U.S. patent application number 14/661391 was filed with the patent office on 2015-10-15 for methods for treating tweak-related conditions.
The applicant listed for this patent is Biogen Idec MA Inc.. Invention is credited to Linda C. Burkly, Kyungmin Hahm, Aniela Jakubowski, Timothy Zheng.
Application Number | 20150291688 14/661391 |
Document ID | / |
Family ID | 29250708 |
Filed Date | 2015-10-15 |
United States Patent
Application |
20150291688 |
Kind Code |
A1 |
Burkly; Linda C. ; et
al. |
October 15, 2015 |
Methods For Treating Tweak-Related Conditions
Abstract
The present invention provides methods and agents for the
treatment of TWEAK-related conditions, including cardiac, liver,
kidney, lung, adipose, skeletal, muscle, neuronal, bone and
cartilage conditions. The invention also provides methods for
identifying TWEAK agonists or antagonists for the treatment of
TWEAK-related conditions. Additionally, the invention provides
transgenic animals that express an exogenous DNA encoding a TWEAK
polypeptide, or fragments, analogs, or muteins thereof, and methods
for using such animals to identify TWEAK agonists or antagonists.
The invention further provides methods for diagnosing a disease
based on TWEAK expression. The invention also provides methods for
affecting cellular differentiation of progenitor cells using TWEAK
polypeptides, agonists, or antagonists.
Inventors: |
Burkly; Linda C.; (Monument
Beach, MA) ; Jakubowski; Aniela; (Brookline, MA)
; Zheng; Timothy; (Boston, MA) ; Hahm;
Kyungmin; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biogen Idec MA Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
29250708 |
Appl. No.: |
14/661391 |
Filed: |
March 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13860983 |
Apr 11, 2013 |
9011859 |
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14661391 |
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13179096 |
Jul 8, 2011 |
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13860983 |
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12417755 |
Apr 3, 2009 |
8506958 |
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13179096 |
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10510804 |
Sep 13, 2005 |
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PCT/US2003/011350 |
Apr 9, 2003 |
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12417755 |
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60371611 |
Apr 9, 2002 |
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Current U.S.
Class: |
424/133.1 ;
424/158.1 |
Current CPC
Class: |
A61P 9/04 20180101; C07K
14/70575 20130101; C07K 2319/00 20130101; A61K 38/00 20130101; A61K
2039/505 20130101; C07K 16/241 20130101; A61P 29/00 20180101; C07K
16/22 20130101; A61P 1/04 20180101; A61P 19/04 20180101; A61K 45/06
20130101; A61P 3/04 20180101; A61P 1/16 20180101; A61P 9/00
20180101; A61P 17/00 20180101; A61P 15/08 20180101; C07K 2319/30
20130101; A61P 13/04 20180101; A61P 13/02 20180101; A61P 19/00
20180101; A61K 39/3955 20130101; A61P 25/00 20180101; A61P 43/00
20180101; A61P 15/00 20180101; A61P 1/00 20180101; A61P 13/12
20180101; A61P 13/00 20180101; C07K 14/525 20130101; A61P 11/00
20180101; A61P 21/00 20180101; A01K 2217/05 20130101; A61P 1/18
20180101; C07K 16/2875 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395 |
Claims
1.-12. (canceled)
13. A method for treating muscle atrophy in a human subject in need
thereof, the method comprising administering to the human subject a
therapeutically effective amount of a TWEAK antagonist, wherein the
TWEAK antagonist is an antibody, or an antigen-binding fragment
thereof, that binds human TWEAK.
14. The method of claim 13, wherein the muscle atrophy is muscular
dystrophy.
15. The method of claim 13, wherein the muscle atrophy is a
mitochondrial myopathy, a lipid myopathy, a central tubular
myopathy, rhabdomyolysis, or an alcoholic myopathy.
16. The method of claim 13, wherein the muscle atrophy is the
result of a neuronal disease.
17. The method of claim 41, wherein the neuronal disease is
amyotrophic lateral sclerosis (ALS).
18. The method of claim 13, wherein the muscle atrophy is
cachexia.
19. The method of claim 13, wherein the muscle atrophy is the
result of polymyositis, an inflammatory myopathy, or a
glucocorticoid induced atrophy.
20. The method of claim 13, wherein the human subject has
cancer.
21. The method of claim 13, wherein the anti-human TWEAK antibody
or the antigen-binding fragment thereof is human, humanized, or
chimeric.
22. The method of claim 13, wherein the TWEAK antagonist is used in
combination with progenitor cell therapy.
23. The method of claim 13, wherein the TWEAK antagonist is used in
combination with tissue transplantation therapy.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to methods and agents for the
treatment of TWEAK-related conditions, including cardiac, liver,
kidney, lung, adipose, skeletal muscle, neural, bone, cartilage,
skin, gastrointestinal, pancreatic, reproductive organ and
connective tissue diseases. The invention also relates to methods
for identifying TWEAK agonists or antagonists for the treatment of
TWEAK-related conditions. Additionally, the invention relates to
transgenic animals that express an exogenous DNA encoding a TWEAK
polypeptide, or fragments, analogs, or muteins thereof and methods
for using such animals to identify TWEAK agonists or antagonists.
The invention further relates to methods for diagnosing a disease
based on TWEAK expression. The invention also relates to methods
for affecting cellular proliferation or differentiation of
progenitor cells using TWEAK polypeptides, agonists or
antagonists.
BACKGROUND OF THE INVENTION
[0002] Members of the Tumor Necrosis Factor (TNF) family of
ligands, so named for their structural similarity to TNF-.alpha.,
are key components in diverse processes, such as inflammatory
responses, cellular immunity and apoptosis. TNF ligands may act
locally as type II membrane-bound proteins through direct
cell-to-cell contact or as secreted proteins having autocrine,
paracrine or endocrine functions. TNF family members bind TNF
receptor (TNF-R) family members via their C-terminal extracellular
domain. Various TNF family members include TNF, lymphotoxins (LT),
Fas, CD27, CD30, CD40, 4-1BB, OX-40, TRAMP, CAR-1, TRAIL, GITR,
HVEM, osteoprotegrin, NGF, TRAIN, Kay (BAFF), APRIL and TWEAK (TNF
relatedness and weak ability to induce cell death).
[0003] A defining feature of this family of cytokine receptors is
found in the cysteine rich extracellular domain, initially revealed
by the molecular cloning of two distinct TNF receptors. This family
of genes encodes glycoproteins characteristic of Type I
transmembrane proteins having an extracellular ligand binding
domain, a single membrane spanning region and a cytoplasmic region
involved in activating cellular functions. The cysteine-rich ligand
binding region exhibits a tightly knit disulfide linked core
domain, which, depending upon the particular family member, is
repeated multiple times. Most receptors have four domains, although
there may be as few as one, or as many as six.
[0004] TNF family members play a role in the regulation of the
immune system, controlling cell survival and differentiation, as
well as acute host defense systems, such as inflammation. Continued
efforts in the art to manipulate members of the TNF family for
therapeutic benefit may provide unique means to control disease.
For instance, some of the ligands of this family can directly
induce the apoptotic death of many transformed cells, e.g., LT,
TNF, Fas ligand and TRAIL. Fas and possibly TNF and CD30 receptor
activation can induce cell death in nontransformed lymphocytes
which may display an immunoregulatory function.
[0005] The ability to induce programmed cell death is an important
and well-studied feature of several members of the TNF family. Fas
mediated apoptosis appears to play a role in the regulation of
autoreactive lymphocytes in the periphery and possibly the thymus.
Also, the TNF and CD30 systems have been implicated in the survival
of T cells and large cell anaplastic lymphoma lines. Death in this
cell line in response to TNF, Fas or LT-.beta. receptor signaling
has features of apoptosis.
[0006] The TNF family of ligands may be categorized into three
groups based on their ability to induce cell death. First, TNF, Fas
ligand and TRAIL can efficiently induce cell death in many lines
and their receptors most likely have good canonical death domains.
Presumably the ligand to DR-3 (TRAMP/WSL-1) would also fall into
this category. Next there are those ligands, such as TWEAK, CD30
ligand, and LTalb2, which trigger a weaker death signal limited to
a few cells. Studies in these systems have suggested that a
separate weaker death signaling mechanism exists. Lastly, there
those members that cannot efficiently deliver a death signal.
Probably all groups may exert antiproliferative effects on some
cell types consequent to inducing cell differentiation, e.g.,
CD40.
[0007] In general, death is triggered following the aggregation of
death domains which reside on the cytoplasmic side of the TNF
receptors. The death domain orchestrates the assembly of various
signal transduction components which lead to activation of the
caspase cascade. Some receptors lack canonical death domains, e.g.
LTb receptor and CD30, yet can induce cell death, albeit more
weakly. Conversely, signaling through other pathways such as CD40
is required to maintain cell survival. There remains a need to
further identify and characterize the functions of the TNF family
members, thereby facilitating the development of new therapies for
TNF family-related diseases.
[0008] TWEAK was isolated in a screen for RNA that hybridized to an
erythropoietin probe. Chicheportiche et al., J. Biol. Chem.
272:32401-32410 (1997). The mouse and human peptides have an
unusually high degree of conservation, including 93% amino acid
identity in the receptor binding domain. TWEAK, shown to be
efficiently secreted from cells, is abundantly expressed in many
tissues, including heart, brain, placenta, lung, liver, skeletal
muscle, kidney, pancreas, spleen, lymph nodes, thymus, appendix,
and peripheral blood lymphocytes.
[0009] One known TWEAK receptor is Fn14, a growth factor-regulated
immediate-early response gene that decreases cellular adhesion to
the extracellular matrix and reduces serum-stimulated growth and
migration (Meighan-Mantha et al., J. Biol. Chem. 274:33166-33176
(1999)). Fn14 has been shown to be induced by FGF, calf serum and
phorbol ester treatment and is expressed at relatively high levels
in heart, kidney, lung, skin, skeletal muscle, ovary and pancreas
tissues, as well as in hepatocellular carcinoma modules and other
cancer cell lines, and at lower levels in normal liver tissues.
[0010] TWEAK has been implicated in many biological processes. For
instance, HT29 cells treated with IFN-.gamma. and TWEAK were shown
to undergo apoptosis; although TWEAK's ability to induce apoptosis
is weak and only a small number of cell types are susceptible.
Chicheportiche et al., J. Biol. Chem. 272:32401-32410 (1997). In
contrast, TWEAK has also been shown to induce angiogenesis and
proliferation of endothelial cells in a VEGF-independent pathway.
Lynch et al., J. Biol. Chem. 274:8455-8459 (1999). Astrocytes are
specifically bound and stimulated by TWEAK. TWEAK can infiltrate an
inflamed brain to influence astrocyte behavior. Astrocytes exposed
to TWEAK secrete high levels of IL-6 and IL-8, as well as
upregulate ICAM-1 expression. Saas et al., GLIA 32:102-107
(2000).
[0011] TWEAK has also been implicated in immune system regulation.
Upon stimulation with IFN-.gamma., monocytes rapidly express TWEAK,
and anti-TWEAK antibodies partially inhibited their cytotoxic
activity against human squamous carcinoma cells. A combination of
anti-TWEAK and anti-TRAIL antibodies almost completely inhibited
cytotoxicity. Nakayama et al., J. Exp. Med. 192:1373-1379 (2000).
In contrast, TWEAK mRNA rapidly disappeared in mice treated with
lipopolysaccharide (LPS), an inducer of the immuno-inflammatory
responses. Furthermore, TWEAK mRNA was also reduced in autoimmune
hemolytic anemia and systemic lupus erythematosus in mouse models.
These data suggest that the down-regulation of TWEAK expression is
an important event in acute and chronic inflammation.
Chicheportiche et al., Biochem. Biophys. Res. Comm. 279:162-165
(2000).
[0012] Currently, the art lacks a complete understanding of what
conditions or diseases are related to TWEAK expression and
function, including the role of TWEAK in both inflammatory and
non-inflammatory conditions.
SUMMARY OF THE INVENTION
[0013] The present invention relates to the role of TWEAK in
contributing to the severity and progression of various
pathological conditions, including diseases of multiple tissues and
organ systems. Such pathological conditions include acute cardiac
injury, chronic heart failure, non-inflammatory dilated
cardiomyopathy, congestive heart failure, liver epithelial cell
hyperplasia, hepatocyte death, liver fibrosis, hepatocyte
vacuolation, other liver injuries, bile duct conditions, including
bile duct hyperplasia, inflammatory kidney conditions, such as
multifocal inflammation, non-inflammatory kidney conditions such as
tubular nephropathy, tubular hyperplasia, glomerular cysts,
glomerular nephropathy, Alport Syndrome, kidney tubular
vacuolation, kidney hyaline casts, kidney fibrosis and inflammatory
lung disease. The present invention establishes a causal link
between the TWEAK molecule and certain diseases of the heart,
liver, kidney and lungs. The invention disclosed herein also
establishes a link between TWEAK and the behavior of progenitor
cells for liver tissue, kidney tubules, skin cells, adipocytes,
skeletal muscle, cartilage and bone, as well as connective tissue
cell types, such as stromal cells in the bone marrow and
fibroblasts.
[0014] In one embodiment, the invention relates to methods for
treating TWEAK-related conditions, i.e. diseases, settings of
injury or other pathological conditions of tissues wherein a
receptor for TWEAK, e.g. FN14, is expressed. Those conditions
include fibrosis, cardiomyopathies, and diseases of the kidney,
lung, liver, skin, skeletal muscle, lipid metabolism (e.g.
obesity), gastrointestinal tract, pancreas, reproductive organs,
neural tissue (including neurodegeneration), cartilage, bone and
connective tissue. In a preferred embodiment, the TWEAK-related
conditions are non-inflammatory in nature. In another preferred
embodiment, the invention relates to methods for treating
TWEAK-related conditions by interfering with the interaction of the
TWEAK polypeptide with its cellular receptor.
[0015] In other embodiments, the invention relates to TWEAK
agonists or antagonists and pharmaceutical compositions comprising
them for use in treating TWEAK-related conditions. Such TWEAK
agonists or antagonists (i.e. inhibitors) may be anti-TWEAK
antibodies, or derivatives thereof; anti-TWEAK receptor antibodies,
or derivatives thereof; TWEAK polypeptide fragments; TWEAK
polypeptide analogs; TWEAK muteins; TWEAK mimetics; TWEAK fusion
proteins; TWEAK receptor polypeptide fragments; TWEAK receptor
polypeptide analogs; TWEAK receptor muteins; TWEAK receptor
mimetics; TWEAK receptor fusion proteins; organic compounds; and
inorganic compounds.
[0016] In other embodiments, the invention relates to TWEAK
agonists or antagonists and pharmaceutical compositions useful in
treating hosts in need of tissue regeneration or replacement. It
also relates to use of TWEAK agonists or antagonists for modulating
the behavior of populations of progenitor cells in vivo or in
vitro. The progenitor cells may be the precursors of liver cell
types, kidney tubules, cardiomyocytes, lung cell types, skin cell
types, skeletal muscle cell types, adipocytes, gastrointestinal
cell types, pancreatic cell types, neural tissue cell types,
cartilage and bone cell types, connective tissue cell types,
including stromal cells in the bone marrow and fibroblasts. TWEAK
agonists or antagonists and pharmaceutical compositions comprising
them may be administered in vivo to promote tissue regeneration and
replacement in settings of disease or tissue injury, including but
not limited to, toxin, viral, chemotherapy or radiation-induced
damage, and genetic or degenerative disorders. In another
embodiment, TWEAK agonists or antagonists and pharmaceutical
compositions thereof could be used in combination with cellular
therapy with stem cells or progenitor cells to regenerate tissue
and organ systems. In yet another embodiment, stem cells or
progenitor cell populations may be expanded in vitro by TWEAK
agonists or antagonists and pharmaceutical compositions thereof.
Progenitor cell populations expanded through the use of TWEAK
agonists or antagonists may be used for transplantation into hosts
in need of tissue regeneration or replacement.
[0017] In other embodiments, the invention relates to methods for
identifying TWEAK agonists or antagonists useful as therapeutic
agents for the treatment of TWEAK-related conditions. In another
embodiment, the invention relates to transgenic animals expressing
exogenous DNAs encoding TWEAK polypeptides. A further embodiment of
this invention includes the use of TWEAK as a molecular marker for
disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1: The role of TWEAK in dilated cardiomyopathy is
shown. A. A FL-TWEAK transgenic (Tg) mouse shows thrombosis of the
right atrium and ventricle, as well as severe dilation. B. Normal
heart is shown for comparison.
[0019] FIG. 2: TWEAK overexpression in the heart induces cardiac
remodeling. A cross section of the heart is viewed at 10.times.
magnification with hematoxylin/eosin staining on day 20 following
infection of adult C57BL/6 mice with an adenoviral vector
comprising murine sTWEAK DNA compared with an adenovirus-GFP
control construct.
[0020] FIG. 3: TWEAK induces biliary duct and oval cell
hyperplasia, as revealed in FL-TWEAK transgenic (Tg) mice as
compared to non-transgenic (NTg) littermates at 2 weeks of age and
7 months of age.
[0021] FIG. 4: TWEAK induces biliary duct and oval cell
hyperplasia, as revealed by increased staining with the A6 mAb
which is specific for a biliary epithelial and oval cell marker in
FL-TWEAK transgenic (Tg) mice as compared to non-transgenic (NTg)
littermates.
[0022] FIG. 5: TWEAK induces oval cell hyperplasia as revealed by
the presence of large, oval cells in the portal region in FL-TWEAK
transgenic (Tg) mice.
[0023] FIG. 6: TWEAK causes hepatocellular vacuolization in
FL-TWEAK transgenic (Tg) mice as compared to non-transgenic (NTg)
littermates.
[0024] FIG. 7: Serum TWEAK levels in mice infected with an
adenoviral vector comprising murine sTWEAK DNA.
[0025] FIG. 8: TWEAK overexpression in the liver induces hepatocyte
death and ductal hyperplasia. Cross sections of the liver are
viewed at 20.times. magnification with hematoxylin/eosin staining
on days 3 and 11 following infection of adult C57BL/6 mice with an
adenoviral vector comprising murine sTWEAK DNA compared with an
adenovirus-GFP control construct.
[0026] FIG. 9: The TWEAK receptor Fn14 is induced after CCl.sub.4
induced liver injury in mice. In situ hybridization for Fn14 mRNA
in normal mouse liver and CCl.sub.4 induced liver injury shows
little if any detectable expression in normal adult liver and
marked induction of Fn14 expression after injury. Hemotoxylin and
eosin (H&E) stained sections show the corresponding normal
healthy liver and CCl.sub.4 injured liver tissue.
[0027] FIG. 10: Fn14 expression is upregulated in biliary
epithelial cells in a murine model of bile duct ligation, as
revealed by increased staining with the anti-sense mRNA probe
directed against Fn14 using in situ hybridization. Hemotoxylin and
eosin (H&E) stained section shows the corresponding section in
bright field microscopy.
[0028] FIG. 11: Cross section of FL-TWEAK transgenic (Tg) mouse
kidney as compared to non-transgenic (NTg) mouse kidney at 2 weeks,
8 weeks and 7 months of age. The results show tubular basophilia in
the TWEAK Tg kidney at 8 weeks and 7 months of age, and dilatation
of the urinary space in glomeruli, i.e., glomerular cysts, with
adjacent basophilic tubules at 7 months of age.
[0029] FIG. 12: Cross section of FL-TWEAK transgenic (Tg) mouse
kidney with H&E staining. A. Glomerular nephropathy with
basophilia of adjacent proximal tubular epithelium is shown. B.
Segmental mesangial hypercellularity, hypertrophy of capsular
epithelia, and capsular thickening.
[0030] FIG. 13: Serial sections from two FL-TWEAK transgenic (Tg)
mouse kidneys stained with H&E (top) and proliferating cell
nuclear antigen (PCNA) (bottom). Basophilic tubules correspond to
tubules expressing PCNA, i.e. proliferating tubules.
[0031] FIG. 14: Serial sections from a FL-TWEAK transgenic (Tg)
mouse kidney stained with H&E, a lectin from T. Purpureas (a
marker for proximal tubules) and a lectin from A. Hypogaea (a
marker for distal tubules). Results show that the basophilic
tubules do not express either epithelial marker.
[0032] FIG. 15: TWEAK overexpression in the kidney induces tubular
hyperplasia and glomerulopathy. A cross section of the kidney is
viewed at 20.times. and 40.times. magnification with
hematoxylin/eosin staining on day 11 following infection of adult
C57BL/6 mice with an adenoviral vector comprising murine sTWEAK DNA
compared with an adenovirus-GFP control construct.
[0033] FIG. 16: TWEAK mRNA is widely expressed throughout the
kidney in an adult wild-type mouse. A cross section of kidney is
viewed at 5.times. magnification with hematoxylin staining, or
under dark field microscopy following in situ hybridization with
sense or anti-sense TWEAK probes.
[0034] FIG. 17: Fn14 mRNA is expressed in the proximal tubules of
outer medulla in adult wild-type mouse kidney. A cross section of
kidney is viewed at 5.times. magnification with hematoxylin
staining, or under dark field microscopy following in situ
hybridization with sense or anti-sense Fn14 probes.
[0035] FIG. 18: A role for TWEAK in kidney fibrosis is suggested by
the upregulation of Fn14 mRNA in Alports kidneys. The fold increase
in Fn14 mRNA levels is shown in two individual mice carrying the
mutation leading to Alports disease relative to wildtype animals at
4, 5, 6, and 7 weeks of age. mRNA levels were determined by
hybridization to a gene chip containing nucleotide sequence
corresponding to a portion of the Fn14 gene. At the 4 and 7 week
time points, replicate results for each the two mice are shown
(indicated by the mouse 1 repeat and mouse 2 repeat bars
respectively). At the 7 week time point, Fn14 mRNA is shown to be
reduced in two settings where disease is inhibited, i.e.
sTGF.beta.R-Fc treatment and in VLA-1 knockout mice (illustrated in
FIG. 18 by either the two independent mice treated with
sTGF.beta.R-Fc ("sTGFbR treated") or the two independent
Alport/VLA-1 KO mice ("Alport/VLA-1 KO").
[0036] FIG. 19: TWEAK antagonist treatment in Unilateral Ureteral
Obstruction (UUO), a murine model of kidney fibrosis, significantly
reduced kidney fibrosis. Metamorph quantitation of blue-staining
area (fibrotic area) on Trichrome-Masson stained paraffin kidney
sections indicates that collagen content was decreased in AB.G11
(anti-TWEAK monoclonal Ab) treated kidney samples to similar levels
observed in sTGF.beta.R-Ig positive control samples. In contrast,
the isotype-control hamster antibody (HA4/8)-treated kidneys showed
no reduction in kidney fibrosis, similar to vehicle (PBS)-treated
kidneys.
[0037] FIG. 20: The TWEAK transgene causes granulomatous and
lymphohistocytic inflammation in the lung. A. A cross section of a
lung from a FL-TWEAK transgenic (Tg) mouse with H&E staining.
B. A cross section of a lung from a sTWEAK Tg mouse with H&E
staining.
[0038] FIG. 21: TWEAK mRNA is expressed in the cells lining the
bronchioles and alveoli in adult wild-type mouse lung. A cross
section of lung is viewed at 10.times. magnification with
hematoxylin staining, or under dark field microscopy following in
situ hybridization with sense or anti-sense TWEAK probes.
[0039] FIG. 22: Fn14 mRNA is expressed in the bronchioles and
alveoli of adult wild-type mouse lung. A cross section of lung is
viewed at 10.times. magnification with hematoxylin staining, or
under dark field microscopy following in situ hybridization with
sense or anti-sense Fn14 probes.
[0040] FIG. 23: Inhibitory effect of TWEAK on 3T3-L1 cell adipocyte
differentiation in vitro. 3T3-L1 cells were induced to undergo
differentiation using a standard protocol. Cells were untreated,
treated with a control agent (recombinant soluble human CD40L-FLAG
100 ng/ml) or various versions of TWEAK at 100 ng/ml recombinant
soluble human TWEAK-FLAG, recombinant soluble human TWEAK, Fc-human
TWEAK) on day 0, together with dexamethasone and insulin, and were
replenished daily. In one experimental group, the blocking
anti-TWEAK mAb AB.G11 was also added at the same time as Fc-hTWEAK.
The cells were stained with Oil-red O on day 7.
[0041] FIG. 24: Inhibitory effect of TWEAK on myogenesis in vitro.
C2C12 myoblasts were grown to near confluency in a DMEM-based
growth media and on day 0, switched to a low-serum differentiation
media that contained 2% horse serum to trigger differentiation.
Cells were untreated or treated on day 0 with Fc-hTWEAK (100
ng/ml). Myotube formation was examined using a phase-contrast
microscope and pictures were taken on day 6 of differentiation. In
other experimental groups, Fn14-Fc or a neutralizing anti-TNF
antibody were added at the same time as Fc-hTWEAK, thereby
demonstrating that the inhibitory effect of Fc-hTWEAK was
TWEAK-specific and not mediated through TNF.
[0042] FIG. 25: TWEAK can bind to human mesenchymal stem cells.
Human mesenchymal stem cells (hMSCs) were incubated with
recombinant Fc-TWEAK protein followed by PE-conjugated goat
anti-human Fc or goat anti-mouse Fc secondary antibodies. The
ability of Fc-TWEAK to bind to hMSCs was determined using
fluorescence activated cell sorter (FACS) analysis. The background
staining is provided by the secondary antibody staining (2nd only)
alone.
DETAILED DESCRIPTION OF THE INVENTION
[0043] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics, virology and protein and
nucleic acid chemistry and hybridization described herein are those
well known and commonly used in the art. The methods and techniques
of the present invention are generally performed according to
conventional methods well known in the art and as described in
various general and more specific references that are cited and
discussed throughout the present specification unless otherwise
indicated. See, e.g., Sambrook et al. Molecular Cloning: A
Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates
(1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990),
which are incorporated herein by reference. Enzymatic reactions and
purification techniques are performed according to manufacturer's
specifications, as commonly accomplished in the art or as described
herein. The nomenclatures used in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0044] In order that the invention herein described may be more
fully understood, the following detailed description is set forth.
In the description, the following terms are employed:
[0045] "Antibody" refers to an intact immunoglobulin, or to an
antigen-binding portion thereof that competes with the intact
antibody for specific binding. Antigen-binding portions may be
produced by recombinant DNA techniques or by enzymatic or chemical
cleavage of intact antibodies. Antigen-binding portions include,
inter alfa, Fab, Fab', F(ab')2, Fv, dAb, and complementarity
determining region (CDR) fragments, single-chain antibodies (scFv),
chimeric antibodies, diabodies and polypeptides that contain at
least a portion of an immunoglobulin that is sufficient to confer
specific antigen binding to the polypeptide. An Fab fragment is a
monovalent fragment consisting of the VL, VH, CL and CH1 domains; a
F(ab')2 fragment is a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; a Fd
fragment consists of the VH and CH1 domains; an Fv fragment
consists of the VL and VH domains of a single arm of an antibody;
and a dAb fragment (Ward et al., Nature 341:544-546, 1989) consists
of a VH domain. A single-chain antibody (scFv) is an antibody in
which a VL and VH regions are paired to form a monovalent molecules
via a synthetic linker that enables them to be made, as a single
protein chain (Bird et al., Science 242:423-426 (1988) and Huston
et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, (1988)). Diabodies
are bivalent, bispecific antibodies in which VH and VL domains are
expressed on a single polypeptide chain, but using a linker that is
too short to allow for pairing between the two domains on the same
chain, thereby forcing the domains to pair with complementary
domains of another chain and creating two antigen binding sites
(see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993), and Poljak et al., Structure 2:1121-1123
(1994)). One or more CDRs may be incorporated into a molecule
either covalently or noncovalently to make it an immunoadhesin. An
immunoadhesin may incorporate the CDR(s) as part of a larger
polypeptide chain, may covalently link the CDR(s) to another
polypeptide chain, or may incorporate the CDR(s) noncovalently. The
CDRs permit the immunoadhesin to specifically bind to a particular
antigen of interest.
[0046] An antibody may have one or more binding sites. If there is
more than one binding site, the binding sites may be identical to
one another or may be different. For instance, a
naturally-occurring immunoglobulin has two identical binding sites,
a single-chain antibody or Fab fragment has one binding site, while
a "bispecific" or "bifunctional" antibody has two different binding
sites.
[0047] "Antibody repertoire" refers to the sum of every different
antibody species in an animal or human. Diversity in antibody
repertoires results from, inter alia, immunoglobulin gene
recombination, immunoglobulin gene junctional diversity, terminal
deoxytransferase activity, and somatic hypermutation.
[0048] "Chimeric antibodies" are antibodies that have been altered
from their original form to comprise amino acid sequences from
another protein. Chimeric antibodies retain at least a portion of
the original antibody amino acid sequence, typically the portion
comprising the antigen binding region (Fab). Examples of chimeric
antibodies include, but are not limited to, bispecific antibodies
and fusions with other non-immunoglobulin protein sequences.
[0049] "cis regulatory elements" generally refer to sequences that
regulate the inducible or constitutive expression of gene sequences
under specific conditions or in specific cells. Examples of
cellular processes that expression control sequences regulate
include, but are not limited to, gene transcription, protein
translation, messenger RNA splicing, immunoglobulin isotype
switching, protein glycosylation, protein cleavage, protein
secretion, intracellular protein localization and extracellular
protein homing.
[0050] "Cytokines" refer generally to signaling molecules of the
immune system. Cytokines include, but are not limited to,
Interleukins (IL), transforming growth factors (TGF), tumor
necrosis factors (TNF), lymphotoxins (LT), interferons,
granulocyte-macrophage colony stimulating factors (GM-CSF),
macrophage CSF, Granulocyte CSF, and migration inhibition
factors.
[0051] "Embryonic stem (ES) cells" refer to pluripotent or
multipotent cells that can, when injected into a blastocyst,
contribute to many or all tissues of a prenatal, postnatal or adult
animal. Animals that result from blastocyst injections are often
referred to as "chimeric" animals since their somatic and/or germ
cells are often derived from both the blastocyst donors and the
injected ES cells. One important property of ES cells is their
ability to contribute to the germ line of the animals, resulting in
any desired heritable characteristics to be passed to the chimeric
animal's progeny. Immortalized ES cells are a powerful tool for
generating animals with targeted disruptions of endogenous gene
sequences or for generating animals with foreign genes
(transgenes).
[0052] "Expression control sequences" refer to sequences that allow
for the constitutive or inducible expression of gene sequences
under specific conditions or in specific cells. Examples of
cellular processes that expression control sequences regulate
include, but are not limited to, gene transcription, protein
translation, messenger RNA splicing, immunoglobulin isotype
switching, protein glycosylation, protein cleavage, protein
secretion, intracellular protein localization and extracellular
protein homing.
[0053] "Fusion Proteins" refer to chimeric proteins comprising
amino acid sequences of two or more different proteins. Typically,
fusion proteins result from in vitro recombinatory techniques well
known in the art. However, fusion proteins may result from in vivo
crossover or other recombinatory events.
[0054] "Human immunoglobulin molecules" refer to immunoglobulin
proteins that are encoded by human immunoglobulin gene sequences.
The immunoglobulin gene sequences may be expressed in any cell or
animal, human or non-human.
[0055] "Humanized antibodies" are antibodies that are derived from
a non-human species, in which certain amino acids in the framework
and constant domains of the heavy and light chains have been
mutated so as to avoid or abrogate an immune response in humans.
Alternatively, humanized antibodies may be produced by fusing the
constant domains from a human antibody to the variable domains of a
non-human species. Examples of how to make humanized antibodies may
be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.
[0056] "Inflammation" or "inflammatory disease" refers to the
fundamental pathologic process consisting of cytologic and
histologic reactions that occur in blood vessels and adjacent
tissues in response to injury, abnormal stimulation or biological
agents. Likewise, "non-inflammatory conditions" or
"non-inflammatory diseases" refer to any condition or disease that
is not inflammatory in nature, as disclosed herein.
[0057] "Isolated protein" or "isolated polypeptide" refers
generally to a protein or polypeptide that by virtue of its origin
or source of derivation: (1) is not associated with naturally
associated components that accompany it in its native state, (2) is
free of other proteins from the same species, (3) is expressed by a
cell from a different species, or (4) does not occur in nature.
Thus, a polypeptide that is chemically synthesized, synthesized in
a cell-free biological system (e.g., a rabbit reticulocyte lysate),
or synthesized in a cellular system different from the cell from
which it naturally originates will be "isolated" from its naturally
associated components. A protein may also be rendered substantially
free of naturally associated components by isolation, using protein
purification techniques well known in the art.
[0058] "Mimetics" or "peptide mimetics" are non-peptide analogs
that are commonly used in the pharmaceutical industry as drugs with
properties analogous to those of the template peptide. Fauchere, J.
Adv. Drug Res. 15:29 (1986); Veber and Freidinger, TINS p. 392
(1985); and Evans et al., J. Med. Chem. 30:1229 (1987),
incorporated herein by reference. Mimetics are often developed with
the aid of computerized molecular modeling. Peptide mimetics that
are structurally similar to therapeutically useful peptides may be
used to produce an equivalent therapeutic or prophylactic effect.
Generally, mimetics are structurally similar to a paradigm
polypeptide (i.e., a polypeptide that has a desired biochemical
property or pharmacological activity), such as TWEAK, but have one
or more peptide linkages optionally replaced by a linkage selected
from the group consisting of: --CH2NH--, --CH2S--, --CH2-CH2-,
--CH.dbd.CH-- (cis and trans), --COCH2-, --CH(OH)CH2-, and
--CH2SO--, by methods well known in the art. Systematic
substitution of one or more amino acids of a consensus sequence
with a D-amino acid of the same type (e.g., D-lysine in place of
L-lysine) may also be used to generate more stable peptides. In
addition, constrained peptides comprising a consensus sequence or a
substantially identical consensus sequence variation may be
generated by methods known in the art (Rizo and Gierasch, Ann. Rev.
Biochem. 61:387 (1992), incorporated herein by reference); for
example, by adding internal cysteine residues capable of forming
intramolecular disulfide bridges which cyclize the peptide.
[0059] "Polypeptide analogs" refer to polypeptides that are derived
from wild-type polypeptides but differ therefrom in their amino
acid sequences. Polypeptides with changes in their amino acid
sequences may be muteins, fusion proteins, or mimetics. Polypeptide
analogs also refer to polypeptides that have non-amino acid
sequence differences as compared with the wild-type polypeptides.
These differences may be chemical or biochemical, and include, but
are not limited to, the types of modifications specifically
disclosed herein.
[0060] "Polypeptide fragments" refer to polypeptides that have an
amino-terminal and/or carboxy-terminal deletion, but where the
remaining amino acid sequence is identical to the corresponding
positions in the naturally-occurring sequence. Fragments typically
are at least 5, 6, 8 or 10 amino acids long, preferably at least 14
amino acids long, more preferably at least 20 amino acids long,
usually at least 50 amino acids long, and even more preferably at
least 70 amino acids long.
[0061] "Progenitor cells" refer to cells that can give rise to one
or more cell lineages. Included are stem cells, totipotent cells,
pluripotent cells, multipotent cells, bipotent cells, embryonic
cells or adult cells. Also included are tissue-specific cells,
including, but not limited to, cells committed to a particular
lineage capable of undergoing terminal differentiation, cells that
derive from tissue resident cells, and circulating cells that have
homed to specific tissues.
[0062] "Subjects" are humans and non-human subjects. An example of
a subject is a patient.
[0063] "TWEAK-related conditions" refer to any conditions that
result from aberrant TWEAK function or regulation. The term may
also refer to any condition that does not directly result from
aberrant TWEAK function or regulation, but rather arises out of
some other mechanism wherein disrupting, increasing or otherwise
altering TWEAK activity will have a detectable outcome on the
condition. TWEAK-related conditions can be either inflammatory or
non-inflammatory in nature, and include, but are not limited to,
the conditions and diseases specifically disclosed herein.
[0064] "Vectors" refer to DNA molecules that allow DNA sequences of
interest to be cloned, propagated, recombined, mutated, or
expressed outside of their native cells. Often vectors have
expression control sequences that allow for the inducible or
constitutive expression of gene sequences under specific conditions
or in specific cells. Examples of vectors include, but are not
limited to, plasmids, yeast artificial chromosomes (YACs), viruses,
Epstein Bar Virus (EBV)-derived episomes, bacteriophages, cosmids
and phagemids.
[0065] "Xenogeneic animals" refer to animals bearing substantial
portions of human immunoglobulin loci. Often, xenogeneic animals
bear homologously targeted endogenous immunoglobulin loci,
rendering them incapable of expressing their endogenous
immunoglobulin genes. Examples include the mice of the
XenoMouse.TM. line (Abgenix, Inc., Fremont, Calif.), which are
capable of somatic rearrangement of transgenic human immunoglobulin
genes, hypermutation of the human variable genes, immunoglobulin
gene expression, and immunoglobulin isotype switching. Xenogeneic
animals are capable of mounting effective humoral responses to
antigenic challenge utilizing the human immunoglobulin gene
sequences. Antibodies produced in xenogeneic animals are fully
human and can be isolated from the animals themselves or progeny
thereof, from cultured cells extracted from the animals or progeny
thereof, and from hybridomas created from xenogeneic B lymphocytic
lines or progeny thereof. Moreover, the rearranged human gene
sequences encoding immunoglobulins raised against specific
antigenic challenges can be isolated by conventional recombinant
techniques.
[0066] "Xenogeneic antibodies" refer to antibodies that are encoded
by foreign immunoglobulin loci. For example, in mice of the
XenoMouse.TM. line, the human antibody loci encode xenogeneic
antibodies.
[0067] "Xenogeneic monoclonal antibodies" refer to homogenous
populations of antibodies that are produced in cloned, immortalized
cells, e.g. hybridomas, derived from xenogeneic animals. For
example, hybridomas made from mice of the XenoMouse.TM. line
produce xenogeneic antibodies.
[0068] The understanding and treatment of diseases fundamentally
advances upon determination of the molecular mechanisms or
biochemical pathways underlying them. Physicians and researchers
are thereby enabled to tailor therapeutic agents and formulate
pharmaceutical compositions that specifically target those
molecular mechanisms or biochemical pathways.
[0069] Some of the most complex and debilitating diseases
afflicting humans include those of the heart, liver, kidney, lung,
skin, skeletal muscle, lipid metabolism, gastrointestinal tract,
nervous system, pancreas, reproductive organs, cartilage, bone,
connective tissue system, and progenitor or stem cells. The present
invention advantageously provides important advances in the
understanding of these diseases. More particularly, the invention
provides transgenic animals which express exogenous TWEAK proteins
and demonstrate for the first time a correlation between expression
of TWEAK protein and certain pathological conditions of the heart,
liver, kidney and lung. The invention also provides methods for
treating or preventing such pathological conditions, as well as
methods for identifying TWEAK agonists or antagonists for use in
those methods. Pathological conditions that may be treated
according to the methods of this invention include acute cardiac
injury, chronic heart failure, non-inflammatory dilated
cardiomyopathy, congestive heart failure, liver epithelial cell
hyperplasia, hepatocyte death, liver fibrosis, hepatocyte
vacuolation, liver injury, bile duct conditions, including bile
duct hyperplasia, inflammatory kidney conditions, such as renal
multifocal inflammation, non-inflammatory kidney conditions, such
as tubular nephropathy, tubular hyperplasia, glomerular cysts,
renal hyperplasia, renal capsular thickening, glomerular
nephropathy, Alport Syndrome, kidney tubular vacuolation, kidney
hyaline casts, kidney fibrosis and inflammatory lung conditions.
The invention further provides methods for detecting TWEAK
structures or functions as molecular markers of disease, including
TWEAK proteins or their functions, TWEAK antibodies and TWEAK
nucleic acids.
[0070] The TWEAK-related conditions described herein are treated
using TWEAK agonists or antagonists that are capable of altering
TWEAK activity or disrupting the interaction between a
membrane-bound or full-length form of TWEAK polypeptides with its
cellular receptors. Alternatively, the therapeutic agents and
treatment methods disrupt the interaction between a membrane-bound
or full-length form of TWEAK polypeptides with another TWEAK
polypeptide. Such interference may occur on the surface of a cell,
intra-cellularly, extra-cellularly, or in vitro bound to a solid
phase or in solution. In another alternative, the therapeutic
agents and treatment methods disrupt the interaction between
membrane-bound or full-length forms of TWEAK polypeptides and TWEAK
interacting partners. Such interacting partners may be proteins,
nucleic acids, saccharides, lipids, fatty acids, and steroids.
[0071] In a preferred embodiment of this invention, the
TWEAK-related condition is non-inflammatory in nature.
[0072] In another preferred embodiment, the TWEAK-related condition
is fibrosis, cardiomyopathy, kidney disease, lung disease or liver
disease.
[0073] In another preferred embodiment, the TWEAK-related condition
is skeletal muscle disease, adipose tissue disease,
gastrointestinal tract disease, pancreatic disease, a reproductive
organ disease, a neural tissue disease, cellular death, skin
disease, cartilage disease, bone disease, or connective tissue
disease.
[0074] In another embodiment, TWEAK agonists or antagonists may be
used to treat subjects suffering from a condition, disease or
injury that requires tissue replacement or regeneration (e.g. burn
victims or radiation patients) by affecting progenitor cells in
vivo. The TWEAK agonists or antagonists may also be used to treat
subjects in vivo in combination with progenitor cell or tissue
transplantation therapy. The TWEAK agonists or antagonists may also
be used to expand cell populations in vivo or progenitor cell
populations in vitro for subsequent transplantation into subjects
with or without additional treatment. Progenitor cell populations
used for in vivo cell therapy or in vitro expansion followed by
transplantation may be embryonic or adult in origin. Adult-derived
progenitors may be multipotent or tissue-restricted (Lagasse et
al., Immunity 14:425-436 (2001); Jackson et al. J. Clin. Invest.
107:1355-402 (2001); Anversa and Nadal-Ginard, Nature 415:240-243
(2002); Gussoni et al., Nature 401:390-394 (1999); Brazelton et
al., Science 290:1672-1674 (2000); Peterson et al., Science
284:1168-1170 (1999); Lagasse et al., Nature Medicine 6:1229-1234
(2000)).
[0075] Heart disease is the predominant cause of disability and
death in industrialized nations. In the United States, heart
disease causes about 40% of all mortalities, accounting for
approximately 750,000 deaths annually. Most basic to the function
of the heart is the myocardium, composed primarily of branching and
anastomosing striated muscle cells (cardiac myocytes). Cardiac
myocytes are much larger than the intervening interstitial cells,
accounting for more than 90% of the volume of the myocardium.
Inflammatory cells are rare and collagen is sparse in a normal
myocardium.
[0076] Myocardial disease is common but occurs secondarily in a
number of different heart conditions. Examples of myocardial
disease include inflammatory disorders (e.g., myocarditis), and
non-inflammatory heart conditions such as dilated cardiomyopathy,
systemic metabolic disorders, muscular dystrophies, and genetic
abnormalities in cardiac muscle cells.
[0077] The major types of cardiomyopathy include dilated,
hypertrophic and restrictive cardiomyopathies. It is an object of
the invention to provide methods for the treatment of dilated
cardiomyopathy, which is typically non-inflammatory in nature. In
the case of non-inflammatory dilated cardiomyopathy, which accounts
for approximately 90% of the clinical cases of myocardial disease,
the heart is characterized by progressive cardiac hypertrophy,
dilation, and contractile (systolic) dysfunction. Dilated
cardiomyopathy may occur at any age, but is most common in persons
ranging in age from 20 to 60 years old. Diagnosis is often made
through noninvasive cardiac imaging, particularly through
two-dimensional echocardiography. The histopathology of dilated
cardiomyopathy is characterized by degenerating myocytes with mild
to moderate hypertrophy, an absence of inflammatory cells, and
interstitial fibrosis.
[0078] Clinically, dilated cardiomyopathy presents with slowly
progressive congestive heart failure, but patients may slip
precipitously from a compensated to a decompensated functional
state. Cardiac transplantation is frequently required. Fifty
percent of patients die within two years, and seventy five percent
within five years. The cause of death is typically progressive
cardiac failure or arrhythmia, however, embolism caused by
dislodgment of an intracardiac thrombus may occur.
[0079] Hearts characterized by dilated cardiomyopathy are enlarged,
flabby, and weigh two to three times as much as normal hearts. All
chambers are dilated, with wall thinning, fibrosis, and typically
mural thrombi. In a minority of dilated cardiomyopathies, mitral or
tricuspid regurgitation results from left ventricular chamber
dilation. Cardiac muscle cells are hypertrophied with enlarged
nuclei. Some of the causes of dilated cardiomyopathy include
myocarditis, alcohol or other toxin abuse, pregnancy (peripartum
cardiomyopathy), ischemia, coronary artery disease, hypertension,
and genetic influences.
[0080] Idiopathic Dilated Cardiomyopathy (IDC), a disease of
unknown etiology, is characterized by dilation of one or both
ventricles, systolic dysfunction, and often progression to
congestive heart failure. It is noted that the term "IDC" is used
by some persons of skill in the art interchangeably with the term
"dilated cardiomyopathy", suggesting that IDC is in fact a broad
category of dilated cardiomyopathies that are not the result of
alcohol abuse, toxic insult or myocarditis.
[0081] Microscopically, IDC is characterized by myocardiocyte
hypertrophy, karymegaly and interstitial and perivascular fibrosis.
In contrast to myocarditis, necrosis and cellular infiltration are
not typically prominent in IDC patients, an indication of its
non-inflammatory etiology. Consistent with that etiology is the
fact that anti-inflammatory drugs, such as prednisone, are largely
ineffective in treating IDC.
[0082] It is an object of this invention to provide methods of
treating or preventing dilated cardiomyopathy associated with TWEAK
activity. Because the cause of dilated cardiomyopathy (e.g., IDC)
is largely unknown, specific therapies have not been developed.
Patients are typically treated for heart failure using physical,
dietary, and pharmacological interventions (e.g., beta-adrenoceptor
antagonists, calcium antagonists, and anticoagulants) to control
the symptoms. Also, cardiac transplantation is used when
available.
[0083] Those of skill in the art have been unable to identify any
immunological, histochemical, morphological, ultrastructural or
microbiological marker that might be used to diagnose IDC. However,
epidemiological evidence suggests that predisposition to IDC may be
genetically-based. In 20 percent of IDC patients, a first-degree
relative also shows evidence of IDC, suggesting frequent familial
transmission. Those of skill in the art have recognized the need to
determine molecular genetic markers for IDC in subclinical and
clinical heart disease patients.
[0084] To date, the list of genes associated with dilated
cardiomyopathy includes cardiac troponin T, d-sarcoglycan, cardiac
b myosin heavy chain, cardiac actin, a-tropomyosin, Lamin A/C,
Desmin, cardiac ryanodine receptor, desmoplakin, plakoglobin,
dystrophin, and tafazzin. The need still exists to find additional
genetic factors that contribute to dilated cardiomyopathy and to
design therapeutics that target them. The present invention has,
for the first time, demonstrated a causal link between TWEAK and
dilated cardiomyopathy. It is therefore an object of the invention
to provide a method for identifying dilated cardiomyopathy in a
patient by detecting changes in TWEAK protein expression, TWEAK
protein function, TWEAK mRNA expression, or a chromosomal
alteration. Methods and reagents for detecting such molecular
markers of disease are well known in the art, and involve
immunological or immunohistochemical analyses, enzyme or other
protein-function assays, and standard hybridization-based assays
such as northern blots, Southern blots, single nucleotide
polymorphism (SNP) analysis, and fluorescence in situ hybridization
(FISH) analysis.
[0085] It should be noted that non-inflammatory dilated
cardiomyopathy is characterized by progressive cardiac hypertrophy,
dilation, and contractile dysfunction. In contrast, Chagas' disease
is a rare form of myocarditis, which is an inflammatory heart
disease that develops in humans and experimental animals following
chronic Trypanosoma cruzi infection. Studies of Chagas' disease,
which is prevalent in Central and South America, have identified
anti-self antibodies in the sera of Chagas' disease patients.
Joshua Wynne and Eugene Braunwald, Heart Disease, A Textbook of
Cardiovascular Medicine, Volume 2, Ch. 41, pp. 1442-1444 (5th ed.
1997). The methods disclosed herein are directed to the treatment
of the more common, non-inflammatory-type dilated cardiomyopathy
associated with TWEAK activity.
[0086] An adult human kidney processes more than 1,700 liters of
blood per day, resulting in approximately 1 liter of urine. The
kidney functions in waste excrement, metabolism, water, salt and pH
homeostasis, as well as contributing to the endocrine system. Renal
diseases are more likely to cause morbidity than mortality, with
approximately 35,000 deaths annually in the United States. In
contrast, millions of persons are afflicted annually by non-fatal
kidney diseases such as infections, kidney stones, and urinary
obstruction.
[0087] Typically, glomerular diseases are caused by immunological
disorders, whereas tubular and interstitial disorders are usually
caused by toxins or infectious agents. A partial list of kidney
diseases includes acute nephritic syndrome, asymptomatic hematuria
or proteinuria, acute renal failure, chronic renal failure, renal
tubular defects, urinary tract infection, nephrolithiasis, urinary
tract obstruction and kidney fibrosis.
[0088] Kidney injuries that involve the tubules typically involve
the interstitium as well. Diseases of the tubules can be
inflammatory or non-inflammatory in nature and include ischemic or
toxic tubular injury. A partial list of tubule diseases includes
acute tubular necrosis and acute renal failure; inflammatory
reactions of the tubules and interstitium (tubulointerstitial
nephritis); tubular hyperplasia; tubulointerstitial fibrosis (a
scarring disease initiated by tubular epithelial cells with
interstitial fibroblast, mononuclear cell, glomerular
ultrafiltrate, cytokine and chemokine components); and autosomal
dominant polycystic kidney disease (ADPKD)(an inherited disorder
characterized by large, fluid-filled cysts from the tubules and
collecting ducts and caused by a mutation in either the PKD1 or
PKD2 genes).
[0089] Glomerular diseases represent the most daunting of the
kidney diseases. For instance, chronic glomerulonephritis is the
most common cause of chronic renal failure in humans. In the
so-called secondary glomerular diseases, glomeruli may be injured
by immunologic disorders such as systemic lupus erythematosus
(SLE), as well as vascular disorders, e.g., hypertension and
polyarteritis nodosa. Also, metabolic diseases, such as diabetes
mellitus (i.e. diabetic nephropathy) and hereditary conditions,
such as Fabry disease, affect the glomeruli. The primary glomerular
diseases include primary glomerulonephritis and glomerulopathy.
[0090] The group of diseases under the umbrella of hereditary
nepthritis includes familial renal diseases associated primarily
with glomerular injury. Alports syndrome is a form of nephritis
that is accompanied by nerve deafness and various eye disorders,
including lens dislocation, posterior cataracts, and corneal
dystrophy. The disease is more prevalent in males because of its
dominant X-linked genotype. However, some females are afflicted due
to one of either an autosomal dominant and recessive genotype. The
glomeruli of Alport kidneys show segmental proliferation or
sclerosis. Sometimes the epithelial cells acquire a foamy
appearance, due to neutral fat and mucopolysaccharide accumulation.
The glomular and tubular basement membranes show irregular foci of
thickening or attenuation, with splitting and lamination of the
lamina densa.
[0091] Because kidney diseases are of significant clinical
importance, those of skill in the art have recognized the need to
understand their physiological and genetic causes. Skilled artisans
further recognize the need to develop new therapeutic agents for
treatment of chronic and acute kidney diseases. The invention
demonstrates for the first time a causal link between TWEAK and
kidney disease.
[0092] Therefore, in one embodiment, the invention provides methods
for the treatment of kidney diseases. In a more preferred
embodiment, the kidney disease may be Alport syndrome. In other
more preferred embodiments of the invention, the target kidney
diseases may be characterized by multifocal inflammation, tubular
nephropathy, tubular hyperplasia, cysts, glomerular nephropathy,
tubular vacuolation, fibrosis or hyaline casts.
[0093] Lung disease has been, and remains, a prevalent affliction.
Primary respiratory infections, such as bronchitis,
bronchopneumonia and other types of pneumonia, must commonly be
treated by clinicians. Lung diseases may be exacerbated by
environmental factors such as cigarette smoke, air pollution and
other inhalants. Chronic bronchitis, emphysema, pulmonary fibrosis
and malignancy are also quite common. The lungs are also
secondarily involved in many terminal diseases, with pulmonary
edema, atelectasis, or bronchopneumonia found in most
critically-ill patients.
[0094] Asthma is a chronic relapsing inflammatory disorder
characterized by hyper-reactive airways. The symptoms are typically
characterized by an episodic, reversible bronchoconstriction.
Asthma is caused by an increased responsiveness of the
tracheobronchial tree to various stimuli and is often associated
with an IgE response to external allergens.
[0095] There are two major types of asthma. The first type is
extrinsic asthma, which is initiated by a type hypersensitivity
reaction induced by exposure to an extrinsic antigen. The list of
extrinsic asthma conditions includes atopic (allergic) asthma,
occupational asthma, and allergic bronchopulmonary aspergillosis.
The second type is intrinsic asthma, which results from nonimmune
mechanisms, and is triggered by factors, such as aspirin ingestion,
pulmonary infections, stress, cold, inhaled irritants, and
exercise.
[0096] Those of skill in the art have recognized the need for a
better understanding of lung disease, including both
non-inflammatory and inflammation-based diseases, such as asthma.
An increased understanding will facilitate the development of
improved pharmaceutical agents for treating lung diseases. The
present invention demonstrates for the first time a causal link
between TWEAK and lung disease, and methods of treatment thereof.
In more preferred embodiments of this invention, the lung disease
is characterized by inflammation, including granulomatous and/or
lymphohistiocytic inflammation.
[0097] The liver is the primary regulator of digestion and
metabolic homeostasis, including the processing of dietary amino
acids, carbohydrates, lipids, and vitamins, as well as the
synthesis of serum proteins, detoxification, and excretion into the
bile of endogenous waste products and pollutant xenobiotics. Thus,
hepatic disease is typically very serious, sometimes
life-threatening.
[0098] The liver is vulnerable to many types of diseases, including
metabolic, toxic, microbial, circulatory, and neoplastic insults.
Toxins or immunological disorders may cause hepatocytes to swell,
and become edematous in appearance, with irregularly clumped
cytoplasm and large clear spaces. Also, retained biliary material
may cause the hepatocytes to become foamy and swollen. Accumulation
of substances such as iron, copper and fat droplets (steatosis) can
accumulate in hepatocytes. In cases of alcoholic liver disease and
acute fatty liver of pregnancy, tiny droplets that do not displace
the nucleus appear (known as microvesicular steatosis).
[0099] Hepatocyte necrosis, which results from significant liver
injury, can be characterized by, inter alia, ischemic coagulative
necrosis. Often, cell death from toxic or immunologically related
conditions is characterized by rounded up hepatocytes and shrunken,
pyknotic, intensely eosinophilic "Councilman bodies" containing
fragmented nuclei (resulting from apoptosis). Alternatively,
hepatocytes may become osmotically swollen and rupture (lytic
necrosis).
[0100] Hepatitis results from some injury to the liver associated
with an influx of acute or chronic inflammatory cells. Hepatocyte
necrosis may precede the onset of inflammation, or vice versa.
Fibrosis, an irreversible consequence of hepatic damage, usually
results from inflammation or non-inflammatory mechanisms, such as a
direct toxic insult. The characteristic deposition of collagen
affects blood flow and perfusion of hepatocytes. With continuing
fibrosis, the liver subdivides into nodules of regenerating
hepatocytes with surrounding scar tissue (cirrhosis).
[0101] Oval cells are so named because of their morphological
appearance as small, proliferating epithelial cells with an ovoid
nucleus and scant basophilic cytoplasm. Oval cells normally reside
in the terminal bile ductules that connect the intrahepatic ducts
which are located in the portal triad with the hepatocyte cords.
These cells may be derived from resident liver progenitor cells, or
from bone marrow progenitor cells that have circulated and homed to
the liver. These ductular progenitor cells have the potential to
differentiate into both bile duct epithelial cells and hepatocytes.
The present invention demonstrates that expression of a TWEAK
transgene in mice has the capacity to expand the population of
ductular progenitor cells.
[0102] Because of the high levels of morbidity and mortality caused
by liver disease, the art has recognized that the molecular and
genetic underpinnings of that disease must be elucidated.
Identification of causative factors facilitates the discovery of
therapeutic agents for treatment of chronic and acute liver
diseases. The present invention demonstrates a causal link between
TWEAK and liver disease, and advantageously provides methods for
the treatment thereof. In a more preferred embodiment, the liver
disease is epithelial hyperplasia, hepatocyte vacuolation, bile
duct injury resulting in fibrosis, hepatocyte death or liver
injury.
[0103] The skeletomuscular system is critical for posture and
locomotion. Skeletal muscle can atrophy in response to disuse,
which may be secondary to conditions of nerve or blood supply
deprivation and drug exposure such as glucocorticoids. Skeletal
muscle can also atrophy in conditions of genetic or degenerative
disorders. These conditions or diseases can be inflammatory or
noninflammatory in nature. Muscular dystrophy constitutes a large
group of hereditary myopathies characterized by atrophy and loss of
muscle fibers in the absence of nerve disease; one common form that
is included in this group is Duchenne's muscular dystrophy.
Congenital muscle disease may also occur in the context of glycogen
storage diseases, such as acid maltase deficiency, which results in
babies with weak muscles, poor athletes, enlarged hearts, and often
early death from cardiac failure. Congenital disorders leading to
muscle atrophy also include, but are not limited to, mitochondrial
myopathies, lipid myopathies, central tubular myopathies, and
rhabdomyolysis. Myopathic conditions also may develop in adults,
one of the most commonly observed being alcoholic myopathy.
Skeletal muscle wasting also may occur as a component of neuronal
disease, including but not limited to, amyotrophic lateral
sclerosis (ALS). In addition, skeletal muscle wasting, also known
as cachexia, is an important pathological condition seen in most
terminally ill cancer patients and often is directly responsible
for patients' death. Diseases of skeletal muscle that occur in the
context of inflammation or autoimmunity include polymyositis,
inflammatory myopathies, and glucocorticoid induced atrophy. The
present invention establishes a link between TWEAK and the ability
of myoblasts to differentiate into myotubes. It is therefore an
object of the invention to provide methods of treatment of skeletal
muscle disorders by promoting skeletal muscle regeneration using in
vivo or in vitro approaches.
[0104] Accumulation of fat cells occurs in conditions of obesity,
including obesity associated with metabolic disorders such as Type
II diabetes. Ingrowth into organs of fat cells, so-called fatty
infiltration, occurs in a variety of settings, and is a
pathological component of muscular dystrophies. The present
invention has demonstrated a link between TWEAK and the ability of
preadipocytes to differentiate into adipocytes. It is therefore an
object of the invention to provide methods of treatment of
disorders associated with an accumulation or paucity of adipocytes
by modulating adipocyte differentiation with TWEAK agonists or
antagonists or pharmaceutical compositions thereof.
[0105] The methods of treating a TWEAK-related condition according
to the present invention utilize TWEAK agonists or antagonists or
compositions comprising them. TWEAK agonists or antagonists useful
in treating TWEAK-related conditions according to this invention
are described herein and are known in the art. Such agents include
those disclosed in, e.g., PCT International Publication Nos. WO
98/05783, WO 98/35061, WO 99/19490, WO 00/42073, and WO 01/45730,
all of which are incorporated herein by reference. TWEAK
antagonists useful in the methods of the invention include
anti-TWEAK antibodies, such as antibodies that are human,
non-human, humanized or xenogeneic, as described herein, and are
polyclonal, monoclonal, or synthetic. Furthermore, the antibodies
may be full-length, frayments thereof, or fusion proteins that
include antigen recognition sequences.
[0106] TWEAK antagonists useful in the methods of the invention
also include anti-TWEAK receptor antibodies. Here, the TWEAK
receptor may be FN14 or other members of the TNF-R family that are
bound by TWEAK. The antibodies to the TWEAK receptor may be human,
non-human, humanized or xenogeneic, as described herein, and are
polyclonal, monoclonal, or synthetic. Furthermore, the antibodies
may be full-length, fragments thereof, or fusion proteins that
include antigen recognition sequences.
[0107] Immunization of animals with TWEAK or TWEAK receptor
antigens may be carried out by any method known in the art. See,
e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York:
Cold Spring Harbor Press, 1990. Methods for immunizing non-human
animals, such as mice, rats, sheep, goats, pigs, cattle, horses and
the like are well known in the art. See, e.g., Harlow and Lane and
U.S. Pat. No. 5,994,619. In a preferred embodiment, the antigen is
administered with or without an adjuvant to stimulate the immune
response. Such adjuvants include, inter alia, complete or
incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM
(immunostimulating complexes).
[0108] The antigen chosen for immunization can be any one of the
following: a TWEAK polypeptide; a TWEAK polypeptide fragment; a
TWEAK mutein; a TWEAK mimetic; a TWEAK fusion protein; a TWEAK
receptor polypeptide; a TWEAK receptor fragment; a TWEAK receptor
mutein; a TWEAK receptor mimetic; a TWEAK receptor fusion protein;
a cell expressing a TWEAK polypeptide, fragment, mutein, or fusion
protein thereof; or a cell expressing a TWEAK receptor polypeptide,
fragment, mutein, or fusion protein thereof. The immunoglobulins
raised in the animals by immunization may be recovered from various
tissues or fluids of the animals, including serum, milk, ascites,
spleen, thymus, peripheral blood cells, fetal liver, bone marrow,
peritoneum, and any other tissues or fluids having significant
immunoglobulin concentrations. Also, hybridomas can be made and
isolated that produce monoclonal antibodies secreted into the
medium.
[0109] In preferred embodiments of this invention, the antibodies
are polyclonal antibodies, monoclonal antibodies, or humanized
antibodies. In a more preferred embodiment, the humanized
antibodies comprise human antibody constant and/or framework
regions. In another preferred embodiment, the antibodies are
xenogeneic antibodies. In more preferred embodiments, the
xenogeneic antibodies are polyclonal antibodies or monoclonal
antibodies.
[0110] Xenogeneic antibodies are complete antibodies of one species
that are expressed in an entirely different species. For instance,
if a mouse expresses the DNA required to produce complete human
antibodies, the resulting antibodies are xenogeneic (i.e. human
antibodies produced in a mouse). Targeted inactivation (knockout)
technology provides the opportunity to disrupt an animal's normal
expression of endogenous immunoglobulin genes. Transgenic animal
technology provides the opportunity to produce non-human animals
that produce xenogeneic immunoglobulin proteins. Such xenogeneic
animals can be mated to the immunoglobulin knockout animals
described above, resulting in animals that produce only the
xenogeneic immunoglobulins and not endogenous immunoglobulins.
[0111] Expression of xenogeneic immunoglobulin genes allows the
production of a highly diverse repertoire of human antibodies,
including monoclonal antibodies. This is because (1) the exogenous
immunoglobulin genes retain their cis regulatory elements and are
subject to the host animal's normal variable (V), diversity (D),
and joining (J) recombinational events; (2) the exogenous
immunoglobulin genes are expressed in a similar fashion as
endogenous immunoglobulin loci; and (3) the resulting antibodies
apparently support normal B lymphocytic development and humoral
responses.
[0112] The exogenous immunoglobulin genes may be introduced into
the animals as an entire immunoglobulin locus, a part of an
immunoglobulin locus, or as a "minilocus" in which a more complete
exogenous Ig locus is mimicked through the inclusion of a handful
of the individual genes from that Ig locus. Furthermore, transgenic
animals may be engineered to express transgenes that encode
modified antibodies such as single-chain antibodies or chimeric
antibodies.
[0113] TWEAK agonists or antagonists useful in the methods of the
invention may also be TWEAK polypeptides, or fragments, analogs,
muteins, or mimetics thereof, as described herein. Analogs can
differ from the naturally occurring TWEAK amino acid sequence, or
in ways that do not involve the sequence, or both. In a preferred
embodiment, the TWEAK polypeptide analogs are muteins. Methods of
generating muteins are well known in the art of molecular biology,
and include altering DNA molecules by random mutagenesis, site
directed mutagenesis, deletions and truncations. Techniques for
mutagenizing DNA are well known in the art, and include polymerase
chain reaction (PCR) mutagenesis, saturation (i.e. chemical or
radiation) mutagenesis, chemical DNA synthesis, alanine scanning
mutagenesis, oligonucleotide-mediated mutagenesis (hybridization to
a DNA template in vitro followed by enzymatic elongation), cassette
(recombinant) mutagenesis, and combinatorial mutagenesis
(introduction of random degenerate sequences into the TWEAK
DNA).
[0114] The TWEAK polypeptides bind to TWEAK receptors, to other
TWEAK polypeptides, or to other TWEAK-interacting partners. The
TWEAK fragments may be membrane bound, and may be delivered in
pharmaceutical compositions that comprise liposomes or other
cellular or pseudocellular delivery systems. The TWEAK fragments
may also be soluble TWEAK polypeptides that contain either a
truncation or internal deletion that removes the transmembrane
domain. Furthermore, the TWEAK polypeptides useful in the methods
of the invention may result in either no TWEAK response, or an
altered TWEAK response. Examples of such TWEAK polypeptides are
analogs of the TWEAK protein, including deletion or truncation
mutants, peptides containing one or more amino acid substitutions,
TWEAK mimetics, as well as non-amino acid sequence-modified TWEAK
polypeptides.
[0115] TWEAK agonists or antagonists useful in the methods of the
invention may also be TWEAK receptor polypeptides, or fragments,
analogs, muteins, or mimetics thereof, as described herein. The
TWEAK receptor polypeptides are bound by TWEAK polypeptides, to
other TWEAK receptor polypeptides, or to other TWEAK
receptor-interacting partners. The TWEAK receptor fragments may be
membrane bound, and may be delivered in pharmaceutical compositions
that comprise liposomes or other cellular or pseudocellular
delivery systems. The TWEAK receptor fragments may also be soluble
TWEAK receptor polypeptides that contain either a truncation or
internal deletion that removes the transmembrane domain.
Furthermore, the TWEAK receptor polypeptides useful in the methods
of the invention may result in either no TWEAK response, or an
altered TWEAK response. Examples of such TWEAK receptor
polypeptides are analogs of TWEAK receptor proteins, including
deletion or truncation mutants, peptides containing one or more
amino acid substitutions, TWEAK receptor mimetics, as well as
non-amino acid sequence-modified TWEAK receptor polypeptides.
[0116] Moreover, TWEAK agonists or antagonists useful in the
methods of the invention may be organic or inorganic compounds. The
organic compounds may be either small organic compounds, such as
those found in chemical libraries well known in the art. Other
organic compounds include, but are not limited to, nucleic acids,
peptides, saccharides, lipids and fatty acids, steroids, or
derivatives thereof. Inorganic compounds may be silica based or
other minerals and salts. The organic or inorganic compounds may
bind to TWEAK polypeptides, TWEAK receptor polypeptides, or other
TWEAK interacting partners, as described herein.
[0117] Non-sequence modifications of the TWEAK or TWEAK receptor
polypeptides may result from in vivo or in vitro chemical
derivatization the polypeptides, and include, but are not limited
to, changes in acetylation, methylation, phosphorylation,
carboxylation, oxidation state, or glycosylation. In addition,
chemical derivatization may involve coupling to organic polymers
such as polyethylene glycol (PEG) or other polymers known in the
medicinal arts. Therefore, a TWEAK polypeptide analog may result
from a non-amino acid sequence modification.
[0118] The TWEAK or TWEAK polypeptides may be expressed as fusion
proteins. Fusion proteins are well known in the art. A person of
skill in the art may choose from a wide variety of fusion partner
moieties, including those from prokaryotes and eukaryotes.
[0119] According to this invention, any individual, including
humans and animals, may be treated in a pharmaceutically acceptable
manner with a pharmaceutically effective amount of a TWEAK agonist
or antagonist or compositions comprising such an agent, for a
period of time sufficient to treat a TWEAK-related condition in the
individual to whom they are administered over some period of time.
Alternatively, individuals may receive a prophylactically effective
amount of a TWEAK agonist or antagonist, or compositions comprising
such an agent, which is effective to prevent a TWEAK-related
condition in an individual to whom they are administered over some
period of time. TWEAK agonists or antagonists useful in the methods
of the invention may be formulated in pharmaceutical compositions
by the methods disclosed herein and may be delivered by parenteral
route, injection, transmucosal, oral, inhalation, ocular, rectal,
long-acting implantation, topical, sustained-released or
stent-coated means. TWEAK agonists or antagonists may be in a
variety of conventional forms employed for administration. These
include, for example, solid, semi-solid and liquid dosage forms,
such as liquid solutions or suspension, slurries, gels, creams,
balms, emulsions, lotions, powders, sprays, foams, pastes,
ointments, salves, and drops.
[0120] In addition, TWEAK agonists or antagonists may be delivered
via a gene therapy route. Briefly, nucleic acid molecules encoding
proteins or expressing antisense molecules are delivered to a
subject utilizing any of the vectors known in the art to be
suitable for delivering the nucleic acid molecules to the target
tissues or organs. Typical vectors include liposomes, plasmids, and
viral vectors (e.g., retroviruses, adenoviruses and
adeno-associated viruses).
[0121] The most effective mode of administration and dosage regimen
of TWEAK agonists or antagonists, or compositions comprising them,
will depend on the effect desired, previous therapy, if any, the
individual's health status, the status of the condition itself, the
response to the TWEAK agonists or antagonists and the judgment of
the treating physician. TWEAK agonists or antagonists, or
compositions comprising them, may be administered in any dosage
form acceptable for pharmaceuticals or veterinary preparations, at
one time or over a series of treatments.
[0122] The amount of TWEAK agonists or antagonists, or compositions
comprising them, which provides a single dosage will vary depending
upon the particular mode of administration, the specific TWEAK
agonists or antagonists, or composition, dose level and dose
frequency. A typical preparation will contain between about 0.01%
and about 99%, preferably between about 1% and about 50%, of TWEAK
agonists or antagonists or compositions thereof (w/w).
[0123] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a TWEAK agonist or antagonist
is between about 0.005-10.00 mg/kg body weight, more preferably
between about 0.05-1.0 mg/kg body weight.
[0124] TWEAK agonists or antagonists, or compositions comprising
them, may be administered alone, or as part of a pharmaceutical or
veterinary preparation, or as part of a prophylactic preparation,
with or without adjuvant. They may be administered by parenteral or
oral routes. For example, they may be administered by oral,
pulmonary, nasal, aural, anal, dermal, ocular, intravenous,
intramuscular, intraarterial, intrapeutoneal, mucosal, sublingual,
subcutaneous, transdermal, topical or intracranial routes, or into
the buccal cavity. In either pharmaceutical or veterinary
applications, TWEAK agonists or antagonists may be topically
administered to any epithelial surface. Such surfaces include oral,
ocular, aural, anal and nasal surfaces. Pharmaceutical compositions
may be produced by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping or lyophilizing processes.
[0125] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in a conventional manner, using
one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
active compounds into preparations which can be used
pharmaceutically. The appropriate formulation will be dependent
upon the intended route of administration.
[0126] For transmucosal administration, penetrants appropriate to
the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art. For ocular
administration, suspensions in an appropriate saline solution are
used, as is known in the art.
[0127] For oral administration, the TWEAK agonists or antagonists
may be formulated readily by combining the active agents with
conventional pharmaceutically acceptable carriers. TWEAK agonists
or antagonists may be formulated as tablets, pills, liposomes,
granules, spheres, dragees, capsules, liquids, gels, syrups,
slurries, suspensions and the like, for oral ingestion by a patient
to be treated.
[0128] TWEAK agonists or antagonists, or compositions comprising
them, may also comprise any conventional carrier or adjuvant used
in pharmaceuticals or veterinary preparations. These carriers and
adjuvants include, for example, Freund's adjuvant, ion exchanges,
alumina, aluminum stearate, lecithin, buffer substances, such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, waters,
salts or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium, trisilicate, cellulose-based substances and polyethylene
glycol. Adjuvants for topical or gel base forms may include, for
example, sodium carboxymethylcellulose, polyacrylates,
polyoxyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wood wax alcohols.
[0129] Pharmaceutical compositions for oral use can be obtained as
a solid excipient, optionally grinding a resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients include fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0130] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0131] Pharmaceutical compositions which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with fillers such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All compositions for oral administration
should be in dosages suitable for such administration.
[0132] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0133] For administration by inhalation, TWEAK agonists or
antagonists 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.
[0134] TWEAK agonists or antagonists may be formulated for either
parenteral administration by injection, e.g., by bolus injection,
or continuous infusion. The agents may be formulated in aqueous
solutions, aqueous suspensions, oily suspensions, or emulsions, and
may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Formulations for injection may be
presented in unit dosage form, e.g., in ampoules or in multi-dose
containers, with an added preservative
[0135] Typical aqueous solution formulations include
physiologically compatible buffers such as Hanks solution, Ringer's
solution, or physiological saline buffer. Typical oily suspensions
may include lipophilic solvents or vehicles that include fatty oils
such as sesame oil, or synthetic fatty acid esters, such as ethyl
oleate or triglycerides, or liposomes. Aqueous injection
suspensions may contain substances which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, the suspensions may also contain suitable
stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions. Alternatively, TWEAK agonists or antagonists may be in
powder form for constitution with a suitable vehicle, such as
sterile pyrogen-free water, before use.
[0136] The TWEAK agonists or antagonists may also be formulated in
rectal compositions, such as suppositories or retention enemas,
e.g., containing conventional suppository bases such as cocoa
butter or other glycerides.
[0137] In addition to the formulations described, TWEAK agonists or
antagonists may also be formulated as a depot preparation. Such
long acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0138] A pharmaceutical carrier for TWEAK agonists or antagonists
which are hydrophobic is a co-solvent system comprising benzyl
alcohol, a nonpolar surfactant, a water-miscible organic polymer,
and an aqueous phase. The co-solvent system may be the VPD
co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8%
w/v of the non-polar surfactant polysorbate 80, and 65% w/v
polyethylene glycol 300, made up to volume in absolute ethanol. The
VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a
5% dextrose in water solution. This co-solvent system dissolves
hydrophobic compounds well, and itself produces low toxicity upon
systemic administration. Naturally, the proportions of a co-solvent
system may be varied considerably without destroying its solubility
and toxicity characteristics. Furthermore, the identity of the
co-solvent components may be varied: for example, other
low-toxicity nonpolar surfactants may be used instead of
polysorbate 80; the fraction size of polyethylene glycol may be
varied; other biocompatible polymers may replace polyethylene
glycol, e.g., polyvinyl pyrrolidone; and other sugars or
polysaccharides may be substituted for dextrose.
[0139] Alternatively, other delivery systems for hydrophobic
pharmaceutical compounds may be employed. Liposomes and emulsions
are examples of delivery vehicles or carriers for hydrophobic
drugs. Certain organic solvents, such as dimethylsulfoxide also may
be employed, although they may display a greater toxicity.
[0140] Additionally, TWEAK agonists or antagonists may be delivered
using a sustained-release system, such as semipermeable matrices of
solid hydrophobic polymers containing the therapeutic agent.
Various sustained-release materials are available and well known by
those skilled in the art. Sustained-release capsules may, depending
on their chemical nature, release the compounds for a few weeks up
to over 100 days.
[0141] Depending on the chemical nature and the biological
stability of the TWEAK agonist or antagonist, additional strategies
for protein stabilization may be employed.
[0142] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include, but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0143] TWEAK agonists or antagonists may be provided as salts with
pharmaceutically compatible counterions. Pharmaceutically
compatible salts may be formed with many acids, including but not
limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic,
succinic, etc. Salts tend to be more soluble in aqueous or other
protonic solvents that are the corresponding free base forms.
[0144] TWEAK agonists or antagonists may also be formulated into
pharmaceutical compositions useful for coating stents, for the
treatment of the TWEAK-related heart conditions.
[0145] The present invention also relates to a method for
identifying a TWEAK agonist or antagonist. Such TWEAK agonists or
antagonists are useful for the treating TWEAK-related conditions,
i.e., diseases, settings of injury or other pathological conditions
of tissues wherein a receptor for TWEAK, e.g. FN14, is expressed.
Those conditions include fibrosis and diseases of the heart (e.g.
cardiomyopathies), kidney, lung, liver, skin, skeletal muscle,
lipid metabolism (e.g. obesity), gastrointestinal tract, pancreas,
reproductive organs, neural tissue (including neurodegeneration),
cartilage, bone and connective tissue. Such TWEAK agonists or
antagonists are also useful for promoting tissue replacement by
modulating the behavior of progenitor cells in vivo or in vitro
according to the present invention.
[0146] One embodiment of the method for identifying a TWEAK
antagonist comprises the steps of: 1) exposing a transgenic test
animal that expresses an exogenous DNA encoding a TWEAK
polypeptide, or a fragment, analog, mutein, or mimetic thereof, to
a compound which is a candidate TWEAK antagonist; 2) comparing the
fibrotic, cardiac, kidney, liver, lung, skin, skeletal muscle,
lipid, gastrointestinal tract, pancreas, reproductive organs,
neural, cartilage, bone or connective tissue from the transgenic
test animal to the same organ or tissue from a reference animal
that expresses the exogenous DNA but was not exposed to the
compound; and 3) determining whether the compound has affected the
fibrotic, cardiac, kidney, liver, lung, skin, skeletal muscle,
lipid, gastrointestinal tract, pancreas, reproductive organs,
neural, cartilage, bone or connective tissue. In another
embodiment, the transgenic test animal is either a mammal or a
non-mammal, as disclosed herein.
[0147] The transgenic animals disclosed herein express exogenous
DNAs encoding TWEAK polypeptides, wherein the expression results in
a TWEAK-related condition. In the examples, transgenic mice were
generated that express exogenous TWEAK proteins in either a
truncated, soluble form or in a full-length, membrane-bound form.
The mice that express the exogenous TWEAK proteins revealed
phenotypes that include non-inflammatory dilated cardiomyopathy,
congestive heart failure, liver epithelial cell hyperplasia,
hepatocyte vacuolation, liver injury and inflammatory kidney
conditions, such as multifocal inflammation, non-inflammatory
kidney conditions, such as tubular nephropathy, cysts, glomerular
nephropathy, kidney tubular hyperplasia, kidney fibrosis and
inflammatory lung conditions. Furthermore, wild-type mice that were
infected with viral vectors that express exogenous TWEAK proteins
showed ductal hyperplasia, hepatocyte death, liver fibrosis and
liver injury as well.
[0148] Having these animals in hand, persons of skill in the art
have a powerful method for drug discovery. Specifically, the
animals that express exogenous TWEAK proteins represent a model
system for practicing a method for the discovery of TWEAK agonists
or antagonists useful for the prevention or treatment of the
TWEAK-related conditions disclosed herein.
[0149] In preferred embodiments, the animals useful in these model
systems are either mammalian or non-mammalian. In more preferred
embodiments, the mammalian animals are mice, rats, hamsters,
rabbits, dogs, cats, cows, pigs, goats, horses, sheep, guinea pigs
and primates. In other more preferred embodiments, the
non-mammalian animals are birds, fish, amphibians, insects, and
invertebrates.
[0150] The exogenous DNA encoding the TWEAK polypeptide is
expressed in the transgenic animals via expression control
sequences that control the expression of the exogenous DNA in the
animal. Expression control sequences that control transcription
include, e.g., promoters, enhancers transcription termination
sites, locus control regions, RNA polymerase processivity signals,
and chromatin remodeling elements. Expression control sequences
that control post-transcriptional events include splice donor and
acceptor sites and sequences that modify the half-life of the
transcribed RNA, e.g., sequences that direct poly(A) addition or
binding sites for RNA-binding proteins. Expression control
sequences that control translation include ribosome binding sites,
sequences which direct targeted expression of the polypeptide to or
within particular cellular compartments, and sequences in the 5'
and 3' untranslated regions that modify the rate or efficiency of
translation.
[0151] Preferred expression control sequences for TWEAK expression
in the transgenic animals include viral elements that direct high
levels of protein expression, such as promoters and/or enhancers
derived from retroviral LTRs, cytomegalovirus (CMV) (such as the
CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e.g., the adenovirus major late
promoter (AdMLP)), polyoma and strong mammalian promoters such as
native immunoglobulin and actin promoters. In one embodiment, the
DNA encoding a TWEAK polypeptide is driven by the alpha1
anti-trypsin (AAT) promoter. For further descriptions of viral
expression control elements, and sequences thereof, see, e.g., U.S.
Pat. Nos. 5,168,062; 4,510,245; and 4,968,615.
[0152] The exogenous DNA may also be expressed in the transgenic
animals from tissue-specific expression control elements, including
promoters. Tissue-specific expression control elements are known in
the art. Non-limiting examples of suitable tissue-specific
promoters include the liver-specific albumin promoter (Pinkert et
al., Genes Dev. 1:268-277 (1987)), lymphoid-specific promoters
(e.g., Calame and Eaton, Adv. Immunol. 43:235-275(1988); Winoto and
Baltimore, EMBO J. 8:729-733 (1989); Banerji et al., Cell
33:729-740 (1983); and Queen and Baltimore, Cell 33:741-748
(1983)), neuron-specific promoters (e.g., Byrne and Ruddle Proc.
Natl. Acad. Sci. USA 86:5473-5477(1989)), pancreas-specific
promoters (e.g., Edlund et al., Science 230:912-916 (1985)),
mammary gland-specific promoters (e.g., U.S. Pat. No. 4,873,316 and
European patent application 264,166), and developmentally-regulated
promoters (e.g., Kessel and Gruss, Science 249:374-379 (1990);
Campes and Tilghman, Genes Dev. 3:537-546 (1989)). Other
non-limiting examples of tissue-specific promoters include the
cardiac tissue promoter alpha myosin heavy chain promoter (.alpha.
MHC), the skin tissue promoter keratin-14 (K14), the lung tissue
promoter surfactant protein C (SPC), and the kidney tissue
promoters Ksp-cadherin and kidney androgen-regulated protein (KAP).
The exogenous DNA may also be expressed from an inducible
eukaryotic promoter, such as the metallothionine (MT) promoter, or
other inducible eukaryotic promoters known in the art.
[0153] In one embodiment of the present invention, the TWEAK
polypeptide expressed in the transgenic animals of the invention
may be a full-length TWEAK polypeptide. In another embodiment, the
polypeptides expressed in the transgenic animals are fragments of
the TWEAK polypeptide. In a preferred embodiment, the TWEAK
polypeptide fragments are soluble TWEAK polypeptides.
[0154] In another embodiment, the invention relates to transgenic
animals that express an exogenous DNA encoding a TWEAK polypeptide
in a tissue selected from the group consisting of: heart; blood;
vessel; lungs; liver; kidney; brain; placenta; skeletal muscle;
pancreas; spleen; lymph; thymus; appendix; peripheral blood
lymphocyte; gastrointestinal tract; neurons; skin; adipocyte;
cartilage; bone; connective tissue. In one embodiment, the TWEAK
DNA is expressed from a constitutive promoter. In another
embodiment, the DNA is expressed from an inducible promoter. In
another embodiment, the DNA is expressed from a tissue specific
promoter.
[0155] The present invention also relates to methods of identifying
TWEAK agonist compounds that may act as therapeutic agents for
treatment of TWEAK-related conditions or for promoting tissue
replacement by modulating the behavior of progenitor cells in vivo
or in vitro according to the present invention. Agonist candidate
compounds may be administered to normal animals and their effect on
organ systems assessed. Fibrotic, cardiac, liver, kidney, lung,
skin, skeletal muscle, lipid, gastrointestinal tract, pancreas,
reproductive organs, neural, cartilage, bone or connective tissue
from the treated animal is then compared to the same tissue from an
untreated animal; it is thereby determined whether the compound has
induced a biological effect in any of said tissues.
[0156] This invention also relates to methods of identifying TWEAK
regulated genes that may act as therapeutic targets for treatment
of TWEAK-related conditions. For example, RNA profiling could be
carried out in TWEAK transgenic animals as compared to normal
animals in various tissues and drug targets thus identified.
[0157] It is a further objective of the invention to provide
methods of affecting progenitor stem cell proliferation or
differentiation, including that of mesenchymal stem cell types that
give rise to muscle cells, cartilage, bone or connective tissue
cell types such as stromal cells, fibroblasts, adipocytes and
dermal cells. It is also an objective of the invention to provide
methods of affecting the proliferative or differentiative ability
of oval cells, which can give rise to biliary epithelial cells or
hepatocytes and kidney progenitors, which can give rise to tubular
epithelium.
EXAMPLES
[0158] In order that this invention may be better understood, the
following examples are set forth. These examples are for the
purpose of illustration only and are not to be construed as
limiting the scope of the invention in any manner.
Example 1
Generation of TWEAK Transgenic Mice
[0159] In order to identify target organ(s) for TWEAK activity and
the biological consequences of TWEAK signaling in vivo, two murine
TWEAK expression constructs were created and used for the
overexpression of TWEAK peptides in normal (C57Bl/6.times.DBA/2)F1
and (C57Bl/6.times.SJL/J)F2 mice using standard transgenic
techniques. R. S. Williams and P. D. Wagner, J. Applied Physiology
88:1119-1126 (2000). The TWEAK expression constructs used were as
follows: (1) A TWEAK cDNA from amino acids 101-249 of SEQ ID NO:1,
encoding a soluble form of murine TWEAK (designated sTWEAK)
downstream of a murine IgG signal sequence was inserted into the
CH269 expression vector (a derivative of vector PCDEP4 (Invitrogen)
containing the SV40 poly A addition sequence) downstream of the
human alpha1 anti-trypsin (AAT) promoter and a beta-globin intron
and upstream of the human growth hormone (hGH) poly A sequence; and
(2) A cDNA encoding the full-length, transmembrane form of the
protein (designated FL-TWEAK) corresponding to amino acids 1-249 of
SEQ ID NO:1 was inserted into the pBlueScript expression vector (as
described in Desplat-Jego et al., J. Neuroimmunology 133:116-123
(2002)) containing the SV40 poly A addition site. The FL-TWEAK
sequence plus the poly A addition site was then isolated and cloned
into another vector; a fragment containing the ApoE enhancer-human
AAT promoter regulatory region was then inserted upstream to create
the expression vector CA300. The AAT promoter has been shown to
direct transcription primarily in liver and at lower levels in
other tissues including kidney. P. Koopman et al., Genes Dev.
3:16-25 (1989).
[0160] For the sTWEAK transgene construct, 23 independent
transgenic founders were identified by tail DNA PCR using probes
corresponding to the sequence of nucleotides 468 to 488 (5' primer)
and the complementary strand sequence for nucleotides 693 to 713
(3' primer) of SEQ ID NO:2. In addition, a serum ELISA for TWEAK
revealed that 10 of these 23 founder animals had detectable levels
of TWEAK in their serum, ranging from 0.06-3.0 micrograms/ml. The
remaining 13 founders had no detectable serum TWEAK, i.e. <10
ng/ml. Nine of the 10 PCR+, serum TWEAK+ founders began to exhibit
ill health at approximately 4-5 months of age. Weight loss, hunched
posture, unkempt fur and bulging eyes were noted. Five of these
founders died unexpectedly and therefore the remaining four that
showed signs of illness were sacrificed with signs of ill health.
In contrast, only 1 of 13 PCR+ serum negative founders exhibited
ill health or died. Also, 0 of 4 PCR negative littermates exhibited
ill health/died.
[0161] For the FL-TWEAK transgene construct, two independent
transgenic founders were identified by tail DNA PCR and Northern
blot analysis for TWEAK mRNA expression in liver tissue. In
addition, a serum ELISA for TWEAK revealed that neither of these
two founder animals had detectable levels of TWEAK in their serum,
i.e., less than 10 nanograms/ml. The FL-TWEAK Tg founder mice did
not exhibit a clinically observable phenotype.
Example 2
Overexpression of TWEAK in Mice Infected with an Adenoviral Vector
Delivering an Exogenous DNA Encoding sTWEAK
[0162] In order to identify the biological effects of
overexpression of TWEAK in vivo, 8-10 week old C57BL/6 female adult
mice were infected with a replication-defective adenoviral vector
with a cytomegalovirus (CMV) promoter driving the cDNA for either
murine sTWEAK ("Adeno-TWEAK") or jellyfish green fluorescent
protein ("GFP") using standard adenoviral techniques as described
in Tao et al., Molecular Therapy 3:28-35 (2001). An adenoviral
vector comprising GFP ("Adeno-GFP") was used as the negative
control. To determine whether mice were successfully infected with
the Adeno-TWEAK construct, TWEAK protein levels in the serum were
determined and monitored at various time points using standard
ELISA assays.
[0163] Systemic overexpression of murine sTWEAK in the adult mice
induced tissue changes in at least three major organs: liver,
kidney and heart. See, Table 1. The phenotypes of these adenoviral
construct-expressing mice were compared with the phenotypes of
TWEAK Tg mice from Example 1 in Table 1. These observations are
discussed in more detail in the following examples below.
TABLE-US-00001 TABLE 1 TWEAK Overexpression Induces Tissue
Remodeling in Adult Mice Adeno- TWEAK Adeno-GFP in Adult in Adult
ORGAN PHENOTYPE TWEAK Tg Mice Mice Liver Bile Duct + + -
Hyperplasia Hepatocyte - + - Death Kidney Tubular + + - Hyperplasia
Heart Dilated + + - Cardiomyopathy
Example 3
sTWEAK and FL-TWEAK Induce Dilated Cardiomyopathy
[0164] Four of the surviving PCR+ serum sTWEAK+ founders from
Example 1 were sacrificed and examined for gross morphological
abnormalities. See, Table 2. Macroscopic observation at the time of
necropsy revealed enlarged hearts, some 2-3 fold increased in size
as compared to those of normal animals. Since the enlarged heart
phenotype was observed in multiple independent sTWEAK transgenic
founders, it is highly unlikely to be due to independent
insertional events. Furthermore, an analysis of the serum chemistry
of the sTWEAK transgenic mice showed elevated cardiac specific
creatine kinase.
TABLE-US-00002 TABLE 2 sTWEAK Transgenic Mice SERUM FOUNDERS TWEAK
PHENOTYPE 10 PCR+ 0.06-3.0 .mu.g/ml 9/10 dead at 4-5 months 4/5
submitted for histophathological examination had enlarged heart 13
PCR+ <10 ng/ml 1/13 dead at 6 months with enlarged heart 4 PCR-
-- 0/4 dead at 6 months
[0165] The enlarged heart phenotype was also observed in individual
mice from one FL-TWEAK transgenic line that was established through
successive backcross onto the C57BL/6 strain. See, Table 3. The
FL-TWEAK transgene negative littermates showed no heart
abnormalities.
TABLE-US-00003 TABLE 3 FL-TWEAK Transgenic Mice INDIVIDUALS SERUM
TWEAK PHENOTYPE 7 PCR+ -- 7/7 showed enlarged hearts at 5 months 4
PCR- <10 ng/ml 0/4 dead at 5 months
[0166] Taken together, these data strongly indicate that the
enlarged heart phenotype is TWEAK-dependent.
[0167] Histopathological analysis of the hearts from the sTWEAK
transgenic and FL-TWEAK transgenic mice showed similar findings.
Low power microscopy of a FL-TWEAK transgenic heart ("Tg") as
compared to a normal heart from a transgene negative ("NTg")
littermate is shown in FIG. 1. The FL-TWEAK transgenic heart shown
is also representative of TWEAK transgenic hearts from sTWEAK
transgenic mice (PCR+, serum TWEAK+). The transgenic hearts
exhibited dilated cardiomyopathy, characterized by dilation of the
ventricles and atria. Consistent with this defect, atrial and
ventricular thrombosis in many of the animals was noted (FIG. 1).
Analysis of lung and liver tissue revealed congestion of the blood
vessels in some animals.
[0168] Higher power microscopy revealed other histopathological
findings in the heart, including myocardiocyte hypertrophy and
karyomegaly. Notably, the histopathological analysis of the
ventricles in TWEAK transgenic mice showed no signs of
inflammation. Therefore, the observed TWEAK-related cardiomyopathy
is non-inflammatory in nature.
[0169] Serum chemistry analysis on terminal bleeds from sTWEAK
transgenic mice (3 founders and 1 progeny mouse) showed abnormally
high levels of creatine kinase (CK) specifically in the heart (i.e.
the MB type of CK), confirming a significant level of cardiac
stress/injury.
[0170] C57BL/6 female mice of 8-10 weeks of age infected with
Adeno-TWEAK (see, Example 2) showed dilated cardiomyopathy which
was apparent three weeks post infection as compared to mice
infected with the negative control Adeno-GFP virus. In
TWEAK-infected mice, the hearts were characterized by dilated
chambers, as shown by histopathology (FIG. 2).
[0171] Taken together, TWEAK was shown to play a critical role in
cardiomyopathies, including dilated cardiomyopathy, and congestive
heart failure (CHF).
Example 4
TWEAK Causes Liver Epithelial Cell Hyperplasia, Hepatocyte
Vacuolation, Hepatocellular Death, Bile Duct Hyperplasia, Liver
Fibrosis and Liver Injury
[0172] A role for TWEAK in liver epithelial hyperplasia and
hepatocyte vacuolation was revealed in sTWEAK and FL-TWEAK
transgenic mice as well as injury in wild-type mice infected with
an adenovirus harboring a DNA that expresses a sTWEAK
polypeptide.
[0173] The livers of TWEAK Tg mice from Example 1 showed
substantial biliary duct and oval cell hyperplasia by 2 weeks of
age as compared to NTg mice. See, FIG. 3. As shown in Table 4, even
at serum TWEAK levels of <10 ng/ml, the livers of two FL-TWEAK
transgenic mouse founders showed mild biliary duct and oval cell
hyperplasia. FL-TWEAK transgenic mouse backcrosses into the C57BL/6
background revealed substantial biliary duct and oval cell
hyperplasia (Table 4).
TABLE-US-00004 TABLE 4 FL-TWEAK Transgenic Mice MICE SERUM TWEAK
PHENOTYPE 2 Founders <10 ng/ml Mild biliary duct and oval cell
hyperplasia 1 Founder -- Prominent biliary duct backcrossed and
oval cell hyperplasia into C57BL/6
[0174] Similarly, the sTWEAK transgenic founders that have TWEAK
serum levels between 0.2 and 3.0 .mu.g/ml showed considerable
biliary duct and oval cell hyperplasia (Table 5).
TABLE-US-00005 TABLE 5 sTWEAK Transgenic Mice SERUM MICE TWEAK
PHENOTYPE 9 Founders 0.2-3.0 .mu.g/ml Prominent biliary duct and
oval cell hyperplasia and oval cell hyperplasia 1 Founder 0.06
.mu.g/ml Mild biliary duct and oval cell hyperplasia
[0175] This biliary and oval duct hyperplasia was confirmed by
immunohistochemical (IHC) staining of FL-TWEAK Tg liver sections
taken from the Tg mice of Example 1 with the A6 mAb, which
distinguishes biliary epithelial cells and oval cells from
hepatocytes (Engelhardt et al., Differentiation 45:29-37 (1990)).
FIG. 4 shows an increase in A6 positive cells that are associated
with portal regions as well as extend out into the liver parenchyma
in FL-TWEAK Tg as compared to NTg mice. Higher magnification of a
hematoxylin and eosin stained section from a FL-TWEAK Tg mouse also
clearly shows a marked increase in the presence of oval cells
adjacent to the biliary ducts in the portal region (FIG. 5).
Immunohistochemistry for the proliferating cell nuclear antigen
(PCNA) confirmed an increased frequency of proliferating biliary
and oval cells in, TWEAK Tg mice as compared to NTg mice as early
as 2 weeks of age. At later time points, an increase in frequency
of proliferating hepatocytes in TWEAK Tg mice as compared to NTg
mice was observed, i.e., between 8 weeks and 7 months of age (not
shown). Furthermore, both FL-TWEAK and sTWEAK induced
hepatocellular vacuolization in 7 month old Tg mice from Example 1
as compared to NTg littermates (FIG. 6).
[0176] C57BL/6 and BALB/c SCID mice of 8-10 weeks of age
overexpressing sTWEAK using the Adeno-TWEAK virus as in Example 2
showed substantial serum TWEAK levels. See, FIG. 7, which displays
the effect of delivering different doses of adenovirus on the serum
TWEAK levels measured. Mice were infected with either 10.sup.11
particles of adenovirus per mouse intravenously (represented by the
"B" line), 10.sup.10 particles of adenovirus per mouse
intravenously (represented by the "J" line) or 10.sup.11 particles
of adenovirus per mouse intramuscularly (represented by the "H"
line). The Adeno-sTWEAK-infected mice showed liver injury, with
serum jaundice observed on days 3 and 7 in the C57BL/6 mouse
background and on days 3 and 4 of the BALB/c SCID mouse background.
Some of the BALB/c SCID mice died on day 4.
[0177] Furthermore, the Adeno-sTWEAK-infected C57BL/6 mice as
described in Example 2 also developed hepatocellular death which
appeared as early as 2-3 days post administration, as demonstrated
by the high level of the aspartate aminotransferase ("AST") and
alanine aminotransferase ("ALT") liver enzyme markers in
TWEAK-infected livers (Adeno-sTWEAK) compared with control
GFP-infected livers (Adeno-GFP) by day 3 (see, Table 6 and FIG. 8).
By day 7 post infection, both liver enzymes also rose in the
Adeno-GFP treated mice, as would be expected due to the
inflammation induced by the adenovirus vector alone. Hepatocyte
death was also apparent in TWEAK-infected livers, as shown by the
histologic morphology characterized by rounded up hepatocyes and
shrunken, pyknotic, intensely eosinophilic "Councilman bodies"
containing fragmented nuclei (FIG. 8). Adeno-sTWEAK treated mice
further developed a strong hyperplastic ductal response, which
peaked on day 7 post infection and was still readily apparent on
day 11 (FIG. 8). In the TWEAK-infected livers, hyperplastic
structures were observed that expressed the A6 marker specific for
biliary epithelium and oval cells, as identified by bright field
microscopy.
TABLE-US-00006 TABLE 6 Liver Enzyme Values in Ad-TWEAK And Ad-GFP
Animals Adeno-GFP Adeno-sTWEAK DAY AST (U/L) ALT (U/L) AST (U/L)
ALT (U/L) 3 148 110 1715 672 7 2140 1545 1910 1194 11 2540 2304 795
508 20 683 420 451 320
[0178] Fn14, shown to be a cellular TWEAK receptor, was induced
after exposure to liver toxins, such as galactosamine (GalN) and
carbon tetrachloride (CCl.sub.4). FIG. 9 shows that Fn14 is not
detectable in normal adult mouse liver as measured by in situ
hybridization (ISH) using a radiolabeled probe for Fn14 and dark
field microscopy. However, Fn14 is highly induced following
CCl.sub.4 injury. Similar results were obtained after GalN injury
(not shown).
[0179] Adeno-TWEAK-infected C57BL/6 mice as described in Example 2
also revealed upregulation of Fn14 in the hepatocytes and some
hyperplastic structures, as observed in Adeno-sTWEAK livers
compared with Adeno-GFP control livers (data not shown).
[0180] The role of Fn14 was further demonstrated in a bilary duct
model wherein hepatic injury in 10 week old C57BL/6 mice was
induced by ligation of the biliary duct as described by Liu et al.,
Hepatology 28:1260-1268 (1999); Olynyc et al., Am. J. Pathol.
152:347-352 (1998). The common bile duct was ligated on day 0 by
surgery and five C57BL/6 mice of 10 weeks of age were then
euthanized on day 4 and day 8 post surgery. Paraffin sections of
liver were then prepared and the expression of TWEAK and Fn14 were
determined by in situ hybridization using a radiolabeled murine
TWEAK and FN14 anti-sense probe encompassing the complete FN14
gene. As shown in FIG. 10, by day 4, Fn14 was expressed strongly in
biliary epithelial cells in bile ducts but not in hepatocytes. By
day 8, Fn14 expression in biliary epithelial cells decreased
significantly but was still detectable at low levels in some mice
(data not shown). However, TWEAK expression did not change and was
not detectable in this bile duct ligation model. These results show
that Fn14 expression is upregulated in biliary epithelial cells in
response to certain liver injuries and, thus, plays an important
role in liver fibrosis.
[0181] Taken together, these observations show that TWEAK is an
important factor in liver epithelial cell hyperplasia, hepatocyte
vacuolation, liver injury, heptocellular death, bile duct
hyperplasia and liver fibrosis.
Example 5
FL-TWEAK and sTWEAK Cause Kidney Disease
[0182] FL-TWEAK transgenic mice from Example 1 showed marked kidney
disease, including mild multifocal inflammation, tubular
nephropathy, cysts, glomerular nephropathy, tubular basophilia,
tubular dilatation, tubular vacuolation and hyaline casts.
[0183] Adeno-TWEAK-infected C57BL/6 mice of 10 weeks of age as
described in Example 2 revealed glomerular nephropathy and tubular
hyperplasia as compared to negative control Adeno-GFP-infected
mice. Also, a role for TWEAK in Alports syndrome was shown by
increased Fn14 expression in a mouse model of Alports disease.
Furthermore, a role for TWEAK in kidney fibrosis was demonstrated
in the murine model of unilateral ureteral obstruction-induced
kidney fibrosis by treatment with a TWEAK antagonist.
[0184] Expansion of the cortical interstitium is typically due to
edema or infiltration with acute or chronic inflammatory cells and
fibrous tissue. FL-TWEAK transgenic mice from Example 1 showed
tubular nephropathy and mild, multifocal interstitial inflammation.
More specifically, kidney cross-sections comparing a non-transgenic
mouse with FL-TWEAK transgenic mice showed pronounced tubular
basophilia at 8 weeks of age (FIG. 11, middle panel).
[0185] Glomerular nephropathy may be characterized by an
infiltration of leukocytes, both neutrophils and monocytes, and
proliferation of endothelial, epithelial and mesangial cells.
FL-TWEAK transgenic mice as described in Example 1 showed marked
glomerular nephropathy as evidenced by hypercellularity of the
mesangial cells and hypertrophy of capsular epithelia and mild
capsular thickening with basophilia of adjacent tubular epithelium
(FIG. 12). Also, FL-TWEAK transgenic mice showed dilation of the
urinary space leading to formation of glomerular cysts with mild
peri-glomerular fibrosis (FIG. 11, lower right panel), as compared
to normal murine glomerular morphology (FIG. 11, upper right
panel).
[0186] The tubular basophilia observed in FL-TWEAK Tg mice is
indicative of increased RNA in the cytoplasm of these tubular
cells, i.e. transcriptional activity, and suggested that these were
proliferating cells. Proliferating Cell Nuclear Antigen (PCNA)
staining confirmed that there was a subset of tubular cells
proliferating in the kidneys of TWEAK-Tg mice as described in
Example 1 and that these corresponded to the basophilic tubules
(FIG. 13). In order to determine whether the basophilic tubules
were proximal or distal tubules, three serial tissue sections from
TWEAK Tg mice were stained (1) with hemotoxylin and eosin (H&E)
to localize the basophilic tubules, (2) with a lectin specific for
proximal tubules (lectin from T. Purpureas) and (3) with a lectin
specific for distal tubules. FIG. 14 shows that the basophilic
(proliferating) tubules in TWEAK Tg mice as described in Example 1
do not express either the proximal or distal tubular epithelial
marker.
[0187] The presence of proliferating tubules that lack at least
some epithelial markers in the TWEAK Tg mice is consistent with a
model for settings of kidney injury where cells derived from the S3
segment of the proximal tubule exhibit the properties of progenitor
cells, i.e. they begin to proliferate and express mesenchymal cell
markers indicative of dedifferentiation. Subsequent differentiation
of these cells may play a role in tissue repair through the
regeneration of new tubules (Witzgall et al., J. Clin. Invest.
93:2175-2188 (1994)). Alternatively, there may be proliferation and
differentiation of a pre-existing progenitor population that
resides in the S3 region.
[0188] The presence of proliferating cells that lack some
epithelial markers in TWEAK Tg mice is also consistent with a model
for kidney development, wherein epithelial tubules are formed from
mesenchymal progenitors that undergo differentiation, thereby
acquiring epithelial markers and properties characteristic of
tubules.
[0189] Similarly, infection of 10 week old C57BL/6 mice with an
Adeno-sTWEAK virus, as described in Example 2, induced glomerular
nephropathy and basophilia of the tubular epithelium as well as
occasional thickening and hyperplasia of the glomerular capsula by
day 11 post infection (FIG. 15). This was in contrast to the normal
histology observed in the negative control Adeno-GFP-infected mice.
Furthermore, the basophilia, which is indicative of epithelial cell
proliferation, was apparent by day 3 but peaked around one week
post administration.
[0190] Consistent with a role for TWEAK in kidney disease, TWEAK
mRNA was shown to be expressed widely in adult C57BL/6 mouse kidney
(FIG. 16), and Fn14 mRNA was shown to be expressed in the proximal
tubules of the inner cortex/outer medulla (FIG. 17), as shown by in
situ hybridization (ISH) using radiolabeled TWEAK and Fn14
antisense probes and revealed by dark field microscopy. Also, Fn14
mRNA was shown to be induced in the kidneys of mouse models for
Alport syndrome. This is shown in FIG. 18 as the fold increase in
Fn14 mRNA in two individual Alport mice relative to wildtype
animals as disease progresses in the Alport mice from 4 to 7 weeks
of age.
[0191] The role of TWEAK in a murine model of Alport disease was
directly tested by treatment with a TWEAK antagonist, a murine
Fn14-Fc fusion protein. Two groups of 5 Alport knockout (KO) mice
prepared according to Cosgrove et al., Genes Dev. 10(23):2981-2992
(1996), were treated with control IgG2a (muP1.17), or muFN14-Fc
fusion protein (prepared by Biogen (Cambridge)). The control IgG2a
used is the murine myeloma protein P1.17 produced from a hybridoma
and purified by standard mAb purification procedure. The muFN14-Fc
is a fusion protein of the extracellular domain of murine Fn14 and
the Fc region of murine IgG2a. The fusion protein was produced
either in human 293 embryonic kidney cells or in Chinese hamster
ovary (CHO) cells and purified by standard mAb purification
procedures. The first treatment was at the age of three weeks with
a dose of one hundred microgram of protein by an intraperitoneal
(IP) route. Treatments continued for the next four weeks with the
same dose administered twice a week. Mice were sacrificed at the
end of the 7th week (7 week old). Kidneys were collected and
embedded in paraffin and frozen. The extent of kidney fibrosis and
inflammation was scored by glomerular morphology from H&E
staining of paraffin sections, activated myofibroblast with smooth
muscle actin staining, and activated monocytes by CDllb staining of
frozen sections. Smooth muscle actin and CD11b stained sections
were used to quantitate positively stained areas to assess extent
of fibrosis and inflammation, respectively, by the MetaMorph
computer program. Results of analysis show health of glomeruli in
FN14-Fc treated mice was greatly improved (59% glomeruli with
pathology in control Ig treated as compared to only 39% with
pathology in Fn14-Fc treated, P value=0.03). Glomerular pathology
is characterized by presence of crescents and/or glomerular
fibrosis. In addition, fibrosis in the cortical area of the kidney
in treated mice was significantly reduced as measured by alpha
smooth muscle actin staining, p value=0.04. There was also a
general trend in reduction of monocyte infiltration in FN14-Fc
treated mice. These results clearly indicate that FN14-Fc treatment
of Alports mice reduces fibrosis in the cortical area of the kidney
and improves the general morphology of glomeruli.
[0192] The role of TWEAK was also tested in the murine model of
unilateral ureteral obstruction-induced kidney fibrosis by
treatment with a TWEAK antagonist, a hamster anti-TWEAK monoclonal
antibody. In the mouse model for renal fibrotic progression, a
ureter is ligated, resulting in unilateral ureteral obstruction
(UUO). (Klahr et al., Am J Kidney Dis 18:689-699 (1991); Moriyama
et al., Kidney Int 54(1):110-119 (1998). UUO causes progressive
nephrosclerosis without near-term renal failure in mice because the
unobstructed kidney can maintain relatively normal renal function.
While the obstructed kidney undergoes rapid global fibrosis, the
unobstructed kidney undergoes adaptive hypertrophy.
[0193] The impact of TWEAK antagonist treatment on UUO-induced
renal fibrosis was quantitated morphometrically. Four groups of
eight viral antigen-free C57BL/6 male mice (Jackson Laboratories,
Bar Harbor Me.), 8-10 weeks of age were used. The mice were divided
into the following groups: PBS alone (VEH), control hamster
anti-Keyhole Limpet Hemocyanin (KLH) antibody (HA4/8; purchased
from BD Biosciences (San Jose)), hamster anti-mouse TWEAK antibody
(AB.G11; prepared by Biogen (Cambridge)), soluble murine TGF-.beta.
receptor Ig (TGF-.beta.R, positive control; prepared by Biogen
(Cambridge)) and unoperated (UNOP).
[0194] To induce kidney fibrosis, the left ureter was aseptically
isolated and tied off in the kidney of the operated side on day 0
as described in Hammad et al., Kidney Int 58:242-250 (2000). The
following groups: PBS, HA4/8 and AB.G11 (anti-TWEAK mAb) were
additionally treated on days 2, 6, and 9 post surgery and the
sTGF-.beta.R-Ig group on days 1, 3, 6 and 8. On day 10 post
surgery, the left ligated kidney was removed and halved
transversely through the center of the renal pelvis and prepared
for paraffin sectioning.
[0195] Paraffin-treated kidney sections were stained with
Trichrome-Masson staining specific for collagen. Using a Metamorph
program, blue-staining areas in Trichrome-Masson slides were
measured to quantitate collagen content in order to assess the
extent of fibrosis in the operated kidneys (FIG. 19).
[0196] Surprisingly, kidney sections from anti-TWEAK monoclonal
(AB.G11) antibody-treated animals demonstrated a 42% decrease in
collagen content compared with PBS-treated animals and a 30%
decrease in collagen content compared with control (HA4/8)
antibody-treated animals. In contrast, the kidneys from soluble
TGF-.beta. receptor Ig-treated (TGF-.beta.R) animals displayed only
a 33% decrease in collagen content compared with PBS-treated
animals and a 19% decrease in collagen content compared with
control (HA4/8) antibody-treated animals. These results clearly
show that treatment with a TWEAK antagonist, such as an anti-TWEAK
monoclonal antibody, significantly reduced kidney fibrosis to a
greater extent than that shown by soluble TGF-.beta. receptor Ig
(TGF-.beta.R).
[0197] Taken together, the results presented herein show that TWEAK
plays an important role in inflammatory kidney conditions, such as
multifocal inflammation, and in non-inflammatory kidney conditions,
such as tubular nephropathy, cysts, glomerular nephropathy, Alports
syndrome, tubular basophilia, tubular dilatation, tubular
vacuolation, hyaline casts, tubular hyperplasia and kidney
fibrosis.
Example 6
TWEAK Causes Lung Inflammation
[0198] In cross sections of lungs from FL-TWEAK transgenic and
control mice as described in Example 1, marked granulomatous and
lymphohistiocytic inflammation was shown in both FL-TWEAK and
sTWEAK Tg mice (FIG. 20). Also, endogenous TWEAK expression was
revealed in lung cells lining the bronchioles and alveoli of normal
mice, as shown by in situ hybridization (ISH) using radio-labeled
TWEAK antisense probes and revealed by dark field (ISH) microscopy
(FIG. 21).
[0199] Consistent with a role for TWEAK in lung disease, Fn14 mRNA
was shown to be expressed widely in adult C57BL/6 mouse lung (FIG.
22) by ISH using radio-labeled Fn14 antisense probes and revealed
by dark field microscopy.
[0200] Taken together, these data show that TWEAK is an important
factor in mediating inflammatory lung conditions, including
granulomatous and lymphohistiocytic inflammation.
Example 7
TWEAK Inhibits both Adipogenesis and Myogenesis
[0201] The effect of TWEAK on cellular differentiation was
investigated using two in vitro models of adipogenesis and
myogenesis well-known in the art. (Green and Meuth, Cell 3:127-133
(1974); Yaffe and Saxel, Nature 270: 725-727 (1977)).
[0202] For adipogenesis, 3T3-L1 cells were first grown to
confluency in a Dulbecco's Modified Eagles Media (DMEM)-based
growth media and then induced to undergo adipogenesis according to
methods known in the art. Green and Kehinde, Cell 5:19-27 (1976).
Briefly, cells were stimulated on day 0 with the DMEM-based MDI
media that contained dexamethasone, insulin and IBMX for two days
followed by insulin-only DMEM media for another two days. On day 5,
cells were cultured in the regular DMEM-based growth media and
adipogenesis was assessed on day 7 by Oil-Red staining.
[0203] For myogenesis, C2C12 cells were grown to near confluency in
a DMEM-based growth media and on day 0, switched to a low-serum
differentiation media that contained 2% horse serum to trigger
differentiation (Yaffe and Saxel, Nature 270: 725-727 (1977)).
Myotube formation was examined using a phase-contrast microscope
and pictures were taken on day 6 of differentiation.
[0204] To examine the effect of TWEAK on these two differentiation
pathways, various versions of recombinant human TWEAK (TWEAK-FLAG,
TWEAK or Fc-TWEAK) were added on day 0 at a final concentration of
100 ng/ml and replenished daily. TWEAK inhibited both adipogenesis
and myogenesis in both systems (FIGS. 23 and 24). The specificity
of TWEAK'S inhibitory effect was confirmed using either the hamster
anti-TWEAK monoclonal antibody AB.G11 or hFn14-Fc as the
neutralizing reagent.
[0205] These results show that TWEAK plays an important role in
cellular differentiation. The present invention therefore provides
methods for affecting cellular differentiation of the progenitor
cells disclosed herein using the TWEAK polypeptides, peptides,
agonists, or antagonists disclosed herein.
Example 8
TWEAK Binds to Human Mesenchymal Stem Cells
[0206] Human mesenchymal stem cells (hMSCs) (Cambrex Corp., East
Rutherford, N.J.) were cultured in MSCGM media (Cambrex) and
harvested by incubating them with PBS containing 5 mM EDTA, and
prepared for fluorescence activated cell sorting (FACS)
analysis.
[0207] The cells were incubated in FACS buffer containing PBS and
1% FBS along with 100 ng/mL of Fc-TWEAK for 1 hour on ice. After
washing twice with FACS buffer, the cells were then incubated with
phycoerythrin-conjugated goat anti-human Fc or goat anti-mouse Fc
secondary antibodies at a dilution of 1:200 (Jackson
ImmunoResearch, West Grove, Pa.) (FIG. 25). The background staining
was measured by secondary antibody staining alone, as shown by the
broken line.
[0208] As shown in FIG. 25, TWEAK binds to human mesenchymal cells,
as demonstrated by the shift in the staining profile of Fc-TWEAK
compared with secondary antibody alone. Thus, the ability of TWEAK
to bind to mesenchymal cells (a progenitor cell type capable of
differentiating into muscle cells as well as cartilage, bone,
connective tissue cell types such as stromal cells, fibroblasts,
adipocytes and dermal cells) shows that TWEAK plays an important
role in the differentiation of these cell types both in normal and
disease models.
Example 9
Fn14 is Expressed on Neural Stem Cells
[0209] The expression of Fn14 was examined in the brains from
embryonic day 13.5 mice on a mixture of both C57BL/6 and 129/Sve
background. The brains were subjected to in situ hybridization with
the Fn14 anti-sense probe. A positive signal was detectable in the
subventricular zone of the embryonic ventricles, correlating with
the position of neural stem cells (data not shown). These results
show that Fn14 plays an important role in neural cellular
differentiation.
Example 10
Methods for Identifying Therapeutic Agents for Treating
TWEAK-Related Conditions
[0210] In order to identify TWEAK antagonist compounds that act as
therapeutic agents for the treatment of TWEAK-related conditions
according to the present invention, a test animal, such as a mouse,
is obtained that expresses an exogenous DNA encoding a TWEAK
polypeptide, or a fragment, analog, mutein, or mimetic thereof. The
animal is then exposed to a candidate compound that may function as
a therapeutic agent for a TWEAK-related condition. Fibrotic,
cardiac, kidney, liver, lung, skin, skeletal muscle, lipid,
gastrointestinal tract, pancreas, reproductive organs, neural,
cartilage, bone or connective tissue from the test animal is then
compared to the same tissue from a reference animal that expresses
the exogenous DNA but has not been exposed to the compound; and it
is determined whether the compound has affected any TWEAK-related
condition of the fibrotic, cardiac, kidney, liver, lung, skin,
skeletal muscle, lipid, gastrointestinal tract, pancreas,
reproductive organs, neural, cartilage, bone or connective
tissues.
[0211] In order to identify TWEAK agonist compounds that act as
therapeutic agents for the treatment of TWEAK-related conditions
according to the present invention, a test animal that either does
or does not express an exogenous DNA encoding a TWEAK polypeptide,
or a fragment, analog, mutein, or mimetic thereof may be exposed to
a candidate compound that may function as a therapeutic agent for a
TWEAK-related condition. Fibrotic, cardiac, kidney, liver, or lung
tissue from the test animal is then compared to the same tissue
from a reference animal that has not been exposed to the compound;
and it is determined whether the compound has induced any
biological change in said tissues as described herein due to TWEAK
signaling in vivo.
[0212] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0213] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the disclosure herein,
including the appended claims.
Sequence CWU 1
1
51249PRTMus musculus 1Met Ala Ala Arg Arg Ser Gln Arg Arg Arg Gly
Arg Arg Gly Glu Pro 1 5 10 15 Gly Thr Ala Leu Leu Ala Pro Leu Val
Leu Ser Leu Gly Leu Ala Leu 20 25 30 Ala Cys Leu Gly Leu Leu Leu
Val Val Val Ser Leu Gly Ser Trp Ala 35 40 45 Thr Leu Ser Ala Gln
Glu Pro Ser Gln Glu Glu Leu Thr Ala Glu Asp 50 55 60 Arg Arg Glu
Pro Pro Glu Leu Asn Pro Gln Thr Glu Glu Ser Gln Asp 65 70 75 80 Val
Val Pro Phe Leu Glu Gln Leu Val Arg Pro Arg Arg Ser Ala Pro 85 90
95 Lys Gly Arg Lys Ala Arg Pro Arg Arg Ala Ile Ala Ala His Tyr Glu
100 105 110 Val His Pro Arg Pro Gly Gln Asp Gly Ala Gln Ala Gly Val
Asp Gly 115 120 125 Thr Val Ser Gly Trp Glu Glu Thr Lys Ile Asn Ser
Ser Ser Pro Leu 130 135 140 Arg Tyr Asp Arg Gln Ile Gly Glu Phe Thr
Val Ile Arg Ala Gly Leu 145 150 155 160 Tyr Tyr Leu Tyr Cys Gln Val
His Phe Asp Glu Gly Lys Ala Val Tyr 165 170 175 Leu Lys Leu Asp Leu
Leu Val Asn Gly Val Leu Ala Leu Arg Cys Leu 180 185 190 Glu Glu Phe
Ser Ala Thr Ala Ala Ser Ser Pro Gly Pro Gln Leu Arg 195 200 205 Leu
Cys Gln Val Ser Gly Leu Leu Pro Leu Arg Pro Gly Ser Ser Leu 210 215
220 Arg Ile Arg Thr Leu Pro Trp Ala His Leu Lys Ala Ala Pro Phe Leu
225 230 235 240 Thr Tyr Phe Gly Leu Phe Gln Val His 245 21239DNAMus
musculus 2atggccgccc gtcggagcca gaggcggagg gggcgccggg gggagccggg
caccgccctg 60ctggccccgc tggtgctgag cctgggcctg gcgctggcct gccttggcct
cctgctggtc 120gtggtcagcc tggggagctg ggcaacgctg tctgcccagg
agccttctca ggaggagctg 180acagcagagg accgccggga gccccctgaa
ctgaatcccc agacagagga aagccaggat 240gtggtacctt tcttggaaca
actagtccgg cctcgaagaa gtgctcctaa aggccggaag 300gcgcggcctc
gccgagctat tgcagcccat tatgaggttc atcctcggcc aggacaggat
360ggagcacaag caggtgtgga tgggacagtg agtggctggg aagagaccaa
aatcaacagc 420tccagccctc tgcgctacga ccgccagatt ggggaattta
cagtcatcag ggctgggctc 480tactacctgt actgtcaggt gcactttgat
gagggaaagg ctgtctacct gaagctggac 540ttgctggtga acggtgtgct
ggccctgcgc tgcctggaag aattctcagc cacagcagca 600agctctcctg
ggccccagct ccgtttgtgc caggtgtctg ggctgttgcc gctgcggcca
660gggtcttccc ttcggatccg caccctcccc tgggctcatc ttaaggctgc
ccccttccta 720acctactttg gactctttca agttcactga ggggccttgc
tctcccagat tccttaaact 780ttccctggct ccaggagcat caccacacct
ccctacccca cccccactcc tccaccccct 840cgctgctcct tggtccagtc
ctgtctctcc tcaaaggcag ccagagcttg ttcacatgtt 900tccattccac
agacgtatcc ttgctcttct taacatccca tcccaccaca actatccacc
960tcactagctc ccaaagcccc tacttatccc tgactccccc acccactcac
ccgaccacgt 1020gtttattgac tttgtgcacc aggcactgag atgggctgga
cctggtggca ggaagccaga 1080gaacctggga ctaggccaga agttcccaac
tgtgaggggg aagagctggg gacaagctcc 1140tccctggatc cctgtggatt
ttgaaaagat actattttta ttattattgt gacaaaatgt 1200taaatggata
ttaaagagaa taaatcatga tttctcttc 12393249PRTHomo sapiens 3Met Ala
Ala Arg Arg Ser Gln Arg Arg Arg Gly Arg Arg Gly Glu Pro 1 5 10 15
Gly Thr Ala Leu Leu Val Pro Leu Ala Leu Gly Leu Gly Leu Ala Leu 20
25 30 Ala Cys Leu Gly Leu Leu Leu Ala Val Val Ser Leu Gly Ser Arg
Ala 35 40 45 Ser Leu Ser Ala Gln Glu Pro Ala Gln Glu Glu Leu Val
Ala Glu Glu 50 55 60 Asp Gln Asp Pro Ser Glu Leu Asn Pro Gln Thr
Glu Glu Ser Gln Asp 65 70 75 80 Pro Ala Pro Phe Leu Asn Arg Leu Val
Arg Pro Arg Arg Ser Ala Pro 85 90 95 Lys Gly Arg Lys Thr Arg Ala
Arg Arg Ala Ile Ala Ala His Tyr Glu 100 105 110 Val His Pro Arg Pro
Gly Gln Asp Gly Ala Gln Ala Gly Val Asp Gly 115 120 125 Thr Val Ser
Gly Trp Glu Glu Ala Arg Ile Asn Ser Ser Ser Pro Leu 130 135 140 Arg
Tyr Asn Arg Gln Ile Gly Glu Phe Ile Val Thr Arg Ala Gly Leu 145 150
155 160 Tyr Tyr Leu Tyr Cys Gln Val His Phe Asp Glu Gly Lys Ala Val
Tyr 165 170 175 Leu Lys Leu Asp Leu Leu Val Asp Gly Val Leu Ala Leu
Arg Cys Leu 180 185 190 Glu Glu Phe Ser Ala Thr Ala Ala Ser Ser Leu
Gly Pro Gln Leu Arg 195 200 205 Leu Cys Gln Val Ser Gly Leu Leu Ala
Leu Arg Pro Gly Ser Ser Leu 210 215 220 Arg Ile Arg Thr Leu Pro Trp
Ala His Leu Lys Ala Ala Pro Phe Leu 225 230 235 240 Thr Tyr Phe Gly
Leu Phe Gln Val His 245 4129PRTHomo sapiens 4Met Ala Arg Gly Ser
Leu Arg Arg Leu Leu Arg Leu Leu Val Leu Gly 1 5 10 15 Leu Trp Leu
Ala Leu Leu Arg Ser Val Ala Gly Glu Gln Ala Pro Gly 20 25 30 Thr
Ala Pro Cys Ser Arg Gly Ser Ser Trp Ser Ala Asp Leu Asp Lys 35 40
45 Cys Met Asp Cys Ala Ser Cys Arg Ala Arg Pro His Ser Asp Phe Cys
50 55 60 Leu Gly Cys Ala Ala Ala Pro Pro Ala Pro Phe Arg Leu Leu
Trp Pro 65 70 75 80 Ile Leu Gly Gly Ala Leu Ser Leu Thr Phe Val Leu
Gly Leu Leu Ser 85 90 95 Gly Phe Leu Val Trp Arg Arg Cys Arg Arg
Arg Glu Lys Phe Thr Thr 100 105 110 Pro Ile Glu Glu Thr Gly Gly Glu
Gly Cys Pro Ala Val Ala Leu Ile 115 120 125 Gln 5309PRTArtificial
Sequencehuman TWEAK receptor fusion protein construct 5Met Ala Arg
Gly Ser Leu Arg Arg Leu Leu Arg Leu Leu Val Leu Gly 1 5 10 15 Leu
Trp Leu Ala Leu Leu Arg Ser Val Ala Gly Glu Gln Ala Pro Gly 20 25
30 Thr Ala Pro Cys Ser Arg Gly Ser Ser Trp Ser Ala Asp Leu Asp Lys
35 40 45 Cys Met Asp Cys Ala Ser Cys Arg Ala Arg Pro His Ser Asp
Phe Cys 50 55 60 Leu Gly Cys Ala Ala Ala Pro Pro Ala Pro Phe Arg
Leu Leu Trp Arg 65 70 75 80 Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala 85 90 95 Glu Gly Ala Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr 100 105 110 Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val 115 120 125 Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 130 135 140 Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 145 150 155
160 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
165 170 175 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala 180 185 190 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro 195 200 205 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln 210 215 220 Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala 225 230 235 240 Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 245 250 255 Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 260 265 270 Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 275 280
285 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
290 295 300 Leu Ser Pro Gly Lys 305
* * * * *