Note: Descriptions are shown in the official language in which they were submitted.
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Description
GROWTH FACTOR HOMOLOG ZVEGF4
BACKGROUND OF THE INVENTION
In multicellular animals, cell growth, differentiation, and migration are
controlled by polypeptide growth factors. These growth factors play a role in
both
normal development and pathogenesis, including the development of solid
tumors.
Polypeptide growth factors influence cellular events by binding to cell-
surface receptors, many of which are tyrosine kinases. Binding initiates a
chain of
signalling events within the cell, which ultimately results in phenotypic
changes, such
as cell division, protease production, and cell migration.
Growth factors can be classified into families on the basis of structural
similarities. One such family, the PDGF (platelet derived growth factor)
family, is
characterized by a dimeric structure stabilized by disulfide bonds. This
family includes
PDGF, the placental growth factors (P1GFs), and the vascular endothelial
growth
factors (VEGFs). The individual polypeptide chains of these proteins form
characteristic higher-order structures having a bow tie-like configuration
about a cystine
knot, formed by disulfide bonding between pairs of cysteine residues.
Hydrophobic
interactions between loops contribute to the dimerization of the two monomers.
See,
Daopin et al., Science 257:369, 1992; Lapthorn et al., Nature 369:455, 1994.
Members
of this family are active as both homodimers and heterodimers. See, for
example,
Heldin et al., EMBO J. 7:1387-1393, 1988; Cao et al., J. Biol. Chem. 271:3154-
3162,
1996. The cystine knot motif and "bow tie" fold are also characteristic of the
growth
factors transforming growth factor-beta (TGF-(3) and nerve growth factor
(NGF), and
the glycoprotein hormones. Although their amino acid sequences are quite
divergent,
these proteins all contain the six conserved cysteine residues of the cystine
knot.
Five vascular endothelial growth factors have been identified: VEGF,
also known as vascular permeability factor (Dvorak et al., Am. J. Pathol.
146:1029-
1039, 1995); VEGF-B (Olofsson et al., Proc. Natl. Acad. Sci. USA 93:2567-2581,
1996; Hayward et al., WIPO Publication WO 96/27007); VEGF-C (Joukov et al.,
EMBO J. 15:290-298, 1996); VEGF-D (Oliviero, WO 97/12972; Achen et al., WO
98/07832), and zvegf3 (SEQ ID NO:32 and NO:33; co-pending U.S. Patent
Applications Nos. 60/111,173, 60/142,576, and 60/161,653). Five VEGF
polypeptides
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(121, 145, 165, 189, and 206 amino acids) arise from alternative splicing of
the VEGF
mRNA.
VEGFs stimulate the development of vasculature through a process
known as angiogenesis, wherein vascular endothelial cells re-enter the cell
cycle,
degrade underlying basement membrane, and migrate to form new capillary
sprouts.
These cells then differentiate, and mature vessels are formed. This process of
growth
and differentiation is regulated by a balance of pro-angiogenic and anti-
angiogenic
factors. Angiogenesis is central to normal formation and repair of tissue,
occuring in
embryo development and wound healing. Angiogenesis is also a factor in the
development of certain diseases, including solid tumors, rheumatoid arthritis,
diabetic
retinopathy, macular degeneration, and atherosclerosis.
A number of proteins from vertebrates and invertebrates have been
identified as influencing neural development. Among those molecules are
members of
the neuropilin family and the semaphorin/collapsin family.
Three receptors for VEGF have been identified: KDR/Flk-1 (Matthews
et al., Proc. Natl. Acad. Sci. USA 88:9026-9030, 1991), Flt-1 (de Vries et
al., Science
255:989-991, 1992), and neuropilin-1 (Soker et al., Cell 92:735-745, 1998).
Neuropilin-1 is also a receptor for P1GF-2 (Migdal et al., J. Biol. Chem. 273:
22272-
22278, 1998).
Neuropilin-1 is a cell-surface glycoprotein that was initially identified in
Xenopus tadpole nervous tissues, then in chicken, mouse, and human. The
primary
structure of neuropilin-1 is highly conserved among these vertebrate species.
Neuropilin-1 has been demonstrated to be a receptor for various members of the
semaphorin family including semaphorin III (Kolodkin et al., Cell 90:753-762,
1997),
Sema E and Sema IV (Chen et al., Neuron 19:547-559, 1997). A variety of
activities
have been associated with the binding of neuropilin-1 to its ligands. For
example,
binding of semaphorin III to neuropilin-1 can induce neuronal growth cone
collapse and
repulsion of neurites in vitro (Kitsukawa et al., Neuron 19: 995-1005, 1997).
Experiments with transgenic mice indicate the involvement of neuropilin-1 in
the
development of the cardiovascular system, nervous system, and limbs. See, for
example, Kitsukawa et al., Development 121:4309-4318, 1995; and Takashima et
al.,
American Heart Association 1998 Meeting, Abstract # 3178.
Semaphorins are a large family of molecules which share the defining
semaphorin domain of approximately 500 amino acids. Dimerization is believed
to be
important for functional activity (Klostermann et al., J. Biol. Chem. 273:7326-
7331,
1998). Collapsin-1, the first identified vertebrate member of the semaphorin
family of
axon guidance proteins, has also been shown to form covalent dimers, with
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dimerization necessary for collapse activity (Koppel et al., J. Biol. Chem.
273:15708-
15713, 1998). Semaphorin III has been associated in vitro with regulating
growth clone
collapse and chemorepulsion of neurites. Semaphorins have been shown to be
responsible for a variety of developmental effects, including effects on
sensory afferent
innervation, skeletal and cardiac development (Fehar et al., Nature 383:525-
528, 1996),
immunosuppression via inhibition of cytokines (Mangasser-Stephan et al.,
Biochem.
Biophys. Res. Comm. 234:153-156, 1997), and promotion of B-cell aggregation
and
differentiation (Hall et al., Proc. Natl. Acad. Sci. USA 93:11780-11785,
1996). CD 100
has also been shown to be expressed in many T-cell lymphomas and may be a
marker
of malignant T-cell neoplasms (Dorfman et al., Am. J. Pathol. 153:255-262,
1998).
Transcription of the mouse semaphorin gene, M-semaH, correlates with
metastatic
ability of mouse tumor cell lines (Christensen et al., Cancer Res. 58:1238-
1244, 1998).
The role of growth factors, other regulatory molecules, and their
receptors in controlling cellular processes makes them likely candidates and
targets for
therapeutic intervention. Platelet-derived growth factor, for example, has
been
disclosed for the treatment of periodontal disease (U.S. Patent No.
5,124,316),
gastrointestinal ulcers (U.S. Patent No. 5,234,908), and dermal ulcers (Robson
et al.,
Lancet 339:23-25, 1992). Inhibition of PDGF receptor activity has been shown
to
reduce intimal hyperplasia in injured baboon arteries (Giese et al.,
Restenosis Summit
VIII, Poster Session #23, 1996; U.S. Patent No. 5,620,687). PDGF has also been
shown to stimulate bone cell replication (reviewed by Canalis et al.,
Endocrinology and
Metabolism Clinics of North America 18:903-918, 1989), to stimulate the
production of
collagen by bone cells (Centrella et al., Endocrinology 125:13-19, 1989) and
to be
useful in regenerating periodontal tissue (U.S. Patent No. 5,124,316; Lynch et
al., J.
Clin. Periodontol. 16:545-548, 1989). Vascular endothelial growth factors
(VEGFs)
have been shown to promote the growth of blood vessels in ischemic limbs
(Isner et al.,
The Lancet 348:370-374, 1996), and have been proposed for use as wound-healing
agents, for treatment of periodontal disease, for promoting endothelialization
in
vascular graft surgery, and for promoting collateral circulation following
myocardial
infarction (WIPO Publication No. WO 95/24473; U.S. Patent No. 5,219,739).
VEGFs
are also useful for promoting the growth of vascular endothelial cells in
culture. A
soluble VEGF receptor (soluble flt-1) has been found to block binding of VEGF
to cell-
surface receptors and to inhibit the growth of vascular tissue in vitro
(Biotechnology
News 16(17):5-6, 1996).
In view of the proven clinical utility of polypeptide growth factors, there
is a need in the art for additional such molecules for use as therapeutic
agents,
diagnostic agents, and research tools and reagents.
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The present invention provides such polypeptides for these and other
uses that should be apparent to those skilled in the art from the teachings
herein.
DESCRIPTION OF THE INVENTION
The present invention provides an isolated polypeptide of at least 15
amino acid residues comprising an epitope-bearing portion of a protein of SEQ
ID
NO:2. Within certain embodiments, the polypeptide comprises a segment that is
at
least 70% identical to residues 52-179 of SEQ ID NO:2 or residues 258-370 of
SEQ ID
NO:2. Within other embodiments, the polypeptide is selected from the group
consisting of residues 19-179 of SEQ ID NO:2, residues 52-179 of SEQ ID NO:2,
residues 19-257 of SEQ ID NO:2, residues 52-257 of SEQ ID NO:2, residues 19-
253 of
SEQ ID NO:2, residues 52-253 of SEQ ID NO:2, residues 19-370 of SEQ ID NO:2,
residues 52-370 of SEQ ID NO:2, residues 258-370 of SEQ ID NO:2, and residues
180-
370 of SEQ ID NO:2.
The invention also provides an isolated polypeptide comprising a
sequence of amino acids of the formula Rl,,-R2y-R3,, wherein R1 is a
polypeptide of
from 100 to 130 residues in length, is at least 70% identical to residues 52-
179 of SEQ
ID NO:2, and comprises a sequence motif
C[KR]Y[DNE][WYF]X{11,15}G[KR][WYF]C (SEQ ID NO:4) corresponding to
residues 109-131 of SEQ ID NO:2; R2 is a polypeptide at least 90% identical to
residues 180-257 of SEQ ID NO:2; R3 is a polypeptide at least 70% identical in
amino
acid sequence to residues 258-370 of SEQ ID NO:2 and comprises cysteine
residues at
positions corresponding to residues 272, 302, 306, 318, 360, and 362 of SEQ ID
NO:2,
a glycine residue at a position corresponding to residue 304 of SEQ ID NO:2,
and a
sequence motif CX { 18,33 } CXGXCX { 6,33 } CX { 20,50 } CXC (SEQ ID NO:3)
corresponding to residues 272-362 of SEQ ID NO:2; and each of x, y, and z is
individually 0 or 1, subject to the limitations that at least one of x and z
is 1, and, if x
and z are each 1, then y is 1. There are thus provided isolated polypeptides
of the above
formula wherein (a) x=l, (b) z=1, and (c) x=1 and z=1. Within certain
embodiments,
x=1 and R1 is at least 90% identical to residues 52-179 of SEQ ID NO:2 or
residues 19-
179 of SEQ ID NO:2. Within related embodiments, x=1 and R1 comprises residues
52-
179 of SEQ ID NO:2. Within other embodiments, z=1 and R3 is at least 90%
identical
to residues 258-370 of SEQ ID NO:2 or R3 comprises residues 258-370 of SEQ ID
NO:2. Within other embodiments, x=1, z=1, and R3 is at least 90% identical to
residues 258-370 of SEQ ID NO:2; and x=l, z=1, R1 is at least 90% identical to
residues 52-179 of SEQ ID NO:2, and R2 is at least 90% identical to residues
180-257
of SEQ ID NO:2. Within additional embodiments, x=1, z=1, and the polypeptide
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comprises residues 19-370 of SEQ ID NO:2 or residues 52-370 of SEQ ID NO:2.
The
isolated polypeptide may further comprise cysteine residues at positions
corresponding
to residues 308 and 316 of SEQ ID NO:2. Within other embodiments, the isolated
polypeptide further comprises an affinity tag. Within a related embodiment,
the
5 isolated polypeptide comprises an immunoglobulin constant domain.
The present invention also provides an isolated protein comprising a first
polypeptide operably linked to a second polypeptide, wherein the first
polypeptide
comprises a sequence of amino acids of the formula R1X-R2y-R3, as disclosed
above.
The protein modulates cell proliferation, apoptosis, differentiation,
metabolism, or
migration. Within one embodiment, the protein is a heterodimer. Within related
embodiments, the second polypeptide is selected from the group consisting of
VEGF,
VEGF-B, VEGF-C, VEGF-D, zvegf3 (SEQ ID NO:33), P1GF, PDGF-A, and PDGF-B.
Within other related embodiments, x=1, z=1, and the second polypeptide
comprises
residues 46-345 of SEQ ID NO:33; x=1 and the second polypeptide comprises
residues
46-170 of SEQ ID NO:33; or z=1 and the second polypeptide comprises residues
235-
345 of SEQ ID NO:33.
Within another embodiment, the protein is a homodimer.
There is also provided an isolated protein produced by a method
comprising the steps of (a) culturing a host cell containing an expression
vector
comprising the following operably linked elements: a transcription promoter; a
DNA
segment encoding a polypeptide selected from the group consisting of (i)
residues 52-
370 of SEQ ID NO:2, (ii) residues 52-253 of SEQ ID NO:2, (iii) residues 180-
370 of
SEQ ID NO:2, and (iv) residues 258-370 of SEQ ID NO:2; and a transcription
terminator, under conditions whereby the DNA segment is expressed; and (b)
recovering from the cell the protein product of expression of the DNA
construct.
Within another aspect of the invention there is provided an isolated
polynucleotide of up to approximately 4.4 kb in length, wherein said
polynucleotide
encodes a polypeptide as disclosed above. Within one embodiment of the
invention,
the polynucleotide is DNA.
Within a further aspect of the invention there is provided an expression
vector comprising the following operably linked elements: (a) a transcription
promoter;
(b) a DNA polynucleotide as disclosed above; and (c) a transcription
terminator. The
vector may further comprise a secretory signal sequence operably linked to the
DNA
polynucleotide.
Also provided by the invention is a cultured cell into which has been
introduced an expression vector as disclosed above, wherein the cell expresses
the
polypeptide encoded by the DNA polynucleotide. The cultured cell can be used
within
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a method of producing a polypeptide, the method comprising culturing the cell
and
recovering the expressed polypeptide.
The proteins provided herein can be combined with a pharmaceutically
acceptable vehicle to provide a pharmaceutical composition.
The invention also provides an antibody that specifically binds to an
epitope of a polypeptide as disclosed above. Antibodies of the invention
include, inter
alia, monoclonal antibodies and single chain antibodies, and may be linked to
a reporter
molecule.
The invention further provides a method for detecting a genetic
abnormality in a patient, comprising the steps of (a) obtaining a genetic
sample from a
patient, (b) incubating the genetic sample with a polynucleotide comprising at
least 14
contiguous nucleotides of SEQ ID NO:1 or the complement of SEQ ID NO:1, under
conditions wherein the polynucleotide will hybridize to a complementary
polynucleotide sequence, to produce a first reaction product, and (c)
comparing the first
reaction product to a control -reaction product, wherein a difference between
the first
reaction product and the control reaction product is indicative of a genetic
abnormality
in the patient.
The invention also provides a polypeptide comprising a sequence
selected from the group consisting of: residues 46-234 of SEQ ID NO:33
operably
linked to residues 258-370 of SEQ ID NO:2; residues 46-170 of SEQ ID NO:33
operably linked to residues 180-370 of SEQ ID NO:2; residues 52-257 of SEQ ID
NO:2
operably linked to residues 235-345 of SEQ ID NO:33; and residues 52-179 of
SEQ ID
NO:2 operably linked to residues 171-345 of SEQ ID NO:33.
The invention also provides a method of activating a cell-surface PDGF
receptor, comprising exposing a cell comprising a cell-surface PDGF receptor
to a
polypeptide or protein as disclosed above, whereby the polypeptide or protein
binds to
and activates the receptor.
The invention also provides a method of inhibiting a PDGF receptor-
mediated cellular process, comprising exposing a cell comprising a cell-
surface PDGF
receptor to a compound that inhibits binding of a polypeptide or protein as
disclosed
above to the receptor.
The invention also provides a method of stimulating the growth of bone
tissue, comprising applying to bone a growth-stimulating amount of a
polypeptide or
protein as disclosed above.
The invention also provides a method of modulating the proliferation,
differentiation, migration, or metabolism of bone cells, comprising exposing
bone cells
to an effective amount of a polypeptide or protein as disclosed above.
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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 accompanying drawings:
Fig. 1 is 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.
Fig. 2 is an illustration of the vector pHB12-8 for use in expressing
cDNAs in transgenic animals.
The term "affinity tag" is used herein to denote a polypeptide segment
that can be attached to a second polypeptide to provide for purification or
detection of
the second polypeptide or provide sites for attachment of the second
polypeptide to a
substrate. In principal, any peptide or protein for which an antibody or other
specific
binding agent is available can be used as an affinity tag. Affinity tags
include a poly-
histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et
al., Methods
Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene
67:31,
1988), maltose binding protein (Kellerman and Ferenci, Methods Enzymol. 90:459-
463,
1982; Guan et al., Gene 67:21-30, 1987), Glu-Glu affinity tag (Grussenmeyer et
al.,
Proc. Natl. Acad. Sci. USA 82:7952-4, 1985; see SEQ ID NO:5), substance P,
F1agTM
peptide (Hopp et al., Biotechnology 6:1204-10, 1988), streptavidin binding
peptide,
thioredoxin, ubiquitin, cellulose binding protein, T7 polymerase, or other
antigenic
epitope or binding domain. See, in general, Ford et al., Protein Expression
and
Purification 2: 95-107, 1991. DNAs encoding affinity tags and other reagents
are
available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ;
New
England Biolabs, Beverly, MA; and Eastman Kodak, New Haven, CT).
The term "allelic variant" is used herein to denote any of two or more
alternative forms of a gene occupying the same chromosomal locus. Allelic
variation
arises naturally through mutation, and may result in phenotypic polymorphism
within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or
may encode polypeptides having altered amino acid sequence. The term allelic
variant
is also used herein to denote a protein encoded by an allelic variant of a
gene.
The terms "amino-terminal" and "carboxyl-terminal" are used herein to
denote positions within polypeptides. Where the context allows, these terms
are used
with reference to a particular sequence or portion of a polypeptide to denote
proximity
or relative position. For example, a certain sequence positioned carboxyl-
terminal to a
reference sequence within a polypeptide is located proximal to the carboxyl
terminus of
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the reference sequence, but is not necessarily at the carboxyl terminus of the
complete
polypeptide.
A "beta-strand-like region" is a region of a protein characterized by
certain combinations of the polypeptide backbone dihedral angles phi (0) and
psi (w).
Regions wherein 0 is less than -60 and Ni is greater than 90 are beta-strand-
like.
Those skilled in the art will recognize that the limits of a (3-strand are
somewhat
imprecise and may vary with the criteria used to define them. See, for
example,
Richardson and Richardson in Fasman, ed., Prediction of Protein Structure and
the
Principles of Protein Conformation, Plenum Press, New York, 1989; and Lesk,
Protein
Architecture: A Practical Approach, Oxford University Press, New York, 1991.
A "complement" of a polynucleotide molecule is a polynucleotide
molecule having a complementary base sequence and reverse orientation as
compared
to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is
complementary to 5' CCCGTGCAT 3'.
"Corresponding to", when used in reference to a nucleotide or amino
acid sequence, indicates the position in a second sequence that aligns with
the reference
position when two sequences are optimally aligned.
The term "degenerate nucleotide sequence" denotes a sequence of
nucleotides that includes one or more degenerate codons (as compared to a
reference
polynucleotide molecule that encodes a polypeptide). Degenerate codons contain
different triplets of nucleotides, but encode the same amino acid residue
(i.e., GAU and
GAC triplets each encode Asp).
The term "expression vector" is used to denote a DNA molecule, linear
or circular, that comprises a segment encoding a polypeptide of interest
operably linked
to additional segments that provide for its transcription. Such additional
segments
include promoter and terminator sequences, and may also include one or more
origins
of replication, one or more selectable markers, an enhancer, a polyadenylation
signal,
etc. Expression vectors are generally derived from plasmid or viral DNA, or
may
contain elements of both.
The term "isolated", when applied to a polynucleotide, denotes that the
polynucleotide has been removed from its natural genetic milieu and is thus
free of
other extraneous or unwanted coding sequences, and is in a form suitable for
use within
genetically engineered protein production systems. Such isolated molecules are
those
that are separated from their natural environment and include cDNA and genomic
clones. Isolated polynucleotide molecules of the present invention are free of
other
genes with which they are ordinarily associated, but may include naturally
occurring 5'
and 3' untranslated regions such as promoters and terminators. The
identification of
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associated regions will be evident to one of ordinary skill in the art (see,
for example,
Dynan and Tijan, Nature 316:774-78, 1985).
An "isolated" polypeptide or protein is a polypeptide or protein that is
found in a condition other than its native environment, such as apart from
blood and
animal tissue. Within one embodiment, the isolated polypeptide or protein is
substantially free of other polypeptides or proteins, particularly other
polypeptides or
proteins of animal origin. The polypeptides or proteins may be provided in a
highly
purified form, i.e. greater than 95% pure or greater than 99% pure. When used
in this
context, the term "isolated" does not exclude the presence of the same
polypeptide or
protein in alternative physical forms, such as dimers or alternatively
glycosylated or
derivatized forms.
A "motif' is a series of amino acid positions in a protein sequence for
which certain amino acid residues are required. A motif defines the set of
possible
residues at each such position.
"Operably linked" means that two or more entities are joined together
such that they function in concert for their intended purposes. When referring
to DNA
segments, the phrase indicates, for example, that coding sequences are joined
in the
correct reading frame, and transcription initiates in the promoter and
proceeds through
the coding segment(s) to the terminator. When referring to polypeptides,
"operably
linked" includes both covalently (e.g., by disulfide bonding) and non-
covalently (e.g.,
by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions)
linked
sequences, wherein the desired function(s) of the sequences are retained.
The term "ortholog" denotes a polypeptide or protein obtained from one
species that is the functional counterpart of a polypeptide or protein from a
different
species. Sequence differences among orthologs are the result of speciation.
A "polynucleotide" is a single- or double-stranded polymer of
deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
Polynucleotides include RNA and DNA, and may be isolated from natural sources,
synthesized in vitro, or prepared from a combination of natural and synthetic
molecules.
Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides
("nt"), or kilobases ("kb"). Where the context allows, the latter two terms
may describe
polynucleotides that are single-stranded or double-stranded. When the term is
applied
to double-stranded molecules it is used to denote overall length and will be
understood
to be equivalent to the term "base pairs". It will be recognized by those
skilled in the
art that the two strands of a double-stranded polynucleotide may differ
slightly in length
and that the ends thereof may be staggered as a result of enzymatic cleavage;
thus all
CA 02370948 2008-05-14
nucleotides within a double-stranded polynucleotide molecule may not be
paired. Such
unpaired ends will in general not exceed 20 nt in length.
A "polypeptide" is a polymer of amino acid residues joined by peptide
bonds, whether produced naturally or synthetically. Polypeptides of less than
about 10
5 amino acid residues are commonly referred to as "peptides".
The term "promoter" is used herein for its art-recognized meaning to
denote a portion of a gene containing DNA sequences that provide for the
binding of
RNA polymerase and initiation of transcription. Promoter sequences are
commonly,
but not always, found in the 5' non-coding regions of genes.
10 A "protean" is a macromolecule comprising one or mom polypeptide
chains. A protein may also comprise non-peptidic components, such as
carbohydrate
groups. Carbohydrates and other non-peptidic substituents may be added to a
protein
by the cell in which the protein is produced, and will vary with the type of
cell.
Proteins are defined herein in terms of their amino acid backbone structures;
substidunts such as carbohydrate groups are generally not specified, but may
be present
nonetheless.
A "secretory signal sequence" is a DNA sequence that encodes a
polypeptide (a "secretory peptide") that, as a component of a larger
polypeptide, directs
the larger polypeptide through a secretory pathway of a cell in which it is
synthesized
The larger polypeptide is commonly cleaved to remove the secretory peptide
during
transit through the secretory pathway.
A "segment'" is a portion of a larger molecule (e.g., polynucleotide or
polypeptide) having specified attributes. For example, a DNA segment encoding
a
specified polypeptide is a portion of a longer DNA molecule, such as a plasmid
or
2S plasmid fragment, that, when read from the 5' to the 3' direction, encodes
the sequence
of amino acids of the specified polypeptide.
Molecular weights and lengths of polymers determined by imprecise
analytical methods (e.g., gel electrophoresis) will be understood to be
approximate
values. When such a value is expressed as "about" X or "approximately" X, the
stated
value of X will be understood to be accurate to 120%.
The present invention is based in part upon the discovery of a novel
DNA molecule that encodes a polypeptide comprising 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 "cystinc 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
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morphogenetic protein-1 (Wozney et al., Science 242:1528-1534, 1988), porcine
seminal plasma protein and bovine acidic seminal fluid protein (Romero et at.,
Nat.
Struct. Biol. 4:783-788, 1997), and X. laevis tolloid-like protein (Lin et
al., Dev. Growth
Differ. 39:43-51, 1997). Analysis of the tissue distribution of the mRNA
corresponding
to this novel DNA showed that expression was widespread in adult human
tissues. The
polypeptide has been designated "zvegf4" in view of its homology to the VEGFs
in the
growth factor domain.
Structural predictions based on the zvegf4 sequence and its homology to
other growth factors suggests that the polypeptide can form homomultimers or
heteromultimers that act on tissues to control organ development by modulating
cell
proliferation, migration, differentiation, or metabolism. Experimental
evidence
supports these predictions. Zvegf4 heteromultimers may comprise a polypeptide
from
another member of the PDGF/VEGF family of proteins, including VEGF, VEGF-B,
VEGF-C, VEGF-D, zvegf3, P1GF (Maglione et al., Proc. Natl. Acad. Sci. USA
88:9267-9271, 1991), PDGF-A (Murray et al., U.S. Patent No. 4,899,919; Heldin
et al.,
U.S. Patent No. 5,219,759), or PDGF-B (Chiu et al., Cell 37:123-129, 1984;
Johnsson
et al., EMBO J. 3:921-928, 1984). Members of this family of polypeptides
regulate
organ development and regeneration, post-developmental organ growth, and organ
maintenance, as well as tissue maintenance and repair processes. These factors
are also
involved in pathological processes where therapeutic treatments are required,
including
cancer, rheumatoid arthritis, diabetic retinopathy, ischemic limb disease,
peripheral
vascular disease, myocardial ischemia, vascular intimal hyperplasia,
atherosclerosis,
and hemangioma formation. To treat these pathological conditions it will often
be
required to develop compounds to antagonize the members of the PDGF/VEGF
family
2S of proteins, or their respective receptors. This may include the
development of
neutralizing antibodies, small molecule antagonists, modified forms of the
growth
factors that maintain receptor binding activity but lack receptor activating
activity,
soluble receptors (including receptor-immunoglobulin fusion proteins) or
antisense or
ribozyme molecules to block polypeptide production.
A representative human zvegf4 polypeptide sequence is shown in SEQ
ID NO:2, and a representative mouse zvegf4 polypeptide sequence is shown in
SEQ ID
NO:53. DNAs encoding these polypeptides are shown in SEQ ID NOS:1 and 52,
respectively. Analysis of the amino acid sequence shown in SEQ ID NO:2
indicates
that residues 1 to 18 form a secretory peptide. The CUB domain extends from
residue
52 to residue 179. A propeptide-like sequence extends from residue 180 to
either
residue 245, residue 249 or residue 257, and includes four potential cleavage
sites at its
carboxyl terminus, monobasic sites at residue 245 and residue 249, a dibasic
site at
uI -I z-zuuu PCT/USOO/4004
CA 02370948 2001-10-16
12
residues 254-255, and a target site for flu in or a furin-like protease at
residues 254-257.
Protein produced in a baculovirus expression system showed cleavage between
residues
250 and 249, as well as longer species with amino termini at residues 19 and
35. The
growth factor domain extends from residue 258 to residue 370, and may include
additional residues at the N-terminus (for instance, this domain may include
residues
250 to 370 or residues 246 to 370). Those skilled in the at will recognize
that domain
boundaries are somewhat imprecise and can be expected to vary by up tot 5
residues
from the specified positions, Cleavage of fall-length zvegf4 with plasmic
resulted in
activation of the zvegf4 polypeptide. By Western analysis, a band migrating at
approximately the same size as the growth factor domain was observed. A
matched,
uncleaved full-length zvegf4 sample demonstrated no activation.
Signal peptide cleavage is predicted to occur in human zvegf4 after
residue 18 ( 3 residues). Upon comparison of human and mouse zvegf4
sequences,
alternative signal peptide cleavage sites arc predicted after residue 23
and/or residue 24,
This analysis suggests that the zvegf4 polypeptide chain may be cleaved to
produce a
plurality of monomeric species, some of which are shown in Table 1. In certain
boot
cells, cleavage after Lys 255 is expected to result in subsequent removal of
residues
254-255, although polypeptides with a carboxyl terminus at residue 255 may
also be
prepared. Cleavage after Lys-257 is expected to result in subsequent removal
of
residue 257. These cleavage sites can be modified to prevent proteolysis and
thus
provide for the production of uncleaved zvegf4 polypeptides and multimers
comprising
them. Actual cleavage patterns are expected to vary among host cells.
AMENDED SHEET
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13
Table 1
Monomer Residues (SEQ ID NO:2)
Cub domain 19 - 179
35 - 179
52- 179
CUB domain + interdomain region
19-257
35 - 257
52 - 257
19-255
35 - 255
52 - 255
19-253
35 - 253
52 - 253
19-249
35 - 249
52 - 249
19-245
35 - 245
52 - 245
Cub domain + interdomain region +
growth factor domain
19 - 370
35 - 370
52-370
Growth factor domain 246 - 370
250 - 370
258 - 370
Growth factor domain +
interdomain region 180 - 370
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14
Also included within the present invention are polypeptides that are at
least 70%, 80%, 90%, and 95% identical to the polypeptides disclosed in Table
1,
wherein these additional polypeptides retain certain characteristic sequence
motifs as
disclosed below.
Zvegf4 polypeptides are designated herein with a subscript indicating the
amino acid residues. For example, the CUB domain polypeptides disclosed in
Table 1
are designated "zvegf419_179", "zvegf435_179", and "zvegf452-179".
Higher order structure of zvegf4 polypeptides can be predicted by
sequence alignment with known homology and computer analysis using available
software (e.g., the Insight II viewer and homology modeling tools; MSI, San
Diego,
CA). Analysis of SEQ ID NO:2 predicts that the secondary structure of the
growth
factor domain is dominated by the cystine knot, which ties together variable
beta strand-
like regions and loops into a bow tie-like structure. Sequence alignment
indicates that
Cys residues within the growth factor domain at positions 272, 302, 306, 318,
360, and
362, and Gly 304 are highly conserved within the family. Further analysis
suggests
pairing (disulfide bond formation) of Cys residues 272 and 318, 302 and 360,
and 306
and 362 to form the cystine knot. This arrangement of conserved residues can
be
represented by the formula CX { 18,33 } CXGXCX { 6,33 } CX { 20,50 } CXC (SEQ
ID
NO:3), wherein amino acid residues are represented by the conventional single-
letter
code, X is any amino acid residue, and { y,z } indicates a region of variable
residues (X)
from y to z residues in length. A consensus bow tie structure is formed as:
amino
terminus to cystine knot --) beta strand-like region 1 --~ variable loop 1 ->
beta strand-
like region 2 -4 cystine knot -4 beta strand-like region 3 - variable loop 2 -
> beta
strand-like region 4 - cystine knot -a beta strand-like region 5 -* variable
loop 3 -
beta strand-like region 6 - cystine knot. Variable loops 1 and 2 form one side
of the
bow tie, with variable loop 3 forming the other side. The structure of the
zvegf4 growth
factor domain appears to diverge from the consensus structure of other family
members
in loop 2 and beta strand-like regions 3 and 4, wherein all are abbreviated
and
essentially replaced by a cysteine cluster comprising residues 307 (Gly)
through 317
(Thr), which includes Cys residues at positions 308 and 316 of SEQ ID NO:2.
The
approximate boundaries of the beta strand-like regions in SEQ ID NO:2 are:
region 1,
residues 273-281; region 2, residues 297-301; region 5, residues 319-324;
region 6,
residues 355-358. Loops separate regions 1 and 2, and regions 5 and 6.
The CUB domain of zvegf4 is believed to form a beta barrel structure
with nine distinct beta strand-like regions. These regions comprise residues
54-57, 61-
65, 79-84, 90-95, 97-99, 112-115, 126-130, 146-150, and 163-170 of SEQ ID
NO:2. A
multiple alignment of CUB domains of Xenopus laevis neuropilin precursor
(Takagi et
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WO 00/66736 PCTIUSOO/40047
al., ibid.), human BMP-1 (Wozney et al., ibid.), and X. laevis tolloid-like
protein (Lin et
al., ibid.) indicates the presence of a conserved motif corresponding to
residues 109-131
of SEQ ID NO:2. This motif is represented by the formula
C[KR]Y[DNE][WYF]X{11,15}G[KR][WYF]C (SEQ ID NO:4), wherein square
5 brackets indicate the allowable residues at a given position and X { y,z }
is as defined
above.
The proteins of the present invention include proteins comprising CUB
domains homologous to the CUB domain of zvegf4. These homologous domains are
from 100 to 120 residues in length and comprise a motif of the sequence
10 C[KR]Y[DNE][WYF]X{11,15}G[KR][WYF]C (SEQ ID NO:4) corresponding to
residues 109-131 of SEQ ID NO:2. These homologous CUB domains are at least 70%
identical to residues 52-179 of SEQ ID NO:2, at least 80% identical, at least
90%
identical, or at least 95% identical to residues 52-179 of SEQ ID NO:2.
CUB domain-containing proteins of the present invention may further
15 include a zvegf4 interdomain region or homolog thereof. The interdomain
region is at
least 70% identical to residues 180 to 253 of SEQ ID NO:2.
As noted above, residues 254-257 of SEQ ID NO:2 are believed to
provide cleavage sites for furin or other proteases. However, polypeptides
comprising a
C-terminal interdomain region (e.g., zvegf452-257) can be prepared with or
without one
or more of residues 254-257 at the amino terminus. In addition, polypeptides
comprising another C-terminal interdomain region (e.g., zvegf452-245) can be
prepared.
Additional proteins of the present invention comprise the zvegf4 growth
factor domain or a homolog thereof. These proteins thus comprise a polypeptide
segment that is at least 70%, 80%, 90% or 95% identical to residues 258-370 of
SEQ
ID NO:2, wherein the polypeptide segment comprises Cys residues at positions
corresponding to residues 272, 302, 306, 318, 360, and 362 of SEQ ID NO:2; a
glycine
at a position corresponding to residue 304 of SEQ ID NO:2; and the sequence
motif
CX { 18,33 } CXGXCX { 6,33 } CX { 20,50 } CXC (SEQ ID NO:3) corresponding to
residues 272-362 of SEQ ID NO:2.
Additional proteins comprising combinations of the CUB domain,
interdomain region, and growth factor domain are shown above in Table 1. In
each
case, the invention also includes homologous proteins comprising homologous
domains
as disclosed above. More particularly, domains or regions in the mouse zvegf4
protein
corresponding to domains or regions in the human zvegf4 protein are included
within
the present invention.
Structural analysis and homology predict that zvegf4 polypeptides
complex with a second polypeptide to form multimeric proteins. These proteins
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
16
include homodimers and heterodimers. In the latter case, the second
polypeptide can be
a truncated or other variant zvegf4 polypeptide or another polypeptide, such
as a P1GF,
PDGF-A, PDGF-B, VEGF, VEGF-B, VEGF-C, VEGF-D, or zvegf3 polypeptide.
Among the dimeric proteins within the present invention are dimers formed by
non-
covalent association (e.g., hydrophobic interactions) with a second subunit,
either a
second zvegf4 polypeptide or other second subunit, or by covalent association
stabilized by intermolecular disulfide bonds between cysteine residues of the
component monomers. Within SEQ ID NO:2, the Cys residues at positions 296,
308,
316, and 364 may form intramolecular or intermolecular disulfide bonds.
The present invention thus provides a variety of multimeric proteins
comprising a zvegf4 polypeptide as disclosed above. These zvegf4 polypeptides
include zvegf419-179, zvegf435-179, zvegf452-179, zvegf419-245, zvegf435-245,
zvegf452-245,
zvegf419-249, zvegf435-249, zvegf452-249, zvegf419.253, zvegf435-253, zvegf452-
253, zvegf419-
255, zvegf435-255, zvegf452-255, zvegf419-257, zvegf435-257, zvegf452-257,
zvegf419-370,
zvegf435-370, zvegf452-370, zvegf4246-370, zvegf4250-370, and zvegf4258-370,
as well as
variants and derivatives of these polypeptides as disclosed herein. These
zvegf4
polypeptides can be prepared as homodimers or as heterodimers with
corresponding
regions of related family members. For example, a zvegf4 CUB domain
polypeptide
can be dimerized with a polypeptide comprising residues 46-170 of SEQ ID
NO:33; a
zvegf4 growth factor domain polypeptide can be dimerized with a polypeptide
comprising residues 235-345 of SEQ ID NO:33; and a zvegf4 CUB domain-
interdomain-growth factor domain polypeptide can be dimerized with a
polypeptide
comprising residues 46-345 of SEQ ID NO:33.
Percent sequence identity is determined by conventional methods. See,
for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986, and Henikoff
and
Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992. Briefly, two amino
acid
sequences are aligned to optimize the alignment scores using a gap opening
penalty of
10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and
Henikoff (ibid.) as shown in Table 2 (amino acids are indicated by the
standard one-
3 0 letter codes). The percent identity is then calculated as:
Total number of identical matches
x 100
[length of the longer sequence plus the
number of gaps introduced into the longer
sequence in order to align the two
sequences]
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
17
b ri
ri N M
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H in N N C)
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d' H m N N
I 1
W N ri H r M N
1 I 1 1
W l0 dl N N H
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.~. in r-i M r-i O H M N N
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I I I 1 I I 1 I 1 I 1 1 I
7-1 l0 r-i m O O C) r-i m M C) N M N ri O N M
1 I I 1 I 1 1 I 1
Q', Ln O N M ri O N O M N N H M N r1 H M N M
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d' ri N N O H H O N r1 H r1 rI N r1 H O M N O
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ri ri N
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
18
The level of identity between amino acid sequences can be determined
using the "FASTA" similarity search algorithm of Pearson and Lipman (Proc.
Natl.
Acad. Sci. USA 85:2444, 1988) and Pearson (Meth. Enzymol. 183:63, 1990).
Briefly,
FASTA first characterizes sequence similarity by identifying regions shared by
the
query sequence (e.g., SEQ ID NO:2) and a test sequence that have either the
highest
density of identities (if the ktup variable is 1) or pairs of identities (if
ktup=2), without
considering conservative amino acid substitutions, insertions, or deletions.
The ten
regions with the highest density of identities are then rescored by comparing
the
similarity of all paired amino acids using an amino acid substitution matrix,
and the
ends of the regions are "trimmed" to include only those residues that
contribute to the
highest score. If there are several regions with scores greater than the
"cutoff' value
(calculated by a predetermined formula based upon the length of the sequence
and the
ktup value), then the trimmed initial regions are examined to determine
whether the
regions can be joined to form an approximate alignment with gaps. Finally, the
highest
scoring regions of the two amino acid sequences are aligned using a
modification of the
Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.
48:444,
1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), which allows for amino acid
insertions and deletions. Illustrative parameters for FASTA analysis are:
ktup=l, gap
opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62.
These parameters can be introduced into a FASTA program by modifying the
scoring
matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, 1990 (ibid.).
FASTA can also be used to determine the sequence identity of nucleic
acid molecules using a ratio as disclosed above. For nucleotide sequence
comparisons,
the ktup value can range between one to six, preferably from four to six.
Within certain embodiments of the invention amino acid substitutions as
compared with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:53 are
conservative substitutions. The BLOSUM62 matrix (Table 2) is an amino acid
substitution matrix derived from about 2,000 local multiple alignments of
protein
sequence segments, representing highly conserved regions of more than 500
groups of
related proteins (Henikoff and Henikoff, ibid.). Thus, the BLOSUM62
substitution
frequencies can be used to define conservative amino acid substitutions that
may be
introduced into the amino acid sequences of the present invention. As used
herein, the
term "conservative amino acid substitution" refers to a substitution
represented by a
BLOSUM62 value of greater than -1. For example, an amino acid substitution is
conservative if the substitution is characterized by a BLOSUM62 value of 0, 1,
2, or 3.
More conservative amino acid substitutions are characterized by a BLOSUM62
value
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
19
of at least 1 (e.g., 1, 2 or 3), while still more conservative amino acid
substitutions are
characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
Polypeptides of the present invention can be prepared with one or more
amino acid substitutions, deletions or additions as compared to SEQ ID NO:2 or
SEQ
ID NO:53. These changes can be of a minor nature, that is conservative amino
acid
substitutions and other changes that do not significantly affect the folding
or activity of
the protein or polypeptide, and include amino- or carboxyl-terminal
extensions, such as
an amino-terminal methionine residue, an amino or carboxyl-terminal cysteine
residue
to facilitate subsequent linking to maleimide-activated keyhole limpet
hemocyanin, a
small linker peptide of up to about 20-25 residues, or an affinity tag as
disclosed above.
Two or more affinity tags may be used in combination. Polypeptides comprising
affinity tags can further comprise a polypeptide linker and/or a proteolytic
cleavage site
between the zvegf4 polypeptide and the affinity tag. Exemplary cleavage sites
include,
without limitation, thrombin cleavage sites and factor Xa cleavage sites.
The present invention further provides a variety of other polypeptide
fusions and related multimeric proteins comprising one or more polypeptide
fusions.
For example, a zvegf4 polypeptide can be prepared as a fusion to a dimerizing
protein
as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Exemplary
dimerizing
proteins in this regard include immunoglobulin constant region domains.
Dimerization
can also be stabilized by fusing a zvegf4 polypeptide to a leucine zipper
sequence
(Riley et al., Protein Eng. 9:223-230, 1996; Mohamed et al., J. Steroid
Biochem. Mol.
Biol. 51:241-250, 1994). Immunoglobulin-zvegf4 polypeptide fusions and leucine
zipper fusions can be expressed in genetically engineered cells to produce a
variety of
multimeric zvegf4 analogs. Auxiliary domains can be fused to zvegf4
polypeptides to
target them to specific cells, tissues, or macromolecules (e.g., collagen).
For example, a
zvegf4 polypeptide or protein can be targeted to a predetermined cell type by
fusing a
zvegf4 polypeptide to a ligand that specifically binds to a receptor on the
surface of the
target cell. In this way, polypeptides and proteins can be targeted for
therapeutic or
diagnostic purposes. A zvegf4 polypeptide can be fused to two or more
moieties, such
as an affinity tag for purification and a targeting domain. Polypeptide
fusions can also
comprise one or more cleavage sites, particularly between domains. See, Tuan
et al.,
Connective Tissue Research 34:1-9, 1996.
Zvegf4 polypeptide fusions will generally contain not more than about
1,500 amino acid residues, often not more than about 1,200 residues, more
often not
3S more than about 1,000 residues, and will in many cases be considerably
smaller. For
example, a zvegf4 polypeptide of 352 residues (residues 19-370 of SEQ ID NO:2)
can
be fused to E. coli /3-galactosidase (1,021 residues; see Casadaban et al., J.
Bacteriol.
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WO 00/66736 PCTIUSOO/40047
143:971-980, 1980), a 10-residue spacer, and a 4-residue factor Xa cleavage
site to
yield a polypeptide of 1,387 residues. In a second example, residues 250-370
of SEQ
ID NO:2 can be fused to maltose binding protein (approximately 370 residues),
a 4-
residue cleavage site, and a 6-residue polyhistidine tag.
5 A polypeptide comprising the zvegf4 growth factor domain (e.g.,
zvegf4258-370 or zvegf4180_370) may be fused to a non-zvegf4 CUB domain.
Within a
related embodiment of the invention, a zvegf4 polypeptide comprising zvegf4
growth
factor and CUB domains is fused to a non-zvegf4 CUB domain, such as a CUB-
domain-comprising neuropilin polypeptide.
10 The present invention further provides polypeptide fusions comprising
the zvegf4 CUB domain (e.g., zvegf452-179)= The CUB domain, with its homology
to
neuropilin-1, may be used to target zvegf4 or other proteins containing it to
cells having
cell-surface semaphorins, including endothelial cells, neuronal cells,
lymphocytes, and
tumor cells. The zvegf4 CUB domain can thus be joined to other moieties,
including
15 polypeptides (e.g., other growth factors, antibodies, and enzymes) and non-
peptidic
moieties (e.g., radionuclides, contrast agents, and the like), to target them
to cells
expressing cell-surface semaphorins. The cleavage sites between the CUB and
growth
factor domains of zvegf4 may allow for proteolytic release of the growth
factor domain
or other moiety through existing local proteases within tissues, or by
proteases added
20 from exogenous sources. The release of the targeted moiety may provide more
localized biological effects.
The polypeptide fusions of the present invention further include fusions
between zvegf4 and zvegf3, wherein a domain of zvegf4 is replaced with the
corresponding domain of zvegf3 or a variant thereof. A representative human
zvegf3
polypeptide sequence is shown in SEQ ID NO:33, and a representative mouse
sequence
is shown in SEQ ID NO:35. Within SEQ ID NO:33, the CUB domain comprises
residues 46-170, the interdomain region comprises residues 171-234, and the
growth
factor domain comprises residues 235-345 (all 5 residues). A secretory
peptide is
predicted to be cleaved from the polypeptide after residue 14 ( 3 residues).
Cleavage
sites are predicted at residue 249, residues 254-255, and residues 254-257.
Domain
boundaries in mouse zvegf3 and other orthologous sequences can be determined
readily
by those of ordinary skill in the art by alignment with the human sequence
disclosed
herein. Of particular interest are fusions in which the zvegf3 CUB domain is
combined
with the zvegf4 growth factor domain, and fusions in which the zvegf4 CUB
domain is
combined with the zvegf3 growth factor domain. Within these polypeptide
fusions the
interdomain region may be derived from either zvegf3 or zvegf4. Polypeptide
fusions
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21
comprising zvegf3 and zvegf4 sequences include both full-length and truncated
sequences, for example sequences analogous to those disclosed in Table 1,
above.
Proteins comprising the wild-type zvegf4 CUB domain and variants
thereof may be used to modulate activities mediated by cell-surface
semaphorins.
While not wishing to be bound by theory, zvegf4 may bind to semaphorins via
its CUB
domain. The observation that semaphorin III is involved in vascular
development
suggests that members of the vascular growth factor family of proteins may
also be
involved, especially due to the co-binding activity of VEGF and semaphorin III
to
neuropilin-1. Zvegf4 may thus be used to design agonists and antagonist of
neuropilin-
semaphorin interactions. For example, the zvegf4 sequence disclosed herein
provides a
starting point for the design of molecules that antagonize semaphorin-
stimulated
activities, including neurite growth, cardiovascular development, cartilage
and limb
development, and T and B-cell function. Additional applications include
intervention
in various pathologies, including rheumatoid arthritis, various forms of
cancer,
autoimmune disease, inflammation, retinopathies, hemangiomas, ischemic events
within tissues including the heart, kidney and peripheral arteries,
neuropathies, acute
nerve damage, and diseases of the central and peripheral nervous systems,
including
stroke.
The isolated CUB domain of either mouse or human zvegf4 (and
multimers thereof) may also be useful to block binding of other zvegf4
molecules (e.g.,
full-length polypeptide, isolated growth factor domain, or multimers thereof)
to cell-
surface molecules and/or extracellular binding sites by itself binding to such
molecules
or sites. In addition, the isolated CUB domain of either mouse or human zvegf4
may be
useful to block zvegf4 binding, and/or more generally vascular endothelial
growth
factor binding, to neuropilin-1 (see M.L. Gagnon et al., Proc. Natl. Acad.
Sci. USA
97:2573-78, 2000). Further, the second major loop of zvegf4 (residues 308-316)
may
represent the receptor-binding loop of zvegf4 (see, for instance, WO 99/13329;
WO
98/10795; and W.J. LaRochelle et al., J. Biol. Chem. 267:17074-77, 1992), and
thus
may be useful as an antagonist of zvegf4 activity. Within this peptide (zvegf4
residues
308-316), Cys308 and Cys316 may or may not be disulfide bonded. Also, dimers
of
this peptide may be constructed such that residue Cys308 is disulfide bonded
to either
Cys308 or Cys316 of the homodimer partner peptide.
Amino acid sequence changes are made in zvegf4 polypeptides so as to
minimize disruption of higher order structure essential to biological
activity. As noted
above, conservative amino acid changes are generally less likely to negate
activity than
are non-conservative changes. Changes in amino acid residues will be made so
as not
to disrupt the cystine knot and "bow tie" arrangement of loops in the growth
factor
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WO 00/66736 PCT/US00/40047
22
domain that is characteristic of the protein family. Conserved motifs will
also be
maintained. The effects of amino acid sequence changes can be predicted by
computer
modeling as disclosed above or determined by analysis of crystal structure
(see, e.g.,
Lapthorn et al., ibid.). A hydrophilicity profile of SEQ ID NO:2 is shown in
Fig. 1.
Those skilled in the art will recognize that this hydrophilicity will be taken
into account
when designing alterations in the amino acid sequence of a zvegf4 polypeptide,
so as
not to disrupt the overall profile. Additional guidance in selecting amino
acid
subsitutions is provided by a comparison of the mouse (SEQ ID NO:53) and human
(SEQ ID NO:2) zvegf4 sequences. The amino acid sequence is highly conserved
between mouse and human zvegf4s, with an overall amino acid sequence identity
of
85.1%.
The polypeptides of the present invention 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
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23
combined with site-directed mutagenesis to further expand the range of
substitutions
(Wynn and Richards, Protein Sci. 2:395-403, 1993).
Essential amino acids in the polypeptides of the present invention can be
identified according to procedures known in the art, such as site-directed
mutagenesis
or alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085,
1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991). In the
latter
technique, single alanine mutations are introduced at every residue in the
molecule, and
the resultant mutant molecules are tested for biological activity of other
properties to
identify amino acid residues that are critical to the activity of the
molecule.
Multiple amino acid substitutions can be made and tested using known
methods of mutagenesis and screening, such as those disclosed by Reidhaar-
Olson and
Sauer (Science 241:53-57, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA
86:2152-2156, 1989). Briefly, these authors disclose methods for
simultaneously
randomizing two or more positions in a polypeptide, selecting for functional
polypeptide, and then sequencing the mutagenized polypeptides to determine the
spectrum of allowable substitutions at each position. Other methods that can
be used
include phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;
Ladner et
al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-
directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA
7:127,
1988).
Variants of the disclosed zvegf4 DNA and polypeptide sequences can be
generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391,
1994
and Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751, 1994. Briefly, variant
genes are generated by in vitro homologous recombination by random
fragmentation of
a parent gene followed by reassembly using PCR, resulting in randomly
introduced
point mutations. This technique can be modified by using a family of parent
genes,
such as allelic variants or genes from different species, to introduce
additional
variability into the process. Selection or screening for the desired activity,
followed by
additional iterations of mutagenesis and assay provides for rapid "evolution"
of
sequences by selecting for desirable mutations while simultaneously selecting
against
detrimental changes.
Mutagenesis methods as disclosed above can be combined with high
volume or high-throughput screening methods to detect biological activity of
zvegf4
variant polypeptides, in particular biological activity in modulating cell
proliferation or
cell differentiation. For example, mitogenesis assays that measure dye
incorporation or
3H-thymidine incorporation can be carried out on large numbers of samples, as
can cell-
based assays that detect expression of a reporter gene (e.g., a luciferase
gene).
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24
Mutagenesis of the CUB domain can be used to modulate its binding to members
of the
semaphorin family, including enhancing or inhibiting binding to selected
family
members. A modified spectrum of binding activity may be desirable for
optimizing
therapeutic and/or diagnostic utility of proteins comprising a zvegf4 CUB
domain.
Direct binding utilizing labeled CUB protein can be used to monitor changes in
CUB
domain binding activity to selected semaphorin family members. Semaphorins of
interest in this regard include isolated proteins, proteins present in cell
membranes, and
proteins present on cell-surfaces. The CUB domain can be labeled by a variety
of
methods including radiolabeling with isotopes, such as 125I, conjugation to
enzymes
such as alkaline phosphatase or horseradish peroxidase, conjugation with
biotin, and
conjugation with various fluorescent markers including FITC. These and other
assays
are disclosed in more detail below. Mutagenized DNA molecules that encode
active
zvegf4 polypeptides can be recovered from the host cells and rapidly sequenced
using
modern equipment. These methods allow the rapid determination of the
importance of
individual amino acid residues in a polypeptide of interest, and can be
applied to
polypeptides of unknown structure.
Using the methods discussed above, one of ordinary skill in the art can
identify and/or prepare a variety of polypeptides that are homologous to the
zvegf4
polypeptides disclosed above in Table 1 and retain the biological properties
of the wild-
type protein. Such polypeptides can also include additional polypeptide
segments as
generally disclosed above.
The present invention also provides polynucleotide molecules, including
DNA and RNA molecules, that encode the zvegf4 polypeptides disclosed above.
The
polynucleotides of the present invention include the sense strand; the anti-
sense strand;
and the DNA as double-stranded, having both the sense and anti-sense strands
annealed
together by hydrogen bonds. A representative DNA sequence encoding human
zvegf4
polypeptides is set forth in SEQ ID NO:1, and a representative DNA sequence
encoding
mouse zvegf4 polypeptides is set forth in SEQ ID NO:52. Additional DNA
sequences
encoding zvegf4 polypeptides can be readily generated by those of ordinary
skill in the
art based on the genetic code. Counterpart RNA sequences can be generated by
substitution of U for T.
Those skilled in the art will readily recognize that, in view of the
degeneracy of the genetic code, considerable sequence variation is possible
among
polynucleotide molecules encoding zvegf4 polypeptides. SEQ ID NO:6 is a
degenerate
DNA sequence that encompasses all DNAs that encode the zvegf4 polypeptide of
SEQ
ID NO: 2. Those skilled in the art will recognize that the degenerate sequence
of SEQ
ID NO:6 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U
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for T. Thus, zvegf4 polypeptide-encoding polynucleotides comprising
nucleotides 1 -
1110, 1 - 537, 55 - 537, 103 - 537, 154 - 537, 55 - 771, 103 - 771, 154 - 771,
55 - 765,
103 - 765, 154 - 765, 55 - 759, 103 - 759, 154 - 759, 55 - 747, 103 - 747, 154
- 747,
55 - 735, 103 - 735, 154 - 735, 55 - 1110, 103 - 1110, 154 - 1110, 772 - 1110,
748 -
5 1110, 736 - 1110, and 538 - 1110 of SEQ ID NO:6 and their RNA equivalents
are
contemplated by the present invention. Table 3 sets forth the one-letter codes
used
within SEQ ID NO:6 to denote degenerate nucleotide positions. "Resolutions"
are the
nucleotides denoted by a code letter. "Complement" indicates the code for the
complementary nucleotide(s). For example, the code Y denotes either C or T,
and its
10 complement R denotes A or G, A being complementary to T, and G being
complementary to C.
Table 3
Nucleotide Resolutions Complement Resolutions
A A T T
C C G G
G G C C
T T A A
R AIG Y CST
Y CIT R AIG
M AIC K GAT
K GIT M AIC
S CMG S CIG
W AIT W AIT
H AICIT D AIGIT
B CIGIT V AICIG
V AICIG B CIGIT
D AIGIT H AICIT
N AICIGIT N AICIGIT
The degenerate codons used in SEQ ID NO:6, encompassing all possible
15 codons for a given amino acid, are set forth in Table 4, below.
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26
TABLE 4
One-Letter
Amino Code Degenerate
Acid Codons Codon
Cys C TGC TGT TGY
Ser S AGC AGT TCA TCC TCG TCT WSN
Thr T ACA ACC ACG ACT CAN
Pro P CCA CCC CCG CCT CCN
Ala A GCA GCC GCG GCT GCN.
Gly G GGA GGC GGG GGT GGN
Asn N AAC AAT AAY
Asp D GAC GAT GAY
Glu E GAA GAG GAR
Gln Q CAACAG CAR
His H CAC CAT CAY
Arg R AGA AGG CGA CGC CGG CGT MGN
Lys K AAA AAG AAR
Met M ATG ATG
Be I ATA ATC ATT ATH
Leu L CTA CTC CTG CTT TTA TTG YTN
Val V GTA GTC GTG GTT GTN
Phe F TTC TTT TTY
Tyr Y TAC TAT TAY
Tip W TGG TGG
Ter TAA TAG TGA TRR
AsnlAsp B RAY
G1ujGln Z SAR
Any X NNN
Gap - One of ordinary skill in the art will appreciate that some ambiguity is
introduced in determining a degenerate codon, representative of all possible
codons
encoding each amino acid. For example, the degenerate codon for serine (WSN)
can, in
some circumstances, encode arginine (AGR), and the degenerate codon for
arginine
(MGN) can, in some circumstances, encode serine (AGY). A similar relationship
exists
between codons encoding phenylalanine and leucine. Thus, some polynucleotides
encompassed by the degenerate sequences may encode variant amino acid
sequences,
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27
but one of ordinary skill in the art can easily identify such variant
sequences by
reference to the amino acid sequence of SEQ ID NO: 2 and of SEQ ID NO:53.
Variant
sequences can be readily tested for functionality as described herein.
Within certain embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID NO:1 or SEQ
ID
NO:52, or a sequence complementary thereto, under stringent conditions. In
general,
stringent conditions are selected to be about 5 C lower than the thermal
melting point
(Tm) for the specific sequence at a defined ionic strength and pH. The Tm is
the
temperature (under defined ionic strength and pH) at which 50% of the target
sequence
hybridizes to a perfectly matched probe. Typical stringent conditions are
those in
which the salt concentration is up to about 0.03 M at pH 7 and the temperature
is at
least about 60 C.
As previously noted, the isolated polynucleotides of the present
invention include DNA and RNA. Methods for preparing DNA and RNA are well
known in the art. Complementary DNA (cDNA) clones are prepared from RNA that
is
isolated from a tissue or cell that produces large amounts of zvegf4 RNA. Such
tissues
and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci.
USA
77:5201, 1980), and include heart, pancreas, stomach, and adrenal gland. Total
RNA
can be prepared using guanidine HCl extraction followed by isolation by
centrifugation
in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)+
RNA is
prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad.
Sci.
USA 69:1408-1412, 1972). Complementary DNA (cDNA) is prepared from poly(A)+
RNA using known methods. In the alternative, genomic DNA can be isolated. For
some applications (e.g., expression in transgenic animals) it may be
advantageous to
use a genomic clone, or to modify a cDNA clone to include at least one genomic
intron.
Methods for identifying and isolating cDNA and genomic clones are well known
and
within the level of ordinary skill in the art, and include the use of the
sequence
disclosed herein, or parts thereof, for probing or priming a library.
Polynucleotides
encoding zvegf4 polypeptides are identified and isolated by, for example,
hybridization
or polymerase chain reaction ("PCR", Mullis, U.S. Patent 4,683,202).
Expression
libraries can be probed with antibodies to zvegf4, receptor fragments, or
other specific
binding partners.
Those skilled in the art will recognize that the sequences disclosed in
SEQ ID NOS:1 and 2 represent a single allele of human zvegf4, and that the
sequences
3S disclosed in SEQ ID NOS:52 and 53 represent a single allele of mouse
zvegf4. Allelic
variants of these sequences can be cloned by probing cDNA or genomic libraries
from
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28
different individuals according to standard procedures. Alternatively spliced
forms of
zvegf4 are also expected to exist.
The zvegf4 polynucleotide sequence disclosed herein can be used to
isolate polynucleotides encoding other zvegf4 proteins. Such other
polynucleotides
include allelic variants, alternatively spliced cDNAs and counterpart
polynucleotides
from other species (orthologs). These orthologous polynucleotides can be used,
inter
alia, to prepare the respective orthologous proteins. Other species of
interest include,
but are not limited to, mammalian, avian, amphibian, reptile, fish, insect and
other
vertebrate and invertebrate species. Of particular interest are zvegf4
polynucleotides
and proteins from other mammalian species, including non-human primate,
murine,
porcine, ovine, bovine, canine, feline, and equine polynucleotides and
proteins.
Orthologs of human zvegf4 can be cloned using information and compositions
provided
by the present invention in combination with conventional cloning techniques.
For
example, a cDNA can be cloned using mRNA obtained from a tissue or cell type
that
expresses zvegf4 as disclosed herein. Suitable sources of mRNA can be
identified by
probing Northern blots with probes designed from the sequences disclosed
herein. A
library is then prepared from mRNA of a positive tissue or cell line. A zvegf4-
encoding cDNA can then be isolated by a variety of methods, such as by probing
with a
complete or partial human cDNA or with one or more sets of degenerate probes
based
on the disclosed sequences. Hybridization will generally be done under low
stringency
conditions, wherein washing is carried out in 1 x SSC with an initial wash at
40 C and
with subsequent washes at 5 C higher intervals until background is suitably
reduced. A
cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis,
U.S.
Patent No. 4,683,202), using primers designed from the representative human
zvegf4
sequence disclosed herein. Within an additional method, the cDNA library can
be used
to transform or transfect host cells, and expression of the cDNA of interest
can be
detected with an antibody to zvegf4 polypeptide. Similar techniques can also
be
applied to the isolation of genomic clones.
For any zvegf4 polypeptide, including variants and fusion proteins, one
of ordinary skill in the art can readily generate a fully degenerate
polynucleotide
sequence encoding that variant using the information set forth in Tables 3 and
4, above.
Conserved regions of zvegf4, identified by alignment with sequences of
other family members, can be used to identify related polynucleotides and
proteins. For
instance, reverse transcription-polymerase chain reaction (RT-PCR) and other
techniques known in the art can be used to amplify sequences encoding the
conserved
motifs present in zvegf4 from RNA obtained from a variety of tissue sources.
In
particular, highly degenerate primers as shown below in Table 5 (designed from
an
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29
alignment of zvegf4 with PDGF A and B chains, VEGF, VEGF-B, VEGF-C, VEGF-D,
and zvegf3) are useful for cloning polynucleotides encoding homologous growth
factor
domains. Primers shown in Table 6, designed from an alignment of zvegf4 with
X.
laevis neuropilin precursor, human BMP-1, human zvegf3, and X. laevis tolloid-
like
protein, are useful for cloning polynucleotides encoding CUB domains. The
primers of
Tables 5 and 6 can thus be used to obtain additional polynucleotides encoding
homologs of the zvegf4 sequence of SEQ ID NO:1 and NO:2.
Table 5
zvegf4 residues 301-305
degenerate: MGN TGY GGN GGN AAY TG (SEQ ID NO:7)
consensus: MGN TGY DSN GGN WRY TG (SEQ ID NO:8)
complement: CAR YWN CCN SHR CAN CK (SEQ ID NO:9)
zvegf4 residues 292-297
degenerate: TTY TTY CCN MGN TGY YT (SEQ IDNO:10)
consensus: NTN DDN CCN NSN TGY BT (SEQ ID NO: 11)
complement: AVR CAN SNN GGN HHN AN (SEQ ID NO:12)
zvegf4 residues 357-362
degenerate CAY GAR MGN TGY GAY TG (SEQ ID NO:13)
consensus: CAY NNN NVN TGY VVN TG (SEQ ID NO: 14)
complement: CAN BBR CAN BNN NNR TG (SEQ ID NO:15)
zvegf4 residues 250-255
degenerate: TGY ACN CCN MGN AAY TA (SEQ ID NO:16)
consensus: TGY HNN MCN MKN RMN DH (SEQ ID NO:17)
complement: DHN KYN MKN GKN NDR CA (SEQ ID NO:18)
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Table 6
zvegf4 residues 110-115
consensus: N TAY GAY TWY GTN GAR GT (SEQ ID NO:19)
complement: N ATR CTR AWR CAN CTY CA (SEQ ID NO:20)
5
zvegf4 residues 68-73
consensus: GN TDB CCN MAN DVN TAY C (SEQ ID NO:21)
complement: CN AHV GGN KTN HBN ATR G (SEQ ID NO:22)
10 zvegf4 residues 126-131
consensus: TN HDN GGN MRN TDB TGY G (SEQ ID NO:23)
complement: AN DHN CCN KYN AHV ACR C (SEQ ID NO:24)
Zvegf4 polynucleotide sequences disclosed herein can also be used as
15 probes or primers to clone 5' non-coding regions of a zvegf4 gene,
including promoter
sequences. A human zvegf4 genomic fragment, comprising 5' non-coding and
coding
sequences, is shown in SEQ ID NO:36. These flanking sequences can be used to
direct
the expression of zvegf4 and other recombinant proteins. In addition, 5'
flanking
sequences can be used as targeting sites for regulatory constructs to activate
or increase
20 expression of endogenous zvegf4 genes as disclosed by Treco et al., U.S.
Patent No.
5,641,670. A human zvegf4 genomic sequence comprising 5' non-coding sequence
and
approximately 100 nucleotides of coding sequence is shown in SEQ ID NO:36.
The polynucleotides of the present invention can also be prepared by
automated synthesis. The production of short, double-stranded segments (60 to
80 bp)
25 is technically straightforward and can be accomplished by synthesizing the
complementary strands and then annealing them. Longer segments (typically >300
bp)
are assembled in modular form from single-stranded fragments that are from 20
to 100
nucleotides in length. Automated synthesis of polynucleotides is within the
level of
ordinary skill in the art, and suitable equipment and reagents are available
from
30 commercial suppliers. See, in general, Glick and Pasternak, Molecular
Biotechnology,
Principles & Applications of Recombinant DNA, ASM Press, Washington, D.C.,
1994;
Itakura et al., Ann. Rev. Biochem. 53: 323-56, 1984; and Climie et al., Proc.
Natl. Acad.
Sci. USA 87:633-7, 1990.
The polypeptides of the present invention, including full-length
polypeptides, biologically active fragments, and fusion polypeptides 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
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31
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, NY, 1989, and Ausubel et
al.,
eds., Current Protocols in Molecular Biology, Green and Wiley and Sons, NY,
1993.
In general, a DNA sequence encoding a zvegf4 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.
To direct a zvegf4 polypeptide into the secretory pathway of a host cell,
a secretory signal sequence (also known as a leader sequence, prepro sequence
or pre
sequence) is provided in the expression vector. The secretory signal sequence
may be
that of zvegf4, or may be derived from another secreted protein (e.g., t-PA;
see, U.S.
Patent No. 5,641,655) or synthesized de novo. The secretory signal sequence is
operably linked to the zvegf4 DNA sequence, i.e., the two sequences are joined
in the
correct reading frame and positioned to direct the newly synthesized
polypeptide into
the secretory pathway of the host cell. Secretory signal sequences are
commonly
positioned 5' to the DNA sequence encoding the polypeptide of interest,
although
certain signal sequences may be positioned elsewhere in the DNA sequence of
interest
(see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S.
Patent No.
5,143,830).
Expression of zvegf4 polypeptides via a host cell secretory pathway is
expected to result in the production of multimeric proteins. As noted above,
such
multimers include both homomultimers and heteromultimers, the latter including
proteins comprising only zvegf4 polypeptides and proteins including zvegf4 and
heterologous polypeptides. For example, a heteromultimer comprising a zvegf4
polypeptide and a polypeptide from a related family member (e.g., VEGF, VEGF-
B,
VEGF-C, VEGF-D, zvegf3, P1GF, PDGF-A, or PDGF-B) can be produced by co-
expression of the two polypeptides in a host cell. Sequences encoding these
other
family members are known. See, for example, Dvorak et al, ibid.; Olofsson et
al, ibid.;
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32
Hayward et al., ibid.; Joukov et al., ibid.; Oliviero et al., ibid.; Achen et
al., ibid.;
Maglione et al., ibid.; Heldin et al., U.S. Patent No. 5,219,759; and Johnsson
et al., ibid.
If a mixture of proteins results from expression, individual species are
isolated by
conventional methods. Monomers, dimers, and higher order multimers are
separated
by, for example, size exclusion chromatography. Heteromultimers can be
separated
from homomultimers by conventional chromatography or by immunoaffinity
chromatography using antibodies specific for individual dimers or by
sequential
immunoaffinity steps using antibodies specific for individual component
polypeptides.
See, in general, U.S. Patent No. 5,094,941.
Cultured mammalian cells are suitable hosts for use within the present
invention. Methods for introducing exogenous DNA into mammalian host cells
include
calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978;
Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology
52:456,
1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran
mediated transfection (Ausubel et al., ibid.), and liposome-mediated
transfection
(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,
1993). The
production of recombinant polypeptides in cultured mammalian cells is
disclosed by,
for example, Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S.
Patent No.
4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S.
Patent No.
4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL
1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570
(ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol.
36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell
lines. Additional suitable cell lines are known in the art and available from
public
depositories such as the American Type Culture Collection, Manassas, Virginia.
Strong
transcription promoters can be used, such as promoters from SV-40 or
cytomegalovirus.
See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those
from
metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the
adenovirus
major late promoter. Expression vectors for use in mammalian cells include pZP-
1 and
pZP-9, which have been deposited with the American Type Culture Collection,
10801
University Blvd., Manassas, VA USA under accession numbers 98669 and 98668,
respectively.
Drug selection is generally used to select for cultured mammalian cells
into which foreign DNA has been inserted. Such cells are commonly referred to
as
"transfectants". Cells that have been cultured in the presence of the
selective agent and
are able to pass the gene of interest to their progeny are referred to as
"stable
transfectants." An exemplary selectable marker is a gene encoding resistance
to the
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33
antibiotic neomycin. Selection is carried out in the presence of a neomycin-
type drug,
such as G-418 or the like. Selection systems can also be used to increase the
expression
level of the gene of interest, a process referred to as "amplification."
Amplification is
carried out by culturing transfectants in the presence of a low level of the
selective
agent and then increasing the amount of selective agent to select for cells
that produce
high levels of the products of the introduced genes. An exemplary amplifiable
selectable marker is dihydrofolate reductase, which confers resistance to
methotrexate.
Other drug resistance genes (e.g. hygromycin resistance, multi-drug
resistance,
puromycin acetyltransferase) can also be used.
Other higher eukaryotic cells can also be used as hosts, including insect
cells, plant cells and avian cells. The use of Agrobacterium rhizogenes as a
vector for
expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci.
(Bangalore) 11:47-58, 1987. Transformation of insect cells and production of
foreign
polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222
and
W1PO publication WO 94/06463.
Insect cells can be infected with recombinant baculovirus, commonly
derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See,
King
and Possee, The Baculovirus Expression System: A Laboratory Guide, London,
Chapman & Hall; O'Reilly et al., Baculovirus Expression Vectors: A Laboratory
Manual, New York, Oxford University Press., 1994; and Richardson, Ed.,
Baculovirus
Expression Protocols. Methods in Molecular Biology, Humana Press, Totowa, NJ,
1995. Recombinant baculovirus can also be produced through the use of a
transposon-
based system described by Luckow et al. (J. Virol. 67:4566-4579, 1993). This
system,
which utilizes transfer vectors, is commercially available in kit form (Bac-to-
BacTM kit;
Life Technologies, Rockville, MD). The transfer vector (e.g., pFastBaclTM;
Life
Technologies) contains a Tn7 transposon to move the DNA encoding the protein
of
interest into a baculovirus genome maintained in E. coli as a large plasmid
called a
"bacmid." See, Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990;
Bonning et
al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk and Rapoport, J. Biol.
Chem.
270:1543-1549, 1995. In addition, transfer vectors can include an in-frame
fusion with
DNA encoding a polypeptide extension or affinity tag as disclosed above. Using
techniques known in the art, a transfer vector containing a zvegf4-encoding
sequence is
transformed into E. coli host cells, and the cells are screened for bacmids
which contain
an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA
containing the recombinant baculovirus genome is isolated, using common
techniques,
and used to transfect Spodopterafrugiperda cells, such as Sf9 cells.
Recombinant virus
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34
that expresses zvegf4 protein is subsequently produced. Recombinant viral
stocks are
made by methods commonly used the art.
For protein production, the recombinant virus is used to infect host cells,
typically a cell line derived from the fall armyworm, Spodopterafrugiperda
(e.g., Sf9
or Sf21 cells) or Trichoplusia ni (e.g., High FiveTM cells; Invitrogen,
Carlsbad, CA).
See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and
Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. See also,
U.S. Patent No. 5,300,435. Serum-free media are used to grow and maintain the
cells.
Suitable media formulations are known in the art and can be obtained from
commercial
suppliers. The cells are grown up from an inoculation density of approximately
2-5 x
105 cells to a density of 1-2 x 106 cells, at which time a recombinant viral
stock is added
at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
Procedures used
are generally described in available laboratory manuals (e.g., King and
Possee, ibid.;
O'Reilly et al., ibid.; Richardson, ibid.).
Fungal cells, including yeast cells, can also be used within the present
invention. Yeast species of particular interest in this regard include
Saccharomyces
cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming
S.
cerevisiae cells with exogenous DNA and producing recombinant polypeptides
therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311;
Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008;
Welch et
al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075.
Transformed cells are selected by phenotype determined by the selectable
marker,
commonly drug resistance or the ability to grow in the absence of a particular
nutrient
(e.g., leucine). An exemplary vector system for use in Saccharomyces
cerevisiae is the
POT] vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373),
which
allows transformed cells to be selected by growth in glucose-containing media.
Suitable promoters and terminators for use in yeast include those from
glycolytic
enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al.,
U.S.
Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol
dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154;
5,139,936 and
4,661,454. Transformation systems for other yeasts, including Hansenula
polymorpha,
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,
Ustilago
maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida
maltosa are known in the art. See, for example, Gleeson et al., J. Gen.
Microbiol.
132:3459-3465, 1986; Cregg, U.S. Patent No. 4,882,279; and Raymond et al.,
Yeast 14,
11-23, 1998. Aspergillus cells may be utilized according to the methods of
McKnight
et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium
chrysogenum
CA 02370948 2007-05-02
are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for
transforming
Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. Production
of
recombinant proteins in Pichia methanolica is disclosed in U.S. Patents No.
5,716,808,
5,736,383, 5,854,039, and 5,888,768.
5 Prokaryotic host cells, including strains of the bacteria Escherichia coli,
Bacillus and other genera are also useful host cells within the present
invention.
Techniques for transforming these hosts and expressing foreign DNA sequences
cloned
therein are well known in the art (see, e.g., Sambrook et al., ibid.). When
expressing a
zvegf4 polypeptide in bacteria such as E. coli, the polypeptide may be
retained in the
10 cytoplasm, typically as insoluble granules, or may be directed to the
periplasmic space
by a bacterial secretion sequence. In the former case, the cells are lysed,
and the
granules are recovered and denatured using, for example, guanidine
isothiocyanate or
urea. The denatured polypeptide can then be refolded and dimerized by diluting
the
denaturant, such as by dialysis against a solution of urea and a combination
of reduced
15 and oxidized glutathione, followed by dialysis against a buffered saline
solution. In the
alternative, the protein may be recovered from the cytoplasm in soluble form
and
isolated without the use of denaturants. The protein is recovered from the
cell as an
aqueous extract in, for example, phosphate buffered saline. To capture the
protein of
interest, the extract is applied directly to a chromatographic medium, such as
an
20 immobilized antibody or heparin-Sepharose column. Secreted polypeptides can
be
Tm
recovered from the periplasmic space in a soluble and functional form by
disrupting the
cells (by, for example, sonication or osmotic shock) to release the contents
of the
periplasmic space and recovering the protein, thereby obviating the need for
denaturation and refolding.
25 Transformed or transfected host cells are cultured according to
conventional procedures in a culture medium containing nutrients and other
components required for the growth of the chosen host cells. A variety of
suitable
media, including defined media and complex media, are known in the art and
generally
include a carbon source, a nitrogen source, essential amino acids, vitamins
and
30 minerals. Media may also contain such components as growth factors or
serum, as
required. The growth medium will generally select for cells containing the
exogenously
added DNA by, for example, drug selection or deficiency in an essential
nutrient which
is complemented by the selectable marker carried on the expression vector or
co-
transfected into the host cell. P. methanolica cells, for example, are
cultured in a
35 medium comprising adequate sources of carbon, nitrogen and trace nutrients
at a
temperature of about 25 C to 35 C. Liquid cultures are provided with
sufficient
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36
aeration by conventional means, such as shaking of small flasks or sparging of
fermentors.
Zvegf4 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, IL, 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.
Covalent, multimeric complexes can also be made by isolating the
desired component polypeptides and combining them in vitro. Covalent complexes
that
can be prepared in this manner include homodimers of zvegf4 polypeptides,
heterodimers of two different zvegf4 polypeptides, and heterodimers of a
zvegf4
polypeptide and a polypeptide from another family member of the VEGF/PDGF
family
of proteins. The two polypeptides are mixed together under denaturing and
reducing
conditions, followed by renaturation of the proteins by removal of the
denaturants.
Removal can be done by, for example, dialysis or size exclusion chromatography
to
provide for buffer exchange. When combining two different polypeptides, the
resulting
renaturated proteins may form homodimers of the individual components as well
as
heterodimers of the two polypeptide components. See, Cao et al., J. Biol.
Chem.
271:3154-3162, 1996.
Non-covalent complexes comprising a zvegf4 polypeptide can be
prepared by incubating a zvegf4 polypeptide and a second polypeptide (e.g., a
zvegf4
polypeptide or another peptide of the PDGF/VEGF family) at near-physiological
pH.
In a typical reaction, polypeptides at a concentration of about 0.1-0.5 g/ l
are
incubated at pH=7.4 in a weak buffer (e.g., 0.01 M phosphate or acetate
buffer); sodium
chloride may be included at a concentration of about 0.1 M. At 37 C the
reaction is
essentially complete with 4-24 hours. See, for example, Weintraub et al.,
Endocrinology 101:225-235, 1997.
Depending upon the intended use, the polypeptides and proteins of the
present invention can be purified to >80% purity, >_90% purity, >_95% purity,
or to a
pharmaceutically pure state, that is greater than 99.9% pure with respect to
contaminating macromolecules, particularly other proteins and nucleic acids,
and free
of infectious and pyrogenic agents.
Zvegf4 proteins (including chimeric polypeptides and polypeptide
multimers) can be purified using fractionation and/or conventional
purification methods
and media, such as by a combination of chromatographic techniques. See, in
general,
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37
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
or cobalt chelate resin. See, for example, Houchuli et al., Bio/Technol. 6:
1321-1325,
1988. 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.
Using methods known in the art, zvegf4 proteins can be prepared as
monomers or multimers, glycosylated or non-glycosylated, pegylated or non-
pegylated,
and may or may not include an initial methionine amino acid residue.
The invention further provides polypeptides that comprise an epitope-
bearing portion of a protein as shown in SEQ ID NO:2. 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. Antibodies that
recognize
short, linear epitopes are particularly useful in analytic and diagnostic
applications that
employ denatured protein, such as Western blotting (Tobin, Proc. Natl. Acad.
Sci. USA
76:4350-4356, 1979). Anti-peptide antibodies are not conformation-dependent
and can
be used to detect proteins in fragmented or otherwise altered forms (Niman et
al., Proc.
Natl. Acad. Sci. USA 82:7924-7928, 1985), such as might occur in body fluids
or cell
culture media. Antibodies to short peptides may also recognize proteins in
native
conformation and will thus be useful for monitoring protein expression and
protein
isolation, and in detecting zvegf4 proteins in solution, such as by ELISA or
in
immunoprecipitation studies.
Antigenic, epitope-bearing polypeptides of the present invention are
useful for raising antibodies, including monoclonal antibodies, that
specifically bind to
a zvegf4 protein. Antigenic, epitope-bearing polypeptides contain a sequence
of at least
six, within other embodiments at least nine, within other embodiments from 15
to about
30 contiguous amino acid residues of a zvegf4 protein (e.g., SEQ ID NO:2).
Polypeptides comprising a larger portion of a zvegf4 protein, i.e., from 30 to
50 or 100
residues or up to the entire sequence are included. It is preferred that the
amino acid
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38
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 of SEQ ID NO:2
include,
for example, residues 39-44, 252-257, 102-107, 264-269, and 339-344. Exemplary
longer peptide immunogens include peptides comprising residues (i) 131-148,
(ii) 230-
253, or (iii) 333-355 of SEQ ID NO:2. Peptide (ii) can be prepared with an
additional
C-terminal cys residue and peptide (iii) with an additional N-terminal cys
residue to
facilitate coupling.
As used herein, the term "antibodies" includes polyclonal antibodies,
affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-
binding
fragments, such as F(ab')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.
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, NY, 1989; and Hurrell, J. G. R. (ed.), Monoclonal Hybridoma
Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL,
1982. As
would be evident to one of ordinary skill in the art, polyclonal antibodies
can be
generated from inoculating a variety of warm-blooded animals such as horses,
cows,
goats, sheep, dogs, chickens, rabbits, mice, and rats with a zvegf4
polypeptide or a
fragment thereof. The immunogenicity of a zvegf4 polypeptide may be increased
through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's
complete or incomplete adjuvant. Polypeptides useful for immunization also
include
fusion polypeptides, such as fusions of zvegf4 or a portion thereof with an
immunoglobulin polypeptide or with maltose binding protein. If the polypeptide
CA 02370948 2001-10-16
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39
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.
Alternative techniques for generating or selecting antibodies useful
herein include in vitro exposure of lymphocytes to zvegf4 protein or peptide,
and
selection of antibody display libraries in phage or similar vectors (for
instance, through
use of immobilized or labeled zvegf4 protein or peptide). Genes encoding
polypeptides
having potential zvegf4 polypeptide binding domains can be obtained by
screening
random peptide libraries displayed on phage (phage display) or on bacteria,
such as E.
coli. Nucleotide sequences encoding the polypeptides can be obtained in a
number of
ways, such as through random mutagenesis and random polynucleotide synthesis.
These random peptide display libraries can be used to screen for peptides that
interact
with a known target, which can be a protein or polypeptide, such as a ligand
or receptor,
a biological or synthetic macromolecule, or organic or inorganic substance.
Techniques for creating and screening such random peptide display libraries
are known
in the art (Ladner et al., US Patent No. 5,223,409; Ladner et al., US Patent
No.
4,946,778; Ladner et al., US Patent No. 5,403,484; and Ladner et al., US
Patent 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, CA),
Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA), and
Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide display
libraries
can be screened using the zvegf4 sequences disclosed herein to identify
proteins which
bind to zvegf4. These "binding proteins", which interact with zvegf4
polypeptides, can
be used for tagging cells or for isolating homologous polypeptides by affinity
purification, or they can be directly or indirectly conjugated to drugs,
toxins,
radionuclides, and the like. Binding proteins can also be used in analytical
methods,
such as for screening expression libraries and for neutralizing zvegf4
activity; for
diagnostic assays for determining circulating levels of polypeptides; for
detecting or
quantitating soluble polypeptides as marker of underlying pathology or
disease; and as
zvegf4 antagonists to block zvegf4 binding and signal transduction in vitro
and in vivo.
Antibodies are determined to be specifically binding if they bind to a
zvegf4 polypeptide, peptide or epitope with an affinity at least 10-fold
greater than the
binding affinity to control (non-zvegf4) polypeptide or protein. In this
regard, a "non-
zvegf4 polypeptide" includes the related molecules VEGF, VEGF-B, VEGF-C, VEGF-
D, zvegf3, P1GF, PDGF-A, and PDGF-B, but excludes zvegf4 polypeptides from non-
human species. Due to the high level of amino acid sequence identity expected
between zvegf4 orthologs, antibodies specific for human zvegf4 may also bind
to
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zvegf4 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.),
5 Fundamental Immunology, Raven Press, 1993; Getzoff et al., Adv. in Immunol.
43:1-
98, 1988; Goding, J.W. (ed.), Monoclonal Antibodies: Principles and Practice,
Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2:67-101, 1984.
A variety of assays known to those skilled in the art can be utilized to
detect antibodies which specifically bind to zvegf4 proteins or peptides.
Exemplary
10 assays are described in detail in Antibodies: A Laboratory Manual, Harlow
and Lane
(Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of
such
assays include: concurrent immunoelectrophoresis, radioimmunoassay,
radioimmuno-
precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western
blot
assay, inhibition or competition assay, and sandwich assay. In addition,
antibodies can
15 be screened for binding to wild-type versus mutant zvegf4 protein or
polypeptide.
Of particular interest are neutralizing antibodies, that is antibodies that
block zvegf4 biological activity. Within the present invention, an antibody is
considered to be neutralizing if the antibody blocks at least 50% of the
biological
activity of a zvegf4 protein when the antibody is present in a 1000-fold molar
excess.
20 Within certain embodiments of the invention the antibody will neutralize
50% of
biological activity when present in a 100-fold molar excess or in a 10-fold
molar
excess. Within other embodiments the antibody neutralizes at least 60% of
zvegf4
activity, at least 70% of zvegf4 activity, at least 80% of zvegf4 activity, or
at least 90%
of zvegf4 activity.
25 Antibodies to zvegf4 may be used for tagging cells that express zvegf4;
for isolating zvegf4 by affinity purification; for diagnostic assays for
determining
circulating levels of zvegf4 polypeptides; for detecting or quantitating
soluble zvegf4 as
a marker of underlying pathology or disease; in analytical methods employing
FACS;
for screening expression libraries; for generating anti-idiotypic antibodies;
and as
30 neutralizing antibodies or as antagonists to block zvegf4 activity in vitro
and in vivo.
Suitable direct tags or labels include radionuclides, enzymes, substrates,
cofactors,
inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles
and the
like; indirect tags or labels may feature use of biotin-avidin or other
complement/anti-
complement pairs as intermediates. Antibodies may also be directly or
indirectly
35 conjugated to drugs, toxins, radionuclides and the like, and these
conjugates used for in
vivo diagnostic or therapeutic applications. Moreover, antibodies to zvegf4 or
fragments thereof may be used in vitro to detect denatured zvegf4 or fragments
thereof
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in assays, for example, Western Blots or other assays known in the art.
Antibodies can
also be used to target an attached therapeutic or diagnostic moiety to cells
expressing
zvegf4 or receptors for zvegf4. Experimental data suggest that zvegf4 may bind
PDGF
alpha and/or beta receptors.
Anti-zvegf4 antibodies may be administered to recipients that would
benefit from a decrease in bone proliferation or differentiation, such as
those recipients
suffering from osteosarcoma or osteopetrosis. In animals overexpressing
zvegf4,
histological analysis showed proliferation of endosteal bone (particularly in
trabecular
bone) that in some instances replaced most of the bone marrow, as well a
proliferation
of stromal cells in bone. Anti-zvegf4 antibodies would interfere with these
processes,
and/or would diminish osteoblast proliferation and bone growth stimulation.
Anti-
zvegf4 antibodies may also be used to antagonize production of cartilage by
interfering
with the ability of zvegf4 to stimulate the development or proliferation of
chondrocytes.
In addition, anti-zvegf4 antibodies may be used to diminish pro-fibrotic
responses. Histological analysis of animals overexpressing zvegf4 detected pro-
fibrotic
responses in certain organs, particularly liver, kidney and lung. Several
diseases or
conditions involve fibrosis in liver, lung and kidney. More particularly,
alcoholism and
viral hepatitis generally involve liver fibrosis, which is often a precursor
to cirrhosis,
which in turn may lead to an irreversible state of liver failure. Lung
fibrosis resulting
from exposure to environmental agents (e.g., asbestosis, silicosis) will often
manifest as
alveolitis or interstitial inflammation. Also, lung fibrosis may occur as a
side effect of
some cancer therapies, such as ionizing radiation or chemotherpeutic agents.
Further,
collagen vascular diseases, such as scleroderma and lupus, may also lead to
lung
fibrosis. In the kidney, the human condition of membranoproliferative
glomerulonephritis may correspond to the pro-fibrotic response observed in
animals
overexpressing zvegf4. Chronic immune complex deposition, as seen in lupus,
hepatitis B and C, and chronic abscesses, may also lead to pro-fibrotic
responses in the
kidney. Administration of anti-zvegf4 antibodies may beneficially interfere
with
zvegf4-stimulated pro-fibrotic responses. Such responses include: sclerosing
peritonitis, adhesions following surgery, particularly laparoscopic surgery,
and
restenosis.
Activity of zvegf4 proteins can be measured in vitro using cultured cells
or in vivo by administering molecules of the claimed invention to an
appropriate animal
model. Target cells for use in zvegf4 activity assays include vascular cells
(especially
endothelial cells, pericytes and smooth muscle cells), hematopoietic (myeloid
and
lymphoid) cells, liver cells (including hepatocytes, fenestrated endothelial
cells, Kupffer
cells, and Ito cells), fibroblasts (including human dermal fibroblasts and
lung
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42
fibroblasts), neurite cells (including astrocytes, glial cells, dendritic
cells, and PC-12
cells), fetal lung cells, articular synoviocytes, pericytes, chondrocytes,
osteoblasts,
kidney mesangial cells, bone marrow stromal cells (see K. Satomura et al., J.
Cell.
Physiol. 177:426-38, 1998), and other cells having cell-surface PDGF
receptors.
Zvegf4 proteins can be analyzed for receptor binding activity by a
variety of methods well known in the art, including receptor competition
assays
(Bowen-Pope and Ross, Methods Enzymol. 109:69-100, 1985), use of soluble
receptors,
and use of receptors produced as IgG fusion proteins (U.S. Patent 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. Cell types
that are
able to bind zvegf4 can be identified through the use of a zvegf4 polypeptide
conjugated to a cytotoxin or other detectable molecule. Suitable detectable
molecules
include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent
markers,
chemiluminescent markers, magnetic particles, and the like. Suitable cytotoxic
molecules include bacterial or plant toxins (for instance, diphtheria toxin,
Pseudomonas
exotoxin, ricin, abrin, saporin, and the like), as well as therapeutic
radionuclides, such
as iodine-131, rhenium- 188 or yttrium-90. These can be either directly
attached to the
polypeptide or indirectly attached according to known methods, such as through
a
chelating moiety. Polypeptides can also be conjugated to cytotoxic drugs, such
as
adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the
detectable or cytotoxic molecule may be conjugated with a member of a
complementary/anticomplementary pair, where the other member is bound to the
polypeptide or antibody portion. For these purposes, biotin/streptavidin is an
exemplary complementary/anticomplementary pair. Binding of a zvegf4-toxin
conjugate by cells, either in tissue culture, in organ culture, or in vivo
will allow for the
incorporation of the conjugate into the cell, causing cell death. This
activity can be
used to identify cell types that are able to bind and internalize zvegf4. In
addition to
allowing for the identification of responsive cell types, toxin conjugates can
be used in
in vivo studies to identify organs and tissues where zvegf4 has a biological
activity by
looking for pathology within the animal following injection of the conjugate.
Activity of zvegf4 proteins can be measured in vitro using cultured cells.
Mitogenic activity can be measured using known assays, including 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.
Exemplary
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mitogenesis assays measure incorporation of 3H-thymidine into (1) 20%
confluent
cultures to look for the ability of zvegf4 proteins to further stimulate
proliferating cells,
and (2) quiescent cells held at confluence for 48 hours to look for the
ability of zvegf4
proteins to overcome contact-induced growth inhibition. 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. Cell
differentiation can be assayed using suitable precursor cells that can be
induced to
differentiate into a more mature phenotype. For example, endothelial cells and
hematopoietic cells are derived from a common ancestral cell, the
hemangioblast (Choi
et al., Development 125:725-732, 1998). Mesenchymal stem cells can also be
used to
measure the ability of zvegf4 protein to stimulate differentiation into
osteoblasts.
Differentiation is indicated by the expression of osteocalcin, the ability of
the cells to
mineralize, and the expression of alkaline phosphatase, all of which can be
measured by
routine methods known in the art. Effects of zvegf4 proteins on tumor cell
growth and
metastasis can be analyzed using the Lewis lung carcinoma model, for example
as
described by Cao et al., J. Exp. Med. 182:2069-2077, 1995. Activity of zvegf4
proteins
on cells of neural origin can be analyzed using assays that measure effects on
neurite
growth. Zvegf4 can also be assayed in an aortic ring outgrowth assay (Nicosia
and
Ottinetti, Laboratory Investigation 63:115, 1990; Villaschi and Nicosia, Am.
J.
Pathology 143:181-190, 1993).
Zvegf4 activity may also be detected using assays designed to measure
zvegf4-induced production of one or more additional growth factors or other
macromolecules. Such assays include those for determining the presence of
hepatocyte
growth factor (HGF), epidermal growth factor (EGF), transforming growth factor
alpha
(TGF(x), interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF),
and
angiogenin. Suitable assays include mitogenesis assays using target cells
responsive to
the macromolecule of interest, receptor-binding assays, competition binding
assays,
immunological assays (e.g., ELISA), and other formats known in the art.
Metalloprotease secretion is measured from treated primary human dermal
fibroblasts,
synoviocytes and chondrocytes. The relative levels of collagenase, gelatinase
and
stromalysin produced in response to culturing in the presence of a zvegf4
protein is
measured using zymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-
106,
1990). Procollagen/collagen synthesis by dermal fibroblasts and chondrocytes
in
response to a test protein is measured using 3H-proline incorporation into
nascent
secreted collagen. 3H-labeled collagen is visualized by SDS-PAGE followed by
autoradiography (Unemori and Amento, J. Biol. Chem. 265: 10681-10685, 1990).
Glycosaminoglycan (GAG) secretion from dermal fibroblasts and chondrocytes is
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44
measured using a 1,9-dimethylmethylene blue dye binding assay (Farndale et
al.,
Biochim. Biophys. Acta 883:173-177, 1986). Collagen and GAG assays are also
carried
out in the presence of IL-1(3 or TGF-(3 to examine the ability of zvegf4
protein to
modify the established responses to these cytokines.
Monocyte activation assays are carried out (1) to look for the ability of
zvegf4 proteins to further stimulate monocyte activation, and (2) to examine
the ability
of zvegf4 proteins to modulate attachment-induced or endotoxin-induced
monocyte
activation (Fuhlbrigge et al., J. Immunol. 138: 3799-3802, 1987). IL-1p and
TNFct
levels produced in response to activation are measured by ELISA (Biosource,
Inc.
Camarillo, CA). Monocyte/macrophage cells, by virtue of CD 14 (LPS receptor),
are
exquisitely sensitive to endotoxin, and proteins with moderate levels of
endotoxin-like
activity will activate these cells.
Hematopoietic activity of zvegf4 proteins can be assayed on various
hematopoietic cells in culture. Suitable assays include primary bone marrow or
peripheral blood leukocyte colony assays, and later stage lineage-restricted
colony
assays, which are known in the art (e.g., Holly et al., WIPO Publication WO
95/21920).
Marrow cells plated on a suitable semi-solid medium (e.g., 50% methylcellulose
containing 15% fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN
antibiotic mix) are incubated in the presence of test polypeptide, then
examined
microscopically for colony formation. Known hematopoietic factors are used as
controls. Mitogenic activity of zvegf4 polypeptides on hematopoietic cell
lines can be
measured using 3H-thymidine incorporation assays, dye incorporation assays, or
cell
counts (Raines and Ross, Methods Enzymol. 109:749-773, 1985 and Foster et al.,
U.S.
Patent No. 5,641,655). For example, cells are cultured in multi-well
microtiter plates.
Test samples and 3H-thymidine are added, and the cells are incubated overnight
at
37 C. Contents of the wells are transferred to filters, dried, and counted to
determine
incorporation of label. Cell proliferation can also be measured using a
colorimetric
assay based on the metabolic breakdown of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl
tetrazolium bromide (MTT) (Mosman, ibid.). Briefly, a solution of MTT is added
to
100 l of assay cells, and the cells are incubated at 37 C. After 4 hours,
200 tl of 0.04
N HCl in isopropanol is added, the solution is mixed, and the absorbance of
the sample
is measured at 570 nm.
Cell migration is assayed essentially as disclosed by Kahler et al.
(Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939, 1997). A
protein is
considered to be chemotactic if it induces migration of cells from an area of
low protein
concentration to an area of high protein concentration. The assay is performed
using
modified Boyden chambers with a polystryrene membrane separating the two
chambers
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(Transwell; Corning Costar Corp.). The test sample, diluted in medium
containing 1%
BSA, is added to the lower chamber of a 24-well plate containing Transwells.
Cells are
then placed on the Transwell insert that has been pretreated with 0.2%
gelatin. Cell
migration is measured after 4 hours of incubation at 37 C. Non-migrating cells
are
5 wiped off the top of the Transwell membrane, and cells attached to the lower
face of the
membrane are fixed and stained with 0.1 % crystal violet. Stained cells are
then counted
directly using a microscope, or extracted with 10% acetic acid and absorbance
is
measured at 600 nm. Migration is then calculated from a standard calibration
curve.
Cell adhesion activity is assayed essentially as disclosed by LaFleur et al.
10 (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter plates are
coated with the
test protein, non-specific sites are blocked with BSA, and cells (such as
smooth muscle
cells, leukocytes, or endothelial cells) are plated at a density of
approximately 104 - 105
cells/well. The wells are incubated at 37 C (typically for about 60 minutes),
then non-
adherent cells are removed by gentle washing. Adhered cells are quantitated by
15 conventional methods (e.g., by staining with crystal violet, lysing the
cells, and
determining the optical density of the lysate). Control wells are coated with
a known
adhesive protein, such as fibronectin or vitronectin.
Assays for angiogenic activity are also known in the art. For example,
the effect of zvegf4 proteins on primordial endothelial cells in angiogenesis
can be
20 assayed in the chick chorioallantoic membrane angiogenesis assay (Leung,
Science
246:1306-1309, 1989; Ferrara, Ann. NY Acad. Sci. 752:246-256, 1995). Briefly,
a small
window is cut into the shell of an eight-day old fertilized egg, and a test
substance is
applied to the chorioallantoic membrane. After 72 hours, the membrane is
examined
for neovascularization. Other suitable assays include microinjection of early
stage quail
25 (Coturnix coturnix japonica) embryos as disclosed by Drake et al. (Proc.
Natl. Acad.
Sci. USA 92:7657-7661, 1995); the rodent model of corneal neovascularization
disclosed by Muthukkaruppan and Auerbach (Science 205:1416-1418, 1979),
wherein a
test substance is inserted into a pocket in the cornea of an inbred mouse; and
the
hampster cheek pouch assay (Hockel et al., Arch. Surg. 128:423-429, 1993).
Induction
30 of vascular permeability, which is indicative of angiogenic activity, is
measured in
assays designed to detect leakage of protein from the vasculature of a test
animal (e.g.,
mouse or guinea pig) after administration of a test compound (Miles and Miles,
J.
Physiol. 118:228-257, 1952; Feng et al., J. Exp. Med. 183:1981-1986, 1996). In
vitro
assays for angiogenic activity include the tridimensional collagen gel matrix
model
35 (Pepper et al. Biochem. Biophys. Res. Comm. 189:824-831, 1992 and Ferrara
et al.,
Ann. NY Acad. Sci. 732:246-256, 1995), which measures the formation of tube-
like
structures by microvascular endothelial cells; and basement membrane matrix
models
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46
(Grant et al., "Angiogenesis as a component of epithelial-mesenchymal
interactions" in
Goldberg and Rosen, Epithelial-Mesenchymal Interaction in Cancer, Birkhauser
Verlag, 1995, 235-248; Baatout, Anticancer Research 17:451-456, 1997), which
are
used to determine effects on cell migration and tube formation by endothelial
cells
seeded in a basement membrane extract enriched in laminin (e.g., Matrigel ;
Becton
Dickinson, Franklin Lakes, NJ). Angiogenesis assays can be carried out in the
presence
and absence of VEGF to assess possible combinatorial effects. VEGF can be used
as a
control within in vivo assays.
The activity of zvegf4 proteins, agonists, antagonists, and antibodies of
the present invention can be measured, and compounds screened to identify
agonists
and antagonists, using assays that measure axon guidance and growth. Of
particular
interest are assays that indicate changes in neuron growth patterns, for
example those
disclosed in Hastings, WIPO Publication WO 97/29189 and Walter et al.,
Development
101:685-96, 1987. Assays to measure the effects on neuron growth are well
known in
the art. For example, the C assay (e.g., Raper and Kapfhammer, Neuron 4:21-9,
1990
and Luo et al., Cell 75:217-27, 1993) can be used to determine collapsing
activity of
zvegf4 on growing neurons. Other methods that can assess zvegf4-induced
inhibition
of neurite extension or divert such extension are also known. See, Goodman,
Annu.
Rev. Neurosci. 19:341-77, 1996. Conditioned media from cells expressing a
zvegf4
protein, a zvegf4 agonist, or a zvegf4 antagonist, or aggregates of such
cells, can by
placed in a gel matrix near suitable neural cells, such as dorsal root ganglia
(DRG) or
sympathetic ganglia explants, which have been co-cultured with nerve growth
factor.
Compared to control cells, zvegf4-induced changes in neuron growth can be
measured
(as disclosed by, for example, Messersmith et al., Neuron 14:949-59, 1995 and
Puschel
et al., Neuron 14:941-8, 1995). Likewise neurite outgrowth can be measured
using
neuronal cell suspensions grown in the presence of molecules of the present
invention.
See, for example, O'Shea et al., Neuron 7:231-7, 1991 and DeFreitas et al.,
Neuron
15:333-43, 1995. PC12 Pheochromocytoma cells (see Banker and Goslin, in
Culturing
Nerve Cells, chapter 6, "Culture and experimental use of the PC12 rat
Pheochromocytoma cell line"; also, see Rydel and Greene, J. Neuroscience
7(11):
3639-53, November 1987) can be grown in the presence of zvegf4 to examine
effects
on neurite outgrowth. PC 12 cells pre-treated with NGF to induce
differentiation into a
neuronal population can also be exposed to zvegf4 to determine the ability of
zvegf4 to
promote survival of neuronal cells.
The biological activities of zvegf4 proteins can be studied in non-human
animals by administration of exogenous protein, by expression of zvegf4-
encoding
polynucleotides, and by suppression of endogenous zvegf4 expression through
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47
antisense or knock-out techniques. Zvegf4 proteins can be administered or
expressed
individually, in combination with other zvegf4 proteins, or in combination
with non-
vegf3 proteins, including other growth factors (e.g., other VEGFs, P1GFs, or
PDGFs).
For example, a combination of zvegf4 polypeptides (e.g., a combination of
zvegf419-179
and zvegf4258-370) can be administered to a test animal or expressed in the
animal. Test
animals are monitored for changes in such parameters as clinical signs, body
weight,
blood cell counts, clinical chemistry, histopathology, and the like.
Stimulation of coronary collateral growth can be measured in known
animal models, including a rabbit model of peripheral limb ischemia and hind
limb
ischemia and a pig model of chronic myocardial ischemia (Ferrara et al.,
Endocrine
Reviews 18:4-25, 1997). Zvegf4 proteins are assayed in the presence and
absence of
VEGFs, angiopoietins, and basic FGF to test for combinatorial effects. These
models
can be modified by the use of adenovirus or naked DNA for gene delivery as
disclosed
in more detail below, resulting in local expression of the test protein(s).
Efficacy of zvegf4 polypeptides in promoting wound healing can be
assayed in animal models. One such model is the linear skin incision model of
Mustoe
et al. (Science 237:1333, 1987). In a typical procedure, a 6-cm incision is
made in the
dorsal pelt of an adult rat, then closed with wound clips. Test substances and
controls
(in solution, gel, or powder form) are applied before primary closure.
Although
administration is commonly limited to a single application, additional
applications can
be made on succeeding days by careful injection at several sites under the
incision.
Wound breaking strength is evaluated between 3 and 21 days post-wounding. In a
second model, multiple, small, full-thickness excisions are made on the ear of
a rabbit.
The cartilage in the ear splints the wound, removing the variable of wound
contraction
2S from the evaluation of closure. Experimental treatments and controls are
applied. The
geometry and anatomy of the wound site allow for reliable quantification of
cell
ingrowth and epithelial migration, as well as quantitative analysis of the
biochemistry
of the wounds (e.g., collagen content). See, Mustoe et al., J. Clin. Invest.
87:694,
1991. The rabbit ear model can be modified to create an ischemic wound
environment,
which more closely resembles the clinical situation (Ahn et al., Ann. Plast.
Surg. 24:17,
1990). Within a third model, healing of partial-thickness skin wounds in pigs
or guinea
pigs is evaluated (LeGrand et al., Growth Factors 8:307, 1993). Experimental
treatments are applied daily on or under dressings. Seven days after wounding,
granulation tissue thickness is determined. This model is commonly used for
dose-
response studies, as it is more quantitative than other in vivo models of
wound healing.
A full thickness excision model can also be employed. Within this model, the
epidermis and dermis are removed down to the panniculus carnosum in rodents or
the
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48
subcutaneous fat in pigs. Experimental treatments are applied topically on or
under a
dressing, and can be applied daily if desired. The wound closes by a
combination of
contraction and cell ingrowth and proliferation. Measurable endpoints include
time to
wound closure, histologic score, and biochemical parameters of wound tissue.
Impaired wound healing models are also known in the art (e.g., Cromack et al.,
Surgery
113:36, 1993; Pierce et al., Proc. Natl. Acad. Sci. USA 86:2229, 1989;
Greenhalgh et
al., Amer. J. Pathol. 136:1235, 1990). Delay or prolongation of the wound
healing
process can be induced pharmacologically by treatment with steroids,
irradiation of the
wound site, or by concomitant disease states (e.g., diabetes). Linear
incisions or full-
thickness excisions are most commonly used as the experimental wound.
Endpoints are
as disclosed above for each type of wound. Subcutaneous implants can be used
to
assess compounds acting in the early stages of wound healing (Broadley et al.,
Lab.
Invest. 61:571, 1985; Sprugel et al., Amer. J. Pathol. 129: 601, 1987).
Implants are
prepared in a porous, relatively non-inflammatory container (e.g.,
polyethylene sponges
or expanded polytetrafluoroethylene implants filled with bovine collagen) and
placed
subcutaneously in mice or rats. The interior of the implant is empty of cells,
producing
a "wound space" that is well-defined and separable from the preexisting
tissue. This
arrangement allows the assessment of cell influx and cell type as well as the
measurement of vasculogenesis/angiogenesis and extracellular matrix
production.
Expression of zvegf4 proteins in animals provides models for study of
the biological effects of overproduction or inhibition of protein activity in
vivo.
Zvegf4-encoding polynucleotides can be introduced into test animals, such as
mice,
using viral vectors or naked DNA, or transgenic animals can be produced. A
zvegf4
protein will commonly be expressed with a secretory peptide. Suitable
secretory
peptides include the zvegf4 secretory peptide (e.g., residues 1-18 of SEQ ID
NO:2) and
heterologous secretory peptides. An exemplary heterologous secretory peptide
is that of
human tissue plasminogen activator (t-PA). The t-PA secretory peptide may be
modified to reduce undesired proteolytic cleavage as disclosed in U.S. Patent
No.
5,641,655.
One in vivo approach for assaying proteins of the present invention
utilizes 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
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49
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.
Using adenovirus vectors where portions of the adenovirus genome are
deleted, inserts are incorporated into the viral DNA by direct ligation or by
homologous
recombination with a co-transfected plasmid. In an exemplary system, the
essential El
gene has been deleted from the viral vector, and the virus will not replicate
unless the
El gene is provided by the host cell (the human 293 cell line is exemplary).
When
intravenously administered to intact animals, adenovirus primarily targets the
liver. If
the adenoviral delivery system has an El 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
secretory signal sequence is present, secrete) the heterologous protein.
Secreted
proteins will enter the circulation in the highly vascularized liver, and
effects on the
infected animal can be determined. Intranasal delivery of adenovirus
expressing zvegf4
will target the zvegf4 protein to lung tissue. Further, adenovirus expressing
zvegf4 can
be administered directly into brain tissue. Adenoviral vectors containing
various
deletions of viral genes can be used in an attempt to reduce or eliminate
immune
responses to the vector. Such adenoviruses are El deleted, and in addition
contain
deletions of E2A or E4 (Lusky et al., J. Virol. 72:2022-2032, 1998; Raper et
al., Human
Gene Therapy 9:671-679, 1998). In addition, deletion of E2b is reported to
reduce
immune responses (Amalfitano, et al., J. Virol. 72:926-933, 1998). Generation
of so-
called "gutless" adenoviruses where all viral transcription units are deleted
is
particularly advantageous for insertion of large inserts of heterologous DNA.
For
review, see Yeh and Perricaudet, FASEB J. 11:615-623, 1997.
In another embodiment, a zvegf4 gene can be introduced in a retroviral
vector as described, for example, by Anderson et al., U.S. Patent No.
5,399,346; Mann
et al., Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et
al., U.S.
Patent No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 1988; Temin et al.,
U.S.
Patent No. 5,124,263; Dougherty et al., WIPO publication WO 95/07358; and Kuo
et
al., Blood 82:845, 1993.
In an alternative method, the vector can be introduced by "lipofection" in
vivo using liposomes. Synthetic cationic lipids can be used to prepare
liposomes for in
vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl.
Acad. Sci.
USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31,
1988).
The use of lipofection to introduce exogenous genes into specific organs in
vivo has
certain practical advantages. Molecular targeting of liposomes to specific
cells
represents one area of benefit. For instance, directing transfection to
particular cell
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types is particularly advantageous in a tissue with cellular heterogeneity,
such as the
pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other
molecules for the purpose of targeting. Targeted peptides (e.g., hormones or
neurotransmitters), proteins such as antibodies, or non-peptide molecules can
be
5 coupled to liposomes chemically.
Within another embodiment target cells are removed from the animal,
and the DNA is introduced as a naked DNA plasmid. The transformed cells are
then re-
implanted into the body of the animal. Naked DNA vectors can be introduced
into the
desired host cells by methods known in the art, e.g., transfection,
electroporation,
10 microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate
precipitation, use of a gene gun or use of a DNA vector transporter. See,
e.g., Wu et al.,
J. Biol. Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.
Mice engineered to express the zvegf4 gene, referred to as "transgenic
mice," and mice that exhibit a complete absence of zvegf4 gene function,
referred to as
15 "knockout mice," can also be generated (Snouwaert et al., Science 257:1083,
1992;
Lowell et al., Nature 366:740-42, 1993; Capecchi, Science 244:1288-1292, 1989;
Palmiter et al., Ann. Rev. Genet. 20:465-499, 1986). Transgenesis experiments
can be
performed using normal mice or mice with genetic disease or other altered
phenotypes.
Transgenic mice that over-express zvegf4, either ubiquitously or under a
tissue-specific
20 or tissue-restricted promoter, can be used to determine whether or not over-
expression
causes a phenotypic change. Exemplary promoters include metallothionein,
albumin,
ApoAl and enolase gene promoters. The metallothionein-1 (MT-1) promoter
provides
expression in liver and other tissues, often leading to high levels of
circulating protein.
Over-expression of a wild-type zvegf4 polypeptide, polypeptide fragment or a
mutant
25 thereof may alter normal cellular processes, resulting in a phenotype that
identifies a
tissue in which zvegf4 expression is functionally relevant and may indicate a
therapeutic target for the zvegf4, its agonists or antagonists. For example, a
transgenic
mouse can be engineered to over-expresses a full-length zvegf4 sequence, which
may
result in a phenotype that shows similarity with human diseases. Similarly,
knockout
30 zvegf4 mice can be used to determine where zvegf4 is absolutely required in
vivo. The
phenotype of knockout mice is predictive of the in vivo effects of zvegf4
antagonists.
Knockout mice can also be used to study the effects of zvegf4 proteins in
models of
disease, including, for example, cancer, atherosclerosis, rheumatoid
arthritis, ischemia,
and cardiovascular disease. The human zvegf4 cDNA can be used to isolate
murine
35 zvegf4 mRNA, cDNA and genomic DNA as disclosed above, which are
subsequently
used to generate knockout mice. These mice may be employed to study the zvegf4
gene
and the protein encoded thereby in an in vivo system, and can be used as in
vivo models
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51
for corresponding human diseases. Moreover, transgenic mice expressing zvegf4
antisense polynucleotides or ribozymes directed against zvegf4, described
herein, can
be used analogously to knockout mice described above.
Antisense methodology can be used to inhibit zvegf4 gene transcription
to examine the effects of such inhibition in vivo. Polynucleotides that are
complementary to a segment of a zvegf4-encoding polynucleotide (e.g., a
polynucleotide as set froth in SEQ ID NO:1) are designed to bind to zvegf4-
encoding
mRNA and to inhibit translation of such mRNA. Such antisense oligonucleotides
can
also be used to inhibit expression of zvegf4 polypeptide-encoding genes in
cell culture.
Zvegf4 proteins may be used therapeutically in human and veterinary
medicine to stimulate tissue development or repair, or cellular
differentiation or
proliferation. Specific applications include, without limitation: the
treatment of full-
thickness skin wounds, including venous stasis ulcers and other chronic, non-
healing
wounds, particularly in cases of compromised wound healing due to diabetes
mellitus,
connective tissue disease, smoking, burns, and other exacerbating conditions;
fracture
repair; skin grafting; within reconstructive surgery to promote
neovascularization and
increase skin flap survival; to establish vascular networks in transplanted
cells and
tissues, such as transplanted islets of Langerhans; to treat female
reproductive tract
disorders, including acute or chronic placental insufficiency (an important
factor
causing perinatal morbidity and mortality) and prolonged bleeding; to promote
the
growth of tissue damaged by periodontal disease; to promote endothelialization
of
vascular grafts and stents; in the treatment of acute and chronic lesions of
the
gastrointestinal tract, including duodenal ulcers, which are characterized by
a deficiency
of microvessels; to promote angiogenesis and prevent neuronal degeneration due
to
acute or chronic cerebral ischemia; to accelerate the formation of collateral
blood
vessels in ischemic limbs; to promote vessel re-endothelialization and to
reduce intimal
hyperplasia following invasive procedures such as balloon angioplasty and
stent
placement; to promote vessel repair and development of collateral circulation
following
myocardial infarction so as to limit ischemic injury; and to stimulate
hematopoiesis.
The polypeptides are also useful additives in tissue adhesives for promoting
revascularization of the healing tissue.
Of particular interest is the use of zvegf4 for the treatment or repair of
liver damage, including damage due to chronic liver disease, including chronic
active
hepatitis 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
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52
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-l-antitrypsin deficiency. Zvegf4 may
also be
useful for the treatment of hepatic chronic passive congestion (CPC) and
central
hemorrhagic necrosis (CHN), which are two circulatory changes representing a
continuum encountered in right-sided heart failure. Other circulatory
disorders that
may be treated with zvegf4 include hepatic vein thrombosis, portal vein
thrombosis,
and cardiac sclerosis. In cases of liver fibrosis, it may be beneficial to
administer a
zvegf4 antagonist to suppress the activation of stellate cells, which have
been
implicated in the production of extracellular matrix in fibrotic liver (Li and
Friedman, J.
Gastroenterol. Hepatol. 14:618-633, 1999). More generally, zvegf4 may be
beneficially used as an anti-fibrotic agent. Conditions that are characterized
by a pro-
fibrotic response include sclerosing peritonitis; adhesions following surgery
(particularly laparoscopic surgery), which may lead to small bowel
obstruction,
difficulties on re-operation, pelvic adhesions and pelvic pain (see N. Panay
and A.M.
Lower, Curr. Opin. Obstet. Gynecol. 11:379-85, 1999); pulmonary fibrosis;
kidney
fibrosis; and restenosis.
Zvegf4 polypeptides can be administered alone or in combination with
other vasculogenic or angiogenic agents, including VEGF and angiopoietins 1
and 2.
For example, basic and acidic FGFs, Ang-1, Ang-2, and VEGF have been found to
play
a role in the development of collateral circulation, and the combined use of
zvegf4 with
one or more of these factors may be advantageous. VEGF has also been
implicated in
the survival of transplanted islet cells (Gorden et al. Transplantation 63:436-
443, 1997;
Pepper, Arteriosclerosis, Throrn. and Vascular Biol. 17:605-619, 1997). Basic
FGF has
been shown to induce angiogenesis and accelerate healing of ulcers in
experimental
animals (reviewed by Folkman, Nature Medicine 1:27-31, 1995). VEGF has been
shown to promote vessel re-endothelialization and to reduce intimal
hyperplasia in
animal models of restenosis (Asahara et al., Circulation 91:2802-2809, 1995;
Callow et
al., Growth Factors 10:223-228, 1994); efficacy of zvegf4 polypeptides can be
tested in
these and other known models. When using zvegf4 in combination with an
additional
agent, the two compounds can be administered simultaneously or sequentially as
appropriate for the specific condition being treated.
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Zvegf4 proteins may be used either alone or in combination with other
hematopoietic factors such as IL-3, G-CSF, GM-CSF, or stem cell factor to
enhance
expansion and mobilization of hematopoietic stem cells, including endothelial
precursor stem cells. Cells that can be expanded in this manner include cells
isolated
from bone marrow, including bone marrow stromal cells (see K. Satomura et al.,
J.
Cell. Physiol. 177:426-38, 1998), or cells isolated from blood. Zvegf4
proteins may
also be given directly to an individual to enhance endothelial stem cell
production and
differentiation within the treated individual. The stem cells, either
developed within the
patient, or provided back to a patient, may then play a role in modulating
areas of
ischemia within the body, thereby providing a therapeutic effect. These cells
may also
be useful in enhancing re-endothelialization of areas devoid of endothelial
coverage,
such as vascular grafts, vascular stents, and areas where the endothelial
coverage has
been damaged or removed (e.g., areas of angioplasty). Zvegf4 proteins may also
be
used in combination with other growth and differentiation factors such as
angiopoietin-
1 (Davis et al., Cell 87:1161-1169, 1996) to help create and stabilize new
vessel
formation in areas requiring neovascularization, including areas of ischemia
(cardiac or
peripheral ischemia), organ transplants, wound healing, and tissue grafting.
Zvegf4 proteins, agonists and antagonists may be used to modulate
neurite growth and development and demarcate nervous system structures. As
such,
Zvegf4 proteins, agonists, and antagonists would be useful as a treatment of
peripheral
neuropathies by increasing spinal cord and sensory neurite outgrowth. A zvegf4
antagonist could be part of a therapeutic treatment for the regeneration of
neurite
outgrowths following strokes, brain damage caused by head injuries and
paralysis
caused by spinal injuries. Application may also be made in treating
neurodegenerative
diseases such as multiple sclerosis, Alzheimer's disease and Parkinson's
disease.
Application may also be made in mediating development and innervation pattern
of
stomach tissue.
Zvegf4 has been found to have PDGF-like activity, including mitogenic
activity on fibroblasts, vascular smooth muscle cells, and pericytes. Zvegf4
has also
been found to stimulate bone growth in an animal model. These results suggest
that
zvegf4 proteins will be useful in promoting the growth of bone and ligament.
Such
uses include, for example, treatment of periodontal disease, fractures
(including non-
union fractures), implant recipient sites, bone grafts, and joint injuries
involving
cartilage and/or ligament damage. Zvegf4 may be used in combination with other
bone
stimulating factors, such as IGF-1, EGF, TGF-(3, PDGF, and BMPs. Methods for
using
growth factors in the treatment of periodontal disease are known in the art.
See, for
example, U.S. Patent No. 5,124,316 and Lynch et al., ibid.
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For pharmaceutical use, zvegf4 proteins, antagonist, and antibodies are
formulated for topical or parenteral, particularly intravenous or
subcutaneous, delivery
according to conventional methods. In general, pharmaceutical formulations
will
include a zvegf4 polypeptide 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, thickeners, gelling
agents, 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. Zvegf4 will ordinarily be used in a
concentration of
about 10 to 100 g/ml of total volume, although concentrations in the range of
1 ng/ml
to 1000 g/ml may be used. For topical application, such as for the promotion
of
wound healing, the protein will be applied in the range of 0.1-10 Rg/cm2 of
wound area,
with the exact dose determined by the clinician according to accepted
standards, taking
into account the nature and severity of the condition to be treated, patient
traits, etc.
Determination of dose is within the level of ordinary skill in the art. The
therapeutic
formulations will generally be administered over the period required for
neovascularization, typically from one 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. In general, a therapeutically effective amount of zvegf4 is an
amount
sufficient to produce a clinically significant change in the treated
condition, such as a
clinically significant reduction in time required by wound closure, a
significant
reduction in wound area, a significant improvement in vascularization, a
significant
reduction in morbidity, or a significantly increased histological score.
Proteins of the present invention are useful for modulating the
proliferation, differentiation, migration, or metabolism of responsive cell
types, which
include both primary cells and cultured cell lines. Of particular interest in
this regard
are hematopoietic cells (including stem cells and mature myeloid and lymphoid
cells),
endothelial cells, neuronal cells, mesenchymal cells (including fibroblasts,
pericytes,
stellate cells, mesangial cells, chondrocytes and smooth muscle cells), and
bone-derived
cells (including osteoblast and osteoclast precursors). Zvegf4 polypeptides
are added to
tissue culture media for these cell types at a concentration of about 10 pg/ml
to about
1000 ng/ml. Those skilled in the art will recognize that zvegf4 proteins can
be
advantageously combined with other growth factors in culture media.
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Within the laboratory research field, zvegf4 proteins can also be used as
molecular weight standards; as reagents in assays for determining circulating
levels of
the protein, such as in the diagnosis of disorders characterized by over- or
under-
production of zvegf4 protein; or as standards in the analysis of cell
phenotype.
5 Zvegf4 proteins can also be used to identify inhibitors of their activity.
Test compounds are added to the assays disclosed above to identify compounds
that
inhibit the activity of zvegf4 protein. In addition to those assays disclosed
above,
samples can be tested for inhibition of zvegf4 activity within a variety of
assays
designed to measure receptor binding or the stimulation/inhibition of zvegf4-
dependent
10 cellular responses. For example, zvegf4-responsive cell lines can be
transfected with a
reporter gene construct that is responsive to a zvegf4-stimulated cellular
pathway.
Reporter gene constructs of this type are known in the art, and will generally
comprise a
zvegf4-activated serum response element (SRE) operably linked to a gene
encoding an
assayable protein, such as luciferase. Candidate compounds, solutions,
mixtures or
15 extracts are tested for the ability to inhibit the activity of zvegf4 on
the target cells as
evidenced by a decrease in zvegf4 stimulation of reporter gene expression.
Assays of
this type will detect compounds that directly block zvegf4 binding to cell-
surface
receptors, as well as compounds that block processes in the cellular pathway
subsequent
to receptor-ligand binding. In the alternative, compounds or other samples can
be
20 tested for direct blocking of zvegf4 binding to receptor using zvegf4
tagged with a
detectable label (e.g., 125I, biotin, horseradish peroxidase, FITC, and the
like). Within
assays of this type, the ability of a test sample to inhibit the binding of
labeled zvegf4 to
the receptor is indicative of inhibitory activity, which can be confirmed
through
secondary assays. Receptors used within binding assays may be cellular
receptors or
2S isolated, immobilized receptors.
The activity of zvegf4 proteins can be measured with a silicon-based
biosensor microphysiometer that measures the extracellular acidification rate
or proton
excretion associated with receptor binding and subsequent physiologic cellular
responses. An exemplary such device is the CytosensorTM Microphysiometer
30 manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular
responses,
such as cell proliferation, ion transport, energy production, inflammatory
response,
regulatory and receptor activation, and the like, can be measured by this
method. See,
for example, McConnell et al., Science 257:1906-1912, 1992; Pitchford et al.,
Meth.
Enzymol. 228:84-108, 1997; Arimilli et al., J. Immunol. Meth. 212:49-59, 1998;
and
3S Van Liefde et al., Eur. J. Phannacol. 346:87-95, 1998. The microphysiometer
can be
used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By
measuring extracellular acidification changes in cell media over time, the
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microphysiometer directly measures cellular responses to various stimuli,
including
zvegf4 proteins, their agonists, and antagonists. The microphysiometer can be
used to
measure responses of a zvegf4-responsive eukaryotic cell, compared to a
control
eukaryotic cell that does not respond to zvegf4 polypeptide. Zvegf4-responsive
eukaryotic cells comprise cells into which a receptor for zvegf4 has been
transfected
creating a cell that is responsive to zvegf4, as well as cells naturally
responsive to
zvegf4 such as cells derived from vascular or neural tissue. Differences,
measured by a
change in extracellular acidification, in the response of cells exposed to
zvegf4
polypeptide relative to a control not exposed to zvegf4, are a direct
measurement of
zvegf4-modulated cellular responses. Moreover, such zvegf4-modulated responses
can
be assayed under a variety of stimuli. The present invention thus provides
methods of
identifying agonists and antagonists of zvegf4 proteins, comprising providing
cells
responsive to a zvegf4 polypeptide, culturing a first portion of the cells in
the absence
of a test compound, culturing a second portion of the cells in the presence of
a test
compound, and detecting a change in a cellular response of the second portion
of the
cells as compared to the first portion of the cells. The change in cellular
response is
shown as a measurable change in extracellular acidification rate. Culturing a
third
portion of the cells in the presence of a zvegf4 protein and the absence of a
test
compound provides a positive control for the zvegf4-responsive cells and a
control to
compare the agonist activity of a test compound with that of the zvegf4
polypeptide.
Antagonists of zvegf4 can be identified by exposing the cells to zvegf4
protein in the
presence and absence of the test compound, whereby a reduction in zvegf4-
stimulated
activity is indicative of antagonist activity in the test compound.
Zvegf4 proteins can also be used to identify cells, tissues, or cell lines
that respond to a zvegf4-stimulated pathway. The microphysiometer, described
above,
can be used to rapidly identify ligand-responsive cells, such as cells
responsive to
zvegf4 proteins. Cells are cultured in the presence or absence of zvegf4
polypeptide.
Those cells that elicit a measurable change in extracellular acidification in
the presence
of zvegf4 are responsive to zvegf4. Responsive cells can than be used to
identify
antagonists and agonists of zvegf4 polypeptide as described above.
Inhibitors of zvegf4 activity (zvegf4 antagonists) include anti-zvegf4
antibodies and soluble zvegf4 receptors, as well as other peptidic and non-
peptidic
agents, including ribozymes, small molecule inhibitors, and angiogenically or
mitogenically inactive receptor-binding fragments of zvegf4 polypeptides. Such
antagonists can be use to block biological activities of zvegf4, including
mitogenic,
chemotactic, or angiogenic effects. These antagonists are therefore useful in
reducing
the growth of solid tumors by inhibiting neovascularization of the developing
tumor or
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57
by directly blocking tumor cell growth; in the treatment of diabetic
retinopathy,
psoriasis, arthritis, and scleroderma; and in reducing fibrosis, including
scar formation.
Inhibitors of zvegf4 may also be useful in the treatment of proliferative
vascular
disorders wherein zvegf4 activity is pathogenic. Such disorders may include
atherosclerosis and intimal hyperplastic restenosis following angioplasty,
endarterectomy, vascular grafting, organ transplant, or vascular stent
emplacement.
These conditions involve complex growth factor-mediated responses wherein
certain
factors may be beneficial to the clinical outcome and others may be
pathogenic.
Inhibitors of zvegf4 may also prove useful in the treatment of ocular
neovascularization, including diabetic retinopathy and age-related macular
degeneration. Experimental evidence suggests that these conditions result from
the
expression of angiogenic factors induced by hypoxia in the retina.
Zvegf4 antagonists are also of interest in the treatment of inflammatory
disorders, such as rheumatoid arthritis and psoriasis. In rheumatoid
arthritis, studies
suggest that VEGF plays an important role in the formation of pannus, an
extensively
vascularized tissue that invades and destroys cartilage. Psoriatic lesions are
hypervascular and overexpress the angiogenic polypeptide IL-8.
Zvegf4 antagonists may also prove useful in the treatment of infantile
hemangiomas, which exhibit overexpression of VEGF and bFGF during the
proliferative phase.
Inhibitors are formulated for pharmaceutical use as generally disclosed
above, taking into account the precise chemical and physical nature of the
inhibitor and
the condition to be treated. The relevant determinations are within the level
of ordinary
skill in the formulation art. Other angiogenic and vasculogenic factors,
including
VEGF and bFGF, have been implicated in pathological neovascularization. In
such
instances it may be advantageous to combine a zvegf4 inhibitor with one or
more
inhibitors of these other factors.
The polypeptides, nucleic acids, and antibodies of the present invention
may be used in diagnosis or treatment of disorders associated with cell loss
or abnormal
cell proliferation (including cancer), including impaired or excessive
vasculogenesis or
angiogenesis, and diseases of the nervous system. Labeled zvegf4 polypeptides
may be
used for imaging tumors or other sites of abnormal cell proliferation. Because
angiogenesis in adult animals is generally limited to wound healing and the
female
reproductive cycle, it is a very specific indicator of pathological processes.
Angiogenesis is indicative of, for example, developing solid tumors,
retinopathies, and
arthritis.
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Zvegf4 polypeptides and anti-zvegf4 antibodies can be directly or
indirectly conjugated to drugs, toxins, radionuclides and the like, and these
conjugates
used for in vivo diagnostic or therapeutic applications. For instance,
polypeptides or
antibodies of the present invention may be used to identify or treat tissues
or organs that
express a corresponding anti-complementary molecule (receptor or antigen,
respectively, for instance). More specifically, zvegf4 polypeptides or anti-
zvegf4
antibodies, or bioactive fragments or portions thereof, can be coupled to
detectable or
cytotoxic molecules and delivered to a mammal having cells, tissues, or organs
that
express the anti-complementary molecule. For example, the CUB domain of zvegf4
can be used to target peptidic and non-peptidic moieties to semaphorins as
disclosed
above. In another embodiment, polypeptide-toxin fusion proteins or
antibody/fragment-toxin fusion proteins may be used for targeted cell or
tissue
inhibition or ablation, such as in cancer therapy. Of particular interest in
this regard are
conjugates of a zvegf4 polypeptide and a cytotoxin, which can be used to
target the
cytotoxin to a tumor or other tissue that is undergoing undesired angiogenesis
or
neovascularization.
In another embodiment, zvegf4-cytokine fusion proteins or
antibody/fragment-cytokine fusion proteins may be used for enhancing in vitro
cytotoxicity (for instance, that mediated by monoclonal antibodies against
tumor
targets) and for enhancing in vivo killing of target tissues (for example,
blood and bone
marrow cancers). See, generally, Hornick et al., Blood 89:4437-4447, 1997). In
general, cytokines are toxic if administered systemically. The described
fusion proteins
enable targeting of a cytokine to a desired site of action, such as a cell
having binding
sites for zvegf4, thereby providing an elevated local concentration of
cytokine. Suitable
cytokines for this purpose include, for example, interleukin-2 and granulocyte-
macrophage colony-stimulating factor (GM-CSF). Such fusion proteins may be
used to
cause cytokine-induced killing of tumors and other tissues undergoing
angiogenesis or
neovascularization.
In yet another embodiment, a zvegf4 polypeptide or anti-zvegf4 antibody
can be conjugated with a radionuclide, particularly with a beta-emitting or
gamma-
emitting radionuclide, and used to reduce restenosis. For instance, iridium-
192
impregnated ribbons placed into stented vessels of patients until the required
radiation
dose was delivered resulted in decreased tissue growth in the vessel and
greater luminal
diameter than the control group, which received placebo ribbons. Further,
revascularisation and stent thrombosis were significantly lower in the
treatment group.
Similar results are predicted with targeting of a bioactive conjugate
containing a
radionuclide, as described herein.
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The bioactive polypeptide or antibody conjugates described herein can
be delivered intravenously, intra-arterially or intraductally, or may be
introduced locally
at the intended site of action.
Polynucleotides encoding zvegf4 polypeptides are useful within gene
therapy applications where it is desired to increase or inhibit zvegf4
activity. For
example, Isner et al., The Lancet (ibid.) reported that VEGF gene therapy
promoted
blood vessel growth in an ischemic limb. Additional applications of zvegf4
gene
therapy include stimulation of wound healing, repopulation of vascular grafts,
stimulation of neurite growth, and inhibition of cancer growth and metastasis.
Gene
delivery systems useful in this regard include adenovirus, adeno-associated
virus, and
naked DNA vectors.
The present invention also provides polynucleotide reagents for
diagnostic use. For example, a zvegf4 gene, a probe comprising zvegf4 DNA or
RNA,
or a subsequence thereof can be used to determine if a mutation has occurred
at the
zvegf4 locus on human chromosome 11. Detectable chromosomal aberrations at the
zvegf4 gene locus include, but are not limited to, aneuploidy, gene copy
number
changes, insertions, deletions, restriction site changes and rearrangements.
Such
aberrations can be detected using polynucleotides of the present invention by
employing
molecular genetic techniques, such as restriction fragment length polymorphism
(RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques,
and
other genetic linkage analysis techniques known in the art (Sambrook et al.,
ibid.;
Ausubel et. al., ibid.; A.J. Marian, Chest 108:255-265, 1995).
The invention is further illustrated by the following non-limiting
examples.
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EXAMPLES
Example 1
Human Multiple Tissue Northern Blots I, II, III and Human RNA Master
5 Blots (Clontech Laboratories, Inc., Palo Alto, CA) were probed to determine
the tissue
expression of zvegf4. Blots were prehybridized for 3 hours at 65 degrees in 10
ml of a
hybridization solution (ExpressHybTM Hybridization Solution; Clontech
Laboratories,
Inc.) containing 1 mg of salmon sperm DNA that had been boiled 5 minutes, then
iced
1 minute. The probe used was a 251-bp PCR fragment generated with 20 pmole
each
10 of primers ZC21,119 (SEQ ID NO:25) and ZC21,120 (SEQ ID NO:26), and 5 l of
a
heart cDNA library prepared from heart RNA using a commercially available kit
(MarathonTM cDNA Amplification Kit from Clontech Laboratories, Inc.). The
reaction
was run as follows: 94 degrees for 1 minute; then 30 cycles of 94 degrees, 20
seconds;
67 degrees, 1 minute; and ended with a 5-minute incubation at 72 degrees. The
PCR
15 product was gel-purified, and the DNA was eluted from the gel slab with a
spin column
containing a silica gel membrane (QlAquickTM Gel Extraction Kit; Qiagen, Inc.,
Valencia, CA).
51 ng of the resulting zvegf4 fragment was labeled with 32P using a
commercially available kit (RediprimeTM II random-prime labeling system;
Amersham
20 Pharmacia Biotech, Piscataway, N.J.). Unincorporated radioactivity was
removed with
a push column (NucTrap column; Stratagene, La Jolla, CA; see U.S. Patent No.
5,336,412). 10 x 106 cpm of the resulting labeled probe and 1 mg of salmon
sperm
DNA were boiled 5 minutes, iced 1 minute, then mixed with 10 ml hybridization
solution (ExpressHybTM) and added to blots. Hybridization took place overnight
at 65
25 degrees, followed by a wash in 2 x SSC, 0.1 % SDS at room temperature,
followed by a
wash in 0.1 x SSC, 0.1% SDS at 50 degrees. Blots were exposed to film at -80
degrees
overnight.
There was an approximately 4.4 kb transcript in every tissue except bone
marrow. Heart, pancreas, stomach and adrenal gland showed the strongest zvegf4
30 expression on the Northern blots, and the dot blot additionally showed
strong
expression in the pituitary gland and the ovary.
Example 2
Zvegf4 was identified from the sequence of a clone from a human
3 5 chronic myelogenous leukemia cell (K562) library by its homology to the
VEGF
family. Additional sequence was elucidated from a long sequence read of a
clone from
a pituitary library. An antisense expressed sequence tag (EST) for zvegf4 was
found,
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61
for which its 5' partner was identified. This 5' EST (EST448186; GenBank)
appeared
to contain the 5' untranslated sequence for zvegf4. A primer was designed from
EST448186 to close the gap in the sequence. 20 pm each of ZC21,987 (SEQ ID
NO:27) and ZC21,120 (SEQ ID NO:26) and 1.93 g of a thyroid library were used
in
the PCR reaction. It was a modified PCR reaction using 5% DMSO and 1/10 volume
of a commercial reagent (GC-Me1tTM; Clontech Laboratories, Inc.). The reaction
was
run for 1 minute at 94 degrees; then 30 cycles of 94 degrees, 20 seconds; 67
degrees, 1
minute; then a final 5-minute incubation at 72 degrees. A resulting 833-bp
product was
sequenced and found to be a zvegf4 fragment containing the remainder of the
coding
sequence with an intiation MET codon, upstream stop codon, and 5' untranslated
sequence. The composite sequence included an open reading frame of 1,110 bp
(SEQ
ID NO:1).
Example 3
To make transgenic animals expressing zvegf4 genes requires adult,
fertile males (studs) (B6C3f1, 2-8 months of age (Taconic Farms, Germantown,
NY)),
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)).
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.
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 7;
all
reagents available from Sigma Chemical Co.) that has been incubated with 5%
CO2,
5% 02, and 90% N2 at 37 C. The eggs are stored in a 37 C/5% CO2 incubator
until
microinjection.
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Table 7
mgs/200 ml mgs/500 ml
NaCl 1280 3200
KCI 72 180
KH2PO4 32 80
MgSO4. 7H20 60 150
Glucose 200 500
Ca2+ Lactate 106 265
Benzylpenicillin 15 37.5
Streptomycin SO4 10 25
NaHCO3 380 950
Na Pyruvate 5 12.5
H2O 200 ml 500 ml
500 mM EDTA 100 l 250 l
5% Phenol Red 200 l 500 l
BSA 600 1500
Zvegf4 cDNA is inserted into the expression vector pHB 12-8 (see Fig.
2). 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.
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.
Plasmid DNA is microinjected into harvested eggs contained in a drop
of W640 medium overlaid by warm, C02-equilibrated mineral oil. The DNA is
drawn
into an injection needle (pulled from a 0.75mm ID, lmm 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.
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
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into an organ tissue-culture dish with pregassed W640 medium for storage
overnight in
a 37 C/5% CO2 incubator.
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.
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, 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.
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 C slide warmer for a minimum of 4 hours.
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.
Genomic DNA is prepared from the tail snips using a commercially
available kit (DNeasyTM 96 Tissue Kit; Qiagen, Valencia, CA) 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:28)
and ZC17,252 (SEQ ID NO:29) amplify a 368-base-pair fragment of hGH. In
addition,
primers ZC17,156 (SEQ ID NO:30) and ZC17,157 (SEQ ID NO:31), 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
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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.
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.
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 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.
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, CA) following the manufacturer's
instructions.
Example 4
An expression plasmid containing all or part of a polynucleotide
encoding zvegf4 is constructed via homologous recombination. A fragment of
zvegf4
cDNA is isolated by PCR using the polynucleotide sequence of SEQ ID NO: 1 with
flanking regions at the 5' and 3' ends corresponding to the vector sequences
flanking
the zvegf4 insertion point. The primers for PCR each include from 5' to 3'
end: 40 bp
of flanking sequence from the vector and 17 bp corresponding to the amino and
carboxyl termini from the open reading frame of zvegf4.
Ten it of the 100 [tl PCR reaction is run on a 0.8% LMP agarose gel
(Seaplaque GTG) with 1 x TBE buffer for analysis. The remaining 90 l of PCR
reaction is precipitated with the addition of 5 [tl 1 M NaCl and 250 l of
absolute
ethanol. The plasmid pZMP6, which has been cut with Smal, is used for
recombination
with the PCR fragment. Plasmid pZMP6 was constructed from pZP9 (deposited at
the
American Type Culture Collection, 10801 University Boulevard, Manassas, VA
20110-
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2209, under Accession No. 98668) with the yeast genetic elements taken from
pRS316
(deposited at the American Type Culture Collection, 10801 University
Boulevard,
Manassas, VA 20110-2209, under Accession No. 77145), an internal ribosome
entry
site (IRES) element from poliovirus, and the extracellular domain of CD8
truncated at
5 the C-terminal end of the transmembrane domain. pZMP6 is a mammalian
expression
vector containing an expression cassette having the mouse metallothionein-1
promoter,
multiple restriction sites for insertion of coding sequences, a stop codon,
and a human
growth hormone terminator. The plasmid also contains an E. coli origin of
replication;
a mammalian selectable marker expression unit comprising an SV40 promoter,
10 enhancer and origin of replication, a DHFR gene, and the SV40 terminator;
as well as
the URA3 and CEN-ARS sequences required for selection and replication in S.
cerevisiae.
One hundred microliters of competent yeast cells (S. cerevisiae) are
independently combined with 10 iil of the various DNA mixtures from above and
15 transferred to a 0.2-cm electroporation cuvette. The yeast/DNA mixtures are
electropulsed at 0.75 kV (5 kV/cm), 00 ohms, 25 F. To each cuvette is added
600 l of
1.2 M sorbitol, and the yeast is plated in two 300- i1 aliquots onto two URA-D
plates
and incubated at 30 C. After about 48 hours, the Ura+ yeast transformants from
a
single plate are resuspended in 1 ml H2O and spun briefly to pellet the yeast
cells. The
20 cell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%
SDS, 100 mM
NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundred microliters of the lysis
mixture is added to an Eppendorf tube containing 300 l acid-washed glass
beads and
200 i l phenol-chloroform, vortexed for 1 minute intervals two or three times,
and spun
for 5 minutes in an Eppendorf centrifuge at maximum speed. Three hundred
25 microliters of the aqueous phase is transferred to a fresh tube, and the
DNA is
precipitated with 600 [tl ethanol (EtOH), followed by centrifugation for 10
minutes at
4 C. The DNA pellet is resuspended in 10 l H2O.
Transformation of electrocompetent E. coli host cells (Electromax
DHIOBTM cells; obtained from Life Technologies, Inc., Gaithersburg, MD) is
done with
30 0.5-2 ml yeast DNA prep and 40 .tl of cells. The cells are electropulsed at
1.7 kV, 25
F, and 400 ohms. Following electroporation, 1 ml SOC (2% BactoTM Tryptone
(Difco, Detroit, MI), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCI, 10
mM
MgC12, 10 mM MgSO4, 20 mM glucose) is plated in 250-i.1 aliquots on four LB
AMP
plates (LB broth (Lennox), 1.8% BactoTM Agar (Difco), 100 mg/L Ampicillin).
35 Individual clones harboring the correct expression construct for zvegf4
are identified by restriction digest to verify the presence of the zvegf4
insert and to
confirm that the various DNA sequences have been joined correctly to one
another.
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The inserts of positive clones are subjected to sequence analysis. Larger
scale plasmid
DNA is isolated using a commercially available kit (QIAGEN Plasmid Maxi Kit,
Qiagen, Valencia, CA) according to manufacturer's instructions. The correct
construct
is designated zvegf4/pZMP6.
Example 5
CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet. 12:555-566,
1986) are plated in 10-cm tissue culture dishes and allowed to grow to
approximately
50% to 70% confluency overnight at 37 C, 5% CO2, in Ham's F12/FBS media (Ham's
F12 medium, Life Technologies), 5% fetal bovine serum (Hyclone, Logan, UT), 1%
L-
glutamine (JRH Biosciences, Lenexa, KS), 1 % sodium pyruvate (Life
Technologies).
The cells are then transfected with the plasmid zvegf4/pZMP6 by liposome-
mediated
transfection using a 3:1 (w/w) liposome formulation of the polycationic lipid
2,3-
dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- l -propaniminium-
trifluoroacetate and the neutral lipid dioleoyl phosphatidylethanolamine in
membrane-
filtered water (LipofectamineTM Reagent, Life Technologies), in serum free
(SF) media
formulation (Ham's F12, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin,
1% L-
glutamine and 1% sodium pyruvate). Zvegf4/pZMP6 is diluted into 15-m1 tubes to
a
total final volume of 640 l with SF media. 35 l of LipofectamineTM is mixed
with
605 l of SF medium. The LipofectamineTM mixture is added to the DNA mixture
and
allowed to incubate approximately 30 minutes at room temperature. Five ml of
SF
media is added to the DNA:LipofectamineTM mixture. The cells are rinsed once
with 5
ml of SF media, aspirated, and the DNA:LipofectamineTM mixture is added. The
cells
are incubated at 37 C for five hours, then 6.4 ml of Ham's F12/10% FBS, 1% PSN
media is added to each plate. The plates are incubated at 37 C overnight, and
the
DNA:LipofectamineTM mixture is replaced with fresh 5% FBS/Ham's media the next
day. On day 3 post-transfection, the cells are split into T-175 flasks in
growth medium.
On day 7 post-transfection, the cells are stained with FITC-anti-CD8
monoclonal
antibody (Pharmingen, San Diego, CA) followed by anti-FITC-conjugated magnetic
beads (Miltenyi Biotec, Auburn, CA). The CD8-positive cells are separated
using
commercially available columns (MiniMACS Separation Unit; Miltenyi Biotec)
according to the manufacturer's directions and put into DMEM/Ham's F12/5% FBS
without nucleosides but with 50 nM methotrexate (selection medium).
Cells are plated for subcloning at a density of 0.5, 1 and 5 cells per well
in 96-well dishes in selection medium and allowed to grow out for
approximately two
weeks. The wells are checked for evaporation of medium and brought back to 200
it
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per well as necessary during this process. When a large percentage of the
colonies in the
plate are near confluency, 100 l of medium is collected from each well for
analysis by
dot blot, and the cells are fed with fresh selection medium. The supernatant
is applied to
a nitrocellulose filter in a dot blot apparatus, and the filter is treated at
100 C in a
vacuum oven to denature the protein. The filter is incubated in 625 mM Tris-
glycine,
pH 9.1, 5mM (3-mercaptoethanol, at 65 C, 10 minutes, then in 2.5% non-fat dry
milk
Western A Buffer (0.25% gelatin, 50 mM Tris-HC1 pH 7.4, 150 mM NaCl, 5 mm
EDTA, 0.05% Igepal CA-630) overnight at 4 C on a rotating shaker. The filter
is
incubated with the anti-CD8 antibody-HRP conjugate in 2.5% non-fat dry milk
Western
A buffer for 1 hour at room temperature on a rotating shaker. The filter is
then washed
three times at room temperature in PBS plus 0.01% Tween 20, 15 minutes per
wash.
The filter is developed with chemiluminescence reagents (ECLTM direct labeling
kit;
Amersham Corp., Arlington Heights, IL) according to the manufacturer's
directions and
TM' TM'
exposed to film (Hyperfilm ECL, Amersham) for approximately 5 minutes.
Positive
clones are trypsinized from the 96-well dish and transferred to 6-well dishes
in selection
medium for scaleup and analysis by Western blot.
Example 6
The protein coding region of zvegf4 is amplified by PCR using primers
that add FseI and AscI restriction sites at the 5' and 3' termini,
respectively. PCR
primers are used with a template containing the full-length zvegf4 cDNA in a
PCR
reaction as follows: one cycle at 95 C for 5 minutes; followed by 15 cycles at
95 C for
1 min., 58 C for 1 min., and 72 C for 1.5 min.; followed by 72 C for 7 min.;
followed
by a 4 C soak. The PCR reaction product is loaded onto a 1.2 % (low melt)
(SeaPlaque
GTGTM; FMC, Rockland, ME) gel in TAE buffer. The zvegf4 PCR product is excised
from the gel and purified using a spin column containing a silica gel membrane
(QIAquickTM Gel Extraction Kit; Qiagen, Inc., Valencia, CA) as per kit
instructions.
The PCR product is then digested, phenol/chloroform extracted, EtOH
precipitated, and
rehydrated in 20m1 TE (Tris/EDTA pH 8). The zvegf4 fragment is then ligated
into the
cloning sites of the transgenic vector pHB12-8 and transformed into E. coli
host cells
(Electromax DH 10BTM cells; obtained from Life Technologies, Inc.,
Gaithersburg, MD)
by electroporation. Clones containing zvegf4 DNA are identified by restriction
analysis. A positive clone is confirmed by direct sequencing.
The zvegf4 cDNA is released from the pTG12-8 vector using Fsel and
3 5 Ascl enzymes. The cDNA is isolated on a 1 % low melt agarose gel, and is
then excised
from the gel. The gel slice is melted at 70 C, extracted twice with an equal
volume of
Tris buffered phenol, and EtOH precipitated. The DNA is resuspended in 10 l
H2O.
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The zvegf4 cDNA is cloned into the Fsel-Ascl sites of a modified
pAdTrack CMV (He et al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998). This
construct contains a GFP marker gene. The CMV promoter driving GFP expression
has been replaced with the SV40 promoter, and the SV40 polyadenylation signal
has
been replaced with the human growth hormone polyadenylation signal. In
addition, the
native polylinker has been replaced with Fsel, EcoRV, and AscI sites. This
modified
form of pAdTrack CMV was named pZyTrack. Ligation is performed using a DNA
ligation and screening kit (Fast-LinkTM; Epicentre Technologies, Madison, WI).
In
order to linearize the plasmid, approximately 5 g of the pZyTrack zvegf4
plasmid is
digested with Pmel. Approximately 1 g of the linearized plasmid is
cotransformed
with 200ng of supercoiled pAdEasy (He et al., ibid.) into BJ5183 cells. The co-
transformation is done using a Bio-Rad Gene Pulser at 2.5kV, 200 ohms and 25
F.
The entire co-transformation is plated on 4 LB plates containing 25 g/ml
kanamycin.
The smallest colonies are picked and expanded in LB/kanamycin, and recombinant
adenovirus DNA identified by standard DNA miniprep procedures. Digestion of
the
recombinant adenovirus DNA with FseI-AscI confirms the presence of zvegf4 DNA.
The recombinant adenovirus miniprep DNA is transformed into E. coli DH l OB
competent cells, and DNA is prepared therefrom.
Approximately 5 g of recombinant adenoviral DNA is digested with
PacI enzyme (New England Biolabs) for 3 hours at 37 C in a reaction volume of
100 l
containing 20-30U of Pacl. The digested DNA is extracted twice with an equal
volume
of phenol/chloroform and precipitated with ethanol. The DNA pellet is
resuspended in
10 l distilled water. A T25 flask of QBI-293A cells (Quantum Biotechnologies,
Inc.,
Montreal, Canada), inoculated the day before and grown to 60-70% confluence,
are
transfected with the PacI digested DNA. The PacI-digested DNA is diluted up to
a total
volume of 50 gl with sterile HBS (150 mM NaCl, 20 mM HEPES). In a separate
tube,
20 gl of lmg/ml N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium
methylsulfate (DOTAP; Boehringer Mannheim) is diluted to a total volume of 100
l
with HBS. The DNA is added to the DOTAP, mixed gently by pipeting up and down,
and left at room temperature for 15 minutes. The media is removed from the
293A
cells and washed with 5 ml serum-free MEM-alpha (Life Technologies,
Gaithersburg,
MD) containing 1 mM sodium pyruvate (Life Technologies), 0.1 mM MEM non-
essential amino acids (Life Technologies) and 25 mM HEPES buffer (Life
Technologies). 5 ml of serum-free MEM is added to the 293A cells and held at
37 C.
The DNA/lipid mixture is added drop-wise to the T25 flask of 293A cells, mixed
gently, and incubated at 37 C for 4 hours. After 4 hours the media containing
the
DNA/lipid mixture is aspirated off and replaced with 5 ml complete MEM
containing
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5% fetal bovine serum. The transfected cells are monitored for Green
Fluorescent
Protein (GFP) expression and formation of foci (viral plaques).
Seven days after transfection of 293A cells with the recombinant
adenoviral DNA, the cells expressing the GFP protein start to form foci. These
foci are
viral "plaques" and the crude viral lysate is collected by using a cell
scraper to collect
all of the 293A cells. The lysate is transferred to a 50m1 conical tube. To
release most
of the virus particles from the cells, three freeze/thaw cycles are done in a
dry
ice/ethanol bath and a 37 C waterbath.
Ten 10-cm plates of nearly confluent (80-90%) 293A cells are set up 20
hours prior to infection. The crude lysate is amplified (primary
amplification) to obtain
a working "stock" of zvegf4 rAdV lysate. 200 ml of crude rAdV lysate is added
to each
10-cm plate, and the plates are monitored for 48 to 72 hours looking for
cytopathic
effect (CPE) under the white light microscope and expression of GFP under the
fluorescent microscope. When all of the 293A cells show CPE, this 1 stock
lysate is
collected, and freeze/thaw cycles performed as described above.
Secondary (2 ) amplification of zvegf4 rAdV is obtained from twenty
15-cm tissue culture dishes of 80-90% confluent 293A cells. All but 20 ml of
5%
MEM media is removed, and each dish is inoculated with 300-500 ml of 1
amplified
rAdv lysate. After 48 hours the 293A cells are lysed from virus production,
the lysate is
collected into 250 ml polypropylene centrifuge bottles, and the rAdV is
purified.
NP-40 detergent is added to a final concentration of 0.5% to the bottles
of crude lysate to lyse all cells. Bottles are placed on a rotating platform
for 10 minutes
and agitated as fast as possible. The debris is pelleted by centrifugation at
20,000 X G
for 15 minutes. The supernatant is transferred to 250-m1 polycarbonate
centrifuge
bottles, and 0.5 volume of 20% PEG8000/2.5M NaCl solution is added. The
bottles are
shaken overnight on ice. The bottles are centrifuged at 20,000 X G for 15
minutes, and
the supernatants are discarded into a bleach solution. A white precipitate
(precipitated
virus/PEG) forms in two vertical lines along the walls of the bottles on
either side of the
spin mark. Using a sterile cell scraper, the precipitate from 2 bottles is
resuspended in
2.5 ml PBS. The virus solution is placed in 2-ml microcentrifuge tubes and
centrifuged
at 14,000 X G in a microcentrifuge for 10 minutes to remove any additional
cell debris.
The supernatants from the 2-ml microcentrifuge tubes are 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 is estimated, and 0.55 g/ml of CsCI added. The
CsCI is
dissolved, and 1 ml of this solution weighed. The solution is transferred to
polycarbonate, thick-walled, 3.2 ml centrifuge tubes (Beckman) and spun at
348,000 X
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G for 3-4 hours at 25 C. The virus forms a white band. Using wide-bore pipette
tips,
the virus band is collected.
The virus from the gradient will have a large amount of CsCI, which
must be removed before it can be used on cells. Pharmacia PD-10 columns
prepacked
5 with Sephadex G-25M (Pharmacia) are used to desalt the virus preparation.
The
column is equilibrated with 20 ml of PBS. The virus is loaded and allowed to
run into
the column. 5 ml of PBS is added to the column, and fractions of 8-10 drops
collected.
The optical density of 1:50 dilutions of each fraction is determined at 260 nm
on a
spectrophotometer, and a clear absorbance peak is identified. These fractions
are
10 pooled, and the optical density (OD) of a 1:25 dilution is determined. OD
is converted
into virus concentration using the formula (OD at 260nm)(25)(1.1 x 1012) =
virions/ml.
To store the virus, glycerol is added to the purified virus to a final
concentration of 15%, mixed gently and stored in aliquots at -80 C.
A protocol developed by Quantum Biotechnologies, Inc. (Montreal,
15 Canada) is followed to measure recombinant virus infectivity. Briefly, two
96-well
tissue culture plates are seeded with 1 x 104 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 x 10-22 to 1 x 10-14 are made in MEM containing
2% fetal
bovine serum. l00 1 of each dilution is placed in each of 20 wells. After 5
days at
20 37 C, wells are read either positive or negative for CPE and PFU/ml is
calculated.
TCID50 formulation used is as per Quantum Biotechnologies, Inc.,
above. The titer (T) is determined from a plate where virus used is diluted
from 10-2 to
10-14, and read 5 days after the infection. At each dilution a ratio (R) of
positive wells
for CPE per the total number of wells is determined. The titer of the
undiluted sample
25 is T = 101+F= TCID50/ml, where F = l+d(S-0.5), S is the sum of the ratios
(R), and d
is Loglo of the dilution series (e.g., d = 1 for a ten-fold dilution series).
To convert
TCID50/ml to pfu/ml, 0.7 is subtracted from the exponent in the calculation
for titer (T).
Example 7
30 Recombinant zvegf4 having a carboxyl-terminal Glu-Glu affinity tag
was produced in a baculovirus expression system according to conventional
methods.
The culture was harvested, and the cells were lysed with a solution of 0.02 M
Tris-HCI,
pH 8.3, 1 mM EDTA, 1 mM DTT, 1 mM 4-(2-Aminoethyl)-benzenesulfonyl fluoride
hydrochloride (Pefabloc SC; Boehringer-Mannheim), 0.5 M aprotinin, 4 mM
35 leupeptin, 4 mM E-64, 1 % NP-40 at 4 C for 15 minutes on a rotator. The
solution was
centrifuged, and the supernatant was recovered. Twenty ml of extract was
combined
with 50 l of anti-Glu-Glu antibody conjugated to Sepharose beads in 50 l
buffer.
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The mixture was incubated on a rotator at 4 C overnight. The beads were
recovered by
centrifugation and washed 3 x 15 minutes at 4 C. Pellets were combined with
sample
buffer containing reducing agent and heated at 98 C for five minutes. The
protein was
analyzed by polyacrylamide gel electrophoresis under reducing conditions
followed by
western blotting on a PVDF membrane using an antibody to the affinity tag. Two
bands were detected, one a M,=49 kD and the other at M,=21 kD. Sequence
analysis
showed the larger band to comprise two sequences, one beginning at Arg-19 of
SEQ ID
NO:2 and the other beginning at Asn-35 of SEQ ID NO:2. The asparagine residue
appeared to have been deamidated to an aspartic acid. The smaller band began
at Ser-
250 of SEQ ID NO:2.
Example 8
The zvegf4 cDNA was cloned into the EcoRV-Ascl sites of a modified
pAdTrack-CMV (He 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-
LinkTM
kit; Epicentre Technologies, Madison, WI).
Zvegf4 was assayed in an aortic ring outgrowth assay (Nicosia and
Ottinetti, ibid.; Villaschi and Nicosia, ibid.). Thoracic aortas were isolated
from 1-2
month old SD male rats and transferred to petri dishes containing HANK's
buffered salt
solution. The aortas were flushed with additional HANK's buffered salt
solution to
remove blood, and adventitial tissue surrounding the aorta was carefully
removed.
Cleaned aortas were transferred to petri dishes containing EBM basal media,
serum free
(Clonetics, San Diego, CA). Aortic rings were obtained by slicing
approximately 1-mm
sections using a scalpel blade. The ends of the aortas used to hold the aorta
in place
were not used. The rings were rinsed in fresh EBM basal media and placed
individually
in a wells of a 24-well plate coated with basement membrane matrix (Matrigel ;
Becton Dickinson, Franklin Lakes, NJ). The rings were overlayed with an
additional 50
l of the matrix solution and placed at 37 C for 30 minutes to allow the matrix
to gel.
Test samples were diluted in EBM basal serum-free media supplemented with 100
units/ml penicillin, 100 g/ml streptomycin and HEPES buffer and added at 1
ml/well.
Background control was EBM basal serum-free media alone. Basic FGF (R&D
Systems, Minneapolis, MN) at 20 ng/ml was used as a positive control. Zvegf4
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adenovirus was added to wells, assuming a cell count of 500,000 cells and a
multiplicity of infection of 5000 particles/cell. A null adenovirus
(designated "zPar")
was used as a control. Samples were added in a minimum of quadruplets. Rings
were
incubated for 5-7 days at 37 C and analyzed for growth. Aortic outgrowth was
scored
by multiple, blinded observers using 0 as no growth and 4 as maximum growth.
Zvegf4
adenovirus produced a significant increase in outgrowth, comparable to the
most potent
control (bFGF).
Example 9
Polyclonal anti-peptide antibodies were prepared by immunizing 2
female New Zealand white rabbits with the peptides huzvegf4-1
(CGHKEVPPRIKSRTNQIK; SEQ ID NO:39), huzvegf4-2
(ESWQEDLENMYLDTPRYRGRSYHDC; SEQ ID NO:40), or huzvegf4-3
(CFEPGHIKRRGRAKTMALVDIQLD; SEQ ID NO:41). The peptides were
synthesized using an Applied Biosystems Model 431A peptide synthesizer
(Applied
Biosystems, Inc., Foster City, CA) according to the manufacturer's
instructions. The
peptides were conjugated to keyhole limpet hemocyanin (KLH) with maleimide
activation. The rabbits were each given an initial intraperitoneal (ip)
injection of 200
tg of peptide in Complete Freund's Adjuvant followed by booster ip injections
of 100
g peptide in Incomplete Freund's Adjuvant every three weeks. Seven to ten days
after
the administration of the second booster injection (3 total injections), the
animals were
bled, and the sea were collected. The animals were then boosted and bled every
three
weeks.
The zvegf4 peptide-specific rabbit sera were characterized by an ELISA
titer check using 1 tg/ml of the peptide used to make the antibody as an
antibody target.
The 2 rabbit sera to the huzvegf4-1 peptide had titer to their specific
peptide at a
dilution of 1:5,000,000. The 2 rabbit sera to the huzvegf4-2 peptide had titer
to their
specific peptide at a dilution of 1:5,000,000. The 2 rabbit seras to the
huzvegf4-3
peptide had titer to their specific peptide at a dilution of 1:500,000.
The zvegf4 peptide-specific polyclonal antibodies were affinity purified
from the sera using CNBr-SEPHAROSE 4B protein columns (Pharmacia LKB) that
were prepared using 10 mg of the specific peptide per gram CNBr-SEPHAROSE,
followed by 20X dialysis in PBS overnight. Zvegf4-specific antibodies were
characterized by an ELISA titer check using 1 g/ml of the appropriate peptide
antigens
as antibody targets. The lower limit of detection (LLD) of the anti-huzvegf4-1
affinity
purified antibody on its specific antigen (huzvegf4-1 peptide) was a dilution
of 0.1
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pg/ml. The LLD of the anti-huzvegf4-2 affinity purified antibody on its
specific antigen
(huzveg4-2 peptide) was a dilution of 5 ng/ml. The LLD of the rabbit anti-
huzvegf4-3
affinity purified antibody on its specific antigen (huzvegf4-3 peptide) was a
dilution of
ng/ml.
5
Example 10
Recombinant carboxyl-terminal Glu-Glu tagged zvegf4 (zvegf4-cee)
was produced from recombinant baculovirus-infected insect cells. Two-liter
cultures
were harvested, and the media were sterile-filtered using a 0.2 tm filter.
Protein was purified from the conditioned media by a combination of
anti-Glu-Glu (anti-EE) peptide antibody affinity chromatography and S-200 gel
exclusion chromatography. Culture media (pH 6.0, conductivity 7 mS) was
directly
loaded onto a 20 x 80 mm (25-m1 bed volume) anti-EE antibody affinity column
at a
flow of 6 ml/minute. The column was washed with ten column volumes of PBS,
then
bound protein was eluted with two column volumes of 0.4 mg/ml EYMPTD peptide
(SEQ ID NO:42) (Princeton BioMolecules Corp., Langhorne, PA). Five-ml
fractions
were collected. Samples from the anti-EE antibody affinity column were
analyzed by
SDS-PAGE with silver staining and western blotting (as disclosed below) for
the
presence of zvegf4-cee. Zvefg4-cee-containing fractions were pooled and
concentrated
to 3.8 ml by filtration using a Biomax- -5 concentrator (Millipore Corp.,
Bedford,
MA), and loaded onto a 16 x 1000 mm gel filtration column (SephacrylTM S-200
HR;
Amersham Pharmacia Biotech, Piscataway, NJ). The fractions containing purified
zvegf4-cee were pooled, filtered through a 0.2 m filter, aliquoted into 100
l each, and
frozen at -80 C. The concentration of the final purified protein was
determined by
colorimetric assay (BCA assay reagents; Pierce, Rockford, IL) and HPLC-amino
acid
analysis.
Recombinant zvegf4-cee was analyzed by SDS-PAGE (NuPAGETM 4-
12% gel; Novex, San Diego, CA) with silver staining (FASTsilverh, Geno
Technology,
Inc., Maplewood, MO) and Western blotting using antibodies to the huzvegf4-1,
huzvegf4-2, and huzvefg4-3 peptides, and anti-EE antibody. Either the
conditioned
media or purified protein was electrophoresed using an electrophoresis mini-
cell (XCell
IITM mini-cell; Novex, San Diego, CA) and transferred to nitrocellulose (0.2
.tm; Bio-
Rad Laboratories, Hercules, CA) at room temperature using an XCell IITM blot
module
(Novex) with stirring according to directions provided in the instrument
manual. The
3S transfer was run at 500 mA for one hour in a buffer containing 25 mM Tris
base, 200
mM glycine, and 20% methanol. The filters were then blocked with 10% non-fat
dry
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milk in PBS for 10 minutes at room temperature. The nitrocellulose was quickly
rinsed, then primary antibody was added in PBS containing 2.5% non-fat dry
milk. The
blots were incubated for two hours at room temperature or overnight at 4 C
with gentle
shaking. Following the incubation, blots were washed three times for 10
minutes each
in PBS. Secondary antibody (goat anti-rabbit IgG conjugated to horseradish
peroxidase; obtained from Rockland Inc., Gilbertsville, PA) diluted 1:2000 in
PBS
containing 2.5% non-fat dry milk was added, and the blots were incubated for
two
hours at room temperature with gentle shaking. The blots were then washed
three
times, 10 minutes each, in PBS, then quickly rinsed in H2O. The blots were
developed
using commercially available chemiluminescent substrate reagents (SuperSignal
ULTRA reagents 1 and 2 mixed 1:1; reagents obtained from Pierce), and the
signal was
captured using image analysis software (Lumi-Imager Lumi Analyst 3.0; Roche
Molecular Biochemicals, Indianapolis, IN) for times ranging from 10 seconds to
5
minutes or as necessary.
The purified zvefg4-cee appeared as a single band at about 85 kDa under
non-reducing conditions with silver staining, but at about 50 kDa under
reducing
conditions, suggesting a dimeric form of zvefg4-cee under non-reducing
conditions.
Using either 4-1, 4-3 or anti-EE antibody, the purified zvegf4-cee
showed the same result as silver staining gel; the 4-3 antibody gave a much
weaker
signal. However, in addition to recognizing the 85-kDa band under non-reducing
conditions and the 50-kDa band under reducing conditions, the 4-2 antibody
recognized two bands at 35 kDa and 32 kDa under non-reducing conditions, and
two
bands at 38 kDa and 35 kDa under reducing conditions. While not wishing to be
bound
by theory, the smaller bands are likely to be cleaved forms of zvefg4-cee
missing the N-
terminal portion of the protein that is recognized by the 4-1 antibody.
Example 11
Recombinant zvegf4 was analyzed for mitogenic activity on rat liver
stellate cells (obtained from N. Fausto, University of Washington), human
aortic
smooth muscle cells (Clonetics Corp., Walkersville, MD), human retinal
pericytes
(Clonetics Corp.) and human hepatic fibroblasts (Clonetics Corp.). Test
samples
consisted of conditioned media (CM) from adenovirally infected HaCaT human
keratinocyte cells (Boukamp et al., J. Cell. Biol. 106:761-771, 1988; Skobe
and
Fusenig, Proc. Natl. Acad. Sci. USA 95:1050-1055, 1998; obtained from Dr.
Norbert E.
Fusenig, Deutsches Krebsforschungszentrum, Heidelberg, Germany) expressing
full
length zvegf-4. Control CM was generated from HaCaT cells infected with a
parental
GFP-expressing adenovirus (zPar). The CM were concentrated 10-fold using a 15
ml
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centrifugal filter device with a 1OK membrane filter (Ultrafree ; Millipore
Corp.,
Bedford, MA), then diluted back to lx with ITS media (serum-free DMEM/Ham's F-
12
medium containing 5 g/ml insulin, 20 tg/ml transferrin, and 16 pg/ml
selenium).
Cells were plated at a density of 2,000 cells/well in 96-well culture plates
and grown for
5 approximately 72 hours in DMEM containing 10% fetal calf serum at 37 C.
Cells were
quiesced by incubating them for approximately 20 hours in serum-free
DMEM/Ham's
F-12 medium containing insulin (5 [,g/ml), transferrin (20 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. For measurement of [3H]thymidine
incorporation,
10 20 l of a 50 tCi/ml stock in DMEM was added directly to the cells, for a
final activity
of 1 [Xi/well. After another 24-hour incubation, 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 (FilterMateTM harvester; Packard
Instrument Co.,
Meriden, CT). The plates were then dried at 65 C for 15 minutes, sealed after
adding
15 40 l/well scintillation cocktail (MicroscintTM 0; Packard Instrument Co.)
and counted
on a microplate scintillation counter (Topcount ; Packard Instrument Co.).
Results,
presented in Table 8, demonstrated that zvegf4 CM had approximately 7-fold
higher
mitogenic activity than control CM on pericytes cells and approximately a 1.5 -
2.4-fold
higher mitogenic activity on the other cell types tested.
Table 8
Sample CPM incorporated
Zvegf4 (lx CM) zPar (1xCM )
Mean St. dev. Mean St. dev.
Human retinal pericytes 3621 223 523 306
Human hepatic fibroblasts 7757 753 3232 264
Human aortic SMC 2009 37 1263 51
Rat liver stellate cells 34707 1411 14413 1939
Example 12
Recombinant, C terminal glu-glu tagged zvegf4 was analyzed for
mitogenic activity on human aortic smooth muscle cells (HAoSMC) (Clonetics),
human
retinal pericytes (Clonetics) and human aortic adventitial fibroblasts (AoAF)
(Clonetics). 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 C. Cells were quiesced by incubating them for 20 hours in ITS medium. At
the
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time of the assay, the medium was removed, and test samples were added to the
wells
in triplicate. Test samples consisted of purified, full-length, tagged zvegf4
expressed in
baculovirus-infected cells. Purified protein in a buffer containing 0.1% BSA
was
serially diluted into ITS medium at concentrations of 1 tg/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 zvegf4 protein and added to the plate. For measurement of
[3H]thymidine incorporation, 20 t1 of a 50 Ci/ml stock in DMEM was added
directly
to the cells, for a final activity of 1 Ci/well. After another 24-hour
incubation,
mitogenic activity was assessed by measuring the uptake of [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
(FilterMateTM
harvester; Packard Instrument Co., Meriden, CT). The plates were then dried at
65 C
for 15 minutes, sealed after adding 40 tl/well scintillation cocktail
(MicroscintTM 0;
Packard Instrument Co.) and counted on a microplate scintillation counter
(Topcount ;
Packard Instrument Co.). Results, presented in Table 9, demonstrated that 80
ng/ml
zvegf4 had approximately 1.7-fold higher mitogenic activity on pericytes, 3.2-
fold
higher activity on aortic SMCs, and 2.6-fold higher activity on aortic
fibroblasts as
compared to the buffer control.
Table 9
Sample CPM Incorporated
Pericytes HAoSMC AoAF
Mean St. dev. Mean St. dev. Mean St. dev.
Zvegf4, 80 96.7 18.2 488.7 29.6 177.0 1.0
ng/ml
Zvegf4, 20 81.7 11.7 211.7 50.8 107.7 20.1
ng/ml
Zvegf4, 5 67.3 6.7 191.7 4.5 123.7 10.5
ng/ml
Buffer control 58.7 8.5 152.3 40.1 68.7 8.3
Example 13
Mice (C57BL6) were infected with a zvegf4-encoding adenovirus vector
(AdZyvegf4) to determine the effects on serum chemistry, complete blood counts
(CBC), body and organ weight changes, and histology. On day -1, the mice were
tagged, individually weighed, and group normalized for separation into
treatment
groups (4 mice per cage). Group 1 mice (n=8 females, 7 males) received GFP
(control)
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77
adenovirus, 1 x 101 l particles. Group 2 mice (n=8 females, 6 males) received
zvegf4
adenovirus, 1 x 101 1 particles. Group 3 mice (n=8 females, 8 males) were
untreated
controls. On day 0, the mice received injections of the appropriate adenovirus
solution.
On day 10, blood was collected (under ether anesthesia) for CBCs and clinical
chemistry measurements. On day 20, mice were weighed and sacrificed by
cervical
dislocation after collecting blood (under ether anesthesia) for CBCs and
clinical
chemistry measurements. Tissues were collected for histopathology.
Observations
were as follows:
Serum chemistry changes: AdZyvegf4 treated mice were hypoglycemic. This effect
increased in magnitude over time (day 10 vs. day 20). Serum cholesterol levels
were significantly increased (2-fold) at both time points. Serum levels of
albumin and the enzymes ALT, AST and alkaline phosphatase were all
significantly increased in AdZyvegf4 treated mice. Serum calcium and total
bilirubin were also significantly increased, and became more elevated over
time.
CBC changes: AdZyvegf4-treated mice had significantly higher lymphocyte count
at
both time points (mean >10). Platelet counts were significantly lower at day
20.
Red blood cell count was significantly higher in females at day 10,
significantly
higher in males at day 20.
Body/organ weights: AdZyvegf4-treated males lost weight over the course of the
experiment. This result was significantly different than control animals,
which
gained weight. There was no difference among the female mice; all groups
gained similar weight. Spleen weight was significantly greater (approximately
4-fold) in all AdZyvegf4-treated mice. Liver weight was also significantly
greater in all AdZyvegf4-treated mice. There was no significant difference in
kidney weight between groups.
Histology: In the liver, proliferation of sinusoidal endothelial cells was
observed. In
the spleen, proliferation of reticuloendothelial cells was observed. In the
kidney, proliferative glomerulopathy was observed. While not wishing to be
bound by theory, this glomerulopathy may have been due to proliferation of
capillary endothelial cells. In the femurs, there was proliferation of
endosteal
bone (mostly in trabecular bone), which in some cases replaced most of the
bone marrow. Proliferation of stromal cells was also observed in bone. In the
lung, there was increased frequency of brochoaveolar lymphoid tissue.
Example 14
90 pg of recombinant zvegf4 protein was dissolved in 500 l PBS
containing 2 mCi Na- 1251 (Amersham Corp.). One derivatized, nonporous
polystyrene
CA 02370948 2007-05-02
78
bead (IODO-Beads ; Pierce, Rockford, IL) was added, and the reaction mixture
was
incubated one minute on ice. The iodinated protein was separated from
unincorporated
1251 by gel filtration using an elution buffer of 10% acetic acid, 150 mM
NaCl, and
0.25% gelatin. The active fraction contained 29 g/m1 1251-zvegf4 with a
specific
activity of 3.0 X 104 cpm/ng.
The following cell lines were plated into the wells of a 24-well tissue
culture dish and cultured in growth medium for three days:
1. Human retinal pericytes, passage 6 (pericytes).
2. Rat stellate cells, passage 8.
3. Human umbilical vein endothelial cells, passage 4 (HUVEC).
4. Human aortic adventicial fibroblasts, passage 5 (AoAF).
5. Human aortic smooth muscle cells, passage 2 (AoSMC).
Cells were washed once with ice-cold binding buffer (HAM'S F-12 containing 2.5
mg/ml BSA, 20 mM HEPES, pH 7.2), then 250 l of the following solutions was
added
to each of three wells of the culture dishes containing the test cells.
Binding solutions
were prepared in 5 mL of binding buffer with 250 pM 125I-zvegf4 and:
1. No addition.
2. 25 nM zvegf4.
3. 25 nM zvegf3.
4. 25 nM PDGF-AA.
5. 25 nM PDGF-BB
The reaction mixtures were incubated on ice for 2 hours, then washed three
times with
one ml of ice-cold binding buffer. The bound 1251-zvegf4 was quantitated by
gamma
counting a Triton-X 100 extract of the cells.
I'A
Results, shown in Table 10, indicate binding of zvegf4 to pericytes,
stellate cells, AoAF, and AoSMC, but not to HUVEC. The first column represents
total
CPM 125I-zvegf4 bound/well. The second column is 125I-zvegf4 bound/well when
blocked with cold ligand. The difference between the two numbers represents
specific
binding.
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79
Table 10
1251-zvegf4 Bound (CPM) 1251-zvegf4 Bound w/cold
Cell Type zvegf4 (CPM)
1. Pericytes 3083 +/- 864 623 +/- 60
2. Stellate Cells 2131 +/- 450 413 +/- 164
3. HUVEC 485 +/- 91 227 +/- 13
4. AoAF 1544+/-131 300 +/- 15
5. AoSMC 1628 +/- 203 440 +/- 46
Example 15
The zvegf4 gene was mapped to human chromosome 11 using the
commercially available version of the Stanford G3 Radiation Hybrid Mapping
Panel
(Research Genetics, Inc., Huntsville, AL). This panel contains PCRable DNAs
from
each of 83 radiation hybrid clones of the whole human genome, plus two control
DNAs
(the RM donor and the A3 recipient). A publicly available WWW server
(http://shgc-
www.stanford.edu) allows chromosomal localization of markers. 20- l reaction
mixtures were set up in a PCRable 96-well microtiter plate (Stratagene, La
Jolla, CA)
and used in a thermal cycler (RoboCycler Gradient 96; Stratagene). Each of
the 85
PCR mixtures consisted of 2 l buffer (l0X KlenTaq PCR reaction buffer,
Clontech
Laboratories, Inc., Palo Alto, CA), 1.6 l dNTPs mix (2.5 mM each, PERKIN-
ELMER,
Foster City, CA), 1 l sense primer, ZC22,685 (SEQ ID NO:37), 1 l antisense
primer,
ZC22,686 (SEQ ID NO:38), 2 l of a density increasing agent and tracking dye
(RediLoad, Research Genetics, Inc., Huntsville, AL), 0.4 l of a commercially
available
DNA polymerase/antibody mix (50X AdvantageTM KlenTaq Polymerase Mix, obtained
from Clontech Laboratories, Inc., Palo Alto, CA), 25 ng of DNA from an
individual
hybrid clone or control, and x it ddH2O for a total volume of 20 l. The
reaction
mixtures were overlaid with an equal amount of mineral oil and sealed. The PCR
cycler conditions were an initial 5-minute denaturation at 94 C; 35 cycles of
45 seconds
denaturation at 94 C, 45 seconds annealing at 64 C. and 75 seconds extension
at 72 C;
followed by a final extension for 7 minutes at 72 C. The reaction products
were
separated by electrophoresis on a 2% agarose gel. The results showed linkage
of zvegf4
2S to the human chromosome 11 framework marker SHGC-34226 with a LOD score of
14.90 and at a distance of 0 cR_10000 from the marker. The use of surrounding
genes/markers positions Zvegf4 in the 11g22.3-g23.1 chromosomal region.
t'U I /U500/4004
ui-i -euuu
CA 02370948 2001-10-16
Examtule 16
The structure of recombinant zvcgf4 was analyzed by Western blotting
using conventional techniques. Protein produced in the HaCaT human
keratinocyte cell
line was electrophoresed under reducing and non reducing conditions,
transferred. to
5 filters, and probed with antibodies to the kftdomain and CUB domain regions
of the
protein. Reduced protein appeared as a single baud having an apparent M of
approximately 53 kD, consistent with a glycosylated, monomeric protein, Non-
reduced
protein appeared as a single band having an apparent M< of approximately 85
kD,
consistent with a disulfide-linked dimer.
Exam a 17
An expression plasmid containing full length zvegf4 was amstuucted,
using the expression vector pEZE2, pEZE2 is derived from pDC312 by the
addition of
additional restriction enzyme recognition sites to the multiple cloning site.
pDC312
and pEZE2 contain an EASE segment, as described in WO 97/25420, which can
improve expression of recombinant proteins two to eight fold in saanunalian
cells,
preferably Chinese Hamster Ovary (CIO) cells. The pEZE2 expression unit
contains
the CMV enhancer/promoter, the adenovirus tripartite leader sequence, a
multiple
cloning site for insertion of the coding region for the recombinant protein,
an
enoephalomyocarditis virus internal ribosome entry site, a coding segment for
mouse
dihydrofolate reductase, and the SV40 transcription terminator. In addition,
pBZE2
contains an . colt origin of replication and a bacterial beta-lactamase gene,
A zvegf4 DNA fragment was generated by PCR (Adv'antage2 PCR. Kit,
Clontech, Palo Alto, CA) with 5' FseI and 3' Ascl sites for direct cloning
into the
expression vector. The 5' primer contained an Fsel site, Kozak sequence, and
the first
21 basepa.irs of the native leader sequence for zvegf4 (ZC26,136; SEQ ID
NO:43). The
3' primer contained the last 21 basepalrs of zvegf4, a stop codcm, and an Ascl
site
(ZC26,137; SEQ II) NO:44). The PCR reaction included 1 pL of template (ESTEP
plasmid zvegf4porl1#3) and was run as follows; 94 C, 1 minute, I cycle; then
25 cycles
of 94 C, 30 seconds; 55 C, 30 seconds; 6811C, 1 minute; then a final extension
cycle of
72 C for 7 minutes.
The ESTEP plasmid zvegf4pcrf#3 contains the full-length humane
zvegf4 fragment. This fragment was generated by PCIt using 20 pm each of
ZC22,341
(SEQ ID NO:45) and ZC22,342 (SEQ ID NO:46) primers and 3 L of a thyroid
library.
The reaction was run as follows: 94 C, 1 minute, 1 cycle; then 30 cycles of 94
C, 20
seconds; 66 C, 1.5 minutes; then a final extension cycle of 72 C for 5
minutes. The
1,272 bp product was gel purified on a I% TBE gel, and the DNA was extracted
from
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U I - I e--L000
CA 02370948 2001-10-16
81
the gel slab using the QlAquick Gel Extraction Kit (Qiagen, Valencia, CA).
This 1,272
bp fragment was subcloned into pCR2.1 vector (Invitrogen, Carlsbad, CA), and
designated zvegf4pcrfl#3,
The PCR. generated fragment was purified (Qiaquick PCR. clean-up kit,
Qiagen, Valencia, CA) and digested with restriction earymes Awl and Fsel (New
England Biolabs, Beverly, MA) in a single 100 L reaction. Five micrograms of
the
expression vector pEZE2 were also digested with Psel and Ascl in a single 100
iL
reaction. The digested DNA was fractionated by agarose gel electrophoresis and
the
DNA fragments were isolated and purified (Qiaquielc Gel Extraction Kit,
Qiagen).
Five micmliters of the zvegf4 DNA fragment and I L of the pEZE2
vector fragment were ligated overnight at room temperature (New England
Biolabs
High Concentrated Ligase and supplied buffer). One microliter of the ligation
reaction
was added to 25 IsL of eIectsocompetant E. coli stain DH14B (Life
Technologies) in a
0.2 cm cuvette. The mixture was electroparated (BioRad E. cols Pulser) at 2.3
kv. To
the eavette, 1 mL of LB broth was added, and 100 gL of the mix was plated onto
LB/Ampicillin agar plates. The plates were incubated overnight at 379C, and 8
isolated
colonies were picked for DNA mini prep (Qiaquick Mini-Prep Kit, Qiagen).
Individual
clones were screened by PCR for the presence of zvegf4 DNA, using the above.
mentioned primers. DNA sequencing was performed on clones #1-6, to verify the
correct full-length sequence. One clone contained the correct expected
sequence and a
Maxi prep of DNA was made (Qiagen P]asmid Maxi Kit, Qiaggen).
CHO DG44 (Chasm at Si., Som. Cell. Malec. Genet. 12:555-666, 1986)
were plated and allowed to grow to appxoximstely 50% to 70% conflue ncy over
night
at 37 C in MEM alpha media (JRH Biosciences, Lenexa, KS), 7.5% fetal bovine
serum
(Hyclone, Logan, UT), 1% L-glutarnine (Life Technologies),1% sodium pyruvate
(Life
Technologies), 1 % HT solution (Life Technologies), and I% P e nicillin/S
Ireptomycin
(Life Tecbnologies). The cells were then transfected with the plasmid
pEZB2/zvegf4
by liposome-mediated transfection, using a 10;1 (w/w) liposom a formulation of
the
polycationie lipid dioctaldecylawidoglycyl spermine, in serum-free (SF) medium
formulation DMEM/F12 - Life Technologies, Non Essential Amino Acids-Life
Technologies, 1% L-glutaminc, 1% sodium pyruvate. The plesmid pEZE2/zvegf4 was
diluted in a final volume of 500 pL of SF mediutxt in a 15 mL conical tube,
and 20 pL
of Transfeotam (Promega, Madison, WI) reagent was added, mixed well and
incubated
at room temperature for 10 minutes. After incubation, 4.5 ai. of SF medium was
added to the DNA mixture and mixed well using a 5 mL pipette, The cells were
rinsed
3 time with SF medium, and the 5 mL of DNA solution was overlayed upon the
cell
monolayer. The cells were incubated at 37 C, 5% CC2 for 2 hours. Then 6 mL of
AMENDED SHEET
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U I - I G-000U
CA 02370948 2001-10-16
82
complete medium (MBM alpha, 7.5% FBS,1% L-glufamine, 1 % sodium pyruvate, 1%
HT,1 % Pen/Strep) and the cells were incubated for a further 48 hours. After
48 hours,
the cells were tx'ypsinized from the plate with I mL of 0.25% Trypsin/ 1 mM
EDTA
(Life Technologies) and quenched with 4 mL of complete medium without
mvcleosides
S (MEM alpha, 7.5% Dialysed PBS, 1% L-glutamine, 10/* sodium pyruvate, 1%
Pen/Strep). Five hundred microliters of the cell suspension were transferred
to plates
containing 10 mL of complete medium without nucleosides. The cultures were
grown
for 14 days, until single colonies that were approxftnately 0.25 cm in
diameter were
present. Cloning rings (Belico Glass, Inc., Vineland, NJ) were used to isolate
24 single
colonies, which were removed with trypsin, transferred to 6 well cell cluster
plates
(Costar, Corning, NY), and incubated 4 days.
The cell wells were rinsed with SF medium and 2 mL of SF medium
was added, and the culture was incubated for 24 hours. The conditioned SF
medium
was concentrated approximately 20 fold using a 10K centrifuge device
(Millipore
Corporation, Bedford, MA). Twenty--Eve microliters of the concentrate was
added to
15 pL of 4X Sample Buffer (Novex, San .Diego, CA) with 50 mM beta-
Mercaptoethanol, and the mixture was run on it 4-12% NuPAGE gel (Novex). The
proteins from the gel were transferred to nitrocellulose membranes (Novex) and
the
blot was blocked with 100/6 non-fat dry milk in Western A (0.25% gelatin, 50
mM Tris-
HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.05% Igepal CA 630) overnight at room
temperature on a rotating shaker platform. The membrane was rinsed 3 times in
Western A. An antibody to the N-terminus of the zvegf4 protein was diluted at
1:3000
in 50 mL 5% non-fat milk in Western A. The antibody solution was overlayed on
the
. _ membrane and incubated at room temperature on a rocking platform for 1
hour. Alter
the i hour incubation, the solution was discarded and the membrane rinsed 3
tinges with
Western A and once with Western B (50 mM Tris pH 7.4, 5 mM EDTA, 0.05% Igepal
CA-630, I M NaCI, 0,25 % Gelatin). The secondary antibody, an F(ab')2 fragment
of
Donkey-Anti-Rabbit=HRP (Amersham Corp., Arlington Heights, IL), was diluted in
Western A at 1:3000, overlayed on. the membrane, and incubated 1 hour at room
temperature on a rocking platform. The secondary antibody solution was
discarded,
and the membrane was washed 3 tunes in Western A and 3 times in Western B.
Chenuluminescence was used to detect the full-length or protease..digested N-
terminus
of zvegf4 according to the manufacturer's instructions (Pierce, Rockford, IL),
and was
-analysed by LumiAnalyser (Roche(Boebringer Mannheim, Mannheim, Germany).
Four of the 12 clones were positive for zvegf4, and numbers 7 and 12 were
trypsi~nized
and transferred to T175 flasks (Costar, Corning, NY) in complete medi= without
nucleosides.
AMENDED SHEET
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CA 02370948 2001-10-16
83
Exam a 18
An expression construct encoding the growth factor domain of zvegf4 is
prepared, A PCR fragment was generated (Clontech Advantage 2 PCR Kit) that
contained a 5' B amHI restriction site, an N-ter inal BE tag, and zvegf4 amino
acid
S residues 258-381 (stop codon included). The 5' oligo primer contains the Ban
I 1 site,
an N-terminal EE tag sequence, and zvegf4 bascpairs cotresponding to the N-
terminus
of the growth factor domain (ZC27,1 I6; SEQ ID NO:47). The 3' oligo primer
contains
the last 21 basepairs of zvegf4 (stop codon included) (ZC27,137; SEQ ID NO-
49). The
expression vector pZMP20 was used, which contains the CMV , immediate early
promoter, a consensus intron from the variable region of mouse immunoglobulin
heavy
chain locus, Kozak sequences, an optimized t-PA secretory signal sequence
(U.S.
Patent No, 5,641,655), multiple restriction sites for insertion of coding
sequences, a
stop codon, and a human growth hormone terminator. The plasxnid also contains
an
IRES element from poliovirus, the extracellular domain of CD8 truncated at the
C-
terminal end of the transmembrane domain, an E. colt origin of replication, a.
DHFR
gene, the SV40 terminator, and the URA3 and CEN-ARS sequences required for
replication in . cerevisiae. The resulting plasmid is designated
pZMP20/GFD.NEE.
A 504 basepair fragment with a 5' Fsel site and 3' AScI site is isolated from
this
plaemid for ligation into the pEZE2 expression vector (5' Fsel, 3' Ascl) for
expression
in CHO DG44 cells.
Example 19
A. Mouse aenomic library screen
A partial mouse zvegf4 sequence was obtained by probing a mouse
genomic library with a human zvegf4 restriction digest fragment containing the
entire
coding sequence. The probe was generated by digesting 8 g of a full length
human
zvegf4 plasmid with EcoRl (Gibco BRL, Gaithersburg, MD). The 1,289 bp fragment
was gel purified on a 2.3% TEE gel and the cDNA was, extracted from the
agarose slab
using the QlAquick Get Extraction Kit (Qiagen). The mouse genomic library was
an
embl3 SP61T7 lambda BamE1 cloned library (Clontech, Palo Alto, CA) plated on a
I(802 host lawn on 24 NZY plates, and represented 7.2 x 105pfus.
Twenty four filter lifts were prehybridized in EXPRESSITY solution
(Clontech) containing 0.1 mg/Tnl salmon sperm DNA which had been boiled 5
minutes,
then iced. Hybridization took place overnight at 50 C. Sixty three ag of the
human
fragment mentioned above were labeled with 32P using the Rediprime: II Random
Prime Labeling System (An=ham Pharmacia, Buekingbamshire, Engiarul).
AMENDED SHEET
u - i c-cuvu l- I /UJUU/4UU4
CA 02370948 2001-10-16
84
Unincorporated radioactivity was removed using it NuoTYap push column
(Stratagene,
La Jolla, CA). Filters were hybridized in EX?RESSITYB solution containing 1.0
x 106
cpmlml zvegf4 probe, 0.1 mglml salmon sperm DNA, and 0.5 tg/ml rnurine cot-1
DNA which had been boiled 5 minutes, then iced, Hybridization took place
overnight
at 50 C. Filter lifts were washed in 2 x SSC, 0.1% SDS at room temperature for
2
hours, then the temperature was raised to 60 C for one hour. Overnight
exposure at -
80 C showed 7 putative primary bits.
A K802 host culture was prepared to plate the primary hits for a
secondary screen. The 7 primary bits were picked with a Pasteur pipet and
eluted in i
nit SM (0.1 M NaCl, 50 mM 'I'ris pH 7.5, 10 mM MMSO4, 0.02% gelatin) with a
few
drops of chlorofoxm overnight at 4 C. Amer plating to determine titers, 10
times the
number of plaques in the original pfu were plated on NZY u>axi plates with 10
mM
MgSO4lNZY top agarose and a lawn of K802 cells for four of the primary bits
and
grown overnight at 37 C. Lifts were done using Ryboad N filters (Amersham
Plzarmacia). The filters were mailed for orientation with a hot needle,
denatured in 1.5
M NaCl and 0.5 M NaOH for 10 minutes, then neutralized in 1.5 M NaCl and OS M
Tris-HO pH 7.2 for 10 minutes. The DNA was affixed to the they using a
STRATALINKER UV erosslinker (Stratagene, La Jolla, CA) at 1200 joules, and
prewashed at 65 C in prewash buffer consisting of 0.25 x SSC, 0.25% SDS and 1
mM
LDTTA, changing solution three tames for a total of 45 minutes to remove cell
debris.
Five lifts were put in each vial, three vials total. Each vial of lifts was
prehybridized
overnight at 50 C in 13 ml of EXPRESSITYB Hybridization Solution (Clontech)
mixed
with 1.3 mg salmon sperm DNA which had been boiled :5 minutes, then iced.
Sixty three ng of the human zvegf4 fragment was labeled for a probe as
described above. Each vial of filters was hybridized in 9 ml of EX?RESSITYB
Hybridization Solution mixed with 0.99 to 1.1 x 106 h=mv zveegf4 probe, U.S
pg/ml
murine cot-1 DNA, and 0.9 mg/nrl salmon sperm DNA which had been boiled 5
minutes, then iced. Hybridization took place overnight at 50 C. Wash
conditions
described above for the primary screen were repeated for this secondary
screen. Two
of the 4 primary putative hits that were tested carne up positive after an
overnight
exposure at -80 C.
Isolated plaques #7c1 and ##18b2 were eluted in 200 1 SM overnight at
4 C, and fresh host K802 cells were prepared. Serial dilutions ranging from 10-
2 to 10`3
were plated to obtain a titer estimate. Only #18b2 gave any plaques (for a
titer of 2.6 to
3.0 x 103 phase per l), and this plaque was further pursued. Two plates with
105 pfus
per pl ate were prepared for a phase DNA prep from plate lysates. Plates were
grown at
AMENDED SHEET
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CA 02370948 2001-10-16
37 C for 6 hours, until the phage Were starting to get confluent, and then 12
m1 of SM
per plate was added to elute the phage overnight at 4 C. At this point, plates
were
shaken at room temperature one hour, the supernatant was removed, 1%
chloroform
was added, and supernatant was shaken for 15 minutes. The DNA was prepped
using
5 the Wizard Lambda Preps DNA Purification System (Promega), sections IV and
VI.
Plaque #18b2 DNA was out with several restriction enzymes to generate
fragments to run on a Southern gel. Digests were run on a 1% TBE agarose gel.
The
gel was soaked in 0.25 M HCl for 30 minutes, rinsed in distilled H1O, soaked
In 0.5 M
NaOH and 1.5 M NaCl for 40 minutes with one solution change, and neutralized
in 1.5
10 M NaC1 and 0S M Tris HCI (pH 72) for 40 minutes with one solution change. A
TURBOBLOTTE1 Rapid Downward Transfer System (Scheicher & Schuell, Keene,
NH) was set up to transfer the DNA onto a Nytrau/BA-S membrane (Schleicher &
Schuell) overnight. The DNA was affixed to the Nytran using a STRATALINKER UV
crosslinker (Stratagene) at 1200 joules. The blot was prehybridized overnight
at 5000
15 in 12 ml EXPRESSITYB Hybridization Solution (Clontech) mixed with 1.2 mg
salmon.
sperm. DNA which had been boiled 5 minutes, then iced. Fifty nine ng of the
human
zvegf4 fragment was labeled for a probe, as described above. Unincorporated
radioactivity was removed by chromatography using a commercially available
push
column (NIJCTRAP column, Stratagene). Ten ml of EXPRESSHYB Hybridization
20 Solution was mixed with 1.0 x 106 cpm/ml of human zvegf4 probe, 0.5 p.g/ral
murine
cot-1 DNA, mad 0.1 mg/ml salmon sperm DNA which had been boiled 5 minutes,
then.
iced, and then added to the blot. Hybridization took place overnight at 500C.
The blot
was washed as described above, and exposed to film overnight as -80 C.
The Southern get had a fragment from the BamHl/Pstl digest which
25 hybridized to the probe in the size range of 2.0 to 2.9 kb, which was
pursued. Plaque
18b2 lambda DNA (2.8 g) was cut with 20 units of BamH1 (Boehringer Mannheim
Indianapolis, IN), and 20 1 Pst1 (Life Technologies) for 2 hours at 37 C. The
digest
was run on a 1% TBE gel, and a 2.0 kb doublet, as well as 2.7 kb/2.9 kb bands,
were
excised from the gel. The DNA was extracted from the agarase using the
Qiaguick Gel
30 Extraction Kit (Qiagen). The 18b2 fragments were ligated into a
pblureseriptllKS+
vector (Stsatagene) cut with BamH1, Pstl and BamHl/Pstl. Three clones with a
?stl
insect, and 4 clones with a BamHl/Pstl insert, from these Iigations were
digested with
their respective insert site restriction enzymes for another Southern blot to
determine
which was the original hybridizing fragment. The 1% TBE gel was treated and
the
35 DNA was transferred to the Nytran blot as described above. The blot was
prehybridized as above in 13 ml of hybridization solution. Fifty nine ng of
the human
zvegf4 fragment was labeled and unincorporated radioactivity was removed as
AMENDED SHEET
V 1 - I G-Guuu F'U I /USUU/4004
CA 02370948 2001-10-16
86
described above. Human zvegf4 probe (8,4 x 105tm1 cpm), 0.1 mg/ml of salmon
sperm DNA, and 0.5 pg/ml of mouse cot-I DNA were boiled 5 minutes, iced 1
minute,
and mixed with 7 ml of EXPRESSITYB hybridization solution, then added to the
blot.
Hybridization took place overnight at 50 C. The same washing procedure was
used as
mentioned above. The blot was exposed to film for 3 hours at -80 C, and both
2.0 kb
band inserts strongly hybridized to the probe. These clones were sequenced and
found
to contain part of the marine zvogf4 cub domain. Primers were designed from
this
sequence for a PCR cDN-A screen.
B. PCR screeo of mouse cDNA panel
A panel of available in house and commercial mouse cDN-As were
screened with 20 pm each of ZG26,317 (SEQ ID NO:49) and Z026,318 (SEQ ID
NO:50) primers. The PCR reaction conditions were as follows: 94 C, 2 raitutes;
then
35 cycles of 94 C, 10 seconds; 651C, 20 seconds; 721C, 30 seconds; then ended
with a
5 minute extension at 72 C. Embryo, salivary gland, neonatal skin and testis
showed
strong products of the predicted 200 bp size.
C. Full Length Mouse zvegX Sequence
The in house mouse testis arrayed library representing 9.6 x 105 clones
was screened by PCR using primers ZG26,317 (SEQ ID NO:49) and ZG26,318 (SEQ
ID NO:50) according to conditions specified above. This library was
deconvoluted
down to a positive pool of 250 clones. R. cal:' DH100 cells (Gibco BRL) were
transformed with this pool by elcctmporation following the manufactmwer's
protocol,
The transformed culture was titered and arrayed out to 96 wells at -20
eells/welL The
cells were grown up in LB+amp overnight at 37 C. An aliquot of the cells was
pelleted
and PCR was used to identify a positive pool. Thermocycler conditions were as
described above. The remaining cells from a positive pool were plated, and
colonies
were steed by PCR to identify a positive clone. Sequence analysis indicated
that
this clone, named "zvegf4mpzp7x-6", was incomplete at the 5' end and appeared
to
contain an intron at the 5' end,
The mouse salivary gland library representing 9.6 x 10'5 clones was
then screened by PCR using primers Z026,3 17 (SEQ ID N0:49) and ZG26,318 (SEQ
ID NO:50) according to conditions specified above. The library was
deconvoluted
down to a positive pool of 250 clones. This 250 clonal pool was verified as
having the
5' end by RACE. Twenty pin each of ZG26,3I8 (SEQ ID NO:50) and ZG14,063 (SEQ
ID N0:51) primers and 3 l of that pool was used. The reaction was run as
follows :
AMENDED SHEET
V I ' I L'GVVV 'L I //UZ~UU/4UU4
CA 02370948 2001-10-16
87
94 C, 2 minutes, then 5 cycles of 94 C, 15 seconds; 70 C, 30 seconds; 30
cycles of
94 C, 15 seconds, 62 C, 20 seconds; 7 C, 30 seconds, and a final extension at
72 C for
7 minutes. The RACE product obtained upon sequencing mimed that this pool
contained the initiation Met. The same protocol as described above was carried
out to
isolate a single clone from the pool. Sequence analysis revealed that this
clone, named
"zvegf4mpzp7x-7", had a 225 bp deletion. in coding compared to clone #6 (bp
865 to
bp 1079 in the final sequence).
The sequences derived from zvegf4mpzp7x-6 and from zvegf4mpzp7x-
7 were combined to obtain a full length mouse zvegf4 polynucleotide sequence
(SEQ
ID NO:52) and mouse zvegf4 polypeptide sequence (SBQ ID NO;53).
D. Full Length Mouse zve f4 Clone
The full length cI)NA clone was generated by a two step ligation of
fragments from done #5 and clone #7 from above. An EcoRl/Mind3 three prime
fragment was generated from clone #6 first. Nine AS of clone #6 were digested
with 15
units of RooRl (Gibco BRL, Gaithersburg, MD) and 15 units of Hindi (Gibco BRL)
for 2 hours at 37 C. The 528 by fragment was gel purified on a 1% TBE gel, and
the
cDNA was extracted from the gel slab using the QlAquick Gel Extraction Kit
(Qiagen).
It was ligated into pbluescriptlIlKS+ (Stratagene) digested with RooRI and
Hindi.
Three g of a clone with this zvegf4 insert was digested with 15 units of EcoR
l (Gibco
BRL), gel purified on a I% TBB gel, and the DNA was extracted using the kit
mentioned above. The 5' EcoRl zvegf4 fragment from clone #7 was ligated into
the
EcoR1-digested clone mentioned above. This EcoR1 fragment was generated by
digesting 8 g of clone #7 with 30 units of EcoRl (Gsbco BRL) for 2 hours at
37 C.
The 754 bp fragment was gel purified on a 1% TBE gel, and the DNA was
extracted
from the gel slab as mentioned above.
Frannie 20
A. Treatment ofNaive PC12 Cells with zvenf4 Conditioned Medium
FlaCat cells were infected with a null adeno virus (zPar) as a, control, or
with adenovirus expressing zvegf4. Conditioned medium (CM) from these
transfected
cells was assayed for its ability to induce neurite outgrowth in the PC12
Pheochromocytoma cell line (see Banker and Goslin, in Culturing Nerve Cells,
chapter
6, "Culture and experimental use of the PC12 rat Pheocluomocytoma cell line ;
also,
see Rydel and Greene, S. Neuroscience 2(111- 3639-53, November 1987).
Briefly, PCI2 cell cultures (ATCC# CRL 1721) were propagated with
RPMI 1640 medium (Cnbco/BRL, Gaithersburg, MD), 10% horse serum (Sigma, St.
AMENDED SHEET
V 1' IG'LVVV r -U
CA 02370948 2001-10-16
88
Louis, MO), and 5% fetal bovine serum (FBS; Hyclone, Logan, Uf). Plastic
culture
dishes (Beekton Dickinson, Bedford, MA) were coated with rat tail collagen
type I, and
PC12 cells were plated into 24 well plates at 2 x 104 cells/ml in R?Ml + 1%
PBS and
incubated overnight at 37CC in 5% CO2. The PC12 culture medium was then
removed,
and replaced with either zvegf4-CM or control-CM added in 2-fold dilutions
(starting at
5x dilution). Recombinant human NOF (R+D, Minneapolis, MN) was added as a
positive control at concentrations of 100 or 30 ng/mL As a negative control,
CM of the
null adcnovirus (zpar) was used. To test for synergy of zvegf4 and NOW,
additional
wells of PC12 cells were treated with zvegf4-CM in combination with a
suboptimal
concentration of NGF (3 ng/ml). The culture medium was replaced every second
day
with RPMI + 1 % FBS, until the total length of incubation reached 7 days.
The NGF-treated PC12 cells exhibited stable neurite outgrowth and
neuronal ctifferenti anion. PC12 cells exposed to zvegf4-CM exhibited
morphological
changes, such as cell flattening and the appearance of cells with short
processes,
suggesting differentiation into neuronal lineage. For PC12 cells incubated
with a
suboptima] dose of NOF plus zvegf3-CM, an increase in a population of cells
bearing
short processes was observed.
B. Treatment of Primed., Neurite-Bearing PC12 Cells with zvegf4
Conditioned Medium
Zvegf4-CM and a control-CM (zpar) (as desc foed in Example 20.A.,
above) were assayed for their ability to promote survival of differentiated
PC12
neurons (see Banker and Goslin, supra, Rydel and Greene, supra).
Briefly, PC12 cells were maintained as described in Example 20.A.,
above, and were treated with appropriate doses of NOF to induce
differentiation into
cells that express the properties of posts-mitotic sympathetic neurons. More
specifically, PC12 cells were treated with recombinant human N OF (R+D,
Minneapolis, MN) at a concentration of 50 ng/ml for 6 days, with a change of
medium
every other day. Cells were plated into 24 well plates overnight, and the
culture
medium was replaced with zvegf4-CM or control-CM (in 2-fold dilutions,
starting at
5x), or with NOF as a positive control (starting with 100 ng/mi in 3-fold
dilutions).
Cultures were set up either with 1% FBS or serum-free culture (SF)
medium Cells were propagated over 9 days, with medium changes on every second
day. Continuous treatment with NOF alone promoted the survival of the entire
neuronal population and produced increasing ueurite outgrowth. Zvegf4-CM
promoted
the survival of a subpopulation of neurons, but did not induce additional
neurite
outgrowth. Cells cultured in control-CM degenerated.
AMENDED SHEET
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
89
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.
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
1
SEQUENCE LISTING
<110> ZymoGenetics, Inc.
<120> GROWTH FACTOR HOMOLOG ZVEGF4
<130> 99-19PC
<150> US 09/304,216
<151> 1999-05-03
<150> US 60/164,463
<151> 1999-11-10
<150> US 60/180,169
<151> 2000-02-04
<160> 53
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 1882
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (226) ... (1338)
<400> 1
ccgtcaccat ttatcagctc agcaccacaa ggaagtgcgg cacccacacg cgctcggaaa 60
gttcagcatg caggaagttt ggggagagct cggcgattag cacagcgacc cgggccagcg 120
cagggcgagc gcaggcggcg agagcgcagg gcggcgcggc gtcggtcccg ggagcagaac 180
ccggcttttt cttggagcga cgctgtctct agtcgctgat cccaa atg cac cgg ctc 237
Met His Arg Leu
1
atc ttt gtc tac act cta atc tgc gca aac ttt tgc agc tgt cgg gac 285
Ile Phe Val Tyr Thr Leu Ile Cys Ala Asn Phe Cys Ser Cys Arg Asp
10 15 20
CA 02370948 2001-10-16
WO 00/66736 PCTIUS00/40047
2
act tct gca acc ccg cag agc gca tcc atc aaa get ttg cgc aac gcc 333
Thr Ser Ala Thr Pro Gln Ser Ala Ser Ile Lys Ala Leu Arg Asn Ala
25 30 35
aac ctc agg cga gat gag agc aat cac ctc aca gac ttg tac cga aga 381
Asn Leu Arg Arg Asp Glu Ser Asn His Leu Thr Asp Leu Tyr Arg Arg
40 45 50
gat gag acc atc cag gtg aaa gga aac ggc tac gtg cag agt cct aga 429
Asp Glu Thr Ile Gln Val Lys Gly Asn Gly Tyr Val Gln Ser Pro Arg
55 60 65
ttc ccg aac agc tac ccc agg aac ctg ctc ctg aca tgg cgg ctt cac 477
Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr Trp Arg Leu His
70 75 80
tct cag gag aat aca cgg ata cag cta gtg ttt gac aat cag ttt gga 525
Ser Gln Glu Asn Thr Arg Ile Gin Leu Val Phe Asp Asn Gln Phe Gly
85 90 95 100
tta gag gaa gca gaa aat gat atc tgt agg tat gat ttt gtg gaa gtt 573
Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp Phe Val Glu Val
105 110 115
gaa gat ata tcc gaa acc agt acc att att aga gga cga tgg tgt gga 621
Glu Asp Ile Ser Glu Thr Ser Thr Ile Ile Arg Gly Arg Trp Cys Gly
120 125 130
cac aag gaa gtt cct cca agg ata aaa tca aga acg aac caa att aaa 669
His Lys Glu Val Pro Pro Arg Ile Lys Ser Arg Thr Asn Gln Ile Lys
135 140 145
atc aca ttc aag tcc gat gac tac ttt gtg get aaa cct gga ttc aag 717
Ile Thr Phe Lys Ser Asp Asp Tyr Phe Val Ala Lys Pro Gly Phe Lys
150 155 160
att tat tat tct ttg ctg gaa gat ttc caa ccc gca gca get tca gag 765
Ile Tyr Tyr Ser Leu Leu Glu Asp Phe Gln Pro Ala Ala Ala Ser Glu
165 170 175 180
acc aac tgg gaa tct gtc aca agc tct att tca ggg gta tcc tat aac 813
Thr Asn Trp Glu Ser Val Thr Ser Ser Ile Ser Gly Val Ser Tyr Asn
185 190 195
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
3
tct cca tca gta acg gat ccc act ctg att gcg gat get ctg gac aaa 861
Ser Pro Ser Val Thr Asp Pro Thr Leu Ile Ala Asp Ala Leu Asp Lys
200 205 210
aaa att gca gaa ttt gat aca gtg gaa gat ctg ctc aag tac ttc aat 909
Lys Ile Ala Glu Phe Asp Thr Val Glu Asp Leu Leu Lys Tyr Phe Asn
215 220 225
cca gag tca tgg caa gaa gat ctt gag aat atg tat ctg gac acc cct 957
Pro Glu Ser Trp Gln Glu Asp Leu Glu Asn Met Tyr Leu Asp Thr Pro
230 235 240
cgg tat cga ggc agg tca tac cat gac cgg aag tca aaa gtt gac ctg 1005
Arg Tyr Arg Gly Arg Ser Tyr His Asp Arg Lys Ser Lys Val Asp Leu
245 250 255 260
gat agg ctc aat gat gat gcc aag cgt tac agt tgc act ccc agg aat 1053
Asp Arg Leu Asn Asp Asp Ala Lys Arg Tyr Ser Cys Thr Pro Arg Asn
265 270 275
tac tcg gtc aat ata aga gaa gag ctg aag ttg gcc aat gtg gtc ttc 1101
Tyr Ser Val Asn Ile Arg Glu Glu Leu Lys Leu Ala Asn Val Val Phe
280 285 290
ttt cca cgt tgc ctc ctc gtg cag cgc tgt gga gga aat tgt ggc tgt 1149
Phe Pro Arg Cys Leu Leu Val Gin Arg Cys Gly Gly Asn Cys Gly Cys
295 300 305
gga act gtc aac tgg agg tcc tgc aca tgc aat tca ggg aaa acc gtg 1197
Gly Thr Val Asn Trp Arg Ser Cys Thr Cys Asn Ser Gly Lys Thr Val
310 315 320
aaa aag tat cat gag gta tta cag ttt gag cct ggc cac atc aag agg 1245
Lys Lys Tyr His Glu Val Leu Gin Phe Glu Pro Gly His Ile Lys Arg
325 330 335 340
agg ggt aga get aag acc atg get cta gtt gac atc cag ttg gat cac 1293
Arg Gly Arg Ala Lys Thr Met Ala Leu Val Asp Ile Gin Leu Asp His
345 350 355
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
4
cat gaa cga tgc gat tgt atc tgc agc tca aga cca cct cga taa 1338
His Glu Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro Pro Arg
360 365 370
gagaatgtgc acatccttac attaagcctg aaagaacctt tagtttaagg agggtgagat 1398
aagagaccct tttcctacca gcaaccaaac ttactactag cctgcaatgc aatgaacaca 1458
agtggttgct gagtctcagc cttgctttgt taatgccatg gcaagtagaa aggtatatca 1518
tcaacttcta tacctaagaa tataggattg catttaataa tagtgtttga ggttatatat 1578
gcacaaacac acacagaaat atattcatgt ctatgtgtat atagatcaaa tgtttttttt 1638
ttttggtata tataaccagg tacaccagag gttacatatg tttgagttag actcttaaaa 1698
tcctttgcca aaataaggga tggtcaaata tatgaaacat gtctttagaa aatttaggag 1758
ataaatttat ttttaaattt tgaaacacga aacaattttg aatcttgctc tcttaaagaa 1818
agcatcttgt atattaaaaa tcaaaagatg aggctttctt acatatacat cttagttgat 1878
tatt 1882
<210> 2
<211> 370
<212> PRT
<213> Homo sapiens
<400> 2
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
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
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
Gln Leu Asp His His Glu Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro
355 360 365
Pro Arg
370
<210> 3
<211> 126
<212> PRT
<213> Artificial Sequence
<220>
<223> polypeptide motif
<221> VARIANT
<222> (2)...(19)
<223> Xaa = Any Amino Acid
<221> VARIANT
<222> (20)...(34)
<223> Xaa = Any Amino Acid or is not present
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
6
<221> VARIANT
<222> (36)...(45)
<223> Xaa = Any Amino Acid
<221> VARIANT
<222> (46)...(72)
<223> Xaa = Any Amino Acid or is not present
<221> VARIANT
<222> (74)...(93)
<223> Xaa = Any Amino Acid
<221> VARIANT
<222> (94) ... (123)
<223> Xaa = Any Amino Acid or is not present
<221> VARIANT
<222> (125) ... (125)
<223> Xaa = Any Amino Acid
<400> 3
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
100 105 110
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys
115 120 125
<210> 4
<211> 24
<212> PRT
<213> Artificial Sequence
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
7
<220>
<223> polypeptide motif
<221> VARIANT
<222> (2)...(2)
<223> Xaa = Lys or Arg
<221> VARIANT
<222> (4)...(4)
<223> Xaa = Asp, Asn or Glu
<221> VARIANT
<222> (5)...(5)
<223> Xaa = Trp, Tyr or Phe
<221> VARIANT
<222> (6)...(16)
<223> Xaa = Any Amino Acid
<221> VARIANT
<222> (17)...(20)
<223> Xaa = Any Amino Acid or is not present
<221> VARIANT
<222> (22)...(22)
<223> Xaa = Lys or Arg
<221> VARIANT
<222> (23)...(23)
<223> Xaa = Trp, Tyr or Phe
<400> 4
Cys Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Gly Xaa Xaa Cys
<210> 5
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
CA 02370948 2001-10-16
WO 00/66736 PCT/USOO/40047
8
<223> polypeptide tag
<400> 5
Glu Tyr Met Pro Met Glu
1 5
<210> 6
<211> 1110
<212> DNA
<213> Artificial Sequence
<220>
<223> degenerate sequence
<221> misc feature
<222> (1)._.(1110)
<223> n = A,T,C or G
<400> 6
atgcaymgny tnathttygt ntayacnytn athtgygcna ayttytgyws ntgymgngay 60
acnwsngcna cnccncarws ngcnwsnath aargcnytnm gnaaygcnaa yytnmgnmgn 120
gaygarwsna aycayytnac ngayytntay mgnmgngayg aracnathca rgtnaarggn 180
aayggntayg tncarwsncc nmgnttyccn aaywsntayc cnmgnaayyt nytnytnacn 240
tggmgnytnc aywsncarga raayacnmgn athcarytng tnttygayaa ycarttyggn 300
ytngargarg cngaraayga yathtgymgn taygayttyg tngargtnga rgayathwsn 360
garacnwsna cnathathmg nggnmgntgg tgyggncaya argargtncc nccnmgnath 420
aarwsnmgna cnaaycarat haarathacn ttyaarwsng aygaytaytt ygtngcnaar 480
ccnggnttya arathtayta ywsnytnytn gargayttyc arccngcngc ngcnwsngar 540
acnaaytggg arwsngtnac nwsnwsnath wsnggngtnw sntayaayws nccnwsngtn 600
acngayccna cnytnathgc ngaygcnytn gayaaraara thgcngartt ygayacngtn 660
gargayytny tnaartaytt yaayccngar wsntggcarg argayytnga raayatgtay 720
ytngayacnc cnmgntaymg nggnmgnwsn taycaygaym gnaarwsnaa rgtngayytn 780
gaymgnytna aygaygaygc naarmgntay wsntgyacnc cnmgnaayta ywsngtnaay 840
athmgngarg arytnaaryt ngcnaaygtn gtnttyttyc cnmgntgyyt nytngtncar 900
mgntgyggng gnaaytgygg ntgyggnacn gtnaaytggm gnwsntgyac ntgyaaywsn 960
ggnaaracng tnaaraarta ycaygargtn ytncarttyg arccnggnca yathaarmgn 1020
mgnggnmgng cnaaracnat ggcnytngtn gayathcary tngaycayca ygarmgntgy 1080
gaytgyatht gywsnwsnmg nccnccnmgn 1110
<210> 7
<211> 17
<212> DNA
<213> Artificial Sequence
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
9
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(17)
<223> n = A,T,C or G
<400> 7
mgntgyggng gnaaytg 17
<210> 8
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1). .(17)
<223> n = A,T,C or G
<400> 8
mgntgydsng gnwrytg 17
<210> 9
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(17)
<223> n = A,T,C or G
<400> 9
carywnccns hrcanck 17
<210> 10
<211> 17
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(17)
<223> n = A,T,C or G
<400> 10
ttyttyccnm gntgyyt 17
<210> 11
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc_feature
<222> (1). .(17)'
<223> n = A,T,C or G
<400> 11
ntnddnccnn sntgybt 17
<210> 12
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc_feature
<222> (1). .(17)
<223> n = A,T,C or G
<400> 12
avrcansnng gnhhnan 17
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
11
<210> 13
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(17)
<223> n = A,T,C or G
<400> 13
caygarmgnt gygaytg 17
<210> 14
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(17)
<223> n = A,T,C or G
<400> 14
caynnnnvnt gyvvntg 17
<210> 15
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(17)
<223> n = A,T,C or G
<400> 15
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
12
canbbrcanb nnnnrtg 17
<210> 16
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(17)
<223> n = A,T,C or G
<400> 16
tgyacnccnm gnaayta 17
<210> 17
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(17)
<223> n = A,T,C or G
<400> 17
tgyhnnmcnm knrmndh 17
<210> 18
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(17)
<223> n = A,T,C or G
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
13
<400> 18
dhnkynmkng knndrca 17
<210> 19
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(18)
<223> n = A,T,C or G
<400> 19
ntaygaytwy gtngargt 18
<210> 20
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(18)
<223> n = A,T,C or G
<400> 20
natrctrawr canctyca 18
<210> 21
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
14
<222> (1)...(18)
<223> n = A,T,C or G
<400> 21
gntdbccnma ndvntayc 18
<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(18)
<223> n = A,T,C or G
<400> 22
cnahvggnkt nhbnatrg 18
<210> 23
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
<221> misc feature
<222> (1)._.(18)
<223> n = A,T,C or G
<400> 23
tnhdnggnmr ntdbtgyg 18
<210> 24
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer
CA 02370948 2001-10-16
WO 00/66736 PCT/USOO/40047
<221> misc feature
<222> (1)._.(18)
<223> n = A,T,C or G
<400> 24
andhnccnky nahvacrc 18
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC21,119
<400> 25
aggacgatgg tgtggacaca agga 24
<210> 26
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC21,120
<400> 26
tccagagcat ccgcaatcag agtg 24
<210> 27
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC21,987
<400> 27
caacctgttg tttgtcccgt cacc 24
<210> 28
<211> 25
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
16
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC17,251
<400> 28
tctggacgtc ctcctgctgg tatag 25
<210> 29
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC17,252
<400> 29
ggtatggagc aaggggcaag ttggg 25
<210> 30
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC17,156
<400> 30
gagtggcaac ttccagggcc aggagag 27
<210> 31
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC17,157
<400> 31
cttttgctag cctcaaccct gactatc 27
<210> 32
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
17
<211> 1760
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (154) ... (1191)
<400> 32
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
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 Gin
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
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
18
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 get gtg agt cct tca gtg cta ccc cct tca get 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 get 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 get 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 Gin
280 285 290 295
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
19
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
<210> 33
<211> 345
<212> PRT
<213> Homo sapiens
<400> 33
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
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
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
<210> 34
<211> 3571
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (1049)...(2086)
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
21
<400> 34
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 get gag tcc 1120
Leu Thr Ser Ala Leu Ala Gly Gln Arg Thr Gly Thr Arg Ala Glu Ser
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
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
22
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 get 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 get 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
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
23
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 get 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
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
24
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
<210> 35
<211> 345
<212> PRT
<213> Mus musculus
<400> 35
Met Leu Leu Leu Gly Leu Leu Leu Leu Thr Ser Ala Leu Ala Gly Gin
1 5 10 15
Arg Thr Gly Thr Arg Ala Glu Ser Asn Leu Ser Ser Lys Leu Gin 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
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
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
<210> 36
<211> 600
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (496) ... (592)
<400> 36
gtgggaagag tcccggccgg cgattaaact gggcatgctc agtggcagag caggtttagg 60
cccggcctgg gaaactgggg agctgaggtg ctcgcgccgc cgctctgagc ccgagtgcgc 120
gcctctcagg ggccgcggcc ggggctggag aacgctgctg ctccgctcgc ctgccccgct 180
agattcggcg ctgcccgccc cctgcagcct gtgctgcagc tgccggccac cggagggggc 240
gaacaaacaa acgtcaacct gttgtttgtc ccgtcaccat ttatcagctc agcaccacaa 300
ggaagtgcgg cacccacacg cgctcggaaa gttcagcatg caggaagttt ggggagagct 360
cggcgattag cacagcgacc cgggccagcg cagggcgagc gcagacggcg agagcgcagg 420
gcggcgcggc gtcggtcccg ggagcagaac ccggcttttt cttggagcga cgctgtctct 480
agtcgctgat cccaa atg cac cgg ctc atc ttt gtc tac act cta atc tgc 531
Met His Arg Leu Ile Phe Val Tyr Thr Leu Ile Cys
1 5 10
gca aac ttt tgc agc tgt cgg gac act tct gca acc ccg cag agc gca 579
Ala Asn Phe Cys Ser Cys Arg Asp Thr Ser Ala Thr Pro Gin Ser Ala
15 20 25
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
26
tcc atc aaa get t gagtattc 600
Ser Ile Lys Ala
<210> 37
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC22,685
<400> 37
gccgtcacca tttatcag 18
<210> 38
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC22,686
<400> 38
cgggtcgctg tgctaatc 18
<210> 39
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> peptide
<400> 39
Cys Gly His Lys Glu Val Pro Pro Arg Ile Lys Ser Arg Thr Asn Gln
1 5 10 15
Ile Lys
<210> 40
<211> 25
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
27
<212> PRT
<213> Artificial Sequence
<220>
<223> peptide
<400> 40
Glu Ser Trp Gln Glu Asp Leu Glu Asn Met Tyr Leu Asp Thr Pro Arg
10 15
Tyr Arg Gly Arg Ser Tyr His Asp Cys
20 25
<210> 41
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> peptide
<400> 41
Cys Phe Glu Pro Gly His Ile Lys Arg Arg Gly Arg Ala Lys Thr Met
5 10 15
Ala Leu Val Asp Ile Gln Leu Asp
<210> 42
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> peptide
<400> 42
Glu Tyr Met Pro Thr Asp
5
<210> 43
<211> 42
<212> DNA
<213> Artificial Sequence
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
28
<220>
<223> oligonucleotide primer ZC26136
<400> 43
taatataggc cggccgccat catgcaccgg ctcatctttg tc 42
<210> 44
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC26137
<400> 44
attatatggc gcgccttatc gaggtggtct tgagct 36
<210> 45
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC22341
<400> 45
cttggagcga cgctgtctct agtc 24
<210> 46
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC22342
<400> 46
ccacttgtgt tcattgcatt gca 23
<210> 47
<211> 60
<212> DNA
<213> Artificial Sequence
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
29
<220>
<223> oligonucleotide primer ZC27116
<400> 47
attataggat ccgagtatat gcctatggag gttgacctgg ataggctcaa tgatgatgcc 60
<210> 48
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC26137
<400> 48
attatatggc gcgccttatc gaggtggtct tgagct 36
<210> 49
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC26317
<400> 49
atcacctcac agacttgtac cagag 25
<210> 50
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC26318
<400> 50
cctacaaatg tcattttctg cttcc 25
<210> 51
<211> 25
<212> DNA
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC14063
<400> 51
caccagacat aatagctgac agact 25
<210> 52
<211> 1472
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (93) ... (1205)
<400> 52
agggactgtg cagtagaaat ccgccgactc aaccctttgg gctttattta tttacttttg 60
gagcaacgcg atccctaggt cgctgagccc as atg caa cgg ctc gtt tta gtc 113
Met Gln Arg Leu Val Leu Val
1 5
tcc att ctc ctg tgc gcg aac ttt agc tgc tat ccg gac act ttt gcg 161
Ser Ile Leu Leu Cys Ala Asn Phe Ser Cys Tyr Pro Asp Thr Phe Ala
10 15 20
act ccg cag aga gca tcc atc aaa get ttg cgc aat gcc aac ctc agg 209
Thr Pro Gln Arg Ala Ser Ile Lys Ala Leu Arg Asn Ala Asn Leu Arg
25 30 35
aga gat gag agc aat cac ctc aca gac ttg tac cag aga gag gag aac 257
Arg Asp Glu Ser Asn His Leu Thr Asp Leu Tyr Gln Arg Glu Glu Asn
45 50 55
att cag gtg aca agc aat ggc cat gtg cag agt cct cgc ttc ccg aac 305
Ile Gln Val Thr Ser Asn Gly His Val Gln Ser Pro Arg Phe Pro Asn
60 65 70
agc tac cca agg aac ctg ctt ctg aca tgg tgg ctc cgt tcc cag gag 353
Ser Tyr Pro Arg Asn Leu Leu Leu Thr Trp Trp Leu Arg Ser Gln Glu
75 80 85
CA 02370948 2001-10-16
WO 00/66736 PCT/US00/40047
31
aaa aca cgg ata caa ctg tcc ttt gac cat caa ttc gga cta gag gaa 401
Lys Thr Arg Ile Gln Leu Ser Phe Asp His Gin Phe Gly Leu Glu Glu
90 95 100
gca gaa aat gac att tgt agg tat gac ttt gtg gaa gtt gaa gaa gtc 449
Ala Glu Asn Asp Ile Cys Arg Tyr Asp Phe Val Glu Val Glu Glu Val
105 110 115
tca gag agc agc act gtt gtc aga gga aga tgg tgt ggc cac aag gag 497
Ser Glu Ser Ser Thr Val Val Arg Gly Arg Trp Cys Gly His Lys Glu
120 125 130 135
atc cct cca agg ata acg tca aga aca aac cag att aaa atc aca ttt 545
Ile Pro Pro Arg Ile Thr Ser Arg Thr Asn Gln Ile Lys Ile Thr Phe
140 145 150
aag tct gat gac tac ttt gtg gca aaa cct gga ttc aag att tat tat 593
Lys Ser Asp Asp Tyr Phe Val Ala Lys Pro Gly Phe Lys Ile Tyr Tyr
155 160 165
tca ttt gtg gaa gat ttc caa ccg gaa gca gcc tca gag acc aac tgg 641
Ser Phe Val Glu Asp Phe Gln Pro Glu Ala Ala Ser Glu Thr Asn Trp
170 175 180
gaa tca gtc aca agc tct ttc tct ggg gtg tcc tat cac tct cca tca 689
Glu Ser Val Thr Ser Ser Phe Ser Gly Val Ser Tyr His Ser Pro Ser
185 190 195
ata acg gac ccc act ctc act get gat gcc ctg gac aaa act gtc gca 737
Ile Thr Asp Pro Thr Leu Thr Ala Asp Ala Leu Asp Lys Thr Val Ala
200 205 210 215
gaa ttc gat acc gtg gaa gat cta ctt aag cac ttc aat cca gtg tct 785
Glu Phe Asp Thr Val Glu Asp Leu Leu Lys His Phe Asn Pro Val Ser
220 225 230
tgg caa gat gat ctg gag aat ttg tat ctg gac acc cct cat tat aga 833
Trp Gln Asp Asp Leu Glu Asn Leu Tyr Leu Asp Thr Pro His Tyr Arg
235 240 245
ggc agg tca tac cat gat cgg aag tcc aaa gtg gac ctg gac agg ctc 881
Gly Arg Ser Tyr His Asp Arg Lys Ser Lys Val Asp Leu Asp Arg Leu
250 255 260
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
32
aat gat gat gtc aag cgt tac agt tgc act ccc agg aat cac tct gtg 929
Asn Asp Asp Val Lys Arg Tyr Ser Cys Thr Pro Arg Asn His Ser Val
265 270 275
aac ctc agg gag gag ctg aag ctg acc aat gca gtc ttc ttc cca cga 977
Asn Leu Arg Glu Glu Leu Lys Leu Thr Asn Ala Val Phe Phe Pro Arg
280 285 290 295
tgc ctc ctc gtg cag cgc tgt ggt ggc aac tgt ggt tgc gga act gtc 1025
Cys Leu Leu Val Gln Arg Cys Gly Gly Asn Cys Gly Cys Gly Thr Val
300 305 310
aac tgg aag tcc tgc aca tgc agc tca ggg aag aca gtg aag aag tat 1073
Asn Trp Lys Ser Cys Thr Cys Ser Ser Gly Lys Thr Val Lys Lys Tyr
315 320 325
cat gag gta ttg aag ttt gag cct gga cat ttc aag aga agg ggc aaa 1121
His Glu Val Leu Lys Phe Glu Pro Gly His Phe Lys Arg Arg Gly Lys
330 335 340
get aag aat atg get ctt gtt gat atc cag ctg gat cat cat gag cga 1169
Ala Lys Asn Met Ala Leu Val Asp Ile Gln Leu Asp His His Glu Arg
345 350 355
tgt gac tgt atc tgc agc tca aga cca cct cga taa aacactatgc 1215
Cys Asp Cys Ile Cys Ser Ser Arg Pro Pro Arg
360 365 370
acatctgtac tttgattatg aaaggacctt taggttacaa aaaccctaag aagcttctaa 1275
tctcagtgca atgaatgcat atggaaatgt tgctttgtta gtgccatggc aagaagaagc 1335
aaatatcatt aatttctata tacataaaca taggaattca cttatcaata gtatgtgaag 1395
atatgtatat atacttatat acatgactag ctctatgtat gtaaatagat taaatacttt 1455
attcagtata tttactg 1472
<210> 53
<211> 370
<212> PRT
<213> Mus musculus
<400> 53
Met Gln Arg Leu Val Leu Val Ser Ile Leu Leu Cys Ala Asn Phe Ser
1 5 10 15
CA 02370948 2001-10-16
WO 00/66736 PCT/USOO/40047
33
Cys Tyr Pro Asp Thr Phe Ala Thr Pro Gln Arg 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 Gln Arg Glu Glu Asn Ile Gln Val Thr Ser Asn Gly His 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 Trp Leu Arg Ser Gln Glu Lys Thr Arg Ile Gln Leu Ser Phe Asp
85 90 95
His Gln Phe Gly Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp
100 105 110
Phe Val Glu Val Glu Glu Val Ser Glu Ser Ser Thr Val Val Arg Gly
115 120 125
Arg Trp Cys Gly His Lys Glu Ile Pro Pro Arg Ile Thr 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 Phe Val Glu Asp Phe Gin Pro Glu
165 170 175
Ala Ala Ser Glu Thr Asn Trp Glu Ser Val Thr Ser Ser Phe Ser Gly
180 185 190
Val Ser Tyr His Ser Pro Ser Ile Thr Asp Pro Thr Leu Thr Ala Asp
195 200 205
Ala Leu Asp Lys Thr Val Ala Glu Phe Asp Thr Val Glu Asp Leu Leu
210 215 220
Lys His Phe Asn Pro Val Ser Trp Gln Asp Asp Leu Glu Asn Leu Tyr
225 230 235 240
Leu Asp Thr Pro His Tyr Arg Gly Arg Ser Tyr His Asp Arg Lys Ser
245 250 255
Lys Val Asp Leu Asp Arg Leu Asn Asp Asp Val Lys Arg Tyr Ser Cys
260 265 270
Thr Pro Arg Asn His Ser Val Asn Leu Arg Glu Glu Leu Lys Leu Thr
275 280 285
Asn Ala 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 Lys Ser Cys Thr Cys Ser Ser
305 310 315 320
Gly Lys Thr Val Lys Lys Tyr His Glu Val Leu Lys Phe Glu Pro Gly
325 330 335
His Phe Lys Arg Arg Gly Lys Ala Lys Asn Met Ala Leu Val Asp Ile
340 345 350
CA 02370948 2001-10-16
WO 00/66736 PCTIUSOO/40047
34
Gln Leu Asp His His Glu Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro
355 360 365
Pro Arg
370
