BACKGROUND. Patients with diffuse midline gliomas (DMGs), including diffuse intrinsic pontine glioma (DIPG), have dismal outcomes. We previously described the H3.3K27M mutation as a shared neoantigen in HLA-A*02.01⁺, H3.3K27M⁺ DMGs. Within the Pacific Pediatric Neuro-Oncology Consortium, we assessed the safety and efficacy of an H3.3K27M-targeted peptide vaccine. METHODS. Newly diagnosed patients, aged 3–21 years, with HLA-A*02.01⁺ and H3.3K27M⁺ status were enrolled in stratum A (DIPG) or stratum B (nonpontine DMG). Vaccine was administered in combination with polyinosinic-polycytidylic acid-poly-I-lysine carboxymethylcellulose (poly-ICLC) every 3 weeks for 8 cycles, followed by once every 6 weeks. Immunomonitoring and imaging were performed every 3 months. Imaging was centrally reviewed. Immunological responses were assessed in PBMCs using mass cytometry. RESULTS. A total of 19 patients were enrolled in stratum A (median age,11 years) and 10 in stratum B (median age, 13 years). There were no grade-4 treatment-related adverse events (TRAEs). Injection site reaction was the most commonly reported TRAE. Overall survival (OS) at 12 months was 40% (95% CI, 22%–73%) for patients in stratum A and 39% (95% CI, 16%–93%) for patients in stratum B. The median OS was 16.1 months for patients who had an expansion of H3.3K27M-reactive CD8⁺ T cells compared with 9.8 months for their counterparts (P = 0.05). Patients with DIPG with below-median baseline levels of myeloid-derived suppressor cells had prolonged OS compared with their counterparts (P < 0.01). Immediate pretreatment dexamethasone administration was inversely associated with H3.3K27M-reactive CD8⁺ T cell responses. CONCLUSION. Administration of the H3.3K27M-specific vaccine was well tolerated. Patients with H3.3K27M-specific CD8⁺ immunological responses demonstrated prolonged OS compared with nonresponders. TRIAL REGISTRATION. ClinicalTrials.gov NCT02960230. FUNDING. The V Foundation, the Pacific Pediatric Neuro-Oncology Consortium Foundation, the Pediatric Brain Tumor Foundation, the Mithil Prasad Foundation, the MCJ Amelior Foundation, the Anne and Jason Farber Foundation, Will Power Research Fund Inc., the Isabella Kerr Molina Foundation, the Parker Institute for Cancer Immunotherapy, and the National Institute of Neurological Disorders and Stroke (NINDS), NIH (R35NS105068).

T cell-based immunotherapies hold promise in treating cancer by leveraging the immune system’s recognition of cancer-specific antigens¹. However, their efficacy is limited in tumours with few somatic mutations and substantial intratumoural heterogeneity^2–4. Here we introduce a previously uncharacterized class of tumour-wide public neoantigens originating from RNA splicing aberrations in diverse cancer types. We identified T cell receptor clones capable of recognizing and targeting neoantigens derived from aberrant splicing in GNAS and RPL22. In cases with multi-site biopsies, we detected the tumour-wide expression of the GNAS neojunction in glioma, mesothelioma, prostate cancer and liver cancer. These neoantigens are endogenously generated and presented by tumour cells under physiologic conditions and are sufficient to trigger cancer cell eradication by neoantigen-specific CD8⁺ T cells. Moreover, our study highlights a role for dysregulated splicing factor expression in specific cancer types, leading to recurrent patterns of neojunction upregulation. These findings establish a molecular basis for T cell-based immunotherapies addressing the challenges of intratumoural heterogeneity.

BACKGROUND. Long-term prognosis of WHO grade II low-grade gliomas (LGGs) is poor, with a high risk of recurrence and malignant transformation into high-grade gliomas. Given the relatively intact immune system of patients with LGGs and the slow tumor growth rate, vaccines are an attractive treatment strategy. METHODS. We conducted a pilot study to evaluate the safety and immunological effects of vaccination with GBM6-AD, lysate of an allogeneic glioblastoma stem cell line, with poly-ICLC in patients with LGGs. Patients were randomized to receive the vaccines before surgery (arm 1) or not (arm 2) and all patients received adjuvant vaccines. Coprimary outcomes were to evaluate safety and immune response in the tumor. RESULTS. A total of 17 eligible patients were enrolled — 9 in arm 1 and 8 in arm 2. This regimen was well tolerated with no regimen-limiting toxicity. Neoadjuvant vaccination induced upregulation of type-1 cytokines and chemokines and increased activated CD8⁺ T cells in peripheral blood. Single-cell RNA/T cell receptor sequencing detected CD8⁺ T cell clones that expanded with effector phenotype and migrated into the tumor microenvironment (TME) in response to neoadjuvant vaccination. Mass cytometric analyses detected increased tissue resident–like CD8⁺ T cells with effector memory phenotype in the TME after the neoadjuvant vaccination. CONCLUSION. The regimen induced effector CD8⁺ T cell response in peripheral blood and enabled vaccine-reactive CD8⁺ T cells to migrate into the TME. Further refinements of the regimen may have to be integrated into future strategies. TRIAL REGISTRATION. ClinicalTrials.gov NCT02549833. FUNDING. NIH (1R35NS105068, 1R21CA233856), Dabbiere Foundation, Parker Institute for Cancer Immunotherapy, and Daiichi Sankyo Foundation of Life Science.

Immune-based therapies have shown limited efficacy in glioma thus far. This might be at least in part due to insufficient numbers of neoantigens, thought to be targets of immune attack. In addition, we hypothesized that dynamic genetic and epigenetic tumor evolution in gliomas might also affect the mutation/neoantigen landscape and contribute to treatment resistance through immune evasion. Here, we investigated changes in the neoantigen landscape and immunologic features during glioma progression using exome and RNA-seq of paired primary and recurrent tumor samples obtained from 25 WHO grade II–IV glioma patients (glioblastoma, IDH-wild-type, n = 8; grade II–III astrocytoma, IDH-mutant, n = 9; and grade II–III oligodendroglioma, IDH-mutant, 1p/19q-codeleted, n = 8). The number of missense mutations, predicted neoantigens, or expressed neoantigens was not significantly different between primary and recurrent tumors. However, we found that in individual patients the ratio of expressed neoantigens to predicted neoantigens, designated the “neoantigen expression ratio,” decreased significantly at recurrence (P = 0.003). This phenomenon was particularly pronounced for “high-affinity,” “clonal,” and “passenger gene–derived” neoantigens. Gene expression and IHC analyses suggested that the decreased neoantigen expression ratio was associated with intact antigen presentation machinery, increased tumor-infiltrating immune cells, and ongoing immune responses. Our findings imply that decreased expression of highly immunogenic neoantigens, possibly due to persistent immune selection pressure, might be one of the immune evasion mechanisms along with tumor clonal evolution in some gliomas.

The efficacy of chimeric antigen receptor T cell (CAR-T) therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28 EGFRvIII gliomas revealed impaired mitochondrial ATP production and a markedly hypoxic status compared with ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of the AMPK activator metformin and the mTOR inhibitor rapamycin (Met+Rap). Met+Rap–pretreated mouse CAR-T cells showed activated PPAR-γ coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap–pretreated CAR-T cells demonstrated persistent and effective antiglioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap–pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28 EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group, with fewer Ly6c ⁺ CD11b ⁺ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions under in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap–pretreated CAR-T cells in human trials.

Neuronal activity-driven mechanisms influence glioblastoma cell proliferation and invasion, while glioblastoma remodels neuronal circuits. Although a subpopulation of malignant cells enhances neuronal connectivity, their impact on the immune system remains unclear. Here, we show that glioblastoma regions with enhanced neuronal connectivity exhibit regional immunosuppression, characterized by distinct immune cell compositions and the enrichment of anti-inflammatory tumor-associated macrophages (TAMs). In preclinical models, knockout of Thrombospondin-1 (TSP1/Thbs1) in glioblastoma cells suppresses synaptogenesis and glutamatergic neuronal hyperexcitability. Furthermore, TSP1 knockout restores antigen presentation-related genes, promotes the infiltration of pro-inflammatory TAMs and CD8 + T-cells in the tumor, and alleviates TAM-mediated T-cell suppression. Pharmacological inhibition of glutamatergic signaling also shifts TAMs toward a less immunosuppressive state, prolongs survival in mice, and shows the potential to enhance the efficacy of immune cell-based therapy. These findings confirm that glioma-neuronal circuit remodeling is strongly linked with regional immunosuppression and suggest that targeting glioma-neuron-immune crosstalk could provide avenues for immunotherapy.