Tobias Weiss

Immunotherapy for glioblastoma: concepts and challenges

PURPOSE OF REVIEW Immunotherapy is an emerging treatment strategy against various cancer types including glioblastoma. It comprises different strategies to induce, boost or restore an antitumor immune response. This review provides an overview of recent preclinical and clinical developments in the field of immunotherapy against glioblastoma. We elucidate the concepts and challenges and point out the strengths and weaknesses of the most promising immunotherapeutic approaches. RECENT FINDINGS Immunotherapy is one of the most active research areas in glioblastoma. Data from preclinical work as well as phase I and phase II clinical trials revealed that immunotherapy against glioblastoma is overall well tolerated and able to promote a potent antitumor immune response. Among the therapeutic approaches that are currently under investigation, vaccination, for example, against the variant III of epidermal growth factor receptor, as well as immune checkpoint inhibition targeting receptors such as cytotoxic T lymphocyte-associated antigen-4 and programmed cell death-1, are among the most promising and advanced treatment strategies. However, there are considerable challenges to overcome such as the identification of novel target molecules for vaccination, appropriate patient selection criteria, strategies to prevent or handle immune-related adverse events, and the implementation of immunotherapy in multimodal treatment regimens together with conventional treatment strategies. SUMMARY Key features of immunotherapy are target specificity, adaptability, and durability. Results from preclinical assessments and clinical trials applying immunotherapy alone or in combination with conventional treatment options are promising. However, intense research and stringent clinical development are required to optimize the available treatment options and to overcome potential pitfalls.

Immunological effects of chemotherapy and radiotherapy against brain tumors

ABSTRACT Introduction: The mainstays of brain tumor therapy are surgery, radiotherapy and chemotherapy. Cancer immunotherapy is explored as an additional treatment modality. However, emerging evidence indicates that also radio- and chemotherapy have immunological effects in addition to their cytotoxic and cytostatic activities. Area covered: We summarize the literature on radio- and chemotherapy-mediated immunological effects in primary and secondary brain tumors and outline open questions within the field. To this end, a literature search was performed using the terms ‘brain tumor’, ‘immune system’, ‘immunogenic cell death’, ‘vaccination’, ‘checkpoint inhibition’, ‘radiotherapy’, ‘chemotherapy’ and derivations thereof. Expert commentary: Immunological effects of chemo- and radiotherapy in brain tumors involve direct immunogenic modulations of tumor cells, changes of the microenvironment and functional alterations of innate and adaptive immune cells. Each treatment modality can exert various effects that comprise both immune-stimulatory and immunosuppressive mechanisms. A detailed knowledge of these mechanisms is indispensable for an optimal combination of conventional anti-tumor treatments and novel immunotherapeutic approaches.

Non-parametric intravoxel incoherent motion analysis in patients with intracranial lesions: Test-retest reliability and correlation with arterial spin labeling

Intravoxel incoherent motion (IVIM) analysis of diffusion imaging data provides biomarkers of true passive water diffusion and perfusion properties. A new IVIM algorithm with variable adjustment of the b-value threshold separating diffusion and perfusion effects was applied for cerebral tissue characterization in healthy volunteers, computation of test-retest reliability, correlation with arterial spin labeling, and assessment of applicability in a small cohort of patients with malignant intracranial masses. The main results of this study are threefold: (i) accounting for regional differences in the separation of the perfusion and the diffusion components improves the reliability of the model parameters; (ii) if differences in the b-value threshold are not accounted for, a significant tissue-dependent systematic bias of the IVIM parameters occurs; (iii) accounting for voxel-wise differences in the b-value threshold improves the correlation with CBF measurements in healthy volunteers and patients. The proposed algorithm provides a robust characterization of regional micro-vascularization and cellularity without a priori assumptions on tissue diffusion properties. The glioblastoma multiforme with its inherently high variability of tumor vascularization and tumor cell density may benefit from a non-invasive clinical characterization of diffusion and perfusion properties.

Addition of lomustine for bevacizumab-refractory recurrent glioblastoma.

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Precise Temporal Profiling of Signaling Complexes in Primary Cells Using SWATH Mass Spectrometry

Summary Spatiotemporal organization of protein interactions in cell signaling is a fundamental process that drives cellular functions. Given differential protein expression across tissues and developmental stages, the architecture and dynamics of signaling interaction proteomes is, likely, highly context dependent. However, current interaction information has been almost exclusively obtained from transformed cells. In this study, we applied an advanced and robust workflow combining mouse genetics and affinity purification (AP)-SWATH mass spectrometry to profile the dynamics of 53 high-confidence protein interactions in primary T cells, using the scaffold protein GRB2 as a model. The workflow also provided a sufficient level of robustness to pinpoint differential interaction dynamics between two similar, but functionally distinct, primary T cell populations. Altogether, we demonstrated that precise and reproducible quantitative measurements of protein interaction dynamics can be achieved in primary cells isolated from mammalian tissues, allowing resolution of the tissue-specific context of cell-signaling events.

NKG2D-Based CAR T Cells and Radiotherapy Exert Synergistic Efficacy in Glioblastoma

Chimeric antigen receptor (CAR) T-cell therapy is an emerging immunotherapy against several malignancies including glioblastoma, the most common and most aggressive malignant primary brain tumor in adults. The challenges in solid tumor immunotherapy comprise heterogenously expressed tumor target antigens and restricted trafficking of CAR T cells to and impaired long-term persistence at the tumor site, as well as the unaddressed integration of CAR T-cell therapy into conventional anticancer treatments. We addressed these questions using a NKG2D-based chimeric antigen receptor construct (chNKG2D) in fully immunocompetent orthotopic glioblastoma mouse models. ChNKG2D T cells demonstrated high IFNγ production and cytolytic activity in vitro Upon systemic administration in vivo, chNKG2D T cells migrated to the tumor site in the brain, did not induce adverse events, prolonged survival, and cured a fraction of glioma-bearing mice. Surviving mice were protected long-term against tumor rechallenge. Mechanistically, this was not solely the result of a classical immune memory response, but rather involved local persistence of chNKG2D T cells. A subtherapeutic dose of local radiotherapy in combination with chNKG2D T-cell treatment resulted in synergistic activity in two independent syngeneic mouse glioma models by promoting migration of CAR T cells to the tumor site and increased effector functions. We thus provide preclinical proof-of-concept of NKG2D CAR T-cell activity in mouse glioma models and demonstrate efficacy, long-term persistence, and synergistic activity in combination with radiotherapy, providing a rationale to translate this immunotherapeutic strategy to human glioma patients.Significance: These findings provide evidence for synergy of conventional anticancer therapy and CAR T cells and heralds future studies for other treatment combinations. Cancer Res; 78(4); 1031-43. ©2017 AACR.

Gamma-hydroxybutyrate (GHB) and topiramate—clinically relevant drug interaction suggested by a case of coma and increased plasma GHB concentration

To the Editor: A 52-year-old woman was hospitalized for worsening chronic cluster headache refractory to all guideline-based medical and invasive treatments. Based on class IV evidence [1] she had regularly taken high-dose gammahydroxybutyrate GHB = sodium oxybate (Xyrem®; UCB-Pharma AG, Bulle, Switzerland) 4.5 g twice-nightly at 2300 and 0300 hours for the last 6 years. This was the only drug that markedly improved her nocturnal headache episodes and insomnia. As an additional therapeutic effort, topiramate (Topamax®; Janssen-Cilag AG, Baar, Switzerland) was added to the therapeutic regime, with the patient taking a single dose of topiramate 25 mg at 1800 hours, followed by the usual two daily doses of GHB. The next morning the patient had developed confusion, followed by intermittent myoclonic jerks, miosis, and a rapid onset of coma [Glasgow Coma Scale (GCS) score was 3 at 0800 hours]. Pulse, blood pressure, respiratory rate, pulse oximetry, electrocardiogram, and laboratory values, including electrolytes and blood glucose, were unremarkable. The plasma GHB concentration, determined by gas chromatography–mass spectrometry (GC-MS) of a blood sample collected at 0800 hours, was 259 mg/L. One hour later electroencephalography (EEG) showed intermittent bifrontal theta activity, a pattern described during sedation with GHB [2]. At 1300 hours the patient awoke from coma and rapidly recovered within a few hours. Topiramate was stopped, but GHB was continued as before. Two days later the plasma GHB concentration was 91 mg/L based on GCMS analysis of a blood sample collected at 0800 hours. In this patient, who was given topiramate concomitant with GHB, the GHB concentration 5 h after the second daily dose of GHB was 2.8-fold higher than without topiramate, and 1.8-fold higher than the peak concentration of 142 mg/L that would be expected 0.5–2 h after the daily second dose according to Xyrem®’s product information. The patient had taken topiramate without concomitant GHB in the past without problems. Based on the rapid onset but short duration of the otherwise unexplained coma and the EEG findings, we suggest that the coma was drug induced due to a pharmacokinetic interaction between GHB and topiramate. GHB has a short half-life of about 30 min [3], and metabolism via GHB-dehydrogenase is its main route of elimination [4]. In vitro studies have demonstrated that GHBdehydrogenase is inhibited by the antiepileptic drugs valproate and ethosuximide [4], but according to Xyrem®’s product information no interaction studies with antiepileptics have been performed in humans. Alternatively, changes in the bioavailability of GHB or other unknown mechanisms of interaction are theoretically possible. In addition, topiramate increases GABA activity at its neuroreceptors, and an additional pharmacodynamic interaction must therefore also be considered. In light of the increasing therapeutic as well as illicit use of GHB, as well as of newer antiepileptic drugs, possible interactions should be evaluated in formal pharmacokinetic studies. In the mean time, we suggest using such combinations only with great care. T. Weiss : I. Marti :C. Happold Department of Neurology, University Hospital Zurich, Zurich, Switzerland

Transcriptional control of O6-methylguanine DNA methyltransferase expression and temozolomide resistance in glioblastoma

O6‐methylguanine DNA methyltransferase (MGMT) promoter methylation is a predictive biomarker for benefit from alkylating chemotherapy, specifically temozolomide (TMZ), in glioblastoma, the most common malignant intrinsic brain tumor. Glioma‐initiating cells (GIC) with stem‐like properties have been associated with resistance to therapy and progression. We assessed the levels of MGMT mRNA and MGMT protein by real‐time PCR and immunoblot and evaluated the impact of MGMT on TMZ sensitivity in clonogenicity assays in GIC sphere cultures (S) or differentiated adherent monolayer cultures (M). Nuclear factor kappa B (NF‐κB) signaling was assessed by reporter assay and immunoblot. Compared to M cells, S cells expressed higher levels of MGMT. Differentiation of GIC induced by S‐to‐M transition resulted in a gradual loss of MGMT expression and increased TMZ sensitivity. This transcriptional regulation of MGMT was restricted to cell lines without MGMT promoter methylation and was not coupled to any specific neurobasal (NB) stem cell medium supplement or loss of cell adhesion. Expression levels of p50/p65 subunits of NF‐κB, a transcriptional regulator of MGMT, were increased in S cells. Inhibition of NF‐κB by the small molecule inhibitor, BAY 11‐7082, or siRNA‐mediated gene silencing, reduced MGMT levels. In summary, alkylator resistance of S cells is mainly promoted by over‐expression of MGMT which results from increased activity of the NF‐κB pathway in this cell culture model of glioma stem‐like cells.

NKG2D-dependent anti-tumor effects of chemotherapy and radiotherapy against glioblastoma

Purpose: NKG2D is a potent activating immune cell receptor, and glioma cells express the cognate ligands (NKG2DL). These ligands are inducible by cellular stress and temozolomide (TMZ) or irradiation (IR), the standard treatment of glioblastoma, could affect their expression. However, a role of NKG2DL for the efficacy of TMZ and IR has never been addressed. Experimental Design: We assessed the effect of TMZ and IR on NKG2DL in vitro and in vivo in a variety of murine and human glioblastoma models, including glioma-initiating cells, and a cohort of paired glioblastoma samples from patients before and after therapy. Functional effects were studied with immune cell assays. The relevance of the NKG2D system for the efficacy of TMZ and IR was assessed in vivo in syngeneic orthotopic glioblastoma models with blocking antibodies and NKG2D knockout mice. Results: TMZ or IR induced NKG2DL in vitro and in vivo in all glioblastoma models, and glioblastoma patient samples had increased levels of NKG2DL after therapy with TMZ and IR. This enhanced the immunogenicity of glioma cells in a NGK2D-dependent manner, was independent from cytotoxic or growth inhibitory effects, attenuated by O6-methylguanine-DNA-methyltransferase (MGMT), and required the DNA damage response. The survival benefit afforded by TMZ or IR relied on an intact NKG2D system and was decreased upon inhibition of the NKG2D pathway. Conclusions: The immune system may influence the activity of convential cancer treatments with particular importance of the NKG2D pathway in glioblastoma. Our data provide a rationale to combine NKG2D-based immunotherapies with TMZ and IR. Clin Cancer Res; 24(4); 882–95. ©2017 AACR.

A tissue-based draft map of the murine MHC class I immunopeptidome

The large array of peptides presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC class I immunopeptidome. Although the MHC class I immunopeptidome is ubiquitous in mammals and represents a critical component of the immune system, very little is known, in any species, about its composition across most tissues and organs in vivo. We applied mass spectrometry (MS) technologies to draft the first tissue-based atlas of the murine MHC class I immunopeptidome in health. Peptides were extracted from 19 normal tissues from C57BL/6 mice and prepared for MS injections, resulting in a total number of 28,448 high-confidence H2Db/Kb-associated peptides identified and annotated in the atlas. This atlas provides initial qualitative data to explore the tissue-specificity of the immunopeptidome and serves as a guide to identify potential tumor-associated antigens from various cancer models. Our data were shared via PRIDE (PXD008733), SysteMHC Atlas (SYSMHC00018) and SWATH Atlas. We anticipate that this unique dataset will be expanded in the future and will find wide applications in basic and translational immunology.