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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4309-4317
Identification of HLA-A2-Restricted T-Cell Epitopes Derived From the
MUC1 Tumor Antigen for Broadly Applicable Vaccine Therapies
By
Peter Brossart,
Kathrin S. Heinrich,
Gernot Stuhler,
Lars Behnke,
Volker L. Reichardt,
Stefan Stevanovic,
Alexandra Muhm,
Hans-Georg Rammensee,
Lothar Kanz, and
Wolfram Brugger
From the Department of Hematology, Oncology and Immunology,
University of Tübingen, Tübingen, Germany; and the
Department of Immunology, Institute for Cell Biology, Tübingen,
Germany.
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ABSTRACT |
The tumor-associated antigen MUC1 is overexpressed on various
hematological and epithelial malignancies and is therefore a suitable
candidate for broadly applicable vaccine therapies. It was demonstrated
that major histocompatibility complex (MHC)-unrestricted cytotoxic T cells can recognize epitopes of the MUC1 protein core localized in the tandem repeat domain. There is increasing evidence now
that MHC-restricted T cells can also be induced after immunization with
the MUC1 protein or segments of the core tandem repeat. Using a
computer analysis of the MUC1 amino acid sequence, we identified two
novel peptides with a high binding probability to the HLA-A2 molecule.
One of the peptides is derived from the tandem repeat region and the
other is derived from the leader sequence of the MUC1 protein,
suggesting that, in contrast to previous reports, the MUC1-directed
immune responses are not limited to the extracellular tandem repeat
domain. Cytotoxic T cells (CTL) were generated from several healthy donors by primary in vitro immunization using peptide-pulsed dendritic cells. The addition of a Pan-HLA-DR binding peptide PADRE as a T-helper epitope during the in vitro priming resulted in an increased cytotoxic activity of the MUC1-specific CTL
and a higher production of cytokines such as interleukin-12 and
interferon- in the cell cultures, demonstrating the importance of
CD4 cells for an efficient CTL priming. The peptide induced CTL lysed
tumors endogenously expressing MUC1 in an antigen-specific and
HLA-A2-restricted fashion, including breast and pancreatic tumor cells
as well as renal cell carcinoma cells, showing that these peptides are
shared among many tumors. The use of MUC1-derived peptides could
provide a broadly applicable approach for the development of dendritic
cell-based vaccination therapies.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MUC1 IS A HIGHLY glycosylated type I
transmembrane glycoprotein with a unique extracellular domain
consisting of a variable number of tandem repeats (VNTR) of 20 amino
acids (PDTRPAPGSTAPPAHGVTSA).1,2 It is abundantly
overexpressed on the cell surface of many human adenocarcinomas such as
breast and ovarian cancers and hematological malignancies, including
multiple myeloma and B-cell lymphoma, making MUC1 an attractive and
broadly applicable target for immunotherapeutic strategies.3-9 It was demonstrated that major
histocompatibility complex (MHC)-unrestricted T cells from
ovarian, breast, pancreatic, and multiple myeloma tumors can recognize
epitopes of the MUC1 protein core localized in the tandem
repeat.10-14 However, there is increasing evidence from
murine and human studies that MHC-restricted T cells can be induced in
mice and humans after immunization with the MUC1 protein or MUC1
antigenic epitopes.15-19
Immunization methods using self-antigens have often resulted in the
induction of low-affinity CTL responses and, consequently, lack of a sufficient recognition of naturally processed antigens by
these CTL.20,21 Presentation of antigens by professional antigen-presenting cells (APC) may be critical for the effectiveness of
an induced immune response, and the nature of the APC can determine the
outcome, ranging from immunity to tolerance.22 Dendritic cells (DC) are recognized to be very potent APC, with the unique capacity to activate naive resting T cells and initiate primary T-cell
responses when pulsed with antigenic peptides or proteins, making them
an interesting tool for cancer vaccine therapies.23-30
CTL recognize antigenic peptides when presented in the groove of the
MHC molecule. The MHC class I peptides are usually derived from
cytosolic antigens by the action of the proteasoms and transported into
the endoplasmatic reticulum (ER) lumen by an adenosine
triphosphate-dependent transporter associated with antigen presentation
(TAP). In the ER, a chaperone-mediated assembly generates a stable
complex containing MHC class I heavy chain,
 2-microglobulin, and an antigenic peptide. This complex
traffics to the cell surface, where it can be recognized by
CD8+ T cells.31-33 The definition of MHC class
I allele-specific motifs allowed investigators to define epitopes
contained within a given antigen and opened new opportunities for
developing vaccine therapies.33 To identify HLA-A2-binding
peptides derived from the MUC1 protein, we performed a computer
analysis of the amino acid sequence of the MUC1 protein to screen for
epitopes with HLA-A2-binding motifs.34,35 Two peptides with
a high binding probability were identified, synthesized, and analyzed
for CTL responses in vitro. For CTL induction, we used autologous DC
generated from peripheral blood monocytes as APC.36,37 One
of the peptides is derived from the tandem repeat region of the MUC1
protein, referred to as M1.1. The second peptide (referred to as M1.2)
is localized within the signal sequence of MUC1, indicating that the
MUC1-directed immune response is not limited to the tandem repeat and
that this peptide can probably be presented by tumor cells independent
of TAP. We show here that the CTL generated from several healthy donors
by primary in vitro immunization using peptide-pulsed DC elicited an
antigen-specific, HLA-A2-restricted cytotoxic activity against targets
pulsed with the cognate synthetic peptide or tumor cells endogenously
expressing MUC1.
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MATERIALS AND METHODS |
Tumor cell lines.
Tumor cell lines used in the experiments were grown in RP10 medium
(RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum
[FCS], 2 mmol/L L-glutamine, 50 µmol/L 2-mercaptoethanol, and
antibiotics). The following HLA-A2-expressing tumors were used: MCF-7
(breast cancer), A 498 (renal cell carcinoma), HPAF (pancreatic cell
line), MZ 1774-RCC, NWI-RCC (renal cell carcinoma lines; kindly
provided by Alexander Knuth, Department of Medicine, Northwest
Hospital, Frankfurt, Germany), HCT 116 (colon carcinoma), 3D7
(Epstein-Barr virus [EBV]-immortalized B-cell line), and T2 cells
(174xCEM.T2 hybridoma, TAP1 and TAP2 deficient). Croft (HLA-A2) is an
EBV-immortalized B-cell line and was kindly donated by O.J. Finn
(Pittsburgh, PA). SK-OV-3 (HLA-A3) is an ovarian cell line, Caki-2
(HLA-A1, HLA-A11) and ACHN (HLA-A26) are derived from renal cell
carcinoma (RCC). The proerythroblastic
HLA-antigen-negative cell line K562 was used as a target cell in
cytotoxicity assays to test for natural killer (NK) activity.
Cell isolation and generation of DC from adherent peripheral blood
mononuclear cells (PBMNC).
Generation of DC from peripheral blood monocytes was performed as
described previously.36-38 In brief, PBMNC were isolated by
Ficoll/Paque (GIBCO-BRL, Grand Island, NY) density gradient centrifugation of heparinized blood obtained from buffy coat
preparations of healthy volunteers (n = 3) from the blood bank of the
University of Tübingen (Tübingen, Germany). Cells were
seeded (1 × 107 cells/3 mL per well) into 6-well
plates (Costar, Cambridge, MA) in RP10 media. After 2 hours of
incubation at 37°C, nonadherent cells were removed and the adherent
blood monocytes (purity >95%) were cultured in RP10 medium
supplemented with the following cytokines: human recombinant
granulocyte-macrophage colony-stimulating factor (GM-CSF; Leukomax; 100 ng/mL; Novartis, Nürnberg, Germany), interleukin-4 (IL-4; 1,000 IU/mL; Genzyme, Cambridge, MA), and tumor necrosis factor- (TNF-; 10 ng/mL; Genzyme). The phenotype of DC was
analyzed by flow cytometry after 7 days of culture.
Immunostaining.
Cell staining was performed using fluorescein isothiocyanate (FITC)- or
phycoerythrin (PE)-conjugated mouse monoclonal antibodies (MoAbs)
against CD86 and CD40 (Pharmingen, Hamburg, Germany); CD80, HLA-DR,
CD54, and CD14 (Becton Dickinson, Heidelberg, Germany); HLA-A, HLA-B,
and HLA-C (W6/32; Dako, Glostrup, Denmark); CD83 (Coulter-Immunotech, Hamburg, Germany); and CD1a (OKT6; Ortho Diagnostic Systems, Seattle, WA). Appropriate mouse IgG
isotypes were used as controls (Becton Dickinson). The level of HLA-A2 expression was analyzed using a purified MoAb specific for HLA-A2 (BB7.2). The MUC1 expression was determined using unlabeled antibodies MAM-6 (IgG2b; BioGenex, San Ramon, CA) and HMFG-1 (IgG1; Novocastra Laboratories, Newcastle, UK), followed by FITC-conjugated goat antimouse antibody (Becton Dickinson). The samples were analyzed on a
FACScan Calibur (Becton Dickinson).
Induction of antigen-specific CTL response using HLA-A2-restricted
synthetic peptides.
MUC1 peptides with a high probability of being presented by HLA-A2 were
predicted using the PAP program and the HLA-A*0201 peptide
motif.34,35 Among the high-scoring peptides were signal sequence or transmembrane region-derived sequences and only one peptide
from the tandem repeat domain. There are 3 of 44 VNTRs in the MUC1
sequence that vary in their amino acid sequence. The M1.1 peptide
(amino acids 950-958: STAPPVHNV) is derived from the last VNTR and
varies in two positions (V in position 6 and N in position 8) from the
previously described STAPPAHGV peptide.15,18 The presence
of the V substitution in position 6 increases the binding of the M1.1
peptide to the HLA-A2 molecule. The MUC1-derived peptide M1.2 (amino
acids 12-20: LLLLTVLTV) is from the leader sequence. Both MUC1 peptides
as well as the Her-2/neu-derived peptide E75 (amino acids 369-377:
KIFGSLAFL), the IMP peptide (influenza matrix protein; amino acids
58-66: GILGFVFTL), and the Pan-HLA-DR binding PADRE peptide
[a(X)VAAWTLKAAa]39 were synthesized using standard Fmoc
chemistry on a peptide synthesizer (432A; Applied Biosystems,
Weiterstadt, Germany) and analyzed by reversed-phase high-performance
liquid chromatography (HPLC) and mass spectrometry. The
HLA-A2 binding of the synthetic peptides was confirmed by the T2
stabilization assay.
For CTL induction, 5 × 105 DC were pulsed with 50 µg/mL synthetic peptide for 2 hours, washed, and incubated with 2.5 × 106 autologous PBMNC in RP10 medium. To analyze the
effect of a helper epitope on antigen-specific CTL induction, DC were
incubated with a Pan-HLA-DR binding peptide PADRE (50 µg/mL) in
addition to the HLA-A2 binding antigenic peptides.38,39
To analyze the influence of MUC1 protein on CTL induction, soluble MUC1
protein (kindly provided by Dr S. Kaul, Heidelberg, Germany) purified from T47D cells by 12H12 affinity
chromotography and gelfiltration40 was added to the
cell cultures at the concentration of 50 U/mL.
After 7 days of culture, cells were restimulated with autologous
peptide-pulsed PBMNC and 1 ng/mL human recombinant IL-2 (Genzyme) was
added on days 1, 3, and 5. The cytolytic activity of induced CTL was
analyzed on day 5 after the last restimulation in a standard 51Cr-release assay.
CTL assay.
The standard 51Cr-release assay was performed as
described.20,38 Target cells were pulsed with 50 µg/mL
peptide for 2 hours and labeled with [51Cr]-sodium
chromate in RP10 for 1 hour at 37°C. Cells (104) were
transferred to a well of a round-bottomed 96-well plate. Varying
numbers of CTL were added to give a final volume of 200 µL and
incubated for 4 hours at 37°C. At the end of the assay, supernatants (50 µL/well) were harvested and counted in a beta-plate counter. The percentage of specific lysis was calculated as follows: 100 × (experimental release  spontaneous release/maximal
release spontaneous release). Spontaneous and maximal release
were determined in the presence of either medium or 1% Triton X-100, respectively.
Antigen specificity of tumor cell lysis was further determined in a
cold target inhibition assay by analyzing the capacity of peptide
pulsed unlabeled T2 cells to block lysis of tumor cells at a ratio of
20:1 (inhibitor to target ratio).
For antibody blocking experiments, cells were incubated for 30 minutes
with 10 µg/mL of MoAb BB7.2 (IgG2b) recognizing HLA-A2, MoAb T8
(IgG1) recognizing CD8 and BMA 031, anti-Pan-TCR- (IgG2b), and
isotype antibodies before seeding in 96-well plates. Antibodies were
purchased from Coulter-Immunotech.
Cytokine determination.
Cytokine concentrations in cell cultures during the primary in vitro
immunizations using peptide-pulsed DC were measured by commercially
available two-site sandwich enzyme-linked immunosorbent assays (ELISAs)
from Genzyme (IL-12, IL-6, interferon- [IFN-], soluble IL-2
receptor [sIL2-R], and TNF-) and R&D Systems
(Minneapolis, MN; IL-10) according to the manufacturers' instructions.
Experiments were performed in triplicates and supernatants were
collected on day 5 of the CTL induction.
Statistical analysis.
Each experiment was performed at least three times. Representative
experiments are shown. The Student's t-test was performed to
evaluate the significance of the results.
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RESULTS |
Induction of antigen-specific CTL using peptide-pulsed DC.
Adherent PBMNC were grown in RP10 medium supplemented with GM-CSF,
IL-4, and TNF-. Analysis of surface markers after 7 days of culture
showed high levels of expression of MHC class I and II molecules, CD83,
CD80, CD86, CD40, and CD54 corresponding to phenotypic characteristics
of mature DC (data not shown).
To identify peptides with a high probability of HLA-A2 binding, we
performed a computer analysis of the amino acid sequence of the MUC1
protein to screen for peptides with HLA-A2-binding motifs.34,35 Two predicted MUC1-derived peptides M1.1 and
M1.2 were synthesized and used for CTL induction in vitro using DC as
APC. As shown in Fig 1, CTL lines obtained
after 3 weekly restimulations demonstrated peptide specific killing. T
cells only exhibited a cytotoxic response against T2 cells coated with
the cognate peptide, whereas they did not lyse T2 cells coated with an
irrelevant HER-2/neu-derived peptide E75. CTL induced in presence of
the Pan-DR binding peptide PADRE39 elicited a higher
cytotoxic activity (Fig 1), demonstrating the importance of CD4 cells
for the induction of a efficient CTL response. In line with these
results, higher concentrations of IL-12, TNF-, IFN-, IL-6, and
sIL-2R were found in supernatants from cultures containing DC pulsed
with the M1.1 and the PADRE peptide (Table
1). Similar results were obtained when the M1.2 peptide was used for
CTL induction (data not shown).
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| Fig 1.
Induction of CTL responses by peptide-pulsed DC. Adherent
PBMNC were grown for 7 days in RP10 medium supplemented with GM-CSF,
IL-4, and TNF- . DC pulsed with the synthetic peptides derived from
the MUC1 protein (M1.1 and M1.2) were used to induce a CTL response in
vitro. In addition to the MUC1 peptide DC were incubated with the
Pan-DR binding peptide PADRE as a T-helper epitope. Cytotoxic activity
of induced CTL was determined in a standard 51Cr-release
assay using T2 cells as targets pulsed for 2 hours with 50 µg of the
cognate (open symbols) or irrelevant Her-2/neu protein-derived E75
peptide (solid symbols).
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Recently, it was demonstrated that the MUC1 protein expression and
secretion in cancer patients is associated with high metastatic potential and poor prognosis.41,42 In vitro studies have
shown that the MUC1 protein can induce apoptosis or inhibit T-cell
proliferation.43,44 The presence of purified MUC1
protein40 in the cell cultures during the primary in vitro
immunization had no inhibitory effects on the induction of MUC1
peptide-specific CTL and their cytotoxic activity (data not shown).
To test the sensitivity of the induced MUC1-specific CTL, T2 cells were
incubated with titrated amounts of the synthetic peptides and effector
cells were added after a preincubation time of 30 minutes at an E:T
ratio of 20:1. As shown in Fig 2, the
MUC1-specific CTL CTL.M1.1 and CTL.M1.2 lysed the target cells in an
antigen concentration-depending fashion. However, the sensitivity of an in vitro-induced CTL line CTL.IMP38 specific for an
influenza matrix protein-derived viral peptide IMP is about 10 times
higher as compared with the CTL specific for the MUC1 self-peptides.
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| Fig 2.
Peptide sensitivity of in vitro-induced MUC1-specific
CTL. T2 cells were incubated with titrated amounts of the MUC1 peptides
M1.1 and M1.2 as well as the influenza matrix protein peptide IMP.
Corresponding specific CTL were added to the target cells incubated
with the cognate peptide at a ratio of 20:1.
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The lysis of allogeneic breast cancer cells by CTL specific for MUC1
is antigen-specific and HLA-A2-restricted.
The expression of MUC1 and HLA-A2 molecules on tumor cells was
determined by flow cytometry, and results are presented in Fig 3. To analyze the ability of
CTL.M1.1 and CTL.M1.2 to lyse endogenously MUC1-expressing tumor cells,
the MUC1-positive, HLA-A2-expressing breast cancer cell line MCF-7 was
used as target cell in a standard 51Cr-release assay. As
shown in Fig 4A and B, both CTL lines were able to efficiently lyse MCF-7 (HLA-A2+/MUC1+)
tumor cells and peptide-pulsed Croft cells
(HLA-A2+/MUC1 ). There was no lysis of
the ovarian cancer cells SK-OV-3
(MUC1+/HLA-A3+) or Croft cells pulsed with an
irrelevant peptide E75 derived from the Her-2/neu protein. These
results indicate the necessity of both the presence of MUC1 epitopes on
the target cells and HLA-A2 restriction of the induced CTL.
Furthermore, these data show that the two novel MUC1 peptides can be
efficiently processed and presented in an HLA-restricted manner by
tumor cells endogenously expressing MUC1 on the cell surface.
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| Fig 3.
Flow cytometric analysis of HLA-A2 and MUC1 expression on
the human tumor cell lines. The level of HLA-A2 expression (dotted
line) was analyzed using a purified MoAb specific for HLA-A2, BB7.2.
The MUC1 expression was determined using unlabeled antibodies HMFG-1
(bold solid line) and MAM-6 (thin solid line), followed by staining
with FITC-conjugated goat antimouse antibody. Solid histograms
represent isotype-matched controls.
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| Fig 4.
Lysis of cancer cells endogenously expressing MUC1 by
CTL.M1.1 (A) and CTL.M1.2 (B). Human breast cancer cell line MCF-7
(HLA-A2+/MUC1+), ovarian cancer cell line
SK-OV-3 (HLA-A2/MUC1+), and the
immortalized B-cell line Croft
(HLA-A2+/MUC1) were used as targets in a
stardard 51Cr-release assay. Croft cells were pulsed with
the MUC1 peptides or an irrelevant Her-2/neu-derived peptide E75.
( ) Croft + E75 peptide; ( ) Croft + M1.1 peptide; ( ) MCF-7;
( ) SK-OV-3.
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Lysis of RCC cells by MUC1-specific CTL.
Flow cytometric analysis of MUC1 expression of different tumor cell
types using two specific MoAbs showed MUC1 expression on several tumor
cell lines, including the proerythroblastic K562 cells and several RCC
cell lines. We therefore analyzed the presentation of MUC1-derived
peptides by these cell lines and used them as targets in a standard
Cr-release assay. As shown in Fig 5A and B
and Table 2, CTL.M1.1 and
CTL.M1.2 did lyse tumor cell lines expressing MUC1 and HLA-A2,
suggesting that the identified MUC1 peptides are presented by these
tumors. In contrast, there was no lysis of MUC1-positive but
HLA-negative K562 cells, demonstrating that the observed cytotoxicity
was not mediated by NK cells and confirming the MHC necessity and
restriction.
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| Fig 5.
Lysis of renal carcinoma cells by MUC1-reactive CTL.M1.1
(A and C) and CTL.M1.2 (B and D). Human RCC cell lines A-498
(HLA-A2+/MUC1+), MZ1774-RCC
(HLA-A2+/MUC1+), Caki-2
(HLA-A2/MUC1+), ovarian cancer cell line
SK-OV-3 (HLA-A2/MUC1+), and K562
(HLA-A2/MUC1+) were used as targets in a
stardard 51Cr-release assay. The antigen specificity of the
CTL lines was tested in the presence of unlabeled cold targets, T2
cells coated with the cognate, or an irrelevant peptide at an
inhibitor:target ratio of 20:1 (C and D). For (A) and (B), ( ) K562;
( ) CAKI-2; ( ) SK-OV-3; ( ) A498; ( ) MZ1774-RCC. For (C) and
(D), () MZ1774-RCC + M1.1 peptide; () MZ1774-RCC; ( )
MZ1774-RCC + T2-M1.1 peptide; () MZ1774-RCC + T2-E75 peptide.
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The antigen specificity and MHC restriction mediated by the in
vitro-induced CTL was confirmed in a cold target inhibition assay. The
lysis of MZ1774-RCC cells could be blocked by the addition of T2 cells
pulsed with the cognate peptide, whereas T2 cells pulsed with an
irrelevant peptide showed no effect on cytotoxic lysis (Fig 5C and D).
Blocking of target cell lysis by MoAbs against HLA-A2, CD8, and TCR.
To further analyze the association of the MUC1 peptide presentation
with HLA-A2, blocking experiments using a monoclonal anti-HLA-A2 antibody BB7.2 were performed. As shown in
Fig 6, incubation of MZ 1774-RCC cells with
the BB7.2 antibody resulted in the inhibition of the target cell lysis,
whereas anti-MUC1 MoAb MAM-6, used as an isotype control (mouse IgG2b),
had no effect. Incubation of the CTL effector cells with anti-CD8 or
anti-TCR Abs before adding to the assay abolished their lytic activity,
suggesting that the target cell lysis is HLA-A2-restricted and
mediated by CD8+ T cells. Isotype-matched control Abs
(MAM-6 and HMFG-1) did not block the lysis of tumor cells.
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| Fig 6.
Inhibition of the cytotoxic activity of MUC1-reactive
CTL.M1.1 (A) and CTL.M1.2 (B) by MoAbs. For HLA-A2 blocking
experiments, target cells (MZ1774-RCC) were incubated for 30 minutes
with 10 µg/mL of MoAbs (BB7.2 (IgG2b) recognizing HLA-A2. For
blocking of CD8 molecules or the TCR on the effector cells, CTL were
incubated with 15 µg/mL of T8 (IgG1) MoAb recognizing CD8 and
anti-Pan-TCR- BMA 031 MoAb (IgG2b) before adding to the assay.
Isotype-matched antibodies (MAM-6, HMFG-1) were used as controls. The
assay was performed at E:T ratio of 20:1. The data are shown as the
mean and standard deviation from 6 replicate wells.
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DISCUSSION |
Mucins are transmembrane type I glycoproteins with a unique
extracellular domain consisting mostly of 20 to 60 tandem
repeats.1,2 The MUC1 protein is overexpressed on many
epithelial and nonepithelial malignant cells and is therefore a
suitable candidate for broadly applicable vaccine
therapies.3-9 Several previous reports have demonstrated
that the tandem repeat of the MUC1 protein is highly immunogenic and
that both antibody-mediated and T-cell-mediated responses recognizing
epitopes derived from this domain can be induced in mice and humans. It
was further reported that MUC1 reactive T cells found in patients with
breast cancer, pancreatic cancer, and multiple myeloma recognize
directly the MUC1 molecule in an MHC-unrestricted
manner.8-14,44-46 It was proposed that
normally cryptic T-cell epitopes from the MUC1 core domain were
recognized by MUC1-specific CTL as a result of underglycosylation in
tumor cells and cross-linking of the T-cell receptor by the highly
multivalent epitopes of tandemly repeated 20 amino acid peptides on a
single MUC1 molecule. However, there is now increasing evidence from mouse and human studies indicating that T cells induced against the
MUC1 protein can be MHC-restricted. In a recently published study using
HLA-A0201/Kb transgenic mice, two HLA-A2-binding peptides
were identified. Interestingly, one of these peptides was also shown to
induce HLA-A11-restricted CTL in humans.15-19
Using a computer-assisted analysis, we screened the MUC1 protein for
HLA-A2-binding peptides.34,35 Using this approach, we were
able to identify two novel 9-mer peptides, M1.1 and M1.2, with a high
binding probability to HLA-A2. The M1.1 peptide is derived from the
VNTR domain of the MUC1 protein. There are 3 of 44 tandem repeats that
differ in their amino acid sequence from the
PDTRPAPGSTAPPAHGVTSA sequence. The M1.1 peptide is localized in the
last VNTR (amino acids 950-958: STAPPVHNV) and varies in two positions
from the previously described STAPPAHGV peptide.15,18 The
presence of the V substitution in position 6 increases the binding of
the M1.1 peptide to the HLA-A2 molecule. To analyze whether these
epitopes are presented by tumor cells endogenously expressing MUC1, we
induced MUC1 peptide-specific CTL responses by primary in vitro
immunization and used these CTL to determine the presentation of MUC1
epitopes on human tumor lines. Autologous DC generated from peripheral
blood monocytes in the presence of GM-CSF, IL-4, and TNF- were
pulsed with the identified peptides M1.1 and M1.2 and used as APC for
CTL priming. DC used here demonstrated a high ability to initiate an
MUC1-specific CTL response. The antigen-specific cytotoxic activity of
the induced CTL could be further increased by the addition of a
T-helper epitope, the Pan-HLA-DR binding peptide PADRE.39
This finding could be of potential importance for the developing of
cancer vaccination protocols.
In a previous report,47 it was demonstrated that a strong
antigen-specific response could be induced in vitro when human peripheral blood lymphocytes (PBL) pulsed with a
liposome-encapsulated MUC1 peptide consisting of 25 amino acids from
the VNTR were used for CTL induction. Proliferative and cytotoxic
T-cell responses as well as the secretion of IFN- were already
detected after 2 weekly restimulations, comparable to the results
obtained in our study and demonstrating that this approach could be
effective and feasible for the development of cancer vaccines.
In our study, we show that MUC1 peptide-specific CTL were able to
recognize tumor cells endogenously expressing the MUC1 protein in an
HLA-A2-restricted manner. Furthermore, the MUC1-specific CTL lysed not
only breast cancer cells, but also pancreatic and RCC cell lines
expressing MUC1 and HLA-A2. The cytotoxicity against tumor cells was
blocked by cold HLA-A2+ targets pulsed with the cognate
peptide in a cold-target inhibition assay and by anti-HLA-A2 MoAb. Our
results extend the list of epithelial tumors that present MUC1-derived
T-cell epitopes that increases the possible clinical use of
peptide-pulsed DC as a cancer vaccine.
Of particular interest is the finding that the M1.2 peptide is derived
from the signal sequence of MUC1, showing that the T-cell-mediated
immune responses to the MUC1 protein are not restricted to the tandem
repeat domain. Furthermore, the M1.2 peptide could be presented in
absence of a functional TAP by malignant cells. It was recently shown
that proteolysis of peptides derived from the signal sequence in the
endoplasmatic reticulum represent a second pathway for the generation
of class I MHC-associated peptides and that this peptides are presented
independent of TAP.48,49
Recently, it was demonstrated that the MUC1 protein expression and
secretion in cancer patients is associated with high metastatic potential and poor prognosis.41,42 In vitro studies have
shown that the MUC1 protein can induce apoptosis or inhibit T-cell
proliferation.43,44 The growth inhibition was mediated by
the whole MUC1 protein or large synthetic tandem repeats of the MUC1
core peptide and was reversible by addition of IL-2, anti-CD28 MoAb, or
short 16-amino acid MUC1 peptide,44 indicating that active
specific immunotherapy using short synthetic peptides in combination
with professional APC and/or IL-2 may overcome the observed
immunosuppression in cancer patients. In MUC1 transgenic mice, the
tolerance to human MUC1 antigen could be reversed by immunizing the
animals with fusions of DC- and MUC1-expressing tumors.50
Interestingly, the presence of purified MUC1 protein in the cell
cultures during primary in vitro immunization with peptide-pulsed DC
did not inhibited the induction of MUC1 peptides-specific CTL responses
or the cytotoxic activity elicited by these CTL (data not shown).
Because protocols for maintenance and expansion of human DC generated
from bone marrow-derived progenitors or peripheral blood monocytes have
recently been established,38,39,51-55 it is now possible to
generate sufficient numbers of DC from patients and apply them in
vaccination therapies. Vaccination with DC pulsed with antigenic
peptides was shown to be effective in patients with malignant lymphoma
and melanoma.56,57 The use of DC- and MUC1-derived peptides
could reverse the observed immunosuppression and MUC1 tolerance in
cancer patients and provide an additional broadly applicable approach
to established therapies of hematologic and epithelial malignancies,
such as multiple myeloma and renal cell, breast, and pancreatic carcinoma.
| |
ACKNOWLEDGMENT |
We thank S. Kurtz and Y. Hoffmann for their excellent technical assistance.
| |
FOOTNOTES |
Submitted December 9, 1998; accepted February 3, 1999.
Supported in part by grants from Deutsche Forschungsgemeinschaft (SFB
510) and Deutsche Krebshilfe.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Wolfram Brugger, MD, Department of
Hematology, Oncology, Rheumatology and Immunology, University of
Tübingen, Otfried-Müller-Strasse-10, D-72076
Tübingen, Germany.
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M. R. Muller, F. Grunebach, K. Kayser, W. Vogel, A. Nencioni, W. Brugger, L. Kanz, and P. Brossart
Expression of Her-2/neu on Acute Lymphoblastic Leukemias: Implications for the Development of Immunotherapeutic Approaches
Clin. Cancer Res.,
August 1, 2003;
9(9):
3448 - 3453.
[Abstract]
[Full Text]
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S. M. Schmidt, K. Schag, M. R. Muller, M. M. Weck, S. Appel, L. Kanz, F. Grunebach, and P. Brossart
Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells
Blood,
July 15, 2003;
102(2):
571 - 576.
[Abstract]
[Full Text]
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M. R. Muller, F. Grunebach, A. Nencioni, and P. Brossart
Transfection of Dendritic Cells with RNA Induces CD4- and CD8-Mediated T Cell Immunity Against Breast Carcinomas and Reveals the Immunodominance of Presented T Cell Epitopes
J. Immunol.,
June 15, 2003;
170(12):
5892 - 5896.
[Abstract]
[Full Text]
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C. Milazzo, V. L. Reichardt, M. R. Muller, F. Grunebach, and P. Brossart
Induction of myeloma-specific cytotoxic T cells using dendritic cells transfected with tumor-derived RNA
Blood,
February 1, 2003;
101(3):
977 - 982.
[Abstract]
[Full Text]
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L. Holtl, C. Zelle-Rieser, H. Gander, C. Papesh, R. Ramoner, G. Bartsch, H. Rogatsch, A. L. Barsoum, J. H. Coggin Jr., and M. Thurnher
Immunotherapy of Metastatic Renal Cell Carcinoma with Tumor Lysate-pulsed Autologous Dendritic Cells
Clin. Cancer Res.,
November 1, 2002;
8(11):
3369 - 3376.
[Abstract]
[Full Text]
[PDF]
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J. Hernandez, F. Garcia-Pons, Y. C. Lone, H. Firat, J. D. Schmidt, P. Langlade-Demoyen, and M. Zanetti
Identification of a human telomerase reverse transcriptase peptide of low affinity for HLA A2.1 that induces cytotoxic T lymphocytes and mediates lysis of tumor cells
PNAS,
September 17, 2002;
99(19):
12275 - 12280.
[Abstract]
[Full Text]
[PDF]
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H. Hebart, S. Daginik, S. Stevanovic, U. Grigoleit, A. Dobler, M. Baur, G. Rauser, C. Sinzger, G. Jahn, J. Loeffler, et al.
Sensitive detection of human cytomegalovirus peptide-specific cytotoxic T-lymphocyte responses by interferon-gamma -enzyme-linked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation
Blood,
May 15, 2002;
99(10):
3830 - 3837.
[Abstract]
[Full Text]
[PDF]
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M. S. von Bergwelt-Baildon, R. H. Vonderheide, B. Maecker, N. Hirano, K. S. Anderson, M. O. Butler, Z. Xia, W. Y. Zeng, K. W. Wucherpfennig, L. M. Nadler, et al.
Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application
Blood,
May 1, 2002;
99(9):
3319 - 3325.
[Abstract]
[Full Text]
[PDF]
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D. W. Mullins, T. N. J. Bullock, T. A. Colella, V. V. Robila, and V. H. Engelhard
Immune Responses to the HLA-A*0201-Restricted Epitopes of Tyrosinase and Glycoprotein 100 Enable Control of Melanoma Outgrowth in HLA-A*0201-Transgenic Mice
J. Immunol.,
November 1, 2001;
167(9):
4853 - 4860.
[Abstract]
[Full Text]
[PDF]
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M. Schnurr, P. Galambos, C. Scholz, F. Then, M. Dauer, S. Endres, and A. Eigler
Tumor Cell Lysate-pulsed Human Dendritic Cells Induce a T-Cell Response against Pancreatic Carcinoma Cells: an in Vitro Model for the Assessment of Tumor Vaccines
Cancer Res.,
September 1, 2001;
61(17):
6445 - 6450.
[Abstract]
[Full Text]
[PDF]
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P. Brossart, A. Schneider, P. Dill, T. Schammann, F. Grunebach, S. Wirths, L. Kanz, H.-J. Buhring, and W. Brugger
The Epithelial Tumor Antigen MUC1 Is Expressed in Hematological Malignancies and Is Recognized by MUC1-specific Cytotoxic T-Lymphocytes
Cancer Res.,
September 1, 2001;
61(18):
6846 - 6850.
[Abstract]
[Full Text]
[PDF]
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M. Gorschluter, C. Ziske, A. Glasmacher, and I. G. H. Schmidt-Wolf
Current Clinical and Laboratory Strategies to Augment the Efficacy of Immunotherapy in Multiple Myeloma
Clin. Cancer Res.,
August 1, 2001;
7(8):
2195 - 2204.
[Abstract]
[Full Text]
[PDF]
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A. Trojan, M. Witzens, J. L. Schultze, R. H. Vonderheide, S. Harig, A. M. Krackhardt, R. A. Stahel, and J. G. Gribben
Generation of Cytotoxic T Lymphocytes against Native and Altered Peptides of Human Leukocyte Antigen-A*0201 Restricted Epitopes from the Human Epithelial Cell Adhesion Molecule
Cancer Res.,
June 1, 2001;
61(12):
4761 - 4765.
[Abstract]
[Full Text]
[PDF]
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M. M. Soares, V. Mehta, and O. J. Finn
Three Different Vaccines Based on the 140-Amino Acid MUC1 Peptide with Seven Tandemly Repeated Tumor-Specific Epitopes Elicit Distinct Immune Effector Mechanisms in Wild-Type Versus MUC1-Transgenic Mice with Different Potential for Tumor Rejection
J. Immunol.,
June 1, 2001;
166(11):
6555 - 6563.
[Abstract]
[Full Text]
[PDF]
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E. M. Jaffee, R. H. Hruban, B. Biedrzycki, D. Laheru, K. Schepers, P. R. Sauter, M. Goemann, J. Coleman, L. Grochow, R. C. Donehower, et al.
Novel Allogeneic Granulocyte-Macrophage Colony-Stimulating Factor-Secreting Tumor Vaccine for Pancreatic Cancer: A Phase I Trial of Safety and Immune Activation
J. Clin. Oncol.,
January 1, 2001;
19(1):
145 - 156.
[Abstract]
[Full Text]
[PDF]
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P. Brossart, S. Wirths, G. Stuhler, V. L. Reichardt, L. Kanz, and W. Brugger
Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with peptide-pulsed dendritic cells
Blood,
November 1, 2000;
96(9):
3102 - 3108.
[Abstract]
[Full Text]
[PDF]
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P. Brossart, A. Zobywalski, F. Grünebach, L. Behnke, G. Stuhler, V. L. Reichardt, L. Kanz, and W. Brugger
Tumor Necrosis Factor {{alpha}} and CD40 Ligand Antagonize the Inhibitory Effects of Interleukin 10 on T-Cell Stimulatory Capacity of Dendritic Cells
Cancer Res.,
August 1, 2000;
60(16):
4485 - 4492.
[Abstract]
[Full Text]
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J. Gong, N. Nikrui, D. Chen, S. Koido, Z. Wu, Y. Tanaka, S. Cannistra, D. Avigan, and D. Kufe
Fusions of Human Ovarian Carcinoma Cells with Autologous or Allogeneic Dendritic Cells Induce Antitumor Immunity
J. Immunol.,
August 1, 2000;
165(3):
1705 - 1711.
[Abstract]
[Full Text]
[PDF]
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M. H. Andersen, L. O. Pedersen, J. C. Becker, and P. t. Straten
Identification of a Cytotoxic T Lymphocyte Response to the Apoptosis Inhibitor Protein Survivin in Cancer Patients
Cancer Res.,
February 1, 2000;
61(3):
869 - 872.
[Abstract]
[Full Text]
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P. Brossart, B. Spahlinger, F. Grunebach, G. Stuhler, V. L. Reichardt, L. Kanz, W. Brugger;, T. Mutis, and E. Goulmy
Induction of Minor Histocompatibility Antigen HA-1-Specific Cytotoxic T Cells for the Treatment of Leukemia After Allogeneic Stem Cell Transplantation
Blood,
December 15, 1999;
94(12):
4374 - 4376.
[Full Text]
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TOP
ABSTRACT
INTRODUCTION
