Semin Immunopathol (2017) 39:225–239

DOI 10.1007/s00281-016-0616-7

REVIEW

The development of dendritic cell vaccine-based

immunotherapies for glioblastoma
David A. Reardon 1 & Duane A. Mitchell 2

Received: 13 December 2016 / Accepted: 20 December 2016 / Published online: 30 January 2017
# Springer-Verlag Berlin Heidelberg 2017

Abstract In this review, we focus on the biologic advantages glioblastoma tumors [3, 4] as well as the evaluation of dose
of dendritic cell-based vaccinations as a therapeutic strategy intensive temozolomide chemotherapy [5], anti-angiogenic
for cancer as well as preclinical and emerging clinical data therapy with bevacizumab [6, 7], and integrin inhibition with
associated with such approaches for glioblastoma patients. cilengitide [8] in large randomized phase 3 clinical trials, none
of these efforts has led to an improvement in survival.
Furthermore, given that all patients ultimately progress fol-
lowing standard therapy, salvage treatments have been woe-
Introduction: glioblastoma overview
fully inadequate with a series of meta-analyses evaluating out-
come across a wide spectrum of salvage therapy clinical trials
Glioblastoma is the most common primary malignant central
revealing progression-free survival rates at 6 months (PFS-6)
nervous system tumor among adults with an incidence rate of
of less than 15% [9]. Although bevacizumab improves PFS-6
3.19 cases per 100,000 person-years, which translates into
to approximately 40%, this improvement translates into min-
approximately 13,000 cases diagnosed in the USA each year
imal overall survival benefit [10]. Two additional points fur-
[1]. The current standard of care for newly diagnosed glioblas-
ther underscore the inadequate outcome associated with cur-
toma patients was established over 10 years ago and includes
rently available therapies for glioblastoma. First, survival data
maximum safe surgical resection, targeted cranial external
quoted for glioblastoma patients reflect outcome for patients
beam radiotherapy administered with daily temozolomide
well enough to participate in prospective clinical trials. A
followed by adjuvant temozolomide chemotherapy adminis-
more accurate representation of outcome derives from
tered for 6 months or longer. This multimodality treatment
population-based studies where outcome for all patients from
approach is associated with a median progression-free surviv-
a given geographic locale is summarized [11]. A recent review
al of only 7.8 months, a median overall survival of
of population-based studies reveals a median survival of ap-
14.6 months, and a 5-year survival rate of under 10% [2].
proximately 8.5 months for newly diagnosed glioblastoma
Despite marked advances that have led to improved under-
patients. [12]. Second, in a recent analysis across a spectrum
standing of the biology and molecular complexity of
of aggressive adult cancers, malignant gliomas including glio-
This article is a contribution to the special issue on Dendritic Cell Subsets
blastoma were rated as having the highest rate of years per life
and Immune-mediated Diseases - Guest Editor: Francisco Quintana lost and earliest age of death, providing further evidence of the
short survival time following diagnosis [13]. Novel, innova-
* David A. Reardon tive treatment strategies are clearly needed for both newly
David_Reardon@dfci.harvard.edu diagnosed and recurrent glioblastoma patients.

1
Center for Neuro-Oncology, Dana-Farber Cancer Institute, 450
Brookline Ave, Dana 2134, Boston, MA 02215-5450, USA The potential of immunotherapy for glioblastoma
2
Preston A. Wells, Jr. Center for Brain Tumor Therapy, Lillian S.
Wells Department of Neurosurgery, McKnight Brain Institute, Although a vision of harnessing the immune system to effec-
University of Florida, Gainesville, FL 32605, USA tively treat cancer dates back over 120 years to initial efforts
226 Semin Immunopathol (2017) 39:225–239

pioneered by William Coley in which he attempted to enhance an ability to eradicate skin allografts at every organ site except
anti-tumor immune responses by administration of bacterial the central nervous system [23]. More recent aggregate data
toxins to bone cancer patients (BColey’s toxins^), [14] disap- refutes limitations associated with immunoprivilege of the
pointing results subsequently associated with various immu- central nervous system and instead supports the presence of
notherapy approaches dampened enthusiasm for decades. a dynamic and effective interaction of systemic immune re-
However, enthusiasm has been markedly revitalized in the sponses within the brain [24, 25].
past few years based on meaningful survival benefits that have
been achieved by a panel of immunotherapeutics across a
spectrum of oncology indications. In April 2010, a historical Preclinical development of dendritic cell vaccines
benchmark was achieved in that the U.S. Food and Drug for glioblastoma
Administration (FDA) approved the first vaccine for the treat-
ment of cancer. This vaccine, sipuleucel-T (Provenge®) was Dendritic cells (DCs) are the professional antigen presenting
approved for the treatment of prostate cancer and consists of cells (APCs) of the immune system, and as such, mediate the
autologous dendritic cells sensitized ex vivo to prostatic acid role of capturing antigens in the periphery and processing and
phosphatase linked to granulocyte-macrophage colony-stimu- presenting them as antigenic peptides to the T cells of the
lating factor (PAP-GM-CSF). Results of a double-blind, immune system [26]. The DC was described as a novel stellate
placebo-controlled randomized, phase 3 clinical trial revealed cell found in the lymphoid organs of mice by Steinman and
that men with metastatic, castration-resistant prostate cancer Cohn in 1973 [27]. The prevailing concept in immunology
who received sipuleucel-T had a 4.1-month longer median through the early 1990s was that CD8+ cytotoxic T lympho-
overall survival than those who received placebo [15]. cytes (CTLs) were activated directly through recognizing
Thereafter, an exciting series of reports were unveiled MHC class I-restricted antigens presented on the surface of
documenting remarkable anti-tumor responses against a mul- all nucleated cells [28–31]. The discovery of intermediate
titude of different cancers that translated into survival benefit professional APCs that were capable of capturing exogenous
following treatment with inhibitory immune checkpoint antigens and presenting them in the class I antigen presenta-
blocking antibodies. Initially, ipilimumab (Yervoy), a human- tion pathway to CD8+ T cells (called Bcross-priming^) and the
ized monoclonal antibody (MAb) against cytotoxic T- identification of these professional APCs as the DC described
lymphocyte antigen-4 (CTLA-4), was approved in by Steinman and colleagues eventually led to an alternate
March 2011 for unresectable, metastatic melanoma [16] understanding that most, if not all, CTL responses against
followed by approval of humanized MAbs against pro- tumor cells and viruses required cross-priming by DCs to
grammed death-1 (PD-1) for metastatic melanoma and ad- initiate productive T cell responses [32, 33]. When culture
vanced lung cancer (nivolumab [Opdivo] and pembrolizumab methods for expanding large numbers of DCs in vitro from
[Keytruda]), as well as renal cell carcinoma (nivolumab myeloid progenitors and monocytes were developed, interest
[Opdivo]) [17–20]. Furthermore, additional cancer indication in using these ex vivo cultured DCs as cellular vaccines for
approvals for CTLA-4 and PD-1 targeting immune check- cancer and infectious disease emerged shortly thereafter [34].
point inhibitors are anticipated in the near future based on Studies in the early and mid-1990s demonstrated that mu-
results of recently completed clinical trials. Finally, rine bone-marrow derived DCs pulsed with peptide antigen
talimogene laherparepvec T-VEC (Imlygic) an oncolytic her- were potent inducers of CD8+ cytotoxic T lymphocyte (CTL)
pes simplex 1 virus was approved in October 2015 for the responses in vivo and more effective than direct peptide im-
treatment of patients with unresectable metastatic melanoma munization in strong adjuvants [35]. These observations led to
based on a significant decrease in lesion size that was main- the investigation of the use of DCs pulsed with exogenous
tained for over 6 months [21]. T-VEC can directly lyse cancer tumor antigens in the form of peptides, tumor lysates, tumor
cells but is also genetically engineered to secrete GM-CSF RNA, and vectors expressing tumor-associated antigens as a
which can trigger anti-tumor immune responses as an addi- novel form of cellular therapeutic vaccines in experimental
tional mechanism of action [22]. model systems. Initial studies were conducted in non-CNS
Based on the dramatic benefit associated with these immu- tumor models and established that small numbers of DCs
notherapy approaches for other cancer indications coupled pulsed with tumor antigens were sufficient to protect against
with the dire need to develop new treatment approaches to tumor challenge, that the effectors generated by DC-
improve outcome for glioblastoma patients, there has been immunization were indeed T cells, and that therapeutic re-
substantial interest to evaluate immunotherapeutics for sponses against established tumors could be engendered by
neuro-oncology patients. Nonetheless, overall enthusiasm to DC immunization [36–41].
advance immunotherapies for brain cancer has historically Shortly thereafter, bone marrow-derived DCs pulsed with
been tainted by dogma articulating immunoprivilege of the tumor lysates or tumor RNA derived from the B16 melanoma
central nervous system including dated studies demonstrating line were demonstrated to be effective in the generation of
Semin Immunopathol (2017) 39:225–239 227

therapeutic anti-tumor immune responses against tumors im- antigens such as myelin basic protein (MBP) to break
planted within the CNS of experimental mice [42]. These tolerance and induce robust EAE in mice, as well as for
studies demonstrated that peripheral immunization with tumor DCs pulsed with unfractionated tumor antigens that con-
antigen-pulsed DCs could engender immune responses that tain normal brain proteins to engender potent and effec-
were effective against tumors growing in the brain and impor- tive anti-tumor immunity without evidence of autoim-
tantly, that intolerable immunologic toxicity within the CNS mune toxicity [54, 55]. Thus, the balance between anti-
was not induced by this immunization strategy. Further exper- tumor reactivity and autoimmunity appears to lie within
imentation using syngeneic murine glioma models confirmed an immunologic hierarchy within these systems where
the capacity for DC-based immunization to expand therapeu- tumor-specific antigens dominate the immunologic re-
tic tumor-specific immune responses against glioma antigens sponse over self-antigens, despite DCs possessing suffi-
without precipitating autoimmune toxicity against normal cient activating capacity to break tolerance to self. In the
brain tissues [43–46]. While these early approaches used development of more effective DC-based therapeutics and
unfractionated tumor antigens in the form of tumor lysates combinatorial strategies, we will need to remain cognizant
or RNA, antigen-specific targeting strategies have also been of the potential for autoimmune manifestations and the
explored in preclinical model systems. DC vaccines targeting rationale for engineering increasing tumor specificity in
glioma relevant antigens such as the epidermal growth factor parallel with the development of increasing potency.
receptor variant 3 (EGFRvIII), gp100, survivin, and telome-
rase have shown efficacy in preclinical glioma models and are DC generation and subtypes
suitable antigen targets in subsets of patients with malignant
glioma [46–48]. These studies laid the foundation for the ad- Through extensive characterization of murine and human
vancement of clinical trials exploring both unfractionated tu- DCs, we now know that several subtypes of these APCs exist
mor antigen loaded DC vaccines as well as DCs pulsed with in vivo with distinct and overlapping functions and varying
single or multiple antigenic targets expressed within malig- lineage-specific differentiation pathways. DCs can be divided
nant brain tumors. broadly into two main cell types, the plasmocytoid DC (pDC),
Several underlying principles have emerged from preclini- potent anti-viral type I interferon producers, and conventional
cal studies of DC-based therapeutics for malignant brain tu- DC (cDC) that specialize in antigen capture and presentation
mors and other cancers, and we now have the benefit of re- [56]. While, cancer vaccine strategies in humans have relied
flection upon the results from the first generation of phase 1 largely on monocyte-derived cDCs generated in presence of
through phase 3 clinical trials of DC vaccines in human GM-CSF and IL-4, followed by a maturation cocktail of cy-
patients. tokines or CD40L stimulation, preclinical studies and human
in vitro studies have demonstrated a variety of culture systems
Toxicity concerns capable of generating DCs with varying phenotypic markers,
cytokine stimulatory capacity, and migratory capacity
A major concern in the development of cancer immuno- [57–60]. As an example, Fujita et al. compared mature murine
therapies is the risk of unleashing intolerable autoimmune DCs generated from bone marrow under standard conditions
or inflammatory toxicity in treated patients through induc- or type 1 polarized DCs (DC1) using exogenous culture in the
tion of tumor-specific or cross reactive immune responses presence of poly-ICLC, IFNγ, IFNα, and IL-4 in a vaccina-
or the activation of non-specific inflammatory pathways. tion protocol targeting the murine glioma line, GL261.
It is notable that DC vaccination has been well-tolerated Polarized DC1s maintained their stimulatory capacity in a
in human clinical trials of patients with GBM thus far, superior fashion to standard DCs in the presence of immuno-
including large-scale phase 3 trials. However, it is also suppressive cytokines and were superior in the induction of
notable that GBM tissues contain antigens that can induce therapeutic anti-tumor immunity in the established tumor
lethal experimental allergic encephalitis (EAE) in pri- model, demonstrating that the culture conditions for DC gen-
mates and guinea pigs [49]. In melanoma models and in eration can be altered to enhance DC function [45]. Similar
patients receiving cellular immunotherapy against mela- studies using genetically engineered DCs have demonstrated
noma, effective anti-tumor immunity can frequently be the capacity to generate superior DC vaccines through specific
associated with autoimmune responses against shared me- modification of DC culture conditions or gene expression
lanocyte antigens leading to the induction of vitiligo [61]. The existence of several DC subsets in vivo and the
[50–53]. Thus, there is some risk that autoimmune reac- observation that various culture conditions can alter the phe-
tions such as EAE may be concomitant with the induction notype and functional properties of DCs in vitro presents a
of truly effective immunization against unfractionated gli- challenge in determining what conditions are optimal for gen-
oma antigens. Preclinical model systems have both dem- eration of DCs for therapeutic use in the treatment of cancer.
onstrated the capacity for DC immunization against self- This is further complicated by the fact that parallels between
228 Semin Immunopathol (2017) 39:225–239

murine and human DC subsets are not applicable across all DC migration
molecular markers or APC functions, limiting the utility of
murine model systems in the optimization of human DC vac- Regardless of the route or immunization site, DC vaccines
cination strategies [62, 63]. As recent studies have developed presumably must migrate to lymphoid organs and activate
the use of humanized mouse models to study human DC resident T cells in order to achieve efficacious anti-tumor re-
function, it may be feasible to construct comparative efficacy sponses. Thus, the in vivo migratory capacity of administered
preclinical models for human DC vaccines against glioblasto- DC vaccines plays a critical role in their function as a cellular
ma and other cancers in the near future [64–66]. therapeutic. Numerous preclinical and clinical studies have
documented that only a fraction of administered DCs success-
fully migrate to vaccine-site draining lymph nodes (VDLNs)
Route of immunization [73–75]. Furthermore, studies aimed at enhancing DC migra-
tion to lymph nodes have demonstrated the capacity to en-
Numerous studies in preclinical systems have examined the hance anti-tumor immunity in preclinical systems. We recent-
route of immunization of antigen-loaded DCs and the impact ly demonstrated in a preclinical model system and randomized
on the induction of anti-tumor immunity. While there is con- clinical trial in patients with GBM that enhancement of DC
sensus agreement that antigen-loaded DCs must migrate to the migration to VDLNs using a tetanus-diphtheria toxoid vaccine
T cell-dependent areas of lymphoid organs in order to produc- (Td) was a potent modality for improving the immunologic
tively engage the host immune system, the best method for and clinical response to DC vaccination [76]. These observa-
achieving this objective is not readily clear despite several tions suggest that further exploration of means to enhance the
studies examining various routes of DC immunization in pre- migratory capacity of tumor-antigen loaded DCs may be a
clinical studies and a few clinical studies examining this var- fruitful axis of manipulation to improve the clinical efficacy
iable. A review of comparative studies would support the con- of this treatment strategy.
clusion that subcutaneous injection and intramuscular injec-
tion routes for DC vaccination are inferior to either intrader- Targeting neoantigens
mal, intralymphatic, intraperitoneal, or intravenous injection
in the induction of T cell-mediated immune responses [57, 67, DC vaccination studies that have advanced into clinical eval-
68]. Intradermal injection (i.d.) has been the most common uation in patients with GBM have utilized DCs pulsed with
route of administration for DC vaccines in patients with unfractionated antigens such as tumor lysates, peptides, or
GBM. Interestingly, the only FDA-approved cellular vaccine tumor RNA, or defined tumor-associated antigens provided
for cancer (Sipuleucel-T) is administered intravenously after as peptides or RNA [77, 78]. However, recent studies have
exogenous pulsing of blood-derived antigen presenting cells highlighted the importance of tumor-specific mutations or
with tumor antigen and GM-CSF. Another interesting varia- neoantigens as the potential drivers of host anti-tumor immu-
tion of DC immunization that has shown promise in preclin- nity and therapeutic immunologic responses to cancer immu-
ical glioma studies has been the intratumoral administration of notherapy [79–82]. The capacity to rapidly assess and identify
cytokine-secreting DCs to condition the tumor microenviron- neoantigens expressed within glial tumors using the advent of
ment [45]. While intranasal administration has not been ex- genomic technology and bioinformatics processing to predict
plored for DC vaccines targeting gliomas, this route of deliv- immunogenicity, lends the ability to derive patient-specific
ery of other cell-based and vector-based therapeutics in rodent vaccines tailored to target tumor neoantigens. Murine gliomas
model systems has been shown to convey unique effects on have been recently characterized with respect to neoantigen
CNS diseases [69, 70]. The deep cervical lymph nodes have expression laying the foundation for neoantigen-directed DC
been shown to be the tumor-draining lymph nodes for CNS vaccines to be explored within these experimental systems
malignancies in murine models, capture tumor-derived anti- [83].
gens that drain from the CNS, and are disproportionately af-
fected by tumor-induced immunosuppression. While it is un- A modified view of immune privilege
clear whether DC vaccines preferentially targeted to the cer-
vical lymph nodes versus other lymphoid organs would be Our current understanding of immune surveillance of the
desirable, a recent study examining proximity of vaccination CNS has shifted considerably from the classic view of the
to an intracranial murine glioma demonstrated inferior T cell brain as an immune privileged site that is largely ignored
activation as immunization was delivered more proximal to by the peripheral immune system. We now know that the
the primary brain tumor [71]. These findings suggest that CNS is drained by classical lymphatic channels in the me-
route and immunization site may be important variables in ninges and that activated lymphocytes readily gain access
the efficacy of DC vaccination in patients with malignant gli- to the brain parenchyma and tumor microenvironment [84,
omas [72]. 85]. There is consensus agreement that while T cell
Semin Immunopathol (2017) 39:225–239 229

priming, or the initiation of immunologic responses, re- evasion. Furthermore, the complex intratumoral heterogene-
quires antigen-presentation to occur in classical lymphoid ity that is characteristic of human GBM tumors is not likely
organs outside of the CNS, the brain has sufficient mech- recapitulated by current transplantable or spontaneously
anisms by which antigens are drained and sampled by the arising GBM models. Thus, we are aware that current murine
peripheral immune system to initiate productive immunity glioma models likely oversimplify the complexity of gener-
within the CNS compartment [24, 86]. Further understand- ating productive anti-tumor immune responses against ma-
ing, however, of the role of and origin of APCs involved lignant gliomas in humans. It is perhaps, therefore, most use-
beyond the initiation of immune responses in the brain may ful to utilize murine models as comparative efficacy screens
impact on future DC vaccine strategies for malignant for prioritizing the merits of advancing one DC-based strat-
gliomas. egy over another approach in stringently controlled model
Classical DCs are not detected in the normal brain pa- systems and for identifying mechanisms of treatment
renchyma, however, MHC class II expressing dendriform resistance.
APCs are readily recruited into inflammatory lesions with- Improving the predictive value of preclinical studies of
in the brain. These DCs may be recruited from the blood, DC-based therapeutics or other treatment approaches may be
derived from the meninges where classical APCs lie at the questionable as a laudable goal for translational neuro-
interface between the periphery and brain circulatory sys- oncology research. Recent studies of the predictive value of
tem, differentiate from perivascular macrophages, or rep- even phase 2 human clinical trial results in oncology have
resent brain APCs that are derived from resident progenitor demonstrated that only about one-third of approaches deemed
cells such as microglia in the context of inflammation [87, appropriate for advancement after phase 2 human trials result
88]. An understanding of the cellular origin and impor- in positive phase 3 clinical trials [90, 91]. This low predictive
tance of APC presentation of tumor antigens in situ within value for earlier stage clinical trials in humans suggests that
the tumor microenvironment may have important implica- utilization of preclinical models to gain mechanistic insights,
tions for continued development of DC vaccines for GBM. identify potential correlative biomarkers and mechanisms of
Whether intratumoral DC vaccines or vaccines adminis- treatment resistance, and to screen treatment approaches for
tered in the proximity of the CNS lesions should be prior- comparative prioritization of clinical development may be
itized may be contingent on a better understanding on the nearer term achievements of continued preclinical model
importance of local APC functions. In contrast, if periph- development.
eral T cell activation is sufficient to generate tumor-
reactive lymphocytes that can migrate efficiently to malig-
nant gliomas and engage tumor cells and resident APCs in
a productive manner, then the expediency of peripheral DC Clinical development of dendritic cell vaccines
vaccination would take precedent. for glioblastoma

Preclinical glioblastoma models for immunotherapy Based on the potent ability of dendritic cells to present
antigens and induce clonal T cell expansion, [92] a vari-
One of the major challenges in evaluating translational im- ety of DC-targeting vaccines have been developed for
munotherapy in neuro-oncology (and other cancers) is the cancer applications including glioblastoma [93, 94].
limitations imposed by contemporary preclinical GBM Proof of concept regarding dendritic cell based vaccina-
models. The majority of preclinical evaluations to date im- tion was recently established for oncology with the FDA
plore the use of syngeneic, immunocompetent murine brain approval of sipuleucel-T (Provenge®) for the treatment
tumor models to examine the efficacy and mechanisms of of metastatic, castration-resistant prostate cancer based
DC-based vaccine strategies against glial tumors [89]. on a 4.1-month survival advantage demonstrated in a
Because of the nature of cell-based therapeutics requiring double-blind, placebo-controlled randomized phase 3
the transfer of DCs generated in one host to be used a vaccine study [15].
in a recipient-host and the desire to have large cohorts of Clinical trials evaluating dendritic cell-based vaccines for
tumor-bearing mice with similarly staged tumors for thera- glioblastoma patients initiated over 15 years ago and can be
peutic evaluation, the use of genetically engineered sponta- classified based on the type of tumor antigens incorporated to
neous mouse models has been limited in favor of the evalu- ex vivo sensitize dendritic cells. A major strategy has utilized
ation of transplantable intracranial tumor lines. These pooled, non-selected tumor antigens collected via either acid
models likely do not recapitulate the host-immunologic evo- elution or tumor cell lysate for dendritic cell sensitization
lutionary process of spontaneous tumor development, and whereas an alternative strategy has utilized synthesized pep-
thus may be lacking in certain physiologic conditions that tides from defined tumor antigens for dendritic cell
contribute to tumor immunosuppression and immune sensitization.
230 Semin Immunopathol (2017) 39:225–239

Dendritic cell vaccination utilizing pooled, Furthermore, intratumoral infiltration of CD3+ T cells, primar-
non-selected tumor antigens ily consisting of CD8+/CD45RO+ memory T cells was noted
in half of the patients who underwent tumor resection follow-
Acid-eluted tumor antigens ing vaccination therapy.

The rationale for sensitizing dendritic cells to a pool of non-

selected tumor antigens is based on the marked heterogeneity Tumor lysate-derived antigens: recurrent malignant
present within glioblastoma tumor cells. Although the term glioma
Bmultiforme^ was originally associated with glioblastoma
based on the marked variability of cell type and morphology While initial studies utilizing acid eluted tumor antigens were
observed microscopically among these tumors, we now ap- ongoing, parallel efforts utilizing whole tumor lysates as a
preciate the striking molecular genetic heterogeneity within source of tumor antigens began to be explored. The whole
and across glioblastoma tumors based on elegant gene muta- tumor lysate approach quickly became preferred due to tech-
tion and expression studies [95]. Theoretically, use of pooled, nical challenges associated with acid elution including re-
non-selected tumor antigens could sensitize the immune sys- quirement for a large tumor sample and difficulties consistent-
tem against a broad repertoire of heterogeneously expressed ly obtaining short-term cultures of resected tumor samples.
tumor associated antigens that could lessen the evolution of The tumor lysate approach involves a series of snap freeze/
immune escape. A theoretical disadvantage associated with thaws (typically 5–6) of homogenized tumor lysate obtained
this approach is that cross reactivity against normal, non- from the operating room which generates tumor fragments.
tumor cellular constituents present within the pooled material Following centrifugation, the cell free supernatant is collected
could lead to potential autoimmunity or experimental allergic and used to pulse cultured autologous dendritic cells. Tumor
encephalomyelitis. The initial studies evaluating this approach antigens are phagocytosed and ultimately presented on MHC
utilized pooled non-specific tumor antigens derived from acid class I and II molecules. Several neuro-oncology immunother-
elution of tumor cells based on the ability of this approach to apy groups have evaluated the tumor lysate dendritic cell vac-
effectively segregate tumor surface peptides bound to MHC cination approach for malignant glioma patients. Initial trials
class I molecules [96]. Liau reported the first glioblastoma focused on patients with recurrent tumors and later including
patient treated with this approach and utilized tumor antigens trials for newly diagnosed patients. All results to date derive
eluted from an allogeneic, MHC class I-matched glioblastoma from primarily single arm, non-randomized clinical trials typ-
tumor to sensitize autologous dendritic cells pulsed ex vivo. ically conducted at a single center.
[97] Importantly, the dendritic cell vaccinations were well Yu initially reported results of a phase 1 study of 12
tolerated with no evidence of autoimmune reactivity. recurrent patients in 2004 (nine with glioblastoma and 3
Furthermore, peripheral blood T cells demonstrated prolifera- with anaplastic astrocytoma) [100] that was followed in
tive responses specific to the allogeneic tumor peptide prepa- 2008 by results of a phase 2 study that included 23 recur-
ration as well as evidence of increased CD3+ T cell infiltration rent glioblastoma patients [101]. There were no grade III/
into the tumor. Despite having a heavily pretreated, recurrent IV adverse events or evidence of autoimmune reactivity
glioblastoma, this patient survived for 6 months after her vac- noted for either study. Over half of the vaccinated patients
cine therapy. Two, parallel prospective phase 1 studies were demonstrated increased interferon-γ expression by periph-
then conducted among malignant glioma patients utilizing eral blood mononuclear cells exposed to tumor lysate after
pooled, non-selected antigens that were acid eluted from au- vaccination. Encouraging evidence of prolonged survival
tologous tumor samples collected following short-term cul- was observed, particularly among patients with evidence of
ture, and then sensitized to autologous dendritic cells interferon-γ cytoresponsiveness.
ex vivo. Yu et al. treated seven patients including five with A group led by De Vleeschouwer and van Gool reported an
glioblastoma and two with anaplastic astrocytoma [98] while initial series of 12 recurrent malignant glioma patients in 2004
Liau treated 12 glioblastoma patients [99]. Patients received [102], which was followed by their report of 56 recurrent
three biweekly vaccinations of up to 1 × 106 dendritic cells glioblastoma patients treated on a phase 2 study in 2008
pulsed with 50–100 μg of acid-eluted tumor peptides. Again [103]. All patients underwent debulking surgery prior to vac-
the dendritic cell vaccinations were well tolerated with no cination and they treated patients who were at least 3 years
evidence of autoimmunity and no dose-limiting toxicities old. Two patients developed grade IV cerebral edema but oth-
were observed among treated patients in either study. Over erwise the vaccinations were overall well tolerated. Median
half of the vaccinated patients demonstrated measurable overall survival on the phase 2 study was 9.6 months and 11%
tumor-specific cytotoxic T cell responses, and some of these of patients were alive at 3 years. Patients who were younger
patients were noted to have a longer survival than those who (<35 years old), and who were able to undergo a gross total
did not develop positive cytotoxic T cell responses. resection, appeared to have a better outcome.
Semin Immunopathol (2017) 39:225–239 231

Hunn and colleagues recently reported preliminary results improved survival was observed for patients with lower recur-
of a phase 1 study of temozolomide re-challenge combined sive partitioning analysis class and MGMT methylation sta-
with autologous tumor lysate pulsed dendritic cell vaccination tus. Although changes in immune cell subsets were measured
among 14 patients who had progressed on standard temozo- before and after vaccination, these data did not correlate with
lomide radiochemotherapy [104]. In this study, tumor lysate outcome.
was derived from surgically resected tumor following progres- Fadul recently reported outcome of ten newly diagnosed
sion. There were no grade ≥ 3 adverse events attributed to the glioblastoma patients treated with administration of autolo-
study vaccine and two patients (14%) demonstrated positive gous, tumor lysate pulsed dendritic cells [110]. A total of three
ELISPOT assays. Two patients achieved a radiographic re- vaccinations were administered at 2-week intervals beginning
sponse and the PFS-6 rate was 22%. 5 weeks following completion of combined temozolomide
Yamanaka and colleagues utilized a different administra- chemoradiotherapy. Each vaccine consisted of 1 × 107 den-
tion strategy in their approach in that patients received a series dritic cells which were injected in equal aliquots into two
of tumor lysate pulsed dendritic cell vaccinations that were bilateral cervical lymph nodes. Patients underwent
administered both intradermally as well as intratumorally leukopheresis prior to beginning vaccination and 2 weeks af-
through an Ommaya reservoir. They initially reported treat- ter the third vaccination at which point they began 12 cycles of
ment of seven recurrent patients in 2003 [105] which was adjuvant temozolomide. No serious adverse events were re-
followed by a report of 24 additional patients in 2005 [106]. ported for vaccinated patients and the median survival was
No series adverse events or evidence of autoimmunity were 28 months. Assessment of immunologic endpoints revealed
reported. Evidence of vaccine immunoreactivity was observed that the proportion of CD4+ interferon-γ and CD8+
in some patients via increased ELISPOT and delayed type interferon-γ producing cells revealed increases but these
hypersensitivity reactions. Overall survival among glioblasto- values did not achieve statistical significance.
ma patients treated on the phase 2 study was approximately Vik-Mo recently reported preliminary results of an adapta-
16 months and interestingly patients who received both intra- tion of the whole tumor lysate approach in which they pulsed
dermal as well as intratumoral vaccination appeared to do autologous dendritic cells with mRNA derived from autolo-
better than those receiving intradermal vaccine alone. gous glioma stem cell cultures established from seven newly
diagnosed glioblastoma patients [111]. The rationale for fo-
Tumor lysate-derived antigens: newly diagnosed cusing on glioma stem cells is based on data demonstrating
glioblastoma this subset of malignant glioma tumors to be associated with
treatment resistance, angiogenesis and tumor growth.
The De Vleeschouwer group led efforts to integrate tumor [112–114] No significant autoimmune or adverse events were
lysate pulsed dendritic cell vaccination with standard temozo- noted. Following vaccination, increased lymphocyte prolifer-
lomide radiochemotherapy as established by Stupp [107] for ation in response to glioma stem cell lysate was observed for
newly diagnosed glioblastoma patients. They initially report- all patients but only one patient demonstrated a delayed type
ed outcome of eight patients treated on a feasibility pilot study hypersensitivity reaction. Progression-free survival was
[108] which was followed by results of a single arm, phase 1/2 1.9 years.
clinical trial that enrolled 77 patients. [109] All patients had
dendritic cells collected by leukopheresis following definitive Tumor lysate-derived antigens: potential biomarkers
surgical resection. Dendritic cell were cultured and tumor ly- of benefit
sate vaccinations were prepared while patients underwent the
standard 6-week course of external beam radiotherapy during Recursive partitioning analysis to integrate pre-treatment pa-
which they received concomitant temozolomide. Thereafter, tient and tumor related parameters has been demonstrated to
they received four vaccine doses administered at weekly in- generate valuable prognostic models for patients with newly
tervals prior to initiating adjuvant temozolomide. Booster vac- diagnosed glioblastoma [115, 116]. De Vleeschouwer devel-
cine doses were administered on day eight of the first, second, oped a four-class recursive partitioning analysis model for 117
third, and sixth adjuvant temozolomide cycles. Of note, all adult recurrent malignant glioma patients undergoing tumor
patients were off corticosteroids at the time of their dendritic lysate pulsed dendritic cell vaccination [117]. The model in-
cell collection and subsequent vaccination. For the phase 2 tegrates age, histopathologic grade, Karnofsky performance
study, the median age was 57 years and 66% of patients status, and mental status. Significant overall survival differ-
underwent a gross total resection. In general, vaccination ther- ences were noted among the four classes including percent
apy was well tolerated and there was no clear evidence of survival for at least 24 months to be 54.5, 26.7, 11.5, and
increased toxicity relative to that typically experienced follow- 0% for classes I, II, III, and IV respectively.
ing standard temozolomide chemoradiotherapy. Median over- Liau, Prins, and colleagues have reported a series of studies
all survival on the phase 2 study was 18.3 months and that provide important insight into potential biomarkers that
232 Semin Immunopathol (2017) 39:225–239

identify patient subsets more likely to derive significant clin- noted to have an improved outcome. Of note, changes in
ical benefit including improved survival following tumor ly- CD8+ T cells did not correlate with survival.
sate pulsed dendritic cell vaccination. In a series of 23 glio-
blastoma patients including 15 newly diagnosed and eight
recurrent patients, patients with a mesenchymal gene expres- Dendritic cell vaccination utilizing selected tumor
sion profile, which in general confers a poorer prognosis [118, antigens
119], had significantly prolonged survival compared to a his-
torical control population (p = 0.0046) [120]. Median overall Single antigen vaccination
survival achieved on this study was also noteworthy including
35.9 months for newly diagnosed patients and 17.9 months for EGFRvIII is an ideal immunotherapeutic target because it re-
those with recurrent disease. sults from an inframe deletion that generates a tumor-specific
In a follow-up study, six specific peripheral blood lympho- antigen that is not found in normal tissues [124]. It is present
cytes subsets were analyzed before and after vaccination from in approximately 30% of glioblastoma tumors [125] and trig-
24 newly diagnosed glioblastoma patients who were treated gers ligand independent, constitutive tyrosine kinase activity
with autologous dendritic cells pulsed with either tumor lysate [126] that is associated with tumor cell resistance to cytotoxic
(n = 19) or glioma-associated antigens (n = 5) [121]. The therapy [127]. Following preclinical demonstration of signif-
subsets included the following: T helpers (CD3+ CD4+); cy- icant anti-tumor activity against intracranial, EGFRvIII-
totoxic T cells (CD3 + CD8+); natural killer T cells (CD3 + positive tumors treated with dendritic cells pulsed with an
CD16+); natural killer cells (CD3-CD16+); B lymphocytes EGFRvIII peptide, an initial phase 1 dose escalation study
(CD3-CD19+); and Tregs (CD3 + CD4 + CD25 + cd127low). was conducted [128]. In this study, patients with newly diag-
The expression level of activation (CD25, CD69) and nega- nosed glioblastoma received increasing dose levels of dendrit-
tive (CTLA-4 and PD-1) costimulatory markers for each lym- ic cells pulsed with an EGFRvIII-specific peptide that spanned
phocyte subset was also assessed. Importantly, across all pa- the fusion junction of EGFRvIII conjugated to keyhole limpet
tients, the median percentage of each lymphocyte subset rela- hemocyanin (KLH) for three doses after maximum safe resec-
tive to total isolated cells did not significantly change after tion and standard radiotherapy. No dose-limiting toxicities or
vaccination. Although changes in the activation status of any serious adverse events were observed and patients received up
lymphocyte subset did not predict outcome, a poorer outcome to 5.7 × 107 dendritic cells. Most patients demonstrated evi-
to vaccination was associated with two measures of immuno- dence of EGFRvIII-specific immunoreactivity after vaccina-
suppression. First , every unit increase in Treg ratio after vac- tion. Specifically, 83% of patients (10 of 12) developed
cination was associated with a 2.623-fold increase risk of EGFRvIII-specific T cell proliferation responses in vitro while
death (p = 0.0228). Second, increased expression of CTLA- 56% (5 of 9 patients) developed positive EGFRvIII-specific
4 by either T helper cells or cytotoxic T cells also predicted delayed type hypersensitivity skin tests. Median time to pro-
poorer survival. gression and overall survival from the date of diagnosis were
More recently, CD8+ T cell cytokine responsiveness after 10.2 and 22.8 months, respectively. Of note, subsequent clin-
dendritic cell vaccination was evaluated among 21 glioblasto- ical trials administering an EGFRvIII-specific peptide conju-
ma patients after vaccination with dendritic cells pulsed with gated to KLH with granulocyte macrophage colony stimulat-
either autologous tumor lysate (n = 17) or glioma-associated ing factor (GM-CSF) without the use of autologous dendritic
antigens (n = 4) [122]. Functional responsiveness before and cells have shown encouraging anti-tumor activity among new-
after vaccination of peripheral blood lymphocytes to the ly diagnosed glioblastoma patients [129, 130]. As further
immunostimulatory cytokines interferon-γ and interleukin-2 proof of principle of the efficacy of targeting EGFRvIII with
was measured by phosphorylation of STAT-1 and STAT-5 via this approach, recurrent tumor samples evaluated following
phospho-specific flow cytometry. In this study, increased ra- administration of the EGFRvIII vaccine are no longer
tios of IL-2 responsiveness/pSTAT5 signaling and decreased EGFRvIII positive [131].
ratios of interferon-γ/pSTAT-1 signaling were associated with In another phase 1 study, Sakai recently reported results
improved overall survival at 2 years. among ten recurrent malignant glioma patients who were
Finally, Pellegatta recently evaluated changes in peripheral treated with autologous dendritic cells pulsed with peptide
blood immune cell subsets as well as circulating immune cy- corresponding to mutant Wilms’ tumor 1 (WT1) antigen
tokines before and after tumor lysate pulsed dendritic cell [132]. Vaccinations were well tolerated and no patients expe-
vaccinations among 15 patients with recurrent glioblastoma rienced grade ≥ 3 adverse events. Six patients (60%) achieved
[123]. They noted that improved survival was noted among a >2-fold increase in WT1-specific cytotoxic T lymphocytes
patients who had an increase in CD56+ natural killer cells as by tetramer analysis. Median overall survival for all patients
well as a decrease in serum TGFβ2 levels after vaccination. and those with glioblastoma on this study were 19 and
Patients with smaller tumors (defined as <20 cm3 were also 7 months, respectively.
Semin Immunopathol (2017) 39:225–239 233

Mitchell et al. recently reported a randomized pilot clinical Positive ELISPOT and skin delayed type hypersensitivity re-
trial in which 12 patients with newly diagnosed GBM re- actions were observed in 6 (67%) and 4 (44%) patients respec-
ceived autologous dendritic cells electoporated with messen- tively. Anti-tumor activity was disappointing however with
ger RNA encoding for the immunodominant cytomegalovirus only one patient achieving durable stable disease while the
(CMV) antigen, pp65 with or without a tetanus-diphtheria remaining patients had a best response of progressive disease.
(Td) toxoid booster vaccine selected to enhance DC migration The third study was conducted among 17 newly diagnosed
[76]. CMV antigens have been reported to be expressed at low HLA-A1/A2-positive, glioblastoma patients and pulsed autol-
levels in a high proportion of GBM tumors [133]. In an em- ogous dendritic cells with synthetic peptides corresponding to
bedded and blinded imaging study examining migration of HER2, TRP-2, gp100, MAGE-1, IL13Rα2, and AIM-2 [136].
Indium-111 labeled RNA-pulsed DCs to vaccine-site draining All patients underwent a gross total resection and received
lymph nodes, this study demonstrated that patients who re- three vaccinations at 2-week intervals beginning after radia-
ceived DC vaccination combined with the Td booster showed tion therapy. Standard adjuvant temozolomide was adminis-
increased migration of DCs to lymph nodes, prolonged immu- tered following the final vaccination. Vaccinations were well
nologic responses, and dramatically enhanced progression- tolerated and no grade >2 adverse events were observed. Post-
free survival (8.5 months vs >32.0 months from time of sur- vaccination increases in antigen-specific CD8+ T cell
gery, P = 0.01) and overall survival (18.5 months vs interferon-γ secretion was observed in 33% of patients.
>36.6 months from time of surgery, P = 0.01). DC migration Median progression-free and overall survivals were 16.9 and
to lymph nodes was strongly correlated with clinical responses 38.4 months, respectively.
in this randomized study, suggesting that enhancement of DC
trafficking may improve clinical responses to DC vaccination
in human patients. A randomized, blinded, and placebo- Remaining questions
controlled phase 2 trial is underway to confirm these results
(clinicaltrials.gov NCT02465268). The use of autologous dendritic cells pulsed with tumor anti-
gens represents a robust strategy to generate potent anti-tumor
Multi-antigen vaccination immune responses for malignant glioma patients. The aggre-
gate clinical data to date (Table 1) clearly demonstrates that
Although glioma associated antigens may induce less robust such an approach is safe and well tolerated. In addition, encour-
immune responses than tumor-specific antigens due to co- aging evidence of immune responses against targeted antigens
expression on normal tissues and host immunotolerance, vac- has been observed among both newly diagnosed and recurrent
cines consisting of synthesized peptides corresponding to a patients. Anti-tumor benefit as reflected by encouraging rates of
number of such antigens and combined with immunoadjuvant progression-free and overall survival have also been observed
can be readily developed and available as Boff the shelf^ prod- although these data typically are derived from small, single arm
ucts for cancer patients. Three studies evaluating autologous studies and hence may be affected by selection bias. Additional
dendritic cells pulsed with cocktails of glioma associated an- ongoing trials, including some randomized placebo controlled
tigens have demonstrated encouraging results for malignant studies (Table 2) will further define the efficacy of such vaccine
glioma patients. In the first study, 22 HLA-A2+ recurrent ma- strategies for malignant glioma patients. Nonetheless, critical
lignant glioma patients received at least four doses of autolo- questions remain to be answered. First and foremost is the
gous dendritic cells loaded with four synthetic peptides question whether ex vivo pulsing of dendritic cells are required
targeting EphA2, interleukin (IL)-13 receptor-α2, YKL-40 or can vaccines capable of activating dendritic cells in vivo
and gp100, in combination with polyinosinic-polycytidylic generate sufficient anti-tumor immune responses. Collection
acid [poly(I:C)] stabilized by lysine and carboxymethylcellu- of autologous dendritic cells requires leukopheresis and cultur-
lose (poly-ICLC) [134]. The vaccine was well tolerated and ing as well as maturation of these cells ex vivo that can be
neither grade ≥3 adverse events nor DLTs were observed. laborious and technically challenging. There are no planned
Encouraging immunogenicity of the vaccine was observed or ongoing trials designed to compare the efficacy and immu-
in that 58% (11 of 19 patients) developed positive nogenicity of ex vivo dendritic cell-based vaccines with vac-
interferon-γ ELISPOT or tetramer assays after the fourth vac- cines that activate dendritic cells in vivo.
cination. Nine patients (75%) remained progression free for at Another highly relevant unanswered question is the deter-
least 12 months. mination of which tumor antigens are most likely to generate
In a small study of nine recurrent malignant glioma pa- the most robust and durable anti-tumor immune responses. As
tients, α-type-1 polarized autologous dendritic cells were reviewed, studies to date have evaluated a full spectrum of
pulsed with synthetic peptides for WT-1, Her2, MAGE-A3, tumor antigens including those derived from whole cell ly-
MAGE-A1, and gp100 [135]. Vaccinations were well tolerat- sates to cocktails of tumor associated antigens to single
ed with no observed grade ≥3 adverse events or autoimmunity. tumor-specific antigens. Along these lines, ongoing studies
234 Semin Immunopathol (2017) 39:225–239

Table 1 Published clinical trials of ex vivo tumor antigen pulsed autologous dendritic cell vaccine treatment of malignant glioma patients

Phase Population Number Antigen Vaccine Immunoreactivity Efficacy Citation

of related
patients grade ≥3
AEs

I R 56 WTL 2% DTH: 2/12 (17%) PFS: 3 months De Vleeschouwer et al.

OS: 9.6 months 2008 [103]
I R 15 WTL NS NS PFS-6: 27% Pellegatta et al. 2013
OS: 8 months [123]
I R 9 WT-1, HER2, MAG-A3, None ELISPOT: 6/9 (67%) SD: 1 (11%) Akiyama et al. 2012
MAGE-A1, gp100 DTH: 4/9 (44%) [135]
I R 14 WTL None ELISPOT: 2/14 (14%) PFS-6: 22% Hunn et al. 2015 [104]
I/II R 10 WTL None DTH: 3/6 (50%) Minor MRI response: Yamanaka et al. 2003
ELISPOT: 5/5 (100%) 2/6 (33%) [105]
I/II ND 12 CMV pp65 None DC migration to lymph DC vaccine vs DC Mitchell et al. 2015 [76]
nodes 6/12 (50%) vaccine + tetanus
PFS 10.8 months vs
>27.0 months
OS: 18.5 months vs
>36.6 months
I/II R 24 WTL None DTH: 8/17 (47%) PR: 1 (4%) Yamanaka et al. 2005
ELISPOT: 7/16 (44%) MR: 3 (13%) [106]
SD: 10 (42%)
II R 10 WTL 8% DTH: 6/8 (75%) PFS: 3 months; Rutkowski et al. 2004
OS: 10.5 months [102]
II R 22 IL-13Rα2; EphA2; None ELISPOT: 6/10 (60%) ORR: 2/22 (9%) Okada et al. 2011 [134]
GP100; YKL-40 Tetramer: 5/9 (56%) TTP: 4 months (GBM)
and 13 months (AG)
II R 117 WTL NS None OS: 6–48.4 months De Vleeschouwer et al.
(varied by RPA class) 2012 [117]
I R and ND 14 WTL None CTL IFN-γ: 6/10 (60%) OS: 133 weeks Yu et al. 2004 [100]
(recurrent GBM)
I R and ND 12 WTL None DTH: 6/12 (50%) TTP: 15.5 months; OS: Liau et al. 2005 [99]
23.4 months
I R and ND 23 WTL None NS OS (newly diagnosed): Prins et al. 2011 [120]
35.9 months
OS (recurrent):
17.9 months
I R and ND 25 AIM-2, MAGE 1, TRP-2, None Cytokine response: 5/15 PFS: 16.9 months; Phuphanich et al. 2012
gp 100, HER2, IL-13Rα2 (33%) OS 38.4 months [136]
(newly diagnosed
GBM)
II R and ND 34 WTL None CTL IFN-γ: 17/34 (50%) TTP: 261 days Wheeler et al. 2008
(immune responders) [101]
I ND 9 WTL None CTL activity: 4/7 (57%) OS: 16. 25 month Yu et al. 2001 [98]
(GBM)
I ND 12 EGFRvIII None DTH: 5/9 (56%) TTP: 6.8 months Sampson et al. 2009
T cell proliferation: 10/12 OS: 18.7 months [128]
(83%)
I ND 7 Autologous glioma stem None CTL: 7/7 (100%) PFS: 1.9 years Vik-Mo et al. 2013 [111]
cell culture mRNA DTH: 1/7 (14%)
I/II ND 8 WTL 13% DTH: 3/6 (50%) PFS: 18 months Ardon et al. 2010 [108]
ELISPOT: 5/8 (63%) OS: 24 months
I/II ND 77 WTL NS None OS: 18.3 months Ardon et al. 2012 [109]
II ND 10 WTL None ELISPOT: 4/10 (40%) PFS: 9.5 months Fadul et al. 2011 [110]
OS: 28 months

AEs adverse events, CTL cytotoxic T lymphocyte, DTH delayed type hypersensitivity, ELISPOT enzyme-linked immunosorbent spot assay, IFN-γ
interferon-gamma, MR minor response, MRI magnetic resonance imaging, N newly diagnosed, NS not specified, OS overall survival, PFS progression-
free survival, PFS-6 progression-free survival rate at 6 months, PR partial response, R recurrent, RPA recursive partitioning analysis, SD stable disease,
TTP time to progression, WTL whole tumor lysate
Semin Immunopathol (2017) 39:225–239 235

Table 2 Ongoing clinical trials of ex-vivo pulsed dendritic cell vaccines for adult glioblastoma patients

Phase Population Number Design Antigens Sponsor Clinicaltrials.gov

of
patients

I Recurrent 20 Open label Whole tumor lysate University of Miami, Miami NCT01808820
FLA USA
I Recurrent 20 Open label CD133 Immunocellular NCT2049489
Therapeutics
I Recurrent and newly 40 Open label Allogeneic glioblastoma stem-like Cedars Sinai, Los Angeles, NCT02010606
diagnosed cell lysate CA USA
I/II Newly diagnosed 20 Open label WT-1 RNA University Hospital, NCT02649582
Antwerp, Belgium
II Newly diagnosed 116 Randomized, CMV pp65-LAMP mRNA University of Florida NCT02465268
single blind
II Newly diagnosed 100 Randomized, Glioma stem-cell like antigens Fudan University, Shanghai, NCT01567202
double blind China
III Newly diagnosed, 414 Randomized, AIM-2, MAGE-1, TRP-1, gp100, Immunocellular NCT02546102
HLA-A2+ double blind HER-2, IL-13Rα2 Therapeutics

are evaluating targeting tumor antigens by peptides, DNA- Combination strategies evaluating potent mechanisms of
and RNA-based strategies and it remains to be seen whether immunosensitization such as dendritic cell vaccination com-
a specific tumor antigen formulation may sensitize autologous bined with immune checkpoint blockade are highly relevant
dendritic cells more effectively. next-generation clinical trials which may significantly en-
There is also much debate regarding choice of vaccine hance therapeutic efficacy relative to that achieved with either
adjuvant and it remains unclear whether there is an optimal approach alone [142].
adjuvant strategy for dendritic cell-based vaccines. A broad Finally, an exciting adaptation for future vaccines includes
array of adjuvants are currently being evaluated in multiple the possibility of targeting specific subsets of dendritic cells in
vaccine formulations including toll-like receptor (TLR) ago- order to enhance particular aspects of anti-tumor immunore-
nists against TLR3 (polyICLC), TLR4 ( monophosphoryl lip- activity. For example, sensitization of tumor antigens to
id A), TLR5 (flagellin), TLR7 (Imiquimod), TLR7/8 CD141+ DCs would be expected to generate particularly po-
(residquimod), and TLR9 (CpG) [137]. In addition, GM- tent cytotoxic T lymphocytes whereas antigen targeting to
CSF has frequently been added based on its ability to attract CD1c+ dendritic cells would be expected to expand
and activate dendritic cells [138], although a severe but CD103 + CD8+ T memory cells that could persist to prevent
reversable hypersensitivity reaction associated with anti- relapse in the CNS microenvironment.
GM-CSC autoantibodies was recently reported in a glioblas-
toma patient undergoing autologous RNA-pulsed dendritic
cell vaccination combined with GM-CSF and dose- Conclusion
intensified temozolomide [139]. Choice of vaccine site and
dosing schedule have also not been carefully evaluated and The use of autologous dendritic cells pulsed ex vivo against
represent additional factors of potentially significant impact. tumor antigens continues to be a very promising avenue of
Which patient populations with regard to underlying tumor immunotherapy for malignant glioma patients. Preclinical
status (newly diagnosed versus recurrent), degree of associat- studies have validated this approach and results of clinical
ed tumor burden (gross total resected or subtotally resected) studies conducted to date are highly encouraging with regard
and use of concurrent corticosteroids may also be relevant to safety, immunogenicity and anti-tumor efficacy.
variables that could impact vaccine efficacy and these consid- Nonetheless, many variables related to patients, vaccine com-
erations also remain to be investigated. ponents, and methodology for vaccine preparation and admin-
A critical question going forward is whether robust tumor- istration remain to be carefully evaluated. Furthermore, given
specific immune responses generated by potent vaccination the remarkable diversity of immunosuppressive mechanisms
strategies can overcome the myriad mechanisms of immuno- employed by many cancers including malignant gliomas,
suppression aggressive tumors such as glioblastoma exploit combination immunotherapy strategies that incorporate potent
[140]. Recent clinical studies have demonstrated highly en- treatments to induce immunosensitization with blockade of
couraging rates of durable tumor response following adminis- critical immunosuppressive mediators will likely be required
tration of immune checkpoint inhibitors as a strategy to over- in order for the full potential of immunotherapy approaches
come immunosuppression mediated by PD-1 signaling [141]. such as dendritic cell vaccination to be achieved.
236 Semin Immunopathol (2017) 39:225–239

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advisory role with Tocagen, Inc.; has patents that have been licensed to 21. Andtbacka RHI et al. 2013 OPTiM: A randomized phase III trial
the Celldex Therapeutics, Inc.; and received research funding from of talimogene laherparepvec (T-VEC) versus subcutaneous (SC)
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