title: Recombinant horseradish peroxidase variants for targeted cancer treatment.
abstract: Cancer is a major cause of death. Common chemo- and radiation-therapies damage healthy tissue and cause painful side effects. The enzyme horseradish peroxidase (HRP) has been shown to activate the plant hormone indole-3-acetic acid (IAA) to a powerful anticancer agent in in vitro studies, but gene directed enzyme prodrug therapy (GDEPT) studies showed ambivalent results. Thus, HRP/IAA in antibody directed enzyme prodrug therapy (ADEPT) was investigated as an alternative. However, this approach has not been intensively studied, since the enzyme preparation from plant describes an undefined mixture of isoenzymes with a heterogenic glycosylation pattern incompatible with the human system. Here, we describe the recombinant production of the two HRP isoenzymes C1A and A2A in a Pichia pastoris benchmark strain and a glyco-engineered strain with a knockout of the α-1,6-mannosyltransferase (OCH1) responsible for hypermannosylation. We biochemically characterized the enzyme variants, tested them with IAA and applied them on cancer cells. In the absence of H2 O2 , HRP C1A turned out to be highly active with IAA, independent of its surface glycosylation. Subsequent in vitro cytotoxicity studies with human T24 bladder carcinoma and MDA-MB-231 breast carcinoma cells underlined the applicability of recombinant HRP C1A with reduced surface glycoslyation for targeted cancer treatment. Summarizing, this is the first study describing the successful use of recombinantly produced HRP for targeted cancer treatment. Our findings might pave the way for an increased use of the powerful isoenzyme HRP C1A in cancer research in the future.
authors.affiliation: Research Area Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
authors.forename: Günther
authors.initials: G
authors.lastname: Bonifert
authors.affiliation: Department of Oncology Oxford Institute for Radiation Oncology, University of Oxford, Northwood, Middlesex, U.K.
authors.forename: Lisa
authors.initials: L
authors.lastname: Folkes
authors.affiliation: Research Area Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Gmeiner
authors.affiliation: Mackenzie Cancer Research Group, Department of Pathology, University of Otago, Christchurch, New Zealand.
authors.forename: Gabi
authors.initials: G
authors.lastname: Dachs
authors.affiliation: Research Area Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
authors.forename: Oliver
authors.identifier: 0000-0003-0916-0644
authors.initials: O
authors.lastname: Spadiut
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D007210:Indoleacetic Acids / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007527:Isoenzymes; D011355:Prodrugs; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D007210:Indoleacetic Acids; D007527:Isoenzymes; D011355:Prodrugs; D011994:Recombinant Proteins; C030737:indoleacetic acid; D006735:Horseradish Peroxidase
keywords: Antibody directed enzyme prodrug therapy (ADEPT); MDA-MB-231 breast carcinoma; Pichia pastoris; T24 bladder carcinoma; horseradish peroxidase; indole-3-acetic acid