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*
Ronald O. Perelman Department of Dermatology, Departments of
Medicine and
Surgery, New York University Medical Center, New York, NY 10016;
§
Epimmune, Inc., San Diego, CA 92121; and
¶
The Rogosin Institute, New York, NY 10021
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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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We have now extended these studies by examining patients’ responses in vivo to a large number of peptides derived from Ags expressed by human melanoma cells. These include MAGE-3 (15), Melan-A/MART-1 (16), gp100 (5), tyrosinase, melanocortin receptor (MC1R)4 (17), and dopachrome tautomerase (TRP-2) (18, 19), all of which are known to be antigenic in vitro. The study focused on HLA-A*0201-restricted peptides, the allele most commonly expressed by patients at risk for developing melanoma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We evaluated 22 sequential, vaccine-treated, HLA-A*02+ patients with resected malignant melanoma. Seven (32%) had American Joint Committee on Cancer (AJCC) stage II, nine (41%) had stage III and six (27%) had stage IV disease. HLA typing was performed by complement-mediated cytotoxicity. HLA-A2 subtyping was performed by the PCR-SSP (sequence specific primers) technique using the set containing 5' and 3' primers for identifying A*0201 to A*0217 alleles (Dynal, Oslo, Norway) (20).
Vaccine and immunization
All patients were immunized to a polyvalent melanoma vaccine prepared, as previously described, from Ags shed into culture medium by a pool of melanoma cell lines (SFM14, SFM20 and SFSKMel28) (21, 22). Briefly, the shed material was collected, concentrated, pooled, treated with 0.5% Nonidet P-40 to break up aggregates, and ultracentrifuged at 100,000 x g for 90 min to deplete alloantigens. The supernatant was filter sterilized, adjusted to a protein concentration of 200 µg/ml, dispensed into sterile glass vials, and frozen at -80°C until used. The vaccine contains MAGE-1, MAGE-3, Melan-A/MART-1, and tyrosinase by Western blotting (14), gp-100 by immune precipitation, and several other melanoma-associated Ags of 45–110 kDa that are expressed by melanoma tissue in vivo and are immunogenic in humans (23). All patients were immunized to vaccine, using alum or liposomes as adjuvant (24). It was administered intradermally split into the four extremities, every 2–3 wk four times. Peripheral blood was collected before immunization and 1 wk following the fourth immunization. Mononuclear cells were separated on Ficoll-hypaque and frozen in liquid N2 until used.
Preparation of peptides
Peptides were prepared on an Applied Biosystems (Foster City,
CA) instrument. Briefly after removal of the
-amino-ter-butylcarbonyl protecting group, the
phenylacetamidomethyl resin peptide was coupled with a 4-fold excess of
preformed symmetrical anhydride (hydroxybenzyltriazole esters for
arginine, histidine, asparagine, and glutamine) for 1 h in
dimethylformamide. For arginine, histidine, asparagine, glutamine, and
histidine residues, the coupling step was repeated to obtain a high
efficiency coupling. Peptides were cleaved by treatment with hydrogen
fluoride in the presence of the appropriate scavengers. Synthetic
peptides were purified by reverse phase HPLC. The purity and identity
of the peptides, which was routinely >95% was determined by amino
acid sequence and mass spectrometric analysis respectively.
Assay of peptide binding affinity to HLA-A*0201
Peptide binding to purified HLA-A*0201 molecules was measured as
previously described (25). Briefly, the assay is based on the
inhibition of binding to detergent-solubilized HLA molecules of a
radiolabeled standard peptide with strong binding affinity for
HLA-A*0201. The standard peptide, FLPSDYFPSV, was radioiodinated by the
chloramine T method using 125I (ICN, Irvine, CA).
HLA-A*0201 concentration yielding 
15% of bound peptide
(approximately in the 10 nM range) was used in the inhibition assays.
Various doses of the test peptides (1 nM to 10 µM) were incubated
with 5 nM radiolabeled standard peptide and HLA-A*0201 molecules for 2
days at room temperature in the presence of a mixture of protease
inhibitors and 1 µM ß2-microglobulin (Scripps
Laboratories, San Diego, CA). At the end of the incubation period, the
percent HLA bound radioactivity was determined by gel filtration. The
peptides were selected based on their capacity to bind to HLA-A*0201
with an affinity greater than an IC50 of 500 nM, which is
known to be in the immunogenic range for cytotoxic T lymphocyte
epitopes (26).
Assay of peptide-specific CD8+ T cells
We determined the number of peptide-specific CD8+ T
cells in peripheral blood by filter spot assay as previously described
(14). Briefly, 96-well polyvinylidene dilfuoride filter plates
(Millipore, Bedford, MA) were precoated with mAbs to human IFN-
,
(Biosource, Camarillo, CA) and seeded with 20,000
HLA-A*0201+ target cells/well (SFM20 · A2) (14). The
target cells were pulsed with 20 nM of test peptide before addition of
effectors. The HLA-A*0201-restricted influenza peptide
FluM158–66 (GILGFVFTL) to which most individuals have
a CD8+ T cell response was added to some wells as a
positive control (27). mAb IVA12 (anti-HLA-DR, DP, and DQ (HB145
from the American Type Culture Collection, Manassas, VA) was added to
all wells to prevent presentation of class II Ags by targets or
residual monocytes. Effector PBMC were thawed, resuspended in RPMI 1640
with 10% FBS and depleted of monocytes on plastic. The cells were then
added to the wells, incubated 48 h at 37°C and 5%
CO2, washed with PBS-Tween 20, incubated overnight with
goat anti-IFN- (R&D, Minneapolis, MN), followed by biotinylated
rabbit anti-goat Ig (Sigma, St. Louis, MO) and extravidin alkaline
phosphatase (Sigma). Spots representing individual T cells that had
been stimulated by peptides and released IFN- were visualized with
BCIP/NBT (Kirkegaard & Perry Laboratories, Gaithersburg, MD)
and counted with a dissecting microscope.
The number of CD8+ T cells was calculated as the
number of IFN--secreting cells in wells without Abs minus those in
wells with anti-CD8 Abs (OKT8 from the American Type Culture
Collection). The number of peptide-specific CD8+ T cells
was determined by subtracting the number of CD8+ cells
reacting to targets pulsed with an A*0201-restricted control peptide
(YLKKIKNSL from falciparum malaria) (28) from the number of
CD8+ T cells reacting to melanoma peptide-pulsed targets.
All assays were conducted twice, once in duplicate and once in
triplicate, and the average value used. The mean SD was ±0.95. The
test was considered positive if there were 
5 peptide-specific
CD8+ T cells per 500,000 PBL, which is twice the maximum
SD. The number of vaccine-induced peptide-specific CD8+ T
cells was calculated by subtracting the number of peptide-specific
CD8+ T cells present before treatment from that after four
vaccine immunizations in the same patient. The vaccine was considered
to have stimulated a CD8+ T cell response to that peptide
if this number was 5.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The peptides used in this study were selected for their potential
ability to stimulate CD8+ T cells in vivo based on being
derived from Ags known to be associated with melanoma, i.e.,
MAGE-3, Melan-A/MART-1, gp100, tyrosinase, MC1R, or TRP-2; having 9–10
amino acid residues; containing an HLA-A*0201 binding motif; and by
having high binding affinity for HLA A*0201 in vitro. A total of 45
melanoma peptides that satisfied these criteria is provided in Table I
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We examined the ability of the 45 HLA-A*0201-restricted peptides selected as described above, an HLA-A*0201-restricted malaria peptide as a negative control (28), and an HLA-A*0201-restricted influenza peptide as a positive control (27) to be recognized by CD8+ T cells from melanoma patients. We measured the number of CD8+ T cells directed to each peptide before and after immunization to a polyvalent melanoma vaccine containing the Ags from which the melanoma peptides were derived. The study was conducted on 22 sequential HLA-A*02+ patients who were immunized to the polyvalent vaccine. All but one patient were A*0201+. Patient 484 was A*0205+.
Before vaccine treatment, elevated numbers of circulating
peptide-specific CD8+ T cells were found in 8 of the 22
patients. These patients reacted to 14 of the 45 melanoma peptides (see
Table I
). One or more of these peptides was derived from each of the 7
Ags studied. However, elevated CD8+ T cells to any one
peptide were present in no more than 1 patient (5%).
Following four vaccine immunizations, a peptide-specific
CD8+ T cell response was induced or augmented to 22
(47.8%) of the peptides, as indicated by a minimum increase of 5
peptide-specific CD8+ T cells over baseline measurement in
the same patient (see Table II). Of the
induced responses, at least one of the inducing peptides was derived
from each of the proteins studied. The most frequently positive
peptides were gp100585–613 IMPGQEAGL) to which
responses were induced in 3 (14%) of the 22 patients (see Table II). The highest vaccine-induced responses were directed to
MAGE-3271–278 (FLWGPRALV),
Melan-A/MART-156–64 (ALMDKSLHV) and
gp10013–21 (AVIGALLAV) which all were >40
peptide-specific CD8+ T cells/500,000 PBL over baseline
measurements. Twenty-one of the 22 patients were also tested for their
ability to recognize the HLA-A*0201-restricted influenza peptide
FluMI58–66 (27) as a positive control. This peptide was
recognized by 76% of the patients. There was no response to the
control malaria peptide in any patient either before or after vaccine
immunization. Overall, vaccine immunization induced a peptide specific
CD8+ T cell response to at least one peptide in 59% of the
22 patients. Responses were induced in 7 of 14 (50%) of patients
without a pre-existing response, and further induced in 4 of 8 (50%)
patients who had a pre-existing response before vaccine treatment.
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The relative responses to each Ag were evaluated based on the
frequency of vaccine-induced, peptide-specific, CD8+ T cell
responses to at least one peptide derived from that Ag. The results are
summarized in Table III. All of the
melanoma Ags stimulated a CD8+ T cell response to at least
one peptide on that Ag. The most frequently recognized Ag was gp100,
which induced CD8+ T cell responses in 27% of patients.
Overall, 59% of the patients had a vaccine-induced CD8+ T
cell response to at least one of the Ags studied.
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There was striking heterogeneity in the ability of
individual patients to develop vaccine-induced CD8+ T cell
responses to individual peptides on the same Ag. An example of this
heterogeneity in responses to peptides of gp100 is illustrated in
Table IV. Individual patients immunized
to the same amount of gp100-containing vaccine developed
CD8+ T cell responses to one gp100 peptide but not to
others, whereas the reverse was true of other patients.
Overall, 27% patients developed a CD8+ T cell response to
at least one of the gp100 peptides, but no more than three
patients (14%) reacted to the same peptide. There was a similar
degree of heterogeneity in vaccine-induced responses to peptides
derived from the other Ags (data not shown). Nine to 18% of the
patients reacted to at least one peptide from each Ag, but no more than
three patients (14%) reacted to the same peptide that Ag.
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, some patients
developed vaccine-induced CD8+ T cell responses to peptides
of one Ag but not to those of another Ag, whereas the reverse was true
for other patients. Though 59% of patients developed CD8+
T cell responses to at least one of seven Ags studied, only 9% to 27%
of patients reacted to the same Ag. Correlation of vaccine-induced CD8+ T cell responses with clinical outcome
The clinical status of the patients in the study is
shown in Table V. All survived beyond 1
year, and over 75% are still surviving. Of those patients who had a
vaccine-induced CD8+ T cell response to at least one of the
Ags tested, a higher proportion (though not statistically significant)
were progression-free after 1 year as compared with those who had no
response (54% vs 22%). This positive correlation was unrelated to the
stage of disease or the type of adjuvant administered with the vaccine
as these two variables were equally distributed in both groups. Most
likely a larger study will be needed to show statistically significant
differences.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing cells/500,000 PBL and required only a
small amount of the blood with minimum manipulation compared with
assays using purified CD8+ T cells. Using this strategy, we
identified multiple peptides presented by HLA-A*0201 and derived from
MAGE-3, Melan-A/MART-1, gp100, tyrosinase, MC1R, and TRP-2 that have
immunogenic potential as evidenced by their ability to stimulate
CD8+ T cell responses in vivo. Several of these peptides
were previously known to be recognized by CD8+ T cells in
vitro, including FLWGPRALV from MAGE-3 (3), AAGIGILTV and GILTVILGV
from Melan-A/MART-1 (4, 29), MLGTHTMEV from gp100 (30) and FLPWHRLFL
from tyrosinase (31). All of the Ags we studied including MAGE-3, Melan-A/MART-1, gp100, tyrosinase, MC1R, and TRP-2 were able to stimulate CD8+ T cell responses in vivo because patients immunized to a vaccine containing these Ags had vaccine-induced responses to peptide epitopes derived from them. There were differences in the frequency with which individual Ags stimulated CD8+ T cell responses, ranging from gp100 which induced CD8+ T cell responses in 27% of the patients to MC1R which induced responses in 9%. However, we could not determine whether this was due to differences in intrinsic immunogenicity in vivo, differences in the relative amount of each Ag present in the vaccine, or a selection bias in the number or type of peptides derived from each Ag selected for testing.
Surprisingly, we did not find any of the peptides to be were clearly immunodominant, as none induced CD8+ T cell responses to a much greater extent than the others. This is in contrast to well-described immunodominance previously reported for one peptide we studied, Melan-A/MART-127–35. The inability to detect frequent immune responses to this particular peptide did not appear to result from lack of sensitivity as we detected responses to the influenza peptide positive control in the majority of patients. Because of its much lower binding affinity than all of the other peptides, it is possible that we would have seen greater recognition had we used more peptide in this specific case or used the decapeptide that has been reported by Romero et al. (32) to have much greater recognition by T cells from different individuals than the nonapeptide.
Our most important finding was the marked HLA-independent heterogeneity in the ability of different patients to develop CD8+ T cell responses to the same peptide or to the same Ag. This was not due to differences in the amount of peptide or Ag used to immunize the patients, since all received the same dose of the same vaccine. Nor was it due to differences in the immunological competence, since all patients were immunologically competent as evidenced by their ability to react to recall Ags and the majority of patients reacted to the control influenza peptide FluM158–66. Nor was it due to differences in the HLA subclass restriction of the peptides or of the patients, since 21 of the 22 patients were A*0201+ and the peptides were all HLA-A*0201-restricted. The one patient who was of the A*0205 subtype did not respond to any peptide. Nor was it due to differences in the solubility or affinity of the peptides used in the assays, since all patients cells were exposed to all of the peptides under the same conditions.
In summary, we have directly demonstrated the ability of seven melanoma-associated Ags to stimulate a CD8+ T cell response in humans and identified multiple CD8+ T cell peptide epitopes on each of these Ags that can be recognized by CD8+ T cells in vivo. These peptides and Ags are attractive candidates for vaccine construction because they can stimulate CD8+ T cell responses. There was marked HLA-independent heterogeneity in the ability of the same peptide or Ag to stimulate CD8+ T cell responses in different patients, and no single peptide or Ag was clearly immunodominant. Regardless of the reasons for this heterogeneity, the implication for cancer vaccine design is that the stimulation of CD8+ T cell responses to cancer will be maximized by constructing vaccines from multiple peptides or multiple Ags.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Immunology, Mayo Clinic, Rochester, MN 55905.
3 Address correspondence and reprint requests to Dr. Jean-Claude Bystryn, Department of Dermatology, New York University Medical Center, 560 First Ave., New York, NY 10016.
4 Abbreviations used in this paper: MC1R, melanocortin receptor; TRP-2, dopachrome tautomerase; AJCC, American Joint Committee on Cancer.
Received for publication May 12, 1998. Accepted for publication August 18, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, J.-C. Bystryn. 1997. Stimulation of CD8+ T cell responses to MAGE-3 and Melan A/MART-1 by immunization to a polyvalent melanoma vaccine. Int. J. Cancer 72:972.[Medline]
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