A Preclinical protocol exploring mRNA-transfected allogeneic
monocytes as a potential cancer vaccine
Li-Jun Mu1,2, Gunnar Kvalheim2, Stein Sæbøe-Larssen1, AnnaCarin Wallgren3, Bengt
Andersson3, Alex Karlsson-Parra3,Gustav Gaudernack1
1
Section for Immunotherapy, 2Lab of Cellular Therapy, The Norwegian Radium Hospital,
University of Oslo, Norway; 3 Department of Clinical Immunology, Sahlgrenska University
Hospital, Göteborg, Sweden
1
Abstract
Aiming to target antigen-presenting cells in vivo, we developed a clinical grade protocol using
allogeneic monocytes as a combined antigen-vehicle and adjuvant. Enriched monocytes were
obtained from leukapheresis product by ElutraTM. Fresh or frozen/thawed monocytes were
transfected with EGFP or hTERT mRNA by square wave electroporation and transferred to
Teflon bags containing X-vivo 20 medium supplemented with GM-CSF 1000 U/ml. After
culturing for 24 hours, cells were treated with Vibrio Cholerae neuraminidase 0.025U/ml for
30 minutes, then concentrated and frozen. Fresh and frozen/thawed transfected monocytes
gave a similar cell yield based on the initial number of loaded cells. Electroporation was
highly efficient in transfecting mRNA into monocytes as tested by flow cytometry of EGFPmRNA transfected cells, or by a TRAP assay after transfection with hTERT mRNA. To prime
T-cell responses, PBMCs (containing T cells as well as antigen-presenting cells) were
incubated with thawed allogeneic transfected monocytes for 7-10 days in vitro. Re-challenge
with transfected autologous monocytes was found to generate a significant T-cell response
specific for transfected mRNA (as measured by proliferation and IFN-gamma ELISPOT
assays). Our results indicate that antigen-loaded allogeneic monocytes may act as antigenvehicles and adjuvant for efficient cross-presentation of transfected antigens by antigenpresenting cells in the “recipient” PBMC-population and thus provide the basis for “on the
shelf” cellular vaccines made from spared products of ordinary blood banking.
2
Introduction
Dendritic cells (DCs) in tumour vaccine therapy can be applied in two approaches. One is to
treat patients with DCs that have been isolated and manipulated in vitro. The other strategy is
to target DCs in vivo (Biragyn et al., 2000). Immature DCs with high capacity to capture
antigens are located in the peripheral tissues but DC-precursors may also be actively recruited
from the circulating pool of phagocytic monocytes (Radolph et al, 1999, Immunity,11,753).
By receiving adequate maturation stimuli, recruited DCs become matured and can migrate to
secondary lymph nodes to prime CTL responses. A microenvironment enriched by
proinflammatory components is thus a prerequisite not only for activation of DC’s and
induction of a potent immune response but also for efficient recruitment of immature DCs and
DC-precursors in situ.
T cells recognizing allogeneic MHC molecules by the direct pathway of allorecognition are
present at very high frequencies in the T-cell repertoire, approximately 1–10% of an
individual's T lymphocytes will respond to intact foreign MHC molecules expressed on
antigen-presenting cells (APCs) from of another, allogeneic, individual (Wang et al., 1999).
By performing conventional allogeneic mixed leukocyte reactions (MLRs) in vitro we
recently showed that primary, and particularly secondary MLR-supernatants, contain high
levels of monocyte/immature DC-recruiting CC-chemokines and pro-inflammatory cytokines
(Wallgren et al, 2005, Scand J Imm. 62, 234 ). Exposure of immature DCs to primary or
secondary MLR-supernatants was found to upregulate CD40-expression and further enhanced
LPS-induced interleukin-12 p70 production. Secondary MLR-supernatants additionally
induced upregulation of CD86 and deviated allogeneic T cells-responses towards Th1
(enhanced IFN-gamma production without concomitant induction of detectable IL-4 or IL-10
production) (Wallgren et al, 2005 ). Taken together, these previous findings predict that the
inflammatory process induced during direct allorecognition in vivo may have the potential to
3
provide a milieu rich in DC-recruiting , DC-maturating and Th1-deviating cytokines and
chemokines without the addition of other immunostimulants or prior clonal expansion of
specific cells. Once recognised by the host T cells, the allogeneic cells are killed and taken up
by resident autologous phagocytic cells, including DCs (Inaba 1998). This opens for the
following novel strategy: To use allogeneic APCs loaded with tumour antigens as a
combined antigen-vehicle and adjuvant to induce potent Th-1 deviated responses against
cancer.
Human monocytes constitute about 10-20% of peripheral blood mononuclear cells and
express both HLA class-I and -II antigens. They are capable of initiating allogeneic mixed
leukocyte responses in culture, particularly when interacting with allogeneic memory T cells (
Thomas et al, J Immunol, 1993, 151, 6840) and can be loaded with antigens by a variety of
mechanisms. The allogenicity of APCs has further been shown to become enhanced by
removing negatively charged surface sialic acid by enzymatic treatment with bacterial
neuraminidases (Taira and Nariuchi, J Immunol, 1988,141,440; Hirayama et al, J Exp Med,
1988, 168,1443; Fanales-Belasi, J Immunol, 1997,159,2203). Treatment of human monocytes
with neuraminidase has further been shown to activate the extracellular signal-related kinases
ERK 1/2 which result in enhanced production of monocyte/immatureDC-recruiting
chemokines, including macrophage inflammatory protein (MIP)- 1 alpha and MIP-1 beta
(Stamatos, et al. 2004). Since monocytes can be loaded with tumour antigens in the form of
mRNA by electroporation, this enables the production of ready made, “on the shelf”
allogeneic vaccines for any cancer where one or more antigens are available .
We here present data demonstrating that neuraminidase-treated allogeneic monocytes loaded
with the “universal” tumour antigen telomerase reversed transcriptase (hTERT) (to a level
similar to that found in cancer cell lines) are able to elicit T-cell responses against autologous
4
monocytes loaded with the same antigen. Aiming to apply this strategy in clinical settings, we
have developed a clinical grade cancer vaccine protocol.
Material and methods
Enrichment of monocytes
Peripheral blood mononuclear cells (PBMCs) were collected by leukapheresis from patients
who were enrolled in our approved ongoing protocol for DC-based vaccination. After
informed consent, excess PBMC were frozen at -80ºC for further use in pre-clinical protocol
development. Monocytes were enriched directly from the leukapheresis products using a cell
separator (Elutra™, Gambro BCT). Cells were washed once and resuspended in X-vivo 20
medium. Samples were taken for enumeration and viability test. The purity of monocytes was
determined by flow cytometry analysis. These monocytes were either cultured directly or
frozen for later use.
Transfection of EGFP-mRNA or hTERT-mRNA and culture of monocytes
Bulk preparations of mRNA for EGFP (Enhanced Green Fluorescence Protein) and hTERT
(Human Telomerase Reverse Transcriptase) were performed as described earlier (SæbøeLarssen et al., 2002). The quality of all mRNA-preparations was controlled by electrophoresis
on agarose gels stained by Gelstar (Cambrex Bio Science, Verviers, Belgium). The RNA
samples were also evaluated on an Agilent Bioanalyser instrument (Agilent Technologies,
Palo Alto, CA, USA), as previously described (Kyte et al., 2005). The mRNA content was
estimated according to the manufacturers protocol. Fresh or frozen/thawed monocytes were
washed and suspended in culture medium, 0.4 ml (107–108) cells were mixed with mRNA
(50–100 μg/ml), transferred to a 4-mm-gap cuvette and pulsed with a BTX ECM 830 square-
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wave electroporator (Genetronics, San Diego, CA) using different parameter settings. Cells
were then immediately transferred into Teflon bags containing X-vivo20 medium
supplemented with GM-CSF (1000U/ml). Mock-transfected monocytes were cultured
following the same electroporation procedure without mRNA. After 24 hours, cells were
treated in medium with neuraminidase (Sigma) 0.025U/ml for 30 minutes. Afterwards, cells
were washed and frozen in culture medium with 50% human albumin and 10% DMSO at 80ºC until use. For monocytes transfected with EGFP mRNA, we tested the transfection
efficacy and cell viability with the FACSCalibur flow cytometer. For monocytes transfected
with hTERT mRNA, expression of hTERT was analysed by TRAP assay (Telomeric Repeat
Amplification Protocol), as described previously (Sæbøe-Larssen et al., 2002).
Monocyte characterization by flow cytometry
Monocytes were phenotyped before incubation, and before and after treatment with
neuraminidase by the use of the following panel of FITC-, PE-, PerP- and APC- conjugated
antibodies: CD1a, CD14, HLA-DR (Dako Cytomation, Glostrup, Denmark), CD40, CD83,
CD86, and CCR7 (Immunotech, Marseilles, France). Irrelevant, matched antibodies were
used as negative control. Cells were analyzed by flow cytometry using a FACSscan (Becton
Dickinson).
Generation of T-cell responses in vitro
Frozen PBMCs and monocytes were thawed in a 37ºC water bath and washed once. After
enumeration, PBMCs were plated in 24-well plate (3x106/well) and stimulated with irradiated
(30Gy) transfected monocytes, either autologous or allogeneic (3x105/well). After 7-10 days
culture in X-vivo 20 medium at 37ºC in a 5% CO2 incubator, cells were collected and washed
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once for further testing. Culture supernatant was harvested at 6 hours, 12 hours, 24 hours, 48
hours, 5 days and 7 days and frozen for later cytokine measurement.
T-cell proliferation assay
PBMCs stimulated by allogeneic monocytes and autologous monocytes were seeded in 96well U-bottomed microtiter plates as responder cells (Tallo, Tauto). Irradiated (30Gy)
autologous transfected and mock- transfected monocytes (tMo, mMo) were used as
stimulators. Responder cells 1x105/well were cultured in triplicate with various numbers of
stimulators for 2-3 days at 37˚C, 5% CO2 in X-vivo 20 medium. After labelling with 3.7 x 104
Bq [3H] thymidine (Laborel, Oslo, Norway) for 18 hours, cells were harvested. Radioactivity
incorporated into proliferating cells was determined using a Packard TopCount microplate
scintillation counter. Data are reported as counts per minute (CPM). Medium only, responder
cells only and stimulator cells only were used as negative controls.
INF γ ELISPOT assay
The same responder and stimulator cells as described above were used. Monocytes as
stimulators were plated with a cell concentration of 5x103/well, while the responder cells were
added in a titration from 1x105 to 2.5x104 cells/well in duplicate. Details have been described
previously (Mu, et al, 2003). Spots were enumerated using an automated ELISPOT counter
(Carl Zeiss Vision, Oberkochen, Germany). Results were recorded as spots per 105 T cells.
Determination of cytokine levels By Bioplex assay
Supernatants were thawed and analysed by Bioplex cytokine assays (Bio-Rad Laboratories,
Hercules, Ca, USA) according to the manufacturers protocol. We analysed the supernatants
for the presence of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, TNF-α, INF-γ, GM-CSF,
7
macrophage inflammatory protein 1β (MIP-1β), and monocyte chemoattractant protein 1
(MCP-1). Standard kits for all cytokines/chemokines were obtained from Bio-Rad
Laboratories (Hercules, Ca, USA).
Statistical analysis
Student’s T test for independent samples comparing CPM in proliferation test and spots in
ELISPOT assay for transfected and non-transfected monocytes was used. Results are
displayed as mean ± standard deviation (SD). Statistical significance was determined at P<
0.05.
Results
Cell yield and Viability
The purity of monocytes following elutriation was 85.7 ± 4.2% (n=3). After transfection and
24 hours incubation, the average cell recovery from fresh monocytes was 34.4±5.1% with a
mean viability of 95.7±4.9%, while from frozen/thawed monocytes the yield was 36.7±7.9%
with a viability of 93.3±3.8% (Table1). The monocytes cultured freshly following elutriation
tended to adhere together and to form large cell aggregates, which were difficult to separate.
Since cell counting is based on single cells in suspension, this accounted for much of the
recorded cell loss in this protocol. The actual cell number present in the cell suspensions may
thus be considerably higher. In contrast, this phenomenon was much less pronounced after
freezing and thawing. The finding that the total cell recovery from fresh and frozen
monocytes was similar indicated that cell loss during freezing and thawing was roughly equal
to cell “loss” due to aggregation.
8
Transfection efficacy and hTERT-mRNA evaluation
Efficiency of RNA transfection was evaluated by transfection with EGFP mRNA using 4-mm
cuvettes. Several parameters with regard to voltages and time of exposure were tested and the
optimal protocol was found to be a single pulse, 500 V at 4ms. By using these parameters, a
transfection efficacy of more than 95% CD14+ cells can be achieved and the mean
fluorescence level using EGFP was increased on an average to 74-fold above background.
The survival rate of transfected monocytes tested by flow cytometry using propidium iodide
staining was similar to that of non-transfected cells. A representative EGFP-transfection
experiment is shown in Figure1. In other experiments, a common tumor antigen, represented
by synthetic mRNA for hTERT, was used for transfection. We analysed the telomerase
enzymatic activity of the transfected monocytes after freezing and thawing, as a measure of
hTERT protein expression, using buffer as a negative control and the tumor cell line K562 as
a positive control. A clear enzymatic activity was detected (Figure2) in mRNA transfected
monocytes compared with mock-transfected cells, showing that mRNA transfection resulted
in telomerase reverse transcriptase activity levels comparable to that found in a tumor cell line
expressing very high levels of the enzyme.
Flow cytometry evaluation of monocytes
Generally, the phenotypic analysis showed little changes in the expression of the HLA-DR,
CD1a, CD40 and CD86 during culturing and treatment of the monocytes. After transfection
and treatment with neuraminidase, cells showed some up regulation of CD83 and CCR7 when
compared with monocytes before culturing (Figure3). This might indicate the presence of
GM-CSF during the 24 hr culture period may to some extent have driven the differentiation of
the monocytes towards dendritic cells. Freezing and thawing of the monocytes had no
apparent effect on their phenotype (data not shown).
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T-cell monitoring and functional study
To prime T-cell responses in vitro, PBMCs were incubated with thawed autologous or
allogeneic transfected monocytes (mRNA either from EGFP or from hTERT) in three
individual experiments for each antigen. In this experimental setting, autologous tMo will
elicit T-cell responses specific for antigens encoded by the transfected mRNA, presented by
autologous HLA molecules. Allogeneic tMo on the other hand, will engage at least two
different sets of reactions: 1) viable tMo (stimulator cells) will elicit a conventional mixed
lymphocyte reaction (MLR) by stimulating allo-specific T cells directly recognizing
allogeneic MHC molecules on stimulator cells, 2) stimulator cells that subsequently become
killed by alloreactive CTLs within the responder population will be phagocytosed by
responder APC’s and their antigens, including epitopes from hTERT or EGFP, will be
presented by autologous HLA molecules (cross presentation). Only the latter set of T-cell
reactivity will be measured if the primed T cells are re-challenged with autologous tMo.
After 7-10 days, specific responses of primed PBMCs were therefore tested by ELISPOT and
proliferation assay using transfected autologous monocytes. Data from the proliferation assays
are shown in Figure 4. A single priming of PBMCs by autologous or allogeneic transfected
monocytes could generate a significant T-cell response specific for transfected mRNA. As
demonstrated in the upper panel, clearly both allogeneic and autologous EGFP transfected
monocytes give rise to specific T-cell responses (4A and B). There was no clear trend
towards auto being better than allo or vice versa. Similar results were seen when hTERT
mRNA was used (Fig. 4C and D). Cells prepared from fresh or frozen monocytes were similar
in stimulating T-cell response (data not shown). The extent of autologous MLR observed was
generally less than seen when autologous mRNA transfected DC were used to prime T cells
(Mu et al., 2003). The background observed with the allo-stimulated PBMC may also be due
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to an auto MLR, or may partly represent continued proliferation by some of the MLR reactive
T cells in the culture.
Very similar data were obtained in the ELISPOT assay. The data in Fig. 5 represent
experiments with cells from five different donors. Clearly, in donor A similar responses to
EGFP were obtained using either way of priming. When mRNA transfection was done with
monocytes that had been frozen, a similar degree of priming was seen. Higher background
was observed with donor B in the EGFP series and with all the donors in the hTERT series,
indicating a considerable auto MLR reactivity, or alternatively reactivity against
neuraminidase. The finding that the reactivity with non-treated/non-transfected monocytes
(nMo) was much less than with mock transfected, neuraminidase treated monocytes, argue in
favour of the latter explanation. Taken together, these experiments strongly indicate that
antigens encoded by the transfected mRNA in allogeneic tMo are taken up and processed in
autologous APC’s and presented to the T cells.
The presentation of tumor antigens in context of an allo MLR rests on two concepts:
generation of an inflammatory response and cross presentation. We accordingly measured the
cytokine concentrations in supernatant from PBMC during priming by autologous tMo and
allogeneic tMo. As shown in Table 2, cytokine levels are much higher when PBMC are
incubated with allogeneic tMo compared with those of autologous, indicating that a strong
MLR reaction develops. No IL-4 could be detected and IL-10 was very low, while GM-CSF,
INFγ, TNFα, MIP-1β and MCP were very high when tested on day 7. IL-12 was also low in
the supernatant, but the peak of the secretion was observed during the first 12 hours to 48
hours of incubation (data not shown). This cytokine/chemokine profile is favourable to drive
APC-maturation into a Th-1 polarizing direction. We also noticed that the concentration of
IL-5 and IL-13 tested on day 7 was high. The secretion was very low during the first 24-48
hours but increased gradually after 2 days of the incubation.
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Discussion
Aiming to target DCs in vivo, we have developed a clinical grade protocol for production of
allogenic monocytes as vehicles to deliver tumor antigens. The rationale is that such cells will
stimulate a potent allo-response when injected into patient and subsequently be killed and
taken up by resident or newly recruited DCs at the site of injection.
Monocytes obtained by Elutra™ are now being applied widely for routine generation of DCs.
We, like others, demonstrate that elutriation of monocytes with Elutra™ provides highly
purified “untouched” monocytes in large quantities within a closed system (Berger, et al.,
2005). We show that these monocytes can be used as antigen carriers in vitro, resulting in
priming of T cells specific for epitopes presented by autologous APCs. The efficiency of
passive antigen loading of cells is greatly dependent on the cell’s capacity for antigen uptake
and on the nature of the antigen used (protein, mRNA, DNA). Antigen loading can be done in
many ways. In a “shotgun approach” using autologous tumor cells as a source of antigen to
obtain a broad and “individualized” immune response, the antigen can be loaded in the form
of tumor extracts/eluates, apoptotic cells or as tumor mRNA. In the context of GMP (Good
Manufacturing Practice) production, the use of mRNA has several advantages. We have
previously shown that square-wave electroporation is a highly efficient way to transfect
mRNA into monocyte-derived DCs and CD34+-derived DCs (Sæbøe-Larssen et al., 2002;
Mu, et al 2003; Mu et al., 2004), and that DCs loaded with tumor mRNA is equal to or better
than DCs that have phagocytized apoptotic cells from the same tumor cell line, in priming Tcell responses in vitro (Jarnjak-Jankovic et al., 2005). In the model experiments presented
here, we accordingly used this method for loading. We demonstrate here that this method
could also be applied successfully to monocytes. By using EGFP mRNA as a reporter gene to
study the optimal transfection conditions by flow cytometry, we could define the optimal
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parameters to balance between transfection efficacy and viability of cells. In addition, hTERT
mRNA was used as a model mRNA for a tumor antigen to allow a direct measurement of
telomerase activity in TRAP assays 24 h after transfection. Protein expression after mRNA
transfection could be confirmed and was comparable to that found in a human cancer cell line.
Based on these expression studies, the mRNA encoding the two antigens were used to study
immune responses in vitro.
Both fresh and frozen monocytes were used in this study. The cell yield based on the initial
number of loaded cells was similar, but probably underestimated in the fresh cell preparations
due to cell aggregation during culturing. Whether this is due to activation of the monocytes
during the elutriation procedure is not clear. This phenomenon is only observed after 24 h of
culture, since monocytes cultured under similar conditions for 5 days generally gave
homogenous suspensions. The cells were difficult to disperse and thus were not included in
the cell count, while they were still present in the preparations. This makes standardization of
the preparations difficult. Cell loss from the freezing/thawing procedure is a common
observation, but this procedure eliminated aggregation and may therefore provide a solution
to the standardization problem. Our study clearly demonstrates that frozen monocytes can be
used and opens for the possibility of using stored monocytes rather than being dependent on
freshly isolated cells from leukopheresis. This represents a logistical simplification of the
vaccine production line.
Tumor antigens can efficiently be targeted to immature DCs and other antigen presenting
cells in vivo by the use of DNA vaccines that encod a chemokine fused to tumor antigen
(Biragyn et al., 1999), or by linking a weakly immunogenic idiotype protein to the immunestimulatory cytokine GM-CSF (Tao and Levy, 1993; Bendandi M et al., 1999). Patients
treated this way have developed strong humoral or cellular Th1 responses. Allogeneic
immunization activates alloreactive T cells that produce high levels of IL-2 and INF- in vivo.
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The release of IL-2 enhances clonal expansion of alloantigen-activated T cells, while IFN-γ
increases the density of MHC class I and II antigens in the microenvironment. In analysis of
the cytokine profile during in vitro priming, we observed greatly increased secretion of
cytokines after stimulation with transfected allogeneic monocytes compared with autologous
cells. The cytokine profile observed favours Th1 polarity since high level of INF-γ and TNF, low level of IL4 and IL10 were observed. High concentration of the chemokines MIP-1
and MCP were also detected. The expression of these pro-inflammatory cytokines/
chemokines might induce recruitment of different subsets of immune cells including
circulating monocytes and bystander DCs at the site of inflammation thus facilitating uptake
and processing of the tumor antigens. A microenvironment enriched with pro-inflammatory
components created by this allogeneic MLR may thus enhance cross presentation of tumour
antigens which is considered to be a prerequisite for effective priming of naive tumour
antigen-specific T cells (Yu et al., 2003).
Interestingly, some uncommon Th2 cytokines IL-5 and IL-13 were present at very high levels.
Similar results were seen in assays of T cells from patients vaccinated with melanoma-mRNA
transfected DCs (Kyte et al., 2005), and we suspect that others assaying an extended panel of
cytokines will see similar results. The role of these cytokines in vivo is not fully understood.
In vitro, however, IL-13 has been found to inhibit the differentiation of monocytes into
macrophages (Sakamoto et al, Blood 1995,85,3487). On the contrary, when combined with
GM-CSF (a growth factor that also was produced during allo-MLR), IL-13 induces monocyte
differentiation into immature DCs ( Piemonti et al, Eur Cytokine Netw, 1995,6,245; Alters et
al, J Immunother, 1999,22,229) . It is therefore tempting to speculate that the presence of
these two factors (IL-13 and GM-CSF) may deviate the differentiation of locally recruited
blood monocytes into DCs and not macrophages in vivo.
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Using elements of the MLR reaction by introducing a component of foreign MHC is not new
in cancer immunotherapy. Several cancer vaccines have applied allogeneic DCs or fusions
between autologous tumor cells and allogeneic DCs. The presence of allo-HLA antigens
enhanced the response rather than interfering negatively (Gong, et al., 1997; Trefzer, et al.,
2005). Generation of DCs in vitro is both expensive, and a time and labour consuming
process, we therefore applied monocytes as an alterative APC for delivery of tumor antigens.
Since pre-treatment of monocytes with bacterial neuraminidase leads to enhanced production
of inflammatory chemokines (Stamatos et al., 2003), it is likely that such monocytes, if
injected subcutaneously, would recruit circulating alloreactive memory T cells and monocytes
as well as bystander resident DCs to the site of injection. The reduced negative charge of the
cells would also improve their interaction with newly recruited alloreactive host T cells. The
treatment with neuraminidase was observed to up-regulate CD83 and CCR7 , indicating a
DC-deviating phenotype that also may contribute to enhanced allogenicity.
The DC maturation step is one of the key elements in generating potent DCs ex vivo. The
optimal factors required to mature DCs for effective -cell priming in vivo is still uncertain.
Studies have shown that under some conditions DCs generated in vitro can direct T cells
towards Th2 response or toleration (Langenkamp et al., 2000). Immature DCs have been
injected into primed or preconditioned cutaneous tissue, resulting in enhanced migration and
potent antitumor immunity (Nair et al., 2003). Allogeneic MHC used in this study function
both as a vehicle for the antigen and as an adjuvant, the adjuvant function is being provided
by the priming of the injection site by the MLR reaction. The in situ priming strategy may
offer considerable advantages over the currently established methods of ex vivo maturation
because this strategy more closely resembles the physiological conditions for DC maturation,
and hence may lead to a more desirable outcome, that is a more potent immune response. In
the setting of patient-specific cell therapy, in situ priming substitutes for the more complex in
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vitro DC culture steps. Experiments in an animal model (accompanying paper) confirm this
notion and provides additional rationale for the initiation of clinical trials.
This study provides a basis for a clinical grade protocol for immunotherapy in cancer patients
with mRNA-pulsed allogeneic monocytes. If successful, this method opens up for a simplified
logistics in cellular therapy of cancer because it allows the production of stocks of readymade vaccines that may be used in a variety of cancers where sets of antigens are available.
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