Background: GPC3 is highly expressed in hepatocellular carcinoma (HCC) and is a promising target for HCC immunotherapy. Earlier phase I trial results demonstrated that second-generation GPC3-specific chimeric antigen receptor (CAR-GPC3) T cells were well tolerated in advanced HCC patients and showed clinical benefits (Shi D, Clin Can Res, 2020). Further preclinical studies showed that CAR-GPC3 T cells combined with multi-tyrosine kinase inhibitors (TKIs) were more effective in killing GPC3⁺ HCC xenografts (Wu X, Mol Ther, 2019), and fourth-generation (4G) CAR-GPC3 T cells co-expressing a transcription factor more effectively killed GPC3⁺ HCC xenografts. Methods: In this single-arm, open-label, first-in-human phase I trial (NCT03980288), we investigated the safety and antitumor activity of autologous 4G-CAR-GPC3 T cells for GPC3⁺ heavily pretreated advanced HCC patients. Patients received 4G-CAR-GPC3 T cell infusion(s) after lymphodepletion pretreatment and 67% patients combined with TKIs. Adverse events (AEs) were graded by CTCAE 5.0. Cytokine release syndrome (CRS) was graded by American Society for Transplantation and Cellular Therapy criteria (2019). Tumor response was assessed by RECISTv1.1. Results: As of January 30th, 2021, 6 subjects with HBV-related metastatic HCC were enrolled. All had progressed on ≥ 2 lines of systemic therapy, with at least one TKI combined with anti-PD-1/PD-L1 immunotherapy or FOLFOX4 chemotherapy. After lymphodepletion, patients were treated with 1-2 cycles (totaling 2.5–5×10⁸ CAR⁺ cells) of 4G-CAR-GPC3 T cell therapy. Among them, 1 patient received half dose of sorafenib and 3 patients received half dose of regorafenib along with cell infusion. No dose limiting toxicity (DLT) occurred. The maximum tolerated dose (MTD) was not observed. No patient withdrew from the trial due to AE. No treatment-related death or neurotoxicity occurred. The most common ≥ grade 3 AEs were hematological toxicity, mainly due to lymphodepletion, and patients recovered within 2 weeks after therapy. All patients developed CRS including 3 at grade 2 and 3 at grade 3. Patients recovered from CRS after tocilizumab therapy with corticosteroid (4 patients) or without. One patient achieved partial response and continued after 18 weeks. The objective response rate (ORR) and disease control rate (DCR) were 16.7% and 50%, respectively. The median progression free survival (mPFS) was 4.2 months. The median peak CAR-GPC3 copies were 5067 copies/ug gDNA, and CAR-GPC3 copies were detectable on day 28 ranging 113–2071 copies/ug gDNA. Conclusions: Our study is the first to report that 4G-CAR-GPC3 T cell therapy in combination with TKIs has a manageable safety profile while demonstrating potential antitumor activity for heavily pretreated advanced HCC patients; however, CRS should be diligently managed. Clinical trial information: NCT03980288