-T cells displayed potent growth suppression activity on GPC3+ASGR1+HCC tumor xenograft while no obvious growth suppression on single or double antigen-negative tumor xenografts. Additionally, the dual targeting T cells exerted superior anti-cancer activity and persistence than single targeting T cells in two GPC3+ASGR1+ HCC xenograft models. Together, T-cells carrying two complementary CARs against GPC3 and ASGR1 might reduce the risk of on-target off-tumor toxicity while remain relative potent anti-tumor activities on GPC3+ASGR1+ HCC