Intratumoral DNX-2401 for High Grade Pediatric Brain Tumors

Tumors of the central nervous system (CNS) represent approximately 20% of childhood cancers and are the leading cause of cancer-related mortality in children. Pediatric high-grade CNS tumors, including gliomas, ependymomas and embryonal CNS tumors (such as medulloblastomas, atypical teratoid/rhabdoid tumors [AT/RT], and embryonal tumors with multilayered rosettes [ETMR]), are associated with a particularly poor prognosis, with median survival after diagnosis often below two years. Despite current conventional treatments, the outcome has not improved, and significant morbidity with impaired quality of life is frequent in survivors. This scenario is even more discouraging at the time of tumor recurrence, where curative options are nonexistent. Therefore, there is a clear need to find novel and alternative therapeutic approaches to improve both survival and quality of life of these children.

In the past years, oncolytic virotherapy has emerged as a promising therapeutic strategy, as it replicates in and destroys only tumor cells while activating the patient's immune system. Among the described oncolytic viruses, DNX-2401, a genetically modified adenovirus type 5, has shown an excellent therapeutic effect in murine models of pediatric high-grade brain tumors, as well as in children with newly diagnosed DIPG. Preclinical studies have demonstrated that Delta-24-RGD, both alone and in combination with radiotherapy, is safe and results in a significant increase in survival in immunodeficient and immunocompetent murine models of pediatric high-grade gliomas, including DIPGs. Moreover, this therapeutic benefit was also validated in AT/RT and other embryonal CNS tumors, resulting in 70% of long-term survivors and a reduction in the dissemination of these tumors. In the clinical setting, intratumoral administration of DNX-2401 has demonstrated to be safe and effective in several phase 1 and 2 clinical trials in adult patients with recurrent high-grade gliomas, both alone and with pembrolizumab, and in children with newly diagnosed DIPG in combination with radiotherapy.

This is an open-label phase 2 trial for pediatric and young adults patients, aged 1 to 25 years old, with three different types of high-grade brain tumors (high-grade gliomas including Diffuse Midline Gliomas, embryonal CNS tumors, and ependymomas), who have relapsed (i.e., tumors that return after treatment) or are refractory (i.e., do not respond to conventional treatment).

A Simon 2-stage optimal design will be used to maximize the probability of detecting the treatment's effectiveness, reducing the probability of continuing with it if it is not effective in the first stage. This statistical methodology will allow testing the null hypothesis that the true response rate is ≤20% versus the alternative hypothesis that the true response rate is ≥50%. A sample size of 13 patients per study group will be predetermined. A response (defined as complete response, partial response, or stable disease for at least 12 weeks) in at least 1 of 5 patients per study group in the first stage will be required for the study to be expanded to the second stage, and a response in 5 of 13 patients will be required to consider the treatment statistically positive.

The study participants will receive a single intratumoral dose of 5 x 10^10 viral particles of DNX-2401 in 1 mL of 0.9% sodium chloride, administered in the brain parenchyma via a SmartFlow Neuro Ventricular Cannula (ClearPoint Neuro) with neuronavigation guidance at a controlled rate over approximately 1 hour (67 minutes at a rate of 0.90 mL/hour). Before virus delivery, a stereotactic biopsy will be performed to obtain tumor tissue.

Clinical follow-up, including neurological examination, functional status and quality of life assessment will be performed at day 3 and during clinical visits at weeks 2, 4, 8, 12, 20, 28, 40 and 52 following DNX-2401 injection. Adverse events will be recorded using the National Cancer Institute - Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5 tool for the first 12 weeks after experimental therapy. Postoperative MRI will be performed in all patients within 72 hours after surgery to detect the biopsy region and the DNX-2401 injection site. In the case of the preoperative Magnetic Resonnace Imaging (MRI), it will be considered the baseline MRI for response assessment, except in patients undergoing debulking surgery, in which early postoperative MRI will be considered the baseline MRI. Follow-up MRI will be performed at weeks 4, 8, 12, 20, 28, 40 and 52 following DNX-2401 injection.

Apart from the overall response rate, the treatment efficacy will also be measured in terms of survival, specifically using the following metrics: progression free survival (PFS) and overall survival (OS). PFS will be defined as the time from treatment to disease progression, determined either clinically or radiologically. OS will be defined as the time from treatment to patient death. Rates of PFS at 6- and 12-months will also be assessed as measures of outcome.

Tumor samples will be analyzed to characterize the immune tumor microenvironment and molecular composition. Blood, cerebrospinal fluid (when available), and microbiome samples will be collected longitudinally during follow-up visits to characterize viral kinetics and immune responses before and after DNX-2401 administration, and to explore correlations with clinical outcomes.

Inclusion criteria

• The participant or participant's parents or legally acceptable representatives (if applicable) provides written informed consent for the trial and the pediatric participant provides writ-ten assent, where applicable, based on age and country requirements.
• Patients with recurrent or refractory high grade malignant brain tumors (high grade glioma, embryonal CNS tumors [medulloblastomas, ATRT, EMTR, pineoblastoma], and ependymomas) diagnosis based on initial histopathological diagnosis and further clinical and radiological fol-low-up, in whom gross total resection is not feasible, and with a life expectancy of at least 16 weeks at the time of consent.
• Recurrences within the radiation field will be considered if there is confirmed growth of the lesion in two consecutive MRI or if they occur at least 12 weeks after completion of ra-diation therapy, or if there is clear histopathological confirmation of tumor recurrence.
• Male and female participants age ≥ 1 years and ≤ 25 years.
• A single measurable lesion longer than 10 mm in two perpendicular diameters, considered by the investigator to be accessible for safe stereotactic biopsy and virus injection.
• Lansky Performance Status (LPS) ≥ 60 for participants < 16 years, or Karnofsky Performance Status (KPS) ≥ 60 for participants ≥ 16 years.
• No other chemotherapy or immunotherapy in the 4 weeks before inclusion.
• Steroids: free off or requiring decreasing or stable corticosteroid dose (≥ 0.05 mg/kg dexa-methasone daily, or equivalent for other steroids) in the 2 weeks before DNX-2401 admin-istration.
• Radiation: no craniospinal irradiation, total body irradiation nor focal irradiation in the 6 weeks before inclusion.
• Autologous Stem Cell Transplantation: patients must be ≥ 3 months post-transplant prior to entry of the study.
• Patients must be fully recovered from all acute treatment related toxicities of all prior therapies.
• Laboratory test: adequate hematological (platelets ≥ 100x10e9/L, neutrophils ≥ 1.0x10e9/L, hemoglobin ≥ 5,6 mmol/L), renal function (creatinine <1.5 times ULN) and liver function (≤3 times ULN) values.
• Negative pregnancy test for female participants of child-bearing potential, where child-bearing potential is defined as a fertile female who is pubertal or post-pubertal and not permanently sterile (hysterectomy, bilateral salpingectomy, bilateral oophorectomy).
• Female participants of child-bearing potential, who are sexually active, agree to use ac-ceptable birth control starting at informed consent and continuing for at least 120 days af-ter DNX-2401 administration. Male participants agree to use acceptable birth control start-ing at informed consent and continuing for at least 90 days after DNX-2401 administration.

Exclusion criteria

• Any medical or psychological condition or disease that might interfere with the subject's ability to participate or give informed consent (if older than 16 years).
• Spinal location, or lesions considered risky for stereotactic injection of virus or that might favor entrance of the virus in the ventricular system.
• Any treatment outside the allowable guidelines outlined in the inclusion criteria.
• Severe acute infection or intercurrent medical condition including, but not limited to se-vere renal, hepatic, heart or bone marrow failure that based on investigator discretion do not permit inclusion in the study.
• Subjects with immunodeficiency or autoimmune conditions, active hepatitis or known HIV. No testing for Hepatitis B, Hepatitis C and HIV is required unless mandated by local health authority.
• Subjects with another primary malignancy.
• Prior history of encephalitis, multiple sclerosis or other CNS infections or primary CNS dis-ease that would interfere with evaluation.
• Li Fraumeni Syndrome or a known germ line deficit in the retinoblastoma gene or its relat-ed pathways.
• Concurrent therapy with any antiviral drug or any immunosuppressive drug (except ster-oids).
• Life or life-attenuated vaccinations within 30 days prior to DNX-2401 administration and while participating in the study. Killed vaccines are permitted.
• Prior participant in experimental viral therapy.
• Inability to undergo MRI scans for any reason.
• Pregnancy or breastfeeding.