Immune checkpoint modulation: Tenets and implications in glioblastoma

John P Lynes; Victoria E Sanchez; Anthony K Nwankwo; Gifty A Dominah; Edjah K Nduom

doi:10.4103/glioma.glioma_47_18

REVIEW

Year : 2019 | Volume : 2 | Issue : 1 | Page : 20-29

Immune checkpoint modulation: Tenets and implications in glioblastoma

John P Lynes, Victoria E Sanchez, Anthony K Nwankwo, Gifty A Dominah, Edjah K Nduom
Brain Tumor Immunology Lab, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA

Date of Web Publication

1-Apr-2019

Correspondence Address:
Dr. Edjah K Nduom
10 Center Drive, 3D-20, Bethesda, MD 20814
USA

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/glioma.glioma_47_18

Abstract

Glioblastoma (GBM) is the most common primary central nervous system tumor, and despite advances made in traditional chemotherapy and radiation, it continues to carry a poor prognosis. The discovery of the profound immunosuppressive microenvironment created by GBM has given insight on the aggressiveness of this recalcitrant disease. This has led many to believe that immune therapy may yield the improvement in survival that the neuro-oncology community is seeking. In other cancers, the targeting of immune checkpoints has been the most promising immunotherapeutic strategy to date. Immune checkpoints modulate the function of the immune system by increasing or decreasing immune activity. Checkpoint inhibitors and more recently agonists target molecules that regulate immune response to increase immune function either directly or by removal of inhibitory signals. These molecules modulate immunity in the physiologic state to maintain homeostasis, but they are co-opted by cancer to avoid immune detection and attack. The use of checkpoint inhibition to improve cancer therapy has revolutionized the field of oncology, leading to unprecedented improvements in survival from many systemic malignancies. Utilizing PubMed and ClinicalTrials.gov to compile published findings and ongoing trials, we review immune checkpoints and their modulators from bench to bedside over several decades. In this review, the discovery of different checkpoint molecules and the development of drugs used to target them are addressed. In addition, the current state of checkpoint inhibition in GBM, presenting completed and ongoing preclinical and clinical studies utilizing these therapies, is discussed. Finally, we conclude by reviewing the current limitations and potential future directions for the use of checkpoint blockade in the treatment of GBM.

Keywords: 4-1BB, blockade, checkpoint, cytotoxic T-lymphocyte-associated protein 4, glioblastoma, glucocorticoid-induced tumor necrosis factor receptor, immunotherapy, inhibitor, lymphocyte-activating gene-3, OX-40, programmed cell death-1, T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain, T-cell immunoglobulin and mucin domain 3

How to cite this article:
Lynes JP, Sanchez VE, Nwankwo AK, Dominah GA, Nduom EK. Immune checkpoint modulation: Tenets and implications in glioblastoma. Glioma 2019;2:20-9

How to cite this URL:
Lynes JP, Sanchez VE, Nwankwo AK, Dominah GA, Nduom EK. Immune checkpoint modulation: Tenets and implications in glioblastoma. Glioma [serial online] 2019 [cited 2023 Jun 4];2:20-9. Available from: http://www.jglioma.com/text.asp?2019/2/1/20/255153

Victoria E. Sanchez and Anthony K. Nwankwo contributed equally.

Introduction

Glioblastoma (GBM) is an aggressive cancer of neoplastic glial cells. It is currently the most common primary brain tumor and has a particularly poor prognosis.^[1],[2] Standard-of-care therapy for GBM involves surgical resection with subsequent radiation and concomitant chemotherapy with temozolomide, followed by adjuvant temozolomide.^[3] Despite this aggressive treatment, the median overall survival for GBM remains disheartening at an average of 14–15 months following new diagnosis of tumor.^[3] Exploration of the tumor microenvironment reveals an immunosuppressive milieu of cells, some of which express immune regulatory checkpoints.^[4] Similar immunosuppressive environments have been found in other cancers, suggesting the potential benefit of treatment paradigms that utilize and optimize the function of the body's innate immune-mediated antitumor response. Since this revelation, multiple immune therapies have begun preclinical development and clinical investigation, including adoptive cell therapies, vaccines, viral therapies, antibodies, and checkpoint inhibitors.^[5] Each of these therapies takes a different strategy to potentiate an antitumor immune response.

Last year, the Nobel Prize in Physiology or Medicine was granted to James Allison of the University of Texas MD Anderson Cancer Center in the USA and Tasuku Honjo of Kyoto University in Japan^[6] for their discovery of checkpoint inhibition for cancer. These groundbreaking developments in cancer immunotherapy provide insight into how these treatments can be applied to GBM.^[7]

Immune checkpoints are molecular modulators of the immune system, which are responsible for balancing the microenvironment between anti-inflammatory and pro-inflammatory responses in the body.^[8] In the body's physiologic state, immune checkpoints play a key role in maintaining homeostasis by immune modulation to preclude autoimmunity.^[8] Typically, during T-lymphocyte co-activation, major histocompatibility complexes (MHCs) I and II found on antigen-presenting cells (APCs) present the target antigen alongside CD28/B7 binding.^[9] On activation, cytotoxic T-cells and T-helper cells prompt an antitumor immune-targeted response to the antigen presented. In addition, this response is augmented by pro-inflammatory cytokines which stimulate the activation of T-cells and memory B-cells.^[10] Immune checkpoints are cell surface proteins that predominantly regulate the potency of the immune response by instigating T-cell anergy and apoptosis,^[11] precluding T-cell co-stimulation and activation by APCs,^[12] and promoting T-regulatory cell (Treg) suppression of effector T-cell performance.^[13] However, other checkpoint molecules assist in immunomodulation by stimulating an immune response. In the tumor microenvironment, cancer cells co-opt these physiologic systems to evade detection and attack by the host immune system [Figure 1]. Checkpoint modulation for cancer therapy involves the use of a targeted small molecule inhibitor, agonist, or antibody that interacts with the checkpoint molecule, inducing an overall immune.stimulating effect at the tumor microenvironment [Figure 2]. Here, we discuss the advances of immune checkpoint inhibition in GBM as well as some challenges as investigators strive to apply these novel therapies to this fatal disease.

Figure 1: Checkpoint molecules have inhibitory and stimulatory effects on the immune system. (A) Inhibitory CTLA-4 outcompetes pro-inflammatory CD28 to bind to co-stimulatory factor B7.1/2 on APC leading to deactivation of the CD8 T-cell. (B) PD-L1 on regulatory T-cell or GBM binds to PD-1 on CD8 T-cell, leading to decreased activation of the CD8 T-cell resulting in immune suppression. PD-L1 can be found on APCs, effector T-cells, regulatory T-cells, and GBM. (C) TRP metabolite KYN secreted from IDO1 expressing GBM induces naive T-cell differentiation into T-regulatory cells which deactivate the CD8 T-cell. KYN also has a direct inhibitory effect on the CD8 T-cell further enhancing the T-regulatory cell immunosuppression. (D) LAG-3 outcompetes CD4 to bind to the MHC II complex on the APC, decreasing its co-stimulatory ability resulting in overall immunosuppression of the CD8 T-cell. (E) CD155 on APC binds to TIGIT on the CD8 T-cell to prevent the homodimerization of CD226, leading to a lack of co-stimulation. This results in a decreased activation of the CD8 T-cell. TIGIT is also found on effector T-cells, T-regulatory cells, and NK cells. (F) Binding of galectin secreted from the GBM to the TIM-3 receptor on Th1 cell decreases pro-inflammatory IFN-γ secretion, thereby decreasing the amount of activation signals the CD8 T-cell receives. TIM-3 is expressed on effector T-cells and NK cells. (G) GITRL on Th1 cell binds to GITR on the CD8 T-cell, inducing NF-kB to increase activation proliferation and cytokine secretion. GITRL is also expressed on T regulatory cells as well as effector T-cells. (H) CD137 L on NK cell activates CD8 T-cell by binding to CD137 to increase co-stimulatory signals. CD137 is also expressed on human effector T-cells, regulatory T-cells, as well as innate immune cells. (I) OX40 L on NK cell binds to the co-stimulatory OX40 on CD8 T-cell which also induces NF-κB, leading to its activation. OX40 is also expressed on effector T-cells. CTLA-4: Cytotoxic T-lymphocyte associated protein 4, APC: Antigen-presenting cell, PD-1: Programmed cell death-1, PD-L1: PD-1 ligand, GBM: Glioblastoma, TRP: Tryptophan, KYN: Kynurenic acid, IDO1: Indoleamine 2,3-dioxygenase-1, LAG-3: Lymphocyte-activating gene-3, MHC: Major histocompatibility complex, TIGIT: T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain, IFN-γ: Interferon-γ, TIM-3: T-cell immunoglobulin and mucin domain 3, NK: Natural killer, GITR: Glucocorticoid-induced tumor necrosis factor receptor, GITRL: GITR-related ligand, OX40: CD134, OX40 L: OX40 ligand, NF-κB: Nuclear factor-κB, Th1: Helper T-cell 1

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Figure 2: Checkpoint targeting inhibits or enhances checkpoint function. (A) Inhibitory anti-CTLA-4 mAbs bind to CTLA-4 blocking its inhibitory function to allow for the B7/CD28 interaction to provide co-stimulatory signals necessary to activate the CD8 T-cell. (B) Inhibitory anti-PD-L1 and anti-PD-1 mAbs bind to PD-L1 on the regulatory T-cell and PD-1 on the CD8 T-cell to decrease immune suppression and promote CD8 T-cell activation. (C) Small molecule inhibitor binds to IDO1 in GBM to inhibit TRP degradation into KYN, therefore decreasing the amount of available KYN to inhibit the CD8 T-cell activation and conversion of naive T-cells to immunosuppressive regulatory T-cells. (D) Inhibitory anti-LAG-3 mAbs bind to LAG-3 to allow for APCs to provide co-stimulatory signals to increase CD8 T-cell response via MHC II. (E) Inhibitory anti-TIGIT mAbs bind to TIGIT to increase CD8 T-cell response by restoring the ability of CD226 to homodimerize and thus provide co-stimulation. (F) Inhibitory anti-TIM-3 mAbs on Th1 cell bind to TIM-3 to increase INF-γ production which will activate the CD8 T-cell. (G) Agonistic anti-GITR mAbs can bind to GITR to induce NF-κB which will then activate the CD8 T-cell. (H) Agonistic anti-CD137 mAbs can bind to CD137 on CD8 T-cell to increase CD8 T-cell activation by providing increased co-stimulation. (I) CD8 T-cells can become more active by using agonistic anti-OX40 mAbs that bind to OX40 to induce more NF-κB. (J) Antagonistic and agonist mAbs enable the CD8 T-cell to become active and attack the GBM. mAbs: Monoclonal antibodies, CTLA-4: Cytotoxic T-lymphocyte associated protein 4, PD-1: Programmed cell death-1, PD-L1: PD-1 ligand, IDO1: Indoleamine 2,3-dioxygenase-1, GBM: Glioblastoma, TRP: Tryptophan, KYN: Kynurenic acid, LAG-3: Lymphocyte-activating gene-3, MHC: Major histocompatibility complex, TIGIT: T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain, TIM-3: T-cell immunoglobulin and mucin domain 3, IFN-γ: Interferon-γ, GITR: Glucocorticoid-induced tumor necrosis factor receptor, NF-κB: Nuclear factor-κB, OX40: CD134

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Methodology

This review was constructed by first outlining the foremost recognized checkpoint inhibitors. The authors then performed literature review via PubMed and ClinicalTrials.gov to find relevant articles with the structure of discussion of basic science, preclinical work, clinical work unrelated to GBM, and clinical work related to GBM where available. Keywords included “immune checkpoint, checkpoint inhibition, glioblastoma, CTLA-4, PD-1, CTLA-4, TIM-3, LAG-3, OX-40, 4-1BB, GITR,” and “TIGIT.”

Immune Checkpoints and Checkpoint Inhibition

Immunosuppressive checkpoints are targeted for inhibition in immune therapy because they attenuate the immune response and allow cancer cells to escape immune surveillance. Herein, we review immunosuppressive checkpoint molecules and their implications in cancer with a focus on glioblastoma.

Cytotoxic T-lymphocyte-associated protein 4

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) was first discovered in 1987,^[14] and its influence over T-cell function was elucidated in 1994.^[15] CTLA-4 is found on APCs and Tregs.^[16] It inhibits T-cell function by competitively binding to the B7 (CD80/CD86) co-stimulatory factor and prevents pro-inflammatory CD28/B7 signaling.^[17],[18] In parallel with this mechanism, CTLA-4 enhances the inhibitory function of Tregs and decreases the activity of T-helper lymphocyte activity.^[19]

After its success in effectively inducing tumor regression in murine models, CTLA-4 blockade was translated to several clinical trials.^[20],[21] Thereafter, a phase III checkpoint inhibition study showed a significant increase in overall survival for metastatic melanoma patients treated with anti-CTLA-4 in 2010.^[22] This led to the first Food and Drug Administration's (FDA) approval of a checkpoint inhibitor as ipilimumab was approved for the use in metastatic melanoma in 2011.^[23]

To support the use of CTLA-4 in the treatment of gliomas, Fecci et al.^[24] demonstrated long-term survival with anti-CTLA-4 monotherapy in an established murine glioma model. In addition, their group noted reversal of CD4 lymphopenia and T-cell dysfunction following anti-CLTA-4 therapy.^[24] In human GBM, levels of CTLA-4 expression on circulating CD4 and CD8 T-cells in patients undergoing immune therapy have been suggested as being predictive of treatment response.^[25] A small phase case series of only five patients treated with ipilimumab alone was promising, albeit with a high rate of immune adverse events.^[26] A subsequent study showed that ipilimumab combined with bevacizumab reduced adverse events while showing a partial therapeutic response in a subset of patients.^[27]

Programmed cell death-1

Programmed cell death-1 (PD-1) is a transmembrane protein on multiple immune cell types including B-cells, macrophages, T-cells, and dendritic cells.^[28] On binding of PD-1 with its ligand, PD-L1, nearby T-cell receptor (TCR) and its downstream components, such as CD28, are dephosphorylated by the cytoplasmic tail of PD-1,^[29] inactivating the stimulatory TCR complex. PD-1 and PD-L1 have recently been reported to interact on a single cell, preventing PD-1 activation on a nearby cell.^[30] While PD-L1 is physiologically expressed on APCs and Tregs, this signal has also been co-opted by disease. The Checkmate 067 trial showed improved survival for melanoma patients when anti-CTLA-4 and anti-PD-1 were used alone or in combination.^[31] In this trial, there was increased response to the drug related to degree of PD-L1 expression,^[32] which has been observed in other studies^[32] as well. The FDA approved pembrolizumab and nivolumab, anti-PD-1 checkpoint inhibitors, for use in treating metastatic melanoma patients as they showed efficacy with minimal toxicity.^[33],[34] Following this breakthrough for the immune-oncology field, nivolumab was then approved by the FDA for use in nonsmall cell lung cancer,^[35] head-and-neck cancer,^[36] renal cell carcinoma,^[37] and Hodgkin's lymphoma.^[38]

PD-1 and PD-L1 appear to play a significant clinical role in GBM with PD-L1 expression being associated with worse survival in patients with GBM.^{[39],[40],[41]} In 2013, Zeng et al.^[42] showed long-term survival in a murine glioma when PD-L1 blockade and radiation therapy were used. Consequently, with the aim of extending these positive findings, Checkmate 143 (NCT02017717), Checkmate 498 (NCT02617589), and Checkmate 548 (NCT02667587) trials began to investigate checkpoint inhibition in high-grade gliomas. Checkmate 143 focused on the use of an anti-PD-1 monoclonal antibody, nivolumab, in recurrent GBM patients, whereas Checkmate 498 and 548 focused on newly diagnosed GBM patients. Overall, Checkmate 143 concluded that nivolumab offers no survival benefit for GBM patients when compared to standard chemotherapy agents.^[43] Checkmate 498 is an ongoing phase III trial investigating nivolumab monotherapy versus temozolomide and radiation in newly diagnosed patients with O-6-methylguanine DNA methyltransferase-unmethylated GBM. Similarly, Checkmate 548 is an ongoing phase III trial comparing nivolumab with temozolomide and radiation with temozolomide and radiation alone in patients with newly diagnosed O-6-methylguanine DNA methyltransferase-methylated tumors. No results have yet been released from these trials.

Indoleamine 2,3-dioxygenase-1

Indoleamine 2,3-dioxygenase-1 (IDO1) is a tryptophan-catabolizing enzyme known to attenuate T-cell activity. Munn et al.^[44] first delineated its role in protecting the fetus from maternal T-lymphocytes which in turn prevented fetal rejection. The immunosuppressive effects of IDO1 expression are attributed to its ability to degrade tryptophan and generate kynurenic acid, a toxic tryptophan metabolite, which inactivates effector T-cells and suppresses dendritic cell function in the tumor microenvironment.^[45] In addition to this, IDO1 was demonstrated to promote differentiation of naive T-cells into Tregs, further enhancing its immunosuppressive effect.^[46] A preclinical application of IDO1 inhibitors in glioma models described by Wainwright et al.^[47] showed that IDO1 inhibitors in conjunction with CTLA-4 and PD-L1 monoclonal antibodies were able to increase median survival of GL261-injected mice and promote primary tumor rejection similar to CTLA-4 and PD-L1 dual blockade alone. In addition to having similar antitumoral responses, the IDO1 inhibitor with dual CTLA-4 and PD-L1 blockade was able to significantly reduce Treg cell frequency in the established brain tumors compared to the dual blockade alone.^[47] Furthermore, Wainwright et al.^[47] then demonstrated prolonged survival when using a combinatorial blockade approach, targeting IDO, PD-L1, and CTLA-4 in mice with gliomas. A recent clinical trial protocol using IDO1 inhibitors along with anti-glucocorticoid-induced tumor necrosis factor receptor (GITR), ipilimumab and nivolumab for the first recurrence of GBM, was published in November 2018, but this trial has not begun recruiting patients (NCT03707457).

Lymphocyte-activating gene-3

Although therapies targeting T-cell immune regulators such as PD-1 and CTLA-4 have shown clinical promise for many cancers, most patients are unresponsive to this approach. This highlights the need for additional treatment approaches. In this way, pathways that work through natural killer (NK) cells have generated increasing interest as an alternative. NK cells are effectors of the innate immune system and are especially important for rapid antitumor and antiviral immune responses via perforin-mediated cytotoxicity.^[48],[49] Lymphocyte-activating gene-3 (LAG-3) is a membrane protein expressed on NK cells, some T-lymphocytes, and APC.^[50] Similar to the mechanism of CTLA-4, LAG-3 competes with CD4 to interact with MHC II molecules preventing typical physiologic T-cell co-stimulation.^[22] By thwarting this co-stimulatory signal, helper T-cell function, including cytokine release, is decreased. Harris-Bookman et al.^[51] recently demonstrated that LAG-3 blockade as a monotherapy alone, and in combination with PD-1 blockade, provides long-term survival benefits in a murine glioma model. In addition, they demonstrate immune memory with re-challenge of these animals with reimplantation.^[51] LAG-3 inhibition was investigated in phase I/II trial for patients with metastatic breast cancer^[52] with low side effect profiles and clinical benefit reported in 90% of patients. Furthermore, a phase I trial in renal cell carcinoma^[53] reported disease stabilization in 88% of patients. Finally, an ongoing study is investigating anti-LAG3 in combination with anti-PD-1 blockade in recurrent GBM patients (NCT02658981) with no preliminary results yet reported.

T-cell immunoglobulin and mucin domain 3

T-cell immunoglobulin and mucin domain 3 (TIM-3) is transmembrane protein found on pro-inflammatory helper T-cells and cytotoxic T-cells as well as NK cells.^[54] TIM-3 is believed to be involved in preventing autoimmunity and loss of function and has been associated with autoimmune diseases, such as multiple sclerosis.^[55] This receptor protein has four active ligands that reportedly both increase and decrease activation of TCR functioning. Activation of this receptor by galectin-9 has been shown in vitro and in vivo to mediate loss of interferon-γ (IFN-γ) producing cells.^[56] However, transient TIM-3 expression has also been reported to increase TCR signaling in one model.^[57] Overall, TIM-3 appears to have a net inhibitory effect. Tomkowicz et al.^[58] showed that activation of TIM-3 inhibits TCR functioning by abrogating the downstream signaling of CD3/CD28 co-stimulation. Conversely, inactivation of TIM-3 via targeted antibodies has been shown to increase proliferation of T-cells and cytokine levels in vitro.^[59] TIM-3 can be found on tumor-infiltrating lymphocytes, particularly on forkhead box P3-positive Tregs, and deactivation of TIM-3 enhances immune targeting in cancer.^[60]

The inhibition of TIM-3 in conjunction with PD-1 has shown potential to be a strong candidate for immunotherapy when treating various cancers. A study conducted by Sakuishi et al.^[60] revealed TIM-3 and PD-1 dual blockade was able to inhibit tumor growth as well as ameliorate T-cell IFN-γ production in CT26 tumor-bearing mice. The study also showed that CD8⁺ tumor-infiltrating lymphocytes were predominately TIM-3+ and PD-1+, and the majority of the exhausted T-cells in the solid tumor mass were of this subset. A similar study by Liu et al.^[61] described the use of combined inhibition of TIM-3 and PD-1 in patients with colorectal cancer. They showed that CD8 T-cells that were TIM-3 and PD-1 positive exhibited a more severe exhausted state than the PD-1+/TIM-3− subset as shown by their attenuated ability to produce various cytokines such as IFN-γ, tumor necrosis factor-a (TNF-a), and interleukin (IL)-2. They also demonstrated that PD-1 blockade alone was not able to reverse T-cell exhaustion and promote antitumor activity as effectively as the dual blockade of both PD-1 and TIM-3. In addition to this, the dual blockade was able to increase tumor antigen-specific CD8 T-cell proliferation as well as decrease the frequency of Tregs which have been associated with poor vaccine efficacy.^[62] A clinical trial started in July 2016 utilizes TSR-022, a TIM-3 monoclonal antibody, along with anti-PD-1 antibodies to fight metastatic solid tumors malignancies, but the study remains in the recruitment stage (NCT02817633). In GBM, TIM-3 is a promising target, with a recent study demonstrating TIM-3 enrichment in these tumors and suggesting that TIM-3 is an independent indicator of poor prognosis.^[63] In addition, Kim et al.^[64] demonstrated modest response to anti-TIM-3 monotherapy in a murine glioma model. However, when combined with PD-1 blockade, radiation, or both, the therapeutic index was significantly increased. Despite promising preclinical data, there are no ongoing clinical trials targeting TIM-3 in GBM.

T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain

T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), a novel immunoreceptor, has gained recent attention as it is expressed on NK cells and T-cells, including Tregs and known for its immunosuppressive phenotype.^[65] TIGIT was first described by Yu et al.^[66] in 2009 as promoting immune regulation by way of poliovirus receptor (PVR or CD155) binding. Here, it was found to modify the expression of cytokines via dendritic cells and specifically led to an upregulation of IL-10.^[55] With continued interest in TIGIT as a potential immune therapy target, the TIGIT/CD226 pathway was elucidated further in the context of several cancer models.^[67] Similar to the co-stimulatory and co-inhibitory relationship of CD28 and CTLA-4, CD226 is the co-stimulatory receptor that competes with TIGIT for PVR binding.^[54],[56] Johnston et al.^[67] reported that TIGIT blocks the homodimerization of CD226, thereby directly inhibiting its T-cell stimulatory function in CT26 colorectal carcinoma murine models. Work therein also demonstrated synergistic anti-TIGIT and anti-PD-1 blockade operating mostly through CD8 T-cell-mediated antitumor immunity. These results showed complete responses for mice treated with the co-blockade of TIGIT/PD-1. The therapeutic response in this model correlated with an increase in IFN-γ production, and similar results were observed in EMT6 breast carcinoma models.^[56] Furthermore, when turning specifically to central nervous system (CNS) solid tumors, Hung et al.^[68] showed complete responses with anti-TIGIT in combination with anti-PD-1 in the GL261-luc glioma murine model. Similar to Johnston et al.'s^[67] findings in lung squamous cell carcinoma, Hung et al.^[68] found that TIGIT and PVR are frequently expressed on tumor-infiltrating CD8 T-cells of GBM patients, suggesting that elucidation of TIGIT may show significant clinical promise. At the time of submission, there are three ongoing trials investigating anti-TIGIT antibodies in conjunction with nivolumab for advanced solid cancers (NCT02913313, NCT03119428), and a third anti-TIGIT therapy is being applied to PD-L1-selected nonsmall cell lung cancer patients in combination with atezolizumab (NCT03563716).

Immune Checkpoints and Checkpoint Agonist

A majority of immune checkpoints are immunosuppressive in normal physiology. However, several checkpoint molecules have been identified that have a stimulatory relationship on multiple immune cell populations.

CD137

CD137 (4-1BB) is a co-stimulatory receptor first identified on murine T-cells by Kwon and Weissman^[69] in 1989. Since that time, this receptor has been detected on human effector T-cells, Tregs, NK cells, as well as innate immune cells.^[70] CD137 interacts with its solitary ligand, 4-1BBL, and activates the transcription factor nuclear factor-κB (NF-κB). NF-κB activation promotes T-cell proliferation and the production of IFN-γ, IL-2,^[71] perforin, and granzyme for potent cytotoxicity.^[72]

The use of agonistic antibodies in conjunction with a checkpoint inhibitor has been shown to generate a strong antitumor response in preclinical models. Kocak et al.^[73] used agonistic anti-CD137 antibodies in combination with inhibitory anti-CTLA-4 antibodies to enhance CD8 T-cell antitumor activity in response to M38 tumors in mice, while preventing autoimmunity. As stated earlier, CTLA-4-blocking antibodies are effective in eliciting a strong immune response to tumor antigen, but a drawback of this strategy is its tendency to induce autoimmunity as reported in multiple animal models^[74],[75] and patients.^[76] Kocak et al.^[73] proposed that the antibodies increased tumor immunity by two independent mechanisms which allow for each antibody to compensate for the side effect of the other. A phase I clinical trial using agonistic CD137 antibodies began in December 2005 and showed promising results. The CD137 antibodies were administered every 3 weeks with escalating dosages to patients with renal cell carcinoma, melanoma, or ovarian or prostate cancer. Results showed an increase in circulating CD8 and CD4 T-cells, INF-inducible genes in the peripheral blood, and serum neopterin levels following a single treatment.^[77] Several studies of combination therapies utilizing 4-1BB agonists have been conducted following this clinical trial. Two trials combining this therapy with ipilimumab (NCT00803374) and chemoradiation (NCT00461110) were withdrawn and terminated, respectively. However, combination with rituximab reported promising results (NCT01775631). Trials combining 4-1BB agonists with elotuzumab (NCT02252263) and cetuximab (NCT02110082) are ongoing, but outcomes have not yet been reported.

Glucocorticoid-induced tumor necrosis factor receptor

Another immunostimulating immune checkpoint, GITR, is a cell surface co-stimulatory molecule found on both effector T-cells and Tregs.^[78] Similar to CD137, activation of this receptor induces NF-κB,^[79] with a subsequent increase in IFN-γ and IL-2 production and T-cells proliferation.^[80] Preclinical studies in a variety of non-CNS solid tumors, including melanoma,^[81] colon cancer,^[82] and bladder cancer,^[83] showed promising survival results. Many of these studies found increased infiltration of CD4 and CD8 cells with decreased Tregs within the tumors.^[84] Miska et al.^[85] reported similar findings in a murine glioma model in different mouse strains with significant improvements in overall survival. Despite promising preclinical studies and ongoing phase I studies in other cancers, there are no active clinical trials targeting GITR in human GBM.

CD134

CD134 (OX40) is another member of the TNF receptor family similar to CD137 and GITR. CD134 is found on CD4 and CD8 effector T-cells^[86] as well as NK cells. Expression on NK cells is dependent on the presence of activated T-lymphocytes and is associated with antitumor killing by NK cells.^[87] CD134 is induced by TCR/CD3 interaction as well as by inflammatory cytokines, but most cells display low levels of expression without these stimulatory signals.^[88] Similar to the effects of 4-1BB or GITR agonist, CD134 agonist induces expansion of CD4 and CD8 T-cells via NF-κB,^[89] although it is not clear if the same downstream molecular machinery is shared by OX40, 4-1BB, and GITR.^[90] In parallel, CD134 agonist decreases Treg-mediated inhibitory signaling by inhibiting IL-10 production.^[91] In preclinical murine studies, significant survival benefit has been seen with several solid tumor models,^[92] including glioma, both as monotherapy^[93] and in combination.^[94] While there are no current clinical trials investigating OX40 in GBM, high OX40 ligand expression has been associated with improved prognosis.^[95]

Agonist antibodies can also be used to target OX40 as a means of provoking an aggressive immune response to tumor antigens. Co-stimulatory OX40 signaling promotes T-cell activation while inhibiting Treg function. A preclinical study led by Redmond et al.^[96] showed the effectiveness of OX40 agonistic monoclonal antibodies in combination with CTLA-4 blockade in augmenting antitumor immunity. Mice with prostate and sarcoma tumors exhibited an increased survival rate, increased effector CD4 and CD8 T-cell expansion, and increased cytokine production of IFN-γ, IL-2, IL-4, IL-5, and IL-13 when treated with dual anti-OX40/anti-CTLA-4.^[96] A phase I clinical trial using OX40 agonistic monoclonal antibodies for patients with advanced metastatic cancer showed regression in at least one lesion in 12 of the 30 patients along with an acceptable toxicity profile (NCT01644968).^[97] The treatment increased the B-cell and T-cell antitumor response in melanoma patients while preferentially upregulating OX40 on the Tregs subset of tumor-infiltrating lymphocytes.^[97]

Checkpoint Inhibition to Augment Adoptive Cell Therapy

Adoptive cell therapy is a process by which tumor-specific T-cells are isolated from a patient, expanded ex vivo, and then re-infused into the patient to aid in the eradication of malignant tumors. Immune modulation with checkpoint inhibition may make adoptive cell therapy more efficacious by abrogating exhaustion of the effector cells. O'Rourke et al.^[98] demonstrated upregulation of immunosuppressive checkpoints PD-L1 and IDO1, as well the immunosuppressive cytokine IL-10 after treatment of recurrent GBM patients with EGFRvIII-directed chimeric antigen receptor (CAR) T-cells, suggesting that a combination of these cells and checkpoint inhibition could be synergistic. In vitro data using small-interfering RNAs (siRNAs) to block PD-1 and CTLA-4 expression were recently investigated by Simon et al.^[99] in melanoma cells, and they found that CAR T-cells transfected with siRNA-targeting PD-1 alone or dual PD-1 and CTLA-4 showed an increase in cytokine secretion as well as overall cytotoxicity. These effects, however, were not observed in CAR T-cells transfected with siRNA-targeting CTLA-4 alone. The novel concept of modifying CAR T-cells to secrete PD-1-blocking antibodies combines the two immune therapy modalities and could be a potential solution to this thwarting limitation to treatment in the CNS. Rafiq et al.^[100] were able to demonstrate that PD-1-blocking CAR T-cells were able to enhance antitumor efficacy in murine models and demonstrated benefits for the co-modified CAR which avoids the systemic toxicities usually associated with checkpoint inhibition. Another study by Cherkassky et al.^[101] utilized CAR T-cells with intrinsic PD-1-targeting short hairpin RNA and demonstrated 60%–70% knockdown of PD-1 receptor expression in murine mesothelioma models. This decrease in PD-1 expression reversed CAR T-cell exhaustion. While the idea of modified CAR T-cells with intrinsic checkpoint blockade has launched as a way to prevent the CAR T-cell exhaustion, a secondary benefit of their use may be an effective strategy for bypassing the blood–brain barrier.

Challenges in Checkpoint Inhibition for Gliomas

As the body of preclinical and clinical work investigating checkpoint inhibition grows, the limitations of this modality to treat gliomas become evident. Although these therapies received FDA approval for several non-CNS cancers, only a subset of patients have a meaningful clinical response to checkpoint inhibition, and the etiology for this phenomenon is incompletely understood.^[102] In 2017, Filley et al.^[103] described several potential factors for the failure of nivolumab to provide clinical benefits for recurrent GBM patients in the Checkmate 143 trial. They posit that the antibody's size may prevent it from traversing the blood–brain barrier, as well as penetrate into the depths of the tumor, ultimately abrogating its efficacy. However, there is evidence that T-lymphocytes can pass through the blood–brain barrier.^[104]

Filley et al.^[103] also suggest that the profoundly immunosuppressive GBM microenvironment as well as the suppressed immune system of these patients may have also contributed to the results of the Checkmate 143 trial. Chen et al.^[105] in studying metastatic melanoma patients suggest that combinatorial treatment regiments, many of which are subjects of ongoing trials, may improve clinical response. This group found that the tumor cells would respond to blockade of one checkpoint by upregulating another in serial melanoma biopsies of patients undergoing checkpoint inhibitor monotherapies.^[105] However, the immunosuppressive GBM microenvironment may be particularly severe. Woroniecka et al.^[106] utilized a murine glioma model to demonstrate that multiple inhibitory checkpoint molecules are upregulated and that lymphocytes isolated from these tumors exhibited reduced efficacy compared to those in breast, lung, and melanoma. In addition to multiple checkpoint upregulation, Hodges et al.^[107] showed that GBM response to checkpoint inhibition does not appear to be related to mutational burden as in some non-CNS tumors. These findings suggest that a more complex relationship exists between GBM and the host immune system which cannot be effectively addressed by monotherapy.

An additional known challenge in immune therapy for GBM is to distinguish therapeutic response from progression in patients undergoing therapy. The immunotherapy response assessment for neuro-oncology criteria was created to direct clinicians in the treatment of patients undergoing immune therapy for glioma.^[108] However, misdiagnosis remains a clinical challenge and may significantly affect the results of trials.^[109] This creates a need to develop new ways to predict an immunotherapeutic response. Neoadjuvant trials may give a window into tumor response following therapy and analysis of which patients respond. Multiple neoadjuvant checkpoint trials are ongoing in non-CNS malignancies.^[110] Two recent neoadjuvant trials in melanoma showed that patients with lower expression of PD-L1 had increased relapse following anti-PD-1/anti-CTLA-4 therapy^[111] and PD-L1 expression was higher in responders.^[112] A single phase II study in neoadjuvant nivolumab for GBM patients has completed, but the results have not yet been released (NCT02550249). A phase I trial led by Orpilla et al.^[113] recently compared checkpoint and immune infiltrate of tumors treated with adjuvant and neoadjuvant PD-1 blockade. The group suggested that focal PD-L1 expression may be induced by CD8 attack directed at the tumor cells and act as an indicator of anti-PD-1 response.^[113] However, studies may be limited to comparing pre- and post-treatment tissue from different patients, rather than longitudinally. The ongoing phase I study by the authors (NCT03493932) is designed to allow extensive immunophenotyping of the tumor before as well as after checkpoint inhibition in each patient. In addition, this study measures cytokine expression within the tumor microenvironment and in surrounding brain tissue via microdialysis, as well as in blood and cerebrospinal fluid, to better understand the host and tumor response to systemic checkpoint inhibition.^[114] The prospect is that these pilot investigations will lead to the development of less invasive immune biomarkers.

Conclusion

The use of immune checkpoint blockade in GBM clinical trials shows promise and has profoundly furthered our understanding of this infiltrative tumor. A potential way to overcome the mechanisms of GBM immune evasion is to combine existing immune checkpoint blockades and/or agonists to effectively downregulate multiple immunosuppressive pathways and/or stimulate pro-inflammatory pathways. As more is uncovered about the immunosuppressive microenvironment of GBM and for effective markers to guide immune therapy, one can expect combination therapies to evolve with the hope that there will 1 day be a safe and effective treatment for GBM.

Financial support and sponsorship

This research was supported by the Intramural Research Program of the NIH, National Institute for Neurological Disorders and Stroke. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. The authors discuss a clinical trial receiving drug-only support from Bristol-Myers Squibb.

Conflicts of interest

The manuscript refers to an ongoing clinical trial by the authors which is being supported with drug-only support from Bristol- Myers Squibb.

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