Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ISOLATED, POLYPEPTIDES WHICH BIND TO HLA-A29
MOLECULES, NUCLEIC ACID, THE MOLECULES
ENCODING THESE, AND USES THEREOF
s RELATED APPLICATION
This application is a continuation-in-part of Serial No. 08/531, 662 filed
September 21,
1995, which is a continuation-in-part of copending application Serial No.
08/370,648, filed
January 10, 1995, which is a continuation in part of copending patent
application Serial No.
08/250,162 filed on May 27, 1994, which is a continuation-in-part of Serial
No. 08/096,039
1. o filed July 22, 1993. All of these applications are incorporated by
reference.
FIELD OF THE INVENTION
This invention relates to a nucleic acid molecule which codes for a tumor
rejection
antigen precursor. More particularly, the invention concerns genes, whose
tumor rejection
15 antigen precursor is processed, in r ' , into at least one tumor rejection
antigen that is
presented by HLA-Cw6 molecules. The genes in question do not appear to be
related to other
known tumor rejection antigen precursor coding sequences. The invention also
relates to
peptides presented by the HLA-Cw6 molecules, and uses thereof. Also a part of
the
inventions are peptides presented by HLA-A29 molecules, and uses thereof.
2~
BACKGROUND AND PRIOR ART
The process by which the mammalian immune system recognizes and reacts to
foreign
or alien materials is a complex one. An important facet of the system is the T
lymphocyte,
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or "T cell" response. This response requires that T cells recognize and
interact with
complexes of cell surface molecules, referred to as human leukocyte antigens
("HLAs"), or
major histocompatibility complexes ("MHCs"), and peptides. The peptides are
derived from
larger molecules which are processed by the cells which also present the
HLA/MHC molecule.
See in this regard Male et al., Advanced Immunology (J.P. Lipincott Company,
1987),
especially chapters 6-10. The interaction of T cells and HLA/peptide complexes
is restricted,
requiring a T cell specific for a particular combination of an HLA molecule
and a peptide.
If a specific T cell is not present, there is no T cell response even if its
partner complex is
present. Similarly, there is no response if the specific complex is absent,
but the T cell is
present. This mechanism is involved in the immune system's response to foreign
materials,
in autoimmune pathologies, and in responses to cellular abnormalities. Much
work has
focused on the mechanisms by which proteins are processed into the HLA binding
peptides.
See, in this regard, Barinaga, Science 257: 880 (1992); Fremont et al.,
Science 257: 919
(1992); Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257:
964 (1992).
:15 Also see Engelhard, Ann. Rev. Immunol. 12: 181-207 (1994).
The mechanism by which T cells recognize cellular abnormalities has also been
implicated in cancer. For example, in PCT application PCT/US92/04354, filed
May 22,
1992, published on November 26, 1992, and incorporated by reference, a family
of genes is
disclosed, which are processed into peptides which, in turn, are expressed on
cell surfaces,
which can lead to lysis of the tumor cells by specific CTLs cytolytic T
lymphocytes, or
"CTLs" hereafter. The genes are said to code for "tumor rejection antigen
precursors" or
"TRAP" molecules, and the peptides derived therefrom are referred to as "tumor
rejection
antigens" or "TRAs" . See Traversari et al. , Immunogenetics 35: 145 ( 1992);
van der Bruggen
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et al., Science 254: 1643 (1991), for further information on this family of
genes. Also, see
U.S. Patent Application Serial Number 807,043, filed December 12, 1991, now
U.S. Patent
No. 5,342,774.
In U.S. Patent Application Serial Number 938,334, now U.S. Patent No.
5,405,940,
the disclosure of which is incorporated by reference, it is explained that the
MAGE-1 gene
codes for a tumor rejection antigen precursor which is processed to
nonapeptides which are
presented by the HLA-A1 molecule. The reference teaches that given the known
specificity
of particular peptides for particular HLA molecules, one should expect a
particular peptide to
bind to one HLA molecule, but not to others. This is important, because
different individuals
possess different HLA phenotypes. As a result, while identification of a
particular peptide as
being a partner for a specific HLA molecule has diagnostic and therapeutic
ramifications, these
are only relevant for individuals with that particular HLA phenotype. There is
a need for
further work in the area, because cellular abnormalities are not restricted to
one particular
HLA phenotype, and targeted therapy requires some knowledge of the phenotype
of the
abnormal cells at issue.
In U.S. Patent Application Serial Number 008,446, filed January 22, 1993 and
incorporated by reference, the fact that the MAGE-1 expression product is
processed to a
second TR is disclosed. This second TRA is presented by HLA-C clone 10
molecules. The
disclosure shows that a given TRAP can yield a plurality of TRAs.
U.S. Patent Application Serial Number 994,928, filed December 22, 1992, and
incorporated by reference herein teaches that tyrosinase, a molecule which is
produced by
some normal cells (e.g., melanocytes), is processed in tumor cells to yield
peptides presented
by HIrA-A2 molecules.
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In U.S. Patent Application Serial Number 08/032,978, filed March 18, 1993, and
incorporated by reference in its entirety, a second TRA, not derived fmm
tyrosinase is taught
to be presented by HI,A-A2 molecules. The TRA is derived from a TRAP, but is
coded for
by a non-MAGE gene. This disclosure shows that a particular HLA molecule may
present
TRAs derived from different sources.
In U.S. Patent Application Serial Number 08/079,110, filed June 17, 1993 and
incorporated by reference herein, an unrelated tumor rejection antigen
precursor, the so-called
"BAGE" precursor, is described. The BAGS precursor is not related to the MAGE
family.
The work which is presented by the papers, patents, and patent applications
cited supra
deals, in large part, with the MAGE family of genes, and the unrelated BAGS
gene. It bas
not been found, however, that additional tumor rejection antigen precursors
are expressed by
cells. These tumor rejection antigen precursors are referred to as "GAGE"
tumor rejection
antigen precursors. They do not show homology to either the MAGE family of
genes or the
BAGS gene. Thus the present invention relates to genes encoding such TRAPs,
the tumor
rejection antigen precursors themselves as well as applications of both.
Thus, another feature of the invention are peptides which are anywhere from 9
to 16
amino acids long, and comprise the sequence:
Xaa Trp Pro Xaa Xaa Xaa Xaa Tyr
(SEQ ID NO: 23)
z 0 where Xaa is any amino acid and Xaa~l,2~ means that 1 or 2 amino acids may
be N-terminal
to the Trp residue. These peptides bind to, and/or are processed to peptides
which bind to
HLA-A29 molecules.
The invention is elaborated upon further in the disclosure which follows.
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BRIEF DESCRIPTION OF THE DRAWIrIGS
Figure 1 sets forth lysis studies using CTL clone 76/6.
Figure 2 shows tumor necrosis factor ("TNF") release assays obtained with
various
transfectants and controls.
Figure 3 compares lysis induced by cytolytic T lymphocytes of clone CTL 7b/6.
Peptides of
varying length were tested, including SEQ ID NO: 4.
Figure 4 presents an alignment of the cDNAs of the six GAGE genes discussed
herein. In the
figure, identical regions are surrounded by boxes. Translation initiation
sites and stop codons
are also indicated. Primers, used in polymerase chain reaction as described in
the examples,
are indicated by arrows.
Figure 5 sets forth the alignment of deduced amino acid sequences for the
members of the
GAGE family. Identical regions are shown by boxes, and the antigenic peptide
of SEQ ID
NO: 4, is shown.
Figure 6 shows the results obtained when each of the GAGE cDNAs was
transfected into COS
2 0 cells, together with HLA-Cw6 cDNA. Twenty-four hours later, samples of CTL
7616 were
added, and TNF release was measured after twenty-four hours.
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Figure 7 compares the stimulation of CTL 22/23 by COS-7 cells, transfected
with HLA-A29
cDNA, a MAGE, BAGS or GAGE sequence, as shown. Control values are provided by
MZ2-MEL.43 and COS cells, as stimulators.
Figure 8 presents results obtained by s'Cr release studies, using various
peptides including
SEQ ID NO: 22 and various peptides derived therefrom.
DETAILED DESCRIPTION OF ~ ~~RRED ~ODnVJ~IVTS
A melanoma cell line, MZ2-MEL was established from melanoma cells taken from
patent MZ2, using standard methodologies. This cell line is described, e.g.,
in PCT
Application PCT/US92/04354, filed May 22, 1992, published November 26, 1992,
and
incorporated by reference in its entirety. Once the cell line was established,
a sample thereof
was irradiated, so as to render it non-proliferative. These irradiated cells
were then used to
isolate cytolytic T cell clones ("CTLs") specific thereto.
A sample of peripheral blood mononuclear cells ("PBMCs") was taken from patent
MZ2, and contacted to the irradiated melanoma cells. The mixture was observed
for lysis of
the melanoma cells, which indicated that CTLs specific for a complex of
peptide and HLA
molecule presented by the melanoma cells were present in the sample.
2 0 The lysis assay employed was a chromium release assay following Herin et
~1. , Int.
J. Cancer 39: 390-396 (1987), the disclosure of which is incorporated by
reference. The
assay, however, is described herein. The target melanoma cells were grown 'fin
vi , and then
resuspended at 10' cells/ml in DMEM, supplemented with 10 mM HEPES and 30
°& FCS, and
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incubated for 45 minutes at 37°C with 200 ~,Ci/ml of Na(s'Cr)04.
Labelleii cells were washed
three times with DMEM, supplemented with 10 mM Hepes. These were then
resuspended
in DMEM supplemented with lOmM Hepes and 10~ FCS, after which 100 ul aliquots
containing 103 cells, were distributed into 96 well microplates. Samples of
PBLs were added
in 100 ul of the same medium, and assays were carried out in duplicate. Plates
were
centrifuged for 4 minutes at 100g, and incubated for four hours at 37°C
in an 8% COZ
atmosphere.
Plates were centrifuged again, and 100 ul aliquots of supernatant were
collected and
counted. Percentage of 5'Cr release was calculated as follows:
°b 5'Cr release = x 100
(MR-SR)
where ER is observed, experimental s'Cr release, SR is spontaneous release
measured by
incubating 103 labeled cells in 200 ul of medium alone, and MR is maximum
release, obtained
by adding 100 ul 0.3 °~ Triton X-100 to target cells.
Those mononuclear blood samples which showed high CTL activity were expanded
and
cloned via limiting dilution, and were screened again, using the same
methodology. The CTL
clone MZ2-CTL 76/6 was thus isolated. The clone is referred to as "76/f"
hereafter.
The same method was used to test target K5f ~ cells, as well as the melanoma
cell line.
2o Figure I shows that this CTL clone r~g~s and lyses the melanoma cell line,
i.e., MZ2-
MP,L b~~t riot K562. The clone was then tested against other melanoma cell
lines and
autologous EBV-transformed B cells in the same manner described supra. Figure
1 shows that
autologous B cells, transformed by Epstein Barn Virus ("EBV") were not lysed,
and that while
MZ2-MEL 3.0 was lysed by CTL clone 76/6, the cell line MZ2-MP.L.4F, a variant
which
2 5 does not express antigen F, was not. Hence, the clone appears to be
specific for this antigen.
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The results presented ~ are inconclusive as to which HLA molecule presents the
TRA. The lysed cell line, i. e. , MZ2-MEL, is known to express HLA-A1, HLA-
A29, HLA-
B37, HLA-B44, HLA-Cw6, and HLA-C clone 10: In experiments not reported here
but
which followed the protocol of this example, a subline of MZ2-MEL was tested,
which had
lost expression of HLA molecules A29, B44, and C clone 10. The subline was
lysed, thus
indicating that the presenting molecule should be one of A1, B37 or Cw6.
Example 2
Further studies were carried out to determine if 76/6 also produced tumor
necrosis
factor {"TNF") when contacted with target cells. The method used was that
described by
Traversari ~ .~l , Immunogenetics 35: 145-152 (1992), the disclosure of which
is incorporated
by reference. Briefly, samples of the CTL line were combined with samples of a
target cell
of interest in culture medium. After 24 hours, supernatant from the cultures
was removed,
and then tested on TNF-sensitive WEHI cells. Cell line MZ2-MEL.43, a subclone
of the
MZ2-MEL cell line discussed supra as well as in the cited references, gave an
extremely
strong response, and was used in the following experiments.
$xample 3
The results from Example 2 indicated that MZ2-MEL.43 presented the target
antigen
2 0 of interest. As such, it was used as a source of total mRNA to prepare a
cDNA library.
Total RNA was isolated from the cell line. The mRNA was isolated using an
oligo-dT
binding kit, following well recognized techniques. Once the mRNA was secured,
it was
transcribed into cDNA, via reverse transcription, using an oligo dT primer
containing a NotI
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site, followed by second strand synthesis. The cDNA was then ligated to a
BstXi adaptor,
digested with NotI, size fractionated by a Sephacryl S-500 HR column, and then
cloned,
undirectionally, into the BstXi and NotI sites of pcDNA I/Amp. The recombinant
plasmid
was then electroporated into DHSa E. coli bacteria. A total of 1500 pools of
100 recombinant
bacteria were seeded in microwells. Each contained about 100 cDNAs, because
nearly all
bacteria contained an insert.
Each pool was amplified to saturation and plasmid DNA was extracted by
alkaline lysis
and potassium acetate precipitation, without phenol extraction.
:L o ~ 4
Following preparation of the library described in Example 3, the cDNA was
transfected into eukaryotic cells. The transfections, described herein, were
carried out in
duplicate. Samples of COS-7 cells were seeded, at 15,000 cells/well into
tissue culture flat
bottom microwells, in Dulbecco's modified Eagles Medium ("DMEM") supplemented
with
109b fetal calf serum. The cells were incubated overnight at 37°C,
medium was removed and
then replaced by 50 ~ 1 /well of DMEM medium containing 10 °6 Nu serum,
400 ~cg/ml DEAE-
dextran, and 100 ~cM chloroquine, plus 100 ng of the plasmids. As was
indicated su~r , the
lysis studies did not establish which HLA molecule presented the antigen. As a
result, cDNA
for each of the HLA molecules which could present the antigen (Al, B37, Cw6)
was used,
2 0 separately, to cotransfect the cells. Specifically, one of 28 ng of the
gene encoding HLA_A 1,
cloned into pCD-SRa, 50 ng of cDNA for HLA-B37 in pcDNA I/Amp, or 75 ng of
cDNA
for HLA-Cw6 in pcDNAI-Amp, using the same protocol as were used for
transfection with
the library.
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Transfection was carried out in duplicate wells, but only 500 pools of the HLA-
Cw6
transfectants could be tested in single wells. Following four hours of
incubation at 37°C, the
medium was removed, and replaced by 50 ~l of PBS containing 10 ~ DMSO. This
medium
was removed after two minutes and replaced by 200 ~.l of DMEM supplemented
with 10 ~
PCS.
Following this change in medium, COS cells were incubated for 24-48 hours at
37°C.
Medium was then discarded, and 1000-3000 cells of CTL clone 76/6 were added,
in 100 ~l
of Iscove's medium containing 10 ~6 pooled human serum supplemented with 20-30
U/ml of
recombinant IL-2. Supernatant was removed after 24 hours, and TNF content was
determined
to in an assay on WEHI cells, as described by Traversari gl ~, Immunogenetics
35: 145-152
(1992), the disclosure of which is incorporated by reference.
The 1500 pools transfected with HLA-Al, and the 1500 pools transfected with
HLA-
B37 stimulated TNF release to a concentration of 15-20 pg/ml, or 2-6 pg/ml,
respectively.
Most of the HLA-Cw6 transfectants yielded 3-20 pg/ml, except for one pool,
which yielded
:15 more than 60 pg/ml. This pool was selected for further work.
Pxample 5
The bacteria of the selected pool were cloned, and 600 clones were tested.
Plasmid
DNA was extracted therefrom, transfected into a new sample of COS cells in the
same manner
2 0 as described supra, and the cells were again tested for stimulation of CTL
clone 76/6. Ninety-
four positive clones were found. One of these, referred to as cDNA clone 2D6,
was tested
further. In a comparative test COS cells were transfected with cDNA clone 2D6
and the
HLA-Cw6 cDNA, HLA-Cw6 cDNA alone, or cDNA 2D6 alone. Control cell lines MZ2-
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MEL F and MZ2-MEL F+ were also used. TNF release into CTL supernatant was
measured
by testing it on WEHI cells, as referred to a ra. The number of surviving WEHI
cells was
measured by optical density after incubation of the cells with MTT. Figure 2
shows that the
COS cells transfected with HI,A-Cw6 and cDNA-2D6, and the cell line MZ2-MEL F+
stimulated TNF release from CTL close 76/b, indicating that HLA-Cw6 presented
the subject
TRA.
Facamnle 66
The cDNA 2D6 was sequenced following art known techniques. A sequence search
revealed that the plasmid insert showed no homology to known genes or
proteins. SEQ >D
NO: 1 presents cDNA nucleotide information for the identified gene, referred
to hereafter as
"GAGE" . A putative open reading frame is located at bases 51-467 of the
molecule. The first
two bases of this sequence are from the vector carrying the cDNA sequence, and
are thus not
part of the cDNA itself.
Example 7
Following sequencing of the cDNA, as per Example 6, experiments were carried
out
to determine if cells of normal tissues expressed the gene. To determine this,
Northern
blotting was carried out on tissues and tumor cell lines, as indicated below.
The blotting
experiments used cDNA for the complete sequence of SEQ ID NO: 1. PCT was then
used
:? 0 to confirm the results.
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Table 1. Expression of gene GAGE -
Normal tissues
PHA activated T cells -
CTL clone 82/30 _
Liver _
Muscle _
Lung _
Brain _
Kidney _
Placenta _
Heart _
Skin _
Testis
Tumor cell lines
Melanoma 7/ 16
2 0 Lung carcinoma 1 /6
Sarcoma 0/ 1
Thyroid medullary carcinoma 0/1
Tumor samples
Melanoma 1/1
Ex~l
3 o Detailed analysis of normal tissues and tumors was carried out by applying
polymerise
chain reaction ("PCR") and the GAGE gene information described supra.
First, total RNA was taken from the particular sample, using art recognized
techniques.
This was used to prepare cDNA. The protocol used to make the cDNA involved
combining
4 ul of reverse transcriptase buffer Sx, 1 ul of each dNTP, (10 mM), 2 ul of
dithiothreitol
3 5 (1001nM), 2 ul of dT-15 primer (20 um), 0.5 ul of RNasin {40 units/ul),
and 1 ul of MoMLV
reverse transcriptase (200 units/ul). Next, 6.5 ul of template RNA (1 ug/3.25
ul water, or 2
ug total template RNA) was added. The total volume of the mixture was 20 ul.
This was
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mixed and incubated ai ~+2 ° C for 60 minutes, after which it
~Vvats°c~lillr~r~d~l' ice:" A tot2llr'tff g0 w
ul of water was then added, to 100 ul total. This mixture was stored at -
20°C until used in
PCR.
To carry out PCR, the primers
5'-AGA CGC TAC GTA GAG CCT-3'
(sense)
and
5'-CCA TCA GGA CCA TCT TCA-3'
(antisense)
l0 SEQ ID NOS: 2 and 3, respectively, were used. The reagents included 30.5 ul
water, 5 ul
of PCR buffer 10x, 1 ul of each dNTP (10 uM), 2.5 ul of each primer (20 uM),
and 0.5 ul
of polymerizing enzyme Dynazyme (2 units/ul). The total volume was 45 ul. A
total of 5 ul
of cDNA was added (this corresponded to 100 ng total RNA). The mixture was
combined,
and layered with one drop of mineral oil. The mixture was transferhed to a
thermocycler
block, preheated to 94°C, and amplification was carried out for 30
cycles, each cycle
consisting of the following:
first denaturation: 94°C, 4 min.
denaturadon: 94°C, 1 min.
annealing: 55 ° C, 2 min.
2 0 extension: 72 ° C, 3 min.
final extension: 72°C, 15 min.
Following the cycling, 10 ul aliquots were run on a 1.5 9b agarose gel,
stained with ethidium
bromide.
cDNA amplified using the primers set forth ~unra yields a 238 base pair
fragment.
2 5 There is no amplification of contaminating genomic DNA, if present.
. ._ _ 13
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The results are presented in Table 2, which follows."°""Ih~y ,;'o~irrri
"'that' thd vi~y
normal tissue which expresses GAGE is testis, whereas a number of tumors,
including
melanoma, lung, breast, larynx, pharynx, sarcoma, testicular seminoma, bladder
and colon
express the gene. Thus, any one of these tumors can be assayed for expression
of the GAGE
gene.
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NORMAL TISSUES
Heart _
Brain _
Liver _
Lung _
Kidney _
Spleen
Lymphocytes _
Bone marrow _
Skin _
Naews _
Melanocytes _
Fibroblasts _
Prostate _
Testis
Ovary _
2 0 Breast _
Adrenals _
Muscle _
Placenta _
TJmbilical cord _
TUMORS
Cell Lines Tumor
Samples
Melanoma 40/63 46/146 (32~)
Lung cancer
Epidermoid carcinoma 10/41 (24~)
Adenocarcinoma 4/ 18
Small Cell Lung Cancer 6/23 0/2
Breast cancer 15/146 (10~)
Head and neck tumor
3 5 Larynx 6/ 15 (40
R&
)
Pharynx 3/13
Sarcoma 1 /4 6/ I 8 (33
&
)
Testicular seminoma 6/6 ( 100
9&
)
Bladder cancer 5/37 ( I4
~
)
Prostate cancer 2/20
Colon cancer 5/13 0/38
Renal cancer 0/6 0/45
Leukemia 3/6 0/ I9
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_Foamole ~ _
The identification of the nucleic acid molecule referred to in the prior
examples led to
further work directed to determination of tumor rejection antigens presented
by HLA-Cw6
molecules, and derived fmm the GAGE gene.
The complete cDNA of GAGE in expression vector pcDNA/Amp was digested with
restriction endonucleases Notl and SpHI, and then with exonuclease III
following supplier's
instruction (Erase-a-base System, Promega). This treatment generated a series
of progressive
deletions, staring at the 3' end.
The deletion products were ligated back into pcDNAI/AMP, and then
electroporated
into ~. ~l'1 strain DHSa'IQ, using well known techniques. The transformants
were selected
with ampicillin (50 micrograms/ml).
Plasmid DNA was extracted fmm each recombinant clone and was then transfected
into
COS-7 cells, together with a vector which coded for HLA-Cw6. The protocols
used follow
the protocols described above.
The transfectants were then tested in the TNF release assay. This permitted
separation
of positive and negative clones. All the negative clones showed a deletion of
the entire GAGE
sequence. The smallest positive clone contained the first 170 nucleotides of
SEQ ID NO: 1.
The analysis of this sequence, supra, notes that the open reading frame starts
at nucleotide 51.
Thus, this fragment contains a sequence which encodes the first 40 amino acids
of the GAGE
TRAP,
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Example 10
Additional experiments were then carried out to define the region encoding the
TRA
peptide more precisely. Polymerise chain reaction ("PCR") amplification was
used to do this.
Two primers were synthesized. The first primer was a 22-mer complementary to a
sequence within the plasmid vector pcDNAI/Amp located upstream of a BalnIiI
site. The
second primer was a 29-mer containing at the 3' end nucleotides 102-119 of SEQ
ID NO: 1,
and at the 5' end an extension of 11 nucleotides containing an Xbal
restriction site.
Following amplification, the PCR product was digested by BamHI and XbaI, and
cloned into the BarnHI-XbaI sites of plasmid pcDNA-3. The recombinant colonies
were
cotransfected into COS-7 cells with cDNA encoding HLA-Cw6, in accordance with
Example
4, and a TNF release assay, also as described sub; was carried out, using CTL
76/6.
TNF release was observed, indicating that the "minigene" was processed to a
TRA.
The minigene, i.e., nucleotides 1-119 of SEQ ID NO: 1, the coding region of
which runs
from nucleotides 51-119, encoded the first 23 amino acids of the cDNA of SEQ
ID NO: 1.
This information served as the basis for the next set of experiments.
Exam In a 11
Two peptides were synthesized, based upon the first 23 amino acids of SEQ ID
NO:
1. These were:
2 0 Met Ser Trp Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg
(SEQ ID NO: 12)
and
2 5 Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val Glu Pro Pro Glu Met Ile
(SEQ ID NO: 13)
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Each peptide was pulsed into COS-7 cells previously transfected with HI;A-Cw6
cDNA, and
combined with CTL 76/6 to determine if TNF release would be induced. Peptides
(20 ug/ml)
were added to COS-7 cells which had been transfected with the HLA-Cw6 cDNA
twenty-four
hours previously After incubation at 37°C for 90 minutes, medium was
discarded, and 3000
CTLs were added in 100 micmliters of medium, containing 25 units/ml of IL-2.
Eighteen
hours later, TNF content of supernatant was tested via determining toxicity on
WEHI-164-13
cells. The second peptide {SEQ ID NO: 13) was found to induce more than 30
pg/ml of TNF,
while the first peptide (SEQ ID NO: 12) was found to induce less than 10 pg/ml
of TNF. The
second peptide was used for further experiments.
Example 12
Various peptides based upon SEQ ID NO: 13 were synthesized, and tested, some
of
which are presented below. To carry out these tests s'Cr labelled LB33-EBV
cells, which are
HL.A-Cw6 positive, were incubated with one of the following peptides:
Tyr Arg Pro Arg Pro Arg Arg Tyr
(SEQ TD NO: 4)
Thr Tyr Arg Pro Arg Pro Arg Arg Tyr
(SEQ ID NO: 5)
Thr Arg Pro Arg Pro Arg Arg Tyr Val
(SEQ ID NO: 6)
Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val
2 5 (SEQ ID NO: 7)
Arg Pro Arg Pro Arg Arg Tyr Val Glu
(SEQ ID NO: 8)
:3 0 Met Ser Trp Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg
{SEQ ID NO: 12)
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The peptide concentration varied, as indicated in figure 3, and the ratio of
CTL: LB33-EBV
("effector: target ratio"), was 10:1. 5'Cr release was determined after four
hours of incubation
at 37°C. Levels of lysis for positive ("F+", MZ2-MEL.3.1), and negative
("F"; MZ2-
MEL.2.2.5) control cells are indicated, in figure 3.
It was found, quite surprisingly that the octamer of SEQ ID NO: 4 was the best
peptide, and appeared to be the tumor rejection antigen. This is the first
time an octamer has
been reported as being involved in presentation by a human MHC molecule. There
is some
precedent for a murine system, as reported by Engelhard, sub, at 199, for H-
2K6 and H-2KK
molecules. The nonamers of SEQ ID NO: 5 and SBQ ID NO: 6 also induced CTL
lysis albeit
1.0 to a lesser extent than the octamer of SEQ ID NO: 4.
In results not reported here, a second CTL was tested (CTL 82/31). This CTL
was
known to lyse cells presenting
MZ2-F. It, too, lysed HLA-Cw6 positive cells following pulsing with the
peptide of SEQ ID
NO: 4.
Exam lp a 13
To fmd out whether the GAGE DNA set forth sub was unique, a cDNA library made
with RNA from MZ2-MEL.43 (the same library that was used for the cloning of
GAGE) was
hybridized with a probe derived from the GAGE cDNA. The probe was a PCR
fragment of
2 0 308 base pairs between positions 20 and 328 of SEQ ID NO: 1. Twenty
positive cDNAs were
obtained. Six of them were entirely sequenced. They were all highly related to
the GAGE
sequence, but they were slightly different from it. Two of the six clones were
identical to
each other, but all the others differed from each other. Thus, five new
sequences different
19
CA 02317492 2000-07-OS
WO 99/37665 PCT/US99/00795
from but highly related to GAGE were identified. They are called GAGE-2, 3, 4,
5 and 6
(Figure 4) and are presented as SEQ m NOS: 14-18, respectively. The fourteen
other clones
were partially sequenced at the S' end and their sequence corresponded to one
of the six
GAGE cDNAs.
The major difference between these cDNAs and GAGE-1 is the absence of a
stretch
of 143 bases located at position 379 to 521 of the GAGE sequence of SEQ 1D NO:
I. The
rest of the sequences shows mismatches only at 19 different positions, with
the exception of
GAGE-3 whose 5' end is totally different from the other GAGE for the first 112
bases. This
region of the GAGE-3 cDNA contains a long repeat and a hairpin structure.
The deduced GAGE-1 protein corresponding to a tumor rejection antigen
precursor is
about 20 amino acids longer than the 5 other proteins, whose last seven
residues also differ
from the homologous residues of GAGE-1 (Figure 5). The rest of the protein
sequences show
only 10 mismatches. One of these is in the region corresponding to the
antigenic peptide of
SEQ ID NO: 4. The sequence of the peptide is modified in GAGE-3, 4 5 and 6 so
that
position 2 is now W instead of R.
Example 14
To assess whether the change at position 2 affected the antigenicity of the
peptide,
cDNA of the 6 GAGE cDNAs were individually transfected into COS cells together
with the
2 0 cDNA of HI.A-Cw6, and the transfectants were tested for recognition by CTL
76/6 as
described, supra. Only GAGE-1 and GAGE-2 transfected cells were recognized,
showing that
the modified peptide encoded by GAGE-3, 4, 5 and 6 was not antigenic in the
context of this
experiment. Sequence analysis of the 5' end of the 14 other clones mentioned
supra showed
CA 02317492 2000-07-OS
WO 99/37665 PCT/US99/00795
that 7 of them contained the sequence encoding the antigenic peptide; and thus
probably
corresponded to either GAGE-1 or GAGE-2.
Exam lp a 15
The PCR primers used supra to test the expression of GAGE in tumor samples do
not
discriminate between GAGE-1 or 2 and the four other GAGE cDNAs that do not
encode
antigen MZ2F. A new set of primers was prepared which specifically amplifies
GAGE-1 and
2, and not GAGE-3, 4, 5 and 6. These primers are:
VDE44 5'-GAC CAA GAC GCT ACG TAG-3' (SEQ ID NO: 9)
VDE24 5'-CCA TCA GGA CCA TCT TCA-3' (SEQ ID NO: 10)
These primers were used as described, supra, in a RT-PCR reaction using a
polymerase
enzyme in the following temperature conditions:
40 min at 94 ° C
30 cycles with 1 min at 94 ° C
2 min at 56 ° C
3 min at 72 ° C
i5 iiiin at i2°i
The results of this analysis are set forth in Table 3.
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WO 99/37665 PCT/US99/00775
Table 3 w
Expression of GAGE genes by tumor samples and tumor cell lines
Histological type Number of GAGE positive tumors
All GAGE genes* GAGE-1 and 2**
Tumor samples
Melanomas
primary lesions 5/39 5/39 (13%)
metastases 47/132 36/131 (27%)
Sarcomas 6/20 6/20 (30%)
Lung carcinoma NSCLC 14/65 12/64 (19%)
Head and neck squamous
cell carcinomas 13/55 10/54 (19%)
Prostatic carcinomas 2/20 2/20
2 Mammary carcinomas 18/162 14/162 (9%)
0
Bladder carcinomas
superficial i/20 1/20
infiltrating 5/26 3/26
Testicular seminomas 6/6 5/6
Colorectal carcinomas 0/43
Leukemias and lymphonas 0/25
Renal carcinomas 0/46
Tumor cell lines
Melanomas 45/74 40/74 (54%)
Sarcomas 1/4 1/4
Lung carcin~nas
SCLC 7/24 7/24 (29%)
3 NSCLC 1/2 1/2
5
Mesotheli~nas 5/19 5/19 (26%)
Head and neck squamous
cell carcin~nas 0/2
Mammary carcinomas 1/4 0/4
4 Bladder carcinomas 0/3
0
Colon carcinomas 5/13 5/13
Leukemias 3/6 1/6
Lymphomas 0/6
Renal carcinomas 0/6
45
*Expression of GAGE was tested VDE-18
by RT-PCR on total RNA with primers and
VDE-24, detecting all GAGE genes. observedwhen these
No PCR product was
primers were assayed on DNA from
MZ2-MEL.
50
**8xpression of GAGE-1 and 2 was th primers
tested by RT-PCR on. total RNA
wi
VDE-44 and VDE-24, which distinguishGAGE-1 and 2 the fourother
from GAGE
genes. No PCR product was observedwhen these primerswere
assayed
on DNA
from MZ2-MEL.
55
zz
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WO 99/37665 PCTNS99/00775
In further work, new primers were designed which amplified all GAGE genes, to
make
sure that there was no expression of any of them in normal tissues. These
primers are
VDE43 S'-GCG GCC CGA GCA GTT CA-3' (SEQ B7 NO: 11)
VDE24 5'-CCA TCA GGA CCA TCT TCA-3 (SEQ ID NO: 10)
These were used exactly as for the PCR using the VDE44 and VDE24 primers. The
results
are shown in Table 4. They confirm that the normal tissues are negative,
except for testis.
23
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WO 99/37665 PCT/US99100795
Table 4
Expression of GAGE genes
in normal adult and fetal tissues
GAGE
Adult tissues expression*
Adrenal gland
Benign naevus
Bone marrow
Brain
Breast
Cerebellum -
Colon
Heart -
Kidney
2 0 Liver -
Lung _
Melanocytes -
Muscle -
Ovary -
2 5 Prostate -
Skin -
Splenocytes -
Stomach -
Testis +
30 Thymocytes -
Urinal bladder -
Uterus -
Placenta -
Umbilical cord
Fetal tissues*
Fibroblasts
Brain
Liver
Spleen
Thymus
Testis +
*Expression of GAGE was tested by RT-PCR amplification on total
RNA with primers VDE43 and VDE24 detecting all GAGE genes (Figure
7). Absence of PCR product is indicated by - and presence by +.
No PCR product Was observed when these primers were assayed on
DNA from MZ2-MEL.
*Fetal tissues derive from fetuses older than 20 weeks.
5
24
CA 02317492 2000-07-OS
wo ~r~~s6s Pcr~s99ioo~~rs
In work not reported here, it had been ascertained that cytolytic T cell clone
CTL
22/23 (Van den Eynde, et al., Int. J. Cancer 44: 634-640 (1989), incorporated
by reference)
did not recognize melanoma cell MZ2-MEL.3.1. This melanoma cell line was
reported by
Van der Bruggen, et al., Eur. J. Immunol. 24: 2134-2140 (1994), to have lost
expression of
MHC molecules HLA-A29, HLA-B24, and HLA-Cw'1601. Studies were undertaken to
determine if transfection with one of these MHC molecules could render the
line sensitive to
CTL 22/23. HLA-A29 was the first molecule tested. To do so, poly A+ RNA was
extracted
from HLA-A29+ cell line MZ2-MEL.43, using a commercially available extraction
kit, and
following the manufacturer's instructions. 'The mRNA was then converted to
cDNA, using
standard methodologies, size fractionated, and then inserted unidirectionally,
into the BstXI
and NotI sites of plasmid pcDNA-I/Amp. The plasmids were electroplated into
E.E. coy strain
DHSaS'IQ, and selected with ampicillin (50 wg/ml). The bacteria were plated
onto
nitrocellulose filters, and duplicated. The filters were prepau~d, and
hybridized overnight in
6xSSC/0.1 °b SDS/lx Denhardt's solution at 40°C, using 32P
labelled probe:
5'-ACTCCATGAGGTATITC-3'
(S&Q ID NO: 19)
The probe is a sequence which surrounds the start colon of most HLA,
sequences.
The filters were washed twice, at room temperature for 5 minutes each time in
6xSSC,
2 0 and twice in 6xSSC at 43 ° C. Positive sequences were then screened
with probe:
5'-TTTCACCACATCCGTGT-3'
(SEQ ID NO: 20)
CA 02317492 2000-07-OS
WO 99137665 PCT/US99/00795
which had been labeDed with ~P. This sequence is specific for HLA-A29, as
determined by
reference to the Kabat Database of sequences and proteins of immunological
interest,
incorporated by reference. This database is available at the NCBI (USA), or on
Web Solte
(Internet) WWW.NCBLNLM.NIH. GOV. The filters were washed twice at room
temperature
for 5 minutes each time, at 6xSSC, followed by two washes, at 6xSSC (5 minutes
per wash),
at 42 ° C
Ex m 7
Once positive HLA-A29 clones were isolated, these were transfected into COS-7
using
the DEAE-dextran chlomquine method out supra. In brief, 1.5 x 104 COS-7 cells
were treated
with SOng of plasmid pcDNA-IlAmp containing HLA-A29, and 100 ng of cDNA
containing
cDNA for one of the GAGE sequences mentioned , or one of the prior art MAGE or
BAGS sequences in plasmid pcDNAa-I/Amp or pcDSR-a, respectively. The
transfectants
were then incubated for 24 hours at 37°C.
The transfectants were then tested for their ability to stimulate TNF
production by
CTLs, using the assay explained at the end of example, 4, .
Figure 7, which presents the results of this study, shows that high levels of
TNF
production were achieved using any of GAGE-3, 4, 5 or 6 and HLA-A29 as
transfectants.
GAGE-1 and GAGE-2, in contrast, do not stimulate CTL clone 22/23, thus leading
to the
2 0 conclusion that GAGE 3, 4, 5 and 6 are processed to an antigen or antigens
presented by
H1.A-A29 molecules and recognized by CTL 22/23.
26
CA 02317492 2000-07-OS
PCTNS99/00775
~?xa ule 18
The fact that GAGE-3, 4, 5 and 6 were processed to peptides presented by HLA-
A29+
cells, while GAGE-1 and GAGE-2 were not, suggested examination of the deduced
amino acid
sequences for those common to GAGE 3, 4, 5 and 6 and absent from GAGE-1 and
GAGE-2.
The sequence:
Arg Ser Thr Tyr Tyr Tip Pro Arg Pro Arg Arg Tyr Val Gln
(SEQ ID NO: 21)
was identified. The peptide was synthesized, lyophilized, and then dissolved
in 1 volume
DMSO, 9 volumes of 10 mM acetic acid in water. This methodology was used for
the other
peptides synthesized, discussed infra.
The peptide (SEQ ID NO: 21) was tested in a 5'Cr release experiment, following
the
method described supra.
It was found that this peptide did provoke lysis. Successive deletions were
prepared,
and tested for their ability to provoke lysis, again using the s'Cr lytic
assay. This work is
depicted in Figure 8. It was found that the shortest peptide to provoke lysis
was
Tyr Tyr Trp Pro Arg Pro Arg Arg Tyr
(SEQ ID NO: 22), which is common to all of GAGE-3 through 6. Specifically,
amino acids
10-18 of GAGE-3, and amino acids 9-17 of GAGE-4, 5 aad 6 correspond to this
peptide.
The members of the peptide family shown in Figure 8, and represented, e.g., by
SEQ
2 0 ID NOS: 21 and 22, do not accord with the data presented by Toubert, et
al. , "HLA-A29
Peptide Binding Motif", Abstract No. 4183, Ninth International Congress of
Immunology,
July 23-29, 1995, San Francisco, CA, incorporated by reference. According to
Toubert, et
27
CA 02317492 2000-07-OS
WO 99/37665 PCTNS99/00775
al., at the least a Phe residue is required at the third position of any
peptide which binds to
HLA-A29. As is shown herein, such is not the case.
The foregoing examples show the isolation of nucleic acid molecules which code
for
tumor rejection antigen precursors and tumor rejection antigens. These
molecules, however,
are not homologous with any of the previously disclosed MACE and BAGS coding
sequences
described in the references set forth supra. Hence, one aspect of the
invention is an isolated
nucleic acid molecule which comprises the nucleotide sequences set forth in
any of SEQ ID
NOS: 1-6 as well as fragments thereof, such as nucleotides 1-170, and 51-170
of SEQ ID NO:
1, or any other fragment which is processed to a tumor rejection antigen. The
sequences of
SEQ ID NOS: 1-6 are neither MAGE nor BAGS coding sequences, as will be seen by
comparing those to the sequence of any of these genes as described in the
cited references.
Also a part of the invention are those nucleic acid molecules which also code
for a non-MAGE
and non-BAGE tumor rejection antigen precursor but which hybridize to a
nucleic acid
molecule containing the described nucleotide sequence of SEQ ID NO: 1, under
stringent
conditions. The term "stringent conditions" as used herein refers to
parameters with which
the art is familiar. More specifically, stringent conditions, as used herein,
refers to
hybridization in 1M NaCI, 1 % SDS, and 10~ dextran sulfate for 18 hours at
65°C. This is
followed by two washes of the filter at room temperature for 5 minutes, in
2xSSC, and one
wash for 30 minutes in 2xSSC, 0.19& SDS, at 65°C. There are other
conditions, reagents,
2 0 and so forth which can be used, which result in the same or higher degree
of stringency. The
skilled artisan will be familiar with such conditions and, thus, they are not
given here.
It will also be seen from the examples that the invention embraces the use of
the
sequences in expression vectors, as well as to transform or transfect host
cells and cell lines,
28
CA 02317492 2000-07-OS
WO 99/37665 PCT/US99/00775
be these prokaryotic (e.g., E. cold, or eukaryotic (e.g., CHO or COS cells).
The expression
vectors require that the pertinent sequence, i.e., those described supra, be
operably linked to
a premoter. As it has been found that both of human leukocyte antigens HLA-Cw6
and HLA-
A29 present tumor rejection antigens derived from these genes, the expression
vector may also
include a nucleic acid molecule coding for one of HLA-Cw6 or HLA-A29. In a
situation
where the vector contains both coding sequences, it can be used to transfect a
cell which does
not normally express either one. The tumor rejection antigen precursor coding
sequence may
be used alone, when, e.g., the host cell already expresses one or both of HLA-
Cw6 and HLA-
A29. Of course, there is no limit on the particular host cell which can be
used. As the
vectors which contain the two coding sequences may be used in HLA-A29 or HLA-
Cw6
presenting cells if desired, and the gene for tumor rejection antigen
precursor can be used in
host cells which do not express HLA-A29 or HLA-Cw6.
The invention also embraces so called expression kits, which allow the artisan
to
prepare a desired expression vector or vectors. Such expression kits include
at least separate
portions of each of the previously discussed coding sequences. Other
components may be
added, as desired, as long as the previously mentioned sequences, which are
required, are
included.
To distinguish the nucleic acid molecules and the TR.APs of the invention from
the
previously described MAGE and BAGE materials, the invention shall be referred
to as the
2 o GAGE family of genes and TRAPS. Hence, whenever "GAGE" is used herein, it
refers to
the tumor rejection antigen precursors coded for by the previously described
sequences.
"GAGE coding molecule" and similar terms are used to describe the nucleic acid
molecules
themselves.
29
CA 02317492 2000-07-OS
'WO 99!37665 PCT/US99/00775
The invention as described herein has a number of uses, some of which are
described
herein. First, the invention permits the artisan to diagnose a disorder such
as melanoma,
characterized by expression of the TRAP, or presentation of the tumor
rejection antigen.
These methods involve determining expression of the TRAP gene, and/or TRAs
derived
therefrom, such as a TRA presented by I3LA-Cw6 or IEiLA-A29. In the former
situation, such
determinations can be carried out via any standard nucleic acid determination
assay, including
the polymerise chain reaction, or assaying with labelled hybridization probes.
In the latter
situation, assaying with binding partners for complexes of TRA and I3LA, such
as antibodies,
is especially preferred. An alternate method for determination is a TNF
release assay, of the
s o type described ~Dr~. To carry out the assay, it is preferred to make sure
that testis cells are
not present, as these normally express GAGE. lfiis is not essential, however,
as one can
routinely differentiate between testis and other cell types. Also, it is
practically impossible
to have testis cells present in non-testicular sample.
The isolation of the TRAP gene also makes it possible to isolate the TRAP
molecule
itself, especially TRAP molecules containing the amino acid sequence coded far
by any of
SEQ ID NOs: 2-6. These isolated molecules when presented as the TRA, or as
complexes of
TRA and I3LA, such as HLA-Cw6 or HLA-A29, may be combined with materials such
as
adjuvants to produce vaccines useful in treating disorders characterized by
expression of the
TRAP molecule.
2 0 Exemplary adjuvants include Freund's complete and incomplete adjuvant,
killed B.
i i organisms, "BCG", or Bacille Calmente-Guerin, Al(OI~3, muramyl dipeptide
and
its derivatives, which may be emulsified in metabolizable oils, such as
squalene,
monophosphoryl lipid A (MPL), keyhole limpet hemocyanin (KLH), saponin
extracts such
CA 02317492 2000-07-OS
1~N0 99/37665 PCT/US99/00735
as QA-7, QA-19, and QA-21 (also referred to as QS-21), these having been
described in U.S.
Patent No. 5,057,540 to Kensil, et al., incorporated by reference, MTP-MF59, N-
[1-(2,3-
dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), the
cationic
amphiphile DOTMA, the neutral phospholipids such as DOPE, and combinations of
these.
This listing is by no means comprehensive, and the artisan of ordinary skill
will be able to
augment this listing. All additional adjuvants are encompassed herein.
In addition, vaccines can be prepared from cells which present the TRA/HLA
complexes on their surface, such as non-proliferative cancer cells, non-
proliferative
transfectants, et cetera. In all cases where cells are used as a vaccine,
these can be cells
1 U transfected with coding sequences for one or both of the components
necessary to provide a
CTL response, or be cells which express both molecules without transfection.
Further, the
TRAP molecule, its associated TRAs, as well as complexes of TRA and HLA, may
be used
to produce antibodies, using standard techniques well known to the art.
When "disorder" is used herein, it refers to any pathological condition where
the tumor
1 ~ rejection antigen precursor is expressed. An example of such a disorder is
cancer, melanoma
in particular. Melanoma is well known as a cancer of pigment producing cells.
As indicate, supra, tumor rejection antigens, such as the one presented in SEQ
ID NO:
4, are also part of the invention. Also a part of the invention are
polypeptides, such as
molecules containing from 8 to 16 amino acids, where the polypeptides contain
the amino acid
2 o sequence set forth in SEQ ID NO: 4. As the examples indicate, those
peptides which are
longer than the octamer of SEQ B~ NO: 4 are processed into the tumor rejection
antigen of
SEQ ID NO: 4 by the HLA-Cw6 presenting cancer cells, and presented thereby.
The
presentation leads to lysis by cytolytic T lymphocytes present in a body fluid
sample contacted
31
CA 02317492 2000-07-OS
-WO 99/37665 PCTNS99/00795
to the cells presenting the complex. Similarly, the peptides longer than SEQ
ID NO: 22, such
as SEQ ID NO: 21, are processed to the appropriate TRA, and are presented by
cancer cells,
such as HI.A-A29 positive cells.
Thus, another feature of the invention are peptides which are anywhere from 9
to 16
amino acids long, and comprise the sequence:
Xaa Xaa Tip Pro Xaa Xaa Xaa Xaa Tyr
(SEQ ID NO: 23)
or
1 C~ Xaa Xaa Trp Xaa Arg Xaa Xaa Xaa Tyr
(SEQ ID NO: 24)
or
Xaa Xaa Trp Xaa Xaa Xaa Xaa Arg Tyr
(SEQ ID NO: 25)
where Xaa in each case is any amino acid.
Especially preferred are peptides which, in accordance with the formula of SEQ
ID
NOS: 23, 24 and 25 also satisfy one or more of the following criteria: the N-
terminal amino
acid position is Tyr, second position is Tyr, fourth position is Pro, fifth
position is Arg, sixth
position is Pro, seventh position is Arg, and eighth position is Arg. Of
course, the fourth
2 n position is already fixed in SEQ ID NO: 23, the fifth is already fixed in
SEQ ID NO: 24, and
the eighth in SEQ ID NO: 25. When all of these criteria are satisfied and this
peptide consists
of 9 amino acids, one has SEQ ID NO: 22. Any or all of the foreign specific
alternatives may
be combined in the peptides of the claimed invention, subject to the motif of
SEQ ID NO: 23,
32
CA 02317492 2000-07-OS
WO 99!37665 PCTNS99/00T~5
24 or 25 and the size of 9-16 amino acids. Especially preferred are peptides
which are 9-14
amino acids long, and which include SEQ ID NO: 23, 24 or 25 subject to the
above preferred
alternatives.
Also a part of the invention are so-called "minigenes", which are isolated
nucleic acid
molecules which encode any of SEQ m NOS: 21, 22, 23, 24 or 25 all of the
especially
preferred embodiments of SEQ ID NO: 23, 24 or 25 being included. There are
only a limited
number of nucleic acid molecules which can encode, e.g., SEQ ID NO: 21 or 22,
and they
can all be deduced from the known rules of degeneracy for codons without any
difficulty. The
use of these minigenes in encoding the peptides of the invention, in both
standard in vivo and
1 c) in vitro methodologies is also encompassed by the invention. These
peptides bind to, and/or
are processed to peptides which bind to HLA-A29 molecules. The fact that these
peptides are
processed to the tumor rejection antigen in indicated by the examples.
This property may be exploited in the context of other parameters in confnming
diagnosis of pathological conditions, such as cancer, melanoma in particular.
For example,
1!, the investigator may study antigens shed into blood or urine, observe
physiological changes,
and then confirm a diagnosis of melanoma using the CTL proliferation
methodologies
described herein.
Also a part of the invention are complexes of HLA-A29 molecules, and one of
the
peptides listed supra, preferably in soluble form. Such soluble complexes can
be used, e.g.,
2 o to determine presence of CTLs in a sample, such as a body fluid sample, by
adding the soluble
complexes to the sample, and then determining reaction with CTLs. Panning for
CTLs using
soluble complexes of MHC molecule and peptide is taught by, e. g. , Bousso et
al. , Immunol.
Lett. 59(2): 85-91 (1997), incorporated by reference. The complexes are
preferably
33
CA 02317492 2000-07-OS
WO 99137665 PCT/US99/00775
immobilized to facilitate the enrichment of the CTLs. Attention is also drawn
to Sakita, et al. ,
J. Immunol. Meth. 192: 105-115 (1996), also incorporated by reference, which
shows that
such complexes can be used to stimulate CTLs in vivo. This is another feature
of the
invention. In an especially preferred embodiment, the soluble complexes
referred to supra are
multimeric, most preferably tetrameric. Altman et al., Science 274: 94-96
(1996),
incorporated by reference, describe how such structures can be made.
On their own, peptides in accordance with the invention may be used to carry
out
HLA-typing assays. It is well lu~own that when a skin graft, organ transplant,
etc., is
necessary one must perform HLA typing so as to minimize the possibility of
graft rejection.
i o 1fie peptides of the invention may be used to determine whether or not an
individual is HLA-
Cw6 or HLA-A29 positive, so that appropriate donors may be selected. This type
of assay
is simple to carry out. The peptides of the invention are contacted to a
sample of interest, and
binding to cells in that sample indicates whether or not the individual from
which the sample
is taken is H1.A-Cw6 or HLA-A29 positive. One may label the peptides
themselves,
conjugate or otherwise bind them to linkers which are labeled, immobilize them
to solid
phases, and so forth, so as to optimize such an assay. Other standard
methodologies will be
clear to the skilled artisan, and need not be presented herein.
Therapeutic approaches based upon the disclosure are premised on a response by
a
subject's immune system, leading to lysis of TRA presenting cells, such as HLA-
A29 or HLA-
2 ~ Cw6 cells. One such approach is the administration of CTLs specific to the
complex to a
subject with abnormal cells of the phenotype at issue. It is within the skill
of the artisan to
develop such CTLs in vitro. Specifically, a sample of cells, such as blood
cells, are contacted
to a cell presenting the complex and capable of provoking a specific CTL to
proliferate. The
34
CA 02317492 2000-07-OS
~WO 99137665 PGT/US99/00775
target cell can be a transfectant, such as COS cell of the type described
supra. These
transfectants present the desired complex on their surface and, when combined
with a CTL
of interest, stimulate its proliferation. COS cells, such as those used
herein, are widely
available, as are other suitable host cells.
To detail the therapeutic methodology, referred to as adoptive transfer
(Greenberg, J.
Immunol. 136(5): 1917 (1986); Riddel et al., Science 257: 238 (7-10-92); Lynch
et al., Eur.
J. Immunol. 21: 1403-1410 (1991); Kast et al., Cell 59: 603-614 (11-17-89)),
cells presenting
the desired complex are combined with CTLs leading to proliferation of the
CTLs specific
thereto. The proliferated CTLs are then administered to a subject with a
cellular abnormality
s o which is characterized by certain of the abnormal cells presenting the
particular complex,
where the complex contains the pertinent HLA molecule: The CTLs then lyre the
abnormal
cells, thereby achieving the desired therapeutic goal.
The foregoing therapy assumes that at least some of the subject's abnormal
cells
present the relevant HLA/TRA complex. This can be determined very easily, as
the art is
very familiar with methods for identifying cells which present a particular
HLA molecule, as
well as how to identify cells expressing RNA of the pertinent sequences, in
this case a GAGE
sequence. Once cells presenting the relevant complex are identified v~'1 the
foregoing
screening methodology, they can be combined with a sample from a patient,
where the sample
contains CTLs. If the complex presenting cells are lysed by the mixed CTL
sample, then it
2 0 can be assumed that a GAGE derived, tumor rejection antigen is being
presented, and the
subject is an appropriate candidate for the therapeutic approaches set forth .
Adoptive transfer is not the only form of therapy that is available in
accordance with
the invention. CTLs can also be provoked in viv , using a number of
approaches. One
CA 02317492 2000-07-OS
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approach, i.e., the use of non-proliferative cells expressing the complex, has
been elaborated
upon supra. The cells used in this approach may be those that normally express
the complex,
such as irradiated melanoma cells or cells transfected with one or both of the
genes necessary
for presentation of the complex. Chen et al., Proc. Natl. Acad. Sci. USA 88:
110-114
(January, 1991 ) exemplifies this approach, showing the use of transfected
cells expressing
HPV E7 peptides in a therapeutic regime. Various cell types may be used.
Similarly, vectors
carrying one or both of the genes of interest may be used. Viral or bacterial
vectors are
especially preferred. In these systems, the gene of interest is carried by,
e.g., a Vaccina vims
or the bacteria BCG, and the materials de facto "infect" host cells. The cells
which result
present the complex of interest, and are recognized by autologous CTLs, which
then
proliferate. A similar effect can be achieved by combining the tumor rejection
antigen or the
precursor itself with an adjuvant to facilitate incorporation into HLAL-Cw6
presenting cells
which then present the HLA/peptide complex of interest. The TRAP is processed
to yield the
peptide partner of the HLA molecule while the TRA is presented without the
need for further
processing.
Other aspects of the invention will be clear to the skilled artisan and need
not be
repeated here.
The terms and expressions which have been employed are used as terms of
description
and not of limitation, and there is no intention in the use of such terms and
expressions of
2 o excluding any equivalents of the features shown and described or portions
thereof, it being
recognized that various modifications are possible within the scope of the
invention.
3b
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( 1 ) GENERAL INFORMATION:
(i) APPLICANTS: Van der Bruggen, Pierre; Van den Eynde, Benoit;
DeBacker, Olivier; Boon-Falleur, Thierry
(ii) ZTTLE OF IIWENTION: Isolated, Polypeptides Which Bind to HLA-A29
Molecules, Nucleic Acid, The Molecules Encoding These, and Uses Thereof
(iii) NUMBER OF SEQUENCES: 25
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Fulbright & Jaworskfi L.L.P.
(B) STREET: 666 Fifth Avenue
(C) CITY: New York City
(D) STATE: New York
(E) COUNTRY: USA
(F~ ZIP: 10103-3198
(v) COMPUTER READABLE FORM:
(A) MEDIITM TYPE: Diskette, 3.5 inch, 1.44 kb storage
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: PC
(D) SOFTWARE: Wordperfect
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: 435
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NiTMBER: 09/012, 818
(B) FIL ING DATE: 23-January-1998
(C) CLASSIFICATION: 435
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NTJMBBR: 08/531,662
(B) FILING DATE: 21-September-1995
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/370,648
(B) FILING DATE: 10-January-1995
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NiJMBER: 08/250,162
(B) FILING DATE: 27-May-1994
37
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(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER.: 08/096,039
(B) FILING DATE: 22-July-1993
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hanson, Norman D.
(B) REGISTRATION NiJIVIBER: 30,946
(C) REFERENCE/DOCKET NUMBER: LUD 5531 PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 318-3168
(B) TELEFAX: (212) 752-5958
(2) INFORMATION FOR SEQ ID NO:
1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 646 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ
ID NO:1:
CTGCCGTCCG GACTCTTTTT CCTCTACTGA GATTCATCTG TGTGAAATAT 50
GAGTTGGCGA GGAAGATCGA CCTATCGGCC TAGACCAAGA CGCTACGTAG 100
AGCCTCCTGA AATGATTGGG CCTATGCGGC CCGAGCAGTT CAGTGATGAA 150
GTGGAACCAG CAACACCTGA AGAAGGGGAA CCAGCAACTC AACGTCAGGA 200
TCCTGCAGCT GCTCAGGAGG GAGAGGATGA GGGAGCATCT GCAGGTCAAG 250
GGCCGAAGCC TGAAGCTGAT AGCCAGGAAC AGGGTCACCC ACAGACTGGG 300
TGTGAGTGTG AAGATGGTCC TGATGGGCAG GAGATGGACC CGCCAAATCC 350
AGAGGAGGTG AAAACGCCTG AAGAAGAGAT GAGGTCTCAC TATGTTGCCC 400
AGACTGGGAT TCTCTGGCTT TTAATGAACA ATTGCTTCTT AAATCTTTCC 450
CCACGGAAAC CTTGAGTGAC TGAAATATCA AATGGCGAGA GACCGTTTAG 500
TTCCTATCAT CTGTGGCATG TGAAGGGCAA TCACAGTGTT AAAAGAAGAC 550
ATGCTGAAAT GTTGCAGGCT GCTCCTATGT TGGAAAATTC TTCATTGAAG 600
TTCTCCCAAT AAAGCTTTAC AGCCTTCTGC AAAGAAAAAA AAAAAA 646
38
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(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
AGACGCTACG TAGAGCCT 18
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CCATCAGGAC CATCTTCA 18
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Tyr Arg Pro Arg Pro Arg Arg Tyr
(2) INFORMATION FOR SBQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Thr Tyr Arg Pro Arg Pro Arg Arg Tyr
5
39
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(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Tyr Arg Pro Arg Pro Arg Arg Tyr Val
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPZTON: SEQ ID NO: 7:
Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val
5 10
(2) INFORMATION FOR SBQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Arg Pro Arg Pro Arg Arg Tyr Val Glu
5
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GACCAAGACG CTACGTAG 18
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(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CCATCAGGAC CATCTTCA 18
(2) INFORMATION FOR S8Q B7 NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GCGGCCCGAG CAGTTCA 17
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SfiQ ID NO: 12:
Met Ser Trp Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg
15
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val Glu Pro Pro Glu Met Ile
5 10 15
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(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 538 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
ACGCCAGGGA GCTGTGAGGC AGTGCTGTGT GGTTCCTGCC GTCCGGACTC 50
TTTTTCCTCT ACTGAGATTC ATCTGTGTGA AATATGAGTT GGCGAGGAAG 100
ATCGACCTAT CGGCCTAGAC CAAGACGCTA CGTAGAGCCT CCTGAAATGA 150
TTGGGCCTAT GCGGCCCGAG CAGTTCAGTG ATGAAGTGGA ACCAGCAACA 200
CCTGAAGAAG GGGAACCAGC AACTCAACGT CAGGATCCTG CAGCTGCTCA 250
GGAGGGAGAG GATGAGGGAG CATCTGCAGG TCAAGGGCCG AAGCCTGAAG 300
CTCATAGCCA GGAACAGGGT CACCCACAGA CTGGGTGTGA GTGTGAAGAT 350
GGTCCTGATG GGCAGGAGAT GGACCCGCCA AATCCAGAGG AGGTGAAAAC 400
GCCTGAAGAA GGTGAAAAGC AATCACAGTG TTAAAAGAAG ACACGTTGAA 450
ATGATGCAGG CTGCTCCTAT GTTGGAAATT TGTTCATTAA AATTCTCCCA 500
ATAAAGCTTT ACAGCCTTCT GCAAAGAAAA AAAAAAAA 538
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 560 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CTCATATTTC ACACAGATGA GTTGGCGAGG AAGATCGACC TATTATTGGT 50
CTAGGCCAAT AATAGGTCGA TCTTCCTCGC CAACTCATAT TTCACACAGA 100
TGAATCTCAG TAGAGGAAAA TCGACCTATT ATTGGCCTAG ACCAAGGCGC 150
TATGTACAGC CTCCTGAAGT GATTGGGCCT ATGCGGCCCG AGCAGTTCAG 200
TGATGAAGTG GAACCAGCAA CACCTGAAGA AGGGGAACCA GCAACTCAAC 250
42
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GTCAGGATCCTGCAGCTGCT CAGGAGGGAG AGGATGAGGG AGCATCTGCA 300
GGTCAAGGGCCGAAGCCTGA AGCTGATAGC CAGGAACAGG GTCACCCACA 350
GACTGGGTGTGAGTGTGAAG ATGGTCCTGA TGGGCAGGAG ATGGACCCGC 400
CAAATCCAGAGGAGGTGAAA ACGCCTGAAG AAGGTGAAAA GCAATCACAG 450
TGTTAAAAGAAGGCACGTTG AAATGATGCA GGCTGCTCCT ATGTTGGAAA 500
~TGTTCATT AAAATTCTCC CAATAAAGCT TTACAGCCTT CTGCAAAGAA 550
560
(2) INFORMATION
FOR SEQ
ID NO:
16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 540 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:
16:
CGCCAGGGAG CTGTGAGGCA GTGCTGTGTG GTTCCTGCCG TCCGGACTCT 50
TTTTCCTCTA CTGAGATTCA TCTGTGTGAA ATATGAGTTG GCGAGGAAGA 100
TCGACCTATT ATTGGCCTAG ACCAAGGCGC TATGTACAGC CTCCTGAAAT 150
GATTGGGCCT ATGCGGCCCG AGCAGTTCAG TGATGAAGTG GAACCAGCAA 200
CACCTGAAGA AGGGGAACCA GCAACTCAAC GTCAGGATCC TGCAGCTGCT 250
CAGGAGGGAG AGGATGAGGG AGCATCTGCA GGTCAAGGGC CGAAGCCTGA 300
AGCTGATAGC CAGGAACAGG GTCACCCACA GACTGGGTGT GAGTGTGAAG 350
ATGGTCCTGA TGGGCAGGAG ATGGACCCGC CAAATCCAGA GGAGGTGAAA 400
ACGCCTGAAG AAGGTGAAAA GCAATCACAG TGTTAAAAGA AGGCACGTTG 450
AAATGATGCA GGCTGCTCCT ATGTTGGAAA TTTGTTCATT AAAATTCTCC 500
CAATAAAGCT TTACAGCCTT CTGCAAAAAA 540
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(2) INFORMATION FOR SBQ ID NO:
17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 532 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTTON: SEQ
ID NO: 17:
AGCTGTGAGG CAGTGCTGTG TGGTTCCTGC CGTCCGGACT CTT'TTTCCTC
50
TACTGAGATT CATCTGTGTG AAATATGAGT TGGCGAGGAA GATCGACCTA 100
TTATTGGCCT AGACCAAGGC GCTATGTACA GCCTCCTGAA GTGATTGGGC 150
CTATGCGGCC CGAGCAGTTC AGTGATGAAG TGGAACCAGC AACACCTGAA 200
GAAGGGGAAC CAGCAACTCA ACGTCAGGAT CCTGCAGCTG CTCAGGAGGG 250
AGAGGATGAG GGAGCATCTG CAGGTCAAGG GCCGAAGCCT GAAGCTGATA 300
GCCAGGAACA GGGTCACCCA CAGACTGGGT GTGAGTGTGA AGATGGTCCT 350
GATGGGCAGG AGATGGACCC GCCAAATCCA GAGGAGGTGA AAACGCCTGA 400
AGAAGGTGAA AAGCAATCAC AGTGTTAAAA GAAGGCACGT TGAAATGATG 450
CAGGCTGCTC CTATGTTGGA AATTTGTTCA TTAAAATTCT CCCAATAAAG 500
CTTTACAGCC TTCTGCAAAG AAAF1,AAAAAAAA 532
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SBQUENCB CHARACTERISTICS:
(A) LENGTH: 539 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
GCCAGGGAGC TGTGAGGCAG TGCTGTGTGG TTCCTGCCGT CCGGACTCTT 50
TTTCCTCTAC TGAGATTCAT CTGTGTGAAA TATGAGTTGG CGAGGAAGAT 100
CGACCTATTA TTGGCCTAGA CCAAGGCGCT ATGTACAGCC TCCTGAAGTG 150
ATTGGGCCTA TGCGGCCCGA GCAGTTCAGT GATGAAGTGG AACCAGCAAC 200
ACCTGAAGAA GGGGAACCAG CAACTCAACG TCAGGATCCT GCAGCTGCTC 250
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AGGAGGGAGA GGATGAGGGA GCATCTGCAG GTCAAGGGCC GAAGCCTGAA 300
GCTGATAGCC AGGAACAGGG TCACCCACAG ACTGGGTGTG AGTGTGAAGA 350
TGGTCCTGAT GGGCAGGAGG TGGACCCGCC AAATCCAGAG GAGGTGAAAA 400
CGCCTGAAGA AGGTGAAAAG CAATCACAGT GTTAAAAGAA GACACGTTGA 450
AATGATGCAG GCTGCTCCTA TGTTGGAAAT TTGTTCATTA AAATTCTCCC 500
AATAAAGCTT TACAGCCTTC TGC'~~P~PrAAAA 5 3 9
(2) INFORMATION FOR SBQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
ACTCCATGAG GTATTTC 17
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
TTTCACCACA TGCGTGT 17
(2) INFORMATION FOR SBQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Arg Ser Thr Tyr Tyr Trp Pro Arg Pro Arg Arg Tyr Val Gln
10
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(2) INFORMATION FOR SBQ ID NO: 22:
(i) SP.QUBNCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Tyr Tyr Trp Pro Arg Pro Arg Arg Tyr
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: Each Xaa may be any amino acid
(xi) SEQUENCE DESCRIPTTON: SEQ ID NO: 23:
Xaa Xaa Trp Pro Xaa Xaa Xaa Xaa Tyr
5
(2) INFORMATION FOR SBQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: Each Xaa may be any amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Xaa Xaa Trp Xaa Arg Xaa Xaa Xaa Tyr
5
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2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ix) FEATCTRE:
(D) Ol~iER INFORMATION: Each Xaa may be any amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Xaa Xaa Trp Xaa Xaa Xaa Xaa Arg Tyr
47
