Genetically engineered Bifidobacterium longum expressing cytosine deaminase for tumor-targeting enzyme-prodrug therapy

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Cancer gene therapy approaches to solid tumor treatment have been limited by the delivering ability to achieve specific high-level expression of the gene product within tumor cells or the tissues following the systemic vector administration. Since hypoxia is characteristic of most solid tumors, gene therapies targeting to hypoxic tumor cells having been investigated. We previously reported that a strain of domestic nonpathogenic and anaerobic bacteria, Bifidobacterium longum (B. longum), selectively localized and proliferated within solid tumors after the systemic administration. Here, we propose a novel approach to cancer therapy in which anaerobic bacteria of the genus B. longum are used to achieve tumor-specific delivery of a desired agent and enzyme-prodrug therapy. We constructed an expression vector, pBLES100-S-eCD, of E. coli cytosine deaminase gene (eCD), transfecting it into B. longum by electroporation. We confirmed that the transformed B. longum (B. longum/S-eCD) localized and grew specifically in solid tumors and that the cytosine deaminase (CD) was produced in the B. longum/ S-eCD detected in the tumors by Gram’s stain and immunostaining for CD. Such cytosine deaminase (CD) production in hypoxic tumors resulted in the tumor site-specific conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). Furthermore, we observed growth suppression of autochthonous mammary tumors chemically-induced in rats by both direct and intravenous administration of B. longum/S-eCD. This method was thus confirmed to be effective irrespective of the systemic administration or the intra-tumoral injection. To further estimate the safety of the non-pathogenic B. longum/S-eCD, the immunogenicity was examined through Active Systemic Anaphylaxis (ASA) reaction and passive cutaneous anaphylaxis (PCA) reaction in guinea pigs. In ASA reaction, no anaphylaxis symptom was observed in any immunized guinea-pigs injected with B.longum/S-eCD. In PCA reaction, no anti-Blongum/S-eCD antibody was detected, either. These results surprised us and also made us convinced that Blongum/S-eCD was safely used for a tumor-specific delivery system. The clinical effectiveness of 5-FU in conventional therapy has been limited by its high degree of the systemic toxicity, particularly toward the bone marrow. However, the enzyme-prodrug therapeutic strategy using B. longum/S-eCD and 5-FC shown here appeared to be safer and more effective, because of the specific generation of 5-FU at the tumor site but not at other normal tissues, than conventional chemotherapies with 5-FU. Taken these findings together, we believe that obligate anaerobic bacteria, such as the genus B. longum, are safe and attractive tools to specifically produce desired anti-tumor agents in enzyme-prodrug therapy of solid tumors.