Genetically Engineered T-Cells for Malignant Glioma: Overcoming the Barriers to Effective Immunotherapy.

Malignant gliomas carry a dismal prognosis. Conventional treatment using chemo- and radiotherapy has limited efficacy with adverse events. Therapy with genetically engineered T-cells, such as chimeric antigen receptor (CAR) T-cells, may represent a promising approach to improve patient outcomes owing to their potential ability to attack highly infiltrative tumors in a tumor-specific manner and possible persistence of the adaptive immune response. However, the unique anatomical features of the brain and susceptibility of this organ to irreversible tissue damage have made immunotherapy especially challenging in the setting of glioma. With safety concerns in mind, multiple teams have initiated clinical trials using CAR T-cells in glioma patients. The valuable lessons learnt from those trials highlight critical areas for further improvement: tackling the issues of the antigen presentation and T-cell homing in the brain, immunosuppression in the glioma microenvironment, antigen heterogeneity and off-tumor toxicity, and the adaptation of existing clinical therapies to reflect the intricacies of immune response in the brain. This review summarizes the up-to-date clinical outcomes of CAR T-cell clinical trials in glioma patients and examines the most pressing hurdles limiting the efficacy of these therapies. Furthermore, this review uses these hurdles as a framework upon which to evaluate cutting-edge pre-clinical strategies aiming to overcome those barriers

Genetically Engineered T-Cells for Malignant Glioma: Overcoming the Barriers to Effective Immunotherapy

Malignant gliomas carry a dismal prognosis. Conventional treatment using chemo- and radiotherapy has limited efficacy with adverse events. Therapy with genetically engineered T-cells, such as chimeric antigen receptor (CAR) T-cells, may represent a promising approach to improve patient outcomes owing to their potential ability to attack highly infiltrative tumors in a tumor-specific manner and possible persistence of the adaptive immune response. However, the unique anatomical features of the brain and susceptibility of this organ to irreversible tissue damage have made immunotherapy especially challenging in the setting of glioma. With safety concerns in mind, multiple teams have initiated clinical trials using CAR T-cells in glioma patients. The valuable lessons learnt from those trials highlight critical areas for further improvement: tackling the issues of the antigen presentation and T-cell homing in the brain, immunosuppression in the glioma microenvironment, antigen heterogeneity and off-tumor toxicity, and the adaptation of existing clinical therapies to reflect the intricacies of immune response in the brain. This review summarizes the up-to-date clinical outcomes of CAR T-cell clinical trials in glioma patients and examines the most pressing hurdles limiting the efficacy of these therapies. Furthermore, this review uses these hurdles as a framework upon which to evaluate cutting-edge pre-clinical strategies aiming to overcome those barriers

ONO-AE3-208 inhibits myeloid derived suppressor cells and glioma growth

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 prostaglandin-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 IFN-g 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

Antigenic expression and spontaneous immune responses support the use of a selected peptide set from the IMA950 glioblastoma vaccine for immunotherapy of grade II and III glioma.

Gliomas are lethal brain tumors that resist standard therapeutic approaches. Immunotherapy is a promising alternative strategy mostly developed in the context of glioblastoma. However, there is a need for implementing immunotherapy for grade II/III gliomas, as these are the most common CNS tumors in young adults with a high propensity for recurrence, making them lethal despite current treatments. We recently identified HLA-A2-restricted tumor-associated antigens by peptide elution from glioblastoma and formulated a multipeptide vaccine (IMA950) evaluated in phase I/II clinical trials with promising results. Here, we investigated expression of the IMA950 antigens in patients with grade II/III astrocytoma, oligodendroglioma or ependymoma, at the mRNA, protein and peptide levels. We report that the BCAN, CSPG4, IGF2BP3, PTPRZ1 and TNC proteins are significantly over-expressed at the mRNA (n = 159) and protein (n = 36) levels in grade II/III glioma patients as compared to non-tumor samples (IGF2BP3 being absent from oligodendroglioma). Most importantly, we detected spontaneous antigen-specific T cell responses to one or more of the IMA950 antigens in 100% and 71% of grade II and grade III patients, respectively (27 patients tested). These patients displayed T cell responses of better quality (higher frequency, broader epitope targeting) than patients with glioblastoma. Detection of spontaneous T cell responses to the IMA950 antigens shows that these antigens are relevant for tumor targeting, which will be best achieved by combination with CD4 epitopes such as the IDH1R132H peptide. Altogether, we provide the rationale for using a selective set of IMA950 peptides for vaccination of patients with grade II/III glioma

Spontaneous immune responses against glioma-associated antigens in a long term survivor with malignant glioma

<p>Abstract</p> <p>Background</p> <p>In patients with high grade glioma, little is known regarding existence of naturally occurring adaptive T cell reactivity against glioma-associated antigens (GAAs). In this report, we characterized GAA-specific CD8⁺T cells and innate immune cells in a patient who has survived with anaplastic astrocytoma (AA) for over 12 years without recurrence.</p> <p>Methods</p> <p>Peripheral blood mononuclear cells (PBMCs) derived from the long term survivor with AA were evaluated for the frequency, cytotoxic T lymphocyte (CTL) activity and differentiation status of CD8⁺cells recognizing GAA-derived epitopes as well as relative numbers of other immune cell subsets. This patient's AA tissue was evaluated for expression of two GAAs EphA2 and interleukin-13 receptor α2 subunit (IL-13Rα2) by immunohistochemistry.</p> <p>Results</p> <p>The patient's tumor expressed both EphA2 and IL-13Rα2, and <it>in vitro </it>stimulated PBMC demonstrated superior EphA2_883–891and IL-13Rα2_345–353-specific CTL reactivity compared to PBMC samples from two other patients with progressing malignant glioma. Unstimulated EphA2_883–891-reactive CD8⁺T cells contained high numbers of CD45RA^-/CCR7^-late effector and CD45RA^-/CCR7⁺central memory cells. Among other leukocyte subsets, elevated numbers of NK-T cells were found.</p> <p>Conclusion</p> <p>To our knowledge, the current study is one of the first demonstrating the presence of antigen-experienced, GAA-reactive CD8⁺T cells in a patient who has survived with AA for over 12 years without recurrence. Further studies are warranted to determine whether the status of GAA-reactive CD8⁺T cells dictates survival of patients and/or response to therapeutic vaccines.</p

Differential activity of interferon-α8 promoter is regulated by Oct-1 and a SNP that dictates prognosis of glioma

We have previously reported that the single nucleotide polymorphism (SNP) rs12553612 in IFNA8 is associated with better overall survival of glioma patients with the AA-genotype compared with patients with the AC-genotype. As rs12553612 is located in the IFNA8 promoter, we hypothesized that the A-allele allows for an enhanced IFNA8 promoter activity compared with the C-allele. Reporter assays in the human monocyte derived THP-1 cell line demonstrated a superior promoter activity of the A-allele compared with the C-allele. Electrophoretic mobility shift assays (EMSA) further demonstrated that the A-genotype specifically binds to more nuclear proteins than the C-genotype, including the transcription factor Oct-1. Further, co-transfection of plasmids encoding Oct-1 and the reporter constructs revealed that Oct-1 enhanced the promoter activity with the A- but not the C-allele. Taken together, our data demonstrate that the A-allele in the rs12553612 SNP, which is associated with better glioma patient survival, allows for IFNA8 transcription by allowing for Oct-1 binding, which is absent in patients with C allele, and suggests a molecular mechanism of IFNA8 mediated immune-surveillance of glioma progression

Vaccination with EphA2-derived T cell-epitopes promotes immunity against both EphA2-expressing and EphA2-negative tumors

BACKGROUND: A novel tyrosine kinase receptor EphA2 is expressed at high levels in advanced and metastatic cancers. We examined whether vaccinations with synthetic mouse EphA2 (mEphA2)-derived peptides that serve as T cell epitopes could induce protective and therapeutic anti-tumor immunity. METHODS: C57BL/6 mice received subcutaneous (s.c.) vaccinations with bone marrow-derived dendritic cells (DCs) pulsed with synthetic peptides recognized by CD8+ (mEphA2(671–679), mEphA2(682–689)) and CD4+ (mEphA2(30–44)) T cells. Splenocytes (SPCs) were harvested from primed mice to assess the induction of cytotoxic T lymphocyte (CTL) responses against syngeneic glioma, sarcoma and melanoma cell lines. The ability of these vaccines to prevent or treat tumor (s.c. injected MCA205 sarcoma or B16 melanoma; i.v. injected B16-BL6) establishment/progression was then assessed. RESULTS: Immunization of C57BL/6 mice with mEphA2-derived peptides induced specific CTL responses in SPCs. Vaccination with mEPhA2 peptides, but not control ovalbumin (OVA) peptides, prevented the establishment or prevented the growth of EphA2+ or EphA2-negative syngeneic tumors in both s.c. and lung metastasis models. CONCLUSIONS: These data indicate that mEphA2 can serve as an attractive target against which to direct anti-tumor immunity. The ability of mEphA2 vaccines to impact EphA2-negative tumors such as the B16 melanoma may suggest that such beneficial immunity may be directed against alternative EphA2+ target cells, such as the tumor-associated vascular endothelial cells

IDH mutation-induced suppression of type-1 anti-glioma immune response

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. Our analysis of LGG cases from The Cancer Genome Atlas (TCGA) database revealed that IDH-mutant (IDH-Mut) cases exhibit decreased expression of type-1 effector T cell response-related genes, which are critical for anti-glioma immunity, including: CD8A, IFNG, OAS2, GZMA, EOMES, CXCL9 and CXCL10, compared with IDH-wild type (IDH-WT) cases. On the other hand, type-2 and regulatory T cell response-related genes, such as IL5 and TGFB1, are not significantly different between IDH-Mut vs. WT cases, indicating that the observed down-regulation of type-1 response-related genes does not merely represent a possible global gene suppression. Furthermore, IDH-Mut cases exhibit increased CXCL10 promotor methylation compared with WT cases. We thus hypothesized that IDH mutation-mediated tumor intrinsic mechanisms occurring within glioma cells may inhibit anti-tumor immunity to promote tumor growth. In vitro, a normal human astrocyte (NHA) cell line transfected with IDH1-Mut cDNA expressed lower levels of CXCL10 compared to NHA cells transfected with WT IDH1. Consistently, C57Bl/6 mouse-syngeneic astrocyte and glioma cell lines transfected with IDH1-Mut expressed lower levels of CXCL10 gene and protein, compared to control cells transfected with IDH-WT, which was restored following 30 day treatment of the cells with the IDH1 inhibitor, IDH-C35. Furthermore, in vivo orthotopic IDH1-Mut gliomas at 21 days post-intracranial injection in syngeneic mice expressed lower levels of T cell chemokines CXCL9 and CXCL10 as determined by RT-PCR and ELISA and reduced infiltration of CD3+CD8+ T cells as determined by flow cytometry and quantitative immunohistochemistry compared with control IDH1-WT gliomas. Further, an in vitro migration assay demonstrated reduced migration of T cells towards culture supernatants from IDH1-Mut cell lines compared with control supernatants derived from IDH1-WT cells. Overall, our data demonstrate that IDH mutations in tumor cells lead to reduced T cell attracting chemokines and reduced T cell accumulation in gliomas. Our analyses of the TCGA 450K gene methylation database suggest that the suppressed expression of OAS2 and CXCL10 in IDH1-Mut cases is associated with hypermethylation of the promoter for these genes. Indeed, treatment of IDH-Mut cell lines with demethylating agent 5-Aza-CdR restored CXCL10 expression levels. Our data suggest that IDH inhibitors and demethylation agents may be used to enhance T cell recruitment to LGG in combination with T cell based immunotherapies