Although we did not investigate further the detailed mechanism in this study, stimulation of interferon- (IFN-) release by oncolytic adenoviruses, as we previously reported, 35 is presumably associated with this PD-L1 upregulation, and PD-L1 upregulation by OBP-502 may be the mechanism underlying the synergistic effect of combination therapy with PD-1 Ab. with OBP-502 (so-called abscopal effect) in CT26 and PAN02 bilateral subcutaneous tumor models, in which active recruitment of CD8-positve lymphocytes was observed even in tumors not treated with OBP-502. This combined efficacy was similar to that observed in a CT26 rectal orthotopic tumor model including liver metastases. In conclusion, telomerase-specific oncolytic adenoviruses are encouraging candidates for combined therapies with ICIs. and genes.17 This gene modification enables OBP-301 to replicate selectively in tumor cells and induce tumor-specific oncolytic cell death. Moreover, following induction of oncolytic cell death, progeny viruses are capable of spreading to surrounding tumor cells to cause continued oncolytic cell death. A US Food and Drug Administration (FDA)-approved phase I clinical study confirmed the security and biological activity of intratumoral administration of OBP-301 in patients with advanced solid tumors in the United States.18 Based Telaprevir (VX-950) on its safety profile and promising preclinical data regarding combination therapy with ionizing radiation, a phase I/II clinical trial is currently ongoing in Japan to evaluate the safety and efficacy of the combination for treating esophageal cancer (UMIN000010158).19 In the present study, we first examined the potential of our telomerase-specific oncolytic adenovirus as an ICD-inducing drug by assessing the secretion of ICD markers such as HMGB1 and ATP and recruitment of CD8-positive TILs on gastrointestinal tumors subcutaneous and orthotopic mouse models, focusing on abscopal effects mediated by the oncolytic adenoviruses via activation of the host immune system. Our findings demonstrate the promise of our telomerase-specific oncolytic adenovirus therapy, particularly in combination with an ICI. Moreover, these findings should facilitate the development of a novel Telaprevir (VX-950) oncolytic virus therapeutic strategy incorporating our adenovirus that will be a more attractive option for malignancy treatment. Results Cytotoxic Activity of OBP-502 against Murine Gastrointestinal Malignancy Cell Lines OBP-502, a variant of OBP-301, was employed in this study. The difference between OBP-301 and OBP-502 is usually that OBP-502 has the gene cassette expressing the RGD peptide in the E3 region (Physique?S1A). The RGD peptide facilitates OBP-502 contamination of murine tumor cells via conversation with the integrin v5 expressed on tumor cells, because OBP-301 contamination of murine cells such as CT26 and PAN02, which do not express the Coxsackie computer virus and adenovirus receptor (CAR) (Physique?S2), is inefficient. OBP-502 killed CT26 and PAN02 cells in a dose-dependent and time-dependent manner (Physique?1A; Physique?S1B), and this cytotoxicity of OBP-502 was higher than OBP-301, especially at high doses, whereas no significant difference was observed between OBP301 and OBP-502 on human malignancy cells such as TE4, GCIY, MIA PaCa-2, and HCT116 (Physique?S1C). This cytotoxicity was associated with induction of apoptosis and autophagy following effective contamination of murine tumor cells, which was shown by induction of E1A, upregulation of cleaved poly(ADP ribose) polymerase (PARP), and downregulation of p62 on western blot analysis (Physique?1B). The cytotoxic mechanism of OBP-502 was considered similar to that of OBP-301, an original oncolytic adenovirus.20 Interestingly, treatment with high doses of OBP-502 upregulated PD-L1 expression by CT26 and PAN02 cells (Physique?1B), particularly around the cell surface (Physique?1C). Open in a separate window Physique?1 Cytotoxic Activity of OBP-502 against Murine Gastrointestinal Malignancy Cell Lines (A) Viability of CT26 and PAN02 cells was assessed using an XTT assay 3?days after OBP-502 treatment at the indicated doses (MOI). The percentage of viable cells relative to non-treated cells (0 MOI) was plotted. Error bars show 95% confidence intervals. (B) Whole-cell lysates of CT26 and PAN02 cells collected 3?days after OBP-502 treatment (0, 10, 100, 500, and 1,000 MOI) were subjected to western blot analysis of E1A, PARP, p62, PD-L1, and -Actin expression. Cleaved PARP (C-PARP) and p62 indicate induction of apoptosis and autophagy, respectively. ITSN2 (C) CT26 Telaprevir (VX-950) and PAN02 cells treated with OBP-502 (1,000 MOI) were subjected to circulation cytometry for analysis of PD-L1 expression around the cell surface 3?days after treatment. Active Release of Immunogenic Molecules and Chemokines by OBP-502 24?h after treatment (Figures 2A and 2B) but did not affect the expression of calreticulin (CRT), another immunogenic molecule, in either cell collection (Physique?S3A). Intracellular HMGB1 levels also increased with OBP-502 treatment (Figures S3B and S3C). OBP-502 decreased -catenin expression in CT26 and PAN02 cells (Physique?S3D), which is reportedly associated with immune exclusion in.