GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy
Authors: Lu Li li , Xiao Shu xiu , Lin Zhi yuan , Bai Jin jin , Li Wei , Song Zheng qing , Zhong Yu hong , Lu Bin , Wu Wei zhong 
Published: April 2023

Abstract
Hepatocellular carcinoma (HCC) patients are mostly diagnosed at advanced stages due to the insidious onset of the disease, making them ineligible for curative therapy. Chimeric antigen receptor (CAR)-T-cell therapy is a novel and revolutionary treatment that has become a mainstay of advanced cancer treatment. However, additional challenges limit the use of this therapy in solid tumors. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes, and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects.We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces coexpression of IL-7 at a moderate level (500 pg/mL) and CCL-19 at a high level (15000 pg/mL) and transduced it into human T cells. In vitro cell killing efficacy was validated by the xCELLigence RTCA system and the LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC cells and HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Peripheral blood mononuclear cells (PBMCs) and tumor, liver and other tissue samples were isolated from the mouse model and analyzed by flow cytometry. Finally, we initiated a phase 1 clinical trial in advanced HCC patients with GPC3 expression to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. GPC3-7-19-CAR-T cells had approximately 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in the humanized NSG mouse xenograft model (highly expressing GPC3) treated with GPC3-7-19-CAR-T cells and in those treated with GPC3-CAR-T cells were 3/5 and 5/5, respectively, and the average tumor volumes were 0.065 cm3±0.061 vs. 0.50 cm3±0.381. Of note, the predominant cell subpopulations in the tumors, CD4+ TEM and CD8+ TEM cells, played a key role in enhancing the antitumor activity of GPC3-7-19-CAR-T cells. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cell (MDSC) and regulatory T (Treg) cell infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues than GPC3-CAR-T cells. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion. In conclusion, all the data show that our engineered GPC3-7-19-CAR-T cells achieve antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets, revealing a promising therapeutic strategy for advanced GPC3+ HCC patients in the clinic. Thus, CD4+ TEM and CD8+ TCM cells are a potential target CAR-T-cell treatment of solid tumors. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic.