Nowadays, cancer immunotherapies such as immune-checkpoint inhibitors, chimeric antigen receptor T-cells and cancer vaccines have achieved great success in cancer therapy. Nevertheless, increasing cancer antigen-specific immune responses is necessary to obtain significant therapeutic effect. Recently, we had developed an oral cancer vaccine utilizing recombinant Bifidobacterium longum displaying murine Wilms’ tumor 1 antigen (B. longum 420) and had showed significant antitumor effect against WT1-positive leukemic cells by induction of WT1-specifc cytotoxic T-cell (CTL)-mediated immunity via activation of gut mucosal immune system in mice. In this study, we constructed B. longum displaying modified-human WT1 (B. longum 440) and investigated the antitumor effect against solid tumors in mice experimental model. Wild-type B. longum 105-A strain was transformed by electroporation by E. coli-B. longum shuttle vector and recombinant B. longum 440 was constructed. B. longum 440 was displaying human WT1 protein that had modified human WT1-CTL epitope to increase the affinity for MHC-class I. 1×106 of TRAMP-C2, a murine prostate cancer, MBT-2, a murine bladder cancer, and 3LL, a murine lung cancer cell lines were subcutaneously inoculated into mice respectively. All the cell lines were endogenously expressing WT1 protein. After tumor formation, 1×109 of B. longum 440, B. longum 2012 (negative control), or PBS were orally administered 5 times a week for following weeks. PD1-antibody therapy was combined with B. longum 440 therapy to investigate synergistic antitumor effect of immune checkpoint inhibitor against MBT-2. We also performed in vitro co-culture experiments by using B. longum 440, human dendritic cells (DCs) and CTLs derived from human peripheral blood mononuclear cells (PBMCs) to investigate the immune-interaction between B. longum 440 and human immune cells responsible for WT1-spcific immunity. As the results, oral vaccination of B. longum 440 induced significantly higher antitumor effect against TRAMP-C2 and 3LL compared with other therapies, and significantly prolonged overall survival (p<0.05). The combination therapy of B. longum 440 and anti-PD-1 induced substantially higher antitumor effect against MBT-2 tumor than B. longum 440 or PD-1 single therapy, and 40% of the mice completely suppressed the tumor growth. Significantly higher WT1-CTL-epitope, Db126 and mp235-specific IFN-γ and IL-2 production in CD4 and CD8 T-cells were seen in spleen cells isolated from B. longum 440-immunized mice, suggesting that human B. longum 440 could induce WT1-specific T-cell responses in mice as well as murine B. longum 420 as previously reported. Regarding in vitro assay, after co-culturing of B. longum 440 and human DCs, matured DCs that ingested WT1 protein were successfully induced. Then, WT1-specific CTLs were induced by co-culturing of the matured DCs and suspended PBMCs in vitro. As the results, those WT1-specific CTLs elicited significantly higher cell cytotoxicity against A549, a human non-small lung cancer cell line, and T24, a grade 3 human bladder cancer cell line compared to control B. longum 2012 (p<0.05), supporting that B. longum 440 might induce functional WT1-specific CTLs in human. In conclusion, we demonstrated that oral cancer vaccination by using B. longum 440 could induced antitumor effect against prostate cancer, lung cancer and bladder cancer in mice experimental model. In addition, we showed the synergistic antitumor effect of combination therapy of anti-PD-1 and B. longum 440 against bladder cancer. Our findings from in vitro co-culture experiments also supported that combining B. longum 440 cancer vaccines with checkpoint inhibitors has the potential to mediate tumor regression in human. Therefore, B. longum could be a promising candidate of combination immunotherapy with immune checkpoint inhibitors including pembrolizumab, an anti-PD-1 antibody therapy for treatment of advanced bladder cancer.
Citation Format: Koichi Kitagawa, Maho Tatsumi, Mako Kato, Shota Komai, Yoshiko Hashii, Takane Katayama, Toshiro Shirakawa. Cancer immunotherapy combining oral vaccination of recombinant Bifidobacterium longum displaying human Wilms’ tumor 1 protein and anti-PD-1 checkpoint blockade for solid tumors in mice experimental model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B114.