Note: Descriptions are shown in the official language in which they were submitted.
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
STAT3 AGONISTS AND ANTAGONISTS
AND THERAPEUTIC USES THEREOF
This application claims priority under 35 U.S.C. ~119(e) to provisional patent
application no. 60/231,212, filed September 8, 2000, which is incorporated by
reference
herein in its entirety.
The development of this invention was supported by grant numbers CA75243,
CA55652 and CA77859 awarded by the National Institutes of Health. The
Government
may therefore have certain rights in this invention.
1. INTRODUCTION
The present invention relates to methods for modulating, i.e., agonizing or
antagonizing, Stat3 (signal transducer and activator of transcription3)
signaling activity for
use in gene therapy. Inhibition and/or activation of Stat3 signaling is an
effective
therapeutic approach to modulate angiogenesis and the immune-response in
various
diseases.
2. BACKGROUND OF THE INVENTION
Signal transducers and activators of transcription (STATs) are Latent
cytoplasmic
transcription factors that function as intracellulaa- effectors of cytokine
and growth factor
signaling pathways (Darne11,1997, Science 277(5332):1630-1635). STAT proteins
were
originally defined in the context of normal cell signaling where STATs have
been
implicated in control of cell proliferation, differentiation, and apoptosis
(Bromberg and
Darnell, 2000, Oncogene, 19:2468-2473; Darnell et al., 1994, Science 264:1415-
1421).
Stat3(3 is a dominant-negative Stat3 variant, which is a truncated form of
Stat3 that
contains the dimerization and DNA binding domain but lacks the transactivation
domain
(Catlett-Falcone et al., 1999, Immunity, 10:105-115). As a consequence,
Stat3/3 can bind
DNA but cannot transactivate gene expression, thus blocking Stat3 signaling in
a trans-
dominant negative fashion in most cases. Blocking Stat3 by Stat3(3 in U266
cells down-
regulated expression of the Stat3-regulated Bcl-XL gene, resulting in a
dramatic sensitization
of cells to Fas-mediated apoptosis is~ vitf°o (Catlett-Falcone et al.,
1999, supra).
Effective gene therapy requires the killing of genetically untransduced cells
("bystander" cells) concomitant with genetically transduced cells. Because
transfection
efficiency is a rate-limiting step for gene therapy, the efficacy of cancer
gene therapy is
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
enhanced by bystander effects. The identification and characterization of
specific
molecules involved in Stat-mediated bystander effects could provide useful
reagents and
techniques for developing novel prophylactic and therapeutic methods.
Citation or discussion of a reference herein shall not be construed as an
admission
that such is prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention provides methods for use of Stat3 agonists and
antagonists for
treatment of disclose involving angiogenesis and immune disorders. The
invention is
based, in part, on the Applicants' discovery that inhibition of Stat3
signaling results in the
induction of a cascade of immunologic danger signals, which are normally
produced only
during inflammation and infection. Thus, the cellular expression of a Stat3
antagonist
results in the production of soluble factors which can induce the expression
of pro-
inflammatory cytokines and chemokines in neighboring cells. The present
invention takes
advantage of this "bystander effect" of Stat3 activity modulators to provide
methods and
compositions for the treatment of a variety of conditions, diseases and
disorders. The
invention further provides methods for identification of such soluble factors,
herein termed
"immunological danger signals". As used herein, the term "immunologic danger
signals"
refers to soluble factors produced as a result of inhibition of Stat3, which
induce an immune
response, such as a pro-inflammatory signal, e.g., a pro-inflammatory
cytokine.
The present invention provides a method for modulating angiogenesis comprising
administering to an individual in need of treatment an effective amount of a
compound that
agonizes or antagonizes the activity of Stat3.
The present invention further provides a method for the treatment or
prevention of a
llypoxic or ischemic condition or disorder, comprising administering to an
individual in
need of treatment an effective amount of a compound that increases the
activity of Stat3, so
that the hypoxic or ischemic condition or disorder is treated or prevented. In
one
embodiment, the compound is Stat3. In another embodiment, the compound is a
constitutive active form of Stat3 (Stat3-C). In one embodiment, the compound
is
interleukin-6. In another embodiment, the condition or disorder is the result
of ischemia,
coronary-atherosclerosis, myocardial infarction, tissue ischemia in the lower
extremities,
infarction, inflammation, trauma, stroke, vascular occlusion, prenatal or
postnatal oxygen
deprivation, suffocation, choking, near drowning, carbon monoxide poisoning,
smoke
inhalation, trauma, including surgery and radiotherapy, asphyxia, epilepsy,
hypoglycemia,
c~°nic obstructive pulmonary disease, emphysema, adult respiratory
distress syndrome,
hypotensive shock, septic shock, anaphylactic shock, insulin shock, cardiac
arrest,
dysrhythmia, or nitrogen narcosis.
_2_
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
In one embodiment, a method is provided for the treatment or prevention of a
proliferative angiopathy with neovascularization, comprising administering to
an individual
in need of treatment an effective amount of a compound that decreases the
activity of Stat3,
so that the a proliferative angiopathy is treated or prevented. In one
embodiment, the
proliferative angiopathy is diabetic microangiopathy. In another embodiment,
the
compound is Stat3(3. In another embodiment, the compound is a negative
regulatory
protein. In another embodiment, the compound is a Stat3 antisense nucleic acid
molecule.
In yet another embodiment, the compound is a ribozyme specific to Stat3. In
yet another
embodiment, the compound is an inhibitor of a positive regulator of Stat3. In
another
embodiment, the compound is an antibody specific to Stat3.
The invention further provides a method for suppressing an immune response,
comprising administering to an individual in need of treatment an effective
amount of a
compound that increases the activity of Stat3. In one embodiment, the compound
is Stat3.
In another embodiment, the compound is a constitutive active form of Stat3
(Stat3-C). In
another embodiment, the compound is interleukin-6. In another embodiment, the
treatment
of the individual ameliorates a symptom of an autoimmune disease. In another
embodiment, the autoimmune disease is insulin dependent diabetes mellitus,
multiple
sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma,
polymyositis,
chronic active hepatitis, mixed connective tissue disease, primary biliary
cirrhosis,
pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease,
vitiligo; gluten-
sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune
neutropenia,
idiopathic thrombocytopenia purpura, rheumatoid arthritis, cirrhosis,
pemphigus vulgaris,
autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid
lupus,
ulcerative colitis, or dense deposit disease.
In another aspect of the invention, a method is provided for activating an
immune
response, comprising administering to an individual in need of treatment an
effective
amount of a compound that decreases the activity of Stat3, with the proviso
that the
treatment is not a cancer treatment. In one embodiment of this method, the
compound is
Stat3[3. In another embodiment, the compound is a negative regulatory protein.
In another
embodiment, the compound is a Stat3 antisense nucleic acid molecule. In yet
another
embodiment, the compound is a ribozyme specific to Stat3. In another
embodiment, the
compound is an inhibitor of a positive regulator of Stat3. In another
embodiment, the
compound is an antibody specific to Stat3.
In various embodiments of the invention, the therapeutic compound is delivered
via
gene therapy. In alternative embodiments, the compound is delivered to the
individual with
a pharmaceutically acceptable carrier.
The invention further provides a method for identifying an immunologic danger
signal comprising: (a) inhibiting Stat3 signaling activity in cells in
culture; (b) separating
-3-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
the supernatant from said cells; (c) adding said supernatant, or fractions
thereof, to immune
cells; and (d) assaying for activation of said irnrnune cells; such that if
immune cells are
activated by a cell supernatant or a fraction thereof, then an immunological
danger signal is
identified. The invention further provides a composition comprising the cell
supernatant or
a fraction thereof that is the product of this method.
In one embodiment of the method, the immune cells are macrophages. In a
specific
embodiment, said assaying for activation of said immune cells comprises
assaying said
macrophages for NO production. In another specific embodiment, said assaying
for
activation of said immune cells comprises assaying said macrophages for iNOS
expression.
In another specific embodiment, said assaying for activation of said immune
cells comprises
assaying said macrophages for RANTES expression.
In another embodiment, the immune cells of the method are neutrophils. In
another
embodiment, said assaying for activation of said immune cells comprises
assaying said
neutrophils for TNF-a expression. In another embodiment, the immune cells are
T cells. In
a specific embodiment said assaying for activation of said immune cells
comprises assaying
said T cells for for IFN-y expression. In another specific embodiment, said
assaying for
activation of said immune cells comprises assaying said T cells for IL-2
expression
In another embodiment, the cells of the method are B16 cells. In another
embodiment, the Stat3 is suppressed by a Stat3 signaling activity antagonist.
In another
embodiment, the antagonist is a dominant negative Stat3 mutant. In yet another
embodiment the antagonist is a negative regulatory protein. In another
embodiment, the
antagonist is a Stat3 antisense nucleic acid molecule. In yet another
embodiment of the
method, the antagonist is a ribozyme specific to Stat3. In another embodiment,
the
antagonist is an inhibitor of a positive regulator of Stat3. In another
embodiment, the
antagonist is an antibody specific to Stat3.
The following standard abbreviations are used herein: Stat, signal transducer
and
activator of transcription; Stat3, signal transducer and activator of
transcription3; TRAIL,
TNF-related apoptosis-inducing ligand; EMSA, electrophoretic mobility shift
assay; hSIE,
high-affnuty sis-inducible element; EGFP, enhanced green fluorescence protein;
FACS,
fluorescence-activated cell sorting; pIRES, vector comprising an internal
ribosome entry
site; IL, interleukin; Stat3[3 or Stat3beta, a dominant negative form of
signal transducer and
activator of transcription3; Stat3-C, a constitutive active form of signal
transducer and
activator of transcription3; VEGF, vascular endothelial growth factor; pIRE,
palindromic
interferon response element.
-4-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
4. BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Inhibition of endogenous Stat3 (SEQ. ID. N0:1; SEQ. ID. N0:2) DNA-
binding
activity in B16 cells by overexpression of Stat3(3 (SEQ. ID. N0:3; SEQ. ID.
N0:4).
EMSA was performed with nuclear extracts prepared from B 16 cells transfected
with no
DNA (lane 1), empty vector (lane 2) or Stat3~3 expression vector (lane 3).
Extracts from
NIH3T3 fibroblasts stimulated with EGF were used as a positive control for
Statl and Stat3
(lane 4). Supershifts were performed using antibodies recognizing either Stat3
(a-ST3) or
Stat3(3 (a-ST3(3) with extracts derived from B16 cells transfected with no DNA
(lanes 5-7),
the empty vector (lanes 8-10), or Stat3(3 vector (lanes 11-13). ST3:3, and
ST1:3, ST1:1
indicate migration of complexes containing Stat3 or Stat3(3 homodimers,
Statl/Stat3
heterodimers and Statl/Statl homodimers, respectively. The asterisk indicates
the position
of supershifted complexes.
Figure 2A-C. Soluble factors produced by Stat3(3-transfected B16 cells induce
growth
inhibition of non-transfected B 16 cells. A. Growth inhibition analysis using
supernatants
derived from either empty vector or Stat3(3 transfected B16 cells collected at
0 h, 12 h, 24 h,
36 h, 48 h after transfection. Growth inhibition of B16 cells by supernatants
from Stat3~3
B 16 at various times was expressed as % inhibition based on the formula, (No.
cells control
- No. cells experimental)/No. cells control x 100. For 3H-TdR incorporation
assays, 0.25
~,Ci 3H-TdR was added during the last 4 h of incubation. For MTT assays, 5 ~,1
MTT (10
mg/ml) was added during the last 4 h of incubation. B. Cell cycle analysis. B
16 cells were
transfected in the lower chambers of Transwell units. Five hours later, non-
transfected B 16
cells were added to upper chambers. Another 48 h later, B 16 cells in the
upper chambers
were harvested for cell cycle analysis. C. Apoptosis assays. After incubating
with
transfected cells in the lower chambers for 48 h, cells in upper chambers were
harvested and
stained with Annexin V-PE and 7-AAD, followed by FACS analysis. FL2-H
represents 7-
AAD and FL3-H represents Annexin V-PE. These experiments (with the exception
of MTT
assays) were repeated at least three times with similar results.
Figure 3A-C. Overexpression of Stat3(3 induces cell cycle arrest and apoptosis
in B16
cells. A. Transfection efficiencies of pIRE-EGFP and PIKE-Stat3(3 in B 16
cells as
determined by GFP expression, and cell viability of B16 cells 48 h after
transfection as
determined by trypan blue exclusion. B. Cell cycle a~.lalysis was performed by
propidium
iodide staining at various times after transfection as indicated. GolGl phase
arrest in Stat3[3-
transfected B 16 cells was detected at 24, 36 and 48 h after transfection. C.
48 h after
~.ansfection, apoptosis was measured by Annexin V-PE staining followed by FACS
analysis. Data shown represent one of three experiments with similar results.
-5-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Figure 4. Stat3(3 overexpression in B16 cells results in induction of TRAIL
mRNA
expression. Ten pg of total cellular RNAs isolated from B 16 (lanes 1 and 4),
and B 16
transfected with either AIRES-EGFP (lanes 2 and 5) or AIRES-Stat3(3 (lanes 3
and 6) were
hybridized with each multiple probe before digestion with RNase. Separation of
protected
fragments was achieved by gel electrophoresis. Fragment assignment was
determined by
migration relative to internal standards. Induction of TRAIL RNA was confirmed
by two
additional RPA analyses.
Figure 5A-B. Blocking Stat3 signaling in B16 cells stimulates production of
soluble
factors capable of inducing iNOS-dependent nitric oxide production by
macrophages. A.
~netics of availability of soluble factors after Stat3(3 transfection.
Supernatants from
transfected B 16 cells were taken out at the times indicated. The data shown
represent one of
two experiments with similar results and expressed as p,M nitrite ~ SD, n=4.
B. NO
production by macrophages is iNOS-dependent. Data shown are representative of
5
independent experiments with similar results. S=supernatant; Mph=macrophage.
Figure 6. Macrophages activated by the supernatant derived from Stat3(3-
transfected B16
cells confers NO-mediated cytostatic activity against untransfected B 16
cells. Macrophages
(1x105) were incubated in 50% supernatants from Stat3(3-transfected (S-
Stat3(i) or GFP-
vector transfected (S-GFP) B 16 cells for 6 h. The supernatants were replaced
by normal
c°mplete medium and wild-type B16 cells (1x104) were added. Cytostatic
activity of
macrophages is determined 48 h later and is expressed as % of inhibition of 3H-
TdR
incorporation. The data shown are the results of one of four similar
experiments ~ SD, n=4.
Figure 7A-B. Stat3(3 expression in B 16 cells upregulates the expression of
pro-
inflammatory chemokines and cytokines, which can stimulate peritoneal
macrophages to
produce NO. Elevated expression of pro-inflammatory cytokines and chemokines
in B 16
cells as a result of Stat3/3 expression (A. TNF-a,, IL-6, IFN-(3; B. IP-10).
Total RNAs were
prepared form mock-transfected, GFP-transfected, Stat3(3-transfected and UV-
irradiated
B 16 cells. Data shown have been confirmed with at least one more experiment,
in which
BAs were prepared from independent transfectants and UV-irradiated B16 tumor
cells.
Figure 8A-B. Factors secreted by Stat3(3-transfected B16 cells upregulate the
expression of
pro-inflammatory cytokines and chemokines by macrophages and neutrophils.
Macrophages and neutrophils were incubated with supernatants derived from
either empty
vector-transfected, Stat3[3-transfected or UV-irradiated (macrophage only) B16
cells. A.
RNAse protection assays using RNAs prepared from macrophages treated with
various
supernatants as indicated. Data shown represent RNAs pooled from two RNA
preparations
-6-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
isolated from macrophages stimulated with supernatants derived from two
independent
transfections. B. TNF-a ELISA assays of neutrophils after incubating with
supernatants or
LPS as indicated. Two independent experiments were performed as shown. Levels
of
TNF-a secreted by neutrophils cultured in the supernatants derived from B16-
pIRES-EGFP
transfectants were assigned as "1".
Figure 9A-B. Expression of Stat3[3 in tumor cells leads to activation of
macrophages and T
lymphocytes in vivo. Mice were injected with B 16 cells (2 X 106/mouse)
transfected with
either GFP or Stat3(3 expression vectors. Five days later, peritoneal
macrophages and
lymphocytes were tested for NO production and IFN-y production, respectively.
A. NO
production by peritoneal macrophages. B. IFN-y ELISPOT of lymphocytes.
Figure l0A-B. Transfection of NIH3T3-Src cells with either: A. Stat3(3
expression vector;
or B. Stat3 anti-sense oligos, results in reduced levels of VEGF protein as
shown by
Western blot. Src tyrosine kinase-mediated VEGF upregulation requires Stat3.
Src
t~osine activity is known to upregulate VEGF expression. In Src-transformed
NIH3T3
cells, VEGF expression is high.
Figure 11. Expression of constitutively-activated Stat3 (SEQ. ID. N0:5)
increases the
production of VEGF in NIH3 fibroblasts. Left panel: Stat3 DNA-binding activity
in
NIH3T3 stable clones transfected with Stat3C, a mutant form of Stat3 that is
constitutively
activated. Nuclear extracts prepared from EGF-induced NIH3T3 cells (EGF), the
wild-type
NIH3T3 cells (WT), and NIH3T3 stable clones transfected with Stat3C clones 1,
3, 6 and 7
(Stat3C-1, -3, -5, -6, -7, respectively) are incubated with the 3ZP-labeled
hSIE
oligonucleotide probe and analyzed by EMSA. Right panel: Western blot analysis
of
VEGF protein levels in the WT and stable transfectants. /3-actin is used as a
standard to
indicate the amount of protein loaded in each lane. Stat3C clones 1, 3, 6 and
7 had more
Stat3 DNA-binding activity and higher levels of VEGF protein compared to those
of wt
NIH3T3 cells and Stat3C clone 5.
Figure 12A-B. Blocking Stat3 signaling in tumor cells inhibits VEGF promoter
activity.
Both B 16 (A) and SCK (B) marine tumor cells were transfected with constructs
containing
the luciferase cDNA in the absence (pluc) or presence of the VEGF promoter
(VEGF).
While VEGF promoter activity was high in both tumor cells, co-transfection
with anti-sense
oligonucleotides (ASO) against Stat3 resulted in a dramatic reduction of VEGF
promoter
3 5 activity.
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Figure 13. Inhibition of Stat3 signaling in tumor cells reduces the expression
of the
endogeneous VEGF gene. B16 tumor cells were transfected with either: A.
Stat3(3; or B.
Stat3 anti-sense oligonucleotides (ASO) at 100 nM, 200 nM, or 300 nM. Western
blot
analyses indicated that a decrease in Stat3 protein is correlated with a
reduction in VEGF
protein. [3-actin is used here to indicate the amount of protein loaded in
each lane.
5. DETAILED DESCRIPTION OF THE INVENTION
The invention described in the subsections below provides therapeutic methods
for
modulating angiogenesis and the immune response by agonizing or antagonizing
Stat3
signaling activity. Stat3 is an essential regulator of several cellular and
physiological
processes, such as cell cycle, apoptosis, the immune response, and
angiogenesis, as
exemplified by the experiments in Section 6, 7 and 8. Based on the discovery
by the
Applicants using a variety of approaches to modulate Stat3 activity, Stat3
activity
modulators can up-regulate or down-regulate cell cycle, apoptosis, immune-
response, and
angiogenesis respectively. Accordingly, agonists and antagonists of Stat3
activity can be
used to modulate cell cycle, apoptosis, immune-response, and angiogenesis to
treat
disorders involving dysfunctions of cell cycle, control of apoptosis, immune-
response, and
angiogenesis.
Such methods and compositions may be used to treat andlor prevent such
diseases or
disorders as, for example, ischemic diseases and proliferative angiopathies
with
neovascularization. The methods and compositions described herein may be used
to
augment the immuneresponse to treat various diseases, such as cancer or
inflammatory
diseases, or to suppress the immune response to treat diseases and disorders
such as auto-
l~~e disorders. Such target diseases and disorders are further described
hereinbelow.
The invention provides methods of treatment and prophylaxis by administration
to a
subject of an effective amount of an agonist or antagonist of Stat3 activity,
which are also
referred to collectively herein as "Stat3 activity modulators" or
"pharmaceuticals of the
invention". Such Stat3 activity modulators include, but are not limited to,
peptides,
p°l~eptides, nucleic acids, and small molecules. In particular,
examples of polypeptide
Stat3 activity modulators include, e.g., Stat3(3, a dominant negative form of
the Stat3 gene
constitutive active Stat3, the wild-type Stat3 gene, product and antibodies
specific to Stat3.
Nucleotide sequences that can be used to inhibit Stat3 gene expression
include, for example,
antisense and ribozyme molecules, as well as gene or regulatory sequence
replacement
c°nstructs designed to enhance the expression of Stat3, Stat3beta, or
constitutive active
Stat3 (e.g., expression constructs that place the Stat3 gene under the control
of a strong
promoter system). Such Stat3 activity modulators are described in detail
hereinbelow.
_g_
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
The invention further provides methods for the identification of "immunologic
danger signals" and compositions comprising such immunologic danger signals,
which may
be used to stimulate an immune response. As used herein, the term "immunologic
danger
signal" refers to a signal which stimulates an immune response, such as a pro-
inflammatory
signal, e.g. pro-inflammatory cytokines and chemokines. Such methods for
identification of
immunological danger signals are further described hereinbelow.
5.1 METHODS FOR TREATMENT OR PREVENTION OF ISCHEMIC DISEASES
In one embodiment of the invention, methods are provided for stimulating
angiogenesis using Stat3 agonists. The therapeutic effect of activating Stat3
signaling in
this embodiment of the invention lies in the promotion of a) de novo formation
of blood
vessels, and b) sprouting from pre-existing vessels. In the context of this
invention, both
phenomenon will be jointly referred to as angiogenesis. The use of Stat3
agonists to
promote angiogenesis may be used, for example, in preventing or treating
ischemic
diseases. Stat3 agonists may be administered to patients in need of such
treatment to
increase stimulated vessel growth, and consequentially increase tissue
perfusion and blood
flow, thereby overcoming the vascular insufficiency characteristic of ischemic
diseases.
Gene therapy approaches and other pharmacological approaches, described in the
subsections below, can be designed and used to augment Stat3 signaling with
relation to the
treatment of vascular insufficiencies.
5.1.1 GENE THERAPY APPROACHES
Angiogenic molecules can be administered by way of gene transfer. With this
strategy, the angiogenic protein, such as Stat3, a constitutive active form of
Stat (Stat3-C),
and agonists of Stat3 signaling, is delivered to the tissue in. form of a
nucleotide sequence
encoding said protein. The gene can be delivered in an expression vector via a
variety of
approaches, including direct injection, electroporation, by way of transfected
cells, or
commercially available liposome preparations. The expression vector, usually
consisting of
a replication-defiecient adenovirus, retrovisus, lentivirus, and/or an adeno-
associated virus,
is taken up by the host cells via receptor-mediated mechanisms and/or
endocytosis (see
5.6.3).
The present invention relates to administering nucleotide sequences encoding
constitutive active Stat3, agonists of Stat3, such as, but not limited to,
interleukin-6 (IL-6),
as well as the normal form of Stat3. A constitutive form of Stat3 is encoded
by the Stat3-C
mutant form of the Stat3 gene. In Stat3-C the substitution of two cysteine
residues within
the C-terminal loop of the SH2 domain of Stat3 produces a molecule that
dimerizes
-9-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
spontaneously, binds to DNA, and activates transcription, thus giving rise to
a constitutive .
active molecule (Bromberg et al., 1999, Cell 98:295-303).
Alternatively, replacing those tyrosine residues in STAT3 that are being
phosphorylated upon activation with aspartic acid residues may result in a
constitutive
active molecule. Dependent on the molecular context, acidic amino acids such
as aspartic
acid can mimic a phosphate. As Stat3 is activated upon phosphorylation at said
tyrosine
residues, mimicking such phosphates constitutively by incorporation of an
aspartic acid can
render the molecule to be constitutively active. In order to replace the
tyrosine residue in
Stat3 with aspartic acid, site directed mutagenesis approaches which are well
known to the
skilled artisan can be used. The present invention also relates to the
expression of proteins
that activate Stat3, such as IL-6. Expression of said protein components via
gene therapy
and resulting activation of Stat3 can be used in order to promote angiogenesis
in ischemic
diseases.
Another embodiment of the invention relates to the expression of the normal
form of
the Stat3 protein component.
The nucleotide sequence to be expressed in a gene therapy approach has to be
operatively linked to a promoter sequence. As it is known to the skilled
artisan,
enhancerlpromoter sequences are essential for the expression of a given gene.
Enhancer/promoter sequences also confer temporal and spatial regulation onto
the
expression pattern of a given gene.
Such enhancer/promoter sequences should be chosen dependent on the indicated
disorder. In some cases tissue specific expression will be the preferred
embodiment of the
invention; in other cases systemic expression of the nucleotide sequence may
be preferred.
This decision will depend on the indicated disorder, and ultimately on the
clinician.
Expression specific to the tissue affected by vascular insufficiency or
ischemia can be
conferred by enhancer/promoter sequences that are active only in that tissue.
Combining
the right promoter sequences with the gene to be expressed will require some
experimentation involving standard techniques known to the skilled artisan. In
other
disorders, inducible expression of the pro-angiogenic molecule, such as
Stat3C, Stat3, or
IL-6, may be indicated. As it is known to the skilled artisan, different
enhancer/promoter
sequences are active only in the absence and/or presence of a particular
factor, which can be
a metabolite, an anorganic molecule or a protein component. In the context of
treating
ischemic diseases, which are characterized by insufficient nutrient and oxygen
supply of
affected tissues, enhancer/promoter sequences that are induced upon hypoxia
are the
preferred embodiment of the invention.
Placing the expression of the nucleotide sequence of the invention under
control of
hypoxia constitutes a self regulatory system. Once the oxygen concentration in
the affected
tissue falls below a certain threshold due to impaired blood-supply resulting
from narrowed
-10-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
or blocked arteries, the expression of the angiogenic gene, i.e. Stat3, Stat3C
or other
constitutive forms of Stat3, IL-6, respectively, is up-regulated. The
resulting newly formed
vascular tissue provides an increased blood-flow in the affected tissue, thus
increasing the
oxygen concentration in said tissue. Consequently, the expression of the
recombinant
angiogenic gene will cease. This method ensures sufficient neovascularization
but prevents
vascular overgrowth that may be associated with too long exposure to or too
high
expression of angiogenic factors.
5.1.2 PHARMACOLOGICAL APPROACHES
Angiogenic molecules can be delivered by administering the recombinant
proteins.
Recombinant Stat3, Stat3-C, or IL-6, respectively, can be synthesized and
purified as fusion
proteins by recombinant DNA techniques. Fusing a "peptide tag" such as a
polyhistidine
tag, glutathione S-transferase (GST), or the E. coli maltose binding protein
(MBP) to the
angiogenic protein facilitates its purification. The fusion proteins can be
synthesized in
different host systems, such as, but not restricted to, bacteria, insects
cells or mammalian
1 S cells. Methods of expressing said proteins in different systems and
purifying them are
described in section 5.6.2.
In another embodiment of the invention, the proteins can be immuno-purified
using
antibodies specific to the respective protein. An exemplary approach comprises
covalently
linking antibodies specific to the protein which is to be purified to a solid
matrix. Protein
extracts of the host cells expressing the desired protein are added to the
matrix under
conditions that allow binding of said protein to the matrix via non-covalent
binding to the
antibodies. After contaminants have been removed by washing under suitable
conditions,
the protein can be eluted.
The recombinant proteins can then be administered by the drug delivery system
of
choice dependent on whether systemic or local administration of the protein is
preferred for
the treatment or prevention, respectively, of the indicated disorder. Various
delivery
systems are known and are described in section 5.6.4.
The administration of agonists of Stat3 signaling, such as but not limited too
Stat3,
Stat3-C, or IL-6 causes increased angiogenesis and subsequent increase in
blood flow, thus
restoring sufficient supply of nutrients and oxygen in the affected tissue.
5.2 METHODS FOR TREATMENT OR PREVENTION OF PROLIFERATIVE
ANGIOPATHIES WITH NEOVASCULARIZATION
A plurality of disorders are caused by the overgrowth of blood vessels, herein
referred to as proliferative angiopathies with neovascularization. An
exemplary disorder of
this kind is diabetic microangiopathy with neovascularization. This disease is
characterized
-11-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
by swollen retinal vessels that leak fluid as well an excess of retinal
vessels which is
diagnosed as diabetic retinopathy and can lead to blindness in the affected
patients.
Because of the regulatory role of Stat3 in angiogenesis through its activating
effect
on VEGF, modulating the activity of Stat3 signaling is a target for treating
diseases
involving neovascularization. The presented invention relates to inhibiting
angiogenesis in
proliferative angiopathies with neovascularization (other than cancer) by
reducing the
activity of Stat3 signaling. The invention relates to inhibiting Stat3 using
negative
regulators of Stat3, such as a dominant negative form of Stat3, Stat3beta. The
invention
comprises inhibiting Stat3 using negative regulators of Stat3, such as SOCS
and PIAS,
inhibitors of Stat3 expression, such as antisense oligonucleotides and
ribozytnes, antibodies
l~ibitors of positive regulators of Stat3, such as inhibitors of the Src
tyrosine kinase. The
effects of the pharmaceuticals of the invention will be referred to in this
context as anti
angiogenic.
5.2.1 GENE THERAPY APPROACHES
In its preferred embodiment, the Stat3 activity modulator is Stat3beta, a
dominant
negative form of Stat3. Compared to STAT3, STAT3beta lacks the C-terminal
transactivation domain. STAT3beta fails to activate a pIRE containing promoter
in
transient transfection assays. Instead, co-expression of STAT3beta inhibits
the
transactivation potential of STAT3, thus effectively inhibiting Stat3 activity
(Caldenhoven
et al., 1996, Journal of Biological Chemistry 271:13221-13227). The dominant
negative
form of Stat3, Stat3beta, can be administered by a gene therapy approach as
described 5.6.3.
With this strategy, Stat3beta is delivered to the targeted tissue in form of a
nucleotide
sequence encoding Stat3beta under conditions that allow Stat3beta expression.
In order for
the Stat3beta gene to be expressed, the gene must be operatively linked to an
e~~cer/promoter sequence. In order to target only certain organs or tissues,
tissue-
specific and/or inducible enhancerlpromoter sequences can be used. For a more
detailed
discussion of tissue-specific gene therapy see section 5.6.3.
Alternative embodiments of the inventions comprise other inhibitors of Stat3
signaling, such as, but not limited to, the SOCS negative regulatory molecues
and the PIAS
family of negative regulatory proteins (Stan and Hilton 1999, Bioessays 21:47-
52). These
factors can also be administered via gene therapy as described in 5.6.3. In
order for these
genes to be expressed, the respective gene must be operatively linked to an
enhancer/promoter sequence. In order to target only certain organs or tissues,
tissue-
specific and/or inducible enhancer/promoter sequences can be employed.
Additionally, the invention relates to suppressing the expression of
endogenous
Stat3. This can be achieved by administering nucleotide sequences that are in
antisense
-12-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
orientation relative to the Stat3 encoding mRNA (hereinafter referred to as
antisense Stat3
nucleotide sequence; see Example 3, Fig. 12). Those nucleotide sequences can
vary in
length from 20 basepairs up to the length of the entire Stat3 cDNA. Antisense
nucleotide
sequences of different length may differ in their efficacy as drugs, and it
may take some
experimentation to fmd the right length to treat the indicated disorder. Said
antisense Stat3
nuleotide sequences can be delivered via gene transfer as described in 5.6.3.
In order for
these antisense nucleotide sequences to be expressed, the anitsense Stat3
nucleotide
sequence must be operatively linked to an enhancer/promoter sequence. For
targeting only
certain organs or tissues, tissue-specific and/or inducible enhancer/promoter
sequences can
be employed.
Furthermore, expression of Stat3 can be suppressed by intracellular expression
of
small RNA therapeutics such as ribozymes. Small RNA therapeutics can be
delivered via
gene therapy by linking the nucleotide sequences encoding said RNA
therapeutics opatively
to an enhancer/promoter sequence. The invention encompasses the administration
of a
vector comprising the nucleotide sequence encoding the Stat3 specific ribozyme
operatively
linked to an enhancer/promoter to the patient by methods described in 5.6.3,
thus resulting
in an antiangiogenic effect.
5.2.2 PHARMACOLOGICAL APPROACHES
Anti-angiogenic molecules can also be delivered by administering the
recombinant
proteins. Recombinant Stat3beta, SOCS, or PIAS respectively, can be
synthesized and
purified as fusion proteins by recombinant DNA techniques. Fusing a "peptide
tag" such as
a polyhistidine tag, glutathione S-transferase (GST), or the E. coli maltose
binding protein
(MBP) to the angiogenic protein facilitates its purification. The fusion
proteins can be
synthesized in different host systems, such as, but not restricted to,
bacteria, insects cells or
mammalian cells. Alternatively the proteins can be immuno-purified using
antibodies
specific to the respective protein.
The recombinant proteins can then be administered by the drug delivery system
of
choice dependent on whether systemic or local administration of the protein is
preferred for
the treatment or prevention, respectively, of the indicated disorder. Various
delivery
systems axe known and are described in 5.8.
Additionally, the invention relates to suppressing the expression of
endogenous
Stat3. This can be achieved by administering antisense Stat3 nucleotide
sequences. Those
nucleotide sequences can vary in length from 20 basepairs up to the length of
the entire
Stat3 cDNA. Antisense nucleotide sequences of different length may differ in
their efficacy
as drugs, and it may take some experimentation to find the right length to
treat the indicated
disorder. Said antisense Stat3 nuleotide sequences can be delivered by
administering
-13-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
directly in vitro synthesized antisense nucleotide sequences. Those antisense
nucleotide
sequences can be modified to increase their stability, thus lengthening their
half life, in a
cell. Antisense Stat3 nucleic acids are described in detailed in Section
5.6.4.
Furthermore, expression of Stat3 can be suppressed by administration of small
RNA
therapeutics such as ribozymes specific to Stat3 RNA. The invention comprises
the in vitro
synthesis of small RNA therapeutics such as ribozymes specific to Stat3 RNA
and
administration of said small RNA therapeutics. Those RNA therapeutics can be
chemically
modified in order to increase their sability and lengthen their half life.
Furthermore, the invention relates to reducing neovascularization by
antagonizing
Stat3 signaling via inhibitors of positive regulators of Stat3 signaling such
as the tyrosine
~nase Src. In a specific embodiment, the invention encompasses the inhibition
of Src by
administration of the drug SU6656 (Blake et al. 2000, Molecular Cellular
Biology 20:9018-
9027).
The invention also comprises reducin neovascularization by antagonizing Stat3
signaling using antibodies specific to the Stat3 protein component. The
antibodies can be
administered by the drug delivery system of choice dependent on whether
systemic or local
administration of the protein is preferred for the treatment or prevention,
respectively, of the
indicated disorder. Various delivery systems are known and are described in
5.8..
5.3. METHODS FOR STIMULATING THE IMMUNE RESPONSE BY INHIBITING
2p STAT3 SIGNALING
In another embodiment, based on the regulatory effect of Stat3 on the
production of
such immunologic danger signals and the immune-response, the invention
provides
methods for stimulating the immune response using antagonists of Stat3
signaling activity.
Immunologic danger signals are factors that attract cells of the immune-system
to the site of
the infection or cancerous growth and activate an immune response. Such
immunologic
danger signals include, but are not limited to: IFN-gamma inducible protein 10
(IP-10),
interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interferon-
beta (IFN-
beta).
In specific embodiments, the invention encompasses administration to a patient
the
supernatant of cells in which Stat3 activity is suppressed by means comprising
Stat3beta
and Stat3 antisense nucleotide sequences. The invention also encompasses
inhibition of
Stat3 signaling in the patient locally or systemically to augment the irmnune
response in
various diseases. The various embodiments of the invention are described in
more detail in
the sections below. The goal of any of these embodiments is to increase the
concentration
of immunologic danger signals either locally or systemically in the patient,
thereby
augmenting the immune response. Such a strengthening of the patient's own
defense
-14-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
system is desirable when the patients natural immune reaction is not
sufficient to eliminate
the pathogen or the malignant cells. More specifically, some tumors evade
immune
surveillance by suppressing the expression of said immunologic danger signals.
5.3.1. APPROACHES FOR ADMINISTERING THE SUPERNATANT OF
STAT3BETA TRANSFECTED CELLS
This embodiment of the invention relates to the inhibition of Stat3 signaling
in cells
such as B16 melanoma cells by such means as expression of Stat3beta,
expression of
negative regulators of Stat3 signaling as for example PIAS and SOCS,
expression of Stat3
antisense nucleotide sequences, administration of in vitro synthesized Stat3
antisense
nucleotide sequences, and antibodies specific to Stat3. In the preferred
embodiment of the
invention, Stat3beta is expressed in B 16 melanoma cells by means of
transfection and
supernatant is obtained from said cell culture. For a detailed description of
the methods
involved refer to Example2 and sections 5.6.1 and 5.6.2.
The supernatant can then be administered to a patient in order to augment the
immune response in various diseases. Such diseases include infectious diseases
and various
malignancies. The supernatant can be administered by any method known in the
art. Some
examples of which are described in section 5.6.4. Said supernatant can be
converted into
solid form by means such as to lyophilization
5.3.2 GENE THERAPY APPROACHES TO AUGMENT THE IIVIMUNE-
RESPONSE 1N VARIOUS DISEASES OTHER THAN CANCER
In a preferred embodiment, the pharmaceutical of the invention is Stat3beta, a
dominant negative form of Stat3. Compared to STAT3, STAT3beta lacks the C-
terminal
transactivation domain. STAT3beta fails to activate a pIRE containing promoter
in
transient transfection assays. Instead, co-expression of STAT3beta inhibits
the
transactivation potential of STAT3, thus effectively inhibiting Stat3 activity
(Caldenhoven
et al. 1996, Journal of Biological Chemistry 271:13221-13227). The dominant
negative
form of Stat3, Stat3beta, can be administered by a gene therapy approach as
described 5.6.3.
With this strategy, Stat3beta is delivered to the targeted tissue in form of a
nucleotide
sequence encoding Stat3beta under conditions that allow Stat3beta expression.
In order for
the Stat3beta gene to be expressed, the gene must be operatively linked to an
enhancer/promoter sequence. In order to target only certain organs or tissues,
tissue-
specific and/or inducible enhancer/promoter sequences can be used. For a more
detailed
discussion of tissue-specific gene therapy see section 5.6.3.
Alternative embodiments of the inventions comprise other inhibitors of Stat3
signaling, such as, but not limited to, the SOCS negative regulatory molecules
and the PIAS
-15-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
family of negative regulatory proteins (Start and Hilton 1999, Bioessays 21:47-
52). These
factors can also be administered via gene therapy as described in 5.6.3. In
order for these
genes to be expressed, the respective gene must be operatively linked to an
enhancer/promoter sequence. In order to target only certain organs or tissues,
tissue-
specific and/or inducible enhancer/promoter sequences can be employed.
Additionally, the invention relates to suppressing the expression of
endogenous
Stat3. This can be achieved by administering nucleotide sequences that are in
antisense
orientation relative to the Stat3 encoding mRNA (hereinafter referred to as
antisense Stat3
nucleotide sequence; see Example 3, Fig. 12). Those nucleotide sequences can
vary in
length from 20 basepairs up to the length of the entire Stat3 cDNA. Antisense
nucleotide
sequences of different length may differ in their efficacy as drugs, and it
may take some
experimentation to find the right length to treat the indicated disorder. Such
antisense Stat3
nuleotide sequences can be delivered via gene transfer as described in 5.6.3.
In order for
these antisense nucleotide sequences to be expressed, the antisense Stat3
nucleotide
sequence must be operatively linked to an enhancer/promoter sequence. For
targeting only
certain organs or tissues, tissue-specific and/or inducible enhancer/promoter
sequences can
be employed.
Furthermore, expression of Stat3 can be suppressed by intracellular expression
of
small RNA therapeutics such as ribozymes. Small RNA therapeutics can be
delivered via
gene therapy by linking the nucleotide sequences encoding RNA therapeutics
operatively to
an enhancer/promoter sequence. The invention encompasses the administration of
a vector
comprising the nucleotide sequence encoding the Stat3 specific ribozyme
operatively linked
to an enhancer/promoter to a patient by methods described in 5.6.3, thus
enhancing the
immune response of the patient.
5.3.3 PHARMACOLOGICAL APPROACHES TO AUGMENT THE IMMUNO-
RESPONSE IN VARIOUS DISEASES OTHER THAN CANCER
Antagonists of Stat3 signaling activity can be delivered by administering the
recombinant proteins. Recombinant Stat3beta, SOCS, or PIAS respectively, can
be
synthesized and purified as fusion proteins by recombinant DNA techniques.
Fusing a
~~peptide tag" such as a polyhistidine tag, glutathione S-transferase (GST),
or the E. coli
maltose binding protein (MBP) to the protein of the invention facilitates its
purification.
The fusion proteins can be synthesized in different host systems, such as, but
not restricted
to, bacteria, insects cells or mammalian cells. Alternatively the proteins can
be immune-
purified using antibodies specific to the respective protein.
The recombinant proteins can then be administered by the drug delivery system
of
choice dependent on whether systemic or local administration of the protein is
preferred for
-16-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
the treatment or prevention, respectively, of the indicated disorder. Various
delivery
systems are known and are described in 5.8.
Additionally, the invention relates to suppressing the expression of
endogenous
Stat3. This can be accomplished by administering antisense Stat3 nucleotide
sequences.
Those nucleotide sequences can vary in length from 20 basepairs up to the
length~of the
entire Stat3 cDNA. Antisense nucleotide sequences of different length may
differ in their
efficacy as drugs, and it may take some experimentation to find the right
length to treat the
indicated disorder. Said antisense Stat3 nuleotide sequences can be delivered
by
administering directly in vitro synthesized antisense nucleotide sequences.
Those antisense
nucleotide sequences can be modified to increase their stability, thus
lengthening their half
life in a cell.
Furthermore, expression of Stat3 can be suppressed by administration of small
RNA
therapeutics such as ribozymes specific to Stat3 RNA. The invention comprises
the in vitro
synthesis of small RNA therapeutics such as ribozymes specific to Stat3 RNA
and
administration of said small RNA therapeutics. Those RNA therapeutics can be
chemically
modified in order to increase their stability and lengthen their half life.
Furthermore, the invention relates to enhancing the immune response by
antagonizing Stat3 signaling via inhibitors of positive regulators of Stat3
signaling such as
the tyrosine kinase Src. In a specific embodiment, the invention encompasses
the inhibition
of Src by administration of the drug SU6656 (Blake et al. 2000, Molecular
Cellular Biology
20:9018-9027).
The invention also comprises augmenting the immune response by antagonizing
Stat3 signaling using antibodies specific to the Stat3 protein. The antibodies
can be
administered by the drug delivery system of choice dependent on whether
systemic or local
administration of the protein is preferred for the treatment or prevention,
respectively, of the
indicated disorder. Various delivery systems are known and are described in
5.8.
5.4. METHODS FOR INHIBITING THE IZVVIMUNE RESPONSE BY ACTIVATING
STAT3 SIGNALING
In another embodiment, based on the regulatory effect of Stat3 on the
production of
such immunologic danger signals and the immune-response, the invention
provides
methods for inhibiting the immune response using agonists of Stat3 signaling
activity.
Immunologic danger signals are factors that attract cells of the immune-system
to the site of
the infection or transplants and activate an immune response. Such
immunologic~danger
signals include, but are not limited to: IFN-gamma inducible protein 10 (IP-
10), interleukin-
6 (~-6), tumor necrosis factor-alpha (TNF-alpha) and interferon-beta (IFN-
beta).
- I7-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
In its specific embodiments, the invention encompasses administration to a
patient
of agonists of Stat3 signaling locally or systemically to suppress the immune
response in
various diseases. The various embodiments of the invention are described in
more detail in
the sections below. The goal of any of these embodiments is to decrease the
concentration
of irnmunologic danger signals either locally or systemically in the patient,
thereby
suppressing the immune response. Such a suppression of the patient's own
defense system
is desirable when the patient is suffering from autoimmune diseases or to
ameliorate
adverse reactions to transplants.
5.4.1 GENE THERAPY APPROACHES TO SUPPRESS THE IMMUNE-
RESPONSE IN VARIOUS DISEASES
Immuno suppressant molecules can be administered by way of gene transfer. With
this strategy, the immuno suppressant protein, such as Stat3, a constitutive
active form of
Stat (Stat3-C), and agonists of Stat3 signaling, is delivered to the tissue in
form of a
nucleotide sequence encoding said protein. The gene can be delivered in an
expression
vector via a variety of approachs, including direct injection,
electroporation, by way of
transfected cells, or commercially available liposome preparations. The
expression vector,
usually consisting of a replication-defiecient adenovirus, retrovisus,
lentivirus, and/or an
adeno-associated virus, is taken up by the host cells via receptor-mediated
mechanisms
and/or endocytosis.
The present invention relates to administering nucleotide sequences encoding
constitutive active Stat3, agonists of Stat3, such as, but not limited to,
interleukin-6 (IL-6),
as well as the normal form of Stat3. A constitutive form of Stat3 is encoded
by the Stat3-C
mutant form of the Stat3 gene. In Stat3-C the substitution of two cysteine
residues within
the C-terminal loop of the SH2 domain of Stat3 produces a molecule that
dimerizes
spontaneously, binds to DNA, and activates transcription, thus giving rise to
a constitutive
active molecule (Bromberg et al., 1999, Cell 98:295-303).
Alternatively, replacing those tyrosine residues in STAT3 that are being
phosphorylated upon activation with aspartic acid residues may result in a
constitutive
active molecule. Dependent on the molecular context, acidic amino acids such
as aspartic
acid can mimic a phosphate. As Stat3 is activated upon phosphorylation at said
tyrosine
residues, mimicking such phosphates constitutively by incorporation of an
aspartic acid can
render the molecule to be constitutively active. In order to replace the
tyrosine residue in
Stat3 with aspartic acid, basic side directed mutagenesis approaches which are
well known
to the skilled artisan can be used. The present invention also relates to the
expression of
proteins that activate Stat3, such as IL-6. Expression of said protein
components via gene
-18-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
therapy and resulting activation of Stat3 can be used in various diseases
where a
suppression of the immune response is desirable.
Another embodiment of the invention relates to the expression of the normal
form of
the Stat3 protein component. Despite the regulation of Stat3 signaling in a
cell, elevating
Stat3 protein levels in a cell can also increase its function thereby
suppressing the immune
response.
The nucleotide sequence to be expressed in a gene therapy approach has to be
operatively linked to a promoter sequence. As it is known to the skilled
artisan,
enhancer/promoter sequences are essential for the expression of a given gene.
.
Enhancer/promoter sequences also confer temporal and spatial regulation onto
the
expression pattern of a given gene.
Such enhancer/promoter sequences should be chosen dependent on the indicated
disorder. In some cases tissue specific expression will be the preferred
embodiment of the
invention; in other cases systemic expression of the nucleotide sequence may
be preferred.
This decision will depend on the indicated disorder, and ultimately on the
clinician.
Expression specific to the tissue affected by the immunologic disorder can be
conferred by
enhancer/promoter sequences that are active only in that tissue. Combining the
right
promoter sequences with the gene to be expressed will require some
experimentation
involving standard techniques known to the skilled artisan. In other
disorders, inducible
expression of the irmnuno-suppressant protein, such as Stat3C, Stat3, or IL-6,
may be
indicated. As it is known to the skilled artisan, different enhancer/promoter
sequences are
active only in the absence and/or presence of a particular factor, which can
be a metabolite,
an anorganic molecule or a protein component.
5.4.2 PHARMACOLOGICAL APPROACHES
I~~o suppressant molecules can be delivered by administering the recombinant
proteins. Recombinant Stat3, Stat3-C, or IL-6, respectively, can be
synthesized and
purified as fusion proteins by recombinant DNA techniques. Fusing a "peptide
tag", such
as a polyhistidine tag, glutathione S-transferase (GST), or the E. coli
maltose binding
protein (MBP) to the angiogenic protein facilitates its purification. The
fusion proteins can
be synthesized in different host systems, such as, but not restricted to,
bacteria, insects cells
or mammalian cells. Methods of expressing said proteins in different systems
and purifying
them are described below.
In another embodiment of the invention, the proteins can be immuno-purified
using
antibodies specific to the respective protein. An examplary approach comprises
covalently
linking antibodies specific to the protein which is to be purified to a solid
matrix. Protein
extracts of the host cells expressing the desired protein are added to the
matrix under
-19-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
conditions that allow binding of said protein to the matrix via non-covalent
binding to the
antibodies. After contaminants have been removed by washing under suitable
conditions,
the protein can be eluted.
The recombinant proteins can then be administered by the drug delivery system
of
choice dependent on whether systemic or local administration of the protein is
preferred for
the treatment or prevention, respectively, of the indicated disorder. Various
delivery
systems are known and are described below.
5.5 METHODS FOR IDENTIFYING IMMC1NOLOGIC DANGER SIGNALS
The invention relates to a method of identifying immunologic danger signals.
Once
those immunologic danger signals have been identified, they can be synthesized
and
administered to patients in order to augment the immune-response in various
diseases.
In its preferred embodiment, the invention encompasses the identification of
immunologic danger signals secreted by melanoma B 16 cells that have been
genetically
engineered to express the dominant negative form of Stat3, Stat3beta. In a
specific
embodiment of the application, Stat3beta is expressed in melanoma B16 cells
using the
pIRES vector system (Clontech; Palo Alto, CA; Catlett-Falcone et al. 1999,
Immunity
10:105-115). The nucleotide sequence encoding Stat3beta can be inserted into
pIRES or
any other vector system suitable for transfection of mammalian cells by
standard molecular
biology techniques. Likewise, the DNA can be transfected into the cells by
standard
techniques known to the skilled artisan. The supernatant of cells expressing
Stat3beta
comprises immunologic danger signals (see Example 2).
In order to identify the individual components of the supernatant that are
responsible
for the immunologic signaling activity of the supernatant, the components of
the
supernatant can be separated by standard biochemical techniques such as, but
not limited to,
gel-filtration chromotography or ion-exchange chromotography. These techniques
are well
known to the skilled artisan, and a minimum of experimentation will be
required to
determine the optimal conditions under which to purify individual components
of the
supernatant. After separation of the constituent components of the supernatant
in individual
components or fractions, said fractions are tested for their immunologic
signaling effects on
different immune cells, such as macrophages, T-cell and neutrophils. Dependent
on the cell
type, different assays can be employed in order to test the immunologic
signaling activity of
a given fraction. Those assays are described in the following subsections.
Once positive
fraction have been identified, the constituent components of a given fraction
can be
analyzed by techniques such as, but not limited to, SDS gel electrophoresis or
mass
spectrometry. In case the fraction of interest contains more than one
component, the
components of the fraction must be seperated from each other and individually
tested for
-20-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
their immunologic signaling activity in the respective assay. Again, standard
biochemical
techniques such as, but not limited to, gel-filtration chromotography or ion-
exchange
chromotography can be used for the isolation of the component of interest.
Once a factor
with immunologic signaling activity is isolated from the supernatant, its
identity can be
determined by using standard techniques such as micro-sequencing or mass-
spectrometry.
In additional embodiments, the invention relates to the identification of
immunologic danger signals released from cells other than melanoma B 16 cells,
but
similarly expressing Stat3beta.
Furthermore, the invention encompasses a method of identifying immunologic
danger signals released from cells, such as but not limited to, melanoma B 16
cells, in which
Stat3beta signaling is inhibited by specific antagonists of Stat3beta
acitivity. Such
antagonists comprise antisense nucleotide sequences specific to Stat3beta and
ribozymes
that act specifically on Stat3beta RNA.
5.5.1 ACTIVATING IMMUNE CELLS SUCH AS MACROPHAGES, T-CELLS,
AND NEUTROPHILS
Immunologic signaling activity can be tested either in cell culture on various
types
of cells of the immune system or in an animal model. Accordingly, the
fractions, which are
obtained from the supernatant as described above, are added either to cells in
culture, such
as cultures of macrophages, T-cells and neutrophils, or, alternatively, are
injected into an
animal, preferrably a mouse. After a sufficient time period said cells are
tested for
immunologic activity. This can be accomplished for example by measuring the
expression
levels of markers of activation. In the case of macrophages such markers
include, but are
not limited to, the nitric oxide synthase, iNOS, and the chemokine RANTES. If
the
activation of T-cells is to be investigated, interferon-gamma (IFN-gamma) and
interleukin-2
(IL-2) can be used as markers. Expression of the tumor necrosis factor alpha
(TNF-alpha)
can be used as a marker if neutrophils are used in this assay system. The
length of the time
period between stimulation and assay of expression of said markers may be
changed and
depends on the precise experimental conditions. A minimum of experimentation
is
necessary to establish the assay system to which the invention relates in such
a way that it
30 ~nctions optimally. The levels of iNOS, RANTES, IFN-gamma, IL-2, and TNF-
alpha can
be determined by immunoblotting, Northern blotting, RNAse protection assays,
immunocytochemistry or similar techniques well known to the skilled artisan.
For any of
those techniques probes specific to iNOS, RANTES, IFN-gamma, IL-2, and TNF-
alpha,
respectively, have to be employed. Such probes comprise antibodies and
antisense RNA
35 molecules. The detection of such probes is well established in the art.
If an animal model is employed, macrophages, T-cells and neutrophils can be
isolated from the animal and subsequently analyzed or, alternatively,
expression levels of
-21 -
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
iNOS, RANTES, IFN-gamma, IL-2, and TNF-alpha can be tested in situ by
immunohistochemistry or in situ hybridization. For any of those techniques
probes specific
to iNOS, RANTES, IFN-gamma, TL-2, and TNF-alpha, respectively, have to be
employed.
Such probes comprise antibodies and antisense RNA molecules. The detection of
such
probes is well established in the art. Quantification and statistical analysis
of the data is
done by standard methods.
5.6 THERAPEUTIC METHODS FOR USE WITH THE INVENTION
5.6.1 RECOMBINANT DNA
In various embodiments of the invention, the Stat3 activity modulator
comprises a
protein which is encoded by a specific nucleotide sequence. In other
embodiments of the
invention, the pharmaceutical comprises a nucleotide sequence which is
transcribed to
generate a biologically active RNA molecule. In even other embodiments of the
invention,
the Stat3 activity modulator comprises a nucleotide sequence which is to be
transcribed and
translated. In either case, said nucleotide sequence is inserted into an
expression vector for
propagation and expression in recombinant cells or in cells of the host in the
case of gene
therapy.
An expression construct, as used herein, refers to a nucleotide sequence
encoding
the Stat3 activity modulator, which can be either an RNA molecule or a
protein, operably
linked to one or more regulatory regions or enhancer/promoter sequences which
enables
expression of the protein of the invention in an appropriate host cell.
"Operably-linked"
refers to an association in which the regulatory regions and the nucleotide
sequence
encoding the Stat3 activity modulator to be expressed are joined and
positioned in such a
way as to permit transcription, and ultimately, translation.
The regulatory regions necessary for transcription of the Stat3 activity
modulator
can be provided by the expression vector. In a compatible host-construct
system, cellular
transcriptional factors, such as RNA polymerase, will bind to the regulatory
regions on the
expression construct to effect transcription of the Stat3 activity modulator
in the host
organism. The precise nature of the regulatory regions needed for gene
expression may
vary from host cell to host cell. Generally, a promoter is required which is
capable of
binding RNA polymerase and promoting the transcription of an operably-
associated nucleic
acid sequence. Such regulatory regions may include those 5'-non-coding
sequences
involved with initiation of transcription and translation, such as the TATA
box, capping
sequence, CART sequence, and the like. The non-coding region 3' to the coding
sequence
may contain transcriptional termination regulatory sequences, such as
terminators and
p°lyadenylation sites.
Both constitutive and inducible regulatory regions may be used for expression
of the
Stat3 activity modulator. It may be desirable to use inducible promoters when
the
-22-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
conditions optimal for growth of the host cells and the conditions for high
level expression
of the Stat3 activity modulator are different. Examples of useful regulatory
regions are
provided below (section 5.6.3).
In order to attach DNA sequences with regulatory functions, such as promoters,
to
the sequence encoding the Stat3 activity modulator or to insert the sequence
encoding the
Stat3 activity modulator into the cloning site of a vector, linkers or
adapters providing the
appropriate compatible restriction sites may be ligated to the ends of the
cDNAs by
techniques well known in the art (Wu et al., 1987, Methods in Enzymol 152:343-
349).
Cleavage with a restriction enzyme can be followed by modification to create
blunt ends by
digesting back or filling in single-stranded DNA termini before ligation.
Alternatively, a
desired restriction enzyme site can be introduced into a fragment of DNA by
amplification
of the DNA by use of PCR with primers containing the desired restriction
enzyme site.
An expression construct comprising a sequence encoding the Stat3 activity
modulator operably linked to regulatory regions (enhancer/promoter sequences)
can be
directly introduced into appropriate host cells for expression and production
of the Stat3
activity modulator without further cloning. The expression constructs can also
contain
DNA sequences that facilitate integration of the sequence encoding the Stat3
activity
modulator into the genome of the host cell, e.g., via homologous
recombination. In this
instance, it is not necessary to employ an expression vector comprising a
replication origin
suitable for appropriate host cells in order to propagate and express the
protein of the
invention in the host cells.
A variety of expression vectors may be used in the present invention which
include,
but are not limited to, plasmids, cosmids, phage, phagemids, or modified
viruses.
Typically, such expression vectors comprise a functional origin of replication
for
propagation of the vector in an appropriate host cell, one or more restriction
endonuclease
sites for insertion of the sequence encoding the Stat3 activity modulator, and
one or more
selection markers. The expression vector must be used with a compatible host
cell which
may be derived from a prokaryotic or an eukaryotic organism including but not
limited to
bacteria, yeasts, insects, mammals, and humans.
Vectors based on E. coli are the most popular and versatile systems for high
level
expression of foreign proteins (Makrides, 1996, Microbiol Rev, 60:512-538).
Non-limiting
examples of regulatory regions that can be used for expression in E. coli may
include but
not limited to lac, trp, lpp, phoA, recA, tac, T3, T7 and ~,PL (Makrides,
1996, Microbiol
Rev, 60:512-538). Non-limiting examples of prokaryotic expression vectors may
include
the 7~gt vector series such as 7~gt11 (Huynh et al., 1984 in "DNA Cloning
Techniques", Vol.
n A Practical Approach (D. Glover, ed.), pp. 49-78, IRL Press, Oxford), and
the pET vector
series (Studier et al., 1990, Methods Enzymol., 185:60-89). However, a
potential drawback
of a prokaryotic host-vector system is the inability to perform many of the
post-translational
- 23 -
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
processing of mammalian cells. Thus, an eukaryotic host-vector system is
preferred, a
mammalian host-vector system is more preferred, and a human host-vector system
is the
most preferred.
For expression of the Stat3 activity modulator in mammalian host cells, a
variety of
regulatory regions can be used, for example, the SV40 early and late
promoters, the
cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus
long
terminal repeat (RSV-LTR) promoter. Inducible promoters that may be useftil in
mammalian cells include but are not limited to those associated with the
metallothionein II
gene, mouse mammary tumor virus glucocorticoid responsive long terminal
repeats
(MMTV-LTR), [3-interferon gene, and hsp70 gene (Williams et al., 1989, Cancer
Res.
49:2735-42 ; Taylor et al., 1990, Mol. Cell Biol., 10:165-75). It may be
advantageous to
use heat shock promoters or stress promoters to drive expression of the Stat3
activity
modulator in recombinant host cells.
In addition, the expression vector may contain selectable or screenable marker
genes
for initially isolating, identifying or tracking host cells that contain DNA
encoding the
elected Stat3 activity modulator. For long term, high yield production of the
elected Stat3
activity modulator, stable expression in mammalian cells is preferred. A
number of
selection systems may be used for mammalian cells, including but not limited
to the Herpes
simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223),
hypoxanthine-guanine
phosphoribosyltransferase (Szybalski and Szybalski, 1962, Proc. Natl. Acad.
Sci. USA
48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell
22:817) genes can
be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite
resistance can be
used as the basis of selection for dihydrofolate reductase (dhfr), which
confers resistance to
methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et
al., 1981, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic
acid
(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neomycin
phosphotransferase (neo), which confers resistance to the aminoglycoside G-418
(Colberre-
Garapin et al., 1981, J. Mol. Biol. 150:1); and hygromycin phosphotransferase
(hyg), which
confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Other
selectable
markers, such as but not limited to histidinol and ZeocinTM can also be used.
5.6.2 PRODUCTION OF RECOMBINANT PROTEINS
5.6.2.1 Peptide Tagging
If the Stat3 activity modulator is a protein (hereinafter: the protein of the
invention),
generating a fusion protein comprising a peptide tag can aid its purification.
In various
embodiments, such a fusion protein can be made by ligating the nucleotide
sequence
encoding the protein of the invention to the sequence encoding the peptide tag
in the proper
reading frame. If genomic sequences are used, care should be taken to ensure
that the
-24-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
modified gene remains within the same translational reading frame,
uninterrupted by
translational stop signals and/or spurious messenger RNA splicing signals.
In a specific embodiment, the peptide tag is fused at its amino terminal to
the
carboxyl terminal of the protein of the invention. The precise site at which
the fusion is
made is not critical. The optimal site can be determined by routine
experimentation.
A variety of peptide tags known in the art may be used in the modification of
the
protein of the invention, such as but not limited to the immunoglobulin
constant regions,
polyhistidine sequence (Petty, 1996, Metal-chelate affinity chromatography, in
Current
Protocols in Molecular Biology, Vol. 2, Ed. Ausubel et al., Greene Publish.
Assoc. & Wiley
Interscience), glutathione S-transferase (GST; Smith, 1993, Methods Mol. Cell
Bio. 4:220-
229), the E. coli maltose binding protein (Guar et al., 1987, Gene 67:21-30),
and various
cellulose binding domains (U.S. patent 5,496,934; 5,202,247; 5,137,819; Tomme
et al.,
1994, Protein Eng. 7:117-123), etc. Other peptide tags are recognized by
specific binding
partners and thus facilitate isolation by affinity binding to the binding
partner, which is
preferably immobilized and/or on a solid support. As will be appreciated by
those skilled in
the art, many methods can be used to obtain the coding region of the above-
mentioned
peptide tags, including but not limited to, DNA cloning, DNA amplification,
and synthetic
methods. Some of the peptide tags and reagents for their detection and
isolation are
available commercially.
5.6.2.2 Expression Systems and Host Cells
Preferred mammalian host cells include but are not limited to those derived
from
humans, monkeys and rodents, (see, for example, Kriegler M. in "Gene Transfer
and
Expression: A Laboratory Manual", New York, Freeman & Co. 1990), such as
monkey
kidney cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic
kidney line (293, 293-EBNA, or 293 cells subcloned for growth in suspension
culture,
Graham et al., J. Gen. Virol., 36:59, 1977; baby hamster kidney cells (BHK,
ATCC CCL
10); Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin. Proc. Natl.
Acad. Sci.
77; 4216, 1980); mouse sertoli cells (Mather, Biol. Reprod. 23:243-251, 1980);
mouse
fibroblast cells (NIH-3T3), monkey kidney cells (CVI ATCC CCL 70); african
green
monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCI~, ATCC CCL 34); buffalo rat
liver
cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human
liver
cells (Hep G2, HB 8065); and mouse mammary tumor cells (MMT 060562, ATCC
CCL51).
~' n~ber of viral-based expression systems may also be utilized with mammalian
cells to produce the Stat3 activity modulator. Vectors using DNA virus
backbones have
been derived from simian virus 40 (SV40) (Hamer et al., 1979, Cell 17:725),
adenovirus
-25-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
(Van Doren et al., 1984, Mol Cell Biol 4:1653), adeno-associated virus
(McLaughlin et al.,
1988, J Virol 62:1963), and bovine papillomas virus (Zinn et aL, 1982, Proc
Natl Acad Sci
79:4897). In cases where an adenovirus is used as an expression vector, the
donor DNA
sequence may be ligated to an adenovirus transcription/translation control
complex, e.g., the
late promoter and tripartite leader sequence. This chimeric gene may then be
inserted in the
adenovirus genome by in vitro or in vivo recombination. Insertion in a non-
essential region
of the viral genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable
and capable of expressing heterologous products in infected hosts. (See e.g.,
Logan and
Shenk, 1984, Proc. Natl. Acad. Sci. (LJSA) 81:3655-3659).
Other useful eukaryotic host-vector system may include yeast and insect
systems. In
yeast, a number of vectors containing constitutive or inducible promoters may
be used with
SacchaYOmyces cerevisiae (baker's yeast), Schizosaccha~omyces pombe (fission
yeast),
Pichia pasto~is, and Hansenula polymorpha (methylotropic yeasts). For a review
see,
Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al.,
Greene Publish.
Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and
Secretion Vectors
for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 1987, Acad. Press,
N.Y., Vol.
153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C.,
Ch. 3; and
Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology,
Eds. Berger
& I~immel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology
of the
Yeast SacclaaYOrnyces, 1982, Eds. Strathern et al., Cold Spring Harbor Press,
Vols. I and II.
In an insect system, Autographa californica nuclear polyhidrosis virus (AcNPV)
a
baculovirus, can be used as a vector to express the protein of the invention
in Spodoptera
frugiperda cells. The sequences encoding the protein of the invention may be
cloned into
non-essential regions (for example the polyhedrin gene) of the virus and
placed under
control of an AcNPV promoter (for example the polyhedrin promoter). These
recombinant
viruses are then used to infect host cells in which the inserted DNA is
expressed. .(See e.g.,
Smith et al., 1983, J Virol 46:584; Smith, U.S. Patent No. 4,215,051.)
Any of the cloning and expression vectors described herein may be synthesized
and
assembled from known DNA sequences by well known techniques in the art. The
regulatory regions and enhancer elements can be of a variety of origins, both
natural and
s~thetic. Some vectors and host cells may be obtained commercially. Non-
limiting
examples of useful vectors are described in Appendix 5 of Current Protocols in
Molecular
Biology, 1988, ed. Ausubel et al., Greene Publish. Assoc. & Wiley
Interscience, which is
incorporated herein by reference; and the catalogs of commercial suppliers
such as Clontech
Laboratories, Stratagene Inc., and Invitrogen, Inc.
Expression constructs containing cloned nucleotide sequence encoding the
protein
of the invention can be introduced into the host cell by a variety of
techniques known in the
art, including but not limited to, for prokaryotic cells, bacterial
transformation (Hanahan,
-26-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
1985, in DNA Cloning, A Practical Approach, 1:109-136), and for eukaryotic
cells, calcium
phosphate mediated transfection (Wigler et al., 1977, Cell 11:223-232),
liposome-mediated
transfection (Schaefer-Ridder et al., 1982, Science 215:166-168),
electroporation (Wolff et
al., 1987, Proc Natl Acad Sci 84:3344), and microinjection (Cappechi, 1980,
Cell 22:479-
488).
For long term, high yield production of the properly processed protein of the
invention, stable expression in mammalian cells is preferred. Cell lines that
stably express
protein of the invention may be engineered by using a vector that contains a
selectable
marker. By way of example but not limitation, following the introduction of
the expression
constructs, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and
then are switched to a selective media. The selectable marker in the
expression construct
confers resistance to the selection and optimally allows cells to stably
integrate the
expression construct into their chromosomes and to grow in culture and to be
expanded into
cell lines. Such cells can be cultured for a long period of time while the
protein of the
invention is expressed continuously.
5.6.2.3 Protein Purification
Generally, the protein of the invention can be recovered and purified from
recombinant cell cultures by known methods, including ammonium sulfate
precipitation,
acid extraction, anion or cation exchange chromatography, phosphocellulose
°~omatography, immunoaffmity chromatography, hydroxyapatite
chromatography, and
lectin chromatography. Before the protein of the invention can be purified,
total protein
has to be prepared from the cell culture. This procedure comprises collection,
washing and
lysis of said cells and is well known to the skilled artisan.
However, the invention provides methods for purification of the protein of the
invention which are based on the properties of the peptide tag present on the
protein of the
invention. One approach is based on specific molecular interactions between a
tag and its
binding partner. The other approach relies on the immunospecific binding of an
antibody to
an epitope present on the tag or on the protein which is to be purified. The
principle of
affinity chromatography well known in the art is generally applicable to both
of these
approaches.
Described below are several methods based on specific molecular interactions
of a
tag and its binding partner.
A method that is generally applicable to purifying protein of the invention
that are
fused to the constant regions of immunoglobulin is protein A affinity
chromatography, a
technique that is well known in the art. Staphylococcus protein A is a 42 kD
polypeptide
that binds specifically to a region located between the second and third
constant regions of
heavy chain immunoglobulins. Because of the Fc domains of different classes,
subclasses
-27-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
and species of immunoglobulins, affinity of protein A for human Fc regions is
strong, but
may vary with other species. Subclasses that are less preferred include human
IgG-3, and
most rat subclasses. For certain subclasses, protein G (of Streptococci) may
be used in
place of protein A in the purification. Protein-A sepharose (Pharmacia or
Biorad) is a
commonly used solid phase for affnuty purification of antibodies, and can be
used
essentially in the same manner for the purification of the protein of the
invention fused to an
immunoglobulin Fc fragment. Bound protein of the invention can be eluted by
various
buffer systems known in the art, including a succession of citrate, acetate
and glycine-HCl
buffers which gradually lowers the pH. This method is less preferred if the
recombinant
cells also produce antibodies which will be copurified with the protein of the
invention.
See, for example, Langone, 1982, J. Immunol. meth. 51:3; Wilchek et al., 1982,
Biochem.
Intl. 4:629; Sjobring et al., 1991, J. Biol. Chem. 26:399; page 617-618, in
Antibodies A
Laboratory Manual, edited by Harlow and Lane, Cold Spring Harbor laboratory,
1988.
Alternatively, a polyhistidine tag may be used, in which case, the protein of
the
invention can be purified by metal chelate chromatography. The polyhistidine
tag, usually a
sequence of six histidines, has a high affinity for divalent metal ions, such
as nickel ions
(Niz+), which can be immobilized on a solid phase, such as nitrilotriacetic
acid-matrices.
Polyhistidine has a well characterized affinity for Ni2+-NTA-agarose, and can
be eluted with
either of two mild treatments: imidazole (0.1-0.2 M) will effectively compete
with the resin
for binding sites; or lowering the pH just below 6.0 will protonate the
histidine sidechains
and disrupt the binding. The purification method comprises loading the cell
culture lysate
onto the Ni2+-NTA-agarose column, washing the contaminants through, and
eluting the
protein of the invention with imidazole or weak acid. Niz+-NTA-agarose can be
obtained
from commercial suppliers such as Sigma (St. Louis) and Qiagen. Antibodies
that
recognize the polyhistidine tag are also available which can be used to detect
and quantitate
the protein of the invention.
Another exemplary peptide tag that can be used is the glutathione-S-
transferase
(GST) sequence, originally cloned from the helininth, Schistosoma japonicurn.
In general, a
protein of the invention-GST fusion expressed in a prokaryotic host cell, such
as E. coli, can
be purified from the cell culture lysate by absorption with glutathione
agarose beads,
followed by elution in the presence of free reduced glutathione at neutral pH.
Since GST is
known to form dimers under certain conditions, dimeric protein of the
invention may be
obtained. See, Smith, 1993, Methods Mol. Cell Bio. 4:220-229.
Another useful peptide tag that can be used is the maltose binding protein
(MBP) of
E. coli, which is encoded by the malE gene. The protein of the invention binds
to amylose
resin while contaminants are washed away. The bound protein of the invention-
MBP
fusion is eluted from the amylose resin by maltose. See, for example, Guan et
al., 1987,
Gene 67:21-30.
-28-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
The second approach for purifying the protein of the invention is applicable
to
peptide tags that contain an epitope for which polyclonal or monoclonal
antibodies are
available. It is also applicable if polyclonal or monoclonal antibodies
specific to the protein
of the invention are available. Various methods known in the art for
purification of protein
by immunospecific binding, such as immunoaffinity chromatography, and
immunoprecipitation, can be used. See, for example, Chapter 13 in Antibodies A
Laboratory Manual, edited by Harlow and Lane, Cold Spring Harbor laboratory,
1988; and
Chapter 8, Sections I and II, in Current Protocols in Immunology, ed. by
Coligan et al.,
John Wiley, 1991; the disclosure of which are both incorporated by reference
herein.
5.6.3 GENE THERAPY APPROACHES
In a specific embodiment, nucleotide sequences encoding Stat3, Stat3beta,
Stat3-C,
IL-6 or nucleotide sequences encoding therapeutic RNA molecules, such as
antisense RNA
and ribozymes specific to Stat3, are administered to treat, or prevent various
diseases.
These nucleotide sequences are collectively referred to as nucleotide
sequences of the
invention. Gene therapy refers to therapy performed by the administration to a
subject of an
expressed or expressible nucleotide sequence. In this embodiment of the
invention, the
nucleotide sequences produce their encoded protein or RNA molecule that
mediates a
therapeutic effect.
Any of the methods for gene therapy available in the art can be used according
to the
present invention. Exemplary methods are described below.
For general reviews of the method of gene therapy, see, Goldspiel et al.,
1993,
Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev,
1993,
Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932;
Morgan
and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH
1,1(5):155-
215. Methods commonly known in the art of recombinant DNA technology which can
be
used are described in Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A
Laboratory
Manual, Stockton Press, NY (1990).
In a specific embodiment, nucleic acid molecules are used in which the
nucleotide
sequence of the invention is flanked by regions that promote homologous
recombination at
a desired site in the genome, thus providing for intrachromosomal expression
of the
nucleotide sequence of the invention (Roller and Smithies, 1989, Proc. Natl.
Acad. Sci.
USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
In a specific embodiment, the nucleic acid sequences are directly administered
ira
vivo, where it is expressed to produce the encoded product. This can be
accomplished by
any of numerous methods known in the art, for example by constructing them as
part of an
appropriate nucleic acid expression vector and administering the vector so
that the nucleic
-29-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
acid sequences become intracellular. Gene therapy vectors can be administered
by infection
using defective or attenuated retrovirals or other viral vectors (see, e.g.,
U.S. Patent No.
4,980,286); direct injection of naked DNA; use of microparticle bombardment
(e.g., a gene
gun; Biolistic, Dupont); coating with lipids or cell-surface receptors or
transfecting agents;
encapsulation in liposomes, microparticles, or microcapsules; administration
in linkage to a
peptide which is known to enter the nucleus; administration in linkage to a
ligand subject to
receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem.
262:4429-4432)
(which can be used to target cell types specifically expressing the
receptors); etc. In another
embodiment, nucleic acid-ligand complexes can be formed in which the ligand
comprises a
fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to
avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be targeted in
vivo for cell
specific uptake and expression, by targeting a specific receptor (see, e.g.,
PCT Publications
WO 92/06 180; WO 92/22635; W092/20316; W093/14188, and WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly and
incorporated within host
cell DNA for expression by homologous recombination (Koller and Smithies,
1989, Proc.
Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
In a specific embodiment, viral vectors that contain the nucleotide sequence
of the
invention are used. For example, a retroviral vector can be used (see Miller
et al., 1993,
Meth. Enzymol. 217:581-599). These retroviral vectors contain the components
necessary
for the correct packaging of the viral genome and integration into the host
cell DNA. The
nucleotide sequences of the invention to be used in gene therapy are cloned
into one or
more vectors, thereby facilitating delivery of the gene into a patient. More
detail about
retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:29 1-302,
which
describes the use of a retroviral vector to deliver the mdr 1 gene to
hematopoietic stem cells
in order to make the stem cells more resistant to chemotherapy. Other
references
illustrating the use of retroviral vectors in gene therapy are: Clowes et al.,
1994, J. Clin.
Invest. 93:644-651; Klein et al., 1994, Blood 83:1467-1473; Salmons and
Gunzberg, 1993,
Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in
Genetics
and Devel. 3:110-114.
The following animal regulatory regions, which exhibit tissue specificity and
have
been utilized in transgenic animals, can be used for expression in a
particular tissue type:
elastase I gene control region which is active in pancreatic acinar cells
(Swift et al., 1984,
Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.
50:399-409;
MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is
active in
pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene
control
region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-
658; Adames
et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.
7:1436-1444),
mouse mammary tumor virus control region which is active in testicular,
breast, lymphoid
-30-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control
region which is
active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-
fetoprotein gene
control region which is active in the liver (Krumlauf et al., 1985, Mol. Cell.
Biol. 5:1639-
1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control
region
which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-
171), beta-globin
gene control region which is active in myeloid cells (Mogram et al., 1985,
Nature 315:338-
340; Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene control
region which is
active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell
48:703-712);
myosin light chain-2 gene control region which is active in skeletal muscle
(Sani, 1985,
Nature 314:283-286), and gonadotropic releasing hormone gene control region
which is
active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).
5.6.4 INHIBITORY ANTISENSE, RIBOZYME AND TRIPLE HELIX
MOLECULES
Among the compounds that may exhibit the ability to modulate the activity of
Stat3
are antisense, ribozyme, and triple helix molecules. Techniques for the
production and use
of such molecules are well known to those of skill in the art. For example,
antisense
targeting Stat3 mRNA inhibits Stat3 signaling, as described in Section 8 (see
Figures 12
and 13).
Antisense RNA and DNA molecules act to directly block the translation of mRNA
by hybridizing to targeted mRNA and preventing protein translation. Antisense
approaches
involve the design of oligonucleotides that are complementary to a target gene
mRNA. The
antisense oligonucleotides will bind to the complementary target gene mRNA
transcripts
and prevent translation. Absolute complementarity, although preferred, is not
required.
A sequence "complementary" to a portion of an RNA, as referred to herein,
means a
sequence having sufficient complementarity to be able to hybridize with the
RNA, forming
a stable duplex; in the case of double-stranded antisense nucleic acids, a
single strand of the
duplex DNA may thus be tested, or triplex formation may be assayed. The
ability to
hybridize will depend on both the degree of complementarity and the length of
the antisense
nucleic acid. Generally, the longer the hybridizing nucleic acid, the more
base mismatches
with an RNA it may contain and still form a stable duplex (or triplex, as the
case may be).
One skilled in the art can ascertain a tolerable degree of mismatch by use of
standard
procedures to determine the melting point of the hybridized complex.
In one embodiment, oligonucleotides complementary to non-coding regions of the
Stat3 gene could be used in an antisense approach to inhibit translation of
endogenous Stat3
~A' ~tisense nucleic acids should be at least six nucleotides in length, and
are
preferably oligonucleotides ranging from 6 to about 50 nucleotides in length.
In specific
-31-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
aspects the oligonucleotide is at least 10 nucleotides, at least 17
nucleotides, at least 25
nucleotides or at least 50 nucleotides.
In an embodiment of the present invention, oligonucleotides complementary to
the
nucleic acids encoding the Stat3 protein as indicated in SEQ ID. NO: 1.
Stat3 antisense molecules complementary to coding or non-coding regions may be
used, members of both are well known in the art. Representative, non-limiting
examples of
Stat3 antisense molecules include the following: 5'- ACTCAAACTGCCCTCCTGCT-3';
5'- TCTGAAGAAACTGCTTGATT-3'; 5'-GCCACAATCCGGGCAATCT-3'; 5'-
TGGCTGCAGTCTGTAGAAGG-3'; 5'-TTTCTGTTCTAGATCCTGCA-3'; 5'-
TAGTTGAAATCAAAGTCATC-3'; 5'-TTCCATTCAGATCTTGCATG-3'; 5'-
TCTGTTCCAGCTGCTGCATC-3'; 5'-TCACTCACGATGCTTCTCCG-3'; 5'-
GAGTTTTCTGCACGTACTCC-3' (see, e.g., U.S. Patent No. 6,159,694, issued December
12, 2000, which is incorporated herein in its entirety).
Regardless of the choice of target sequence, it is preferred that in vitro
studies are
first performed to quantitate the ability of the antisense oligonucleotide to
inhibit gene
expression. It is preferred that these studies utilize controls that
distinguish between
antisense gene inhibition and nonspecific biological effects of
oligonucleotides. It is also
preferred that these studies compare levels of the target RNA or protein with
that of an
internal control RNA or protein. Additionally, it is envisioned that results
obtained using
the antisense oligonucleotide are compared with those obtained using a control
°ligonucleotide. It is preferred that the control oligonucleotide is of
approximately the
same length as the test oligonucleotide and that the nucleotide sequence of
the
oligonucleotide differs from the antisense sequence no more than is necessary
to prevent
specific hybridization to the target sequence.
The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or
modified versions thereof, single-stranded or double-stranded. The
oligonucleotide can be
modified at the base moiety, sugar moiety, or phosphate backbone, for example,
to improve
stability of the molecule, hybridization, etc. The oligonucleotide may include
other
appended groups such as peptides (e.g., for targeting host cell receptors in
vivo), or agents
facilitating transport across the cell membrane (see, e.g., Letsinger, et al.,
1989, Proc. Natl.
Acad. Sci. U.S.A. 86, 6553-6556; Lemaitre, et al., 1987, Proc. Natl. Acad.
Sci. 84, 648-652;
PCT Publication No. W088/09810, published December 15, 1988) or the blood-
brain
barner (see, e.g., PCT Publication No. W089/10134, published April 25, 1988),
hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988,
BioTechniques 6, 958-
976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5, 539-549).
To this end, the
°ligonucleotide may be conjugated to another molecule, e.g., a peptide,
hybridization
triggered cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
-32-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
The antisense oligonucleotide may comprise at least one modified base moiety
which is selected from the group including but not limited to 5-fluorouracil,
5-brornouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-
(caxboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-
carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine, N6-
isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-
methyladenine, 2-methylguaune, 3-methylcytosine, 5-methylcytosine, N6-adenine,
7-
methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-
mannosylqueosine, 5 -methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-
N6-
isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil,
queosine, 2-
thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
methyluracil, uracil-5-
oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil, 3-(3-amino-
3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
The antisense oligonucleotide may also comprise at Ieast one modified sugar
moiety
selected from the group including but not limited to arabinose, 2-
fluoroarabinose, xylulose,
and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at least
one
modified phosphate backbone selected from the group consisting of a
phosphorothioate (S-
ODNs), a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a
formacetal or
analog thereof.
In yet another embodiment, the antisense oligonucleotide is an -anomeric
oligonucleotide. An -anomeric oligonucleotide forms specific double-stranded
hybrids
with complementary RNA in which, contrary to the usual -units, the strands run
parallel to
each other (Gautier, et al., 1987, Nucl. Acids Res. 15, 6625-6641), The
oligonucleotide is a
2 -0-methylribonucleotide (moue, et al., 1987, Nucl. Acids Res. 15, 6131-
6148), or a
chimeric RNA-DNA analogue (moue, et al., 1987, FEBS Lett. 2I5, 327-330).
Oligonucleotides of the invention may be synthesized by standard methods known
in the art, e.g. by use of an automated DNA synthesizer (such as axe
commercially available
from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein, et al. (1988,
Nucl. Acids Res.
16, 3209), methylphosphonate oligonucleotides can be prepared by use of
controlled pore
glass polymer supports (Satin, et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85,
7448-7451),
etc.
While antisense nucleotides complementary to the target gene coding region
sequence could be used, those complementary to the transcribed, untranslated
region are
most preferred.
-33-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
In one embodiment of the present invention, gene expression downregulation is
achieved because specific target mRNAs are digested by RNAse H after they have
hybridized with the antisense phosphorothioate oligonucleotides (S-ODNs).
Since no rules
exist to predict which antisense S-ODNs will be more successful, the best
strategy is
completely empirical and consists of trying several antisense S-ODNs.
Antisense
phosphorothioate oligonucleotides (S-ODNs) will be designed to target specific
regions of
mRNAs of interest. Control S-ODNs consisting of scrambled sequences of the
antisense S-
ODNs will also be designed to assure identical nucleotide content and minimize
differences
potentially attributable to nucleic acid content. All S-ODNs will be
synthesized by Oligos
Etc. (Wilsonville, OR). In order to test the effectiveness of the antisense
molecules when
applied to cells in culture, such as assays for research purposes or ex vivo
gene therapy
protocols, cells will be grown to 60-80% confluence on 100 mm tissue culture
plates, rinsed
with PBS and overlaid with lipofection mix consisting of 8 ml Opti-MEM, 52.8 1
Lipofectin, and a final concentration of 200 nM S-ODNs. Lipofections will be
carried out
using Lipofectin Reagent and Opti-MEM (Gibco BRL). Cells will be incubated in
the
presence of the lipofection mix for 5 hours. Following incubation the medium
will be
replaced with complete DMEM. Cells will be harvested at different time points
post-
lipofection and protein levels will be analyzed by Western blot.
Antisense molecules should be targeted to cells that express the target gene,
either
directly to the subj ect in vivo or to cells in culture, such as in ex vivo
gene therapy
protocols. A number of methods have been developed for delivering antisense
DNA or
RNA to cells; e.g., antisense molecules can be injected directly into the
tissue site, or
modified antisense molecules, designed to target the desired cells (e.g.,
antisense linked to
peptides or antibodies that specifically bind receptors or antigens expressed
on the target
cell surface) can be administered systemically.
However, it is often difficult to achieve intracellular concentrations of the
antisense
sufficient to suppress translation of endogenous mRNAs. Therefore a preferred
approach
utilizes a recombinant DNA construct in which the antisense oligonucleotide is
placed
under the control of a strong pol III or pol II promoter. The use of such a
construct to
transfect target cells in the patient will result in the transcription of
sufficient amounts of
single stranded RNAs that will form complementary base pairs with the
endogenous target
gene transcripts and thereby prevent translation of the target gene mRNA. For
example, a
vector can be introduced e.g., such that it is taken up by a cell and directs
the transcription
of an antisense RNA. Such a vector can remain episomal or become chromosomally
integrated, as long as it can be transcribed to produce the desired antisense
RNA. Such
vectors can be constructed by recombinant DNA technology methods standard in
the art.
Vectors can be plasmid, viral, or others known in the art, used for
replication and expression
in mammalian cells. Expression of the sequence encoding the antisense RNA can
be by any
-34-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
promoter known in the art to act in mammalian, preferably human cells. Such
promoters
can be inducible or constitutive. Such promoters include but are not limited
to: the SV40
early promoter region (Bernoist and Chambon, 1981, Nature 290, 304-310), the
promoter
contained in the 3 long terminal repeat of Rous sarcoma virus (Yamamoto, et
al., 1980,
Cell 22, 787-797), the herpes thymidine kinase promoter (Wagner, et al., 1981,
Proc. Natl.
Acad. Sci. U.S.A. 78, 1441-1445), the regulatory sequences of the
metallothionein gene
(Brinster, et al., 1982, Nature 296, 39-42), etc. Any type of plasmid, cosmid,
YAC or viral
vector can be used to prepare the recombinant DNA construct which can be
introduced
directly into the tissue site. Alternatively, viral vectors can be used that
selectively infect
the desired tissue, in which case administration may be accomplished by
another route (e.g.,
systemically).
Ribozyme molecules designed to catalytically cleave target gene mRNA
transcripts
can also be used to prevent translation of target gene mRNA and, therefore,
expression of
target gene product (see, e.g., PCT International Publication WO90/11364,
published
October 4, 1990; Sarver, et al., 1990, Science 247, 1222-1225). In an
embodiment of the
present invention, oligonucleotides which hybridize to the Stat3 gene are
designed to be
complementary to the nucleic acids encoding the Stat3 protein (SEQ m. NO: 2).
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific
cleavage of RNA. (For a review, see Rossi, 1994, Current Biology 4, 469-471).
The
mechanism of ribozyme action involves sequence specific hybridization of the
ribozyme
molecule to complementary target RNA, followed by an endonucleolytic cleavage
event.
The composition of ribozyme molecules must include one or more sequences
complementary to the target gene mRNA, and must include the well known
catalytic
sequence responsible for mRNA cleavage. For this sequence, see, e.g., U.S.
Patent No.
5,093,246, which is incorporated herein by reference in its entirety.
While ribozymes that cleave mRNA at site specific recognition sequences can be
used to destroy target gene mRNAs, the use of hammerhead ribozymes is
preferred.
Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions
that form
complementary base pairs with the target mRNA. The sole requirement is that
the target
mRNA have the following sequence of two bases: 5'-UG-3'. The construction and
production of hammerhead ribozymes is well known in the art and is described
more fully
in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk
Reference,
VCH Publishers, New York, (see especially Figure 4, page 833) and in Haseloff
& Gerlach,
1988, Nature, 334, 585-591, which is incorporated herein by reference in its
entirety.
Preferably the ribozyme is engineered so that the cleavage recognition site is
located
near the 5' end of the target gene mRNA, i.e., to increase efficiency and
minimize the
intracellular accumulation of non-functional mRNA transcripts.
-35-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
The ribozymes of the present invention also include RNA endoribonucleases
(hereinafter "Cech-type ribozymes") such as the one that occurs naturally in
Tetrahymena
thermophila (known as the IVS, or L-19 IVS RNA) and that has been extensively
described
by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224, 574-578;
Zaug and
Cech, 1986, Science, 231, 470-475; Zaug, et al., 1986, Nature, 324, 429-433;
published
International patent application No. WO 88/04300 by University Patents Inc.;
Been ~ Cech,
1986, Cell, 47, 207-216). The Cech-type ribozymes have an eight base pair
active site
which hybridizes to a target RNA sequence whereafter cleavage of the target
RNA takes
place. The invention encompasses those Cech-type ribozymes which target eight
base-pair
active site sequences that are present in the target gene.
As in the antisense approach, the ribozymes can be composed of modified
oligonucleotides (e.g., for improved stability, targeting, etc.) and should be
delivered to
cells that express the target gene in vivo. A preferred method of delivery
involves using a
DNA construct "encoding" the ribozyme under the control of a strong
constitutive pol III or
pol II promoter, so that transfected cells will produce sufficient quantities
of the ribozyme
to destroy endogenous target gene messages and inhibit translation. Because
ribozymes
unlike antisense molecules, are catalytic, a lower intracellular concentration
is required for
efficiency.
Endogenous target gene expression can also be reduced by inactivating or
"knocking
out" the target gene or its promoter using targeted homologous recombination
(e.g., see
Smithies, et al., 1985, Nature 317, 230-234; Thomas & Capecchi, 1987, Cell 51,
503-512;
Thompson, et al., 1989, Cell 5, 313-321; each of which is incorporated by
reference herein
in its entirety). For example, a mutant, non-functional target gene (or a
completely
unrelated DNA sequence) flanked by DNA homologous to the endogenous target
gene
(either the coding regions or regulatory regions of the target gene) can be
used, with or
without a selectable marker and/or a negative selectable marker, to transfect
cells that
express the target gene in vivo. Insertion of the DNA construct, via targeted
homologous
recombination, results in inactivation of the target gene. Such approaches are
particularly
suited modifications to ES (embryonic stem) cells can be used to generate
animal offspring
with an inactive target gene (e.g., see Thomas & Capecchi, 1987 and Thompson,
1989,
supra). However this approach can be adapted for use in humans provided the
recombinant
DNA constructs are directly administered or targeted to the required site in
vivo using
appropriate viral vectors.
Alternatively, endogenous target gene expression can be reduced by targeting
deoxyribonucleotide sequences complementary to the regulatory region of the
target gene
(i~e., the target gene promoter and/or enhancers) to form triple helical
structures that prevent
transcription of the target gene in target cells in the body. (See generally,
Helene, 1991,
-36-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Anticancer Drug Des., 6(6), 569-584; Helene, et al., 1992, Ann. N.Y. Acad.
Sci., 660, 27-
36; and Maher, 1992, Bioassays 14(12), 807-815).
Nucleic acid molecules to be used in triple helix formation for the inhibition
of
transcription should be single stranded and composed of deoxynucleotides. The
base
composition of these oligonucleotides must be designed to promote triple helix
formation
via Hoogsteen base pairing rules, which generally require sizeable stretches
of either
purines or pyrimidines to be present on one strand of a duplex. Nucleotide
sequences may
be pyrimidine-based, which will result in TAT and CGC+ triplets across the
three
associated strands of the resulting triple helix. The pyrimidine-rich
molecules provide base
complementarity to a purine-rich region of a single strand of the duplex in a
parallel
°rientation to that strand. In addition, nucleic acid molecules may be
chosen that are
purine-rich, for example, contain a stretch of G residues. These molecules
will form a triple
helix with a DNA duplex that is rich in GC pairs, in which the majority of the
purine
residues are located on a single strand of the targeted duplex, resulting in
GGC triplets
across the three strands in the triplex.
Alternatively, the potential sequences that can be targeted for triple helix
formation
may be increased by creating a so called "switchback" nucleic acid molecule.
Switchback
molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that
they base pair with
first one strand of a duplex and then the other, eliminating the necessity for
a sizeable
stretch of either purines or pyrimidines to be present on one strand of a
duplex.
In instances wherein the antisense, ribozyme, and/or triple helix molecules
described
herein are utilized to inhibit mutant gene expression, it is possible that the
technique may so
efficiently reduce or inhibit the transcription (triple helix) and/or
translation (antisense,
ribozyme) of mRNA produced by normal target gene alleles that the possibility
may arise
wherein the concentration of normal target gene product present may be lower
than is
necessary for a normal phenotype. In such cases, to ensure that substantially
normal levels
of target gene activity are maintained, therefore, nucleic acid molecules that
encode and
express target gene polypeptides exhibiting normal target gene activity may,
be introduced
into cells via gene therapy methods such as those described, below, in Section
5.7.2 that do
not contain sequences susceptible to whatever antisense, ribozyme, or triple
helix treatments
are being utilized. Alternatively, in instances whereby the target gene
encodes an
extracellular protein, it may be preferable to co-administer normal target
gene protein in
order to maintain the requisite level of target gene activity.
Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention
may be prepared by any method known in the art for the synthesis of DNA and
RNA
molecules, as discussed above. These include techniques for chemically
synthesizing
oligodeoxyribonucleotides and oligoribonucleotides well known in the art such
as for
example solid phase phosphoramidite chemical synthesis. Alternatively, RNA
molecules
-37-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
may be generated by in vitro and in vivo transcription of DNA sequences
encoding the
antisense RNA molecule. Such DNA sequences may be incorporated into a wide
variety of
vectors that incorporate suitable RNA polymerase promoters such as the T7 or
SP6
polymerase promoters. Alternatively, antisense cDNA constructs that synthesize
antisense
RNA constitutively or inducibly, depending on the promoter used, can be
introduced stably
into cell lines.
5.6.5 ANTIBODIES TO STAT3 AND DERIVATIVES
According to the invention, Stat3, its fragments or other derivatives, or
analogs
thereof, may be used as an immunogen to generate antibodies which
immunospecifically
bind such an immunogen.
Antibodies of the invention include, but are not limited to, polyclonal,
monoclonal,
multispecific, human, humanized or chimeric antibodies, single chain
antibodies, Fab
fragments, F(ab') fragments, fragments produced by a Fab expression library,
anti-idiotypic
(anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the
invention), and
epitope-binding fragments. The term "antibody," as used herein, refers to
immunoglobulin
molecules and immunologically active portions of immunoglobulin molecules,
i.e.,
molecules that contain an antigen binding site that immunospecifically binds
an antigen.
The immunoglobulin molecules of the invention can be of any type (e.g., IgG,
IgE, lgM,
IgD, IgA and IgY), class (e.g., IgGI, IgG2, IgG3, IgG~, IgAI and IgAz) or
subclass of
immunoglobulin molecule. Examples of immunologically active portions of
immunoglobulin molecules include Flab) and F(ab')2 fragments which can be
generated by
treating the antibody with an enzyme such as pepsin or papain. In a specific
embodiment,
antibodies to a human Stat3 protein are produced. In another embodiment,
antibodies to a
domain of Stat3 are produced.
Various procedures known in the art may be used for the production of
polyclonal
antibodies to Stat3 or derivative or analog. In a particular embodiment,
rabbit polyclonal
antibodies to an epitope of Stat3 encoded by a sequence or fragment of SEQ ID
NO: 2, or a
subsequence thereof, can be obtained. For the production of antibody, various
host animals
can be immunized by injection with the native Stat3, or a synthetic version,
or derivative
(e.g., fragment) thereof, including but not limited to rabbits, mice, rats,
etc. Various
adjuvants may be used to increase the immunological response, depending on the
host
species, and including but not limited to Freund's (complete and incomplete),
mineral gels
such as aluminum hydroxide, surface active substances such as lysolecithin,
pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,-
dinitrophenol,
and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin)
and
corynebacterium parvuxn.
-38-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
For preparation of monoclonal antibodies directed toward an Stat3 sequence or
analog thereof, any technique which provides for the production of antibody
molecules by
continuous cell lines in culture may be used. For example, the hybridoma
technique
originally developed by Kohler and Milstein (1975, Nature 256:495-497), as
well as the
trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983,
Immunology
Today 4:72), and the EBV-hybridoma technique to produce human monoclonal
antibodies
(Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp.
77-96). In an additional embodiment of the invention, monoclonal antibodies
can be
produced in germ-free animals utilizing recent technology (PCT/US90/02545).
According
to the invention, human antibodies may be used and can be obtained by using
human
hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or
by
transforming human B cells with EBV virus in vitro (Cole et al., 1985, in
Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact, according to
the
invention, techniques developed for the production of "chimeric antibodies"
(Morrison et
al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984,
Nature
312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes
from a mouse
antibody molecule specific for Stat3 together with genes from a human antibody
molecule
of appropriate biological activity can be used; such antibodies are within the
scope of this
invention.
According to the invention, techniques described for the production of single
chain
antibodies (U.S. Patent No. 4,946,778) can be adapted to produce Stat3-
specific single
chain antibodies. An additional embodiment of the invention utilizes the
techniques
described for the construction of Fab expression libraries (Huse et al., 1989,
Science
246:1275-1281) to allow rapid and easy identification of monoclonal Fab
fragments with
the desired specificity for Stat3s, derivatives, or analogs.
~tibody fragments which contain the idiotype of the molecule can be generated
by
known techniques. For example, such fragments include but are not limited to:
the F(ab')2
fragment which can be produced by pepsin digestion of the antibody molecule;
the Fab'
fragments which can be generated by reducing the disulfide bridges of the
F(ab')2 fragment,
the Fab fragments which can be generated by treating the antibody molecule
with.papain
and a reducing agent, and Fv fragments.
In the production of antibodies, screening for the desired antibody can be
accomplished by techniques known in the art, e.g. ELISA (enzyme-linked
immunosorbent
assay). For example, to select antibodies which recognize a specific domain of
a STAT,
e.g., the transcriptional activation domain, DNA binding domain, dimerization
domain, SH2
domain, or SH3 domain, one may assay generated hybridomas for a product which
binds to
a Stat3 fragment containing such domain. For selection of an antibody that
specifically
binds a first Stat3 homolog but which does not specifically bind a different
Stat3 homolog,
-39-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
one can select on the basis of positive binding to the first Stat3 homolog and
a lack of
binding to the second Stat3 homolog.
Antibodies specific to a domain of Stat3 are also provided, such as to a
transcriptional activation domain, DNA binding domain, a dimerization domain,
SH2
domain, SH3 domain.
The foregoing antibodies can be used in methods known in the art relating to
the
localization and activity of the Stat3 sequences of the invention, e.g., for
imaging these
proteins, measuring levels thereof in appropriate physiological samples, in
diagnostic
methods, etc.
In another embodiment of the invention (see irzfYa), anti-Stat3 antibodies and
fragments thereof containing the binding domain are used as therapeutics.
Anti-Stat3 antibodies can be obtained from Santa Cruz Biotechnology, Inc.
(Santa
Cruz, CA), Research Diagnostics, Inc. (Flanders, NJ) or Zymed Laboratories
(South San
Francisco, CA). Alternatively, anti-Stat3 antibodies antibodies can be
produced by any
method known in the art for the synthesis of antibodies, in particular, by
chemical synthesis
or preferably, by recombinant expression techniques.
5.7 TARGET DISEASES AND DISORDERS
In one embodiment, Stat3 agonists may be used to stimulate angiogenesis for
the
~eatment or prevention of ischemic diseases. Ischemia is caused by an impaired
blood
supply resulting from narrowed or blocked arteries that starve tissues of
needed nutrients
and oxygen. Thus, any condition which reduces the availability of nutrients or
oxygen to a
tissue, resulting in stress, damage, and finally, cell death, may be treated
by the methods of
the present invention. Ischemic disorders that may be treated by the methods
described
herein include, but are not limited to, coronary-atherosclerosis induced
myocardial
infarction and tissue ischemia in the lower extremities. In another
embodiment, Stat3
agonists may be used to protect cardiac tissue from injury sustained during
ischemia,
infarction, inflammation, or trauma. These conditions arise from or include,
but are not
limited to stroke, vascular occlusion, prenatal or postnatal oxygen
deprivation, suffocation,
choking, near drowning, carbon monoxide poisoning, smoke inhalation, trauma,
including
surgery and radiotherapy, asphyxia, epilepsy, hypoglycemia, chronic
obstructive pulmonary
disease, emphysema, adult respiratory distress syndrome, hypotensive shock,
septic shock,
anaphylactic shock, insulin shock, sickle cell crisis, cardiac arrest,
dysrhytlunia, and
nitrogen narcosis.
In another embodiment, autoimmune diseases that can be treated by the methods
of
the present invention include, but are not limited to, insulin dependent
diabetes mellitus
(i.e., IDDM, or autoimmune diabetes), multiple sclerosis, systemic lupus
erythematosus,
-40-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed
connective
tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune
thyroiditis,
idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves'
disease,
myastheiua gravis, autoimmune neutropenia, idiopathic thrombocytopenia
purpura,
rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility,
Goodpasture's
disease, bullous pemphigoid, discoid lupus, ulcerative colitis, and dense
deposit disease.
The diseases set forth above, as referred to herein, include those exhibited
by animal models
for such diseases, such as, for example non-obese diabetic (NOD) mice for IDDM
and
experimental autoimmune encephalomyelitis (EAE) mice for multiple sclerosis.
The methods of the present invention can be used to treat such autoimmune
diseases
by reducing or eliminating the immune response to the patient's own (self)
tissue, or,
alternatively, by reducing or eliminating a pre-existing autoimmune response
directed at
tissues or organs transplanted to replace self tissues or organs damaged by
the autoimmune
response.
Inflammation caused by infectious diseases may also be treated or prevented
using
the methods and compositions of the present invention. Such infectious
diseases include
those caused by intracellular pathogens such as viruses, bacteria, protozoans,
and
intracellular parasites. Viruses include, but are not limited to viral
diseases such as those
caused by hepatitis type B virus, parvoviruses, such as adeno-associated virus
and
cytomegalovirus, papovaviruses such as papilloma virus, polyoma viruses, and
SV40,
adenoviruses, herpes viruses such as herpes.simplex type I (HSV-I), herpes
simplex type II
(HSV-II), and Epstein-Barr virus, poxviruses, such as variola (smallpox) and
vaccinia virus,
RNA viruses, including but not limited to human immunodeficiency virus type I
(HIV-I),
human immunodeficiency virus type II (HIV-II), human T-cell lymphotropic virus
type I
(HTLV-I), and human T-cell lymphotropic virus type II (HTLV-II); influenza
virus,
measles virus, rabies virus, Sendai virus, picornaviruses such as
poliomyelitis virus,
coxsackieviruses, rhinoviruses, reoviruses, togaviruses such as rubella virus
(German
measles) and Semliki forest virus, arboviruses, and hepatitis type A virus.
In another embodiment, bacterial infections can be treated or prevented such
as, but
not limited to disorders caused by pathogenic bacteria including, but not
limited to,
streptococcus pyogenes, Streptococcus pneunzozziae, Neisseria gonorrhoea,
Neisseria
nzenizzgitidis, Cozynebacteriunz diphtheriae, Clostridium botulinuzn,
Clostridium
perfringens, Clostz°idiunz tetani, Haemoplzilus izzfluenzae, Klebsiella
przeuzzzoniae,.
Klebsiella ozaenae, Klebsiella rlzinosclerozzzotis, Staphylococcus aureus,
Vibrio clzolerae,
Eschericlzia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus ,
Campylobacter
~e~ujzi, Aeronzonas hydroplzila, Bacillus cereus, Edwardsiella tarda, Yersinia
enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella
dysenteriae, Shigella
flexneri, Shigella sonnei, Salnzonella typhiimurium, Salmonella typhii,
Treponezna
-41 -
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii,
Borrelia
burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis,
Toxoplasma
gondii, Pneumocystis carinii, Francisella tularensis, Brucella abortus,
Brucella suis,
Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia
tsutsugumushi,
Chlamydia spp., and Helicobacter pylori.
In another preferred embodiment, the methods can be used to treat or prevent
infections caused by pathogenic protozoans such as, but not limited to,
Entomoeba
histolytica, Trichomonas tenas, Trichomonas hominis, Trichotrortas vaginalis,
Trypanosoma gambiense, Tiypanosoma rhodesiense, Trypanosonta cruzi,
Leishmaytia
donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis
pneumoyzia,
Plasmodium vivax, Plasmodium falciparum, and Plasmodium malaria.
With respect to specific proliferative and oncogenic disease, the diseases
that can be
treated or prevented by the methods of the present invention include, but are
not limited to:
human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
l~phangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,
Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,
breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell
carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma,
seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular
tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma,
neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and
acute
myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic
and
erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia
and
chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's
disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and
heavy
chain disease.
Diseases and disorders involving a deficiency in cell proliferation or in
which cell
proliferation is desired for treatment or prevention, and that can be treated
or prevented by
antagonizing Stat3, include but are not limited to degenerative disorders,
growth .
deficiencies, hypoproliferative disorders, physical trauma, lesions, and
wounds; for
example, to promote wound healing, or to promote regeneration in degenerated,
lesioned or
injured tissues, etc.
-42-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
5.8 PHARMACEUTICAL FORMULATIONS AND MODES OF ADMINISTRATION
In a preferred aspect, a pharmaceutical of the invention comprises a
substantially
purified protein, nucleic acid, or chemical (e.g., substantially free from
substances that limit
its effect or produce undesired side-effects). The subj ect is preferably an
animal, including
but not limited to animals such as cows, pigs, horses, chickens, cats, dogs,
etc., and is
preferably a mammal, and most preferably human.
Various delivery systems are known and can be used to administer the
pharmaceutical of the invention, e.g., encapsulation in liposomes,
microparticles,
microcapsules, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J.
Biol. Chem.
262:4429-4432), construction of a nucleic acid as part of a retroviral or
other vector, etc.
Methods of introduction include but are not limited to intradermal,
intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral
routes. Nucleic
acids and proteins of the invention may be administered by any convenient
route, for
example by infusion or bolus injection, by absorption through epithelial or
mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and rnay be
administered
together with other biologically active agents such as chemotherapeutic
agents.
Administration can be systemic or local.
In a specific embodiment, it may be desirable to administer the nucleic acid
or
protein of the invention by injection, by means of a catheter, by means of a
suppository, or
by means of an implant, said implant being of a porous, non-porous, or
gelatinous material,
including a membrane, such as a sialastic membrane, or a fiber. Preferably,
when
administering a protein, including an antibody, of the invention, care must be
taken to use
materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a
vesicle,
in particular a liposome (see Larger, 1990, Science 249:1527-1533; Treat et
al., 1989, in
Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler
(eds.), Liss, New York, pp. 353- 365; Lopez-Berestein, ibid., pp. 317-327; see
generally,
ibid.)
In yet another embodiment, the compound or composition can be delivered in a
controlled release system. In one embodiment, a pump may be used (see
Langer,~supra;
Sefton, 1989, CRC Crit. Ref. Biomed. Erg. 14:201; Buchwald et al., 1980,
Surgery 88:507;
Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment,
polymeric
materials can be used (see Medical Applications of Controlled Release, 1974,
Larger and
Wise (eds.), CRC Pres., Boca Raton, Florida; Controlled Drug Bioavailability,
Drug
Product Design and Performance, 1984, Smolen and Ball (eds.), Wiley, New York;
Ranger
and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et
al., 1985,
- 43 -
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al.,
1989, J.
Neurosurg. 71:105).
Other controlled release systems are discussed in the review by Langer, 1990,
Science 249:1527-1533.
In a specific embodiment where a nucleic acid of the invention is
administered, the
nucleic acid can be administered ih vivo to promote expression of its encoded
protein or
RNA molecule, by constructing it as part of an appropriate nucleic acid
expression vector
and administering it so that it becomes intracellular, e.g., by use of a
retroviral vector (see
U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle
bombardment
(e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface
receptors or
transfecting agents, or by administering it in linkage to a homeobox- like
peptide which is
known to enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.
Sci. USA
88:1864-1868), etc. Alternatively, a nucleic acid can be introduced
intracellularly and
incorporated within host cell DNA for expression, by homologous recombination:
For a
more detailed description of gene therapy approaches, see section 5.6.3.
As alluded to above, the present invention also provides pharmaceutical
compositions (pharmaceuticals of the invention). Such compositions comprise a
therapeutically effective amount of a nucleic acid, chemical or protein of the
invention, and
a pharmaceutically acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency of the
Federal or a
state government or listed in the U.S. Pharmacopeia or other generally
recognized
pharmacopeia for use in animals, and more particularly in humans. The term
"carrier"
refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids, such as
water and oils,
including those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil,
soybean oil, mineral oil, sesame oil and the like. Water is a preferred
carrier when the
pharmaceutical composition is administered intravenously. Saline solutions and
aqueous
dextrose and glycerol solutions can also be employed as liquid carriers,
particularly for
injectable solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,
glycerol monostearate,
talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the
like. The composition, if desired, can also contain minor amounts of wetting
or emulsifying
agents, or pH buffering agents. These compositions can take the form of
solutions,
suspensions, emulsion, tablets, pills, capsules, powders, sustained-release
formulations and
the like. The composition can be formulated as a suppository, with traditional
binders and
cers such as triglycerides. Oral formulation can include standard Garners such
as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine,
cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical
carriers are
-44-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such
compositions
will contain a therapeutically effective amount of the nucleic acid or protein
of the
invention, preferably in purified form, together with a suitable amount of
carrier so as to
provide the form for proper administration to the patient. The formulation
should suit the
mode of administration.
In a preferred embodiment, the pharmaceutical of the invention is formulated
in
accordance with routine procedures as a pharmaceutical composition adapted for
intravenous administration to human beings. Typically, compositions for
intravenous
administration are solutions in sterile isotonic aqueous buffer. Where
necessary, the
pharmaceutical of the invention may also include a solubilizing agent and a
local anesthetic
such as lignocaine to ease pain at the site of the inj ection. Generally, the
ingredients are
supplied either separately or mixed together in unit dosage form, for example,
as a dry
lyophilized powder or water free concentrate in a hermetically sealed
container such as an
ampoule or sachette indicating the quantity of active agent. Where the
pharmaceutical of
the invention is to be administered by infusion, it can be dispensed with an
infusion bottle
containing sterile pharmaceutical grade water or saline. Where the
pharmaceutical of the
invention is administered by injection, an ampoule of sterile water for
injection or saline can
be provided so that the ingredients may be mixed prior to administration.
For buccal administration the compositions may take the form of tablets or
lozenges
formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present
invention are conveniently delivered in the form of an aerosol spray
presentation from
pressurized packs or a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the dosage unit
may be
determined by providing a valve to deliver a metered amount. Capsules and
cartridges of
e.g. gelatin for use in an inhaler or insufflator may be formulated containing
a powder mix
of the compound and a suitable powder base such as lactose or starch.
The amount of the nucleic acid or protein of the invention which will be
effective in
the treatment or prevention of the indicated disease can be determined by
standard clinical
techniques. In addition, ifZ vitro assays may optionally be employed to help
identify optimal
dosage ranges. The precise dose to be employed in the formulation will also
depend on the
route of administration, and the stage of indicated disease, and should be
decided 'according
to the judgment of the practitioner and each patient's circumstances.
Effective doses may
be extrapolated from dose-response curves derived from in vitro or animal
model test
systems.
- 45
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
The present invention may be better understood by reference to the following
non-
limiting Examples, which are provided as exemplary of the invention. The
following
examples are presented in order to more fully illustrate the preferred
embodiments of the
invention. They should in no way be construed, however, as limiting the broad
scope of the
invention.
As is described hereinbelow, the studies that were performed by the inventors
herein
are standard, universally-accepted tests in animal models predictive of
prophylactic and
therapeutic benefit.
6' EXAMPLE 1: OVEREXPRESSION OF A DOMINANT-NEGATIVE~STAT3
VARIANT LEADS TO PRODUCTION OF SOLUBLE
FACTORS THAT INDUCE CELL CYCLE ARREST AND
APOPTOSIS
In this example, gene therapy of B 16 tumors with a dominant-negative Stat3
variant,
designated Stat3(3, resulted in inhibition of tumor growth and tumor
regression. Ten to
fifteen percent of the tumor cells were transfected in vivo. The Stat3(3-
induced anti-tumor
effect was associated with massive apoptosis of B 16 tumor cells, indicating a
potent
bystander effect. Overexpression of Stat3(3 in B16 cells resulted in both
apoptosis and cell
cycle arrest. Importantly, apoptosis and cell cycle arrest also occurred in
non-transfected
B 16 cells when they were co-cultured in separate chambers with Stat3 (3-
transfected B 16
cells, demonstrating that soluble factors mediated the bystander effect. RNase
protection
assays using mufti-template probes specific for key physiologic regulators of
apoptosis
revealed that overexpression of Stat3(3 in B16 tumor cells induced the
expression of the
apoptotic effector, TRAIL. These ih vitro results demonstrated that the
observed ih. vivo
bystander effect leading to tumor cell growth inhibition was mediated by
soluble factors
produced as a result of overexpression of Stat3 (3 in the B 16 tumor cells.
6.1 INTRODUCTION
Effective cancer gene therapies require the killing of genetically
untransduced tumor
cells ("bystander" cells) concomitant with genetically transduced tumor cells.
Because
30 ~.~sfection efficiency is a rate-limiting step for gene therapy, the
efficacy of cancer gene
therapy is enhanced by bystander effects.
It has recently been demonstrated that ih vivo transgenic expression of Stat3
J3 in
marine B 16 tumors results in tumor regression involving massive apoptosis of
tumor cells
despite relatively low transfection efficiencies (10 to 15%). To demonstrate
the cellular and
35 molecular mechanisms underlying the Stat3(3-mediated bystander effects
observed in vivo,
this example describes in vitro studies. This example shows that inhibition of
Stat3 activity
-46-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
in B 16 cells leads to production of soluble factors that induce both
apoptosis and cell cycle
arrest. Consistent with the finding that soluble factors are involved in the
bystander effects,
induction of mRNA encoding the apoptosis effector, TRAIL, was detected in
Stat3 [3-
transfected B 16 cells.
6.2 MATERIALS AND METHODS .
Plasmids. The bicistronic green fluorescent protein vector, pIRES-EGFP, was
obtained from Clontech (Palo Alto, CA). Insertion of Stat3(3 cDNA into the
pIRES-EGFP
plasmid to construct pIRES-Stat3 j3 was as described previously Catlett-
Falcone et al., 1999,
sups°a.
Cell culture and transfection. B 16 marine melanoma cells were grown in RPMI
1640 containing 10% FBS. Transfections were performed using GenePORTERTM
Transfection Reagent (Gene Therapy Systems, San Diego, CA) according to the
manufacturer's instructions. To determine transfection efficiency,
fluorescence intensities of
B 16 cells transfected with either pIRES-EGFP or pIRES-Stat3(3 were measured
by FACS
(Becton Dickinson Immunocytometry, CA) 24 h after transfection.
Nuclear extracts and EMSA. Nuclear extract preparation and EMSA analysis of
STAT DNA-binding activity were performed as previously described Catlett-
Falcone et al.,
1999, supra.
Cell growth inhibition assay. Cells were plated at 1.7 x 105 cells/well in 6-
well
plates, followed by transfection with either pIRES-EGFP or pIRES-Stat3(3
plasmids 24 h
later. Cells were harvested at 24 h, 4~ h or 72 h to determine the numbers of
live cells. Cell
viability was determined by trypan blue exclusion.
A~optosis assay. B 16 cells transfected with pIRES-EGFP or AIRES-Stat3(3 were
washed with CellScrubTM buffer (Gene Therapy Systems, San Diego, CA) 24 h
after
tr'ansfection. Apoptosis of transiently-transfected B 16 cells was analyzed
after staining with
Annexin V-PE by two-color flow cytometry. Apoptosis of non-transfected tumor
cells in
the upper chambers of Transwell units was analyzed after staining with Annexin
V-PE and
VIA-PROBETM 7-AAD (Pharmingen, San Diego, CA) by two-color flow cytometry.
Cell cycle analysis. Cell cycle analysis based on DNA content was perforined.
Cells were harvested, washed twice in PBS and resuspended in 70% ethanol on
ice for at
least 30 min. After centrifugation, cells were resuspended in 1 ml of
propidium iodide
staining solution (50 ~,g propidium iodide, 1 mg RNase A and 1 mg glucose per
ml PBS)
and incubated at room temperature for 30 min. The cells were analyzed by FACS
using
ModFit LT cell cycle analysis software (Verity Software, Topsham, ME). Cells
transfected
with vectors encoding EGFP were fixed in 1 ml of 0.5% formaldehyde on ice for
~10 min.
before adding 70% ethanol.
-47-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Supernatant studies. The supernatants derived from either empty vector or
Stat3[3
transfected B16 cells were collected at 12 h, 24 h, 36 h and 48 h after
transfection and
filtered through a 0.22 ~,m filter. Meanwhile, B 16 cells were plated 5 x 103/
well in 96-well
plate in triplicates. After cells were attached to the wells, 100 ~,1 of fresh
culture medium
and 100 ~1 supernatant from each time point were added to each well. Cells in
supernatants
were cultured for 48 h before analysing. For direct cell number counting,
cells were
harvested and counted by trypan blue exclusion. For 3H-thymidine (3H-TdR)
incorporation
assay, 0.25 ~,ci 3H-TdR was added to each well during the last 4 h of
incubation, transferred
to glassfiber filters by an automated cell harvester (Tomtec, Hamden, CT) and
3H-TdR
incorporation was determined with a liquid scintillation J3-counter (Pharmacia
Wallac,
Finland). For MTT assays, 5 ~l MTT (10 mg/ml) was added to each well during
the last 4 h
of incubation. Cells were lysed in 100 ~1 DMSO and metabolic activity was
quantified
spectrophotometrically.
Co-culturing studies in Transwell units. B 16 cells in the lower chambers of
Transwell units were transfected with either AIRES-EGFP or AIRES-Stat3(3
plasmids. Five
hours later, Sx104 of either B 16 or MethA cells were added to upper chambers.
After 48 h
co-culturing, cells in the upper chambers were harvested for both cell cycle
analysis and
apoptosis assays.
RNA isolation and RNase protection assax. Total RNA was isolated from 5.0 x
106
cells by TRIzoI reagent (Gibco BRL, Grand Island, NY). RNase protection assays
(RPA)
were carried out using the PharMingen Riboquant mAPO- 3 (TRAIL, Fast, CD95,
and
other death receptor associated genes) and mAPO- 2 (Bcl- 2 family members)
mufti- probe
templates according to the manufacturer's protocol (PharMingen, San Diego,
CA). Briefly,
the mufti-probe template was synthesized by ih vitro transcription with
incorporation of
[s2P]-a UTP and purified on a G50 Sephadex column (5- Prime to 3- Prime,
Boulder, CO).
Specific activity was quantitated in a Beckman LS 6500 scintillation counter
(Beckman,
Schaumburg, IL). Purified probe (0.8- 1.5 x 106 cpm/~.1) was hybridized with
10 ~g of total
RNA for 16 h, followed by RNase digestion at 37° C for 1 h. Protected
RNA fragments
were separated on a 5 % polyacrylamide denaturing gel and quantified with
Image Quant
software (Molecular Dynamics, Sunnyvale, CA). RPAs are representative of three
individual experiments.
6.3 RESULTS
Stat3J3 overexpression in B16 cells disrupts Stat3 DNA-binding activity.
To show that Stat3[3 expression in transfected B16 cells inhibits endogenous
Stat3
DNA-binding activity, EMSA with the 32P-labeled hSIE probe that binds to Stat3
and
Stat3[3 with high affinity was performed using nuclear extracts. Fig. 1 shows
specific DNA-
-48-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
binding activities of endogenous Stat3 (lanes 1, 2) and ectopic Stat3(3 (lane
3). EGF-
induced Stat3 binding activity in NIII3T3 (lane 4) was used as a positive
control. By,
supershift analysis with antibody that recognizes Stat3 but not Stat3(3 or
antibody that
recognizes Stat3(3 but not Stat3, it was shown that there were Stat3-Stat3
homodimers in
mock-transfected B 16 cells and empty vector-transfected B 16 cells.
Overexpression of
Stat3(3 in Stat3(3-transfected B16 cells results in mostly Stat3~3-Stat3(3
homodimer
formation. These data suggest that Stat3(3 disrupts Stat3-specific gene
regulation in B16
cells.
Stat3(3-mediated B16 cell growth inhibition involves both cell cycle arrest
and apoptosis.
To show that transient transfection of pIRES-Stat3 (i leads to B 16 cell
growth
inhibition, pIRES-EGFP or AIRES-Stat3(3 vectors were transfected into B16
cells,
respectively. While their transfection efficiencies were similar within each
experiment as
determined by the percentage of cells that exhibit green fluorescence at 24
hours post
transfection (by FACS analysis), the number of live B16 cells decreases
dramatically 48 h
later in the Stat3[3-transfected population (Fig. 2A). To show that Stat3(3-
induced growth
inhibition was mediated by cell cycle arrest and apoptosis, the effect of
Stat3(3 on cell cycle
progression and survival of B 16 cells was examined. The cell cycle
distributions of empty
vector and Stat3[3-transfected cells were shown in Fig. 2B. The Stat3[3
transfected B16 cells
show progressive accumulation in Go/G, phase, with concomitant decrease of the
population
in S and GZ/M phase. This Go/Gl phase arrest was observed as early as 24
hourspost
transfection. At 48 hours post transfection with Stat3(3 vector, Annexin V-PE
staining
followed by FACS analysis to detect apoptotic activity was performed with
transfected B 16
cells. A high level of apoptosis in Stat3(3 transfected cells (75%) relative
to empty vector
transfected cells (25%) was observed, as shown in Fig. 2C. Increased levels of
apoptosis as
a result of Stat3(3 transfection was confirmed by confocal laser scanning
microscope
analysis using rhodamine-labelled TLTNEL assay. This example demonstrates that
Stat3[3-
mediated growth inhibition of B 16 cells in vitro involves both cell cycle
arrest and
apoptosis.
Stat3(3 overexpression leads to production of soluble factors capable of
inducing both cell
cycle arrest and apoptosis.
Many of the GFP-negative B 16 cells (non-transfected) in the B 16 cell culture
transiently transfected with Stat3[3 also undergo apoptosis (Fig. 2C). This
indicated that
overexpression of Stat3(3 in B16 cells leads to bystander effects in. vitro.
To show that
Stat3[3-dependent bystander effects were not mediated by cell-cell contact,
but were
mediated via soluble factors, supernatants were collected 24 h, 36 h, 48 h
after Stat3(3 vector
transfection and subsequently used as conditioned medium for non-transfected B
16 cells.
-49-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Different assays for growth inhibition (cell number counts, MTT assays and 3H-
TdR
incorporation assays) were performed to show that the supernatants from
Stat3(3-transfected
B 16 cells inhibit the growth of non-transfected B 16 cells. Fig. 3A shows
that the
conditioned media obtained from Stat3 (3-transfected B 16 cells inhibit B 16
cell growth,
while that obtained from wild-type B16 cells or empty vector-transfected B16
cells does
not. To rule out the possibility that Stat3[3-induced growth inhibition of non-
transfected
tumor cells was due to apoptosis or stress in general, supernatant derived
from UV-
irradiated, apoptotic B 16 cells was tested for its ability to inhibit B 16
cell growth. Results
show that supernatant derived from UV-irradiated, apoptotic B 16 cells failed
to induce any
growth inhibition of B 16 cells.
To show that soluble factor-induced growth inhibition was through apoptosis
and
cell cycle arrest, experiments using Transwell units were performed. A
significant increase
in the percentage of B 16 cells arrested in Go/GI was observed when cultured
in conditioned
medium derived from Stat3[3-transfected B16 cells was compared to those
cultured in
conditioned media from mock or vector-transfected B 16 cells. Furthermore, non-
~ansfected B 16 cells cultured in upper chambers in which the lower chambers
contained
Stat3(3-transfected B16 cells undergo apoptosis as demonstrated by Annexin V-
PE and 7-
AAD staining followed by FAGS analysis (Fig. 3C). These Stat3(3-induced
soluble factors
produced by transfected-B 16 cells were also capable of inducing apoptosis of
non-
transfected Meth A cells (Fig. 3C).
Expression of the apoptosis effector, TRAIL, was induced in Stat3(3-
transfected B16 cells.
To demonstrate the identity of factors that cause apoptosis of untransfected
tumor
cells as a result of Stat3/3 expression in B16 cells, RNase protection assays
(RPAs) using
mufti-template probes were performed. Thirty hours after transfection, total
RNA was
isolated from various cell cultures and RPAs were carried out using probes
specific for key
physiologic regulators of apoptosis. An induction of TRAIL RNA expression in B
16 cells
as a result of Stat3(3 overexpression was detected (Fig. 4). This induction of
TRAIL was
specific, as none of the other genes examined was induced (Fig. 4).
6.4 DISCUSSION
The potential of Stat3(3 gene therapy as an effective cancer therapeutic
approach is
supported by the finding that ih. vivo the number of dying tumor cells greatly
exceeds the
number of tumor cells transfected with Stat3(3. Ih vitro results presented
herein demonstrate
that overexpression of Stat3(3 leads to apoptosis and cell cycle arrest of
marine melanoma
B 16 cells. Importantly, disruption of Stat3 signaling in B 16 cells also
results in the
production of soluble factors. The soluble factors were capable of inducing
apoptosis and
-SO-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
cell cycle arrest of non-transfected B 16 tumor cells, showing that killing of
bystander B 16
tumor cells ifa vivo is mediated by one or more soluble factors.
Constitutively-activated Stat3 correlates with elevated levels of members of
the Bcl-
2 family of anti-apoptotic regulatory proteins, Bcl-XL and Mcl-1 in human
malignancies.
Inhibition of Stat3 activity by Stat3 (3 down regulates the expression of
these anti-apoptotic
proteins, resulting in apoptosis. In addition to inducing anti-apoptotic
proteins, constitutive
activation of Stat3 promotes the expression of proteins that were important
for cell
proliferation. In particular, cyclin Dl, which controls progression from Gl to
S phase, is
elevated in cells expressing the constitutively-activated mutant form of
Stat3, Stat3C, or
endogenous Stat3 activated by the Src oncoprotein. Down-regulation of these
and other
anti-apoptotic and pro-proliferation proteins by Stat3(3 could, without being
limited by
theory, explain why overexpression of Stat3(3 in B16 tumor cells leads to both
apoptosis and
cell cycle arrest. Again, without being limited by theory, activated Stat3
could contribute to
oncogenesis by preventing apoptosis and promoting proliferation by down
regulating pro-
apoptotic and anti-proliferative genes. This example shows that inhibition of
Stat3 activity
in B16 cells induces expression of the pro-apoptotic effector, TRAIL. TRAIL is
a type II
membrane protein but various cell types produce a soluble form of TRAIL.
Taken together, the experiments described in this example demonstrate a role
for
soluble factors produced by tumor cells in mediating Stat3[3-dependent
bystander~effects, as
a result of disrupting endogenous Stat3 activity.
7. EXAMPLE 2: INHIBITION OF STAT3 SIGNALING IN B16 CELLS
INDUCES SECRETION OF IMMUNOLOGIC DANGER
SIGNALS
This example shows that blocking Stat3 signaling induces the secretion of
immunologic danger signals. B 16 tumors treated ira vivo with a Stat3 dominant-
negative
variant, Stat3(3, become infiltrated with iNOS-positive macrophages and T
cells. Inhibition
of Stat3 signaling in B 16 tumor cells results in secretion of soluble
factors, which activate
macrophages to produce additional inflammatory and tmnoricidal mediators,
including
mmc oxide. Furthermore, transfection of B 16 cells with the Stat3(3 gene
upregulates
expression of pro-inflammatory factors, including IL-6, IP-10, IFN-(3 and TNF-
a, capable
of stimulating nitric oxide production by macrophages. Significantly, this in
vivo study
demonstrates that expression of Stat3(3 in tumor cells leads to systemic
activation of
macrophages and T cells. This example shows that inhibition of Stat3 signaling
can
generate a cascade of immunologic danger signals important for activating
immune
responses, thus enhancing the utility of the present invention.
-51-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
7.1 INTRODUCTION
This study demonstrates that Stat3(3 gene therapy of B 16 tumors was
accompanied
by heavy infiltration of immune cells, including macrophages, neutrophils and
T cells.
Significantly, this example demonstrates that tumor-infiltrating macrophages
after Stat3(3
gene therapy were strongly positive for iNOS expression, showing that the
macrophages
were activated in vivo. A critical role of iNOS induction in mediating the
Stat3~3-induced
bystander effects iya vivo was shown by detection of macrophage-stimulating
soluble factors
as a result of Stat3(3 expression in B16 tumor cells. These factors stimulate
peritoneal
macrophages to synthesize NO, which in turn has a strong cytostastic effect on
B 16 cells.
Blockade of iNOS production, either in the presence of an iNOS inhibitor, NMA,
or using
iNOS deficient macrophages, abrogates soluble factor-induced, macrophage-
mediated anti-
B 16 activity. Furthermore, our results demonstrate that inhibition of Stat3
signaling in B 16
tumor cells results in elevated expression of IP-10, IL-6, TNF-a and IFN-(3.
Production of
the soluble factors, including these pro-inflammatory cytokines and
chemokines, in turn
upregulates the expression of RANTES in macrophages and TNF-a in neutrophils.
Thus,
l~ibition of Stat3 signaling results in the induction of a cascade of
immunologic danger
signals, which are normally produced only during inflammation and infection.
Activation
of local inflammatory responses in the tumor microenvironment is known to be
critical in
stimulating antitumor T cells. This example shows that inhibiting Stat3
signaling in tumors,
via activated innate immunity, leads to activation of T cells. This example
thus provides
support for an immunologic basis for the observed strong bystander effect that
increases the
antitumor efficacy of Stat3 (3 gene therapy.
7.2 METHODS
Tumor cells and supernatants. The B 16 melanoma cells were cultured in RPMI
medium
with 10 % FBS. Transfections were performed using GenePORTERTM Transfection
Reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer's
instructions. To determine transfection efficiency, fluorescence intensities
of B 16 cells
transfected with either pIRES-EGFP or pIRES-Stat3 were measured by FACS
(Becton
Dickinson Immunocytometry, CA) 24 h after transfection. Supernatants were
collected at
v~ous time points as indicated in figures and figure legends. Supernatants
were also
collected from B 16 cells treated UV-irradiation at various time points.
Mice. Six- to eight-week old female C57/B6 mice were obtained from the
National Cancer
Institute (Frederick, MD). Cohorts of 3-5 mice per group were used for these
experiments.
To induce tumor, mice were shaved on the left flank and injected s.c. with 5 x
105 of B16
cells in 100 p,1 of PBS. Gene therapy with Stat3(3 of established B16 tumors
was described
previously CNiu, 1999 #72].
-52-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Antibodystaining of iNOS in B16 tumors. 3 wm paraffin sections were
deparaffmized and
endogenous peroxidase was blocked with 3% aqueous hydrogen peroxide. To
minimize
non-specific binding, the sections were incubated for 20 min in normal goat
serum in PBS,
followed by overnight incubation at 4° C with rabbit anti-iNOS
polyclonal antibody
(Transduction Laboratories). After washing with PBS, sections were incubated
for 2 min
with diaminobenzidine tetrahydrochloride, rinsed with tap water and
counterstained with
modified Mayer's hematoxylin. Sections were dehydrated, cleared and mounted.
Peritoneal macrophages and neutrophils. Peritoneal macrophages were obtained
and
enriched. Neutrophils were obtained from peritoneal cavity 4 h after i.p.
injection of 1 ml
of 3% thioglycollate. The percentage of macrophages and neutrophils was
estimated by
morphological criteria using Giemsa staining (>98%). Macrophages were
incubated for 48 h
in conditioned medium containing 50 % supernatants from either non-
transfected, or
AIRES-Stat3 (3 or AIRES-EGFP transfected, or UV-irradiated B 16 cells.
Macrophage
supernatants (0.1 ml) were collected and examined for nitric oxide
accumulation using
Griess reagent. Neutrophil supernatants were tested for TNF-a, production
using ELISA (R
~ D Systems, MN). MTT assay was performed to ensure that the viability of
macrophages
and neutrophils cultivated in different supernatants was not affected.
Macrophage-mediated antitumor c otoxicitx. Antitumor cytotoxic activity of
macrophages
against B 16 cells was determined by inhibition of DNA synthesis. Briefly,
peritoneal
macrophages (1 x 105) were incubated in 50 % supernatants derived from either
wild type,
AIRES-Stat3 (3 or AIRES-GFP transfected, or UV-irradiated B 16 cells for 6 h.
After
replacing the supernatants with normal complete medium, B16 cells (1.0 x
104/well) were
added and co-cultured for 48 h with and without peritoneal macrophages. For
some
experiments, NOS inhibitor, N-monomethyl-L-arginine (NMA) (0.5 mM, Sigma) or
H202
quencher, catalase (500 U/ml, Boehringer Mannheim, Indianapolis, III were
added to
macrophages before adding supernatant from Stat3 (3-transfected B 16 cells.
The co-cultured
cells were pulsed with 3H-thymidine (3H-TdR) (0.25 ~.Ci/well) during the last
6 h, of
incubation to estimate DNA synthesis. 3H-TdR incorporation was determined
using a liquid
scintillation (3-counter (Pharmacia Wallac, Finland).
RNase protection assay. 10 ~,g of total RNA isolated from either AIRES-EGFP-
or pIRES-
Stat3(3-transfected B16 cells (36 h after transfection) was hybridized to
multi-template
probes from PharMingen (mCK-5, Top Panel; mCK-3, Lower Panel) that were
labeled with
3zP-dUTP using ih vitro transcription. The RNA encoded by GAPDH housekeeping
gene
was used to normalize the amounts of RNAs loaded in each lane. Similar
protocols were
used to determine RNA expression profiles of macrophages treated with
supernatants
derived from B 16 cells transfected with either Stat3 (3 or GFP vector. For
the mCK-5, Top
Panel; mCK-3 RPAs, RNAs prepared from mock transfected B 16 cells and UV-
irradiated,
-53-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
apoptotic B 16 cells were also included to serve as negative controls. For
macrophage RNA
analysis, RNAs prepared from macrophages treated with supernatants from mock
transfected and W-irradiated B 16 cells were also included.
7.3 RESULTS
B 16 tumors treated with Stat3b gene transfer were infiltrated with immune
cells, including
iNOS-positive macrophages
The bystander effect was demonstrated by complete regression of tumors despite
ih
vivo transduction of <15% of tumor cells. To show that immune cells have a
role in the
antitumor bystander effect, histochemical or immunohistochemical staining of B
16 tumors
treated with either pIRES-Stat3(3 or the control expression vector, pIRES-
EGFP, was
performed. Tissue sections from control vector-treated and Stat3(3-treated
tumors were
stained with H&E and anti-iNOS antibodies. A dramatic increase in number of
inflammatory cells, including neutrophils, macrophages, and T cells in Stat3(3-
treated, but
not control vector-treated, B16 tumors was observed. These results
demonstrated that
Stat3[3 gene therapy in B16 tumors lead to induction of iNOS gene expression
in the tumor-
infiltrating macrophages.
Inhibition of Stat3 si nalin~ in B 16 cells stimulates production of soluble
factors that
induce NO production by macropPhage.
The observed inflammatory infiltrate in Stat3(3-treated B16 tumors ih vivo
results
from factors secreted by transfected B 16 cells. To show that Stat3 (3-
transfected B 16 cells
produce factors that contribute to activation of macrophages, the effects of
the supernatants
derived from B 16 transfectants on NO production is demonstrated. Conditioned
medium
collected from macrophages treated with supernatant from Stat3(3-transfected B
16 cells, but
not control B 16 cells (mock transfected and GFP-control vector transfected),
contained high
levels of NO (Fig. 2). To rule out the possibility that production of soluble
factors capable
of activating macrophages produced by B 16 tumor cells was due to apoptosis or
stress in
general, supernatant collected from UV-irradiated, apoptotic B16 cells was
tested for its
ability to induce macrophage NO production. In contrast to supernatant from
Stat3(3-
transfected B 16 cells, supernatant from UV-irradiated, apoptotic B 16 cells
fails to induce
NO production (Fig. 5A). Results in Fig. 5B show that macrophage production of
NO was
iNOS-dependent, since blocking iNOS activity leads to abrogation of NO
production.
Nitric oxide-dependent cytotoxic activity against B16 tumor cells by soluble
factor-
activated macrophages.
-54-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Nitric oxide was the key mediator of the tumoricidal activity of macrophages.
This
example demonstrates that soluble factor-induced macrophage NO production
leads to
cytotoxic activity against B 16 tumor cells. 3H-thymidine incorporation to
estimate DNA
synthesis and cell proliferation was performed. In the results summarized in
Fig. 6,
supernatants derived from various B 16 cells were removed from macrophages
after 6 hours
of incubation. B 16 cells have little effect on the proliferation of non-
transfected B 16 cells,
pre-incubation of macrophages in supernatant collected from pIRES-Stat3~i-
transfected, but
not control vector-transfected B 16 cells, induce strong cytostasis of non-
transfected B 16
cells (Fig. 6). Macrophage-mediated cytostasis of B 16 cells was significantly
blocked by a
specific inhibitor of iNOS, NMA (Fig. 6). In contrast, addition of catalase,
which inhibits
H202 production, does not influence macrophage cytotoxic effects against B 16
cells.
Furthermore, soluble factor-induced macrophage cytostasis against B 16 cells
was abrogated
when macrophages derived from iNOS knockout mice were used instead of those
from
wild-type mice (Fig. 6).
Blocking Stat3 signaling in B 16 cells elevates the expression of pro-
inflammatorX
chemokines and cytokines, which in turn activates inflammatory cells to
produce additional
danger signals.
RNA expression profiles of a number of cytokines and chemokines in pIRES-
Stat3(3
transfected B 16 cells were determined. In addition, RNAs prepared from mock-
and control
vector-transfected, as well as UV-irradiated B 16 cells were included as
negative controls.
Results from these RNase protection assays using mufti-template RNA probes
indicated that
the expression levels of IFN-(3, TNF-a, IL-6 and IP-10 mRNAs, but not IL-4 and
IL-10,
were elevated in Stat3(3-transfected B16 cells in comparison with mock-
transfected, control
vector-transfected and IJV-irradiated B 16 cells (Fig. 7).
To show that these pro-inflammatory cytokines and chemokines participate in
the
activation of macrophages, macrophages were activated to produce NO by these
cytokines
ira vitro. ' Peritoneal macrophages were able to synthesize NO when stimulated
by IFN-[3 and
TNF-a simultaneously. Moreover, supernatant derived from Stat3(3-transfected
B16 cells
was capable of stimulating enhanced expression of RANTES by macrophages (Fig.
8A) and
T~-a by neutrophils (Fig. 8B). These chemokine and cytokines in turn further
attract and
activate macrophages.
Inhibition of Stat3 si ng aling in B 16 cells leads to systemic activation of
macropha es T
cells.
In addition to direct tumoricidal effect shown herein with production of NO by
macrophages, innate immunity critically impacts the development of adaptive
immune
responses. To show that blocking Stat3 signaling in tumor cells causes
macrophage and
-55-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Thl T cell activation, B16 cells transiently transfected with Stat3(3 were
injected s.c. into
mice. Compared to both naive mice and mice injected with control-vector
transfected B 16
cells, a clear induction of NO production by peritoneal macrophages was
observed (Fig.
9A). Furthermore, a four-fold increase in IFN-y production by lymphocytes
derived from
mice inj ected with Stat3 [3-transfected B 16 cells was also detected (Fig.
9B).
7.4 DISCUSSION
This example demonstrates that blocking Stat3 signaling in tumor cells results
in
secretion of immunologic danger signals, including pro-inflammatory cytokines
and
chemokines, which stimulate macrophages and neutrophils to produce additional
inflammatory and tumoricidal mediators. Production of iNOS-dependent NO by
macrophages stimulated by the soluble factors was shown to induce potent
cytotoxic
activity against non-transfected B 16 tumor cells. Among the identified pro-
inflammatory
factors is also the T cell chemotractant, IP-10, which attracts T cells to the
tumor site iya
vivo. This example shows that blocking Stat3 signaling in tumor cells causes a
cascade of
Immune responses, leading to activation of T cells. Importantly, this example
supports a
mechanistic basis for the heavy infiltration of immune cells in tumors treated
with Stat3 (3
gene transfer and indicate a critical immune component to the potent bystander
effect of
gene therapy targeting Stat3 signaling in tumor cells.
This example has shown that targeting Stat3 signaling in tumor cells induces
secretion of macrophage-activating factors that lead to production of iNOS-
dependent NO,
which in turn exerts potent, direct cytotoxic activity on tumor cells. Of the
four pro-
inflammatory cytokines and chemokines identified here, TNF-a and IFN-/3 were
elicited
upon ingestion of most microbes, showing the importance of these cytokines in
activating
iNOS for NO production. W duction of iNOS activity and subsequently the high-
output
pathway of NO production by macrophages under physiological conditions is only
observed
during inflammation and tissue damage due to viral or bacterial infections.
Our
demonstration that a direct antitumor effect is afforded by macrophage-
produced, iNOS-
dependent NO induced by inhibition of Stat3 signaling in tumor cells is
therefore of great
significance. Further, the importance of induction of iNOS and availability of
NO at the
30 ~mor site is not limited to direct cytotoxic activity against tumor cells.
There is a critical
role of iNOS and NO in mediating T cell-dependent antitumor responses: GM-CSF
vaccine-induced antitumor T cell immune response requires NO/iNOS, and IL-12-
induces
antitumor T-cell responses that were also iNOS/NO dependent .
Activation of innate immune responses demonstrated here can also be translated
into
35 stimulation of T cells. Among the pro-inflammatory factors resulting from
inhibiting Stat3
signaling in B 16 tumor cells were factors that can directly impact on tumor
cell growth.
TNF-a, for example, causes necrosis of tumor cells. Death of tumor cells ira
vivo 'itself
-56-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
promotes immunogenicity, because the release of tumor antigens under
inflammatory
conditions allows cross-priming of antigen-presenting cells, from which a
tumor-specific T
cell immunity can be elicited [Huang et al., 1994, Science 264:961-965; Huang
et al., 1994,
Ciba Found. Symp. 187:229-240; Dranoff et al., 1993, Proc. Natl. Acad. Sci.
USA 90:3539-
3543; Levitsky et al., 1994, J. Exp. Med. 179:1215-1224; Banchereau and
Steinman, 1998,
Nature 392:245-252; Maass et al., 1995, Int. J. Immunopharmacol. 17: 65-73;
Maass et al.,
1995, Proc. Natl. Acad. Sci. USA 92:5540-5544; Cayeux et al., 1997, Eur. J.
Immunol.
27:1657-1662; Cayeux et al., 1997, J. Immunol. 158:2834-2841]. Targeting Stat3
signaling
may generate tumor-specific T cell immunity.
Constitutive activation of Stat3 contributes to oncogenesis by helping tumor
cells
evade immune surveillance. The ability of normal cells to produce immunologic
danger
signals during infection and tissue destruction is well known, and Stat3 in
hematopoietic
cells is the regulator of macrophage activation. Stat3 mediates immune
suppression by IL-
10 signaling, which antagonizes the production of inflammatory cytokines such
as TNF-a,
IL-1 and IL-6, and suppresses iNOS activity. Blockade of Stat3 signaling, as
in Stat3-/-
macrophages, severely impairs the inhibitory activity of IL-10 on production
of .
inflammatory cytokines. As a result, mice with Stat3-/- macrophage and
neutrophils were
highly susceptible to endotoxin shock, with increased production of TNF-a, IL-
1, IL-6 and
IFN-y, and showed an enhanced T-helper 1 cell activity. Furthermore, Stat3-/-
macrophages
display increased expression of MHC class II and B7-1 molecules, thus Stat3
signaling
suppress macrophage activation. Our present examples show a novel role for
Stat3
signaling in nonhematopoietic tumor cells in blocking release of danger
signals that activate
a cascade of immune responses. As such, Stat3 activation in tumor cells may
serve to cloak
them from immune surveillance.
A critical role for Stat3 signaling in suppressing immune responses in normal
nonhematopoietic cells during wound healing has also been suggested.
Inflammatory
cytokines and immune mediators, including TNF-a, IL-1, IL-6 and NO, were in
reduced
amounts in naturally healing wounds compared to non-healing wounds [Trengove
et al.,
2000, Wound Repair Regen. 8: 13-25; Cao et al., 2000, Am. J. Sports Med.
28:176-182]. In
the absence of Stat3 signaling, as shown in mice with epidermal and
keratinocytes that lack
fictional Stat3, pronounced inflammatory infiltration is observed throughout
the dermis
while wound healing is impaired [Sano et al., 1999, EMBO J. 18:4657-4668].
Complementary to these findings were our current results in which blocking
Stat3 signaling
in tumor cells leads to upregulation of inflammatory factors, including
production of TNF-
a, IFN-(3, IP-10, IL-6, and subsequent production of iNOS-dependent NO, RANTES
by
macrophages and TNF-a by neutrophils. Collectively, our results, together with
recent
studies using Stat3-/- inflammatory cells and skin cells [Takeda et al., 1999,
Immunity
10:39-49; Sano et al., 1999, EMBO J. 18:4657-4668], suggest that Stat3
signaling down-
-57-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
regulates immunologic danger signals. Furthermore, constitutive activation of
Stat3 may
promote tumorigenesis by suppressing danger signals, thereby helping tumor
cells escape
immune recognition of their antigens.
8. EXAMPLE 3: STAT3 SIGNALING IN TUMOR CELLS PROMOTES
ANGIOGENESIS THROUGH UPREGULATION OF VEGF
Stat3 signaling is required for cell transformation by v-Src. Activity of Src
tyrosine
kinase has been shown to regulate the expression of VEGF, a potent stimulator
of
angiogenesis, which is crucial for tumor growth and metastasis formation. In
this third
example, it was shown that blocking Stat3 signaling inhibits v-Src-mediated
VEGF
upregulation, and expression of constitutively-activated Stat3 increases the
production of
VEGF in fibroblasts. In tumor cells, blocking Stat3 signaling inhibits
transcriptional
activity of the VEGF promoter and downregulates expression of the endogeneous
VEGF
gene. This example shows that constitutive Stat3 signaling upregulates VEGF
expression,
which in turn induces angiogenesis. Therefore, in the present invention,
inhibition of Stat3
signaling inhibits angiogenesis mediated by downregulation of VEGF expression.
And
activation of Stat3 signaling promotes angiogenesis mediated by upregulation
of VEGF
expression.
8.1 INTRODUCTION
~giogenesis plays a critical role in a wide variety of disorders, such as
ischemic
diseases and proliferative angiopathies with neovascularization. Vascular
endothelial
growth factor (VEGF) has been shown to be a potent endothelial cell-specific
mitogen that
stimulates angiogenesis. An essential role of VEGF in tumorigenesis has been
shown when
systemic treatment of tumor-bearing animals with a neutralizing antibody to
VEGF inhibits
t~or growth, which correlates with reduced tumor vascularity.
Constitutive activation of Stat3 in numerous human solid tumors was caused by
deregulated activities of c-Src tyrosine kinase. In hemotopoietic
malignancies, such as
multiple myeloma, Stat3 was constitutively activated by IL-6 mediated
signaling. Because
Src tyrosine kinase activity and IL-6 mediated signaling can lead to both
Stat3 activation
and VEGF upregulation in tumor cells, it is demonstrated herein that Stat3
regulates VEGF
expression in tumor cells. This example shows that Stat3 signaling was
required for Src-
induced VEGF upregulation and that Stat3 activity induces VEGF expression in
tumor cells
and in fibroblasts, showing that Stat3 plays an important role in VEGF
expression and thus
in angiogenesis.
-58-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
8.2 RESULTS
Stat3 signaling was reduired for v-Src-induced VEGF upre 1~.
Src tyrosine kinase activity upregulates VEGF expression. Because Src-induced
transformation requires Stat3 signaling, this example demonstrates that Stat3
is a requisite
intermediary step for VEGF upregulation by Src activity. NIH3T3 fibroblasts
transformed
by v-Src were transiently transfected with a dominant-negative variant of
Stat3, Stat3[3. The
transfection efficiency was approximately 40% based on the number of cells
that were
fluorescent due to the presence of GFP. The presence of Stat3(3 in the cells
was
accompanied by loss of Stat3 DNA binding activity as shown by an EMSA (Fig.
10A).
Forty-eight hours later, VEGF expression in v-Src-NIH3T3 fibroblasts with or
without
Stat3(3 expression was compared at both RNA and protein levels. Figure lOB
shows that
blocking Stat3 signaling in v-SrcNIH3T3 fibroblasts inhibits VEGF. Anti-sense
oligonuclotides against Stat3 as well as control oligonucleotides were also
transfected into
v-Src-NIH3T3 cells. A reduction of endogenous Stat3 protein as a result of
Stat3 anti-sense
oligonucleotide also caused inhibition of VEGF expression.
Expression of a constitutively-activated mutant form of Stat3 in fibroblasts
stimulate the
production of VEGF
It was also shown herein that persistent Stat3 signaling by itself can
upregulate
VEGF expression. Transfection of a mutant form of Stat3 that was
constitutively activated,
Stat3-C (Bromberg et al., 1999, Cell 98:295-303), led to increased Stat3 DNA
binding
activity in several clones of NIH3T3 cells, which correlated with increased
expression of
VEGF at both protein and RNA levels (Fig. 11).
Blocking Stat3 si aling in tumor cells inhibits VEGF promoter activitx.
To show that transcriptional activity of VEGF promoter was regulated by the
endogeneous Stat3 activity in tumor cells, B 16 marine melanoma and SCK marine
tumor
cells were transiently transfected with Stat3(3 and a reporter construct
containing luciferase
cDNA under the control of the VEGF promoter. A plasmid construct containing
the
luciferase cDNA in the absence of the VEGF promoter was also transfected into
3T3
f broblasts. Both of B 16 and SCK tumor cells harbor constitutively activated
Stat3. In the
absence of Stat3(3, VEGF promoter activity was readily detectable in both
tumor cells as
indicated by the high expression levels of luciferase protein. However,
cotransfection with
Stat3(3, but not the control vector, greatly inhibited the transcriptional
activity of the VEGF
promoter. The inhibitory effect of Stat3 (3 on the transcriptional activity of
the VEGF
promoter was also observed in the tumor cells transfected with Stat3 anti-
sense
oligonucleotides (Fig. 12A-B). It was also shown that blocking Stat3 signaling
in tumor
cells inhibits the expression of the endogeneous VEGF gene. B 16 tumor cells
were
-59-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
transiently transfected with Stat3(3 and the expression of endogeneous VEGF
gene was
determined at the RNA and protein levels. As shown in Fig. 13, inhibition of
constitutive
activation of Stat3 in tumor cells downregulates expression of the endogeneous
VEGF gene.
8.3 DISCUSSION
This example clearly shows that Stat3 is a requisite intermediary in the v-Src-
induced VEGF expression, showing that Stat3 is an important regulator of VEGF-
mediated
angiogenesis. The present example establishes that constitutive signaling of
Stat3
upregulates VEGF expression. Thus, a novel role of constitutive activation of
Stat3 as an
angiogenic regulator is shown.
Blocking Stat3 signaling, either by a Stat3 dominant-negative variant or
antisense
oligos, leads to antiangiogensis via down regulation of VEGF, thus adding a
new~dimension
to the therapeutic effect of anti-Stat3 signaling.
In addition to antiangiogenesis, downregulation of VEGF may also contribute to
increased immune responses associated with inhibition of Stat3 signaling in
tumor cells.
VEGF produced by tumor cells have been shown to inhibit the functional
maturation of
dendritic cells, the most potent antigen presenting cells. Dendritic cells
incubated with
tumor cells in the presence of Stat3 antisense oligos, but not control oligos,
undergo normal
functional maturation.
The invention is not to be limited in scope by the specific embodiments
described
which are intended as single illustrations of individual aspects of the
invention, and
functionally equivalent methods and components are within the scope of the
invention.
Indeed various modifications of the invention, in addition to those shown and
described
herein will become apparent to those skilled in the art from the foregoing
description and
accompanying drawings. Such modifications are intended to fall within the
scope of the
appended claims.
All references cited herein, including patent applications, patents, and other
publications, are incorporated by reference herein in their entireties for all
purposes.
35
-60-
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
SEQUENCE LISTING
<110> Yu, Hua et al.
<120> Stat3 Agonists and Antagonists and Therapeutic Uses Thereof
<130> 10873-009-228
<140> To Be Assigned
<141> 2001-09-10
<150> 60/231,212
<151> 2000-09-08
<160> 5
<170> PatentIn version 3.0
<210> 1
<211> 2631
<212> DNA
<213> Homo Sapiens
<400>
1
ggatcctggacaggcaccccggcttggcgctgtctctccccctcggctcggagaggccct 60
tcggcctgagggagcctcgccgcccgtccccggcacacgcgcacgcccggCCtCtCggCC 120
tctgccggagaaacaggatggcccaatggaatcagctacagcagcttgacacacggtacc 180
tggagcagctccatcagctctacagtgacagcttcccaatggagctgcggcagtttctgg 240
ccccttggattgagagtcaagattgggcatatgcggccagcaaagaatcacatgccactt 300
tggtgtttcataatctcctgggagagattgaccagcagtatagccgcttcctgcaagagt 360
cgaatgttctctatcagcacaatctacgaagaatcaagcagtttcttcagagcaggtatc 420
1
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
ttgagaagccaatggagattgcccggattgtggcccggtgcctgtgggaagaatcacgcc480
ttctacagactgcagccactgcggcccagcaagggggccaggccaaccaccccacagcag540
ccgtggtgacggagaagcagcagatgctggagcagcaccttcaggatgtccggaagagag600
tgcaggatctagaacagaaaatgaaagtggtagagaatctccaggatgactttgatttca660
actataaaaccctcaagagtcaaggagacatgcaagatctgaatggaaacaaccagtcag720
tgaccaggcagaagatgcagcagctggaacagatgctcactgcgctggaccagatgcgga780
gaagcatcgtgagtgagctggcggggcttttgtcagcgatggagtacgtgcagaaaactc840
tcacggacgaggagctggctgactggaagaggcggcaacagattgcctgcattggaggcc900
cgcccaacatctgcctagatcggctagaaaactggataacgtcattagcagaatctcaac960
ttcagacccgtcaacaaattaagaaactggaggagttgcagcaaaaagtttcctacaaag1020
gggaccccattgtacagcaccggccgatgctggaggagagaatcgtggagctgtttagaa1080
acttaatgaaaagtgcctttgtggtggagcggcagccctgcatgcccatgcatcctgacc1140
ggcccctcgtcatcaagaccggcgtccagttcactactaaagtcaggttgctggtcaaat1200
tccctgagttgaattatcagcttaaaattaaagtgtgcattgacaaagactctggggacg1260
ttgcagctctcagaggatcccggaaatttaacattctgggcacaaacacaaaagtgatga1320
acatggaagaatccaacaacggcagcctctctgcagaattcaaacacttgaccctgaggg1380
agcagagatgtgggaatgggggccgagccaattgtgatgcttccctgattgtgactgagg1440
agctgcacctgatcacctttgagaccgaggtgtatcaccaaggcctcaagattgacctag1500
agacccactccttgccagttgtggtgatctccaacatctgtcagatgccaaatgcctggg1560
cgtccatcctgtggtacaacatgctgaccaacaatcccaagaatgtaaacttttttacca1620
agcccccaattggaacctgggatcaagtggccgaggtcctgagctggcagttctcctcca1680
ccaccaagcgaggactgagcatcgagcagctgactacactggcagagaaactcttgggac1740
ctggtgtgaattattcagggtgtcagatcacatgggctaaattttgcaaagaaaacatgg1800
ctggcaagggcttctccttctgggtctggctggacaatatcattgaccttgtgaaaaagt1860
acatcctggccctttggaacgaagggtacatcatgggctttatcagtaaggagcgggagc1920
gggccatcttgagcactaagcctccaggcaccttcctgctaagattcagtgaaagcagca1980
aagaaggaggcgtcactttcacttgggtggagaaggacatcagcggtaagacccagatcc2040
agtccgtggaaccatacacaaagcagcagctgaacaacatgtcatttgctgaaatcatca2100
tgggctataagatcatggatgctaccaatatcctggtgtctccactggtctatctctatc2160
ctgacattcccaaggaggaggcattcggaaagtattgtcggccagagagccaggagcatc2220
ctgaagctgacccaggcgctgccccatacctgaagaccaagtttatctgtgtgacaccaa2280
2
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
cgacctgcag caataccatt gacctgccga tgtccccccg cactttagat tcattgatgc 2340
agtttggaaataatggtgaaggtgctgaaccctcagcaggagggcagtttgagtccctca2400
cctttgacatggagttgacctcggagtgcgctacctcccccatgtgaggagctgagaacg2460
gaagctgcagaaagatacgactgaggcgcctacctgcattctgccacccctcacacagcc2520
aaaccccagatcatctgaaactactaactttgtggttccagattttttttaatctcctac2580
ttctgctatctttgagcaatctgggcacttttaaaaatagagaaatgagtg 2631
<210> 2
<211> 769
<212> PRT
<213> Homo Sapiens
<400> 2
Met Ala Gln Trp Asn Gln Leu Gln Gln Leu Asp Thr Arg Tyr Leu Glu
1 5 10 15
Gln Leu His Gln Leu Tyr Ser Asp Ser Phe Pro Met Glu Leu Arg Gln
20 25 30
Phe Leu Ala Pro Trp Ile Glu Ser Gln Asp Trp Ala Tyr Ala Ala Ser
35 40 45
Lys Glu Ser His Ala Thr Leu Val Phe His Asn Leu Leu Gly Glu Ile
50 55 60
Asp Gln Gln Tyr Ser Arg Phe Leu Gln Glu Ser Asn Val Leu Tyr Gln
65 70 75 80
His Asn Leu Arg Arg Ile Lys Gln Phe Leu Gln Ser Arg Tyr Leu Glu
85 90 95
Lys Pro Met Glu Ile Ala Arg Ile Val Ala Arg Cys Leu Trp Glu Glu
100 105 110
Ser Arg Leu Leu Gln Thr Ala Ala Thr Ala Ala Gln Gln Gly Gly Gln
115 120 125
Ala Asn His Pro Thr Ala Ala Val Val Thr Glu Lys Gln Gln Met Leu
130 135 140
Glu Gln His Leu Gln Asp Val Arg Lys Arg Val Gln Asp Leu Glu Gln
145 150 155 160
Lys Met Lys Val Val Glu Asn Leu Gln Asp Asp Phe Asp Phe Asn Tyr
165 170 175
Lys Thr Leu Lys Ser Gln Gly Asp Met Gln Asp Leu Asn Gly Asn Asn
180 185 190
3
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Gln Ser Val Thr Arg Gln Lys Met Gln Gln Leu Glu Gln Met Leu Thr
195 200 205
Ala Leu Asp Gln Met Arg Arg Ser Ile Val Ser Glu Leu Ala Gly Leu
210 215 220
Leu Ser Ala Met Glu Tyr Val Gln Lys Thr Leu Thr Asp Glu Glu Leu
225 230 235 240
Ala Asp Trp Lys Arg Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro Pro
245 250 255
Asn Ile Cys Leu Asp Arg Leu Glu Asn Trp Ile Thr Ser Leu Ala Glu
260 265 270
Ser Gln Leu Gln Thr Arg Gln Gln Ile Lys Lys Leu Glu Glu Leu Gln
275 280 285
Gln Lys Val Ser Tyr Lys Gly Asp Pro Ile Val Gln His Arg Pro Met
290 295 300
Leu Glu Glu Arg Ile Val Glu Leu Phe Arg Asn Leu Met Lys Ser Ala
305 310 315 320
Phe Val Val Glu Arg Gln Pro Cys Met Pro Met His Pro Asp Arg Pro
325 330 335
Leu Val Ile Lys Thr Gly Val Gln Phe Thr Thr Lys Val Arg Leu Leu
340 345 350
Val Lys Phe Pro Glu Leu Asn Tyr Gln Leu Lys Ile Lys Val Cys Ile
355 360 365
Asp Lys Asp Ser Gly Asp Val Ala Ala Leu Arg Gly Ser Arg Lys Phe
370 375 380
Asn Ile Leu Gly Thr Asn Thr Lys Val Met Asn Met Glu Glu Ser Asn
385 390 395 400
Asn Gly Ser Leu Ser Ala Glu Phe Lys His Leu Thr Leu Arg Glu Gln
405 410 415
Arg Cys Gly Asn Gly Gly Arg Ala Asn Cys Asp Ala Ser Leu Ile Val
420 425 430
Thr Glu Glu Leu His Leu Ile Thr Phe Glu Thr Glu Val Tyr His Gln
435 440 445
Gly Leu Lys Ile Asp Leu Glu Thr His Ser Leu Pro Val Val Val Ile
450 455 460
Ser Asn Ile Cys Gln Met Pro Asn Ala Trp Ala Ser Ile Leu Trp Tyr
465 470 475 480
Asn Met Leu Thr Asn Asn Pro Lys Asn Val Asn Phe Phe Thr Lys Pro
485 490 495
Pro Tle Gly Thr Trp Asp Gln Val Ala Glu Val Leu Ser Trp Gln Phe
500 505 510
4
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Ser Ser Thr Thr Lys Arg Gly Leu Ser Ile Glu Gln Leu Thr Thr Leu
515 520 525
Ala Glu Lys Leu Leu Gly Pro Gly Val Asn Tyr Ser Gly Cys Gln Ile
530 535 540
Thr Trp Ala Lys Phe Cys Lys Glu Asn Met Ala Gly Lys Gly Phe Ser
545 550 555 560
Phe Trp Val Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile
565 570 575
Leu Ala Leu Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu
580 585 590
Arg Glu Arg Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu
595 600 605
Arg Phe Ser Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val
610 615 620
Glu Lys Asp Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr
625 630 635 640
Thr Lys Gln Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly
645 650 655
Tyr Lys Ile Met Asp Ala Thr Asn Ile Leu Val Ser Pro Leu Val Tyr
660 665 670
Leu Tyr Pro Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg
675 680 685
Pro Glu Ser Gln Glu His Pro Glu A1a Asp Pro Gly Ala Ala Pro Tyr
690 695 700
Leu Lys Thr Lys Phe Ile Cys Val Thr Pro Thr Thr Cys Ser Asn Thr
705 710 715 720
Ile Asp Leu Pro Met Ser Pro Arg Thr Leu Asp Ser Leu Met Gln Phe
725 730 735
Gly Asn Asn Gly G1u Gly Ala Glu Pro Ser Ala Gly Gly Gln Phe Glu
740 745 750
Ser Leu Thr Phe Asp Met Glu Leu Thr Ser Glu Cys Ala Thr Ser Pro
755 760 765
Met
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
<210> 3
<211> 2303
<212> DNA
<213> Homo Sapiens
<400>
3
ggatcctggacaggcaccccggcttggcgctgtctctccccctcggctcggagaggccct60
tcggcctgagggagcctcgccgcccgtccccggcacacgcgcacgcccggcctctcggcc120
tctgccggagaaacaggatggcccaatggaatcagctacagcagcttgacacacggtacc180
tggagcagctccatcagctctacagtgacagcttcccaatggagctgcggcagtttctgg240
ccccttggattgagagtcaagattgggcatatgcggccagcaaagaatcacatgccactt300
tggtgtttcataatctcctgggagagattgaccagcagtatagccgcttcctgcaagagt360
cgaatgttctctatcagcacaatctacgaagaatcaagcagtttcttcagagcaggtatc420
ttgagaagccaatggagattgcccggattgtggcccggtgcctgtgggaagaatcacgcc480
ttctacagactgcagccactgcggcccagcaagggggccaggccaaccaccccacagcag540
ccgtggtgacggagaagcagcagatgctggagcagcaccttcaggatgtccggaagagag600
tgcaggatctagaacagaaaatgaaagtggtagagaatctccaggatgactttgatttca660
actataaaaccctcaagagtcaaggagacatgcaagatctgaatggaaacaaccagtcag720
tgaccaggcagaagatgcagcagctggaacagatgctcactgcgctggaccagatgcgga780
gaagcatcgtgagtgagctggcggggcttttgtcagcgatggagtacgtgcagaaaactc840
tcacggacgaggagctggctgactggaagaggcggcaacagattgcctgcattggaggcc900
cgcccaacatctgcctagatcggctagaaaactggataacgtcattagcagaatctcaac960
ttcagacccgtcaacaaattaagaaactggaggagttgcagcaaaaagtttcctacaaag1020
gggaccccattgtacagcaccggccgatgctggaggagagaatcgtggagctgtttagaa1080
acttaatgaaaagtgcctttgtggtggagcggcagccctgcatgcccatgCatCCtgaCC1140
ggcccctcgtcatcaagaccggcgtccagttcactactaaagtcaggttgctggtcaaat1200
tccctgagttgaattatcagcttaaaattaaagtgtgcattgacaaagactctggggacg1260
ttgcagctctcagaggatcccggaaatttaacattctgggcacaaacacaaaagtgatga1320
acatggaagaatccaacaacggcagcctctctgcagaattcaaacacttgaccctgaggg1380
agcagagatgtgggaatgggggccgagccaattgtgatgcttccctgattgtgactgagg1440
agctgcacctgatcacctttgagaccgaggtgtatcaccaaggcctcaagattgacctag1500
6
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
agacccactccttgccagttgtggtgatctccaacatctgtcagatgccaaatgcctggg1560
cgtccatcctgtggtacaacatgctgaccaacaatcccaagaatgtaaacttttttacca1620
agcccccaattggaacctgggatcaagtggccgaggtcctgagctggcagttctcctcca1680
ccaccaagcgaggactgagcatcgagcagctgactacactggcagagaaactcttgggac1740
ctggtgtgaattattcagggtgtcagatcacatgggctaaattttgcaaagaaaacatgg1800
ctggcaagggcttctccttctgggtctggctggacaatatcattgaccttgtgaaaaagt1860
acatcctggccctttggaacgaagggtacatcatgggctttatcagtaaggagcgggagc1920
gggccatcttgagcactaagcctccaggcaccttcctgctaagattcagtgaaagcagca1980
aagaaggaggcgtcactttcacttgggtggagaaggacatcagcggtaagacccagatcc2040
agtccgtggaaccatacacaaagcagcagctgaacaacatgtcatttgctgaaatcatca2100
tgggctataagatcatggatgctaccaatatcctggtgtctccactggtctatctctatc2160
ctgacattcccaaggaggaggcattcggaaagtattgtcggccagagagccaggagcatc2220
ctgaagctgacccaggcgctgccccatacctgaagaccaagtttatctgtgtgacaccat2280
tcattgatgcagtttggaaataa 2303
<210>4
<211>720
<212>PRT
<213>Homo Sapiens
<400> 4
Met Ala Gln Trp Asn Gln Leu Gln Gln Leu Asp Thr Arg Tyr Leu Glu
1 5 10 15
Gln Leu His Gln Leu Tyr Ser Asp Ser Phe Pro Met Glu Leu Arg Gln
20 25 30
Phe Leu Ala Pro Trp Ile G1u Ser Gln Asp Trp Ala Tyr Ala Ala Ser
35 40 45
Lys Glu Ser His Ala Thr Leu Val Phe His Asn Leu Leu Gly Glu Ile
50 55 60
Asp Gln Gln Tyr Ser Arg Phe Leu Gln Glu Ser Asn Val Leu Tyr Gln
65 70 75 80
His Asn Leu Arg Arg Ile Lys Gln Phe Leu Gln Ser Arg Tyr Leu Glu
85 90 95
Lys Pro Met Glu Ile Ala Arg Ile Val Ala Arg Cys Leu Trp Glu Glu
100 105 110
7
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Ser Arg Leu Leu Gln Thr Ala Ala Thr Ala Ala Gln Gln Gly Gly Gln
115 120 125
Ala Asn His Pro Thr Ala Ala Val Val Thr Glu Lys Gln Gln Met Leu
130 135 140
Glu Gln His Leu Gln Asp Val Arg Lys Arg Val Gln Asp Leu Glu Gln
145 150 155 160
Lys Met Lys Val Val Glu Asn Leu Gln Asp Asp Phe Asp Phe Asn Tyr
165 170 175
Lys Thr Leu Lys Ser Gln Gly Asp Met Gln Asp Leu Asn Gly Asn Asn
180 185 190
Gln Ser Val Thr Arg Gln Lys Met Gln Gln Leu Glu Gln Met Leu Thr
195 200 205
Ala Leu Asp Gln Met Arg Arg Ser Ile Val Ser Glu Leu Ala Gly Leu
210 215 220
Leu Ser Ala Met Glu Tyr Val Gln Lys Thr Leu Thr Asp Glu Glu Leu
225 230 235 240
Ala Asp Trp Lys Arg Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro Pro
245 250 255
Asn Ile Cys Leu Asp Arg Leu Glu Asn Trp Ile Thr Ser Leu Ala Glu
260 265 270
Ser Gln Leu Gln Thr Arg Gln Gln Ile Lys Lys Leu Glu Glu Leu Gln
275 280 285
Gln Lys Val Ser Tyr Lys Gly Asp Pro Ile Val Gln His Arg Pro Met
290 295 300
Leu Glu Glu Arg Ile Val Glu Leu Phe Arg Asn Leu Met Lys Ser Ala
305 310 315 320
Phe Val Val Glu Arg Gln Pro Cys Met Pro Met His Pro Asp Arg Pro
325 330 335
Leu Val Ile Lys Thr Gly Val Gln Phe Thr Thr Lys Val Arg Leu Leu
340 345 350
Val Lys Phe Pro Glu Leu Asn Tyr Gln Leu Lys Ile Lys Val Cys Ile
355 ~ 360 365
Asp Lys Asp Ser Gly Asp Val Ala Ala Leu Arg Gly Ser Arg Lys Phe
370 375 380
Asn Ile Leu GIy Thr Asn Thr Lys Val Met Asn Met Glu Glu Ser Asn
385 390 395 400
Asn Gly Ser Leu Ser Ala Glu Phe Lys His Leu Thr Leu Arg Glu Gln
405 410 415
Arg Cys Gly Asn Gly Gly Arg Ala Asn Cys Asp Ala Ser Leu Ile Val
420 425 430
8
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Thr Glu Glu Leu His Leu Ile Thr Phe Glu Thr Glu Val Tyr His Gln
435 440 445
Gly Leu Lys Ile Asp Leu Glu Thr His Ser Leu Pro Val Val Val Ile
450 455 460
Ser Asn Ile Cys Gln Met Pro Asn Ala Trp Ala Ser Ile Leu Trp Tyr
465 470 475 480
Asn Met Leu Thr Asn Asn Pro Lys Asn Val Asn Phe Phe Thr Lys Pro
485 490 495
Pro Ile Gly Thr Trp Asp Gln Val Ala Glu Val Leu Ser Trp Gln Phe
500 505 510
Ser Ser Thr Thr Lys Arg Gly Leu Ser Ile Glu Gln Leu Thr Thr Leu
515 520 525
Ala Glu Lys Leu Leu Gly Pro Gly Val Asn Tyr Ser Gly Cys Gln Ile
530 535 540
Thr Trp Ala Lys Phe Cys Lys Glu Asn Met Ala Gly Lys Gly Phe Ser
545 550 555 560
Phe Trp Val Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile
565 570 575
Leu Ala Leu Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu
580 585 590
Arg Glu Arg Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu
595 600 605
Arg Phe Ser Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val
610 615 620
Glu Lys Asp Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr
625 630 635 640
Thr Lys Gln Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly
645 650 655
Tyr Lys Ile Met Asp Ala Thr Asn Ile Leu Val Ser Pro Leu Val Tyr
660 665 670
Leu Tyr Pro Asp Ile Pro Lys G1u Glu Ala Phe Gly Lys Tyr Cys Arg
675 680 685
Pro Glu Ser Gln Glu His Pro Glu Ala Asp Pro Gly Ala Ala Pro Tyr
690 695 700
Leu Lys Thr Lys Phe Ile Cys Val Thr Phe Ile Asp Ala Val Trp Lys
705 710 715 720
9
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
<210> 5
<211> 769
<212> PRT
<213> Homo Sapiens
<400> 5
Met Ala Gln Trp Asn Gln Leu Gln Gln Leu Asp Thr Arg Tyr Leu Glu
l 5 10 15
Gln Leu His Gln Leu Tyr Ser Asp Ser Phe Pro Met Glu Leu Arg Gln
20 25 30
Phe Leu Ala Pro Trp Ile Glu Ser Gln Asp Trp Ala Tyr Ala Ala Ser
35 40 45
Lys Glu Ser His Ala Thr Leu Val Phe His Asn Leu Leu Gly Glu Ile
50 55 60
Asp Gln Gln Tyr Ser Arg Phe Leu Gln Glu Ser Asn Val Leu Tyr Gln
65 70 75 80
His Asn Leu Arg Arg Ile Lys Gln Phe Leu Gln Ser Arg Tyr Leu Glu
85 90 95
Lys Pro Met Glu Ile Ala Arg Ile Val Ala Arg Cys Leu Trp Glu Glu
100 105 110
Ser Arg Leu Leu Gln Thr Ala Ala Thr Ala Ala Gln Gln Gly Gly Gln
115 120 125
Ala Asn His Pro Thr Ala Ala Val Val Thr Glu Lys Gln Gln Met Leu
130 135 140
Glu Gln His Leu Gln Asp Val Arg Lys Arg Val Gln Asp Leu Glu Gln
145 150 155 160
Lys Met Lys Val Val Glu Asn Leu Gln Asp Asp Phe Asp Phe Asn Tyr
165 170 175
Lys Thr Leu Lys Ser Gln Gly Asp Met Gln Asp Leu Asn Gly Asn Asn
180 185 190
Gln Ser Val Thr Arg Gln Lys Met Gln Gln Leu Glu Gln Met Leu Thr
195 200 205
Ala Leu Asp Gln Met Arg Arg Ser Ile Val Ser Glu Leu Ala Gly Leu
210 215 220
Leu Ser Ala Met Glu Tyr Val Gln Lys Thr Leu Thr Asp Glu Glu Leu
225 230 235 240
Ala Asp Trp Lys Arg Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro Pro
245 250 255
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Asn Ile Cys Leu Asp Arg Leu Glu Asn Trp Ile Thr Ser Leu Ala G1u
260 265 270
Ser Gln Leu Gln Thr Arg Gln Gln Ile Lys Lys Leu Glu Glu Leu Gln
275 280 285
Gln Lys Val Ser Tyr Lys Gly Asp Pro Ile Val Gln His Arg Pro Met
290 295 300
Leu Glu Glu Arg Ile Val Glu Leu Phe Arg Asn Leu Met Lys Ser Ala
305 310 315 320
Phe Val Val Glu Arg Gln Pro Cys Met Pro Met His Pro Asp Arg Pro
325 330 335
Leu Val Ile Lys Thr Gly Val Gln Phe Thr Thr Lys Val Arg Leu Leu
340 345 350
Val Lys Phe Pro Glu Leu Asn Tyr Gln Leu Lys Ile Lys Val Cys Ile
355 360 365
Asp Lys Asp Ser Gly Asp Val Ala Ala Leu Arg Gly Ser Arg Lys Phe
370 375 380
Asn Ile Leu Gly Thr Asn Thr Lys Val Met Asn Met Glu Glu Ser Asn
385 390 395 400
Asn Gly Ser Leu Ser Ala Glu Phe Lys His Leu Thr Leu Arg Glu Gln
405 410 415
Arg Cys Gly Asn Gly Gly Arg Ala Asn Cys Asp Ala Ser Leu Ile Val
420 425 430
Thr Glu Glu Leu His Leu Ile Thr Phe Glu Thr Glu Val Tyr His Gln
435 440 445
Gly Leu Lys Ile Asp Leu Glu Thr His Ser Leu Pro Val Val Val Ile
450 455 460
Ser Asn Ile Cys Gln Met Pro Asn Ala Trp Ala Ser Ile Leu Trp Tyr
465 470 475 480
Asn Met Leu Thr Asn Asn Pro Lys Asn Val Asn Phe Phe Thr Lys Pro
485 490 495
Pro Ile Gly Thr Trp Asp Gln Val Ala Glu Val Leu Ser Trp Gln Phe
500 505 510
Ser Ser Thr Thr Lys Arg Gly Leu Ser Ile Glu Gln Leu Thr Thr Leu
515 520 525
Ala Glu Lys Leu Leu Gly Pro Gly Val Asn Tyr Ser Gly Cys Gln Ile
530 535 540
Thr Trp Ala Lys Phe Cys Lys Glu Asn Met Ala Gly Lys Gly Phe Ser
545 550 555 560
Phe Trp Val Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile
565 570 575
11
CA 02421723 2003-03-10
WO 02/20032 PCT/USO1/28254
Leu Ala Leu Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu
580 585 590
Arg Glu Arg Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu
595 600 605
Arg Phe Ser Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val
610 615 620
Glu Lys Asp Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr
625 630 635 640
Thr Lys Gln Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly
645 650 655
Tyr Lys Ile Met Asp Cys Thr Cys Ile Leu Val Ser Pro Leu Val Tyr
660 665 670
Leu Tyr Pro Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg
675 680 685
Pro Glu Ser Gln Glu His Pro Glu Ala Asp Pro Gly Ala Ala Pro Tyr
690 695 700
Leu Lys Thr Lys Phe Ile Cys Val Thr Pro Thr Thr Cys Ser Asn Thr
705 710 715 720
Ile Asp Leu Pro Met Ser Pro Arg Thr Leu Asp Ser Leu Met Gln Phe
725 730 735
Gly Asn Asn Gly Glu Gly Ala Glu Pro Ser Ala Gly Gly Gln Phe Glu
740 745 750
Ser Leu Thr Phe Asp Met Glu Leu Thr Ser Glu Cys Ala Thr Ser Pro
755 760 765
Met
12
