U.S. patent application number 13/210672 was filed with the patent office on 2012-05-17 for antibodies as a cancer diagnostic.
This patent application is currently assigned to Purdue Research Foundation. Invention is credited to Michael S. KINCH, Nicole D. Zantek.
Application Number | 20120122112 13/210672 |
Document ID | / |
Family ID | 37863795 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120122112 |
Kind Code |
A1 |
KINCH; Michael S. ; et
al. |
May 17, 2012 |
ANTIBODIES AS A CANCER DIAGNOSTIC
Abstract
Method and kits are provided for the detection and diagnosis of
metastatic disease. More particularly, the methods and kits employ
compounds that can detect EphA2, a specific epithelial cell
tyrosine kinase that is overexpressed in metastatic tumor cells. In
one embodiment the compound is an antibody capable of binding to an
epitope of EphA2.
Inventors: |
KINCH; Michael S.;
(Laytonsville, MD) ; Zantek; Nicole D.; (Edina,
MN) |
Assignee: |
Purdue Research Foundation
West Lafayette
IN
|
Family ID: |
37863795 |
Appl. No.: |
13/210672 |
Filed: |
August 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11716792 |
Mar 12, 2007 |
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13210672 |
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09640952 |
Aug 17, 2000 |
7192698 |
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11716792 |
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60149259 |
Aug 17, 1999 |
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Current U.S.
Class: |
435/6.14 ;
435/7.23 |
Current CPC
Class: |
G01N 33/57419 20130101;
G01N 33/57423 20130101; G01N 33/57496 20130101; C12Q 1/6886
20130101; G01N 33/57415 20130101; G01N 33/57434 20130101; C12Q
2600/112 20130101; G01N 33/57407 20130101; C12Q 2600/158 20130101;
G01N 33/57438 20130101; Y10S 436/813 20130101 |
Class at
Publication: |
435/6.14 ;
435/7.23 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/577 20060101 G01N033/577; G01N 33/574 20060101
G01N033/574 |
Goverment Interests
GOVERNMENT FUNDING
[0002] The present invention was made with government support under
Grant No. DAMD17-98-1-8146, awarded by the United States Army
Medical Research and Materiel Command. The Government has certain
rights in this invention.
Claims
1-66. (canceled)
67. A method for detecting the presence of metastatic or
potentially metastatic cells in a cell population comprising the
steps of: contacting the cells with a compound capable of specific
binding to EphA2 to allow compound binding to EphA2; and detecting
compound-EphA2 binding.
68. The method of claim 67 further comprising lysing at least a
portion of the cell population, and contacting the lysed cells with
the compound.
69. The method of claim 67 wherein the compound comprises an
antibody specific for a domain of the EphA2 protein, a natural or
artificial ligand, a peptide, an anti-sense molecule, an ATP
analogue, or a small molecule capable of specifically targeting
EphA2.
70. The method of claim 67 wherein the compound is a monoclonal
antibody.
71. The method of claim 70 further comprising lysing at least a
portion of the cell population, and contacting the lysed cells with
the antibody, wherein the antibody binds to an intracellular
epitope of EphA2.
72. The method of claim 70 wherein the antibody is produced by
hybridoma cell line D7 (ATCC number PTA 2755).
73. The method of claim 67 further comprising detecting whether
EphA2 is overexpressed, wherein overexpression of EphA2 relative to
normal cells is indicative of the presence of metastatic or
potentially metastatic cells in the cell population.
74. The method of claim 67 wherein the compound is labeled with a
detectable label, and the detecting step includes detecting the
label.
75. The method of claim 67 wherein the cell population is selected
from the group consisting of breast, kidney, prostate, lung, colon
and epithelial cells.
76. The method of claim 67 wherein the cell population comprises
cells from a tissue biopsy.
77. The method of claim 67 wherein the cell population comprises
cells from a body fluid.
78. The method of claim 77 wherein the body fluid is selected from
the group consisting of blood, plasma, spinal fluid, saliva, and
urine.
79. The method of claim 67 further comprising detecting whether the
EphA2 is phosphorylated; wherein detection of unphosphorylated
EphA2 is indicative of the presence of metastatic cancer cells in
the cell population.
80. The method of claim 79 wherein detecting whether the EphA2 is
phosphorylated comprises contacting the EphA2 with an
anti-phosphotyrosine antibody to allow binding of the
anti-phosphotyrosine antibody to EphA2; and detecting
anti-phosphotyrosine antibody-EphA2 binding.
81. The method of claim 67 further comprising detecting whether
EphA2 is localized to sites of cell-cell contact; wherein detecting
that EphA2 is not localized to sites of cell-cell contact is
indicative of the presence of metastatic cancer cells in the cell
population.
82. A method for distinguishing between metastatic and
non-metastatic cancers comprising assaying a population of cancer
cells for EphA2 expression, wherein assaying the cell population
comprises: contacting the cells with a compound capable of binding
to EphA2 to allow binding of the compound to EphA2; and detecting
compound-EphA2 binding; wherein EphA2 overexpression is indicative
of metastatic cancer, and absence of EphA2 expression is indicative
of non-metastatic cancer.
83. The method of claim 82 wherein the compound is an antibody.
84. An antibody which specifically binds to an intracellular
epitope of EphA2.
85. The antibody of claim 84 bound to a detectable label.
86. The antibody of claim 84 which is a monoclonal antibody.
Description
CONTINUING APPLICATION DATA
[0001] This application is a continuation application under 37 CFR
.sctn.1.53(b) of U.S. patent application Ser. No. 11/716,792, filed
on Mar. 12, 2007, which is a continuation of U.S. Ser. No.
09/640,952, filed on Aug. 17, 2000, which claims the benefit under
35 U.S.C. .sctn.119(e) of U.S. Provisional Application No.
60/149,259, filed Aug. 17, 1999, each of which is incorporated by
reference herein.
FIELD OF THE INVENTION
[0003] The present invention relates to diagnosis of metastatic
disease. More particularly, this invention relates to reagents that
can detect a specific epithelial cell tyrosine kinase that is
overexpressed in metastatic tumor cells. Most particularly, this
invention relates to reagents which bind to an intracellular
epitope of the epithelial cell tyrosine kinase, and the use of
these reagents for cancer diagnosis.
BACKGROUND AND SUMMARY OF THE INVENTION
[0004] Cancer cell metastasis requires cellular capacity to 1)
detach from a primary tumor, 2) migrate and invade through local
tissues, 3) translocate to distant sites in the body (via lymph or
blood), 4) colonize a foreign site, and 5) grow and survive in this
foreign environment. All of these behaviors are linked to cell
adhesions. Cell adhesions control the physical interactions of
cells with their microenvironment. Cell adhesions also initiate
signals that dictate tumor cell growth, death, and
differentiation.
[0005] Various cancer cells, including breast cancer cells, are
known to exhibit altered cell adhesion. As compared to normal
breast epithelia, transformed human breast epithelial cells have
decreased cell-cell contacts and increased interactions with the
surrounding extracellular matrix. These changes facilitate
increased detachment and migration of cancer cells away from cell
colonies and are directly linked with alteration in tyrosine
phosphorylation of cell membrane proteins. Tyrosine phosphorylation
is a potent form of cell signal transduction, and alteration in
levels of tyrosine phosphorylation is believed to be important for
tumor cell invasiveness. Thus, regulation of tyrosine
phosphorylation represents a promising target for therapeutic
intervention against metastatic cancer. Tyrosine phosphorylation is
controlled by cell membrane tyrosine kinases, and increased
expression of tyrosine kinases is known to occur in metastatic
cancer cells.
[0006] Identification of increased expression of cell membrane
tyrosine kinases would aid in the diagnosis and treatment of
metastatic diseases. One such tyrosine kinase is EphA2. A member of
the Eph family of tyrosine kinases known as Ephrins, EphA2 is a
transmembrane receptor tyrosine kinase with a cell-bound ligand.
Although cloned a decade ago, see Lindberg, R. A. and Hunter, T.,
"cDNA Cloning and Characterization of Eck, an Epithelial Cell
Receptor Protein-tyrosine Kinase in the Eph/elk Family of Protein
Kinases," Mol. Cell. Biol. 10 (12), 6316-6324 (1990), rather little
is known about EphA2 function, largely because EphA2-specific
antibodies previously have been difficult to generate.
[0007] To facilitate research on EphA2, an improved method for
generating a panel of monoclonal antibodies specific for tyrosine
phosphorylated proteins has been developed. Using this method, a
multiplicity of EphA2 recognizing monoclonal antibodies has been
generated. These antibodies have been used to show that EphA2 is
overexpressed in metastatic breast, lung, colon, and prostate
cells. Because EphA2 is expressed differently in normal and
metastatic cells, EphA2-specific antibodies are useful in the
diagnosis of metastatic disease. Antibodies produced by one
particular hybridoma recognize an intracellular epitope of EphA2
and have been shown to be highly specific in binding to EphA2.
[0008] Thus, one aspect of this invention is a compound which
specifically binds to an intracellular epitope of EphA2. In a
preferred embodiment, the compound is an antibody specific for a
domain of the EphA2 protein. However, natural or artificial
ligands, peptides, anti-sense, ATP analogues, or other small
molecules capable of specifically targeting EphA2 may be employed.
A second aspect of this invention is a method for generating
antibodies which recognize EphA2 intracellular epitopes. Another
aspect of this invention is the use of EphA2-specific antibodies in
the diagnosis of metastatic disease. An additional aspect of this
invention is a diagnostic reagent specific for detecting EphA2, any
fragment thereof, or DNA or RNA coding for the EphA2 protein.
[0009] Additional features of the present invention will become
apparent to those skilled in the art upon consideration of the
following detailed description of preferred embodiments
exemplifying the best mode of carrying out the invention as
presently perceived.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1A-C show a series of western blots showing EphA2
expression in cell lines derived from human prostate cells;
[0011] FIG. 1A is a western blot showing EphA2 expression in
various human prostate cancer cell lines;
[0012] FIG. 1B is a western blot showing EphA2 expression in human
prostatic epithelial cell line MLC and expression in that cell line
after transformation by oncogenic K-Ras or X-irradiation;
[0013] FIG. 1C is similar to FIG. 1B, except showing expression in
human prostatic epithelial cell line 267B1 and expression in that
cell line after transformation by oncogenic K-Ras or
X-irradiation;
[0014] FIG. 2 is a western blot showing EphA2 expression in various
human mammary epithelial cell lines;
[0015] FIG. 3A-B shows EphA2 localization in the cell membranes of
various mammary epithelial cell lines, as seen by
immunofluorescence microscopy;
[0016] FIG. 3A shows EphA2 localization in sites of cell adhesion
in normal MCF-10A cells; and
[0017] FIG. 3B shows EphA2 redistribution in malignant cells.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Antibodies specific for EphA2 have been isolated through an
improved method. The method employed is designed for increased
sensitivity and diversity of responding hybridomas. According to
this method, tyrosine phosphorylated proteins from Ras-transformed
human epithelial cells are isolated by affinity chromatography
using existing phosphotyrosine-specific antibodies. The tyrosine
phosphorylated proteins are then used as an immunogen for producing
monoclonal antibodies. Low-dose amounts of tyrosine phosphorylated
proteins are injected proximal to lymph nodes, every other day,
over a ten day period (the RIMMS strategy). B cells from engorged
lymph nodes are then isolated and fused with a Bcl-2-overexpressing
myeloma, to minimize apoptosis after fusion. This method results in
increased diversity, specificity, and cost-effectiveness of
hybridoma production. The hybridomas are first screened to identify
those hybridomas producing antibodies capable of distinguishing
malignant from normal cancer cells. To date, at least 450 such
hybridomas have been identified.
[0019] Hybridomas which are specific to EphA2 have been selected.
Use of the RIMMS strategy has resulted in the production of various
monoclonal antibodies that specifically bind EphA2. Of the first
four hybridomas characterized, two recognize independent epitopes
on EphA2. The first, D7, recognizes an intracellular epitope. The
second, B2D6, binds to an extracellular epitope. D7 has proven to
be highly specific for an intracellular epitope of EphA2 and this
specificity provides much of the current basis for diagnosis of
metastatic tumors. Hybridoma D7, identified as "murine hybridoma
D7," was deposited with the American Type Culture Collection
(ATCC), 10801 University Blvd., Manassas, Va., 20110-2209, USA, on
Dec. 8, 2000, and assigned ATCC number PTA 2755. Hybridoma B2D6,
identified as "murine hybridoma B2D6," was deposited with the
American Type Culture Collection (ATCC), 10801 University Blvd.,
Manassas, Va., 20110-2209, USA, on Dec. 8, 2000, and assigned ATCC
number PTA 2754.
[0020] It is known in the art to use antibodies to detect the
presence or overexpression of a specific protein. Because EphA2 is
overexpressed in metastatic cells, EphA2-specific antibodies of
this invention may be used to detect this overexpression and, thus,
to detect metastatic disease. Such techniques include but are not
limited to western blotting, dot blotting, precipitation,
agglutination, ELISA assays, immunohistochemistry, in situ
hybridization, flow cytometry on a variety of tissues or bodily
fluids, and a variety of sandwich assays. These techniques are well
known in the art. See, for example, U.S. Pat. No. 5,876,949, hereby
incorporated by reference. When antibodies specific for an
intracellular epitope of EphA2 are used, the cells must be lysed
and incubated with the antibody. The above techniques may be
performed on whole-cell lysates, or EphA2 may be separated out for
testing, such as by immunoprecipitation. The D7 antibodies of this
invention are highly specific for an intracellular epitope of EphA2
and have proven to be sensitive to differential expression of EphA2
in metastatic cells. Other techniques, such as immunohistological
staining, require whole cells, and may further require cell layers
of a particular cell density. Such tests require an antibody
specific for an extracellular epitope of EphA2.
[0021] The antibodies of this invention may be used to detect
metastatic disease in a wide variety of tissue samples. For
instance, research using EphA2-specific antibodies has revealed
that altered EphA2 expression occurs in breast, kidney, prostate,
lung, and colon cells, and it is believed that altered EphA2
expression occurs in other types of cell metastasis, particularly
epithelial malignancies. EphA2-specific antibodies may be used to
detect metastasis in biopsied tumor tissue. Also, samples of a
variety of body fluid samples, such as blood, plasma, spinal fluid,
saliva, and urine, can be tested with the antibodies of the present
invention. Altered EphA2 expression in these samples indicates the
presence of metastatic disease.
[0022] Additionally, other antibodies may be used in combination
with the antibodies of the present invention to provide further
information concerning metastatic disease state. For example, the
EphA2 of metastatic cells exhibits altered tyrosine
phosphorylation. In normal breast epithelial cells, EphA2 is
expressed and is tyrosine phosphorylated. However, in metastatic
breast epithelial cells, EphA2 is overexpressed, and the EphA2 is
not tyrosine phosphorylated. Because a test quantifying EphA2
expression sometimes may lead to an ambiguous result, it may be
desirable to determine tyrosine phosphorylation, as well as the
magnitude of EphA2 expression. Thus, a method of diagnosis using
the antibodies of this invention in combination with
phosphotyrosine-specific antibodies provides data for determining
the state of metastatic disease.
[0023] Moreover, the EphA2-specific antibodies of this invention
can be exploited to detect changes in EphA2 localization which are
associated with metastasis. In normal breast and prostate
epithelial cells, EphA2 is enriched in within sites of cell
adhesion. Conversely, in metastatic prostate cells EphA2 is
diffusely distributed, and in metastatic breast cancer cells EphA2
is redistributed into the membrane ruffles. Techniques such as
immunohistological staining or immunofluorescent microscopy are
well known in the art and may be used to visualize EphA2
distribution. See, for example, U.S. Pat. No. 5,514,554, hereby
incorporated by reference. EphA2 expression can be detected by
using antibodies capable of detecting whole EphA2 or fragments of
the EphA2 protein. Other methods of detecting altered EphA2
expression include detecting DNA or RNA sequences coding for the
EphA2 protein.
[0024] In order to detect overexpression or altered distribution of
EphA2, the EphA2-specific antibodies may be labeled covalently or
non-covalently with any of a number of known detectable labels,
such fluorescent, radioactive, or enzymatic substances, as is known
in the art. Alternatively, a secondary antibody specific for the
antibodies of this invention is labeled with a known detectable
label and used to detect the EphA2-specific antibodies in the above
techniques.
[0025] Preferred labels include chromogens dyes. Among the most
commonly used are 3-amino-9-ethylcarbazole (AEC) and
3,3'-diaminobenzidine tetrahydrocholoride (DAB). These can be
detected using light microscopy. Also preferred are fluorescent
labels. Among the most commonly used fluorescent labeling compounds
are fluorescein isothiocyanate, rhodamine, phycoerythrin,
phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
Chemiluminescent and bioluminescent compounds such as luminol,
isoluminol, theromatic acridinium ester, imidazole, acridinium
salt, oxalate ester, luciferin, luciferase, and aequorin also may
be used. When the fluorescent-labeled antibody is exposed to light
of the proper wavelength, its presence can be detected due to its
fluorescence.
[0026] Also preferred are radioactive labels. Radioactive isotopes
which are particularly useful for labeling the antibodies of the
present invention include .sup.3H, .sup.125I, .sup.131I, .sup.35S,
.sup.32P, and .sup.14C. The radioactive isotope can be detected by
such means as the use of a gamma counter, a scintillation counter,
or by autoradiography.
[0027] Another method in which the antibodies can be detectably
labeled is by linking the antibodies to an enzyme and subsequently
using the antibodies in an enzyme immunoassay (EIA) or
enzyme-linked immunosorbent assay (ELISA). The enzyme, when
subsequently exposed to its substrate, reacts with the substrate
and generates a chemical moiety which can be detected, for example,
by spectrophotometric, fluorometric, or visual means. Enzymes which
can be used to detectably label antibodies include, but are not
limited to malate dehydrogenase, staphylococcal nuclease,
delta-5-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase, and
acetylcholinesterase. Other methods of labeling and detecting
antibodies are known in the art and are within the scope of this
invention.
Example 1
[0028] The antibodies produced by the D7 hybridoma are used to
detect differential expression of EphA2 between normal prostate
epithelial cells and metastatic cells. FIG. 1 shows EphA2
expression in various human prostate cell lines. Referring first to
FIG. 1A, three metastatic cell lines, LNCAP, DU145, and PC3, are
tested for levels of EphA2 expression. It is known that, of these
three cell lines, LNCAP is the least invasive, DU145 is somewhat
more invasive, and PC3 is the most invasive. EphA2 expression is
determined by western blotting with D7 antibodies. As can be seen
in FIG. 1A, EphA2 expression positively correlates with
invasiveness.
[0029] In FIG. 1B, D7 antibodies are used to test EphA2 expression
in normal MLC cells as compared to expression in transformed cells.
Normal MLC cells, MLC cells which have been transformed by K-Ras,
and MLC cells with have been transformed by X-irradiation are
studied. As can be seen in FIG. 1B, EphA2 is overexpressed in both
of the transformed cell lines. FIG. 1C shows results similar to
FIG. 1B, except the normal cell line is 267B1. As with FIG. 1B,
FIG. 1C shows that EphA2 is overexpressed in the transformed cells.
In sum, FIG. 1 demonstrates that EphA2-specific antibodies detect
changes in metastatic cells, and that tests using these antibodies
indicate the level of metastatic invasiveness.
Example 2
[0030] EphA2 antibodies are used to detect altered EphA2 expression
in metastatic mammary cells. EphA2 is expressed in normal mammary
epithelial cells. FIG. 2 illustrates altered EphA2 expression in
mammary tumor cell lines. As can be seen in FIG. 2, western blots
from whole cell lysates using D7 antibodies reveal that EphA2
expression is completely absent in cells derived from
non-metastatic breast tumors (ZR75-1, BT474, SKBR3, MDA-MB-435). By
contrast, EphA2 is overexpressed in metastatic breast cancer cell
lines (MDA-MB-435, MDA-MB-231). Thus, EphA2 antibodies detect
altered EphA2 expression in breast cancer cells, which can be used
to diagnose metastasis. Moreover, in non-metastatic breast
epithelial cells, loss of EphA2 occurs early in the disease, and
testing with EphA2-specific antibodies provide information relevant
to invasiveness even when other known markers remain normal. Thus,
D7 antibodies are useful as a diagnostic, even in early stages of
disease.
Example 3
[0031] EphA2 antibodies in combination with other antibodies are
used to detect further alterations in EphA2 expression. As
discussed above in Example 2, western blots using D7 can
distinguish between non-metastatic and metastatic tumors, with
non-metastatic tumors failing to express EphA2, and metastatic
cells overexpressing EphA2. However, different results are found
when tyrosine phosphorylation is studied. Using a
phosphotyrosine-specific antibody, it has been found that EphA2 is
phosphorylated in normal cells, but it is not phosphorylated in
metastatic cells. Thus, while EphA2 specific antibodies can
qualitatively detect a difference between metastatic and
non-metastatic mammary tumor cells, diagnostics incorporating both
an EphA2-specific antibody and a phosphotyrosine-specific antibody
provides a sensitive test for distinguishing between normal,
non-metastatic, and metastatic mammary cells.
Example 4
[0032] Immunofluorescently labeled EphA2-specific antibodies detect
redistribution of EphA2 expression in transformed cells. The
EphA2-specific antibodies used in this example are produced by a
cell line known as B2D6, and these antibodies are specific for an
extracellular epitope of EphA2. As seen in FIG. 3A,
immunofluorescence with B2D6 demonstrates that EphA2 is found
within sites of cell-cell contact in normal cells. However, in
transformed cells, shown in FIG. 3B, EphA2 is redistributed.
Furthermore, in metastatic cells EphA2 is found in the membrane
ruffles. Similarly, in normal prostate epithelial cells, EphA2 is
found within sites of cell-cell adhesion, but in metastatic
prostate epithelial cells, EphA2 is overexpressed and the
expression is diffusely distributed. Therefore, immunofluorescence
using EphA2-specific antibodies provides an additional means for
diagnosing the transformation and metastatic state of tumor
cells.
[0033] As shown in Examples 1-4, overexpression, redistribution,
and phosphorylation of EphA2 in metastatic cells provide various
bases for diagnosis of metastatic tumors using EphA2-specific
antibodies. Immunohistochemistry or Western blotting may be used to
monitor the change of EphA2 expression in biopsied samples of
patient breast tissue, prostate tissue, or tissue from other
tumors. Additionally, D7 and other EphA2-specific antibodies can be
used to monitor plasma, urine, and other body fluids to detect
altered expression of EphA2, which would signal metastasis.
Detection of altered tyrosine phosphorylation of EphA2 in
combination with information concerning an alteration of EphA2
expression further aids in diagnosis of metastatic disease.
[0034] Although the invention has been described in detail with
reference to preferred embodiments, variations and modifications
exist within the scope and spirit of the invention as described and
defined in the following claims.
* * * * *