U.S. patent application number 10/917517 was filed with the patent office on 2005-04-07 for claudins as markers for early detection, diagnosis, prognosis and as targets of therapy for breast and metastatic brain or bone cancer.
Invention is credited to Kominsky, Scott L., Sukumar, Saraswati V..
Application Number | 20050074798 10/917517 |
Document ID | / |
Family ID | 27737556 |
Filed Date | 2005-04-07 |
United States Patent
Application |
20050074798 |
Kind Code |
A1 |
Sukumar, Saraswati V. ; et
al. |
April 7, 2005 |
Claudins as markers for early detection, diagnosis, prognosis and
as targets of therapy for breast and metastatic brain or bone
cancer
Abstract
Methods of diagnosis, prognosis, and treatment of breast cancer,
and of metastatic brain cancer, are provided The diagnostic and
prognostic methods involve the immunohistochemical detection of the
level of expression of the proteins claudin 1, 3, 4, and 7 in
tissue or cell samples. Claudins 1 and 7 are underexpressed in the
majority of breast cancers, and claudins 3 and 4 are overexpressed.
The methods of treatment involve the use of Clostridium perfringens
enterotoxin (or a variant thereof) to lyse metastatic cancer cells
in the brain and bone that overexpress claudins 3 and 4.
Inventors: |
Sukumar, Saraswati V.;
(Columbia, MD) ; Kominsky, Scott L.; (Columbia,
MD) |
Correspondence
Address: |
Whitham, Curtis & Christofferson, PC
Suite 340
11491 Sunset Hills Road
Reston
VA
20190
US
|
Family ID: |
27737556 |
Appl. No.: |
10/917517 |
Filed: |
August 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10917517 |
Aug 13, 2004 |
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PCT/US03/04371 |
Feb 14, 2003 |
|
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60356860 |
Feb 14, 2002 |
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60424222 |
Nov 6, 2002 |
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Current U.S.
Class: |
435/6.14 ;
435/7.23 |
Current CPC
Class: |
G01N 33/57484 20130101;
G01N 33/57415 20130101; A61K 38/4886 20130101; G01N 33/57407
20130101; A61P 35/04 20180101; A61K 38/164 20130101; G01N 2333/705
20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/006 ;
435/007.23 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Goverment Interests
[0002] This invention was made using funds from grants from the
Department of Defense having grant numbers DAMD17-01-0285 and
DAMD17-02-1-0429. The United States government may have certain
rights in this invention.
Claims
We claim:
1. A method for diagnosing cancer or metastasis in a patient in
need thereof, comprising the steps of determining a level of
expression of at least one claudin in cells of interest, wherein
said at least one claudin is selected from the group consisting of
claudin 1, claudin 3, claudin 4, and claudin 7, and assessing
whether claudins 3 or 4 are expressed at a level which is higher
than a predetermined level, or whether or not claudins 1 or 7 are
expressed at a level which is lower than a predetermined level,
where cancer or metastasis is implicated when claudins 3 or 4 are
at or above said level which is higher that said predetermined
level, or when claudins 1 or 7 are at or below said level which is
lower than said predetermined level.
2. The method of claim 1, wherein said cancer or metastasis is
selected from the group consisting of breast cancer, lung cancer,
colon cancer, kidney cancer, prostate cancer, pancreas cancer,
ovarian cancer, thyroid cancer, gastric cancer, head and neck
cancer, and skin cancer.
3. The method of claim 1 wherein said step of determining is
carried out by exposing said cells of interest to at least one
antibody recognizing a claudin.
4. The method of claim 3, wherein said at least one antibody
includes an antibody recognizing claudin 1, claudin 3, claudin 4,
or combinations thereof.
5. The method of claim 3, wherein said at least one antibody to a
claudin includes an antibody recognizing a C-terminal region of
CLDN-7.
6. The method of claim 3, wherein said antibody is an antibody
recognizing SEQ ID NO. 1.
7. The method of claim 1 further comprising the step of obtaining a
sample of said cells of interest from said patient.
8. The method of claim 7, wherein said sample is a biopsy tissue
sample.
9. The method of claim 7, wherein said sample comprises cells from
ductal lavage fluid.
10. The method of claim 7, wherein said at least one claudin is
claudin 3 or claudin 4, or both claudins 3 and 4, and said sample
is blood.
11. The method of claim 1, further comprising the step of
determining a grade of a sample containing said cells of interest
based on an assessment made in said assessing step, and wherein
said grade is high if claudin 7 expression is low, or low if
claudin 7 expression is high.
12. A method of killing cancer cells that express claudin-3 or
claudin-4 or both claudin-3 and claudin-4, comprising the step of
exposing said cancer cells to molecules recognizing claudin-3 or
claudin-4, wherein said molecules kill said cancer cells or deliver
cytotoxic agents that kill said cancer cells.
13. The method of claim 12, wherein said molecules include
Clostridium perfringens enterotoxin in quantities sufficient to
lyse said cancer cells.
14. The method of claim 13, wherein said Clostridium perfringens
enterotoxin is truncated by 45 amino acids at the amino
terminus.
15. The method of claim 13, wherein said Clostridium perfringens
enterotoxin is encapsulated in vessels selected from the group
consisting of liposomes, biodegradable synthetic polymer wafers,
and micro-spheres.
16. The method of claim 13, wherein said Clostridium perfringens
enterotoxin is in the form of a chimeric protein comprising a
matrix metalloprotease that is over-expressed by breast tumors.
17. The method of claim 12, wherein said molecules include
antibodies recognizing claudin-3 or claudin-4.
18. The method of claim 17, wherein said antibodies recognizing
claudin-3 or claudin-4 are attached to cytotoxic agents that kill
cancer cells.
19. The method of claim 12 wherein said cytotoxic agents that kill
cancer cells are contained in vessels selected from the group
consisting of liposomes, biodegradable synthetic polymer wafers,
and micro-spheres.
20. The method of claim 19, wherein antibodies recognizing
claudin-3 or claudin-4 are attached to said vessels.
21. The method of claim 12, wherein said cancer cells are primary
or metastatic cancer cells selected from the group consisting of
breast cancer cells, lung cancer cells, colon cancer cells, kidney
cancer cells, prostate cancer cells, pancreas cancer cells, ovarian
cancer cells, thyroid cancer cells, gastric cancer cells, head and
neck cancer cells, and skin cancer cells.
22. The method of claim 12, wherein said cancer cells are
metastatic cancer cells.
23. The method of claim 22, wherein said metastatic cancer cells
are located in a patient's brain or bone.
Description
[0001] This application claims priority to international patent
application PCT/US03/04371, filed Feb. 14, 2003, which in turn
claims priority to U.S. provisional patent application 60/356,860,
filed Feb. 14, 2002 and 60/424,222, filed Nov. 6, 2002, the
complete contents of which are hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The invention generally relates to the diagnosis, prognosis,
and treatment of cancer. In particular, the invention provides the
use of claudin proteins as targets for detection and treatment of
primary epithelial cancers and metastatic brain and bone
cancer.
[0005] 2. Background of the Invention
[0006] Breast cancer therapies have shown limited efficacy in
patients with advanced disease making early diagnosis essential for
long-term survival. Although many advances in diagnostic,
prognostic, and therapeutic methods have been made over the last
several years, breast cancer remains the second leading cause of
cancer death in women and the leading cause of death in women
between the ages of 40 and 55. Thus, there is an ongoing need for
new and improved diagnostic, prognostic, and therapeutic techniques
related to this disease.
[0007] Further, few effective therapies for metastatic brain or
bone cancer are currently available, and there is an ongoing need
for promising therapy for these diseases as well.
SUMMARY OF THE INVENTION
[0008] It is an object of this invention to provide methods of
diagnosis, prognosis, and treatment of breast cancer, and of
metastatic brain and bone cancer. The diagnostic and prognostic
methods involve the immunohistochemical detection of the level of
expression of the proteins claudin 1, 3, 4, and 7 in tissue or cell
samples. Claudins 1 and 7 are underexpressed in the majority of
breast cancers, and claudins 3 and 4 are overexpressed. The methods
of treatment involve the use of Clostridium perfringens enterotoxin
(or a variant thereof) or other cytotoxic agents targeted against
claudins 3 and/or 4 to lyse cancer cells that express claudins 3
and 4.
[0009] The invention thus provides a method for diagnosing breast
cancer or metastasis in a patient, comprising the steps of
determining a level of expression of at least one claudin in a
tissue or cell sample. In the method, the claudin may be claudin 1,
claudin 3, claudin 4, or claudin 7. The second step of the method
is to assess whether claudins 3 or 4 are expressed at a level which
is higher than a predetermined level, or whether or not claudins 1
or 7 are expressed at a level which is lower than a predetermined
level. Cancer or metastasis is implicated when claudins 3 or 4 are
at or above the predetermined level, or when claudins 1 or 7 are at
or below the predetermined level. The cancer of metastasis may be
breast cancer, lung cancer, colon cancer, kidney cancer, prostate
cancer, pancreas cancer, ovarian cancer, thyroid cancer, gastric
cancer, head and neck cancer, and skin cancer. In one embodiment,
the method is carried out by exposing the sample to at least one
antibody to a claudin (for example, an antibody to claudins 1, 3,
or 4, or any combination of these). The antibody may be directed to
a C-terminal region of CLDN-7, for example, an antibody to SEQ ID
NO. 1 (described below).
[0010] The method may further include the step of obtaining a
sample of cells of interest from a patient, e.g. as a biopsy tissue
sample or from ductal lavage fluid. In one embodiment of the
invention, the claudins are claudins-3 or -4, or both, and said
sample is blood.
[0011] The method may further comprise the step of determining a
grade of a sample containing cells of interest. Such a
determination is based on an assessment made in said assessing
step, and wherein the tumor grade is low if staining for claudin-7
is high, or the tumor grade is high is staining for claudin-7 is
low.
[0012] The present invention further provides a method of killing
cancer cells that express claudins-3 or -4, or both claudins-3 and
-4. The method comprises the step of exposing the cancer cells to
molecules the recognize claudin-3 or claudin-4, and the molecules
either kill the cancer cells or deliver cytotoxic agents that kill
the cancer cells. In one embodiment of the invention, the molecules
include Clostridium perfringens enterotoxin in sufficient
quantities to lyse the cancer cells. The Clostridium perfringens
enterotoxin may be truncated by 45 amino acids at the amino
terminus, and may be encapsulated in vessels such as liposomes,
biodegradable synthetic polymer wafers, or micro-spheres. In one
embodiment, the Clostridium perfringens enterotoxin is part of a
chimeric protein comprising a matrix metalloprotease that is
over-expressed by breast tumors. In another embodiment, the
molecules include antibodies that recognize claudin-3 or claudin-4,
or both. The antibodies that recognize claudin-3 or claudin-4, or
both may be attached to cytotoxic agent the kill cancer cells. In
yet another embodiment, the cytotoxic agents may be contained
within vessels such as liposomes, biodegradable synthetic polymer
wafers, or micro-spheres. In another embodiment, antibodies that
recognize claudin-3 or claudin-4 (or both) are attached to the
vessels.
[0013] The cancer cells may be breast cancer cells, lung cancer
cells, colon cancer cells, kidney cancer cells, prostate cancer
cells, pancreas cancer cells, ovarian cancer cells, thyroid cancer
cells, gastric cancer cells, head and neck cancer cells, and skin
cancer cells. The cancer cells may be metastatic, and in some
embodiments are located in a patient's brain or bone.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1: CLDN-7 mRNA expression in invasive ductal
carcinomas, uncultured luminal (Lum) and myoepithelial (Myo) human
mammary epithelial cells (uncultured HMEC), and human mammary
epithelial cells cultured in vitro (cultured HMEC).
[0015] Total RNA was extracted and cDNA was generated by reverse
transcription. CLDN-7 and GAPDH (a "housekeeping" gene) were
amplified individually by real-time PCR. CLDN-7 expression levels
were normalized to levels of GAPDH, multiplied by (10).sup.3, and
reported in arbitrary units +/-s.d. Data are from experiments
performed in triplicate.
[0016] FIG. 2: Claudin-7 polyclonal antibody production and
detection by ELISA.
[0017] Rabbit anti-human Claudin-7 pAb was raised against the
synthetic polypeptide (CKAGYRAPRSYPKSNSSKEYV) (SEQ ID NO:1), which
corresponds to the C-terminus of human Claudin-7. The presence of
Claudin-7 polyclonal antibody in rabbit sera was determined by
ELISA. 96-well plates were coated over night with synthetic
polypeptide at a concentration of 600 ng/50 ul. Plates were dried
and incubated at room temperature (RT) in blocking buffer (50 g
Milk/L PBS) for 2 hrs. Rabbit antisera was diluted in PBS, added to
plate, and incubated for 2 hours at RT. Presence of Claudin-7 pAb
was visualized using anti-rabbit secondary antibody conjugated to
horse radish peroxidase (HRP) at 490 nm.
[0018] FIGS. 3A and B. Clostridium perfringens enterotoxin
efficiently lyses human breast cancer cells expressing Claudins 3
and 4 while it has no effect on human breast cancer cells lacking
Claudin 3 and 4 expression. Cells were plated at 3.times.10.sup.5
cells/well in 6-well plates and grown to 80% confluence. Media was
then removed and replaced with fresh media with or without CPE at
concentration ranging from 0.05 to 4 ug/ml. Cells were incubated at
37.degree. C. for 60 min. Floating and attached cells were
collected and counted using a hemocytometer. Cell viability was
determined by trypan blue (0.4%) dye exclusion. Data from
representative experiments are expressed as % cytotoxicity as
compared to media control +/-S.D. A, results from CLDN-3,4 positive
cells; B, results from CLDN-3,4 negative cells.
[0019] FIGS. 4A and B. Treatment of T47D human breast cancer cell
xenografts with Clostridium perfringens enterotoxin results in a
reduction of tumor volume. T47D cells (1.times.10.sup.7) were
resuspended in matrigel and subcutaneously injected bilaterally in
the flank of 6-8 week old SCID mice. Tumors were grown to
approximately 100 mm.sup.3 prior to CPE treatment. CPE (A=2 ug,
B=10 ug) was administered intratumorally on days 1, 3, 5, 7, 9, 11,
and 13. Tumor volumes were measured using a caliper and reported
+/-sd. Each experiment is representative of 6 animals. Reduction in
CPE-treated tumor size (B) on day 14 relative to day 1 was found to
be significant by Student's t-test (p=0.007).
[0020] FIG. 5. Clostridium perfringens enterotoxin efficiently
lyses rat breast cancer cells (NMU 36/NMU 58) expressing Claudins 3
and 4 while it has no effect on NIH 3T3 cells lacking Claudin 3 and
4 expression. Cells were plated at 3.times.10.sup.5 cells/well in
6-well plates and grown to 80% confluence. Media was then removed
and replaced with fresh media with or without CPE at concentration
ranging from 0.05 to 4 ug/ml. Cells were incubated at 37.degree. C.
for 60 min. Floating and attached cells were collected and counted
using a hemocytometer. Cell viability was determined by trypan blue
(0.4%) dye exclusion. Data from representative experiments are
expressed as % cytotoxicity as compared to media control
+/-S.D.
[0021] FIG. 6A-D. Recombinant CPE treatment of Sprague-Dawley
NMU-induced breast tumor results in a reduction in tumor volume.
Sprague-Dawley NMU-induced tumors were injected intraductally with
(A, B) 17 ug CPE on days 1, 5, and 11 or (C, D) 30 ug CPE on days
1, 7, 9, 14, 16, 19, 22, and 26 versus PBS alone. Tumor volumes
were measured using a caliper. Each graph is representative of 1
tumor.
[0022] FIG. 7. Intraductal administration of native CPE
significantly inhibits the growth of NMU-induced rat mammary
tumors. Female Sprague-Dawley rats (3-6 weeks old) were injected
with 50 mg/kg NMU i.p. CPE was administered ID at 3 or 5
.mu.g/injection once every three days for 30 days once tumors grew
to a size of 100 mm.sup.3. Tumor size was measured using calipers
three times per week. Significant differences in tumor size between
CPE-treated and Control tumors was determined by Student's
t-test.
[0023] FIG. 8. Intracranial administration of CPE significantly
increases the survival of mice with established breast cancer
metastasis to the brain. MDA-MB-468 human breast cancer cells were
injected intracranially into athymic nude mice. Following the
establishment of solid tumor, mice were administered intracranial
injections of either 0.5 .mu.g CPE or PBS three times per week for
two weeks. Animals were observed for signs of neurological
complications resulting from tumor burden and sacrificed when
moribund.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0024] The present invention provides diagnostic and prognostic
methods for the detection of breast cancer. The methods are based
on the discovery that certain tight junction proteins, namely
claudins 1, 3, 4, and 7, are differentially expressed in breast
cancer cells. In particular, claudins 1 and 7 are underexpressed in
breast cancer cells and tissue, compared to normal breast cells and
tissue, while claudins 3 and 4 are overexpressed in breast cancer
cells and tissue, compared to normal breast cells and tissue. Thus,
the detection of the expression (or lack thereof) of one or more of
claudins 1, 3, 4 and 7 provides a means of determining whether or
not cells or tissue from the breast are malignant. Such detection
methods may be used, for example, for early diagnosis of the
disease, to monitor the progress of the disease or the progress of
treatment protocols, or to assess the grade of the cancer. The
grade of the cancer is related to the progression of disease and
thus aides in diagnosis, prognosis, and treatment
considerations.
[0025] The detection of the expression profile of claudins 1, 3, 4
and/or 7 in breast cells may be carried out by any of several means
well known to those of skill in the art. In a preferred embodiment
of the present invention, the method of detecting claudins 1, 3, 4
and/or 7 is immunological in nature. By `immunological`, we mean
that antibodies (e.g., monoclonal antibodies) specific for claudins
1, 3, 4 or 7, will be used. By "specific for claudins 1, 3, 4 or 7"
we mean antibodies that recognize claudin 1, 3, 4, or 7 while not
cross-reacting with samples containing other proteins.
[0026] The antibodies which can be used according to the method of
the invention can be monoclonal antibodies prepared using hybridoma
fusion techniques or can be derived from known secreting myeloma
cell lines. Those of skill in the art will recognize that many
techniques are available for the production of monoclonal
antibodies.
[0027] While in a preferred embodiment of the present invention the
method of detection of claudins is by utilizing monoclonal
antibodies, those of skill in the art will recognize that other
methods of detection can also be used in the practice of the
invention. For example, polyclonal antibodies raised against a
claudin (or fragment thereof) might also be used in various immuno
assays. For example, in a preferred embodiment of the invention,
for the detection of claudin 7, a polyclonal antibody against a
synthetic polypeptide with the sequence CKAGYRAPRSYPKSNSSKEYV, (SEQ
ID NO: 1) which corresponds to the C-terminus of human claudin-7 is
used.
[0028] Many antibody detection systems are known to those of skill
in the art and can be employed within the scope of the present
invention. For example, the antibody may be diagnostically labeled.
The term "diagnostically labeled" means that the antibody has
attached to it a diagnostically detectable label. Many such labels
and methods of conjugating labels to antibodies are well-known to
those of skill in the art. Examples of types of labels which can be
used in the practice of the present invention include but are not
limited to fluorescent labels, enzyme labels, and radionuclide
labels, specific binding pair components, colloidal dye substances,
fluorochromes, reducing substances, latexes, digoxigenin, metals,
particulates, dansyl lysine, antibodies, protein A, protein G,
electron dense materials, chromophores and the like. Any suitable
label, whether directly or indirectly detectable, may be employed.
One skilled in the art will recognize that these labels set forth
above are merely illustrative of the different labels that could be
utilized in this invention.
[0029] Many methods employing antibodies which specifically bind
target substances are known in the art. Preferred methods include
immunochemical methods, such as enzyme-linked immunosorbent assay
(ELISA) methods, immunonophelometry methods, agglutination methods,
precipitation methods, immunodiffusion methods,
immunoelectrophoresis methods, immunofluorescent methods, and
radioimmunoassay methods. Assays for detecting the presence of
proteins and/or peptides with antibodies have been previously
described and follow known formats, such as a standard blot and
ELISA formats. These formats are normally based on incubating an
antibody with a sample suspected of containing the protein or
peptide and detecting the presence of a complex between the
antibody and the protein or peptide. The antibody is labeled either
before, during or after the incubation step. Immobilization is
usually required and may be accomplished by immobilizing the
protein or peptide to a solid surface, such as a microtiter well,
or by binding the protein to immobilized antibodies.
[0030] In a preferred embodiment, the claudin(s) is bound to an
immobilized first antibody. A second labeled antibody, also
specific for the claudin, or specific for the first antibody, is
then bound, unbound material is washed away, and the complex is
detectable due to the immobilized label of the second antibody.
Such assays are well-known to those of skill in the art and include
such assays as simultaneous sandwich, forward sandwich and reverse
sandwich immunoassays, terms which are well-known to those of skill
in the art.
[0031] Many solid phase immunoabsorbents for immobilization are
known and can be used in the practice of the present invention.
Well-known immunoabsorbents include beads formed from glass,
polystyrene, polypropylene, dextran, nylon and other material; and
tubes formed from or coated with such materials, and the like. The
immobilized antibodies may be covalently or physically linked to
the solid phase immunosorbent by techniques such as covalent
bonding via an amide or ester linkage or by absorption.
[0032] In each of the above assays, the details of the assay
protocol, such as time and temperature of incubation, may vary
according to the concentration of antibodies used, the source and
form of the sample, the affinity of the antibodies for their target
molecules, etc. In a preferred embodiment of the present invention,
an ELISA assay may be carried out as follows: 96-well microtiter
plates are coated with a monoclonal first antibody specific for a
claudin 1, 3, 4 and/or 7. The first antibody is immobilized in the
wells. Standards and samples are pipetted into wells in, for
example, duplicate or triplicate, and any claudin present in the
standards and samples will be bound by the immobilized antibody.
The standards are composed of known concentrations of claudin,
which is known to be crossreactive with the first antibody. After
incubation at room temperature for 2 hours, the wells are washed
with an appropriate buffer to remove any unbound substances. Then a
second enzyme-linked polyclonal (or monoclonal) antibody specific
for claudin (or for the primary antibody) is added to the wells.
After a 1 hour incubation at room temperature, the wells are again
washed with an appropriate buffer to remove unbound antibody-enzyme
reagent, and a solution which contains a substrate for the enzyme
is added to the wells. The substrate is such that when it is acted
on by the enzyme, a characteristic color is produced. Color will
develop in proportion to the amount of enzyme present in the wells,
which is directly proportional to the amount of bound claudin.
After an appropriate period of time, the color development is
stopped and the intensity of the color will be measured
spectrophotometrically. The amount of claudin in the samples will
be determined by comparing the color intensity of the sample wells
to that of the control wells which contain a known amount of
claudin.
[0033] The antibodies to be employed in the practice of the present
invention are specific for claudin 1, 3, 4, and/or 7 and may be
specific for any epitope of claudin 1, 3, 4, and/or 7. The
antibodies may be raised against purified samples of a synthetic
peptide having a sequence identical to that of claudin 1, 3, 4,
and/or 7, either the full-length native form of the protein, or to
proteolytic or synthetic fragments thereof. Those of skill in the
art will readily recognize that there are numerous established
protocols available for generating antibodies to specific peptides
and proteins.
[0034] Various types of immuno assays which might be utilized in
the practice of the present invention include but are not limited
to immunoelectrophoresis, nephelometry, gel electrophoresis
followed by Western blot, dot blots, affinity chromatography,
immuno-fluorescence, and the like. In addition, other methods of
detection of peptides known to those of skill in the art may be
used in the practice of the current invention, such as gas
chromatography/mass spectrometry, HPLC, and gel electrophoresis
followed by sequencing.
[0035] In general, such methods involve obtaining a sample to be
tested. Such samples may be obtained by any of many methods known
to those of skill in the art. For example, the cells and/or tissue
may be from a biopsy sample. More preferably, since the method is
designed to detect breast cancer at very early stages, the sample
may be ductal lavage fluid which may contain cells with altered
claudin expression before a recognizable tumor mass has developed.
Further, the sample may also be blood since the detection of
claudins 3 and 4 in blood may be indicative of the presence of
tumors or metastasis.
[0036] The sample of cells or tissue is prepared and exposed to the
antibody or a mixture of antibodies according to means which are
known to those of skill in the art. Briefly, tissue sample may be
obtained by biopsy, lumpectomy, or mastectomy. Samples are then
paraffin embedded and sectioned for immunohistochemical analysis of
gene expression. cells may be obtained by ductal lavage and/or
collection of blood. Red blood cells are then removed from whole
blood by lysis in H.sub.2O. Protein is then extracted from the
remaining mixture of leukocytes and cancer cells or ductal lavage
fluid. Equal amounts of protein are then absorbed to a 96-well
plate over night for ELISA based assay. Alternatively, serum may be
separated from blood samples. Proteins are then immobilized to a
solid support for ELISA based assay.
[0037] The present invention further provides a kit for use in, for
example, the screening, diagnosis or monitoring of breast cancer.
Such a kit may comprise antibodies to claudins 1, 3, 4 and/or 7, a
reaction container, various buffers, secondary antibodies,
directions for use, and the like. In these kits, antibodies may be
provided with means for binding to detectable marker moieties or
substrate surfaces. Alternatively, the kits may include antibodies
already bound to marker moieties or substrates. The kits may
further include positive and/or negative control reagents as well
as other reagents for carrying out diagnostic techniques. For
example, kits containing antibody bound to multiwell microtiter
plates can be provided. The kit may include a standard or multiple
standard solutions containing a known concentrations of claudins or
other proteins for calibration of the assays. A large number of
control samples will be assayed to establish the threshold, mode
and width of the distribution of claudins 1, 3, 4 and 7 in normal
cells and tissues against which test samples will be compared.
These data will be provided to users of the kit.
[0038] In general, in order to be considered significantly over
expressed (i.e. to be considered "positive" for the presence of the
claudin), a claudin will be detected as present or in an amount of
about 10 to about 100% or more above the known, standardized level
of normal control tissue, or more preferably from about 25 to about
100% or more above the known, standardized level of normal control
tissue. In general, in order to be considered significantly under
expressed, a claudin will be detected as present or in an amount of
about 25 to about 75% or more below the known, standardized level
of normal control tissue, or more preferably from about 50 to about
100% or more lower than the known, standardized level of normal
control tissue. By "known, standardized level of normal control
tissue" we mean that level detected in equivalent tissue derived
from disease-free individuals. Further, such comparisons are
typically made in comparison to a known negative control, such as
tissue known to be devoid of the antigen being detected.
[0039] Other means of detecting the expression profile of claudins
1, 3, 4 and/or 7 include but are not limited to, for example,
detection of mRNA encoding one or more of the proteins. Those of
skill in the art are well acquainted with methods of mRNA
detection, e.g. via the use of complementary hybridizing primers
(e.g. labeled with radioactivity or fluorescent dyes) with or
without polymerase chain reaction (PCR) amplification of the
detected products, followed by visualization of the detected mRNA
via, for example, by electrophoresis (e.g gel or capillary); by
mass spectroscopy; etc. Any means of detecting the presence of the
mRNA in excess over a normal or baseline control (or to detect the
absence of the mRNA compared to such a control) may be used in the
practice of the present invention.
[0040] Further, in an assay designed to detect the expression
profile or pattern of claudins 1, 3, 4 and/or 7, in breast cells or
tissue, at least one but possibly two, three or all four of
claudins 1, 3, 4 and 7 may be assayed. This may be true, for
example, where the detection of claudin 7 is involved since it is
expressed at very low levels or not at all in about 70% of breast
carcinomas. Thus, advantages may accrue by assaying for claudin 7
together with one or more of the claudins simultaneously as a
panel. Such a profile or panel may be designed according to
guidelines which are well-known to those of skill in the art.
[0041] Since claudin 7 is expressed at very low levels or not at
all in about 70% of breast carcinomas, it is possible that its
reintroduction into breast cancer cells would impair the ability of
breast cancer cells to metastasize. Thus, the invention also
encompasses a method of preventing the metastasis of breast cancer
cells by reintroducing claudin 7 into breast tumor cells, or
alternatively, by inducing expression of claudin 7 in breast cancer
cells. Means of carrying out this aspect of the invention are known
to those of skill in the art. For example, a vector containing DNA
encoding claudin 7 may be introduced into the breast cancer cells
via gene therapy techniques. Alternatively, the claudin 7 protein
may be introduced into the cells via tagging the claudin protein to
a Trojan peptide (e.g. HOX proteins, TGF-.beta., etc.) or attaching
claudin 7 to ligands or receptors expressed on breast cancer cells,
etc. Administering there agents intraductally would confine the
uptake to the breast epithelial cells lining the ducts, and tumors
arising from these epithelia.
[0042] Claudins 3 and 4 are known to be overexpressed in breast
cancer cells. Therefore, in yet another aspect, the present
invention also provides targeted antibody and T-cell immunotherapy
for breast cancer. Humanized monoclonal antibodies specific for
claudin 3 and 4 are generated. These antibodies are administered
systemically or locally. The binding of the antibody to cancer
cells expression claudin 3 and 4 will result in cancer cell death
by subsequent recognition and attack by cytotoxic T-cells or other
immune cells participating in antibody dependent cell cytotoxicity
(ADCC) reactions (e.g. natural killer cells, macrophages, etc.).
Alternatively, cytotoxic molecules may be conjugated to claudin 3
and 4 antibodies allowing targeted delivery of cytotoxic compounds
to the cancer cells. Examples of such cytotoxic agents include but
are not limited to Doxil, Pseudomonas exotoxin, and paclitaxel.
Additionally, claudin-3 and -4 antibodies can be attached to the
exterior of vessels containing cytotoxic agents, which are designed
for slow agent release, including liposomes and biodegradable
synthetic polymer wafers or micro-spheres. Vaccines consisting of
irradiated tumor cells overexpressing claudins, with or without
augmentation with cytokines, could also result in the generation of
T-cells that specifically recognize over-expressed claudins on
tumor cells and cause cytotoxicity.
[0043] Claudins 3 and 4 are known to function as receptors for
Clostridium perfringens enterotoxin (CPE). When cells which express
claudins 3 and 4 are exposed to CPE, the toxin binds to the cells,
induces formation of a pore in the cell membrane, and causes lysis
of the cells. Because claudins 3 and 4 are expressed in epithelial
cancer cells, CPE can be used to treat epithelial cancers. Exposure
of epithelial cancer cells (e.g. breast cancer cells, prostate
cancer cells, pancreas cancer cells, etc.) to CPE results in
binding of the toxin to the cells, internalization and lysis of the
cancer cells. In the case of breast cancer, the reduced sensitivity
of normal mammary epithelial cells relative to breast cancer cells
allows CPE to preferentially destroy breast cancer cells. In many
cases, lysis will cause death of the cells. However, those of skill
in the art will recognize that the method may still be valuable if
all cells are not necessarily killed outright, but are damaged so
as to slow their rate of replication and/or growth, or made more
susceptible to other types of treatment such as radiation or
chemotherapy. Further, it is understood that the methods of the
present invention may be practiced in conjunction with other cancer
treatment protocols such as radiation, chemotherapy, etc.
[0044] In one embodiment of the invention, the entire, native CPE
toxin molecule (GenBank Accession #M98037) is utilized for the
treatment of breast cancer. However, those of skill in the will
recognize that the practice of the present invention need not be
limited to the use of the entire native sequence. Several active
forms or variants of CPE are known or can be designed by
well-known, routine, genetic engineering techniques and can also be
utilized in the practice of the present invention. For example,
with respect to amino acid sequences, variants may exist or be
constructed which display: conservative amino acid substitutions;
non-conservative amino acid substitutions; truncation by, for
example, deletion of amino acids at the amino or carboxy terminus,
or internally within the molecule; or by addition of amino acids at
the amino or carboxy terminus, or internally within the molecule
(e.g. the addition of a histidine tag for purposes of facilitating
protein isolation, the substitution of residues to alter solubility
properties, the replacement of residues which comprise protease
cleavage sites to eliminate cleavage and increase stability, the
addition or elimination of glycosylation sites, and the like, or
for any other reason). Such variants may be naturally occurring
(e.g. as a result of natural variations between species or between
individuals); or they may be purposefully introduced (e.g. in a
laboratory setting using genetic engineering techniques). All such
variants of the sequences disclosed herein are intended to be
encompassed by the teaching of the present invention, provided the
variant CPE retains the ability to bind to claudin 3 and 4
receptors, and to lyse the cell to which it binds. Preferably,
amino acid sequence identity of such variants when compared to
native CPE will be in the range of about 50 to about 100%, and more
preferably in the range of about 75 to about 100%, and most
preferably in the range of about 80 to about 100%. The identity is
with reference to the portion of the amino acid sequence that
corresponds to the original native sequence, i.e. not including
additional elements that might be added, such as those described
below for chimeric proteins. Further, such a variant will retain at
least from about 50 to 100% or more of the ability to bind to
claudins 3 and 4 and preferably will retain about 75 to 100% or
more of the ability to bind to claudins 3 and 4. Further, such a
variant will retain cell lysing activity of native CPE in the range
of about 50 to 100% or more, or preferably in the range of about 75
to 100% or more. By "cell lysing activity" we mean the ability to
initiate the production of pores in the cell membrane, leading to
rupture and lysis of the cell, and typically cell death.
[0045] In a preferred embodiment of the invention, the CPE variant
is one in which the N-terminal 45 amino acids of CPE have been
removed to generate a CPE protein known to exhibit twice the
cytotoxicity of native CPE. The use of a variant with enhanced
cytotoxicity will allow an increased level of drug activity thereby
increasing therapeutic efficacy at anatomical sites where injection
volume is limiting (e.g. injection of solid tumors, brain, bone,
etc.).
[0046] The invention also encompasses chimeric CPE toxin molecules,
for example, CPE proteins comprised of native CPE (or a variant as
described above) plus additional sequences which are not
necessarily associated with CPE, but the addition of which conveys
some additional benefit. For example, such benefit may have utility
in isolation and purification of the protein, (e.g. histidine tag,
GST, and maltose binding protein); or in directing the protein to a
particular intracellular location (e.g. yeast secretory protein).
All such chimeric constructs are intended to be encompassed by the
present invention, provided the portion of such construct that is
based on CPE is present in at least the indicated level of
homology. In a preferred embodiment, CPE will be linked to a
macromolecule (e.g. a protein, or a polypeptide) that is expressed
exclusively by malignant epithelial cells or tissue in order to
more specifically target epithelial cancer cells and allow for
systemic delivery of the CPE. Examples of such macromolecules
include but are not limited to: proteases such as matrix
metalloproteases which are known to be over-expressed by various
epithelial tumors; breast tumor-associated stromal elements, breast
tumor vasculature, etc.
[0047] For the purposes of treating breast cancer, delivery of the
CPE or CPE variant can be carried out by any suitable means, many
of which are known to those of skill in the art. Because some
non-cancerous cells also express claudin 3 and 4, in preferred
embodiments of the invention, the CPE is delivered directly to the
site of a tumor to avoid systemic lysis of otherwise healthy
tissue. Examples of methods of direct delivery include but are not
limited to direct injection into a tumor bed, topical application,
and the like. In a preferred embodiment of the invention, delivery
to breast tumors will be intraductal, for example, as described in
U.S. Pat. No. 6,330,472 (Sukumar et al., issued Dec. 11, 2001), the
entire contents of which is hereby incorporated by reference. The
CPE may be delivered in any suitable form, many of which are known
to those of skill in the art. For example, the CPE may be contained
within vessels including but not limited to liposomes and
biodegradable synthetic polymer wafers or micro-spheres designed
for slow drug release.
[0048] The invention further encompasses the treatment of
metastasized cancer which has metastasized to any site in the body.
In one embodiment of the invention, the metastasis is located in
the brain and/or the bone. It is well-recognized that patients
afflicted with breast, lung, colon, kidney, prostate, pancreas, and
skin cancer frequently die from neurological complications
resulting from metastases to the brain or bone, or both. Most
carcinomas express claudins 3 and 4 (e.g. breast, lung, colon,
kidney, prostate, pancreas, etc.) whereas the cell types of the
brain and bone do not. Thus, intracranial or intraosteal
administration of CPE may be used to eliminate cancer cells or
tumors, especially metastatic tumors originating from these sources
while leaving non-malignant brain and bone cells unharmed. In other
words, targeting claudins 3 and 4 with CPE may provide a means of
eliminating metastatic cancer from the brain and bone without
damaging the brain itself. Drug delivery may be accomplished by any
of several means which are well known to those of skill in the art,
including but not limited to stereotactic injection, implantation
of drug saturated wafers, micropump, etc.
[0049] Those of skill in the art will recognize that the amount of
CPE which must be administered in order to treat primary epithelial
cancer and cancer metastases will vary from case to case depending
on several factors (e.g. the size, gender, age and general health
of the patient, the stage of the disease, etc) and is best
determined by a skilled practitioner such as a physician. The
details of the dosage are typically determined during clinical
trials. However, in general, the quantity to be administered will
be in the range of from about 0.05 mg/kg to about 20 mg/kg, and
preferably from about 0.01 mg/kg to about 1 mg/kg.
EXAMPLES
Background for Examples 1 to 7
[0050] Metastasis is the primary cause of fatality in breast cancer
patients. Although there are believed to be numerous events
contributing to the process of metastasis, it is widely accepted
that the loss of cell-to-cell adhesion in neoplastic epithelium is
necessary for invasion of surrounding stromal elements and
subsequent metastatic events. Cell-to-cell adhesion in epithelial
cell sheets is maintained mainly through two types of junctions:
adherens junctions and tight junctions.
[0051] Numerous studies have focused their attention on the
transmembrane protein of the adherens junction, E-cadherin. These
studies have shown that impairing the function of E-cadherin can
cause cell dispersion and confer invasive properties in various
cell types. Owing to these abilities, E-cadherin is believed to
function as a tumor suppressor in numerous tissues and has been
shown to be a useful prognostic indicator for some tumors,
illustrating the importance of cell-to-cell adhesion proteins in
cancer progression (Soler et al., 1995; Wheelock et al., 2001).
[0052] Tight junctions, unlike adherens junctions, are solely
involved in cell-to-cell adhesion and serve two main functions in
epithelial cell layers. First, they prevent the paracellular
transport of solutes and ions, maintaining concentration gradients
driving transcellular transport. Second, tight junctions prevent
the diffusion of membrane proteins and lipids from the apical layer
to the basolateral layer of an epithelial cell sheet, helping to
maintain cell polarity (Mitic and Anderson, 1998).
[0053] Although tight junctions have clearly been shown to play a
role in cell-to-cell adhesion, their potential role in cancer
progression has been scarcely studied. This may be due, in part, to
the lack of knowledge concerning the protein components of these
junctions. However, in 1998, Tsukita et al. discovered a new family
of tight junction proteins named Claudins (CLDNs) (Furuse et al.,
1998). Currently, there are 20 known members of the CLDN family
(Mitic et al., 2000). CLDNs contain four transmembrane domains and
two extracellular loops through which they bind to CLDNs on
adjacent cells (Morita et al., 1999). CLDNs have also been shown to
bind to the tight junction protein ZO-1 through their carboxyl
terminus (Itoh et al., 1999). Interestingly, ZO-1 is believed to
interact with several proteins involved in cell signaling and
transcriptional regulation (Balda and Matter, 2000; Mitic et al.,
2000). These studies suggest that CLDNs may play an indirect role
in cell signaling and transcriptional regulatory events. Most
importantly, studies have shown CLDNs to be the main sealing
proteins of the tight junction (Tsukita and Furuse, 1999).
[0054] Although changes in the permeability of tight junctions have
been observed in several types of cancer, little is known about the
role of CLDNs in cancer. In one such investigation, CLDN-1 cDNA
levels were found to be decreased in a number of breast tumors and
breast cancer cell lines (Kramer et al., 2000). Kramer et al.
(2000) went on to examine the genetic status of CLDN-1 in a large
number of sporadic and hereditary breast cancers, but found no
genetic alterations that could explain this loss or provide
evidence supporting the involvement of aberrant CLDN-1 in breast
tumorigenesis.
[0055] Here we present, for the first time, data showing that
expression of the tight junction protein CLDN-7 is lost in ductal
carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and
invasive ductal carcinoma (IDC) of the breast relative to normal
mammary epithelium. Loss of CLDN-7 closely associates with the
discohesive architecture typically observed in high-grade lesions,
suggesting a potential functional role for CLDN-7 in breast cancer
progression. While the mechanism of loss of CLDN-7 in breast cancer
cell lines could be ascribed to promoter hypermethylation (Jones
and Baylin, 2002), this was not found to be the case in primary
IDCs. Taken together, these studies suggest that the loss of CLDN-7
may aid the dissemination of cancer cells. Further, a second
claudin, claudin-1, has been shown to exhibit similar
properties.
Materials and Methods for Examples 1-7
[0056] Cell Lines, Organoids and Tumors
[0057] Most cell lines were obtained from American Type Culture
Collection (Manassas, Va., USA), and cultured according to
conditions specified. Finite lifespan HMECs 9F1403, 04372, 16637
were purchased from Clonetics (Rockville, Md., USA). Breast cancer
cell lines 21 PT and 21 MT; New England 184, immortalized HMECs
184A 1 (early and late passages), 184B5; HMEC strain 11-24; were
provided as gifts. Mammary organoid samples, N1, N34, N65, and N74
were prepared from reduction mammoplasty specimens of women with no
abnormalities in the breast as described (Bergstraessar and
Weitzman, 1993). Briefly, the specimens were enzymatically digested
into duct-like structures (organoids), filtered, histologically
confirmed to contain greater than 80% epithelial cells, and frozen
at -70.degree. C. until use (Bergstraessar and Weitzman, 1993).
Highly purified (95-99%) luminal and myoepithelial cells were
isolated by differential centrifugation and fluorescence-activated
cell sorting of enzymatically digested normal mammoplasty specimens
(Gomm et al., 1995). Paraffin blocks of DCIS, LCIS, and IDCs of the
breast were obtained from the Surgical Pathology files of the Johns
Hopkins Hospital, observing institutional guidelines for
acquisition of such specimens.
[0058] Generation of CLDN-7 Antibody
[0059] A synthetic peptide corresponding to the C-terminus of
CLDN-7 protein conjugated to the carrier protein, keyhole limpet
hemocyanin (KLH) was generated by Mimotopes (Raleigh, N.C., USA).
Polyclonal rabbit antipeptide antibodies were raised and sera were
collected. CLDN-7 polyclonal antibody was then affinity purified
using the Aminolink Immobilization kit (Pierce, Rockford, Ill.,
USA) and the peptide against which the antibody was raised. To test
the affinity-purified CLDN-7 antibody for crossreactivity with
other CLDN proteins, human CLDN-3-, -4, and -7 were cloned into pCR
3.1 (Invitrogen, Carlsbad, Calif., USA). CLDN-3, -4, and -7
proteins were generated in vitro using cDNA clones in the TnT Quick
Coupled Transcription/Translation System (Promega, Madison, Wis.,
USA).
[0060] Immunofluorescence Microscopy
[0061] Cells (1.times.10.sup.5) were plated in eight-chamber slides
(Nunc, Naperville, Ill., USA) and cultured until confluent. Cells
were rinsed in phosphate-buffered saline (PBS) and fixed in 2%
paraformaldehyde diluted in PBS for 15 min. Cells were then
permeabilized in 0.5% Triton-X diluted in PBS for 5 min. Following
permeabilization, cells were incubated in 20 mg/ml bovine serum
albumin for 1 h at room temperature. Rabbit polyclonal CLDN-7
antibody diluted at 1:500 was then added to the cells and incubated
at room temperature for 1 h. Subsequently, cells were incubated
with mouse monoclonal 0-1 antibody (Zymed, San Francisco, Calif.,
USA) for 1 h at room temperature. Cells were then incubated with
anti-rabbit IgG conjugated to Alexafluor 568 and anti-mouse IgG
conjugated to Alexafluor 488 (Molecular Probes, Eugene, Oreg., USA)
for 1 h at room temperature. Before visualizing the cells, sections
were coverslipped and sealed.
[0062] Confocal Microscopy
[0063] Images were obtained using a Nikon PCM 2000.
[0064] Immunohistochemistry
[0065] Paraffin-embedded sections and breast tumor array sections
were deparaffinized in xylene and rehydrated through graded
ethanols. Antigen retrieval was performed by immersing sections in
0.01 m sodium citrate, pH 6.0, and boiling by microwave for 20 min.
Sections were then cooled to room temperature and endogenous
peroxidase activity was quenched by immersing in 0.3% hydrogen
peroxide for 30 min. Blocking was then performed by incubation in
diluted normal goat serum (Vectastain kit, Vector, Burlingame,
Mich., USA) as per the manufacturer's instructions. Sections were
then incubated with rabbit polyclonal CLDN-7 at a 1:500 dilution
for a period of 16 h. Diluted biotinylated anti-rabbit IgG
(Vectastain kit) was added to the sections and incubated for 30
min. Vectastain ABC reagent was then added for 30 min. CLDN-7
protein was visualized using 3,30-diaminobenzamidine (DAB) as per
the manufacturer's instructions (Vector). Sections were then
counterstained in hematoxylin (Richard-Allan Scientific, Kalamazoo,
Mich., USA) for 10 s. Lastly, sections were dehydrated through
graded ethanols, cleared in xylene, mounted, and coverslipped.
Images were acquired by light microscopy.
[0066] Statistical Analysis of CLDN-7 Expression
[0067] IHC staining of CLDN-7 in DCIS, LCIS, and IDC lesions was
scored relative to adjacent normal mammary epithelium as positive
(no change in expression) or negative (loss of expression).
Comparisons of CLDN-7 expression across grade were made by
tabulating scores for CLDN-7 staining according to histological
grade (Nuclear or Elston grades 1, 2, or 3). Two-sided Fisher's
exact tests were used to assess statistical significance. Although
grade is an ordinal variable, the analysis treated it as nominal
categorical. As such, P values are slightly conservative. Inverse
correlation implies that as histological grade tends to higher
values, CLDN-7 is less likely to be expressed.
[0068] Methylation-Specific PCR
[0069] Genomic DNA (1 .mu.g) was treated with sodium bisulfite as
previously described (Ferguson et al., 2000) and was analysed by
MSP using primer sets located within a CpG-rich area in the CLDN-7
promoter (GenBank Accession #11425795) Primers specific for
unmethylated DNA were 5'-TGGGGAAAGGGTGGTGTTG-3' (SEQ ID NO: 2)
(sense, -831 to -812) and 5'-TTACCCAATTTTAACCACCAC-3' (SEQ ID NO:
3) (antisense, -670 to -649) yielding a 182 bp product. Primers
specific for methylated DNA were 5'-GACGTTAGGTTATTTTCGGTC-3' (SEQ
ID NO: 4) (sense, -550 to -529) and 5'-AAACGCGTTTCTAAACGCCG-3' (SEQ
ID NO: 5) (antisense, -350 to -330) yielding a 220 bp product. The
PCR conditions were as follows: one cycle of 95.degree. C. for 5
min `hot start,` then addition of 1 ml Taq polymerase (RedTaq,
Sigma, St Louis, Mo., USA); 35 cycles of 95.degree. C. for 30 s,
56.degree. C. for 30 s, and 72.degree. C. for 45 s; and one cycle
of 72.degree. C. for 5 min. PCR samples were resolved by
electrophoresis on a 1.5% agarose gel.
[0070] 5-aza-dC Treatment
[0071] Cells were seeded in a 100 mm plate at a density of
1.times.10.sup.6 cells. After 24 h, cells were treated with 0.75 mm
5-aza-dC (Sigma) (Ferguson et al., 2000; Evron et al., 2001a, b).
Total cellular DNA and RNA were isolated at 0, 3, and 5 days after
addition of 5-aza-dC.
[0072] RT-PCR.
[0073] Total RNA was extracted using TR1 REAGENT BD by the
manufacturer's protocol (Molecular Research Center, Cincinnati,
Ohio, USA). cDNA was generated by reverse transcription. Reactions
contained 2 mg DNAse-treated RNA, 0.25 mg/.mu.l pdN6 random primers
(Life Technologies, Rockville, Md., USA), 1.times. first-strand
buffer (Life Technologies), 1 mm of each deoxynucleotide
triphosphate (Life Technologies), 200 units Superscript reverse
transcriptase (Life Technologies), and were incubated for 1 h at
37.degree. C., followed by heat inactivation at 70.degree. C. for
15 min. PCR was performed using the primers
5'-CCACTCGAGCCCTAATGGTG-3- ' (SEQ ID NO: 6) (sense) and
5'-GGTACCCAGCCTTGCTCTCA-3' (SEQ ID NO: 7) (anti-sense) for CLDN-7
(Accession #AJ011497). Coamplified products of 36B4, a
`housekeeping` ribosomal protein gene, were used as an internal
control, using primers 5'-GATTGGCTACCCAACTGTTGCA-3' (SEQ ID NO: 8)
and 5'-CAGGGGCAGCAGCCACAAAGGC-3' (SEQ ID NO: 9) for sense and
antisense, respectively. The 25 ml reactions contained 1.times.
buffer (2.times. reaction mix, Life Technologies), 1 .mu.l cDNA,
and 100 nm of each primer. The PCR conditions were: one cycle of
94.degree. C. for 1 min, `hot start,` followed by addition of one
unit of Taq polymerase (RedTaq, Sigma), 35 cycles of 94.degree. C.
for 30 s, 59.degree. C. for 30 s, 72.degree. C. for 45 s, and
finally one cycle of 72.degree. C. for 5 min. PCR samples were
resolved by electrophoresis on a 1.5% agarose gel.
[0074] Real-Time PCR
[0075] Total RNA was extracted and cDNA was generated by reverse
transcription as described above. CLDN-7 and GAPDH (a
`housekeeping` gene) were amplified individually using a 96-well
plate and optical caps (PE Applied Biosystems, Foster City, Calif.,
USA) with a 25 .mu.l final reaction volume containing 250 nmol/l
sense and antisense primer, 200 nmol/l probe, 2.5 mm MgCl.sub.2,
one unit Amplitaq Gold, 200 mmol/l each of dATP, dCTP, dTTP, and
dGTP in 1.times. Taqman Buffer A. Reaction mixtures were preheated
to 95.degree. C. for 10 min, followed by 40 cycles at 95.degree. C.
for 15 s and 60.degree. C. for 1 min. The primer and probe
sequences are as follows: CLDN-7 (sense) 5'-AAAG
TGAAGAAGGCCCGTATAGC-3' (SEQ ID NO: 10), CLDN-7 (antisense)
5'-GCTACCAAGGCGGCAAGAC-3' (SEQ ID NO: 11), CLDN-7 (probe) 5'-CC
ACGATGAAAATTATGCCTCCACCCA-3' (SEQ ID NO: 12), GAPDH (sense)
5'-CCCATGTTCGTCATGGGTGT-3' (SEQ ID NO: 13), GAPDH (antisense)
5'-TGGTCATGAGTCCTTCCACGATA-3' (SEQ ID NO: 14), and GAPDH (probe)
5'-CTGCACCACCAACTGCTTAG-3' (SEQ ID NO: 15). All PCR reagents,
including primers and probes, were purchased from PE Applied
Biosystems.
[0076] Sequencing of Sodium-Bisulfite-Treated DNA
[0077] DNA from peripheral white blood cells, IDCs, and breast
cancer cell lines was treated with sodium bisulfite as previously
described (Ferguson et al., 2000). Briefly, the DNA was purified
and a CpG-rich promoter region was amplified by PCR using the
following primers: 5'-GTGATTTTGGTGTTTAGGT-3' (SEQ ID NO: 16) (sense
primer with start at -675) and 5'-ATCCCAAAATATCCTAAACTA-3' (SEQ ID
NO: 17) (antisense primer with start at -375), which generated a
300 bp PCR product. The product was purified using a Qiagen PCR
purification kit (Qiagen Corp) and sequenced using the antisense
primer.
[0078] Western Blotting
[0079] IDC of the breast and normal mammary organoid tissue was
homogenized and total protein was extracted using lysis buffer
consisting of 15% glycerol, 5% SDS, and 250 mm Tris-HCl, pH 6.7.
Equal amounts of protein from cell lysates were resolved using 12%
SDS-PAGE (Invitrogen, Carlsbad, Calif., USA). Protein was then
transferred to ECL nitrocellulose membranes (Amersham, Arlington
Heights, Ill., USA). Following Western transfer, membranes were
probed with CLDN-3, 4 (Zymed), CLDN-7, or b-actin (Amersham)
antibody diluted 1:1000 (CLDN-3, 4, and 7) or 1:5000 (bactin).
Horseradish peroxidase-conjugated antibody against rabbit or mouse
IgG (Amersham) was used at 1:1000 and binding was revealed using
enhanced chemiluminescence (Amersham).
[0080] Abbreviations
[0081] CLDN, Claudin; IDC, invasive ductal carcinoma; RT-PCR,
reverse transcription-polymerization chain reaction; IHC,
immunohistochemical analysis; DCIS, ductal carcinoma in situ; LCIS,
lobular carcinoma in situ; HGF/scatter factor, hepatocyte growth
factor/scatter factor; KLH, keyhole limpet hemocyanin; MSP,
methylation-specific PCR; DAB, 3 3'-diaminobenzamidine; GAPDH,
glyceraldehyde phosphate dehydrogenase; 5-aza-dC,
5'-aza-2'-deoxycytidine; HMEC, human mammary epithelial cells.
Example 1
Expression of CLDN-7 mRNA in IDC and Normal Mammary Epithelium
[0082] A SAGE and cDNA microarray analysis performed previously in
our laboratory had suggested that CLDN-7 was overexpressed in
breast cancer cell lines and IDCs of the breast relative to
cultured finite lifespan human mammary epithelial cells (HMEC)
(Nacht et al., 1999). We initiated validation studies by directly
comparing the expression of a number of differentially expressed
mRNAs in IDCs using semiquantitative RT-PCR analysis. Total RNA was
extracted and cDNA was generated by reverse transcription. CLDN-7
and 36B4, a `housekeeping` ribosomal protein gene, were amplified
individually by PCR. PCR products were resolved by electrophoresis
on a 1.5% agarose gel. Samples were: 16637- and 04372-cultured
HMECs from Clonetics; Lum 1-4 and Myo 1-3-immunobead purified
luminal and myoepithelial cells from normal mammoplasty specimens;
and 10 invasive ductal carcinomas. Confirming data from microarray
analysis (Nacht et al., 1999), CLDN-7 expression was undetectable
by RT-PCR in finite lifespan HMECs expanded in tissue culture,
16637 and 04372, and in HMEC 184, 184A1 (early and late passage)
and 184B1 (data not shown). Contrary to our expectation, however,
easily detectable to high levels of CLDN-7 mRNA expression were
seen in seven of seven uncultured luminal and myoepithelial cell
populations derived from normal mammoplasty specimens. Also, nine
of ten IDCs showed low or undetectable levels of CLDN-7 mRNA. These
observations were in direct contrast to our published data (Nacht
et al., 1999), where we had reported that at least 50% of primary
tumors express levels of CLDN-7 mRNA that were significantly higher
than cultured finite lifespan HMEC.
[0083] One possible explanation for these contradictory findings
could be the choice of HMEC used to compare expression profiles
between normal and tumor samples. In our study, as in many other
comparative gene expression profiling studies (Fujii et al., 2002;
Iacobuzio-Donahue et al., 2002), we had used mortal HMEC expanded
in tissue culture as our source of normal breast epithelium. We
considered the possibility that placing the cells in tissue
culture, albeit short term, may have altered their expression
profile and resulted in a loss of CLDN-7 expression. To test this
possibility, we determined the expression of CLDN-7 in two
immortalized and four finite lifespan HMEC cultured in vitro, six
uncultured HMEC derived from three normal mammoplasty specimens,
and 10 breast cancer cell lines by realtime PCR analysis (FIG. 1).
A striking difference in CLDN-7 mRNA expression was observed
between the six tissue cultured cell lines (n=2) and strains (n=4)
and the six uncultured HMEC. HMEC cultured in vitro showed very low
to undetectable levels of CLDN-7 mRNA expression, while an average
of nearly 1000-fold higher levels were observed in uncultured HMEC,
of both luminal and myoepithelial subfractions. Thus, the erroneous
conclusion of CLDN-7 overexpression in primary tumors likely arose
as a consequence of using cultured HMEC (which expressed extremely
low levels of CLDN-7 mRNA) as a basis for comparison. Relative to
CLDN-7 mRNA levels in uncultured HMEC, however, CLDN-7 expression
in all 10 breast cancer cell lines was lower by 10-1000-fold. Thus,
although immaterial for many other genes (Ferguson et al., 2000;
Evron et al., 2001a, b; Loeb et al., 2001), placing HMEC in tissue
culture had the profound effect of silencing CLDN-7 expression.
When used as controls for comparative gene expression studies, such
tissue-culture-based alterations could lead to inaccurate
interpretation of data.
[0084] This example demonstrates that CLDN-7 is consistently
expressed in normal mammary epithelium which CLDN-7 expression is
lost in approximately 70% of primary breast carcinomas.
Example 2
Generation and Characterization of CLDN-7 Polyclonal Antibody
[0085] To study expression of CLDN-7 protein in breast tissues, we
generated a rabbit polyclonal antibody against the synthetic
polypeptide CKAGYRAPRSYPKSNSSKEYV (SEQ ID NO: 1) corresponding to
the C-terminus of CLDN-7. This region of the protein shares little
sequence similarity with other members of the CLDN family. Human
CLDN-3, -4, and -7 were cloned into pCR 3.1 (Invitrogen) and
proteins were generated in vitro using cDNA clones in the TnT Quick
Coupled Transcription/Translation System as determined by Western
analysis using antibodies specific for CLDN-3 and -4 (Zymed). Next,
we used the C-terminal CLDN-7 peptide in enzyme-linked
immunosorbent assay (ELISA) to test for the presence of CLDN-7
antibody in rabbit sera. The results are shown in FIG. 2 where
CLDN-7 antibody is detected in rabbit sera from bleeds 1-3 as
indicated by a linearly increased level of absorbance over several
folds dilution versus no absorbance detected in rabbit sera from
prebleed (blood drawn prior to antigen delivery). The CLDN-7
antibody was affinity purified using the peptide against which it
was raised. Western analysis was performed on equal amounts of
protein from TnT reactions using CLDN-7 antibody producing a single
band at the predicted size of approximately 23 kDa, while not
detecting CLDN-3 or -4. Conversely, antibodies to CLDN-3 and -4 did
not detect CLDN-7 protein, but detected their cognate protein.
Further, preincubation of CLDN-7 antibody with the C-terminal
peptide was able to compete out binding to CLDN-7 protein in
Western analysis (data not shown).
[0086] To perform immunofluorescence studies using the affinity
purified antibody, MCF-7 cells were grown to confluence on a
chambered slide, and probed with CLDN-7 and ZO-1 antibodies. CLDN-7
and ZO-1 proteins were visualized both individually and as a
composite by confocal microscopy at a magnification of .times.600.
The results showed colocalization of CLDN-7 with the tight junction
protein ZO-1 at the cell membrane. Unique red spots were observed
in the cytoplasm. Whether they represent nonspecific staining or
CLDN-7 localized in cell organelles is not yet known. These spots
were not localized to mitochondria, however, since they did not
colocalize with organelles stained by using the MitoTracker Red dye
(Molecular Probes, Eugene Oreg.).
[0087] This example demonstrates that affinity purified CLDN-7
antibody recognizes CLDN-7 at the cell membrane and more
specifically at the tight junction.
Example 3
Expression of CLDN-7 Protein in IDC and Normal Mammary
Epithelium
[0088] To determine whether protein expression reflected that of
CLDN-7 mRNA expression as obtained by RT-PCR, we performed Western
analysis on a panel of 10 breast cancer cell lines, eight IDCs, and
four samples of mammary organoids isolated from reduction
mammoplasty specimens of normal women. Western analysis was
performed on equal amounts of protein from total cell lysates using
CLDN-7 and b-actin antibodies. Consistent with real-time
quantitative RT-PCR results (FIG. 1), Western analysis of a panel
of 10 breast cancer cell lines showed a close correlation between
CLDN-7 protein and CLDN-7 mRNA expression. Cell lines that showed
low or no detectable mRNA (MDA-MB-435, MDA-MB-231, and HS578T) had
no detectable protein, while the remaining seven cell lines showed
detectable CLDN-7 expression. Also consistent with RT-PCR, CLDN-7
expression in six of eight IDCs was significantly lower than in
four samples of epithelial organoids obtained by enzymatic
digestion of normal mammoplasty specimens. Lastly, Claudin-1
expression was found to be down-regulated in 6 out of 10 breast
cancer cell lines as compared to immortalized and finite life-span
normal human mammary epithelial cells. This is consistent with
reports of frequent done-regulation of Claudin-1 in primary breast
carcinoma.
[0089] Owing to the heterogeneity of cell types in breast tissue
and the fact that only the epithelial cell component expresses
CLDNs, it was necessary to determine if the loss of CLDN-7
expression observed in breast cancer tissues relative to normal
mammary epithelium was simply because of a difference in epithelial
cell content. Therefore, we performed immunohistochemical (IHC)
analysis on several of the same IDC cases that had been tested by
Western analysis. IHC analysis was performed on paraffin-embedded
sections of human breast cancer tissues 079, 126, and 973 using
CLDN-7 antibody. CLDN-7 protein in human breast cancer tissues (T)
and adjacent normal mammary epithelium (N) were visualized using
DAB. Membrane staining was observed in normal breast epithelium.
Sections were counterstained with hematoxylin and visualized by
light microscopy (.times.200) In each case, the CLDN-7 staining
pattern was compared to that in adjacent normal epithelium as an
internal positive control. Surrounding fibroblasts and adipocytes
served as negative controls since these cells do not express CLDN
proteins. The analysis was performed on equal amounts of protein
from cell lysates using CLDN-7 and b-actin antibodies.
[0090] As expected for a tight junction protein, CLDN-7 staining
was restricted to epithelial cells with the strongest expression
concentrated at the cell membrane, although diffuse staining in the
cytoplasm was also observed. Consistent with the Western analysis
results, the level of CLDN-7 staining was greatly reduced in all
three IDCs tested as compared to adjacent normal epithelium.
[0091] This example demonstrates that CLDN-7 protein expression is
lost in primary breast carcinoma cells relative to normal mammary
epithelium.
Example 4
Expression of CLDN-7 in Ductal Carcinoma In Situ and IDC
[0092] To assess the potential value of loss of CLDN-7 as a
prognostic indicator for breast cancer, we determined its
expression pattern in a series of in situ and invasive breast
carcinomas by IHC analysis. As DCIS is believed to be a direct
precursor to IDC, we first examined the CLDN-7 staining pattern in
a range of DCIS cases, from nuclear grade 1 (low grade) through 3
(high grade). In each case, the staining pattern of CLDN-7 in DCIS
was compared to that in adjacent normal epithelium, where staining
was predominantly membranous. IHC analysis showed no changes in
CLDN-7 expression in either grade 1 (0/10) or grade 2 (0/14) cases,
while 71% of grade 3 cases (10/14) showed a loss of its expression
(Table 1). Thus, we observed that CLDN-7 expression in DCIS was
inversely correlated with nuclear grade (P<0.001).
[0093] We next examined the CLDN-7 staining pattern in IDCs ranging
from Elston grade 1 (low grade) through 3 (high grade), which was
compared in each case to that seen in the normal epithelium present
on the same section. IHC analysis was performed on
paraffin-embedded sections of human breast tissue using CLDN-7
antibody. CLDN-7 protein was visualized using DAB. Sections were
counterstained in hematoxylin and visualized by light microscopy
(.times.200). Membrane staining of normal breast epithelium was
noted. Few grade 1 (1/6) or grade 2 (3/12) IDC cases showed a loss
of CLDN-7 expression, while 77% of grade 3 cases (10/13) showed a
significant loss of staining (Table 1). Thus, CLDN-7 expression in
IDC was found to be inversely correlated with histological grade
(P=0.014).
[0094] CLDN-7 immunoreactivity in IDC was further studied by tissue
array analysis. IHC analysis was performed on tissue arrays
containing 612 paraffin-embedded sections of human breast tissue
using CLDN-7 antibody. CLDN-7 protein was visualized using DAB.
Sections were counterstained in hematoxylin and visualized by light
microscopy (.times.200) Of the 612 total cases of IDC on the tissue
array, 100 Elston grade 1, 140 Elston grade 2, and 115 Elston grade
3 cases were evaluable and showed an inverse correlation between
CLDN-7 expression and histological grade (P=0.03). This finding was
consistent with the results of the case-by-case analysis
(summarized in Table 1).
1TABLE 1 IHC analysis of CLDN-7 expression.sup.a Cases with loss of
Histological expression/total Histology grade cases P.sup.b DCIS
Nuclear grade 0/10 1 0/14 2 10/14 <0.001 3 DC Elston Grade 1 1/6
2 3/12 3 10/13 0.014 LCIS NA 13/17 .sup.aData are compiled from IHC
analysis of whole paraffin-embedded sections .sup.bComparisons of
CLDN-7 expression across grade were made by tabulating scores for
CLDN-7 staining according to histological grade (nuclear or Elston
grades 1, 2, or 3) Two-sided Fisher's exact tests were used to
assess statistical significance
[0095] No correlation between CLDN-7 expression and
estrogen/progesterone receptor status, age, tumor size, or lymph
node status was found by tissue array analysis. This last result
was contrary to our case-by-case analysis, where seven of ten IDCs
with a positive lymph node status showed a loss of CLDN-7
expression. While the utility of tissue arrays cannot be
underestimated since it allows for very high sample throughput, the
lack of an internal control (normal epithelium) for each tumor
sample, combined with the small sampling represented in each tissue
punch could lead to a greater error in determining gene expression.
In our study, these factors may be responsible for the lack of
correlation with lymph node status in tissue arrays when compared
to case-by-case analysis. This source of error is being minimized
in newer generations of tissue arrays, which contain several
punches from the same tumor tissue, and also from their normal
margins. Thus, at the present time, performing a case-by-case
analysis alongside tissue array analysis is preferred.
[0096] This example demonstrates that expression of CLDN-7 is
inversely correlated with tumor grade, being lost in the majority
of high grade DCIS and high grade IDC lesions, suggesting that
CLDN-7 will be useful as a prognostic marker for breast cancer.
Example 5
Expression of CLDN-7 in LCIs
[0097] If CLDNs play a functional role in cell-to-cell adhesion,
breast lesions that are typified by scattered cells should express
very low levels of CLDN-7. In agreement with this notion, IHC
analysis of LCIS, a lesion whose defining and characteristic
feature is discohesion, was carried out. IHC analysis was performed
on paraffin-embedded sections of human breast tissue using CLDN-7
antibody. CLDN-7 protein was visualized using DAB. Sections were
counterstained in hematoxylin and visualized by light microscopy
(.times.200). The results showed a loss of CLDN-7 expression in 76%
(13/17) of cases. This contrasted significantly (P=0.001) with
DCIS, where its loss is seen in only 26% (10/38) of cases
irrespective of grade (Table 1).
[0098] This example demonstrates that loss of CLDN-7 expression
correlates with cellular discohesiveness.
Example 6
Effect of HGF/Scatter Factor on CLDN-7 Expression
[0099] A direct demonstration of the inverse correlation between
CLDN-7 expression and cell-to-cell adhesion was sought by the
treatment of breast cancer cell lines with hepatocyte growth
factor/scatter factor (HGF/scatter factor). HGF is well known for
its ability to decrease cell-to-cell adhesion and stimulate cell
migration (Jiang et al., 1999). Breast cancer cell lines MCF-7 and
T47D, which express high levels of CLDN-7 localized at the tight
junction were treated with HGF/scatter factor for a period of 24 h.
Western analysis was performed on equal amounts of total cell
lysate using CLDN-7 and b-actin antibodies. The results showed a
dramatic downregulation of CLDN-7 was observed in MCF-7, and to a
lesser extent in T47D cells.
[0100] These data provide further direct evidence that loss of
CLDN-7 occurs concurrently with loss of cell-to-cell adhesion.
Example 7
Mechanism of Loss of CLDN-7 Expression in Breast Cancer
[0101] To investigate the mechanism responsible for the loss of
CLDN-7 expression, we first wanted to rule out the presence of
mutations in the CLDN-7 mRNA (Accession #AJ011497) sequence.
Nucleotide sequencing of the full-length cDNA revealed no mutations
in the CLDN-7 coding sequences in all 11 primary IDCs tested (data
not shown). Among the 11 tumors, six expressed very low or no
CLDN-7 mRNA as determined by semiquantitative RT-PCR analysis.
[0102] The presence of CG-dinucleotide-rich sequences in the
promoter region of genes is quite often a signature denoting that
hypermethylation may be a potential mechanism for gene silencing
(Ferguson et al., 2000; Evron et al., 2001a, b; Loeb et al., 2001;
Jones and Baylin, 2002). The CLDN-7 promoter contains a CpG-rich
region extending from -20 to -900 bp upstream of the translational
start site (Accession #11425795). Therefore, we investigated the
promoter region of the CLDN-7 gene. Methylation-specific PCR (MSP)
analysis was performed on DNA from six breast cancer cell lines.
Methylated (M) and unmethylated (UM) gene sequences were amplified
individually by MSP using sodium-bisulfite-treated DNA from breast
cancer cell lines. WBC (peripheral blood cells) served as a
negative control. MSP products were resolved by electrophoresis on
a 1.5% agarose gel. The three breast cancer cell lines that show no
detectable CLDN-7 expression (HS578T, MDAMB-231, and MDA-MB-435)
contained hypermethylated promoter sequences, while the three that
express CLDN-7 (T47D, MCF-7, and MDA-MB-468) were unmethylated in
the same region.
[0103] This correlation between the loss of CLDN-7 expression and
promoter hypermethylation was further confirmed by sequencing a 300
bp region (containing a dense region of 25 CpG dinucleotides, and
included the CG-rich region analysed by MSP) of the CLDN-7
promoter, PCR-amplified from sodium-bisulfite-treated DNA. All 25
CpGs were methylated in HS578T, MDA-MB-231, and MDA-MB-435 cells,
while MCF-7 cells contained no methylated CpGs (data not
shown).
[0104] Lastly, treatment of HS578T and MDA-MB-435 cells with the
demethylating agent, 5-aza-dC, resulted in the re-expression of
CLDN-7. RT-PCR was performed for CLDN-7 and -36B4, a `housekeeping`
ribosomal protein gene. PCR products were resolved by
electrophoresis on a 1.5% agarose gel. The results clearly showed
that hypermethylation is a major mechanism responsible for
silencing expression of CLDN-7 in breast cancer cell lines, and
provides another line of evidence supporting the premise that
hypermethylation is a major mechanism responsible for silencing
expression of CLDN-7 in breast cancer cell lines.
[0105] Next, to determine if hypermethylation-mediated silencing of
CLDN-7 expression is functional in primary breast cancer as well,
we performed MSP analysis on DNA from IDCs. As expected, the sample
of normal mammary organoid, N65, and two IDC samples that express
CLDN-7 were unmethylated in this region. However, contrary to our
findings in breast cancer cell lines, MSP analysis of the CLDN-7
promoter in the five IDCs that have lost CLDN-7 expression also
showed completely unmethylated promoter sequences. Since MSP
analyses only a few CpGs in the promoter, we sequenced the 300 bp
segment of the promoter described above. Sequencing of
sodium-bisulfite-treated DNA from IDCs 079 and 973 showed no
methylated CpGs (data not shown).
[0106] Thus, the evidence provided by MSP, nucleotide sequencing
analysis, and re-expression of genes following 5-aza-dC treatment,
strongly support the notion that promoter hypermethylation of
CLDN-7 is the underlying mechanism for loss of its expression in
breast cancer cell lines.
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Background for Examples 8 to 15
[0133] Breast cancer therapies have shown limited efficacy in
patients with advanced disease. Although many advances in
diagnostic, prognostic, and therapeutic methods have been made over
the last several years, breast cancer remains the second leading
cause of cancer death in women and the leading cause of death in
women between the ages of 40 and 55. Thus preventative and new
therapeutic techniques are needed.
[0134] Clostridium perfringens enterotoxin (CPE) is a common cause
of food poisoning. Following ingestion of CPE, the toxin binds to
its receptors on intestinal epithelial cells resulting in cell
lysis. The receptors for CPE were identified in 1999 as the tight
junction proteins Claudin 3 and 4. Claudin 3 functions as the low
affinity receptor and Claudin 4 as the high affinity receptor. We
propose that CPE targeting through claudins 3 and 4 may provide an
effective preventative measure as well as therapy for breast
cancer. Further, patients afflicted with breast as well as lung,
colon, kidney, and skin cancer frequently die from neurological
complications resulting from metastases to the brain or bone.
Several other varieties of carcinoma have been known to metastasize
to the brain and bone as well including those of prostate and
pancreas. Targeting claudins 3 and 4 with CPE may provide a means
of eliminating these metastases from the brain and bone without
damaging healthy tissues as normal brain and tissue does not
express these proteins.
Materials and Methods for Examples 8-15
[0135] Reagents
[0136] Purified Clostridium perfringens enterotoxin was obtained as
a gift. Antibodies against Claudin 3 and 4 were obtained from Zymed
Laboratories.
[0137] Cell Lines
[0138] HBL-100 cells were maintained in growth medium consisting of
McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS).
SKBr3 cells were maintained in growth medium consisting of McCoy's
5A medium supplemented with 15% fetal bovine serum (FBS). HS578T,
MCF-7, MDA-MB 435, and NIH 3T3 cells were maintained in growth
medium consisting of Dulbeccos modified eagles medium (DMEM)
supplemented with 10% fetal bovine serum (FBS). T47D and MDA-MB 231
cells were maintained in growth medium consisting of RPMI medium
supplemented with 10% fetal bovine serum (FBS). 21MT and 21PT cells
were maintained in growth medium consisting of alpha minimal
essential medium supplemented with 10% fetal bovine serum (FBS), 10
mM HEPES, 1.times. non-essential amino acids, 1 mM sodium pyruvate,
2 mM glutamine, 1 .mu.g/ml insulin, 25 ng/ml EGF, and 1 .mu.g/ml
hydrocortisol. MW cells were maintained in growth medium consisting
of 45% DM-EM and 45% Ham's F 12 supplemented with 10% fetal bovine
serum (FBS). MCF-10A cells were maintained in growth medium
consisting of 47.5% DMEM and 47.5% Ham's F12 supplemented with 5%
horse serum, 100 ng/ml cholera toxin, 10 .mu.g/ml insulin, 0.5
.mu.g/ml hydrocortisol, and 20 ng/ml EGF. NMU 36 and NMU 58 cells
were maintained in growth medium consisting of 45% DMEM and 45%
Ham's F 12 supplemented with 10% fetal bovine serum (FBS).
[0139] CPE Cytotoxicity
[0140] Cells were plated at 3.times.10.sup.5 cells/well in 6-well
plates and grown to 80% confluence. Media was then removed and
replaced with fresh media with or without CPE at concentration
ranging from 0.05 to 2 .mu.g/ml. Cells were incubated at 37.degree.
C. for 60 min. Floating and attached cells were collected and
counted using a hemocytometer. Cell viability was determined by
trypan blue (0.4%) dye exclusion.
[0141] Detection of Claudin-3 and 4 in Blood and Ductal Lavage
Fluid
[0142] Blood and ductal lavage fluid is collected from breast
cancer patients. Red blood cells are then removed from whole blood
by lysis in H.sub.2O. Protein is then extracted from the remaining
mixture of leukocytes ind cancer cells or ductal lavage fluid.
Equal amounts of protein are then absorbed to a 96-well plate over
night. Plates are then dried and incubated at room temperature (RT)
in blocking buffer (50 g Milk/L PBS) for 2 hrs. Claudin-3 or 4
antibody (Zymed) is added to the plate and incubated for 2 hours at
RT. Claudin-3 or 4 is detected following the addition of HRP
substrate using a plate reader at 490 nm. HRP-labeled secondary
antibody is then added and incubated for 1 hour. The presence of
Claudin-3 or 4 is detected following the addition of HRP substrate
using a plate reader at 490 nm.
[0143] Mouse Toxicity Studies
[0144] 6-8 week old athymic nude and Balb/C mice were be
anesthetized and a 2 mm burr hole centered 2 mm posterior to the
coronal suture and 2 mm lateral to the sagittal suture was made.
Mice were transferred to a stereotactic frame and administered
either PBS or CPE (0.05-10 ug) as a single injection into the
cerebral cortex at a depth of 3 mm three times per week for two
weeks. One group of six mice were observed for any symptoms of
toxicity or neurological complications including sluggishness, lack
of grooming, hemiparesis, and weight loss on a daily basis for two
months. A second group of six mice were sacrificed at two weeks,
brains were removed and fixed in 10% neutral buffered formalin, and
sections of brain tissue stained with hematoxylin and eosin.
[0145] ELISA
[0146] 96-well plates were coated over night with synthetic
polypeptide at a concentration of 600 ng/50 ul. Plates were dried
and incubated at room temperature (RT) in blocking buffer (50 g
Milk/L PBS) for 2 hrs. Rabbit antisera was diluted in PBS, added to
plate, and incubated for 2 hours at room temperature. The presence
of Claudin-7 pAb was visualized using anti-rabbit secondary
antibody conjugated to horse radish peroxidase (HRP) at 490 nm.
[0147] Generation of Claudin-7 Polyclonal Antibody
[0148] Anti-human Claudin-7 pAb was raised in rabbits against the
synthetic polypeptide (CKAGYRAPRSYPKSNSSKEYV) (SEQ ID NO. 1)
(Mimotopes), which corresponds to the C-terminus of human
Claudin-7. The presence of Claudin-7-polyclonal antibody in
successive bleeds was determined by ELISA. 96-well plates were
coated over night with synthetic polypeptide at a concentration of
600 ng/50 .mu.l. Plates were dried and incubated at room
temperature (RT) in blocking buffer (50 g Milk/L PBS) for 2 hrs.
Rabbit antisera was diluted in PBS, added to plate, and incubated
for 2 hours at RT. The presence of Claudin-7 pAb was visualized
using anti-rabbit secondary antibody conjugated to horse radish
peroxidase (HRP) at 490 nm.
[0149] Immunohistochemistry
[0150] Sections of human breast cancer tissue embedded in paraffin
were obtained. Sections were deparaffinized in xylene and
rehydrated through graded EtOH. Antigen retrieval was performed by
microwaving sections in 0.01M Citrate buffer, pH 6.0 for 20 min.
Sections were cooled for 1 hour and then immersed in 0.3%
H.sub.2O.sub.2 in MeOH to quench endogenous peroxidase activity for
30 min. Blocking was performed in diluted normal blocking serum
(Vectastain kit, Vector Labs). Sections were probed using Claudin-3
and 4 antibody, followed by biotinylated secondary antibody
(Vectastain kit, Vector Labs), incubation with ABC reagent
(Vectastain kit, Vector Labs), and DAB (Vector kit, Vector Labs).
Finally, sections were counterstained in Hematoxylin, dehydrated,
cleared in xylene, and visualized by light microscopy.
[0151] RT-PCR
[0152] Total RNA was extracted using Trizol per instructions (Sigma
Co, St. Louis, Mo.). cDNA was generated by reverse transcription.
Reactions contained 2 .mu.g DNAse-treated RNA, 0.25 .mu.g/.mu.l
pdN6 random primers (Pharmacia), 1.times. first-strand buffer (Life
Technologies), 1 mM of each deoxynucleotide triphosphate
(Pharmacia), 200 units Superscript reverse transcriptase (Life
Technologies), and were incubated for 1 hr at 37.degree. C.,
followed by heat inactivation at 70.degree. C. for 15 min. PCR was
performed using the primers: 5'-CCACTCGAGCCCTAATGGTG-3' (sense)
(SEQ ID NO: 18) and 5' GGTACCCAGCCTTGCTCTCA-3' (anti-sense) (SEQ ID
NO: 19) for Claudin-7. Coamplified products of 36B4, a
"housekeeping" ribosomal protein gene, were used as an internal
control, using primers 5'-GATTGGCTACCCAACTGTTGCA-3' (sense) (SEQ ID
NO:20) and 5' AGGGGCAGCAGCCACAAAGGC-3' (anti-sense) (SEQ ID NO:
21). The 25 .mu.l reactions contained 1.times. buffer (2.times.
reaction mix, BRL), 1 .mu.l cDNA, and 100 nm of each primer. The
PCR conditions were: 1 cycle of 94.degree. C. for 1 min, "hot
start," followed by addition of 1 unit of Taq polymerase (RedTaq,
Sigma), 35 cycles of 94.degree. C. for 30 sec, 59.degree. C. for 30
sec, 72.degree. C. for 45 sec, and finally 1 cycle of 72.degree. C.
for 5 min. PCR samples were resolved by electrophoresis on a 1.5%
agarose gel.
[0153] Western Blotting
[0154] Primary breast cancer tissue, normal, and benign (B) mammary
organoid tissue was homogenized. Total protein was extracted from
tissue and cells using lysis buffer consisting of 15% glycerol, 5%
SDS, and 250 mM Tris-HCl, pH 6.7. Equal amounts of protein from
cell lysates were resolved using 12% SDS-PAGE (Invitrogen). Protein
was then transferred to ECL nitrocellulose membranes (Amersham).
Following Western transfer, membranes were probed with Claudin-3
and 4 (Zymed) and Actin (Amersham) antibodies, and developed using
ECL (Amersham).
Example 8
Expression of Claudins 3 and 4 Protein in Primary Breast
Carcinomas, Breast Cancer Cell Lines, and Normal Mammary
Epithelium
[0155] The expression of Claudin 3 and 4 proteins in primary breast
carcinomas, breast cancer cell lines, and normal mammary organoids
was determined by Western blot analysis. We found Claudin 3 and 4
proteins to be expressed in the majority of breast cancer cell
lines (7/10) and, more importantly, in all primary breast tumors
tested (15/15). Further, Claudin 3 and 4 proteins were
over-expressed by more than 2-fold in 12/15 (p=0.008) and 5/15
(p=0.046) primary breast tumors, respectively, relative to human
mammary epithelial cells and normal epithelial organoids obtained
from reduction mammmoplasty specimens as determined by
densitometric scanning.
[0156] These results showed that primary breast carcinomas
consistently express the receptors for CPE, Claudins 3 and 4, and
have increased expression relative to normal mammary epithelial
cells.
[0157] Although Western analysis showed expression of Claudins 3
and 4 in all primary breast carcinomas tested it was important to
determine the cellular localization of Claudins 3 and 4 as
CPE-mediated cytolysis requires expression of its receptors at the
cell membrane. Immunohistochemical analysis of Claudin 3 and 4
expression was performed on 10 primary breast carcinoma cases, 4 of
which were included in our Western blot analysis. It was found that
Claudin 3 and 4 proteins were both expressed at the cell membrane
although some amount of cytoplasmic staining was also observed.
Consistent with Western blot analysis, expression of Claudin 3 and
4 proteins in tumor epithelium was increased in 5/10 and 3/10
cases, respectively, relative to that seen in adjacent normal
mammary epithelium.
Example 9
CPE Specifically and Efficiently Lyses Claudin 3 and 4 Expressing
Breast Cancer Cells
[0158] As CPE is known to efficiently destroy cells bearing Claudin
3 and/or 4 the ability of CPE to destroy several breast cancer cell
lines was tested. Cells were plated at 3.times.10.sup.5 cells/well
in 6-well plates and grown to 80% confluence. Media was then
removed and replaced with fresh media with or without recombinant
CPE at concentrations ranging from 0.05 to 2 .mu.g/ml. Cells were
incubated at 37.degree. C. for 60 min. Floating and attached cells
were collected and counted using a hemocytometer. Cell viability
was determined by trypan blue (0.4%) dye exclusion. Data from
representative experiments are expressed as % cytotoxicity as
compared to media control .+-.S.D. Treatment of breast cancer cell
lines with various concentrations of CPE resulted in rapid and
dose-dependent cytolysis specific for cells expressing Claudins 3
and 4 (FIG. 3). Treatment with CPE at a concentration of 1 ug/ml
resulted in maximum cytolysis killing virtually 100% of the
cells.
Example 10
Treatment of T47D Breast Cancer Cell Xenografts with CPE
[0159] T47D cells (1.times.10.sup.7) were resuspended in matrigel
and subcutaneously injected bilaterally in the flank of 6-8 week
old SCID mice. Tumors were grown to approximately 100 mm.sup.3
prior to CPE treatment. Tumors treated with recombinant CPE showed
a significant dose-dependent reduction in tumor volume (FIG. 4).
Further, Hematoxylin and Eosin staining of CPE-treated tumors
revealed high levels of tumor necrosis accounting for approximately
30-80% of the total tumor area.
[0160] This example demonstrates that CPE effectively destroys
Claudin 3 and 4 positive breast cancer cell lines established as
xenografts in vivo.
Example 11
Expression of Claudin 3 and 4 in Rat NMU-Induced Breast Cancer Cell
Lines
[0161] We next wanted to determine the effectiveness of recombinant
CPE against spontaneously occurring breast tumors. To accomplish
this we choose to use the Sprague-Dawley rat NMU-induced model of
breast cancer. To determine if the cells from these tumors would be
susceptible to CPE we first determined the expression of Claudin 3
and 4 proteins in two breast cancer cell lines established from
these tumors by Western analysis. We found that both NMU 36 and NMU
58 breast cancer cell lines expressed Claudins 3 and 4, although
NMU 36 expressed lower levels.
Example 12
CPE Specifically and Efficiently Lyses Claudin 3 and 4 Expressing
Rat Breast Cancer Cells
[0162] To determine whether rat NMU-induced breast cancer cell
lines were sensitive to the cytolytic effects of recombinant CPE we
treated NMU 36 and NMU 58 with various concentrations of CPE for a
period of 60 min (FIG. 5). Consistent with our results in human
breast cancer cell lines, CPE treatment resulted in rapid and
dose-dependent cytolysis specific for cells expressing claudins 3
and 4 as the claudin 3 and 4 negative NIH3T3 cells were unaffected.
Further, NMU-36 cells were not as sensitive to CPE-mediated
cytolysis as NMU 58, consistent with their lower level of claudin 3
and 4 expression. Despite their reduced sensitivity, virtually 100%
cytolysis of NMU 36 cells was achieved at higher CPE
concentrations.
Example 13
CPE Treatment of Sprague-Dawley NMU-Induced Breast Tumor by
Intraductal Injection
[0163] We further tested the therapeutic potential of CPE against
breast cancer in the Sprague-Dawley NMU-induced model of breast
cancer. Because numerous tissues in the body express claudins 3 and
4, systemic administration of CPE by routes such as intravenous,
intramuscular, and oral may result in high systemic toxicity and
greatly limit therapeutic efficacy. Therefore, the anti-tumor
potential of CPE in vivo was tested using a novel intraductal (ID)
administration approach (U.S. Pat. No. 6,330,472 to Sukumar et al.,
issued Dec. 11, 2001) through the teat in order to limit systemic
toxicity and provide more direct access of CPE to the tumor thereby
increasing therapeutic efficacy. At 3-6 weeks of age, female
Sprague-Dawley rats were injected with 50 mg/kg NMU i.p. Mammary
tumors developed within several months. CPE was administered ID at
various concentrations once tumors grew to a size of 100 mm.sup.3.
Three injections of 17 ug CPE over the course of 2 weeks prevented
tumor growth (FIG. 6a) while an untreated tumor in the same animal
grew to approximately 12.5 times the size (FIG. 6b). Increasing the
concentration of CPE to 30 ug per injection while delivering 8
injections over the course of 4 weeks resulted in a reduction in
tumor volume (FIGS. 6c,d). Concurrently, an untreated tumor in the
same animal grew to more than 100 times the size. Further, CPE
treatment led to significant tumor necrosis as evidenced by
histological examination following Hematoxylin and Eosin staining.
This experiment was subsequently repeated using native CPE, which
has 10-fold higher activity that the recombinant CPE used
previously. CPE was administered ID at 3 or 5 .mu.g per injection
once every three days for 30 days beginning once tumors reached a
size of 100 mm.sup.3. Treatment with 5 .mu.g CPE significantly
inhibited the growth of tumors relative to untreated controls (FIG.
7). Although CPE administration of doses used in this experiment
given i.p. are known to elicit system toxicity, no evidence of
adverse reaction was observed in any of the treated animals. This
example demonstrates that ID administration of CPE results in
cytolysis of rat mammary tumors concurrent with a reduction in
tumor volume without any evidence of systemic toxicity.
Example 14
Treatment of Metastatic Epithelial Cancers
[0164] Patients afflicted with cancer frequently die from
metastasis to vital organs. Breast, lung, colon, kidney, and skin
cancer patients frequently die from neurological complications
resulting from metastases to the brain or bone. Other carcinomas
have been reported to metastasize to the brain and bone as well,
including those of prostate, pancreas, etc. Breast, lung, colon,
kidney, prostate, and pancreas cancer cells all express claudins 3
and 4 whereas the cell types of the brain and bone do not. Thus,
intracranial and intraosteal administration of CPE is used to
eliminate cancer cells or metastatic tumors expressing claudin 3
and/or 4 while leaving brain or bone cells unharmed. In other
words, targeting claudins 3 and 4 with CPE may provide a means of
eliminating these metastases from the brain without damaging the
brain itself. For example, in a case of breast cancer metastasis to
the brain, a stereotactic injection of purified full-length CPE is
administered directly to the site of the tumor. Injection is made
through a small opening in the skull and guided with the aid of
imaging equipment (e.g. CT scan, etc.). Tumor volume is measured
via imaging (e.g. CT scan, PET scan, etc.) and repeated injections
are administered as needed. Similarly, a micropump or CPE-saturated
wafer may be implanted for slow drug release at the tumor site.
[0165] By immunohistochemical analysis, we found that claudin 3
and/or 4 were expressed in brain metastases from various primary
tumors including those of breast (9/9), lung (7/7), colon (3/4),
ovarian (1/2), and prostate (1/1). In contrast, sections of normal
brain tissue obtained from hemispherectomy was negative for claudin
3 and 4 expression. To further explore the expression of CLDN 3 and
4 in brain cells we obtained a culture of primary human astrocytes.
As determined by Western blot analysis, normal human astrocytes
showed no expression of CLDN 3 or 4. Correspondingly, we found that
these cells were unaffected by CPE treatment in vitro.
[0166] We next tested the toxicity of CPE to the brain using
athymic nude and Balb/C mice. Mice were administered doses of CPE
ranging from 0.05-10 ug three days a week for two weeks by direct
intracranial injection 2 mm posterior to the coronal suture and 2
mm lateral to the sagittal suture. The maximum tolerated dose of
CPE was 0.5 ug native toxin. One group of mice were observed for a
period of two months for any signs of illness or neurological
complications including sluggishness, lack of grooming, huddling,
weight loss, and hemiparesis. A second group of mice were
sacrificed at two weeks, brains were removed, and sections of brain
tissue stained with hematoxylin and eosin were analyzed by a
neuropathologist. Mice treated with 0.5 ug CPE did not show any
signs of toxicity or neurological damage as determined by daily
observation or analysis of brain tissue sections.
[0167] Next, a mouse model of human breast cancer metastasis to the
brain in athymic nude mice was generated. The injection of 250,000
MDA-MB-468 cells intracranially 2 mm posterior to the coronal
suture and 2 mm lateral to the sagittal suture resulted in death at
18 days on average. Using this model, the efficacy of CPE in the
treatment of breast cancer metastasis to the brain was tested.
MDA-MB-468 breast cancer cells were injected into the brains of
athymic nude mice. Following solid tumor formation, mice received
intracranial injections of 0.5 ug CPE or PBS three times per week
for 2 weeks. Mice were observed on a daily basis for signs of
neurological complication resulting from tumor burden and were
sacrificed when moribund. Mice receiving PBS began to show signs of
excessive tumor burden on day 10 and did not survive beyond day 17.
Mice that received CPE did not begin to show signs of illness until
day 14 and 20% of mice survived beyond 40 days (FIG. 8). Thus,
intracranial administration of CPE eliminated or delayed the growth
of brain metastases.
Example 15
Development of Variant Forms of CPE for Use in Breast Cancer
Treatment
[0168] Using PCR, a CPE cDNA molecule encoding all but the
N-terminal 45 amino acids of CPE is generated This variant is known
to exhibit twice the cytotoxicity of full-length CPE and is tested
in all studies previously using full-length CPE as outlined in
previous examples.
[0169] CPE or a variant of CPE is conjugated to a linker rendering
it unable to lyse cells expressing claudin 3 and/or 4 until the
linker is enzymatically cleaved. The inactivation takes place by
preventing binding to claudin 3 and/or 4, preventing cytotoxic
activity following receptor binding, etc. The linker contains a
sequence cleaved by a protease (e.g. matrix metalloproteases,
serine proteases, etc.) that is over-expressed by tumors expressing
Claudin 3 and/or 4 (including, but not limited to breast, lung,
prostate, kidney, pancreas, etc.), tumor stromal elements, or tumor
vasculature. This system of activation allows systemic
administration of CPE and specific targeting of cancer cells while
preventing systemic toxicity.
[0170] While the invention has been described in terms of its
preferred embodiments, those skilled in the art will recognize that
the invention can be practiced with modification within the spirit
and scope of the appended claims. Accordingly, the present
invention should not be limited to the embodiments as described
above, but should further include all modifications and equivalents
thereof within the spirit and scope of the description provided
herein.
Sequence CWU 1
1
21 1 21 PRT Artificial synthetic polypeptide 1 Cys Lys Ala Gly Tyr
Arg Ala Pro Arg Ser Tyr Pro Lys Ser Asn Ser 1 5 10 15 Ser Lys Glu
Tyr Val 20 2 19 DNA Artificial synthetic oligonucleotide primer 2
tggggaaagg gtggtgttg 19 3 21 DNA Artificial synthetic
oligonucleotide primer 3 ttacccaatt ttaaccacca c 21 4 21 DNA
Artificial synthetic oligonucleotide primer 4 gacgttaggt tattttcggt
c 21 5 20 DNA Artificial synthetic oligonucleotide primer 5
aaacgcgttt ctaaacgccg 20 6 20 DNA Artificial synthetic
oligonucleotide primer 6 ccactcgagc cctaatggtg 20 7 20 DNA
Artificial synthetic oligonucleotide primer 7 ggtacccagc cttgctctca
20 8 22 DNA Artificial synthetic oligonucleotide primer 8
gattggctac ccaactgttg ca 22 9 22 DNA Artificial synthetic
oligonucleotide primer 9 caggggcagc agccacaaag gc 22 10 23 DNA
Artificial synthetic oligonucleotide primer 10 aaatgtaaga
aggcccgtat agc 23 11 19 DNA Artificial synthetic oligonucleotide
primer 11 gctaccaagg cggcaagac 19 12 27 DNA Artificial synthetic
oligonucleotide primer 12 ccacgatgaa aattatgcct ccaccca 27 13 20
DNA Artificial synthetic oligonucleotide primer 13 cccatgttcg
tcatgggtgt 20 14 23 DNA Artificial synthetic oligonucleotide primer
14 tggtcatgag tccttccacg ata 23 15 20 DNA Artificial synthetic
oligonucleotide primer 15 ctgcaccacc aactgcttag 20 16 19 DNA
Artificial synthetic oligonucleotide primer 16 gtgattttgg tgtttaggt
19 17 21 DNA Artificial synthetic oligonucleotide primer 17
atcccaaaat atcctaaact a 21 18 20 DNA Artificial synthetic
oligonucleotide primer 18 ccactcgagc cctaatggtg 20 19 20 DNA
Artificial synthetic oligonucleotide primer 19 ggtacccagc
cttgctctca 20 20 22 DNA Artificial synthetic oligonucleotide primer
20 gattggctac ccaactgttg ca 22 21 21 DNA Artificial synthetic
oligonucleotide primer 21 aggggcagca gccacaaagg c 21
* * * * *