Except for melanomas, tumor antigens recognized by cytotoxic T lymphocytes (CTLs) are yet
unidentified. We have identified a gene encoding antigenic peptides of human squamous cell
carcinomas (SCCs) recognized by human histocompatibility leukocyte antigens (HLA)-
A2601-restricted CTLs. This gene showed no similarity to known sequences, and encoded
two (125- and 43-kilodalton [kD]) proteins. The 125-kD protein with the leucine zipper motif
was expressed in the nucleus of the majority of proliferating cells tested, including normal and
malignant cells. The 43-kD protein was expressed in the cytosol of most SCCs from various
organs and half of lung adenocarcinomas, but was not expressed in other cancers nor in a panel
of normal tissues. The three nonapeptides shared by the two proteins were recognized by the
KE4 CTLs, and one of the peptides induced in vitro from peripheral blood mononuclear cells
(PBMCs) the CTLs restricted to the autologous tumor cells. The 43-kD protein and this nonapeptide (KGSGKMKTE) may be useful for the specific immunotherapy of HLA-A2601+ epithelial cancer patients.
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Introduction |
Many genes encoding tumor-rejection antigens recognized by CTLs were identified from cDNA of
melanomas (1). Further, a large number of nonapeptides
recognized by HLA class I-restricted CTLs cytotoxic to
melanoma cells have been identified in the past five years
(5). Some of them are under clinical trials as cancer vaccines, and major tumor regression in several HLA-A1+
melanoma patients was reported in the vaccine therapy
with the melanoma antigen (MAGE)-3 peptide (16). Therefore, these nonapeptides recognized by the CTLs could be
a new tool for the specific immunotherapy of melanoma.
However, no peptides are yet identified from human squamous cell carcinomas (SCCs)1 one of the major human cancers, except for a mutated CASP-8 (17). We previously reported the HLA-A2601-restricted and tumor-specific CTL
line recognizing peptide antigen(s) expressed on SCCs (18). In this study, we have investigated a gene encoding tumor
antigen recognized by this CTL line, and report a new gene
encoding two novel proteins and three nonapeptides as the
antigens recognized by the HLA-A2601-restricted CTLs.
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Materials and Methods |
Identification of 6A1-1D7 Genes.
Expression-gene cloning methods developed by T. Boon and colleagues (4, 6) were used in this
study to identify a gene coding tumor antigen recognized by the
KE4 CTLs (18). In brief, messenger RNA (mRNA) of the KE4
tumor cells was converted to cDNA, ligated to SalI adapter, and
inserted into the expression vector pSV-SPORT-1 (GIBCO
BRL, Gaithersburg, MD). cDNA of HLA-A2601 or HLA-A0201
was obtained by reverse transcription PCR (RT-PCR), and was
cloned into the eukaryotic expression vector pCR3 (Invitrogen, San Diego, CA). Both 200 ng of plasmid DNA pools or clones of the KE4 cDNA library and 200 ng of the HLA-A2601 cDNA
were mixed with 1 µl of lipofectin in 70 µl of OPTI-MEM®
(GIBCO BRL) for 15 min. 30 µl of the mixture was then added
to the VA13 (2 × 104) cells and incubated for 5 h. 200 µl of the
RPMI-1640 medium containing 20% FCS was then added and
cultured for 2 d followed by adding the KE4 CTLs (104 cells/
well; reference 8). After a 18-h incubation, 100 µl of supernatant
was collected to measure IFN-
by an ELISA kit (Otsuka Pharm.
Co., Tokyo, Japan) in a duplicate assay. DNA sequencing was
performed with dideoxynucleotide sequencing method using
DNA Sequencing kit (Perkin-Elmer Corp., Foster, CA) and analyzed by ABI PRISMTM 377 DNA Sequencer (Perkin-Elmer).
Northern Blot Analysis.
Nylon membranes (Hybond-N+; Amersham, Buckinghamshire, UK) with UV-fixed total RNAs (5 µg/lane) extracted from the various cells or UV-fixed poly A+
RNA (2 µg/lane) from various tissues were prehybridized for
10 min and hybridized overnight at 65°C in the same solution
(7% SDS, 1 mM EDTA, 0.5 M NaH2PO4, pH 7.2) containing
32P-labeled 6A1-1D7 probe. The membranes were washed three
times at 65°C in a washing buffer (1% SDS, 40 mM Na2HPO4,
pH 7.2), and then autoradiographed. The relative expression level
of the squamous cell carcinoma antigen recognized by T cells
1 (SART-1) mRNA was calculated by the following formula:
index = (SART-1 density of a sample/
-actin density of a sample) × (-actin density of the KE4 tumor/SART-1 density of the
KE4 tumor).
Cloning of the SART-1 Gene.
We tentatively named this gene
encoding a tumor antigen recognized by the KE4 CTLs as the
SART-1 gene. The SART-1 clone was obtained from both PBMCs
(SuperScriptTM Human Leukocyte cDNA Library in pCMV-SPORT; GIBCO BRL) and KE4 cDNA libraries by the standard
colony hybridization method with the 32P-labeled 6A1-1D7 cDNA
as a probe. Sequence data of the SART-1 are available from
EMBL/GenBank/DDBJ under accession number AB006198. The
difference of the sequence at nucleotide (nt) position 812 of the
SART-1 between PBMCs and KE4 was further analyzed by treatment of the PCR products with an SecI restriction enzyme. Amplification was performed for 30 cycles (1 min at 94°C, 2 min at
56°C, and 2 min at 72°C) with the primers of 5
-CCAAGTTACTGGAGGAGATGG-3 (forward primer) and 5-TTGGACAGGATAGAGCGAGG-3 (reverse primer).
Preparation of Glutathion S-transferase Fusion Proteins and Rabbit
Antisera.
For SART-1800/GST (GST, glutathione s-transferase) protein, the full length of SART-1 was digested with EcoRI and NotI
at the multiple cloning site of pCMV-SPORT, and then ligated
into the pGEX-4T-2 vector (Pharmacia Biotech AB, Uppsala,
Sweden). For SART-16A1-1D7/GST protein, the SART-1 cDNA
fragment (nt 1,663-2,449) was amplified by PCR using a forward
primer 5-TGGGAATTCGATGAGGATCCCGAGC-3 (sf-1),
and a reverse primer 5-TACGGGCGGCCGCTGTCACTTGGT-3 (sr-1). Amplified product was digested with EcoRI and
NotI, and ligated to the pGEX-5X-3 (Pharmacia Biotech AB).
For SART-167/GST protein, the SART-1 cDNA fragment (nt 1,663-
1,866) was amplified by PCR using a sf-1 primer and a reverse
primer 5-CGTGAATTCTCACCGTGCTCCAGCC-3. Amplified product was digested with EcoRI and ligated to the pGEX-5X-3. For SART-1219/GST protein, the SART-1 cDNA fragment
(1,781-2,449) was amplified by PCR using a forward primer
5-GAGAATTCCATGGACTTTGAACGGGATG-3 (sf-2) and
a sr-1 primer. Amplified product was digested with EcoRI and NotI,
and ligated to the pGEX-5X-3. Polyclonal anti-SART-1800/GST, anti-SART-16A1-1D7/GST, anti-SART-167/GST, and anti-SART-1219/GST Abs were prepared by immunization of rabbits with purified
SART-1800/GST, SART-16A1-1D7/GST, SART-167/GST, and SART-1219/GST proteins, respectively, by the methods previously reported (19).
Preparation of SART-1-tag Fusion Protein in Expression Vector
Constructs.
For preparation of the SART-1800/myc, the SART-1
of positions 29-2,449 was amplified by PCR using a forward
primer 5-GCTCGGAATTCACGTGCCACTATGGG-3 and
a reverse primer 5-AGGGAATTCTCGCTTGGTGATGGTGTTC-3 (sr-2). Amplified product was digested with EcoRI,
and ligated to pcDNA3.1/Myc-His vector (Invitrogen). The
gene encoding a tag peptide was ligated to the 2,438 position before the stop codon of the third frame, which was used as the
SART-1800/myc. Similarly, the SART-1 fragment of positions 1,663-
2,449 or 1,782-2,449 was amplified by PCR using a sf-1 and a
sr-2 primer or a sf-2 primer and a sr-2 primer, and the amplified
product was used for preparation of the SART-16A1-1D7/myc or
SART-1219/myc, respectively.
Western Blot Analysis.
The samples were lysed with a buffer
consisting of 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Triton
X-100, 0.2 mM PMSF (Sigma Chemical Co., St. Louis, MO),
and 0.03 one trypsin inhibitor unit (TIU)/ml aprotinin, sonicated, and centrifuged at 14,000 rpm for 20 min, and the supernatant was used as the cytosol fraction. Then, the pellet was lysed
with a buffer consisting of 7.2 M urea, 1.6% Triton X-100, 0.8%
dithiothreitol, and 2% lithium dodecyl sulfate, and was centrifuged, and the supernatant was used as the nuclear fraction. The
lysate was separated by SDS-PAGE. The proteins in acrylamide
gel were blotted to HybondTM-polyvinylidene difluoride (PVDF)
membrane (Amersham) and were incubated with appropriate Abs
for 4 h at room temperature. The other methods of Western blot
analysis were previously described (19).
KE4 CTL, Its Sublines, and CTL Assay.
HLA-A2601-restricted
and SCC-specific CTL line (KE4 CTL) established from an
esophageal cancer patient (18) was used in this study as effector
cells for identification of the peptide antigens encoded by the
SART-1 gene. KE4 CTL sublines were established from the parental KE4 CTL line by the limiting dilution culture as reported
(20). In brief, 1 cell/well (round bottomed 96-well microculture
plate) of the parental KE4 CTL line was incubated with the culture medium (45% RPMI-1640 medium, 45% AIM-V® medium
[GIBCO BRL], and 10% FCS [EQUITECH BIO, Ingram, TX]
with 100 units/ml of IL-2 [Shionogi Pharm. Co., Osaka, Japan]
and 0.1 mM MEM nonessential amino acids solution [GIBCO
BRL]; termed as the culture medium) in the presence of irradiated (50 gray) allogenic PBMCs (2 × 105 cells/well) donated
from three healthy volunteers as feeder cells. The proliferating
CTL sublines were expanded in wells of 24-well microculture
plates in the culture medium alone for up to 30 d. The sublines
were tested for their cytotoxicity to the KE4 (A2402/2601), KE3
(A2402/A0201), and VA13 fibroblast cell lines in a 6-h 51Cr-
release assay as reported (18) at an E/T ratio of 5:1, and the 80 sublines showing cytotoxicity against the KE4, but not either
KE3 or VA13, were used in this study.
Constructions of Deletion Mutants.
The pcDNA3/6A1-1D7 plasmid, a derivative of the pcDNA3 vector containing a 990-bp
DNA fragment of the 6A1-1D7 gene corresponding to the nucleotide positions 1,517-2,506 of the SART-1 gene and a CMV
promoter for directing transcription, was digested with NotI for
preparation of deletion mutants. The linear lysed DNA was subjected to the second restriction enzyme ApaI digestion to generate one end sensitive to ExoIII. ExoIII nuclease/Mung bean nuclease was performed according to the manufacturer's instructions (TaKaRa, Ootsu, Japan) to obtain five deletion mutants of the 6A1-1D7 (6A11-492 corresponding to nucleotide positions 1-492 of the 6A1-1D7 gene, 6A11-625, 6A11-736, 6A11-839, and 6A11-951). The SART-11-1,668 fragment was prepared by digestion of the
SART-1 in pSV-SPORT with the KpnI and BamHI, separated
by agarose gel electrophoresis and purified by Qiaex gel extraction kit (Qiagen, Hilden, Germany). This fragment was ligated to
the KpnI and BamHI sites of 6A11-492, 6A11-625, 6A11-736, 6A11-839,
and 6A11-951 in pcDNA3 vector, respectively, and five mutants
(SART-11-2,008 corresponding to nucleotide positions 1-2,008,
SART-11-2,141, SART-11-2,252, SART-11-2,355, and SART-11-2,467)
were obtained. Further, the SART-1 gene in pCMV-SPORT
was digested with BamHI, ApaI, or SmaI, respectively, and each
band was separated, purified, and ligated to prepare the three mutants (SART-11-793, SART-11-1,190, and SART-11-1,668).
Peptides and Assays.
In this manuscript, amino acid (aa) positions were named based on the sequence of the predicted SART-1800
protein because all the synthesized peptides are located in the region shared by both the SART-1800 and SART-1259 proteins that
was translated in the third frame. A series of 22 different 10 mer,
according to the predicted aa sequences corresponding to a part
of deduced SART-1800 protein (aa positions 730-800: SHRFHGKGSGKMKTERRMKKLDEEALLKKMSSSDTPLGTVALLQEKQKAQKTPYIVLSGSGKSMNANTITK), were prepared.
Each peptide is a 10 mer that shares the same aa with the following peptide at positions 4-10. Six different nonapeptides were also
prepared in which each of the three 10 mer (SART-1736-745, SART-1748-757, SART-1784-793) was deleted at position 1 or 10. Further, each aa of the three nonapeptides (SART-1736-744,
SART-1749-757, SART-1785-793) was substituted by glycine (G)
when it was not glycine or by threonine (T) when it was glycine
to determine aa residues critical for binding to HLA-A2601 and
CTL-mediated recognition. These peptides were purchased from
Biologica (Nagoya, Japan). The purity was >70% in most of the
peptides, and >95 % in those used for induction of CTLs. For
detection of antigenic peptides, the HLA-A2601 or -A0201 cDNA
(as a control) were transfected to the VA13 (2 × 104) cells and incubated for 5 h. Then, 200 µl of the RPMI-1640 medium containing 20% FCS was added and cultured for 2 d, followed by adding the peptides at the concentration of 10 µM in most experiments, or 10 nM to 50 µM in certain experiments. 2 h later, the
supernatant was removed and the KE4 CTLs (104 cells/well)
were added, incubated for 18 h, and 100 µl of supernatant was
collected to measure IFN- by an ELISA kit in a duplicate assay.
Induction of CTL by Nonapeptides.
PBMCs (2 × 106) from a
KE4 patient that had been cryopreserved in a nitrogen tank were
thawed in the morning of experiments, and were incubated with
a nonapeptide (10 µM) in a well of a 24-well plate containing
2 ml of the culture medium. PBMCs from HLA-A2601+ healthy
volunteers were also used. At days 7 and 14 of culture, cells were
collected, washed, and stimulated with antigen presenting cells
consisting of the irradiated autologous PBMCs that had been preincubated with the same nonapeptide (10 µM) for 2 h followed by washing with PBS. The ratio of the responder to stimulator cell was 10:1. Cells were harvested at day 21 of the culture, and
most of them were tested for their CTL activity in a 6-h 51Cr-
release assay. Some of them were provided for preparation of the
CTL sublines by incubation of 10 cells/well (round bottomed 96-well microculture plate) with the culture medium in the presence of irradiated allogenic PBMCs as feeder cells. These cells
from the microculture were tested for their activity at 10 d of culture to produce IFN- in response to tumor cells by an ELISA.
Several sublines from well-proliferating wells were further expanded in the culture medium alone in wells of a 24-well plate,
and were tested for their cytotoxicity to the KE4 and KE3 tumor
cells at an E/T ratio of 5:1 in a 6-h 51Cr-release assay at 15 d of
the culture.
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Results |
Identification of the 6A1-1D7 Gene.
The total of 105 cDNA
clones from cDNA library of the KE4 tumor cells were
tested for their ability to stimulate IFN- production by
the KE4 CTLs after cotransfection with the HLA-A2601
into the VA13 human fibroblast cells. This method allows
identification of genes encoding tumor-rejection antigens
(1). After repeated experiments for the several candidate
clones, one clone (6A1-1D7) was confirmed to encode a
tumor antigen recognized by the KE4 CTLs when cotransfected with HLA-A2601 (Fig. 1 A). The sequence of this
cDNA clone proved to be 990 bp long. Expression of this gene was investigated by Northern blot analysis. A band of
~2.6 kb was observed in all the normal tissues and tumor
cell lines tested (Fig. 2). The relative level of mRNA expression was within the range of 2.3 ± 2.9 in all the samples except for testis (the expression level: 7.5) and pancreas
(17.4) (Fig. 2). These results suggest that this gene was
ubiquitously expressed at the mRNA level with higher expression in testis and pancreas, and that the 990-bp-long
cDNA was incomplete.
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Fig. 1.
Recognition of the SART-1 gene products by the KE4
CTLs. Different amounts of the 6A1-1D7 (Fig. 1 A) or the SART-1
cloned from the KE4 tumor (Fig. 1 B) and 100 ng of HLA-A2601 or
HLA-A0201 cDNA were cotransfected into VA13 cells, followed by testing their ability to stimulate IFN- production by the KE4 CTLs. The
background of IFN- production by the KE4 CTLs in response to VA13
cells (~200 pg/ml) was subtracted in the figure. Similar results were obtained in the SART-1 cloned from the PBMCs (data not shown).
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Fig. 2.
Expression of the SART-1. 21 tumor cell lines (KE4, KE3,
TE8, Kuma-1, HSC4, QG56, Sq-1, A549, MKN28, Colo201, SW620,
KMG-A, R-28, 86-2, LK79, LC65A, KIM-1, KYN-1, M36, M73,
NALM-1; reference 18), PBMCs, Bec-1, COS, or 16 tissues (heart,
brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas on human multiple tissue Northern blot, and spleen, thymus, prostate, testis,
uterus, small intestine, colon, and peripheral blood leukocyte on human multiple tissue Northern blot IV; Clontech Lab., Inc., Palo Alto, CA)
were provided for Northern blot analysis with the 6A1-1D7 as a probe.
Some of the results are shown in the figure.
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Identification of the SART-1 Gene.
A 2,506-bp-long gene
was independently cloned from the cDNA libraries of KE4
tumor and PBMCs of healthy donors using the 6A1-1D7 as a probe (Fig. 3). The (nt) sequences of these clones were
identical with the exception of the position 812 (cytosine
in the KE4 versus thymine in PBMCs). This would be due
to a genetic polymorphism, but not due to a point mutation, since the samples in which the nt position 812 was
cytosine were the KE4 CTLs, a B cell line from the KE4
patient (Bec-1), fetal liver, COS cells, and 16 of 22 solid
tumor cell lines tested, whereas it was thymine in testis, VA13 cells, and the other six tumor cell lines. An aa translated from these codons in the third frame is identical
(ACC, ACU = threonine). KE4 CTLs also recognized
VA13 cells cotransfected with these new genes and HLA-A2601 (Fig. 1 B). Both clones contained the 6A1-1D7 at
positions 1,517-2,506. This 2,506-bp-long gene showed
no similarity to known sequences, and was tentatively named as the SART-1 gene. Although the SART-1 mRNA was
ubiquitously expressed, the KE4 CTLs did not recognize
nonmalignant cells including Bec-1 cells (18) or VA13 cells
transfected with HLA-A2601 alone (Fig. 1). This might be
due to preferential expression of tumor antigens on the malignant cells by the mechanism of posttranscriptional regulation.
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Fig. 3.
Nucleotide sequence of the SART-1. The cloned cDNA
(6A1-1D7) was initially provided for the nucleotide sequencing. The sequence of 6A1-1D7 is 990 bp long (positions 1,517-2,506, underlined by the solid line) that has an ORF of 201 bp long encoding 67 aa if the first
AUG codon (1,663-1,665, underlined by the bold line) and stop codon
(1,864-1,866, underlined by the dotted line) in the first frame are used for
protein synthesis. One S-D and each of the two different S-D-like sequences are marked by the dot on the top. The 2,506-bp-long SART-1
was then cloned from cDNA libraries of the KE4 tumor and human
PBMCs. The SART-1 has an ORF 2,400 bp long encoding 800 aa when
the first AUG codon (39-41, underlined by the bold line) and stop codon
(2,439-2,441, underlined by the dotted line) are used for protein synthesis in
the third frame. There was only one nt difference at the position 812 (marked by  ) between the SART-1 of KE4 tumor and PBMCs (cytosine in the KE4 tumor versus thymine in PBMCs). These sequence data
are available from EMBL/GenBank/DDBJ under accession number
AB006198.
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We then intended to investigate the SART-1 protein
expression in various cells and tissues by Western blot analysis with anti-SART-1800/GST and -SART-16A1-1D7/GST Abs,
since both the SART-1 and 6A1-1D7 encoded a tumor
antigen recognized by the KE4 CTLs. The first AUG
codon resided at positions 39-41 of the SART-1 in the
third frame with suitable context (CCACUAUGG; Fig. 3)
for initiation of protein synthesis (21, 22). The SART-1
thus contains an open reading frame (ORF) of 2,400 bp
encoding a protein of 800 aa residues (SART-1800). In
contrast, the first AUG codon of the 6A1-1D7 exists at
positions 1,663-1,665 with unfavorable content (GGAGGAUGA) in the first frame. Between this first AUG and the stop UGA (1,864-1,866) codon of the 6A1-1D7, there is
one Shine-Dalgarno (S-D) sequence (AGGAGG, 1,771-
1,776), one S-D-like sequence (AGGGGG, 1,681-1,686),
and the other S-D like sequence (GGAG at seven different regions) that are known to induce frame shifting in prokaryotic mRNAs (23, 24). A protein of 259 aa (SART-1259)
could be translated if any of these S-D sequences induces
1 frame shifting and change the stop codon from the positions 1,864-1,866 of the first frame to the positions
2,439-2,441 of the third frame. If not, a peptide of 67 aa
could be translated in the first frame.
Expression of the SART-1800 Protein.
An Ab to the SART-1800/GST recognized a 125-kD band of SART-1800 protein
after cleavage of GST with thrombin (data not shown), and
recognized a 125-kD band in the nuclear fraction of
PBMCs activated with 10 µg/ml of PHA (PHA blasts),
KE4 tumor, and Bec-1, but not unstimulated PBMCs (Fig.
4 A). No protein in the cytosol was recognized by this Ab
in any samples tested. The 125-kD band was also expressed
in the nucleus of the majority of tumor tissues, tumor cell
lines, and normal cell lines tested, but was not expressed in
normal tissues except for testis and fetal liver. The summary is shown in Table 1. When the SART-1 of positions 29-
2,449 (SART-1800) was transfected to VA13, intensities of
the 125-kD band in both the nuclear and cytosol fractions
increased (Fig. 4 A). Furthermore, this Ab and anti-myc
monoclonal Ab recognized a 132-kD band of the VA13
cells transfected with the SART-1 of positions 29-2,449 in
conjunction with pcDNA3.1/Myc-His vector (SART-1800/myc; Fig. 4 A). The different migration of these bands (125 and
132 kD) will be due to a tag peptide (theoretically ~5 kD).
These results suggest that the 125 kD of the SART-1800 protein was expressed in the nucleus of proliferating cells including normal and malignant cells, but not in nonproliferating
cells, nor any normal tissues except for testis and fetal liver.
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Fig. 4.
Expression of SART-1800 and SART-1259 proteins. Tumor
cell lines used for Western blot analysis were head and neck SCCs (Ca9-22, HSC3, HSC4, Kuma-1, and Kuma-3), esophageal SCCs (KE4, KE3,
TE8, TE9, TE10, and TE11), lung adenocarcinomas (1-87, LK87, PC-9, A549, 11-18, and RERF-LC-MS), lung SCCs (Sq-1, RERF-LC-AI, and QG56), leukemia cells ( MOLT-4, HPB-ALL, HPB-MLT, HUT-102,
BALL-1, NALM16, ARH77, THP1, U937, HL60, ML-1, ML-2,
NALL-1, SPI-801, K562, and HEL), and melanomas (M36, M73) (18).
PBMCs, PHA blasts, fibroblast cells (WI-38, VA13), and tumor tissues
from various organs were also studied. (A) Expression of the SART-1800
protein was investigated by Western blot analysis with anti-SART-1800/GST Ab. Anti-myc monoclonal Ab (Invitrogen) was also used for analysis of
VA13 transfected with the SART-1800/myc. (B) Expression of the SART-1259
was investigated with anti-SART-16A1-1D7/GST Ab. In the left gel, 70-, 43-, and 27-kD bands corresponded to the SART-16A1-1D7/GST, SART-16A1-1D7,
and GST, respectively. The data of the cytosol fraction were shown. No
bands were detected by this Ab in the nucleus of any samples tested (data
not shown). (C) Expression of the SART-1259 was investigated with anti-
SART-167/GST and anti-SART-1219/GST Abs. The data of the cytosol fraction were shown. (D) Anti-myc and anti-SART-1219/GST Abs were used
for analysis of COS cells transfected with the SART-16A1-1D7/myc or SART-1219/myc. The total lysate was used for experiments.
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Expression of the SART-1259 Protein.
An Ab to the SART-16A1-1D7/GST recognized a 43-kD band of the recombinant
SART-16A1-1D7 protein after cleavage of GST with factor
Xa (Fig. 4 B). Therefore, the SART-1259 could be translated by the mechanism of 1 frame shifting in the
prokaryotic mRNA, and be recognized by anti-SART-16A1-1D7/GST Ab. This Ab also recognized a 43-kD protein
in the cytosol of KE4 and TE9 esophageal SCC cell lines,
fresh esophageal SCCs, and lung SCCs and adenocarcinomas, but not PBMCs (Fig. 4 B). No protein in the nucleus
was detected by this Ab in any samples tested (data not
shown). The 43-kD protein was expressed in the cytosol of all the head and neck SCC tissues tested, 60% of esophageal
SCCs, and half of the lung SCCs and lung adenocarcinomas, but not observed in leukemia, melanomas, nor any
normal tissues, normal cell lines, or normal cells except for
fetal liver and testis (Table 1). These results suggest that the
SART-1259 protein was translated by the mechanism of 1
frame shifting using an internal ribosomal entry site in human mRNAs primarily from SCCs and adenocarcinomas, and was recognized by this Ab.
To investigate this possibility, we developed rabbit Abs
against GST fusion protein with a peptide of 67 aa in the
first frame (SART-167/GST) and a protein of 219 aa in the
third frame (SART-1219/GST), since each of the two proteins is necessary for construction of the SART-1259. Both
anti-SART-167/GST and anti-SART-1219/GST Abs recognized a 43-kD band in the cytosol of the KE4, but not of PBMCs or PHA blasts (Fig. 4 C). These Abs also recognized a 43-kD protein of the other SCCs and lung adenocarcinomas, and the pattern of the reactivity was almost
identical to that of anti-SART-16A1-1D7/GST Ab shown in
Table 1. The results suggest that this 43-kD protein consists
of both a peptide of 67 aa in the first frame and a protein of
219 aa in the third frame. Furthermore, we prepared the plasmid construct in which the part of the SART-1 at nt positions
1,663-2,449 or 1,782-2,449 was ligated into the pcDNA3.1/
Myc-His vector (SART-16A1-1D7/myc and SART-1219/myc, respectively). When the SART-16A1-1D7/myc was transfected
to COS cells, two bands (48 and 43 kD) were detected with both anti-myc monoclonal and anti-SART-1219/GST
Abs, whereas only a 43-kD band was detected in COS cells
transfected with the SART-1219/myc (Fig. 4 D). The 48-kD
protein might consist of 43 kD of SART-1259 plus 5 kD of
a tag peptide that would be initiated by the AUG codon at
positions 1,663-1,665 with the mechanism of 1 frame
shifting. On the other hand, the 43-kD protein might consist of the 38-kD protein of the SART-1219 plus 5 kD of a
tag peptide that would be initiated by the AUG codon at
positions 1,782-1,784 in the third frame.
Identification of Regions Containing Antigenic Peptides for
CTLs.
To identify antigenic peptides encoded by the
SART-1 gene, we investigated the capability of deletion
mutants of both the 6A1-1D7 and SART-1 genes to stimulate IFN- production by the KE4 CTLs, since both
genes encoded tumor antigens recognized by the KE-4 CTL
as shown in Fig. 1. Higher levels of IFN- production were observed in the 6A11-990 (full length), 6A11-951, and
6A11-839 when cotransfected with HLA-A2601 into VA13
cells (Fig. 5 A). In contrast, no IFN- production was observed in the 6A11-736 or any of the two mutants. Similarly,
higher levels of IFN- production were observed in the
SART-11-2,506 (full length), SART-11-2,467, and SART-11-2,355
(Fig. 5 B). In contrast, a very low level or no IFN- production was observed in the SART-11-2,252 or any of the
other five mutants, respectively. These results suggest that antigenic peptide(s) mainly resided within the 254-bp region of the 3 end of both the 6A1-1D7 and SART-1. This
region encodes 62 deduced aa of the SART-1 protein at
the positions of 737-800 in the third frame, which was
shared by both the 6A1-1D7-derived SART-1259 protein
and the SART-1-derived SART-1800 protein.
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Fig. 5.
Identification of regions containing antigenic peptides for CTL.
Deletion mutants of 6A1-1D7 gene (6A11-492, 6A11-625, 6A11-736, 6A11-839,
and 6A11-951) and the full length of 6A1-1D7 in A or mutants of SART-1
gene (SART-11-2,008, SART-11-2,141, SART-11-2,252, SART-11-2,355, and
the others) and the full length of SART-1 in B were cotransfected to
VA13 cells (2 × 104) with HLA-A2601 or -A0201, and 2 d later these
cells were tested for their ability to stimulate IFN- production by the
KE4 CTLs. The background of IFN- production by the KE4 CTLs in
response to VA13 cells transfected with both each mutant and HLA-A0201 (~50 pg/ml) was subtracted in the figure.
|
|
Determination of Peptide Antigens.
A series of 22 SART-1
oligopeptides (10 mer) corresponding to the region shown
above were loaded to the VA13 cells that had been transfected with HLA-A2601 or -A0201, and tested for their ability both to stimulate IFN- production and to be recognized by the KE4 CTLs in a 51Cr-release assay. Representative results are shown in Fig. 6 A. The three 10 mer
(SART-1736-745 [KGSGKMKTER], SART-1748-757 [KKLDEEALLK], and SART-1784-793 [IVLSGSGKSM]) possessed the activity to stimulate significant levels of IFN-
production (>50 pg/ml), whereas none of the other 10 mer did. The SART-1736-745 or SART-1748-757 peptide had
the high (45% lysis at an E/T ratio of 5:1) or low (10% lysis) activity to be recognized when loaded on VA13 cells
transfected with HLA-A2601, respectively. None of the
other 10 mer, including the SART-1784-793, had the significant level (>10% lysis) of activity in a 51Cr-release assay.
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Fig. 6.
Determination of peptide antigens. A series of 22 SART-1 oligopeptides (10 mer; 10 µM) in A or 6 different nonapeptides (10 µM) from
three 10 mer (SART-1736-745, SART-1748-757, and SART-1784-793) with deletion of one aa at position 1 or 10 in B were loaded for 2 h to the VA13 cells
(2 × 104) transfected with HLA-A2601 or -A0201. For IFN- production, the KE4 CTLs (104) were added, incubated for 18 h, and the culture supernatant was collected for measurement of IFN- by the ELISA in duplicate assays. The background of IFN- production by the KE4 CTLs in response to each
peptide loaded to the VA13 cells transfected with HLA-A0201 (~50 pg/ml) was subtracted in the figure. In a 51Cr-release assay, these VA13 cells were
labeled with Na2 51CrO4 for 1 h followed by adding the KE4 CTLs (5 × 104). 6 h later, the supernatant was harvested for measurement of the radioactivity in triplicate assays as reported (18). The background of percent lysis by the KE4 CTLs of the VA13 cells that were transfected with HLA-A0201 and
loaded by each peptide was <5%.
|
|
Six different nonapeptides from these three 10 mer with
deletion of one aa at position 1 or 10 were tested for their ability to stimulate IFN- production by the parental KE4 CTL
(Fig. 6 B). Each nonapeptide (SART-1736-744 [KGSGKMKTE], SART-1749-757 [KLDEEALLK], and SART-1785-793
[VLSGSGKSM]) had higher activity to stimulate IFN-
production than had the parental 10 mer. In contrast, each
of the remaining nonapeptides failed to stimulate IFN-
production.
To confirm the presence of a peptide-specific CTL, 80 KE4 CTL sublines were tested for their reactivity to each
of the three 10 mer (SART-1736-745, SART-1748-757, and
SART-1784-793). 4, 5, or 6 of 80 of the KE4 CTL sublines
showed the SART-1736-745, SART-1748-757, or SART-1784-793
peptide-specific reactivity, respectively. The representative
results are shown in Fig. 7.
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Fig. 7.
CTL sublines recognizing each nonapeptide. 80 KE4 CTL sublines were tested
for their ability to produce IFN-
by recognition of each of the
three 10 mer (SART-1736-745,
SART-1748-757, and SART-1784-793)
that was loaded at 10 µM on the
VA13 cells for 2 h transfected
with HLA-A2601 or -A0201.
Detailed methods are shown in
the legend for Fig. 6. Four, five,
or six of 80 of the KE4 CTL
sublines reacted to the SART-1736-745, SART-1748-757, or
SART-1784-793, respectively.
The representative results from the peptide-specific CTL sublines (No. 48 and 53: the SART-1736-745-specific CTLs; No. 3 and
60: the SART-1748-757-specific
CTLs; and No. 13 and 36: the
SART-1784-793-specific CTLs)
are shown in the figure. The
other CTL sublines were mostly
not reactive to any of the 10 mer, and only a few sublines
were reactive to two of the three 10 mer (data not shown). None
of the sublines were reactive to all the three peptides.
|
|
In the SART-1736-744 peptide, the ability to stimulate
IFN- production was observed at 50 nM with the maximal level at 3 µM (Fig. 8). This ability was observed as low
as 10 nM with the maximal level at 0.78 µM in the cases of
both the SART-1748-757 and SART-1785-793 peptides.
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Fig. 8.
Dose dependency of
nonapeptides. Various doses of
each of the nonapeptides
(SART-1736-744, SART-1749-757,
and SART-1785-793) were loaded
for 2 h on VA13 cells transfected
with HLA-A2601 or -A0201 followed by testing their ability to
stimulate IFN- production by
the parental KE4 CTLs. Detailed
methods are shown in the legend for Fig. 6.
|
|
Determination of aa Required for CTL-mediated Recognition.
Each aa of the three nonapeptides (SART-1736-744,
SART-1749-757, and SART-1785-793) was substituted by glycine (G) when it was not glycine or by threonine when it
was glycine. These peptides, along with the parental nonapeptides, were tested for their ability to stimulate IFN-
production by the parental KE4 CTLs. The representative results are shown in Fig. 9. The ability of the SART-1736-744 disappeared or extremely decreased when glutamic acid (E)
or threonine (T) at position 9 (9E-G in Fig. 9) or 8 (8T-G
in Fig. 9) was substituted, respectively. It also decreased
when glycine or methionine (M) at position 4 or 6 was
substituted, whereas it slightly increased when glycine or
serine (S) at position 2 or 3 was substituted to threonine or
glycine, respectively. The ability of SART-1749-757 disappeared when leucine (L) or alanine (A) at position 2 or 6 was substituted. It also decreased when glutamic acid or
leucine at position 5 or 8 was substituted. In a case of the
SART-1785-793, the ability disappeared when glycine or
serine at position 4 or 8 was substituted. It decreased when
leucine or serine at position 2 or 5 was substituted, whereas
slightly increased when serine at position 3 was substituted.
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Fig. 9.
Determination of aa required for CTL-mediated recognition.
Each aa of the three nonapeptides (SART-1736-744, SART-1749-757, and
SART-1785-793) was substituted by glycine when it was not glycine, or
threonine when it was glycine. These substituents along with the parental
nonapeptides (10 µM) were loaded for 2 h to the VA13 cells transfected
with HLA-A2601 or -A0201 followed by testing their ability to stimulate
IFN- production by the parental KE4 CTLs. Detailed methods are
shown in the legend for Fig. 6.
|
|
Induction of CTLs by the Nonapeptides.
The three nonapeptides (SART-1736-744, SART-1749-757, and SART-1785-793)
were tested for their ability to induce the CTLs against the
autologous tumor cells from PBMCs of a KE4 patient. PBMCs stimulated with the SART-1736-744 and their subline No. 1 showed higher levels of the KE4 autologous
tumor cell lysis than those of the KE3 allogenic tumor cell
lysis (Table 2). In contrast, PBMCs cultured with IL-2 alone
or stimulated with SART-1749-757 or SART-1785-793 equally
lysed both the tumor cells. The cells from the total of 144 microcultures (48 microcultures from PBMCs alone, 48 stimulated with the SART-1736-744, and 48 with the
SART-1749-757) were independently tested for their activity
to produce IFN- by recognition of the KE3, KE4 tumor,
and VA13 cells. The cells from 10 of 48 of the microcultures from the PBMCs stimulated with the SART-1736-744
produced higher levels of IFN- by recognition of the
KE4, but not the other cells. The representative result of
the one microculture showing positive IFN- production
is shown in Fig. 10. In contrast, the cells from none of 48 of the microcultures besides one from PBMCs alone or
PBMCs stimulated with the SART-1749-757 produced
higher IFN- by recognition of the KE4 tumor cells. The representative result of one microculture showing negative
IFN- production is shown in Fig. 10.
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Fig. 10.
Induction of CTLs by the nonapeptides. The cells from total of 144 microcultures (48 microcultures from PBMCs alone, 48 stimulated with the SART-1736-744, and 48 with the SART-1749-747) were independently tested for their activity to produce IFN- in response to the KE3, KE4 tumor, and VA13 cells. The cells from 10 of 48 of the microcultures from the PBMCs stimulated with the SART-1736-744 produced
IFN- by recognition of the KE4, but not the other cells. The representative result of one microculture (subline 11) showing positive IFN- production is shown in the figure. In contrast, the cells from none of 48 of
the microcultures besides one from either PBMCs alone or PBMCs stimulated with the SART-1749-757 produced IFN- by recognition of the
KE4 cells. The representative result of one microculture (subline 6 from PBMCs alone or subline 14 from PBMCs with the SART-1749-757) showing
negative IFN- production is shown in the figure.
|
|
| |
Discussion |
The results of this study suggest that SART-1 gene is a
bicistronic gene encoding two proteins, SART-1800 (125 kD)
in the nucleus and SART-1259 (43 kD) in the cytosol. Most
eukaryotic mRNAs have a single ORF and a single functional initiation site, which is usually the AUG codon that
lies closest to the 5 end (21, 22). However, there are some
viral mRNAs that break this rule; two proteins are translated from either the same or a different ORF (25).
Several human genes are also suggested to be bicistronic. liver-enriched transcriptional-activator protein (LAP) mRNA
was found to be translated into two proteins, LAP and the
liver-enriched transcriptional-inhibitory protein (LIP; 28, 29). The LIP contains the DNA-binding and dimerization
domains, but is devoid of the transcription-activation domain. LAP and LIP seem to exhibit antagonistic activities.
Another example is the glycoprotein (gp) 75 encoding two
different polypeptides, gp75 recognized by sera from cancer patients and a peptide with 24 aa recognized by CTLs (30).
However, the mechanism of posttranscriptional regulation
in human mRNAs is scarcely understood at the present
time. Therefore, SART-1 shall be a novel tool to explore
the mechanism.
It is of note that SART-1 encodes a leucine zipper motif
around nt positions 1,125-1,202 in the third frame (corresponding peptide: RELEEIRAKLRLQAQSLSTVGPRLAS). The leucine zipper motif is known to form
homo- or heterodimers that can bind DNA and modulate transcription of many genes (31, 32). Indeed, the SART-1
gene product bound to DNA (our unpublished results). Although its biological functions are currently unknown, the
SART-1800 protein might be involved in regulation of gene
transcription, because it was localized in the nucleus of proliferating cells, possessed a leucine zipper motif, and bound
to DNA. In contrast, the SART-1259 protein without leucine zipper motif expressed in the cytosol of SCCs and adenocarcinomas might inhibit the activity of the SART-1800.
If this is the case, these proteins might be involved in regulation of proliferation of epithelial cells and their malignant
transformation.
The region of antigenic peptides encoded by the 6A1-1D7 and SART-1 genes was 62 aa from the COOH terminus shared by the SART-1259 and SART-1800. Therefore,
both proteins could be used for antigen processing to
present the antigenic peptides on the groove of the HLA-A2601 molecule, although the SART-1259 protein, but not the SART-1800, is expected to be used as a major source of
the antigenic peptides recognized by the KE4 CTL because
of its preferential expression in the cytosol of tumor cells.
The three 10 mer and their nonapeptides in the region
of SART-1 protein at positions of 730-800 were identified
by an IFN- assay as antigenic peptides recognized by the
HLA-A2601-restricted KE4 CTLs. Because of the presence of CTL sublines reacting to each of the three 10 mer
among the 80 sublines tested, the parental KE4 CTL line
would consist of the mixtures of these peptide-specific CTL
clones. The other sublines were either not reactive to any of the 10 mer or reactive to two of the three 10 mer.
Among these 10 mer, SART-1736-745, and also SART-1748-757
to some extent, but not SART-1785-794, had the activity in a
51Cr-release assay. SART-1736-744, but not the others, possessed the ability to induce CTLs in PBMCs against the autologous tumor cells. Although the molecular basis for this
discrepancy is presently unclear, SART-1736-744 might be
naturally expressed on the HLA-A2601 allele of the KE4
tumor cells.
Our results suggest that threonine and glutamic acid at
positions 8 and 9 of SART-1736-744 (KGSGKMKTE), leucine and alanine at positions 2 and 6 of SART-1749-757
(KLDEEALLK), and glycine and serine at positions 4 and 8 of SART-1785-793 (VLSGSGKSM) are critical for the recognition of each peptide by the parental KE4 CTLs. In addition, glycine and methionine at positions 4 and 6 of
SART-1736-744, glutamic acid and leucine at positions 5 and 8 of SART-1749-757, and leucine and serine at positions 2 and 5 of SART-1785-793 are important for the recognition.
The binding motif for HLA-A2601 has not been determined as far as we know, and the KE4 CTL did not react
to HLA-A2603+ SCC and thus seemed to be HLA-A2601
restricted (18). The F pocket residues of these two subtypes
are different (33), and therefore a binding motif at position
9 for them may be different from each other. Subsequently,
it is difficult to compare our results of aa residues at position 9 to others, showing that valine or a hydrophobic residue at position 9 is important for binding to HLA-A26 (34,
35). With regard to the position 2, threonine, leucine, or
valine was reported as the motif for binding to HLA-A26. Our results indicated that leucine of both SART-1749-757
and SART-1785-793 was required for binding to HLA-A2601 allele, and substitution of glycine to threonine at
position 2 of the SART-1736-744 rather increased its activity
to induce IFN- production by the parental KE4 CTLs.
From the results of the experiments of dose-dependent reactions, both SART-1749-757 and SART-1785-793 seemed to
have higher affinity for binding to the groove of HLA-A2601 molecule than that of SART-1736-744. Modified
gp100 nonapeptides are reported to be more potent for
induction from PBMCs of HLA-A2-restricted CTLs cytotoxic to melanoma cells (36). Therefore, a modified SART-1736-744 peptide at position 2 from glycine to threonine, or probably to leucine or valine, may increase affinity
of the binding to HLA-A2601, which in turn increase the
ability to induce CTLs restricted to HLA-A2601+ SCCs.
This issue needs to be tested for development of better cancer vaccines.
The SART-1736-744 peptide, but not the others, induced
from the patient's PBMCs the CTLs restricted to the autologous tumor cells. CTL precursors in the patient's PBMCs
increased by 10-fold after three rounds of stimulation with
the peptide in vitro. This peptide failed to induce CTLs in
PBMCs from any of three HLA-A2601+ healthy donors
tested under the conditions used in this study (data not
shown). The SART-1259 protein was expressed in the cytosol of the majority of SCCs tested and half of lung adenocarcinomas. Because of its preferential expression in
the cytosol of SCCs and adenocarcinomas, the SART-1259
protein, but not the SART-1800, could be a major source of
antigenic peptides recognized by CTLs. The HLA-A26 allele is found in ~22% of Japanese, 17% of Caucasians, and
14% of Africans (37). The A2601 subtype is found most
frequently among the A26 subtypes (38). Therefore, the
SART-1259 protein along with the SART-1736-744 peptide
could be useful for specific immunotherapy of relatively
large numbers of HLA-A2601 patients with SCCs or adenocarcinomas as a cancer vaccine and also an antigen in
vitro to induce CTLs for adoptive cellular therapy.
Address correspondence to Kyogo Itoh, Department of Immunology, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830, Japan. Phone: 81-942-31-7551; Fax: 81-942-31-7699; E-mail: kitoh{at}kutume.ktarn.or.jp
We thank Dr. Teruo Kakegawa of Kurume University for critical discussion.
This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture (Japan) and a grant from the Science Research Promotion
Fund of the Japanese Private School Promotion Foundation.
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Abstract
Introduction
