U.S. patent application number 10/854485 was filed with the patent office on 2005-02-24 for novel fgf homologs.
Invention is credited to Conklin, Darrell C., Deisher, Theresa A..
Application Number | 20050043234 10/854485 |
Document ID | / |
Family ID | 34199407 |
Filed Date | 2005-02-24 |
United States Patent
Application |
20050043234 |
Kind Code |
A1 |
Deisher, Theresa A. ; et
al. |
February 24, 2005 |
Novel FGF homologs
Abstract
The present invention relates to methods of using zFGF5
compositions to proliferate chondrocytes and their progenitors, and
to induce deposition of cartilage. zFGF5 compositions are disclosed
for treating disorders associated with chondrocytes, such as
cartilage injuries and defects. In addition, methods for treating
neurological disorders, such as stroke, are disclosed, and methods
for using zFGF5 compositions to stimulate growth of cells
associated with neurological injury and disease are disclosed.
Inventors: |
Deisher, Theresa A.;
(Seattle, WA) ; Conklin, Darrell C.; (Seattle,
WA) |
Correspondence
Address: |
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Family ID: |
34199407 |
Appl. No.: |
10/854485 |
Filed: |
May 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10854485 |
May 26, 2004 |
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10315431 |
Dec 9, 2002 |
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10315431 |
Dec 9, 2002 |
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09634318 |
Aug 9, 2000 |
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09634318 |
Aug 9, 2000 |
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09613708 |
Jul 11, 2000 |
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09613708 |
Jul 11, 2000 |
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09574750 |
May 18, 2000 |
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09574750 |
May 18, 2000 |
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09229947 |
Jan 13, 1999 |
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6518236 |
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09229947 |
Jan 13, 1999 |
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08951822 |
Oct 16, 1997 |
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5989866 |
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60028646 |
Oct 16, 1996 |
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Current U.S.
Class: |
514/9.1 ;
514/12.2; 514/16.8 |
Current CPC
Class: |
C07K 2319/00 20130101;
C07K 14/50 20130101; Y10S 514/825 20130101; A61K 38/1825
20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/18 |
Claims
We claim:
1. A method of increasing cartilage deposition in a patient
comprising: administering a pharmaceutical composition comprising a
fibroblast growth factor (FGF) homolog polypeptide to a
fibrocartilage, hyaline, or elastic cartilage injury in said
patient, wherein said polypeptide comprises a sequence of amino
acids that is at least 80% identical to the amino acid sequence as
shown in SEQ ID NO: 2 from amino acid residue 28 (Glu) to 175
(Met).
2. The method of claim 1 wherein the pharmaceutical composition
comprises a polypeptide that is at least 80% identical to the amino
acid sequence as shown in SEQ ID NO: 2 from residue 28 (Glu) to 196
(Lys).
3. The method of claim 1 wherein the pharmaceutical composition
comprises a polypeptide that is at least 80% identical to the amino
acid sequence as shown in SEQ ID NO:2 from residue 28 (Glu) to 207
(Ala).
4. The method of claim 1 wherein the pharmaceutical composition
comprises a polypeptide that is at least 90% identical to the amino
acid sequence as shown in SEQ ID NO: 2 from amino acid residue 28
(Glu) to residue 175 (Met).
5. The method of claim 1 wherein the pharmaceutical composition
comprises a polypeptide that is at least 90% identical to the amino
acid sequence as shown in SEQ ID NO: 2 from residue 28 (Glu) to 196
(Lys).
6. The method of claim 1 wherein the pharmaceutical composition
comprises a polypeptide that is at least 90% identical to the amino
acid sequence as shown in SEQ ID NO:2 from residue 28 (Glu) to 207
(Ala).
7. The method of claim 1 wherein the pharmaceutical composition
comprises a polypeptide having the amino acid sequence as shown in
SEQ ID NO: 2 from residue 28 (Glu) to residue 175 (Met).
8. The method of claim 1 wherein the pharmaceutical composition
comprises a polypeptide having the amino acid sequence as shown in
SEQ ID NO: 2 from residue 28 (Glu) to residue 196 (Lys).
9. The method of claim 1 wherein the pharmaceutical composition
comprises a polypeptide having the amino acid sequence as shown in
SEQ ID NO: 2 from residue 28 (Glu) to residue 207 (Ala).
10. The method of claim 1, wherein the administration of the
composition is selected from the group consisting of
intracartilaginous administration; intraarticular administration;
intravenous; intramuscular; and topical administration.
11. The method of claim 2, wherein the administration of the
composition is selected from the group consisting of
intracartilaginous administration; intraarticular administration;
intravenous; intramuscular; and topical administration.
12. The method of claim 3, wherein the administration of the
composition is selected from the group consisting of
intracartilaginous administration; intraarticular administration;
intravenous; intramuscular; and topical administration.
13. The method of claim 1 wherein the pharmaceutical composition
further comprises an anti-inflammatory agent.
14. The method of claim 2 wherein the pharmaceutical composition
further comprises an anti-inflammatory agent.
15. The method of claim 3 wherein the pharmaceutical composition
further comprises an anti-inflammatory agent.
16. A method of treating a condition involving reduced cartilage
deposition in a patient in need of such treatment comprising:
administering a pharmaceutical composition comprising a fibroblast
growth factor (FGF) homolog polypeptide to a fibrocartilage,
hyaline, or elastic cartilage injury in said patient, wherein said
polypeptide comprises a sequence of amino acids that is at least
80% identical to the amino acid sequence as shown in SEQ ID NO: 2
from amino acid residue 28 (Glu) to 175 (Met).
17. The method of claim 1 wherein the pharmaceutical composition
comprises a polypeptide having the amino acid sequence as shown in
SEQ ID NO: 2 from residue 28 (Glu) to residue 175 (Met).
18. The method of claim 16 wherein the condition involving reduced
cartilage deposition is osteoarthritis.
19. The method of claim 16, wherein the administration of the
composition is selected from the group consisting of
intracartilaginous administration; intraarticular administration;
intravenous; intramuscular; and topical administration.
20. The method of claim 16 wherein the pharmaceutical composition
further comprises an anti-inflammatory agent.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 10/315,431, filed Dec. 9, 2002, which is a
continuation of U.S. application Ser. No. 09/634,318, filed on Aug.
9, 2000, which is a continuation-in-part of U.S. patent application
Ser. No. 09/613,708, filed on Jul. 11, 2000 which is a continuation
of U.S. patent application Ser. No. 09/574,750, filed May 18, 2000,
which is a continuation-in-part of U.S. patent application Ser. No.
09/229,947 (U.S. Pat. No. 6,518,236), filed on Jan. 13, 1999, which
is a continuation-in-part of U.S. application Ser. No. 08/951,822
(U.S. Pat. No. 5,989,866), filed on Oct. 16, 1997 and U.S.
application Ser. No. 10/081,347, filed on Feb. 21, 2002, a
continuation of U.S. Pat. No. 5,989,866, for which claims of
benefit are made under 35 U.S.C. .sctn. 120, and Provisional
Application 60/028,646, filed on Oct. 16, 1996 for which claims of
benefit are made under 35 U.S.C. .sctn. 119(e)(1), and are
incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] The fibroblast growth factor (FGF) family consists of at
least eighteen distinct members (Basilico et al., Adv. Cancer Res.
59:115-165, 1992 and Fernig et al., Prog. Growth Factor Res.
5(4):353-377, 1994) which generally act as mitogens for a broad
spectrum of cell types. For example, basic FGF (also known as
FGF-2) is mitogenic in vitro for endothelial cells, vascular smooth
muscle cells, fibroblasts, and generally for cells of mesoderm or
neuroectoderm origin, including cardiac and skeletal myocytes
(Gospodarowicz et al., J. Cell. Biol. 70:395-405, 1976;
Gospodarowicz et al., J. Cell. Biol. 89:568-578, 1981 and Kardami,
J. Mol. Cell. Biochem. 92:124-134, 1990). In vivo, bFGF has been
shown to play a role in avian cardiac development (Sugi et al.,
Dev. Biol. 168:567-574, 1995 and Mima et al., Proc. Nat'l. Acad.
Sci. 92:467-471, 1995), and to induce coronary collateral
development in dogs (Lazarous et al., Circulation 94:1074-1082,
1996). In addition, non-mitogenic activities have been demonstrated
for various members of the FGF family. Non-proliferative activities
associated with acidic and/or basic FGF include: increased
endothelial release of tissue plasminogen activator, stimulation of
extracellular matrix synthesis, chemotaxis for endothelial cells,
induced expression of fetal contractile genes in cardiomyocytes
(Parker et al., J. Clin. Invest. 85:507-514, 1990), and enhanced
pituitary hormonal responsiveness (Baird et al., J. Cellular
Physiol. 5:101-106, 1987.)
[0003] Several members of the FGF family do not have a signal
sequence (aFGF, bFGF and possibly FGF-9) and thus would not be
expected to be secreted. In addition, several of the FGF family
members have the ability to migrate to the cell nucleus (Friesel et
al., FASEB 9:919-925, 1995). All the members of the FGF family bind
heparin based on structural similarities. Structural homology
crosses species, suggesting a conservation of their
structure/function relationship (Ornitz et al., J. Biol. Chem.
271(25):15292-15297, 1996.)
[0004] There are four known cellular FGF receptor genes (FGFRs),
and they are all tyrosine kinases. In general, the FGF family
members bind to all of the known FGFRs, however, specific FGFs bind
to specific receptors with higher degrees of affinity. Another
means for specificity within the FGF family is the spatial and
temporal expression of the ligands and their receptors during
embryogenesis. Evidence suggests that the FGFs most likely act only
in autocrine and/or paracrine manner, due to their heparin binding
affinity, which limits their diffusion from the site of release
(Flaumenhaft et al., J. Cell. Biol. 111(4):1651-1659, 1990.) Basic
FGF lacks a signal sequence, and is therefore restricted to
paracrine or autocrine modes of action. It has been postulated that
basic FGF is stored intracellularly and released upon tissue
damage. Basic FGF has been shown to have two receptor binding
regions that are distinct from the heparin binding site (Abraham et
al.,. EMBO J. 5(10):2523-2528, 1986.)
[0005] It has been shown that FGFR-3 plays a role in bone growth.
Mice made homozygous null for the FGFR-3 (-/-) resulted in
postnatal skeletal abnormalities (Colvin et al., Nature Genet.
12:309-397, 1996 and Deng et al., Cell 84:911-921, 1996). The
mutant phenotype suggests that in normal mice, FGFR-3 plays a role
in regulation of chondrocyte cell division in the growth plate
region of the bone (Goldfarb, Cytokine and Growth Factor Rev.
7(4):311-325, 1996). The ligand for the FGFR-3 in the bone growth
plate has not been identified.
[0006] Although four FGFRs have been identified, all of which have
been shown to have functional splice variants, the possibility that
novel FGF receptors exist is quite likely. For example, no receptor
has been identified for the FGF-8a isoform (MacArthur et al., J.
Virol. 69(4):2501-2507, 1995.).
[0007] FGF-8 is a member of the FGF family that was originally
isolated from mammary carcinoma cells as an androgen-inducible
mitogen. It has been mapped to human chromosome 10q25-q26 (White et
al., Genomics 30:109-11, 1995.) FGF-8 is involved in embryonic limb
development (Vogel et al., Development 122:1737-1750, 1996 and
Tanaka et al., Current Biology 5(6):594-597, 1995.) Expression of
FGF-8 during embryogenesis in cardiac, urogenital and neural tissue
indicates that it may play a role in development of these tissues
(Crossley et al., Development 121:439-451, 1995.) There is some
evidence that acrocephalosyndactylia, a congenital condition marked
by peaked head and webbed fingers and toes, is associated with
FGF-8 point mutations (White et al., 1995, ibid.)
[0008] FGF-8 has five exons, in contrast to the other known FGFs,
which have only three exons. The first three exons of FGF-8
correspond to the first exon of the other FGFs (MacArthur et al.,
Development 121:3603-3613, 1995.) The human gene for FGF-8 codes
for four isoforms which differ in their N-terminal regions: FGF
isoforms a, b, e, and f; in contrast to the murine gene which gives
rise to eight FGF-8 isoforms (Crossley et al., 1995, ibid.) Human
FGF-8a and FGF-8b have 100% homology to the murine proteins, and
FGF-8e and FGF-8f proteins are 98% homologous between human and
mouse (Gemel et al., Genomics 35:253-257, 1996.)
[0009] Heart disease is the major cause of death in the United
States, accounting for up to 30% of all deaths. Myocardial
infarction (MI) accounts for 750,000 hospital admissions per year
in the U.S., with more than 5 million people diagnosed with
coronary disease. Risk factors for MI include diabetes mellitus,
hypertension, truncal obesity, smoking, high levels of low density
lipoprotein in the plasma or genetic predisposition.
[0010] Cardiac hyperplasia is an increase in cardiac myocyte
proliferation, and has been demonstrated to occur with normal aging
in the human and rat (Olivetti et al., J. Am. Coll. Cardiol.
24(1):140-9, 1994 and Anversa et al., Circ. Res. 67:871-885, 1990),
and in catecholamine-induced cardiomyopathy in rats (Deisher et
al., Am. J. Cardiovasc. Pathol. 5(1):79-88, 1994.) Whether the
increase in myocytes originate with some progenitor cell, or are a
result of proliferation of a more terminally differentiated cell
type, remains controversial.
[0011] However, because infarction and other causes of myocardial
necrosis appear to be irreparable, it appears that the normal
mechanisms of cardiac hyperplasia cannot compensate for extensive
myocyte death, and there remains a need for exogenous factors that
promote hyperplasia and ultimately result in renewal of the heart's
ability to function.
[0012] Stroke is caused by either cerebral thrombosis, embolism, or
subarachnoid or cerebral hemorrhage, and results in ischemia in
approximately 80% of occurrences. Stroke is a major health problem
disabling over three million people in the United States, with
550,0000 Americans suffering stroke each year, of which 150,000 of
those affected will die. The current treatments to prevent tissue
damage resulting from stroke are very limited and require
administration within an hour of onset of the stroke. While there
are more drugs available to try to prevent reoccurrence of stroke,
they are not without some serious drawbacks, including the
development of intracranial hemorrhaging, gastrointestinal bleeding
and neutropenia. Therefore, any therapeutics that promote
angiogenesis, promote neurite outgrowth, or survival of neurons in
necrotic areas of the central nervous system with some specificity
will be valuable. The molecules of the present invention have been
shown to promote growth in specific tissues, including neuronal
tissue.
[0013] Bone remodeling is the dynamic process by which tissue mass
and skeletal architecture are maintained. The process is a balance
between bone resorption and bone formation, with two cell types
thought to be the major players. These cells are the osteoblast and
osteoclast. Osteoblasts synthesize and deposit matrix to become new
bone. The activities of osteoblasts and osteoclasts are regulated
by many factors, systemic and local, including growth factors.
[0014] While the interaction between local and systemic factors has
not been completely elucidated, there does appear to be consensus
that growth factors play a key role in the regulation of both
normal skeletal remodeling and fracture repair. Some of the growth
factors that have been identified in bone include: IGF-I, IGF-II,
TGF-.beta..sub.1, TGF-.beta..sub.2, bFGF, aFGF, PDGF and the family
of bone morphogenic proteins (Baylink et al., J. Bone Mineral Res.
8 (Supp. 2):S565-S572, 1993).
[0015] When bone resorption exceeds bone formation, a net loss in
bone results, and the propensity for fractures is increased.
Decreased bone formation is associated with aging and certain
pathological states. In the U.S. alone, there are approximately 1.5
million fractures annually that are attributed to osteoporosis. The
impact of these fractures on the quality of the patient's life is
immense. Associated costs to the health care system in the U.S. are
estimated to be $5-$10 billion annually, excluding long-term care
costs.
[0016] Other therapeutic applications for growth factors
influencing bone remodeling include, for example, the treatment of
injuries which require the proliferation of osteoblasts to heal,
such as fractures, as well as stimulation of mesenchymal cell
proliferation and the synthesis of intramembraneous bone which have
been indicated as aspects of fracture repair (Joyce et al. 36th
Annual Meeting, Orthopaedic Research Society, Feb. 5-8, 1990. New
Orleans, La.).
[0017] Replacement of damaged articular cartilage caused either by
injury or defect is a major challenge for physicians, and available
treatments are considered unpredictable and effective for only a
limited time. Therefore, the majority of younger patients either do
not seek treatment or are counseled to postpone treatment for long
as possible. When treatment is required, the standard procedure is
a total joint replacement or penetration of the subchondral bone to
stimulate fibrocartilage deposition by chondrocytes. While
deposition of fibrocartilage is not a functional equivalent of
articular cartilage, it is at the present the best available
treatment because there has been little success in replacing
articular cartilage. Two approaches to stimulating deposition of
articular cartilage that are being investigated are: stimulating
chondrocyte activity in vivo and ex vivo expansion of chondrocytes
and their progenitors for transplantation (Jackson et al.,
Arthroscopy: The J. of Arthroscopic and Related Surg. 12:732-738,
1996). In addition, regeneration or repair of elastic cartilage is
valuable for treating injuries and defects to ear and nose. Any
growth factor with specificity for chondrocytes lineage cells that
stimulates those cells to growth, differentiate or induce cartilage
production would be valuable for maintaining, repairing or
replacing articular cartilage.
[0018] The present invention provides such polypeptides for these
and other uses that should be apparent to those skilled in the art
from the teachings herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 and FIG. 2 illustrate a multiple alignment of human
fibroblast growth factor homologous factor 1 (FHF-1; SEQ ID NO:
21), human myocyte-activating factor (FGF-10; SEQ ID NO: 22), human
fibroblast growth factor homologous factor 4 (FHF-4; SEQ ID NO:
23), human fibroblast growth factor homologous factor 2 (FHF-2; SEQ
ID NO: 24), human fibroblast growth factor homologous factor 3
(FHF-3; SEQ ID NO: 25), human FGF-4 (SEQ ID NO: 26), human FGF-6
(SEQ ID NO: 27), human FGF-2 (basic; SEQ ID NO: 28), human FGF-1
(acidic; SEQ ID NO: 29), human keratinocyte growth factor 2 (KGF-2;
SEQ ID NO: 30), human keratinocyte growth factor precursor (FGF-7;
SEQ ID NO: 31), human zFGF5 (SEQ ID NO: 2), human FGF-8 (SEQ ID NO:
32) human FGF-5 (SEQ ID NO: 33), human FGF-9 (SEQ ID NO: 34), and
human FGF-3 (SEQ ID NO: 35). "*" designates conserved amino acids;
":" designates conserved amino acid substitutions; and "."
designates less stringently conserved amino acid substitutions.
[0020] FIG. 3 is an inter-family similarity matrix illustrating the
percent identity between: (1) human FGF-5 (SEQ ID NO: 33), (2)
human FGF-6 (SEQ ID NO: 27), (3) human FGF-7 (SEQ ID NO: 31), (4)
human FGF-8 (SEQ ID NO: 32), (5) human FGF-9 (SEQ ID NO: 34), (6)
human zFGF5 (SEQ ID NO: 2), (7) human FGF-10 (SEQ ID NO: 22), (8)
human FGF-1 (SEQ ID NO: 29), (9) human FHF-1 (SEQ ID NO: 21), (10)
human FGF-2 (SEQ ID NO: 28), (11) human FHF-2 (SEQ ID NO: 24), (12)
human FHF-4 (SEQ ID NO: 23), (13) human FGF-3 (SEQ ID NO: 35), (14)
human KGF-2 (SEQ ID NO: 30), (15) human FHF-3 (SEQ ID NO: 25), and
(16) human FGF4 (SEQ ID NO: 26).
[0021] FIG. 4 is a multiple alignment of the amino acid sequences
for mature human zFGF5 and mouse zFGF5 (SEQ ID NOS: 2 and 39,
respectively).
DETAILED DESCRIPTION OF THE INVENTION
[0022] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0023] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,
Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and
Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et
al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P,
Flag.TM. peptide (Hopp et al., Biotechnology 6:1204-10, 1988),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2: 95-107, 1991. DNAs encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0024] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0025] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0026] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complem- ent pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0027] The term "complements of a polynucleotide molecule" is a
polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to 5'
CCCGTGCAT 3'.
[0028] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0029] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription. Such additional segments include
promoter and terminator sequences, and may also include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0030] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0031] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term "isolated" does
not exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0032] The term "operably linked", when referring to DNA segments,
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in the promoter and proceeds through the coding segment to the
terminator. The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speciation.
[0033] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired. Such unpaired ends will
in general not exceed 20 nt in length.
[0034] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0035] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0036] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0037] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-peptide structure comprising an
extracellular ligand-binding domain and an intracellular effector
domain that is typically involved in signal transduction. Binding
of ligand to receptor results in a conformational change in the
receptor that causes an interaction between the effector domain and
other molecule(s) in the cell. This interaction in turn leads to an
alteration in the metabolism of the cell. Metabolic events that are
linked to receptor-ligand interactions include gene transcription,
phosphorylation, dephosphorylation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids. In general, receptors can be membrane
bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating
hormone receptor, beta-adrenergic receptor) or multimeric (e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF
receptor, G-CSF receptor, erythropoietin receptor and IL-6
receptor).
[0038] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0039] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a protein encoded by a splice
variant of an mRNA transcribed from a gene.
[0040] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
understood to be approximate values. When such a value is expressed
as "about" X or "approximately" X, the stated value of X will be
understood to be accurate to .+-.10%.
[0041] All references cited herein are incorporated by reference in
their entirety.
[0042] The present invention is based in part upon the discovery of
a novel DNA sequence that encodes a fibroblast growth factor (FGF)
homolog polypeptide having homology to FGF-8 and FGF-17 (Hoshikawa
et al., Biochem. Biophys. Res. Comm. 244:187-191, 1998). Analysis
of the tissue distribution of the human mRNA corresponding to this
novel DNA showed that expression was highest in fetal heart tissue
and adult heart tissue, followed by apparent but decreased
expression levels in fetal lung, skeletal muscle, smooth muscle
tissues such as small intestine, colon and trachea. The FGF homolog
polypeptide has been designated zFGF5.
[0043] Tissue distribution in murine species does not appear to
completely correspond with expression in human tissues. Northern
analysis of mouse tissues revealed that expression of mouse zFGF5
is highest in spleen and day 17 embryo, followed by relatively
lower expression in heart, lung, kidney and testis. Mouse heart
tissue analysis found expression highest in day 16 fetal heart
tissue, with expression in adult heart present in most mouse
strains. It also appears that there may be variability within
murine expression levels and tissues (Hu et al., Mol. Cell. Biol.
18:6063-6074, 1998; Ohbayashi et al., J. Biol. Chem.
273:18161-18164, 1998 and Maruoka et al., Mech. Develop.
74:175-175, 1998).
[0044] The nucleotide sequence of the zFGF5 cDNA is described in
SEQ ID NO. 1, and its deduced amino acid sequence is described in
SEQ ID NO. 2. When amino acid residue 28 (Glu) to amino acid
residue 181 (Gln) of SEQ ID NO: 2 is compared to the corresponding
region of FGF-8 (See FIGS. 1 and 2) the aligned and deduced amino
acid sequence has approximately 56% identity. FGF-17 (Hoshiwara et
al., Biochem. Biophys. Res. Comm. 244:187-191, 1998) has recently
been identified, and has the highest degree of homology to zFGF5.
The region of highest identity is .about.66% over a 123 amino acid
overlap which corresponds to the region of SEQ ID NO: 2 from
residue 55 (Tyr) to residue 177 (Arg).
[0045] The novel polypeptide encoded by the polynucleotide
described herein contains the CXFXE{6}Y motif present in all
members of the FGF family. The CXFXE{6}Y motifs (SEQ ID NO: 36) are
highly conserved. A consensus amino acid sequence of the CXFXEX{6}Y
domain (SEQ ID NO: 36) includes human fibroblast growth factor
homologous factor 1 (FHF-1; Smallwood et al., Proc. Natl. Acad.
Sci. USA 93:9850-9857, 1996), human myocyte-activating factor
(FGF-10; HSU76381, GENBANK identifier), human fibroblast growth
factor homologous factor 4 (FHF-4; Smallwood et al., 1996, ibid.),
human fibroblast growth factor homologous factor 2 (FHF-2;
Smallwood et al., 1996, ibid.), human fibroblast growth factor
homologous factor 3 (FHF-3; Smallwood et al., 1996, ibid.), human
FGF-4 (Basilico et al., Adv. Cancer Res. 59:115-165,1992), human
FGF-6 (Basilico et al., 1992, ibid.), human FGF-2 (basic; Basilico
et al., 1992, ibid.), human FGF-1 (acidic; Basilico et al., 1992,
ibid.), human keratinocyte growth factor 2 (KGF-2; HSU67918 GENBANK
identifier), human keratinocyte growth factor precursor (FGF-7;
Basilico et al., 1992, ibid.), human zFGF5, human FGF-8 (Gemel et
al., Genomics 35:253-257, 1996), human FGF-5 (Basilico et al.,
1992, ibid.), human FGF-9 (Miyamoto et al., Mol. Cell. Biol.
13:4251-4259, 1993), human FGF-3 (Basilico et al., 1992, ibid.),
and FGF-17 (Hoshiwara et al., 1998, ibid.).
[0046] Analysis of the cDNA encoding a zFGF5 polypeptide (SEQ ID
NO: 1) revealed an open reading frame encoding 207 amino acids (SEQ
ID NO: 2) comprising a mature polypeptide of 180 amino acids
(residue 28 to residue 207 of SEQ ID NO: 2). Multiple alignment of
zFGF5 with other known FGFs revealed a block of high percent
identity corresponding to amino acid residue 127 (Cys) to amino
acid residue 138 (Tyr), of SEQ ID NO: 2 and is shown in FIG. 1.
Several of the members of the FGF family do not have signal
sequences.
[0047] The mouse zFGF5 polynucleotide sequence as shown in SEQ ID
NO: 38 and corresponding amino acid sequence as shown in SEQ ID NO:
39 were found to have a high degree of homology to that of the
human ortholog. At the amino acid level, the mouse and human
polypeptides are approximately 98% identical, with three amino acid
changes. The changes as shown in FIG. 4, correspond to a Val.sub.26
in SEQ ID NO: 2 being Ala.sub.26 in SEQ ID NO: 39 in the mouse
polypeptide, Pro.sub.183 in SEQ ID NO: 2 to Ala.sub.183 in SEQ ID
NO: 39 and Ala.sub.207 in SEQ ID NO: 2 to Gly.sub.207 in SEQ ID NO:
39. As is noted previously, Ala.sub.26 (mouse) and the
corresponding Val.sub.26 (human) are in the secretory signal
sequence, leaving only two amino acid differences in the mature
polypeptide. Based on the high identity between the mouse and human
sequences, it is predicted that function will be equivalent as
well. However, based on differences in tissue distribution for the
mouse and human expression, zFGF5 may have a wider organ target
distribution, and more diverse biological functions in the mouse
than in the human.
[0048] Members of the FGF family are characterized by heparin
binding domains. A putative heparin-binding domain for zFGF5 has
been identified in the region of amino acid residue 148 (Gly) to
amino acid residue 169 (Gln) of SEQ ID NO: 2 and SEQ ID NO: 39.
[0049] It is postulated that receptor-mediated signaling is
initiated upon binding of FGF ligand complexed with cell-surface
heparin sulfate proteoglycans. Many FGF family members can be
placed into one of two related families on the basis of their
structures and functions. aFGF and bFGF consist of three exons
separated by two introns of variable length. FGF-8 consists of five
exons, the first three of which correspond to the first exon of
aFGF and bFGF. All the known FGF family members are spliced to form
single polypeptides.
[0050] Analysis of the ligand-receptor complex of zFGF-5 has
demonstrated that zFGF-5 has specificity for the FGFR3.alpha.-IIIc
and FGFR4 receptors. Using cells that do not normally express any
of the FGF receptors but had been transfected to express
FGFR1.alpha.-IIIb,-IIIc, FGFR2.alpha.-IIIb,-IIIc, or
FGFR3.alpha.-IIIb, -IIIc, a high affinity cell proliferative
response (EC50 .about.1-2 ng/ml) was seen with cells expressing the
FGFR3.alpha.IIIc. A lower affinity response (EC50 .about.10-20
ng/ml) of activation was observed in cells expressing
FGFR2.alpha.-IIIc. Moreover, the activation of the FGFR3.alpha.IIIc
and FGFR2.alpha.-IIIc expressing cells by zFGF-5 was completely
dependent on exogenous heparin. Addition of zFGF-5 did not enhance
proliferation of FGFR1 nor any of the FGFR b splice variant
expressing cells. A comparison of FGF receptor specificity of FGF-1
and FGF-2 suggests that the receptor binding specificity of zFGF-5
is considerably more restricted than either FGF-1 or -2.
[0051] SEQ ID NO: 6 is a degenerate polynucleotide sequence that
encompasses all polynucleotides that could encode the zFGF5
polypeptide of SEQ ID NO: 2 (amino acids 1 or 28 to 207). Thus,
zFGF5 polypeptide-encoding polynucleotides ranging from nucleotide
1 or 82 to nucleotide 621 of SEQ ID NO: 6 are contemplated by the
present invention. Also contemplated by the present invention are
fragments and fusions as described above with respect to SEQ ID NO:
1, which are formed from analogous regions of SEQ ID NO: 6, wherein
nucleotides 82 to 621 of SEQ ID NO: 6 correspond to nucleotides 82
to 621 of SEQ ID NO: 1, for the encoding a mature zFGF5
molecule.
[0052] The symbols in SEQ ID NO: 6 are summarized in Table 1
below.
1 TABLE 1 Nucleotide Resolutions Complement Resolutions A A T T C C
G G G G C C T T A A R A.vertline.G Y C.vertline.T Y C.vertline.T R
A.vertline.G M A.vertline.C K G.vertline.T K G.vertline.T M
A.vertline.C S C.vertline.G S C.vertline.G C.vertline.G
A.vertline.T W A.vertline.T H A.vertline.C.vertline.T D
A.vertline.G.vertline.T B C.vertline.G.vertline.T V
A.vertline.C.vertline.G V A.vertline.C.vertline.G B
C.vertline.G.vertline.T D A.vertline.G.vertline.T H
A.vertline.C.vertline.T N A.vertline.C.vertline.G.vertline.T N
A.vertline.C.vertline.G.vertline.T The degenerate codons used in
SEQ ID NO: 6, encompassing all possible codons for a given amino
acid, are set forth in Table 2 below.
[0053]
2TABLE 2 Amino Degenerate Acid Letter Codons Codon Cys C TGC TGT
TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro
P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG
GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q
CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys
K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG
CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y
TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn.vertline.Asp B
RAY Glu.vertline.Gln Z SAR Any X NNN Gap - - - -
[0054] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding each amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may have some incorrect amino acids, but one of
ordinary skill in the art can easily identify such erroneous
sequences by reference to the amino acid sequence of SEQ ID NO:
2.
[0055] The highly conserved amino acids in zFGF5 can be used as a
tool to identify new family members. To identify new family members
the conserved CXFXEX{6}Y motif (SEQ ID NO: 36) can be used. In
another method using polynucleotide probes and hybridization
methods, RNA obtained from a variety of tissue sources can be used
to generate cDNA libraries and probe these libraries for new family
members. In particular, reverse transcription-polymerase chain
reaction (RT-PCR) can be used to amplify sequences encoding highly
degenerate DNA primers designed from the sequences corresponding to
amino acid residue 127 (Cys) to amino acid residue 138 (Tyr) of SEQ
ID NO: 2.
[0056] Within certain embodiments of the invention the isolated
polynucleotides will serve as a probe and hybridize to similar
sized regions of SEQ ID NO: 1 or a sequence complementary thereto,
under stringent conditions. In general, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Typical stringent
conditions are those in which the salt concentration is at least
about 0.02 M at pH 7 and the temperature is at least about
60.degree. C.
[0057] As previously noted, the isolated polynucleotides of the
present invention include DNA and RNA. Methods for isolating DNA
and RNA are well known in the art. It is generally preferred to
isolate RNA from cardiac tissue, although DNA can also be prepared
using RNA from other tissues or isolated as genomic DNA. Total RNA
can be prepared using guanidine HCl extraction followed by
isolation by centrifugation in a CsCl gradient (Chirgwin et al.,
Biochemistry 18:52-94, 1979). Poly (A).sup.+ RNA is prepared from
total RNA using the method of Aviv and Leder (Proc. Natl. Acad.
Sci. USA 69:1408-1412, 1972). Complementary DNA (cDNA) is prepared
from poly(A).sup.+ RNA using known methods. Polynucleotides
encoding zFGF5 polypeptides are then identified and isolated by,
for example, hybridization or PCR.
[0058] The present invention further provides counterpart
polypeptides and polynucleotides from other species (orthologs). Of
particular interest are zFGF5 polypeptides from other mammalian
species, including murine, rat, porcine, ovine, bovine, canine,
feline, equine and other primate proteins. Identification of
variants of the human sequence are particularly interesting because
while eight variants of murine FGF-8 have been identified, only
four human variants are known. Human variants or orthologs of the
human proteins can be cloned using information and compositions
provided by the present invention in combination with conventional
cloning techniques. For example, a cDNA can be cloned using mRNA
obtained from a tissue or cell type that expresses the protein.
Suitable sources of mRNA can be identified by probing Northern
blots with probes designed from the sequences disclosed herein. A
library is then prepared from mRNA of a positive tissue or cell
line. A zFGF5-encoding cDNA can then be isolated by a variety of
methods, such as by probing with a complete or partial human cDNA
or with one or more sets of degenerate probes based on the
disclosed sequences. A cDNA can also be cloned using the polymerase
chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using
primers designed from the sequences disclosed herein. Within an
additional method, the cDNA library can be used to transform or
transfect host cells, and expression of the cDNA of interest can be
detected with an antibody to zFGF5. Similar techniques can also be
applied to the isolation of genomic clones.
[0059] Those skilled in the art will recognize that the sequences
disclosed in SEQ ID NO: 1 or SEQ ID NO: 38 and SEQ ID NO: 2 and SEQ
ID NO: 39 represent a single allele of the human and mouse zFGF5
gene and polypeptide, respectively, and that allelic variation and
alternative splicing are expected to occur. Allelic variants can be
cloned by probing cDNA or genomic libraries from different
individuals according to standard procedures. Allelic variants of
the DNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 38, including
those containing silent mutations and those in which mutations
result in amino acid sequence changes, are within the scope of the
present invention, as are proteins which are allelic variants of
SEQ ID NO: 2 or SEQ ID NO: 39.
[0060] The present invention also provides isolated zFGF5
polypeptides that are substantially homologous to the polypeptides
of SEQ ID NO: 2 and their orthologs. The term "substantially
homologous" is used herein to denote polypeptides having 50% to
60%, with certain embodiments having at least 80%, sequence
identity to the sequences shown in SEQ ID NO: 2 or their orthologs.
In other embodiments, polypeptides may also be at least 90%
identical to 95% or more identical to SEQ ID NO: 2 or its
orthologs. Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:
603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915-10919, 1992. Briefly, two amino acid sequences are aligned
to optimize the alignment scores using a gap opening penalty of 10,
a gap extension penalty of 1, and the "blosum 62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: 1 Total number of identical matches [ length
of the longer sequence plus the number of gaps introduced into the
longer sequence in order to align the two sequences ] .times.
100
3 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0
6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0
-2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3
-4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1
-3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3
-3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1
-1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1
-1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3
-2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3
-3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0061] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0062] Substantially homologous proteins and polypeptides are
characterized as having one or more amino acid substitutions,
deletions or additions. These changes are preferably of a minor
nature, that is conservative amino acid substitutions (see Table 4)
and other substitutions that do not significantly affect the
folding or activity of the protein or polypeptide; small deletions,
typically of one to about 30 amino acids; and small amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine
residue, a small linker peptide of up to about 20-25 residues, or a
small extension that facilitates purification (an affinity tag),
such as a poly-histidine tract, protein A (Nilsson et al., EMBO J.
4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991),
glutathione S transferase (Smith and Johnson, Gene 67:31, 1988),
maltose binding protein (Kellerman and Ferenci, Methods Enzymol.
90:459-463, 1982; Guan et al., Gene 67:21-30, 1987), or other
antigenic epitope or binding domain. See, in general Ford et al.,
Protein Expression and Purification 2: 95-107, 1991, which is
incorporated herein by reference. DNAs encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.; New England Biolabs, Beverly, Mass.).
4TABLE 4 Conservative amino acid substitutions Basic: arginine
lysine histidine Acidic: glutamic acid aspartic acid Polar:
glutamine asparagine Hydrophobic: leucine isoleucine valine
Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine
serine threonine methionine
[0063] The proteins of the present invention can also comprise, in
addition to the 20 standard amino acids, non-naturally occurring
amino acid residues. Non-naturally occurring amino acids include,
without limitation, trans-3-methylproline, 2,4-methanoproline,
cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl-glycine,
allo-threonine, methylthreonine, hydroxyethyl-cysteine,
hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic
acid, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenyl-alanine, 4-fluorophenylalanine,
4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,
isovaline and .alpha.-methyl serine. Several methods are known in
the art for incorporating non-naturally occurring amino acid
residues into proteins. For example, an in vitro system can be
employed wherein nonsense mutations are suppressed using chemically
aminoacylated suppressor tRNAs. Methods for synthesizing amino
acids and aminoacylating tRNA are known in the art. Transcription
and translation of plasmids containing nonsense mutations are
carried out in a cell free system comprising an E. coli S30 extract
and commercially available enzymes and other reagents. Proteins are
purified by chromatography. See, for example, Robertson et al., J.
Am. Chem. Soc. 113:2722, 1991; Ellman et al., Meth. Enzymol.
202:301, 1991; Chung et al., Science 259:806-09, 1993; and Chung et
al., Proc. Natl. Acad. Sci. USA 90:10145-49, 1993). In a second
method, translation is carried out in Xenopus oocytes by
microinjection of mutated mRNA and chemically aminoacylated
suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-98,
1996). Within a third method, E. coli cells are cultured in the
absence of a natural amino acid that is to be replaced (e.g.,
phenylalanine) and in the presence of the desired non-naturally
occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470-76, 1994. Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395-403,
1993).
[0064] Essential amino acids in the zFGF5 polypeptides of the
present invention can be identified according to procedures known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244: 1081-1085, 1989).
In the latter technique, single alanine mutations are introduced at
every residue in the molecule, and the resultant mutant molecules
are tested for biological activity (e.g., receptor binding activity
using .sup.125I-zFGF5 (Moscatelli, J. Cell Physio. 131:123-130.
1987), activation of receptor tyrosine kinase (Panek et al., J.
Pharm. Exp. Therapeutics 286:569-577, 1998 and Schafer et al.,
Anal. Biochem. 261:100-112, 1998), generation of cardiac myocytes
or fibroblasts, or stimulation of bone formation) to identify amino
acid residues that are critical to the activity of the molecule.
See also, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. Sites
of ligand-receptor interaction can also be determined by physical
analysis of structure, as determined by such techniques as nuclear
magnetic resonance, crystallography, electron diffraction or
photoaffinity labeling, in conjunction with mutation of putative
contact site amino acids. See, for example, de Vos et al., Science
255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992;
Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of
essential amino acids can also be inferred from analysis of
homologies with related FGFs and are shown in FIGS. 1 and 2.
[0065] Analyses of the amino acid sequence of human and mouse zFGF5
revealed a dibasic site at the C-terminus of the polypeptide (amino
acid residue 196-197 (Lys-Arg)). A C-terminally truncated
polypeptide comprising an amino acid sequence as shown in SEQ ID
NO: 2, from amino acid residue 28 (Glu) to amino acid residue 196
(Lys) was demonstrated to have biological activity. Dibasic amino
acids, such as, Arg-X-X-Arg (wherein X is any amino acid residue;
SEQ ID NO: 37), Arg-Arg or Lys-Arg; are subject to cleavage by
several enzymes, including, but not limited to, thrombin and
carboxypeptidases. Therefore, it is within the scope of the claims
to make conservative changes at dibasic amino acid residues, in
particular the dibasic residues at amino acid residues 196 and 197
(Lys and Arg, respectively) of SEQ ID NO: 2 or SEQ ID NO: 39.
[0066] Based on analyses of the FGF family a C-terminally truncated
molecule that comprises amino acid residue 28 (Glu) to residue 175
(Met) of SEQ ID NO: 2 will be biologically active. An
intramolecular disulfide bond is predicted to occur between amino
acid residue 109 (Cys) and residue 127 (Cys) of SEQ ID NO: 2 or SEQ
ID NO: 39.
[0067] Based on homology alignments with FGF-1 and FGF-2 crystal
structures (Eriksson et al., Prot. Sci. 2:1274, 1993), secondary
structure predictions for beta strand structure of zFGF5 correlates
to amino acid residues 56-59, 64-69, 73-76, 85-92, 96-102, 106-111,
115-119, 128-134, 138-144, 149-155, and 173-177 of SEQ ID NO: 2 or
SEQ ID NO: 39. Amino acids critical for zFGF5 binding to receptors
can be identified by site-directed mutagenesis of the entire zFGF5
polypeptide. More specifically, they can be identified using
site-directed mutagenesis of amino acids in the zFGF5 polypeptide
which correspond to amino acid residues in acidic FGF (FGF1) and
basic FGF (FGF2) identified as critical for binding of these FGFs
to their receptors (Blaber et al., Biochem. 35:2086-2094, 1996).
These amino acids include Tyr33, Arg53, Asn110, Tyr112, Lys119,
Trp123, Leu149 and Met151 in human FGF2, and Tyr30, Arg50, Asn107,
Tyr109, Lys116, Trp122, Leu148 and Leu150 in human FGF1, as shown
in FIG. 1 and FIG. 2. The corresponding amino acids in zFGF5, as
shown in FIG. 1 and FIG. 2, would be Tyr58, Gly77, Asn136, Tyr138,
Lys145, Trp149, Met175 and Arg177. One skilled in the art will
recognize that other members, in whole or in part, of the FGF
family may have structural or biochemical similarities to zFGF5,
and be substituted making such analyses. Such regions would be
important for biological functions of the molecule.
[0068] An alignment based on homology of zFGF5 with FGF-17 revealed
the highest percent identity region consists of a 123 amino acid
overlap found between residue 55 (Tyr) and residue 177 (Arg) of SEQ
ID NO: 2 with .about.66% identity. When conservative amino acid
changes are calculated over the same region, the percent homology
is .about.92%.
[0069] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or
Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner
et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO
92/06204) and region-directed mutagenesis (Derbyshire et al., Gene
46:145, 1986; Ner et al., DNA 7:127, 1988).
[0070] Mutagenesis methods as disclosed above can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode active polypeptides (e.g., cell
proliferation) can be recovered from the host cells and rapidly
sequenced using modern equipment. These methods allow the rapid
determination of the importance of individual amino acid residues
in a polypeptide of interest, and can be applied to polypeptides of
unknown structure.
[0071] Using the methods discussed above, one of ordinary skill in
the art can identify and/or prepare a variety of polypeptides that
are substantially homologous to residues 28 (Glu) to 175 (Met),
residues 28 (Glu) to 196 (Lys) or residues 28 (Glu) to 207 (Ala) of
SEQ ID NO: 2, allelic variants thereof, or biologically active
fragments thereof, and retain the proliferative properties of the
wild-type protein. Such polypeptides may also include additional
polypeptide segments as generally disclosed above.
[0072] The polypeptides of the present invention, including
full-length proteins, fragments thereof and fusion proteins, can be
produced in genetically engineered host cells according to
conventional techniques. Suitable host cells are those cell types
that can be transformed or transfected with exogenous DNA and grown
in culture, and include bacteria, fungal cells, and cultured higher
eukaryotic cells. Eukaryotic cells, particularly cultured cells of
multicellular organisms, are preferred. Techniques for manipulating
cloned DNA molecules and introducing exogenous DNA into a variety
of host cells are disclosed by Sambrook et al., Molecular Cloning:
A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989, and Ausubel et al. (eds.), Current
Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y.,
1987, which are incorporated herein by reference.
[0073] In general, a DNA sequence encoding a zFGF5 polypeptide of
the present invention is operably linked to other genetic elements
required for its expression, generally including a transcription
promoter and terminator within an expression vector. The vector
will also commonly contain one or more selectable markers and one
or more origins of replication, although those skilled in the art
will recognize that within certain systems selectable markers may
be provided on separate vectors, and replication of the exogenous
DNA may be provided by integration into the host cell genome.
Selection of promoters, terminators, selectable markers, vectors
and other elements is a matter of routine design within the level
of ordinary skill in the art. Many such elements are described in
the literature and are available through commercial suppliers.
[0074] To direct a zFGF5 polypeptide into the secretory pathway of
a host cell, a secretory signal sequence (also known as a leader
sequence, prepro sequence or pre sequence) is provided in the
expression vector. The secretory signal sequence may be the native
sequence, or a chimera comprising a signal sequence derived from
another secreted protein (e.g., t-PA and .alpha.-pre-pro secretory
leader) or synthesized de novo. The secretory signal sequence is
joined to the zFGF5 DNA sequence in the correct reading frame.
Secretory signal sequences are commonly positioned 5' to the DNA
sequence encoding the polypeptide of interest, although certain
signal sequences may be positioned elsewhere in the DNA sequence of
interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland
et al., U.S. Pat. No. 5,143,830).
[0075] Fungal cells, including yeast cells, can also be used within
the present invention. Yeast species of particular interest in this
regard include Saccharomyces cerevisiae, Pichia pastoris, and
Pichia methanolica. Methods for transforming S. cerevisiae cells
with exogenous DNA and producing recombinant polypeptides therefrom
are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311;
Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No.
4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et
al., U.S. Pat. No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient (e.g., leucine). A preferred vector system for use in
Saccharomyces cerevisiae is the POT1 vector system disclosed by
Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed
cells to be selected by growth in glucose-containing media.
Suitable promoters and terminators for use in yeast include those
from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No.
4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter,
U.S. Pat. No. 4,977,092), and alcohol dehydrogenase genes. See also
U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0076] The use of Pichia methanolica as host for the production of
recombinant proteins is disclosed in WIPO Publications WO 97/17450,
WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in
transforming P. methanolica will commonly be prepared as
double-stranded, circular plasmids, which are preferably linearized
prior to transformation. For polypeptide production in P.
methanolica, it is preferred that the promoter and terminator in
the plasmid be that of a P. methanolica gene, such as a P.
methanolica alcohol utilization gene (AUG1 or AUG2). Other useful
promoters include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. A preferred selectable
marker for use in Pichia methanolica is a P. methanolica ADE2 gene,
which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), which allows ade2 host cells to grow in the absence of
adenine. For large-scale, industrial processes where it is
desirable to minimize the use of methanol, it is preferred to use
host cells in which both methanol utilization genes (AUG1 and AUG2)
are deleted. For production of secreted proteins, host cells
deficient in vacuolar protease genes (PEP4 and PRB1) are preferred.
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. It is preferred to transform P. methanolica
cells by electroporation using an exponentially decaying, pulsed
electric field having a field strength of from 2.5 to 4.5 kV/cm,
preferably about 3.75 kV/cm, and a time constant (.omega.) of from
1 to 40 milliseconds, most preferably about 20 milliseconds.
[0077] Other methods for transforming yeast cells with exogenous
DNA and producing recombinant polypeptides therefrom are disclosed
by, for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et
al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch
et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No.
4,845,075. Transformed cells are selected by phenotype determined
by the selectable marker, commonly drug resistance or the ability
to grow in the absence of a particular nutrient (e.g., leucine). An
alternative preferred vector system for use in yeast is the POT1
vector system disclosed by Kawasaki et al. (U.S. Pat. No.
4,931,373), which allows transformed cells to be selected by growth
in glucose-containing media. Suitable promoters and terminators for
use in yeast include those from glycolytic enzyme genes (see, e.g.,
Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No.
4,615,974; and Bitter, U.S. Pat. No. 4,977,092, which are
incorporated herein by reference) and alcohol dehydrogenase genes.
See also U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and
4,661,454, which are incorporated herein by reference.
Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
guillermondii, and Candida maltosa are known in the art. A
particularly preferred system utilizes Pichia methanolica (see, PCT
application WO 9717450). For alternative transformation systems,
see, for example, Gleeson et al.; J. Gen. Microbiol. 132:3459-3465,
1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be
utilized according to the methods of McKnight et al., U.S. Pat. No.
4,935,349, which is incorporated herein by reference. Methods for
transforming Acremonium chrysogenum are disclosed by Sumino et al.,
U.S. Pat. No. 5,162,228, which is incorporated herein by reference.
Methods for transforming Neurospora are disclosed by Lambowitz,
U.S. Pat. No. 4,486,533, which is incorporated herein by
reference.
[0078] Cultured mammalian cells are also preferred hosts within the
present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et
al., eds., Current Protocols in Molecular Biology, John Wiley and
Sons, Inc., NY, 1987), and liposome-mediated transfection
(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus
15:80, 1993), which are incorporated herein by reference. The
production of recombinant polypeptides in cultured mammalian cells
is disclosed, for example, by Levinson et al., U.S. Pat. No.
4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al.,
U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134,
which are incorporated herein by reference. Preferred cultured
mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC
No. CRL 1651), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL
1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese
hamster ovary (e.g. CHO-K1; ATCC No. CCL 61 or DG44) cell lines.
Additional suitable cell lines are known in the art and available
from public depositories such as the American Type Culture
Collection, Rockville, Md. In general, strong transcription
promoters are preferred, such as promoters from SV-40 or
cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitable
promoters include those from metallothionein genes (U.S. Pat. Nos.
4,579,821 and 4,601,978, which are incorporated herein by
reference) and the adenovirus major late promoter.
[0079] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is a gene
encoding resistance to the antibiotic neomycin. Selection is
carried out in the presence of a neomycin-type drug, such as G-418
or the like. Selection systems may also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification" Amplification is carried out by culturing
transfectants in the presence of a low level of the selective agent
and then increasing the amount of selective agent to select for
cells that produce high levels of the products of the introduced
genes. A preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate. Other drug
resistance genes (e.g., hygromycin resistance, multi-drug
resistance, puromycin acetyltransferase) can also be used.
[0080] Other higher eukaryotic cells can also be used as hosts,
including insect cells, plant cells and avian cells. Transformation
of insect cells and production of foreign polypeptides therein is
disclosed by Guarino et al., U.S. Pat. No. 5,162,222; Bang et al.,
U.S. Pat. No. 4,775,624; and WIPO publication WO 94/06463, which
are incorporated herein by reference. The use of Agrobacterium
rhizogenes as a vector for expressing genes in plant cells has been
reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,
1987.
[0081] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell.
[0082] Expressed recombinant zFGF5 polypeptides (or chimeric zFGF5
polypeptides) can be purified using fractionation and/or
conventional purification methods and media. Ammonium sulfate
precipitation and acid or chaotrope extraction may be used for
fractionation of samples. Exemplary purification steps may include
hydroxyapatite, size exclusion, FPLC and reverse-phase high
performance liquid chromatography. Suitable anion exchange media
include derivatized dextrans, agarose, cellulose, polyacrylamide,
specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives
are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,
Piscataway, N.J.) being particularly preferred. Exemplary
chromatographic media include those media derivatized with phenyl,
butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),
Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.),
Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins,
such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid
supports include glass beads, silica-based resins, cellulosic
resins, agarose beads, cross-linked agarose beads, polystyrene
beads, cross-linked polyacrylamide resins and the like that are
insoluble under the conditions in which they are to be used. These
supports may be modified with reactive groups that allow attachment
of proteins by amino groups, carboxyl groups, sulfhydryl groups,
hydroxyl groups and/or carbohydrate moieties. Examples of coupling
chemistries include cyanogen bromide activation,
N-hydroxysuccinimide activation, epoxide activation, sulfhydryl
activation, hydrazide activation, and carboxyl and amino
derivatives for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Methods for binding receptor
polypeptides to support media are well known in the art. Selection
of a particular method is a matter of routine design and is
determined in part by the properties of the chosen support. See,
for example, Affinity Chromatography: Principles & Methods,
Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.
[0083] The polypeptides of the present invention can also be
isolated by exploitation of their heparin binding properties. For a
review, see, Burgess et al., Ann. Rev. of Biochem. 58:575-606,
1989. Members of the FGF family can be purified to apparent
homogeneity by heparin-Sepharose affinity chromatography
(Gospodarowicz et al., Proc. Natl. Acad. Sci. 81:6963-6967, 1984)
and eluted using linear step gradients of NaCl (Ron et al., J.
Biol. Chem. 268(4):2984-2988, 1993; Chromatography: Principles
& Methods, pp. 77-80, Pharmacia LKB Biotechnology, Uppsala,
Sweden, 1993; in "Immobilized Affinity Ligand Techniques",
Hermanson et al., eds., pp. 165-167, Academic Press, San Diego,
1992; Kjellen et al., Ann. Rev. Biochem.Ann. Rev. Biochem.
60:443-474, 1991; and Ke et al., Protein Expr. Purif. 3(6):497-507,
1992.)
[0084] Other purification methods include using immobilized metal
ion adsorption (IMAC) chromatography to purify histidine-rich
proteins. Briefly, a gel is first charged with divalent metal ions
to form a chelate (E. Sulkowski, Trends in Biochem. 3:1-7, 1985).
Histidine-rich proteins will be adsorbed to this matrix with
differing affinities, depending upon the metal ion used, and will
be eluted by competitive elution, lowering the pH, or use of strong
chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (Methods in
Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher,
(ed.), Acad. Press, San Diego, 1990, pp.529-39). Alternatively, a
fusion of the polypeptide of interest and an affinity tag (e.g.,
polyhistidine, maltose-binding protein, an immunoglobulin domain)
may be constructed to facilitate purification.
[0085] Protein refolding (and optionally reoxidation) procedures
may be advantageously used. It is preferred to purify the protein
to >80% purity, more preferably to >90% purity, even more
preferably >95%, and particularly preferred is a
pharmaceutically pure state, that is greater than 99.9% pure with
respect to contaminating macromolecules, particularly other
proteins and nucleic acids, and free of infectious and pyrogenic
agents. Preferably, a purified protein is substantially free of
other proteins, particularly other proteins of animal origin.
[0086] zFGF5 polypeptides or fragments thereof may also be prepared
through chemical synthesis. zFGF5 polypeptides may be monomers or
multimers; glycosylated or non-glycosylated; pegylated or
non-pegylated; and may or may not include an initial methionine
amino acid residue. PEGylation is one method commonly used that has
been demonstrated to increase plasma half-life, increased
solubility, and decreased antigenicity and decreased immunogenicity
(Nucci et al., Advanced Drug Delivery Reviews 6:133-155, 1991 and
Lu et al., Int. J. Peptide Protein Res. 43:127-138, 1994). Several
procedures have been reported to create and purify PEGylated
proteins (See, e.g., Abuchowski et al., J. Biol. Chem.
252:3582-3586, 1977 and Becauchamp et al., Anal. Biochem.
131:25-33, 1983.) PEGylation may be achieved by modification of
carboxyl amino acid residues of a polypeptide or protein. In
particular acid amino acid residues (e.g. glutamic and aspartic
acids) and amino acids at the carboxyl-terminus are amenable to
PEGylation (Zalipsky, Bioconjugate Chem. 6:150-165, 1995).
[0087] The activity of molecules of the present invention can be
measured using a variety of assays that, for example, measure
neogenesis or hyperplasia (i.e., proliferation) of cardiac cells
based on the tissue specificity in adult heart. Additional
activities likely associated with the polypeptides of the present
invention include proliferation of endothelial cells,
cardiomyocytes, chondrocytes, fibroblasts, skeletal myocytes
directly or indirectly through other growth factors; action as a
chemotaxic factor for endothelial cells, fibroblasts and/or
phagocytic cells; osteogenic factor; and factor for expanding
mesenchymal stem cell and precursor populations.
[0088] Proliferation can be measured using cultured cardiac cells
or in vivo by administering molecules of the claimed invention to
the appropriate animal model. Generally, proliferative effects are
seen as an increase in cell number and therefore, may include
inhibition of apoptosis, as well as mitogenesis. Cultured cells
include cardiac fibroblasts, cardiac myocytes, skeletal myocytes,
chondrocytes, human umbilical endothelial vein cells from primary
cultures. Established cell lines include: NIH 3T3 fibroblast (ATCC
No. CRL-1658), CHH-1 chum heart cells (ATCC No. CRL-1680), H9c2 rat
heart myoblasts (ATCC No. CRL-1446), Shionogi mammary carcinoma
cells (Tanaka et al., Proc. Natl. Acad. Sci. 89:8928-8932, 1992)
and LNCap.FGC adenocarcinoma cells (ATCC No. CRL-1740.) Assays
measuring cell proliferation are well known in the art. For
example, assays measuring proliferation include such assays as
chemosensitivity to neutral red dye (Cavanaugh et al.,
Investigational New Drugs 8:347-354, 1990, incorporated herein by
reference), incorporation of radiolabelled nucleotides (Cook et
al., Analytical Biochem. 179:1-7, 1989, incorporated herein by
reference), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the
DNA of proliferating cells (Porstmann et al., J. Immunol. Methods
82:169-179, 1985, incorporated herein by reference), and use of
tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983;
Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth
Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833,
1988; all incorporated herein by reference).
[0089] Differentiation is a progressive and dynamic process,
beginning with pluripotent stem cells and ending with terminally
differentiated cells. Pluripotent stem cells that can regenerate
without commitment to a lineage express a set of differentiation
markers that are lost when commitment to a cell lineage is made.
Progenitor cells express a set of differentiation markers that may
or may not continue to be expressed as the cells progress down the
cell lineage pathway toward maturation. Differentiation markers
that are expressed exclusively by mature cells are usually
functional properties such as cell products, enzymes to produce
cell products and receptors. The stage of a cell population's
differentiation is monitored by identification of markers present
in the cell population. Myocytes, osteoblasts, adipocytes,
chondrocytes, fibroblasts and reticular cells are believed to
originate from a common mesenchymal stem cell (Owen et al., Ciba
Fdn. Symp. 136:42-46, 1988). Markers for mesenchymal stem cells
have not been well identified (Owen et al., J. of Cell Sci.
87:731-738, 1987), so identification is usually made at the
progenitor and mature cell stages. The existence of early stage
cardiac myocyte progenitor cells (often referred to as cardiac
myocyte stem cells) has been speculated, but not demonstrated, in
adult cardiac tissue. However, recent evidence confirms the
presence of myocyte proliferation in end-stage cardiac failure in
humans (Kajstura et al., Proc. Natl. Assoc. Science, 95:8801-8805,
1998). The novel polypeptides of the present invention are useful
to isolate mesenchymal stem cells and cardiac myocyte progenitor
cells, both in vivo and ex vivo.
[0090] There is evidence to suggest that factors that stimulate
specific cell types down a pathway towards terminal differentiation
or dedifferentiation, affects the entire cell population
originating from a common precursor or stem cell. Thus, the present
invention includes stimulating inhibition-or proliferation of
myocytes, smooth muscle cells, osteoblasts, adipocytes,
chondrocytes and endothelial cells. Molecules of the present
invention may, while stimulating proliferation or differentiation
of cardiac myocytes, inhibit proliferation or differentiation of
adipocytes, by virtue of the affect on their common precursor/stem
cells. Thus molecules of the present invention, have use in
inhibiting osteosarcomas, chondro-sarcomas, atherosclerosis,
restenosis, osteoporosis and obesity.
[0091] Assays measuring differentiation include, for example,
measuring cell-surface markers associated with stage-specific
expression of a tissue, enzymatic activity, functional activity or
morphological changes (Watt, FASEB, 5:281-284, 1991; Francis,
Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol.
Technol. Bioprocesses, 161-171, 1989; all incorporated herein by
reference).
[0092] In vivo assays for evaluating cardiac neogenesis or
hyperplasia include treating neonatal and mature rats with the
molecules of the present invention. The animals cardiac function is
measured as heart rate, blood pressure, and cardiac output to
determine left ventricular function. Post-mortem methods for
assessing cardiac improvement include: increased cardiac weight,
nuclei/cytoplasmic volume, staining of cardiac histology sections
to determine proliferating cell nuclear antigen (PCNA) vs.
cytoplasmic actin levels (Quaini et al., Circulation Res.
75:1050-1063, 1994 and Reiss et al., Proc. Natl. Acad. Sci.
93:8630-8635, 1996.)
[0093] In vivo assays for measuring changes in bone formation rates
include performing bone histology (see, Recker, R., eds. Bone
Histomorphometry: Techniques and Interpretation. Boca Raton: CRC
Press, Inc., 1983) and quantitative computed tomography (QCT;
Ferretti,J. Bone 17:353S-364S, 1995; Orphanoludakis et al.,
Investig. Radiol. 14:122-130,, 1979 and Durand et al., Medical
Physics 19:569-573, 1992). An ex vivo assay for measuring changes
in bone formation would be, for example, a calavarial assay (Gowen
et al., J. Immunol. 136:2478-2482, 1986).
[0094] With regard to modulating energy balance, particularly as it
relates to adipocyte metabolism, proliferation and differentiation,
zFGF5 polypeptides modulate effects on metabolic reactions. Such
metabolic reactions include adipogenesis, gluconeogenesis,
glycogenolysis, lipogenesis, glucose uptake, protein synthesis,
thermogenesis, oxygen utilization and the like. Among other methods
known in the art or described herein, mammalian energy balance may
be evaluated by monitoring one or more of the aforementioned
metabolic functions. These metabolic functions are monitored by
techniques (assays or animal models) known to one of ordinary skill
in the art, as is more fully set forth below. For example, the
glucoregulatory effects of insulin are predominantly exerted in the
liver, skeletal muscle and adipose tissue. In skeletal muscle and
adipose tissue, insulin acts to stimulate the uptake, storage and
utilization of glucose.
[0095] Art-recognized methods exist for monitoring all of the
metabolic functions recited above. Thus, one of ordinary skill in
the art is able to evaluate zFGF5 polypeptides, fragments, fusion
proteins, antibodies, agonists and antagonists for metabolic
modulating functions. Exemplary modulating techniques are set forth
below.
[0096] Insulin-stimulated lipogenesis, for example, may be
monitored by measuring the incorporation of .sup.14C-acetate into
triglyceride (Mackall et al. J. Biol. Chem. 251:6462-6464, 1976) or
triglyceride accumulation (Kletzien et al., Mol. Pharmacol.
41:393-398, 1992).
[0097] zFGF5-stimulated uptake may be evaluated, for example, in an
assay for insulin-stimulated glucose transport. Primary adipocytes
or NIH 3T3 L1 cells (ATCC No. CCL-92.1) are placed in DMEM
containing 1 g/l glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mM
glutamine. After two to five hours of culture, the medium is
replaced with fresh, glucose-free DMEM containing 0.5 or 1.0% BSA,
20 mM Hepes, 1 mM pyruvate, and 2 mM glutamine. Appropriate
concentrations of zFGF5, insulin or IGF-1, or a dilution series of
the test substance, are added, and the cells are incubated for
20-30 minutes. .sup.3H or .sup.14C-labeled deoxyglucose is added to
.apprxeq.50 .mu.M final concentration, and the cells are incubated
for approximately 10-30 minutes. The cells are then quickly rinsed
with cold buffer (e.g. PBS), then lysed with a suitable lysing
agent (e.g. 1% SDS or 1 N NaOH). The cell lysate is then evaluated
by counting in a scintillation counter. Cell-associated
radioactivity is taken as a measure of glucose transport after
subtracting non-specific binding as determined by incubating cells
in the presence of cytochalasin b, an inhibitor of glucose
transport. Other methods include those described by, for example,
Manchester et al., Am. J. Physiol. 266 (Endocrinol. Metab.
29):E326-E333, 1994 (insulin-stimulated glucose transport).
[0098] Protein synthesis may be evaluated, for example, by
comparing precipitation of .sup.35S-methionine-labeled proteins
following incubation of the test cells with .sup.35S-methionine and
.sup.35S-methionine and a putative modulator of protein
synthesis.
[0099] Thermogenesis may be evaluated as described by B. Stanley in
The Biology of Neuropeptide Y and Related Peptides, W. Colmers and
C. Wahlestedt (eds.), Humana Press, Ottawa, 1993, pp. 457-509; C.
Billington et al., Am. J. Physiol. 260:R321, 1991; N. Zarjevski et
al., Endocrinology 133:1753, 1993; C. Billington et al., Am. J.
Physiol. 266:R1765, 1994; Heller et al., Am. J. Physiol. 252(4 Pt
2): R661-7, 1987; and Heller et al., Am. J. Physiol. 245(3):
R321-8, 1983. Also, metabolic rate, which may be measured by a
variety of techniques, is an indirect measurement of
thermogenesis.
[0100] Oxygen utilization may be evaluated as described by Heller
et al., Pflugers Arch. 369(1): 55-9, 1977. This method also
involved an analysis of hypothalmic temperature and metabolic heat
production. Oxygen utilization and thermoregulation have also been
evaluated in humans as described by Haskell et al., J. Appl.
Physiol. 51(4): 948-54, 1981.
[0101] zFGF5 polypeptides can also be used to prepare antibodies
that specifically bind to zFGF5 epitopes, peptides or polypeptides.
Methods for preparing polyclonal and monoclonal antibodies are well
known in the art (see, for example, Sambrook et al., Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor,
N.Y., 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma
Antibodies: Techniques and Applications, CRC Press, Inc., Boca
Raton, Fla., 1982, which are incorporated herein by reference). As
would be evident to one of ordinary skill in the art, polyclonal
antibodies can be generated from a variety of warm-blooded animals,
such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice,
and rats.
[0102] The immunogenicity of a zFGF5 polypeptide may be increased
through the use of an adjuvant, such as alum (aluminum hydroxide)
or Freund's complete or incomplete adjuvant. Polypeptides useful
for immunization also include fusion polypeptides, such as fusions
of zFGF5 or a portion thereof with an immunoglobulin polypeptide or
with maltose binding protein. The polypeptide immunogen may be a
full-length molecule or a portion thereof. If the polypeptide
portion is "hapten-like", such portion may be advantageously joined
or linked to a macromolecular carrier (such as keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
[0103] As used herein, the term "antibodies" includes polyclonal
antibodies, affinity-purified polyclonal antibodies, monoclonal
antibodies, and antigen-binding fragments, such as F(ab').sub.2 and
Fab proteolytic fragments. Genetically engineered intact antibodies
or fragments, such as chimeric antibodies, Fv fragments, single
chain antibodies and the like, as well as synthetic antigen-binding
peptides and polypeptides, are also included. Non-human antibodies
may be humanized by grafting only non-human CDRs onto human
framework and constant regions, or by incorporating the entire
non-human variable domains (optionally "cloaking" them with a
human-like surface by replacement of exposed residues, wherein the
result is a "veneered" antibody). In some instances, humanized
antibodies may retain non-human residues within the human variable
region framework domains to enhance proper binding characteristics.
Through humanizing antibodies, biological half-life may be
increased, and the potential for adverse immune reactions upon
administration to humans is reduced. Alternative techniques for
generating or selecting antibodies useful herein include in vitro
exposure of lymphocytes to zFGF5 protein or peptide, and selection
of antibody display libraries in phage or similar vectors (for
instance, through use of immobilized or labeled zFGF5 protein or
peptide).
[0104] Antibodies are defined to be specifically binding if they
bind to a zFGF5 polypeptide with a binding affinity (K.sub.a) of
10.sup.6 M.sup.-1 or greater, preferably 10.sup.7 M.sup.-1 or
greater, more preferably 10.sup.8 M.sup.-1 or greater, and most
preferably 10.sup.9 M.sup.-1 or greater. The binding affinity of an
antibody can be readily determined by one of ordinary skill in the
art (for example, by Scatchard analysis).
[0105] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which specifically bind to zFGF5
proteins or peptides. Exemplary assays are described in detail in
Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold
Spring Harbor Laboratory Press, 1988. Representative examples of
such assays include: concurrent immunoelectrophoresis,
radioimmunoassay, radioimmuno-precipitation, enzyme-linked
immunosorbent assay (ELISA), dot blot or Western blot assay,
inhibition or competition assay, and sandwich assay. In addition,
antibodies can be screened for binding to wild-type versus mutant
zFGF5 protein or peptide.
[0106] Antibodies to zFGF5 may be used for tagging cells that
express zFGF5; to target another protein, small molecule or
chemical to heart tissue; for isolating zFGF5 by affinity
purification; for diagnostic assays for determining circulating
levels of zFGF5 polypeptides; for detecting or quantitating soluble
zFGF5 as marker of underlying pathology or disease; in analytical
methods employing FACS; for screening expression libraries; for
generating anti-idiotypic antibodies; and as neutralizing
antibodies or as antagonists to block zFGF5 mediated proliferation
in vitro and in vivo. Antagonists will be useful for inhibiting
bone formation where such formation results in premature closure of
the growth plate, for example in craniosyntosis.
[0107] zFGF5 has been identified in endothelial and smooth muscle
cells by immunocytochemistry. In addition, there is evidence that
zFGF5 plays a role in chemotaxis of macrophage. Therefore,
antagonists to zFGF5 could be useful for inhibition of restenosis
and artherosclerosis.
[0108] Suitable direct tags or labels include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent markers,
chemiluminescent markers, magnetic particles and the like; indirect
tags or labels may feature use of biotin-avidin or other
complement/anti-complement pairs as intermediates. Antibodies
herein may also be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like, and these conjugates used for
in vivo diagnostic or therapeutic applications.
[0109] Molecules of the present invention can be used to identify
and isolate receptors involved in cardiac myocyte, cardiac
fibroblast, or cardiac progenitor cell proliferation. For example,
proteins and peptides of the present invention can be immobilized
on a column and membrane preparations run over the column
(Immobilized Affinity Ligand Techniques, Hermanson et al., eds.,
Academic Press, San Diego, Calif., 1992, pp.195-202). Proteins and
peptides can also be radiolabeled (Methods in Enzymol., vol. 182,
"Guide to Protein Purification", M. Deutscher, ed., Acad. Press,
San Diego, 1990, 721-737) or photoaffinity labeled (Brunner et al.,
Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem.
Pharmacol. 33:1167-1180, 1984) and specific cell-surface proteins
can be identified.
[0110] Antagonists will be useful for inhibiting the proliferative
activities of zFGF5 molecules, in cell types such as cardiac cells,
including myocytes, fibroblasts and endothelial cells, osteoblasts
and chondrocytes. Genes encoding zFGF5 polypeptide binding domains
can be obtained by screening random peptide libraries displayed on
phage (phage display) or on bacteria, such as E. coli. Nucleotide
sequences encoding the polypeptides can be obtained in a number of
ways, such as through random mutagenesis and random polynucleotide
synthesis. These random peptide display libraries can be used to
screen for peptides which interact with a known target which can be
a protein or polypeptide, such as a ligand or receptor, a
biological or synthetic macromolecule, or organic or inorganic
substances. Techniques for creating and screening such random
peptide display libraries are known in the art (Ladner et al., U.S.
Pat. No.:5,223,409; Ladner et al., U.S. Pat. No.:4,946,778; Ladner
et al., U.S. Pat. No.:5,403,484 and Ladner et al., U.S. Pat.
No.:5,571,698) and random peptide display libraries and kits for
screening such libraries are available commercially, for instance
from Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego,
Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia
LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the zFGF5 sequences disclosed
herein to identify proteins which bind to zFGF5. These "binding
proteins" which interact with zFGF5 polypeptides may be used for
tagging cells; for isolating homolog polypeptides by affinity
purification; they can be directly or indirectly conjugated to
drugs, toxins, radionuclides and the like. These binding proteins
can also be used in analytical methods such as for screening
expression libraries and neutralizing activity. The binding
proteins can also be used for diagnostic assays for determining
circulating levels of polypeptides; for detecting or quantitating
soluble polypeptides as marker of underlying pathology or disease.
These binding proteins can also act as zFGF5 "antagonists" to block
zFGF5 binding and signal transduction in vitro and in vivo. These
anti- zFGF5 binding proteins would be useful for inhibiting
expression of genes which result in proliferation or
differentiation. Such anti-zFGF5 binding proteins can be used for
treatment, for example, in rhabdomyosarcoma, cardiac myxoma, bone
cancers of osteoblast origin, and dwarfism, arthritis, ligament and
cartilage repair, alone or combination with other therapies.
[0111] The molecules of the present invention will be useful for
proliferation of cardiac tissue cells, such as cardiac myocytes,
myoblasts or progenitors; skeletal myocytes or myoblasts and smooth
muscle cells; chondrocytes; endothelial cells; adipocytes and
osteoblasts in vitro. For example, molecules of the present
invention are useful as components of defined cell culture media,
and may be used alone or in combination with other cytokines and
hormones to replace serum that is commonly used in cell culture.
Molecules of the present invention are particularly useful in
specifically promoting the growth and/or development of myocytes in
culture, and may also prove useful in the study of cardiac myocyte
hyperplasia and regeneration.
[0112] The polypeptides, nucleic acid and/or antibodies of the
present invention may be used in treatment of disorders associated
with heart disease, i.e., myocardial infarction, coronary artery
disease, congestive heart failure, hypertrophic cardiomyopathy,
myocarditis, congenital heart defects and dilated cardiomyopathy.
Molecules of the present invention may also be useful for limiting
infarct size following a heart attack, promoting angiogenesis and
wound healing following angioplasty or endarterectomy, to develop
coronary collateral circulation, for revascularization in the eye,
for complications related to poor circulation such as diabetic foot
ulcers, for stroke, following coronary reperfusion using
pharmacologic methods and other indications where angiogenesis is
of benefit. Molecules of the present invention may be useful for
improving cardiac function, either by inducing cardiac myocyte
neogenesis and/or hyperplasia, by inducing coronary collateral
formation, or by inducing remodelling of necrotic myocardial
area.
[0113] An ischemic event is the disruption of blood flow to an
organ, resulting in necrosis or infarct of the non-perfused region.
Ischemia-reperfusion is the interruption of blood flow to an organ,
such as the heart or brain, and subsequent restoration (often
abrupt) of blood flow. While restoration of blood flow is essential
to preserve functional tissue, the reperfusion itself is known to
be deleterious. In fact, there is evidence that reperfusion of an
ischemic area compromises endothelium-dependent vessel relaxation
resulting in vasospasms, and in the heart compromised coronary
vasodilation, that is not seen in an ischemic event without
reperfusion (Cuevas et al., Growth Factors 15:29-40, 1997). Both
ischemia and reperfusion are important contributors to tissue
necrosis, such as a myocardial infarct or stroke. The molecules of
the present invention will have therapeutic value to reduce damage
to the tissues caused by ischemia or ischemia-reperfusion events,
particularly in the heart or brain.
[0114] Molecules of the present invention for be useful for the
treatment of injuries to the central nervous system. In particular,
pharmaceutical compositions of zFGF5 will be useful in the
treatment of ischemic events, such as stroke. The effects of zFGF5
compositions in ischemic cerebrovascular disease (i.e., stroke)
have been demonstrated using a middle cerebral artery occlusion
model of stroke in mice (Huang Z et al., Am J Physiol 272:
H1401-H1405, 1996). As measured by densitometry, administration of
zFGF5 resulted in a 50% reduction in infarct volume over vehicle in
animals where cerebral blood flow had been reduced by 75%, after
transient occlusion of the anterior communicating and middle
cerebral arteries. Reduction in infarct volume was greater using
zFGF5 than vehicle, and was dose dependent. The administration of
zFGF5 did not affect arterial blood pressure, .sub.pO.sub.2,
pCO.sub.2, or pH of the animals.
[0115] Generally, administration of drugs intended to limit damage
caused by ischemia and reperfusion are administered within hours of
diagnosis of the ischemic event. The pharmaceutical composition
comprising zFGF5 may be given by intra-arterially, intravenously,
or intracerebrovascularly, or other methods for administration as
determined by one skilled in the art. When determining efficacy of
a therapeutic treatment for stroke, various tests are used
clinically. Generally, these tests are directed toward evaluation
of neurological function and patient progress toward recovery from
neurological deficiencies. The effects of stroke severity and
recovery can be determined using scales designed to measure either
impairment or disability. Impairment is measured as the effect of
the disease at the organ level. Impairment scales for stroke can
include measurement of arm and leg motor function, speech,
consciousness, facial paresis, muscle tone, orientation, and
reflexes. Disability scales measure limitations in daily living or
performance at a general level. Disability scales can include the
patient's ability to walk, dress, feed themselves, bowel and
bladder control and groom themselves. Outcomes can be measured at
predetermined increments, for example, 1, 3, 6, and 12 months. Two
stroke scales that are used, particularly in clinical
investigation, are the Rankin stroke scale and Barthel scale. The
Rankin scale uses five grades: 1="no significant disability";
2="slight disability"; 3="moderate disability"; 4="moderately
severe disability"; and 5="severe disability". The Barthel scale
consists of ten activities that include: feeding, transfer,
grooming, toilet use, bathing, mobility, stair climbing, dressing
and continence for bowel and bladder. The score range is 0-100,
with 100 being maximal or normal function. Other measurements for
recovery from stroke include the NIH stoke scale, Mathew scale and
Scandinavian scale. See, e.g., Ginsberg and Bogousslavsky, eds.
Cardiovascular Disease: Pathophysiology, Diagnosis and Management.
Malden, Mass. Blackwell Science, 1998; and Duncan et al., Stroke
30:2131-2140, 1999.
[0116] zFGF5 induced coronary collateral development is measured in
rabbits, dogs or pigs using models of chronic coronary occlusion
(Landau et al., Amer. Heart J. 29:924-931, 1995; Sellke et al.,
Surgery 120(2):182-188, 1996 and Lazarous et al., 1996, ibid.)
zFGF5 benefits for treating stroke is tested in vivo in rats
utilizing occlusion of the middle cerebral artery or carotid artery
occlusion and measuring histological changes, as well as maze
performance (Gage et al., Neurobiol. Aging 9:645-655, 1988). zFGF5
efficacy in hypertension is tested in vivo utilizing spontaneously
hypertensive rats (SHR) for systemic hypertension (Marche et al.,
Clin. Exp. Pharmacol. Physiol. Suppl. 1:S114-116, 1995). Other
therapeutic uses for the present invention include induction of
skeletal muscle neogenesis and/or hyperplasia, kidney regeneration
and/or for treatment of systemic and pulmonary hypertension.
[0117] Additional uses for zFGF5 compositions include treatment of
neuronal degenerative diseases such as Alzheimer's disease,
Parkinson's disease, amyotrophic lateral sclerosis, multiple
sclerosis, Huntington's disease; and traumatic injuries to brain or
spinal cord, nervous system tumors, infection, dementia, epilepsy,
and peripheral nerve injury.
[0118] Molecules of the present invention can be used to target the
delivery of agents or drugs to the heart. For example, the
molecules of the present invention will be useful limiting
expression to the heart, by virtue of the tissue specific
expression directed by the zFGF5 promoter. For example,
heart-specific expression can be achieved using a zFGF5-adenoviral
discistronic construct (Rothmann et al., Gene Therapy 3:919-926,
1996). In addition, the zFGF5 polypeptides can be used to restrict
other agents or drugs to heart tissue by linking zFGF5 polypeptides
to another protein (Franz et al., Circ. Res. 73:629-638, 1993) by
linking a first molecule that is comprised of a zFGF5 homolog
polypeptide with a second agent or drug to form a chimera.
Proteins, for instance antibodies, can be used to form chimeras
with zFGF5 molecules of the present invention (Narula et al., J.
Nucl. Cardiol. 2:26-34, 1995). Examples of agents or drugs include,
but are not limited to, bioactive-polypeptides, genes, toxins,
radionuclides, small molecule pharmaceuticals and the like. Linking
may be direct or indirect (e.g., liposomes), and may occur by
recombinant means, chemical linkage, strong non-covalent
interaction and the like.
[0119] Polynucleotides encoding zFGF5 polypeptides are useful
within gene therapy applications where it is desired to increase or
inhibit zFGF5 activity. If a mammal has a mutated or absent zFGF5
gene, the zFGF5 gene can be introduced into the cells of the
mammal. In one embodiment, a gene encoding a zFGF5 polypeptide is
introduced in vivo in a viral vector. Such vectors include an
attenuated or defective DNA virus, such as, but not limited to,
herpes simplex virus (HSV), papillomavirus, Epstein Barr virus
(EBV), adenovirus, adeno-associated virus (AAV), and the like.
Defective viruses, which entirely or almost entirely lack viral
genes, are preferred. A defective virus is not infective after
introduction into a cell. Use of defective viral vectors allows for
administration to cells in a specific, localized area, without
concern that the vector can infect other cells. Examples of
particular vectors include, but are not limited to, a defective
herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell.
Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as
the vector described by Stratford-Perricaudet et al., J. Clin.
Invest. 90:626-30, 1992; and a defective adeno-associated virus
vector (Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et
al., J. Virol. 63:3822-8, 1989).
[0120] In another embodiment, a zFGF5 gene can be introduced in a
retroviral vector, e.g., as described in Anderson et al., U.S. Pat.
No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S.
Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289;
Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S. Pat.
No. 5,124,263; International Patent Publication No. WO 95/07358,
published Mar. 16, 1995 by Dougherty et al.; and Kuo et al., Blood
82:845, 1993. Alternatively, the vector can be introduced by
lipofection in vivo using liposomes. Synthetic cationic lipids can
be used to prepare liposomes for in vivo transfection of a gene
encoding a marker (Felgner et al., Proc. Natl. Acad. Sci. USA
84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA
85:8027-31, 1988). The use of lipofection to introduce exogenous
genes into specific organs in vivo has certain practical
advantages. Molecular targeting of liposomes to specific cells
represents one area of benefit. More particularly, directing
transfection to particular cells represents one area of benefit.
For instance, directing transfection to particular cell types in a
tissue with cellular heterogeneity, such as the heart, brain, lungs
or liver. Lipids may be chemically coupled to other molecules for
the purpose of targeting. Targeted peptides (e.g., hormones or
neurotransmitters), proteins such as antibodies, or non-peptide
molecules can be coupled to liposomes chemically.
[0121] It is possible to remove the target cells from the body; to
introduce the vector as a naked DNA plasmid; and then to re-implant
the transformed cells into the body. Naked DNA vectors for gene
therapy can be introduced into the desired host cells by methods
known in the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun or use of a DNA vector
transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992;
Wu et al., J. Biol. Chem. 263:14621-4, 1988.
[0122] Antisense methodology can be used to inhibit zFGF5 gene
transcription, such as to inhibit cell proliferation in vivo.
Polynucleotides that are complementary to a segment of a
zFGF5-encoding polynucleotide (e.g., a polynucleotide as set froth
in SEQ ID NO:1) are designed to bind to zFGF5-encoding mRNA and to
inhibit translation of such mRNA. Such antisense polynucleotides
are used to inhibit expression of zFGF5 polypeptide-encoding genes
in cell culture or in a subject.
[0123] The present invention also provides reagents which will find
use in diagnostic applications. For example, the zFGF5 gene, a
probe comprising zFGF5 DNA or RNA or a subsequence thereof can be
used to determine if the zFGF5 gene is present on chromosome 5 and
if a mutation in the zFGF5 gene locus has occurred including, but
not limited to, aneuploidy, gene copy number changes, insertions,
deletions, restriction site changes and rearrangements. Such
aberrations can be detected using polynucleotides of the present
invention by employing molecular genetic techniques, such as
restriction fragment length polymorphism (RFLP) analysis, short
tandem repeat (STR) analysis employing PCR techniques, and other
genetic linkage analysis techniques known in the art (Sambrook et
al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65,
1995).
[0124] Mice engineered to express the zFGF5 gene, referred to as
"transgenic mice," and mice that exhibit a complete absence of
zFGF5 gene function, referred to as "knockout mice," may also be
generated (Snouwaert et al., Science 257:1083, 1992; Lowell et al.,
Nature 366:740-42, 1993; Capecchi, M. R., Science 244: 1288-1292,
1989; Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499, 1986).
For example, transgenic mice that over-express zFGF5, either
ubiquitously or under a tissue-specific or tissue-restricted
promoter can be used to ask whether over-expression causes a
phenotype. For example, over-expression of a wild-type zFGF5
polypeptide, polypeptide fragment or a mutant thereof may alter
normal cellular processes, resulting in a phenotype that identifies
a tissue in which zFGF5 expression is functionally relevant and may
indicate a therapeutic target for the zFGF5, its agonists or
antagonists. For example, a preferred transgenic mouse to engineer
is one that over-expresses the zFGF5 (approximately amino acid
residue 28 to residue 207 of SEQ ID NO:2). Moreover, such
over-expression may result in a phenotype that shows similarity
with human diseases. Similarly, knockout zFGF5 mice can be used to
determine where zFGF5 is absolutely required in vivo. The phenotype
of knockout mice is predictive of the in vivo effects of a zFGF5
antagonist, such as those described herein, may have. These mice
may be employed to study the zFGF5 gene and the protein encoded
thereby in an in vivo system, and can be used as in vivo models for
corresponding human diseases.
[0125] In one embodiment of the present invention, a composition
comprising zFGF5 protein is used as a therapeutic agent to enhance
osteoblast-mediated bone formation. The compositions and methods
using the compositions of the invention may be applied to promote
the repair of bone defects and deficiencies, such as those
occurring in closed, open and non-union fractures; to promote bone
healing in plastic surgery; to stimulate bone ingrowth into
non-cemented prosthetic joints and dental implants; in the
treatment of periodontal disease and defects; to increase bone
formation during distraction osteogenesis; and in treatment of
other skeletal disorders that may be treated by stimulation of
osteoblastic activity, such as osteoporosis and arthritis. De novo
bone formation provided by the methods of the present invention
will have use in repair of congenital, trauma-induced, oncologic
resection of bone or healing bone following radiation-induced
osteonecrosis (Hart et al, Cancer 37:2580-2585, 1976). The methods
of the present invention may also find use in plastic surgery.
[0126] The molecules of the present invention provide a method for
stimulating the proliferation of chondrocytes, in particular
differentiated chondrocytes capable of inducing specialized cell
functions, normally associated with terminally differentiated
cells. When zFGF5 was administered locally to chondrocytes isolated
from articular cartilage, cartilage explants, proliferation of the
cells and concomitant synthesis of glycosaminoglycans was
increased. Additionally, an increase in cartilaginous tissue in ear
has been measured in mice ears injected with adenovirus expressing
zFGF5. These results indicate that zFGF5 can play a therapeutic
role in maintaining or repairing cartilaginous tissue, such as
joints damaged by osteoarthritis, rheumatoid arthritis or traumatic
injury.
[0127] ZFGF5 have been shown to increase cartilage deposition both
in vivo and in vitro. Generation of hyaline cartilage, elastic
cartilage, and fibrocartilage are valuable both as a therapeutic
and as component for biological matrices. zFGF5 compositions will
be useful in treating articular cartilage defects in synovial
joints that are due to age-related superficial fibrillation,
cartilage degeneration due to osteoarthritis, and focal chondral
and osteochondral defects due to injury or disease. ZFGF5
compositions will also be useful for treating joint disease caused
by osteochondritis dissecans and degenerative joint disease. In the
field of reconstructive and plastic surgery, zFGF5 compositions
will be useful for autogenous or allogenic cartilage expansion and
transfer for reconstruction of extensive tissue defects. Expansions
of cells and induction of elastic cartilage production will be
useful for generation and repair of ear and nose tissue.
[0128] ZFGF5 compositions can be applied by direct injection into
the synovial fluid of the joint or directly into the defect, either
alone or complexed with a suitable carrier for extended release of
protein. ZFGF5 can also be used to expand chondrocyte populations
in culture for autogenous or allogenic chondrocyte transplantation.
In these procedures, for example, chondrocytes can be harvested
arthroscopically from an uninjured minor load-bearing area of the
damaged joint, and can be cultured in the presence of zFGF5
compositions to increase the number of cells prior to
transplantation. The expanded cultures will then be admixed with
zFGF5 compositions and placed in the joint space or directly into
the defect. ZFGF5 compositions can be used in combination with
periosteal or perichondrial grafts that contain cells that can form
cartilage and/or help to hold the transplanted chondrocytes or
their precursor cells in place. ZFGF5 compositions can be used to
repair cartilage damage in conjunction with lavage of the joint,
stimulation of bone marrow, abrasion arthroplasty, subchondral
drilling, or microfracture of the subchondral bone.
[0129] For pharmaceutical use, the proteins of the present
invention are formulated for parenteral, particularly intravenous
or subcutaneous, administration according to conventional methods.
Intravenous administration will be by bolus injection or infusion
over a typical period of one to several hours. In general,
pharmaceutical formulations will include a zFGF5 protein in
combination with a pharmaceutically acceptable vehicle, such as
saline, buffered saline, 5% dextrose in water or the like.
Formulations may further include one or more excipients,
preservatives, solubilizers, buffering agents, albumin to prevent
protein loss on vial surfaces, extend half-life, etc. Methods of
formulation are well known in the art and are disclosed, for
example, in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack
Publishing Co., Easton Pa., 1990, which is incorporated herein by
reference. Therapeutic doses will generally be in the range of 0.1
to 100 .mu.g/kg of patient weight per day, preferably 0.5-20
.mu.g/kg per day, with the exact dose determined by the clinician
according to accepted standards, taking into account the nature and
severity of the condition to be treated, patient traits, etc.
Determination of dose is within the level of ordinary skill in the
art. The proteins may be administered for acute treatment, over one
week or less, often over a period of one to three days or may be
used in chronic treatment, over several months or years.
[0130] In other embodiments, a pharmaceutical zFGF5 composition
will comprise a formulation for timed-release of the protein.
Time-release formulations generally include a monolithic delivery
device comprising biocompatible solutions, gels, pastes, and
putties in a matrix, in which the composition is entrapped or
dissolved. Release from such a timed-release composition occurs by
diffusion through the matrix and/or erosion of the matrix. A
reservoir system, where the pharmaceutical composition diffuses
through a membrane, may also be used.
[0131] Delivery devices can include, but are not limited to,
medical devices; sutures; and solid matrices, such as collagen
sponges; ethylene-vinyl acetate copolymers; and acrylonitrile-vinyl
chloride copolymers. Liquid delivery devices would include, for
example, hydrogels. Examples of biodegradable polymers include:
poly(DL-lactide), poly(DL-lactice co-glycolide),
poly(DL-lactide-co-caprolactone) and polyanhydrides and
ATRIGEL.RTM. (Atrix Laboratories, Fort Collins, Colo.). Another
delivery device can include poloxamers, for example, Pluronic.RTM.
F-127 (BASF, Parsippany, N.Y.), which are liquid at room
temperature and form gels at 37.degree. C. Additional delivery
devices include negatively charged macromolecules such as
hyaluronic acid, sulfated proteoglycans, B-cyclodextrin
tetradecasulphate; or hydroxyapatite, alginate microspheres, and
methylcellulose. For review of delivery systems for growth factors,
see, e.g., Minmi, Biomaterials 18:1201-1225, 1997; and Langer et
al., J. Cell. Biochem. 45:340-345, 1991.
[0132] Although administration of zFGF5 alone is sufficient to
provide the delivery of the chondrogenic peptides of the present
method, there may be clinical situations where additional drugs are
combined in the admixture. Examples of other drugs which may be
clinically indicated include anti-inflammatory drugs such as
nonspecific and specific cyclooxygenase-2 inhibitors, non-steriodal
and steroidal anti-inflammatory drugs. Some of the nonspecific COX
inhibitors that could be used in the present invention include
salicylic acid and derivatives, such as aspirin or sulfasalazine,
para-aminophenol derivatives, such as acetaminophen, indole and
indene acetic acids, such as indomethacin or sulindac, arylprpionic
acids, such as ibuprofen, naproxen, or oxaprozin, anthranilic
acids, such as mefenamic acid, enolic acids including oxicams, or
alkanonoes, such as nabumentone. Specific COX-2 inhibitors would be
diaryl-substituted fuanonoes (Refecoxib), diaryl-substituted
pyrazoles (Celecoxib), indole acetic acids (Etodolac) and
sulfonaildes (Nimesulide). Additionally, steroids, such as
dexamethazone, prednisone, triamcinolone, or methylprednisone, are
among the drugs that could be used. Other types of drugs suitable
for the present invention would be inhibitors of the tumor necrosis
factor family, such as Enbrel or TACI-Ig, IL-1 antagonists such as
Kinaret, antagonists of IL-18 and IL-15, and immunosuppressive
drugs such as cyclosporine. In addition, zFGF5 may be administered
with inhibitors of the CC (MCP-1, RANTES, MIP-1alpha, and
MIP-1beta) and CXC (IL-8 and GRO-alpha) chemokine family.
[0133] In one embodiment, a therapeutically effective amount of
zFGF5 is an amount sufficient to produce a clinically significant
change in myocyte proliferation, heart function, brain function,
bone formation or increases in specific cell types associated with
mesenchymal stem cells and progenitors for myocytes, osteoblasts
and chondrocytes. In particular, a clinically significant
improvement in cardiac performance may be an increase in the number
of myocytes or myocyte progenitor cells. Improvements in cardiac
performance can be determined by methods well known and accepted by
clinicians and those skilled in the art. Such determinations
include, but are not limited to, measuring the left ventricular
ejection fraction, prior to, and after administration of zFGF5
molecules, and determining at least a 5% increase, preferably 10%
or more, in the total ejection fraction, increases in -dP/dt or
+dP/dt, greater exercise tolerance, a decrease in vascular
resistance, and increased blood flow to the heart. A reduction in
symptoms may also be indication of a significant improvement in
cardiac performance, and include, for example, reduction in angina
pectoris, breathlessness, leg swelling, heart or respiratory rates,
edema, fatigue and weakness.
[0134] Thus, in summary, certain aspects the present invention
includes: Methods of reducing infarct volume in a mammal diagnosed
as having a cerebrovascular ischemic stroke comprising: (1)
determining infarct volume in the mammal; (2) administering a
pharmaceutical composition comprising a polypeptide that is at
least 80% identical to an amino acid sequence as shown in SEQ ID
NO: 2 from amino acid residue 28 (Glu) to amino acid residue 175
(Met) sufficient to reduce infarct volume; (3) determining infarct
volume in the mammal; and (4) comparing the infarct volume of step
1 to step 4. In further embodiments the pharmaceutical composition
is a polypeptide that is at least 80% identical to the amino acid
sequence as shown in SEQ ID NO: 2 from residue 28 (Glu) to 196
(Lys) and is a polypeptide is the amino acid sequence as shown in
SEQ ID NO: 2 from residue 28 (Glu) to residue 196 (Lys).
[0135] In another aspect, the present invention includes methods
for treating a patient who has suffered an injury to the central
nervous system, comprising administering to the patient a
pharmaceutical composition comprising a fibroblast growth factor
(FGF) homolog polypeptide, wherein said polypeptide comprises a
sequence of amino acids that is at least 80% identical to the amino
acid sequence as shown in SEQ ID NO: 2 from amino acid residue 28
(Glu) to 175 (Met), in amount sufficient to improve functional
recovery in the patient. In other embodiments, the injury to the
central nervous system is an ischemic event such as a stroke. In
further embodiments, improved functional recovery is defined as a
patient score of at least 3 on a Rankin stroke scale, or is defined
as a patient score of lower than 75 on a Barthel's scale.
Furthermore, certain embodiment include methods wherein said
polypeptide encoded by said polynucleotide is at least 90%
identical to the amino acid sequence as shown in SEQ ID NO: 2 from
amino acid residue 28 (Glu) to residue 196 (Lys) or is at least 80%
identical to the amino acid sequence as shown in SEQ ID NO: 2 from
amino acid residue 28 (Glu) to residue 207 (Ala).
[0136] In another aspect, the present invention can be summarized
as methods of increasing cartilage deposition in a patient
comprising: administering a pharmaceutical composition comprising a
fibroblast growth factor (FGF) homolog polypeptide to a
fibrocartilage, hyaline, or elastic cartilage injury in said
patient, wherein said polypeptide comprises a sequence of amino
acids that is at least 80%, 90%, or identical to the amino acid
sequence as shown in SEQ ID NO: 2 from amino acid residue 28 (Glu)
to 175 (Met), from amino acid 28 (Glu) to 196 (Lys), and from amino
acid residue 28 (Glu) to 207 (Ala). Furthermore, additional
embodiments include when the administration of the composition is
selected from the group consisting of intracartilaginous
administration; intraarticular, intravenous and intramuscular
administration; and topical administration or addition of an
anti-inflammatory to the pharmaceutical composition.
[0137] A final aspect of the present invention is a method of
treating a condition involving reduced cartilage deposition in a
patient in need of such treatment comprising administering a
pharmaceutical composition comprising a fibroblast growth factor
(FGF) homolog polypeptide to a fibrocartilage, hyaline, or elastic
cartilage injury in said patient, wherein said polypeptide
comprises a sequence of amino acids that is at least 80%, 90% or
identical to the amino acid sequence as shown in SEQ ID NO: 2 from
amino acid residue 28 (Glu) to 175 (Met); from amino acid residue
28 (Glu) to 196 (Lys); and from amino acid residue 28 (Glu) to 207
(Ala). Furthermore, additional embodiments include when the
administration f the composition is selected from the group
consisting of intracartilaginous administration; intraarticular,
intravenous and intramuscular administration; and topical
administration or addition of an anti-inflammatory to the
pharmaceutical composition.
[0138] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Extension of EST Sequence
[0139] Scanning of a translated DNA database using a query for
growth factors resulted in identification of an expressed sequence
tag (EST) sequence found to be a novel member of the FGF family,
and designated zFGF5.
[0140] Oligonucleotide primers ZC11676 (SEQ ID NO: 3) and ZC11677
(SEQ ID NO: 4) were designed from the sequence of an expressed
sequence tag (EST). The primers were used for priming internally
within the EST, and when PCR was performed using MARATHON READY
cDNA (Clontech, Palo Alto, Calif.) from adult heart tissue as
template in polymerase chain reaction (PCR).
[0141] The conditions used for PCR were 1 cycle at 94.degree. C.
for 90 seconds, 35 cycles at 94.degree. C. for 15 seconds;
68.degree. C. for 1 minute; followed by 1 cycle for 10 minutes at
72.degree. C. and 4.degree. C. incubation period. The PCR reaction
recreated 160 bp of the EST sequence, and confirmed that EST
sequence was correct.
[0142] Other libraries that could be amplified with the
oligonucleotide primers included skeletal muscle, lung, stomach,
small intestine and thyroid.
Example 2
Tissue Distribution
[0143] Northerns were performed using Human Multiple Tissue Blots
from Clontech (Palo Alto, Calif.). The 160 bp DNA fragment
described in Example 1 was electrophoresed on a 1% agarose gel, the
fragment was electroeluted, and then radioactively labeled using a
random priming MEGAPRIME DNA labeling system (Amersham, Arlington
Heights, Ill.) according to the manufacturer's specifications. The
probe was purified using a NUCTRAP push column (Stratagene Cloning
Systems, La Jolla, Calif.). EXPRESSHYB (Clontech, Palo. Alto,
Calif.) solution was used for prehybridization and as a
hybridrizing solution for the Northern blots. Hybridization took
place overnight at 8.degree. C., and the blots were then washed in
2.times.SSC and 0.05% SDS at RT, followed by a wash in
0.1.times.SSC and 0.1% SDS at 50.degree. C. A single band was
observed at approximately 2.0 kb. Signal intensity was highest for
adult heart with relatively less intense signals in skeletal muscle
and stomach. Dot blots were probed essentially as described above,
confirming that expression for human zFGF5 was highest in heart
tissue followed by lung and skeletal muscle.
Example 3
Assay for In Vitro Activity of zFGF5
[0144] A.
[0145] The mitogenic activity of zFGF5 is assayed using cell lines
and cells from a primary culture. Conditioned medium from cells
expressing the recombinant protein and/or purified protein is added
to cultures of the following cell lines: NIH 3T3 fibroblast (ATCC
No. CRL-1658), CHH-1 chum heart cells (ATCC No. CRL-1680), H9c2 rat
heart myoblasts (ATCC No. CRL-1446), Shionogi mammary carcinoma
cells (Tanaka et al., 1992, ibid.) and LNCaP.FGC adenocarcinoma
cells. Freshly isolated cells useful for testing the proliferative
activity of zFGF5 include: cardiac fibroblasts, cardiac myocytes,
skeletal myocytes and human umbilical vein endothelial cells.
[0146] Mitogenic activity is assayed by measurement of
.sup.3H-thymidine incorporation based on the method of Raines and
Ross (Meth. Enzymology 109:749-773, 1985). Briefly, quiescent cells
are plated cells at a density of 3.times.10.sup.4 cells/ml in an
appropriate medium. A typical growth medium is Dulbecco's Growth
Medium (GIBCO-BRL, Gaithersburg, Md.) containing 10% fetal calf
serum (FCS). The cells are cultured in 96-well plates and allowed
to grow for 3-4 days. The growth medium is removed, and 180 .mu.l
of DFC (Table 5) containing 0.1% FCS is added per well. Half the
wells have zFGF5 protein added to them and the other half are a
negative control, without zFGF5. The cells are incubated for up to
3 days at 37.degree. C. in 5% CO.sub.2, and the medium is removed.
One hundred microliters of DFC containing 0.1% FCS and 2 .mu.Ci/ml
.sup.3H-thymidine is added to each well, and the plates are
incubated an additional 1-24 hours at 37.degree. C. The medium is
aspirated off, and 150 .mu.l of trypsin is added to each well. The
plates are incubated at 37.degree. C. until the cells detached (at
least 10 minutes). The detached cells are harvested onto filters
using an LKB Wallac 1295-001 Cell Harvester (LKB Wallac, Pharmacia,
Gaithersburg, Md.). The filters are dried by heating in a microwave
oven for 10 minutes and counted in an LKB Betaplate 1250
scintillation counter (LKB Wallac) as described by the
supplier.
5TABLE 5 250 ml Dulbecco's Modified Eagle's Medium (DMEM,
Gibco-BRL) 250 ml Ham's F12 medium (Gibco-BRL) 0.29 mg/ml
L-glutamine (Sigma, St. Louis, MO) 1 mM sodium pyruvate (Sigma, St.
Louis, MO) 25 mM Hepes (Sigma, St. Louis, MO) 10 .mu.g/ml fetuin
(Aldrich, Milwaukee, WI) 50 .mu.g/ml insulin (Gibco-BRL) 3 ng/ml
selenium (Aldrich, Milwaukee, WI) 20 .mu.g/ml transferrin (JRH,
Lenexa, KS)
[0147] B.
[0148] Hearts were isolated from 1 day old neonatal mice and then
disrupted by repeat collagenase digestions, following the protocol
of Brand et al., (J. Biol. Chem. 268:11500-11503, 1993). Individual
myocytes were isolated over a Percoll gradient, and 2 ml were
plated in 6 well tissue culture dishes at 0.5.times.10.sup.6
cells/ml. Three days later the wells were washed 3 times with PBS
without calcium or magnesium, and refed with 1 ml serum free medium
(Table 6). The wells were inoculated with 10.sup.11 particles
AdCMV-zFGF5 per well or AdCMV-GFP (green fluorescent protein) as a
control, and incubated at 37.degree. C. for 8 hours. The wells were
then washed again 3 times with PBS without calcium or magnesium,
and then refed with 2 mls serum free media.
[0149] Within 48 hours after inoculation with the AdCMV-zFGF5, the
cultured myocytes have ceased to beat and have undergone a
morphologic alteration, while the wells inoculated with the
AdCMV-GFP continued to beat spontaneously and are unaffected
morphologically by the inoculation. Wells inoculated with
AdCMV-zFGF5 also contained, after 48, hours, a confluent layer of
viable, non-adherent cells, without any loss in confluence of the
adherent myocyte layers, indicating the proliferative activity of
the adCMV-zFGF5 on cultured murine myocytes.
6TABLE 6 DMEM Ham's Nutrient Mixture F12 (Gibco-BRL; 1:1 mixture
with DMEM) 17 mM NaHCO.sub.3 (Sigma) 2 mM L-glutamine (Sigma) 1%
PSN (Sigma) 1 .mu.g/ml insulin 5 .mu.g/ml transferrin 1 nM LiCl
(Sigma) 1 nM selenium 25 .mu.g/ml ascorbic acid(Sigma) 1 nM
thyroxine (Sigma)
[0150] C.
[0151] zFGF5 fused to a maltose binding protein (MBP), as described
in Example 9A and purified as described in Example 10, was added to
myocytes (Example 3B) at a concentration of 0.1 ng/ml. MBP-zFGF5
was shown to stimulate proliferation of myocytes, as well.
Example 4
Assay for Ex Vivo Activity of zFGF5
[0152] Cardiac mitogenesis is measured ex vivo by removing entire
hearts from neonatal or 8-week old mice or rats. The excised heart
is placed in Joklik's (Sigma, St. Louis, Mo.) or Dulbecco's medium
at 37.degree. C., 5% CO.sub.2 for 4-24 hours. During the incubation
period zFGF5 polypeptide is added at a concentration range of 1
pg/ml to 100 .mu.g/ml. Negative controls are using buffer only.
.sup.3H-thymidine is added and the samples are incubated for 1-4
hours, after which the heart is sectioned and mitogenesis is
determined by autoradiography. Sections are used for
histomorphometry to determine the nuclei/cytoplasmic volume
(McLaughlin, Am. J. Physiol. 271:R122-R129, 1996.)
[0153] Alternatively, the heart was lyophilized and resuspended in
1 ml 0.1 N NaOH. The DNA was precipitated using ice cold 10%
trichloroacetic acid (TCA). The supernatant was added to 9 ml
scintillation fluid to measure non-specific .sup.3H-thymidine
incorporation. The resulting pellet was resuspended in 1 ml BTS-450
tissue solubilizer (Beckman, Fullerton, Calif.) and added to 9 ml
of scintillation fluid to measure specific DNA incorporation of
.sup.3H-thymidine.
[0154] Left and right ventricles were isolated from 1 day old CD-1
mice (Jackson Labs, Bar Harbor, Me.), and incubated for 4 hours
with 3 ng/ml zFGF5Hep2 (n=13; see Example 10) or control (n=10).
.sup.3H-thymidine was added for 1 hour. The ventricles were washed
several times and then homogenized in 1 ml Joklik's medium. The
resulting homogenate was added to 9 ml scintillation cocktail and
analyzed for total .sup.3H-thymidine uptake and DNA
incorporation.
[0155] zFGF5-Hep2 increased .sup.3H-thymidine uptake and
incorporation in DNA 2.068.+-.0.489 fold over control, indicating
that zFGF5 is mitogenic for a cardiac cell.
Example 5
Assay for In Vivo Activity of zFGF5
[0156] The proliferative effects of zFGF5 are assayed in vivo using
two-week old neonatal rats and/or two-month old adult rats. The
rats are injected intraperiocardially either acutely or
chronically.
[0157] A.
[0158] Neonatal rats are treated with zFGF5 for 1 to 14 days over a
dose range of 50 ng/day to 100 .mu.g/day. After treatment, the
effects of zFGF5 versus the sham-treated animals is evaluated by
measuring increased cardiac weight, improved in vivo and ex vivo
left ventricular function, and by increased cardiac nuclear to
cytosolic volume fractions, that are determined
histomorphometrically.
[0159] B.
[0160] Rats with cardiomyopathy induced by chronic catecholamine
infusion, by coronary ligation or for models of cardiomyopathy such
as the Syrian Cardiomyopathic hamster (Sole et al., Amer. J.
Cardiol. 62(11):20G-24G, 1988) are also used to evaluate the
effects of zFGF5 on cardiac function and tissue.
[0161] To induce cardiomyopathy using catecholamine, 7-8 week old
rats are infused continuously with epinephrine for 2 weeks via
osmotic minipumps implanted subcutaneously between their shoulder
blades. The epinephrine infusion results in an increase in the left
ventricular fibrotic lesion score from 0.005.+-.0.005 to
2.11.+-.0.18, scale from 0-3); increased left ventricular myocyte
cell width from 17.36.+-.0.46 .mu.m to 23.05.+-.0.62 .mu.m; and
negligible left ventricular papillary muscle contractile responses
to isoproterenol (0.2 vs 1.1 grams tension compared to
saline-infused rats. After the two week treatment period, the rats
are injected intraperiocardially daily with either vehicle, zFGF5,
bFGF, IGF-I or IGF-II for up to 14 days. The rats are sacrificed
and histomorphometry and histocytochemistry are performed.
[0162] Rats, treated as described above, are also evaluated at the
end of the cathecholamine treatment, and again after growth factor
treatment, where cardiac regeneration is measured as decreased left
ventricular fibrotic lesion scores, reduced myocyte cell width and
increased left ventricular papillary contractile responses to
isoproterenol.
Example 6
Chromosomal Mapping of zFGF5
[0163] ZFGF5 was mapped to chromosome 5 using the commercially
available version of the Whitehead Institute/MIT Center for Genome
Research's "GeneBridge 4 Radiation Hybrid Panel" (Research
Genetics, Inc., Huntsville, Ala.). The GeneBridge 4 Radiation
Hybrid Panel contains DNAs suitable for PCR use from each of 93
radiation hybrid clones, plus two control DNAs (the HFL donor and
the A23 recipient). A publicly available WWW server allows mapping
relative to the Whitehead Institute/MIT Center for Genome
Research's radiation hybrid map of the human genome (the "WICGR"
radiation hybrid map) which was constructed with the GeneBridge 4
Radiation Hybrid Panel.
[0164] For the mapping of zFGF5 with the "GeneBridge 4 RH Panel",
25 .mu.l reactions were set up in a 96-well microtiter plate
(Stratagene, La Jolla, Calif.) and used for PCR in a "RoboCycler
Gradient 96" thermal cycler (Stratagene). Each of the 95 PCR
reactions consisted of 2.5 .mu.l 50.times. "Advantage KlenTaq
Polymerase Mix" (Clontech), 2 .mu.l dNTPs mix (2.5 mM each;
Perkin-Elmer, Foster City, Calif.), 1.25 .mu.l sense primer,
ZC11677 (SEQ ID NO: 4) 1.25 .mu.l antisense primer, ZC12053 (SEQ ID
NO: 5).
[0165] 2.5 .mu.l "RediLoad" (Research Genetics, Inc), 0.5 .mu.l
"Advantage KlenTaq Polymerase Mix" (Clontech Laboratories, Inc.),
25 ng of DNA from an individual hybrid clone or control and ddH2O
for a total volume of 25 .mu.l. The reactions were overlaid with an
equal amount of mineral oil and sealed. The PCR cycler conditions
were as follows: an initial 1 cycle of 4 minutes at 94.degree. C.,
35 cycles of 1 minute at 94.degree. C., 1.5 minute annealing at
66.degree. C. and 1.5 minute extension at 72.degree. C., followed
by a final 1 cycle extension of 7 minutes at 72.degree. C. The
reactions were separated by electrophoresis on a 3% NuSieve GTG
agarose gel (FMC Bioproducts, Rockland, Me.).
[0166] The results showed that zFGF5 maps 541.12 cR from the top of
the human chromosome 5 linkage group on the WICGR radiation hybrid
map. Relative to the centromere, its nearest proximal marker was
WI-16922 and its nearest distal marker was WI-14692. The use of
surrounding CHLC map markers also helped position zFGF5 in the
5q34-q35 region on the CHLC chromosome 5 version v8c7 integrated
marker map (The Cooperative Human Linkage Center).
Example 7
zFGF5 Effects on Bone
[0167] A. Adenovirus Expressed zFGF5
[0168] An adenovirus vector containing the cDNA for zFGF5 was
constructed using methods described by Becker et al. (Methods in
Cell Biology 43:161-189, 1994). Briefly, the cDNA for zFGF5 (as
shown in SEQ ID NO: 1) was cloned as a Xba I-Sal I fragment into
pACCMV (Gluzman et al., In Eucaryotic Viral Vectors, Gluzman (eds.)
pp.187-192, Cold Spring Harbor Press, Cold Springs Harbor N.Y.,
1982). The pACCMV vector contains part of the adenovirus 5 genome,
the CMV promoter and an SV40 terminator sequence. The plasmid
containing the vector and cDNA insert was cotransfected with a
plasmid containing the the adenovirus 5 genome, designated pJM17,
(McGrory et al., Virology 163:614-617, 1988) into 293 cells (ATCC
No. CRL-1573; American Type Culture Collection, Rockville, Md.),
leading to a recombination event and the production of a
recombinant adenovirus containing zFGF5, designated AdCMV-zFGF5.
The presence of the zFGF5 cDNA was confirmed by PCR.
[0169] The adenovirus vector AdCMV-zFGF5 was used for gene tranfer
in vivo by intravenous injection of between 1.times.10.sup.11 and
5.times.10.sup.11 particles/mouse. It has been shown that after
intravenous injection, the majority of the virus targets the liver
and very efficiently transduces hepatocytes (Herz et al., Proc.
Natl. Acad. Sci. USA 90:2812-2816, 1993). It has been demonstrated
that the cells produce the protein encoded by the cDNA, and in the
case of secreted proteins, secret them into the circulation. High
levels of expression and physiological effects have been
demonstrated (Ohwada et al., Blood 88:768-774, 1996; Stevenson et
al., Arteriosclerosis, Thrombosis and Vascular Biology, 15:479-484,
1995; Setoguchi et al., Blood 84:2946-2953, 1994; and Sakamoto et
al., Proc. Natl. Acad. Sci. USA 91:12368-12372, 1994).
[0170] Six week old CD-1 mice (Jackson Labs, Bar Harbor, Me.) were
treated with adenovirus containing no cDNA insert (AdCMV-null) or
AdCMV-zFGF5 either IV through the tail vein or intrapericardially
(IPC). A total of 5.times.10.sup.11 viral particles/100 .mu.l/mouse
were given. 14 days after injection, the animals were sacrificed,
and tibias and femurs were removed without being separated to
examine any potential inflammatory response. The bones were fixed
in 10% neutral buffered formalin and processed. They were
decalcified in 5% formic acid with 10% sodium citrate, washed in
water, dehydrated in a series of 70%-100% ethanol, cleared in
xylene and embedded in paraffin. The specimens were cut
longitudinally through both tibial and femoral metaphyses and
stained with hematoxylin and eosin for identification of bone
cells. Osteoblasts were identified by central negative Golgi area
and eccentric nucleus, while osteoclasts were identified by
multinucleation, non-uniform shape and the Howship's lacunae
associated with these resorbing cells.
[0171] For bone histomorphometry, femur samples were chosen.
Cancellous bone volume was not measured due to variation in the
sampling site (i.e., femur samples were not sectioned exactly at
the same plane). Three bone parameters were evaluated for
histomorphometric changes.
[0172] 1. Number of endosteal osteoblasts: measured along the
endosteal surface of cancellous bone at 180.times. magnification in
an area 1.22 mm proximal to the growth plate.
[0173] 2. Number of endosteal osteoclasts: measured along the
endosteal surface of cancellous bone at 180.times. magnification in
an area 1.22 mm proximal to the growth plate.
[0174] 3. Growth plate width: measured every 72 .mu.m at 90.times.
magnification across the entire growth plate except at the
peripheral ends to determine the growth plate activity.
[0175] Analyses of the data (mean.+-.SD, n=4-7/group) demonstrated
the following:
[0176] 1. There appeared to be no detectable inflammatory response
at the joint between tibia and femur.
[0177] 2. AdCMV-zFGF5 given IV or IPC in mice significantly
increased osteogenic activity in the distal femural metaphysis,
when examined at 2 weeks. This stimulation of osteogenic activity
was indicated by:
[0178] a) significant increases in the number of endosteal
osteoblasts in the cancellous bone of distal femurs following IV
infusion or IPC injection of AdCMV-zFGF5, 530% and 263%,
respectively, when compared with their relative vector only
controls; and
[0179] b) the observation of increased osteogenic tissues on the
bone surface, suggesting increased differentiation of bone marrow
stromal cells toward the osteoblast lineage.
[0180] 3. The number of endosteal osteoclasts was not significantly
affected by IV or IPC administration of AdCMV-zFGF5,when compared
with their relative vector only controls.
[0181] 4. The growth plate width was significantly decreased by IV
infusion, but not IPC injection, of AdCMV-zFGF5, suggesting
depressed growth plate activity following IV infusion. The
differential effects of AdCMV-zFGF5 administrations have not been
elucidated.
[0182] These results suggest that zFGF5 is a strong mitogen for
stimulation of osteoblast proliferation and that zFGF5 has the
capacity to induce new bone formation.
[0183] Using essentially the same procedures described above in
7.A. QCT was done on female CD-1 (Jackson Labs) that were injected
with 1.times.10.sup.11 particles AdCMV-zFGF5 per mouse. The mice
were sacrificed 30 days after injection and heart/tibial length
ratios were increased compared to controls (injected with empty
adenorvirus or saline). There were no differences between the
groups in tibial lengths to account for the change, nor were there
differences in any other organ weights among the groups. Thus, the
indication is that zFGF5 adenovirus selectively increases total
bone density, trabecular bone density, and cortical thickness in
the femur, as measured by QCT.
[0184] B. Effect of zFGF5 Pure Protein on Osteogenesis
[0185] An experimental pharmacology study was undertaken to
evaluate the acute safety of zFGF5 over a large dose range.
Individual male Sprague-Dawley rats were given a single bolus IV
injection of zFGF5 via an indwelling venous catheter at dose levels
of 8 mg/kg, 40 mg/kg and 76 mg/kg. Two additional animals were
given vehicle alone. On day 14 post dose, these animals as well as
two untreated male animals of the same age were euthanized by
CO.sub.2, necropsied and tissues collected and preserved in 10%
buffered formalin. When present, the following tissues were
trimmed, processed, and examined microscopically from each animal:
brain, liver, kidney, lung, heart, thymus, spleen, mesenteric lymph
node, pancreas, salivary gland, stomach, small intestine, large
intestine, testis, epididymis, urinary bladder, seminal vesicle,
trachea, esophagus, thyroid, adrenal, pituitary, skeletal muscle,
femur, and bone marrow.
[0186] Microscopically, the only test-related findings were in
sections of the femur. The femurs from animals given the highest
doses of zFGF5 had evidence of hyperostosis and osteoblast
hyperplasia. These findings were accompanied by an apparent
premature closing of the growth plate, seen as a loss of epiphysial
cartilage; as well as an increased deposition of new bone along the
diaphysis or shaft. There were no other test-material related
findings in any of the remaining tissues examined, nor were any
significant changes noted in the CBCs or serum chemistries. There
were no changes noted in the kidney similar to those reported for
FGF-2. These results suggest that zFGF5 will be useful as an
osteogenic factor for treatment of fracture repair and osteoporosis
and will be useful as an osteogenic factor in reconstructive
surgery.
Example 8
Effects of zFGF5 on Heart
[0187] As described in 7.B. CD-1 mice were given a single IV
injection of AdCMV-zFGF5, sacrificed after four weeks, and the
heart/tibial length ratios were found to be increased compared to
empty adenovirus or saline treated mice. The results showed that
there were no differences between the groups in tibial lengths to
account for this change, nor were there differences in any other
organ weights among the groups. This result suggests that
AdCMV-zFGF5 selectively increased cardiac growth, when administered
as an IV adenoviral construct.
Example 9
Expression of zFGF5
[0188] A. Construction of zFGF5-Encoding Plasmids
[0189] zFGF5, a fibroblast growth factor homolog, was expressed in
E. coli using the MBP (maltose binding protein) fusion system from
New England Biolabs (NEB; Beverly, Mass.). In this system, the
zFGF5 cDNA was attached to the 3' end of the malE gene to form an
MBP-zFGF5 fusion protein. Fusion protein expression was driven by
the tac promoter; expression is "off" until the promoter is induced
by addition of 1 mmol IPTG (isopropyl b-thiogalactosylpyranoside).
Three variations of this fusion protein were made, differing only
in their cleavage site for liberating zFGF5 from MBP. One construct
had a thrombin cleavage site engineered between the MBP and zFGF5
domains. The second construct had a Factor Xa cleavage site,
instead of a thrombin cleavage site. The third construct had an
enterokinase cleavage site, instead of the thrombin cleavage
site.
[0190] The constructs were built as in-frame fusions with MBP in
accordance with the Multiple Cloning Site (MCS) of the pMAL-c2
vector (NEB), and according to the manufacturer's specifications.
zFGF5 was amplified via PCR using primers which introduced
convenient cloning sites, as well as cleavage sites using the
following oligonucleotide primers: 1) for the thrombin construct:
zc12652 (SEQ ID NO: 7) and zc12631 (SEQ ID NO: 8); 2) for the
Factor Xa construct: zc15290 (SEQ ID NO: 9) and zc12631 (SEQ ID NO:
8); and 3) for the enterokinase construct: zc15270 (SEQ ID NO: 10)
and zc12631 (SEQ ID NO: 8). In each case, the native zFGF5 signal
sequence was not amplified; the zFGF5 as expressed begins at amino
acid residue 26 of SEQ ID NO: 2 (Val was changed to an Ala). The
thrombin construct was built by inserting an Xba I-Sal I zFGF5
fragment into the Xba I-Sal I sites of pMAL-c2. The Factor Xa
construct was built by inserting a blunt-Sal I fragment into the
Xmn I-Sal I sites of the MCS. The enterokinase construct was built
by inserting an Xba I-Sal I fragment into the Xba-Sal I sites of
pMAL-c2. Once the constructs were built, they were transformed into
a variety of E. coli host strains and analyzed for high-level
expression. The thrombin construct (designated pSDH90.5) was
transfected into DH10B cells (GIBCO-BRL), while both the Factor Xa
construct (designated pSDH117.3) and the enterokinase construct
(designated pSDH116.3) were transfected into TOP10 cells
(Invitrogen, San Diego, Calif.). All three MBP fusions are about 63
kD (43 kD in the MBP domain, and approximately 20 kD in the zFGF5
domain).
[0191] B. Homologous Recombination/zFGF5
[0192] Expression of zFGF5 in Pichia methanolica utilizes the
expression system described in co-assigned PCT publication
WO97/17450, incorporated herein by reference. An expression plasmid
containing all or part of a polynucleotide encoding zFGF5 is
constructed via homologous recombination. The expression vector is
built from pCZR204, which contains the AUG1 promoter, followed by
the .alpha.Fpp leader sequence, followed by an amino-terminal
peptide tag, a blunt-ended SmaI restriction site, a
carboxy-terminal peptide tag, a translational STOP codon, followed
by the AUG1 terminator, the ADE2 selectable marker, and finally the
AUG1 3' untranslated region. Also included in this vector are the
URA3 and CEN-ARS sequences required for selection and replication
in S. cerevisisiae, and the Amp.sup.R and colE1 ori sequences
required for selection and replication in E. coli. The zFGF5
sequence inserted into this vector begins at residue 27 (Ala) of
the zFGF amino acid sequence.
[0193] To construct pSDH114, a plasmid for expression of zFGF5 in
P. methanolica, the following DNA fragments were transformed into
S. cerevisisae: 100 ng of the `acceptor vector` pCZR204 that has
been digested with SmaI; 1 .mu.g of an XbaI-SalI restriction
fragment liberated from pSDH90.5 and encompassing zFGF5 coding
sequence.; 1 .mu.g of a synthetic, PCR-generated, double-stranded
linker segment that spans 70 base pairs of the aFpp coding sequence
on one end and joins it to the 70 base pairs of the amino-terminus
coding sequence from the mature zFGF5 sequence on the other was
generated from the four oligonucleotides zc13497 (SEQ ID NO: 11);
zc15131 (SEQ ID NO: 12); zc15132; (SEQ ID NO: 18); zc15134 (SEQ ID
NO: 13), of which the sense strand of a double stranded sequence is
shown in SEQ ID NO: 19 (5' linker sequence (aFpp.fwdarw.zFGF5
N-terminus)) and 1 .mu.g of of a synthetic, PCR-generated,
double-stranded linker segment that spans 70 base pairs of
carboxy-terminus coding sequence from zFGF5 on one end with 70 base
pairs of AUG1 terminator sequence was generated from the four
oligonucleotides 13529 (SEQ ID NO: 14); zc13525 (SEQ ID NO: 15)
zc13526 (SEQ ID NO: 16); zc13528 (SEQ ID NO: 17) of which the sense
strand of a double stranded sense is shown in the SEQ ID NO: 20 (3'
linker sequence (zFGF5 C-terminus.fwdarw.AUG1 terminator)).
Ura.sup.+ colonies were selected, and DNA from the resulting yeast
colonies was extracted and transformed into E. coli. Individual
clones harboring the correct expression construct were identified
by PCR screening with oligonucleotides zc13497 (SEQ ID NO: 11) and
zc13528 (SEQ ID NO: 12) followed by restriction digestion to verify
the presence of the zFGF5 insert and DNA sequencing to confirm the
desired DNA sequences had been enjoined with one another. Larger
scale plasmid DNA is isolated for one of the correct clones, and
the DNA is digested with Sfi I to liberate the Pichia-zFGF5
expression cassette from the vector backbone. The Sfi I-cut DNA is
then transformed into a Pichia methanolica expression host,
designated PMAD16, and plated on ADE D plates for selection. A
variety of clones are picked and screened via Western blot for
high-level zFGF5 expression.
[0194] More specifically, for small-scale protein production (e.g.,
plate or shake flask production), P. methanolica transformants that
carry an expression cassette comprising a methanol-regulated
promoter (such as the AUG1 promoter) are grown in the presence of
methanol and the absence of interfering amounts of other carbon
sources (e.g., glucose). For small-scale experiments, including
preliminary screening of expression levels, transformants may be
grown at 30.degree. C. on solid media containing, for example, 20
g/L Bacto-agar (Difco), 6.7 g/L yeast nitrogen base without amino
acids (Difco), 10 g/L methanol, 0.4 mg/L biotin, and 0.56 g/L of
-Ade -Thr -Trp powder. Because methanol is a volatile carbon source
it is readily lost on prolonged incubation. A continuous supply of
methanol can be provided by placing a solution of 50% methanol in
water in the lids of inverted plates, whereby the methanol is
transferred to the growing cells by evaporative transfer. In
general, not more than 1 ml of methanol is used per 100-mm plate.
Slightly larger scale experiments can be carried out using cultures
grown in shake flasks. In a typical procedure, cells are cultivated
for two days on minimal methanol plates as disclosed above at
30.degree. C., then colonies are used to inoculate a small volume
of minimal methanol media (6.7 g/L yeast nitrogen base without
amino acids, 10 g/L methanol, 0.4 mg/L biotin) at a cell density of
about 1.times.10.sup.6 cells/ml. Cells are grown at 30.degree. C.
Cells growing on methanol have a high oxygen requirement,
necessitating vigorous shaking during cultivation. Methanol is
replenished daily (typically 1/100 volume of 50% methanol per
day).
[0195] For production scale culturing, fresh cultures of high
producer clones are prepared in shake flasks. The resulting
cultures are then used to inoculate culture medium in a fermenter.
Typically, a 500 ml culture in YEPD grown at 30.degree. C. for 1-2
days with vigorous agitation is used to inoculate a 5-liter
fermenter. The cells are grown in a suitable medium containing
salts, glucose, biotin, and trace elements at 28.degree. C., pH
5.0, and >30% dissolved O.sub.2. After the initial charge of
glucose is consumed (as indicated by a decrease in oxygen
consumption), a glucose/methanol feed is delivered into the vessel
to induce production of the protein of interest. Because
large-scale fermentation is carried out under conditions of
limiting carbon, the presence of glucose in the feed does not
repress the methanol-inducible promoter.
Example 10
Purification of zFGF5
[0196] E.coli fermentation medium was obtained from a strain
expressing zFGF5 as a Maltose Binding protein fusion (pSDH90.5, as
described above). The MBPzFGF5 fusion was solubilized during
sonication or French press rupture, using a buffer containing 20 mM
Hepes, 0.4 M Nacl, 0.01 M EDTA, 10 mM DTT, at pH 7.4. The
extraction buffer also included 5 .mu.g/ml quantities of Pepstatin,
Leupeptin, Aprotinin, Bestatin. Phenyl methyl sulfonylfluoride
(PMSF) was also included at a final concentration of 0.5 mM.
[0197] The extract was spun at 18,000.times.g for 30 minutes at
4.degree. C. The resulting supernatent was processed on an Amylose
resin (Pharmacia LKB Biotechnology, Piscataway, N.J.) which binds
the MBP domain of the fusion. Upon washing the column, the bound
MBPzFGF5 fusion was eluted in the same buffer as extraction buffer
without DTT and protease inhibitors but containing 10 mM
Maltose.
[0198] The eluted pool of MBPzFGF5 was treated with 1:100 (w/w)
Bovine thrombin to MBPzFGF5 fusion. The cleavage reaction was
allowed to proceed for 6 to 8 hours at room temperature, after
which the reaction mixture was passed over a bed of Benzamidine
sepharose (Pharmacia LKB Biotechnology, Piscataway, N.J.) to remove
the thrombin, using the same elution buffer as described above for
Amylose affinity chromatography.
[0199] The passed fraction, containing the cleaved product zFGF5
and free MBP domain were applied to a Toso Haas Heparin affinity
matrix (Toso Haas, Montgomeryville, Pa.) equilibrated in 0.5 M
NaCl, 20 mM Hepes, 0.01 M EDTA at pH 7.4. The MBP and zFGF5 both
bound to heparin under these conditions. The bound proteins were
eluted with a 2 to 3 column volume gradient formed between 0.5M
NaCl and 2.0 M NaCl in column buffer.
[0200] The MBP eluted early, at about 0.7 M NaCl, and the cleaved
zFGF5 eluted at about 1.3 M NaCl. The pooled zFGF5 fractions were
passed through the amylose step once again to remove any residual
MBPzFGF5 that is a minor contaminant. The purified material was
designated zFGF5-Hep2, and shows a single highly pure species at
.about.20 kDa on reducing SDS-PAGE analysis.
[0201] Amino acid N-terminal sequencing yielded the native
N-Terminal sequence but Mass Spectrophotometry data revealed
molecular masses indicating that the C-Terminus must be truncated
at residue 196 (Lys) of SEQ ID NO: 2, where a "dibasic site" is
present.
[0202] zFGF5 protein was very stable in 1.3 M NaCl. Upon dialysis
into PBS, the zFGF5 aggregated and left the solution phase.
Therefore, formulations that include heparin and other "polyanions"
may be used to prevent the aggregation of pure zFGF5.
Example 11
Production of Antibodies
[0203] Antibodies for ZFGF5 were produced, using standard
techniques known in the art and described previously, by immunizing
guinea pigs, rabbits and mice with peptides QTRARDDVSRKQLRLYC (SEQ
ID NO: 2 amino acid residue 40 to residue 56), designated zFGF-1;
YTTVTKRSRRIRPTHRAC (SEQ ID NO: 2 amino acid residue 191 to residue
207, with an additional Cys at the C-terminus), designated zFGF5 or
the full-length zFGF5 polypeptide as shown in SEQ ID NO: 2, plus
the MPB fusion protein, and designated MBP-FGF5. Peptides were
conjugated through Cys residues using Maleimide-activated KLH
(Pierce Chemical Co., Rockford, Ill.).
[0204] Table 7 is a description of the animals, immunization levels
and antibody separations.
7TABLE 7 Peptide or Protein animal immun. level Ab produced ZFGF5-1
G.P. 50 ug/animal initial Affinity purified 25 ug/animal boost and
IgG fractionated. Rabbit 100 ug/animal initial Affinity purified 50
ug/animal boost and IgG fractionated. ZFGF5-2 G.P. 50 ug/animal
initial Affinity purified 25 ug/animal boost and IgG fractionated.
Rabbit 100 ug/animal initial Affinity purified. 50 ug/animal boost
and IgG fractionated. ZFGF5-MBP Mouse 20 ug/animal initial 10
ug/animal boost Rabbit 200 ug/animal initial Affinity purified 100
ug/animal boost
Example 12
[0205] A. Effects of zFGF5 on ob/ob Mice
[0206] The effects of zFGF5 on adipocytes and fat metabolism were
examined using female ob/ob mice (C57B1/6J, Jackson Labs, Bar
Harbor, Me.). The mice are obese, insulin resistant and have "fatty
bone". The mice were weighed and all were found to be the same
weight, and were injected IV with 10.sup.11 particles per mouse of
AdCMVzFGF5 or either saline or Ad5CMV-GFP for controls, as
described in Example 7. 17 days after injection, the control mice
injected with Ad5CMV-GFP had gained 5.342.+-.0.5 grams of body
weight compared to the day of injection, while the AdCMVzFGF5
treated mice lost 3.183.+-.0.743 grams of body weight.
[0207] B. Effect of Pure zFGF5 Protein on Sprague-Dawley Rats
[0208] In addition, zFGF5 produced a dose dependent reduction in
food intake within 24 h of treatment. Individual male
Sprague-Dawley rats were given a single bolus IV injection of zFGF5
via an indwelling venous catheter at dose levels of 8 mg/kg, 40
mg/kg and 76 mg/kg. Two additional animals were given vehicle
alone. On day 14 post dose, these animals as well as two untreated
male animals of the same age were euthanized by CO2, necropsied and
tissues collected and preserved in 10% buffered formalin. Food
intake was reduced 30% and 80% by the injection of 9.0 and 40.0
mg/kg zFGF5, respectively. No further reduction in food intake was
seen with the 76 mg/kg dose. Food intake increased slowly over the
next ten days to near normal levels. The reduction in food intake
was associated with a drop in body weight in the zFGF5 treated
animals that began with 24 h and remained suppressed for 10 days in
the high dose zFGF5 groups. The maximal drop in body weight over
the course of these experiments was 20-30% of the initial weight. A
normal rate of increase in body weight was seen between days 10 and
14 in these animals.
[0209] These data suggest that zFGF5 will act as a satiety factor
and would be useful for treating obesity disorders.
Example 13
[0210] A. Cloning of Mouse zFGF5
[0211] A cDNA for the mouse ortholog of zFGF5 was isolated from a
mouse embryo library. Oligonucleotide primers were designed from
the full length human zFGF5 sequence (ZC17578 and ZC17579, SEQ ID
NOS: 37 and 38, respectively). A PCR reaction was done using 2
.mu.l of library as template and ExTaq polymerase (PanVera,
Madison, Wis.) under the following conditions 1 cycle at 94.degree.
C. for 15 seconds; 35 cycles at 94.degree. C. for 15 seconds,
60.degree. C. for 20 seconds, 72.degree. C. for 30 seconds; and 1
cycle at 72.degree. C. for 10 minutes. The reaction mixture was
incubated a 4.degree. C. overnight. After the first reaction was
screened, no positive clones were identified and the procedure was
repeated until a positive clone was identified. The positive clones
were identified by transforming ElectroMAZ DH10B cells (GibcoBRL)
with 1 .mu.l of reaction mixture at 2.3 kV. The cells were plated
on culture plates containing ampicillin and methicillin and
incubated at room temperature for 3 days.
[0212] A DNA fragment obtained by PCR as described above was
radiolabeled using a Multiprime DNA Labeling System (Amersham) and
used as a probe for filters lifted from culture plates. The filter
lifts were hybridized overnight at 65.degree. C. in EXPRESS HYB
(Clontech). After hybridiziation, the filters were washed in buffer
of 0.25.times.SSC, 0.25% SDS, 1 mM EDTA at 65.degree. C., 6
times.
[0213] Positive clones were identified and cDNA inserts were
screened. The clones identified had truncations at the 5' end,
complete at the 3' ends and included 3' UTR. One clone, designated
LC 7-2 had the longest 5' end when compared to the human zFGF5
sequence. Sequence analysis verified that approximately 52 bp of 5'
sequence were missing and that this sequence was in the signal
sequence and that the entire nucleotide sequence encoding the
mature polypeptide was intact.
[0214] B. Northern Analysis
[0215] Northern analyses were performed using Mouse Multiple Tissue
Blots from Clontech (Palo Alto, Calif.), mouse heart blots
(prepared at ZymoGenetics, Inc.) and mouse dot blots (Clontech).
Using oligonucleotides ZC17579 (SEQ ID NO: 29) and ZC17578 (SEQ ID
NO: 40) and the mouse zFGF5 as a template, a probe was generated.
The DNA probe was radioactively labeled using a random priming
MEGAPRIME DNA labeling system (Amersham, Arlington Heights, Ill.)
according to the manufacturer's specifications. The probe was
purified using a NUCTRAP push column (Stratagene Cloning Systems,
La Jolla, Calif.). EXPRESSHYB (Clontech, Palo Alto, Calif.)
solution was used for prehybridization and as a hybridrizing
solution for the Northern blots. Hybridization took place overnight
at 68.degree. C., and the blots were then washed in 2.times.SSC and
0.05% SDS at RT, followed by a wash in 0.1.times.SSC and 0.1% SDS
at 50.degree. C. Multiple bands were observed at with predominate
bands at approximately 0.6-0.8 kb, 1.2 kb and 2.2-2.4 kb bands
depending on the blot used. Signal intensity was highest for spleen
with slightly lower intensity signals in heart, lung, liver,
skeletal muscle, kidney and testis. Mouse dot blots with the same
probe were positive only for spleen and day 17 mouse embryo. Mouse
heart mRNA northerns were probed and results were positive for C57
Black, CD1, neonatal heart, and day 16 and day 20 embryo, with
strongest signal present in the day 16 embryo. BALB C mouse heart
did not have a signal present.
[0216] Because the results in the mouse tissue did not directly
correlate with results seen in the human tissue, a new probe was
designed. The new probe was designed specifically to exclude the
possibility that any members of the FGF family other than zFGF5
were positive by Northern analysis. The probe was prepared using
PCR with oligonucleotides ZC195687 (SEQ ID NO: 41) and ZC19633 (SEQ
ID NO: 42) and template DNA from the mouse cDNA of zFGF5. The
reactions were essentially the same as described above. The mouse
heart blot was positive for C57 Black mouse, neonatal mouse, days
16 and 20 mouse embryo, with signals strongest in the neonatal
heart and day 16 mRNA. The dot blots were positive for spleen and
epididymus. It appeared that there was some variability for mouse
mRNA expression, unlike human tissue, where heart mRNA consistently
was the primarily tissue in which zFGF5 was expressed in humans.
Similar variability was seen with rat northern analysis.
Example 14
In vivo Study of Cardiomyopathic Rats
[0217] Rats infused subcutaneously with epinephrine for 2 weeks
develop a cardiomyopathy quite similar to human idiopathic dilated
cardiomyopathy (Deisher et al., Am. J. Cardiovasc. Pathol.
5(1):79-88, 1994 and Deisher et al., J. Pharmacol. Exp. Ther.
266(1):262-269, 1993.)
[0218] The effect of zFGF5 on the initiation and progression of the
catecholamine-induced cardiomyopathy was evaluated by administering
zFGF5 by intra-pericardial injection to male, Sprague-Dawley rats
receiving subcutaneous infusions of epinephrine or saline.
[0219] In one protocol, rats (300 gms) were implanted with
subcutaneous saline- or epinephrine-filled osmotic mini-pumps under
light ether anesthesia. 96 hours following minipump implantation, a
single intra-pericardial injection of vehicle (n=25) or zFGF5 at
25, 250 or 500 .mu.g/kg was given (n=10 per dose). Mortality was
monitored for an additional two weeks, at the end of which the rats
were sacrificed, the hearts were weighed wet, and fixed in 10%
neutral buffered formalin for histology.
[0220] The zFGF5 had no effect on mortality, body weight, heart
weight or cardiac fibrosis in saline-infused rats.
[0221] In epinephrine-infused rats, the 25 .mu.g/kg and 250
.mu.g/kg doses reduced mortality from 32% in vehicle injected rats
to 0% in 25 .mu.g/kg and 10% in 250 .mu.g/kg injected rats. The
highest zFGF5 dose, 500 .mu.g/kg, reduced mortality to 20% compared
to vehicle injected rats, however this was not statistically
significant. Cardiac fibrosis was determined by scoring Masson's
Trichrome stained heart sections. Three sections were scored for
each heart, and the average score taken. The fibrosis score for the
vehicle-infused hearts was 1.26.+-.0.25, while the score for the 25
.mu.g/kg zFGF5 injection was 1.74.+-.0.23, the 250 .mu.g/kg
injection was 1.38.+-.0.29, and the 500 .mu.g/kg injection was
0.81.+-.0.10. The dose of zFGF5 which completely prevented
mortality increased the cardiac fibrosis score (25 .mu.g/kg), while
the dose which had no effect on mortality reduced the cardiac
fibrosis score (500 .mu.g/kg). These results indicate that a
pro-fibrotic activity can be beneficial in the setting of heart
failure of varying etiologies, of which can include myocardial
infarct (MI), idiopathic dilated cardiomyopathy (IDCM),
hypertrophic cardiomyopathy, viral myocarditis, congenital
abnormalities, and obstructive diseases.
[0222] In another protocol, the rats (300 gms) were anesthetized by
an intra-muscular injection of an anesthetic cocktail
ketamine:rompun:acepromazine (1:1:0.1). Subcutaneous
epinephrine-filled osmotic mini-pumps were implanted, and either
vehicle or zFGF5 was injected intra-pericardially immediately
afterward at 25 .mu.g/kg (n=25 per group). For the vehicle injected
rats, 21% had died within 6 days following the epinephrine-filled
minipump implantation, while none of the zFGF5 injected rats had
died. By the end of the 2 week epinephrine infusion period, 25% of
the vehicle-injected rats had died, while only 22% of the
zFGF5-injected rats had died. In this model, zFGF5 co-treatment at
the time of minipump implantation delayed mortality by at least 7
days.
Example 15
Mammalian Expression Constructs
[0223] An expression plasmid containing all or part of a
polynucleotide encoding zFGF5 is constructed via homologous
recombination. A fragment of zFGF5 cDNA is isolated using PCR that
includes the polynucleotide sequence from nucleotide 1 to
nucleotide 621 of SEQ ID NO: 1 or SEQ ID NO: 37, with flanking
regions at the 5' and 3' ends corresponding to the vectors
sequences flanking the zFGF5 insertion point. The primers for PCR
each include from 5' to 3' end: 40 bp of flanking sequence from the
vector and 17 bp corresponding to the amino and carboxyl termini
from the open reading frame of zFGF5.
[0224] Ten .mu.l of the 100 .mu.l PCR reaction is run on a 0.8% LMP
agarose gel (Seaplaque GTG) with 1.times.TBE buffer for analysis.
The remaining 90 .mu.l of PCR reaction is precipitated with the
addition of 5 .mu.l 1 M NaCl and 250 .mu.l of absolute ethanol. The
plasmid pZMP6 which has been cut with SmaI is used for
recombination with the PCR fragment. Plasmid pZMP6 was constructed
from pZP9 (deposited at the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209, and is designated
No. 98668) with the yeast genetic elements taken from pRS316
(deposited at the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209, and designated No.
77145), an IRES element from poliovirus, and the extracellular
domain of CD8, truncated at the carboxyl terminal end of the
transmembrane domain. pZMP6 is a mammalian expression vector
containing an expression cassette having the cytomegalovirus
immediate early promoter, immunoglobulin signal peptide intron,
multiple restriction sites for insertion of coding sequences, a
stop codon and a human growth hormone terminator. The plasmid also
has an E. coli origin of replication, a mammalian selectable marker
expression unit having an SV40 promoter, enhancer and origin of
replication, a DHFR gene, the SV40 terminator, as well as the URA3
and CEN-ARS sequences required for selection and replication in S.
cerevisiae.
[0225] One hundred microliters of competent yeast cells (S.
cerevisiae) are independently combined with 10 .mu.l of the various
DNA mixtures from above and transferred to a 0.2 cm electroporation
cuvette. The yeast/DNA mixtures are electropulsed at 0.75 kV (5
kV/cm), .infin. ohms, 25 .mu.F. To each cuvette is added 600 .mu.l
of 1.2 M sorbitol and the yeast is plated in two 300 .mu.l aliquots
onto two URA-D plates and incubated at 30.degree. C. After about 48
hours, the Ura+ yeast transformants from a single plate are
resuspended in 1 ml H.sub.2O and spun briefly to pellet the yeast
cells. The cell pellet is resuspended in 1 ml of lysis buffer (2%
Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
Five hundred microliters of the lysis mixture is added to an
Eppendorf tube containing 300 .mu.l acid washed glass beads and 200
.mu.l phenol-chloroform, vortexed for 1 minute intervals two or
three times, followed by a 5 minute spin in a Eppendorf centrifuge
at maximum speed. Three hundred microliters of the aqueous phase is
transferred to a fresh tube, and the DNA precipitated with 600
.mu.l ethanol (EtOH), followed by centrifugation for 10 minutes at
4.degree. C. The DNA pellet is resuspended in 10 .mu.l
H.sub.2O.
[0226] Transformation of electrocompetent E. coli cells (DH10B,
GibcoBRL) is done with 0.5-2 ml yeast DNA prep and 40 ul of DH10B
cells. The cells are electropulsed at 1.7 kV, 25 .mu.F and 400
ohms. Following electroporation, 1 ml SOC (2% Bacto` Tryptone
(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) is plated in
250 .mu.l aliquots on four LB AMP plates (LB broth (Lennox), 1.8%
Bacto Agar (Difco), 100 mg/L Ampicillin).
[0227] Individual clones harboring the correct expression construct
for zFGF5 are identified by restriction digest to verify the
presence of the zFGF5 insert and to confirm that the various DNA
sequences have been joined correctly to one another. The insert of
positive clones are subjected to sequence analysis. Larger scale
plasmid DNA is isolated using the Qiagen Maxi kit (Qiagen)
according to manufacturer's instruction.
Example 16
Mammalian Expression of zFGF5
[0228] CHO DG44 (Chasin et al., Som. Cell. Molec. Genet.
12:555-666, 1986) are plated in 10 cm tissue culture dishes and
allowed to grow to approximately 50 to 70% confluency overnight at
37.degree. C., 5% CO.sub.2, in Ham's F12/FBS media (Ham's F12
medium, (Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum
(Hyclone, Logan, Utah), 1% L-glutamine (JRH Biosciences, Lenexa,
Kans.), 1% sodium pyruvate (Gibco BRL)). The cells are then
transfected with the plasmid zFGF5/pZMP6, using Lipofectamine.TM.
(Gibco BRL), in serum free (SF) media formulation (Ham's F12, 10
mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine
and 1% sodium pyruvate). ZFGF5/pZMP6 is diluted into 15 ml tubes to
a total final volume of 640 .mu.l with SF media. 35 .mu.l of
Lipofectamine.TM. (Gibco BRL) is mixed with 605 .mu.l of SF medium.
The Lipofectamine.TM. mix is added to the DNA mix and allowed to
incubate approximately 30 minutes at room temperature. Five
milliliters of SF media is added to the DNA:Lipofectamine.TM.
mixture. The cells are rinsed once with 5 ml of SF media,
aspirated, and the DNA:Lipofectamine.TM. mixture is added. The
cells are incubated at 37.degree. C. for five hours, then 6.4 ml of
Ham's F12/10% FBS, 1% PSN media is added to each plate. The plates
are incubated at 37.degree. C. overnight and the
DNA:Lipofectamine.TM. mixture is replaced with fresh 5% FBS/Ham's
media the next day. On day 3 post-transfection, the cells are split
into T-175 flasks in growth medium. On day 7 postransfection the
cells are stained with FITC-anti-CD8 monoclonal antibody
(Pharmingen, San Diego) followed by anti-FITC-conjugated magnetic
beads (Miltenyi Biotec, Auburn, Calif.). The CD8 positive cells are
separated by Miltenyi mini-MACS columns according to manufacturer's
directions (Miltenyi Biotec), and put into DMEM/Ham's F12/5% FBS
without nucleosides but with 50 nM methotrexate (selection
medium).
[0229] Cells are plated for subcloning at a density of 0.5, 1 and 5
cells per well in 96 well dishes in selection medium and allowed to
grow out for approximately two weeks. The wells are checked for
evaporation of medium and brought back to 200 .mu.l per well as
necessary during this process. When a large percentage of the
colonies in the plate are near confluency, 100 .mu.l of medium is
collected from each well for analysis by dot blot, and the cells
are fed with fresh selection medium. The supernatant is applied to
a nitrocellulose filter in a dot blot apparatus, and the filter is
treated at 100.degree. C. in a vacuum oven to denature the protein.
The filter is incubated in 625 mM tris glycine, pH 9.1, 5 mM
.beta.mercaptoethanol, at 65.degree. C., 10 minutes, then in 2.5%
non-fat dry milk Western A Buffer (0.25% gelatin, 50 mM TrisHCl pH
7.4, 150 mM NaCl, 5 mM EDTA, 0.05% Igepal, Sigma) overnight at
4.degree. C. on a rotating shaker. The filter is incubated with the
antibody-HRP conjugate in 2.5% non-fat dry milk Western A buffer
for 1 hour at room temperature on a rotating shaker. The filter is
washed three times at room temperature in PBS plus 0.01% Tween 20,
15 minutes per wash. The filter was developed with ECL reagent
according to manufacturer's directions (Amersham, Arlington
Heights, Ill.), and exposed to film (Hyperfilm ECL, Amersham)
approximately 5 minutes. Positive clones are trypsinized from the
96 well dish and transferred to 6 well dishes in selection medium
for scaleup and analysis by Western blot.
Example 17
Expansion of Cells From Bone Marrow
[0230] Assays were performed to measure the frequency of fibroblast
colony forming units from monkey low density, non-adherent cells
isolated from bone marrow. This assay is indicative of mesenchymal
stem cell frequency.
[0231] One half of a 96 well microtiter plate is inoculated with
cells at a density of 10,000 cells/well and the other half of the
plate is inoculated with cells at a density of 1,000 cells/well.
The culture medium is .alpha.MEM (GIBCO-BRL, Gaithersburg, Md.), 2%
bovine serum albumin, 10 .mu.g/ml insulin, 200 .mu.g/ml
transferrin, antibiotic and 50 .mu.M ?-Mercaptoethanol. The cells
are incubated at 37.degree. C. in 5% CO.sub.2 for 14 days and then
stained with toluidine blue to improve cell visibility and examined
microscopically. Positive wells have at least 50 cells exhibiting a
"stromal" morphology, i.e., large, spread out cells. The positive
control is medium containing 20% fetal bovine serum. Results
demonstrated that zFGF5, at a concentration of 100 ng/ml increased
the frequency of CFU-F to levels equivalent to the positive control
of 20% FBS.
Example 18
Effects of zFGF5 on Neural Cells
[0232] A. Neurite Outgrowth Assay-Relative Efficacy of zFGF5
[0233] The effect of zFGF5 on PC12, rat pheochromocytoma cells with
neural potential (ATCC No. CRL-1721, American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209)
examined using the following growth factors, each at 3
dilutions:
[0234] 1 .mu.g/ml, 100 ng/ml, and 10 ng/ml neural growth factor
(NGF; source and location) in medium containing RPMI 1640 (R&D
Systems, Minneapolis, Minn.)
[0235] 1 .mu.g/ml, 100 ng/ml, and 10 ng/ml human basic FGF (R&D
Systems, Minneapolis, Minn.)
[0236] 1 .mu.g/ml, 100 ng/ml, and 10 ng/ml zFGF5 (recombinantly
produced in E. coli.)
[0237] 1 .mu.g/ml, 100 ng/ml, and 10 ng/ml zFGF5 (recombinantly
produced in CHO cells)
[0238] The PC12 cells were plated at a concentration of
5.times.10.sup.4/ml onto collagen coated 24 well culture plates and
incubated for 48 hours in the appropriate medium. After 48 hours,
the medium was changed to include one of the cytokines described
above and then changed again every 2 days. The wells were scored
for relative neurite outgrowth on days 6 and 9.
[0239] Neurite outgrowth was induced with each of the cytokines.
NGF and bFGF appeared to have similar affinity, while zFGF5 had
significantly lower affinity. NGF exerted the greatest extent of
neural outgrowth activity, followed by bFGF, with significantly
lower activity seen with zFGF5.A second experiment showed that
zFGF5 has activity on neural-derived cells PC-12 cells.
[0240] B. Specificity and Efficacy of zFGF5 For Neural-Derived
Cells
[0241] Under a variety of culture conditions, in naive and primed
cells, zFGF5 promoted neurite outgrowth. Survival of these cultures
under serum-free conditions was also enhanced by the addition of
zFGF5. ZFGF5 protein was active over a concentration range of
0.03-1.0 ug/ml, suggesting that PC-12 cells contain a receptor that
recognizes zFGF5.
[0242] To evaluate whether PC-12 cells express any of the known FGF
receptors, mRNA was isolated from cultures of PC-12 cells and the
levels of FGFR mRNA were measured by PCR using primers specific for
FGFR3c. A band of the correct size was observed only in the samples
amplified with FGFR3c primers indicating that FGFR3c is a high
affinity zFGF5 receptor on these cells.
[0243] The expression of zFGF5 mRNA was also examined in mouse
brain tissue by in situ hybridization with probes specific for
zFGF5. Expression of zFGF5 mRNA was observed in neurons throughout
the brain, expression was exceptionally high in Purkinje cells of
the cerebellum, neurons in the cerebrum, thalamus, and hippocampus.
Staining was also observed in astrocytes throughout the brain.
These results suggest that zFGF5 plays a role in normal
brain-homeostasis.
[0244] These data suggest that zFGF5 will be useful for treating a
variety of neurological conditions such as stroke, spinal cord
injuries, dementia, and other neurological syndromes.
Example 19
Identification of a Target Cell
[0245] Identification of a putative mesenchymal stem cell as a
target for zFGF5 was made using FITC-labeled protein and neonatal
mouse heart tissue.
[0246] ZFGF5, purified as described above, was dialyzed into 0.1 M
sodium bicarbonate pH 9.0. Fluorescein isothiocyanate (FITC;
Molecular Probes, Eugene, Oreg.) was dissolved at 1 mg/ml in the
same buffer without exposure to strong light. The mixture was
prepared containing 1 mg FIFC/1 mg zFGF5, and reacted for 1-2 hours
in the dark at room temperature. The reaction was stopped by adding
1 M glycine to a final concentration of 0.1 M, then reacted for 1
hour at room temperature. The mixture was then dialyzed against 0.1
M sodium biocarbonate to make a 1:500-1:1000 dilution for 3 hours.
The dialysis solution was changed and the process repeated for 3-18
hours to remove unlabeled FITC.
[0247] Neonatal mouse heart ventricles were isolated, minced, and
repeatedly washed in phosphate buffered solution (PBS) until all
red blood cells and debris were removed. The minced ventricles were
placed in a solution containing 18 ml PBS and 1% glucose and 1 ml
of 2% DNAse/Collagenase solution was added. The mixture was
incubated on a shaker for 30 minutes at 37.degree. C. The
supernatant was discarded and the process was repeated once more.
After incubation, the supernatant (.about.20 ml) was transferred to
a tube containing 20 ml DF 20 (Dulbecco's Modified Eagle's
Medium/Ham's Nutrient Mixture F12, 1:1 (GIBCO-BRL, Gaithersburg,
Md.) and 20% fetal bovine serum). After mixing, the tubes were
centrifuged at 1650 rpms in a Beckman CS-6R centrifuge (Beckman,
Fullerton, Calif.) at 4.degree. C. for 10 minutes. The supernatant
was discarded and the pellet was resuspended in DF 10 (10% FBS).
The cells were kept cold and spun again and resuspended in 40 ml of
DF 10. The cell mixture was passed over a 40 .mu.m filter (Becton
Dickinson, Detroit, Mich.) and counted using a hemacytometer.
[0248] The cells were incubated in FITC-labeled zFGF5 at 4.degree.
C. for 30 minutes at a concentration of 2.times.10.sup.6 cells/1
.mu.g zFGF5. After incubation, the cells were spun at 1650 rpms in
a Beckman CS-6R centrifuge (Beckman) for 5 minutes. The supernatant
was discarded and the pellet washed once in 10 ml of DF 10 and
resuspended in 4 ml DF 10.
[0249] 10 .mu.l of MACS anti-FITC microbeads (Miltenyi Biotech,
Auburn, Calif.) were mixed with 10.sup.7 cells in 4 ml of DF10 and
incubated at 4.degree. C. for 30 minutes.
[0250] MACS positive selection type LS+ separation columns
(Miltenyi Biotech) were washed with 3 ml of MAC buffer (PBS, 0.5%
BSA, 2 mM EDTA) and the cell/bead mixture was washed in 10 ml MAC
buffer and then resuspended in 6 ml MAC buffer. The cell/bead
mixture was divided between the two columns and the first negative
fraction was discarded. 1.5 ml of 0.6 M NaCl was added to each
column and eluted but not collected. The columns were then washed
with 1.5 ml MAC buffer. The cells bound with FITC-labeled zFGF5
were collected by adding 3 ml MAC buffer, removing the column from
the magnet and flushing out the positive cells using the plunger.
The positive cell fraction was plated in a T75 flask and 50 ml of
plating medium was added (DF with 15% FBS and antibiotics). The
cells were incubated at 37.degree. C. for 1 week and counted. The
yield of positive cells was approximately 0.1% of original total
cells counted.
[0251] Cells binding FITC-labeled zFGF5 were examined by
transmission electronmicroscopy (TEM). The cells were between 3-5
microns in diameter. The cell nuclei occupied the majority of the
cell volume, and few cytoplasmic organelles were apparent. The
phenotype identified by TEM identifies the zFGF5-isolated cells as
primitive mesenchymal stem cells.
Example 20
Identification of an FGF Receptor With Specificity For zFGF5
[0252] A panel of six BAF 3 cell lines were transfected to express
each of the known FGF receptor splice variants
(FGFR1.alpha.-IIIb,-IIIc, FGFR2.alpha.-IIIb,-IIIc,
FGFR3.alpha.-IIIb, -IIIc). The transfected cell lines were used to
assess the receptor specificity of zFGF5. BAF3 cells do not express
their own FGF receptors and are dependent on IL-3 for growth. Wild
type BAF3 cells, thus, do not respond to FGF, and removal of IL-3
from the growth media abolishes cell proliferation. In transfected
cell lines the addition of 2.0 ug heparin/ml and an FGF that
activates a specific FGF receptor stimulates cell proliferation.
Each cell line was tested in mitogenesis assays using zFGF5.
[0253] These data were confirmed and extended using FGFR3-IIIc
expressing cells and soluble FGFRs. The soluble FGFRs contained the
ligand binding domain of each FGF receptor fused to the Fc portion
of human immunoglobulin G. In competition studies using zFGF5 to
stimulate cell proliferation, only FGFR3.alpha.-IIIc-Fc and
FGFR4-Fc produced a high affinity blockade of cell growth. Some
inhibition was also observed with FGFR2.alpha.-III-Fc but the
affinity was about 100-fold lower. Table 8 illustrates the effects
of zFGF5 on cell proliferation of FGF receptor-expressing
cells.
8TABLE 8 zFGF5 FGFR1 FGFR2 FGFR3 Concentration -IIIb -IIIc -IIIb
-IIIc -IIIb -IIIc (ng/ml) Alamar Blue Units 1.6 0 2.6 0.2 241 0
1402 3.1 1.7 4.2 0.06 480 0 2003 6.2 1.3 14.2 0 715 0 2581 12.5 0
29.9 0.4 977 0 3201 25 10.9 86 0.2 1307 1.4 3764 50 0 224 0.5 1523
19 3958 100 0 488 1.3 1723 189 4117 200 0 912 1.3 1794 472 4205
[0254] The only high affinity response (EC50.about.1-2 ng/ml) was
seen with BAF3 cells expressing FGFR3.alpha. IIIc. A lower affinity
response (EC50.about.10-20 ng/ml) of activation was observed in
cells expressing FGFR2.alpha.-IIIc. Activation of the 3c and 2c
cells by zFGF5 was, moreover, completely dependent on exogenous
heparin. Addition of zFGF5 did not enhance proliferation of any of
the FGFR "b" splice variant expressing cells.
[0255] To further explore the receptor selectivity of zFGF5, the
ability of a series of FGF receptor-human Fc fusion proteins
(soluble receptors) was tested for their ability to compete for
zFGF5-mediated cell proliferation in BAF3 cells expressing the
FGFR3.alpha.-IIIc receptor. Of the soluble receptors tested, only
the FGFR4/Fc and FGFR3.alpha.-IIIc/Fc receptors exhibited a high
affinity competition for zFGF5-mediated cell proliferation. The
FGFR2.alpha.-IIIc/Fc receptor did compete for zFGF5 binding,
however, the EC50 for this effect was more than 100-fold higher
than that seen with FGFR4/Fc and FGFR3.alpha.-IIIc/Fc. Competition
was not observed using the FGFR1.alpha.-IIIc/Fc or the
FGFR2.alpha.-IIIb/Fc receptors. Table 9 illustrates the inhibition
of zFGF-5-mediated cell proliferation by FGF receptor-Fc Fusion
proteins.
9 TABLE 9 FGFR1 FGFR2 FGFR3 Concentration IIIc-Fc IIIb-Fc IIIc-Fc
IIIc-Fc FGFR4 (fold-excess) Cell Proliferation (percent of control)
0 100 100 100 100 100 0.8 85 79 1.6 66 64 3.2 37 27 6.3 5 8 7.5 92
96 12.5 1 2 15.4 96 93 25 .2 1 31 99 91 50 0 0 62.5 104 101 77 100
0 0 125 101 90 61 250 100 91 39 500 102 101 22 1000 92.3 99 6
[0256] Table 10 is a comparison of the receptor specificity of
FGF-1, FGF-2, and zFGF-5.
10 TABLE 10 FGF receptor FGF-1 FGF-2 zFGF5 1b + + - 1c + + - 2b + -
- 2c + + .+-. 3b + - - 3c + + + 4 + + +
[0257] These data demonstrate that of the known FGF receptors,
zFGF5 appears to preferentially bind to FGFR4 and FGFR3-IIIc. A
comparison of FGF receptor specificity of FGF-1, FGF-2, and zFGF5,
suggests that the receptor binding specificity of zFGF5 is
considerably more restricted than either FGF-1 or -2.
Example 21
Stimulation of Chrondocytes and Cartilage Matrix
[0258] Adenovirus expressing zFGF5 was injected into the external
ear tissue of nude mice. Compared to infection with the control
"empty" adenovirus, ears infected with adenovirus expressing zFGF5
were visibly thicker. Histologic analysis revealed that the
increase in thickness was due in part to the proliferation of
chondrocyte cells within the ear cartilage. Cells within this
region stained positive for PCNA, a marker of cell proliferation,
and were stained by both Alcian blue and Toluidine blue, markers of
collagen synthesis. There was little or no staining with PCNA in
contrast, in the chondrocyte zone of mouse ears infected with the
control adenoviral vector. These data suggested that zFGF5 is a
mitogen for chondrocytes.
[0259] To test this directly, chondrocytes were isolated from
porcine articular cartilage of the knee joint and cultured in the
presence and absence of zFGF5 protein. Proliferation of these
cells, measured by [.sup.3H]thymidine incorporation was enhanced
more than three-fold by incubation with zFGF5 over a concentration
range of 10-500 ng/ml. In addition, the synthesis of
glycosaminoglycans, measured by .sup.35S incorporation which are
characteristic of cartilage tissue, was enhanced in these cultures
more than 4-fold by the addition of zFGF5 over a similar
concentration range. Table 11 illustrates the effects of zFGF5 on
chondrocyte proliferation and glycosaminoglycan production.
11TABLE 11 zFGF5 [.sup.3H]Thymidine [.sup.35S]Sulfate concentration
incorporation incorporation (ng/ml) cpm cpm 0 35805 4834 10 43777
4242 50 65519 100 100896 16457 200 107174 18612 500 125294
[0260] To assess whether zFGF5 mRNA is expressed in normal human
tracheal and articular cartilage, probes specific for zFGF5 mRNA
were prepared and zFGF5 mRNA levels were assessed by in situ
hydridization techniques. These analyses revealed intense staining
for zFGF5 mRNA in the cytoplasm of mature chondrocytes in both
tissues. No expression was detected in immature chondrocytes on the
same section. The results demonstrate that zFGF5 can stimulate the
proliferation of mature chondrocytes in vivo and in vitro, can
stimulate matrix production by these cells, and is expressed by
chondrocytes in human cartilage.
[0261] Further analysis of the role of zFGF5 in cartilage biology
in normal and diseased states examined expression of mRNA for zFGF5
and FGFR3.alpha.-IIIc in human chondrosarcoma tissue. Expression of
zFGF5 and FGFR3.alpha.-IIIc mRNAs was observed in chondrocytes
throughout the tumor tissue sections suggesting that zFGF5 may
regulate cell proliferation in these tumors.
[0262] Further confirmation of zFGF5-induced proliferation of
mesenchymal-derived tumor cells, in particular osteoblast- and
chondrocyte-derived tumors, was shown by conjugating zFGF5 to the
cell toxin saporin. Conjugates of cytotoxic polypeptides and zFGF5
components were prepared using standard techniques for conjugating
polypeptides, and are described, for example, by Lappi et al.,
Biochem. Biophys. Res. Commun. 160:917 (1989), Soria et al.,
Targeted Diagn. Ther. 7:193 (1992), Buechler et al., Eur. J.
Biochem. 234:706 (1995), Behar-Cohen et al., Invest. Ophthalmol.
Vis. Sci. 36:2434 (1995), Lappi and Baird, U.S. Pat. No. 5,191,067,
Calabresi et al., U.S. Pat. No. 5,478,804, and Lappi and Baird,
U.S. Pat. No. 5,576,288. Additional approaches to conjugating
polypeptide are known to those of skill in the art. For example,
Lam and Kelleher, U.S. Pat. No. 5,055,291, describe the production
of antibodies conjugated with either diphtheria toxin fragment A or
ricin toxin.
[0263] When human osteosarcoma cells were incubated with a
zFGF5-saporin conjugate and cell viability was determined,
zFGF5-saporin conjugate produced a dose-dependent killing of the
cells with an EC50 of about 1.0 nM. Saporin alone or other saporin
conjugated to several unrelated proteins were without effect,
suggesting that zFGF5 conjugated to a cytotoxin has specificity for
mesenchymal-derived tumor cells and may be used to inhibit such
tumors.
Example 22
zFGF5 Affects Wound Healing
[0264] Investigation of whether zFGF5 would promote wound healing
was made using bFGF for comparison and PDGF as a positive control
in male, 19 weeks old, db/db mice. In the study, the mice received
a dorsal full-thickness skin excision of approximately 1 cm square.
The wound was covered with a semi-occlusive, self-adhesive
dressing. The wounds were treated topically, daily, with one of the
following: vehicle, PDGF, bFGF or zFGF5. The treatments, 10.mu.g of
a growth factor in 0.1 ml of PBS, were injected through the
dressing onto the wound bed daily for 7 treatments. The mice were
sacrificed on Day 18 and the whole wound bed plus surrounding
normal skin were harvested for histology.
[0265] Wound closure was measured on Days 6, 10 and 18 by tracing
the edge of the scar (the originally cut edge) and the advancing
edge of the new epithelium onto acetate transparencies. The areas
defined by the two edge tracings were measured and calculations of
% Closure, % Re-epithelialization and % Contraction compared to Day
0 were done at the three timepoints.
[0266] Wound fluid accumulated over the healing wound beds was
removed and measured daily as another index of the rate of wound
healing. The volume of wound fluid collected from the bFGF treated
wounds was clearly greater than any other group. This is consistent
with the observation that the bFGF treated wound beds appeared much
more highly vascularized than the other 3 groups. The bFGF wounds
were very bright red in comparison with the other groups. The open
and re-epithelialized areas of the wounds were measured using the
planimetry program, Optimas. The statistical analyses were done
using the program InStat.
[0267] On Day (-2), the mice had the hair clipped from their dorsum
and were then depilated. On Day 0, the mice were anesthetized with
Metofane anesthetic; the denuded dorsum wiped with Povidone Iodine
then 70% Isopropyl alcohol, and the corners of a 1 cm square
template were marked and the skin within the defined area excised
(approximately) under aseptic conditions. The mice were
administered through the dressing topically onto the wound bed
daily for 7 treatments, starting immediately following surgery. On
Days 6, 10, 14 and 18 the wounds were traced.
12 Group 1 vehicle n = 8 Group 2 PDGF 10 .mu.g n = 8 Group 3 bFGF5
10 .mu.g n = 8 Group 4 zFGF5 10 .mu.g n = 8
[0268] Wound healing occurs by a combination of contraction and
re-epithelialization. Percent Closure is the union of the %
Re-epithelialization and % Contraction. In this study, the %
Contraction was equivalent in the groups treated with bFGF and
PDGF. The group treated with zFGF5 had only slightly higher %
Contraction than the vehicle treated group on Day 18. The earlier
timepoints showed equivalent % Contraction between vehicle and
zFGF5 groups.
[0269] There was a statistically significant increase in % Closure
compared to the vehicle treated wounds in the zFGF5-, FGF-2-, and
PDGF-treated wounds on Day 18. Similar changes were observed for
each group for the percent re-epithelialization parameter. The
changes observed in the zFGF5-treated animals did not quite achieve
statistical significance.
[0270] Wound fluid accumulates in the wound from vessels that are
rendered "leaky" by cytokines locally released from various cell
types, such as histamine released from infiltrating macrophages.
The comparatively "hyper-vascular" appearance of the wounds in the
bFGF group is consistent with more extravasation of fluid in that
group than any other group. The zFGF5 group had more fluid
accumulation than the vehicle group, but significantly less than
the PDGF group, which was intermediate in accumulated fluid
volume.
[0271] The volumes of wound fluid collected were clearly different
between the groups. A table of the total volume collected per
treatment group per day follows.
13 TABLE 12 wound fluid accumulations: ml/day Day Day Day Day Day
Day Day Day 4 5 6 7 8 9 10 11 Vehicle 0 0 0 0.06 0.02 0 0 0 PDGF
0.1 0.29 0.35 0.5 0.42 0 0 0 FGF-2 0.05 0.72 1.04 0.81 0.54 0.05
0.45 0.01 zFGF5 0 0 0.12 0.1 0.07 0 0 0
[0272] The wounds treated with bFGF, however, looked extremely red
and also accumulated much more fluid than the other three groups.
Grossly, the wounds had a "hyper-vascularized" and inflamed
appearance. Histologic evaluation will reveal whether they actually
contained more infiltrating white blood cells than the other
groups.
Example 23
Reduction in Cerebral Infarct Volume in Mouse Stroke Model
[0273] Tests for the effects of zFGF5 on mice were done using the
middle cerebral artery occlusion (MCAO) model of stroke. C57BL6
mice were subjected to cerebral ischemia by a 1.0 h occlusion of
the anterior communicating and middle cerebral arteries, followed
by 24 h of reperfusion. Administration of vehicle or zFGF5 was by
one of two routes: intracerebrovascular (ICV) injection (0, 0.05,
0.5, or 5.0 ug per mouse) given 2 h prior to the onset of ischemia,
or by iv infusion (0, 50, 100, 200 ug/kg for 2h) starting 15 min
after the onset of ischemia.
[0274] Behavioral analysis was performed on each mouse 24 h after
the onset of ischemia, the animals were sacrificed after the 24 h
behavioral test, and infarct volume was measured by TTC staining
(Koketsu et al., Ann. Neurol. 35:451-457, 1994). Infarct volume in
mm.sup.3 was measured by computer-assisted videodensitometry in
seven sections taken rostral to caudal through the infarct.
Neurological deficits were graded from none (0) to most severe (3)
as follows: 0, no observable deficit; 1, failure to extend right
forepaw upon lifting of tail; 2, circling to the contralateral
side; 3, leaning to the contralateral side at rest or no
spontaneous motor activity.
[0275] For each animal, thermistor probes were inserted into the
rectum and temporalis muscle to monitor body and brain temperatures
was maintained at 37.degree. C. Mean arterial blood pressure and
arterial blood pH, pO2, pCO2 were analyzed 10 min before occlusion,
10 min after occlusion and 10 min after reperfusion. Regional
cerebral blood flow was measured by laser-Doppler flowmetry with a
fiber-optic probe inserted 2 mm posterior, 6 mm lateral to the
bregma on the ipsilateral hemisphere, the site supplied by the
proximal segment of the middle cerebral artery (MCA). Table 13
illustrates the effect of IV infusion of zFGF5 on infarct volume.
Table 14 illustrates the occlusion of the MCA reduced regional
blood flow in all groups.
14 TABLE 13 zFGF5 dose Infarct Volume .+-. SD .mu.g/kg/hour
mm.sup.3 0 88 .+-. 25 50 63 .+-. 31 100 52 .+-. 22* 200 39 .+-.
17** *P < 0.008, **p < 0.001
[0276]
15 TABLE 14 Cerebral blood flow (% of control) zFGF5 dose before
during during (.mu.g/kg/h) ischemia ischemia reperfusion 0 100 19
96 50 100 21 97 100 100 22 95 200 100 24 98
[0277] Administration of zFGF5 by ICV injection 2.0 h prior to the
onset of cerebral ischemia or by iv infusion starting 15 min after
ischemia, produced concentration-dependent reductions in infarct
volume. Compared to injection of vehicle, infarct volume was
reduced more than 50% by treatment with zFGF5 (p<0.003, n=8 mice
for each experimental condition). In addition to the reduction in
infarct volume, zFGF5 appeared to reduce ischemia-induced
neurological deficits.
[0278] There were no significant changes in mean arterial blood
pressure, pO.sub.2, pCO.sub.2, or pH in any of the groups under any
of the conditions tested. Compared with cerebral blood flow prior
to ischemia, regional blood flow during ischemia was reduced about
75% confirming that cerebral blood flow was reduced under these
conditions.
Example 23
Immunocytochemistry of zFGF5
[0279] Immunocytochemistry to detect zFGF5 protein was carried out
on human brain tissue. The subjects and tissues used were the
temporal cortex of a 70-year old male and an 83 year-old female,
the cerebrum of a 47 year-old female and brain tissue (no region
specified) from a 71 year-old male. All subjects died of
cerebrovascular accidents. In the uninvolved areas of the grey
matter, capillaries stained positive for zFGF5, but neurons were
negative for staining. In areas of old infarct, capillaries,
vascular endothelium, and macrophages stained positive for zFGF5
protein. Both the astrocytes surrounding the capillaries as well as
the endothelium were positively stained. Occasional microglial
cells were seen within the white matter that stained positive, as
well as subpial glial cells. Regions adjacent to the infarcted area
showed increased staining of the capillary endothelium, astrocytes,
and macrophages.
[0280] Normal cerebral cortical tissue from a 55 year-old female
who died of an aortic aneurysm was also examined for zFGF5
immunoreactivity. Sections of grey matter showed neurons and
astrocytes that were largely negative for staining with antibody.
Sections of white matter showed oligodendrocytes and astrocytes
that were negative for staining. Smooth muscle of cerebral vessels
and capillaries, in contrast, were strongly positive for staining
within both the grey and white matter.
[0281] Thus, in cerebrovascular accident cases, staining for zFGF5
was seen within vessels adjacent to the infarcted area, with
decreased staining of the infarct itself. Staining was increased in
areas of healing particularly within astrocytes surrounding
capillaries and the capillary endothelium itself. Macrophages and
microglial cells were also positively stained within the infarct
and the adjacent areas. Neurons in these regions were largely
negative for staining. In normal cerebral cortical tissue, smooth
muscle of cerebral vessels and capillaries were strongly
positive.
[0282] The pattern of detection of zFGF5 protein and mRNA within
the brain suggests that zFGF5 might play a role in control of brain
microvasuclature. In addition, zFGF5 may regulate communication
between neuronal and non-neuronal cell types within this tissue, or
may be upregulated in specific cell types, such as astrocytes and
macrophages, in response to injury. The apparent differences in
cellular localization between zFGF5 protein and zFGF5 mRNA suggests
either that zFGF5 is transported from its site of synthesis to
depot sites within the vicinity or that significant species
differences exist in the expression of the zFGF5 gene in the
central nervous system.
Example 24
Cartilage Repair in Goats
[0283] The effect of zFGF5 delivered in a slow degrading polymer
matrix on healing of a cartilage defect is preformed using a goat
cartilage defect model. The experiment will demonstrate whether the
polymer matrix also acts as a scaffold for cellular and matrix
ingrowth. The dosage of zFGF5 is 0, 0.5, 5, 50, or 500 ng/mm.sup.3
defect volume. Efficacy of the combined factor and slow release
polymer is assessed using gross and histological examination of the
repaired tissue in the cartilage defect site.
[0284] The following doses are administered:
[0285] Atrix Slow Release Polymer with Vehicle (0.0 .mu.gram zFGF5)
n=3
[0286] Atrix Slow Release Polymer with 0.3 .mu.gram zFGF5 n=3
[0287] Atrix Slow Release Polymer with 3.0 .mu.gram zFGF5 n=3
[0288] Atrix Slow Release Polymer with 30.0 .mu.gram zFGF5 n=3
[0289] Atrix Slow Release Polymer with 300.0 .mu.gram zFGF5 n=3
[0290] The control and test materials are supplied as a sterile
packaged implant containing the polymer and either vehicle or
premixed solution containing the appropriate zFGF5
concentration.
[0291] A total of 15 skeletally mature female Spanish goats will be
used for this study. They will be acquired from an approved, USDA
source. The goats will weigh between 50-80 lbs. at the start of the
study. The contralateral joint from all animals are not be operated
on and serve for baseline normal measurements.
[0292] Animal housing conditions conform with applicable laws and
regulations relating to laboratory animals, i.e., Animal Welfare
Act, Public Law 89-544 as amended in Public Law 99-198, Federal
Register 52:16, United States Department of Agriculture--Animal and
Plant Inspection Service (USDA-APHIS), 1985 and Public Health
Service Policy on Humane Care of Laboratory Animals, Office for
Protection Against Research Risks/National Institutes of Health
(OPRR/NIH), September, 1986. The animals are examined for any
evidence of disease or lameness, and are Q-Fever negative, T/B
negative, Brucellosis negative, CAE negative.
[0293] The goats are maintained in large indoor runs (pens) or in
outdoor runs for several days following surgery, and when
appropriate, the goats are moved to a large outdoor (fenced)
paddock area for the remainder of the study. The goats have
unrestricted activity at all times. The goats are fed a mixture of
baled grass hay and alfalfa cubes. Tap water is provided ad
libitum. However, feed is withheld 24 hours prior to surgery.
[0294] Bodyweight measurements are taken from all animals once
during the quarantine period, prior to surgery (Day 0) and at the
end of the study (Day 56). Blood (approximately 5 ml) is taken from
all animals prior to surgery (Day 0) and at the end of the study
(Day 56). The serum is harvested, placed in appropriately labeled
screw top freezer tubes, and stored at -20.degree. C.
[0295] The basic surgical procedure is identical for all subjects.
All surgeries are performed under strict asepsis. Peri-operative
antibiotics are dosed IM at 2.4 million units of Bicillin.RTM. at
the beginning of the procedure. Anesthesia is induced with a
mixture of ketamine-xylazine, the subject intubated, and anesthesia
maintained with a gaseous mixture of Isoflurane and oxygen.
[0296] A lateral or medial trochlear defect is created in the right
femoral condyle. The defects are approximately 6 mm in diameter and
approximately 2.5 mm in depth, and pass into the subchondral bone.
The defects are made on either the lateral or medial wall of the
distal trochlear sulcus dependent on individual anatomy. Following
creation of the defect, the wall may be undercut to provide for a
mechanical interlock with the polymer matrix. Each defect is then
filled with the appropriate test article.
[0297] The surgical approach consists of a curved, lateral skin
incision made from the distal one-third of the left femur to the
level of the tibial plateau and across to the medial side of the
tibial spine. Using this method, the skin is bluntly dissected and
retracted to allow a lateral parapatellar approach into the stifle
joint. An incision is made parallel to the lateral border of the
patella and patellar ligament. This extends from the lateral side
of the fascia lata along the cranial border of the biceps femoris
and into the lateral fascia of the stifle joint. The biceps femoris
and attached lateral fascia are retracted allowing an incision into
the joint capsule. The joint is extended and the patella luxated
medially exposing the stifle joint.
[0298] With the knee joint fully flexed, the appropriate location
for the point of drilling the defect on the trochlear sulcus is
identified and marked with a surgical marker. A specially designed
cartilage cutter is used to slice through the cartilage outer layer
and prevent tearing of the cartilage. The approximate 6 mm diameter
core cutter is used under power to create a fixed depth of
approximately 2.5 mm, maintaining a plane perpendicular to the
tangent of the sulcus. The core of subchondral bone and cartilage
is carefully removed.
[0299] The cartilage defect is copiously flushed with sterile
saline and then dried prior to insertion of the test article. The
polymer materials will set-up in situ once it comes into contact
with fluid. The appropriate test material is then injected into the
defect until the surface is flush with the surrounding cartilage.
Buffer is flooded over the surface of the polymer and the polymer
is allowed to set for 5 minutes prior to carefully flushing the
joint. The patella is then reduced and the joint moved through a
complete range-of-motion to ensure that there is no impingement due
to the implant. This is followed by routine closure of the joint in
three or four layers using appropriate suture material. Post
operative checks are made for any animal displaying signs of post
operative discomfort. Post operative analgesics will be given if
the animals display any signs of distress of discomfort. All
treatments will be recorded in the appropriate study
documentation.
[0300] All remaining animals are humanely sacrificed at Day 56 (8
weeks) postoperatively. Bodyweights are recorded immediately prior
to sacrifice. Gross evaluation is performed on the heart, lungs,
liver, spleen, kidneys, and popliteal and superficial inguinal
lymph nodes for signs of any systemic toxicity from the implant
material. Lymph nodes in close proximity to the joint are examined.
The articulating surfaces opposing the defect sites are examined
for any abnormal joint surface. Additionally, gross evaluations of
the knee joints are made to determine the cartilage repair based on
previous scoring criteria listed in Table 15. Femora, patellae,
synovium, and popliteal lymph nodes are harvested and placed into
appropriately labeled containers. Immediately following tissue
harvest, gross morphological examination of the cartilage surface
is done and photographic records made of each specimen.
[0301] After collection of the knee joints, the joints will be
opened, photographed and the surface of the defect site scored as
indicated in Table 15 and with standardized grading sheets. The
synovial membrane will be examined for any inflammation.
16TABLE 15 Scoring Criteria for Gross Morphological Evaluations
characteristic grading score Edge Integration Full 2 (new tissue
relative Partial 1 to native cartilage) None 0 Smoothness of the
Smooth 2 cartilage surface Intermediate 1 Rough 0 Cartilage
surface, Flush 2 degree of filling Slight depression 1
Depressed/overgrown 0 Color of cartilage, Transparent 2 opacity or
translucency Translucent 1 of the neocartilage Opaque 0
[0302] Immediately after dissection and following gross joint
surface observations, the joints are placed in 10% phosphate
buffered formalin (at least ten-fold volume) for at least 48 hours
and histologically processed. After fixation in 10% phosphate
buffered formalin, the specimens are grossly trimmed to remove
extra tissue. The tissue blocks are cut approximately 1/3 of the
distance in from the exterior implant/tissue interface in order to
examine them grossly. Contact radiographs are taken prior to the
commencement of decalcification.
[0303] The tissues are decalcified in 10% EDTA until radiographs of
the decalcified sections assures complete decalcification. Once
complete decalcification is determined, the specimens are
dehydrated through an ethanol series and paraffin embedded. The
specimens are sectioned to 5-10 .mu.m. One section is stained with
H&E and another sequential section with Safranin O
counter-stained with Fast Green. For histologic analysis of the
sections, the scoring scale shown in Table 16 is used.
17TABLE 16 characteristic grading score I. Nature of predominant
hyaline cartilage 4 tissue mostly hyaline cartilage 3 mixed hyaline
and 2 fibrocartilage mostly fibrocartilage 1 some fibrocartilage,
mostly 0 nonchondrocytic cells II. Structural smooth and intact 3
characteristics A. Surface regularity superficial horizontal 2
lamination fissures 1 severe disruption, including 0 fibrillation
B. Structural integrity normal 2 slight disruption, including 1
cysts severe disintegration 0 C. Thickness 100% of normal adjacent
2 cartilage 50-100% of normal cartilage 1 0-50% of normal cartilage
0 D. Bonding to adjacent Bonded at both ends of graft 2 cartilage
Bonded at one end or 1 partially at both ends Not bonded 0 III.
Freedom from cellular normal cellularity 2 changes of degeneration
slight hypocellularity 1 A. Hypocellularity moderate
hypocellularity or 0 hypercellularity B. Chondrocyte clustering No
clusters 2 <25% of the cells 1 25-100% of the cells 0 IV.
Freedom from degenera- Normal cellularity, no 3 tive changes in
adjacent clusters, normal staining cartilage Normal cellularity,
mild 2 clusters, moderate staining Mild or moderate hypocellu- 1
larity, slight staining Severe hypocellularity, 0 poor or no
staining V. A. Reconstitution of normal 3 subchondral bone reduced
2 minimal 1 none 0 B. Inflammatory response none/mild 2 in
subchondral bone moderate 1 severe 0 VI. Safranin-O staining normal
3 moderate 2 slight 1 none 0
[0304] Histologic Scoring Scale is modified for scoring the "Nature
of predominant tissue", Section I of the scale. In the 16 week goat
study, if the tissue is scored as "4=hyaline cartilage" it
essentially consists of only hyaline cartilage, no trace of
fibrocartilage. Scoring the nature of the repair tissue as
"3=mostly hyaline cartilage" is given to sections which have some
trace of fibrocartilage, but less than 25% as determined visually.
A score of "2=mixed hyaline and fibrocartilage" is given to repair
tissue which has both hyaline and fibrous tissue, varying from
approximately 75% hyaline/25% fibrous to 25% hyaline/75% fibrous. A
score of "1=mostly fibrocartilage" is given to repair tissue which
show some traces (less than 25%) of hyaline, but is primarily
fibrous in nature. A score of "0=some fibrocartilage, mostly
non-chondrocytic" is given to repair tissue which does not exhibit
any hyaline tissue at all.
[0305] A total of 39 adult female goats were used and were divided
into thirteen groups of three goats each. A full thickness
cartilage lesion (6.25 mm wide.times.2.5 mm deep) was created in
the distal femoral trochlear sulcus of each goat. zFGF5 (0, 0.04,
0.4, 4.0 or 40.0 ug) was delivered directly into the defects either
alone or suspended in a bio-degradable fast release (degradation
over 1-2 weeks) or slow release (degradation over 2-4 weeks)
poly(lactide-co-glycolide) matrix that solidified in situ
(Atrigel). Eight weeks after treatment, the defect sites were
scored for gross morphology and harvested. Sections were taken
through the center of the lesions and repair of subcondral bone was
evaluated by contact radiography and by staining of adjacent
sections with H&E. Formation of chondral tissue was evaluated
by staining of adjacent sections with safranin-O. Degeneration of
adjacent articular cartilage was assessed microscopically as
decreased chondrocyte cell density and loss of safranin-O staining
within cartilage adjacent to the lesions. Sections were scored in
two ways: initially they were scored in a blinded fashion using the
a semi-quantitative scoring scale (Frenkel S R et al., J Bone Joint
Surg Br 1997, 79: 831-6); this was followed with an unblinded
qualitative comparative analysis of slides fromeach group.
[0306] All surgeries were uneventful and all animals recovered
without incident. The repair tissue at the lesion surface was
concave in a few specimens or had a central zone that was concave.
The repair tissue at the periphery of the lesion was often level or
nearly level with the surrounding cartilage and often appeared
articular cartilage-like. Fine blood spots were sometimes observed
on the surface of the repair tissue and occasionally on the
opposing fat pad surface suggesting a vascular supply and/or
connection was present. As a group there was slightly more synovial
fluid in operated knees, 0.25 ml (range 0.03-0.6 ml) compared to
contralateral control knees, 0.17 ml (range 0.05-0.45 ml) but the
viscosity was normal. Delivery of zFGF5 produced little or no
excess loss of bone or cartilage around the defect site. In
addition, little or no inflammatory response was seen at the defect
site in any of the groups indicating good biocompatibility of the
delivery matrix and the recombinant human zFGF5. No synovitis was
apparent with any dose of zFGF5 and the synovial fluid had normal
viscosity in all groups. A mild osteophytosis along the medial or
lateral aspect of the patellar groove was observed in some of the
animals treated with the highest dose (40 ug) of zFGF5. Relative to
defects treated with vehicle or slow release matrix alone, zFGF5
produced dose-dependent increases in gross morphology scores that
were maximal between 0.4 and 4.0 ug of zFGF5. Little or no
consistent effects were observed, in contrast, with zFGF5 delivered
in the fast-release matrix. Relative to lesions treated with
vehicle or either polymer matrix alone, the highest scores for
reconstitution of subchondral bone were observed with the 4.0 ug
and 0.04-4.0 ug doses of zFGF5 administered either in vehicle alone
or in the slow release matrix, respectively. Animals treated with
either the slow release matrix alone or with slow release matrix
containing 0.4 or 4.0 ug doses of zFGF5 also exhibited the least
degenerative changes in cartilage adjacent to the lesions. ZFGF5
had little or no consistent effects on subchondral bone repair when
delivered in the fast release matrix. In addition, these animals
also exhibited the most degenerative changes in adjacent cartilage.
Histologic analysis revealed that with vehicle or either polymer
alone, the defects were composed of mainly fibrous fill. Modest
amounts of safranin-O-stained chondral tissue was occasionally seen
at the edge of the defects with a small amount of lamellar bone
formation at the base and endochondral bone formation at the edges
of the defect. A modest increase in histologic score was seen with
dose of zFGF5 delivered in vehicle alone. Semi-quantitative
analysis of the H&E and safranin-O stained sections revealed
that the highest overall histologic scores were observed with 0.4
and 4.0 ug doses of zFGF5 delivered in the slow release matrix. In
these animals, the size of the lesions appeared to decrease and the
relative amount of safranin-O-stained chondral tissue appeared to
increase with dose of zFGF5. Chondrocytes could be seen embedded in
the extracellular matrix within the repair tissue. zFGF5 had little
or no consistent effects on histologic scores when delivered in the
fast release matrix. In general, these results were confirmed in
the qualitative group assessment. These data demonstrate that
zFGF5, delivered in a bio-degradable poly(lactide-co-glycolide)
Atrigel matrix, can increase the formation of chondral tissue with
ongoing endochondral and lamellar bone formation in a large animal
model of full-thickness articular cartilage defects.
EXAMPLE 25
Partial Thickness Injury Repair in Goats
[0307] The effect of zFGF5 delivered by intra-articular injection
on healing of a partial thickness cartilage defect is preformed
using a goat cartilage defect model. The dosage of zFGF5 is either
0 (carrier alone=0.5% hyaluronic acid) or 100 ug zFGF5 (in 0.5%
hyaluronan) injected into the knee joint space twice per week for
three weeks. Efficacy of zFGF5 is assessed using gross and
histological examination of the repaired tissue in the cartilage
defect site.
[0308] The following doses are administered:
18 0.5% hyaluronic acid (0.0 .mu.gram zFGF5) n = 2 0.5% hyaluronic
acid with 100 .mu.gram zFGF5 n = 2
[0309] The control and test materials are supplied as a sterile
packaged solution containing the hyaluronic acid alone or
hyaluronic acid containing the appropriate concentration of
zFGF5.
[0310] A total of 4 skeletally mature female Spanish goats will be
used for this study. They will be acquired from an approved, USDA
source. The goats will weigh between 50-80 lbs. at the start of the
study. The contralateral joint from all animals are not be operated
on and serve for baseline normal measurements.
[0311] Animal housing conditions conform with applicable laws and
regulations relating to laboratory animals, i.e., Animal Welfare
Act, Public Law 89-544 as amended in Public Law 99-198, Federal
Register 52:16, United States Department of Agriculture--Animal and
Plant Inspection Service (USDA-APHIS), 1985 and Public Health
Service Policy on Humane Care of Laboratory Animals, Office for
Protection Against Research Risks/National Institutes of Health
(OPRR/NIH), September, 1986. The animals are examined for any
evidence of disease or lameness, and are Q-Fever negative, T/B
negative, Brucellosis negative, CAE negative.
[0312] The goats are maintained in large indoor runs (pens) or in
outdoor runs for several days following surgery. The goats have
unrestricted activity at all times. The goats are fed a mixture of
baled grass hay and alfalfa cubes. Tap water is provided ad
libitum. However, feed is withheld 24 hours prior to surgery.
[0313] Bodyweight measurements are taken from all animals once
during the quarantine period, prior to surgery (Day 0) and at the
end of the study (Day 21).
[0314] The basic surgical procedure is identical for all subjects.
All surgeries are performed under strict asepsis. Peri-operative
antibiotics are dosed IM at 2.4 million units of Bicillin.RTM. at
the beginning of the procedure. Anesthesia is induced with a
mixture of ketamine-xylazine, the subject intubated, and anesthesia
maintained with a gaseous mixture of Isoflurane and oxygen.
[0315] Defect Creation Procedure (Day 0): The surgical approach
will consist of a curved, lateral skin incision made from the
distal one-third of the left femur to the level of the tibial
plateau and across to the medial side of the tibial spine. Using
this method, the skin is bluntly dissected and retracted to allow a
lateral parapatellar approach into the stifle joint. An incision is
made parallel to the lateral border of the patella and patellar
ligament, This extends from the lateral side of the fascia lata
along the cranial border of the biceps femoris and into the lateral
fascia of the stifle joint. The biceps femoris and attached lateral
fascia are retracted allowing an incision into the joint capsule.
The joint is extended and the patella luxated medially exposing the
stifle joint. The harvest site will be the same as the location of
the planned trochlear defect which will be created in the right
femoral condyle. The defects will be made on either the lateral or
medial wall of the distal trochlear sulcus dependent on individual
anatomy. The cartilage defects will be approximately 3 mm in
diameter and approximately 0.5 mm in depth. Immediately after its
creation, the defect will be treated by dripping the test solution
into the defect site. After 1 min, the wound site will be closed in
layers using appropriate suture and patterns. The remainder of the
1.0 ml of test solution will be injected into the joint.
Thereafter, the test articles will be administered to the animals
twice per week for three weeks by intra-articular injection of 1.0
ml.
[0316] All remaining animals are humanely sacrificed at Day 21 (3
weeks) postoperatively. Bodyweights are recorded immediately prior
to sacrifice. Gross evaluation is performed on the left and right
popliteal lymph nodes and both knees. The articulating surfaces
opposing the defect sites are examined for any abnormal joint
surface. Additionally, gross evaluations of the knee joints are
made to determine the cartilage repair. Femora, patellae, synovium,
and popliteal lymph nodes are harvested and placed into
appropriately labeled containers. Immediately following tissue
harvest, gross morphological examination of the cartilage surface
is done and photographic records made of each specimen.
[0317] After collection of the knee joints, the joints will be
opened, photographed and the surface of the defect site visually
assessed. The synovial membrane will be examined for any
inflammation.
[0318] Immediately after dissection and following gross joint
surface observations, the joints are placed in 10% phosphate
buffered formalin (at least ten-fold volume) for at least 48 hours
and histologically processed. After fixation in 10% phosphate
buffered formalin, the specimens are grossly trimmed to remove
extra tissue. The tissue blocks are cut approximately 1/3 of the
distance in from the exterior implant/tissue interface in order to
examine them grossly.
[0319] The tissues are decalcified in 10% EDTA until radiographs of
the decalcified sections assures complete decalcification. Once
complete decalcification is determined, the specimens are
dehydrated through an ethanol series and paraffin embedded. The
specimens are sectioned to 5-10 .mu.m. One section is stained with
H&E and another sequential section with toluidine blue. The
sections are examined microscopically for evidence of chondrocyte
cloning, a measure of chondrocyte proliferation, and measured for
cartilage thickness with the aid of a micrometer. The sections are
assessed visually for proteoglycan accumulation by the intensity of
toluidine blue staining.
[0320] Goat defects treated with vehicle alone had decreased
toluidine blue staining in the cartilage surrounding the cartilage
defect especially in the superficial layer and the upper one-third
of the cartilage surface. Fragments of acellular or poorly cellular
collagenous matrix, devoid of proteoglycan were present at the
edges of the defect. There was no evidence of repair tissue in the
defect. The marginal zone and periosteum has an approximately 330
um thick layer of fibroblast proliferation with metachromatic
stained matrix deposition and irregular periosteal new bone
trabeculae lined with osteoblasts extended from the cortical bone.
Small areas of synovium had fibroplasias and mild mononuclear
inflammatory cell infiltration were also observed. Thus, little
repair of cartilage with little or no periosteal bone formation was
observed in the patellar groove of the vehicle treated goats.
[0321] Animals treated with vehicle containing 100 ug of zFGF5 had
little or no decreased toluidine blue staining in the cartilage
surrounding the defect suggesting that proteoglycan synthesis was
maintained by zFGF5. A marked cloning response was present in the
deep zone of the lesion and individual chondrocytes had increased
toluidine blue staining around the cells, indicative of increased
proteoglycan synthesis. In addition, chondrocytes in the
surrounding matrix had cloning responses. Taken together, these
data suggest that zFGF5 increased chondrocyte proliferation in the
lesion sites and in the matrix surrounding the defects. These
changes in matrix accumulation and chondrocyte proliferation
resulted in increased thickness of the cartilage adjacent to the
lesion sites. Cartilage thickness in the patellar groove increased
from 322 um to 385 um in goats treated with vehicle or zFGF5,
respectively. In contrast, cartilage thickness in the contralateral
untreated patellar groove was similar in the two groups of goats at
330 um and 345 um for the vehicle and zFGF5 treated animals,
respectively. Thus, goats treated with zFGF5 had increased
cartilage thickness in the patellar groove.
[0322] The marginal zone and periosteum from the zFGF5 treated
animals had an approximately 350 um thick layer of fibroblast
proliferation with metachromatic stained matrix deposition and an
outer layer of mononuclear inflammatory cells. Subjacent to this
layer was periosteal new bone formation approximately 600 um thick.
These data demonstrate that zFGF5 increased synthesis of bone in
the periosteum of the goats. The marginal zones on both sides of
the patella also showed enhanced chondrogenesis and fibrous tissue
proliferation in animals injected with zFGF5. These results show
that intra-articular injection of zFGF5 can increase cartilage
proliferation and matrix deposition within and around partial
thickness cartilage lesions in the non-load bearing region of the
patellar groove of goats.
[0323] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
43 1 917 DNA Homo sapiens CDS (1)...(621) 1 atg tat tca gcg ccc tcc
gcc tgc act tgc ctg tgt tta cac ttc ctg 48 Met Tyr Ser Ala Pro Ser
Ala Cys Thr Cys Leu Cys Leu His Phe Leu 1 5 10 15 ctg ctg tgc ttc
cag gta cag gtg ctg gtt gcc gag gag aac gtg gac 96 Leu Leu Cys Phe
Gln Val Gln Val Leu Val Ala Glu Glu Asn Val Asp 20 25 30 ttc cgc
atc cac gtg gag aac cag acg cgg gct cgg gac gat gtg agc 144 Phe Arg
Ile His Val Glu Asn Gln Thr Arg Ala Arg Asp Asp Val Ser 35 40 45
cgt aag cag ctg cgg ctg tac cag ctc tac agc cgg acc agt ggg aaa 192
Arg Lys Gln Leu Arg Leu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys 50
55 60 cac atc cag gtc ctg ggc cgc agg atc agt gcc cgc ggc gag gat
ggg 240 His Ile Gln Val Leu Gly Arg Arg Ile Ser Ala Arg Gly Glu Asp
Gly 65 70 75 80 gac aag tat gcc cag ctc cta gtg gag aca gac acc ttc
ggt agt caa 288 Asp Lys Tyr Ala Gln Leu Leu Val Glu Thr Asp Thr Phe
Gly Ser Gln 85 90 95 gtc cgg atc aag ggc aag gag acg gaa ttc tac
ctg tgc atg aac cgc 336 Val Arg Ile Lys Gly Lys Glu Thr Glu Phe Tyr
Leu Cys Met Asn Arg 100 105 110 aaa ggc aag ctc gtg ggg aag ccc gat
ggc acc agc aag gag tgt gtg 384 Lys Gly Lys Leu Val Gly Lys Pro Asp
Gly Thr Ser Lys Glu Cys Val 115 120 125 ttc atc gag aag gtt ctg gag
aac aac tac acg gcc ctg atg tcg gct 432 Phe Ile Glu Lys Val Leu Glu
Asn Asn Tyr Thr Ala Leu Met Ser Ala 130 135 140 aag tac tcc ggc tgg
tac gtg ggc ttc acc aag aag ggg cgg ccg cgg 480 Lys Tyr Ser Gly Trp
Tyr Val Gly Phe Thr Lys Lys Gly Arg Pro Arg 145 150 155 160 aag ggc
ccc aag acc cgg gag aac cag cag gac gtg cat ttc atg aag 528 Lys Gly
Pro Lys Thr Arg Glu Asn Gln Gln Asp Val His Phe Met Lys 165 170 175
cgc tac ccc aag ggg cag ccg gag ctt cag aag ccc ttc aag tac acg 576
Arg Tyr Pro Lys Gly Gln Pro Glu Leu Gln Lys Pro Phe Lys Tyr Thr 180
185 190 acg gtg acc aag agg tcc cgt cgg atc cgg ccc aca cac cct gcc
621 Thr Val Thr Lys Arg Ser Arg Arg Ile Arg Pro Thr His Pro Ala 195
200 205 taggccaccc cgccgcggcc ctcaggtcgc cctggccaca ctcacactcc
cagaaaactg 681 catcagagga atatttttac atgaaaaata aggattttat
tgttgacttg aaacccccga 741 tgacaaaaga ctcacgcaaa gggactgtag
tcaacccaca ggtgcttgtc tctctctagg 801 aacagacaac tctaaactcg
tccccagagg aggacttgaa tgaggaaacc aacactttga 861 gaaaccaaag
tcctttttcc caaaggttct gaaaaaaaaa aaaaaaaaaa ctcgag 917 2 207 PRT
Homo sapiens 2 Met Tyr Ser Ala Pro Ser Ala Cys Thr Cys Leu Cys Leu
His Phe Leu 1 5 10 15 Leu Leu Cys Phe Gln Val Gln Val Leu Val Ala
Glu Glu Asn Val Asp 20 25 30 Phe Arg Ile His Val Glu Asn Gln Thr
Arg Ala Arg Asp Asp Val Ser 35 40 45 Arg Lys Gln Leu Arg Leu Tyr
Gln Leu Tyr Ser Arg Thr Ser Gly Lys 50 55 60 His Ile Gln Val Leu
Gly Arg Arg Ile Ser Ala Arg Gly Glu Asp Gly 65 70 75 80 Asp Lys Tyr
Ala Gln Leu Leu Val Glu Thr Asp Thr Phe Gly Ser Gln 85 90 95 Val
Arg Ile Lys Gly Lys Glu Thr Glu Phe Tyr Leu Cys Met Asn Arg 100 105
110 Lys Gly Lys Leu Val Gly Lys Pro Asp Gly Thr Ser Lys Glu Cys Val
115 120 125 Phe Ile Glu Lys Val Leu Glu Asn Asn Tyr Thr Ala Leu Met
Ser Ala 130 135 140 Lys Tyr Ser Gly Trp Tyr Val Gly Phe Thr Lys Lys
Gly Arg Pro Arg 145 150 155 160 Lys Gly Pro Lys Thr Arg Glu Asn Gln
Gln Asp Val His Phe Met Lys 165 170 175 Arg Tyr Pro Lys Gly Gln Pro
Glu Leu Gln Lys Pro Phe Lys Tyr Thr 180 185 190 Thr Val Thr Lys Arg
Ser Arg Arg Ile Arg Pro Thr His Pro Ala 195 200 205 3 24 DNA
Artificial Sequence oligonucleotide primer ZC11676 3 ggacttgact
accgaaggtg tctg 24 4 23 DNA Artificial Sequence oligonucleotide
primer ZC11677 4 gtcgatgtga gccgtaagca gct 23 5 26 DNA Artificial
Sequence oligonucleotide primer ZC12053 5 gcatacttgt ccccatcctc
gccgcg 26 6 621 DNA Artificial Sequence degenerate sequence 6
atgtaywsng cnccnwsngc ntgyacntgy ytntgyytnc ayttyytnyt nytntgytty
60 cargtncarg tnytngtngc ngargaraay gtngayttym gnathgaygt
ngaraarcar 120 acnmgngcnm gngaygaygt nwsnmgnaar carytnmgny
tntaycaryt ntaywsnmgn 180 acnwsnggna arcayathca rgtnytnggn
mgnmgnathw sngcnmgngg ngargayggn 240 gayaartayg cncarytnyt
ngtngaracn gayacnttyg gnwsncargt nmgnathaar 300 ggnaargara
cngarttyta yytntgyatg aaymgnaarg gnaarytngt nggnaarccn 360
gayggnacnw snaargartg ygtnttyath garaargtny tngaraayaa ytayacngcn
420 ytnatgwsng cnaartayws nggntggtay gtnggnttya cnaaraargg
nmgnccnmgn 480 aarggnccna aracnmgnga raaycarcar gaygtncayt
tyatgaarmg ntayccnaar 540 ggncarccng arytncaraa rccnttyaar
tayacnacng tnacnaarmg nwsnmgnmgn 600 athmgnccna cncayccngc n 621 7
47 DNA Artificial Sequence oligonucleotide primer ZC12652 7
tatttatcta gactggttcc gcgtgccgcc gaggagaacg tggactt 47 8 33 DNA
Artificial Sequence oligonucleotide primer ZC12631 8 gtatttgtcg
actcaggcag ggtgtgtggg ccg 33 9 22 DNA Artificial Sequence
oligonucleotide primer ZC15290 9 gccgaggaga acgtggactt cc 22 10 47
DNA Artificial Sequence oligonucleotide primer ZC15270 10
tatttatcta gagatgacga tgacaaggcc gaggagaacg tggactt 47 11 41 DNA
Artificial Sequence oligonucleotide primer ZC13497 11 agcattgcta
aagaagaagg tgtaagcttg gacaagagag a 41 12 63 DNA Artificial Sequence
oligonucleotide primer ZC15131 12 ggtgtaagct tggacaagag agaggagaac
gtggacttcc gcatccacgt ggagaaccag 60 acg 63 13 39 DNA Artificial
Sequence oligonucleotide primer ZC15134 13 ccggctgtag agctggtaca
gccgcagctg cttacggct 39 14 42 DNA Artificial Sequence
oligonucleotide primer ZC13529 14 cttcagaagc ccttcaagta cacgacggtg
accaagaggt cc 42 15 61 DNA Artificial Sequence oligonucleotide
primer ZC13525 15 acgacggtga ccaagaggtc ccgtcggatc cggcccacac
accctgccta gggggaattc 60 g 61 16 61 DNA Artificial Sequence
oligonucleotide primer ZC13526 16 caaacaggca gccctagaat actagtgtcg
actcgaggat ccgaattccc cctaggcagg 60 g 61 17 44 DNA Artificial
Sequence oligonucleotide primer ZC13528 17 ctcaaaaatt ataaaaatat
ccaaacaggc agccctagaa tact 44 18 186 DNA Artificial Sequence
oligonucleotide primer ZC15132 18 gtaccgcgag cagttcccgt caatccctcc
ccccttacac aggatgtcca tattaggaca 60 tctgcgtctc gaggccaccg
tggttgagcc cgacactcat tcataaaacg cttgttataa 120 aagcagtggc
tgcggcgcct cgtactccaa ccgcatctgc agcgagcaac tgagaagcca 180 aggatc
186 19 141 DNA Artificial Sequence 5' linker sequence 19 agcattgctg
ctaaagaaga aggtgtaagc ttggacaaga gagaggagaa cgtggacttc 60
cgcatccacg tggagaacca gacgcgggct cgggacgatg tgagccgtaa gcagctgcgg
120 ctgtaccagc tctacagccg g 141 20 144 DNA Artificial Sequence 3'
linker sequence 20 cttcagaagc ccttcaagta cacgacggtg accaagaggt
cccgtcggat ccggcccaca 60 caccctgcct agggggaatt cggatcctcg
agtcgacact agtattctag ggctgcctgt 120 ttggatattt ttataatttt tgag 144
21 243 PRT Homo sapiens 21 Met Ala Ala Ala Ile Ala Ser Ser Leu Ile
Arg Gln Lys Arg Gln Ala 1 5 10 15 Arg Glu Ser Asn Ser Asp Arg Val
Ser Ala Ser Lys Arg Arg Ser Ser 20 25 30 Pro Ser Lys Asp Gly Arg
Ser Leu Cys Glu Arg His Val Leu Gly Val 35 40 45 Phe Ser Lys Val
Arg Phe Cys Ser Gly Arg Lys Arg Pro Val Arg Arg 50 55 60 Arg Pro
Glu Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Phe Ser Gln 65 70 75 80
Gln Gly Tyr Phe Leu Gln Met His Pro Asp Gly Thr Ile Asp Gly Thr 85
90 95 Lys Asp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu Ile Pro Val
Gly 100 105 110 Leu Arg Val Val Ala Ile Gln Gly Val Lys Ala Ser Leu
Tyr Val Ala 115 120 125 Met Asn Gly Glu Gly Tyr Leu Tyr Ser Ser Asp
Val Phe Thr Pro Glu 130 135 140 Cys Lys Phe Lys Glu Ser Val Phe Glu
Asn Tyr Tyr Val Ile Tyr Ser 145 150 155 160 Ser Thr Leu Tyr Arg Gln
Gln Glu Ser Gly Arg Ala Trp Phe Leu Gly 165 170 175 Leu Asn Lys Glu
Gly Gln Ile Met Lys Gly Asn Arg Val Lys Lys Thr 180 185 190 Lys Pro
Ser Ser His Phe Val Pro Lys Pro Ile Glu Val Cys Met Tyr 195 200 205
Arg Glu Pro Ser Leu His Glu Ile Gly Glu Lys Gln Gly Arg Ser Arg 210
215 220 Lys Ser Ser Gly Thr Pro Thr Met Asn Gly Gly Lys Val Val Asn
Gln 225 230 235 240 Asp Ser Thr 22 168 PRT Homo sapiens 22 Met Ala
Ser Lys Glu Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Phe 1 5 10 15
Ser Gln Gln Gly Tyr Phe Leu Gln Met His Pro Asp Gly Thr Ile Asp 20
25 30 Gly Thr Lys Asp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu Ile
Pro 35 40 45 Val Gly Leu Arg Val Val Ala Ile Gln Gly Val Lys Ala
Ser Leu Tyr 50 55 60 Val Ala Met Asn Gly Glu Gly Tyr Leu Tyr Ser
Ser Asp Val Phe Thr 65 70 75 80 Pro Glu Cys Lys Phe Lys Glu Ser Val
Phe Glu Asn Tyr Tyr Val Ile 85 90 95 Tyr Ser Ser Thr Leu Tyr Arg
Gln Gln Glu Ser Gly Arg Ala Trp Phe 100 105 110 Leu Gly Leu Asn Lys
Glu Gly Gln Ile Met Lys Gly Asn Arg Val Glu 115 120 125 Lys Thr Lys
Pro Ser Ser His Phe Val Pro Lys Pro Ile Glu Val Cys 130 135 140 Met
Tyr Arg Glu Pro Ser Leu His Glu Ile Gly Glu Asn Lys Gly Val 145 150
155 160 Gln Gly Lys Phe Trp Thr Pro Pro 165 23 247 PRT Homo sapiens
23 Met Ala Ala Ala Ile Ala Ser Gly Leu Ile Arg Gln Lys Arg Gln Ala
1 5 10 15 Arg Glu Gln His Trp Asp Arg Pro Ser Ala Ser Arg Arg Arg
Ser Ser 20 25 30 Pro Ser Lys Asn Arg Gly Leu Cys Asn Gly Asn Leu
Val Asp Ile Phe 35 40 45 Ser Lys Val Arg Ile Phe Gly Leu Lys Lys
Arg Arg Leu Arg Arg Gln 50 55 60 Asp Pro Gln Leu Lys Gly Ile Val
Thr Arg Leu Tyr Cys Arg Gln Gly 65 70 75 80 Tyr Tyr Leu Gln Met His
Pro Asp Gly Ala Leu Asp Gly Thr Lys Asp 85 90 95 Asp Ser Thr Asn
Ser Thr Leu Phe Asn Leu Ile Pro Val Gly Leu Arg 100 105 110 Val Val
Ala Ile Gln Gly Val Lys Thr Gly Leu Tyr Ile Ala Met Asn 115 120 125
Gly Glu Gly Tyr Leu Tyr Pro Ser Glu Leu Phe Thr Pro Glu Cys Lys 130
135 140 Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser Ser
Met 145 150 155 160 Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe
Leu Gly Leu Asn 165 170 175 Lys Glu Gly Gln Ala Met Lys Gly Asn Arg
Val Lys Lys Thr Lys Pro 180 185 190 Ala Ala His Phe Leu Pro Lys Pro
Leu Glu Val Ala Met Tyr Arg Glu 195 200 205 Pro Ser Leu His Asp Val
Gly Glu Thr Val Pro Lys Pro Gly Val Thr 210 215 220 Pro Ser Lys Ser
Thr Ser Ala Ser Ala Ile Met Asn Gly Gly Lys Pro 225 230 235 240 Val
Asn Lys Ser Lys Thr Thr 245 24 245 PRT Homo sapiens 24 Met Ala Ala
Ala Ile Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln Ala 1 5 10 15 Arg
Glu Arg Glu Lys Ser Asn Ala Cys Lys Cys Val Ser Ser Pro Ser 20 25
30 Lys Gly Lys Thr Ser Cys Asp Lys Asn Lys Leu Asn Val Phe Ser Arg
35 40 45 Val Lys Leu Phe Gly Ser Lys Lys Arg Arg Arg Arg Arg Pro
Glu Pro 50 55 60 Gln Leu Lys Gly Ile Val Thr Lys Leu Tyr Ser Arg
Gln Gly Tyr His 65 70 75 80 Leu Gln Leu Gln Ala Asp Gly Thr Ile Asp
Gly Thr Lys Asp Glu Asp 85 90 95 Ser Thr Tyr Thr Leu Phe Asn Leu
Ile Pro Val Gly Leu Arg Val Val 100 105 110 Ala Ile Gln Gly Val Gln
Thr Lys Leu Tyr Leu Ala Met Asn Ser Glu 115 120 125 Gly Tyr Leu Tyr
Thr Ser Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys 130 135 140 Glu Ser
Val Phe Glu Asn Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr 145 150 155
160 Arg Gln Gln Gln Ser Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu
165 170 175 Gly Glu Ile Met Lys Gly Asn His Val Lys Lys Asn Lys Pro
Ala Ala 180 185 190 His Phe Leu Pro Lys Pro Leu Lys Val Ala Met Tyr
Lys Glu Pro Ser 195 200 205 Leu His Asp Leu Thr Glu Phe Ser Arg Ser
Gly Ser Gly Thr Pro Thr 210 215 220 Lys Ser Arg Ser Val Ser Gly Val
Leu Asn Gly Gly Lys Ser Met Ser 225 230 235 240 His Asn Glu Ser Thr
245 25 225 PRT Homo sapiens 25 Met Ala Ala Leu Ala Ser Ser Leu Ile
Arg Gln Lys Arg Glu Val Arg 1 5 10 15 Glu Pro Gly Gly Ser Arg Pro
Val Ser Ala Gln Arg Arg Val Cys Pro 20 25 30 Arg Gly Thr Lys Ser
Leu Cys Gln Lys Gln Leu Leu Ile Leu Leu Ser 35 40 45 Lys Val Arg
Leu Cys Gly Gly Arg Pro Ala Arg Pro Asp Arg Gly Pro 50 55 60 Glu
Pro Gln Leu Lys Gly Ile Val Thr Lys Leu Phe Cys Arg Gln Gly 65 70
75 80 Phe Tyr Leu Gln Ala Asn Pro Asp Gly Ser Ile Gln Gly Thr Pro
Glu 85 90 95 Asp Thr Ser Ser Phe Thr His Phe Asn Leu Ile Pro Val
Gly Leu Arg 100 105 110 Val Val Thr Ile Gln Ser Ala Lys Leu Gly His
Tyr Met Ala Met Asn 115 120 125 Ala Glu Gly Leu Leu Tyr Ser Ser Pro
His Phe Thr Ala Glu Cys Arg 130 135 140 Phe Lys Glu Cys Val Phe Glu
Asn Tyr Tyr Val Leu Tyr Ala Ser Ala 145 150 155 160 Leu Tyr Arg Gln
Arg Arg Ser Gly Arg Ala Trp Tyr Leu Gly Leu Asp 165 170 175 Lys Glu
Gly Gln Val Met Lys Gly Asn Arg Val Lys Lys Thr Lys Ala 180 185 190
Ala Ala His Phe Leu Pro Lys Leu Leu Glu Val Ala Met Tyr Gln Glu 195
200 205 Pro Ser Leu His Ser Val Pro Glu Ala Ser Pro Ser Ser Pro Pro
Ala 210 215 220 Pro 225 26 206 PRT Homo sapiens 26 Met Ser Gly Pro
Gly Thr Ala Ala Val Ala Leu Leu Pro Ala Val Leu 1 5 10 15 Leu Ala
Leu Leu Ala Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala Pro 20 25 30
Thr Ala Pro Asn Gly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu 35
40 45 Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gln
Pro 50 55 60 Lys Glu Ala Ala Val Gln Ser Gly Ala Gly Asp Tyr Leu
Leu Gly Ile 65 70 75 80 Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly
Ile Gly Phe His Leu 85 90 95 Gln Ala Leu Pro Asp Gly Arg Ile Gly
Gly Ala His Ala Asp Thr Arg 100 105 110 Asp Ser Leu Leu Glu Leu Ser
Pro Val Glu Arg Gly Val Val Ser Ile 115 120 125 Phe Gly Val Ala Ser
Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys 130 135 140 Leu Tyr Gly
Ser Pro Phe Phe Thr Asp Glu Cys Thr Phe Lys Glu Ile 145 150 155 160
Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly
165 170 175 Met Phe Ile Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly
Asn Arg 180 185 190 Val Ser Pro Thr Met Lys Val Thr His Phe Leu Pro
Arg Leu 195 200 205 27 208 PRT Homo sapiens 27 Met Ala Leu Gly Gln
Lys Leu Phe Ile Thr Met Ser Arg Gly Ala Gly 1 5 10 15 Arg Leu Gln
Gly Thr Leu Trp Ala Leu Val Phe Leu Gly Ile Leu Val 20 25 30 Gly
Met Val Val Pro Ser Pro Ala Gly Thr Arg Ala Asn Asn Thr Leu 35 40
45 Leu Asp Ser Arg Gly Trp Gly Thr Leu Leu Ser Arg Ser Arg Ala Gly
50 55 60 Leu Ala Gly Glu Ile Ala Gly Val Asn Trp Glu Ser Gly Tyr
Leu Val 65 70 75 80 Gly Ile Lys Arg Gln Arg Arg Leu Tyr Cys Asn Val
Gly Ile Gly Phe 85 90 95 His Leu Gln Val Leu Pro Asp Gly Arg Ile
Ser Gly Thr His Glu Glu 100 105 110 Asn Pro Tyr Ser Leu Leu Glu Ile
Ser Thr Val Glu Arg Gly Val Val 115 120 125 Ser Leu Phe Gly Val Arg
Ser Ala Leu Phe Val Ala Met Asn Ser Lys 130 135 140 Gly Arg Leu Tyr
Ala Thr Pro Ser Phe Gln Glu Glu Cys Lys Phe Arg 145 150 155 160 Glu
Thr Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Asp Leu Tyr 165 170
175 Gln Gly Thr Tyr Ile Ala Leu Ser Lys Tyr Gly Arg Val Lys Arg Gly
180 185 190 Ser Lys Val Ser Pro Ile Met Thr Val Thr His Phe Leu Pro
Arg Ile 195 200 205 28 155 PRT Homo sapiens 28 Met Ala Ala Gly Ser
Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 Gly Ser Gly
Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr
Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg 35 40
45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu
50 55 60 Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys
Ala Asn 65 70 75 80 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu
Ala Ser Lys Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg
Leu Glu Ser Asn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr
Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys
Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe
Leu Pro Met Ser Ala Lys Ser 145 150 155 29 155 PRT Homo sapiens 29
Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5
10 15 Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys
Ser 20 25 30 Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr
Val Asp Gly 35 40 45 Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu
Gln Leu Ser Ala Glu 50 55 60 Ser Val Gly Glu Val Tyr Ile Lys Ser
Thr Glu Thr Gly Gln Tyr Leu 65 70 75 80 Ala Met Asp Thr Asp Gly Leu
Leu Tyr Gly Ser Gln Thr Pro Asn Glu 85 90 95 Glu Cys Leu Phe Leu
Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110 Ile Ser Lys
Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125 Asn
Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala 130 135
140 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 30 208
PRT Homo sapiens 30 Met Trp Lys Trp Ile Leu Thr His Cys Ala Ser Ala
Phe Pro His Leu 1 5 10 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu
Leu Phe Leu Val Ser Ser 20 25 30 Val Pro Val Thr Cys Gln Ala Leu
Gly Gln Asp Met Val Ser Pro Glu 35 40 45 Ala Thr Asn Ser Ser Ser
Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly 50 55 60 Arg His Val Arg
Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 Lys Leu
Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly 85 90 95
Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu 100
105 110 Ile Thr Ser Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn
Ser 115 120 125 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr
Gly Ser Lys 130 135 140 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg
Ile Glu Glu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Ala Ser Phe Asn
Trp Gln His Asn Gly Arg Gln Met 165 170 175 Tyr Val Ala Leu Asn Gly
Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr 180 185 190 Arg Arg Lys Asn
Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 195 200 205 31 194
PRT Homo sapiens 31 Met His Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr
Leu Leu Tyr Arg 1 5 10 15 Ser Cys Phe His Ile Ile Cys Leu Val Gly
Thr Ile Ser Leu Ala Cys 20 25 30 Asn Asp Met Thr Pro Glu Gln Met
Ala Thr Asn Val Asn Cys Ser Ser 35 40 45 Pro Glu Arg His Thr Arg
Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile 50 55 60 Arg Val Arg Arg
Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp 65 70 75 80 Lys Arg
Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn 85 90 95
Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly 100
105 110 Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu
Tyr 115 120 125 Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu
Leu Ile Leu 130 135 140 Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys
Trp Thr His Asn Gly 145 150 155 160 Gly Glu Met Phe Val Ala Leu Asn
Gln Lys Gly Ile Pro Val Arg Gly 165 170 175 Lys Lys Thr Lys Lys Glu
Gln Lys Thr Ala His Phe Leu Pro Met Ala 180 185 190 Ile Thr 32 233
PRT Homo sapiens 32 Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu
Leu His Leu Leu 1 5 10 15 Val Leu Cys Leu Gln Ala Gln Glu Gly Pro
Gly Arg Gly Pro Ala Leu 20 25 30 Gly Arg Glu Leu Ala Ser Leu Phe
Arg Ala Gly Arg Glu Pro Gln Gly 35 40 45 Val Ser Gln Gln His Val
Arg Glu Gln Ser Leu Val Thr Asp Gln Leu 50 55 60 Ser Arg Arg Leu
Ile Arg Thr Tyr Gln Leu Tyr Ser Arg Thr Ser Gly 65 70 75 80 Lys His
Val Gln Val Leu Ala Asn Lys Arg Ile Asn Ala Met Ala Glu 85 90 95
Asp Gly Asp Pro Phe Ala Lys Leu Ile Val Glu Thr Asp Thr Phe Gly 100
105 110 Ser Arg Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile Cys
Met 115 120 125 Asn Lys Lys Gly Lys Leu Ile Ala Lys Ser Asn Gly Lys
Gly Lys Asp 130 135 140 Cys Val Phe Thr Glu Ile Val Leu Glu Asn Asn
Tyr Thr Ala Leu Gln 145 150 155 160 Asn Ala Lys Tyr Glu Gly Trp Tyr
Met Ala Phe Thr Arg Lys Gly Arg 165 170 175 Pro Arg Lys Gly Ser Lys
Thr Arg Gln His Gln Arg Glu Val His Phe 180 185 190 Met Lys Arg Leu
Pro Arg Gly His His Thr Thr Glu Gln Ser Leu Arg 195 200 205 Phe Glu
Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser 210 215 220
Gln Arg Thr Trp Ala Pro Glu Pro Arg 225 230 33 268 PRT Homo sapiens
33 Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu Ile Leu
1 5 10 15 Ser Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro Lys Gly
Gln Pro 20 25 30 Gly Pro Ala Ala Thr Asp Arg Asn Pro Ile Gly Ser
Ser Ser Arg Gln 35 40 45 Ser Ser Ser Ser Ala Met Ser Ser Ser Ser
Ala Ser Ser Ser Pro Ala 50 55 60 Ala Ser Leu Gly Ser Gln Gly Ser
Gly Leu Glu Gln Ser Ser Phe Gln 65 70 75 80 Trp Ser Pro Ser Gly Arg
Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly 85 90 95 Ile Gly Phe His
Leu Gln Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser 100 105 110 His Glu
Ala Asn Met Leu Ser Val Leu Glu Ile Phe Ala Val Ser Gln 115 120 125
Gly Ile Val Gly Ile Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met 130
135 140 Ser Lys Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp
Cys 145 150 155 160 Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn
Thr Tyr Ala Ser 165 170 175 Ala Ile His Arg Thr Glu Lys Thr Gly Arg
Glu Trp Tyr Val Ala Leu 180 185 190 Asn Lys Arg Gly Lys Ala Lys Arg
Gly Cys Ser Pro Arg Val Lys Pro 195 200 205 Gln His Ile Ser Thr His
Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln 210 215 220 Pro Glu Leu Ser
Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro 225 230 235 240 Ser
Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr 245 250
255 Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265 34 208
PRT Homo sapiens 34 Met Ala Pro Leu Gly Glu Val Gly Asn 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 35 239
PRT Homo sapiens 35 Met Gly Leu Ile Trp Leu Leu Leu Leu Ser Leu Leu
Glu Pro Gly Trp 1 5 10 15 Pro Ala Ala Gly Pro Gly Ala Arg Leu Arg
Arg Asp Ala Gly Gly Arg 20 25 30 Gly Gly Val Tyr Glu His Leu Gly
Gly Ala Pro Arg Arg Arg Lys Leu 35 40 45 Tyr Cys Ala Thr Lys Tyr
His Leu Gln Leu His Pro Ser Gly Arg Val 50 55 60 Asn Gly Ser Leu
Glu Asn Ser Ala Tyr Ser Ile Leu Glu Ile Thr Ala 65 70 75 80 Val Glu
Val Gly Ile Val Ala Ile Arg Gly Leu Phe Ser Gly Arg Tyr 85 90 95
Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr Ser 100
105 110 Ala Glu Cys Glu Phe Val Glu Arg Ile His Glu Leu Gly Tyr Asn
Thr 115 120 125 Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser Ser Thr Pro
Gly Ala Arg 130 135 140 Arg Gln Pro Ser Ala Glu Arg Leu Trp Tyr Val
Ser Val Asn Gly Lys 145 150 155 160 Gly Arg Pro Arg Arg Gly Phe Lys
Thr Arg Arg Thr Gln Lys Ser Ser 165 170 175 Leu Phe Leu Pro Arg Val
Leu Asp His Arg Asp His Glu Met Val Arg 180 185 190 Gln Leu Gln Ser
Gly Leu Pro Arg Pro Pro Gly Lys Gly Val Gln Pro 195 200 205 Arg Arg
Arg Arg Gln Lys Gln Ser Pro Asp Asn Leu Glu Pro Ser His 210 215 220
Val Gln Ala Ser Arg Leu Gly Ser Gln Leu Glu Ala Ser Ala His 225 230
235 36 11 PRT Artificial Sequence FGF family motif 36 Cys Xaa Phe
Xaa Glu Glu Glu Glu Glu Glu Tyr 1 5 10 37 4 PRT Artificial Sequence
dibasic cleavage peptide 37 Arg Xaa Xaa Arg 1 38 1023 DNA Mus
musculus CDS (1)...(624) 38 atg tat tca gcg ccc tcc gcc tgc act tgc
ctg tgt tta cac ttt cta 48 Met Tyr Ser Ala Pro Ser Ala Cys Thr Cys
Leu Cys Leu His Phe Leu 1 5 10 15 ctg ctg tgc ttc cag gtt cag gtg
ttg gca gcc gag gag aat gtg gac 96 Leu Leu Cys Phe Gln Val Gln Val
Leu Ala Ala Glu Glu Asn Val Asp 20 25 30 ttc cgc atc cac gtg gag
aac cag acg cgg gct cga gat gat gtg agt 144 Phe Arg Ile His Val Glu
Asn Gln Thr Arg Ala Arg Asp Asp Val Ser 35 40 45 cgg aag cag ctg
cgc ttg tac cag ctc tat agc agg acc agt ggg aag 192 Arg Lys Gln Leu
Arg Leu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys 50 55 60 cac att
caa gtc ctg ggc cgt agg atc agt gcc cgt ggc gag gac ggg 240 His Ile
Gln Val Leu Gly Arg Arg Ile Ser Ala Arg Gly Glu Asp Gly 65 70 75 80
gac aag tat gcc cag ctc cta gtg gag aca gat acc ttc ggg agt caa 288
Asp Lys Tyr Ala Gln Leu Leu Val Glu Thr Asp Thr Phe Gly Ser Gln 85
90 95 gtc cgg atc aag ggc aag gag aca gaa ttc tac ctg tgt atg aac
cga 336 Val Arg Ile Lys Gly Lys Glu Thr Glu Phe Tyr Leu Cys Met Asn
Arg 100 105 110 aaa ggc aag ctc gtg ggg aag cct gat ggt act agc aag
gag tgc gtg 384 Lys Gly Lys Leu Val Gly Lys Pro Asp Gly Thr Ser Lys
Glu Cys Val 115 120 125 ttc att gag aag gtt ctg gaa aac aac tac acg
gcc ctg atg tct gcc 432 Phe Ile Glu Lys Val Leu Glu Asn Asn Tyr Thr
Ala Leu Met Ser Ala 130 135 140 aag tac tct ggt tgg tat gtg ggc ttc
acc aag aag ggg cgg cct cgc 480 Lys Tyr Ser Gly Trp Tyr Val Gly Phe
Thr Lys Lys Gly Arg Pro Arg 145 150 155 160 aag ggt ccc aag acc cgc
gag aac cag caa gat gta cac ttc atg aag 528 Lys Gly Pro Lys Thr Arg
Glu Asn Gln Gln Asp Val His Phe Met Lys 165 170 175 cgt tac ccc aag
gga cag gcc gag ctg cag aag ccc ttc aaa tac acc 576 Arg Tyr Pro Lys
Gly Gln Ala Glu Leu Gln Lys Pro Phe Lys Tyr Thr 180 185 190 aca gtc
acc aag cga tcc cgg cgg atc cgc ccc act cac ccc ggc tag 624 Thr Val
Thr Lys Arg Ser Arg Arg Ile Arg Pro Thr His Pro Gly * 195 200 205
gtccggccac actcaccccc ccagagaact acatcagagg aatattttta catgaaaaat
684 aaggaagaat ctctattttt gtacattgtg tttaaaagaa gacaaaaact
gaacctaaag 744 tcttgggagg aggggcgata ggattccact gttgacctga
accccatgac aaaggactca 804 cacaagggga ccgctgtcaa cccacaggtg
cttgcctctc tctaggaggt gacaattcaa 864 aactcatccc cagaggagga
cttgaacgag gaaactgcga gaaaccaaag tcctttcccc 924 ccaaaggttc
tgaaagcaaa caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 984
aaaaaaaaaa aaaaaaaaaa gggcggccgc tctagagga 1023 39 207 PRT Mus
musculus 39 Met Tyr Ser Ala Pro Ser Ala Cys Thr Cys Leu Cys Leu His
Phe Leu 1 5 10 15 Leu Leu Cys Phe Gln Val Gln Val Leu Ala Ala Glu
Glu Asn Val Asp 20 25 30 Phe Arg Ile His Val Glu Asn Gln Thr Arg
Ala Arg Asp Asp Val Ser 35 40 45 Arg Lys Gln Leu Arg Leu Tyr Gln
Leu Tyr Ser Arg Thr Ser Gly Lys 50 55 60 His Ile Gln Val Leu Gly
Arg Arg Ile Ser Ala Arg Gly Glu Asp Gly 65 70
75 80 Asp Lys Tyr Ala Gln Leu Leu Val Glu Thr Asp Thr Phe Gly Ser
Gln 85 90 95 Val Arg Ile Lys Gly Lys Glu Thr Glu Phe Tyr Leu Cys
Met Asn Arg 100 105 110 Lys Gly Lys Leu Val Gly Lys Pro Asp Gly Thr
Ser Lys Glu Cys Val 115 120 125 Phe Ile Glu Lys Val Leu Glu Asn Asn
Tyr Thr Ala Leu Met Ser Ala 130 135 140 Lys Tyr Ser Gly Trp Tyr Val
Gly Phe Thr Lys Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Pro Lys
Thr Arg Glu Asn Gln Gln Asp Val His Phe Met Lys 165 170 175 Arg Tyr
Pro Lys Gly Gln Ala Glu Leu Gln Lys Pro Phe Lys Tyr Thr 180 185 190
Thr Val Thr Lys Arg Ser Arg Arg Ile Arg Pro Thr His Pro Gly 195 200
205 40 21 DNA Artificial Sequence Oligonucleotide primer ZC17579 40
aaaggcaagc tcgtggggaa g 21 41 22 DNA Artificial Sequence
Oligonucleotide primer ZC17578 41 tcgcttggtg actgtggtgt at 22 42 19
DNA Artificial Sequence Oligonucleotide primer ZC19567 42
atgtattcag cgccctccg 19 43 19 DNA Artificial Sequence
Oligonucleotide primer ZC19633 43 cgagcccgcg tctggttct 19
* * * * *