title: Antibody-guided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells.
abstract: A number of enzyme/prodrug activation approaches for the treatment of cancer have been reported to date with varying success. We describe progress in the development of a system based on a beta-glucosidase enzyme in combination with a naturally occurring "prodrug," the sugar linamarin, which releases the cytotoxin cyanide. A recombinant fusion protein, composed of an scFv (MFE-23) reactive against carcinoembryonic antigen (CEA) and a plant-derived beta-glucosidase (linamarase), was produced and its cytotoxic potential was investigated. The fusion protein was expressed in a supersecretory mutant strain of Saccharomyces cerevisiae and purified by affinity chromatography. Extensive functional in vitro characterisation of the fusion protein showed that it retained antigen binding activity but that its catalytic activity was impaired, a problem not related to its fusion with the scFv. Nevertheless, we demonstrated complete tumour cell killing at doses of prodrug that are completely nontoxic to nontargeted cells. Preliminary in vivo characterisation showed that extensive glycosylation of the fusion protein caused its rapid clearance through the hepatic route. Aggregational properties also led to poor pharmacokinetics. Furthermore, we present some data analysing the mode of cell death resulting from exposure to this system. Enzymic catalysis of the substrate generates cyanide, a metabolic poison that asphyxiates cells and leads them to a necrotic-like cell death. This system has been called antibody-guided enzyme nitrile therapy (AGENT).
authors.affiliation: Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, United Kingdom.
authors.forename: Christina A
authors.initials: CA
authors.lastname: Kousparou
authors.forename: Agamemnon A
authors.initials: AA
authors.lastname: Epenetos
authors.forename: Mahendra P
authors.initials: MP
authors.lastname: Deonarain
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002272:Carcinoembryonic Antigen / Q000276:immunology* / Q000378:metabolism; D002470:Cell Survival; D003486:Cyanides / Q000627:therapeutic use*; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009336:Necrosis; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics / Q000627:therapeutic use*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D002272:Carcinoembryonic Antigen; D003486:Cyanides; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C021514:cyanogenic beta-glucosidase; D001617:beta-Glucosidase