The oncolytic adenovirus Delta-24-RGD in combination with ONC201 induces a potent antitumor response in pediatric high-grade and diffuse midline glioma models.

BACKGROUND: Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated. METHODS: The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq, and multiplexed immunofluorescence staining. RESULTS: The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype. CONCLUSIONS: The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone.

Radiotherapy protocols for mouse cancer model.

Radiotherapy is a crucial treatment modality for cancer patients, with approximately 60% of individuals undergoing ionizing radiation as part of their disease management. In recent years, there has been a growing trend toward minimizing irradiation fields through the use of image-guided dosimetry and innovative technologies. These advancements allow for selective irradiation, delivering higher local doses while reducing the number of treatment sessions. Consequently, computer-assisted methods have significantly enhanced the effectiveness of radiotherapy in the curative and palliative treatment of various cancers. Although radiation therapy alone can effectively achieve local control in some cancer types, it may not be sufficient for others. As a result, further preclinical research is necessary to explore novel approaches including new schedules of radiotherapy treatments. Unfortunately, there is a concerning lack of correlation between clinical outcomes and experiments conducted on mouse models. We hypothesize that this disparity arises from the differences in irradiation strategies employed in preclinical studies compared to those used in clinical practice, which ultimately affects the translatability of findings to patients. In this study, we present two comprehensive radiotherapy protocols for the treatment of orthotopic melanoma and glioblastoma tumors. These protocols utilize a small animal radiation research platform, which is an ideal radiation device for delivering localized and precise X-ray doses to the tumor mass. By employing these platforms, we aim to limit the side effects associated with irradiating healthy surrounding tissues. Our detailed protocols offer a valuable framework for conducting preclinical studies that closely mimic clinical radiotherapy techniques, bridging the gap between experimental results and patient outcomes.

TIM-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain stem tumor and the leading cause of pediatric cancer-related death. To date, these tumors remain incurable, underscoring the need for efficacious therapies. In this study, we demonstrate that the immune checkpoint TIM-3 (HAVCR2) is highly expressed in both tumor cells and microenvironmental cells, mainly microglia and macrophages, in DIPG. We show that inhibition of TIM-3 in syngeneic models of DIPG prolongs survival and produces long-term survivors free of disease that harbor immune memory. This antitumor effect is driven by the direct effect of TIM-3 inhibition in tumor cells, the coordinated action of several immune cell populations, and the secretion of chemokines/cytokines that create a proinflammatory tumor microenvironment favoring a potent antitumor immune response. This work uncovers TIM-3 as a bona fide target in DIPG and supports its clinical translation.

Galectin-3 inhibition boosts the therapeutic efficacy of Semliki Forest virus in pediatric osteosarcoma.

The outcomes of metastatic and nonresponder pediatric osteosarcoma patients are very poor and have not improved in the last 30 years. These tumors harbor a highly immunosuppressive environment, making existing immunotherapies ineffective. Here, we evaluated the use of Semliki Forest virus (SFV) vectors expressing galectin-3 (Gal3) inhibitors as therapeutic tools, since both the inhibition of Gal3, which is involved in immunosuppression and metastasis, and virotherapy based on SFV have been demonstrated to reduce tumor progression in different tumor models. In vitro, inhibitors based on the Gal3 amino-terminal domain alone (Gal3-N) or fused to a Gal3 peptide inhibitor (Gal3-N-C12) were able to block the binding of Gal3 to the surface of activated T cells. In vivo, SFV expressing Gal3-N-C12 induced strong antitumor responses in orthotopic K7M2 and MOS-J osteosarcoma tumors, leading to complete regressions in 47% and 30% of mice, respectively. Pulmonary metastases were also reduced in K7M2 tumor-bearing mice after treatment with SFV-Gal3-N-C12. Both the antitumor and antimetastatic responses were dependent on modulation of the immune system, primarily including an increase in tumor-infiltrating lymphocytes and a reduction in the immunosuppressive environment inside tumors. Our results demonstrated that SFV-Gal3-N-C12 could constitute a potential therapeutic agent for osteosarcoma patients expressing Gal3.

Oncolytic DNX-2401 Virus for Pediatric Diffuse Intrinsic Pontine Glioma.

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 1×1010 (the first 4 patients) or 5×1010 (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.).

Exploiting 4-1BB immune checkpoint to enhance the efficacy of oncolytic virotherapy for diffuse intrinsic pontine gliomas.

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors, and patient survival has not changed despite many therapeutic efforts, emphasizing the urgent need for effective treatments. Here, we evaluated the anti-DIPG effect of the oncolytic adenovirus Delta-24-ACT, which was engineered to express the costimulatory ligand 4-1BBL to potentiate the antitumor immune response of the virus. Delta-24-ACT induced the expression of functional 4-1BBL on the membranes of infected DIPG cells, which enhanced the costimulation of CD8+ T lymphocytes. In vivo, Delta-24-ACT treatment of murine DIPG orthotopic tumors significantly improved the survival of treated mice, leading to long-term survivors that developed immunological memory against these tumors. In addition, Delta-24-ACT was safe and caused no local or systemic toxicity. Mechanistic studies showed that Delta-24-ACT modulated the tumor-immune content, not only increasing the number, but also improving the functionality of immune cells. All of these data highlight the safety and potential therapeutic benefit of Delta-24-ACT the treatment of patients with DIPG.

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.

CD137 and PD-L1 targeting with immunovirotherapy induces a potent and durable antitumor immune response in glioblastoma models.

BACKGROUND: Glioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising strategy for these tumors. Our group constructed a new OV named Delta-24-ACT, which was based on the Delta-24-RGD platform armed with 4-1BB ligand (4-1BBL). In this study, we evaluated the antitumor effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor (ICI) in preclinical models of glioma. METHODS: The in vitro effect of Delta-24-ACT was characterized through analyses of its infectivity, replication and cytotoxicity by flow cytometry, immunofluorescence (IF) and MTS assays, respectively. The antitumor effect and therapeutic mechanism were evaluated in vivo using several immunocompetent murine glioma models. The tumor microenvironment was studied by flow cytometry, immunohistochemistry and IF. RESULTS: Delta-24-ACT was able to infect and exert a cytotoxic effect on murine and human glioma cell lines. Moreover, Delta-24-ACT expressed functional 4-1BBL that was able to costimulate T lymphocytes in vitro and in vivo. Delta-24-ACT elicited a more potent antitumor effect in GBM murine models than Delta-24-RGD, as demonstrated by significant increases in median survival and the percentage of long-term survivors. Furthermore, Delta-24-ACT modulated the tumor microenvironment, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by increases in the expression of Programmed Death 1 (PD-1) on T cells and Programmed Death-ligand 1 (PD-L1) on different myeloid cell populations. Because Delta-24-ACT did not induce an immune memory response in long-term survivors, as indicated by rechallenge experiments, we combined Delta-24-ACT with an anti-PD-L1 antibody. In GL261 tumor-bearing mice, this combination showed superior efficacy compared with either monotherapy. Specifically, this combination not only increased the median survival but also generated immune memory, which allowed long-term survival and thus tumor rejection on rechallenge. CONCLUSIONS: In summary, our data demonstrated the efficacy of Delta-24-ACT combined with a PD-L1 inhibitor in murine glioma models. Moreover, the data underscore the potential to combine local immunovirotherapy with ICIs as an effective therapy for poorly infiltrated tumors.

Local administration of IL-12 with an HC vector results in local and metastatic tumor control in pediatric osteosarcoma.

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.

Delta-24-RGD, an Oncolytic Adenovirus, Increases Survival and Promotes Proinflammatory Immune Landscape Remodeling in Models of AT/RT and CNS-PNET.

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.

The oncolytic adenovirus VCN-01 promotes anti-tumor effect in primitive neuroectodermal tumor models.

Last advances in the treatment of pediatric tumors has led to an increase of survival rates of children affected by primitive neuroectodermal tumors, however, still a significant amount of the patients do not overcome the disease. In addition, the survivors might suffer from severe side effects caused by the current standard treatments. Oncolytic virotherapy has emerged in the last years as a promising alternative for the treatment of solid tumors. In this work, we study the anti-tumor effect mediated by the oncolytic adenovirus VCN-01 in CNS-PNET models. VCN-01 is able to infect and replicate in PNET cell cultures, leading to a cytotoxicity and immunogenic cell death. In vivo, VCN-01 increased significantly the median survival of mice and led to long-term survivors in two orthotopic models of PNETs. In summary, these results underscore the therapeutic effect of VCN-01 for rare pediatric cancers such as PNETs, and warrants further exploration on the use of this virus to treat them.

The oncolytic virus Delta-24-RGD elicits an antitumor effect in pediatric glioma and DIPG mouse models.

Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors in desperate need of a curative treatment. Oncolytic virotherapy is emerging as a solid therapeutic approach. Delta-24-RGD is a replication competent adenovirus engineered to replicate in tumor cells with an aberrant RB pathway. This virus has proven to be safe and effective in adult gliomas. Here we report that the administration of Delta-24-RGD is safe in mice and results in a significant increase in survival in immunodeficient and immunocompetent models of pHGG and DIPGs. Our results show that the Delta-24-RGD antiglioma effect is mediated by the oncolytic effect and the immune response elicited against the tumor. Altogether, our data highlight the potential of this virus as treatment for patients with these tumors. Of clinical significance, these data have led to the start of a phase I/II clinical trial at our institution for newly diagnosed DIPG (NCT03178032).

Assessment of metabolic patterns and new antitumoral treatment in osteosarcoma xenograft models by [18F]FDG and sodium [18F]fluoride PET.

BACKGROUND: Osteosarcoma is the most common malignant bone tumor in children and young adults that produces aberrant osteoid. The aim of this study was to assess the utility of 2-deoxy-2-[18F-] fluoro-D-glucose ([18F] FDG) and sodium [18F] Fluoride (Na [18F] F) PET scans in orthotopic murine models of osteosarcoma to describe the metabolic pattern of the tumors, to detect and diagnose tumors and to evaluate the efficacy of a new treatment based in oncolytic adenoviruses. METHODS: Orthotopic osteosarcoma murine models were created by the injection of 143B and 531MII cell lines. [18F]FDG and Na [18F] F PET scans were performed 30 days (143B) and 90 days (531MII) post-injection. The antitumor effect of two doses (107 and 108 pfu) of the oncolytic adenovirus VCN-01 was evaluated in 531 MII model by [18F] FDG PET studies. [18F] FDG uptake was quantified by SUVmax and Total Lesion Glycolysis (TLG) indexes. For Na [18F] F, the ratio tumor SUVmax/hip SUVmax was calculated. PET findings were confirmed by histopathological techniques. RESULTS: The metabolic pattern of tumors was different between both orthotopic models. All tumors showed [18F] FDG uptake, with a sensitivity and specificity of 100%. The [18F] FDG uptake was significantly higher for the 143B model (p < 0.001). Sensitivity for Na [18F] F was around 70% in both models, with a specificity of 100%. 531MII tumors showed a heterogeneous Na [18F] F uptake, significantly higher than 143B tumors (p < 0.01). Importantly, [18F] FDG and Na [18F] F uptake corresponded to highly cellular or osteoid-rich tumors in the histopathological analysis, respectively. [18F] FDG data confirmed that the oncolytic treatment of 531MII tumors produced a significant reduction in growth even with the 107 pfu dose. CONCLUSIONS: PET studies demonstrated that the different osteosarcoma xenograft models developed tumors with diverse metabolic patterns that can be described by multitracer PET studies. Since not all tumors produced abundant osteoid, [18F] FDG demonstrated a better sensitivity for tumor detection and was able to quantitatively monitor in vivo response to the oncolytic adenovirus VCN-01.

The Oncolytic Adenovirus VCN-01 as Therapeutic Approach Against Pediatric Osteosarcoma.

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.

The oncolytic adenovirus Δ24-RGD in combination with cisplatin exerts a potent anti-osteosarcoma activity.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. The presence of metastases and the lack of response to conventional treatment are the major adverse prognostic factors. Therefore, there is an urgent need for new treatment strategies that overcome both of these problems. Our purpose was to elucidate whether the use of the oncolytic adenovirus Δ24-RGD alone or in combination with standard chemotherapy would be effective, in vitro and in vivo, against osteosarcoma. Our results showed that Δ24-RGD exerted a potent antitumor effect against osteosarcoma cell lines that was increased by the addition of cisplatin. Δ24-RGD osteosarcoma treatment resulted in autophagy in vitro that was further enhanced when combined with cisplatin. Of importance, administration of Δ24-RGD and/or cisplatin, in novel orthotopic and two lung metastatic models in vivo resulted in a significant reduction of tumor burden meanwhile maintaining a safe toxicity profile. Together, our data underscore the potential of Δ24-RGD to become a realistic therapeutic option for primary and metastatic pediatric osteosarcoma. Moreover, this study warrants a future clinical trial to evaluate the safety and efficacy of Δ24-RGD for this devastating disease.

Methotrexate in pediatric osteosarcoma: response and toxicity in relation to genetic polymorphisms and dihydrofolate reductase and reduced folate carrier 1 expression.

OBJECTIVE: To determine the influence of the genotype and the level of expression of different enzymes involved in folate metabolism on the response to and toxicity of high-dose methotrexate treatment in pediatric osteosarcomas. STUDY DESIGN: DHFR and Reduced folate carrier 1 (RFC1) semiquantitative expression was analyzed in 34 primary and metastatic osteosarcoma tissues by real-time polymerase chain reaction. The following polymorphisms were also analyzed in peripheral blood from 96 children with osteosarcoma and 110 control subjects: C677T, A1298C (MTHFR), G80A (RFC1), A2756G (MTR), C1420T (SHMT), the 28bp-repeat polymorphism, and 1494del6 of the TYMS gene. Treatment toxicity was scored after each cycle according to criteria from the World Health Organization. RESULTS: DHFR and RFC1 expression was lower in initial osteosarcoma biopsy specimens than in metastases (P = .024 and P = .041, respectively). RFC1 expression was moderately decreased in samples with poor histologic response to preoperative treatment (P = .053). Patients with osteosarcoma with G3/G4 hematologic toxicity were more frequently TT than CT/CC for C677T/MTHFR (P = .023) and GG for A2756G/MTR (P = .048 and P = .057 for gastrointestinal and hematologic toxicity, respectively). CONCLUSIONS: The role of C677T/MTHFR and A2756G/MTR on chemotherapy-induced toxicity should be further investigated in pediatric osteosarcomas receiving high-dose methotrexate. Altered expression of DHFR and RFC1 is a feasible mechanism by which osteosarcoma cells become resistant to methotrexate.