PT - JOURNAL ARTICLE AU - Sun, Luan AU - Gao, Fang AU - Gao, Zhanhui AU - Ao, Lei AU - Li, Na AU - Ma, Sujuan AU - Jia, Meng AU - Li, Nan AU - Lu, Peihua AU - Sun, Beicheng AU - Ho, Mitchell AU - Jia, Shaochang AU - Ding, Tong AU - Gao, Wei TI - Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma AID - 10.1136/jitc-2020-001875 DP - 2021 Apr 01 TA - Journal for ImmunoTherapy of Cancer PG - e001875 VI - 9 IP - 4 4099 - http://jitc.bmj.com/content/9/4/e001875.short 4100 - http://jitc.bmj.com/content/9/4/e001875.full SO - J Immunother Cancer2021 Apr 01; 9 AB - Background Glypican-3 (GPC3), a cell surface glycoprotein that is pathologically highly expressed in hepatocellular carcinoma (HCC), is an attractive target for immunotherapies, including chimeric antigen receptor (CAR) T cells. The serum GPC3 is frequently elevated in HCC patients due to the shedding effect of cell surface GPC3. The shed GPC3 (sGPC3) is reported to block the function of cell-surface GPC3 as a negative regulator. Therefore, it would be worth investigating the potential influence of antigen shedding in anti-GPC3 CAR-T therapy for HCC.Methods In this study, we constructed two types of CAR-T cells targeting distinct epitopes of GPC3 to examine how sGPC3 influences the activation and cytotoxicity of CAR-T cells in vitro and in vivo by introducing sGPC3 positive patient serum or recombinant sGPC3 proteins into HCC cells or by using sGPC3-overexpressing HCC cell lines.Results Both humanized YP7 CAR-T cells and 32A9 CAR-T cells showed GPC3-specific antitumor functions in vitro and in vivo. The existence of sGPC3 significantly inhibited the release of cytokines and the cytotoxicity of anti-GPC3 CAR-T cells in vitro. In animal models, mice carrying Hep3B xenograft tumors expressing sGPC3 exhibited a worse response to the treatment with CAR-T cells under both a low and high tumor burden. sGPC3 bound to CAR-T cells but failed to induce the effective activation of CAR-T cells. Therefore, sGPC3 acted as dominant negative regulators when competed with cell surface GPC3 to bind anti-GPC3 CAR-T cells, leading to an inhibitory effect on CAR-T cells in HCC.Conclusions We provide a proof-of-concept study demonstrating that GPC3 shedding might cause worse response to CAR-T cell treatment by competing with cell surface GPC3 for CAR-T cell binding, which revealed a new mechanism of tumor immune escape in HCC, providing a novel biomarker for patient enrolment in future clinical trials and/or treatments with GPC3-targeted CAR-T cells.All data relevant to the study are included in the article or uploaded as online supplemental information.