To deplete the CD8+ T?cells and NK cells, mice were injected peritoneally with 500?g of anti-CD8a (Bioxcell, West Lebanon, NH, USA) or anti-NK1.1 (Bioxcell, West Lebanon, NH, USA) on day 10 and received a second injection 1?week later. tumor rechallenges at both and distant sites. Thus, Ad5-PC harnesses several distinct functions to efficiently overcome several major hurdles of viro-immunotherapy. administration of agonistic CD137 antibodies led to regression of some tumors.18 However, systemic immune toxicity limited its clinical application.19 Here, we introduce a novel replication-competent adenovirus expressing a bispecific fusion protein, which contains the extracellular domain of PD-1 on one end and the extracellular domain of CD137L on another (PD1/CD137L). Our study suggests that this genetically engineered adenovirus promotes immune cell infiltration into the tumor sites. Moreover, PD1/CD137L activates tumor-specific CTLs by signaling through the CD137 pathway and blocks?the PD-L1/PD-1 pathway in CTLs to diminish T?cell exhaustion. Ultimately, the recombinant adenovirus mediates a markedly enhanced antitumor immune response and durable tumor regression. Results Replicative Adenovirus Improves the Immune Responses in TME but Fails to Prolong Survival in an HCC Ascites Mouse Model To investigate whether the oncolytic adenovirus has a therapeutic effect on malignant tumors, we constructed a replication-competent type V adenovirus (Ad5con) with an additional early region 1A (E1A) replication element (Figure?1A). The replication and oncolytic capabilities of Ad5con were confirmed in several types of tumor cells (Figure?S1). The H22-based hepatocellular carcinoma (HCC) ascites mouse model was also employed to evaluate the antitumor activity of Ad5con and its influence on the TME KLF1 (Figure?1B). We further monitored the dynamic changes in ascites associated with the therapeutic outcome in each mouse. As a result, both the concentration of IFN- and the number of IFN-Cproducing cells in ascites were significantly increased when H22-bearing mice were treated with oncolytic adenovirus (Figures 1C and 1D). In addition, CD8+ T?cells and NK1.1 cells were recruited into ascites after Ad5con treatment (Figure?1E). However, survival did not improve in the Teneligliptin hydrobromide hydrate Ad5con group compared with the untreated group (Figure?1G). Interestingly, PD-L1, an immunosuppressive molecule, was dramatically upregulated on the surfaces of ascitic cells in mice treated with Ad5con (Figure?1H). Open in a separate window Figure?1 Replication-Competent Adenovirus Improves Immune Responses in TME but Fails to Prolong Survival in an Ascitic HCC Murine Model Male C57BL/6 mice were injected peritoneally with 5? 106 H22 cells. On days 7, 12, and 17, mice were treated i.p. with Ad5con (5? 108 PFUs), with saline used as a control (n?= 10 for each group). (A) Construction of the replication-competent adenovirus. (B) Schematic diagram of the experimental setup for adenovirus therapy. (C) Mouse ascites were collected on day 14, and the IFN–producing lymphocytes were determined by ELISpot. The representative results for ELISpot are shown in the left panel, and the associated plot counts are shown in the right panel. (D) IFN- concentrations in the ascites were determined by ELISA. (E and F) Frequencies of (E) CD8+ T?cells and (F) NK?cells were determined by flow cytometry. (G) Survival curves of mice with or without Ad5con treatment. (H) PD-L1-positive cells in ascites were detected by flow cytometry. Data shown are the means? SD. Data are representative of at least three independent Teneligliptin hydrobromide hydrate experiments. *p?< 0.05, **p?< 0.01, ***p?< 0.001. These data suggest that the oncolytic Ad5con induces a moderate immune response at the tumor sites, which may transform the tumor from cold into hot. Unfortunately, Ad5con treatment was insufficient to produce an effective antitumor effect, which may?be substantially due to the elevated expression of PD-L1 on tumor cells. Generation of a Novel Recombinant Adenovirus Regulating PD-L1/PD-1 Negative Feedback Signaling and CD137L/CD137 Costimulatory Signaling To elicit a therapeutic antitumor immune response, we aimed to generate a novel recombinant adenovirus that blocks PD-L1/PD-1 negative feedback and provides a costimulatory Teneligliptin hydrobromide hydrate signal to immune Teneligliptin hydrobromide hydrate effector cells. To this end, a soluble fusion protein, PD-1/CD137L, was designed that contains the extracellular domain of PD-1 in the N terminus and the extracellular domain of CD137L in the C terminus (Figure?2A). The soluble fusion protein readily conjugates PD-L1 on tumor cells; hence the PD-1 signal in immune effector cells is blocked, and their antitumor activity can be sustained. Soluble CD137L (sCD137L) was employed to enhance T?cell proliferation and cytolytic activity. The expression fragments were inserted into the genome of a replicative adenovirus. First, we confirmed that the soluble PD-1.