Original article 1

Peptide-pulsed dendritic cell vaccine in combination withcarboplatin and paclitaxel chemotherapy for stage IV
melanoma
Keitaro Fukudaa, Takeru Funakoshia, Toshiharu Sakuraib, Yoshio Nakamuraa,
Mariko Moria, Keiji Tanesea, Akiko Tanikawaa, Junichi Taguchic,
Tomonobu Fujitab, Masato Okamotob, Masayuki Amagaia and
Yutaka Kawakamib
In this study, we aimed to evaluate the feasibility andefficacy of peptide-pulsed dendritic cell (DC) vaccine in
combination with carboplatin and paclitaxel chemotherapy(DCCP) for patients with stage IV melanoma previously
treated with dacarbazine-containing regimen. Six HLA-A24+
and 3 HLA-A02+ patients were treated with carboplatin(area under the curve 5) and paclitaxel (175 mg/m2) on day
1 and DCs (2 × 107 cells) pulsed with Wilms tumor gene 1(WT1), gp100, tyrosinase, and either MAGE-A3 (for HLAA24+) or MAGE-A2 (for HLA-A02+) peptides on days 8 and
22 in 28-day cycle for up to three cycles. DCCP was welltolerated, and median progression-free survival and medianoverall survival were 2.3 and 12.0 months, respectively. In
four of nine patients, a WT1-specific immune response(WT1-IR) was detected using the interferon-γ enzymelinked ImmunoSpot assay and WT1/HLA tetramer assay.
DCCP was more likely to elicit a WT1-IR in patients who
received DCs pulsed with the HLA-A24-restricted peptide(75%) compared with patients who received DCs pulsedwith the HLA-A02-restricted peptide (0%, P = 0.058).

Furthermore, three (75%) of four patients with a WT1-IR
survived longer than 12 months, whereas only one (20%) of
five patients without a WT1-IR who received the BRAF
inhibitor after DCCP survived longer than 12 months. These
results suggest that DCCP may be beneficial for HLA-A24+
melanoma patients with a WT1-IR. Melanoma Res
00:000–000 Copyright © 2017 Wolters Kluwer Health, Inc. All
rights reserved.

Introduction

with an objective response rate of 8.5% [10]. Although
DC vaccines induce tumor-reactive T cells in the
majority of the patients [11], in-vivo tumor reduction isdifficult because of immunosuppression mediated by thetumor microenvironment, including exposure to immune
checkpoint molecules such as PD-1/PD-L1 [12].
Therefore, combined treatment with a DC vaccine isrequired to attenuate immunosuppression and increase
efficacy [13].

Melanoma is a highly aggressive skin cancer that is particularly immunogenic, evident by its ability to undergo
spontaneous regression [1]. The observation that vitiligo,
an autoimmune skin disease characterized by patchy
depigmentation, occasionally appears simultaneouslywith tumor regression supports this consideration [2,3].
Vitiligo is caused by the destruction of melanocytes by
CD8+ T cells and can be induced by immunotherapy
such as administration of a cancer vaccine to melanoma
patients. CD8+ T cells that cause vitiligo are specific formelanocyte/melanoma-shared antigens such as gp100
and can reduce melanoma mass and prevent tumor
recurrence [4–6]. Indeed, vitiligo induced by immunotherapy is a favorable prognostic factor [3,7].
To induce melanocyte/melanoma antigen-specific effector and memory T cells similar to the induction of vitiligo, numerous trials of dendritic cell (DC) vaccines havebeen performed for patients with advanced melanoma[8,9]. However, the efficacy of the DC vaccine is limited,0960-8931 Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.

Melanoma Research 2017, 00:000–000
Keywords: carboplatin and paclitaxel chemotherapy, dendritic cell vaccine,
HLA-A24, melanoma, WT1
a
Department of Dermatology, bDivision of Cellular Signaling, Institute forAdvanced Medical Research, Keio University School of Medicine and cTokyo
Midtown Clinic, Tokyo, Japan

Correspondence to Yutaka Kawakami, MD, PhD, Division of Cellular Signaling,
Institute for Advanced Medical Research, Keio University School of Medicine, 35
Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Tel: + 81 3 5363 3778; fax: + 81 3 5362 9259; e-mail: yutakawa@keio.jp
Received 30 October 2016 Accepted 26 January 2017

Carboplatin and paclitaxel chemotherapy (CP therapy)
is a commonly used second-line chemotherapy formelanoma. In addition to causing direct cytotoxicity,
carboplatin downregulates the inhibitory molecule PDL2 expressed by DCs and melanoma cells to enhance
T-cell activation [14]. Furthermore, paclitaxel reduces
the number of regulatory T cells (Tregs), decreases
production of the cytokines interleukin (IL)-10 andtransforming growth factor-β by Treg, and stimulates
DC-mediated antigen presentation [15–17]. Therefore,
DOI: 10.1097/CMR.0000000000000342

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2 Melanoma Research 2017, Vol 00 No 00

the addition of CP therapy would likely enhance theefficacy of a DC vaccine.
The selection of tumor antigens is an important component of developing DC vaccines. The National Cancer
Institute pilot project was conducted to prioritize 75
cancer antigens and concluded that Wilms tumor gene 1(WT1), a zinc-finger transcription factor expressed inmelanomas, which promotes proliferation, is the mostpromising cancer antigen [18–20]. Furthermore, melanoma differentiation antigen gp100 and tyrosinase as wellas cancer/testis antigen MAGE-A3 are ranked within the
top 20 most promising cancer antigens [18]. Moreover,
several peptide vaccines comprising MAGE-A proteinsinduce antivaccine T cells as well as a far larger number ofantitumor T cells that recognize other antigens expressedin the melanoma [21,22].
Accordingly, we hypothesized that DCs pulsed with peptides derived from WT1, gp100, tyrosinase, and either
MAGE-A3 (for HLA-A24+) or MAGE-A2 (for HLA-A02+)
peptides, which represents a multiple peptide cocktailpulsed DC vaccine not yet explored, in combination withCP therapy (DCCP), may show higher efficacy comparedwith available DC vaccines used to treat patients withmelanoma. To test this hypothesis, we conducted a phase
I/II pilot study aimed to investigate the feasibility andefficacy of DCCP for patients previously treated withdacarbazine-based chemotherapy. Furthermore, we investigated immunological factors that may correlate with theinduction of immune responses and prognosis.

Methods
Study design

This trial was a phase I/II pilot study conducted at Keio
University School of Medicine (Tokyo, Japan) and Tokyo
Midtown Clinic (Tokyo, Japan). The primary endpoint of thisstudy was to explore the feasibility of DCCP. Adverse eventswere assessed according to the Common Terminology
Criteria for Adverse Events, version 4.0. The secondary
endpoints were overall survival (OS), progression-free survival(PFS), response rate, and induction of immunity against the
WT1, gp100, tyrosinase, MAGE-A3, and MAGE-A2 peptides. The clinical response was evaluated according to the
Response Evaluation Criteria in Solid Tumors (version 1.1).
Events were measured from the first day of CP chemotherapy. This study was approved by the Institutional Review
Board (IRB) of Keio University and fulfilled the principles of
Declaration of Helsinki. All patients provided written
informed consent. As a peptide-pulsed DC vaccine is not anapproved therapy and CP chemotherapy is off-label use inmelanoma, we obtained the review and approval of investigational use of DCCP by the IRB of Keio University andOncology Board of Keio University Hospital. The trial was
registered with the University Hospital Medical Information
Network Clinical Trials Registry (http://www.umin.ac.jp/ctr/,
number: UMIN-000006629).

Patients

Patients with histologically confirmed stage IV melanoma
previously treated with dacarbazine-containing regimen
were eligible. Other inclusion criteria were as follows: (a)20–75 years of age; (b) HLA-A24+ or HLA-A02+ phenotype; (c) lesion that can be evaluated using Response
Evaluation Criteria in Solid Tumors; (d) more than
4 weeks passed since prior treatment for melanoma; (e)Eastern Cooperative Oncology Group clinical performance status of 0 or 1; (f) adequate hematologic, hepatic,
renal, and cardiac function; and (g) life expectancy ofmore than 4 months. Exclusion criteria were as follows: (i)other active primary malignancies; (ii) history of severe
allergy; (iii) severe comorbidity (infections, cardiovasculardisease, renal disease, liver disease, or uncontrolled diabetes); (iv) pleural effusion or pericardial fluid requiring
treatment, (v) pregnancy or nursing; (vi) desiring tobecome pregnant; (vii) carrier of hepatitis virus, humanT-cell lymphotropic virus type 1, or HIV; (viii) severe
psychiatric disease; (ix) history of autoimmune disease;and (x) ongoing treatment with immunosuppressive
agents or steroids.
DCCP treatment protocol

Intravenous carboplatin area under the curve 5 plus
intravenous paclitaxel (175 mg/m2) was administered on
day 1 of a 28-day cycle. Patients received intradermal
injections of 2 × 107 DCs pulsed with WT1, gp100, tyrosinase, and either MAGE-A3 (for HLA-A24+) or
MAGE-A2 (for HLA-A02+) peptides close to the axillary
or inguinal lymph nodes on days 8 and 22 (Fig. 1).
Patients continued treatment for up to three cycles unlessthey developed progressive disease (PD). After thecompletion of the protocol, DC vaccination continued
with the patient’s consent.
Pretreatment assessment and clinical monitoring

All patients underwent complete history taking, physicalexamination, blood tests, and computed tomography
before treatment commenced. During DCCP treatment,
patients underwent physical examinations and blood
tests at least every 2 weeks to assess toxicities. In addition, computed tomography was repeated before eachcycle and 4 weeks after the third cycle of DCCP toevaluate clinical response.
Generation of peptide-pulsed DC vaccine

DCs were generated from peripheral blood mononuclear
cells (PBMCs) prepared from leukapheresis products aspreviously described [23]. Briefly, after leukapheresis,
monocytes were enriched through their adherence toplastic and cultured using AIM-V Medium (Thermo Fisher
Scientific, Waltham, Massachusetts, USA) containing granulocyte macrophage colony-stimulating factor (50 ng/ml;
Primmune, Kobe, Japan) and IL-4 (50 ng/ml; R&D
Systems, Minneapolis, Minnesota, USA) for 5 days togenerate immature DCs. On day 6, the immature DCs

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DC vaccine plus CP chemotherapy for melanoma Fukuda et al. 3

Fig. 1

Immunological monitoring and PBMC preparation

PBMCs were collected and cryopreserved from patientson day 1 of each cycle before CP therapy and 1 week
after the third cycle (day 91) for immunological monitoring. The immune responses to the WT1, gp100, tyrosinase, MAGE-A3, and MAGE-A2 peptides wereanalyzed using the interferon (IFN)-γ ELISPOT assay,
HLA tetramer assay, and delayed-type hypersensitivity(DTH) skin test.
Cryopreserved PBMCs were thawed and pulsed with 10
μg/ml of WT1, gp100, tyrosinase, and MAGE-A3 peptides
restricted to HLA-A24 or with WT1, gp100, tyrosinase, andMAGE-A2 peptides restricted to HLA-A02 in AIM-V CTS
Medium (Thermo Fisher Scientific) supplemented with
10% human AB serum (MP Biomedicals, Solon, Ohio,USA), 20 U/ml IL-2 (Shionogi, Osaka, Japan), and 10 ng/ml
IL-7 (Peprotech, Rocky Hill, New Jersey, USA). After
9 days, the cells were analyzed using the IFN-γ ELISPOT
assay and HLA tetramer assay.

Protocol of DC vaccination combined with carboplatin and paclitaxel
chemotherapy. WT1, gp100, tyrosinase, and either MAGE-A3 (for HLAA24+) or MAGE-A2 (for HLA-A02+) peptide-pulsed DCs (2 × 107 cells)
were injected on days 8 and 22 every 4 weeks. Intravenous carboplatin atarea under the curve 5 (AUC 5) and intravenous paclitaxel (175 mg/m2)
were administered on day 1 of a 28-day cycle. Immunological monitoring
was performed on day 1 of each cycle before CP therapy and 1 week
after the third cycle. CP, CP, carboplatin and paclitaxel; DC, dendritic cell.

were incubated with prostaglandin E2 (50 ng/ml; Daiichi
Fine Chemical, Tokyo, Japan) and penicillin-killed andlyophilized preparations of a low-virulence strain (Su) of
Streptococcus pyogenes (OK-432, 10 μg/ml; Chugai Pharmaceutical, Tokyo, Japan) for 24 h to generate mature DCs.
The mature DCs were subjected to quality control
according to a published method [24] or cryopreserved
until the day of administration. The phenotypes CD11c+,
CD14−, CD40+, CD80+, CD83+, CD86+, CCR7+, HLADR+, and HLA-ABC+ were defined as those of mature
DCs, according to the quality criteria for DC vaccines.
For each vaccination, cryopreserved DCs were thawed
and pulsed with a mixture of WT1 peptide(CYTWNQMNL; PolyPeptide, San Diego, California,USA), gp100 peptide (VWKTWGQYW; AnyGen,
Samtae,South
Korea),
tyrosinase
peptide(AFLPWHRLF; PolyPeptide), and MAGE-A3 peptide(IMPKAGLLI; PolyPeptide) that were restricted to
HLA-A24 or with a mixture of WT1 peptide(RMFPNAPYL; PolyPeptide), gp100 peptide (IMDQ
VPFSV; PolyPeptide), tyrosinase peptide (YMDGTMSQV; AnyGen), and MAGE-A2 peptide (YLQLVF
GIEV; PolyPeptide) that were restricted to HLA-A02 for
60 min and washed twice before their use. All peptides
used for generating peptide-pulsed DC vaccine were
products synthesized under GMP conditions.

WT1-specific, gp100-specific, tyrosinase-specific, MAGEA3-specific, and MAGE-A2-specific IFN-γ ELISPOT
assaysThe IFN-γ ELISPOT assay was performed as previously
described [25]. PBMCs were defined to be specificallysensitized by peptides when the number of spots in the
ELISPOT assay indicating IFN-γ release in response to
the WT1, gp100, tyrosinase, MAGE-A3, or MAGE-A2
peptide was at least 15 spots and of at least 1.5-times the
response to HIVenv (for HLA-A24+) or HIVgag (for
HLA-A02+) peptide-pulsed PBMCs.
HLA tetramer assay of PBMCs cultured with peptide
cocktails

WT1-specific, gp100-specific, tyrosinase-specific, MAGEA3-specific, and MAGE-A2-specific CD8+ T cells in patients’
PBMCs were assessed using HLA tetramers (T-Select MHC
Tetramer; Medical & Biological Laboratories, Nagoya,Japan), as previously described [26]. The results were defined
as positive when WT1-, gp100-, tyrosinase-, MAGE-A3-or
MAGE-A2-tetramer-positive CD3+CD8+ T cells weredetected in clusters and not in a diffused population as well as
no detectable HIVenv/HLA-A24 or HIVgag/HLA-A02
tetramer-positive CD3+CD8+ T cells.
DTH test

The DTH skin test was performed as previously described[27]. The diameters of the erythemas and indurations were
measured 48 h after the injection of the WT1, gp100, tyrosinase, MAGE-A3, or MAGE-A2 peptide on day 1 of eachcycle and at 4 weeks after the third cycle. An erythema diameter greater than 5 mm was defined as a positive reaction.
Flow cytometric analysis of cell phenotypes

PBMC samples were incubated with fluorescent dyeconjugated monoclonal antibodies for 45 min at 4°C in

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4 Melanoma Research 2017, Vol 00 No 00

the dark. After they were washed with FACS buffer (2%fetal bovine serum in PBS), the cells were fixed withstabilizing fixative (BD Biosciences, San Jose, California,
USA) and analyzed using a flow cytometer (Gallios;
Beckman Coulter, Pasadena, California, USA). Data wereanalyzed using Kaluza software (Beckman Coulter).

Adverse events

Adverse events that occurred within the protocol treatment period are summarized in Table 2. There were no
adverse skin reactions at the site of DC vaccination.
DCCP was well tolerated and adverse events that showed
grade 3 or higher toxicity were related to hematotoxicity
associated with CP therapy. One patient who developed
grade 4 neutropenia was treated with granulocyte-colony
stimulating factor and recovered promptly. No patient
received blood or platelet transfusion. The nonhematological toxicities arthralgia and myalgia were treated with NSAIDs for several days during each cycle.
Overall, treatment with the DC vaccine did not appear to
exacerbate the adverse effects of CP therapy.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 6(GraphPad Software, La Jolla, California, USA). The survival curve was generated using the Kaplan–Meier method.
Univariate analysis was conducted using the log-rank test.
The immune response was analyzed using the t-test.
Probability (P) values less than 0.05 were two sided andconsidered statistically significant.

Results

Clinical outcomes

Patient characteristics

One of nine (11%) patients achieved a partial response(PR), four (44%) had stable disease (SD), and the disease
control rate was 55%. The median PFS and OS were 2.3
and 12.0 months, respectively (Fig. 2a and b). Furthermore,
the median PFS and OS of patients treated with DCs
pulsed with HLA-A24-restricted peptides were 2.9 and14.1 months, respectively. The OS of patients with PR or
SD was significantly better compared with those of patientswith PD (P = 0.0027, Fig. 2c). Among six patients treatedwith DCs pulsed with HLA-A24-restricted peptides, onepatient exhibited PR, three patients exhibited SD, and four
patients survived longer than 12 months. However, of thethree patients treated with DCs pulsed with HLAA02-restricted peptides, one patient who had been treatedwith vemurafenib, a BRAF inhibitor, after DCCP, exhibited SD and survived longer than 12 months (Table 2).
Furthermore, there were no significant differences in thevalues of various inflammatory markers and cytokines suchas the neutrophil/lymphocyte ratio, lactate dehydrogenase,C-reactive protein, IL-6, IL-8, and tumor necrosis factor-α
between patients who achieved PR or SD and those who
exhibited PD (data not shown).

From October 2011 to October 2013, nine patients wereenrolled. Patient clinical characteristics are listed in Table 1.
The median age was 57 years (range: 43–68 years). Amongthe nine patients, the primary lesions of three patients were
mucosal melanomas. Three of the nine patients had lungmetastasis (stage IV-M1b) and the other six patients had
metastasis to visceral organs other than the lung (stage IVM1c). Six patients were positive for HLA-A24, and their
HLA-A+ genotypes were HLA-A2402. All nine patientswere positive for HLA-A02 and those three (33%) patients
who were not HLA-A2402 received DCs pulsed with HLAA02-restricted peptides. Five patients completed the protocol, and four (44%) patients terminated the protocol
because of disease progression during the second cycle. One
patient was administered postprotocol DC vaccination. The
median frequency of DC vaccine administration was six
times (range: 3–10 times). Three patients received CP
therapy and four patients received postprotocol dacarbazinebased chemotherapy. One patient received vemurafenib, a
BRAF inhibitor, and one patient received mogamulizumab,
a humanized anti-CCR4 monoclonal antibody, as postprotocol therapy.
Table 1

Patient characteristics and clinical outcomesCase
numberAge/sexPrimary sites

Metastatic sites

LDH

AJCC M
category

12

54/female
47/maleRectumEsophagus

> ULN
≤ ULN

M1c
M1c

2402
2402

3

67/female

Lung

Distant LN
Retroperitoneum,
distant LN
Peritoneum

≤ ULN

M1c

4
5
6
7

43/male68/male57/female
67/maleShoulder
Chest
Toe
Chest

≤ ULN
≤ ULN
> ULN
≤ ULN

8
9

66/female
44/maleAbdomenShoulderLiver
Liver, lung
Liver
Lung, distant LN,
distant skin
Liver, lung
Lung, distant LN

≤ ULN
≤ ULN

Genotypes of
HLA-A*DC (times)

Postprotocol
therapy

Types ofresponse

PFS(months)

OS (months)

–
0206

6
6

CP, DAC-Tam
CP

PR
SD

2.9
10.2

16.8
24.4

2402

2601

6

SD

5.2

12.0

M1c
M1b
M1c
M1b

2402
2402
2402
0201

–
2601
0206
3101

6
4
3
10

DAC-Tam,mogamulizumab
CP, DAC-Tam
None
None
DC, vemurafenib

SD
PD
PD
SD

2.8
1.2
1.0
2.3

14.1
4.4
2.9
21.6

M1c
M1b

0207
0201

2602
0206

4
4

DTIC
None

PD
PD

1.2
1.4

4.6
2.2

AJCC, American Joint Committee on Cancer; CP, carboplatin and paclitaxel; DAC-Tam, chemotherapy combined with dacarbazine, nimustine hydrochloride, cisplatin, andtamoxifen; DC, dendritic cell vaccine; DTIC, dacarbazine; LDH, lactate dehydrogenase; LN, lymph node; OS, overall survival; PD, progressive disease; PFS, progressionfree survival; PR, partial response; SD, stable disease; ULN, upper limit normal.

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DC vaccine plus CP chemotherapy for melanoma Fukuda et al. 5

Table 2Adverse events
N = 9 [n (%)]
Grade 1/2

Grade 3

Grade 42 (22)
8 (89)
6 (67)
5 (56)
5 (56)
5 (56)
4 (44)
3 (33)
3 (33)

5 (56)
1 (11)
2 (22)
1 (11)–––––

1 (11)
–
–
–
–
–
–
–
–

Neutrophil count decreased
AnemiaWhite blood cell decreased
Platelet count decreased
Arthralgia/myalgia
Alopecia
Fatigue
Peripheral sensory neuropathy
Diarrhea

Immunological monitoring

In terms of induction of WT1-specific, gp100-specific,
tyrosinase-specific, MAGE-A3-specific, and MAGE-A2specific T-cell responses, DCCP elicited a WT1-specificimmune response (WT1-IR) in four patients (cases 1, 2, 4 and5), which was detected using the IFN-γ ELISPOT and HLA
tetramer assay (Table 3). In addition, case 1 showed a
gp100-specific immune response in the IFN-γ ELISPOT
assay, and case 9, who received the DC vaccine pulsed with
HLA-A02-restricted peptides, showed a gp100-specificimmune response in the IFN-γ ELISPOT and HLA tetramer
assay. Interestingly, DCCP was more likely to elicit a WT1-IR

in patients treated with DCs pulsed with HLA-A24-restricted
peptides (66%, 4/6 patients) compared with patients treatedwith DCs pulsed with HLA-A02-restricted peptides (0/3patients) (P = 0.058). Furthermore, the number of WT1
tetramer-positive CD8+ T cells significantly increased after
DC vaccination of patients treated with DCs pulsed with
HLA-A24-restricted peptides (P = 0.047), in contrast to thosetreated with DCs pulsed with HLA-A02-restricted peptides(Fig. 3a). Moreover, the analysis of OS, excluding the patient
who showed significant tumor regression induced using
BRAF inhibitor, showed possible longer survival in patientswith a WT1-IR compared with those without WT1-IR(P = 0.051, Fig. 2d). Given that patients with a WT1-IR weretreated with DCs pulsed with HLA-A24-restricted peptides,
these results suggest that DCCP may be beneficial forpatients with HLA-A24+ melanomas that mount a WT1-IR.
As regards the DTH test, no patient treated with DCs
pulsed with HLA-A02-restricted peptides exhibited DTH
positivity to any of the peptides. Among the six patientstreated with DCs pulsed with HLA-A24-restricted peptides,
three patients reacted with gp100, tyrosinase, and MAGEA3 at baseline (cases 2, 4, and 5), and one patient developed
responses to gp100, tyrosinase, and MAGE-A3 peptides(case 1) after immunization (Table 3). Interestingly, no

Fig. 2Kaplan–Meier analysis for (a) progression-free survival and (b) overall survival of melanoma patients treated with DCCP. Comparison of OS (c) inpatients who achieved PR or SD (N = 5) and PD (N = 4), (d) in patients with a WT1-IR (N = 4) and without a WT1-IR (N = 4). A patient treated withvemurafenib (BRAF inhibitor) was excluded from the analysis. DCCP, dendritic cell vaccination combined with carboplatin and paclitaxel
chemotherapy; PD, progressive disease; PR, partial response; SD, stable disease.

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6 Melanoma Research 2017, Vol 00 No 00

Table 3

Case
numberClinical outcomes and immunological monitoring data

HLA-A of DC
vaccineTypes ofresponse

A24
A24
A24
A24
A24
A24
A02
A02
A02

PR
SD
SD
SD
PD
PD
SD
PD
PD

1
2
3
4
5
6
7
8
9

HLA tetramer assay

IFN-γ ELISPOT assay

DTH test

Post-treatment

Post-treatment

Pretreatment/post-treatment

OS (m) gp100 TYR

MAGE-A3/
A2

−
−
−
−
−
−
−
−
+

−
−
−
−
−
−
−
−
−

16.8
24.4
12.0
14.1
4.4
2.9
21.6
4.6
2.2

−
−
−
−
−
−
−
−
−

WT1 gp100 TYR
+
+
−
+
+
−
−
−
−

+
−
−
−
−
−
−
−
+

−
−
−
−
−
−
−
−
−

MAGE-A3/
A2
+
−
−
−
−
−
−
−
−

WT1 gp100

TYR

MAGE-A3/
A2

WT1

−/+
+/−
−/−
+/+
+/−
−/−
−/−
−/−
−/−

−/+
+/+
−/−
+/+
+/+
−/−
−/−
−/−
−/−

−/+
+/−
−/−
+/+
+/−
−/−
−/−
−/−
−/−

−/−
−/−
−/−
−/−
−/−
−/−
−/−
−/−
−/−

+
+
−
+
+
−
−
−
−

DC, dendritic cell vaccine; DTH, delayed-type hypersensitivity; IFN, interferon; PD, progressive disease; PR, partial response; SD, stable disease; TYR, tyrosinase;+, positive; −, negative; +/−, positive at pretreatment DTH test and turned to negative in post-treatment DTH test.

patient exhibited DTH reactivity to WT1 peptides, whereas
WT1-specific T-cell responses were detected in four
patients in IFN-γ ELISPOT assay and HLA tetramer assay.
This discrepancy suggests that these four patients were in
immunosuppressed state and were unable to mount a strong
immune response sufficiently robust to develop a DTH skinreaction in vivo, although there were WT1-specific T cells inblood that could induce WT1-specific T-cell responses
ex vivo.
Prognostic factors

To identify factors predictive of the immune response to
DC vaccination, we evaluated the various subsets ofimmune cells in pretreatment peripheral blood. Because
there was no WT1-specific immune response among the
HLA-A02+ patients, we investigated the immune
response among patients who were HLA-A24 positiveand found that a WT1-IR significantly correlated with alow percentage of Th17 (CD3+CD4+CXCR3−CCR6+)
cells (P = 0.036, Fig. 3b). In contrast, there was no significant difference in the percentages of naïve, effector,
central memory CD8+ T cells, that of naïve, effector,
central memory CD4 T cells, that of Th1, Th2, Treg,
monocytic myeloid-derived suppressor cells (MDSCs),
and granulocytic MDSCs between WT1-IR-positive and
WT1-IR-negative patients (data not shown). These
results suggest that DCCP is more likely to be effectivein patients with HLA-A24+ melanoma with a low percentage of Th17 cells before treatment.

Discussion
In this study, we evaluated the feasibility and efficacy of
DCCP for treating patients with stage IV melanoma
patients previously treated with dacarbazine-containing
regimen. The most common adverse events were
hematologic abnormalities, and their frequencies werecomparable to those described in other studies of CP
therapy [28–30]. Nonhematologic adverse events weremild (grade 1 or 2). Overall, the toxicity profile of DCCP
was clinically acceptable.

Until the recent application of BRAF inhibitors, MEK
inhibitors, and immune checkpoint blockers such as
antibodies against CTLA-4 and PD-1, stage IV melanoma had an extremely poor prognosis because of its
resistance to chemotherapy. Dacarbazine chemotherapy
had been the standard first-line therapy; however,
because of the low objective response rate of ∼ 12%,which is often transient, CP therapy had been commonlyused as a second-line regimen [31]. Given that theobjective response rate and median PFS and OS ofpatients treated with CP are 5–22% and 2.3–4.1 and5.2–9.7 months, respectively [28–30], DCCP seems to
improve OS compared with CP therapy, but not response
rate or PFS. These results suggest that the benefit of the
combination of peptide-pulsed DCs with CP therapy ischaracterized by a delayed clinical effect, which is typically shown by various immunotherapeutic drugs [32–34]. Indeed, the metastatic lesions of all patients who
survived longer than 12 months with a WT1-IR slowly
progressed after they were evaluated as PD.
In the present study, none of the patients treated with DCs
pulsed with HLA-A02-restricted peptides elicited a WT1-IR.
DCCP was more likely to elicit a WT1-IR in patients treatedwith DCs pulsed with HLA-A24-restricted WT1 peptide
compared with those treated with DCs pulsed with HLAA02-restricted WT1 peptide (P =0.058). Furthermore, themedian OS of patients with HLA-A24+ melanoma with a
WT1-IR was 15.0 months, whereas the median OS of other
patients (patients with HLA-A24+ melanoma without a
WT1-IR and patients with HLA-A02+ melanoma) was
4.6 months. These results suggest that DCCP may be beneficial for HLA-A24+ melanoma patients with the inductionof WT1-IR.
In humans and mice, the ability of the gp100 peptide tobind MHC class I molecules increases and enhances theinduction of melanoma-reactive CD8+ T cells whenamino acid residue substitutions are introduced into thewild-type gp100 peptide [35,36]. Similarly, the modified
9-mer HLA-A24-restricted WT1 peptide, a peptide with

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DC vaccine plus CP chemotherapy for melanoma Fukuda et al. 7

Fig. 3affinity of the peptide for the HLA molecule appears toaffect the induction of a WT1-IR and improved OS.
Numerous studies report that the DTH response isinduced after DC vaccination and that DTH positivity
correlates with improved OS [38]. In contrast, there arereports that DTH outcomes do not correlate with clinicalresponse [39]. Here, only one patient developed DTH
reactivity after immunization and the DTH outcome did
not correlate with clinical response. However, four
patients reacted with the WT1 peptide in both ex-vivo
assays, IFN-γ ELISPOT assay and HLA tetramer assay,
and these patients tended to have a more favorable
prognosis compared with others, although none of thesefour patients reacted in the DTH assay to the WT1
peptide.

(a) Frequency of WT1-specific CD8+ T cells in peripheral blood of HLAA24+ patients (N = 6, upper panel) and HLA-A02+ patients (N = 3,
lower panel) before and after DCCP. (b) Pretreatment frequencyof Th17 cells in peripheral blood of HLA-A24+ melanoma patients(N = 6). Th17 cells were identified to be CD3+CD4+CXCR3−CCR6+.
*P < 0.05. DCCP, dendritic cell vaccination combined with carboplatin
and paclitaxel chemotherapy.

a single amino acid residue substitution of the wild-type
WT1 peptide, significantly increases the binding affinity
to the HLA-A24 molecule and elicits WT1-specificCD8+ T cells more effectively compared with the wildtype WT1 peptide [37]. Here, we used a modified 9-mer
HLA-A24-restricted WT1 peptide for treating HLAA24+ patients, whereas we used the wild-type WT1
peptide for HLA-A02+ patients. Thus, the binding

In a recent study, the WT1 peptide-pulsed DC vaccination combined with gemcitabine to treat pancreatic cancer
was positive in the DTH assays of three of 10 patients,
and all patients with liver metastasis were DTH-negative,
although some were positive in the IFN-γ ELISPOT
assay and HLA tetramer assay [27]. Similarly, here wefound that all patients with liver metastasis had no DTH
reactivity to WT1, and, among the four patients who had a
WT1-specific T-cell response in IFN-γ ELISPOT assay
and HLA tetramer assay, two had liver metastasis.
Furthermore, immunosuppressive agents such as steroids
can decrease DTH reactivity and, less frequently, causeanergy, which leads to the absence of a detectable DTH
skin reaction [40]. Here we used steroids for longer times(3 days each cycle) to prevent emesis induced by CPtherapy compared with a study of WT1 peptide-pulsed
DC vaccination combined with gemcitabine administeredto patients with pancreatic cancer (1 day each cycle) [27].
Therefore, another possible explanation for the lack of
DTH positivity to the WT1 peptide in the current study
is the prolonged use of steroids. Indeed, one (case 2) offour patients with a WT1-IR lost DTH reactivity to gp100
and MAGE-A3 peptides after the initiation of DCCP,
although the patient did not have liver metastasis andachieved the longest OS. These results indicate theimportance of patient selection and consideration of theperiod of using steroids to evaluate whether the DTH test
serves as a useful indicator of prognosis.
We evaluated various immunological biomarkers in ourpatients before treatment, and we found that, among
patients who received DCs pulsed with HLAA24-restricted peptides, WT1-IR significantly correlatedwith a low percentage of Th17 cells. IL-17A is a key
cytokine produced by Th17 cells, and the delayed
growth of subcutaneously transplanted B16 melanoma
cells in mice deficient in IL-17A reveal that the Th17 cell
response can promote tumor growth [41]. However, when
B16-F10 melanoma cells are intravenously injected,
metastasis of melanoma to the lung increases in IL-17Adeficient mice, suggesting that Th17 suppresses the

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8 Melanoma Research 2017, Vol 00 No 00

progression of melanoma [42]. Furthermore, in asurvivin–peptide vaccination trial for patients with previously treated stage IV melanoma, high frequencies ofTh17 cells before vaccination were more likely todevelop survivin-specific T-cell reactivity after vaccination, which is opposite to our results [43]. Thus, Th17
cells may play different roles depending on tumor stage
and the antigenicity of a peptide. The relevance of Th17
cells for melanoma is controversial and warrants further
investigation.
Conclusion

DCCP was well tolerated in patients with stage IV
melanoma previously treated with dacarbazine-containing
regimen and may be beneficial for HLA-A24+ patientswith a WT1-IR. The induction of a WT1-IR seemed to
be associated with the effect of a modified WT1 peptide
with higher affinity for HLA-A24 and induces more
WT1-specific CD8+ T cells compared with the wild-type
WT1 peptide. Moreover, it has been reported recently
that patients with metastatic melanoma who responds to a
CTLA-4 or PD-1 inhibitor, which are immune checkpoint
inhibitors and are recommended for first-line and secondline treatment of stage IV melanoma [44], had significantly higher numbers of CD8+ T cells in the tumor
after two to three courses of treatment compared withthose of nonresponders [45]. The HLA-A24-restricted
peptide-pulsed DC vaccine of DCCP induced
WT1-specific CD8+ T cells in four (67%) of six cases.
Therefore, we believe that our results provide justification for the continued investigations of combination
therapy comprising a DC vaccine and immune checkpoint inhibitors in patients with HLA-A24+ melanoma.

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Acknowledgements

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The authors thank the patients who participated in thisstudy, A. Iguchi (Keio University School of Medicine) for
clinical assistance, and S. Noji (Keio University School of
Medicine) for technical assistance.

15

This work was supported by the Project for Development of
Innovative Research on Cancer Therapeutics (P-DIRECT)and the Project for Cancer Research And Therapeutic
Evolution (P-CREATE) from the Japan Agency for Medical
Research and Development (AMED); Grants‐in‐aid for
Scientific Research (S) (26221005) from the Ministry of
Education, Culture, Sports, Science and Technology-Japan(MEXT); a grant from Tokyo Biochemical Research
Foundation; and grants from Tella Inc.

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Conflicts of interest

Y. Kawakami and M. Amagai received the grant from
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supported in part by these grants. The salary of
K. Fukuda was provided by a grant from Tella Inc. thatwas provided to M. Amagai’s institution. For theremaining authors there are no conflicts of interest.

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