U.S. patent application number 11/318327 was filed with the patent office on 2006-08-03 for human fgf-20 gene and gene expression products.
This patent application is currently assigned to Chiron Corporation. Invention is credited to Nobuyuki Itoh, Michael Kavanaugh.
Application Number | 20060172386 11/318327 |
Document ID | / |
Family ID | 32995790 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060172386 |
Kind Code |
A1 |
Itoh; Nobuyuki ; et
al. |
August 3, 2006 |
Human FGF-20 gene and gene expression products
Abstract
This invention relates to human fibroblast growth factor
(hFGF-20), and to variants thereof and to polynucleotides encoding
FGF-20. The invention also relates to diagnostic and therapeutic
agents related to the polynucleotides and proteins, including
probes and antibodies, to methods of treating neuronal degenerative
disease such as Parkinson's disease and to methods of treating
disorders of the cochlea including those causing hearing loss. The
invention also relates to rat fibroblast growth factor (rFGF-20),
and to variants thereof and polynucleotides encoding rFGF-20.
Inventors: |
Itoh; Nobuyuki; (Kyoto,
JP) ; Kavanaugh; Michael; (Mill Valley, CA) |
Correspondence
Address: |
Chiron Corporation
P.O. Box 8097
Emeryville
CA
94608
US
|
Assignee: |
Chiron Corporation
Emeryville
CA
|
Family ID: |
32995790 |
Appl. No.: |
11/318327 |
Filed: |
December 27, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10639117 |
Aug 12, 2003 |
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11318327 |
Dec 27, 2005 |
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09692945 |
Oct 20, 2000 |
6797695 |
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11318327 |
Dec 27, 2005 |
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60187856 |
Mar 8, 2000 |
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60161162 |
Oct 22, 1999 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 514/18.2; 514/8.3; 514/9.1; 530/350;
530/388.24; 536/23.5 |
Current CPC
Class: |
C12N 2799/026 20130101;
C07K 14/50 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/069.1 ;
530/350; 530/388.24; 435/320.1; 435/325; 514/012; 536/023.5 |
International
Class: |
C07K 14/50 20060101
C07K014/50; A61K 38/18 20060101 A61K038/18; C07K 16/22 20060101
C07K016/22; C12P 21/06 20060101 C12P021/06; C07H 21/04 20060101
C07H021/04 |
Claims
1-11. (canceled)
12. An isolated polypeptide comprising amino acids at least 95%
identical to amino acids selected from the group consisting of: (a)
amino acids from about 1 to about 211 of SEQ ID NO:4; (b) amino
acids from about 2 to about 211 of SEQ ID NO:4; (c) amino acids
from about 170 to about 186 of SEQ ID NO:4; (d) amino acids from
about 1 to about 169 and from about 187 to about 211 of SEQ ID
NO:4, wherein said amino acids at positions about 169 and about 187
are joined by a peptide bond; and (e) amino acids from about 59 to
about 193 of SEQ ID NO:4.
13. An isolated polypeptide wherein, except for at least one
conservative amino acid substitution, said polypeptide has an amino
acid sequence selected from the group consisting of: (a) amino
acids from about 1 to about 211 of SEQ ID NO:4; (b) amino acids
from about 2 to about 211 of SEQ ID NO:4; (c) amino acids from
about 170 to about 186 of SEQ ID NO:4; (d) amino acids from about 1
to about 169 and from about 187 to about 211 of SEQ ID NO:4,
wherein said amino acids at positions about 169 and about 187 are
joined by a peptide bond; and (e) amino acids from about 59 to
about 193 of SEQ ID NO:4.
14. An isolated polypeptide comprising amino acids selected from
the group consisting of: (a) amino acids from about 1 to about 211
of SEQ ID NO:4; (b) amino acids from about 2 to about 211 of SEQ ID
NO:4; (c) amino acids from about 170 to about 186 of SEQ ID NO:4;
(d) amino acids from about 1 to about 169 and from about 187 to
about 211 of SEQ ID NO:4, wherein said amino acids at positions
about 169 and about 187 are joined by a peptide bond; and (e) amino
acids from about 59 to about 193 of SEQ ID NO:4.
15. An epitope-bearing portion of the polypeptide of SEQ ID
NO:4.
16. The epitope-bearing portion of claim 15, which comprises
between 10 and 50 contiguous amino acids of SEQ ID NO:4.
17. The epitope-bearing portion of claim 15, which comprises a
polypeptide selected from the group consisting of amino acids
RDGARSKRHQKFTH (SEQ ID NO:5) and QLAHLHGILRRRQLY (SEQ ID NO:6).
18. An isolated antibody that binds specifically to the polypeptide
of claim 12.
19. An isolated antibody that binds specifically to the polypeptide
of claim 13.
20. An isolated antibody that binds specifically to the polypeptide
of claim 14.
21. A pharmaceutical composition comprising the polypeptide of
claim 12, in combination with a pharmaceutically acceptable
carrier.
22-44. (canceled)
45. An isolated polypeptide comprising amino acids at least 95%
identical to amino acids selected from the group consisting of: (a)
amino acids from about 1 to about 212 of SEQ ID NO:2; (b) amino
acids from about 2 to about 212 of SEQ ID NO:2; (c) amino acids
from about 170 to about 186 of SEQ ID NO:2; (d) amino acids from
about 1 to about 169 and from about 187 to about 212 of SEQ ID
NO:2, wherein said amino acids at positions about 169 and about 187
are joined by a peptide bond; and (e) amino acids from about 59 to
about 193 of SEQ ID NO:2.
46. An isolated polypeptide wherein, except for at least one
conservative amino acid substitution, said polypeptide has an amino
acid sequence selected from the group consisting of: (a) amino
acids from about 1 to about 212 of SEQ ID NO:2; (b) amino acids
from about 2 to about 212 of SEQ ID NO:2; (c) amino acids from
about 170 to about 186 of SEQ ID NO:2; (d) amino acids from about 1
to about 169 and from about 187 to about 212 of SEQ ID NO:2,
wherein said amino acids at positions about 169 and about 187 are
joined by a peptide bond; and (e) amino acids from about 59 to
about 193 of SEQ ID NO:2.
47. An isolated polypeptide comprising amino acids selected from
the group consisting of: (a) amino acids from about 1 to about 212
of SEQ ID NO:2; (b) amino acids from about 2 to about 212 of SEQ ID
NO:2; (c) amino acids from about 170 to about 186 of SEQ ID NO:2;
(d) amino acids from about 1 to about 169 and from about 187 to
about 212 of SEQ ID NO:2, wherein said amino acids at positions
about 169 and about 187 are joined by a peptide bond; and (e) amino
acids from about 59 to about 193 of SEQ ID NO:2.
48. An epitope-bearing portion of the polypeptide of SEQ ID
NO:2.
49. The epitope-bearing portion of claim 48, which comprises
between 10 and 50 contiguous amino acids of SEQ ID NO:2.
50. An isolated antibody that binds specifically to the polypeptide
of claim 45.
51. An isolated antibody that binds specifically to the polypeptide
of claim 46.
52. An isolated antibody that binds specifically to the polypeptide
of claim 47.
53. A polypeptide encoded by a nucleic acid molecule comprising the
polynucleotide of SEQ ID NO:1.
54. The polypeptide of claim 53, comprising the amino acid sequence
of SEQ ID NO:2.
55. A polypeptide encoded by a nucleic acid molecule comprising the
polynucleotide of SEQ ID NO:3.
56. The polypeptide of claim 55, comprising the amino acid sequence
of SEQ ID NO:4.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/639,117 filed Aug. 12, 2003, which is a divisional application
of U.S. Ser. No. 09/692,945 filed Oct. 20, 2000, and claims
priority from U.S. Patent Application No. 60/161,162 filed Oct. 22,
1999, and U.S. Patent Application No. 60/187,856 filed Mar. 8,
2000, which are incorporated by reference herein in their
entirety.
TECHNICAL FIELD
[0002] 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
[0003] 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 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.
[0004] Glial 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.
[0005] 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 (Baird, 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. NY Acad. Sci. 638:18-26 (1991); 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); 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 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
.about.120-amino acid residue core with .about.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
[0006] The present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
[0007] (a) a polynucleotide comprising at least eight contiguous
nucleotides of SEQ ID NO:1 or 3; [0008] (b) a polynucleotide that
encodes a variant of the polypeptide encoded by (a); and [0009] (c)
a polynucleotide encoding a protein expressed by a polynucleotide
having the sequence of SEQ ID NO:1 or 3.
[0010] The invention further provides for the use of the isolated
polynucleotides or fragments thereof as diagnostic probes or as
primers.
[0011] The present invention also provides a composition comprising
a polypeptide, wherein said polypeptide is selected from the group
consisting of: [0012] (a) a polypeptide comprising at least 6
contiguous amino acids encoded by SEQ ID NO:1 or 3; [0013] (b) a
polypeptide encoded by a polynucleotide comprising SEQ ID NO:1 or
3; and [0014] (c) a variant of the polypeptide of (a) or (b).
[0015] Polypeptides of the invention are shown in SEQ ID NO:2 and
4. Other polypeptides comprise fragments of SEQ ID NO:2 and 4.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] The invention further provides for the isolation,
self-renewal and survival of mammalian neural stem cells and the
differentiation of their progeny.
[0020] 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.
[0021] 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
[0022] FIG. 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.
[0023] FIG. 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.
[0024] FIG. 3. Localization of FGF-20 mRNA in rat brain. (A, B)
Coronal sections were hybridized with .sup.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 B are shown in C and D,
respectively. White grains in the dark-field photograph show the
localization of FGF-20 mRNA. Scale bar=50 .mu.m. SNC, substantial
nigra pars compacta.
[0025] FIG. 4. Expression of FGF-20 mRNA in rat brain. Rat brain
poly (A).sup.+RNA (10 .mu.g) was electrophoresed on a denaturing
agarose gel (1%) containing formaldehyde and transferred onto a
nitrocellulose membrane. Hybridization was performed with a
.sup.32P labeled rat FGF-20 or .beta.-actin cDNA probe. 28S and 18S
indicate the positions of 28 and 18S rRNAs, respectively.
[0026] FIG. 5. FGF-20 enhances survival of midbrain dopaminergic
neurons. (A) Effect of FGF-20 on survival of midbrain dopaminergic
neurons in serum-free 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.
[0027] FIG. 6. FIG. 6 provides the DNA sequence (SEQ ID NO:1) of
rat FGF-20.
[0028] FIG. 7. FIG. 7 provides the amino acid sequence (SEQ ID
NO:2) of rat FGF-20.
[0029] FIG. 8. FIG. 8 provides the DNA (SEQ ID NO:3) and amino acid
(SEQ ID NO:4) sequences of human FGF-20.
[0030] FIG. 9. FIG. 9 provides an alignment of the amino acid
sequences of human (SEQ ID NO:4) and rat (SEQ ID NO:2) FGF-20.
[0031] FIG. 10. FIG. 10 provides codon usage for E. coli.
[0032] FIG. 11. FIG. 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. 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.
[0033] FIG. 12. FIG. 12 provides codon usage for Drosophila.
DETAILED DESCRIPTION OF THE INVENTION
[0034] 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 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.
[0035] 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 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 (1996); and
Ghosh et al., Cell Growth and Differentiation 7:1425-1434
(1996).
[0036] 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.N.A.S. 89:8928-8932 (1992)).
[0037] 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 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. 41:279-288 (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.
[0038] 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 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
NO: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
polynucleotides and genes encoding known FGF proteins.
[0039] 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.
[0040] 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.
[0041] As discussed herein, the knowledge about the roles played by
various FGF proteins continues to grow, but is by far
incomplete.
[0042] The present invention adds to this knowledge by disclosing
that the FGF of SEQ ID NO:1 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.
[0043] The invention therefore is based upon the identification,
isolation, sequencing and expression patterns of a new fibroblast
growth factor (FGF-20).
[0044] Isolation and Analysis of Rat cDNA encoding FGF-20 Members
of the FGF family have a conserved .about.120-amino acid residue
core with .about.30 to 70% amino 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. FGF-20 is most similar to
FGF-9 and FGF-16 (70 and 62% amino acid identity) among 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.
[0045] 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).sup.+ RNA was examined
by Northern blotting analysis using a .sup.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).sup.+ RNA, the
hybridized probe was washed from the membrane, and the membrane was
rehybridized with a .sup.32P-labeled rat .beta.-actin cDNA probe. A
strong and discrete signal of .beta.-actin mRNA was detected
indicating that the poly (A).sup.+ 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 .sup.35S-labeled FGF-20
anti-sense and sense cRNA probes (FIG. 10).
[0046] 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 .sup.35S-labeled antisense or sense FGF-20 cRNA probe. Discrete
specific labeling was observed only in the substantia nigra pars
compacta (FIG. 3A, C). No specific labeling was observed in other
brain regions examined. The 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, Calif., Vol. 1, pp. 79-97 (1989)). Black grains of labeled
probes were found in most neurons of these brain areas (FIG. 3E).
Dopaminergic neurons in the substantial nigra are preferentially
localized in the substantial nigra pars compacta (Fallon, J. H. et
al., The Rat Nervous System, 2.sup.nd Ed., Academic Press, San
Diego, Calif., 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.sup.nd Ed., Academic Press, San Diego, Calif., pp. 215-238
(1995)). It is expected that FGF-20 is preferentially expressed in
dopaminergic neurons in the substantia nigra pars compacta.
[0047] Preparation of Recombinant Rat FGF-20 To produce recombinant
rat FGF-20, High Five insect cells were infected with recombinant
baculovirus containing the rat FGF-20 cDNA with the 3'-terminal
extension encoding E and His.sub.6 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. 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.
[0048] 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 medium for 4
days, numbers of surviving dopaminergic neurons were greatly
reduced. 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).
[0049] 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.
[0050] Degeneration of dopaminergic neurons in the substantia nigra
causes Parkinson's disease (Fallon, J. H. et al., The Rat Nervous
System, 2.sup.nd Ed., Academic Press, San Diego, Calif., 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. 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, 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.
[0051] 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. 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.
[0052] 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.
[0053] In vivo imaging of dopaminergic activity in the basal
ganglia, using [.sup.18F]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.
[0054] FGF-20 Expression in Rat Embryo Cochlea FGF-20 is
preferentially 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 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 suggests its importance
in development of this tissue.
[0055] 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
(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.
[0056] Reference to FGF-20 herein is intended to be construed to
include 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.
[0057] 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.
[0058] 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.
[0059] Sequence identity or percent identity is intended to mean
the percentage of same residues between two sequences, referenced
to human FGF when determining percent identity with non-human
FGF-20, referenced to FGF-20 when determining 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, Wis.). 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 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).
[0060] Percent conservation is calculated from the above alignment
by adding the percentage of identical residues to the percentage of
positions at which the two 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 are: R-K; E-D, Y-F,
L-M; V-I, Q-H.
[0061] 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
blocking the formation of disulfide bonds and aggregation when the
protein is exposed 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.
[0062] 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.
[0063] 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.
[0064] 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 fragment thereof and a
signal sequence of a heterologous protein to promote secretion of
the protein product.
[0065] 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 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.
[0066] 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.
[0067] The invention also includes fragments of FGF-20. Preferred
fragments of SEQ ID NO: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 NO:2); amino acids from about 1 to about 169
and about 187 to about 211 (212 for SEQ ID NO: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.
[0068] 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 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. Pat. 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. Pat. No. 5,876,969, incorporated
by reference herein.
[0069] 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 residues
170-186 are inserted at the desired position(s) 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.
[0070] Also included within the meaning of substantially homologous
is any 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 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.
[0071] 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 growth factor
families. FGF-20 binds to FGF receptor 2 and FGF receptor 3, but
does not bind to FGF receptor 1. Thus, its receptor binding profile
differs from FGF-2 and FGF-4, which bind to FGF receptor 1.
[0072] 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.
[0073] 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.
[0074] 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 FIG. 11, and codon usage in
Drosophila is show in FIG. 12.
[0075] 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
Sf9 cells, using baculovirus vector.
[0076] 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 known and standard procedures. Such modified
nucleic acid sequences are included within the scope of this
invention.
[0077] 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 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.
[0078] 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 present invention.
[0079] 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 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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 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 FGF-20 antisense
oligonucleotides comprise from about 15 to about 30 nucleotides.
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.
[0085] 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 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.
[0086] 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), 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.)
[0087] 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 solution, sterile water or the like may
also be used. It may also be desirable that a 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.
[0088] 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
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.
[0089] Dose administration can be repeated depending upon the
pharmacokinetic parameters of the dosage formulation and the route
of administration used.
[0090] 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 active agents.
[0091] 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.
[0092] 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 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).
[0093] 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.
[0094] FGF-20 bound heparin-Sepharose beads may be encapsulated
into calcium alginate microspheres to permit the controlled release
of the heparin-stabilized 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.
[0095] 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 NaCl and functional
activity assays may be performed.
[0096] 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 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 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.
[0097] In one embodiment of this invention, FGF-20 may be
therapeutically 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 FGF-20 or a precursor thereof or
the cells can be transformed to express FGF-20 or a 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.
[0098] 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
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.
[0099] 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 function related to cell growth and
survival. Indeed, other neurotrophic factors are known to play a
role in the function of neuronal and non-neuronal tissues. (Scully
and Otten, Cell Bol. Int. 19:459-469 (1995); Otten 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 4:563-565 (1993), which is incorporated by
reference).
[0100] 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.
[0101] 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.
[0102] 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, serum, CSF or the like. FGF-20 is
expressed in neural tissues as discussed in 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.
[0103] 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 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, but may be generated in any
manner including for example, chemical synthesis or DNA replication
or reverse transcription or transcription.
[0104] 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 AluI. 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.
[0105] 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 hybridized DNA-probe to detect
chromosomal DNA containing at least a portion of a human FGF-20
gene.
[0106] 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.
[0107] 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.
[0108] Hybridization is typically carried out at
25.degree.-45.degree. C., more preferably at 32.degree.-40.degree.
C. and more preferably at 37.degree.-38.degree. 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.
[0109] 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 primers are typically
from about 20 to about 30 base pairs in length and hybridize to the
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.degree. C. to 105.degree. C. for times ranging from about
1 to about 10 minutes. The process is repeated for the desired
number of cycles.
[0110] 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.
[0111] 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 which might change
activity or expression levels or the like.
[0112] 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 a
patient suspected of having an abnormality in the FGF-20 gene or in
the FGF-20 gene of particular cells.
[0113] 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 nucleic acid
from the sample is subjected to gel electrophoresis or other size
separation techniques.
[0114] 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.
[0115] 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.
[0116] 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-20 specific primers.
(Belyavsky et al., Nucl. Acid Res. 17:2919-2932 (1989); Krug and
Berger, Methods in Enzymology 152:316-325, Academic Press, NY,
1987, which are incorporated by reference).
[0117] The polymerase 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.
[0118] Following amplification, the PCR product is then
electrophoresed and detected by ethidium bromide staining or by
phosphoimaging.
[0119] 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
assays, immunohistochemical techniques, agglutination and
complement assays. (For example, see Basic and Clinical Immunology,
217-262, Sites and Terr, eds., Appleton & Lange, Norwalk,
Conn., 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.
[0120] 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 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.
[0121] Numerous competitive and non-competitive protein binding
immunoassays are well known in the art. Antibodies employed in such
assays may be 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.
[0122] 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 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.
[0123] 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 a mouse (see
Example 11).
[0124] 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 NO:4. These oligopeptides are RDGARSKRHQKFTH
(SEQ ID NO:5) and QLAHLHGILRRRQLY (SEQ ID NO: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.
[0125] Antibodies to FGF-20 can also be raised against
oligopeptides that 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.
[0126] 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, Del.) (Caprino and Han, J. Org. Chem.
37:3404 (1972), which is incorporated by reference).
[0127] 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 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.
[0128] 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.
[0129] 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.
[0130] Polypeptides encoded by the instant polynucleotides and
corresponding full-length genes can be used to screen peptide
libraries, protein libraries, small molecule libraries, and phage
display libraries, and other known methods, to identify analogs or
antagonists.
[0131] 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.
[0132] A library of peptides may be synthesized following the
methods disclosed in U.S. Pat. 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. According to the method of the '175 patent, a suitable
peptide synthesis support (e.g., a 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.
[0133] 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 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.
[0134] 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 that exhibit a high
binding constant. These peptides can be tested for their ability to
inhibit or enhance the native activity. The methods described in
PCT No. WO 91/7823 and U.S. Pat. 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.
[0135] 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.
[0136] 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-20 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 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.
[0137] 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 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.
[0138] 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 1:51-64 (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.
[0139] The present invention can employ recombinant retroviruses
which are constructed to carry or express a selected nucleic acid
molecule of interest. Retrovirus 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. Pat. 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. Pat. 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.
[0140] 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 production of recombinant vector particles. Within
particularly preferred embodiments 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.
[0141] 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 described in U.S. Pat. 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.
[0142] Gene delivery vehicles of the present invention can also
employ parvovirus such as adeno-associated virus (AAV) vectors.
Representative examples 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.N.A.S. 90:10613-10617 (1993).
[0143] 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 obtain in vivo expression of a
foreign gene in the cochlea of guinea pigs.
[0144] 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-Eisleret al., P.N.A.S.
90:11498-11502 (1993); Guzman et al., Circulation 88:2838-2848
(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 described in WO 94/12649, WO
93/03769; WO 93/19191; WO 94/28938; 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.
[0145] 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. Ser. No. 08/240,030, filed May 9, 1994, and
U.S. Ser. No. 08/404,796; deposition of photopolymerized hydrogel
materials; hand-held gene transfer particle gun, as described in
U.S. Pat. No. 5,149,655; ionizing radiation as described in U.S.
Pat. 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).
[0146] Naked DNA may also be employed. Exemplary naked DNA
introduction methods are described in WO 90/11092 and U.S. Pat. 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 cytoplasm. Liposomes that can act
as gene delivery vehicles are described in U.S. Pat. No. 5,422,120,
PCT Patent Publication Nos. WO 95/13796, WO 94/23697, and WO
91/14445, and EP No. 0 524 968.
[0147] 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. Pat. No. 5,149,655; use of ionizing radiation for
activating transferred gene, as described in U.S. Pat. No.
5,206,152 and PCT Patent Publication No. WO 92/11033.
[0148] 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).
[0149] 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 cells. Examples include skeletal myopathies, bone
disease, and arthritis.
[0150] 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.
[0151] 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 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.
[0152] 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.
[0153] 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.
[0154] 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 1
[0155] Preparation of RNA--RNA was prepared from adult rat brain
using an RNA extraction kit (Pharmacia Biotech, Uppsala, Sweden).
Poly (A).sup.+ RNA was prepared using oligo (dT)-cellulose (Type 2,
Collaborative Biomedical Products, Bedford, Mass.).
Example 2
[0156] 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 .mu.l of a reaction mixture containing 5 pmole/.mu.l
of each of the sense and antisense degenerate 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, Wis.). The nucleotide sequence of the
cloned DNA was determined by a DNA sequencer (Applied Biosystems,
Foster, Calif.). To determine the coding region of a novel FGF
cDNA, the coding region was amplified from cDNA synthesized from
rat brain poly (A).sup.+ RNA by adaptor-ligation mediated
polymerase chain reaction using a Marathon cDNA amplification kit
(Clontech, Palo Alto, Calif.). 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 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
[0157] 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 .sup.35S-labeled FGF-20 anti-sense and sense
cRNA probes. The sections were counterstained with hematoxylin 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
[0158] Northern Blotting Analysis--Poly (A).sup.+ RNA (10 .mu.g)
from rat adult brain was dissolved on a denaturing agarose gel (1%)
containing formaldehyde, and transferred to a nitrocellulose
membrane in 20.times.SSC (1.times.SSC:0.15 M NACI/0.015 M sodium
citrate) overnight. A .sup.32P-labeled FGF-20 cDNA probe
(.about.650 base pairs) was labeled with a random primer labeling
kit (Pharmacia Biotech, Uppsala, Sweden) and deoxycytidine
5'-[.alpha.-.sup.32P-] triphosphate (.about.110 TBq/mmol) (ICN
Biomedicals Inc., Costa Mesa, Calif.). 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).sup.+ RNA, the hybridized probe on the membrane was washed with
0.5.times.SSC containing 0.01 M EDTA (pH 8.0) at 100.degree. C. for
5 min and with 0.05.times.SSC containing 0.01 M EDTA (pH 8.0) and
0.1% SDS at 60.degree. C. for 15 min. The washed membrane was
rehybridized with a .sup.32P-labeled rat Pactin cDNA probe
(.about.410 base pairs) (Nudel, U. et al., Nucleic Acids Res.
11:1759-1771 (1983)).
Example 5
[0159] In Situ Hybridization--Adult Wistar rat brain was frozen in
powdered dry ice, and sagittal sections were cut at 16 .mu.m with a
cryostat, thaw-mounted onto poly-L-lysine-coated slides, and stored
at -85.degree. C. until hybridization. A .sup.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'-.alpha.[.sup.35S]thiotriphosphate (.about.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)).
Example 6
[0160] Preparation of Recombinant Rat FGF-20--The rat FGF-20 cDNA
with a DNA fragment (75 BP) encoding an E-tag (GAPVPYPDPLEPR) and a
His.sub.6 tag (HHHHHH) at the 3'-terminus of the coding region was
constructed in a transfer vector DNA, pBacPAK9 (Clontech, Palo
Alto, Calif.). 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, Calif.). High Five insect
cells were infected with the resultant recombinant baculovirus and
incubated at 27.degree. C. for 65 h in serum-free medium EX-CELL
400 (JRH Biosciences, Lenexa, Kans.). 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 NaCl. After washing the column
with PBS containing 20 mM imidazole and 0.5 M NaCl, recombinant
FGF-20 was eluted from the column with PBS containing 250 mM
imidazole and 0.5 M NaCl, and applied to a column of Bio-Gel P-6 DG
(Bio-Rad Lab., Hercules, Calif.) in PBS containing 100
.mu.g/mlBSA.
EXAMPLE 7
[0161] Detection of Recombinant FGF-20 by Western Blotting
Analysis--The culture medium or rat recombinant FGF-20 was
separated by sodium dodecyl sulfate (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. 244:187-191 (1998)).
Example 8
[0162] 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.degree. C. in the culture
medium containing 10% fetal calf serum. From the 5.sup.th day of
culture, the cells were incubated in the culture medium containing
10% horse serum.
Example 9
[0163] Examination of Neurotrophic Activity of FGF-20 for Midbrain
Dopaminergic Neurons--Cells on the 8.sup.th 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.sup.th day. Cells on
the 8.sup.th 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
incubated in medium without FGF-20 and 1 mM glutamate and then
fixed with 4% paraformaldehyde for 30 min on the 12.sup.th 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, N.J.) 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.
Example 10
[0164] Isolation and Analysis of Human FGF-20--The coding region of
human FGF-20 DNA was amplified from human brain cDNA library
(.lamda.gt10) by PCR using primers specific for FGF-20 and
.lamda.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 FIG. 7. An alignment of rat and human FGF-20 amino
acid sequences is shown in FIG. 8.
Example 11
[0165] Preparation of Antisera to FGF-20 by Immunization of Rabbits
with an 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, N.Y.). 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.
[0166] Exemplary peptide sequences are: TABLE-US-00001 1.
RDGARSKRHQKFTH (SEQ ID NO:5) 2. QLAHLHGILRRRQLY (SEQ ID NO:6)
Example 12
[0167] Binding of FGF-20 to the Recombinant Extracellular Domains
of FGFR-1c, FGFR-2c, and FGFR-3c--Recombinant FGF-20 was fixed on
the sensor tip CM5 (Amersham Pharmacia Biotech). Binding of the
recombinant extracellular domain of FGFR-1c, FGFR-2c, or FGFR-3c to
FGF-20 on the tip was analyzed using the BIACORE 2000 System
(Amersham Pharmacia Biotech). The equilibrium dissociation constant
was determined by the BIA evaluation software (Amersham Pharmacia
Biotech). TABLE-US-00002 TABLE 1 BINDING OF FGF-20 TO FGF RECEPTORS
Receptor K.sub.diss (S.sup.-1) K.sub.ass (M.sup.-1 S.sup.-1)
K.sub.d (M) FGFR-1c nd nd FGFR-2c 1.67 .times. 10.sup.-2 5.95
.times. 10.sup.5 2.81 .times. 10.sup.-8 FGFR-3c 2.47 .times.
10.sup.-2 1.15 .times. 10.sup.5 2.17 .times. 10.sup.-7 nd: not
detected
[0168] 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.
[0169] All patents, published patent applications and publications
cited herein are incorporated by reference as if set forth fully
herein.
[0170] 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.
Sequence CWU 1
1
17 1 648 DNA Rattus norvegicus 1 ccttccatgg ctcccttgac cgaagtcggt
gccttcttgg gcggcctgga gggcttgggc 60 cagcaggtgg ggtcgcactt
cttgctgcct cctgcagggg agcgaccgcc gctgctaggg 120 gagcggcggg
gcgcgttgga gcggggcgcc cgcggcgggc cgggttccgt ggagctggcg 180
cacctgcacg gcatcctgcg ccgccggcag ctctactgcc gcaccggctt ccacctgcag
240 atcctgcccg acggcagtgt gcagggcacc cggcaggatc acagcctctt
cggtatcctg 300 gaattcatca gtgtggcggt ggggctggtc agtatcagag
gtgtggacag cggcctgtac 360 cttggcatga atggcaaagg agagctttat
ggctcagaga aattgacttc tgaatgcatc 420 ttcagggaac aatttgaaga
gaactggtat aatacctatt catccaacat atacaaacac 480 ggagacacag
gtcgcaggta ttttgtagca cttaacaaag acgggactcc aagggacggt 540
gccaggtcca aaagacacca aaagtttacc cattttttac ccagaccagt ggacccagag
600 agagtcccag agttatacaa agacctactg gtgtacactg gatgaacc 648 2 212
PRT Rattus norvegicus 2 Met Ala Pro Leu Thr Glu Val Gly Ala Phe Leu
Gly Gly Leu Glu Gly 1 5 10 15 Leu Gly Gln Gln Val Gly Ser His Phe
Leu Leu Pro Pro Ala Gly Glu 20 25 30 Arg Pro Pro Leu Leu Gly Glu
Arg Arg Gly Ala Leu Glu Arg Gly Ala 35 40 45 Arg Gly Gly Pro Gly
Ser Val Glu Leu Ala His Leu His Gly Ile Leu 50 55 60 Arg Arg Arg
Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Gln Ile Leu 65 70 75 80 Pro
Asp Gly Ser Val Gln Gly Thr Arg Gln Asp His Ser Leu Phe Gly 85 90
95 Ile Leu Glu Phe Ile Ser Val Ala Val Gly Leu Val Ser Ile Arg Gly
100 105 110 Val Asp Ser Gly Leu Tyr Leu Gly Met Asn Gly Lys Gly Glu
Leu Tyr 115 120 125 Gly Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Arg
Glu Gln Phe Glu 130 135 140 Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn
Ile Tyr Lys His Gly Asp 145 150 155 160 Thr Gly Arg Arg Tyr Phe Val
Ala Leu Asn Lys Asp Gly Thr Pro Arg 165 170 175 Asp Gly Ala Arg Ser
Lys Arg His Gln Lys Phe Thr His Phe Leu Pro 180 185 190 Arg Pro Val
Asp Pro Glu Arg Val Pro Glu Leu Tyr Lys Asp Leu Leu 195 200 205 Val
Tyr Thr Gly 210 3 636 DNA Homo sapiens 3 atggctccct tagccgaagt
cgggggcttt ctgggcggcc tggagggctt gggccagcag 60 gtgggttcgc
atttcctgtt gcctcctgcc ggggagcggc cgccgctgct gggcgagcgc 120
aggagcgcgg cggagcggag cgcgcgcggc gggccggggg ctgcgcagct ggcgcacctg
180 cacggcatcc tgcgccgccg gcagctctat tgccgcaccg gcttccacct
gcagatcctg 240 cccgacggca gcgtgcaggg cacccggcag gaccacagcc
tcttcggtat cttggaattc 300 atcagtgtgg cagtgggact ggtcagtatt
agaggtgtgg acagtggtct ctatcttgga 360 atgaatgaca aaggagaact
ctatggatca gagaaactta cttccgaatg catctttagg 420 gagcagtttg
aagagaactg gtataacacc tattcatcta acatatataa acatggagac 480
actggccgca ggtattttgt ggcacttaac aaagacggaa ctccaagaga tggcgccagg
540 tccaagaggc atcagaaatt tacacatttc ttacctagac cagtggatcc
agaaagagtt 600 ccagaattgt acaaggacct actgatgtac acttga 636 4 211
PRT Homo sapiens 4 Met Ala Pro Leu Ala Glu Val Gly Gly Phe Leu Gly
Gly Leu Glu Gly 1 5 10 15 Leu Gly Gln Gln Val Gly Ser His Phe Leu
Leu Pro Pro Ala Gly Glu 20 25 30 Arg Pro Pro Leu Leu Gly Glu Arg
Arg Ser Ala Ala Glu Arg Ser Ala 35 40 45 Arg Gly Gly Pro Gly Ala
Ala Gln Leu Ala His Leu His Gly Ile Leu 50 55 60 Arg Arg Arg Gln
Leu Tyr Cys Arg Thr Gly Phe His Leu Gln Ile Leu 65 70 75 80 Pro Asp
Gly Ser Val Gln Gly Thr Arg Gln Asp His Ser Leu Phe Gly 85 90 95
Ile Leu Glu Phe Ile Ser Val Ala Val Gly Leu Val Ser Ile Arg Gly 100
105 110 Val Asp Ser Gly Leu Tyr Leu Gly Met Asn Asp Lys Gly Glu Leu
Tyr 115 120 125 Gly Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Arg Glu
Gln Phe Glu 130 135 140 Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile
Tyr Lys His Gly Asp 145 150 155 160 Thr Gly Arg Arg Tyr Phe Val Ala
Leu Asn Lys Asp Gly Thr Pro Arg 165 170 175 Asp Gly Ala Arg Ser Lys
Arg His Gln Lys Phe Thr His Phe Leu Pro 180 185 190 Arg Pro Val Asp
Pro Glu Arg Val Pro Glu Leu Tyr Lys Asp Leu Leu 195 200 205 Met Tyr
Thr 210 5 14 PRT Artificial Sequence Oligopeptides for raising
antibodies 5 Arg Asp Gly Ala Arg Ser Lys Arg His Gln Lys Phe Thr
His 1 5 10 6 15 PRT Artificial Sequence Oligopeptides for raising
antibodies 6 Gln Leu Ala His Leu His Gly Ile Leu Arg Arg Arg Gln
Leu Tyr 1 5 10 15 7 10 PRT Artificial Sequence Residues which can
be incorporated into FGF-20 to allow myc monoclonal antibody-based
affinity purification. 7 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1
5 10 8 5 PRT Artificial Sequence Preferred thrombin cleavage site.
8 Leu Val Pro Arg Gly 1 5 9 10 PRT Artificial Sequence Sequence
which can be incorporated to allow for puficiation of FGF-20
because of its ablility to bind to paramagentic streptavidin beads.
9 Ser Ala Trp Arg His Pro Gln Phe Gly Gly 1 5 10 10 6 PRT
Artificial Sequence Consensus amino acid sequences used to create
sense and anti-sense PCR primers. 10 Phe Glu Glu Asn Trp Tyr 1 5 11
6 PRT Artificial Sequence Consensus amino acid sequences used to
create sense and anti-sense PCR primers. 11 Thr His Phe Leu Pro Arg
1 5 12 6 PRT Artificial Sequence Consensus amino acid sequences
used to create sense and anti-sense PCR primers. 12 Glu Asn Trp Tyr
Asn Thr 1 5 13 6 PRT Artificial Sequence Consensus amino acid
sequences used to create sense and anti-sense PCR primers. 13 His
Gln Lys Phe Thr His 1 5 14 13 PRT Artificial Sequence E-tag 14 Gly
Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg 1 5 10 15 6 PRT
Artificial Sequence His tag 15 His His His His His His 1 5 16 208
PRT Rattus norvegicus 16 Met Ala Pro Leu Gly Glu Val Gly Ser Tyr
Phe Gly Val Gln Asp Ala 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val
Leu Pro Val Asp Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Gly
Gln Ser Glu Ala Gly Gly Leu Pro Arg Gly 35 40 45 Pro Ala Val Thr
Asp Leu Asp His Leu Lys Gly Ile Leu Arg Arg Arg 50 55 60 Gln Leu
Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly 65 70 75 80
Thr Ile Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu 85
90 95 Phe Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp
Ser 100 105 110 Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr
Gly Ser Glu 115 120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln
Phe Glu Glu Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr
Lys His Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu
Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg
His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro
Asp Lys Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205
17 207 PRT Rattus norvegicus 17 Met Ala Glu Val Gly Gly Val Phe Ala
Ser Leu Asp Trp Asp Leu Gln 1 5 10 15 Gly Phe Ser Ser Ser Leu Gly
Asn Val Pro Leu Ala Asp Ser Pro Gly 20 25 30 Phe Leu Asn Glu Arg
Leu Gly Gln Ile Glu Gly Lys Leu Gln Arg Gly 35 40 45 Ser Pro Thr
Asp Phe Ala His Leu Lys Gly Ile Leu Arg Arg Arg Gln 50 55 60 Leu
Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly Thr 65 70
75 80 Val His Gly Thr Arg His Asp His Ser Arg Phe Gly Ile Leu Glu
Phe 85 90 95 Ile Ser Leu Ala Val Gly Leu Ile Ser Ile Arg Gly Val
Asp Ser Gly 100 105 110 Leu Tyr Leu Gly Met Asn Glu Arg Gly Glu Leu
Phe Gly Ser Lys Lys 115 120 125 Leu Thr Arg Glu Cys Val Phe Arg Glu
Gln Phe Glu Glu Asn Trp Tyr 130 135 140 Asn Thr Tyr Ala Ser Thr Leu
Tyr Lys His Ser Asp Ser Glu Arg Gln 145 150 155 160 Tyr Tyr Val Ala
Leu Asn Lys Asp Gly Ser Pro Arg Glu Gly Tyr Arg 165 170 175 Thr Lys
Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val Asp 180 185 190
Pro Ser Lys Leu Pro Ser Met Ser Arg Asp Leu Phe Arg Tyr Arg 195 200
205
* * * * *