Purpose of review

Chimeric antigen receptor (CAR) T cell therapy has been successful in some haematologic malignancies, but the central nervous system (CNS) presents unique obstacles to its use against tumours arising therein. This review discusses recent improvements in the delivery and design of these cells to improve the efficacy and safety of this treatment against malignant gliomas.

Recent findings

The immunosuppressive environment of the CNS affects the functionality of CAR T cells, but recent developments using metabolic manipulation and cytokine delivery have shown that the performance of CAR T cells can be improved in this environment. Emerging techniques can improve the delivery of CAR T cells to the CNS parenchyma, which is normally well protected from peripheral immune cells. The implementation of novel antigens and CAR-expression regulation strategies will improve the specificity and efficacy of these cells. Finally, although autologous T cells have historically been the standard, recent developments have made the use of allogeneic T cells or natural killer (NK) cells more clinically feasible.

Summary

The discoveries highlighted in this review will aid the development of CAR cells that are safer, more resilient against immunosuppressive signals in the CNS, and able to specifically target intracranial tumour cells.

Dendritic cells (DCs) that orchestrate mucosal immunity have been studied in mice. Here we characterized human gut DC populations and defined their relationship to previously studied human and mouse DCs. CD103(+)Sirpα(-) DCs were related to human blood CD141(+) DCs and to mouse intestinal CD103(+)CD11b(-) DCs and expressed markers of cross-presenting DCs. CD103(+)Sirpα(+) DCs aligned with human blood CD1c(+) DCs and mouse intestinal CD103(+)CD11b(+) DCs and supported the induction of regulatory T cells. Both CD103(+) DC subsets induced the TH17 subset of helper T cells, while CD103(-)Sirpα(+) DCs induced the TH1 subset of helper T cells. Comparative analysis of transcriptomes revealed conserved transcriptional programs among CD103(+) DC subsets and identified a selective role for the transcriptional repressors Bcl-6 and Blimp-1 in the specification of CD103(+)CD11b(-) DCs and intestinal CD103(+)CD11b(+) DCs, respectively. Our results highlight evolutionarily conserved and divergent programming of intestinal DCs.

BACKGROUND:Tumor-associated macrophages (TAMs) are abundant in gliomas and immunosuppressive TAMs are a barrier to emerging immunotherapies. It is unknown to what extent macrophages derived from peripheral blood adopt the phenotype of brain-resident microglia in pre-treatment gliomas. The relative proportions of blood-derived macrophages and microglia have been poorly quantified in clinical samples due to a paucity of markers that distinguish these cell types in malignant tissue. RESULTS:We perform single-cell RNA-sequencing of human gliomas and identify phenotypic differences in TAMs of distinct lineages. We isolate TAMs from patient biopsies and compare them with macrophages from non-malignant human tissue, glioma atlases, and murine glioma models. We present a novel signature that distinguishes TAMs by ontogeny in human gliomas. Blood-derived TAMs upregulate immunosuppressive cytokines and show an altered metabolism compared to microglial TAMs. They are also enriched in perivascular and necrotic regions. The gene signature of blood-derived TAMs, but not microglial TAMs, correlates with significantly inferior survival in low-grade glioma. Surprisingly, TAMs frequently co-express canonical pro-inflammatory (M1) and alternatively activated (M2) genes in individual cells. CONCLUSIONS:We conclude that blood-derived TAMs significantly infiltrate pre-treatment gliomas, to a degree that varies by glioma subtype and tumor compartment. Blood-derived TAMs do not universally conform to the phenotype of microglia, but preferentially express immunosuppressive cytokines and show an altered metabolism. Our results argue against status quo therapeutic strategies that target TAMs indiscriminately and in favor of strategies that specifically target immunosuppressive blood-derived TAMs.

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.

The median overall survival for children with diffuse intrinsic pontine glioma (DIPG) is less than one year. The majority of diffuse midline gliomas, including more than 70% of DIPGs, harbor an amino acid substitution from lysine (K) to methionine (M) at position 27 of histone 3 variant 3 (H3.3). From a CD8⁺ T cell clone established by stimulation of HLA-A2⁺ CD8⁺ T cells with synthetic peptide encompassing the H3.3K27M mutation, complementary DNA for T cell receptor (TCR) α- and β-chains were cloned into a retroviral vector. TCR-transduced HLA-A2⁺ T cells efficiently killed HLA-A2⁺H3.3K27M⁺ glioma cells in an antigen- and HLA-specific manner. Adoptive transfer of TCR-transduced T cells significantly suppressed the progression of glioma xenografts in mice. Alanine-scanning assays suggested the absence of known human proteins sharing the key amino acid residues required for recognition by the TCR, suggesting that the TCR could be safely used in patients. These data provide us with a strong basis for developing T cell-based therapy targeting this shared neoepitope.

Abstract: For the past several decades, chimeric antigen receptor T cell (CAR T) therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells in vivo. Using synthetic biology, we engineered T cells with a chimeric receptor SyNthetic Intramembrane Proteolysis Receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette upon recognition of specific tumor markers. We then applied a SNIPR-based positron emission tomography (PET) reporter system to two cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors respectively. Antigen-specific reporter induction of the SNIPR-PET T cells was confirmed in vitro using GFP fluorescence, luciferase luminescence, and the HSV-TK PET reporter with [18F]FHBG. T cells associated with their target antigens were successfully imaged using PET in dual xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII-animal models, with > 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. The main innovation described is therefore PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.

Abstract

BACKGROUND

The prognosis of WHO grade II low-grade gliomas (LGG) is varied with potential for long survival.Given their relatively intact immune system and slow growth rate, vaccines are an attractive treatment strategy for LGG in an attempt to defer more toxic treatments. The goals of this pilot study were to evaluate safety and immunological effects of vaccination with GBM6-AD, an allogeneic glioblastoma stem cell line lysate, with poly-ICLC in LGG.

METHODS

Eligible patients were ≥ 18 years old, ≥ 70 KPS, with recurrent LGG or imaging consistent with LGG, and amenable to resection. Patients were randomized to vaccine prior to surgery (Arm 1) or not (Arm 2) and all received adjuvant vaccine. Co-primary outcomes were safety and immune response in the tumor, with exploratory outcomes of survival and immunologic effects in peripheral blood.

RESULTS

A total of 17 eligible patients were evaluable – nine into Arm 1 and eight into Arm 2. Median age was 33 years, with median time from initial diagnosis of 4.7 years (0 – 20). Two patients (11.8%) previously received radiotherapy and seven (41.2%) prior systemic therapy. No dose limiting toxicities or grade 3 AEs were observed. Neoadjuvant vaccination induced up regulation of type-1 cytokines and chemokines in peripheral blood, and CD8+ T cell clones that reacted to the vaccine were also detected in the tumor. Median follow-up time from first post-operative vaccine was 20.8 months with median PFS of 11.0 months and time to change in therapy of 23.7 months. Of the six patients to receive additional treatment, three had second surgery only one confirming malignant progression to anaplastic oligodendroglioma.

CONCLUSION

Treatment was well-tolerated with no regimen-limiting toxicity. GBM6-AD plus poly-ICLC induced effector CD8+ T cell response in peripheral blood and enables some vaccine-reactive CD8+ T cells to migrate into the TME. Further investigation is warranted.

For the past several decades, chimeric antigen receptor T-cell therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells in vivo. Methods: Using synthetic biology, we engineered T cells with a chimeric receptor synthetic intramembrane proteolysis receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette on recognition of specific tumor markers. We then applied a SNIPR-based PET reporter system to 2 cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors, respectively. Results: Antigen-specific reporter induction of the SNIPR PET T cells was confirmed in vitro using green fluorescent protein fluorescence, luciferase luminescence, and the HSV-TK PET reporter with 9-(4-¹⁸F-fluoro-3-[hydroxymethyl]butyl)guanine ([¹⁸F]FHBG). T cells associated with their target antigens were successfully imaged using PET in dual-xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII- animal models, with more than 10-fold higher [¹⁸F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. Conclusion: The main innovation found in this work was PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.