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Inflammation is a common feature in neurodegenerative diseases that contributes to neuronal loss. Previously, we demonstrated that the basal inflammatory tone differed between brain regions and, consequently, the reaction generated to a pro-inflammatory stimulus was different. In this study, we assessed the innate immune reaction in the midbrain and in the striatum using an experimental model of Parkinson's disease. An adeno-associated virus serotype 9 expressing the α-synuclein and mCherry genes or the mCherry gene was administered into the substantia nigra. Myeloid cells (CD11b

Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2–20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% CI 40.1–69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5 months (10.7–13.5 months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05–0.87). A total of 56.2% (95% CI 41.1–70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60 months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406).

Supplementary Data from Local Treatment of a Pediatric Osteosarcoma Model with a 4-1BBL Armed Oncolytic Adenovirus Results in an Antitumor Effect and Leads to Immune Memory

Supplementary Figure from Local Treatment of a Pediatric Osteosarcoma Model with a 4-1BBL Armed Oncolytic Adenovirus Results in an Antitumor Effect and Leads to Immune Memory

Osteosarcoma is an aggressive bone tumor occurring primarily in pediatric patients. Despite years of intensive research, the outcomes of patients with metastatic disease or those who do not respond to therapy have remained poor and have not changed in the last 30 years. Oncolytic virotherapy is becoming a reality to treat local and metastatic tumors while maintaining a favorable safety profile. Delta-24-ACT is a replicative oncolytic adenovirus engineered to selectively target cancer cells and to potentiate immune responses through expression of the immune costimulatory ligand 4-1BB. This work aimed to assess the antisarcoma effect of Delta-24-ACT. MTS and replication assays were used to quantify the antitumor effects of Delta-24-ACT in vitro in osteosarcoma human and murine cell lines. Evaluation of the in vivo antitumor effect and immune response to Delta-24-ACT was performed in immunocompetent mice bearing the orthotopic K7M2 cell line. Immunophenotyping of the tumor microenvironment was characterized by immunohistochemistry and flow cytometry. In vitro, Delta-24-ACT killed osteosarcoma cells and triggered the production of danger signals. In vivo, local treatment with Delta-24-ACT led to antitumor effects against both the primary tumor and spontaneous metastases in a murine osteosarcoma model. Viral treatment was safe, with no noted toxicity. Delta-24-ACT significantly increased the median survival time of treated mice. Collectively, our data identify Delta-24-ACT administration as an effective and safe therapeutic strategy for patients with local and metastatic osteosarcoma. These results support clinical translation of this viral immunotherapy approach.

Supp Figure 4 Detection of Delta-24-RGD replication at late stages of disease.

Purpose: Osteosarcoma is the most common malignant bone tumor in children and adolescents. Despite aggressive chemotherapy, more than 30% of patients do not respond and develop bone or lung metastasis. Oncolytic adenoviruses engineered to specifically destroy cancer cells are a feasible option for osteosarcoma treatment. VCN-01 is a replication-competent adenovirus specifically engineered to replicate in tumors with a defective RB pathway, presents an enhanced infectivity through a modified fiber and an improved distribution through the expression of a soluble hyaluronidase. The aim of this study is to elucidate whether the use of VCN-01 would be an effective therapeutic strategy for pediatric osteosarcoma.Experimental Design: We used osteosarcoma cell lines established from patients with metastatic disease (531MII, 678R, 588M, and 595M) and a commercial cell line (143B). MTT assays were carried out to evaluate the cytotoxicity of VCN-01. Hexon assays were used to evaluate the replication of the virus. Western blot analysis was performed to assess the expression levels of viral proteins and autophagic markers. The antitumor effect of VCN-01 was evaluated in orthotopic and metastatic osteosarcoma murine animal models.Results: This study found that VCN-01, a new generation genetically modified oncolytic adenovirus, administered locally or systemically, had a potent antisarcoma effect in vitro and in vivo in mouse models of intratibial and lung metastatic osteosarcoma. Moreover, VCN-01 administration showed a safe toxicity profile.Conclusions: These results uncover VCN-01 as a promising strategy for osteosarcoma, setting the bases to propel a phase I/II trial for kids with this disease. Clin Cancer Res; 22(9); 2217–25. ©2015 AACR.

A, Quantification of the tumor bioluminescence in the mice from the indicated treatment groups. D24-RGDOX: Delta-24-RGDOX. B, Waterfall plots showing tumor burden change from baseline in mice of indicated treatment groups base on the bioluminescence from the tumor. Baseline time point: Day 8 for s.c. tumor, Day 15 for i.c. tumor. Evaluation time point: Day 57 or the last time point before euthanasia.

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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.

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Purpose:Atypical teratoid/rhabdoid tumors (AT/RT) and central nervous system primitive neuroectodermal tumors (CNS-PNET) are pediatric brain tumors with poor survival and life-long negative side effects. Here, the aim was to characterize the efficacy and safety of the oncolytic adenovirus, Delta-24-RGD, which selectively replicates in and kills tumor cells.Experimental Design:Delta-24-RGD determinants for infection and replication were evaluated in patient expression datasets. Viral replication and cytotoxicity were assessed in vitro in a battery of CNS-PNET and AT/RT cell lines. In vivo, efficacy was determined in different orthotopic mouse models, including early and established tumor models, a disseminated AT/RT lesion model, and immunocompetent humanized mouse models (hCD34+-NSG-SGM3).Results:Delta-24-RGD infected and replicated efficiently in all the cell lines tested. In addition, the virus induced dose-dependent cytotoxicity [IC50 value below 1 plaque-forming unit (PFU)/cell] and the release of immunogenic markers. In vivo, a single intratumoral Delta-24-RGD injection (107 or 108 PFU) significantly increased survival and led to long-term survival in AT/RT and PNET models. Delta-24-RGD hindered the dissemination of AT/RTs and increased survival, leading to 70% of long-term survivors. Of relevance, viral administration to established tumor masses (30 days after engraftment) showed therapeutic benefit. In humanized immunocompetent models, Delta-24-RGD significantly extended the survival of mice bearing AT/RTs or PNETs (ranging from 11 to 27 days) and did not display any toxicity associated with inflammation. Immunophenotyping of Delta-24-RGD–treated tumors revealed increased CD8+ T-cell infiltration.Conclusions:Delta-24-RGD is a feasible therapeutic option for AT/RTs and CNS-PNETs. This work constitutes the basis for potential translation to the clinical setting.

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Supp Figure 2 Replication and cytolytic activity of Delta24-RGD in AT/RT, PNET, and PNET-like cell cultures.

Supp Figure 3 Therapeutic effect of Delta-24-RGD in mice bearing PNET-like tumors.

Supp Figure 5 Safety and early effects on immune landscape of Delta-24-RGD in humanized mice bearing CHLA-06 and PFSK-1 supratentorial tumors.

Supp Figure 1 Infectivity of Delta24-RGD in PNET-like cell cultures.

Supplementary Figure 1a movie from Transgenic E2F1 Expression in the Mouse Brain Induces a Human-Like Bimodal Pattern of Tumors

The Rb/E2F pathway is deregulated in most human brain tumors, and the finding that loss of E2F1 reduced pituitary tumorigenesis in Rb+/− mice suggests that loss of pRb induces brain tumors by activating E2F1. We therefore investigated the role of E2F1 in the development and maintenance of brain cancer using a transgenic mouse model engineered to express E2F1 specifically within glial cells (GFAP-tgE2F1). GFAP-tgE2F1 mice developed a highly penetrant phenotype characterized by neurologic defects, and examination of the brains revealed the presence of brain tumors in 20% of these animals. Importantly, the distribution of tumors according to mouse age suggests the existence of a bimodal pattern of tumor development, forcing a comparison with the human disease. Mice, at an early age, with deregulated E2F1 show the formation of embryonal brain tumors such as medulloblastoma, choroid plexus carcinoma, and primary neuroectodermal tumor. Conversely, at an older age, mice escaping embryonal tumor formation present with malignant gliomas, which are typically identified in the human adult population. Thus, this study offers the first evidence for a global role of E2F1 in the formation and maintenance of multilineage brain tumors, irrefutably establishing E2F1 as an oncogene in the brain. [Cancer Res 2007;67(9):4005–9]

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.

Osteosarcoma is the most frequent and aggressive bone tumor in children and adolescents, with a long-term survival rate of 30%. Interleukin-12 (IL-12) is a potent cytokine that bridges innate and adaptive immunity, triggers antiangiogenic responses, and achieves potent antitumor effects. In this work, we evaluated the antisarcoma effect of a high-capacity adenoviral vector encoding mouse IL-12. This vector harbored a mifepristone-inducible system for controlled expression of IL-12 (High-Capacity adenoviral vector enconding the EF1α promoter [HCA-EFZP]-IL-12). We found that local administration of the vector resulted in a reduction in the tumor burden, extended overall survival, and tumor eradication. Moreover, long-term survivors exhibited immunological memory when rechallenged with the same tumor cells. Treatment with HCA-EFZP-IL-12 also resulted in a significant decrease in lung metastasis. Immunohistochemical analyses showed profound remodeling of the osteosarcoma microenvironment with decreases in angiogenesis and macrophage and myeloid cell numbers. In summary, our data underscore the potential therapeutic value of IL-12 in the context of a drug-inducible system that allows controlled expression of this cytokine, which can trigger a potent antitumor immune response in primary and metastatic pediatric osteosarcoma., Graphical Abstract, Osteosarcoma is the most aggressive bone tumor in children and adolescents. In this work, the authors show the potential therapeutic value of IL-12 in the context of a drug-inducible system that allows controlled expression of this cytokine, which led to a potent antitumor immune response in primary and metastatic pediatric osteosarcoma model.

Purpose of the work: Pediatric High Grade Gliomas (pHGGs) and Diffuse Midline Gliomas (DMGs) are two of the most aggressive tumors developed during childhood. Its poor overall survival emphasizes the need of alternative treatments. ONC201, an imipiridone small molecule, and the oncolytic virus Delta-24-RGD have shown safety and effectiveness in preclinical and clinical settings for these diseases. In this work, we set to characterize the antitumor effect of these two agents combined for the treatment of pHGGs and DMGs. Experimental procedures: A panel of human pHGG (CHLA-03-AA, SF188, SJ-GBM2), DMG (SU-DIPG IV, SF8628, TP-54, HSJD-DIPG-007, UMPED83), and murine DMG cell lines (XFM, NP-53) were used. We assessed viral replication by hexon titration, protein expression by immunoblotting/immunohistochemistry, and oxygen consumption by Seahorse analyses. Viability was measured by MTS. Mitochondrial DNA (mtDNA) were measured by qPCR. CHLA-03-AA/XFM were orthotopically engrafted in athymic nude/Balb/c mice. For in vivo experiments, animals were treated with PBS/Delta-24-RGD (107 pfu/mice) intratumorally, followed by ONC201 oral gavage administration (125 mg/kg/twice/weekly). The tumor microenvironment immune infiltration was evaluated by flow cytometry. Results: We checked the potential negative interaction between both agents in vitro. Adenovirus replication was not affected by ONC201 cotreatment, indicating the suitability of this combination. In vitro citotoxycity analyses showed the cotreatment is either synergistic or additive in 80% of the cells evaluated. Then, we set to characterize the mechanistic effects of the combination. Analysis of mitochondria status showed the decrease in oxygen consumption previously reported as ONC201 effect is maintained with Delta-24-RGD presence. Confirmation of higher mitochondria damage was assessed by a reduction of mtDNA in the combination. Independently, endoplasmic reticulum stress (ERS) previously described in both agents was found potentiated in cotreatment via ATF4. Altogether, these data suggest an improved response in the combination by mitochondria and ERS perturbation. In vivo, cotreatment led to a significant increase in the median OS (Control: 48 days; ONC201: 54.5 days; Delta-24-RGD: 62 days; Delta-24-RGD/ONC201: 95 days (P=0.0008)) in CHLA-03-AA bearing mice, leading to 20% long-term survivors, free of disease. Of importance, a reshaping of the tumor microenvironment towards a proinflammatory phenotype were described (increases of lymphoid (T and NK cells) and myeloid (macrophages, granulocytes, monocytes, denditric cells and microglia) linage. Conclusions: Our data support the combination ONC201/Delta-24-RGD leads to a superior therapeutic effect due to the generation of a proinflammatory microenvironment in pHGG and DMGs preclinical models. Citation Format: Daniel de la Nava, Virginia Laspidea, Iker Ausejo-Mauleón, Marc García-Moure, Sabine Mueller, Javad Nazarian, Joshua E. Allen, Oren Becher, Juan Fueyo, Candelaria Gomez-Manzano, Ana Patiño-Garcia, Carl Koschmann, Marta M. Alonso. Depicting ONC201/Delta-24-RGD combination for the treatment of pHGGs and DMGs reveals a therapeutic benefit and a proinflammatory tumor microenvironment remodelation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4659.

Epigenetic modulators in combination with proapoptotic drugs have become a standard of treatment for acute myeloid leukemia. However, the clinical efficacy of these combinations in solid tumors has shown to be negligible. Our group has demonstrated that the epigenetic drug CM272, which targets DNMT and G9a, induces synergistic responses in combination with proapoptotic drugs in hematological malignancies (Cancer Discovery 2021). We have also reported that CM272 induces an immunogenic cell death that sensitizes tumor cells to immune checkpoint blockade (ICB) (Nature Medicine 2020). Here, we hypothesized that CM272 in combination with proapoptotic drugs may sensitize tumor cells to ICB, resulting in profound anti-tumor responses. To test this hypothesis, we evaluated in vitro the sensitivity of cancer cells to different combinations of epigenetic and proapoptotic drugs. The most efficient combination was tested in various immunocompetent mouse models, followed by an immunophenotypic characterization of the tumor microenvironment. We found that CM272 in combination with inhibitors of BCL-XL, BCL2 or MCL1, yielded synergistic in vitro responses in a large collection of human and mouse cell lines derived from solid tumors. Mechanistically, the treatments induced the expression of endogenous retroelements/retroviruses, which led to the activation of RIG-1 and MDA5 viral sensors, ATP hydrolysis and tumor cell death. Remarkably, we found that CM272 in combination with BCL-XL inhibition had more potent anti-tumor effects than that found with BCL2 or MCL1 inhibitors. In vivo, the triple combination CM272, A1331852 (BCL-XL inhibitor) and an anti-PD-1 moAb significantly reduced tumor growth and increased overall survival in three subcutaneous lung cancer models (LLC, 393P, Lacun-3) in comparison to double treatments. Moreover, this triple combination also induced significant anti-tumor responses in subcutaneous mouse models of colon cancer (MC38) and melanoma (B16), as well as in orthotopic models of lung cancer (LLC), glioblastoma (CT-2A) and breast cancer (ANV5), leading to prolonged survival and cure in a fraction of animals. The triple therapy was associated with a significant increase in the ratio of CD8 T versus immunosuppressive Treg cells, and M1 versus M2 macrophages. In conclusion, we report a novel regimen combining a dual epigenetic inhibitor, an anti-BCL-XL, and an anti-PD-1 moAb that results in potent responses in multiple pre-clinical models of solid tumors. The mechanisms underneath the antitumor responses include the modulation of the energy metabolism in tumor cells, leading to cell death boosted by an anti-BCL-XL pro-apoptotic drug, along with fostering the immune system to generate an efficient anti-tumor response assisted by ICB. This study reveals the potential of epigenetic therapeutics to treat and cure patients with solid tumors. Citation Format: Yaiza Senent, Vicente Fresquet, Victoria Jimenez, Karmele Valencia, Ana Remirez, Francisco Exposito, Marisol Gonzalez-Huarriz, Borja Ruiz-Fernandez de Cordoba, Haritz Moreno, Daniel Ajona, Marta M Alonso, Fernando Lecanda, Antonio Pineda-Lucena, Felipe Prosper, Alfonso Calvo, Jose Angel Martinez-Climent, Ruben Pio. Combined treatment with the epigenetic drug CM272 and an anti-BCL-XL proapoptotic drug sensitizes solid tumors to immune checkpoint blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5117.

Oncolytic virotherapy shows great potential for treating brain tumors and other solid tumors that metastasize to the brain. A phase I clinical trial testing the oncolytic adenovirus Delta-24-RGD (DNX-2401) in patients with recurrent malignant gliomas demonstrated significant clinical benefits for a subset of patients, and another phase I trial testing Delta-24-RGD in patients with pediatric brain tumors also indicated similar clinical benefits. However, virus injection in tumors initiates both anti-virus and anti-tumor immune responses by activating respective clones of CD8+ T-cells, which compete for limited resources for clonal expansion. Expansion of T-cells against highly immunogenic viral antigens might limit the expansion of subdominant clones against tumor antigens. We hypothesized that inducing immune tolerance for the dominant viral antigens will allow the expansion of previously subdominant tumor-specific T-cells. In this work, we observed that nanoparticles encapsulating adenoviral antigens successfully induced immune tolerance for viral antigens. These nanoparticles were taken up by liver resident macrophages, which are involved in maintaining peripheral immune tolerance. RNA-sequencing revealed that T-cells recognizing the encapsulated antigens had unique transcriptome signatures with upregulation of genes specifically involved in peripheral immune tolerance, including immune coinhibitory molecules. Virus-specific immune tolerance using nanoparticles redirected the focus of the immune response towards antigens for melanoma as measured by interferon-gamma secretion (P < 0.0001). Reduction of virus-specific T-cells and simultaneous expansion of tumor-specific T-cell clones were confirmed with tetramer staining (P < 0.05). Importantly, virotherapy in combination with nanoparticle-induced immune tolerance towards viral antigens increased the percentage of long-term survivors compared to virus treatment alone (100% versus 50%). Our data provide the basis to develop future clinical trials that aim to maximize the potential of cancer virotherapy in solid tumors.

Diffuse midline glioma (DMG), including those of the brainstem (diffuse intrinsic pontine glioma), are pediatric tumors of the central nervous system (CNS). Recognized as the most lethal of all childhood cancers, palliative radiotherapy remains the only proven treatment option, however, even for those that respond, survival is only temporarily extended. DMG harbor an immunologically “cold” tumor microenvironment (TME) with few infiltrating immune cells. The mechanisms underpinning the cold TME are not well understood. Low expression levels of immune checkpoint proteins, including PD-1, PD-L1, and CTLA-4, are recurring features of DMG and likely contribute to the lack of response to immune checkpoint inhibitors (ICIs). The unique epigenetic signatures (including stem cell-like methylation patterns), a low tumor mutational burden, and recurring somatic mutations (H3K27M, TP53, ACVR1, MYC, and PIK3CA), possibly play a role in the reduced efficacy of traditional immunotherapies. Therefore, to circumvent the lack of efficacy thus far seen for the use of ICIs, adoptive cell transfer (including chimeric antigen receptor T cells) and the use of oncolytic viruses, are currently being evaluated for the treatment of DMG. It remains an absolute imperative that we improve our understanding of DMG’s intrinsic and TME features if patients are to realize the potential benefits offered by these sophisticated treatments. Herein, we summarize the limitations of immunotherapeutic approaches, highlight the emerging safety and clinical efficacy shown for sophisticated cell-based therapies, as well as the evolving knowledge underpinning the DMG-immune axis, to guide the development of immunotherapies that we hope will improve outcomes.

BACKGROUND: Pediatric patients with diffuse intrinsic pontine glioma (DIPG) have a poor prognosis, with a median survival of less than 1 year. Oncolytic viral therapy has been evaluated in patients with pediatric gliomas elsewhere in the brain, but data regarding oncolytic viral therapy in patients with DIPG are lacking. METHODS: We conducted a single-center, dose-escalation study of DNX-2401, an oncolytic adenovirus that selectively replicates in tumor cells, in patients with newly diagnosed DIPG. The patients received a single virus infusion through a catheter placed in the cerebellar peduncle, followed by radiotherapy. The primary objective was to assess the safety and adverse-event profile of DNX-2401. The secondary objectives were to evaluate the effect of DNX-2401 on overall survival and quality of life, to determine the percentage of patients who have an objective response, and to collect tumor-biopsy and peripheral-blood samples for correlative studies of the molecular features of DIPG and antitumor immune responses. RESULTS: A total of 12 patients, 3 to 18 years of age, with newly diagnosed DIPG received 1×10(10) (the first 4 patients) or 5×10(10) (the subsequent 8 patients) viral particles of DNX-2401, and 11 received subsequent radiotherapy. Adverse events among the patients included headache, nausea, vomiting, and fatigue. Hemiparesis and tetraparesis developed in 1 patient each. Over a median follow-up of 17.8 months (range, 5.9 to 33.5), a reduction in tumor size, as assessed on magnetic resonance imaging, was reported in 9 patients, a partial response in 3 patients, and stable disease in 8 patients. The median survival was 17.8 months. Two patients were alive at the time of preparation of the current report, 1 of whom was free of tumor progression at 38 months. Examination of a tumor sample obtained during autopsy from 1 patient and peripheral-blood studies revealed alteration of the tumor microenvironment and T-cell repertoire. CONCLUSIONS: Intratumoral infusion of oncolytic virus DNX-2401 followed by radiotherapy in pediatric patients with DIPG resulted in changes in T-cell activity and a reduction in or stabilization of tumor size in some patients but was associated with adverse events. (Funded by the European Research Council under the European Union's Horizon 2020 Research and Innovation Program and others; EudraCT number, 2016-001577-33; ClinicalTrials.gov number, NCT03178032.).