Malignant gliomas, including glioblastoma (GBM) as the most aggressive type of adult CNS tumors, are notoriously resistant to current standard of care treatments, including surgery, systemic chemotherapy, and radiation therapy (RT). This lack of effective treatment options highlights the urgent need for novel therapies, including immunotherapies. The overarching goal of immunotherapy is to stimulate and activate the patient's immune system in a targeted manner to kill tumor cells. The success of immunotherapeutic interventions in other cancer types has led to interest in and evaluation of various experimental immunotherapies in patients with malignant gliomas. However, these primary malignant brain tumors present a challenge because they exist in a vital and sensitive organ with a unique immune environment. The challenges and current status of experimental immunotherapeutic approaches, including vaccines, immune-checkpoint blockade, chimeric antigen receptor T-cell therapy, and oncolytic viruses will be discussed, as well as the potential for combinatorial therapies.

Glioblastoma (GBM) is the most common primary malignant brain tumor, with a median overall survival of less than 2 years and a nearly 100% mortality rate under standard therapy that consists of surgery followed by combined radiochemotherapy. Therefore, new therapeutic strategies are urgently needed. The success of chimeric antigen receptor (CAR) T cells in hematological cancers has prompted preclinical and clinical investigations into CAR-T-cell treatment for GBM. However, recent trials have not demonstrated any major success. Here, we delineate existing challenges impeding the effectiveness of CAR-T-cell therapy for GBM, encompassing the cold (immunosuppressive) microenvironment, tumor heterogeneity, T-cell exhaustion, local and systemic immunosuppression, and the immune privilege inherent to the central nervous system (CNS) parenchyma. Additionally, we deliberate on the progress made in developing next-generation CAR-T cells and novel innovative approaches, such as low-intensity pulsed focused ultrasound, aimed at surmounting current roadblocks in GBM CAR-T-cell therapy.

BACKGROUND: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade. METHODS: An IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade. RESULTS: The HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone. CONCLUSION: The development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.

Background

Rigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART) cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII).

Methods

We first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T cells and evaluated the function of the CART cells both in vitro and in vivo. To inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208).

Results

Both RV- and Tg-CART cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28.

Conclusion

Our new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies.