U.S. patent application number 13/184269 was filed with the patent office on 2012-02-16 for diagnostic and therapeutic methods and compositions involving pten and breast cancer.
This patent application is currently assigned to Board of Regents of The University of Texas System. Invention is credited to Francisco J. Esteva, Yoichi Nagata, Aysegul A. Sahin, Dihua Yu, Xiaoyan Zhou.
Application Number | 20120039875 13/184269 |
Document ID | / |
Family ID | 34572954 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120039875 |
Kind Code |
A1 |
Yu; Dihua ; et al. |
February 16, 2012 |
DIAGNOSTIC AND THERAPEUTIC METHODS AND COMPOSITIONS INVOLVING PTEN
AND BREAST CANCER
Abstract
Patients with ErbB2-overexpressing cancers can be given an ErbB2
targeting agent as a therapeutic regimen but not all patients are
responsive. The present invention concerns the diagnostic,
prognostic and therapeutic methods and compositions for evaluating
potential efficacy of an ErbB2 targeting agent in an
ErbB2-overexpressing cancers by evaluating PTEN expression, which
is predictive of responsiveness or resistance to ErbB2 targeting
agents such as trastuzumab. Low PTEN expression is predictive of a
patient who will respond poorly to trastuzumab.
Inventors: |
Yu; Dihua; (Houston, TX)
; Zhou; Xiaoyan; (Shanghai, CN) ; Nagata;
Yoichi; (Tosu, JP) ; Esteva; Francisco J.;
(Bellaire, TX) ; Sahin; Aysegul A.; (Houston,
TX) |
Assignee: |
Board of Regents of The University
of Texas System
Austin
TX
|
Family ID: |
34572954 |
Appl. No.: |
13/184269 |
Filed: |
July 15, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10983011 |
Nov 5, 2004 |
7981618 |
|
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13184269 |
|
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|
|
60517559 |
Nov 5, 2003 |
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Current U.S.
Class: |
424/133.1 ;
435/6.11 |
Current CPC
Class: |
G01N 33/573 20130101;
Y10T 436/25 20150115; A61P 35/00 20180101; G01N 33/57415 20130101;
C12Q 1/42 20130101; G01N 33/5011 20130101; C12Q 2600/106 20130101;
C12Q 1/6886 20130101 |
Class at
Publication: |
424/133.1 ;
435/6.11 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
[0002] The government may own rights in the present invention
pursuant to funding from the United States Army Medical Research
and Material Command No. DAMD17-02-1-0462.
Claims
1. A method of determining whether a patient has a lower
probability of response to treatment comprising an ErbB2-targeting
anti-cancer agent, the method comprising: determining the PTEN
nucleic acid status of a sample obtained from said patient and
correlating PTEN negative or PTEN low status to a lower probability
of response to said treatment.
2. The method of claim 1, wherein determining the PTEN nucleic acid
status of said sample comprises determining the expression level of
PTEN transcript in said sample.
3. The method of claim 2, wherein said sample is contacted with an
oligonucleotide hybridizing under high stringency conditions to a
PTEN transcript or a complementary DNA derived therefrom.
4. The method of claim 1, wherein determining the PTEN nucleic acid
status of said sample comprises determining the genomic copy number
of PTEN in said sample.
5. The method of claim 1, wherein determining the PTEN nucleic acid
status of said sample comprises determining whether cells in said
sample have mutations in PTEN.
6. The method of claim 1, wherein said ErbB2-targeting anti-cancer
agent is trastuzumab.
7. The method of claim 1, wherein said patient's cancer
overexpresses ErbB2.
8. The method of claim 1, further comprising determining ErbB2
status in a sample from said patient.
9. The method of claim 8, wherein determining ErbB2 status in a
sample from said patient comprises determining whether cells in
said sample have increased ErbB2 gene expression, increased ErbB2
protein expression, increased ErbB2 gene copy number, or any
combination thereof.
10. A method of treating a patient comprising identifying said
patient as a candidate for a treatment regimen comprising an
ErbB2-targeting anti-cancer agent, determining the PTEN nucleic
acid status of a sample obtained from said patient, and
administering said treatment regimen if said sample is determined
to have PTEN negative or PTEN low status.
11. The method of claim 10, wherein identifying said patient as a
candidate for a treatment regimen comprising an ErbB2-targeting
anti-cancer agent comprises determining ErbB2 status in a sample
from said patient.
12. The method of claim 11, wherein determining the status of ErbB2
in a sample from said patient comprises determining whether cells
in said sample have increased ErbB2 gene expression, increased
ErbB2 protein expression, increased ErbB2 gene copy number, or any
combination thereof.
Description
[0001] The present application is a divisional application of U.S.
Utility application Ser. No. 10/983,011, filed 5 Nov. 2004, which
claims the benefit of U.S. Provisional Application Ser. No.
60/517,559 filed on Nov. 5, 2003. Each of these applications is
incorporated herein by reference in their entirety.
SEQUENCE LISTING
[0003] The instant application was filed with a formal Sequence
Listing submitted electronically as a text file. This text file,
which was named "1203-50-1D-2011-07-15-SEQ-LIST-PRJ-DFB-ST25.txt",
was created on Jul. 15, 2011, and is 10,368 bytes in size. Its
contents are incorporated by reference herein in their
entirety.
BACKGROUND OF THE INVENTION
[0004] I. Field of the Invention
[0005] The present invention relates generally to the fields of
molecular biology and oncology. More particularly, it concerns
diagnostic, prognostic, and therapeutic methods and compositions
involving ErbB2-overexpressing cancers and potential efficacy of
ErbB2 targeting agents to treat such cancers. The invention
involves evaluating PTEN expression and/or activity to evaluate
and/or predict efficacy or possible resistance to such agents.
[0006] II. Description of Related Art
[0007] Overexpression of ErbB2, a 185 kDa membrane receptor
tyrosine kinase, is found in approximately 20-30% of human breast
cancers and many other cancer types (Slamon et al., 1987; Yu and
Hung, 2000). ErbB2 overexpression leads to a very aggressive cancer
phenotype and poor patient survival (Yu and Hung, 2000). Numerous
efforts have been directed at developing ErbB2-targeting cancer
therapies (Bange et al., 2001). One successful example is the
recombinant humanized anti-ErbB2 monoclonal antibody trastuzumab
(Herceptin) that specifically binds to the extracellular domain of
ErbB2 (Shepard et al., 1991). The currently known mechanisms
underlying trastuzumab's anti-tumor activity include the
down-regulation of p185.sup.ErbB2 and the subsequent inhibition of
its down-stream PI3K-Akt signaling pathway (Hudziak et al., 1989;
Yakes et al., 2002), the induction of G.sub.1 arrest and
cyclin-dependent kinase inhibitor p27.sup.kip1 (Sliwkowski et al.,
1999), and the inhibition of ErbB2 ectodomain cleavage (Molina et
al., 2001). Despite these and other reported functions resulting
from ErbB2 down-regulation (Petit et al., 1997), the mechanism of
trastuzumab's anti-tumor activity remains a fundamental question to
be clearly addressed (Albanell and Baselga, 2001).
[0008] As the first FDA approved therapeutic antibody for
metastatic breast cancer, trastuzumab has demonstrated durable
responses as a single agent and striking therapeutic efficacy in
combination with other chemotherapeutics (Baselga et al., 1996;
Cobleigh et al., 1999; Esteva et al., 2002; Pegram et al., 1998;
Seidman et al., 2001; Slamon et al., 2001; Vogel et al., 2002).
However, only less than 35% of patients with ErbB2-overexpressing
metastatic breast cancer respond to trastuzumab as a single agent
whereas .about.5% patients suffer from severe side effects (e.g.,
cardiac dysfunction) and 40% of patients experience other adverse
effects from trastuzumab treatment (Cobleigh et al., 1999; Vogel et
al., 2002). Thus, there is an urgent need to identify patients who
are unlikely to respond to trastuzumab treatment to spare them the
potential side effects and unnecessary cost. More importantly,
trastuzumab resistance-conferring factors may serve as molecular
targets for overcoming trastuzumab resistance. Unfortunately, there
is very limited information on mechanisms of trastuzumab resistance
of breast cancer cells. Currently, there is no clinically verified
factor that can be used to predict trastuzumab resistance (Albanell
and Baselga, 2001).
[0009] Consequently, there is a need for a screen to evaluate
whether a patient may be resistant to trastuzumab in order to
prevent unnecessary side effects and costs in breast cancer
patients who might otherwise receive the treatment even though they
are resistant. Methods and kits for determining whether a patient
might have resistance or susceptibility to an ErbB2-overexpressing
cancer have not been previously known.
SUMMARY OF THE INVENTION
[0010] The present invention is based on the observation that a
deficiency in PTEN activity in cancer cells is significantly
associated with the resistance of those cells to ErbB2-targeting
agents that are used to treat cancer. Thus, the present invention
concerns diagnostic, prognostic, and therapeutic methods and
compositions for cancers that involve ErbB2 expression, and
consequently, ErbB2 targeting agents. It provides information about
whether a particular cancer patient is more or less likely to
respond or be resistant to an ErbB2 targeting agent that is being
considered as a therapeutic agent for an ErbB2-overexpressing
cancer.
[0011] Methods of the invention concern evaluating the potential
efficacy of an ErbB2-targeting therapeutic agent for the treatment
of cancer in a patient comprising evaluating the expression of PTEN
in cells of the cancer. The term "ErbB2-targeting therapeutic
agent" refers to a compound or composition that has a therapeutic
application based on its ability to affect directly or indirectly
ErbB2 expression or activity in a cell. "Potential efficacy" refers
to possible therapeutic effectiveness of a therapeutic agent. It is
contemplated that evaluating the expression of PTEN can involve, in
some cases, comparing a level or amount observed in the cancer
cells with a level of amount observed in noncancerous or normal
cells or in cells that are not ErbB2-overexpressing cells. The term
"evaluate" is used according to its plain and ordinary meaning to
refer to "examine and judge carefully" or "consider."
[0012] A patient whose cancer cells have PTEN expression that is
lower than PTEN expression in a normal (noncancerous) cell or in
cancer cells expressing PTEN at levels similar to that in a normal
cell (considered normal PTEN expression levels) is predictive of a
patient more likely not to respond to (or be resistant to) an ErbB2
targeting agent than a patient whose PTEN expression is at a normal
or above-normal level. It is contemplated that one or more
standards may be generated in which a normal level of PTEN
expression is defined or identified. That standard may then be
referred to as a way of determining whether PTEN expression in a
given patient is normal or below-normal. The type of standard
generated will depend upon the assay or test employed to evaluate
PTEN expression. In some embodiments of the invention, a score is
assigned to a sample based on certain criteria and numbers within
or below a certain number or range are deemed "below normal." In
preferred embodiments, PTEN expression is considered below normal
if an assay indicates that a particular measurement, amount or
level is at about or at most about 80%, 75%, 70%, 65%, 60%, 55%,
50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the
measurement, amount or level observed in cells that have normal
levels of PTEN expression. In other words, for example, a cell with
normal PTEN expression exhibit a level of PTEN transcript that is
x; the sample from the patient being tested may be 0.5.times., in
which case, in some embodiments that patient may be considered to
have a below normal level of PTEN transcript and thus a below
normal level of PTEN expression. Alternatively, in some
embodiments, PTEN expression is considered below normal if an assay
indicates that a particular measurement, amount or level is about
or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more standard
deviations below the measurement, amount or level observed in cells
that have normal levels of PTEN expression. In other cases, PTEN
expression may be considered below normal if a measurement, amount
or level indicative of PTEN expression is or is at most 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more times less
than the measurement, amount, or level indicative of PTEN
expression in normal cells.
[0013] Methods of the invention that involve evaluating the
expression of PTEN in cancer cells can be achieved by a number of
ways that directly or indirectly provide information regarding PTEN
expression. Thus, ways of evaluating PTEN expression include, but
are not limited to, assessing or measuring PTEN protein, assessing
or measuring PTEN transcript, sequencing PTEN transcript or genomic
sequence, measuring PTEN gene copy number, assaying PTEN gene
methylation status, and assaying PTEN activity.
[0014] It is contemplated that methods and compositions of the
invention could be implemented with respect to cancer patients,
particularly to patients with ErbB2-overexpressing cancers. It is
understood that the term "ErbB2-overexpressing cancer" refers to a
cancer whose etiology or cause is believed to be related to cancer
cells that express higher levels of ErbB2 protein compared to
noncancerous cells or cancer cells whose etiology or cause is not
related to ErbB2 protein levels. Therefore, in some embodiments of
the invention, the cancer being treated involves cancerous cells of
the breast, lung, ovary, brain, gastrointestinal tract, salivary
duct, endometrium, prostate, head & neck, glioma, pancreas,
hepatocyte, myeloma, soft tissue sarcoma, or non-small cell lung
cancer, but is not limited to such.
[0015] The invention can be used with respect to any agent that
targets ErbB2 so as to reduce, inhibit, eliminate, or ameliorate
its activity. Such agents may work by directly affecting ErbB2
activity or they may work indirectly by affecting ErbB2
transcription, translation, post-translational modification,
transcript or protein stability, transcript or protein
localization, or some other mechanism that ultimately affects the
amount of a protein's activity. In particular embodiments of the
invention, the ErbB2-targeting therapeutic agent is trastuzumab,
which is commercially known as Herceptin.TM..
[0016] In some embodiments, the expression of PTEN in cancer cells
is evaluated by steps that include, but are not limited to, (a)
obtaining from the patient a sample comprising cancer cells; and
(b) determining the level of PTEN expression in the sample.
Alternatively, the level or amount of PTEN expression in a
particular patient may already be known, and consequently, that
level or amount would be evaluated to make a determination
regarding efficacy of a therapeutic agent in embodiments of the
invention.
[0017] A sample from a patient refers to a biological sample, which
includes, but is not limited to a tissue biopsy or section, blood
sample, lavage, swab, scrape, nipple aspirate, or other composition
that may be extracted from the body and that contains cancer cells.
In particular embodiments, the present invention concerns a sample
that contains all or part of a tissue biopsy. In further
embodiments, the sample contains all or part of a breast tissue
biopsy, which may be from a bilateral biopsy or a unilateral
biopsy.
[0018] In some embodiments of the invention, methods involve
evaluating PTEN expression in cancer cells by assessing PTEN
protein, PTEN transcript, or PTEN gene copy number. The term
"assessing" is used according to its ordinary and plain meaning to
refer to "determining the extent of." In certain embodiments, PTEN
protein or transcript is assessed by assaying (measuring) the
amount of PTEN protein, transcript, or gene copy in the cancer
cells. In specific aspects of the invention, PTEN expression is
evaluated by assessing PTEN protein. An anti-PTEN antibody can be
used in some cases to assess PTEN protein. Such methods may involve
using immunohistochemistry, Western blotting, ELISA,
immunoprecipitation, or an antibody array. In particular
embodiments, PTEN protein is assessed using immunohistochemistry.
The use of immunohistochemistry allows for quantitation and
characterization of PTEN protein. It also allows an immunoreactive
score for the sample to be determined. The term "immunoreactive
score" (IRS) refers to a number that is calculated based on a scale
reflecting the percentage of PTEN positive cells (on a scale of
1-4, where 0=0%, 1=<10%, 2=10%-50%, 3=>50%-80%, and
4=>80%) multiplied by the intensity of PTEN staining (on a scale
of 1-3, where 1=weak, 2=moderate, and 3=strong). Immunoreactive
scores range from 0-12.
[0019] In some embodiments of the invention, PTEN expression is
evaluated by assessing PTEN transcription. PTEN transcription can
be assessed by a variety of methods including those that involve
amplifying PTEN transcripts or performing Northern blotting on PTEN
transcripts. Amplification of PTEN transcripts can be utilized in
quantitative polymerase chain reactions, which are well known to
those of ordinary skill in the art. Alternatively, nuclease
protection assays may be implemented to quantify transcripts. Other
methods that take advantage of hybridization between a probe and
target are also contemplated, such as fluorescence in situ
hybridization (FISH) and RNA in situ hybridization (RISH).
[0020] Further embodiments of the invention involve evaluating PTEN
expression by assaying the level of PTEN activity. PTEN is a
phosphatase and its activity can be observed and measured using a
phosphatase assay involving a PTEN substrate, such as PIP3, or
measured indirectly by measuring Akt phosphorylation. Thus, the
phosphorylation level of Akt can be determined or analyzed.
Alternatively, when the level of PTEN activity is down, the level
of the lipid PIP3 is relatively elevated. Thus, PTEN activity can
be assayed by measuring the level of PIP3. Any other compound
affected by PTEN activity can be evaluated as a way of assaying for
PTEN activity.
[0021] In some embodiments of the invention, PTEN expression is
determined by sequencing a PTEN transcript or PTEN genomic sequence
or evaluating any modifications of such sequences. Sequencing can
be done to determine whether there has been loss of heterozygosity
(LOH) of the PTEN gene. Alternatively, sequencing can provide
information regarding the nature of any mutations in the PTEN gene,
such as deletions, insertions, frame-shifts, translocations, or
truncations, which may result in mutations in the PTEN protein.
Such mutations can affect PTEN expression and/or activity and thus
are relevant to the claimed invention. Moreover, in some cases,
modification of a PTEN-encoding sequence provides information
regarding the level of PTEN expression or activity in a cell.
Moreover, the occurrence of any epigenetic silencing of the PTEN
genomic sequence can be evaluated. An example of such silencing
involves methylation, and thus, in some methods of the invention,
the evaluation of a PTEN genomic sequence involves determining
whether one or both PTEN genes is methylated.
[0022] Assessment of PTEN expression, protein, sequence, transcript
or activity may involve labels, tags, colorimetric indicators
including fluorescence, enzyme indicators, radioactivity, or other
means to quantify or characterize such levels or amounts.
[0023] In yet further aspects, compositions or methods of the
invention may assess, detect, affect or result in the reduced
phosphorylation of the PTEN polypeptide, thus assessing the
phosphorylation state or increasing the activity of PTEN. In one
aspect, trastuzumab may inhibit Src tyrosine kinase. Src activation
has recently been reported to increase PTEN tyrosine phoshorylation
(Lu et al., 2003). Therapeutic or diagnostic methods may inhibit or
detect inhibition of Src kinase activity and reduced Src
phosphorylation on Y416, an indicator of Src activity (Frame,
2002). Reduced Src-Y416 phosphorylation typically corresponds to
reduced PTEN tyrosine phosphorylation. Reduced Src-Y416
phosphorylation may be assessed by using an antibody that
specifically binds a phosphorylated tyrosine of PTEN. In other
aspects, trastuzumab-mediated Src inhibition may be assessed by
determining Src binding to ErbB2, which, for example, is inhibited
by trastuzumab treatment. Trastuzumab may induce a reduction of
ErbB2 bound Src, indicating that trastuzumab inhibited Src activity
in ErbB2 overexpressing cells most likely by inhibiting Src binding
to ErbB2.
[0024] Src kinase inhibitors, such as PP2, may reduce PTEN tyrosine
phosphoryulation in the ErbB2-overexpressing breast cancer cells
(Yu et al., 1998a). These inhibitors may be used in combination
with other methods and compositions of the invention for assessment
or regulation of PTEN status in a cell or subject of interest. For
example, PP2 treatment may effectively inhibited Src
phosphorylation on Y416 and also dramatically reduced tyrosine
phosphorylation of PTEN, similar to trastuzumab's effect.
Trastuzumab treatment inhibits Src binding to ErbB2 in breast
cancer cells, thus inhibiting Src kinase activity, which leads to
reduced PTEN tyrosine phosphorylation and increased PTEN membrane
localization and activity.
[0025] Methods of the invention also include, in some embodiments,
a step of administering the ErbB2 targeting therapeutic agent to
the patient. Such a step is particularly contemplated after it has
been determined that there is potential efficacy for the agent in a
particular patient. In certain embodiments, the therapeutic agent
is trastuzumab. In others it can be inhibitors of PI3K family
proteins. Thus, it is contemplated that another agent for use with
the invention is LY294002 and/or Wortmannin, which inhibit PI3K. In
other cases, the patient can be given a composition comprising any
combination of trastuzumab, LY294002, and Wortmannin. Furthermore,
methods can also involve treating the patient with other
anti-cancer therapy, which can be selected from the group
consisting of radiotherapy, chemotherapy, immune therapy, or gene
therapy.
[0026] Other methods of the invention include a method for
evaluating potential efficacy of a therapeutic agent for the
treatment of an ErbB2-overexpressing cancer comprising: (a)
obtaining from the patient a sample comprising cancer cells; (b)
evaluating the level of PTEN expression or activity in the sample.
In some cases, the ErbB2-overexpressing cancer is breast cancer. In
other embodiments, the therapeutic agent is trastuzumab.
[0027] The present invention also concerns kits for evaluating
potential efficacy of an anti-cancer agent for the treatment of
breast cancer. In some embodiments, the kits include (a) an ErbB2
reagent for evaluating the level of ErbB2 expression or activity in
a sample; (b) a PTEN reagent for evaluating the level of PTEN
expression or activity in a sample. It is contemplated that the
reagents for evaluating the level of expression or activity of
either ErbB2 or PTEN can be one or more nucleic acids. The nucleic
acids may be complementary to all or part of ErbB2 or PTEN and they
can be used in hybridization reactions, such as for amplification
(primers), primer extensions, nuclease protection assays, Northern
blotting, or with an array or other structure. Alternatively,
antibodies against either ErbB2 or PTEN can be used, for example,
in Western blotting, ELISAs, other sandwich assays, antibody
arrays, immunohistochemistry, or FACS analysis. The antibody may be
a monoclonal or a polyclonal antibody. In other embodiments, the
kit comprises a ErbB2 and/or PTEN reagent that is an ErbB2 nucleic
acid or PTEN nucleic acid. The term "PTEN nucleic acid" refers to a
nucleic acid that is complementary or identical to all or part of a
PTEN nucleic acid sequence in order to identify or detect
specifically the PTEN nucleic acid, as opposed to other nucleic
acids. Likewise is the case for a "ErbB2 nucleic acid." Such
nucleic acids may be further characterized as discussed herein. It
is contemplated that kits of the invention may comprise 1, 2, 3, 4,
5, 6 or more ErbB2 reagents and 1, 2, 3, 4, 5, 6 or more PTEN
reagents.
[0028] While the present invention is discussed with respect to the
treatment of cancer, it is contemplated that the present invention
has applications generally to any disease or condition involving
ErbB2 activity, particularly any diseases or conditions
characterized by a relatively high activity or expression level of
ErbB2. Furthermore, any method used or discussed herein with
respect to the detection of ErbB2 overexpression in cancer cells
may be implemented with respect to the detection of PTEN
expression, and vice versa.
[0029] It is specifically contemplated that any embodiment of any
method or composition of the invention may be used with respect to
any other method or composition of the invention.
[0030] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternative are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0031] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device or method being employed to determine the value.
[0032] Following long-standing patent law, the words "a" and "an,"
when used in conjunction with the word "comprising" in the claims
or specification, denotes one or more, unless specifically
noted.
[0033] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0035] FIGS. 1A-1F. Rapid Akt dephosphorylation and PTEN activation
by trastuzumab FIG. 1A: Immunoblotting (IB) of ErbB2,
Tyr1248-phosphorylated ErbB2 (p-ErbB2), phosphorylated Akt (p-Akt),
and total Akt in trastuzumab (+, 2 .mu.g/ml) or control IgG-treated
(-, 2 .mu.g/ml) BT474 cells and SKBr3 cells. .beta.-actin blot
served as loading controls in all IB experiments. All in vitro
experiments were repeated at least three times. Reproducible
representative results are shown. FIG. 1B: PI3K assay after 2
.mu.g/ml of trastuzumab treatment of SKBr3 cells for the indicated
time. Lysates were immunoprecipitated with the PY20 antibody
followed by incubation in kinase buffer. Numbers below the panel
indicate relative PI3K activity to untreated cells. IP with IgG
served as a control. FIG. 1C: Trastuzumab transiently increased
association of the p85 subunit of PI3K with the ErbB2 complex.
ErbB2 immunoprecipitates and total cell lysates from untreated or
trastuzumab-treated (2 .mu.g/ml) cells were immunoblotted with
indicated antibodies. FIG. 1D: Increased PTEN phosphatase
activities in trastuzumab-treated (2 .mu.g/ml) SKBr3 cells. Upper
right insert shows, from left to right, immunoprecipitated PTEN
proteins from untreated and trastuzumab-treated (20 or 60 min)
cells. Immunoprecipitates by rabbit IgG from trastuzumab treated
cells were the negative control. FIG. 1E: Trastuzumab increased
membrane bound PTEN in BT474 cells. PTEN protein in the cytosolic
pool (sol) and the membrane bound pool (mem) of untreated (-) or
trastuzumab-treated (+, 2 .mu.g/ml) cell lysates were blotted with
indicated antibodies. E-cadherin and ErbB2 were membrane protein
markers. FIG. 1F: Reduction of PTEN tyrosine phosphorylation after
trastuzumab treatment in BT474 cells. BT474 cells were treated
without or with 2 .mu.g/ml of trastuzumab for the indicated time.
PTEN IP using the A2B1 PTEN antibody was followed by IB with PY99
phosphotyrosine antibody (top). The membrane was then stripped and
reprobed with A2B1 PTEN antibody to show the IP efficiency. IgG was
used as a negative control. Numbers below the panel indicate
relative levels of PTEN tyrosine phosphorylation with 0 min
trastuzumab treatment defined as 1.
[0036] FIGS. 2A-2B. ErbB2 remains localized on the cell membrane up
to 15 h of trastuzumab treatment. FIG. 2A: Immunofluorescent flow
cytometry analysis of ErbB2 on the cell membrane. BT474 and SKBR3
human breast cancer cells were treated for 1 h and 15 h with 2
mg/ml trastuzumab. After washing cells with PBS buffer,
membrane-localized ErbB2 was detected with anti-ErbB2 antibody
(Ab5, Calbiochem, La Jolla, Calif.), which recognizes the
extracellular domain of ErbB2, followed by FITC-conjugated
secondary antibody. The shaded and unshaded areas represent the
distribution of ErbB2 in trastuzumab-treated and untreated cells,
respectively. The upper right percentage indicates the number of
ErbB2 receptors on the membrane in trastuzumab-treated cells as the
percentage of those in untreated cells. FIG. 2B: Immunofluorescence
staining of ErbB2 in SKBR3 cells. The cells were plated into glass
chambers. After 24 h, the cells were treated for 1 h with 2 mg/ml
trastuzumab, fixed with 4% paraformaldehyde, and were perforated
with 0.3% TritonX/PBS. ErbB2 protein was probed with anti-ErbB2
antibody (Ab3) and FITC-conjugated secondary antibody.
[0037] FIGS. 3A-3G. PTEN reduction by PTEN AS oligonucleotides
confers resistance to trastuzumab's antiproliferative effects in
vitro and in vivo FIG. 3A: PTEN protein is reduced in SKBr3 cells
transfected with PTEN AS (25 nM) compared to mock or mismatched
(MIS) oligonucleotidetransfected cells. FIG. 3B:
Trastuzumab-mediated Akt dephosphorylation is attenuated in PTEN
AS-transfected SKBr3 cells. MIS or PTEN AS-transfected SKBr3 cells
(as in FIG. 3A) were treated with trastuzumab (2 .mu.g/ml) for the
indicated times, and cell lysates were immunoblotted. FIG. 3C:
Trastuzumab inhibited cell proliferation less in PTEN AS-treated
SKBr3 cells compared to MIS transfected cells. MIS or PTEN
AS-transfected SKBr3 cells (as in FIG. 3A) were treated with
trastuzumab (2 .mu.g/ml) for 3 and 5 days. Cell mass was determined
by MTS assay. Percent cell number represents % cell counts under
each treatment relative to untreated cells. FIG. 3D: Similar cell
growth in MIS- or PTEN AS-transfected SKBr3 cells without
trastuzumab. Percent cell numbers represents % cell counts compared
to Day 1 (defined as 100%). FIG. 3E: PTEN-reduced BT474 cells are
more resistant to trastuzumab plus paclitaxel than PTEN normal
cells but have a similar paclitaxel response to PTEN normal cells.
BT474 cells were transfected with 25 nM MIS or PTEN AS, treated by
paclitaxel (1, 2, and 4 nM) without or with 2 .mu.g/ml of
trastuzumab (Ttzm) for 3 days. Percent cell viability are cell mass
as a % of untreated MIS-transfected cells. FIG. 3F: PTEN expression
is reduced in BT474 xenografts injected with PTEN AS. BT474 cells
were inoculated into the mfp of female nude mice. After tumors
reached 150 mm3, MIS or PTEN AS were injected intratumor (15
.mu.g/injection, twice a week) for 1 week. Removed tumors were
stained with antibodies to PTEN and ErbB2. FIG. 3G: PTEN-deficient
BT474 xenografts are more resistant to trastuzumab than PTEN normal
BT474 xenografts. After 1 week of PTEN AS or MIS treatment (as in
FIG. 3F), mice were treated with trastuzumab (10 mg/kg) or vehicle
twice a week. The arrows indicate the starting days of AS/MIS
treatment (AS) and trastuzumab/vehicle treatment (Ttzm). The
results shown are the mean tumor volume+SE; *p<0.05.
[0038] FIGS. 4A-4B. Trastuzumab reduces BrdU incorporation of PTEN
normal expressing (MIS) but not of PTEN-deficient (PTEN AS) BT474
cells. FIG. 4A: BT474 cells were treated with PTEN AS or MIS
Control oligonucleotides plated on glass chamber slides, and
incubated overnight with 2 mg/ml of trastuzumab for 48 h. The cells
were then incubated with 10 mM of BrdU for 30 min, fixed with
ethanol, and incubated with anti-BrdU antibody following the
manufacturer's protocol (Cell Proliferation ELISA, BrdU
colorimetric kit, (Roche Molecular Biochemicals, Indianapolis,
Ind.). BrdU-positive cells were visualized under an
immunofluorescence microscope. Cells were counterstained with DAPI
as a control. FIG. 4B: BrdU positivity in BT474 cells were counted
and compared with total cell numbers determined by DAPI positive
cells. The results shown are representative of three repeated
experiments.
[0039] FIGS. 5A-5C. PI3K inhibitors enhance trastuzumab's
antiproliferation function in PTEN-reduced BT474 cells in vitro and
in vivo FIG. 5A: BT474 cells were treated with MIS or PTEN AS
oligonucleotides as FIG. 3A. Cells were then treated with
trastuzumab (Ttzm, 2 .mu.g/ml), LY294002 (LY, 0.8 .mu.M), or
trastuzumab plus LY294002 (Ttzm+LY) for 3 days. Percent inhibition
is % growth reduction under each treatment relative to untreated
cells. **p<0.01. FIG. 5B: BT474 cells were treated with MIS or
PTEN AS as in FIG. 3A. Cells were then treated with trastuzumab (2
.mu.g/ml), Wortmannin (WN, 1 .mu.M), or tras-tuzumab plus
Wortmannin (Ttzm+WN) for 3 days. Percent inhibition is % growth
reduction under each treatment relative to untreated cells.
**p<0.01. FIG. 5C: BT474 xenografts were treated with PTEN AS as
in FIG. 3G. Mice were treated with trastuzumab (10 mg/kg) twice a
week or/and LY294002 (100 mg/kg) 3 times a week for 3 weeks. The
arrows indicate the starting days of AS treatment (AS) and
trastuzumab and/or LY294002 treatment (Ttzm, LY). The results are
the mean tumor volume+SE. *p<0.05.
[0040] FIGS. 6A-6D. Patients with PTEN deficient breast tumors have
a poor response to trastuzumab-based therapy. FIG. 6A: PTEN
expression patterns in human breast tumors. Forty-seven
ErbB2-overexpressing (3+, 2+, and/or FISH positive) primary breast
carcinomas were collected from patients who subsequently developed
metastatic breast cancer and received trastuzumab plus taxane
chemotherapy (paclitaxel or docetaxel) in clinical trials conducted
(Esteva et al., 2002; Seidman et al., 2001). PTEN expression in
these tumors was examined by immunohistochemistry and
semi-quantified using immunoreactive scores (IRS). IRS was
calculated by the percent of PTEN positive cells (0%=0, <10%=1,
10%-50%=2, 51%-80%=3, >80%=4) multiplied by the intensity of the
staining (weak=1, moderate=2, strong=3), resulting in IRS ranging
from 0-12. Representative PTEN staining (IRS 12, 9, 3, and 0) of
tumor samples was shown. FIG. 6B: Worse responses to
trastuzumab-based therapy in patients with PTEN low and negative
tumors than those with PTEN positive tumors. PTEN low and negative
tumors were defined by PTEN IRS<9 (left panel), or <4 (right
panel). *, P<0.05; **, P<0.01. Complete response (CR),
partial response (PR), stable disease (SD), and progressive disease
(PD), number of patients studied (n). FIG. 6C: Trastuzumab plus
taxane response rates in patients with ErbB2 FISH positive and/or
IHC 3+ ErbB2 overexpression breast cancers versus patients with
ErbB2 FISH negative and/or 2+ ErbB2 overexpression cancers
(P>0.05). FIG. 6D: Among patients with ErbB2 FISH positive
and/or IHC 3+ ErbB2 overexpressing breast cancers, patients with
PTEN low and negative tumors responded less to trastuzumab-based
therapy than those with PTEN positive tumors (**, P<0.01). Data
in FIGS. 4A-4D were obtained in a double blind manner.
[0041] FIG. 7. The response rate to trastuzumab decreases as PTEN
expression (IRS score) decreases. The clinical response rate to
trastuzumab includes either complete response or partial response
in patients with breast cancers expressing three ranges of PTEN IRS
of 0-3, 4-7, and 8-12. The Cochran-Armitage test was performed on
the data which supports the trend hypothesis that the probability
of response to trastuzumab decreases as PTEN IRS decreases. **
P<0.01
[0042] FIG. 8. Ki67 expression levels do not correlate with
clinical response to trastuzumab plus taxane treatment. Ki67 high
expression was defined as .sup.325% Ki67 positive cells. Ki67
staining was performed in 43 samples due to limited sample
availability. CR, complete response; PR, partial response; SD,
stable disease; PD, progressive disease
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0043] The present invention concerns methods and kits for
evaluating or determining the potential efficacy of an ErbB2
targeting agent in a patient who may be given the agent for the
treatment of an ErbB2-overexpressing disease such as cancer. More
particularly, the invention concerns evaluating PTEN expression or
activity in cancer cells of the patient in order to predict whether
the patient will be resistant to the ErbB2 targeting agent. In
addition to diagnostic applications, the present invention concerns
therapeutic and prognostic applications
I. PTEN
[0044] PTEN (phosphatase and tensin homologue, also named
MMAC1/TEP) is a dual phosphatase that mainly dephosphorylates
position D3 of membrane phosphatidylinositol-3,4,5 trisphosphate
(PI3,4,5P3), which is the site for recruiting the
pleckstrin-homology domain of Akt to the cell membrane (Cantley and
Neel, 1999; Parsons and Simpson, 2003; Vivanco and Sawyers, 2002).
Since the phosphatidylinositol 3-Kinase (PI3K) catalyzes the
production of PI3,4,5P3 (Cantley, 2002), PTEN antagonizes this PI3K
function and negatively regulates Akt activities. Loss of PTEN
function due to PTEN mutations, PTEN haploinsufficiency from LOH at
the PTEN locus, and epigenetic down-modulation of PTEN have been
reported in nearly 50% of breast cancers and in many other cancer
types (Li et al., 1997; Mutter et al., 2000; Parsons and Simpson,
2003; Teng et al., 1997; Whang et al., 1998). Restoration of PTEN
expression in PTEN-null cells leads to inhibition of Akt activities
and tumor suppression (Li and Sun, 1998; Lu et al., 1999).
Therefore, PTEN is an important tumor suppressor gene. However,
possible functional interactions of PTEN activity with ErbB2
receptor tyrosine kinase signaling and potential roles of PTEN in
the anti-tumor activity of trastuzumab have never been
explored.
[0045] The nucleic acid and amino acid sequences for human PTEN can
be found at GenBank Accession number U93051 and AAC51183,
respectively, which are specifically incorporated herein by
reference.
II. ERBB2 AND ERBB2 TARGETING AGENTS
[0046] ErbB2 is a polypeptide also known as HER2 (Human Epidermal
growth factor Receptor 2), which is encoded by a proto-oncogene
referred to as c-erbB2, HER2, or HER2/neu. It is overexpressed as a
result of a gene amplification in about 25% of breast cancers
(Slamon et al., 1989). Patients are evaluated to determine whether
they overexpress ErbB2. This can be accomplished by several ways
including the use of the DAKO HercepTest.TM., an
immunohistochemical test for detecting ErbB2 protein
overexpression, or the Vysis PathVysion.TM. HER2DNA Probe, a FISH
test for detecting ErbB2 gene amplification.
[0047] A therapeutic regimen of trastuzumab (Herceptin) in
combination with paclitaxel is generally indicated for treatment of
patients with metastatic breast cancer whose tumors overexpress the
ErbB2 protein and who have not received chemotherapy for their
metastatic disease. Trastuzumab is a humanized anti-ErbB2
monoclonal antibody, and thus, it qualifies as an ErbB2 targeting
agent. Because the present invention concerns ErbB2-overexpressing
cancers, it has applications with respect to any agent used to
target ErbB2, and should not be limited to evaluating resistance to
trastuzumab.
III. PROTEINACEOUS COMPOSITIONS
[0048] The present invention concerns evaluating the expression
and/or activity of the polypeptide PTEN, as well as determining
whether a cancer is a ErbB2-overexpressing cancer.
[0049] As used herein, a "proteinaceous molecule," "proteinaceous
composition," "proteinaceous compound," "proteinaceous chain" or
"proteinaceous material" generally refers, but is not limited to, a
protein of greater than about 200 amino acids or the full length
endogenous sequence translated from a gene; a polypeptide of
greater than about 100 amino acids; and/or a peptide of from about
3 to about 100 amino acids. All the "proteinaceous" terms described
above may be used interchangeably herein.
[0050] In certain embodiments the size of the at least one
proteinaceous molecule may be at least, at most or may comprise,
but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,
240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,
550, 575, 582, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825,
850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500,
1750, 2000, 2250, 2500 or greater amino molecule residues, and any
range derivable therein. It is specifically contemplated that such
lengths of contiguous amino acids from SEQ ID NO:2 (amino acid
sequence of human PTEN) are part of the invention.
[0051] As used herein, an "amino molecule" refers to any amino
acid, amino acid derivative or amino acid mimic as would be known
to one of ordinary skill in the art. In certain embodiments, the
residues of the proteinaceous molecule are sequential, without any
non-amino molecule interrupting the sequence of amino molecule
residues. In other embodiments, the sequence may comprise one or
more non-amino molecule moieties. In particular embodiments, the
sequence of residues of the proteinaceous molecule may be
interrupted by one or more non-amino molecule moieties.
[0052] Proteinaceous compositions may be made by any technique
known to those of skill in the art, including the expression of
proteins, polypeptides or peptides through standard molecular
biological techniques, the isolation of proteinaceous compounds
from natural sources, or the chemical synthesis of proteinaceous
materials. The nucleotide and protein, polypeptide and peptide
sequences for various genes have been previously disclosed, and may
be found at computerized databases known to those of ordinary skill
in the art. One such database is the National Center for
Biotechnology Information's Genbank and GenPept databases found on
the internet at the National Institutes of Health website. The
coding regions for these known genes may be amplified and/or
expressed using the techniques disclosed herein or as would be know
to those of ordinary skill in the art. Alternatively, various
commercial preparations of proteins, polypeptides and peptides are
known to those of skill in the art.
[0053] When the present application refers to the function or
activity of PTEN, it is meant that the molecule in question has at
least the ability to catalyze the dephosphorylation of another
polypeptide. Determination of which molecules possess this activity
and what level of activity there is may be achieved using assays
familiar to those of skill in the art, and include those described
in the Examples.
[0054] Certain aspects of the present invention concern the
purification, and in particular embodiments, the substantial
purification, of an encoded protein or peptide. The term "purified
protein or peptide" as used herein, is intended to refer to a
composition, isolatable from other components, wherein the protein
or peptide is purified to any degree relative to its
naturally-obtainable state. A purified protein or peptide therefore
also refers to a protein or peptide, free from the environment in
which it may naturally occur.
[0055] Generally, "purified" will refer to a protein or peptide
composition that has been subjected to fractionation to remove
various other components, and which composition substantially
retains its expressed biological activity. Where the term
"substantially purified" is used, this designation will refer to a
composition in which the protein or peptide forms the major
component of the composition, such as constituting about 50%, about
60%, about 70%, about 80%, about 90%, about 95% or more of the
proteins in the composition.
[0056] Various methods for quantifying the degree of purification
of the protein or peptide will be known to those of skill in the
art in light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
assessing the amount of polypeptides within a fraction by SDS/PAGE
analysis. A preferred method for assessing the purity of a fraction
is to calculate the specific activity of the fraction, to compare
it to the specific activity of the initial extract, and to thus
calculate the degree of purity, herein assessed by a "-fold
purification number." The actual units used to represent the amount
of activity will, of course, be dependent upon the particular assay
technique chosen to follow the purification and whether or not the
expressed protein or peptide exhibits a detectable activity.
[0057] Various techniques suitable for use in protein purification
will be well known to those of skill in the art. These include, for
example, precipitation with ammonium sulfate, PEG, antibodies and
the like or by heat denaturation, followed by centrifugation;
chromatography steps such as ion exchange, gel filtration, reverse
phase, hydroxylapatite and affinity chromatography; isoelectric
focusing; gel electrophoresis; and combinations of such and other
techniques. As is generally known in the art, it is believed that
the order of conducting the various purification steps may be
changed, or that certain steps may be omitted, and still result in
a suitable method for the preparation of a substantially purified
protein or peptide.
[0058] Another embodiment of the present invention are antibodies.
In some cases, the antibody is an ErbB2 targeting agent, while in
others, it is used to evaluate, assess, or determine PTEN or ErbB2
expression. It is understood that antibodies can be used to
quantify polypeptides. Such antibodies, polyclonal or monoclonal,
can be generated. Means for preparing and characterizing antibodies
are also well known in the art (See, e.g., Harlow and Lane, 1988;
incorporated herein by reference). Alternatively, they can be
obtained commercially. For example, PTEN antibodies can be readily
obtained from Santa Cruz Biotechnology (A2B1, Santa Cruz, Calif.)
and Lab Vision Corp. (Ab-2, Fremont, Calif.).
[0059] As discussed, in some embodiments, the present invention
concerns immunodetection methods for assessing, evaluating,
determining, quantifying and/or otherwise detecting biological
components such as PTEN polypeptides.
[0060] Immunodetection methods include enzyme linked immunosorbent
assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay,
fluoroimmunoassay, chemiluminescent assay, bioluminescent assay,
western blot, and screening an antibody array, though several
others are well known to those of ordinary skill. The steps of
various useful immunodetection methods have been described in the
scientific literature, such as, e.g., Doolittle et al., 1999;
Gulbis and Galand, 1993; De Jager et al., 1993; and Nakamura et
al., 1987, each incorporated herein by reference.
[0061] In general, the immunobinding methods include obtaining a
sample suspected of containing a protein, polypeptide and/or
peptide, and contacting the sample with a first antibody,
monoclonal or polyclonal, in accordance with the present invention,
as the case may be, under conditions effective to allow the
formation of immunocomplexes.
[0062] The immunobinding methods include methods for detecting and
quantifying the amount of an antigen component in a sample and the
detection and quantification of any immune complexes formed during
the binding process. Here, one would obtain a sample suspected of
containing an antigen or antigenic domain, and contact the sample
with an antibody against the antigen or antigenic domain, and then
detect and quantify the amount of immune complexes formed under the
specific conditions.
[0063] In terms of antigen detection, the biological sample
analyzed may be any sample that is suspected of containing an
antigen or antigenic domain, such as, for example, a cancer cell or
tissue, or any biological fluid that comes into contact with the
cell or tissue, including blood and/or serum.
[0064] Contacting the chosen biological sample with the antibody
under effective conditions and for a period of time sufficient to
allow the formation of immune complexes (primary immune complexes)
is generally a matter of simply adding the antibody composition to
the sample and incubating the mixture for a period of time long
enough for the antibodies to form immune complexes with, i.e., to
bind to, any antigens present. After this time, the sample-antibody
composition, such as a tissue section, ELISA plate, dot blot or
Western blot, will generally be washed to remove any
non-specifically bound antibody species, allowing only those
antibodies specifically bound within the primary immune complexes
to be detected.
[0065] In general, the detection of immunocomplex formation is well
known in the art and may be achieved through the application of
numerous approaches. These methods are generally based upon the
detection of a label or marker, such as any of those radioactive,
fluorescent, biological and enzymatic tags. U.S. patents concerning
the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each
incorporated herein by reference. Of course, one may find
additional advantages through the use of a secondary binding ligand
such as a second antibody and/or a biotin/avidin ligand binding
arrangement, as is known in the art.
[0066] One method of immunodetection designed by Charles Cantor
uses two different antibodies (see, Sano et al., 1992). A first
step biotinylated, monoclonal or polyclonal antibody is used to
detect the target antigen(s), and a second step antibody is then
used to detect the biotin attached to the complexed biotin. In that
method the sample to be tested is first incubated in a solution
containing the first step antibody. If the target antigen is
present, some of the antibody binds to the antigen to form a
biotinylated antibody/antigen complex. The antibody/antigen complex
is then amplified by incubation in successive solutions of
streptavidin (or avidin), biotinylated DNA, and/or complementary
biotinylated DNA, with each step adding additional biotin sites to
the antibody/antigen complex. The amplification steps are repeated
until a suitable level of amplification is achieved, at which point
the sample is incubated in a solution containing the second step
antibody against biotin. This second step antibody is labeled, for
example, with an enzyme that can be used to detect the presence of
the antibody/antigen complex by histoenzymology using a chromogen
substrate. With suitable amplification, a conjugate can be produced
which is macroscopically visible.
[0067] Another known method of immunodetection takes advantage of
the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR.TM.
method is similar to the Cantor method up to the incubation with
biotinylated DNA, however, instead of using multiple rounds of
streptavidin and biotinylated DNA incubation, the
DNA/biotin/streptavidin/antibody complex is washed out with a low
pH or high salt buffer that releases the antibody. The resulting
wash solution is then used to carry out a PCR.TM. reaction with
suitable primers with appropriate controls. At least in theory, the
enormous amplification capability and specificity of PCR.TM. can be
utilized to detect a single antigen molecule.
[0068] As detailed above, immunoassays, in their most simple and/or
direct sense, are binding assays. Certain preferred immunoassays
are the various types of enzyme linked immunosorbent assays
(ELISAs) and/or radioimmunoassays (RIA) known in the art.
Immunohistochemical detection using tissue sections is also
particularly useful. However, it will be readily appreciated that
detection is not limited to such techniques, and/or western
blotting, dot blotting, FACS analyses, and/or the like may also be
used.
[0069] Irrespective of the format employed, ELISAs have certain
features in common, such as coating, incubating and binding,
washing to remove non-specifically bound species, and detecting the
bound immune complexes. These are described below.
[0070] In coating a plate with either antigen or antibody, one will
generally incubate the wells of the plate with a solution of the
antigen or antibody, either overnight or for a specified period of
hours. The wells of the plate will then be washed to remove
incompletely adsorbed material. Any remaining available surfaces of
the wells are then "coated" with a nonspecific protein that is
antigenically neutral with regard to the test antisera. These
include bovine serum albumin (BSA), casein or solutions of milk
powder. The coating allows for blocking of nonspecific adsorption
sites on the immobilizing surface and thus reduces the background
caused by nonspecific binding of antisera onto the surface.
[0071] In ELISAs, it is probably more customary to use a secondary
or tertiary detection means rather than a direct procedure. Thus,
after binding of a protein or antibody to the well, coating with a
non-reactive material to reduce background, and washing to remove
unbound material, the immobilizing surface is contacted with the
biological sample to be tested under conditions effective to allow
immune complex (antigen/antibody) formation. Detection of the
immune complex then requires a labeled secondary binding ligand or
antibody, and a secondary binding ligand or antibody in conjunction
with a labeled tertiary antibody or a third binding ligand.
[0072] "Under conditions effective to allow immune complex
(antigen/antibody) formation" means that the conditions preferably
include diluting the antigens and/or antibodies with solutions such
as BSA, bovine gamma globulin (BGG) or phosphate buffered saline
(PBS)/Tween. These added agents also tend to assist in the
reduction of nonspecific background.
[0073] The "suitable" conditions also mean that the incubation is
at a temperature or for a period of time sufficient to allow
effective binding. Incubation steps are typically from about 1 to 2
to 4 hours or so, at temperatures preferably on the order of
25.degree. C. to 27.degree. C., or may be overnight at about
4.degree. C. or so.
[0074] Following all incubation steps in an ELISA, the contacted
surface is washed so as to remove non-complexed material. An
example of a washing procedure includes washing with a solution
such as PBS/Tween, or borate buffer. Following the formation of
specific immune complexes between the test sample and the
originally bound material, and subsequent washing, the occurrence
of even minute amounts of immune complexes may be determined.
[0075] To provide a detecting means, the second or third antibody
will have an associated label to allow detection. This may be an
enzyme that will generate color development upon incubating with an
appropriate chromogenic substrate. Thus, for example, one will
desire to contact or incubate the first and second immune complex
with a urease, glucose oxidase, alkaline phosphatase or hydrogen
peroxidase-conjugated antibody for a period of time and under
conditions that favor the development of further immune complex
formation (e.g., incubation for 2 h at room temperature in a
PBS-containing solution such as PBS-Tween).
[0076] After incubation with the labeled antibody, and subsequent
to washing to remove unbound material, the amount of label is
quantified, e.g., by incubation with a chromogenic substrate such
as urea, or bromocresol purple, or
2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or
H.sub.2O.sub.2, in the case of peroxidase as the enzyme label.
Quantification is then achieved by measuring the degree of color
generated, e.g., using a visible spectra spectrophotometer.
[0077] The antibodies of the present invention may also be used in
conjunction with both fresh-frozen and/or formalin-fixed,
paraffin-embedded tissue blocks prepared for study by
immunohistochemistry (IHC). For example, immunohistochemistry may
be utilized to characterize PTEN or to evaluate the amount PTEN in
a cell. The method of preparing tissue blocks from these
particulate specimens has been successfully used in previous IHC
studies of various prognostic factors, and/or is well known to
those of skill in the art (Brown et al., 1990; Abbondanzo et al.,
1990; Allred et al., 1990). Other details are provided in the
Examples section.
IV. PTEN NUCLEIC ACIDS
[0078] The present invention concerns polynucleotides and
oligonucleotides, isolatable from cells, that are free from total
genomic DNA and that are capable of expressing all or part of a
protein or polypeptide. The polynucleotides or oligonucleotides may
be identical or complementary to all or part of a nucleic acid
sequence encoding a PTEN amino acid sequence. These nucleic acids
may be used directly or indirectly to assess, evaluate, quantify,
or determine PTEN expression.
[0079] As used in this application, the term "PTEN polynucleotide"
refers to a PTEN-encoding nucleic acid molecule that has been
isolated essentially or substantially free of total genomic nucleic
acid to permit hybridization and amplification, but is not limited
to such. Therefore, a "polynucleotide encoding PTEN" refers to a
DNA segment that contains wild-type (SEQ ID NO:1), mutant, or
polymorphic PTEN polypeptide-coding sequences isolated away from,
or purified free from, total mammalian or human genomic DNA. A PTEN
oligonucleotide refers to a nucleic acid molecule that is
complementary or identical to at least 5 contiguous nucleotides of
a PTEN-encoding sequence, such as SEQ ID NO:1, which is the cDNA
sequence encoding human PTEN.
[0080] It also is contemplated that a particular polypeptide from a
given species may be represented by natural variants that have
slightly different nucleic acid sequences but, nonetheless, encode
the same protein.
[0081] Similarly, a polynucleotide comprising an isolated or
purified wild-type, polymorphic, or mutant polypeptide gene refers
to a DNA segment including wild-type, polymorphic, or mutant
polypeptide coding sequences and, in certain aspects, regulatory
sequences, isolated substantially away from other naturally
occurring genes or protein encoding sequences. In this respect, the
term "gene" is used for simplicity to refer to a functional
protein, polypeptide, or peptide-encoding unit. As will be
understood by those in the art, this functional term includes
genomic sequences, cDNA sequences, and smaller engineered gene
segments that express, or may be adapted to express, proteins,
polypeptides, domains, peptides, fusion proteins, and mutants. A
nucleic acid encoding all or part of a native or modified
polypeptide may contain a contiguous nucleic acid sequence encoding
all or a portion of such a polypeptide of the following lengths:
about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,
400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510,
520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,
650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,
780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,
910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020,
1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000,
2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500,
8000, 9000, 10000, or more nucleotides, nucleosides, or base pairs,
including such sequences from SEQ ID NO:1, other PTEN encoding
sequences, or ErbB2-encoding sequences.
[0082] In particular embodiments, the invention concerns isolated
DNA segments and recombinant vectors incorporating DNA sequences
that encode all or part of a wild-type, polymorphic, or mutant PTEN
polypeptide or peptide that includes within its amino acid sequence
a contiguous amino acid sequence in accordance with, or essentially
corresponding to a native polypeptide. The term "recombinant" may
be used in conjunction with a polypeptide or the name of a specific
polypeptide, and this generally refers to a polypeptide produced
from a nucleic acid molecule that has been manipulated in vitro or
that is the replicated product of such a molecule.
[0083] In other embodiments, the invention concerns isolated DNA
segments and recombinant vectors incorporating DNA sequences that
encode a polypeptide or peptide that includes within its amino acid
sequence a contiguous amino acid sequence in accordance with, or
essentially corresponding to the polypeptide.
[0084] The nucleic acid segments used in the present invention,
regardless of the length of the coding sequence itself, may be
combined with other nucleic acid sequences, such as promoters,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length may vary considerably. It is therefore
contemplated that a nucleic acid fragment of almost any length may
be employed, with the total length preferably being limited by the
ease of preparation and use in the intended recombinant DNA
protocol.
[0085] The sequence of a PTEN polypeptide will substantially
correspond to a contiguous portion of that shown in SEQ ID NO:2,
and have relatively few amino acids that are not identical to, or a
biologically functional equivalent of, the amino acids shown in SEQ
ID NO:2. The term "biologically functional equivalent" is well
understood in the art and is further defined in detail herein.
[0086] Accordingly, sequences that have between about 70% and about
80%; or more preferably, between about 81% and about 90%; or even
more preferably, between about 91% and about 99%; of amino acids
that are identical or functionally equivalent to the amino acids of
SEQ ID NO:2 will be sequences that are "essentially as set forth in
SEQ ID NO:2."
[0087] In certain other embodiments, the invention concerns
isolated DNA segments and recombinant vectors that include within
their sequence a contiguous nucleic acid sequence from that shown
in SEQ ID NO:1. This definition is used in the same sense as
described above and means that the nucleic acid sequence
substantially corresponds to a contiguous portion of that shown in
SEQ ID NO:1 and has relatively few codons that are not identical,
or functionally equivalent, to the codons of SEQ ID NO:1. The term
"functionally equivalent codon" is used herein to refer to codons
that encode the same amino acid, such as the six codons for
arginine or serine, and also refers to codons that encode
biologically equivalent amino acids.
[0088] The various probes and primers designed around the
nucleotide sequences of the present invention may be of any length.
By assigning numeric values to a sequence, for example, the first
residue is 1, the second residue is 2, etc., an algorithm defining
all primers can be proposed:
n to n+y
where n is an integer from 1 to the last number of the sequence and
y is the length of the primer minus one, where n+y does not exceed
the last number of the sequence. Thus, for a 10-mer, the probes
correspond to bases 1 to 10, 2 to 11, 3 to 12 . . . and so on. For
a 15-mer, the probes correspond to bases 1 to 15, 2 to 16, 3 to 17
. . . and so on. For a 20-mer, the probes correspond to bases 1 to
20, 2 to 21, 3 to 22 . . . and so on.
[0089] It also will be understood that this invention is not
limited to the particular nucleic acid and amino acid sequences of
SEQ ID NO:1 and SEQ ID NO:2 respectively. Recombinant vectors and
isolated DNA segments may therefore variously include the
PTEN-coding regions themselves, coding regions bearing selected
alterations or modifications in the basic coding region, or they
may encode larger polypeptides that nevertheless include
PTEN-coding regions or may encode biologically functional
equivalent proteins or peptides that have variant amino acids
sequences.
[0090] Moreover, the nucleic acids are not limited to coding
sequences. In some embodiments of the invention, genomic PTEN
sequences can be used to determine directly or indirectly PTEN
expression.
[0091] Encompassed by certain embodiments of the present invention
are DNA segments encoding relatively small peptides, such as, for
example, peptides of from about 15 to about 50 amino acids in
length, and more preferably, of from about 15 to about 30 amino
acids in length; and also larger polypeptides up to and including
proteins corresponding to the full-length sequences set forth in
SEQ ID NO:2, or to specific fragments of SEQ ID NO:1 that
correspond to differences as compared to the published sequence for
human PTEN.
[0092] Nucleic acid molecules may be comprised in a vector. The
term "vector" is used to refer to a carrier nucleic acid molecule
into which a nucleic acid sequence can be inserted for introduction
into a cell where it can be replicated. A nucleic acid sequence can
be "exogenous," which means that it is foreign to the cell into
which the vector is being introduced or that the sequence is
homologous to a sequence in the cell but in a position within the
host cell nucleic acid in which the sequence is ordinarily not
found. Vectors include plasmids, cosmids, viruses (bacteriophage,
animal viruses, and plant viruses), and artificial chromosomes
(e.g., YACs). One of skill in the art would be well equipped to
construct a vector through standard recombinant techniques, which
are described in Sambrook et al. (2001) and Ausubel et al. (1996),
both incorporated herein by reference. In addition to encoding a
modified polypeptide such as modified gelonin, a vector may encode
non-modified polypeptide sequences such as a tag or targeting
molecule. In addition to control sequences that govern
transcription and translation, vectors and expression vectors may
contain nucleic acid sequences that serve other functions as well.
Such sequences may include one or more of the following: promoters,
enhancers, initiation signals, internal ribosome binding sites,
multiple cloning sites, splicing sites, termination signals,
polyadenylation signals, origins of replication, and selectable and
screenable markers.
[0093] The nucleic acid sequences disclosed herein have a variety
of uses as probes or primers for embodiments involving nucleic acid
hybridization in methods of the invention.
[0094] The use of a probe or primer of between 13 and 100
nucleotides, preferably between 17 and 100 nucleotides in length,
or in some aspects of the invention up to 1-2 kilobases or more in
length, allows the formation of a duplex molecule that is both
stable and selective. Such probes or primers can be of lengths
described above from SEQ ID NO:1. Molecules having complementary
sequences over contiguous stretches greater than 20 bases in length
are generally preferred, to increase stability and/or selectivity
of the hybrid molecules obtained. One will generally prefer to
design nucleic acid molecules for hybridization having one or more
complementary sequences of 20 to 30 nucleotides, or even longer
where desired. Such fragments may be readily prepared, for example,
by directly synthesizing the fragment by chemical means or by
introducing selected sequences into recombinant vectors for
recombinant production.
[0095] Accordingly, the nucleotide sequences of the invention may
be used for their ability to selectively form duplex molecules with
complementary stretches of DNAs and/or RNAs or to provide primers
for amplification of DNA or RNA from samples. Depending on the
application envisioned, one would desire to employ varying
conditions of hybridization to achieve varying degrees of
selectivity of the probe or primers for the target sequence.
[0096] For applications requiring high selectivity, one will
typically desire to employ relatively high stringency conditions to
form the hybrids. For example, relatively low salt and/or high
temperature conditions, such as provided by about 0.02 M to about
0.10 M NaCl at temperatures of about 50.degree. C. to about
70.degree. C. Such high stringency conditions tolerate little, if
any, mismatch between the probe or primers and the template or
target strand and would be particularly suitable for isolating
specific genes or for detecting specific mRNA transcripts. It is
generally appreciated that conditions can be rendered more
stringent by the addition of increasing amounts of formamide.
[0097] For certain applications, for example, site-directed
mutagenesis, it is appreciated that lower stringency conditions are
preferred. Under these conditions, hybridization may occur even
though the sequences of the hybridizing strands are not perfectly
complementary, but are mismatched at one or more positions.
Conditions may be rendered less stringent by increasing salt
concentration and/or decreasing temperature. For example, a medium
stringency condition could be provided by about 0.1 to 0.25 M NaCl
at temperatures of about 37.degree. C. to about 55.degree. C.,
while a low stringency condition could be provided by about 0.15 M
to about 0.9 M salt, at temperatures ranging from about 20.degree.
C. to about 55.degree. C. Hybridization conditions can be readily
manipulated depending on the desired results.
[0098] In other embodiments, hybridization may be achieved under
conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3
mM MgCl.sub.2, 1.0 mM dithiothreitol, at temperatures between
approximately 20.degree. C. to about 37.degree. C. Other
hybridization conditions utilized could include approximately 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, at temperatures
ranging from approximately 40.degree. C. to about 72.degree. C.
[0099] In certain embodiments, it will be advantageous to employ
nucleic acids of defined sequences of the present invention in
combination with an appropriate means, such as a label, for
determining hybridization. A wide variety of appropriate indicator
means are known in the art, including fluorescent, radioactive,
enzymatic or other ligands, such as avidin/biotin, which are
capable of being detected. In preferred embodiments, one may desire
to employ a fluorescent label or an enzyme tag such as urease,
alkaline phosphatase or peroxidase, instead of radioactive or other
environmentally undesirable reagents. In the case of enzyme tags,
colorimetric indicator substrates are known that can be employed to
provide a detection means that is visibly or spectrophotometrically
detectable, to identify specific hybridization with complementary
nucleic acid containing samples.
[0100] In general, it is envisioned that the probes or primers
described herein will be useful as reagents in solution
hybridization, as in PCR.TM., for detection of expression of
corresponding genes, as well as in embodiments employing a solid
phase. In embodiments involving a solid phase, the test DNA (or
RNA) is adsorbed or otherwise affixed to a selected matrix or
surface. This fixed, single-stranded nucleic acid is then subjected
to hybridization with selected probes under desired conditions. The
conditions selected will depend on the particular circumstances
(depending, for example, on the G+C content, type of target nucleic
acid, source of nucleic acid, size of hybridization probe, etc.).
Optimization of hybridization conditions for the particular
application of interest is well known to those of skill in the art.
After washing of the hybridized molecules to remove
non-specifically bound probe molecules, hybridization is detected,
and/or quantified, by determining the amount of bound label.
Representative solid phase hybridization methods are disclosed in
U.S. Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods of
hybridization that may be used in the practice of the present
invention are disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and
5,851,772. The relevant portions of these and other references
identified in this section of the Specification are incorporated
herein by reference.
[0101] Nucleic acids used as a template for amplification may be
isolated from cells, tissues or other samples according to standard
methodologies (Sambrook et al., 2001). In certain embodiments,
analysis is performed on whole cell or tissue homogenates or
biological fluid samples without substantial purification of the
template nucleic acid. The nucleic acid may be genomic DNA or
fractionated or whole cell RNA. Where RNA is used, it may be
desired to first convert the RNA to a complementary DNA.
[0102] The term "primer," as used herein, is meant to encompass any
nucleic acid that is capable of priming the synthesis of a nascent
nucleic acid in a template-dependent process. Typically, primers
are oligonucleotides from ten to twenty and/or thirty base pairs in
length, but longer sequences can be employed. Primers may be
provided in double-stranded and/or single-stranded form, although
the single-stranded form is preferred.
[0103] Pairs of primers designed to selectively hybridize to a
nucleic acid corresponding to SEQ ID NO:1 or other PTEN nucleic
acid sequence are contacted with the template nucleic acid under
conditions that permit selective hybridization. Depending upon the
desired application, high stringency hybridization conditions may
be selected that will only allow hybridization to sequences that
are completely complementary to the primers. In other embodiments,
hybridization may occur under reduced stringency to allow for
amplification of nucleic acids contain one or more mismatches with
the primer sequences. Once hybridized, the template-primer complex
is contacted with one or more enzymes that facilitate
template-dependent nucleic acid synthesis. Multiple rounds of
amplification, also referred to as "cycles," are conducted until a
sufficient amount of amplification product is produced.
[0104] The amplification product may be detected or quantified. In
certain applications, the detection may be performed by visual
means. Alternatively, the detection may involve indirect
identification of the product via chemiluminescence, radioactive
scintigraphy of incorporated radiolabel or fluorescent label or
even via a system using electrical and/or thermal impulse signals
(Bellus, 1994).
[0105] A number of template dependent processes are available to
amplify the oligonucleotide sequences present in a given template
sample. One of the best known amplification methods is the
polymerase chain reaction (referred to as PCR.TM.) which is
described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and
4,800,159, and in Innis et al., 1988, each of which is incorporated
herein by reference in their entirety.
[0106] A reverse transcriptase PCR.TM. amplification procedure may
be performed to quantify the amount of mRNA amplified. Methods of
reverse transcribing RNA into cDNA are well known (see Sambrook et
al., 2001). Alternative methods for reverse transcription utilize
thermostable DNA polymerases. These methods are described in WO
90/07641. Polymerase chain reaction methodologies are well known in
the art. Representative methods of RT-PCR are described in U.S.
Pat. No. 5,882,864.
[0107] Another method for amplification is ligase chain reaction
("LCR"), disclosed in European Application No. 320 308,
incorporated herein by reference in its entirety. U.S. Pat. No.
4,883,750 describes a method similar to LCR for binding probe pairs
to a target sequence. A method based on PCR.TM. and oligonucleotide
ligase assay (OLA), disclosed in U.S. Pat. No. 5,912,148, may also
be used.
[0108] Alternative methods for amplification of target nucleic acid
sequences that may be used in the practice of the present invention
are disclosed in U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783,
5,849,546, 5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776,
5,922,574, 5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291
and 5,942,391, GB Application No. 2 202 328, and in PCT Application
No. PCT/US89/01025, each of which is incorporated herein by
reference in its entirety. Qbeta Replicase, described in PCT
Application No. PCT/US87/00880, may also be used as an
amplification method in the present invention. In this method, a
replicative sequence of RNA that has a region complementary to that
of a target is added to a sample in the presence of an RNA
polymerase. The polymerase will copy the replicative sequence which
may then be detected.
[0109] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide
5'-[alpha-thio]-triphosphates in one strand of a restriction site
may also be useful in the amplification of nucleic acids in the
present invention (Walker et al., 1992). Strand Displacement
Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779, is
another method of carrying out isothermal amplification of nucleic
acids which involves multiple rounds of strand displacement and
synthesis, i.e., nick translation.
[0110] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA) and 3SR (Kwoh et al.,
1989; PCT Application WO 88/10315, incorporated herein by reference
in their entirety). European Application No. 329 822 disclose a
nucleic acid amplification process involving cyclically
synthesizing single-stranded RNA ("ssRNA"), ssDNA, and
double-stranded DNA (dsDNA), which may be used in accordance with
the present invention.
[0111] PCT Application WO 89/06700 (incorporated herein by
reference in its entirety) disclose a nucleic acid sequence
amplification scheme based on the hybridization of a promoter
region/primer sequence to a target single-stranded DNA ("ssDNA")
followed by transcription of many RNA copies of the sequence. This
scheme is not cyclic, i.e., new templates are not produced from the
resultant RNA transcripts. Other amplification methods include
"RACE" and "one-sided PCR" (Frohman, 1990; Ohara et al., 1989).
[0112] Following any amplification or step such as primer
extension, it may be desirable to separate the amplification or
primer extension product from the template and/or the excess
primer. In one embodiment, amplification products are separated by
agarose, agarose-acrylamide or polyacrylamide gel electrophoresis
using standard methods (Sambrook et al., 2001). Separated
amplification products may be cut out and eluted from the gel for
further manipulation. Using low melting point agarose gels, the
separated band may be removed by heating the gel, followed by
extraction of the nucleic acid.
[0113] Separation of nucleic acids may also be effected by
chromatographic techniques known in art. There are many kinds of
chromatography which may be used in the practice of the present
invention, including adsorption, partition, ion-exchange,
hydroxylapatite, molecular sieve, reverse-phase, column, paper,
thin-layer, and gas chromatography as well as HPLC.
[0114] In certain embodiments, the amplification products are
visualized. A typical visualization method involves staining of a
gel with ethidium bromide and visualization of bands under UV
light. Alternatively, if the amplification products are integrally
labeled with radio- or fluorometrically-labeled nucleotides, the
separated amplification products can be exposed to x-ray film or
visualized under the appropriate excitatory spectra.
[0115] In one embodiment, following separation of amplification
products, a labeled nucleic acid probe is brought into contact with
the amplified marker sequence. The probe preferably is conjugated
to a chromophore but may be radiolabeled. In another embodiment,
the probe is conjugated to a binding partner, such as an antibody
or biotin, or another binding partner carrying a detectable
moiety.
[0116] In particular embodiments, detection is by Southern blotting
and hybridization with a labeled probe. The techniques involved in
Southern blotting are well known to those of skill in the art (see
Sambrook et al., 2001). One example of the foregoing is described
in U.S. Pat. No. 5,279,721, incorporated by reference herein, which
discloses an apparatus and method for the automated electrophoresis
and transfer of nucleic acids. The apparatus permits
electrophoresis and blotting without external manipulation of the
gel and is ideally suited to carrying out methods according to the
present invention.
[0117] Other methods of nucleic acid detection that may be used in
the practice of the instant invention are disclosed in U.S. Pat.
Nos. 5,840,873, 5,843,640, 5,843,651, 5,846,708, 5,846,717,
5,846,726, 5,846,729, 5,849,487, 5,853,990, 5,853,992, 5,853,993,
5,856,092, 5,861,244, 5,863,732, 5,863,753, 5,866,331, 5,905,024,
5,910,407, 5,912,124, 5,912,145, 5,919,630, 5,925,517, 5,928,862,
5,928,869, 5,929,227, 5,932,413 and 5,935,791, each of which is
incorporated herein by reference.
[0118] Other methods for genetic screening may be used within the
scope of the present invention, for example, to detect mutations in
genomic DNA, cDNA and/or RNA samples. Methods used to detect point
mutations include denaturing gradient gel electrophoresis ("DGGE"),
restriction fragment length polymorphism analysis ("RFLP"),
chemical or enzymatic cleavage methods, direct sequencing of target
regions amplified by PCR.TM. (see above), single-strand
conformation polymorphism analysis ("SSCP") and other methods well
known in the art.
[0119] One method of screening for point mutations is based on
RNase cleavage of base pair mismatches in RNA/DNA or RNA/RNA
heteroduplexes. As used herein, the term "mismatch" is defined as a
region of one or more unpaired or mispaired nucleotides in a
double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This
definition thus includes mismatches due to insertion/deletion
mutations, as well as single or multiple base point mutations.
[0120] U.S. Pat. No. 4,946,773 describes an RNase A mismatch
cleavage assay that involves annealing single-stranded DNA or RNA
test samples to an RNA probe, and subsequent treatment of the
nucleic acid duplexes with RNase A. For the detection of
mismatches, the single-stranded products of the RNase A treatment,
electrophoretically separated according to size, are compared to
similarly treated control duplexes. Samples containing smaller
fragments (cleavage products) not seen in the control duplex are
scored as positive.
[0121] Other investigators have described the use of RNase I in
mismatch assays. The use of RNase I for mismatch detection is
described in literature from Promega Biotech. Promega markets a kit
containing RNase I that is reported to cleave three out of four
known mismatches. Others have described using the MutS protein or
other DNA-repair enzymes for detection of single-base
mismatches.
[0122] Alternative methods for detection of deletion, insertion or
substitution mutations that may be used in the practice of the
present invention are disclosed in U.S. Pat. Nos. 5,849,483,
5,851,770, 5,866,337, 5,925,525 and 5,928,870, each of which is
incorporated herein by reference in its entirety.
[0123] Other methods involve fluorescent in situ hybridization
(FISH), which refers to process that vividly paints chromosomes or
portions of chromosomes with fluorescent molecules. Such techniques
are well known to those of skill in the art (Weier et al., 2002;
Moter et al., 2000; Nath et al., 1998). Another method that may
also be employed involves RNA in situ hybridization (RISH). This
technique may utilize nonradioactive probes such as
digoxigenin-labeled copy RNA (cRNA) probes for the examination of
mRNA expression, and is well known to one of ordinary skill in the
art.
[0124] Reverse transcription (RT) of RNA to cDNA followed by
relative quantitative PCR.TM. (RT-PCR) can be used to determine the
relative concentrations of specific mRNA species isolated from a
cell, such as a PTEN-encoding transcript. By determining that the
concentration of a specific mRNA species varies, it is shown that
the gene encoding the specific mRNA species is differentially
expressed.
[0125] Specifically contemplated are chip-based DNA technologies
such as those described by Hacia et al. (1996) and Shoemaker et al.
(1996). Briefly, these techniques involve quantitative methods for
analyzing large numbers of genes rapidly and accurately. By tagging
genes with oligonucleotides or using fixed probe arrays, one can
employ chip technology to segregate target molecules as high
density arrays and screen these molecules on the basis of
hybridization (see also, Pease et al., 1994; and Fodor et al.,
1991). It is contemplated that this technology may be used in
conjunction with evaluating the expression level of PTEN with
respect to diagnostic methods of the invention.
V. COMBINATION TREATMENTS
[0126] The compounds and methods of the present invention may be
used in the context of cancer and other diseases and conditions
that are caused or aggravated by ErbB2 overexpression. In order to
increase the effectiveness of a treatment with the compositions of
the present invention, such as trastuzumab, it may be desirable to
combine these compositions with other agents effective in the
treatment of those diseases and conditions. For example, the
treatment of a cancer may be implemented with therapeutic compounds
of the present invention and other anti-cancer therapies, such as
anti-cancer agents or surgery.
[0127] Various combinations may be employed; for example, where the
ErbB2 targeting agent is "A" and the secondary anti-cancer therapy
is "B" as follows:
TABLE-US-00001 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B
A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0128] Administration of the ErbB2 targeting agent to a patient
will follow general protocols for the administration of that
particular secondary therapy, taking into account the toxicity, if
any, of the ErbB2 targeting agent. It is expected that the
treatment cycles would be repeated as necessary. It also is
contemplated that various standard therapies, as well as surgical
intervention, may be applied in combination with the described
cancer therapy.
[0129] An "anti-cancer" agent is capable of negatively affecting
cancer in a subject, for example, by killing cancer cells, inducing
apoptosis in cancer cells, reducing the growth rate of cancer
cells, reducing the incidence or number of metastases, reducing
tumor size, inhibiting tumor growth, reducing the blood supply to a
tumor or cancer cells, promoting an immune response against cancer
cells or a tumor, preventing or inhibiting the progression of
cancer, or increasing the lifespan of a subject with cancer.
Anti-cancer agents include biological agents (biotherapy),
chemotherapy agents, and radiotherapy agents. More generally, these
other compositions would be provided in a combined amount effective
to kill or inhibit proliferation of the cell. This process may
involve contacting the cells with an expression construct and the
agent(s) or multiple factor(s) at the same time. This may be
achieved by contacting the cell with a single composition or
pharmacological formulation that includes both agents, or by
contacting the cell with two distinct compositions or formulations,
at the same time, wherein one composition includes an expression
construct and the other includes a second agent(s).
[0130] Tumor cell resistance to chemotherapy and radiotherapy
agents represents a major problem in clinical oncology. One goal of
current cancer research is to find ways to improve the efficacy of
chemo- and radiotherapy by combining it with gene therapy. For
example, the herpes simplex-thymidine kinase (HS-tK) gene, when
delivered to brain tumors by a retroviral vector system,
successfully induced susceptibility to the antiviral agent
ganciclovir (Culver et al., 1992). In the context of the present
invention, it is contemplated that ErbB2 targeting therapy could be
used similarly in conjunction with chemotherapeutic,
radiotherapeutic, immunotherapeutic or other biological
intervention, in addition to other pro-apoptotic or cell cycle
regulating agents.
[0131] Alternatively, the ErbB2 targeting therapy may precede or
follow the other agent treatment by intervals ranging from minutes
to weeks. In embodiments where the other agent and expression
construct are applied separately to the cell, one would generally
ensure that a significant period of time did not expire between the
time of each delivery, such that the agent and expression construct
would still be able to exert an advantageously combined effect on
the cell. In such instances, it is contemplated that one may
contact the cell with both modalities within about 12-24 h of each
other and, more preferably, within about 6-12 h of each other. In
some situations, it may be desirable to extend the time period for
treatment significantly, however, where several days (2, 3, 4, 5, 6
or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations.
[0132] A. Chemotherapy
[0133] Cancer therapies also include a variety of combination
therapies with both chemical and radiation based treatments. The
use of trastuzumab has been employed with paclitaxel, or
anthracyclines and cyclophosphamide. It is contemplated that other
combination chemotherapies include, for example, cisplatin (CDDP),
carboplatin, procarbazine, mechlorethamine, cyclophosphamide,
camptothecin, ifosfamide, melphalan, chlorambucil, busulfan,
nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin,
plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene,
estrogen receptor binding agents, taxol, gemcitabien, navelbine,
farnesyl-protein transferase inhibitors, transplatinum,
5-fluorouracil, vincristine, vinblastine and methotrexate,
Temazolomide (an aqueous form of DTIC), or any analog or derivative
variant of the foregoing. The combination of chemotherapy with
biological therapy is known as biochemotherapy.
[0134] B. Radiation Therapy
[0135] Other factors that cause DNA damage and that have been used
extensively generally for cancer include what are commonly known as
.gamma.-rays, X-rays, and/or the directed delivery of radioisotopes
to tumor cells. Other forms of DNA damaging factors are also
contemplated such as microwaves and UV-irradiation. It is most
likely that all of these factors effect a broad range of damage on
DNA, on the precursors of DNA, on the replication and repair of
DNA, and on the assembly and maintenance of chromosomes. Dosage
ranges for X-rays range from daily doses of 50 to 200 roentgens for
prolonged periods of time (3 to 4 wk), to single doses of 2000 to
6000 roentgens. Dosage ranges for radioisotopes vary widely, and
depend on the half-life of the isotope, the strength and type of
radiation emitted, and the uptake by the neoplastic cells.
[0136] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
construct and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing or stasis, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0137] C. Immunotherapy
[0138] Immunotherapeutics, generally, rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
Trastuzumab is such an example. The immune effector may be, for
example, an antibody specific for some marker on the surface of a
tumor cell. The antibody alone may serve as an effector of therapy
or it may recruit other cells to actually effect cell killing. The
antibody also may be conjugated to a drug or toxin
(chemotherapeutic, radionuclide, ricin A chain, cholera toxin,
pertussis toxin, etc.) and serve merely as a targeting agent.
Alternatively, the effector may be a lymphocyte carrying a surface
molecule that interacts, either directly or indirectly, with a
tumor cell target. Various effector cells include cytotoxic T cells
and NK cells. The combination of therapeutic modalities, i.e.,
direct cytotoxic activity and inhibition or reduction of ErbB2
would provide therapeutic benefit in the treatment of ErbB2
overexpressing cancers.
[0139] Another immunotherapy could also be used as part of a
combined therapy with trastuzumab or another antibody to ErbB2 may
be employed. The general approach for combined therapy is discussed
below. In one aspect of immunotherapy, the tumor cell must bear
some marker that is amenable to targeting, i.e., is not present on
the majority of other cells. Many tumor markers exist and any of
these may be suitable for targeting in the context of the present
invention. Common tumor markers include carcinoembryonic antigen,
prostate specific antigen, urinary tumor associated antigen, fetal
antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis
Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb
B and p155. An alternative aspect of immunotherapy is to combine
anticancer effects with immune stimulatory effects. Immune
stimulating molecules also exist including: cytokines such as IL-2,
IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1,
IL-8 and growth factors such as FLT3 ligand. Combining immune
stimulating molecules, either as proteins or using gene delivery in
combination with a tumor suppressor such as mda-7 has been shown to
enhance anti-tumor effects (Ju et al., 2000).
[0140] As discussed earlier, examples of immunotherapies currently
under investigation or in use are immune adjuvants e.g.,
Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene
and aromatic compounds (U.S. Pat. No. 5,801,005; U.S. Pat. No.
5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998),
cytokine therapy e.g., interferons .alpha., .beta. and .gamma.;
IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998;
Hellstrand et al., 1998) gene therapy e.g., TNF, IL-1, IL-2, p53
(Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. No.
5,830,880 and U.S. Pat. No. 5,846,945) and monoclonal antibodies
e.g., anti-ganglioside GM2, anti-HER-2, anti-p185; Pietras et al.,
1998; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). Herceptin
(trastuzumab) is a chimeric (mouse-human) monoclonal antibody that
blocks the HER2-neu receptor. It possesses anti-tumor activity and
has been approved for use in the treatment of malignant tumors
(Dillman, 1999). Combination therapy of cancer with herceptin and
chemotherapy has been shown to be more effective than the
individual therapies. Thus, it is contemplated that one or more
anti-cancer therapies may be employed with the ErbB2 targeting
therapies described herein.
[0141] A number of different approaches for passive immunotherapy
of cancer exist. They may be broadly categorized into the
following: injection of antibodies alone; injection of antibodies
coupled to toxins or chemotherapeutic agents; injection of
antibodies coupled to radioactive isotopes; injection of
anti-idiotype antibodies; and finally, purging of tumor cells in
bone marrow.
[0142] Preferably, human monoclonal antibodies are employed in
passive immunotherapy, as they produce few or no side effects in
the patient. However, their application is somewhat limited by
their scarcity and have so far only been administered
intralesionally. Human monoclonal antibodies to ganglioside
antigens have been administered intralesionally to patients
suffering from cutaneous recurrent melanoma (Irie and Morton,
1986). Regression was observed in six out of ten patients,
following, daily or weekly, intralesional injections. In another
study, moderate success was achieved from intralesional injections
of two human monoclonal antibodies (Irie et al., 1989).
[0143] It may be favorable to administer more than one monoclonal
antibody directed against two different antigens or even antibodies
with multiple antigen specificity. Treatment protocols also may
include administration of lymphokines or other immune enhancers as
described by Bajorin et al. (1988). The development of human
monoclonal antibodies is described in further detail elsewhere in
the specification.
[0144] In active immunotherapy, an antigenic peptide, polypeptide
or protein, or an autologous or allogenic tumor cell composition or
"vaccine" is administered, generally with a distinct bacterial
adjuvant (Ravindranath and Morton, 1991; Morton et al., 1992;
Mitchell et al., 1990; Mitchell et al., 1993). In melanoma
immunotherapy, those patients who elicit high IgM response often
survive better than those who elicit no or low IgM antibodies
(Morton et al., 1992). IgM antibodies are often transient
antibodies and the exception to the rule appears to be
anti-ganglioside or anticarbohydrate antibodies.
[0145] In adoptive immunotherapy, the patient's circulating
lymphocytes, or tumor infiltrated lymphocytes, are isolated in
vitro, activated by lymphokines such as IL-2 or transduced with
genes for tumor necrosis, and readministered (Rosenberg et al.,
1988; 1989). To achieve this, one would administer to an animal, or
human patient, an immunologically effective amount of activated
lymphocytes in combination with an adjuvant-incorporated anigenic
peptide composition as described herein. The activated lymphocytes
will most preferably be the patient's own cells that were earlier
isolated from a blood or tumor sample and activated (or "expanded")
in vitro. This form of immunotherapy has produced several cases of
regression of melanoma and renal carcinoma, but the percentage of
responders were few compared to those who did not respond.
[0146] D. Gene Therapy
[0147] In yet another embodiment, the secondary treatment is a gene
therapy in which a therapeutic polynucleotide is administered
before, after, or at the same time as an ErbB2 targeting agent is
administered. Delivery of an ErbB2 targeting agent in conjunction
with a vector encoding one of the following gene products may have
a combined anti-hyperproliferative effect on target tissues. A
variety of proteins are encompassed within the invention, some of
which are described below. Table 1 lists various genes that may be
targeted for gene therapy of some form in combination with the
present invention.
[0148] 1. Inducers of Cellular Proliferation
[0149] The proteins that induce cellular proliferation further fall
into various categories dependent on function. The commonality of
all of these proteins is their ability to regulate cellular
proliferation. For example, a form of PDGF, the sis oncogene, is a
secreted growth factor. Oncogenes rarely arise from genes encoding
growth factors, and at the present, sis is the only known
naturally-occurring oncogenic growth factor. In one embodiment of
the present invention, it is contemplated that anti-sense mRNA or
siRNA directed to a particular inducer of cellular proliferation is
used to prevent expression of the inducer of cellular
proliferation.
[0150] The proteins FMS and ErbA are growth factor receptors, like
ErbB. Mutations to these receptors result in loss of regulatable
function. For example, a point mutation affecting the transmembrane
domain of the Neu receptor protein results in the neu oncogene. The
erbA oncogene is derived from the intracellular receptor for
thyroid hormone. The modified oncogenic ErbA receptor is believed
to compete with the endogenous thyroid hormone receptor, causing
uncontrolled growth.
[0151] The largest class of oncogenes includes the signal
transducing proteins (e.g., Src, Abl and Ras). The protein Src is a
cytoplasmic protein-tyrosine kinase, and its transformation from
proto-oncogene to oncogene in some cases, results via mutations at
tyrosine residue 527. In contrast, transformation of GTPase protein
ras from proto-oncogene to oncogene, in one example, results from a
valine to glycine mutation at amino acid 12 in the sequence,
reducing ras GTPase activity.
[0152] The proteins Jun, Fos and Myc are proteins that directly
exert their effects on nuclear functions as transcription
factors.
[0153] 2. Inhibitors of Cellular Proliferation
[0154] The tumor suppressor oncogenes function to inhibit excessive
cellular proliferation. The inactivation of these genes destroys
their inhibitory activity, resulting in unregulated proliferation.
The tumor suppressors p53, mda-7, FHIT, p16 and C-CAM can be
employed.
[0155] In addition to p53, another inhibitor of cellular
proliferation is p16. The major transitions of the eukaryotic cell
cycle are triggered by cyclin-dependent kinases, or CDK's. One CDK,
cyclin-dependent kinase 4 (CDK4), regulates progression through the
G.sub.1. The activity of this enzyme may be to phosphorylate Rb at
late G.sub.1. The activity of CDK4 is controlled by an activating
subunit, D-type cyclin, and by an inhibitory subunit, the
p16.sup.INK4 has been biochemically characterized as a protein that
specifically binds to and inhibits CDK4, and thus may regulate Rb
phosphorylation (Serrano et al., 1993; Serrano et al., 1995). Since
the p16.sup.INK4 protein is a CDK4 inhibitor (Serrano, 1993),
deletion of this gene may increase the activity of CDK4, resulting
in hyperphosphorylation of the Rb protein. p16 also is known to
regulate the function of CDK6.
[0156] p16.sup.INK4 belongs to a newly described class of
CDK-inhibitory proteins that also includes p16.sup.B, p19,
p21.sup.WAF1, and p27.sup.KIP1. The p16.sup.INK4 gene maps to 9p21,
a chromosome region frequently deleted in many tumor types.
Homozygous deletions and mutations of the p16.sup.INK4 gene are
frequent in human tumor cell lines. This evidence suggests that the
p16.sup.INK4 gene is a tumor suppressor gene. This interpretation
has been challenged, however, by the observation that the frequency
of the p16.sup.INK4 gene alterations is much lower in primary
uncultured tumors than in cultured cell lines (Caldas et al., 1994;
Cheng et al., 1994; Hussussian et al., 1994; Kamb et al., 1994;
Kamb et al., 1994; Mori et al., 1994; Okamoto et al., 1994; Nobori
et al., 1995; Orlow et al., 1994; Arap et al., 1995). Restoration
of wild-type p16.sup.INK4 function by transfection with a plasmid
expression vector reduced colony formation by some human cancer
cell lines (Okamoto, 1994; Arap, 1995).
[0157] Other genes that may be employed according to the present
invention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II,
zac1, p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16
fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1,
TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp,
hst, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF,
FGF, thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.
[0158] 3. Regulators of Programmed Cell Death
[0159] Apoptosis, or programmed cell death, is an essential process
for normal embryonic development, maintaining homeostasis in adult
tissues, and suppressing carcinogenesis (Kerr et al., 1972). The
Bcl-2 family of proteins and ICE-like proteases have been
demonstrated to be important regulators and effectors of apoptosis
in other systems. The Bcl-2 protein, discovered in association with
follicular lymphoma, plays a prominent role in controlling
apoptosis and enhancing cell survival in response to diverse
apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985;
Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce,
1986). The evolutionarily conserved Bcl-2 protein now is recognized
to be a member of a family of related proteins, which can be
categorized as death agonists or death antagonists.
[0160] Subsequent to its discovery, it was shown that Bcl-2 acts to
suppress cell death triggered by a variety of stimuli. Also, it now
is apparent that there is a family of Bcl-2 cell death regulatory
proteins which share in common structural and sequence homologies.
These different family members have been shown to either possess
similar functions to Bcl-2 (e.g., Bcl.sub.XL, Bcl.sub.W, Bcl.sub.S,
Mcl-1, A1, Bfl-1) or counteract Bcl-2 function and promote cell
death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
[0161] E. Surgery
[0162] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0163] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
microscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present invention may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0164] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0165] F. Other Agents
[0166] It is contemplated that other agents may be used in
combination with the present invention to improve the therapeutic
efficacy of treatment. These additional agents include
immunomodulatory agents, agents that affect the upregulation of
cell surface receptors and GAP junctions, cytostatic and
differentiation agents, inhibitors of cell adhesion, agents that
increase the sensitivity of the hyperproliferative cells to
apoptotic inducers, or other biological agents. Immunomodulatory
agents include tumor necrosis factor; interferon alpha, beta, and
gamma; IL-2 and other cytokines; F42K and other cytokine analogs;
or MIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines. It is
further contemplated that the upregulation of cell surface
receptors or their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL
(Apo-2 ligand) would potentiate the apoptotic inducing abilities of
the present invention by establishment of an autocrine or paracrine
effect on hyperproliferative cells. Increases intercellular
signaling by elevating the number of GAP junctions would increase
the anti-hyperproliferative effects on the neighboring
hyperproliferative cell population. In other embodiments,
cytostatic or differentiation agents can be used in combination
with the present invention to improve the anti-hyerproliferative
efficacy of the treatments. Inhibitors of cell adhesion are
contemplated to improve the efficacy of the present invention.
Examples of cell adhesion inhibitors are focal adhesion kinase
(FAKs) inhibitors and Lovastatin. It is further contemplated that
other agents that increase the sensitivity of a hyperproliferative
cell to apoptosis, such as the antibody c225, could be used in
combination with the present invention to improve the treatment
efficacy.
[0167] Apo2 ligand (Apo2L, also called TRAIL) is a member of the
tumor necrosis factor (TNF) cytokine family. TRAIL activates rapid
apoptosis in many types of cancer cells, yet is not toxic to normal
cells. TRAIL mRNA occurs in a wide variety of tissues. Most normal
cells appear to be resistant to TRAIL's cytotoxic action,
suggesting the existence of mechanisms that can protect against
apoptosis induction by TRAIL. The first receptor described for
TRAIL, called death receptor 4 (DR4), contains a cytoplasmic "death
domain"; DR4 transmits the apoptosis signal carried by TRAIL.
Additional receptors have been identified that bind to TRAIL. One
receptor, called DR5, contains a cytoplasmic death domain and
signals apoptosis much like DR4. The DR4 and DR5 mRNAs are
expressed in many normal tissues and tumor cell lines. Recently,
decoy receptors such as DcR1 and DcR2 have been identified that
prevent TRAIL from inducing apoptosis through DR4 and DR5. These
decoy receptors thus represent a novel mechanism for regulating
sensitivity to a pro-apoptotic cytokine directly at the cell's
surface. The preferential expression of these inhibitory receptors
in normal tissues suggests that TRAIL may be useful as an
anticancer agent that induces apoptosis in cancer cells while
sparing normal cells. (Marsters et al., 1999).
[0168] There have been many advances in the therapy of cancer
following the introduction of cytotoxic chemotherapeutic drugs.
However, one of the consequences of chemotherapy is the
development/acquisition of drug-resistant phenotypes and the
development of multiple drug resistance. The development of drug
resistance remains a major obstacle in the treatment of such tumors
and therefore, there is an obvious need for alternative approaches
such as gene therapy.
[0169] Another form of therapy for use in conjunction with
chemotherapy, radiation therapy or biological therapy includes
hyperthermia, which is a procedure in which a patient's tissue is
exposed to high temperatures (up to 106.degree. F.). External or
internal heating devices may be involved in the application of
local, regional, or whole-body hyperthermia. Local hyperthermia
involves the application of heat to a small area, such as a tumor.
Heat may be generated externally with high-frequency waves
targeting a tumor from a device outside the body. Internal heat may
involve a sterile probe, including thin, heated wires or hollow
tubes filled with warm water, implanted microwave antennae, or
radiofrequency electrodes.
[0170] A patient's organ or a limb is heated for regional therapy,
which is accomplished using devices that produce high energy, such
as magnets. Alternatively, some of the patient's blood may be
removed and heated before being perfused into an area that will be
internally heated. Whole-body heating may also be implemented in
cases where cancer has spread throughout the body. Warm-water
blankets, hot wax, inductive coils, and thermal chambers may be
used for this purpose.
[0171] Hormonal therapy may also be used in conjunction with the
present invention or in combination with any other cancer therapy
previously described. The use of hormones may be employed in the
treatment of certain cancers such as breast, prostate, ovarian, or
cervical cancer to lower the level or block the effects of certain
hormones such as testosterone or estrogen. This treatment is often
used in combination with at least one other cancer therapy as a
treatment option or to reduce the risk of metastases.
TABLE-US-00002 TABLE 1 Oncogenes Gene Source Human Disease Function
Growth Factors HST/KS Transfection FGF family member INT-2 MMTV
promoter FGF family member Insertion INTI/WNTI MMTV promoter
Factor-like Insertion SIS Simian sarcoma virus PDGF B Receptor
Tyrosine Kinases ERBB/HER Avian erythroblastosis Amplified, deleted
EGF/TGF-.alpha./ Virus; ALV promoter Squamous cell Amphiregulin/
Insertion; amplified Cancer; glioblastoma Hetacellulin receptor
Human tumors ERBB-2/NEU/HER-2 Transfected from rat Amplified
breast, Regulated by NDF/ Glioblastomas Ovarian, gastric Heregulin
and EGF- cancers Related factors FMS SM feline sarcoma virus CSF-1
receptor KIT HZ feline sarcoma virus MGF/Steel receptor
Hematopoieis TRK Transfection from NGF (nerve growth Human colon
cancer Factor) receptor MET Transfection from Scatter factor/HGF
Human osteosarcoma Receptor RET Translocations and point Sporadic
thyroid cancer; Orphan receptor Tyr mutations familial medullary
Kinase thyroid cancer; multiple endocrine neoplasias 2A and 2B ROS
URII avian sarcoma Orphan receptor Tyr Virus Kinase PDGF receptor
Translocation Chronic TEL(ETS-like Myelomonocytic transcription
factor)/ Leukemia PDGF receptor gene Fusion TGF-.beta. receptor
Colon carcinoma mismatch mutation target NONRECEPTOR TYROSINE
KINASES ABI. Abelson Mul. V Chronic myelogenous Interact with RB,
RNA leukemia translocation polymerase, CRK, with BCR CBL FPS/FES
Avian Fujinami SV; GA FeSV LCK Mul. V (murine leukemia Src family;
T cell Virus) promoter signaling; interacts Insertion CD4/CD8 T
cells SRC Avian Rous sarcoma Membrane-associated Virus Tyr kinase
with signaling function; activated by receptor kinases YES Avian
Y73 virus Src family; signaling SER/THR PROTEIN KINASES AKT AKT8
murine retrovirus Regulated by PI(3)K?; regulate 70-kd S6 k? MOS
Maloney murine SV GVBD; cystostatic factor; MAP kinase kinase PIM-1
Promoter insertion Mouse RAF/MIL 3611 murine SV; MH2 Signaling in
RAS avian SV Pathway MISCELLANEOUS CELL SURFACE APC Tumor
suppressor Colon cancer Interacts with catenins DCC Tumor
suppressor Colon cancer CAM domains E-cadherin Candidate tumor
Breast cancer Extracellular homotypic Suppressor binding;
intracellular interacts with catenins PTC/NBCCS Tumor suppressor
and Nevoid basal cell cancer 12 transmembrane Drosophilia homology
syndrome (Gorline domain; signals syndrome) through Gli homogue CI
to antagonize hedgehog pathway TAN-1 Notch Translocation T-ALI.
Signaling homologue MISCELLANEOUS SIGNALING BCL-2 Translocation
B-cell lymphoma Apoptosis CBL Mu Cas NS-1 V Tyrosine-
Phosphorylated RING finger interact Abl CRK CT1010 ASV Adapted
SH2/SH3 interact Abl DPC4 Tumor suppressor Pancreatic cancer
TGF-.beta.-related signaling Pathway MAS Transfection and Possible
angiotensin Tumorigenicity Receptor NCK Adaptor SH2/SH3 GUANINE
NUCLEOTIDE EXCHANGERS AND BINDING PROTEINS BCR Translocated with
ABL Exchanger; protein in CML Kinase DBL Transfection Exchanger GSP
NF-1 Hereditary tumor Tumor suppressor RAS GAP Suppressor
neurofibromatosis OST Transfection Exchanger Harvey-Kirsten, N-RAS
HaRat SV; Ki RaSV; Point mutations in many Signal cascade
Balb-MoMuSV; human tumors Transfection VAV Transfection S112/S113;
exchanger NUCLEAR PROTEINS AND TRANSCRIPTION FACTORS BRCA1
Heritable suppressor Mammary Localization unsettled cancer/ovarian
cancer BRCA2 Heritable suppressor Mammary cancer Function unknown
ERBA Avian erythroblastosis Thyroid hormone Virus receptor
(transcription) ETS Avian E26 virus DNA binding EVII MuLV promotor
AML Transcription factor Insertion FOS FBI/FBR murine Transcription
factor osteosarcoma viruses with c-JUN GLI Amplified glioma Glioma
Zinc finger; cubitus interruptus homologue is in hedgehog signaling
pathway; inhibitory link PTC and hedgehog HMGI/LIM Translocation
t(3:12) Lipoma Gene fusions high t(12:15) mobility group HMGI-C
(XT-hook) and transcription factor LIM or acidic domain JUN ASV-17
Transcription factor AP-1 with FOS MLL/VHRX + ELI/MEN
Translocation/fusion Acute myeloid leukemia Gene fusion of DNA- ELL
with MLL binding and methyl Trithorax-like gene transferase MLL
with ELI RNA pol II elongation factor MYB Avian myeloblastosis DNA
binding Virus MYC Avian MC29; Burkitt's lymphoma DNA binding with
Translocation B-cell MAX partner; cyclin Lymphomas; promoter
regulation; interact Insertion avian RB?; regulate leukosis
apoptosis? Virus N-MYC Amplified Neuroblastoma L-MYC Lung cancer
REL Avian NF-.kappa.B family Retriculoendotheliosis transcription
factor Virus SKI Avian SKV770 Transcription factor Retrovirus VHL
Heritable suppressor Von Hippel-Landau Negative regulator or
syndrome elongin; transcriptional elongation complex WT-1 Wilm's
tumor Transcription factor CELL CYCLE/DNA DAMAGE RESPONSE ATM
Hereditary disorder Ataxia-telangiectasia Protein/lipid kinase
homology; DNA damage response upstream in P53 pathway BCL-2
Translocation Follicular lymphoma Apoptosis FACC Point mutation
Fanconi's anemia group C (predisposition leukemia FHIT Fragile site
3p14.2 Lung carcinoma Histidine triad-related diadenosine 5',3''''-
P.sup.1.p.sup.4 tetraphosphate asymmetric hydrolase hMLI/MutL HNPCC
Mismatch repair; MutL Homologue HMSH2/MutS HNPCC Mismatch repair;
MutS Homologue HPMS1 HNPCC Mismatch repair; MutL Homologue hPMS2
HNPCC Mismatch repair; MutL Homologue INK4/MTS1 Adjacent INK-4B at
Candidate MTS1 p16 CDK inhibitor 9p21; CDK complexes suppressor and
MLM melanoma gene INK4B/MTS2 Candidate suppressor p15 CDK inhibitor
MDM-2 Amplified Sarcoma Negative regulator p53 p53 Association with
SV40 Mutated >50% human Transcription factor; T antigen tumors,
including checkpoint control; hereditary Li-Fraumeni apoptosis
syndrome PRAD1/BCL1 Translocation with Parathyroid adenoma; Cyclin
D Parathyroid hormone B-CLL or IgG RB Hereditary Retinoblastoma;
Interact cyclin/cdk; Retinoblastoma; osteosarcoma; breast regulate
E2F Association with many cancer; other sporadic transcription
factor DNA virus tumor cancers Antigens XPA xeroderma Excision
repair; photo- pigmentosum; skin product recognition; cancer
predisposition zinc finger
VI. THERAPEUTIC USES: FORMULATIONS AND ROUTES OF ADMINISTRATION
[0172] ErbB-2 is overexpressed in a number of cancer types
including those involving the female genital tract (e.g.,
endometrial cancer), gastric cancer and prostate cancer. A
preliminary clinical study using a variant of Herceptin.TM. showed
some improvement in patients with prostate and kidney cancer.
However, a primary target for ErbB-2 targeted therapies is breast
cancer, of which 20-30% show overexpression of this marker. Thus,
in accordance with the present invention, there are provided
therapeutic methods designed to intervene in such ErbB-2 related
cancers. These therapies may facilitate tumor growth inhibition,
reduction in tumor size, induction of apoptosis in tumor cells,
inhibition or reduction in metastasis formation, or otherwise
result in an improvement in the clinical situation of a cancer
patient, including improving one or more symptoms of cancer.
[0173] Trastuzumab is commercially available as HERCEPTIN from
Genentech, which provides it in a vial as a sterile, white to pale
yellow, preservative-free lyophilized powder for intravenous (IV)
administration. Each vial has 440 mg Trastuzumab, 9.9 mg
L-histidine HCl, 6.4 mg L-histidine, 400 mg trehalose dihydrate,
and 1.8 mg polysorbate 20, USP, which is a pharmaceutically
acceptable formulation.
[0174] The number of mg of Herceptin needed can be determined based
on a loading dose of 4 mg/kg body weight, or a maintenance dose of
2 mg/kg body weight. The weekly maintenance dose can be continued
until disease progression. For outpatient administration, patients
are given a 4-mg/kg loading dose as an infusion over 90 minutes. A
2-mg/kg weekly maintenance dose can be administered as a 30-minute
infusion if the prior dose was well tolerated.
[0175] The phrases "pharmaceutically or pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce an adverse, allergic or other untoward reaction when
administered to a human. As used herein, "pharmaceutically
acceptable carrier" includes any and all solvents, dispersion
media, antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions. The compositions will be
sterile, be fluid to the extent that easy syringability exists,
stable under the conditions of manufacture and storage.
[0176] For non-Herceptin ErbB2 targeting agents, the actual dosage
amount of a composition of the present invention administered to a
patient can be determined by physical and physiological factors
such as body weight, severity of condition, idiopathy of the
patient and on the route of administration. With these
considerations in mind, the dosage of a lipid composition for a
particular subject and/or course of treatment can readily be
determined.
[0177] Although it is most preferred that compositions of the
present invention be prepared in sterile water containing other
non-active ingredients, made suitable for injection, solutions of
such active ingredients can also be prepared in water suitably
mixed with a surfactant, such as hydroxypropylcellulose, if
desired. Dispersions can also be prepared in liquid polyethylene
glycols, and mixtures thereof and in oils. The carrier can also be
a solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, propylene glycol, and liquid
polyethylene glycol, and the like), suitable mixtures thereof, and
vegetable oils. The proper fluidity can be maintained, for example,
by the use of a coating, such as lecithin, by the maintenance of
the required particle size in the case of dispersion and by the use
of surfactants.
[0178] The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0179] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. For parenteral administration in an
aqueous solution, for example, the solution should be suitably
buffered if necessary and the liquid diluent first rendered
isotonic with sufficient saline or glucose. These particular
aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal administration. In
this connection, sterile aqueous media which can be employed will
be known to those of skill in the art in light of the present
disclosure. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject.
[0180] The compositions of the present invention can be
administered intravenously, intradermally, intraarterially,
intraperitoneally, intralesionally, intracranially,
intraarticularly, intraprostaticaly, intrapleurally,
intratracheally, intranasally, intravitreally, intravaginally,
rectally, topically, intratumorally, intramuscularly,
intraperitoneally, subcutaneously, intravesicularlly, mucosally,
intrapericardially, orally, topically, locally using aerosol,
injection, infusion, continuous infusion, localized perfusion
bathing target cells directly or via a catheter or lavage. In
particular, the invention may provide local, regional or systemic
administration with respect to the tumor location. Typically, such
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for preparing
solutions or suspensions upon the addition of a liquid prior to
injection can also be prepared; and the preparations can also be
emulsified.
VII. KITS
[0181] Any of the compositions described herein may be comprised in
a kit. In a non-limiting example, one or more reagents for
evaluating PTEN expression can be provided in a kit, alone or in
combination with reagents for evaluating whether a cancer is an
ErbB2-overexpressing cancer. The kits may thus comprise, in
suitable container means, nucleic acids, antibodies, or other
polypeptides that can be used to determine PTEN and/or ErbB2
expression in a sample. Such reagents that can be included in kits
of the invention are discussed herein. In some embodiments, the
reagents are attached or fixed to a support, such as a plate, chip
or other non-reactive substance. For example, a reagent can be
fixed to a microtiter well, and the sample placed in the well to
determine the expression level of PTEN and/or ErbB2.
[0182] The kits may comprise a suitably aliquoted nucleic acids
that can be used as probes or primers; alternatively, it may
comprise a suitably aliquoted antibody that can be used in
immunohistochemical detection methods or any other method discussed
herein or known to those of skill in the art.
[0183] The components of the kits may be packaged either in aqueous
media or in lyophilized form. The container means of the kits will
generally include at least one vial, test tube, flask, bottle,
syringe or other container means, into which a component may be
placed, and preferably, suitably aliquoted. Where there is more
than one component in the kit, the kit also will generally contain
a second, third or other additional container into which the
additional components may be separately placed. However, various
combinations of components may be comprised in a vial. The kits of
the present invention also will typically include a means for
containing the containers in close confinement for commercial sale.
Such means may include injection or blow-molded plastic containers
into which the desired vials are retained.
VIII. EXAMPLES
[0184] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Experimental Procedures
[0185] Antibodies and reagents. PTEN antibodies were from Santa
Cruz Biotechnology (A2B1, Santa Cruz, Calif.) and Lab Vision Corp.
(Ab-2, Fremont, Calif.), antibodies to Akt, phospho-Akt and
Y416-phosphorylated Src were from Cell Signaling Technology
(Beverly, Mass.), antibodies to ErbB2 and v-Src (Ab-1) were from
Calbiochem (La Jolla, Calif.), antibodies to the 85-kDa subunit of
PI3K, phosho-ErbB2 and phosph-tyrosine (4G10) were from Upstate
Biotechnology Inc. (Waltham, Mass.); antibody to phospho-tyrosine
(PY20) was from BD Biosciences (San Diego, Calif.); and trastuzumab
from Genentech Inc. (South San Francisco, Calif.). LY294002 was
from Cell Signaling Technology, Wortmannin, PP2, and AG1879 were
from Calbiochem; Enolase was from Sigma (St. Louis, Mo.); and Taxol
was from Bristol Myers-Squibb (Princeton, N.J.). Src/CA mutant
expression vector was provided by Dr. David Shalloway (Cornell
University).
[0186] Cell culture. SKBr3 cells were from the American Type
Culture Collection (Manassas, Va.) and maintained in McCoy's 5A
containing 15% fetal bovine serum (FBS), BT474 sub-line was
maintained in DMEM/F12 with 10% FBS. Both lines have the wild-type
PTEN gene (Li et al., 1997).
[0187] PI3K Activity assay. PI3K activities were determined in the
PY20 antibody immunoprecipitates of untreated or
trastuzumab-treated lysates as done previously (Tan et al.,
1999).
[0188] Src kinase assay. Equal amounts of cell lysates were
immunoprecipitated with anti-Src antibody (OP07, Oncogene Tesearch
Products, Boston, Mass.). The precipitates were washed and
incubated with 250 .mu.g/ml enolase, 2 .mu.Ci of
[.gamma..sup.32P]ATP, and 3 .mu.l of 100 mM ATP in 40 .mu.l of
kinase buffer. After 30 min at 37.degree. C., the samples were
boiled in 15 .mu.l of 6.times.SDS sample buffer for 5 min and then
subjected to SDS-PAGE. The .sup.32P-labeled enolase was visualized
by autoradiography.
[0189] Antisense Delivery in vitro. Antisense oligonucleotides
against PTEN (ISIS 116847: 5'-CTGCTAGCCTCTGGATTTGA-3') (SEQ ID
NO:3) and control oligonucleotides (ISIS116848:
5'-CTTCTGGCATCCGGTTTAGA-3' (SEQ ID NO:4), with six base mismatches
to ISIS116847) have been described (Butler et al., 2002). Cells
were plated in 100-mm dishes at 30% confluence and transfected with
oligonucleotides (12.5 nM for BT474 and 25 nM for SKBr3 cells)
twice using Oligofectamine (Life Technologies, Rockville, Md.) 24
and 72 h post-plating. Ninety-six hours post-plating, cells were
re-plated for individual assays described in this report. PTEN
expression was determined 120 h post-plating (FIG. 2A).
[0190] Cell viability assay. Cells were seeded at
1-5.times.10.sup.3 cells/well in 96-well plates after
oligonucleotide transfection. Viable cells were determined by
CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay
(Promega, Madison, Wis.).
[0191] Preparation of cell lysates, immunoprecipitation, and
Western blot analysis. Cells at 70%-80% confluence were treated
with each reagent indicated in the individual experiments. For PTEN
phosphotyrosine immunoblots, ErbB2 IP, and Src IP, cells were
treated with 30 mM pervanadate for 10 min as described (Lu et al.,
2003). Unless noted, cells were washed with PBS and lysed in IP
buffer with protease inhibitor cocktail (Sigma Chemical Co., St.
Louis, Mo.). Immunoprecipitation and Western blot analysis were
performed (Yu et al., 1998).
[0192] PTEN phosphatase assay. PTEN phosphatase activity was
measured as described with minor modifications (Georgescu et al.,
1999). Each assay was performed in 50-.mu.l buffer containing 100
mM Tris-HCl (pH 8.0), 10 mM DTT, and 100 .mu.M water-soluble
diC8-PIP.sub.3 (Echelon, Salt Lake City, Utah). Reactions contained
PTEN immunoprecipitated from cell lysates on protein G-agarose
beads. Prior to immunoprecipitation with PTEN antibody (Ab-2),
lysates were precleared with protein G-agarose beads to eliminate
trastuzumab used for treatment. After immunoprecipitation, the
beads were washed once in lysis buffer; five times in
low-stringency buffer containing 20 mM Hepes (pH 7.7), 50 mM NaCl,
0.1 mM EDTA, and 2.5 mM MgCl.sub.2; and once in phosphatase assay
buffer lacking PIP.sub.3. Reactions were incubated for 40 min at
37.degree. C. and transferred to a 96-well plate. Release of
phosphate from substrate was measured using Biomol Green Reagent
(Biomol Research Laboratories, Inc., Plymouth Meeting, Pa.).
[0193] Separation of membrane-bound and cytosolic proteins. Cells
were lysed in saponin buffer (0.01% saponin, 10 mM Tris-HCL at pH
7.4, 140 mM NaCl, 5 mM EDTA, 2 mM EGTA, 0.2 mM sodium vanadate, 50
mM NaF, 25 mM .beta.-glycerophosphate) containing protease
inhibitor cocktail (Sigma). Proteins were extracted for 20 min on
ice, and samples centrifuged at 14,000 g for 30 min at 4.degree. C.
The saponin soluble pool (cytosolic pool) was collected (Palka and
Green, 1997). The remaining pellet was rinsed with saponin buffer
once, extracted in IP buffer, and centrifuged to remove insoluble
materials. The supernatant was collected (membrane-bound pool).
[0194] Analysis of internalization using flow cytometry.
Immunofluorescent flow cytometry was performed as described (Drebin
et al., 1985; van Leeuwen et al., 1990) using anti-ErbB2 (Ab5,
Calbiochem) as the primary antibody.
[0195] BrdU incorporation assay. S-phase cells were detected using
5-bromo-2'-deoxyuridine (BrdUrd) Labeling and Detection Kit I
(Roche Molecular Biochemicals, Indianapolis, Ind.).
[0196] Immunofluorescence staining. It was performed as described
in (Tan et al., 2002).
[0197] Animal experiment. Female athymic nude mice (Harlan Sprague
Dawley, Indianapolis, Ind.) were implanted with 0.72-mg
60-day-release 17.beta.-estradiol pellets (Innovative Research,
Sarasota, Fla.) and irradiated at 3.5 Gy prior to inoculation.
BT474 cells (8.times.10.sup.6) in 200 .mu.l of PBS and Matrigel (BD
Biosciences, Bedford, Mass.) mixture (1:1 ratio) were injected
subcutaneously into a mouse mammary fat pad (mfp). When the mfp
tumor volume reached >150 mm.sup.3, the tumor was treated with
PTEN antisense or control mismatched oligonucleotides (30
.mu.g/week) in saline. One week later, mice received trastuzumab
(10 mg/kg) or vehicle (PBS) twice/week, combined with LY294002 (100
mg/kg) or vehicle (DMSO) three times/week. Tumor diameters were
serially measured with calipers and tumor volumes calculated:
volume=width.sup.2.times.length/2. Each treatment group contained
8-12 mice. Unpaired Student's t-test was used to assess statistical
significance.
[0198] Patient samples. Forty-seven ErbB2-overexpressing primary
breast carcinomas were collected from patients who subsequently
developed metastatic breast cancer and received trastuzumab plus
taxane chemotherapy (paclitaxel or docetaxel) (Esteva et al., 2002;
Seidman et al., 2001). Primary breast carcinomas not selected for
ErbB2-overexpressing tumors from 37 patients who subsequently
developed metastatic breast cancer and received taxane without
trastuzumab were collected as controls (Holmes et al., 1991; Valero
et al., 1995). The 47 patients were treated between 1998-2001 and
the 37 patients were treated between 1990 and 1991 under
IRB-approved clinical trials at The University of Texas M.D.
Anderson Cancer Center. Association between clinical response to
therapy and PTEN status was tested using chi-square and Fisher's
tests.
[0199] PTEN Immunohistochemistry. Slides of formalin-fixed,
paraffin-embedded tissue sections (4 .mu.m) were incubated with
primary antibody (1:500) against PTEN (Podsypanina et al. 2001)
(Ab-2, Lab Vision Corp.). Immunodetection was performed with LSAB2
kit (DAKO, Carpinteria, Calif.), color development with
3-3'-diaminobenzidine, and counterstaining with hematoxylin. PTEN
expression level was scored semiquantitatively based on staining
intensity and distribution using the immunoreactive score (IRS) as
described (Chui et al., 1996; Fredrichs et al., 1993) and as
following: IRS=SI (staining intensity).times.PP (percentage of
positive cells). SI was determined as 0=negative; 1=weak;
2=moderate; and 3=strong. PP was defined as 0, <1%; 1, 1%-10%;
2, 11%-50%; 3, 51%-80%; and 4, >80% positive cells. Ten visual
fields from different areas of each tumor were used for the IRS
evaluation. Negative control slides without primary antibody were
included for each staining Normal breast epithelium or vascular
endothelium knoen to express normal PTEN was used as positive
controls.
[0200] ErbB2 status. ErbB2 gene amplification in patients' samples
was determined by fluorescence in situ hybridization (FISH) using
PathVysion FISH assay (Vysis, Inc., Downers Grove, Ill.). ErbB2
protein overexpression was determined by IHC as previously
described (Jacobs et al., 1999).
Example 2
Trastuzumab Activates PTEN in Breast Cancer Cells
[0201] In our initial investigation of the mechanisms of
trastuzumab's anti-tumor function, the inventors observed that
>20 h trastuzumab treatment of BT474 and SKBr3 human breast
cancer cell lines overexpressing endogenous ErbB2 led to ErbB2
downregulation, dephosphorylation, and receptor internalization as
expected (Lee et al., 2002). However, 1 h trastuzumab treatment
(2-10 .mu.g/ml) did not induce ErbB2 downregulation,
dephosphorylation, and receptor internalization (FIGS. 1A and 2A
and 2B). Surprisingly, 1 h trastuzumab treatment quickly reduced
the level of phosphorylated and activated Akt in BT474 and SKBr3
cells when the Akt protein level remained unchanged (FIG. 1A). It
is known that Akt is constitutively activated in
ErbB2-overexpressing cancer cells and its inhibition is critical
for trastuzumab's anti-tumor effect (Ignatoski et al., 2000; Yakes
et al., 2002; Zhou et al., 2000). Thus, mechanisms of the rapid Akt
dephosphorylation by trastuzumab before ErbB2 downregulation,
dephosphorylation, and internalization were investigated. It was
determined whether trastuzumab induced rapid Akt dephosphorylation
by inhibition of PI3K activation which is the major upstream
signaling event leading to Akt phosphorylation (Cantley, 2002).
PI3K activity was not inhibited within 1 h of trastuzumab treatment
(FIG. 1B). Contrarily, the p85 subunit of PI3K had a transiently
increased association with the ErbB2 complex (FIG. 1C), which
paralleled a moderate increase of ErbB2 tyrosine phosphorylation by
short0time trastuzumab treatment (FIG. 1A). The apparently
paradoxical results indicated that the rapid Akt dephosphorylation
by 1 h trastuzumab treatment was not due to PI3K inhibition, nor by
ErbB2 degradation, dephosphorylation, and internalization, but by
some other mechanisms.
[0202] The tumor suppressor PTEN is a dual-phosphatase that
negatively regulates Akt activity (Cantley and Neel, 1999; Di
Cristofano and Pandolfi, 2000; Leslie and Downes, 2002). Thus, it
was determined whether PTEN may be involved in the
trastuzumab-mediated rapid Akt dephosphorylation before PI3K
inhibition. The phosphatase activity of PTEN was examined in
untreated and trastuzumab-treated (20 min or 60 min) SKBr3 cells
after immunoprecipitation of a similar amount of PTEN (FIG. 1D,
insert). Compared with untreated cells, PTEN activity dramatically
increased in cells after 20 min trastuzumab treatment, which
continued to increase 60 min after treatment (FIG. 1D). Therefore,
trastuzumab treatment indeed led to a rapid increase of PTEN
phosphatase activity that could account for the rapid Akt
dephosphorylation before PI3K is inhibited by trastuzumab.
[0203] Membrane-associated PTEN is known to be biologically active
in cells (Comer and Parent, 2002; Iijima and Devreotes, 2002; Wu et
al., 2000). To investigate if PTEN activation by trastuzumab is due
to recruitment of PTEN to its biologically active sub-cellular
location, it was examined whether membrane localization of PTEN is
increased by trastuzumab treatment. After treating BT474 cells with
or without trastuzumab for 20 min, membrane-bound proteins were
separated from cytosolic proteins in cell lysates and PTEN proteins
examined by Western blotting (FIG. 1E). In the untreated cells, the
majority of PTEN protein was in the cytosolic pool of the lysates,
while a very low level of PTEN was detected in the membrane-bound
pool. However, upon trastuzumab treatment, PTEN protein in the
membrane-bound pool was noticeably enriched. Similar results were
also observed in SKBr3 cells (data not shown). This suggested that
trastuzumab treatment facilitates PTEN localization to the cell
membrane, where this phosphatase is active and functions to
dephosphorylate PIP.sub.3 to PIP.sub.2, leading to Akt
dephosphorylation (Cantley and Neel, 1999). Taken together,
activation of PTEN is an early molecular event after trastuzumab
treatment.
[0204] PTEN binds to the cellular membrane through its C2 domain
and there are two tyrosine phosphorylation sites in the C2 domain
of PTEN(Y240 and Y315) important for PTEN phophatase activity and
tumor inhibition function (Koul et al., 2002). Tyrosine
phosphorylation of PTEN reduces the capacity of the PTEN C2 domain
to bind to the cellular membrane (Lu et al., 2003). Since increased
membrane localization of PTEN by trastuzumab treatment in
ErbB2-overexpressing cells was observed, the inventors investigated
whether this may result from reduced PTEN tyrosine phosphorylation.
PTEN was immunoprecipitated from untreated or trastuzumab treated
BT474 cells with the A2B1 PTEN antibody and immunoblotted with the
PY99 phosphotyrosine antibody (FIG. 1F). Consistent with the notion
that PTEN is functionally inactive in ErbB2-overexpressing cells
(Lu et al., 1999), the inventors found that PTEN is highly
phosphorylated on tyrosine in untreated BT474 cells. However,
trastuzumab treatment rapidly reduced PTEN tyrosine phosphorylation
in BT474 cells (FIG. 1F), which paralleled the increased PTEN
membrane localization and PTEN activation by trastuzumab in these
cells (FIGS. 1D and 1E). Thus, transtuzumab induces PTEN activation
by increasing the translocation of PTEN from the cytoplasm to the
membreane through reducing the inhibitory tyrosine phosphorylation
of PTEN. Notably, these events occur before ErbB2 is downregulated
and PI3K is inhibited. Therefore, activation of PTEN is an early
molecular event after trastuzumab treatment independent of ErbB2
downregulation and PI3K inhibition.
Example 3
PTEN Activation Contributes to Trastuzumab's Anti-Proliferation
Function
[0205] To determine if PTEN activation indeed caused Akt
dephosphorylation in response to trastuzumab treatment, SKBr3
breast cancer cells were transfected with PTEN antisense (AS)
oligonucleotides (Butler et al., 2002), which inhibited endogenous
PTEN expression compared with mock-transfected cells or cells
transfected with control mismatched (MIS) oligonucleotides (FIG.
3A). Compared with MIS control-treated cells showing rapid Akt
dephosphorylation by trastuzumab treatment (FIG. 3B, top), Akt
dephosphorylation was attenuated in PTEN AS-treated cells (FIG. 3B,
bottom). This data indicated that PTEN expression is required for
the rapid Akt dephosphorylation by trastuzumab.
[0206] It was investigated whether PTEN activation contributes to
the anti-proliferation function of trastuzumab in MIS control and
PTEN AS-transfected SKBr3 cells. Inhibition of cell proliferation
by trastuzumab was found to be significantly less effective in PTEN
AS-treated cells with reduced PTEN than in MIS control-transfected
SKBr3 cells having normal PTEN expression (FIG. 3C). Similar
results in BT474 cells were observed (FIGS. 4A-4B). This data
demonstrated that normal PTEN expression levels are required for
the anti-proliferation function of trastuzumab. Without trastuzumab
treatment, PTEN reduction by PTEN AS alone had no significant
effect on cell proliferation (FIG. 3D), which was consistent with
the similar Akt phosphorylation levels in cells treated with PTEN
AS alone to that treated with MIS control (FIG. 3B, time 0). That
blocking PTEN by PTEN AS only reduced trastuzumab's
anti-proliferative function but had no effect on cells without
trastuzumab treatment reiterated that PTEN activity is important
for trastuzumab function, which reconciles well with the earlier
finding that PTEN is activated by trastuzumab (FIGS. 1C and 1D).
Thus, PTEN reduction led to resistance to the anti-proliferation
effect of trastuzumab in these cells.
[0207] Trastuzumab is mostly used in combination with other
chemotherapeutics in the clinic (Slamon et al., 2001). Therefore,
it was investigated whether PTEN reduction may confer breast cancer
cell resistance to trastuzumab plus paclitaxel chemotherapy. BT474
cells were transfected with PTEN AS or MIS control
oligonucleotides, then treated them with or without trastuzumab (2
.mu.g/ml) plus paclitaxel (1 nm), and cell viability was measured 3
days later. MIS control-treated cells with normal PTEN expression
showed significant growth inhibition by trastuzumab plus paclitaxel
(P<0.0001), whereas PTEN AS-transfected cells were not
significantly inhibited (FIG. 3E). Similar results were observed at
higher concentrations (2 and 4 nm) of paclitaxel. However,
paclitaxel treatment alone without trastuzumab showed no
statistically significant difference in cell viability between PTEN
AS-transfected and MIS control-transfected cells (FIG. 3E),
indicating that PTEN status does not significantly impact breast
cancer cell sensitivity to paclitaxel but specifically impacts
trastuzumab sensitivity. Together, these results indicated that
breast cancer cells with reduced PTEN are resistant to growth
inhibition by trastuzumab as a single agent (FIG. 3C) as well as in
combination therapy with paclitaxel (FIG. 3E).
Example 4
PTEN Deficiency Contributes to Trastuzumab Resistance In Vivo
[0208] To investigate if reduced PTEN expression may confer breast
cancer cell resistance to trastuzumab treatment in vivo, a
tumorigenic subline of BT474 human breast cancer cells was injected
into the mammary fat pad (mfp) of athymic nude mice. When tumor
xenografts reached >150 mm.sup.3, PTEN AS or MIS
oligonucleotides were injected into the tumor xenografts. One week
later, mice were treated with trastuzumab (10 mg/kg) or vehicle
twice per week. PTEN expression in tumor xenografts was effectively
inhibited by PTEN AS when ErbB2 expression remained the same (FIG.
3F). Trastuzumab treatment only inhibited MIS control-treated
tumors expressing normal PTEN, but not PTEN AS-treated tumors with
reduced PTEN (FIG. 3G). Without trastuzumab treatment, PTEN AS and
MIS treated tumors had similar tumor growth rates (FIG. 3G). These
results demonstrated that PTEN reduction confers breast tumor
xenografts resistance to the anti-tumor function of trastuzumab in
vivo.
Example 5
PI3K Inhibitors Rescue PTEN Loss-Induced Trastuzumab Resistance
[0209] The above data clearly demonstrate that PTEN deficiency
confers trastuzumab resistance in breast cancer. In search of
strategies for overcoming trastuzumab resistance from the loss of
PTEN, it was reasoned that since PI3K antagonizes PTEN function,
inactivation of PI3K may rescue trastuzumab resistance from PTEN
deficiency. The inventors investigated whether PI3K inhibitors that
are known to quickly induce Akt dephosphorylation in
ErbB2-overexpressing cells may reverse trastuzumab resistance in
PTEN-deficient breast cancer cells (Xiong et al., 2001). BT474
cells were transfected with MIS and PTEN AS oligonucleotides, then
treated them with trastuzumab, the PI3K inhibitor LY294002, or
trastuzumab plus LY294002. The inventors then determined cell
viability 3 days later (FIG. 5A). The MIS-treated cells are more
sensitive to trastuzumab-mediated growth inhibition than the PTEN
AS-treated cells, and the combination treatment inhibited the
viability of MIS-treated cells slightly better than trastuzumab
alone or LY294002 alone (p>0.05). However, the LY294002 plus
trastuzumab combination inhibited cell viability significantly
better than trastuzumab alone or LY294002 alone in PTEN AS-treated
cells (p<0.01). Similarly, another PI3K inhibitor, Wortmannin,
in combination with trastuzumab, also inhibited PTEN AS-treated
BT474 cells significantly better than either treatment alone
(p<0.01), whereas the combination had a similar inhibitory
effect as trastuzumab alone in MIS-treated cells (FIG. 5B). In
vivo, trastuzumab combined with the PI3K inhibitor LY294002 was
also significantly (p<0.05) more effective than trastuzumab
alone or LY294002 alone in inhibiting PTEN AS-treated BT474 tumor
xenografts in mice (FIG. 4C). These results indicate that PI3K
inhibitors could overcome trastuzumab resistance in PTEN-deficient
breast cancer cells.
Example 6
Patients with PTEN-Deficient Breast Cancers have a Poor Clinical
Response to Trastuzumab
[0210] To explore the clinical significance of PTEN deficiency in
predicting a poor response to trastuzumab-based therapy in
patients, PTEN expression was evaluated in 47 ErbB2-overexpressing
primary breast carcinomas from patients who subsequently developed
metastatic breast cancer and received trastuzumab plus taxane
chemotherapy (paclitaxel or docetaxel) (Esteva et al., 2002;
Seidman et al., 2001). As controls, PTEN expression was evaluated
in primary breast carcinomas not selected for ErbB2 overexpression
from 37 patients who subsequently developed metastatic breast
cancer and received taxane without trastuzumab (Holmes et al.,
1991; Valero et al., 1995). Immunohistochemical staining using
anti-PTEN antibodies revealed that PTEN expression in these tumors
was heterogeneous with different intensities. Thus, PTEN expression
levels were semi-quantified using immunoreactive scores (IRS)
calculated by multiplying the percentage of PTEN positive cells
(scored 0 to 4) with the PTEN staining intensity (1 to 3) (Joki et
al., 2000). IRS of 0 to 12 represents PTEN staining from a
non-detectable level in PTEN-lost tumors to positive full
expression seen in normal (non-cancerous) individuals (FIG. 6A). A
loss of PTEN (IRS<9) was observed in 36.2% of the tumor samples
examined (17/47), which is consistent with previous reports on loss
of PTEN expression in approximately 40% breast cancers using IHC
staining (Depowski et al., 2001; Perren et al., 1999). Remarkably,
breast cancer patients with PTEN low or negative tumors (IRS 6-0)
had significantly lower complete and partial response (CR+PR) rates
to trastuzumab plus taxane therapy than patients with PTEN positive
tumors (35.7% vs. 66.7%, P<0.05) (FIG. 6B, left). Using more
stringent criteria for loss of PTEN (IRS<4), patients with PTEN
negative tumors demonstrated a striking, highly significant
(P<0.01) worse response to trastuzumab plus taxane therapy than
patients with PTEN positive tumors (11.1% versus 65.8% CR+PR) (FIG.
6B, right). Furthermore, a statistically significant trend was
identified, suggesting that the probability of response to
trastuzumab decreases as PTEN IRS decreases (P<0.01) (FIG. 7).
However, among the 37 patients treated by taxane without
trastuzumab, patients with PTEN-deficient tumors had similar
response (CR+PR) rates to those with PTEN-positive tumors (p=0.04
when PTEN loss is defined as IRS 0-6; p=0.74 when PTEN loss is
defined as IRS<4. These data clearly demonstrate that PTEN loss
does not confer breast cancer resistance to taxane but to
trastuzmab. To confirm that PTEN is a specific marker for
trastuzumab responsiveness, Ki67, a known prognostic marker for
breast cancers, was examined (Faneyte et al., 2003), which may also
predict trastuzumab responsiveness. Ki67 did not correlate with
clinical response to trastuzumab plus taxane treatment in this
group (FIG. 8). Interestingly, although patients with ErbB2 FISH
positive and/or IHC 3+ ErbB2 overexpression breast cancers seemed
to respond to trastuzumab plus taxane better than patients with
ErbB2 FISH negative and/or 2+ ErbB2 overexpression cancer (59% vs.
37%), the difference was not statistically significant (FIG. 6C).
However, among patients with ErbB2 FISH positive and/or IHC 3+
ErbB2 overexpressing breast cancers, PTEN low or negative tumors
(IRS 3-0) had significantly lower response (CR+PR) rates than
patients with PTEN positive tumors (12.5% vs. 71%, P<0.01) (FIG.
4D).
Example 7
Trastuzumab Activates PTEN by Inhibiting Src Association with
ErbB2
[0211] To investigate how trastuzumab inhibits PTEN tyrosine
phosphorylation, the inventors examined whether trastuzumab may
inhibit Src tyrosine kinase, since Src activation has recently been
reported to increase PTEN tyrosine phoshorylation (Lu et al.,
2003). Compared to untreated cells, trastuzumab treatment rapidly
inhibited Src kinase activity and rapidly reduced Src
phosphorylation on Y416, an indicator of Src activity (Frame,
2002). Reduced Src-Y416 phosphorylation corresponded to reduced
PTEN tyrosine phosphorylation.
[0212] Src is known to bind to ErbB2 and is thus activated in
ErbB2-overexpressing cancer cells (Belsches-Jablonski et al., 2001;
Muthuswamy and Muller, 1995). To explore the mechanisms of
trastuzumab-mediated Src inhibition, the inventors examined whether
Src binding to ErbB2 is inhibited by trastuzumab treatment in BT474
cells. Trastuzumab induced a very rapid and dramatic reduction of
ErbB2 bound Src, indicating that trastuzumab inhibited Src activity
in ErbB2 overexpressing cells most likely by inhibiting Src binding
to ErbB2.
[0213] To further confirm that Src inhibition can reduce PTEN
tyrosine phosphorylation in ErbB2-overexpressing breast cancer
cells, the inventors examined whether the Src kinase inhibitor PP2
may reduce PTEN tyrosine phosphoryulation in the
ErbB2-overexpressing 435.eB breast cancer cells (Yu et al., 1998b).
Compared to the solvent 10% dimethyl sulfoxide (DMSO)-treated
435.eB cells, PP2 treatment effectively inhibited Src
phosphorylation on Y416 and also dramatically reduced tyrosine
phosphorylation of PTEN, similar to trastuzumab's effect.
Contrarily, the eB/SrcCA cells, which are 435.eB cells stably
expressing a constitutively activated Src (Chan et al., 2003) and
having higher levels of Src-Y416 phosphorylation and Src activity,
showed a dramatic increase of PTEN tyrosine phosphorylation
compared to the 435.eB cells. Furthermore, although trastuzumab
treatment effectively increased the PTEN membrane translocation in
the 435.eB cells, PTEN membrane translocation by trastuzumab in the
eB/SrcCA cells expressing constitutively activated Src was less
effective. Together, the data indicate that trastuzumab treatment
inhibits Src binding to ErbB2 in ErbB2-overexpressing breast cancer
cells, thus inhibiting Src kinase activity, which leads to reduced
PTEN tyrosine phosphorylation and increased PTEN membrane
localization and activity.
[0214] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents that are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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[0215] The following references are specifically incorporated
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Sequence CWU 1
1
411212DNAHomo sapiensCDS(1)..(1212) 1atg aca gcc atc atc aaa gag
atc gtt agc aga aac aaa agg aga tat 48Met Thr Ala Ile Ile Lys Glu
Ile Val Ser Arg Asn Lys Arg Arg Tyr1 5 10 15caa gag gat gga ttc gac
tta gac ttg acc tat att tat cca aac att 96Gln Glu Asp Gly Phe Asp
Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile 20 25 30att gct atg gga ttt
cct gca gaa aga ctt gaa ggc gta tac agg aac 144Ile Ala Met Gly Phe
Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn 35 40 45aat att gat gat
gta gta agg ttt ttg gat tca aag cat aaa aac cat 192Asn Ile Asp Asp
Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His 50 55 60tac aag ata
tac aat ctt tgt gct gaa aga cat tat gac acc gcc aaa 240Tyr Lys Ile
Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys65 70 75 80ttt
aat tgc aga gtt gca caa tat cct ttt gaa gac cat aac cca cca 288Phe
Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro 85 90
95cag cta gaa ctt atc aaa ccc ttt tgt gaa gat ctt gac caa tgg cta
336Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu
100 105 110agt gaa gat gac aat cat gtt gca gca att cac tgt aaa gct
gga aag 384Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala
Gly Lys 115 120 125gga cga act ggt gta atg ata tgt gca tat tta tta
cat cgg ggc aaa 432Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu
His Arg Gly Lys 130 135 140ttt tta aag gca caa gag gcc cta gat ttc
tat ggg gaa gta agg acc 480Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe
Tyr Gly Glu Val Arg Thr145 150 155 160aga gac aaa aag gga gta act
att ccc agt cag agg cgc tat gtg tat 528Arg Asp Lys Lys Gly Val Thr
Ile Pro Ser Gln Arg Arg Tyr Val Tyr 165 170 175tat tat agc tac ctg
tta aag aat cat ctg gat tat aga cca gtg gca 576Tyr Tyr Ser Tyr Leu
Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala 180 185 190ctg ttg ttt
cac aag atg atg ttt gaa act att cca atg ttc agt ggc 624Leu Leu Phe
His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly 195 200 205gga
act tgc aat cct cag ttt gtg gtc tgc cag cta aag gtg aag ata 672Gly
Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile 210 215
220tat tcc tcc aat tca gga ccc aca cga cgg gaa gac aag ttc atg tac
720Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met
Tyr225 230 235 240ttt gag ttc cct cag ccg tta cct gtg tgt ggt gat
atc aaa gta gag 768Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp
Ile Lys Val Glu 245 250 255ttc ttc cac aaa cag aac aag atg cta aaa
aag gac aaa atg ttt cac 816Phe Phe His Lys Gln Asn Lys Met Leu Lys
Lys Asp Lys Met Phe His 260 265 270ttt tgg gta aat aca ttc ttc ata
cca gga cca gag gaa acc tca gaa 864Phe Trp Val Asn Thr Phe Phe Ile
Pro Gly Pro Glu Glu Thr Ser Glu 275 280 285aaa gta gaa aat gga agt
cta tgt gat caa gaa atc gat agc att tgc 912Lys Val Glu Asn Gly Ser
Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys 290 295 300agt ata gag cgt
gca gat aat gac aag gaa tat cta gta ctt act tta 960Ser Ile Glu Arg
Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu305 310 315 320aca
aaa aat gat ctt gac aaa gca aat aaa gac aaa gcc aac cga tac 1008Thr
Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr 325 330
335ttt tct cca aat ttt aag gtg aag ctg tac ttc aca aaa aca gta gag
1056Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu
340 345 350gag ccg tca aat cca gag gct agc agt tca act tct gta aca
cca gat 1104Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr
Pro Asp 355 360 365gtt agt gac aat gaa cct gat cat tat aga tat tct
gac acc act gac 1152Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser
Asp Thr Thr Asp 370 375 380tct gat cca gag aat gaa cct ttt gat gaa
gat cag cat aca caa att 1200Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu
Asp Gln His Thr Gln Ile385 390 395 400aca aaa gtc tga 1212Thr Lys
Val2403PRTHomo sapiens 2Met Thr Ala Ile Ile Lys Glu Ile Val Ser Arg
Asn Lys Arg Arg Tyr1 5 10 15Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr
Tyr Ile Tyr Pro Asn Ile 20 25 30Ile Ala Met Gly Phe Pro Ala Glu Arg
Leu Glu Gly Val Tyr Arg Asn 35 40 45Asn Ile Asp Asp Val Val Arg Phe
Leu Asp Ser Lys His Lys Asn His 50 55 60Tyr Lys Ile Tyr Asn Leu Cys
Ala Glu Arg His Tyr Asp Thr Ala Lys65 70 75 80Phe Asn Cys Arg Val
Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro 85 90 95Gln Leu Glu Leu
Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu 100 105 110Ser Glu
Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys 115 120
125Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys
130 135 140Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val
Arg Thr145 150 155 160Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln
Arg Arg Tyr Val Tyr 165 170 175Tyr Tyr Ser Tyr Leu Leu Lys Asn His
Leu Asp Tyr Arg Pro Val Ala 180 185 190Leu Leu Phe His Lys Met Met
Phe Glu Thr Ile Pro Met Phe Ser Gly 195 200 205Gly Thr Cys Asn Pro
Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile 210 215 220Tyr Ser Ser
Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr225 230 235
240Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu
245 250 255Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met
Phe His 260 265 270Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu
Glu Thr Ser Glu 275 280 285Lys Val Glu Asn Gly Ser Leu Cys Asp Gln
Glu Ile Asp Ser Ile Cys 290 295 300Ser Ile Glu Arg Ala Asp Asn Asp
Lys Glu Tyr Leu Val Leu Thr Leu305 310 315 320Thr Lys Asn Asp Leu
Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr 325 330 335Phe Ser Pro
Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu 340 345 350Glu
Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr Pro Asp 355 360
365Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp
370 375 380Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr
Gln Ile385 390 395 400Thr Lys Val320DNAHomo sapiens 3ctgctagcct
ctggatttga 20420DNAHomo sapiens 4cttctggcat ccggtttaga 20
* * * * *