title: A monoclonal antibody-beta-glucuronidase conjugate as activator of the prodrug epirubicin-glucuronide for specific treatment of cancer.
abstract: The anti-pan carcinoma monoclonal antibody (MAb) 323/A3, linked to E. coli-derived beta-glucuronidase (GUS) was used to study the tumour-site-selective activation of the prodrug Epirubicin-glucuronide (Epi-glu). Epi-glu was isolated from the urine of patients treated with Epirubicin (Epi) by reversed phase chromatography on a silica-C18 column. Epi-glu was stable in human blood and was not converted into Epi by A2780, MCF-7, or OVCAR-3 cancer cells, despite the presence of intracellular GUS. The stability of the prodrug was confirmed in BALB/c mice. MAb 323/A3 and GUS were linked through a stable thioether bond. The conjugate (1:1) was purified by ion exchange and gel filtration chromatography. Binding to target cells revealed an immunoreactivity of at least 60% and good retention of enzyme activity. A protein dye (sulforhodamine B) assay was used to analyse cytotoxicity. Epi (IC50 of 0.003-0.2 microM) was 100-1,000 times more toxic than Epi-glu (IC50 of greater than 20 microM), when cancer cells were exposed for 4 or 24 h to the drugs. The low cytotoxicity of Epi-glu was most likely due to the reduced cellular uptake rate of the prodrug (2.7 pmol 10(-6) cells min-1) as compared to that of the parent compound (25 pmol 10(-6) cells min-1). Pretreatment of antigen-positive cells with the 323/A3-GUS conjugate prior to prodrug exposure completely restored cytotoxicity as a result from hydrolysis of Epi-glu into Epi. Our results demonstrate that the 323/A3-GUS conjugate can specifically activate the stable non-toxic prodrug Epi-glu at the tumour cell level.
authors.affiliation: Department of Medical Oncology, Free University Hospital, Amsterdam, The Netherlands.
authors.forename: H J
authors.initials: HJ
authors.lastname: Haisma
authors.forename: E
authors.initials: E
authors.lastname: Boven
authors.forename: M
authors.initials: M
authors.lastname: van Muijen
authors.forename: J
authors.initials: J
authors.lastname: de Jong
authors.forename: W J
authors.initials: WJ
authors.lastname: van der Vijgh
authors.forename: H M
authors.initials: HM
authors.lastname: Pinedo
mesh_terms: D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D015251:Epirubicin / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use; D005260:Female; D005965:Glucuronates / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use; D005966:Glucuronidase / Q000627:therapeutic use*; D006801:Humans; D000922:Immunotoxins / Q000627:therapeutic use*; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D005965:Glucuronates; D000922:Immunotoxins; D011355:Prodrugs; D015251:Epirubicin; D005966:Glucuronidase