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Clinical Trials |
Departments of Urology [T. N., M. T., Y. H., M. M.], Radiology [K. N.], and Internal Medicine [Y. I.], School of Medicine, Keio University, Tokyo 160-8582, and Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Otsu, Shiga 520-21 [K. T.], Japan
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bladder cancers constitute a broad spectrum of malignancies at clinical presentation. Various chemotherapies with multiple anticancer reagents have only limited efficacy against highly advanced disease and have considerable systemic side effects (25) . On the other hand, superficial bladder cancer, especially carcinoma in situ disease, is well known to respond to immunotherapy such as intravesical treatment by Bacillus Calmette-Guérin, and thus tumor-specific immunotherapy has been suggested as a potentially useful strategy against bladder cancer (26) . In the present study, we were able to establish a MAGE-3+ bladder cancer cell line from a HLA-A24+ patient with advanced bladder cancer. Recently, Tanaka et al. (14) reported the successful induction of antitumor CTLs with a MAGE-3-encoded synthetic peptide presented by HLA-A24. In this previous report, five peptides of nine amino acids were found to contain the binding motif for HLA-A24 in the known sequence of MAGE-3. These peptides were synthesized and tested for their binding ability to purified HLA-A24 molecules. Of the five peptides studied, one high MHC binder (IMPKAGLLI) was found to be capable of eliciting CTLs. Using this particular model, we explored the possibility of inducing a significant MAGE-specific CTL response by the in vitro stimulation of PBMCs with autologous DCs pulsed with the MAGE-3 synthetic epitope peptide, IMPKAGLLI, which binds to HLA-A24 molecules with a very high affinity. On the basis of this achievement, a pilot clinical trial of DC vaccination targeting MAGE-3 has been conducted in selected HLA-A24+ patients with advanced bladder cancer proven to be MAGE-3+.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clinical Tumor Samples.
Specimens of the 28 bladder cancers and 8 upper tract urothelial
cancers as well as normal bladder mucosa samples were obtained from the
patients at the time of surgery performed at Keio University Hospital.
A routine histopathological evaluation was conducted, and a portion of
each sample was immediately frozen in liquid nitrogen and stored at
-80°C for later RNA extraction.
Analysis of mRNA Expression.
Total RNA was isolated from each of the samples using the acid
guanidinium thiocyanate-phenol chloroform extraction procedure
(30)
. As described by De Smet et al.
(31)
, cDNA synthesis from 2.5 µg of total RNA was
accomplished by extension with oligo(dT)15
in a 20-µl reaction volume. Each of the MAGE cDNA samples was
detected by PCR amplification by 33 cycles (30 s at 94°C and 40 s at 72°C) using oligonucleotide primers specific for the different
exons of each MAGE gene. The sequences were as
follows: (A)5'-CGGCCGAAGGAACCTGACCCAG-3' (CHO-14) and
5'-GCTGGAACCCTCACTGGGTTGCC-3' (CHO-12) for the MAGE-1 gene
(16)
; (B) 5'-AAGTAGGACCCGAGGCACTG-3' (CDS-9)
and 5'-GAAGAGGAAGAAGCGGTCTG-3' (CDS-7) for the MAGE-2
gene (31)
; and (c) 5'-TGGAGGACCAGAGGCCCCC-3'
(AB-1197) and 5'-GGACGATTATCAGGAGGCCTGC-3' (BLE-5) for the
MAGE-3 gene (17)
. To ensure that the RNA had
not degraded, a PCR assay for the glyceraldehyde-3-phosphate
dehydrogenase gene also was carried out; the sense primer was
5'-GTCAACGGATTTGGTCGTATT-3' and the antisense primer was
5'-AGTCTTCTGGGTGGCAGTGAT-3' (32)
. The PCR product was
size-fractionated on 1% agarose gel.
Statistical evaluations regarding the relationship between the MAGE gene expression and the disease stages were analyzed by the Mann-Whitney nonparametric U test.
Generation of Dendritic Cells.
PBMCs were isolated from the heparinized venous blood of the patient
from whom the FY cell line was established, by Ficoll-Hypaque
(Lymphoprep; Nycomed Labs, Oslo, Norway) gradient centrifugation at
580 x g for 20 min and then washed with PBS three
times. The adherent monocytes were cultured in RPMI 1640
supplemented with 5% human AB serum, 2 mM
L-glutamine, 1 mM sodium
pyruvate, 0.1 mM nonessential amino acids, 20
µg/ml gentamicin, 2,000 units/ml recombinant IL-4 (R & D Systems,
Inc., Minneapolis, MN), and 1,000 units/ml recombinant GM-CSF (R & D
Systems, Inc.) for 7 days at 37°C. At the time of CTL induction
in vitro, the DCs were pulsed with 10 µg/ml of the MAGE-3
epitope peptide for 4 h at 37°C in a serum-free medium, washed
extensively, and then added to the bulk cultures of the PBMCs.
Phenotyping of DCs.
The expressions of the cell surface antigens, MHC classes I and II,
CD3, CD14, CD83, and CD86, were analyzed by flow cytometry using mAbs
before and after the generation of DCs to evaluate the populations of
the DCs generated from the PBMCs. The mAbs used were G46–2.6 (IgG1,
anti-HLA-A, B, C), L243 (IgG2a, anti-HLA-DR; BD Biosciences, San Jose,
CA), SK7 (IgG1, anti-CD3), SK3 (IgG1, anti-CD4), SK1 (IgG1, anti-CD8),
M
P9 (IgG2b, anti-CD14; Becton Dickinson, San Jose, CA), HB-15e
(IgG1, anti-CD83), and BU63 (IgG1, anti-CD86; Ancell, Bayport, MN). All
of the data were analyzed using the CellQuest software (Becton
Dickinson).
Synthetic Epitope Peptide.
A nine amino acids-peptide with a sequence of IMPKAGLLI (amino acid
position in MAGE-3, 195–203) was synthesized and provided by Takara
Shuzo, Shiga, Japan. This MAGE-3 synthetic epitope peptide was
evaluated previously and identified to bind to HLA-A24 molecules with a
very high affinity (14)
.
Induction of CTL Responses.
The PBMCs were collected from patient F. Y. and from HLA-A24+
healthy donors by the centrifugation of blood samples on a
Ficoll-Hypaque density gradient as described above and cultured in RPMI
1640 containing 5% heat-inactivated human AB serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin sulfate (Life Technologies,
Inc.). As a stimulator, autologous DCs pulsed with MAGE-3 epitope
peptide (PBMC:DC, 10:1) were added to the media every 7 days.
Irradiated FY cells or nonpulsed autologous DCs were used as a control
stimulator (PBMC:FY cells, 10:1). Recombinant IL-2 (R & D Systems,
Inc.) was added to the media at a concentration of 300 units/ml every 3
days. The whole culture media was replaced on days 9 and 16. After 21
days of incubation, the cells were harvested, and the CTL activity was
assessed. A flow cytometric analysis of the cell-surface antigens of
the effector cells was performed. The cells were stained with mouse
antihuman mAbs against CD3, CD4, and CD8 (Becton Dickinson).
Isotype-matched mouse antibodies (Becton Dickinson) served as a
negative control.
Cytotoxicity Assay.
The target cells were labeled with 51Cr by
incubating with 100 µCi of
Na51CrO4 (New England
Nuclear, Boston, MA) for 1 h at 37°C, washed four times, and
plated onto round-bottomed, 96-well microtiter plates at a
concentration of 5 x 103
cells/0.1 ml/well.
Effector cells were then added at a concentration of 2.5 x
104
cells/0.1 ml/well (effector:target ratio
5:1). To eliminate nonspecific lysis, the cytotoxic activity was tested
in the presence of a 30-fold excess of unlabeled K562 cells. After
incubation for 4 h, the release of 51Cr in
the supernatant was measured by an automated gamma counter. The
percentage of specific 51Cr release was
calculated by the following formula: 100 x
([release by CTL] - [spontaneous
release])/([maximum release] - [spontaneous
release]).
Spontaneous release was generally 15–20% of the maximum release in our experiments.
Statistical evaluations between the relationship of the CTL activity and the methods of stimulation of effector cells were analyzed using Student’s t test.
Inhibition of Cytotoxicity by Blocking mAbs.
The target cells were incubated with 10 µg/ml of anti-HLA class I
mAbs (Immunotech, Marseille, France) for 1 h at 4°C before the
CTL assay.
Patient Eligibility and Clinical Protocol.
The protocol was reviewed and approved by the Keio University Hospital
Institutional Protocol Review Committee, and all of the patients
provided signed informed consent that fulfilled the committee’s
guidelines. A total of four HLA-A24+ patients with advanced bladder
cancers were eligible and enrolled in our pilot clinical study. The
diseases of these patients were progressive despite the fact that
previously they had had surgery, chemotherapy, and radiotherapy.
The expression of the MAGE-3 gene in all four cancers was
proved by RT-PCR in at least one excised tumor. None of these patients
received other treatments within the 4 weeks preceding the initial DC
vaccination. The autologous DCs were prepared from PBMCs obtained from
leukapheresis and were cryopreserved at -80°C. While preparing the
DCs, samples were collected from each step and tested for sterility,
including bacterial, Mycoplasma, and endotoxin
contamination. The DCs were pulsed with the MAGE-3-encoded HLA-A24
binding epitope (IMPKAGLLI) just before each vaccination, and the
patients received a s.c. injection of 1 x
107 to 1 x 108
autologous DCs at each vaccination. The treatments were conducted once
every 2 weeks for a minimum of 6 and a maximum of 18 times. No other
concomitant treatments were provided during the protocol. The patients
were carefully observed, and vital signs, clinical symptoms, and
laboratory examinations were monitored and evaluated throughout the
experiments.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A total of 9 of 28 (32%) bladder cancers expressed any of the three
MAGE genes. The MAGE+ bladder cancer was
significantly more frequent in muscle invasive disease (pT2
; 73%)
than in superficial disease (<pT1; 6%). In upper tract urothelial
cancers, all of the invasive disease (pT2) expressed at least one of
the MAGE genes, whereas two of four (50%) of the
superficial diseases (<pT1) did; however, the difference was not
statistically significant. In the established human bladder
cancer cell lines, all but KU-1 expressed MAGE genes (Table 2)
. None of the samples that we examined, which were obtained from renal
cell carcinoma and adrenal tumors, expressed MAGE genes,
whereas in five testicular cancers, all of the samples were positive
for at least one of the MAGE
genes.3
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Induction of Autologous CTL Response against FY Cells by DCs Pulsed
with HLA-A24-binding MAGE-3 Epitope Peptide.
Compared with the CTL response stimulated only by FY cells or nonpulsed
autologous DCs, the cytotoxicity was significantly higher in the
stimulation with autologous DCs pulsed with HLA-A24-binding MAGE-3
epitope peptide. The cytotoxic effect of this CTL was significantly
blocked by the pretreatment of target FY cells with anti-HLA class I
mAbs (Fig. 1
A). In addition, the induced CTL did not recognize both KU-7
and KU-19-19, MAGE-3+, and HLA-A24- bladder cancer cells, but
in contrast, moderately recognized T24, HLA-A24+, and MAGE-3 weakly
positive bladder cancer cells (Fig. 1
B). PBMCs were also
collected from a HLA-A24+ healthy donor, and the effector cells were
prepared according to the same method. These HLA-matched, but
heterologous, CTLs were also shown to respond to target FY cells as
well as to autologous
CTLs.4
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. The number of CD3+ cells gradually increased during the incubation.
CD4+ cells decreased after 2 weeks, whereas the number of CD8+ cells
kept increasing. CD3+ and CD8+ cells accounted for 98% and 66% of the
effectors, respectively, after 3 weeks of induction.
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. These patients had already been treated intensively with surgery,
chemotherapy, and radiotherapy. All of the bladder cancers resected
from these patients were examined by RT-PCR and thus were proven to be
MAGE-3+. None of the patients showed any unfavorable side
effects throughout the DC vaccinations. A complete response of lymph
node metastases was achieved in one patient, and a partial response was
observed in two other patients. However, one patient died because of a
progression of local recurrence and pleural dissemination. The first
case (F. Y.) developed disseminated intravascular coagulation and died
of sepsis attributable to a perforation of the small intestine 2 months
after the DC vaccination; however, autopsy showed a complete remission
of lymph node metastases, which were histologically evident before the
DC vaccination. The second case (G. K.) showed the disappearance of a
solitary liver metastasis and a >50% reduction in the size of the
para-aortic lymph node metastasis evaluated by a computed tomography
scan (Figs. 3
and 4)
. However, a solitary brain metastasis was apparent 6 months after the
treatment, and was surgically removed. The third case (N. H.)
showed a significant reduction in the size of inguinal lymph node
metastases. A biopsy taken from the lymph node 3 months after the DC
vaccination demonstrated significant necrotic changes in the lesions
(Fig. 5)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DCs are well known to be professional antigen-presenting cells for the
induction of a T cell-mediated immune response (33)
. High
expression levels of adhesion molecules and costimulators, such as B7,
on the cell surface and intracellular vesicles critical for antigen
presentation are believed to prime CD4+ and CD8+ T cells (34
, 35)
. Another means by which DCs induce potent T cell responses
is via the release of IL-12. Using murine T cell receptor transgenic
CD4+ T cells, Macatonia et al. (36)
showed that
DCs induced the differentiation of naive T cells into IFN-
producing
Th cells by stimulating them with IL-12 production. In addition, DCs
induce potent human antiviral CD8+ CTL responses without any need for
CD4+ T cells or exogenous cytokines (37)
. As these
previous reports have indicated, DCs and macrophages have the ability
to induce CTL and Th cell responses in vivo. DCs are shown
to process exogenous antigens conventionally for presentation on MHC
class II molecules. Bachmann et al. (38)
reported that a cloned DC line is able to present cell
debris-associated exogenous viral proteins to MHC class I-restricted
CTLs in vitro. Before using DCs in anticancer immunotherapy,
the epitope peptide that is most essential to anticancer CTL induction
must be identified to avoid the risk of developing autoimmune diseases
(13)
.
The antigenic peptides encoded by the MAGE-3 gene were shown
to be presented by either HLA-A1 (39)
, -A2
(13)
, or -B44 (40)
molecules
(41)
, and Tanaka et al. (14)
recently showed tumor-specific CTLs to be induced from healthy donors’
PBMCs by stimulation with HLA-A24-binding MAGE-3- derived synthetic
peptide, IMPKAGLLI. HLA-A24 is positive in 
61% of the Japanese
population (42)
. As a result, it makes sense to develop
current DC-based immunotherapy using this specific synthetic epitope
peptide.
In our current experiment, we have shown the successful induction of the CTL response by stimulating PBMCs with autologous DCs pulsed with a HLA-A24-binding MAGE-3 epitope peptide. This CTL was considered to recognize MAGE-3+ tumor cells in a MHC class I-restricted manner because the cytotoxic effect of this CTL against FY cells was significantly blocked by the pretreatment of FY cells with anti-HLA class I mAbs. In addition, the induced CTL did not recognize either KU-7 or KU-19-19, both of which are MAGE-3+ but HLA-A24- bladder cancer cells. On the other hand, CTL activity against FY cells was seen in the effector cells prepared from a nonrelated healthy donor with HLA-A24 typing, thus indicating that the basic mechanism underlying this modality requires matching for both HLA typing and tumor-specific peptide, which is expressed in target cells and is also presented to effector cells by DCs in vitro. These achievements using in vitro experiments formed the basis for conducting clinical trials with this DC-based immunotherapy in patients with advanced bladder cancers and histologically proven, measurable metastatic lesions. Our initial findings of clinical trials with only the four cases presented herein indicated that tumor-specific, DC-based cancer immunotherapy may be useful as a new additional treatment modality for advanced bladder cancer that is not curable by surgery, chemotherapy, or radiotherapy.
Numerous concerns still exist regarding the application of DCs in clinical treatment. Most of the patients with progressive cancers are likely to be of advanced age and to have a poor immunological status. The combination therapies with DCs and the systemic administration of cytokines are possible solutions. IL-12 is known to induce CTLs in patients with a poor immune status, and the concomitant use of IL-12 may reduce the need for large doses of DCs (43) . In our preliminary experiments in vitro, when IL-12 was used to stimulate effector cells from PBMCs along with the autologous DCs pulsed with MAGE-3-encoded HLA-A24-binding epitope peptide, the CTL activity was significantly higher than the effector cells prepared without IL-12.5 Additional studies based on these recent findings are now underway. Such studies should focus on the safety and the feasibility of using this DC-based active immunotherapy against advanced bladder cancers.
In summary, autologous DCs pulsed with HLA-A24-binding MAGE-3 epitope peptide have been shown to successfully induce MHC class I-restricted MAGE-3 specific autologous CTLs in vitro in MAGE-3+ bladder cancer. The efficacy of DCs pulsed with the epitope peptide remains to be elucidated, because its clinical application has just been initiated. However, our results clearly showed a good potential for the development of a DC-based, tumor-specific immunotherapy for the treatment of bladder cancer.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Tokyo Medical University, 6-7-1 Nishishinjuku,
Shinjuku-ku, Tokyo 160-0023, Japan. Phone: 81-3-3342-6111; Fax:
81-3-3342-8587. E-mail: tachi{at}tokyo-med.ac.jp 
2 The abbreviations used are: Th, T-helper; DC,
dendritic cell; MAGE, melanoma antigens, RT-PCR, reverse
transcriptase-PCR; HLA, human lymphocytic antigen; PBMC,
peripheral blood mononuclear cell; IL, interleukin; GM-CSF, granulocyte
macrophage colony-stimulating factor; mAb, monoclonal antibody.
3 T. Nishiyama, unpublished data.
4 T. Nishiyama, unpublished data.
5 T. Nishiyama, unpublished data.
Received 1/19/00; revised 9/12/00; accepted 10/30/00.
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