We had developed a H(2)O(2) generating enzyme, polyethylene glycol conjugated D-amino acid oxidase (PEG-DAO), which exhibited potent antitumor activity by generating toxic reactive oxygen species, namely oxidation therapy, subsequently showed remarkable antitumor effect on murine Sarcoma 180 solid tumor, by taking advantage of the enhanced permeability and retention effect. Along this line, we report here the preparation of PEG-DAO by use of recombinant DAO and its antitumor activity by using various tumor cell lines and tumor models. Recombinant DAO (rDAO) was obtained from E. coli BL21 (DE3) carrying the porcine DAO expression vector with high yield (20 mg/l) and high enzyme activity (5.3 U/mg). Pegylated rDAO (PEG-rDAO) showed high stability against sonication, repeated freezing/thawing, lyophilization and exhibited superior in vivo pharmacokinetics. PEG-rDAO had a molecular size of 65 kDa and existed as nanoparticles in aqueous solution with mean particle diameter of 119 nm. In vitro experiments showed strong cytotoxicity of PEG-rDAO against various tumor cells, whereas less cytotoxicity was found against various normal cells. In vivo antitumor treatment was carried out using 2 mice tumor models, namely colon 38 tumor and Meth A tumor model. PEG-rDAO was administered i.v. and after an adequate lag time, D-proline (the substrate of DAO) was injected i.p. to the tumor-bearing mice. Consequently, preferential generation of H(2)O(2) in the tumor was successfully achieved, which resulted in remarkable suppression of tumor growth without any visible side effects. These findings suggest a potential of PEG-rDAO as a novel anticancer strategy toward clinical development.
(c) 2007 Wiley-Liss, Inc.