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Volume 588, Issue 2 p. 377-382
Review article
Free Access

Glypican-3 antibodies: A new therapeutic target for liver cancer

Mingqian Feng

Mingqian Feng

Antibody Therapy Section, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

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Mitchell Ho

Corresponding Author

Mitchell Ho

Antibody Therapy Section, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

Corresponding author.Search for more papers by this author
First published: 15 October 2013
Citations: 82

Abstract

Glypican-3 (GPC3) is an emerging therapeutic target in hepatocellular carcinoma (HCC), even though the biological function of GPC3 remains elusive. Currently human (MDX-1414 and HN3) and humanized mouse (GC33 and YP7) antibodies that target GPC3 for HCC treatment are under different stages of preclinical or clinical development. Humanized mouse antibody GC33 is being evaluated in a phase II clinical trial. Human antibodies MDX-1414 and HN3 are under different stages of preclinical evaluation. Here, we summarize current evidence for GPC3 as a new target in liver cancer, discuss both its oncogenic function and its mode of actions for current antibodies, and evaluate potential challenges for GPC3-targeted anti-cancer therapies.

1 Hepatocellular carcinoma (HCC) is a high-ranking malignancy with limited treatment modalities

Liver cancer is the fifth most prevalent cancer in the world and the third most frequent cause of cancer-related death [1-3]. Both the incidence and associated-mortality of liver cancer are rising. Hepatocellular carcinoma (HCC) is the major form of liver cancer, accounting for 90% of all liver cancers, and resulting in at least 500 000 deaths per year [4]. The overall 5-year relative survival rate for patients with liver cancer is about 15% in the US (www.cancer.org). Liver cancer is usually resistant to most chemotherapy drugs [5]. Potential curative therapeutic approaches are available only for cases in which the diagnosis is done at an asymptomatic early stage [3], which constitutes only 37% of patients. In vivo imaging and surveillance of individuals with high risk are most utilized strategies for early detection of hepatic nodules [6], while histological examination of biopsy samples for tumor markers (such as HSP70) could significantly increase the diagnosis accuracy [7]. The major risk factors of HCC include cirrhosis, hepatitis B- and hepatitis C-virus infection, non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), obesity, and diabetes [8, 9].

2 GPC3 is a potential biomarker for both targeted therapy and diagnosis of HCC

Glypican-3 (GPC3, also called as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS, SGBS1, or heparan sulfate proteoglycan (HSPG) is a member of the glypican family that attaches to the cell surface by a glycosylphosphatidylinositol (GPI) anchor [10] (Fig. 1 A). The GPC3 core protein is a 70-kD protein, with a furin cleavage site in the middle. Furin cleavage results in the formation of the 40-kD N-terminal fragment and the 30-kD C-terminal fragment [11] (Fig. 1A). Recently, the crystal structure of Drosophila glypican Dally-like (Dlp, a homolog to human GPC1) and human GPC1 showed that the N- and C-terminal domains of GPC1 are linked by disulfide bonds [12, 13]. Given that the glypican family (including six members from GPC1 to GPC6) contains 14 highly conserved cysteine residues, their three-dimensional structures are presumed to be similar, indicating that the N- and C-terminal fragments of GPC3 are very likely to be associated at the cell surface by intra-molecular disulfide bonds.

figure image
Therapeutic antibodies targeting GPC3 for liver cancer treatment. (A) Diagram illustration of GPC3 structure and the binding sites of the current antibodies. N, amino-terminal domain. C, carboxyl-terminal domain. HS, heparan sulfate. (B) The mechanism of HN3 antibody function. In the absence of HN3, growth factors bind to GPC3 and promote cell proliferation. In the presence of HN3, HN3 blocks the binding of growth factors and triggers intracellular signaling, leading to inactivation of YAP and inhibition of cell proliferation in HCC.

2.1 GPC3 expression in HCC

Several studies have confirmed that GPC3 is a potential liver cancer therapeutic target because it is over-expressed in HCC but not expressed or expressed at low levels in normal adult tissue [14-16].

Hsu et al. performed pioneering work to identify GPC3 as a potential biomarker for HCC [17]. By using an mRNA differential display method with paired HCC and non-tumor liver samples, Hsu et al. found GPC3 mRNA highly expressed in 9 out of 14 HCC samples, whereas none were detected in eight non-tumor liver samples. This HCC specificity was further confirmed by Northern blot analysis in an expanded number of HCC samples, fetal and adult normal tissues, as well as other adult tumor types. From 154 patients, 143 out of 191 (74.8%) primary and recurrent HCC samples were GPC3 positive, but only 5 out of 154 (3.2%) non-tumor liver samples had detectable GPC3 mRNA. In fetal tissues, GPC3 mRNA level was high in lung, liver, kidney, and placenta, and low in pancreas. In adult tissues, GPC3 mRNA only had low expression in heart, lung, kidney, and ovary, and in trace amounts in skeleton muscle, pancreas, small intestine, and colon [17]. Comparison of GPC3 with another established HCC marker, alphafetoprotein (AFP), revealed a higher frequency of GPC3 mRNA expression than serum AFP level (71.7% versus 51.3%) based on the analysis of 113 patients with unicentric primary HCC. The difference was even more significant when tumor size was below 3 cm (77% for GPC3 versus 43% for AFP) [17].

By using Northern blot and in situ hybridization, Zhu et al. also found that GPC3 mRNA was either low or absent in normal liver, focal nodular hyperplasia (FNH), and in liver cirrhosis [16]. In contrast, expression of GPC3 mRNA was markedly increased in 20 out of 30 HCC samples and moderately increased in five out of 30 HCC samples. The average increase in GPC3 mRNA expression in HCC was 21.7-fold compared with expression in normal liver, and 7.2- and 10.8-fold, respectively in comparison with FNH or liver cirrhosis.

Filmus et al. later on confirmed GPC3 expression in HCC patients at the protein level by a mouse monoclonal antibody (1G12) against a GPC3 C-terminal peptide [18]. By using immunohistochemistry staining and ELISA method, Filmus et al. found GPC3 over-expressed in 72% of HCC (21 out of 29) based on immunohistochemistry, and 53% (18 out of 34) of HCC patients had elevated GPC3 level in serum (151–2924 ng/ml), while it is undetectable in healthy donors. Since then, more and more studies, majorly based on immunohistochemistry, convinced that GPC3 could be a marker for routine histological examination and potentially as targets in monoclonal antibody-based hepatocellular carcinoma therapy. Yamauchi et al. developed two other GPC3 monoclonal antibodies, GPC3-C02 and A1836A, and performed GPC3-immunohistochemistry in the pathological diagnosis of benign and malignant hepatocellular lesions with formalin-fixed and paraffin-embedded specimens [19]. Diffusely positive staining of GPC3 was observed in malignant hepatocytes in hepatoblastomas and in hepatocellular carcinomas (47/56, 84%), and the expression of GPC3 was independent of the differentiation and size of the hepatocellular carcinoma [19].

GPC3 may also be used as an ancillary tool in the histopathologic diagnostic process to distinguish HCC from cirrhosis, dysplastic nodules, and focal nodular hyperplasia-like nodules [20]. Libbrecht et al. performed immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction studies on 59 HCCs with a diameter less than or equal to 3 cm present in the cirrhotic liver of 66 patients and in patients from 16 low-grade dysplastic nodules, 33 high-grade dysplastic nodules, and 13 focal nodular hyperplasia-like nodules [20]. It was found that GPC3 expression was higher in small HCCs than in cirrhosis and other types of small focal lesions, indicating that the transition from premalignant lesions to small HCC is associated with a sharp increase of GPC3 expression in a majority of cases. The sensitivity and specificity of a positive GPC3-staining for the diagnosis of HCC in small focal lesions was 77% and 96%, respectively, in resected cases, and 83% and 100%, respectively, for needle biopsies. [20]. Similar results were also reported by Wang et al. who used immunohistochemistry on 54 HCCs and adjacent liver tissues (21 developing from cirrhosis and 33 from normal liver) and 94 cirrhotic macronodules [21]. GPC3 staining was observed in 19 (90%) of 21 HCC cases with cirrhosis and in 18 (64%) of 28 HCC cases with normal liver. Among the 94 macronodules, GPC3 immunostaining was noted in 48% (14/29) of high-grade dysplastic or early HCC and in 3% (2/65) of benign or low-grade dysplastic macronodules, indicating GPC3 may be used to identify some cirrhotic macronodules with malignant potential. Baumhoer et al. used tissue microarray immunohistochemical technology to investigate 4387 tissue samples from 139 tumor categories and 36 non-neoplastic and preneoplastic tissue types [14]. GPC3 expression was detected in 9.2% of non-neoplastic liver samples (11/119), 16% of preneoplastic nodular liver lesions (6/38), and 63.6% of HCCs (140/220), indicating GPC3 could differentiate HCC from non-neoplastic and preneoplastic liver disease. Furthermore, several other tumors also revealed GPC3 expression, including squamous cell carcinoma of the lung (27/50, 54%), testicular non-seminomatous germ cell tumors (32/62, 52%), and liposarcoma (15/29, 52%) [14]. Coston et al. studied the expression of GPC3 and CD34 in 107 cases of HCC, 19 cases of hepatic adenoma (HA), 16 cases of focal nodular hyperplasia (FNH), and 225 cases of non-hepatic human tumors with epithelial differentiation [22]. Ninety-four of 107 cases (88%) of HCC showed focal or diffuse cytoplasmic GPC3 staining, whereas all HA and FNH cases were GPC3-negative, and only 7 of 225 cases (3%) of non-hepatic tumors with epithelial differentiation expressed GPC3. This data indicates that GPC3 is a very specific marker not only for differentiating HCC from non-hepatic tumors with epithelial differentiation, but also for differentiating HCC from HA and FNH [22]. Wang et al. studied 111 HCCs, 48 hepatocellular adenomas, 30 focal nodular hyperplasias, and 32 large regenerative nodules in the background of cirrhosis [23]. It was found that cytoplasmic, membranous, and canalicular GPC3 staining was detected in 84 (75.7%) of the 111 HCCs. In contrast, none of the 110 cases of hepatocellular adenoma, focal nodular hyperplasia, and large regenerative nodule showed detectable GPC3 staining. GPC3 expression in HCCs did not correlate with the size, differentiation, or stage of the tumors, the presence or absence of cirrhotic background, or the underlying etiologies [23]. Many other studies have similarly shown that GPC3 is highly and specifically expressed in 70–100% cases of HCC, and could be used as a maker to differentiate HCC from benign liver tissues [24-28].

2.2 Combination of GPC3 with other markers for histological diagnosis of HCC

Since one marker may not be sufficient to detect all HCC cases, adoption of a panel of HCC markers may be a good choice. The most common combination of HCC markers is GPC3, HSP70 and glutamine synthetase (GS) [7, 29, 30]. Di Tommaso analyzed 52 surgically removed non-malignant hepatocellular nodules (15 large regenerative (LRN), 15 low grade dysplastic (LGDN), 22 high grade dysplastic (HGDN) and 53 HCCs (10 early, 22 grade 1, and 21 grade 2–3) by immunostaining them for HSP70, GPC3, and GS [29]. The sensitivity and specificity of the individual markers for the detection of early HCC grade 1 (eHCC-G1) were 59% and 86% for GS, 69% and 91% for GPC3, and 78% and 95% for HSP70. Using a 3-marker panel with a minimum of two positives, (regardless of which), the sensitivity and specificity for the detection of eHCC-G1 were respectively 72% and 100%. The most sensitive combination was HSP70+/GPC3+ (59%) when a 2-marker panel was used. In contrast, the combination of three positive markers revealed a negative detection of 100% LRN and LGDN, 73% HGDN and 3% eHCC-G1 [29].

2.3 GPC3 as a serum marker

Although GPC3 is a cell-surface marker, it can also be released into the serum by a lipase called Notum that cleaves the GPI anchor [31]. Therefore, GPC3 also showed diagnostic values as a serum marker [32, 33]. Qiao et al. compared the serum level of three markers, GPC-3, Human-Cervical-Cancer-Oncogene (HCCR) and α-fetoprotein (AFP), for diagnosing HCC in 189 samples (101 cases of HCC, 40 cases of cirrhosis, 18 cases of hepatitis and 30 cases of control healthy donors). It was found that GPC-3 was the best marker. Using 26.8 ng/ml as the cut-off for HCC diagnosis, GPC-3 had a sensitivity of 51.5% and a specificity of 92.8%. HCCR reached a sensitivity of 22.8% and a specificity of 90.9% if the cut-off was set as 58.8 mAU/ml. The efficacy and sensitivity of AFP were 36.6% and 98.5% when using 199.3 ng/ml as the cut-off. No significant correlation was found between these three markers. Simultaneous detection of three markers significantly increased the sensitivity to 80.2%, much higher than AFP alone [33]. Chen et al. also measured serum GPC3 in a total of 1037 subjects, including 155 patients with HCC, 180 with chronic hepatitis, 124 with liver cirrhosis, 442 with non-HCC cancer and 136 healthy controls. The average level of serum GPC3 (sGPC3) in HCC patients was 99.94 ± 267.2 ng/ml, which was significantly higher than in patients with chronic hepatitis (10.45 ± 46.02 ng/ml), liver cirrhosis (19.44 ± 50.88 ng/ml), non-HCC cancer (20.50 ± 98.33 ng/ml) and healthy controls (4.14 ± 31.65 ng/ml) [32]. In addition to full length GPC3, Hippo et al. found the N-terminal portion of GPC3 (GPC3N) is cleaved and secreted into serum, therefore the GPC3N fragment may also serve as a serological marker [34]. However, the serum level of GPC3N fragment (4.84 ± 8.91 ng/ml in HCC versus 1.09 ± 0.74 ng/ml in liver cirrhosis, and 0.65 ± 0.32 ng/ml in healthy controls) is much lower than that of the full-length protein.

3 GPC3 promotes the growth of HCC by stimulating oncogenic signaling pathways

GPC3 was initially discovered from a patient with Simpson–Golabi–Behmel syndrome (SGBS), a rare X-linked overgrowth disease [35], which is caused by loss-of-function mutations. GPC3-deficient mice display developmental overgrowth and some of the abnormalities typical of SGBS [36]. In transgenic mice, over-expression of GPC3 suppresses hepatocyte proliferation and liver regeneration [37].

At the cellular level, GPC3 may act as a co-receptor or storage pocket for several growth factors, including Wnts [38, 39], Hedgehogs [40, 41], fibroblast growth factors (FGFs) [42], and bone morphogenetic proteins [43], therefore regulating the interaction of these growth factors to their cell-surface receptors. This may explain the observation that cell-surface GPC3 promoted the growth of HCC cells in the following two studies [44, 45]. First, HCC cells infected with lentivirus expressing soluble GPC3 (a secreted form that lacks the GPI anchoring domain) have a lower cell proliferation rate [44], suggesting that the soluble GPC3 protein secreted by infected cells may inhibit cell proliferation in an autocrine manner by competing the binding of grow factors with endogenous cell-surface GPC3. To confirm this, our group produced recombinant GPC3 (GPC3ΔGPI, amino acid residues Q25-H559) and found that recombinant GPC3 protein, functioning as a dominant-negative form, inhibited the growth of HCC in vitro [45]. In addition, silencing GPC3 expression by siRNA or shRNA in HCC cell lines HepG2, Hep3B, Huh-7 and Huh-4 can inhibit cell proliferation [46, 47]. Sun et al. performed transient transfection of Huh-7 and HepG2 cells with GPC3 siRNA, and found suppression of GPC3 induced upregulation of TGF-β2 [46]. Furthermore, addition of human recombinant TGF-β2 to HCC cells in culture prevented cell growth, and cotransfection of siRNA-GPC3 with siRNA-TGF-β2 partially attenuated the effects of GPC3 suppression on cell proliferation, cell cycle progression, apoptosis, and replicative senescence, indicating the involvement of TGF-β2 in siRNA-GPC3-mediated growth suppression [46].

4 Antibodies that are therapeutically targeting GPC3

To date, several mouse monoclonal antibodies (mAb) against GPC3 have been produced [18, 19, 34, 48-52], and almost all of them target a peptide derived from GPC3. However, none of these antibodies have shown the ability to inhibit HCC cell proliferation or induce apoptosis. Consequently, they are used as a research tool, with the exception of GC33, which is currently being developed as a potential therapeutic agent. Together, four GPC3 antibodies including GC33 are being developed for liver cancer therapy (Table 1 ).

Table Table 1. Current therapeutic monoclonal antibodies targeting GPC3 in liver cancer
Antibody name Species Antibody form Epitope Mechanism of action Development status Antibody developer References
GC33 Mouse (humanized) IgG Linear, C-terminal (residues 524–563) ADCC Phase II Chugai Pharmaceutical Co. Ltd. and Roche [50-54]
YP7 Mouse (humanized) IgG Linear, C-terminal (residues 511–560) ADCC Preclinical NCI [49]
HN3 Human VH-hFc Conformational, both N- and C-terminal domains Inhibition of YAP signaling; direct inhibition of HCC cell proliferation; ADCC Preclinical NCI [47]
MDX-1414 Human IgG N/A N/A Preclinical Bristol-Myers Squibb Co. AACR.2009 (Abstract# 1223), 58

4.1 GC33: the first humanized mouse antibody being evaluated in clinical trial

GC33 recognizes a C-terminal peptide of GPC3 [50, 51] (Fig. 1A). Humanized GC33, as a single agent, has passed phase I clinical trials for advanced or metastatic HCC (ClinicalTrials.gov Identifier: NCT00746317) [53]. More clinical trials for GC33 in combination with FDA-approved chemo-drug sorafenib (phase I, ClinicalTrials.gov Identifier: NCT00976170) and GC33 alone (phase II, ClinicalTrials.gov Identifier: NCT01507168) are currently recruiting volunteers. The mechanism of GC33 function is though antibody-dependent cell cytotoxicity (ADCC), and GC33 can bring cytotoxic infiltrating T-lymphocytes into tumor tissues [50, 52, 54].

GC33 exhibited marked tumor growth inhibition of subcutaneously transplanted HepG2 and Huh-7 xenografts [50]. In a phase I trial, patients with measurable, histologically proven, advanced HCC were enrolled to a dose-escalation study of GC33 (2.5–20 mg/kg) given intravenously weekly. The results of 20 patients showed that no maximum tolerated dose was reached as there were no dose-limiting toxicities up to the highest planned dose level. Mean half-life (t 1/2) was 2.94, 3.46, 5.16, and 6.47 days, at 2.5, 5, 10, and 20 mg/kg respectively. Median time to progression was 26.0 weeks in the GPC3 high expression group and 7.1 weeks in the GPC3 low expression group. Stable disease of more than 26 weeks was observed in 4 of 15 (26.7%) patients and all of them were in the GPC3 high expression group. Common adverse events with all grades included fatigue (50%), constipation (35%), headache (35%), and hyponatremia (35%). The incidence of adverse events seemed not to be dose dependent. Overall, this phase I study showed that GC33 was well tolerated in advanced HCC and the preliminary clinical benefit of GC33 warrants prospective evaluation [53]. In the ongoing phase II clinical trial, the dosing of GC33 is set as 1600 mg iv Day 1 and 8, and every 2 weeks thereafter (ClinicalTrials.gov Identifier: NCT01507168).

4.2 YP7: a new mouse anti-GPC3 antibody with high affinity

Our lab generated a new mouse antibody (named YP7) via GPC3 peptide (a.a. 510–560) immunization and selected by high-throughput cell binding screening by flow cytometry [49]. YP7 has a single-digit picomolar affinity for GPC3, and has much more sensitive detection limit than the commercially available antibody, 1G12, in immunohistochemistry and Western blot. The epitope of YP7 overlaps with that of GC33 (Fig. 1A). YP7 has in vivo tumor-suppression activity and holds great potential for in vivo tumor imaging as well as antibody therapies.

4.3 HN3: a human single-domain antibody that can directly inhibit HCC cell proliferation

Ideally, therapeutic antibodies should have direct cell-growth inhibition function by blocking important signaling pathways (e.g., Herceptin), plus ADCC and CDC (complement-dependent cytotoxicity). Conventional antibodies (i.e., Y-shaped antibodies with full-length heavy chains and light chains) targeting GPC3 do not have direct inhibition properties, due speculatively to the difficulty of targeting the potentially cryptic functional epitope of GPC3 by a conventional antibody. Domain antibodies (VH or VL domains only) are able to target cryptic epitopes on antigens (e.g., the clefts of enzymes and receptors) due to their small size [55-57]. Based on this rationale, a human heavy chain variable (VH) domain antibody (HN3) targeting GPC3 was isolated and identified by using phage display technology [47].

HN3 binds a unique conformational epitope in the native form of GPC3 core protein on cancer cells with high affinity (Fig. 1A). HN3 binding requires both the N-terminus and C-terminus domains of GPC3, and is independent of the HS chains on GPC3. This feature distinguishes HN3 from all the known mAbs that recognize either the N- or C-terminus of GPC3. Furthermore, the conformation of the HN3 binding site may affect a newly-discovered GPC3 function, since HN3 can directly inhibit HCC cell growth in several HCC cell models and exhibited significant HCC xenograft tumor growth inhibition in nude mice. Our HN3 study suggests that it is possible to inhibit HCC cell proliferation with an antibody that neutralizes the proliferative function of GPC3 by targeting the appropriate epitope. Due to the fact that HN3 is the first mAb that shows direct inhibition of cell proliferation, future GPC3 structural studies are worthwhile to reveal the precise structure that HN3 recognizes.

In addition to the unique epitope that it targets, the mechanism of HN3 function is also novel. The down-stream signaling pathway of HN3 involves YAP inactivation and cell cycle arrest. YAP is an oncogene that when knocked down in HCC cells, cell proliferation becomes significantly decreased. Over-expression of YAP-S127A, a constitutively active form of YAP, completely abolished HN3 activity and promoted cell proliferation. How GPC3 regulates the YAP pathway has not been described and only speculation may be made on the extracellular event of HN3 function. A reasonable assumption is that HN3 blocks the interaction of GPC3 with undetermined growth factors, triggering the intracellular inactivation of YAP signaling, and eventually leading to cell proliferation inhibition (Fig. 1B).

HN3 has three unique properties that GC33 lacks – it can directly inhibit HCC cell proliferation, it is a single-domain antibody, and it is a fully human protein. Due to these characteristics and its unique mechanism, HN3 is an attractive addition to the existing GPC3-targeted liver cancer therapies. Side-by-side studies to compare GC33 and HN3 in both preclinical and clinical settings are needed.

4.4 MDX-1414: human anti-GPC3 antibody in pre-clinical studies

Medarex, Inc. (Princeton, US, now owned by Bristol-Myers Squibb Corporate, New York, US) generated a panel of fully human anti-glypican-3 antibodies, from which a lead candidate, MDX-1414, was selected for its high affinity, specificity, effector function and internalization properties. MDX-1414 demonstrated anti-tumor efficacy leading to significant and durable suppression of established subcutaneous tumors in a liver cancer xenograft model with no evidence of toxicity (Li-Sheng Lu, Jon Terrett, Chin Pan, Dapeng Yao, Colin Chong, Jerry Jiang, Pina Cardarelli, Yi Wu, Haichun Huang, Tim Chen, Alasdair Bell, Mohan Srinivasan, Karla Henning. Development of anti-Glypican-3 therapeutic antibodies. Annual Proceedings of the AACR.2009, Abstract #1223). MDX-1414 is being evaluated for preclinical development [58].

5 Potential problems for GPC3-targeted antibody therapies

Based on the knock-down and siRNA experimental results, GPC3 is not a lethal gene to HCC cells. Therefore, it remains to be established whether anti-GPC3 antibodies can cause complete regression of tumor growth. Both GC33 and HN3 cannot completely eliminate tumors, nor does knocking down GPC3 work, therefore indicating that the therapeutics of a naked antibody may not be potent enough for curative treatment of HCC. Strategies to overcome this problem include combination with chemotherapy (e.g., sorafenib that has been approved for HCC), and armed antibodies, such as antibody-drug conjugates, bi-specific antibodies (e.g., anti-GPC3/anti-CD3), and chimeric antigen receptor-T cell adoptive therapy.

Another question that has not been studied is the stability of GPC3 expression after antibody treatment. It is possible that treatment-survived cells may lose GPC3 expression to gain drug resistance. To address this issue, adiditonal future studies on GPC3 expression regulation are needed.

6 Concluding remarks

GPC3 is an attractive target for developing therapeutic antibodies to treat HCC patients. However, its structure–function relationship is not clear. Most GPC3-targeting antibodies, excluding HN3, did not directly inhibit HCC cell growth. Our HN3 study suggests that the direct tumor growth-inhibition is dependent on the functional epitope of GPC3. Therefore, further structure-biology studies of GPC3 will facilitate the discovery of new antibodies that may have more potent tumor-suppression activity.

Acknowledgments

This research was supported by the . We thank the NIH Fellows Editorial Board for editorial assistance. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.