Note: Descriptions are shown in the official language in which they were submitted.
CA 02304368 2000-03-15
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REAGENTS AND METHODS USEFUL FOR DETECTING DISEASES OF
THE PROSTATE BREAST AND OVARY
Bac ,ground of the Invention
This invention relates generally to detecting diseases of the prostate,
breast,
and ovary. Furthermore, the invention also relates to reagents and methods for
detecting diseases of the prostate, breast, and ovary. More particularly, the
present
invention relates to reagents such as polynucleotide sequences and the
polypeptide
sequences encoded thereby, as well as methods which utilize these sequences.
The
polynucleotide and polypeptide sequences are useful for detecting, diagnosing,
staging, monitoring, prognosticating; in vivo imaging, preventing or treating,
or
determining predisposition to diseases or conditions of the prostate, breast,
and
ovary such as prostate cancer, breast cancer, and ovarian cancer.
Prostate cancer is the most common form of cancer occurnng in males
in the United States, with projections of 184,500 new cases diagnosed and
39,200 related deaths to occur during 1998 {American Cancer Society
statistics). Prostate cancer also has shown the largest increase in incidence
as
compared to other types of cancer, increasing 142% from 1992 to 1996.
Breast cancer is the most common form of cancer occurnng in females
in the U.S. The incidence of breast cancers in the United States is projected
to
be 180,300 cases diagnosed and 43,900 breast cancer-related deaths to occur
during 1998 (American Cancer Society statistics). Worldwide, the incidence of
breast cancer increased from 700,000 in 1985 to about 900,000 in 1990. G.N.
Hortobagyi et al., CA Cancer J Clin 45:199-226 { 1995).
Ovarian cancer is the leading cause of death from gynecological cancer
in the United States with 25,400 new cases and 14,500 deaths projected for
1998 (American Cancer Society statistics). In the U.S. and other
industrialized
countries, the age-adjusted death rate of this cancer has been steadily
increasing
during the past 25 years. R.C. Young et al. In: Cancer: Principles and
Practice
of Oncology, Fourth Edition, V.T. DeVita et al., eds., pp. 1226-1263,
Philadelphia, PA, J.B. Lippincott Co. (1993).
Procedures used for detecting, diagnosing, staging, monitoring,
prognosticating, in vivo imaging, preventing or treating, or determining
predisposition to diseases or conditions of the prostate such as prostate
cancer
are of critical importance to the outcome of the patient. For example,
patients
diagnosed with localized prostate cancer have greater than a 90% five-year
survival rate compared to a rate of 25 to 31% for patients diagnosed with
CA 02304368 2000-03-15
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distant metastasis. (American Cancer Society statistics). A diagnostic
procedure for early detection of prostate cancer should, therefore,
specifically
detect this disease and be capable of detecting the presence of prostate
cancer
before symptoms appear.
Such procedures could include assays based upon the appearance of
various disease markers in test samples such as blood, plasma, serum, or urine
obtained by minimally invasive procedures which are detectable by
immunological methods. These procedures would provide information to aid
the physician in managing the patient with disease of the prostate and at low
cost to the patient. Markers such as the prostate specific antigen (PSA) exist
and are used clinically for screening patients for prostate cancer. Elevated
levels of PSA protein in serum can be used as a marker in the early detection
of
prostate cancer in asymptomatic men. G.E. Hanks, et al., In: Cancer:
Principles and Practice of Oncology. Vol. 1, Fourth Edition, pp. 1073-1113,
Philadelphia, PA: J.B. Lippincott Co. (1993.). PSA normally is secreted by
the prostate at high levels into the seminal fluid, but is present in very low
levels in the blood of men with normal prostates. However, in patients with
diseases of the prostate including benign prostatic hyperplasia (BPH) and
adenocarcinoma of the prostate, the level of PSA can be markedly elevated in
the blood and thus be useful as an indicator of prostate disease. PSA,
however, cannot differentiate between BPH and prostate cancer, which reduces
its specificity as a marker for prostate cancer. M.K. Schwartz, et al., In:
Cancer: Principles and Practice of Oncology, Vol. 1, Fourth Edition, pp. 531-
542, Philadelphia, PA: J.B. Lippincott Co. 1993. New markers which are
more specific for prostate cancer thus would be beneficial in the initial
detection
of this disease.
A critical step in managing patients with prostate cancer is the
presurgical staging of the cancer to provide prognostic value and criteria for
designing optimal therapy. Improved procedures for accurately staging
prostate cancer prior to surgery are needed. One study demonstrated that
current methods of staging prostate cancer prior to surgery were incorrect
approximately fifty percent (50%) of the time. F. Labrie, et al., Urolo~v 44
(Symposium Issue):29-37 (1994). Prostate cancer management also could be
improved by utilizing new markers found in an inappropriate body
compartment. Such markers could be mRNA or protein markers expressed by
cells originating from the primary prostate tumor but residing in blood, bone
marrow or lymph nodes and could be sensitive indicators for metastasis to
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these distal organs. For example, in patients with metastatic prostate cancer,
PSA protein has been detected by immunohistochemical techniques in bone
marrow, and PSA mRNA has been detected by RT-PCR in cells of blood,
lymph nodes and bone marrow. K. Pantel, et al., Onkologie 18:394-401
( 1995).
New markers which could predict the biologic behavior of early
prostate cancers would also be of significant value. Early prostate cancers
that
threaten or will threaten the life of the patient are more clinically
important than
those that do not or will not be a threat. G.E. Hanks, supra. A need therefore
exists for new markers which can differentiate between the clinically
important
and unimportant prostate cancers. Such markers would allow the clinician to
accurately identify and effectively treat early cancers localized to the
prostate
which could otherwise metastasize and kill the patient. Further, if one could
show that such a marker characteristic of aggressive cancer was absent, the
patient could be spared expensive and non-beneficial treatment.
It also would be beneficial to find a prostate associated marker which is
more sensitive in detecting recurrence of prostate cancer than PSA and which
is not
affected by androgens. To date, PSA has proven to be the most sensitive marker
for detecting recurrent disease. However, in some cases tumor progression
occurs
without PSA elevation due to hormonal therapy utilized for treating the
cancer.
Although the decrease in androgen results in a concomitant decrease in PSA, it
does not necessarily reflect a decrease in tumor metastasis. This complication
is
the result of androgen-stimulated PSA expression. Part of the decline in PSA
observed after androgen ablation is due not to tumor cell death but to
diminished
PSA expression. G.E. Hanks, supra.
Patients diagnosed with early breast cancer have greater than a 90%
five-year relative survival rate as compared to a survival rate of about 20%
for
patients diagnosed with distantly metastasized breast cancers. (American
Cancer Society statistics). Currently, the best initial indicators of early
breast
cancer are physical examination of the breast and mammography. J.R. Harris
et al. In: Cancer: Principles and Practice of Oncology, Fourth Edition, pp.
1264-1332, Philadelphia, PA: J.B. Lippincott Co. (1993). Mammography
may detect a breast tumor before it can be detected by physical examination,
but
it has limitations. For example, mammography's predictive value depends on
the observer's skill and the quality of the mammogram. In addition, 80 to 93%
of suspicious mammograms are false positives, and 10 to 15% of women with
breast cancer have false negative mammograms. C.J. Wright et al., Lancet
3
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346:29-32 ( 1995). New diagnostic methods which are more sensitive and
specific for detecting early breast cancer are clearly needed.
Breast cancer patients are closely monitored following initial therapy
and during adjuvant therapy to determine response to therapy, and to detect
S persistent or recurrent disease, or early distant metastasis. Current
diagnostic
procedures for monitoring breast cancer include mammography, bone scan,
chest radiographs, liver function tests and tests for serum markers. The serum
tumor markers most commonly used for monitoring patients are
carcinoembryonic antigen (CEA) and CA 15-3. Limitations of CEA include
absence of elevated serum levels in about 40% of women with metastatic
disease. In addition, CEA elevation during adjuvant therapy may not be related
to recurrence but to other factors that are not clinically important. CA 15-3
can
also be negative in a significant number of patients with progressive disease
and, therefore, fail to predict metastasis. Both CEA and CA 15-3 can be
elevated in nonmalignant, benign conditions giving rise to false positive
results. Therefore, it would be clinically beneficial to find a breast
associated
marker which is more sensitive and specific in detecting cancer recurrence. J.
R. Harris et al., su~r_a. M. K. Schwartz, In: Cancer: Principles and Practice
of
Oncology. Vol. 1, Fourth Edition, pp. 531 - 542, Philadelphia, PA: J.B.
Lippincott Co. 1993.
Another important step in managing breast cancer is to determine the
stage of the patient's disease because stage determination has potential
prognostic value and provides criteria for designing optimal therapy.
Currently, pathological staging of breast cancer is preferable over clinical
staging because the former gives a more accurate prognosis. J. R. Harris et
al., supra. On the other hand, clinical staging would be preferred were it at
least as accurate as pathological staging because it does not depend on an
invasive procedure to obtain tissue for pathological evaluation. Staging of
breast cancer could be improved by detecting new markers in serum or urine
which could differentiate between different stages of invasion. Such markers
could be mRNA or protein markers expressed by cells originating from the
primary tumor in the breast but residing in blood, bone marrow or lymph
nodes and could serve as sensitive indicators for metastasis to these distal
organs. For example, specific protein antigens and mRNA, associated with
breast epithelial cells, have been detected by immunohistochemical techniques
and RT-PCR, respectively, in bone marrow, lymph nodes and blood of breast
cancer patients suggesting metastasis. K. Pantel et al., Onk o ie 18:394-401
4
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( 1995).
New markers which could predict the biologic behavior of early breast
cancers would also be of significant value. Early breast cancers that threaten
or
will threaten the life of the patient are more clinically important than those
that
do not or will not be a threat. G.E. Hanks, supra. Such markers are needed to
predict which patients with histologically negative lymph nodes will
experience
recurrence of cancer and also to predict which cases of ductal carcinoma in
situ
will develop into invasive breast carcinoma. More accurate prognostic markers
would allow the clinician to accurately identify early cancers localized to
the
breast which will progress and metastasize if not treated aggressively.
Additionally, the absence of a marker for an aggressive cancer in the patient
could spare the patient expensive and non-beneficial treatment. J. R. Harris
et
al., su,Pr_a_. E. R. Frykberg et al., C cer 74:350-361 ( 1994).
Patients diagnosed with early stage ovarian cancer have about a 90%
five-year survival rate as compared to survival rates of less than 50% for
patients initially presenting with more advanced stage cancer (American Cancer
Society statistics). Despite the favorable prognosis of early stage disease,
only
25% of patients diagnosed with ovarian cancer initially present with early
stage
disease. R.C. Young, et al., supra. The failure to detect ovarian cancer while
it is still localized and therefore more treatable is due to the absence of
clinical
symptoms at the early stage of disease and the lack of a sensitive and
specific
marker for ovarian cancer. Detection of the CA-125 tumor marker in serum
has been evaluated as a screening marker for ovarian cancer; however its
usefulness is limited by a lack of overall sensitivity and its absence at an
early
stage. J. R. van Nagell Jr. et al., ancer 76: 2086-2091 ( 1995). New
diagnostic methods which are sensitive and specific for detecting ovarian
cancer at an early stage are needed.
Current diagnostic procedures for monitoring ovarian cancer include
second-look laparotomy, laparoscopy, computerized tomography (CT) scans
and repeated monitoring of CA-125 serum levels. Second-look laparotomy is a
surgical procedure which has poor sensitivity; 30% to 50% of patients
negative by this procedure later have cancer recurrence. S.D. Thompson et al.,
r'n Net (Internet) (October 1996). Further, 48% of patients with normal
CA-125 serum levels later were found to have residual cancer. In addition,
CA-125 serum levels can be elevated in other types of cancers and in benign
diseases. M.K. Schwartz, In: Cancer Principles and Practice of Oncology,
Fourth Edition, supra, pp. 531-542. Therefore, it would be advantageous to
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provide an ovary-associated marker which could be more sensitive and specific
in detecting ovarian cancer.
A critical factor in managing ovarian cancer is determining the stage of the
patient's disease. Staging has potential prognostic value and provides
criteria for
designing optimal therapy. Currently, accurate staging of ovarian cancer
primarily
depends upon surgical exploration and pathological evaluation, which are
crucial
for proper management of the disease. R.C. Young, et al., supra. Clinical
staging
which does not involve an invasive procedure for pathological evaluation would
be
preferable if it also was sensitive. Thus, the staging of ovarian cancer could
be
10 improved by utilizing new markers in test samples which could differentiate
invasive stages of the disease. Such markers could be nucleic acids or
proteins
which are expressed by cells originating from the primary ovarian tumor but
residing in, for example, blood, bone marrow or lymph nodes. These markers
thus would serve as sensitive indicators for metastasis to distal sites.
15 It therefore would be advantageous to provide specific methods and
reagents for detecting, diagnosing, staging, monitoring, prognosticating, in
vivo
imaging, preventing or treating, or determining predisposition to diseases and
conditions of the prostate, breast, or ovary. Such methods would include
assaying
a test sample for products of a gene which are overexpressed in prostate,
breast, or
20 ovarian diseases and conditions such as cancer. Such methods may also
include
assaying a test sample for products of a gene alteration associated with
prostate,
breast, or ovarian disease or condition. Such methods may further include
assaying a test sample for products of a gene whose distribution among the
various
tissues and compartments of the body have been altered by a prostate-, breast-
, or
25 ovary-associated disease or condition such as cancer. Useful reagents
include
polynucleotide(s), or fragments) thereof which may be used in diagnostic
methods
such as reverse transcriptase-polymerase chain reaction (RT-PCR), PCR, or
hybridization assays using mRNA extracted from biopsied tissue, blood or other
test samples; polypeptides or proteins which are the translation products of
such
30 mRNAs; or antibodies directed against these polypeptides or proteins. Drug
treatment or gene therapy for diseases or conditions of the prostate, breast,
and/or
ovary can then be based on these identified gene sequences or their expressed
proteins and efficacy of any particular therapy can be monitored. Furthermore,
it
would be advantageous to have available alternative, non-surgical diagnostic
35 methods capable of detecting early stage prostate, breast, or ovarian
disease such
as cancer.
6
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~mmarXof the Invention
The present invention provides a method of detecting a target PS214
polynucleotide in a test sample which comprises contacting the test sample
with at
least one PS214-specific polynucleotide and detecting the presence of the
target
5 PS214 polynucleotide in the test sample. The PS214-specific polynucleotide
has at
least 50% identity with a polynucleotide selected from the group consisting of
SEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3,
SEQUENCE ID NO 4, SEQUENCE ID NO 5, SEQUENCE ID NO 6,
SEQUENCE ID NO 7, SEQUENCE lZ7 NO 8, SEQUENCE ID NO 9
10 ("SEQUENCE ID NOS 1-9"), and fragments or complements thereof. Also, the
PS214-specific polynucleotide may be attached to a solid phase prior to
performing
the method.
The present invention also provides a method for detecting PS214 mRNA
in a test sample, which comprises performing reverse transcription (RT) with
at
15 least one primer in order to produce cDNA, amplifying the cDNA so obtained
using PS214 oligonucleotides as sense and antisense primers to obtain PS214
amplicon, and detecting the presence of the PS214 amplicon as an indication of
the
presence of PS214 mRNA in the test sample, wherein the PS214 oligonucleotides
have at least 50% identity with a sequence selected from the group consisting
of
20 SEQUENCE m NOS 1-9, and fragments or complements thereof. Amplification
can be performed by the polymerise chain reaction. Also, the test sample can
be
reacted with a solid phase prior to performing the method, prior to
amplification or
prior to detection. This reaction can be a direct or an indirect reaction.
Further, the
detection step can comprise utilizing a detectable label capable of generating
a
25 measurable signal. The detectable label can be attached to a solid phase.
The present invention further provides a method of detecting a target PS214
polynucleotide in a test sample suspected of containing target PS214
polynucleotides, which comprises (a) contacting the test sample with at least
one
PS214 oligonucleotide as a sense primer and at least one PS214 oligonucleotide
as
30 an anti-sense primer, and amplifying same to obtain a first stage reaction
product;
(b) contacting the first stage reaction product with at least one other PS214
oligonucleotide to obtain a second stage reaction product, with the proviso
that the
other PS214 oligonucleotide is located 3' to the PS214 oligonucleotides
utilized in
step (a) and is complementary to the first stage reaction product; and (c)
detecting
35 the second stage reaction product as an indication of the presence of a
target PS214
polynucleotide in the test sample. The PS214 oligonucleotides selected as
reagents
in the method have at least 50% identity with a sequence selected from the
group
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consisting of SEQUENCE m NOS 1-9, and fragments or complements thereof.
Amplification may be performed by the polymerase chain reaction. The test
sample
can be reacted either directly or indirectly with a solid phase prior to
performing the
method, or prior to amplification, or prior to detection. The detection step
also
comprises utilizing a detectable label capable of generating a measurable
signal;
further, the detectable label can be attached to a solid phase.
Test kits useful for detecting target PS214 polynucleotides in a test sample
are also provided which comprise a container containing at least one PS214-
specific polynucleotide selected from the group consisting of SEQUENCE Il7 NOS
1-9, and fragments or complements thereof. These test kits further comprise
containers with tools useful for collecting test samples (such as, for
example,
blood, urine, saliva and stool). Such tools include lancets and absorbent
paper or
cloth for collecting and stabilizing blood; swabs for collecting and
stabilizing
saliva; and cups for collecting and stabilizing urine or stool samples.
Collection
materials, such as papers, cloths, swabs, cups, and the like, may optionally
be
treated to avoid denaturation or irreversible adsorption of the sample. The
collection materials also may be treated with or contain preservatives,
stabilizers or
antimicrobial agents to help maintain the integrity of the specimens.
The present invention also provides a purified polynucleotide or fragment
thereof derived from a PS214 gene. The purified polynucleotide is capable of
selectively hybridizing to the nucleic acid of the PS214 gene, or a complement
thereof. The polynucleotide has at least 50% identity with a polynucleotide
selected from the group consisting of SEQUENCE ID NOS 1-6, 8, 9, a fragment
of the polynucleotide occurnng at positions 1-650 of SEQUENCE 1D NOS 8 or 9,
and complements thereof. Further, the purified polynucleotide can be produced
by
recombinant and/or synthetic techniques. The purified recombinant
polynucleotide
can be contained within a recombinant vector. The invention further comprises
a
host cell transfected with the recombinant vector.
The present invention further provides a recombinant expression system
comprising a nucleic acid sequence that includes an open reading frame derived
from PS214. The nucleic acid sequence has at least 50% identity with a
sequence
selected from the group consisting of SEQUENCE 1D NOS 1-9, and fragments or
complements thereof. The nucleic acid sequence is operably linked to a control
sequence compatible with a desired host. Also provided is a cell transfected
with
this recombinant expression system.
The present invention also provides a polypeptide encoded by PS214. The
polypeptide can be produced by recombinant technology, provided in purified
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form, or produced by synthetic techniques. The polypeptide comprises an amino
acid sequence which has at least 50% identity with an amino acid sequence
selected
from the group consisting of SEQUENCE )D NO 29, SEQUENCE ID NO 30,
SEQUENCE ID NO 31, SEQUENCE m NO 32, and fragments thereof.
S Also provided is a specific binding member, such as an antibody, which
specifically binds to at least one PS214 epitope. The antibody can be a
polyclonal
or monoclonal antibody. The epitope is derived from an amino acid sequence
selected from the group consisting of SEQUENCE ID NO 29, SEQUENCE ID
NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
Assay kits for determining the presence of PS214 antigen or anti-PS214
antibody
in a test sample are also included. In one embodiment, the assay kits comprise
a
container containing at least one PS214 polypeptide having at least 50%
identity
with an amino acid sequence selected from the group consisting of SEQUENCE ID
NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32,
and fragments thereof. Further, the test kit can comprise a container with
tools
useful for collecting test samples (such as blood, urine, saliva, and stool).
Such
tools include lancets and absorbent paper or cloth for collecting and
stabilizing
blood; swabs for collecting and stabilizing saliva; and cups for collecting
and
stabilizing urine or stool samples. Collection materials such as papers,
cloths,
swabs, cups, and the Iike, may optionally be treated to avoid denaturation or
irreversible adsorption of the sample. These collection materials also may be
treated with or contain preservatives, stabilizers or antimicrobial agents to
help
maintain the integrity of the specimens. Also, the polypeptide can be attached
to a
solid phase.
In another embodiment of the invention, specific binding members, such as
antibodies or fragments thereof against the PS214 antigen, can be used to
detect or
image localization of the antigen in a patient for the purpose of detecting or
diagnosing a disease or condition. Such antibodies can be polyclonal or
monoclonal, or made by molecular biology techniques, and can be labeled with a
variety of detectable labels, including but not limited to radioisotopes and
paramagnetic metals. Furthermore, antibodies or fragments thereof, whether
monoclonal, polyclonal, or made by molecular biology techniques, can be used
as
therapeutic agents for the treatment of diseases characterized by expression
of the
PS214 antigen. In the case of therapeutic applications, the antibody may be
used
without derivitization, or it may be derivitized with a cytotoxic agent such
as a
radioisotope, enzyme, toxin, drug, prodrug, or the like.
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Another assay kit for determining the presence of PS214 antigen or anti-
PS214 antibody in a test sample comprises a container containing an antibody
which specifically binds to a PS214 antigen, wherein the PS214 antigen
comprises
at least one PS214-encoded epitope. The PS214 antigen has at least about 60%
sequence similarity to a sequence of a PS214-encoded antigen selected from the
group consisting of, SEQUENCE ID NO 29, SEQUENCE 1D NO 30,
SEQUENCE 1D NO 31, SEQUENCE 1D NO 32, and fragments thereof. These
test kits can further comprise containers with tools useful for collecting
test
samples (such as blood, urine, saliva, and stool). Such tools include lancets
and
absorbent paper or cloth for collecting and stabilizing blood; swabs for
collecting
and stabilizing saliva; cups for collecting and stabilizing urine or stool
samples.
Collection materials, such as papers, cloths, swabs, cups and the like, may
optionally be treated to avoid denaturation or irreversible adsorption of the
sample.
These collection materials also may be treated with, or contain,
preservatives,
stabilizers or antimicrobial agents to help maintain the integrity of the
specimens.
The antibody can be attached to a solid phase.
A method for producing a polypeptide which contains at least one epitope
of PS214 is provided, which method comprises incubating host cells transfected
with an expression vector. This vector comprises a polynucleotide sequence
encoding a polypeptide, wherein the polypeptide comprises an amino acid
sequence having at least 50% identity with a PS214 amino acid sequence
selected
from the group consisting of SEQUENCE 1D NO 29, SEQUENCE 1D NO 30,
SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
A method for detecting PS214 antigen in a test sample suspected of
containing PS214 antigen also is provided. The method comprises contacting the
test sample with an antibody or fragment thereof which specifically binds to
at least
one epitope of PS214 antigen, for a time and under conditions sufficient for
the
formation of antibody/antigen complexes; and detecting the presence of such
complexes containing the antibody as an indication of the presence of PS214
antigen in the test sample. The antibody can be attached to a solid phase and
may
be either a monoclonal or polyclonal antibody. Furthermore, the antibody
specifically binds to at least one PS214 antigen selected from the group
consisting
of SEQUENCE m NO 29, SEQUENCE 1D NO 30, SEQUENCE ID NO 31,
SEQUENCE m NO 32, and fragments thereof.
Another method is provided which detects antibodies which specifically
bind to PS214 antigen in a test sample suspected of containing these
antibodies.
The method comprises contacting the test sample with a poiypeptide which
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
contains at least one PS214 epitope, wherein the PS214 epitope comprises an
amino acid sequence having at least 50% identity with an amino acid sequence
encoded by a PS214 polynucleotide, or a fragment thereof. Contacting is
performed for a time and under conditions sufficient to allow antigen/antibody
S complexes to form. The method further entails detecting complexes which
contain
the polypeptide. The polypeptide can be attached to a solid phase. Further,
the
polypeptide can be a recombinant protein or a synthetic peptide having at
least 50%
identity with an amino acid sequence selected from the group consisting of
SEQUENCE B7 NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31,
SEQUENCE ID NO 32, and fragments thereof.
The present invention provides a cell transfected with a PS214 nucleic acid
sequence that encodes at least one epitope of a PS214 antigen, or fragment
thereof.
The nucleic acid sequence is selected from the group consisting of SEQUENCE ID
NOS 1-9, and fragments or complements thereof.
A method for producing antibodies to PS214 antigen also is provided,
which method comprises administering to an individual an isolated immunogenic
polypeptide or fragment thereof, wherein the isolated immunogenic polypeptide
comprises at least one PS214 epitope. The immunogenic polypeptide is
administered in an amount sufficient to produce an immune response. The
isolated, immunogenic polypeptide comprises an amino acid sequence selected
from the group consisting of SEQUENCE ID NO 29, SEQUENCE ID NO 30,
SEQUENCE m NO 31, SEQUENCE ID NO 32, and fragments thereof.
Another method for producing antibodies which specifically bind to PS214
antigen is disclosed, which method comprises administering to an individual a
plasmid comprising a nucleic acid sequence which encodes at least one PS214
epitope derived from an amino acid sequence selected from the group consisting
of
SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID NO 31,
SEQUENCE ID NO 32, and fragments thereof. The plasmid is administered in an
amount such that the plasmid is taken up by cells in the individual and
expressed at
levels sufficient to produce an immune response.
Also provided is a composition of matter that comprises a
PS214 polynucleotide of at least about 10-12 nucleotides having at least 50%
identity with a polynucleotide selected from the group consisting of SEQUENCE
ID NOS 1-6, 8, 9, a fragment of the polynucleotide occurring at positions 1-
650 of
SEQUENCE ID NOS 8 or 9, and complements thereof. The PS214
polynucleotide encodes an amino acid sequence having at least one PS214
epitope.
Another composition of matter provided by the present invention comprises a
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polypeptide with at least one PS214 epitope of about 8-10 amino acids. The
polypeptide comprises an amino acid sequence having at least 50% identity with
an
amino acid sequence selected from the group consisting of SEQUENCE ID NO
29, SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and
fragments thereof. Also provided is a gene, or a fragment thereof, coding for
a
PS214 polypeptide which has at least 50% identity with SEQUENCE ID NO 29;
and a gene, or a fragment thereof, comprising DNA having at least 50% identity
with SEQUENCE m NO 8 or SEQUENCE ID NO 9.
Brief Description of the Drawings
Figures lA-1C show the nucleotide alignment of clones 3039477
(SEQUENCE ID NO 1), 4277853 (SEQUENCE ID NO 2), 1689288
(SEQUENCE ID NO 3), 3186729 (SEQUENCE ID NO 4), 3407934
(SEQUENCE ID NO 5), 2624832 (SEQUENCE ID NO 6), g2584130
(SEQUENCE ID NO 7), the full-length sequence of clone 3039477 [designated as
clone 3039477IH (SEQUENCE ID NO 8)], and the consensus sequence
(SEQUENCE ID NO 9) derived therefrom.
Figure 2 shows the contig map depicting the formation of the consensus
nucleotide sequence (SEQUENCE ID NO 9) from the nucleotide alignment of
overlapping clones 3039477 (SEQUENCE ID NO 1 ), 4277853 (SEQUENCE m
NO 2), 1689288 (SEQUENCE ID NO 3), 3186729 (SEQUENCE ID NO 4),
3407934 (SEQUENCE ID NO 5), 2624832 (SEQUENCE ID NO 6), g2584130
(SEQUENCE ID NO 7), and 3039477IH (SEQUENCE ID NO 8).
Figure 3 is a scan of a SYBR~ Green stained agarose gel of PS214 RNA
specific RT-PCR amplification products from BPH and prostate cancer tissue
RNAs.
Figure 4 is a scan of a SYBR~ Green stained agarose gel of PS214 RNA
specific RT-PCR amplification products from normal breast and breast cancer
tissue
RNAs.
Figure 5 is a scan of a SYBR~ Green stained agarose gel of PS214 RNA
specific RT-PCR amplification products from RNAs of normal or cancer tissues
from
placenta, breast, prostate, colon, bladder, and lung.
Figure 6 shows the results of the Western blot performed on a panel of tissue
protein extracts probed with antiserum against PS214 synthetic peptide
(SEQUENCE
ID NO 32).
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WO 99/14357 PCT/US98/19496
Detailed Description of the Invention
The present invention provides a gene, or a fragment thereof, which codes
for a PS214 polypeptide having at least about 50% identity with SEQUENCE ID
NO 29. The present invention further encompasses a PS214 gene, or a fragment
thereof, comprising DNA which has at least about 50% identity with SEQUENCE
ID NO 8 or SEQUENCE ID NO 9.
The present invention also provides methods for assaying a test sample for
products of a gene associated with prostate, breast and ovary diseases,
including
cancer, designated as PS214. The method comprises making cDNA from mRNA
in the test sample, and detecting the cDNA as an indication of the presence of
the
PS214 gene. The method may include an amplification step, wherein one or more
portions of the mRNA from PS214 corresponding to the gene or fragments
thereof, is amplified. Methods also are provided for assaying for the
translation
products of PS214. Test samples which may be assayed by the methods provided
herein include tissues, cells, body fluids and secretions. The present
invention
also provides reagents such as oligonucleotide primers and polypeptides which
are
useful in performing these methods.
Portions of the nucleic acid sequences disclosed herein are useful as
primers for the reverse transcription of RNA or for the amplification of cDNA;
or
as probes to determine the presence of certain mRNA sequences in test samples.
Also disclosed are nucleic acid sequences which permit the production of
encoded
polypeptide sequences which are useful as standards or reagents in diagnostic
immunoassays, as targets for pharmaceutical screening assays and/or as
components or as target sites for various therapies. Monoclonal and polyclonal
antibodies directed against at least one epitope contained within these
polypeptide
sequences are useful as delivery agents for therapeutic agents as well as for
diagnostic tests and for screening for diseases or conditions associated with
PS214, especially prostate, breast, and/or ovarian cancer. Isolation of
sequences
of other portions of the gene of interest can be accomplished utilizing probes
or
PCR primers derived from these nucleic acid sequences. This allows additional
probes of the mRNA or cDNA of interest to be established, as well as
corresponding encoded polypeptide sequences. These additional molecules are
useful in detecting, diagnosing, staging, monitoring, prognosticating, ~ vivo
imaging, preventing or treating, or determining the predisposition to diseases
and
conditions of the prostate, breast, and/or ovary, such as prostate, breast,
and
ovarian cancer, characterized by PS214, as disclosed herein.
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The compositions and methods described herein will enable the
identification of certain markers as indicative of a prostate, breast, or
ovary tissue
disease or condition; the information obtained therefrom will aid in the
detecting,
diagnosing, staging, monitoring, prognosticating, i~r vivo imaging, preventing
or
5 treating, or determining diseases or conditions associated with PS214,
especially
prostate, breast, and ovarian cancer. Test methods include, for example, probe
assays which utilize the sequences) provided herein and which also may utilize
nucleic acid amplification methods such as the polymerase chain reaction
(PCR),
the ligase chain reaction (LCR), and hybridization.
10 In addition, the nucleotide sequences provided herein contain open reading
frames from which an immunogenic epitope may be found. This epitope is
believed to be unique to the disease state or condition associated with PS214.
It
also is thought that the polynucleotides or polypeptides and protein encoded
by the
PS214 gene are useful as a marker. This marker is either elevated in disease
such
15 as prostate, breast, or ovarian cancer, altered in disease such as
prostate, breast, or
ovarian cancer, or present as a normal protein but appearing in an
inappropriate
body compartment. The uniqueness of the epitope may be determined by (i) its
immunological reactivity and specificity with antibodies directed against
proteins
and polypeptides encoded by the PS214 gene, and (ii) its nonreactivity with
any
20 other tissue markers. Methods for determining immunological reactivity are
well-
known and include, but are not limited to, for example, radioimmunoassay
(RIA),
enzyme-linked immunoabsorbent assay (ELISA), hemagglutination (HA),
fluorescence polarization immunoassay (FPIA), chemiluminescent immunoassay
(CLIA) and others. Several examples of suitable methods are described herein.
25 Unless otherwise stated, the following terms shall have the following
meanings:
A polynucleotide "derived from" or "specific for" a designated sequence
refers to a polynucleotide sequence which comprises a contiguous sequence of
approximately at least about 6 nucleotides, preferably at least about 8
nucleotides,
30 more preferably at least about 10-12 nucleotides, and even more preferably
at least
about 15-20 nucleotides corresponding, i.e., identical or complementary to, a
region of the designated nucleotide sequence. The sequence may be
complementary or identical to a sequence which is unique to a particular
polynucleotide sequence as determined by techniques known in the art.
35 Comparisons to sequences in databanks, for example, can be used as a method
to
determine the uniqueness of a designated sequence. Regions from which
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sequences may be derived, include but are not limited to, regions encoding
specific
epitopes, as well as non-translated and/or non-transcribed regions.
The derived polynucleotide will not necessarily be derived physically from
the nucleotide sequence of interest under study, but may be generated in any
manner, including, but not limited to, chemical synthesis, replication,
reverse
transcription or transcription, which is based on the information provided by
the
sequence of bases in the regions) from which the polynucleotide is derived. As
such, it may represent either a sense or an antisense orientation of the
original
polynucleotide. In addition, combinations of regions corresponding to that of
the
designated sequence may be modified in ways known in the art to be consistent
with the intended use.
A "fragment" of a specified polynucleotide refers to a polynucleotide
sequence which comprises a contiguous sequence of approximately at least about
6
nucleotides, preferably at least about 8 nucleotides, more preferably at least
about
1 S 10-12 nucleotides, and even more preferably at least about 15-20
nucleotides
corresponding, i.e., identical or complementary to, a region of the specified
nucleotide sequence.
The term "primer" denotes a specific oligonucleotide sequence which is
complementary to a target nucleotide sequence and used to hybridize to the
target
nucleotide sequence. A primer serves as an initiation point for nucleotide
polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse
transcriptase.
The term "probe" denotes a defined nucleic acid segment (or nucleotide
analog segment, e.g., PNA as defined hereinbelow) which can be used to
identify
a specific polynucleotide present in samples bearing the complementary
sequence.
"Encoded by" refers to a nucleic acid sequence which codes for a
polypeptide sequence, wherein the polypeptide sequence or a portion thereof
contains an amino acid sequence of at least 3 to 5 amino acids, more
preferably at
least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino
acids
from a polypeptide encoded by the nucleic acid sequence. Also encompassed are
polypeptide sequences which are immunologically identifiable with a
polypeptide
encoded by the sequence. Thus, a "polypeptide," "protein," or "amino acid"
sequence has at least about 50% identity, preferably about 60% identity, more
preferably about 75-85% identity, and most preferably about 90-95% or more
identity with a PS214 amino acid sequence. Further, the PS214 "polypeptide,"
"protein," or "amino acid" sequence may have at least about 60% similarity,
preferably at least about 75% similarity, more preferably about 85%
similarity, and
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most preferably about 95% or more similarity to a polypeptide or amino acid
sequence of PS214. This amino acid sequence can be selected from the group
consisting of SEQUENCE ID NO 29, SEQUENCE ID NO 30, SEQUENCE ID
NO 31, SEQUENCE ID NO 32, and fragments thereof.
5 Techniques for determining amino acid sequence "similarity" are well-
known in the art. In general, "similarity" means the exact amino acid to amino
acid
comparison of two or more polypeptides at the appropriate place, where amino
acids are identical or possess similar chemical and/or physical properties
such as
charge or hydrophobicity. A so-termed "percent similarity" then can be
determined
10 between the compared polypeptide sequences. Techniques for determining
nucleic
acid and amino acid sequence identity also are well known in the art and
include
determining the nucleotide sequence of the mRNA for that gene (usually via a
cDNA intermediate) and determining the amino acid sequence encoded thereby,
and comparing this to a second amino acid sequence. In general, "identity"
refers
15 to an exact nucleotide to nucleotide or amino acid to amino acid
correspondence of
two polynucleotides or polypeptide sequences, respectively. Two or more
polynucleotide sequences can be compared by determining their "percent
identity."
Two or more amino acid sequences likewise can be compared by determining their
"percent identity." The percent identity of two sequences, whether nucleic
acid or
20 peptide sequences, is the number of exact matches between two aligned
sequences
divided by the length of the shorter sequences and multiplied by 100. An
approximate alignment for nucleic acid sequences is provided by the local
homology algorithm of Smith and Waterman, Advances in Apnlie~ Mathematics
2:482-489 (1981). This algorithm can be extended to use with peptide sequences
25 using the scoring matrix developed by Dayhoff,~tlas of Protein Secfuences
and
Structure, M.O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research
Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids
Res. 14(6):6745-6763 (1986). An implementation of this algorithm for nucleic
acid and peptide sequences is provided by the Genetics Computer Group
30 (Madison, WI) in their BestFit utility application. The default parameters
for this
method are described in the Wisconsin Sequence Analysis Package Program
Manual, Version 8 (1995) (available from Genetics Computer Group, Madison,
WI). Other equally suitable programs for calculating the percent identity or
similarity between sequences are generally known in the art.
35 A "recombinant polypeptide," "recombinant protein," or "a polypeptide
produced by recombinant techniques," which terms may be used interchangeably
herein, describes a polypeptide which by virtue of its origin or manipulation
is not
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associated with all or a portion of the polypeptide with which it is
associated in
nature and/or is linked to a polypeptide other than that to which it is linked
in
nature. A recombinant or encoded polypeptide or protein is not necessarily
translated from a designated nucleic acid sequence. It also may be generated
in any
manner, including chemical synthesis or expression of a recombinant expression
system.
The term "synthetic peptide" as used herein means a polymeric form of
amino acids of any length, which may be chemically synthesized by methods well-
known to the routineer. These synthetic peptides are useful in various
applications.
The term "polynucleotide" as used herein means a polymeric form of
nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
This
term refers only to the primary structure of the molecule. Thus, the term
includes
double- and single-stranded DNA, as well as double- and single-stranded RNA.
It
I S also includes modifications, such as methylation or capping and unmodified
forms
of the polynucleotide. The terms "polynucleotide," "oligomer,"
"oligonucleotide,"
and "oligo" are used interchangeably herein.
"A sequence corresponding to a cDNA" means that the sequence contains a
polynucleotide sequence that is identical or complementary to a sequence in
the
designated DNA. The degree (or "percent") of identity or complementarity to
the
cDNA will be approximately 50% or greater, preferably at least about 70% or
greater, and more preferably at least about 90% or greater. The sequence that
corresponds to the identified cDNA will be at least about 50 nucleotides in
length,
preferably at least about 60 nucleotides in length, and more preferably at
least about
25 70 nucleotides in length. The correspondence between the gene or gene
fragment
of interest and the cDNA can be determined by methods known in the art and
include, for example, a direct comparison of the sequenced material with the
cDNAs described, or hybridization and digestion with single strand nucleases,
followed by size determination of the digested fragments.
30 "Purified polynucleotide" refers to a polynucleotide of interest or
fragment
thereof which is essentially free, e.g., contains less than about 50%,
preferably
less than about 70%, and more preferably less than about 90%, of the protein
with
which the polynucleotide is naturally associated. Techniques for purifying
polynucleotides of interest are well-known in the art and include, for
example,
35 disruption of the cell containing the polynucleotide with a chaotropic
agent and
separation of the polynucleotide(s) and proteins by ion-exchange
chromatography,
affinity chromatography and sedimentation according to density.
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"Purified polypeptide" or "purified protein" means a polypeptide of interest
or fragment thereof which is essentially free of, e.g., contains less than
about
50%, preferably less than about 70%, and more preferably less than about 90%,
cellular components with which the polypeptide of interest is naturally
associated.
Methods for purifying polypeptides of interest are known in the art.
The term "isolated" means that the material is removed from its original
environment (e.g., the natural environment if it is naturally occurring). For
example, a naturally-occurring polynucleotide or polypeptide present in a
living
animal is not isolated, but the same polynucleotide or DNA or polypeptide,
which
10 is separated from some or all of the coexisting materials in the natural
system, is
isolated. Such polynucleotide could be part of a vector and/or such
polynucleotide
or polypeptide could be part of a composition, and still be isolated in that
the vector
or composition is not part of its natural environment.
"Polypeptide" and "protein" are used interchangeably herein and indicate at
15 least one molecular chain of amino acids linked through covalent and/or non-
covalent bonds. The terms do not refer to a specific length of the product.
Thus
peptides, oligopeptides and proteins are included within the definition of
polypeptide. The terms include post-translational modifications of the
polypeptide,
for example, glycosylations, acetylations, phosphorylations and the like. In
20 addition, protein fragments, analogs, mutated or variant proteins, fusion
proteins
and the like are included within the meaning of polypeptide.
A "fragment" of a specified polypeptide refers to an amino acid sequence
which comprises at least about 3-5 amino acids, more preferably at least about
8-10
amino acids, and even more preferably at least about 15-20 amino acids derived
25 from the specified polypeptide.
"Recombinant host cells " "host cells " "cells " "cell lines " "cell cultures
"
> > >
and other such terms denoting microorganisms or higher eukaryotic cell lines
cultured as unicellular entities refer to cells which can be, or have been,
used as
recipients for recombinant vector or other transferred DNA, and include the
30 original progeny of the original cell which has been transfected.
As used herein "replicon" means any genetic element, such as a plasmid, a
chromosome or a virus, that behaves as an autonomous unit of polynucleotide
replication within a cell.
A "vector" is a replicon in which another polynucleotide segment is
35 attached, such as to bring about the replication and/or expression of the
attached
segment.
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The term "control sequence" refers to a polynucleotide sequence which is
necessary to effect the expression of a coding sequence to which it is
ligated. The
nature of such control sequences differs depending upon the host organism. In
prokaryotes, such control sequences generally include a promoter, a ribosomal
5 binding site and terminators; in eukaryotes, such control sequences
generally
include promoters, terminators and, in some instances, enhancers. The term
"control sequence" thus is intended to include at a minimum all components
whose
presence is necessary for expression, and also may include additional
components
whose presence is advantageous, for example, leader sequences.
10 "Operably linked" refers to a situation wherein the components described
are in a relationship permitting them to function in their intended manner.
Thus,
for example, a control sequence "operably linked" to a coding sequence is
ligated
in such a manner that expression of the coding sequence is achieved under
conditions compatible with the control sequence.
15 The term "open reading frame" or "ORF" refers to a region of a
polynucleotide sequence which encodes a polypeptide. This region may represent
a portion of a coding sequence or a total coding sequence.
A "coding sequence" is a polynucleotide sequence which is transcribed into
mRNA and translated into a polypeptide when placed under the control of
20 appropriate regulatory sequences. The boundaries of the coding sequence are
determined by a translation start codon at the 5' -terminus and a translation
stop
codon at the 3' -terminus. A coding sequence can include, but is not limited
to,
mRNA, cDNA and recombinant polynucleotide sequences.
The term "immunologically identifiable with/as" refers to the presence of
25 epitope(s) and polypeptide(s) which also are present in and are unique to
the
designated polypeptide(s). Immunological identity may be determined by
antibody
binding and/or competition in binding. These techniques are known to the
routineer and also are described herein. The uniqueness of an epitope also can
be
determined by computer searches of known data banks, such as GenBank, for the
30 polynucleotide sequence which encodes the epitope and by amino acid
sequence
comparisons with other known proteins.
As used herein, "epitope" means an antigenic determinant of a polypeptide
or protein. Conceivably, an epitope can comprise three amino acids in a
spatial
conformation which is unique to the epitope. Generally, an epitope consists of
at
35 least five such amino acids and more usually, it consists of at least eight
to ten
amino acids. Methods of examining spatial conformation are known in the art
and
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include, for example, x-ray crystallography and two-dimensional nuclear
magnetic
resonance.
A "conformational epitope" is an epitope that is comprised of a specific
juxtaposition of amino acids in an immunologically recognizable structure,
such
amino acids being present on the same polypeptide in a contiguous or non-
contiguous order or present on different polypeptides.
A polypeptide is "immunologically reactive" with an antibody when it binds
to an antibody due to antibody recognition of a specific epitope contained
within
the polypeptide. Immunological reactivity may be determined by antibody
binding,
10 more particularly, by the kinetics of antibody binding, and/or by
competition in
binding using as competitors) a known polypeptide(s) containing an epitope
against which the antibody is directed. The methods for determining whether a
polypeptide is immunologically reactive with an antibody are known in the art.
As used herein, the term "immunogenic polypeptide containing an epitope
15 of interest" means naturally occurring polypeptides of interest or
fragments thereof,
as well as polypeptides prepared by other means, for example, by chemical
synthesis or the expression of the polypeptide in a recombinant organism.
The term "transfection" refers to the introduction of an exogenous
polynucleotide into a prokaryotic or eucaryotic host cell, irrespective of the
method
20 used for the introduction. The term "transfection" refers to both stable
and
transient introduction of the polynucleotide, and encompasses direct uptake of
polynucleotides, transformation, transduction, and f-mating. Once introduced
into
the host cell, the exogenous polynucleotide may be maintained as a non-
integrated
replicon, for example, a plasmid, or alternatively, may be integrated into the
host
25 genome.
"Treatment" refers to prophylaxis and/or therapy.
The term "individual" as used herein refers to vertebrates, particularly
members of the mammalian species and includes, but is not limited to, domestic
animals, sports animals, primates and humans; more particularly, the term
refers to
30 humans.
The term "sense strand" or "plus strand" (or "+") as used herein denotes a
nucleic acid that contains the sequence that encodes the polypeptide. The term
"antisense strand" or "minus strand" (or ' =") denotes a nucleic acid that
contains a
sequence that is complementary to that of the "plus" strand.
35 The term "test sample" refers to a component of an individual's body which
is the source of the analyte (such as antibodies of interest or antigens of
interest).
These components are well known in the art. A test sample is typically
anything
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suspected of containing a target sequence. Test samples can be prepared using
methodologies well known in the art such as by obtaining a specimen from an
individual and, if necessary, disrupting any cells contained thereby to
release target
nucleic acids. These test samples include biological samples which can be
tested
by the methods of the present invention described herein and include human and
animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid,
sputum, bronchial washing, bronchial aspirates, urine, lymph fluids, and
various
external secretions of the respiratory, intestinal and genitourinary tracts,
tears,
saliva, milk, white blood cells, myelomas and the like; biological fluids such
as cell
culture supernatants; tissue specimens which may be fixed; and cell specimens
which may be fixed.
"Purified product" refers to a preparation of the product which has been
isolated from the cellular constituents with which the product is normally
associated and from other types of cells which may be present in the sample of
interest.
"PNA" denotes a "peptide nucleic acid analog" which may be utilized in a
procedure such as an assay described herein to determine the presence of a
target.
"MA" denotes a "morpholino analog" which may be utilized in a procedure such
as
an assay described herein to determine the presence of a target. See, for
example,
U.S. Patent No. 5,378,841. PNAs are neutrally charged moieties which can be
directed against RNA targets or DNA. PNA probes used in assays in place of,
for
example, the DNA probes of the present invention, offer advantages not
achievable
when DNA probes are used. These advantages include manufacturability, large
scale labeling, reproducibility, stability, insensitivity to changes in ionic
strength
and resistance to enzymatic degradation which is present in methods utilizing
DNA
or RNA. These PNAs can be labeled with ("attached to") such signal generating
compounds as fluorescein, radionucleotides, chemiluminescent compounds and the
like. PNAs or other nucleic acid analogs such as MAs thus can be used in assay
methods in place of DNA or RNA. Although assays are described herein utilizing
DNA probes, it is within the scope of the routineer that PNAs or MAs can be
substituted for RNA or DNA with appropriate changes if and as needed in assay
reagents.
"Analyte," as used herein, is the substance to be detected which may be
present in the test sample. The analyte can be any substance for which there
exists
a naturally occurring specific binding member (such as an antibody), or for
which
a specific binding member can be prepared. Thus, an analyte is a substance
that
can bind to one or more specific binding members in an assay. "Analyte" also
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includes any antigenic substances, haptens, antibodies and combinations
thereof.
As a member of a specific binding pair, the analyte can be detected by means
of
naturally occurring specific binding partners (pairs) such as the use of
intrinsic
factor protein as a member of a specific binding pair for the determination of
5 Vitamin B 12, the use of folate-binding protein to determine folic acid, or
the use of
a lectin as a member of a specific binding pair for the determination of a
carbohydrate. The analyte can include a protein, a polypeptide, an amino acid,
a
nucleotide target and the like. The analyte can be soluble in a body fluid
such as
blood, blood plasma or serum, urine or the like. The analyte can be in a
tissue,
either on a cell surface or within a cell. The analyte can be on or in a cell
dispersed
in a body fluid such as blood, urine, breast aspirate, or obtained as a biopsy
sample.
The terms "diseases of the prostate," "prostate disease," and "condition of
the prostate," are used interchangeably herein to refer to any disease or
condition of
15 the prostate including, but not limited to, benign prostatic hyperplasia
(BPH),
prostatitis, prostatic intraepithelial neoplasia (PIN) and cancer.
The terms "diseases of the breast," "breast disease," and "condition of the
breast" are used interchangeably herein to refer to any disease or condition
of the
breast including, but not limited to, atypical hyperplasia, fibroadenoma,
cystic
breast disease, and cancer.
The terms "diseases of the ovary," "ovarian disease," and "condition of the
ovary," are used interchangeably herein to refer to any disease or condition
of the
ovary including, but not limited to, ovarian cysts, ovarian cyst adenomas,
ovarian
endometriomas, and cancer.
25 "Prostate cancer," as used herein, refers to any malignant disease of the
prostate including but not limited to, adenocarcinoma, small cell
undifferentiated
carcinoma and mutinous (colloid) cancer.
"Breast cancer," as used herein, refers to any malignant disease of the
breast including, but not limited to, ductal carcinoma in situ, lobular
carcinoma in
situ, infiltrating ductal carcinoma, medullary carcinoma, tubular carcinoma,
mutinous carcinoma, infiltrating lobular carcinoma, infiltrating
comedocarcinoma
and inflammatory carcinoma.
"Ovarian cancer," as used herein, refers to any malignant disease of the
ovary including but not limited to, adenocarcinoma, serous neoplasms,
rnucinous
neoplasms, endometroid neoplasms, clear cell neoplasms, ovarian borderline
tumors, ovarian germ cell tumors, and ovarian sex-cord tumors.
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An "Expressed Sequence Tag" or "EST" refers to the partial sequence of a
cDNA insert which has been made by reverse transcription of mRNA extracted
from a tissue followed by insertion into a vector.
A "transcript image" refers to a table or list giving the quantitative
distribution of ESTs in a library and represents the genes active in the
tissue from
which the library was made.
. The present invention provides assays which utilize specific binding
members. A "specific binding member," as used herein, is a member of a
specific
binding pair. That is, two different molecules where one of the molecules,
through
10 chemical or physical means, specifically binds to the second molecule.
Therefore,
in addition to antigen and antibody specific binding pairs of common
immunoassays, other specific binding pairs can include biotin and avidin,
carbohydrates and lectins, complementary nucleotide sequences, effector and
receptor molecules, cofactors and enzymes, enzyme inhibitors, and enzymes and
15 the like. Furthermore, specific binding pairs can include members that are
analogs
of the original specific binding members, for example, an analyte-analog.
Immunoreactive specific binding members include antigens, antigen fragments,
antibodies and antibody fragments, both monoclonal and polyclonal and
complexes
thereof, including those formed by recombinant DNA molecules.
20 Specific binding members include "specific binding molecules." A
"specific binding molecule" intends any specific binding member, particularly
an
immunoreactive specific binding member. As such, the term "specific binding
molecule" encompasses antibody molecules (obtained from both polyclonal and
monoclonal preparations), as well as, the following: hybrid (chimeric)
antibody
25 molecules [see, for example, Winter, et al., Nature 349:293-299 (1991), and
U.S.
Patent No. 4,816,567); F(ab')2 and Flab) fragments; Fv molecules (non-covalent
heterodimers, see, for example, Inbar, et al., Proc. Natl. Acad. Sci. USA
69:2659-2662 (1972), and Ehrlich, et al., Biochem. 19:4091-4096 (1980)];
single
chain Fv molecules {sFv) [see, for example, Huston, et al., Proc. Natl. Acad.
Sci.
30 USA 85:5879-5883 (1988)]; humanized antibody molecules (see, for example,
Riechmann, et al., Na a 332:323-327 (1988), Verhoeyan, et al., Science
239:1534-1536 ( 1988), and UK Patent Publication No. GB 2,276,169, published
21 September 1994); and, any functional fragments obtained from such
molecules,
wherein such fragments retain immunological binding properties of the parent
35 antibody molecule.
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The term "hapten," as used herein, refers to a partial antigen or non-protein
binding member which is capable of binding to an antibody, but which is not
capable of eliciting antibody formation unless coupled to a Garner protein.
A "capture reagent," as used herein, refers to an unlabeled specific binding
5 member which is specific either for the analyte as in a sandwich assay, for
the
indicator reagent or analyte as in a competitive assay, or for an ancillary
specific
binding member, which itself is specific for the analyte, as in an indirect
assay.
The capture reagent can be directly or indirectly bound to a solid phase
material
before the performance of the assay or during the performance of the assay,
10 thereby enabling the separation of immobilized complexes from the test
sample.
The "indicator reagent" comprises a "signal-generating compound"
("label") which is capable of generating and generates a measurable signal
detectable by external means, conjugated ("attached") to a specific binding
member. In addition to being an antibody member of a specific binding pair,
the
15 indicator reagent also can be a member of any specific binding pair,
including
either hapten-anti-hapten systems such as biotin or anti-biotin, avidin or
biotin, a
carbohydrate or a lectin, a complementary nucleotide sequence, an effector or
a
receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an
enzyme and the like. An immunoreactive specific binding member can be an
20 antibody, an antigen, or an antibody/antigen complex that is capable of
binding
either to the polypeptide of interest as in a sandwich assay, to the capture
reagent as
in a competitive assay, or to the ancillary specific binding member as in an
indirect
assay. When describing probes and probe assays, the term "reporter molecule"
may be used. A reporter molecule comprises a signal generating compound as
25 described hereinabove conjugated to a specific binding member of a specific
binding pair, such as carbazole or adamantine.
The various "signal-generating compounds" (labels) contemplated include
chromagens, catalysts such as enzymes, luminescent compounds such as
fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes,
30 acridiniums, phenanthridiniums and luminol, radioactive elements and direct
visual
labels. Examples of enzymes include alkaline phosphatase, horseradish
peroxidase, beta-galactosidase and the like. The selection of a particular
label is
not critical, but it must be capable of producing a signal either by itself or
in
conjunction with one or more additional substances.
35 "Solid phases" ("solid supports") are known to those in the art and include
the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic
or
non-magnetic beads, nitrocellulose strips, membranes, microparticles such as
latex
24
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
particles, sheep (or other animal) red blood cells and Duracytes~ (red blood
cells
"fixed" by pyruvic aldehyde and formaldehyde, available from Abbott
Laboratories, Abbott Park, IL) and others. The "solid phase" is not critical
and can
be selected by one skilled in the art. Thus, latex particles, microparticles,
magnetic
5 or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells,
glass
or silicon chips, sheep (or other suitable animal's) red blood cells and
Duracytes~
are all suitable examples. Suitable methods for immobilizing peptides on solid
phases include ionic, hydrophobic, covalent interactions and the like. A
"solid
phase," as used herein, refers to any material which is insoluble, or can be
made
10 insoluble by a subsequent reaction. The solid phase can be chosen for its
intrinsic
ability to attract and immobilize the capture reagent. Alternatively, the
solid phase
can retain an additional receptor which has the ability to attract and
immobilize the
capture reagent. The additional receptor can include a charged substance that
is
oppositely charged with respect to the capture reagent itself or to a charged
15 substance conjugated to the capture reagent. As yet another alternative,
the
receptor molecule can be any specific binding member which is immobilized upon
(attached to) the solid phase and which has the ability to immobilize the
capture
reagent through a specific binding reaction. The receptor molecule enables the
indirect binding of the capture reagent to a solid phase material before the
20 performance of the assay or during the performance of the assay. The solid
phase
thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal,
glass or
silicon surface of a test tube, microtiter well, sheet, bead, microparticle,
chip,
sheep (or other suitable animal's) red blood cells, Duracytes~ and other
configurations known to those of ordinary skill in the art.
25 It is contemplated and within the scope of the present invention that the
solid phase also can comprise any suitable porous material with sufficient
porosity
to allow access by detection antibodies and a suitable surface affinity to
bind
antigens. Microporous structures generally are preferred, but materials with a
gel
structure in the hydrated state may be used as well. Such useful solid
supports
30 include, but are not limited to, nitrocellulose and nylon. It is
contemplated that
such porous solid supports described herein preferably are in the form of
sheets of
thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm. The pore size
may vary within wide limits and preferably is from about 0.025 to 15 microns,
especially from about 0.15 to 15 microns. The surface of such supports may be
35 activated by chemical processes which cause covalent linkage of the antigen
or
antibody to the support. The irreversible binding of the antigen or antibody
is
obtained, however, in general, by adsorption on the porous material by poorly
CA 02304368 2000-03-15
WO 99/14357 PCTNS98/19496
understood hydrophobic forces. Other suitable solid supports are known in the
art.
Rae
The present invention provides reagents such as polynucleotide sequences
derived from a prostate, breast, or ovary tissue of interest and designated as
PS214, polypeptides encoded thereby and antibodies specific for these
polypeptides. The present invention also provides reagents such as
oligonucleotide
fragments derived from the disclosed polynucleotides and nucleic acid
sequences
complementary to these polynucleotides. The polynucleotides, polypeptides, or
10 antibodies of the present invention may be used to provide information
leading to
the detecting, diagnosing, staging, monitoring, prognosticating, in vivo
imaging,
preventing or treating of, or determining the predisposition to, diseases and
conditions of the prostate, breast, or ovary, such as prostate, breast, or
ovarian
cancer. The sequences disclosed herein represent unique polynucleotides which
15 can be used in assays or for producing a specific profile of gene
transcription
activity. Such assays are disclosed in European Patent Number 0373203B 1 and
International Publication No. WO 95/11995.
Selected PS214-derived polynucleotides can be used in the methods
described herein for the detection of normal or altered gene expression. Such
20 methods may employ PS214 polynucleotides or oligonucleotides, fragments or
derivatives thereof, or nucleic acid sequences complementary thereto.
The polynucleotides disclosed herein, their complementary sequences, or
fragments of either, can be used in assays to detect, amplify or quantify
genes,
nucleic acids, cDNAs or mRNAs relating to prostate, breast, or ovary disease
and
25 conditions associated therewith. They also can be used to identify an
entire or
partial coding region of a PS214 polypeptide. They further can be provided in
individual containers in the form of a kit for assays, or provided as
individual
compositions. If provided in a kit for assays, other suitable reagents such as
buffers, conjugates and the like may be included.
30 The polynucleotide may be in the form of RNA or DNA. Polynucleotides
in the form of DNA, cDNA, genomic DNA, nucleic acid analogs and synthetic
DNA are within the scope of the present invention. The DNA may be double-
stranded or single-stranded, and if single stranded, may be the coding (sense)
strand or non-coding (anti-sense) strand. The coding sequence which encodes
the
35 polypeptide may be identical to the coding sequence provided herein or may
be a
different coding sequence which coding sequence, as a result of the redundancy
or
26
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
degeneracy of the genetic code, encodes the same polypeptide as the DNA
provided herein.
This polynucleotide may include only the coding sequence for the
polypeptide, or the coding sequence for the polypeptide and an additional
coding
sequence such as a leader or secretory sequence or a proprotein sequence, or
the
coding sequence for the polypeptide (and optionally an additional coding
sequence)
and non-coding sequence, such as a non-coding sequence 5' and/or 3' of the
coding sequence for the polypeptide.
In addition, the invention includes variant polynucleotides containing
10 modifications such as polynucleotide deletions, substitutions or additions;
and any
polypeptide modification resulting from the variant polynucleotide sequence. A
polynucleotide of the present invention also may have a coding sequence which
is a
naturally occurring allelic variant of the coding sequence provided herein.
In addition, the coding sequence for the polypeptide may be fused in the
15 same reading frame to a polynucleotide sequence which aids in expression
and
secretion of a polypeptide from a host cell, for example, a leader sequence
which
functions as a secretory sequence for controlling transport of a polypeptide
from
the cell. The polypeptide having a leader sequence is a preprotein and may
have
the leader sequence cleaved by the host cell to form the polypeptide. The
20 polynucleotides may also encode for a proprotein which is the protein plus
additional 5' amino acid residues. A protein having a prosequence is a
proprotein
and may, in some cases, be an inactive form of the protein. Once the
prosequence
is cleaved, an active protein remains. Thus, the polynucleotide of the present
invention may encode for a protein, or for a protein having a prosequence, or
for a
25 protein having both a presequence (leader sequence) and a prosequence.
The polynucleotides of the present invention may also have the coding
sequence fused in frame to a marker sequence which allows for purification of
the
polypeptide of the present invention. The marker sequence may be a hexa-
histidine
tag supplied by a pQE-9 vector to provide for purification of the polypepdde
fused
30 to the marker in the case of a bacterial host, or, for example, the marker
sequence
may be a hemagglutinin (HA) tag when a mammalian host, e.g. a COS-7 cell line,
is used. The HA tag corresponds to an epitope derived from the influenza
hemagglutinin protein. See, for example, I. Wilson et al., ~l 37:767 ( 1984).
It is contemplated that polynucleotides will be considered to hybridize to the
35 sequences provided herein if there is at least 50%, preferably at least
70%, and
more preferably at least 90% identity between the polynucleotide and the
sequence.
27
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
The degree of sequence identity between two nucleic acid molecules greatly
affects the efficiency and strength of hybridization events between such
molecules.
A partially identical nucleic acid sequence is one that will at least
partially inhibit a
completely identical sequence from hybridizing to a target molecule.
Inhibition of
hybridization of the completely identical sequence can be assessed using
hybridization assays that are well known in the art (e.g., Southern blot,
Northern
blot, solution hybridization, in situ hybridization, or the like, see
Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring
Harbor, N.Y.). Such assays can be conducted using varying degrees of
10 selectivity, for example, using conditions varying from low to high
stringency. If
conditions of low stringency are employed, the absence of non-specific binding
can be assessed using a secondary probe that lacks even a partial degree of
sequence identity (for example, a probe having less than about 30% sequence
identity with the target molecule), such that, in the absence of non-specific
binding
events, the secondary probe will not hybridize to the target.
When utilizing a hybridization-based detection system, a nucleic acid probe
is chosen that is complementary to a target nucleic acid sequence, and then by
selection of appropriate conditions the probe and the target sequence
"selectively
hybridize," or bind, to each other to form a hybrid molecule. In one
embodiment
20 of the present invention, a nucleic acid molecule is capable of hybridizing
selectively to a target sequence under moderately stringent hybridization
conditions. In the context of the present invention, moderately stringent
hybridization conditions allow detection of a target nucleic acid sequence of
at least
14 nucleotides in length having at least approximately 70% sequence identity
with
25 the sequence of the selected nucleic acid probe. In another embodiment,
such
selective hybridization is performed under stringent hybridization conditions.
Stringent hybridization conditions allow detection of target nucleic acid
sequences
of at least 14 nucleotides in length having a sequence identity of greater
than 90%
with the sequence of the selected nucleic acid probe. Hybridization conditions
30 useful for probe/target hybridization where the probe and target have a
specific
degree of sequence identity, can be determined as is known in the art (see,
for
example, Nucleic Acid Hybridization: A Practical Approach, editors B.D. Hames
and S.J. Higgins, ( 1985) Oxford; Washington, DC; IRL Press). Hybrid
molecules can be formed, for example, on a solid support, in solution, and in
35 tissue sections. The formation of hybrids can be monitored by inclusion of
a
reporter molecule, typically, in the probe. Such reporter molecules, or
detectable
28
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
elements include, but are not limited to, radioactive elements, fluorescent
markers,
and molecules to which an enzyme-conjugated ligand can bind.
With respect to stringency conditions for hybridization, it is well known in
the art that numerous equivalent conditions can be employed to establish a
5 particular stringency by varying, for example, the following factors: the
length and
nature of probe and target sequences, base composition of the various
sequences,
concentrations of salts and other hybridization solution components, the
presence
or absence of blocking agents in the hybridization solutions (e.g., formamide,
dextran sulfate, and polyethylene glycol), hybridization reaction temperature
and
10 time parameters, as well as, varying wash conditions. The selection of a
particular
set of hybridization conditions is well within the skill of the routineer in
the art
(see, for example, Sambrook, et al., Molecular Cloning A Laboratory Manual,
Second Edition, (1989) Cold Spring Harbor, N.Y.).
The present invention also provides an antibody produced by using a
15 purified PS214 polypeptide of which at least a portion of the polypeptide
is
encoded by a PS214 polynucleotide selected from the polynucleotides provided
herein. These antibodies may be used in the methods provided herein for the
detection of PS214 antigen in test samples. The presence of PS214 antigen in
the
test samples is indicative of the presence of a prostate, breast, or ovary
disease or
20 condition. The antibody also may be used for therapeutic purposes, for
example,
in neutralizing the activity of PS214 polypeptide in conditions associated
with
altered or abnormal expression.
The present invention further relates to a PS214 polypeptide which has the
deduced amino acid sequence as provided herein, as well as fragments, analogs
25 and derivatives of such polypeptide. The polypeptide of the present
invention may
be a recombinant polypeptide, a natural purified polypeptide or a synthetic
polypeptide. The fragment, derivative or analog of the PS214 polypeptide may
be
one in which one or more of the amino acid residues is substituted with a
conserved or non-conserved amino acid residue (preferably a conserved amino
acid
30 residue) and such substituted amino acid residue may or may not be one
encoded
by the genetic code; or it may be one in which one or more of the amino acid
residues includes a substituent group; or it may be one in which the
polypeptide is
fused with another compound, such as a compound to increase the half life of
the
polypeptide (for example, polyethylene glycol); or it may be one in which the
35 additional amino acids are fused to the polypeptide, such as a leader or
secretory
sequence or a sequence which is employed for purification of the polypeptide
or a
proprotein sequence. Such fragments, derivatives and analogs are within the
scope
29
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
of the present invention. The polypeptides and polynucleotides of the present
invention are provided preferably in an isolated form and preferably purified.
Thus, a polypeptide of the present invention may have an amino acid
sequence that is identical to that of the naturally occurring polypeptide or
that is
S different by minor variations due to one or more amino acid substitutions.
The
variation may be a "conservative change" typically in the range of about 1 to
5
amino acids, wherein the substituted amino acid has similar structural or
chemical
properties, e.g., replacement of leucine with isoleucine or threonine with
serine.
In contrast, variations may include nonconservative changes, e.g., replacement
of
a glycine with a tryptophan. Similar minor variations may also include amino
acid
deletions or insertions, or both. Guidance in determining which and how many
amino acid residues may be substituted, inserted or deleted without changing
biological or immunological activity may be found using computer programs well
known in the art, for example, DNASTAR software (DNASTAR Inc., Madison
WI).
Probes constructed according to the polynucleotide sequences of the
present invention can be used in various assay methods to provide various
types of
analysis. For example, such probes can be used in fluorescent in ~
~bridization
(FISH) technology to perform chromosomal analysis, and used to identify cancer-
20 specific structural alterations in the chromosomes, such as deletions or
translocations that are visible from chromosome spreads or detectable using
PCR-
generated and/or allele specific oligonucleotides probes, allele specific
amplification
or by direct sequencing. Probes also can be labeled with radioisotopes,
directly- or
indirectly- detectable haptens, or fluorescent molecules; and utilized for in
situ
hybridization studies to evaluate the mRNA expression of the gene comprising
the
polynucleotide in tissue specimens or cells.
This invention also provides teachings as to the production of the
polynucleotides and polypeptides provided herein.
Probe Assavs
30 The sequences provided herein may be used to produce probes which can
be used in assays for the detection of nucleic acids in test samples. The
probes
may be designed from conserved nucleotide regions of the polynucleotides of
interest or from non-conserved nucleotide regions of the polynucleotide of
interest.
The design of such probes for optimization in assays is within the skill of
the
35 routineer. Generally, nucleic acid probes are developed from non-conserved
or
unique regions when maximum specificity is desired, and nucleic acid probes
are
developed from conserved regions when assaying for nucleotide regions that are
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
closely related to, for example, different members of a multi-gene family or
in
related species like mouse and man.
The polymerase chain reaction (PCR) is a technique for amplifying a
desired nucleic acid sequence (target) contained in a nucleic acid or mixture
thereof.
In PCR, a pair of primers are employed in excess to hybridize to the
complementary strands of the target nucleic acid. The primers are each
extended
by a polymerase using the target nucleic acid as a template. The extension
products
become target sequences themselves, following dissociation from the original
target strand. New primers then are hybridized and extended by a polymerase,
and
10 the cycle is repeated to geometrically increase the number of target
sequence
molecules. PCR is disclosed in U.S. Patents 4,683,195 and 4,683,202.
The Ligase Chain Reaction (LCR) is an alternate method for nucleic acid
amplification. In LCR, probe pairs are used which include two primary (first
and
second) and two secondary (third and fourth) probes, all of which are employed
in
15 molar excess to target. The first probe hybridizes to a first segment of
the target
strand, and the second probe hybridizes to a second segment of the target
strand,
the first and second segments being contiguous so that the primary probes abut
one
another in 5'phosphate-3'hydroxyl relationship, and so that a ligase can
covalently
fuse or ligate the two probes into a fused product. In addition, a third
(secondary)
20 probe can hybridize to a portion of the first probe and a fourth
(secondary) probe
can hybridize to a portion of the second probe in a similar abutting fashion.
Of
course, if the target is initially double stranded, the secondary probes also
will
hybridize to the target complement in the first instance. Once the ligated
strand of
primary probes is separated from the target strand, it will hybridize with the
third
25 and fourth probes which can be ligated to form a complementary, secondary
ligated
product. It is important to realize that the ligated products are functionally
equivalent to either the target or its complement. By repeated cycles of
hybridization and ligation, amplification of the target sequence is achieved.
This
technique is described more completely in EP-A- 320 308 to K. Backman
30 published June 16, 1989 and EP-A-439 182 to K. Backman et al., published
July
31, 1991.
For amplification of mRNAs, it is within the scope of the present invention
to reverse transcribe mRNA into cDNA followed by polymerase chain reaction
(RT-PCR); or, to use a single enzyme for both steps as described in U.S.
Patent
35 No. 5,322,770; or reverse transcribe mRNA into cDNA followed by asymmetric
gap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall et al.,
PAR
Methods and A~nlications 4:80-84 ( 1994).
31
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WO 99/14357 PCT/US98/19496
Other known amplification methods which can be utilized herein include
but are not limited to the so-called "NASBA" or "3SR" technique described by
J.
C. Guatelli et al., Proc. Natl. Acad Sci USA 87:1874-1878 (1990) and also
described by J. Compton, Nature 350 {No. 6313):91-92 (1991); Q-beta
5 amplification as described in published European Patent Application (EPA)
No.
4544610; strand displacement amplification (as described in G. T. Walker et
al.,
Clin. Chem. 42:9-13 (1996]) and European Patent Application No. 684315; and
target mediated amplification, as described in International Publication No.
WO
93/22461.
10 Detection of PS214 may be accomplished using any suitable detection
method, including those detection methods which are currently well known in
the
art, as well as detection strategies which may evolve later. See, for example,
Caskey et al., U.S. Patent No. 5,582,989, Gelfand et al., U.S. Patent No.
5,210,015. Examples of such detection methods include target amplification
15 methods as well as signal amplification technologies. An example of
presently
known detection methods would include the nucleic acid amplification
technologies
referred to as PCR, LCR, NASBA, SDA, RCR and TMA. See, for example,
Caskey et al., U.S. Patent No. 5,582,989, Gelfand et al., U.S. Patent No.
5,210,015. Detection may also be accomplished using signal amplification such
as
20 that disclosed in Snitman et al., U.S. Patent No. 5,273,882. While the
amplification of target or signal is preferred at present, it is contemplated
and
within the scope of the present invention that ultrasensitive detection
methods
which do not require amplification can be utilized herein.
Detection, both amplified and non-amplified, may be performed using a
25 variety of heterogeneous and homogeneous detection formats. Examples of
heterogeneous detection formats are disclosed in Snitman et al., U.S. Patent
No.
5,273,882, Albarella et al in EP-84114441.9, Urdea et al., U.S. Patent No.
5,124,246, Ullman et al. U.S. Patent No. 5,185,243 and Kourilsky et al., U.S.
Patent No. 4,581,333. Examples of homogeneous detection formats are disclosed
30 in, Caskey et al., U.S. Patent No. 5,582,989, Gelfand et al., U.S. Patent
No.
5,210,015. Also contemplated and within the scope of the present invention is
the
use of multiple probes in the hybridization assay, which use improves
sensitivity
and amplification of the PS214 signal. See, for example, Caskey et al., U.S.
Patent No. 5,582,989, Gelfand et al., U.S. Patent No. 5,210,015.
35 In one embodiment, the present invention generally comprises the steps of
contacting a test sample suspected of containing a target polynucleotide
sequence
with amplification reaction reagents comprising an amplification primer, and a
32
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
detection probe that can hybridize with an internal region of the amplicon
sequences. Probes and primers employed according to the method provided herein
are labeled with capture and detection labels, wherein probes are labeled with
one
type of label and primers are labeled with another type of label.
Additionally, the
5 primers and probes are selected such that the probe sequence has a lower
melt
temperature than the primer sequences. The amplification reagents, detection
reagents and test sample are placed under amplification conditions whereby, in
the
presence of target sequence, copies of the target sequence (an amplicon) are
produced. In the usual case, the amplicon is double stranded because primers
are
10 provided to amplify a target sequence and its complementary strand. The
double
stranded amplicon then is thermally denatured to produce single stranded
amplicon
members. Upon formation of the single stranded amplicon members, the mixture
is cooled to allow the formation of complexes between the probes and single
stranded amplicon members.
i 5 As the single stranded amplicon sequences and probe sequences are cooled,
the probe sequences preferentially bind the single stranded amplicon members.
This finding is counterintuitive given that the probe sequences generally are
selected to be shorter than the primer sequences and therefore have a lower
melt
temperature than the primers. Accordingly, the melt temperature of the
amplicon
20 produced by the primers should also have a higher melt temperature than the
probes. Thus, as the mixture cools, the re-formation of the double stranded
amplicon would be expected. As previously stated, however, this is not the
case.
The probes are found to preferentially bind the single stranded amplicon
members.
Moreover, this preference of probe/single stranded amplicon binding exists
even
25 when the primer sequences are added in excess of the probes.
After the probe/single stranded amplicon member hybrids are formed, they
are detected. Standard heterogeneous assay formats are suitable for detecting
the
hybrids using the detection labels and capture labels present on the primers
and
probes. The hybrids can be bound to a solid phase reagent by virtue of the
capture
30 label and detected by virtue of the detection label. In cases where the
detection
label is directly detectable, the presence of the hybrids on the solid phase
can be
detected by causing the label to produce a detectable signal, if necessary,
and
detecting the signal. In cases where the label is not directly detectable, the
captured
hybrids can be contacted with a conjugate, which generally comprises a binding
35 member attached to a directly detectable label. The conjugate becomes bound
to the
complexes and the conjugate's presence on the complexes can be detected with
the
directly detectable label. Thus, the presence of the hybrids on the solid
phase
33
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
reagent can be determined. Those skilled in the art will recognize that wash
steps
may be employed to wash away unhybridized amplicon or probe as well as
unbound conjugate.
In one embodiment, the heterogeneous assays can be conveniently
performed using a solid phase support that carries an array of nucleic acid
molecules. Such arrays are useful for high-throughput and/or multiplexed assay
formats. Various methods for forming such arrays from pre-formed nucleic acid
molecules, or methods for generating the array using in situ synthesis
techniques,
are generally known in the art. [See, for example, Dattagupta, et al., EP
10 Publication No. 0 234, 726A3; Southern, U.S. Patent No. 5,700,637; Pirrung,
et
al., U.S. Patent No. 5,143,854; PCT International Publication No. WO 92/10092;
and, Fodor, et al., c'enc 251:767-777 ( 1991 )].
Although the target sequence is described as single stranded, it also is
contemplated to include the case where the target sequence is actually double
15 stranded but is merely separated from its complement prior to hybridization
with
the amplification primer sequences. In the case where PCR is employed in this
method, the ends of the target sequences are usually known. In cases where LCR
or a modification thereof is employed in the preferred method, the entire
target
sequence is usually known. Typically, the target sequence is a nucleic acid
20 sequence such as, for example, RNA or DNA.
The method provided herein can be used in well-known amplification
reactions that include thermal cycle reaction mixtures, particularly in PCR
and gap
LCR (GLCR). Amplification reactions typically employ primers to repeatedly
generate copies of a target nucleic acid sequence, which target sequence is
usually a
25 small region of a much larger nucleic acid sequence. Primers are themselves
nucleic acid sequences that are complementary to regions of a target sequence.
Under amplification conditions, these primers hybridize or bind to the
complementary regions of the target sequence. Copies of the target sequence
typically are generated by the process of primer extension and/or ligation
which
30 utilizes enzymes with polymerase or Iigase activity, separately or in
combination,
to add nucleotides to the hybridized primers and/or ligate adjacent probe
pairs. The
nucleotides that are added to the primers or probes, as monomers or preformed
oligomers, are also complementary to the target sequence. Once the primers or
probes have been sufficiently extended and/or ligated, they are separated from
the
35 target sequence, for example, by heating the reaction mixture to a "melt
temperature" which is one in which complementary nucleic acid strands
dissociate.
Thus, a sequence complementary to the target sequence is formed.
34
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
A new amplification cycle then can take place to further amplify the number
of target sequences by separating any double stranded sequences, allowing
primers
or probes to hybridize to their respective targets, extending and/or ligating
the
hybridized primers or probes and re-separating. The complementary sequences
5 that are generated by amplification cycles can serve as templates for primer
extension or filling the gap of two probes to further amplify the number of
target
sequences. Typically, a reaction mixture is cycled between 20 and 100 times,
more typically, a reaction mixture is cycled between 25 and 50 times. The
numbers of cycles can be determined by the routineer. In this manner, multiple
copies of the target sequence and its complementary sequence are produced.
Thus,
primers initiate amplification of the target sequence when it is present under
amplification conditions.
Generally, two primers which are complementary to a portion of a target
strand and its complement are employed in PCR. For LCR, four probes, two of
15 which are complementary to a target sequence and two of which are similarly
complementary to the target's complement, are generally employed. In addition
to
the primer sets and enzymes previously mentioned, a nucleic acid amplification
reaction mixture may also comprise other reagents which are well known and
include but are not limited to: enzyme cofactors such as manganese; magnesium;
20 salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide
triphosphates
(dNTPs) such as, for example, deoxyadenine triphosphate, deoxyguanine
triphosphate, deoxycytosine triphosphate and deoxythymine triphosphate.
While the amplification primers initiate amplification of the target sequence,
the detection (or hybridization) probe is not involved in amplification.
Detection
25 probes are generally nucleic acid sequences or uncharged nucleic acid
analogs such
as, for example, peptide nucleic acids which are disclosed in International
Publication No. WO 92/20702; morpholino analogs which are described in U.S.
Patents Nos 5,1$5,444, 5,034,506 and 5,142,047; and the like. Depending upon
the type of label carned by the probe, the probe is employed to capture or
detect the
30 amplicon generated by the amplification reaction. The probe is not involved
in
amplification of the target sequence and therefore may have to be rendered
"non-
extendible" in that additional dNTPs cannot be added to the probe. In and of
themselves, analogs usually are non-extendible and nucleic acid probes can be
rendered non-extendible by modifying the 3' end of the probe such that the
35 hydroxyl group is no longer capable of participating in elongation. For
example,
the 3' end of the probe can be functionalized with the capture or detection
label to
thereby consume or otherwise block the hydroxyl group. Alternatively, the 3'
CA 02304368 2000-03-15
WO 99/14357 PCTNS98/19496
hydroxyl group simply can be cleaved, replaced or modified. U.S. Patent
Application Serial No. 07/049,061 filed April 19, 1993 describes modifications
which can be used to render a probe non-extendible.
The ratio of primers to probes is not important. Thus, either the probes or
primers can be added to the reaction mixture in excess whereby the
concentration
of one would be greater than the concentration of the other. Alternatively,
primers
and probes can be employed in equivalent concentrations. Preferably, however,
the primers are added to the reaction mixture in excess of the probes. Thus,
primer
to probe ratios of, for example, 5:1 and 20:1, are preferred.
10 While the length of the primers and probes can vary, the probe sequences
are selected such that they have a lower melt temperature than the primer
sequences. Hence, the primer sequences are generally longer than the probe
sequences. Typically, the primer sequences are in the range of between 20 and
50
nucleotides long, more typically in the range of between 20 and 30 nucleotides
long. The typical probe is in the range of between 10 and 25 nucleotides long.
Various methods for synthesizing primers and probes are well known in
the art. Similarly, methods for attaching labels to primers or probes are also
well
known in the art. For example, it is a matter of routine to synthesize desired
nucleic acid primers or probes using conventional nucleotide phosphoramidite
20 chemistry and instruments available from Applied Biosystems, Inc., (Foster
City,
CA), DuPont (Wilmington, DE), or Milligen (Bedford MA). Many methods have
been described for labeling oligonucleotides such as the primers or probes of
the
present invention. Enzo Biochemical (New York, NY) and Clontech (Palo Alto,
CA) both have described and commercialized probe labeling techniques. For
25 example, a primary amine can be attached to a 3' oligo terminus using 3'-
Amine-
ON CPGTM (Clontech, Palo Alto, CA). Similarly, a primary amine can be attached
to a 5' oligo terrminus using Aminomodifier II~ (Clontech). The amines can be
reacted to various haptens using conventional activation and linking
chemistries.
In addition, copending applications U.S. Serial Nos. 625,566, filed December
11,
30 1990 and 630,908, filed December 20, 1990, teach methods for labeling
probes at
their S' and 3' termini, respectively. International Publication Nos WO
92/10505,
published 25 June 1992, and WO 92/11388, published 9 July 1992, teach methods
for labeling probes at their 5' and 3' ends, respectively. According to one
known
method for labeling an oligonucleotide, a label-phosphoramidite reagent is
prepared
35 and used to add the label to the oligonucleotide during its synthesis. See,
for
example, N.T. Thuong et al., Tet. Letters 29(46):5905-5908 (1988); or J.S.
Cohen et al., published U.S. Patent Application 07/246,688 (NTIS ORDER No.
36
CA 02304368 2000-03-15
WO 99/14357 PCTlUS98/19496
PAT-APPL-7-246,688) ( 1989). Preferably, probes are labeled at their 3' and 5'
ends.
A capture label is attached to the primers or probes and can be a specific
binding member which forms a binding pair with the solid phase reagent's
specific
5 binding member. It will be understood that the primer or probe itself may
serve as
the capture label. For example, in the case where a solid phase reagent's
binding
member is a nucleic acid sequence, it may be selected such that it binds a
complementary portion of the primer or probe to thereby immobilize the primer
or
probe to the solid phase. In cases where the probe itself serves as the
binding
member, those skilled in the art will recognize that the probe will contain a
sequence or "tail" that is not complementary to the single stranded amplicon
members. In the case where the primer itself serves as the capture label, at
least a
portion of the primer will be free to hybridize with a nucleic acid on a solid
phase
because the probe is selected such that it is not fully complementary to the
primer
sequence.
Generally, probe/single stranded amplicon member complexes can be
detected using techniques commonly employed to perform heterogeneous
immunoassays. Preferably, in this embodiment, detection is performed according
to the protocols used by the commercially available Abbott LCx~
instrumentation
(Abbott Laboratories, Abbott Park, IL,).
The primers and probes disclosed herein are useful in typical PCR assays,
wherein the test sample is contacted with a pair of primers, amplification is
performed, the hybridization probe is added, and detection is performed.
Another method provided by the present invention comprises contacting a
test sample with a plurality of polynucleotides, wherein at least one
polynucleotide
is a PS214 molecule as described herein, hybridizing the test sample with the
plurality of polynucleotides and detecting hybridization complexes.
Hybridization
complexes are identified and quantitated to compile a profile which is
indicative of
prostate, breast, or ovary tissue diseases, such as prostate, breast, or
ovarian
30 cancer. Expressed RNA sequences may further be detected by reverse
transcription and amplification of the DNA product by procedures well-known in
the art, including polymerise chain reaction (PCR).
Drug Screening and Gene Therapy.
The present invention also encompasses the use of gene therapy methods
for the introduction of anti-sense PS214 derived molecules, such as
polynucleotides or oligonucleotides of the present invention, into patients
with
conditions associated with abnormal expression of polynucleotides related to a
37
CA 02304368 2000-03-15
WO 99/14357 PCTNS98/19496
prostate, breast, or ovary tissue diseases or condition especially prostate,
breast, or
ovarian cancer. These molecules, including antisense RNA and DNA fragments
and ribozymes, are designed to inhibit the translation of PS214 mRNA, and may
be used therapeutically in the treatment of conditions associated with altered
or
abnormal expression of PS214 polynucleotide.
Alternatively, the oiigonucleotides described above can be delivered to cells
by procedures known in the art such that the anti-sense RNA or DNA may be
expressed in vivo to inhibit production of a PS214 polypeptide in the manner
described above. Antisense constructs to a PS214 polynucleotide, therefore,
10 reverse the action of PS214 transcripts and may be used for treating
prostate,
breast, or ovary tissue disease conditions, such as prostate, breast, or
ovarian
cancer. These antisense constructs may also be used to treat tumor metastases.
The present invention also provides a method of screening a plurality of
compounds for specific binding to PS214 polypeptide(s), or any fragment
thereof,
to identify at least one compound which specifically binds the PS214
polypeptide.
Such a method comprises the steps of providing at least one compound;
combining
the PS214 polypeptide with each compound under suitable conditions for a time
sufficient to allow binding; and detecting the PS214 polypeptide binding to
each
compound.
20 The polypeptide or peptide fragment employed in such a test may either be
free in solution, affixed to a solid support, borne on a cell surface or
located
intracellularly. One method of screening utilizes eukaryotic or prokaryotic
host
cells which are stably transfected with recombinant nucleic acids which can
express
the polypeptide or peptide fragment. A drug, compound, or any other agent may
25 be screened against such transfected cells in competitive binding assays.
For
example, the formation of complexes between a polypeptide and the agent being
tested can be measured in either viable or fixed cells.
The present invention thus provides methods of screening for drugs,
compounds, or any other agent which can be used to treat diseases associated
with
30 PS214. These methods comprise contacting the agent with a polypeptide or
fragment thereof and assaying for either the presence of a complex between the
agent and the polypeptide, or for the presence of a complex between the
polypeptide and the cell. In competitive binding assays, the poiypeptide
typically
is labeled. After suitable incubation, free (or uncomplexed) polypeptide or
35 fragment thereof is separated from that present in bound form, and the
amount of
free or uncomplexed label is used as a measure of the ability of the
particular agent
to bind to the polypeptide or to interfere with the polypeptide/cell complex.
38
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
The present invention also encompasses the use of competitive screening
assays in which neutralizing antibodies capable of binding polypeptide
specifically
compete with a test agent for binding to the polypeptide or fragment thereof.
In
this manner, the antibodies can be used to detect the presence of any
polypeptide in
the test sample which shares one or more antigenic determinants with a PS214
polypeptide as provided herein.
Another technique for screening provides high throughput screening for
compounds having suitable binding affinity to at least one polypeptide of
PS214
disclosed herein. Briefly, large numbers of different small peptide test
compounds
10 are synthesized on a solid phase, such as plastic pins or some other
surface. The
peptide test compounds are reacted with polypeptide and washed. Polypeptide
thus bound to the solid phase is detected by methods well-known in the art.
Purified polypeptide can also be coated directly onto plates for use in the
screening
techniques described herein. In addition, non-neutralizing antibodies can be
used
to capture the polypeptide and immobilize it on the solid support. See, for
example, EP 84/03564, published on September 13, 1984.
The goal of rational drug design is to produce structural analogs of
biologically active polypeptides of interest or of the small molecules
including
agonists, antagonists, or inhibitors with which they interact. Such structural
20 analogs can be used to design drugs which are more active or stable forms
of the
polypeptide or which enhance or interfere with the function of a polypeptide
i~
vivo. J. Hodgson, Bio/TechnolQgy 9:19-21 ( 1991 ).
For example, in one approach, the three-dimensional structure of a
polypeptide, or of a polypeptide-inhibitor complex, is determined by x-ray
25 crystallography, by computer modeling or, most typically, by a combination
of the
two approaches. Both the shape and charges of the polypeptide must be
ascertained to elucidate the structure and to determine active sites) of the
molecule.
Less often, useful information regarding the structure of a polypeptide may be
gained by modeling based on the structure of homologous proteins. In both
cases,
30 relevant structural information is used to design analogous polypeptide-
like
molecules or to identify efficient inhibitors
Useful examples of rational drug design may include molecules which have
improved activity or stability as shown by S. Braxton et aL, Biochemistpr
31:7796-7801 ( 1992), or which act as inhibitors, agonists, or antagonists of
native
35 peptides as shown by S.B.P. Athauda et al., J Biochem. lTok~ 113 (6):742-
746
( 1993 ).
39
CA 02304368 2000-03-15
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It also is possible to isolate a target-specific antibody selected by an assay
as described hereinabove, and then to determine its crystal structure. In
principle
this approach yields a pharmacophore upon which subsequent drug design can be
based. It further is possible to bypass protein crystallography altogether by
5 generating anti-idiotypic antibodies ("anti-ids") to a functional,
pharmacologically
active antibody. As a minor image of a mirror image, the binding site of the
anti-id
is an analog of the original receptor. The anti-id then can be used to
identify and
isolate peptides from banks of chemically or biologically produced peptides.
The
isolated peptides then can act as the pharmacophore (that is, a prototype
pharmaceutical drug).
A sufficient amount of a recombinant polypeptide of the present invention
may be made available to perform analytical studies such as X-ray
crystallography.
In addition, knowledge of the polypeptide amino acid sequence which is
derivable
from the nucleic acid sequence provided herein will provide guidance to those
15 employing computer modeling techniques in place of, or in addition to, x-
ray
crystallography.
Antibodies specific to a PS214 polypeptide (e.g., anti-PS214 antibodies)
further may be used to inhibit the biological action of the polypeptide by
binding to
the polypeptide. In this manner, the antibodies may be used in therapy, for
20 example, to treat prostate, breast, and/or ovary tissue diseases including
prostate,
breast, or ovarian cancer and its metastases.
Further, such antibodies can detect the presence or absence of a PS214
polypeptide in a test sample and, therefore, are useful as diagnostic markers
for the
diagnosis of a prostate, breast, or ovary tissue diseases. or condition
especially
25 prostate, breast, or ovarian cancer. Such antibodies may also function as a
diagnostic marker for prostate, breast, or ovary tissue disease conditions,
such as
prostate, breast, or ovarian cancer.
The present invention also is directed to antagonists and inhibitors of the
polypeptides of the present invention. The antagonists and inhibitors are
those
30 which inhibit or eliminate the function of the polypeptide. Thus, for
example, an
antagonist may bind to a polypeptide of the present invention and inhibit or
eliminate its function. The antagonist, for example, could be an antibody
against
the polypeptide which eliminates the activity of a PS214 polypeptide by
binding a
PS214 polypeptide, or in some cases the antagonist may be an oligonucleotide.
35 Examples of small molecule inhibitors include, but are not limited to,
small
peptides or peptide-like molecules.
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
The antagonists and inhibitors may be employed as a composition with a
pharmaceutically acceptable carrier including, but not limited to, saline,
buffered
saline, dextrose, water, glycerol, ethanol and combinations thereof.
Administration of PS214 polypeptide inhibitors is preferably systemic. The
S present invention also provides an antibody which inhibits the action of
such a
polypeptide.
Antisense technology can be used to reduce gene expression through triple-
helix formation or antisense DNA or RNA, both of which methods are based on
binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion
10 of the polynucleotide sequence, which encodes for the polypeptide of the
present
invention, is used to design an antisense RNA oligonucleotide of from 10 to 40
base pairs in length. A DNA oligonucleotide is designed to be complementary to
a
region of the gene involved in transcription, thereby preventing transcription
and
the production of the PS214 polypeptide. For triple helix, see, for example,
Lee et
1 S al., Nuc. Acids Rgs. 6:3073 ( 1979); Cooney et al., fence 241:456 ( 1988);
and
Dervan et al., i a 251:1360 ( 1991 ) The antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of a mRNA molecule into
the PS214 polypeptide. For antisense, see, for example, Okano, J. Neurochem.
56:560 ( 1991 ); and "Oligodeoxynucleotides as Antisense Inhibitors of Gene
20 Expression," CRC Press, Boca Raton, Fla. (1988). Antisense oligonucleotides
act
with greater efficacy when modified to contain artificial internucleotide
linkages
which render the molecule resistant to nucleolytic cleavage. Such artificial
internucleotide linkages include, but are not limited to, methylphosphonate,
phosphorothiolate and phosphoroamydate internucleotide linkages
25 Recombinant Techno~gy.
The present invention provides host cells and expression vectors
comprising PS214 polynucleotides of the present invention and methods for the
production of the polypeptide(s) they encode. Such methods comprise culturing
the host cells under conditions suitable for the expression of the PS214
30 polynucleotide and recovering the PS214 polypeptide from the cell culture.
The present invention also provides vectors which include PS214
polynucleotides of the present invention, host cells which are genetically
engineered with vectors of the present invention and the production of
polypeptides
of the present invention by recombinant techniques.
35 Host cells are genetically engineered (transfected, transduced or
transformed) with the vectors of this invention which may be cloning vectors
or
expression vectors. The vector may be in the form of a plasmid, a viral
particle, a
41
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
phage, etc. The engineered host cells can be cultured in conventional nutrient
media modified as appropriate for activating promoters, selecting transfected
cells,
or amplifying PS214 gene(s). The culture conditions, such as temperature, pH
and the like, are those previously used with the host cell selected for
expression,
and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for
producing a polypeptide by recombinant techniques. Thus, the polynucleotide
sequence may be included in any one of a variety of expression vehicles, in
particular, vectors or plasmids for expressing a polypeptide. Such vectors
include
10 chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives
of Simian Virus 40 (SV40); bacterial plasmids; phage DNA; yeast plasmids;
vectors derived from combinations of plasmids and phage DNA, viral DNA such
as vaccinia, adenovirus, fowl pox virus and pseudorabies. However, any other
plasmid or vector may be used so long as it is replicable and viable in the
host.
15 The appropriate DNA sequence may be inserted into the vector by a variety
of procedures. In general, the DNA sequence is inserted into appropriate
restriction endonucIease sites by procedures known in the art. Such procedures
and others are deemed to be within the scope of those skilled in the art. The
DNA
sequence in the expression vector is operatively linked to an appropriate
expression
20 control sequences) (promoter) to direct mRNA synthesis. Representative
examples of such promoters include, but are not limited to, the LTR or the
SV40
promoter, the E. coli lac or trp, the phage lambda P sub L promoter and other
promoters known to control expression of genes in prokaryotic or eukaryotic
cells
or their viruses. The expression vector also contains a ribosome binding site
for
25 translation initiation and a transcription terminator. The vector may also
include
appropriate sequences for amplifying expression. In addition, the expression
vectors preferably contain a gene to provide a phenotypic trait for selection
of
transfected host cells such as dihydrofolate reductase or neomycin resistance
for
eukaryotic cell culture, or such as tetracycline or ampicillin resistance in ~
~.
30 The vector containing the appropriate DNA sequence as hereinabove
described, as well as an appropriate promoter or control sequence, may be
employed to transfect an appropriate host to permit the host to express the
protein.
As representative examples of appropriate hosts, there may be mentioned:
bacterial
cells, such as ~ coli, Salmonella Iyt~himurium; Strentomyces sue; fungal
cells,
35 such as yeast; insect cells, such as Drosophila and Sf9; animal cells, such
as CHO,
COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host
is
42
CA 02304368 2000-03-15
WO 99/14357 PCTNS98/19496
deemed to be within the scope of those skilled in the art from the teachings
provided herein.
More particularly, the present invention also includes recombinant
constructs comprising one or more of the sequences as broadly described above.
The constructs comprise a vector, such as a plasmid or viral vector, into
which a
sequence of the invention has been inserted, in a forward or reverse
orientation. In
a preferred aspect of this embodiment, the construct further comprises
regulatory
sequences including, for example, a promoter, operably linked to the sequence.
Large numbers of suitable vectors and promoters are known to those of skill in
the
10 art and are commercially available. The following vectors are provided by
way of
example. Bacterial: pINCY (Incyte Pharmaceuticals Inc., Palo Alto, CA),
pSPORTI (Life Technologies, Gaithersburg, MD), pQE70, pQE60, pQE-9
(Qiagen) pBs, phagescript, psiX174, pBluescript SK, pBsKS, pNHBa, pNHl6a,
pNHl8a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540,
15 pRITS (Pharmacia); Eukaryotic: pWLneo, pSV2cat, pOG44, pXTI, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other
plasmid or vector may be used as long as it is replicable and viable in the
host.
Plasmid pINCY is generally identical to the plasmid pSPORTI (available
from Life Technologies, Gaithersburg, MD) with the exception that it has two
20 modifications in the polylinker (multiple cloning site). These
modifications are ( 1 )
it lacks a HindIII restriction site and (2) its EcoRI restriction site lies at
a different
location. pINCY is created from pSPORT1 by cleaving pSPORTI with both
HindllI and EcoRI and replacing the excised fragment of the polylinker with
synthetic DNA fragments (SEQUENCE 1D NO 10 and SEQUENCE ID NO 11 ).
25 This replacement may be made in any manner known to those of ordinary skill
in
the art. For example, the two nucleotide sequences, SEQUENCE ID NO I O and
SEQUENCE ID NO I 1, may be generated synthetically 5' terminal phosphates,
mixed together, and then ligated under standard conditions for performing
staggered end ligations into the pSPORTI plasmid cut with HindIll and EcoRI.
30 Suitable host cells (such as E cold DHSp, cells) then are transfected with
the ligated
DNA and recombinant clones are selected for ampicillin resistance. Plasmid DNA
then is prepared from individual clones and subjected to restriction enzyme
analysis
or DNA sequencing in order to confirm the presence of insert sequences in the
proper orientation. Other cloning strategies known to the ordinary artisan
also may
35 be employed.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with selectable
markers.
43
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial
promoters include lacI, lacZ, T3, SP6, T7, gpt, lambda P sub R, P sub L and
trp.
Eukaryotic promoters include cytomegalovirus (CMV) immediate early, herpes
simplex virus (HSV) thymidine kinase, early and late SV40, LTRs from
5 retroviruses and mouse metallothionein-I. Selection of the appropriate
vector and
promoter is well within the level of ordinary skill in the art.
In a further embodiment, the present invention provides host cells
containing the above-described construct. The host cell can be a higher
eukaryotic
cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast
cell, or
10 the host cell can be a prokaryotic cell, such as a bacterial cell.
Introduction of the
construct into the host cell can be effected by calcium phosphate
transfection,
DEAF-Dextran mediated transfection, or electroporation [L. Davis et al.,
"Basic
Methods in Molecular Biology," 2nd edition, Appleton and Lang, Paramount
Publishing, East Norwalk, CT ( 1994)].
15 The constructs in host cells can be used in a conventional manner to
produce the gene product encoded by the recombinant sequence. Alternatively,
the
polypeptides of the invention can be synthetically produced by conventional
peptide synthesizers.
Recombinant proteins can be expressed in mammalian cells, yeast, bacteria,
20 or other cells, under the control of appropriate promoters. Cell-free
translation
systems can also be employed to produce such proteins using RNAs derived from
the DNA constructs of the present invention. Appropriate cloning and
expression
vectors for use with prokaryotic and eukaryotic hosts are described by
Sambrook
et al., Molecular Cloning,_,A Laboratory Manual, Second Edition, (Cold Spring
25 Harbor, NY, 1989).
Transcription of a DNA encoding the polypeptide(s) of the present
invention by higher eukaryotes is increased by inserting an enhancer sequence
into
the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to
300 bp, that act on' a promoter to increase its transcription. Examples
include the
30 SV40 enhancer on the late side of the replication origin (bp 100 to 270), a
cytomegalovirus early promoter enhancer, a polyoma enhancer on the late side
of
the replication origin and adenovirus enhancers.
Generally, recombinant expression vectors will include origins of
replication and selectable markers permitting transfection of the host cell,
e.g., the
35 ampicillin resistance gene of E ~ and S . cerevisiae TRP1 gene, and a
promoter
derived from a highly-expressed gene to direct transcription of a downstream
structural sequence. Such promoters can be derived from operons encoding
44
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha factor, acid
phosphatase, or heat shock proteins, among others. The heterologous structural
sequence is assembled in appropriate phase with translation initiation and
termination sequences, and preferably, a leader sequence capable of directing
5 secretion of translated protein into the periplasmic space or extracellular
medium.
Optionally, the heterologous sequence can encode a fusion protein including an
N-
terminal identification peptide imparting desired characteristics, e.g.,
stabilization
or simplified purification of expressed recombinant product.
Useful expression vectors for bacterial use are constructed by inserting a
10 structural DNA sequence encoding a desired protein together with suitable
translation initiation and termination signals in operable reading phase with
a
functional promoter. The vector will comprise one or more phenotypic
selectable
markers and an origin of replication to ensure maintenance of the vector and
to, if
desirable, provide amplification within the host. Suitable prokaryotic hosts
for
15 transfection include ~ ~, Ba ' us s b 'lis, Salmonella typhimurium and
various
species within the genera Pseudomonas, Streptomyces and Staphylococcus,
although others may also be employed as a routine matter of choice.
Useful expression vectors for bacterial use comprise a selectable marker
and bacterial origin of replication derived from plasmids comprising genetic
20 elements of the well-known cloning vector pBR322 (ATCC 37017). Other
vectors
include but are not limited to PKK223-3 (Pharmacia Fine Chemicals, Uppsala,
Sweden) and GEM 1 (Promega Biotec, Madison, WI). These pBR322 "backbone"
sections are combined with an appropriate promoter and the structural sequence
to
be expressed.
25 Following transfection of a suitable host and growth of the host to an
appropriate cell density, the selected promoter is derepressed by appropriate
means
(e.g., temperature shift or chemical induction), and cells are cultured for an
additional period. Cells are typically harvested by centrifugation, disrupted
by
physical or chemical means, and the resulting crude extract retained for
further
30 purification. Microbial cells employed in expression of proteins can be
disrupted
by any convenient method including freeze-thaw cycling, sonication, mechanical
disruption, or use of cell lysing agents. Such methods are well-known to the
ordinary artisan.
Various mammalian cell culture systems can also be employed to express
35 recombinant protein. Examples of mammalian expression systems include the
COS-7 lines of monkey kidney fibroblasts described by Gluzman, ~g~l 23:175
( 1981 ), and other cell lines capable of expressing a compatible vector, such
as the
CA 02304368 2000-03-15
WO 99/14357 PCT/US9$/19496
C 127, HEK-293, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression
vectors will comprise an origin of replication, a suitable promoter and
enhancer and
also any necessary ribosome binding sites, polyadenylation sites, splice donor
and
acceptor sites, transcriptional termination sequences and 5' flanking
nontranscribed
5 sequences. DNA sequences derived from the SV40 viral genome, for example,
SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may
be
used to provide the required nontranscribed genetic elements. Representative,
useful vectors include pRc/CMV and pcDNA3 (available from Invitrogen, San
Diego, CA).
10 PS214 polypeptides are recovered and purified from recombinant cell
cultures by known methods including affinity chromatography, ammonium sulfate
or ethanol precipitation, acid extraction, anion or cation exchange
chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
hydroxyapatite chromatography or lectin chromatography. It is preferred to
have
IS low concentrations (approximately 0.1-5 mM) of calcium ion present during
purification [Price, et al., .1. Biol. Chem. 244:917 ( 1969)]. Protein
refolding steps
can be used, as necessary, in completing configuration of the polypeptide.
Finally,
high performance liquid chromatography (HPLC) can be employed for final
purification steps.
20 Thus, polypeptides of the present invention may be naturally purified
products expressed from a high expressing cell line, or a product of chemical
synthetic procedures, or produced by recombinant techniques from a prokaryotic
or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and
mammalian cells in culture). Depending upon the host employed in a recombinant
25 production procedure, the polypeptides of the present invention may be
glycosylated with mammalian or other eukaryotic carbohydrates or may be non-
glycosylated. The polypeptides of the invention may also include an initial
methionine amino acid residue.
The starting plasmids can be constructed from available plasmids in accord
30 with published, known procedures. In addition, equivalent plasmids to those
described are known in the art and will be apparent to one of ordinary skill
in the
art.
The following is the general procedure for the isolation and analysis of
cDNA clones. in a particular embodiment disclosed herein, mRNA was isolated
35 from prostate, breast, or ovary tissue and used to generate the cDNA
library.
Prostate, breast, or ovary tissue was obtained from patients by surgical
resection
and was classified as tumor or non-tumor tissue by a pathologist.
46
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WO 99/14357 PCT/US98/19496
The cDNA inserts from random isolates of the prostate, breast, or ovary
tissue libraries were sequenced in part, analyzed in detail as set forth in
the
Examples, and are disclosed in the Sequence Listing as SEQUENCE ID NOS 1-7.
Aiso analyzed in detail as set forth in the Examples, and disclosed in the
Sequence
5 Listing, is the full-length sequence of clone 3039477 [referred to herein as
clone
3039477IH (SEQUENCE )D NO 8)]. The consensus sequence of these inserts is
presented as SEQUENCE ID NO 9. These polynucleotides may contain an entire
open reading frame with or without associated regulatory sequences for a
particular
gene, or they may encode only a portion of the gene of interest. This is
attributed
10 to the fact that many genes are several hundred and sometimes several
thousand
bases in length and, with current technology, cannot be cloned in their
entirety
because of vector limitations, incomplete reverse transcription of the first
strand, or
incomplete replication of the second strand. Contiguous, secondary clones
containing additional nucleotide sequences may be obtained using a variety of
15 methods known to those of skill in the art.
Methods for DNA sequencing are well known in the art. Conventional
enzymatic methods employ DNA polymerase, Klenow fragment, Sequenase (US
Biochemical Corp, Cleveland, OH) or Taq polymerase to extend DNA chains from
an oligonucleotide primer annealed to the DNA template of interest. Methods
have
20 been developed for the use of both single-stranded and double-stranded
templates.
The chain termination reaction products may be electrophoresed on
urea/polyacrylamide gels and detected either by autoradiography (for
radionucleotide labeled precursors) or by fluorescence (for fluorescent-
labeled
precursors). Recent improvements in mechanized reaction preparation,
sequencing
25 and analysis using the fluorescent detection method have permitted
expansion in
the number of sequences that can be determined per day using machines such as
the Applied Biosystems 377 DNA Sequencers (Applied Biosystems, Foster City,
CA).
The reading frame of the nucleotide sequence can be ascertained by several
30 types of analyses. First, reading frames contained within the coding
sequence can
be analyzed for the presence of start codon ATG and stop codons TGA, TAA or
TAG. Typically, one reading frame will continue throughout the major portion
of
a cDNA sequence while other reading frames tend to contain numerous stop
codons. In such cases, reading frame determination is straightforward. In
other
35 more difficult cases, further analysis is required.
Algorithms have been created to analyze the occunence of individual
nucleotide bases at each putative codon triplet. See, for example J.W.
Fickett,
47
CA 02304368 2000-03-15
WO 99/14357 PCTNS98/19496
Nuc. Acids Res. 10:5303 ( 1982). Coding DNA for particular organisms
(bacteria,
plants and animals) tends to contain certain nucleotides within certain
triplet
periodicities, such as a significant preference for pyrimidines in the third
codon
position. These preferences have been incorporated into widely available
software
which can be used to determine coding potential (and frame) of a given stretch
of
DNA. The algorithm-derived information combined with start/stop codon
information can be used to determine proper frame with a high degree of
certainty.
This, in turn, readily permits cloning of the sequence in the correct reading
frame
into appropriate expression vectors.
The nucleic acid sequences disclosed herein may be joined to a variety of
other polynucleotide sequences and vectors of interest by means of well-
established recombinant DNA techniques. See J. Sambrook et al., supra. Vectors
of interest include cloning vectors, such as plasmids, cosmids, phage
derivatives,
phagemids, as well as sequencing, replication and expression vectors, and the
like.
1 S In general, such vectors contain an origin of replication functional in at
least one
organism, convenient restriction endonuclease digestion sites and selectable
markers appropriate for particular host cells. The vectors can be transferred
by a
variety of means known to those of skill in the art into suitable host cells
which
then produce the desired DNA, RNA or polypeptides.
Occasionally, sequencing or random reverse transcription errors will mask
the presence of the appropriate open reading frame or regulatory element. In
such
cases, it is possible to determine the correct reading frame by attempting to
express
the polypeptide and determining the amino acid sequence by standard peptide
mapping and sequencing techniques. See, F.M. Ausubel et al., Current Protocols
in Molecular Biology, John Wiley & Sons, New York, NY ( 1989). Additionally,
the actual reading frame of a given nucleotide sequence may be determined by
transfection of host cells with vectors containing all three potential reading
frames.
Only those cells with the nucleotide sequence in the correct reading frame
will
produce a peptide of the predicted length.
The nucleotide sequences provided herein have been prepared by current,
state-of-the-art, automated methods and, as such, may contain unidentified
nucleotides. These will not present a problem to those skilled in the art who
wish
to practice the invention. Several methods employing standard recombinant
techniques, described in J. Sambrook su ra) or periodic updates thereof, may
be
used to complete the missing sequence information. The same techniques used
for
obtaining a full length sequence, as described herein, may be used to obtain
nucleotide sequences.
48
.. , .._ _.. _.
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
Expression of a particular cDNA may be accomplished by subcloning the
cDNA into an appropriate expression vector and transfecting this vector into
an
appropriate expression host. The cloning vector used for the generation of the
prostate, breast, or ovary tissue cDNA library can be used for transcribing
mRNA
5 of a particular cDNA and contains a promoter for beta-galactosidase, an
amino-
terminal met and the subsequent seven amino acid residues of beta-
galactosidase.
Immediately following these eight residues is an engineered bacteriophage
promoter useful for artificial priming and transcription, as well as a number
of
unique restriction sites, including EcoRI, for cloning. The vector can be
transfected into an appropriate host strain of E. coli.
Induction of the isolated bacterial strain with isopropylthiogalactoside
(IPTG) using standard methods will produce a fusion protein which contains the
first seven residues. of beta-galactosidase, about I S residues of linker and
the
peptide encoded within the cDNA. Since cDNA clone inserts are generated by an
15 essentially random process, there is one chance in three that the included
cDNA
will lie in the correct frame for proper translation. If the cDNA is not in
the proper
reading frame, the correct frame can be obtained by deletion or insertion of
an
appropriate number of bases by well known methods including i~r, vitro
mutagenesis, digestion with exonuclease III or mung bean nuclease, or
oligonucleotide linker inclusion.
The cDNA can be shuttled into other vectors known to be useful for
expression of protein in specific hosts. Oligonucleotide primers containing
cloning
sites and segments of DNA sufficient to hybridize to stretches at both ends of
the
target cDNA can be synthesized chemically by standard methods. These primers
25 can then be used to amplify the desired gene segments by PCR. The resulting
new
gene segments can be digested with appropriate restriction enzymes under
standard
conditions and isolated by gel electrophoresis. Alternately, similar gene
segments
can be produced by digestion of the cDNA with appropriate restriction enzymes
and filling in the nussing gene segments with chemically synthesized
30 oligonucleotides. Segments of the coding sequence from more than one gene
can
be ligated together and cloned in appropriate vectors to optimize expression
of
recombinant sequence.
Suitable expression hosts for such chimeric molecules include, but are not
limited to, mammalian cells, such as Chinese Hamster Ovary (CHO) and human
35 embryonic kidney (HEK) 293 cells, insect cells, such as Sf9 cells, yeast
cells,
such as Saccharom~rces cerevisiae and bacteria, such as E. coli. For each of
these
cell systems, a useful expression vector may also include an origin of
replication to
49
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
allow propagation in bacteria and a selectable marker such as the beta-
lactamase
antibiotic resistance gene to allow selection in bacteria. In addition, the
vectors
may include a second selectable marker, such as the neomycin
phosphotransferase
gene, to allow selection in transfected eukaryotic host cells. Vectors for use
in
eukaryotic expression hosts may require the addition of 3' poly A tail if the
sequence of interest lacks poly A.
Additionally, the vector may contain promoters or enhancers which
increase gene expression. Such promoters are host specific and include, but
are
not limited to, MMTV, SV40, or metallothionine promoters for CHO cells; trp,
lac,
10 tac or T7 promoters for bacterial hosts; or alpha factor, alcohol oxidase
or PGH
promoters for yeast. Adenoviral vectors with or without transcription
enhancers,
such as the Rous sarcoma virus (RSV) enhancer, may be used to drive protein
expression in mammalian cell lines. Once homogeneous cultures of recombinant
cells are obtained, large quantities of recombinantly produced protein can be
15 recovered from the conditioned medium and analyzed using chromatographic
methods well known in the art. An alternative method for the production of
large
amounts of secreted protein involves the transfection of mammalian embryos and
the recovery of the recombinant protein from milk produced by transgenic cows,
goats; sheep, etc. Polypeptides and closely related molecules may be expressed
20 recombinantly in such a way as to facilitate protein purification. One
approach
involves expression of a chimeric protein which includes one or more
additional
polypeptide domains not naturally present on human polypeptides. Such
purification-facilitating domains include, but are not limited to, metal-
chelating
peptides such as histidine-tryptophan domains that allow purification on
25 immobilized metals, protein A domains that allow purification on
immobilized
immunoglobulin and the domain utilized in the FLAGS extension/affmity
purification system (Immunex Corp, Seattle, WA). The inclusion of a cleavable
linker sequence such as Factor XA or enterokinase from Invitrogen (San Diego,
CA) between the polypeptide sequence and the purification domain may be useful
30 for recovering the polypeptide.
Immunoassay,,
PS214 polypeptides, including fragments, derivatives, and analogs thereof,
or cells expressing such polypeptides, can be utilized in a variety of assays,
many
of which are described herein, for the detection of antibodies to prostate,
breast, or
35 ovary tissue. They also can be used as immunogens to produce antibodies.
These
antibodies can be, for example, polyclonal or monoclonal antibodies, chimeric,
single chain and humanized antibodies, as well as Fab fragments, or the
product of
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
an Fab expression library. Various procedures known in the art may be used for
the production of such antibodies and fragments.
For example, antibodies generated against a polypeptide comprising a
sequence of the present invention can be obtained by direct injection of the
5 polypeptide into an animal or by administering the polypeptide to an animal
such as
a mouse, rabbit, goat or human. A mouse, rabbit or goat is preferred. The
polypeptide is selected from the group consisting of SEQUENCE m NO 29,
SEQUENCE 1D NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and
fragments thereof. The antibody so obtained then will bind the polypeptide
itself.
10 In this manner, even a sequence encoding only a fragment of the polypeptide
can
be used to generate antibodies that bind the native polypeptide. Such
antibodies
then can be used to isolate the polypeptide from test samples such as tissue
suspected of containing that polypeptide. For preparation of monoclonal
antibodies, any technique which provides antibodies produced by continuous
cell
15 line cultures can be used. Examples include the hybridoma technique as
described
by Kohler and Milstein, Nature 256:495-497 ( 1975), the trioma technique, the
human B-cell hybridoma technique as described by Kozbor et al., Immun. Todav
4:72 (1983) and the EBV-hybridoma technique to produce human monoclonal
antibodies as described by Cole et al., in Monoclonal Antibodies and Cancer
20 Theranv, Alan R. Liss, Inc, New York, NY, pp. 77-96 ( 1985). Techniques
described for the production of single chain antibodies can be adapted to
produce
single chain antibodies to immunogenic polypeptide products of this invention.
See, for example, U.S. Patent No. 4,946,778.
Various assay formats may utilize the antibodies of the present invention,
25 including "sandwich" immunoassays and probe assays. For example, the
antibodies of the present invention, or fragments thereof, can be employed in
various assay systems to determine the presence, if any, of PS214 antigen in a
test
sample. For example, in a first assay format, a polyclonal or monoclonal
antibody
or fragment thereof, or a combination of these antibodies, which has been
coated
30 on a solid phase, is contacted with a test sample, to form a first mixture.
This first
mixture is incubated for a time and under conditions sufficient to form
antigen/antibody complexes. Then, an indicator reagent comprising a monoclonal
or a polyclonal antibody or a fragment thereof, or a combination of these
antibodies, to which a signal generating compound has been attached, is
contacted
35 with the antigen/antibody complexes to form a second mixture. This second
mixture then is incubated for a time and under conditions sufficient to form
antibody/antigen/antibody complexes. The presence of PS214 antigen in the test
S1
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
sample and captured on the solid phase, if any, is determined by detecting the
measurable signal generated by the signal generating compound. The amount of
PS214 antigen present in the test sample is proportional to the signal
generated.
In an alternative assay format, a mixture is formed by contacting: ( 1 ) a
5 polyclonal antibody, monoclonal antibody, or fragment thereof, which
specifically
binds to PS214 antigen, or a combination of such antibodies bound to a solid
support; (2) the test sample; and (3) an indicator reagent comprising a
monoclonal
antibody, polyclonal antibody, or fragment thereof, which specifically binds
to a
different PS214 antigen (or a combination of these antibodies) to which a
signal
10 generating compound is attached. This mixture is incubated for a time and
under
conditions sufficient to form antibody/antigen/antibody complexes. The
presence,
if any, of PS214 antigen present in the test sample and captured on the solid
phase
is determined by detecting the measurable signal generated by the signal
generating
compound. The amount of PS214 antigen present in the test sample is
15 proportional to the signal generated.
In another assay format, one or a combination of at least two monoclonal
antibodies of the invention can be employed as a competitive probe for the
detection of antibodies to PS214 antigen. For example, PS214 poiypeptides such
as the recombinant antigens disclosed herein, either alone or in combination,
are
20 coated on a solid phase. A test sample suspected of containing antibody to
PS214
antigen then is incubated with an indicator reagent comprising a signal
generating
compound and at least one monoclonal antibody of the invention for a time and
under conditions sufficient to form antigen/antibody complexes of either the
test
sample and indicator reagent bound to the solid phase or the indicator reagent
25 bound to the solid phase. The reduction in binding of the monoclonal
antibody to
the solid phase can be quantitatively measured.
In yet another detection method, each of the monoclonal or polyclonal
antibodies of the present invention can be employed in the detection of PS214
antigens in tissue sections, as well as in cells, by irnmunohistochemical
analysis.
30 The tissue sections can be cut from either frozen or chemically fixed
samples of
tissue. If the antigens are to be detected in cells, the cells can be isolated
from
blood, urine, breast aspirates, or other bodily fluids. The cells may be
obtained by
biopsy, either surgical or by needle. The cells can be isolated by
centrifugation or
magnetic attraction after labeling with magnetic particles or ferrofluids so
as to
35 enrich a particular fraction of cells for staining with the antibodies of
the present
invention. Cytochemical analysis wherein these antibodies are labeled directly
(with, for example, fluorescein, colloidal gold, horseradish peroxidase,
alkaline
52
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
phosphatase, etc.) or are labeled by using secondary labeled anti-species
antibodies
(with various labels as exemplified herein) to track the histopathology of
disease
also are within the scope of the present invention.
In addition, these monoclonal antibodies can be bound to matrices similar
5 to CNBr-activated Sepharose and used for the affinity purification of
specific
PS214 polypeptides from cell cultures or biological tissues such as to purify
recombinant and native PS214 proteins.
The monoclonal antibodies of the invention also can be used for the
generation of chimeric antibodies for therapeutic use, or other similar
applications.
The monoclonal antibodies or fragments thereof can be provided
individually to detect PS214 antigens. Combinations of the monoclonal
antibodies
(and fragments thereof) provided herein also may be used together as
components
in a mixture or "cocktail" of at least one PS214 antibody of the invention,
along
with antibodies which specifically bind to other PS214 regions, each antibody
15 having different binding specificities. Thus, this cocktail can include the
monoclonal antibodies of the invention which are directed to PS214
polypeptides
disclosed herein and other monoclonal antibodies specific to other antigenic
determinants of PS214 antigens or other related proteins.
The polyclonal antibody or fragment thereof which can be used in the assay
20 formats should specifically bind to a PS214 polypeptide or other PS214
polypeptides additionally used in the assay. The polyclonal antibody used
preferably is of mammalian origin such as, human, goat, rabbit or sheep
polyclonal antibody which binds PS214 polypeptide. Most preferably, the
polyclonal antibody is of rabbit origin. The polyclonal antibodies used in the
25 assays can be used either alone or as a cocktail of polyclonal antibodies.
Since the
cocktails used in the assay formats are comprised of either monoclonal
antibodies
or polyclonal antibodies having different binding specificity to PS214
polypeptides, they are useful for the detecting, diagnosing, staging,
monitoring,
prognosticating, in vivo imaging, preventing or treating, or determining the
30 predisposition to, diseases and conditions of the prostate, breast, or
ovary, such as
prostate, breast, or ovarian cancer.
It is contemplated and within the scope of the present invention that PS214
antigen may be detectable in assays by use of a recombinant antigen as well as
by
use of a synthetic peptide or purified peptide, which peptide comprises an
amino
35 acid sequence of PS214. The amino acid sequence of such a polypeptide is
selected from the group consisting of SEQUENCE ID NO 29, SEQUENCE ID
NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and fragments thereof.
53
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
It also is within the scope of the present invention that different synthetic,
recombinant or purified peptides, identifying different epitopes of PS214, can
be
used in combination in an assay for the detecting, diagnosing, staging,
monitoring,
prognosticating, in vivo imaging, preventing or treating, or determining the
5 predisposition to diseases and conditions of the prostate, breast, or ovary,
such as
prostate, breast, or ovarian cancer. In this case, all of these peptides can
be coated
onto one solid phase; or each separate peptide may be coated onto separate
solid
phases, such as microparticles, and then combined to form a mixture of
peptides
which can be later used in assays. Furthermore, it is contemplated that
multiple
10 peptides which define epitopes from different antigens may be used for the
detection, diagnosis, staging, monitoring, prognosis, prevention or treatment
of,
or determining the predisposition to, diseases and conditions of the prostate,
breast, or ovary, such as prostate, breast, or ovarian cancer. Peptides coated
on
solid phases or labeled with detectable labels are then allowed to compete
with
I S those present in a patient sample (if any) for a limited amount of
antibody. A
reduction in binding of the synthetic, recombinant, or purified peptides to
the
antibody (or antibodies) is an indication of the presence of PS214 antigen in
the
patient sample. The presence of PS214 antigen indicates the presence of
prostate,
breast, or ovary tissue diseases, especially prostate, breast, ovarian cancer,
in the
20 patient. Variations of assay formats are known to those of ordinary skill
in the art
and many are discussed herein below.
In another assay format, the presence of anti-PS214 antibody and/or PS214
antigen can be detected in a simultaneous assay, as follows. A test sample is
simultaneously contacted with a capture reagent of a first analyte, wherein
said
25 capture reagent comprises a first binding member specific for a first
analyte
attached to a solid phase and a capture reagent for a second analyte, wherein
said
capture reagent comprises a first binding member for a second analyte attached
to a
second solid phase, to thereby form a mixture. This mixture is incubated for a
time
and under conditions sufficient to form capture reagent/first analyte and
capture
30 reagent/second analyte complexes. These so-formed complexes then are
contacted
with an indicator reagent comprising a member of a binding pair specific for
the
first analyte labeled with a signal generating compound and an indicator
reagent
comprising a member of a binding pair specific for the second analyte labeled
with
a signal generating compound to form a second mixture. This second mixture is
35 incubated for a time and under conditions sufficient to form capture
reagent/first
analyte/indicator reagent complexes and capture reagent/second
analyte/indicator
reagent complexes. The presence of one or more analytes is determined by
54
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
detecting a signal generated in connection with the complexes formed on either
or
both solid phases as an indication of the presence of one or more analytes in
the
test sample. In this assay format, recombinant antigens derived from the
expression systems disclosed herein may be utilized, as well as monoclonal
5 antibodies produced from the proteins derived from the expression systems as
disclosed herein. For example, in this assay system, PS214 antigen can be the
first analyte. Such assay systems are described in greater detail in EP
Publication
No. 0473065.
In yet other assay formats, the polypeptides disclosed herein may be
10 utilized to detect the presence of antibody against PS2I4 antigen in test
samples.
For example, a test sample is incubated with a solid phase to which at least
one
polypeptide such as a recombinant protein or synthetic peptide has been
attached.
The polypeptide is selected from the group consisting of SEQUENCE m NO 29,
SEQUENCE ID NO 30, SEQUENCE )D NO 31, SEQUENCE ID NO 32, and
15 fragments thereof. These are reacted for a time and under conditions
sufficient to
form antigen/antibody complexes. Following incubation, the antigen/antibody
complex is detected. Indicator reagents may be used to facilitate detection,
depending upon the assay system chosen. In another assay format, a test sample
is
contacted with a solid phase to which a recombinant protein produced as
described
20 herein is attached, and also is contacted with a monoclonal or polyclonal
antibody
specific for the protein, which preferably has been labeled with an indicator
reagent. After incubation for a time and under conditions sufficient for
antibody/antigen complexes to form, the solid phase is separated from the free
phase, and the label is detected in either the solid or free phase as an
indication of
25 the presence of antibody against PS214 antigen. Other assay formats
utilizing the
recombinant antigens disclosed herein are contemplated. These include
contacting
a test sample with a solid phase to which at least one antigen from a first
source has
been attached, incubating the solid phase and test sample for a time and under
conditions sufficient to form antigen/antibody complexes, and then contacting
the
30 solid phase with a labeled antigen, which antigen is derived from a second
source
different from the first source. For example, a recombinant protein derived
from a
first source such as E. ~ is used as a capture antigen on a solid phase, a
test
sample is added to the so-prepared solid phase, and following standard
incubation
and washing steps as deemed or required, a recombinant protein derived from a
35 different source (i.e., non-E Vii) is utilized as a part of an indicator
reagent which
subsequently is detected. Likewise, combinations of a recombinant antigen on a
solid phase and synthetic peptide in the indicator phase also are possible.
Any
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
assay format which utilizes an antigen specific for PS214 produced or derived
from a first source as the capture antigen and an antigen specific for PS214
from a
different second source is contemplated. Thus, various combinations of
recombinant antigens, as well as the use of synthetic peptides, purified
proteins
5 and the like, are within the scope of this invention. Assays such as this
and others
are described in U.S. Patent No. 5,254,458.
Other embodiments which utilize various other solid phases also are
contemplated and are within the scope of this invention. For example, ion
capture
procedures for immobilizing an immobilizable reaction complex with a
negatively
10 charged polymer (described in EP publication 0326100 and EP publication No.
0406473), can be employed according to the present invention to effect a fast
solution-phase immunochemical reaction. An immobilizable immune complex is
separated from the rest of the reaction mixture by ionic interactions between
the
negatively charged poly-anion/immune complex and the previously treated,
15 positively charged porous matrix and detected by using various signal
generating
systems previously described, including those described in chemiluminescent
signal measurements as described in EPO Publication No. 0 273,115.
Also, the methods of the present invention can be adapted for use in
systems which utilize microparticle technology including automated and semi-
20 automated systems wherein the solid phase comprises a microparticle
(magnetic or
non-magnetic). Such systems include those described in, for example, published
EPO applications Nos. EP 0 425 633 and EP 0 424 634, respectively.
The use of scanning probe microscopy (SPM) for immunoassays also is a
technology to which the monoclonal antibodies of the present invention are
easily
25 adaptable. In scanning probe microscopy, particularly in atomic force
microscopy,
the capture phase, for example, at least one of the monoclonal antibodies of
the
invention, is adhered to a solid phase and a scanning probe microscope is
utilized
to detect antigen/antibody complexes which may be present on the surface of
the
solid phase. The use of scanning tunneling microscopy eliminates the need for
30 labels which normally must be utilized in many immunoassay systems to
detect
antigen/antibody complexes. The use of SPM to monitor specific binding
reactions
can occur in many ways. In one embodiment, one member of a specific binding
partner (analyte specific substance which is the monoclonal antibody of the
invention) is attached to a surface suitable for scanning. The attachment of
the
35 analyte specific substance may be by adsorption to a test piece which
comprises a
solid phase of a plastic or metal surface, following methods known to those of
ordinary skill in the art. Or, covalent attachment of a specific binding
partner
56
CA 02304368 2000-03-15
WO 99/14357 PCTNS98/19496
(analyte specific substance) to a test piece which test piece comprises a
solid phase
of derivatized plastic, metal, silicon, or glass may be utilized. Covalent
attachment
methods are known to those skilled in the art and include a variety of means
to
irreversibly link specific binding partners to the test piece. If the test
piece is
5 silicon or glass, the surface must be activated prior to attaching the
specific binding
partner. Also, polyelectrolyte interactions may be used to immobilize a
specific
binding partner on a surface of a test piece by using techniques and
chemistries.
The preferred method of attachment is by covalent means. Following attachment
of a specific binding member, the surface may be further treated with
materials
10 such as serum, proteins, or other blocking agents to minimize non-specific
binding. The surface also may be scanned either at the site of manufacture or
point
of use to verify its suitability for assay purposes. The scanning process is
not
anticipated to alter the specific binding properties of the test piece.
While the present invention discloses the preference for the use of solid
15 phases, it is contemplated that the reagents such as antibodies, proteins
and
peptides of the present invention can be utilized in non-solid phase assay
systems.
These assay systems are known to those skilled in the art, and are considered
to be
within the scope of the present invention.
It is contemplated that the reagent employed for the assay can be provided
20 in the form of a test kit with one or more containers such as vials or
bottles, with
each container containing a separate reagent such as a probe, primer,
monoclonal
antibody or a cocktail of monoclonal antibodies, or a polypeptide (e.g.
recombinantly, synthetically produced or purified) employed in the assay. The
polypeptide is selected from the group consisting of SEQUENCE ID NO 29,
25 SEQUENCE ID NO 30, SEQUENCE ID NO 31, SEQUENCE ID NO 32, and
fragments thereof. Other components such as buffers, controls and the like,
known to those of ordinary skill in art, may be included in such test kits. It
also is
contemplated to provide test kits which have means for collecting test samples
comprising accessible body fluids, e.g., blood, urine, saliva and stool. Such
tools
30 useful for collection ("collection materials") include lancets and
absorbent paper or
cloth for collecting and stabilizing blood; swabs for collecting and
stabilizing
saliva; cups for collecting and stabilizing urine or stool samples. Collection
materials, papers, cloths, swabs, cups and the like, may optionally be treated
to
avoid denaturation or irreversible adsorption of the sample. The collection
35 materials also may be treated with or contain preservatives, stabilizers or
antimicrobial agents to help maintain the integrity of the specimens. Test
kits
designed for the collection, stabilization and preservation of test specimens
57
CA 02304368 2000-03-15
WO 99/14357 PCT1US98/19496
obtained by surgery or needle biopsy are also useful. It is contemplated that
all kits
may be configured in two components which can be provided separately; one
component for collection and transport of the specimen and the other component
for the analysis of the specimen. The collection component, for example, can
be
5 provided to the open market user while the components for analysis can be
provided to others such as laboratory personnel for determination of the
presence,
absence or amount of analyte. Further, kits for the collection, stabilization
and
preservation of test specimens may be configured for use by untrained
personnel
and may be available in the open market for use at home with subsequent
transportation to a laboratory for analysis of the test sample.
In Vivo Antibody U,~g.
Antibodies of the present invention can be used in vivo; that is, they can be
injected into patients suspected of having diseases of the prostate, breast,
or ovary
for diagnostic or therapeutic uses. The use of antibodies for iQ vivo
diagnosis is
15 well known in the art. Sumerdon et al., Nucl. Med. Biol. 17:247-254 (1990)
have
described an optimized antibody-chelator for the radioimmunoscintographic
imaging of carcinoembryonic antigen (LEA) expressing tumors using Indium-111
as the label. Griffin et al., J. Clin. Onc. 9:631-640 (1991) have described
the use
of this agent in detecting tumors in patients suspected of having recurrent
colorectal
20 cancer. The use of similar agents with paramagnetic ions as labels for
magnetic
resonance imaging is known in the art (R. B. Lauffer, Magnetic Resonance in
edicine 22:339-342 (1991). It is anticipated that antibodies directed against
PS214 antigen can be injected into patients suspected of having a disease of
the
prostate, breast, or ovary such as prostate, breast, or ovarian cancer for the
25 purpose of diagnosing or staging the disease status of the patient. The
label used
will depend on the imaging modality chosen. Radioactive labels such as Indium-
111, Technetium-99m, or Iodine-131 can be used for planar scans or single
photon
emission computed tomography (SPELT). Positron emitting labels such as
Fluorine-19 can also be used for positron emission tomography (PET). For MRI,
30 paramagnetic ions such as Gadolinium (III) or Manganese (II) can be used.
Localization of the label within the prostate, breast, or ovary or external to
the
prostate, breast, or ovary, may allow determination of spread of the disease.
The
amount of label within the prostate, breast, or ovary may allow determination
of
the presence or absence of cancer of the prostate, breast, or ovary.
35 For patients known to have a disease of the prostate, breast, or ovary,
injection of an antibody directed against PS214 antigen may have therapeutic
benefit. The antibody may exert its effect without the use of attached agents
by
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CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
binding to PS214 antigen expressed on or in the tissue or organ.
Alternatively, the
antibody may be conjugated to cytotoxic agents such as drugs, toxins, or
radionuclides to enhance its therapeutic effect. Garnett and Baldwin, Cancer
~,~~earch 46:2407-2412 ( 1986) have described the preparation of a drug-
s monoclonal antibody conjugate. Pastan et al., dell 47:641-648 (1986) have
reviewed the use of toxins conjugated to monoclonal antibodies for the therapy
of
various cancers. Goodwin and Meares, Cancer Supplement 80:2675-2680 ( 1997)
have described the use of Yttrium-90 labeled monoclonal antibodies in various
strategies to maximize the dose to tumor while limiting normal tissue
toxicity.
10 Other known cytotoxic radionuclides include Copper-67, Iodine-131, and
Rhenium-186 all of which can be used to label monoclonal antibodies directed
against PS214 antigen for the treatment of cancer of the prostate, breast, or
ovary.
E. ~ bacteria (clone 3039477) was deposited on March 9, 1998 with the
American Type Culture Collection (A.T.C.C.), 12301 Parklawn Drive, Rockville,
15 Maryland 20852. The deposit was made under the terms of the Budapest Treaty
and will be maintained for a period of thirty (30) years from the date of
deposit, or
for five (5) years after the last request for the deposit, or for the
enforceable period
of the U.S. patent, whichever is longer. The deposit and any other deposited
material described herein are provided for convenience only, and are not
required
20 to practice the present invention in view of the teachings provided herein.
The
cDNA sequence in all of the deposited material is incorporated herein by
reference.
Clone 3039477 was accorded A.T.C.C. Deposit No. 98678.
The present invention will now be described by way of examples, which
are meant to illustrate, but not to limit, the scope of the present invention.
EXAMPLES
Example 1 ~ Identification of Prostate Breast or Ovar~r Tissue Library PS214
Gene-S~ific Clones
A Library Cornparison of Expressed S~c~uence Tags (EST'sl or
30 Transcript Images. Partial sequences of cDNA clone inserts, so-called
"expressed
sequence tags" (EST's), were derived from cDNA libraries made from prostate,
breast, or ovary tumor tissues, non-tumor tissues and numerous other tissues,
both tumor and non-tumor and entered into a database (LIFESEQTM database,
available from Incyte Pharmaceuticals, Palo Alto, CA) as gene transcript
images.
35 S,~e International Publication No. WO 95/20681. (A transcript image is a
listing of
the number of EST's for each of the represented genes in a given tissue
library.
EST's sharing regions of mutual sequence overlap are classified into clusters.
A
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cluster is assigned a clone number from a representative 5' EST. Often, a
cluster
of interest can be extended by comparing its consensus sequence with sequences
of
other EST's which did not meet the criteria for automated clustering. The
alignment of all available clusters and single EST's represent a contig from
which a
5 consensus sequence is derived.) The transcript images then were evaluated to
identify EST sequences that were representative primarily of the prostate,
breast, or
ovary tissue libraries. These target clones then were ranked according to
their
abundance (occurrence) in the target libraries and their absence from
background
libraries. Higher abundance clones with low background occurrence were given
10 higher study priority. EST's corresponding to the consensus sequence of
PS214
were found in prostate, breast and ovarian libraries. EST's corresponding to
the
consensus sequence of PS214 were found in 55.2% (21 of 38) of prostate tissue
libraries, 18.9% (7 of 37) of breast tissue libraries, and 14.2% (3 of 21) of
ovary
tissue libraries. EST's corresponding to the consensus sequence SEQUENCE ID
15 NO 9 (or fragments thereof) were found in only 5.1 % (35 of 681 ) of the
other,
non-prostate , libraries of the data base. Therefore, the consensus sequence
or
fragment thereof was found more than 10.7 times more often in prostate than
non-
prostate tissues. In addition, EST's corresponding to the consensus sequence
SEQUENCE ID NO 9 (or fragments thereof] were found in only 7.1 % (49 of 682)
20 of the other, non-breast, libraries of the data base. Therefore, the
consensus
sequence or fragment thereof was found more than 2.6 times more often in
breast
than non-breast tissues. Furthermore, EST's corresponding to the consensus
sequence SEQUENCE ID NO 9 (or fragments thereof) were only found in 7.5%
(53 of 698) of the other, non-ovary, libraries of the data base. Therefore,
the
25 consensus sequence or fragment thereof was found 1.8 times more often in
ovary
than non-ovary tissues. Overlapping clones 3039477 (SEQUENCE B7 NO 1 ),
4277853 (SEQUENCE 117 NO 2), 1689288 (SEQUENCE ID NO 3), 3186729
(SEQUENCE ID NO 4), 3407934 (SEQUENCE ID NO 5), 2624832
(SEQUENCE ID NO 6), and g2584130(SEQUENCE ID NO 7), respectively,
30 were identified for further study. These represented the minimum number of
clones that, along with the full-length sequence of clone 3039477IH
[hereinafter
referred to as clone 3039477IH (SEQUENCE ID NO 8)], were needed to form the
contig and from which the consensus sequence provided herein (SEQUENCE ID
NO 9) was derived.
35 B. Generation Qf a Consensus Sequence. The nucleotide sequences of
clones 3039477 (SEQUENCE 1D NO 1 ), 4277853 (SEQUENCE ID NO 2),
1689288 (SEQUENCE m NO 3), 3186729 (SEQUENCE ID NO 4), 3407934
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
(SEQUENCE ID NO 5), 2624832 (SEQUENCE ID NO 6), g2584130
(SEQUENCE II7 NO 7), and 3039477IH (SEQUENCE 1T~ NO 8) were entered in
the SequencherTM Program (available from Gene Codes Corporation, Ann Arbor,
MI) in order to generate a nucleotide alignment (contig map) and then generate
5 their consensus sequence (SEQUENCE ID NO 9). Figures lA-1C show the
nucleotide sequence alignment of these clones and their resultant nucleotide
consensus sequence (SEQUENCE ID NO 9). Figure 2 presents the contig map
depicting the clones 3039477 (SEQUENCE ID NO 1 ), 4277853 (SEQUENCE 1D
NO 2), 1689288 (SEQUENCE l~ NO 3), 3186729 (SEQUENCE 1D NO 4},
10 3407934 (SEQUENCE 1D NO 5), 2624832 (SEQUENCE ID NO 6), g2584130
(SEQUENCE ID NO 7), and 3039477IH (SEQUENCE 1D NO 8) which form
overlapping regions of the PS214 gene, and the resultant consensus nucleotide
sequence (SEQUENCE ID NO 9) of these clones in a graphic display. Following
this, a three-frame translation was performed on the consensus sequence
15 (SEQUENCE ID NO 9).
The first forward frame was found to have an open reading frame encoding
an 395 residue amino acid sequence which is presented as SEQUENCE ID NO 29.
The open reading frame corresponds to nucleotides 55 to 1239 of SEQUENCE ll~
NO 9. There is a TIC polymorphism at position 337 in consensus sequence
20 SEQUENCE ff~ NO 9. The ratio of T to C at position 337 was found to be 1:3.
This results in an amino acid shift from serine (TCC) to proline (CCC). The T
was
found in the consensus sequence (SEQUENCE 1D NO 24). The amino acid
sequence was compared with published sequences using software and techniques
known to those skilled in the art. A portion of the polypeptide sequence of
the
25 murine bZIP protein, LZIP-1, was found to be partially homologous with the
PS214 polypeptide. The LZIP-1 polypeptide sequence is deposited with
GenBank under Accession No. L22167, and described by Burbelo et al., Gene
139:241-245 (1994). Similarly, the amino acid sequence was found to show
partial homology to the luman protein, described in Lu et al., Mol. Cell.
Biol.
30 17:5117-5126 {Sept. 1997).
Example 2: Seduencing of PS214 EST-Specific Clones
The full-length DNA sequence of clone 3039477 of the PS214 gene contig
35 was determined (clone 3039477IH, SEQUENCE ID NO 9) using dideoxy
termination sequencing with dye terminators following known methods [F. Sanger
et al., Proc. Natl. Acad. Sci. USA 74:5463 ( 1977)].
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Because vectors such as pSPORTl (Life Technologies, Gaithersburg, MD)
and pINCY (available from Incyte Pharmaceuticals, Inc., Palo Alto, CA) contain
universal priming sites just adjacent to the 3' and 5' ligation junctions of
the
inserts, the inserts were sequenced in both directions using two universal
primers
5 (SEQUENCE B7 NO 12 and SEQUENCE ID NO 13, available from New England
Biolabs, Beverly, MA, and Applied Biosystems Inc, Foster City, CA,
respectively). The sequencing reactions were run on a polyacrylamide
denaturing
gel, and the sequences were determined by an Applied Biosystems 377 Sequencer
{available from Applied Biosystems, Foster City, CA). Additional sequencing
10 primers (SEQUENCE ID NOS 14-26) were designed from sequence information
of the consensus sequence (SEQUENCE B7 NO 9). These primers then were used
to determine the remaining DNA sequence of the cloned insert from each DNA
strand, as previously described.
15 Example 3: Nucleic Acid
A. RNA Extraction from Tissue. Total RNA is isolated from prostate,
breast, or ovary tissues and from non-prostate, breast, or ovary tissues.
Various
methods are utilized, including, but not limited to, the lithium chloride/urea
technique, known in the art and described by Kato et al., J. Virol. 61:2182-
2191
20 ( 1987), and TRIzoITM (Gibco-BRL, Grand Island, NY).
Briefly, tissue is placed in a sterile conical tube on ice and 10-15 volumes
of 3 M LiCI, 6 M urea, 5 mM EDTA, 0.1 M (3-mercaptoethanol, 50 mM Tiis-HCl
(pH 7.5) are added. The tissue is homogenized with a Polytron~ homogenizer
(Brinkman Instruments, Inc., Westbury, NY) for 30-50 sec on ice. The solution
25 is transferred to a 15 ml plastic centrifuge tube and placed overnight at -
20°C. The
tube is centrifuged for 90 min at 9,000 x g at 0-4°C and the
supernatant is
immediately decanted. Ten ml of 3 M LiCI are added and the tube is vortexed
for 5
sec. The tube is centrifuged for 45 min at 11,000 x g at 0-4°C. The
decanting,
resuspension in LiCI, and centrifugation is repeated and the final pellet is
air dried
30 and suspended in 2 ml of 1 mM EDTA, 0.5% SDS, 10 mM Tris (pH 7.5).
Twenty microliters (20 ~.1) of Proteinase K (20 mg/ml) are added, and the
solution
is incubated for 30 min at 37°C with occasional mixing. One-tenth
volume (0.22-
0.25 ml) of 3 M NaCI is added and the solution is vortexed before transfer
into
another tube containing 2 ml of phenoUchloroform/isoamyl alcohol (PCI). The
35 tube is vortexed for 1-3 sec and centrifuged for 20 min at 3,000 x g at
10°C. The
PCI extraction is repeated and followed by two similar extractions with
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chloroform/isoamyl alcohol (CI). The final aqueous solution is transferred to
a
prechilled 15 ml Corex glass tube containing 6 ml of absolute ethanol, the
tube is
covered with parafilm, and placed at -20°C overnight. The tube is
centrifuged for
30 min at 10,000 x g at 0-4°C and the ethanol supernatant is decanted
immediately.
5 The RNA pellet is washed four times with 10 ml of 75% ice-cold ethanol and
the
final pellet is air dried for 15 min at room temperature. The RNA is suspended
in
0.5 ml. of 10 mM TE (pH 7.6, 1 mM EDTA) and its concentration is determined
spectrophotometrically. RNA samples are aliquoted and stored at -70°C
as ethanol
precipitates.
10 The quality of the RNA is determined by agarose gel electrophoresis (see
Example 5, Northern Blot Analysis) and staining with 0.5 p,g/ml ethidium
bromide for one hour. RNA samples that do not contain intact rRNAs are
excluded from the study.
Alternatively, for RT-PCR analysis, 1 ml of Ultraspec RNA reagent was
15 added to 120 mg of pulverized tissue in a 2.0 ml polypropylene microfuge
tube,
homogenized with a Polytron~ homogenizes (Brinkman Instruments, Inc.,
Westbury, NY) for 50 sec and placed on ice for S nun. Then, 0.2 ml of
chloroform was added to each sample, followed by vortexing for 15 sec. The
sample was placed on ice for another 5 min, followed by centrifugation at
12,000 x
20 g for 15 min at 4°C. The upper layer was collected and transferred
to another
RNase-free 2.0 ml microfuge tube. An equal volume of isopropanol was added to
each sample, and the solution was placed on ice for 10 min. The sample was
centrifuged at 12,000 x g for 10 min at 4°C, and the supernatant was
discarded.
The remaining pellet was washed twice with cold 75% ethanol, resuspended by
25 vortexing, and the resuspended material was then pelleted by centrifugation
at 7500
x g for 5 min at 4°C. Finally, the RNA pellet was dried in a Speedvac
(Savant,
Farmingdale, NY) for 5 min and reconstituted in RNase-free water.
B. RNA Extraction from Blood Mononuclear Cells. Mononuclear cells are
isolated from blood samples from patients by centrifugation using Ficoll-
Hypaque
30 as follows. A 10 ml volume of whole blood is mixed with an equal volume of
RPMI Medium (Gibco-BRL, Grand Island, NY). This mixture is then underlayed
with 10 ml of Ficoll-Hypaque (Pharmacia, Piscataway, NJ) and centrifuged for
30
minutes at 200 x g. The buffy coat containing the mononuclear cells is
removed,
diluted to 50 ml with Dulbecco's PBS (Gibco-BRL, Grand Island, NY) and the
35 mixture centrifuged for 10 minutes at 200 x g. After two washes, the
resulting
pellet is resuspended in Dulbecco's PBS to a final volume of 1 ml.
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RNA is prepared from the isolated mononuclear cells as described by N.
Kato et al., it 1"ggY 61:2182-2191 ( 1987). Briefly, the pelleted mononuclear
cells are brought to a final volume of 1 ml and then are resuspended in 250
p.L of
PBS and mixed with 2.5 ml of 3 M LiCI, 6 M urea, 5 mM EDTA, 0.1 M 2-
5 mercaptoethanol, 50 mM Tris-HCl (pH 7.5). The resulting mixture is
homogenized and incubated at -20°C overnight. The homogenate is
centrifuged at
8,000 RPM in a Beckman J2-21M rotor for 90 minutes at 0-4°C. The pellet
is
resuspended in 10 ml of 3 M LiCI by vortexing and then centrifuged at 10,000
RPM in a Beckman J2-21M rotor centrifuge for 45 minutes at 0-4.°C.
The
10 resuspending and pelleting steps then are repeated. The pellet is
resuspended in 2
ml of 1 mM EDTA, 0.5% SDS, 10 mM Tris (pH 7.5) and 400 pg Proteinase K
with vortexing and then it is incubated at 37°C for 30 minutes with
shaking. One
tenth volume of 3 M NaCI then is added and the mixture is vortexed. Proteins
are
removed by two cycles of extraction with phenol/ chloroform/ isoamyl alcohol
15 (PCI) followed by one extraction with chloroform/ isoamyl alcohol {CI). RNA
is
precipitated by the addition of 6 ml of absolute ethanol followed by overnight
incubation at -20°C. After the precipitated RNA is collected by
centrifugation, the
pellet is washed 4 times in 75% ethanol. The pelleted RNA is then dissolved in
solution containing 1 mM EDTA; 10 mM Tris-HCl (pH 7.5).
20 Non-prostate, breast, or ovary tissues were used as negative controls. The
mRNA can be further purified from total RNA by using commercially available
kits
such as oligo dT cellulose spin columns (RediCoITM from Pharmacia, Uppsala,
Sweden) for the isolation of poly-adenylated RNA. Total RNA or mRNA can be
dissolved in lysis buffer (5 M guanidine thiocyanate, 0.1 M EDTA, pH 7.0) for
25 analysis in the ribonuclease protection assay.
C. RNA Extraction from nolvsomes. Tissue is minced in saline at
4°C and
mixed with 2.5 volumes of 0.8 M sucrose in a TK,SOM (150 mM KCI, 5 mM
MgClz, 50 mM Tris-HCI, pH 7.4) solution containing 6 mM 2-mercaptoethanol.
The tissue is homogenized in a Teflon-glass Potter homogenizes with five
strokes
30 at 100-200 rpm followed by six strokes in a Dounce homogenizes, as
described by
B. Mechler, Methods in Enzymo~ 152:241-248 (1987). The homogenate then
is centrifuged at 12,000 x g for 15 min at 4°C to sediment the nuclei.
The
polysomes are isolated by mixing 2 ml of the supernatant with 6 ml of 2.5 M
sucrose in TK,~M and layering this mixture over 4 ml of 2.5 M sucrose in
35 TK~s~IVi in a 38 ml polyallomer tube. Two additional sucrose TK,SOM
solutions
are successively layered onto the extract fraction; a first layer of 13 ml
2.05 M
sucrose followed by a second layer of 6 ml of 1.3 M sucrose. The polysomes are
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isolated by centrifuging the gradient at 90,000 x g for 5 hr at 4°C.
The fraction
then is taken from the 1.3 M sucrose/2.05 M sucrose interface with a
siliconized
pasteur pipette and diluted in an equal volume of TE ( 10 mM Tris-HCI, pH 7.4,
1
mM EDTA). An equal volume of 90°C SDS buffer ( 1 % SDS, 200 mM NaCI, 20
5 mM Tris-HCI, pH 7.4) is added and the solution is incubated in a boiling
water
bath for 2 min. Proteins next are digested with a Proteinase K digestion (50
mg/ml) for 15 min at 37°C. The mRNA is purified with 3 equal volumes of
phenol-chloroform extractions followed by precipitation with 0.1 volume of 2 M
sodium acetate (pH 5.2) and 2 volumes of 100% ethanol at -20°C
overnight. The
10 precipitated RNA is recovered by centrifugation at 12,000 x g for 10 min at
4°C.
The RNA is dried and resuspended in TE (pH 7.4) or distilled water. The
resuspended RNA then can be used in a slot blot or dot blot hybridization
assay to
check for the presence of PS214 mRNA (see Example 6).
The quality of nucleic acid and proteins is dependent on the method of
15 preparation used. Each sample may require a different preparation technique
to
maximize isolation efficiency of the target molecule. These preparation
techniques
are within the skill of the ordinary artisan.
Example 4: Ribonuclease Protection Assay
20 A. Synthesis of Labeled Complementanr RNA fcRNA) H, bridiza~jon
P~Qbe and Unlabeled Sense Strand Labeled antisense and unlabeled sense
riboprobes are transcribed from the PS214 gene cDNA sequence which contains a
5' RNA polymerase promoter such as SP6 or T7. The sequence may be from a
vector containing the appropriate PS214 cDNA insert, or from a PCR-generated
25 product of the insert using PCR primers which incorporate a 5' RNA
polymerase
promoter sequence. For example, clone 3039477, or another comparable clone
containing the PS214 gene cDNA sequence flanked by opposed SPb and T7 or
other RNA polymerase promoters, is purified using a Qiagen Plasmid
Purification
Kit (Qiagen, Chatsworth, CA). Then 10 ~,g of the plasmid DNA are linearized by
30 cutting with an appropriate restriction enzyme such as DdeI for 1 hr at
37°C. The
linearized plasmid DNA is purified using the QIAprep Kit (Qiagen, Chatsworth,
CA) and used for the synthesis of antisense transcript from the appropriate
promoter using the Riboprobe~ in v' ro Transcription System (Promega
Corporation, Madison, WI), as described by the supplier's instructions,
35 incorporating either (alpha32P) CTP (Amersham Life Sciences, Inc. Arlington
Heights, IL) or biotinylated CTP as a label. To generate the sense strand, 10
P.g of
the purified plasmid DNA are cut with restriction enzymes, such as XbaI and
NotI,
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WO 99/14357 PCT/US98/19496
and transcribed as above from the appropriate promoter. Both sense and
antisense
strands are isolated by spin column chromatography. Unlabeled sense strand is
quantitated by UV absorption at 2b0 nm.
B. Hvbridization of .~beled Probe t~ Target. Frozen tissue is pulverized
to powder under liquid nitrogen and 100-500 mg are dissolved in 1 ml of lysis
buffer, available as a component of the Direct Protecf'~"'' Lysate RNase
Protection
Kit (Ambion, Inc., Austin, TX). Further dissolution can be achieved using a
tissue homogenizer. In addition, a dilution series of a known amount of sense
strand in mouse liver lysate is made for use as a positive control. Finally,
45 pl of
10 solubilized tissue or diluted sense strand are mixed directly with either:
( 1 ) 1 x 105
cpm of radioactively labeled probe; or (2) 250 pg of non-isotopically labeled
probe
in 5 p.l of lysis buffer. Hybridization is allowed to proceed overnight at
37°C.
See, T. Kaabache et al., Anal. Biochem. 232:225-230 (1995).
C. RNase Dige lion. RNA that is not hybridized to probe is removed
from the reaction as per the Direct Protecf'~"'' protocol using a solution of
RNase A
and RNase T1 for 30 min at 37°C, followed by removal of RNase by
Proteinase K
digestion in the presence of sodium sarcosyl. Hybridized fragments protected
from digestion are then precipitated by the addition of an equal volume of
isopropanol and placed at -70°C for 3 hr. The precipitates are
collected by
centrifugation at 12,000 x g for 20 min.
D. Fragment Analysis. The precipitates are dissolved in denaturing gel
loading dye (80% formamide, 10 mM EDTA (pH 8.0), 1 mg/ml xylene cyanol, 1
mg/ml bromophenol blue), heat denatured, and electrophoresed in 6%
polyacrylamide TBE, 8 M urea denaturing gels. The gels are imaged and analyzed
using the STORM~'~"'' storage phosphor autoradiography system (Molecular
Dynamics, Sunnyvale, CA). Quantitation of protected fragment bands, expressed
in femtograms (fg), is achieved by comparing the peak areas obtained from the
test
samples to those from the known dilutions of the positive control sense strand
(see
Section B, supra). The results are expressed in molecules of PS214 RNA/cell
and
as a image rating score. In cases where non-isotopic labels are used, hybrids
are
transferred from the gels to membranes (nylon or nitrocellulose) by blotting
and
then analyzed using detection systems that employ streptavidin alkaline
phosphatase conjugates and chemiluminescence or chemifluorescence reagents.
Detection of a product comprising a sequence selected from the group
consisting of SEQUENCE ID NOS 1-9, and fragments or complements thereof, is
indicative of the presence of PS214 mRNAs, suggesting a diagnosis of a
prostate,
66
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WO 99/14357 PCT/US98/19496
breast, or ovary tissue disease or condition, such as prostate, breast, or
ovarian
cancer.
Example 5: Northern Blotting
The Northern blot technique is used to identify a specific size RNA
fragment from a complex population of RNA using gel electrophoresis and
nucleic
acid hybridization. Northern blotting is well-known technique in the art.
Briefly,
5-10 p.g of total RNA (see Example 3) are incubated in 15 pl of a solution
containing 40 mM morphilinopropanesulfonic acid (MOPS) (pH 7.0), 10 mM
sodium acetate, 1 mM EDTA, 2.2 M formaldehyde, 50% v/v formamide for 15
min at 65°C. The denatured RNA is mixed with 2 p.l of loading buffer
(50%
glycerol, 1 mM EDTA, 0.4% bromophenol blue, 0.4% xylene cyanol) and loaded
into a denaturing 1.0% agarose gel containing 40 mM MOPS (pH 7.0), 10 mM
sodium acetate, 1 mM EDTA and 2.2 M formaldehyde. The gel is electrophoresed
at 60 V for 1.5 hr and rinsed in RNAse free water. RNA is transferred from the
gel onto nylon membranes (Brightstar-Plus, Ambion, Inc., Austin, TX) for 1.5
hours using the downward alkaline capillary transfer method (Chomczynski,
Anal.
Biochem. 201:134-139, 1992). The filter is rinsed with 1X SSC, and RNA is
crosslinked to the filter using a Stratalinker~'~"' (Stratagene, Inc., La
Jolla, CA) on
the autocrosslinking mode and dried for 15 min. The membrane is then placed
into
a hybridization tube containing 20 ml of preheated prehybridizadon solution
(SX
SSC, 50% formamide, SX Denhardt's solution, 100 p,g/ml denatured salmon
sperm DNA) and incubated in a 42°C hybridization oven for at least 3
hr. While
the blot is prehybridizing, a 32P-labeled random-primed probe is generated
using
the PS214 insert fragment (obtained by digesting clone 3039477 or another
comparable clone with XbaI and NotI) using Random Primer DNA Labeling
System (Life Technologies, Inc., Gaithersburg, MD) according to the
manufacturer's instructions. Half of the probe is boiled for 10 min, quick
chilled
on ice and added to the hybridization tube. Hybridization is performed at
42°C for
at least 12 hr. The hybridization solution is discarded and the filter is
washed in 30
ml of 3X SSC, 0.1 % SDS at 42°C for 15 min, followed by 30 ml of 3X
SSC,
0.1 % SDS at 42°C for 15 min. The filter is wrapped in Saran Wrap,
exposed to
Kodak XAR-Omat film for 8-96 hr, and the film is developed for analysis. High
level of expression of mRNA corresponding to a sequence selected from the
group
consisting of SEQUENCE ID NOS 1-9, and fragments or complements thereof, is
an indication of the presence of PS214 mRNA, suggesting a diagnosis of a
67
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WO 99/14357 PCT/US98/19496
prostate, breast, or ovary tissue disease or condition, such as prostate,
breast, or
ovarian cancer.
Examl?le 6' Dot Blot/Slot Blot
5 Dot and slot blot assays are quick methods to evaluate the presence of a
specific nucleic acid sequence in a complex mix of nucleic acid. To perform
such
assays, up to SO ~.g of RNA are mixed in 50 p,l of SO% formamide, 7%
formaldehyde, 1X SSC, incubated IS min at 68°C, and then cooled on ice.
Then,
100 p,l of 20X SSC are added to the RNA mixture and loaded under vacuum onto a
10 manifold apparatus that has a prepared nitrocellulose or nylon membrane.
The
membrane is soaked in water, 20X SSC for I hour, placed on two sheets of 20X
SSC prewet Whatman #3 filter paper, and loaded into a slot blot or dot blot
vacuum manifold apparatus. The slot blot is analyzed with probes prepared and
labeled as described in Example 4, supra. Detection of mRNA corresponding to a
15 sequence selected from the group consisting of SEQUENCE ID NOS I-9, and
fragments or complements thereof, is an indication of the presence of PS214,
suggesting a diagnosis of a prostate, breast, or ovary tissue diseases or
condition,
such as prostate, breast, or ovarian cancer.
Other methods and buffers which can be utilized in the methods described
20 in Examples 5 and 6, but not specifically detailed herein, are known in the
art and
are described in J. Sambrook et al., supra.
Example 7' In Situ Hybridization
This method is useful to directly detect specific target nucleic acid
25 sequences in cells using detectable nucleic acid hybridization probes.
Tissues are prepared with cross-linking fixative agents such as
paraformaldehyde or glutaraldehyde for maximum cellular RNA retention. See, L.
Angerer et al., Methods in Cell Biol. 35:37-71 ( 1991 ). Briefly, the tissue
is placed
in greater than 5 volumes of 1 % glutaraldehyde in 50 mM sodium phosphate, pH
30 7.5 at 4°C for 30 min. The solution is changed with fresh
glutaraldehyde solution
( 1 % glutaraldehyde in SO mM sodium phosphate, pH 7.5) for a further 30 min
fixing. The fixing solution should have an osmolality of approximately 0.375%
NaCI. The tissue is washed once in isotonic NaCI to remove the phosphate.
The fixed tissues then are embedded in paraffin as follows. The tissue is
35 dehydrated though a series of increasing ethanol concentrations for 15 min
each:
50% (twice), 70% (twice), 85%, 90% and then 100% (twice). Next, the tissue is
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WO 99/14357 PCT/US98/19496
soaked in two changes of xylene for 20 min each at room temperature. The
tissue
is then soaked in two changes of a 1:1 mixture of xylene and paraffin for 20
min
each at 60°C; and then in three final changes of paraffin for 15 min
each.
Next, the tissue is cut in 5 ~.m sections using a standard microtome and
placed on a slide previously treated with a tissue adhesive such as 3-
aminopropyltriethoxysilane.
Paraffin is removed from the tissue by two 10 min xylene soaks and
rehydrated in a series of decreasing ethanol concentrations: 99% (twice), 95%,
85%, 70%, 50%, and 30%; and then in distilled water (twice). The sections are
pre-treated with 0.2 M HCl for 10 min and permeabilized with 2 p,g/ml
Proteinase
K at 37°C for 15 min.
Labeled riboprobes transcribed from the PS214 gene plasmid (see Example
4) are hybridized to the prepared tissue sections and incubated overnight at
56°C in
3X standard saline extract and 50% formamide. Excess probe is removed by
15 washing in 2X standard saline citrate and 50% formamide followed by
digestion
with 100 ~.g/ml RNase A at 37°C for 30 min. Probe fluorescence is
visualized by
illumination with ultraviolet (UV) light under a microscope. Fluorescence in
the
cytoplasm is indicative of PS214 mRNA. Alternatively, the sections can be
visualized by autoradiography.
Example 8: Reverse Transcription P R
A. One Step RT-PCR Assav. Target-specific primers are designed to
detect the above-described target sequences by reverse transcription PCR using
methods known in the art. One step RT-PCR is a sequential procedure that
25 performs both RT and PCR in a single reaction mixture. The procedure is
performed in a 200 p,l reaction mixture containing 50 mM (N,N,-bis[2-
Hydroxyethyl]glycine), pH 8.15, 81.7 mM KOAc, 33.33 mM KOH, 0.01 mg/ml
bovine serum albumin, 0.1 mM ethylene diaminetetraacetic acid, 0.02 mg/ml
NaN3~ 8% w/v glycerol, 150 p.M each of dNTP, 0.25 p.M each primer, SU rTth
30 polymerise, 3.25 mM Mn(OAc)Z and 5 p.l of target RNA (see Example 3). Since
RNA and the rTth polymerise enzyme are unstable in the presence of Mn(OAc)2,
the Mn(OAc)2 should be added just before target addition. Optimal conditions
for
cDNA synthesis and thermal cycling readily can be determined by those skilled
in
the art. The reaction is incubated in a Perkin-Elmer Thermal Cycler 480.
35 Conditions which may be found useful include cDNA synthesis at 60°-
70°C for 15-
45 min and 30-45 amplification cycles at 94°C, 1 min; 55°-
70°C, 1 min; 72°C, 2
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WO 99/14357 PCTNS98/19496
min. One step RT-PCR also may be performed by using a dual enzyme procedure
with Taq polymerise and a reverse transcriptase enzyme, such as MMLV
(Moloney murine leukemia virus) or AMV (avian myeloblastosis virus) RT
(reverse transcriptase) enzymes.
S B. Traditional RT-PCR. A traditional two-step RT-PCR reaction was
performed, as described by K.Q. Hu et al., Virology 181:721-726 ( 1991 ).
Briefly, O.S ~.g of extracted mRNA (see Example 3) was reverse transcribed in
a
20 p.l reaction mixture containing 1X PCR II buffer (Perkin-Elmer), S mM
MgCl2,
1 mM each dNTP, 20 U RNasin, 2.S p,M random hexamers, and SO U MMLV
10 RT. Reverse transcription was performed at room temperature for 10 min,
42°C
for 30 min in a PE-480 thermal cycler (Perkin-Elmer), followed by further
incubation at 9S°C for S min to inactivate the RT. PCR was performed
using 2 p,l
of the cDNA reaction in a final PCR reaction volume of SO p,l containing 1X
PCR
II buffer (Perkin-Elmer), SO mM KCI, 1.S mM MgCl2, 200 p,M dNTPs, O.S p,M
1 S of each sense and antisense primer, (SEQUENCE m NO 27 and SEQUENCE ID
NO 28, respectively), and 2.S U of Taq Gold polymerise. The reaction was
incubated in a MJ Research Model PTC-200, as follows: 3S cycles of
amplification (94°C, 4S sec; 6S°C, 4S sec; 72°C, 2 min.);
a final extension at (94°C,
15 min.) then (72°C, S min); and a soak at 4°C.
20 C. PCR Fragment Analysis. The correct products were verified by size
determination using gel electrophoresis with a SYBR~ Green I nucleic acid gel
stain (Molecular Probes, Eugene, OR). Gels were stained with SYBR~ Green I at
a 1:10,000 dilution in 1X TBE for 4S min. The gels, Figures 3-S, were imaged
using a STORMTM imaging system (Molecular Dynamics, Sunnyvale, CA).
2S Figure 3 shows a 596 by PS214-specific PCR amplification product in lanes 3-
7
and 9-10 indicating that PS214 mRNA was present in 7 of 8 BPH (lanes S-7 and
9) and prostate cancer (lanes 3, 4, 8 and 10) samples tested. Figure 4 shows a
S96
by PS214-specific PCR amplicon in lanes 3 through 10, indicating that PS214
mRNA was present in 8 of 8 normal breast (lanes 6, 7 and 9) and breast cancer
30 (lanes 3, 4, S, 8 and 10) samples tested. In Figure S, the S96 by PS214-
specific
PCR amplification product was observed in normal breast and breast cancer
(lanes
3 and S), BPH (lane 4), prostate cancer (lane 6), colon cancer (lanes 7 and
8),
bladder cancer (lane 12), normal lung (lane 14) and lung cancer (lanes 13 and
1S).
In Figures 3, 4 and S, lane 1 represents molecular weight markers and lane 2
3S placental DNA.
Detection of a product comprising a sequence selected from the group
consisting of SEQUENCE ID NOS 1-9, and fragments or complements thereof, is
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
indicative of the presence of PS214 mRNA(s), suggesting a diagnosis of a
prostate, breast, or ovary tissue disease or condition, such as prostate,
breast, or
ovarian cancer.
Examr~]e 9: OH-P _R
A. Probe selection and Lab~jn~. Target-specific primers and probes are
designed to detect the above-described target sequences by oligonucleotide
hybridization PCR. International Publication Nos WO 92/10505, published June
25, 1992, and WO 92/11388, published July 9, 1992, teach methods for labeling
10 oligonucleotides at their 5' and 3' ends, respectively. According to one
known
method for labeling an oligonucleotide, a label-phosphoramidite reagent is
prepared
and used to add the label to the oligonucleotide during its synthesis. For
example,
seg N. T. Thuong et al., Tet. Letters 29(46):5905-5908 (1988); or J. S. Cohen
et
al., published U.S. Patent Application 07/246,688 (NTIS ORDER No. PAT-
15 APPL-7-246,688) ( 1989). Preferably, probes are labeled at their 3' end to
prevent
participation in PCR and the formation of undesired extension products. For
one
step OH-PCR, the probe should have a TM at least 1 S°C below the TM of
the
primers. The primers and probes are utilized as specific binding members, with
or
without detectable labels, using standard phosphoramidite chemistry and/or
post-
20 synthetic labeling methods which are well-known to one skilled in the art.
B. One Sten Oli,go Hybridization PCR. OH-PCR is performed on a 200
p.l reaction containing 50 mM (N,N,-bis[2-HydroxyethylJglycine), pH 8.15, 81.7
mM KOAc, 33.33 mM KOH, 0.01 mg/ml bovine serum albumin, 0.1 mM
ethylene diaminetetraacetic acid, 0.02 mg/ml NaN;~ 8% w/v glycerol, 150 ~,M
each
25 of dNTP, 0.25 ~,M each primer, 3.75 nM probe, SU rTth polymerase, 3.25 mM
Mn(OAc)Z and 5 p.l blood equivalents of target (see Example 3). Since RNA and
the rTth polymerase enzyme are unstable in the presence of Mn(OAc)2, the
Mn(OAc)2 should be added just before target addition. The reaction is
incubated in
a Perkin-Elmer Thermal Cycler 480. Optimal conditions for cDNA synthesis and
30 thermal cycling can be readily determined by those skilled in the art.
Conditions
which may be found useful include cDNA synthesis (60°C, 30 min), 30-45
amplification cycles (94°C, 40 sec; 55-70°C, 60 sec), oligo-
hybridization (97°C, 5
min; 15°C, 5 min; 15°C soak). The correct reaction product
contains at least one of
the strands of the PCR product and an internally hybridized probe.
35 C. OH-PCR Product Analvsic, Amplified reaction products are detected
on an LCx~ Analyzer system (available from Abbott Laboratories, Abbott Park,
71
r _ _ _._ _~__
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
IL). Briefly, the correct reaction product is captured by an antibody labeled
microparticle at a capturable site on either the PCR product strand or the
hybridization probe, and the complex is detected by binding of a detectable
antibody conjugate to either a detectable site on the probe or the PCR strand.
Only
5 a complex containing a PCR strand hybridized with the internal probe is
detectable.
The detection of this complex then is indicative of the presence of PS214
mRNA,
suggesting a diagnosis of a prostate, breast, or ovary disease or condition,
such as
prostate, breast, or ovarian cancer.
Many other detection formats exist which can be used and/or modified by
those skilled in the art to detect the presence of amplified or non-amplified
PS214-
derived nucleic acid sequences including, but not limited to, ligase chain
reaction
(LCR, Abbott Laboratories, Abbott Park, IL); Q-beta replicase (Gene-TrakTM,
Naperville, Illinois), branched chain reaction (Chiron, Emeryville, CA) and
strand
displacement assays (Becton Dickinson, Research Triangle Park, NC).
Example 10: Synthetic Peptide Production
Synthetic peptides were modeled and then prepared based upon the
predicted amino acid sequence of the PS214 polypeptide consensus sequence (see
Example 1 ). In particular, a number of PS214 peptides derived from SEQUENCE
20 ID NO 29 were prepared, including the peptides of SEQUENCE ID NO 30,
SEQUENCE ID NO 31, and SEQUENCE ID NO 32. All peptides were
synthesized on a Symphony Peptide Synthesizer (available from Rainin
Instrument
Co, Emeryville, CA) using FMOC chemistry, standard cycles and in- ' HBTU
activation. Cleavage and deprotection conditions were as follows: a volume of
2.5
25 ml of cleavage reagent (77.5% v/v trifluoroacetic acid, 15% v/v
ethanedithiol,
2.5% v/v water, 5% v/v thioanisole, 1-2% w/v phenol) were added to the resin,
and agitated at room temperature for 2-4 hours. The filtrate was then removed
and
the peptide was precipitated from the cleavage reagent with cold diethyl
ether. Each
peptide was filtered, purified via reverse-phase preparative HPLC using a
30 water/acetonitrile/0.1 % TFA gradient, and lyophilized. The product was
confirmed by mass spectrometry.
The purified peptides were used to immunize animals (see Example 14).
Example 11 a: Expression of Protein in a Cell Line Using Plasmid 577
35 A. Construction of a PS214 Expression Plasmid. Plasmid 577, described
in U.S. patent application Serial No. 08/478,073, filed June 7, 1995, has been
constructed for the expression of secreted antigens in a permanent cell line.
This
72
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
plasmid contains the following DNA segments: (a) a 2.3 kb fragment of pBR322
containing bacterial beta-lactamase and origin of DNA replication; (b) a 1.8
kb
cassette directing expression of a neomycin resistance gene under control of
HSV-
1 thymidine kinase promoter and poly-A addition signals; (c) a 1.9 kb cassette
5 directing expression of a dihydrofolate reductase gene under the control of
an
SV40 promoter and poly-A addition signals; (d) a 3.5 kb cassette directing
expression of a rabbit immunoglobulin heavy chain signal sequence fused to a
modified hepatitis C virus (HCV) E2 protein under the control of the SV40 T-Ag
promoter and transcription enhancer, the hepatitis B virus surface antigen
(HBsAg)
10 enhancer I followed by a fragment of Herpes Simplex Virus-1 (HSV-1) genome
providing poly-A addition signals; and (e) a residual 0.7 kb fragment of SV40
genome late region of no function in this plasmid. All of the segments of the
vector were assembled by standard methods known to those skilled in the art of
molecular biology.
15 Plasmids for the expression of secretable PS214 proteins are constructed
by replacing the hepatitis C virus E2 protein coding sequence in plasmid 577
with
that of a PS214 polynucleotide sequence selected from the group consisting of
SEQUENCE ID NOS 1-9, and fragments or complements thereof, as follows.
Digestion of plasmid 577 with XbaI releases the hepatitis C virus E2 gene
20 fragment. The resulting plasmid backbone allows insertion of the PS214 cDNA
insert downstream of the rabbit immunoglobulin heavy chain signal sequence
which directs the expressed proteins into the secretory pathway of the cell.
The
PS214 cDNA fragment is generated by PCR using standard procedures. Encoded
in the sense PCR primer sequence is an XbaI site, immediately followed by a 12
25 nucleotide sequence that encodes the amino acid sequence Ser-Asn-Glu-Leu
("SNEL") to promote signal protease processing, efficient secretion and final
product stability in culture fluids. Immediately following this 12 nucleotide
sequence the primer contains nucleotides complementary to template sequences
encoding amino acids of the PS214 gene . The antisense primer incorporates a
30 sequence encoding the following eight amino acids just before the stop
codons:
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQUENCE >D NO 33). Within this
sequence is incorporated a recognition site to aid in analysis and
purification of the
PS214 protein product. A recognition site (termed "FLAG") that is recognized
by
a commercially available monoclonal antibody designated anti-FLAG M2 (Eastman
35 Kodak, Co., New Haven, CT) can be utilized, as well as other comparable
sequences and their corresponding antibodies. For example, PCR is performed
using GeneAmp~ reagents obtained from Perkin-Elmer-Cetus, as directed by the
73
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
supplier's instructions. PCR primers are used at a final concentration of 0.5
~.M.
PCR is perfonmed on the PS214 plasmid template in a 100 E.tl reaction for 35
cycles
(94°C, 30 seconds; 55°C, 30 seconds; 72°C, 90 seconds)
followed by an extension
cycle of 72°C for 10 min.
5 B Transfection of Dihydrofolate Reductase Deficient Chinese Hamster
Ov~r,~Cells. The plasmid described ~ is transfected into CHO/dhfr- cells
[DXB-11 l, Uriacio et al., Proc Natl Acad Sci USA 77:4451-4466 (1980)].
These cells are available from the A.T.C.C., 12301 Parklawn Drive, Rockville,
MD 20852, under Accession No. CRL 9096. Transfection is performed using the
10 cationic liposome-mediated procedure described by P. L. Felgner et al.,
Proc.
Natl. Acad. Sci. USA 84:7413-7417 (1987). Particularly, CHO/dhfr- cells are
cultured in Ham's F-12 media supplemented with 10% fetal calf serum, L-
glutamine ( 1 mM) and freshly seeded into a flask at a density of 5-8 x 105
cells per
flask. The cells are grown to a confluency of between 60 and 80% for
15 transfection. Twenty micrograms (20~.g) of plasmid DNA are added to 1.5 ml
of
Opti-MEM I medium and 100 ~,1 of Lipofectin Reagent (Gibco-BRL; Grand Island,
NY) are added to a second 1.5 ml portion of Opti-MEM I media. The two
solutions are mixed and incubated at room temperature for 20 min. After the
culture medium is removed from the cells, the cells are rinsed 3 times with 5
ml of
20 Opti-MEM I medium. The Opti-MEM I-Lipofection-plasmid DNA solution then is
overlaid onto the cells. The cells are incubated for 3 hr at 37°C,
after which time
the Opti-MEM I-Lipofectin-DNA solution is replaced with culture medium for an
additional 24 hr prior to selection.
C. Selection and Amplification. One day after transfection, cells are
25 passaged 1:3 and incubated with dhfr/G418 selection medium (hereafter, "F-
12
minus medium G"). Selection medium is Ham's F-12 with L-glutamine and
without hypoxanthine, thymidine and glycine (JRH Biosciences, Lenexa, Kansas)
and 300 ~,g per ml 6418 (Gibco-BRL; Grand Island, NY). Media volume-to-
surface area ratios of 5 ml per 25 cmz are maintained. After approximately two
30 weeks, DHFR/G418 cells are expanded to allow passage and continuous
maintenance in F-12 minus medium G.
Amplification of each of the transfected PS214 cDNA sequences is
achieved by stepwise selection of DHFR+, 6418+ cells with methotrexate
(reviewed by R. Schimke, Cel[ 37:705-713 [1984]). Cells are incubated with F-
12
35 minus medium G containing 150 nM methotrexate (MTX) (Sigma, St. Louis, MO)
for approximately two weeks until resistant colonies appear. Further gene
amplification is achieved by selection of 150 nM adapted cells with 5 N,M MTX.
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WO 99/14357 PCTNS98/19496
D. Antigen Production. F-12 minus medium G supplemented with 5 N.M
MTX is overlaid onto just confluent monolayers for 12 to 24 hr at
37°C in 5%
CO2. The growth medium is removed and the cells are rinsed 3 times with
Dulbecco's phosphate buffered saline (PBS) (with calcium and magnesium)
5 (Gibco-BRL; Grand Island, NY) to remove the remaining media/serum which may
be present. Cells then are incubated with VAS custom medium (VAS custom
formulation with L-glutamine with HEPES without phenol red, available from
JRH Bioscience; Lenexa, KS, product number 52-08678P), for 1 hr at
37°C in 5%
CO2. Cells then are overlaid with VAS for production at 5 ml per T flask.
10 Medium is removed after seven days of incubation, retained, and then frozen
to
await purification with harvests 2, 3 and 4. The monolayers are overlaid with
VAS
for 3 more seven day harvests.
E. Analysis of Prostate. Breast or Ovary Tissue Gene PS214 Antieen
ssion. Aliquots of VAS supernatants from the cells expressing the PS214
15 protein construct are analyzed, either by SDS-polyacrylamide gel
electrophoresis
(SDS-PAGE) using standard methods and reagents known in the art (Laemmli
discontinuous gels), or by mass spectrometry.
F. Purification. Purification of the PS214 protein containing the FLAG
sequence is performed by immunoaffmity chromatography using an affinity matrix
20 comprising anti-FLAG M2 monoclonal antibody covalently attached to agarose
by
hydrazide linkage (Eastman Kodak Co., New Haven, CT). Prior to affinity
purification, protein in pooled VAS medium harvests from roller bottles is
exchanged into 50 mM Tris-HCl (pH 7.5), 150 mM NaCI buffer using a Sephadex
G-25 (Pharmacia Biotech Inc., Uppsala, Sweden) column. Protein in this buffer
25 is applied to the anti-FLAG M2 antibody affinity column. Non-binding
protein is
eluted by washing the column with 50 mM Tris-HCI (pH 7.5), 150 mM NaCI
buffer. Bound protein is eluted using an excess of FLAG peptide in 50 mM Tris-
HCI (pH 7.5), 150 mM NaCI. The excess FLAG peptide can be removed from the
purified PS214 protein by gel electrophoresis or HPLC.
30 Although plasmid 577 is utilized in this example, it is known to those
skilled in the art that other comparable expression systems, such as CMV, can
be
utilized herein with appropriate modifications in reagent and/or techniques
and are
within the skill of the ordinary artisan.
The largest cloned insert containing the coding region of the PS214 gene is
35 then sub-cloned into either (i) a eukaryotic expression vector which may
contain,
for example, a cytomegalovirus (CMV) promoter and/or protein fusible sequences
which aid in protein expression and detection, or (ii) a bacterial expression
vector
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
containing a superoxide-dismutase (SOD) and CMP-KDO synthetase (CKS) or
other protein fusion gene for expression of the protein sequence. Methods and
vectors which are useful for the production of polypeptides which contain
fusion
sequences of SOD are described in EPO 0196056, published October 1, 1986, and
5 those containing fusion sequences of CKS are described in EPO Publication
No.
0331961, published September 13, 1989. This so-purified protein can be used in
a variety of techniques, including, but not limited to animal immunization
studies,
solid phase immunoassays, etc.
Example 1 lb: Expression of Protein in a Cell Line Llsing pcDNA3 1/Myc His
A. Construction of a PS214 Expression Plasmid. Plasmid
pcDNA3.1/Myc-His (Cat.# V855-20, Invitrogen, Carlsbad, CA) has been
constructed, in the past, for the expression of secreted antigens by most
mammalian cell lines. Expressed protein inserts are fused to a myc-his peptide
tag.
15 The myc-his tag is a 21 residue amino acid sequence having the following
sequence: Glu-Gln-Lys-Leu-Ile-Ser-Glu- Glu-Asp-Leu-Asn-Met-His-Thr-Glu-
His-His-His-His-His-His (SEQUENCE m NO 34) and comprises a c-myc
oncoprotein epitope and a polyhistidine sequence which are useful for the
purification of an expressed fusion protein by using either anti-myc or anti-
his
affinity columns, or metalloprotein binding columns.
Plasmids for the expression of secretable PS214 proteins are constructed
by inserting a PS214 polynucleotide sequence selected from the group
consisting
of SEQUENCE ID NOS 1-9, and fragments or complements thereof. Prior to
construction of a PS214 expression plasmid, the PS214 cDNA sequence is first
cloned into a pCR~-Blunt vector as follows:
The PS214 cDNA fragment is generated by PCR using standard
procedures. For example, PCR is performed procedures and reagents from
Stratagene~, Inc. (La Jolla, CA), as directed by the manufacturer. PCR primers
are used at a final concentration of 0.5 pM. PCR using 5 U of pfu polymerise
30 (Stratagene, La Jolla, CA) is performed on the PS214 plasmid template (see
Example 2) in a SO ~,l reaction for 30 cycles (94°C, 1 min;
65°C, 1.5 min; 72°C, 3
min) followed by an extension cycle of 72°C for 8 min. {The sense PCR
primer
sequence comprises nucleotides which are either complementary to the pINCY
vector directly upstream of the PS214 gene insert or which incorporate a 5'
EcoRI
35 restriction site, an adjacent downstream protein translation consensus
initiator, and
a 3' nucleic acid sequence which is the same sense as the 5'-most end of the
PS214
cDNA insert. The antisense PCR primer incorporates a 5' NotI restriction
76
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
sequence and a sequence complementary to the 3' end of the PS214 cDNA insert
just upstream of the 3'-most, in-frame stop codon.) Five microliters (5 ~.l)
of the
resulting blunted-ended PCR product are ligated into 25 ng of linearized pCR~-
Blunt vector (Invitrogen, Carlsbad, CA) interrupting the lethal ccdB gene of
the
5 vector. The resulting ligated vector is transformed into TOP10 E ~
(Invitrogen,
Carlsbad, CA) using a One ShotT"' Transformation Kit (Invitrogen, Carlsbad,
CA)
following manufacturer's instructions. The transformed cells are grown on LB-
Kan (50 p,g/ml kanamycin) selection plates at 37°C. Only cells
containing a
plasmid with an interrupted ccdB gene will grow after transformation [Grant,
10 S.G.N., Proc. Natl. Acad Sci USA 87:4645-4649 (1990)). Transformed
colonies are picked and grown up in 3 ml of LB-Kan broth at 37°C.
Plasmid DNA
is isolated by using a QIAprep~ (Qiagen Inc., Santa Clarita, CA) procedure, as
directed by the manufacturer. The DNA is cut with EcoRI or SnaBI, and NotI
restriction enzymes to release the PS214 insert fragment. The fragment is run
on
15 1 % Seakem~ LE agarose/0.5 p.g/ml ethidium bronvdeftE gel, visualized by UV
irradiation, excised and purified using QIAquickT"' (Qiagen Inc., Santa
Clarita,
CA) procedures, as directed by the supplier's instructions.
The pcDNA3.1/Myc-His plasmid DNA is linearized by digestion with
EcoRI or SnaBI, and NotI in the polylinker region of the plasmid DNA. The
20 resulting plasmid DNA backbone allows insertion of the PS214 purified cDNA
fragment, supra, downstream of a CMV promoter which directs expression of the
proteins in mammalian cells. The ligated plasmid is transformed into DHS
alpha~'~"''
cells (GibcoBRL Grand Island, NY), as directed by the manufacturer. Briefly,
10
ng of pcDNA3.1/Myc-His containing a PS214 insert are added to 50 pl of
25 competent DHS alpha cells, and the contents are mixed gently. The mixture
is
incubated on ice for 30 min, heat shocked for 20 sec at 37°C, and
placed on ice for
an additional 2 min. Upon addition of 0.95 ml of LB medium, the mixture is
incubated for 1 hr at 37°C while shaking at 225 rpm. The transformed
cells then are
plated onto 100 mm LB/Amp (SOpg/ml ampicillin) plates and grown at
37°C.
30 Colonies are picked and grown in 3 ml of LB/Amp broth. Plasmid DNA is
purified using a QIAprep Kit. The presence of the insert is confirmed using
techniques known to those skilled in the art, including, but not limited to
restriction
digestion and gel analysis. (J. Sambrook et al., ra.)
B. Transfection of Human Embryonic Kidney Cell 293 Cells. The PS214
35 expression plasmid described in section A, supra, is retransformed into DHS
alpha
cells, plated onto LB/ampicillin agar, and grown up in 10 ml of LB/ampicillin
broth, as described hereinabove. The plasmid is purified using a QIAfilter~
Maxi
77
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
Kit (Qiagen, Chatsworth, CA) and is transfected into HEK293 cells [F.L. Graham
et al., J. Gen. Vir. 36:59-72 (1977)]. These cells are available from the
A.T.C.C.,
12301 Parkiawn Drive, Rockville, MD 20852, under Accession No. CRL 1573.
Transfection is performed using the cationic Iipofectamine-mediated procedure
described by P. Hawley-Nelson et al., Focus 15:73 (1993). Particularly, HEK293
cells are cultured in 10 ml DMEM media supplemented with 10% fetal bovine
serum (FBS), L-glutamine (2 mM) and freshly seeded into 100 mm culture plates
at a density of 9 x 106 cells per plate. The cells are grown at 37 °C
to a confluency
of between 70% and 80% for transfection. Eight micrograms (8 p,g) of plasmid
10 DNA are added to 800 p,l of Opti-MEM I~ medium (Gibco-BRL, Grand Island,
NY), and 48-96 ~.l of Lipofectamine'~'' Reagent (Gibco-BRL, Grand Island, NY)
are added to a second 800 ~,l portion of Opti-MEM I media. The two solutions
are
mixed and incubated at room temperature for 15-30 min. After the culture
medium
is removed from the cells, the cells are washed once with 10 ml of serum-free
15 DMEM. The Opti-MEM I-Lipofectamine-plasmid DNA solution is diluted with
6.4 ml of serum-free DMEM and then overlaid onto the cells. The cells are
incubated for 5 hr at 37°C, after which time, an additional 8 ml of
DMEM with
20% FBS are added. After 18-24 hr, the old medium is aspirated, and the cells
are
overlaid with 5 ml of fresh DMEM with 5% FBS. Supernatants and cell extracts
20 are analyzed for PS214 gene activity 72 hr after transfection.
C. Analvsis of Prostate Breast or Ovay Tissue Gene PS214 Antigen
x r 'fin. The culture supernatant, sera, is transferred to cryotubes and
stored
on ice. HEK293 cells are harvested by washing twice with 10 ml of cold
Dulbecco's PBS and lysing by addition of 1.5 ml of CAT lysis buffer
(Boehringer
25 Mannheim, Indianapolis,1N), followed by incubation for 30 min at room
temperature. Lysate is transferred to 1.7 ml polypropylene microfuge tubes and
centrifuged at 1000 x g for 10 min. The supernatant is transferred to new
cryotubes and stored on ice. Aliquots of supernatants from the cells and the
lysate
of the cells expressing the PS214 protein construct are analyzed for the
presence of
30 PS214 recombinant protein. The aliquots can be run on SDS-polyacrylamide
gel
electrophoresis (SDS-PAGE) using standard methods and reagents known in the
art. (J. Sambrook et al., supra) These gels can then be blotted onto a solid
medium such as nitrocellulose, nytran, etc., and the PS214 protein band can be
visualized using Western blotting techniques with anti-myc epitope or anti-
histidine
35 monoclonal antibodies (Invitrogen, Carlsbad, CA) or anti-PS214 polyclonal
serum
(see Example 14). Alternatively, the expressed PS214 recombinant protein can
be
analyzed by mass spectrometry (see Example 12).
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CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
D. Purification. Purification of the PS214 recombinant protein containing
the myc-his sequence is performed using the Xpress~ affinity chromatography
system (Invitrogen, Carlsbad, CA) containing a nickel-charged agarose resin
which specifically binds polyhistidine residues. Supernatants from 10 x 100 mm
5 plates, prepared as described supra, are pooled and passed over the nickel-
charged
column. Non-binding protein is eluted by washing the column with 50 mM Tris-
HCl (pH 7.5)/150 mM NaCI buffer, leaving only the myc-his fusion proteins.
Bound PS214 recombinant protein then is eluted from the column using either an
excess of imidazole or histidine, or a low pH buffer. Alternatively, the
10 recombinant protein can also be purified by binding at the myc-his sequence
to an
affinity column consisting of either anti-myc or anti-histidine monoclonal
antibodies conjugated through a hydrazide or other linkage to an agarose resin
and
eluting with an excess of myc peptide or histidine, respectively.
The purified recombinant protein can then be covalently cross-linked to a
15 solid phase, such as N-hydroxysuccinimide-activated sepharose columns
(Pharmacia Biotech, Piscataway, NJ), as directed by supplier's instructions.
These columns containing covalently linked PS214 recombinant protein, can then
be used to purify anti-PS214 antibodies from rabbit or mouse sera (see
Examples
13 and 14).
20 E. oa ing Microtiter Plates with PS214 Expressed Proteins Supernatant
from a 100 mm plate, as described , is diluted in an appropriate volume of
PBS. Then, 100 pl of the resulting mixture is placed into each well of a
Reacti-
Bind~ metal chelate microtiter plate (Pierce, Rockford, IL), incubated at room
temperature while shaking, and followed by three washes with 200 ~t.l each of
PBS
25 with 0.05°lo Tween~ 20. The prepared microtiter plate can then be
used to screen
polyclonal antisera for the presence of PS214 antibodies (see Example 17).
Although pcDNA3.1/Myc-His is utilized in this example, it is known to
those skilled in the art that other comparable expression systems can be
utilized
herein with appropriate modifications in reagent and/or techniques and are
within
30 the skill of one of ordinary skill in the art. The largest cloned insert
containing the
coding region of the PS214 gene is sub-cloned into either (i) a eukaryotic
expression vector which may contain, for example, a cytomegalovirus (CMV)
promoter and/or protein fusible sequences which aid in protein expression and
detection, or (ii) a bacterial expression vector containing a superoxide-
dismutase
35 (SOD) and CMP-KDO synthetase (CKS) or other protein fusion gene for
expression of the protein sequence. Methods and vectors which are useful for
the
production of polypeptides which contain fusion sequences of SOD are described
79
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
in published EPO application No. EP 0 196 056, published October l, 1986, an_d
vectors containing fusion sequences of CKS are described in published EPO
application No. EP 0 331 961, published September 13, 1989. The purified
protein can be used in a variety of techniques, including, but not limited to
animal
immunization studies, solid phase immunoassays, etc.
Example 12: Chemical Analysis of Prostate Breast or Ovary Tissue Proteins
A. Analysis of Try tiD c Perztide Fragmen' is Using,~S. Sera from patients
with prostate, breast, or ovary diseases, such as prostate, breast, or ovarian
10 cancer, sera from patients with no prostate, breast, or ovary diseases,
extracts of
prostate, breast, or ovary tissues or cells from patients with prostate,
breast, or
ovary diseases, such as prostate, breast, or ovarian cancer, extracts of
prostate,
breast, or ovary tissues or cells from patients with no prostate, breast, or
ovary
diseases, and extracts of tissues or cells from other non-diseased or diseased
15 organs of patients, are run on a polyacrylamide gel using standard
procedures and
stained with Coomassie Blue. Sections of the gel suspected of containing the.
unknown polypeptide are excised and subjected to an in-gel reduction,
acetamidation and tryptic digestion. P. Jeno et al., pal io. 224:451-455
( 1995) and J. Rosenfeld et al., al io. 203:173-179 ( 1992). The gel sections
20 are washed with 100 mM NH4HC0~ and acetonitrile. The shrunken gel pieces
are
swollen in digestion buffer (50 mM NH4HCOj, 5 mM CaCl2 and 12.5 p,g/ml
trypsin) at 4°C for 45 min. The supernatant is aspirated and replaced
with 5 to 10
p,l of digestion buffer without trypsin and allowed to incubate overnight at
37°C.
Peptides are extracted with 3 changes of 5% formic acid and acetonitrile and
25 evaporated to dryness. The peptides are adsorbed to approximately 0.1 ~tl
of
POROS R2 sorbent (Perseptive Biosystems, Framingham, Massachusetts) trapped
in the tip of a drawn gas chromatography capillary tube by dissolving them in
10 pl
of 5% formic acid and passing it through the capillary. The adsorbed peptides
are
washed with water and eluted with 5% formic acid in 60% methanol. The eluant
is
30 passed directly into the spraying capillary of an API III mass spectrometer
(Perkin-
Elmer Sciex, Thornhill, Ontario, Canada) for analysis by nano-electrospray
mass
spectrometry. M. Wilm et al., Int J Mass Spectrom Ion Process 136:167-180
( 1994) and M. Wilm et al., Anal. Chem. 66:1-8 ( 1994). The masses of the
tryptic
peptides are determined from the mass spectrum obtained off the first
quadrupole.
35 Masses corresponding to predicted peptides can be further analyzed in MS/MS
mode to give the amino acid sequence of the peptide.
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B. Pe tide Fragment Analysis Using C/M, . The presence of
polypeptides predicted from mRNA sequences found in hyperplastic disease
tissues also can be confirmed using liquid chromatography/tandem mass
spectrometry (LC/MS/MS). D. Hess et al., METHODS. A Companion to
5 Methods in Enzymology 6:227-238 (1994). The senam specimen or tumor extract
from the patient is denatured with SDS and reduced with dithiothreitol (1.5
mg/ml)
for 30 min at 90°C followed by alkylation with iodoacetamide (4 mg/ml)
for I S min
at 25°C. Following acrylamide electrophoresis, the polypeptides are
electroblotted
to a cationic membrane and stained with Coomassie Blue. Following staining,
the
10 membranes are washed and sections thought to contain the unknown
polypeptides
are cut out and dissected into small pieces. The membranes are placed in S00
~.1
microcentrifuge tubes and immersed in 10 to 20 ~.1 of proteolytic digestion
buffer
( 100 mM Tris-HCI, pH 8.2, containing 0.1 M NaCI, 10% acetonitrile, 2 mM
CaCh and 5 ~.g/ml trypsin) (Sigma, St. Louis, MO). After IS hr at 37°C,
3 ~.1 of
15 saturated urea and 1 ~,1 of 100 p,g/ml trypsin are added and incubated for
an
additional 5 hr at 37°C. The digestion mixture is acidified with 3 ~,1
of 10%
trifluoroacetic acid and centrifuged to separate supernatant from membrane.
The
supernatant is injected directly onto a microbore, reverse phase HPLC column
and
eluted with a linear gradient of acetonitrile in 0.05% trifluoroacetic acid.
The eluate
20 is fed directly into an electrospray mass spectrometer, after passing
though a
stream sputter if necessary to adjust the volume of material. The data is
analyzed
following the procedures set forth in Example 12, Section A.
Example 13: Gene Immunization Protocol
25 A. In Vivo Antigen Ex ression. Gene immunization circumvents protein
purification steps by directly expressing an antigen in vivo after inoculation
of the
appropriate expression vector. Also, production of antigen by this method may
allow correct protein folding and glycosylation since the protein is produced
in
mammalian tissue. The method utilizes insertion of the gene sequence into a
30 plasmid which contains a CMV promoter, expansion and purification of the
plasmid and injection of the plasmid DNA into the muscle tissue of an animal.
Preferred animals include mice and rabbits. See, for example, H. Davis et al.,
Human Molecular Genetics 2:1847-1851 ( 1993). After one or two booster
immunizations, the animal can then be bled, ascites fluid collected, or the
animal's
35 spleen can be harvested for production of hybridomas.
B. Plasmid Pret~aration and Purification. PS214 cDNA sequences are
generated from the PS214 cDNA-containing vector using appropriate PCR primers
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containing suitable 5' restriction sites following the procedures described in
Example 11. The PCR product is cut with appropriate restriction enzymes and
inserted into a vector which contains the CMV promoter (for example, pRc/CMV
or pcDNA3 vectors from Invitrogen, San Diego, CA). This plasmid then is
5 expanded in the appropriate bacterial strain and purified from the cell
lysate using a
CsCI gradient or a Qiagen plasmid DNA purification column. All these
techniques
are familiar to one of ordinary skill in the art of molecular biology.
C. Immunization Protocol. Anesthetized animals are immunized
intramuscularly with 0.1-100 pg of the purified plasmid diluted in PBS or
other
DNA uptake enhancers (Cardiotoxin, 25% sucrose). See, for example, H. Davis
et al., Human Gene Theranv_ 4:733-740 (1993); and P. W. Wolff et al.,
Biotec, hniaues 11:474-485 (1991 ). One to two booster injections are given at
monthly intervals.
D. Testing_and Use of Antitenim. Animals are bled and the resultant sera
tested for antibody using peptides synthesized from the known gene sequence
(see
Example 16) using techniques known in the art, such as Western blotting or EIA
techniques. Antisera produced by this method can then be used to detect the
presence of the antigen in a patient's tissue or cell extract or in a
patient's serum by
ELISA or Western blotting techniques, such as those described in Examples 15
through 18.
Example 14: Production of Antibodies Against PS214
A. Production of Polyclonal Antisera Antiserum against PS214 was
prepared by injecting rabbits with peptides whose sequences were derived from
that of the predicted amino acid sequence of the PS214 consensus nucleotide
sequence (SEQUENCE ID NO 9). The synthesis of peptides (SEQUENCE ID
NO 30, SEQUENCE B7 NO 31, and SEQUENCE ID NO 32) is described in
Example 10. Peptides used as immunogens were not conjugated to a carrier such
30 as keyhole limpet hemocyanine, KLH, (i.e., they were unconjugated.).
Animal Immunization. Female white New Zealand rabbits
weighing 2 kg or more were used for raising polyclonal antiserum. One animal
was immunized per unconjugated peptide (SEQUENCE m NO 30, SEQUENCE
ID NO 31, and SEQUENCE B7 NO 32). One week prior to the first
immunization, 5 to 10 ml of blood were obtained from the animal to serve as a
non-immune prebleed sample.
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Unconjugated PS214 peptides, SEQUENCE ID NO 30, SEQUENCE B7
NO 31, and SEQUENCE ID NO 32, were used to prepare the primary immunogen
by emulsifying 0.5 ml of the peptide at a concentration of 2 mg/ml in PBS (pH
7.2) which contained 0.5 ml of complete Freund's adjuvant (CFA) (Difco,
Detroit,
5 MI). The immunogen was injected into several sites of the animal via
subcutaneous, intraperitoneal, and intramuscular routes of administration.
Four
weeks following the primary immunization, a booster immunization was
administered. The immunogen used for the booster immunization dose was
prepared by emulsifying 0.5 ml of the same unconjugated peptide used for the
10 primary immunogen, except that the peptide now was diluted to 1 mg/ml with
0.5
ml of incomplete Freund's adjuvant (IFA) (Difco, Detroit, MI). Again, the
booster
dose was administered into several sites via subcutaneous, intraperitoneal and
intramuscular types of injections. The animals were bled (5 ml) two weeks
after
the booster immunizations and each serum was tested for immunoreactivity to
the
15 peptide as described below. The booster and bleed schedule was repeated at
4
week intervals until an adequate titer was obtained. The titer or
concentration of
antiserum was determined by using unconjugated peptides in a microtiter EIA as
described in Example 17, below. An antibody titer of 1:500 or greater was
considered an adequate titer for further use and study.
20
Table 1 Titer of rabbit anti PS214 peptide antisera (6 week bleed)
Peptide Immunog~ Titer
SEQUENCE 1D NO 30 >62,500
SEQUENCE ID NO 31 >62,500
SEQUENCE ID NO 32 40,000
>3. Production of Monoclonal Antibodv
25 1. Immunization Protocol. Mice are immunized using peptides
which can either be conjugated to a carrier such as KLH [prepared as described
hereinbelow, or unconjugated (i.e., not conjugated to a carrier such as KLH))
except that the amount of the unconjugated or conjugated peptide for
monoclonal
antibody production in mice is one-tenth the amount used to produce polyclonal
30 antisera in rabbits. Thus, the primary immunogen consists of 100 p,g of
unconjugated or conjugated peptide in 0.1 ml of CFA emulsion while the
immunogen used for booster immunizations consists of 50 ~,g of unconjugated or
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conjugated peptide in 0.1 ml of IFA. Hybridomas for the generation of
monoclonal antibodies are prepared and screened using standard techniques. The
methods used for monoclonal antibody development follow procedures known in
the art such as those detailed in Kohler and Milstein, Na a 256:494 ( 1975)
and
S reviewed in J.G.R. Hurrel, ed., Monoclonal Hybridoma Antibodies' Techniaues
and Applications, CRC Press; Inc., Boca Raton, FL ( 1982). Another method of
monoclonal antibody development which is based on the Kohler and Milstein
method is that of L.T. Mimms et al., Vir o 176:604-619 (1990).
The immunization regimen (per mouse) consists of a primary
10 immunization with additional booster immunizations. The primary immunogen
used for the primary immunization consists of 100 pg of unconjugated or
conjugated peptide in 50 p,l of PBS (pH 7.2) previously emulsified in 50 p,l
of
CFA. Booster immunizations performed at approximately two weeks and four
weeks post primary immunization consist of 50 p.g of unconjugated or
conjugated
15 peptide in 50 p,l of PBS (pH 7.2) emulsified with 50 p.l IFA. A total of
100 p.l of
this immunogen are inoculated intraperitoneally and subcutaneously into each
mouse. Individual mice are screened for immune response by microtiter plate
enzyme immunoassay (EIA) as described in Example 17 approximately four weeks
after the third immunization. Mice are inoculated either intravenously,
20 intrasplenically or intraperitoneally with 50 pg of unconjugated or
conjugated
peptide in PBS (pH 7.2) approximately fifteen weeks after the third
immunization..
Three days after this intravenous boost, splenocytes are fused with,
for example, Sp2/0-Agl4 myeloma cells (Milstein Laboratories, England) using
the polyethylene glycol (PEG) method. The fusions are cultured in Iscove's
25 Modified Dulbecco's Medium (IMDM) containing 10% fetal calf serum (FCS),
plus 1 % hypoxanthine, aminopterin and thymidine (HAT). Bulk cultures were
screened by miciotiter plate EIA following the protocol in Example 17. Clones
reactive with the peptide used an immunogen and non-reactive with other
peptides
(i.e., peptides of PS214 not used as the immunogen) are selected for final
30 expansion. Clones thus selected are expanded, aliquoted and frozen in IMDM
containing 10% FCS and 10% dimethyl sulfoxide, (DMSO).
2. Peptide Conjugation. Peptide is conjugated to maleimide
activated KLH (commercially available as Imject~, available from Pierce
Chemical
Company, Rockford, IL). Imject° contains about 250 moles of reactive
maleinude
35 groups per mole of hemocyanine. The activated KLH is dissolved in phosphate
buffered saline (PBS, pH 8.4) at a concentration of about 7.7 mg/ml. The
peptide
is conjugated through cysteines occurnng in the peptide sequence, or to a
cysteine
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previously added to the synthesized peptide in order to provide a point of
attachment. The peptide is dissolved in DMSO (Sigma Chemical Company, St.
Louis, MO) and reacted with the activated KLH at a mole ratio of about 1.5
moles
of peptide per mole of reactive maleimide attached to the KLH. A procedure for
the conjugation of peptide is provided hereinbelow. It is known to the
ordinary
artisan that the amounts, times and conditions of such a procedure can be
varied to
optimize peptide conjugation.
The conjugation reaction described hereinbelow is based on
obtaining 3 mg of KLH peptide conjugate {"conjugated peptide"), which contains
about 0.77 pmoles of reactive maleimide groups. This quantity of peptide
conjugate usually is adequate for one primary injection and four booster
injections
for production of polyclonal antisera in a rabbit. Briefly, peptide is
dissolved in
DMSO at a concentration of 1.16 p.moles/100 ~.1 of DMSO. One hundred
microliters ( 100 p.l) of the DMSO solution are added to 380 p,l of the
activated
15 KLH solution prepared as described hereinabove, and 20 p,l of PBS (pH 8.4)
are
added to bring the volume to S00 pl. The reaction is incubated overnight at
room
temperature with stirring. The extent of reaction is determined by measuring
the
amount of unreacted thiol in the reaction mixture. The difference between the
starting concentration of thiol and the final concentration is assumed to be
the
20 concentration of peptide which has coupled to the activated KLH. The amount
of
remaining thiol is measured using Ellman's reagent (5,5'-dithiobis(2-
nitrobenzoic
acid), Pierce Chemical Company, Rockford, IL). Cysteine standards are made at
a
concentration of 0, 0.1, 0.5, 2, 5 and 20 mM by dissolving 35 mg of cysteine
HCl
(Pierce Chemical Company, Rockford, IL) in 10 ml of PBS (pH 7.2) and diluting
25 the stock solution to the desired concentration(s). The photometric
determination
of the concentration of thiol is accomplished by placing 200 p.l of PBS (pH
8.4) in
each well of an Immulon 2~ microwell plate (Dynex Technologies, Chantilly,
VA).
Next, 10 p.l of standard or reaction mixture are added to each well. Finally,
20 p.l
of Ellman's reagent at a concentration of 1 mg/ml in PBS (pH 8.4) are added to
30 each well. The wells are incubated for 10 minutes at room temperature, and
the
absorbance of all wells is read at 415 nm with a microplate reader (such as
the
BioRad Model 3550, BioRad, Richmond, CA). The absorbance of the standards
is used to construct a standard curve and the thiol concentration of the
reaction
mixture is determined from the standard curve. A decrease in the concentration
of
35 free thiol is indicative of a successful conjugation reaction. Unreacted
peptide is
removed by dialysis against PBS (pH 7.2) at room temperature for 6 hours. The
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conjugate is stored at 2-8°C if it is to be used immediately;
otherwise, it is stored at
-20°C or colder.
3. Production of Ascites Fluid Containing Monoclonal Antibodies.
Frozen hybridoma cells prepared as described hereinabove are thawed and placed
into expansion culture. Viable hybridoma cells are inoculated
intraperitoneally into
Pristane treated mice. Ascitic fluid is removed from the mice, pooled,
filtered
through a 0.2 ~t filter and subjected to an immunoglobulin class G (IgG)
analysis
to determine the volume of the Protein A column required for the purification.
4. Purification of Monoclonal Antibodies From Ascites Fluid.
10 Briefly, filtered and thawed ascites fluid is mixed with an equal volume of
Protein
A sepharose binding buffer (1.5 M glycine, 3.0 M NaCI, pH 8.9) and refiltered
through a 0.2 p, filter. The volume of the Protein A column is determined by
the
quantity of IgG present in the ascites fluid. The eluate then is dialyzed
against PBS
(pH 7.2) overnight at 2-8°C. The dialyzed monoclonal antibody is
sterile filtered
15 and dispensed in aliquots. The immunoreactivity of the purified monoclonal
antibody is confirmed by determining its ability to specifically bind to the
peptide
used as the immunogen by use of the EIA microtiter plate assay procedure of
Example 17. The specificity of the purified monoclonal antibody is confirmed
by
determining its lack of binding to irrelevant peptides such as peptides of
PS214 not
20 used as the immunogen. The purified anti-PS214 monoclonal thus prepared and
characterized is placed at either 2-8°C for short term storage or at -
80°C for long
term storage.
5. Further Characterization of Monoclonal Antibody. The isotype
and subtype of the monoclonal antibody produced as described hereinabove can
be
25 determined using commercially available kits (available from Amersham.
Inc.,
Arlington Heights, II,). Stability testing also can be performed on the
monoclonal
antibody by placing an aliquot of the monoclonal antibody in continuous
storage at
2-8°C and assaying optical density (OD) readings throughout the course
of a given
period of time.
30 ~ Use of Recombinant Proteins as ImmunoQens It is within the scope of
the present invention that recombinant proteins made as described herein can
be
utilized as immunogens in the production of polyclonal and monoclonal
antibodies,
with corresponding changes in reagents and techniques known to those skilled
in
the art.
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Example 15: Purification of Serum Antibodies Which Specif callvcallv
Bind to PS214 Pe tp ides
5 Immune sera, obtained as described hereinabove in Examples 13 andlor
14, is affinity purified using immobilized synthetic peptides prepared as
described
in Example 10, or recombinant proteins prepared as described in Example 1 I .
An
IgG fraction of the antiserum is obtained by passing the diluted, crude
antiserum
over a Protein A column (Affi-Gel protein A, Bio-Rad, Hercules, CA). Elution
10 with a buffer (Binding Buffer, supplied by the manufacturer) removes
substantially
all proteins that are not immunoglobulins. Elution with O.IM buffered glycine
(pH
3) gives an immunoglobulin preparation that is substantially free of albumin
and
other serum proteins.
Immunoaffinity chromatography is performed to obtain a preparation with a
I S higher fraction of specific antigen-binding antibody. The peptide used to
raise the
antiserum is immobilized on a chromatography resin, and the specific
antibodies
directed against its epitopes are adsorbed to the resin. After washing away
non-
binding components, the specific antibodies are eluted with 0.1 M glycine
buffer,
pH 2.3. Antibody fractions are immediately neutralized with 1.0 M Tris buffer
20 (pH 8.0) to preserve immunoreactivity. The chromatography resin chosen
depends on the reactive groups present in the peptide. If the peptide has an
amino
group, a resin such as Affi-Gel 10 or Affi-Gel 15 is used (Bio-Rad, Hercules,
CA). If coupling through a carboxy group on the peptide is desired, Affi-Gel
102
can be used (Bio-Rad, Hercules, CA). If the peptide has a free sulfhydryl
group,
25 an organomercurial resin such as Affi-Gel 501 can be used (Bio-Rad,
Hercules,
CA).
Alternatively, spleens can be harvested and used in the production of
hybridomas to produce monoclonal antibodies following routine methods known
in the art as described hereinabove.
Example 16: Western Blotting of Tissue Samples
Protein extracts were prepared by homogenizing tissue samples in 0.1 M
Tris-HCl (pH 7.5), 15% (w/v) glycerol, 0.2 mM EDTA, I.0 mM 1,4-
dithiothreitol, 10 p.g/ml leupeptin and 1.0 mM phenylmethylsulfonylfluoride
[S. R.
Kain et al., Biotechniques 17:982 ( 1994)]. Following homogenization, the
homogenates were centrifuged at 4°C for 5 minutes to separate
supernatant from
87
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WO 99/14357 PCT/US98/19496
debris. Debris was re-extracted by homogenization with a buffer that was
similar to
above but also contained 0.1 M Tricine and 0.1 % SDS. The supernatant from the
second extraction was used for Western blotting. For protein quantitation, 3-
10 p.l
of supernatant were added to 1.5 ml of bicinchoninic acid reagent (Sigma, St.
Louis, MO), and the resulting absorbance at 562 nm were measured.
For SDS-PAGE, samples were adjusted to desired protein concentration
with Tricine Buffer (Novex, San Diego, CA), mixed with an equal volume of 2X
Tricine sample buffer (Novex, San Diego, CA), and heated for 5 minutes at
100°C
in a thermal cycler. Samples were then applied to a Novex 10-20% Precast
Tricine
Gel for electrophoresis. Following electrophoresis samples were transferred
from
the gels to nitrocellulose membranes in Novex Tris-Glycine Transfer buffer.
Membranes were then probed with specific anti-peptide antibodies using the
reagents and procedures provided in the Western Lights Plus or Western Lights
(Tropix, Bedford, MA) chemiluminesence detection kits. Chemiluminesent bands
IS were visualized by exposing the developed membranes to Hyperfilm ECL
(Amersham, Arlington Heights, IL,). After visualization of the bands on film,
the
bands were also visualized directly on the membranes by the addition and
development of chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate
(BCIP). This chromogenic solution contained 0.016% BCIP, 100 mM NaCI, 5
mM MgCl2 in a 100 mM Tris-HCI, pH 9.5 buffer. The filter was incubated in the
solution at room temperature until the bands developed to the desired
intensity.
Molecular mass determination was made based upon the mobility of pre-stained
molecular weight standards (Novex, San Diego, CA) and biotinylated molecular
weight standards (Tropix, Bedford, MA).
Figure 6 shows the results of the Western blot performed on a panel of
tissue extracts using antiserum against PS214 synthetic peptide (SEQUENCE m
NO 32; see Example 14). Each lane of Figure 6 represents a different tissue
protein extract : 1, colon cancer; 2, bladder cancer; 3, lung cancer; 4-6,
breast
cancer; 7 and 8, BPH; 9-11, prostate cancer; 12, molecular weight markers. A
band near 45 kD (indicated with an arrow) was observed in all samples but was
of
lower intensities in the BPH tissues. There was a strong band just below the
45
kD marker that was pronounced in the prostate cancers (lanes 9-11) but was
weaker in the BPH tissues (lanes 7 and 8). A very weak band was also observed
in one of the three breast cancers (lane 6) while strong bands were observed
in the
other cancer tissues (colon, bladder, lung, breast; lanes 1-5, respectively).
88
_.__.~_._ _T..........-___ _. .~..._
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Example 17: EIA Microtiter,Plate Assav
The immunoreactivity of antiserum preferably obtained from rabbits as
described in Example 14 was determined by means of a microtiter plate EIA, as
follows. Briefly, synthetic peptides, SEQUENCE m NO 30, SEQUENCE ID NO
5 31, and SEQUENCE 117 NO 32, prepared as described in Example 10, were
dissolved in carbonate buffer (50 mM, pH 9.6) to a final concentration of 2
p.g/ml.
Next, 100 p.l of the peptide or protein solution were placed in each well of
an
Immulon 2~ microtiter plate (Dynex Technologies, Chantilly, VA). The plate was
incubated overnight at room temperature and then washed four times with
10 deionized water. The wells were blocked by adding 125 p.l of a suitable
protein
blocking agent, such as Superblock~ (Pierce Chemical Company, Rockford, IL),
to each well and then immediately discarding the solution. This blocking
procedure was performed three times. Antiserum obtained from immunized rabbits
or mice, prepared as previously described, was diluted in a protein blocking
agent
15 (e.g., a 3% Superblock~ solution) in PBS containing 0.05% Tween-20~
(monolaurate polyoxyethylene ether) (Sigma Chemical Company, St. Louis, MO)
and 0.05% sodium azide at dilutions of 1:100, 1:500, 1:2500, 1:12,500, and
1:62,500 and placed in each well of the coated microtiter plate. The wells
then
were incubated for three hours at room temperature. Each well was washed four
20 times with deionized water. One hundred microliters of alkaline phosphatase-
conjugated goat anti-rabbit IgG or goat anti-mouse IgG antiserum (Southern
Biotech, Birmingham, AB) diluted 1:2000 in 3% Superblock~ solution in
phosphate buffered saline containing 0.05% Tween 200 and 0.05% sodium azide,
were added to each well. The wells were incubated for two hours at room
25 temperature. Next, each well was washed four times with deionized water.
One
hundred microliters of paranitrophenyl phosphate substrate (Kirkegaard and
Perry
Laboratories, Gaithersburg, MD) then were added to each well. The wells were
incubated for thirty minutes at room temperature. The absorbance at 405 nm was
read in each well. Positive reactions were identified by an increase in
absorbance
30 at 405 nm in the test well above that absorbance given by a non-immune
serum
(negative control). A positive reaction was indicative of the presence of
detectable
anti-PS214 antibodies. Titers of the anti-peptide antisera were calculated
from the
previously described dilutions of antisera and defined as the calculated
dilution,
where A~S~m = 0.5 OD.
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Example 18: Coatine of Solid Phase Particles
A. Coating of Micronarticles with Antibodies Which S ifical~',y-Bind to
5 PS214 A~~, i en. Affinity purified antibodies which specifically bind to
PS214
protein (see Example 15) are coated onto microparticles of polystyrene,
carboxylated polystyrene, polymethylacrylate or similar particles having a
radius in
the range of about 0.1 to 20 p.m. Microparticles may be either passively or
actively
coated. One coating method comprises coating EDAC (1-(3-
10 dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride {Aldrich Chemical
Co.,
Milwaukee, WI) activated carboxylated latex microparticles with antibodies
which
specifically bind to PS214 protein, as follows. Briefly, a final 0.375% solid
suspension of resin washed carboxylated latex microparticles (available from
Bangs Laboratories, Carmel, IN or Serodyn, Indianapolis, IN) is mixed in a
15 solution containing 50 mM MES buffer, pH 4.0 and 150 mg/1 of affinity
purified
anti-PS214 antibody (see Example 14) for 15 min in an appropriate container.
EDAC coupling agent is added to a final concentration of 5.5 ~tg/ml to the
mixture
and mixed for 2.5 hr at room temperature.
The microparticles then are washed with 8 volumes of a Tween 20~/sodium
20 phosphate wash buffer (pH 7.2) by tangential flow filtration using a 0.2
p,m
Microgon Filtration module. Washed microparticles are stored in an appropriate
buffer which usually contains a dilute surfactant and irrelevant protein as a -
blocking agent, until needed.
B. Coating of 1/4 Inch Beads. Antibodies which specifically bind to
25 PS214-antigen also may be coated on the surface of 1/4 inch polystyrene
beads by
routine methods known in the art (Snitman et al., US Patent 5,273,882) and
used
in competitive binding or EIA sandwich assays.
Polystyrene beads first are cleaned by ultrasonicating them for about 15
seconds in 10 mM NaHCO, buffer at pH 8Ø The beads then are washed in
30 deionized water until all fines are removed. Beads then are immersed in an
antibody solution in 10 mM carbonate buffer, pH 8 to 9.5. The antibody
solution
can be as dilute as 1 p,g/ml in the case of high affinity monoclonal
antibodies or as
concentrated as about 500 pg/ml for polyclonal antibodies which have not been
affinity purified. Beads are coated for at least 12 hours at room temperature,
and
35 then they are washed with deionized water. Beads may be air dried or stored
wet
(in PBS, pH 7.4). They also may be overcoated with protein stabilizers (such
as
CA 02304368 2000-03-15
WO 99/14357 PCTlUS98/19496
sucrose) or protein blocking agents used as non-specific binding blockers
(such as
irrelevant proteins, Carnation skim milk, Superblock~, or the like).
Example 19' Microparticle Enzyme Immunoassay jMEIA)
5 PS214 antigens are detected in patient test samples by performing a
standard antigen competition EIA or antibody sandwich EIA and utilizing a
solid
phase such as microparticles (MEIA). The assay can be performed on an
automated analyzer such as the IMx~ Analyzer (Abbott Laboratories, Abbott
Park,
IL).
I O A. Antibody Sandwich EIA. Briefly, samples suspected of containing
PS214 antigen are incubated in the presence of anti-PS214 antibody-coated
microparticles (prepared as described in Example 17) in order to form
antigen/antibody complexes. The microparticles then are washed and an
indicator
reagent comprising an antibody conjugated to a signal generating compound
(e.g.,
15 enzymes such as alkaline phosphatase or horseradish peroxide) is added to
the
antigen/antibody complexes or the nucroparticles and incubated. The
microparticles are washed and the bound antibody/antigen/antibody complexes
are
detected by adding a substrate (e.g., 4-methyl umbelliferyl phosphate (MUP),
or
OPD/peroxide, respectively), that reacts with the signal generating compound
to
20 generate a measurable signal. An elevated signal in the test sample,
compared to
the signal generated by a negative control, detects the presence of PS214
antigen.
The presence of PS214 antigen in the test sample is indicative of a diagnosis
of a
prostate, breast, or ovary disease or condition, such as prostate, breast, or
ovarian
cancer.
25 B. Competitive Bindin Assay. The competitive binding assay uses a
peptide or protein that generates a measurable signal when the labeled peptide
is
contacted with an anti-peptide antibody coated microparticle. This assay can
be
performed on the IMx~ Analyzer (available from Abbott Laboratories, Abbott
Park,
IL,). The labeled peptide is added to the PS214 antibody-coated microparticles
30 (prepared as described in Example 17) in the presence of a test sample
suspected of
containing PS214 antigen, and incubated for a time and under conditions
sufficient
to form labeled PS214 peptide (or labeled protein) / bound antibody complexes
and/or patient PS214 antigen / bound antibody complexes. The PS214 antigen in
the test sample competes with the labeled PS214 peptide (or PS214 protein) for
35 binding sites on the microparticle. PS214 antigen in the test sample
results in a
lowered binding of labeled peptide and antibody coated microparticles in the
assay
since antigen in the test sample and the PS214 peptide or PS214 protein
compete
91
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for antibody binding sites. A lowered signal (compared to a control} indicates
the
presence of PS214 antigen in the test sample. The presence of PS214 antigen
suggests the diagnosis of a prostate, breast, or ovary disease or condition,
such as
prostate, breast, or ovarian cancer.
5 The PS214 polynucleotides and the proteins encoded thereby which are
provided and discussed hereinabove are useful as markers of prostate, breast,
or
ovary tissue disease, especially prostate, breast, or ovarian cancer. Tests
based
upon the appearance of this marker in a test sample such as blood, plasma or
serum
can provide low cost, non-invasive, diagnostic information to aid the
physician to
10 make a diagnosis of cancer, to help select a therapy protocol, or to
monitor the
success of a chosen therapy. This marker may appear in readily accessible body
fluids such as blood, urine or stool as antigens derived from the diseased
tissue
which are detectable by immunological methods. This marker may be elevated in
a
disease state, altered in a disease state, or be a normal protein of the
prostate,
1 S breast, or ovary which appears in an inappropriate body compartment.
Example 20: Immunohistochemical Detection of PS214 Protein
Antiserum against a PS214 synthetic peptide derived from the consensus
peptide sequence (SEQUENCE ID NO 29) described in Example 14, above, is
20 used to immunohistochemically stain a variety of normal and diseased
tissues using
standard procedures. Briefly, frozen blocks of tissue are cut into 6 micron
sections, and placed on microscope slides. After fixation in cold acetone, the
sections are dried at room temperature, then washed with phosphate buffered
saline
and blocked. The slides are incubated with the antiserum against a synthetic
25 peptide derived from the consensus PS214 peptide sequence (SEQUENCE )D NO
29) at a dilution of 1:500, washed, incubated with biotinylated goat anti-
rabbit
antibody, washed again, and incubated with avidin labeled with horseradish
peroxidase. After a final wash, the slides are incubated with 3-amino-9-
ethylcarbazole substrate which gives a red stain. The slides are
counterstained with
30 hematoxylin, mounted, and examined under a microscope by a pathologist.
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Sequence Listing
<110> Abbott Laboratories
<120> Reagents and Methods Useful for Detecting Diseases of the
Prostate, Breast and Ovary
<130> 6159.PC.01
<150> 08/938,383
<151> 1997-09-19
<160> 34
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 273
<212> DNA
<213> Homo sapiens
<220>
<221> base-polymorphism
<222> 48
<223> /note = "'n' represents an a or g or t or c polymorphism at
this position
<400> 1
ggcggtcaac gggctatgct ggcttgacag ggctgggntc ttcagaacag aagcatggat 60
ctcggaatcc ctgacctgct ggacgcgtgg ctggagcccc cagaggatat cttctcgaca 120
ggatccgtcc tggagctggg actccactgc ccccctccag aggttccggt aactaggcta 180
caggaacagg gactgcaagg ctggaagtcc ggtggggacc gtggctgtgg ccttcaagag 240
agtgagcctg aagatttctt gaagcttttc att 273
<210> 2
<211> 270
<212> DNA
<213> Homo sapiens
<400> 2
ctgcaaggctggaagtccggtggggaccgtggctgtggccttcaagagagtgagcctgaa 60
gatttcttgaagcttttcattgatcccaatgaggtgtactgctcagaagcatctcctggc 120
agtgacagtggcatctctgaggacccctgccatccagacagtccccctgcccccagggca 180
accagttctcctatgctctatgaggttgtctatgaggcaggggccctggagaggatgcag 240
ggggaaactgggccaaatgtaggccttatc 270
<210> 3
<211> 218
<212> DNA
<213> Homo sapiens
<220>
<221> base_polymorphism
<222> 86
<223> /note = "'n' represents an a or g or t or c polymorphism at
this position
<400> 3
gaagatttct tgaagctttt cattgatccc aatgaggtgt actgctcaga agcatctcct 60
ggcagtgaca gtggcatctc tgaggncccc tgccatccag acagtccccc tgcccccagg 120
gcaaccagtt ctcctatgct ctatgaggtt gtctatgagg caggggccct ggagaggatg 180
cagggggaaa ctgggccaaa tgtaggcctt atctccat 218
1
CA 02304368 2000-03-15
WO 99/14357 PCT/US98/19496
<210> 4
<211> 329
<212> DNA
<213> Homo sapiens
<220>
<221> base~olymorphism
<222> 34
<223> /note = "'n' represents an a or g or t or c polymorphism at
this position
<400> 4
ccctgttcctgaccgatgaggagaagcgtctgcnggggcaggaaggggtttccctgccct 60
ctcacctgcccctcaccaaggcagaggagagggtcctcaagaaggtcaggaggaaaatcc 120
gtaacaagcagtcagctcaggacagtcggcggcggaagaaggagtacattgatgggctgg 180
agagcagggtggcagcctgttctgcacagaaccaagaattacagaaaaaagtccaggagc 240
tggagaggcacaacatctccttggtagctcagctccgccagctgcagacgctaattgctc 300
aaacttccaacaaagctgcccagaccagc 329
<210>5
<211>263
<212>DNA
<213>Homo sapiene
<220>
<221>base~olymorphism
<222>142
<223>/note = "'n' representsan a or g or t or c polymorphism
at
this position
<220>
<221>base_polymorphism
<222>215
<223>/note = "'n' representsan a or g or t or c polymorphism
at
this position
<220>
<221>base~olymorphism
<222>226
<223>/note = "'n' representsan a or g or t or c polymorphism
at
this position
<220>
<221>baserpolymorphism
<222>261
<223>/note = "'n' representsan a or g or t or c polymorphism
at
this position
<400> 5
gctgcccagaccagcacttgtgttttgattcttcttttttccctggctctcatcatcctg60
cccagcttcagtccattccagagtcgaccagaagctgggtctgaggattaccagcctcac120
ggagtgacttccagaaatatcntgacccacaaggacgtaacagaaaatctggagacccaa180
gtggtagagtccagactgagggagccacctggagncaaggatgcanatggctcaacaagg240
acatgcttgagaagatgggangg 263
<210> 6
<211> 234
<212> DNA
<213> Homo sapiens
<400> 6
caaggacgta acagaaaatc tggagaccca agtggtagag tccagactga gggagccacc 60
tggagccaag gatgcaaatg gctcaacaag gacactgctt gagaagatgg gagggaagcc 120
aagacccagt gggcgcatcc ggtccgtgct gcatgcagat gagatgtgag ctggaacaga 180
ccttcctggc ccacttcctg atcacaagga atcctgggct tccttatggc tttg 234
<210> 7
2
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WO 99/14357 PCTNS98/19496
<211> 426
<212> DNA
<213> Homo sapiens
<400> 7
tttttttttttttacaatttcagctaaaacgtttatttctcactcaaaacttatttcccc 60
tcaaccctatacccaaagaagaaataaaatcacagatacataacagaagtatttgaggta 120
ccctctcatatatgcaaacaaatgcagactaggcctcaggcagagactaaaggacatctc 180
ttggggtgtcctgaagtgatttggacccctgagggcagacacctaagtaggaatcccagt 240
gggaagcaaagccataaggaagcccaggattccttgtgatcaggaagtgggccaggaagg 300
tctgttccagctcacatctcatctgcatgcagcacggaccggatgcgcccactgggtctt 360
ggcttccctcccatcttctcaagcagtgtccttgttgagccatttgcatccttggctcca 420
ggtggc
426
<210> 8
<211> 1486
<212> DNA
<213> Homo sapiens
<220>
<400> B
ggcggtcaacgggctatgctggcttgacagggctgggctcttcagaacagaagcatggat60
ctcggaatccctgacctgctggacgcgtggctggagcccccagaggatatcttctcgaca120
ggatccgtcctggagctgggactccactgcccccctccagaggttccggtaactaggcta180
caggaacagggactgcaaggctggaagtccggtggggaccgtggctgtggccttcaagag240
agtgagcctgaagatttcttgaagcttttcattgatcccaatgaggtgtactgctcagaa300
gcatctcctggcagtgacagtggcatctctgaggactcctgccatccagacagtccccct360
gcccccagggcaaccagttctcctatgctctatgaggttgtctatgaggcaggggccctg420
gagaggatgcagggggaaactgggccaaatgtaggccttatctccatccagctagatcag480
tggagcccagcatttatggtgcctgattcctgcatggtcagtgagctgccctttgatgct540
catgcccacatcctgcccagagcaggcaccgtagccccagtgccctgtacaaccctgctg600
ccctgtcaaaccctgttcctgaccgatgaggagaagcgtctgctggggcaggaaggggtt660
tccctgccctctcacctgcccctcaccaaggcagaggagagggtcctcaagaaggtcagg720
aggaaaatccgtaacaagcagtcagctcaggacagtcggcggcggaagaaggagtacatt780
gatgggctggagagcagggtggcagcctgttctgcacagaaccaagaattacagaaaaaa840
gtccaggagctggagaggcacaacatctccttggtagctcagctccgccagctgcagacg900
ctaattgctcaaacttccaacaaagctgcccagaccagcacttgtgttttgattcttctt960
ttttccctggctctcatcatcctgcccagcttcagtccattccagagtcgaccagaagct1020
gggtctgaggattaccagcctcacggagtgacttccagaaatatcctgacccacaaggac1080
gtaacagaaaatctggagacccaagtggtagagtccagactgagggagccacctggagcc1140
aaggatgcaaatggctcaacaaggacactgcttgagaagatgggagggaagccaagaccc1200
agtgggcgcatccggtccgtgctgcatgcagatgagatgtgagctggaacagaccttcct1260
ggcccacttcctgatcacaaggaatcctgggcttccttatggctttgcttcccactggga1320
ttcctacttaggtgtctgccctcaggggtccaaatcacttcaggacaccccaagagatgt1380
cctttagtctctgcctgaggcctagtctgcatttgtttgcatatatgagagggtacctca1440
aatacttctgttatgtatctgtgattttatttcttctttgggtata 1486
<210> 9
<211> 1553
<212> DNA
<213> Homo Sapiens
<400>
9
ggcggtcaacgggctatgctggcttgacagggctgggctcttcagaacagaagcatggat 60
ctcggaatccctgacctgctggacgcgtggctggagcccccagaggatatcttctcgaca 120
ggatccgtcctggagctgggactccactgcccccctccagaggttccggtaactaggcta 180
caggaacagggactgcaaggctggaagtccggtggggaccgtggctgtggccttcaagag 240
agtgagcctgaagatttcttgaagcttttcattgatcccaatgaggtgtactgctcagaa 300
gcatctcctggcagtgacagtggcatctctgaggacccctgccatccagacagtccccct 360
gcccccagggcaaccagttctcctatgctctatgaggttgtctatgaggcaggggccctg 420
gagaggatgcagggggaaactgggccaaatgtaggccttatctccatccagctagatcag 480
tggagcccagcatttatggtgcctgattcctgcatggtcagtgagctgccctttgatgct 540
catgcccacatcctgcccagagcaggcaccgtagccccagtgccctgtacaaccctgctg 600
ccctgtcaaaccctgttcctgaccgatgaggagaagcgtctgctggggcaggaaggggtt 660
tccctgccctctcacctgcccctcaccaaggcagaggagagggtcctcaagaaggtcagg 720
aggaaaatccgtaacaagcagtcagctcaggacagtcggcggcggaagaaggagtacatt 780
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WO 99/14357 PCT/US98/19496
gatgggctggagagcagggtggcagcctgttctgcacagaaccaagaattacagaaaaaa840
gtccaggagctggagaggcacaacatctccttggtagctcagctccgccagctgcagacg900
ctaattgctcaaacttccaacaaagctgcccagaccagcacttgtgttttgattcttctt960
ttttccctggctctcatcatcctgcccagcttcagtccattccagagtcgaccagaagct1020
gggtctgaggattaccagcctcacggagtgacttccagaaatatcctgacccacaaggac1080
gtaacagaaaatctggagacccaagtggtagagtccagactgagggagccacctggagcc1140
aaggatgcaaatggctcaacaaggacactgcttgagaagatgggagggaagccaagaccc1200
agtgggcgcatccggtccgtgctgcatgcagatgagatgtgagctggaacagaccttcct1260
ggcccacttcctgatcacaaggaatcctgggcttccttatggctttgcttcccactggga1320
ttcctacttaggtgtctgccctcaggggtccaaatcacttcaggacaccccaagagatgt1380
cctttagtctctgcctgaggcctagtctgcatttgtttgcatatatgagagggtacctca1440
aatacttctgttatgtatctgtgattttatttcttctttgggtatagggttgaggggaaa1500
taagttttgagtgagaaataaacgttttagctgaaaaaaaaaaaaaaaaaaaa 1553
<210> 10
<211> 68
<212> DNA
<213> Artificial Sequence
<220>
<223> Restriction Site
<400> 10
agctcggaat tccgagcttg gatcctctag agcggccgcc gactagtgag ctcgtcgacc 60
cgggaatt 68
<210> 11
<211> 68
<212> DNA
<213> Artificial Sequence
<220>
<223> Restriction site
<400> 11
aattaattcc cgggtcgacg agctcactag tcggcggccg ctctagagga tccaagctcg 60
gaattccg 68
<210> 12
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Universial Primer
<400> 12
agcggataac aatttcacac agga 24
<210> 13
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Universal Primer
<400> 13
tgtaaaacga cggccagt 1B
<210> 14
<211> 24
<212> DNA
<213> Homo sapiens
<400> 14
gctctatgag gttgtctatg aggc 24
4
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WO 99/14357 PCTNS98/19496
<210> 15
<211> 20
<212> DNA
<213> Homo sapiens
<400> 15
ttcagtccat tccagagtcg 20
<210> 16
<211> 20
<212> DNA
<213> Homo sapiens
<400> 16
atccgtaaca agcagtcagc 20
<210> 17
<211> 20
<212> DNA
<213> Homo sapiens
<400> 17
atgcttctga gcagtacacc 20
<210> 18
<211> 20
<212> DNA
<213> Homo sapiens
<400> 18
tgtcctgagc tgactgcttg 20
<210> 19
<211> 21
<212> DNA
<213> Homo sapiens
<400> 19
cgactctgga atggactgaa g 21
<210> 20
<211> 26
<212> DNA
<213> Homo sapiens
<400> 20
tttttttttt tttttttttt tttttc 26
<210> 21
<211> 20
<212> DNA
<213> Homo sapiens
<400> 21
gcggaagaag gagtacattg 20
<210> 22
<211> 20
<212> DNA
<213> Homo sapiens
<400> 22
tatcctgacc cacaaggacg 20
<210> 23
<211> 20
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WO 99/14357 PCTNS98/19496
<212> DNA
<213> Homo sapiens
<400> 23
acttcaggac accccaagag 20
<210> 24
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 24
tttcccctca accctatacc 20
<210> 25
<211> 20
<212> DNA
<213> Homo sapiens
<400> 25
actccgtgag gctggtaatc 20
<210> 26
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 26
ggtcaggaac agggtttgac 20
<210> 27
<211> 22
<212> DNA
<213> Homo sapiens
<400> 27
acattgatgg gctggagagc 22
ag
<210> 28
<211> 22
<212> DNA
<213> Homo sapiens
<400> 28
tggggtgtcc tgaagtgatt 22
tg
<210> 29
<211> 395
<212> PRT
<213> Homo sapiens
<400> 29
Met Asp Leu Gly Ile AspLeu LeuAspAla TrpLeuGlu ProPro
Pro
1 5 10 15
Glu Asp Ile Phe Ser GlySer ValLeuGlu LeuGlyLeu HisCys
Thr
20 25 30
Pro Pro Pro Glu Val ValThr ArgLeuGln GluGlnGly LeuGln
Pro
35 40 45
Gly Trp Lys Ser Gly AspArg GlyCysGly LeuGlnGlu SerGlu
Gly
50 55 60
Pro Glu Asp Phe Leu LeuPhe IleAspPro AsnGluVal TyrCys
Lys
65 70 75 80
Ser Glu Ala Ser Pro SerAsp SerGlyIle SerGluAsp SerCys
Gly
85 90 95
His Pro Asp Ser Pro AlaPro ArgAlaThr SerSerPro MetLeu
Pro
100 105 110
6
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Tyr Glu Val Val Tyr Glu Ala Gly Ala Leu Glu Arg Met Gln Gly Glu
115 120 125
Thr Gly Pro Asn Val Gly Leu Ile Ser Ile Gln Leu Asp Gln Trp Ser
130 135 140
Pro Ala Phe Met Val Pro Asp Ser Cys Met Val Ser Glu Leu Pro Phe
145 150 155 160
Asp Ala His Ala His Ile Leu Pro Arg Ala Gly Thr Val Ala Pro Val
165 170 175
Pro Cys Thr Thr Leu Leu Pro Cys Gln Thr Leu Phe Leu Thr Asp Glu
180 185 190
Glu Lys Arg Leu Leu Gly Gln Glu Gly Val Ser Leu Pro Ser His Leu
195 200 205
Pro Leu Thr Lys Ala Glu Glu Arg Val Leu Lys Lys Val Arg Arg Lys
210 215 220
Ile Arg Asn Lys Gln Ser Ala Gln Asp Ser Arg Arg Arg Lys Lys Glu
225 230 235 240
Tyr Ile Asp Gly Leu Glu Ser Arg Val Ala Ala Cys Ser Ala Gln Asn
245 250 255
Gln Glu Leu Gln Lys Lys Val Gln Glu Leu Glu Arg His Asn Ile Ser
260 265 270
Leu Val Ala Gln Leu Arg Gln Leu Gln Thr Leu Ile Ala Gln Thr Ser
275 280 285
Asn Lys Ala Ala Gln Thr Ser Thr Cys Val Leu Ile Leu Leu Phe Ser
290 295 300
Leu Ala Leu Ile Ile Leu Pro Ser Phe Ser Pro Phe Gln Ser Arg Pro
305 310 315 320
Glu Ala Gly Ser Glu Asp Tyr Gln Pro His Gly Val Thr Ser Arg Asn
325 330 335
Ile Leu Thr His Lys Asp Val Thr Glu Asn Leu Glu Thr Gln Val Val
340 345 350
G1u Ser Arg Leu Arg Glu Pro Pro Gly Ala Lys Asp Ala Asn Gly Ser
355 360 365
Thr Arg Thr Leu Leu Glu Lys Met Gly Gly Lys Pro Arg Pro Ser Gly
370 375 380
Arg Ile Arg Ser Val Leu His Ala Asp Glu Met
385 390 395
<210> 30
<211> 29
<212> PRT
<213> Homo sapiens
<400> 30
Phe Ile Asp Pro Asn Glu Val Tyr Cys Ser Glu Ala Ser Pro Gly Ser
1 5 10 15
Asp Ser Gly Ile Ser Glu Asp Pro Cys His Pro Asp Ser
20 25
<210> 31
<211> 41
<212> PRT
<213> Homo sapiens
<400> 31
Glu Asp Tyr Gln Pro His G1y Va1 Thr Ser Arg Asn Ile Leu Thr His
1 5 10 15
Lys Asp Val Thr Glu Asn Leu Glu Thr Gln Val Val Glu Ser Arg Leu
20 25 30
Arg Glu Pro Pro Gly Ala Lys Asp Ala
35 40
<210> 32
<211> 28
<212> PRT
<213> Homo Sapiens
<400> 32
7
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Ser Thr Arg Thr Leu Leu Glu Lys Met Gly Gly Lys Pro Arg Pro Ser
1 5 10 15
Gly Arg Ile Arg Ser Val Leu His Ala Asp Glu Met
20 25
<210> 33
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> Affinitiy purification system recognition site
<400> 33
Ala Ser Pro Thr Tyr Arg Leu Tyr Ser Ala Ser Pro Ala Ser Pro Ala
1 5 10 15
Ser Pro Ala Ser Pro Leu Tyr Ser
<210> 34
<211> 57
<212> PRT
<213> Artificial Sequence
<220>
<223> Affinitiy purification system recognition site
<400> 34
Gly Leu Gly Leu Asn Leu Tyr Ser Leu Glu Ile Leu Glu Ser Glu Arg
1 5 10 15
Gly Leu Gly Leu Ala Ser Pro Leu Glu Ala Ser Asn Met Glu Thr His
20 25 30
Ile Ser Thr His Arg Gly Leu His Ile Ser His Ile Ser His Ile Ser
35 40 45
His Ile Ser His Ile Ser His Ile Ser
50 55
8
