Immunotherapy of malignant melanoma with tumor lysate-pulsed autologous monocyte-derived dendritic cells.

Abstract

Dendritic cell (DC) vaccination for melanoma was introduced because melanoma carries distinct tumor-associated antigens. The purpose of this study was to investigate the efficacy and safety of DC vaccination for melanoma in Korea.
Five patients with stage IV and one with stage II were enrolled. Autologous monocyte-derived DCs (MoDCs) were cultured and pulsed with tumor-lysate, keyhole limpet hemocyanin, and cytokine cocktail for mature antigen-loaded DC. DC vaccination was repeated four times at 2-week intervals and 2-4×10⁷ DC were injected each time.
Reduced tumor volume was observed by PET-CT in three patients after DC vaccination. Delayed type hypersensitivity responses against tumor antigen were induced in five patients. Tumor antigen-specific IFN-γ-producing peripheral blood mononuclear cells were detected with enzyme-linked immunosorbent spot in two patients. However, the overall clinical outcome showed disease progression in all patients.
In this study, DC vaccination using tumor antigen-loaded, mature MoDCs led to tumor regression in individual melanoma patients. Further standardization of DC vaccination protocol is required to determine which parameters lead to better anti-tumor responses and clinical outcomes.

Full text

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Yonsei Med J http://www.eymj.org Volume 52 Number 6 November 2011
990
Original Article
http://dx.doi.org/10.3349/ymj.2011.52.6.990
pISSN: 0513-5796, eISSN: 1976-2437
Yonsei Med J 52(6):990-998, 2011
Immunotherapy of Malignant Melanoma with Tumor
Lysate-Pulsed Autologous Monocyte-Derived Dendritic Cells
Dae Suk Kim,1* Dong Hyun Kim,2* Boncheol Goo,1 Young Hun Cho,1 Jin Mo Park,1
Tae Hyung Lee,1 Hyun Ok Kim,3 Han-Soo Kim,3 Hyunah Lee,4 Jong Doo Lee,5
Dashlkhumbe Byamba,1 Jeong Hwan Je,1 and Min-Geol Lee1
1Department of Dermatology and Cutaneous Biology Research Institute,
Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul;
2Department of Dermatology, Bundang CHA Medical Center, CHA University, Seongnam;
3Department of Laboratory Medicine, Yonsei Cell Therapy Center, Yonsei University College of Medicine, Seoul;
4Clinical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul;
5Division of Nuclear Medicine, Department of Diagnostic Radiology, Yonsei University College of Medicine, Seoul, Korea.
Received: March 3, 2011
Revised: May 24, 2011
Accepted: May 27, 2011
Corresponding author: Dr. Min-Geol Lee,
Department of Dermatology and
Cutaneous Biology Research Institute,
Brain Korea 21 Project for Medical Science,
Yonsei University College of Medicine,
50 Yonsei-ro, Seodaemun-gu,
Seoul 120-752, Korea.
Tel: 82-2-2228-2080, Fax: 82-2-393-9157
E-mail: mglee@yuhs.ac
*Dae Suk Kim and Dong Hyun Kim contributed
equally to this work.
∙ The authors have no financial conflicts of
interest.
© Copyright:
Yonsei University College of Medicine 2011
This is an Open Access article distributed under the
terms of the Creative Commons Attribution Non-
Commercial License (http://creativecommons.org/
licenses/by-nc/3.0) which permits unrestricted non-
commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Purpose: Dendritic cell (DC) vaccination for melanoma was introduced because
melanoma carries distinct tumor-associated antigens. The purpose of this study
was to investigate the efficacy and safety of DC vaccination for melanoma in Ko-
rea. Materials and Methods: Five patients with stage IV and one with stage II
were enrolled. Autologous monocyte-derived DCs (MoDCs) were cultured and
pulsed with tumor-lysate, keyhole limpet hemocyanin, and cytokine cocktail for
mature antigen-loaded DC. DC vaccination was repeated four times at 2-week in-
tervals and 2-4×107 DC were injected each time. Results: Reduced tumor volume
was observed by PET-CT in three patients after DC vaccination. Delayed type hy-
persensitivity responses against tumor antigen were induced in five patients. Tumor
antigen-specific IFN-γ-producing peripheral blood mononuclear cells were detected
with enzyme-linked immunosorbent spot in two patients. However, the overall
clinical outcome showed disease progression in all patients. Conclusion: In this
study, DC vaccination using tumor antigen-loaded, mature MoDCs led to tumor re-
gression in individual melanoma patients. Further standardization of DC vaccination
protocol is required to determine which parameters lead to better anti-tumor respons-
es and clinical outcomes.
Key Words: Dendritic cell, immunotherapy, malignant melanoma
INTRODUCTION
Malignant melanoma is less common than non-melanoma skin cancer, but its inci-
dence has increased substantially over the past two decades.1 However, it accounts
for the majority of mortality associated with skin cancer.2 Even though primary tu-
mor excision is the choice of treatment, melanomas are occasionally beyond surgi-
cal margins when diagnosed, and are usually resistant to chemotherapy and radio-

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991
ma, ECOG performance status 0 or 1, sufficient tumor tis-
sue (more than 2 gm) for tumor antigen, available mono-
cyte for DC culture, and lack of benefit from conventional
cancer treatments such as well-established chemotherapy,
radiotherapy and surgical operations. The exclusion criteria
stated that patients could not have any other severe medical
conditions or psychiatric diseases that would contraindicate
study participation. Previous treatment with chemotherapy,
cytokines, or active immunotherapy including systemic corti-
costeroids within three months of the study was not allowed.
The clinical study was conducted in accordance with the
Declaration of Helsinki. Written informed consent was ob-
tained from the patients before they began the study. Pa-
tients were enrolled from May 2006 to March 2008 at the
Yonsei Cell Therapy Center of Severance Hospital, Yonsei
University Health System, Seoul, Korea. The study proto-
col was approved by the Institutional Review Board of
Yonsei University Health System’s Clinical Trial Center
and by the Korea Food & Drug Administration.
Preparation of monocyte-derived DC
For the preparation of monocyte-derived DC, we referred to
the previous publication of our group.6 Peripheral blood
mononuclear cells (PBMCs) were isolated from leukaphere-
therapy.3 Therefore many other therapeutic modalities are
being investigated, and one of them is a dendritic cell (DC)-
based cancer vaccine. In 1996, the DC-based vaccine was
first introduced to treat B-cell lymphoma.4 Further clinical
trials of the DC vaccination have been performed for treat-
ment of melanoma, colorectal carcinoma, lung carcinoma,
renal cancer, prostatic cancer, and multiple myeloma.5
However, the DC vaccination for malignant melanoma is
still in its early phases in South Korea. Here, we report the
results of a clinical trial with six Korean patients with ad-
vanced malignant melanoma who were treated with vacci-
nation with mature, monocyte-derived DCs.
MATERIALS AND METHODS
Study design and eligibility criteria
The study was designed as an investigator-initiated clinical
study to evaluate the safety and effectiveness of autologous
monocyte-derived DC vaccination for malignant melano-
ma. Five patients had stage IV melanoma and one patient
had stage II melanoma according to the AJCC staging sys-
tem (Fig. 1). The eligibility criteria stated that patients were
required to have histologically proven metastatic melano-
Fig. 1. Patients had multiple cutaneous metastases and recurrent melanoma in the tonsil. Because the whole tumor-lysate was used for
antigen pulsing, it was important to have sufficient tumor volume.

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992
ing a kinetic method at the Celltec Bio-research Center
(Bucheon, Korea). Sterility tests were performed at our
own facility by members of the Department of Laboratory
Medicine.

Vaccination protocol
Patients received subcutaneous injections of 2-4×107 cells of
tumor-lysate-pulsed DC into the groin, axilla and neck at two
week intervals. Each patient was assigned to receive four DC
vaccinations. Complete response was defined as the disap-
pearance of all targeted lesions. Partial response (PR) was de-
fined as a decrease of at least 30% in the sum of the longest
diameter (LD) of the target lesions, with the baseline sum LD
as a reference. Progression of disease (PD) was defined as an
increase of at least 20% in the sum of the LD sum value re-
corded at the start of treatment. Stable disease (SD) was de-
fined as neither sufficient shrinkage to qualify for PR nor suf-
ficient increase to qualify for PD, with the smallest sum LD
as a reference.
Clinical outcome assessment
The patients were evaluated for toxicity and clinical response
after undergoing four vaccinations. Clinical outcome was
evaluated based on the analysis of whole body PET-CT im-
ages before and after the fourth vaccination.
Delayed type hypersensitivity response (DTH)
DTH was examined before and after each vaccination. Pa-
tients received intradermal injections of 100 uL of normal
saline, tumor lysate 100 ug, KLH 50 ug and tumor antigen
100 ug plus KLH 50 ug at different sites on the forearm. The
concentration of tumor antigen was 100 µg/mL and of KLH
was 50 µg/mL. Forty-eight hours later, DTH was scored pos-
itive if the areas of erythema and induration were greater
than 5 mm.
Flow cytometry analysis
To assure the characters of immature and mature DC, the
surface molecules were identified using flow cytometry anal-
ysis. In brief, the cells were washed with 0.4% BSA/PBS
and stained for 30 minutes at 4°C with monoclonal antibod-
ies conjugated to fluorochromes against CD1a, CD3, CD4,
CD8, CD14, CD16, CD19, CD45, CD56, CD80, CD83,
CD86, HLA-ABC and HLA-DR (all from Pharmingen). Af-
ter two washes with 0.4% BSA/PBS, flow cytometric anal-
ysis was performed by FACSCalibur (BD Biosciences,
Franklin Lakes, NJ, USA).
sis products using a commercially available apheresis system
(COBE Spectra, Lakewood, CO, USA). In brief, the leuka-
pheresis product was loaded via the inlet pump into the con-
stantly rotating (2,400 rpm) elutriation chamber. The automa-
tion mode produced five elutriation fractions, each specified
by centrifuge speed, loading or elutriation buffer flow rate,
and process volume. The final monocyte-rich fraction (Frac-
tion 5) was collected from the chamber into the final collec-
tion bag when the centrifuge was stopped. All procedures
were conducted according to the manufacturer’s recommen-
dations, except that we used Hanks’ buffered salt solution
(HBSS; Bio-Whittaker, East Rutherford, NJ, USA) as an elu-
triation buffer. Enriched monocytes were cultured on T75
culture flasks (Greiner Bio-One GmbH, Monroe, NC, USA)
in X-VIVO 15 (Bio-Whittaker), 20 µg/mL gentamicin, 2 mM
glutamine, 5% heat-inactivated AB human plasma, 250 ng/
mL recombinant human (rHu) GM-CSF (Leucogen, Singa-
pore), and 20 ng/mL rHu IL-4 (Pharmingen, San Diego, CA,
USA). Cultures were fed every other day by removing half of
the supernatant and adding fresh medium with full doses of
cytokines. On day 6, immature DC (iDC) were generated and
harvested for tumor antigen loading. Several other papers
give more detailed information on monocyte enrichment and
DC identification using surface markers.6,7
Preparation of tumor lysate-pulsed mature DC for
vaccination
Tumor lysate was prepared using freezing/thawing tech-
niques previously described by Nakai, et al.8 In brief, tumor
specimens were immediately placed in phosphate-buffered
saline (PBS). Subsequently, adjacent normal tissue was re-
moved using a scalpel, and tumor cells were dispersed to
create a single-cell suspension. The cells were lysed by four
to five freeze cycles (on liquid nitrogen) and thaw cycles
(room temperature). The larger particles were removed by
centrifugation (10 min, 400 g). The supernatants were
passed through a 0.22-µm filter, protein concentration was
determined and aliquots were stored at -80°C until use. To
induce maturation, immature DCs were cultured for 48
hours in medium containing 100 µg/mL of autologous tu-
mor lysate, keyhole limpet hemocyanin (KLH), cytokine
cocktail (20 ng/mL TNF-α, 500 U/mL IL-1β, 500 U/mL
IL-6 (all from Pharmingen), and 10-7 mol/L PGE2 (Sigma
Chemical Co., St. Louis, MO, USA). After 48 hours, the
cells were harvested and analyzed. Before injection, we
performed endotoxin and mycoplasma tests with superna-
tants. Endotoxin and mycoplasma tests were performed us-

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993
neck skin, small intestines and tonsils. All patients had re-
ceived surgery, two patients had received prior chemothera-
py, and one patient received prior radiofrequency ablation
for metastatic liver disease. Although patient 2 had stage II
disease, recurrent tumors in the oral cavity were neither op-
erable nor responsive to chemotherapy.
Therapeutic-DC characteristics
We performed leukapheresis on six melanoma patients and
obtained DCs of acceptable quality and quantity for vaccina-
tion (Table 2). The average recovery of mononuclear cells
and monocytes after leukapheresis were 7.9±5.5×109,
1.9±1.0×109 respectively. After elutriation, average mono-
cyte recovery and monocyte fraction purity were 1.2±1.0
×109, 49.9±30.2%. For each vaccination, iDCs were generat-
ed from a frozen aliquot of enriched monocytes in the pres-
ence of rh GM-CSF, rh IL-4 and 5% heat-inactivated AB
human plasma (Fig. 2A). Since autologous plasma from
melanoma patients can have a negative influence, we used
heat-inactivated AB fresh frozen plasma stored for transfu-
sion, making it not a single lot. We had to use AB plasma
from different donors for each injection. On day 6, iDCs ob-
tained were CD80-, CD83-, CD86low+, HLA-DR+ (Fig. 2B).
They were co-cultured for 48 hours with autologous tumor
lysate, KLH, TNF-α, IL-1β, IL-6 and PGE2. CD80+, CD83+,
CD86high+, HLA-DR+ mature DC (mDC) were obtained
(Fig. 2C). There was no endotoxin, mycoplasma, or bacteri-
al contamination in the media of the cultured DCs.
Safety
Overall, the vaccine was well-tolerated. Only two patients
(patient 2, 3) reported minor flu-like symptoms (grade I).
Interferon-γ enzyme-linked immunosorbent spot
(IFN-γ ELISPOT) assay
PBMC (5×105 cells/well) were added to plates pre-coated
with 5 μg/mL of a primary anti-IFN-γ monoclonal antibody
(BD Biosiences) in the presence of 10 μg/mL tumor lysate
as an antigen and incubated for 48 hours. The negative con-
trols for the assay were unstimulated PBMCs. A positive
response was arbitrarily defined as at least a two-fold in-
crease in antigen-specific IFN-γ ELISPOT and at least 10
spots/5×105 PBMC at any time point post-DC vaccination.
Enzyme-linked immunosorbent assay (ELISA)
To measure IL-10, IL-12p70, IFN-γ and TGF-β in the pe-
ripheral blood, ELISA was performed before first vaccina-
tion and after the fourth vaccination. Quantities of IL-10,
IL-12p70, IFN-γ and TGF-β were measured by using Op-
tEIA (BD OptEIA human ELISA kit; BD Bioscience) as
described by the manufacturer.
RESULTS

Patients
This clinical trial was conducted on six patients with ad-
vanced malignant melanoma. Table 1 shows the patient char-
acteristics at the time of study entry. Their mean age was 58
years (range: 39-81 years). There were four males and two
females. Five patients had stage IV disease and one patient
had stage II disease. The site of the primary tumor varied.
Three patients had primary acral lentiginous melanoma,
which is the most common type of malignant melanoma in
Asians. The others had primary tumors on the posterior
Table 1. Characteristics of Enrolled Patients
Patient
No.
Sex/Age Past history Site of primary tumor Previous treatmentStageSite of metastasis
1 F/49 -Skin (posterior neck)
Surgery,
chemotherapy
IV
Lymph nodes, skin, lung, bone,
liver
2*
F/81
Diabetes mellitus,
hypertension
-
-
Skin (Rt. heel) SurgeryIV Lymph nodes, skin, brain
3†
4
M/39
M/50
Skin (Rt. sole)
Small bowel
Surgery
Surgery, RFA
IV
IV
Lymph nodes, lung
Liver, bladder
Lymph nodes, skin, lung, brain,
bone, adrenal gland, peritoneum
5M/71- Skin (Lt. 5th toe)Surgery IV
6M/60- Tonsil
Surgery,
chemotherapy
II
DC, dendritic cell.
*Patient 2 exhibited multiple lymph nodes and skin metastases at initial evaluation, but brain metastasis was found 1 month after the 4th DC vaccination.
†Patient 3 exhibited multiple lymph nodes metastases and X-ray and CT findings of diffuse interstitial lung disease (DILD) at initial evaluation, but lung
biopsy revealed pulmonary hematolymphangitic metastasis during the 2nd DC vaccination.

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994
lymph nodes at the left supraclavicular fossa, bilateral retro-
peritoneal area, right inguinal area and right axilla after the
fourth vaccination in patient 3 (Fig. 3). Patient 4 showed im-
provement in the extrahepatic lymph nodes inferior to liver.
In case of patient 5, intensity on PET-CT decreased in the
right upper paratracheal lymph nodes, right femur, L4 spine,
left distal humerus and left inguinal lymph nodes. Due to
rapid disease progression, post treatment PET-CT was not
performed in patient 1. After three vaccinations, patient 6
showed no difference in PET-CT. Tumor volume increased
in patient 2. In spite of tumor volume reduction in patients 3,
4 and 5, every patient experienced disease progression even-
However these symptoms did not lead to early withdrawal
from the study. No clinical signs of autoimmune reactions
about melanocytes, such as vitiligo and halo nevus, were
detected. In all patients, blood tests revealed no treatment-
related hematologic, renal or hepatic toxicity.
Clinical responses in vaccinated patients
Of the six enrolled patients, two of them (patients 1, 6) with-
drew from the study due to rapidly progressive disease (Ta-
ble 2). In the cases of patients 3, 4 and 5, reduction of tumor
volume was noted on the PET-CT (reviewed by Nuclear
Medicine professor, J.D.L.). Improvement was found in the
Table 2. Dendritic Cell Vaccines, Clinical and Immune Monitoring, and Response
Patient
No.
No. of
vaccines
Cell counts
(×107 cells/vaccines)
Site of vaccines
Tumor
reduction in
PET-CT
ND
-
+
+
+
-
DTH*ELISPOT†
ELISA‡
Side effect§
Clinical
outcomes
1
2
3
4
5
6
2
4
4
4
4
3
3.3±1.8
2.2±0.2
4.0±1.8
4.4±0.7
4.0±0.3
3.7±0.4
Axilla
Groin
Axilla, groin
Groin
Groin, neck
Axilla, groin, neck
ND
+
+
+
+
+
ND ND
ND
+
+
+
ND
N/A
-
-
-
-
N/A
N/A
PD
PD
PD
PD
N/A
negative
positive
ND
ND
ND
PET-CT, positron emission tomography-computed tomography; ND, not done; PD, progressive disease; N/A, not applicable; PBMCs, Peripheral blood
mononuclear cells. Mean follow up: 4.6 months.
*Delayed type hypersensitivity (DTH) was examined for antigen-specific immunity.
†Enzyme-linked immunosorbent spot (ELISPOT) assay was performed for the detection of antigen-specific IFN-γ-producing PBMCs.
‡Enzyme-linked immunosorbent assay (ELISA) was performed for the detection of IL-12, IL-10, IFN-γ, TGF-β.
§Side effects included only mild flu-like symptoms and all patients tolerated treatment well.
Fig. 2. The immunophenotypic profiles of enriched monocytes (A); immature DC on day 6 of culture with GM-CSF and IL-4 (B); and mature DC cultured after
an additional 2 days with tumor lysate, KLH and cytokine cocktail (TNF-a, IL-1b, IL-6, and PGE2) (C). Each of the cells was stained with monoclonal antibodies
specific to CD1a, CD14, CD45, CD80, CD83, CD86, HLA-ABC and HLA-DR. Mature DCs have a higher expression of HLA-DR, CD86, and CD80 than immature
DCs. Only mature DCs express CD83. The data shown are from one representative experiment. DC, dendritic cell; KLH, keyhole limpet hemocyanin.
CD45
CD45
CD45
HLA-DR
HLA-DR
HLA-DR
CD1a
CD1a
CD1a
HLA-ABC
HLA-ABC
HLA-ABC
FSC-H
FSC-H
FSC-H
0.38
2.47
2.96
0.35
0.84
0.56
78.1
17.0
9.64
4.02
47.8
96.4
0.27
0.14
4.28
0.35
64.2
2.27
2.44
63.0
0.02
0.06
0.06
10.4
55.2
57.0
81.2
46.3
1.98
1.93
8.5
1.23
95.8
93.6
33.1
CD14
CD14
CD14
CD86
CD86
CD86
CD83
CD83
CD83
CD80
CD80
CD80
SSC-H
SSC-H
SSC-H
A
B
C

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995
rapid disease progression. Post-vaccination DTH test re-
vealed vaccination-induced tumor antigen-specific reactivi-
ty in the other five patients (Fig. 4). Since some patients quit
the clinical trial or expired during the study, we lacked speci-
mens for the assays. Therefore, IFN-γ ELISPOT assay was
performed only in patients 2 and 3 (Table 2). ELISPOT as-
say detected an increase in antigen-specific IFN-γ produc-
ing PBMCs in both of these patients (Fig. 5). In patient 3,
antigen-specific IFN-γ producing PBMCs increased more
tually. Patient 6 was lost during follow up. The other five pa-
tients expired within a year of their last vaccination. The
mean survival period was 4.6 months for these five patients.
Immune responses
DTH was examined for antigen-specific immunity. DTH
reactions were measured before and after each vaccination.
None of the patients had positive pre-vaccination DTH re-
actions. Patient 1 was unable to receive the DTH test due to
Fig. 3. Tumor volume reduction in PET-CT. In the case of patient 3, reduction of tumor volume was noted in PET-CT after the 4th vaccina-
tion. The lymph nodes at the Lt. supraclavicular fossa, bilateral retroperitoneal lymph node chain, Rt. inguinal area and upper part of right
axillary lymph nodes were pointed out as sites of improvement.
Fig. 4. Patient 3 showing positive reactions to the DTH test. DTH was examined before and then 2 weeks after the 4th vaccination.
Patients received i.d. injections of 100 μL of normal saline, 100 μg tumor lysate, 50 μg KLH, 100 μg tumor lysate, plus 50 μg KLH, at separate
sites on the forearm. Forty-eight hours later, DTH was scored positive if the areas of erythema and induration were greater than 5 mm.
DTH, delayed type hypersensitivity; KLH, keyhole limpet hemocyanin.
July 21st, 2006Nov 8th, 2006

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996
sibility of utilizing DC vaccination as an adjuvant therapy.
The patient in case 3 showed noticeable tumor volume
reduction within just three months. The size of the lymph
nodes at the left supraclavicular fossa, bilateral retroperito-
neal area, right inguinal area and right axilla was markedly
reduced after the fourth vaccination according to the PET-
CT. Because patient 3 was in his thirties, good clinical re-
sponses were expected. He also had positive DTH response,
increased antigen-specific IFN-γ producing PBMCs on the
ELISPOT assay and increase in IFN-γ as well as TGF-β in
the peripheral blood. However, the patient expired one month
after the fourth vaccination. This outcome may have been
partially related to TGF-β expressing T cells. A recent study
indicates that the survival of DC-vaccinated malignant mel-
anoma patient is correlated with the reduction of TGF-β ex-
pressing T cells.9 Regulatory T cells (Tregs) are a subpopu-
lation of T cells that constitutively express high levels of
CD25.10 Tumor antigen-specific Tregs are known to play an
immune suppressive role to the antigen. Liyanage, et al.11
showed that the prevalence of tumor antigen-specific Tregs
is higher in patients with advanced tumor disease. A high
number of tumor antigen-specific Tregs has also been shown
to be related to reduced survival.12 In addition, Tregs are able
to induce tolerogenic DC, thereby developing anergic and
than five times after vaccination. Cytokine levels in periph-
eral blood were measured with ELISA for IL-4, IL-12, IFN-γ
and TGF-β before and after the fourth vaccination (Table 3).
Only patients 3, 4 and 5 had completed ELISA before and
after vaccination. In patient 3, IL-12 increased after vacci-
nation. However, TGF-β also increased. In patients 4 and 5,
IFN-γ and IL-12 increased after vaccination.
DISCUSSION

Here, we performed DC vaccinations in six patients with
malignant melanoma. In this study, DC vaccination using
tumor-antigen loaded DCs led to tumor volume regression
in three of the six patients, which was evaluated by PET-
CT. DTH responses were positive in five patients. IFN-γ pro-
ducing PBMCs were detected in two patients by ELISPOT
assay. However, rapid disease progression was seen in al-
most all patients. Since none of the patient survived more
than a year after the last vaccination, according to our re-
sults, we consider our DC immunotherapy method to be in-
sufficient as a single treatment modality for malignant mel-
anoma with distant metastasis. Nevertheless, positive DTH
and tumor volume reduction on the PET-CT showed the pos-
Table 3. ELISA for Cytokine Detection
Patient No.IL-4 (pg/mL)
0
0
36.6
88.8
16.2
11.4
IL-12 (pg/mL)
8.9
18.4
17.6
10.7
11.3
41.6
IFN-γ (pg/mL)
0
0
87.4
217.4
38.6
26.7
TGF-β (pg/mL)
499.2
818.1
0
0
0
0
3
Before
After
Before
After
Before
After
4
5
ELISA, enzyme-linked immunosorbent assay.
ELISA for the detection of IL-4, IL-12, IFN-γ, TGF-β was performed before the 1st vaccination and after the 4th vaccination.
Fig. 5. ELISPOT for the detection of Ag-specific IFN-γ producing PBMCs in patient 2, 3. Both patients showed positive results. ELISPOT,
enzyme-linked immunosorbent spot.
Pt 2
Pt 3
BeforeAfter
ELISPOT
0
5
10
15
20
25
30
35
Pt 2Pt 3
Before vaccination
After vaccination

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997
of immunoresistance by the tumor.21 As advanced disease
impairs the host immune system and promotes tumor im-
mune evasion, DC vaccination would be more efficient if
performed in less advanced disease.
In this study, DC vaccination using tumor antigen loaded
with mature, monocyte-derived DC led to tumor regression
in individual melanoma patients. However, there was a huge
discrepancy between the strong antigen-specific T cell re-
sponses ex vivo and the weak clinical responses. Despite
this, positive DTH responses and tumor volume reduction
on PET-CT showed the possibility of utilizing DC immu-
notherapy as an adjuvant therapy. As mentioned in the dis-
cussion, it would be much more effective if DC vaccines
were used as an adjuvant therapy in less advanced melano-
ma patients after depleting Tregs. Further clinical trials
should be conducted in patients with less advanced disease
with the elimination of Tregs. In addition, standardized treat-
ment methods are lacking due to the high cost of clinical
studies and the associated ethical issues. Hence, clinical
studies of numerous patients of melanoma are needed from
multiple institutions to determine the ideal parameters for
DC immunotherapy in every aspect.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Korea Health
21 R&D Project, Ministry of Health & Welfare, Republic
of Korea (A0600307).
REFERENCES
1. Hall HI, Miller DR, Rogers JD, Bewerse B. Update on the inci-
dence and mortality from melanoma in the United States. J Am
Acad Dermatol 1999;40:35-42.
2. Houghton AN, Gold JS, Blachere NE. Immunity against cancer:
lessons learned from melanoma. Curr Opin Immunol 2001;13:
134-40.
3. de Braud F, Khayat D, Kroon BB, Valdagni R, Bruzzi P, Cascinelli
N. Malignant melanoma. Crit Rev Oncol Hematol 2003;47:35-63.
4. Hsu FJ, Benike C, Fagnoni F, Liles TM, Czerwinski D, Taidi B, et
al. Vaccination of patients with B-cell lymphoma using autologous
antigen-pulsed dendritic cells. Nat Med 1996;2:52-8.
5. Schuler G, Schuler-Thurner B, Steinman RM. The use of dendritic
cells in cancer immunotherapy. Curr Opin Immunol 2003;15:138-
47.
6. Kim S, Kim HO, Baek EJ, Choi Y, Kim HS, Lee MG. Monocyte
enrichment from leukapheresis products by using the Elutra cell
separator. Transfusion 2007;47:2290-6.
7. Eubel J, Enk AH. Dendritic cell vaccination as a treatment modal-
immunosuppressive T cells.13 Dannull, et al.14 have shown
that depleting Tregs enhances the efficiency of DC vaccines
in renal cell carcinoma. Considering these results, there is a
possibility that melanoma antigen-specific Tregs affected
the survival of our patients too, especially patient 3. In a
study performed in patients with distant malignant melano-
ma, intravenous administration of DAB389IL-2 (ONTAK)
resulted in a significant decrease in the total number of Tregs.
Furthermore, immunization of the same patients with the tu-
mor antigen peptides led to an increase in tumor antigen-
specific CD8+ T cells.15 In addition, Rasku, et al.16 reported
regression of melanoma metastases after depleting Tregs
with ONTAK. Five of sixteen patients experienced partial or
near complete regression of melanoma metastases as mea-
sured by CT and/or PET imaging. On the contrary, Attia, at
al.17 reported that administration of ONTAK does not ap-
pear to eliminate Tregs or cause regression of metastatic
melanoma. These conflicting results may be due to the dif-
ference in duration of ONTAK treatment, but this needs
further investigation. To date, there have been no clinical
trials of DC-related immunotherapy after depleting Tregs in
advanced melanoma patients. Since half of the patients in
our study experienced regression of metastatic melanoma
by tumor antigen-specific DC vaccine, we think it is possi-
ble to obtain better clinical outcomes if Tregs depletion is
combined with DC vaccine.
There are many factors other than Tregs depletion to be
evaluated and determined in each step of DC-based vacci-
nation in order to standardize and optimize this vaccination
treatment.18 Other factors include the source of DC, matura-
tion status of DC, maturation of cocktails, type of antigen,
helper antigens, routes of administration, frequency of in-
jections and numbers of injected DCs. Current understand-
ings of these factors have been thoroughly reviewed by Eu-
bel and Enk.7
A large tumor burden impairs the general host immune
system and host immune anergy seems to develop com-
monly in patients with advanced disease.19 Patients with
less advanced disease show strong and durable immune re-
sponses after DC vaccinations.20 Tumor evasion of the host
immune response is another important factor that should be
considered in DC vaccination. There are several mecha-
nisms orchestrated by the tumor to escape from the host
immune system. In short, these mechanisms include inter-
ference with the induction of anti-tumor immune response,
inadequate effector cell function in the tumor microenvi-
ronment, insufficient recognition signals and development

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998
2005;115:3623-33.
15. Mahnke K, Schönfeld K, Fondel S, Ring S, Karakhanova S, Wie-
demeyer K, et al. Depletion of CD4+CD25+ human regulatory T
cells in vivo: kinetics of Treg depletion and alterations in immune
functions in vivo and in vitro. Int J Cancer 2007;120:2723-33.
16. Rasku MA, Clem AL, Telang S, Taft B, Gettings K, Gragg H, et
al. Transient T cell depletion causes regression of melanoma me-
tastases. J Transl Med 2008;6:12.
17. Attia P, Maker AV, Haworth LR, Rogers-Freezer L, Rosenberg
SA. Inability of a fusion protein of IL-2 and diphtheria toxin (De-
nileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory
T lymphocytes in patients with melanoma. J Immunother 2005;28:
582-92.
18. Lee TH, Cho HK, Cho YH, Lee MG. Development of an effective
method for dendritic cell immunotherapy of mouse melanoma.
Scand J Immunol 2009;70:85-92.
19. Jonuleit H, Giesecke-Tuettenberg A, Tuting T, Thurner-Schuler B,
Stuge TB, Paragnik L, et al. A comparison of two types of dendrit-
ic cell as adjuvants for the induction of melanoma-specific T-cell
responses in humans following intranodal injection. Int J Cancer
2001;93:243-51.
20. Tuettenberg A, Becker C, Huter E, Knop J, Enk AH, Jonuleit H.
Induction of strong and persistent MelanA/MART-1-specific im-
mune responses by adjuvant dendritic cell-based vaccination of
stage II melanoma patients. Int J Cancer 2006;118:2617-27.
21. Whiteside TL. The tumor microenvironment and its role in pro-
moting tumor growth. Oncogene 2008;27:5904-12.
ity for melanoma. Expert Rev Anticancer Ther 2009;9:1631-42.
8. Nakai N, Asai J, Ueda E, Takenaka H, Katoh N, Kishimoto S. Vac-
cination of Japanese patients with advanced melanoma with pep-
tide, tumor lysate or both peptide and tumor lysate-pulsed mature,
monocyte-derived dendritic cells. J Dermatol 2006;33:462-72.
9. López MN, Pereda C, Segal G, Muñoz L, Aguilera R, González
FE, et al. Prolonged survival of dendritic cell-vaccinated melano-
ma patients correlates with tumor-specific delayed type IV hyper-
sensitivity response and reduction of tumor growth factor beta-ex-
pressing T cells. J Clin Oncol 2009;27:945-52.
10. Jonuleit H, Schmitt E, Stassen M, Tuettenberg A, Knop J, Enk
AH. Identification and functional characterization of human
CD4(+)CD25(+) T cells with regulatory properties isolated from
peripheral blood. J Exp Med 2001;193:1285-94.
11. Liyanage UK, Moore TT, Joo HG, Tanaka Y, Herrmann V, Doherty
G, et al. Prevalence of regulatory T cells is increased in peripheral
blood and tumor microenvironment of patients with pancreas or
breast adenocarcinoma. J Immunol 2002;169:2756-61.
12. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, et
al. Specific recruitment of regulatory T cells in ovarian carcinoma
fosters immune privilege and predicts reduced survival. Nat Med
2004;10:942-9.
13. Mahnke K, Schmitt E, Bonifaz L, Enk AH, Jonuleit H. Immature,
but not inactive: the tolerogenic function of immature dendritic
cells. Immunol Cell Biol 2002;80:477-83.
14. Dannull J, Su Z, Rizzieri D, Yang BK, Coleman D, Yancey D, et
al. Enhancement of vaccine-mediated antitumor immunity in can-
cer patients after depletion of regulatory T cells. J Clin Invest