Glypican-3 (GPC3) has emerged as a candidate therapeutic target in hepatocellular carcinoma (HCC), but the oncogenic role of GPC3 in HCC is poorly understood. Here, we report a human heavy-chain variable domain antibody, HN3, with high affinity (K d = 0.6 nM) for cell-surface-associated GPC3 molecules. The human antibody recognized a conformational epitope that requires both the amino and carboxy terminal domains of GPC3. HN3 inhibited proliferation of GPC3-positive cells and exhibited significant inhibition of HCC xenograft tumor growth in nude mice. The underlying mechanism of HN3 action may involve cell-cycle arrest at G1 phase through Yes-associated protein signaling. This study suggests a previously unrecognized mechanism for GPC3-targeted cancer therapy.heparan sulfate proteoglycans | liver cancer | monoclonal antibodies | phage display | cell growth

Glypican-3 is a cell surface glycoprotein that associates with Wnt in liver cancer. We develop two antibodies targeting glypican-3, HN3 and YP7. The first antibody recognizes a functional epitope and inhibits Wnt signaling, whereas the second antibody recognizes a C-terminal epitope but does not inhibit Wnt signaling. Both are fused to a fragment of Pseudomonas exotoxin A (PE38) to create immunotoxins. Interestingly, the immunotoxin based on HN3 (HN3-PE38) has superior anti-tumor activity as compared to YP7 (YP7-PE38) both in vitro and in vivo. Intravenous administration of HN3-PE38 alone, or in combination with chemotherapy, induces regression of Hep3B and HepG2 liver tumor xenografts in mice. This study establishes glypican-3 as a promising candidate for immunotoxin-based liver cancer therapy. Our results demonstrate immunotoxin-induced tumor regression via dual mechanisms: inactivation of cancer signaling via the antibody and inhibition of protein synthesis via the toxin.

Wnt signaling is important for cancer pathogenesis and is often upregulated in hepatocellular carcinoma (HCC). Heparan sulfate proteoglycans (HSPGs) function as co-receptors or modulators of Wnt activation. Glypican-3 (GPC3) is a HSPG that is highly expressed in HCC, where it can attract Wnt proteins to the cell surface and promote cell proliferation. Thus, GPC3 has emerged as a candidate therapeutic target in liver cancer. While monoclonal antibodies to GPC3 are currently being evaluated in preclinical and clinical studies, none have shown an effect on Wnt signaling. Here, we first document the expression of Wnt3a, multiple Wnt receptors and GPC3 in several HCC cell lines, and demonstrate that GPC3 enhanced the activity of Wnt3a/β-catenin signaling in these cells. Then, we report the identification of HS20, a human monoclonal antibody against GPC3, which preferentially recognized the heparan sulfate chains of GPC3, both the sulfated and non-sulfated portions. HS20 disrupted the interaction of Wnt3a and GPC3 and blocked Wnt3a/β-catenin signaling. Moreover, HS20 inhibited Wnt3a-dependent cell proliferation in vitro and HCC xenograft growth in nude mice. In addition, HS20 had no detectable undesired toxicity in mice. Taken together, our results show that a monoclonal antibody primarily targeting the heparin sulfate chains of GPC3 inhibited Wnt/β-catenin signaling in HCC cells and had potent anti-tumor activity in vivo. Conclusion Here, we provide one of the first examples of an antibody directed against the heparan sulfate of a proteoglycan that showed efficacy in blocking Wnt signaling and HCC growth, suggesting a novel strategy for liver cancer therapy.

Wnt signaling is one of the key regulators of hepatocellular carcinoma (HCC) tumor progression. In addition to the classical receptor frizzled (FZD), various coreceptors including heparan sulfate proteoglycans (HSPGs) are involved in Wnt activation. Glypican‐3 (GPC3) is an HSPG that is overexpressed in HCC and functions as a Wnt coreceptor that modulates HCC cell proliferation. These features make GPC3 an attractive target for liver cancer therapy. However, the precise interaction of GPC3 and Wnt and how GPC3, Wnt, and FZD cooperate with each other are poorly understood. In this study, we established a structural model of GPC3 containing a putative FZD‐like cysteine‐rich domain at its N‐terminal lobe. We found that F41 and its surrounding residues in GPC3 formed a Wnt‐binding groove that interacted with the middle region located between the lipid thumb domain and the index finger domain of Wnt3a. Mutating residues in this groove significantly inhibited Wnt3a binding, β‐catenin activation, and the transcriptional activation of Wnt‐dependent genes. In contrast with the heparan sulfate chains, the Wnt‐binding groove that we identified in the protein core of GPC3 seemed to promote Wnt signaling in conditions when FZD was not abundant. Specifically, blocking this domain using an antibody inhibited Wnt activation. In HCC cells, mutating residue F41 on GPC3 inhibited activation of β‐catenin in vitro and reduced xenograft tumor growth in nude mice compared with cells expressing wild‐type GPC3. Conclusion: Our investigation demonstrates a detailed interaction of GPC3 and Wnt3a, reveals the precise mechanism of GPC3 acting as a Wnt coreceptor, and provides a potential target site on GPC3 for Wnt blocking and HCC therapy.

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BackgroundShark new antigen receptor variable domain (VNAR) antibodies can bind restricted epitopes that may be inaccessible to conventional antibodies.MethodsHere, we developed a library construction method based on polymerase chain reaction (PCR)-Extension Assembly and Self-Ligation (named “EASeL”) to construct a large VNAR antibody library with a size of 1.2 × 1010 from six naïve adult nurse sharks (Ginglymostoma cirratum).ResultsThe next-generation sequencing analysis of 1.19 million full-length VNARs revealed that this library is highly diversified because it covers all four classical VNAR types (Types I–IV) including 11% of classical Type I and 57% of classical Type II. About 30% of the total VNARs could not be categorized as any of the classical types. The high variability of complementarity determining region (CDR) 3 length and cysteine numbers are important for the diversity of VNARs. To validate the use of the shark VNAR library for antibody discovery, we isolated a panel of VNAR phage binders to cancer therapy-related antigens, including glypican-3, human epidermal growth factor receptor 2 (HER2), and programmed cell death-1 (PD1). Additionally, we identified binders to viral antigens that included the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) spike proteins. The isolated shark single-domain antibodies including Type I and Type II VNARs were produced in Escherichia coli and validated for their antigen binding. A Type II VNAR (PE38-B6) has a high affinity (Kd = 10.1 nM) for its antigen.ConclusionsThe naïve nurse shark VNAR library is a useful source for isolating single-domain antibodies to a wide range of antigens. The EASeL method may be applicable to the construction of other large diversity gene expression libraries.

Background: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two most common primary liver cancers, yet there have been no significant advances in effective therapeutics. Mesothelin has been reported as a new therapeutic target in various types of cancer. Here, we investigated the expression of mesothelin in liver cancer and its potential role as a novel therapeutic target for immunotherapy.Methods: HCC and CCA specimens were examined by immunohistochemistry for mesothelin expression. Protein expression was assessed by immunoblotting and flow cytometry. The SS1P immunotoxin targeting mesothelin was evaluated in the well-established CCA cell lines HuCCT1, HuH-28, KMBC, KMCH, Mz-ChA-1 and OZ.Results: We showed strong immunochemical mesothelin staining in 33% of the surgically resected CCA specimens and 3 of 6 CCA cell lines (OZ, KMBC and KMCH). No mesothelin staining was found in HCC or normal liver tissue. Mesothelin was primarily localized to the cellular plasma membrane and the mature form (molecular weight, ~40 kDa) was expressed at a high level in CCA tissues. Moreover, 22% of CCA specimens had a high mesothelin expression level which was comparable to the CCA cell line models. Interestingly, SS1P showed very high and specific growth inhibition when added to mesothelin-expressing CCA cells with IC50 values ranging from 0.5 to 11 ng/mL.Conclusions: Mesothelin is overexpressed in one-third of CCA tissues. SS1P targeting mesothelin reveals a remarkable single agent activity against CCA in vitro. These findings indicate a potential for SS1P in the immunotherapeutic treatment of CCA.

Glypican-3 (GPC3) represents an attractive target for hepatocellular carcinoma (HCC) therapy because it is highly expressed in HCC but not in adult normal tissue. Recently, high affinity anti-GPC3 antibodies have been developed; however, full antibodies may not penetrate evenly into tumor parenchyma, reducing their effectiveness. In this study, we compared a whole IgG antibody, anti-GPC3 YP7, with an anti-GPC3 human heavy chain antibody, HN3, with regard to their relative therapeutic effects. Both YP7 and HN3 bound to GPC3-positive A431/G1 cells and were internalized by the cells by in vitro evaluation with (125)I- and (111)In-radiolabeling antibodies. In vivo biodistribution and tumor accumulation was performed with (111)In-labeled antibodies, and intratumoral microdistribution was evaluated using fluorescently labeled antibodies (IR700). HN3 showed similar high tumor accumulation but superior homogeneity within the tumor compared with YP7. Using the same IR700 conjugated antibodies photoimmunotherapy (PIT) was performed in vitro and in a tumor-bearing mouse model in vivo. PIT with IR700-HN3 and IR700-YP7 demonstrated that comparable results could be achieved despite of low reaccumulation 24 h after the first NIR light exposure. These results indicated that a heavy-chain antibody, HN3, showed more favorable characteristics than YP7, a conventional IgG, as a therapeutic antibody platform for designing molecularly targeted agents against HCC.