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Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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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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-catenin (7), CDK-4 (8), and MUM-1
(9). The third group of Ags are widely expressed in a
variety of normal tissues but are selectively overexpressed on tumors.
These include p15 (10) and PRAME (11). The fourth group includes the melanoma differentiation Ags such as MART-1 (12, 13), gp100 (14), and tyrosinase (15). Other melanoma differentiation Ags include gp75/tyrosinase-related protein (TRP)2-1 (16) and TRP-2, which have recently been identified as melanoma Ags and contain antigenic epitopes recognized by specific CTL in the context of HLA-A31 and HLA-A33 alloantigens (17, 18). In other studies, HLA-A2-restricted melanoma-specific CTL epitopes in TRP-2 were identified using lymphocytes from repeated in vitro stimulation of PBMC from donors (19, 20, 21, 22).
We conducted a melanoma vaccine trial that enrolled 16 patients with metastatic disease. Patients were vaccinated s.c. with peptides (gp100: 209–217 (210 M), gp100: 280–288 (288V), MART-1: 27–35, and tyrosinase 368–376) emulsified in IFA. Two patients experienced dramatic tumor regression. In this report, we studied and analyzed the immunologic reactivity of one of these patients, MM, and have isolated, for the first time to our knowledge, melanoma-specific tumor-infiltrating lymphocytes (TIL) that recognized TRP-2 and NY-ESO-1 epitopes restricted by HLA-A2. In addition, we also identified a novel TRP-2 mRNA isoform that contained an HLA-A2-restricted CTL epitope recognized by TIL. This mRNA transcript was derived from alternative splicing of the TRP-2 gene in which two novel exons were spliced from noncontiguous regions of the sixth intron between exons 6 and 7.
Surprisingly, the immunologic reactivity directed against NY-ESO-1 and the two TRP-2 Ags was present in patient MM even before treatment and thus may have played a role in the dramatic tumor regression seen in this patient.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MM is a 63-year-old female who underwent a wide local excision
of a primary melanoma on her back in 1981. In May 1997, she developed a
s.c. metastasis on her left chest wall, which was resected, and she was
started on a chemoimmunotherapy regimen consisting of cisplatin,
vinblastine, dacarbazine, IL-2, and IFN-
. Because of disease
progression, she was referred to the Surgery Branch of the National
Cancer Institute (Bethesda, MD) in June 1998. At that time, she had
developed metastatic disease in multiple sites including the lungs,
liver, intrapelvic area, left abdominal wall, and left thigh. She was
started on a four-peptide vaccination protocol using 1 mg each of
gp100: 209–217 (210 M), gp100: 280–288 (288V), MART-1: 27–35, and
tyrosinase: 368–376 in IFA s.c. every 3 wk. Most of her tumors
completely regressed after 2 cycles of treatment (day 45), including
complete resolution of a large tumor in her left thigh, an intrapelvic
mass, a liver lesion, and most of the nodules in her lungs. She
completed a total of six cycles of vaccinations in October 1998. A year
later (October 1999), she developed a frontal lobe metastatic brain
lesion and a s.c. nodule on her right chest wall, both of which were
resected. Bulk TIL 1790 used in this study was grown from the chest
wall lesion. In March 2000, she underwent a second right temporal
craniotomy for resection of recurrent disease at the prior resection
site, followed by whole brain irradiation. The patient is doing well at
this time, with disease limited to three small lung lesions (
1
x 1 cm) that are slow growing.
Cell lines
Melanoma-reactive CTL were derived from bulk TIL cultures grown in IMDM (Life Technologies, Gaithersburg, MD) containing 6000 IU/ml human rIL-2 (Chiron, Emeryville, CA) as previously described (23). CTL clones were derived from bulk TIL cultures by limiting dilution with the addition of anti-CD3 Ab (OKT-3; Ortho-McNeil Pharmaceuticals, Raritan, NJ). Briefly, 5 x 104 irradiated (3000–3400 cG) PBMC from three allogeneic donors were plated in round-bottom 96-well plates with 0.5–90 T cells per well. Cells were cultured in RPMI 1640 medium (Life Technologies) containing 20% heat-inactivated FBS (Life Technologies) and 30 ng/ml OKT-3 Ab with 300 IU/ml IL-2. The same dose of IL-2 was added on day 7, and clones were tested for recognition of HLA-A2+ vs HLA-A2- melanoma cell lines 14 days after stimulation. After testing, the remainder of T cells were expanded by plating them in a T25 flask with 2.5 x 107 irradiated PBMC from three allogeneic donors in 25 ml of RPMI 1640 medium containing 10% FBS and 30 ng/ml OKT-3 Ab. Subsequent expansion was similarly done, but with 1–2 x 106 CTL and 2.5 x 108 allogeneic PBMC plated in an upright T162 flask in 150 ml of medium.
The melanoma cell lines 624-28, 624-38, 938, 526, 888, 888A2, F002S, and F002R were established in the Surgery Branch (National Cancer Institute). 624-28 and 624-38 cell lines were derived from the same parental cell line, 624-mel, and share a similar pattern of Ag and HLA allele expression, except for HLA-A0201 Ag, which was expressed by 624-38 cells but not by 624-28 cells because of aberrant pre-mRNA splicing (24). 888A2-mel cell line was obtained by stable transfection of 888-mel with HLA-A2 cDNA (pCDNA3 plasmid; Invitrogen, San Diego, CA). F002S cell line is deficient in gp100 expression. F002R cell line was derived from F002S cell line by in vitro immunoselection for loss of MART-1 expression (22). T2 cells, deficient in transporter-associated protein, were used to test HLA-A2-restricted peptides for CTL activity. 293 cell line was derived from primary human embryonal kidney cells and was used for transfection purposes. 293A2 cell line was obtained by stable transfection of 293 cells with HLA-A2 cDNA. All cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 10 mM HEPES buffer, 100 U/ml penicillin-streptomycin (Biofluids, Rockville, MD), 2 mM L-glutamine (Biofluids). This medium is referred to as complete medium (CM) in this paper.
Cytokine release assays
CTL cells (5 x 104) were plated with
1 x 105 target cells in 96-well
round-bottom plates in 200 µl of CM. After 18–24 h of incubation at
37°C, the supernatant was harvested for detection of IFN-
release
using ELISA kits (Endogen, Cambridge, MA). For the MHC blocking assays,
target cells were incubated with the appropriate mAb at a final
concentration of 50 µg/ml (except for BB7.2, which was used at 25%
final volume) for 1 h at 37°C before the addition of CTLs. W6/32
(anti-HLA class I) was obtained from the American Type Culture
Collection (Manassas, VA). TU39 (anti-HLA class II) and L243
(anti-HLA-DR) mAbs were purchased from BD PharMingen (San Diego,
CA). Anti-HLA-A2 mAbs, SB00-60 and BB7.2, were a kind gift from Dr. F.
Marincola (Department of Transfusion Medicine, National Cancer
Institute) and Dr. E. Bergmann-Leitner (Department of Immunology,
Walter Reed Army Institute of Research, Silver Spring, MD),
respectively. For the 12-day in vitro sensitization assay, PBMC were
plated at 3 x 106 cells in 24-well plates
in 2 ml of IMDM, prepared as the CM but with 10% heat-inactivated
human AB serum. On day 0, peptides were added at a final concentration
of 1 µM and the plates were incubated at 37°C. On day 1 and every
3–4 days thereafter, 300 IU/ml human rIL-2 was added. On day 12, cells
were harvested for testing. Targets were T2 cells that had been pulsed
with peptides at 1 µM for 2 h at 37°C and washed once.
Detection of IFN- release was done as described above, but with
1 x 105 CTL instead of 5 x
104.
cDNA library screening
624-mel cDNA expression library was kindly prepared by Dr. R.-F.
Wang (Baylor College of Medicine, Houston, TX). Briefly, total RNA was
extracted from 624-mel cells using TRIzol reagent (Life Technologies).
Poly(A) RNA was purified from total RNA by the poly(A) tract isolation
system (Promega, Madison, WI) and converted to cDNA using an oligo(dT)
primer. The cDNA was ligated to BstXI adapters and digested
with NotI, then ligated to the expression vector pEAK 8. The
cDNA was electroporated into DH10B cells (Life Technologies). Pools
containing 100 cDNA clones were prepared from bacteria. The plasmid
DNA (200 ng) were transfected into 293A2 cells with Effectene reagent
(Qiagen, Valencia, CA). Briefly, 4 x 104
cells per well were plated in flat-bottom 96-well plates in complete
medium the day before transfection. On the day of transfection, plasmid
DNA (200 ng) from each pool were diluted in 29 µl of buffer
EC. Next, 0.8 µl of Enhancer was added and mixed. The
mixture was incubated at room temperature for 5 min. After the
incubation, 5 µl of Effectene reagent was diluted in 17.5 µl of
buffer EC and then added to and mixed with the DNA-Enhancer mixture.
The mixture was incubated for 10 min at room temperature. During this
time, old medium from 293A2 plates was removed and replaced with 100
µl of fresh CM. After the incubation, the DNA-Enhancer-Effectene
mixture was added drop-wise onto the cells. The plates were then
incubated at 37°C/5% CO2 for 24 h. The
following day, the old medium was removed and replaced with 100 µl of
fresh CM. A total of 5 x 104 CTL in 100
µl of CM were added to each well, and the plates were incubated at
37°C for 24 h. ELISA for IFN- was performed as described.
DNA sequencing
Sequencing of the isolated cDNA clone was performed with an ABI Prism 310 automated capillary electrophoresis instrument (PerkinElmer, Foster City, CA) using the Dye Terminator Cycle Sequencing Ready Reaction kit (PerkinElmer). Searches for sequence homology were done with the GenBank database using the basic local alignment search tool algorithm.
Peptide synthesis
Peptides were synthesized using a solid-phase method based on standard F-moc chemistry on a multiple peptide synthesizer (Gilson, Worthington, OH). Peptide identity was verified by laser desorption mass spectrometry (Biosynthesis, Lewisville, TX). Lyophilized peptides were solubilized in DMSO at a 10 mg/ml concentration.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 1790 TIL line was isolated from a metastatic s.c. lesion in
the right chest wall of patient MM, who was HLA-typed as
A0201, A0301, B15, B35, C3, and C4. The 1790 TIL
line, in four separate experiments, recognized a panel of melanoma cell
lines matched for HLA-A2 but did not recognize non-HLA-A2 cell lines
(Table I
). In addition, TIL 1790 was
tested against peptide-pulsed T2 cells and was found to recognize
MART-1 27–35 but not the tyrosinase or two gp100 peptides against
which patient MM was immunized. Of interest, TIL 1790 recognized both
MART-1 peptide and F002R cells, a melanoma cell line that does not
express MART-1. The lack of MART-1 expression on F002R cells was
confirmed in the same experiment by the lack of recognition of F002R
cells by a control anti-MART-1 CTL clone (clone V2C8) (data not
shown). This demonstrated that besides MART-1, 1790 TIL line recognized
at least one other melanoma Ag.
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In an attempt to identify the Ag(s) recognized by TIL 1790, a
number of CTL clones were generated from this TIL line by limiting
dilution. CTL clones MR7, MB4, and M8 were selected for further study
based on their ability to specifically release cytokine (IFN-) in
response to HLA-A2 melanoma cell lines but not to cell lines that did
not express HLA-A2 (Table II), and based
on the lack of recognition of T2 cells pulsed with peptides from
MART-1, gp-100, and tyrosinase (data not shown). The fact that
non-HLA-A2 melanoma cell lines such as 888 stimulated cytokine release
from clones MR7 and MB4 after being stably transfected with the cDNA
encoding HLA-A2 Ag indicated that they expressed the Ag(s) of interest
and that MR7 and MB4 recognized a melanoma Ag restricted by HLA-A2.
Clone M8 recognized neither 888 nor 888A2; however, it reacted with
624-38, but not the HLA-A2 negative variant, 624-28. This suggested
that M8 also recognized a melanoma Ag restricted by HLA-A2. This HLA-A2
restriction was confirmed with Ab blocking studies performed on clone
MR7. Anti-class I Ab (W6/32) caused a 100% reduction in the activity
of clone MR7, whereas anti-class II and anti-DR Abs had a
minimal effect (6 and 13%, respectively). In addition, anti-HLA-A2
Abs, SB00-60 and BB7.2, blocked the activity of clone MR7 at 99 and
77% efficiency, respectively. These same Abs exerted a minimal effect
(20 and 0% reduction, respectively) on a control, HLA-A24-restricted
CTL clone (clone G5G10), which was specific for a mutated -catenin
epitope. Clones MR7, M8, and MB4 were tested for recognition of
previously described Ags in a coculture assay using 293A2 cells
transfected with cDNA encoding these Ags (Table III). Clone MR7 recognized TRP-2, and
clone M8 recognized NY-ESO-1, whereas clone MB4 did not recognize any
of the known Ags tested. In addition, a cDNA clone coding for TRP-2 was
isolated and sequenced when CTL clone MR7 was used to screen the
624-mel library (data not shown). The nonapeptide TRP-2: 180–188, and
the overlapping NY-ESO-1 peptides NY-ESO-1: 157–167, NY-ESO-1:
157–165, and NY-ESO-1: 155–163 were found to be the epitopes
recognized by CTL MR7 and CTL M8, respectively (Table IV).
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A cDNA library prepared from 624-mel cells was next used to screen
for a gene encoding reactivity with CTL MB4. Plasmid DNA from pools
containing 100 cDNA clones was transiently transfected into 293A2
cells, a human embryonal kidney cell line transfected with the cDNA
encoding HLA-A2 Ag. From 400 pools, the cDNA in one pool stimulated
strong IFN- release from CTL MB4. By subcloning the positive pool,
one cDNA clone (cDNA 198) was identified which induced a high level of
IFN- secretion from TIL clone MB4 (Table V).
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The sequence of cDNA 198 was identical to that of the
TRP-2 gene with the exception of a 99-nt insert between exon
6 and exon 7 of the TRP-2 gene (Fig. 1). This extra insert was composed of two
separate sequences, named 6b and 6c, and each showed complete homology
with two noncontiguous sequences in intron 6 of the TRP-2
gene. The deduced amino acid sequence was translated in-frame
with the TRP-2 product. The two polypeptides encoded by cDNA 198 and
the cDNA encoding the previously described TRP-2 were identical except
for an in-frame insert of a 33-aa sequence following amino acid 393. 6b
and 6c were coding sequences and their flanking regions
conformed to the intron consensus motif GT-AG (data not shown);
therefore, 6b and 6c were identified as novel exons, alternatively
spliced from intron 6 of TRP-2. In addition to the insertion in the
coding region, the sequence of cDNA 198 differed from TRP-2 in the
noncoding regions. The initial 122 bp (of 414 bp) of the 5'
untranslating region (5' UTR) of TRP-2 was deleted in cDNA 198.
However, the 3' UTR of TRP-2 was further extended with a 10-bp sequence
in cDNA 198. The protein encoded by cDNA 198 was named TRP-2-6b.
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The fact that CTL MB4 did not recognize 293A2 cells transfected
with TRP-2 cDNA suggested that the antigenic epitope may be encoded by
the 99-bp sequence of the new exons 6b and 6c. Using a peptide-HLA
motif search program, 22 peptides were generated with predicted HLA-A2
binding, from the new 33-aa sequence (encoded by exons 6b and 6c) and
10 amino acids upstream and downstream of it. These peptides were
pulsed on T2 cells and tested for recognition by CTL MB4. Two peptides,
ATTNILEHV and NATTNILEHV, designated TRP-2-6b: 403–411 and TRP-2-6b:
402–411, respectively, were recognized by CTL MB4 when pulsed on T2
cells (Table VI). The decapeptide
TRP-2-6b: 402–411 was able to sensitize target cells for recognition
by CTL MB4 at 5 ng/ml, whereas the nonapeptide TRP-2-6b: 403–411 was
recognized at 50 pg/ml (Fig. 2).
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Patient MM’s PBMC collected before and during the course of
treatment were tested for reactivity against NY-ESO-1, TRP-2, TRP-2-6b,
and the four peptides that patient MM was immunized with, by
stimulating the PBMC in vitro for 12 days with the unmodified epitopes
from these Ags (Fig. 3). The nonapeptide
from TRP-2-6b was used in this experiment. HLA-A2-restricted
telomerase: 540–548 was used as an irrelevant peptide control. The
patient’s pretreatment PBMC (day 0) exhibited strong reactivity
against NY-ESO-1 and the two TRP-2 isoforms, none of which she had been
vaccinated against (Fig. 3). The reactivity against the TRP-2
Ags increased and persisted over time. In contrast, the immune
responses directed against MART-1, gp100: 209–217, gp100: 280–288,
and tyrosinase, the peptides she had been vaccinated with, were not
present in the prevaccination PBMC (Fig. 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50% of patients whose tumors expressed
NY-ESO-1 (6, 26). TIL clone M8 recognized the overlapping
epitopes from NY-ESO-1 previously identified by Jager et al.
(6). In addition, TIL-1790 recognized TRP-2-6b, a novel TRP-2 mRNA isoform. The original TRP-2 protein is a membrane-bound melanosomal enzyme (DOPAchrome tautomerase) involved in melanin biosynthesis. Of the TRP-2 isoforms reported so far, TRP-2-6b is unique in that it contains an in-frame insertion of two extra exons, with no deletion in the coding regions. Because exon 8 encodes a putative transmembrane domain (27, 28, 29), which seems to be critical for the DOPAchrome tautomerase activity of TRP-2 (30), the novel isoform TRP-2-6b, which contains an intact exon 8, is most likely localized to the melanosomal membrane and to have functional DOPAchrome tautomerase activity. However, the full functional status of TRP-2-6b is still not known. In contrast, the isoform TRP-2-INT2, which contains an HLA-A68-restricted CTL epitope, encodes a shortened protein of 237 amino acids because of an early stop site located in the retained intron 2 (31). Two other isoforms, TRP-2-8b and TRP-2-LT, have been described but have not been shown to contain any novel CTL epitopes (30). TRP-2-8b contains a novel exon 8b but lacks exon 8 and therefore, like TRP-2-INT2, is devoid of transmembrane domain as well as the DOPAchrome tautomerase activity. The isoform TRP-2-LT is identical to TRP-2 except for a long stretch of nucleotides (2315 bp) in the 3' UTR.
The novel exon 6b of TRP-2-6b encoded an antigenic epitope recognized by melanoma-specific HLA-A2-restricted TIL. Both the nonapeptide (TRP-2-6b: 403–411) and decapeptide (TRP-2-6b: 402–411) of this epitope sensitized target cells for recognition by TIL clone MB4 at low concentrations, 50 pM and 5 nM, respectively. Even though the predicted HLA-A2 binding affinity of TRP-2-6b: 403–411 was low, the extremely low concentration it required to stimulate TIL clone MB4 indicated that clone MB4 might be high-affinity effectors.
It is interesting that patient MM was never vaccinated against NY-ESO-1
or TRP-2, and that she presented to us with a high level of immune
reactivity against NY-ESO-1 and two different TRP-2 peptides before
receiving the four-peptide treatment with peptides from gp100, MART-1,
and tyrosinase (Fig. 3
). The overall response directed against all Ags
may have been responsible for the eradication of her tumors; however,
anti-NY-ESO-1, anti-TRP-2, and anti-TRP-2-6b reactivity may
have played a more dominant role in mediating the regression of her
tumors. Vaccination with gp100, MART-1, and tyrosinase appeared to
stimulate immunologic responses against the two TRP-2 Ags. Before
treatment, patient MM had circulating precursors directed against
NY-ESO-1, TRP-2, and TRP-2-6b, as evidenced from the immunologic
reactivity seen in her prevaccination PBMC, even though she continued
to have progression of her metastatic disease at multiple sites. We
hypothesize that repeated vaccination with gp-100, MART-1, and
tyrosinase peptides emulsified in IFA induced cytokine cascades both
locally and systemically, resulting in activation and proliferation of
anti-melanoma Ag precursors such as those against NY-ESO-1 and the
two TRP-2 Ags, and infiltration of these effectors into tumors. This
may explain the initial decrease in anti-NY-ESO-1 reactivity
observed in PBMC collected at the second time point (day 45), when her
tumors were decreasing. In our prior studies, we have shown an increase
in IFN- message in tumors in patients immunized with the gp100:
209–217 (210 M) peptide (32), which was one of the
peptides used to immunize this patient. Of interest, the immune
reactivity against the two TRP-2 Ags persisted over time, whereas that
against NY-ESO-1 waned over the course of follow-up. One possible
explanation for the continued decrease in reactivity against NY-ESO-1
over time is that residual tumors were immunoselected in vivo for loss
of NY-ESO-1 expression, but sufficient tissues were unavailable for
this analysis.
These findings indicate that alternative splicing of TRP-2 or other Ags such as gp-100 may generate novel melanoma Ags, and that the immunologic responses directed against NY-ESO-1, TRP-2, and TRP-2-6b may have played a role in the dramatic tumor regression seen in this patient. This suggests that the HLA-A2-restricted CTL peptide epitopes from NY-ESO-1 and the TRP-2 Ags may be useful in the active immunotherapy of patients with melanoma.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: TRP, tyrosinase-related protein; TIL, tumor-infiltrating lymphocyte; UTR, untranslating region; CM, complete medium.
Received for publication August 1, 2001. Accepted for publication November 21, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes. J. Exp. Med. 183:1185.This article has been cited by other articles:
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