PiggyBac-engineered T cells expressing a glypican-3-specific chimeric antigen receptor show potent activities against hepatocellular carcinoma.

Glypican-3 (GPC3) is an attractive target for chimeric antigen receptor (CAR)-T cell therapy, as it is overexpressed in most hepatocellular carcinoma (HCC) tissues but shows restricted expression in healthy adult tissues. Herein, we generated GPC3-specific CAR-T cells for HCC therapy by electroporation with plasmid DNA encoding the piggyBac (PB) transposon and the hyperactive piggyBac transposase simultaneously instead of by commonly-used viral vectors. Our results demonstrated that GPC3CAR gene was efficiently integrated into the genome of T cells utilizing the PB transposon system. Upon stimulation with GPC3 antigen, GPC3CAR-T cells could be effectively activated, proliferate strongly and secrete high levels of cytokines. It also was demonstrated that GPC3CAR-T cells displayed potent cytotoxicity against GPC3-positive HCC cell lines in vitro by using real-time cell analyser (RTCA) system and the JuLI™ Stage Cell History Recorder. More importantly, in a Huh-7 xenograft mouse model, GPC3CAR-T cells significantly reduced the tumour burden companied with the secretion of high levels of IFN-γ. Moreover, T cells in mice treated with GPC3CAR-T cells could infiltrate into tumour tissues and persist as effector memory T cells (TEM). Overall, our study suggests that the use of PB system-based GPC3CAR-T cell therapy could be a promising clinical strategy for patients with HCC.

Wang P ，Qin W ，Liu T ，Jiang D ，Cui L ，Liu X ，Fang Y ，Tang X ，Jin H ，Qian Q ... -  《-》

Development of T cells carrying two complementary chimeric antigen receptors against glypican-3 and asialoglycoprotein receptor 1 for the treatment of hepatocellular carcinoma.

Chen C ，Li K ，Jiang H ，Song F ，Gao H ，Pan X ，Shi B ，Bi Y ，Wang H ，Wang H ，Li Z ... -  《-》

Development of T cells redirected to glypican-3 for the treatment of hepatocellular carcinoma.

The aim of our study is to elucidate whether T cells expressing GPC3-targeted chimeric antigen receptor (CAR) can efficiently eliminate GPC3-positive HCC cells and their potential in the treatment of HCC. T cells expressing a first-generation and third-generation GPC3-targeted CAR were prepared using lentiviral vector transduction. The in vitro and in vivo cytotoxic activities of the genetically engineered CAR T cells were evaluated against various HCC cell lines. GPC3-targeted CAR T cells could efficiently kill GPC3-positive HCC cells but not GPC3-negative cells in vitro. These cytotoxic activities seemed to be positively correlated with GPC3 expression levels in the target cells. In addition, T cells expressing the third-generation GPC3-targeted CAR could eradicate HCC xenografts with high level of GPC3 expression and efficiently suppress the growth of HCC xenografts with low GPC3 expression level in vivo. The survival of the mice bearing established orthotopic Huh-7 xenografts was significantly prolonged by the treatment with the third-generation GPC3-targeted CAR T cells. GPC3-targeted CAR T cells could potently eliminate GPC3-positive HCC cells, thereby providing a promising therapeutic intervention for GPC3-positive HCC.

Gao H ，Li K ，Tu H ，Pan X ，Jiang H ，Shi B ，Kong J ，Wang H ，Yang S ，Gu J ，Li Z ... -  《-》

Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma.

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. 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. 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. 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.

Sun L ，Gao F ，Gao Z ，Ao L ，Li N ，Ma S ，Jia M ，Li N ，Lu P ，Sun B ，Ho M ，Jia S ，Ding T ，Gao W ... -  《Journal for ImmunoTherapy of Cancer》

T Cells Engineered to Express a T-Cell Receptor Specific for Glypican-3 to Recognize and Kill Hepatoma Cells In Vitro and in Mice.

Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican-3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC), but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice. We used mass spectrometry to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of human leukocyte antigen (HLA)-A2 and bioinformatics to identify immunodominant peptides. To circumvent GPC3 tolerance resulting from fetal expression, dendritic cells from HLA-A2-negative donors were cotransfected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells. Peptide GPC3367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3367 multimer and secreted interferon-γ when cultured with GPC3367, but not with control peptide-loaded cells. By genomic sequencing of these T-cell clones, we identified a gene encoding a dominant T-cell receptor. The gene was cloned and the sequence was codon optimized and expressed from a retroviral vector. Primary CD8(+) T cells that expressed the transgenic T-cell receptor specifically bound GPC3367 on HLA-A2. These T cells killed GPC3-expressing hepatoma cells in culture and slowed growth of HCC xenograft tumors in mice. We identified a GPC3367-specific T-cell receptor. Expression of this receptor by T cells allows them to recognize and kill GPC3-positive hepatoma cells. This finding could be used to advance development of adoptive T-cell therapy for HCC.

Dargel C ，Bassani-Sternberg M ，Hasreiter J ，Zani F ，Bockmann JH ，Thiele F ，Bohne F ，Wisskirchen K ，Wilde S ，Sprinzl MF ，Schendel DJ ，Krackhardt AM ，Uckert W ，Wohlleber D ，Schiemann M ，Stemmer K ，Heikenwälder M ，Busch DH ，Richter G ，Mann M ，Protzer U ... -  《-》