JMBD Journal

Introduction

Hepatocellular carcinoma (HCC) is a prevalent and highly fatal malignancy worldwide, particularly in East Asia and parts of Africa [1,2]. Although systemic therapies such as sorafenib and other tyrosine kinase inhibitors can extend survival to some degree, the prognosis of advanced HCC remains poor [3].

Glypican-3 (GPC3) is a membrane-bound heparan sulfate proteoglycan that is typically absent in normal adult tissue but is overexpressed in most HCCs [4]. Research suggests that GPC3 may promote tumor progression via modulation of the Wnt and other key signalling pathways [5,6]. Given its relatively high tumor specificity, GPC3 has emerged as a promising immunotherapeutic target. Early-phase clinical studies on GPC3-directed monoclonal antibodies and chimeric antigen receptor (CAR) T cell therapies have shown an acceptable safety profile, although with modest

objective response rates so far [7,8]. Moreover, reports on the detailed toxicity and efficacy profile of GPC3 CAR T cells in HCC patients are limited, especially in patients with high tumor burden.

We herein reported a case of advanced, recurrent HCC in a patient who received GPC3-targeted CAR T cell therapy after multiple previous treatments had failed. We described his clinical course, adverse effects, pharmacokinetics, early efficacy assessment, and change of biomarkers, providing insights into both the feasibility and limitations of this approach in heavily pre-treated patients.

Case Presentation

A 66-year-old male (weight, 58.9 kg) was diagnosed with primary HCC 15 years ago. Over the course of his disease, he underwent repeated local ablative therapies (radiofrequency ablation and chemical ablation), hepatic arterial infusion chemotherapy (HAIC), transcatheter arterial chemoembolization (TACE), surgical resection, and multiple systemic therapies (antiangiogenic therapy and immune checkpoint therapy). In late 2020, computed tomography (CT) revealed multiple intrahepatic and peritoneal lesions with a maximum diameter of approximately 7.3 cm (Table 1, Table 2). Serum alpha-fetoprotein (AFP) exceeded 3,000 ng/mL. Pathological analysis of newly biopsied tumor tissue demonstrated high GPC3 expression (Figure 1A). According to the inclusion criteria of an ongoing phase I trial (NCT05123209), the patient’s Eastern Cooperative Oncology Group (ECOG) performance status was 1, and baseline liver, kidney, and hematologic parameters were within acceptable limits. He provided written informed consent.

Figure 1: Immunohistochemical examination of GPC3 expression, study timeline and the quality control parameters of the GPC3 CAR T formulation. A. Immunohistochemical staining for Glypican-3 (GPC3) in tumour tissue obtained before enrolment (upper = negative control; lower = GPC3-positive; scale bar = 100 µm). B. Clinical timeline showing enrolment, leukapheresis (Day -19), lymphodepletion chemotherapy (Days -5 to -3), GPC3-CAR-T infusion (Day 0) and planned follow-up (Days 7, 14, 28, 56). C. In-vitro expansion profile during manufacture: blue line = total viable T-cell number; red line = percentage of CAR-positive (CAR⁺) T cells (flow-cytometry) on culture Days 5–11. D. Quality control of the final GPC3-CAR-T product (30.2 % transduction efficiency, 75 % viability, 8 × 10⁶ kg⁻¹ dose; sterility, endotoxin and mycoplasma all negative).

Table 1: Baseline characteristics of the patient.

Table 2: Previous Treatments of the Patient.

Results

CAR T Cell Manufacturing and Infusion

Peripheral blood mononuclear cells were collected by leukapheresis. T cells were activated with anti-CD3/CD28 magnetic beads, followed by lentiviral transduction with a construct encoding the humanized GPC3-specific single-chain variable fragment (scFv), CD28 costimulatory domain, and CD3ζ signaling domain. After 11 days of ex vivo expansion, CAR T cells were harvested, with a transduction efficiency of approximately 30.2% and viability of 75% (Figure 1B–1D). The patient received cyclophosphamide (250 mg/ m²/day) plus fludarabine (25 mg/m²/day) for 3 days as lymphodepletion therapy, and then received a single CAR T cell infusion (total dose of 4.71×10⁸).

Adverse Events and Efficacy Assessment

The patient was closely monitored for changes in vital signs, laboratory parameters, and serum cytokines including interleukin (IL)-2, IL6, IL-10, interferon (IFN)-γ, etc. On Day 4 post-infusion, he experienced low-grade fever (<38.5°C) alongside elevated IL-6 and IL-10, consistent with Grade 1 cytokine release syndrome (CRS) (Figure 2A, 2B). Symptoms were resolved promptly following administration of tocilizumab (IL-6 receptor blocker) and dexamethasone. No neurotoxicity or other severe adverse events were observed (Table 3). Flow cytometry revealed an obvious expansion of CAR T cells in the peripheral blood, with the peak number on Day 7 and subsequent decrease to a low level (Figure 2C).

Table 3: CAR T Cell Therapy Related Adverse Events and Management.

Figure 2: Safety, pharmacodynamics and early efficacy after GPC3 CAR T infusion. A. Left: C-reactive protein (CRP, purple) and interleukin-6 (IL-6, red) kinetics; right: vital-sign trends (body temperature, Temp; systolic/diastolic blood pressure, SBP/DBP; peripheral oxygen saturation, SpO₂) before and after tocilizumab + dexamethasone given for Grade 1 cytokine-release syndrome (dotted lines). B. Additional serum cytokines (IL-2, IL-4, IL-10, TNF-α, IFN-γ, IL-17A) from Day -1 to Day 26. C. Circulating GPC3-CAR-T cells (% CAR⁺ of CD3⁺, blue; absolute CAR-T count × 10³ µL⁻¹, red) peaking on Day 7 and declining thereafter. D. Contrast-enhanced CT at baseline (left) and Day 28 (right) showing enlargement of the dominant hepatic lesion. E. Serum tumour-marker dynamics (AFP, CEA, CA19-9) from baseline to Day 56; AFP rose continuously, consistent with progressive disease.