Isolated peptides derived from tumor rejection antigens, and their use

A new family of tumor rejection antigen precursors, and the nucleic acid molecules which code for them, are disclosed. These tumor rejection antigen precursors are referred to as GAGE tumor rejection antigen precursors, and the nucleic acid molecules which code for them are referred to as GAGE coding molecules. Various diagnostic and therapeutic uses of the coding sequences and the tumor rejection antigens, and their precursor molecules are described. Tumor rejection antigens are also shown.

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 antigen precursor is processed, inter alia, 
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. 
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, 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 ("HLA"), 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). 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 Nov. 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 et al., Science 254: 
1643 (1991), for further information on this family of genes. Also, see 
U.S. patent application Ser. No. 807,043, filed Dec. 12, 1991, now U.S. 
Pat. No. 5,342,774. 
In U.S. patent application Ser. No. 938,334 now U.S. Pat. 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 Ser. No. 008,446, filed Jan. 22, 1993 and 
incorporated by reference, the fact that the MAGE-1 expression product is 
processed to a second TRA 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 Ser. No. 994,928, filed Dec. 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 HLA-A2 molecules. 
In U.S. patent application Ser. No. 08/032,978, filed Mar. 18, 1993, and 
incorporated by reference in its entirety, a second TRA, not derived from 
tyrosinase is taught to be presented by HLA-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 Ser. No. 08/079,110, filed Jun. 17, 1993 and 
incorporated by reference herein, an unrelated tumor rejection antigen 
precursor, the so-called "BAGE" precursor is described. The BAGE precursor 
is not related to the MAGE family. 
The work which is presented by the papers, patent, and patent applications 
cited supra deals, in large part, with the MAGE family of genes, and the 
unrelated BAGE gene. It has now 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 BAGE gene. Thus the present invention relates to genes encoding 
such TRAPs, the tumor rejection antigen precursors themselves as well as 
applications of both. 
The invention is elaborated upon further in the disclosure which follows.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
Example 1 
A melanoma cell line, MZ2-MEL was established from melanoma cells taken 
from patient MZ2, using standard methodologies. This cell line is 
described, e.g., in PCT Application PCT/US92/04354, filed May 22, 1992, 
published Nov. 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 
patient 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. 
The lysis assay employed was a chromium release assay following Herin et 
al., 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 in vitro, and then resuspended at 
10.sup.7 cells/ml in DMEM, supplemented with 10 mM HEPES and 30% FCS, and 
incubated for 45 minutes at 37.degree. C. with 200 .mu.Ci/ml of Na(.sup.51 
Cr)O.sub.4. Labelled cells were washed three times with DMEM, supplemented 
with 10 mM Hepes. These were then resuspended in DMEM supplemented with 10 
mM Hepes and 10% FCS, after which 100 ul aliquots containing 10.sup.3 
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 100 g, and incubated 
for four hours at 37.degree. C. in a 8% CO.sub.2 atmosphere. 
Plates were centrifuged again, and 100 ul aliquots of supernatant were 
collected and counted. Percentage of .sup.51 Cr release was calculated as 
follows: 
##EQU1## 
where ER is observed, experimental .sup.51 Cr release, SR is spontaneous 
release measured by incubating 10.sup.3 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 "76/6" hereafter. 
The same method was used to test target K562 cells, as well as the melanoma 
cell line. FIG. 1 shows that this CTL clone recognizes and lyses the 
melanoma cell line, i.e. MZ2-MEL but not K562. The clone was then tested 
against other melanoma cell lines and autologous EBV-transformed B cells 
in the same manner described supra. FIG. 1 shows that autologous B cells, 
transformed by Epstein Barr Virus ("EBV") were not lysed, and that while 
MZ2-MEL 3.0 was lysed by CTL clone 76/6, the cell line MZ2-MEL.4F.sup.-, a 
variant which does not express antigen F, was not. Hence, the clone 
appears to be specific for this antigen. 
The results presented supra 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 et al., 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. 
Example 3 
The results from Example 2 indicated that MZ2.MEL.43 presented the target 
antigen 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 site, followed by second strand 
synthesis. The cDNA was then ligated to a BstXI adaptor, digested with 
NotI, size fractionated on a Sephacryl S-500 HR column, and then cloned, 
undirectionally, into the BstXI and Not I sites of pcDNAI/Amp. The 
recombinant plasmid was then electroporated into DH5.alpha. 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. 
Example 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 10% fetal 
calf serum. The cells were incubated overnight at 37.degree. C., medium 
was removed and then replaced by 50 .mu.l/well of DMEM medium containing 
10% Nu serum, 400 .mu.g/ml DEAE-dextran, and 100 .mu.M chloroquine, plus 
100 ng of the plasmids. As was indicated supra, 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 (A1, B37, Cw6) 
was used, separately, to cotransfect the cells. Specifically, one of 28 ng 
of the gene encoding HLA-A1, cloned into pCD-SR.alpha., 50 ng of cDNA for 
HLA-B37 in pcDNA-I-Amp, or 75 ng of cDNA for HLA-Cw6 in pcDNA-I-Amp, using 
the same protocol as was used for transfection with the library, were 
used. 
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.degree. C., the medium was removed, and replaced 
by 50 .mu.l of PBS containing 10% DMSO. This medium was removed after two 
minutes and replaced by 200 .mu.l of DMEM supplemented with 10% FCS. 
Following this change in medium, COS cells were incubated for 24-48 hours 
at 37.degree. C. Medium was then discarded, and 1000-3000 cells of CTL 
clone 76/6 were added, in 100 .mu.l of Iscove's medium containing 10% 
pooled human serum supplemented with 20-30 U/ml of IL-2. Supernatant was 
removed after 24 hours, and TNF content was determined in an assay on WEHI 
cells, as described by Traversari et al., Immunogenetics 35: 145-152 
(1992), the disclosure of which is incorporated by reference. 
The 1500 pools transfected with HLA-A1, 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 more than 60 pg/ml. This pool was 
selected for further work. 
Example 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 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-MEL F.sup.- and MZ2-MEL F.sup.+ were also used. TNF release into CTL 
supernatant was measured by testing it on WEHI cells, as referred to 
supra. The number of surviving WEHI cells was measured by optical density 
after incubation of the cells with MTT. FIG. 2 shows that the COS cells 
transfected with HLA-Cw6 and cDNA-2D6, and the cell line MZ2-MEL F.sup.+ 
stimulated TNF release from CTL clone 76/6, indicating that HLA-Cw6 
presented the subject TRA. 
Example 6 
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. ID 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. PCR was then used to confirm the 
results. 
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 
Lung Carcinoma 1/6 
Sarcoma 0/1 
Thyroid medullary carcinoma 
0/1 
Tumor samples 
Melanoma 1/1 
______________________________________ 
Example 8 
Detailed analysis of normal tissues and tumors was carried out by applying 
polymerase 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 5.times., 1 
ul of each dNTP, (10 mM), 2 ul of dithiothreitol (100 mM), 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 mixed and incubated at 42.degree. C. for 60 
minutes, after which it was chilled on ice. A total of 80 ul of water was 
then added, to 100 ul total. This mixture was stored at -20.degree. C. 
until used in PCR. 
##STR1## 
SEQ ID NOS: 2 and 3, respectively, were used. The reagents included 30.5 
ul water, 5 ul of PCR buffer 10.times., 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 transferred to a 
thermocycler block, preheated to 94.degree. C., and amplification was 
carried out for 30 cycles, each cycle consisting of the following: 
______________________________________ 
first denaturation: 94.degree. C., 4 min. 
denaturation: 94.degree. C., 1 min. 
annealing: 55.degree. C., 2 min. 
extension: 72.degree. C., 3 min. 
final extension: 72.degree. C., 15 min. 
______________________________________ 
Following the cycling, 10 ul aliquots were run on a 1.5% agarose gel, 
stained with ethidium bromide. 
cDNA amplified using the primers set forth supra yields a 238 base pair 
fragment. There is no amplification of contaminating genomic DNA, if 
present. 
The results are presented in Table 2, which follows. They confirm that the 
only 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 by assaying for expression of the GAGE 
gene. 
TABLE 2 
______________________________________ 
RT-PCR analysis of the expression of gene GAGE 
______________________________________ 
NORMAL TISSUES 
______________________________________ 
Heart - 
Brain - 
Liver - 
Lung - 
Kidney - 
Spleen - 
Lymphocytes - 
Bone marrow - 
Skin - 
Naevus - 
Melanocytes - 
Fibroblasts - 
Prostate - 
Testis + 
Ovary - 
Breast - 
Adrenals - 
Muscle - 
Placenta - 
Umbilical 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 
Larynx 6/15 (40%) 
Pharynx 3/13 
Sarcoma 1/4 6/18 (33%) 
Testicular seminoma 6/6 (100%) 
Bladder cancer 5/37 (14%) 
Prostate cancer 2/20 
Colon carcinoma 5/13 0/38 
Renal cancer 0/6 0/45 
Leukemia 3/6 0/19 
______________________________________ 
Example 9 
The identification of the nucleic acid molecule referred to in the prior 
examples led to further work directed to the determination of tumor 
rejection antigens presented by HLA-Cw6 molecules, and derived from the 
GAGE gene. 
The complete cDNA of GAGE in expression vector pcDNAI/Amp was digested with 
restriction endonucleases NotI and SpHI, and then with exonuclease III 
following supplier's instruction (Erase-a-base System, Promega). This 
treatment generated a series of progressive deletions, starting at the 
3'end. 
The deletion products were ligated back into pcDNAI/Amp, and then 
electroporated into E. coli strain DH5alpha'IQ, using well known 
techniques. The transformants were selected with ampicillin (50 
micrograms/ml). 
Plasmid DNA was extracted from 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. 
Example 10 
Additional experiments were then carried out to define the region encoding 
the TRA peptide more precisely. Polymerase 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 
BamHI 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 XbaI restriction site. 
Following amplification, the PCR product was digested by BamHI and XbaI, 
and cloned into the BamHI-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 
supra, 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. 
Example 11 
Two peptides were synthesized, based upon the first 23 amino acids of SEQ 
ID NO: 1. These were: 
##STR2## 
Each peptide was pulsed into COS-7 cells previously transfected with 
HLA-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.degree. C. for 90 minutes, medium was discarded, and 3000 
CTLs were added in 100 microliters 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, .sup.51 Cr 
labelled LB33-EBV cells, which are HLA-Cw6 positive, were incubated with 
one of the following peptides: 
##STR3## 
The peptide concentration varied, as indicated in FIG. 3, and the ratio of 
CTL: LB33-EBV ("effector: target ratio"), was 10:1. .sup.51 Cr release was 
determined after four hours of incubation at 37.degree. C. Levels of lysis 
for positive ("F.sup.+ ", MZ2-MEL.3.1), and negative ("F.sup.- "; 
MZ2-MEL.2.2.5) control cells are indicated, in FIG. 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, supra, at 199, for H-2K.sup.b and H-2K.sup.K 
molecules. The nonamers of SEQ ID NO: 5 and SEQ ID NO: 6 also induced CTL 
lysis albeit 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. 
Example 13 
To find out whether the GAGE DNA set forth supra 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 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 from but highly related to GAGE were identified. They 
are called GAGE-2, 3, 4, 5 and 6 (FIG. 4), and are presented as SEQ ID 
NOS: 14-18, respectively. The fourteen other clones were partially 
sequenced at the 5' 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 ID NO: 1. 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 
(FIG. 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 cDNA of HLA-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 that 7 of them contained the sequence encoding the 
antigenic peptide, and thus probably corresponded to either GAGE-1 or 
GAGE-2. 
Example 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: 
##STR4## 
These primers were used as described, supra, in a RT-PCR reaction using a 
polymerase enzyme in the following temperature conditions: 
______________________________________ 
4 min at 94.degree. C. 
30 cycles with 1 min at 94.degree. C. 
2 min at 56.degree. C. 
3 min at 72.degree. C. 
15 min at 72.degree. C. 
______________________________________ 
The results of this analysis are set forth in Table 3. 
TABLE 3 
______________________________________ 
Expression of GAGE genes by tumor samples and tumor cell lines 
Number of GAGE positive tumors 
Histological type 
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 carcinomas NSCLC 
14/65 12/64 (19%) 
Head and neck squamous cell 
13/55 10/54 (19%) 
carcinomas 
Prostatic carcinomas 
2/20 2/20 
Mammary carcinomas 
18/162 14/162 (9%) 
Bladder carcinomas 
superficial 1/20 1/20 
infiltrating 5/26 3/26 
Testicular seminomas 
6/6 5/6 
Colorectal carcinomas 
0/43 
Leukemiss and lymphomas 
0/25 
Renal carinomas 0/46 
Tumor cell lines 
Melanomas 45/74 40/74 (54%) 
Sarcomas 1/4 1/4 
Lung carcinomas 
SCLC 7/24 7/24 (29%) 
NSCLC 1/2 1/2 
Mesotheliomas 5/19 5/19 (26%) 
Head and neck squamous cell 
0/2 
carcinomas 
Mammary carcinomas 
1/4 0/4 
Bladder carcinomas 
0/3 
Colon carcinomas 
5/13 5/13 
Leukemias 3/6 1/6 
Lymphomas 0/6 
Renal carcinomas 
0/6 
______________________________________ 
*Expression of GAGE was tested by RTPCR on total RNA with primers VDE18 
and VDE24, detecting all GAGE genes. No PCR product was observed when 
these primers were assayed on DNA from MZ2MEL. 
**Expression of GAGE1 and 2 was tested by RTPCR on total RNA with primers 
VDE44 and VDE24, which distinguish GAGE1 and 2 from the four other GAGE 
genes. No PCR product was observed when these primers were assayed on DNA 
from MZ2MEL. 
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 
##STR5## 
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. 
TABLE 4 
______________________________________ 
Expression of GAGE genes 
in normal adult and fetal tissues 
GAGE 
expression* 
______________________________________ 
Adult tissues 
Adrenal gland - 
Benign naevus - 
Bone marrow - 
Brain - 
Breast - 
Cerebellum - 
Colon - 
Heart - 
Kidney - 
Liver - 
Lung - 
Melanocytes - 
Muscle - 
Ovary - 
Prostate - 
Skin - 
Splenocytes - 
Stomach - 
Testis + 
Thymocytes - 
Urinal bladder - 
Uterus - 
Placenta - 
Umbilical - 
cord 
Fetal tissues* 
Fibroblasts - 
Brain - 
Liver - 
Spleen - 
Thymus - 
Testis + 
______________________________________ 
*Expression of GAGE was tested by RTPCR amplification on total RNA with 
primers VDE43 and VDE24 detecting all GAGE genes (FIG. 7). Absence of PCR 
product is indicated by - and presence by +. No PCR product was observed 
when these primers were assayed on DNA from MZ2MEL. 
*Fetal tissues derive from fetuses older than 20 weeks. 
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 MAGE and BAGE 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 sequence set forth in SEQ ID 
NO: 1 as well as fragments thereof, such as nucleotides 1-170, and 51-170, 
and any other fragment which is processed to a tumor rejection antigen. 
The sequence of SEQ ID NO: 1 is neither a MAGE nor a BAGE coding sequence, 
as will be seen by comparing it 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, 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 NaCl, 1% SDS, and 10% dextran 
sulfate. This is followed by two washes of the filter at room temperature 
for 5 minutes, in 2.times.SSC, and one wash for 30 minutes in 2.times.SSC, 
0.1% SDS. There are other conditions, reagents, 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, be these prokaryotic (e.g., E. coli), 
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 promoter. As it has been found that human leukocyte antigen 
HLA-Cw6 presents a tumor rejection antigen derived from these genes, the 
expression vector may also include a nucleic acid molecule coding for 
HLA-Cw6. 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 HLA-Cw6. 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-Cw6 
presenting cells if desired, and the gene for tumor rejection antigen 
precursor can be used in host cells which do not express 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 TRAPs of the invention 
from the previously described MAGE and BAGE materials, the invention shall 
be referred to as the 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. 
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 HLA-Cw6. In the former situation, such 
determinations can be carried out via any standard nucleic acid 
determination assay, including the polymerase chain reaction, or assaying 
with labelled hybridization probes. In the latter situation, assaying with 
binding partners for complexes of TRA and HLA, such as antibodies, is 
especially preferred. An alternate method for determination is a TNF 
release assay, of the type described supra. To carry out the assay, it is 
preferred to make sure that testis cells are not present, as these 
normally express GAGE. This 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 for by SEQ ID NO: 1. These isolated molecules when 
presented as the TRA, or as complexes of TRA and HLA, such as HLA-Cw6, may 
be combined with materials such as adjuvants to produce vaccines useful in 
treating disorders characterized by expression of the TRAP molecule. 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, etcetera. In all cases where cells are 
used as a vaccine, these can be cells 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 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 indicated, supra, tumor 
rejection antigens, such as the one presented in SEQ ID NO: 4 are also a 
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 sequence set forth in SEQ ID NO: 4. As the examples 
indicate, those peptides which are longer than the octamer of SEQ ID 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 to the cells presenting the complex. The fact that these 
peptides are processed to the tumor rejection antigen, is indicated by the 
examples. 
This property may be exploited in the context of other parameters in 
confirming diagnosis of pathological conditions, such as cancer, melanoma 
in particular. For example, 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. 
On their own, peptides in accordance with the invention may be used to 
carry out HLA-typing assays. It is well known that when a skin graft, 
organ transplant, etc., is necessary one must perform HLA typing so as to 
minimize the possibility of graft rejection. The peptides of the invention 
may be used to determine whether or not an individual is HLA-Cw6 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 HLA-Cw6 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-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 target cell can be a transfectant, such as a 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 (Jun. 10, 1992); Lynch et al., Eur. J. Immunol. 21: 1403-1410 (1991); 
Kast et al., Cell 59: 603-614 (Nov. 17, 1989)), 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 which is characterized by certain of 
the abnormal cells presenting the particular complex, where the complex 
contains the pertinent HLA molecule. The CTLs then lyse 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 via 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 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 supra. 
Adoptive transfer is not the only form of therapy that is available in 
accordance with the invention. CTLs can also be provoked in vivo, using a 
number of approaches. One 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 Vaccinia virus 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 HLA-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 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. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 18 
(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: 
CTGCCGTCCGGACTCTTTTTCCTCTACTGAGATTCATCTGTGTGAAATAT50 
GAGTTGGCGAGGAAGATCGACCTATCGGCCTAGACCAAGACGCTACGTAG100 
AGCCTCCTGAAATGATTGGGCCTATGCGGCCCGAGCAGTTCAGTGATGAA150 
GTGGAACCAGCAACACCTGAAGAAGGGGAACCAGCAACTCAACGTCAGGA200 
TCCTGCAGCTGCTCAGGAGGGAGAGGATGAGGGAGCATCTGCAGGTCAAG250 
GGCCGAAGCCTGAAGCTGATAGCCAGGAACAGGGTCACCCACAGACTGGG300 
TGTGAGTGTGAAGATGGTCCTGATGGGCAGGAGATGGACCCGCCAAATCC350 
AGAGGAGGTGAAAACGCCTGAAGAAGAGATGAGGTCTCACTATGTTGCCC400 
AGACTGGGATTCTCTGGCTTTTAATGAACAATTGCTTCTTAAATCTTTCC450 
CCACGGAAACCTTGAGTGACTGAAATATCAAATGGCGAGAGACCGTTTAG500 
TTCCTATCATCTGTGGCATGTGAAGGGCAATCACAGTGTTAAAAGAAGAC550 
ATGCTGAAATGTTGCAGGCTGCTCCTATGTTGGAAAATTCTTCATTGAAG600 
TTCTCCCAATAAAGCTTTACAGCCTTCTGCAAAGAAAAAAAAAAAA646 
(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: 
AGACGCTACGTAGAGCCT18 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
CCATCAGGACCATCTTCA18 
(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: 
TyrArgProArgProArgArgTyr 
(2) INFORMATION FOR SEQ 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: 
ThrTyrArgProArgProArgArgTyr 
5 
(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: 
TyrArgProArgProArgArgTyrVal 
5 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
ThrTyrArgProArgProArgArgTyrVal 
510 
(2) INFORMATION FOR SEQ 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: 
ArgProArgProArgArgTyrValGlu 
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: 
GACCAAGACGCTACGTAG18 
(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: 
CCATCAGGACCATCTTCA18 
(2) INFORMATION FOR SEQ ID 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: 
GCGGCCCGAGCAGTTCA17 
(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: SEQ ID NO: 12: 
MetSerTrpArgGlyArgSerThrTyrArgProArgProArgArg 
51015 
(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: 
ThrTyrArgProArgProArgArgTyrValGluProProGluMetIle 
51015 
(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: 
ACGCCAGGGAGCTGTGAGGCAGTGCTGTGTGGTTCCTGCCGTCCGGACTC50 
TTTTTCCTCTACTGAGATTCATCTGTGTGAAATATGAGTTGGCGAGGAAG100 
ATCGACCTATCGGCCTAGACCAAGACGCTACGTAGAGCCTCCTGAAATGA150 
TTGGGCCTATGCGGCCCGAGCAGTTCAGTGATGAAGTGGAACCAGCAACA200 
CCTGAAGAAGGGGAACCAGCAACTCAACGTCAGGATCCTGCAGCTGCTCA250 
GGAGGGAGAGGATGAGGGAGCATCTGCAGGTCAAGGGCCGAAGCCTGAAG300 
CTCATAGCCAGGAACAGGGTCACCCACAGACTGGGTGTGAGTGTGAAGAT350 
GGTCCTGATGGGCAGGAGATGGACCCGCCAAATCCAGAGGAGGTGAAAAC400 
GCCTGAAGAAGGTGAAAAGCAATCACAGTGTTAAAAGAAGACACGTTGAA450 
ATGATGCAGGCTGCTCCTATGTTGGAAATTTGTTCATTAAAATTCTCCCA500 
ATAAAGCTTTACAGCCTTCTGCAAAGAAAAAAAAAAAA538 
(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: 
CTCATATTTCACACAGATGAGTTGGCGAGGAAGATCGACCTATTATTGGT50 
CTAGGCCAATAATAGGTCGATCTTCCTCGCCAACTCATATTTCACACAGA100 
TGAATCTCAGTAGAGGAAAATCGACCTATTATTGGCCTAGACCAAGGCGC150 
TATGTACAGCCTCCTGAAGTGATTGGGCCTATGCGGCCCGAGCAGTTCAG200 
TGATGAAGTGGAACCAGCAACACCTGAAGAAGGGGAACCAGCAACTCAAC250 
GTCAGGATCCTGCAGCTGCTCAGGAGGGAGAGGATGAGGGAGCATCTGCA300 
GGTCAAGGGCCGAAGCCTGAAGCTGATAGCCAGGAACAGGGTCACCCACA350 
GACTGGGTGTGAGTGTGAAGATGGTCCTGATGGGCAGGAGATGGACCCGC400 
CAAATCCAGAGGAGGTGAAAACGCCTGAAGAAGGTGAAAAGCAATCACAG450 
TGTTAAAAGAAGGCACGTTGAAATGATGCAGGCTGCTCCTATGTTGGAAA500 
TTTGTTCATTAAAATTCTCCCAATAAAGCTTTACAGCCTTCTGCAAAGAA550 
AAAAAAAAAA560 
(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: 
CGCCAGGGAGCTGTGAGGCAGTGCTGTGTGGTTCCTGCCGTCCGGACTCT50 
TTTTCCTCTACTGAGATTCATCTGTGTGAAATATGAGTTGGCGAGGAAGA100 
TCGACCTATTATTGGCCTAGACCAAGGCGCTATGTACAGCCTCCTGAAAT150 
GATTGGGCCTATGCGGCCCGAGCAGTTCAGTGATGAAGTGGAACCAGCAA200 
CACCTGAAGAAGGGGAACCAGCAACTCAACGTCAGGATCCTGCAGCTGCT250 
CAGGAGGGAGAGGATGAGGGAGCATCTGCAGGTCAAGGGCCGAAGCCTGA300 
AGCTGATAGCCAGGAACAGGGTCACCCACAGACTGGGTGTGAGTGTGAAG350 
ATGGTCCTGATGGGCAGGAGATGGACCCGCCAAATCCAGAGGAGGTGAAA400 
ACGCCTGAAGAAGGTGAAAAGCAATCACAGTGTTAAAAGAAGGCACGTTG450 
AAATGATGCAGGCTGCTCCTATGTTGGAAATTTGTTCATTAAAATTCTCC500 
CAATAAAGCTTTACAGCCTTCTGCAAAAAAAAAAAAAAAA540 
(2) INFORMATION FOR SEQ ID NO: 17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 532 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 
AGCTGTGAGGCAGTGCTGTGTGGTTCCTGCCGTCCGGACTCTTTTTCCTC50 
TACTGAGATTCATCTGTGTGAAATATGAGTTGGCGAGGAAGATCGACCTA100 
TTATTGGCCTAGACCAAGGCGCTATGTACAGCCTCCTGAAGTGATTGGGC150 
CTATGCGGCCCGAGCAGTTCAGTGATGAAGTGGAACCAGCAACACCTGAA200 
GAAGGGGAACCAGCAACTCAACGTCAGGATCCTGCAGCTGCTCAGGAGGG250 
AGAGGATGAGGGAGCATCTGCAGGTCAAGGGCCGAAGCCTGAAGCTGATA300 
GCCAGGAACAGGGTCACCCACAGACTGGGTGTGAGTGTGAAGATGGTCCT350 
GATGGGCAGGAGATGGACCCGCCAAATCCAGAGGAGGTGAAAACGCCTGA400 
AGAAGGTGAAAAGCAATCACAGTGTTAAAAGAAGGCACGTTGAAATGATG450 
CAGGCTGCTCCTATGTTGGAAATTTGTTCATTAAAATTCTCCCAATAAAG500 
CTTTACAGCCTTCTGCAAAGAAAAAAAAAAAA532 
(2) INFORMATION FOR SEQ ID NO: 18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 539 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 
GCCAGGGAGCTGTGAGGCAGTGCTGTGTGGTTCCTGCCGTCCGGACTCTT50 
TTTCCTCTACTGAGATTCATCTGTGTGAAATATGAGTTGGCGAGGAAGAT100 
CGACCTATTATTGGCCTAGACCAAGGCGCTATGTACAGCCTCCTGAAGTG150 
ATTGGGCCTATGCGGCCCGAGCAGTTCAGTGATGAAGTGGAACCAGCAAC200 
ACCTGAAGAAGGGGAACCAGCAACTCAACGTCAGGATCCTGCAGCTGCTC250 
AGGAGGGAGAGGATGAGGGAGCATCTGCAGGTCAAGGGCCGAAGCCTGAA300 
GCTGATAGCCAGGAACAGGGTCACCCACAGACTGGGTGTGAGTGTGAAGA350 
TGGTCCTGATGGGCAGGAGGTGGACCCGCCAAATCCAGAGGAGGTGAAAA400 
CGCCTGAAGAAGGTGAAAAGCAATCACAGTGTTAAAAGAAGACACGTTGA450 
AATGATGCAGGCTGCTCCTATGTTGGAAATTTGTTCATTAAAATTCTCCC500 
AATAAAGCTTTACAGCCTTCTGCAAAAAAAAAAAAAAAA539 
__________________________________________________________________________