U.S. patent number 7,785,814 [Application Number 11/575,732] was granted by the patent office on 2010-08-31 for method of detecting cancer based on immune reaction to boris.
This patent grant is currently assigned to N/A, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Ziedulla Abdullaev, Victor V. Lobanenkov, Dmitri Loukinov, Svetlana Pack.
United States Patent |
7,785,814 |
Lobanenkov , et al. |
August 31, 2010 |
Method of detecting cancer based on immune reaction to BORIS
Abstract
The invention provides a method of detecting a proliferative
disease, such as a disease associated with the abnormal expression
of BORIS, in a mammal comprising detecting antibodies to BORIS in a
sample obtained from the mammal. The invention also provides BORIS
polypeptides as well as compositions and kits comprising the BORIS
polypeptides and methods of using the same. The invention further
provides a method of inducing an immune response in a mammal using
BORIS polypeptides.
Inventors: |
Lobanenkov; Victor V.
(Rockville, MD), Loukinov; Dmitri (Germantown, MD),
Abdullaev; Ziedulla (Damascus, MD), Pack; Svetlana (N.
Potomac, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
N/A (N/A)
|
Family
ID: |
36090647 |
Appl.
No.: |
11/575,732 |
Filed: |
September 21, 2005 |
PCT
Filed: |
September 21, 2005 |
PCT No.: |
PCT/US2005/033796 |
371(c)(1),(2),(4) Date: |
August 27, 2007 |
PCT
Pub. No.: |
WO2006/034335 |
PCT
Pub. Date: |
March 30, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20080095805 A1 |
Apr 24, 2008 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60611798 |
Sep 21, 2004 |
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Current U.S.
Class: |
435/7.23;
435/7.21 |
Current CPC
Class: |
G01N
33/57407 (20130101) |
Current International
Class: |
G01N
33/574 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 03/072799 |
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Sep 2003 |
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WO |
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WO 2005/021029 |
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Mar 2005 |
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WO |
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Other References
Skolnick et al. (Trends in Biotech., 18(1):34-39, 2000). cited by
examiner .
Dong et al., "Identification of two novel CT antigens and their
capacity to elicit antibody response in hepatocellular carcinoma
patients," British Journal of Cancer, 89, 291-297 (2003). cited by
other .
Klenova et al., "The novel BORIS + CTCF gene family is uniquely
involved in the epigenetics of normal biology and cancer," Seminars
in Cancer Biology, 12, 399-414 (2002). cited by other .
Lobanenkov et al., "CTCG and BORIS in Cancer (Epi)genetics,"
Keystone Symposia, 1, (2004). cited by other .
Loukinov et al., "BORIS, a novel male germ-line-specific protein
associated with epigenetic reprogramming events, shares the same
11-zinc-finger domain with CTCF, the insulator protein involved in
reading imprinting marks in the soma," Proc. Natl. Acad. Sci.
U.S.A., 99(10), 6806-6811 (2002). cited by other .
Masutomi et al., "Identification of serum anti-human telomerase
reverse transcriptase (hTERT) auto-antibodies during progression to
hepatocellular carcinoma," Oncogene, 21, 5946-5950 (2002). cited by
other .
Rockland Immunochemicals for Research, retrieved from the internet
at
www.rockland-inc.com/obects/catalog/product/extras/12282.sub.--600-401-90-
7.pdf on May 29, 2006, pp. 1-2. cited by other.
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Primary Examiner: Huff; Sheela J
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Claims
What is claimed is:
1. A method of detecting a cancer characterized by abnormal BORIS
expression in a mammal comprising detecting antibodies to a BORIS
polypeptide in a sample obtained from the mammal, wherein the BORIS
polypeptide comprises at least 100 amino acids of the amino acid
sequence encoded by (a) SEQ ID NO: 1 or (b) SEQ ID NO: 2, and
wherein the presence of antibodies to the BORIS polypeptide in the
sample indicates the presence of the cancer in the mammal.
2. The method of claim 1, wherein the antibodies to BORIS are
detected by (a) contacting the sample with a BORIS polypeptide
comprising at least 100 amino acids of the amino acid sequence
encoded by (a) SEQ ID NO: 1 or (b) SEQ ID NO: 2, and (b) detecting
the binding of the BORIS polypeptide with an antibody.
3. The method of claim 1, wherein the cancer is breast cancer,
glioma, lung cancer, or prostate cancer.
4. A method of detecting anti-BORIS antibodies in a sample from a
mammal comprising: (a) contacting the sample with a BORIS
polypeptide comprising at least 100 amino acids of the amino acid
sequence encoded by (a) SEQ ID NO: 1 or (b) SEQ ID NO: 2, and (b)
detecting the binding of the BORIS polypeptide with an antibody,
wherein the binding of the BORIS polypeptide with the antibody
indicates the presence of anti-BORIS antibodies in the sample.
5. The method of claim 1, wherein the mammal is a human.
6. The method of claim 1, wherein the sample is blood, serum,
plasma, lymph, or interstitial fluid.
7. The method of claim 2, wherein the BORIS polypeptide comprises a
full-length native BORIS polypeptide.
8. The method of claim 2, wherein the BORIS polypeptide comprises
at least 200 amino acids of the amino acid sequence encoded by SEQ
ID NO: 1.
9. The method of claim 8, wherein the BORIS polypeptide comprises
the amino acid sequence encoded by SEQ ID NO: 1.
10. The method of claim 2, wherein the BORIS polypeptide comprises
at least 100 amino acids of the amino acid sequence encoded by SEQ
ID NO: 2.
11. The method of claim 10, wherein the BORIS polypeptide comprises
the amino acid sequence encoded by SEQ ID NO: 2.
12. The method of claim 2, wherein the BORIS polypeptide further
comprises a flexible linker.
13. The method of claim 12, wherein the flexible linker comprises
an amino acid sequence of SEQ ID NO: 5.
14. The method of claim 2, wherein the BORIS polypeptide does not
comprise the zinc-finger region of BORIS.
15. The method of claim 2, wherein the BORIS polypeptide comprises
the amino acid sequence encoded by SEQ ID NO: 3.
16. The method of claim 2, further comprising determining the class
of the antibody that binds with the BORIS polypeptide.
17. The method of claim 2, wherein the antibody detected has a
binding affinity for BORIS that is greater than its binding
affinity for CCCTC-binding-factor (CTCF).
18. The method of claim 4, wherein the mammal is a human.
19. The method of claim 4, wherein the sample is blood, serum,
plasma, lymph, or interstitial fluid.
20. The method of claim 2 comprising contacting the sample with a
BORIS polypeptide that comprises at least 200 amino acids from the
N-terminal domain of BORIS of the amino acid sequence encoded by
SEQ ID NO: 1.
21. The method of claim 2 comprising contacting the sample with a
BORIS polypeptide that comprises the amino acid sequence encoded by
SEQ ID NO: 1.
22. The method of claim 2 comprising contacting the sample with a
BORIS polypeptide that comprises at least 100 amino acids from the
C-terminal domain of BORIS of the amino acid sequence encoded by
SEQ ID NO: 2.
23. The method of claim 2 comprising contacting the sample with a
BORIS polypeptide that comprises the amino acid sequence encoded by
SEQ ID NO: 2.
24. The method of claim 2 comprising contacting the sample with a
BORIS polypeptide that further comprises a flexible linker.
25. The method of claim 24, wherein the flexible linker comprises
an amino acid sequence of SEQ ID NO: 5.
26. The method of claim 2 comprising contacting the sample with a
BORIS polypeptide that does not comprise the zinc-finger region of
BORIS.
27. The method of claim 2 comprising contacting the sample with a
BORIS polypeptide that comprises the amino acid sequence encoded by
SEQ ID NO: 3.
28. The method of claim 2 comprising contacting the sample with a
BORIS polypeptide that comprises a full-length native BORIS
polypeptide.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application is the U.S. national phase of International Patent
Application No. PCT/US05/033796, which was filed on Sep. 21,
2005.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
on Mar. 21, 2007 and identified as follows: One 4,056 Byte ASCII
(Text) file named "701191_SEQUENCE.TXT," created on Mar. 15,
2007.
FIELD OF THE INVENTION
This invention pertains to novel BORIS polypeptides and the use of
such polypeptides for the detection of antibodies specific for
BORIS in a sample obtained from a mammal. The detection of
antibodies in a mammal specific for BORIS is useful, inter alia,
for the detection of proliferative diseases, such as cancer.
BACKGROUND OF THE INVENTION
The identification of tumor-associated antigens recognized by a
mammalian immune system is useful for the diagnosis and treatment
of cancer. A variety of tumor-associated antigens have been
identified, including cancer/testis antigens that are expressed in
cancer cells, but not in normal tissues other than testis. Only a
minority of tumor-associated antigens, however, are immunogenic to
the mammal that produces them.
BORIS (Brother of the Regulator of Imprinted Sites) is a
tumor-associated antigen, which is activated in a wide range of
human cancers. In fact, aberrant synthesis of the BORIS gene
product has been found in over 300 primary tumors and cancer cell
lines representing all major types of human cancers with recurrent
20q13 chromosomal gains. BORIS activation has also been found in
all of the standard NCI-60 cancer cell lines, which are maintained
by the National Cancer Institute (NCI), and which are thought to be
a reasonably complete representative set of human cancers. One
mechanism of action by which BORIS is thought to cause cancer
through interference with the maintenance of an appropriate
methylation pattern in the genome mediated by CCCTC binding factor
(CTCF) (see Klenova et al., Seminars in Cancer Biology 12, 399-414
(2002)).
BORIS is also a CTCF paralog, which contains all eleven zinc
fingers of CTCF, and has been shown to promote cell growth leading
to transformation (see Loukinov et al, Proc. Natl. Acad. Sci. (USA)
99, 6806-6811 (2002), and International Patent Application
Publication WO 03/072799 (PCT/US03/05186)). The BORIS gene is
believed to map to the cancer-associated amplification region of
chromosome 20q13.
The detection of aberrant expression of cancer markers, such as
prostate specific antigen (PSA) and carcinoembryonic antigen (CEA),
are known in the art. These assays, however, detect only a limited
number of cancers, and have limited positive predictive value for
the detection of new or recurring cancer, or for the prognosis of
cancer. Accordingly, there is a need in the art to identify
additional antigens whose expression can be linked to
hyperproliferative diseases, such as cancer, as well as methods of
detecting the presence of such antigens to aid in the detection,
diagnosis, prognostication, or research of such disease states.
The invention provides methods and compositions useful for the
detection, diagnosis, prognostication, or research of diseases
associated with abnormal BORIS expression, such as cancer. These
and other advantages of the invention, as well as additional
inventive features, will be apparent from the description of the
invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The invention provides a method of detecting a disease associated
with abnormal BORIS expression, including but not limited to
cancer, which method comprises detecting antibodies (e.g.,
autoantibodies) to BORIS in a sample obtained from a mammal. The
invention also provides a method of detecting antibodies to BORIS
in a sample obtained from a mammal, which method comprises
contacting the sample with a BORIS polypeptide and detecting the
presence of antibodies that bind to the BORIS polypeptide.
The invention further provides BORIS polypeptides (e.g.,
polypeptides comprising BORIS epitopes), compositions, and kits
comprising the BORIS polypeptides. In addition, the invention
provides a method of inducing an immune response in a mammal
comprising administering a BORIS polypeptide to the mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph depicting results of indirect ELISA assays for
anti-BORIS antibodies in human sera.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a method of detecting a disease
characterized by abnormal gene expression, such as a
hyperproliferative disease involving abnormal BORIS expression
(e.g., cancer), in a mammal, which method comprises providing a
sample from a mammal and detecting antibodies to BORIS in the
sample obtained from the mammal. The presence of anti-BORIS
antibodies in the sample indicates the presence of the disease in
the mammal. The terms "proliferative disease" and
"hyperproliferative disease" are used synonymously herein. Without
wishing to be bound to any particular theory, it is believed that
diseases characterized by abnormal gene expression, such as cancer,
are associated with the abnormal expression of BORIS in tissues
where BORIS is not normally found (e.g., tissues other than the
testes). As a result, the immune system of the mammal produces
antibodies to BORIS that can be detected in a sample (e.g., the
tissues, sera, or bloodstream) obtained from the diseased mammal.
In the absence of such a disease, BORIS is confined to the tissues
and organs in which it is normally found (e.g., the testes), and
the immune system of the mammal does not produce antibodies against
BORIS. Thus, a sample taken from a non-diseased mammal (e.g., a
mammal without a disease characterized by abnormal gene expression)
will not contain anti-BORIS antibodies. Accordingly, by detecting
the presence or absence of anti-BORIS antibodies in the sample of a
mammal, the method of the invention enables a determination as to
whether the mammal has a disease characterized by abnormal gene
expression, especially abnormal BORIS expression, such as
cancer.
The terms "BORIS" and "native BORIS" are used synonymously herein
to refer to the BORIS polypeptide as found in nature (e.g., as
produced by or isolated from a non-genetically engineered mammal).
Thus, these terms encompass any natural isoforms or homologues of
BORIS. The terms "anti-BORIS antibody" and "antibodies to BORIS"
are used synonymously herein to refer to any antibody that binds to
BORIS, including autoantibodies to BORIS.
The method of the invention can be used to detect any disease
characterized by or associated with the abnormal production of
BORIS, and consequent production of anti-BORIS antibodies,
including, but not limited, to the detection of cancer. As
mentioned above, BORIS mRNA has been detected in several hundred
cancer and tumor cell lines representing most of the major forms of
cancer. Thus, the method of the invention can be used to detect any
type of cancer. Such cancers include, but are not limited to,
cancer of the oral cavity and pharynx, the digestive system (e.g.,
the esophagus, stomach, small intestine, colon, rectum, anus,
liver, gall bladder, and pancreas), the respiratory system (e.g.,
the larynx, lung, and bronchus, including non-small cell lung
carcinoma), bones and joints (e.g., bony metastases), soft tissue,
the skin (e.g., melanoma), breast, the genital system (e.g., the
uterine cervix, uterine corpus, ovary vulva, vagina, prostate,
testis, and penis), the urinary system (e.g., the urinary bladder,
kidney, renal pelvis, and ureter), the eye and orbit, the brain and
nervous system (e.g., glioma), or the endocrine system (e.g.,
thyroid). The cancer also can be a lymphoma (e.g., Hodgkin's
disease and Non-Hodgkin's lymphoma), multiple myeloma, or leukemia
(e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia,
acute myeloid leukemia, chronic myeloid leukemia, and the
like).
Any suitable sample can be used in the invention. The types of
samples that can contain antibodies (e.g., autoantibodies) are
known in the art. The sample can be a solid sample, such as a
tissue sample, or the sample can be fluid, such as a sample of body
fluid. For instance, a section of whole tissue can be used for
immunohistochemistry-based analysis or can be homogenized to
liquefy the components found in the tissue. The sample is
preferably a fluid. Suitable fluid samples include, but are not
limited to, blood, serum, plasma, lymph, and interstitial
fluid.
Any suitable method of detecting anti-BORIS antibodies in a sample
can be used. Methods for detecting the presence of antibodies to a
known antigen in a sample are available in the art.
In a related aspect, the invention provides a method of detecting
antibodies specific for BORIS in a sample obtained from a mammal,
which method comprises contacting the sample with a BORIS
polypeptide (e.g., a polypeptide comprising a suitable portion of
BORIS that comprises one or more BORIS epitopes) and detecting
whether the sample contains anti-BORIS antibodies. Of course, the
binding of the BORIS polypeptide to an antibody in the sample
indicates the presence of anti-BORIS antibodies in the sample. The
method of detecting anti-BORIS antibodies can be used for any
suitable purpose, and is especially useful for detecting,
prognosticating, monitoring, or researching diseases characterized
by abnormal gene expression, especially abnormal BORIS expression,
including without limitation hyperproliferative diseases such as
cancer. Accordingly, the method of detecting anti-BORIS antibodies
provided by the invention can be used in conjunction with the
method of detecting a disease provided by the invention.
In this regard, the term "BORIS polypeptide" as used herein is
defined as a polypeptide to which an anti-BORIS antibody binds.
Thus, the term "BORIS polypeptide" as defined for the purposes of
this invention encompasses, but is not limited to, native BORIS. As
discussed in greater detail below, a BORIS polypeptide can, for
example, comprise, consist essentially of, or consist of BORIS, a
portion or fragment of BORIS recognized by an anti-BORIS antibody,
or a variant of BORIS or portion or fragment thereof recognized by
an anti-BORIS antibody.
The BORIS polypeptide can comprise, consist essentially of, or
consist of the native BORIS protein or a portion thereof.
Full-length BORIS proteins' are known in the art and disclosed, for
example, in Loukinov et al., supra, Klenova et al., supra, and
GenBank Accession No. AF336042. Other isoforms, homologs, and
paralogs can be isolated using the information provided herein and
routine techniques.
Any suitable portion of BORIS can be used to detect antibodies
specific for BORIS. As used herein, the term "portion" is
synonymous with the term "fragment" both of which are used to refer
to contiguous part of a polypeptide, preferably comprising 5 or
more amino acids. The portion of BORIS can be provided by the full
length BORIS protein (e.g., the full-length native BORIS
polypeptide); however, in some embodiments it is convenient to use
a shorter portion of BORIS. Accordingly, the portion of BORIS can
be any portion of the full-length native BORIS protein that can
recognize and bind to an anti-BORIS antibody in vitro or in vivo.
One can determine whether any given portion of BORIS binds to an
anti-BORIS antibodies using routine techniques in view of the
disclosures provided herein. For example, anti-BORIS antibodies can
be obtained from a mammal using native BORIS. Subsequently, the
given "test" portion of BORIS can be contacted with the anti-BORIS
antibodies to determine whether the anti-BORIS antibodies bind to
the given "test" portion of BORIS. Other methods of determining
whether a given portion of BORIS can bind to an anti-BORIS antibody
will be apparent to those of ordinary skill in the art.
Suitable portions of BORIS include the amino-terminal portion of
BORIS (the "N-terminal domain"), defined as the region extending
from the amino-terminal up to the zinc finger domain (the
"N-terminal domain"), or at least some portion thereof comprising
about 100 or more amino acids (e.g., 200 or more, 250 or more, 300
or more, 400 or more, or 500 or more amino acids) of the BORIS
N-terminal domain. A preferred portion of BORIS is the N-terminal
domain of BORIS encoded by a nucleic acid sequence comprising,
consisting of, or consisting essentially of SEQ ID NO: 1.
Other suitable portions of BORIS include the carboxyl-terminal
portion of BORIS, defined as the region starting after the
zinc-finger domain and terminating at the carboxyl-terminus of
BORIS (the "C-terminal domain"), or at least some portion thereof
comprising about 75 or more amino acids (e.g., about 100 or more,
200 or more, 300 or more, or 400 or more amino acids) of the BORIS
C-terminal domain. A preferred portion of BORIS includes the
C-terminal domain encoded by a nucleic acid sequence comprising,
consisting essentially of, or consisting of SEQ ID NO: 2.
In a particularly preferred embodiment of the invention, the BORIS
polypeptide comprises at least a portion of each of the N-terminal
domain and the C-terminal domain, as described herein. More
preferably, the BORIS polypeptide comprises the entire N-terminal
domain and C-terminal domain. It is further preferred that the
BORIS polypeptide excludes the zinc finger domain. A preferred
BORIS polypeptide is encoded by a nucleic acid sequence comprising,
consisting of, or consisting essentially of SEQ ID NO: 3.
Even smaller portions of BORIS can be used, provided that a BORIS
epitope is present in the portion or fragment. By "epitope" is
meant a sequence on an antigen that is recognized by an antibody or
an antigen receptor. Epitopes also are referred to in the art as
"antigenic determinants." For example, the portion of BORIS, or the
BORIS polypeptide comprising, consisting essentially of, or
consisting of the portion of BORIS, can be less than about 660,
200, 150, 100, 60, 50, 30, 20, 15, or 12 amino acid residues in
length, so long as it can be specifically bound by an antibody that
binds to BORIS, and preferably an antibody that binds only to BORIS
(i.e., a "BORIS-specific" antibody). The BORIS polypeptide
preferably comprises at least about 10, 11, or 12 amino acids. Of
course, the preferred number of amino acids also can be expressed
in terms of ranges within any of the above described preferred
limits (e.g., 10-200 amino acids, 10-100 amino acids, 10-50 amino
acids, 10-20 amino acids, etc.). However, BORIS polypeptides
comprising fewer than 11 amino acids (e.g., about 4, 6, 8, or 10 or
more amino acids) are also within the scope of the invention.
Any epitope of BORIS that is reactive with an anti-BORIS antibody
can be used alone, in the context of a larger BORIS polypeptide,
inserted into another (i.e., "non-BORIS") protein, or attached to
another (i.e., "non-BORIS") protein. Alternatively, more than one
BORIS epitope can be used. While not desiring to be bound by any
particular theory, it is believed that different individuals will
have immunogenic responses to BORIS based on the MHC molecules
expressed on their antigen presenting cells (e.g., macrophages).
Accordingly, the reactive epitopes of BORIS can vary from
individual to individual. Moreover, an autoreactive antibody
response directed against both the N-terminal and C-terminal
domains of BORIS has been detected in some cancer patients. Thus,
the method preferably involves the use of more than one BORIS
epitope. When more than one BORIS epitope is used, the different
BORIS epitopes can be provided, for example, by several
discontiguous portions of BORIS used simultaneously (e.g., two or
more BORIS polypeptides each comprising a different portion of
BORIS) or linked together and used as a single reagent (e.g., a
single BORIS polypeptide comprising two or more different portions
of BORIS).
The BORIS polypeptide can be joined to other biomolecules, such as,
for example, proteins, polypeptides, lipids, carbohydrates, prenyl,
and acyl moieties, and nucleic acids. Where another protein or
polypeptide is linked with a portion of BORIS, it is preferably
unrelated to BORIS (e.g., the other polypeptide will have less than
40% amino acid sequence identity with BORIS). In a preferred
embodiment of the invention, the BORIS polypeptide comprises a
flexible linker amino acid sequence. Flexible linkers are used in
the art to join two distinct polypeptides, such as, for example, in
the construction of fusion or chimeric proteins. In embodiments
where the BORIS polypeptide comprises two or more portions of
BORIS, for example, an N-terminal domain portion and a C-terminal
domain portion of BORIS, a flexible linker can be used to join the
N-terminal domain and the C-terminal domain to form a single
polypeptide molecule. The flexible linker can be any suitable amino
acid sequence that can be used to join to separate polypeptide
domains. In this regard, the flexible linker preferably comprises
about 5 or more amino acids (e.g., about 6 or more, 7 or more, or 9
or more amino acids), more preferably about 10 or more amino acids
(e.g., about 11 or more, 12 or more, or 14 or more amino acids),
and most preferably about 15 or more amino acids (e.g., about 17 or
more, 20 or more, or 25 or more amino acids). The flexible linker
for use in the present invention preferably comprises the amino
acid sequence of SEQ ID NO: 5, however, other linker sequences as
well as methods for joining polypeptide domains using flexible
linkers are known in the art (see, e.g., Imanishi et al., Biochem.
Biophys. Res. Commun., 333(1), 167-73 (2005); Lin-et al., Eur.
Cytokine Netw., 15(3), 240-6 (2004)).
The BORIS polypeptide can be attached to a signaling moiety (also
known as a detectable label). The identity and use of signaling
moieties is well-known in the art. A signaling moiety is a molecule
capable of indicating the presence of an analyte or reagent in a
sample, usually after manipulation of the sample. Such
manipulations often include incubating a sample and appropriate
detection reagents under conditions allowing two moieties to bind
together, if present, and then removing any of the labeled moiety
from the sample via washing, filtration, or other suitable
techniques. Other methods of working with signaling moieties are
well-known in the art. Suitable signaling moieties include, but are
not limited to, fluorescent molecules (e.g., green fluorescent
protein), fluorescent quenchers, epitopes and haptens for
antibodies that do not recognize BORIS (e.g., the well-known FLAG
epitope), enzymes (e.g., chromogenic or luminescent (such as horse
radish peroxidase or .beta.-galactosidase)), a nucleic acid that
can be amplified or specifically hybridized to a probe, biotin,
avidin or streptavidin, lectins and colloids. Methods for linking
proteins with detectable labels and solid supports are well-known
in the art.
As the primary function of the BORIS polypeptide is to bind with
anti-BORIS antibodies in the sample, the BORIS polypeptide need not
comprise a portion of BORIS per se. Rather, the BORIS polypeptide
can be a polypeptide that binds with an antibody that binds to
BORIS. In this respect, the polypeptide can comprise, consist
essentially of, or consist of a variant of BORIS. As used herein,
the term "variant of BORIS" means a variant of the full-length
native BORIS protein or a variant of a portion of BORIS, as
described herein. The skilled artisan can generate and characterize
BORIS variants for their ability to bind with an anti-BORIS
antibody or a functional fragment thereof (e.g., a Fab or
F'(ab).sub.2) using the information provided herein and routine
methods known in the art. For example, BORIS variants can be
generated using any suitable method known in the art, including,
but not limited to, site-directed or random mutagenesis of a
nucleic acid sequence encoding BORIS. The binding characteristics
of a BORIS variant thus produced can be determined, for example,
using antibodies to BORIS obtained from the serum of a mammal with
cancer. Such antibodies can be obtained, for instance, using a
native BORIS protein.
A variant of BORIS desirably shares one or more regions of amino
acid sequence identity with a native BORIS protein. In this regard,
the variant preferably comprises an amino acid sequence that is at
least about 50% identical (e.g., at least about 60%, at least about
70%, at least about 80%, or at least about 90% identical) to a
native BORIS polypeptide. More preferably, the polypeptide
comprises an amino acid sequence that is at least about 75%
identical (e.g., at least about 85%, or at least about 95%
identical) to an amino acid sequence of native BORIS. Most
preferably, the polypeptide comprises an amino acid sequence that
is at least about 90% identical (e.g., at least about 95%, at least
about 97%, or at least about 99% identical) to an amino acid
sequence of native BORIS. As used herein, the sequence identity is
that determined using the well-known BLAST algorithms (e.g.,
BLASTp, BLAST 2.1, BL2SEQ, and later versions thereof)). Variants
of BORIS capable of binding to anti-BORIS antibodies preferably
have at least 5, 6, or 7 amino acid residues that are identical to
BORIS over a window of eight amino acid residues.
BORIS variants in which an alteration destroys the ability of the
variant to bind with, or bind specifically with, an anti-BORIS
antibody can be used as a negative control.
The antibody detected in the invention preferably binds to BORIS
better than to CCCTC-binding-factor (CTCF), or does not bind CTCF
at all. Thus, the method of the invention preferably comprises
detecting an anti-BORIS antibody with a binding affinity for BORIS
that is greater than its binding affinity for CTCF. This is
particularly true where the portion of BORIS used comprises the
zinc-finger domain of BORIS. More preferably, the dissociation
constant (K.sub.d) of binding under standard conditions between the
antibody detected by the method of the invention and BORIS is at
least 10-fold less, more preferably at least 100-fold less, or even
1000-fold less than the K.sub.d of binding between the same
antibody and CTCF. In some embodiments, binding of antibodies in a
patient's serum to CTCF can be used as a negative control.
The mammal used in conjunction with the methods described herein
can be any suitable mammal, such as dogs, cats, cows, goats, pigs,
mice, rats, guinea pigs, rabbits, gerbils, monkeys, and hamsters.
The mammal preferably is a mouse, and more preferably a human.
The sample can be contacted with a BORIS polypeptide using any
suitable method known in the art. Preferably, the sample is
contacted with a BORIS polypeptide in vitro. In vitro methods for
detecting antibodies in a sample are well known in the art and
include, for example, enzyme-linked immunosorbent assay (ELISA),
affinity chromatography, and radioimmunoassay (RIA).
In a preferred embodiment, the method of detecting cancer or method
of detecting anti-BORIS antibodies includes determining the class
and/or subclass of the antibodies present in the patient's body, or
sample derived therefrom, that are reactive with BORIS. One of
ordinary skill in the art will appreciate that the five major human
immunoglobulin classes (or "isotypes") are immunoglobulin M (i.e.,
IgM), IgD, IgG, IgA, and IgE, which are typically defined by the
structure of the constant regions of the antibody heavy chain. The
light chain of a human antibody molecule is typically classified in
the art as either a lambda (.lamda.) chain or a kappa (.kappa.)
chain. IgG antibodies can be subdivided further into four subtypes
(i.e., IgG1, IgG2, IgG3, and IgG4), whereas IgA antibodies
typically are subdivided into two subtypes (i.e., IgA1 and IgA2).
It is well-known in the art how to determine the class and subclass
of isolated or purified antibodies. For example, BORIS-reactive
antibodies can be isolated from a human's serum by
immunochromatography. Wells of microtiter plates can be coated with
10 .mu.g/ml of anti-human immunoglobin overnight at 4.degree. C.
After blocking with 5% BSA, the plates are reacted with 10 .mu.g/ml
of a monoclonal antibody or purified isotype controls, at ambient
temperature for two hours. The wells can then be reacted with human
IgG 1-specific, IgG2-specific, IgG3-specific or IgG4-specific or
human IgM-specific alkaline phosphatase-conjugated probes. After
washing, the plates can be developed with a luminogenic or
chromogenic substrate and analyzed for light or color
development.
The methods of detecting a disease and detecting anti-BORIS
antibodies can be used in different ways. For example, the method
can be used simply to establish the existence of a disease state
for the purposes of diagnosis or screening. In addition, the method
can be used, for example, to monitor the status (e.g., progression
or regression) of a disease state, such as by comparing the level
of anti-BORIS antibodies (or BORIS expression levels) from
different samples over time. Such a use would be helpful in
monitoring the response of patients to a particular therapeutic
regimen.
The invention also provides an isolated immunoreactive portion of
BORIS. By "immunoreactive portion" is meant a portion of the BORIS
protein that can generate an immune response in a mammal in vivo,
binds to an anti-BORIS antibody in vivo or in vitro, or comprises
one or more BORIS epitopes. A portion of BORIS is isolated if it is
synthesized, or if it is removed from its natural environment. The
isolated immunoreactive portion of BORIS can comprise, consist
essentially of, or consist of any portion of BORIS or any BORIS
polypeptide described herein as useful in conjunction with the
method of detecting a disease or method of detecting an anti-BORIS
antibody, including without limitation the amino acid sequences
encoded by any of SEQ ID NOs: 1-3.
The BORIS polypeptide or portion of BORIS, as described herein, can
be generated using any suitable method. In this regard, nucleic
acid sequences encoding BORIS fragments can be synthetically
produced and expressed in an appropriate host cell, thereby
resulting in production of a portion of a BORIS polypeptide. In
addition, BORIS peptide fragments can be synthesized using protein
synthesis methods known in the art. Alternatively, BORIS
polypeptide fragments can be generated using proteases that cleave
within the full-length BORIS polypeptide. As discussed above, the
portion of BORIS can be labeled with a signaling moiety or
detectable label, or linked to a solid support.
The invention further provides a composition comprising a BORIS
polypeptide, as described herein, wherein the BORIS polypeptide can
optionally be labeled with a detectable label. Ideally, the
composition comprises a BORIS polypeptide and a pharmaceutically
acceptable (e.g., physiologically acceptable) carrier, diluent, or
excipient. The phrase "pharmaceutically acceptable carrier," as
used herein, refers to a carrier medium which does not interfere
with the effectiveness of the biological activity of the active
ingredients and which is not toxic to the host or patient. The
pharmaceutical compositions of the present invention can be in a
variety of forms. These include, for example, solid, semi-solid and
liquid dosage forms, such as tablets, pills, powders, liquid
solutions or suspensions, liposomes, injectable and infusible
solutions. Inhalable preparations, such as aerosols, are also
included. Preferred formulations are those directed to oral,
intranasal and parenteral applications, but it will be appreciated
that the preferred form will depend on the particular diagnostic or
therapeutic application. The methods for the formulation and
preparation of pharmaceutical compositions are well known in the
art and are described in, for example, Remington's Pharmaceutical
Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed.
(1985), The Merck Index, 11th ed., (Merck & Co. 1989), and
Langer, Science, 249, 1527-1533 (1990).
The BORIS polypeptide as described herein, including a full-length
native BORIS protein or isolated portions, fragments, chimeras,
fusions, and derivatives thereof, can be used for any purpose. In
addition to being useful in the method of detecting a disease and
method of detecting anti-BORIS antibodies, the BORIS polypeptide
can be used for other purposes, such as for inducing an immune
response in a mammal. Such immune responses have multiple uses. For
example, antibodies and other immunity-related molecules specific
for BORIS can be isolated and used for research, control reagents
useful in a method for detecting BORIS expression in a mammal, and
as a method for destroying cancer cells that are present or could
arise in a mammal. In this regard, the invention provides, as a
related aspect, a method of inducing an immune response in a mammal
comprising administering to a mammal a BORIS polypeptide as defined
herein. Suitable methods of administration are known in the art.
All other aspects of the method of inducing an immune response are
as previously described herein.
A BORIS polypeptide as described herein, including a full-length
native BORIS protein or isolated portions, fragments, chimeras,
fusions, and derivatives thereof, can also be supplied in a kit.
Thus, in a related aspect, the invention provides a kit comprising
a BORIS polypeptide as described herein and a reagent that
generates a signal when an antibody binds to a portion of a BORIS
polypeptide. Such reagents are known in the art. The kit preferably
further comprises instructions for determining whether a sample
obtained from a mammal comprises antibodies specific for BORIS,
which instructions optionally include criteria for determining
whether a cancer cell is likely present in the mammal from which
the sample was obtained. The kit can be used for any purpose, such
as in researching BORIS expression and diseases associated
therewith, and for the detection of BORIS antibodies and the
detection of cancer in a mammal.
The following examples further illustrate the invention but, of
course, should not be construed as in any way limiting its
scope.
Example 1
This example demonstrates that indirect ELISA detection of serum
antibodies to BORIS can be used to diagnose cancer in human
patients.
In order to detect antibodies to BORIS present in human sera, an
indirect ELISA was used, employing regions of BORIS that do not
have homology to CTCF as bait. In this regard, three different
plasmid constructs encoding portions of the N-terminal and/or
C-terminal domains of BORIS were generated using the pGEX-5.times.1
plasmid (Amersham Biosciences, Piscataway, N.J.). The sequence of
the full-length BORIS protein is provided in Loukinov et al.,
supra, Klenova et al., supra, and GenBank Accession No. AF336042.
The first plasmid contained the N-terminal domain of BORIS
corresponding to nucleotides 90-867 of the published BORIS nucleic
acid sequence (GenBank Accession No. AF336042) and a flexible
linker (SEQ ID NO: 5). The second plasmid contained the C-terminal
domain of BORIS corresponding to nucleotides 1761-2098 of the
published BORIS nucleic acid sequence. The third plasmid contained
both the N-terminal domain and the C-terminal domain of BORIS of
the second plasmid joined together via the flexible linker. Each
BORIS sequence was expressed as part of a fusion protein with
glutathione-5-transferase (GST) to facilitate purification.
Suitable concentrations (for example, about 1 .mu.g/mL) of each of
the BORIS fusion proteins in a suitable coating buffer (e.g., 15 mM
Na.sub.2CO.sub.3, 30 mM NaHCO.sub.3, pH 9.6 with 0.02% NaN.sub.3)
were absorbed onto 96-well microtititer plates for a suitable time
and at a suitable temperature (e.g., overnight at 4.degree. C.).
Plates were then washed with a suitable solution (e.g., 0.05%
Tween-20/PBS) and blocked (e.g., with 100 .mu.l per well of a BSA
or casein solution for about 1 hour at room temperature (RT)).
After washing, serial dilutions of human serum in a suitable
blocking reagent, such as BSA or casein, were added and incubated
under suitable conditions (e.g., for about 2 hours at 37.degree. C.
in a water-saturated incubator). Serum was obtained from human
patients suffering from glioma, lung cancer, breast cancer, and
prostate cancer.
Plates were washed and diluted secondary antibody (including, e.g.,
anti-human IgG 1-specific, IgG2-specific, IgG3-specific,
IgG4-specific or human IgM-specific alkaline
phosphatase-conjugates) in a blocking agent was added and incubated
for a suitable period of time under suitable conditions (e.g.,
about 1 hour at 37.degree. C.) The plates were then washed,
incubated with visualizing reagents under suitable conditions for
suitable times, the reaction was stopped, and the results
determined.
Using the BORIS fusion protein comprising the N-terminal domain
linked to the C-terminal domain of BORIS, sera from forty seven
cancer patients were positive for anti-BORIS antibodies, while sera
from six normal humans were negative for anti-BORIS antibodies (see
FIG. 1). FIG. 1 shows that the ELISA of the sera from six normal
subjects had a mean microtiter plate reading of about 0.1. The sera
from seventeen patients with gliomas had a mean microtiter plate
reading of greater than 0.15, from nine patients with lung cancer a
mean of about 0.3, from fifteen patients with breast cancer a mean
of over 0.4, and from six patients with prostate cancer a mean of
over 0.2. The differences between each cancer group's mean reading
and that of normal human sera were statistically significant.
Antibodies to the N-terminal fragment of the BORIS fusion protein
encoded by the first plasmid (e.g., without the C-terminal domain)
were detected in human sera, but at lower levels than the
antibodies specific to the BORIS polypeptide encoded by the third
plasmid. Antibodies to the C-terminal fragment of the BORIS fusion
protein encoded by the second plasmid (e.g., without the N-terminal
domain) were not detectable.
This example demonstrates that the use of a BORIS polypeptide for
the detection of anti-BORIS antibodies in serum is a highly
specific method for the diagnosis of a wide variety of cancers.
All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference to the
same extent as if each reference were individually and specifically
indicated to be incorporated by reference and were set forth in its
entirety herein.
The use of the terms "a" and "an" and "the" and similar referents
in the context of describing the invention (especially in the
context of the following claims) are to be construed to cover both
the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. The terms "comprising," "having,"
"including," and "containing" are to be construed as open-ended
terms (i.e., meaning "including, but not limited to,") unless
otherwise noted. Recitation of ranges of values herein are merely
intended to serve as a shorthand method of referring individually
to each separate value falling within the range, unless otherwise
indicated herein, and each separate value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those preferred embodiments may become
apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
SEQUENCE LISTINGS
1
51778DNAArtificialSynthetic 1atggcagcca ctgagatctc tgtcctttct
gagcaattca ccaagatcaa agaactcgag 60ttgatgccgg aaaaaggcct gaaggaggag
gaaaaagacg gagtgtgcag agagaaagac 120catcggagcc ctagtgagtt
ggaggccgag cgtacctctg gggccttcca ggacagcgtc 180ctggaggaag
aagtggagct ggtgctggcc ccctcggagg agagcgagaa gtacatcctg
240accctgcaga cggtgcactt cacttctgaa gctgtggagt tgcaggatat
gagcttgctg 300agcatacagc agcaagaagg ggtgcaggtg gtggtgcaac
agcctggccc tgggttgctg 360tggcttgagg aagggccccg gcagagcctg
cagcagtgtg tggccattag tatccagcaa 420gagctgtact ccccgcaaga
gatggaggtg ttgcagttcc acgctctaga ggagaatgtg 480atggtggcca
gtgaagacag taagttagcg gtgagcctgg ctgaaactgc tggactgatc
540aagctcgagg aagagcagga gaagaaccag ttattggctg aaagaacaaa
ggagcagctc 600ttttttgtgg aaacaatgtc aggagatgaa agaagtgacg
aaattgttct cacagtttca 660aattcaaatg tggaagaaca agaggatcaa
cctacagctg gtcaagcaga tgctgaaaag 720gccaaatcta caaaaaatca
aagaaagaca aagggagcaa aaggaacctt ccactgtg
7782338DNAArtificialSynthetic 2tggattaacc tgcacagaca ttcggagaag
tgtggatcag gggaagcaaa gtcggctgct 60tcaggaaagg gaagaagaac aagaaagagg
aagcagacca tcctgaagga agccacaaag 120ggtcagaagg aagctgcgaa
gggatggaag gaagccgcga acggagacga agctgctgct 180gaggaggctt
ccaccacgaa gggagaacag ttcccaggag agatgtttcc tgtcgcctgc
240agagaaacca cagccagagt caaagaggaa gtggatgaag gcgtgacctg
tgaaatgctc 300ctcaacacga tggataagtg agagggattc gggttgcg
33831161DNAArtificialSynthetic 3atggcagcca ctgagatctc tgtcctttct
gagcaattca ccaagatcaa agaactcgag 60ttgatgccgg aaaaaggcct gaaggaggag
gaaaaagacg gagtgtgcag agagaaagac 120catcggagcc ctagtgagtt
ggaggccgag cgtacctctg gggccttcca ggacagcgtc 180ctggaggaag
aagtggagct ggtgctggcc ccctcggagg agagcgagaa gtacatcctg
240accctgcaga cggtgcactt cacttctgaa gctgtggagt tgcaggatat
gagcttgctg 300agcatacagc agcaagaagg ggtgcaggtg gtggtgcaac
agcctggccc tgggttgctg 360tggcttgagg aagggccccg gcagagcctg
cagcagtgtg tggccattag tatccagcaa 420gagctgtact ccccgcaaga
gatggaggtg ttgcagttcc acgctctaga ggagaatgtg 480atggtggcca
gtgaagacag taagttagcg gtgagcctgg ctgaaactgc tggactgatc
540aagctcgagg aagagcagga gaagaaccag ttattggctg aaagaacaaa
ggagcagctc 600ttttttgtgg aaacaatgtc aggagatgaa agaagtgacg
aaattgttct cacagtttca 660aattcaaatg tggaagaaca agaggatcaa
cctacagctg gtcaagcaga tgctgaaaag 720gccaaatcta caaaaaatca
aagaaagaca aagggagcaa aaggaacctt ccactgtggg 780tggcggtggc
tccggtggcg gtggctccgg tggcggtggc tcctggatta acctgcacag
840acattcggag aagtgtggat caggggaagc aaagtcggct gcttcaggaa
agggaagaag 900aacaagaaag aggaagcaga ccatcctgaa ggaagccaca
aagggtcaga aggaagctgc 960gaagggatgg aaggaagccg cgaacggaga
cgaagctgct gctgaggagg cttccaccac 1020gaagggagaa cagttcccag
gagagatgtt tcctgtcgcc tgcagagaaa ccacagccag 1080agtcaaagag
gaagtggatg aaggcgtgac ctgtgaaatg ctcctcaaca cgatggataa
1140gtgagaggga ttcgggttgc g 1161445DNAArtificialSynthetic
4ggtggcggtg gctccggtgg cggtggctcc ggtggcggtg gctcc
45515PRTArtificialSynthetic 5Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser1 5 10 15
* * * * *
References