A New Immune-Stimulating Gene Therapy for High-Grade Glioma Brain Tumors

Advances in technology and knowledge are enabling new, effective treatments to be developed every day. In September 2023, collaborative work by Yoshie Umemura and many other scientists introduced a promising new treatment approach. The study targets high-grade gliomas, which are common in adults and have a poor prognosis.

Virus-Mediated Gene Therapy

New gene therapies developed for high-grade gliomas have used viral vectors (carrier viruses) determined to be safe for humans.

However, challenges include the failure to kill cancer cells effectively at the treatment site or to distribute genetic material sufficiently. This prevents the approach from stopping malignant cells from infiltrating other areas.

This clinical study at Michigan Medical School combines the enzymes herpes simplex virus type 1 thymidine kinase (HSV1-TK) and FMS-like tyrosine kinase 3 ligand (Flt3L). HSV1-TK phosphorylates the prodrug valacyclovir, converting it into a nucleoside analog that causes cell death in dividing cells, as in high-grade gliomas. Flt3L recruits dendritic cells into the tumor microenvironment .

Combining Flt3L and HSV1-TK brings dendritic cells into the tumor microenvironment and increases endogenous glioma antigens. These antigens are released through tumor-cell cytotoxicity caused by HSV1-TK. The released antigens are activated together with the HMGB1 enzyme, a TLR2 agonist, and dendritic cells induce a specific anti-glioma immune response against malignant glioma cells remaining after surgical excision.

In preclinical animal studies, this approach demonstrated a cytotoxic T-cell response, acquisition of immune memory, and destruction of much of the tumor. Although the safety of dual-vector treatment has been demonstrated in animals, it has not yet been established in humans.

The hypothesis of the study published in September was that HSV1-TK and Flt3L could be coadministered as adenoviruses under CMV promoter control after tumor removal in patients with newly diagnosed high-grade glioma, then safely combined with standard oral valacyclovir chemoradiotherapy. The combination was expected to generate an immune response against high-grade glioma.

The primary objective was to identify the maximum tolerated dose of Ad-hCMV-TK and Ad-hCMV-Flt3L. Secondary objectives were to assess the potential benefit of dual-vector therapy by measuring overall survival, and to investigate histopathological indices of inflammatory responses using immune responses in resection samples from a second operation as a biomarker.

Groundbreaking Clinical Research and Initial Results

Designed as a phase 1 study, this clinical trial was open-label, meaning that investigators and patients knew all the procedures being performed. It was conducted at Michigan Medical School in 21 adult patients diagnosed after resection with supratentorial brain lesions consistent with high-grade glioma. They had not previously received gene therapy, chemotherapy, or radiotherapy. Disease was assessed using the immunotherapy response assessment system in neuro-oncology. Among high-grade gliomas, this system included grade 4 glioblastoma multiforme, grade 4 gliosarcomas, grade 3 anaplastic astrocytomas, grade 3 anaplastic oligodendrogliomas, and grade 3 anaplastic ependymomas. Patients' condition was assessed with the Karnofsky Performance Status (KPS), expressed as a number from 0 to 100, where 0 indicates a healthy patient and 100 indicates that the patient has died. All trial participants had KPS scores of 70 or higher.

The trial's viral vectors were manufactured at the Center for Cell and Gene Therapy at Baylor College of Medicine. Patients were to receive injections of 6 different vectors at escalating doses. The first group of 3 patients would receive 1x10^10 Ad-hCMV-TK and 1x10^9 Ad-hCMV-Flt3L. Subsequent doses increased by cohort. The procedure specified dose escalation if none of the 3 patients at a given dose experienced dose-limiting toxicity; if 2 of 3 patients experienced this toxicity, the trial would stop and the next lower dose would be considered the maximum tolerated dose. The starting dose of HSV1-TK was higher because it had been used previously, whereas Flt3L was being used in humans for the first time. Once doses were established, the vectors were injected into the post-resection cavity using 2 tuberculin syringes of 1 mL each, delivering 0.1 mL at each of 20 sites for a total of 2 mL. After vector injection, patients began two 14-day courses of valacyclovir, starting on days 1-3 and at weeks 10-12. Patients received valacyclovir at a dose of 2 grams, 3 times daily. The dose could be modified if circumstances required. Patients began standard chemotherapy 15-35 days after these procedures. Adverse effects were graded using Common Terminology Criteria for Adverse Events (CTCAE) version 4. If any condition in that classification occurred within 21 days of dual-vector injection, it was considered a dose-limiting factor. To identify changes, patients' histories, vital signs, blood results, radiological images, physical examinations, KPS results, and psychiatric examinations were recorded within the 14 days before surgery. Tumor type was also confirmed by intraoperative biopsy, and postoperative MRI determined the extent of resection. Tests were repeated at weeks 2, 4, 6, 8, 10, 12, 14, and 18 for comparison with earlier results. Clinical survival follow-up took place at months 12 and 24. Patients were not included if they refused treatment during the study, did not complete treatment, or did not receive the 2nd valacyclovir course. Patients who withdrew consent or died were also not included.

The study ran from 8 April 2014 to 13 March 2019, with 21 patients initially considered eligible. One was subsequently excluded because of pregnancy and another because metastatic carcinoma was detected. A further patient withdrew voluntarily later in the study, so the dual-vector investigation was completed with 18 patients: 8 women and 10 men. The research ended in 2019, but data collection continued until 14 June 2021. Of the 18 tumors, 17 were glioblastomas and one was an anaplastic ependymoma. All tumors were an aggressive gene type associated with glioblastoma. Six patients experienced adverse effects thought to be treatment-related, including nausea, confusion, and acute kidney disease. None was considered dose-limiting. More serious adverse effects occurred in 10 patients. Among these 10, one developed pneumocystis pneumonia (PCP, a lung infection) and unfortunately died. This death was not attributed to the study. Another patient had bleeding in the tumor bed after surgery and dual-vector administration and was discharged in stable condition after appropriate treatment. The bleeding was not attributed to the study. Despite numerous adverse effects, no dose-limiting factors were identified. However, the maximum dose for Ad-hCMV-TK and Ad-hCMV-Flt3L could not be established. Overall survival was calculated as 21.3 months. In the study, 14 patients died from disease progression, 2 from causes unrelated to the study, and 1 from an undetermined cause. Unfortunately, mortality is high in such an aggressive and lethal cancer. There were nevertheless promising results: survival was 72.2% at 12 months and 38.8% at 24 months. Particularly striking was that 3 of the 18 patients had survived for 4 years, while one patient remained alive 59 months after treatment. These results offer a positive perspective for the future in glioblastoma. Differences between primary and recurrent tumors were examined by immunohistochemistry in 8 patients. Although CD45+ cells did not differ significantly, CD3e+ cells (T cells) and CD8+ cells increased significantly. Regulatory T cells in the recurrent tumor microenvironment decreased in 6 of these 8 patients. Cell-to-cell interactions between several T-cell subgroups and macrophages increased in recurrent tumors, while interactions between tumor cells and macrophages decreased. Regional analysis found increased density of immune cells, including T cells, double-positive T cells, and plasmacytoid dendritic cells, within the recurrent tumor region compared with other areas. Analyses in 5 of 8 patients also found increased HSV1-TK cells in recurrent tumors. In one patient, effects were observed even 17 months after injection. Serum concentration analysis showed that Flt3L increased significantly 2 weeks after treatment compared with baseline.

The Importance of the Phase 1 Study and Future Expectations

The maximum tolerated dose of the HSV1-TK and Flt3L vector combination was not reached in this phase 1 study. Although adverse effects thought to be caused by valacyclovir were observed, the study was too small to determine whether valacyclovir's contribution to overall toxicity was beneficial or harmful. Some survival rates were obtained, but the study was insufficient for survival analysis, making further studies necessary. Future work could examine adding immune checkpoint blockade and reducing immunosuppressive factors in the tumor microenvironment, potentially producing much more effective results. The increase in blood antibodies after vector administration suggests that injecting adenoviral vectors into the resection site can initiate or enhance a systemic humoral immune response. Analyses showed increased numbers of CD8+ T cells and plasmacytoid dendritic cells. This increase is thought to help patients generate an effective anti-glioma immune response. Larger phase 2 or phase 3 trials are needed to confirm this important hypothesis. Cell-neighborhood analyses were performed by quantifying immune-cell subsets. The subset proportions differed from those in other studies, and immune infiltration occurred in areas dense with tumor cells, both within and around the tumor. This difference is thought to result from vector administration. As reported in 2021, the study also indicates that immunosuppressive macrophages may reduce the effect of increased CD8+ T cells and plasmacytoid dendritic cells. Using viral vectors for transgene expression and immune infiltration is also thought to turn cold tumors, lacking immune and inflammatory cells, into hot tumors enriched with these cells. This study demonstrated that this transition occurred.

Conclusion and Future Vision

In conclusion, this important study investigated valacyclovir administration while demonstrating dual-vector use and the distribution of dendritic cells in the tumor microenvironment. The findings suggest that valacyclovir's applications could be expanded and that adenoviral vector-mediated transgene expression can persist for up to 17 months. The promising survival data and evidence of immune-cell recruitment indicate that this treatment has significant future potential. Large-scale phase 2 and phase 3 trials are needed to enhance its effects and obtain more detailed, definitive results.