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Review
Skin Pharmacol Physiol 2006;19:124–131
DOI: 10.1159/000092592
Current State and Perspectives of
Dendritic Cell Vaccination in Cancer
Immunotherapy
A. Farkas
a, b
C. Conrad
a
G. Tonel
a
Z. Borbenyi
c
L. Kemeny
b
A. Dobozy
b
F.O. Nestle
a
a
Department of Dermatology, University Hospital of Zurich, Zurich , Switzerland; b
Department of
Dermatology and Allergology, University of Szeged, and c
2nd Department of Medicine and
Cardiology Center, Medical Faculty, Albert Szent-Györgyi Medical and Pharmaceutical Center,
University of Szeged, Szeged , Hungary
Introduction
In the past decades, many immunotherapeutic ap-
proaches for cancer have been developed. Recent scien-
tifi c progress included the discovery of tumor-associated
antigens (TAAs), new tools for monitoring an anticancer
immune response and application of novel adjuvants for
amplifi cation of the immune response such as dendritic
cells (DCs) [1] . DCs are potent antigen-presenting cells
recognizing microorganisms, secreting proinfl ammatory
cytokines and stimulating primary T-cell immune re-
sponses [2] . DC-based vaccines have shown effi cacy in
animal models and are now being tested in the clinic.
There are many parameters which should be taken into
account in DC vaccination studies including optimal DC
source and type, optimal maturation status, optimal cell
dose, optimal antigen preparation, optimal route, opti-
mal timing and optimal means of assessing immune and
clinical responses [3] . Furthermore, possible immune es-
cape mechanisms of tumors affecting DCs should also be
investigated [4]
.
Key Words
Cancer Immunotherapy Dendritic cell vaccination
Abstract
Recent progress in the approach towards immunother-
apy of cancer consists in molecular defi nition of tumor
antigens, new tools for phenotypical and functional char-
acterization of tumor-specifi c effector cells and clinical
use of novel adjuvants for optimal stimulation of a can-
cer-specifi c immune response such as dendritic cells. In
spite of these advances and immunological as well as
clinical responses in selected patients, mechanisms in-
volved in dendritic-cell-based cancer immunotherapy
are still poorly understood. Therefore, a standardized
study design and small pilot trials are needed to explore
open scientifi c questions in future clinical trials. This re-
view focuses on the different parameters of dendritic cell
biology relevant to cancer immunotherapy and on inno-
vative approaches to hopefully enhance the effi cacy of
dendritic cell vaccination.
Copyright © 2006 S. Karger AG, Basel
Received: May 2, 2005
Accepted after revision: June 23, 2005
Published online: April 6, 2006
Arpad Farkas, MD, PhD
Department of Dermatology, University Hospital of Zurich
Gloriastrasse 31, CH–8091 Zurich (Switzerland)
Tel. +41 44 255 3977, Fax +41 44 255 4418
E-Mail arpad.farkas@usz.ch or farkas_arpad@yahoo.com
© 2006 S. Karger AG, Basel
1660–5527/06/0193–0124$23.50/0
Accessible online at:
www.karger.com/spp
Dendritic-Cell-Based Cancer
Immunotherapy
Skin Pharmacol Physiol 2006;19:124–131 125
Source, Type of DCs and Their in vitro
Manipulation for Clinical Applications
The fi rst DC studies utilized whole blood leukapher-
esis products and gradient-based methods to enrich DC
precursors. Vaccination with these cells induced some
immunological and clinical responses in non-Hodgkin’s
lymphoma [5] , multiple myeloma [6] and prostate cancer
[7] . The potential problem with peripherial blood DC iso-
lation is the low yield. There are current attempts to mo-
bilize DCs using systemic fms -related tyrosine kinase 3
(FLT3) ligand [8] . The expansion of DCs from bone mar-
row precursors is also under investigation. CD34+ pro-
genitor-derived DC preparations lead to the induction of
T-cell responses and clinical responses in metastatic mel-
anoma [9] or multiple myeloma [10] .
Although DC numbers in peripherial blood are very
small, it is possible to generate acceptable numbers of cells
in vitro. The common way to generate DC vaccines is to
culture monocytes in the presence of interleukin-4 (IL-4)
and granulocyte macrophage colony-stimulating factor
(GM-CSF) for 5–7 days which results in the generation of
immature monocyte-derived DCs (moDCs). Immature
moDCs may be loaded with antigen and given to the pa-
tient or they may be subjected to a maturation step for
2–3 days. Usually, the maturation cocktail contains IL-4,
GM-CSF, in combination with IL-1
, IL-6, tumor necrosis
factor-
and prostaglandin E
2
(PGE
2
), referred to as the
gold standard maturation cocktail [11] . These standard
moDCs are homogenous, have high viability, migrate well
and induce cytotoxic T-lymphocyte (CTL) responses.
There is evidence that less time is required for DC differ-
entiation in vivo [12, 13] and there are studies which show
that fully functional DCs can be generated within 48 h
from monocytes in vitro, so-called ‘fast DC’ [14] . Most of
the clinical studies indicate that mature DCs are superior
to immature DCs in stimulating T cells [15, 16] , but there
are some clinical trials which clearly show immune re-
sponses and selected clinical responses using immature
DCs [17, 18] . Furthermore, some data indicate that ‘semi-
mature’ moDCs can prime strong CD4 T-cell responses
with a T-helper 1 (Th1)-type cytokine profi le in cancer pa-
tients [19] . The production of bioactive IL-12 by DCs is
crucial for the development of naïve T-cell precursors into
Th1-dominant cells, which will ensure the generation of
CTLs [20] . Thus, the IL-12 production of the ex-vivo-gen-
erated DCs provides information on their functional ma-
turity. There is a list of agents that inhibit IL-12 production
while leaving other aspects of the DC maturation process
unaffected, one of them being PGE
2
[21] . Indeed it has
been shown that DCs matured in the presence of the gold
standard maturation cocktail produce low levels of IL-12
[22] . DCs that fail to produce suffi cient IL-12 might prime
Th2 responses [23] ; therefore, some studies focus on en-
hancing the effi cacy of DC-based immunotherapy by
transduction of the IL-12 gene
into moDCs [24] . Not only
the maturation signals but also the kinetics of DC activa-
tion are very important in infl uencing their IL-12 produc-
tion. DCs produce IL-12 within 8–16 h after IL-12-induc-
ing stimuli, and later become resistant to further stimula-
tion. Thus, only properly activated DCs can effi ciently
prime Th1 responses, whereas so-called ‘exhausted’ DCs
may prime Th2 or nonpolarized T-cell responses [23] .
Many other protocols have been investigated for the
generation of DCs. For example, moDCs can also be gen-
erated in the presence of IL-13 or IL-15 instead of IL-4,
and for DC maturation, simply tumor necrosis factor-
without additional cytokines has been used [11] . Other
protocols may contain CD40 ligand, FLT3 ligand, stem
cell factor [25] , transforming growth factor-
[25] , lipo-
polysaccharide, poly-I:C, interferon-
(IFN-
), IFN-
,
and there are methods where leukemic cells were used as
DC sources [11] .
For clinical purposes, the development of DC vaccines
requires standardized methods that comply with Good
Manufacturing Practice (GMP) guidelines. Cytokines
and many Toll-like receptor ligands are now available in
GMP quality. Fetal calf serum (FCS) was part of initial
DC protocols, but DCs are now mostly generated in the
absence of FCS using autologous plasma [26] or in serum-
free cultures [27] in order to avoid unwanted loading with
xenoantigens being part of FCS [28] . A future approach
to the safe clinical use of DCs might be closed culture
system. The investigation of such systems is under way
in clinical trials [29–35] . Cryopreservation of DCs might
be an advantage to obtain standardized DC preparations
during vaccination studies. Many freezing and thawing
methods have been described in the literature [36–40] ,
and it seems that cryopreservation does not cause signif-
icant changes in the phenotype or function of DCs, indi-
cating that cryopreserved aliquots of DCs are suitable for
clinical application [41] .
Antigen Preparation and Loading
Tumors express many antigens which are potential tar-
gets for immunotherapy. DCs must obtain tumor anti-
gens to initiate antigen-specifi c immune responses. The
selection of tumor antigens and the way of loading ex-
Farkas /Conrad /Tonel /Borbenyi /Kemeny /
Dobozy /Nestle
Skin Pharmacol Physiol 2006;19:124–131
126
vivo-generated DCs is an important step to develop an
effi cient cancer vaccine. Antigen presentation strategies
use peptides, proteins, tumor lysates, tumor-derived
RNA or genetically modifi ed recombinant viruses for de-
livery of DNA. Other trials use exosomes plus peptides,
apoptotic bodies, DC/tumor fusions, tumoral idiotype or
-galactosylceramide. The majority of vaccinations were
carried out with tumor lysate or peptide-loaded DCs, but
there is a tendency to use RNA-loaded DCs [11] .
Whole tumor cell preparations aim to deliver a wide
range of tumor antigens to DCs. For this purpose, whole
apoptotic [42–44] or necrotic tumor [45, 46] cells, entire
tumor lysates [47–49] , and DC/tumor cell fusion prod-
ucts [50–52] have been used. Identifi cation of specifi c
TAAs is not necessary when we use whole tumor cell prep-
arations, but this strategy depends on the availability of
suffi cient tumor tissue from the patient. In the case of
unfractionated tumor material, there is a potential risk of
inducing autoimmunity. One of the strategies with whole
tumor cells is the use of hybridomas created by the fusion
of DCs with tumor cells. The standardization of hybrid
generation, using electrofusion or polyethylene glycol, is
not easy to achieve. To address this problem, a new meth-
od was developed to generate hybrid cell vaccines, based
on gene transfer of a viral fusogenic membrane glycopro-
tein into tumor cells. These DC/tumor cell hybrids can
be rapidly isolated, are highly potent in vitro in antigen
presentation assays, target lymph nodes in vivo and are
powerful immunogens against metastatic disease in mice
[53] . There are clinical trials published with DC/tumor
fusions in patients with solid tumors [51, 54, 55] and he-
matological malignancies [56–58] . Recently, investiga-
tors have used allogeneic DCs to generate DC/tumor fu-
sion vaccines. In this case, DCs are generated from nor-
mal donors which may have better functional properties
[59, 60] . On the other hand, T-cell responses to alloge-
neic DC/tumor fusions are dependent on tumor MHC
class I molecule expression, which is often inconsistent.
In a recent report, 24 patients underwent serial vaccina-
tion with
allogeneic DC/tumor hybrids. Two patients ex-
perienced a partial response, and 8 patients
demonstrated
stable disease [61] .
There is no need for tumor tissue if peptide combina-
tions are used and more accurate peptide-specifi c T-cell
response monitoring is possible [62] . As the induced im-
mune response is largely epitope-specifi c, no cross-reac-
tivity to normal tissue antigens is expected. For this meth-
od, it is a prerequisite to know the tumor epitopes, patient
HLA type and amino acid sequence of relevant peptides.
To avoid the generation of tumor cell escape variants, a
broad spectrum of tumor antigens can be used. Another
possibility is to use an unfractionated mixture of peptides
eluted from tumor cells to target a broad repertoire of tu-
mor antigens [63, 64] .
Purifi ed or recombinant proteins can also be a choice
for loading DCs [5] . Loading DCs with soluble protein
antigens circumvents the need of peptide epitopes bind-
ing to specifi c MHC surface molecules. The potential to
induce a broad repertoire of antigen-specifi c T cells might
be advantageous and should minimize immune escape.
As the immature phenotype of DCs is associated with ef-
fi cient protein uptake, the costimulatory molecule expres-
sion should be carefully monitored before loading [65] .
During a study of patients with
hormone-refractory pros-
tate cancer, autologous human DCs were exposed to a
recombinant fusion protein, linking full-length human
prostatic
acid phosphatase and GM-CSF and then in-
fused intravenously. The postvaccination
proliferative
responses that did develop were specifi c for the
immuniz-
ing antigen. Delayed time to disease progression also
cor-
related with development of a proliferative response to
prostatic
acid phosphatase [66] .
The RNA-based strategy to load DCs with tumor cell
antigens is receiving more attention [11, 67] . Even when
the amount of tumor material is limited, suffi cient
amounts of total RNA can be prepared and transfected
into DCs resulting in MHC class I-restricted antigen pre-
sentation [37, 68] . However, obtaining effective MHC
class II presentation is a challenge. Tumor-specifi c RNA
can be enriched by subtractive hybridization with normal
cell RNA; thus, the risk of autoimmunity can be decreased
[69] . Recently, it has been demonstrated that not only
immature DCs but alternatively mature DCs can be di-
rectly transfected with RNAs [70] . In one comparative
study, it was demonstrated that DCs loaded with tumor
mRNA were more potent in inducing T-cell responses
than DCs loaded with apoptotic cancer cells [44] .
Genetically modifi ed recombinant viruses can be used
to transfect genes of interest into DCs [71, 72] . However,
the potential adverse effects such as cell toxicity and the
induction of antivirus immune responses have so far lim-
ited the use of such vaccines [73, 74] . It has also been
shown that viral transduction of DCs may downregulate
their costimulatory molecule expression [73] .
Biodegradable poly(lactide-co-glycolide) microspheres
(PLGA-MS) are under investigation as delivery tools for
synthetic peptide antigen loading of human moDCs. Im-
mature moDCs readily take up PLGA-MS and present
epitopes from encapsulated proteins or peptides both on
MHC class I and class II. These DCs have good migra-
Dendritic-Cell-Based Cancer
Immunotherapy
Skin Pharmacol Physiol 2006;19:124–131 127
tion, cytokine secretion, survival and allostimulatory
properties and their antigen presentation capacity is pro-
longed [75] . However, PLGA polymers undergo bulk hy-
drolysis, resulting in a dramatic drop in pH value that can
signifi cantly degrade encapsulated DNA molecules, and
their DNA release rate is diffi cult to regulate. To solve
this problem, two alternative polyortho esters (POEs)
have recently been tested, and in contrast to the bulk deg-
radation seen in PLGA polymers, POEs exhibit surface-
restricted hydrolysis, causing a milder pH shift, less like-
ly to result in DNA degradation [76] .
Delivery of DCs
The route of vaccine administration is a very impor-
tant issue [77] . Various vaccination routes have been uti-
lized such as intravenous, intradermal, subcutaneous or
intranodal. A study which compared the intravenous, in-
tradermal and intranodal routes in melanoma patients
has suggested that the intravenous administration may
not work as well and that intranodal administration as
fi rst applied by our group [47] seems to result in superior
T-cell function [78] . Recent studies in mice suggest that
the number of DCs migrating from the skin to draining
lymph nodes after gene gun use is hundred times higher
than estimated earlier and these DCs persist for 2 weeks
[79] . Myeloid DC migration is controlled by the chemo-
kine receptor CCR7 and its ligands [80] . PGE
2
, which is
in most cases part of the maturation cocktail, induces a
very weak bioactive IL-12 production, but is important
for functional CCR7 expression of DCs [13, 81] . Cur-
rently, alternative efforts are being employed to facilitate
DC migration following intradermal injection such as the
distribution of DCs to more than one areas of the body
and pretreatment of the skin with proinfl ammatory cyto-
kines or Toll-like receptor ligands [82, 83] .
The direct injection of DCs into the lymph nodes cir-
cumvents the skin migration problem [49] . Intranodal
injection might destroy the fi ne architecture of the lymph
node; therefore, intralymphatic injection of DCs might
be an alternative.
Ex-vivo-derived DCs can be directly injected into the
tumor site [84] . In one early clinical study, patients with
metastatic melanoma and breast cancer were treated with
intratumoral DC vaccination and regression of the in-
jected tumors was seen in 6 out of 10 patients [85] . There
are attempts to enhance the effi cacy of intratumoral vac-
cination by engineering proinfl ammatory cytokine-ex-
pressing DCs [86–88] .
In vivo DC Manipulation
In vivo targeting of DCs would be an ideal situation
to circumvent demanding and time-consuming in vitro
cell manipulation. Several DC-associated C-type lectin
receptors such as DEC205 and DC-SIGN have been
shown to endocytose antigens coupled to antibodies di-
rected against them [89, 90] . The antigen DEC205 com-
plex leads to both CD4 and CD8 T-cell responses indicat-
ing cross-presentation of endocytosed antigens [90] , but
only the simultaneous delivery of a DC maturation stim-
ulus via CD40,
together with an antigen coupled to
DEC205, results in immunostimulation [90] . The ma-
nipulation of CD40 on DCs not only helps to overcome
peripheral T-cell tolerance [91] but also enhances their
costimulatory molecule expression, cytokine secretion
and induces anti-apoptotic signals [92] .
The combination of FLT3
ligand with a DC activator,
immunostimulatory DNA,
and a tumor antigen to acti-
vate and load DCs in vivo showed that DCs can be acti-
vated, and potent antitumor
immunity can be induced in
mice [93, 94] .
Epidermal DCs or Langerhans cells (LCs) migrate to
draining lymph nodes and undergo maturation. LC-tar-
geted delivery of gene products has been accomplished by
plasmid DNA encoding TAAs fused to GM-CSF or che-
mokines [95] . Migratory LCs might also be entrapped by
subcutaneous implantation of an ethylene vinyl acetate
polymer rod releasing macrophage infl ammatory protein
3
and hapten. The entrapped LCs may be in situ antigen
loaded by a second ethylene vinyl acetate polymer rod re-
leasing TAAs. This technology induced tumor-specifi c im-
munity without the use of ex vivo DC manipulation [96] .
Another immunization strategy has recently been de-
scribed which uses a plasmid DNA expressing HIV pro-
teins. The DNA is formulated to a mannosylated particle
to target antigen-presenting cells and to protect the DNA
from intracellular degradation. This topically applied vac-
cination represents an effective in situ DC-based immuni-
zation possibility providing new therapeutic options [97] .
Future Challenges and Conclusions
Until now, a large number of DC vaccination studies
have been performed [98] ; however, the comparison of
these trials is diffi cult because of the lack of standardiza-
tion. It will be important to standardize the parameters
of DC vaccination and to test the many different vari-
ables in defi nitive trials with clear endpoints.
Farkas /Conrad /Tonel /Borbenyi /Kemeny /
Dobozy /Nestle
Skin Pharmacol Physiol 2006;19:124–131
128
Closed culture systems may simplify the handling pro-
cedures and will circumvent the use of expensive GMP
facilities. The efforts focusing on the in vivo application
of DCs will also hopefully facilitate the laborious and
time-consuming in vitro cell manipulation. The optimal
DC type for vaccination remains to be determined in the
future; therefore, other DC types should be tested for ther-
apeutic use such as plasmacytoid DCs (pDC) [99] and
IFN-
-derived DCs (IFN-DC) [100] . pDCs are known to
infi ltrate tumor tissues [101] and they have the capacity
to produce high amounts of type I IFNs [99] , which may
induce the maturation of local myeloid DCs. Highly ac-
tive IFN-DCs can be generated from monocytes in the
presence of IFN-
and GM-CSF [100] and may be useful
in future vaccination studies (e.g. chronic myelogenous
leukemia) [102] .
Preferably, vaccination against multiple tumor anti-
gens and TAAs expressed by the tumor and also by the
stroma should be used. Many tumors such as malignant
melanoma are characterized by high genetic diversity and
instability; therefore, it would be desirable to analyze the
immunogenic profi le of the primary tumor and of the
metastatic lesions with the help of microarrays to design
and choose the specifi c vaccine [103] .
It will be important to remove regulatory T cells or to
block their function, e.g. with the help of anti-CTLA-4
antibody [104, 105] . Clinical studies have just started ex-
ploiting this option. Other combination possibilities need
to be investigated such as DC vaccination with concurrent
cytokine administration, with chemotherapy and in com-
bination with therapies targeting the tumor stroma [106] .
The stage of cancer determines the effi cacy of immu-
notherapy. Until now, end-stage patients have been treat-
ed with a weak immune system; therefore, it is suggested
that in future studies DC vaccination should be extended
for treatment of patients with low tumor burden.
In conclusion, future controlled clinical trials will
hopefully provide new insight into immune activation,
tumor regression and tumor immune escape during DC
vaccination.
Acknowledgements
We apologize that many excellent papers could not be cited due
to space limitations. Arpad Farkas is on the leave from the Depart-
ment of Dermatology and Allergology in Szeged, Hungary, and was
supported by an International Union against Cancer (UICC) fel-
lowship.
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