U.S. patent application number 09/975856 was filed with the patent office on 2003-01-30 for antibodies specific for ssx proteins.
Invention is credited to Chen, Yao-Tseng, Gure, Ali O., Knuth, Alexander, Old, Lloyd J., Pfreundschuh, Michael, Sahin, Ugur, Scanlan, Matthew J., Tsang, Solam, Tureci, Ozlem.
Application Number | 20030023057 09/975856 |
Document ID | / |
Family ID | 25310065 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030023057 |
Kind Code |
A1 |
Gure, Ali O. ; et
al. |
January 30, 2003 |
Antibodies specific for SSX proteins
Abstract
The invention relates to members of the SSX family of genes, as
well as their uses.
Inventors: |
Gure, Ali O.; (New York,
NY) ; Tureci, Ozlem; (Homburg, DE) ; Sahin,
Ugur; (Homburg, DE) ; Tsang, Solam; (New York,
NY) ; Scanlan, Matthew J.; (New York, NY) ;
Knuth, Alexander; (Frankfurt am Main, DE) ;
Pfreundschuh, Michael; (Homburg, DE) ; Old, Lloyd
J.; (New York, NY) ; Chen, Yao-Tseng; (New
York, NY) |
Correspondence
Address: |
Fulbright & Jaworski LLP
666 Fifth Avenue
New York
NY
10103
US
|
Family ID: |
25310065 |
Appl. No.: |
09/975856 |
Filed: |
October 11, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09975856 |
Oct 11, 2001 |
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09796780 |
Mar 1, 2001 |
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6339140 |
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09796780 |
Mar 1, 2001 |
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08851138 |
May 5, 1997 |
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6291658 |
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Current U.S.
Class: |
536/23.2 |
Current CPC
Class: |
C07K 14/47 20130101 |
Class at
Publication: |
536/23.2 |
International
Class: |
C07H 021/04 |
Claims
What is claimed is:
1. Isolated nucleic acid molecule which encodes a protein, the
amino acid sequence of which consists of the amino acid sequence
encoded by SEQ ID NO: 1, SEQ ID NO: 2, or nucleotides 1-330
concatenated to nucleotides 467-576 of SEQ ID NO: 1.
2. The isolated nucleic acid molecule of claim 1, wherein said
isolated nucleic acid molecule encodes the protein encoded by SEQ
ID NO: 1.
3. The isolated nucleic acid molecule of claim 1, wherein said
isolated nucleic acid molecule encodes the protein encoded by SEQ
ID NO: 2.
4. The isolated nucleic acid molecule of claim 1, wherein said
isolated nucleic acid molecule encodes the protein encoded by
nucleotides 1-330 concatenated to nucleotides 467-576 of SEQ ID NO:
1.
5. The isolated nucleic acid molecule of claim 1, having the
nucleotide sequence of SEQ ID NO: 1.
6. The isolated nucleic acid molecule of claim 1, having the
nucleotide sequence of SEQ ID NO: 2.
7. The isolated nucleic acid molecule of claim 1, having the
nucleotide sequence defined by nucleotides 1-330 concatenated to
nucleotides 467-576, as set forth in SEQ ID NO: 1.
8. Expression vector comprising the isolated nucleic acid molecule
of claim 1, operably linked to a promoter.
9. Cell line or cell strain, transformed or transfected with the
isolated nucleic acid molecule of claim 1.
10. Cell line or cell strain, transformed or transfected with the
expression vector of claim 8.
11. Isolated protein encoded by the isolated nucleic acid molecule
of claim 1
12. Isolated protein encoded by the isolated nucleic acid molecule
of claim 2
13. Isolated protein encoded by the isolated nucleic acid molecule
of claim 3
14. Isolated protein encoded by the isolated nucleic acid molecule
of claim 4
15. Isolated nucleic acid molecule useful in determining expression
of an SSX gene in a sample said isolated nucleic acid molecule
consisting of the nucleotide sequence set forth in SEQ ID NO: 3,
SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:
8 SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 SEQ ID NO: 12, SEQ ID
NO: 13, SEQ ID NO: 14.
16. Composition useful in determining expression of an SSX gene in
a sample, comprising (a) SEQ ID NOS: 3 and SEQ ID NO: 4, (b) SEQ ID
NO: 5 and SEQ ID NO: 6, (c) SEQ ID NO: 7 and SEQ ID NO: 8, (d) SEQ
ID NO: 9 and SEQ ID NO: 10, (e) SEQ ID NO: 11 and SEQ ID NO: 12,
and (f) SEQ ID NO: 13 and SEQ ID NO: 14.
17. Method for determining expression of an SSX gene in a sample,
comprising contacting said sample with at least one isolated
nucleic acid molecule of claim 14 and determining hybridization of
said isolated nucleic acid molecule to a target as a determination
of expression of an SSX gene in said sample.
18. Isolated antibody which specifically bonds to the isolated
protein of claim 11.
19. Method for determining presence of an expression product of an
SSX gene in a sample, comprising contacting saw sample with the
isolated antibody of claim 17 and determining binding of said
antibody to a target as a determination of presence of expression
product of an SSX gene in said sample.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the isolation and cloning of genes
which are members of the "SSX" family, which is discussed herein,
and the uses thereof.
BACKGROUND AND PRIOR ART
[0002] It is fairly well established that many pathological
conditions, such as infections, cancer, autoimmune disorders, etc.,
are characterized by the inappropriate expression of certain
molecules. These molecules thus serve as "markers" for a particular
pathological or abnormal condition. Apart from their use as
diagnostic "targets", i.e., materials to be identified to diagnose
these abnormal conditions, the molecules serve as reagents which
can be used to generate diagnostic and/or therapeutic agents. A by
no means limiting example of this is the use of cancer markers to
produce antibodies specific to a particular marker. Yet another
non-limiting example is the use of a peptide which complexes with
an MHC molecule, to generate cytolytic T cells against abnormal
cells.
[0003] Preparation of such materials, of course, presupposes a
source of the reagents used to generate these. Purification from
cells is one laborious, far from sure method of doing so. Another
preferred method is the isolation of nucleic acid molecules which
encode a particular marker, followed by the use of the isolated
encoding molecule to express the desired molecule.
[0004] To date, two strategies have been employed for the detection
of such antigens, in e.g., human tumors. These will be referred to
as the genetic approach and the biochemical approach. The genetic
approach is exemplified by, e.g., dePlaen et al., Proc. Natl. Sci.
USA 85: 2275 (1988), incorporated by reference. In this approach,
several hundred pools of plasmids of a cDNA library obtained from a
tumor are transfected into recipient cells, such as COS cells, or
into antigen-negative variants of tumor cell lines. Transfectants
are screened for the expression of tumor antigens via their ability
to provoke reactions by anti-tumor cytolytic T cell clones. The
biochemical approach, exemplified by, e.g., Mandelboim, et al.,
Nature 369: 69 (1994) incorporated by reference, is based on acidic
elution of peptides which have bound to MHC-class I molecules of
tumor cells, followed by reversed-phase high performance liquid
chromography (HPLC). Antigenic peptides are identified after they
bind to empty MHC-class I molecules of mutant cell lines, defective
in antigen processing, and induce specific reactions with cytotoxic
T-lymphocytes. These reactions include induction of CTL
proliferation, TNF release, and lysis of target cells, measurable
in an MTT assay, or a .sup.51Cr release assay.
[0005] These two approaches to the molecular definition of antigens
have the following disadvantages: first, they are enormously
cumbersome, time-consuming and expensive; second, they depend on
the establishment of cytotoxic T cell lines (CTLs) with predefined
specificity; and third, their relevance in vivo for the course of
the pathology of disease in question has not been proven, as the
respective CTLs can be obtained not only from patients with the
respective disease, but also from healthy individuals, depending on
their T cell repertoire.
[0006] The problems inherent to the two known approaches for the
identification and molecular definition of antigens is best
demonstrated by the fact that both methods have, so far, succeeded
in defining only very few new antigens in human tumors. See, e.g.,
van der Bruggen et al., Science 254: 1643-1647 (1991); Brichard et
al., J. Exp. Med. 178: 489-495 (1993); Coulie, et al., J. Exp. Med.
180: 35-42 (1994); Kawakami, et al., Proc. Natl. Acad. Sci. USA 91:
3515-3519 (1994).
[0007] Further, the methodologies described rely on the
availability of established, permanent cell lines of the cancer
type under consideration. It is very difficult to establish cell
lines from certain cancer types, as is shown by, e.g., Oettgen, et
al., Immunol. Allerg. Clin. North. Am. 10: 607-637 (1990). It is
also known that some epithelial cell type cancers are poorly
susceptible to CTLs in vitro, precluding routine analysis. These
problems have stimulated the art to develop additional
methodologies for identifying cancer associated antigens.
[0008] One key methodology is described by Sahin, et al., Proc.
Natl. Acad. Sci. USA 92: 11810-11913 (1995), incorporated by
reference. Also, see U.S. patent applications Ser. No. 08/580,980,
and application Ser. No. 08/479,328, filed on Jun. 7, 1995 and Jan.
3, 1996, respectively. All three of these references are
incorporated by reference. To summarize, the method involves the
expression of cDNA libraries in a prokaryotic host. (The libraries
are secured from a tumor sample). The expressed libraries are then
immunoscreened with absorbed and diluted sera, in order to detect
those antigens which elicit high titer humoral responses. This
methodology is known as the SEREX method ("Serological
identification of antigens by Recombinant Expression Cloning"). The
methodology has been employed to confirm expression of previously
identified tumor associated antigens, as well as to detect new
ones. See the above referenced patent applications and Sahin, et
al., supra, as well as Crew, et al., EMBO J 144: 2333-2340
(1995).
[0009] The SEREX methodology has been applied to esophageal cancer
samples, and an esophageal cancer associated antigen has now been
identified, and its encoding nucleic acid molecule isolated and
cloned, as per U.S. patent application Ser. No. 08/725,182, filed
Oct. 3, 1996, incorporated by reference herein.
[0010] The relationship between some of the tumor associated genes
and a triad of genes, known as the SSX genes, is under
investigation. See Sahin, et al., supra; Tureci, et al., Cancer Res
56:4766-4772 (1996). One of these SSX genes, referred to as SSX2,
was identified, at first, as one of two genes involved in a
chromosomal translocation event (t(X; 18)(p11.2; q 11.2)), which is
present in 70% of synovial sarcomas. See Clark, et al., Nature
Genetics 7:502-508 (1994); Crew et al., EMBO J 14:2333-2340 (1995).
It was later found to be expressed in a number of tumor cells, and
is now considered to be a tumor associated antigen referred to as
HOM-MEL-40 by Tureci, et al, supra. Its expression to date has been
observed in cancer cells, and normal testis only. Thus parallels
other members of the "CT" family of tumor antigens, since they are
expressed only in cancer and testis cells. Crew et al. also
isolated and cloned the SSX1 gene, which has 89% nucleotide
sequence homology with SSX2. See Crew et al., supra. Additional
work directed to the identification of SSX genes has resulted in
the identification of SSX3, as is described by DeLeeuw, et al.,
Cytogenet. Genet 73:179-183 (1996). The fact that SSX presentation
parallels other, CT antigens suggested to the inventors that other
SSX genes might be isolated.
[0011] Application of a modification of the SEREX technology
described supra has been used, together with other techniques, to
clone two, additional SSX genes, referred to as SSX4 and SSX5
hereafter as well as an alternate splice variant of the SSX4 gene.
Specifically, while the SEREX methodology utilizes autologous
serum, the methods set forth infra use allogenic serum. This, as
well as other features of the invention, are set forth in the
disclosure which follows.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
[0012] A human testicular cDNA expression library was obtained, and
screened, with serum from a melanoma patient identified as MZ2. See
e.g., parent application U.S. patent application Ser. No.
08,479,328 incorporated by reference; also see U.S. patent
application Ser. No. 08/725,182 also incorporated by reference;
Sahin, et al., Proc. Natl. Acad. Sci. USA 92:11810-11813 (1995).
This serum had been treated using the methodology described in
these references. Briefly, serum was diluted 1:10, and then
preabsorbed with transfected E. coli lysate. Following this
preabsorption step, the absorbed serum was diluted 1:10, for a
final dilution of 1:100. Following the final dilution the samples
were incubated overnight at room temperature, with nitrocellulose
membranes containing phage plaques prepared using the methodology
referred to supra. The nitrocellulose membranes were washed,
incubated with alkaline phosphatase conjugated goat anti-human
Fc.sub..gamma. secondary antibodies, and the reaction was observed
with the substrates 5-bromo-4-chloro-3-indolyl phosphate and
nitroblue tetrazolium. In a secondary screen, any phagemids which
encoded human immunoglobulin were eliminated.
[0013] A total of 3.6.times.10.sup.5 pfus were screened, resulting
in eight positive clones. Standard sequencing reactions were
carried out, and the sequences were compared to sequence banks of
known sequences.
[0014] Of the eight clones, two were found to code for known
autoimmune disease associated molecules, i.e., Golgin--95
(Fritzler, et al., J. Exp. Med.178:49-62 (1993)), and human
upstream binding factor (Chan, et al., J. Exp. Med. 174:1239-1244
(1991)). Three other clones were found to encode for proteins which
are widely expressed in human tissue, i.e., ribosomal receptor,
collagen type VI globular domain, and rapamycin binding protein. Of
the remaining three sequences, one was found to be non-homologous
to any known sequence, but was expressed ubiquitously in human
tissues (this was found via RT-PCR analysis, but details are not
provided herein). The remaining two were found to be identical to
full length HOM-MEL-40, described in Ser. No. 08/479,328, while the
eighth clone was found to be almost identical to "SSX3," as
described by DeLeeuw, et al., Cytogenet. Cell Genet 73:179-183
(1996), differing therefrom in only two base pair differences in
the coding region. These differences are probably artifactual in
nature; however, the clone also included a 43 base pair
3'-untranslated region.
EXAMPLE 2
[0015] In order to carry out Southern blotting experiments,
described infra, the SSX genes were amplified, using RT-PCR.
[0016] To do this, two primers were prepared using the published
SSX2 sequence i.e., MEL-40A:
1 5'-CACACAGGAT CCATGAACGG AGA (SEQ ID NO:3) and MEL-40B:
5'-CACACAAAGC TTTGAGGGGA GTTACTCGTC ATC (SEQ. ID NO:4)
[0017] See Crew, et al., EMBO J 14:2333-2340 (1995). Amplification
was then carried out using 0.25 U Taq polymerase in a 25 .mu.l
reaction volume, using an annealing temperature of 60.degree. C. A
total of 35 cycles were carried out.
EXAMPLE 3
[0018] The RT-PCR methodology described supra was carried out on
testicular total RNA, and the amplification product was used in
southern blotting experiments.
[0019] Genomic DNA was extracted from non-neoplastic tissue
samples, and then subjected to restriction enzyme digestion, using
BamHI, Eco RI, or HindIII in separate experiments and then
separated on a 0.7% agarose gel, followed by blotting on to
nitrocellulose filters. The amplification products described supra
were labeled with .sup.32P, using well-known methods, and the
labeled materials were then used as probes under high stringency
conditions (65.degree. C., aqueous buffer), followed by high
stringency washes, ending with a final wash at 0.2.times.SSC, 0.2%
SDS, 65.degree. C.
[0020] The Southern blotting revealed more than 10 bands, in each
case (i.e., each of the BamHI, EcoRI, and HindIII digests),
strongly suggesting that there is a family of SSX genes which
contained more than the three identified previously. In view of
this observation, an approach was designed which combined both PCR
cloning, and restriction map analysis, to identify other SSX
genes.
EXAMPLE 4
[0021] When the sequences of SSX1, 2 and 3 were compared, it was
found that they shared highly conserved 5' and 3' regions, which
explained why the olignucleotides of SEQ ID NOS: 3 and 4 were
capable of amplifying all three sequences under the recited
conditions, and suggested that this homology was shared by the
family of SSX genes, whatever its size. Hence, the oligonucleotides
of SEQ ID NOS: 3 and 4 would be sufficient to amplify the other
members of the SSX gene family.
[0022] An analysis of the sequences of SSX1, 2 and 3 revealed that
SSX1 and 2 contained a BglII site which was not shared by SSX3.
Similarly, SSX3 contained an EcoRV site not shared by the other
genes.
[0023] In view of this information, testicular cDNA was amplified,
using SEQ ID NOS: 3 and 4, as described supra, and was then
subjected to BglII digestion. Any BglII resistant sequences were
then cloned, sequenced, and compared with the known sequences.
[0024] This resulted in the identification of two previously
unidentified sequences, referred to hereafter as SSX4 and SSX5,
presented as SEQ ID NOS: 1 and 2 herein. A search of the GenBank
database found two clones, identified by Accession Number N24445
and W00507, both of which consisted of a sequence-tag--derived cDNA
segments. The clone identified by N24445 contained the
3'-untranslated region of SSX4, and part of its coding sequence,
while the one identified, as W00507 contained a shorter fragment of
the 3'-untranslated region of SSX4, and a longer part of the coding
sequence. Specifically, N24445 consists of base 344 of SSX4 (SEQ ID
NO: 1), through the 3-end, plus 319 bases 3' of the stop codon. The
W00507 sequence consists of a 99 base pair sequence, showing no
homology to SSX genes followed by a region identical to nucleotides
280 through the end of SEQ ID NO:1, through 67 bases 3' of the stop
codon of SEQ ID NO:1.
[0025] Two forms of SSX4 (SEQ ID NO: 1) were identified. One of
these lacked nucleotides 331 to 466 but was otherwise identical to
SSX4 as presented in SEQ ID NO: 1. As is described infra, the
shorter form is an alternatively spliced variant.
[0026] In Table 1, which follows, the nucleotide and amino acid
sequences of the 5 known members of the SSX family are compared.
One reads the table horizontally for nucleotide homology, and
vertically for amino acid homology.
2TABLE 1 Nucleotide and amino acid homology among SSX family
members Nuclcotide Sequence Homology (%) SSX1 SSX2 SSX3 SSX4 SSX5
SSX1 89.1 89.6 89.4 88.7 SSX2 78.2 95.1 91.5 92.9 SSX3 77.7 91.0
91.1 92.7 SSX4 79.3 79.8 80.9 89.8 SSX5 76.6 83.5 84.0 77.7 Amino
Acid Sequence Homology (%)
[0027] Hence, SSX1 and SSX4 share 89.4% homology on the nucleotide
level, and 79.3% homology on the amino acid level.
[0028] When the truncated form of SSX4 is analyzed, it has an amino
acid sequence completely different from others, due to alternate
splicing and shifting of a downstream open reading frame. The
putative protein is 153 amino acids long, and the 42 carboxy
terminal amino acids show no homology to the other SSX
proteins.
EXAMPLE 5
[0029] The genomic organization of the SSX2 genes was then studied.
To do this, a genomic human placental library (in lambda phage) was
screened, using the same protocol and probes described supra in the
discussion of the southern blotting work. Any positive primary
clones were purified, via two additional rounds of cloning.
[0030] Multiple positive clones were isolated, one of which was
partially sequenced, and identified as the genomic clone of SSX2. A
series of experiments carrying out standard subcloning and
sequencing work followed, so as to define the exon--intron
boundaries.
[0031] The analysis revealed that the SSX2, gene contains six
exons, and spans at least 8 kilobases. All defined boundaries were
found to observe the consensus sequence of exon/intron junctions,
i.e. GT/AG.
[0032] The alternate splice variant of SSX4, discussed supra, was
found to lack the fifth exon in the coding region. This was
ascertained by comparing it to the SSX2 genomic clone, and drawing
correlations therefrom.
EXAMPLE 6
[0033] The expression of individual SSX genes in normal and tumor
tissues was then examined. This required the construction of
specific primers, based upon the known sequences, and these follow,
as SEQ ID NOS: 5-14:
3TABLE 2 Gene-specific PCR primer sequences for individual SSX
genes SSX 1A (5'): 5'-CTAAAGCATCAGAGAAGAGAAGC [nt.44-66] SSX 1B
(3'): 5'-AGATCTCTTATTAATCTTCTCAGAAA [nt.440-65] SSX 2A (5'):
5'-GTGCTCAAATACCAGAGAAGATC [nt.41-63] SSX 2B (3'):
5'-TTTTGGGTCCAGATCTCTCGTG [nt.102-25] SSX 3A (5'):
5'-GGAAGAGTGGGAAAAGATGAAAGT [nt.454-75] SSX 3B (3'):
5'-CCCCTTTTGGGTCCAGATATCA [nt.458-79] SSX 4A (5'):
5'-AAATCGTCTATGTGTATATGAAGCT [nt.133-58 SSX 4B (3'):
5'-GGGTCGCTGATCTCTTCATAAAC [nt.526-48] SSX 5A (5'):
5'-GTTCTCAAATACCACAGAAGATG [nt.39-63] SSX 5B (3'):
5'-CTCTGCTGGCTTCTCGGGCG [nt.335-54]
[0034] The specificity of the clones was confirmed by amplifying
the previously identified cDNA for SSX1 through SSX5. Taq
polymerase was used, at 60.degree. C. for SSX1 and 4, and
65.degree. C. for SSX2, 3 and 5. Each set of primer pairs was found
to be specific, except that the SSX2 primers were found to amplify
minute (less than {fraction (1/20)} of SSX2) amounts of SSX3
plasmid DNA.
[0035] Once the specificity was confirmed, the primers were used to
analyze testicular mRNA, using the RT-PCR protocols set forth
supra.
[0036] The expected PCR products were found in all 5 cases, and
amplification with the SSX4 pair did result in two amplification
products, which is consistent with alternative splice variants.
[0037] The expression of SSX genes in cultured melanocytes was then
studied. RT-PCR was carried out, using the protocols set forth
supra. No PCR product was found. Reamplification resulted in a
small amount of SSX4 product, including both alternate forms,
indicating that SSX4 expression in cultured melanocytes is
inconsistent and is at very low levels when it occurs.
[0038] This analysis was then extended to a panel of twelve
melanoma cell lines. These results are set forth in the following
table.
4TABLE 3 SSX expression in melanoma cell lines detected by RT-PCR*
SSX1 SSX2 SSX3 SSX4 SSX5 MZ2-Mel 2.2 + + - - - MZ2-Mel 3.1 + + - -
- SK-MEL-13 - - - - - SK-MEL-19 - - - - - SK-MEL-23 - - - - -
SK-MEL-29 - - - - - SK-MEL-30 -* -* - -* - SK-MEL-31 - - - - -
SK-MEL-33 - - - - - SK-MEL-37 + + - + + SK-MEL-179 - - - - -
M24-MET - - - - - *Positive (+) denotes strong expression. Weak
positivity was observed inconsistently in SK-MEL-30 for SSX 1, 2,
and 4, likely representing low level expression.
[0039] The foregoing examples describe the isolation and cloning of
nucleic acid molecules for the SSX4, splice variant of SSX4, and
SSX5 genes. As was indicated, supra, these genes are expressed in
tumor cells, thereby enabling the skilled artisan to utilize these
for, e.g., assaying for cancer, such as melanoma. As the genes
express a protein which, in turn, would provoke generation of
antibodies in vivo, these proteins are also a part of the invention
as are the isolated antibodies specific for them.
[0040] The isolated nucleic acid molecules of the invention
encompass those degenerate sequences which, though not identical to
SEQ ID NO: 1, its splice variant, or SEQ ID NO: 2, do encode the
same proteins which these sequences encode. Also a part of the
invention are expression vectors which comprise these molecules,
operably linked to a promoter, and the cell lines or cell strains
which are transformed or transfected with these vectors, or the
nucleic acid molecules themselves. These are all useful in making
the protein, e.g., as well as for producing amplified copies of the
relevant sequences.
[0041] Also, a part of the invention are those nucleic acid
molecules defined herein by SEQ ID NOS: 5 through 14, compositions
containing these, and the use thereof in assaying for expression of
one or more SSX gene. Any nucleic acid hybridization methodology
can be used, including, e.g., PCR methodologies. The antibodies of
the invention may also be used in assays, but in this case the
target is the expression product of the SSX genes.
[0042] Other features of the invention will be clear to the skilled
artisan, and need not be repeated here.
[0043] 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.
5 SSX4 DNA; 576BP. ATGAACGGAG ACGACGCCTT TGCAAGGAGA CCCAGGGATG
ATGCTCAAAT SEQ ID NO:1 ATCAGAGAAG TTACGAAAGG CCTTCGATGA TATTGCCAAA
TACTTCTCTA AGAAAGAGTG GGAAAAGATG AAATCCTCGG AGAAAATCGT CTATGTGTAT
ATGAAGCTAA ACTATGAGGT CATGACTAAA CTAGGTTTCA AGGTCACCCT CCCACCTTTC
ATGCGTAGTA AACGGGCTGC AGACTTCCAC GGGAATGATT TTGGTAACGA TCGAAACCAC
AGGAATCAGG TTGAACGTCC TCAGATGACT TTCGGCAGCC TCCAGAGAAT CTTCCCGAAG
ATCATGCCCA AGAAGCCAGC AGAGGAAGAA AATGGTTTGA AGGAAGTGCC AGAGGCATCT
GGCCCACAAA ATGATGGGAA ACAGCTGTGC CCCCCGGGAA ATCCAAGTAC CTTGGAGAAG
ATTAACAAGA CATCTGGACC CAAAAGGGGG AAACATGCCT GGACCCACAG ACTGCGTGAG
AGAAAGCAGC TGGTGGTTTA TGAAGAGATC AGCGACCCTG AGGAAGATGA CGAGTAACTC
CCCTCG SSX5 DNA; 576BP. ATGAACGGAG ACGACGCCTT TGTACGGAGA CCTAGGGTTG
GTTCTCAAAT SEQ ID NO:2 ACCACAGAAG ATGCAAAAGG CCTTCGATGA TATTGCCAAA
TACTTCTCTG AGAAAGAGTG GGAAAAGATG AAAGCCTCGG AGAAAATCAT CTATGTGTAT
ATGAAGAGAA AGTATGAGGC CATGACTAAA CTAGGTTTCA AGGCCACCCT CCCACCTTTC
ATGCGTAATA AACGGGTCGC AGACTTCCAG GGGAATGATT TTGATAATGA CCCTAACCGT
GGGAATCAGG TTGAACATCC TCAGATGACT TTCGGCAGGC TCCAGGGAAT CTTCCCGAAG
ATCACGCCCG AGAAGCCAGC AGAGGAAGGA AATGATTCAA AGGGAGTGCC AGAAGCATCT
GGCCCACAGA ACAATGGGAA ACAGCTGCGC CCCTCAGGAA AACTAAATAC CTCTGAGAAG
GTTAACAAGA CATCTGGACC CAAAAGGGGG AAACATGCCT GGACCCACAG AGTGCGTGAG
AGAAAGCAAC TGGTGATTTA TGAAGAGATC AGCGACCCTC CGGAAGATGA CGAGTAACTC
CCCTCA
* * * * *