Recombinant immunotoxin targeting GPC3 is cytotoxic to H446 small cell lung cancer cells

Abstract

Glypican-3 (GPC3) is a cell membrane glycoprotein that regulates cell growth and proliferation. Aberrant expression or distribution of GPC3 underlies developmental abnormalities and the development of solid tumours. The strongest evidence for the participation of GPC3 in carcinogenesis stems from studies on hepatocellular carcinoma and lung squamous cell carcinoma. To the best of our knowledge, the role of the GPC3 protein and its potential therapeutic application have never been studied in small cell lung carcinoma (SCLC), despite the known involvement of associated pathways and the high mortality caused by this disease. Therefore, the aim of the present study was to examine GPC3 targeting for SCLC immunotherapy. An immunotoxin carrying an anti-GPC3 antibody (hGC33) and Pseudomonas aeruginosa exotoxin A 38 (PE38) was generated. This hGC33-PE38 protein was overexpressed in E. coli and purified. ADP-ribosylation activity was tested in vitro against eukaryotic translation elongation factor 2. Cell internalisation ability was confirmed by confocal microscopy. Cytotoxicity was analysed by treating liver cancer (HepG2, SNU-398 and SNU-449) and lung cancer (NCI-H510A, NCI-H446, A549 and SK-MES1) cell lines with hGC33-PE38 and estimating viable cells number. A BrdU assay was employed to verify anti-proliferative activity of hGC33-PE38 on treated cells. Fluorescence-activated cell sorting was used for the detection of cell membrane-bound GPC3. The hGC33-PE38 immunotoxin displayed enzymatic activity comparable to native PE38. The protein was efficiently internalised by GPC3-positive cells. Moreover, hGC33-PE38 was cytotoxic to HepG2 cells but had no effect on known GPC3-negative cell lines. The H446 cells were sensitive to hGC33-PE38 (IC₅₀, 70.6±4.6 ng/ml), whereas H510A cells were resistant. Cell surface-bound GPC3 was abundant on the membranes of H446 cells, but absent on H510A. Altogether, the present findings suggested that GPC3 could be considered as a potential therapeutic target for SCLC immunotherapy.

Keywords: Wnt/β-catenin pathway; glypican-3; hepatocellular carcinoma; immunotherapy; immunotoxin; small cell lung carcinoma.

Figure 1.

Molecular analysis of purified hGCPE38 and N-PE38. Proteins from pooled and concentrated size-exclusion chromatography fractions were loaded onto SDS-PAGE gels and analysed. (A) SDS-PAGE in reducing conditions: 2,000 or 500 ng of the hGCPE38 protein per lane. (B) Western blotting. The toxins were detected using anti-exotoxin A-specific primary antibodies; 50 ng of each protein per lane. (C) Enzyme-linked immunosorbent assay analysis of hGC33-PE38 binding affinity to GPC3 antigen. Absorbance values are given as the mean ± SD from three independent replicates. BSA (0 µg/ml of GPC3 antigen) was the negative control. PE38, Pseudomonas aeruginosa exotoxin A 38; hGC33, anti-GPC3 antibody; GPC3, glypican-3.

Figure 2.

Internalisation of hGC33-PE38 immunotoxin in A549 and HepG2 cells visualised using confocal microscopy. Merged views are shown in parts A and E on both panels. Fluorescent signal corresponding to the hGC33-PE38 immunotoxin is marked as green (parts B and F). The cells were treated with fluorescently labelled hGC33-PE38 immunotoxin (Alexa Fluor 488), fixed and additionally labelled with Alexa Fluor 594 Phalloidin for actin visualisation (parts C and G), and with NucRed Live 647 Ready Probes Reagent (parts D and H) for nuclei visualisation. Magnification, ×60. PE38, Pseudomonas aeruginosa exotoxin A 38; hGC33, anti-GPC3 antibody.

Figure 3.

Internalisation of hGC33-PE38 immunotoxin in NCI-H446 and NCI-H510A cells visualised using confocal microscopy. Blue channel, nuclei (Hoechst 33342); green channel, live cells (calcein-AM); red channel, hGC33-PE38 (ATTO 542). (A) Intracellular localization of ATTO 542-stained hGC33-PE38 immunotoxin within NCI-H446 cells and no specific signal in NCI-H510A cells. (B) Calcein-AM staining indicates shape of the cells and tightness of cellular membrane. On the upper right corner of the right panels, non-specific binding within a dead cell is shown. Scale bar, 10 µm. Magnification, ×60. PE38, Pseudomonas aeruginosa exotoxin A 38; hGC33, anti-GPC3 antibody.

Figure 4.

Treatment of H446 and H510A cells with increasing concentrations of the hGC33-PE38 immunotoxin. (A) Cytotoxicity curve for H446 cells. Dashed line represents the logarithmic dose-response logistic curve fitted to data. (B) No effect on H510A cells' survival. Dotted line represents the baseline of cytotoxicity. Cytotoxicity was calculated based on the analysis of neutral red uptake by viable cells. Mean ± SD (n=3). Statistical significance of differences vs. control (fixed value, 0) was calculated with ANOVA and Dunnett's post hoc test; *P≤0.05, ***P≤0.001. PE38, Pseudomonas aeruginosa exotoxin A 38; hGC33, anti-GPC3 antibody.

Figure 5.

Inhibition of H446 cell proliferation treated for 48 h with increasing concentrations of the hGC33-PE38 immunotoxin. Proliferation inhibition was determined using the BrdU assay. Mean ± SD (n=3). Statistical significance of differences vs. control (point 0.0) was calculated with ANOVA and Tukey's post hoc test; *P≤0.05. PE38, Pseudomonas aeruginosa exotoxin A 38; hGC33, anti-GPC3 antibody.

Figure 6.

Graph showing a normalized estimation of the GPC3 gene expression across 67 lung cancer cell lines. Data obtained from the EMBL-EBI Expression Atlas tool. The x-axis presents expression values in FPKM. The accession number of primary dataset: E-MTAB-2706. LAD, lung adenocarcinoma; SCLC, small cell lung carcinoma; GPC3, glypican-3; FPKM, fragments per kilobase of exon model per million reads mapped.

Figure 7.

Cell surface expression of GPC3 in HepG2, H510A and H446 cell lines measured by flow cytometry. Grey curves/numbers on the histograms represent cell staining with isotype control; black curves/numbers represent cells stained by antibodies recognising GPC3. Boxes represent interval gates threshold based on respective isotype control. GPC3, glypican-3.