title: Immunotoxin-mediated targeting of claudin-4 inhibits the proliferation of cancer cells.
abstract: Immunotoxins are engineered chimeric proteins that consist of a fragment of a toxin fused to a modified antibody or growth factor capable of targeting specific cells. Furthermore, these proteins can be targeted to receptors that are commonly overexpressed on cancer cells. The majority of immunotoxins function by binding to cells, translocating into the cytosol and inhibiting protein synthesis. In this study, the expression of claudin‑4 (CLDN4) in various cancer cells was analysed as a potential target for immunotoxins. To target CLDN4-expressing cancer cells, the c-terminal CLDN4‑binding domain of Clostridium perfringens enterotoxin (CPE) was fused to the Pseudomonas aeruginosa exotoxin A (ETA) domain to create an immunotoxin (CPE‑ETA'). Subsequently, the capacity of such an immunotoxin in suppressing the proliferation of CLDN4-positive cancer cells was investigated. We report that head and neck squamous carcinoma cells (HN5) have an elevated CLDN4 expression compared to the other cell lines tested. Our findings further demonstrate that CPE‑ETA' is highly potent against MCF-7 breast [50% inhibitory concentration (IC50) 9.8 ng/ml] and HN5 head/neck (IC50 8.8 ng/ml) cancer cell lines, while it has no cytotoxic effects on HeLa cells (CLDN4‑negative). The immunotoxin was subsequently expressed in the tumour colonising oncolytic strain, Clostridium ghonii. Most importantly, the strictly anaerobic Clostridium ghonii was able to overexpress and secrete a functional CPE‑ETA' fusion protein. Our findings open the possibility of the targeted delivery of the immunotoxin locally to tumour sites at a high concentration using strictly anaerobic Clostridium ghonii for the treatment of CLDN4-positive cancer cells.
authors.affiliation: Division of Molecular and Gene Therapies, Griffith Health Institute and School of Medical Science, Griffith University, Gold Coast, Queensland 4215, Australia.
authors.forename: S M
authors.initials: SM
authors.lastname: Hashimi
authors.forename: S
authors.initials: S
authors.lastname: Yu
authors.forename: N
authors.initials: N
authors.lastname: Alqurashi
authors.forename: D S
authors.initials: DS
authors.lastname: Ipe
authors.forename: M Q
authors.initials: MQ
authors.lastname: Wei
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000276:immunology / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D001943:Breast Neoplasms / Q000188:drug therapy / Q000473:pathology; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D062506:Claudin-4 / Q000378:metabolism*; D003013:Clostridium / Q000235:genetics; D004768:Enterotoxins / Q000235:genetics; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism; D005260:Female; D006367:HeLa Cells; D006258:Head and Neck Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D006801:Humans; D000922:Immunotoxins / Q000235:genetics* / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D058990:Molecular Targeted Therapy / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D062506:Claudin-4; D004768:Enterotoxins; D005098:Exotoxins; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C568157:CLDN4 protein, human; C035598:enterotoxin, Clostridium