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With a 2-year survival less than 20%, Diffuse Intrinsic Pontine Glioma (DIPG) is the principal cause of pediatric death. Despite recent advances in the current treatments, the outcome for children with DIPGs remains dismal. Since the approval of T-VEC for melanoma by the FDA, oncolytic adenoviruses have emerged as a promising therapeutic strategy for brain tumors. Thus, our group launched the first world clinical trial phase I with the oncolytic adenovirus Delta-24-RGD (DNX-2401 in the clinic) for newly diagnosed DIPG (NCT03178032), which has shown safety and feasibility. Despite DNX-2401 increases the recruitment of T cells into the tumor, they usually become inactive due to the non-responsive tumor microenvironment evidencing the urgent need to improve this strategy focusing on the generation of effective long-term immune responses. Therefore, we decided to combine the Delta-24-RGD with the targeting of the costimulatory molecule CD40 in immunocompetent mice bearing orthotopic DIPG. The activation of the CD40 receptor, which is expressed by antigen presenting cells (APC) such as microglia, macrophages, and dendritic cells, is known to increase antigen presentation and enable T-cell priming and activation. Here, we observed that in addition to Delta-24-RGD anti-tumor effects, the stimulation of CD40 (using an agonistic antibody) on the tumor APCs results in a remodeling of the tumor immune compartment towards a proinflammatory scenario and a more efficient T-cell infiltration. Of importance, the combination therapy extends survival of treated mice as compared to single treatments or non-treated mice. In addition, we observe a complete regression of tumors in more than 40% of treated mice and the development of long-term anti-tumor immunity. We believe that these results provide a translational breakthrough in the treatment of these lethal tumors and open the door for a future innovative clinical trial.

Purpose of Study: Diffuse Midline Gliomas (DMG) are aggressive pediatric brain tumors that arise in the brainstem of children, with a peak of incidence between 5-10 years old. The median survival of DMG patients is only 9 months, being the leading cause of pediatric death caused by a brain tumor. On this project we set to characterize the efficacy of Delta-24-RGDOX, an oncolytic adenovirus based on Delta-24-RGD platform, which has demonstrated safety and a therapeutic benefit in different pediatric tumors, armed with the ligand of OX40. The binding of OX40 to OX40L leads to the co-stimulation of CD4 and CD8 cells, generating effector and memory T cells. Therefore, the aim of this project is to improve the antitumor effect of the virus, providing a greater co-stimulation in the tumor. Experimental Procedures: Murine and human DMG cell lines were used. Viral protein expression was measured by western blot, viral replication was analyzed using a method based on hexon detection and the oncolytic effect by MTS assay. OX40L expression was measured by flow cytometry and qPCR. For in vivo experiments, cells were injected in the pons of mice using a screw-guided system. The adenovirus was administered once into the tumor using the same procedure. Tumor immune populations were analyzed by flow cytometry. Results: We first confirmed the oncolytic effect of Delta-24-RGDOX in DMG murine and human cell lines in vitro. The virus was able to infect the cells, produce viral proteins, and to cause cell death in a dose-dependent manner. In addition, we observed effective viral replication in human cell lines but not in mouse cells, as previously described. We then measured OX40L expression in infected cells both at protein and mRNA level, observing that at 50 PFU/cell almost 100% of cells expressed the ligand on their membrane. More importantly, the OX40L was functional as it activated CD8 lymphocytes in vitro. Once we confirmed that the viral administration in vivo was safe, we assess the efficacy of the virus in murine DMG models. We observed a significant survival benefit in mice bearing NP53 tumor treated with Delta-24-RGDOX, which lead to 30% of long-term survivors (P=0.003, median OS PBS 25.5 days vs 35.5 days for treated mice). More importantly, we administered the virus in an already stablished tumor model using XFM cells and also obtained a significant improve of survival (P=0.018, median OS PBS 9 days vs 12.5 treated mice). Finally, we analyzed the immune mechanisms underlying the survival benefit, both in the tumor and in spleen. We observed a significant increase of activated immune cells in the tumor microenvironment 7 days after the viral administration. In addition, splenocytes from virus-treated mice were also significantly more activated. Conclusions: These data show that Delta-24-RGDOX adenovirus expresses a functional OX40L that can modulate the immune response, leading to a significantly improved survival outcome in DMG models. Citation Format: Virginia Laspidea, Sara Labiano, Sumit Gupta, Hong Jiang, Iker Ausejo-Mauleon, Daniel de la Nava, Marc García-Moure, Javier Marco-Sanz, Reyes Hernández-Osuna, Oren J Becher, Juan Fueyo, Ana Patiño-García, Candelaria Gomez-Manzano, Marta M Alonso. Delta-24-RGDOX oncolytic adenovirus improves the survival by modulating the immune system in DMG models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2001.

Purpose of the work: Pediatric High Grade Gliomas (pHGGs), including Diffuse Midline Gliomas (DMGs), are very aggressive solid tumors developed during childhood with a poor overall survival underscoring the need for effective treatments. The adenovirus Delta-24RGD and the imipridone ONC201 have demonstrated safety and effectiveness in preclinical models and clinical trials. Thus, we evaluated the therapeutic efficacy of the Delta-24-RGD/ONC201 combination and analyzed possible changes in the tumor microenvironment in preclinical models of pHGGs and DMGs. Experimental procedures: A battery of pHGGs (SF188 and CHLA-03-AA), human DMG (SU-DIPG IV), and murine DMG cell lines (XFM and NP-53) were used. We assessed the viral replication by hexon titration, the protein expression amount (E1A, fiber, and mTORC1 key proteins)by immunoblotting, and oxygen consumption by Seahorse Analyses. Viability was measured by MTS. CHLA-03-AA and XFM were engrafted orthotopically in athymic nude mice and Balb/c mice, respectively. To assess in vivo efficacy, the animals were treated with Delta-24-RGD (107 PFU/animal) or PBS, followed by ONC201administration (125 mg/kg/twice/weekly). The tumor immune microenvironment was evaluated by flow cytometry. Results: First, the potential adverse interactions between both agents were checked in vitro, showing that ONC201 cotreatment did not interfere with the Delta-24-RGD replication capacity. As expected, ONC201 treatment led to a reduction in oxygen consumption, which was also observed in the combination treatment. Evaluation of cytotoxicity in a panel of cell lines showed that the combination was significantly better than either agent alone. In vivo, CHLA-03-AA bearing mice treated with the combination showed a significant increase in the median overall survival (PBS: 48 days; ONC201: 54.5 days; Delta-24-RGD: 62 days; Delta-24-RGD/ONC201: 95 days (P=0.0008)), leading to 20%long-term survivors, free of disease. Evaluation of the combination in an immunocompetent orthotopic DMG model also revealed the superior efficacy of the Delta-24-RGD/ONC201 (PBS: 11 days; ONC201: 12 days; Delta-24-RGD: 12 days; ONC201+Delta-24-RGD: 15 days (P=0.02)). Of importance, the combination treatment resulted in the reshaping of the tumor microenvironment towards a proinflammatory phenotype (T cells, NK cells, macrophages, granulocytes, monocytes, dendritic cells, and microglia). Conclusions: Our data indicate that the combination Delta-24-RGD/ONC201 generates a pro-inflammatory microenvironment that ultimately leads to a superior therapeutic effect in preclinical pHGG and DMGs models. Citation Format: Daniel de la Nava, Javier Marco-Sanz, Virginia Laspidea, Sara Labiano, Iker Ausejo-Mauleon, Reyes Hernandez-Osuna, Sabine Mueller, Javad Nazarian, Joshua E. Allen, Oren Becher, Juan Fueyo, Candelaria Gomez-Manzano, Ana Patiño-Garcia, Marta M. Alonso. The adenovirus Delta-24-RGD in combination with ONC201 provides a therapeutic benefit and a proinflammatory recruitment in preclinical models of pHGGs and DMGs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2000.

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