Strong enhancement of recombinant cytosine deaminase activity in Bifidobacterium longum for tumor-targeting enzyme-prodrug therapy

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In our previous studies, a strain of the nonpathogenic, anaerobic, domestic bacterium, Bifidobacterium longum (B. longum), was shown to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the pro-drug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU) and induced cancer recession in chemically induced rat mammary tumors with enzyme/pro-drug therapy. This therapeutic strategy is named BifidobactErial Selective Targeting-Cytosine Deaminase (BEST-CD) therapy. Here, we constructed a novel shuttle-plasmid pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity. In addition, the plasmid-transfected B. longum could produce mutant CD and strongly increased (10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid would increase the effectiveness of our enzyme/pro-drug strategy.