Targeting glypican-3 as a new frontier in liver cancer therapy

Abstract

Glypican-3 (GPC3) is a tumor-associated antigen that is specifically expressed in hepatocellular carcinoma (HCC) and having relatively low levels in normal tissues. This unique expression pattern positions GPC3 as a potential target for precision therapy and drug development in HCC. Recent studies have shown significant advancements in GPC3-targeted therapies and immunotherapies, particularly for patients with advanced or treatment-resistant HCC. Although certain clinical trials have yielded suboptimal results, numerous ongoing studies continue to explore its therapeutic efficacy. This mini-review focuses on the latest research developments regarding GPC3 as a therapeutic target across various HCC treatment strategies, including monoclonal antibodies, bispecific antibodies, chimeric antigen receptor-T-cell therapies, and other innovative approaches. In addition, the limitations of GPC3-targeted therapies and their future application prospects in HCC treatment are discussed. The review particularly emphasizes the unmet need for future research directions, such as combination immunotherapy strategies and novel drug designs. Through the integration of innovative technologies and clinical validation, GPC3 holds strong potential as a promising breakthrough in the treatment of HCC, offering new opportunities for enhancing patient outcomes and improving therapeutic efficacy.

Key Words: Glypican-3; Hepatocellular carcinoma; Chimeric antigen receptor T-cell; Wnt; Immunotherapy

Core Tip: This review provides a comprehensive overview of glypican-3 (GPC3)-targeted strategies in hepatocellular carcinoma (HCC), including monoclonal and bispecific antibodies, chimeric antigen receptor-T cell therapies, vaccines, and photodynamic approaches. It further explores GPC3’s role in molecular imaging, radiomics, and liquid biopsy. Despite challenges in clinical translation, ongoing trials and novel combination therapies highlight the potential of GPC3-based approaches to improve treatment specificity, overcome resistance, and guide personalized therapy in HCC.

INTRODUCTION

The glypican (GPC) family belongs to the heparan sulfate (HS) proteoglycan family and is characterized by its unique structural components, including a core protein, HS glycosaminoglycan chains, and a glycosylphosphatidylinositol anchor that tethers the protein to the cell surface[1]. Among the six members of this family (GPC1 to GPC6), glypican-3 (GPC3) stands out as a key molecular marker and therapeutic target in hepatocellular carcinoma (HCC), due to its tumor-specific overexpression and oncogenic functions. In normal adult liver, the expression level of GPC3 is extremely low[2]. Notably, GPC3 expression is markedly upregulated in HCC and melanoma[3], while it is significantly downregulated or completely silenced in several malignancies, including ovarian cancer[4], breast cancer[5], and clear cell renal cell carcinoma[6]. These contrasting expression patterns highlight the critical role of GPC3 in tumorigenesis, emphasizing its relevance as a potential target in cancer therapy.

GPC3 is not merely a biomarker but also an active driver of HCC progression, contributing to tumor proliferation, immune evasion, and poor prognosis. Its oncogenic effects, primarily through activation of the c-Myc signaling pathway, make it a crucial target for both diagnostic and therapeutic interventions. Given the high incidence and mortality rates seen in HCC patients, developing effective GPC3-targeted strategies could very well address the significantly unmet clinical need currently seen. In HCC, GPC3 is overexpressed in more than 70% of cases when compared to adjacent normal tissue, with this upregulation being strongly associated with adverse clinical outcomes[7]. Moreover, GPC3 promotes tumor progression primarily through activation of the c-Myc signaling pathway[8], establishing it as a key molecular marker for both diagnostic and therapeutic applications in HCC. Despite advancements in HCC treatment, current options such as surgical resection, liver transplantation, tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) remain limited due to high recurrence rates and insufficient long-term efficacy[9,10]. Therefore, there remains an urgent need for novel molecularly targeted therapies to help improve patient survival. Unlike other biomarkers with overlapping expression in normal liver or non-malignant diseases, GPC3 demonstrates exceptional specificity for HCC, making it not only an ideal diagnostic marker but also an attractive therapeutic target. This unique expression profile has propelled the development of multiple GPC3-targeted therapies, all of which hold great promise for transforming HCC treatment.

GPC3 is primarily expressed on the surface of HCC cells but undergoes proteolytic cleavage by the enzyme Furin at Arg358 and Ser359, resulting in the release of soluble GPC3 (sGPC3) into the bloodstream[11]. Additionally, GPC3 has been detected in small extracellular vesicles (EVs) isolated from the serum of HCC patients, highlighting its potential as a blood-based biomarker for HCC diagnosis and prognosis[12]. Although sGPC3 alone has a sensitivity of only 40%, its high specificity for HCC makes it an indispensable component of multi-marker diagnostic panels, enhancing early detection and improving diagnostic accuracy when combined with other biomarkers[13]. While sGPC3 demonstrates high specificity for HCC, its presence has also been detected in other malignancies, including lung cancer (13.5%) and thyroid cancer (13.2%), further highlighting the importance of using it as part of a multi-marker diagnostic panel rather than as a single diagnostic indicator[14]. To overcome the limitations of single biomarkers, combining multiple serum markers has emerged as a promising strategy to enhance HCC detection and monitoring. Our studies highlighted the added value of combining Protein induced by vitamin K absence-II (PIVKA-II) with alpha-fetoprotein (AFP), particularly in AFP-negative patients, as well as its potential to predict recurrence[15]. Additionally, a highly sensitive glycan microarray has enabled more reliable detection of AFP-L3 in hepatitis B virus (HBV)-related HCC, even at low AFP levels[16]. These findings suggest the value of integrating novel detection platforms and complementary biomarkers, such as GPC3, lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) and PIVKA-II, in order to develop more accurate and clinically applicable multi-marker diagnostic panels.

Importantly, GPC3-targeted therapies offer a new frontier in HCC treatment by leveraging its tumor-specific expression. Given the limitations of current HCC treatments, GPC3-targeted therapies represent a promising development in the field. Various strategies, including monoclonal antibodies, antibody-drug conjugates (ADCs) and chimeric antigen receptor (CAR)-T-cell therapies, are all in active development, aiming to harness GPC3’s tumor-specific expression for precise treatment. However, challenges such as optimizing delivery, overcoming tumor heterogeneity, and minimizing immune evasion must be addressed to fully realize GPC3-targeted therapy as a transformative clinical solution. These strategies aim to enhance antitumor efficacy while also minimizing off-target toxicity, addressing a major limitation to the current treatments. However, challenges remain in translating these therapies into clinical practice, including optimizing drug delivery, overcoming tumor heterogeneity, and mitigating resistance mechanisms. Further research is still needed in order to refine these approaches and better develop combinatorial strategies that will enhance treatment effectiveness.

This review highlights the pivotal role of GPC3 in HCC, from its fundamental oncogenic mechanisms to its emerging role as a critical biomarker and therapeutic target. As novel GPC3-based strategies advance, they hold the potential to reshape the current landscape of HCC diagnosis and treatment, addressing the urgent need for more effective therapeutic solutions. We have critically examined the molecular mechanisms underlying GPC3's biological functions and subsequently highlighted its significance as a therapeutic target in HCC diagnosis and treatment. Additionally, we analyzed the current challenges in translating GPC3-based therapies from bench to bedside so as to optimize their clinical implementation.

MOLECULAR AND BIOLOGICAL FUNCTIONS OF GPC3

GPC3 is essential for embryonic development and organogenesis. During embryogenesis, GPC3 regulates multiple regulatory pathways by modulating growth factor signaling cascades, including Wnt, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and insulin-like growth factor (IGF)[17-21] (Figure 1). These interactions influence downstream pathways involved in cellular proliferation, differentiation and tissue morphogenesis.

Figure 1 Diverse growth factor signaling pathways coordinated by glypican-3 in hepatocellular carcinoma. Glypican-3 (GPC3) plays a crucial role in hepatocellular carcinoma (HCC) by interacting with multiple ligands through modulation by GPC3-conjugated heparan sulfate. These ligands include Wnt, fibroblast growth factor 2 (FGF 2), hepatocyte growth factor (HGF), bone morphogenetic protein (BMP), and insulin-like growth factor (IGF). Through these interactions, GPC3 regulates key oncogenic signaling pathways, thereby contributing to tumor proliferation, migration, invasion, and angiogenesis. Specifically, FGF2, HGF, BMP, and IGF activate their respective receptors (FGFR, C-MET, BMPR, and IGF1R), triggering downstream signaling cascades such as AKT/mTOR, c-Myc, and SMAD pathways, which ultimately drive tumor progression. Moreover, GPC3 enhances Wnt signaling by facilitating Wnt ligand binding to Frizzled receptors, leading to the activation of the β-catenin and Hippo/YAP pathways, which further promote oncogenic signaling in HCC. GPC3: Glypican-3; HGF: Hepatocyte growth factor; IGF: Insulin-like growth factor.

Under physiological conditions, GPC3 is essential for the development of various organ systems. In liver development, it regulates hepatocyte proliferation and differentiation, ensuring proper liver formation[17,22]. In kidney development, GPC3 influences the migration and differentiation of nephron progenitor cells[19,23]. It also plays a critical role in skeletal and limb formation through BMP signaling[20] In the gastrointestinal tract, GPC3 contributes to both intestinal cell differentiation and tissue organization, while in the neural system, it is thought to affect brain and spinal cord development by regulating neural stem cell differentiation and migration[24,25]. Through these diverse functions, GPC3 serves as an essential regulator of embryonic tissue patterning and organogenesis.

In pathological conditions such as HCC, GPC3 is aberrantly re-expressed or overexpressed, serving as a critical regulator of multiple oncogenic signaling pathways. Through its core protein and HS side chains, GPC3 interacts with both ligands and receptors, functioning as a co-receptor and scaffold to facilitate signal complex formation, thereby activating downstream effectors such as c-Myc, AKT, and ERK that drive proliferation, survival, and metastasis in HCC cells.

Among the multiple oncogenic pathways regulated by GPC3, the Wnt/β-catenin pathway is one of the most frequently activated (Figure 2). GPC3 enhances Wnt signaling by directly binding to Wnt ligands and increasing their local concentration near receptors[26,27]. This process facilitates the assembly of Wnt-Frizzled (FZD)-LRP5/6 receptor complexes[28], involving both the GPC3 core protein and HS side chains for stabilizing Wnt-FZD interactions[29]. Upon activation, this pathway leads to β-catenin nuclear translocation and transcription of cell cycle-related genes like cyclin D1. Notably, mutations within the N-terminal Wnt-binding domain of GPC3 impair its Wnt-binding capacity and suppress tumor growth in murine HCC models[26], while sGPC3 acts as a decoy receptor that sequesters Wnt ligands to inhibit signal transmission[30].

Figure 2 Glypican-3-mediated Wnt/β-catenin and YAP/TAZ signaling axes in hepatocellular carcinoma. GPC3 acts as a central signaling molecule that regulates two key pathways in hepatocellular carcinoma. One is the canonical Wnt/β-catenin pathway, where activated β-catenin translocates to the nucleus and induces the expression of oncogenic genes. The other is the alternative Wnt–YAP/TAZ pathway, in which activated YAP/TAZ translocate to the nucleus and promote tumor progression through transcriptional regulation. GPC3: Glypican-3.

GPC3 also modulates the Hippo/YAP signaling cascade. Certain Wnt ligands, upon binding to FZD receptors, can bypass β-catenin and suppress LATS1/2 kinase activity, promoting YAP nuclear translocation and transcriptional activity[31,32]. The Hippo pathway, generally tumor-suppressive in liver tissue, is frequently inactivated during hepatocarcinogenesis[33]. In HCC, GPC3 knockdown reduces YAP activity, while monoclonal antibodies targeting GPC3 (e.g., HN3) further suppress YAP-mediated transcription and tumor cell proliferation[34]. Activated YAP can coactivate TEAD and TCF transcription factors with nuclear β-catenin, inducing oncogenesis-associated genes and mediating recruitment of M2-polarized tumor-associated macrophages, enhancing angiogenesis and immune evasion[35].

GPC3 regulates multiple growth factor pathways through its core protein and HS chains[36]. For example, GPC3 binds FGF2 through its HS chains to promote FGFR activation and downstream MAPK, PKC, and PI3K/AKT pathways[36], with SULF2 enhancing GPC3 expression and FGF signaling in HCC[37]. The HS chains of GPC3 regulate BMP signaling, which typically activates the intracellular SMAD pathway upon receptor binding, leading to nuclear translocation and transcriptional regulation[38]. GPC3 is frequently overexpressed in HCC and regulates both FGF2 and BMP-7 signaling pathways[39]. In the hepatocyte growth factor (HGF)/c-Met axis, GPC3 promotes cell motility and invasion through HS chain-mediated interactions, with antibodies targeting the HS domain of GPC3 (e.g., HS20) significantly inhibiting HGF-induced migration and 3D spheroid formation in HCC cells[40]. GPC3 also supports IGF pathway signaling by binding IGF-II and IGF-1R via a proline-rich N-terminal domain, promoting downstream MAPK/ERK and PI3K/AKT signaling that facilitates proliferation, metabolic reprogramming, and apoptosis resistance[21]. Additionally, GPC3 inhibits ubiquitination and degradation of IGF-1R by sequestering the adapter protein Grb10, sustaining prolonged receptor activation[41].

GPC3 in hypoxia and oncogenic signaling

Recent studies have shown that under hypoxic conditions, hypoxia-inducible factor 1-alpha upregulates GPC3 mRNA expression and promotes its secretion via exosomes in HCC cells[42] (Figure 3). These GPC3-enriched exosomes have been shown to enhance HCC cell proliferation, migration, and epithelial-mesenchymal transition (EMT), primarily through activation of the β-catenin signaling cascade[42]. This activation is accompanied by upregulation of β-catenin and c-Myc, and downregulation of GSK-3β. Moreover, exosomal GPC3 facilitates angiogenic activity in HUVECs, indicating its role in remodeling the tumor microenvironment[42]. Notably, GPC3 knockdown attenuates these tumor-promoting effects, suggesting that targeting exosomal GPC3 may offer a promising therapeutic strategy to limit HCC progression both in vitro and in vivo[42]. These findings establish GPC3 as a master regulator of multiple oncogenic pathways in HCC, suggesting that targeting GPC3, particularly its exosomal form, may offer a multifaceted therapeutic approach to simultaneously inhibit tumor growth, metastasis, and angiogenesis in HCC patients.

Figure 3 Hypoxia-induced exosomal release of glypican-3 and its effects on hepatocellular carcinoma progression and angiogenesis. Under hypoxic conditions, hepatocellular carcinoma cells upregulate glypican-3 expression and release it via exosomes, contributing to tumor progression and angiogenesis in the tumor microenvironment. GPC3: Glypican-3; HCC: Hepatocellular carcinoma; EMT: Epithelial-mesenchymal transition.

GPC3 in metabolic adaptation and tumor stemness

In addition to its role in oncogenic signaling, GPC3 plays a pivotal role in metabolic adaptation under hypoxic conditions. It promotes lactylation and cellular glycolysis, thus enhancing tumor stemness, metabolic plasticity and immune evasion[43] (Figure 4). By increasing lactate accumulation and c-Myc signaling, GPC3 facilitates metabolic reprogramming that supports tumor adaptation to the microenvironment[43]. The relationship between GPC3 and c-Myc appears bidirectional, forming a positive feedback regulatory loop that drives HCC progression[8]. Luciferase reporter assays and chromatin immunoprecipitation analyses have demonstrated that c-Myc directly binds to the GPC3 promoter region, enhancing its transcriptional activity and protein expression[8]. Conversely, GPC3 enhances c-Myc expression and signaling. This relationship is further evidenced by functional studies where c-Myc overexpression rescues the diminished proliferative capacity, stemness, and glycolytic activity caused by GPC3 silencing, while GPC3 knockdown reduces c-Myc expression and lactate-driven post-translational modifications, leading to diminished tumor cell viability[43]. These findings suggest that GPC3 is not only a key player in signal transduction but also a critical regulator of tumor metabolism. Its ability to enhance metabolic adaptation further strengthens its role in promoting tumor aggressiveness and resistance to therapy. Understanding how GPC3 influences the metabolic landscape of HCC may help open new avenues for targeted therapeutic strategies that aim to disrupt its role in tumor cell survival.

Figure 4 Glypican-3-mediated regulation of c-Myc stability and metabolic adaptation under hypoxia. Glypican-3 (GPC3) regulates tumor metabolic adaptation and stemness by modulating c-Myc protein stability under normoxic and hypoxic conditions. Under normoxic conditions, c-Myc is more prone to ubiquitination and degradation when GPC3 expression is low. Under hypoxia, GPC3 is upregulated and promotes global protein lactylation, which specifically prevents c-Myc degradation, thereby stabilizing c-Myc protein and enhancing glycolysis and stemness. GPC3: Glypican-3.

Differential functions of GPC3 across cancer types

In cervical cancer, elevated GPC3 expression promotes tumor progression. Studies have shown that GPC3 antisense long non-coding RNA (GPC3-AS1) is upregulated and cooperates with ELK1 to regulate GPC3 expression, enhancing cell proliferation and migration, thereby revealing its regulatory mechanism in cervical cancer[44]. However, the functionality of GPC3 exhibits marked heterogeneity across different tumor types. In breast cancer, GPC3 acts as a tumor suppressor by inhibiting EMT through p38 MAPK signaling pathway activation, subsequently preventing metastasis and inducing tumor dormancy[45,46]. Notably, certain studies have demonstrated the capacity of GPC3 to trigger apoptotic responses in cancer cells, suggesting its potential role as a tumor suppressor gene[47]. This tumor-suppressive function is further evidenced by the significant downregulation of GPC3 expression in papillary thyroid cancer and non-small cell lung cancer[48,49]. These findings indicate the tumor-specific nature of GPC3 expression, which is likely modulated by tissue-specific signaling networks and microenvironmental factors.

The role of GPC3 in the cancer immune microenvironment

In the tumor immune microenvironment, the correlation between elevated GPC3 expression and therapeutic response has emerged as a significant area of investigation. Studies have revealed that high GPC3 expression correlates with diminished clinical efficacy and may contribute to resistance against combination therapy involving anti-PD-1 (atezolizumab) and anti-VEGF (bevacizumab) in HCC patients, potentially through interference with immune and angiogenic co-regulatory mechanisms[50]. Furthermore, research has demonstrated that combining GPC3-targeted therapy with TGF-β signaling pathway inhibition significantly enhances the anti-tumor efficacy of natural killer (NK) cells against HCC, suggesting the potential benefits of multi-targeted therapeutic approaches in immunotherapy[51]. Mechanistically, this synergistic effect occurs because TGF-β signaling normally suppresses NK cell cytotoxicity, limiting the effectiveness of GPC3-CAR NK cells alone[51]. By genetically disrupting TGFBR2 or employing dominant-negative inhibition strategies to block TGF-β signaling, NK cells can retain their cytolytic function even within highly immunosuppressive tumor microenvironments[51]. This dual-targeting approach creates a sustained and potent anti-tumor response that neither strategy could achieve independently, demonstrating significant therapeutic synergy against HCC. Parallel investigations have identified significant GPC3 upregulation in the cancer-associated fibroblasts (CAFs) of advanced gastric cancer patients, correlating strongly with poor prognosis and reduced responsiveness to PD-1 treatment[52]. These findings suggest that targeting GPC3-high CAFs could potentially enhance the therapeutic efficacy of PD-1 blockade in gastric cancer patients[52]. GPC3 secreted by CAFs induces the expression of multiple immunosuppressive and oncogenic markers, such as PD-L1, TIM-3, CD24, Cyclin D1, and c-Myc, in gastric cancer cells, thereby promoting tumor immune evasion[52]. These findings suggest that immune checkpoint blockade alone, such as anti-PD-1 therapy, may be insufficient in GPC3-enriched tumor microenvironments. In vivo models have further confirmed that neutralizing GPC3 in CAFs significantly restores CD8⁺ T cell activity and enhances the therapeutic efficacy of PD-1 blockade[52]. Thus, a multi-targeted strategy combining anti-GPC3 therapy with immune checkpoint inhibition may help overcome CAF-mediated immunosuppression and improve treatment outcomes in GPC3-high gastric cancer. These collective findings underscore the crucial role of GPC3 in modulating the tumor microenvironment and immune escape mechanisms, highlighting its significance in cancer immunotherapy development.

INNOVATIVE APPLICATIONS OF GPC3 IN HCC MOLECULAR IMAGING AND DIAGNOSTICS

Molecular imaging

Recent advances have enhanced GPC3-based molecular imaging for HCC diagnosis. A single-domain antibody probe (ssHN3) conjugated for selective GPC3 binding and labeled with [89Zr] demonstrated improved tumor uptake and reduced liver accumulation, optimizing PET imaging for HCC detection[53]. Another novel PET probe, ^[18F]AlF-NOTA-IPB-GPC3P, outperformed ^[18F]AlF-GPC263, showing higher cellular uptake, better internalization, and prolonged tumor retention[54]. Additionally, ^[68Ga]Ga-T2P, a dual-specific probe targeting GPC3 and PSMA, significantly enhanced both diagnostic sensitivity and imaging precision, underscoring the potential of multi-target probes in liver cancer detection[55].

Radiomics

Advanced radiomic models have improved non-invasive prediction of GPC3 expression in HCC. A multi-sequence magnetic resonance imaging-based radiomic scoring model integrating tumor morphology and microvascular invasion achieved high predictive accuracy [area under the curve (AUC) = 0.979][56]. Similarly, a contrast-enhanced computed tomography radiomics model demonstrated superior performance over serum AFP testing (AUC = 0.867), while integrating clinical risk factors further improved diagnostic accuracy to AUC = 0.895[57]. These methods offer effective, non-invasive approaches to assess GPC3 expression.

Serum biomarker detection

Technological advancements have significantly improved serum-based GPC3 detection. A sandwich-type ultrasensitive electrochemical aptamer sensor using both gold-polyaniline nanomaterials and graphene oxide-platinum palladium nanoparticles (PrGO-Hemin-PdNP) showed exceptional sensitivity for GPC3 detection, thus enhancing early HCC diagnosis[58]. A single-tube immuno-RPA and CRISPR-Cas13a system enables rapid, extraction-free detection of GPC3 in EVs, offering a promising diagnostic tool[59]. Additionally, time-resolved fluorescence immunochromatography (TRFIS) allows 15-minute plasma exosome-based GPC3 detection, demonstrating the potential for rapid HCC screening[60].

Circulating tumor cell detection

Circulating tumor cell (CTC)-based technologies have advanced GPC3 detection in HCC. A dual fluorescence aptamer-based sensing system achieved 92% sensitivity and 100% specificity, with a detection limit of just 2 cells/mL[61]. The d-SCOUT technology, capable of quantifying multiple HCC CTC biomarkers including GPC3, offers high sensitivity and reproducibility, further enhancing diagnostic precision[61].

Together, these advancements demonstrate that GPC3 has been innovatively applied across molecular imaging, radiomics, serum detection and CTC analysis, significantly improving the sensitivity and accuracy of HCC diagnosis. These technologies provide novel, non-invasive tools for early detection and disease monitoring, reinforcing GPC3’s clinical potential as both a diagnostic and prognostic marker in liver cancer.

TRANSLATIONAL PROGRESS OF GPC3-DIRECTED THERAPIES IN HCC

Given its selective expression in HCC, various GPC3-targeting strategies have been developed, including monoclonal antibodies, ADCs, photodynamic therapy (PDT), DNA vaccines, and CAR-T-cell therapies (Figure 5). Although still under development, these approaches have demonstrated promising antitumor activity in preclinical models. In particular, targeting the C-terminal domain of GPC3 helps prevent loss of tumor specificity due to proteolytic cleavage. However, while several Phase I clinical trials have been completed, multiple trials are still ongoing (Table 1).

Figure 5 Applications of glypican-3-targeted therapies. Glypican-3 (GPC3) serves as a promising target for hepatocellular carcinoma (HCC) therapy, with various therapeutic strategies under development. I-124 codrituzumab, a radiolabeled monoclonal antibody targeting GPC3, enables tumor imaging and potential radioimmunotherapy applications. Antibody-drug conjugates utilize GPC3-targeting antibodies conjugated with cytotoxic agents to selectively eliminate cancer cells. Photodynamic therapy, an emerging approach, employs light-activated GPC3-targeting molecules to induce localized cytotoxicity. GPC3-specific chimeric antigen receptor (CAR)-T cells have been engineered to target tumor cells, with their efficacy further enhanced by the secretion of cytokines such as IL-15, IL-21, and IL-7, which improve CAR-T cell persistence and strengthen antitumor activity. Bispecific CAR-T cells, designed to simultaneously recognize GPC3 and B7-H3, offer a dual-targeting strategy that enhances tumor specificity, boosts immune activation, and mitigates antigen escape, ultimately improving the effectiveness of CAR-T-cell therapy. In DNA vaccine development, PLGA/PEI nanoparticles are utilized to encapsulate and deliver GPC3 and HMGB1, aiming to stimulate immune responses and activate cytotoxic T lymphocyte. ERY974, a bispecific T cell-redirecting antibody, is designed to target both GPC3 on tumor cells and CD3 on T cells, facilitating potent T cell-mediated tumor killing. Collectively, these GPC3-targeted strategies offer significant potential for advancing HCC treatment by leveraging multiple mechanisms to enhance tumor specificity, immune activation, and therapeutic efficacy. CTL: Cytotoxic T lymphocyte; CAR-T: Chimeric antigen receptor-T.

Table 1 Current clinical trials targeting glypican-3.

No.	NCT number	Study title	Study status	Stage	Enrollment (estimated)	Primary endpoint
1	NCT05352542	GPC3-targeting CART Cell in Treatment of Advanced Hepatocellular Carcinoma	Terminated	Phase 1	10	Incidence, severity, and type of TEAEs; DLT rate; RP2D finding; CAR positive T cells; CAR transgene levels in peripheral blood
2	NCT02395250	Anti-GPC3 CAR T for Treating Patients with Advanced HCC	Completed	Phase 1	13	AEs attributed to the administration of the anti-GPC3 CAR T cells
3	NCT03146234	CAR-GPC3 T Cells in Patients with Refractory Hepatocellular Carcinoma	Completed	NA	7	Safety and tolerance
4	NCT03980288	4th Generation Chimeric Antigen Receptor T Cells Targeting Glypican-3	Completed	Phase 1	6	DLT and MTD
5	NCT02905188	Glypican 3-specific Chimeric Antigen Receptor Expressing T Cells for Hepatocellular Carcinoma (GLYCAR)	Completed	Phase 1	9	Number of patients with DLT; RP2D
6	NCT03884751	Chimeric Antigen Receptor T Cells Targeting Glypican-3	Completed	Phase 1	9	DLT, MTD
7	NCT02748837	A Study of ERY974 in Patient with Advanced Solid Tumors	Completed	Phase 1	29	Dose escalation: MTD determination; cohort expansion: Preliminary assessment of change in tumor size
8	NCT05070156	B010-A Injection for Treating Patients with GPC3 Positive Advanced Hepatocellular Carcinoma	Active not recruiting	Phase 1	3	Evaluate incidence of TEAEs [safety and tolerability] after B010-A injection
9	NCT02932956	Glypican 3-specific Chimeric Antigen Receptor Expressed in T Cells for Patients with Pediatric Solid Tumors (GAP)	Active not recruiting	Phase 1	10	Number of patients with DLT
10	NCT04405778	A Study of TAK-102 in Adult with Previously-Treated Solid Tumors	Active not recruiting	Phase 1	11	Number of participants with first cycle DLTs; number of participants with one or more TEAE; number of participants with AEs of clinical interest
11	NCT03198546	GPC3-CAR-T Cells for Immunotherapy of Cancer with GPC3 Expression	Recruiting	Phase 1	30	Number of patients with DLT
12	NCT05003895	GPC3 Targeted CAR-T Cell Therapy in Advanced GPC3 Expressing Hepatocellular Carcinoma (HCC)	Recruiting	Phase 1	38	To determine the safety and feasibility of T-cells, expressing a novel humanized anti-GPC3 chimeric antigen receptor, in patients with advanced HCC, expressing GPC3
13	NCT04928677	A Study of Codrituzumab in Children and Young Adults with Solid Tumors and Have Not Responded to Treatment or Have Come Back After Treatment	Recruiting	Phase 1	50	Estimate the MTD
14	NCT06196294	GPC3/Mesothelin-CAR-γδT Cells Against Cancers	Recruiting	Phase 1	30	Number of patients with DLT
15	NCT06461624	Clinical Trial of Autologous GPC3 CAR-T Cells (CBG166) Therapy for Advanced Hepatocellular Carcinoma	Recruiting	Phase 1	15	DLT; AEs; MTD
16	NCT05620706	Study of GPC-3 CAR-T Cells in Treating with Hepatocellular Carcinoma	Recruiting	NA	20	AE/SAE
17	NCT05410717	CLDN6/GPC3/Mesothelin/AXL-CAR-NK Cell Therapy for Advanced Solid Tumors	Recruiting	Phase 1	200	Safety by CTCAE V5.0
18	NCT05047510	GPC3 Targeted Fluorescence Image Guided Surgery of Hepatocellular Carcinoma	Recruiting	NA	60	HCC lesions (numbers of intraoperatively detected hepatocellular carcinoma lesions)
19	NCT05779917	Mesothelin/GPC3/GUCY2C-CAR-T Cells Against Cancers	Recruiting	Phase 1	30	Number of patients with DLT
20	NCT06383520	Study on the Clinical Application Value of PET Imaging Targeting GPC3 in Hepatocellular Carcinoma	Recruiting	Early Phase 1	100	Visual and standardized uptake values assessment of lesions and biodistribution
21	NCT05926726	GPC3-directed CAR-T in the Treatment Amongst Subjects With Advanced Hepatocellular Carcinoma	Recruiting	NA	12	TRAEs; DLT; RP2D of JWATM214 in HCC patients
22	NCT06687941	A Study to Evaluate the Tolerability, Safety, and PK of AST-201 in Patients With GPC3-positive Advanced Solid Tumors	Recruiting	Phase 1	70	DLT
23	NCT06726161	Study of the Theranostic Pair RYZ811 (diagnostic) and RYZ801 (therapeutic) to Identify and Treat Subjects with GPC3+ Unresectable HCC	Recruiting	Phase 1	70	Evaluate RYZ801 dose and safety; Evaluate RYZ811 safety and distribution
24	NCT03198052	GPC3/Mesothelin/Claudin18.2/GUCY2C/B7-H3/PSCA/PSMA/MUC1/TGFβ/HER2/Lewis-Y/AXL/EGFR-CAR-T Cells Against Cancers	Recruiting	Phase 1	30	Number of patients with DLT
25	NCT06652243	Clinical Study of SN301A Injection in the Treatment of Hepatocellular Carcinoma	Recruiting	Phase 1	12	DLT; Incidence and severity of AEs and SAEs (safety and tolerability)
26	NCT06478693	A Study of MT-303 in Adults With Advanced or Metastatic GPC3-Expressing Cancers, Including HCC	Recruiting	Phase 1	48	Type, incidence and severity of AEs; RP2D; change from baseline in vital signs; change in laboratory parameters; change from baseline in ECG parameters
27	NCT06084884	A Phase I/II Study to Evaluate AZD5851 in GPC3+ Advanced/Recurrent Hepatocellular Carcinoma	Recruiting	Phase 1 Phase 2	94	Incidence of participants with DLTs, AEs, including AESIs and SAEs; determination of the recommended dose of AZD5851 for expansion phase
28	NCT05120271	BOXR1030 T Cells in Subjects With Advanced GPC3-Positive Solid Tumors	Recruiting	Phase 1; Phase 2	110	Dose limiting toxicity; MTD; RP2D; TEAEs
29	NCT05783570	To Evaluate the Safety, Tolerability and Preliminary Efficacy of EU307	Recruiting	Phase 1	12	AEs (including DLT); production of replication competent lentiviruses; development of anti-drug antibodies
30	NCT04377932	Interleukin-15 Armored Glypican 3-specific Chimeric Antigen Receptor Expressed in T Cells for Pediatric Solid Tumors	Recruiting	Phase 1	24	Number of patients with DLT
31	NCT04715191	Interleukin-15 and -21 Armored Glypican-3-specific Chimeric Antigen Receptor Expressed in T Cells for Pediatric Solid Tumors	Recruiting	Phase 1	24	Number of patients with DLT
32	NCT06427941	A Phase 1 Study of BGB-B2033, Alone or in Combination With Tislelizumab, in Participants With Advanced or Metastatic Solid Tumors	Recruiting	Phase 1	70	DLT
33	NCT05103631	Interleukin-15 Armored Glypican 3-specific Chimeric Antigen Receptor Expressed in Autologous T Cells for Solid Tumors	Recruiting	Phase 1	27	Number of patients with DLT
34	NCT06088459	NWRD06 DNA Plasmid for HCC After Radical Resection	Recruiting	Phase 1	9	DLT; all AEs
35	NCT04756648	Phase I Clinical Trial of CT0180 Cells in the Treatment of Hepatocellular Carcinoma	Recruiting	Phase 1	21	DLT; MTD; AE; AESI
36	NCT06560827	CT011 Autologous CAR-T Cells in Patients With Hepatocellular Carcinoma at Risk of Recurrence After Surgical Resection	Recruiting	Phase 1	30	Incidence and severity of TEAE; incidence and severity of TRAE; incidence and severity of AESI
37	NCT04842812	Engineered TILs/CAR-TILs to Treat Advanced Solid Tumors	Recruiting	Phase 1	40	Safety of TILs/CAR-TILs treatment in advanced solid cancers
38	NCT06715839	Target-specific immunoPET Imaging of Digestive System Carcinoma	Recruiting	NA	400	Biodistribution; standardized uptake value; radiation dosimetry; diagnostic value in patients with digestive system malignancy; the correlation between the expression of specific target and tracer uptake value
39	NCT05263830	Glypican-3 as a Prognostic Factor in Patients With Hepatocellular Carcinoma Treated by Immunotherapy	Recruiting	NA	120	Evaluate concentration of circulating GPC-3
40	NCT04506983	GPC3-CAR-T Cells for the Hepatocellular Carcinoma	Suspended	Phase 1	12	Percentage of AEs
41	NCT06815432	GPC-3 CAR T CELLS for Recurrent GPC-3 Positive Glioblastoma	Not yet recruiting	Phase 1	27	Number of patients with DLT
42	NCT06641453	CAR-T Cell Therapy Targeting GPC3 in Patients with Advanced GPC3-Positive Hepatocellular Carcinoma	Not yet recruiting	Phase 1; Phase 2	30	Incidence of TRAEs; incidence of DLTs
43	NCT06198296	Immunotherapy for Adults with GPC3-Positive Solid Tumors Using IL-15 and IL-21 Armored GPC3-CAR T Cells	Not yet recruiting	Phase 1	21	Number of patients with DLT
44	NCT06590246	A Study to Evaluate C-CAR031 in Glypican-3 (GPC3)+ Advanced/recurrent Hepatocellular Carcinoma (HCC)	Not yet recruiting	Phase 1; Phase 2	121	Safety and tolerability; anti-tumor activity
45	NCT06795022	First in Human Study to Evaluate AZD9793 in Participants With Advanced or Metastatic Solid Tumours	Not yet recruiting	Phase 1; Phase 2	304	Number of patients with AEs; number of patients with SAEs; number of patients with AESIs; number of patients with DLT; objective response rate
46	NCT02723942	CAR-T Cell Immunotherapy for HCC Targeting GPC3	Withdrawn	Phase 1; Phase 2	0	Radiological assessment
47	NCT04093648	T Cells co- Expressing a Second Generation Glypican 3-specific Chimeric Antigen Receptor With Cytokines Interleukin-21 and 15 as Immunotherapy for Patients With Liver Cancer (TEGAR)	Withdrawn	Phase 1	0	DLT rate
48	NCT04121273	GPC3-targeted CAR-T Cell for Treating GPC3 Positive Advanced HCC	Unknown	Phase 1	20	Number of patient with dose limiting toxicity
49	NCT03084380	Anti-GPC3 CAR-T for Treating GPC3-positive Advanced Hepatocellular Carcinoma (HCC)	Unknown	Phase 1; Phase 2	20	Safety: Measured by occurrence of study related adverse effects defined by NCI CTCAE 4.0
50	NCT03130712	A Study of GPC3-targeted T Cells by Intratumor Injection for Advanced HCC (GPC3-CART)	Unknown	Phase 1; Phase 2	10	Safety of CAR-T cell infusion mediated by intratumoral injection as measured by number of participants with AEs
51	NCT02715362	A Study of GPC3 Redirected Autologous T Cells for Advanced HCC	Unknown	Phase 1; Phase 2	30	Safety of CAR-T cell infusion mediated by TAI as measured by number of participants with AEs
52	NCT05344664	Novel GPC3 CAR-T Cell Therapy for Hepatocellular Carcinoma	Unknown	Phase 1	12	Percentage of AEs
53	NCT02876978	Anti-GPC3 CAR T for Recurrent or Refractory Lung Squamous Cell Carcinoma	Unknown	Phase 1	20	Safety and tolerance: Occurrence of study related AEs
54	NCT04076137	Targeted T-cell Therapy in Solid Tumors	Unknown	Early Phase 1	10	Overrall survival
55	NCT03146637	Study of Activated CIK Armed With Bispecific Antibody for Advanced Liver Cancer	Unknown	Phase 2	80	Overrall survival
56	NCT04728139	Assessment of Glypican 3 as Apredictive Marker in Colorectal Cancer Patients	Unknown	NA	90	Evaluation of Glypican 3 role as a predictive and diagnostic marker of colorectal marker
57	NCT05059821	Personalized Cancer Vaccine in Egyptian Cancer Patients	Unknown	Phase 1	10	Assessment of the safety of the personalized cancer vaccine; assessment of immunological response

DLT: Dose limiting toxicity; RP2D: Recommended Phase 2 dose; MTD: Maximum tolerated dose; AE: Adverse event; SAE: Serious adverse event; AESI: Adverse event of special interest; TEAE: Treatment-emergent adverse event; TRAE: Treatment-related adverse event; GPC3: Glypican-3

Among the earliest GPC3-targeting therapeutic approaches, codrituzumab, a humanized monoclonal antibody against GPC3, was developed to leverage antibody-dependent cytotoxicity through its interaction with CD16/FcγRIIIa[62]. However, despite its promising preclinical efficacy, codrituzumab did not demonstrate any significant clinical benefit in a randomized Phase II placebo-controlled trial involving previously treated advanced HCC patients[62] (NCT01507168). Several factors may have contributed to this outcome. First, the clinical dosing only achieved approximately 50%-60% GPC3 target saturation, which may have been insufficient to trigger effective antitumor responses. Second, therapeutic benefit was observed primarily in patients with both high GPC3 and high CD16 expression. The absence of prospective biomarker-based patient selection likely resulted in a diluted overall treatment effect. Furthermore, interindividual variation in CD16 expression may have influenced the efficiency of NK cell-mediated antibody-dependent cellular cytotoxicity, further impacting response. These findings underscore the importance of tumor and immune microenvironment heterogeneity in determining therapeutic efficacy. Building on these insights, I-124 codrituzumab (GC33) was designed as a radiolabeled diagnostic tool for detecting GPC3-expressing HCC. In clinical evaluations, I-124 codrituzumab successfully localized tumors in most HCC patients without exhibiting cross-reactivity with normal tissues, demonstrating its potential as a non-invasive diagnostic tool[63]. Beyond monoclonal antibodies, GPC3 has emerged as a promising target for ADCs. Two GPC3-specific ADCs, hYP7-DC and hYP7-PC, were developed using duocarmycin SA and pyrrolobenzodiazepine dimer as cytotoxic payloads[7]. These ADCs demonstrated potent anti-tumor activity at picomolar concentrations against GPC3-positive cancer cell lines while sparing GPC3-negative cells[7]. Recently, GPC3-targeting peptide-drug conjugates have been explored for PDT, offering a selective tumor-killing strategy. Among them, conjugate 8b, designed by linking a GPC3-targeting peptide to chlorin e6, demonstrated strong tumor-targeting ability, potent anti-tumor efficacy, and minimal toxicity to normal cells[64]. In a HepG2 xenograft model, light-activated conjugate 8b achieved complete tumor elimination without harming surrounding tissues, highlighting its clinical potential[64].

ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody that simultaneously targets GPC3 on tumor cells and CD3 on T cells to promote T cell-mediated tumor killing[65]. In preclinical studies, ERY974 demonstrated strong antitumor activity against various GPC3-positive tumors and was capable of converting non-inflamed tumor microenvironments into inflamed ones, thereby enhancing T cell infiltration[65]. In addition, combination therapy with ERY974 and chemotherapeutic agents such as paclitaxel, cisplatin, and capecitabine showed synergistic efficacy by promoting T cell infiltration into the tumor core and enhancing drug activation, significantly improving responses in poorly immunogenic tumors[66]. The phase I clinical trial has been completed, where a starting dose of 3.0 ng/kg was selected, which is considered to be within a safe and potentially effective range based on both safety and efficacy perspectives[67]. As GPC3-targeted therapies continue to evolve, the focus has increasingly shifted toward immune cell-based treatments. In particular, CAR-T-cell therapy has shown impressive potential against GPC3-positive tumors in preclinical studies.

Preclinical evaluation of GPC3-CAR-T cells

In preclinical mouse models, GPC3-CAR-T cells demonstrated significant anti-tumor activity, eliminating approximately 66% of the tumor burden. Beyond direct tumor cell killing, these cells also influence Wnt signaling, which is critical in HCC progression. However, challenges such as limited persistence and the immunosuppressive tumor microenvironment hinder their therapeutic efficacy. To address these limitations, researchers are actively refining GPC3-CAR-T cell design in order to enhance both their functionality and clinical potential.

Patients in clinical trials NCT02905188 and NCT02932956 received GPC3 CAR-T cell therapy, which was found to be safe but did not generate a significant antitumor response[68]. However, challenges such as limited persistence and the immunosuppressive tumor microenvironment hindered its therapeutic efficacy. To overcome these limitations, researchers developed a modified version co-expressing IL-15 (15.CAR), as IL-15 has been shown to enhance expansion, intratumoral persistence, and antitumor activity in GPC3-CAR-T cells. In clinical trials NCT05103631 and NCT04377932, 15.CAR-T cells exhibited significantly improved expansion, resulting in a disease control rate of 66% and an objective response rate of 33%[68].

Cytokine co-expression to boost CAR-T cell persistence

A major limitation of CAR-T therapy in HCC is the absence of essential survival signals, such as IL-15 and IL-21, within the tumor microenvironment[69,70]. GPC3-CAR-T cells co-expressing IL-15 and IL-21 exhibited enhanced proliferation and anti-tumor activity, significantly improving therapeutic outcomes in HCC mouse models[71]. Additionally, a novel GPC3-7-19-CAR-T construct, engineered to secrete IL-7 and CCL19, demonstrated increased tumor-killing efficiency and promoted immune cell infiltration within the tumor microenvironment, further strengthening therapeutic efficacy[72].

Metabolic regulation for CAR-T enhancement

Metabolic adaptation is critical for CAR-T cell persistence and function. Overexpression of GLUT1 or AGK enhanced glycolytic metabolism and anti-apoptotic capacity, improving GPC3-CAR-T cell persistence and cytotoxicity via PI3K/Akt pathway activation in HCC xenograft models[73]. In addition to metabolic adaptation, T-cell exhaustion markers such as CD39 also play a crucial role in CAR-T cell persistence and function[74,75]. Optimal anti-tumor efficacy was observed in moderate CD39-expressing GPC3-CAR-T cells, as excessive or insufficient CD39 expression impaired function[76]. Targeting CD39 with either Mitochondrial Division Inhibitor 1 (Mdivi-1) or shRNA led to the highest proportion of CD39int CAR-T cells, resulting in improved tumor infiltration and robust anti-tumor activity in vivo[76].

Multi-target CAR-T therapies for enhanced efficacy

One of the major obstacles in CAR-T therapy is antigen escape, which occurs due to antigen loss, shedding, or low expression, as well as limited T-cell homing and immunosuppressive tumor microenvironments[77,78].

A recent preclinical study developed GPC3-BiTE CAR-T cells, which not only express a GPC3-directed CAR but also secrete BiTE molecules targeting B7H3[79]. These BiTEs engage CD3 on non-transduced T cells, expanding their recruitment and cytotoxic activity against HCC. This dual-targeting approach enabled non-transduced T cells to attack GPC3-/B7H3+ tumor cells, compensating for the limitations of single-antigen targeting and addressing antigen heterogeneity. Compared to conventional GPC3-CAR-T cells, BiTE-secreting CAR-T cells exhibited superior cytotoxicity against GPC3+/B7H3+ tumor cells and effectively targeted GPC3-/B7H3+ HCC cells, enhancing CD69 expression and IFN-γ production[79]. These findings highlight the potential of GPC3-BiTE CAR-T therapy in mitigating antigen escape and improving therapeutic outcomes.

To further enhance T-cell homing and infiltration, researchers engineered GPC3-CAR-T cells co-expressing IL-21 and CXCL9 in combination with PD-1 blockade, significantly improving tumor infiltration and tumor suppression in HCC xenograft models[80]. Mechanistically, this study demonstrates that GPC3-CAR-T cells engineered to co-express IL-21 and CXCL9 exhibit enhanced proliferation, cytokine secretion (including IL-2, IFN-γ, and TNF-α), and improved tumor infiltration[80]. When combined with PD-1 blockade, these modified CAR-T cells further increase their effector differentiation, such as cytotoxic T lymphocytes, natural killer T cells, and effector memory T (TEM) cells, and their cytotoxic activity by alleviating T cell exhaustion[80]. This dual approach not only promotes T cell recruitment via CXCL9-mediated chemotaxis but also overcomes immune suppression through PD-1 pathway inhibition. Together, the combination therapy markedly enhances antitumor efficacy in both in vitro and in vivo HCC models, offering a promising strategy to overcome the immunosuppressive tumor microenvironment and improve immunotherapeutic outcomes for GPC3-positive liver cancer patients.

Allogeneic and novel T-cell therapies

Conventional autologous CAR-T cell therapy faces challenges in manufacturing, T-cell homing, antigen heterogeneity, and tumor immune evasion. To overcome these limitations, researchers developed GPC3-CAR/sIL-15 Vδ1 T cells, leveraging the unique properties of Vδ1 γδ T cells, which exhibit MHC-independent cytotoxicity and a reduced risk of graft-versus-host disease (GvHD)[81]. By engineering these CAR-T cells to secrete soluble IL-15 (sIL-15), researchers improved both T-cell survival and persistence, counteracting the immunosuppressive tumor microenvironment. In vitro studies demonstrated that GPC3-CAR/sIL-15 Vδ1 T cells exhibited potent cytotoxicity against both high (HepG2) and low (PLC/PRF/5) GPC3-expressing HCC cells, suggesting their ability to overcome antigen heterogeneity[81]. In a HepG2 xenograft mouse model, a single intravenous injection of GPC3-CAR/sIL-15 Vδ1 T cells significantly inhibited tumor growth while predominantly accumulating at tumor sites, highlighting strong tumor infiltration capabilities. Compared to non-sIL-15-expressing CAR-T cells, those expressing sIL-15 demonstrated a prolonged expansion and sustained tumor suppression without requiring IL-2 supplementation. Moreover, this strategy reduced T-cell exhaustion markers (PD-1, TIM-3, LAG-3) and promoted memory T-cell formation, enhancing long-term immune responses. Importantly, no GvHD-related toxicity was observed, indicating a high safety profile[81]. These findings suggest that GPC3-CAR/sIL-15 Vδ1 T cells hold promise as an off-the-shelf therapy for HCC and other GPC3-expressing tumors.

GPC3-targeted DNA vaccine for enhanced T-Cell response

Beyond CAR-T cell therapy, a novel PLGA/PEI-HMGB1/GPC3 dual-target DNA vaccine was developed to enhance CD8+ T-cell responses and counteract immune suppression in HCC[82].

HMGB1, an inflammatory mediator overexpressed in solid tumors, contributes to cancer progression and immune modulation[83]. While blocking extracellular HMGB1 has been shown to inhibit tumor growth and enhance immune checkpoint blockade efficacy, HMGB1 also plays a role in adaptive immunity by promoting dendritic cell maturation, Th1 differentiation, and T-cell proliferation[84,85]. By leveraging HMGB1 as an immune adjuvant, researchers designed a GPC3-targeted vaccine that enhances dendritic cell activation and antigen presentation, leading to a stronger T-cell response against HCC. The PLGA/PEI nanoparticle delivery system further improved vaccine expression, antigen release, and T-cell efficacy. Preclinical studies in Hepa1-6-hGPC3 mouse models demonstrated significant tumor growth inhibition, along with increased CD8+ T-cell and dendritic cell infiltration[82]. Compared to a GPC3-only vaccine, the dual-target vaccine enhanced IFN-γ secretion and cytotoxic activity, resulting in better tumor control[82]. However, the precise role of HMGB1 in vaccine-induced immunity remains to be elucidated, necessitating further investigation in additional preclinical models.

COMPARISON TO OTHER HCC THERAPIES AND FUTURE PERSPECTIVES

The treatment of advanced HCC has evolved from TKIs, such as sorafenib and lenvatinib, to ICIs, which have improved clinical outcomes to some extent[86,87]. However, tumor heterogeneity, an immunosuppressive microenvironment, and adaptive resistance mechanisms continue to hinder treatment efficacy, leading to limited tumor responses and patient survival[9,10]. These challenges highlight the urgent need for novel, tumor-specific therapeutic strategies.

As a tumor-specific antigen highly expressed in HCC, GPC3 represents a promising therapeutic target with the potential to overcome current treatment limitations. Multiple clinical trials are evaluating GPC3-directed approaches, including CAR-T cell therapy, DNA vaccines, T-cell redirecting antibodies, and allogeneic strategies. Among these, CAR-T cell therapy has seen major advancements through cytokine modulation, metabolic reprogramming, dual-targeting strategies, and combination therapies with ICIs, significantly improving persistence, tumor infiltration and antitumor efficacy. Additionally, emerging modalities such as vaccines and multifunctional antibody therapies continue to expand the immunotherapy landscape, offering new opportunities for enhancing immune responses against HCC.

Although there has been significant progress, further improvements are needed to boost effectiveness, overcome resistance, and better identify the right patients for treatment. Future research should focus on integrating GPC3-targeted therapies into personalized treatment plans, exploring combination approaches, and enhancing drug delivery to maximize effectiveness. HBV-related HCC progression involves the epigenetic silencing of tumor-suppressive miRNAs, such as let-7c, through HBx-induced EZH2 upregulation, leading to HMGA2-mediated metastasis[88]. This underscores the complexity of HCC oncogenesis and highlights the potential of combinatorial strategies that target both surface antigens like GPC3 and intracellular oncogenic pathways.

CONCLUSION

With ongoing advancements in both CAR-T technology and clinical trials, GPC3-targeted therapies are poised to revolutionize HCC treatment, addressing key limitations within the existing therapies. By integrating innovative dual-targeting designs, metabolic enhancements, and immune checkpoint blockade, these therapies have the potential to overcome antigen escape, enhance T-cell function, and improve treatment durability. As research continues, GPC3-based immunotherapies may offer breakthrough solutions for HCC, bringing new hope for improved patient outcomes and long-term disease control.

ACKNOWLEDGEMENTS

The authors thank Mr. Tsung-Hung Hung from the Medical Art Department, IBMS, Academia Sinica, for figure preparation.