GPC3 Human

What is GPC3 and what is its molecular structure?

GPC3 is a glycophosphatidylinositol (GPI)-anchored cell surface heparan sulfate proteoglycan. The protein core consists of two subunits: an N-terminal subunit (~40 kDa) and a C-terminal subunit (~30 kDa) . The full protein has a predicted molecular weight of approximately 21.5 kDa for specific fragments like the Arg438-Asn554 region . GPC3 undergoes post-translational modifications including the addition of heparan sulfate glycan chains, which are important for some of its biological functions, particularly in growth factor binding .

GPC3 is encoded by the GPC3 gene in humans, with several synonyms including DGSX, GTR2-2, MXR7, OCI-5, SGBS, and SGBS1 . It is classified as a membrane-bound heparan sulfate proteoglycan that plays crucial roles in cell signaling and growth regulation.

Where and when is GPC3 expressed in human tissues?

GPC3 shows a distinct temporospatial expression pattern:

• Expressed during early development in human embryo, fetus, and placental tissues

• Not expressed in normal adult tissue

• Highly expressed in hepatocellular carcinoma (HCC)

• Also expressed to varying degrees in other cancers including melanoma, ovarian clear-cell carcinomas, yolk sac tumors, neuroblastoma, hepatoblastoma, and Wilms' tumor cells

This oncofetal expression pattern makes GPC3 an interesting candidate as both a biomarker and therapeutic target for cancer, particularly HCC.

How is GPC3 implicated in hepatocellular carcinoma (HCC)?

GPC3 is highly expressed in HCC but not in normal liver tissue, making it a specific marker for this cancer type . Evidence suggests that GPC3 is actively involved in HCC tumorigenesis through several mechanisms:

• Stimulation of canonical Wnt signaling by forming complexes with Wnt molecules

• Modulation of Hedgehog signaling pathway

• Interaction with growth factors like FGF-2 in HCC cells

• Possible roles in other signaling pathways including TGF-β2, HGF, and Yap

The specificity of GPC3 expression in HCC (versus cholangiocarcinoma or normal liver) has established it as a promising diagnostic marker and therapeutic target for HCC .

What is Simpson-Golabi-Behmel Syndrome and how is it related to GPC3?

Simpson-Golabi-Behmel Syndrome (SGBS) is a rare X-linked overgrowth disorder characterized by pre- and postnatal overgrowth of multiple tissues and organs, and an increased risk for developing embryonic tumors . This syndrome is caused by loss-of-function mutations in the GPC3 gene .

The phenotype of SGBS patients aligns with observations from GPC3 knockout mice, which also show overgrowth phenotypes . These findings indicate that GPC3 normally functions to regulate cell survival and inhibit cell proliferation during development . The association between GPC3 dysfunction and overgrowth syndromes highlights its critical role in growth regulation, providing insight into how its dysregulation might contribute to cancer development.

What other cancers show abnormal GPC3 expression?

While GPC3 is most prominently associated with HCC, several other cancer types show altered GPC3 expression:

• Melanoma

• Ovarian clear-cell carcinomas

• Yolk sac tumors (YST)

• Neuroblastoma

• Hepatoblastoma

• Wilms' tumor

• Lung adenocarcinoma (LUAD)

In LUAD, approximately 1.4% to 2.2% of patients exhibit copy number amplifications in GPC3 . The significance of GPC3 in these various cancer types may differ, with evidence suggesting its involvement in specific signaling pathways relevant to each cancer type.

What recombinant GPC3 proteins are available for research?

Several recombinant GPC3 proteins are available for research purposes, including:

• Human GPC3 (438-554) Protein-VLP expressed from HEK293 cells

• Full-length recombinant human Glypican-3 protein

When selecting a recombinant protein for research, important considerations include:

• Expression system (e.g., HEK293 cells for proper post-translational modifications)

• Protein fragment vs. full-length protein

• Purity (typically >95% as determined by HPLC)

• Activity validation (e.g., binding assays with known antibodies)

• Endotoxin levels (important for cell-based and in vivo experiments)

For binding studies, researchers should note that different regions of GPC3 interact with different partners, with the C-terminal region being particularly important for some antibody recognition .

What methods are used to study GPC3 interactions with signaling pathways?

Several experimental approaches can be employed to investigate GPC3's interactions with signaling pathways:

• Protein-protein interaction studies:

  • Co-immunoprecipitation to detect direct binding between GPC3 and pathway components

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

  • Proximity ligation assays for visualizing interactions in situ

• Signaling pathway activation assays:

  • Luciferase reporter assays for Wnt/β-catenin pathway activation

  • Western blotting for phosphorylation of downstream effectors

  • Transcriptional profiling of pathway target genes

• Functional studies with GPC3 variants:

  • Use of GPC3 lacking the GPI anchoring domain to block Wnt signaling

  • Comparison of wild-type GPC3 versus mutants lacking heparan sulfate chains to distinguish HS-dependent from HS-independent interactions

  • Structure-function analysis using different GPC3 domains

When designing these experiments, researchers should consider the cellular context, as GPC3's effects may vary between different cell types and developmental stages.

How does proteolytic processing affect GPC3 function?

GPC3 undergoes endoproteolytic processing by proprotein convertases, which is required for its modulation of Wnt signaling . This processing results in two subunits (N-terminal ~40 kDa and C-terminal ~30 kDa) that remain associated through disulfide bonds .

Methodological approaches to study this processing include:

• Western blotting with antibodies specific to different domains to monitor processing

• Site-directed mutagenesis of cleavage sites to generate non-cleavable variants

• Inhibition of proprotein convertases to assess processing-dependent functions

• Comparison of signaling activities between processed and unprocessed forms

Understanding this processing is critical for designing therapeutic strategies and interpreting experimental results, as antibodies targeting different epitopes may recognize processed or unprocessed forms differently.

What is the role of GPC3 in cancer immune evasion?

While GPC3 is primarily studied for its direct roles in signaling, emerging evidence suggests potential roles in cancer immune interactions. Researchers investigating this aspect should consider:

• GPC3's impact on tumor microenvironment:

  • Analysis of immune cell infiltration in GPC3-expressing versus non-expressing tumors

  • Correlation between GPC3 expression and immune checkpoint molecule expression

  • Assessment of cytokine profiles in the presence of GPC3

• Immunotherapeutic targeting approaches:

  • Antibody-dependent cell-mediated cytotoxicity (ADCC) with anti-GPC3 antibodies

  • Complement-dependent cytotoxicity (CDC) effects

  • Development of chimeric antigen receptor (CAR) T cells targeting GPC3

  • Bispecific antibodies linking GPC3-expressing tumor cells to immune effectors

The humanized anti-GPC3 antibodies hYP7 and hYP9.1b have been shown to induce both ADCC and CDC in GPC3-positive cancer cells but not in GPC3-negative cells, demonstrating the potential of GPC3 as an immunotherapeutic target .

How are anti-GPC3 antibodies developed and optimized for clinical use?

The development of therapeutic anti-GPC3 antibodies involves several critical steps:

• Generation of high-affinity mouse monoclonal antibodies:

  • Immunization with GPC3 peptides or proteins (e.g., C-terminal peptide)

  • Screening for high-affinity binders using techniques like ELISA

  • Characterization of binding specificity and epitope mapping

• Humanization strategies:

  • Grafting of combined KABAT/IMGT complementarity determining regions (CDRs) into human IgG germline frameworks

  • Identification of critical non-CDR residues (e.g., proline at position 41 in VH)

  • Back-mutation of selected framework residues if needed for affinity preservation

• Functional validation:

  • Testing binding affinity to cell surface GPC3

  • Evaluation of ADCC and CDC activities

  • Assessment of in vivo antitumor efficacy in xenograft models

For example, humanized antibodies hYP7 and hYP9.1b have shown specific binding to GPC3-positive cells with EC50 values of 0.7 nM and 0.4 nM respectively, while demonstrating no binding to GPC3-negative cells . These antibodies have also demonstrated effective ADCC and CDC activities against GPC3-expressing cancer cells .

Methodological approaches for evaluating GPC3-targeted therapies include:

• In vitro assessments:

  • Cell viability and proliferation assays in GPC3-positive versus GPC3-negative cell lines

  • Pathway inhibition assays (e.g., Wnt reporter assays)

  • ADCC and CDC assays using various effector cell sources

  • 3D organoid models for improved physiological relevance

• In vivo evaluations:

  • Xenograft tumor growth inhibition in nude mice

  • Patient-derived xenograft (PDX) models

  • Assessment of tumor microenvironment changes

  • Pharmacokinetic and biodistribution studies

• Biomarker strategies:

  • Monitoring target engagement through biopsies

  • Assessing pathway inhibition through surrogate markers

  • Evaluation of immune infiltration changes in immunotherapeutic approaches

For example, the hYP7 antibody has demonstrated inhibition of HCC xenograft tumor growth in nude mice, providing validation for its potential therapeutic efficacy .