Purpose: Intratumoral injection of oncolytic adenovirus Delta-24-RGDOX induces efficacious antiglioma immunity in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is effective against disseminated melanomas.Experimental Design: We tested the therapeutic effect of injecting Delta-24-RGDOX into primary subcutaneous (s.c.) B16-Red-FLuc tumors in s.c./s.c. and s.c./intracranial (i.c.) melanoma models in C57BL/6 mice. Tumor growth and in vivo luciferase-expressing ovalbumin-specific (OT-I/Luc) T cells were monitored with bioluminescence imaging. Cells were profiled for surface markers with flow cytometry.Results: In both s.c./s.c. and s.c./i.c. models, 3 injections of Delta-24-RGDOX significantly inhibited the growth of both the virus-injected s.c. tumor and untreated distant s.c. and i.c. tumors, thereby prolonging survival. The surviving mice were protected from rechallenging with the same tumor cells. The virus treatment increased the presence of T cells and the frequency of effector T cells in the virus-injected tumor and mediated the same changes in T cells from peripheral blood, spleen, and brain hemispheres with untreated tumor. Moreover, Delta-24-RGDOX decreased the numbers of exhausted T cells and regulatory T cells in the virus-injected and untreated tumors. Consequently, the virus promoted the in situ expansion of tumor-specific T cells and their migration to tumors expressing the target antigen.Conclusions: Localized intratumoral injection of Delta-24-RGDOX induces an in situ antovaccination of the treated melanoma, the effect of which changes the immune landscape of the treated mice, resulting in systemic immunity against disseminated s.c. and i.c. tumors.

BackgroundOncolytic viruses are considered part of immunotherapy and have shown promise in preclinical experiments and clinical trials. Results from these studies have suggested that tumor microenvironment remodeling is required to achieve an effective response in solid tumors. Here, we assess the extent to which targeting specific mechanisms underlying the immunosuppressive tumor microenvironment optimizes viroimmunotherapy.MethodsWe used RNA-seq analyses to analyze the transcriptome, and validated the results using Q-PCR, flow cytometry, and immunofluorescence. Viral activity was analyzed by replication assays and viral titration. Kyn and Trp metabolite levels were quantified using liquid chromatography–mass spectrometry. Aryl hydrocarbon receptor (AhR) activation was analyzed by examination of promoter activity. Therapeutic efficacy was assessed by tumor histopathology and survival in syngeneic murine models of gliomas, including Indoleamine 2,3-dioxygenase (IDO)-/- mice. Flow cytometry was used for immunophenotyping and quantification of cell populations. Immune activation was examined in co-cultures of immune and cancer cells. T-cell depletion was used to identify the role played by specific cell populations. Rechallenge experiments were performed to identify the development of anti-tumor memory.ResultsBulk RNA-seq analyses showed the activation of the immunosuppressive IDO-kynurenine-AhR circuitry in response to Delta-24-RGDOX infection of tumors. To overcome the effect of this pivotal pathway, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors. The combination therapy increased the frequency of CD8+ T cells and decreased the rate of myeloid-derived suppressor cell and immunosupressive Treg tumor populations in animal models of solid tumors. Functional studies demonstrated that IDO-blockade-dependent activation of immune cells against tumor antigens could be reversed by the oncometabolite kynurenine. The concurrent targeting of the effectors and suppressors of the tumor immune landscape significantly prolonged the survival in animal models of orthotopic gliomas.ConclusionsOur data identified for the first time the in vivo role of IDO-dependent immunosuppressive pathways in the resistance of solid tumors to oncolytic adenoviruses. Specifically, the IDO-Kyn-AhR activity was responsible for the resurface of local immunosuppression and resistance to therapy, which was ablated through IDO inhibition. Our data indicate that combined molecular and immune therapy may improve outcomes in human gliomas and other cancers treated with virotherapy.

Background Viroimmunotherapy is evolving as a strong alternative for the standard treatment of malignant gliomas. Promising results from a recent clinical trial testing the anticancer effect of Delta-24-RGD in patients with glioblastoma suggested the induction of antitumoral immunity after viral administration. To further enhance the anti-glioma immune effect, we have armed Delta-24-RGD with the costimulatory ligand GITRL (Delta-24-GREAT [Glucocorticoid Receptor Enhanced Activity of T cells]). Methods We tested the infectivity and replication of Delta-24-GREAT, and the expression of ectopic GITRL in human and murine glioma cell lines. In vivo experiments involved the intracranial implantation of glioma cells into an immunocompetent model to study the anticancer effect, and rechallenging experiments to study long-term protection. Phenotypic and functional characterization of lymphocyte populations were performed by FACS and ELISA for Th1 cytokines expression, respectively. Results Our results showed that Delta-24-GREAT infects and induces the expression of GITRL. Delta-24-GREAT prolonged the survival of glioma-bearing immunocompetent mice and resulted in both anti-viral and anti-glioma immune responses, including increased frequency of central memory CD8+ T cells. Rechallenging the surviving mice with a second implantation of glioma cells did not lead to tumor growth; however, the surviving mice developed lethal tumors when B16/F10 melanoma cells were implanted intracranially, strongly indicating that the immune response was specific for glioma antigens. Conclusions GITRL-armed Delta-24-RGD treatment results in an antigen-restricted antitumor memory, an enhanced anti-glioma effect, and the generation of central immune memory. Our results strongly indicate that this strategy represents a vertical advance in virotherapy designed to treat patients with malignant brain tumors.