Previously, we constructed a recombinant Bifidobacterium longum displaying a partial mouse Wilms' tumor 1 (WT1) protein (B. longum 420) as an oral cancer vaccine using a bacterial vector and demonstrated that oral administration of B. longum 420 significantly inhibited tumor growth compared with the Db126 WT1 peptide vaccine in the TRAMP-C2, mouse castration-resistant prostate cancer (CRPC) syngeneic tumor model. The present study demonstrated that oral administration of 1.0 Â 10 9 colony-forming units of B. longum 420 induced significantly higher cytotoxicity against TRAMP-C2 cells than intraperitoneal injection of 100 mg of Db126, and the in vivo antitumor activity of B. longum 420 in the TRAMP-C2 tumor model could be augmented by intraperitoneal injections of 250 mg of anti-PD-1 antibody. For the clinical development, we produced the B440 pharmaceutical formulation, which is lyophilized powder of inactivated B. longum 440 displaying the partially modified human WT1 protein. We confirmed that B. longum 440 could induce cellular immunity specific to multiple WT1 epitopes. In a preclinical dosage study, B440 significantly inhibited growth of the TRAMP-C2 tumors compared with that of the control groups (PBS and B. longum not expressing WT1) at all dosages (1, 5, and 10 mg/body of B440). These mouse doses were considered to correspond with practical oral administration doses of 0.2, 1, and 2 g/body for humans. Taken together, these results suggest that the B440 WT1 oral cancer vaccine can be developed as a novel oral immuno-oncology drug to treat CRPC as a monotherapy or as an adjunct to immune checkpoint inhibitors.

Cancer immunotherapy using immune-checkpoint inhibitors (ICIs) such as PD-1/PD-L1 inhibitors has been well established for various types of cancer. Monotherapy with ICIs, however, can achieve a durable response in only a subset of patients. There is a great unmet need for the ICI-resistant-tumors. Since patients who respond to ICIs should have preexisting antitumor T cell response, combining ICIs with cancer vaccines that forcibly induce an antitumor T cell response is a reasonable strategy. However, the preferred administration sequence of the combination of ICIs and cancer vaccines is unknown. In this study, we demonstrated that combining an oral WT1 cancer vaccine using a Bifidobacterium vector and following anti-PD-1 antibody treatment eliminated tumor growth in a syngeneic mouse model of bladder cancer. This vaccine induced T cell responses specific to multiple WT1 epitopes through the gut immune system. Moreover, in a tumor model poorly responsive to an initial anti-PD-1 antibody, this vaccine alone significantly inhibited the tumor growth, whereas combination with continuous anti-PD-1 antibody could not inhibit the tumor growth. These results suggest that this oral cancer vaccine alone or as an adjunct to anti-PD-1 antibody could provide a novel treatment option for patients with advanced urothelial cancer including bladder cancer.

<p>Supplementary Fig. S1. Representative dot blots of Intracellular cytokine staining (ICCS) in Figure 1B. After vaccination, mice splenocytes were obtained and re-stimulated with TRAMP-C2 in vitro. ICCS was performed for IFN-γ, IL-2 and TNF-α-producing CD4T and CD8T cells. Splenocytes were gated by front scatter (FSC) and side scatter (SSC). After gating with CD3, splenocytes were separated CD4T and CD8T that were producing each cytokine. Representative dot plots were shown in this figure. Supplementary Fig. S2. Representative dot plots of Intracellular cytokine staining (ICCS) of the significant data in Figure 4. After vaccination, mice splenocytes were obtained and re-stimulated with following peptides; Db126, WH187, mp235, Db332 and A24 in vitro. ICCS was performed for IFN-γ, IL-2 and TNF-α-producing CD4T and CD8T cells. Splenocytes were gated by front scatter (FSC) and side scatter (SSC). After gating with CD3, splenocytes were separated CD4T and CD8T that were producing each cytokine. Representative dot plots of the significant data were shown in this figure.</p>

<div>Abstract<p>Previously, we constructed a recombinant <i>Bifidobacterium longum</i> displaying a partial mouse Wilms' tumor 1 (WT1) protein (<i>B. longum</i> 420) as an oral cancer vaccine using a bacterial vector and demonstrated that oral administration of <i>B. longum</i> 420 significantly inhibited tumor growth compared with the Db126 WT1 peptide vaccine in the TRAMP-C2, mouse castration-resistant prostate cancer (CRPC) syngeneic tumor model. The present study demonstrated that oral administration of 1.0×10⁹ colony-forming units of <i>B. longum</i> 420 induced significantly higher cytotoxicity against TRAMP-C2 cells than intraperitoneal injection of 100 μg of Db126, and the <i>in vivo</i> antitumor activity of <i>B. longum</i> 420 in the TRAMP-C2 tumor model could be augmented by intraperitoneal injections of 250 μg of anti–PD-1 antibody. For the clinical development, we produced the B440 pharmaceutical formulation, which is lyophilized powder of inactivated <i>B. longum</i> 440 displaying the partially modified human WT1 protein. We confirmed that <i>B. longum</i> 440 could induce cellular immunity specific to multiple WT1 epitopes. In a preclinical dosage study, B440 significantly inhibited growth of the TRAMP-C2 tumors compared with that of the control groups (PBS and <i>B. longum</i> not expressing WT1) at all dosages (1, 5, and 10 mg/body of B440). These mouse doses were considered to correspond with practical oral administration doses of 0.2, 1, and 2 g/body for humans. Taken together, these results suggest that the B440 WT1 oral cancer vaccine can be developed as a novel oral immuno-oncology drug to treat CRPC as a monotherapy or as an adjunct to immune checkpoint inhibitors.</p></div>

<p>Supplementary Fig. S1. Representative dot blots of Intracellular cytokine staining (ICCS) in Figure 1B. After vaccination, mice splenocytes were obtained and re-stimulated with TRAMP-C2 in vitro. ICCS was performed for IFN-γ, IL-2 and TNF-α-producing CD4T and CD8T cells. Splenocytes were gated by front scatter (FSC) and side scatter (SSC). After gating with CD3, splenocytes were separated CD4T and CD8T that were producing each cytokine. Representative dot plots were shown in this figure. Supplementary Fig. S2. Representative dot plots of Intracellular cytokine staining (ICCS) of the significant data in Figure 4. After vaccination, mice splenocytes were obtained and re-stimulated with following peptides; Db126, WH187, mp235, Db332 and A24 in vitro. ICCS was performed for IFN-γ, IL-2 and TNF-α-producing CD4T and CD8T cells. Splenocytes were gated by front scatter (FSC) and side scatter (SSC). After gating with CD3, splenocytes were separated CD4T and CD8T that were producing each cytokine. Representative dot plots of the significant data were shown in this figure.</p>