U.S. patent application number 10/938375 was filed with the patent office on 2005-03-03 for method for treating fibrosis.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Gilbertson, Debra G..
Application Number | 20050049218 10/938375 |
Document ID | / |
Family ID | 27496469 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050049218 |
Kind Code |
A1 |
Gilbertson, Debra G. |
March 3, 2005 |
Method for treating fibrosis
Abstract
Materials and methods for treating fibrosis in a mammal are
disclosed. The methods comprise administering to a mammal a
composition comprising a therapeutically effective amount of a
zvegf3 antagonist in combination with a pharmaceutically acceptable
delivery vehicle. Zvegf3 antagonists include anti-zvegf3
antibodies, mitogenically inactive receptor-binding zvegf3 variant
polypeptides, and inhibitory polynucleotides. Within one embodiment
of the invention the fibrosis is liver fibrosis.
Inventors: |
Gilbertson, Debra G.;
(Seattle, WA) |
Correspondence
Address: |
Gary E. Parker
Patent Department
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
27496469 |
Appl. No.: |
10/938375 |
Filed: |
September 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10938375 |
Sep 10, 2004 |
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09695121 |
Oct 23, 2000 |
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60161653 |
Oct 21, 1999 |
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60165255 |
Nov 12, 1999 |
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60222223 |
Aug 1, 2000 |
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Current U.S.
Class: |
514/44A ;
424/145.1; 514/15.4; 514/19.5; 514/19.8; 514/9.4 |
Current CPC
Class: |
C12N 2799/022 20130101;
C07K 14/52 20130101; C12N 2710/10343 20130101; A61P 1/16 20180101;
C12N 15/86 20130101; C07K 16/22 20130101; C07K 16/24 20130101; A01K
2217/052 20130101; A01K 2217/05 20130101; A01K 2227/105 20130101;
C12N 2840/007 20130101; A61K 38/00 20130101; A01K 67/0275 20130101;
C07K 14/475 20130101; A61K 2039/505 20130101 |
Class at
Publication: |
514/044 ;
514/012; 424/145.1 |
International
Class: |
A61K 048/00; A61K
039/395 |
Claims
What is claimed is:
1. A method of treating fibrosis in a mammal comprising
administering to the mammal a composition comprising a
therapeutically effective amount of a zvegf3 antagonist in
combination with a pharmaceutically acceptable delivery vehicle,
wherein the zvegf3 antagonist is selected from the group consisting
of anti-zvegf3 antibodies, mitogenically inactive receptor-binding
zvegf3 variant polypeptides, and inhibitory polynucleotides.
2. The method of claim 1 wherein the fibrosis is liver
fibrosis.
3. The method of claim 1 wherein the fibrosis is kidney
fibrosis.
4. The method of claim 1 wherein the antagonist is an anti-zvegf3
antibody.
5. The method of claim 4 wherein the antibody is a monoclonal
antibody.
6. The method of claim 1 wherein the antagonist is selected from
the group consisting of antisense polynucleotides,
ribozyme-encoding polynucleotides, and external guide
sequence-encoding polynucleotides.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/695,121, filed Oct. 23, 2000, which is incorporated herein
by reference and which claims the benefit of U.S. Provisional
Applications Ser. No. 60/161,653, filed Oct. 21, 1999; 60/165,255,
filed Nov. 12, 1999; and 60/222,223, filed Aug. 1, 2000.
BACKGROUND OF THE INVENTION
[0002] Fibrosis is the abnormal accumulation of fibrous tissue that
can occur as a part of the wound-healing process in damaged tissue.
Such tissue damage may result from physical injury, inflammation,
infection, exposure to toxins, and other causes. Examples of
fibrosis include dermal scar formation, keloids, liver fibrosis,
lung fibrosis (e.g., silicosis, asbestosis), kidney fibrosis
(including diabetic nephropathy), and glomerulosclerosis.
[0003] Liver (hepatic) fibrosis, for example, occurs as a part of
the wound-healing response to chronic liver injury. Fibrosis occurs
as a complication of haemochromatosis, Wilson's disease,
alcoholism, schistosomiasis, viral hepatitis, bile duct
obstruction, exposure to toxins, and matabolic disorders. This
formation of scar tissue is believed to represent an attempt by the
body to encapsulate the injured tissue. Liver fibrosis is
characterized by the accumulation of extracellular matrix that can
be distinguished qualitatively from that in normal liver. Left
unchecked, hepatic fibrosis progresses to cirrhosis (defined by the
presence of encapsulated nodules), liver failure, and death.
[0004] In recent years there have been significant advances in the
understanding of the cellular and biochemical mechanisms underlying
liver fibrosis (reviewed by Li and Friedman, J. Gastroenterol.
Hepatol. 14:618-633, 1999). Stellate cells are believed to be a
major source of extracellular matrix in the liver. Stellate cells
respond to a variety of cytokines present in the liver, some of
which they also produce (Friedman, Seminars in Liver Disease
19:129-140, 1999).
[0005] As summarized by Li and Friedman (ibid.), actual and
proposed therapeutic strategies for liver fibrosis include removal
of the underlying cause (e.g., toxin or infectious agent),
suppression of inflammation (using, e.g., corticosteroids, IL-1
receptor antagonists, or other agents), down-regulation of stellate
cell activation (using, e.g., gamma interferon or antioxidants),
promotion of matrix degradation, or promotion of stellate cell
apoptosis. Despite recent progress, many of these strategies are
still in the experimental stage, and existing therapies are aimed
at suppressing inflammation rather than addressing the underlying
biochemical processes. Thus, there remains a need in the art for
materials and methods for treating fibrosis, including liver
fibrosis.
DESCRIPTION OF THE INVENTION
[0006] The present invention provides materials and methods for
reducing cell proliferation or extracellular matrix production,
treating fibrosis, and reducing stellate cell activation in a
mammal.
[0007] Within one aspect of the invention there is provided a
method of reducing cell proliferation or extracellular matrix
production in a mammal comprising administering to the mammal a
composition comprising a zvegf3 antagonist in combination with a
pharmaceutically acceptable delivery vehicle, wherein the zvegf3
antagonist is selected from the group consisting of anti-zvegf3
antibodies, mitogenically inactive receptor-binding zvegf3 variant
polypeptides, and inhibitory polynucleotides, in an amount
sufficient to reduce cell proliferation or extracellular matrix
production. Within certain embodiments of the invention,
proliferation of mesangial, endothelial, smooth muscle, fibroblast,
osteoblast, osteoclast, stellate, or interstitial cells is reduced.
Within other embodiments, the mammal is suffering from a
fibroproliferative disorder of the liver, kidney, or bone.
[0008] Within another aspect of the present invention there is
provided a method of treating fibrosis in a mammal comprising
administering to the mammal a composition comprising a
therapeutically effective amount of a zvegf3 antagonist in
combination with a pharmaceutically acceptable delivery vehicle,
wherein the zvegf3 antagonist is selected from the group consisting
of anti-zvegf3 antibodies, mitogenically inactive receptor-binding
zvegf3 variant polypeptides, and inhibitory polynucleotides. Within
certain embodiment of the invention the fibrosis is liver fibrosis
or kidney fibrosis.
[0009] Within a third aspect of the invention there is provided a
method of reducing stellate cell activation in a mammal comprising
administering to the mammal a composition comprising a zvegf3
antagonist in combination with a pharmaceutically acceptable
delivery vehicle, wherein the zvegf3 antagonist is selected from
the group consisting of anti-zvegf3 antibodies, mitogenically
inactive receptor-binding zvegf3 variant polypeptides, and
inhibitory polynucleotides, in an amount sufficient to reduce
stellate cell activation. Within one embodiment, the stellate cells
are liver stellate cells.
[0010] Within a fourth aspect of the invention there are provided
pharmaceutical compositions for use within the above methods. In
general, the compositions comprise a zvegf3 antagonist in
combination with a pharmaceutically acceptable delivery vehicle,
wherein the zvegf3 antagonist is selected from the group consisting
of anti-zvegf3 antibodies, mitogenically inactive receptor-binding
zvegf3 variant polypeptides, and inhibitory polynucleotides.
[0011] These and other aspects of the invention will become evident
upon reference to the following detailed description of the
invention and the attached drawings. In the drawings:
[0012] FIGS. 1A-1G are a Hopp/Woods hydrophilicity profile of the
amino acid sequence shown in SEQ ID NO:2. The profile is based on a
sliding six-residue window. Buried G, S, and T residues and exposed
H, Y, and W residues were ignored. These residues are indicated in
the figure by lower case letters.
[0013] FIG. 2 is an alignment of human (SEQ ID NO:2) and mouse (SEQ
ID NO:4) amino acid sequences.
[0014] The term "antagonist" is used herein to denote a compound
that reduces a biological activity of another compound. Within the
present invention, a "zvegf3 antagonist" is a compound that reduces
the receptor-mediated biological activity (e.g., mitogenic
activity) of zvegf3 on a target cell. Antagonists may exert their
action by competing with zvegf3 for binding sites on a cell-surface
receptor, by binding to zvegf3 and preventing it from binding to a
cell-surface receptor, by otherwise interfering with receptor
function, by reducing production of zvegf3, or by other means.
[0015] "Extracellular matrix" (ECM) is a complex mixture of
macromolecules that accumulates within tissues in close apposition
to cell surfaces. ECM contains secreted macromolecules such as
collagens I, III and IV, fibronectin, laminins, and various
proteoglycans. These macromolecules can be organized to provide
cohesion to the tissue and can contribute to its structural and
mechanical properties. ECM can act as a depository for, and release
site of, potent secreted growth factors, and is known to influence
growth, survival and differentiation of the cells it surrounds.
Pathologic ECM accumulation, if unchecked, can restrict access of
nutrients, growth factors and other physiologically important
molecules to cells and can lead to the creation of areas of low
live cell density. Over time, this accumulation can result in the
inability of a tissue to perform its specific metabolic and
structural roles, and may ultimately lead to overt cell and tissue
death.
[0016] An "inhibitory polynucleotide" is a DNA or RNA molecule that
reduces or prevents expression (transcription or translation) of a
second (target) polynucleotide. Inhibitory polynucleotides include
antisense polynucleotides, ribozymes, and external guide sequences.
The term "inhibitory polynucleotide" further includes DNA and RNA
molecules that encode the actual inhibitory species, such as DNA
molecules that encode ribozymes.
[0017] The terms "treat" and "treatment" are used broadly to denote
therapeutic and prophylactic interventions that favorably alter a
pathological state. Treatments include procedures that moderate or
reverse the progression of, reduce the severity of, prevent, or
cure a disease.
[0018] All references cited herein are incorporated by reference in
their entirety.
[0019] The present invention provides methods for treating fibrosis
in a patient using zvegf3 antagonists. Zvegf3 is a protein that is
structurally related to platelet-derived growth factor (PDGF) and
the vascular endothelial growth factors (VEGF). This protein has
also been designated "VEGF-R" (WIPO Publication WO 99/37671) and,
more recently, "PDGF-C" (WO 00/18212). Zvegf3/PDGF-C is a
multi-domain protein with significant homology to the PDGF/VEGF
family of growth factors. Representative amino acid sequences of
human and mouse zvegf3 are shown in SEQ ID NO:2 and SEQ ID NO:4,
respectively. DNAs encoding these polypeptides are shown in SEQ ID
NOS: 1 and 3, respectively.
[0020] The term "zvegf3 protein" is used herein to denote proteins
comprising the growth factor domain of a zvegf3 polypeptide (e.g.,
residues 235-345 of human zvegf3 (SEQ ID NO:2) or mouse zvegf3 (SEQ
ID NO:4)), wherein said protein is mitogenic for cells expressing
cell-surface PDGF .alpha.-receptor subunit. Zvegf3 has been found
to bind to the .alpha..alpha. and .alpha..beta. isoforms of PDGF
receptor. Zvegf3 proteins include homodimers and heterodimers as
disclosed below. Using methods known in the art, zvegf3 proteins
can be prepared in a variety of forms, including glycosylated or
non-glycosylated, pegylated or non-pegylated, with or without an
initial methionine residues, and as fusion proteins as disclosed in
more detail below.
[0021] Structural predictions based on the zvegf3 sequence and its
homology to other growth factors suggests that the polypeptide can
form homomultimers or heteromultimers having growth factor
activity, i.e., modulating one or more of cell proliferation,
migration, differentiation, and metabolism. Experimental evidence
confirms that biologically active zvegf3 is a dimeric protein.
While not wishing to be bound by theory, the similarity of zvegf3
to other members of the PDGF/VEGF family suggests that zvegf3 may
also form heteromultimers with other members of the family,
including VEGF, VEGF-B, VEGF-C, VEGF-D, zvegf4 (SEQ ID NO:5), PIGF
(Maglione et al., Proc. Natl. Acad. Sci. USA 88:9267-9271, 1991),
PDGF-A (Murray et al., U.S. Pat. No. 4,899,919; Heldin et al., U.S.
Pat. No. 5,219,759), or PDGF-B (Chiu et al., Cell 37:123-129, 1984;
Johnsson et al., EMBO J. 3:921-928, 1984).
[0022] The zvegf3 polypeptide chain comprises a growth factor
domain and a CUB domain. The growth factor domain is characterized
by an arrangement of cysteine residues and beta strands that is
characteristic of the "cystine knot" structure of the PDGF family.
The CUB domain shows sequence homology to CUB domains in the
neuropilins (Takagi et al., Neuron 7:295-307, 1991; Soker et al.,
ibid.), human bone morphogenetic protein-i (Wozney et al., Science
242:1528-1534, 1988), porcine seminal plasma protein and bovine
acidic seminal fluid protein (Romero et al., Nat. Struct. Biol.
4:783-788, 1997), and X. laevis tolloid-like protein (Lin et al.,
Dev. Growth Differ. 39:43-51, 1997).
[0023] An alignment of mouse and human zvegf3 polypeptide sequences
is shown in FIG. 2. Analysis of the amino acid sequence shown in
SEQ ID NO:2 indicates that residues 1 to 14 form a secretory
peptide. The CUB domain extends from residue 46 to residue 163. A
propeptide-like sequence extends from residue 164 to residue 234,
and includes two potential cleavage sites at its carboxyl terminus,
a dibasic site at residues 231-232 and a target site for furin or a
furin-like protease at residues 231-234. The growth factor domain
extends from residue 235 to residue 345. Those skilled in the art
will recognize that domain boundaries are somewhat imprecise and
can be expected to vary by up to .+-.5 residues from the specified
positions. Potential proteolytic cleavage sites occur at residues
232 and 234. Processing of recombinant zvegf3 produced in BHK cells
has been found to occur between residues 225 and 226. Signal
peptide cleavage is predicted to occur after residue 14 (+3
residues). This analysis suggests that the zvegf3 polypeptide chain
may be cleaved to produce a plurality of monomeric species as shown
in Table 1. Cleavage after Arg-234 is expected to result in
subsequent removal of residues 231-234, with possible conversion of
Gly-230 to an amide. Cleavage after Lys-232 is expected to result
in subsequent removal of residue 231, again with possible
conversion of Gly-230 to an amide. In addition, it may be
advantageous to include up to seven residues of the interdomain
region at the carboxyl terminus of the CUB domain. The interdomain
region can be truncated at its amino terminus by a like amount. See
Table 1. Corresponding domains in mouse and other non-human zvegf3s
can be determined by those of ordinary skill in the art from
sequence alignments.
1TABLE 1 Monomer Residues (SEQ ID NO: 2) Cub domain 15-163 46-163
15-170 46-170 CUB domain + interdomain region 15-234 46-234 15-229
amide 15-230 Cub domain + interdomain region + growth 15-345 factor
domain 46-345 Growth factor domain 235-345 226-345 Growth factor
domain + interdomain 164-345 region 171-345
[0024] Zvegf3 can thus be prepared in a variety of multimeric forms
comprising a zvegf3 polypeptide as disclosed above. These zvegf3
polypeptides include zvegf3.sub.15-345, zvegf3.sub.46-345,
zvegf3.sub.226-345, and zvegf3.sub.235-345. Variants and
derivatives of these polypeptides can also be prepared as disclosed
herein.
[0025] Zvegf3 proteins can be prepared as fusion proteins
comprising amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue, an affinity tag, or a targetting
polypeptide. For example, a zvegf3 protein can be prepared as a
fusion with an affinity tag to facilitate purification. 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, for example, 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 82:79524, 1985), substance P,
Flag.TM. peptide (Hopp et al., Biotechnology 6:1204-1210, 1988),
streptavidin binding peptide, maltose binding protein (Guan et al.,
Gene 67:21-30, 1987), cellulose binding protein, thioredoxin,
ubiquitin, T7 polymerase, or other antigenic epitope or binding
domain. Fusion of zvegf3 to, for example, maltose binding protein
or glutatione S transferase can be used to improve yield in
bacterial expression systems. In these instances the non-zvegf3
portion of the fusion protein ordinarily will be removed prior to
use. Separation of the zvegf3 and non-zvegf3 portions of the fusion
protein is facilitated by providing a specific cleavage site
between the two portions. Such methods are well known in the art.
Zvegf3 can also be fused to a targetting peptide, such as an
antibody (including polyclonal antibodies, monoclonal antibodies,
antigen-binding fragments thereof such as F(ab').sub.2 and Fab
fragments, single chain antibodies, and the like) or other peptidic
moiety that binds to a target tissue.
[0026] Variations can be made in the zvegf3 amino acid sequences
shown in SEQ ID NO:2 and SEQ ID NO:4 to provide mitogenically
inactive, receptor-binding polypeptides that act as zvegf3
antagonists. As used herein, the term "mitogenically inactive"
means that the protein does not show statistically significant
activity in a standard mitogenesis assay as compared to a wild-type
zvegf3 control. Such variations include amino acid substitutions,
deletions, and insertions. While not wishing to be bound by theory,
it is believed that residues Arg260-Trp271 of human zvegf3 (SEQ ID
NO:2) form a loop that define the ability of the protein to bind to
PDGF-.beta. receptors, although binding is also permitted to alpha
receptors. It is thus predicted that binding to either receptor
subunit can be blocked or enhanced by mutations in this region. In
addition, residues Leu311-His321 of SEQ ID NO:2 are predicted to
form a loop (loop3) that may be mutated to block receptor binding.
Peptides that mimic this region of the molecule may act as
antagonists.
[0027] The effects of amino acid sequence changes can be predicted
by computer modeling (using, e.g., the Insight II.RTM. viewer and
homology modeling tools; MSI, San Diego, Calif.) or determined by
analysis of crystal structure (see, e.g., Lapthorn et al., Nature
369:455, 1994), and can be assessed using art-recognized
mutagenesis procedures in combination with activity assays.
Representative mutagenesis procedures include, for example,
site-directed mutagenesis and alanine-scanning mutagenesis
(Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al.,
Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991). Multiple amino acid
substitutions can be made and tested using known methods, 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). 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),
region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986;
Ner et al., DNA 7:127, 1988), and DNA shuffling as disclosed by
Stemmer (Nature 370:389-391, 1994) and Stemmer (Proc. Natl. Acad.
Sci. USA 91:10747-10751, 1994). The resultant mutant molecules are
tested for mitogenic activity or other properties (e.g., receptor
binding) to identify amino acid residues that are critical to these
functions. Mutagenesis can be combined with high volume or
high-throughput screening methods to detect biological activity of
zvegf3 variant polypeptides, in particular biological activity in
modulating cell proliferation. For example, mitogenesis assays that
measure dye incorporation or .sup.3H-thymidine incorporation can be
carried out on large numbers of samples. Competition assays can be
employed to confirm antagonist activity.
[0028] Zvegf3 proteins, including full-length polypeptides,
fragments, 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
(including cultured cells of multicellular organisms). 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, Green and Wiley
and Sons, NY, 1993. See, WO 00/34474. In general, a DNA sequence
encoding a zvegf3 polypeptide 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. See, WO 00/34474.
[0029] Zvegf3 proteins can also comprise 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-methylglycine,
allo-threonine, methylthreonine, hydroxyethylcysteine,
hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic
acid, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
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 is 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., Methods Enzymol. 202:301, 1991;
Chung et al., Science 259:806-809, 1993; and Chung et al., Proc.
Natl. Acad. Sci. USA 90:10145-10149, 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. 271:19991-19998, 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. 33:7470-7476,
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).
[0030] Zvegf3 polypeptides or fragments thereof can also be
prepared through chemical synthesis according to methods known in
the art, including exclusive solid phase synthesis, partial solid
phase methods, fragment condensation or classical solution
synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149,
1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition),
Pierce Chemical Co., Rockford, Ill., 1984; Bayer and Rapp, Chem.
Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide
Synthesis: A Practical Approach, IRL Press, Oxford, 1989.
[0031] Zvegf3 proteins are purified by conventional protein
purification methods, typically by a combination of chromatographic
techniques. See, in general, Affinity Chromatography: Principles
& Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988;
and Scopes, Protein Purification: Principles and Practice,
Springer-Verlag, New York, 1994. Proteins comprising a
polyhistidine affinity tag (typically about 6 histidine residues)
are purified by affinity chromatography on a nickel chelate resin.
See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988.
Furthermore, the growth factor domain itself binds to nickel resin
at pH 7.0-8.0 and 25 mM Na phosphate, 0.25 M NaCl. Bound protein
can be eluted with a descending pH gradient down to pH 5.0 or an
imidazole gradient. Recombinant zvegf3 growth factor domain protein
can be purified using a combination of chromatography on a strong
cation exchanger followed by a tandem column array comprising a
strong anion exchanger followed by an immobilized metal affinity
column in series. It has also been found that zvegf3 binds to
various dye matrices (e.g., BLUE1, BLUE 2, ORANGE 1, ORANGE 3, and
RED3 from Lexton Scientific, Signal Hill, Calif.) in PBS at pH 6-8,
from which the bound protein can be eluted in 1-2 M NaCl in 20 mM
boric acid buffer at pH 8.8. Protein eluted from RED3 may be passed
over RED2 (Lexton Scientific) to remove remaining contaminants.
Proteins comprising a glu-glu tag can be purified by immunoaffinity
chromatography according to conventional procedures. See, for
example, Grussenmeyer et al., ibid. Maltose binding protein fusions
are purified on an amylose column according to methods known in the
art.
[0032] As disclosed in more detail below, overexpression of zvegf3
in the livers of transgenic mice led to marked stellate cell
activation and proliferation. At 8 weeks of age there was an
accumulation of perisinusiodal extracellular matrix (ECM) that
progressed to a perivenular ECM deposition at 22 weeks and possible
eary stages of cirrhosis characterized by fibrotic banding and
hepatic nodule formation at 33 weeks of age. Thus, zvegf3 dimers
appear to resemble the previously described PDGF isoforms in being
potent mitogens of hepatic stellate cells and appearing to play a
role in liver fibrosis. These transgenic mice thus provide a model
for testing zvegf3 antagonists as well as other antifibrotic
agents. In view of these and other experiments disclosed herein, it
is expected that altered zvegf3 expression may initiate or
exacerbate a variety of fibrotic conditions. In this context,
inhibiting the action of zvegf3 using a zvegf3 antagonist will
limit the progress of such conditions. While not wishing to be
bound by theory, it is believed that the pro-fibrotic effects of
zvegf3 are due at least in part to the induction of TGF-.beta.
production.
[0033] Zvegf3 antagonists include, without limitation, anti-zvegf3
antibodies (including neutralizing antibodies), inhibitory
polynucleotides (including antisense polynucleotides, ribozymes,
and external guide sequences), and other peptidic and non-peptidic
agents, including small molecule inhibitors and mitogenically
inactive receptor-binding zvegf3 polypeptides. Such antagonists can
be use to block the mitogenic effects of zvegf3 and thereby reduce,
inhibit, prevent, or otherwise treat fibrosis, including, without
limitation, scar formation, keloids, scleroderma, liver fibrosis,
lung fibrosis, kidney fibrosis, pancreatic fibrosis, myelofibrosis,
post-surgical fibrotic adhesions, fibroproliferative disorders of
the vasculature, fibroproliferative disorders of the prostate,
fibroproliferative disorders of bone, fibromatosis, fibroma,
fibrosarcoma, and the like.
[0034] Of particular interest is the use of zvegf3 or zvegf3
antagonists for the treatment or repair of liver damage, including
damage due to chronic liver disease, including chronic active
hepatitis (including hepatitis C) and many other types of
cirrhosis. Widespread, massive necrosis, including destruction of
virtually the entire liver, can be caused by, inter alia, fulminant
viral hepatitis; overdoses of the analgesic acetaminophen; exposure
to other drugs and chemicals such as halothane, monoamine oxidase
inhibitors, agents employed in the treatment of tuberculosis,
phosphorus, carbon tetrachloride, and other industrial chemicals.
Conditions associated with ultrastructural lesions that do not
necessarily produce obvious liver cell necrosis include Reye's
syndrome in children, tetracycline toxicity, and acute fatty liver
of pregnancy. Cirrhosis, a diffuse process characterized by
fibrosis and a conversion of normal architecture into structurally
abnormal nodules, can come about for a variety reasons including
alcohol abuse, post necrotic cirrhosis (usually due to chronic
active hepatitis), biliary cirrhosis, pigment cirrhosis,
cryptogenic cirrhosis, Wilson's disease, and alpha-1-antitrypsin
deficiency. In cases of liver fibrosis it may be beneficial to
administer a zvegf3 antagonist to suppress the activation of
stellate cells, which have been implicated in the production of
extracellular matrix in fibrotic liver (Li and Friedman,
ibid.).
[0035] Fibrotic disorders of the kidney include, without
limitation, glomerulonephritis (including membranoproliferative,
diffuse proliferative, rapidly progressive, and chronic forms),
diabetic glomerulosclerosis, focal glomerulosclerosis, diabetic
nephropathy, lupus nephritis, tubulointerstitial fibrosis,
membranous nephropathy, amyloidosis (which affects the kidney among
other tissues), renal arteriosclerosis, and nephrotic syndrome. The
glomerulus is a major target of many types of renal injury,
including immunologic (e.g., immune-complex- or T-cell-mediated),
hemodynamic (systemic or renal hypertension), metabolic (e.g.,
diabetes), "atherosclerotic" (accumulation of lipids in the
glomerulus), infiltrative (e.g., amyloid), and toxic (e.g., snake
venom) (Johnson, Kidney Int. 45:1769-1782, 1994). The renal
structural changes in patients with diabetic nephropathy include
hypertrophy of the glomerulus, thickening of the glomerular and
tubular membranes (due to accumulated matrix), and increased
amounts of matrix in the measangium and tubulointerstitium (Ziyadeh
et al., Proc. Natl. Acad. Sci. USA 97:8015-8020, 2000). Glomerular
hypertension due to intrarenal hemodynamic changes in diabetes can
contribute to the progression of diabetic nephropathy (Ishida et
al., Diabetes 48:595-602, 1999). Autoimmune nephritis can also lead
to altered mesangial cell growth responses (Liu and Ooi, J.
Immunol. 151:2247-2251, 1993). Infection by hepatitis-C virus can
also result in idiopathic membranoproliferative glomerulonephritis
(Johnson et al., N. Engl. J. Med. 328:465-470, 1993). While not
wishing to be bound by theory, experiments have shown that the
activity of zvegf3 is mediated by the .alpha..alpha. and
.alpha..beta. PDGF receptor isoforms. PDGF receptors are widely
expressed in most renal cell types, and their expression is
upregulated in a number of kidney pathologies (e.g., Iida et al.,
Proc. Natl. Acad. Sci. USA 88:6560-6564, 1991). Stimulation of PDGF
receptors has been implicated in fibroproliferative diseases of the
kidney in a variety of animal models (e.g., Ooi et al., P.S.E.B.M.
213:230-237, 1996; Lindahl et al., Development 125:3313-3322, 1998;
Lindahl and Betsholtz, Curr. Op. Nephr. Hypert. 7:21-26, 1998; and
Betsholtz and Raines, Kidney Int. 51:1361-1369, 1997).
[0036] Fibrotic disorders of the lung include, for example,
silicosis, asbestosis, idiopathic pulmonary fibrosis, bronchiolitis
obliterans-organizing pneumonia, pulmonary fibrosis associated with
high-dose chemotherapy, idiopathic pulmonary fibrosis, and
pulmonary hypertension. These diseases are characterized by cell
proliferation and increased production of extracellular matrix
components, such as collagens, elastin, fibronectin, and
tenascin-C.
[0037] Pancreatic fibrosis occurs in chronic pancreatitis. This
condition is characterized by duct calcification and fibrosis of
the pancreatic parenchyma. Like liver cirrhosis, chronic
pancreatitis is associated with alcohol abuse. See, Fogar et al.,
J. Medicine 29:277-287, 1998.
[0038] Diseases of the skeleton that are due to modified growth and
matrix production in the bone include, but are not limited to,
osteopetrosis, hyperostosis, osteosclerosis, osteoarthritis, and
endosteal bone formation in metastatic prostate cancer.
Fibroproliferative disorders of bone are characterized by aberrant
and ectopic bone formation, commonly seen as active proliferation
of the major cell types participating in bone formation as well as
elaboration by those cells of a complex bone matrix. Exemplary of
such bone disorders is the fibrosis that occurs with prostate tumor
metastases to the axial skeleton. In prostate tumor-related
cancellous bone growth, prostate carcinoma cells can interact
reciprocally with osteoblasts to produce enhanced tumor growth and
osteoblastic action when they are deposited in bone (Zhau et al.,
Cancer 88:2995-3001, 2000; Ritchie et al., Endocrinology
138:1145-1150, 1997). Fibroproliferative responses of the bone
originating in the skeleton per se include ostepetrosis and
hyperstosis. A defect in osteoblast differentiation and function is
thought to be a major cause in osteopetrosis, an inherited disorder
characterized by bone sclerosis due to reduced bone resorption,
marrow cavities fail to develop, resulting in extramedullary
hematopoiesis and severe hematologic abnormalities associated with
optic atrophy, deafness, and mental retardation (Lajeunesse et al.,
J. Clin Invest. 98:1835-1842, 1996). In osteoarthritis, bone
changes are known to occur, and bone collagen metabolism is
increased within osteoarthritic femoral heads. The greatest changes
occur within the subchondral zone, supporting a greater proportion
of osteoid in the diseased tissue (Mansell and Bailey, J. Clin.
Invest. 101:1596-1603, 1998). As shown in more detail below, zvegf3
has been found to be produced by prostate cells and to stimulate an
osteoblast cell line.
[0039] Fibroproliferative disorders of the vasculature include, for
example, transplant vasculopathy, which is a major cause of chronic
rejection of heart transplantation. Transplant vasculopathy is
characterized by accelerated atherosclerotic plaque formation with
diffuse occlusion of the coronary arteries, which is a "classic"
fibroproliferative disease. See, Miller et al., Circulation
101:1598-1605, 2000).
[0040] Antibodies used as zvegf3 antagonists include antibodies
that specifically bind to a zvegf3 protein and, by so binding,
reduce or prevent the binding of zvegf3 protein to the receptor
and, consequently, reduce or block the receptor-mediated activity
of zvegf3. 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
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.
Monoclonal antibodies can also be produced in mice that have been
genetically altered to produce antibodies that have a human
structure.
[0041] Methods for preparing and isolating polyclonal and
monoclonal antibodies are well known in the art. See, for example,
Cooligan et al. (eds.), Current Protocols in Immunology, National
Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et
al., Molecular Cloning: A Laboratory Manual, second edition, Cold
Spring Harbor, N.Y., 1989; and Hurrell (ed.), Monoclonal Hybridoma
Antibodies: Techniques and Applications, CRC Press, Inc., Boca
Raton, Fla., 1982. As would be evident to one of ordinary skill in
the art, polyclonal antibodies can be generated by inoculating a
variety of warm-blooded animals such as horses, cows, goats, sheep,
dogs, chickens, rabbits, mice, and rats with a zvegf3 polypeptide
or a fragment thereof.
[0042] Immunogenic polypeptides will comprise an epitope-bearing
portion of a zvegf3 polypeptide (e.g., as shown in SEQ ID NO:2) or
receptor. An "epitope" is a region of a protein to which an
antibody can bind. See, for example, Geysen et al., Proc. Natl.
Acad. Sci. USA 81:3998-4002, 1984. Epitopes can be linear or
conformational, the latter being composed of discontinuous regions
of the protein that form an epitope upon folding of the protein.
Linear epitopes are generally at least 6 amino acid residues in
length. Relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, Sutcliffe et
al., Science 219:660-666, 1983. Immunogenic, epitope-bearing
polypeptides contain a sequence of at least six, often at least
nine, more often from 15 to about 30 contiguous amino acid residues
of a zvegf3 protein. Polypeptides comprising a larger portion of a
zvegf3 protein, i.e. from 30 to 50 residues up to the entire
sequence are included. It is preferred that the amino acid sequence
of the epitope-bearing polypeptide is selected to provide
substantial solubility in aqueous solvents, that is the sequence
includes relatively hydrophilic residues, and hydrophobic residues
are substantially avoided. Such regions include residues 43-48,
96-101, 97-102, 260-265, and 330-335 of SEQ ID NO:2. As noted
above, it is generally preferred to use somewhat longer peptides as
immunogens, such as a peptide comprising residues 80-104, 299-314,
and 299-326 of SEQ ID NO:2. The latter peptide can be prepared with
an additional N-terminal Cys residue to facilitate coupling.
[0043] The immunogenicity of a polypeptide immunogen 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 a zvegf3 polypeptide or a portion
thereof with an immunoglobulin polypeptide or with maltose binding
protein. 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.
[0044] Alternative techniques for generating or selecting
antibodies include in vitro exposure of lymphocytes to a
polypeptide immunogen, and selection of antibody display libraries
in phage or similar vectors (for instance, through use of
immobilized or labeled polypeptide). Techniques for creating and
screening such random peptide display libraries are known in the
art (e.g., 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 Laboratories (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 zvegf3 sequences disclosed herein to identify
proteins which bind to zvegf3.
[0045] Antibodies are determined to be specifically binding if they
bind to their intended target (e.g., zvegf3 protein or receptor)
with an affinity at least 10-fold greater than the binding affinity
to control (e.g., non-zvegf3 or non-receptor) polypeptide or
protein. In this regard, a "non-zvegf3 polypeptide" includes the
related molecules VEGF, VEGF-B, VEGF-C, VEGF-D, PIGF, PDGF-A, and
PDGF-B, but excludes zvegf3 polypeptides from non-human species.
Due to the high level of amino acid sequence identity expected
between zvegf3 orthologs, antibodies specific for human zvegf3 may
also bind to zvegf3 from other species. The binding affinity of an
antibody can be readily determined by one of ordinary skill in the
art, for example, by Scatchard analysis (Scatchard, G., Ann. NY
Acad. Sci. 51: 660-672, 1949). Methods for screening and isolating
specific antibodies are well known in the art. See, for example,
Paul (ed.), Fundamental Immunology, Raven Press, 1993; Getzoff et
al., Adv. in Immunol. 43:1-98, 1988; Goding (ed.), Monoclonal
Antibodies: Principles and Practice, Academic Press Ltd., 1996;
Benjamin et al., Ann. Rev. Immunol. 2:67-101, 1984.
[0046] Binding affinity can also be determined using a commercially
available biosensor instrument (BIAcore.TM., Pharmacia Biosensor,
Piscataway, N.J.), wherein protein is immobilized onto the surface
of a receptor chip. See, Karlsson, J. Immunol. Methods 145:229-240,
1991 and Cunningham and Wells, J. Mol. Biol. 234:554-563, 1993.
This system allows the determination of on- and off-rates, from
which binding affinity can be calculated, and assessment of
stoichiometry of binding.
[0047] A variety of assays known to those skilled in the art can be
utilized to detect antibodies that specifically bind to zvegf3
proteins or receptors. 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 assays,
inhibition or competition assays, and sandwich assays.
[0048] For therapeutic applications it is generally preferred to
use neutralizing antibodies. As used herein, the term "neutralizing
antibody" denotes an antibody that inhibits at least 50% of the
biological activity of the cognate antigen when the antibody is
added at a 1000-fold molar access. Those of skill in the art will
recognize that greater neutralizing activity is sometimes
desirable, and antibodies that provide 50% inhibition at a 100-fold
or 10-fold molar access may be advantageously employed.
[0049] Zvegf3 antagonists further include antisense
polynucleotides, which can be used to inhibit zvegf3 gene
transcription and thereby inhibit cell activation and/or
proliferation in vivo. Polynucleotides that are complementary to a
segment of a zvegf3-encoding polynucleotide (e.g., a polynucleotide
as set forth in SEQ ID NO:1) are designed to bind to
zvegf3-encoding mRNA and to inhibit translation of such mRNA.
Antisense polynucleotides can be targetted to specific tissues
using a gene therapy approach with specific vectors and/or
promoters, such as viral delivery systems as disclosed in more
detail below.
[0050] Ribozymes can also be used as zvegf3 antagonists within the
present invention. Ribozymes are RNA molecules that contain a
catalytic center and a target RNA binding portion. The term
includes RNA enzymes, self-splicing RNAs, self-cleaving RNAs, and
nucleic acid molecules that perform these catalytic functions. A
ribozyme selectively binds to a target RNA molecule through
complementary base pairing, bringing the catalytic center into
close proximity with the target sequence. The ribozyme then cleaves
the target RNA and is released, after which it is able to bind and
cleave additional molecules. A nucleic acid molecule that encodes a
ribozyme is termed a "ribozyme gene." Ribozymes can be designed to
express endonuclease activity that is directed to a certain target
sequence in a mRNA molecule (see, for example, Draper and Macejak,
U.S. Pat. No. 5,496,698, McSwiggen, U.S. Pat. No. 5,525,468,
Chowrira and McSwiggen, U.S. Pat. No. 5,631,359, and Robertson and
Goldberg, U.S. Pat. No. 5,225,337). An expression vector can be
constructed in which a regulatory element is operably linked to a
nucleotide sequence that encodes a ribozyme.
[0051] In another approach, expression vectors can be constructed
in which a regulatory element directs the production of RNA
transcripts capable of promoting RNase P-mediated cleavage of mRNA
molecules that encode a zvegf3 polypeptide. According to this
approach, an external guide sequence can be constructed for
directing the endogenous ribozyme, RNase P, to a particular species
of intracellular mRNA, which is subsequently cleaved by the
cellular ribozyme (see, for example, Altman et al., U.S. Pat. No.
5,168,053; Yuan et al., Science 263:1269, 1994; Pace et al., WIPO
Publication No. WO 96/18733; George et al., WIPO Publication No. WO
96/21731; and Werner et al., WIPO Publication No. WO 97/33991). An
external guide sequence generally comprises a ten- to
fifteen-nucleotide sequence complementary to zvegf3 mRNA, and a
3'-NCCA nucleotide sequence, wherein N is preferably a purine. The
external guide sequence transcripts bind to the targeted mRNA
species by the formation of base pairs between the mRNA and the
complementary external guide sequences, thus promoting cleavage of
mRNA by RNase P at the nucleotide located at the 5'-side of the
base-paired region.
[0052] The growth factor domain of zvegf3 has been found to be the
active (PDGF receptor-binding) species of the molecule. Proteolytic
processing to remove the N-terminal portion of the molecule is
required for activation. Thus, inhibitors of this proteolytic
activation can also be used as zvegf3 antagonists within the
present invention.
[0053] For pharmaceutical use, zvegf3 antagonists are formulated
for topical or parenteral, particularly intravenous or
subcutaneous, delivery according to conventional methods. In
general, pharmaceutical formulations will include a zvegf3
antagonist 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, etc. Methods of formulation are well
known in the art and are disclosed, for example, in Remington: The
Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing
Co., Easton, Pa., 19th ed., 1995. A "therapeutically effective
amount" of a composition is that amount that produces a
statistically significant effect, such as a statistically
significant reduction in disease progression or a statistically
significant improvement in organ function. The exact dose will be
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 therapeutic formulations
will generally be administered over the period required to achieve
a beneficial effect, commonly up to several months and, in
treatment of chronic conditions, for a year or more. Dosing is
daily or intermittently over the period of treatment. Intravenous
administration will be by bolus injection or infusion over a
typical period of one to several hours. Sustained release
formulations can also be employed. For treatment of pulmonary
fibrosis, a zvegf3 antagonist can be delivered by aerosolization
according to methods known in the art. See, for example, Wang et
al., U.S. Pat. No. 5,011,678; Gonda et al., U.S. Pat. No.
5,743,250; and Lloyd et al., U.S. Pat. No. 5,960,792.
[0054] Other mitogenic factors, including EGF, TGF.beta., and FGF,
have been implicated in the initiation or perpetuation of fibrosis.
It may therefore be advantageous to combine a zvegf3 inhibitor with
one or more inhibitors of these other factors.
[0055] Antibodies are preferably administered parenterally, such as
by bolus injection or infusion (intravenous, intramuscular,
intraperitoneal or subcutaneous) over the course of treatment.
Antibodies are generally administered in an amount suficient to
provide a minimum circulating level of antibody throughout the
treatment period of between approximately 20 .mu.g and 1 mg/kg body
weight. In this regard, it is preferred to use antibodies having a
circulating half-life of at least 12 hours, preferably at least 4
days, more preferably up to 14-21 days. Chimeric and humanized
antibodies are expected to have circulatory half-lives of up to
four and up to 14-21 days, respectively. In many cases it will be
preferable to administer daily doses during a hospital stay,
followed by less frequent bolus injections during a period of
outpatient treatment. Antibodies can also be delivered by
slow-release delivery systems, pumps, and other known delivery
systems for continuous infusion. Dosing regimens may be varied to
provide the desired circulating levels of a particular antibody
based on its pharmacokinetics. Thus, doses will be calculated so
that the desired circulating level of therapeutic agent is
maintained. Daily doses referred to above may be administered as
larger, less frequent bolus administrations to provide the recited
dose averaged over the term of administration.
[0056] Those skilled in the art will recognize that the same
principles will guide the use of other zvegf3 antagonists. The
dosing regimen for a given antagonist will be determined by a
number of factors including potency, pharmacokinetics, and the
physicochemical nature of the antagonist. For example, non-peptidic
zvegf3 antagonists may be administered enterally.
[0057] Therapeutic polynucleotides, such as antisense
polynucleotides, can be delivered to patients or test animals by
way of viral delivery systems. Exemplary viruses for this purpose
include adenovirus, herpesvirus, retroviruses, vaccinia virus, and
adeno-associated virus (AAV). Adenovirus, a double-stranded DNA
virus, is currently the best studied gene transfer vector for
delivery of heterologous nucleic acids. For review, see Becker et
al., Meth. Cell Biol. 43:161-89, 1994; and Douglas and Curiel,
Science & Medicine 4:44-53, 1997. The adenovirus system offers
several advantages. Adenovirus can (i) accommodate relatively large
DNA inserts; (ii) be grown to high-titer; (iii) infect a broad
range of mammalian cell types; and (iv) be used with many different
promoters including ubiquitous, tissue specific, and regulatable
promoters. Because adenoviruses are stable in the bloodstream, they
can be administered by intravenous injection.
[0058] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. When
intravenously administered to intact animals, adenovirus primarily
targets the liver. If the adenoviral delivery system has an E1 gene
deletion, the virus cannot replicate in the host cells. However,
the host's tissue (e.g., liver) will express and process (and, if a
signal sequence is present, secrete) the heterologous protein.
[0059] An alternative method of gene delivery comprises removing
cells from the body and introducing a vector into the cells as a
naked DNA plasmid. The transformed cells are then re-implanted in
the body. Naked DNA vectors are introduced into host cells by
methods known in the art, including transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun, or use of a DNA vector
transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988;
Wu et al., J. Biol. Chem. 267:963-967, 1992; and Johnston and Tang,
Meth. Cell Biol. 43:353-365, 1994.
[0060] Activity of zvegf3 antagonists can be measured in vitro
using assays (including cell-based assays) designed to measure
zvegf3 activity. Antagonists will reduce the effects of zvegf3
within the assay. Ligand-receptor binding can be assayed by a
variety of methods well known in the art, including receptor
competition assays (Bowen-Pope and Ross, Methods Enzymol.
109:69-100, 1985) and through the use of soluble receptors,
including receptors produced as IgG fusion proteins (U.S. Pat. No.
5,750,375). Receptor binding assays can be performed on cell lines
that contain known cell-surface receptors for evaluation. The
receptors can be naturally present in the cell, or can be
recombinant receptors expressed by genetically engineered cells.
Mitogenic activity can be measured using known assays, including
.sup.3H-thymidine incorporation assays (as disclosed by, e.g.,
Raines and Ross, Methods Enzymol. 109:749-773, 1985 and Wahl et
al., Mol. Cell Biol. 8:5016-5025, 1988), dye incorporation assays
(as disclosed by, for example, Mosman, J. Immunol. Meth. 65:55-63,
1983 and Raz et al., Acta Trop. 68:139-147, 1997) or cell counts.
Suitable mitogenesis assays measure incorporation of
.sup.3H-thymidine into (1) 20% confluent cultures to look for the
ability of zvegf3 proteins to further stimulate proliferating
cells, and (2) quiescent cells held at confluence for 48 hours to
look for the ability of zvegf3 proteins to overcome contact-induced
growth inhibition. Suitable dye incorporation assays include
measurement of the incorporation of the dye Alamar blue (Raz et
al., ibid.) into target cells. See also, Gospodarowicz et al., J.
Cell. Biol. 70:395-405, 1976; Ewton and Florini, Endocrinol.
106:577-583, 1980; and Gospodarowicz et al., Proc. Natl. Acad. Sci.
USA 86:7311-7315, 1989.
[0061] The biological activities of zvegf3 antagonists can be
studied in non-human animals by administration of exogenous
compounds, by expression of zvegf3 antisense polynucleotides, and
by suppression of endogenous zvegf3 expression through knock-out
techniques. Viral delivery systems (disclosed above) can be
employed. Zvegf3 antagonists can be administered or expressed
individually, in combination with other zvegf3 antagonists, or in
combination other compounds, including other growth factor
antagonists. Test animals are monitored for changes in such
parameters as clinical signs, body weight, blood cell counts,
clinical chemistry, histopathology, and the like.
[0062] Effects of zvegf3 antagonists on liver and kidney fibrosis
can be tested in known animal models, such as the db/db mouse model
disclosed by Cohen et al., Diabetologia 39:270-274, 1996 and Cohen
et al., J. Clin. Invest. 95:2338-2345, 1995, transgenic animal
models (Imai et al., Contrib. Nephrol. 107:205-215, 1994), and the
CCl.sub.4-induced cirrhosis model (Rojkind and Greenwel, Adv. Vet.
Sci. Comp. Med. 37:333-355, 1993; Daz-Gil et al., J. Hepatol.
30:1065-1072, 1999).
[0063] Effects on lung fibrosis can be assayed in a mouse model
using bleomycin. The chemotherapy agent bleomycin is a known
causative agent of pulmonary fibrosis in humans and can induce
interstitial lung disease in mice, including an increase in the
number of fibroblasts, enhanced collagen deposition, and
dysregulated matrix remodeling. C57B1/6 mice are administered
bleomycin by osmotic minipump for 1 week. There follows a period of
inflammation, with cutaneous toxicity beginning approximately 4-7
days after bleomycin administration and continuing for about a
week, after which the mice appear to regain health. About 3-4 weeks
after the finish of bleomycin delivery, the mice are sacrificed,
and the lungs are examined histologically for signs of fibrosis.
Scoring is based on the extent of lung fibrotic lesions and their
severity. Serum is assayed for lactic dehydrogenase, an
intracellular enzyme that is released into the circulation upon
general cell death or injury. Lung tissue is assayed for
hydroxyproline as a measure of collagen deposition.
[0064] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
[0065] A human salivary gland library was screened for a
full-length clone of zvegf3 by PCR. This library was an arrayed
library representing 9.6.times.10.sup.5 clones made in the vector
pZP5x. The vector pZP5x is the same as vector pZP-9 (deposited with
American Type Culture Collection, 10801 University Blvd., Manassas,
Va. under Accession Number 98668), but contains a cytomegalovirus
promoter instead of a metallothionein promoter between the Asp718
and BamHI sites. The plasmid thus comprises a dihyrofolate
reductase gene under control of the SV40 early promoter and SV40
polyadenylation site, and a cloning site to insert the gene of
interest under control of the CMV promoter and the human growth
hormone (hGH) gene polyadenylation site. The working plate
containing 80 pools of 12,000 colonies each was screened by PCR
using oligonucleotide primers ZC19,045 (SEQ ID NO:6) and ZC19,047
(SEQ ID NO:7) with an annealing temperature of 60.degree. C. for 35
cycles. There were two strong positives, pools 58 (T-8 F1-F12) and
77 (T-7 H1-H12). The corresponding pools in the transfer plate were
then screened by PCR using the same conditions. Two positives were
obtained at the transfer level. The positives were T-7H11 and T-8
F10. 5' RACE reactions were done on the transfer plate pools, and
the fragments were sequenced to check zvegf3 sequence and determine
if a full-length clone was present. For PCR, oligonucleotide
primers ZC12,700 (SEQ ID NO:8) and ZC19,045 (SEQ ID NO:6) were used
at an annealing temperature of 61.degree. C. for 5 cycles, then
55.degree. C. for 30 cycles. Sequencing showed that the pool T-7H
11 had a frameshift. Transfer plate 8 pool F10 sequence appeared to
be correct, so this pool of DNA was used in filter lifts.
[0066] Pool F10 from transfer plate 8 was plated and filter lifted
using nylon membranes (Hybond-N.TM.; Amersham Corporation).
Approximately 1200 colonies per plate on each of 5 filters were
lifted for a total of approximately 6000 colonies. The filters were
marked with a hot needle for orientation, then denatured for 6
minutes in 0.5 M NaOH and 1.5 M Tris-HCl, pH 7.2. The filters were
then neutralized in 1.5 M NaCl and 0.5 M Tris-HCl, pH 7.2 for 6
minutes. The DNA was affixed to the filters using a UV crosslinker
(Stratalinker.RTM., Stratagene, La Jolla, Calif.) at 1200 joules.
The filters were prewashed at 65.degree. C. in prewash buffer
consisting of 0.25.times.SSC, 0.25% SDS, and 1 mM EDTA. The
solution was changed a total of three times over a 45-minute period
to remove cell debris. Filters were prehybridized for approximately
3 hours at 65.degree. C. in 25 ml of ExpressHyb.TM.. The probe was
generated using an approximately 400-bp fragment produced by
digestion of the first proprietary database clone with EcoRI and
BglII and gel-purified using a spin column as disclosed above. The
probe was radioactively labeled with .sup.32P by random priming as
disclosed above and purified using a push column. ExpressHyb.TM.
solution was used for the hybridizing solution for the filters.
Hybridization took place overnight at 65.degree. C. Blots were
rinsed 2.times.in 65.degree. C. solution 1 (2.times.SSC, 0.1% SDS),
then washed 4 times in solution 1 at 65.degree. C. The filters were
exposed to film overnight at -80.degree. C. There were 14 positives
on the filters. 85 clones were picked from the positive areas and
screened by PCR using oligonucleotide primers ZC19,045 (SEQ ID
NO:6) and ZC19,047 (SEQ ID NO:7) and an annealing temperature of
60.degree. C. Thirteen positives were obtained and streaked out for
individual clones. Twenty-four colonies were picked and checked by
PCR as previously described. Six positives were obtained, two of
which were sequenced. Both sequences were the same and full length.
The sequence is shown in SEQ ID NO:1.
Example 2
[0067] A PCR panel was screened for mouse zvegf3 DNA. The panel
contained 8 cDNA samples from brain, bone marrow, 15-day embryo,
testis, salivary gland, placenta, 15-day embryo (Clontech
Laboratories), and 17-day embryo (Clontech Laboratories)
libraries.
[0068] PCR mixtures contained oligonucleotide primers ZC21,222 (SEQ
ID NO:9) and ZC21,224 (SEQ ID NO:10). The reaction was run at an
annealing temperature of 66.degree. C. with an extension time of 2
minutes for a total of 35 cycles using Ex Taq.TM. DNA polymerase
(PanVera, Madison, Wis.) plus antibody. DNA samples found to be
positive for zvegf3 by PCR and confirmed by sequencing included
mouse 15-day embryo library total pool cDNA, mouse 15-day embryo
(Clontech Laboratories) and 17-day embryo (both obtained from
Clontech Laboratories), mouse salivary gland library total pool
cDNA, and mouse testis library total pool cDNA. Fragments of about
600 bp from each of the mouse 15-day embryo library total pool
cDNA, mouse 15-day embryo mcDNA, and mouse 17-day embryo mcDNA PCR
products were sequenced. Sequence from the mouse 17-day embryo
mcDNA and mouse 15-day embryo library total pool cDNA products
confirmed the fragments to be mouse zvegf3 DNA.
[0069] The mouse 15-day embryo library was screened for full-length
zvegf3 DNA. This library was an arrayed library representing
9.6.times.10.sup.5 clones in the pCMV.cndot.SPORT 2 vector (Life
Technologies, Gaithersburg, Md.). The working plate, containing 80
pools of 12,000 colonies each, was screened by PCR using
oligonucleotide primers ZC21,223 (SEQ ID NO:11) and ZC21,224 (SEQ
ID NO:10) with an annealing temperature of 66.degree. C. for 35
cycles. Eighteen positives were obtained. Fragments from four pools
(A2, A10, B2, and C4) were sequenced; all were confirmed to encode
zvegf3. Additional rounds of screening using the same reaction
conditions and pools from the working and source plates identified
one positive pool (5D).
[0070] Positive colonies were screened by hybridization. Pool 5D
from original source plate #5 was plated at about 250 colonies per
plate and transferred to nylon membranes (Hybond-N.TM.; Amersham
Corporation, Arlington Heights, Ill.). Five filters were lifted for
a total of .about.1250 colonies. The filters were marked with a hot
needle for orientation, then denatured for 6 minutes in 0.5 M NaOH
and 1.5 M Tris-HCl, pH 7.2. The filters were then neutralized in
1.5 M NaCl and 0.5 M Tris-HCl, pH 7.2 for 6 minutes. The DNA was
fixed to the filters using a a UV crosslinker (Stratalinker.RTM.,
Stratagene, La Jolla, Calif.) at 1200 joules. A probe was generated
by PCR using oligonucleotide primers ZC21,223 (SEQ ID NO:11) and
ZC21,224 (SEQ ID NO:10), and a mouse 15-day embryo template at an
annealing temperature of 66.degree. C. for 35 cycles. The PCR
fragment was gel purified using a spin column containing a silica
gel membrane (QIAquick.TM. Gel Extraction Kit; Qiagen, Inc.,
Valencia, Calif.). The DNA was radioactively labeled with p using a
commercially available kit (Rediprime.TM. II random-prime labeling
system; Amersham Corp., Arlington Heights, Ill.) according to the
manufacturer's specifications. The probe was purified using a
commercially available push column (NucTrap.RTM. column;
Stratagene, La Jolla, Calif.; see U.S. Pat. No. 5,336,412). The
filters were prewashed at 65.degree. C. in prewash buffer
consisting of 0.25.times.SSC, 0.25% SDS and 1 mM EDTA. The solution
was changed a total of three times over a 45-minute period to
remove cell debris. Filters were prehybridized overnight at
65.degree. C. in 25 ml of a hybridization solution (ExpressHyb.TM.
Hybridization Solution; Clontech Laboratories, Inc., Palo Alto,
Calif.), then hybridized overnight at 65.degree. C in the same
solution. Filters were rinsed twice at 65.degree. C. in pre-wash
buffer (0.25.times.SSC, 0.25% SDS, and 1 mM EDTA), then washed
twice in pre-wash buffer at 65.degree. C. Filters were exposed to
film for 2 days at -80.degree. C. There were 10 positives on the
filters. 3 clones were picked from the positive areas, streaked
out, and 15 individual colonies from these three positives were
screened by PCR using primers ZC21,223 (SEQ ID NO:11) and ZC21,334
(SEQ ID NO:12) at an annealing temp of 66.degree. C. Two positives
were recovered and sequenced. Both sequences were found to be the
same and encoded full-length mouse zvegf3 (SEQ ID NO:4).
[0071] The amino acid sequence is highly conserved between mouse
and human zvegf3s, with an overall amino acid sequence identity of
87%. The secretory peptide, CUB domain, inter-domain, and growth
factor domain have 82%, 92%, 79% and 94% amino acid identity,
respectively.
Example 3
[0072] A mammalian cell expression vector for the growth factor
domain of zvegf3 was constructed by joining the zvegf3 fragment to
a sequence encoding an optimized t-PA secretory signal sequence
(U.S. Pat. No. 5,641,655) in the linearized pZMP11 vector
downstream of the CMV promoter. The plasmid pZMP11 is a mammalian
expression vector containing an expression cassette having the CMV
immediate early promoter, a consensus intron from the variable
region of mouse immunoglobulin heavy chain locus, Kozak sequences,
multiple restriction sites for insertion of coding sequences, a
stop codon, and a human growth hormone terminator. The plasmid also
contains an IRES element from poliovirus, the extracellular domain
of CD8 truncated at the C-terminal end of the transmembrane domain,
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, and the URA3 and
CEN-ARS sequences required for selection and replication in S.
cerevisiae. The resulting vector was designated
pZMP111/zv3GF-otPA.
[0073] BHK 570 cells were transfected with pZMP11/zv3GF-otPA by
liposome-mediated transfection (using (Lipofectamine.TM.; Life
Technologies) and cultured according to conventional
procedures.
[0074] BHK cell-conditioned media was adjusted to 20 mM MES at pH
5.5. A column of cation exchange resin (Poros.RTM. HS 50;
PerSeptive Biosystems, Framingham, Mass.) (2-cm diameter; 50 ml bed
volume) was equilibrated in 20 mM MES, 150 mM NaCl, pH 5.5. The
adjusted media was pumped into the column at 20 ml/minute. When
loading was completed, the column was washed, first in 20 mM MES,
150 mM NaCl, pH 5.5, then with the same composition buffer at pH
6.0. Once the baseline was back to zero absorbance, the column was
eluted with a 10 column volume gradient to 20 mM MES, 1M NaCl, pH
6.0. The zvegf3 growth factor domain eluted at 25 to 50 mS
conductivity during the evolving gradient. Reducing SDS-PAGE
revealed a distinct band at 20 kD, which was confirmed as zvegf3 by
Western blotting., This material was pooled and prepared for
loading to a tandem column array comprising a strong anion exchange
resin (Poros.RTM. HQ 50; PerSeptive Biosystems) followed by an
immobilized metal (nickel) affinity column in series. The system of
columns was equilibrated in 20 mM MOPS buffer at pH 7.0. The vegf3
pool was in-line diluted at 1:10 (V:V) with the MOPS equilibration
buffer while loading. After loading was completed the column series
was washed with 20 mM MOPS pH 7.0 buffer until baseline absorbance
was obtained. The nickel column was then disconnected fron the
anion exchanger and washed with 20 mM MOPS pH 7.0 containing 150 mM
NaCl. The columnwais then eluted with a 1 column volume gradient
between the last washing buffer and the same buffer containing 20
mM imidazole at pH 7.0. The fractions containing the zvegf3 growth
factor domain were pooled and concentrated using 5 kD cuttoff
membrane in preparation for buffer exchange and polishing on a size
exclusion column equilibrated in PBS.
Example 4
[0075] To make transgenic animals expressing zvegf3 genes requires
adult, fertile males (studs) (B6C3f1,2-8 months of age (Taconic
Farms, Germantown, N.Y.)), vasectomized males (duds) (B6D2f1, 2-8
months, (Taconic Farms)), prepubescent fertile females (donors)
(B6C3f1, 4-5 weeks, (Taconic Farms)) and adult fertile females
(recipients) (B6D2f1, 2-4 months, (Taconic Farms)).
[0076] The donors are acclimated for 1 week, then injected with
approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin
(Sigma, St. Louis, Mo.) I.P., and 46-47 hours later, 8 Iu/mouse of
human Chorionic Gonadotropin (hCG (Sigma)) I.P. to induce
superovulation. Donors are mated with studs subsequent to hormone
injections. Ovulation generally occurs within 13 hours of hCG
injection. Copulation is confirmed by the presence of a vaginal
plug the morning following mating.
[0077] Fertilized eggs are collected under a surgical scope (Leica
MZ12 Stereo Microscope, Leica, Wetzlar, Germany). The oviducts are
collected and eggs are released into urinanalysis slides containing
hyaluronidase (Sigma Chemical Co.). Eggs are washed once in
hyaluronidase, and twice in Whitten's W640 medium (Table 2; all
reagents available from Sigma Chemical Co.) that has been incubated
with 5% CO.sub.2, 5% O.sub.2, and 90% N.sub.2 at 37.degree. C. The
eggs are stored in a 37.degree. C./5% CO.sub.2 incubator until
microinjection.
2 TABLE 2 mgs/200 ml mgs/500 ml NaCl 1280 3200 KCl 72 180
KH.sub.2PO.sub.4 32 80 MgSO.sub.4.7H.sub.2O 60 150 Glucose 200 500
Ca.sup.2+ Lactate 106 265 Benzylpenicillin 15 37.5 Streptomycin
SO.sub.4 10 25 NaHCO.sub.3 380 950 Na Pyruvate 5 12.5 H.sub.20 200
ml 500 ml 500 mM EDTA 100 .mu.l 250 .mu.l 5% Phenol Red 200 .mu.l
500 .mu.l BSA 600 1500
[0078] Zvegf3 cDNA is inserted into the expression vector pHB12-8.
Vector pHB12-8 was derived from p2999B4 (Palmiter et al., Mol. Cell
Biol. 13:5266-5275, 1993) by insertion of a rat insulin II intron
(ca. 200 bp) and polylinker (Fse I/Pme I/Asc I) into the Nru I
site. The vector comprises a mouse metallothionein (MT-1) promoter
(ca. 750 bp) and human growth hormone (hGH) untranslated region and
polyadenylation signal (ca. 650 bp) flanked by 10 kb of MT-1 5'
flanking sequence and 7 kb of MT-1 3' flanking sequence. The cDNA
is inserted between the insulin II and hGH sequences.
[0079] 10-20 micrograms of plasmid DNA is linearized, gel-purified,
and resuspended in 10 mM Tris pH 7.4, 0.25 mM EDTA pH 8.0, at a
final concentration of 5-10 nanograms per microliter for
microinjection.
[0080] Plasmid DNA is microinjected into harvested eggs contained
in a drop of W640 medium overlaid by warm, CO.sub.2-equilibrated
mineral oil. The DNA is drawn into an injection needle (pulled from
a 0.75 mm ID, 1 mm OD borosilicate glass capillary) and injected
into individual eggs. Each egg is penetrated with the injection
needle into one or both of the haploid pronuclei.
[0081] Picoliters of DNA are injected into the pronuclei, and the
injection needle is withdrawn without coming into contact with the
nucleoli. The procedure is repeated until all the eggs are
injected. Successfully microinjected eggs are transferred into an
organ tissue-culture dish with pregassed W640 medium for storage
overnight in a 37.degree. C./5% CO.sub.2 incubator.
[0082] The following day, 2-cell embryos are transferred into
pseudopregnant recipients. The recipients are identified by the
presence of copulation plugs, after copulating with vasectomized
duds. Recipients are anesthetized and shaved on the dorsal left
side and transferred to a surgical microscope. A small incision is
made in the skin and through the muscle wall in the middle of the
abdominal area outlined by the ribcage, the saddle, and the hind
leg, midway between knee and spleen. The reproductive organs are
exteriorized onto a small surgical drape. The fat pad is stretched
out over the surgical drape, and a baby serrefine (Roboz,
Rockville, Md.) is attached to the fat pad and left hanging over
the back of the mouse, preventing the organs from sliding back
in.
[0083] With a fine transfer pipette containing mineral oil followed
by alternating W640 and air bubbles, 12-17 healthy 2-cell embryos
from the previous day's injection are transferred into the
recipient. The swollen ampulla is located and holding the oviduct
between the ampulla and the bursa, and a nick in the oviduct is
made with a 28 g needle close to the bursa, making sure not to tear
the ampulla or the bursa.
[0084] The pipette is transferred into the nick in the oviduct, and
the embryos are blown in, allowing the first air bubble to escape
the pipette. The fat pad is gently pushed into the peritoneum, and
the reproductive organs are allowed to slide in. The peritoneal
wall is closed with one suture, and the skin is closed with a wound
clip. The mice recuperate on a 37.degree. C. slide warmer for a
minimum of 4 hours.
[0085] The recipients are returned to cages in pairs, and allowed
19-21 days gestation. After birth, 19-21 days postpartum is allowed
before weaning. The weanlings are sexed and placed into separate
sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off
the tail with clean scissors.
[0086] Genomic DNA is prepared from the tail snips using a
commercially available kit (DNeasy.TM. 96 Tissue Kit; Qiagen,
Valencia, Calif.) following the manufacturer's instructions.
Genomic DNA is analyzed by PCR using primers designed to the human
growth hormone (hGH) 3' UTR portion of the transgenic vector. The
use of a region unique to the human sequence (identified from an
alignment of the human and mouse growth hormone 3' UTR DNA
sequences) ensures that the PCR reaction does not amplify the mouse
sequence. Primers ZC17,251 (SEQ ID NO:13) and ZC17,252 (SEQ ID
NO:14) amplify a 368-base-pair fragment of hGH. In addition,
primers ZC17,156 (SEQ ID NO:15) and ZC17,157 (SEQ ID NO:16), which
hybridize to vector sequences and amplify the cDNA insert, may be
used along with the hGH primers. In these experiments, DNA from
animals positive for the transgene will generate two bands, a
368-base-pair band corresponding to the hGH 3' UTR fragment and a
band of variable size corresponding to the cDNA insert.
[0087] Once animals are confirmed to be transgenic (TG), they are
back-crossed into an inbred strain by placing a TG female with a
wild-type male, or a TG male with one or two wild-type female(s).
As pups are born and weaned, the sexes are separated, and their
tails snipped for genotyping.
[0088] To check for expression of a transgene in a live animal, a
partial hepatectomy is performed. A surgical prep is made of the
upper abdomen directly below the xiphoid process. Using sterile
technique, a small 1.5-2 cm incision is made below the sternum, and
the left lateral lobe of the liver is exteriorized. Using 4-0 silk,
a tie is made around the lower lobe securing it outside the body
cavity. An atraumatic clamp is used to hold the tie while a second
loop of absorbable Dexon (American Cyanamid, Wayne, N.J.) is placed
proximal to the first tie. A distal cut is made from the Dexon tie,
and approximately 100 mg of the excised liver tissue is placed in a
sterile petri dish. The excised liver section is transferred to a
14-ml polypropylene round bottom tube, snap frozen in liquid
nitrogen, and stored on dry ice. The surgical site is closed with
suture and wound clips, and the animal's cage is placed on a
37.degree. C. heating pad for 24 hours post-operatively. The animal
is checked daily post-operatively, and the wound clips are removed
7-10 days after surgery.
[0089] Analysis of the mRNA expression level of each transgene is
done using an RNA solution hybridization assay or real-time PCR on
an ABI Prism 7700 (PE Applied Biosystems, Inc., Foster City,
Calif.) following the manufacturer's instructions.
[0090] An adenovirus vector was prepared using a liver-specific
albumin gene enhancer and basal promoter (designated "AEO
promoter"). The albumin promoter construct (designated pAEO) was
constructed by inserting a 2.2 kb NotI/EcoRV fragment from
pALBdelta2L (Pinkert et al., Genes Dev. 1:268-276, 1987) and an 850
bp NruI/NotI DNA segment comprising the rat insulin II intron, an
FseI/PmeI/AscI polylinker, and the human growth hormone poly A
sequence into a commercially available phagemid vector
(pBluescript.RTM. KS(+); Stratagene, La Jolla, Calif.). For
microinjection, the plasmid is digested with Not1 to liberate the
expression cassette.
[0091] An additional adenovirus vector was constructed using an
epithelial cell-specific keratin gene (K14) promoter (Vassar et
al., Proc. Natl. Acad. Sci. USA 86:1563-1567, 1989). The 1038-bp
open reading frame encoding full-length human zvegf3 was amplified
by PCR so as to introduce an optimized initiation codon and
flanking 5' PmeI and 3' AscI sites using the primers ZC20,180 (SEQ
ID NO:17) and ZC20,181 (SEQ ID NO:18). The resulting PmeI/AscI
fragment was subcloned into the polylinker of pKFO114, a basal
keratinocyte-restricted transgenic vector comprising the human
keratin 14 (K14) promoter (an approximately 2.3 Kb fragment
amplified from human genomic DNA [obtained from Clontech
Laboratories, Inc.] based on the sequence of Staggers et al.,
"Sequence of the promoter for the epidermal keratin gene, K14",
GenBank accession #U11076, 1994), followed by a heterologous intron
(a 294-bp BstXI/PstI fragment from pIRES1hyg (Clontech
Laboratories, Inc.; see, Huang and Gorman, Nucleic Acids Res.
18:937-947, 1990), a PmeI/AscI polylinker, and the human growth
hormone gene polyadenylation signal (a 627 bp SmaI/EcoRI fragment;
see, Seeburg, DNA 1:239-249, 1982). The transgene insert was
separated from the plasmid backbone by NotI digestion and agarose
gel purification, and fertilized ova from matings of B6C3FVB/NTac
mice or inbred FVB/NTac mice were microinjected and implanted into
pseudopregnant females essentially as described by Malik et al.,
Molec. Cell. Biol. 15:2349-2358, 1995. Transgenic founders were
identified by PCR on genomic tail DNA using primers specific for
the human growth hormone poly A signal (ZC17,252, SEQ ID NO:14; and
ZC17,251, SEQ ID NO:13) to amplify a 368-bp diagnostic product.
Transgenic lines were initiated by breeding founders with
C57BL/6Tac or FVB/NTac mice.
[0092] Transgenic mice were generated essentially as disclosed
above using MT-1, K14, and AEO promoters. Four MT-1/zvegf3
transgenic mice were generated. In one animal (female)
approximately 800 molecules zvegf3 mRNA/cell were produced in the
liver after zinc induction. This animal had enlargement of the
liver and spleen. Also observed were proliferation of hepatic
sinusoidal cells and extra-medullary hematopoiesis. One K14/zvegf3
transgenic mouse (female) showed a low level of expression with low
body weight, low hematocrit, and low platelet count. One AEO/zvegf3
transgenic mouse (male) with a low level of expression exhibited
liver sinusoidal cell proliferation.
[0093] Histological analysis was carried out on livers from Ni
transgenic mice overexpressing full-length zvegf3 from the AEO
promoter and nontransgenic littermate controls at 8, 16, 22, and 33
weeks of age. Similar changes were seen in male and female animals.
H & E and trichrome stains indicate a definite increase in
number of liver sinusoidal (stellate) cells and increased
perisinusoidal extracellular matrix (EMC) deposition at 8 weeks of
age. There was a persistent increase in number of stellate cells
and in the amount and thickness of perisinusoidal EMC as well as
some perivenular EMC deposition by 16 weeks. At 22 weeks similar
changes were seen, but with an increase in incidence and severity
of perivenular fibrosis (steato fibrosis). Changes similar to those
at 16 and 22 weeks were observed at 33 weeks, however some animals
had fibrotic banding and multiple, encapsulated areas in which
hepatocytes appeared enlarged and vacuolated. These areas tended to
be surrounded by a fibrous tissue capsule, and the hepatocytes
within these areas appeared normal. These changes were consistent
with early cirrhosis.
[0094] Similar changes were seen in 8-week-old N2 mice and in
breeder males sacrificed at approximately 32-34 weeks.
Example 5
[0095] For construction of adenovirus vectors, the protein coding
region of human zvegf3 was amplified by PCR using primers that
added PmeI and AscI restriction sties at the 5' and 3' termini
respectively. PCR primers ZC20,180 (SEQ ID NO:17) and ZC20,181 (SEQ
ID NO:18) were used with a full-length zvegf3 cDNA template in a
PCR reaction as follows: incubation at 95.degree. C. for 5 minutes;
followed by 15 cycles at 95.degree. C. for 1 min., 61.degree. C.
for 1 min., and 72.degree. C. for 1.5 min.; followed by 72.degree.
C. for 7 min.; followed by a 4.degree. C. soak. The reaction
product was loaded onto a 1.2% low-melting-temperature agarose gel
in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA). The zvegf3 PCR
product was excised from the gel and purified using a commercially
available kit comprising a silica gel mambrane spin column
(QIAquick.TM. PCR Purification Kit and gel cleanup kit; Qiagen,
Inc.) as per kit instructions. The zvegf3 product was then digested
with PmeI and AscI, phenol/chloroform extracted, EtOH precipitated,
and rehydrated in 20 ml TE (Tris/EDTA pH 8). The 1038 bp zvegf3
fragment was then ligated into the PmeI-AscI sites of the
transgenic vector pTG12-8 (also known as pHB12-8; see Example 4)
and transformed into E. coli DH10B.TM. competent cells by
electroporation. Clones containing zvegf3 were identified by
plasmid DNA miniprep followed by digestion with PmeI and AscI. A
positive clone was sequenced to insure that there were no deletions
or other anomalies in the construct. The sequence of zvegf3 cDNA
was confirmed.
[0096] DNA was prepared using a commercially available kit (Maxi
Kit, Qiagen, Inc.), and the 1038 bp zvegf3 cDNA was released from
the pTG12-8 vector using PmeI and AscI enzymes. The cDNA was
isolated on a 1% low melting temperature agarose gel and was
excised from the gel. The gel slice was melted at 70.degree. C.,
and the DNA was extracted twice with an equal volume of
Tris-buffered phenol and precipitated with EtOH. The DNA was
resuspended in 10 .mu.L H.sub.2O.
[0097] The zvegf3 cDNA was cloned into the EcoRV-AscI sites of a
modified pAdTrack-CMV (He, T-C. et al., Proc. Natl. Acad. Sci. USA
95:2509-2514, 1998). This construct contains the green fluorescent
protein (GFP) marker gene. The CMV promoter driving GFP expression
was replaced with the SV40 promoter, and the SV40 polyadenylation
signal was replaced with the human growth hormone polyadenylation
signal. In addition, the native polylinker was replaced with FseI,
EcoRV, and AscI sites. This modified form of pAdTrack-CMV was named
pZyTrack. Ligation was performed using a commercially available DNA
ligation and screening kit (Fast-Link.TM. kit; Epicentre
Technologies, Madison, Wis.). Clones containing zvegf3 were
identified by digestion of mini prep DNA with FseI and AscI. In
order to linearize the plasmid, approximately 5 .mu.g of the
resulting pZyTrack zvegf3 plasmid was digested with PmeI.
Approximately 1 .mu.g of the linearized plasmid was cotransformed
with 200 ng of supercoiled pAdEasy (He et al., ibid.) into E. coli
BJ5183 cells (He et al., ibid.). The co-transformation was done
using a Bio-Rad Gene Pulser at 2.5 kV, 200 ohms and 25 .mu.Fa. The
entire co-transformation mixture was plated on 4 LB plates
containing 25 .mu.g/ml kanamycin. The smallest colonies were picked
and expanded in LB/kanamycin, and recombinant adenovirus DNA was
identified by standard DNA miniprep procedures. Digestion of the
recombinant adenovirus DNA with FseI and AscI confirmed the
presence of the zvegf3 insert. The recombinant adenovirus miniprep
DNA was transformed into E. coli DH10B.TM. competent cells, and DNA
was prepared using a Maxi Kit (Qiagen, Inc.) according to kit
instructions.
[0098] Approximately 5 .mu.g of recombinant adenoviral DNA was
digested with PacI enzyme (New England Biolabs) for 3 hours at
37.degree. C. in a reaction volume of 100 .mu.l containing 20-30U
of PacI. The digested DNA was extracted twice with an equal volume
of phenol/chloroform and precipitated with ethanol. The DNA pellet
was resuspended in 10 .mu.l distilled water. A T25 flask of
QBI-293A cells (Quantum Biotechnologies, Inc. Montreal, Qc.
Canada), inoculated the day before and grown to 60-70% confluence,
were transfected with the PacI digested DNA. The PacI-digested DNA
was diluted up to a total volume of 50 .mu.l with sterile HBS (150
mM NaCl, 20 mM HEPES). In a separate tube, 20 .mu.l of 1 mg/ml
N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts
(DOTAP) (Boehringer Mannheim, Indianapolis, Ind.) was diluted to a
total volume of 100 .mu.l with HBS. The DNA was added to the DOTAP,
mixed gently by pipeting up and down, and left at room temperature
for 15 minutes. The media was removed from the 293A cells and
washed with 5 ml serum-free minimum essential medium (MEM) alpha
containing 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino
acids, and 25 mM HEPES buffer (reagents obtained from Life
Technologies, Gaithersburg, Md.). 5 ml of serum-free MEM was added,
and the cells were held at 37.degree. C. The DNA/lipid mixture was
added drop-wise to the flask of cells, mixed gently, and incubated
at 37.degree. C. for 4 hours. The media containing the DNA/lipid
mixture was then aspirated off and replaced with 5 ml complete MEM
containing 5% fetal bovine serum. The transfected cells were
monitored for GFP expression and formation of foci (viral
plaques).
[0099] Seven days after transfection of 293A cells with the
recombinant adenoviral DNA, the cells expressed GFP and started to
form foci. The crude viral lysate was collected using a cell
scraper to collect all of the 293A cells. The lysate was
transferred to a 50-ml conical tube. To release most of the virus
particles from the cells, three freeze/thaw cycles were done in a
dry ice/ethanol bath and a 37.degree. waterbath.
[0100] The crude lysate was amplified (Primary (10) amplification)
to obtain a working stock of zvegf3 rAdV lysate. Ten 10-cm plates
of nearly confluent (80-90%) 293A cells were set up 20 hours
previously, 200 .mu.l of crude rAdV lysate added to each 10-cm
plate and monitored for 48 to 72 hours looking for CPE under the
white light microscope and expression of GFP under the fluorescent
microscope. When all of the cells showed CPE (Cytopathic Effect)
this 1.degree. stock lysate was collected and freeze/thaw cycles
performed as described above.
[0101] Secondary (2.degree.) amplification of zvegf3 rAdV was
obtained as follows: Twenty 15-cm tissue culture dishes of 293A
cells were prepared so that the cells were 80-90% confluent. All
but 20 ml of 5% MEM media was removed, and each dish was inoculated
with 300-500 .mu.l of the 1.degree. amplified rAdv lysate. After 48
hours the cells were lysed from virus production, the lysate was
collected into 250-ml polypropylene centrifuge bottles, and the
rAdV was purified.
[0102] NP-40 detergent was added to a final concentration of 0.5%
to the bottles of crude lysate in order to lyse all cells. Bottles
were placed on a rotating platform for 10 minutes agitating as fast
as possible without the bottles falling over. The debris was
pelleted by centrifugation at 20,000.times.G for 15 minutes. The
supernatant was transferred to 250-ml polycarbonate centrifuge
bottles, and 0.5 volume of 20% PEG8000/2.5 M NaCl solution was
added. The bottles were shaken overnight on ice. The bottles were
centrifuged at 20,000.times.G for 15 minutes and, the supernatant
was discarded into a bleach solution. Using a sterile cell scraper,
the white, virus/PEG precipitate from 2 bottles was resuspended in
2.5 ml PBS. The resulting virus solution was placed in 2-ml
microcentrifuge tubes and centrifuged at 14,000.times.G in the
microcentrifuge for 10 minutes to remove any additional cell
debris. The supernatant from the 2-ml microcentrifuge tubes was
transferred into a 15-ml polypropylene snapcap tube and adjusted to
a density of 1.34 g/ml with CsCl. The volume of the virus solution
was estimated, and 0.55 g/ml of CsCl was added. The CsCl was
dissolved, and 1 ml of this solution weighed 1.34 g. The solution
was transferred to 3.2-ml, polycarbonate, thick-walled centrifuge
tubes and spun at 348,000.times.G for 3-4 hours at 25.degree. C.
The virus formed a white band. Using wide-bore pipette tips, the
virus band was collected.
[0103] The virus recovered from the gradient had a large amount of
CsCl which had to be removed before it was used on cells.
Commercially available ion-exchange columns (PD-10 columns
prepacked with Sephadex.RTM. G-25M; Pharmacia Biotech, Piscataway,
N.J.) were used to desalt the virus preparation. The column was
equilibrated with 20 ml of PBS. The virus was loaded and allowed to
run into the column. 5 ml of PBS was added to the column, and
fractions of 8-10 drops were collected. The optical density of a
1:50 dilution of each fraction was determined at 260 nm on a
spectrophotometer. A clear absorbance peak was present between
fractions 7-12. Peak fractions were pooled, and the optical density
(OD) of a 1:25 dilution was determined. OD was converted to virus
concentration using the formula: (OD at 260 nm)(25)(1.1.times.10
2)=virions/ml. The OD of a 1:25 dilution of the zvegf3 rAdV was
0.145, giving a virus concentration of 4.times.10.sup.12
virions/ml.
[0104] To store the virus, glycerol was added to the purified virus
to a final concentration of 15%, mixed gently but effectively, and
stored in aliquots at -80.degree. C.
[0105] A protocol developed by Quantum Biotechnologies, Inc.
(Montreal, Canada) was followed to measure recombinant virus
infectivity. Briefly, two 96-well tissue culture plates were seeded
with 1.times.10.sup.4 293A cells per well in MEM containing 2%
fetal bovine serum for each recombinant virus to be assayed. After
24 hours 10-fold dilutions of each virus from 1.times.10.sup.-2 to
1.times.10.sup.-14 were made in MEM containing 2% fetal bovine
serum. 100 .mu.l of each dilution was placed in each of 20 wells.
After 5 days at 37.degree. C., wells were read either positive or
negative for Cytopathic Effect (CPE), and a value for plaque
forming units (pfu)/ml was calculated.
[0106] TCID.sub.50 formulation used was as per Quantum
Biotechnologies, Inc., above. The titer (T) was determined from a
plate where virus was diluted from 10.sup.-2 to 10.sup.-14, and
read 5 days after the infection. At each dilution a ratio (R) of
positive wells for CPE per the total number of wells was
determined.
[0107] To calculate titer of the undiluted virus sample: the
factor, "F"=1+d(S-0.5); where "S" is the sum of the ratios (R); and
"d" is Log10 of the dilution series, for example, "d" is equal to 1
for a ten-fold dilution series. The titer of the undiluted sample
is T=10.sup.(1+F)=TCID.sub.50/ml. To convert TCID.sub.50/ml to
pfu/ml, 0.7 is subtracted from the exponent in the calculation for
titer (T).
[0108] The zvegf3 adenovirus had a titer of 1.8.times.10.sup.10
pfu/ml.
Example 6
[0109] Treatment of mice with zvegf3-adenovirus led to changes in
liver and spleen. The livers were pale and very enlarged, with
enlarged vessels at the tips of the lobes. The livers also showed
sinusoidal cell proliferation. Changes were also seen in
hepatocytes (hypertrophy, degeneration, and necrosis) and were most
likely non-specific effects of adenovirus infection. Splenic change
consisted of increased extramedulary hematopoiesis, which was
correlated with enlarged splenic size.
Example 7
[0110] Polyclonal anti-peptide antibodies were prepared by
immunizing two female New Zealand white rabbits with the peptides
huzvegf3-1 (residues 80-104 of SEQ ID NO:2), huzvegf3-2 (residues
299-314 of SEQ ID NO:2), huzvegf3-3 (residues 299-326 of SEQ ID
NO:2 with an N-terminal cys residue), or huzvegf3-4 (residues
195-225 of SEQ ID NO:2 with a C-terminal cys residue). The peptides
were synthesized using an Applied Biosystems Model 431A peptide
synthesizer (Applied Biosystems, Inc., Foster City, Calif.)
according to the manufacturer's instructions. The peptides
huzvegf3-1, huzvegf3-3, and huzvegf3-4 were then conjugated to the
carrier protein maleimide-activated keyhole limpet hemocyanin (KLH)
through cysteine residues (Pierce Chemical Co., Rockford, Ill.).
The peptide huzvefg3-2 was conjugated to the carrier protein KLH
using gluteraldehyde. The rabbits were each given an initial
intraperitoneal (IP) injection of 200 .mu.g of conjugated peptide
in Complete Freund's Adjuvant (Pierce Chemical Co.) followed by
booster IP injections of 100 .mu.g conjugated peptide in Incomplete
Freund's Adjuvant every three weeks. Seven to ten days after the
administration of the third booster injection, the animals were
bled and the serum was collected. The rabbits were then boosted and
bled every three weeks.
[0111] The huzvegf3 peptide-specific antibodies were affinity
purified from the rabbit serum using an CNBr-Sepharose.RTM. 4B
peptide column (Pharmacia Biotech) that was prepared using 10 mg of
the respective peptides per gram CNBr-Sepharose.RTM., followed by
dialysis in PBS overnight. Peptide specific-huzvegf3 antibodies
were characterized by an ELISA titer check using 1 .mu.g/ml of the
appropriate peptide as an antibody target. The huzvegf3-1
peptide-specific antibodies had a lower limit of detection (LTD) of
500 pg/ml by ELISA on the appropriate antibody target and recognize
full-length recombinant protein (MBP-fusion) by ELISA. The
huzvegf3-2 peptide-specific antibodies had an LLD of 1 ng/ml by
ELISA. The huzvegf3-3 peptide-specific antibodies had an LLD of 50
pg/ml by ELISA and recognized recombinant protein by Western Blot
analysis. The huzvegf3-4 peptide-specific antibodies had an LLD of
50 pg/ml by ELISA and recognized recombinant protein by Western
Blot analysis.
Example 8
[0112] Mouse hybridomas producing monoclonal antibodies (MAbs)
specific for recombinant human zvegf3 growth factor domain (GFD)
protein were generated using purified, untagged, recombinant human
zvegf3 GFD produced in BHK cells (huzvegf3-GFD-BHK). Ten BALB/c
mice were each injected IP on day 1 with 20 .mu.g of
huzvegf3-GFD-BHK mixed 1:1 (v/v) in complete Freund's adjuvant.
Each mouse was subsequently injected IP with 10 .mu.g of
huzvegf3-GFD-BHK mixed 1:1 in incomplete Freund's adjuvant on days
15, 29, 41, 57, 71, 89 and 115. On day 118, splenocytes and
lymphocytes from enlarged lymph nodes of two mice with the highest
anti-huzvegf3 antibody titer (as determined in a biotinylated
huzvegf3-GFD capture ELISA; see below) were fused at a 2.76:1 ratio
with the X63-Ag8.653 mouse myeloma cell line (Kearney et al., J.
Immunol. 123:1548-1550, 1979) essentially as disclosed by Lane (J.
Immunol. Methods 81:223-228, 1985). The fusion mixture was plated
into 24 96-well plates at an average density of 1.2.times.10.sup.5
total cells/well in Iscove's modified Dulbecco's medium (IMDM; Life
Technologies, Inc., Gaithersburg, Md.) containing 10% fetal clone I
serum (HyClone Laboratories, Inc., Logan, Utah), 10% hybridoma
cloning supplement (BM Condimed.RTM. H1; Roche Diagnostics Corp.,
Indianapolis, Ind.), 2 mM L-glutamine (Life Technologies, Inc.),
100 U/mL penicillin G sodium (Life Technologies, Inc.), and 100
.mu.g/mL streptomycin sulfate (Life Technologies, Inc.). Wells were
fed on days 4 and 7 by aspiration and replacement of approximately
three-fourths of the media contents in each well. This fusion was
designated HH1.
[0113] Anti-huzvegf3 mAbs of the IgG class were detected on days
9/10 post-fusion using a biotinylated huvegf3-GFD capture ELISA.
Wells of plates (Immulon.RTM. II; Dynex Technologies, Chantilly,
Va.) were coated with 1 .mu.g/mL of goat anti-mouse IgG (obtained
from Kirkegaard & Perry Laboratories, Gaithersburg, Md.) in
0.05 M carbonate/bicarbonate buffer (Sigma, St. Louis, Mo.), 50
.mu.L/well. Plates were incubated at 4.degree. C. overnight, then
washed three times with phosphate buffered saline containing 0.05%
polyoxyethylenesorbitan monolaurate (Tween 20) (PBST) to remove
unbound antibody. Wells were blocked with PBST containing 1% (w/v)
bovine serum albumin (Sigma) (PTB buffer), 200 .mu.L/well, for 1
hour at room temperature (RT), then washed as above. Culture
supernatants from the fusion plates were replica-plated onto
antibody-coated plates, 100 .mu.L/well, and incubated at RT for 1
hour. Human zvegf3-GFD protein was biotinylated with
sulfo-NHS-LC-biotin (Pierce Chemical Company, Rockford, Ill.)
according to the manufacturer's instructions for 45 minutes at RT.
The reaction was stopped with 2M glycine. Biotinylated protein was
diluted to 1 .mu.g/mL in PTB buffer. The plates were washed four
times with PBST, biotinylated huzvegf3-GFD was added at 100
.mu.L/well, and the plates were incubated at RT for 1 hour. The
plates were then washed four times with PBST, and HRP-conjugated
streptavidin (Pierce Chemical Company) was diluted to 0.5 .mu.g/mL
in PTB buffer and added 100 to the plates at uL/well. The plates
were incubated at RT for 1 hour, then washed five times with PBST.
TMB substrate (EIA chromagen, Cat. # R6R; Genetic Systems Corp.)
was diluted 1:100 in substrate buffer (Genetic Systems Corp.) and
added to the plates at 100 .mu.L/well. The plates were incubated
10-15 minutes at RT in the dark. Color development was stopped by
the addition of 100 .mu.L/well of 1N H.sub.2SO.sub.4. Optical
density was read on an ELISA plate reader (Molecular Devices,
Sunnyvale, Calif.) at wavelengths 450 nm (L1) and 650 nm (L2).
Final OD measurement was determined by the formula L1-L2.
[0114] 78 master wells contained antibody that was capable of
capturing biotinylated huzvegf3-GFD. These master wells were
expanded for hybridoma cryopreservation and additional supernatant
generation. ELISA analysis of the expanded master well supernatants
demonstrated that 27 of the original 78 master wells retained
antibody specific for huzvegf3-GFD, with 20 of 27 possessing
significant reactivity with the antigen.
[0115] Six of the designated master wells were cloned by limiting
dilution at a density of less than one cell per well. Monoclonality
was assessed by microscopic examination of wells for a single focus
of cell growth. Clones were tested for specific antibody by the
same assay used in the master screen. Five to six of the strongest
clones were briefly expanded. Supernatants from these clones was
then serially diluted and tested by ELISA to identify the three
best antibody-producing clones. Upon verification, the best clone
from each master well was subsequently recloned and assayed as
described above.
[0116] Six of the highest titered second round clones were adapted
to growth in fusion/cloning medium minus the addition of hybridoma
cloning supplement. Each of the clones was subsequently adapted to
growth in a production medium formulation consisting essentially of
Dulbecco's modified Eagle's medium+2.5% fetal clone I serum and
various supplements. Supernatants were again serially diluted and
tested by ELISA on huzvegf3-GFD to identify the highest titered and
next highest titered clones (designated the primary and secondary
clones, respectively). MAb produced by each set of clones was then
evaluated for IgG subclass using the Mouse Hybridoma Subtyping Kit
(Cat. #1183117; Roche Diagnostics Corp.). Clones HH1-24, -40, -57
and -76 were all found to produce an IgG.sub.1 antibody. Clones
HH1-58 and -78 produced an IgG.sub.2b antibody. All antibodies
possessed a K light chain.
Example 9
[0117] Recombinant zvegf3 was analyzed for mitogenic activity on
rat stellate cells (Greenwel et al., Laboratory Invest. 65:644,
1991; Greenwel et al., Laboratory Invest. 69:210, 1993). Stellate
cells were plated at a density of 2,000 cells/well in 96-well
culture plates and grown for approximately 72 hours in DMEM
containing 10% fetal calf serum at 37.degree. C. Cells were
quiesced by incubating them for 20 hours in serum-free DMEM/Ham's
F-12 medium containing insulin (5 .mu.g/ml), transferrin (20
.mu.g/ml), and selenium (16 pg/ml) (ITS). At the time of the assay,
the medium was removed, and test samples were added to the wells in
triplicate. Test samples consisted of either conditioned media (CM)
from adenovirally-infected HaCaT human keratinocyte cells (Boukamp
et al., J. Cell. Biol. 106:761-771, 1988) expressing full-length
zvegf3, purified growth factor domain expressed in BHK cells, or
control media from cells infected with parental adenovirus (Zpar)
containing an expression unit for green fluorescent protein. The CM
was concentrated 10-fold using a 15-ml centrifugal filter device
with a 10K membrane filter (Ultrafree.RTM.; Millipore Corp.,
Bedford, Mass.), then diluted back to 3.times. with ITS medium and
added to the cells. Purified protein in a buffer containing 0.1%
BSA was serially diluted into ITS medium at concentrations of 1
.mu.g/ml to 1 ng/ml and added to the test plate. A control buffer
of 0.1% BSA was diluted identically to the highest concentration of
zvegf3 protein and added to the plate. For measurement of
[.sup.3H]thymidine incorporation, 20 .mu.l of a 50 .mu.Ci/ml stock
in DMEM was added directly to the cells, for a final activity of 1
.mu.Ci/well. After another 24-hour incubation, mitogenic activity
was assessed by measuring the uptake of [.sup.3H]thymidine. Media
were removed, and cells were incubated with 0.1 ml of trypsin until
cells detached. Cells were harvested onto 96-well filter plates
using a sample harvester (FilterMate.TM. harvester; Packard
Instrument Co., Meriden, Conn.). The plates were then dried at
65.degree. C. for 15 minutes, sealed after adding 40 .mu.l/well
scintillation cocktail (Microscint.TM. O; Packard Instrument Co.)
and counted on a microplate scintillation counter (Topcount.RTM.;
Packard Instrument Co.).
[0118] Results, presented in Table 3, demonstrated that zvegf3 CM
had approximately 4.4-fold higher mitogenic activity on stellate
cells over control CM, and purified protein at 100 ng/ml caused a
maximal 14-fold increase in [.sup.3H]thymidine incorporation over
the buffer control.
3 TABLE 3 CPM Incorporated Sample Mean St. dev. zvegf3 (2 .times.
CM) 42489 1306 Zpar (2 .times. CM) 9629 540 zvegf3 GF domain, 100
ng/ml 77540 4142 zvegf3 GF domain, 33.3 ng/ml 74466 18142 zvegf3 GF
domain, 11.1 ng/ml 52462 6239 zvegf3 GF domain, 3.7 ng/ml 15128
4989 Buffer control 5618 573 PDGF-BB 20 ng/ml 19741 2075 PDGF-AA 20
ng/ml 33133 3325 Media alone (basal response) 6765 226
Example 10
[0119] Northern blot analysis was performed on 2 .mu.g samples of
poly(A)+ RNA from five mouse prostate cell lines (designated
Jakotay, Nelix, Paris, Torres, and Tuvak). Total RNA from mouse and
rat liver were used as controls (20 .mu.g each). An approximately
680-bp DNA probe was generated by PCR using oligonucleotide primers
ZC21,222 (SEQ ID NO:9) and ZC21,224 (SEQ ID NO:10) and a mouse
zvegf3 full-length cDNA clone as a template. The DNA probe was
purified by conventional procedures using a commercially available
kit (QIAquick.TM. Gel Extraction Kit; Qiagen, Inc., Valencia,
Calif.). The probe was radioactively labeled with .sup.32P using a
commercially available kit (Rediprime.TM. II DNA Labeling system;
Amersham, Arlington Heights, Ill.) according to the manufacturer's
specifications. The probe was purified using a push column
(NucTrap.RTM. column; Stratagene, La Jolla, Calif.; see U.S. Pat.
No. 5,336,412). A commercially available hybridization solution
(ExpressHyb.TM. Hybridization Solution; Clontech Laboratories,
Inc., Palo Alto, Calif.) was used for the blots. Hybridization took
place overnight at 65.degree. C. The blots were then washed four
times in 2.times.SCC and 0.1% SDS at room temperature, followed by
two washes in 0.1.times.SSC and 0.1% SDS at 55.degree. C. One
transcript size was detected at approximately 4 kb in Jakotay,
Nelix, Paris and Tuvak cell lines. Signal intensity was highest for
Jakotay, then Nelix and Tuvak.
Example 11
[0120] Recombinant human zvegf3 GFD produced in BHK cells was
assayed for growth factor activity using an osteoblast cell line
containing a luciferase reporter gene construct. CCC4 cells
(derived from p53 knock-out mice) transfected with the reporter
construct were plated in 96-well plates (BioCoat.TM.; Becton
Dickinson, Franklin Lakes, N.J.) at 1.times.10.sup.4 cells/well in
1% FBS alpha MEM (JRH Biosciences, Lexena, Kans.) containing
L-Glutamine and sodium pyruvate (Life Technologies, Inc.). The
cells were cultured for 24 hours, the medium was removed, and the
wells were washed twice with wash buffer (1% BSA in PBS), 100
.mu.l/well at 37.degree. C. Zvegf3, PDGF-AA, and PDGF-BB were
diluted in assay medium (alpha MEM containing 1% BSA, 10 mM Hepes,
L-Glutamate, and sodium pyruvate), and 100-.mu.l samples were added
to the wells. The plates were incubated 4 hours at 37.degree. C.,
then the wells were washed with wash buffer. Lysis reagent (Cell
Culture Lysis Reagent; Promega Corporation, Madison, Wis.) was
diluted 1:5 in water, and 25 .mu.l was added to each well. The
plates were incubated 10 minutes at room temperature, then
transferred to -20.degree. C. Luciferase substrate (Luciferase
Assay System; Promega Corporation) was diluted 1:1 with assay
medium and added to the wells. The plates were read on a
luminometer. Results, shown in Table 4, indicted that all three
growth factors exhibited mitogenic activity on the CCC4 cells.
4 TABLE 4 Concentration Activity (Luminometer Units) (ng/ml) Zvegf3
PDGF-BB PDGF-AA 0 3.5 3.1 3.2 0.4 5.2 7.7 8.4 0.8 5.6 10.7 9.5 1.5
7.6 14 9.4 3.1 10.6 19.9 11.4 6.2 12.5 25.1 12 12.5 14.2 26.1 12.3
25 16 25.7 11.9 50 19.4 25.8 12.6 100 20.6 23.8 14
Example 12
[0121] A study was undertaken to test whether adenovirally
delivered zvegf3 stimulated cell proliferation as determined by
incorporation of bromodeoxyuridine (BrdU) into tissues.
[0122] Mice (male, C57B1, 7 weeks old) were divided into three
groups. On day 0, parental or zvegf3 adenovirus was administered to
the first (n=11) and second (n=12) groups, respectively, via the
tail vein, with each mouse receiving a dose of
.about.1.times.10.sup.11 particles in .about.0.1 ml volume. The
third group (n=8) received no treatment. Each mouse was given two
intraperitoneal doses of 3 mg of freshly made BrdU solution at
approximately 24 and 12 hours prior to sacrifice. On days 2, 4, 6,
8, and 10, two mice from each treatment group and one or two
untreated mice were sacrificed, and tissues and blood were
harvested. Samples were analyzed for complete blood count (CBC) and
serum chemistry, and slides were prepared for manual differential
blood and marrow progenitor cell analysis. One femur, lung, heart,
thymus, liver, kidney, spleen, pancreas, duodenum, and mesenteric
lymph nodes were submitted for standard histology and assessment of
BrdU incorporation. The lining of the duodenum served as the
control tissue for BrdU incorporation.
[0123] In addition, two mice that received approximately half the
dose of zvegf3 adenovirus particles and one mouse that received the
full dose of parental adenovirus were sacrificed and analyzed as
described above on day 16.
[0124] A piece of liver from each mouse was saved for mRNA assay of
adenovirus protein to examine the time course of expression of the
adenovirus preparations.
[0125] Beginning on day 6, most of the animals treated with either
adenovirus had visibly enlarged livers and spleens compared to the
untreated mice. The livers of the zvegf3 adenovirus-treated mice
tended to look more pale than animals treated with the parental
virus. Proliferation of sinusoidal cells was observed in liver.
Visual inspection suggested that these cells were stellate cells
and/or fibroblasts. Spleen color was the same in both groups. Most
of the animals that received the zvegf3 adenovirus had paler femur
shafts, with the marrow lighter in color.
[0126] Peripheral blood CBCs showed a possible difference in
platelet counts, but not in RBC or WBC counts between zvegf3 and
parental virus-treated animals. In comparison to the untreated and
parental virus-treated groups, the zvegf3 group had lower platelet
counts on days 2, 4, 6, and 8, but not on day 10. The mean platelet
volume (average size of individual platelets) in the zvegf3 group
also tended to be greater, consistent with a relative increase in
the larger, immature platelet population.
[0127] BrdU labeling showed increased cell proliferation in kidney,
mainly in the medulla and to a lesser extent in the cortex.
Proliferating cells appeared to be interstitial cells, which may
have included fibroblasts and/or mesangial cells.
Example 13
[0128] Human zvegf3 growth factor domain protein produced in BHK
cells was tested for the ability to stimulate production of
TGF-.beta. in stellate cells. Rat hepatic stellate cells (obtained
from Dr. Nelson Fausto, University of Washington) were plated in
48-well tissue culture clusters (Costar.RTM.; Coming, Corning,
N.Y.) in DMEM growth medium (Life Technologies, Inc.) supplemented
with pyruvate and 10% serum (HyClone Laboratories, Inc.). At
confluence, the medium was changed to serum-free medium by
substituting 0.1% BSA (Fraction V; Sigma, St. Louis, Mo.) for
serum. 48 hours later, the medium was changed, replaced with the
same serum-free medium, and the zvegf3 GFD protein was added at 100
ng/ml in the wells. 48 hours later, conditioned media were
collected and spun down to get rid of any floating cells or debris.
Total TGF-.beta.1 levels were determined in these media using a
commercially available ELISA kit (R&D Systems, St Paul, Minn.).
Stimulation of stellate cells with 100 ng/ml zvegf3 GFD resulted in
an approximately 5-fold increase in the production of TGF-.beta.
compared to a BSA control.
Example 14
[0129] Recombinant human zvegf3 GFD was analyzed for mitogenic
activity on human mesangial cells (Clonetics, San Diego, Calif.).
Mesangial cells were plated at a density of 2,000 cells/well in
96-well culture plates and grown for approximately 72 hours in DMEM
containing 10% fetal calf serum at 37.degree. C. Cells were
quiesced by incubating them for 20 hours in serum-free DMEM/Ham's
F-12 medium containing insulin (5 .mu.g/ml), transferrin (20
.mu.g/ml), and selenium (16 pg/ml) (ITS). At the time of the assay,
the medium was removed, and test samples were added to the wells in
triplicate. Test samples consisted of purified zvegf3 GFD at 3, 10,
30 and 100 ng/ml, and PDGF-AA, PDGF-AB and PDGF-BB at 30 ng/ml in a
buffer containing 0.1% BSA. Samples were serially diluted into ITS
medium and added to the test plate. A control buffer of 0.1% BSA
was diluted identically to the highest concentration of zvegf3
protein and added to the plate. 10% fetal bovine serum (FBS) was
used as a positive control. For measurement of [.sup.3H]thymidine
incorporation, 20 .mu.l of a 50 .mu.Ci/ml stock in DMEM was added
directly to the cells, for a final activity of 1 .mu.Ci/well. After
another 24-hour incubation, mitogenic activity was assessed by
measuring the uptake of [.sup.3H]thymidine. Media were removed, and
cells were incubated with 0.1 ml of trypsin until cells detached.
Cells were harvested onto 96-well filter plates using a sample
harvester (FilterMate.TM. harvester; Packard Instrument Co.,
Meriden, Conn.). The plates were then dried at 65.degree. C. for 15
minutes, sealed after adding 40 .mu.l/well scintillation cocktail
(Microscint.TM. 0; Packard Instrument Co.), and counted on a
microplate scintillation counter (Topcount.RTM.; Packard Instrument
Co.). Results, shown in Table 5, demonstrated that zvegf3 GFD had
approximately 3-fold higher mitogenic activity cells over PDGF-AA
and comparable activity to PDGF-AB and PDGF-BB at 30 ng/ml on
mesangial cells.
5 TABLE 5 CPM [.sup.3H] Thymidine Incorporated Sample Mean St. dev.
PDGF-AA, 30 ng/ml 492 120 PDGF-AB, 30 ng/ml 1554 114 PDGF-BB, 30
ng/ml 1852 464 Zvegf3 GFD, 3 ng/ml 1321 91 Zvegf3 GFD, 10 ng/ml
1615 325 Zvegf3 GFD, 30 ng/ml 1545 237 Zvegf3 GFD, 100 ng/ml 1677
88 10% FBS 1447 174 Buffer control 392 109
[0130] 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
18 1 1760 DNA Homo sapiens CDS (154)...(1191) 1 attatgtgga
aactaccctg cgattctctg ctgccagagc aggctcggcg cttccacccc 60
agtgcagcct tcccctggcg gtggtgaaag agactcggga gtcgctgctt ccaaagtgcc
120 cgccgtgagt gagctctcac cccagtcagc caa atg agc ctc ttc ggg ctt
ctc 174 Met Ser Leu Phe Gly Leu Leu 1 5 ctg ctg aca tct gcc ctg gcc
ggc cag aga cag ggg act cag gcg gaa 222 Leu Leu Thr Ser Ala Leu Ala
Gly Gln Arg Gln Gly Thr Gln Ala Glu 10 15 20 tcc aac ctg agt agt
aaa ttc cag ttt tcc agc aac aag gaa cag aac 270 Ser Asn Leu Ser Ser
Lys Phe Gln Phe Ser Ser Asn Lys Glu Gln Asn 25 30 35 gga gta caa
gat cct cag cat gag aga att att act gtg tct act aat 318 Gly Val Gln
Asp Pro Gln His Glu Arg Ile Ile Thr Val Ser Thr Asn 40 45 50 55 gga
agt att cac agc cca agg ttt cct cat act tat cca aga aat acg 366 Gly
Ser Ile His Ser Pro Arg Phe Pro His Thr Tyr Pro Arg Asn Thr 60 65
70 gtc ttg gta tgg aga tta gta gca gta gag gaa aat gta tgg ata caa
414 Val Leu Val Trp Arg Leu Val Ala Val Glu Glu Asn Val Trp Ile Gln
75 80 85 ctt acg ttt gat gaa aga ttt ggg ctt gaa gac cca gaa gat
gac ata 462 Leu Thr Phe Asp Glu Arg Phe Gly Leu Glu Asp Pro Glu Asp
Asp Ile 90 95 100 tgc aag tat gat ttt gta gaa gtt gag gaa ccc agt
gat gga act ata 510 Cys Lys Tyr Asp Phe Val Glu Val Glu Glu Pro Ser
Asp Gly Thr Ile 105 110 115 tta ggg cgc tgg tgt ggt tct ggt act gta
cca gga aaa cag att tct 558 Leu Gly Arg Trp Cys Gly Ser Gly Thr Val
Pro Gly Lys Gln Ile Ser 120 125 130 135 aaa gga aat caa att agg ata
aga ttt gta tct gat gaa tat ttt cct 606 Lys Gly Asn Gln Ile Arg Ile
Arg Phe Val Ser Asp Glu Tyr Phe Pro 140 145 150 tct gaa cca ggg ttc
tgc atc cac tac aac att gtc atg cca caa ttc 654 Ser Glu Pro Gly Phe
Cys Ile His Tyr Asn Ile Val Met Pro Gln Phe 155 160 165 aca gaa gct
gtg agt cct tca gtg cta ccc cct tca gct ttg cca ctg 702 Thr Glu Ala
Val Ser Pro Ser Val Leu Pro Pro Ser Ala Leu Pro Leu 170 175 180 gac
ctg ctt aat aat gct ata act gcc ttt agt acc ttg gaa gac ctt 750 Asp
Leu Leu Asn Asn Ala Ile Thr Ala Phe Ser Thr Leu Glu Asp Leu 185 190
195 att cga tat ctt gaa cca gag aga tgg cag ttg gac tta gaa gat cta
798 Ile Arg Tyr Leu Glu Pro Glu Arg Trp Gln Leu Asp Leu Glu Asp Leu
200 205 210 215 tat agg cca act tgg caa ctt ctt ggc aag gct ttt gtt
ttt gga aga 846 Tyr Arg Pro Thr Trp Gln Leu Leu Gly Lys Ala Phe Val
Phe Gly Arg 220 225 230 aaa tcc aga gtg gtg gat ctg aac ctt cta aca
gag gag gta aga tta 894 Lys Ser Arg Val Val Asp Leu Asn Leu Leu Thr
Glu Glu Val Arg Leu 235 240 245 tac agc tgc aca cct cgt aac ttc tca
gtg tcc ata agg gaa gaa cta 942 Tyr Ser Cys Thr Pro Arg Asn Phe Ser
Val Ser Ile Arg Glu Glu Leu 250 255 260 aag aga acc gat acc att ttc
tgg cca ggt tgt ctc ctg gtt aaa cgc 990 Lys Arg Thr Asp Thr Ile Phe
Trp Pro Gly Cys Leu Leu Val Lys Arg 265 270 275 tgt ggt ggg aac tgt
gcc tgt tgt ctc cac aat tgc aat gaa tgt caa 1038 Cys Gly Gly Asn
Cys Ala Cys Cys Leu His Asn Cys Asn Glu Cys Gln 280 285 290 295 tgt
gtc cca agc aaa gtt act aaa aaa tac cac gag gtc ctt cag ttg 1086
Cys Val Pro Ser Lys Val Thr Lys Lys Tyr His Glu Val Leu Gln Leu 300
305 310 aga cca aag acc ggt gtc agg gga ttg cac aaa tca ctc acc gac
gtg 1134 Arg Pro Lys Thr Gly Val Arg Gly Leu His Lys Ser Leu Thr
Asp Val 315 320 325 gcc ctg gag cac cat gag gag tgt gac tgt gtg tgc
aga ggg agc aca 1182 Ala Leu Glu His His Glu Glu Cys Asp Cys Val
Cys Arg Gly Ser Thr 330 335 340 gga gga tag ccgcatcacc accagcagct
cttgcccaga gctgtgcagt 1231 Gly Gly * 345 gcagtggctg attctattag
agaacgtatg cgttatctcc atccttaatc tcagttgttt 1291 gcttcaagga
cctttcatct tcaggattta cagtgcattc tgaaagagga gacatcaaac 1351
agaattagga gttgtgcaac agctcttttg agaggaggcc taaaggacag gagaaaaggt
1411 cttcaatcgt ggaaagaaaa ttaaatgttg tattaaatag atcaccagct
agtttcagag 1471 ttaccatgta cgtattccac tagctgggtt ctgtatttca
gttctttcga tacggcttag 1531 ggtaatgtca gtacaggaaa aaaactgtgc
aagtgagcac ctgattccgt tgccttgctt 1591 aactctaaag ctccatgtcc
tgggcctaaa atcgtataaa atctggattt tttttttttt 1651 tttttgctca
tattcacata tgtaaaccag aacattctat gtactacaaa cctggttttt 1711
aaaaaggaac tatgttgcta tgaattaaac ttgtgtcgtg ctgatagga 1760 2 345
PRT Homo sapiens 2 Met Ser Leu Phe Gly Leu Leu Leu Leu Thr Ser Ala
Leu Ala Gly Gln 1 5 10 15 Arg Gln Gly Thr Gln Ala Glu Ser Asn Leu
Ser Ser Lys Phe Gln Phe 20 25 30 Ser Ser Asn Lys Glu Gln Asn Gly
Val Gln Asp Pro Gln His Glu Arg 35 40 45 Ile Ile Thr Val Ser Thr
Asn Gly Ser Ile His Ser Pro Arg Phe Pro 50 55 60 His Thr Tyr Pro
Arg Asn Thr Val Leu Val Trp Arg Leu Val Ala Val 65 70 75 80 Glu Glu
Asn Val Trp Ile Gln Leu Thr Phe Asp Glu Arg Phe Gly Leu 85 90 95
Glu Asp Pro Glu Asp Asp Ile Cys Lys Tyr Asp Phe Val Glu Val Glu 100
105 110 Glu Pro Ser Asp Gly Thr Ile Leu Gly Arg Trp Cys Gly Ser Gly
Thr 115 120 125 Val Pro Gly Lys Gln Ile Ser Lys Gly Asn Gln Ile Arg
Ile Arg Phe 130 135 140 Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gly
Phe Cys Ile His Tyr 145 150 155 160 Asn Ile Val Met Pro Gln Phe Thr
Glu Ala Val Ser Pro Ser Val Leu 165 170 175 Pro Pro Ser Ala Leu Pro
Leu Asp Leu Leu Asn Asn Ala Ile Thr Ala 180 185 190 Phe Ser Thr Leu
Glu Asp Leu Ile Arg Tyr Leu Glu Pro Glu Arg Trp 195 200 205 Gln Leu
Asp Leu Glu Asp Leu Tyr Arg Pro Thr Trp Gln Leu Leu Gly 210 215 220
Lys Ala Phe Val Phe Gly Arg Lys Ser Arg Val Val Asp Leu Asn Leu 225
230 235 240 Leu Thr Glu Glu Val Arg Leu Tyr Ser Cys Thr Pro Arg Asn
Phe Ser 245 250 255 Val Ser Ile Arg Glu Glu Leu Lys Arg Thr Asp Thr
Ile Phe Trp Pro 260 265 270 Gly Cys Leu Leu Val Lys Arg Cys Gly Gly
Asn Cys Ala Cys Cys Leu 275 280 285 His Asn Cys Asn Glu Cys Gln Cys
Val Pro Ser Lys Val Thr Lys Lys 290 295 300 Tyr His Glu Val Leu Gln
Leu Arg Pro Lys Thr Gly Val Arg Gly Leu 305 310 315 320 His Lys Ser
Leu Thr Asp Val Ala Leu Glu His His Glu Glu Cys Asp 325 330 335 Cys
Val Cys Arg Gly Ser Thr Gly Gly 340 345 3 3571 DNA Mus musculus CDS
(1049)...(2086) 3 gaattcccgg gtcgacccac gcgtccgggc gcccagggga
aaggaagctg ggggccgcct 60 ggcggcattc ctcgccgcag tgtgggctcc
gtctgccgcg gggcccgcag tgccccctgt 120 ctgcgccagc acctgttggc
ccgccagctg gccgcccgcg ccccccgcgc cccccgcgcc 180 cgcccggccg
ccagccccgc gccccgcgcg ccgcccgctg ggggaaagtg gagacgggga 240
ggggacaaga gcgatcctcc aggccagcca ggccttccct tagccgcccg tgcttagccg
300 ccacctctcc tcagccctgc gtcctgccct gccttagggc aggcatccga
gcgctcgcga 360 ctccgagccg cccaagctct cccggcttcc cgcagcactt
cgccggtacc cgagggaact 420 tcggtggcca ccgactgcag caaggaggag
gctccgcggt ggatccgggc cagtcccgag 480 tcgtccccgc ggcctctctg
cccgcccggg acccgcgcgg cactcgcagg gcacggtccc 540 ctccccccag
gtgggggtgg ggcgccgcct gccgccccga tcagcagctt tgtcattgat 600
cccaaggtgc tcgcctcgct gccgacctgg cttccagtct ggcttggcgg gaccccgagt
660 cctcgcctgt gtcctgtccc ccaaactgac aggtgctccc tgcgagtcgc
cacgactcat 720 cgccgctccc ccgcgtcccc accccttctt tcctccctcg
cctaccccca ccccccgcac 780 ttcggcacag ctcaggattt gtttaaacct
tgggaaactg gttcaggtcc aggttttgct 840 ttgatccttt tcaaaaactg
gagacacaga agagggctct aggaaaaact tttggatggg 900 attatgtgga
aactaccctg cgattctctg ctgccagagc cggccaggcg cttccaccgc 960
agcgcagcct ttccccggct gggctgagcc ttggagtcgt cgcttcccca gtgcccgccg
1020 cgagtgagcc ctcgccccag tcagccaa atg ctc ctc ctc ggc ctc ctc ctg
1072 Met Leu Leu Leu Gly Leu Leu Leu 1 5 ctg aca tct gcc ctg gcc
ggc caa aga acg ggg act cgg gct gag tcc 1120 Leu Thr Ser Ala Leu
Ala Gly Gln Arg Thr Gly Thr Arg Ala Glu Ser 10 15 20 aac ctg agc
agc aag ttg cag ctc tcc agc gac aag gaa cag aac gga 1168 Asn Leu
Ser Ser Lys Leu Gln Leu Ser Ser Asp Lys Glu Gln Asn Gly 25 30 35 40
gtg caa gat ccc cgg cat gag aga gtt gtc act ata tct ggt aat ggg
1216 Val Gln Asp Pro Arg His Glu Arg Val Val Thr Ile Ser Gly Asn
Gly 45 50 55 agc atc cac agc ccg aag ttt cct cat aca tac cca aga
aat atg gtg 1264 Ser Ile His Ser Pro Lys Phe Pro His Thr Tyr Pro
Arg Asn Met Val 60 65 70 ctg gtg tgg aga tta gtt gca gta gat gaa
aat gtg cgg atc cag ctg 1312 Leu Val Trp Arg Leu Val Ala Val Asp
Glu Asn Val Arg Ile Gln Leu 75 80 85 aca ttt gat gag aga ttt ggg
ctg gaa gat cca gaa gac gat ata tgc 1360 Thr Phe Asp Glu Arg Phe
Gly Leu Glu Asp Pro Glu Asp Asp Ile Cys 90 95 100 aag tat gat ttt
gta gaa gtt gag gag ccc agt gat gga agt gtt tta 1408 Lys Tyr Asp
Phe Val Glu Val Glu Glu Pro Ser Asp Gly Ser Val Leu 105 110 115 120
gga cgc tgg tgt ggt tct ggg act gtg cca gga aag cag act tct aaa
1456 Gly Arg Trp Cys Gly Ser Gly Thr Val Pro Gly Lys Gln Thr Ser
Lys 125 130 135 gga aat cat atc agg ata aga ttt gta tct gat gag tat
ttt cca tct 1504 Gly Asn His Ile Arg Ile Arg Phe Val Ser Asp Glu
Tyr Phe Pro Ser 140 145 150 gaa ccc gga ttc tgc atc cac tac agt att
atc atg cca caa gtc aca 1552 Glu Pro Gly Phe Cys Ile His Tyr Ser
Ile Ile Met Pro Gln Val Thr 155 160 165 gaa acc acg agt cct tcg gtg
ttg ccc cct tca tct ttg tca ttg gac 1600 Glu Thr Thr Ser Pro Ser
Val Leu Pro Pro Ser Ser Leu Ser Leu Asp 170 175 180 ctg ctc aac aat
gct gtg act gcc ttc agt acc ttg gaa gag ctg att 1648 Leu Leu Asn
Asn Ala Val Thr Ala Phe Ser Thr Leu Glu Glu Leu Ile 185 190 195 200
cgg tac cta gag cca gat cga tgg cag gtg gac ttg gac agc ctc tac
1696 Arg Tyr Leu Glu Pro Asp Arg Trp Gln Val Asp Leu Asp Ser Leu
Tyr 205 210 215 aag cca aca tgg cag ctt ttg ggc aag gct ttc ctg tat
ggg aaa aaa 1744 Lys Pro Thr Trp Gln Leu Leu Gly Lys Ala Phe Leu
Tyr Gly Lys Lys 220 225 230 agc aaa gtg gtg aat ctg aat ctc ctc aag
gaa gag gta aaa ctc tac 1792 Ser Lys Val Val Asn Leu Asn Leu Leu
Lys Glu Glu Val Lys Leu Tyr 235 240 245 agc tgc aca ccc cgg aac ttc
tca gtg tcc ata cgg gaa gag cta aag 1840 Ser Cys Thr Pro Arg Asn
Phe Ser Val Ser Ile Arg Glu Glu Leu Lys 250 255 260 agg aca gat acc
ata ttc tgg cca ggt tgt ctc ctg gtc aag cgc tgt 1888 Arg Thr Asp
Thr Ile Phe Trp Pro Gly Cys Leu Leu Val Lys Arg Cys 265 270 275 280
gga gga aat tgt gcc tgt tgt ctc cat aat tgc aat gaa tgt cag tgt
1936 Gly Gly Asn Cys Ala Cys Cys Leu His Asn Cys Asn Glu Cys Gln
Cys 285 290 295 gtc cca cgt aaa gtt aca aaa aag tac cat gag gtc ctt
cag ttg aga 1984 Val Pro Arg Lys Val Thr Lys Lys Tyr His Glu Val
Leu Gln Leu Arg 300 305 310 cca aaa act gga gtc aag gga ttg cat aag
tca ctc act gat gtg gct 2032 Pro Lys Thr Gly Val Lys Gly Leu His
Lys Ser Leu Thr Asp Val Ala 315 320 325 ctg gaa cac cac gag gaa tgt
gac tgt gtg tgt aga gga aac gca gga 2080 Leu Glu His His Glu Glu
Cys Asp Cys Val Cys Arg Gly Asn Ala Gly 330 335 340 ggg taa
ctgcagcctt cgtagcagca cacgtgagca ctggcattct gtgtaccccc 2136 Gly *
345 acaagcaacc ttcatcccca ccagcgttgg ccgcagggct ctcagctgct
gatgctggct 2196 atggtaaaga tcttactcgt ctccaaccaa attctcagtt
gtttgcttca atagccttcc 2256 cctgcaggac ttcaagtgtc ttctaaaaga
ccagaggcac caagaggagt caatcacaaa 2316 gcactgcctt ctagaggaag
cccagacaat ggtcttctga ccacagaaac aaatgaaatg 2376 aatgtagatc
gctagcaaac tctggagtga cagcatttct tttccactga cagaatggtg 2436
tagcttagtt gtcttgatat gggcaagtga tgtcagcaca agaaaatggt gaaaaacaca
2496 cacttgattg tgaacaatgc agaaatactt ggatttctcc aacctgtttg
catagataga 2556 cagatgctct gttttctaca aactcaaagc ttttagagag
cagctatgtt aataggaatt 2616 aaatgtgcca tgctgaaagg aaagactgaa
gttttcaatg cttggcaact tctccgcaat 2676 ttggaggaaa ggtgcggtca
tggtttggag aaagcacacc tgcacagagg agtggccttc 2736 ccttcccttc
cctctgaggt ggcttctgtg tttcattgtg tatattttta tattctcctt 2796
ttgacattat aactgttggc ttttctaatc ttgttaaata tttctatttt taccaaaggt
2856 atttaatatt cttttttatg acaacctaga gcaattattt ttagcttgat
aatttttttt 2916 tctaaacaaa attgttatag ccagaagaac aaagatgatt
gatataaaaa tcttgttgct 2976 ctgacaaaaa catatgtatt tcttccttgt
atggtgctag agcttagcgt catctgcatt 3036 tgaaaagatg gaatggggaa
gtttttagaa ttggtaggtc gcagggacag tttgataaca 3096 actgtactat
catcaattcc caattctgtt cttagagcta cgaacagaac agagcttgag 3156
taaatatgga gccattgcta acctacccct ttctatggga aataggagta tagctcagag
3216 aagcacgtcc ccagaaacct cgaccatttc taggcacagt gttctgggct
atgctgcgct 3276 gtatggacat atcctattta tttcaatact agggttttat
tacctttaaa ctctgctcca 3336 tacacttgta ttaatacatg gatattttta
tgtacagaag tatatcattt aaggagttca 3396 cttattatac tctttggcaa
ttgcaaagaa aatcaacata atacattgct tgtaaatgct 3456 taatctgtgc
ccaagttttg tggtgactat ttgaattaaa atgtattgaa tcatcaaata 3516
aaataatctg gctattttgg ggaaaaaaaa aaaaaaaaaa aaaaagggcg gccgc 3571 4
345 PRT Mus musculus 4 Met Leu Leu Leu Gly Leu Leu Leu Leu Thr Ser
Ala Leu Ala Gly Gln 1 5 10 15 Arg Thr Gly Thr Arg Ala Glu Ser Asn
Leu Ser Ser Lys Leu Gln Leu 20 25 30 Ser Ser Asp Lys Glu Gln Asn
Gly Val Gln Asp Pro Arg His Glu Arg 35 40 45 Val Val Thr Ile Ser
Gly Asn Gly Ser Ile His Ser Pro Lys Phe Pro 50 55 60 His Thr Tyr
Pro Arg Asn Met Val Leu Val Trp Arg Leu Val Ala Val 65 70 75 80 Asp
Glu Asn Val Arg Ile Gln Leu Thr Phe Asp Glu Arg Phe Gly Leu 85 90
95 Glu Asp Pro Glu Asp Asp Ile Cys Lys Tyr Asp Phe Val Glu Val Glu
100 105 110 Glu Pro Ser Asp Gly Ser Val Leu Gly Arg Trp Cys Gly Ser
Gly Thr 115 120 125 Val Pro Gly Lys Gln Thr Ser Lys Gly Asn His Ile
Arg Ile Arg Phe 130 135 140 Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro
Gly Phe Cys Ile His Tyr 145 150 155 160 Ser Ile Ile Met Pro Gln Val
Thr Glu Thr Thr Ser Pro Ser Val Leu 165 170 175 Pro Pro Ser Ser Leu
Ser Leu Asp Leu Leu Asn Asn Ala Val Thr Ala 180 185 190 Phe Ser Thr
Leu Glu Glu Leu Ile Arg Tyr Leu Glu Pro Asp Arg Trp 195 200 205 Gln
Val Asp Leu Asp Ser Leu Tyr Lys Pro Thr Trp Gln Leu Leu Gly 210 215
220 Lys Ala Phe Leu Tyr Gly Lys Lys Ser Lys Val Val Asn Leu Asn Leu
225 230 235 240 Leu Lys Glu Glu Val Lys Leu Tyr Ser Cys Thr Pro Arg
Asn Phe Ser 245 250 255 Val Ser Ile Arg Glu Glu Leu Lys Arg Thr Asp
Thr Ile Phe Trp Pro 260 265 270 Gly Cys Leu Leu Val Lys Arg Cys Gly
Gly Asn Cys Ala Cys Cys Leu 275 280 285 His Asn Cys Asn Glu Cys Gln
Cys Val Pro Arg Lys Val Thr Lys Lys 290 295 300 Tyr His Glu Val Leu
Gln Leu Arg Pro Lys Thr Gly Val Lys Gly Leu 305 310 315 320 His Lys
Ser Leu Thr Asp Val Ala Leu Glu His His Glu Glu Cys Asp 325 330 335
Cys Val Cys Arg Gly Asn Ala Gly Gly 340 345 5 370 PRT Homo sapiens
5 Met His Arg Leu Ile Phe Val Tyr Thr Leu Ile Cys Ala Asn Phe Cys 1
5 10 15 Ser Cys Arg Asp Thr Ser Ala Thr Pro Gln Ser Ala Ser Ile Lys
Ala 20 25 30 Leu Arg Asn Ala Asn Leu Arg Arg Asp Glu Ser Asn His
Leu Thr Asp 35 40 45 Leu Tyr Arg Arg Asp Glu Thr Ile Gln Val Lys
Gly Asn Gly Tyr Val 50 55 60 Gln Ser Pro Arg Phe Pro Asn
Ser Tyr Pro Arg Asn Leu Leu Leu Thr 65 70 75 80 Trp Arg Leu His Ser
Gln Glu Asn Thr Arg Ile Gln Leu Val Phe Asp 85 90 95 Asn Gln Phe
Gly Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp 100 105 110 Phe
Val Glu Val Glu Asp Ile Ser Glu Thr Ser Thr Ile Ile Arg Gly 115 120
125 Arg Trp Cys Gly His Lys Glu Val Pro Pro Arg Ile Lys Ser Arg Thr
130 135 140 Asn Gln Ile Lys Ile Thr Phe Lys Ser Asp Asp Tyr Phe Val
Ala Lys 145 150 155 160 Pro Gly Phe Lys Ile Tyr Tyr Ser Leu Leu Glu
Asp Phe Gln Pro Ala 165 170 175 Ala Ala Ser Glu Thr Asn Trp Glu Ser
Val Thr Ser Ser Ile Ser Gly 180 185 190 Val Ser Tyr Asn Ser Pro Ser
Val Thr Asp Pro Thr Leu Ile Ala Asp 195 200 205 Ala Leu Asp Lys Lys
Ile Ala Glu Phe Asp Thr Val Glu Asp Leu Leu 210 215 220 Lys Tyr Phe
Asn Pro Glu Ser Trp Gln Glu Asp Leu Glu Asn Met Tyr 225 230 235 240
Leu Asp Thr Pro Arg Tyr Arg Gly Arg Ser Tyr His Asp Arg Lys Ser 245
250 255 Lys Val Asp Leu Asp Arg Leu Asn Asp Asp Ala Lys Arg Tyr Ser
Cys 260 265 270 Thr Pro Arg Asn Tyr Ser Val Asn Ile Arg Glu Glu Leu
Lys Leu Ala 275 280 285 Asn Val Val Phe Phe Pro Arg Cys Leu Leu Val
Gln Arg Cys Gly Gly 290 295 300 Asn Cys Gly Cys Gly Thr Val Asn Trp
Arg Ser Cys Thr Cys Asn Ser 305 310 315 320 Gly Lys Thr Val Lys Lys
Tyr His Glu Val Leu Gln Phe Glu Pro Gly 325 330 335 His Ile Lys Arg
Arg Gly Arg Ala Lys Thr Met Ala Leu Val Asp Ile 340 345 350 ln Leu
Asp His His Glu Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro 355 360 365
Pro Arg 370 6 20 DNA Artificial Sequence oligonucleotide primer 6
agcaggtcca gtggcaaagc 20 7 21 DNA Artificial Sequence
oligonucleotide primer 7 cgtttgatga aagatttggg c 21 8 21 DNA
Artificial Sequence oligonucleotide primer 8 ggaggtctat ataagcagag
c 21 9 20 DNA Artificial Sequence oligonucleotide primer 9
tgagccctcg ccccagtcag 20 10 25 DNA Artificial Sequence
oligonucleotide primer 10 acatacagga aagccttgcc caaaa 25 11 25 DNA
Artificial Sequence oligonucleotide primer 11 aaactaccct gcgattctct
gctgc 25 12 21 DNA Artificial Sequence oligonucleotide primer 12
ggtaaatgga gcttggctga g 21 13 25 DNA Artificial Sequence
oligonucleotide primer 13 tctggacgtc ctcctgctgg tatag 25 14 25 DNA
Artificial Sequence oligonucleotide primer 14 ggtatggagc caggggcaag
ttggg 25 15 27 DNA Artificial Sequence oligonucleotide primer 15
gagtggcaac ttccagggcc aggagag 27 16 27 DNA Artificial Sequence
oligonucleotide primer 16 cttttgctag cctcaaccct gactatc 27 17 35
DNA Artificial Sequence oligonucleotide primer ZC20,180 17
cgcgcggttt aaacgccacc atgagcctct tcggg 35 18 32 DNA Artificial
Sequence oligonucleotide primer ZC20,181 18 cgtatcggcg cgccctatcc
tcctgtgctc cc 32
* * * * *