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Triple Immunotherapy Completely Eradicates Glioma in Animal Models

Key findings

  • In glioma-bearing mice, dual immunotherapy that combined anti–PD-1 immunotherapy with either whole tumor cell vaccination and agonist anti-OX40 immunotherapy improved survival compared with using any of those approaches alone
  • Dual immunotherapy achieved long-term survival for 30% to 50% of the animals
  • Triple immunotherapy with vaccination, anti–PD-1 antibody and agonist anti-OX40 led to long-term survival in 100% of glioma-bearing mice
  • Triple immunotherapy augmented systemic Th1 immune responses to tumor and shifted the CD4+/CD8+ T cell ratio within the tumor microenvironment toward Th1 immunity

For the treatment of malignant glioblastoma, initial results from several high-profile trials of vaccination and immune checkpoint inhibition have been negative.

A team led by William T. Curry, MD, co-director of Mass General Neuroscience, believes no single-agent immunotherapy is likely to overcome the multiple types of immunosuppression associated with glioblastoma. In Oncoimmunology, they report that triple combination immunotherapy with whole tumor cell vaccination, a PD-1 inhibitor and an agonist anti-OX40 antibody cured all glioma-bearing mice treated and was associated with long-term survival.

Dual Immunotherapy

The researchers started by exploring dual combination immunotherapy. They injected glioma cells into the brains of wild-type C57BL/6 mice, then treated them with an anti-PD-1 antibody plus whole tumor cell vaccination (GVAX). Vaccination consisted of subcutaneous implantation of irradiated glioma cells engineered to express granulocyte macrophage colony-stimulating factor.

The combination therapy led to long-term survival (at least 120 days) in 50% of mice, compared with about 20% of mice treated with either component as monotherapy.

Next, the researchers combined the PD-1 inhibitor with an agonist anti-OX40 monoclonal antibody. They explain that OX40 receptor activation can induce proliferation of memory T cells, render effector T cells insensitive to suppression by regulatory T cells and directly inhibit the suppressive capacity of regulatory T cells.

Dual immunotherapy also improved survival when compared with its components used alone, although not always significantly so. It was associated with a 30%-40% cure rate.

Triple Immunotherapy

In previous studies, researchers had found that a third combination of dual immunotherapy, GVAX plus agonist anti-OX40, was significantly more effective than its single components. This accumulation of positive data prompted them to study triple immunotherapy: simultaneous delivery of GVAX, anti–PD-1 antibody and agonist anti-OX40 on days 3, 6 and 9 after intracranial injection of glioma cells.

Triple immunotherapy led to complete eradication of glioma and long-term survival in all seven mice treated. Splenocytes from those animals expressed significantly higher amounts of interferon gamma and interleukin-2 than those from other treatment groups, indicating a stronger systemic Th1 immune response.

Furthermore, triple immunotherapy increased the CD4+/CD8+ T cell ratio within the tumor microenvironment by a factor of 3.7 compared with baseline, nearly double the increase in any other treatment group. This ratio is a biomarker for the vigor of a Th1 immune response, so the increase suggests intratumoral shifting toward Th1 immunity.

Mice that had been cured with triple immunotherapy or with anti–PD-1 plus agonist anti-OX40 were rechallenged with glioma cells at five-times higher load, 120 days after the initial injection. All of them continued to survive, whereas age-matched tumor-naïve mice died within 25 days. After sacrifice, there was visibly less tumor volume in the brains of triple-treated mice than in those treated with anti–PD-1 plus agonist anti-OX40.

Implications for Further Research

Dr. Curry's group describes "a likely oversimplified, but reasonable" summary of the mechanism of the triple therapy: GVAX expands the number and diversity of activated tumor-specific T cells and increases the number of intratumor CD8+ T cells, while PD-1 inhibition further invigorates the effects of those mobilized cells. Meanwhile, agonist anti-OX40 continues to skew systemic and tumor microenvironments toward Th1 immunity, suppress regulatory T cells and prevent T cell exhaustion.

T cell response is not the only arm of the immune system impaired in glioblastoma. Tumor-associated macrophages, myeloid-derived suppressor cells and innate immunity are also involved and may need to be engaged. Still, tumor-specific activated CD8+ T cells, whose activity is promoted by activated CD4+ Th1 cells, have the ultimate tumoricidal impact.

The glioma cells used (GL261) have responded to a variety of immunotherapeutic approaches in previous research; however, the complete eradication of glioma as seen in this study has been rare. These results underscore the likelihood that glioma immunotherapy should be multipronged.

Learn more about the Department of Neurosurgery at Mass General

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Mass General has developed a genotype-targeted therapy for glioma, designed to be given at the tumor site during surgery, and an accompanying rapid diagnostic.


Immunotherapy for glioma is under active investigation, but tumor-associated immunosuppression is a major obstacle. A Mass General team studied an investigational monoclonal antibody in mice with glioma, alone and in combination with tumor cell vaccination.