Joseph P. Antonios

pH-weighted molecular imaging of gliomas using amine chemical exchange saturation transfer MRI

BACKGROUND Interstitial tissue acidosis resulting from abnormal perfusion and metabolism is a hallmark of cancer. The current study demonstrates that chemical exchange saturation transfer (CEST) MRI can be used as a noninvasive pH-weighted molecular imaging technique by targeting the chemical exchange between amine protons and protons in extracellular bulk water. METHODS First, the sensitivity of amine CEST was validated in phantoms under a variety of conditions, including different magnetic field strengths, amino acid concentrations, and pH values. Amine CEST was compared with histology in both a preclinical GL261 intracranial glioma model at 7T and human patients at 3T. The association between physiologic and pH-weighted MRI was explored, along with the ability to predict time to progression to radiochemotherapy in 20 glioblastoma patients. RESULTS z-Spectral asymmetry increased at 3 ppm (amine range) on CEST MRI with decreasing pH within the range observed in tumors for both 3T and 7T scanners. Lesions with acidic signatures showed active tumor and pseudopalisading tumor on histology and showed elevated FDOPA PET uptake, lactate on MR spectroscopy, and perfusion abnormalities. Patients with acidic lesions after surgery or stable/growing acidic lesions had a shorter time to progression following radiochemotherapy compared with patients with lesions demonstrating relatively low acidity (P < .001). CONCLUSION Results suggest pH-weighted MRI may provide new insight into brain tumor physiology beyond traditional imaging technologies.

Immunosuppressive tumor-infiltrating myeloid cells mediate adaptive immune resistance via a PD-1/PD-L1 mechanism in glioblastoma

Background Adaptive immune resistance in the tumor microenvironment appears to attenuate the immunotherapeutic targeting of glioblastoma (GBM). In this study, we identified a tumor-infiltrating myeloid cell (TIM) population that expands in response to dendritic cell (DC) vaccine treatment. The aim of this study was to understand how this programmed death ligand 1 (PD-L1)-expressing population restricts activation and tumor-cytolytic function of vaccine-induced tumor-infiltrating lymphocytes (TILs). Methods To test this hypothesis in our in vivo preclinical model, we treated mice bearing intracranial gliomas with DC vaccination ± murine anti-PD-1 monoclonal antibody (mAb) blockade or a colony stimulating factor 1 receptor inhibitor (CSF-1Ri) (PLX3397) and measured overall survival. We then harvested and characterized the PD-L1+ TIM population and its role in TIL activation and tumor cytolysis in vitro. Results Our data indicated that the majority of PD-L1 expression in the GBM environment is contributed by TIMs rather than by tumor cells themselves. While PD-1 blockade partially reversed the TIL dysfunction, targeting TIMs directly with CSF-1Ri altered TIM expression of key chemotactic factors associated with promoting increased TIL infiltration after vaccination. Neither PD-1 mAb nor CSF-1Ri had a demonstrable therapeutic benefit alone, but when combined with DC vaccination, a significant survival benefit was observed. When the tripartite regimen was given (DC vaccine, PD-1 mAb, PLX3397), long-term survival was noted together with an increase in the number of TILs and TIL activation. Conclusion Together, these studies elucidate the role that TIMs play in mediating adaptive immune resistance in the GBM microenvironment and provide evidence that they can be manipulated pharmacologically with agents that are clinically available. Development of immune resistance in response to active vaccination in GBM can be reversed with dual administration of CSF-1Ri and PD-1 mAb.

Efficacy of systemic adoptive transfer immunotherapy targeting NY-ESO-1 for glioblastoma.

BACKGROUND Immunotherapy is an ideal treatment modality to specifically target the diffusely infiltrative tumor cells of malignant gliomas while sparing the normal brain parenchyma. However, progress in the development of these therapies for glioblastoma has been slow due to the lack of immunogenic antigen targets that are expressed uniformly and selectively by gliomas. METHODS We utilized human glioblastoma cell cultures to induce expression of New York-esophageal squamous cell carcinoma (NY-ESO-1) following in vitro treatment with the demethylating agent decitabine. We then investigated the phenotype of lymphocytes specific for NY-ESO-1 using flow cytometry analysis and cytotoxicity against cells treated with decitabine using the xCelligence real-time cytotoxicity assay. Finally, we examined the in vivo application of this immune therapy using an intracranially implanted xenograft model for in situ T cell trafficking, survival, and tissue studies. RESULTS Our studies showed that treatment of intracranial glioma-bearing mice with decitabine reliably and consistently induced the expression of an immunogenic tumor-rejection antigen, NY-ESO-1, specifically in glioma cells and not in normal brain tissue. The upregulation of NY-ESO-1 by intracranial gliomas was associated with the migration of adoptively transferred NY-ESO-1-specific lymphocytes along white matter tracts to these tumors in the brain. Similarly, NY-ESO-1-specific adoptive T cell therapy demonstrated antitumor activity after decitabine treatment and conferred a highly significant survival benefit to mice bearing established intracranial human glioma xenografts. Transfer of NY-ESO-1-specific T cells systemically was superior to intracranial administration and resulted in significantly extended and long-term survival of animals. CONCLUSION These results reveal an innovative, clinically feasible strategy for the treatment of glioblastoma.

TCR sequencing can identify and track glioma-infiltrating T cells after DC vaccination

A clinically translatable platform was developed to track T-cell populations without prior knowledge of their specificity. TCR sequencing data could be used to distinguish patients with glioblastoma who will benefit and are benefitting from immunotherapy. Although immunotherapeutic strategies are emerging as adjunctive treatments for cancer, sensitive methods of monitoring the immune response after treatment remain to be established. We used a novel next-generation sequencing approach to determine whether quantitative assessments of tumor-infiltrating lymphocyte (TIL) content and the degree of overlap of T-cell receptor (TCR) sequences in brain tumors and peripheral blood were predictors of immune response and overall survival in glioblastoma patients treated with autologous tumor lysate–pulsed dendritic cell immunotherapy. A statistically significant correlation was found between a higher estimated TIL content and increased time to progression and overall survival. In addition, we were able to assess the proportion of shared TCR sequences between tumor and peripheral blood at time points before and after therapy, and found the level of TCR overlap to correlate with survival outcomes. Higher degrees of overlap, or the development of an increased overlap following immunotherapy, was correlated with improved clinical outcome, and may provide insights into the successful, antigen-specific immune response. Cancer Immunol Res; 4(5); 412–8. ©2016 AACR.

Epithelial membrane protein-2 (EMP2) promotes angiogenesis in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor and is associated with an extremely poor clinical prognosis. One pathologic hallmark of GBM is excessive vascularization with abnormal blood vessels. Extensive investigation of anti-angiogenic therapy as a treatment for recurrent GBM has been performed. Bevacizumab, a monoclonal anti-vascular endothelial growth factor A (VEGF-A), suggests a progression-free survival benefit but no overall survival benefit. Developing novel anti-angiogenic therapies are urgently needed in controlling GBM growth. In this study, we demonstrate tumor expression of epithelial membrane protein-2 (EMP2) promotes angiogenesis both in vitro and in vivo using cell lines from human GBM. Mechanistically, this pro-angiogenic effect of EMP2 was partially through upregulating tumor VEGF-A levels. A potential therapeutic effect of a systemic administration of anti-EMP2 IgG1 on intracranial xenografts was observed resulting in both significant reduction of tumor load and decreased tumor vasculature. These results suggest the potential for anti-EMP2 IgG1 as a promising novel anti-angiogenic therapy for GBM. Further investigation is needed to fully understand the molecular mechanisms how EMP2 modulates GBM pathogenesis and progression and to further characterize anti-EMP2 therapy in GBM.

Detection of immune responses after immunotherapy in glioblastoma using PET and MRI

Significance The inability to accurately monitor glioblastoma tumor progression vs. pseudoprogression has severely limited clinical treatment decisions, especially in the setting of immunotherapy. We have identified a novel noninvasive imaging combination that could distinguish intracranial immune responses from tumor progression in mice bearing orthotopic gliomas and in patients with glioblastomas. We combined the use of advanced MRI with PET imaging of deoxycytidine kinase, an enzyme overexpressed in immune cells. This combination resulted in superior differentiation between immune responses and tumors within the brain, and identified peripheral lymph nodes in which immune responses occurred after immunotherapy combinations. This combined imaging approach may provide a useful method to clinically monitor patients with glioblastomas treated with immune-based therapies, and to distinguish tumor progression from pseudoprogression. Contrast-enhanced MRI is typically used to follow treatment response and progression in patients with glioblastoma (GBM). However, differentiating tumor progression from pseudoprogression remains a clinical dilemma largely unmitigated by current advances in imaging techniques. Noninvasive imaging techniques capable of distinguishing these two conditions could play an important role in the clinical management of patients with GBM and other brain malignancies. We hypothesized that PET probes for deoxycytidine kinase (dCK) could be used to differentiate immune inflammatory responses from other sources of contrast-enhancement on MRI. Orthotopic malignant gliomas were established in syngeneic immunocompetent mice and then treated with dendritic cell (DC) vaccination and/or PD-1 mAb blockade. Mice were then imaged with [18F]-FAC PET/CT and MRI with i.v. contrast. The ratio of contrast enhancement on MRI to normalized PET probe uptake, which we term the immunotherapeutic response index, delineated specific regions of immune inflammatory activity. On postmortem examination, FACS-based enumeration of intracranial tumor-infiltrating lymphocytes directly correlated with quantitative [18F]-FAC PET probe uptake. Three patients with GBM undergoing treatment with tumor lysate-pulsed DC vaccination and PD-1 mAb blockade were also imaged before and after therapy using MRI and a clinical PET probe for dCK. Unlike in mice, [18F]-FAC is rapidly catabolized in humans; thus, we used another dCK PET probe, [18F]-clofarabine ([18F]-CFA), that may be more clinically relevant. Enhanced [18F]-CFA PET probe accumulation was identified in tumor and secondary lymphoid organs after immunotherapy. Our findings identify a noninvasive modality capable of imaging the host antitumor immune response against intracranial tumors.

Cell-Based Immunotherapy of Gliomas

Current cell-based immunotherapeutic strategies attempt to produce and maintain an immune response against glioma cells by artificially stimulating the immune system using passive and/or active approaches. Cellular immunotherapy is taken to mean the administration of live immune cells that either have immune effector capabilities themselves (passive immunotherapy) or engender a downstream antitumor response (active immunotherapy). Passive cellular immunotherapy most often takes the form of the adoptive transfer of a range of cell types, whereby antitumor immune cells from a patient (or allogeneic donor) are created, activated, and/or expanded ex vivo and subsequently administered back to the patient to directly attack the neoplasm. Active cellular immunotherapy approaches for the treatment of malignant gliomas have most often taken the form of dendritic cell (DC)-based vaccines.

Dendritic Cell Therapy for Brain Tumors

Abstract Glioblastomas are characterized by immunosuppression, rapid proliferation, angiogenesis, and invasion into the surrounding brain parenchyma. The limitations of current treatment options have prompted the development of alternate modalities, such as immunotherapy. Among these, active immunotherapy using dendritic cells (DCs) has emerged as an attraction option for glioma treatment. A variety of vaccine formulations have been used to take advantage of the specialized antigen presenting abilities of these cells to potentiate antitumor immune responses. Data acquired in multiple preclinical models and clinical trials have shown evidence of immune responses correlate with prolonged survival. Here we provide an overview of the current status of DC therapy for the treatment of malignant gliomas. Recent research has shown that DC vaccination relies on the complicated relationship between tumor cells, their surrounding microenvironment, and the immune system. This chapter aims to explain the concept and various forms of DC therapy, completed and ongoing clinical trials, current data on the safety, and clinical effects of DC vaccination areas of ongoing and future research.

Abstract 767: TCR sequencing can identify and track tumor-specific T cell populations and is a predictive biomarker of response to DC vaccination in glioblastoma patients

While immunotherapeutic strategies are emerging adjunctive treatments for cancer, sensitive methods of monitoring the immune response after treatment remain to be established. We used a novel next generation sequencing (NGS) approach to determine whether quantitative assessments of tumor infiltrating lymphocyte (TIL) content and the degree of overlap of T cell receptor (TCR) sequences in brain tumors and peripheral blood were predictors of immune response and overall survival in glioblastoma (GBM) patients treated with autologous tumor lysate-pulsed dendritic cell (DC) immunotherapy. A significant correlation was found between a higher estimated TIL content and increased time to progression (TTP) and overall survival (OS). In addition, we were able to assess the proportion of shared TCR sequences between tumor and peripheral blood at time points before and after therapy, and found the level of TCR overlap to correlate with survival outcomes. Higher degrees of overlap, or the development of an increased overlap following immunotherapy, correlated with improved clinical outcome, and may provide insights into the successful, antigen-specific immune response. Citation Format: Shaina Sedighim, Melody Hsu, Tina Wang, Richard G. Everson, Alex Tucker, Joseph P. Antonios, Lin Du, Ryan Emerson, Erik Yusko, Catherine Sanders, Harlan Robins, William Yong, Tom B. Davidson, Gang Li, Linda M. Liau, Robert Prins. TCR sequencing can identify and track tumor-specific T cell populations and is a predictive biomarker of response to DC vaccination in glioblastoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 767.

Adjuvant inhibition of iAPC function in the tumor microenvironment promotes therapeutic immunity in the setting of vaccination-induced T cell anti-tumor response.

Meeting abstracts Glioma tumor lysate-pulsed dendritic cell (DC) vaccination is an effective treatment modality. However, cure rates in the established tumor setting are not therapeutically significant in our preclinical models. We inferred that immunosuppressive antigen presenting cells (iAPCs)