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

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 (IC(50), 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.