Note: Descriptions are shown in the official language in which they were submitted.
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REAGENTS AND METHODS USEFUL FOR
DETECTING DISEASES OF THE BREAST
Background of the Invention
The invention relates generally to detecting diseases of the breast, and more
particularly, relates to reagents such as polynucleotide sequences and the
polypeptide
to sequences encoded thereby, as well as methods which utilize these
sequences, which
are useful for detecting, diagnosing, staging, monitoring, prognosticating,
preventing
or treating, or determining predisposition to diseases or conditions of the
breast such as
breast cancer.
Breast cancer is the most common form of cancer occurring in females in the
US. The incidence of breast cancers in the United States is projected to be
175,000
cases diagnosed and 43,300 breast cancer related deaths to occur during 1999
(American Cancer Society statistics). Worldwide, the incidence of breast
cancer has
increased from 700,000 in 1985 to about 900,000 in 1990. G.N. Hortobagyi et
al., CA
Cancer J Clin 45: 199-226 (11995).
Procedures used for detecting, diagnosing, staging, monitoring,
prognosticating, preventing or treating, or determining predisposition to
diseases or
conditions of the breast such as breast cancer are of critical importance to
the outcome
of the patient. For example, 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:
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Principles and Practice of Oncology, Fourth Edition, pp. 1264-1332,
Philadelphia, PA:
JIB. 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 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
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, fails 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.,
sue. 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 it 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
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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.,
Onkologie 18: 394-401 (1995).
Such procedures also 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 breast at low cost to the patient. Markers such as prostate
specific antigen
(PSA) and human chorionic gonadotropin (hCG) exist and are used clinically for
screening patients for prostate cancer and testicular cancer, respectively.
For example,
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. Elevated
levels of
PSA protein in serum are used in the early detection of prostate cancer or
disease in
asymptomatic men. See, for example, 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. 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. Likewise, the management of breast diseases could be improved by the use
of new
markers normally expressed in the breast but found in elevated amounts in an
inappropriate body compartment as a result of the disease of the breast.
Further, 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
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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., supra. E. R. Frykberg, et al., Cancer 74: 350-
361
(1994).
It would be advantageous, therefore, to provide specific methods and reagents
useful for detecting, diagnosing, staging, monitoring, prognosticating,
preventing or
treating, or determining predisposition to diseases or conditions of the
breast. Such
methods would include assaying a test sample for products of a gene which are
overexpressed in diseases and conditions associated with the breast including
cancer.
Such methods may also include assaying a test sample for products of a gene
which
have been altered by the disease or condition associated with the breast
including
cancer. For example, these assays would include methods for detecting the gene
products (proteins) in light of possible post-translational modifications that
can occur
in the body. Such post-translational modifications can include proteolytic
processing,
alteration of the chain termini, glycosylation, lipid attachment, sulfation,
gamma-
carboxylation, hydroxylation, phosphorylation, ADP-ribosylation, disulfide
bond
formation, transglutamination, and multiple non-covalent interactions with
molecules
such as co-factors, inhibitors (both small molecule and protein), activators
(both small
molecule and protein), and other proteins in formation of multi-subunit
complexes.
See, for example, T. E. Creighton et al., In: Proteins: Structures and
Molecular
Properties, Second Edition, pp. 78-102, New York, NY:W. H. Freeman and Co.
1993.
Some modifications are sequence specific and are therefore predictive whereas
others
are not and are observed by empirical data only. 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 breast-associated
disease
or condition including cancer. Such methods would comprise making cDNA from
mRNA in the test sample, amplifying, when necessary, portions of the cDNA
corresponding to the gene or a fragment thereof, and detecting the cDNA
product as an
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indication of the presence of the disease or condition including cancer or
detecting
translation products of the mRNAs comprising gene sequences as an indication
of the
presence of the disease. Useful reagents include polynucleotides, or fragments
thereof
which may be used in diagnostic methods such as reverse transcriptase-
polymerase
5 chain reaction (RT-PCR), PCR, or hybridization assays of mRNA extracted from
biopsied tissue, blood or other test samples; or proteins which are the
translation
products of such mRNAs; or antibodies directed against these proteins. Such
assays
would include methods for assaying a sample for products of the gene and
detecting
the products as an indication of disease of the breast. Drug treatment or gene
therapy
for diseases and conditions of the breast including cancer can 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 methods capable of detecting early stage
breast
disease such as cancer.
Summary of the Invention
The present invention provides a method of detecting a target B S 106
polynucleotide in a test sample which comprises contacting the test sample
with at least
one BS 106-specific polynucleotide and detecting the presence of the target BS
106
polynucleotide in the test sample. The BS106-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, and fragments or complements thereof. Also, the BS 106-
specific polynucleotide may be attached to a solid phase prior to performing
the method.
The present invention also provides a method for detecting BS 106 mRNA in a
test
sample, which comprises performing reverse transcription (RT) with at least
one primer in
order to produce cDNA, amplifying the cDNA so obtained using BS106
oligonucleotides
as sense and antisense primers to obtain BS 106 amplicon, and detecting the
presence of
the BS106 amplicon as an indication of the presence of BS106 mRNA in the test
sample,
wherein the BS 106 oligonucleotides have at least 50% identity to a sequence
selected from
the group consisting of SEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID
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NO 3, SEQUENCE ID NO 4, SEQUENCE ID NO 5, SEQUENCE ID NO 6, and
fragments or complements thereof. Amplification can be performed by the
polymerase
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 measurable signal. The detectable label can be
attached to a solid
phase.
The present invention further provides a method of detecting a target BS 106
polynucleotide in a test sample suspected of containing target BS106
polynucleotides,
which comprises (a) contacting the test sample with at least one BS106
oligonucleotide as
a sense primer and at least one BS106 oligonucleotide as 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 BS 106 oligonucleotide to obtain a
second stage
reaction product, with the proviso that the other BS106 oligonucleotide is
located 3' to the
BS106 oligonucleotides utilized in step (a) and is complementary to the first
stage reaction
product; and (c) detecting the second stage reaction product as an indication
of the
presence of a target BS106 polynucleotide in the test sample. The BS106
oligonucleotides
selected as reagents in the method have at least 50% identity to a sequence
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, 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 BS 106 polynucleotides in a test sample are also
provided which
comprise a container containing at least one BS106-specific 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, 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).
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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 provides a purified polynucleotide or fragment thereof
derived from a BS106 gene. The purified polynucleotide is capable of
selectively
hybridizing to the nucleic acid of the BS106 gene, or a complement thereof.
The
polynucleotide has at least 50% identity to 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, and fragments or
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 said vector.
The present invention further provides a recombinant expression system
comprising a nucleic acid sequence that includes an open reading frame derived
from
BS106. The nucleic acid sequence has at least 50% identity with a sequence
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, 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 polypeptides encoded by BS 106. The
polypeptides can be produced by recombinant technology, provided in purified
form, or
produced by synthetic techniques. The polypeptides comprise amino acid
sequences
which have at least 50% identity to an amino acid sequence selected from the
group
consisting of SEQUENCE ID NOS. 20-33.
Also provided is an antibody which specifically binds to at least one BS 106
epitope. The antibody can be a polyclonal or monoclonal antibody. The epitope
is
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derived from an amino acid sequence selected from the group consisting of
SEQUENCE
ID NOS. 20-33, and fragments thereof. Assay kits for determining the presence
of BS106
antigen or anti-B S 106 antibody in a test sample are also included. In one
embodiment, the
assay kits comprise a container containing at least one BS 106 polypeptide
having at least
50% identity to an amino acid sequence selected from the group consisting of
SEQUENCE ID NOS. 20-33, 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 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. Also, the polypeptide can be attached to a solid phase.
In another embodiment of the invention, antibodies or fragments thereof
against
the BS 106 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 BS106
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.
Another assay kit for determining the presence of B S 106 antigen or anti-B S
106
antibody in a test sample comprises a container containing an antibody which
specifically
binds to a BS106 antigen, wherein the BS106 antigen comprises at least one B S
106-
encoded epitope. The BS106 antigen has at least about 60% sequence similarity
to a
sequence of a BS 106-encoded antigen selected from the group consisting of
SEQUENCE
ID NOS. 20-33, and fragments thereof. These test kits can further comprise
containers
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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, 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
BS106 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 to a BS106 amino acid sequence selected from the group consisting
of
SEQUENCE ID NOS. 20-33, and fragments thereof.
A method for detecting B S 106 antigen in a test sample suspected of
containing
BS 106 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 a B S 106
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 BS 106 antigen in the test sample. The antibody
can be
attached to a solid phase and be either a monoclonal or polyclonal antibody.
Furthermore,
the antibody specifically binds to at least one BS 106 antigen selected from
the group
consisting of SEQUENCE ID NOS. 20-33, and fragments thereof.
Another method is provided which detects antibodies which specifically bind to
BS 106 antigen in a test sample suspected of containing these antibodies. The
method
comprises contacting the test sample with a polypeptide which contains at
least one BS 106
epitope, wherein the BS 106 epitope comprises an amino acid sequence having at
least
50% identity with an amino acid sequence encoded by a BS106 polynucleotide, or
a
fragment thereof. Contacting is carried out for a time and under conditions
sufficient to
allow antigen/antibody complexes to form. The method further entails detecting
complexes which contain the polypeptide. The polypeptide can be attached to a
solid
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phase. Further, the polypeptide can be a recombinant protein or a synthetic
peptide having
at least 50% identity to an amino acid sequence selected from the group
consisting of
SEQUENCE ID NOS. 20-33, and fragments thereof.
The present invention provides a cell transfected with a BS 106 nucleic acid
5 sequence that encodes at least one epitope of a BS 106 antigen, or fragment
thereof. The
nucleic acid sequence is 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, and fragments or complements thereof.
A method for producing antibodies to BS 106 antigen also is provided, which
10 method comprises administering to an individual an isolated immunogenic
polypeptide or
fragment thereof, wherein the isolated immunogenic polypeptide comprises at
least one
BS106 epitope 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 NOS. 20-33, and fragments thereof.
Another method for producing antibodies which specifically bind to BS106
antigen
is disclosed, which method comprises administering to a mammal a plasmid
comprising a
nucleic acid sequence which encodes at least one BS 106 epitope derived from
an amino
acid sequence selected from the group consisting of SEQUENCE ID NOS. 20-33,
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 BS106 polynucleotide
of
at least about 10-12 nucleotides having at least 50% identity to 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, and
fragments or complements thereof. The BS106 polynucleotide encodes an amino
acid
sequence having at least one B S 106 epitope. Another composition of matter
provided by
the present invention comprises a polypeptide with at least one BS 106 epitope
of about 8-
10 amino acids. The polypeptide comprises an amino acid sequence having at
least 50%
identity to an amino acid sequence selected from the group consisting of
SEQUENCE ID
NOS. 20-33, and fragments thereof. Also provided is a gene, or fragment
thereof, coding
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for a BS106 polypeptide which has at least 50% identity to SEQUENCE ID NO 20;
and a
gene, or a fragment thereof, comprising DNA having at least 50% identity to
SEQUENCE
ID NO 5 or SEQUENCE ID NO 6.
Brief Description of the Drawings
Figure 1 shows the nucleotide alignment of clones 1662885 (SEQUENCE ID NO
1), 893988 (SEQUENCE ID NO 2), 901429 (SEQUENCE ID NO 3), 1209814
(SEQUENCE ID NO 4), the full-length sequence of clone 1662885 [designated as
1662885inh (SEQUENCE ID NO 5)], and the consensus sequence (SEQUENCE ID NO
6) derived therefrom.
Figure 2 shows the contig map depicting the formation of the consensus
nucleotide
sequence (SEQUENCE ID NO 6) from the nucleotide alignment of overlapping
clones
1662885 (SEQUENCE ID NO 1), 892988 (SEQUENCE ID NO 2), 901429 (SEQUENCE
ID NO 3), 1209814 (SEQUENCE ID NO 4), 1662885inh (SEQUENCE ID NO 5).
Figure 3A contains a scan of an ethidium bromide stained agarose gel of RNA
and
the corresponding Northern blot of RNA following hybridization with a BS 106
radiolabeled probe. Samples include from breast tissues and prostate tissue
extracts.
Figure 3B contains a scan of an ethidium bromide stained agarose gel of RNA
and
the corresponding Northern blot of RNA following hybridization with a BS106
radiolabeled probe. Samples include from normal breast tissues and breast
cancer tissue
extracts.
Figure 4 shows the results of a BS106 probe against a Clontech (Clontech
Laboratories, Inc., Palo Alto, CA) Multiple Tissue Expression ArrayTM
containing polyA
RNA from 76 different human tissues.
Figure 5A is a scan of a SYBR Green stained agarose gel of BS106 RNA-
specific PCR amplification products. It shows a DNA band at 201 bases that is
indicative
of a BS 106 mRNA-specific RT-PCR product in normal breast tissue samples and
in breast
cancer tissue samples.
Figure 5B is. a scan of a SYBR Green stained agarose gel of BS 106 RNA-
specific PCR amplification products. The 201 bases band which is indicative of
a BS106
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mRNA-specific RT-PCR product is absent in colon tissue samples and lung tissue
samples.
Figure 6 shows the BS 106 amplicon was easily detected at 18 picograms of RNA
from the MDA 361 cell line using a LCx system (Abbott Laboratories, Abbott
Park, IL)
assay.
Figure 7 shows the overall tissue distribution for the BS 106 marker using a
LCx"
system assay.
Figure 8 shows a Western blot of HEK293 cell products with a BS106 anti-myc
epitope monoclonal antibody.
Figure 9 shows a dot blot of fractions collected from Nickel chelate column
using
a monoclonal antibody recognizing a myc epitope.
Figure 10 shows a Western blot of pooled, dialyzed, semi-purified supernatant
analyzed for the presence of B S 106 M/H.
Figure 11 shows pooled, purified BS 106 M/H analysed by Western blot using
both
an anti-myc monoclonal antibody, in panel A and an anti-BS 106 polyclonal
antisera in
panel B.
Figure 12 shows the results of a Western blot performed on a panel of tissue
extracts using a monoclonal antibody (H9C29) directed against BS106 peptide
SEQUENCE ID NO 26. Each lane of Figure 12 contains a different tissue extract.
Figure 13 shows Western blots of reduced and unreduced human milk fractions
against the affinity purified rabbit anti-BS 106 antibody. Panel B shows the
results of a
competitive reaction with the disappearance of the bands seen in Panel A in
the presence
of BS106 peptide (SEQUENCE ID NO 26).
Figure 14 shows Western blots of biological samples with BS 106 antibodies.
Figure 15 A-C shows samples of the BS106 fluorescein conjugates titrated with
the
affinity purified rabbit anti-BS 106 and the mouse BS 106 monoclonal
antibodies H39C51
and H9C29.
Detailed Description of the Invention
The present invention provides a gene or a fragment thereof which codes for a
BS106 polypeptide having at least about 50% identity to SEQUENCE ID NO 20. The
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present invention further encompasses a BS106 gene or a fragment thereof
comprising
DNA which has at least about 50% identity to SEQUENCE ID NO 5 or SEQUENCE ID
NO 6.
The present invention provides methods for assaying a test sample for products
of
a breast tissue gene designated as BS106, which method comprises making cDNA
from
mRNA in the test sample and detecting the cDNA as an indication of the
presence of
breast tissue gene BS106. The method may include an amplification step,
wherein one or
more portions of the mRNA from BS106 corresponding to the gene or fragments
thereof,
is amplified. Methods also are provided for assaying for the translation
products of
BS106. 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
BS 106,
especially breast 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,
preventing or treating, or determining the predisposition to, diseases and
conditions of the
breast such as breast cancer, characterized by BS106, as disclosed herein.
The compositions and methods described herein will enable the identification
of
certain markers as indicative of a breast tissue disease or condition; the
information
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obtained therefrom will aid in the detecting, diagnosing, staging, monitoring,
prognosticating, preventing or treating, or determining diseases or conditions
associated
with BS106, especially breast cancer. Test methods include, for example, probe
assays
which utilize the sequence(s) 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.
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 BS 106. It also is thought
that the
polynucleotides or polypeptides and protein encoded by the BS106 gene are
useful as a
marker. This marker is either elevated in disease such as breast cancer,
altered in disease
such as breast cancer, or present as a normal protein but appearing in an
inappropriate
body compartment. The uniqueness of the epitope may be determined by its
immunological reactivity and specificity with antibodies directed against
proteins and
polypeptides encoded by the BS106 gene, and (ii) its nonreactivity with any
other tissue
markers. Methods for determining immunological reactivity are well-known and
include
but are not limited to, for example, radioimmunoassay (RIA), enzyme-linked
immunosorbent assay (ELISA), hemagglutination (HA), fluorescence polarization
immunoassay (FPIA), chemiluminescent immunoassay (CLIA) and others. Several
examples of suitable methods are described herein.
Unless otherwise stated, the following terms shall have the following
meanings:
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 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,
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"identity" refers 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
5 determining their "percent identity." The percent identity of two sequences,
whether
nucleic acid or peptide sequences, is the number of exact matches between two
aligned
sequences divided by the length of the shorter sequence and multiplied by 100.
An
approximate alignment for nucleic acid sequences is provided by the local
homology
algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489
(1981).
10 This algorithm can be extended to use with peptide sequences using the,
scoring matrix
developed by Dayhoff, Atlas of Protein Sequences 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
15 Genetics Computer Group (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.
For example, percent identity of a particular nucleotide sequence to a
reference
sequence can be determined using the homology algorithm of Smith and Waterman
with a
default scoring table and a gap penalty of six nucleotide positions. Another
method of
establishing percent identity in the context of the present invention is to
use the MPSRCH
package of programs copyrighted by the University of Edinburgh, developed by
John F.
Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc.
(Mountain View,
CA). From this suite of packages, the Smith-Waterman algorithm can be employed
where
default parameters are used for the scoring table (for example, gap open
penalty of 12, gap
extension penalty of one, and a gap of six). From the data generated, the
"Match" value
reflects "sequence identity." Other suitable programs for calculating the
percent identity
or similarity between sequences are generally known in the art, such as the
alignment
program BLAST, which can also be used with default parameters. For example,
BLASTN
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and BLASTP can be used with the following default parameters: genetic code =
standard;
filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62;
Descriptions
50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL
+ DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR.
One of skill in the art can readily determine the proper search parameters to
use for
a given sequence in the above programs. For example, the search parameters may
vary
based on the size of the sequence in question. Thus, for example, a
representative
embodiment of the present invention would include an isolated BS 106
polynucleotide
having X contiguous nucleotides, wherein (i) the X contiguous nucleotides have
at least
about 50% identity to Y contiguous nucleotides derived from any 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, (ii) X equals Y, and (iii) X is greater than or equal
to 6
nucleotides and up to 5000 nucleotides, preferably greater than or equal to 8
nucleotides
and up to 5000 nucleotides, more preferably 10-12 nucleotides and up to 5000
nucleotides,
and even more preferably 15-20 nucleotides, up to the number of nucleotides
present in
the full-length BS 106 sequence described herein, including all integer values
falling
between the above-described ranges.
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, 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. Comparisons to sequences in databanks, for example, can be used as a
method to
determine the uniqueness of a designated sequence. Regions from which
sequences may
be derived, include but are not limited to, regions encoding specific
epitopes, as well as
non-translated and/or non-transcribed regions.
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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
region(s) 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 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 to a BS106 amino acid sequence.
Further, the
BS106 "polypeptide," "protein," or "amino acid" sequence may have at least
about 60%
similarity, preferably at least about 75% similarity, more preferably about
85% similarity,
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18
and most preferably about 95% or more similarity to a polypeptide or amino
acid sequence
of BS106. This amino acid sequence can be selected from the group consisting
of
SEQUENCE ID NOS 20-33, and fragments thereof.
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 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 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 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
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19
the sequenced material with the cDNAs described, or hybridization and
digestion with
single strand nucleases, followed by size determination of the digested
fragments.
"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, 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.
"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 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
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 addition, protein fragments, analogs,
mutated or variant
proteins, fusion proteins and the like are included within the meaning of
polypeptide.
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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 from the
specified
polypeptide.
5 "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 vectors or other transferred DNA, and include the original progeny
of the
original cell which has been transfected.
10 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 attached,
such
as to bring about the replication and/or expression of the attached segment.
15 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 binding site, and
terminators;
in eukaryotes, such control sequences generally include promoters, terminators
and, in
20 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.
"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.
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. Rare errors in translation may occur,
termed
CA 02399047 2009-08-25
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translational frameshifting, or programmed frameshifting, that allow the
ribosome to
translate two partially overlapping reading frames as a single polypeptide
I.P. Ivanov et al.
RNA 4(10):1230-1238 (1998); and P.J. Farabaugh Annu Rev Genet 30:507-528
(1996).
A "coding sequence" is a polynucleotide sequence which is transcribed into
mRNA and translated into a polypeptide when placed under the control of
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
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 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 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 include, for example,
x-ray
crystallography and two-dimensional nuclear magnetic resonance.
The methods for identifying epitopes in a novel peptide sequence are well
known
and described in both the scientific, commercial, and patent literature. For
example, M. H.
Van Regenmortel describes how to predict epitopes from the primary sequence of
a
protein. (See "Protein structure and antigenicity", Int JRad Appl Instrum B.,
14(4):277-
80, 1987.)
Perkin-Elmer Biosystems, a major provider of DNA sequencing and peptide
synthesizing instruments has established a public website which describes how
to select
peptides which reflect the epitopes of a protein.
CA 02399047 2009-08-25
22
Patent application WO 97/25426 describes in detail how to identify epitopes
from peptide sequences. The sequence can be scanned for hydrophobicity and
hydrophilicity values by the method of Hopp, Prog. Clin. Biol. Res. 172B: 367-
377 (1985)
or the method of Cease et al, J. Exp. Med. 164: 1779-1784 (1986) or the method
of
Spouge et al, J. Immunol. 138: 204-212 (1987). Commercial software programs to
implement these methods are available.
A "conformational epitope" is an epitope that is comprised of 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,
more
particularly, by the kinetics of antibody binding, and/or by competition in
binding using as
competitor(s) 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 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 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 genome.
"Treatment" refers to prophylaxis and/or therapy.
CA 02399047 2009-08-25
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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 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.
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
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,
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24
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 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 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.
"Diseases of the breast" or "breast disease," or "condition of the breast," as
used
herein, refer to any disease or condition of the breast including, but not
limited to, atypical
hyperplasia, fibroadenoma, cystic breast disease, and 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,
mucinous
carcinoma, infiltrating lobular carcinoma, infiltrating comedocarcinoma and
inflammatory
carcinoma.
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.
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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
5 "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 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,
10 effector and receptor molecules, cofactors and enzymes, enzyme inhibitors
and enzymes
and 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
15 formed by recombinant DNA molecules.
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
20 as, the following: hybrid (chimeric) antibody molecules (see, for example,
Winter, et al.,
Nature 349:293-299 (1991), and U.S. Patent No. 4,816,567); F(ab')2 and F(ab)
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.
25 USA 85:5879-5883 (1988)); humanized antibody molecules (see, for example,
Riechmann, et al., Nature 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 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 carrier protein.
A "capture reagent," as used herein, refers to an unlabeled specific binding
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, 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 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 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 described
hereinabove
conjugated to a specific binding member of a specific binding pair, such as
carbazole or
adamantane.
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, 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
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producing a signal either by itself or in conjunction with one or more
additional
substances.
"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
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 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 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 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
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.
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
CA 02399047 2009-08-25
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may be used as well. Such useful solid supports 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 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
understood
hydrophobic forces. Other suitable solid supports are known in the art.
Reagents
The present invention provides reagents such as polynucleotide sequences
derived
from a breast tissue of interest and designated as BS106, 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 antibodies of the present invention may be used to provide
information
leading to the detecting, diagnosing, staging, monitoring, prognosticating,
preventing or
treating of, or determining the predisposition to, diseases and conditions of
the breast such
as cancer. The sequences disclosed herein represent unique polynucleotides
which 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 BS 106-derived polynucleotides can be used in the methods described
herein for the detection of normal or altered gene expression. Such methods
may employ
BS 106 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 breast tissue disease and conditions
associated
therewith. They also can be used to identify an entire or partial coding
region of a BS 106
polypeptide. They further can be provided in individual containers in the form
of a kit for
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29
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.
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 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 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 a non-coding
sequence,
is 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
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 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 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 protein having both a presequence (leader sequence) and a prosequence.
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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 polypeptide fused to the
marker in the case
5 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., Cell 37:767 (1984).
It is contemplated that polynucleotides will be considered to hybridize to the
10 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.
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
15 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
20 varying degrees of 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
25 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 of the present
invention, a
30 nucleic acid molecule is capable of hybridizing selectively to a target
sequence under
moderately stringent hybridization conditions. In the context of the present
invention,
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31
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 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 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 tissue sections. The formation of hybrids
can be
monitored by inclusion of a reporter molecule, typically, in the probe. Such
reporter
molecules, or detectable 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 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 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 purified
BS 106 polypeptide of which at least a portion of the polypeptide is encoded
by a BS 106
polynucleotide selected from the polynucleotides provided herein. These
antibodies may
be used in the methods provided herein for the detection of BS106 antigen in
test samples.
The presence of BS106 antigen in the test samples is indicative of the
presence of a breast
disease or condition. The antibody also may be used for therapeutic purposes,
for
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32
example, in neutralizing the activity of BS 106 polypeptide in conditions
associated with
altered or abnormal expression.
The present invention further relates to a BS 106 polypeptide which has the
deduced amino acid sequence as provided herein, as well as fragments, analogs
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 BS106 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 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
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 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
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).
CA 02399047 2009-08-25
33
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 situ hybridization (FISH)
technology to
perform chromosomal analysis, and used to identify cancer-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 Assays
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 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 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 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.
CA 02399047 2009-08-25
34
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
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) 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 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 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 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., PCR Methods and Applications
4:80-84 (1994).
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., PNAS USA 87:1874-1878 (1990) and also described by J. Compton, Nature 350
(No.
6313):91-92 (1991); Q-beta 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;
CA 02399047 2009-08-25
and target mediated amplification, as described in International Publication
No. WO
93/22461.
Detection of BS 106 may be accomplished using any suitable detection method,
including those detection methods which are currently well known in the art,
as well as
5 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 methods as well as signal
amplification technologies. An example of presently known detection methods
would
10 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 that
disclosed in Snitman et al., U.S. Patent No. 5,273,882. While the
amplification of target
15 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 (combined) carried out
using
a variety of heterogeneous and homogeneous detection formats. Examples of
20 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 in, Caskey et al., U.S. Patent No. 5,582,989,
Gelfand et al.,
25 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 BS 106 signal. See, for example, Caskey
et al., U.S.
Patent No. 5,582,989, and Gelfand et al., U.S. Patent No. 5,210,015.
30 In one embodiment, the present invention generally comprises the steps of
contacting a test sample suspected of containing a target polynucleotide
sequence with
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36
amplification reaction reagents comprising an amplification primer, and a
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 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 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.
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 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 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 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
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37
is not directly detectable, the captured hybrids can be contacted with a
conjugate, which
generally comprises a binding member attached to a directly detectable label.
The
conjugate becomes bound to the complexes and the conjugates presence on the
complexes
can be detected with the directly detectable label. Thus, the presence of the
hybrids on the
solid phase 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 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., Science 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
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 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 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 utilizes enzymes with polymerase or ligase activity, separately
or in
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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
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.
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 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 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; 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
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;
CA 02399047 2009-08-25
39
morpholino analogs which are described in U.S. Patents Nos 5,185,444,
5,034,506 and
5,142,047; and the like. Depending upon the type of label carried by the
probe, the probe
is employed to capture or detect the 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
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 T hydroxyl group simply can be
cleaved,
replaced or modified.
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.
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 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
CA 02399047 2009-08-25
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 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
5 to a 5' oligo terminus using Aminomodifier II (Clontech). The amines can be
reacted to
various haptens using conventional activation and linking chemistries. In
addition, U.S.
Patent No. 5,290,925, issued March 1, 1994, teaches methods for labeling
probes at their 5'
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
to 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. See, for example, N.T. Thuong et
al., Tet. Letters
29 (46):5905-5908 (1988). 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
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
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41
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 B S 106
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 breast
tissue disease,
such as breast 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 polymerase 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 BS 106 derived molecules, such as polynucleotides
or
oligonucleotides of the present invention, into patients with conditions
associated with
abnormal expression of polynucleotides related to a breast tissue disease or
condition
especially breast cancer. These molecules, including antisense RNA and DNA
fragments
and ribozymes, are designed to inhibit the translation of BS 106-mRNA, and may
be used
therapeutically in the treatment of conditions associated with altered or
abnormal
expression of a BS106 polynucleotide.
Alternatively, the oligonucleotides 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 BS106 polypeptide in the manner described
above.
Antisense constructs to a BS 106 polynucleotide, therefore, reverse the action
of BS 106
transcripts and may be used for treating breast tissue disease conditions,
such as breast
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 BS106 polypeptide(s), or any fragment
thereof, to
identify at least one compound which specifically binds the BS106 polypeptide.
Such a
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42
method comprises the steps of providing at least one compound; combining the
BS106
polypeptide with each compound under suitable conditions for a time sufficient
to allow
binding; and detecting the BS 106 polypeptide binding to each compound.
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 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 BS
106. 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 polypeptide typically is labeled. After suitable
incubation, free (or
uncomplexed) polypeptide or 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.
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 BS 106 polypeptide as
provided herein.
Another technique for agent screening provides high throughput screening for
compounds having suitable binding affinity to at least one polypeptide of BS
106 disclosed
herein. Briefly, large numbers of different small peptide test compounds 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
CA 02399047 2009-08-25
43
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 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 in vivo. J. Hodgson, Bio/Technology 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 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 site(s) 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, 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., Biochemistry
31:7796-7801
(1992), or which act as inhibitors, agonists, or antagonists of native
peptides as shown by
S.B.P. Athauda et al., J. Biochem. (Tokyo) 113 (6):742-746 (1993).
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 generating
anti-idiotypic
antibodies ("anti-ids") to, a functional, pharmacologically active antibody.
As a mirror
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
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44
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 employing
computer
modeling techniques in place of, or in addition to, x-ray crystallography.
Antibodies specific to a BS106 polypeptide (e.g., anti-BS106 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
example, to treat
breast tissue diseases including breast cancer and its metastases.
Further, such antibodies can detect the presence or absence of the B S 106
polypeptide in a test sample and, therefore, are useful as diagnostic markers
for the
diagnosis of a breast tissue disease or condition especially breast cancer.
Such antibodies
may also function as a diagnostic marker for breast tissue disease conditions
such as breast
cancer.
The present invention also is directed to antagonists and inhibitors of the
polypeptides of the present invention. The antagonists and inhibitors are
those 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 the BS106 polypeptide by binding the BS106 polypeptide, or in some
cases the
antagonist may be an oligonucleotide. Examples of small molecule inhibitors
include, but
are not limited to, small peptides or peptide-like molecules.
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
BS 106
polypeptide inhibitors is preferably systemic. The 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
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polynucleotide to DNA or RNA. For example, the 5' coding portion 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
5 transcription, thereby preventing transcription and the production of the
BS106
polypeptide. For triple helix, see, for example, Lee et al, Nuc. Acids Res.
6:3073 (1979);
Cooney et al, Science 241:456 (1988); and Dervan et al, Science 251:1360
(1991) The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of a
mRNA molecule into the BS106 polypeptide. For antisense, see, for example,
Okano, J.
10 Neurochem. 56:560 (1991); and "Oligodeoxynucleotides as Antisense
Inhibitors of Gene
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
15 phosphoroamydate internucleotide linkages.
Recombinant Technology.
The present invention provides host cells and expression vectors comprising
BS 106 polynucleotides of the present invention and methods for the production
of the
polypeptide(s) they encode. Such methods comprise culturing the host cells
under
20 conditions suitable for the expression of the BS106 polynucleotide and
recovering the
BS106 polypeptide from the cell culture.
The present invention also provides vectors which include BS106
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
25 recombinant techniques.
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 phage, etc. The
engineered host
cells can be cultured in conventional nutrient media modified as appropriate
for activating
30 promoters, selecting transfected cells, or amplifying BS 106 gene(s). The
culture
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46
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 chromosomal, nonchromosomal
and
synthetic DNA sequences, e.g., derivatives of 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.
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
endonuclease 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 control
sequence(s)
(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 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
E. coli.
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 E. coli,
Salmonella
typhimurium; Streptomyces sue.; fungal cells, such as yeast; insect cells such
as Drosophila
and Sf9; animal cells such as CHO, COS or Bowes melanoma; plant cells, etc.
The
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47
selection of an appropriate host is 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 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,
pNH8a, pNH16a, pNHl8a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,
pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, 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
modifications in
the polylinker (multiple cloning site). These modifications are (1) it lacks a
Hindlll
restriction site and (2) its EcoRl restriction site lies at a different
location. pINCY is
created from pSPORTI by cleaving pSPORTI with both Hindlll and EcoRI and
replacing
the excised fragment of the polylinker with synthetic DNA fragments (SEQUENCE
ID
NO 7 and SEQUENCE ID NO 8). 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 7 and SEQUENCE ID NO 8, may be generated synthetically with 5'
terminal phosphates, mixed together, and then ligated under standard
conditions for
performing staggered end ligations into the pSPORTI plasmid cut with Hindlll
and
EcoRl. Suitable host cells (such as E. coli DH5oc 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
CA 02399047 2009-08-25
48
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
be employed.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with selectable
markers. 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 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 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, DEAE-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)).
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, 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 Harbor, N.Y., 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 SV40 enhancer on
the late
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49
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
ampicillin resistance
gene of E. coli 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 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 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
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 transfection include E. coli, Bacillus
subtilis, 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
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 GEM1
(Promega
Biotec, Madison, WI). These pBR322 "backbone" sections are combined with an
appropriate promoter and the structural sequence to be expressed.
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
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typically harvested by centrifugation, disrupted by physical or chemical
means, and the
resulting crude extract retained for further 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
5 well-known to the ordinary artisan.
Various mammalian cell culture systems can also be employed to express
recombinant protein. Examples of mammalian expression systems include the COS-
7
lines of monkey kidney fibroblasts described by Gluzman, Cell 23:175 (1981),
and other
cell lines capable of expressing a compatible vector, such as the C127, HEK-
293, 3T3,
10 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 sequences. DNA sequences derived from
the
SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice,
and
15 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).
BS106 polypeptides are recovered and purified from recombinant cell cultures
by
known methods including affinity chromatography, ammonium sulfate or ethanol
20 precipitation, acid extraction, anion or cation exchange chromatography,
phosphocellulose
chromatography, hydrophobic interaction chromatography, hydroxyapatite
chromatography or lectin chromatography. It is preferred to have low
concentrations
(approximately 0.1-5 mM) of calcium ion present during purification (Price, et
al., J. Biol.
Chem. 244:917 (1969)). Protein refolding steps can be used, as necessary, in
completing
25 configuration of the polypeptide. Finally, high performance liquid
chromatography
(HPLC) can be employed for final purification steps.
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
30 example, by bacterial, yeast, higher plant, insect and mammalian cells in
culture).
Depending upon the host employed in a recombinant production procedure, the
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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
with
published, known procedures. In addition, equivalent plasmids to those
described are
known in the art and will be apparent to the ordinarily skilled artisan.
The following is the general procedure for the isolation and analysis of cDNA
clones. In a particular embodiment disclosed herein, mRNA was isolated from
breast
tissue and used to generate the cDNA library. Breast tissue was obtained from
patients by
surgical resection and was classified as tumor or non-tumor tissue by a
pathologist.
The cDNA inserts from random isolates of the breast 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 NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3,
and SEQUENCE ID NO 4. The consensus sequence of these inserts is presented as
SEQUENCE ID NO 6. The full-length sequence is SEQUINCE ID NO 5. 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 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
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, OBI) or Taq polymerase to extend DNA chains from an oligonucleotide
primer
annealed to the DNA template of interest. Methods have 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
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52
preparation, sequencing 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
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 more difficult cases, further
analysis is required.
Algorithms have been created to analyze the occurrence of individual
nucleotide
bases at each putative codon triplet. See, for example J.W. Fickett, 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. 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
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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
(supra) 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.
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 breast
tissue cDNA
library can be used for transcribing mRNA 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 EcoRl, 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 15 residues of linker and the peptide
encoded within
the cDNA. Since cDNA clone inserts are generated by an 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
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methods including in 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 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 missing gene
segments with
chemically synthesized 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 embryonic
kidney
(HEK) 293 cells, insect cells such as Sf9 cells, yeast cells such as
Saccharomyces
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 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, 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 recovered from the conditioned medium and analyzed
using
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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
recombinantly in
5 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 immobilized metals, protein A domains that allow purification
on
10 immobilized immunoglobulin and the domain utilized in the FLAGS
extension/affinity
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 for recovering
the,
polypeptide.
15 Immunoassays
BS106 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 breast tissue. They also
can be used as
immunogens to produce antibodies. These antibodies can be, for example,
polyclonal or
20 monoclonal antibodies, chimeric, single chain and humanized antibodies, as
well as Fab
fragments, or the product of 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 polypeptide
into an animal
25 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 ID NOS 20-33, and fragments thereof. The antibody so
obtained then will bind the polypeptide itself. In this manner, even a
sequence encoding
only a fragment of the polypeptide can be used to generate antibodies that
bind the native
30 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
CA 02399047 2009-08-25
56
antibodies, any technique which provides antibodies produced by continuous
cell 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. Today 4:72 (1983) and
the
EBV-hybridoma technique to produce human monoclonal antibodies as described by
Cole
et al., in Monoclonal Antibodies and Cancer Therapy, 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,
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 BS 106 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 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
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 BS106 antigen in the test 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 BS 106 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
polyclonal
antibody, monoclonal antibody, or fragment thereof, which specifically binds
to BS 106
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 BS106 antigen (or a
combination
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57
of these antibodies) to which a signal 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 BS106 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 BS106 antigen present in the test
sample is
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 BS106 antigen. For example, BS106 polypeptides such as the
recombinant
antigens disclosed herein, either alone or in combination, are coated on a
solid phase. A
test sample suspected of containing antibody to BS 106 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 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 B S 106 antigens
in tissue
sections, as well as in cells, by immunohistochemical analysis. Cytochemical
analysis
wherein these antibodies are labeled directly (with, for example, fluorescein,
colloidal
gold, horseradish peroxidase, alkaline 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 to
CNBr-activated Sepharose and used for the affinity purification of specific BS
106
polypeptides from cell cultures or biological tissues such as to purify
recombinant and
native BS 106 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 BS 106 antigens. Combinations of the monoclonal antibodies (and
fragments
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58
thereof) provided herein also may be used together as components in a mixture
or
"cocktail" of at least one B S 106 antibody of the invention, along with
antibodies which
specifically bind to other BS106 regions, each antibody having different
binding
specificities. Thus, this cocktail can include the monoclonal antibodies of
the invention
which are directed to BS106 polypeptides disclosed herein and other monoclonal
antibodies specific to other antigenic determinants of BS 106 antigens or
other related
proteins.
The polyclonal antibody or fragment thereof which can be used in the assay
formats should specifically bind to a B S 106 polypeptide or other B S 106
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
BS106
polypeptide. Most preferably, the polyclonal antibody is of rabbit origin. The
polyclonal
antibodies used in the 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
BS 106 polypeptides, they are useful for the detecting, diagnosing, staging,
monitoring,
prognosticating, preventing or treating, or determining the predisposition to,
diseases and
conditions of the breast such as breast cancer.
It is contemplated and within the scope of the present invention that B S 106
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 acid
sequence of
BS 106. The amino acid sequence of such a polypeptide is selected from the
group
consisting of SEQUENCE ID NOS 20-33, and fragments thereof. It also is within
the
scope of the present invention that different synthetic, recombinant or
purified peptides,
identifying different epitopes of BS106, can be used in combination in an
assay for the
detecting, diagnosing, staging, monitoring, prognosticating, preventing or
treating, or
determining the predisposition to diseases and conditions of the breast such
as breast
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 peptides which define epitopes
from
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59
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 breast, such as breast cancer. Peptides coated on solid phases or labeled
with
detectable labels are then allowed to compete with 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 BS 106 antigen in the patient sample. The presence of BS 106
antigen indicates
the presence of breast tissue disease, especially breast cancer, in the
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-BS106 antibody and/or BS106
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 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 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 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 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 antibodies produced from the
proteins
derived from the expression systems as disclosed herein. For example, in this
assay
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system, BS 106 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 utilized
to
detect the presence of antibody against BS106 antigen in test samples. For
example, a test
5 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 ID NOS 20-33, and 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
10 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 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
15 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
the presence of
antibody against BS106 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
20 solid phase and test sample for a time and under conditions sufficient to
form
antigen/antibody complexes, and then contacting the 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. coli is
used as a
capture antigen on a solid phase, a test sample is added to the so-prepared
solid phase, and
25 following standard incubation and washing steps as deemed or required, a
recombinant
protein derived from a different source (i.e., non-E. coli) 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 assay format which utilizes an antigen specific for BS106
produced or
30 derived from a first source as the capture antigen and an antigen specific
for BS 106 from
a different second source is contemplated. Thus, various combinations of
recombinant
CA 02399047 2009-08-25
61
antigens, as well as the use of synthetic peptides, purified proteins 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, which enjoys common ownership.
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 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, 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-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
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 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 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
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those of ordinary skill in the art. Or, covalent attachment of a specific
binding partner
(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 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 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
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 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 NOS 20-33, 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
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
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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. Test kits designed for the collection, stabilization and
preservation of test
specimens 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
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 Use
Antibodies of the present invention can be used in vivo; that is, they can be
injected into patients suspected of having or having diseases of the breast
for diagnostic or
therapeutic uses. The use of antibodies for in vivo diagnosis is 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
(CEA) expressing tumors using Indium-111 as the label. Griffin et al., J Clin
Ong 9:631-
640 (1991) have described the use of this agent in detecting tumors in
patients suspected
of having recurrent colorectal cancer. The use of similar agents with
paramagnetic ions as
labels for magnetic resonance imaging is know in the art (R. B. Lauffer,
Magnetic
Resonance in Medicine 22:339-342 (1991). It is anticipated that antibodies
directed
against B S 106 antigen can be injected into patients suspected of having a
disease of the
breast such as breast cancer for the 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 (SPECT). Positron emitting labels
such as
Fluorine-19 can also be used for positron emission tomography (PET). For MRI,
paramagnetic ions such as Gadolinium (III) or Manganese (II) can be used.
Localization
CA 02399047 2009-08-25
64
of the label within the breast or external to the breast may allow
determination of spread of
the disease. The amount of label within the breast may allow determination of
the
presence or absence of cancer of the breast.
For patients known to have a disease of the breast, injection of an antibody
directed
against BS106 antigen may have therapeutic benefit. The antibody may exert its
effect
without the use of attached agents by binding to BS106 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 Research 46:2407-2412 (1986) have described the preparation of a drug-
monoclonal antibody conjugate. Pastan et al., Cell 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
Yittrium-90 labeled monoclonal antibodies in various strategies to maximize
the dose to
tumor while limiting normal tissue toxicity. Other known cytotoxic
radionuclides include
Copper-67, Iodine-131, and Rhenium-186 all of which can be used to label
monoclonal
antibodies directed against BS106 antigen for the treatment of cancer of the
breast.
E. coli bacteria (clone 1662885) was deposited on November 20, 1996 with the
American Type Culture Collection (A.T.C.C.), 10801 University Blvd., Manassas,
VA.
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 to practice the present invention in
view of the
teachings provided herein.
Clone 1662885 was accorded A.T.C.C. Deposit No.
98256.
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.
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EXAMPLES
Example 1: Identification of Breast Tissue Library BS 106 Gene-Specific Clones
A. Library Comparison of Expressed Sequence Tags (EST's) or Transcript
Images. Partial sequences of cDNA clone inserts, so-called "expressed sequence
tags"
5 (EST's), were derived from cDNA libraries made from breast tumor tissues,
breast 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. See 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
10 tissue library. EST's sharing regions of mutual sequence overlap are
classified into
clusters. A 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 that did not meet the criteria for automated clustering. The
alignment of all
available clusters and single EST's represents a contig from which a consensus
sequence
15 is derived.) The transcript images then were evaluated to identify EST
sequences that
were representative primarily of the breast 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 higher study priority. EST's corresponding to the consensus
sequence of
20 BS106 were found in 50.8% (30 of 59) of breast tissue libraries. EST's
corresponding to
the consensus sequence, SEQUENCE ID NO 6 (or fragments thereof), were found in
only
0.509% (5 of 983) of the other non-breast tissue libraries of the database.
Therefore, the
consensus sequence or fragment thereof was found more than 100 times more
often in
breast tissue than non-breast tissues. Overlapping clones 1662885 (SEQUENCE ID
NO
25 1), 893988 (SEQUENCE ID NO 2), 901429 (SEQUENCE ID NO 3), 1209814
(SEQUENCE ID NO 4), respectively, were identified for further study. These
represented
the minimum number of clones that (along with the full-length sequence of
clone 1662885
[designated as 1662885inh (SEQUENCE ID NO 5)] were needed to form the contig
and
from which the consensus sequence provided herein (SEQUENCE ID NO 6) was
derived.
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B. Generation of a Consensus Sequence. The nucleotide sequences of clones
1662885 (SEQUENCE ID NO 1), 893988 (SEQUENCE ID NO 2), 901429 (SEQUENCE
ID NO 3), 1209814 (SEQUENCE ID NO 4), and the full-length sequence of clone
1662885 [designated as 1662885inh (SEQUENCE ID NO 5)] 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 their
consensus
sequence (SEQUENCE ID NO 6). Figure 1 shows the nucleotide sequence alignment
of
these clones and their resultant nucleotide consensus sequence (SEQUENCE ID NO
6).
Figure 2 presents the contig map depicting the clones 1662885 (SEQUENCE ID NO
1),
893988 (SEQUENCE ID NO 2), 901429 (SEQUENCE ID NO 3), 1209814 (SEQUENCE
ID NO 4), and the full-length sequence of clone 1662885 [designated as
1662885inh
(SEQUENCE ID NO 5)] which form overlapping regions of the BS106 gene and the
resultant consensus nucleotide sequence (SEQUENCE ID NO 6) of these clones in
a
graphic display. Following this, a three-frame translation was performed on
the consensus
sequence (SEQUENCE ID NO 6). The first forward frame was found to have an open
reading frame encoding a 90 residue amino acid sequence which is presented as
SEQUENCE ID NO 20. The open reading frame corresponds to nucleotides 55 - 324
of
SEQUENCE ID NO 6.
Analysis of the LIFESEQTM database indicates a possible T/G polymorphism at
position 45 in the consensus nucleotide sequence (SEQUENCE ID NO 6).
Example 2: Sequencing of BS106 EST-Specific Clones
The DNA sequence of clone 1662885inh of the BS106 gene contig was determined
(SEQUENCE ID NO 5) using dideoxy termination sequencing with dye terminators
following known methods [F. Sanger et al., PNAS U.S.A. 74:5463 (1977)].
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 universal primers, SEQUENCE ID NO 9 and
SEQUENCE ID NO 10 (New England Biolabs, Beverly, MA and Applied Biosystems
Inc, Foster City, CA, respectively). The sequencing reactions were run on a
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polyacrylamide denaturing gel, and the sequences were determined by an Applied
Biosystems 377 Sequencer (available from Applied Biosystems, Foster City, CA).
Additional sequencing primers, SEQUENCE ID NO 11 and SEQUENCE ID NO 12 were
designed from sequence information of the consensus sequence, SEQUENCE ID NO
6.
These primers then were used to determine the remaining DNA sequence of the
cloned
insert from each DNA strand, as previously described.
Example 3: Nucleic Acid
A. RNA Extraction from Tissue. Total RNA was isolated from breast tissues and
from non-breast tissues. Various methods were 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, 1987), and TRIzoITM (Gibco-BRL, Grand Island, NY).
Briefly, tissue was placed in a sterile conical tube on ice and 10-15 volumes
of 3 M
LiCl, 6 M urea, 5 mM EDTA, 0.1 M 2-mercaptoethanol and 50 mM Tris-HCl (pH 7.5)
were added. The tissue was homogenized with a Polytrono homogenizer (Brinkman
Instruments, Inc., Westbury, NY) for 30-50 sec on ice. The solution was
transferred to a
15 ml plastic centrifuge tube and placed overnight at -20 C. The tube was
centrifuged for
90 min at 9,000 x g at 0-4 C and the supernatant was immediately decanted.
Ten ml of 3
M LiCI were added and the tube was vortexed for 5 sec. The tube was
centrifuged for 45
min at 11,000 x g at 0-4 C. The decanting, resuspension in LiCI, and
centrifugation were
repeated and the final pellet was air dried and suspended in 2 ml of 1 mM
EDTA, 0.5%
SDS, 10 mM Tris (pH 7.5). Twenty microliters (20 l) of Proteinase K (20
mg/ml) were
added, and the solution was incubated for 30 min at 37 C with occasional
mixing. One-
tenth volume (0.22-0.25 ml) of 3 M NaCl was added and the solution was
vortexed before
transfer into another tube containing 2 ml of phenol/chloroform/isoamyl
alcohol (PCI).
The tube was vortexed for 1-3 sec and centrifuged for 20 min at 3,000 x g at
10 C. The
PCI extraction was repeated and followed by two similar extractions with
chloroform/isoamyl alcohol (CI). The final aqueous solution was transferred to
a
prechilled 15 ml Corex glass tube containing 6 ml of absolute ethanol. The
tube was
covered with parafiim and placed at -20 C overnight. The tube was centrifuged
for 30
min at 10,000 x g at 0-4 C and the ethanol supernatant was decanted
immediately. The
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RNA pellet was washed four times with 10 ml of 75% ice-cold ethanol and the
final pellet
was air dried for 15 min at room temperature. The RNA was suspended in 0.5 ml
of 10
mM TE (10 mM Tris-HC1, pH 7.6, 1 mM EDTA) and its concentration was determined
spectrophotometrically. RNA samples were aliquoted and stored at -70 C as
ethanol
precipitates.
The quality of the RNA was determined by agarose gel electrophoresis (see
Example 5) and staining with 0.5 pg/ml ethidium bromide for one hour. RNA
samples
that did not contain intact ribosomal RNAs were excluded from the study.
Alternatively, for RT-PCR analysis, 1 ml of Ultraspec RNA reagent was added to
120 mg of pulverized tissue in a 2.0 ml polypropylene microfuge tube,
homogenized with
a Polytron homogenizer (Brinkman Instruments, Inc., Westbury, NY) for 50 sec
and
placed on ice for 5 min. 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 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
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 were
isolated from blood samples from patients by centrifugation using Ficoll-
Hypaque as
follows. A 10 ml volume of whole blood was mixed with an equal volume of RPMI
Medium (Gibco BRL, Grand Island, NY). This mixture was 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 was removed, diluted to 50 ml
with
Dulbecco's PBS (Gibco BRL, Grand Island, NY) and the mixture centrifuged for
10
minutes at 200 x g. After two washes, the resulting pellet was resuspended in
Dulbecco's
PBS to a final volume of 1 ml.
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RNA was prepared from the isolated mononuclear cells by the TRIZOL Reagent
following the manufacturer's (Gibco BRL, Grand Island, NY) instructions.
Briefly, the
pelleted cells were resuspended in TRIZOL Reagent and lysed by repetitive
pipetting.
The mixture was homogenized and incubated at 15 to 30 C for 5 minutes.
Chloroform
was added to the homogenate and the tubes were vigorously shaken by hand for
15
seconds, then incubated at 15 to 30 C for 2 to 3 minutes. The samples were
centrifuged
at 12,000 x g for 15 minutes at 2 to 8 C and the aqueous phase containing the
RNA was
removed to a fresh tube. The RNA was precipitated by mixing with isopropanol,
incubated for 10 minutes at 15 to 30 C, and centrifuged at 12,000 x g for 10
minutes at 2
to 8 C. The supernatant was removed and the RNA pellet was washed once with
75%
ethanol. The pellet was briefly dried and resuspended in RNAse-free water by
passing the
solution through a pipette tip and incubating for 10 minutes at 55 to 60 C.
The RNA was
quantitated by UV spectrophotometry.
C. RNA Extraction from pol s~ omes. Tissue is minced in saline at 4 C and
mixed
1s with 2.5 volumes of 0.8 M sucrose in a TK150M (150 mM KC1, 5 mM MgC12, 50
mM
Tris-HCI, pH 7.4) solution containing 6 mM 2-mercaptoethanol. The tissue is
homogenized in a Teflon-glass Potter homogenizer with five strokes at 100-200
rpm
followed by six strokes in a Dounce homogenizer, as described by B. Mechler,
Methods in
Enzymology 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 TK15OM and layering this mixture
over 4 ml of
2.5 M sucrose in TK150M in a 38 ml polyallomer tube. Two additional sucrose
TK150M
solutions are successively layered onto the extract fraction; a first layer of
13 ml of 2.05 M
sucrose is followed by a second layer of 6 ml of 1.3 M sucrose. The polysomes
are
isolated by centrifuging the gradient at 90,000 x g for 5 h 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 NaCl, 20 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
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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 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 (10 mM Tris-HC1, pH 7.4, 1 mM
5 EDTA) 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 BS 106 mRNA (see Example 6).
The quality of nucleic acid and proteins is dependent on the method of
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
10 ordinary artisan.
Example 4: Ribonuclease Protection Assay
A. Synthesis of Labeled Complementary RNA (cRNA) Hybridization Probe and
Unlabeled Sense Strand. A pINCY plasmid containing the BS106 gene cDNA
sequence
insert (clone 1662885), flanked by opposed SP6 and T7 polymerase promoters,
was
15 purified using Qiagen Plasmid Purification Kit (Qiagen, Chatsworth, CA).
Then, 10 g of
the plasmid were cut with 10 U Dde I restriction enzyme for 1 h at 37 C. The
cut plasmid
was purified using QlAprep kits (Qiagen, Chatsworth, CA). The purified plasmid
was
used for the synthesis of antisense transcript labeled with 10 M (alpha32P)
CTP (800
Ci/mmol)(Amersham Life Sciences, Inc. Arlington Heights, IL) starting from the
T7
20 promoter using the Riboprobe in vitro Transcription System (Promega
Corporation,
Madison, WI), as described by the supplier's instructions. To generate the
sense strand,
10 pg of the purified plasmid were cut with restriction enzymes IOU Xba I and
10 U Not
I, and 250 ng were transcribed with unlabeled CTP from the SP6 promoter. Both
sense
and antisense strands were isolated by spin column chromatography. Unlabeled
sense
25 strand was quantitated by UV absorption at 260 nm.
B. Hybridization of Labeled Probe to Target. Frozen tissue was pulverized to
powder under liquid nitrogen and 100-500 mg were dissolved in lysis buffer
available as a
component of the RNAqueousTM RNA Isolation Kit (Ambion, Inc., Austin, TX).
Further
dissolution was achieved using a tissue homogenizer and RNA isolated as
described by the
30 supplier's instructions. In addition, a dilution series of a known amount
of sense strand in
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yeast RNA was made for use as a positive control. Finally, up to 5.0 g of
purified tissue
RNA or diluted sense strand were co-precipitated with 1 x105 cpm of
radioactively labeled
probe (1.5 x109 cpm/ g) in ammonium acetate and ethanol. Pellets were air
dried and
then resuspended in 20 l of hybridization buffer supplied as a component of
the RPA IITM
Ribonuclease Protection Assay Kit, (Ambion, Inc., Austin, TX). After heat
denaturation at
90 C for 5 minutes, hybridization was allowed to proceed overnight at 45 C.
C. RNase Digestion. RNA that was not hybridized to probe was removed from
the reaction as per the RPA IITM protocol using a solution of RNase A and
RNase Tl for
30 min at 37 C. Hybridized fragments protected from digestion were then
precipitated
according to the supplier's instructions and by centrifugation at 12,000 x g
for 15 min.
D. Fragment Analysis. The precipitates were 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 were imaged and analyzed using the STORMTM
storage
phosphor autoradiography system (Molecular Dynamics, Sunnyvale, CA).
Quantitation of
protected fragment bands, expressed in femtograms (fg), was 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 as an
image rating
score in Table 1. Samples with no detectable protected fragment were scored "-
"; samples
with detectable protected fragment, the fg values of which were within the
standard curve,
were scored "+".
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Table 1: RNase Protection Results
Tissue Score Tissue Score
Placental DNA - Malignant Breast -
Normal Breast - Malignant Breast +
Normal Breast + Malignant Breast +
Normal Breast + Malignant Breast +
Normal Breast + Normal Lung -
Normal Breast + Malignant Lung -
Malignant Breast - Normal Colon -
Malignant Breast - Malignant Colon -
Example 5: Northern Blotting
Northern blotting is a well known technique in the art. The Northern blot
technique was used to identify a specific size RNA fragment from a complex
population
of RNA using gel electrophoresis and nucleic acid hybridization. Briefly, 5-10
gg of total
RNA (see Example 3) were incubated in 15 l of a solution containing 40 mM
morpholinopropanesulfonic 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
was
mixed with 2 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
was electrophoresed at 60 V for 1.5 h and rinsed in RNAse free water. RNA was
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 was rinsed with 1X SSC, and RNA
was
crosslinked to the filter using a Stratalinker (Stratagene, Inc., La Jolla,
CA) on the
autocrosslinking mode and dried for 15 min. The membrane was then placed into
a
hybridization tube containing 20 ml of preheated prehybridization solution (5X
SSC, 50%
formamide, 5X Denhardt's solution, 100 gg/ml denatured salmon sperm DNA) and
incubated in a 42 C hybridization oven for at least 3 h. While the blot was
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prehybridizing, a 32P-labelled random-primed probe was generated using the
BS106 insert
fragment (obtained by digesting clone 603148H1 with Xbal and Notl) using
Random
Primer DNA Labelling System (Life Technologies, Inc., Gaithersburg, MD)
according to
the manufacturer's instructions. Half of the probe was boiled for 10 min,
quick chilled on
ice and added to the hybridization tube. Hybridization was carried out at 42
C for at least
12 h. The hybridization solution was discarded and the filter was 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 was wrapped in plastic wrap, exposed to Kodak XAR-Omat
film for
8-96 h, and the film was developed for analysis.
Results of the analysis of RNA quality using an ethidium bromide stained
agarose
gel and the corresponding northern blot using BS106 probe hybridized to RNAs
from
breast tissues and prostate tissues, and from normal breast and breast cancer
tissues are
shown in Figures 3A and B, respectively. The positions of RNA size standards
(in kb) are
shown to the left of each panel. As shown in Figure 3A, the BS 106 probe
hybridized to an
RNA band of approximately 0.7 kb in 3 of 3 normal breast tissues (lanes 1 -
3), and in 1
of 3 prostate cancer tissues (lane 7). The RNA band was not detected in 3
normal prostate
tissues (lanes 4 - 6) nor in a prostate or breast cancer cell line (lanes 10
and 11). Figure
3B shows that the BS106 probe hybridized to an RNA of approximately 0.7 kb in
5 of 6
normal breast RNA samples (lanes 1- 4 and 6) and 2 of 6 breast cancer RNA
samples
(lanes 7 and 11).
Detection of a product comprising a sequence 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, and fragments or complements
thereof, is indicative of the presence of BS106 mRNA, suggesting a diagnosis
of a breast
tissue disease or condition, such as breast cancer.
Example 6: Dot Blot/Slot Blot
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. Commercially available
kits
make these assays especially attractive.
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A dot blot analysis was performed using a Clontech (Clontech Laboratories,
Inc.,
Palo Alto, CA) Multiple Tissue Expression ArrayTM containing polyA RNA from 76
different human tissues. BS106 plasmid DNA was labeled by nick translation
with
[32P]dCTP (Amersham Pharmacia Biotech) to a specific activity of 108 cpm/g.
Approximately 63 ng of 32P-labeled probe was used to hybridize dots on the
Clontech
array. Hybridization was carried out according to instructions supplied with
the Clontech
kit. Briefly, labeled probe was hybridized to the expression array membrane at
65 C for
hours. The membrane was washed five times with 2X SSC, 1% SDS at 65 C
followed
by two washes with 0.1X SSC, 0.5% SDS at 55 C. The hybridization image was
10 analyzed using the Molecular Dynamics STORM 840 Phosphor Imaging systemTM
(Molecular Dynamics, Sunnyvale, CA).
Results of the analysis are shown in Figure 4. Identification of the dots was
provided with the kit. Positive signals were detected with mRNA isolated from
human
salivary gland and mammary gland and none of the 74 remaining human tissues.
Positive
15 signals were also obtained for E.coli DNA and human genomic DNA.
Hybridization to the
E. coli DNA was expected since the BS 106 plasmid was isolated directly from
E. coli
without any subsequent purification away from residual E. coli DNA. The
hybridization
to human genomic DNA could indicate that BS106 might belong to a multi-gene
family.
Example 7: In Situ Hybridization
This method is useful to directly detect specific target nucleic acid
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 7.5 at 4 C for 30 min. The
solution is
changed with fresh glutaraldehyde solution (1% glutaraldehyde in 50 mM sodium
phosphate, pH 7.5) for a further 30 min fixing. The fixing solution should
have an
osmolality of approximately 0.375% NaCl. The tissue is washed once in isotonic
NaCl to
remove the phosphate.
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The fixed tissues then are embedded in paraffin as follows. The tissue is
dehydrated though a series of ethanol concentrations for 15 min each: 50%
(twice), 70%
(twice), 85%, 90% and then 100% (twice). Next, the tissue is soaked in two
changes of
xylene for 20 min each at room temperature. The tissue is then soaked in two
changes of a
5 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
10 a series of ethanol concentrations: 99% twice, 95%, 85%, 70%, 50%, 30%, and
then
distilled water twice. The sections are pre-treated with 0.2 M HCl for 10 min
and
permeabilized with 2 g/ml Proteinase-K at 37 C for 15 min.
Labeled riboprobes transcribed from the BS106 gene plasmid (see Example 4) are
hybridized to the prepared tissue sections and incubated overnight at 56 C in
3X standard
15 saline extract and 50% formamide. Excess probe is removed by washing in 2X
standard
saline citrate and 50% formamide followed by digestion with 100 g/ml RNase A
at 37 C
for 30 min. Fluorescence probe is visualized by illumination with ultraviolet
(UV) light
under a microscope. Fluorescence in the cytoplasm is indicative of BS 106
mRNA.
Alternatively, the sections can be visualized by autoradiography.
Example 8: Reverse Transcription PCR
A. One Step RT-PCR Assay. 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 performs both RT and
PCR in a
single reaction mixture. The procedure is performed in a 200 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 M each of dNTP, 0.25 pM each primer, 5U
rTth polymerase, 3.25 mM Mn(OAc)2 and 5 pl of target RNA (see Example 3).
Since
RNA and the rTth polymerase enzyme are unstable in the presence of Mn(OAc)2,
the
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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. 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, I min; 72 C, 2 min. One step RT-PCR also
may be
performed by using a dual enzyme procedure with Taq polymerase and a reverse
transcriptase enzyme, such as MMLV (Moloney murine leukemia virus) or AMV
(avian
myeloblastosis virus) RT (reverse transcriptase) enzymes.
B. Traditional RT-PCR. Alternatively, a traditional two-step RT-PCR reaction
was performed, as described by K.Q. Hu et al., Virology 181:721-726 (1991).
Briefly, 0.5
g of extracted mRNA (see Example 3) was reverse transcribed in a 20 l
reaction
mixture containing 1X PCR II buffer (Perkin-Elmer), 5 mM MgC12, 1 mM dNTP, 20
U
RNasin, 2.5 pM random hexamers, and 50 U MMLV (Moloney murine leukemia virus)
reverse transcriptase (RT). Reverse transcription was performed at 42 C for
60 min in an
MJ Research Cycler Model PTC-200, followed by further incubation at 95 C for
5 min to
inactivate the RT. PCR was performed using 2 l of the cDNA reaction in a
final PCR
reaction volume of 50 l containing 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 2 mM
MgC12, 200 M dNTP, 0.5 gM of each sense and antisense primer, SEQUENCE ID NO
13 and SEQUENCE ID NO 14, respectively, and 2.5 U of Taq polymerase. The
reaction
was incubated in an MJ Research Model PTC-200 as follows: 40 cycles of
amplification
(94 C, 20 see; 58 C, 30 sec; 72 C, 30 sec); a final extension (72 C, 10
min); and a soak
at 4 C.
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 45 min. Gels were then destained in 1X TBE for 30 min.
and
imaged using a STORM imaging system (Figures 5A and 5B). Figure 5A shows a DNA
band at 201 bases which is indicative of a BS106 mRNA-specific RT-PCR product.
This
band is present in RNA of 5 of 5 normal breast tissue samples (lanes 3-7) and
in RNA of 4
of 5 breast cancer tissue samples (lanes 8 - 11). It is absent in placental
DNA (lane 2). As
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shown in Figure 513, the product was not detected using RNA isolated from
colon tissue
(lanes 1-5), lung tissue (lanes 6-10), nor in placental DNA (lane 11).
Detection of a product comprising a sequence 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, and fragments or complements
thereof, is indicative of the presence of BS 106 mRNA, suggesting a diagnosis
of a breast
tissue disease or condition, such as breast cancer.
Example 9: OH-PCR
A. Probe selection and Labeling. Target-specific primers (SEQUENCE ID NO 15
and SEQUENCE ID NO 16) and a probe (SEQUENCE ID NO 17) were 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 oligonucleotides at their
5' and 3' ends,
respectively. The label-phosphoramidite reagent was prepared and used to add
the label to
the oligonucleotide during its synthesis. [See N. T. Thuong et al., Tet.
Letters
29(46):5905-5908 (1988); J. S. Cohen et al., published U.S. Patent Application
07/246,688
(NTIS ORDER No. PAT-APPL-7-246,688) (1989)]. Probes were labeled at their 3'
end to
prevent participation in PCR and the formation of undesired extension
products. For one-
step OH-PCR, the probe had a TM at least 15 C below the TM of the primers.
The
primers and probes were utilized as specific binding members, with or without
detectable
labels, using standard phosphoramidite chemistry and/or post-synthetic
labeling methods
which are well-known to one skilled in the art.
B. One Step Oligo Hybridization PCR. The cDNA was prepared from total RNA
(see Example 3) using 2.5 U MMLV Reverse Transcriptase in a buffer of 50 mM
KCI, 10
mM Tris-HCl (pH 8.3), 5 mM MgC12, 1 mM dGTP, 1 mM dATP, 1 mM dCTP, 1 mM
dTTP, 1.0 U RNase Inhibitor, 2.5 M Oligo d(T) 16 at 42 C for 30 minutes
followed by
99 C for 5 minutes and 4 C for 5 minutes. The prepared cDNA was then added
to the
OH-PCR reaction which contained 190 l of 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
CA 02399047 2002-07-30
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78
ethylene diaminetetraacetic acid, 0.02 mg/ml NaN3, 8% w/v glycerol, 150 M
each of
dNTPs, 0.335 M forward primer (SEQUENCE ID NO 15), 0.586 M reverse primer
(SEQUENCE ID NO 16), 20.0 nM probe (SEQUENCE ID NO 17), 5U rTth polymerase,
and 3.25 mM MnC12. The MnC12 was added just prior to target addition, since
rTth
polymerase enzyme is unstable in the presence of MnC12. Alternatively, total
RNA.(see
Example 3) can be used as a target instead of cDNA. The reaction was incubated
in a
Perkin-Elmer Thermal Cycler 480. Optimal conditions for eDNA synthesis and
thermal
cycling can be readily determined by those skilled in the art. Conditions
which were
found useful included cDNA synthesis (60 C, 30 min) when starting with RNA,
and 37
amplification cycles (94 C, 40 sec; 62 C, 80 sec). Oligo hybridization was
performed
subsequent to amplification (97 C, 5 min; 12 C, 5 min; 12 C soak). The
correct
reaction product contained at least one of the strands of the PCR product and
an internally
hybridized probe.
C. OH-PCR Product Analysis. Amplified reaction products were detected on an
LCx Analyzer system (available from Abbott Laboratories, Abbott Park, IL).
Briefly, the
specific reaction product was captured by an antibody labeled microparticle at
a capturable
site on the hybridization probe and the complex was detected by binding of a
detectable
antibody conjugate to the PCR product. Only the complex containing a PCR
strand
hybridized with the internal probe was detectable. Figure 6 shows the BS106
amplicon
was easily detected at 18 picograms of RNA from the MDA 361 cell line. The MDA-
361
cell line (#HTB27) originated from a metastatic human breast adenocarcinoma
(A.T.C.C.,
10801 University Blvd., Manassas, VA). PolyA RNA was used as a negative
control and
undetectable even at 400 nanograms.
Ten picograms of RNA from various tissues were assayed for BS106 activity
using
the LCx" system. Figure 7 shows the overall tissue distribution for the BS 106
marker. All
5 normal breast (columns 1 - 5) and 3 of 6 breast cancer tissue RNAs (columns
6, 9, and
11) were reactive in the BS106 LCx" assay, while lung cancer, normal colon,
and colon
cancer tissue RNAs (columns 13-15) were non-reactive. Normal lung tissue RNA
(column 12) was slightly reactive.
The detection of this complex is indicative of the presence of B S 106 mRNA,
suggesting a diagnosis of a breast disease or condition, such as breast
cancer.
CA 02399047 2009-08-25
79
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 BS 106-
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 BS 106 polypeptide consensus sequence (see Example
1). In
particular, a number of BS 106 peptides derived from SEQUENCE ID NO 20 were
prepared, including the peptides of SEQUENCE ID NOS 21 - 33. All peptides were
synthesized on a Symphony Peptide Synthesizer (available from Rainin
Instrument Co,
Emeryville California), using FMOC chemistry, standard cycles and in-situ HBTU
activation. Cleavage and deprotection conditions were as follows: a volume of
2.5 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) was 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 cleaveage reagent with cold diethyl ether. Each peptide was then
filtered,
purified via reverse-phase preparative HPLC using a water/acetonitrile/0.1 %
TFA
gradient, and lyophilized. The product was confirmed by mass spectrometry
(data not
shown).
The purified peptides were mixed with adjuvant, and injected into rabbits (see
Example 14). Alternatively, the purified peptides may be conjugated to Keyhole
Limpet
Hemocyanin with glutaraldehyde, mixed with adjuvant, and injected into rabbits
(see
Example 14).
Example 11 a: Expression of Protein in a Cell Line Using Plasmid 577
A. Construction of a BS 106 Expression Plasmid. Plasmid 577, described in U.S.
Patent No. 6,020,122, issued February 1, 2000, has been constructed for the
expression of
secreted antigens in a permanent cell
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line. This 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
directing
5 expression of a dihydrofolate reductase gene under the control of an Simian
Virus 40
(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 Simian Virus 40 T-Ag promoter and
transcription enhancer, the hepatitis B virus surface antigen (HBsAg) enhancer
I followed
10 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.
Plasmids for the expression of secretable BS 106 proteins are constructed by
15 replacing the hepatitis C virus E2 protein coding sequence in plasmid 577
with that of a
BS 106 polynucleotide sequence 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, and fragments or complements thereof, as follows.
Digestion of plasmid 577 with Xbal releases the hepatitis C virus E2 gene
fragment. The
20 resulting plasmid backbone allows insertion of the BS 106 cDNA insert
downstream of the
rabbit immunoglobulin heavy chain signal sequence which directs the expressed
proteins
into the secretory pathway of the cell. The BS 106 cDNA fragment is generated
by PCR
using standard procedures. Encoded in the sense PCR primer sequence is an Xbal
site,
immediately followed by a 12 nucleotide sequence that encodes the amino acid
sequence
25 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 BS106 gene. The antisense primer incorporates a sequence encoding
the
following eight amino acids just before the stop codons: Asp-Tyr-Lys-Asp-Asp-
Asp-Asp-
30 Lys (SEQUENCE ID NO 34). Within this sequence is incorporated a recognition
site to
aid in analysis and purification of the BS 106 protein product. A recognition
site (termed
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81
"FLAG") that is recognized by a commercially available monoclonal antibody
designated
anti-FLAG M2 (Eastman Kodak, Co., New Haven, CT) can be utilized, as well as
other
comparable sequences and their corresponding antibodies. For example, PCR is
performed using GeneAmporeagents obtained from Perkin-Elmer-Cetus, as directed
by
the supplier's instructions. ,PCR primers are used at a final concentration of
0.5 PM. PCR
is performed on the BS106 plasmid template in a 100 l 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.
B. Transfection of Dihydrofolate Reductase Deficient Chinese Hamster Ovary
Cells. The plasmid described supra is transfected into CHO/DHFR- cells [DXB-1
11,
Uriacio et al., Proc. Natl. Acad. Sci. USA 77:4451-4466 (1980)]. These cells
are
available from the A.T.C.C., 10801 University Blvd., Manassas, VA, under
Accession No.
CRL 9096. Transfection is carried out using the cationic liposome-mediated
procedure
described by P. L. Feigner 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
transfection. Twenty micrograms (20 g) of plasmid DNA are added to 1.5 ml of
Opti-
MEM I medium and 100 pl 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 Opti-MEM I medium. The Opti-
MEM I-
Lipofection-plasmid DNA solution then is overlaid onto the cells. The cells
are incubated
for 3 h at 37 C, after which time the Opti-MEM I-Lipofectin-DNA solution is
replaced
with culture medium for an additional 24 h prior to selection.
C. Selection and Amplification. One day after transfection, cells are 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 G418 (Gibco-
BRL;
Grand Island, NY). Media volume-to-surface area ratios of 5 ml per 25 cm2 are
CA 02399047 2009-08-25
82
maintained. After approximately two weeks, DHFR/G418 cells are expanded to
allow
passage and continuous maintenance in F-12 minus medium G.
Amplification of each of the transfected BS 106 cDNA sequences is achieved by
stepwise selection of DHFR+, G418+ cells with methotrexate (reviewed by R.
Schimke,
Cell 37:705-713 [1984]). Cells are incubated with F-12 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 M MTX.
D. Antigen Production. F-12 minus medium G supplemented with 5 M MTX is
overlaid onto just confluent monolayers for 12 to 24 hat 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) (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 h at
37 C in 5% CO2. Cells then are overlaid with VAS for production at 5 ml per T
flask.
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 Breast Tissue Gene BS 106 Antigen Expression. Aliquots of VAS
supernatants from the cells expressing the BS106 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 BS 106 protein containing the FLAG
sequence
is performed by immunoaffmity chromatography using an affinity matrix
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-HC1(pH
7.5),
150 mM NaCl buffer using a Sephadex G-25 (Pharmacia Biotech Inc., Uppsala,
Sweden)
column. Protein in this buffer is applied to the anti-FLAG M2 antibody
affinity column.
CA 02399047 2009-08-25
83
Non-binding protein is eluted by washing the column with 50 mM Tris-HC1(pH
7.5), 150
mM NaCl buffer. Bound protein is eluted using an excess of FLAG peptide in 50
mM
Tris-HC1(pH 7.5), 150 mM NaCl. The excess FLAG peptide can be removed from the
purified BS 106 protein by gel electrophoresis or HPLC.
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 BS 106 gene is
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 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 those containing fusion sequences of
CKS are
described in EPO Publication No. 0331961, published September 13, 1989. This
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 Using pcDNA3.1/Myc-His
A. Construction of a BS106 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. The myc-his tag (SEQUENCE ID NO 35)
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.
A plasmid for the expression of secretable BS 106 protein was constructed by
inserting a BS 106 polynucleotide sequence from clone 1662885 (SEQUENCE ID NO
5)
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84
into the pcDNA3.1 /Myc-His vector. Prior to construction of the BS 106
expression
plasmid, the BSI 06 cDNA sequence was first cloned into a pCR -Blunt vector.
The
BS106 cDNA fragment was generated by PCR performed using Stratagene reagents
obtained from Stratagene, as directed by the supplier's instructions. PCR
primers are used
at a final concentration of 0.5 M. PCR using 5 U of pfu polymerase
(Stratagene, La
Jolla, CA) was performed on the BS 106 plasmid template (see Example 2) in a
50 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 (SEQUENCE ID
NO
18) comprises nucleotides which are identical to the pINCY vector directly
upstream of
the B S 106 gene insert. The antisense primer (SEQUENCE ID NO 19) incorporated
a 5'
Notl restriction sequence and a sequence complementary to the 3' end of the BS
106
cDNA insert just upstream of the 3'-most, in-frame stop codon. Five
microliters (5 l) of
the resulting blunted-ended PCR product were ligated with 25 ng of linearized
pCR -
Blunt vector (Invitrogen, Carlsbad, CA) interrupting the lethal ccdB gene of
the vector.
The resulting ligated vector was transformed into TOP 10 E. coli (Invitrogen,
Carlsbad,
CA) using a One ShotTM transformation kit (Invitrogen, Carlsbad, CA) following
supplier's directions. The transformed cells were grown on LB-Kan (50 g/ml
kanamycin) selection plates at 37 C. Only cells containing a plasmid with an
interrupted
ccdB gene grew after transformation (Grant, S.G.N., PNAS 87:4645-4649 (1990)).
Transformed colonies were picked and grown in 3 ml of LB-Kan broth at 37 C.
Plasmid
DNA was isolated by using a QIAprep"(Qiagen Inc., Santa Clarita, CA)
procedure, as
directed by the supplier's instructions. The DNA was digested with EcoRI and
NotI
restriction enzymes to release the BS106 insert fragment. The fragment was
electrophoresed on 1% Seakem LE agarose (FMC, Rockland, ME)/0.5 g/ml ethidium
bromide/TE gel, visualized by UV illumination, excised and purified using
QlAquickTM
(Qiagen Inc., Santa Clarita, CA) procedures, as directed by the supplier's
instructions.
The pcDNA3.1/Myc-His plasmid DNA was linearized by digestion with EcoRl
and NotI, sites present in the polylinker region of the plasmid DNA. The B S
106 purified
fragment, sura_, was ligated with the resulting plasmid DNA downstream from a
CMV
promoter, and transformed into DH5 alphaTM cells (GibcoBRL Gaithersburg, Md),
as
directed by the supplier's instructions. Briefly, 10 ng of pcDNA3. I /Myc-His
containing
CA 02399047 2009-08-25
the BS106 insert were added to 50 pl of competent DH5 alpha cells, and the
contents were
mixed gently. The mixture was incubated on ice for 30 min, heated 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 was incubated for 1 h at 37 C while shaking at 225 rpm. The
transformed cells
5 then were plated onto 100 mm LB/Amp (50 pg/ml ampicillin) plates and grown
at 37 C.
Colonies were picked and grown in 3 ml of LB/ampicillin broth. Plasmid DNA was
purified using a QIAprep kit. The presence of the insert was confirmed using
restriction
enzyme digestion and gel analysis (J. Sambrook et al., supra.).
B. Transfection of Human Embryonic Kidney 293 Cells. The BS106 expression
to plasmid described in Section A, supra, was retransformed into DH5 alpha
cells, plated
onto LB/ampicillin agar, and grown in 10 ml of LB/ampicillin broth, as
described
hereinabove. The plasmid was purified using a QIAfilterTM Maxi kit (Qiagen,
Chatsworth,
CA) and 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., 10801 University Blvd., Manassas,
VA, under
15 Accession No. CRL 1573. Transfection was carried out using the cationic
lipofectamine-
mediated procedure described by P. Hawley-Nelson et al., Focus 15.73 (1993).
Particularly, HEK293 cells were cultured in 10 ml DMEM media, supplemented
with 10%
fetal bovine serum (FBS), L-glutamine (2 mM), and freshly seeded into 12 x 100
mm
culture plates at a density of 8'x 106 cells per plate . The cells were grown
at 37 C to a
20 confluency of between 70% and 80% for transfection. Eight micrograms (8 g)
of
plasmid DNA were added to 800 l of Opti-MEM I medium (Gibco-BRL, Grand
Island,
NY), and 48-96 l of LipofectamineTM Reagent (Gibco-BRL, Grand Island, NY)
were
added to a second 800 l portion of Opti-MEM I medium. The two solutions were
mixed
and incubated at room temperature for 15-30 min. After the culture medium was
removed
25 from the cells, the cells were washed once with 10 ml of serum-free DMEM.
The Opti-
MEM I-Lipofectamine-plasmid DNA solution was diluted with 6.4 ml of serum-free
DMEM and then overlaid onto the cells. The cells were incubated for 5 h at 37
C, after
which time an additional 8 ml of DMEM with 20% FBS were added. After 18-24 h,
the
old medium was aspirated, and the cells were overlaid with 5 ml of fresh DMEM
with 5%
30 FBS. Supernatants and cell extracts were analyzed for BS106 gene activity
72 h after
transfection.
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86
C. Production of stable cell line, HEK293 - 106C1. The BS 106 expression
plasmid described in section A, su r was retransformed into DH5 alpha cells,
plated onto
LB/ampicillin agar, and grown up in 10 ml of LB/ampicillin broth. The plasmids
were
purified using a QIAfilterTM Maxi Kit (Qiagen, Chatsworth, CA) and were
transfected into
HEK293 cells [F.L. Graham et al., J. Gen. Vir. 36:59-72 (l977)].
The purified expression plasmid, as described supra, was 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., 10801 University Blvd., Manassas, VA, under Accession No. CRL 1573.
Transfection of the expression plasmid was performed using the cationic
lipofectamine
mediated procedure described by P. Hawley-Nelson et al., Focus 15.73 (1993).
Particularly, HEK293 cells were cultured in 10 ml DMEM media supplemented with
10%
fetal bovine serum (FBS), L-glutamine (2 mM), sodium pyruvate (1 mM) and
essential
amino acids and freshly seeded into 60 mm culture plates at a density of 9 x
106 cells per
plate. The cells were grown at 37 C to a confluency of between 70% and 80%
for
is transfection. Two micrograms (2 g) of plasmid DNA were added to 800 l of
unsupplemented DMEM medium (Gibco-BRL, Grand Island, NY). Eight microliters (8
1) of Plus Reagent (Gibco-BRL, Grand Island, NY) were added to this solution,
which
was then mixed briefly. Twelve microliters (12 l) of Lipofectamine (LTI) were
added to
a second 800 l portion of unsupplemented DMEM media. After a 15 minute
incubation,
the two solutions were mixed and incubated at room temperature for an
additional 15-30
minutes. During this time the culture medium was removed from the plates
containing the
HEK293 cells. The DMEM containing the Plus reagent:Lipofectamine plasmid DNA
complex was then overlaid onto the cells. The cells were incubated for 5 h at
37 C and
5% CO2, after which time, an additional 2 - 8 ml of DMEM with 20% FBS were
added.
After 18-24 h, the old medium was aspirated, and the cells were overlaid with
5 ml of
fresh DMEM with 5% FBS containing 400 g/ml G418, and the incubation was
continued
until 72 h had elapsed. Supernatants were analyzed for BS106 M/H (Myc/His)
polypeptide
expression by Western blot analysis.
At 72 hours post transfection, the cells were released from the dish by
limited
trypsinization and reseeded into 100 mm culture dishes in DMEM, 10% FBS, and
400
gg/ml G418 at dilutions of 1:100, 1:1000 and 1:10000. These cultures were
allowed to
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grow for 5-7 days, until well-isolated foci of cells were identified by
microscopy. These
foci were isolated by cloning cylinders, their cells released by limited
trypsinization, and
individual foci were transferred to separate wells in 24-well dishes, again in
DMEM, 10%
FB S, 400 g/ml G418. After growth for 7-10 days, the supernatants of each
well were
analysed for BS106 M/H expression by Western blot analysis, as described
hereinbelow.
The clonal line labeled 106C1 was found to express BS106 M/H in the
supernatant. This
line was expanded into 75 cm2 flasks, and then passaged 1:30 three times,
following
expression of BS106 M/H to ensure stability of the insertion event. The final
product of
this procedure was a cell line derived from HEK293 cell line that expresses
BS106 M/H,
which we have labeled 106C1 (HEK293-106C1).
D. Analysis of Breast Tissue Gene BS106 Antigen Expression. The culture
supernatant, (Example l lb, Subsection B), was transferred to cryotubes and
stored on ice.
HEK293 cells were 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 Mannheim,
Indianapolis,
IN), followed by incubation for 30 min at room temperature. Lysate was
transferred to
1.7 ml polypropylene microfuge tubes and centrifuged at 1000 x g for 10 min.
The
supernatant was transferred to new cryotubes and stored on ice. Aliquots of
supernatants
from the cells and the lysate of the cells expressing the B S 106 protein
construct were
analyzed for the presence of BS 106 recombinant protein. The aliquots were run
on SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) using standard methods and
reagents
known in the art (J. Sambrook et al., supra). These gels were then blotted
onto
nitrocellulose, and the BS106 protein band was visualized using Western
blotting
techniques with an anti-myc epitope monoclonal antibody (Invitrogen, Carlsbad,
CA).
Figure 8 shows the resultant Western blot. Lane 1 contains biotinylated
molecular weight
markers. Lane 2 contains colored molecular weight markers. Lane 3 contains the
cell
lysate of transiently transfected HEK293 cells. Lane 4 contains the
supernatant from the
transiently transfected HEK293 cells. Lane 5 contains the cell lysate of a
negative control
(HEK293 cells not transiently transfected). Lane 6 contains the supernatant of
a negative
control (HEK293 cells not transiently transfected). Lane 4 shows BS106 M/H as
a broad
band at approximately 40 kD. Lane 3 shows BS106 M/H as a sharp band at
approximately 17 kD. The difference in molecular weight between the two
samples is
CA 02399047 2009-08-25
88
attributed to glycosylation. Specifically, BS106 M/H in lane 3 is found inside
the cell,
which has yet to be fully post-translationally modified. The BS 106 M/H of
lane 4, which
has been secreted from the cell, is a more mature, more fully modified
protein.
Heterogeneity in glycosylation results in molecular weight differences among
the
molecules contributing to the broadness of the band observed in lane 4.
E. Purification. Purification of the BS 106 recombinant protein containing the
myc-his sequence was performed using Chelating Sepharose Fast Flow (Pharmacia
Biotech, Piscataway, NJ) charged with nickel which specifically binds
polyhistidine
residues. Three hundred fifty milliliters (350 ml) of supernatant from the
transiently
transfected HEK293 cells, prepared as described in subsection B, were pooled
and passed
over the 40 ml nickel-charged column. Non-binding protein was eluted by
washing the
column with 10 mM Tris-HC1 (pH 7.4)/500 mM NaCl buffer. Bound BS106
recombinant
protein then was eluted from the column using an imidazole gradient. The flow
rate was 2
ml/min; the gradient was 3.1 mM imidazole per milliliter of buffer; and the
elution time
was 80 minutes, creating an elution profile that went from 0 to 500 mM
imidazole.
Each 2 ml fraction was sampled. One hundred microliters of each fraction was
applied to a well of a dot blot apparatus and the volume was suctioned through
a piece of
nitrocellulose. The nitrocellulose filter was then developed with the same
procedure to
develop Western blots, as described supra, using a monoclonal antibody
recognizing a
myc epitope. Figure 9 illustrates the developed dot blot, which shows
immunorecognition
of material in fractions 20 - 31 by the anti-myc monoclonal antibody. These
fractions
correspond to elution conditions of 125 - 200 mM imidazole indicating the
successful
binding of the histidine tagged proteins to the nickel column and their
elution with a
histidine analogue. Fractions 20 - 31 were pooled and dialysed for a minimum
of 4 hours
each, against 2 x 4 1 of phosphate buffered saline (PBS, 50 mM phosphate, 150
mM
sodium chloride, pH 7.4) using Slide-a-Lyzers'm (3500 MWCO).
The pooled, dialyzed, semi-purified supernatant was analyzed for the presence
of
BS 106 M/H recombinant protein by Western blot, as described in subsection D.
Figure 10
illustrates the blot where lane 1 contains colored molecular weight markers;
lane 2
contains the supernatant prior to chromatography; lane 3 contains the flow
through
material during sample loading; lane 4 contains the material eluting during
column
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washing; and lane 5 contains the pooled, dialyzed, eluted material. BS106 M/H
appeared
in lane 5 (and faintly in lane 2) as a broad band with an apparent molecular
weight of 40
kD, higher than the theoretical molecular weight of the peptide backbone of
10.7 kD. The
added molecular mass of the secreted protein product is attributed to
glycosylation.
In a similar manner, 400 ml of supernatant from the growth of the 106C1 cells
were purified using nickel chelation chromatography. Briefly, a 1.6 cm x 20 cm
Hi-Trap
Chelate column (Pharmacia Biotech, Piscataway, NJ) was charged with 50 ml of
nickel
chloride and equilibrated with buffer, 15 mM Tris (pH 7.5)/ 450 mM NaCl. NaCl
(300
mM) was added to the crude supernatant prior to application to the column.
After sample
application, the column was washed with buffer containing 25 mM imidazole. The
myc/his tagged protein was then eluted with buffer containing 300 mM
imidazole. Eight
milliliter (8 ml) fractions were collected. These fractions were tested for
the presence of
the myc epitope by Western blot, and those fractions found to be positive were
pooled.
The pooled, purified BS 106 M/H was analysed by Western blot using both an
anti-
myc monoclonal antibody (Figure 11 panel A) and an anti-BS 106 polyclonal
antisera
(Figure 11 panel B). In panels A and B, lane 1 contains an unrelated protein;
lane 2
contains BS106 expressed in E. coli; lane 3 contains biotinylated molecular
markers; lane
4 contains colored molecular markers; lane 5 contains the pooled, purified
BS106 M/H
from 106C 1 cells; lane 6 contains colored molecular markers; and lane 7
contains
biotinylated molecular markers. BS106 M/H from the 106C1 cells was recognized
by
both anti-myc monoclonal antibody (lane 5, panel A) and the anti-BS 106
polyclonal
antisera (lane 5, panel B) and had an apparent molecular weight of 40 kD,
consistent with
the material provided by the transient transfection of HEK293 cells.
F. Coating Microtiter Plates with BS106 Expressed Proteins. Supernatant from a
100 mm plate, as described supra, is diluted in an appropriate volume of PBS.
Then, 100
l of the resulting mixture is placed into each well of a Reacti-Bind TM metal
chelate
microtiter plate (Pierce, Rockford, IL), incubated at room temperature while
shaking, and
followed by three washes with 200 l each of PBS with 0.05% Tween" 20. The
prepared
microtiter plate can then be used to screen polyclonal antisera for the
presence of BS 106
antibodies (see Example 17).
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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 the
skill of one of
ordinary skill in the art. The largest cloned insert containing the coding
region of the
5 BS106 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 (SOD) and CMP-KDO synthetase (CKS) or other
protein fusion genes for expression of the protein sequence. Methods and
vectors which
io are useful for the production of polypeptides which contain fusion
sequences of SOD are
described in published EPO application No. EP 0 196 056, published October 1,
1986, and
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
is phase immunoassays, etc.
Example 11 c: Expression of Protein in a Cell Line Using RcDNA3.1
A. Construction of a BS 106 Expression Plasmid. Plasmid pcDNA3. l (Cat.#
20 V790-20, Invitrogen, Carlsbad, CA) has been used, in the past, for the
expression of
secreted antigens by most mammalian cell lines. A plasmid for the expression
of
secretable BS 106 protein was constructed by inserting the BS 106
polynucleotide sequence
from clone 1662885inh into the pcDNA3.1 vector. In order to construct the
BS106
expression plasmid, the BS 106 cDNA fragment was excised with EcoRI and NotI
25 restriction enzymes. The fragment was electrophoresed on 1% Seakem LE
agarose
(FMC, Rockland, ME)/0.5 .tg/ml ethidium bromide/TE gel, visualized by UV
illumination, excised and purified using QlAquickt (Qiagen Inc., Santa
Clarita, CA)
procedures, as directed by the supplier's instructions.
The pcDNA3.1 plasmid DNA was linearized by digestion with EcoRl and Nod,
30 sites present in the polylinker region of the plasmid DNA. The BS 106
purified fragment
was ligated with the resulting plasmid DNA and transformed into DH5 alphatm
cells
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(GibcoBRL Gaithersburg, Md), as directed by the supplier's instructions.
Briefly, 10 ng of
pcDNA3.1 containing the BS 106 insert were added to 50 tl of competent DH5
alpha cells,
and the contents were mixed gently. The mixture was incubated on ice for 30
min, heated
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 was incubated for 1 h at 37 C while shaking at 225
rpm. The
transformed cells then were plated onto 100 mm LB/Amp (50 gg/ml ampicillin)
plates and
grown at 37 C. Colonies were picked and grown in 3 ml of LB/ampicillin broth.
Plasmid
DNA was purified using a QlAprep kit. The presence of the insert was confirmed
using
restriction enzyme digestion and gel analysis (J. Sambrook et al., supra.).
B. Transfection of Human Embryonic Kidney Cell 293 Cells. The BS106
expression plasmid described in section A, supra, was retransformed into DH5
alpha cells,
plated onto LB/ampicillin agar, and grown up in 100 ml of LB/ampicillin broth,
as
described hereinabove. The plasmid was purified using a QlAfilterTM Maxi Kit
(Qiagen,
Chatsworth, CA) and was transfected into HEK293 cells [F.L. Graham et al., J.
Gen. Vir.
1s 36:59-72 (1977)]. These cells were available from the A.T.C.C., 10801
University
Boulevard, Manassas, Virginia 20110-2209, under Accession No. CRL 1573.
Transfection was carried out using the cationic lipofectamine-mediated
procedure [P.
Hawley-Nelson et al., Focus 15.73 (1993)]. In particular, HEK293 cells were
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 2 x 106
cells per plate
for 72 hour incubation or 7 x 106 cells per plate for 24 hour incubation. The
cells were
grown at 37 C to a confluency of between 70% and 80% for transfection. Eight
micrograms (8 g) of plasmid DNA were added to 800 l of Opti-MEM I medium
(Gibco-BRL, Grand Island, NY), and 48 l of LipofectamineTM Reagent (Gibco-
BRL,
Grand Island, NY) were added to a second 800 1 portion of Opti-MEM I media.
The two
solutions were mixed gently and incubated at room temperature for 15-30 min.
After the
culture medium was removed from the cells, the cells were washed once with 10
ml of
serum-free DMEM. The Opti-MEM I-Lipofectamine-plasmid DNA solution was diluted
with 6.4 ml of serum-free DMEM and then overlaid onto the cells. The cells
were
incubated for 5 h at 37 C, after which time, an additional 8 ml of DMEM with
20% FBS
were added. After 18-24 h, the old medium was aspirated, and the cells were
overlaid
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with 5 ml of fresh DMEM with 5% FBS. Supernatants and cell extracts were
analyzed for
BS 106 gene activity 72 h after transfection.
C. Analysis of Breast Tissue Gene BS106 Antigen Expression. The culture
supernatant, supra, is transferred to cryotubes and stored on ice. HEK293
cells are
harvested by washing twice with 10 ml of cold Dulbecco's PBS and lysed by
addition of
1.5 ml of CAT lysis buffer (Boehringer Mannheim, Indianapolis, IN), 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 BS106 protein construct are analyzed for the presence
of BS 106
recombinant protein. The aliquots can be electrophoresed on SDS-polyacrylamide
gels
(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 BS106 protein band can be visualized using Western blotting
techniques with
anti-myc epitope or anti-histidine monoclonal antibodies (Invitrogen,
Carlsbad, CA) or
anti-BS 106 polyclonal serum (see Example 14). Alternatively, the expressed BS
106
recombinant protein can be analyzed by mass spectrometry (see Example 12).
Example 11 d: Expression of Protein in a Cell Line Using pGEX4T/Myc-His
A. Construction of a BS 106 Expression Plasmid. A plasmid suitable for the
production of protein BS 106 as a fusion protein in E. coli was produced. The
fusion
protein partner was glutathione-S-transferase, and the vector was designed so
that the
fused protein could be released by limited proteolysis with thrombin. All
cloning
modifications were made with the method of J. Sambrook et al, supra.
Plasmid pG4-BS106 was constructed from the BS106 polynucleotide sequence
[clone 1662885inh (SEQUENCE ID NO 5)] from the pcDNA 3.1 myc/his vector and
the
bacterial expression vector pGEX4T (Invitrogen, Carlsbad, CA). The B S 106
polynucleotide sequence [clone 1662885inh (SEQUENCE ID NO 5)] including the
myc/his tag was obtained from the pcDNA3. I /Myc-His vector construct (as
described in
Example l lb subsection A). The BamH I restriction site was inserted into the
BS106
pcDNA 3.1 myc/his vector construct to separate the coding sequence of the
protein signal
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93
sequence and the coding sequence of the mature protein. The BS106
polynucleotide
sequence [clone 1662885inh (SEQUENCE ID NO 5)] including the myc/his tag was
excised from this modified vector construct with BamH I and Pme I.
The pGEX4T vector (Invitrogen, Carlsbad, CA) was modified by inserting a Pme I
site (SEQUENCE ID NO 37) using oligonucleotide primers. The BS106
polynucleotide
sequence [clone 1662885inh (SEQUENCE ID NO 5)] including the myc/his tag was
cloned into the BamH I/Pme I sites of the modified pGEX4T vector. The BamH I
site was
then removed using oligonucleotides of SEQUENCE ID NO 36 to restore a native
amino
acid sequence to the released fusion protein product. The resultant pG4-BS 106
vector was
transformed into DH5 alphaTM cells (GibcoBRL Gaithersburg, MD), as directed by
the
supplier's instructions.
B. Expression of GST-BS 106 M/H. Four liters of Super Broth media (24 g/l
yeast
extract, 12 g/l tryptone, 5 g/l glycerol, 100 mM K phosphate pH 7.4)
containing 100 g/ml
ampicillin were inoculated with 200 ml LB media in which E coli DH5 alpha
cells
containing vector pG4-106 had been grown to stationary phase. This mixture was
then
allowed to grow with aeration (101pm air) and agitation (300 rpm) at 37 C for
3 h. IPTG
was added to 100 M, and growth continued for another 3 h. Cells (100 g) were
harvested by centrifugation (-5000 x g), and lysed by sonication (45 min) on
ice in 250 ml
1%. Triton X-100 in phosphate buffered saline. After cellular debris was
pelleted by
centrifugation, the supernatant containing the expressed protein from the
cellular lysate
was saved.
C. Purification of BS 106 M/H. The lysate supernatant was passed over a
glutathione-agarose column (50 ml). The column was washed with phosphate
buffered
saline until the baseline as detected by absorbance at 280 nm was stable (--
300 ml). The
GST-BS 106 M/H fusion protein was then eluted with 0.15% glutathione in
phosphate
buffered saline.
This material was further purified using a Mono Q 10/10 column (Pharmacia,
Piscataway, NJ) with 25 mM Tris buffer pH 8.8 and a linear gradient of NaCl
from 0 -
1000 mM. Fractions eluting between 200 to 300 mM NaCl were found to contain
full-
length fusion protein as detected by Western blotting using a myc specific
monoclonal
antibody. These fractions were pooled and treated with bovine thrombin in a
ratio of 50 U
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thrombin per mole fusion protein (calculated using an A280 1% -1, and a
molecular mass
-50 kD) for 4 h at 37 C. PMSF was added to 1 mM, and the sample was again
purified
by ion exchange chromatography under identical conditions. The BS106 M/H
eluted at
approximately 100 mM NaCl. The fractions were pooled and dialyzed against
phosphate
buffered saline.
This preparation was subjected to Western blot analysis using both myc
specific
and BS106 specific antibodies. Lane 2 of Figure 11, panels A and B, which was
described
in Example 11b, subsection E, demonstrates the material in this pool. The anti-
myc
monoclonal antibody (panel A) recognized a major band at approximately 13 - 20
kD as
well as two other higher molecular weight species. The anti-BS106 polyclonal
antibody
recognized more bands. The extra bands are attributed to some fusion protein
missing the
myc-his tag (since these bands were not detected in the myc specific blot),
non-fusion
protein missing the myc-his tag, and some degradation products from thrombin
cleavage.
Example 12: Chemical Analysis of Breast Tissue Proteins
A. Analysis of Tryptic Peptide Fragments Using MS. Sera from patients with
breast disease, such as breast cancer, sera from patients with no breast
disease, extracts of
breast tissues or cells from patients with breast disease, such as breast
cancer, extracts of
breast tissues or cells from patients with no breast disease, and extracts of
tissues or cells
from other non-diseased or diseased 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., Anal. Bio. 224:451-455
(1995) and J.
Rosenfeld et al., Anal. Bio. 203:173-179 (1992). The gel sections are washed
with 100
mM NH4HCO3 and acetonitrile. The shrunken gel pieces are swollen in digestion
buffer
(50 mM NH4HCO3, 5 mM CaC12 and 12.5 g/ml trypsin) at 4 C for 45 min. The
supernatant is aspirated and replaced with 5 to 10 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 evaporated to dryness. The peptides are
adsorbed to
approximately 0.1 l of POROS R2 sorbent (Perseptive Biosystems, Framingham,
Massachusetts) trapped in the tip of a drawn gas chromatography capillary tube
by
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dissolving them in 10 .il 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 passed directly into the spraying capillary of an API
III mass
spectrometer (Perkin-Elmer Sciex, Thornhill, Ontario, Canada) for analysis by
nano-
5 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.
Masses corresponding to predicted peptides can be further analyzed in MS/MS
mode to
give the amino acid sequence of the peptide.
10 B. Peptide Fragment Analysis Using LC/MS. 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 Methods in Enzymology 6:227-238 (1994). The
serum specimen or tumor extract from the patient is denatured with SDS and
reduced with
15 dithiothreitol (1.5 mg/ml) for 30 min at 90 C followed by alkylation with
iodoacetamide
(4 mg/ml) for 15 min at 25 C. Following acrylamide electrophoresis, the
polypeptides
are electroblotted to a cationic membrane and stained with Coomassie Blue.
Following
staining, the membranes are washed and sections thought to contain the unknown
polypeptides are cut out and dissected into small pieces. The membranes are
placed in
20 500 l microcentrifuge tubes and immersed in 10 to 20 l of proteolytic
digestion buffer
(100 mM Tris-HC1, pH 8.2, containing 0.1 M NaCl, 10% acetonitrile, 2 mM CaC12
and 5
g/ml trypsin) (Sigma, St. Louis, MO). After 15 h at 37 C, 3 l of saturated
urea and 1
l of 100 g/ml trypsin are added and incubated for an additional 5 h at 37 C.
The
digestion mixture is acidified with 3 l of 10% trifluoroacetic acid and
centrifuged to
25 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 is fed directly into an electrospray
mass
spectrometer, after passing though a stream splitter if necessary to adjust
the volume of
material. The data is analyzed following the procedures set forth in Example
12, Section
30 A.
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Example 13: Gene Immunization Protocol
A. In Vivo Antigen pression. 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 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 spleen can be harvested for production of hybridomas.
B. Plasmid Preparation and Purification. BS106 cDNA sequences are generated
from the BS106 cDNA-containing vector using appropriate PCR primers 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 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 g of the purified plasmid diluted in PBS or other DNA uptake
enhancers
(Cardiotoxin, 25% sucrose). See, for example, H. Davis et al., Human Gene
Therapy
4:733-740 (1993); and P. W. Wolff et al., Biotechniques 11:474-485 (1991). One
to two
booster injections are given at monthly intervals.
D. Testing and Use of Antiserum. 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.
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Example 14: Production of Antibodies Against BS106
A. Production of Polyclonal Antisera. Antiserum against BS 106 was prepared by
injecting rabbits with peptides whose sequences were derived from that of the
predicted
amino acid sequence of the BS106 consensus nucleotide sequence (SEQUENCE ID NO
6). The synthesis of peptides (SEQUENCE ID NO 21 - 33) is described in Example
10.
Peptides used as immunogens were either conjugated to a carrier protein such
as keyhole
limpet hemocyanine, KLH, or not conjugated to a carrier (i.e., they were
unconjugated.).
1. Peptide Conjugation. Peptide was conjugated to maleimide activated keyhole
limpet hemocyanine (KLH, commercially available as linject , available from
Pierce
Chemical Company, Rockford, IL). Imjectocontains about 250 moles of reactive
maleimide groups per mole of hemocyanine. The activated KLH was dissolved in
phosphate buffered saline (PBS, pH 8.4) at a concentration of about 7.7 mg/ml.
The
peptides were conjugated through cysteines occurring in the peptide sequence,
or to a
cysteine previously added to the synthesized peptide in order to provide a
point of
attachment. The peptides were dissolved in dimethyl sulfoxide (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 peptides SEQUENCE ID NO 23, SEQUENCE ID NO 25, SEQUENCE
ID NO 26, and SEQUENCE ID NOS 28 - 32 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 was based on obtaining 3 mg of
KLH peptide conjugate ("conjugated peptide"), which contains about 0.77 moles
of
reactive maleimide groups. This quantity of peptide conjugate usually was
adequate for
one primary injection and four booster injections for production of polyclonal
antisera in a
rabbit. Briefly, the peptides (SEQUENCE ID NO 23, SEQUENCE ID NO 25,
SEQUENCE ID NO 26, and SEQUENCE ID NOS 28 - 32) were dissolved in DMSO at a
concentration of 1.16 moles/100 l of DMSO. One hundred microliters (100 l)
of the
DMSO solution was added to 380 1 of the activated KLH solution prepared as
described
hereinabove, and 20 l of PBS (pH 8.4) was added to bring the volume to 500
l. The
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reaction was incubated overnight at room temperature with stirring. The extent
of
reaction was determined by measuring the amount of unreacted thiol in the
reaction
mixture. The difference between the starting concentration of thiol and the
final
concentration was assumed to be the concentration of peptide which had coupled
to the
activated KLH. The amount of remaining thiol was measured using Ellman's
reagent
(5,5'-dithiobis(2-nitrobenzoic acid), Pierce Chemical Company, Rockford, IL).
Cysteine
standards were 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 the stock solution to the desired concentration(s). The
photometric
determination of the concentration of thiol was accomplished by placing 200 l
of PBS
(pH 8.4) in each well of an Immulon 2 microwell plate (Dynex Technologies,
Chantilly,
VA). Next, 10 l of standard or reaction mixture was added to each well.
Finally, 20 l
of Ellman's reagent at a concentration of 1 mg/ml in PBS (pH 8.4) was added to
each well.
The wells were incubated for 10 minutes at room temperature, and the
absorbance of all
is wells was read at 415 nm with a microplate reader (such as the BioRad Model
3550,
BioRad, Richmond, CA). The absorbance of the standards was used to construct a
standard curve and the thiol concentration of the reaction mixture was
determined from
the standard curve. A decrease in the concentration of free thiol was
indicative of a
successful conjugation reaction. Unreacted peptide was removed by dialysis
against PBS
(pH 7.2) at room temperature for 6 hours. The conjugate was stored at 2-8 C
if it is to be
used immediately; otherwise, it was stored at -20 C or colder.
2. Animal Immunization. Female white New Zealand rabbits weighing 2 kg or
more were used for raising polyclonal antiserum. One animal was immunized per
conjugated peptide (SEQUENCE ID NO 23, SEQUENCE ID NO 25, SEQUENCE ID NO
26, and SEQUENCE ID NOS 28 - 32 ) or unconjugated peptide (SEQUENCE ID NOS 21
- 24 ). 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.
Conjugated, (SEQUENCE ID NO 23, SEQUENCE ID NO 25, SEQUENCE ID
NO 26, and SEQUENCE ID NOS 28 - 32 ), or unconjugated peptides, (SEQUENCE ID
NOS 21 - 24 ), were used to prepare the primary immunogen by emulsifying 0.5
ml of the
conjugated or unconjugated peptide at a concentration of 2 mg/ml in PBS (pH
7.2) which
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contained 0.5 ml of complete Freund's adjuvant (CFA) (Difco, Detroit, 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
conjugated or
unconjugated peptide used for the primary immunogen, except that the peptide
or
peptide/carrier complex 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 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. Table 2 below shows
the titers
obtained with the peptide immunized rabbits.
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Table 2. Titer of rabbit anti-BS106 peptide antisera (12 week bleed)
Peptide Conjugated to a
Peptide Immunogen Carrier Protein? Titer
SEQUENCE ID NO 21 no 200
SEQUENCE ID NO 22 no 5000
SEQUENCE ID NO 23 no 44,000
SEQUENCE ID NO 23 yes >62,500
SEQUENCE ID NO 24 no <100
SEQUENCE ID NO 25 yes 62,500
SEQUENCE ID NO 26 yes 258,000
SEQUENCE ID NO 28* yes 39,000
SEQUENCE ID NO 29 yes 47,000
SEQUENCE ID NO 30 yes 200
SEQUENCE ID NO 31 yes 1000
SEQUENCE ID NO 32 yes 400
* Rabbit antisera to BS106.8 was tested using SEQUENCE ID NO 29 peptide
B. Production of Monoclonal Antibody.
1. Immunization Protocol. Mice were immunized using peptides conjugated
(SEQUENCE ID NO 23, SEQUENCE ID NO 26, and SEQUENCE ID NO 29) to a carrier
such as KLH [prepared as described hereinabove] except that the amount of the
conjugated peptide for monoclonal antibody production in mice was one-tenth
the amount
used to produce polyclonal antisera in rabbits. Thus, the primary immunogen
consisted of
100 g of conjugated peptide in 0.1 ml of CFA emulsion while the immunogen
used for'
booster immunizations consisted of 50 g conjugated peptide in 0.1 ml of IFA.
Hybridomas for the generation of monoclonal antibodies were prepared and
screened
using standard techniques. The methods used for monoclonal antibody
development
followed procedures known in the art such as those detailed in Kohler and
Milstein,
Nature 256:494 (1975) and reviewed in J.G.R. Hurrel, ed., Monoclonal Hybridoma
Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL
(1982).
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Another method of monoclonal antibody development which is based on the Kohler
and
Milstein method is that of L.T. Minims et al., Virology 176:604-619 (1990).
The immunization regimen (per mouse) consisted of a primary immunization with
additional booster immunizations. The primary immunogen used for the primary
immunization consisted of 100 g of conjugated peptide in 50 l of PBS (pH
7.2)
previously emulsified in 50 l of CFA. Booster immunizations performed at
approximately two weeks and four weeks post primary immunization consisted of
50 g
of conjugated peptide in 50 l of PBS (pH 7.2) emulsified with 50 l IFA. A
total of 100
l of this immunogen was inoculated intraperitoneally and subcutaneously into
each
mouse. Individual mice were screened for immune response by microtiter plate
enzyme
immunoassay (EIA) as described in Example 17 approximately four weeks after
the third
immunization. Mice were inoculated intravenously with 50 g of conjugated
peptide in
PBS (pH 7.2) approximately fifteen weeks after the third immunization.
Three days after this intravenous boost, splenocytes were fused with Sp2/0-
Ag14
myeloma cells (Milstein Laboratories, England) using the polyethylene glycol
(PEG)
method. The fusions were cultured in Dulbecco's Modified Eagle's Medium (DMEM)
with the addition of L-glutamine, L-asparagine, L-arginine, folic acid, and
containing 10%
fetal calf serum (FCS), plus 1% hypoxanthine, aminopterin and thymidine (HAT).
Bulk
cultures were screened by microtiter 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 B S 106 not used as the immunogen) were selected for final
expansion.
Supernatant from the final expansion was harvested and used for further
characterization.
Hybridoma cells from the expansion growth were harvested, aliquoted and frozen
in
DMEM containing 10% FCS and 10% dimethyl sulfoxide, (DMSO).
2. 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 g
filter and subjected
to an immunoglobulin class G (IgG) analysis to determine the volume of the
Protein A
column required for the purification.
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3. Purification of Monoclonal Antibodies From Ascites Fluid or Cell Culture
Supernatant. Monoclonal antibodies can be purified from ascites fluid or cell
culture
supernatant using a variety of methods including Protein A, Protein G, and
Protein L
column chromatography or precipitation. 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
NaCl, pH 8.9) and refiltered through a 0.2 .t 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 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 BS 106 not used as the
immunogen. The
purified anti-BS 106 monoclonal antibody thus prepared and characterized is
placed at
either 2-8 C for short term storage or at -80 C for long term storage.
4. Further Characterization of Monoclonal Antibody. The isotype and subtype of
the monoclonal antibody produced as described hereinabove were determined
using an
EIA microtiter assay. Briefly, the peptide immunogen was prepared at 2 g/ml
in 50 mM
carbonate, pH 9.6 and 100 l was placed in each well of an Immunlon 2 High
Binding
microtiter plate (Dynex Technologies, Chantilly, VA). The plate was incubated
for 14-18
hours at room temperature and then washed four times with deionized water. The
wells
were blocked by adding 125 l of Superblock (Pierce Chemical Company,
Rockford, IL)
to each well and then immediately discarding the solution. This blocking
procedure was
performed three times. One hundred microliters (100 l) of culture supernatant
was
diluted 1:2 in a protein blocking agent of PBS containing 3% Superblock, 0.05%
Tween
20 (monolaurate polyoxyethlene ether) (Sigma Chemical Company, St. Louis, MO),
and
0.05% sodium azide and placed in each well of the coated microtiter plate. The
wells
were incubated for one hour at room temperature. Each well was washed four
times with
deionized water. One hundred microliters (100 l) of alkaline phosphatase-
conjugated
goat anti-mouse IgG (H+L), or IgGl, or IgG2a, or IgG2b, or IgG3 (Southern
Biotech,
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Birmingham, AL) diluted 1:2000 in PBS containing 3% Superblock, 0.05% Tween
20,
and 0.05% sodium azide were added to the appropriate wells. The wells were
incubated
for one hour at room temperature. Next, each well was washed four times with
deionized
water. One hundred microliters (100 l) of para-nitrophenyl phosphate
substrate
s (Kirkegaard and Perry Laboratories, Gaithersburg, MD) then were added to
each well.
The wells were incubated for thirty minutes at room temperature and the
absorbance at
405 nm was read. The results of the isotyping are presented in Table 3.
Table 3. Characterization of Monoclonal Antibodies
Study Experiment Hybridoma Peptide Immunogen Isotype
392 3 H6C52 SEQUENCE ID NO 26 IgGi
392 3 H9C29 SEQUENCE ID NO 26 IgGl
392 3 H39C51 SEQUENCE ID NO 26 IgGl
392 3 H80C32 SEQUENCE ID NO 26 IgGl
392 7 H84C55 SEQUENCE ID NO 26 IgGl
392 7 H121C68 SEQUENCE ID NO 26 IgG1
392 7 H144C20 SEQUENCE ID NO 26 IgG2a
392 7 H24C16 SEQUENCE ID NO 26 IgGl
392 14 H17C77 SEQUENCE ID NO 23 IgGl
392 14 H59C34 SEQUENCE ID NO 23 IgGl
392 14 H121C19 SEQUENCE ID NO 23 IgGl
392 14 H131C50 SEQUENCE ID NO 23 IgGi
392 14 H147C64 SEQUENCE ID NO 23 ND
392 14 H184C51 SEQUENCE ID NO 23 IgGi
392 14 H195C62 SEQUENCE ID NO 23 IgGi
392 14 H212C68 SEQUENCE ID NO 23 IgGl
392 14 H218C31 SEQUENCE ID NO 23 IgG2a
392 15 H119C17 SEQUENCE ID NO 26 IgGi
392 15 H150C31 SEQUENCE ID NO 26 IgGi
392 15 H182C63 SEQUENCE ID NO 26 IgG2b
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392 15 H188C82 SEQUENCE ID NO 26 IgG1
392 15 H203C46 SEQUENCE ID NO 26 IgG2a
392 15 H205C33 SEQUENCE ID NO 26 IgG1
400 3 H24C29 SEQUENCE ID NO 29 IgG1
400 3 H51 C42 SEQUENCE ID NO 29 IgG3
400 3 H62C52 SEQUENCE ID NO 29 IgG2b
400 3 H102C47 SEQUENCE ID NO 29 ND
400 3 H30C35 SEQUENCE ID NO 29 ND
400 3 H26C39 SEQUENCE ID NO 29 ND
400 4 H16C70 SEQUENCE ID NO 29 IgG1
400 4 H45C35 SEQUENCE ID NO 29 IgG1
400 4 H62C59 SEQUENCE ID NO 29 IgGl
400 4 H76C43 SEQUENCE ID NO 29 ND
400 4 H4C33 SEQUENCE ID NO 29 ND
400 4 H57C43 SEQUENCE ID NO 29 ND
400 4 H44C56 SEQUENCE ID NO 29 ND
400 4 H94C45 SEQUENCE ID NO 29 ND
400 4 H34C78 SEQUENCE ID NO 29 ND
400 4 H2C 15 SEQUENCE ID NO 29 ND
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.
s C. Use of Recombinant Proteins as Immunogens. 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 Specifically Bind to BS106 Peptides
A. Preparation of Affinity Column for Purification of Rabbit Anti-BS 106.
Immune sera, obtained as described hereinabove in Example 14, was affinity
purified
using an immobilized synthetic peptide prepared as described in Example 10.
SEQUENCE ID NO 26 was coupled to SulfoLinkTM gel (Pierce Chemical, Rockford,
IL)
according to the manufacturer's instructions. Briefly, 2.0 ml peptide,
dissolved in 2.0 ml
coupling buffer was poured into 2 ml SulfoLinkTM gel previously washed with 10
ml
coupling buffer. After 18 h at room temperature the gel was washed with 4 ml
coupling
buffer, then blocked with 10 mg L-cysteine hydrochloride in 2.0 ml coupling
buffer. After
30 min the gel was washed with 8 ml coupling buffer, then 7 ml 0.2% BSA in
PBS, then 7
ml PBS. The gel was conditioned by washing with 7 ml of 100 mM glycine pH
2.08, then
ml PBS.
B. Affinity Purification of Rabbit Anti-B S 106 Antisera. Ten milliliters (10
ml) of
15 rabbit anti-BS106 antiserum was passed through a column containing 2 ml of
the affinity
gel, as described supra. The gel was washed with PBS until the absorbance at
280 nm was
less than 0.05, then the bound antibody was eluted with 100 mM glycine pH
2.08. The
eluted material was collected as 500 1 fractions in tubes containing 30 l of
1.0 M Tris
base. Fractions 4-9 were combined and passed through a 10 ml gel filtration
column
(Pierce KwikTM column), exchanging the buffer to PBS. Fractions collected from
this
column were pooled into a 3.0 ml volume at 3.0 mg/ml and a 1.5 ml volume at
0.32
mg/ml.
Example 16: Western Blotting of Tissue Samples
A. Tissue Specificity. Tissue extracts were prepared by homogenizing tissue
samples in 0.1M Tris-HC1(pH 7.5), 15% (w/v) glycerol, 0.2 mM EDTA, 1.0 mM 1,4-
dithiothreitol, 10 g/ml leupeptin and 1.0 mM phenylmethylsulfonylfluoride
(Kain et al.,
Biotechniques, 17:982 (1994). The homogenates were centrifuged at 4 C for 5
minutes to
separate supernate from debris. For protein quantitation, 3-10 l of supernate
were added
to 1.5 ml of bicinchoninic acid reagent (Sigma, St. Louis, MO), and the
resulting
absorbance at 562 nm was measured.
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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 Protein G purified anti-peptide monoclonal antibody H9C29 at 1
gg/ml. The
blots were then incubated with commercially obtained anti-mouse alkaline
phosphatase
(Tropix, Bedford, MA). The bands were visualized directly on the membranes by
the
addition of 5-bromo-4-chloro-3-indolyl phosphate (BCIP). This chromogenic
solution
contains 0.016% BCIP in a solution containing 100 mM NaCl, 5 mM MgCl2 and 100
mM Tris-HCl (pH 9.5). Molecular mass determination was preformed using pre-
stained
molecular weight standards (Novex, San Diego, CA).
Competition experiments were also carried out in an analogous manner as above,
with the following exception; the primary antibody (anti-peptide monoclonal
antibody)
was pre-incubated overnight in the refrigerator with 1.3 M, 0.43 M and 0.14
M of
BS106 peptide SEQUENCE ID NO 26 prior to exposure to the nitrocellulose
membrane.
Figure 12 shows the results of a Western blot performed on a panel of tissue
extracts using a monoclonal antibody (H9C29) directed against BS106 peptide
SEQUENCE ID NO 26. Each lane of Figure 12 contains a different tissue extract:
1,
testicle cancer; 2, endometrial cancer; 3, ovarian cancer; 4, bladder; 5,
colon; 6, prostate;
7, lung ; 8, breast cancer; 9, blank and 10 molecular weight markers (kD). The
extract
from the breast cancer tissue (lane 8) shows a strong broad band consistent
with a
glycoprotein at approximately 40 kD, whereas the other tissues are negative.
Several other
breast tissue specimens were analyzed including extracts form seven other
breast cancer
tissues and six normals tissues. Together, five of the eight breast cancers
and two of the
six normals were positive for BS106. Competition experiments with a BS106
peptide
(SEQUENCE ID NO 26) resulted in complete inhibition of the band seen with
breast
cancer tissue.
B. Analysis of Human Milk. Aqueous and fat portions of a sample of human milk,
separated as in Example 22D, were prepared for electrophoresis as indicated in
the Table
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4 below. Milk fat was diluted with an equal volume of PBS prior to preparation
for
electrophoresis. The samples were incubated for 10 minutes at room temperature
after
addition of the TCEP (Tris-carboxyethylphosphine hydrochloride). PBS and 2X
SDS
Sample Buffer (Novex, San Diego, CA) were then added. The mixtures were heated
5
minutes at 100 C, then sodium carbonate was added to neutralize the TCEP
acidity.
Twelve microliters (12 l) of the prepared samples were added to the wells of
a 10-20%
Tris-glycine gel (Novex, San Diego,CA). The samples were run in duplicate with
molecular weight markers between the two sets of samples. After
electrophoresis was
complete, the gels were blotted to nitrocellulose, as described in Example
16A. The
nitrocellulose filters were blocked for 60 minutes with IblockTM (Tropix,
Bedford MA)
(0.2% in PBS containing 0.1% Tween-20). The blot was then cut to separate the
replicated samples. Both blots (labeled A and B) were incubated for 60 minutes
with 10
ml of IblockTM solution containing 2.5 p1 of affinity purified rabbit anti-
BS106.6.
However, the B blot antibody solution included 100 l of 0.1 mM BS106.6
peptide,
which was mixed with the antibody before adding to the blot. After a 60 min
incubation,
while rocking at room temperature, the blots were washed three times with 20
ml PBS
containing 0.1 % Tween 20. The blots were then incubated for 60 min while
rocking with
5 ml of a 1:10000 dilution of alkaline phosphatase anti-rabbit conjugate
(Tropix, Bedford
MA). The blots were developed with 2 ml of NBT/BLIP substrate (Pierce One
StepTM,
Pierce Chemical, Rockford, IL).
Table 4: Preparation of aqueous and fat portions of human milk samples
Sample 10% 2X Sample Sodium
Sample Volume TCEP PBS Buffer Carbonate
Milk Aqueous 20 0 10 30 0
Milk Aqueous 20 5 0 30 8
Milk Fat 20 0 10 30 0
Milk Fat 20 5 0 30 8
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Figure 13 shows that the affinity purified rabbit anti-BS 106 antibody detects
bands
at several molecular weights, and that all of these bands disappear in the
presence of
BS106 peptide (SEQUENCE ID NO 26), suggesting that multiple proteins present
in both
aqueous and fatty fractions of milk contain amino acid sequence identical to
or strongly
related to that of BS 106 peptide (SEQUENCE ID NO 26). In the absence of
reducing
agent the aqueous and fatty fractions show a high molecular weight band (>200
kD), and
another band at about 120 kD (A, lanes 2 and 4). On reduction the band at 120
kD
remains, however the high molecular weight band (>200 kD) disappears, and
bands at 80
kD, 65 kD, and 40 kD appear (A, lanes 3 and 5). The sample containing the
peptide for
competition was run on blot B. The 80 kD and 65 kD bands appear there (B, lane
3) and
are therefore considered not specific for BS106, unlike the 40 kD band.
The band at 40 kD is consistent with a single chain BS106 protein molecule
containing 0-linked glycosylation as predicted from its sequence (as described
in
Example 1 lb). The sole cysteine residue and its penultimate position in the
sequence
suggest that it forms disulfide links with other proteins in the course of its
biological
function. This is consistent with the disappearance of the high MW band (>200
kD) and
the appearance of the 40 kD band after exposure to conditions which reduce the
disulfide
bonds.
The 120 kD band, however, is not affected by reduction. Apparently BS106
protein forms reduction resistant links with other proteins present in milk.
One suggestion
is that one or both of the lysines near the C-terminus may be coupled to other
proteins by
transglutamination.
C. Western Blotting of Biological Samples with BS106 Antibodies. Various
biological samples were analyzed by Western blot under reducing and non-
reducing
conditions. Aqueous and fatty fractions of human milk were prepared as in
Example 22D.
To 6 l of the milk aqueous fraction was added 24 1 of PBS and 30 1 of 2X
SDS
Sample Buffer (Novex, San Diego, CA) with and without 5% dithiothreitol. The
milk
fatty fraction (6% dispersion in PBS) and an extract of breast tumor tissue
(from Example
16A) were similarly treated.
A sample of saliva was added to 18 l of PBS and centrifuged for 10 min at
15,000
x g. Twelve microliters (12 l) of the supernate were added to 30 12X Sample
Buffer
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(Novex, San Diego, CA) with and without 5% dithiothreitol. Twelve microliters
(12 l)
of a suspension of the sediment from the above saliva preparation in its
original volume of
PBS were similarly treated. To 3 l of a sample of BS106 protein, affinity
purified from
human milk as in Example 22D, were added 27 l PBS and 30 l 2X SDS Sample
Buffer
(Novex, San Diego, CA) with and without 5% dithiothreitol.
All samples were heated 5 minutes at 100 C and electrophoresed as described
in
Example 16A. After electrophoresis, the gels were blotted to nitrocellulose as
directed by
the manufacturer's instructions. The blots were blocked for 60 minutes with
IblockTM
(0.2% in PBS containing 0.1% Tween-20). The blots were incubated for 60
minutes with
10 ml IblockTM solution containing either 8.5 l affinity purified rabbit anti-
BS106
peptide (SEQUENCE ID NO 26) from Example 15B, or, 280 l of the culture
supernate
containing H24C16 monoclonal antibody, both either with or without 50 g BS106
peptide (SEQUENCE ID NO 26) added to compete for the antibody. After a 60 min
incubation, while rocking at room temperature, the blots were washed three
times with 20
ml of PBS containing 0.1% Tween 20. They were then incubated for 60 min while
rocking with 5 ml of a 1:10000 dilution of alkaline phosphatase anti-rabbit or
anti-mouse
conjugate (Tropix, Bedford MA), and developed with 2 ml of NBT/BCIP substrate
(Pierce One StepTM, Pierce Chemical, Rockford, IL).
The results are shown in Figure 14. According to the manufacturer, the colored
MW markers move differently on Tricine gels than on the Tris-glycine gels used
in
Example 16A, resulting in different molecular weights attributed to them The
>200 kD
band is recognized by both the affinity purified rabbit antibody and the
monoclonal
antibody H24C16. The band is present (under non-reducing conditions) in the
aqueous
milk fraction, breast tumor, saliva supernate, and the BS 106 protein affinity
purified from
milk.
The 120 kD band is also recognized by both the affinity purified rabbit
antibody
and the monoclonal antibody H24C16. The band is observed in the milk samples
including the aqueous milk fraction, the fatty fraction, and the BS 106
protein affinity
purified from milk. However, it does not appear to be present in the saliva
samples and is
difficult to conclude its presence in the breast tumor sample due to the
intense staining.
The breast tumor specimen and the BS 106 protein affinity purified from milk
further
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contain some material ranging in molecular weight from 45 - 70 kD that is
recognized by
both antibodies.
Upon reduction, the high molecular weight band (>200 kD) and the bands of
molecular weight (45 - 70 kD) disappear and a band at 40 kD is produced. This
is best
observed with the BS 106 protein affinity purified from milk, as the sample is
cleaner and
more concentrated. The 120 kD band is unaffected by the reduction.
These results are consistent with the presence of a disulfide-linked complex
involving BS106 with a molecular weight greater than 200 kD. BS106 also
appears to be
involved in a non-reduceable complex with a molecular weight of approximately
120 kD.
Example 17: EIA Microtiter Plate Assay
The immunoreactivity of monoclonal antibodies or antiserum obtained from
rabbits or mice as described in Example 14 was determined by means of a
microtiter plate
EIA, as follows. Microtiter plates were coated with either synthetic peptides
or semi-
purified expressed protein material (purified as described in Example l lb,
Part E).
Briefly, synthetic peptides, SEQUNENCE ID NO 21 - 33, prepared as described in
Example 10, were dissolved in carbonate buffer (50 mM, pH 9.6) to a final
concentration
of 2 g/ml. Semi-purified expressed protein was diluted in PBS 1:20. Next, 100
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 deionized water. The wells were
blocked by
adding 125 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. Monoclonal antibodies or
antisera
obtained from immunized rabbits or mice, prepared as previously described,
were diluted
in a protein blocking agent (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
(polyclonal antisera) or 1:10, 1:100, 1:1000, and 1:10,000 (hybridoma
supernatants) 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 times with deionized
water. One
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hundred microliters (100 l) 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 20 and
0.05%
sodium azide, were added to each well. The wells were incubated for two hours
at room
temperature. Next, each well was washed four times with deionized water. One
hundred
microliters (100 l) 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 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-B S 106 antibodies.
Titers of the
anti-peptide antisera or monoclonal antibodies were calculated from the
previously
described dilutions of antisera and defined as the calculated dilution, where
A405nm = 0.5
OD. Table 5 shows titers of the monoclonal antibody culture supernatants which
were
generated using synthetic peptide (peptide used as immunogen) or the semi-
purified
expressed protein material (B S 106 M/H).
Table 5: Binding Properties of Monoclonal Antibodies
Peptide
Peptide Immunogen Immunogen BS106M/H BS106M/H
Study Experiment Hybridoma SEQUENCE ID NO Titer Titer Kd(app)
392 3 H6C52 26 800 660 118
392 3 H9C29 26 250 250 231
392 3 H39C51 26 380 350 241
392 3 H80C32 26 900 900 70
392 7 H84C55 26 600 650 170
392 7 H121C68 26 100 270 143
392 7 H144C20 26 600 700 164
392 7 H24C16 26 300 125 89
392 14 H17C77 23 <10 <10 ND
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392 14 H59C34 23 300 100 358
392 14 H121C19 23 90 <10 ND
392 14 H131C50 23 800 <10 ND
392 14 H147C64 23 <10 <10 ND
392 14 H184C51 23 325 75 132
392 14 H195C62 23 700 100 448
392 14 H212C68 23 825 100 193
392 14 H218C31 23 725 500 509
392 15 H119C17 26 760 70 201
392 15 H150C31 26 50 60 478
392 15 H182C63 26 800 <10 ND
392 15 H188C82 26 700 100 271
392 15 H203C46 26 540 100 236
392 15 H205C33 26 1500 <10 28
400 3 H24C29 29 900 ND ND
400 3 H51C42 29 3600 ND ND
400 3 H62C52 29 40 ND ND
400 3 H102C47 29 ND ND ND
400 3 H30C35 29 ND ND ND
400 3 H26C39 29 ND ND ND
400 4 H16C70 29 680 ND ND
400 4 H45C35 29 2800 ND ND
400 4 H62C59 29 350 ND ND
400 4 H76C43 29 ND ND ND
400 4 H4C33 29 ND ND ND
400 4 H57C43 29 ND ND ND
400 4 H44C56 29 ND ND ND
400 4 H94C45 29 ND ND ND
400 4 H34C78 29 ND ND ND
400 4 H2C15 29 ND ND ND
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In addition to titers, apparent affinities [Kd(app)] were also determined for
some of
the antibodies. In this case, pooled and dialysed semi-purified expressed
protein material
(purified as described in Example 1 lb, Part E) were prepared at dilutions of
1:3, 1:9, 1:27,
1:81, 1:243, 1:729, 1:2187, 1:6561, and 1:19683 in PBS and 100 l were placed
in each
well of an Immulon 20High Binding microtiter plate (Dynex Technologies,
Chantilly,
VA). The plate was incubated for 14-18 hours at room temperature and then
washed four
times with deionized water. The wells were blocked by adding 125 l of
Superblock (Pierce Chemical Company, Rockford, IL) to each well and then
immediately
discarding the solution. The blocking procedure was performed three times.
Monoclonal
antibodies obtained from hybridoma culture supernatant or antisera obtained
from
immunized rabbits or mice, as described hereinabove in Example 14, were
diluted at an
appropriate dilution in a protein blocking agent (i.e., 3% Superblock"
solution) in PBS
containing 0.05% Tween-20 (monolaurate polyoxyethylene ether) (Sigma Chemical
Company, St. Louis, MO) and 0.05% sodium azide and placed in each well of the
coated
microtiter plate. The wells were then incubated for one hour at room
temperature. Each
well was washed four times with deionized water. One hundred microliters (100
l) of
alkaline phosphatase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG
(Southern
Biotech, Birmingham, AL), diluted 1:2000 in 3% Superblock solution were added
to each
well. The wells were incubated for one hour at room temperature. Next, each
well was
washed four times with deionized water. One hundred microliters (100 l) of
para-
nitrophenyl 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. EIA microtiter
plate assay
results were used to derive the apparent dissociation constants [Kd(app)]
based on an analog
of the Michaelis-Menten equation (V. Van Heyningen, Methods in Enzymology,
Vol. 121,
p. 472 (1986) and further described in X. Qiu, et al, Journal of Immunology,
Vol. 156, p.
3350 (1996)):
[Ab]
[Ag-Ab] = [Ag-Ab]max X [Ab] + Kd
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where [Ag-Ab] was the antigen-antibody complex concentration, [Ag-Ab]m. was
the maximum complex concentration, [Ab] was the antibody concentration, and Kd
was
the dissociation constant. During the curve fitting, the [Ag-Ab] was replaced
with the
background subtracted value of the OD405nm at the given concentration of Ab.
Both Kd,
which corresponds to Kd(app), and [OD405nm]max, which corresponds to the [Ag-
Ab]m ,
were treated as fitted parameters. The software program Origin was used for
the curve
fitting. Apparent affinities [Kd(app)] were determined for the monoclonal
antibodies
(Table 6) and the rabbit polyclonal antibodies (Table 5) using semi-purified
expressed
protein material (BS106 M/H).
Table 6: Binding Properties of Rabbit Polyclonal Antibodies
Peptide Conjugated BS106M/H
Rabbit # Peptide Immunogen to a Carrier Protein? Kd(app)
11537 SEQUENCE ID NO 23 no 287
11538 SEQUENCE ID NO 23 no 197
11539 SEQUENCE ID NO 23 no 270
11540 SEQUENCE ID NO 23 no 166
11492 SEQUENCE ID NO 23 yes 370
11494 SEQUENCE ID NO 26 yes 576
Example 18: Coating of Solid Phase Particles
A. Coating of Microparticles with Antibodies Which Specifically Bind to BS106
Antigen. One hundred microliters (100 l) of carboxymethyl latex microspheres
(Interfacial Dynamics 1.0 micron diameter, 4.2% solids) were suspended in 1.0
ml of 5
mM morpholinoethanesulfonic acid (MES) buffer (pH 5.6) with 0.1 % Triton 'X
100. The
mixture was vortexed and then centrifuged for 1 minute at approximately 500 x
g to
sediment the microspheres. The supernatant was discarded and the pellet
containing the
microspheres was resuspended in 1.0 ml of the MES buffer. Forty microliters
(40 1) of
antibody (affinity purified rabbit anti-BS106 antibody at 3 mg/ml in PBS) and
20 l of 1.0
mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDAC) were added to the
resuspended microspheres. The mixture was vortexed and rotated for 5 h at room
CA 02399047 2009-08-25
115
temperature. To the mixture was added 400 l of BSA buffer (0.2% bovine serum
albumin (Sigma, St. Louis, MO) and 0.08% Tween-20 (Sigma, St. Louis, MO) in
MEIA
buffer (Abbott Laboratories, Abbott Park, IL). The mixture was vortexed,
centrifuged as
above, and the supernatant was discarded. The pellet was washed twice with 1.0
ml of the
BSA buffer prior to resuspension in 400 l of the same buffer.
B. Coating of 1/4 Inch Beads. Antibodies which specifically bind to BS106-
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 carbonate buffer at pH 8Ø The beads then are washed in 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 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 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 (MEIA)
BS106 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 Aayyzer (Abbott Laboratories, Abbott Park, IL).
A. Antibody Sandwich EIA. Briefly, samples suspected of containing BS106
antigen are incubated in the presence of anti-B S 106 antibody-coated
microparticles
(prepared as described in Example 18) 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 (i.e., enzymes such as alkaline
phosphatase or
horseradish peroxide) is added to the antigen/antibody complexes or the
microparticles
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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 generate a measurable signal. An elevated signal in the test sample,
compared to the
signal generated by a negative control, detects the presence of BS106 antigen.
The
presence of BS 106 antigen in the test sample is indicative of a diagnosis of
a breast
disease or condition, such as breast cancer.
B. Competitive Binding 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 (Abbott Laboratories, Abbott Park, IL). The labeled peptide is
added to the
BS106 antibody-coated microparticles (prepared as described in Example 18) in
the
presence of a test sample suspected of containing BS 106 antigen, and
incubated for a time
and under conditions sufficient to form labeled BS 106 peptide (or labeled
protein) / bound
antibody complexes and/or patient BS106 antigen / bound antibody complexes.
The
BS106 antigen in the test sample competes with the labeled BS106 peptide (or
BS106
protein) for binding sites on the microparticle. BS106 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 BS106 peptide or BS106 protein compete for
antibody
binding sites. A lowered signal (compared to a control) indicates the presence
of BS106
antigen in the test sample. The presence of BS 106 antigen suggests the
diagnosis of a
breast disease or condition, such as breast cancer.
The B S 106 polynucleotides and the proteins encoded thereby which are
provided
and discussed hereinabove are useful as markers of breast tissue disease,
especially breast
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 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,
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altered in a disease state, or be a normal protein of the breast, which
appears in an
inappropriate body compartment.
Example 20: Immunohistochemical Detection of BS106 Protein
Antiserum against a BS106 synthetic peptide derived from the consensus peptide
sequence (SEQUENCE ID NO 20) described in Example 14, above, is used to
immunohistochemically stain a variety of normal and diseased tissues using
standard
proceedures. 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 peptide derived from the
consensus
BS106 peptide sequence (SEQUENCE ID NO 20) 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 hematoxylin, mounted, and examined under a microscope by a
pathologist.
Example 21: Fluorescence Polarization Immunoassay of BS 106
A. Conjugation of BS106 Peptide to Fluorescein Derivatives. The peptide,
SEQUENCE ID NO 26 was coupled to fluorescein-5-maleimide using the following
procedure. One and one tenth milligram (1.1 mg) of Tris-carboxyethylphosphine
hydrochloride and 0.3 mole of fluorescein-5-maleimide (Molecular Probes,
Eugene, OR)
were dissolved in 17 1 of dimethylformamide. This mixture was added to 0.75
moles of
SEQUENCE ID NO 26 peptide in 50 l of 600 mM triethanolamine buffer (pH 7.6).
The
reaction was allowed to proceed for 30 minutes at room temperature after which
the
mixture was passed over a Biogel P2 column (8 ml) to separate the higher
molecular
weight conjugate from the unreacted fluorescein. The column was equilibrated
with a 10-
fold dilution of PBS in water. The first colored band was collected, giving
500 l of
yellow solution.
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The peptide, SEQUENCE ID NO 26 was coupled to 5-carboxyfluorescein using
the following procedure. Three tenths micromole (0.3 mole) 5-
carboxyfluorescein NHS
ester (Molecular Probes, Eugene, OR) was dissolved in 21 l of
dimethylformamide. This
mixture was added to 0.75 mole of SEQUENCE ID NO 26 peptide in 50 l of 600
mM
triethanolamine buffer (pH 7.6). The reaction was allowed to proceed for 30
minutes at
room temperature after which the mixture was passed over a Biogel P2 column (8
ml) to
separate the higher molecular weight conjugate from the unreacted fluorescein.
The
column was equilibrated with a 10-fold dilution of PBS in water. The first
colored band
was collected, giving 500 l of yellow solution.
B. Purification of BS106 Fluorescein Conjugates. The isomeric conjugates in
the
above products were separated by isoelectric focusing using prepared gels and
buffers
from Novex (Novex, San Diego,CA). To 150 l IEF sample buffer containing 1.5
mg
dithiothreitol was added 150 l of the product of the above conjugation
reaction. The
mixtures were placed in the sample compartments of the IEF gels from which the
lane
dividers had been removed to give a single well for each gel. The gels were
electrophoresed for 60 minutes at 100 V and then for 60 minutes at 200 V,
during which
each of the samples were visibly resolved into several narrow bands. The gel
cassettes
were opened, and the major bands were cut out with a razor blade and
transferred to
culture tubes containing 1.0 ml of PBS. After 3 hours of incubation at room
temperature,
most of the color appeared to be in solution. The fluorescein concentration of
each sample
was measured by absorbance at 492 nm, assuming an extinction coefficient of
73000 M-
1cm1
The fluorescence intensity and polarization of the conjugates were measured
using
the fluorescence polarization format of the IMx"Analyzer (Abbott Laboratories,
Abbott
Park, IL). Each conjugate was diluted to 10 nM and the measurements were taken
after
which 1.0 l of the 3.0 mg/ml affinity purified rabbit anti-BS 106 was added.
The mixture
was vortexed and the measurement was repeated. Results are presented in Table
7.
Table 7 lists the approximate isoelectric point (pI), absorbance measurement
(A492), fluorescence intensity (Intensity), and positive (Pol+) and negative
(Pol-)
polarization values of each conjugate. Conjugates Nl-N5 were derived from 5-
carboxyfluorescein while Ml-M3 were derived from fluorescein-5-maleimide.
Isoelectric
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pH standards were not used, therefore the pIs listed were estimated from
comparison with
a published chart (Novex, San Diego,CA) and are only approximate. A492 refers
to the
absorbance of the undiluted solution, and is a measure of the yield of the
procedure.
Intensity is in arbitrary fluorescence units. Pol- is the fluorescence
polarization in mP
units in the absence of anti BS 106 antibody, and Pol+ is the polarization
after addition of
the antibody.
It is first notable that conjugates N5 and M3, those with the lowest p1, show
no
change in fluorescence polarization on addition of the antibody. These
compounds are
attributed to be the unconjugated fluorescein derivatives. Conjugates M1 and
M2, both of
which show significant elevation of the fluorescence polarization in the
presence of
antibody, are consistent with derivatization at either of the two cysteines in
the peptide.
The situation for N1-N4 is less clear. Since the SEQUENCE ID NO 26 peptide
contains 3
amines available for reaction with the fluorescein derivative (two lysines
plus the N-
terminal), only three major conjugates should be present. However, all 4 show
significant
binding to the antibody.
Table 7: Fluorescence Intensity and Polarization Results
Conjugate PI A492 Intensity Pol- Pol+
Ni 6.5 0.2406 4357 48.59 95.58
N2 6.0 0.2464 4370 64.55 182.47
N3 5.5 0.2519 4171 47.92 114.54
N4 5.3 0.2589 3391 53.92 149.82
N5 3.5 0.2693 4507 31.07 31.61
Ml 6.5 0.1884 5329 57.83 142.07
M2 5.7 0.1323 4886 53.77 136.91
M3 3.5 0.2169 8044 24.80 24.15
These conjugates were useful in testing antibodies directed against the C-
terminal
end of BS106, and in competitive immunoassays measuring BS106 concentrations
in
biological fluids. In Figure 15, 1.0 ml samples containing 2.0 nM of the
conjugates were
titrated with the affinity purified rabbit anti-BS 106 and the mouse
monoclonal antibodies
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H39C51 and H9C29. The polarizations increased rapidly with addition of
antibody,
leveling off as the solutions become saturated. The maximum polarization is
characteristic
of both the conjugate and the antibody, depending on the environment of the
fluorescein
moiety in the complex. Interestingly, conjugate N2, which showed the highest
limiting
polarization with the rabbit antibody, showed one of the lowest with both the
monoclonal
antibodies. Although they were derived from separate fusions, the two
monoclonal
antibodies showed nearly identical titration plots with all the conjugates,
suggesting strong
similarity of the binding sites.
C. Fluorescence Polarization Immunoassay of BS106 Using Fluorescein
Conjugate. A competitive assay was carried out using the SEQUENCE ID NO 26
peptide-fluorescein conjugate (Ml) as competitor. The antibody used was
monoclonal
antibody H24C16. The assay was set up such that the antibody [0.4 g/ml in 400
[tl of
FPIA diluent (Abbott Laboratories, Abbott Park, IL)] and the sample or
standard were
mixed and incubated at room temperature for 20 minutes. The competitor
(conjugate M1)
was then added and the polarization measured. The standard curve was obtained
using
known amounts of SEQUENCE ID NO 26 peptide. The indicated test samples were
products of affinity purification of human milk (Example 22D). The results are
presented
in Table S.
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Table 8: Competitive Assay Results
Sample l Pol, mP pmoles from M sample from
curve curve
Standard 0.0 159.24 0.00
1.0 M 0.5 140.99 0.53 1.060
SEQUENCE ID NO 26 1.0 129.93 0.92 0.920
2.0 110.54 2.11 1.055
5.0 94.72 4.88 0.976
10.0 86.91 10.08 1.008
11B 0.1 133.77 0.77 7.740
1.0 92.83 5.60 5.598
10.0 79.75
24A 0.1 131.44 0.86 8.630
1.0 93.44 5.35 5.345
24B 0.1 158.26 0.04 0.390
1.0 143.75 0.45 0.446
10.0 96.45 4.35 0.435
26A 10.0 145.95 0.38 0.038
26B 10.0 152.32 0.21 0.021
The polarization results were evaluated using a four parameter log-logit curve
fitting routine to give the pmoles of analyte in the cuvette, which was
divided by the
volume of sample added to give its concentration in M. The results are
calculated with
the assumption that the assay is insensitive to differences between BS 106
protein isolated
from milk and the SEQUENCE ID NO 26 peptide-fluorescein conjugate, which
competes
with it for binding sites on the antibody. Isolation and measurement of the
concentration
of BS106 protein by an unambiguous means would provide standards, which could
be
used in this immunoassay format to give reliably accurate concentrations of
unknown
samples.
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Example 22: Affinity Purification of BS106
A. Preparation of Amino-LinkTM Affinity Column for Purification of BS106.
Affinity purified rabbit anti-BS 106 antibody was coupled to Amino-LinkTM gel
(Pierce
Chemical, Rockford, IL) as directed by the manufacturer's instructions.
Briefly, 6.5 mg of
affinity purified rabbit anti-BS 106 antibody (in 4.5 ml of PBS) were passed
through a
desalting column equilibrated with citrate/carbonate buffer (pH 10). The
eluent was
placed on a column containing 2.0 ml AminoLink gel equilibrated with
citrate/carbonate
buffer. The column containing the antibody and gel was rotated for 4 hours at
room
temperature, drained, and washed with 5 ml of PBS. After draining the column,
2.0 ml of
PBS and 40 l of 5 M sodium cyanoborohydride were added. The column was
rotated for
4 hours at room temperature, drained, and washed with 5 ml of Pierce wash
buffer. The
column was ready for use.
B. Preparation of Sulfo-LinkTM Affinity Column for Purification of BS 106.
Affinity purified rabbit anti-BS 106 antibody was coupled to Sulfo-LinkTM gel
(Pierce
Chemical, Rockford, IL) as directed by the manufacturer's instructions.
Briefly, 8.6 mg of
affinity purified rabbit anti-BS 106 antibody (in 2 ml of 100 mM sodium
phosphate, 5 mM
EDTA, pH 6.0) were reduced with 14 mg of mercaptoethylamine hydrochloride for
90
minutes at 37 C. The reaction mixture was desalted on a 10 ml KwikTM column
and
placed into vacuum degassed coupling buffer. The reduced protein was
transferred to a 2
ml Sulfo-LinkTM column previously washed with degassed coupling buffer. The
gel-
protein mixture was mixed by inversion for 15 minutes at room temperature, and
allowed
to settle for 30 minutes. The buffer containing uncoupled protein was
collected and the
column was washed with 6 ml of coupling buffer. The remaining binding sites on
the
column were blocked by agitating the gel for 15 minutes with 2.0 ml of
coupling buffer
containing 15.8 mg cysteine hydrochloride. The gel was allowed to settle for
30 minutes,
and was then drained, and washed with 10 ml of PBS. The column was ready for
use.
C. Affinity Purification of BS 106 from Transient Transfection. Fifteen
milliliters
(15 ml) of culture supernate from the transient transfection of BS 106 (as
described in
Example l lc) were passed through the rabbit anti-BS106 Amino-LinkTM column.
The
column was washed with 5 ml of PBS and the protein was eluted with 4 ml of 100
mM
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glycine, pH 2.1. The eluting protein was collected in tubes containing 300 l
of 1.0 M
Tris base for neutralization.
The protein was further purified using a lectin which binds O-linked sugars.
The
eluate was passed through a column containing 5.0 ml ofjacalin agarose
equilibrated in
PBS (Sigma, St. Louis, MO) to bind the glycosylated protein. The column was
washed
with 20 ml of PBS, and the product was eluted with 10% melibiose (Sigma, St.
Louis,
MO) in PBS. The product, which was concentrated in fractions at 4.0-7.0 ml,
was passed
through a 10 ml desalting column equilibrated with PBS. The desalted product
was
concentrated to 0.5 ml using a Centricon 10 centrifugal concentrator (Amicon,
Beverly
MA) resulting in an absorbance reading at 280 nm of 0.91.
D. Affinity Purification of BS 106 from Human Milk. One hundred milliliters
(100 ml) of frozen human milk was thawed, transferred to two 50 ml conical
tubes, and
centrifuged for 60 min at 2000 x g. The tubes were punctured near the bottom
to drain the
turbid aqueous portion from the fatty layer at the top and the small amount of
solid at the
bottom. Fifty milliliters of the aqueous portion was passed through the rabbit
anti-BS 106
Amino-LinkTM column. The column was washed with PBS until the absorbance at
280
urn was below 0.005. The protein was eluted with 100 mM glycine, pH 2.15. The
eluent
was collected in 400 l fractions, which were neutralized with 50 1 of 1.0 M
Tris base.
The fractions with an absorbance reading at 280 nm of at least 0.03 were
combined and
passed through a column containing 0.5 ml jacalin agarose (Sigma, St. Louis,
MO)
equilibrated with PBS. This column was washed with 5 ml of PBS and the product
was
eluted with 10% melibiose in PBS. The fraction eluting at 400 1 to 1400 l
was passed
through a desalting column with PBS, and then concentrated with a Microcon 10
centrifugal concentrator (Amicon, Beverly MA) to 250 l. The absorbance at 280
urn of
the solution was 0.080.
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SEQUENCE LISTING
<110> Abbott Laboratories
Billing-Medel, Patricia A.
Cohen, Maurice
Colpitts, Tracey L.
Friedman, Paula N.
Gordon, Julian
Granados, Edward N.
Hodges, Steven C.
Klass, Michael R.
Kratochvil, Jon D.
Roberts-Rapp, Lisa
Russell, John C.
Stroupe, Stephen D.
<120> Reagents And Method Useful For Detecting
Diseases Of The Breast
<130> 5995.US.P2
<140> 09/516,444
<141> 2000-02-29
<150> US 08/962,094
<151> 1997-10-31
<150> US 08/742,067
<151> 1996-10-31
<160> 39
<170> FastSEQ for Windows version 4.0
<210> 1
<211> 201
<212> DNA
<213> Artificial Sequence
<220>
<223> EST Clone 1662885
<221> misc feature
<222> (26) _.. (26)
<223> n = a or g or c or t/u, unknown or other at
position 26
<221> misc feature
<222> (98) ... (98)
<223> n = a or g or c or t/u, unknown or other at
position 98
<221> misc feature
<222> (133) ... (133)
<223> n = a or g or c or t/u, unknown or other at
CA 02399047 2002-07-30
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position 133
<221> misc feature
<222> (145) ... (145)
<223> n = a or g or c or t/u, unknown or other at
position 145
<221> misc feature
<222> (183) ... (183)
<223> n = a or g or c or t/u, unknown or other at
position 183
<400> 1
ctcttaggct ttgaagcatt tttgtntgtg ctccctgatc ttcatgtcac caccatgaag 60
ttcttagcag tcctggtact cttgggagtt tccatctntc tggtctctgc ccagaatccg 120
acaacagctg ctncagctga cacgnatcca gctactggtc ctgctgatga tgaagcccct 180
gangctgaaa ccactgctgc t 201
<210> 2
<211> 308
<212> DNA
<213> Homo sapiens
<400> 2
taggctttga agcatttttg tctgtgctcc ctgatcttca ggtcaccacc atgaagttct 60
tagcagtcct ggtactcttg ggagtttcca tctttctggt ctctgcccag aatccgacaa 120
cagctgctcc agctgacacg tatccagcta ctggtcctgc tgatgatgaa gcccctgatg 180
ctgaaaccac tgctgctgca accactgcga ccactgctgc tcctaccact gcaaccaccg 240
ctgcttctac cactgctcgt aaagacattc cagttttacc caaatgggtt ggggatcttc 300
cgaatggt 308
<210> 3
<211> 292
<212> DNA
<213> Artificial Sequence
<220>
<223> EST Clone 901429
<221> misc_feature
<222> (236) ... (236)
<223> n = a or g or c or t/u, unknown or other at
position 236
<221> misc feature
<222> (259) ... (259)
<223> n = a or g or c or t/u, unknown or other at
position 259
<400> 3
gcatttttgt ctgtgctccc tgatcttcat gtcaccacca tgaagttctt agcagtcctg 60
gtactcttgg gagtttccat ctttctggtc tctgcccaga atccgacaac agctgctcca 120
gctgacacgt atccagctac tggtcctgct gatgatgaag cccctgatgc tgaaaccact 180
gctgctgcaa ccactgcgac cactgctgct cctaccactg caaccaccgc tgcttntacc 240
actgctcgta aagacattnc agttttaccc aaatgggttg gggatctccc ga 292
<210> 4
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<211> 197
<212> DNA
<213> Homo sapiens
<400> 4
gttttaccca aatgggttgg ggatctcccg aatggtagag tgtgtccctg agatggaatc 60
agcttgagtc ttctgcaatt ggtcacaact attcatgctt cctgtgattt catccaacta 120
cttaccttgc ctacgatatc ccctttatct ctaatcagtt tattttcttt caaataaaaa 180
ataactatga gcaacat 197
<210> 5
<211> 472
<212> DNA
<213> Homo sapiens
<400> 5
ctcttaggct ttgaagcatt tttgtctgtg ctccctgatc ttcatgtcac caccatgaag 60
ttcttagcag tcctggtact cttgggagtt tccatctttc tggtctctgc ccagaatccg 120
acaacagctg ctccagctga cacgtatcca gctactggtc ctgctgatga tgaagcccct 180
gatgctgaaa ccactgctgc tgcaaccact gcgaccactg ctgctcctac cactgcaacc 240
accgctgctt ctaccactgc tcgtaaagac attccagttt tacccaaatg ggttggggat 300
ctcccgaatg gtagagtgtg tccctgagat ggaatcagct tgagtcttct gcaattggtc 360
acaactattc atgcttcctg tgatttcatc caactactta ccttgcctac gatatcccct 420
ttatctctaa tcagtttatt ttctttcaaa taaaaaataa ctatgagcaa ca 472
<210> 6
<211> 473
<212> DNA
<213> Homo sapiens
<400> 6
ctcttaggct ttgaagcatt tttgtctgtg ctccctgatc ttcatgtcac caccatgaag 60
ttcttagcag tcctggtact cttgggagtt tccatctttc tggtctctgc ccagaatccg 120
acaacagctg ctccagctga cacgtatcca gctactggtc ctgctgatga tgaagcccct 180
gatgctgaaa ccactgctgc tgcaaccact gcgaccactg ctgctcctac cactgcaacc 240
accgctgctt ctaccactgc tcgtaaagac attccagttt tacccaaatg ggttggggat 300
ctcccgaatg gtagagtgtg tccctgagat ggaatcagct tgagtcttct gcaattggtc 360
acaactattc atgcttcctg tgatttcatc caactactta ccttgcctac gatatcccct 420
ttatctctaa tcagtttatt ttctttcaaa taaaaaataa ctatgagcaa cat 473
<210> 7
<211> 68
<212> DNA
<213> Artificial Sequence
<220>
<223> Restriction site
<400> 7
agctcggaat tccgagcttg gatcctctag agcggccgcc gactagtgag ctcgtcgacc 60
cgggaatt 68
<210> 8
<211> 68
<212> DNA
<213> Artificial Sequence
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<220>
<223> Restriction site
<400> 8
aattaattcc cgggtcgacg agctcactag tcggcggccg ctctagagga tccaagctcg 60
gaattccg 68
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Universal Primer
<400> 9
agcggataac aatttcacac agga 24
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Universal Primer
<400> 10
tgtaaaacga cggccagt 18
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 11
actgctcgta aagacattcc 20
<210> 12
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 12
gggacacact ctaccattc 19
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
CA 02399047 2002-07-30
WO 01/65262 PCT/US01/06516
5/11
<223> Sense Primer
<400> 13
aagcccctga tgctgaaacc 20
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Antisense Primer
<400> 14
tgcagaagac tcaagctgat tcc 23
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Target-Specific Forward Primer
<400> 15
aagcccctga tgctgaaacc 20
<210> 16
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Target-Specific Reverse Primer
<400> 16
tgcagaagac tcaagctgat tcc 23
<210> 17
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe
<400> 17
gaccactgct gctcc 15
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Sense Primer
CA 02399047 2002-07-30
WO 01/65262 PCT/US01/06516
6/11
<400> 18
actgctcgta aagacattcc 20
<210> 19
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Antisense Primer
<400> 19
gggacacact ctaccattc 19
<210> 20
<211> 90
<212> PRT
<213> Homo sapiens
<400> 20
Met Lys Phe Leu Ala Val Leu Val Leu Leu Gly Val Ser Ile Phe Leu
1 5 10 15
Val Ser Ala Gln Asn Pro Thr Thr Ala Ala Pro Ala Asp Thr Tyr Pro
20 25 30
Ala Thr Gly Pro Ala Asp Asp Glu Ala Pro Asp Ala Glu Thr Thr Ala
35 40 45
Ala Ala Thr Thr Ala Thr Thr Ala Ala Pro Thr Thr Ala Thr Thr Ala
50 55 60
Ala Ser Thr Thr Ala Arg Lys Asp Ile Pro Val Leu Pro Lys Trp Val
65 70 75 80
Gly Asp Leu Pro Asn Gly Arg Val Cys Pro
85 90
<210> 21
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 21
Ala Gln Asn Pro Thr Thr Ala Ala Pro Ala Asp Thr Tyr Pro Ala Thr
1 5 10 15
Gly Pro Ala Asp Asp Glu Ala Pro Asp Ala Glu Thr Thr Ala Ala Ala
20 25 30
Thr Thr Ala Thr Thr Ala Ala
<210> 22
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
CA 02399047 2002-07-30
WO 01/65262 PCT/US01/06516
7/11
<223> Synthetic Peptide
<400> 22
Thr Thr Ala Thr Thr Ala Ala Pro Thr Thr Ala Thr Thr Ala Ala Ser
1 5 10 15
Thr Thr Ala Arg Lys Asp Ile Pro Val Leu Pro Lys Trp Val Gly Asp
20 25 30
Leu Pro Asn Gly Arg Val Cys
<210> 23
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 23
Ala Arg Lys Asp Ile Pro Val Leu Pro Lys Trp Val Gly Asp Leu Pro
1 5 10 15
Asn Gly Arg Val Cys
<210> 24
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 24
Ala Ala Pro Ala Asp Thr Tyr Pro Ala Thr Gly Pro Ala Asp Asp Glu
1 5 10 15
Ala Pro Asp Ala Glu
<210> 25
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 25
Ala Gln Asn Pro Thr Thr Ala Ala Cys
1 5
<210> 26
<211> 23
<212> PRT
CA 02399047 2002-07-30
WO 01/65262 PCT/US01/06516
8/11
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 26
Cys Ala Arg Lys Asp Ile Pro Val Leu Pro Lys Trp Val Gly Asp Leu
1 5 10 15
Pro Asn Gly Arg Val Cys Pro
<210> 27
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 27
Gly Gly Trp Val Gly Asp Leu Pro Asn Gly Arg Val Cys Pro
1 5 10
<210> 28
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 28
Gly Pro Ala Asp Asp Glu Ala Pro Asp Ala Glu Cys
1 5 10
<210> 29
<211> 40
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 29
Ala Gln Asn Pro Thr Thr Ala Ala Pro Ala Asp Thr Tyr Pro Ala Thr
1 5 10 15
Gly Pro Ala Asp Asp Glu Ala Pro Asp Ala Glu Thr Thr Ala Ala Ala
20 25 30
Thr Thr Ala Thr Thr Ala Ala Cys
35 40
<210> 30
<211> 11
CA 02399047 2002-07-30
WO 01/65262 PCT/US01/06516
9/11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 30
Gln Asn Pro Thr Thr Ala Ala Pro Ala Asp Cys
1 5 10
<210> 31
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 31
Asn Pro Thr Thr Ala Ala Pro Ala Asp Cys
1 5 10
<210> 32
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 32
Pro Thr Thr Ala Ala Pro Ala Asp Thr Tyr Cys
1 5 10
<210> 33
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Peptide
<400> 33
Ala Arg Lys Asp Ile Pro Val Leu Pro Lys Trp Val Gly Asp Leu Pro
1 5 10 15
Asn Gly Arg Val Cys Pro
<210> 34
<211> 24
<212> PRT
<213> Artificial Sequence
CA 02399047 2002-07-30
WO 01/65262 PCT/US01/06516
10/11
<220>
<223> Affinity Purification System Recognition Site
<400> 34
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> 35
<211> 57
<212> PRT
<213> Artificial Sequence
<220>
<223> Affinity Purification System Recognition Site
<400> 35
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 Net 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
<210> 36
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> BamH I site
<400> 36
tccatctttc tggtcggatc ccagaatccg acaaca 36
<210> 37
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Pme I site
<400> 37
gagcggccgc atcgtttaaa ctgacgatct gcctc 35
<210> 38
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
CA 02399047 2002-07-30
WO 01/65262 PCT/US01/06516
ll/ll
<223> XbaI site plus 12 nucleotide sequences that
encode the four amino acid sequences
<400> 38
Ser Asn Glu Leu
1
<210> 39
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Antisense primer incorporates a sequence encoding
the eight amino acids just before the stop codons
<400> 39
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
