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Improved Immunogenicity of an
Immunodominant Epitope of the Her-2/neu
Protooncogene by Alterations of MHC
Contact Residues
Simona Vertuani, Alessandro Sette, John Sidney, Scott
Southwood, John Fikes, Elissa Keogh, Jan Alvar
Lindencrona, Glenn Ishioka, Jelena Levitskaya and Rolf
Kiessling
J Immunol 2004; 172:3501-3508; ;
doi: 10.4049/jimmunol.172.6.3501
http://www.jimmunol.org/content/172/6/3501
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References
The Journal of Immunology
Improved Immunogenicity of an Immunodominant Epitope of
the Her-2/neu Protooncogene by Alterations of MHC Contact
Residues1
Simona Vertuani,* Alessandro Sette,‡ John Sidney,‡ Scott Southwood,† John Fikes,†
Elissa Keogh,† Jan Alvar Lindencrona,* Glenn Ishioka,† Jelena Levitskaya,* and
Rolf Kiessling2*
D
uring the last decade, a large number of tumor-associated Ags (TAA),3 which can be recognized by T lymphocytes and therefore constitute tumor vaccine candidates, have been identified (1). Numerous immunodominant
epitopes recognized by CTL or Th cells have been defined from
human melanomas, and several of them are currently being evaluated in clinical trials. Promising results have been reported for
some of them (2– 4). Much less is known, however, about the TAA
expressed in carcinomas. Among the few TAA defined to date,
HER-2/neu (hereafter termed HER-2) is a prominent example.
HER-2 is a nonmutated, overexpressed oncogene that encodes a
185-kDa transmembrane, receptor-like glycoprotein with tyrosine
kinase activity and is expressed in a broad spectrum of human
carcinomas. Several properties render HER-2 a promising candidate for T cell-based tumor vaccination strategies (5). Of particular
importance, HER-2 overexpression appears to contribute not only
*Cancer Center Karolinska, Karolinska Hospital, Stockholm, Sweden; and †Epimmune and ‡La Jolla Institute for Allergy and Immunology, San Diego, CA 92121
Received for publication September 29, 2003. Accepted for publication January
13, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Swedish Cancer Society, the Cancer
Society of Stockholm, the King Gustaf V Jubilee Fund, and the European Community; a gene therapy grant (to R.K.) from the Swedish Research Council; the Wallenberg Foundation; the Swedish Foundation for Strategic Research (Stockholm,
Sweden); and National Institutes of Health Contract AI95362.
2
Address correspondence and reprint requests to Dr. Rolf Kiessling, Cancer Center
Karolinska, R8:01, Karolinska Hospital, S-17176 Stockholm, Sweden. E-mail address: [email protected]
3
Abbreviations used in this paper: TAA, tumor-associated Ag; PSA, prostate-specific
Ag; SM, standard medium.
Copyright © 2004 by The American Association of Immunologists, Inc.
to disease initiation and progression, but also to the transformation
of human mammary epithelium. Therefore, the risk that HER-2overexpressing tumors escape immune responses by Ag loss is
relatively low.
HER-2 is a typical example of a self tumor Ag, expressed not
only by tumors, but also at low levels in a variety of healthy fetal
and adult tissues derived from all three germ layers (6). Therefore,
it is expected that this tumor Ag may be weakly immunogenic, and
that most T cells specific for this Ag are clonally eliminated or
subjected to tolerance in the periphery. Yet accounts from experimental models and clinical trials confirm that HER-2 can be immunogenic and generate Ab, CTL, and Th cell responses in subjects bearing HER-2-overexpressing tumors (5). Results from
murine neu transgenic experimental models further illustrate that
HER-2-based vaccines can induce tumor protection (7).
Because of the possible repertoire limitations related to T cell
tolerance alluded to above, we and others are interested in enhancing the immunogenicity of the HER-2 molecule and achieving
more efficient tumor-specific CTL responses. One approach entails
agonist analogues of HER-2 epitopes associated with the ability to
induce higher levels of responses than wild-type peptides. These
epitope analogues may ultimately be more efficient in inducing
anti-tumor responses in patients with HER-2-expressing tumors.
Two general approaches have been taken to design agonist
epitopes from tumor Ags. One approach entails modification of
HLA anchor residues, resulting in higher HLA binding. This approach has been applied with success for several HLA-A2 peptides
derived from melanoma Ags and for HER-2-derived epitopes (8 –
12). Alternatively, the replacement of residues involved in the
TCR contact may also result in an increased response by T cells
(13–16). In this study we have applied the first approach and tested
a series of variants with enhanced HLA-A2 binding derived from
0022-1767/04/$02.00
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The HER-2/neu (HER-2) oncogene is expressed in normal epithelial surfaces at low levels and overexpressed in several types of
tumors. The low immunogenicity against this self tumor Ag can be improved by developing epitopes with amino acid replacements
in their sequences. In this study, three HER-2/neu.369 (HER-2.369) analogue peptides, produced by modifying both anchor
positions by introducing L, V, or T at position 2 and V at the C terminus, were analyzed for their capacity to induce CTLs in vitro
from human PBMC and in vivo in HLA-A2.1/Kb transgenic mice. One of the analogues (HER-2.369 V2V9) sensitized target cells
for HER-2-specific recognition by human CTLs and induced specific CTLs in vitro at 100-fold lower concentrations than the
HER-2.369 wild-type epitope. These CTLs were also able to recognize the wild-type epitope and HER-2-expressing tumors in an
MHC-restricted manner. Furthermore, a 100-fold lower amount of the HER-2.369 V2V9 analogue compared with the wild-type
epitope was required to induce CTLs in HLA-A2.1/Kb transgenic mice. However, the V2V9 analogue demonstrated only marginally better binding to the MHC class I A2 allele compared with wild type. To establish thermodynamic parameters, we
developed radiolabeled F3*Y analogues from both the HER-2.369 epitope and the V2V9 analogue. Our results indicate that the
high biological activity of the HER-2.369 V2V9 epitope is associated with a slower dissociation kinetic profile, resulting in an
epitope with greater HLA-A2 stability. The Journal of Immunology, 2004, 172: 3501–3508.
3502
IMPROVED Her2/neu IMMUNOGENICITY AND MHC-PEPTIDE INTERACTION
the immunodominant HER-2.369 –377 epitope (17). We found that
one of the analogues (HER-2.369 V2V9) can induce wild-type
specific CTLs in vitro in HLA-A2-positive human donors and in
vivo in HLA A2.1/Kb transgenic mice (HLA-A2.1/Kb tg) at markedly lower concentrations than the HER-2.369 wild-type epitope.
This analogue bound HLA-A2 only marginally better than the
wild-type peptide, but resulted in HLA complexes associated with
higher stability. We speculate that the higher biological activity of
the HER-2.369 V2V9 epitope could result from a higher HLA-A2
stability and/or subtle conformation alterations of the peptide/HLA
complex.
Materials and Methods
Cell lines
Peptides and HLA-A2 binding assays
Peptides were synthesized by a solid phase method using a multiple peptide
synthesizer and were analyzed by reverse phase HPLC as previously described (18, 19).
HLA-A2.1 molecules were purified as previously described (20).
Briefly, cell lysates from JY cells were first depleted of HLA-B and -C
molecules by repeated passage over a B1.23.2 (anti-HLA-B and -C) column. Remaining HLA-A molecules were captured on a W6/32 (antiHLA-A, B, C) column. The protein content and purity of class I preparations were monitored by SDS-PAGE analysis.
Quantitative assays to measure the binding of peptides to soluble class
I molecules are based on the inhibition of binding of a radiolabeled standard peptide. These assays were performed as previously described (21).
Briefly, 1–10 nM radiolabeled peptide was coincubated at room temperature with 1 ␮M to 1 nM purified MHC in the presence of 1 ␮M human
␤2-microglubulin (The Scripps Laboratories, San Diego, CA) and a mixture of protease inhibitors. After a 2-day incubation, the percentage of
MHC bound radioactivity was determined by size exclusion gel filtration
chromatography using a TSK 2000 column. Alternatively, the percentage
of MHC-bound radioactivity was determined by capturing MHC/peptide
complexes on W6/32 Ab-coated Optiplates (Packard Instrument, Meriden,
CT), and determining bound counts per minute using the TopCount (Packard Instrument) microscintillation counter.
The radiolabeled standard peptide used for HLA-A2.1 assays was an
F6⬎Y analogue of the HBV core 18 –27 epitope (sequence FLPSDYF
PSV). The average IC50 of this peptide for HLA-A2.1 was 5.0 nM. In the
case of competitive assays, the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled peptide was calculated. Peptides
were initially tested at one or two high doses. The IC50 of peptides yielding
positive inhibition were then determined in subsequent experiments, in
which two to six further dilutions were tested. Under the conditions used,
where [label] ⬍ [MHC] and IC50 ⱖ [MHC], the measured IC50 values are
reasonable approximations of the true Kd values. Each competitor peptide
was tested in two to four independent experiments. As a positive control,
the unlabeled version of the radiolabeled probe was also tested in each
experiment.
To allow the performance of kinetic of association (Ka) experiments
using radiolabeled peptides, analogues representing F371 to Y substitutions
of Her2/neu peptides were designed, and peptide-MHC assays were set-up
as described above, and then incubated for various times ranging between
0 and 60 min at either room temperature or 37°C. Further association
between HLA-A2.1 and the radiolabeled ligand was stopped by dilution,
and the fraction of peptide bound was determined as described above by
size exclusion chromatography. The initial linear part of the binding iso-
HLA typing
mAb recognizing HLA-A2 was derived from culture supernatants of
American Type Culture Collection MA2.1 hybridomas. PBLs from healthy
donors were incubated with either primary mAb or murine IgG of appropriate class as a control for 30 min at 4°C and then stained with secondary
FITC-conjugated rabbit anti-mouse Ig (DAKO, Glostrup, Denmark). Data
acquisition was performed on FACScan flow cytometer (BD Biosciences,
Mountain View, CA). For data shown in Fig. 3, donors were class I typed
serologically (One Lambda, Canoga Park, CA), and HLA-A2-positive individuals were A2.1 subtyped by standard PCR methods.
Peptide- and tumor-specific CTL lines
For data shown in Fig. 2, PBMCs were isolated by centrifugation of venous
blood from HLA-A2.1-positive healthy donors on Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden). PBMC were resuspended in
X-VIVO (BioWhittaker, Walkersville, MD) medium supplemented with
1% autologous plasma (hereafter termed standard medium (SM)). Twenty
million cells were incubated in six-well plates for 2 h at 37°C (20 ⫻ 106
cells/3 ml/well), and the nonadherent cells were removed by gentle pipetting. The nonadherent cells (responder cells) were cryopreserved in FCS
supplemented by 10% DMSO (Sigma-Aldrich, St. Louis, MO).
The adherent cells (stimulator cells) were cultured in SM containing 800
U/ml GM-CSF (Schering-Plough, Stockholm, Sweden) and 500 U/ml IL-4
(Schering-Plough) added days 2 and 5 after the initiation of the cultures.
TNF-␣ (Cetus, Emeryville, CA; 50 ng/ml) was added during the last 24 h
of culture. Stimulator cells were harvested and resuspended in SM containing 20 ␮g/ml peptide and 3 ␮g/ml ␤2-microglobulin (Sigma-Aldrich).
After culturing for 4 h at 37°C, stimulator cells were irradiated with 25 Gy,
cultured in SM containing 2000 U/ml IL-7 (R&D Systems, Abingdon, UK)
and 0.01 U/ml of IL12 (R&D Systems), and mixed with the thawed nonadherent responder cells at a responder-to-stimulator ratio of 10:1. After 7
days of in vitro culture, IL-7 (R&D Systems) was added to a final concentration of 2000 U/ml. Cultures were restimulated four times at weekly
intervals with autologous adherent PBMC irradiated with 60 Gy and cultured for 2 h in SM containing 20 ␮g/ml peptide and 3 ␮g/ml ␤2-microglobulin (Sigma-Aldrich). IL-2 (Chiron, Emeryville, CA) at a concentration of 20 U/ml was added at regular intervals 2 days after each
restimulation.
In Fig. 3, tumor-specific CTL lines from HLA-A2-positive healthy donors were generated with some variations on the protocol given above as
described previously (22).
Human IFN-␥ in situ capture ELISA
Ninety-six-well ELISA plates (Costar, Corning, NY) were coated overnight at 4°C with 50 ␮l/well mouse mAb specific for human IFN-␥ (clone
NIB42; BD PharMingen, San Diego, CA) at a concentration of 4 ␮g/ml in
coating buffer (100 mM NaHCO3, pH 8.2). Plates were then washed and
blocked for 2 h, after which the CTLs (100 ␮l/well) and targets (100 ␮l/
well) were added to each well, leaving empty wells for the standards (recombinant human IFN-␥; BD PharMingen) and blanks (medium only). The
plates were incubated for 48 h at 37°C with 5% CO2.
The cells were removed by washing the plates with PBS/0.05% Tween
20, and the amount of IFN-␥ that was secreted and captured by the bound
clone NIB42 mAb was measured using a sandwich format ELISA. A biotinylated mouse mAb specific for human (clone 4S.B3; BD PharMingen)
was used to detect the secreted IFN-␥. HRP-coupled strepavidin (Zymed
Laboratories, South San Francisco, CA), 3,3⬘,5,5⬘-tetramethylbenzidine,
and H2O2 (ImmunoPure TMB Substrate Kit; Pierce, Rockford, IL) were
used according to the manufacturer’s directions for color development. The
absorbance was read at 450 nm on a Multiskan RC ELISA plate reader
(Labsystems, Helsinki, Finland). A culture was considered positive if it
measured at least 50 pg of IFN-␥/well above background and was twice the
background level of expression.
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The TAP-defective HLA-A2.1 T2 cell line derived from the human T cell
leukemia/B cell LCL hybrid 174 was a gift from Dr. P. Cresswell (Yale
University School of Medicine, New Haven, CT). The .221A2.1 cell line
was produced by transferring the HLA-A2.1 gene into the HLA-A, -B,
-C-null mutant human B lymphoblastoid cell line .221. The colon adenocarcinoma cell lines SW403 and HT-29 were obtained from American
Type Culture Collection (Manassas, VA). Cells were grown in RPMI 1640
medium supplemented with antibiotics, sodium pyruvate, nonessential
amino acids, and 10% (v/v) heat-inactivated FCS. Jurkat A*0201/Kb, a
human T cell leukemia HLA A2.1-negative cell line stably transfected with
an HLA-A*0201/Kb chimeric gene, was provided by W. M. Kast (Loyola
University, Maywood, IL). Cells were maintained in RPMI 1640 (Life
Technologies, Grand Island, NY) supplemented with 10% heat-inactivated
FCS, 100 IU/ml penicillin, and 100 ␮g/ml streptomycin (complete
medium).
therm was used to estimate Ka values. To determine the kinetics of dissociation, large volumes of HLA-A2.1-peptide complexes were generated
using the basic assay protocol described above, and using excess amounts
of radiolabeled peptide and purified MHC. After a 2-day incubation, complexes were isolated using size exclusion chromatography and then incubated at 37°C for various times in the presence of a 1000-fold excess of
unlabeled peptide (to prevent reassociation of radiolabeled peptide). The
percentage of radiolabeled peptide bound at various time points was determined using size exclusion chromatography. Kd values were calculated
as the slope of the regression line of ln (% bound) vs time.
The Journal of Immunology
3503
Human IFN-␥ ELISPOT assay
Ninety-six-well filtration plates (MAIPS45 10; Millipore, Bedford, MA)
were coated overnight at 4°C with 75 ␮l/well of 2 ␮g/ml anti-human IFN-␥
mAb. Wells were then washed six times with PBS and blocked with 100
␮l/well RPMI 1640/1% human serum albumin for 2 h at 37°C. Stimulator
cells were washed once, resuspended in RPMI 1640 at 25 ⫻ 104 cells/ml,
plated at 80 ␮l/well, then mixed with 20 ␮l of 0.5 ␮g/ml peptide solution.
CTLs were added at the concentration of 2 ⫻ 104 cells/well in 100 ␮l.
After 4 h of incubation at 37°C, the plates were washed six times with
PBS/0.05% Tween 20. Wells were incubated overnight at 4°C with 75
␮l/well mouse biotinylated anti-human IFN-␥ mAb (7-B6-1-biotin;
Mabtech, Nacka, Sweden) at a concentration of 0.75 ␮g/ml in PBS, 1%
BSA, and 0.02% NaN3. After washing six times with PBS/0.05% Tween
20, 75 ␮l of streptavidin-alkaline phosphatase conjugate solution
(Mabtech, Nacka, Sweden) diluted 1/1000 in PBS (1% BSA/0.02% NaN3)
was added for 2 h at room temperature in the dark. After another washing
step with PBS, 75 ␮l/well substrate solution was added to each well for
5–10 min. Color development was stopped by washing under running tap
water. After drying at room temperature, IFN-␥-secreting T cells were
counted using the automated image analysis system ELISPOT reader.
HLA-A2.1/Kb mice (23) were coimmunized with varying doses of the
HER-2 wild-type or analogue peptide and 50 ␮g/mouse of the PADRE Th
epitope prepared in IFA. The dose of immunizing peptide ranged from 100
␮g/mouse to 10 ng/mouse, in log intervals. As a control, mice were injected with an IFA emulsion without peptide. Eleven days after immunization, splenocytes from injected animals were cultured with 1 ␮g/ml of
the immunizing peptide and irradiated LPS/dextran sulfate-activated
splenocytes (as additional APCs) to expand CTLs in vitro. Five days later,
peptide-specific IFN-␥ production by effector cells was measured using an
in situ capture ELISA or the ELISPOT assay. The assays were performed
according to standard protocols (24, 25).
Results
Fixed anchor analogues derived from the HER-2.369 epitope
can sensitize target cells at low peptide concentrations for
recognition by HER-2.369-specific CTLs
Three HER-2.369 analogue peptides, produced by modifying both
main anchor positions by introducing L, V, or T at position 2 and
V at the C terminus, were previously demonstrated to have improved HLA-A2.1 binding and/or supertype cross-reactive binding
compared with the wild-type epitope (22). We first analyzed to
what extent these HER-2/neu.369 (HER-2.369) variants would be
able to sensitize HLA-A2-expressing T2 cells for recognition by
HER-2.369 wild-type-specific CTLs, as measured in ELISPOT
IFN-␥ production assays. Both V2V9 (KVFGSLAFV) and L2V9
(KLFGSLAFV) analogues more efficiently sensitized T2 cells for
HER-2.369-specific CTL lysis compared with the T2V9 (KT
FGSLAFV) and wild-type (KIFGSLAFL) epitopes (Fig. 1). In the
case of the L2V9 analogue, this result could be explained by the
superior HLA-A2.1 binding compared with the T2V9 analogue or
the wild-type 369 epitope. The V2V9 analogue, however, bound
only marginally better to HLA-A2.1 compared with the wild-type
HER-2.369 epitope (23 vs 37 nM), but nevertheless was associated
with increased ability to induce HER-2.369-specific CTL
recognition.
The HER-2.369 V2V9 analogue can induce specific CTLs in
vitro at lower concentrations than the wild-type epitope
We next determined the capacity of the HER-2.369 peptide variants V2V9 and L2V9 to induce peptide-specific CTLs. A range of
concentrations was used for each peptide to determine the minimal
concentration required to elicit specific CD8⫹ T cell responses
from HLA-A2-positive healthy donors stimulated with autologous
DC cells pulsed with various concentrations of wild-type (Fig. 2A),
V2V9 analogue (Fig. 2B), or L2V9 analogue (Fig. 2C). Seven days
after the third stimulation, the CTL cultures were tested in IFN-␥
FIGURE 1. HER-2.369 analogues show improved capacity to sensitize
T2 cells for recognition by a HER-2.369 wild-type induced CTL clone. The
release of IFN-␥ was tested in an ELISPOT assay against T2 cells pulsed
with decreasing concentrations of the wild-type peptide (A) or HER-2.369
V2V9 (B), HER2.369 L2V9 (C), and HER-2.369 T2V9 (D) analogues. The
arrows indicate the concentration of peptide required for production of
IFN-␥ in 25% of effector CTLs.
release ELISPOT assays against T2 cells pulsed with 10 ␮g of
wild-type peptide, V2V9 analogue, L2V9 analogue, or irrelevant
peptide (Pol476 – 484, peptide from the reverse transcriptase of HIV1). The wild-type HER-2 epitope was found to generate specific
CTLs, but only at the concentration of 1 ␮g/ml, whereas no specific CTL responses were detected at the lower concentrations (10
or 1 ng/ml). In contrast, the variant peptide V2V9 was able to
efficiently elicit CTL responses to the wild-type 369 epitope when
cultures were stimulated with 1 ␮g/ml and 10 ng/ml peptide. Of
particular interest, these CTLs raised by the V2V9 analogue were
able to recognize not only the V2V9 analogue, but also the 369
wild-type epitope as well as the L2V9 analogue. The L2V9 variant
peptide, which had the highest HLA-A2.1 binding affinity, did not
generate CTL responses at any of the tested concentrations. Similar results were obtained with PBL from two other HLA-A2-positive donors (data not shown).
CTLs induced by the HER-2.369 V2V9 analogue can recognize
HER-2-expressing, HLA-A2.1-positive tumor cells
The HER-2.369 epitope was previously demonstrated to be naturally processed and expressed on HER-2-expressing, HLA-A2positive tumor targets (17, 26). The experiments described above
showed that the CTLs raised against the V2V9 peptide were also
able to recognize low concentrations of the wild-type HER-2 peptide, indicating that these CTLs may also recognize HER-2-expressing tumor targets. To test this hypothesis, microcultures of
CTLs were induced by either the HER-2.369 V2V9 peptide or the
wild-type control peptide (data not shown) from two healthy HLAA2.1-positive donors. Nineteen of 26 V2V9-specific cultures from
both donors were able to recognize the wild-type 369 epitope, as
measured in IFN-␥ release assays (data not shown). Of importance, five of the CTL cultures specific for the wild-type 369
epitope were able to recognize the SW403 HER-2-expressing,
HLA-A2.1-positive tumor targets, but not HER-2-expressing,
HLA-A2.1-negative HT29 tumor targets (Fig. 3). We therefore
conclude that CTLs raised by the V2V9 peptides are capable of
recognizing not only the wild-type HER-2.369 epitope, but also
HER-2-expressing tumor targets.
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Analysis of in vivo immunogenicity in HLA-A2.1/Kb
transgenic mice
3504
IMPROVED Her2/neu IMMUNOGENICITY AND MHC-PEPTIDE INTERACTION
the HLA-A2.1/Kb transgenic mice (Fig. 4). Specifically, the HER2.369 V2V9 analogue was able to induce a specific CTL response
in HLA-A2.1/Kb mice immunized with 10 ng of peptide or more.
In contrast, the wild-type HER-2.369 epitope was only able to do
so at an immunizing dose of 100 ng of peptide, not at any of the
lower doses. Comparable results were obtained with both the
IFN-␥ ELISA and the ELISPOT assay (Fig. 4). Taken together,
the results demonstrate that the higher immunogenicity in vitro of
the HER-2.369 V2V9 peptide is paralleled by a markedly increased immunogenicity in vivo of this analogue compared with
the wild-type HER-2.369 epitope. Next, we further analyzed the
mechanism behind the enhanced immunogenicity of the V2V9
peptide.
High stability of HER-2.369 V2V9 HLA-A2.1 complexes
FIGURE 2. The HER-2.369 V2V9 analogue induces more efficiently
CTLs that can recognize also wild-type (WT) peptide. The HER-2.369 wt
(A), V2V9 (B), and L2V9 (C) peptides were used at the indicated concentrations to stimulate PBMC from healthy HLA-A2-positive donors three
times at weekly intervals according to the CTL induction protocol in Materials and Methods. Cultures were tested in IFN-␥ ELISPOT assays using
T2 cells prepulsed with the WT, V2V9, L2V9, or HIV control peptides.
The HER-2.369 V2V9 analogue can induce specific CTLs in
vivo in HLA-A2.1/Kb transgenic mice at lower concentrations
than the HER-2.369 wild-type epitope
We next investigated weather the HER-2.369 V2V9 analogue
would be more efficient than the HER-2.369 wild-type epitope in
inducing immunity in vivo. HLA-A2.1/Kb transgenic mice were
immunized with varying doses of the HER-2 wild-type or analogue peptide coemulsified in IFA with the PADRE Th epitope.
Eleven days after immunization, splenocytes were cultured with
the immunizing peptide and APC for 5 days, and peptide-specific
IFN-␥ production was measured using an in situ capture ELISA or
the ELISPOT assay. The results demonstrated that the HER-2.369
V2V9 analogue possessed a markedly higher immunogenicity in
Both the association and the dissociation rates contribute to the
steady state binding equilibrium of peptide/MHC complexes and,
ultimately, are also expected to affect that of the TCR/peptide/
MHC complexes. To further characterize the mechanisms of the
increased immunogenicity of theV2V9 analogue peptide in comparison with its wild-type counterpart, the kinetics of association
and dissociation of these two epitopes were analyzed. The results
are summarized in Table I and Fig. 5. Previous data compared the
equilibrium binding capacity at room temperature of the wild-type
epitope and the V2V9 analogue peptide. As mentioned above, the
V2V9 analogue bound marginally better than the wild type (23 vs
37 nM), but still less strongly than the control peptide HBV core
18 –27 F6*Y (5 nM). This assay was used because room temperature binding affinity values have been found to correlate well with
immunogenicity (27) and because the empty MHC molecules rapidly unfold at 37°C, making 37°C equilibrium competition analysis somewhat impractical and inaccurate. In our experience, to accurately estimate 37°C binding constants, it is most effective to
separately radiolabel each peptide and to independently determine
on-off rate parameters. In the current set of experiments we first
developed two F3*Y analogues to allow for radiolabeling and direct establishment of thermodynamic parameters. As measured by
the equilibrium assay (see first column of Table I), the binding at
room temperature of the F3 peptides are similar (within 2-fold) to
the corresponding Y3 analogues (3.6 ⫻ 10⫺8 vs 1.9 ⫻ 10⫺8 M for
HER-2.369; 2.3 ⫻ 10⫺8 vs 3.3 ⫻ 10⫺8 M for the V2V9 analogue),
even though the V2F3V9 analogue is slightly less than that of the
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FIGURE 3. The Her2/neu.369 V2V9 analogue induces CTL cultures
that recognize wild-type peptide-pulsed and HER2/neu-positive tumor target cells. Individual CTL cultures induced with Her2/neu.369 V2V9 were
tested against .221A2.1 without peptide (䡺) or pulsed with Her2/neu.369
peptide (f) or against HT29 (A2⫺, Her2/neu⫹; 1) or SW403 (A2⫹, Her2/
neu⫹; _) tumor lines in an in situ capture ELISA. The colon cancer cells
were treated with 100 U/ml IFN-␥ for 48 h at 37°C before use as targets in
the in situ IFN-␥ assays.
The Journal of Immunology
3505
Discussion
FIGURE 4. The HER-2.369 V2V9 analogue can induce specific CTLs
in vivo in HLA-A2.1/Kb transgenic mice at lower concentrations than the
HER-2.369 wild-type epitope. HLA-A2.1/Kb mice were coimmunized
with varying doses of the HER-2 wild-type or analogue peptide and 50 ␮g
of the PADRE Th epitope prepared in IFA. As a control, mice were injected with an IFA emulsion without peptide (IFA). Mice were sacrificed
11 days after immunization, and splenocytes from the injected animals
were cultured with 1 ␮g/ml of the immunizing peptide to expand CTLs as
described in Materials and Methods. Five days later, peptide-specific
IFN-␥ production by the CTLs was measured using an in situ capture
ELISA (A) or the ELISPOT assay (B). The background IFN-␥ levels in
assay wells tested in the absence of peptide or in the presence of an irrelevant peptide ranged from 0 –59 pg/well and 0.1–3 spots/2 ⫻ 104 cells.
Values shown represent net CTL responses with background subtracted.
F3 analogue (3.3 vs 1.9 ⫻ 10⫺8 M). The on rates of these two
peptides (see second and third columns of Table I), measured after
125
I labeling, were essentially identical regardless of whether they
were measured at room temperature or 37°C (3.4 vs 3.7 ⫻ 102
s⫺1M⫺1 at 23°C and 2.6 vs 2.4 ⫻ 103 s⫺1M⫺1 at 37°C). These
values were substantially lower (⬃3-fold) than those obtained with
the control HBV core 18 –27 peptide.
The results presented in this study demonstrate that the HER-2.369
V2V9 variant is a more potent immunogen than the wild-type
epitope, and that the T cell responses activated by this analogue are
able to recognize the natural processed epitope on tumor target
cells. However, this increased immunogenicity is associated with
only a marginal increase in HLA class I binding of the variant
epitope. When the thermodynamic parameters for these peptides
were established, the result demonstrated that HER-2 peptides, in
particular the HER-2.369 V2V9 peptide, were associated with a
slow on, slow off kinetic profile, whereas the HBV core epitope
demonstrated a much lower stability. These data therefore indicate
that the strong immunogenicity associated with the HER-2 V2V9
peptide could be explained by a marked stability of binding of
these peptides to MHC class I molecules.
Previously, we reported the identification of 11 new epitopes
derived from HER-2, CEA, p53, and MAGE2/3, which included
the three epitopes analyzed in this study (22). These epitopes were
produced by modifying both the main anchor positions by introducing L, V, or T at position 2 and V at the C terminus, and were
demonstrated to have improved HLA-A2.1 binding and/or supertype cross-reactive binding compared with the wild-type epitope.
These substitutions were introduced based on the results reported
by Rupert et al. (28), who showed that these residues are associated with optimal HLA-A2 binding capacity. Although the main
finding in our earlier report was to confirm the importance of a
threshold of HLA binding as a necessary criterion for epitopes to
Table I. Summary of thermodynamic parametersa
Peptide
Kd at RTa
Ka at RTb
Ka at 37°Cb
Kd at 37°Cc
Kd at 37°Cd
HBV core 18 –27 F6⬎Y
HER-2.369
HER-2.369 F3⬎Y
HER-2.369 I2⬎V, L9⬎V
HER-2.369 I2⬎V, F3⬎Y, L9⬎V
5 ⫻ 10⫺9
3.6 ⫻ 10⫺8
1.9 ⫻ 10⫺8
2.3 ⫻ 10⫺8
3.3 ⫻ 10⫺8
1.0 ⫻ 103
ND
3.4 ⫻ 102
ND
3.7 ⫻ 102
7.1 ⫻ 103
ND
2.6 ⫻ 103
ND
2.4 ⫻ 103
4 ⫻ 10⫺5
ND
2 ⫻ 10⫺5
ND
7 ⫻ 10⫺6
5.6 ⫻ 10⫺9
ND
7.7 ⫻ 10⫺9
ND
2.9 ⫻ 10⫺9
a
Kd molar values from the inhibition assay.
M⫺1 s⫺1 values calculated from data in Fig. 5.
s values calculated from data in Fig. 5.
d
Kd at 37°C molar values calculated as Kd/Ka ratios.
b
c ⫺1
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Next, off-rate values at 37°C were determined (fourth column of
Table I). Surprisingly, the F3 and V2F3V9 epitopes were remarkably more stable than the HBV core 18 –27 F6*Y epitope (4 ⫻
10⫺5 s⫺1 for HBV core 18 –27 F6*Y, 2 ⫻ 10⫺5 s⫺1 for F3, and
7 ⫻ 10⫺6 for V2F3V9). Thus, when the 37°C Kd values were
calculated as the ratio of Kd/Ka (fifth column of Table I), both the
wild-type epitope and the analogue were associated with affinities
similar to that of the HBV core 18 –27 epitope (note that the Kd of
the HBV core 18 –27 did not change significantly with temperature, but that of the HER-2 epitopes did). The gain in affinity was
particularly remarkable for the V2F3V9 peptide, which increased
⬎10-fold (33 vs 2.9 nM), compared with the F3 analogue, which
increased only 2- to 3-fold. Thus, these HER-2-derived epitopes
achieved binding affinities similar to that of HBV core 18 –27, but
with a different thermodynamic profile (the HBV core is “fast on,
fast off,” whereas the HER-2 peptides are associated with a “slow
on, slow off” kinetic profile). It is reasonable to speculate that the
higher affinity for A2.1 at 37°C of these peptides (both F3 wild
type and V2F3V9 analogue) and their stability (especially the
V2F3V9) could be linked to their high biological activity.
3506
IMPROVED Her2/neu IMMUNOGENICITY AND MHC-PEPTIDE INTERACTION
be immunogenic, no side-by-side comparison with the corresponding wild-type epitope with regard to immunogenicity was conducted. In this study we have compared the HER-2 analogues and
the wild-type epitope using both human CTL induction protocols
and the mouse HLA-A2.1/Kb transgenic model.
Several reports have demonstrated that amino acid modifications of CTL epitopes from tumor Ags associated with increased
affinity of peptide/MHC binding can enhance their immunogenicity (8 –11, 17, 29, 30). Terasawa et al. (30) designed a human
agonist CTL epitope of the prostate-specific Ag (PSA). Compared
with the corresponding native PSA epitope, this agonist epitope
demonstrated enhanced binding to the HLA-A2.1 allele as well as
enhanced stability of the peptide-MHC complex. In line with our
data, this PSA agonist was shown to induce higher levels of T cell
activation compared with the native PSA peptide in HLA-A2.1/Kb
mice. Also, the gp100.209(2M) analogue, which is an MHC anchor residue modification of the antigenic peptide derived from the
gp100209 –217 melanoma Ag, was demonstrated to be 100-fold
more potent in activating naive T cells than its wild-type peptide
(31). This approach has been applied to HER-2-derived epitopes,
in which modifications of the p654 – 662 HER-2-derived epitope,
which has a low MHC class I binding affinity, was found to improve induction of peptide- and tumor-specific CTLs (32). Baratin
et al. (29) demonstrated that amino acid substitutions of a p53-
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FIGURE 5. Kinetics of peptide-HLA-A2.1 binding. Determination of
the kinetics of peptide binding to HLA-A2.1 molecules was performed as
described in Materials and Methods for HBV core 18 –27. f, F6⬎Y; F,
Her2/neu.369 F3⬎Y; E, Her2/neu.369 V2V9 F3⬎Y. Dissociation (A) was
measured at 37°C, and Kd values were calculated as the slope of the regression line of ln (percent bound) as a function of time. The kinetics of
association were determined at 37°C (B) and room temperature (C), and Ka
values were calculated using the initial linear part of the binding isotherm.
derived murine CTL epitope associated with 10- to 100-fold enhanced MHC binding affinity could overcome its poor immunogenicity. Thus, especially in the case of low on rate epitopes,
increased binding affinity can be associated with increased immunogenicity. In this study we analyzed the effect of substituting the
main anchor positions of an epitope that is already classified as a
good binder, as it binds HLA molecules with an affinity ⬍50 mM.
Accordingly, the affinity of the HER-2 V2V9 analogue described
in this study was only marginally increased compared with wildtype epitope (23 vs 37 nM). Despite this modest increase in MHC
class I binding, the V2V9 analogue was still associated with a
strong increase in immunogenicity, which motivated our analysis
of the kinetics of the association and dissociation of this peptide.
When the thermodynamic parameters for the HER-2.369
epitopes were established, the result, particularly for the V2V9
epitope, demonstrated an association with a slow on, slow off kinetic profile. The enhanced stability of the MHC binding of this
epitope could be an important parameter explaining the stronger
immunogenicity. Other studies using whole-cell, flow cytometrybased techniques and measuring the half-time of the peptide-MHC
complex have found that high stability is associated with enhanced
immunogenicity (30). Because of the indirect techniques used,
these reports did not assess on/off rates of the peptide analogues.
Restifo found that the dissociation rate of the gp100.209(2M) analogue was ⬎100-fold slower than that of the wild-type peptide
(N. Restifo, personal communication). Of particular interest, in a
clinical trial where this anchor-modified gp100 peptide was used to
immunize melanoma patients, a dramatic increase in tumor-reactive T cells was observed (4). One can speculate that peptides that
posses enhanced stability may remain bound to MHC class I on the
APCs for a longer period, allowing this peptide/MHC class I complex to reach the local lymph nodes and there activate a more
potent CTL response. This consideration might be of particular
relevance in immunization strategies when the antigenic epitope is
exogenously provided, and epitope/MHC complexes cannot be replenished by synthesis of new epitope molecules from within the
APC. It is also possible that the increased immunogenicity of the
peptide analogue might be related to increased functional binding
activity for T cells. This might be the indirect result of the increased stability of the complexes, allowing more avid TCR contact. Alternatively, this might also originate from subtle conformational alterations in the peptide residues interacting with the
TCR induced by the alterations in the MHC anchor motifs. If this
were the case, our analogue would be classified as a heteroclitic
analogue, associated with increased TCR binding (13).
The risk that these substitution analogues may alter the conformation of the MHC class I/peptide complex and thereby induce a
different set of CTLs that do not cross-react with the wild-type
epitope must be considered. Results from melanoma patients vaccinated with the gp100 epitope, which had been modified at the
second position (g9 –209(2M)) to enhance MHC binding affinity,
support this idea (33). Their data suggest that the T cell repertoire
in the vaccinated patients had been altered by expanding an array
of T cells with different fine specificities, only some of which
recognized melanoma cells. Our results showed that a subset of the
microcultures of CTLs induced by the analogue peptide that were
able to recognize the wild-type peptide also were able to recognize
the HLA-A2.1-positive SW403 HER-2-expressing tumor line. We
conclude that this recognition was MHC class I-restricted, as a
HER-2 expressing, but HLA-A2.1-negative, tumor was not recognized. In line with these findings, Kawashima et al. (34) were able
to identify double-substitution analogues from CEA with strong
tumor-specific immunogenicity in vitro, and others have found that
The Journal of Immunology
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double or single substitution of CTL epitopes is able to induce
tumor-specific T cells (8 –11, 17, 29, 30).
Taken together, our results demonstrate that the CTLs induced
by the V2V9 analogue also recognize the processed wild-type
epitope expressed on tumor cells, but do not rule out that the induced T cell repertoire may be altered compared with the repertoire induced by the wild-type epitope.
Our data also indicate that epitope analogues may be useful to
develop more sensitive assays to monitor immune responses in
patients in tumor vaccination trials based on wild-type recombinant proteins or epitopes. Thus, we demonstrate that a HER-2 specific CTL line generated against the wild-type epitope responded
with a higher number of IFN-␥-producing cells at low peptide
concentrations when stimulated with the V2V9 analogue peptide
compared with the wild-type peptide (Fig. 2). In line with these
data, T cells generated against a native epitope from a PSA showed
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Several clinical trials with HER-2-based vaccines have been
conducted or are in progress in patients with HER-2-overexpressing tumors (35–37). The T cell responses in patients vaccinated
with the HER-2.369 epitope in combination with GM-CSF were
reported to be weak and short-lived (38). Our results therefore
suggest that the HER-2.369 V2V9 analogue should be tested in
clinical trials, which may lead to stronger and more persistent T
cell responses, in analogy with our results observed in HLA-A2
transgenic mice. This analog could be used in a peptide-based
format in the presence of an appropriate adjuvant, such as GMCSF, or in a vaccine based on DC pulsed ex vivo with peptides
(36, 37). Protective HER-2-based vaccines based on plasmid DNA
vaccination have shown promising results in experimental animal
models (39, 40). Therefore, the alternative strategy based on the
construction of vectors that contain multiple epitope genes or
the entire HER-2 gene, but with altered amino acid sequences of
the V2V9 analog, could also be considered.
3507
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IMPROVED Her2/neu IMMUNOGENICITY AND MHC-PEPTIDE INTERACTION
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