Glycoengineered horseradish peroxidase for targeted cancer treatment

After diseases affecting the circulation, cancer is currently the second most frequent cause of death in Europe. In 2011, ~1.3 million people in Europe died as a consequence of tumors. The most common strategies in the battle against cancer are chemo- and radiation-therapies, which have a strong impact on the human body and cause unpleasant and painful side-effects. A more gentle and targeted method to fight tumor cells, like an enzyme/prodrug therapy, would significantly reduce damage of healthy tissue and make the lives of cancer patients more comfortable. The plant enzyme horseradish peroxidase (HRP; EC 1.11.1.7) is a very useful agent for targeted cancer therapies. The isoenzyme HRP C1A oxidizes the plant hormone indole-3-acetic acid, which then induces cellular oxidative stress and decreases the cell viability of carcinoma cells by activating apoptotic pathways. However, despite this very useful application of HRP, it has not been extensively employed for targeted cancer treatment yet, due to the quite cumbersome production and purification of the enzyme from plant and its heterogenic glycosylation pattern. Also an alternative production of HRP in yeast poses a hurdle due to the nature of yeast to hyper-mannosylate glycoproteins, which significantly hampers the downstream processing and causes immune responses in humans. The goal of the inter- and multidisciplinary study proposed here, is to glycoengineer HRP to obtain variants with high catalytic activity and stability, but with a significantly reduced amount of attached glycan chains, thus allowing a facilitated downstream processing. These variants will be purified to homogeneity and tested for potential applications in medical diagnostics and targeted cancer treatment.

The goal of project P24861-B19 was to glyco-engineer the plant enzyme horseradish peroxidase to obtain variants with high catalytic activity and stability, but with a significantly reduced amount of attached glycan chains, allowing facilitated downstream processing and potentially applications of the enzyme in targeted cancer treatment. We tackled the different hurdles in the project in an integrated and multidisciplinary approach. We can summarize our efforts shortly as: single HRP isoenzymes can be produced in the yeast P. pastoris and purified efficiently it is possible to reduce the heterogeneous surface glycosylation of recombinant HRP by protein and strain engineering surface glycosylation is not a prerequisite for HRP activity, but reduction causes reduced enzyme kinetics and stability glyco-engineered HRP variants are catalytically active and thus potentially interesting for medical applications Furthermore, we reviewed the yeast P. pastoris as expression host for biopharmaceuticals, investigated the freezing characteristics of HRP in aqueous solution, searched for novel NADH:quinone oxidoreductases in extremophiles, which could potentially boost the energy metabolism in P. pastoris, and developed new mixed feed strategies for P. pastoris strains producing with a de-repression promoter. The efforts we undertook in P24861-19 resulted in 16 published, peer-reviewed papers, 8 invited talks at international congresses and several poster presentations so far. In all these contributions the FWF was acknowledged as funding agency, creating public awareness. Two other publications are currently in preparation. Fruitful collaborations with Dr. Lisa Folkes (Gray Institute for Radiation Oncology and Biology at Oxford University, Oxford, UK), Prof. Gabi Dachs (Department of Pathology, University of Otago, Christchurch, New Zealand) and Prof. Anton Glieder (University of Technology Graz, Austria) were established and the students employed on P24861-B19 (1 PhD and 4 MSc students) were able to visit the facilities abroad and perform experiments there. Overall, project P24861-B19 can be regarded as a rather successful project.