PEG-DAAO conjugate: A promising tool for cancer therapy optimized by protein engineering

The flavoenzyme D-amino acid oxidase (DAAO) represents a potentially good option for cancer enzyme prodrug therapy as it produces H₂O₂ using D-amino acids as substrates, compounds present at low concentration in vivo and that can be safely administered to regulate H₂O₂ production. We optimized the cytotoxicity of the treatment by: i) using an efficient enzyme variant active at low O₂ and D-alanine concentrations (mDAAO); ii) improving the stability and half-life of mDAAO and the enhanced permeability and retention effect by PEGylation; and iii) inhibiting the antioxidant cellular system by a heme oxygenase-1 inhibitor (ZnPP). A very low amount of PEG-mDAAO (10 mU, 50 ng of enzyme) induces cytotoxicity on various tumor cell lines. Notably, PEG-mDAAO seems well suited for in vivo evaluation as it shows the same cytotoxicity at air saturation (21%) and 2.5% O₂, a condition resembling the microenvironment found in the central part of tumors.