The CD39(+) HBV surface protein-targeted CAR-T and personalized tumor-reactive CD8(+) T cells exhibit potent anti-HCC activity

CD39, expressed by tumor-infiltrating lymphocytes (TILs), is a marker to identify tumor-reactive T cells, which is frequently associated with stronger antitumor activity than bystander T cells in a variety of malignancies. Therefore, CD39 could be a promising marker for identifying the active antitumor immune cells used for cellular immunotherapy. To test this possibility, we constructed the hepatitis B virus (HBV) surface protein-specific chimeric antigen receptor T cells (HBVs-CAR-T cells) and generated the personalized tumor-reactive CD8(+) T cells. We subsequently assessed their antitumor efficiency mainly with a co-culture system for autologous HBVs(+) HCC organoid and T cells. We found that both CD39(+) HBVs-CAR-T and CD39(+) personalized tumor-reactive CD8(+) T cells induced much more apoptosis in HCC organoids. Although the exhaustion status of CAR-T cells increased in CD39(+) CAR-T cells, triple knockdown of PD-1, Tim-3, and Lag-3 with shRNAs further enhanced antitumor activity in CD39(+) CAR-T cells. Furthermore, these CD39(+) CAR-T cells exerted an increased secretion of interferon-γ and stronger antitumor effect in a patient-derived xenograft mouse model. Our findings demonstrated that CD39 could be a promising biomarker to enrich active immune cells and become an indicator marker for evaluating the prognosis of immunotherapy for HCC patients.