Note: Descriptions are shown in the official language in which they were submitted.
CA 02388637 2002-04-19
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HUMAN FGF-20 GENE AND GENE EXPRESSION PRODUCTS
TECHNICAL FIELD
The present invention relates to nucleic acid sequences encoding
members of the fibroblast growth factor (FGF) family, and to polypeptides
encoded by
the nucleic acid sequences.
BACKGROUND OF THE INVENTION
The substantia nigra is an area of the brain that has generated intensive
research. Interest in the substantia nigra was originally based on the finding
that
degeneration of dopaminergic neurons in the area causes Parkinson's disease.
In
1 o addition, the substantia nigra has been strongly implicated in thought and
affective
disorders (1). Therefore, neurotrophic factors for dopaminergic neurons in the
substantia nigra are of substantial clinical interest.
filial cell line-derived neurotrophic factor (GDNF) is the first
neurotrophic factor documented to enhance the survival of midbrain
dopaminergic
neurons (Lin, L.-F.H. et al., Science 260:1130-1132 (1993)). Persephin,
Artemin,
BDGF and NT-3 also enhance the survival of midbrain dopaminergic neurons and
have
clinical potential in the treatment of Parkinson's disease (Milbrandt, J. et
al., Neuron
20:245-253 (1998); Baloh, R.H. et al., Neuron 21:1291-1302 (1998); Hyman, C.
et al.,
Nature 350:230-232 (1991); Hyman, C. et al., J. Neurosci. 14:335-347 (1994)).
However, GDNF was reported to be widely expressed in neurons of the brain
(Pochon,
N.A. et al., Eur. J. Neurosci. 9:463-471 ( 1997)). Persephin was also widely
expressed
in several major tissues including heart, kidney, liver and brain (Milbrandt,
J. et al.,
Neuron 20:245-253 (1998)). Artemin in brain was expressed in the basal ganglia
and
thalamus, suggesting that it influences the subcortical motor system (Baloh,
R.H. et al.,
Neuron 21:1291-1302 (1998)). BDNF and NT-3 were predominantly expressed in the
hippocampus (Ernfors, P. et al., Neuron 5:511-526 (1990)). Therefore, these
neurotrophic factors appear not to be specific for dopaminergic neurons in the
substantia nigra.
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The prototypic fibroblast growth factors (FGFs), FGF-1 (aFGF) and
FGF-2 (bFGF), were originally isolated from brain and pituitary as mitogens
for
fibroblasts. However, FGF-1 and FGF-2 are widely expressed in developing and
adult
tissues, and are polypeptides with multiple biological activities including
angiogenesis,
mitogenesis, cellular differentiation and repair of tissue injury (Baud, A. et
al., Cancer
Cells 3:239-243 ( 1991 ); Burgess, W.H. et al., Annu. Rev. Biochem. 58:575-606
( 1989)).
According to the published literature, the FGF family now consists of at least
nineteen
members, FGF-1 to FGF-19 (Dickson, C. et al., Ann. NYAcad. Sci. 638:18-26
(1991);
Yoshida, T. et al., Ann. NY Acad. Sci. 638:27-37 ( 1991 ); Goldfarb, M. et
al., Ann. NY
l0 Acad. Sci. 638:38-52 ( 1991 ); Coulier, F. et al., Ann. NY Acad. Sci.
638:53-61 ( 1991 );
Aaronson, S.A. et al., Ann. NY Acad. Sci. 638:62-77 (1991); Tanaka, A. et al.,
Proc.
Natl. Acad. Sci. USA 89:8928-8932 (1992); Miyamoto, M. et al., Mol. Cell.
Biol.
13:4251-4259 (1993); Yamasaki, M. et al., J. Biol. Chem. 271:15918-15921
(1996);
Smallwood, P.M. et al., Proc. Natl. Acad. Sci. USA 93:9850-9857 (1996);
McWhirter,
J.R. et al., Development 124:3221-3232 (1997); Miyake, A. et al., Biochem.
Biophys.
Res. Commun. 243:148-152 (1998); Hoshikawa, M. et al., Biochem. Biophys. Res.
Commun. 244:187-191 (1998); Ohbayashi, N. et al., J. Biol. Chem. 273:18161-
18164
(1998); Nishimura, T. et al., Biochim. Biophys. Acta 1444:148-151 (1999)). FGF-
3 was
identified to be a common target for activation by the mouse mammary tumor
virus
(Dickson, C. et al., Ann. NY Acad. Sci. 638:18-26 ( 1991 )). FGF-4 to FGF-6
were
identified as oncogene products (Yoshida, T. et al., Ann. NY Acad. Sci. 638:27-
37
(1991); Goldfarb, M. et al., Ann. NY Acad. Sci. 638:38-52 (1991); Coulier, F.
et al.,
Ann. NY Acad. Sci. 638:53-61 (1991)). FGF-10 was identified from rat lung by
homology-based polymerase chain reaction (PCR) (Yamasaki, M. et al., J. Biol.
Chem.
271:15918-15921 (1996)). FGF-11 to FGF-14 (FGF homologous factors (FHFs) 1 to
4)
were identified from human retina by a combination of random cDNA sequencing,
data
base searches and homology-based PCR (Smallwood, P.M. et al., Proc. Natl.
Acad. Sci.
USA 93:9850-9857 (1996)). FGF-15 was identified as a downstream target of a
chimeric homeodomain oncoprotein (McWhirter, J.R. et al., Development 124:3221-
3232 (1997)). FGF-16, FGF-17, and FGF-18 were identified from rat heart and
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embryos by homology-based PCR, respectively (Miyake, A. et al., Biochem.
Biophys.
Res. Commun. 243:148-152 (1998); Hoshikawa, M. et al., Biochem. Biophys. Res.
Commun. 244:187-191 (1998); Ohbayashi, N. et al., J. Biol. Chem. 273:18161-
18164
(1998)). Recently, FGF-19 was identified from human fetal brain by data base
search
(Nishimura, T. et al., Biochim. Biophys. Acta 1444:148-151 (1999)). They have
a
conserved N 120-amino acid residue core with ~~30 to 60% amino acid identity.
These
FGFs also appear to play important roles in both developing and adult tissues.
Thus,
there is a need in the art for additional FGF molecules having functions and
activities
that differ from the known FGFs and for FGF molecules specifically expressed
in
regions of the brain implicated in human disease.
SUMMARY OF THE INVENTION
The present invention provides a composition comprising an isolated
polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising at least eight contiguous nucleotides
of SEQ ID NO:1 or 3;
(b) a polynucleotide that encodes a variant of the polypeptide
encoded by (a); and
(c) a polynucleotide encoding a protein expressed by a
polynucleotide having the sequence of SEQ ID NO:1 or 3.
The invention further provides for the use of the isolated polynucleotides
or fragments thereof as diagnostic probes or as primers.
The present invention also provides a composition comprising a
polypeptide, wherein said polypeptide is selected from the group consisting
of:
(a) a polypeptide comprising at least 6 contiguous amino acids
encoded by SEQ ID NO:1 or 3;
(b) a polypeptide encoded by a polynucleotide comprising SEQ ID
NO:1 or 3; and
(c) a variant of the polypeptide of (a) or (b).
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Polypeptides of the invention are shown in SEQ ID N0:2 and 4. Other
polypeptides comprise fragments of SEQ ID N0:2 and 4.
In certain preferred embodiments of the invention, the polynucleotide is
operably linked to an expression control sequence. The invention further
provides a
host cell, including bacterial, yeast, insect and mammalian cells, transformed
with the
polynucleotide sequence. The invention also provides full-length cDNA and full-
length
polynucleotides corresponding to SEQ ID NO:1 or 3.
Protein and polypeptide compositions of the invention may further
comprise a pharmaceutically acceptable carrier. Compositions comprising an
antibody
that specifically reacts with such protein or polypeptide are also provided by
the present
invention.
The invention also provides for the production of large amounts of
otherwise minor cell populations of cells to be used for generation of cDNA
libraries for
the isolation of rare molecules expressed in the precursors cell or progeny;
cells
produced by treatment may directly express growth factors or other molecules,
and
conditioned media is screened in assays for novel activities.
The invention further provides for the isolation, self renewal and
survival of mammalian neural stem cells and the differentiation of their
progeny.
The invention also provides for compositions and methods of preventing
or slowing degeneration of or increasing the numbers of dopaminergic neurons,
such as
in the substantial nigra, in disease states including Parkinson's disease.
The invention further provides for compositions and methods of
preventing or slowing degeneration of, or for enhancing the growth of, cells
in the inner
ear.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Amino acid sequence comparison of rat FGF-20 with rat FGF-
9 and FGF-16. Numbers refer to amino acid positions of FGF-9, FGF-16 and FGF-
20.
Asterisks indicate identical amino acid residues of the sequences.
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Figure 2. The apparent evolutionary relationships of 20 members of the
FGF family. The length of each horizontal line is proportional to the degree
of amino
acid sequence divergence. mFGF, rFGF, and hFGF indicate mouse FGF, rat FGF,
and
human FGF, respectively.
Figure 3. Localization of FGF-20 mRNA in rat brain. (A, B) Coronal
sections were hybridized with 35S-labeled FGF-20 antisense (A) and sense (B)
probes,
and exposed to X-ray film for 10 days. Section A is adjacent to section B.
Scale bar =
0.5 cm. (C, D) Sections A and B were dipped in liquid emulsion and
counterstained
with Cresyl violet after 3 weeks. Dark-field photographs of the SNC in
sections A and
1 o B are shown in C and D, respectively. White grains in the dark-field
photograph show
the localization of FGF-20 mRNA. Scale bar = SO Vim. SNC, substantial nigra
pars
compacta.
Figure 4. Expression of FGF-20 mRNA in rat brain. Rat brain poly
(A)+RNA (10~g) was electrophoresed on a denaturing agarose gel (1%) containing
formaldehyde and transferred onto a nitrocellulose membrane. Hybridization was
performed with a 32P labeled rat FGF-20 or ~3-actin cDNA probe. 28S and 18S
indicate
the positions of 28 and 18S rRNAs, respectively.
Figure 5. FGF-20 enhances survival of midbrain dopaminergic neurons.
(A) Effect of FGF-20 on survival of midbrain dopaminergic neurons in serum-
free
2o medium. Midbrain cultured cells were incubated for 4 days in medium
supplemented
with 10% horse serum (control) or serum-free medium supplemented with FGF-20,
and
then the numbers of surviving dopaminergic neurons were determined. (B) Effect
of
FGF-20 for 24 h, and then treated with no (control) or 1 mM glutamate for 10
min. The
cultured cells were further incubated in the medium in the absence of
glutamate and
FGF-20 for 3 days, and then the numbers of surviving dopaminergic neurons were
determined.
Figure 6. Figure 6 provides the DNA sequence (SEQ ID NO:1 ) of rat
FGF-20.
Figure 7. Figure 7 provides the amino acid sequence (SEQ ID N0:2) of
rat FGF-20.
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Figure 8. Figure 8 provides the DNA (SEQ ID N0:3) and amino acid
(SEQ ID N0:4) sequences of human FGF-20.
Figure 9. Figure 9 provides an alignment of the amino acid sequences of
human (SEQ ID N0:4) and rat (SEQ ID N0:2) FGF-20.
Figure 10. Figure 10 provides codon usage for E. coli.
Figure 11. Figure 11 provides codon usage for yeast. The first field of
information on each line of the table contains a three-letter code for an
amino acid. The
second field contains an unambiguous codon for that amino acid. The third
field lists
the number of occurrences of that codon in the genes from which the table is
compiled.
1o The fourth field lists the expected number of occurrences of that codon per
1,000
codons in genes whose codon usage is identical to that compiled in the codon
frequency
table. The last field contains the fraction of occurrences of the codon in its
synonymous
codon family.
Figure 12. Figure 12 provides codon usage for Drosophila.
~5 DETAILED DESCRIPTION OF THE INVENTION
Because of their potent activities for promoting growth, proliferation,
survival and differentiation of a wide variety of cells and tissue types, FGFs
continue to
be pursued as therapeutic agents for a number of different indications,
including wound
healing, such as musculo-skeletal conditions, for example, bone fractures,
ligament and
20 tissue repair, tendonitis, bursitis, etc.; skin conditions, for example,
burns, cuts,
lacerations, bed sores, slow healing ulcers, etc.; tissue protection and
repair during
myocardial infarction and ischemia, in the treatment of neurological
conditions, for
example, neuro-degenerative disease and stroke, in the treatment of eye
disease,
including macular degeneration, and the like.
25 The fibroblast growth factor (FGF) proteins identified to date belong to a
family of signaling molecules that regulate growth and differentiation of a
variety of
cell types. The significance of FGF proteins to human physiology and pathology
relates
in part to their key roles in embryogenesis, in blood vessel development and
growth,
and in bone growth. In vitro experiments have demonstrated a role for FGF in
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regulating cell growth and division of endothelial cells, vascular smooth
muscle cells,
fibroblasts, and cardiac and skeletal myocytes. Other members of the FGF
family and
their biological roles are described in Crossley et al., Development 121:439-
451 (1995);
Ohuchi et al., Development 124:2235-2244 (1997); Gemel et al., Genomics 35:253-
257
s (1996); and Ghosh et al., Cell Growth and Differentiation 7:1425-1434
(1996).
FGF proteins are also significant to human health and disease because of
a role in cancer cell growth. For example, FGF-8 was identified as an androgen-
induced growth factor in breast and prostate cancer cells. (Tanaka et al.,
FEBS Lett.
363:226-230 (1995) and P.NA.S. 89:8928-8932 (1992)).
The role of FGF in normal development is being elucidated in part
through studies of FGF receptors. Wilke, T. et al., Dev. Dynam. 210:41-52
(1997)
found that FGFR1, FGFR2, and FGFR3 transcripts were localized to specific
regions of
the head during embryonic development in chickens. The expression pattern
correlated
with areas affected by human FGFR mutations in Crouzon syndrome, a condition
of
~ s abnormal intramembranous bone formation. Belluardo, N. et al., Jour. Comp.
Neur.
379:226-246 (1997) studied localization of FGFR 1, 2, and 3 mRNAs in rat
brain, and
found cellular specificity in several brain regions. Furthermore, FGFR1 and
FGFR2
mRNAs were expressed in astroglial reactive cells after brain lesion,
supporting a role
of certain FGF's in brain disease and injury. Ozawa, K. et al., Mol. Brain
Res. 41:279-
20 288 (1996) reported that FGF1 and FGF-5 expression increased after birth,
whereas
FGF3, FGF-6, FGF-7, and FGF-8 genes showed higher expression in late embryonic
stages than in postnatal stages.
New members of the FGF family are described here, wherein the FGF
protein is expressed in dopaminergic neurons of the substantial nigra and in
cochlear
2s tissue of rat embryos. A polynucleotide encoding the rat FGF of the
invention has the
sequence as shown in SEQ ID NO:1. A polynucleotide encoding the human FGF of
the
invention has the sequence as shown in SEQ ID N0:3. The rat polynucleotide was
identified as encoding a member of the FGF family by the conserved regions
throughout the amino acid sequence and by the regions of homology shared by
the
3o polynucleotides and genes encoding known FGF proteins.
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The inventors believe that FGF-20 is a previously unidentified member
of the FGF family. To date, over 19 human FGF proteins have been identified.
In most
cases, homologous proteins in the other mammals, particularly mice and rats,
have also
been identified. The human proteins vary to different degrees in terms of
amino acid
sequence, receptor specificity, tissue expression patterns, and biological
activity.
The present FGF-20 differs in sequence from all the FGF proteins
described to date in publications. FGF-20 shares some homology with FGF-9 and
FGF-16.
As discussed herein, the knowledge about the roles played by various
FGF proteins continues to grow, but is by far incomplete.
The present invention adds to this knowledge by disclosing that the FGF
of SEQ ID NO:I is highly expressed in dopaminergic neurons of the substantia
nigra of
brain, and human FGF-20 may play a role in development of and recovery from a
neural disease, such as Parkinson's disease. FGF-20 is also preferentially
expressed in
rat embryo (E14.5) cochlea of the inner ear.
The invention therefore is based upon the identification, isolation,
sequencing and expression patterns of a new fibroblast growth factor (FGF-20).
Isolation and Analysis of Rat cDNA encoding FGF 20 Members of the
FGF family have a conserved N 120 -amino acid residue core with N30 to 70%
amino
2o acid identity. Among the members of the FGF family, FGF-9 and FGF-16 are
highly
homologous (73% amino acid identity). According to the invention, DNA encoding
a
novel rat FGF has been identified. The nucleotide sequence of the entire
coding region
was determined by adaptor-ligation mediated polymerase chain reaction using
rat-brain
cDNA as a template and cassette-ligation mediated polymerase chain reaction
using rat
genomic DNA as a template. The nucleotide sequence of the coding region
allowed for
the elucidation of the complete amino acid sequence of the FGF (212 amino
acids),
which has a conserved amino acid residue core (amino acids 62 to 197) (Fig. 1
). Two
cysteine residues that are well conserved in the FGF family are also conserved
in the
protein (amino acids 71 and 137) (Fig. 1). This protein is tentatively named
FGF-20.
3o FGF-20 is most similar to FGF-9 and FGF-16 (70 and 62% amino acid identity)
among
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19 members of the FGF family, respectively (Fig. 1 ). The apparent
evolutionary
relationships of twenty members of the FGF family are shown in Fig. 2. FGF-20
was
closest to FGF-9 and FGF-16.
Expression of FGF 20 mRNA in Rat Tissues FGF-9 and FGF-16
mRNAs are preferentially expressed in rat kidney and heart, respectively
(Miyamoto,
M. et al., Mol. Cell. Biol. 13:4251-4259 (1993); Miyake, A. et al., Biochem.
Biophys.
Res. Commun. 243:148-152 (1998)). The expression of FGF-20 mRNA was examined
in adult rat major tissues including brain, heart, lung, liver, kidney, and
small intestine
by polymerase chain reaction. FGF-20 mRNA was detected in the brain, but was
undetectable or present in very low levels in other tissues. To confirm the
expression of
FGF-20 mRNA in rat brain, rat brain poly (A)+ RNA was examined by Northern
blotting analysis using a 32P-labeled rat FGF-20 cDNA probe. A faint but
definite
signal of FGF-20 mRNA was detected (Fig. 4). To confirm the integrity of the
poly
(A)+ RNA, the hybridized probe was washed from the membrane, and the membrane
t5 was rehybridized with a 3zP-labeled rat f3-actin cDNA probe. A strong and
discrete
signal of 13-actin mRNA was detected indicating that the poly (A)+ RNA was not
degraded (Fig. 4). FGF-20 mRNA was also detected in rat embryos (E14.5),
specifically in the cochlea of the inner ear, using 35S-labeled FGF-20 anti-
sense and
sense cRNA probes (Fig. 10).
2o Expression of FGF 20 mRNA in Rat Brain To examine the expression
of FGF-20 mRNA in rat brain, consecutive coronal sections of rat brain were
analyzed
by in situ hybridization with an 35S-labeled antisense or sense FGF-20 cRNA
probe.
Discrete specific labeling was observed only in the substantia nigra pans
compacta (Fig.
3A, C). No specific labeling was observed in other brain regions examined. The
25 cellular localization of FGF-20 mRNA was examined by microscopy at higher
magnification. By Nissle staining of brain sections, glial cells can be
identified as small
intensely stained (dark) cells, while neurons are generally larger and less
intensely
stained (lighter) owing to their larger volume (Gerfen, C.R., Methods in
Neurosciences,
Academic Press, San Diego, CA, Vol. l, pp. 79-97 (1989)). Black grains of
labeled
3o probes were found in most neurons of these brain areas (Fig. 3E).
Dopaminergic
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neurons in the substantial nigra are preferentially localized in the
substantial nigra pars
compacta (Fallon, J.H. et al., The Rat Nervous System, 2"d Ed., Academic
Press, San
Diego, CA, pp. 215-238 (1995)). Furthermore, neurons in the substantia nigra
pars
compacta predominantly consist of dopaminergic neurons (Fallon, J.H. et al.,
The Rat
Nervous System, 2"d Ed., Academic Press, San Diego, CA, pp. 215-238 (1995)).
It is
expected that FGF-20 is preferentially expressed in dopaminergic neurons in
the
substantia nigra pars compacta.
Preparation of Recombinant Rat FGF 20 To produce recombinant rat
FGF-20, High Five insect cells were infected with recombinant baculovirus
containing
1 o the rat FGF-20 cDNA with the 3'-terminal extension encoding E and Hiss
tags. To
detect recombinant FGF-20 in the culture medium, the medium was examined by
Western blotting analysis with anti-E tag antibodies. A major band of 26.5 kDa
was
detected in the culture medium. The observed molecular mass of the major band
was
consistent with the calculated molecular mass of recombinant FGF-20 (26,247).
This
result indicates that FGF-20 is secreted, although on hydropathy plot analysis
(Nielsen,
H. et al., Protein Engineering 10:1-6 (1997)) the value of the amino-terminal
region of
FGF-20 was low, suggesting that FGF-20 has no signal sequence. Although FGF-9
and
FGF-16 have no typical signal sequence in their amino termini, they are also
secreted
(Miyamoto, M. et al., Mol. Cell. Biol. 13:4251-4259 (1993), Miyake, A. et al.,
Biochem.
2o Biophys. Res. Commun. 243:148-152 (1998)). Recombinant FGF-20 was purified
from
the culture medium by affinity chromatograph with Ni-NTA agarose and was
analyzed
by SDS-polyacrylamide gel electrophoresis under reducing conditions. A 26.5
kDa
protein of FGF-20 was detected.
Neurotrophic Activity of FGF 20 for Rat Midbrain Dopaminergic
Neurons FGFs are local signal molecules that act on proximal cells (Burgess,
W.H. et
al., Annu. Rev. Biochem. 58:575-606 (1989)). Therefore, it was expected that
FGF-20
acts on dopaminergic neurons in the substantia nigra in autocrine and/or
paracrine
manner. The neurotrophic activity of FGF-20 for cultured rat midbrain
dopaminergic
neurons was examined. When the dopaminergic neurons were cultured in a serum-
free
3o medium for 4 days, numbers of surviving dopaminergic neurons were greatly
reduced.
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FGF-20 significantly enhanced survival of the dopaminergic neurons in the
serum-free
medium (Fig. 5A). The effect of FGF-20 on glutamate-induced neuronal death in
cultured rat midbrain dopaminergic neurons was also examined. When the
cultured
cells were exposed to 1 mM glutamate for 10 min, the numbers of surviving
dopaminergic neurons were reduced. FGF-20 also significantly enhanced survival
of
the dopaminergic neurons exposed to toxic concentrations of glutamate (Fig.
5B).
Several FGFs are expressed in brain and expected to play important roles
as neutrophic factors. FGF-1 and FGF-2 are abundant in brain (Gospodarowicz,
D.,
Methods Enzymol. 147:106-119 (1987)) and exert survival enhancing effects on
primary
cultures from various regions of the brain (Walicke, P.A., J. Neurosci. 8:2618-
2627
(1988)). FGF-1 is expressed predominantly in motor and sensory neurons of the
midbrain and brainstem (Elde, R. et al., Neuron 7:349-364 ( 1991 )). In
contrast, FGF-2
is preferentially expressed in neurons in restricted regions including the
cingulate
cortex, industium grieum, fasciola cinererum and hippocampus, and in
astrocytes in
widespread regions of the brain (Emoto, N. et al., Growth Factors 2:21-29
(1989);
Woodward, W.R. et al., J. Neurosci. 12:142-152 (1992)). FGF-5 is weakly
expressed in
the cerebral cortex, hippocampus and thalamus (Haub, O. et al., Proc. Natl.
Acad. Sci.
USA 87:8022-8026 (1990)). FGF-9 and FGF-11 to FGF-14 are expressed in neurons
of
restricted regions including the hippocampus, thalamus, midbrain and brainstem
(Yamamoto, S. et al., Biochim. Biophys. Acta 1398:38-41 (1998)). In contrast,
FGF-20
of the invention was preferentially expressed in dopaminergic neurons of the
substantia
nigra. The expression profile of FGF-20 was quite distinct from those of other
FGFs,
indicating that FGF-20 plays a unique role in the brain.
Degeneration of dopaminergic neurons in the substantia nigra causes
Parkinson's disease (Fallon, J.H. et al., The Rat Nervous System, 2"d Ed.,
Academic
Press, San Diego, CA, pp. 215-238 (1995)). Therefore, neurotrophic factors for
dopaminergic neurons in the substantia nigra have received substantial
attention.
GDNF, Persephin, Artemin, BDNF, and NT-3 enhance survival of midbrain
dopaminergic neurons (Lin, L.-F.H. et al., Science 260:1130-1132 (1993);
Milbrandt, J.
3o et al., Neuron 20:245-253 (1998); Baloh, R.H. et al., Neuron 21:1291-1302
(1998);
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Hyman, C. et al., Nature 350:230-232 (1991); Hyman, C. et al., J. Neurosci.
14:335-347
(1994)). However, their expression is not restricted to the substantia nigra
(Pochon,
N.A. et al., Eur. J. Neurosci. 9:463-471 (1997); Milbrandt, J. et al., Neuron
20:245-253
(1998); Baloh, R.H. et al., Neuron 21:1291-1302 (1998); Ernfors, P. et al.,
Neuron
5:511-526 (1990)). In contrast, the expression of FGF-20, which also enhanced
the
survival of midbrain dopaminergic neurons, was highly restricted in
dopaminergic
neurons in the substantia nigra pars compacta. Therefore, FGF-20 is expected
to play
an important role as a neurotrophic factor for dopaminergic neurons in the
substantia
nigra. It is therefore an important finding of the invention that FGF-20 is
the first
neurotrophic factor documented to be expressed preferentially in dopaminergic
neurons
of the substantia nigra.
It is believed that dopamine neurons are dysfunctional for, perhaps,
years, before they are irreversibly damaged. (Dunnett, S.B. et al., Nature
399:A32-A39
(1999)) Thus, neurotrophic agents such as FGF-20 may be useful in preventing
cell
death or restoring function The FGF-20 may be administered using gene transfer
methods to block degeneration. Such methods have been used with neurotrophic
factor
GDNF (glial cell line-derived neutrophic factor). In a rat Parkinson's model,
nanogram
amounts of BDNF and GDNF were measured from transduced cells, and the
neuroprotective effect was in the order of 40-70% rescue of nigral dopamine
neurons.
2o Thus, transplants using fibroblasts or fibroblast cell lines engineered to
secrete FGF-20
of the invention can allow secretion of the factor and rescue of nigral
dopamine
neurons. Alternatively, injection of the striatum or the substantia nigra
region with
viral vectors carrying the FGF-20 gene may also have a neuroprotective effect.
In Parkinson's Disease, neuronal degeneration in the substantial nigra
generally is slow and protracted. This suggests that early intervention could
block or
slow down the degenerative process, perhaps up to 4 or 5 years before clinical
symptoms appear. A decline in striatal dopamine function can be detected by
PET and
SPECT imaging before the appearance of clinical symptoms, providing an
opportunity
for neuroprotective intervention at this early stage.
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In vivo imaging of dopaminergic activity in the basal ganglia, using
[~8F]fluorodopa PET, can be used to monitor progress of the disease as well as
the
impact of treatment. A progressive reduction in fluorodopa signal is seen in
brain tissue
of pre-symptomatic and symptomatic individuals. After treatment with nigral
tissue
implant, the fluorodopa signal increases over time. (Dunnett et al., supra. )
This and
other techniques known in the art can be used to measure the effect of
treatments
described herein using FGF-20, and the clinician will be skilled in the art of
determining appropriate treatment levels and regimens.
FGF 20 Expression in Rat Embryo Cochlea FGF-20 is preferentially
t0 expressed in the cochlea of the inner ear in rat embryos (E14.5). This
supports a role
for FGF-20 in the development and maintenance of normal ear function. Other
previously-identified members of the FGF family contribute to normal ear
growth and
development. For example, sensory cells in the cochlea of the rat transiently
express
FGF-1 during the time of terminal innervation in the sensory epithelium
(Dazert et al.,
J. Cell Physiol. 177:123-129 (1998)). These authors also found that in vitro,
spiral
ganglion explants cultured in the presence of FGF-1 exhibited a dose-dependent
increase in the number and length of neurites. In chick cochlea, FGF-1 mRNA
levels
increased in sensory epithelium of the cochlea in response to ototoxic damage,
suggesting that the FGF system may be involved in the response of the cochlear
2o epithelium to ototoxic damage. Pickles et al., Dev. Neuroscience 19:476-487
( 1997).
FGF-2 may help to regulate the proliferation step during hair cell development
and
regeneration after trauma in rats. Zheng et al., J. Neuroscience 17:216-226
(1997).
Thus, FGF molecules play several roles in maintaining normal development and
function of the cochlea, and recovery of the cochlea from ototoxic damage. The
absence of FGF receptor 3 contributes to inner ear defects in mice homozygous
for
skeletal and inner ear defects, including failure of pillar cell
differentiation and tunnel of
Corti formation, and profound deafness. Colvin et al., Nat. Genet. 12:390-397
(1996).
It is of interest that FGF-20 of the invention binds to FGF receptor 3c
(Example 12).
The fact that FGF-20 is expressed at a specific stage in rat inner ear
development further
3o suggests its importance in development of this tissue.
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FGF-20 therefore may be suitable for treating a variety of conditions
related to the ear. Currently, about 7.8 million Americans have mild hearing
loss, 10
million have moderate hearing loss, and 2.7 million have profound or severe
hearing
loss. The causes include, but are not limited to, otosclerosis; Cogan's
syndrome;
Meniere's disease; Pendred's syndrome; diabetes-associated hearing loss (non-
insulin-
dependent diabetes mellitus in combination with obesity can cause tissue
changes in the
cochlea, McQueen et al., J. Laryngol. Otol. 113:113-118 (1999)); congenital
malformations; autoimmune disease-related hearing loss; age-related hearing
loss;
deafness associated with lack of FGF receptor (Colvin et al., Nat. Genet.
12:390-397
t o ( 1996)); ischemia-related hearing disturbance; and other conditions in
which cochlear
structure and function plays a role. Administration of FGF-20 protein or
polynucleotide
may be used to treat inherited, congenital and acquired diseases of hearing
and balance,
by promoting the survival, proliferation or differentiation of cells of the
inner ear.
Reference to FGF-20 herein is intended to be construed to include
t 5 growth factors of any origin which are substantially homologous to and
which are
biologically equivalent to the FGF-20 characterized and described herein. Such
substantially homologous growth factors may be native to any tissue or species
and,
similarly, biological activity can be characterized in any of a number of
biological assay
systems.
20 The term "biologically equivalent" is intended to mean that the
compositions of the present invention are capable of demonstrating some or all
of the
same growth properties in a similar fashion, not necessarily to the same
degree as the
FGF-20 isolated as described herein or recombinantly produced human FGF-20 of
the
invention.
25 By "substantially homologous" it is meant that the degree of homology
of human FGF-20 to FGF-20 from any species is greater than that between FGF-20
and
any previously reported member of the FGF family.
Sequence identity or percent identity is intended to mean the percentage
of same residues between two sequences, referenced to human FGF when
determining
3o percent identity with non-human FGF-20, referenced to FGF-20 when
determining
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WO 01/31008 PCT/US00/29237
percent identity with non-FGF-20 growth factors, when the two sequences are
aligned
using the Clustal method (Higgins et al., Cabios 8:189-191 (1992)) of multiple
sequence alignment in the Lasergene biocomputing software (DNASTAR, INC,
Madison, WI). In this method, multiple alignments are carried out in a
progressive
manner, in which larger and larger alignment groups are assembled using
similarity
scores calculated from a series of pairwise alignments. Optimal sequence
alignments are
obtained by finding the maximum alignment score, which is the average of all
scores
between the separate residues in the alignment, determined from a residue
weight table
representing the probability of a given amino acid change occurring in two
related
proteins over a given evolutionary interval. Penalties for opening and
lengthening gaps
in the alignment contribute to the score. The default parameters used with
this program
are as follows: gap penalty for multiple alignment=10; gap length penalty for
multiple
alignment=10; k-tuple value in pairwise alignment=1; gap penalty in pairwise
alignment=3; window value in pairwise alignment=5; diagonals saved in pairwise
~ 5 alignment=5. The residue weight table used for the alignment program is
PAM250
(Dayhoff et al., in Atlas of Protein Sequence and Structure, Dayhoff, Ed.,
NDRF,
Washington, Vol. 5, suppl. 3, p. 345, 1978).
Percent conservation is calculated from the above alignment by adding
the percentage of identical residues to the percentage of positions at which
the two
2o residues represent a conservative substitution (defined as having a log
odds value of
greater than or equal to 0.3 in the PAM250 residue weight table). Conservation
is
referenced to human FGF-20 when determining percent conservation with non-
human
FGF-20, and referenced to FGF-20 when determining percent conservation with
non
FGF-20 growth factors. Conservative amino acid changes satisfying this
requirement
25 are: R-K; E-D, Y-F, L-M; V-I, Q-H.
The invention provides FGF-20 proteins or variants thereof having one
or more polymers covalently attached to one or more reactive amino acid side
chains.
By way of example, not limitation, such polymers include polyethylene glycol
(PEG),
which can be attached to one or more free cysteine sulfhydryl residues,
thereby
3o blocking the formation of disulfide bonds and aggregation when the protein
is exposed
CA 02388637 2002-04-19
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to oxidizing conditions. In addition, pegylation of FGF-20 proteins and/or
muteins is
expected to provide such improved properties as increased half life,
solubility, and
protease resistance. FGF-20 proteins and/or muteins may alternatively be
modified by
the covalent addition of polymers to free amino groups such as the lysine
epsilon or the
N-terminal amino group. Preferred cysteines and lysines for covalent
modification will
be those not involved in receptor or heparin binding. In both human and rat
FGF-20,
the heparin binding site comprises amino acids 170-186. It will be apparent to
one
skilled in the art that the methods for assaying FGF-20 biochemical and/or
biological
activity may be employed in order to determine if modification of a particular
amino
acid residue affects the activity of the protein as desired.
It may be advantageous to improve the stability of FGF-20 by modifying
one or more protease cleavage sites. Thus, the present invention provides FGF-
20
variants in which one or more protease cleavage site has been altered by, for
example,
substitution of one or more amino acids at the cleavage site in order to
create as FGF-20
variant with improved stability. Such improved protein stability may be
beneficial
during protein production and/or therapeutic use.
Suitable protease cleavage sites for modification are well known in the
art and likely will vary depending on the particular application contemplated.
For
example, typical substitutions would include replacement of lysines or
arginines with
other amino acids such as alanine. The loss of activity, such as receptor
binding or
heparin binding, can be tested for as described herein.
FGF-20 can also include hybrid and modified forms of FGF-20
including fusion proteins and FGF-20 fragments and hybrid and modified forms
in
which certain amino acids have been deleted or replaced and modifications such
as
where one or more amino acids have been changed to a modified amino acid or
unusual
amino acid and modifications such as glycosylations so long as the hybrid or
modified
form retains the biological activity of FGF-20. By retaining the biological
activity, it is
meant that neuronal survival is promoted, although not necessarily at the same
level of
potency as that of the FGF-20 isolated as described herein or that of the
recombinantly
produced human FGF-20. Fusion proteins can consist of the FGF-20 of the
invention or
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WO 01/31008 PCT/US00/29237
fragment thereof and a signal sequence of a heterologous protein to promote
secretion
of the protein product.
Fusion proteins comprising FGF-20 or a biologically active or antigenic
fragment thereof can be produced using methods known in the art. Such fusion
proteins
can be used therapeutically or can be produced in order to simplify the
isolation and
purification procedures. Histidine residues can be incorporated to allow
immobilized
metal affinity chromatography purification. Residues EQKLISEEDL contain the
antigenic determinant recognized by the myc monoclonal antibody and can be
incorporated to allow myc monoclonal antibody-based affinity purification. A
thrombin
1o cleavage site can be incorporated to allow cleavage of the molecule at a
chosen site; a
preferred thrombin cleavage site consists of residues LVPRG. Purification of
the
molecule can be facilitated by incorporating a sequence, such as residues
SAWRHPQFGG, which binds to paramagnetic streptavidin beads. Such embodiments
are described in WO 97/25345, which is incorporated by reference.
The invention further includes chimeric molecules between FGF-20 and
keratinocyte growth factor (KGF) (Reich-Slotky, R. et al., J. Biol. Chem.
270:29813-
29818 (1995)). The chimeric molecule can contain specific regions or fragments
of one
or both of the FGF-20 and KGF molecules, such as the FGF-20 fragments
described
below.
2o The invention also includes fragments of FGF-20. Preferred fragments
of SEQ ID N0:4 and 2 include: amino acids from about 170 to about 186; amino
acids
from about 1 to about 169; amino acids 2-211 (212 for SEQ ID N0:2); amino
acids
from about 1 to about 169 and about 187 to about 211 (212 for SEQ ID N0:2),
wherein
amino acids about 169 and about 187 are joined by a peptide bond; and amino
acids
from about 59 to about 193. Such fragments can be prepared from the protein by
standard biochemical methods or by expressing a polynucleotide encoding the
fragment.
FGF-20, or a fragment thereof, can be produced as a fusion protein
comprising human serum albumin (HSA) or a portion thereof. Such fusion
constructs
are suitable for enhancing expression of the FGF-20, or fragment thereof, in
an
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WO 01/31008 PCT/US00/29237
eukaryotic host cell. Exemplary HSA portions include the N-terminal
polypeptide
(amino acids 1-369, 1-419, and intermediate lengths starting with amino acid
1), as
disclosed in U.S. Patent No. 5,766,883, and publication WO 97/24445,
incorporated by
reference herein. Other chimeric polypeptides can include a HSA protein with
FGF-20,
or fragments thereof, attached to each of the C-terminal and N-terminal ends
of the
HSA. Such HSA constructs are disclosed in U.S. Patent No. 5,876,969,
incorporated by
reference herein.
Also included with the scope of the invention are FGF-20 molecules that
differ from native FGF-20 by virtue of changes in biologically active sites.
FGF-20 has
a putative heparin binding site at amino acid residues 170-186. An FGF-20
molecule
that does not bind heparin can be prepared by expressing DNA encoding FGF-20,
wherein the corresponding codons for amino acid residues 170-186 have been
deleted.
Conversely, one or more additional heparin binding sites can be added to FGF-
20 by,
for example, expressing DNA encoding FGF-20 wherein the codons corresponding
to
~5 residues 170-186 are inserted at the desired positions) in the reading
frame. DNA
encoding FGF-20 with altered receptor binding can likewise be produced. For
example,
it may be desirable to alter receptor specificity of FGF-20 by substituting
the receptor
binding regions of a different FGF for that of FGF-20.
Also included within the meaning of substantially homologous is any
2o FGF-20 which may be isolated by virtue of cross-reactivity with antibodies
to the FGF-
20 described herein or whose encoding nucleotide sequences including genomic
DNA,
mRNA or cDNA may be isolated through hybridization with the complementary
sequence of genomic or subgenomic nucleotide sequences or cDNA of the FGF-20
herein or fragments thereof. It will also be appreciated by one skilled in the
art that
25 degenerate DNA sequences can encode human FGF-20 and these are also
intended to be
included within the present invention, as are mammalian allelic variants of
FGF-20.
Growth factors are thought to act at specific receptors. According to the
invention, FGF-20 and as yet unknown members of this family of growth factors
act
through specific receptors having distinct distributions as has been shown for
other
30 growth factor families. FGF-20 binds to FGF receptor 2 and FGF receptor 3,
but does
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WO 01/31008 PCT/US00/29237
not bind to FGF receptor 1. Thus, its receptor binding profile differs from
FGF-2 and
FGF-4, which bind to FGF receptor 1.
A preferred hFGF-20 of the present invention has been identified and
isolated in purified form as described. Also preferred is hFGF-20 prepared by
recombinant DNA technology. By "pure form" or "purified form" or
"substantially
purified form" it is meant that an FGF-20 composition is substantially free of
other
proteins which are not FGF-20.
Recombinant human FGF-20 may be made by expressing the DNA
sequences encoding FGF-20 in a suitable transformed host cell. Using methods
well
known in the art, the DNA encoding FGF-20 may be linked to an expression
vector,
transformed into a host cell and conditions established that are suitable for
expression of
FGF-20 by the transformed cell.
The DNA encoding FGF-20 can be engineered to take advantage of
preferred codon usage of host cells. Codon usage in Pseudomonas aeruginosa is
described in, for example, West et al., Nucleic Acids Res. 11:9323-9335
(1988). Codon
usage in Saccharomyces cerevisiae is described in, for example, Lloyd et al.,
Nucleic
Acids Res. 20:5289-5295 (1992). Codon preference in Corynebacteria and a
comparison with E. coli preference is provided in Malubres et al., Gene 134:15-
24
(1993). Codon usage in Drosophila melanogaster is described in, for example,
Akashi,
Genetics 136:927-935 (1994). Codon usage in yeast is also shown in Figure 11,
and
codon usage in Drosophila is show in Figure 12.
Any suitable expression vector may be employed to produce
recombinant human FGF-20 such as expression vectors for use in insect cells.
Baculovirus expression systems can also be employed. A preferable method is
expression in insect cells, such as Tr5 or Sf~ cells, using baculovirus
vector.
The present invention includes nucleic acid sequences including
sequences that encode human FGF-20. Also included within the scope of this
invention
are sequences that are substantially the same as the nucleic acid sequences
encoding
FGF-20. Such substantially the same sequences may, for example, be substituted
with
codons more readily expressed in a given host cell such as E. coli according
to well
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WO 01/31008 PCT/US00/29237
known and standard procedures. Such modified nucleic acid sequences are
included
within the scope of this invention.
Specific nucleic acid sequences can be modified by those skilled in the
art and, thus, all nucleic acid sequences that code for the amino acid
sequences of FGF-
20 can likewise be so modified. The present invention thus also includes
nucleic acid
sequence which will hybridize with all such nucleic acid sequences or
complements of
the nucleic acid sequences where appropriate and encode a polypeptide having
the
neuronal cell survival promoting activities disclosed herein. The present
invention also
includes nucleic acid sequences that encode polypeptides that have neuronal
cell
1 o survival promoting activity and that are recognized by antibodies that
bind to FGF-20.
Preferred methods and epitopes for raising antibodies are described in Example
10.
The present invention also encompasses vectors comprising expression
regulatory elements operably linked to any of the nucleic acid sequences
included
within the scope of the invention. This invention also includes host cells of
any variety
that have been transformed with vectors comprising expression regulatory
elements
operably linked to any of the nucleic acid sequences included within the scope
of the
presentinvention.
Methods are also provided herein for producing FGF-20. Preparation
can be by isolation from conditioned medium from a variety of cell types so
long as the
2o cell type produces FGF-20. A second and preferred method involves
utilization of
recombinant methods by isolating or obtaining a nucleic acid sequence encoding
FGF-
20, cloning the sequence along with appropriate regulatory sequences into
suitable
vectors and cell types, and expressing the sequence to produce FGF-20.
Although FGF-20 has been described on the basis of its ability to
enhance the survival of midbrain dopaminergic neurons, this factor may act on
other
cell types as well. Thus, it is likely that FGF-20 can act on other neural
cells.
It is also likely that FGF-20 will act on non-neuronal cells to promote
their survival, growth or function. This expectation is based upon the
activity of known
growth factors. Members of the FGF family act on many cell types of different
function
and embryologic origin.
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The inventors herein have identified that FGF-20 is expressed in the
brain, but not in other adult tissues, including heart, lung, liver, kidney
and small
intestine. This suggests a role for FGF-20 in, for example, Parkinson's
disease and other
diseases of neural tissue.
The present invention also includes therapeutic or pharmaceutical
compositions comprising FGF-20 in an effective amount for treating patients
with
neuronal disease including Parkinson's disease, and a method comprising
administering
a therapeutically effective amount of FGF-20. These compositions and methods
are
useful for treating a number of diseases. The compositions and methods herein
can also
be useful to prevent degeneration and/or promote survival in other non-
neuronal tissues
as well. One skilled in the art can readily use a variety of assays known in
the art to
determine whether FGF-20 would be useful in promoting survival or functioning
in a
particular cell type, such as neuronal cells.
In certain circumstances, it may be desirable to modulate or decrease the
amount of FGF-20 expressed. Thus, in another aspect of the present invention,
FGF-20
anti-sense oligonucleotides can be made and a method utilized for diminishing
the level
of expression of FGF-20 by a cell comprising administering one or more FGF-20
anti-
sense oligonucleotides. By FGF-20 anti-sense oligonucleotides reference is
made to
oligonucleotides that have a nucleotide sequence that interacts through base
pairing
2o with a specific complementary nucleic acid sequence involved in the
expression of
FGF-20 such that the expression of FGF-20 is reduced. Preferably, the specific
nucleic
acid sequence involved in the expression of FGF-20 is a genomic DNA molecule
or
mRNA molecule that encodes FGF-20. This genomic DNA molecule can comprise
regulatory regions of the FGF-20 gene, or the coding sequence for mature FGF-
20
protein. The term complementary to a nucleotide sequence in the context of FGF-
20
antisense oligonucleotides and methods therefor means sufficiently
complementary to
such a sequence as to allow hybridization to that sequence in a cell, i.e.,
under
physiological conditions. The FGF-20 antisense oligonucleotides preferably
comprise a
sequence containing from about 8 to about 100 nucleotides and more preferably
the
3o FGF-20 antisense oligonucleotides comprise from about 15 to about 30
nucleotides.
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The FGF-20 antisense oligonucleotides can also contain a variety of
modifications that
confer resistance to nucleolytic degradation such as, for example, modified
internucleoside linages (Uhlmann and Peyman, Chemical Reviews 90:543-548 1990;
Schneider and Banner, Tetrahedron Lett. 31:335 (1990), which are incorporated
by
reference), modified nucleic acid bases and/or sugars and the like.
The therapeutic or pharmaceutical compositions of the present invention
can be administered by any suitable route known in the art including for
example
intravenous, subcutaneous, intramuscular, transdermal, intrathecal or
intracerebral.
Administration can be either rapid as by injection or over a period of time as
by slow
1o infusion or administration of slow release formulation. For treating
tissues in the
central nervous system, administration can be by injection or infusion into
the
cerebrospinal fluid (CSF). When it is intended that FGF-20 be administered to
cells in
the central nervous system, administration can be with one or more agents
capable of
promoting penetration of FGF-20 across the blood-brain barrier.
t5 FGF-20 can also be linked or conjugated with agents that provide
desirable pharmaceutical or pharmacodynamic properties. For example, FGF-20
can be
coupled to any substance known in the art to promote penetration or transport
across the
blood-brain barrier such as an antibody to the transferrin receptor, and
administered by
intravenous injection (see, for example, Friden et al., Science 259:373-377
(1993),
2o which is incorporated by reference). Furthermore, FGF-20 can be stably
linked to a
polymer such as polyethylene glycol to obtain desirable properties of
solubility,
stability, half life and other pharmaceutically advantageous properties. (See,
for
example, Davis et al., Enzyme Eng. 4:169-73 (1978); Burnham, Am. J. Hosp.
Pharm.
51:210-218 (1994), which are incorporated by reference.)
25 The compositions are usually employed in the form of pharmaceutical
preparations. Such preparations are made in a manner well known in the
pharmaceutical art. One preferred preparation utilizes a vehicle of
physiological saline
solution, but it is contemplated that other pharmaceutically acceptable
carriers such as
physiological concentrations of other non-toxic salts, five percent aqueous
glucose
3o solution, sterile water or the like may also be used. It may also be
desirable that a
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suitable buffer be present in the composition. Such solutions can, if desired,
be
lyophilized and stored in a sterile ampoule ready for reconstitution by the
addition of
sterile water for ready injection. The primary solvent can be aqueous or
alternatively
non-aqueous. FGF-20 can also be incorporated into a solid or semi-solid
biologically
compatible matrix which can be implanted into tissues requiring treatment.
The carrier can also contain other pharmaceutically-acceptable excipients
for modifying or maintaining the pH, osmolarity, viscosity, clarity, color,
sterility,
stability, rate of dissolution, or odor of the formulation. Similarly, the
carrier may
contain still other pharmaceutically-acceptable excipients for modifying or
maintaining
1 o release or absorption or penetration across the blood-brain barrier. Such
excipients are
those substances usually and customarily employed to formulate dosages for
parenteral
administration in either unit dosage or multi-dose form or for direct infusion
into the
cerebrospinal fluid by continuous or periodic infusion.
Dose administration can be repeated depending upon the
pharmacokinetic parameters of the dosage formulation and the route of
administration
used.
It is also contemplated that certain formulations containing FGF-20 are
to be administered orally. Such formulations are preferably encapsulated and
formulated with suitable carriers in solid dosage forms. Some examples of
suitable
carriers, excipients, and diluents include lactose, dextrose, sucrose,
sorbitol, mannitol,
starches, gum acacia, calcium phosphate, alginates, calcium silicate,
microcrystalline
cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose,
methyl- and
propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the
like.
The formulations can additionally include lubricating agents, wetting agents,
emulsifying and suspending agents, preserving agents, sweetening agents or
flavoring
agents. The compositions may be formulated so as to provide rapid, sustained,
or
delayed release of the active ingredients after administration to the patient
by employing
procedures well known in the art. The formulations can also contain substances
that
diminish proteolytic degradation and promote absorption such as, for example,
surface
3o active agents.
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Depending on the treatment regimen contemplated, it may be desired to
control the rate of release of FGF-20 protein or variant thereof to provide
long-term
treatment while minimizing the frequency of administration. Such treatment
regimens
may be desired, for example, where the FGF-20 protein is found to be
relatively
unstable such that the localized concentration of active protein is at an
efficacious level
for an insufficient period of time. Thus, for example, for certain diseases,
it may not be
desired or practical to perform repeated and frequent injections. The major
advantages
of such sustained release systems include targeted local delivery of drugs at
a constant
rate, less drug required to treat the disease state, minimization of possible
side effects,
and enhanced efficacy of treatment. Also, these forms of delivery systems are
capable
of protecting drugs that are unstable in vivo and that would normally require
a frequent
dosing interval. Under such circumstances, sustained release may be achieved
by one
of the methods readily available in the art such as the encapsulation of FGF-
20
conjugated heparin-Sepharose beads to form heparin-alginate microspheres or
the
preparation of FGF-20 PLG microspheres.
Heparin-alginate microspheres have been successfully employed for the
delivery of Basic Fibroblast Growth Factor to tissue (Lopez et al., Journal of
Pharmacology and Experimental Therapeutics 282( 1 ):385-390 ( 1997)).
Similarly,
Alginate/heparin-Sepharose microspheres and films have been used as drug
carriers to
2o control the release of a basic FGF-saponin conjugate in order to control
its release in
small doses. Addition of heparin to solutions of bFGF prevents losses in
activity that
accompany changes in pH or elevation in temperature. See, for example,
Gospodarowicz et al., J. Cell. Physiol. 128:475-484 ( 1986).
As disclosed herein, FGF-20 has a heparin binding domain at residues
170-186. Accordingly, binding of FGF-20 to heparin may be employed in order to
enhance its stability either during in vivo expression or administration or in
vitro during
various stages of protein purification. Thus, by the present invention,
heparin may be
added to a solution of FGF-20 and the activity assayed by the methods
disclosed herein.
FGF-20 bound heparin-Sepharose beads may be encapsulated into
3o calcium alginate microspheres to permit the controlled release of the
heparin-stabilized
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FGF-20 protein. For example, microspheres may be constructed by dropping a
mixed
solution of sodium alginate with FGF-20 bound heparin-Sepharose beads into a
hardening solution of calcium chloride. Spheres are formed instantaneously as
the
mixture enters the hardening solution. The size of the microsphere may be
adjusted by
passing the FGF-20 bound heparin-Sepharose beads through a cylinder of reduced
cross-sectional area such as through a hypodermic needle.
Encapsulation efficiency may be determined by comparing the amount
of encapsulated growth factor with that initially present in solution. For
example, the
FGF-20 may be stripped from the heparin-Sepharose beads with a solution of 3 M
NaCI
I o and functional activity assays may be performed.
The specific dose is calculated according to the approximate body weight
or body surface area of the patient or the volume of body space to be
occupied. The
dose will also be calculated dependent upon the particular route of
administration
selected. Further refinement of the calculations necessary to determine the
appropriate
I5 dosage for treatment is routinely made by those of ordinary skill in the
art. Such
calculations can be made without undue experimentation by one skilled in the
art in
light of the activity disclosed herein in assay preparations of target cells.
Exact dosages
are determined in conjunction with standard dose-response studies. It will be
understood that the amount of the composition actually administered will be
determined
20 by a practitioner, in the light of the relevant circumstances including the
condition or
conditions to be treated, the choice of composition to be administered, the
age, weight,
and response of the individual patient, the severity of the patient's
symptoms, and the
chosen route of administration.
In one embodiment of this invention, FGF-20 may be therapeutically
25 administered by implanting into patients vectors or cells capable of
producing a
biologically-active form of FGF-20 or a precursor of FGF-20, i.e., a molecule
that can
be readily converted to a biological-active form of FGF-20 by the body. In one
approach cells that secrete FGF-20 may be encapsulated into semipermeable
membranes for implantation into a patient. The cells can be cells that
normally express
30 FGF-20 or a precursor thereof or the cells can be transformed to express
FGF-20 or a
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precursor thereof. It is preferred that the cell be of human origin and that
the FGF-20 be
human FGF-20 when the patient is human. However, the formulations and methods
herein can be used for veterinary as well as human applications and the term
"patient"
as used herein is intended to include human and veterinary patients.
Cells can be grown ex vivo for use in transplantation or engraftment into
patients (Muench et al., Leuk. & Lymph. 16:1-11 (1994), which is incorporated
by
reference). In another embodiment of the present invention, FGF-20 is used to
promote
the ex vivo expansion of a cells for transplantation or engraftment. Current
methods
have used bioreactor culture systems containing factors such as
erythropoietin, colony
stimulating factors, stem cell factor, and interleukins to expand
hematopoietic
progenitor cells for erythrocytes, monocytes, neutrophils, and lymphocytes
(Verfaillie,
Stem Cells 12:466-476 ( 1994), which is incorporated by reference). These stem
cells
can be isolated from the marrow of human donors, from human peripheral blood,
or
from umbilical cord blood cells. The expanded blood cells are used to treat
patients
~ 5 who lack these cells as a result of specific disease conditions or as a
result of high dose
chemotherapy for treatment of malignancy (George, Stem Cells 12(Suppl 1):249-
255
( 1994), which is incorporated by reference). In the case of cell transplant
after
chemotherapy, autologous transplants can be performed by removing bone marrow
cells
before chemotherapy, expanding the cells ex vivo using methods that also
function to
purge malignant cells, and transplanting the expanded cells back into the
patient
following chemotherapy (for review, see Rummel and Van Zant, J. Hematotherapy
3:213-218 (1994), which is incorporated by reference). Since FGF-20 is
expressed in
neural cells, it is believed that FGF-20 can function to prevent or slow the
degeneration
of dopaminergic neurons, such as substantia nigra.
In a number of circumstances it would be desirable to determine the
levels of FGF-20 in a patient. The identification of FGF-20 along with the
data herein
showing expression of FGF-20 provides the basis for the conclusion that the
presence of
FGF-20 serves a normal physiological fiznction related to cell growth and
survival.
Indeed, other neurotrophic factors are known to play a role in the function of
neuronal
3o and non-neuronal tissues. (Scully and Otten, Cell Bol. Int. 19:459-469
(1995); Otten
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WO 01/31008 PCT/US00/29237
and Gadient, Int. J. Devl. Neurosciences 13:147-151 (1995), which are
incorporated by
reference.) Endogenously produced FGF-20 may also play a role in certain
disease
conditions, particularly where there is cellular degeneration such as in
neurodegenerative conditions or diseases. Other neurotrophic factors are known
to
change during disease conditions. For example, in multiple sclerosis, levels
of NGF
protein in the cerebrospinal fluid are increased during acute phases of the
disease
(Bracci-Laudiero et al., Neuroscience Lett. 147:9-12 (1992), which is
incorporated by
reference) and in systemic lupus erythematosus there is a correlation between
inflammatory episodes and NGF levels in sera (Bracci-Laudiero et al.,
NeuroReport
l0 4:563-565 (1993), which is incorporated by reference).
Given that FGF-20 is expressed in adult neural cells and in cochlear cells
during embryonic development, it is likely that the level of FGF-20 may be
altered in a
variety of conditions and that quantification of FGF-20 levels would provide
clinically
useful information. Furthermore, in the treatment of degenerative conditions,
compositions containing FGF-20 can be administered and it would likely be
desirable to
achieve certain target levels of FGF-20 in sera, in cerebrospinal fluid or in
any desired
tissue compartment. It would, therefore, be advantageous to be able to monitor
the
levels of FGF-20 in a patient. Accordingly, the present invention also
provides methods
for detecting the presence of FGF-20 in a sample from a patient.
The term "detection" as used herein in the context of detecting the
presence of FGF-20 in a patient is intended to include the determining of the
amount of
FGF-20 or the ability to express an amount of FGF-20 in a patient, the
distinguishing of
FGF-20 from other growth factors, the estimation of prognosis in terms of
probable
outcome of a degenerative disease and prospect for recovery, the monitoring of
the
FGF-20 levels over a period of time as a measure of status of the condition,
and the
monitoring of FGF-20 levels for determining a preferred therapeutic regimen
for the
patient.
To detect the presence of FGF-20 in a patient, a sample is obtained from
the patient. The sample can be a tissue biopsy sample or a sample of blood,
plasma,
3o serum, CSF or the like. FGF-20 is expressed in neural tissues as discussed
in
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WO 01/31008 PCT/US00/29237
Example 8. Samples for detecting FGF-20 can be taken from this tissue. When
assessing peripheral levels of FGF-20, it is preferred that the sample be a
sample of
blood, plasma or serum. When assessing the levels of FGF-20 in the central
nervous
system a preferred sample is a sample obtained from cerebrospinal fluid or
neural
tissue.
In some instances it is desirable to determine whether the FGF-20 gene
is intact in the patient or in a tissue or cell line within the patient. By an
intact FGF-20
gene it is meant that there are no alterations in the gene such as point
mutations,
deletions, insertions, chromosomal breakage, chromosomal rearrangements and
the like
t 0 wherein such alteration might alter production of FGF-20 or alter its
biological activity,
stability or the like to lead to disease processes or susceptibility to
cellular degenerative
conditions. Thus, in one embodiment of the present invention a method is
provided for
detecting and characterizing any alterations in the FGF-20 gene. The method
comprises
providing an oligonucleotide that contains the FGF-20 cDNA, genomic DNA or a
fragment thereof or a derivative thereof. By a derivative of an
oligonucleotide, it is
meant that the derived oligonucleotide is substantially the same as the
sequence from
which it is derived in that the derived sequence has sufficient sequence
complementarily
to the sequence from which it is derived to hybridize to the FGF-20 gene. The
derived
nucleotide sequence is not necessarily physically derived from the nucleotide
sequence,
2o but may be generated in any manner including for example, chemical
synthesis or DNA
replication or reverse transcription or transcription.
Typically, patient genomic DNA is isolated from a cell sample from the
patient and digested with one or more restriction endonucleases such as, for
example,
TaqI and AIuI. Using the Southern blot protocol, which is well known in the
art, this
assay determines whether a patient or a particular tissue in a patient has an
intact FGF-
20 gene or an FGF-20 gene abnormality.
Hybridization to an FGF-20 gene would involve denaturing the
chromosomal DNA to obtain a single-stranded DNA; contacting the single-
stranded
DNA with a gene probe associated with the FGF-20 gene sequence; and
identifying the
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WO 01/31008 PCT/US00/29237
hybridized DNA-probe to detect chromosomal DNA containing at least a portion
of a
human FGF-20 gene.
The term "probe" as used herein refers to a structure comprised of a
polynucleotide that forms a hybrid structure with a target sequence, due to
complementarity of probe sequence with a sequence in the target region.
Oligomers
suitable for use as probes may contain a minimum of about 8-12 contiguous
nucleotides
which are complementary to the targeted sequence and preferably a minimum of
about 20.
The FGF-20 gene probes of the present invention can be DNA or RNA
oligonucleotides and can be made by any method known in the art such as, for
example,
excision, transcription or chemical synthesis. Probes may be labeled with any
detectable label known in the art such as, for example, radioactive or
fluorescent labels
or enzymatic marker. Labeling of the probe can be accomplished by any method
known
in the art such as by PCR, random priming, end labeling, nick translation or
the like.
One skilled in the art will also recognize that other methods not employing a
labeled
probe can be used to determine the hybridization. Examples of methods that can
be
used for detecting hybridization include Southern blotting, fluorescence in
situ
hybridization, and single-strand conformation polymorphism with PCR
amplification.
Hybridization is typically carried out at 25° - 45°C, more
preferably at
32° - 40°C and more preferably at 37° - 38°C. The
time required for hybridization is
from about 0.25 to about 96 hours, more preferably from about one to about 72
hours,
and most preferably from about 4 to about 24 hours.
FGF-20 gene abnormalities can also be detected by using the PCR
method and primers that flank or lie within the FGF-20 gene. The PCR method is
well
known in the art. Briefly, this method is performed using two oligonucleotide
primers
which are capable of hybridizing to the nucleic acid sequences flanking a
target
sequence that lies within an FGF-20 gene and amplifying the target sequence.
The
terms "oligonucleotide primer" as used herein refers to a short strand of DNA
or RNA
ranging in length from about 8 to about 30 bases. The upstream and downstream
3o primers are typically from about 20 to about 30 base pairs in length and
hybridize to the
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WO 01/31008 PCT/US00/29237
flanking regions for replication of the nucleotide sequence. The
polymerization is
catalyzed by a DNA-polymerase in the presence of deoxynucleotide triphosphates
or
nucleotide analogs to produce double-stranded DNA molecules. The double
strands are
then separated by any denaturing method including physical, chemical or
enzymatic.
Commonly, a method of physical denaturation is used involving heating the
nucleic
acid, typically to temperatures from about 80°C to 105°C for
times ranging from about
I to about 10 minutes. The process is repeated for the desired number of
cycles.
The primers are selected to be substantially complementary to the strand
of DNA being amplified. Therefore, the primers need not reflect the exact
sequence of
the template, but must be sufficiently complementary to selectively hybridize
with the
strand being amplified.
After PCR amplification, the DNA sequence comprising FGF-20 or pre-
pro FGF-20 or a fragment thereof is then directly sequenced and analyzed by
comparison of the sequence with the sequences disclosed herein to identify
alterations
t 5 which might change activity or expression levels or the like.
In another embodiment, a method for detecting FGF-20 is provided
based upon an analysis of tissue expressing the FGF-20 gene, as described in
the
Examples. The method comprises hybridizing a polynucleotide to mRNA from a
sample of tissue that normally expresses the FGF-20 gene. The sample is
obtained from
2o a patient suspected of having an abnormality in the FGF-20 gene or in the
FGF-20 gene
of particular cells.
To detect the presence of mRNA encoding FGF-20 protein, a sample is
obtained from a patient. The sample can be from blood or from a tissue biopsy
sample.
The sample may be treated to extract the nucleic acids contained therein. The
resulting
25 nucleic acid from the sample is subjected to gel electrophoresis or other
size separation
techniques.
The mRNA of the sample is contacted with a DNA sequence serving as a
probe to form hybrid duplexes. The use of a labeled probes as discussed above
allows
detection of the resulting duplex.
CA 02388637 2002-04-19
WO 01/31008 PCT/US00/29237
When using the cDNA encoding FGF-20 protein or a derivative of the
cDNA as a probe, high stringency conditions can be used in order to prevent
false
positives, that is the hybridization and apparent detection of FGF-20
nucleotide
sequences when in fact an intact and functioning FGF-20 gene is not present.
When
using sequences derived from the FGF-20 cDNA, less stringent conditions could
be
used, however, this would be a less preferred approach because of the
likelihood of
false positives. The stringency of hybridization is determined by a number of
factors
during hybridization and during the washing procedure, including temperature,
ionic
strength, length of time and concentration of formamide.
1o In order to increase the sensitivity of the detection in a sample of mRNA
encoding the FGF-20 protein, the technique of reverse
transcription/polymerization
chain reaction (RT/PCR) can be used to amplify cDNA transcribed from mRNA
encoding the FGF-20 protein. The method of RT/PCR is well known in the art,
and can
be performed as follows. Total cellular RNA is isolated by, for example, the
standard
guanidium isothiocyanate method and the total RNA is reverse transcribed. The
reverse
transcription method involves synthesis of DNA on a template of RNA using a
reverse
transcriptase enzyme and a 3' end primer. Typically, the primer contains an
oligo(dT)
sequence. The cDNA thus produced is then amplified using the PCR method and
FGF-
specific primers. (Belyavsky et al., Nucl. Acid Res. 17:2919-2932 ( 1989);
Krug and
2o Bergen Methods in Enzymology 152:316-325, Academic Press, NY, 1987, which
are
incorporated by reference).
The polymerise chain reaction method is performed as described above
using two oligonucleotide primers that are substantially complementary to the
two
flanking regions of the DNA segment to be amplified.
Following amplification, the PCR product is then electrophoresed and
detected by ethidium bromide staining or by phosphoimaging.
The present invention further provides for methods to detect the presence
of FGF-20 protein in a sample obtained from a patient. Any method known in the
art
for detecting proteins can be used. Such methods include, but are not limited
to
immunodiffusion, immunoelectrophoresis, immunochemical methods, binder-ligand
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WO 01/31008 PCT/US00/29237
assays, immunohistochemical techniques, agglutination and complement assays.
(For
example, see Basic and Clinical Immunology, 217-262, Sites and Terr, eds.,
Appleton
& Lange, Norwalk, CT, 1991 which is incorporated by reference). Preferred are
binder-
ligand immunoassay methods including reacting antibodies with an epitope or
epitopes
of the FGF-20 protein and competitively displacing a labeled FGF-20 protein or
derivative thereof. Preferred antibodies are prepared according to Example 11.
As used herein, a derivative of the FGF-20 protein is intended to include
a polypeptide in which certain amino acids have been deleted, replaced, or
changed to
modified or unusual amino acids wherein the FGF-20 derivative is biologically
l0 equivalent to FGF-20 and wherein the polypeptide derivative cross-reacts
with
antibodies raised against the FGF-20 protein. By cross-reaction it is meant
that an
antibody reacts with an antigen other than the one that induced its formation.
Numerous competitive and non-competitive protein binding
immunoassays are well known in the art. Antibodies employed in such assays may
be
~ 5 unlabeled, for example as used in agglutination tests, or labeled for use
in a wide variety
of assay methods. Labels that can be used include radionuclides, enzymes,
fluorescers,
chemiluminescers, enzyme substrates or co-factors, enzyme inhibitors,
particles, dyes
and the like for use in radioimmunoassay (RIA), enzyme immunoassays, e.g.,
enzyme-
linked immunosorbent assay (ELISA), fluorescent immunoassays and the like.
2o Polyclonal or monoclonal antibodies to the FGF-20 protein or an epitope
thereof can be made for use in immunoassays by any of a number of methods
known in
the art. By epitope reference is made to an antigenic determinant of a
polypeptide. An
epitope could comprise 3 amino acids in a spatial conformation which is unique
to the
epitope. Generally an epitope consists of at least 5 such amino acids. Methods
of
25 determining the spatial conformation of amino acids are known in the art,
and include,
for example, x-ray crystallography and 2 dimensional nuclear magnetic
resonance.
One approach for preparing antibodies to a protein is the selection and
preparation of an amino acid sequence of all or part of the protein,
chemically
synthesizing the sequence and injecting it into an appropriate animal, usually
a rabbit or
3o a mouse (see Example 11 ).
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Oligopeptides can be selected as candidates for the production of an
antibody to the FGF-20 protein based upon the oligopeptides lying in
hydrophilic
regions, which are thus likely to be exposed in the mature protein.
Oligopeptides for
raising antibodies include the contiguous amino acids at positions 176-189, or
56-70 of
SEQ ID N0:4. These oligopeptides are RDGARSKRHQKFTH (SEQ ID NO:S) and
QLAHLHGILRRRQLY (SEQ ID N0:6). Additional oligopeptides can be determined
using, for example, the Antigenicity Index of Welling, G.W. et al., FEBS Lett.
188:215-
218 (1985), incorporated herein by reference.
Antibodies to FGF-20 can also be raised against oligopeptides that
1o include one or more of the conserved regions identified herein such that
the antibody
can cross-react with other family members. Such antibodies can be used to
identify and
isolate the other family members.
Methods for preparation of the FGF-20 protein or an epitope thereof
include, but are not limited to chemical synthesis, recombinant DNA techniques
or
isolation from biological samples. Chemical synthesis of a peptide can be
performed,
for example, by the classical Merrifeld method of solid phase peptide
synthesis
(Merrifeld, J., Am. Chem. Soc. 85:2149 (1963), which is incorporated by
reference) or
the FMOC strategy on a Rapid Automated Multiple Peptide Synthesis system (E.
I. du
Pont de Nemours Company, Wilmington, Delaware) (Caprino and Han, J. Org. Chem.
37:3404 (1972), which is incorporated by reference).
Polyclonal antibodies can be prepared by immunizing rabbits or other
animals by injecting antigen followed by subsequent boosts at appropriate
intervals.
The animals are bled and sera assayed against purified FGF-20 protein usually
by
ELISA or by bioassay based upon the ability to block the action of FGF-20 on
neurons
or other cells. When using avian species, e.g., chicken, turkey and the like,
the antibody
can be isolated from the yolk of the egg. Monoclonal antibodies can be
prepared after
the method of Milstein and Kohler by fusing splenocytes from immunized mice
with
continuously replicating tumor cells such as myeloma or lymphoma cells.
(Milstein and
Kohler, Nature 256:495-497 ( 1975); Gulfre and Milstein, Methods in
Enzymology:
Immunochemical Techniques 73:1-46, Langone and Banatis eds., Academic Press,
1981
33
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WO 01/31008 PCT/US00/29237
which are incorporated by reference). The hybridoma cells so formed are then
cloned
by limiting dilution methods and supernates assayed for antibody production by
ELISA,
RIA or bioassay.
The unique ability of antibodies to recognize and specifically bind to
target proteins provides an approach for treating an overexpression of the
protein. Thus,
another aspect of the present invention provides for a method for preventing
or treating
diseases involving overexpression of the FGF-20 protein by treatment of a
patient with
specific antibodies to the FGF-20 protein.
Specific antibodies, either polyclonal or monoclonal, to the FGF-20
protein can be produced by any suitable method known in the art as discussed
above.
For example, murine or human monoclonal antibodies can be produced by
hybridoma
technology or, alternatively, the FGF-20 protein, or an immunologically active
fragment
thereof, or an anti-idiotypic antibody, or fragment thereof can be
administered to an
animal to elicit the production of antibodies capable of recognizing and
binding to the
FGF-20 protein. Such antibodies can be from any class of antibodies including,
but not
limited to IgG, IgA, IgM, IgD, and IgE or in the case of avian species, IgY
and from
any subclass of antibodies.
Polypeptides encoded by the instant polynucleotides and corresponding
full-length genes can be used to screen peptide libraries, protein libraries,
small
2o molecule libraries, and phage display libraries, and other known methods,
to identify
analogs or antagonists.
Native FGF polypeptides may play a role in cancer. For example, FGF
family members can induce marked morphological transformation of NIH 3T3
cells,
and exhibit strong tumorigenicity in nude mice. Angiogenic activity has been
exhibited
by FGF family members. Thus, inhibitors of FGF can be used to treat cancer.
A library of peptides may be synthesized following the methods
disclosed in U.S. Patent No. 5,010,175, and in PCT No. WO 91/17823. As
described
below in brief, a mixture of peptides is prepared, which is then screened to
identify the
peptides exhibiting the desired signal transduction and receptor binding
activity.
3o According to the method of the ' 175 patent, a suitable peptide synthesis
support (e.g., a
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WO 01/31008 PCT/US00/29237
resin) is coupled to a mixture of appropriately protected, activated amino
acids. The
concentration of each amino acid in the reaction mixture is balanced or
adjusted in
inverse proportion to its coupling reaction rate so that the product is an
equimolar
mixture of amino acids coupled to the starting resin. The bound amino acids
are then
deprotected, and reacted with another balanced amino acid mixture to form an
equimolar mixture of all possible dipeptides. This process is repeated until a
mixture of
peptides of the desired length (e.g., hexamers) is formed. Note that one need
not
include all amino acids in each step: one may include only one or two amino
acids in
some steps (e.g., where it is known that a particular amino acid is essential
in a given
position), thus reducing the complexity of the mixture. After the synthesis of
the
peptide library is completed, the mixture of peptides is screened for binding
to the
selected polypeptide. The peptides are then tested for their ability to
inhibit or enhance
activity. Peptides exhibiting the desired activity are then isolated and
sequenced.
The method described in PCT No. WO 91/17823 is similar. However,
instead of reacting the synthesis resin with a mixture of activated amino
acids, the resin
is divided into twenty equal portions (or into a number of portions
corresponding to the
number of different amino acids to be added in that step), and each amino acid
is
coupled individually to its portion of resin. The resin portions are then
combined,
mixed, and again divided into a number of equal portions for reaction with the
second
2o amino acid. In this manner, each reaction may be easily driven to
completion.
Additionally, one may maintain separate "subpools" by treating portions in
parallel,
rather than combining all resins at each step. This simplifies the process of
determining
which peptides are responsible for any observed receptor binding or signal
transduction
activity.
In such cases, the subpools containing, e.g., 1-2,000 candidates each are
exposed to one or more polypeptides of the invention. Each subpool that
produces a
positive result is then resynthesized as a group of smaller subpools (sub-
subpools)
containing, e.g., 20-100 candidates, and reassayed. Positive sub-subpools may
be
resynthesized as individual compounds, and assayed finally to determine the
peptides
3o that exhibit a high binding constant. These peptides can be tested for
their ability to
CA 02388637 2002-04-19
WO 01/31008 PCT/US00/29237
inhibit or enhance the native activity. The methods described in PCT No. WO 91
/7823
and U.S. Patent No. 5,194,392 (herein incorporated by reference) enable the
preparation
of such pools and subpools by automated techniques in parallel, such that all
synthesis
and resynthesis may be performed in a matter of days.
Peptide agonists or antagonists are screened using any available method,
such as signal transduction, antibody binding, receptor binding and mitogenic
assays.
The assay conditions ideally should resemble the conditions under which the
native
activity is exhibited in vivo, that is, under physiologic pH, temperature, and
ionic
strength. Suitable agonists or antagonists will exhibit strong inhibition or
enhancement
of the native activity at concentrations that do not cause toxic side effects
in the subject.
Agonists or antagonists that compete for binding to the native polypeptide may
require
concentrations equal to or greater than the native concentration, while
inhibitors capable
of binding irreversibly to the polypeptide may be added in concentrations on
the order
of the native concentration.
t 5 The availability of hFGF-20 and rFGF-20 allows for the identification of
small molecules and low molecular weight compounds that inhibit the binding of
FGF-
to its receptor, through routine application of high-throughput screening
methods
(HTS). HTS methods generally refer to technologies that permit the rapid
assaying of
lead compounds for therapeutic potential. HTS techniques employ robotic
handling of
2o test materials, detection of positive signals, and interpretation of data.
Lead
compounds may be identified via the incorporation of radioactivity or through
optical
assays that rely on absorbance, fluorescence or luminescence as read-outs.
Gonzalez,
J.E., et al., Curr. Opin. Biotech. 9:624-631 (1998). Assays for detecting
interaction
between an FGF molecule and FGF receptor are described in, for example, Blunt,
A. G.
et al., J. Biol. Chem. 272:3733-3738 (1997), and such assays can be adapted
for
determining if a candidate molecule can inhibit the interaction between FGF-20
and its
receptor.
Model systems are available that can be adapted for use in high
throughput screening for compounds that inhibit the interaction of FGF-20 with
receptors to which it binds (see Example 12), for example by competing with
FGF-20
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WO 01/31008 PCT/US00/29237
for receptor binding. Sarubbi et al., Anal. Biochem. 237:70-75 (1996),
describe cell-
free, non-isotopic assays for discovering molecules that compete with natural
ligands
for binding to the active site of IL-1 receptor. Martens, C. et al., Anal.
Biochem.
273:20-31 (1999), describe a generic particle-based nonradioactive method in
which a
labeled ligand binds to its receptor immobilized on a particle; label on the
particle
decreases in the presence of a molecule that competes with the labeled ligand
for
receptor binding.
The therapeutic FGF-20 polynucleotides and polypeptides of the present
invention may be utilized in gene delivery vehicles. The gene delivery vehicle
may be
of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy I:51-
64 (1994);
Kimura, Human Gene Therapy 5:845-852 (1994); Connelly, Human Gene Therapy
1:185-193 (1995); and Kaplitt, Nature Genetics 6:148-153 (1994)). Gene therapy
vehicles for delivery of constructs including a coding sequence of a
therapeutic of the
invention can be administered either locally or systemically. These constructs
can
utilize viral or non-viral vector approaches. Expression of such coding
sequences can
be induced using endogenous mammalian or heterologous promoters. Expression of
the
coding sequence can be either constitutive or regulated.
The present invention can employ recombinant retroviruses which are
constructed to carry or express a selected nucleic acid molecule of interest.
Retrovirus
2o vectors that can be employed include those described in EP 0 415 731; WO
90/07936;
WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740;
WO 93/11230; WO 93/10218; Vile and Hart, Cancer Res. 53:3860-3864 (1993); Vile
and Hart, Cancer Res. 53:962-967 (1993); Ram et al., Cancer Res. 53:83-88
(1993);
Takamiya et al., J. Neurosci. Res. 33:493-503 (1992); Baba et al., J.
Neurosurg. 79:729-
735 (1993); U.S. Patent No. 4,777,127; GB Patent No. 2,200,651; and EP 0 345
242.
Preferred recombinant retroviruses include those described in WO 91/02805.
Packaging cell lines suitable for use with the above-described retroviral
vector constructs may be readily prepared (see PCT publications WO 95/30763
and WO
92/05266), and used to create producer cell lines (also termed vector cell
lines) for the
3o production of recombinant vector particles. Within particularly preferred
embodiments
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WO 01/31008 PCT/US00/29237
of the invention, packaging cell lines are made from human (such as HT1080
cells) or
mink parent cell lines, thereby allowing production of recombinant
retroviruses that can
survive inactivation in human serum.
The present invention also employs alphavirus-based vectors that can
function as gene delivery vehicles. Such vectors can be constructed from a
wide variety
of alphaviruses, including, for example, Sindbis virus vectors, Semliki forest
virus
(ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246)
and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR
1249; ATCC VR-532). Representative examples of such vector systems include
those
to described in U.S. Patent Nos. 5,091,309; 5,217,879; and 5,185,440; and PCT
Publication Nos. WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; and WO
95/07994.
Gene delivery vehicles of the present invention can also employ
parvovirus such as adeno-associated virus (AAV) vectors. Representative
examples
t 5 include the AAV vectors disclosed by Srivastava in WO 93/09239, Samulski
et al., J.
Vir. 63:3822-3828 (1989); Mendelson et al., Virol. 166:154-165 (1988); and
Flotte et
al., P.NA.S. 90:10613-10617 (1993).
AAV vectors may be suitable for administering FGF-20 to treat hearing
disorders. For example, Lalwani et al., Gene Ther. 3:588-592 (1996), used AAV
to
20 obtain in vivo expression of a foreign gene in the cochlea of guinea pigs.
Representative examples of adenoviral vectors include those described
by Berkner, Biotechniques 6:616-627 (Biotechniques); Rosenfeld et al., Science
252:431-434 (1991); WO 93/19191; Kolls et al., P.N.A.S. :215-219 (1994); Kass-
Eider et al., P.NA.S. 90:11498-11502 (1993); Guzman et al., Circulation
88:2838-2848
25 (1993); Guzman et al., Cir. Res. 73:1202-1207 (1993); Zabner et al., Cell
75:207-216
(1993); Li et al., Hum. Gene Ther. 4:403-409 (1993); Cailaud et al., Eur. J.
Neurosci.
5:1287-1291 (1993); Vincent et al., Nat. Genet. 5:130-134 (1993); Jaffe et
al., Nat.
Genet. 1:372-378 (1992); and Levrero et al., Gene 101:195-202 (1992).
Exemplary
adenoviral gene therapy vectors employable in this invention also include
those
3o described in WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938;
38
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WO 01/31008 PCT/US00/29237
WO 95/11984 and WO 95/00655. Administration of DNA linked to killed adenovirus
as described in Curiel, Hum. Gene Ther. 3:147-154 (1992), may be employed.
Other gene delivery vehicles and methods may be employed, including
polycationic condensed DNA linked or unlinked to killed adenovirus alone, for
example
Curiel, Hum. Gene Ther. 3:147-154 (1992); ligand-linked DNA, for example see
Wu, J.
Biol. Chem. 264:16985-16987 (1989); eukaryotic cell delivery vehicles cells,
for
example see U.S. Serial No. 08/240,030, filed May 9, 1994, and U.S. Serial No.
08/404,796; deposition of photopolymerized hydrogel materials; hand-held gene
transfer particle gun, as described in U.S. Patent No. 5,149,655; ionizing
radiation as
described in U.S. Patent No. 5,206,152 and in WO 92/11033; nucleic charge
neutralization or fusion with cell membranes. Additional approaches are
described in
Philip, Mol. Cell Biol. 14:2411-2418 (1994), and in Woffendin, Proc. Natl.
Acad. Sci.
91:1581-1585 (1994).
Naked DNA may also be employed. Exemplary naked DNA
introduction methods are described in WO 90/11092 and U.S. Patent No.
5,580,859.
Uptake efficiency may be improved using biodegradable latex beads. DNA coated
latex
beads are efficiently transported into cells after endocytosis initiation by
the beads. The
method may be improved further by treatment of the beads to increase
hydrophobicity
and thereby facilitate disruption of the endosome and release of the DNA into
the
2o cytoplasm. Liposomes that can act as gene delivery vehicles are described
in U.S.
Patent No. 5,422,120, PCT Patent Publication Nos. WO 95/13796, WO 94/23697,
and
WO 91/14445, and EP No. 0 524 968.
Further non-viral delivery suitable for use includes mechanical delivery
systems such as the approach described in Woffendin et al., Proc. Natl. Acad.
Sci. USA
91(24):11581-11585 (1994). Moreover, the coding sequence and the product of
expression of such can be delivered through deposition of photopolymerized
hydrogel
materials. Other conventional methods for gene delivery that can be used for
delivery
of the coding sequence include, for example, use of hand-held gene transfer
particle
gun, as described in U.S. Patent No. 5,149,655; use of ionizing radiation for
activating
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WO 01/31008 PCT/US00/29237
transferred gene, as described in U.S. Patent No. 5,206,152 and PCT Patent
Publication
No. WO 92/11033.
FGF has been implicated in diseases characterized by loss of function,
inadequate function/number, abnormal function or death of cells, tissues or
organs for
which function or survival can be prolonged/rescued, and abnormalities
reversed or
prevented by therapy with FGF.
Loss of pulmonary, bronchia or alveolar cells or function, healing of
pulmonary or bronchia wounds, pulmonary infraction, emphysema/chronic
obstructive
pulmonary disease, asthma, sequelae of infectious or autoimmune disease,
sequelae of
pulmonary arterial or venous hypertension, pulmonary fibrosis, pulmonary
disease of
immaturity, and cystic fibrosis are conditions amenable to treatment with FGF.
Ischemic vascular disease may be amenable to FGF-20 treatment,
wherein the disease is characterized by inadequate blood flow to an organ(s).
Treatment may induce therapeutic angiogenesis or preserve function/survival of
cells
(myocardial ischemia/infarction, peripheral vascular disease, renal artery
disease,
stroke). Cardiomyopathies characterized by loss of function or death of
cardiac
myocytes or supporting cells in the heart (congestive heart failure,
myocarditis) may
also be treated using FGF-20, as can musculoskeletal disease characterized by
loss of
function, inadequate function or death of skeletal muscle cells, bone cells or
supporting
2o cells. Examples include skeletal myopathies, bone disease, and arthritis.
FGF-20 polynucleotides and polypeptides may aid in correction of
congenital defects due to loss of FGF-20 molecule or its function (heart,
lung, brain,
limbs, kidney, etc.). FGF-20 polynucleotides and polypeptides may also aid in
the
correction of such defects wherein the defects lead to hearing loss due to
cochlear
defects.
Treatment of wound healing is yet another use of FGF-20 polypeptides
and polynucleotides, either due to trauma, disease, medical or surgical
treatment,
including regeneration of cell populations and tissues depleted by these
processes.
Examples include liver regeneration, operative wound healing, re-
endothelialization of
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injured blood vessels, healing of traumatic wounds, healing of ulcers due to
vascular,
metabolic disease, etc., bone fractures, loss of cells due to inflammatory
disease, etc.
FGF-20 may also be used in screens to identify drugs for treatment of
cancers which involve over activity of the molecule, or new targets which
would be
useful in the identification of new drugs.
For all of the preceding embodiments, the clinician will determine, based
on the specific condition, whether FGF-20 polypeptides or polynucleotides,
antibodies
to FGF-20, or small molecules such as peptide analogues or antagonists, will
be the
most suitable form of treatment. These forms are all within the scope of the
invention.
Preferred embodiments of the invention are described in the following
examples. Other embodiments within the scope of the claims herein will be
apparent to
one skilled in the art from consideration of the specification or practice of
the invention
as disclosed herein. It is intended that the specification, together with the
examples, be
considered exemplary only, with the scope and spirit of the invention being
indicated by
the claims which follow the examples.
EXAMPLES
EXAMPLE I
Preparation of RNA --- RNA was prepared from adult rat brain using an
RNA extraction kit (Pharmacia Biotech, Uppsala, Sweden). Poly (A)+ RNA was
2o prepared using oligo (dT)-cellulose (Type 2, Collaborative Biomedical
Products,
Bedford, Massachusetts).
EXAMPLE 2
Isolation and Analysis of Rat FGF 20 cDNA --- DNA was amplified
from rat genomic DNA by polymerase chain reaction (PCR) for 30 cycles in 25 ~l
of a
reaction mixture containing 5 pmole/~l of each of the sense and antisense
degenerate
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WO 01/31008 PCT/US00/29237
primers representing all possible codons corresponding to the consensus amino
acid
sequences of rat FGF-9 (17) and FGF-16 (21), FEENWY and THFLPR, respectively.
The amplified product was further amplified by PCR with each of the sense and
antisense degenerate primers representing all possible codons corresponding to
another
consensus amino acid sequences of rat FGF-9 (17) and FGF-16 (21), ENWYNT and
HQKFTH, respectively. The amplified DNA of expected size (approximately 150
base
pairs) was cloned into the pGEM-T DNA vector (Promega, Madison, Wisconsin).
The
nucleotide sequence of the cloned DNA was determined by a DNA sequencer
(Applied
Biosystems, Foster, California). To determine the coding region of a novel FGF
cDNA,
1o the coding region was amplified from cDNA synthesized from rat brain poly
(A)+ RNA
by adaptor-ligation mediated polymerase chain reaction using a Marathon cDNA
amplification kit (Clontech, Palo Alto, California). To determine the amino-
terminal
region, DNA encoding the region was amplified from rat genomic DNA by cassette-
ligation mediated polymerase chain reaction (Isegawa, Y. et al., Mol. Cell.
Probes
6:467-475 ( 1992)) using a LA PCR in vitro cloning kit (TaKaRa, Kyoto, Japan).
The
cDNA encoding the entire coding region of the FGF was amplified from rat brain
cDNA by polymerase chain reaction in the presence of 5% dimethyl sulfoxide
(Villarreal, X.C. et al., Anal. Biochem. 197:362-367 (1991)) using the FGF-
specific
primers including the 5' and 3' noncoding sequences, and cloned into the pGEM-
T
2o DNA vector. The apparent evolutionary relationships of members of the FGF
family
were examined by the unweighted pair-group method with arithmetic mean method
using the sequence analysis software, Genetyx (Software Development Co.,
Tokyo,
Japan).
EXAMPLE 3
Expression of FGF 20 mRNA in Rat Embryonic Inner Ear ---
Expression of FGF-20 in rat embryos was examined. Consecutive transverse
sections
of rat embryos (E14.5) were examined by in situ hybridization with 35S-labeled
FGF-20
anti-sense and sense cRNA probes. The sections were counterstained with
hematoxylin
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WO 01/31008 PCT/US00/29237
and eosin. Bright-field and dark-field photographs of the sections reveal that
FGF-20 is
preferentially expressed in the cochlea of the inner ear.
EXAMPLE 4
Northern Blotting Analysis --- Poly (A)+ RNA (10 fig) from rat adult
brain was dissolved on a denaturing agarose gel (1%) containing formaldehyde,
and
transferred to a nitrocellulose membrane in 20X SSC (1X SSC:0.15 M NACI/0.015
M
sodium citrate) overnight. A 3zP-labeled FGF-20 cDNA probe 0650 base pairs)
was
labeled with a random primer labeling kit (Pharmacia Biotech, Uppsala, Sweden)
and
deoxycytidine 5'-[a 3ZP-] triphosphate (~ 110 TBq/mmol) (ICN Biomedicals Inc.,
Costa
Mesa, California). The membrane was incubated in hybridization solution
containing
the labeled probe as described (22), and analyzed with a radio-imaging
analyzer (BAS
2000, Fuji Photo Film Co., Tokyo, Japan). To confirm the integrity of the poly
(A)+
RNA, the hybridized probe on the membrane was washed with 0.5X SSC containing
0.01 M EDTA (pH 8.0) at 100°C for 5 min and with O.OSX SSC containing
0.01 M
EDTA (pH 8.0) and 0.1 % SDS at 60°C for 15 min. The washed
membrane was
rehybridized with a 3zP-labeled rat ~3actin cDNA probe 0410 base pairs)
(Nudel, U. et
al., Nucleic Acids Res. 11:1759-1771 (1983)).
EXAMPLE S
In Situ Hybridization --- Adult Wistar rat brain was frozen in powdered
dry ice, and sagittal sections were cut at 16 ~m with a cryostat, thaw-mounted
onto
poly-L-lysine-coated slides, and stored at -85°C until hybridization. A
35S-labeled rat
FGF-20 antisense or sense cRNA probe was transcribed using SP6 RNA polymerase
or
T7 RNA polymerase (TaKaRa, Kyoto, Japan) with uridine 5'-
a[355]thiotriphosphate
(~30 TBq/mmol) (Amersham, Buckinghamshire, England), respectively. The
sections
were examined by in situ hybridization with the labeled probe as described
(Yamasaki,
M. et al., J. Biol. Chem. 271:15918-15921 (1996)).
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EXAMPLE 6
Preparation of Recombinant Rat FGF 20 --- The rat FGF-20 cDNA
with a DNA fragment (75 BP) encoding an E-tag (GAPVPYPDPLEPR) and a Hisb tag
(HHHHHH) at the 3'-terminus of the coding region was constructed in a transfer
vector
DNA, pBacPAK9 (Clontech, Palo Alto, California). Recombinant baculovirus
containing the FGF-20 cDNA with the tag sequences was obtained by
cotransfection of
Sf9 cells with the recombinant pBacPAK9 and a Bsu36 I-digested expression
vector,
BacPAK6 (Clontech, Palo Alto, California). High Five insect cells were
infected with
the resultant recombinant baculovirus and incubated at 27°C for 65 h in
serum-free
I o medium EX-CELL 400 (JRH Biosciences, Lenexa, Kansas). The culture medium
was
dialyzed against phosphate-buffered saline (PBS), and applied to a column of
Ni-NTA
agarose (QIAGEN GmbH, Hilden, Germany) in PBS containing 20 mM imidazole and
0.5 M NaCI. After washing the column with PBS containing 20 mM imidazole and
0.5
M NaCI, recombinant FGF-20 was eluted from the column with PBS containing 250
IS mM imidazole and 0.5 M NaCI, and applied to a column of Bio-Gel P-6 DG (Bio-
Rad
Lab., Hercules, California) in PBS containing 100 pg/mIBSA.
EXAMPLE 7
Detection of Recombinant FGF 20 by Western Blotting Analysis --- The
culture medium or rat recombinant FGF-20 was separated by sodium dodecyl
sulfate
20 (SDS)-polyacrylamide gel (12.5%) electrophoresis under reducing conditions
and
transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham,
Buckinghamshire, England). The membrane was incubated with anti-E tag
antibodies
(1:500) (Pharmacia Biotech, Uppsala, Sweden). The protein with the E-tag was
visualized as described (Hoshikawa, M. et al., Biochem. Biophys. Res. Commun.
25 244:187-191 (1998)).
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EXAMPLE 8
Rat Midbrain Cultured Cells --- The ventral mesencephalon was resected
from rat embryos (E16.5). The mesencephalic blocks were washed 10 times with
Hanks' solution and mechanically dissociated without enzymatic treatment. The
midbrain cultured cells were prepared essentially as described (Sawada, H. et
al., J.
Neurosci. Res. 43:503-510 (1996)). The culture medium consisted of Eagle's
minimum
essential medium (EMEM) supplemented with 0.2% sodium carbonate, 0.1 %
glucose,
0.029% L-glutamine and 0.238% HEPES. The cultured cells were incubated at
37°C in
the culture medium containing 10% fetal calf serum. From the 5'" day of
culture, the
cells were incubated in the culture medium containing 10% horse serum.
EXAMPLE 9
Examination of Neurotrophic Activity of FGF 20 for Midbrain
Dopaminergic Neurons --- Cells on the 8'" day of culture were incubated in
Eagle's
minimum essential medium supplemented with 0.2% sodium hydrogen carbonate, 0.1
glucose, 0.029% L-glutamine, 0.238% HEPES and 10% horse serum or 0.1% bovine
serum albumin in the presence or absence of FGF-20 for 4 days and then fixed
with
fresh 4% paraformaldehyde for 30 min on the 12'" day. Cells on the 8'" day of
culture
were also incubated in the presence or absence of recombinant rat FGF-20 for
24 h and
then were treated with 1 mM glutamate for 10 min. The cultured cells were
further
2o incubated in medium without FGF-20 and 1mM glutamate and then fixed with 4%
paraformaldehyde for 30 min on the 12'" day. The fixed cells were washed with
PBS
for 15 min, and then treated with 0.2% Triton X-10 for 30 min. The cells were
immunostained with anti-tyrosine hydroxylase (TH) antibody (Eugene Tech,
Ridgefield
Park, New Jersey) essentially as described (Sawada, H. et al., J. Neurosci.
Res. 43:503-
510 (1996)). Numbers of cultured dopaminergic neurons were evaluated by
counting
cells stained with anti-TH antibody.
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EXAMPLE 1O
Isolation and Analysis of Human FGF 20 --- The coding region of
human FGF-20 DNA was amplified from human brain cDNA library (7~gt10) by PCR
using primers specific for FGF-20 and 7~gt10 DNA. The nucleotide sequence of
the
cDNA encoding the carboxy-terminal 112 amino acids of human FGF-20 was
determined, and is shown in Figure 7. An alignment of rat and human FGF-20
amino
acid sequences is shown in Figure 8.
EXAMPLE 1 I
Preparation of Antisera to FGF 20 by Immunization of Rabbits with an
to FGF 20 Peptide --- A peptide sequence corresponding to selected contiguous
amino
acids of the human FGF-20 protein is synthesized and coupled to keyhole limpet
hemocyanin (KLH) as described (Harlow and Land, Antibodies: A Laboratory
Manual,
1988. Cold Spring Harbor Laboratory, New York, NY). The KLH-coupled peptide is
used to immunize rabbits. Antisera are tested for specificity to FGF-20, and
for cross-
reactivity with other FGF proteins.
Exemplary peptide sequences are:
1. RDGARSKRHQKFTH (SEQ ID NO:S)
2. QLAHLHGILRRRQLY (SEQ ID N0:6)
EXAMPLE 12
Binding of FGF 20 to the Recombinant Extracellular Domains of
FGFR-1 c, FGFR-2c, and FGFR-3c -- Recombinant FGF-20 was fixed on the sensor
tip
CMS (Amersham Pharmacia Biotech). Binding of the recombinant extracellular
domain of FGFR-Ic, FGFR-2c, or FGFR-3c to FGF-20 on the tip was analyzed using
the BIACORE 2000 System (Amersham Pharmacia Biotech). The equilibrium
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dissociation constant was determined by the BIA evaluation software (Amersham
Pharmacia Biotech).
TABLE 1
BINDING OF FGF-ZO TO FGF RECEPTORS
Receptor Ka~ss (S Kris (M ~ K~, (M)
~) ' S ~)
FGFR-I c nd nd
FGFR-2c I .67 x 10-25.95 x 105 2.81 x 10-g
FGFR-3c 2.47 x 10-Z 1.15 x 105 2.17 x 10-'
nd: not detected
As shown in Table 1, FGF-20 binds to FGF receptors 2 and 3, but not to
FGF receptor 1. Thus, FGF-20 may exhibit biological effects not found in
members of
the FGF family that bind to FGF receptor 1, such as FGF-2 and FGF-4.
t o All patents, published patent applications and publications cited herein
are incorporated by reference as if set forth fully herein.
Although certain preferred embodiments have been described herein, it is
not intended that such embodiments be construed as limitations on the scope of
the
invention except as set forth in the following claims.
47
