L-RNA aptamer-based CXCL12 inhibition combined with radiotherapy in newly-diagnosed glioblastoma: dose escalation of the phase I/II GLORIA trial.

The chemokine CXCL12 promotes glioblastoma (GBM) recurrence after radiotherapy (RT) by facilitating vasculogenesis. Here we report outcomes of the dose-escalation part of GLORIA (NCT04121455), a phase I/II trial combining RT and the CXCL12-neutralizing aptamer olaptesed pegol (NOX-A12; 200/400/600 mg per week) in patients with incompletely resected, newly-diagnosed GBM lacking MGMT methylation. The primary endpoint was safety, secondary endpoints included maximum tolerable dose (MTD), recommended phase II dose (RP2D), NOX-A12 plasma levels, topography of recurrence, tumor vascularization, neurologic assessment in neuro-oncology (NANO), quality of life (QOL), median progression-free survival (PFS), 6-months PFS and overall survival (OS). Treatment was safe with no dose-limiting toxicities or treatment-related deaths. The MTD has not been reached and, thus, 600 mg per week of NOX-A12 was established as RP2D for the ongoing expansion part of the trial. With increasing NOX-A12 dose levels, a corresponding increase of NOX-A12 plasma levels was observed. Of ten patients enrolled, nine showed radiographic responses, four reached partial remission. All but one patient (90%) showed at best response reduced perfusion values in terms of relative cerebral blood volume (rCBV). The median PFS was 174 (range 58-260) days, 6-month PFS was 40.0% and the median OS 389 (144-562) days. In a post-hoc exploratory analysis of tumor tissue, higher frequency of CXCL12+ endothelial and glioma cells was significantly associated with longer PFS under NOX-A12. Our data imply safety of NOX-A12 and its efficacy signal warrants further investigation.

H3K27M neoepitope vaccination in diffuse midline glioma induces B and T cell responses across diverse HLA loci of a recovered patient.

H3K27M, a driver mutation with T and B cell neoepitope characteristics, defines an aggressive subtype of diffuse glioma with poor survival. We functionally dissect the immune response of one patient treated with an H3K27M peptide vaccine who subsequently entered complete remission. The vaccine robustly expanded class II human leukocyte antigen (HLA)-restricted peripheral H3K27M-specific T cells. Using functional assays, we characterized 34 clonally unique H3K27M-reactive T cell receptors and identified critical, conserved motifs in their complementarity-determining region 3 regions. Using detailed HLA mapping, we further demonstrate that diverse HLA-DQ and HLA-DR alleles present immunogenic H3K27M epitopes. Furthermore, we identified and profiled H3K27M-reactive B cell receptors from activated B cells in the cerebrospinal fluid. Our results uncover the breadth of the adaptive immune response against a shared clonal neoantigen across multiple HLA allelotypes and support the use of class II-restricted peptide vaccines to stimulate tumor-specific T and B cells harboring receptors with therapeutic potential.

[Immunotherapy against gliomas]. / Immuntherapie gegen Gliome.

BACKGROUND: Gliomas represent the most frequent malignant primary brain tumors in adults. Despite multimodal treatment concepts involving surgery, irradiation and chemotherapy, the prognosis remains poor and they are incurable. Recent insights into the interactions between the immune system and the central nervous system as well as breakthroughs in the results of other cancer types have led to the fact that various immunotherapeutic approaches against gliomas have also been investigated and in some cases specifically developed. OBJECTIVE: This article provides an overview of the current status of different immunotherapeutic concepts against gliomas, highlighting the advantages, disadvantages, and challenges. Additionally, it provides an overview of currently ongoing immunotherapeutic clinical trials in Germany and neighboring countries. RESULTS: Previous randomized studies on antibodies against programmed cell death protein 1 (anti-PD1) immune checkpoint inhibition, viral treatment and peptide vaccination targeting the variant III of the epidermal growth factor receptor (EGFRvIII) in glioblastomas were negative with respect to survival benefits. Conversely, other immunotherapeutic approaches, such as multivalent or driver mutation-based vaccinations, cytokine-based therapy and cell therapy, demonstrated a robust scientific foundation, with at least early studies showing promising safety and pharmacodynamic effects on the tumors. DISCUSSION: Currently, immunotherapies against gliomas should only be applied within the framework of well-designed clinical studies. There are still many knowledge gaps regarding the mechanisms of action and resistance of various immunotherapies. Accompanying translational research is essential to address these gaps and develop more effective therapies.

Cerebrospinal Fluid cfDNA Sequencing for Classification of Central Nervous System Glioma.

PURPOSE: Primary central nervous system (CNS) gliomas can be classified by characteristic genetic alterations. In addition to solid tissue obtained via surgery or biopsy, cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is an alternative source of material for genomic analyses. EXPERIMENTAL DESIGN: We performed targeted next-generation sequencing of CSF cfDNA in a representative cohort of 85 patients presenting at two neurooncological centers with suspicion of primary or recurrent glioma. Copy-number variation (CNV) profiles, single-nucleotide variants (SNV), and small insertions/deletions (indel) were combined into a molecular-guided tumor classification. Comparison with the solid tumor was performed for 38 cases with matching solid tissue available. RESULTS: Cases were stratified into four groups: glioblastoma (n = 32), other glioma (n = 19), nonmalignant (n = 17), and nondiagnostic (n = 17). We introduced a molecular-guided tumor classification, which enabled identification of tumor entities and/or cancer-specific alterations in 75.0% (n = 24) of glioblastoma and 52.6% (n = 10) of other glioma cases. The overlap between CSF and matching solid tissue was highest for CNVs (26%-48%) and SNVs at predefined gene loci (44%), followed by SNVs/indels identified via uninformed variant calling (8%-14%). A molecular-guided tumor classification was possible for 23.5% (n = 4) of nondiagnostic cases. CONCLUSIONS: We developed a targeted sequencing workflow for CSF cfDNA as well as a strategy for interpretation and reporting of sequencing results based on a molecular-guided tumor classification in glioma. See related commentary by Abdullah, p. 2860.

Concurrent gliomas in patients with multiple sclerosis.

BACKGROUND: Concurrent malignant brain tumors in patients with multiple sclerosis (MS) constitute a rare but paradigmatic phenomenon for studying neuroimmunological mechanisms from both molecular and clinical perspectives. METHODS: A multicenter cohort of 26 patients diagnosed with both primary brain tumors and multiple sclerosis was studied for disease localization, tumor treatment-related MS activity, and molecular characteristics specific for diffuse glioma in MS patients. RESULTS: MS neither predisposes nor protects from the development of gliomas. Patients with glioblastoma WHO grade 4 without isocitratdehydrogenase (IDH) mutations have a longstanding history of MS, whereas patients diagnosed with IDH-mutant astrocytoma WHO grade 2 receive multiple sclerosis diagnosis mostly at the same time or later. Concurrent MS is associated with a lesser extent of tumor resection and a worse prognosis in IDH-mutant glioma patients (PFS 32 vs. 64 months, p = 0.0206). When assessing tumor-intrinsic differences no distinct subgroup-defining methylation pattern is identified in gliomas of MS patients compared to other glioma samples. However, differential methylation of immune-related genetic loci including human leukocyte antigen locus on 6p21 and interleukin locus on 5q31 is found in MS patients vs. matched non-MS patients. In line, inflammatory disease activity increases in 42% of multiple sclerosis patients after brain tumor radiotherapy suggesting a susceptibility of multiple sclerosis brain tissue to pro-inflammatory stimuli such as ionizing radiation. CONCLUSIONS: Concurrent low-grade gliomas should be considered in multiple sclerosis patients with slowly progressive, expansive T2/FLAIR lesions. Our findings of typically reduced extent of resection in MS patients and increased MS activity after radiation may inform future treatment decisions.

Brain tumors such as gliomas can evade attacks by the immune system. In contrast, some diseases of the central nervous system such as multiple sclerosis (MS) are caused by an overactive immune system. Our study looks at a cohort of rare patients with both malignant glioma and concurrent MS and examines how each disease and their treatments affect each other. Our data suggest that even in patients with known MS, if medical imaging findings are unusual, a concurrent brain tumor should be excluded at an early stage. Radiotherapy, as is the standard of care for malignant brain tumors, may worsen the inflammatory disease activity in MS patients, which may be associated with certain genetic risk factors. Our findings may help to inform treatment of patients with brain tumors and MS.

In vivo nanoparticle-based T cell imaging can predict therapy response towards adoptive T cell therapy in experimental glioma.

Rationale: Intrinsic brain tumors, such as gliomas are largely resistant to immunotherapies including immune checkpoint blockade. Adoptive cell therapies (ACT) including chimeric antigen receptor (CAR) or T cell receptor (TCR)-transgenic T cell therapy targeting glioma-associated antigens are an emerging field in glioma immunotherapy. However, imaging techniques for non-invasive monitoring of adoptively transferred T cells homing to the glioma microenvironment are currently lacking. Methods: Ultrasmall iron oxide nanoparticles (NP) can be visualized non-invasively by magnetic resonance imaging (MRI) and dedicated MRI sequences such as T2* mapping. Here, we develop a protocol for efficient ex vivo labeling of murine and human TCR-transgenic and CAR T cells with iron oxide NPs. We assess labeling efficiency and T cell functionality by flow cytometry and transmission electron microscopy (TEM). NP labeled T cells are visualized by MRI at 9.4 T in vivo after adoptive T cell transfer and correlated with 3D models of cleared brains obtained by light sheet microscopy (LSM). Results: NP are incorporated into T cells in subcellular cytoplasmic vesicles with high labeling efficiency without interfering with T cell viability, proliferation and effector function as assessed by cytokine secretion and antigen-specific killing assays in vitro. We further demonstrate that adoptively transferred T cells can be longitudinally monitored intratumorally by high field MRI at 9.4 Tesla in a murine glioma model with high sensitivity. We find that T cell influx and homogenous spatial distribution of T cells within the TME as assessed by T2* imaging predicts tumor response to ACT whereas incomplete T cell coverage results in treatment resistance. Conclusion: This study showcases a rational for monitoring adoptive T cell therapies non-invasively by iron oxide NP in gliomas to track intratumoral T cell influx and ultimately predict treatment outcome.

Targeting the aryl hydrocarbon receptor (AhR) with BAY 2416964: a selective small molecule inhibitor for cancer immunotherapy.

BACKGROUND: The metabolism of tryptophan to kynurenines (KYN) by indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase is a key pathway of constitutive and adaptive tumor immune resistance. The immunosuppressive effects of KYN in the tumor microenvironment are predominantly mediated by the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor that broadly suppresses immune cell function. Inhibition of AhR thus offers an antitumor therapy opportunity via restoration of immune system functions. METHODS: The expression of AhR was evaluated in tissue microarrays of head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). A structure class of inhibitors that block AhR activation by exogenous and endogenous ligands was identified, and further optimized, using a cellular screening cascade. The antagonistic properties of the selected AhR inhibitor candidate BAY 2416964 were determined using transactivation assays. Nuclear translocation, target engagement and the effect of BAY 2416964 on agonist-induced AhR activation were assessed in human and mouse cancer cells. The immunostimulatory properties on gene and cytokine expression were examined in human immune cell subsets. The in vivo efficacy of BAY 2416964 was tested in the syngeneic ovalbumin-expressing B16F10 melanoma model in mice. Coculture of human H1299 NSCLC cells, primary peripheral blood mononuclear cells and fibroblasts mimicking the human stromal-tumor microenvironment was used to assess the effects of AhR inhibition on human immune cells. Furthermore, tumor spheroids cocultured with tumor antigen-specific MART-1 T cells were used to study the antigen-specific cytotoxic T cell responses. The data were analyzed statistically using linear models. RESULTS: AhR expression was observed in tumor cells and tumor-infiltrating immune cells in HNSCC, NSCLC and CRC. BAY 2416964 potently and selectively inhibited AhR activation induced by either exogenous or endogenous AhR ligands. In vitro, BAY 2416964 restored immune cell function in human and mouse cells, and furthermore enhanced antigen-specific cytotoxic T cell responses and killing of tumor spheroids. In vivo, oral application with BAY 2416964 was well tolerated, induced a proinflammatory tumor microenvironment, and demonstrated antitumor efficacy in a syngeneic cancer model in mice. CONCLUSIONS: These findings identify AhR inhibition as a novel therapeutic approach to overcome immune resistance in various types of cancers.

INTERCEPT H3: a multicenter phase I peptide vaccine trial for the treatment of H3-mutated diffuse midline gliomas.

INTRODUCTION: Diffuse midline gliomas (DMG) are universally lethal central nervous system tumors that carry almost unanimously the clonal driver mutation histone-3 K27M (H3K27M). The single amino acid substitution of lysine to methionine harbors a neoantigen that is presented in tumor tissue. The long peptide vaccine H3K27M-vac targeting this major histocompatibility complex class II (MHC class II)-restricted neoantigen induces mutation-specific immune responses that suppress the growth of H3K27M+ flank tumors in an MHC-humanized rodent model. METHODS: INTERCEPT H3 is a non-controlled open label, single arm, multicenter national phase 1 trial to assess safety, tolerability and immunogenicity of H3K27M-vac in combination with standard radiotherapy and the immune checkpoint inhibitor atezolizumab (ATE). 15 adult patients with newly diagnosed K27M-mutant histone-3.1 (H3.1K27M) or histone-3.3 (H3.3K27M) DMG will be enrolled in this trial. The 27mer peptide vaccine H3K27M-vac will be administered concomitantly to standard radiotherapy (RT) followed by combinatorial treatment with the programmed death-ligand 1 (PD-L1) targeting antibody ATE. The first three vaccines will be administered bi-weekly (q2w) followed by a dose at the beginning of recovery after RT and six-weekly administrations of doses 5 to 11 thereafter. In a safety lead-in, the first three patients (pts. 1-3) will be enrolled sequentially. PERSPECTIVE: H3K27M-vac is a neoepitope targeting long peptide vaccine derived from the clonal driver mutation H3K27M in DMG. The INTERCEPT H3 trial aims at demonstrating (1) safety and (2) immunogenicity of repeated fixed dose vaccinations of H3K27M-vac administered with RT and ATE in adult patients with newly diagnosed H3K27M-mutant DMG. TRIAL REGISTRATION: NCT04808245.

A H3K27M-targeted vaccine in adults with diffuse midline glioma.

Substitution of lysine 27 to methionine in histone H3 (H3K27M) defines an aggressive subtype of diffuse glioma. Previous studies have shown that a H3K27M-specific long peptide vaccine (H3K27M-vac) induces mutation-specific immune responses that control H3K27M+ tumors in major histocompatibility complex-humanized mice. Here we describe a first-in-human treatment with H3K27M-vac of eight adult patients with progressive H3K27M+ diffuse midline glioma on a compassionate use basis. Five patients received H3K27M-vac combined with anti-PD-1 treatment based on physician's discretion. Repeat vaccinations with H3K27M-vac were safe and induced CD4+ T cell-dominated, mutation-specific immune responses in five of eight patients across multiple human leukocyte antigen types. Median progression-free survival after vaccination was 6.2 months and median overall survival was 12.8 months. One patient with a strong mutation-specific T cell response after H3K27M-vac showed pseudoprogression followed by sustained complete remission for >31 months. Our data demonstrate safety and immunogenicity of H3K27M-vac in patients with progressive H3K27M+ diffuse midline glioma.

Activity-regulated cytoskeleton-associated protein/activity-regulated gene 3.1 (Arc/Arg3.1) enhances dendritic cell vaccination in experimental melanoma.

Dendritic cell (DC) vaccination has proven to be an effective and safe adjuvant for cancer immunotherapies. As the presence of DCs within the tumor microenvironment promotes adaptive antitumor immunity, enhancement of DC migration toward the tumor microenvironment following DC vaccination might represent one possible approach to increase its therapeutic efficacy. While recent findings suggest the activity-regulated cytoskeleton-associated protein/activity-regulated gene 3.1 (Arc/Arg3.1) as critical regulator of DC migration in the context of autoimmune diseases, we aimed to investigate the impact of Arc/Arg3.1 expression for DC-based cancer vaccines. To this end, DC migration capacity as well as the induction of T cell-mediated antitumor immunity was assessed in an experimental B16 melanoma model with Arc/Arg3.1-/- and Arc/Arg3.1-expressing BMDCs applied as a subcutaneous vaccine. While antigen presentation on DCs was critical for unleashing effective T cell mediated antitumor immune responses, Arc/Arg3.1 expression enhanced DC migration toward the tumor and secondary lymphoid organs. Moreover, Arc/Arg3.1-expressing BMDCs shape the tumor immune microenvironment by facilitating tumor recruitment of antigen-specific effector T cells. Thus, Arc/Arg3.1 may represent a novel therapeutic target in DCs in order to increase the therapeutic efficacy of DC vaccination.

Molecular profiling-based decision for targeted therapies in IDH wild-type glioblastoma.

BACKGROUND: Molecular profiling allows tumor classification as well as assessment of diagnostic, prognostic, and treatment-related molecular changes. Translation into clinical practice and relevance for patients has not been demonstrated yet. METHODS: We analyzed clinical and molecular data of isocitrate dehydrogenase wild-type glioblastoma patients with sufficient clinical follow-up from the Heidelberg Neuro-Oncology Center and with molecular analysis of tumor tissue that consisted of DNA methylation array data, genome-scale copy number variations, gene panel sequencing, and partly mTOR immunohistochemistry between October 2014 and April 2018. RESULTS: Of 536 patients screened, molecular assessment was performed in 253 patients (47%) in a prospective routine clinical setting with further clinical appointments. Therapy decision was directly based on the molecular assessment in 97 (38%) patients. Of these, genetic information from MGMT (n = 68), EGFR (n = 7), CDKN2A/B (n = 8), alterations of the PI3K-AKT-mTOR pathway (n = 5), and BRAF (n = 3) have been the most frequently used for decision making with a positive overall survival signal for patients with glioblastoma harboring an unmethylated MGMT promoter treated according to the molecular assignment. Based on detected molecular alterations and possible targeted therapies, we generated an automated web-based prioritization algorithm. CONCLUSION: Molecular decision making in clinical practice was mainly driven by MGMT promoter status in elderly patients and study inclusion criteria. A reasonable number of patients have been treated based on other molecular aberrations. This study prepares for complex molecular decisions in a routine clinical decision making.

Autoimmune diseases and immunosuppressive therapy in relation to the risk of glioma.

Effectors from the immune system can modulate the course and possibly the early development of gliomas. We, therefore, hypothesized that autoimmune diseases associated with increased immune-surveillance may also modulate the risk of human glioma. To test this hypothesis, we used data from the well-validated Clinical Practice Research Datalink (CPRD) GOLD from the UK to analyze the association of immune-related disorders or use of immunosuppressive drugs and the risk of glioma. We identified 3112 incident glioma cases diagnosed between 1995 and 2017. We randomly selected up to 10 controls, matching them to glioma cases on age, sex, index date, general practice, and number of years of active history in the database prior to the index date. We performed conditional logistic regression analyses to estimate Odds Ratios (ORs) of glioma among those exposed to allergies, autoimmune diseases, and immunosuppressive drugs. Overall, we found no materially altered association between a history of any autoimmune disease (OR 0.98, 95% CI 0.86-1.11), allergy (OR 0.97, 95% CI 0.89-1.05), or use of immunosuppressive drugs and the risk of glioma. However, subgroup analyses among younger patients found a statistically significant increased risk of glioma in patients with a history of inflammatory bowel disease (IBD) (OR 2.59, 95% CI 1.31-5.12). There was also an inverse association between asthma and risk of glioma in patients with longer survival (OR 0.73, 95% CI 0.58-0.91) and between long-term duration diabetes and risk of glioma (OR 0.71, 95% CI 0.53-0.96).