4-1BB, also known as CD137, is a costimulatory receptor expressed on the surface of activated T cells as well as natural killer (NK) cells. 4-1BB plays a role in cytokine section, antiapoptotic signaling, NK cytotoxicity and promotion of T cell effector function.17 After binding its natural ligand 4-1BBL, 4-1BB induces intracellular signaling through TNFR-associated factor 2.18 Murine glioma models have shown in vivo antitumor activity from treatment with 4-1BB agonists. Radiotherapy combined with anti-4-1BB agonists can induce tumor eradication and prolong survival.19 Furthermore, this antitumor response correlates with increased tumor-infiltrating lymphocyte (TIL) density and Interferon gamma (IFNy). The combination of a 4-1BB agonist and a CTLA-4 inhibitor, plus radiation treatment has also been shown to improve survival and TIL trafficking in an intracranial model.20 Interestingly, depletion of CD4 T cells abrogated this effect to an even greater extent than CD8 depletion. Given this preclinical evidence for antitumoral efficacy and immune modulation, 4-1BB represents an attractive target for GBM and indeed is being explored in a clinical trial, NCT02658981, in patients with recurrent GBM.

Glucocorticoid-induced tumor necrosis factor (TNF)-related receptor (GITR) is highly expressed on T regulatory cells (Tregs) and induced by FOXP3 and NFkB signaling.21 Tregs are immunosuppressive lymphocytes which act to inhibit recognition and clearance of tumor cells.22 GITR ligand (TNFSF18) is expressed on APC. When GITR binds GITR ligand in concert with TCR stimulation, naïve T cells are activated, eventually leading to NF-kB mediated proliferation and cytokine production such as IL-2 and IFNy.23–25 Murine studies have shown that treatment with GITR agonists result in improved survival, increased immune cell infiltrates and robust cytokine production by TILs, but structural and functional differences between murine and human GITR exist.26 27 One study that evaluated the use of intracranial injections of a GITR agonist found improved overall survival (OS) and selective Treg depletion.28 Systemic administration of these anti-GITR monoclonal antibody (mab) had limited effects on mouse survival. Another study combining anti-GITR mab with stereotactic radiosurgery showed increased effector CD4 infiltration, as well as elevated IFNy, IL2, and TNFa production but this did not translate into survival benefit.29 The significance of the expression of GITR on Tregs is less clear. One study found no difference in the expression of GITR on peripheral blood cells between patients with GBM and healthy controls.30 In contrast, another report suggests that tumor growth upregulates GITR on intratumoral Tregs.31 There are multiple early phase trials evaluating GITR, but most are excluding patients with active central nervous system (CNS) metastasis and to date, there are no trials for patients with primary brain tumors. The GITR agonist BMS-986156 has recently demonstrated a favorable safety profile both alone and in combination with nivolumab. In a study of 66 patients, the most common side effect, occurring in 30%, was fever.32 A phase I GBM trial, NCT03707457 combining an anti-GITR agent with PD-1 blockade is currently recruiting.

Inducible costimulatory (ICOS) is expressed by T cells following TCR crosslinking and CD28 costimulation.5 Through its binding with the ICOS ligand (B7-H2), ICOS plays a role in a variety of immune processes including the regulation of T cell helper cells. The ICOS ligand protein and corresponding mRNA are expressed by gliomas and the neutralization of ICOS ligand subsequently reduces Th1 and Th2 cytokines.33 Chimeric antigen receptor (CAR) T cells targeting ICOS and epidermal growth factor receptor variant III (EGFRvIII) have demonstrated cytotoxicity against glioma (U87) cells in vitro, although concerns about the relevance of the U87 model to human GBM remain.34 35 These CAR T cells secreted IFNy as determined by cytokine release assay and suppressed tumor growth in a xenograph mouse model.

OX40 is a transmembrane glycoprotein expressed by Tregs and transiently expressed on T cells following TCR stimulation by viral antigen.36 37 OX40 agonism inhibits Treg immunosuppression, thereby leading to effector T cell proliferation.38 OX40 ligand is expressed on GBM tumor cells and high levels of OX40L mRNA are associated with prolonged progression-free survival in patients with GBM.39 Murine glioma models have shown that OX40 agonists can induce tumor regression and increase TILs.40 Similarly, combination immunotherapies have been investigated. One study combining anti-OX40 antibody plus dendritic cell (DC) vaccine plus local cranial radiation demonstrated tumor regression, TIL infiltration, and improved survival compared with mice treated with only two of the three modalities.41 Another study combining granulocyte macrophage colony stimulating factor-expressing cells and an OX40 agonist, showing that the combined therapy promoted Th1 responses while also decreasing Th2 response.42 Of note, coexpression of PD-1, TIM-3, and lymphocyte-activation gene 3 (LAG-3) was also reduced.

CD27, a member of the TNF receptor family, is expressed on naïve CD4 and CD8 T cells and upregulated with T cell activation. On activation by binding CD70, a surface antigen expressed on meningioma and glioma cells,43 CD27 promotes proliferation by facilitating entry into the cell cycle as well as promoting effector T cell differentiation.44 In vitro studies have shown that glioma cells respond to CD70 signaling. Cell lines engineered to produce soluble CD70 increase proliferation and IFN secretion of cocultured lymphocytes.45 Treating in vivo murine glioma models with both a CD70 specific CAR T therapy46 and a CD70 antibody-drug conjugate produced tumor regression.47 However, other studies have suggested that the CD27 pathway may actually suppress T lymphocytes. One study found that glioma cells expressing CD70 could induce T cell apoptosis while simultaneously inhibiting glioma growth.48 This finding may be explained by lytic NK activity or a mechanistic difference in the apoptosis-regulating Siva pathway between human and mice cells. Furthermore, CD70 overexpression has been shown to increase recruitment of immunosuppressive tumor-associated macrophages.49 NCT02924038 is currently examining the CD27 agonist varlilumab in patients with low-grade glioma. Another study, NCT02335918, is looking at the combination of varlilumab with PD-1 blockade among a variety of solid tumors including GBM. Finally, the clinical trial NCT03688178 is investigating varilumab in combination with a DC vaccine and is scheduled to begin soon pending new testing requirements from the FDA.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is constitutively expressed on activated Tregs.50 51 Additionally, CTLA-4 expression can be induced on cytotoxic T cells after their stimulation. CTLA-4 competes with the costimulatory molecule CD28 for the CD80 and CD86 ligands (B7-1 and B7-2, respectively), ultimately leading to T cell inactivation if CTLA-4 engagement predominates. Prognostically, CTLA-4 expression on CD4 and CD8 T cells may positively correlate with survival in patients with GBM.52 Several murine glioma models have shown antitumor efficacy and improved survival using species-specific mab blockade of CTLA-4. Although long-term tumor-free survival in mice is only modest with single agent anti-CTLA-4 therapy, combinational studies with anti-PD-1 or anti-PD-L1 have demonstrated cure rates as high as 75%.53 CTLA-4 antibody blockade has also been shown to enhance whole tumor-cell vaccine efficacy and improve survival.54 55 In mice treated with a herpes simplex oncolytic virus, the combination of anti-CTLA-4 and anti PD-1 therapy was found to improve survival.56 Furthermore, combined with intratumoral administration of IL-12, CTLA-4 inhibitors were shown to cause a reduction in tumor burden, decreasing Tregs and increasing effector T cells.57 Several early phase studies are examining CTLA-4 inhibitors in GBM. CheckMate 143, a phase I study of 40 patients with recurrent GBM treated with the PD-1 inhibitor nivolumab, alone or in combination with the CTLA inhibitor ipilimumab, showed a survival of only 7 to 10 months and response rates of 0%–11%.58 Additionally, nivolumab did not improve OS when compared with bevacizumab in this population.59 There remain several trials studying ipilimumab in conjunction with nivolumab. NCT03367715 is examining nivolumab and ipilimumab in conjunction with short-course radiotherapy. Similarly, NCT02829931 and NCT03425292 combine CTLA-4, PD-1 and bevacizumab with radiation therapy. NCT03233152 is investigating intratumoral administration of ipilimumab in conjunction with nivolumab after resection of recurrent GBM. NCT03430791 uses nivolumab and ipilimumab with tumor-treating fields. NCT03422094 combines dual checkpoint blockade with a personalized neoantigen-based vaccine (NeoVax). The pediatric study, CheckMate 908, is examining ipilimumab and nivolumab in childhood CNS malignancies. NCT02794883 is a study of the CTLA-4 inhibitor tremelimumab plus the PD-1 inhibitor durvalumab in recurrent glioma. Additionally, an expanded access program of ipilimumab is available in countries worldwide although it is currently not available in the USA and many European nations.

PD-1, is expressed on activated T cells. When PD-1 binds its ligand, programmed death ligand 1 (PD-L1), TCR signaling is downregulated, which in turn decreases T cell proliferation and activation. PD-1 has also been implicated in the activation of epithelial-mesenchymal transition60 and prevention of Treg expansion in glioma.61 Estimates of PD-L1 expression on human GBM have ranged between 19% and 88%, with some variation ascribed to the technique and antibody use.62–64 Prognostically, PD-1 expression on TILS and PD-L1 expression on tumor cells both correlate with glioma grade.65 Zeng et al, described improved survival in murine models with the combination of PD-1 inhibitor plus radiotherapy.66 Combining anti-PD-1 with temozolomide, the standard of care for treatment of GBM, produces decreased tumor growth, increased TILs and improved survival when compared with monotherapy.67–69 PD-1 blockade combined with oncolytic viral therapy has also been shown to increase survival in mice.70 71 DC vaccines in combination with PD-1 inhibitors likewise demonstrate improved murine survival.72 Finally, the combination of a PD-1 inhibitor and toll-like receptor (TLR-3) agonist therapy increased DC activation and T cell proliferation.73 A phase I study of 16 patients with recurrent GBM treated with atezolizumab reported a favorable safety profile but a disappointing median survival of only 4.2 months.74 Similarly, the phase I trial KEYNOTE-028 described 26 patients with recurrent PD-L1+GBM treated with pembrolizumab, observing an OS of 14.4 months and objective response rate of only 4%.75 However, a pilot trial of 35 patients with recurrent GBM recently found neoadjuvant pembrolizumab treatment arm to have an OS of 13.7 months, a statistically significant difference to the 7.5 month survival of those receiving adjuvant (post-surgical) pembrolizumab.3 While the OS is in keeping with historical expectations, the molecular findings suggest that treatment effect may actually be responsible for the discrepancies between the two cohort arms. In this study, neoadjuvant PD-1 blockade upregulated T-cell-γ and IFN-γ signals and downregulated cell-cycle-related transcripts. Focal expression of PD-L1 on tumor cells was also inducible by neoadjuvant treatment. TCR sequencing demonstrated enhanced clonal expansion in this cohort as well. A companion paper detailing 30 patients with GBM treated with neoadjuvant nivolumab likewise showed increases in chemokine transcripts, immune infiltration and TCR clonal diversity.76 Two patients on this study remained alive over 28 months later. Another study performed genomic and transcriptomic analysis of 66 patients with GBM treated with PD-1 inhibitors, highlighting alterations of PTEN in non-responders and the MAPK pathway in responders.77

There are numerous early phase trials examining PD-1 and PD-L1 targeting agents in gliomas, often in conjunction with radiation or bevacizumab. In addition to these, Checkmate 548 and 498 are phase III clinical trials examining checkpoint inhibitor therapy in MGMT methylated and unmethylated populations, respectively. A recent press release from CheckMate498 indicates that the study did not meet its primary endpoint of OS.78 NCT03491683 combines a PD-1 inhibitor with an IL-12 and antigen-stimulation strategy delivered by intramuscular injection and electroporation. Another interesting trial, NCT03347097, is examining the use of pluripotent immune killer cells constructed from transgenic-modified TILs to highly express PD-1. Combinational approaches with tyrosine kinase inhibitors (TKI) are also under investigation. GLIAVAX, a phase II trial of 54 patients with recurrent GBM treated with avelumab and axitinib demonstrated favorable tolerability but did not meet the primary endpoint goal with a 6-month progression-free survival (PFS-6) of only 18%.79 The addition of pembrolizumab to bevacizumab, a VEGF inhibitor, also did not improve PFS-6.80 Other TKI combinations being studied with PD-1 blockade include TTAC-001 (VEGFR-2/KDR) and lenvatinib (VEGFR1/2/3). Small molecule inhibitors such as vorinostate (histone deacetylase inhibitor), ipatasertib (AKT), olaparib (PARP), and epacadostat (indoleamine 2,3-dixoygenase-1) are also being under investigation in GBM. The NCT Neuro Master Match employs molecular characterization and bioinformatic evaluation to stratify patients toward multiple therapeutic arms including atezolizumab. Vaccine combinations currently under investigation include the IMA950 (multipeptide vaccine), HSPPC-96 (heat shock protein peptide complex), DNX-2401 (genetically modified oncolytic adenovirus), SurVaxM (survivin tumor-specific antigen), DCVax-L (autologous DC pulsed with tumor lysate antigen), VXM01 (VEGFR-2 DNA vaccine), NeoVax, and a IDH1 R132H-specific vaccine. Additionally, there are also CAR T strategies targeting EGFRvIII and PD-1. NCT02359565 and NCT03173950 are examining PD-1 inhibitors among rare or pediatric CNS tumors populations. Finally, NCT02311582 and NCT03341806 are studies focused on the effects of PD-1 inhibition in conjunction with MRI-guided laser ablation and laser interstitial thermal therapy, respectively.

LAG-3 is an immunoglobulin expressed on NK, DC, and B cells that plays an inhibitory role in T cell proliferation and cytokine secretion.81 In glioma samples analyzed by flow cytometry, 30% of CD8 TIL express LAG-3.82 Much lower expression (1.25%) was observed in CD4 TILs. One phase I study, NCT03493932, is examining LAG-3 inhibitor therapy after cytokine microdialysis and tumor resection in patients with recurrent GBM.

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a transmembrane protein which binds to CD155 and inhibits NK cell activity.83 TIGIT expression is also found on CD8 T cells, CD4 T cells, Tregs, and NK cells. Pediatric glial tumors contain CD4 and CD8 T cells which express TIGIT as well as TIM3, OX40, and 4-1BB.84 Combination treatment with TIGIT and PD-1 blockade was shown to increase cytotoxic CD8 cells, reduce Tregs and improved survival in a murine glioma model.85 PD-1 and TIGIT coblockage increased IFNy- and TNFa-producing CD8 (and CD4) T cells as compared with monotherapy groups. Tregs were also decreased but with no significant difference observed between combination and monotherapy groups. Reimplantation of tumor in the surviving animals demonstrated immune memory with no deaths at 90 days compared with a median survival of 21 days in a control group. There are currently no trials of TIGIT agents specifically in glioma or GBM. However, NCT03119428 is examining the anti-TIGIT agent OMP-313M32 in locally advanced, recurrent or metastatic solid tumor malignancies.

T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) is present on T cells, DC, macrophages, NK, and tumor cells. On binding galectin-9 and phosphatidylserine, TIM-3 induces T cell apoptosis, increasing phagocytosis and upregulating proinflammatory cytokine secretion.86 TIM-3 also binds carcinoembryonic antigen cell adhesion molecule 1 (CAECAM1) on activated T cells thereby inhibiting them.86 TIM-3 is expressed in higher grade glioma and negatively correlates with T-cell-mediated immune responses.87 Peripheral T lymphocyte expression of TIM-3 is also increased and correlates with worsened grade.88 89 Furthermore, this expression may be even higher in intratumoral effector lymphocytes.90 Combined antibody blockade of TIM-3 and its CAECAM ligand have been shown to prolong survival in GBM mouse models.91 This therapy increased the ratio of CD4/CD8 to Tregs among brain-infiltrating lymphocytes (BIL) and selective depletion of these CD4 and CD8 cell extinguished the survival effect. IFNy and TGF-β were upregulated and decreased, respectively, reflecting shift in the cytokine milieu. Triple therapy with PD-1 blockade, TIM-3 blockade, and radiation has also been demonstrated to improved survival compared with dual or monotherapy.92 Combination therapy also increased the CD8 effector/Treg ratio, IFNγ-producing CD4 cells and IFNγ-producing CD8 cells. Interestingly, BIL coexpression of PD-1 and TIM-3 were noted to increase with time. This suggests that the natural history of glioma may upregulate TIM-3, representing an immunosuppressive adaptation that can be countered with inhibitors. Following reimplantation, none of these long-term survivors established tumors, thus demonstrating that the mice had achieved immunological memory. A clinical trial, NCT03961971, combining the anti-TIM-3 inhibitor MBG453 with the PD-1 inhibitor spartalizumab and stereotactic radiosurgery is scheduled to begin August 2019.

Adenosine A2 receptor (A2AR) is a G protein-coupled receptor. Tissue breakdown and hypoxia promote extracellular adenosine production which in turn leads to anti-inflammatory effects mediated by these G protein-coupled receptors.93 Increased cyclic AMP upregulates immunosuppressive cytokines, increases PD-1, induces T cell anergy and promotes Treg differentiation. Taken together, these events create a more immunosuppressive tumor microenvironment. Inhibition of A2AR is, therefore, a potential method to stimulate the immune system. Several purine derivatives related to this have been identified as having in vivo activity against glioma. The surface ectoenzyme CD39 catabolizes proinflammatory ATP into AMP, and glioma cells have been shown to induce CD4 T cell suppression via CD39, an effect which is preventable with the introduction of A2AR antagonists.94

B7-H3, an immunomodulatory protein expressed on both lymphocytes and tumor cells, appears to have a host of complex effects on T lymphocytes. Initial reports of human B7-H3 identified a role in upregulating T cell proliferation, and it was noted that cytotoxicity with antibody blockade of B7-H3, or its potential ligand TLT-2, in turn suppresses T cell activation.95 96 However, more recent experiments of murine B7-H3 conversely suggest that B7-H3 may actually have suppressive effects on cytotoxic T cells by downregulating proinflammatory Th1 cells.97 98 B7-H3 expression in glioma correlates with higher grade and worsened prognosis.99 100 One study in murine glioma models found that B7-H3 gene silencing of B7-H3 leads to a less invasive phenotype.101 Together, these suggest that B7-H3 upregulation promotes tumor growth and invasion which, if true, could be through immune or non-immune related mechanisms.

V-domain Ig suppressor of T cell activation (VISTA), also known as PD-1H, is an inhibitory molecule expressed on hematopoietic cells and tumor cells. On binding an unknown receptor VISTA suppresses CD4 and CD8 T cell activation and promotes Treg proliferation.102 103 In murine glioma models (GL261), VISTA knockout was shown to prolong survival and synergism with radiation was observed.104 VISTA is not currently being studied in human trials.

There are a host of other immune checkpoint molecules which have been shown to influence the tumor microenviroment but remain early in development. B7-H4 (B7S1) is an inhibitory molecule expressed on APC which inhibits T cells.105 106 Additionally, B7-H4 is expressed by glioma stem-like cells (U251).107 B7-H4 expression has been shown to be associated with prognosis in GBM where it appears to mediate cross talk between glioma and macrophages via the IL-6/STAT3 signaling pathway.108 Other checkpoint molecules such as B and T lymphocyte attenuator (BTLA) have yet to be sufficiently described in glioma.