PDF file, 29K, Vaccine Administration Schedule and Characteristics of Adverse Events.

PDF file, 23K, Adjuvant Therapies Prior to and After Vaccination.

PDF file, 48K, Overall Survival Characteristics of Treated Patients.

PDF file, 9905K, Representative phenotypic analysis of circulating lymphocytes in a clinical responder (Patient #009).

Supplementary Figure 2. Immunohistochemistry evaluating expression of vaccine-targeted antigens in resected tumor tissues.

Supplementary Table 2. Comparison of ELISPOT responses between the current study and our recent study in pediatric glioma patients

PDF file, 17162K, Model of HSPPC-96 mechanism of action.

PDF file, 8781K, Representative phenotypic analysis of circulating lymphocytes in a clinical non-responder (Patient #003).

Supplementary Figure 1. T2-Flair MRI images of 2 cases (Patient 9 in Cohort 1 and Patient 1 in Cohort 2)

Supplementary Table 1. Associations between baseline IFN-γ ELISPOT values vs. PFS or subsequent IFN-γ ESLIPOT responses

PDF file, 50K, Descriptive Characteristics of the Study Population.

Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 are among the first genetic alterations observed during the development of lower-grade glioma (LGG). LGG-associated IDH mutations confer gain-of-function activity by converting α-ketoglutarate to the oncometabolite R-2-hydroxyglutarate (2HG). Clinical samples and gene expression data from The Cancer Genome Atlas (TCGA) demonstrate reduced expression of cytotoxic T lymphocyte-associated genes and IFN-γ-inducible chemokines, including CXCL10, in IDH-mutated (IDH-MUT) tumors compared with IDH-WT tumors. Given these findings, we have investigated the impact of IDH mutations on the immunological milieu in LGG. In immortalized normal human astrocytes (NHAs) and syngeneic mouse glioma models, the introduction of mutant IDH1 or treatment with 2HG reduced levels of CXCL10, which was associated with decreased production of STAT1, a regulator of CXCL10. Expression of mutant IDH1 also suppressed the accumulation of T cells in tumor sites. Reductions in CXCL10 and T cell accumulation were reversed by IDH-C35, a specific inhibitor of mutant IDH1. Furthermore, IDH-C35 enhanced the efficacy of vaccine immunotherapy in mice bearing IDH-MUT gliomas. Our findings demonstrate a mechanism of immune evasion in IDH-MUT gliomas and suggest that specific inhibitors of mutant IDH may improve the efficacy of immunotherapy in patients with IDH-MUT gliomas.

We evaluated whether isocitrate dehydrogenase (IDH) mutations could directly alter the immune landscape of gliomas, possibly through production of the onco-metabolite, 2-hydroxygluterate (2HG). TCGA RNAseq data demonstrate reduced expression of CD8 T-cell, type-1 effector and chemokine genes in IDH-mutated (Mut) patients compared to IDH-wild-type (Wt) cases. Conversely, we observed no differences in B-cell, type-2 or regulatory T-cell-related genes between IDH-Wt and IDH-Mut cases. Normal human astrocyte (NHA) cells and syngeneic GL261 glioma models expressing the R132H mutated IDH1 (NHA-Mut and GL261-Mut, respectively) further demonstrated that the IDH1 mutation and 2HG directly reduce CXCL10 protein and migration of T-cells to the tumor site, which was reversed by an IDH-specific inhibitor, IDH-C35. Further, while mice receiving prophylactic peptide vaccination targeting glioma-associated antigens resulted in clearance of GL261-Wt tumors, vaccinated mice could not reject GL261-Mut tumors. Treatment of vaccinated mice bearing GL261-Mut tumors with IDH-C35 enhanced anti-tumor immunity and prolonged survival compared to mice receiving vaccine alone. Interestingly, both parental GL261 cells treated with 2HG and GL261-Mut cells demonstrated decreased amounts of STAT1 protein but not mRNA compared with untreated GL261-Wt cells, suggesting that STAT1, a known regulator of CXCL10, is regulated at the post-transcriptional level in IDH-Mut gliomas. Consistent with these findings, TCGA microRNA data demonstrated increased levels of miR-145, a known regulator of STAT1, in IDH-Mut compared with IDH-Wt cases. We next evaluated direct tumor extrinsic implications of the IDH1 mutation. 2HG, which is known to be present in the tumor microenvironment, directly induced a ~10 fold increase in miR-145-5p and reduced both STAT1 levels and CXCL10 secretion from primary and BV2 murine microglia. Our findings demonstrate both glioma-intrinsic and extrinsic mechanisms of immunosuppression in IDH-Mut gliomas and suggest that IDH inhibitors can be used to enhance T-cell migration to the tumor site in patients with IDH-Mut tumors.

Meeting abstracts Isocitrate dehydrogenase (IDH) mutations are the first mutations that occur during the oncogenic process of lower-grade glioma (LGG) and confers a novel gain-of-function activity by converting α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), promoting DNA hyper-methylation.

Abstract Purpose: WHO grade 2 low-grade gliomas (LGG) with high risk factors for recurrence are mostly lethal despite current treatments. We conducted a phase I study to evaluate the safety and immunogenicity of subcutaneous vaccinations with synthetic peptides for glioma-associated antigen (GAA) epitopes in HLA-A2+ adults with high-risk LGGs in the following three cohorts: (i) patients without prior progression, chemotherapy, or radiotherapy (RT); (ii) patients without prior progression or chemotherapy but with prior RT; and (iii) recurrent patients. Experimental Design: GAAs were IL13Rα2, EphA2, WT1, and Survivin. Synthetic peptides were emulsified in Montanide-ISA-51 and given every 3 weeks for eight courses with intramuscular injections of poly-ICLC, followed by q12 week booster vaccines. Results: Cohorts 1, 2, and 3 enrolled 12, 1, and 10 patients, respectively. No regimen-limiting toxicity was encountered except for one case with grade 3 fever, fatigue, and mood disturbance (cohort 1). ELISPOT assays demonstrated robust IFNγ responses against at least three of the four GAA epitopes in 10 and 4 cases of cohorts 1 and 3, respectively. Cohort 1 patients demonstrated significantly higher IFNγ responses than cohort 3 patients. Median progression-free survival (PFS) periods since the first vaccine are 17 months in cohort 1 (range, 10–47+) and 12 months in cohort 3 (range, 3–41+). The only patient with large astrocytoma in cohort 2 has been progression-free for more than 67 months since diagnosis. Conclusion: The current regimen is well tolerated and induces robust GAA-specific responses in WHO grade 2 glioma patients. These results warrant further evaluations of this approach. Clin Cancer Res; 21(2); 286–94. ©2014 AACR.

To elucidate mechanisms underlying epidemiological findings of decreased risk of glioma development in patients with allergies and asthma, gliomas were induced in mice deficient for histidine decarboxylase (HDC), the enzyme responsible for histamine production. These mice exhibited shortened survival and enhanced tumor growth compared to wild-type (WT) mice. Previous studies have shown a pivotal role of HDC in maturation of bone marrow (BM)-derived myeloid cells. In our glioma models, brain-infiltrating leukocytes (BIL) demonstrated an increased frequency of CD11b(+)Gr1(+) immature myeloid cells (IMC; both CD11b(+)Ly6G(+) and CD11b(+)Ly6C(+) subpopulations) as well as diminished CD8(+) T cell infiltration and their effector functions in HDC-/- mice compared with WT mice. Furthermore, HDC-/- IMC demonstrated a more profound immune suppression of CD8(+) T cell proliferation and functions associated with increased prostaglandin E2 (PGE2) expression levels. Celecoxib, a cyclooxygenase-2 inhibitor, which is vital for PGE2 production, abrogated suppressive capabilities of HDC-/- IMC. In addition, glioma-bearing HDC-eGFP mice, in which HDC promoter drives green fluorescence protein (GFP) expression, exhibited decreased HDC promoter activities in CD11b(+)Gr1(+) cells in the BM, spleen, and intracranial tumor site compared with non-tumor bearing HDC-eGFP mice. Additionally, in vitro culture with glioma supernatants decreased GFP expression in CD11b(+)Gr1(+), CD11b(+)Ly6G(+), and CD11b(+)Ly6C(+) IMC. HDC expression levels inversely correlated with suppressive functions of CD11b(+)Gr1(+) IMC, as GFP(+) CD11b(+)Gr1(+) more profoundly inhibited CD8(+) T cell proliferation compared with CD11b(+)Gr1(+)GFP(+) cells. Taken together, these data show a significant role of HDC in the glioma microenvironment via maturation of myeloid cells and resulting activation of CD8(+) T cells.

Myeloid-Derived Suppressor Cells (MDSCs) heavily infiltrate in a variety of solid tumors and suppress anti-tumor T-cell activity. Our recent studies have demonstrated the ability of monocytic, Ly6C+ MDSCs to promote glioma growth through the activation of cyclooxygenase (COX)-2 pathway, which is responsible for plostaglandin-synthesis. ONO-AE3-208 is an antagonist of the prostaglandin E (EP)-4 receptor, which is an important positive feedback regulator of the COX-2 pathway. We thus examined the ability of ONO-AE3-208 to suppress MDSC activity in gliomas. ONO-AE3-208 treatment in mice bearing established GL261-quad glioma in the brain resulted in complete and persistent rejection of the tumors. Flow cytometric analysis revealed that gliomas in the ONO-AE3-208-treated mice were infiltrated by fewer numbers of Ly6C+ MDSCs compared with non-treated animals. We subsequently isolated glioma-infiltrating Ly6C+ MDSCs by flow-sorting to address their functions. RT-PCR analysis revealed that the Ly6C+ MDSCs derived from ONO-AE3-208 treated mice expressed lower levels of the Arg1 and Cox2 expression compared to control animals. Consistently, brain infiltrating leukocytes in ONO-AE3-208 treated tumor-bearing mice demonstrated enhanced Ifng expression compared with control mice, suggestive of enhanced T-cell activity. Importantly, ONO-AE3-208 inhibited glioma growth and promoted immune activity in 2 additional murine glioma models: the Sleeping Beauty de novo glioma model and the SB28 glioma cell line model. Our data demonstrate that ONO-AE3-208 may be useful in the treatment of glioma patients to suppress Ly6C+ MDSCs and promote anti-tumor immunity.

PURPOSE: WHO grade II low-grade gliomas (LGGs) with high risk factors for recurrence are mostly lethal despite current treatments. We conducted a phase I study to evaluate the safety and immunogenicity of subcutaneous vaccinations with synthetic peptides for glioma-associated antigen (GAA) epitopes in HLA-A2+ adults with high-risk LGGs in the following three cohorts: 1) patients without prior progression, chemotherapy or radiation therapy (RT); 2) patients without prior progression or chemotherapy but with prior RT, and 3) recurrent patients. METHODS: GAAs were IL-13Rα2, EphA2, WT1, and Survivin. Synthetic peptides were emulsified in Montanide-ISA-51 and given every 3 weeks for 8 courses with intramuscular injections of poly-ICLC, followed by q12week booster vaccines. RESULTS: Cohorts 1, 2, and 3 enrolled 12, 1, and 10 patients, respectively. No regimen-limiting toxicity was encountered except for one case with Grade 3 fever, fatigue and mood disturbance (Cohort 1). ELISPOT assays demonstrated robust IFN-γ responses against at least 3 of the 4 GAA epitopes in 10 and 4 cases of Cohorts 1 and 3, respectively. Cohort 1 patients demonstrated significantly higher IFN-γ responses than Cohort 3 patients. Median progression-free survival (PFS) periods since the 1st vaccine are 17 months in Cohort 1 (range 10-42+) and 12 months in Cohort 3 (range 3-37+). The only patient with large astrocytoma in Cohort 2 has been progression-free for over 60 months since diagnosis. CONCLUSION: The current regimen is well tolerated and induces robust GAA-specific responses in WHO grade II glioma patients. These results warrant further evaluations of this approach.

Abstract Multiple epidemiological studies have demonstrated an association between allergic conditions and protection from glioma development. Also, use of anti-histamines has shown up to a 3.5-fold increase in the risk of malignant glioma. Previous studies have shown deviations in myeloid cell maturation in mice deficient for histidine decarboxylase (HDC-/-), the key enzyme in histamine production. HDC-/- mice exhibited increased levels of CD11b+GR1+ immature myeloid cells (IMC). This cellular phenotype is associated with myeloid-derived suppressor cells (MDSC), which are thought to be IMC that can inhibit T cell function and have been shown to promote tumor development. From this data, we hypothesize that histamine is important to normal myeloid maturation, whereas a lack thereof can promote accumulation of immunosuppressive IMC, which can stimulate glioma development. Utilizing our de novo tumor model, we observed faster tumor induction as well as increased growth in the HDC-/- mice compared to wild type (WT). Also, HDC-/- mice exhibited decreased symptom-free survival (SFS) compared to WT (74 vs 86 days, respectively; p=0.04). Analysis of brain-infiltrating leukocytes (BIL) a time of symptoms demonstrated increased frequency of CD11b+Gr1+ IMC in HDC-/- mice compared to WT (52% vs 34%; p=0.02). While not reaching statistical significance, there was a trend towards increased CD11b+GrLo compartment (28% vs 11%; p=0.08). Furthermore, SB-derived glioma cell lines from both HDC-/- and WT mice were generated. No significant differences were observed in in vitro growth dependent upon HDC expression or histamine concentration. These cell lines were utilized for in vivo studies via intracranial orthotopic injections. No differences in survival or growth were observed when HDC-expressing cell lines were utilized, whereas significantly increased tumor volume at day 14 and decreased SFS was observed in HDC-/- mice compared to WT when the HDC-/- glioma cells were used (15 vs 23 days; p=0.04). HDC-/- mice exhibited increased CD11b+Gr1Lo compared to WT in the BIL (45% vs 28%; p=0.03). Also, HDC-/- mice possessed decreased CD8+CD107a+ cells at the tumor site, suggesting a decreased T cell response in the absence of histamine. This data suggests a more crucial role for local compared to systemic histamine on glioma development. Additionally, we believe that the presence of glioma can inhibit HDC expression further leading to the accumulation of IMC. By use of HDC-eGFP mice, in vitro studies showed that the addition of glioma supernatant prevented the upregulation of HDC in bone marrow cells by growth factors. In vivo HDC expression in CD11b+Gr1+ myeloid cells was decreased in bone marrow, spleen, and BIL in mice with glioma compared to non-tumor bearing mice. These collective data suggest that disruption of histamine signaling can promote glioma development by augmenting the accumulation of immunosuppressive IMC leading to decreased T cell response. Citation Format: Brian Ahn, Gary Kohanbash, Akemi Kosaka, Takayuki Ohkuri, Hideho Okada. Histamine in myeloid cell maturation and malignant glioma development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4846. doi:10.1158/1538-7445.AM2014-4846

AbstractPurpose: Cancer immunotherapy offers hope of a highly specific nontoxic adjuvant treatment. Heat shock protein peptide complexes (HSPPCs) found in cancer cells carry tumor-specific antigenic proteins and can facilitate adaptive and innate immune responses. Here we show that peptides bound to a 96 kD chaperone protein (HSP-96) from brain tissue containing glioblastoma multiforme (GBM) can be used to safely immunize patients with recurrent GBM.Experimental Design: Multimodality immunomonitoring was completed on 12 patients with recurrent GBM before and after immunization with an autologous HSPPC vaccine derived from surgically resected tumor. Clinical endpoints included safety assessments and overall survival.Results: No adverse events attributable to the vaccine were found. Testing of peripheral blood leukocytes before and after vaccination revealed a significant peripheral immune response specific for the peptides bound to HSP-96, in 11 of the 12 patients treated. Brain biopsies of immune responders after vaccination revealed focal CD4, CD8, and CD56 IFNγ positive cell infiltrates, consistent with tumor site specific immune responses. Immune responders had a median survival of 47 weeks after surgery and vaccination, compared with 16 weeks for the single nonresponder.Conclusions: These data provide the first evidence in humans of individual patient-specific immune responses against autologous tumor derived peptides bound to HSP-96. Clin Cancer Res; 19(1); 205–14. ©2012 AACR.