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Immunology |
Center for Biological Therapy and Melanoma Research, University of California, San Diego, La Jolla, California 92093, and the University of Southern California-Norris Cancer Center, University of Southern California, Los Angeles, California 90025
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2b, considerable impetus was given to the determination
of which antigens were recognized by the immune response. Van der
Bruggen et al. (1)
, drawing upon their work in
the P815 mastocytoma in mice, demonstrated in 1991 an epitope called
MAGE-1, the first such epitope recognized by human CTLs. Since then,
other investigators have found a variety of melanoma antigens,
including MART-1/Melan A, gp 100, and tyrosinase. Boon et
al. (2)
showed that the screening of melanoma
expression libraries with CTLs derived from autologous
vaccine-immunized patients was an effective method of detecting
neoantigens. Other groups have also used this procedure in their
successful quest for new melanoma antigens (3)
. In the late 1980s, we began our attempts to isolate melanoma genes with a method known as subtractive hybridization, which had been used successfully by Hedrick et al. (4) to isolate the T-cell receptor. This procedure depends upon the hybridization of cDNA from one type of cell with an excess of mRNA from another cell that is closely related but is likely to differ in the critical gene of interest. Single-stranded cDNAs, which presumably represent gene fragments unique to the cell type of interest, can be separated from double-stranded hybrids. Our strategy was to identify novel rare genes with a frequency <1:20,000 that might encode tumor neoantigens, which appeared to be among the less frequent cellular antigens. By subtractive hybridization with mRNA from a squamous lung carcinoma cell line (LU-1) as the partner, we identified 12 cDNAs that were found only in a melanoma cell line MSM-M-1 (5) , a component of our allogeneic lysate vaccine (6) . We chose the squamous lung carcinoma because it was another tumor but was unrelated histologically to the melanoma. We purposely avoided using normal melanocytes as a partner, because we did not want to rule out rare genes that encoded melanocyte differentiation antigens. Of the 12 cDNAs, 6 had sequences that were at that time unreported in any of the gene sequence databases such as GenBank and were, therefore, considered "novel."
We decided to explore two clones in more detail, because of their distribution among tumor cells and normal cells. One of the cDNAs ["clone 159" in our original paper (5) ] was found to be identical with PMP-22, a gene found in Schwann cells and involved in the pathogenesis of Charcot-Marie-Tooth disease (Uchiyama, C., Ph.D. thesis, University of Southern California, 1994). A second cDNA, "clone 50," has been studied in more detail, requiring almost a decade because of its large size and complex structure. The gene from which clone 50 was derived, which we have called MG50 (registered in GenBank as D2S448) has been localized to chromosome 2p25.3 by fluorescence in situ hybridization (7) .
In our laboratory, Weiler (8) was able to determine the sequence of 1.3 kb at the 3' end of the cDNA molecule but could not go beyond that point. Only recently have we been successful in sequencing nearly all of MG50, through a variety of approaches. Here we present that sequence, as well as the protein encoded by the large open reading frame. Their identical sequence in the 3' region indicates that MG50 may be closely related to or identical with the IL-1 receptor intracellular binding protein (IL-1 receptor antagonist). We further present evidence that MG50 encodes a new melanoma antigen containing at least six naturally expressed melanoma epitopes that are recognized by CTLs in the context of HLA-A2.1.
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MATERIALS AND METHODS |
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The first long extension to the 1.3-kb clone was 1200 bp toward the 5'
end. We verified that the 5' sequences hypothetically constructed were
linked to the 1.3-kb cDNA clone, and in the proper order, by
constructing sense primers from the GenBank-derived sequence, and
antisense primers from within our 1.3-kb cDNA sequence. The first sense
and antisense primers were 22 bases in length. cDNA from melanoma cell
line MSM-M3 (hereafter abbreviated M3), which contains MG50,
was then used as a template for these primers in a PCR. The PCR was
also performed with the same cDNA using sense and antisense primers
from sequences contained entirely within the 1.3-kb cDNA clone. cDNA
from the M3 melanoma cell line was amplified in a PCR with the sense
and antisense primers located within the 1.3-kb cDNA, and more
importantly, cDNA from the M3 melanoma cell line was also amplified
with a sense primer located upstream from the 1.3-kb cDNA and an
antisense primer located within our 1.3-kb cDNA clone (Fig. 1)
. These results indicate that the sequence generated from GenBank DNA
clones is an additional 5' sequence contiguous with the original
1.3-kb cDNA clone.
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This approach of constructing a hypothetical sequence based upon fragment homology and then testing the validity of the extension with PCR was used as a means of extending the sequence well into the 5' region upstream from the 22-kb sequence we first added to the 1.3-kb cDNA.
When Nomura and colleagues reported in the gene database a number of large-sized cDNAs obtained from the human genome by random priming PCR,4 we used the sequence of bases that overlapped our known MG50 sequence to deduce a long extension toward the 5' end. These sequences were from a human myeloblast line (KG-1) derived from bone marrow and were located on chromosome 2, where MG50 is found. We confirmed by PCR that we had the full sequence from the 3' end to 6.8 kb, or 1.3 kb from the 5' end of the molecule. Unfortunately, none of the methods that we used subsequently, such as anchored PCR and marathon PCR, could extend the sequence further. However, a long open reading frame was obtained from which the amino acid sequence of a large protein was deduced.
Quantitative PCR for Determining the Presence of
MG50 in Archived Specimens and Cell Lines.
The method was as described by Kan-Mitchell et al.
(9)
. In brief, cDNA libraries were constructed from each
specimen and cell line, with avian myeloblastosis virus reverse
transcriptase (Seikagaku American, Inc., St. Petersburg, FL), from
acidified guanidinium thiocyanate-extracted total RNA. Each was then
incubated with 23-base sense and antisense primers from within the
1.3-kb sequence, subjected to the PCR for 30 cycles, and the reaction
was analyzed in 6% polyacrylamide gels with 7 M
urea, 89 mM Tris-borate, and 2
mM EDTA. The T7 polymerase promoter sequence was
attached to the 5' primer to convert amplified cDNA to cRNA, for an
additional 500-fold amplification. In parallel, reverse
transcription-PCR for ß-actin was also performed, with bases
2104–2127 of exon 3 (sense) and bases 2409–2432 of exon 4 (antisense)
genomic sequence as primers. Radioactive RNA products (associated with
[-32]CTP) were visualized by
autoradiography, and the amount of radioactivity was determined by
liquid scintillation. By the calculations of Danenberg et
al. (10)
, we estimated the initial amount of cDNA
template. A comparison of cpm/µl cDNA of MG50 RNA with
that of ß-actin provided a measurement of the relative expression of
MG50 DNA among the specimens. A ratio of MG50 cDNA to
ß-actin cDNA 
0.06, 2 SD, was considered a significant amount of
MG50.
Deducing Putative Epitopes Binding to HLA-A2.1.
The principal algorithm used was that of Falk and Rotzschke
(11)
, where the motif most suggestive of an
HLA-A2.1-binding peptide was *[LM]***[CFILMVWY]**[VL]. A
computer program selected regions within the deduced protein product of
MG50 with this motif. Motifs for HLA-A1 binding,
**[TSM][DEAS]****Y (12)
, and HLA-B7 binding,
*P******Y, were also used to scan the molecule for possible binding to
other common HLA class I molecules.
Binding Inhibition (Competitive Binding) Assay for Putative
Epitopes.
The method used for measuring the binding of putative epitopes to HLA
class I molecules was that of del Guercio et al.
(13)
. Briefly, 2 x 106
HLA-A*0201-transfected Drosophila
cells were incubated for 24 h with copper sulfate to induce HLA
expression. HBVc peptide 18–27 (FLPSDFFPSV) was radiolabeled with
125I as a standard. Unlabeled putative epitopes
at various concentrations were incubated with the Drosophila
cells at 26°C for 4 h, together with radiolabeled HBVc. The
cells were then layered on fetal bovine serum and centrifuged to
separate free and cell-bound peptide. Binding inhibition was calculated
as 100 x (1 - binding of HBVc in the
absence of unlabeled peptide/binding of HBVc in the presence of
unlabeled peptide). Optimal inhibition occurred at a concentration of
100 µg/ml of peptide. More than 20% inhibition of binding of the
HBVc standard at 100 µg/ml of peptide was considered sufficient for
the peptide to be examined further for immunogenicity.
Generation of CTLs with Peptides (Method 1).
A modification of the method of Rivoltini et al.
(14)
was used. PBMCs were plated at a concentration of
1.5 x 106
/ml in 2-ml capacity
wells, on 24-well plates, in Iskove’s modified DMEM + 10%
human AB serum (CM). One µg/ml of peptide was added to this
preparation. Two days later, 12 IU/ml of IL-2 were added. Weekly
restimulation of the PBMCs was performed by harvesting, washing, and
replating responders in new 24-well plates at a concentration of
2.5 x 105
cells/ml in CM
(5 x 105
cells/well). Autologous
PBMCs were thawed, washed twice, resuspended at 5–8 x 106
cells/ml, and pulsed with 10 µg/ml of
peptide in 15-ml tubes for 3 h at 37°C. These PBMCs were
irradiated at 3000 cGy, washed once, and added to the responders in
24-well plates at stimulator:responder ratios ranging from 3:1 to 10:1.
The next day, 12 IU of IL-2 were added. This weekly stimulation was
performed two to three times, judging by the activity of the CTLs in a
4-h 51Cr release assay and by IFN-
release.
After the final stimulation, the CTLs were stored frozen in 10% DMSO
in CM for further studies.
Generation of CTLs with Peptides (Method 2).
Drosophila cells were also used as APCs in an alternative
and more sensitive method (15)
. The Schneider S2
Drosophila cell line (deposited with the American Type
Culture Collection as CRL 10974) was transduced with HLA-A2.1, CD80
(B7-1), and CD54 (intercellular adhesion molecule-1) with a pRmHa-3
plasmid vector. The Drosophila cells were grown in
Schneider’s medium (106
cells/ml) with 10%
fetal bovine serum and CuSO4 at 27°C, the
optimal temperature for these insect cells. They were harvested,
washed, and resuspended in X-press medium (BioWhittaker) containing 100
µg/ml of the putative MG50 epitope.
CD8+ T cells were obtained from the PBMCs of a
normal donor by positive selection with anti-CD8 immunobeads
(Dynabeads; Dynal, Lake Success, NY) and a second antibody
(16)
. At least 92% of the resultant population were
CD8+, with the 
4% CD16+
and 4% CD4+. After incubation with the
MG50 peptide at 27°C for 3 h, the S2
Drosophila cells were incubated with the
CD8+ T cells at 37°C at a ratio of 1:10 in RPMI
1640 containing 10% autologous serum. Two days later, 20 IU of IL-2
and 30 IU of IL-7 were added to the growth medium. Incubation was
continued for 1 week, after which the Drosophila cells were
replaced with autologous, irradiated PBMCs (3000 cGy) and peptide. This
was repeated for one further round of stimulation, after which the
CD8+ T cells were tested for cytotoxicity by a
4-h 51Cr release assay.
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RESULTS |
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Through these methods, we were able to obtain the sequence of the
MG50 gene, which is shown in Fig. 2A
. A number of internal poly(A) sequences were found, which
may have accounted for regions of compression in the molecule and
caused many of the difficulties that we encountered in sequencing. 6.8
of the 8.1-kb were identified, continuous from the 3' end to 1.3 kb
from the 5' end. The remaining 1.3 kb at the 5' end were unidentifiable
by any of the methods we used. Similarly, the investigators who
reported a cDNA sequence overlapping with that of MG50 in
the 5' region did not obtain sequence at the very end of the
molecule.5
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. Many leucine-serine regions were found
within the molecule, which suggested that it might bind to chondroitin
sulfate. There were regions within the protein that had similarities to
several different molecules. Among these is a considerable homology in
regions 965-1057 and 1084–1102 with human peroxidase precursors,
including eosinophil peroxidase precursor, myeloperoxidase precursor,
and anti-thyroid peroxidase precursor. Perhaps the most striking
similarity was the 75% homology with a molecule related to human
peroxidases: peroxidasin. Peroxidasin is a newly described protein
combining peroxidase and extracellular matrix motifs, which is involved
in the formation of extracellular matrix during the embryogenesis of
hemocytes in Drosophila. It is also involved in phagocytosis
and defense in that organism (17)
. MG50 had
other regions with homology to human thrombospondin, human contactin,
human heparin sulfate core protein, and human axonin-1.
Probable Identity of MG50 as the Gene Encoding the
IL-1 Receptor Antagonist Protein.
Most recently, in the course of review for a patent for
MG50, we were made aware of information from the United
States Patent Office, suggesting that it might be closely homologous
with another gene. A cDNA clone encoding a portion of IL-1 receptor
intracellular binding protein (18)
was isolated by Drs.
L.-L Lin and J. Graham. The entire clone was 1571 bp, comprising 528 bp
in the coding region. These were identical with the 3' terminal portion
of the MG50 gene. The amino acid sequence of the encoded
protein fragment of 176 amino acids was also identical with a portion
of the MG50 protein. The doubly underlined region in Fig. 2A
shows the sequence of bases 3961 to 4488 in MG50 and in Fig. 2
B, the corresponding amino acid sequence of peptides 1321
to 1496 in our gene, which match exactly with the cDNA and encoded
peptide of Lin and Graham (18)
. The contiguous downstream
noncoding region of the Lin-Graham cDNA contained a sequence identical
with that of MG50, completing its 3' end. Thus, we think
that it is likely that MG50 is the full-length IL-1 receptor
binding protein (IL-1 receptor antagonist; IL-1Ra) gene.
Distribution of MG50 by Reverse Transcription-PCR.
"Clone 50" was originally chosen for further study because it was
expressed in four of five melanoma cell lines (MSM-M1, MSM-M3, MSM-M4,
and UCLA-M-21), a breast cancer cell line (734B), and a glioblastoma
(U138 MG) but not in myeloid (K562) or lymphoid (Daudi) tumor cell
lines, concanavalin A-induced lymphoblasts, or normal spleen. This
distribution was determined by in situ colony hybridization
with radiolabeled cDNA probes from human cells and tissues
(5)
. By PCR, we also found MG50 in an embryonal
rhabdomyosarcoma and in normal placenta (data not shown).
We repeated studies of the distribution of the gene with a quantitative
PCR (9)
on archived tissues and cell lines. By a
quantitative reverse transcription-PCR, comparing cpm/µl cDNA
library, of MG50 and ß-actin as an internal control,
MG50 was demonstrable mainly in melanoma and breast cancer
cells (Table 1)
. The skin melanoma cell line M3, ocular melanoma cell line OCM-3, and
two of four metastatic skin melanoma specimens were "positive" by
this assay. In addition, specimens of two of three mammary carcinomas,
an ovarian carcinoma, and one of two specimens of colon carcinoma (a
sigmoid carcinoma) were "positive." Specimens of soft tissue
sarcoma, pancreatic carcinoma, squamous cell carcinoma of the lung, and
pseudomyxoma peritonei were negative. Of the six normal tissues tested,
a specimen of skin was "positive," although a congenital nevus
specimen was negative. In addition, EBV-transformed lymphoblastoid
cells from patient M14 were negative.
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Examination of the Secondary Structure of the Protein.
The protein sequence encoded by the large open reading frame was 1496
amino acids. Its secondary structure was kindly examined by Drs. A. F.
Kirkin and J. Zeuthen, (Danish Cancer Society Institute of Cancer
Biology, Copenhagen, Denmark) with the GOR algorithm for secondary
structure prediction of Garnier et al. (19)
.
From their analysis, the protein was predicted to contain a paucity
(23%) of long -helical structures and, therefore, was thought to be
likely to unfold without difficulty. The other percentages of the
residues were: sheet, 28.6%; turns, 27.9%; and coil, 21.6%. (Full
data of the analysis are available upon request.) Consequently, Kirkin
and Zeuthen further predicted that the protein was likely to be
degraded by antigen-processing cells, exposing its epitopes to
CTL.6
We also analyzed the protein for "PEST" sequences, i.e., those rich in proline, glutamic acid, serine, threonine, and aspartic acid residues (20) . Such sequences are characteristic of regulatory proteins with a high turnover rate. They may also improve the potential for expression of epitopes if found outside the region of the epitopes themselves (21) . A 15-amino acid peptide in the untranslated region, 1793–1807, (RPEQEPLPDGSSQGR) had a very high PEST score of 8.05, with a PEST mole fraction of 38.68% and a hydrophobicity index of 26.44, but no PEST-rich sequence within the 1496 amino acid translated protein was identified. In fact, there were 18 regions of PEST-poor amino acids, two of which (68–87 and 614–635) included sequences we later found to be immunogenic.
Binding of Putative Epitopes Encoded by MG50 to HLA
Class I Molecules.
We examined the sequence of the MG50 protein with several algorithms
based upon motifs known to bind to various HLA class I molecules. A
decamer (1801–1810), which we now know is from the untranslated
region, was studied early in the course of this investigation. Its
sequence (RPRPEQEPLP) suggested that it might bind to HLA-B7
histocompatibility antigen; therefore, it was tested for binding to
that molecule (13)
through the courtesy of Dr. Esteban
Celis, then at Cytel Corporation (San Diego, CA). The decamer was found
to have an extremely high binding affinity for HLA-B7, 1.96
nM. Despite, or perhaps because of, the exceedingly strong
binding affinity, the peptide was not found to be immunogenic for CTLs,
as tested by in vitro immunization with matched
autologous PBMCs. Whether alternative in vitro immunization
procedures, such as with dendritic or Drosophila cells as
APCs, will give different results has not been tested. One potential
HLA-A1 binding motif, DVTSGNTVY, was also identified (peptide
272–280), but the peptide has not yet been studied further.
However, there were 12 peptides whose structure suggested binding to
HLA-A2.1, an HLA class I molecule found in 40–50% of Caucasians and
Asians. Each of the peptides had a leucine or methionine in position 2
and a leucine or valine in position 9. Table 2
illustrates the amino acid sequence of the 12 peptides. Their binding
to HLA-A2.1 was assayed by competition against the strongly binding
HBVc peptide 18–27 (HBc18–27; FLPSDFFPSV) as
the standard (13)
. No binding was found with five of the
peptides in a preliminary assay. Fig. 3
shows the results of the binding assay on the remaining seven peptides.
One of the seven (844–852) bound only weakly and was eliminated from
further consideration, whereas the other six had a moderate to strong
binding affinity that provided an impetus for their testing as
immunogens.
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All six peptides were capable of immunizing CTLs de novo to
recognize APCs presenting those peptides when presented on
HLA-A2.1+ T2 or HLA-A2.1-transduced Jurkat cells
(Figs. 4
and 5)
. Two of these peptides, 65–73 and 624–632, were identified by the
method of Rivoltini et al. (14)
and confirmed
with Drosophila as APCs; the other four were identified by
the more sensitive procedure with Drosophila APC. Fig. 4
shows the results of immunization with peptides 65–73, 68–76,
624–632, and 1050–1058 with autologous PBMCs as APCs. Testing was
performed with T2 cells after 3 weeks of stimulation in
vitro. Peptides 65–73 and 624–632 were able to generate immunity
against themselves, whereas by this method peptides 68–73 and
1050–1058 were not.
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shows the results of immunization by each of the six peptides
with Drosophila cells as APCs. After 3 weeks of
immunization, CD8+ CTLs were tested against the
corresponding peptide as presented on HLA-A2.1+
T2 or HLA-A2.1-transduced Jurkat (JA2) cells over a range of E:T
ratios. At a 50:1 ratio, the background reactivity against T2 or JA2
cells alone was generally high, but at ratios of 10:1 and 2:1 a clear
distinction of reactivity against peptide-bearing cells was obtained.
All six putative epitopes were found to be immunogenic by this method.
Reactivity of Peptide-immunized CTLs against Melanoma Cell Lines.
Most importantly, all six peptides, 65–73, 68–76, 209–217, 624–632,
1050–1058 and 1243–1251, immunized CD8+ T cells
to react against the long-term HLA-A2.1+ melanoma
cell lines MSM-M7, MSM-M14, and MALME-3M and against the short-term
(<6 months) HLA-A2.1+ IW melanoma cell line
(Fig. 6)
. Immunization against the melanoma cell lines was accomplished equally
well when the peptides were introduced singly or together in
Drosophila APCs (data not shown). There was no reactivity of
CTLs that lysed MALME-3M melanoma cells against control MALME-3
fibroblasts. These data indicated that the peptides were epitopes that
were naturally expressed on melanoma cells. We did not have a melanoma
cell line that was HLA-A2.1+ but did not express
MG50, which would have been a final control for specificity
of the reaction of CTLs.
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, we also tested the epitopes against a "control"
melanoma cell line, M3, which was a high expresser of MG50
mRNA but was HLA-A2.1 negative. By serotyping, M3 bears the following
alleles: HLA-A68, HLA-A30, HLA-B13, HLA-B27, HLA-C6, and
HLA-C8. None of these alleles was found in the
HLA-A2.1+ melanoma cell lines to which the CTLs
otherwise reacted. An epitope that was "classically" restricted
solely by HLA-A2.1 (HLA-A*0201) was 624–632, against which
CTLs were elicited that did not react against M3 cells. Reactivity of
CTLs directed against peptide 65–73 to M3 cells was low but only
slightly less than to the other melanoma cell lines. However, the other
four putative epitopes, 68–76, 209–217, 1050–1058, and 1243–1251,
although leading to a similar degree of lysis of the HLA-A2.1 melanoma
cell lines as 65–73 and 624–632, also elicited CTL reactivity against
the M3 melanoma cell line (Fig. 6)
. We assume that the reactivity of CTLs against epitopes 68–76, 209–27, and 1243–1251 as expressed on M3 cells was restricted by HLA-A*6802, which is closely related in amino acid sequence to HLA-A*0201. Although we have not formally tested whether cross-reactivity involved HLA-B and HLA-C alleles, no HLA-B or HLA-C alleles of M3 were common to the other melanoma cell lines
Also, because we did not clone the CTLs, we cannot be certain whether cross-reactivity was characteristic of the majority of the CTLs or was attributable to a subpopulation of "nonfastidious" (degenerate) CTLs.
Sequence of the Six Likely Epitopes.
To summarize, the sequences of the HLA-A2.1-restricted epitopes are:
65–73, CMHLLLEAV; 624–632, VLSVNVPDV; 68–76, LLLEAVPAV; 209–217,
TLHCDCEIL; 1243–1251, RLGPTLMCL; and 1050–1058, WLPKILGEV. The last
four sequences may also be recognized in the context of other HLA class
I alleles.
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DISCUSSION |
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Although we have not yet investigated the distribution of MG50 in a large number of normal tissues and tumor cells, evidence from this report and our previous studies (5) suggests that the gene is relatively restricted to tumors such as melanoma, breast cancer, ovarian cancer, and glioblastoma. MG50 was absent from hematopoietic (lymphocytic and myelocytic) tumor cell lines and archived specimens of normal tissues, with the exception of skin, but a cDNA sequence from MG50 was identified by others in fetal liver and spleen. Because MG50 appeared in our early work to be relatively tumor associated and potentially a useful immunogen and target for immunotherapy, we persisted in our attempts to sequence MG50 over nearly a decade.
We are hopeful that the MG50 protein will serve as a useful source of
immunogenic peptides for vaccination against several types of cancer,
overcoming some of the problems with immunization with the
cancer/testis (e.g., MAGE) group of antigens. The
disappointing results thus far with those peptides may have several
causes. Kirkin et al. (21)
suggested that
antigens recognized by CTLs have a structure that permits efficient
unfolding of the molecule, denaturation, and ubiquitination. The
cancer/testis group are highly organized stable structures, with
enrichment of long stretches of helices, and may not easily be
subject to degradation and proteasomal cleavage. In contrast, the
melanoma differentiation antigens, such as tyrosinase, are less highly
organized, which may explain their improved immunogenicity over the
MAGE group. Examination of the structure of the MG50 protein with the
GOR algorithm for stable structural proteins (19)
revealed
that it had a paucity of long -helical structures. It was concluded
that MG50 was likely to unfold easily and be degraded, presumably into
peptides recognizable by CTLs.6
The presence of
PEST-rich sequences outside the region of the epitopes would have
further strengthened the possibility of efficient and rapid degradation
of the protein. Unfortunately, the MG50 protein contains only one
PEST-rich sequences at its 3' end, and no others within the translated
region. Nevertheless, we have some confidence from its structure alone
that the whole MG50 protein, or the gene encoding it, will offer a
feasible method of immunization against a large variety of epitopes of
various HLA restrictions. There are undoubtedly additional epitopes
binding to HLA molecules other than HLA-A2.1, beyond the six we have
identified for that common haplotype.
Its CTL-defined epitopes, which were naturally represented on melanoma cells, indicated that the novel gene MG50 encoded a previously undescribed melanoma antigen. We found six epitopes recognized by CTLs within the MG50 protein, which is very different from the paucity of CTL-defined epitopes even on large-sized viruses (22) . Two epitopes, 65–73 (CMHLLLEAV) and 624–632 (VLSVNVPDV), were demonstrable on melanoma cells and were classically HLA-A2.1 restricted. Four other epitopes, 68–76 (LLLEAVPAV), 209–217 (TLHCDCEIL), 1050–1058 (WLPKILGEV), and 1243–1251 (RLGPTLMCL), were recognized not only on HLA-A2.1+ melanoma cell lines but also on the M3 melanoma cell line (HLA-A68, HLA-30). This is not entirely surprising because HLA-A*0201 and HLA-A*6802 are members of the same HLA supertype (23) . We have also described "nonfastidious" CTLs in patients with skin or ocular melanoma that cross-reacted against melanoma cell lines with HLA class I haplotypes outside the HLA-A2 supertype (24 , 25) . However, we have not determined whether the four novel epitopes we have described here can be recognized outside the HLA-A2 supertype family.
For active immunotherapy, our bias has been toward the use of polyvalent or multiepitopic immunogens rather than a single peptide (26 , 27) . Whether the gene, the whole protein, or selected epitopes will prove to be most useful in creating a synthetic melanoma or breast cancer vaccine is uncertain. The methods we used to demonstrate the presence of epitopes, especially the use of purified APCs such as Drosophila or dendritic cells, can be adapted for use in in vivo or in vitro vaccination. Several methods of incorporating epitopes into dendritic cells have been described (28) , including our own procedure for adding signal sequences that conduct nonamers into the endoplasmic reticulum (29) . Likewise, several viral vectors conducting minigenes or entire genes into dendritic cells have been developed. We favor the use of lentiviral vectors, some of which have the capacity to transduce genes as large as 10 kb into the DNA of nonreplicating cells. The characteristics of translated MG50 protein should permit it to unfold and be degraded within APCs and then be recognized by CD4+ and CD8+ T cells.
Besides cancer vaccines, melanoma epitopes could be used for in vitro immunization of CTLs, to be administered as adoptive immunotherapy. In a Phase I trial, 5 x 106 CTLs immunized ex vivo against a tyrosinase epitope, YMNGTMSQV, with Drosophila cells as APCs elicited a clinical partial remission lasting over 1 year in 1 of 10 patients.7 A comparison of these strategies will enable us to determine which construct and therapeutic approach with MG50 are most useful for specific immunotherapy.
Sequence data at the 3' end of the gene strongly suggest that MG50 is identical with the IL-1 receptor antagonist protein (IL-1Ra) gene, cDNA of which was identified independently by Lin and Graham of Genetics Institute (Cambridge, MA; PCT/US96/06363; Ref. 18 ). Like the panoply of other genes for tumor-associated antigens, such as tyrosinase and gp100 in melanoma, and c-erbB2 and the mucins in adenocarcinomas, MG50 is not unique to tumors. However, like the others, it may be overexpressed in tumors and thus identifiable by CTLs as a potential target for cellular immunotherapy, regardless of its expression in normal cells.
The function of the IL-1Ra in a melanoma cell is uncertain but may
represent another example of the antagonism of the tumor cell to
cytokines that aid the host in rejecting it. IL-1 is a proinflammatory
cytokine produced by bone marrow and bone marrow-derived cells such as
macrophages. A component of what was originally called
"lymphocyte-activating factor" (30, 31, 32)
, IL-1
production is stimulated from macrophages by immunological adjuvants
such as Bacillus Calmette Guérin, in vivo
and in vitro (33, 34, 35)
. IL-1 is important in the
inflammatory cascade for up-regulating the expression of adhesion
molecules, recruitment of neutrophils, and inducing other cytokines
(36)
. These activities and others are blocked efficiently
by the IL-1 receptor antagonist protein. After administration of
lipopolysaccharide (endotoxin), IL-1Ra levels increase within 2 h
in the serum of normal human subjects (37)
. The secretory
IL-1Ra gene is controlled through three
lipopolysaccharide-responsive elements, one of which was found to be an
nuclear factor-
B binding site (38)
. In contrast, IL-18,
which induces IL-1, does not induce anti-inflammatory cytokines such as
IL1-Ra or IL-10 (39)
.
Among its positive activities, IL-1Ra was useful in overcoming
experimental "postmenopausal osteoporosis," decreasing bone loss
and bone resorption in ovariectomized rats (40)
. In the
tumor setting, however, it is plausible that, similar to IL-10,
transforming growth factor-ß, and prostaglandin
E2, IL-1Ra functions to subvert the activity of
antitumor lymphocytes. IL-1 released in the vicinity of the tumor by
macrophages may be blocked in its activity if IL-1Ra binds to IL-1R in
antitumor lymphocytes. Burger et al. (41)
found
high levels of IL-1Ra in the ascites of 18 patients with advanced
ovarian cancer. Ascites tumor cells spontaneously released IL-1Ra and
TNF-/ß binding protein BP55, and tumor-associated macrophages
released IL-1Ra and the TNF-/ß binding protein BP-75.
Lymphocytes from the ascites of these patients released no TNF- or
TNF-ß. Although our major interest has been in T cell-defined
epitopes, it is possible that targeting of MG50 with
monoclonal antibodies or antisense oligonucleotides will be useful for
selective inhibition of tumor-induced immunosuppression mediated by
IL-1Ra.
|
ACKNOWLEDGMENTS |
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|
FOOTNOTES |
|---|
1 Supported by USPHS Grant RO1-CA57846 and a
grant from the A-H Foundation. 
2 To whom requests for reprints should be
addressed, at Hudson-Webber Cancer Research Center, Room 740.2,
Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, MI 48201.
Phone: (313) 966-7852; Fax: (313) 966-7558; E-mail: mitchell{at}karmanos.org
3 The abbreviations used are: IL, interleukin;
MAGE, melanoma antigen; MG, melanoma gene; HBVc, hepatitis B virus
core; PBMC, peripheral blood mononuclear cell; CM, complete medium;
APC, antigen-presenting cell; TNF, tumor necrosis factor.
4 T. Nagase, N. Seki, K. Ishikawa, O. Ohara, and
N. Nomura. Prediction of the coding sequences of unidentified human
genes. VI. The coding sequences of 80 new genes (KIAA 0201–KIAA
0280) deduced by analysis of cDNA clones from human cell line
KG-1 and brain, August 2, 1996.
5 N. Nomura, personal communication, October 4,
1996.
6 A. F. Kirkin and J. Zeuthen, personal
communication, 1998.
7 M. S. Mitchell, D. Darrah, D. Yeung, S.
Halpern, A. Wallace, V. Jones, and J. Kan-Mitchell. Adoptive
immunotherapy of melanoma with cytolytic T lymphocytes immunized
in vitro against a tyrosinase epitope: Phase I trial,
submitted for publication.
Received 4/10/00. Accepted 9/19/00.
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