Advanced immunotherapies for glioblastoma: tumor neoantigen vaccines in combination with immunomodulators.

Glial-origin brain tumors, including glioblastomas (GBM), have one of the worst prognoses due to their rapid and fatal progression. From an oncological point of view, advances in complete surgical resection fail to eliminate the entire tumor and the remaining cells allow a rapid recurrence, which does not respond to traditional therapeutic treatments. Here, we have reviewed new immunotherapy strategies in association with the knowledge of the immune micro-environment. To understand the best lines for the future, we address the advances in the design of neoantigen vaccines and possible new immune modulators. Recently, the efficacy and availability of vaccine development with different formulations, especially liposome plus mRNA vaccines, has been observed. We believe that the application of new strategies used with mRNA vaccines in combination with personalized medicine (guided by different omic's strategies) could give good results in glioma therapy. In addition, a large part of the possible advances in new immunotherapy strategies focused on GBM may be key improving current therapies of immune checkpoint inhibitors (ICI), given the fact that this type of tumor has been highly refractory to ICI.

On optimal temozolomide scheduling for slowly growing glioblastomas.

Background: Temozolomide (TMZ) is an oral alkylating agent active against gliomas with a favorable toxicity profile. It is part of the standard of care in the management of glioblastoma (GBM), and is commonly used in low-grade gliomas (LGG). In-silico mathematical models can potentially be used to personalize treatments and to accelerate the discovery of optimal drug delivery schemes. Methods: Agent-based mathematical models fed with either mouse or patient data were developed for the in-silico studies. The experimental test beds used to confirm the results were: mouse glioma models obtained by retroviral expression of EGFR-wt/EGFR-vIII in primary progenitors from p16/p19 ko mice and grown in-vitro and in-vivo in orthotopic allografts, and human GBM U251 cells immobilized in alginate microfibers. The patient data used to parametrize the model were obtained from the TCGA/TCIA databases and the TOG clinical study. Results: Slow-growth "virtual" murine GBMs benefited from increasing TMZ dose separation in-silico. In line with the simulation results, improved survival, reduced toxicity, lower expression of resistance factors, and reduction of the tumor mesenchymal component were observed in experimental models subject to long-cycle treatment, particularly in slowly growing tumors. Tissue analysis after long-cycle TMZ treatments revealed epigenetically driven changes in tumor phenotype, which could explain the reduction in GBM growth speed. In-silico trials provided support for implementation methods in human patients. Conclusions: In-silico simulations, in-vitro and in-vivo studies show that TMZ administration schedules with increased time between doses may reduce toxicity, delay the appearance of resistances and lead to survival benefits mediated by changes in the tumor phenotype in slowly-growing GBMs.

IDP-410: a Novel Therapeutic Peptide that Alters N-MYC Stability and Reduces Angiogenesis and Tumor Progression in Glioblastomas.

Glioblastomas (GBMs) are the most frequent and highly aggressive brain tumors, being resistant to all cytotoxic and molecularly targeted agents tested so far. There is, therefore, an urgent need to find novel therapeutic approaches and/or alternative targets to bring treatment options to patients. Here, we first show that GBMs express high levels of N-MYC protein, a transcription factor involved in normal brain development. A novel stapled peptide designed to specifically target N-MYC protein monomer, IDP-410, is able to impair the formation of N-MYC/MAX complex and reduce the stability of N-MYC itself. As a result, the viability of GBM cells is compromised. Moreover, the efficacy is found dependent on the levels of expression of N-MYC. Finally, we demonstrate that IDP-410 reduces GBM growth in vivo when administered systemically, both in subcutaneous and intracranial xenografts, reducing the vascularization of the tumors, highlighting a potential relationship between the function of N-MYC and the expression of mesenchymal/angiogenic genes. Overall, our results strengthen the view of N-MYC as a therapeutic target in GBM and strongly suggest that IDP-410 could be further developed to become a first-in-class inhibitor of N-MYC protein, affecting not only tumor cell proliferation and survival, but also the interplay between GBM cells and their microenvironment.

Impact of depatuxizumab mafodotin on health-related quality of life and neurological functioning in the phase II EORTC 1410/INTELLANCE 2 trial for EGFR-amplified recurrent glioblastoma.

BACKGROUND: In the EORTC 1410/INTELLANCE 2 randomised, phase II study (NCT02343406), with the antibody-drug conjugate depatuxizumab mafodotin (Depatux-M, ABT-414) in patients with recurrent EGFR-amplified glioblastoma, the primary end-point (overall survival) was not met, and the drug had ocular dose-limiting toxicity. This study reports results from the prespecified health-related quality of life (HRQoL) and neurological deterioration-free survival (NDFS) exploratory analysis. PATIENTS AND METHODS: Patients (n = 260) were randomised 1:1:1 to receive either Depatux-M 1.25 mg/kg or 1.0 mg/kg intravenously every 2 weeks with oral temozolomide (TMZ) 150 mg/m2, Depatux-M alone, or TMZ or oral lomustine (CCNU) 110 mg/m2 (TMZ/CCNU). HRQoL outcomes were recorded using the EORTC core Quality of Life QLQ-C30, and brain cancer-specific QLQ-BN20 questionnaires. Questionnaires were completed at baseline, weeks 8 and 16, and month 6, and changes from baseline to each time point were calculated. NDFS was defined as time to first deterioration in World Health Organisation performance status. RESULTS: Compliance with HRQoL was 88.1% at baseline and decreased to 37.9% at month 6. Differences from baseline between Depatux-M arms and TMZ/CCNU in global health/QoL status throughout treatment did not reach clinical relevance (≥10 points). Self-reported visual disorders deteriorated to a clinically relevant extent with Depatux-M arms versus TMZ/CCNU at all timepoints (mean differences range: 24.6-35.1 points). Changes from baseline for other HRQoL scales and NDFS were generally similar between treatment arms. CONCLUSIONS: Depatux-M had no impact on HRQoL and NDFS in patients with EGFR-amplified recurrent glioblastoma, except for more visual disorders, an expected side-effect of the study drug. CLINICAL TRIAL REGISTRATION: NCT02343406.

Immune Profiling of Gliomas Reveals a Connection with IDH1/2 Mutations, Tau Function and the Vascular Phenotype.

BACKGROUND: Gliomas remain refractory to all attempted treatments, including those using immune checkpoint inhibitors. The characterization of the tumor (immune) microenvironment has been recognized as an important challenge to explain this lack of response and to improve the therapy of glial tumors. METHODS: We designed a prospective analysis of the immune cells of gliomas by flow cytometry. Tumors with or without isocitrate dehydrogenase 1/2 (IDH1/2) mutations were included in the study. The genetic profile and the presence of different molecular and cellular features of the gliomas were analyzed in parallel. The findings were validated in syngeneic mouse models. RESULTS: We observed that few immune cells infiltrate mutant IDH1/2 gliomas whereas the immune content of IDH1/2 wild-type tumors was more heterogeneous. Some of them contained an important immune infiltrate, particularly enriched in myeloid cells with immunosuppressive features, but others were more similar to mutant IDH1/2 gliomas, with few immune cells and a less immunosuppressive profile. Notably, we observed a direct correlation between the percentage of leukocytes and the presence of vascular alterations, which were associated with a reduced expression of Tau, a microtubule-binding protein that controls the formation of tumor vessels in gliomas. Furthermore, overexpression of Tau was able to reduce the immune content in orthotopic allografts of GL261 cells, delaying tumor growth. CONCLUSIONS: We have confirmed the reduced infiltration of immune cells in IDH1/2 mutant gliomas. By contrast, in IDH1/2 wild-type gliomas, we have found a direct correlation between the presence of vascular alterations and the entrance of leukocytes into the tumors. Interestingly, high levels of Tau inversely correlated with the vascular and the immune content of gliomas. Altogether, our results could be exploited for the design of more successful clinical trials with immunomodulatory molecules.

The IDH-TAU-EGFR triad defines the neovascular landscape of diffuse gliomas.

Gliomas that express the mutated isoforms of isocitrate dehydrogenase 1/2 (IDH1/2) have better prognosis than wild-type (wt) IDH1/2 gliomas. However, how these mutant (mut) proteins affect the tumor microenvironment is still a pending question. Here, we describe that the transcription of microtubule-associated protein TAU (MAPT), a gene that has been classically associated with neurodegenerative diseases, is epigenetically controlled by the balance between wt and mut IDH1/2 in mouse and human gliomas. In IDH1/2 mut tumors, we found high expression of TAU that decreased with tumor progression. Furthermore, MAPT was almost absent from tumors with epidermal growth factor receptor (EGFR) mutations, whereas its trancription negatively correlated with overall survival in gliomas carrying wt or amplified (amp) EGFR We demonstrated that the overexpression of TAU, through the stabilization of microtubules, impaired the mesenchymal/pericyte-like transformation of glioma cells by blocking EGFR, nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and the transcriptional coactivator with PDZ-binding motif (TAZ). Our data also showed that mut EGFR induced a constitutive activation of this pathway, which was no longer sensitive to TAU. By inhibiting the transdifferentiation capacity of EGFRamp/wt tumor cells, TAU protein inhibited angiogenesis and favored vascular normalization, decreasing glioma aggressiveness and increasing their sensitivity to chemotherapy.

Ocoxin Modulates Cancer Stem Cells and M2 Macrophage Polarization in Glioblastoma.

Glioblastoma (GBM) is the most common and devastating primary brain tumor. The presence of cancer stem cells (CSCs) has been linked to their therapy resistance. Molecular and cellular components of the tumor microenvironment also play a fundamental role in the aggressiveness of these tumors. In particular, high levels of hypoxia and reactive oxygen species participate in several aspects of GBM biology. Moreover, GBM contains a large number of macrophages, which normally behave as immunosuppressive tumor-supportive cells. In fact, the presence of both, hypoxia and M2-like macrophages, correlates with malignancy and poor prognosis in gliomas. Antioxidant agents, as nutritional supplements, might have antitumor activity. Ocoxin® oral solution (OOS), in particular, has anti-inflammatory and antioxidant properties, as well as antitumor properties in several neoplasia, without known side effects. Here, we describe how OOS affects stem cell properties in certain GBMs, slowing down their tumor growth. In parallel, OOS has a direct effect on macrophage polarization in vitro and in vivo, inhibiting the protumoral features of M2 macrophages. Therefore, OOS could be a feasible candidate to be used in combination therapies during GBM treatment because it can target the highly resilient CSCs as well as their supportive immune microenvironment, without adding toxicity to conventional treatments.

Biomarker and Histopathology Evaluation of Patients with Recurrent Glioblastoma Treated with Galunisertib, Lomustine, or the Combination of Galunisertib and Lomustine.

Galunisertib, a Transforming growth factor-ßRI (TGF-ßRI) kinase inhibitor, blocks TGF-ß-mediated tumor growth in glioblastoma. In a three-arm study of galunisertib (300 mg/day) monotherapy (intermittent dosing; each cycle =14 days on/14 days off), lomustine monotherapy, and galunisertib plus lomustine therapy, baseline tumor tissue was evaluated to identify markers associated with tumor stage (e.g., histopathology, Ki67, glial fibrillary acidic protein) and TGF-ß-related signaling (e.g., pSMAD2). Other pharmacodynamic assessments included chemokine, cytokine, and T cell subsets alterations. 158 patients were randomized to galunisertib plus lomustine (n = 79), galunisertib (n = 39) and placebo+lomustine (n = 40). In 127 of these patients, tissue was adequate for central pathology review and biomarker work. Isocitrate dehydrogenase (IDH1) negative glioblastoma patients with baseline pSMAD2⁺ in cytoplasm had median overall survival (OS) 9.5 months vs. 6.9 months for patients with no tumor pSMAD2 expression (p = 0.4574). Eight patients were IDH1 R132H⁺ and had a median OS of 10.4 months compared to 6.9 months for patients with negative IDH1 R132H (p = 0.5452). IDH1 status was associated with numerically higher plasma macrophage-derived chemokine (MDC/CCL22), higher whole blood FOXP3, and reduced tumor CD3⁺ T cell counts. Compared to the baseline, treatment with galunisertib monotherapy preserved CD4⁺ T cell counts, eosinophils, lymphocytes, and the CD4/CD8 ratio. The T-regulatory cell compartment was associated with better OS with MDC/CCL22 as a prominent prognostic marker.

A Phase II randomized study of galunisertib monotherapy or galunisertib plus lomustine compared with lomustine monotherapy in patients with recurrent glioblastoma.

BACKGROUND: The combination of galunisertib, a transforming growth factor (TGF)-ß receptor (R)1 kinase inhibitor, and lomustine was found to have antitumor activity in murine models of glioblastoma. METHODS: Galunisertib (300 mg/day) was given orally 14 days on/14 days off (intermittent dosing). Lomustine was given as approved. Patients were randomized in a 2:1:1 ratio to galunisertib + lomustine, galunisertib monotherapy, or placebo + lomustine. The primary objective was overall survival (OS); secondary objectives were safety, pharmacokinetics (PKs), and antitumor activity. RESULTS: One hundred fifty-eight patients were randomized: galunisertib + lomustine (N = 79), galunisertib (N = 39), and placebo + lomustine (N = 40). Baseline characteristics were: male (64.6%), white (75.3%), median age 58 years, ECOG performance status (PS) 1 (63.3%), and primary glioblastoma (93.7%). The PKs of galunisertib were not altered with lomustine, and galunisertib had a median half-life of ∼8 hours. Median OS in months (95% credible interval [CrI]) for galunisertib + lomustine was 6.7 (range: 5.3-8.5), 8.0 (range: 5.7-11.7) for galunisertib alone, and 7.5 (range: 5.6-10.3) for placebo + lomustine. There was no difference in OS for patients treated with galunisertib + lomustine compared with placebo + lomustine [P (HR < 1) = 26%]. Median progression-free survival of ∼2 months was observed in all 3 arms. Among 8 patients with IDH1 mutation, 7 patients were treated with galunisertib (monotherapy or with lomustine); OS ranged from 4 to 17 months. Patients treated with galunisertib alone had fewer drug-related grade 3/4 adverse events (n = 34) compared with lomustine-treated patients (10% vs 26%). Baseline PS, post-discontinuation of bevacizumab, tumor size, and baseline levels of MDC/CCL22 were correlated with OS. CONCLUSIONS: Galunisertib + lomustine failed to demonstrate improved OS relative to placebo + lomustine. Efficacy outcomes were similar in all 3 arms. CLINICAL TRIAL REGISTRATION: NCT01582269, ClinicalTrials.gov.

First-in-human dose study of the novel transforming growth factor-ß receptor I kinase inhibitor LY2157299 monohydrate in patients with advanced cancer and glioma.

PURPOSE: TGFß signaling plays a key role in tumor progression, including malignant glioma. Small-molecule inhibitors such as LY2157299 monohydrate (LY2157299) block TGFß signaling and reduce tumor progression in preclinical models. To use LY2157299 in the treatment of malignancies, we investigated its properties in a first-in-human dose (FHD) study in patients with cancer. EXPERIMENTAL DESIGN: Sixty-five patients (58 with glioma) with measurable and progressive malignancies were enrolled. Oral LY2157299 was given as a split dose morning and evening on an intermittent schedule of 14 days on and 14 days off (28-day cycle). LY2157299 monotherapy was studied in dose escalation (part A) first and then evaluated in combination with standard doses of lomustine (part B). Safety was assessed using Common Terminology Criteria for Adverse Events version 3.0, echocardiography/Doppler imaging, serum troponin I, and brain natriuretic peptide (BNP) levels. Antitumor activity was assessed by RECIST and Macdonald criteria. RESULTS: In part A, 16.6% (5/30) and in part B, 7.7% (2/26) of evaluable patients with glioma had either a complete (CR) or a partial response (PR). In both parts, 15 patients with glioma had stable disease (SD), 5 of whom had SD ≥ 6 cycles of treatment. Therefore, clinical benefit (CR+PR+SD ≥ 6 cycles) was observed in 12 of 56 patients with glioma (21.4%). LY2157299 was safe, with no cardiac adverse events. CONCLUSIONS: On the basis of the safety, pharmacokinetics, and antitumor activity in patients with glioma, the intermittent administration of LY2157299 at 300 mg/day is safe for future clinical investigation.

Cardiac Safety of TGF-ß Receptor I Kinase Inhibitor LY2157299 Monohydrate in Cancer Patients in a First-in-Human Dose Study.

Transforming growth factor-beta (TGF-ß) signaling plays an important role in the fetal development of cardiovascular organs and in the repair mechanisms of the heart. Hence, inhibitors of the TGF-ß signaling pathway require a careful identification of a safe therapeutic window and a comprehensive monitoring of the cardiovascular system. Seventy-nine cancer patients (67 glioma and 12 solid tumor) enrolled in a first-in-human dose study and received the TGF-ß inhibitor LY2157299 monohydrate (LY2157299) as monotherapy (n = 53) or in combination with lomustine (n = 26). All patients were monitored using 2D echocardiography/color and Spectral Doppler (2D Echo with Doppler) every 2 months, monthly electrocardiograms, thorax computer tomography scans every 6 months, and monthly serum brain natriuretic peptide (BNP), troponin I, cystatin C, high-sensitivity C-reactive protein (hs-CRP). Administration of LY2157299 was not associated with medically relevant cardiovascular toxicities, including patients treated ≥6 months (n = 13). There were no increases of troponin I, BNP, or hs-CRP or reduction in cystatin C levels, which may have been considered as signs of cardiovascular injury. Blood pressure was generally stable during treatment. Imaging with echocardiography/Doppler showed an increase in mitral and tricuspid valve regurgitation by two grades of severity in only one patient with no concurrent clinical symptoms of cardiovascular injury. Overall, this comprehensive cardiovascular monitoring for the TGF-ß inhibitor LY2157299 did not detect medically relevant cardiac toxicity and hence supports the evaluation of LY2157299 in future clinical trials.

Pharmacokinetic, pharmacodynamic and biomarker evaluation of transforming growth factor-ß receptor I kinase inhibitor, galunisertib, in phase 1 study in patients with advanced cancer.

Purpose Transforming growth factor-beta (TGF-ß) signaling plays a key role in epithelial-mesenchymal transition (EMT) of tumors, including malignant glioma. Small molecule inhibitors (SMI) blocking TGF-ß signaling reverse EMT and arrest tumor progression. Several SMIs were developed, but currently only LY2157299 monohydrate (galunisertib) was advanced to clinical investigation. Design The first-in-human dose study had three parts (Part A, dose escalation, n = 39; Part B, safety combination with lomustine, n = 26; Part C, relative bioavailability study, n = 14). Results A preclinical pharmacokinetic/pharmacodynamic (PK/PD) model predicted a therapeutic window up to 300 mg/day and was confirmed in Part A after continuous PK/PD. PK was not affected by co-medications such as enzyme-inducing anti-epileptic drugs or proton pump inhibitors. Changes in pSMAD2 levels in peripheral blood mononuclear cells were associated with exposure indicating target-related pharmacological activity of galunisertib. Twelve (12/79; 15%) patients with refractory/relapsed malignant glioma had durable stable disease (SD) for 6 or more cycles, partial responses (PR), or complete responses (CR). These patients with clinical benefit had high plasma baseline levels of MDC/CCL22 and low protein expression of pSMAD2 in their tumors. Of the 5 patients with IDH1/2 mutation, 4 patients had a clinical benefit as defined by CR/PR and SD ≥6 cycles. Galunisertib had a favorable toxicity profile and no cardiac adverse events. Conclusion Based on the PK, PD, and biomarker evaluations, the intermittent administration of galunisertib at 300 mg/day is safe for future clinical investigation.