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Current state and future prospects of
immunotherapy for glioma
Neha Kamran1,2, Mahmoud S Alghamri1,2, Felipe J Nunez1,2,DianaShah
1,2, Antonela S
Asad3, Marianela Candol3, David Altshuler1,2, Pedro R Lowenstein1,2 &MariaGCastro*
,1,2
1Department of Neurosurgery, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center
Drive, Ann Arbor, MI 48109-5689, USA
2Department of Cell & Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West
Medical Center Drive, Ann Arbor, MI 48109-5689, USA
3Instituto de Investigaciones Biom ′
edicas (CONICET-UBA), Facultad de Medicina, Universidad de Buenos Aires, Argentina
* Author for correspondence: Tel: +1 734 764 0850; Fax: +1 734 764 705; mariacas@umich.edu
There is a large unmet need for effective therapeutic approaches for glioma, the most malignant brain
tumor. Clinical and preclinical studies have enormously expanded our knowledge about the molecular as-
pects of this deadly disease and its interaction with the host immune system. In this review we highlight the
wide array of immunotherapeutic interventions that are currently being tested in glioma patients. Given
the molecular heterogeneity, tumor immunoediting and the profound immunosuppression that charac-
terize glioma, it has become clear that combinatorial approaches targeting multiple pathways tailored to
the genetic signature of the tumor will be required in order to achieve optimal therapeutic efcacy.
First draft submitted: 29 August 2017; Accepted for publication: 30 November 2017; Published online:
9 February 2018
Keywords: cancer vaccines ?checkpoint blockade ?gene therapy ?glioma ?immunotherapy ?passive immunother-
apy
Glioma subtypes & molecular classication
Gliomas are the most common primary brain tumors with an estimated incidence of approximately 7 per 100,000
people in USA, representing 27% of all CNS tumors and 80% of malignant tumors [1]. Until 2016, the WHO
classi?ed gliomas based on histology and categorized them into three principal groups: astrocytoma, oligoden-
droglioma and oligoastrocytoma [2,3]. Gliomas are further separated based on their degree of anaplasia into WHO
grades I-IV, of which WHO grade I is assigned to lesions with slow growth and better prognosis; and WHO grade
IV is assigned to the most malignant tumors represented by high-grade gliomas (HGG) or glioblastomas [2–4].
With the advancement of genomics, transcriptomics and epigenomics, together with the incorporation of
high-throughput technology and histological methods in the analysis of the glioma specimens, several molecular
markers have been identi?ed, for example, TERT,PDGFR,PTEN,IDH,PI3K,ATRX,EGFR andH3histone
family member 3A (Figure 1)[5–7]. These markers are associated with speci?c tumor phenotypes and indicate the
need to de?ne new glioma subtypes [6–10]. With the introduction of molecular data to tumor classi?cation, the
WHO 2016 classi?cation underwent a notable improvement from the classical histological classi?cation [3,11].One
of the more signi?cant criteria is the mutation status of IDH1. Mutation of IDH1 at arginine 132 (R132H) is
present in around 80% of low-grade gliomas (LGG; WHO grade II) and anaplastic astrocytomas (WHO grade
III), as well as in a subset of HGG (WHO grade IV) [12,13]. IDH1-R132H results in the production of the
oncometabolite R-2-hydroxyglutarate, which can inhibit a variety of α-ketoglutarate-dependent dioxygenases, such
as prolyl-4 hydroxylase, prolyl hydroxylase and the ten-eleven translocation family of DNA hydroxylases, which
also function as histone demethylases [14,15]. 2-hydroxyglutarate also induces histone 3 hypermethylation, and is
suf?cient for formation of a glioma CpG island methylator phenotype thus causing a global hypermethylation
phenotype in glioma cells [16,17]. Generally, patients with IDH1 mutation have better prognosis and better response
to treatment [7,9,10,18]. Therefore, gliomas can be separated into two large groups: mutant IDH1 and wild-type
IDH1 (wt-IDH1) (Figure 1). In turn, mutant IDH1 LGG can be further dissected into two subgroups according
to 1p/19q or ATR X status, which are mutually exclusive (Figure 1)[3,9,10,12]. Mutant IDH1 with 1p/19q co-
Immunotherapy (2018) 10(4), 317–339 ISSN 1750-743X 31710.2217/imt-2017-0122 C
2018 Future Medicine Ltd
Review Kamran, Alghamri, Nunez et al.
Grade
IDH1
ATR X
Other
alterations
Age
Histology
OS (months)
Diffuse glioma, WHO grade II or III WHO grade IV
wt mIDH1
wt-IDH1
ATR X r e tain e d ATR X l o ss
ATR X r e tain e d
mPTEN
mNF1
1p/19q codel
mTERTp
mCIC
mTP53
mTP53
CDKN2A
del
H3K27M H3G34
mDAXX
RTK I RTK II
Mesenchymal
EGFRaPDGFRaPDGFRa
mNF1; mPTEN
Adult >45 Young adult 20–45 (Y/A) Children <20
Y/A Adult >45
~15
~30 ~12
~24
~12–14
Glioblastoma
<AS<AS <OD
mTP53
mTERTp
Figure 1. Overview of the major subtypes of glioma.
AS: Astrocytoma; OD: Oligodendroglioma; OS: Overall survival.
deletion is associated with oligodendroglioma phenotype in diffuse LGG [10,19].Inthissubgroup,TERTp and CIC
mutations are also present (Figure 1)[9,10,12]. Mutant IDH1 with ATRX loss and TP53 mutation is associated with
astrocytoma and oligoastrocytoma phenotypes (Figure 1)[9,10,12,19]. This particular subtype of glioma can progress
in malignancy to reach WHO IV grade [20]. For this reason, these molecular markers can also be found in the most
aggressive forms of glioma [3]. On the other hand, gliomas harboring wt-IDH1 represent most of the WHO grade IV
gliomas. Gliomas expressing wt-IDH1, with loss of TP53 and ATRX, and mutations in H3 histone family member
3A, including H3K27M and H3G34, are typically found in pediatric and young adult patients [19,21]. Gliomas
with wt-IDH1 that have retained ATRX typically co-express TERTp mutations and alterations in regulators of the
RTK-RAS-PI3K signaling cascade and are typically encountered in adult patients (Figure 1)[3,4,6,11].RTKIisa
molecular subgroup of glioblastomas that generally arises in young adults, characterized by TERTp mutation and
PDGFRA ampli?cation [4,11]. Glioblastoma can also be divided in primary and secondary. Primary glioblastomas are
generated de novo and represent almost 90% of glioblastoma patients [3,22]. Secondary glioblastomas develop from
diffuse lower grade glioma [22]. They also harbor different molecular alterations. For example, EGFR overexpression
is prevalent in primary glioblastoma, but is rare in secondary [23].Incontrast,TP53 mutation is rare in primary
glioblastoma; however, is a characteristic of secondary glioblastoma [23]. In addition, IDH1 mutation and ATRX
inactivation are typically found in secondary glioblastoma together with TP53 mutation [3,22]. Therefore, primary
and secondary glioblastoma correspond to a distinctive brain-tumor entities differing in origin and molecular
characteristics.
In summary, gliomas represent a heterogeneous group of brain tumors that can be classi?ed according to histology,
malignancy, age range and genetic/epigenetic alterations. The molecular features of these tumors are crucial for
accurate diagnosis, and also for designing therapeutic strategies tailored to tumor subtypes. We hypothesized speci?c
molecular alterations can impact glioma responses to therapies.
Glioma prognosis & treatment
Glioma treatment modalities include surgical resection, radiation therapy and/or chemotherapy. Treatment strate-
gies are in?uenced by the recently revised 2016 WHO brain tumor classi?cation guidelines [3]. Maximal safe surgical
resection is the primary treatment strategy for LGG. The most common LGG in adults is oligodendroglioma, a
grade II tumor by the 2016 WHO classi?cation. Molecular and genetic characteristics, such as IDH mutation and
codeletion of the 1p/19q chromosomal arms are becoming increasingly important for stratifying patients based
on response to treatment. In most cases, the standard treatment for oligodendroglioma beyond surgery is radio-
therapy followed by procarbazine, lomustine and vincristine chemotherapy [24]. For those patients with anaplastic
astrocytomas, the standard of care (SOC) involves maximal safe resection or biopsy followed by involved-?eld ra-
diotherapy to 60 Gy given in 1.8-2 Gy fractions [25]. Median survival time is doubled in patients receiving adjuvant
radiotherapy versus surgery alone in randomized trials [26]. However, whole brain radiation therapy can signi?cantly
318 Immunotherapy (2018) 10(4) future science group
Current state & future prospects of immunotherapy for glioma Review
impact patient cognitive functions. First-line treatment also includes the use of chemotherapeutics with modest
increases in 1- and 2-year survival times (58–63% and 31–37%, respectively) [27]. Glioma (WHO IV) carries the
poorest prognosis, and surgical resection is a key element for initial management versus diagnostic biopsy. However,
the bene?t is mitigated if the patient is left with a neurologic de?cit which signi?cantly impairs daily function.
Radiotherapy and adjuvant temozolomide (TMZ) have been the SOC for glioblastoma (GBM) patients based
on the treatment protocol per Stupp et al. in a 2005 randomized controlled trial [28]. Patients receiving TMZ in
addition to radiotherapy after surgery experienced a 2.5-month survival advantage compared with those receiving
adjuvant radiotherapy alone [28]. Several important prognostic indicators exist, the most important of which include
patient age and functional status, measured commonly on the Karnofsky Performance Scale and MGMT promoter
methylation [29]. Current treatments for anaplastic oligodendroglioma and oligoastrocytoma, both WHO grade III
tumors, represent a more rapidly evolving paradigm and insight into future glioma treatment effort. The molecular
biology of these tumors and signi?cance of well-recognized genetic aberrancies are leading to new treatment options
and targeted therapies [25]. Evidence challenging the central dogma of the brain as an immune privileged organ and
the success of immunotherapeutic approaches in other cancers has driven the investigation of several immune-based
approaches in preclinical models and in numerous clinical trials [30–34].
Innate immune responses in glioma
Although the brain was originally considered an immune privileged organ, studies showed that due to disturbance of
the blood–brain barrier integrity during in?ammation or cancer and the presence of lymphatic out?ow channels, the
immune system is able to interact with cells within the CNS [30–32]. Immune cell in?ltration has been demonstrated
in patients with malignant glioma; however, endogenous immune responses fail to control the disease [35].This
is mainly due to glioma-mediated suppression of the in?ltrating immune cells by mechanisms such as TGF-β
secretion, release of LDH5 or expression of galectin-1 (gal-1) [36–38]. Immune surveillance by innate immune cells
is the ?rst line of defense against malignant cells. Natural killer (NK) cells are the main effector cells of the innate
immune system mediating antitumor responses. Activation of NK cells is tightly regulated based on germline-
encoded activating and inhibitory receptors [39,40]. Data from our laboratory showed that NK cells can mediate
an antiglioma immune response which is suppressed by gal-1 expression by glioma cells [41]. Additional studies
demonstrated that glioma-derived LDH5 and TGF-βabrogate the antiglioma activity of NK cells. LDH5 induces
expression of activating receptor NKG2D ligands on myeloid cells, resulting in downregulation of NKG2D on NK
cells and thereby decreased antiglioma reactivity [36]. Moreover, glioma cell-derived TGF-βhas been shown to inhibit
expression of activating NKG2D ligands MICA and ULBP2 on glioma cells, which facilitates downregulation of
NKG2D receptor on immune cells, including NK cells and CD8+T cells [37,42].
It was recently demonstrated that a myeloid cell population (Gr-1+CD11b+) is required for NK cell-mediated
glioma eradication in the absence of immunosuppressive gal-1 [43]. Immunodepletion of Gr-1+or Ly6C+ex-
pressing cells in an orthotopic glioma mouse model resulted in abrogation of tumor rejection, demonstrating the
importance of this myeloid cell population for the innate antiglioma immune response [43]. Additionally, studies
demonstrated a role for TLR-mediated immune activation in the context of glioma-targeting immunotherapeutic
approaches. TLRs are germline-encoded receptors, some of which are expressed on the cell surface while others are
expressed in the endosomal compartment [44]. Natural TLR ligands are common pathogen-associated molecular
patterns, such as viral ssRNA (TLR7) or dsRNA (TLR3) or bacterial components like lipopolysaccharide (LPS;
TLR4) or CpG-containing dsDNA (TLR9) [44]. TLR activation can also be induced by recognition of endoge-
nous damage-associated molecular patterns released upon cell death [45]. Once activated, TLR signaling results
in production of proin?ammatory cytokines and upregulation of co-stimulatory molecules, thereby mediating
activation of the adaptive immune response. We demonstrated in vivo that release of the endogenous TLR2 ligand
HMGB1 (high mobility group box 1) from dying glioma cells mediates tumor regression following combined
immunotherapy/cytotoxic therapy [46]. In this therapeutic approach, adenoviral vectors encoding for herpes sim-
plex type 1-thymidine kinase (TK) and FMS-like Tyrosine kinase 3 ligand (Flt3L) are delivered into the brain
where glioma cell death is induced upon systemic ganciclovir treatment [47]. This in turn facilitates the release of
tumor antigens and the endogenous TLR2 ligand HMGB1, which then activates dendritic cells (DCs) that were
recruited into the brain by Flt3L. Activation of TLR signaling in recruited DCs induces CD8+T cell-dependent
glioma regression as well as antiglioma immunological memory [46]. In addition to TLR-induced immune acti-
vation by endogenous TLR ligands, administration of synthetic TLR ligands as single agents or as adjuvants in
combination with peptide vaccines is under investigation. Studies in glioma mouse models showed that topical
future science group www.futuremedicine.com 319
Review Kamran, Alghamri, Nunez et al.
Inhibition of
immunosuppressive
cells
Basiliximab
M2 macrophages
MDSCs NK cells
Nivolumab
Pembrolizumab
Pidilizumab
Tregs
IL-10, TGF-E, PGE2,
Galectins, GM-CSF,
PAMPs and DAMPs
CD8+
T cells
PD-1
Ipilimumab
Tremelimumab
Activation
of effector
cells
CAR T cells targeting
EGFRvIII, IL-13RD2,
HER2, EphA2, CD133
and MUC I
DCs
SL-701 Ad-TK +Ad-Flt3L
ICT-107
DCVax-L
ICT-121
AVO113
Rindopepimut
Cetuximab
Nimotuzumab
Antibody–drug
conjugate ABT414
Durvalumab
IMA950
HSPP-96
Glioma cell
PEPIDH1M
NOA-16
VEGF-A
Bevacizumab
Inhibition of
secretory factors
EphA2
IL13RD2
EGFRvIII
PD-L1
Genetic lesions,
neoantigens and TAA
targeting
TUMAPs
HSP
IDH1
neo-Ag
CD25
CTLA-4
CAR
T cells
Figure 2. Immunotherapy strategies in clinical trials. Glioma evades the immune system through a variety of ways. Antiglioma
immunotherapeutic strategies are targeting T-cell exhaustion, immunosuppressive cells and angiogenesis and testing peptide or dendritic
cell vaccines-based approaches to enhance the antitumor adaptive immune response or the activity of effector cells using chimeric
antigen receptor T cells.
application of imiquimod (TLR7/8 ligand) can convert plasmacytoid dendritic cells (pDCs) into antiglioma ef-
fector cells [48]. In a different glioma mouse model, topical imiquimod administration exerted therapeutic effect
by inducing glioma-reactive CD8+T cells and reducing the number of CD4+Foxp3+Treg cells [49]. Importantly,
synthetic TLR agonists have entered clinical trials as adjuvant to amplify the therapeutic effect of peptide vaccines
(NCT01920191, NCT02454634).
Together, these studies demonstrate that glioma can be in?ltrated and recognized by cells of the innate as well
as adaptive immune system; however, in most of the cases these immune interactions are effectively suppressed
by glioma tumor cells. Therefore, counteracting glioma-mediated immune suppression is a prerequisite for the
development of new and more effective immunotherapies for this devastating disease.
Immunosuppression in glioma
Glioma-mediated immunosuppression depends on the local production of cytokines and chemokines and the
recruitment of regulatory, immunosupressive cells (Figure 2)[50].TGF-βand IL-10 are central to maintaining
the immunosuppressive glioma microenvironment. These cytokines are not only secreted by tumor-in?ltrating
immune cells, but also by glioma cells themselves [50]. IL-10 inhibits the activation and effector functions of DCs,
macrophages and T cells, modulates the growth and differentiation of immune cells, and limits the expression of
320 Immunotherapy (2018) 10(4) future science group
Current state & future prospects of immunotherapy for glioma Review
MHC class II in monocytes [50,51]. IL-10 has also been shown to upregulate PD-L1 in circulating monocytes and
tumor-associated macrophages in glioma [52]. Interestingly, IL-10 is not always a mediator of immunosuppression
and also exerts proin?ammatory and antitumor effects. It has been found that T cells’ inhibition of glioma growth
relies on high levels of IL-10 [53]. Furthermore, genetic ablation of IL-10 facilitates tumor growth and metastasis
development in mouse models of colon cancer, an effect that was associated with the expansion of myeloid-derived
suppressor cells (MDSCs) and Tregs [54]. In addition, IL-10 has been shown to stimulate macrophages to produce
antiangiogenic cytokines and to promote antitumor NK-cell responses [50].
TGF-βis a pleiotropic cytokine involved in many biological functions, including the blockade of T-cell activation
and proliferation, as well as the induction of Tregs [55,56]. It was ?rst isolated from patients with glioma and is
considered an important immunosuppressive factor [57–59].TGF-βexpression levels correlate with higher tumor
grades and worse prognosis [60]. Nevertheless, this cytokine plays a complex role in glioma. In the early stages of
tumor growth, TGF-βacts as a tumor suppressor [61]. At later stages, however, glioma cells stop responding to these
TGF-β-mediated inhibitory signals and instead TGF-βenhances tumor progression through different mechanisms
such as increasing angiogenesis and promoting the expansion of Tregs [62].
Glioma secrete other immunosuppressive factors such as CSF-1, VEGF, PGE2, NO, Arg I, IDO and Gal-
1[63] . VEGF does not only induce angiogenesis, but also inhibits the functional maturation of DCs [64].PGE2
downregulates the production of Th1 cytokines (IL-2, IFN-γand TNF-α) and upregulates Th2 cytokines (IL-10,
IL-6, IL-4) [65,66]. Both TGF-βand PGE2 can act synergistically to induce a regulatory phenotype in DCs [67].
Studies performed in mice harboring subcutaneous glioma have shown that systemic immunosuppression associated
with growing gliomas is partly mediated by the overproduction of NO in splenocytes [68]. Inhibition of inducible
NO synthase, using either mercaptoethylguanidine or l-N(6)-(1-Iminoethyl)-l-lysine (l-NIL) could boost IFN-γ-
based immunotherapy approach in rats with intracranial tumors [69,70]. Arg I has been proposed as a mechanism
of monocyte-mediated inhibition of T-cell function in glioma patients and researchers have found an expanded
population of circulating degranulated neutrophils, which is associated with elevated levels of serum Arg I and
decreased T-cell CD3ζexpression in the peripheral blood of glioma patients [71]. IDO expression is higher in HGG
than in LGG and correlates with worse overall survival rates [72]. IDO expression has been shown to increase
the recruitment of immunosuppressive Tregs, promoting tumor escape [73]. Another immunosuppressive ligand
expressed by glioma cells, Gal-1, enhances tumor cell migration, induces T-cell apoptosis and inhibition of T-cell
proliferation, expansion and accumulation of Tregs and protumoral DCs, and its expression was found to correlate
with the grade of astrocytic tumors and with a dismal prognosis [38,74,75].
Soluble factors that bind to pattern recognition receptors, such as TLRs on microglia and activate them, have also
been involved in the maintenance of an immunosuppressive microenvironment in glioma [76,77]. Although glioma-
derived chemokines (i.e., CSF-1 or CCL2) attract and activate microglia, locally produced TGF-βand PGE2
induces an anti-in?ammatory phenotype in these cells, reducing their antigen-presenting activity and facilitating
the in?ltration of immunosuppressive cells such as Th2 cells and Tregs [78–81]. Glioma-derived CCL22 and CCL2
also recruit Tregs that express CCR4 into the tumor microenvironment [82,83]. Thus, blockade of these chemokines
could improve antitumor immunity.
Tumor progression depends on the accumulation of genetic and epigenetic alterations in cancer cells result-
ing in a complex and heterogeneous cellular composition at the site of tumor growth [84,85]. This heteroge-
neous environment contains an immunosuppressive network of immune cells such as MDSCs, tumor-associated
macrophages/microglia (TAMs) and Tregs [86]. Of particular interest are the MDSCs which promote tumor growth
by variety of mechanisms including inhibition of T-cell proliferation and effector functions, suppression of NK
and NKT activity, recruitment of Tregs, secretion of immune suppressive cytokines and upregulation of checkpoint
receptor ligands such as PD-L1 [87,88]. MDSCs can also promote tumor angiogenesis via the secretion of VEGF as
well as matrix metallopeptidase 9 [89,90].
In mice, MDSCs are characterized by the dual expression of CD11b and Gr-1 surface markers. They are further
distinguished into polymorphonuclear (PMN-MDSCs) or monocytic (M-MDSCs). The two subtypes differ not
only in the surface makers (i.e., CD11b+,Ly6G
+,Ly6C
low for the PMN-MDSCs, and CD11b+,Ly6G
-,Ly6C
high
for the M-MDSCs) but also in the main mechanism involved in immunosuppression. The generation of Arg I
and NO is the main suppressive mechanism of M-MDSC, whereas the PMN-MDSC suppresses CD8+T-c ell
responses mainly by producing reactive oxygen species [91–93]. There are multiple strategies for targeting MDSCs
in glioma [94,95]. MDSCs depletion and/or blockade of their inhibitory mechanisms seem to be the most effective
method. We have recently shown that depletion of immune-suppressive MDSCs in glioma-bearing mice markedly
future science group www.futuremedicine.com 321
Review Kamran, Alghamri, Nunez et al.
enhances the ef?cacy of immunostimulatory/cytotoxic gene therapy [95]. Another possible strategy for targeting
MDSCs is by promoting their differentiation into mature cells. Using an in vitro cell differentiation model, two
groups showed that the macrophage migration inhibitory factor can be targeted to enhance DC differentiation
from MDSCs [96,97]. TAMs are another dominant immune cell type in glioma [98,99]. Glioma can activate M2
polarized macrophages, which secrete IL-10 and TGF-β, and inhibit T-cell proliferation [100]. The exact mechanism
for such effect is still not clear, although the CSF-1 receptor could be a possible mediator [101].Gabrusiewiczet al.
showed that in glioma patients, TAMs resembled the phenotype of nonpolarized M0 macrophages with partially
immune-suppressive phenotype [98]. More recently, TAM recruitment has been connected to NF1 gene expression
in glioma and the resistance to radiotherapy in some glioma patients may be associated with the presence of M2
macrophages [102].
In addition to MDSCs and TAMs, glioma cells promote the accumulation of Tregs [103]. Tregs restrain antitumor
immune response through numerous mechanisms [102]. They can mediate inhibition of functional T cells through
the interaction of CTLA-4 (on Tregs) with CD80/86 (on T cells) [104], by the activation of the perforin/granzyme
B pathway to cause target cell death [105], by suppressing the release of IL-2 and IFN-γ[106,107], or by expression
of TGF-β[108] which can also inhibit NK cells activity [109]. Many reports have illustrated the correlation between
blocking the activity of Tregs with more effective antiglioma effector response and improved survival [103,104].This
can be done by a variety of mechanisms such as blocking CTLA-4 [110], inhibiting the signaling pathways that lead
to FoxP3 activation (i.e., STAT3) [111–113], or by targeting CD25 [114]. The use of chemotherapeutic alkylating
agents (such as temozolomide and cyclophosphamide) has also been reported to inhibit Treg activity [113,115,116].
In summary, glioma cells secrete numerous chemokines, cytokines and growth factors that promote in?ltration
and expansion of MDSCs, microglia, macrophages and Tregs that directly enhance the invasion of glioma cells,
dampen antitumor immune response and accelerate tumor progression. Such immunosuppressive networks are
crucial targets for the development of effective immunotherapies.
T-cell exhaustion in glioma
Acquisition of effector functions by naive T cells in response to acute infections is accompanied by robust
proliferation, transcriptional, metabolic and epigenetic reprogramming [117–119]. Memory T cells arise from a small
subset of these activated T cells upon the resolution of infection, while the majority of the T cells die [117]. However,
during chronic infections or in?ammation, such as in cancer, an altered state of exhaustion is generated in T
cells. Hallmarks of exhausted T cells include loss of effector functions, expression of various inhibitory receptors,
metabolic and transcriptional derangements [117,120]. Typically as exhaustion develops, the ability of the T cells
to release IL-2 and undergo proliferation is lost, followed by the failure to produce IFN-γ,TNFαand undergo
degranulation which results in the release of cytolytic granules from the T cells and is essential for granzyme-
dependent killing [117,120]. There is also an increase in the amount and diversity of inhibitory receptors expressed by
the T cells. Some of the common inhibitory receptors associated with T-cell exhaustion are CTLA-4, PD-1, T-cell
immunoreceptor with immunoglobulin and ITIM domains, LAG-3, 2B4, B and T lymphocyte attenuator and
TIM3 [117,118,121,122]. It has been shown that patients with primary intracranial tumors have impaired cell-mediated
immunity, with the majority of patients failing to respond to common recall skin test antigens and to neoantigens
in vivo [123–125]. T-cell receptor (TCR)-mediated signaling in peripheral blood lymphocytes was also shown to
be defective in T cells obtained from patients with primary brain tumors [126]. In addition, T cells from these
patients showed a marked reduction in tyrosine phosphorylation in response to mitogens. Reduction in CD4 and
CD8 T cells has been reported in the tumor and circulation of GBM patients [127]. Using immunohistochemistry
and ?ow cytometry, PD-L1 expression was analyzed in 94 GBM patients. A total of 60.6% of GBM patients
had tumors with at least 1% or more PD-L1-positive cells and 5.32% had 50% or greater PD-L1-positive cells.
Higher PD-L1 expression was also observed to be correlated with a worse outcome [128]. Interestingly, since tumors
with high mutational burdens may be more immunogenic, immune checkpoint inhibition using nivolumab was
tested in two siblings with recurrent multifocal biallelic mismatch repair de?ciency GBM with clinically signi?cant
responses [129].
Thus approaches targeting T-cell exhaustion could provide clinical bene?t in glioma. CTLA-4 and PD-1 have
been identi?ed as the two major inhibitory receptors/checkpoints involved in T-cell exhaustion and monoclonal
antibodies targeting CTLA-4 (ipilimumab and tremelimumab) and PD-1 (nivolumab and pembrolizumab) have
been tested for safety and ef?cacy in clinical trials for melanoma [34,130,131], non-small-cell lung cancer [132,133] and
322 Immunotherapy (2018) 10(4) future science group
Current state & future prospects of immunotherapy for glioma Review
renalcellcarcinoma[134]. Based on the impressive clinical bene?t observed in the treatment of melanoma by the
use of checkpoint inhibitors a number of preclinical and clinical studies are investigating their use in glioma.
Immunotherapeutic approaches in clinical trials
Various preclinical studies have demonstrated the success of immunotherapy-based approaches in animal models and
many Phase I and II clinical trials have shown immunotherapy to be safe and in some cases improve progression-free
survival (PFS) and overall survival (OS) [135–139]. Below, we provide an overview of the immunological approaches
which yielded promising results in the preclinical setting and are currently being tested in the clinic (Figure 2).
Targeting immune checkpoints
Preclinical studies using murine models with orthotopic-transplanted gliomas have shown great bene?t with
checkpoint inhibitors used individually or with other immunotherapeutic strategies [95,110,140,141]. Administration
of CTLA-4 blocking antibodies improved the survival of animals bearing intracranial SMA-560 tumors and in
combination with IL-12 also caused tumor regression in GL261 tumor models [110,140]. Tumor eradication in
both studies was accompanied by reduction of Tregs in the tumor microenvironment and an increase in effector
CD8 T cells. Promising results from clinical trials for metastatic melanoma using ipilimumab in combination with
gp100 vaccine or dacarbazine and the US FDA approval for ipilimumab’s use in malignant melanoma have fueled
research into its use for the treatment of other cancers [34]. Currently two clinical trials are assessing the use of
anti-CTLA-4 antibodies (ipilimumab and tremelimumab) in the treatment of recurrent glioma (NCT02794883
and NCT02017717). The NCT02794883 trial evaluates the combination of tremelimumab with durvalumab,
a PD-L1 blocking antibody and the NCT02017717 evaluates the combination of ipilimumab with nivolumab,
a PD-1 blocking antibody. Preclinical studies investigating the PD-1 checkpoint blockade in glioma have shown
antitumor ef?cacy [142]. Combination of PD-1 blocking antibodies with radiotherapy enhanced the survival of
GL261 glioma-bearing mice [142,143]. PD-1 blocking antibodies, pembrolizumab and nivolumab were approved by
the FDA for use in metastatic melanoma in 2014 and for non-small-cell lung cancer in 2015. Multiple clinical
trials are investigating the ef?cacy of anti-PD-1 and anti-PD-L1 antibodies in malignant glioma. In addition to
NCT02794883 and NCT02017717, two Phase I/II studies will test the use of pembrolizumab with or without
bevacizumab (NCT02337491) or pembrolizumab in combination with laser ablation (NCT02311582) in patients
with recurrent glioma. NCT01952769 will evaluate the use of pidilizumab (humanized anti-PD-1 monoclonal
antibody) against diffuse intrinsic pontine glioma and recurrent glioma and NCT02336165 is testing MEDI4736
(anti-PD-L1 monoclonal antibody) in combination with radiotherapy and bevacizumab. As shown in Table 1,
several other clinical trials are also testing the use of immune checkpoint inhibitors in malignant glioma.
Preclinical studies are investigating the potential of other checkpoints such as TIM-3, IDO, LAG-3 and adenosine
A2a receptor as therapeutic targets in glioma [144]. Combination of anti-PD-1, anti-TIM-3 and focal radiation
resulted in regression of murine gliomas and combination of IDO, CTLA-4 and PD-L1 blockade induced long-
term survival in 100% of the glioma-bearing animals [145,146].
Immune checkpoint blockade appears to be an exciting avenue to develop based on the preclinical studies. An
important consideration, however, is the use of GL261 as a tumor model in a variety of these studies. Tumors
generated by implantation of GL261 cells mimic many of the features of GBM including neovascularization,
pseudopalisading necrosis, perivascular organization and angiogenesis [147]. These characteristics have therefore
prompted the use of this model to test a variety of antitumor strategies. With a high mutational burden, however,
GL261 tumors may potentially generate neoantigens leading to the development of a large T-cell repertoire and
a high response rate to immunotherapies. Human GBM is highly immunosuppressive though and it is therefore
important to validate the ef?cacy of immunotherapeutic approaches including checkpoint inhibition with other
rodent models (Table 2) and with tumors containing the mutations commonly found in human GBM (Figure 1).
This would also allow for the development of personalized immunotherapy strategies.
Immunotherapy with peptide vaccines
Numerous glioma-associated antigens such as IL-13Rα2, HER2, EphA2, gp100 and AIM-2 are being targeted in
glioma [148–150]. Additionally tumor-speci?c neoantigens such as EGFRvIII are being used to target tumor cells
speci?cally [150,151]. EGFRvIII is expressed in about 20–30% of glioma patients [151]. Evaluation of a combination
of EGFRvIII-speci?c peptide (PEP-3, rindopepimut) keyhole limpet hemocyanin conjugate vaccine plus GM-CSF
with standard radiotherapy and chemotherapy in 18 patients expressing EGFRvIII showed a median survival of
future science group www.futuremedicine.com 323
Review Kamran, Alghamri, Nunez et al.
Table 1. Current clinical trials evaluating checkpoint inhibition.
NCT# Phase Study title Current status Inclusion diagnosis Intervention Target
NCT02017717 Phase III A Study of the Effectiveness and Safety of
Nivolumab Compared to Bevacizumab and
of Nivolumab With or Without Ipilimumab
in Glioblastoma Patients (CheckMate 143)
Recruiting Recurrent glioblastoma Nivolumab alone vs
bevacizumab or
nivolumab alone or
with ipilimumab
PD-1, CTLA-4
NCT02327078 Phase I/II with
glioblastoma
cohort in Phase
II
A Study of the Safety, Tolerability, and
Efcacy of Epacadostat Administered in
Combination With Nivolumab in Select
Advanced Cancers (ECHO-204)
Recruiting Recurrent glioblastoma Nivolumab +
epacadostat
PD-1, IDO1
NCT02311582 Phase I/II MK-3475 in Combination With MRI-guided
Laser Ablation in Recurrent Malignant
Gliomas
Recruiting Recurrent malignant
gliomas
MK-3475 in
combination with
mri-guided laser
ablation
PD-1
NCT02313272 Phase I Hypofractionated Stereotactic Irradiation
(HFSRT) With Pembrolizumab and
Bevacizumab for Recurrent High Grade
Gliomas
Recruiting Recurrent high grade
gliomas
Pembrolizumab
with radiation
therapy and
bevacizumab
PD-1 +VEGF
NCT02335918 Phase I/II with
Phase II only for
glioblastoma
A Dose Escalation and Cohort Expansion
Study of Anti-CD27 (Varlilumab) and
Anti-PD-1 (Nivolumab) in Advanced
Refractory Solid Tumors
Recruiting Glioblastoma Combination of
varlilumab and
nivolumab
CD27 +PD-1
NCT02337686 Phase II Pharmacodynamic Study of Pembrolizumab
in Patients With Recurrent Glioblastoma
Recruiting Recurrent glioblastoma Pembrolizumab PD-1
NCT02550249 Phase II Neoadjuvant Nivolumab in Glioblastoma
(Neo-nivo)
Recruiting Primary and recurrent
Glioblastoma
Nivolumab PD-1
NCT02529072 Phase I Nivolumab With DC Vaccines for Recurrent
Brain Tumors (AVERT)
Recruiting Recurrent grade iii and
grade iv brain tumors
Nivolumab with DC
vaccines
PD-1
NCT02526017 Phase Ia/Ib Study of FPA008 in Combination With
Nivolumab in Patients With Selected
Advanced Cancers (FPA008-003)
Recruiting Malignant glioma FPA008 in
combination with
nivolumab
CSF-1R +PD-1
NCT02617589 Phase III An Investigational Immuno-therapy Study
of Nivolumab Compared to Temozolomide,
Each Given With Radiation Therapy, for
Newly-diagnosed Patients With
Glioblastoma (GBM, a Malignant Brain
Cancer) (CheckMate 498)
Recruiting Newly diagnosed
adults with
unmethylated MGMT
glioblastoma
Nivolumab +radia-
tion vs
temozolomide +ra-
diation
PD-1
NCT02648633 Phase I Stereotactic Radiosurgery With Nivolumab
and Valproate in Patients With Recurrent
Glioblastoma
Recruiting Recurrent glioblastoma Stereotactic
radiosurgery with
nivolumab and
concurrent
valproate
PD-1
NCT02658279 Proof-of-
concept, pilot
study
Pembrolizumab (MK-3475) in Patients With
Recurrent Malignant Glioma With a
Hypermutator Phenotype
Recruiting Recurrent malignant
glioma with a
hypermutator
phenotype
Pembrolizumab
(MK-3475)
PD-1
NCT02658981 Phase I Anti-LAG-3 or Urelumab Alone and in
Combination With Nivolumab in Treating
Patients With Recurrent Glioblastoma
Recruiting Recurrent GBM Anti-LAG-3 or
urelumab alone
and in combination
with nivolumab
LAG-
3+CD137 +PD-
1
NCT02667587 Phase III An Investigational Immuno-therapy Study
of Temozolomide Plus Radiation Therapy
With Nivolumab or Placebo, for Newly
Diagnosed Patients With Glioblastoma
(GBM, a Malignant Brain Cancer)
(CheckMate548)
Recruiting MGMT-methylated
glioblastoma
Temozolomide +ra-
diation therapy
with nivolumab or
placebo
PD-1
NCT02798406 Phase II Combination Adenovirus +Pembrolizumab
to Trigger Immune Virus Effects (CAPTIVE)
Recruiting Recurrent glioblastoma
or gliosarcoma
DNX-2401 +Pem-
brolizumab
PD-1
NCT02829723 Phase I/II Phase I/II Study of BLZ945 Single Agent or
BLZ945 in Combination With PDR001 in
Advanced Solid Tumors
Recruiting Glioblastoma BLZ945 single agent
or BLZ945 in
combination with
PDR001
CSF1R +PD-1
DC: Dendritic cell; GBM: Glioblastoma.
324 Immunotherapy (2018) 10(4) future science group
Current state & future prospects of immunotherapy for glioma Review
Table 1. Current clinical trials evaluating checkpoint inhibition (cont.).
NCT# Phase Study title Current status Inclusion diagnosis Intervention Target
NCT02852655 Pilot A Pilot Surgical Trial To Evaluate Early
Immunologic Pharmacodynamic
Parameters For The PD-1 Checkpoint
Inhibitor, Pembrolizumab (MK-3475), In
Patients With Surgically Accessible
Recurrent/Progressive Glioblastoma
Recruiting Recurrent/progressive
glioblastoma
Pembrolizumab
(MK-3475)
PD-1
NCT02336165 Phase II Phase II Study of MEDI4736 in Patients With
Glioblastoma
Not recruiting Unmethylayed MGMT
GBM and recurrent
GBM
MEDI4736 alone or
with radiotherapy
or with
bevacizumab
PD-L1
NCT02794883 Phase II Tremelimumab and Durvalumab in
Combination or Alone in Treating Patients
With Recurrent Malignant Glioma
Recruiting Recurrent malignant
glioma
Tremelimumab and
durvalumab
(MEDI4736) alone
and in combination
CTLA-4 +PD-
L1
NCT02937844 Phase I Pilot Study of Autologous Chimeric Switch
Receptor Modied T Cells in Recurrent
Glioblastoma Multiforme
Recruiting Glioblastoma
multiforme
Anti-PD-L1 CSR T
cells +cyclophos-
phamide +udara-
bine
PD-L1
NCT02866747 Phase I/II A Study Evaluating the Association of
Hypofractionated Stereotactic Radiation
Therapy and Durvalumab for Patients With
Recurrent Glioblastoma (STERIMGLI)
Recruiting Recurrent glioblastoma Durvalumab +radi-
ation
therapy
PD-L1
NCT02968940 Phase II Avelumab With Hypofractionated
Radiation Therapy in Adults With IDH
Mutant Glioblastoma
Recruiting Transformed IDH
mutant glioblastoma
Avelumab +hy-
pofractionated
radiation therapy
PD-L1
DC: Dendritic cell; GBM: Glioblastoma.
26 months (Phase II multicenter study, ACTIVATe, NCT00643097) [152]. After adjustment for age and Karnofsky
performance status, the OS of vaccinated patients was greater than that observed in a matched control group.
Interestingly 82% of the tumors had lost EGFRvIII expression indicating treatment-induced immunoediting [152].
Immunoediting was also observed in a subsequent Phase II (ACT III, NCT00458601) trial using the combination of
rindopepimut, GM-CSF and standard and dose-intensi?ed TMZ with a decrease in EGFRvIII immunoreactivity in
67% of the patients and an OS of 21.8 months [138]. Phase III study of rindopepimut/GM-CSF with adjuvant TMZ
in patients with newly diagnosed glioma (ACT IV, NCT01480479) has been discontinued as the median OS with
rindopepimut was 20.4 months compared with 21.1 months in the control arm [153]. ReACT (NCT01498328)
is a Phase II study of rindopepimut/GM-CSF plus bevacizumab in patients with relapsed EGFRvIII-positive
glioma [154]. Interim analysis shows rindopepimut induces potent EGFRvIII-speci?c immune response and tumor
regression, and appears to signi?cantly prolong survival when administered with bevacizumab in patients with
relapsed glioma [155].
To overcome the risk of immunoediting and disease recurrence following single peptide vaccinations, investiga-
tions testing peptide combinations are underway. Results from the NCT01130077 trial using the combination of
EphA2, IL-13Rα2 and survivin with tetanus toxoid and Poly I:C in pediatric brain stem and HGG showed the
development of peptide-speci?c immune responses and indications of immune cell in?ltrates. Five out of twenty-six
children showed pseudoprogression that was manageable, nineteen showed stable disease and two showed progres-
sive disease [156]. The NCT02078648 study is testing the SL-701 vaccine (IL13Rα2, EphA2 and survivin) in
combination with bevacizumab in patients with newly diagnosed glioma. A Phase I trial of IMA950 (consisting
of peptides derived from the following proteins: brevican; chondroitin sulfate proteoglycan 4; fatty acid binding
protein 7; insulin-like growth factor 2 mRNA binding protein 3; neuroligin 4, X-linked; neuronal cell adhesion
molecule; protein tyrosine phosphatase, receptortype, Z polypeptide 1; tenascin C; Met proto-oncogene; baculovi-
ral inhibitor of apoptosis repeat-containing 5; and HBV core antigen) in 45 patients with newly diagnosed glioma
receiving maintenance TMZ showed 36 of 40 patients as single-peptide responders and 20 patients as multipeptide
responders. However, the median OS was 15.3 months [157]. NCT01920191 tested the combination of intradermal
IMA950 with intra muscular Poly-ICLC as an adjuvant in combination with TMZ in newly diagnosed HLA-A2
glioma patients. Preliminary results showed improvement in the median OS with two of the six patients showing
induction of both peptide-speci?c CD4 and CD8 T cells [158].
future science group www.futuremedicine.com 325
Review Kamran, Alghamri, Nunez et al.
Table 2. Rodent models for brain tumors.
Induction Species/strain Source Pathology Applications Ref.
Cell line inoculation C57BL/6 GL26 cells GBM/
ependymoblastoma
DC vaccines engineered to express IL-12.
Treg depletion. Metronomic
chemotherapy. Combined conditionally
cytotoxic and immunostimulatory gene
therapy
[1–5]
GL26.1 cells GBM/
ependymoblastoma
Immunological checkpoint blockade.
Antitumor DC vaccines +Treg blockade
with anti-CD25 antibody. Peptide
vaccinations +TGF-?neutralizing
antibody
[6–10]
CT-2A cells Anaplastic
astrocytoma
Genetically modied T cells targeting
EGFRvIII and IL13R?2
[11,12]
VM-Dk SMA-560 cells Anaplastic
astrocytoma
Overexpression of soluble CD70 ligand.
Inhibition of TGF-?signaling. EGFRvIII
CAR-modied T-cell therapy. Antitumor
DC vaccines
[13–17]
B6D2F1 4C8 cells
Oligodendroglioma,
astrocytoma
Cationic liposome–DNA complexes. HSV
vaccines encoding IL-12
[18,19]
Cell line inoculation LEWIS CNS-1 cells GBM Antitumor DC vaccines. TLR agonists.
Metronomic chemotherapy. Combined
conditionally cytotoxic and
immunostimulatory gene therapy
[20–23]
FISHER 344 F98 cells GBM Combined conditionally cytotoxic and
immunostimulant gene therapy.
Cellular vaccinations +GM-CSF.
Upregulation of costimulatory
molecules
[5,24,25]
RG2 cells Anaplastic
astrocytoma
Gene therapy-mediated delivery of
chemokines and cytokines. Metronomic
chemotherapy
[26,27]
9L cells Gliosarcoma Gene therapy-mediated delivery of
proinammatory cytokines. Tumor
vaccination +TGF-?inhibition
[5,28,29]
Genetic
engineering
GFAP-Cre Lentiviral-mediated knock down of NF1
and p53
Mesenchymal GBM [30]
p53 KO Lentiviral-mediated delivery of Ras and
AKT
GBM [31]
C57BL/6FVB/nBalb/c
Sleeping beauty transposon plasmids
encoding for NRAS, AKT, SV40-LgT,
EGFRvIII, shp53
Grade III
astrocytoma, GBM
[32,33]
p53, Arf or
Ink4a-Arf KO Gtv-a
mice
RCAS-mediated delivery of PDGF GBM/
oligodendroglioma
TAM targeting with a CSF-1R inhibitor [34,35]
Ntva-a mice RCAS-mediated delivery of PDGF, Ras
and AKT
GBM [36]
Ink4a-Arf KO Gtv-a
mice
RCAS-mediated delivery of Ras and AKT GBM/gliosarcoma [37]
Genetic
engineering
Sprague Dawley Retroviral-mediated delivery of PDGF GBM [38]
Lentiviral-mediated delivery of PDGF,
H-RAS, AKT
GBM [39]
DC: Dendritic cell; GBM: Glioblastoma; HSV: Herpes simplex virus; RCAS: Retroviral vectors derived from the SR-A strain of Rous sarcoma virus; TAM: Tumor-associated microglia.
NCT02149225 is a GAPVAC Phase I trial in newly diagnosed glioblastoma patients testing vaccines using both
tumor-associated peptides and tumor-speci?c peptides, derived by expression pro?ling of tumors from individual
patients. A Phase I clinical trial is testing the safety and ef?cacy of personalized neoantigen vaccines with radiotherapy
for patients with MGMT unmethylated, newly diagnosed glioma (NCT02287428).
The genetic makeup of glioma seems to affect its response to immunotherapeutic strategies. Our lab has recently
shown that tumors with ATRX loss have increased genetic instability [159]. Genome wide data analysis of human
gliomas showed that ATRX mutation is associated with increased mutation rate at the single nucleotide variant level.
326 Immunotherapy (2018) 10(4) future science group
Current state & future prospects of immunotherapy for glioma Review
Such tumors may therefore be more immunogenic. IDH1 and IDH2 have been found to be mutated in >80% of
WHO grade II/III astrocytomas, oligodendrogliomas and oligoastrocytomas [9,12]. The study by Schumacher et al.
has shown that immunotherapeutic approaches can target this neoantigen [160].
Currently NCT02193347 (RESIST) is testing the use of IDH1 peptide vaccine (PEPIDH1M) in combination
with GM-CSF and tetanus toxoid in recurrent grade II glioma positive for IDH1-R132H in adults. NOA-16
(NCT02454634) is another Phase I study testing an IDH1 peptide vaccine in IDH1 mutant WHO grade III-IV
tumors that also show ATRX loss without 1p/19q codeletion.
Certain HSP such as HSP70 and HSP90 have been shown to bind glioma antigens and induce innate and
adaptive immune responses. Most trials using HSPs as vaccines have used the HSP–peptide complex 96. A Phase
I trial (NCT00293423) using HSP vaccine in recurrent glioma showed tumor-speci?c responses [161].APhaseII
trial showed median PFS and OS as 19.1 and 42.6 weeks, respectively in patients given the vaccine postsurgical
resection [135]. NCT01814813 is a Phase II study testing the combination of HSP–peptide complex 96 with
bevacizumab postsurgical resection in patients with recurrent disease.
Immunotherapy with DC vaccines
Successful preclinical studies have prompted a number of clinical studies using DC vaccines. DCs can be loaded
with peptides, tumor cell lysates, tumor-derived mRNA, viral antigens and cancer stem cells, all of which can be
tailored to the individual makeup of a tumor. An autologous DC vaccine, ICT-107, consisting of six peptides
(AIM-2, MAGE1, TRP-2, gp100, HER2 and IL-13Ra2) was tested in combination with radiochemotherapy in
a Phase I trial in glioma patients [137]. An OS of 38.4 months was noted along with an increased production of
IFNγand TNFαin stimulated CD8 T cells, and a Phase III trial (NCT02546102) is currently recruiting patients
to further investigate this treatment. In a Phase I/II trial of 22 patients with malignant glioma, administration of a
type 1-polarized DCs pulsed with synthetic peptides (EphA2, IL-13Rα2, YKL-40 and gp100) and poly IC, 58%
of the patients developed an immune response speci?c to at least one antigen, and IL-12 production by DCs was
observed to positively correlate with PFS [162]. A Phase I study evaluated the safety and ef?cacy of an autologous
tumor lysate-based DC vaccine. The median survival time for patients with recurrent glioma was determined to
be 133 weeks with a signi?cant increase in CD8 T cell activity [163]. An ongoing Phase III study (NCT00045968)
is using an autologous DC vaccine (DCVax-L) prepared by loading the DCs with proteins from the patient’s own
tumor.
APhaseI/II study (NCT00846456) was conducted using DCs loaded with mRNA ampli?ed from the cancer
stem cells isolated from the patient’s tumor. No severe side effects were observed and encouragingly the PFS in treated
patients was 2.9-fold longer than the matched controls [139]. An ongoing Phase I trial (NCT02049489) in recurrent
glioma is testing the safety of ICT-121, a DC vaccine targeting CD133, the antigen expressed on glioma stem
cells [164]. With the identi?cation of cytomegalovirus (CMV) and its gene products in glioma, preclinical studies
have utilized this feature to develop targeted immunotherapy. NCT00626483 is a study evaluating the combination
of anti-CD25 antibody, basiliximab with autologous DCs loaded with CMV pp65-lysosomal-associated membrane
protein mRNA. Another study (NCT00639639) showed that preconditioning with tetanus/diphtheria toxoid prior
to vaccination with pp65 RNA-pulsed DCs improves DC migration and survival [136]. ELEVATE is a Phase II
randomized study (NCT02366728) currently recruiting patients with newly diagnosed glioma, is investigating
preconditioning with tetanus toxoid or basiliximab prior to a CMV-targeted DC vaccine.
Immunological checkpoint blockade could improve the ef?cacy of antitumor DC vaccines in glioma patients, as
it has been shown in preclinical studies [95,145,146,165]. AVERT, a Phase I clinical trial (NCT02529072) is evaluating
the combination of CMV-targeted DC vaccine with PD-1-blocking antibody, nivolumab in patients with recurrent
HGG. Another Phase II study combining the SOC with the DC vaccine, AVO113 and bevacizumab showed an
increase in the median OS compared with the vaccine alone or bevacizumab alone group [166].
Immunotherapy with antibodies
This form of immunotherapy relies on targeting antigens uniquely expressed on glioma cells or molecules that
are overexpressed by tumor cells. Several Phase II trials have tested the ef?cacy of anti-VEGF therapies because
gliomas are highly vascularized tumors that express high amounts of VEGF. Most commonly used anti-VEGF
antibody is bevacizumab that has been tested either alone (NCT00345163) or in combination with irinotecan
(NCT00345163), etoposide (NCT00612430) or with concurrent radiotherapy (NCT00595322) [155,167–170].The
RTOG0825 and the AVAglio studies are prospective Phase III studies that tested the ef?cacy of TMZ-based
future science group www.futuremedicine.com 327
Review Kamran, Alghamri, Nunez et al.
radiochemotherapy with bevacizumab. No signi?cant bene?t in PFS or OS was seen in the RTOG 0825 study,
while the AVAglio study showed an improvement of 4.4 months in the PFS with no change in the OS in the
bevacizumab arm [171,172].
Monoclonal antibody therapy has been used to target EGFR using cetuximab. Combination of cetuximab with
bevacizumab/irinotecan was not superior to bevacizumab/irinotecan alone [173]. A Phase I study (NCT01238237)
showed that intra-arterial cerebral infusion of cetuximab and/or bevacizumab was safe for the treatment of recurrent
gliomas in adults, and a Phase I/II trial is now evaluating the safety and ef?cacy of intra-arterial cetuximab and
bevacizumab for the treatment of relapsed/refractory glioma in patients <22 years (NCT01884740). A Phase II
study tested the combination of nimotuzumab (anti-EGFR monoclonal antibody) with concomitant radiation and
vinorelbine in childhood diffuse pontine glioma [174].
A Phase III open label trial (NCT00753246) showed no signi?cant bene?t in OS by the addition of nimotuzumab
to standard therapy for newly diagnosed glioma [175]. ABT414 is an antibody–drug conjugate that delivers the
cytotoxic microtubule inhibitor, monomethyl auristatin F to cells with active EGFR or EGFRvIII [176].APhaseI
study (NCT02573324) tested the use of ABT414 alone or in combination with chemotherapy or chemotherapy
and radiation and showed responses in 4 out of 12 patients [177].
Immunotherapy with adoptive T-cell transfer & chimeric antigen receptor T cells
Adoptive T cell transfer (ACT) involves the ex vivo production of autologous tumor reactive T cells that are directly
transferred back to the patients. Initial studies using ACT for glioma involved the ex vivo expansion of T cells
induced by culturing with tumor cells or the isolation of T cells from the draining lymph nodes (dLNs) following
immunization with irradiated tumor cells and GM-CSF [178]. In a Phase I study in patients with recurrent glioma
and CMV-positive serology, 4 out of 10 patients who received at least three T-cell infusions showed PFS at the time
of data compilation and a median OS of 403 days, when infused with ex vivo expanded CMV-speci?c autologous
T cells (Australia New Zealand Clinical Trial Registry; ACTRN12609000338268). While the therapy was deemed
to be safe, no correlation was observed between the phenotype and functionality of T cells with PFS and further
investigations are warranted [179].
Chimeric antigen receptor (CAR) T cells consist of the antigen-binding region of a monoclonal antibody fused
with the signal transduction domain of CD3ζor FcR1γ, and thus combine the speci?city and avidity of a
monoclonal antibody to the signaling pathways for T-cell effector functions [180]. Preclinical studies have used CAR
T cells to target EGFRvIII, IL-13Rα2, HER2 and EphA2. The approach has also been shown to be safe with
minimal side effects in a ?rst-in-human pilot safety and feasibility trial (NCT00730613) targeting IL-13Rα2in
recurrent glioma [181]. Two out of three patients also developed transient antiglioma responses. Based on these
?ndings, the IL13Rα2-targeted CAR T cells were further modi?ed to incorporate 4-1BB (CD137) costimulation
and a mutated IgG4-Fc linker. Central memory T cells were lentivirally transduced to produce these IL13BBζCAR
T cells. Early ?ndings from one patient who received intracavity and intraventricular infusions showed clinical
regression that was sustained for 7.5 months post the initiation of the CAR T-cell therapy. Disease recurrence was,
however, observed at new locations after the last cycle, possibly due to the decreased expression of IL13Rα2maybe
responsible. Additionally accumulation and expansion of the CAR T cells in the CSF in later cycles and over the
7-day infusion cycle were limited. The results from this patient have prompted the expansion of the Phase I study
to evaluate intraventricular administration in a larger cohort of patients [181].
Ongoing clinical trials are evaluating the safety and ef?cacy of CAR T cells against EGFRvIII, IL-13Rα2, HER2,
EphA2, CD133 and MUC I in malignant glioma. A Phase I study (NCT02209376) evaluated the feasibility and
safety of manufacturing and administering CAR T cells redirected to EGFRvIII (CART-EGFRvIII) to patients with
EGFRvIII-expressing recurrent GBM. No evidence of off-target toxicity or cytokine release syndrome was observed.
The data showed evidence of CART-EGFRvIII traf?cking to the brain tumor, proliferation of the CAR T cells
and antitumor activity; however, robust compensatory immunosuppressive mechanisms including upregulation of
IDO1 and PD-L1 and recruitment of Tregs were observed to develop [182].
A Phase I trial is also investigating the use of CMV-speci?c cytotoxic T lymphocytes expressing a CAR targeting
HER2 in patients with glioma (NCT01109095). Infusion of HER2-speci?c CAR-modi?ed CMV-speci?c T cells
was shown to be well tolerated with no dose-limiting side effects. The study also showed clinical bene?t in 8 out
of 17 patients (partial response in one and stable disease in seven patients) thus warranting further trials [183].
Numerous investigations are looking into enhancing the speci?city and antitumor activity of CAR T cells
including the generation of tandem CARs, balanced-signal CAR, dual-receptor circuit CARs and CARs containing
328 Immunotherapy (2018) 10(4) future science group
Current state & future prospects of immunotherapy for glioma Review
chimeric switch receptors [184–188]. The extracellular domain in chimeric switch receptor consists of PD-1 and
the intracellular domain is stimulatory so that upon binding PD-L1 a stimulatory rather than inhibitory signal is
generated [189]. Efforts are also being made to manage the toxicities associated with the administration of CAR T
cells [190].
Oncolytic viral therapy
Viruses hijack host cells’ replication, eventually leading to host cell death and infection of the surrounding healthy
cells. Some studies have shown how viruses can be targeted speci?cally to tumor cells [191,192]. In addition to causing
cell death, virus infection also leads to the activation of innate and adaptive immune responses and therefore
become attractive immunotherapeutic agents [193]. Two variants of HSV-1 containing mutations in ICP34.5
and ribonucleotide reductase (RNR) have been shown to be safe in Phase I trials and are currently in Phase II
testing [194,195]. Other variants of HSV-1, such as M032 and rQNestin34.5 are in preclinical testing. ONYX-015
is an E1B mutant adenovirus that was shown be safe in Phase I study [196]. Another variant AdDelta24-RGD
is currently in preclinical and clinical development [197–200]. Reovirus selectively infects cells with activated Ras
pathways and when tested in Phase I study with recurrent glioma demonstrated safety and antiglioma activity [201].
An attenuated poliovirus PVS-RIPO showed ef?cacy in preclinical testing and is currently being tested in Phase
I study (NCT01491893) [202,203]. Interim analysis from this study using historical controls seems to confer a
survival advantage to the patients infused with PVS-RIPO [204]. H-1 is a parvovirus variant, that was shown to be
oncolytic in rat and human GBM cell lines and is in a Phase I/IIa study for recurrent glioma (NCT01301430) [205].
NCT00390299 is a Phase I trial testing the measles virus variant, MV-CEA, in patients with recurrent glioma.
Since the receptor for measles virus is expressed on healthy brain tissue and T cells, further work is ongoing
to enhance the selectivity and safety of this virus [206,207]. Avian Newcastle disease virus has been tested in a
Phase I/II trial (NCT01174537), with no serious side effects and a complete response in one patient [208].To
further enhance the therapeutic ef?cacy of the oncolytic virus and to reverse tumor-induced immunosuppression,
NCT02798406 (CAPTIVE/KEYNOTE-192) is a Phase II trial of a conditionally replicative adenovirus, DNX-
2401, in combination with anti-PD-1 monoclonal antibody, pembrolizumab in patients of recurrent glioma or
gliosarcoma.
Immunostimulatory gene therapy
The aim of immune stimulatorygene therapy is to modulate the tumor environment such that a robust and
effective antitumor immune response can be generated. In a Phase I trial, combination of suicide gene therapy
using HSV-TK and IL-2 resulted in minimal side effects and partial response in 2 out of 12 patients [209]. IL-4 has
been tested in Phase I study of IL-4-HSV-TK gene-modi?ed autologous tumor to elicit an immune response [210].
A Phase I study conducted in Japan in patients with malignant glioma showed minimal toxicity and a 50%
reduction in tumor size in two out of ?ve patients that were given liposomal-mediated delivery of IFNβ[211].A
second Phase I trial that directly delivered Ad-hIFNβinto the tumor cavity and the surrounding area demonstrated
safety and tumor cell apoptosis [212]. Adeno-associated viral vectors have been also developed to locally deliver
adeno-associated virus (AAV)-IFN-βand tested in combination with chemotherapy. Since DNA replication is
required for the synthesis of the second strand of DNA in order to activate the transcription of single-stranded
AAV vector, chemotherapy was administered after viral gene therapy, improving the median survival of murine
GBM models when compared with single treatments [213]. AAV vectors have been also employed to deliver IL-12
in rodent GBM models [214]. Our team has developed a conditionally cytotoxic immune stimulatory gene therapy
mediated through the delivery of adenoviruses encoding HSV1-TK and Flt3L. This therapeutic approach results in
tumor regression, long-term survival and a robust memory T-cell response in numerous preclinical glioma models.
Importantly, concomitant treatment with temozolomide enhances the ef?cacy of this gene therapeutic approach in
murine models of brain cancer [215]. This strategy is currently under investigation in a Phase I clinical trial in GBM
patients (NCT01811992) [47,216–218].
The combination of immune gene therapeutic strategies with the blockade of immunosuppressive mechanisms
could improve their ef?cacy in GBM patients. We have recently found that antibody-mediated blockade of
immunological checkpoints and depletion of MDSCs, which constitute 40% of tumor-in?ltrating immune cells,
enhances the antitumor immune response induced by TK/Flt3L gene therapy in GBM mouse models [95].Armed
oncolytic HSV (oHSV G47), which encodes for IL-12 has been shown to exhibit robust antitumor effects in
murine models of GBM when combined with anti-CTLA-4, anti-PD-1 antibodies [219].
future science group www.futuremedicine.com 329
Review Kamran, Alghamri, Nunez et al.
Conclusion
The extensive molecular characterization of gliomas, coupled with the 2016 WHO histological classi?cation, have
been instrumental in improving our understanding of glioma progression and the response to therapeutics. Also, the
genetic lesions encountered within the glioma cells, play a critical role in reprogramming the immune TME. This
has opened the horizon for scientists to investigate novel glioma treatment strategies. Work in the ?eld has led to the
conclusion that there is a need for combinatorial treatments, in order to elicit higher ef?cacy and better outcomes in
the clinic. In particular, immunotherapies offer very promising approaches for prolonging patient survival; in several
ongoing clinical trials immunotherapies have shown evidence of signi?cant anti-tumor outcomes, i.e., circulating
speci?c anti-glioma T cells and higher in?ltration of activated immune cells into the TME.
Future perspective
Glioma is a devastating disease and despite many years of research the prognosis remains dismal. Signi?cant progress
has been made in developing immunotherapeutic regimens and these may soon be included in the SOC. Several
challenges, however, need to be overcome, the chief among which is the intratumor heterogeneity [220].
Given the enormous increase in availability of gene expression, epigenetic and molecular pathway analysis, a
personalized therapeutic approach tailored to the tumor would be ideal. A second point to consider is the standard-
ization of diagnostic, therapeutic response and ef?cacy criteria for clinical trials, making it easier to interpret results
and compare outcomes across different clinical trials. The immunotherapy response assessment in neuro-oncology
criteria is being established in this regard [221]. Repeat tissue sampling is extremely challenging for CNS tumors
and the assessment of therapeutic ef?cacy is further complicated by the associated edema and pseudoprogression.
Efforts are also being made to identify unique biomarkers to serve as inclusion criterion or that can be of prognostic
value to predict the response to a particular therapy using tumor-derived DNA from the cerebrospinal ?uid [222].It
is also apparent that given the tumor heterogeneity and immunoediting resulting from treatment, a single approach
will not be suf?cient and successful treatment will require the combination of multipronged therapies such as those
combining multiple checkpoint inhibitors with radiation, the combination of checkpoint inhibitors with IDO
inhibitors or the combination of checkpoint inhibitors with immune stimulatory gene therapy or with vaccination
strategies [95,145,146]. Ongoing clinical trials are testing combinatorial approaches to achieve broad and durable
clinical ef?cacy. Another important factor to consider for the integration of immunotherapy with the current SOC,
is the effect of radiation and TMZ on cells of the immune system. Hyperfractionated radiation has been found
to correlate with CD4 T-cell depletion [223]. TMZ also causes lymphopenia and it is therefore critical to evaluate
the novel immunotherapeutic approaches in the context of SOC [224,225]. Of note, our lab has shown that TMZ
administration does not affect the therapeutic ef?cacy of the TK/Flt3L immunotherapy approach currently in a
Phase I study [215].
Executive summary
rRecent molecular characterization of several glioma subtypes, raises the possibility of tailoring treatments to
specic genetic lesions encountered in these tumors. This will give rise to precision medicine-based therapies for
glioma patients.
rThe presence of the blood–brain barrier hampers the efcacy of chemotherapies for brain tumors.
Immunotherapies, which rely on the migration of activated, tumor antigen-specic cytotoxic T cells, could yield
efcacious therapeutic options, as activated immune cells can migrate across the blood–brain barrier.
rThe use of oncolytic therapeutic approaches, which induce the release of tumor-derived ligands capable of
stimulating the immune system of the host, provides an exciting therapeutic modality, triggering immunogenic
tumor cell death.
rImprovements in genetically engineered chimeric antigen receptor T cells, in order to improve their survival,
tumor penetration and in vivo expansion, may provide an attractive therapeutic modality.
rCombination therapies, including standard of care together with immunotherapies provide improved efcacy.
rAdding immune checkpoint blockade to immunotherapies, would provide another layer of enhancement of
therapeutic efcacy.
rA coordinated, multi-institutional approach would be required to analyze the results from multicentric Phase I
clinical trials, which would enable to move these exciting novel therapies in a timelier fashion into the clinical
arena in order to benet glioma patients.
330 Immunotherapy (2018) 10(4) future science group
Current state & future prospects of immunotherapy for glioma Review
Supplementary data
Supplementary information includes one table. To view the supplementary data that accompany this paper please visit the journal
website at: www.futuremedicine.com/doi/suppl/10.2217/imt-2017-0122
Financial & competing interests disclosure
This work was supported by the NIH/National Institute of Neurological Disorders & Stroke (NIH/NINDS) Grants R37NS094804,
R01NS074387 and R21NS091555, to M G Castro; NIH/NINDS Grants R01NS076991, R01NS082311 and R01NS096756 to P R
Lowenstein; NIH/NCI U01CA224160-01 and NIH/NIBIB R01EB022563 to M G Castro and P R Lowenstein; D Shah and M G
Castro are supported in part by NIH/NCI T32CA009676; University of Michigan M-Cubed and the Center for RNA Biomedicine
to M G Castro; the University of Michigan Medical School Department of Neurosurgery and the Comprehensive Cancer Center;
Leah’s Happy Hearts, Chad Tough Foundation, and the Phase One Foundation; Consejo Nacional de Investigaciones Cient′
?cas
y Tecnol ′
ogicas (CONICET PIP 114-201101-00353, Cooperaci ′
on Internacional CONICET-NIH, doctoral fellowship to AS Asad) and
Agencia Nacional de Promoci ′
on Cient′
?ca y Tecnol ′
ogica (PICT-2013-0310 and PICT-2015-3309) to M Candol. The authors have
no other relevant afliations or nancial involvement with any organization or entity with a nancial interest in or nancial conict
with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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