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* Institut National de la Santé et de la Recherche Médicale, Unité 487, Institut Gustave Roussy, Villejuif, France; and
Unité d’Immunité Cellulaire Antivirale, Département SIDA-Retrovirus, Institut Pasteur, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MAGE-A is a multigene family that consists of 12 homologous genes (MAGE-A1 to -A12) located in the q28 region of chromosome X (8). Among the different family members, MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 are abundantly expressed by tumors but not by normal tissues except testis and placenta (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). Each one of the seven MAGE-A Ags is detected in primary and metastatic tumors of various histological types including melanoma, lung, bladder, ovarian, and breast carcinomas. Individual MAGE-A expression varies from one tumor type to the other, but overall the large majority of tumors expresses at least one MAGE-A. Targeting epitopes shared by all MAGE-A Ags would be of interest against a broad spectrum of cancers. Moreover, the use of these epitopes will not come up against the tolerance of their CTL repertoire and the risks of autoimmunity. MAGE-A do not belong to the immunological self and the MAGE-A-expressing testis is considered as an immunoprivileged organ and cannot be the target of an autoimmune response.
To find common MAGE-A epitopes susceptible to induce MAGE-A-specific HLA-A*0201-restricted CTL, we first selected peptides likely to share the same antigenicity, even if they lack the HLA-A*0201-specific anchor residues. Sequence modifications were introduced in some of these peptides to improve their binding capacity and their immunogenicity.
In this work we describe one high-affinity heteroclitic peptide (p248V9) corresponding to two HLA-A*0201-restricted, cross-recognized epitopes, derived from MAGE-A2, -A3, -A4, -A6, -A10, -A12 (p248G9) and MAGE-A1 (p248D9). This heteroclitic peptide stimulates CTL that recognize each MAGE-A Ag individually and kill MAGE-A-expressing tumor cells. Therefore, it may be implemented for a broad-spectrum, efficient, but nonetheless safe, vaccination.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The HLA-A*0201 transgenic HHD mice were previously described (24).
Cells
Murine RMAS/HHD cells were obtained by transfection of murine RMAS cells with the HHD construct as previously described (24). COS-7 cells and WEHI-164 clone 13 cells were kindly provided by Dr. F. Jotereau (Institut National de la Santé et de la Recherche Médicale, Unité 463, Nantes, France). The HLA-A*0201-expressing human tumor cells were T2 (deficient in TAP1 and TAP2 transporters); M44 and M113 (melanoma; kindly provided by Dr. F. Jotereau), OBR (bladder cancer; kindly provided by Dr. D. Zeliszewsli, Centre National de la Recherche Scientifique UPRES-A8067, Paris, France), MCF-7 (breast cancer), and Caco-2 (colon cancer). Cells were grown in RPMI 1640 medium supplemented with 10% FCS.
Peptides and plasmids
Peptides were synthesized by Synt:em (Nîmes, France). Plasmids containing the cDNA for MAGE-A (pcD-Sra/MAGE-A1, pcD-Sra/MAGE-A2, pcDNAI/MAGE-A3, pcDNAI/MAGE-A4, pcDNAI/MAGE-A6, pcDNAI/MAGE-A10, and pcDNAI/MAGE-A12) were kindly provided by Dr. P. van der Bruggen (Ludwig Institute for Cancer Research, Brussels, Belgium). The plasmid containing the HHD construct was described previously (24).
Generation of CTL in HHD mice
HHD mice were injected s.c. with 100 µg of peptide emulsified in IFA in the presence of 140 µg of the I-Ab-restricted HBV core-derived Th epitope (128–140: TPPAYRPPNAPIL). After 11 days, 5 x 107 spleen cells were stimulated in vitro with peptide (10 µM) in RPMI 1640 medium supplemented with 10% FCS, 2 µM glutamine, and antibiotics. On day 6 of culture, the bulk responder populations were tested for specific cytotoxicity. Upon response, CTL cultures were restimulated weekly in vitro with 2 x 107 irradiated spleen cells in the presence of 1 to 0.1 µM peptide and 50 U/ml rIL-2 (Proleukin; Chiron, Suresnes, France).
Generation of CTL from human PBMC
PBMC were collected by leukapheresis from healthy HLA-A*0201
donors. Dendritic cells (DC) were produced from adherent cells cultured
for 7 days (2 x 106 cells/ml) in the
presence of 500 IU/ml GM-CSF (Leucomax; Schering-Plough, Levallois
Perret, France) and 500 IU/ml IL-4 (R&D Systems, Minneapolis, MN) in
complete medium (RPMI 1640 supplemented with 10% heat inactivated
human AB serum, 2 µM L-glutamine, and antibiotics). On
day 7 maturation agents poly(I:C) at 100 ng/ml and anti-CD40 mAb
(kindly provided by Dr. J. P. Abastado, Immuno-Designed Molecules,
Paris, France) at 2 µg/ml were added in the culture for 24 h.
Mature DC were pulsed with 10 µM peptide in the presence of 5 µg/ml
2-microglobulin for 2 h at 37°C and
then irradiated (3500 rad). CD8+ cells were
purified by positive selection with CD8 MicroBeads (Miltenyi Biotec,
Paris, France) according to the manufacturer’s instructions. A total
of 2 x 105 CD8+ cells
were stimulated with 2 x 104 peptide-pulsed
DC in complete culture medium supplemented with 1000 IU/ml IL-6 (R&D
Systems) and 5 IU/ml IL-12 (R&D Systems) in a final volume of 100
µl/well in 96-well round-bottom plates. From day 7, cultures were
weekly restimulated with peptide-loaded DC in the presence of 20 IU/ml
IL-2 and 10 ng/ml IL-7 (R&D Systems). After the third in vitro
restimulation, CD8 cells were collected and stimulated with allogeneic
HLA-A*0201 EBV-transformed B cells in the presence of 10 µM peptide
for 16 h. IFN-
-producing CD8+ cells were
purified using the IFN- secretion assay cell enrichment and
detection kit (Miltenyi Biotec) according to the manufacturer’s
instructions. Purified CD8+ cells were then
cultivated in complete medium supplemented with 20 IU/ml IL-2 and 10
ng/ml IL-7 for 1 wk.
Cytotoxic assay
Targets were labeled with 100 µCi of 51Cr for 90 min, washed twice, and plated in 96-well round-bottom plates (3 x 103 cells/well in 100 µl of RPMI 1640 plus 5% FCS). When RMAS/HHD cells were used as targets they were pulsed with various concentrations of peptides at 37°C for 90 min. A total of 100 µl of effectors were then added to the wells. After a 4-h incubation, 100 µl of supernatant were collected and radioactivity was measured in a gamma counter. The percentage of specific lysis was determined as follows: lysis = (experimental release - spontaneous release)/(maximal release - spontaneous release) x 100.
Intracellular IFN- staining
T cells were stimulated by T2 cells loaded with peptide (10
µM) and by tumor cells in the presence of 20 µg/ml brefeldin A
(Sigma, Oakville, Canada). In blocking experiments tumor cells were
preincubated with an anti-HLA-A*0201 (BB7.2) and an
anti-HLA-B/C (B1.23.2) mAb for 1 h. Six hours later T cells
were washed, stained with PE-conjugated anti-CD8 mAb (Caltag
Laboratories, Burlingame, CA) in PBS for 25 min at 4°C, washed, and
fixed with 4% paraformaldehyde. Then, cells were permeabilized
with PBS/0.2% saponin/0.5% BSA (Sigma) and stained with
allophycocyanin-conjugated anti-IFN- mAb (BD PharMingen,
Mississauga, Canada). Cells were analyzed on FACSCalibur (BD
Biosciences, Mountain View, CA).
Peptide processing assay on COS-7 transfected cells
A total of 2.2 x 104 simian COS-7
cells were plated in flat-bottom 96-well plates in DMEM 10% FCS, in
triplicate per condition. Eighteen hours later, the medium was
discarded and 100 ng of each DNA plasmid was put in contact with COS
cells in DMEM, 10% Nuserum (Collaborative Biochemical Products,
Belford, MA), 10 mM chloroquine, and 10 mg/ml DEAE dextran.
After a 4-h incubation at 37°C, transfection medium was discarded and
50 µl PBS 10% DMSO was added for 2 min. Transfected COS-7 cells were
incubated in DMEM 10% FCS during 40 h and then used with 5
x 104 murine CTL in a TNF-
secretion
assay.
TNF- secretion assay
Transfected COS-7 cells at day 4 and human tumor cells were used
as stimulating cells. When necessary, they were incubated with 10 µM
peptide for 2 h. A total of 5 x 104 T
cells were then added in 50 µl RPMI and 10% FCS and incubated for
6 h. Each condition was tested in triplicate. A total of 50 µl
of the supernatant was collected. Standard dilutions were prepared in
50 µl with final doses of TNF- ranging from
104 to 0 pg/ml. On both the supernatants and the
standard dilutions, 3 x 104 TNF-
sensitive WEHI-164c13 cells in 50 µl were added. They were incubated
for 16 h at 37°C. Inhibition of cell proliferation was evaluated
by the MTT colorimetric method (25).
MAGE-A expression by human tumor cells
Isolation of total RNA from tumor cells was performed using
TRIzol (Invitrogen, Cergy Pontoise, France) reagent. For cDNA
synthesis, RNA (2 µg) was diluted in water, 4 µl of 5x reverse
transcriptase buffer (Invitrogen), 1 µl of 10 mM dNTP, 2 µl of a 20
µM solution of oligo(dT), 20 U of RNasin (Promega, Madison, WI), 2
µl of 0.1 M DTT, and 200 U of Moloney murine leukemia virus
reverse transcriptase (Invitrogen) in a 20-µl reaction volume, and
incubated at 42°C for 60 min. PCR amplification was performed with
DNA polymerase TaKaRa Taq (Takara Biomedicals, Shiga, Japan)
using pairs of oligonucleotide primers that are highly specific for
each MAGE-A gene (8, 26, 27, 28). To ensure that the RNA is
not degraded, a PCR assay with primers specific for -actin was
conducted. Samples were scored positive when a band of appropriate size
was visible on an agarose gel in the presence of ethidium bromide.
Measurement of peptide RA to HLA-A*0201
The protocol used has been described previously (29). Briefly, T2 cells were incubated with various concentrations of peptides ranging from 100 to 0.1 µM at 37°C for 16 h, and then stained with the BB7.2 mAb to quantify the expression of HLA-A*0201. For each peptide concentration, the HLA-A*0201-specific staining was calculated as the percentage of staining obtained with 100 µM of the reference peptide HIVpol 589 (IVGAETFYV). The relative affinity (RA) was determined as follows: RA = (concentration of each peptide that induces 20% of HLA-A*0201 expression/concentration of the reference peptide that induces 20% of HLA-A*0201 expression).
Assessment of peptide/HLA-A*0201 complex stability
As previously described (29), T2 cells were incubated overnight with 100 µM of each peptide at 37°C in serum-free medium. Cells were washed, incubated with brefeldin A (10 µg/ml) for 1 h, washed again, and incubated at 37°C during 0, 2, 4, or 6 h in the presence of brefeldin A at 0.5 µg/ml. Cells were stained with the BB7.2 mAb. Dissociation complex (DC50) was defined as the time required for the loss of 50% of the HLA-A*0201/peptide complexes stabilized at t = 0 h.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 are expressed in a
large variety of tumors with a frequency ranging from 5 to 76% (Table I
). However, a very high percentage of
tumors express at least one MAGE-A.
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. Peptides were designed by the
position of the first residue in the MAGE-A1 sequence. p262 had a
complete sequence homology in all MAGE-A, whereas p174 was identical in
all MAGE-A Ags except MAGE-A10. The homology group p248 was composed by
two peptides, p248G9 (MAGE-A2, -A3, -A4, -A6, -A10, and -A12) and
p248D9 (MAGE-A1), differing at the C-terminal position. The fourth
group (p264) was excluded from this study because it corresponds to the
already known HLA-A*0201-restricted MAGE-A3 epitope (FLWGPRALV) that is
reported to be not efficiently processed by tumor cells
(30).
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We then evaluated the capacity of selected peptides to bind (RA)
and to stabilize (DC50) the HLA-A*0201 molecule
(Table II
). As we reported previously, these parameters allow for
distinguishing high-affinity peptides (RA < 5 and
DC50 > 2 h) that are immunogenic in both
HHD mice and humans from low-affinity peptides (RA > 5 and
DC50 < 2 h) that are nonimmunogenic
(29). According to these criteria, peptides p174, p262,
p248G9, and p248D9 had a low affinity for HLA-A*0201. To enhance
affinity of p174, p262, p248G9, and p248D9 and thus increase their
immunogenicity, we modified their sequences at HLA-A*0201-specific
primary (P2 and P9/10) and/or secondary anchor positions. p262, p248G9,
and p248D9 lacked the anchor residues at P2 or C-terminal position. The
heteroclitic variants p262L2 and p248V9 exhibited high binding affinity
(RA = 0.2 and 1.8, respectively) and stabilization capacity
(DC50 = 6 and 4 h, respectively). For the
p174 we produced the p174Y1V10 variant by replacing the residue at P1
with a tyrosine as previously described (29) and the
residue at P10 with the strongest anchor valine. P174Y1V10 was a strong
binder (RA = 2.5) but weakly stabilized the HLA-A*0201 molecule
(DC50 < 2 h).
Generation of native peptide-specific CTL by vaccination with heteroclitic variants
To study whether heteroclitic peptides can induce CTL able to
recognize the corresponding native peptides, we vaccinated HHD mice
with heteroclitic peptides, stimulated their spleen cells with 10 µM
peptide in vitro, and tested cultures for killing RMAS/HHD cells loaded
with the heteroclitic or the native peptides (Fig. 1A). p174Y1V10 was unable to
elicit a CTL response, probably because of its weak stabilization
capacity. CTL generated in p262L2-vaccinated mice recognized the
heteroclitic variant but not the native p262. In contrast,
p248V9-vaccinated mice developed a CTL response directed against the
heteroclitic but also against the native p248G9 and p248D9 peptides. We
established a CTL line (CTL248) from HHD spleen cells primed in vivo
with p248V9 and in vitro repetitively restimulated with decreasing
concentrations of p248G9 (10 to 1 µM). CTL248 was maintained in
culture with 1 µM p248G9 and recognized the p248G9 and p248D9 with a
relatively high avidity (Fig. 1B).
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To test whether p248V9-induced CTL recognize endogenously
processed MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12, we first
stimulated the CTL248 line with COS cells cotransfected with cDNA
encoding the HHD construct (1 and
2 domains of HLA-A*0201 and the
3 and intracellular domains of
Db) and each one of the seven MAGE-A. CTL
activation was evaluated by measure of TNF- secretion. Results show
that the CTL248 line responded to COS cells coexpressing HHD and any of
the MAGE-A tested but not to COS cells expressing HHD or MAGE-A
separately (Fig. 2). This demonstrates
that CTL248 recognize all the endogenous MAGE-A presented by
HLA-A*0201.
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. CTL248 responded to all the
MAGE-A+ cells but not to MCF-7 and Caco-2 cells
(Fig. 3). These results were further
confirmed by measuring the number of IFN--producing CTL248 cells
upon stimulation (data not shown). To demonstrate that tumor cell
recognition was HLA-A*0201 restricted, we stimulated CTL248 with M44
cells in the presence of an anti-HLA-A*0201 (BB7.2) and an
anti-HLA-B/C (B1.23.2) mAb. CTL248 response was inhibited by the
BB7.2 but not by the B1.23.2 mAb (Fig. 3B).
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P248V9 stimulates CTL from healthy donors’ PBMC
We then assessed the capacity of p248V9 to generate CTL in vitro
from HLA-A*0201 healthy donors’ PBMC. CTL were induced by three weekly
in vitro stimulations with the p248V9. IFN--producing cells upon
peptide stimulation were isolated and cultured for 7–10 days before
testing. CTL were generated in seven of nine healthy donors tested.
Results from one donor are presented in Fig. 4. CTL responded to T2 cells loaded with
p248V9, p248G9, and p248D9 but not to T2 cells loaded with an
irrelevant peptide (HIVgag76) (Fig. 4A). They responded also to
HLA-A*0201+MAGE-A+ M44 and
M113 but not to the
HLA-A*0201+MAGE-A- Caco-2
cells (Fig. 4B). We verified the HLA-A*0201
restriction of M44 cell recognition in an anti-HLA-A*0201 Ab
blocking experiment. The response of CTL to M44 cells was blocked by
the HLA-A*0201-specific BB7.2 Ab but not by the HLA-B/C-specific
B1.23.2 Ab (Fig. 4C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), >80% of all tumors
express at least one MAGE-A Ag and could, therefore, be targeted by the
p248V9-induced CTL. Second, the use of p248V9 peptide for tumor
immunotherapy will lower the risk of tumor escape due to loss of
nominal Ag (31, 32). Considering that very often tumors
simultaneously express several MAGE-A Ags (33), to
escape p248V9-specific CTL response these tumors will have to lose all
the MAGE-A they express, which is less likely. Third, because MAGE-A is
not expressed in normal tissues except testis, the p248G9- and
p248D9-specific CTL repertoire recruited by the heteroclitic p248V9
epitope should not have been negatively selected and the high-avidity
CTL providing the strongest antitumor effect (34) should
not have been eliminated. Fourth, the absence of MAGE-A in normal
tissues limits the risk of autoimmunity that could be a great problem
for other universal TA, like the human TERT, which is expressed in
activated T and B cells and in the CD34+
hematopoietic progenitors (5). The three latter points
underline the advantage of targeting the p248G9 and p248D9 MAGE-A
epitope compared with targeting epitopes from other universal TA like
human TERT or p53. The homology between the different MAGE-A varies from 48% (MAGE-A10 vs MAGE-A12) to 95% (MAGE-A3 vs MAGE-A6). As a result, the alignment of all the MAGE-A sequences reveals only one completely identical nonameric peptide (p262). However, the complete homology is not necessary for peptides to be cross-recognized by CTL. The homology can be limited to the central segment of peptides (P3–P8/9) that contains residues contacting the TCR (35). Moreover, the presence of the HLA-A*0201-specific primary anchor motifs, which is a prerequisite for a high affinity, and immunogenicity were not necessary either. The p248G9 and p248D9 epitopes did not have an anchor motif at P9 in any of the MAGE-A sequences and exhibited a low affinity for HLA-A*0201. The substitution of the P9 residue with the anchor motif V gave an heteroclitic peptide with high affinity, strong immunogenicity, and the same antigenicity as the native peptides. The preservation of the native peptide antigenicity has been reported for several heteroclitic peptides, having substituted the anchor residues (36, 37, 38, 39). The rare exceptions of this rule must concern very nonconservative substitutions, as is the case for the p262L2, where an E is substituted by an L at P2. In conclusion, the search of cross-recognized HLA-A*0201-restricted peptides from sequences with relatively high homology can be exclusively based on their homology in the P3/C-terminal P segment. This strategy can effectively be applied to other multigenic families of TA like HER, BAGE, and GAGE to enrich the pool of universal tumor epitopes. The search for a common HER epitope is currently under way in our laboratory.
The p248G9/D9 is the unique common MAGE-A epitope presented by HLA-A*0201. The p262, which is identical to all MAGE-A sequences, is unlikely to be naturally processed because of the unfavorable residue (E) at the anchor P2. Moreover, the previously described MAGE-A3 epitope FLWGPRALV, which differs in the seven MAGE-A only at the C-terminal position, has recently been shown to be ineffectively processed by tumor cells (30). Concerning the numerous other HLA-A*0201-restricted MAGE-A epitopes, their sequences are either unique to the MAGE-A they belong to or they are shared by very few MAGE-A (40, 41, 42). One of the rare exceptions is the HLA-B*3701-restricted promiscuous epitope described by Tanzarella et al. (23). This epitope is shared by MAGE-A1, -A2, -A3, and -A6 but not by MAGE-A4, -A10, and -A12.
In conclusion, in this paper we describe an MAGE-A-derived HLA-A*0201-restricted heteroclitic peptide (p248V9) that induces CTL able to recognize each MAGE-A Ag individually and subsequently to kill MAGE-A-expressing tumor cells. This heteroclitic peptide can be used for a broad-spectrum tumor immunotherapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kostas Kosmatopoulos, Institut National de la Santé et de la Recherche Médicale, Unité 487, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94800, Villejuif, France. E-mail address: kostas{at}igr.fr
3 Abbreviations used in this paper: TA, tumor Ag; TERT, telomerase reverse transcriptase; RA, relative affinity; DC, dendritic cell; DC50, dissociation complex.
Received for publication February 15, 2002. Accepted for publication April 29, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 microglobulin (2m) HLA-A2.1 monochain transgenic H-2Db 2m double knockout mice. J. Exp. Med. 185:2043.This article has been cited by other articles:
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) and p248D9 (
) peptides. Lysis of RMAS/HHD cells loaded
with the irrelevant HIVgag76 peptide (10 µM) was 3%. The
E:T ratio was 10:1.
