Karolina Woroniecka

Prospect of rindopepimut in the treatment of glioblastoma

ABSTRACT Introduction: Rindopepimut (CDX-110) is a peptide vaccine that targets epidermal growth factor receptor variant III (EGFRvIII), a tumor-specific epitope expressed in the most common and lethal primary malignant neoplasm of the brain – glioblastoma (GBM). Areas covered: The EGFRvIII mutation introduces an 801 base pair in-frame deletion of the extracellular domain of the transmembrane tyrosine kinase, resulting in constitutive kinase activity, amplification of cell growth, and inhibition of apoptosis. Rindopepimut contains a 14mer amino acid peptide spanning the EGFRvIII mutation site that is conjugated to keyhole limpet hemocyanin (KLH). The EGFRvIII neoantigen is exclusively present on GBM cells, providing rindopepimut tumor-specific activity. The authors review rindopepimut’s clinical efficacy, administration, safety, and prospects in the treatment of GBM. Expert opinion: Rindopepimut showed clinical benefit and significant efficacy in phase II clinical trials, including as part of a multi-immunotherapy approach. A phase III clinical trial was terminated early, however, as it was deemed likely the study would fail to meet its primary endpoint. Longer term and sub-group analyses will be necessary to better understand rindopepimut’s future role in GBM therapy.

T-Cell Exhaustion Signatures Vary with Tumor Type and Are Severe in Glioblastoma

Purpose: T-cell dysfunction is a hallmark of glioblastoma (GBM). Although anergy and tolerance have been well characterized, T-cell exhaustion remains relatively unexplored. Exhaustion, characterized in part by the upregulation of multiple immune checkpoints, is a known contributor to failures amid immune checkpoint blockade, a strategy that has lacked success thus far in GBM. This study is among the first to examine, and credential as bona fide, exhaustion among T cells infiltrating human and murine GBM. Experimental Design: Tumor-infiltrating and peripheral blood lymphocytes (TILs and PBLs) were isolated from patients with GBM. Levels of exhaustion-associated inhibitory receptors and poststimulation levels of the cytokines IFNγ, TNFα, and IL2 were assessed by flow cytometry. T-cell receptor Vβ chain expansion was also assessed in TILs and PBLs. Similar analysis was extended to TILs isolated from intracranial and subcutaneous immunocompetent murine models of glioma, breast, lung, and melanoma cancers. Results: Our data reveal that GBM elicits a particularly severe T-cell exhaustion signature among infiltrating T cells characterized by: (1) prominent upregulation of multiple immune checkpoints; (2) stereotyped T-cell transcriptional programs matching classical virus-induced exhaustion; and (3) notable T-cell hyporesponsiveness in tumor-specific T cells. Exhaustion signatures differ predictably with tumor identity, but remain stable across manipulated tumor locations. Conclusions: Distinct cancers possess similarly distinct mechanisms for exhausting T cells. The poor TIL function and severe exhaustion observed in GBM highlight the need to better understand this tumor-imposed mode of T-cell dysfunction in order to formulate effective immunotherapeutic strategies targeting GBM. Clin Cancer Res; 24(17); 4175–86. ©2018 AACR. See related commentary by Jackson and Lim, p. 4059

Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors

T cell dysfunction contributes to tumor immune escape in patients with cancer and is particularly severe amidst glioblastoma (GBM). Among other defects, T cell lymphopenia is characteristic, yet often attributed to treatment. We reveal that even treatment-naïve subjects and mice with GBM can harbor AIDS-level CD4 counts, as well as contracted, T cell–deficient lymphoid organs. Missing naïve T cells are instead found sequestered in large numbers in the bone marrow. This phenomenon characterizes not only GBM but a variety of other cancers, although only when tumors are introduced into the intracranial compartment. T cell sequestration is accompanied by tumor-imposed loss of S1P1 from the T cell surface and is reversible upon precluding S1P1 internalization. In murine models of GBM, hindering S1P1 internalization and reversing sequestration licenses T cell–activating therapies that were previously ineffective. Sequestration of T cells in bone marrow is therefore a tumor-adaptive mode of T cell dysfunction, whose reversal may constitute a promising immunotherapeutic adjunct.Patients with glioblastoma experience lymphopenia and sequestration of T cells in the bone marrow, which is recapitulated in mice with brain tumors, where the reversible nature of this effect is demonstrated by an approach that enables the efficacy of other immunotherapeutics.

T-Cell Dysfunction in Glioblastoma: Applying a New Framework

A functional, replete T-cell repertoire is an integral component to adequate immune surveillance and to the initiation and maintenance of productive antitumor immune responses. Glioblastoma (GBM), however, is particularly adept at sabotaging antitumor immunity, eliciting severe T-cell dysfunction that is both qualitative and quantitative. Understanding and countering such dysfunction are among the keys to harnessing the otherwise stark potential of anticancer immune-based therapies. Although T-cell dysfunction in GBM has been long described, newer immunologic frameworks now exist for reclassifying T-cell deficits in a manner that better permits their study and reversal. Herein, we divide and discuss the various T-cell deficits elicited by GBM within the context of the five relevant categories: senescence, tolerance, anergy, exhaustion, and ignorance. Categorization is appropriately made according to the molecular bases of dysfunction. Likewise, we review the mechanisms by which GBM elicits each mode of T-cell dysfunction and discuss the emerging immunotherapeutic strategies designed to overcome them. Clin Cancer Res; 24(16); 3792–802. ©2018 AACR.

MTAP loss correlates with an immunosuppressive profile in GBM and its substrate MTA stimulates alternative macrophage polarization

Glioblastoma (GBM) is a lethal brain cancer known for its potent immunosuppressive effects. Loss of Methylthioadenosine Phosphorylase (MTAP) expression, via gene deletion or epigenetic silencing, is one of the most common alterations in GBM. Here, we show that MTAP loss in GBM cells is correlated with differential expression of immune regulatory genes. In silico analysis of gene expression profiles in GBM samples revealed that low MTAP expression is correlated with reduced proportions of γδT cells, fewer activated CD4 cells, and an increased proportion of M2 macrophages. Using in vitro macrophage models, we found that methylthioadenosine (MTA), the metabolite that accumulates as a result of MTAP loss in GBM cells, promotes the immunosuppressive alternative activation (M2) of macrophages. We show that this effect of MTA on macrophages is independent of IL4/IL3 signaling, is mediated by the adenosine A2B receptor, and can be pharmacologically reversed. This study suggests that MTAP loss in GBM cells contributes to the immunosuppressive microenvironment, and that MTAP status should be a factor for consideration in understanding GBM immune states and devising immunotherapy-based approaches for treating MTAP-null GBM.

T-cell exhaustion in glioblastoma

Glioblastoma (GBM) is the most common primary malignant brain tumor and remains universally lethal. Median survival is persistently less than two years, despite modest improvements to standard of care. Immunotherapies, though FDA-approved in other solid tumors, are hampered in GBM by the tumor’s marked heterogeneity and immunosuppressive influences. Propagating multiple modes of T-cell dysfunction facilitates GBM’s ability to escape immunotherapeutic targeting [1, 2]. T-cell anergy [3] and tolerance [4, 5] are well-characterized in GBM patients, while other contributing manners of T-cell dysfunction, including sequestration [6], senescence, and exhaustion [7], are beginning to be further explored [8]. We uncovered recently that T-cell exhaustion likely makes substantial contributions to dysfunction among T-cells that successfully arrive at GBM, and may provide direct mechanistic limitations to the efficacy of checkpoint blockade [7]. Exhaustion is a hypo-responsive T-cell state resulting from chronic antigenic exposure under sub-optimal conditions. It was initially described amidst chronic viral infection (such as with chronic lymphocytic choriomeningitis virus (LCMV)) but is increasingly appreciated in cancer. Exhaustion represents a specific transcriptional program that is often characterized by up-regulation of the various co-inhibitory receptors constituting immune checkpoints. Blocking the classical immune checkpoints, PD-1 and CTLA-4, to rejuvenate T-cell function is an FDA-approved strategy in many cancers, yet a phase III clinical trial of PD-1 blockade demonstrated limited efficacy against GBM. Recent work has shown that one mode of resistance to PD-1 blockade involves the emergence of alternative immune checkpoints / exhaustion markers on T-cells, such as TIM-3 and LAG3 [9]. We therefore sought to determine the prevalence of exhaustion and these alternative immune checkpoints among T-cells in GBM. We found that GBM does indeed elicit a severe exhaustion signature amidst T-cells [7]. T-cells infiltrating human GBM tumors (TIL) were found to express multiple immune checkpoints, including PD-1, TIM-3, LAG-3, TIGIT, and CD39. TIL were likewise less able to secrete the cytokines IFN-γ, IL-2, or TNF-α than T-cells isolated from patient or control blood. Among patient samples, triple-positive PD-1+TIM-3+LAG-3+ CD8+ T-cells were least functional compared to PD-1 negative and PD-1 single-positive cells, showing that mounting expression of the alternative immune checkpoints TIM-3 and LAG3 resulted in loss of function. Importantly, we noted that CD8+ TIL expressing PD-1 alone remained functional, highlighting that expression of PD-1 alone may represent a state of activation rather than exhaustion. In seeking to determine whether T-cell exhaustion arises preferentially in tumor-specific T-cells in human GBM, we found a surprising and disappointing lack of clonal expansion in human GBM-infiltrating CD8+ T-cells. Interestingly, CD4+ TIL demonstrated ample clonal expansion and, despite expressing multiple immune checkpoints, remained notably functional. These findings suggest a high level of CD8+ T-cell dysfunction that evolves immediately upon tumor infiltration (i.e. not after chronic exposure), imply differing susceptibilities of CD8+ and CD4+ T-cells to tumor-imposed dysfunction, and underscore the importance of functional studies over phenotype in proclaiming T-cell exhaustion. We recapitulated our patient findings in two orthotopic murine models of GBM (SMA-560 and CT2A). Both models demonstrated high levels of tripleEditorial

Flow Cytometric Identification of Tumor-Infiltrating Lymphocytes from Glioblastoma

We describe an isolation method of tumor-infiltrating lymphocytes (TILs) from glioblastoma tumors for the purpose of analysis by flow cytometry. This protocol is unique from many others in that the use of a selective lymphocyte isolation procedure, such as a Ficoll or Percoll gradient, is not used. We find that staining of TILs and analysis by flow cytometry is not affected by the presence of heterogeneous populations, while other selective isolation procedures can significantly decrease lymphocyte yield from already rare populations.