Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL FIBROBLAST GROWTH FACTORS AND METHODS OF USE THEREOF
[001] This application claims the benefit of U.S. Provisional Application
Serial No.
60/469,353, filed May 9, 2003, which is incorporated herein by reference in
its entirety.
1. FIELD OF INVENTION
[002] The present invention relates to compositions and methods of treatment
of
various conditions, including but are not limited to, stroke, wound healing,
and joint diseases
(e.g., osteoarthritis and rheumatoid arthritis). More particularly, the
present invention relates
to compositions comprising a member of the fibroblast growth factor family,
FGF-CX (also
known as CG53135-05 or FGF-20), its related polypeptides, nucleic acids
encoding such
polypeptides, and their uses for treating a condition, such as but is not
limited to, stroke,
would healing, and joint diseases (e.g., osteoarthritis and rheumatoid
arthritis).
2. BACKGROUND OF THE INVENTION
[003] The FGF family of proteins, whose prototypic members include acidic FGF
(FGF-1) and basic FGF (FGF-2), bind to four related receptor tyrosine kinases.
These FGF
receptors are expressed on most types of cells in tissue culture. Dimerization
of FGF receptor
monomers upon ligand binding has been reported to be a requisite for
activation of the kinase
domains, leading to receptor trans phosphorylation. FGF receptor-1 (FGFR-1),
which shows
the broadest expression pattern of the four FGF receptors, contains at least
seven tyrosine
phosphorylation sites. A number of signal transduction molecules are affected
by binding
with different affinities to these phosphorylation sites.
[004] Expression of FGFs and their receptors in brains of perinatal and adult
mice
has been examined. Messenger RNA of all FGF genes, with the exception of FGF-
4, is
detected in these tissues. FGF-3, FGF-6, FGF-7 and FGF-8 genes demonstrate
higher
expression in the late embryonic stages than in postnatal stages, suggesting
that these
members are involved in the late stages of brain development. In contrast,
expression of
FGF-1 and FGF-5 increased after birth. In particular, FGF-6 expression in
perinatal mice has
been reported to be restricted to the central nervous system and skeletal
muscles, with intense
signals in the developing cerebrum in embryos but in cerebellum in 5-day-old
neonates.
FGF-receptor (FGFR)-4, a cognate receptor for FGF-6, demonstrates similar
spatiotemporal
expression, suggesting that FGF-6 and FGFR-4 play significant roles in the
maturation of
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nervous system as a ligand-receptor system. According to Ozawa et al., these
results strongly
suggest that the various FGFs and their receptors are involved in the
regulation of a variety of
developmental processes of brain, such as proliferation and migration of
neuronal progenitor
cells, neuronal and glial differentiation, neurite extensions, and synapse
formation. See, e.g.,
Ozawa et al., Brain Res. Mol. Brain Res. 1996 41(1-2):279-88.
[005] Other members of the FGF polypeptide family include the FGF receptor
tyrosine kinase (FGFRTK) family and the FGF receptor heparan sulfate
proteoglycan
(FGFRHS) family. These members interact to regulate active and specific FGFR
signal
transduction complexes. These regulatory activities are diversified throughout
a broad range
of organs and tissues, and in both normal and tumor tissues, in mammals.
Regulated
alternative messenger RNA (mRNA) splicing and combination of variant
subdomains give
rise to diversity of FGFRTK monomers. Divalent cations cooperate with the
FGFRHS to
conformationally restrict FGFRTK trans-phosphorylation, which causes
depression of kinase
activity and facilitates appropriate activation of the FGFR complex by FGF.
For example, it
is known that different point mutations in the FGFRTK commonly cause
craniofacial and
skeletal abnormalities of graded severity by graded increases in FGF-
independent activity of
total FGFR complexes. Other processes in which FGF family exerts important
effects are
liver growth and function, and prostate tumor progression.
[006] Glia-activating factor (GAF), another FGF family member, is a heparin-
binding growth factor that was purified from the culture supernatant of a
human glioma cell
line. See, Miyamoto et al., 1993, Mol. Cell Biol. 13(7): 4251-4259. GAF shows
a spectrum
of activity slightly different from those of other known growth factors, and
is designated as
FGF-9. The human FGF-9 cDNA encodes a polypeptide of 208 amino acids. Sequence
similarity to other members of the FGF family was estimated to be around 30%.
Two
cysteine residues and other consensus sequences found in other family members
were also
well conserved in the FGF-9 sequence. FGF-9 was found to have no typical
signal sequence
in its N-terminus like those in acidic FGF and basic FGF. Acidic FGF and basic
FGF are
known not to be secreted from cells in a conventional manner. However, FGF-9
was found
to be secreted efficiently from cDNA-transfected COS cells despite its lack of
a typical signal
sequence. It could be detected exclusively in the culture medium of cells. The
secreted
protein lacked no amino acid residues at the N-terminus with respect to those
predicted by the
cDNA sequence, except the initiation methionine. The rat FGF-9 cDNA was also
cloned,
and the structural analysis indicated that the FGF-9 gene is highly conserved.
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[007] FGFs have been shown to induce neuronal sprouting. See Proc. Natl. Acad.
Sci. U.S.A. 1997 94 (15): 8179-84. Kawamata et al. (Journal of Cerebral Blood
Flow and
Metabolism, 16:542-547, 1996) proposed that basic FGF, an 18 kDa and 154 amino
acid long
polypeptide, supports the survival and outgrowth of a wide variety of brain
neurons. US
Patent Application US 2002/0151496 Al suggests that FGF-20 is a neurotrophic
factor and
stimulate survival of cells of neuronal origin.
2.1. Inflammation: Osteoarthritis and Rheumatoid Arthritis
[008] Osteoarthritis ("OA") is a degenerative joint disease and a frequent
cause of
joint pain that affects a large and growing population. OA is estimated to be
the most
common cause of disability in adults. The disease typically manifests itself
in the 2nd to 3rd
decades, with most people over forty years exhibiting some pathologic change
in weight
bearing joints, although the change may be asymptomatic. A systematic review
of incidence
and prevalence of OA of the knee in people older than 55 years in the United
Kingdom
reported an incidence of 25 percent per year, a prevalence of disability of 10
percent, and
severe disability in about two to three percent. The National Health and
Nutrition
Examination Survey (Center for Health Statistics, Centers for Disease Control
and
Prevention) found the prevalence of this disease to be over 80 percent in
people over age 55,
compared to less than 0.1 percent in those aged 25 to 34 years old. OA results
from a
complex interplay of multiple factors, including joint integrity, genetics,
local inflammation,
mechanical forces, and cellular and biochemical processes. Characteristic
features of the
disease are degradation of articular cartilage, hypertrophy of bone at the
margins, and
changes in the synovial membrane, typically accompanied by pain and stiffness
of the joint.
For the majority of patients, OA is linked to one or more factors, such as
aging, occupation,
trauma, and repetitive and small insults over time. The pathophysiologic
process of OA is
almost always progressive.
[009] CG53135-05 and their variants belong to the FGF family that regulates
proliferation (see U.S. Application Serial No. 10/174,394, which is
incorporated herein by its
entirety). The identification of a polymorphism (CG53135-12) in the gene
encoding an
FGF20-like protein, CG53135-O1, in humans, and a method for identifying
individuals who
are carriers of the genetic risk-altering factor for OA have been described in
U.S. Application
Serial No. 10/702,126 ("the '126 application"), which is incorporated herein
by its entirety.
The '126 application describes a DNA-based diagnostic test for identifying
individuals with
increased risk for OA and resultant musculoskeletal complications.
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[0010] There are several well-established treatment modes for OA, ranging from
non-
pharmaceutical to pharmaceutical intervention. Non-pharmaceutical
interventions include
behaviour modification, weight loss, exercise, walking aids, avoidance of
aggravating
activities, as well as joint irrigation, and arthroscopic and surgical
interventions. Current
pharmaceutical interventions include nonsteroidal antiinflammatory drugs,
intraarticular
corticosteroids, and colchicine. In addition, FGF-18 has been shown to repair
damaged
cartilage in a rat meniscal tear model for OA (See Paper #0199, 50th Annual
Meeting of the
Orthopaedic Research Society, San Francisco CA, 2004).
[0011] However, satisfactory treatment of OA is an unmet medical need, as
existing
therapeutics have not been successful in curtailing the incidence or the
severity of the disease.
Consequently, a therapeutic that can successfully treat osteoarthritis has the
beneficial effects
of decreasing morbidity, while potentially saving the healthcare system
millions of dollars in
costs associated with invasive surgical procedures, disability and ancillary
support services.
[0012] Citation or discussion of a reference herein shall not be construed as
an
admission that such is prior art to the present invention.
3. SUMMARY OF THE INVENTION
[0013] The present invention provides methods of preventing or treating a
disease
(e.g., stroke, joint diseases, and trauma) comprising administering to a
subject in need thereof
a FGF-CX polypeptide, which has homology to Fibroblast Crrowth Factor (FGF)
protein.
.The present invention also encompasses FGF-CX polynucleotide sequences and
the FGF-CX
polypeptides encoded by these nucleic acid sequences, and fragments, homologs,
analogs,
and derivatives thereof.
[0014] In accordance with the present invention, the diseases to be prevented
or
treated include, but are not limited to, joint diseases (non-limiting examples
being arthritis,
osteoarthritis, joint pathology, ligament and tendon injuries, and meniscal
injuries), ischemic
stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin
poisoning, and
neurodegenerative diseases (non-limiting examples being Alzheimer's,
Parkinson's Disease,
Amyotrophic Lateral Sclerosis, Huntington's Disease).
[0015] In one aspect, the invention encompasses an isolated FGF-CX nucleic
acid
(SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,
as shown in
Table A), that encodes a FGF-CX polypeptide, or a fragment, homolog, analog or
derivative
thereof. The nucleic acid can include, but not limited to, nucleic acid
sequence encoding a
polypeptide at least 85% identical to a polypeptide comprising the amino acid
sequence of
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Table A (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 36). The
invention also encompasses the polypeptides resulting from the proteolytic
cleavage. of
CG53135-05 (SEQ m NO: 2) that includes SEQ m Nos: 37, 38, 39, 40. The nucleic
acid
cari be, but is not limited to, a genomic DNA fragment, and a cDNA molecule.
[0016] The present invention also encompasses a vector containing one or more
of the
nucleic acids described herein, and a cell containing the vectors or nucleic
acids described
herein.
[0017] The present invention further encompasses host cells transformed with a
recombinant expression vector comprising any of the nucleic acid molecules
described above.
[0018] In one embodiment, the invention provides a pharmaceutical composition
that
comprises a FGF-CX nucleic acid and a pharmaceutically acceptable carrier. In
another
embodiment, the invention provides a substantially purified FGF-CX
polypeptide, e.g., any of
the FGF-CX polypeptides encoded by a FGF-CX nucleic acid, and fragments,
homologs,
analogs, and derivatives thereof. The invention also provides a pharmaceutical
composition
that comprises a FGF-CX polypeptide and a pharmaceutically acceptable carrier.
[0019] In another embodiment, the invention provides an antibody that binds
specifically to a FGF-CX polypeptide. The antibody can be, but is not limited
to, a
monoclonal or polyclonal antibody, and fragments, homologs, analogs, and
derivatives
thereof. The invention also provides a pharmaceutical composition including
FGF-CX
antibody and a pharmaceutically acceptable carrier. The present invention also
emcompasses
isolated antibodies that bind to an epitope on a polypeptide encoded by any of
the nucleic
acid molecules described above.
[0020] The present invention further provides kits comprising antibodies that
bind to
a polypeptide encoded by any of the nucleic acid molecules described above and
a negative
control antibody.
[0021] The invention encompasses a method for producing a FGF-CX polypeptide.
The method includes providing a cell containing a FGF-CX nucleic acid, e.g., a
vector that
includes a FGF-CX nucleic acid, and culturing the cell under conditions
sufficient to express
the FGF-CX polypeptide encoded by the nucleic acid. The expressed FGF-CX
polypeptide is
then recovered from the cell. Preferably, the cell produces little or no
endogenous FGF-CX
polypeptide. The cell can be, e.g., a prokaryotic cell or eukaryotic cell.
[0022] The present invention provides a method of inducing an immune response
in a
subject against a polypeptide encoded by any of the nucleic acid molecules
disclosed above
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by administering to the mammal an amount of the polypeptide sufficient to
induce the
immune response.
[0023] The present invention also provides methods of identifying a compound
that
binds to FGF-CX polypeptide by contacting the FGF-CX polypeptide with a
compound and
determining whether the compound binds to the FGF-CX polypeptide.
[0024] The invention provides a prophylactic treatment with FGF-CX polypeptide
wherein an injury that predisposes the subject to osteoarthritis has occurred
but the cartilage
is intact.
[0025] The invention also provides a therapeutic treatment with FGF-CX
polypeptide
wherein intrinsic or extrinsic factors (e.g. genetic predisposition or
meniscal injury,
respectively) has led to osteoarthritic changes and cartilage damage.
4. BRIEF DESCR1PTTON OF THE FIGURES
[0026] Figure 1 shows Liquid Chromatography and Mass Spectrometry analysis of
CG53135-05. CG53135-05 was injected onto the phenyl-hexyl column in an aqueous
mobile
phase containing 95% water, 5% acetonitrile, and 0.1% trifluoroacetic acid.
The protein was
then eluted by using a non-linear gradient with an organic mobile phase
containing 95%
acetonitrile, 5% water, and 0.085% trifluoroacetic acid. Each of the 4 peaks
was
characterized using LC/ESI/MS, MALDI-TOF MS, and N-terminal amino acid
sequencing.
[0027] Figure 2A and 2B depict Peptide Map of CG531~35-05. The upper tracing
in
each panel represents that of CG53135-05 and the lower tracing in each panel
represents an
identical sample treated similarly but without CG53135-05. Figure 2A:
Detection at 214 nm
to monitor CG53I35 peptides. Figure 2B: Detection at 295 nm to monitor
tryptophan-
containing peptides.
[0028] Figure 3 shows Receptor Binding Specificity of CG53135. NlH 3T3 cells
were serum-starved, incubated with the indicated factor (green
squares=platelet derived
growth factor; blue triangle=FGF-1; red circle=CG53135) either alone or
together with the
indicated soluble FGFR, and DNA synthesis in response to CG53135 was measured
in a
BrdU incorporation assay. Data points represent the average obtained from
triplicate wells,
and are represented as the percent BrdU incorporation relative to cells
receiving factor alone.
[0029] Figure 4 shows the results of Forelimb Placing Test. The mean and
standard
error of the score for groups receiving vehicle (diamonds), 1.0 ~,g/injection
CG53135-05
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(square), and 2.5 p,g/injection CG53135-05 (triangles) are represented over
time. Asterisks
indicate significant difference from vehicle control as assessed by one-way
ANOVA.
[0030] Figure 5 shows the results of Hindlimb Placing Test. The mean and
standard
error of the score for groups receiving vehicle (diamonds), 1.0 p,g/injection
CG53135-05
(square), and 2.5 pglinjection CG53135-05 (triangles) are represented over
time. Asterisks
indicate significant difference from vehicle control as assessed by one-way
ANOVA.
[0031] Figure 6 shows the results of Body Swing Test. The mean and standard
error
of the score for groups receiving vehicle (diamonds), 1.0 ~.glinjection
CG53135-05 (square),
and 2.5 pg/injection CG53135-05 (triangles) are represented over time. A score
range of
~50% swings to the right indicates no impairment, whereas 0% swings to the
right swing
indicates maximal impairment. Asterisks indicate significant difference from
vehicle control
as assessed by one-way ANOVA.
[0032] Figure 7 shows the results of Cylinder Test. The mean and standard
error of
the score for groups receiving vehicle (diamonds), 1.0 ~,g/injection CG53135-
05 (square),
and 2.5 ~,g/injection CG53135-05 (triangles) are represented over time.
[0033] Figure 8 shows the results of Body Weight. The mean and standard errors
of
the weights for groups receiving vehicle (diamonds), 1.0 p.g/injection CG53135-
05 (square),
and 2.5 ~.g/injection CG53135-05 (triangles) is represented over time.
[0034] Figure 9 shows the effect of CG53135-05 on Pro-MMP production in
SW1353 cells in the presence of IL-1 beta.
[0035] Figure 10 shows the effect of CG53135-05 on Pro-MMP production in
SW1353 cells in the presence of TNF-alpha.
[0036] ~ Figure 11 shows the effect of CG53135-05 on TIMP production in SW1353
cells.
[0037] Figure 12 shows the effect of intra-articular injection of CG53135-05
in the
Meniscal Tear Model of Rat Osteoarthritis (Prophylactic Dosing): Mean Tibial
Cartilage
Degeneration.
[0038] Figure 13 shows results of infra-articular injection of CG53135-05 in
the
Meniscal Tear Model of Rat Osteoarthritis: (Prophylactic Dosing): Total
Cartilage
Degeneration Width.
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[0039] Figure 14 shows results of infra-articular injection of CG53135-05 in
the
Meniscal Tear Model of Rat Osteoarthritis: (Prophylactic Dosing): Significant
Tibial
Cartilage Degeneration Width.
[0040] Figure 15 shows results of infra-articular injection CG53135-05 in the
Meniscal Tear Model of Rat Osteoarthritis (Therapeutic Dosing): Mean Tibial
Degeneration.
[0041] Figure 16 shows results of infra-articular injection of CG53135-05 in
the
Meniscal Tear Model of Rat Osteoarthritis (Therapeutic Dosing): Total
Cartilage
Degeneration Width.
[0042] Figure 17 shows results of infra-articular injection of CG53135-05 in
Meniscal Tear Model of Rat Osteoarthritis (Therapeutic Dosing): Significant
Tibial Cartilage
Degeneration Width.
5. DETAILED DESCRIPTION OF THE INVENTION
[0043] The present invention provides methods of preventing or treating a
joint
disease (e.g., osteoarthritis, other related joint pathologies, such as but
are not limited to,
ligament and tendon injuries within the ligament and tendon itself, or within
their respective
insertion sites, meniscal tears, other joint disorders where matrix deposition
occurs, joint
disorders where remodeling and repair are required, and cartilage and joint
pathology
occurred as a result of an inflammatory disease (e.g., rheumatoid arthritis))
in a subject
comprising administering to the subject a composition comprising an FGF-CX
polypeptide.
[0044] The present invention also encompasses methods of using FGF-CX to
improve
functional recovery following middle cerebral artery (MCA) occlusion in rats.
As stroke may
result in disturbances of motor strength and coordination, sensory
discrimination, visual ,
function, speech, memory or other intellectual abilities, the present
invention evaluates the
efficacy and safety of FGF-CX in a model that assesses these parameters. In
accordance with
the present invention, administering FGF-CX will be beneficial in the
treatment of
pathological conditions including, but are not limited to, ischemic stroke,
hemorrhagic stroke,
trauma, spinal cord damage, heavy metal or toxin poisoning, and
neurodegenerative diseases
(such as Alzheimer's, Parkinson's Disease, Amyotrophic Lateral Sclerosis,
Huntington's
Disease).
[0045] FGF-CX polypeptides, nucleic acids encoding the polypeptides, and
methods
of making such polypeptides are described in U.S. Application Serial Nos.
091494,585 and
10/174,394, both of which are incorporated herein by reference in their
entireties. FGF-CX is
used interchangeably with the term "CG53135," "CG53135-05," and "FGF-20."
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[0046] Included within the invention are FGF-CX nucleic acids, isolated
nucleic acids
that encode FGF-CX polypeptide or a portion thereof, FGF-CX polypeptides,
vectors
containing these nucleic acids, host cells transformed with the FGF-CX nucleic
acids, anti-
FGF-CX antibodies, and pharmaceutical compositions. Also disclosed are methods
of
making FGF-CX polypeptides, as well as methods of screening, diagnosing,
treating
conditions using these compounds, and methods of screening compounds that
modulate FGF-
CX polypeptide activity. Table A provides a summary of the FGF-CX nucleic
acids and their
encoded polypeptides.
Table A
FGF-CXX Internal i SEQ ID NO SEQ ID NO gomolo
Assignment Identification ~ (nucleic acid) '~ (amino acid)
~FGF-CXla CG53135-05 il 2 Fibroblast growth factor-
20 (FGF-20) - Homo
sapiens
~FGF-CXlb CG53135-01 ~3 4 Fibroblast growth factor-
20 (FGF-20) - Homo
i
~sapiens
~FGF-CXlc CG53135-04 '5 '6 Fibroblast growth factor-
t 20 (FGF-20) - Homo
sapiens
F CXld 250059596 7 8 Fibroblast growth factor-
i 20 (FGF-20) - Homo
sapiens
~FGF-CXle 250059629 ~9 10 Fibroblast growth factor-
~20 (FGF-20) - Homo
~sapiens
FGF-CXlf 250059669 jll 12 Fibroblast growth factor-
i !, ' 20 (FGF-20) - Homo
sapiens
~FGF-CXlg 316351224 i13 14 Fibroblast growth factor-
20 (FGF-20) - Homo
sapiens
'FGF-CXlh 317459553 15 16 Fibroblast growth factor-
3 20 (FGF-20) - Homo
i_ ~ a sapiens
~FGF-CXli 317459571 ~17 18 Fibroblast growth factor-
20 (FGF-20) - Homo
_ I sapiens
F -CXlj CG53135-02 19 20 ~Fibroblast growth factor-
20 (FGF-20) - Homo
sapiens
FGF-CXlk CG53135-03 '~21 22 Fibroblast growth factor-
20 (FGF-20) - Homo
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FGF-CX11 CG53135-06 X23 24 Fibroblast
growth factor-
20 (FGF-20) - Homo
'sapiens
FGF-CXlm CG53135-07 X25 26 Fibroblast growth
factor-
20 (FGF-20) - Homo
sapiens
FGF-CXln CG53135-08 i27 28 Fibroblast growth
factor-
i ~20 (FGF-20) - Homo
sapiens
~FGF-CXloCG53135-09 29 '~30 Fibroblast
growth
factor-
20 (FGF-20) - Homo
i ~ sapiens
IFGF-CXlpCG53135-10 X31 32 Fibroblast growth
factor-
20 (FGF-20) - Homo
~~ sapiens
~GF-CXlq ~34 Fibroblast growth
CG53135-11 factor-
X33
20 (FGF-20) - Homo
t
a sapiens
FGF-CXlr CG53135-12 i35 'l36 Fibroblast growth
factor-
20 (FGF-20) - Homo
sapiens
[0047] As used herein, the termsand "subjects"refer to an animal,
"subject"
preferably a mammal, including a non-primate (e.g., a cow, pig, horse, cat,
dog, rat, and
mouse) and a primate (e.g., a monkey, such as a cynomolgous monkey,
chimpanzee, and a
human), and more preferably a human. In a certain embodiment, the subject is a
mammal,
preferably a human, who is suffering from a joint disease (e.g.,
osteoarthritis, other
osteoarthritis-related disorders), ischemic stroke, hemorrhagic stroke,
trauma, spinal cord
damage, heavy metal or toxin poisoning, or neurodegenerative diseases (non-
limiting
examples being Alzheimer's, Parkinson's Disease, Amyotrophic Lateral
Sclerosis,
Huntington's Disease). In another embodiment, the subject is a mammal,
preferably a human,
who axe at risk for a joint disease, ischemic stroke, hemorrhagic stroke,
trauma, spinal cord
damage, heavy metal or toxin poisoning, or neurodegenerative diseases. In one
embodiment,
the subject is a mammal, preferably a human, who is suffering from a joint
disease, but who
is not suffering from stroke or a neurodegenerative disease. The term
"subject" is used
interchangeably with "patient" in the present invention.
[0048] As used herein, the term "therapeutically effective amount" refers to
the
amount of a therapy (e.g., FGF-CX polypeptide), which is sufficient to reduce
the severity of
a disease (e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma,
spinal cord
damage, heavy metal or toxin poisoning, or neurodegenerative diseases), reduce
the duration
of a disease, prevent the advancement of a disease, cause regression of a
disease, ameliorate
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one or more symptoms associated with a disease, or enhance or improve the
therapeutic
effects) of another therapy.
[0049] Compositions comprising FGF-CX can also be administered in combination
with one or more other therapies to prevent, treat, or ameliorate a disease
(e.g., a joint
disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage,
heavy metal or
toxin poisoning, or neurodegenerative diseases) or one or more symptoms
thereof. In a
preferred embodiment, compositions comprising FGF-CX is administered in
combination
with one or more other therapies known to be used in preventing, treating, or
ameliorating a
disease such as a joint disease, ischemic stroke, hemorrhagic stroke, trauma,
spinal cord
damage, heavy metal or toxin poisoning, or neurodegenerative diseases, or one
or more
symptoms thereof.
[0050] In one embodiment, during a combination therapy, FGF-CX polypeptide
and/or another therapy are administered in a sub-optimal amount, e.g., an
amount that does
not manifest detectable therapeutic benefits when administered alone, as
determined by
methods known in the art. In such methods, co-administration of FGF-CX
polypeptide and
another therapy results in an overall improvement in effectiveness of
treatment.
[0051] In one embodiment, FGF-CX polypeptide and one or more other therapies
are
administered within the same patient visit. In another embodiment, FGF-CX
polypeptide is
administered prior to the administration of one or more other therapies. In
yet another
embodiment, the FGF-CX polypeptide is administered subsequent to the
administration of
one or more other therapies. In a specific embodiment, FGF-CX polypeptide and
one or
more other therapies are cyclically administered to a subject. Cycling therapy
involves the
administration of FGF-CX polypeptide for a period of time, followed by the
administration of
one or more other therapies for a period of time and repeating this sequential
administration.
Cycling therapy can reduce the development of resistance to one or more of the
therapies,
avoid or reduce the side effects of one of the therapies, and/or improve the
efficacy of the
treatment.
[0052] Toxicity and therapeutic efficacy of a composition of the invention
(e.g., FGF-
CX polypeptide) can be determined by standard pharmaceutical procedures in
cell cultures or
n
experimental animals, e.g., for determining the LDSO (the dose lethal to 50%
of the
population) and the EDso (the dose therapeutically effective in 50% of the
population). The
dose ratio between toxic and therapeutic effects is the therapeutic index and
it can be
expressed as the ratio of LDSO/EDso. Compositions that exhibit large
therapeutic indices are
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preferred. While compositions that exhibit toxic side effects may be used,
care should be
taken to design a delivery system that targets such composition to the site of
affected tissue in
order to minimize potential damage to uninfected cells and, thereby, reduce
side effects.
[0053] In one embodiment, the data obtained from the cell culture assays and
animal
studies can be used in formulating a range of dosage for use in humans. The
dosage of
complexes lies preferably within a range of circulating concentrations that
include the EDso
with little or no toxicity. The dosage may vary within this range depending
upon the dosage
form employed, the route of administration utilized, the severity of the
disease, age and
weight of the subject, and other factors normally considered by a medical
professional (e.g., a
physician). For any composition used in the method of the invention, the
therapeutically
effective dose can be estimated initially from cell culture assays. A dose may
be formulated
in animal models to achieve a circulating plasma concentration range that
includes the IC50
(i.e., the concentration of the test compound that achieves a half maximal
inhibition of
symptoms) as determined in cell cultures. Such information can be used to more
accurately
determine useful doses in humans. Levels in plasma may be measured, for
example, by high
performance liquid chromatography. [INSERT PRFERRED DOSAGE RANGES HERE]
[0054] The appropriate and recommended dosages, formulation and routes of
administration for treatment modalities such as chemotherapeutic agents,
radiation therapy
and biological/immunotherapeutic agents such as cytokines are known in the art
and
described in such literature as the Physician's Desk Reference (5th ed.,
2004).
[0055] Various delivery systems are known and can be used to administer a
composition of the invention. Such delivery systems include, but are not
limited to,
encapsulation in liposomes, microparticles, microcapsules, expression by
recombinant cells,
receptor-mediated endocytosis, construction of the nucleic acids of the
invention as part of a
retroviral or other vectors, etc. Methods of introduction include, but are not
limited to,
intradermal, intramuscular, intraperitoneal, intrathecal,
intracerebroventricular, epidural,
intravenous, subcutaneous intranasal, intratumoral, transdermal, rectal, and
oral routes. The
compositions of the invention may be administered by any convenient route, for
example, by
infusion or bolus injection, by absorption through epithelial or mucocutaneous
linings (e.g.,
oral mucosa, virginal mucosa, rectal and intestinal mucosa, etc.), and may be
administered
together with other biologically active agents. Administration can be systemic
or local.
[0056] In some embodiments, it may be desirable to administer the
pharmaceutical
compositions of the invention locally to the area in need of treatment. This
may be achieved
12
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WO 2004/100892 PCT/US2004/014623
by, for example, local infusion during surgery, or topical application, e.g.,
in conjunction with
a wound dressing after surgery, by injection, by means of a catheter, by means
of a
suppository, or by means of an implant (said implant being of a porous, non-
porous, or
gelatinous material, including membranes, such as sialastic membranes, or
fibers). In one
embodiment, administration can be by direct injection at the site (or former
site) of rapidly
proliferating tissues which are most sensitive to an insult such radiation,
chemotherapy, or
chemical warfare agent.
[0057] In some embodiments, where the composition of the invention is a
nucleic
acid encoding a prophylactic or therapeutic agent, the nucleic acid can be
administered in
vivo to promote expression of their encoded proteins (e.g., FGF-CX
polypeptide), by
constructing the nucleic acid as part of an appropriate nucleic acid
expression vector and
administering it so that it becomes intracellular, e.g., by use of a
retroviral vector, or by direct
injection, or by use of microparticle bombardment (e.g., a gene gun), or
coating with lipids or
cell-surface receptors or transfecting agents, or by administering it in
linkage to a homeobox-
like peptide which is known to enter the nucleus, etc. Alternatively, a
nucleic acid of the
invention can be introduced intracellularly and incorporated within host cell
DNA for
expression, by homologous recombination.
[0058] ~ The compositions of the invention include bulk drug compositions
useful in
the manufacture of pharmaceutical compositions that can be used in the
preparation of unit
dosage forms. In a preferred embodiment, a composition of the invention is a
pharmaceutical
composition. Such compositions comprise a prophylactically or therapeutically
effective
amount of one or more compositions (e.g., FGF-CX polypeptide) of the
invention, and a
pharmaceutically acceptable carrier. Preferably, the pharmaceutical
compositions are
formulated to be suitable for the route of administration to a subject.
[0059] In one embodiment, the term "pharmaceutically acceptable" means
approved
by a regulatory agency of the Federal or a state government or listed in the
IT.S.
Pharmacopeia or other generally recognized pharmacopeia for use in animals,
and more
particularly in humans. The term "carrier" refers to a diluent, adjuvant
(e.g., Freund's
adjuvant (complete and incomplete)), excipient, or vehicle with which the
prophylactic or
therapeutic agent is administered. Such pharmaceutical carriers can be sterile
liquids, such as
water and oils (e.g., oils of petroleum, animal, vegetable or synthetic
origins, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like), or solid Garners,
such as one or more
substances which may also act as diluents, flavoring agents, solubilizers,
lubricants,
suspending agents, binders, tablet disintegrating agents, or encapsulating
material. Water is a
13
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WO 2004/100892 PCT/US2004/014623
preferred carrier when the pharmaceutical composition is administered
intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be employed as
liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical excipients
include, but are not
limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,
chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim mills,
glycerol, propylene,
glycol, water, ethanol, or a combination thereof. The composition, if desired,
can also
contain minor amounts of wetting or emulsifying agents, or pH buffering
agents.
[0060] The compositions of the present invention may be formulated into any of
many possible dosage forms such as, but not limited to, liquid syrups,
tablets, capsules, gel
capsules, soft gels, pills, powders, enemas, sustained-release formulations
and the like. The
compositions of the present invention may also be formulated as suspensions in
aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain substances
that
increase the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may also
contain
stabilizers. The composition can also be formulated as a suppository, with
traditional binders
and carriers such as triglycerides. Oral formulation can include standard
carriers, such as
pharmaceutical 'grades of mannitol, lactose, starch, magnesium stearate,
sodium saccharine,
cellulose, magnesium carbonate, etc.
[0061] In some embodiments, the compositions of the present invention may be
formulated and used as foams. Pharmaceutical foams include formulations such
as, but not
limited to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar
in nature, these formulations vary in the components and the consistency of
the final product.
The preparation of such compositions and formulations is generally known to
those skilled in
the pharmaceutical and formulation arts and may be applied to the formulation
of the
compositions of the present invention.
[0062] A pharmaceutical composition of the invention is formulated to be
compatible
with its intended route of administration. In a specific embodiment, the
composition is
formulated in accordance with routine procedures as a pharmaceutical
composition adapted
for intravenous, subcutaneous, intramuscular, oral, intranasal, intratumoral
or topical
administration to human beings. Typically, compositions for intravenous
administration are
solutions in sterile isotonic aqueous buffer. Where necessary, the composition
may also
include a solubilizing agent and a local anesthetic such as lidocaine to ease
pain at the site of
the inj ection.
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[0063] If the compositions of the invention are to be administered topically,
the
compositions can be formulated in the form of transdermal patches, ointments,
lotions,
creams, gels, drops, suppositories, sprays, liquids and powders. Conventional
pharmaceutical
carriers, aqueous, powder or oily bases, thickeners and the like may be
necessary or desirable.
Coated condoms, gloves and the like may also be useful. Preferred topical
formulations
include those in which the polypeptides of the invention are in admixture with
a topical
delivery agent, such as but not limited to, lipids, liposomes, fatty acids,
fatty acid esters,
steroids, chelating agents and surfactants. Preferred lipids and liposomes
include, but are not
limited to, neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,
dimyristoylphosphatidyl
choline DMPC, distearolyphosphatidyl choline), negative (e.g.
dimyristoylphosphatidyl
glycerol DMPG), and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and
dioleoylphosphatidyl ethanolamine DOTMA). The polypeptides of the invention
may be
encapsulated within liposomes or may form complexes thereto, in particular to
cationic
liposomes. Alternatively, the polypeptides may be complexed to lipids, in
particular to
cationic lipids. Preferred fatty acids and esters include, but are not limited
to, arachidonic
acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid,
myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,
monoolein, dilaurin,
glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an
acylcholine, or
a C1-10 allcyl ester (e.g, isopropylmyristate IPM), monoglyceride,
diglyceride, or
pharmaceutically acceptable salt thereof. For non-sprayable topical dosage
forms, viscous to
semi-solid or solid forms comprising a carrier or one or more excipients
compatible with
topical application and having a dynamic viscosity preferably greater than
water are typically
employed. Other suitable topical dosage forms include sprayable aerosol
preparations .
wherein the active ingredient, preferably in combination with a solid or
liquid inert carrier, is
packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant,
such as freon) or
in a squeeze bottle. Moisturizers or humectants can also be added to
pharmaceutical
compositions and dosage forms if desired. Examples of such additional
ingredients are well-
known in the art.
[0064] If the method of the invention comprises intranasal administration of a
composition, the composition can be formulated in an aerosol form, spray, mist
or in the form
of drops. In particular, prophylactic or therapeutic agents for use according
to the present
invention can be conveniently delivered in the form of an aerosol spray
presentation from
pressurized packs or a nebuliser, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon dioxide
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
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
(composed 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.
[0065] If the method of the invention comprises oral administration,
compositions can
be formulated in the form of powders, granules, microparticulates,
nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules, gel
capsules, sachets,
tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers,
dispersing aids or
binders may be desirable. Tablets or capsules can be prepared by conventional
means with
pharmaceutically acceptable excipients such as binding agents (e.g.,
pregelatinised maize
starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers
(e.g., lactose,
microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g.,
magnesium
stearate, talc, or silica); disintegrants (e.g., potato starch or sodium
starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets maybe coated by
methods well-
known in the art. Liquid preparations for oral administration may be prepared
by
conventional means with pharmaceutically acceptable additives such as
suspending agents
(e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);
emulsifying agents
(e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily
esters, ethyl alcohol, or
fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-
hydroxybenzoates or
sorbic acid). The preparations may also contain buffer salts, flavoring,
coloring, and
sweetening agents as appropriate. Preparations for oral administration may be
suitably
formulated for slow release, controlled release, or sustained release of a
prophylactic or,
therapeutic agent(s).
[0066] In one embodiment, the compositions of the invention are orally
administered
in conjunction with one or more penetration enhancers, e.g., surfactants and
chelators.
Preferred surfactants include, but are not limited to, fatty acids and esters
or salts thereof, bile
acids and salts thereof. In some embodiments, combinations of penetration
enhancers are
used, e.g., fatty acids/salts in combination with bile acids/salts. In a
specific embodiment,
sodium salt of lauric acid, capric acid is used in combination with UDCA.
Further
penetration enhancers include, but are not limited to, polyoxyethylene-9-
lauryl ether,
polyoxyethylene-20-cetyl ether. Compositions of the invention may be delivered
orally in
granular form including, but is not limited to, sprayed dried particles, or
complexed to form
micro or nanoparticles. Complexing agents that can be used for complexing with
the peptide
of the invention (e.g., FGF-CX polypeptide)'include, but are not limited to,
poly-amino acids,
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WO 2004/100892 PCT/US2004/014623
polyimines, polyacrylates, polyalkylacrylates, polyoxethanes,
polyalkylcyanoacrylates,
cationized gelatins, albumins, acrylates, polyethyleneglycols (PEG),
polyalkylcyanoacrylates,
DEAF-derivatized polyimines, pollulans, celluloses, and starches. Particularly
preferred
complexing agents include, but are not limited to, chitosan, N-
trimethylchitosan, poly-L-
lysine, polyhistidine, polyornithine, polyspermines, protamine,
polyvinylpyridine,
polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino),
poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate),
poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate,
DEAE-
hexylacrylate, DEAF-acrylamide, DEAF-albumin and DEAE-dextran,
polymethylacrylate,
polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid
(PLGA), alginate,
and polyethyleneglycol (PEG).
[0067] The method of the invention may comprise pulmonary administration,
e.g., by
use of an inhaler or nebulizer, of a composition formulated with an
aerosolizing agent.
[0068] The method of the invention may comprise administration of a
composition
formulated for parenteral administration by injection (e.g., by bolus
injection or continuous
infusion). Formulations for injection may be presented in unit dosage form
(e.g., in ampoules
or in mufti-dose containers) with an added preservative. The compositions may
take such
forms as suspensions; solutions or emulsions in oily or aqueous vehicles, and
may contain
formulatory agents such as suspending, stabilizing and/or dispersing agents.
Alternatively,
the active ingredient may be in powder form for constitution with a suitable
vehicle (e.g.,
sterile pyrogen-free water) before use.
[0069] In a preferred embodiment, the composition is formulated in accordance
with
routine procedures as a pharmaceutical composition adapted for intravenous
administration to
human beings. Typically, compositions for intravenous administration are
solutions in sterile
isotonic aqueous buffer. Where necessary, the composition may also include a
solubilizing
agent and a local anesthetic such as lignocaine to ease pain at the site of
the injection.
Generally, the ingredients are supplied either separately or mixed together in
unit dosage
form, for example, as a dry lyophilized powder or water free concentrate in a
hermetically
sealed container, such as an ampoule or sachette, indicating the quantity of
active agent.
Where the composition is to be administered by infusion, it can be dispensed
with an infusion
bottle containing sterile pharmaceutical grade water or saline. Where the
composition is
administered by injection, an ampoule of sterile water for injection or saline
can be provided
so that the ingredients may be mixed prior to administration.
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[0070] The compositions of the invention can be formulated as neutral or salt
forms.
Pharmaceutically acceptable salts include, but are not limited to, those
formed with free
amino groups such as those derived from hydrochloric, phosphoric, acetic,
oxalic, tartaric
acids, etc., and those formed with free carboxyl groups such as those derived
from sodium,
potassium, ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-
ethylamino ethanol, histidine, procaine, etc. Non-limiting examples of
pharmaceutically
acceptable salts are acetate, benzenesulfonate, benzoate, bicarbonate,
bitartrate, bromide,
calcium acetate, camsylate, carbonate, chloride, citrate, dihydrochloride,
edetate, edisylate,
estolate, esylate, fumarate, glucaptate, gluconate, glutamate,
glycollylarsanilate,
hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate,
iodide,
isethionate, lactate, lactobionate, malate, maleate, mandelate mesylate,
methylbromide,
methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate (embonate),
pantothenate,
phosphateldiphosphate, polygalacturonate, salicylate, stearate, subacetate,
succinate, sulfate,
tannate, tartrate, teoclate, triethiodide, benzathine, chloroprocaine,
choline, diethanolamine,
ethylenediamine, meglumine, procaine, aluminium, calcium, lithium, magnesium,
potassium,
sodium, and zinc.
[0071] In addition to the formulations described previously, the compositions
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 compositions 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. Liposomes and emulsions are well known examples of delivery
vehicles or
carriers for hydrophilic drugs.
[0072] In one embodiment, the ingredients of the compositions of the invention
(e.g.,
FGF-CX) are derived from a subject that is the same species origin or species
reactivity as
recipient of such compositions.
[0073] The invention also provides kits for carrying out the therapeutic
regimens of
the invention. Such kits comprise in one or more containers prophylactically
or
therapeutically effective amounts of the composition of the invention (e.g.,
FGF-CX
polypeptide) in pharmaceutically acceptable form. The composition in a vial of
a kit of the
invention may be in the form of a pharmaceutically acceptable solution, e.g.,
in combination
with sterile saline, dextrose solution, or buffered solution, or other
pharmaceutically
acceptable sterile fluid. Alternatively, the composition may be lyophilized or
desiccated; in
18
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WO 2004/100892 PCT/US2004/014623
this instance, the kit optionally' further comprises in a container a
pharmaceutically acceptable
solution (e.g., saline, dextrose solution, etc.), preferably sterile, to
reconstitute the
composition to form a solution for injection purposes.
[0074] In another embodiment, a kit of the invention further comprises a
needle or
syringe, preferably packaged in sterile form, for injecting the formulation,
and/or a packaged
alcohol pad. Instructions are optionally included for administration of the
formulations of the
invention by a clinician or by the patient.
[0075] In some embodiments, the present invention provides kits comprising a
plurality of containers each comprising a pharmaceutical formulation or
composition
comprising a dose of the composition of the invention (e.g., FGF-CX
polypeptide) sufficient
for a single administration.
[0076] As with any pharmaceutical product, the packaging material and
container are
designed to protect the stability of the product during storage and shipment.
In one
embodiment, compositions of the invention are stored in containers with
biocompatible
detergents, including but not limited to, lecithin, taurocholic acid, and
cholesterol; or with
other proteins, including but not limited to, gamma globulins and serum
albumins. Further,
the products of the invention include instructions for use or other
informational material that
advise the physician, technician, or patient on how to appropriately prevent
or treat the
disease or disorder in question.
[0077] The present invention is further illustrated by the following Examples.
5.1. Example 1: Seauence Anal, sis
[0078] Details of the sequence relatedness and domain analysis for each FGF-CX
are
presented in Table 1A. The FGF-CX1 clone was analyzed, and the nucleotide and
encoded
19
polypeptide sequences are shown in Table 1A.
CA 02525107 2005-11-07
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llenCe
MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHLHGILRRR
LYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTS
CIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPEL
KDLLMYT
~Xlb, CG53135-01 ~SEQ m NO: 3 (633 by
Sequence ORF Start: ATG at l ORF S
CTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGGCAGCGTGCAGGGCAC
CCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTA
ACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTT
TGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATA
ACTGATGTACACT
b, CG53135-01 ~SEQ m NO: 4 211 as BMW at 23498.4kD
:auence
MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHLHGILRRRQ
LYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGN.~TDKGELYGSEKLTSE
CIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELY
KDLLMYT
CG53135-04 PSEQ m NO: 5
Sequence ~ORF Start: ATG at 1 ORF Stop: end of sequence
GCAGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGC
AGATCCTGCCCGACGGCAGCGCGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTC
ATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGA
CAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGA
ACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCA
c, CG53135-04 ~SEQ m NO: 6 ~ 179 as BMW at 20118.6kD
MAPLAEVGGFLGGLEGLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSAQGTRQDHSLFGILE
ISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRR.YFV
LNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLLMYT
~Xld, 250059596 'SEQ 1D NO: 7 x556 r
Sequence IORF Start: IORF
CACCAGATCTATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGC
CGGGGGCAGCGCAGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGC
CTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTG
GAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAG
TTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTA
TTTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTA
CACATTTCTTACCTAGACCAGTGGATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTAC
d, 250059596 ~SEQ m NO: 8 X185 as BMW at 20762.3kD
TRSMAPLAEVGGFLGGLEGLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGI
LEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRY
FVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLLMYTVDG
~Xle, 250059629 ~SEQ m N_O: 9 (415 by _
Sequence ~, ORF Start: ORF Stop:
TCTATCCTGCGCCGCCGGCAGCTCTA
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WO 2004/100892 PCT/US2004/014623
TATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTA
CTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACA
GAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGA
e, 250059629 (SEQ m NO: 10 138 as MW at 15847.7kD
;quence
QLYCRTGFHLQILPDGSVQGTRQDHSLFGTLEFISVAVGLVSIRGVDSGLYLGM
ECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTH
~Xlf, 250059669 ~SEQ m N0: 11 X466 by
Sequence OR_F Start: ~ORF Stop:
~GATCTATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCC
TGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTAT
ATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAA
ACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGAC
TCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACACTGTCGACGGC
1f, 250059669 ~SEQ m NO: 12 X155 as BMW at 17911.1kD
~equence
RQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGM
SECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTH
LYKDLLMYTVDG
~Xlg, 316351224 iSEQ m NO: 13 X549 by
Sequence ~RF Star't: ' ORF Stop:
'.TATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCA
iCGCAGCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCG
TCAGTGTGGCAGTGGGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAAT
AAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTG
CTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTT
CAGTGGATCCAGAAAGAGTTCCAGAA
Klg, 316351224 ,~SEQ m NO: 14 X183 as MW at 20632.2kI~
Sequence ~ , ~ ~
~AEVGGFLGGLEGLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGIL
..VGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENVVYNTYSSNIYKHGDTGRRYF
~GTPRDGARSKRHOKFTHFLPRPVDPERVPELYKDLLMYTLE
F-CXlh, 317459553 ~SEQ m NO: 15 X408 by
A Sequence ORF Start: ORF Stop:
~TCTATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACG
4CGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTG
'TGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAATGAATGACAAAGGAGAACTCTA
TCATCTAACATATATAAACATGAAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGAA
TCCAAGAGATGGCGCCAGGTCCAAGAGGCA'T'CAGAA.ATTTACACATTTCTTACCTAGACCACTCGAG
GF-CXlq, 317459553 -~~~~SEQ m NO: 16 136 as ~MW at 15789.6kD
rotein Se uence '
SILRR'E2QLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELY
SEKLTSECIFREQFEENWYNTYSSNIYKHEDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPLE
GF-CX~li, 317459571 ~~ ~ ~~~~~~SEQ m NO: 17 ~~T ~T~408~bpT~TT.....__._.~.-
ANA Sequence ORF Start: '1 ORF Stop:
GATCTATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGCCCGACGG
AGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGG
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GGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCT
TTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTTTGTGGCACTTAACAAAGACGGA
CTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCACTCGA
FGF-CXli, 317459571 SEQ m N0: 18 ,136 as ~MW~at 15717.6kD
Protein Seauence
RSILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGEL
GSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPL
FGF-CXlj, CG53135-Q2 ~.~~~ ;SEQ m ~NO: 19 ~ ;477 by
DNA Sequence jp~' Start: ATG at 1 10RF Ston: end of seauence
CTGCAGATCCTGCCGGATGGTTCTGTTCAGGGTACCCGTCAGGACCACTCTCTGTTCGGTATC
ATTCATCTCTGTTGCTGTTGGTCTGGTTTCTATCCGTGGTGTTGACTCTGGCCTGTACCTGGG
ACTCTTCCAACATCTACAAACATGGTGACACCGGCCGTCGCTACTTC
CGCGCCCAGTTGACCCGGAGCGTGTTCCAGAACTGT
1j, CG53135-02 fSEQ m NO: 20 X158 as BMW at 18254.6kD
MAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGL
NDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQ
FLPRPVDPERVPELYKDLLMYT
FGF-CXlk,~CG53135-03 ~~~ ~' SEQ~m N0: 21~ '636 by ~
DNA Sequence ' ORF Start: ATG at 1 ' ORF Ston: end of seauenc
TCTATTGCCGCACCGGCTTCCACCTGCAGATCCTGC
CACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTG
'1'GG':1'C'1'C'1'ATCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTC
TCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATATATAAAC
AAGGACCTACTGATGTACACTTGA
FGF-CXlk, CG53135-03 SEQ m N0: 22 211 as MW at 23498.4kD
Protein Sequence
MA.PLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHLHGILRRR
LYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTS
CIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPEL
F-CX11, CG53135-06 ~SEQ ~ NO: 23 ;540 by
A Sequence ~ORF Start: ATG at 1 IORF Ston: end of
GCTGGCGCACCTGCACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCCACCTGC
TCCTGCCCGACGGCAGCGTGCAGGGCACCCGGCAGGACCACAGCCTCTTCGGTATCTTGGAATTC
CAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCGAATGCATCTTTAGGGAGCAG
ACTGGTATAACACCTATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTA
CTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGTCCAAGAGGCATCAGAAATTTA
S
l, CG53135-06 SEQ m NO: 24 X179 as BMW at 20146.7kD
MAPLAEVGGFLGGLEGLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILE
ISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREOFEENWYNTYSSNIYKHGDTGRRYFV
.l~G'1'PR17GARSKRHQKFTHFLPRPVDPERVPELYKDLLMYT
F-CXlm, CG53135-07~~~~~ ~~4~SEQ m NO: 25~~~~~~~ ; 54 by
A Sequence ~ORF Start: ATG at 1 ' ORF Stop:
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PGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGC
3F-CXlm, CG53135-07 SEQ 117 NO: 26[18 as MW at~1b88.OkD~~
~EVGGFLGGLEGLG
~Xln, CG53135-08 SEQ >D NO: 27 X63 by
Sequence ___. O~ Start~~ I pRF Stop:
n, CG53135-08~SEQ ll~ NO: 28,21 aa~MW at 2262
ERPPLLGERRSAAERSARGGP _
FGF-CXlo, CG53135-09 sSEQ ll~ NO: 29 63 by
DNA Sequence ORF Start: ORF Ston:
o, CG53135-09~SEQ D7 NO: 3022 aafMW at 2463
TALNKDGTPRDGARSKR
~Xlp, CG53135-10~~~~~EQ-ID NO: 31~~~~~~'~60 by
Sequence ORF Start: ,~ORF Stop:
FGF-CXlp, CG53135-lO~SEQ m NO: 3220 aaMW at 2431.8
Protein Sequence
PRPVDPERVPELYKDLLMYT
FGF-CXlq, CG53135-11 SEQ ID NO: 33 51 by
DNA Sequence O~ Start: ATG at 1 ~ ORF Stop:
'FGF-CXlq, CG53135-I1SEQ ID NO: 3417 aaMW at 1904.1kD
Protein Sequence
MNDKGELYGSEKLTSEC
FGF-CXlr, CG53135-12 ~EQ ID NO: 35~~ ~ 633 by
DNA Sequence ORF Start ATG at 1 ORF Stop:
ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGGGCTTGGGCCAGCAGGTC
GCATTTCCTGTTGCCTCCTGCCGGGGAGCGGCCGCCGCTGCTGGGCGAGCGCAGGAGCGCGGC
GGAGCGCGCGCGGCGGGCCGGGGGCTGCGCAGCTGGCGCACCTGCACGGCATCCTGCGCCGCC
CTCTATTGCCGCACCGGCTTCCACCTGC__ACATt~rmc~rrrr ArrrrAr_~nmrr n rrn~ a r~~~~r-
GGTATCTTGGAATTCATCAGTGTGGCAGTGGGACTGGTCAGTATT
TCTTGGAATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACT
AGCAGTTTGAAGAGAACTGGTATAACACCTATTCATCTAACATAT
AGAACCTACTGATGTACACT
~GF-CXlr, CG53135-I2 SEQ ID NO: 36 '221 as MW at 23497.4kD
'rotein Sequence
APLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHLHGTLRRR
YCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTS
LFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPEL
[0079] A ClustalW comparison of the above protein sequences yields the
following
sequence alignment shown in Table 1B.
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Table 1B. Comparison of the FGF-CX1 protein sequences.
FGF-CXla MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL
FGF-CXlb. MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL
FGF-CXlc _-__________________________________________________________
FGF-CXld ____________________________________________________________
FGF-CXle ___________________________________________________________T
FGF-CXlf -------------------------------TRSILRRR.QLYCRTGFHLQILPDGSVQGT
FGF-CXlg ____________________________________________________________
FGF-CXlh ____________________________________________________________
FGF-CXli ____________________________________________________________
FGF-CXlj -------------------------MAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGT
FGF-CXlk MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL
FGF-CX11 ----MAPLAEVGGFLGGLEGLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGT
FGF-CXlm ____________________________________________________________
FGF-CXln ____________________________________________________________
FGF-CXlo ____________________________________________________________
FGF-CXlp _-__________________________________________________________
FGF-CXlq _-__________________________________________________________
FGF-CXlr MAPLAEVGGFLGGLEGL'GQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL
FGF-CXla HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
FGF-CXlb HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
FGF-CXlc ____________________________________________________________
FGF-CXld ____________________________________________________________
FGF-CXle RSILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
FGF-CXlf RQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWY
FGF-CXlg ____________________________________________________________
FGF-CXlh RSILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
FGF-CXli RSILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
FGF-CXlj RQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWY
FGF-CXlk HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
FGF-CX11 RQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWY
FGF-CXlm _____-______________________________________________________
FGF-CXln ____________________________________________________________
FGF-CXlo ____________________________________________________________
FGF-CXlp ____________________________________________________________
FGF-CXIq ____________________________________________________________
FGF-CXlr HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
FGF-CXla MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGAR
FGF-CXlb MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRR.YFVALNKDGTPRDGAR
FGF-CXlc _____________________________________________~PLAEVGGFLGGLE
FGF-CXld ------------------------------------------TRSMAPLAEVGGFLGGLE
FGF-CXle MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGAR
FGF-CXlf NTYSSNIYKHGDTGRRYFVALNKDGTPRI7GARSKRHQKFTHFLPRPVDPERVPELYKDLL
FGF-CXlg -------------------------------------------RSMAPLAEVGGFLGGLE
FGF-CXIh MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHEDTGRRYFVALNKDGTPRDGAR
FGF-CXli MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGAR
FGF-CXlj NTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLL
FGF-CXlk MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRR.YFVALNKDGTPRDGAR
FGF-CX11 NTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLL
FGF-CXlm ---------------------------------------------MAPLAEVGGFLGGLE
FGF-CXIn ------------------------------------------ERPPLLGERRSAAERSAR
FGF-CXlo _-___-____________________________________gRYFVALNKDGTPRDGAR
FGF-CXlp -------------------------------------------PRPVDPERVPELYKDLL
FGF-CXlq ________________________________________________~DKGELYGSEK
FGF-CXlr MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGAR
FGF-CXla SKRHQKFTHFLPRPVDPERVPELYKDLLMYT-----------------------------
FGF-CXlb SKRHQKFTHFLPRPVDPERVPELYKDLLMYT-----------------------------
FGF-CX1C GLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSAQGTRQDHSLFGILEFISVAVGL
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FGF-CXld GLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGL
FGF-CXle SKRHQKFTHFLPRPVDG-_________________________________,________
FGF-CXlf MYTVDG-_____________________________________________________
FGF-CXlg GLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGL
FGF-CXlh SKRHQKFTHFLPRPLE-___________________________________________
FGF-CXli SKRHQKFTHFLPRPLE--------------------------------------------
FGF-CXlj MYT-________________________________________________________
FGF-CXlk SKRHQKFTHFLPRPVDPERVPELYKDLLMYT-----------------------------
FGF-CX17. MYT-________________________________________________________
FGF-CXlm GLG-________________________________________________________
FGF-CXln GGP-________________________________________________________
'FGF-CXlo SKR-__________-_____________________________________________
FGF-CXlp MYT-________________________________________________________
FGF-CXlq LTSEC-_________________________________-____________________
'FGF-CXlr SKRHQKFTHFLPRPVDPERVPELYKNLLMYT-----------------------------
FGF-CXla ___________________________________________________________
'FGF-CXlb _-_________________________________________________________
FGF-CXlc VSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFV
FGF-CXId VSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFV
'FGF-CXle _-_________________________________________________________
'FGF-CXlf ____________________________________________________________'
',FGF-CXlg VSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFV,
'FGF-CXlh ___________________________________________________________
FGF CXli ____________________________________-_______________________'
I'FGF-CXl~ ____________________________________________________________
'FGF-CXlk ____________________________________________________________
IFGF-CX11 ____________________________________________________________',
FGF-CXlm ____________________________________________________________)
'FGF-CXln ____________________________________________________________'
'FGF-CXlo ____________________________________________________________',
i
FGF-CXlp ___________________________________________________________
',FGF-CXlq ____________________________________________________________,
'FGF-CXlr ______________-_____________________________________________'
FGF-CXla _______________________________________-_______
'FGF-CXlb _______________________________________-_______
'FGF-CXlc ALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLLMYT---
FGF-CXld ALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLLMYTVDG
',FGF-CXle _-_____________________________________________
'FGF-CXlf _______________________________________________
FGF-CXlg ALNKDGTPRDGARSKRHQKFTHFLPRPVDPERVPELYKDLLMYTLE-
'FGF-CXlh _______________________________________________
',FGF-CXli _______________________________________________
'FGF-CXIj _______________________________________________
'FGF-CXIk __________-_________________.__________________
FGF-CX11 _______________________________________________
~FGF-CXlm __________________________________,_____________
FGF-CXln _-_____________________________________________
'FGF-CXlo _______________________________________________
',FGF-CXlp ________________________________________-______
FGF-CXlq _______________________________________________
FGF-CXlr _-_____________________________________________
FGF-CXla (SEQ ID N0: 2)
FGF-CXlb (SEQ ID NO: 4)
FGF-CX1C (SEQ ID N0: 6)
'FGF-CXld (SEQ ID NO: 8)
FGF-CXle (SEQ ID N0: 10)
FGF-CXlf (SEQ ID N0: 12)
'FGF-CXlg (SEQ ID NO: 14)
FGF-CXlh (SEQ ID NO: 16)
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FGF-CXli(SEQ ID 18).
N0:
FGF=CXlj(SEQ ID 20)
NO:
FGF-CXlk(SEQ ID 22)
NO:
FGF-CX11(SEQ ID 24)
NO:
FGF-CXIm(SEQ ID 26)
NO:
FGF-CXln(SEQ ID 28)
N0:
FGF-CXIo(SEQ ID 30)
NO:
',FGF-CXlp(SEQ ID 32)
N0:
'FGF-CXlq(SEQ ID 34)
N0:
FGF-CXlr( ID 36 ) ~..~~.. ...._._.~_~ ...____ ___..
SEQ NO v ___~,.j
:
[000] Further analysis of the FGF-CXla protein yielded the following
properties
shown in Table 1 C.
Table 1C. Protein Sequence Properties FGF-CXla
SignalP analysis: No Known Signal Sequence Predicted
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 6; pos.chg 0; neg.chg 1
H-region: length 8; peak value 0.00
PSG score: -4.40
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): -5.49
possible cleavage site: between 16 and 17
»> Seems to have no N-terminal signal peptide
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 1
Tentative number of TMS(s) for the threshold 0.5: 1
Number of TMS(s) for threshold 0.5: 1
INTEGRAL Likelihood = -6.42 Transmembrane 9.4 - 110
PERIPHERAL Likelihood = 5.20 (at 1)
ALOM score: -6.42 (number of TMSs: 1)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 101
Charge difference: 0.5 C( 0.0) - N(-0.5)
C > N: C-terminal side will be inside
»> membrane topology: type 1b (Cytoplasmic tail 94 to 211)
MITDISC: discrimination of mitochondrial targeting seq
R content: 0 Hyd Moment(75): 3.24
Hyd Moment(95): 6.56 G content: 4
D/E Content: 2 S/T content: 0
Score: -9.30
Gavel: prediction of cleavage sites for mitochondrial preseq
cleavage site motif not found
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 12.3%
NLS Score: -0.47
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'KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals: none
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: none
'RNA-binding motif: none
',Actinin-type actin-binding motif:
type 1: none
type 2: none
',NMYR: N-myristoylation pattern : none
',Prenylation motif: none
'memY~RL: transport motif from cell surface to Golgi: none
ITyrosines in the tail: too long tail
'Dileucine motif in the tail: found
LL at 207
',checking 63 PROSITE DNA binding motifs: none
i
(checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
IINNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: cytoplasmic
Reliability: 89
',COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9/23):
34.8 %: nuclear
21.7 %: mitochondrial
21.7 %: cytoplasmic
8.7 %: vesicles of secretory system
4.3 %: vacuolar
4.3 %: peroxisomal
4.3 %: endoplasmic reticulum
» prediction for CG53135-05 is nuc (k=23)
[0081] A search of the FGF-CXla protein against the Geneseq database, a
proprietary
database that contains sequences published in patents and patent publication,
yielded several
homologous proteins shown in Table 1D.
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Table 1D. Geneseq Results for FGF-CXla
FGF- Identities/
Geneseq Protein/Organism/Length CXla Similarities Expect
[Patent for
Identifier#, Date] Residues/the Matched Value
Match Region
Residues
ABP54435 Human fibroblast growth 1..211 211/211 (100%.)e-123
factor
(FGF) CX protein - Homo 1..211 211/211 (100%)
Sapiens,
2I1 aa. [W0200277266-A2,
03-
OCT-2002]
ABP54434 Xenopus XFGF-CX amino acid1..211 211/211 (100%)e-123
sequence SEQ m N0:24 - 1..211 211/211 (100%)
Xenopus
laevis, 211 aa. [W0200277266-A2,
03-OCT-2002]
ABP54429 Human fibroblast growth 1..211 211/211 (100%)e-123
factor
(FGF) CX protein SEQ ID 1..211 211/211 (100%)
N0:2 -
Homo sapiens, 211 aa.
[W0200277266-A2, 03-OCT-2002]
AAU75323 Human fibroblast growth 1..211 211/211 (100%)e-123
factor,
FGF-CX - Homo Sapiens, 1..211 211/211 (100%)
211 aa.
[W0200202625-A2, 10-JAN-2002]
ABB07261 Human FGF-20 polypeptide 1..211 211/211 (100%)e-123
- Homo
Sapiens, 2I1 aa. [WO200192522-A2,1..211 211/211 (I00%)
06-DEC-2001]
[0082] In a BLAST search of public sequence databases, the FGF-CXIa protein
was
found to have homology to the proteins shown in the BLASTP data in Table 1E.
Table 1E. Public BLASTP Results for FGF-CXla
Protein ~ FGF-CXla Identities/
Residues/Similarities Expect
AccessionProtein/Organism/Length for
~
Match the Matched Value
Number
~ Residues Portion
Q9NP95 ' Fibroblast growth factor-201..211 211/211 (100%)e-122
'
j (FGF-20) - Homo sapiens1..2I 211/211 (100%)
~ 1
;, (Human), 211 aa.
Q8C7A8 Fibroblast growth factor 1..211 201/211 (95%) e-117
20 - Mus ~
. ; musculus (Mouse), 1..211 204/211 (96%)
211 aa.
Q9EST9 ~ FGF-20 - Rattus norvegicus 201/211 (95%) e-117
(Rat), ' 1..211 ~
212 aa. 1..211 204/211 (96%)
Q9ESL9 ' Fibroblast growth factor1..211 200/211 (94%) e-116
20 - Mus .'
musculus (Mouse), 212 1..211 204/211 (9S%)
aa.
Q9PVY1 1..211 170/211 (80%) 5e-97
, XFGF-20
- Xenopus
laevis
~
(African clawed frog), 1..208 189/211 (89%)
208 aa. ~
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[0083] PFam analysis predicts that the FGF-CXla protein contains the domains
shown in the Table 1F.
Table 1F. Domain Analysis of FGF-CXla
Identities/
Pfam Domain ! FGF-CXla Match Region Similarities Expect Value
for the Matched Region
FGF 63..194 ~ 83/147 (56%) 7.4e-83
122/147 (83%)
5.2. Example 2: Proteolytic Cleavage Products of CG53135-05 (FGF-20)
[0084] Liquid Chromatography, Mass spectrometry and N-terminal sequencing of
CG53135-05 resulted in variants that have high activity in the proliferation
assays. Thus
these variants detailed in this section are expected to have same utility as
that of CG53135-
05.
Liauid Chromatography (LC) and Mass Spectromety (MS) analysis of CG53135-05
[0085] Purified CG53135-05 was injected onto a phenyl-hexyl column (tuna 5 mm,
250 mm X 3 mm, Phenomenex) using a standard HPLC system (Agilent 1100,
Agilent) in a
mobile phase containing acetonitrile, water and trifluoroacetic acid. The
resulting analysis
revealed detectable levels of micro-.heterogeneity, showing 1 major peak (#3)
and 3 minor
peaks (#1, 2, 4, and 5) related to CG53135-05 (Figure 1). In order to
characterize these
species, fractions were collected using an automated fraction-collector
(Agilent 1100) and
the fractions characterized by liquid chromatography electrospray ionization
ion trap mass
spectrometry (LC/ESI/MS), matrix-assisted laser desorption ionization time of
flight mass
spectrometry (MALDI-TOF-MS), and N-terminal amino acid sequencing.
[0086] Peak 0 is not a protein, is not product-related, and is not a residual
process
impurity (i.e., it is not DNA, endotoxin, kanamycin, or IPTG). The CG53135-05-
related
species (Peaks l, 2, 3, and 4) eluted in mobile phase of 36% acetonitrile, 63%
water, and
0.1% trifluoroacetic acid. Similar fractions were then pooled and the samples
concentrated
using an Amicon 10,000 dalton cut-off filter (Millipore, Bedford, MA) in a
4°C centrifuge.
The samples were diluted 16-fold in 200 mM arginine, 40 mM sodium acetate, and
3%
glycerol and then concentrated to a volume of approximately 500 ~.1. The
concentration of
the pooled fractions was determined. using amino acid analysis.
[0087] All four CG53135-05-related species (Peaks 1, 2, 3, and 4) were
proteolytically digested using trypsin and the peptides analyzed using liquid
chromatography
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with mass spectrometry detection. Mass Spectrometry was performed using a XP
DECA
nanospray/ion trap instrument (ThermoFinnigan, San Jose, CA) interfaced with
an Ultimate
Nanoflow Chromatography System (LC Packings, Amsterdam, Netherlands). Data
were
collected via Xcalibur Software (Thermofinngan) using automated MS to MS/MS
switching.
In the Instrument Method files, the XP DECA was set to acquire a full MS scan
between 400
and 1400 m/z followed by full MS/MS scans between 400 and 2000 m/z of the top
3 ions
from the preceding MS scan. Data were processed using TurboSequest
(Thermofinngan).
Database searching and protein identification was performed using MASCOT
(Matrix
Sciences, Manchester, UK). MASCOT reports a probability-based MOWSE score and
percent coverage for the identified protein based on molecular mass of the
peptides, MS/MS
sequence information, mass accuracy, and number of peptides detected. Table 2
contains the
peak number for the CG53135-05-related species, confidence score provide by
MASCOT,
and percent coverage obtained from MS/MS spectra.
Table 2: Characterization of CG53135-OS (DEV10) by MASCOT
Retention % of Total Probability-based% Coverage
Peak # Time Peak Area MOWSE score b MS/MS
0 10.13 1.12 NA NA
1 16.29 2.84 434. 46%
2 18.03 79.94 562 59%
3 20.17 13.59 708 80%
4 ~ 22.25 ~- 2.50 - ~ 428 ~ 54%
NA = data not available
[0088] The fractions collected were analyzed by MALDI-TOF and N-terminal
sequencing to determine the identity of CG53135-05-related species. N-terminal
amino.acid
sequence of purified CG53135-05 was determined qualitatively. CG53135-05
protein was
resolved by SDS-PAGE and electrophoretically transferred to a
polyvinylidenefluoride
membrane; the Coomassie-stained ~23 kDa major band was excised from the
membrane and
analyzed by an automated Edman sequencer (Procise, Applied Biosystems, Foster
City, CA).
Table 3 presents the molecular weight obtained for each species and variant
determined by N-
terminus sequencing (where N = full-length CG53135):
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Table 3: Characterization of CG53135-05 (DEV10) by MALDI-TOF and Sequence
Analysis
Peak # Molecular Weight Variant
(by MALDI-TOF) (determined by N-terminal
sequence)
0 NA NA
1 21343 N-23
N-14
22380 N-11
N8
2 23247 N-2
3 23473 N
4 23300 N-1
NA =, data not available, not a protein, not product-related
[0089] The molecular weight determined by MALDI-TOF and N-terminus
sequencing can identify the 4 species. For peak 1, 4 different species were
detected via N-
terminal sequencing, 2 of which were also detected by MALDI-TOF. These results
are also
in agreement with the coverage obtained using LC/MS. The polypeptide sequences
of each
species derived by N-terminal sequencing are given in Table 4.
Table 4: Polypeptide Sequences of the proteolytic cleavage products
N-8:
GFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL
HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
MNDKGELYGSEKLTSECIFREQFEENVVYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGAR
SKRHQKFTHFLPRPVDPERVPELYKDLLMYT (SEQ ID NO: 37)
N-11:
GGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL
HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGAR
SKRHQKFTHFLPRPVDPERVPELYKDLLMYT (SEQ ID NO: 38)
N-14:
EGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL
HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGAR
SKRHQKFTHFLPRPVDPERVPELYKDLLMYT (SEQ ID NO: 39)
N-23:
HFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL
HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGAR
SKRHQKFTHFLPRPVDPERVPELYKDLLMYT (SEQ ID NO: 40)
31
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WO 2004/100892 PCT/US2004/014623
Table 5: Clustal analysis of CG53135-05 (SEQ ID NO: 2) versus the novel
proteolytic
cleavage products
Multiple Alig~unent:
CG53135-05 1 MAPLAEVG 60
N-11 1 ________ 49
N-14 1 -------- 46
N-8 1 _______- 52
N-23 1 -------- 37
CG53135-05 61 120
N-11 50 109
N-14 47 106
N-8 53 112
N-23 38 97
CG53135-05 121 180
N-11 110 169
N-14 107 166
N-8 113 172
N-23 98 157
CG53135-05181 211
N-11 17o I zoo
N-14 167 197
N-8 173 203
N-23 158 188
Total Amino Acid Anal.~sis
[0090] The amino acid composition of CG53135-05 was determined. Samples of
CG53135-05 were hydrolyzed for 16 h at 115°C in 100 mL of 6 N HCl, 0.2%
phenol
containing 2 nmol norleucine as an internal standard. Samples were dried in a
Speed Vac
Concentrator and dissolved in 100 mL sample buffer containing 2 nmol
homoserine as an
internal standard. The amino acids in each sample were separated on a Beckman
Model 7300
ion-exchange instrument. The amino acid composition of CG53135-05 was
consistent with
the theoretical amino acid composition.
[0091] The experimental amino acid composition was used to derive the
extinction
coeffeicient used in estimation of concentration via UV absorbance (protein
estimation using
the Bradford method). The extinction coefficient at ~,max is 0.97 mL/mg~cm.
32
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WO 2004/100892 PCT/US2004/014623
Table 6: Amino Acid Analysis of CG53135-05 (DEV10)
Amino Acid Theoretical Mole PercentEx erimental Mole Percent
ala 5.69 5.69
ar 9.00 ND
asx 6.63 6.68
cys 0.95 ND
1 12.80 13.46
glx 10.90 8.67
his 3.79 4.69
ile 3.32 3.46
leu 12.32 13.18
1 s 3.32 3.55
met 1.42 0.80
he 4.74 4.93
ro 5.69 5.69
ser 6.16 4.98
thr 3.79 5.97
0.47 ND
tyr 4.26 4.55
val 4.74 5.26
A Not determined because of excess arg in the formulation;
~ Not determined because cys is destroyed in the acid hydrolysis during
analysis;
C Not determined because trp is destroyed in the acid hydrolysis during
analysis
D During acid hydrolysis asn will be converted to asp and gln to glu acid.
Therefore, asx
represents the sum of asn and asp while glx represents the sum of gln and glu.
Peptide Mapping
[0092] Purified CG53135-05 (25 mg) was denatured and reduced in urea and
dithiothreitol at 50°C and then alkylated with iodoacetate. After
lowering the concentration
of urea, the samples were treated with trypsin for 40 h at 20°C. The
resulting peptide
fragments were separated by RP-HPLC (using a C-18 column with an acetonitrile
gradient in
trifluoroacetate) to obtain a peptide map (Figure 2A and 2B). The chromatogram
in Figure
2A is consistent with the 20 peptides expected from the digestion of CG53135-
05 with
trypsin, and the chromatogram in Figure 2B reveals a single peak as expected
for the single
tryptophan residue in CG53135-05.
Bioassay
[0093] The biological activity of CG53135-05 related species collected from
the 4
peaks identified by LC and MS was measured by treatment of serum-starved
cultured N1H
3T3 murine embryonic fibroblast cells with various doses of the isolated
CG53135-05 related
33
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WO 2004/100892 PCT/US2004/014623
species and measurement of incorporation of bromodeoxyuridine (BrdU) during
DNA
synthesis. For this assay, cells were cultured in Dulbecco's modified Eagle's
medium
supplemented with 10% fetal bovine serum. Cells were grown in 96-well plates
to
confluence at 37°C in 10% COz/air and then starved in Dulbecco's
modified Eagle's medium
for 24 - 72 h. CG53135-05-related species were added and incubated for 18 h at
37°C in
10% C02lair. BrdU (10 mM final concentration) was added and incubated with the
cells for
2 h 37°C in 10% C02/air. Incorporation of BrdU was measured by enzyme-
linked
immunosorbent assay according to the manufacturer's specifications (Roche
Molecular
Biochemicals, Indianapolis, IN).
[0094] Peak 4 was not included in this assay since insufficient material was
collected
(Peak 4 is less than 3% of the total peak area for CG53135-05). CG53135-05 and
material
collected from all 3 remaining fractions (i.e., Peak l, 2, and 3) induced DNA
synthesis in
NIH 3T3 mouse fibroblasts in a dose-dependent manner (Table 7). The PI2oo was
defined as
the concentration of protein that resulted in incorporation of BrdU at 2 times
the background.
CG53135-05 and CG53135-05 related species recovered from all 3 measurable
peaks
demonstrated similar biological activity with a PI2oo of 0.7 - 11 ng/mL (Table
9).
Table 7: Biological Activity of CG53135-05 (DEV10): Induction of DNA Synthesis
PI2oo (ng/mL)CG53135-05 Peak 1 Peak 2 Peak 3
(DEV 10
- - -
-
1.0 ~0.7 ~ 11 8.6
~
5.3. Example 3: Receptor Binding Specificity of CG53135 (Study L-116.01)
[0095] FGF family members transduce signals intracellularly via high affinity
interactions with cell surface immunoglobulin (Ig) domain-containing tyrosine
kinase FGF
receptors (FGFRs). Four distinct human genes encode FGFRs (Powers et al.,
Endocr Relat
Cancer 2000, 7:165-97; Klint and Claesson-Welsh, Front Biosci 1999, 4:D165-77;
Xu et al.,
Cell Tissue Res 1999, 296:33-43). A related fifth human sequence lacking a
kinase domain
has recently been identified and named FGFR-5 (Kim et al., Biochim Biophys
Acta 2001,
1518:152-6). These receptors can each bind several different members of this
family (Kim et
al., Biochim Biophys Acta 2001, 1518:152-6; Ornitz et al., J Biol Chem 1996,
271:15292-7).
FGFs also bind, albeit with low affinity, to heparin sulfate proteoglycans
(HSPGs) present on
most cell surfaces and extracellular matrices (ECM). Interactions between FGFs
and HSPGs
serve to stabilize FGF/FGFR interactions and to sequester FGF and protect it
from
degradation (Powers et al., Endocr Relat Cancer 2000, 7:165-97; Szebenyi and
Fallon, Int
34
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WO 2004/100892 PCT/US2004/014623
Rev Cytol 1999,185:45-106). Dimerization of FGF receptor monomers upon ligand
binding
is reported to be a requisite for activation of the kinase domains, leading to
receptor trans-
phosphorylation. FGF receptor-1 (FGFR-1), which shows the broadest expression
pattern of
the four FGF receptors, contains at least seven tyrosine phosphorylation
sites. A number of
signal transduction molecules are affected by binding with different
affinities to these
phosphorylation sites.
[0096] FGFR-1, FGFR-2 and FGFR-3 each recognize FGF-l, FGF-2, FGF-4 and
FGF-8. In addition, FGFR-1 & FGFR-2 bind FGF-3, FGF-5, FGF-6, FGF-10 and FGF-
17
(Powers et al., Endocr Relat Cancer 2000, 7:165-97). Binding of various FGF
ligands varies
with each receptor splice form, thus allowing a wide repertoire of FGF-
mediated signaling
events through a limited number of receptor coding genes. Tissue-specific
alternate splicing
permits cells expressing a single FGFR gene to significantly diversify their
biological
response by generating distinct receptor isoforms that may exhibit different
ligand specificity
and function. FGFR-4, binds FGF-1, FGF-2, FGF-4, FGF-6, FGF-8 and FGF-9 but
not FGF-
3, FGF-5 or FGF-7. FGF-7, or keratinocyte growth factor-1 (KGF-1) is only
recognized by
FGFR-2, whereas FGF-9 binds to FGFR-2, FGFR-3 and FGFR-4. Receptor specificity
of
FGFs-11 to -19 is not well understood (Powers et al., Endocr Relat Cancer
2000, 7:165-97;
Ornitz et al., J Biol Chem 1996, 271:15292-7).
[0097] Immunobistochemistry studies (Hughes, J Histochem Cytochem 1997,
45:1005-19),in normal human adult tissues from the major organ systems
indicated that
FGFR-1, FGFR-2 and FGFR-3 are widely expressed, suggesting an important
functional role
in tissue homeostasis. Protein expression patterns for tissue-specific
isoforms have not yet
been determined. FGFR-4 has a more limited expression pattern being notably
absent from
lung, oviduct, placenta, testis, prostate, thyroid, parathyroid, and
sympathetic ganglia, tissues
where all three other receptors are predominantly expressed (Hughes, J
Histochem Cytochem
1997, 45:1005-19).
[0098] To determine the receptor binding specificity of CG53135, we examined
the
effect of soluble FGFRs on the induction of DNA synthesis in N1H 3T3 cells by
recombinant
CG53135-01 produced in E. coli.
Materials and Methods
[0099] Protein Purification from Escherichia coli: For production in E. coli,
plasmid pETMY-hFGF20X was transformed into the E. coli expression host BL21
(Novagen,
Madison, WI) and the induction of protein CG53135 expression was carried out
according to
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
the manufacturer's instructions. pETMYhFGF20X/BL21 E. coli bacteria were grown
in LB
medium at 37°C. At an OD of 0.6, bacteriophage lambda (CE6) was added
to a final
multiplicity of infection of 5. The infected culture was further incubated at
27°C for 3 hours.
After induction, total cells were harvested, and proteins were analyzed by
Western blotting
using anti-HisGly antibody (Invitrogen). Cells were harvested by low-speed
centrifugation
(5000 rpm in a GS-3 rotor for 15 minutes at 4°C), suspended in
phosphate-buffered saline
(PBS) containing 0.5M NaCl and 1M arginine, and disrupted with two passes
through a
microfluidizer. Cell debris was removed by low-speed centrifugation and the
soluble protein
fraction (supernatant) was clarified by filtration through a 0.2 micron low-
protein binding
membrane. The protein sample was then loaded onto a metal chelation column
(pre-charged
with nickel sulfate). The nickel column was washed with PBS/0.5M NaCl + 1M L-
arginine
and bound proteins were eluted with a linear gradient of imidazole (0-0.5 M).
Fractions
containing CG53135 (100-150 mM imidazole) were pooled and dialyzed against 1 X
106
volumes of PBS pH 8.0 containing 1M L-arginine. The protein sample was stored
at -80°C.
[00100] Receptor Specificity: NIH 3T3 cells were cultured in 96-well plates to
approximately.100% confluence, washed and fed with DMEM without supplements
(Life
Technologies), and incubated for 24 h. Recombinant CG53135-O1 or control
protein was then
added to the cells for 18 h. Control proteins used were aFGF (positive
control) and platelet
derived growth factor-BB (PDGFBB) (negative control). To analyze the effect of
soluble
FGFRs on CG53135 activity, recombinant CG53135-O1, aFGF, or PDGF-BB (final
concentrations of 10, 5 and 3 ng/mL, respectively), were mixed with soluble
receptors (final
concentrations of 0.2, 1 and 5 ug/mL), and incubated for 30 min at 37°C
prior to addition to
serum-starved NIH 3T3 cells. Factor concentrations represent the amount of
ligand needed to
generate a half maximal BrdU response in NIH 3T3 cells. Soluble FGFRs were Fc
chimeras
of the following receptor forms (FGFR1~3(IIIc); FGFR2(3(IIIb); FGFR2a(IIIb);
FGFR2oc(IIIc); FGFR3oc(IIIc); FGFR4) and were.obtained from R&D Systems
(Minneapolis,
MN). The BrdU assay was performed according to the manufacturer's
specifications (Roche
Molecular Biochemicals, Indianapolis, IN) using a 4 h BrdU incorporation time.
Results and Conclusions
[00101] To determine the receptor binding specificity of CG53135, we examined
the
effect of soluble FGFRs on the induction of DNA synthesis in NIH 3T3 cells by
recombinant
CG53135-O1 produced in E. coli. Soluble receptors for FGFR1 (3(IIIc), FGFR2(3
(IITb),
FGFR2oc(IIIb), FGFR2cc(IIIc), FGFR3a(IIIc), and FGFR4 were utilized. We found
that
36
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
soluble forms of each of these FGFRs were able to specifically inhibit the
biological activity
of CG53135 (Figure 3).
[00102] Complete or nearly complete inhibition was obtained with soluble
FGFR2oc(IIIb), FGFR2~i(IIIb), FGFR2oc (lIIc), and FGFR3oc (IIIc), whereas
partial
inhibition was achieved with soluble FGFR1 (3 (llIc) and FGFR4. None of the
soluble
receptor reagents interfered with the induction of DNA synthesis by PDGF-BB
(Figure 2),
thereby demonstrating their specificity. The integrity of each soluble
receptor reagent was
demonstrated by showing their ability to inhibit the induction of DNA
synthesis by aFGF, a
factor known to interact with all of the FGFR's under analysis (Figure 3).
5.4. Example 4: Treatment of Stroke
[00103] Thirty male Sprague Dawley rats were allocated to treatment groups as
indicated in the study design Table 8 below.
Table 8: Experimental Design
Number of Dose * Volume ~
Animals
Treatment Males L
Vehicle 1 10 0 SO
CG53135-05 10 1 50
CG53135-05 10 2.S 50
*Administered dose and volume is based on an average bodyweight of 330 g.
Experimental Procedures
[00104] Middle cerebral artery (MCA) Surgery and Intracisternal Injections:
Animals were handled for 7 days prior to surgery. Cefazolin sodium (40 mg/kg,
i.p) was
administered on the day before surgery and just after surgery. At the time of
surgery, the rats
were anesthetized with 2% halothane in a 2:1 N20 :02 mixture. Body temperature
was
maintained at 37 ~ 0.50°C. The proximal right MCA was electrocoagulated
from just
proximal to the olfactory tract to the inferior cerebral vein and was then
transected. For
intracisternal injections, animals were re-anesthetized as above and placed in
a stereotaxic
frame. Rats were given CG53135-05 or vehicle [40mM acetate, 200 mM mannitol
(pH 5.3)]
by percutaneous injection into the cisterna magna, once at 1 day,
(approximately 24 hours)
and once at 3 days, (approximately 72 hours) after MCA. Animals were given
test article (2
dose groups) or vehicle treatment according to the study design.
37
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WO 2004/100892 PCT/US2004/014623
Clinical Observations/Si~ns
[00105] Animals were observed immediately over a 1 hour period following
injections
for signs of seizure (indicated by tremor and violent motion about the cage),
pain (indicated
by loud vocalization), and lethargy. Animals were also observed daily for
mortality and
moribundity.
[00106] Body Weight: Animals were weighed on Days 1, 3, 7, 14 and 21.
[00107] ~ Limb Placing Test: limb placing tests were carned out on all animals
on Day
-1 (pre-operation), Day 1 (just prior to injection), Day 3 and then every 7
days thereafter
(Days 7, 14, 21).
[00108] Forelimb Placing Test Assessment Score: The forelimb placing test
measures sensorimotor function in each forelimb as the animal places the limb
on a table top
in response to visual, tactile, and proprioceptive stimuli. The forelimb
placing test consists of
the following evaluations and scoring, where the combined total score for the
forelimb
placing test reflects a range from 0 (no impairment) to 10 (maximal
impairment):
visual placing (forward, sideways):, 0 - 4
tactile placing (dorsal,lateral): 0 - 4
proprioceptive placing: 0 - 2
Total score for all forelimb tests: 0 - 10
[00109] Hindlimb Placing Test Assessment Score: Similarly, the hindlimb
placing
test measures sensorimotor function of the hindlimb as the animal places it on
a tabletop in
response to tactile and proprioceptive stimuli. The hindlimb placing test
consists of the
following evaluations and scoring, where the combined total score for the
hindlimb placing
test reflects a range from 0 (no impairment) to 6 (maximal impairment):
tactile placing (dorsal,lateral): 0 - 4
proprioceptive placing: 0 - 2
Total score for all hindlimb tests: 0 - 6
[00110] Body Swing Test: the body swing test was carried out on all animals on
Day
-1 (pre-operation), Day 1 (just prior to injection), Day 3 and then every 7
days thereafter
(Days 7, 14, 21).
[00111] The body swing test examines side preference as the animal is held
approximately one inch above the surface of the table, and swings to the right
or the left side.
Thirty swings axe counted, and the score is then calculated based on the
percentage of swings
38
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
to the right. (score range = ~50% right swing (no impairment) - 0% right swing
(maximal
impairment))
[00112] Cylinder Test: the cylinder test was carried out on all animals on Day
-1
(pre-operation) and 7 days thereafter (Days 7, 14, 21). The cylinder test
measures
spontaneous motor activity of the forelimbs. Animals are placed in a narrow
glass cylinder
(16.5 x 25 cm) and videotaped for 5 min on the day before stroke surgery and
at weekly
intervals thereafter. Videotapes are then scored independently by one
experienced observer
and up to 50 spontaneous movements will be counted (~ 5 min per rat per day).
Spontaneous
movements include those made by each forelimb to initiate rearing, to land on
or to move
laterally along the wall of the cylinder, or to land on the floor after
rearing.
[00113] Macroscopic and Histomorphology: on the day of scheduled termination
(Day 3), animals were euthanized by an intraperitoneal injection of Chloral
hydrate
(500mg/Kg). Brains were examined grossly and removed, postfixed in formalin,
dehydrated
and embedded in paraffin. Coronal sections (5 mm) will be cut on a microtome
mounted on
to glass slides, and stained with hematoxylin/eosin (H&E). The area of
cerebral infarcts on
each of seven slices (+4.7, +2.7, +0.7, -1.3, -3.3, -5.3, and -7.3 mm compared
with Bregma)
was determined using a computer interface imaging system using the indirect
method (area of
the intact contralateral hemisphere - area of the intact ipsilateral
hemisphere) to correct for
brain shrinkage during processing. Infarct volume was then expressed as a
percentage of the
intact contralateral hemispheric volume. Volumes of the infarction in the
cortex and striatum
were also determined separately using these same methods. H&E stained section
was
examined for histological changes such as hemorrhage, abscess or tumor
formation.
[00114] Statistical Analysis: all intracisternal injections, behavioral
testing, and
subsequent histological analyses were done by investigators blinded to the
treatment
assignment of each animal. Data are then expressed as means +/- SEM, and will
be analyzed
by one or two way (ANOVA) followed by appropriate pairwise post hoc tests with
correction
for multiple comparisons.
Results
[00115] Forelimb Placing Test: on days -1, 1, 3, 7, 14, and 21 relative to MCA
occlusion, animals were examined by using a limb placing test to assess
sensorimotor
function in the forelimb in response to visual, tactile and proprioceptive
stimuli (Kawamata,
T., Dietrich, W. D., Schallert, T., Gotts, E., Cocke, R. R., Benowitz, L. I. &
Finklestein, S. P.
(1997) Proc. Natl. Acad. Sci. USA 94, 8179-8184; De Ryck, M., Van Reempts, J.,
39
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Duytschaever, H., Van Deuren, B. & Clincke, G. (1992) Brain Res. 573, 44-60.)
Visual
placing (scored 0-4), tactile placing (scored 0-4), and proprioceptive placing
(scored 0-2)
were summed to generate a range of potential total scores from 0 to 12, with
12 representing
maximal impairment (Figure 4).
[00116] Hindlimb Placing: on days -1, 1, 3, 7, 14, and 21 relative to MCA
occlusion,
animals were examined by using a limb placing test to assess sensorimotor
function in the
hindlimb in response to tactile and proprioceptive stimuli [Kawamata, T.,
Dietrich, W. D.,
Schallert, T., Gotts, E., Cocke, R. R., Benowitz, L. I. & Finklestein, S. P.
(1997) Proc. Natl.
Acad. Sci. USA 94, 8179-8184; De Ryck, M., Van Reempts, J., Duytschaever, H.,
Van
Deuren, B. & Clincke, G. (1992) Brain Res. 573, 44-60]. Tactile placing
(scored 0-4), and
proprioceptive placing (scored 0-2) were summed to generate a range of
potential total scores
from 0 to 6, with 6 representing maximal impairment (Figure 5).
[00117] Body Swing Test: On days -1, 1, 3, 7, 14, and 21 relative to MCA
occlusion,
animals were examined by using a body swing test to assess side preference as
the animal is
held approximately one inch above the surface of the table, and swings to the
right or the left
side. (Kawamata, T., Dietrich, W. D., Schallert, T., Gotts, E., Cocke, R. R.,
Benowitz, L. I. &
Finklestein, S. P. (1997) Proc. Natl. Acad. Sci. USA 94, 8179-8184; De Ryck,
M., Van
Reempts, J., Duytschaever, H., Van Deuren, B. & Clincke, G. (1992) Brain Res.
573, 44-
60.) Thirty swings were counted, and the score calculated based on the
percentage of swings
to the right (Figure 6).
[00118] Cylinder Test: On days -l, 1, 3, 7, 14, and 21 relative to MCA
occlusion,
animals were examined by cylinder test to assess spontaneous motor activity of
the forelimbs
(Kawamata, T., Dietrich, W. D., Schallert, T., Gotts, E., Cocke, R. R.,
Benowitz, L. I. &
Finklestein, S. P. (1997) Proc. Natl. Acad. Sci. USA 94, 8179-8184; De Ryck,
M., Van
Reempts, J., Duytschaever, H., Van Deuren, B. & Clincke, G. (1992) Brain Res.
573, 44-
60.) Briefly, animals are placed in a narrow glass cylinder (16.5 x 25 cm) and
videotaped for
min on the day before stroke surgery and at weekly intervals thereafter.
Videotapes are
then scored independently by one experienced observer and up to 50 spontaneous
movements
will be counted (~ 5 min per rat per day). Spontaneous movements include those
made by
each forelimb to initiate rearing, to land on or to move laterally along the
wall of the cylinder,
or to land on the floor after rearing (Figure 7).
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
[00119] Body Weight: animals were weighed on days -l, 1, 3, 7, 14, and 21
relative
to MCA occlusion and the results indicate no significant difference between
the vehicle and
CG53135-05 treatment (Figure 8).
Conclusion
[00120] Administering CG53135-05 following MCA occlusion suggested that both
the
low and high doses produced a significant enhancement of recovery~on forelimb
(Figure 4)
and hindlimb placing tests (Figure 5) for the contralateral (affected) limbs,
and improvement
on the body swing test (Figure 6). This pattern of activity with other
therapeutics in this
model has generally been shown to reflect improvement in cerebrocortical and
subcortical
(striatal) function, respectively (Dijkhuizen RM, Ren J, Mandeville JB, Wu O,
Ozdag FM,
Moskowitz MA, Rosen BR, Finklestein SP. 2001, Proc Natl Acad Sci U S A
98(22):12766-
71). No apparent differences were seen on the cylinder test (Figure 7) of
spontaneous limb
use or on animal body weight (Figure 8).
[00121] Therefore, CG53135-05 administration will be useful in the treatment
of
pathological conditions including ischemic stroke, hemorrhagic stroke, trauma,
spinal cord
damage, heavy metal or toxin poisoning and neurodegenerative diseases (such as
Alzheimer's, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Huntington's
Disease).
5.5. Example 5: Matrix Metallo~roteinase Production Assay
[00122] The matrix metalloproteinases (MMPs) are a family of related enzymes
that
degrade the extracellular matrix in bone and cartilage. These enzymes operate
during normal
development in tissues differentiation and remodeling. In arthritic diseases,
such as
Osteoarthritis (OA) and Rheumatoid Arthritis (RA), elevated expression of
these enzymes
contribute to irreversible matrix degradation. Thus, effect of CG53135-05 on
MMP
production was assayed.
[00123] The activity of CG53135 on matrix metalloproteinase (MMP) production
was
assessed using the SW1353 chondrosarcoma cell line (ATCC HTB-94). This cell
line is a
well-established chondrocytic cellular model for matrix metalloproteinases
(MMP)
production. SW1353 cells were plated in a 24-well plate at 1 x105 cells/ml (1
ml) in DMEM
medium-10 % FBS. Following overnight incubation, the medium was replaced with
DMEM
+ 0.2 % Lactabulmin serum. CG53135-05 was added to the wells at doses ranging
from 10 to
5000 ng/ml, in the absence or presence of IL-1 beta (0.1 to 1 ng/ml, R&D
systems
Minneapolis, MN), TNF-alpha (10 ng/ml, R&D systems) or vehicle control to a
final volume
of 0.5 ml. IL-1 beta and TNF-alpha are both potent stimulators of MMP
activity. All
41
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treatments were done in triplicate wells. After 24 h, the supernatants were
collected and Pro-
MMP-l, and -13, as well as TIMP-I (tissue inhibitor of matrix
metalloproteinase), a natural
inhibitor of MMP activity, was measured by ELISA (R&D systems). The
measurements were
normalized to the number of cells by an MTS assay.
Results
[00124] CG53135-05 significantly decreased MMP-13 production in the presence
of
either IL-1 beta or TNF-alpha as demonstrated in Figure 14 and Figurel5
respectively. IL-1
beta and TNF-alpha are both potent stimulators of MMP activity. MMP-13
affinity for type II
collagen, the main collagen that is degraded in OA, is ten times higher that
of MMP-1. Since
MMP-13 expression increases in OA and RA, the decrease of MMP-13 observed with
addition of CG53135-05 indicates that the protein can be used as an OA and RA
therapeutic
(Figure 10). Furthermore, CG53135-05 up-regulated the production of TIMP-1, a
natural
inhibitor of MMP activity (Figure 11). This enhancement of TIMP-1 production
by
CG53I35-05 is beneficial in reducing the matrix breakdown by MMP-1 and -13
observed in
OA and RA. In addition, CG53135-05 had no effect on MMP-3 production
constitutively or
after IL-1 induction (data not shown.). Similarly, CG53135-05 (FGF-20) showed
increase in
basal expression of MMP-1 in SW1353 cells (data not shown).
5.6. Example 6: Effect of CG53135-05 on Normal Rats: Proof of Principle to the
Meniscal
Tear Model
[00125] The effect of CG53135-05 on the normal rats was studied as a proof of
principle to drive further studies in disease model (ex: rneniscal tear model
of osteoarthritis in
rats). The effect of CG53135-05 on synovium and cartilage was assessed by
injecting the
protein into normal male Lewis rats.
Effects of Intra-articular imiection of CG53135-05 in Normal Rats
[00126] The rats were injected intra-articularly three times per week for 2
weeks with
vehicle solution (8mM acetate, 40 mM arginine, and 0.6% glycerol (pH 5.3) in
approximately
1% hyaluronic acid), 10 ~g CG 53135-05 or 100 ~g CG 53135-05.
[00127] Study Design: Male Lewis rats weighing 293-325 grams on day 0 were
obtained from Harlan Sprague Dawley (Indianapolis, Indiana) and acclimated for
8 days.
The rats were divided into three treatment groups with three animals in each
group: two
groups received CG53135 and one received only the vehicle control. The rats
were
anesthetized with Isoflurane and injected through the patellar tendon into the
area of the
cruciate attachments of both knees. CG53135 was injected at doses of 0.1 mglml
(0.01
42~
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mg/joint) or 1.0 mg/ml (0.1 mg/joint). Controls were injected with the vehicle
solution as
described above. Injections were done Monday, Wednesday and Friday for 2
weeks. The
animals were terminated on day 15 at which time they were injected ip with
BRDU (100
mg/kg) in order to pulse label proliferating cells.
Observations and Analysis of Markers of Pathology
[00128] Gross observations Rats were observed daily for abnormal swelling or
gait
alterations and were weighed weekly.
[00129] Histopathology Preserved and decalcified (5% formic acid) knees were
trimmed into 2 approximately equal longitudinal (ankles) or frontal (knees)
halves, processed
through graded alcohols and a clearing agent, infiltrated and embedded in
paraffin, sectioned,
and stained with toluidine blue (knees). Multiple sections (3 levels) of right
knee were
analyzed microscopically with attention to the parameters of interest listed
below. Each
parameter was graded as normal, minimal, mild, moderate, marked or severe.
Evaluation of
the cartilage was done using descriptive parameters rather than the scoring
criteria generally
used in the osteoarthritis model because of the type of alterations generated
by the repetitive
injection of the protein. Although animals were injected with BRDU prior to
termination, the
proliferative changes were readily observed in toluidine blue stained
sections.
RESULTS
Table 9: Microscopically Monitored Parameters
Synovial AlterationsCartilage Central CruciateChondrogenesis
AlterationsAttachment Area
Alterations
-hyperplasia -cartilage -inflammation -marginal zone
and or
-infiltration of proteoglycanfibroplasia periosteal
synovium
with macrophages loss -bone or cartilagechondrogenesis
-fibroplasia -cartilage damage
-matrix (proteoglycanfibrillation
deposition in fibrotic
synovium)
[00130] Live Phase Parameters Body weights were similar in vehicle and protein
injected animals throughout the study (Table 12). Knees injected with 100 ~g
of protein had
some evidence of fibrosis clinically during the injection process beginning
with the 3rd
injection.
[00131] Morphologic Pathology Vehicle injected rats had minimal to mild
synovial
hyperplasia, inflammation and fibroplasia with none to minimal matrix
deposition in fibrotic
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synovium. Articular cartilage had no proteoglycan loss or fibrillation. The
central area of the
joint where the cruciates attach and in which the infra-articular injections
are made had none
to minimal fibroplasia and cartilage/bone damage. No marginal zone
chondrogenesis Was
present.
[00132] Knees injected with 10 ~g CG 53135-05 had mild to moderate synovial
hyperplasia, inflammation and fibroplasia with minimal to moderate matrix
deposition in
fibrotic synovium. Articular cartilage had no proteoglycan loss or
fibrillation. The central
area of the joint where the cruciates attach and in which the infra-articular
injections are made
had none to minimal fibroplasia and cartilage/bone damage. One knee had
minimal marginal
zone chondrogenesis.
[00133] Knees injected with 100 ~g CG 53135-05 had moderate to marked synovial
hyperplasia, inflammation and fibroplasia with moderate matrix deposition in
fibrotic
synovium. Articular cartilage had none to minimal proteoglycan loss or
fibrillation. The
central area of the joint where the cruciates attach and in which the infra-
articular injections
are made had minimal to marked fibroplasia and cartilage/bone damage. All
knees had mild
to moderate marginal zone chondrogenesis. One animal had chondrogenesis in
areas
associated with articular cartilage.
Conclusion
[00134] These results demonstrate that repetitive infra-articular injection of
CG53135-
05 induces synovial fibroplasia and chondrogenesis. Vehicle injections
resulted in mild
inflammation and fibroplasia thus suggesting that this vehicle has some
irritant potential.
Concentration responsive increases in synovial proliferative response as well
as marginal
zone chondrogenesis occurred in animals injected with protein. The area of the
cruciate
attachment where injections occurred had areas of bone resorption and
fibroplasia which also
increased in severity with increasing concentrations of the protein as did the
synovial
inflanunation. The potentially adverse effects of observed synovial
fibroplasia and bone
resorption could have been due to either FGF-20 activity or endotoxin levels
within the non-
clinical grade hyaluronic acid used to formulate the protein. In addition,
inflammation in the
joint can induce bone resorption and marginal zone chondrogenesis so these
results need to
be interpreted in light of the possibility that the inflammatory response to
the protein injection
contributed to the proliferative response. The morphologic appearance of the
proliferative
changes and chondrogenesis clearly indicates that the biological activity of
this protein
(CG53135-05) is important in generating the response.
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[00135] The results of the experiments reported herein indicate repetitive
intra-
articular injection of CG53135-05 induces synovial fibroplasia and
chondrogenesis.
5.7. Example 7: Intra-axticular infection of CG53135-05 in Meniscal Tear model
of Rat
Osteoarthritis: Prophylactic and Therapeutic Dosing
[00136] Example 6 utilized CG53135-05 administration into the joints of normal
rats
to identify effects on relevant cell populations by histomorphometric
analysis. At the dose of
100 ug/joint, CG53135-05 induced significant marginal zone chondrogenesis
similar to that
seen with other growth factors such as TGF-beta, suggesting an effect on
pluripotent stem
cells within the marginal zone. There was no apparent effect on mature
chondrocytes as
evidenced by the lack of a response in the mature cartilage areas of the
joints. The potentially
adverse effects of observed synovial fibroplasia and bone resorption could
have been due to
either FGF-20 activity or endotoxin levels within the non-clinical grade
hyaluronic acid used
to formulate the protein.
[00137] Further studies in osteoarthritic animals performe addressed the
following: 1)
synergy with an anti-inflammatory drug (standard approach for osteoarthritis
patients), 2)
whether CG53135-05 (FGF-20) can induce functional repair or protection of
joint cartilage
layers, and 3) whether synovial fibroplasia and bone resorption were FGF-20-
induced or due
to contaminating endotoxin within the formulation.
[00138] Thus one aspect of this study was to evaluate the protective and
therapeutic
effects of intra-articular injection of CG53135-05 on joint damage in
osteoarthritis in the
meniscal tear model of rat osteoarthritis. This relatively new model of OA has
been shown to
have morphologic alterations of cartilage degeneration and osteophyte
formation that
resemble changes occurring in spontaneous disease and surgically induced
disease in other
species (Bendele, A.M., Animal Models of Osteoarthritis. J. Musculoskel.
Neuron Interact.
2001; 1:363-376, Bendele, A. M. and Hulman, J. F. Spontaneous cartilage
degeneration in
guinea pigs. Arthritis Rheum. 1988; 31:561-565). The model can be used to
evaluate
potential beneficial effects of anti-degenerative as well as regenerative
therapies.
Experimental Design
[00139] Animals (10/group), housed 2/cage, were anesthetized with Isoflurane
and the
right knee area is prepared for surgery. A skin incision was made over the
medial aspect of
the knee and the medial collateral ligament was exposed by blunt dissection,
and then
transected. The medial meniscus was then reflected medially with a fine
scissor and a cut was
made through the full thickness to simulate a complete tear. The skin was
closed with suture.
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[00140] Prophylactic Dosing: intra-articular dosing (CG53135-05) of the right
knee
joint was initiated on the day of surgery and is continued for 2 weeks post-
surgery with intra-
articular injections given Thursday, Saturday, and Monday (day 0, 2, 4, 7, 9,
and 11) with rats
under Isoflurane anesthesia. Indomethacin, a nonsteroidal anti-inflammatory
drug, was dosed
(1mg/kglday) daily by the oral route starting on the day of surgery to reduce
any potential
inflammation due to the injection. Body weights were recorded on days 0, 7 and
14. After
animal termination on day 14 post-surgery, both knees were collected for
histopathologic
evaluation. The study design is shown in Table 10.
Table 10. Prophylactic Dosing Study Design
CG53135-05 Co-therapy Number of
Group Treatments Treatmentb Animals
Males
Vehicle Vehicle 10
1 (intra-articular)
2 Vehicle Indomethacin 10
(intra-articular)
3 CG53135-05 Vehicle 10
(intra-articular)
4 CG53135-05 Indomethacin 10
(intra-articular)
None None 10
sAdministration 3 times per week for 2 weeks (100 ~g/joint, intra-articular)
bAdministration daily for 2 weeks (0.5 mg/kg, PO)
[00141] Therapeutic Dosing: intra-articular dosing (CG53135-05) of the right
knee
joint is initiated on day 21 of post-surgery and is continued for 2 weeks with
intra-articular
injections given Friday, Sunday, and Tuesday (day 22, 25, 27, 29, 32, and 34)
with rats under
Isoflurane anesthesia. Indomethacin is dosed daily by the oral route starting
on the day of
surgery. Body weights are recorded on days 0, 7, 14, 21, 28, and 35. On day
35, both knees
are collected for histopathologic evaluation. The study design is shown in
Table 11.
Table 11. Therapeutic Dosing Study Design
CG53135-05 Co-therapy Number of
Group Treatments Treatmentb Animals
F Males
1 Vehicle Vehicle 10
(intra-articular)
2 Vehicle Indomethacin 10
(intra-articular)
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3 CG53135-05 Vehicle 10
(infra-articular)
4 CG53135-05 Indomethacin 10
(infra-articular)
None None 10
aAdministration 3 times per week for 2 weelcs (100 p.g/joint, infra-articular)
bAdministration daily far 2 weeks (0.5 mg/kg, PO)
[00142] Results of prophylactic dosing study: Observations made include the
standards followed for this model. Multiple sections (3 levels) of right knee
were analyzed
microscopically and scored according to the following methods. In scoring the
3 sections, the
worst case scenario for the 2 halves on each of the 3 slides representing 3
levels was
determined for cartilage degeneration and osteophyte formation. This value for
each
parameter for each slide was then averaged to determine overall subjective
cartilage
degeneration scores for tibia and femur and osteophyte scores for tibia.
[00143] Cartilage degeneration was scored none to severe (numerical values 0-
5) for
depth and area (surface divided into thirds) using the following criteria:
0=no degeneration
1=minimal degeneration, chondracyte and proteoglycan loss with or without
fibrillation
involving the superficial zone
2= mild degeneration, chondrocyte and proteoglycan loss with ar without
fibrillation
involving the upper 1/3
3=moderate degeneration, chondrocyte and proteoglycan loss with fibrillation
extending well
into the midzone and generally affecting 1/2 of the total cartilage thickness
4=marked degeneration, chondrocyte and proteoglycan loss with fibrillation
extending well
into the deep zone but without complete (to the tidemark) lass of matrix
5=severe degeneration, matrix loss to tidemark
[00144] Strict attention to zones (outside, middle, inside thirds) was adhered
to in this
scoring method and the summed scores reflect a global summation of severity of
tibial
degeneration.
[00145] In addition to this overall subjective analysis of cartilage
degeneration, an
additional subjective assessment was done using similar criteria to evaluate
severity of
degeneration but with attention to specific regional differences across the
tibial plateau. In
this OA model, generally the outside 1/3 of the tibia is most severely
affected by the meniscal
47
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tear injury with lesions often extending to the tidemark by 3 weeks post-
surgery. The middle
1l3 is usually a transition zone where severe or marked change becomes
moderate or mild
and the inner 1/3 seldom has changes greater than mild or minimal. In an
attempt to
determine potential differences of treatment on the severe lesion of the
outside 1/3 vs. the
milder lesions of the middle 1/3 and inside 1/3, these regions were each
scored separately.
The sum of the regional values was calculated and expressed as sum of 3 zones.
[00146] In addition to the above subjective scoring, a micrometer measurement
of total
extent of tibial plateau affected by any severity of degeneration (Total
Tibial Cartilage
Degeneration Width Vim) extended from the origination of the osteophyte or
marginal zone if
no osteophyte was present with adjacent cartilage degeneration (outside 1/3)
across the
surface to the point where tangential layer and underlying cartilage appeared
histologically
normal.
[00147] An additional measurement (Significant Cartilage Degeneration Width
pm)
reflected areas of tibial cartilage degeneration in which chondrocyte and
matrix loss extended
through greater than 50% of the cartilage thickness.
[00148] Finally, a micrometer depth of any type of lesion (cell/proteoglycan
loss,
change in metachromasia, but may have good retention of collagenous matrix and
no
fibrillation) expressed as a ratio of depth of changed area vs. depth to
tidemark was included
and taken over 4 equally spaced points on the tibial surface. These
measurements were taken
(1st) matrix adjacent to osteophyte (2nd) 1/4 of the distance across the
tibial plateau (3rd) 1/2
of the distance across the tibial plateau (4th) 3/4 of the distance across the
tibial plateau. This
measurement was the most critical analysis of any type of microscopic change
present. The
depth to tidemark measurement (denominator) also gives an indication of
cartilage thickness
across the tibial plateau and therefore allows comparisons across groups when
trying to
determine if hypertrophy or hyperplasia has occurred.
[00149] A single tibial growth plate measurement was taken for each section in
an area
thought to best represent the overall width in the non tangential plane of the
section.
[00150] Scoring of the osteophytes and categorization into small, medium and
large
was done with an ocular micrometer.
None = 0 no measurable proliferative response at marginal zone
Small osteophytes =1 (up to 299 pm)
Medium osteophytes=2 (300-399 Vim)
Large osteophytes=3 (>400~m)
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[00151] The score (0-3) was included in the overall joint score. In addition,
the
mean~SE for the actual osteophyte measurement (average for 3 sections) was
also
determined.
[00152] Generally, in doing the surgery, attempts were made to transect the
collateral
ligament at a location that results in the meniscus being reflected proximally
toward the
femur. The cut was then made by inserting the scissors tip toward the femur
rather than the
tibia. Some mechanical damage may then be detected in the femoral condylar
cartilage but is
rarely encountered on the tibia, thus making the tibia the most appropriate
site for assessment
of chondroprotection.
[00153] Focal small areas .of proteoglycan and cell loss that were likely a
result of
physical trauma to the femoral cartilage were described but not included in
the score with
larger more diffuse areas receiving subjective scores according to methods
described for the
tibia. These larger areas were more consistent with non traumatic
degeneration. Because of
the possibility of iatrogenic lesions on the femur, overall joint scores were
expressed both
with and without femoral cartilage degeneration scores.
[00154] Damage to the calcified cartilage layer and subchondral bone was
scored using
the following criteria:
0=No changes
1=Increased basophilic at tidemark: no fragmentation of tidemark or marrow
changes
2=Increased basophilic at tidemark: minimal to mild fragmentation of calcified
cartilage of
tidemark, mesenchymal change in marrow involves 1/4 of total area but
generally is restricted
to subchondral region under lesion
3=Increased basophilic at tidemark: Mild to marked fragmentation of calcified
cartilage,
Mesenchymal change in marrow is up to 3/4 of total area, Areas of marrow
chondrogenesis
may be evident but no collapse of articular cartilage into epiphyseal bone
4=Increased basophilic at tidemark: Marked to severe fragmentation of
calcified cartilage,
Marrow mesenchymal change involves up to 3/4 of area and articular cartilage
has collapsed
into the epiphysis to a depth of 250pm or less from tidemark
5=Increased basophilic at tidemark: Marked to severe fragmentation of
calcified cartilage,
Marrow mesenchymal change involves up to 3/4 of area and Articular cartilage
has collapsed
into the epiphysis to a depth of greater than 250~m from tidemark
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[00155] Descriptive comments were made on degree of synovial inflammation,
synovial fibrosis, marginal zone chondrogenesis, bone resorption, fibrous
overgrowth with or
without chondrogenesis/incorcoration into existing cartilage
[00156] Statistical Analysis: statistical analysis of histopathologic
parameters was
done by comparing group means using the Student's two-tailed t-test with
significance set at
p<0.05. Because of the nature of the data, a non Parametric ANOVA (Kruskal-
Wallis test)
was used to analyze the scored parameters and a parametric ANOVA was used to
analyze the
measurements. The appropriate post test used was Dunnett's multiple
comparisons test on the
parametric data and a Dunn's test was used on the non parametric data.
Significance was set
at p<_0.05'for all parameters.
[00157] Results: intra-articular injection of 100 ~g CG53135-05 with or
without
concurrent indomethacin administration resulted in significant inhibition
(39%) of tibial
cartilage degeneration on the middle 1/3 (40-43% for zone 1) and an overall
insignificant
inhibition of the summed 3 zones of 41 % (Figure 12). Total cartilage
degeneration width was
significantly decreased 35-37% (Figure 13) and significant degeneration was
reduced 70-89%
with this inhibition being significant only in the group treated with protein
and indomethacin
(Figure 14).
[00158] Results of the prophylactic dosing study: the data described indicate
that
intra-articular injection of 100 ~g of CG53135-05 in knee joints of rats with
medial meniscal .
tear results in chondroprotective effects as a result of both inhibition of
cartilage degeneration
and stimulation of cartilage repair. Some joints had layering of proliferated
new cartilage
over existing normal appearing or damaged cartilage. This observation is
particularly exciting
as it demonstrates the potential for resurfacing to occur.
[00159) These beneficial effects were always associated with diffuse synovial
fibroplasia, bone resorption and increased synovial inflammation. Concurrent
indomethacin
treatment (1 mglkg/day) had minimal if any effect on the disease process in
knees injected
with Synvisc alone or the disease process and reaction to the protein in knees
injected with
Synvisc containing protein. The single exception to this statement is
reflected in the data for
osteophyte,measurements where all groups had similar measurements except the
group
treated with protein and vehicle po. This group had greater measurements thus
suggesting
greater marginal zone stimulation, not an uncommon occurrence in inflamed
joints.
[00160] The morphologic changes induced by injection of 100 ~g of this protein
demonstrate the potential for CG53135-05 to be effective in cartilage repair
processes. It has
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
the capacity to induce fibrous tissue proliferation with differentiation to
cartilage and
importantly, integration of that newly proliferated tissue. The proliferative
processes are
somewhat disorganized and counter productive in areas such as the marginal
zone and
subchondral bone. However, rodents definitely have much greater propensity to
exhibit
marginal zone, periosteal and marrow proliferation from a variety of stimuli
including
inflammatory mediators so some of the excessive and counter productive
responses seen in
rats might not occur in dogs or primates. Also, there may have been some
induction of an
antibody response thus leading to enhanced knee inflammation that would not
occur in
humans or other animals that did not have an antibody response.
[00161] Additional studies that are useful in delineating the potential
efficacy of
CG53135-05 in osteoarthritis include:
[00162] 1. Evaluation in a dog model of OA-this would allow evaluation in a
larger joint with cartilage and bone structure that is more similar to humans
and this is a
species that has less tendency to exhibit hyperproliferative responses such as
those that occur
in rodents.
[00163] 2. Evaluation of is injections for 3-4 weeks, possibly with more
aggressive anti-inflammatory systemic therapy followed by a recovery period to
see how the
new tissue remodels would be interesting. It may be that allowing the joint to
remodel with
no further proliferative stimulus would result in a more pleasing morphologic
endpoint.
Cycles of treatment with periods of remodeling might be the way to achieve the
most
satisfactory repair. Studies such as these would also answer the question of
whether the repair
tissue will hold up long term. Generally fibrocartilage has less of a tendency
to do this.
[00164] Results: Intra-articular injection of 100 ~g CG53135-05 with or
without
concurrent oral indomethacin administration did not result in significant
inhibition of tibial
cartilage degeneration scores (Figure 15). Total or significant cartilage
degeneration width
was not decreased (Figures 16, 17).
[00165] Results of the therapeutic dosing study: The data described
demonstrated
the potential chondroproliferative activities of CG53135-05 administered intra-
articularly.
However, protein injected joints had markedly increased inflammation,
fibroplasia and
connective tissue resorptive process.
[00166] The most important difference between the prophylactic and therapeutic
dosing studies was the nature of the OA lesion at the time of initiation of
dosing. Rats in the
therapeutic dosing study had an area of severe matrix loss in the outer to
middle 1/3 of the
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cartilage thus exposing the calcified cartilage/subchondral bone to the
protein. Effective
repair thus required filling of this defect with newly proliferated tissue
coming from the
marginal zone or exposed marrow pleuripotential cells. In the prophylactic
dosing study,
beneficial effects required inhibition of matrix degradation and stimulation
of repair on a
degenerating scaffold with repair tissue originating from the marginal zone
only. Since the
filling of a defect would be much more difficult than repairing a damaged
scaffold, it may be
that a longer duration of treatment would be required in a therapeutic model
in order to see
beneficial effects.
[00167] Indomethacin treatment was not effective in reducing the inflammatory
changes and it had no beneficial effects on inhibiting the resorptive
processes occurring in
bone. In order to achieve effective proliferation and differentiation to
cartilage in the absence
of inflammation and tissue destruction, following modification to the
therapeutic dosing
study can be attempted: Increasing the dosing interval to once or twice weekly
and/or
increasing the study duration to allow time for the proliferative tissue to
fill the large cartilage
defects induced by this disease process. Another possibility is to investigate
the effects of
CG53135-05 in a larger species such as the dog as dogs have less of a tendency
to proliferate
connective tissue and resorb bone in response to various stimuli than rodents.
[00168] The results detailed herein (both prophylactic and therapeutic dosing
studies)
indicate that CG53135-05 has specific utility in severely osteoarthritic
joints that are destined
for joint replacement. These types of agents would be injected into joints
that have little or no
normal cartilage remaining and are in need of resurfacing. In this situation,
repair could
originate from pleuripotential cells in the marginal zones or bone marrow.
Repair originating
from these locations will likely result in production of fibrocartilage rather
than hyaline
cartilage. However, some cartilage would be preferable to no cartilage and it
may be that an
injectable method of sustaining a cartilage surface would be acceptable even
though
treatments would likely have to be repeated over time to sustain the repair.
Treatments with
injectable anabolic agents will likely require some kind of cyclical process
in conjunction
with continuous passive motion rather than sustained active load bearing
motion.
6. EQUTVALENCE AND REFERENCE CITED
[00169] All references cited herein are incorporated herein by reference in
their
entirety and for all purposes to the same extent as if each individual
publication or patent or
patent application was specifically and individually indicated to be
incorporated by reference
in its entirety for all purposes.
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[00170] Many modifications and variations of this invention can be made
without
departing from its spirit and scope, as will be apparent to those skilled in
the art. The specific
embodiments described herein are offered by way of example only. We therefore
do not
wish to be limited to the precis terms set forth, but desire to avail
ourselves of such changes
and alterations that may be made for adapting the invention to various usages
and conditions.
Such alterations and changes may include, but not limited to, different
compositions for the
administration of the polypeptides according to the present invention to a
subject; different
amounts of the polypeptide; different times and means of administration;
different materials
contained in the administration dose including, for example, combinations of
different
peptides, or combinations of peptides with different biologically active
compounds. Such
changes and alterations also are intended to include modifications in the
amino acid sequence
of the specific polypeptides described herein in which such changes alter the
sequence in a
manner as not to change the functionality of the polypeptide, but as to change
solubility of
the peptide in the composition to be administered to a subject, absorption of
the peptide by
the body, protection of the polypeptide for either shelf life or within the
body until such time
as the biological action of the peptide is able to bring about the desired
effect, and such
similar modifications. Accordingly, such changes and alterations are properly
intended to be
within the full range of equivalents of the present invention.
53
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 57oA Sequence Listing PCT
<110> CuraGen Corporation
<120> NOVEL FIBROBLAST GROWTH FACTORS AND METHODS OF USE THEREOF
<130> Cura 57oA
<140> *'"'Enter Current Patent Application ID*'~'r
<141> ***Enter Current filing date as 2001-03-25'~'~*
<160> 40
<170> CuraSeqList version 0.1
<210> 1
<211> 636
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (1)..(633)
<400> 1
atg get ceg ctg get gaa gtt ggt ggt tte etg ggc ggt ctg gag ggt 48
Met Ala Pro Leu Ala Glu Val Gly Gly Phe Leu Gly Gly Leu Glu Gly
1 5 10 15
ctg ggt cag cag gtt ggt tct cac ttc ctg ctg ccg ccg get ggt gaa 96
Leu Gly Gln Gln Val Gly Ser His Phe Leu Leu Pro Pro Ala Gly Glu
20 25 30
egt ccg cea etg ctg ggt gaa cgt ege tec gca get gaa cgc tcc get 144
Arg Pro Pro Leu Leu Gly Glu Arg Arg Ser Ala Ala Glu Arg Ser Ala
35 40 45
cgt ggt ggc ecg ggt get get cag ctg get cae etg cat ggt ate ctg 192
Arg Gly Gly Pro Gly Ala Ala Gln Leu Ala His Leu His Gly Ile Leu
50 55 60
cgt cgc cgt cag ctg tac tgc cgt act ggt ttc cac ctg cag atc ctg 240
Arg Arg Arg Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Gln Il.e Leu
65 70 75 80
ccg gat ggt tct gtt cag ggt acc cgt cag gac cac tct ctg ttc ggt 288
Pro Asp Gly Ser Val Gln Gly Thr Arg Gln Asp His Ser Leu Phe Gly
85 90 95
atc ctg gaa ttc ate tet gtt get gtt ggt etg gtt tet ate cgt ggt 336
Ile Leu Glu Phe Ile Ser Val Ala Val Gly Leu Val Ser Ile Arg Gly
100 105 110
gtt gac tct ggc ctg tac ctg ggt atg aac gac aaa ggc gaa ctg tac 384
Val Asp Ser Gly Leu Tyr Leu Gly Met Asn Asp Lys G1y Glu Leu Tyr
115 120 125
ggt tct gaa aaa ctg acc tct gaa tgc atc ttc cgt gaa cag ttt gaa 432
Gly Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Arg Glu Gln Phe Glu
130 135 140
gag aac tgg tac aac acc tac tct tcc aac atc tac aaa cat ggt gac 480
Glu Asn Trp Tyr Asn Thr Tyr 5er Ser Asn Ile Tyr Lys His Gly Asp
145 150 155 160
acc gge cgt ege tae ttc gtt get etg aac aaa gac ggt acc ecg cgt 528
Thr Gl.y Arg Arg~Tyr Phe Val Ala Leu Asn Lys Asp G1y Thr Pro Arg
165 170 175
Page 1
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 570A Sequence Listing PCT
gat ggt get cgt tct aaa cgt cac cag aaa ttc acc cac ttc ctg ccg 576
Asp Gly Ala Arg Ser Lys Arg His Gln Lys Phe Thr His Phe Leu Pro
180 18 5' 190
cgc cca gtt gac ccg gag cgt gtt cca gaa ctg tat aaa gac ctg ctg 624
Arg Pro Val Asp Pro Glu Arg Val Pro Glu Leu Tyr Lys Asp Leu Leu
195 200' 205
atg tac acc taa 636
Met Tyr Thr
210
<210> 2
<211> 211
<212> PRT
<213> Homo Sapiens
<400> 2
Met Ala Pro Leu Ala Glu Val Gly Gly Phe Leu Gly Gly Leu Glu Gly
1 5 10 ~ 15
Leu Gly Gln Gln Val Gly Ser His Phe Leu Leu Pro Pro Ala Gly Glu
20 25 30
Arg Pro Pro Leu Leu Gly Glu Arg Arg Ser Ala Ala Glu Arg Ser Ala
35 40 45
Arg Gly Gly Pro Gly Ala Ala Gln Leu Ala His Leu His Gly Ile Leu
50 55 60
Arg Arg Arg Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Gln Ile Leu
65 70 75 80
Pro Asp Gly Ser Val Gln Gly Thr Arg Gln Asp His Ser Leu Phe Gly
.85 90 95
Ile Leu Glu Phe Ile Ser Val Ala Val Gly Leu Val Ser Ile Arg Gly
100 105 110
Val Asp Ser Gly Leu Tyr Leu Gly Met Asn Asp Lys Gly Glu Leu Tyr
115 120 125
Gly Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Arg Glu Gln Phe Glu
130 135 140
Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile Tyr Lys His.Gly Asp
145 150 155 160
Thr Gly Arg Arg Tyr Phe Val Ala Leu Asn Lys Asp Gly Thr Pro Arg
165 170 175
Asp Gly Ala Arg Ser Lys Arg His Gln Lys Phe Thr His Phe Leu Pro
180 185 190
Arg Pro Val Asp Pro Glu Arg Val Pro Glu Leu Tyr Lys Asp Leu Leu
195 200. 205
Met Tyr Thr
210
<210> 3
<211> 633
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (1) . . (633)
Page 2
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 57oA sequence Listing PCT
<400>
3'
atg getcccttagcc gaagtcggg ggctttctgggc ggcctg gagggc 48
Met AlaProLeuAla GluValGly GlyPheLeuGly GlyLeu GluGly
1 5 10 15
ttg ggccagcaggtg ggttcgcat ttcctgttgcct cctgcc ggggag 96
Leu GlyGlnGlnVal GlySerHis PheLeuLeuPro ProAla GlyGlu
20 25 30
cgg ccgccgctgctg ggcgagcgc aggagcgcggcg gagcgg agcgcg 144
Arg ProProLeuLeu GlyGluArg ArgSerAlaAla GluArg SerAla
35 40 45
cgc ggcgggccgggg getgcgcag ctggcgcacctg cacggc atcctg 192
Arg GlyGlyProGly AlaAlaGln LeuAlaHisLeu HisGly IleLeu
50 55 60
cgc cgccggcagctc tattgccgc accggcttccac ctgcag atcctg 240
Arg ArgArgGlnLeu TyrCysArg ThrG1yPheHis LeuGln IleLeu
65 70 75 80
ccc gacggcagcgtg cagggcacc cggcaggaccac agcctc ttcggt 288
Pro AspGlySerVa1 GlnGlyThr ArgGlnAspHis SerLeu PheGly
85 90 95
atc ttggaattcatc agtgtggca gtgggactggtc agtatt agaggt 336
Ile LeuGluPheIle SerValAla ValGlyLeuVal SerIle ArgGly
100 105 110
gtg gacagtggtctc tatcttgga atgaatgacaaa ggagaa ctctat 384
Val AspSerGlyLeu TyrLeuGly MetAsnAspLys GlyGlu LeuTyr
115 120 125
gga tcagagaaactt acttccgaa tgcatctttagg gagcag tttgaa 432
Gly SerGluLysLeu ThrSerGlu CysIlePheArg GluGln PheGlu
130 135 140
fag aactggtataac acctattca tctaacatatat aaacat ggagac 480
Glu AsnTrpTyrAsn ThrTyrSer SerAsnIleTyr LysHis GlyAsp
145 150 155 160
act ggccgcaggtat tttgtggca cttaacaaagac ggaact ccaaga 528
Thr GlyArgArgTyr PheValAla LeuAsnLysAsp GlyThr ProArg
165 17 0 175
gat ggcgccaggtcc aagaggcat cagaaatttaca catttc ttacct 576
Asp GlyAlaArgSer LysArgHis GlnLysPheThr HisPhe LeuPro
180 185 190
aga ccagtggatcca gaaagagtt ccagaattgtac aaggac ctactg 624
Arg ProValAspPro GluArgVal ProGluLeuTyr LysAsp LeuLeu
195 200 205
atg tacact 633
Met TyrThr
210
<210>
4
<211>
211
<212>
PRT
<213> sapiens
Homo
<400>
4
Met AlaProLeuAla GluValGly GlyPheLeuGly GlyLeu GluGly
1 5 10 15
Leu GlyGlnGlnVal GlySerHis PheLeuLeuPro ProAla GlyGlu
Page 3
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 570A Sequence Listing PCT
20 25 30
Arg ProProLeu LeuGly.GluArg ArgSerAla GluArg SerAla
Ala
35 40 45
Arg GlyGlyPro GlyAlaAlaGln LeuAlaHis LeuHisGly IleLeu
50 55 60
Arg ArgArgGln LeuTyrCysArg ThrGlyPhe HisLeuGln IleLeu
65 70 75 80
Pro AspGlySer ValGlnGlyThr ArgGlnAsp HisSerLeu PheGly
85 90 95
Ile LeuGluPhe IleSerValAla ValGlyLeu ValSerIle ArgGly
100 105 110
Val AspSerGly LeuTyrLeuGly MetAsnAsp LysGlyGlu LeuTyr
115 120 125
Gly SerGluLys LeuThrSerGlu CysIlePhe ArgGluGln PheGlu,
130 135 140
Glu AsnTrpTyr AsnThrTyrSer SerAsnIle TyrLysHis GlyAsp
14 5 150 155 160
Thr GlyArgArg TyrPheValAla LeuAsnLys AspGlyThr ProArg
165 170 175
Asp GlyAlaArg SerLysArgHis GlnLysPhe ThrHisPhe LeuPro
180 185 190
Arg ProValAsp ProGluArgVal ProGluLeu TyrLysAsp LeuLeu
195 200 205
Met TyrThr
210
<210>
<211>
540
<212>
ANA
<213>
Homo
sapiens
<220>
<221>
CDS
<222>
(1)..(537)
<400>
5
atg getccctta gccgaagtcggg ggctttctg ggcggcctg gagggc 48
Met AlaProLeu AlaGluValGly GlyPheLeu GlyGlyLeu GluGly
1 5 10 15
ttg ggccagccg ggggcagcgcag ctggcgcac ctgcacggc atcctg 96
Leu GlyGlnPro GlyAlaAlaGln LeuAlaHis LeuHisGly IleLeu
20 25 30
cgc cgccggcag ctctattgccgc accggcttc cacctgcag atcctg 144
Arg ArgArgGln LeuTyrCysArg ThrGlyPhe HisLeuGln IleLeu
35 40 45
ccc gacggcagc gcgcagggcacc cggcaggac cacagcctc ttcggt 1.92
Pro AspGlySer AlaGlnGlyThr ArgGlnAsp HisSerLeu PheG1y
50 55 60
atc ttggaattc atcagtgtggca gtgggactg gtcagtatt agaggt 240
Ile LeuGluPhe IleSerVa1Ala ValGlyLeu ValSerIle ArgGly
65 70 75 80
Page 4
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 570ASequence
Listing
PCT
gtggacagtggt ctctatctt ggaatgaat gacaaaggagaa ctctat 288
ValAspSerGly LeuTyrLeu GlyMetAsn AspLysGlyGlu LeuTyr
85 90 95
ggatcagagaaa cttacttcc gaatgcatc tttagggagcag tttgaa 336
GlySerGluLys LeuThrSer GluCysIle PheArgGluGln PheGlu
100 105 110
gagaactggtat aacacctat tcatctaac atatataaacat ggagac 384
GluAsnTrpTyr AsnThrTyr 5erSerAsn IleTyrLysHis GlyAsp
115 120 125
actggccgcagg tattttgtg gcacttaac aaagacggaact ccaaga 432
ThrGlyArgArg TyrPheVal AlaLeuAsn LysAspGlyThr ProArg
130 135 140
gatggcgccagg tccaagagg catcagaaa tttacacatttc ttacct 480
AspGlyAlaArg SerLysArg HisGlnLys PheThrHisPhe LeuPro
145 150 155 160
agaccagtggat ccagaaaga gttccagaa ttgtacaaggac ctactg 528
ArgProValAsp ProGluArg ValProGlu LeuTyrLysAsp LeuLeu
165 170 175
atgtacacttag 540
MetTyrThr
<210> 6
<211> 179
<212> PRT
<213> Homo sapiens
<400> 6
Met Ala Pro Leu Ala Giu Val Giy Gly Phe Leu Giy Giy Leu Glu Gly
1 5 10 15
Leu Gly Gln Pro Gly Ala Ala Gln Leu Ala His Leu His Gly Ile Leu
20 25 30
Arg Arg Arg Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Gln Ile Leu
35 40 45
Pro Asp Gly Se~r Ala Gln Gly Thr Arg Gln Asp His Ser Leu Phe Gly
50 55 60
Ile Leu Glu Phe Ile Ser Val Ala Val Gly Leu Val Ser Ile Arg Gly
65 70 75 80
Val Asp Ser Giy Leu Tyr Leu Gly Met Asn Asp Lys Gly Glu Leu Tyr
85 90 95
Gly Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Ar.g Glu Gln Phe Glu
100 105 110
Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile Tyr Lys His Gly Asp
115 120 125
Thr Gly Arg Arg Tyr Phe Val Ala Leu Asn Lys Asp Gly Thr Pro Arg
130 135 140
Asp Gly Ala Arg Ser Lys Arg His Gln Lys Phe Thr His Phe Leu Pro
145 150 155 160
Arg Pro Val Asp Pro Glu Arg Val Pro Glu Leu Tyr Lys Asp Leu Leu
165 170 175
Met Tyr Thr
Page 5
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 570A Sequence Listing PCT
<210> 7
<211> 556
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (2)..(556)
<400> 7
c acc aga tct atg get ccc tta gcc gaa gtc ggg ggc ttt ctg ggc g c 49
Thr Arg Ser Met Ala Pro Leu Ala Glu Val Gly Gly Phe Leu Gly G~y
1 5 10 15
ctg gag ggc ttg ggc cag ccg ggg gca gcg cag ctg gcg cac ctg cac 97
Leu Glu Gly Leu Gly Gln Pro Gly Ala Ala Gln Leu Ala His Leu His
20 25 30
ggc atc ctg cgc cgc cgg cag ctc tat~tgc cgc acc ggc ttc cac ctg 145
Gly Ile Leu Arg Arg Arg Gln Leu Tyr Cys Arg Thr Gly Phe His Leu
35 40 45
cag atc ctg ccc gac ggc agc gtg cag ggc acc cgg cag gac cac agc 193
Gln Ile Leu Pro Asp Gly Ser Val Gln Gly Thr Arg Gln Asp His Ser
50 55 60
ctc ttc ggt atc ttg gaa ttc atc agt gtg gca gtg gga ctg gtc agt 241
Leu Phe Gly Ile Leu Glu Phe Ile Ser Val Ala Val Gly Leu Val Ser
65 70 75 80
att aga ggt gtg gac agt ggt ctc tat ctt gga atg aat gac aaa gga 289
Ile Arg G1y Val Asp Ser G1y Leu Tyr Leu G1y Met Asn Asp Lys Gly
85 90 95
gaa ctc tat gga tca gag aaa ctt act tcc gaa tgc atc ttt agg gag 337
Glu Leu Tyr Gly Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Arg Glu
100 105 110
cag ttt gaa gag aac tgg tat aac acc tat tca tct aac ata tat aaa 385
Gln Phe Glu Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile Tyr Lys
115 120 125
cat gga gac act ggc cgc agg tat ttt gtg gca ctt aac aaa gac gga - 433
His Gly Asp Thr Gly Arg Arg Tyr Phe Val Ala Leu Asn Lys Asp G1y
130 135 140
act cca aga gat ggc gcc agg tcc aag agg cat cag aaa ttt aca cat 481
Thr Pro Arg Asp Gly Ala Arg Ser Lys Arg His Gln Lys Phe Thr His
145 150 155 160
ttc tta cct aga cca gtg gat cca~gaa aga gtt cca gaa ttg tac aag 529
Phe Leu Pro Arg Pro Val Asp Pro Giu Arg Val Pro Glu Leu Tyr Lys
165 170 175
gac cta ctg atg tac act gtc gac ggc 556
Asp Leu Leu Met Tyr Thr Val Asp Gly
180 185
<210> 8
<211> 185
<212> PRT
<213> Homo Sapiens
<400> 8
Thr Arg Ser Met Ala Pro Leu Ala Glu Val Gly Gly Phe Leu Gly Gly
1 5 10 15
Page 6
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 57oA Sequence Listing PCT
LeuGluGlyLeu GlyGlnPro GlyAla Leu~AlaHis LeuHis
Ala
Gln
20 25 30
GlyIieLeuArg ArgArgGln LeuTyrCysArg ThrGlyPhe HisLeu
35 40 45
GlnIleLeuPro AspGlySer ValGlnGlyThr ArgGlnAsp HisSer
50 55 60
LeuPheGlyIle LeuGluPhe IleSerValAla ValGlyLeu ValSer
65 70 75 80
IleArgGlyVal AspSerGly LeuTyrLeuGly MetAsnAsp LysGly
85 90 95
GluLeuTyrGly SerGluLys LeuThrSerGlu CysIlePhe ArgGlu
100 105 110
GlnPheGiuGlu AsnTrpTyr AsnThrTyrSer SerAsnIle TyrLys
115 120 125
HisGlyAspThr GlyArgArg TyrPheValAla LeuAsnLys AspGly
130 135 140
ThrProArgAsp GlyAlaArg SerLysArgHis GlnLysPhe ThrHis
145- 150 155 160
PheLeuProArg ProValAsp ProGluArgVal ProGluLeu TyrLys
165 170 175
AspLeuLeuMet TyrThrVal AspGly
180 185
<210>
9
<211>
415
<212>
DNA
<213> sapiens
Homo
<220>
<221>
CDS
<222> (415)
(2)..
<400>
9
c ct tc 49
ace atc tat
aga ctg tgc
t cgc cgc
cgc acc
cgg ggc
cag ttc
c
Thr er eu
Arg Ile Tyr
S Leu Cys
Arg Arg
Arg Thr
Arg Gly
Gln Phe
L
1 5 10 15
cacctgcagatc ctgcccgac ggcagcgtgcag ggcacccgg caggac 97
HisLeuGlnIle LeuProAsp GlySerValGln GlyThrArg GlnAsp
20 25 30
cacagcctcttc ggtatcttg gaattcatcagt gtggcagtg ggactg 145
HisSerLeuPhe GlyIleLeu GluPheIleSer ValAlaVal GlyLeu
35 40 45
gtcagtattaga ggtgtggac agtggtctctat cttggaatg aatgac 193
ValSerIleArg GlyValAsp SerGlyLeuTyr LeuGlyMet AsnAsp
50 55 60
aaaggagaactc tatggatca gagaaacttact tccgaatgc atcttt Z41
LysGlyGluLeu TyrGlySer GluLysLeuThr SerGluCys IlePhe
65 70 75 80
agggagcagttt gaagagaac tggtataacacc tattcatct aacata 289
ArgGluGlnPhe GluGluAsn TrpTyrAsnThr TyrSerSer AsnIle
85 90 95
Page 7
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura Sepuence PCT
570A Listing
tat aaa gga gacactggc cgcaggtatttt gtggca cttaacaaa 337
cat
Tyr Lys Gly AspThrGly ArgArgTyrPhe ValAla LeuAsnLys
His
100 105 110
gac gga cca agagatggc gccaggtccaag aggcat cagaaattt 385
act
Asp Gly Pro ArgAspGly AlaArgSerLys ArgHis GlnLysPhe
Thr
115 120 125
aca cat tta cctagacca gtcgacggc 415
ttc
Thr His Leu ProArgPro ValAspGly
Phe
130 135
<210>
<211>
138
<212>
PRT
<213>
Homo
Sapiens
<400>
10
Thr Arg Ile LeuArgArg ArgGlnLeuTyr CysArg ThrGlyPhe
Ser
1 5 10 15
His Leu Ile LeuProAsp GlySerValGln GlyThr ArgGlnAsp
Gln
20 25 30
His Ser Phe GlyIleLeu GluPheIleSer ValAla ValGlyLeu
Leu
35 40 45
Val Ser Arg GlyValAsp SerGlyLeuTyr LeuGly MetAsnAsp
Ile
50 55 60
Lys Gly Leu TyrGlySer GluLysLeuThr SerGlu CysIlePhe
Glu
65 70 75 80
Arg Glu Phe GluGluAsn TrpTyrAsnThr TyrSer SerAsnIle
Gln
85 90 95
Tyr Lys Gly AspThrGly ArgArgTyrPhe ValAla LeuAsnLys
His
100 105 110
Asp Gly Pro ArgAspGly AlaArgSerLys ArgHis GlnLysPhe
Thr
115 120 125
Thr His Leu ProArgPro ValAspGly
Phe
130 135
<210>
11
<211>
466
<212>
DNA
<213> Sapiens
Homo
<220>
<221>
CDS
<222> (466)
(2)..
<400>
11
c acc ct at 49
aga t atc tgc
ctg cgc
cgc acc
cgc ggc
cgg ttc
cag
ctc
t
Thr Arg er eu y
S Ile Tyr Phe
Leu Cys
Arg Arg
Arg Thr
Arg Gl
Gln
L
1 5 10 15
cac ctg atc ctgcccgac ggcagcgtgcag ggcacc cggcaggac 97
cag
His Leu Ile LeuProAsp GlySerValGln GlyThr ArgGlnAsp
Gln
20 25 30
cac agc ttc ggtatcttg gaattcatc~agt gtggca gtgggactg 145
ctc
His Ser Phe GlyIleLeu GluPheIleSer ValAla ValGlyLeu
Leu
35 40 45
gtc agt aga ggtgtggac agtggtctctat cttgga atgaatgac 193
att
P age 8
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura Sequence Listing PCT
570A
ValSerIleArg GlyValAsp SerGlyLeuTyr LeuGly MetAsnAsp
50 55 60
aaaggagaactc tatggatca gagaaacttact tccgaa tgcatcttt 241
LysGlyGluLeu TyrGlySer GluLysLeuThr SerGlu CysIlePhe
65 70 75 80
agggagcagttt gaagagaac tggtataacacc tattca tctaacata 289
ArgGluGlnPhe GluGluAsn TrpTyrAsnThr TyrSer SerAsnIle
85 90 95
tataaacatgga gacactggc cgcaggtatttt gtggca cttaacaaa 337
TyrLysHisGly AspThrGly ArgArgTyrPhe ValAla LeuAsnLys
100 105 110
gacggaactcca agagatggc gccaggtccaag aggcat cagaaattt 385
AspGlyThrPro ArgAspGly AlaArgSerLys ArgHis GlnLysPhe
115 120 125
acacatttctta cctagacca gtggatccagaa agagtt ccagaattg 433
ThrHisPheLeu ProArgPro ValAspProGlu ArgVal ProGluLeu
130 135 140
tacaaggaccta ctgatgtac actgtcgacggc 466
TyrLysAspLeu LeuMetTyr ThrValAspGly
145 150 155
<210>
12
<211>
155
<212>
PRT
<213>
Homo
Sapiens
<400>
12
ThrArgSerIle LeuArgArg ArgGlnLeuTyr CysArg ThrGlyPhe
1 5 10 15
HisLeuGlnIle LeuProAsp GlySerValGln GlyThr ArgGlnAsp
20 25 30
HisSerLeuPhe GlyIleLeu GluPheIleSer ValAla ValGlyLeu
35 40 45
ValSerIleArg GlyValAsp SerGlyLeuTyr LeuGly MetAsnAsp
50 . 55 60
LysGlyGluLeu TyrGlySer GluLysLeuThr SerGlu CysIlePhe
65 70 75 80
ArgGluGlnPhe GluGluAsn TrpTyrAsnThr TyrSer SerAsnIle
85 90 95
TyrLysHisGly AspThrGly ArgArgTyrPhe ValAla LeuAsnLys
100 105 110
AspGlyThrPro ArgAspGly AlaArgSerLys ArgHis GlnLysPhe
115 120 125
ThrHisPheLeu ProArgPro ValAspProGlu ArgVal ProGluLeu
130 135 140
TyrLysAspLeu LeuMetTyr ThrValAspGly
145 150 155
<210> 3
1
<211>
549
<212>
DNA
<213> Sapiens
Homo
Page 9
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 57oA Sequence Listing PCT
<220>
<221>
CDS
<222>
(1)..(549)
<400>
13
agatctatgget cccttagcc gaagtcgggggc tttctgggc ggcctg 48
ArgSerMetAla ProLeuAla GluValGlyGly PheLeuGly GlyLeu
1 5 10 15
gagggcttgggc cagccgggg gcagcgcagctg gcgcacctg cacggc 96
GluGlyLeuGly GlnProGly AlaAlaGlnLeu AlaHisLeu HisGly
20 25 30
atcctgcgccgc cggcagctc tattgccgcacc ggcttccac ctgcag 144
IleLeuArgArg ArgGlnLeu TyrCysArgThr GlyPheHis LeuGln
35 40 45
atcctgcccgac ggcagcgtg cagggcacccgg caggaccac agcctc 192
IleLeuProAsp GlySerVal GlnGlyThrArg GlnAspHis SerLeu
50 55 60
ttcggtatcttg gaattcatc agtgtggcagtg ggactggtc agtatt 240
PheGlyIleLeu GluPheIle SerValAlaVal GlyLeuVal SerIle
65 70 75 80
agaggtgtggac agtggtctc tatcttggaatg ~aatgacaaa ggagaa 288
ArgGlyValAsp SerGlyLeu TyrLeuGlyMet AsnAspLys GlyGlu
85 90 95
ctctatggatca gagaaactt acttccgaatgc atctttagg gagcag 336
LeuTyrGlySer GluLysLeu ThrSerGluCys IlePheArg GluGln
100 105 110
tttgaagagaac tggtataac acctattcatct aacatatat aaacat 384
PheGluGluAsn TrpTyrAsn ThrTyrSerSer AsnIleTyr LysHis
115 120 125
ggagacactggc cgcaggtat tttgtggcactt aacaaagac ggaact 432
GlyAspThrGly ArgArgTyr PheValAlaLeu AsnLysAsp GlyThr
130 135 140
ccaagagatggc gccaggtcc aagaggcatcag aaatttaca catttc 480
ProArgAspGly AlaArgSer LysArgHisGln LysPheThr HisPhe
145 150 155 160
ttacctagacca gtggatcca gaaagagttcca gaattgtac aaggac 528
LeuProArgPro ValAspPro GluArgValPro GluLeuTyr LysAsp
165 170 175
ctactgatgtac actctcgag 549
LeuLeuMetTyr ThrLeuGlu
180
<210>
14
<211>
183
<212>
PRT
<213> Sapiens
Homo
<400>
14
ArgSerMetAla ProLeuAla GluValGlyGly PheLeuGly GlyLeu
1 5 10 15
GluGlyLeuGly GlnProGly AlaAlaGlnLeu AlaHisLeu HisGly
20 25 30
IleLeuArgArg ArgGlnLeu TyrCysArgThr GlyPheHis LeuGln
35 40 45
Page 10
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura Sequence PCT
570A Listing
IleLeuProAsp GlySerVal GlnGlyThrArg GlnAsp HisSerLeu
50 55 60
PheGlyIleLeu GluPheIle SerValAlaVal GlyLeu ValSerIle
65 70 75 80
ArgGlyValAsp SerGlyLeu TyrLeuGlyMet AsnAsp LysGlyGlu
85 90 95
LeuTyrGlySer GluLysLeu ThrSerGluCys IlePhe ArgGluGln
100 105 110
PheGluGluAsn TrpTyrAsn ThrTyrSerSer AsnIle TyrLysHis
115 120 125
GlyAspThrGly ArgArgTyr PheValAlaLeu AsnLys AspGlyThr
130 135 140
ProArgAspGly AlaArgSer LysArgHisGln LysPhe ThrHisPhe
145 150 155 160
LeuProArgPro ValAspPro GluArgValPro GluLeu TyrLysAsp
165 170 175
LeuLeuMetTyr ThrLeuGlu
180
<210>
15
<211>
408
<212>
DNA
<213>
Homo
sapiens
<220>
<221>
CDS
<22Z>
(1)..(408)
<400>
15
agatctatcctg cgccgccgg cagctctattgc cgcacc ggcttccac 48
ArgSerIleLeu ArgArgArg GlnLeuTyrCys ArgThr GlyPheHis
1 5 10 15
ctgcagatcctg cccgacggc agcgtgcagggc acccgg caggaccac 96
LeuGlnIleLeu ProAspGly SerValGlnGly ThrArg GlnAspHis
20 25 30
agcctcttcggt atcttggaa ttcatcagtgtg gcagtg ggactggtc 144
SerLeuPheGly IleLeuGlu PheIleSerVal AlaVal GlyLeuVal
35 40 45
agtattagaggt gtggacagt ggtctctatctt ggaatg aatgacaaa 192
SerIleArgGly ValAspSer GlyLeuTyrLeu GlyMet AsnAspLys
50 55~ 60
ggagaactctat ggatcagag aaacttacttcc gaatgc atctttagg 240
GlyGluLeuTyr GlySerGlu LysLeuThrSer GluCys IlePheArg
65 70 75 80
gagcagtttgaa gagaactgg tataacacctat tcatct aacatatat 288
GluGlnPheGlu GluAsnTrp TyrAsnThrTyr SerSer AsnIleTyr
85 90 95
aaacatgaagac actggccgc aggtattttgtg gcactt aacaaagac 336
LysHisGluAsp ThrGlyArg ArgTyrPheVal AlaLeu AsnLysAsp
100 105 110
ggaactccaaga gatggcgcc aggtccaagagg catcag aaatttaca 384
GlyThrProArg AspGly-AlaArgSerLysArg HisGln LysPheThr
115 120 125
Page 11
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 57oA Sequence Listing PCT
cat ttc cct agaccactc gag 408
tta
His Phe Pro ArgProLeu Glu
Leu
130 135
<210>
16
<211>
136
<212>
PRT
<213>
Homo
sapiens
<400>
16
Arg Ser Leu ArgArgArg GlnLeuTyrCys Arg.ThrGly PheHis
Ile
1 5 10 15
Leu G1n Leu ProAspGly SerValGlnGly ThrArgGln AspHis
Ile
20 25 30
Ser Leu Gly IleLeuGlu PheIleSerVal AlaValGly LeuVal
Phe
35 40 45
Ser Ile Gly ValAspSer GlyLeuTyrLeu GlyMetAsn AspLys
Arg
50 55 60
Gly Glu Tyr GlySerGlu LysLeuThrSer GluCys.IlePheArg
Leu
65 70 75 80
Glu Gln Glu GluAsnTrp TyrAsnThrTyr SerSerAsn IleTyr
Phe
85 90 95
Lys His Asp ThrGlyArg ArgTyrPheVal AlaLeuAsn LysAsp
Glu
100 105 110
Gly Thr Arg AspGlyAla ArgSerLysArg HisGlnLys PheThr
Pro
115 120 125
His Phe Pro ArgProLeu Glu
Leu
130 135
<Z10>
17
<211>
408
<212>
DNA
<213> sapiens
Homo
<220>
<221>
CDs
<222> (408)
(1)..
<400>
17
aga tct ctg cgccgccgg cagctctattgc cgcaccggc ttccac 48
atc
Arg Ser Leu ArgArgArg GlnLeuTyrCys ArgThrGly PheHis
Ile
1 5 10 15
ctg cag ctg cccgacggc agcgtgcagggc acccggcag gaccac 96
atc
Leu Gln Leu ProAspGly SerValGlnGly ThrArgGln AspHis
Ile
20 25 30
agc ctc ggt atcttggaa ttcatcagtgtg gcagtggga ctggtc 144
ttc
Ser Leu Gly IleLeuGlu PheIleSerVal AlaVa1Gly LeuVal
Phe
35 40 45
agt att ggt gtggacagt ggtctctatctt ggaatgaat gacaaa 192
aga
Ser Ile Gly ValAspSer GlyLeuTyrLeu GlyMetAsn AspLys'
Arg
50 55 60
gga gaa tat ggatcagag aaacttacttcc gaatgcatc tttagg 240
ctc
Gly Glu Tyr GlySerGlu LysLeuThrSer GluCysIle PheArg
Leu
65 70 75 80
Page 12
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura Sequence
570A Listing
PCT
gag cag gaa gagaactgg tataacacctat tcatctaac atatat 288
ttt
Glu Gln Glu GluAsnTrp TyrAsnThrTyr SerSerAsn IleTyr
Phe
85 90 95
aaa cat gac actggccgc aggtattttgtg gcacttaac aaagac 336
gga
Lys His Asp ThrGlyArg ArgTyrPheVal AlaLeuAsn LysAsp
Gly
100 - 105 110
gga act aga gatggcgcc aggtccaagagg catcagaaa tttaca 384
cca
Gly Thr Arg AspGlyAla ArgSerLysArg HisGlnLys PheThr
Pro
115 120 125
cat ttc cct agaccactc gag 408
tta
His Phe Pro ArgProLeu Glu
Leu
130 135
<210>
18
<211>
136
<212>
PRT
<213> Sapiens
Homo
<400>
18
Arg Ser Leu ArgArgArg GlnLeuTyrCys ArgThrGly PheHis
Ile
1 5 10 15
Leu Gln Leu ProAspGly SerValGlnGly ThrArgGln AspHis
Ile
20 25 30
Ser Leu Gly IleLeuGlu PheIleSerVal AlaValGly LeuVal
Phe
35 40 45
Ser Ile Gly ValAspSer GlyLeuTyrLeu GlyMetAsn AspLys
Arg
50 55 60
Gly Glu Tyr GlySerGlu LysLeuThrSer GluCysIle PheArg
Leu
65 70 75 80
Glu Gln Glu GluAsnTrp TyrAsnThrTyr SerSerAsn IleTyr
Phe
85 90 95
Lys His Asp ThrGlyArg ArgTyrPheVal AlaLeuAsn LysAsp
Gly
100 105 110
Gly Thr Arg AspGlyAla ArgSerLysArg HisGlnLys PheThr
Pro
115 120 125
His Phe Pro ArgProLeu Glu
Leu
130 _ 135
<210>
19
<211>
477
<212>
DNA
<213> Sapiens
Homo
<220>
<221>
CDS
<222> (474)
(1)..
<400>
19
atg get ctg getcacctg catggtatcctg cgtcgccgt cagctg 48
cag
Met Ala Leu AlaHisLeu HisGlyIleLeu ArgArgArg GlnLeu
Gln
1 5 10 15
tac tgc act ggtttccac ctgcagatcctg ccggatggt tctgtt 96
cgt
Tyr Cys Thr GlyPheHis LeuGlnIleLeu ProAspGly SerVal
Arg
20 25 30
cag ggt cgt caggaccac tctctgttcggt atcctggaa ttcatc 144
acc
Page
13
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura Sequence PCT
570A Listing
GlnGlyThrArg GlnAspHis SerLeuPheGly IleLeu GluPheIle
35 40 45
tctgttgetgtt ggtctggtt tctatccgtggt gttgac tctggcctg 192
SerValAlaVai GlyLeuVal SerIleArgGly ValAsp SerGlyLeu
50 55 60
tacctgggtatg aacgacaaa ggcgaactgtac ggttct gaaaaactg 240
TyrLeuGlyMet AsnAspLys GlyGluLeuTyr GlySer GluLysLeu
65 70 75 80
acctctgaatgc atcttccgt gaacagtttgaa gagaac tggtacaac 288
ThrSerGluCys IlePheArg GluGlnPheGlu GluAsn TrpTyrAsn
85 90 95
acctactcttcc aacatctac aaacatggtgac accggc cgtcgctac 336
ThrTyrSerSer AsnIleTyr LysHisGlyAsp ThrGly ArgArgTyr
100 105 110
ttcgttgetctg aacaaagac ggtaccccgcgt gatggt getcgttct 384
PheValAlaLeu AsnLysAsp GlyThrProArg AspGly AlaArgSer
115 120 125
aaacgtcaccag aaattcacc cacttcctgccg cgccca gttgacccg 432
LysArgHisGln LysPheThr HisPheLeuPro ArgPro ValAspPro
130 135 140
gagcgtgttcca gaactgtat aaagacctgctg atgtac acctaa 477
GluArgValPro GluLeuTyr LysAspLeuLeu MetTyr Thr
145 150 155
<210>
20
<211>
158
<212>
PRT
<213>
Homo
sapiens
<400>
20
MetAlaGlnLeu AlaHisLeu HisGlyIleLeu ArgArg ArgGlnLeu
1 5 10 ~ 15
TyrCysArgThr GlyPheHis LeuGlnIleLeu ProAsp GlySerVal
20 25 30
GlnGlyThrArg Gl~nAspHis SerLeuPheGly IleLeu GluPheIle
35 40 45
SerValAlaVal GlyLeuVal SerIleArgGly ValAsp SerGlyLeu
50 55 60
TyrLeuGlyMet AsnAspLys GlyGluLeuTyr GlySer GluLysLeu
65 70 75 80
ThrSerGluCys IlePheArg GluGlnPheGlu GluAsn TrpTyrAsn
85 90 95
ThrTyrSerSer AsnIleTyr LysHisGlyAsp ThrGly ArgArgTyr
100 105 110
PheValAlaLeu AsnLysAsp GlyThrProArg AspGly AlaArgSer
115 120 125
LysArgHisGln LysPheThr HisPheLeuPro ArgPro ValAspPro
130 135 140
GluArgValPro GluLeuTyr LysAspLeuLeu MetTyr Thr
145 150 155
<210>
21
Page 14
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 57oA Sequence Listing PCT
<211> 636
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> ~(1) . . (633)
<400> 21
atg get ccc tta gcc gaa gtc ggg ggc ttt ctg ggc ggc ctg gag ggc 48
Met Ala Pro Leu Ala Glu Val Gly Gly Phe Leu Gly Gly Leu Glu Gly
1 5 10 15
ttg ggc cag cag gtg ggt tcg cat ttc ctg ttg cct cct gcc ggg gag 96
Leu Gly Gln Gln Val Gly Ser His Phe Leu Leu Pro Pro Ala Gly Glu.
20 25 30
cgg ccg ccg ctg ctg ggc gag cgc agg agc gcg gcg gag cgg agc gcg ~ 144
Arg Pro Pro Leu Leu Gly Glu Arg Arg Ser Ala Ala Glu Arg Ser Ala
35 40 45
cgc ggc ggg ccg ggg get gcg cag ctg gcg cac ctg cac ggc atc ctg 192
Arg Gly Gly Pro Gly Ala Ala Gln Leu Ala His Leu His Gly Ile Leu
50 55 60
cgc cgc cgg cag ctc tat tgc cgc acc ggc ttc cac ctg cag atc ctg 240
Arg Arg Arg Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Gln Ile Leu
65 70 75 80
ccc gac ggc agc gtg cag ggc acc cgg cag gac cac agc ctc ttc ggt 288
Pro Asp Gly Ser Val Gln Gly Thr Arg Gln Asp His Ser Leu Phe Gly
85 90 95
atc ttg gaa ttc atc agt gtg gca gtg gga ctg gtc agt att aga ggt 336
Ile Leu Giu Phe Iie Ser Val Aia vai Giy Leu vai ser Iie Arg Giy
100 105 110
gtg gac agt ggt ctc tat ctt gga atg aat gac aaa gga gaa ctc tat 384
Val Asp Ser Gly Leu Tyr Leu Gly Met Asn Asp Lys Gly Glu Leu Tyr
115 120 125
gga tca gag aaa ctt act tcc gaa tgc atc ttt agg gag cag ttt gaa 432
Gly Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Arg Glu Gln Phe Glu
130 135 140
gag aac tgg tat aac acc tat tca tct aac ata tat aaa cat gga gac 480
Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile Tyr Lys His Gly Asp
145 150 155 160
act ggc cgc agg tat ttt gtg gca ctt aac aaa gac gga act cca aga 528
Thr Gly Arg Arg Tyr Phe Val Ala Leu Asn Lys Asp Gly Thr Pro Arg
165 170 175
gat ggc gcc agg tcc aag agg cat cag aaa ttt aca cat ttc tta cct 576
Asp Gly Ala Arg Ser Lys Arg His Gln Lys Phe Thr His Phe Leu Pro
180 185 190
aga cca gtg gat cca gaa aga gtt cca gaa ttg tac aag gac cta ctg 624
Arg Pro Val Asp Pro Glu Arg Val Pro Glu Leu Tyr Lys Asp Leu Leu
195 200 205
atg tac act tga 636
Met Tyr Thr
210
<210> 22
<211> 211
<212> PRT
Page 15
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura Sequence PCT
570A Listing
<213>
Homo
sapiens
<400>
22
Met ProLeu AlaGluVal GlyGlyPhe LeuGlyGly LeuGluGly
Ala
1 5 10 15
Leu GlnGln ValGlySer HisPheLeu LeuProPro AlaGlyGlu
Gly
20 25 30
Arg ProLeu LeuGlyGlu ArgArgSer AlaAlaGlu ArgSerAla
Pro
35 40 45
Arg GlyPro GlyAlaAla GlnLeuAla HisLeuHis GlyIleLeu
Gly
50 55 60
Arg ArgGln LeuTyrCys ArgThrGly PheHisLeu GlnIleLeu
Arg
65 70 75 80
Pro GlySer ValGlnGly ThrArgGln AspHisSer LeuPheGly
Asp
85 90 95
Ile GluPhe IleSerVal AlaValGly LeuValSer IleArgGly
Leu
100 105 110
Val SerGly LeuTyrLeu GlyMetAsn AspLysGly GluLeuTyr
Asp
115 1zo 125
Gly GluLys LeuThrSer GluCysIle PheArgGlu GlnPheGlu
Ser
130 135 140
Glu TrpTyr AsnThrTyr SerSerAsn IleTyrLys NisGlyAsp
Asn
145 150 155 160
Thr ArgArg TyrPheVal AlaLeuAsn LysAspGly ThrProArg
Gly
165 170 175
Asp AlaArg SerLysArg HisGlnLys PheThrHis PheLeuPro
Gly
18 0 185 190
Arg ValAsp ProGluArg ValProGlu LeuTyrLys AspLeuLeu
Pro
195 200 205
Met Thr
Tyr
210
<210>
23
<211> 0
54
<212> A
DN
<213> sapiens
Homo
<220>
<221>
CDS
<222> (537)
(1)..
<400>
23
atg ccctta gccgaagtc gggggcttt ctgggcggc ctggagggc 48
get
Met ProLeu AlaGluVal GlyGlyPhe LeuGlyGly LeuGluGly
Ala
1 5 . 10 15
ttg cagccg ggggcagcg cagctggcg cacctgcac ggcatcctg 96
ggc
Leu GlnPro GlyAlaAla GlnLeuAla HisLeuHis GlyIleLeu
Gly
20 25 30
cgc cggcag ctctattgc cgcaccggc ttccacctg cagatcctg 144
cgc
Arg ArgGln LeuTyrCys ArgThrGly PheHisLeu GlnIleLeu
Arg
35 40 45
ccc ggcagc gtgcagggc acccggcag gaccacagc ctcttcggt 192
gac
Page 6
1
CA 02525107 2005-11-07
WO PCT/US2004/014623
2004/100892
Cura 570ASequence PCT
Listing
ProAspGlySer ValGlnGly ThrArgGlnAsp HisSer LeuPheGly
50 55 60
atcttggaattc atcagtgtg gcagtgggactg gtcagt attagaggt 240
IleLeuGluPhe IleSerVal AlaValGlyLeu ValSer IleArgGly
65 70 75 80
gtggacagtggt ctctatctt ggaatgaatgac aaagga gaactctat 288
ValAspSerGly LeuTyrLeu GlyMetAsnAsp LysGly GluLeuTyr
85 90 95
ggatcagagaaa cttacttcc gaatgcatcttt agggag cagtttgaa 336
GlySerGluLys LeuThrSer GluCysIlePhe ArgGlu GlnPheGlu
100 105 110
gagaactggtat aacacctat tcatctaacata tataaa catggagac 384
GluAsnTrpTyr AsnThrTyr SerSer'AsnIle TyrLys HisGlyAsp
115 120 125
actggccgcagg tattttgtg gcacttaacaaa gacgga actccaaga 432
ThrGlyArgArg TyrPheVal AlaLeuAsnLys AspGly ThrProArg
130 135 140
gatggcgccagg tccaagagg catcagaaattt acacat ttcttacct 480
AspGlyAlaArg SerLysArg HisGlnLysPhe ThrHis PheLeuPro
145 150 155 160
agaccagtggat ccagaaaga gttccagaattg tacaag gacctactg 528
ArgProValAsp ProGluArg ValProGIuLeu TyrLys AspLeuLeu
165 170 175
atgtacacttag 540
MetTyrThr
<210> 24
<211> 179
<212> PRT
<213> Homo sapiens
<400> 24
Met Ala Pro Leu Ala Glu Val Gly Gly Phe L.eu Gly Gly Leu Glu Gly
1 ~ 5 10 15
Leu Gly Gln Pro Gly Ala Ala Gln Leu Ala His Leu His Gly Ile Leu
20 25 30
Arg Arg Arg Gln_Leu Tyr Cys Arg Thr Gly Phe His Leu Gln Ile Leu
35 40 45
Pro Asp Gly Ser Val Gln Gly Thr Arg Gln Asp His Ser Leu Phe Gly
50 55 60
Ile Leu Glu Phe Ile Ser Val Ala Val Gly Leu Val Ser Ile Arg Gly
65 70 75 80
Val Asp Ser Gly Leu Tyr Leu Gly Met Asn Asp Lys Gly Glu Leu Tyr
85 90 95
Gly Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Arg Glu Gln Phe Glu
100 10S 110
Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile Tyr Lys His Gly Asp
115 120 125
Thr Gly Arg Arg Tyr Phe Val Ala Leu Asn Lys Asp Gly Thr Pro Arg
130 135 140
Page 17
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 57oA Sequence Listing PCT
Asp Gly Ala Arg Ser Lys Arg His Gln Lys Phe Thr His Phe Leu Pro
145 150 155 160
Arg Pro Val Asp Pro Glu Arg Val Pro Glu Leu Tyr Lys Asp Leu Leu
165 170 175
Met Tyr Thr
<210> 25
<211> 54
<212> DNA
<213> Homo Sapiens
<220>
<221> CDs.
<222> (1) . . (54)
<400> 25
atg get ccc tta gcc gaa gtc ggg ggc ttt ctg ggc ggc ctg gag ggc 48
Met Ala Pro Leu Ala Glu Val Gly Gly Phe Leu Gly Gly Leu Glu Gly
1 5 10 15
ttg ggc 54
Leu Gly
<210> 26
<211> 18
<212> PRT
<213> Homo Sapiens
<400> 26
Met Ala Pro Leu Ala Glu Val Gly Gly Phe Leu Gly Gly Leu Glu Gly
1 5 10 15
Leu Gly
<210> 27
<211> 63
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (1)..(63)
<400> 27
gag cgg ccg ccg ctg ctg ggc gag cgc agg agc gcg gcg gag cgg agc 48
Glu Arg Pro Pro Leu Leu Gly Glu Arg Arg Ser Ala Ala Glu Arg Ser
1 5 10 15
c c c c cc 63
Ala Agg Gly Ggy Pro
<210> 28
<211> 21
<212> PRT
<213> Homo Sapiens
<400> 28
Glu Arg Pro Pro Leu Leu Gly Glu Arg Arg Ser Ala Ala Glu Arg Ser
1 5 10 15
Ala Arg Gly Gly Pro
Page 18
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
cura 57oA Sequence Listing PCT
<210> 29
<211> 63
<212> DNA
<213> Homo Sapiens
<220>
<221> cos
<222> (1),.(63)
<400> 29
cgc agg tat ttt gca ctt aac gacgga actcca gatggc 48
gtg aaa aga
Arg Arg Tyr Phe Ala Leu Asn AspGly ThrPro AspGly
Val Lys Arg
1 5 10 15
gcc agg tcc aag 63
agg
A1 a Arg Ser
Lys Arg
20
<210> 30
<211> 21
<212> PRT
<213> Homo Sapiens
<400> 30
Arg Arg Tyr Phe Ala Leu Asn AspGly ThrPro AspGly
Val Lys Arg
1 5 10 15
Ala Arg Ser Lys
Arg
20
<210> 31
<211> 60
<212> DNA
<213> Homo Sapiens
<220>
<221> CDs
<222> (1) . .
(60)
<400> 31
cct aga cca gtg cca gaa aga ccagaa ttgtac gaccta 48
gat gtt aag
Pro Arg Pro Val Pro Glu Arg ProGlu LeuTyr AspLeu
Asp Val Lys
1 5 10 15
ctg atg tac act &0
Leu Met Tyr Thr
20
<220> 3Z
<211> 20
<212> PRT
<213> Homo Sapiens
<400> 32
Pro Arg Pro Val Pro Glu Arg ProGlu LeuTyr AspLeu
Asp Val Lys
1 5 10 15
Leu Met Tyr Thr
20
<210> 33
<211> 51
<212> DNA
<213> Homo Sapiens
<220>
<221> CDs
Page 19
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 57oA Sequence Listing PCT
<222> (1),.(51)
<400> 33
atg aac gac aag ggc gag ctg tac ggc agc gag aag ctg acc agc gag 48
Met Asn Asp Lys Gly Glu Leu Tyr Gly Ser Glu Lys Leu Thr Ser Glu
1 5 10 15
tgc 51
Cys
<210> 34
<211> 17
<212> PRT
<213> Homo sapiens
<400> 34
Met Asn Asp Lys Gly Glu Leu Tyr Gly Ser Glu Lys Leu Thr Ser Glu
1 5 10 15
Cys
<210>
35
<211> 3
63
<212>
DNA
<213>
Homo
sapiens
<220>
<221> S
CD
<222>
(1)..(633)
<400>
35
atg ccctta gccgaagtc gggggctttctg ggcggcctg gagggc 48
get
Met ProLeu AlaGiuVal GiyGlyPheLeu GiyGlyLeu GluGiy
Aia
1 5 10 15
ttg cagcag gtgggttcg catttcctgttg cctcctgcc ggggag 96
ggc
Leu GlnGln ValGlySer HisPheLeuLeu ProProAla GlyGlu
Gly
20 25 30
cgg ccgctg ctgggcgag cgcaggagcgcg gcggagcgg agcgcg 144
ccg
Arg ProLeu LeuGlyGlu ArgArgSerAla AlaGluArg SerAla
Pro
35 40 45
cgc gggccg ggggetgcg cagctggcgcac ctgcacggc atcctg 192
ggc
Arg GlyPro GlyAlaAla GlnLeuAlaHis LeuHisGly IleLeu
Gly
50 55 60
cgc cggcag ctctattgc cgcaccggcttc cacctgcag atcctg 240
cgc
Arg ArgGln LeuTyrCys ArgThrGlyPhe HisLeuGln IleLeu
Arg
65 70 75 80
ccc ggcagc gtgcagggc acccggcaggac c~cagcctc ttcggt 288
gac
Pro GlySer ValGlnGly ThrArgGlnAsp HisSerLeu PheGly
Asp
85 90 95
atc gaattc atcagtgtg gcagtgggactg gtcagtatt agaggt 336
ttg
Ile GluPhe IleSerVal AlaValGlyLeu ValSerIle ArgGly
Leu
100 105 110
gtg agtggt ctctatctt ggaatgaatgac aaaggagaa ctctat 384
gac
Val SerGly LeuTyrLeu GlyMetAsnAsp LysGlyGlu LeuTyr
Asp
115 120 125
gga gagaaa cttacttcc gaatgcatcttt agggagcag tttgaa 432
tca
Gly GluLys LeuThrSer GluCysIlePhe ArgGluGln PheGlu
Ser
130 135 140
Pa ge 0
2
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 57oA Sequence Listing PCT
gagaactggtat aacacctat tcatctaac atatataaacat ggagac 480
GluAsnTrpTyr AsnThrTyr SerSerAsn IleTyrLysHis GlyAsp
145 150 155 160
actggccgcagg tattttgtg gcacttaac aaagacggaact ccaaga 528
ThrGlyArgArg TyrPheVal AlaLeuAsn LysAspGlyThr ProArg
165 170 175
gatggcgccagg tccaagagg catcagaaa tttacacatttc ttacct 576
AspGlyAlaArg SerLysArg HisGlnLys PheThrHisPhe LeuPro
180 185 190
agaccagtggat ccagaaaga gttccagaa ttgtacaagaac ctactg 624
ArgProValAsp ProGluArg ValProGlu LeuTyrLysAsn LeuLeu
195 200 205
atgtacact 633
MetTyrThr
210
<210>
36
<211>
211
<212>
PRT
<213>
Homo
sapiens
<400>
36
MetAlaProLeu AlaGluVal GlyGlyPhe LeuGlyGlyLeu GluGly
1 5 10 15
LeuGlyGlnGln ValGlySer HisPheLeu LeuProProAla GlyGlu
20 25 30
ArgProProLeu LeuGlyGlu ArgArgSer AlaAlaGlu.ArgSerAla
35 40 45
ArgGlyGlyPro GlyAlaAla GlnLeuAla HisLeuHisGly IleLeu
50 55 60
ArgArgArgGln LeuTyrCys ArgThrGly PheHisLeuGln IleLeu
65 70 75 80
ProAspGlySer ValGlnGly ThrArgGln AspHisSerLeu PheGly
85 90 95
IleLeuGluPhe IleSerVal AlaValGly LeuValSerIle ArgGly
100 105 110
ValAspSerGly LeuTyrLeu GlyMetAsn AspLysGlyGlu LeuTyr
115 120 125
GlySerGluLys LeuThrSer GluCysIle PheArgGluGln PheGlu
130 135 140
GluAsnTrpTyr AsnThrTyr SerSerAsn IleTyrLysHis GlyAsp
145 150 155 160
ThrGlyArgArg TyrPheVal AlaLeuAsn LysAspGlyThr ProArg
165 170 175
AspGlyAlaArg SerLysArg HisGlnLys PheThrHisPhe LeuPro
180 185 190
ArgProValAsp ProGluArg ValProGlu LeuTyrLysAsn LeuLeu
195 200 205
MetTyrThr
210
Page 21
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 57oA Sequence Listing PCT
<210> 37
<211> 203
<212> PRT
<213> Homo sapiens
<400> 37
Gly Phe Leu Gly Gly Leu Glu Gly Leu Gly Gln Gln Val Gly Ser His
1 5 10 15
Phe Leu Leu Pro Pro Ala Gly Glu Arg Pro Pro Leu Leu Gly Glu Arg
20 25 30
Arg Ser Ala Ala Glu Arg Ser Ala Arg Gly Gly Pro Gly Ala Ala Gln
35 40 45
Leu Ala His Leu His Gly Ile Leu Arg Arg Arg Gln Leu Tyr Cys Arg
50 55 60
Thr Gly Phe His Leu Gin Ile Leu Pro Asp Giy Ser Val Gln Giy Thr
65 70 75 80
Arg Gln Asp His Ser Leu Phe Gly Ile Leu Glu Phe Ile Ser Val Ala
85 90 95
Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser Gly Leu Tyr Leu Gly
100 105 110
Met Asn Asp Lys Gly Glu Leu Tyr Gly Ser Glu Lys Leu Thr Ser Glu
1l5 120 125
Cys Ile Phe Arg Glu Gln Phe Glu Glu Asn Trp Tyr Asn Thr Tyr Ser
130 135 140
Ser Asn Ile Tyr Lys His Giy Asp Thr Gly Arg Arg Tyr Phe Val Aia
145 150 155 160
Leu Asn Lys Asp Gly Thr Pro Arg Asp Gly Ala Arg Ser Lys Arg His
165 170 175
Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val Asp Pro Glu Arg Val
180 185 190
Pro Glu Leu Tyr Lys Asp Leu Leu Met Tyr Thr
195 200
<210> 38
<211> 200
<212> PRT
<213> Homo sapiens
<400> 38
Gly Gly Leu Glu Gly Leu Gly Gln Gln Val Gly Ser His Phe Leu Leu
1 5 10 15
Pro Pro Ala Gly Glu Arg Pro Pro Leu Leu Gly Glu Arg Arg Ser Ala
20 25 30
Ala Glu Arg Ser Ala Arg Gly Gly Pro Gly Ala Ala Gln Leu Ala His
35 40 45
Leu His Gly Ile Leu Arg Arg.Arg Gln Leu Tyr Cys Arg Thr Gly Phe
50 55 60
His Leu Gln Ile Leu Pro Asp Gly Ser Val Gln Gly Thr Arg Gln Asp
65 70 75 80
His Ser Leu Phe Gly Ile Leu Glu Phe Ile Ser Val Ala Val Gly Leu
Page 22
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura 570A Sequence Listing PCT
85 90 95
Val Ser Ile Arg Gly Val Asp Ser Gly Leu Tyr Leu Gly Met Asn Asp
100 105 110
Lys Gly Glu Leu Tyr Gly Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe
115 120 125
Arg Glu Gln Phe Glu Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile
130 135 140
Tyr Lys His Gly Asp Thr Gly Arg Arg Tyr Phe Val Ala Leu Asn Lys
145 150 155 160
Asp Gly Thr Pro Arg Asp Gly Ala Arg Ser Lys Arg His Gln Lys Phe
165 170 175
Thr His Phe Leu Pro Arg Pro Val Asp Pro Glu Arg Val Pro Glu Leu
180 185 190
Tyr Lys Asp Leu Leu Met Tyr Thr
195 Z00
<210> 39
<211> 197
<212> PRT
<213> Homo sapiens
<400> 39
Glu Gly Leu Gly Gln Gln Val Gly Ser His Phe Leu Leu Pro Pro Ala
1 5 10 15
Gly Glu Arg Pro Pro Leu Leu Gly Glu Arg Arg Ser Ala Ala Glu Arg
20 25 30
Ser Ala Arg Gly Gly Pro Gly Ala Ala Gln Leu Ala His Leu His Gly
35 40 45
Ile Leu Arg Arg Arg Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Gln
50 55 60
Ile Leu Pro Asp Gly Ser Val Gln Gly Thr Arg Gln Asp His Ser Leu
65 70 75 80
Phe Gly Ile Leu Glu Phe Ile Ser Val Ala Val Gly Leu Val Ser Ile
85 90 95
Arg Gly Val Asp Ser Gly Leu Tyr Leu Gly Met Asn Asp Lys Gly Glu
100 105 110
Leu Tyr Gly Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Arg Glu Gln
115 120 125
Phe Glu Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile Tyr Lys His
130 135 140
Gly Asp Thr Gly Arg Arg Tyr Phe Val Ala Leu Asn Lys Asp Gly Thr
145 150 155 160
Pro Arg Asp Gly Ala Arg Ser Lys Arg His Gln Lys Phe Thr His Phe
165 170 175
Leu Pro Arg Pro Val Asp Pro Glu Arg Val Pro Glu Leu Tyr Lys Asp
180 185 190
Leu Leu Met Tyr Thr
195
Page 23
CA 02525107 2005-11-07
WO 2004/100892 PCT/US2004/014623
Cura- 570ASequence PCT
Listing
<210>
40
<211>
188
<212>
PRT
<213> sapiens
Homo
<400>
40
His Phe Leu ProProAla GlyGluArgPro ProLeu LeuGlyGlu
Leu
1 5 10 15
Arg Arg Ala AlaGluArg SerAlaArgGly GlyPro GlyAlaAla
Ser
20 25 30
Gln Leu His LeuHisGly IleLeuArgArg ArgGln LeuTyrCys
Ala
35 40 45
Arg Thr Phe HisLeuGln IleLeuProAsp GlySer ValGlnGly
Gly
50 55 60
Thr Arg Asp His~SerLeu PheGlyIleLeu GluPhe IleSerVal
Gln
65 70 75 80
Ala Val Leu ValSerIle ArgGlyValAsp SerGly LeuTyrLeu
Gly
85 90 95
Gly Met Asp LysGlyGlu LeuTyrGlySer GluLys LeuThrSer
Asn
100 105 110
Glu Cys Phe ArgGluGln PheGluGluAsn TrpTyr AsnThrTyr
Ile
115 120 125
Ser Ser Ile TyrLysHis ~GlyAspThrGly ArgArg TyrPheVal
Asn
130 135 140
Ala Leu Lys AspGlyThr ProArgAspGly AlaArg SerLysArg
Asn
145 150 155 160
His Gln Phe ThrHisPhe LeuProArgPro ValAsp ProGluArg
Lys
165 170 175
Val Pro Leu TyrLysAsp LeuLeuMetTyr Thr
Glu
180 185
Page 24
