Yoshinori Hamaji

Genetically engineered Bifidobacterium longum for tumor‐targeting enzyme‐prodrug therapy of autochthonous mammary tumors in rats

A fundamental obstacle in systemic therapy for cancer patients is the specific targeting of therapy directly to solid tumors. A strain of the domestic bacterium Bifidobacterium longum, which is non‐pathogenic and anaerobic, showed selective localization to and proliferation within solid tumors after systemic application. Here, we propose a novel approach to cancer gene therapy in which anaerobic and non‐pathogenic bacteria of the genus B. longum are used to achieve tumor‐specific gene delivery and enzyme‐prodrug therapy. We constructed a plasmid, pBLES100‐S‐eCD, which included eCD. Transfected B. longum produced CD in hypoxic tumors and achieved tumor site‐specific conversion of 5‐FC to 5‐FU. Furthermore, we demonstrated antitumor efficacy in rat bearing autochthonous mammary tumors injected with the transfected B. longum directly or intravenously. This method was confirmed to be effective for enzyme‐prodrug therapy not only by intratumoral injection but also by systemic administration. To estimate the toxicity of this bacterial vector, the systemic immunogenicity was evaluated by ASA reaction and the anaphylactic activity of IgG was evaluated by PCA reaction in guinea pigs. In the ASA reaction, no anaphylaxis symptoms were observed in any immunized guinea pigs injected with transfected B. longum. In the PCA reaction, B. longum/S‐eCD specific‐PCA‐induced antibody was not detected. Thus, we proposed that anaerobic bacteria of the genus B. longum were an attractive and safe tumor‐targeting vector and transfected B. longum was a potential anticancer agent that could effectively and specifically treat solid tumors. (Cancer Sci 2006; 97: 649–657)

Strong Enhancement of Recombinant Cytosine Deaminase Activity in Bifidobacterium longum for Tumor-Targeting Enzyme/Prodrug Therapy

In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found 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 prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, 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-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.

Exogeneous Cytosine Deaminase Gene Expression in Bifidobacterium breve I-53-8w for Tumor-Targeting Enzyme/Prodrug Therapy

Bifidobacteria are nonpathogenic, anaerobic domestic bacteria with health-promoting properties for the host. In our previous study, Bifidobacterium longum (B. longum) were found to be localized selectively and to proliferate within solid tumors after systemic application. Additionally, B. longum transformed by shuttle-plasmid including the cytosine deaminase (CD) gene expressed active CD, converted the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). We also demonstrated antitumor efficacy with a transformant of B. longum in rats. In this study, we found that Bifidobacterium breve (B. breve), the smallest species of human-derived bifidobacterium, expressed the exogenous transgene (CD), that CD enzymatic activity in the transformant of B. breve was much higher, and that the segregational stability of the plasmid was greater than that of B. longum. Thus, numerous transformants of B. breve were detected solely in the tumors after systemic administration. We consider the transformant of B. breve to be more beneficial in our enzyme/prodrug therapy.