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* Institut National de la Santé et de la Recherche Médicale Unité 487, Institut Gustave Roussy, Villejuif, France;
Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115; and
Unité d’Immunité Cellulaire Antivirale, Departement 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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However, like the majority of TuAgs, HER-2/neu and hTERT are also expressed on normal tissues, including the thymus (20). This raises the problem of the tolerance of their specific CTL repertoire, and consequently their inability to trigger a strong and efficient antitumor response, as it has been demonstrated for other TuAgs (21, 22, 23, 24, 25). Breaking tolerance to TuAgs is, indeed, one of the major goals of tumor vaccination (26). Tolerance to self Ags involves dominant epitopes and much lesser cryptic epitopes (26, 27, 28). Hence, we can hypothesize that the recruitment of the CTL repertoire specific for cryptic determinants should circumvent immunologic tolerance. Results obtained in the murine p53 and more recently in the transgenic adenocarcinoma mouse prostate mouse model fit in favor of this hypothesis (21, 24, 25). Cryptic tumor epitopes might, therefore, be considered good candidates for tumor immunotherapy provided that they are efficiently presented by tumor cells.
In this study, we used the heteroclitic peptide approach (29) to identify six low affinity cryptic HER-2/neu and hTERT epitopes presented by HLA-A*0201. These epitopes, in their P1Y heteroclitic form, stimulated CTL that specifically lysed HER-2/neu- or hTERT-expressing tumor cells of various histological origins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HHD mice have been previously described (30). They
are 
2-microglobulin
(2m)-/-,
Db-/- and express an HLA-A*0201 monochain
composed of a chimeric heavy chain (
1 and 2 domains of HLA-A*0201
and the 3 and intracellular domains of Db)
linked by its N terminus to the C terminus of the human
2m by a 15-aa peptidic arm.
Cell lines
Murine RMA/HHD and RMAS/HHD cells were obtained by transfection of RMA and RMAS cells with the HHD construct (30). Human tumor cells used as targets for HER-2/neu-specific CTL were the HLA-A*0201+ HER-2/neu+ MCF-7 (breast cancer), HCT-116 (colon cancer), PUB/N (lung cancer) kindly provided by M. Chouaib (Institut Gustave Roussy, Villejuif, France), LAW (renal cell carcinoma) kindly provided by F. Triebel (Institut Gustave Roussy), and the HLA-A*0201+ HER-2/neu- ZR75.1 (breast cancer) and SUP/M2 (anaplastic lymphoma) kindly provided by C. Gambacorti (Instituto di Tumori, Milan, Italy). The level of HER-2/neu expression by these lines has been reported previously (18). Human tumor cells used as targets for hTERT-specific CTL were the HLA-A*0201+ hTERT+ U266 (myeloma), 36 M (ovarian carcinoma), KO29 (melanoma) and SKW6.4 (EBV-transformed lymphoblasts), and the HLA-A*0201+ hTERT- U20S (osteosarcoma) and the HLA-A*0201- hTERT+ HSS (myeloma) and Calu-1 (lung cancer). The hTERT expression by these lines has been described previously (10). Unless indicated otherwise, the human cell lines were obtained from American Type Culture Collection (Manassas, VA) or as previously reported (10). The TAP1/2 mutant T2 cells and the NK-sensitive K562 cells were also used. All cells were grown in RPMI 1640 medium supplemented with 10% FCS.
Peptides
Peptides were synthesized by Synt:em S.A. (Nimes, France).
Viruses
T39 (vac-neu) HER-2/neu-expressing recombinant and TBC-Wy wild-type (vac-wt) vaccinia viruses were kindly provided by G. Mazzara (Therion Biologics, Cambridge, MA).
Generation of CTL in HHD mice
HHD mice were injected s.c. at the base of the tail with 100 µg peptide emulsified in IFA in the presence of 140 µg of the I-Ab-restricted hepatitis B virus core-derived Th epitope (128–140; sequence TPPAYRPPNAPIL). After 11 days, spleen cells (5 x 107 cells in 10 ml) were stimulated in vitro with 10 µM peptide. On day 6 of culture, the bulk responder populations were tested for specific cytotoxicity.
Generation of CTL from human PBMC
Dendritic cells were prepared by cultivating plastic adherent PBMC in the presence of 700 U/ml GM-CSF (R&D Systems, Minneapolis, MN) and 100 U/ml IL-4 (R&D Systems) for 7 days. Immunofluorescence staining of this dendritic cell-enriched population showed that >80% were CD80/B7-1+, CD86/B7-2+, and HLA-DR+ cells. Activated B cells were prepared by culture of Percoll-isolated B cells over a monolayer of CD40 ligand-expressing fibroblasts in B cell medium supplemented with 2.8 µg/ml cyclosporin A (31). After 14 days, greater than 85% of the cells were CD19+, CD80+, CD86+, HLA-DR+.
Dendritic cells were harvested after 7 days, pulsed overnight with peptide (10 µM) in the presence of soluble CD40 ligand to induce their maturation, irradiated at 36 Gy, and added to autologous T cells at a T:dendritic cell ratio of 20:1 in culture medium (RPMI 1640 supplemented with 10% human AB serum, 10 nM L-glutamine, and gentamicin). Three days later, primed T cells were harvested, isolated by Ficoll centrifugation, and replated in culture medium supplemented with 20 IU/ml IL-2. On day 7, and weekly thereafter, T cells were restimulated with peptide-pulsed irradiated dendritic cells (first restimulation) or activated B cells (second, third, and fourth restimulations). Cytotoxicity was tested 5–7 days after the fourth restimulation.
Cytotoxic assay
Murine RMA, RMAS/HHD, and RMA/HHD cells, and human tumor cells were used as targets. For virus infection, RMA and RMA/HHD cells were incubated with vac-wt or vac-neu recombinant viruses (10 PFU/cell) for 16 h. Targets were labeled with 100 µCi 51Cr for 90 min, washed four times, and plated in 96-well round-bottom plates (104 cells/well in 100 µl RPMI 1640 + 3% FCS). RMAS/HHD and T2 cells were pulsed with 1 µM peptides at 37°C for 90 min. Variable numbers of effector cells (in 100 µl) were then added and incubated at 37°C for 4 h. After incubation, 100 µl supernatant was collected, and radioactivity was measured in a gamma counter. Percentage of specific lysis was determined as: percentage of specific lysis = (experimental release - spontaneous release)/(maximal release - spontaneous release) x 100. Spontaneous release was always <20% of maximal release induced by 3 N HCl.
Measurement of peptide relative affinity to HLA-A*0201
T2 cells (3 x 105 cells/ml) were
incubated with various concentrations of peptides ranging from 100 to
0.1 µM in serum-free RPMI 1640 medium supplemented with 100 ng/ml
human 2m at 37°C for 16 h. Cells were
then washed twice and stained with the BB7.2 mAb, followed by
FITC-conjugated goat anti-mouse Ig 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 the staining obtained with
100 µM of the reference peptide HIVpol589
(IVGAETFYV). The relative affinity (RA) is determined as: 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. The lower the RA value, the
stronger is the peptide binding to HLA-A*0201. The definitive RA value
for each peptide was determined from at least three independent
experiments. In all experiments, 20% of HLA-A*0201 expression using
the reference peptide was obtained at 1–3 µM.
Assessment of peptide/HLA-A*0201 complex stability
T2 cells (106/ml) were incubated overnight
with 100 µM of each peptide in serum-free RPMI 1640 medium
supplemented with 100 ng/ml 2m at 37°C.
Cells were then washed four times to remove free peptides, incubated
with brefeldin A (10 µg/ml) for 1 h to block cell surface
expression of newly synthesized HLA-A*0201 molecules, washed, and
incubated at 37°C for 0, 2, 4, 6, or 8 h. Subsequently, cells
were stained with the BB7.2 mAb, followed by FITC-conjugated goat
anti-mouse Ig mAb. For each time point, peptide-induced HLA-A*0201
expression was calculated as: mean fluorescence of peptide-preincubated
T2 cells - mean fluorescence of T2 cells treated in similar
conditions in the absence of peptide. DC50
(dissociation complex, DC) was defined as the time required for the
loss of 50% of the HLA-A*0201/peptide complexes stabilized at
t = 0. The definitive DC50 value
for each peptide was determined from at least three independent
experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four previously described HER-2/neu peptides with low
affinity for HLA-A*0201 (neu391,
neu402,
neu466, and
neu650) (18, 29) were used in
this study. Two hTERT peptides predicted by the Bioinformatics and
Molecular Analysis Section algorithm (32) to have
low HLA-binding affinity were also included. Their capacity to bind and
stabilize HLA-A*0201 molecules was assessed using the T2-binding assay.
As shown in Table I
, all these peptides
form unstable complexes with HLA*0201 (DC50 <
2 h). Analysis of the relative binding affinity showed that
neu391,
neu402, hTERT572,
and hTERT988 were weak binders to HLA-A*0201
(RA > 5), whereas the peptides
neu466 and
neu650 exhibited a strong binding affinity
(RA < 5).
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The generation of specific CTL is a prerequisite for the identification of CTL epitopes. However, low affinity peptides are generally nonimmunogenic in either humans or HLA-A*0201 transgenic mouse models (33).
To generate CTL against low affinity peptides, we used the P1Y
heteroclitic peptide approach we described previously
(29). It consists of substituting the amino acid at
position 1 with a tyrosine. Indeed, P1Y variants of
neu391,
neu402,
neu466,
neu650, hTERT572,
and hTERT988 exhibited a high affinity (RA
< 5 and DC50 
4 h), fulfilling the
criteria of peptides predicted to be immunogenic (Table I
). We then
used the heteroclitic P1Y variants of the low affinity
HER-2/neu and hTERT peptides to induce CTL from the PBMC of
healthy donors. Four to five donors were tested for each peptide. The
generation of CTL was evaluated on cognate peptide-pulsed T2 cell
targets after the fourth in vitro restimulation. Peptide-specific CTL
were generated from PBMC stimulated with all these heteroclitic
peptides. CTL killed T2 cells pulsed with the cognate peptide, but not
T2 cells pulsed with an irrelevant peptide
(HIVgag76 or MAGE3271)
(Fig. 1).
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To evaluate whether these low affinity
HER-2/neu and hTERT peptides are naturally processed and
presented by tumor cells, peptide-specific CTL were tested for
their ability to lyse HER-2/neu- or hTERT-expressing
HLA-A*0201+ tumor cells. For the
HER-2/neu-derived peptides, CTL generated were tested as
effector cells in cytotoxic assays using MCF-7
(HLA-A*0201+,
HER-2/neu+), ZR75.1
(HLA-A*0201+,
HER-2/neu-), and K562 (NK-sensitive) cells
as targets. As shown in Fig. 2A for a representative case,
CTL specific for neu391Y,
neu402Y,
neu466Y, and
neu650Y lysed the MCF-7 cells, but not the
ZR75.1 nor the K562 cells. Similarly, for the hTERT-derived peptides,
CTL generated by stimulation with hTERT572Y and
hTERT988Y lysed the SKW6.4 cells
(HLA-A*0201+, hTERT+), but
failed to lyse the U20S (HLA-A*0201+
hTERT-) and the K562 cells (Fig. 2B).
We further demonstrated that the MCF-7 lysis by
HER-2/neu-reactive CTL involves the specific recognition of
the HLA-A*0201/neu peptide complexes in cold target
inhibition experiments. MCF-7 killing was inhibited by T2 cells loaded
with the corresponding native HER-2/neu peptide, but not by
T2 cells loaded with the irrelevant HIVgag76
peptide (Fig. 3). Inhibition was
dependent on the cold-hot target ratio. These studies demonstrate that
the low affinity, cryptic epitopes neu391,
neu402,
neu466,
neu650, hTERT572,
and hTERT988 are naturally processed and
presented by human tumor cells expressing the relevant TuAg.
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HER-2/neu and hTERT are appealing TuAgs because of their wide expression on multiple cancers. To determine the potential of low affinity, cryptic HER-2/neu and hTERT epitopes for broad spectrum immunotherapy, we tested the capacity of CTL induced by the PY1 variants of these epitopes to lyse malignant cells derived from tumors of different histological origins.
For the HER-2/neu epitopes, CTL specific for the four
cryptic epitopes were used as effectors, as well as CTL generated using
the dominant peptide neu369
(12). The CTL were tested for their capacity to lyse the
HLA-A*0201+
HER-2/neu+ MCF-7, HCT116, LAW, and PUB/N
cells. These cell lines express variable amounts of
HER-2/neu (18). The
HLA-A*0201+
HER-2/neu- cell lines ZR75.1 and SUP/M2
were used as controls. As shown in Fig. 4, CTL generated using the PY1 variants
of the cryptic epitopes lysed all
HER-2/neu+ tumor cells (black symbols)
irrespective of their tissue of origin, including those with low
expression of HER-2/neu (LAW) (18).
Importantly, these CTL showed a cytotoxic efficacy equivalent to that
mediated by neu369-specific CTL (Fig. 4, donors 1 and 2), thus demonstrating that presentation of low affinity
epitopes does not require high expression of the endogenous
Ag.
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). Four
HLA-A*0201+ hTERT+ tumor
cells (U266, 36 M, KO29, and SKW6.4) were used as targets for CTL
specific for the two hTERT cryptic epitopes or the dominant peptide
hTERT540 (10, 11). The
HLA-A*0201+ hTERT- cell
line U20S and two HLA-A*0201-
hTERT+ cells (HSS and Calu-1) were used as
negative controls. CTL lysed all the HLA-A*0201+
hTERT+ cells (filled symbols) irrespective of
their histological origin. In contrast, they did not lyse targets that
exclusively express hTERT or HLA-A*0201 (open symbols). CTL specific
for the cryptic epitopes and those specific for the dominant epitope
hTERT540 recognized hTERT+
tumor cells with equivalent efficacy (Fig. 5, donors 1 and 2).
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P1Y heteroclitic low affinity HER-2/neu and hTERT peptides induce in vivo tumor-specific CTL
The capacity of the cryptic HER-2/neu and hTERT
epitopes to induce in vivo tumor-specific CTL was assessed using the
HLA-A*0201-transgenic HHD mouse model. Mice were vaccinated with the
native peptides and their P1Y heteroclitic variants. After 11 days,
their spleens were removed and the splenic cells were restimulated in
vitro with the corresponding native peptide. Results in Fig. 6 show that native peptide-vaccinated
mice did not develop CTL, confirming previous results showing that low
affinity peptides are not immunogenic in HHD mice (29). In
contrast, CTL were generated in mice vaccinated with
HER-2/neu (Fig. 6A) and hTERT (Fig. 6B) heteroclitic peptides. These CTL lysed RMAS/HHD targets
pulsed with the cognate and the P1Y heteroclitic, but not an irrelevant
peptide.
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A), thus demonstrating the
HLA-A*0201 restriction of the endogenous HER-2/neu
recognition. To evaluate the endogenous TERT recognition by
TERT-specific CTL, we used the HLA-A*0201+
mTERT+ RMA/HHD cells as targets, since the
hTERT572 and hTERT988
epitopes are identical in mouse and human TERT sequences. CTL induced
in mice vaccinated with hTERT572Y and
hTERT988Y lysed RMA/HHD cells, but failed to lyse
TERT+ HLA-A*0201-negative RMA cells (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This observation, if extended to low affinity epitopes of other widely expressed TuAgs, is of great importance for a broad spectrum tumor immunotherapy. In fact, the majority of TuAgs, including HER-2/neu and hTERT, corresponds to nonmutated self proteins overexpressed in tumors, and the CTL repertoire against their high affinity immunodominant epitopes may be tolerized. Such tolerance mainly concerns high avidity CTL. This has been demonstrated in HLA-A*0201/Kb mice for HLA-A*0201-restricted p53-specific and tyrosinase-specific CTL (21, 22, 23), and more recently in HHD mice for the HLA-A*0201-restricted CTL specific for the mTERT (D.-A. Gross, unpublished observations). Low affinity cryptic epitopes are weakly or not at all involved in tolerance induction. For instance, B6 mice possess a fully functional CTL repertoire against low affinity Db-restricted mgp100 and Kb-restricted gp75 epitopes (25, 34). Moreover, in the transgenic adenocarcinoma mouse prostate model, specific CTL can be generated against a subdominant/cryptic SV40T epitope, but not against a dominant SV40T epitope (24). Given that the efficacy of tumor immunotherapy depends on the avidity of recruited CTL (35, 36), these results point out the potential of using low affinity tumor epitopes, provided that they are able to mobilize their specific CTL repertoire and that they are presented by tumor cells efficiently enough to be recognized by CTL.
An effective strategy to mobilize CTL targeting low affinity epitopes is the use of heteroclitic variants that have a higher affinity and more stable binding to HLA molecules (25, 34, 37). We have previously described two heteroclitic variant approaches that enabled the generation of CTL directed to low affinity Db-restricted and HLA-A*0201-restricted epitopes (29, 37). Heteroclitic variants of low affinity Db epitopes from flu nucleoprotein induced a potent protective immunity against lethal challenge of mice with flu virus (37). In this study, we demonstrate in HHD mice that this strategy can be used to mobilize antitumor CTL specific for cryptic HER-2/neu and hTERT epitopes. We are presently evaluating the potential of P1Y heteroclitic peptides as tumor vaccines in HHD mouse tumor models.
The low affinity peptides we described are presented by tumor cells at a level sufficient to be targets of antitumor CTL, an observation that raises several issues. First, low affinity HER-2/neu and hTERT epitopes are presented by tumor cells of different origins; they can, therefore, be used alone or in association with other previously described HER-2/neu and hTERT epitopes for a broad spectrum immunotherapy (38, 39, 40). The vaccine potential of a polyepitopic vector containing the four HER-2/neu P1Y variants along with intermediate or high affinity HER-2/neu epitopes described by us and by others is being currently studied in HHD mice. Second, low affinity epitopes, even those with the lowest affinity, such as neu391 and neu402, are efficiently presented by tumor cells expressing low amounts of HER-2/neu such as LAW. The level of HER-2/neu expression is not, therefore, a barrier to the use of low affinity epitopes for tumor immunotherapy. This is most likely explained by the fact that CTL effectors require a small number of peptide/HLA complexes on the target surface to be activated (41). Third, six of six low affinity peptides we have studied in this work are naturally processed by tumor cells. Although the small number of peptides does not permit any conclusion about the diversity of the epitopic repertoire of an Ag, these results suggest that the epitopic repertoire contains a very large number of cryptic epitopes. This is strengthened by our previous observation that three of five flu nucleoprotein-derived peptides with low or intermediate Db affinity tested are processed by flu-infected murine cells (42) and by recent results showing that low HLA-A*0201 affinity peptides from MAGE are presented by tumor cells.6 It is noteworthy that all these peptides had the appropriate HLA primary anchor motifs (P5N and P9 M for Db molecule; P2L and P9/10V/L for HLA-A*0201 molecule). This raises the question of whether the presence of anchor motifs is by itself sufficient to ensure a natural presentation independently of the overall HLA affinity. Investigation of a large panel of randomly selected peptides having the anchor motifs or not and exhibiting variable affinities is necessary before drawing any definitive conclusion.
In summary, we provide results demonstrating that low affinity tumor epitopes for HER-2/neu and hTERT are efficiently presented by tumor cells and can, therefore, be used for tumor immunotherapy.
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Acknowledgments |
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Footnotes |
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2 A.S. and D.-A.G. equally contributed to this work.
3 R.H.V. and A.A.C. equally contributed to this work.
4 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
5 Abbreviations used in this paper: TuAg, tumor Ag; 2m, 2-microglobulin; DC, dissociation complex; MAGE, melanoma Ag; RA, relative affinity; wt, wild type; hTERT, human telomerase reverse transcriptase; mTERT, murine telomerase reverse transcriptase.
6 S. Graff-Dubois et al. Generation of CTL recognizing an HLA-A*0201 restricted epitope shared by MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 tumor antigens: implication in a broad-spectrum tumor immunotherapy. Submitted for publication.
Received for publication September 27, 2001. Accepted for publication March 21, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-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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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), the
native cognate (•), and the P1Y variant (
) peptides.
); and B, U20S (HLA-A*0201+
hTERT-) (
), SKW6.4 (HLA-A*0201+
hTERT+) (
), and K562 (
) targets.
), and RMA/HHD (
)
targets at a 30:1 E:T ratio.