Recombinant horseradish peroxidase for targeted cancer treatment

Cancer is the second most frequent cause of death in Europe. Chemo- and radiation-therapies, which are commonly used to fight cancer, cause unpleasant and painful side-effects. A targeted enzyme/prodrug therapy allows a more specific treatment. It was shown that a combination of the plant enzyme horseradish peroxidase C1A (HRP) and the plant hormone indole-3-acetic acid (IAA) can be used for targeted cancer therapy. However, HRP has not been employed for this purpose yet, due to the cumbersome production and purification of the enzyme from plant and its heterogenic glycosylation pattern. In our project P24861-B19, funded by the Austrian Science Fund (FWF), we successfully performed different tasks to tackle the existing hurdles for using recombinant HRP for medical purposes and found that surface glycosylation was actually not a prerequisite for enzyme activity. Consequently, in this follow-up project we propose to express HRP and mutated HRP variants in Escherichia coli both 1) as soluble enzyme in the periplasm of E. coli allowing straight-forward screening of engineered HRP variants, and 2) as inclusion bodies (IBs) in the cytoplasm of E. coli in high amounts, which are then processed and refolded, biochemically characterize the recombinantly produced, unglycosylated enzyme variants, test them with IAA and paracetamol, develop a strategy to efficiently conjugate the mutated HRP lead candidate with antibodies and lectins as recognition ligands for cell-specific delivery approaches and evaluate the mutated HRP lead candidate and its conjugates on a panel of human cancer cell lines grown in 2D and as 3D models in vitro to investigate their potential in targeted cancer treatment. Summarizing, in this project the use of recombinant HRP in combination with IAA and paracetamol for targeted cancer treatment is assessed.

In this FWF project, we managed to recombinantly produce a plant enzyme in the bacterium Escherichia coli. We were able to produce it in amounts and quality to use it for medical applications. We stabilized the enzyme by mutations and increased its catalytic activity. Furthermore, we successfully conjugated it to other proteins, like antibodies. Finally, we were able to demonstrate that this enzyme in combination with a plant acid can be used to fight tumor cells. This FWF project resultated in 2 international patents and 8 scientific publications.