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 ␤-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 ␤-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).

Glioma Regression In vitro and In vivo by a Suicide Combined Treatment

“…The cyanide produced in the lis/lin system induces severe mitochondrial damage, blocking oxidative phosphorylation that causes a fast ATP depletion and promoting the disruption of the mitochondrial filament pattern. In the conditions used in this study, the lis/lin system induces necrosis according to what would be expected from the conditions of a complete ATP depletion (19), in agreement with previous studies in which linamarase-conjugated antibodies were used for tumor therapy (36). The observed death acceleration by the lis/lin/GO combination is not a consequence of a faster mitochondrial fragmentation or ATP depletion, confirming that the cyanide oxidative phosphorylation blockage is responsible for both effects.…”

Histopathological evaluation of tumor necrosis and volume after cyanogenic chemotherapy

“…The presence of coagulative necrosis in SET is frequently observed, particularly in larger tumors, where necrosis is characterized by extensive cell lysis that occurs during the course of acute injury 19,20 . In the present experiment a sharper ACG necrosis was observed in relation to the CG; such lesions are consistent with exposure to cyanide, where ATP levels are suspected to fall sharply due to inhibition of oxidative phosphorylation, being this decrease below the level required for apoptosis, thus resulting in cell death by necrosis 12 .…”

“…The ability of cyanide to penetrate the cell without the action of a receiver is due to the cyanide ion being a small molecule and its detoxification in mammals occur for the most part mediated by the enzyme rodanase 12 . Animals bearing solid tumors undergoing distance treatment with cyanide showed a clear regression of tumors compared to controls, demonstrating that cyanide was diffused through the tissues into the bloodstream 9,13 ; in our experiment using SET inoculated subcutaneously in animals, cyanogenic chemotherapy released cyanide outside the tumor, abdominal cavity, but causing accentuated necrosis and tumor growth inhibition.…”

“…Cyanide toxicity occurs in two ways: ATP depletion caused by blockage of oxidative phosphorylation and production of reactive oxygen species 12 .…”

Evaluation of acetone cyanohydrin effect in "in vitro" inativation of the Ehrlich ascites tumor cells

“…The comparison of the effect of Acetone Cyanohydrin in normal cells was satisfactory, showing greater cytotoxic capacity occurring on the Ehrlich Ascites tumor cells as also described in other studies using cyanoglucosides 14,15 , demonstrating absence of intoxication and of inviability of normal cells.…”