395. Development of the Novel Oral Tumor Vaccine Using Bifidobacterium longum Displaying Wilms’ Tumor 1 Protein

Abstract

INTRODUCTION: The Wilms’ Tumor 1 gene (WT1), firstly reported as a gene responsible for pediatric renal cancer, is highly expressed in leukemia and various types of solid tumors. Therefore, WT1 gene products are considered as ideal targets for the cancer immunotherapy. WT1 peptide-based immunotherapies are currently being developed for treatment of pediatric and adult cancers in Japan and other countries. However the efficacy of the peptide-based immunotherapy is restricted by patients’ HLA types, and multiple injections of the peptides are painful especially in children and often cause scarring and swelling on the injection sites. In this study, we developed a genetically modified Bifidobacterium longum displaying a WT1 protein as an oral vaccine for HLA-nonrestrictive and less-invasive cancer immunotherapy, and confirmed its therapeutic efficacy in tumor bearing mouse model. Furthermore, we also demonstrated the synergistic anti-tumor effect of combination therapy of this oral vaccine and interleukin-2 immunotherapy. EXPERIMENTAL SUMMARY: For optimized expression of WT1 protein on bacterial cell surface, a shortened murine WT1 gene (a.a. 117-419) including several CD8+ and CD4+ T cell epitopes was fused to galacto-N-biose/lacto-N-biose binding protein (GLBP) gene that coded ABC transporter of B. longum. We transferred the WT1-GLBP fusion gene into B. longum 105A strain using our E-coli-B. longum shuttle vector system, and constructed the B. longum 420 vaccine strain, which is displaying GLBP-WT1 fusion protein. We confirmed that B. longum 420 displayed WT1 protein on cell surface and its stability by immunofluorescence staining and western blotting. For in vivo vaccination study, 1×106 of C1498 murine leukemia cells stably expressing full-length murine WT1 protein were inoculated subcutaneously into the right flanks of 8 week old C57BL/6N mice (n=15). Following tumor inoculation, mice were randomly assigned into 3 treatment groups; B. longum 420, B. longum 2012 which was transfected with control vector, and PBS. 100 µL of PBS with or without 1×109 CFU of each recombinant B. longum was orally administered every other day for 4 weeks (14 times totally). Furthermore, to enhance the anti-tumor effect, we added subcutaneous injections of 2000 IU of mouse IL-2 for five days a week for 4 weeks. As the result, B. longum 420 oral vaccination significantly inhibited the tumor growth compared with PBS group at the day 29 after tumor inoculation (5455±891 mm3 vs 8953±777 mm3; p<0.05). Also the additional IL-2 treatment significantly decreased the mean tumor volume of B. longum 420 group as early as day 19 (289±73 mm3 vs 1063±252 mm3; p<0.01), and at day 29 (1196±507 mm3 vs 5455±891 mm3; p<0.05). These results suggested that B. longum 420 could induce WT1-specific CTLs in mice and an additional IL-2 treatment boosted their anti-tumor effects. CONCLUSION: We confirmed that oral vaccination with B. longum displaying WT1 protein successfully induced WT1 specific anti-tumor immunity in mice, and it could be enhanced by IL-2 treatment. These findings guaranteed the feasibility of our oral tumor vaccine, which would create a new paradigm of cancer immunotherapy.