Note: Descriptions are shown in the official language in which they were submitted.
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VASCULAR ENDOTHELIAL GROWTH FACTOR 2
Background of the Invention
The present invention relates to newly identified polynucleotides,
polypeptides
encoded by such polynucleotides, the use of such polynucleotides and
polypeptides,
as well as the production of such polynucleotides and polypeptides. The
polypeptides
of the present invention have been identified as members of the vascular
endothelial
growth factor family. More particularly, the polypeptides of the present
invention are
1o human vascular endothelial growth factor 2 (VEGF-2). The invention also
relates to
inhibiting the action of such polypeptides.
The formation of new blood vessels, or angiogenesis, is essential for
embryonic development, subsequent growth, and tissue repair. Angiogenesis is
also
an essential part of certain pathological conditions, such as neoplasia (i.e.,
tumors and
gliomas). Abnormal angiogenesis is associated with other diseases such as
inflammation, rheumatoid arthritis, psoriasis, and diabetic retinopathy
(Folkman, J .
and Klagsbrun, M., Science 235:442-447( 1987)).
Both acidic and basic fibroblast growth factor molecules are mitogens for
endothelial cells and other cell types. Angiotropin and angiogenin can induce
angiogenesis, although their functions are unclear (Folkman, J., Cancer
Medicine,
Lea and Febiger Press, pp. 153-170 (1993)). A highly selective mitogen for
vascular
endothelial cells is vascular endothelial growth factor or VEGF (Ferrara, N.
et al.,
Endocr. Rev. 13:19-32 ( 1992)), which is also known as vascular permeability
factor
(VPF).
Vascular endothelial growth factor is a secreted angiogenic mitogen whose
target cell specificity appears to be restricted to vascular endothelial
cells. The murine
VEGF gene has been characterized and its expression pattern in embryogenesis
has
been analyzed. A persistent expression of VEGF was observed in epithelial
cells
adjacent to fenestrated endothelium, e.g., in choroid plexus and kidney
glomeruli.
The data was consistent with a role of VEGF as a multifunctional regulator of
endothelial cell growth and differentiation (Breier, G. et al., Development
114:521-
532 ( 1992)).
VEGF shares sequence homology with human platelet-derived growth factors,
PDGFa and PDGFb (Leung, D.W., et al., Science 246:1306-1309, (1989)). The
3s extent of homology is about 21% and 23%, respectively. Eight cysteine
residues
contributing to disulfide-bond formation are strictly conserved in these
proteins.
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Although they are similar, there are specific differences between VEGF and
PDGF.
While PDGF is a major growth factor for connective tissue, VEGF is highly
specific
for endothelial cells. Alternatively spliced mRNAs have been identified for
both
VEGF, PLGF, and PDGF and these different splicing products differ in
biological
activity and in receptor-binding specificity. VEGF and PDGF function as homo-
dimers or hetero-dimers and bind to receptors which elicit intrinsic tyrosine
kinase
activity following receptor dimerization.
VEGF has four different forms of 121, 165, 189 and 206 amino acids due to
alternative splicing. VEGF121 and VEGF165 are soluble and are capable of
t0 promoting angiogenesis, whereas VEGF189 and VEGF-206 are bound to heparin
containing proteoglycans in the cell surface. The temporal and spatial
expression of
VEGF has been correlated with physiological proliferation of the blood vessels
{Gajdusek, C.M., and Carbon, S.J., Cell Physio1.139:570-579 ( 1989); McNeil,
P.L., et al., J. Cell. Biol. 109:811-822 (1989)). Its high affinity binding
sites are
localized only on endothelial cells in tissue sections (Jakeman, L.B., et al.,
Clin.
Invest. 89:244-253 ( 1989)). The factor can be isolated from pituitary cells
and several
tumor cell lines, and has been implicated in some human gliomas (Plate, K. H .
,
Nature 359:845-848 (1992)). Interestingly, expression of VEGF121 or VEGF165
confers on Chinese hamster ovary cells the ability to form tumors in nude mice
(Ferrara, N. et al., J. Clin. Invest. 91:160-170 (1993)). The inhibition of
VEGF
function by anti-VEGF monoclonal antibodies was shown to inhibit tumor growth
in
immune-deficient mice (Kim, K.J., Nature 362:841-844 (1993)). Further, a
dominant-negative mutant of the VEGF receptor has been shown to inhibit growth
of
glioblastomas in mice.
Vascular permeability factor (VPF) has also been found to be responsible for
persistent microvascular hyperpermeability to plasma proteins even after the
cessation
of injury, which is a characteristic feature of normal wound healing. This
suggests
that VPF is an important factor in wound healing. Brown, L.F. et al., J. Exp.
Med.176:1375-1379 ( 1992).
The expression of VEGF is high in vascularized tissues, (e. g. , lung, heart,
placenta and solid tumors) and correlates with angiogenesis both temporally
and
spatially. VEGF has also been shown to induce angiogenesis in vivo. Since
angiogenesis is essential for the repair of normal tissues, especially
vascular tissues,
VEGF has been proposed for use in promoting vascular tissue repair (e.g., in
atherosclerosis).
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U.S. Patent No. 5,073,492, issued December 17, 1991 to Chen et al.,
discloses a method for synergistically enhancing endothelial cell growth in an
appropriate environment which comprises adding to the environment, VEGF,
effectors and serum-derived factor. Also, vascular endothelial cell growth
factor C
sub-unit DNA has been prepared by polymerase chain reaction techniques. The
DNA
encodes a protein that may exist as either a heterodimer or homodimer. The
protein is
a mammalian vascular endothelial cell mitogen and, as such, is useful for the
promotion of vascular development and repair, as disclosed in European Patent
Application No. 92302750.2, published September 30, 1992.
to
Summary of the Invention
The polypeptides of the present invention have been putatively identified as a
novel vascular endothelial growth factor based on amino acid sequence homology
to
~ 5 human VEGF.
In accordance with one aspect of the present invention, there are provided
novel mature polypeptides, as well as biologically active and diagnostically
or
therapeutically useful fragments, analogs, and derivatives thereof. The
polypeptides of
the present invention are of human origin.
2o In accordance with another aspect of the present invention, there are
provided
isolated nucleic acid molecules comprising polynucleotides encoding full
length or
truncated VEGF-2 polypeptides having the amino acid sequences shown in SEQ ID
NOS:2 or 4, respectively, or the amino acid sequences encoded by the cDNA
clones
deposited in bacterial hosts as ATCC Deposit Number 97149 on May 12, 1995 or
25 ATCC Deposit Number 75698 on March 4, 1994.
The present invention also relates to biologically active and diagnostically
or
therapeutically useful fragments, analogs, and derivatives of VEGF-2.
In accordance with still another aspect of the present invention, there are
provided processes for producing such polypeptides by recombinant techniques
30 comprising culturing recombinant prokaryotic and/or eukaryotic host cells,
containing
a nucleic acid sequence encoding a polypeptide of the present invention, under
conditions promoting expression of said proteins and subsequent recovery of
said
proteins.
In accordance with yet a further aspect of the present invention, there are
35 provided processes for utilizing such polypeptides, or polynucleotides
encoding such
polypeptides for therapeutic purposes, for example, to stimulate angiogenesis,
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wound-healing, growth of damaged bone and tissue, and to promote vascular
tissue
repair. In particular, there are provided processes for utilizing such
polypeptides, or
polynucleotides encoding such polypeptides, for treatment of peripheral artery
disease, such as critical limb ischemia and coronary disease.
In accordance with yet another aspect of the present invention, there are
provided antibodies against such polypeptides and processes for producing such
polypeptides.
In accordance with yet another aspect of the present invention, there are
provided antagonists to such polypeptides, which may be used to inhibit the
action of
such polypeptides, for example, to prevent tumor angiogenesis and thus inhibit
the
growth of tumors, to treat diabetic retinopathy, inflammation, rheumatoid
arthritis and
psoriasis.
In accordance with another aspect of the present invention, there are provided
nucleic acid probes comprising nucleic acid molecules of sufficient length to
specifically hybridize to nucleic acid sequences of the present invention.
In accordance with another aspect of the present invention, there are provided
methods of diagnosing diseases or a susceptibility to diseases related to
mutations in
nucleic acid sequences of the present invention and proteins encoded by such
nucleic
acid sequences.
2o In accordance with yet a further aspect of the present invention, there is
provided a process for utilizing such polypeptides, or polynucleotides
encoding such
polypeptides, for in vitro purposes related to scientific research, synthesis
of DNA
and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those
skilled in the art from the teachings herein.
Brief Description of the Figures
The following drawings are illustrative of embodiments of the invention and
are not meant to limit the scope of the invention as encompassed by the
claims.
Figures lA-lE show the full length nucleotide (SEQ ID NO:I) and the
deduced amino acid (SEQ )D N0:2) sequence of VEGF-2. The polypeptide
comprises approximately 419 amino acid residues of which approximately 23
represent the leader sequence. The standard one letter abbreviations for amino
acids
are used. Sequencing was performed using the Model 373 Automated DNA
Sequencer (Applied Biosystems, Inc.). Sequencing accuracy is predicted to be
greater
than 97%.
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Figures 2A-2D show the nucleotide (SEQ ID N0:3) and the deduced amino
acid (SEQ ID N0:4) sequence of a truncated, biologically active form of VEGF-
2.
The polypeptide comprises approximately 350 amino acid residues of which
approximately the first 24 amino acids represent the leader sequence.
Figures 3A-3B are an illustration of the amino acid sequence homology
between PDGFa (SEQ ID NO:S), PDGFb (SEQ ID N0:6), VEGF (SEQ )D N0:7),
and VEGF-2 (SEQ ID N0:4). The boxed areas indicate the conserved sequences and
the location of the eight conserved cysteine residues.
Figure 4 shows, in table-form, the percent homology between PDGFa,
1o PDGFb, VEGF, and VEGF-2.
Figure 5 shows the presence of VEGF-2 mRNA in human breast tumor cell
lines.
Figure 6 depicts the results of a Northern blot analysis of VEGF-2 in human
adult tissues.
Figure 7 shows a photograph of an SDS-PAGE gel after in vitro transcription,
translation and electrophoresis of the polypeptide of the present invention.
Lane l:
~°C and rainbow M.W. marker; Lane 2: FGF control; Lane 3: VEGF-2
produced by
M13-reverse and forward primers; Lane 4: VEGF-2 produced by M13 reverse and
VEGF-F4 primers; Lane 5: VEGF-2 produced by M13 reverse and VEGF-F5
2o primers.
Figures 8A and 8B depict photographs of SDS-PAGE gels. VEGF-2
polypeptide was expressed in a baculovirus system consisting of Sf9 cells.
Protein
from the medium and cytoplasm of cells were analyzed by SDS-PAGE under non-
reducing (Figure 8A) and reducing (Figure 8B) conditions.
Figure 9 depicts a photograph of an SDS-PAGE gel. The medium from Sf9
cells infected with a nucleic acid sequence of the present invention was
precipitated.
The resuspended precipitate was analyzed by SDS-PAGE and stained with
Coomassie
brilliant blue.
Figure 10 depicts a photograph of an SDS-PAGE gel. VEGF-2 was purified
from the medium supernatant and analyzed by SDS-PAGE in the presence or
absence
of the reducing agent b-mercaptoethanol and stained by Coomassie brilliant
blue.
Figure 11 depicts reverse phase HPLC analysis of purified VEGF-2 using a
RP-300 column (0.21 x 3 cm, Applied Biosystems, Inc.). The column was
equilibrated with 0.1 % trifluoroacetic acid (Solvent A) and the proteins
eluted with a
7.5 min gradient from 0 to 60% Solvent B, composed of acetonitrile containing
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0.07% TFA. The protein elution was monitored by absorbance at 215 nm ("red"
line)
and 280 nm ("blue" line). The percentage of Solvent B is shown by the "green"
line.
Figure 12 is a bar graph illustrating the effect of partially-purified VEGF-2
protein on the growth of vascular endothelial cells in comparison to basic
fibroblast
growth factor.
Figure 13 is a bar graph illustrating the effect of purified VEGF-2 protein on
the growth of vascular endothelial cells.
Figure 14 depicts expression of VEGF-2 mRNA in human fetal and adult
tissues.
Figure 15 depicts expression of VEGF-2 mRNA in human primary culture
cells.
Figure 16 depicts transient expression of VEGF-2 protein in COS-7 cells.
Figure 17 depicts VEGF-2 stimulated proliferation of human umbilical vein
endothelial cells (HUVEC).
Figure 18 depicts VEGF-2 stimulated proliferation of dermal microvascular
endothelial cells.
Figure 19 depicts the stimulatory effect of VEGF-2 on proliferation of
microvascular, umbilical cord, endometrial, and bovine aortic endothelial
cells.
Figure 20 depicts inhibition of PDGF-induced vascular (human aortic) smooth
2o muscle cell proliferation.
Figure 21 depicts stimulation of migration of HUVEC and bovine
microvascular endothelial cells (BMEC) by VEGF-2.
Figure 22 depicts stimulation of nitric oxide release of HUVEC by VEGF-2
and VEGF-1.
25 Figure 23 depicts inhibition of cord formation of microvascular endothelial
cells (CADMEC) by VEGF-2.
Figure 24 depicts stimulation of angiogenesis by VEGF, VEGF-2, and bFGF
in the CAM assay.
Figure 25 depicts restoration of certain parameters in the ischemic limb by
3o VEGF-2 protein (Figure 25, top panels) and naked expression plasmid (Figure
25,
middle panels): BP ratio (Figure 25a); Blood Flow and Flow Reserve (Figure
25b);
Angiographic Score (Figure 25c); Capillary density (Figure 25d).
Figures 26 A-G depicts ability of VEGF-2 to affect the diastolic blood
pressure in spontaneously hypertensive rats (SHR). Figures 26a and b depict
the
35 dose-dependent decrease in diastolic blood pressure achieved with VEGF-2.
(Figures
26c and d depict the decreased mean arterial pressure (MAP) observed with VEGF-
2.
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Panel E shows the effect of increasing doses of VEGF-2 on the mean arterial
pressure
(MAP) of SHR rats. Panel F shows the effect of VEGF-2 on the diastolic
pressure of
SHR rats. Panel G shows the effect of VEGF-2 on the diastolic blood pressure
of
SHR rats.
Figure 27 depicts inhibition of VEGF-2N= and VEGF-2-induced
proliferation.
Figure 28 shows a schematic representation of the pHE4a expression vector
(SEQ ID N0:16). The locations of the kanamycin resistance marker gene, the
multiple cloning site linker region, the oriC sequence, and the lacIq coding
sequence
i o are indicated.
Figure 29 shows the nucleotide sequence of the regulatory elements of the
pHE4a promoter (SEQ ID N0:17). The two lac operator sequences, the Shine-
Delgarno sequence (S/D), and the terminal HindIII and NdeI restriction sites
(italicized) are indicated.
Detailed Description of the Preferred Embodiments
In accordance with one aspect of the present invention, there are provided
isolated nucleic acid molecules comprising a polynucleotide encoding a VEGF-2
polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID N0:2),
2o which was determined by sequencing a cloned cDNA. The nucleotide sequence
shown in SEQ >Z7 NO:1 was obtained by sequencing a cDNA clone, which was
deposited on May 12, 1995 at the American Type Tissue Collection (ATCC), 10801
University Boulevard, Manassas, VA 20110-2209, and given ATCC Deposit No.
97149.
In accordance with another aspect of the present invention, there are provided
isolated nucleic acid molecules comprising a polynucleotide encoding a
truncated
VEGF-2 polypeptide having the deduced amino acid sequence of Figure 2 (SEQ ID
N0:4), which was determined by sequencing a cloned cDNA. The nucleotide
sequence shown in SEQ ID N0:3 was obtained by sequencing a cDNA clone, which
3o was deposited on March 4, 1994 at the American Type Tissue Collection
(ATCC),
10801 University Boulevard, Manassas, VA 20110-2209, and given ATCC Deposit
Number 75698.
Unless otherwise indicated, all nucleotide sequences determined by
sequencing a DNA molecule herein were determined using an automated DNA
sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino
acid
sequences of polypeptides encoded by DNA molecules determined herein were
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predicted by translation of a DNA sequence determined as above. Therefore, as
is
known in the art for any DNA sequence determined by this automated approach,
any
nucleotide sequence determined herein may contain some errors. Nucleotide
sequences determined by automation are typically at least about 90% identical,
more
typically at least about 95% to at least about 99.9% identical to the actual
nucleotide
sequence of the sequenced DNA molecule. The actual sequence can be more
precisely determined by other approaches including manual DNA sequencing
methods
well known in the art. As is also known in the art, a single insertion or
deletion in a
determined nucleotide sequence compared to the actual sequence will cause a
frame
shift in translation of the nucleotide sequence such that the predicted amino
acid
sequence encoded by a determined nucleotide sequence will be completely
different
from the amino acid sequence actually encoded by the sequenced DNA molecule,
beginning at the point of such an insertion or deletion.
A polynucleotide encoding a polypeptide of the present invention may be
obtained from early stage human embryo (week 8 to 9) osteoclastomas, adult
heart or
several breast cancer cell lines. The polynucleotide of this invention was
discovered in
a cDNA library derived from early stage human embryo week 9. It is
structurally
related to the VEGF/PDGF family. It contains an open reading frame encoding a
protein of about 419 amino acid residues of which approximately the first 23
amino
2o acid residues are the putative leader sequence such that the mature protein
comprises
396 amino acids, and which protein exhibits the highest amino acid sequence
homology to human vascular endothelial growth factor (30% identity), followed
by
PDGFa (24%) and PDGFb (22%). (See Figure 4). It is particularly important that
all
eight cysteines are conserved within all four members of the family (see boxed
areas
of Figure 3). In addition, the signature for the PDGF/VEGF family,
PXCVXXXRCXGCCN, (SEQ )D N0:8) is conserved in VEGF-2 (see Figure 3).
The homology between VEGF-2, VEGF and the two PDGFs is at the protein
sequence level. No nucleotide sequence homology can be detected, and
therefore, it
would be difficult to isolate the VEGF-2 through simple approaches such as low
stringency hybridization.
The VEGF-2 polypeptide of the present invention is meant to include the full
length polypeptide and polynucleotide sequence which encodes for any leader
sequences and for active fragments of the full length polypeptide. Active
fragments
are meant to include any portions of the full length amino acid sequence which
have
less than the full 419 amino acids of the full length amino acid sequence as
shown in
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SEQ ID N0:2, but still contain the eight cysteine residues shown conserved in
Figure
3 and that still have VEGF-2 activity.
There are at least two alternatively spliced VEGF-2 mRNA sequences present
in normal tissues. The two bands in Figure 7, lane 5 indicate the presence of
the
alternatively spliced mRNA encoding the VEGF-2 polypeptide of the present
invention.
The polynucleotide of the present invention may be in the form of RNA or in
the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
The DNA may be double-stranded or single-stranded, and if single stranded may
be
the coding strand or non-coding (anti-sense) strand. The coding sequence which
encodes the mature polypeptide may be identical to the coding sequence shown
in
Figure 1 or Figure 2, or that of the deposited clones, or may be a different
coding
sequence which, as a result of the redundancy or degeneracy of the genetic
code,
encodes the same, mature polypeptide as the DNA of Figure 1, Figure 2, or the
~ 5 deposited cDNAs.
The polynucleotide which encodes for the mature polypeptide of Figure 1 or
Figure 2 or for the mature polypeptides encoded by the deposited cDNAs may
include: only the coding sequence for the mature polypeptide; the coding
sequence for
the mature polypeptide and additional coding sequences such as a leader or
secretory
20 sequence or a proprotein sequence; the coding sequence for the mature
polypeptide
(and optionally additional coding sequences) and non-coding sequences, such as
introns or non-coding sequence 5' and/or 3' of the coding sequence for the
mature
polypeptide.
Thus, the term "polynucleotide encoding a polypeptide" encompasses a
25 polynucleotide which includes only coding sequences for the polypeptide as
well as a
polynucleotide which includes additional coding and/or non-coding sequences.
The present invention further relates to variants of the hereinabove described
polynucleotides which encode for fragments, analogs, and derivatives of the
polypeptide having the deduced amino acid sequence of Figures 1 or 2, or the
30 polypeptide encoded by the cDNA of the deposited clones. The variant of the
polynucleotide may be a naturally occurring allelic variant of the
polynucleotide or a
non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same
mature polypeptide as shown in Figures 1 or 2 or the same mature polypeptide
35 encoded by the cDNA of the deposited clones as well as variants of such
polynucleotides which variants encode for a fragment, derivative, or analog of
the
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polypeptides of Figures I or 2, or the polypeptide encoded by the cDNA of the
deposited clones. Such nucleotide variants include deletion variants,
substitution
variants, and addition or insertion variants.
As hereinabove indicated, the polynucleotide may have a coding sequence
which is a naturally occurring allelic variant of the coding sequence shown in
Figures
1 or 2, or of the coding sequence of the deposited clones. As known in the
art, an
allelic variant is an alternate form of a polynucleotide sequence which have a
substitution, deletion or addition of one or more nucleotides, which does not
substantially alter the function of the encoded polypeptide.
1 o The present invention also includes polynucleotides, wherein the coding
sequence for the mature polypeptide may be fused in the same reading frame to
a
polynucleotide which aids in expression and secretion of a polypeptide from a
host
cell, for example, a leader sequence which functions as a secretory sequence
for
controlling transport of a polypeptide from the cell. The polypeptide having a
leader
sequence is a preprotein and may have the leader sequence cleaved by the host
cell to
form the mature form of the polypeptide. The polynucleotides may also encode
for a
proprotein which is the mature protein plus additional 5' amino acid residues.
A
mature protein having a prosequence is a proprotein and is an inactive form of
the
protein. Once the prosequence is cleaved an active mature protein remains.
2o Thus, for example, the polynucleotide of the present invention may encode
for
a mature protein, or for a protein having a prosequence or for a protein
having both a
prosequence and presequence (leader sequence).
The polynucleotides of the present invention may also have the coding
sequence fused in frame to a marker sequence which allows for purification of
the
polypeptide of the present invention. The marker sequence may be a hexa-
histidine
tag supplied by a pQE-9 vector to provide for purification of the mature
polypeptide
fused to the marker in the case of a bacterial host, or, for example, the
marker
sequence may be a hemagglutinin (HA) tag when a mammalian host, e. g. COS-7
cells, is used. The HA tag corresponds to an epitope derived from the
influenza
3o hemagglutinin protein (Wilson, L, et al., Cell 37:767 ( 1984)).
Further embodiments of the invention include isolated nucleic acid molecules
comprising a polynucieotide having a nucleotide sequence at least 95%
identical, and
more preferably at least 96%, 97%, 98% or 99% identical to (a) a nucleotide
sequence
encoding the polypeptide having the amino acid sequence in SEQ ID N0:2; (b) a
nucleotide sequence encoding the polypeptide having the amino acid sequence in
SEQ
ll) N0:2, but lacking the N-terminal methionine; (c) a nucleotide sequence
encoding
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the polypeptide having the amino acid sequence at positions from about 1 to
about 396
in SEQ ID N0:2; (d) a nucleotide sequence encoding the polypeptide having the
amino
acid sequence encoded by the cDNA clone contained in ATCC Deposit No.97149;
(e)
a nucleotide sequence encoding the mature VEGF-2 polypeptide having the amino
acid
sequence encoded by the cDNA clone contained in ATCC Deposit No.97149; or (f)
a
nucleotide sequence complementary to any of the nucleotide sequences in (a),
(b), (c),
(d), or (e).
Further embodiments of the invention include isolated nucleic acid molecules
comprising a polynucleotide having a nucleotide sequence at least 95%
identical, and
1o more preferably at least 96%, 97%, 98% or 99% identical to (a) a nucleotide
sequence
encoding the polypeptide having the amino acid sequence in SEQ ID N0:4; (b) a
nucleotide sequence encoding the polypeptide having the amino acid sequence in
SEQ
117 N0:4, but lacking the N-terminal methionine; (c) a nucleotide sequence
encoding
the polypeptide having the amino acid sequence at positions from about 1 to
about 326
in SEQ ID N0:4; (d) a nucleotide sequence encoding the polypeptide having the
amino
acid sequence encoded by the cDNA clone contained in ATCC Deposit No.75698;
(e)
a nucleotide sequence encoding the mature VEGF-2 polypeptide having the amino
acid
sequence encoded by the cDNA clone contained in ATCC Deposit No.75698; or (f)
a
nucleotide sequence complementary to any of the nucleotide sequences in (a),
(b), (c),
(d), or (e).
By a polynucleotide having a nucleotide sequence at least, for example, 95%
"identical" to a reference nucleotide sequence encoding a VEGF-2 polypeptide
is
intended that the nucleotide sequence of the polynucleotide is identical to
the reference
sequence except that the polynucleotide sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleotide sequence
encoding the
VEGF-2 polypeptide. In other words, to obtain a polynucleotide having a
nucleotide
sequence at least 95% identical to a reference nucleotide sequence, up to 5%
of the
nucleotides in the reference sequence may be deleted or substituted with
another
nucleotide, or a number of nucleotides up to 5% of the total nucleotides in
the
3o reference sequence may be inserted into the reference sequence. These
mutations of
the reference sequence may occur at the 5N or 3N terminal positions of the
reference
nucleotide sequence or anywhere between those terminal positions, interspersed
either
individually among nucleotides in the reference sequence or in one or more
contiguous
groups within the reference sequence.
As a practical matter, whether any particular nucleic acid molecule is at
least
95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence
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shown in SEQ ID NOS:1 or 3, or to the nucleotides sequence of the deposited
cDNA
clones) can be determined conventionally using known computer programs such as
the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science Drive, Madison,
WI 53711 ). Bestfit uses the local homology algorithm of Smith and Waterman,
Advances in Applied Mathematics 2: 482-489 ( 198 I ), to find the best segment
of
homology between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is, for instance,
95%
identical to a reference sequence according to the present invention, the
parameters are
t 0 set, of course, such that the percentage of identity is calculated over
the full length of
the reference nucleotide sequence and that gaps in homology of up to 5% of the
total
number of nucleotides in the reference sequence are allowed.
As described in detail below, the polypeptides of the present invention can be
used to raise polyclonal and monoclonal antibodies, which are useful in
diagnostic
assays for detecting VEGF-2 protein expression as described below or as
agonists and
antagonists capable of enhancing or inhibiting VEGF-2 protein function.
Further,
such polypeptides can be used in the yeast two-hybrid system to "capture" VEGF-
2
protein binding proteins which are also candidate agonist and antagonist
according to
the present invention. The yeast two hybrid system is described in Fields and
Song,
2o Nature 340:245-246 ( 1989).
In another aspect, the invention provides a peptide or polypeptide comprising
an epitope-bearing portion of a polypeptide of the invention. As to the
selection of
peptides or polypeptides bearing an antigenic epitope (i.e., that contain a
region of a
protein molecule to which an antibody can bind), it is well known in that art
that
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,
for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A.
(1983)
Antibodies that react with predetermined sites on proteins. Science 219:660-
666.
Peptides capable of eliciting protein-reactive sera are frequently represented
in the
3o primary sequence of a protein, can be characterized by a set of simple
chemical rules,
and are confined neither to immunodominant regions of intact proteins (i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that
are
extremely hydrophobic and those of six or fewer residues generally are
ineffective at
inducing antibodies that bind to the mimicked protein; longer, soluble
peptides,
especially those containing proline residues, usually are effective. Sutcliffe
et al.,
supra, at 661. For instance, 18 of 20 peptides designed according to these
guidelines,
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containing 8-39 residues covering 75% of the sequence of the influenza virus
hemagglutinin HA1 polypeptide chain, induced antibodies that reacted with the
HAI
protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18
from the rabies glycoprotein induced antibodies that precipitated the
respective
proteins.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore useful to raise antibodies, including monoclonal antibodies, that
bind
specifically to a polypeptide of the invention. Thus, a high proportion of
hybridomas
obtained by fusion of spleen cells from donors immunized with an antigen
epitope-
to bearing peptide generally secrete antibody reactive with the native
protein. Sutcliffe et
al., supra, at 663. The antibodies raised by antigenic epitope-bearing
peptides or
polypeptides are useful to detect the mimicked protein, and antibodies to
different
peptides may be used for tracking the fate of various regions of a protein
precursor
which undergoes post-translational processing. The peptides and anti-peptide
antibodies may be used in a variety of qualitative or quantitative assays for
the
mimicked protein, for instance in competition assays since it has been shown
that even
short peptides (e.g., about 9 amino acids) can bind and displace the larger
peptides in
immunoprecipitation assays. See, for instance, Wilson et al., Cell 37:767-778
(1984)
at 777. The anti-peptide antibodies of the invention also are useful for
purification of
2o the mimicked protein, for instance, by adsorption chromatography using
methods well
known in the art.
Antigenic epitope-bearing peptides and polypeptides of the invention designed
according to the above guidelines preferably contain a sequence of at least
seven,
more preferably at least nine and most preferably between about 15 to about 30
amino
acids contained within the amino acid sequence of a polypeptide of the
invention.
However, peptides or polypeptides comprising a larger portion of an amino acid
sequence of a polypeptide of the invention, containing about 30, 40, 50, 60,
70, 80,
90, 100, or 150 amino acids, or any length up to and including the entire
amino acid
sequence of a polypeptide of the invention, also are considered epitope-
bearing
peptides or polypeptides of the invention and also are useful for inducing
antibodies
that react with the mimicked protein. Preferably, the amino acid sequence of
the
epitope-bearing peptide is selected to provide substantial solubility in
aqueous solvents
(i.e., the sequence includes relatively hydrophilic residues and highly
hydrophobic
sequences are preferably avoided); and sequences containing proline residues
are
particularly preferred.
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-14
Non-limiting examples of antigenic polypeptides or peptides that can be used
to generate VEGF-2-specific antibodies include the following: a polypeptide
comprising amino acid residues from about leu-37 to about glu-45 in SEQ ID
N0:2,
from about Tyr-58 to about Gly-66 in SEQ ID N0:2, from about Gln-73 to about
Glu-81 in SEQ ID N0:2, from about Asp-100 to about Cys-108 in SEQ >D N0:2,
from about Gly-140 to about Leu-148 in SEQ ID N0:2, from about Pro-168 to
about
Val-176 in SEQ >D N0:2, from about His-183 to about Lys-191 in SEQ ID N0:2,
from about Ile-201 to about Thr-209 in SEQ ID N0:2, from about Ala-216 to
about
Tyr-224 in SEQ ID N0:2, from about Asp-244 to about His-254 in SEQ ID N0:2,
from about Gly-258 to about Glu-266 in SEQ ID N0:2, from about Cys-272 to
about
Ser-280 in SEQ ID N0:2, from about Pro-283 to about Ser-291 in SEQ lD N0:2,
from about Cys-296 to about Gln-304 in SEQ ID N0:2, from about Ala-307 to
about
Cys-316 in SEQ ID N0:2, from about Val-319 to about Cys-335 in SEQ ID N0:2,
from about Cys-339 to about Leu-347 in SEQ ID N0:2, from about Cys-360 to
about
Glu-373 in SEQ ID N0:2, from about Tyr-378 to about Val-386 in SEQ ID N0:2,
and from about Ser-388 to about Ser-396 in SEQ ll~ N0:2. These polypeptide
fragments have been determined to bear antigenic epitopes of the VEGF-2
protein by
the analysis of the Jameson-Wolf antigenic index.
The epitope-bearing peptides and polypeptides of the invention may be
produced by any conventional means for making peptides or polypeptides
including
recombinant means using nucleic acid molecules of the invention. For instance,
a
short epitope-bearing amino acid sequence may be fused to a larger polypeptide
that
acts as a carrier during recombinant production and purification, as well as
during
immunization to produce anti-peptide antibodies. Epitope-bearing peptides also
may
be synthesized using known methods of chemical synthesis. For instance,
Houghten
has described a simple method for synthesis of large numbers of peptides, such
as 10-
20 mg of 248 different 13 residue peptides representing single amino acid
variants of a
segment of the HA 1 polypeptide which were prepared and characterized (by
ELISA-
type binding studies) in less than four weeks. Houghten, R. A. ( 1985) General
method for the rapid solid-phase synthesis of large numbers of peptides:
specificity of
antigen-antibody interaction at the level of individual amino acids. Proc.
Natl. Acad.
Sci. USA 82:5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)"
process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (
1986).
In this procedure the individual resins for the solid-phase synthesis of
various
peptides are contained in separate solvent-permeable packets, enabling the
optimal use
of the many identical repetitive steps involved in solid-phase methods. A
completely
CA 02322748 2000-09-07
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-15
manual procedure allows 500-1000 or more syntheses to be conducted
simultaneously. Houghten et al., supra, at 5134.
Epitope-bearing peptides and polypeptides of the invention are used to induce
antibodies according to methods well known in the art. See, for instance,
Sutcliffe et
al., supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA
82:910
914; and Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985). Generally,
animals
may be immunized with free peptide; however, anti-peptide antibody titer may
be
boosted by coupling of the peptide to a macromolecular earner, such as keyhole
limpet
hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine
may
1 o be coupled to carrier using a linker such as m-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier
using a more general linking agent such as glutaraldehyde. Animals such as
rabbits,
rats and mice are immunized with either free or carrier-coupled peptides, for
instance,
by intraperitoneal and/or intradermal injection of emulsions containing about
100 mg
i 5 peptide or carrier protein and Freund's adjuvant. Several booster
injections may be
needed, for instance, at intervals of about two weeks, to provide a useful
titer of anti-
peptide antibody which can be detected, for example, by ELISA assay using free
peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in
serum from
an immunized animal may be increased by selection of anti-peptide antibodies,
for
20 instance, by adsorption to the peptide on a solid support and elution of
the selected
antibodies according to methods well known in the art.
Immunogenic epitope-bearing peptides of the invention, i.e., those parts of a
protein that elicit an antibody response when the whole protein is the
immunogen, are
identified according to methods known in the art. For instance, Geysen et al.,
supra,
25 discloses a procedure for rapid concurrent synthesis on solid supports of
hundreds of
peptides of sufficient purity to react in an enzyme-linked immunosorbent
assay.
Interaction of synthesized peptides with antibodies is then easily detected
without
removing them from the support. In this manner a peptide bearing an
immunogenic
epitope of a desired protein may be identified routinely by one of ordinary
skill in the
30 art. For instance, the immunologically important epitope in the coat
protein of foot-
and-mouth disease virus was located by Geysen et ul. with a resolution of
'even
amino acids by synthesis of an overlapping set of all 208 possible
hexapeptides
covering the entire 213 amino acid sequence of the protein. Then, a complete
replacement set of peptides in which all 20 amino acids were substituted in
turn at
35 every position within the epitope were synthesized, and the particular
amino acids
conferring specificity for the reaction with antibody were determined. Thus,
peptide
CA 02322748 2000-09-07
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-16
analogs of the epitope-bearing peptides of the invention can be made routinely
by this
method. U.S. Patent No. 4,708,781 to Geysen ( 1987) further describes this
method
of identifying a peptide bearing an immunogenic epitope of a desired protein.
Further still, U.S. Patent No. 5,194,392 to Geysen ( 1990) describes a
general method of detecting or determining the sequence of monomers (amino
acids or
other compounds) which is a topological equivalent of the epitope (i.e., a
Amimotope)
which is complementary to a particular paratope (antigen binding site) of an
antibody
of interest. More generally, U.S. Patent No. 4,433,092 to Geysen ( 1989)
describes a
method of detecting or determining a sequence of monomers which is a
topographical
equivalent of a ligand which is complementary to the ligand binding site of a
particular
receptor of interest. Similarly, U.S. Patent No. 5,480,971 to Houghten, R. A.
et al.
( 1996) on Peralkylated Oligopeptide Mixtures discloses linear C,-C~-alkyl
peralkylated oligopeptides and sets and libraries of such peptides, as well as
methods
for using such oligopeptide sets and libraries for determining the sequence of
a
~5 peralkylated oligopeptide that preferentially binds to an acceptor molecule
of interest.
Thus, non-peptide analogs of the epitope-bearing peptides of the invention
also can be
made routinely by these methods.
As one of skill in the art will appreciate, VEGF-2 polypeptides of the present
invention and the epitope-bearing fragments thereof described above can be
combined
2o with parts of the constant domain of immunoglobulins (IgG), resulting in
chimeric
polypeptides. These fusion proteins facilitate purification and show an
increased
half life in vivo. This has been shown, e.g., for chimeric proteins consisting
of the
first two domains of the human CD4-polypeptide and various domains of the
constant
regions of the heavy or light chains of mammalian immunoglobulins (EPA
394,827;
25 Traunecker et al., Nature 331:84- 86 ( 1988)).
In accordance with the present invention, novel variants of VEGF-2 are also
described. These can be produced by deleting or substituting one or more amino
acids
of VEGF-2. Natural mutations are called allelic variations. Allelic variations
can be
silent (no change in the encoded polypeptide) or may have altered amino acid
3o sequence.
In order to attempt to improve or alter the characteristics of native VEGF-2,
protein engineering may be employed. Recombinant DNA technology known to those
skilled in the art can be used to create novel polypeptides. Muteins and
deletions can
show, e.g., enhanced activity or increased stability. In addition, they could
be purified
35 in higher yield and show better solubility at least under certain
purification and storage
conditions. Set forth below are examples of mutations that can be constructed.
CA 02322748 2000-09-07
WO 99/46364 PCTNS99105021
-17
Amino terminal and carboxy terminal deletions
Furthermore, VEGF-2 appears to be proteolytically cleaved upon expression
resulting in polypeptide fragments of the following sizes when run on a SDS-
PAGE
gel (sizes are approximate) (See, Figures 6-8, for example): 80, 59, 45, 43,
4I, 40,
39, 38, 37, 36, 31, 29, 21, and 15 kDa. These polypeptide fragments are the
result
of proteolytic cleavage at both the N-terminal and C-terminal portions of the
protein.
These proteolytically generated fragments appears to have activity,
particularly the 21
lcDa fragment.
In addition, protein engineering may be employed in order to improve or alter
one or more characteristics of native VEGF-2. The deletion of carboxyterminal
amino
acids can enhance the activity of proteins. One example is interferon gamma
that
shows up to ten times higher activity by deleting ten amino acid residues from
the
carboxy terminus of the protein (Dobeli et al., J. of Biotechnology 7:199-216
( 1988)).
Thus, one aspect of the invention is to provide polypeptide analogs of VEGF-2
and
nucleotide sequences encoding such analogs that exhibit enhanced stability
(e.g.,
when exposed to typical pH, thermal conditions or other storage conditions)
relative to
the native VEGF-2 polypeptide.
Particularly preferred VEGF-2 polypeptides are shown below (numbering
starts with the first amino acid in the protein (Met) (Figure 1 (SEQ 1D
N0:18)): Ala
(residue 24) to Ser (residue 419); Pro (25) to Ser (419); Ala (26) to Ser
(419); Ala
(27) to Ser (419); Ala (28) to Ser (419); Ala (29) to Ser (419); Ala (30) to
Ser (419);
Phe (31 ) to Ser (419); Glu (32) to Ser (419); Ser (33) to Ser (419); Gly (34)
to Ser
(419); Leu (35) to Ser (419); Asp (36) to Ser (419); Leu (37) to (Ser (419);
Ser (38)
to Ser (419); Asp (39)to Ser (419); Ala (40) to Ser (419); Glu (41 ) to Ser
(419); Pro
(42) to Ser (419); Asp (43) to Ser (419); Ala (44) to Ser (419); Gly (45) to
Ser (419);
Glu (46) to Ser (419); Ala (47) to Ser (419); Thr (48) to Ser (419); Ala (49)
to Ser
(419); Tyr (50) to Ser (419); Ser (52) to Ser (419); Asp (54) to Ser (419);
Val (62) to
Ser (419); Val (65) to Ser (419); Met( 1 ), Glu (23), or Ala (24) to Met
(418); Met ( 1 ),
Glu (23), or Ala (24) to Gln (417); Met (1), Glu (23), or Ala (24) to Pro
(416);
3o Met(1), Glu (23), or Ala (24} to Arg (415); Met(1), Glu (23), or Ala (24)
to Gln
(414); Met( 1 ), Glu (23), or Ala (24) to Trp (413}; Met( 1 ), Glu (23), or
Ala (24) to
Tyr (412); Met( 1 ), Glu (23), or Ala (24) to Ser (411 ); Met( 1 ), Glu (23),
or Ala (24)
to Pro (410); Met ( 1 ), Glu (23), or Ala(24) to Val (409); Met ( 1 ), Glu
(23), or Ala
(24) to Cys (408); Met( 1 ), Glu (23), or Ala (24) to Arg (407); Met( 1 ), Glu
(23), or
Ala (24) to Cys (406); Met ( 1 ), Glu (23), or Ala (24) to Val (405); Met( 1
), Glu (23),
or Ala (24) to Glu (404); Met( 1 ), Glu (23), or Ala (24) to Glu (403); Met( 1
), Glu
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-I 8
(23), or Ala (24) to Ser (402); Met( 1 ), Glu (23), or Ala (24) to Gly (398);
Met( 1 ),Glu
(23), or Ala (24) to Pro (397); Met( 1 ), Glu (23 ), or Ala (24) to Lys (393);
Met( 1 },
Glu (23), or Ala (24) to Met{263); Met( 1 ), Glu (23), or Ala (24) to Asp(311
); Met( I ),
Glu (23), or Ala (24) to Pro (367); Met(1) to Ser (419); Met(1) to Ser(228);
Glu(47)
to Ser(419); Ala( 1 I 1 ) to Lys(214); Ala( 112) to Lys(214); His( 1 I 3) to
Lys(214);
Tyr( 114) to Lys(214); Asn( 115) to Lys(214); Thr( 116) to Lys(214); Thr( 103)
to
Leu(215); Glu( 104) to Leu(215); Glu( 105) to Leu(215); Thr( 106) to Leu(215);
Ile( 107) to Leu(215); Lys( 108) to Leu(2 I5); Phe( 109) to Leu(215); Ala(
110) to
Leu(215); Ala( 11 I ) to Leu(215); Ala( 112) to Leu(215); His( 113) to
Leu(215);
to Tyr( 114) to Leu(215); Asn( 115) to Leu(215); Thr( 116) to Leu(215); Thr(
103) to
Ser(228); Glu( 104) to Ser(228); Glu( 105) to Ser(228); Thr( 106) to Ser(228);
Ile( 107) to Ser(228); Lys( 108) to Ser(228); Phe( 109) to Ser(228); Ala( 110)
to
Ser(228); Ala( 111 ) to Ser(228); Ala( 112) to Ser(228); His( 113) to
Ser(228);
Tyr( 114) to Ser(228); Asn( 115) to Ser(228); Thr( 116) to Ser(228); Thr( 103)
to
~ 5 Leu(229); Glu( 104) to Leu(229); Thr( 103) to Arg(227); Glu( 104) to
Arg(227);
Glu( 105) to Arg (227); Thr( 106) to Arg (227); Ile( 107) to Arg (227); Lys{
108) to
Arg (227); Phe( 109) to Arg (227); Ala( 110) to Arg (227); Ala( 111 ) to Arg
(227);
Ala( 112) to Arg (227); His( 113) to Arg (227); Tyr( 114} to Arg (227); Asn(
115) to
Arg (227); Thr( 116) to Arg (227); Thr( 103) to Ser(213); Glu( 104) to
Ser(213);
2o Glu(105) to Ser(213); Thr(106) to Ser(213); Ile(107) to Ser(213); Lys(108)
to
Ser(213); Phe(109) to Ser(213); Ala(110) to Ser(213); Ala(111) to Ser(213);
Ala( 112) to Ser(213); His( 113) to Ser{213); Tyr( 114) to Ser(213); Asn( 115)
to
Ser(213); Thr( 116) to Ser(213); Thr( 103) to Lys(214); Glu( 104) to Lys(214);
Glu( 105) to Lys(214); Thr( 106) to Lys(214); Ile( 107) to Lys(214); Lys( 108}
to
25 Lys(214); Phe{ 109) to Lys(214); Ala( 110) to Lys(214); Glu( 105) to
Leu(229);
Thr( 106) to Leu(229); Ile( 107) to Leu{229); Lys( 108) to Leu(229); Phe( 109)
to
Leu(229); Ala( 110) to Leu(229); Ala( 111 ) to Leu(229); Ala( 112) to
Leu(229);
His( 113) to Leu(229); Tyr( 114) to Leu(229); Asn( 115) to Leu(229); Thr( 116)
to
Leu(229).
30 Preferred embodiments include the following deletion mutants: Thr( 103) -
Arg(227); Glu( 104) -- Arg(227); Ala(112) -- Arg (227); Thr( 103) -- Ser(213);
Glu( 104) -- Ser(213); Thr( 103) -- Leu(215); Glu(47) -- Ser(419}; Met( 1 ),
Glu (23),
or Ala (24) -- Met(263); Met( 1 ), Glu (23), or Ala (24) -- Asp(311 ); Met( 1
), Glu (23),
or Ala (24) -- Pro (367); Met( 1 ) -- Ser(419); and Met( 1 ) -- Ser(228) of
(Figure I
35 (SEQ ID N0:18)).
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-19
Also included by the present invention are deletion mutants having amino acids
deleted from both the NB terminus and the C-terminus. Such mutants include all
combinations of the N-terminal deletion mutants and C-terminal deletion
mutants
described above. Those combinations can be made using recombinant techniques
known to those skilled in the art.
Particularly, N-terminal deletions of the VEGF-2 polypeptide can be described
by the general formula m-396, where m is an integer from -23 to 388, where m
corresponds to the position of the amino acid residue identified in SEQ ID
N0:2.
Preferably, N-terminal deletions retain the conserved boxed area of Figure 3
(PXCVXXXRCXGCCN)(SEQ ID NO: 8), and include polypeptides comprising the
amino acid sequence of residues: N-terminal deletions of the polypeptide of
the
invention shown as SEQ ID NO:1 include polypeptides comprising the amino acid
sequence of residues: : E-1 to S-396; A-2 to S-396; P-3 to S-396; A-4 to S-
396; A-S
to S-396; A-6 to S-396; A-7 to S-396; A-8 to S-396; F-9 to S-396; E-10 to S-
396; S-
I1 to S-396; G-12 to S-396; L-13 to S-396; D-14 to S-396; L-IS to S-396; S-16
to S-
396; D-17 to S-396; A-18 to S-396; E-19 to S-396; P-20 to S-396; D-21 to S-
396; A-
22 to S-396; G-23 to S-396; E-24 to S-396; A-25 to S-396; T-26 to S-396; A-27
to S-
396; Y-28 to S-396; A-29 to S-396; S-30 to S-396; K-31 to S-396; D-32 to S-
396; L-
33 to S-396; E-34 to S-396; E-35 to S-396; Q-36 to S-396; L-37 to S-396; R-38
to S-
396; S-39 to S-396; V-40 to S-396; S-41 to S-396; S-42 to S-396; V-43 to S-
396; D-
44 to S-396; E-45 to S-396; L-46 to S-396; M-47 to S-396; T-48 to S-396; V-49
to S-
396; L-50 to S-396; Y-51 to S-396; P-52 to S-396; E-53 to S-396; Y-54 to S-
396; W-
55 to S-396; K-56 to S-396; M-57 to S-396; Y-58 to S-396; K-59 to S-396; C-60
to
S-396; Q-61 to S-396; L-62 to S-396; R-63 to S-396; K-64 to S-396; G-65 to S-
396;
G-66 to S-396; W-67 to S-396; Q-68 to S-396; H-69 to S-396; N-70 to S-396; R-
71
to S-396; E-72 to S-396; Q-73 to S-396; A-74 to S-396; N-75 to S-396; L-76 to
S-
396; N-77 to S-396; S-78 to S-396; R-79 to S-396; T-80 to S-396; E-81 to S-
396; E-
82 to S-396; T-83 to S-396; I-84 to S-396; K-85 to S-396; F-86 to S-396; A-87
to S-
396; A-88 to S-396; A-89 to S-396; H-90 to S-396; Y-91 to S-396; N-92 to S-
396; T-
93 to S-396; E-94 to S-396; I-95 to S-396; L-96 to S-396; K-97 to S-396; S-98
to S-
396; I-99 to S-396; D-100 to S-396; N-101 to S-396; E-102 to S-396; W-103 to S-
396; R-104 to S-396; K-105 to S-396; T-106 to S-396; Q-107 to S-396; C-108 to
S-
396; M-109 to S-396; P-110 to S-396; R-111 to S-396; E-112 to S-396; V-113 to
S-
396; C-I 14 to S-396; I-115 to S-396; D-I 16 to S-396; V-117 to S-396; G-118
to S-
396; K-119 to S-396; E- I 20 to S-396; F-121 to S-396; G-122 to S-396; V-123
to S-
396; A-124 to S-396; T-125 to S-396; N-126 to S-396; T-127 to S-396; F-128 to
S-
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396; F-129 to S-396; K-130 to S-396; P-131 to S-396; P-132 to S-396; C-133 to
S-
396; V-134 to S-396; S-135 to S-396; V-136 to S-396; Y-137 to S-396; R-138 to
S-
396; C-139 to S-396; G-140 to S-396; G-141 to S-396; C-142 to S-396; C-143 to
S-
396; N-144 to S-396; S-145 to S-396; E-146 to S-396; G-147 to S-396; L-148 to
S-
396; Q-149 to S-396; C-150 to S-396; M-151 to S-396; N-152 to S-396; T-153 to
S-
396; S-154 to S-396; T-155 to S-396; S-156 to S-396; Y-157 to S-396; L-158 to
5-
396; S-159 to S-396; K-160 to S-396; T-161 to S-396; L-162 to S-396; F-163 to
S-
396; E-164 to S-396; I-165 to S-396; T-166 to S-396; V-167 to S-396; P-168 to
S-
396; L-169 to S-396; S-170 to S-396; Q-171 to S-396; G-172 to S-396; P-173 to
S-
t o 396; K-174 to S-396; P-175 to S-396; V-176 to S-396; T-177 to S-396; I-178
to S-
396; S-179 to S-396; F-180 to S-396; A-181 to S-396; N-182 to S-396; H-183 to
S-
396; T-184 to S-396; S-185 to S-396; C-186 to S-396; R-187 to S-396; C-188 to
S-
396; M-189 to S-396; S-190 to S-396; K-191 to S-396; L-192 to S-396; D-193 to
S-
396; V-194 to S-396; Y-195 to S-396; R-196 to S-396; Q-I97 to S-396; V-198 to
S-
ts 396; H-199 to S-396; S-200 to S-396; I-201 to S-396; I-202 to S-396; R-203
to S-
396; R-204 to S-396; S-205 to S-396; L-206 to S-396; P-207 to S-396; A-208 to
S-
396; T-209 to S-396; L-210 to S-396; P-211 to S-396; Q-212 to S-396; C-213 to
S-
396; Q-214 to S-396; A-215 to S-396; A-216 to S-396; N-217 to S-396; K-218 to
S-
396; T-219 to S-396; C-220 to S-396; P-221 to S-396; T-222 to S-396; N-223 to
S-
20 396; Y-224 to S-396; M-225 to S-396; W-226 to S-396; N-227 to S-396; N-228
to S-
396; H-229 to S-396; I-230 to S-396; C-231 to S-396; R-232 to S-396; C-233 to
S-
396; L-234 to S-396; A-235 to S-396; Q-236 to S-396; E-237 to S-396; D-238 to
S-
396; F-239 to S-396; M-240 to S-396; F-241 to S-396; S-242 to S-396; S-243 to
S-
396; D-244 to S-396; A-245 to S-396; G-246 to S-396; D-247 to S-396; D-248 to
S-
2s 396; S-249 to S-396; T-250 to S-396; D-251 to S-396; G-252 to S-396; F-253
to S-
396; H-254 to S-396; D-255 to S-396; I-256 to S-396; C-257 to S-396; G-258 to
S-
396; P-259 to S-396; N-260 to S-396; K-261 to S-396; E-262 to S-396; L-263 to
5-
396; D-264 to S-396; E-265 to S-396; E-266 to S-396; T-267 to S-396; C-268 to
5-
396; Q-269 to S-396; C-270 to S-396; V-271 to S-396; C-272 to S-396; R-273 to
S-
30 396; A-274 to S-396; G-275 to S-396; L-276 to S-396; R-277 to S-396; P-278
to S-
396; A-279 to S-396; S-280 to S-396; C-281 to S-396; G-282 to S-396; P-283 to
S-
396; H-284 to S-396; K-285 to S-396; E-286 to S-396; L-287 to S-396; D-288 to
5-
396; R-289 to S-396; N-290 to S-396; S-291 to S-396; C-292 to S-396; Q-293 to
S-
396; C-294 to S-396; V-295 to S-396; C-296 to S-396; K-297 to S-396; N-298 to
S-
35 396; K-299 to S-396; L-300 to S-396; F-301 to S-396; P-302 to S-396; S-303
to S-
396; Q-304 to S-396; C-305 to S-396; G-306 to S-396; A-307 to S-396; N-308 to
S-
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WO 99/46364 PCT/US99/05021
-21
396; R-309 to S-396; E-310 to S-396; F-311 to S-396; D-312 to S-396; E-313 to
S-
396; N-314 to S-396; T-315 to S-396; C-316 to S-396; Q-317 to S-396; C-318 to
S-
396; V-319 to S-396; C-320 to S-396; K-321 to S-396; R-322 to S-396; T-323 to
S-
396; C-324 to S-396; P-325 to S-396; R-326 to S-396; N-327 to S-396; Q-328 to
S-
396; P-329 to S-396; L-330 to S-396; N-33I to S-396; P-332 to S-396; G-333 to
S-
396; K-334 to S-396; C-335 to S-396; A-336 to S-396; C-337 to S-396; E-338 to
5-
396; C-339 to S-396; T-340 to S-396; E-341 to S-396; S-342 to S-396; P-343 to
S-
396; Q-344 to S-396; K-345 to S-396; C-346 to S-396; L-347 to S-396; L-348 to
S-
396; K-349 to S-396; G-350 to S-396; K-351 to S-396; K-352 to S-396; F-353 to
S-
to 396; H-354 to S-396; H-355 to S-396; Q-356 to S-396; T-357 to S-396; C-358
to S-
396; S-359 to S-396; C-360 to S-396; Y-361 to S-396; R-362 to S-396; R-363 to
S-
396; P-364 to S-396; C-365 to S-396; T-366 to S-396; N-367 to S-396; R-368 to
5-
396; Q-369 to S-396; K-370 to S-396; A-371 to S-396; C-372 to S-396; E-373 to
S-
396; P-374 to S-396; G-375 to S-396; F-376 to S-396; S-377 to S-396; Y-378 to
S-
396; S-379 to S-396; E-380 to S-396; E-381 to S-396; V-382 to S-396; C-383 to
S-
396; R-384 to S-396; C-385 to S-396; V-386 to S-396; P-387 to S-396; S-388 to
S-
396; Y-389 to S-396; W-390 to S-396; Q-391 to S-396 of SEQ ID N0:2. One
preferred embodiment comprises amino acids S-205 to S-396 of SEQ ID N0:2. Also
preferred are polynucleotides encoding these polypeptides.
Moreover, C-terminal deletions of the VEGF-2 polypeptide can also be
described by the general formula -23-n, where n is an integer from -15 to 395
where n
corresponds to the position of amino acid residue identified in SEQ ID N0:2.
Preferably, C-terminal deletions retain the conserved boxed area of Figure 3
(PXCVXXXRCXGCCN)(SEQ ID NO: 8), and include polypeptides comprising the
amino acid sequence of residues: Likewise, C-terminal deletions of the
polypeptide of
the invention shown as SEQ ID N0:2 include polypeptides comprising the amino
acid
sequence of residues: E-1 to M-395; E-1 to Q-394; E-I to P-393; E-1 to R-392;
E-1 to
Q-391; E-1 to W-390; E-1 to Y-389; E-1 to S-388; E-1 to P-387; E-1 to V-386; E-
1 to
C-385; E-1 to R-384; E-1 to C-383; E-1 to V-382; E-1 to E-381; E-1 to E-380; E-
1 to
3o S-379; E-1 to Y-378; E-1 to S-377; E-1 to F-376; E-1 to G-375; E-1 to P-
374; E-1 to
E-373; E-1 to C-372; E-1 to A-371; E-1 to K-370; E-1 to Q-369; E- I to R-368;
E- I to
N-367; E-1 to T-366; E-1 to C-365; E-1 to P-364; E-1 to R-363; E-1 to R-362; E-
1 to
Y-361; E-1 to C-360; E-I to S-359; E-1 to C-358; E-1 to T-357; E-1 to Q-356; E-
1 to
H-355; E-1 to H-354; E-1 to F-353; E-1 to K-352; E-1 to K-351; E-1 to G-350; E-
1
to K-349; E-1 to L-348; E-1 to L-347; E-1 to C-346; E-1 to K-345; E-1 to Q-
344; E-l
to P-343; E-1 to S-342; E-1 to E-341; E-1 to T-340; E-1 to C-339; E-1 to E-
338; E-1
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-22
to C-337; E-1 to A-336; E-1 to C-335; E-I to K-334; E-I to G-333; E-1 to P-
332; E-I
to N-331; E-I to L-330; E-I to P-329; E-1 to Q-328; E-1 to N-327; E-1 to R-
326; E-1
to P-325; E- I to C-324; E-1 to T-323; E-1 to R-322; E- I to K-32 I ; E- I to
C-320; E- I
to V-319; E- I to C-318; E-I to Q-317; E-1 to C-316; E-1 to T-315; E-1 to N-
314; E-1
to E-313; E-1 to D-312; E-1 to F-311; E-1 to E-310; E- I to R-309; E-1 to N-
308 ; E- I
to A-307; E-1 to G-306; E-1 to C-305; E-1 to Q-304; E-1 to S-303; E- I to P-
302; E- I
to F-301; E-I to L-300; E-I to K-299; E-I to N-298; E-I to K-297; E-I to C-
296; E-1
to V-295; E- I to C-294; E-1 to Q-293 ; E- I to C-292; E-1 to S-29 I ; E-1 to
N-290; E- I
to R-289; E-I to D-288; E-1 to L-287; E-1 to E-286; E-1 to K-285; E-1 to H-
284; E-1
to P-283; E-1 to G-282; E-I to C-281; E-I to S-280; E-I to A-279; E-1 to P-
278; E-1
to R-277; E- I to L-276; E-1 to G-275; E- I to A-274; E- I to R-273; E- I to C-
272; E- I
to V-271; E- I to C-270; E-1 to Q-269; E-1 to C-268; E- I to T-267; E-1 to E-
266; E-1
to E-265; E-1 to D-264; E-1 to L-263; E-1 to E-262; E-1 to K-26 I ; E-1 to N-
260; E- I
to P-259; E-1 to G-258; E-1 to C-257; E-1 to I-256; E-1 to D-255; E-I to H-
254; E-1
to F-253 ; E-1 to G-252; E-1 to D-251; E-1 to T-250; E-1 to S-249; E- I to D-
248; E- I
to D-247; E-I to G-246; E-1 to A-245; E-1 to D-244; E-I to S-243; E-I to S-
242; E-I
to F-241; E-1 to M-240; E-1 to F-239; E-I to D-238; E-I to E-237; E-1 to Q-
236; E-1
to A-235; E-I to L-234; E-1 to C-233; E-1 to R-232; E-I to C-231; E-I to I-
230; E-I
to H-229; E-1 to N-228 ; E- I to N-227; E-1 to W-226; E- I to M-225; E- I to Y-
224; E-
1 to N-223 ; E-1 to T-222; E-1 to P-221; E-1 to C-220; E-1 to T-219; E- I to K-
218 ; E-
1 to N-217; E-1 to A-216; E-1 to A-215; E-I to Q-214; E-I to C-213; E-1 to Q-
212;
E-1 to P-211; E-1 to L-210; E-1 to T-209; E- I to A-208; E- I to P-207 ; E- I
to L-206;
E- I to S-205; E- I to R-204; E- I to R-203 ; E- I to I-202; E- I to I-201; E-
1 to S-200;
E-1 to H-199; E-I to V-198; E-1 to Q-197; E-1 to R-196; E-1 to Y-195; E-I to V-
194;
E-1 to D-193; E-I to L-192; E-1 to K-191; E-1 to S-190; E-1 to M-189; E-1 to C-
188;
-. E-1 to R-187; E-I to C-186; E-1 to S-185; E-1 to T-184;8-1 to H-183; E-I to
N-182;
E-1 to A-181; E-1 to F-180; E-1 to S-179; E-1 to I-178; E-1 to T-177; E-I to V-
176;
E-I to P-175; E-1 to K-174; E-1 to P-173; E-1 to G-172; E-I to Q-171; E-1 to S-
170;
E-I to L-169; E-I to P-168; E-I to V-167; E-1 to T-166; E-I to I-165; E-1 to E-
164;
3o E-I to F-163; E-I to L-162; E-1 to T-161; E-I to K-160; E-1 to S-159; E-1
to L-158;
E-1 to Y-157; E-1 to S-156; E-1 to T-155; E-1 to S-154; E-I to T-153; E-i to N-
152;
E-I to M-151; E-1 to C-150; E-1 to Q-149; E-1 to L-148; E-1 to G-147; E-1 to E-
146;
E-i to S-145; E-1 to N-144; E-1 to C-143; E-1 to C-142; E-I to G-141; E-1 to G-
140;
E-1 to C-139; E-1 to R-I38; E-i to Y-137; E-1 to V-136; E-I to S-135; E-i to V-
i34;
3s E-I to C-133; E-I to P-132; E-I to P-131; E-I to K-130; E-I to F-129; E-1
to F-128;
E-I to T-127; E-1 to N-126; E-i to T-125; E-I to A-124; E-I to V-123; E-1 to G-
122;
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E-1 to F-121; E-1 to E-120; E-1 to K-119; E-1 to G-118; E-1 to V-117; E-1 to D-
116;
E-1 to I-115; E-1 to C-114; E-1 to V-113; E-1 to E-112; E-1 to R-111; E-1 to P-
110;
E-1 to M-109; E-I to C-108; E-1 to Q-107; E-1 to T-106; E-1 to K-105; E-1 to R-
104;
E-1 to W-103 ; E-1 to E-102; E-1 to N-10 I ; E-1 to D-100; E-1 to I-99; E-1 to
S-98 ; E-
s 1 to K-97; E-1 to L-96; E-1 to I-95; E-1 to E-94; E-1 to T-93; E-1 to N-92;
E-1 to Y-
91; E-1 to H-90; E-1 to A-89; E-1 to A-88; E-1 to A-87; E-1 to F-86; E-1 to K-
85; E-1
to I-84; E-1 to T-83; E-1 to E-82; E-1 to E-81; E-1 to T-80; E-1 to R-79; E-I
to S-78;
E-1 to N-77; E-1 to L-76; E-1 to N-75; E-I to A-74; E-1 to Q-73; E-1 to E-72;
E-1 to
R-71; E-1 to N-70; E-1 to H-69; E-1 to Q-68; E-1 to W-67 ; E-1 to G-66; E-1 to
G-65 ;
E-1 to K-64; E-1 to R-63; E-1 to L-62; E-1 to Q-61; E-1 to C-60; E-1 to K-59;
E-1 to
Y-58; E-1 to M-57; E-I to K-56; E-1 to W-55; E-1 to Y-54; E-1 to E-53; E-1 to
P-52;
E-1 to Y-51; E-1 to L-50; E-1 to V-49; E-1 to T-48; E-1 to M-47; E-1 to L-46;
E-1 to
E-45; E-1 to D-44; E-I to V-43; E-1 to S-42; E-I to S-41; E-1 to V-40; E-1 to
S-39;
E-1 to R-38; E-1 to L-37; E-I to Q-36; E-1 to E-35; E-1 to E-34; E-1 to L-33;
E-1 to
15 D-32; E-1 to K-31; E-1 to S-30; E-I to A-29; E-1 to Y-28; E-1 to A-27; E-1
to T-26;
E- I to A-25; E-1 to E-24; E-1 to G-23; E- I to A-22; E- I to D-21; E-1 to P-
20; E-1 to
E-19; E-1 to A-18; E-1 to D-17; E-1 to S-16; E-1 to L-15; E-I to D-14; E-1 to
L-13;
E- I to G-12; E-1 to S-11; E-1 to E-10; E-1 to F-9; E-1 to A-8; E-1 to A-7 of
SEQ >D
N0:2. Also preferred are polynucleotides encoding these polypeptides.
2o Moreover, the invention also provides polypeptides having one or more amino
acids deleted from both the amino and the carboxyl termini, which may be
described
generally as having residues m-n of SEQ ID N0:2, where n and m are integers as
described above.
Likewise, also preferred are C-terminal deletions of the VEGF-2 polypeptide
25 of the invention shown as SEQ ID N0:2 which include polypeptides comprising
the
amino acid sequence of residues: F-9 to M-395; F-9 to Q-394; F-9 to P-393; F-9
to R-
392; F-9 to Q-391; F-9 to W-390; F-9 to Y-389; F-9 to S-388; F-9 to P-387; F-9
to
V-386; F-9 to C-385; F-9 to R-384; F-9 to C-383; F-9 to V-382; F-9 to E-381; F-
9 to
E-380; F-9 to S-379; F-9 to Y-378; F-9 to S-377; F-9 to F-376; F-9 to G-375; F-
9 to
3o P-374; F-9 to E-373; F-9 to C-372; F-9 to A-371; F-9 to K-370; F-9 to Q-
369; F-9 to
R-368; F-9 to N-367; F-9 to T-366; F-9 to C-365; F-9 to P-364; F-9 to R-363; F-
9 to
R-362; F-9 to Y-361; F-9 to C-360; F-9 to S-359; F-9 to C-358; F-9 to T-357; F-
9 to
Q-356; F-9 to H-355; F-9 to H-354; F-9 to F-353; F-9 to K-352; F-9 to K-351; F-
9 to
G-350; F-9 to K-349; F-9 to L-348; F-9 to L-347; F-9 to C-346; F-9 to K-345; F-
9 to
35 Q-344; F-9 to P-343; F-9 to S-342; F-9 to E-341; F-9 to T-340; F-9 to C-
339; F-9 to
E-338; F-9 to C-337; F-9 to A-336; F-9 to C-335; F-9 to K-334; F-9 to G-333; F-
9 to
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WO 99/46364 PCTNS99/05021
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P-332; F-9 to N-331; F-9 to L-330; F-9 to P-329; F-9 to Q-328; F-9 to N-327; F-
9 to
R-326; F-9 to P-325; F-9 to C-324; F-9 to T-323; F-9 to R-322; F-9 to K-321; F-
9 to
C-320; F-9 to V-319; F-9 to C-318; F-9 to Q-317; F-9 to C-316; F-9 to T-315; F-
9 to
N-314; F-9 to E-313; F-9 to D-312; F-9 to F-311; F-9 to E-3 I0; F-9 to R-309;
F-9 to
s N-308; F-9 to A-307; F-9 to G-306; F-9 to C-305; F-9 to Q-304; F-9 to S-303;
F-9 to
P-302; F-9 to F-301; F-9 to L-300; F-9 to K-299; F-9 to N-298; F-9 to K-297; F-
9 to
C-296; F-9 to V-295; F-9 to C-294; F-9 to Q-293; F-9 to C-292; F-9 to S-291; F-
9 to
N-290; F-9 to R-289; F-9 to D-288; F-9 to L-287; F-9 to E-286; F-9 to K-285; F-
9 to
H-284; F-9 to P-283; F-9 to G-282; F-9 to C-281; F-9 to S-280; F-9 to A-279; F-
9 to
1o P-278; F-9 to R-277; F-9 to L-276; F-9 to G-275; F-9 to A-274; F-9 to R-
273; F-9 to
C-272; F-9 to V-271; F-9 to C-270; F-9 to Q-269; F-9 to C-268; F-9 to T-267; F-
9 to
E-266; F-9 to E-265; F-9 to D-264; F-9 to L-263; F-9 to E-262; F-9 to K-261; F-
9 to
N-260; F-9 to P-259; F-9 to G-258; F-9 to C-257; F-9 to I-256; F-9 to D-255; F-
9 to
H-254; F-9 to F-253; F-9 to G-252; F-9 to D-251; F-9 to T-250; F-9 to S-249; F-
9 to
15 D-248; F-9 to D-247; F-9 to G-246; F-9 to A-245; F-9 to D-244; F-9 to S-
243; F-9 to
S-242; F-9 to F-241; F-9 to M-240; F-9 to F-239; F-9 to D-238; F-9 to E-237; F-
9 to
Q-236; F-9 to A-235; F-9 to L-234; F-9 to C-233; F-9 to R-232; F-9 to C-23I; F-
9 to
I-230; F-9 to H-229; F-9 to N-228; F-9 to N-227; F-9 to W-226; F-9 to M-225; F-
9
to Y-224; F-9 to N-223; F-9 to T-222; F-9 to P-221; F-9 to C-220; F-9 to T-
219; F-9
20 to K-218; F-9 to N-217; F-9 to A-2I6; F-9 to A-215; F-9 to Q-214; F-9 to C-
213; F-9
to Q-212; F-9 to P-2I 1; F-9 to L-210; F-9 to T-209; F-9 to A-208; F-9 to P-
207; F-9
to L-206; F-9 to S-205; F-9 to R-204; F-9 to R-203; F-9 to I-202; F-9 to I-
201; F-9 to
S-200; F-9 to H-199; F-9 to V-I98; F-9 to Q-197; F-9 to R-196; F-9 to Y-195; F-
9 to
V -194; F-9 to D-193; F-9 to L- I 92; F-9 to K-191; F-9 to S- I 90; F-9 to M-
189; F-9 to
25 C-188; F-9 to R-187; F-9 to C-186; F-9 to S-185; F-9 to T-184; F-9 to H-
183; F-9 to
N=X82; F-9 to A-I81; F-9 to F-180; F-9 to S-179; F-9 to I-178; F-9 to T-177; F-
9 to
V-176; F-9 to P-175; F-9 to K-174; F-9 to P-I73; F-9 to G-172; F-9 to Q-171; F-
9 to
S-170; F-9 to L-169; F-9 to P-168; F-9 to V-167; F-9 to T- I 66; F-9 to I-165;
F-9 to
E-164; F-9 to F- I 63 ; F-9 to L- I 62; F-9 to T-161; F-9 to K-160; F-9 to S-
159; F-9 to
3o L-158; F-9 to Y-157; F-9 to S-156; F-9 to T-155; F-9 to S-154; F-9 to T-
153; F-9 to
N-152; F-9 to M-151; F-9 to C-150; F-9 to Q-149; F-9 to L-148 ; F-9 to G-147;
F-9 to
E-146; F-9 to S-145; F-9 to N-144; F-9 to C-143; F-9 to C-142; F-9 to G-141; F-
9 to
G-140; F-9 to C-139; F-9 to R-138; F-9 to Y-137; F-9 to V-136; F-9 to S-135; F-
9 to
V-134; F-9 to C-133; F-9 to P-132; F-9 to P-131; F-9 to K-130; F-9 to F-129; F-
9 to
35 F-128; F-9 to T-127; F-9 to N-I26; F-9 to T-125; F-9 to A-124; F-9 to V-
123; F-9 to
G-122; F-9 to F-121; F-9 to E- I 20; F-9 to K-119; F-9 to G-118; F-9 to V- I
17; F-9 to
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D- I 16; F-9 to I-1 I 5; F-9 to C-1 I 4; F-9 to V-113; F-9 to E- I 12; F-9 to
R- I 1 I ; F-9 to
P- I 10; F-9 to M-109; F-9 to C-108; F-9 to Q-107; F-9 to T-106; F-9 to K-105;
F-9 to
R-104; F-9 to W-103; F-9 to E- I 02; F-9 to N-10 I ; F-9 to D-100; F-9 to I-
99; F-9 to
S-98; F-9 to K-97; F-9 to L-96; F-9 to I-95; F-9 to E-94; F-9 to T-93; F-9 to
N-92; F-
9 to Y-91; F-9 to H-90; F-9 to A-89; F-9 to A-88; F-9 to A-87; F-9 to F-86; F-
9 to K-
85; F-9 to I-84; F-9 to T-83; F-9 to E-82; F-9 to E-81; F-9 to T-80; F-9 to R-
79; F-9
to S-78; F-9 to N-77; F-9 to L-76; F-9 to N-75; F-9 to A-74; F-9 to Q-73; F-9
to E-
72; F-9 to R-71; F-9 to N-70; F-9 to H-69; F-9 to Q-68; F-9 to W-67; F-9 to G-
66; F-
9 to G-65; F-9 to K-64; F-9 to R-63; F-9 to L-62; F-9 to Q-61; F-9 to C-60; F-
9 to K-
59; F-9 to Y-58; F-9 to M-57; F-9 to K-56; F-9 to W-55; F-9 to Y-54; F-9 to E-
53; F-
9 to P-52; F-9 to Y-51; F-9 to L-50; F-9 to V-49; F-9 to T-48; F-9 to M-47; F-
9 to L-
46; F-9 to E-45; F-9 to D-44; F-9 to V-43; F-9 to S-42; F-9 to S-41; F-9 to V-
40; F-9
to S-39; F-9 to R-38; F-9 to L-37; F-9 to Q-36; F-9 to E-35; F-9 to E-34; F-9
to L-33;
F-9 to D-32; F-9 to K-3I; F-9 to S-30; F-9 to A-29; F-9 to Y-28; F-9 to A-27;
F-9 to
T-26; F-9 to A-25; F-9 to E-24; F-9 to G-23; F-9 to A-22; F-9 to D-21; F-9 to
P-20;
F-9 to E-19; F-9 to A-18; F-9 to D-17; F-9 to S-16; F-9 to L-15; of SEQ ID
N0:2.
Specifically preferred is the polypeptide fragment comprising amino acid
residues F-9
to R-203 of SEQ 1D N0:2, as well as polynucleotides encoding this polypeptide.
This F-9 to R-203 of SEQ ID N0:2 polypeptide preferably is associated with a S-
205
to S-396 of SEQ 117 N0:2 polypeptide. Association may be through disulfide,
covalent or noncovalent interactions, by linkage via a linker (e.g. serine,
glycine,
proline linkages), or by an antibody.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID NO: l and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
3o general formula of a-b, where a is any integer between 1 to 1660 of SEQ ID
NO:1, b
is an integer of 15 to 1674, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:1, and where the b is greater than or
equal
to a + 14.
Thus, in one aspect, N-terminal deletion mutants are provided by the present
invention. Such mutants include those comprising the amino acid sequence shown
in
Figure 1 (SEQ ID N0:18) except for a deletion of at least the first 24 N-
terminal
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amino acid residues (i.e., a deletion of at least Met (1) -- Glu (24)) but not
more than
the first 115 N-terminal amino acid residues of Figure 1 (SEQ >D N0:18).
Alternatively, first 24 N-terminal amino acid residues (i.e., a deletion of at
least Met
( 1 ) -- Glu (24)) but not more than the first 103 N-terminal amino acid
residues of
Figure 1 (SEQ ID N0:18), etc.
In another aspect, C-terminal deletion mutants are provided by the present
invention. Such mutants include those comprising the amino acid sequence shown
in
Figure 1 (SEQ ID N0:18) except for a deletion of at least the last C-terminal
amino
acid residue (Ser (419)) but not more than the last 220 C-terminal amino acid
residues
(i.e., a deletion of amino acid residues Val ( 199) - Ser (419)) of Figure 1
(SEQ ID
N0:18). Alternatively, the deletion will include at least the last C-terminal
amino acid
residue but not more than the last 216 C-terminal amino acid residues of
Figure 1
(SEQ ID N0:18). Alternatively, the deletion will include at least the last C-
terminal
amino acid residue but not more than the last 204 C-terminal amino acid
residues of
Figure 1 (SEQ ID N0:18). Alternatively, the deletion will include at least the
last C-
terminal amino acid residues but not more than the last 192 C-terminal amino
acid
residues of Figure 1 (SEQ ID N0:18). Alternatively, the deletion will include
at least
the last C-terminal amino acid residues but not more than the last 156 C-
terminal
amino acid residues of Figure 1 (SEQ 117 N0:18). Alternatively, the deletion
will
include at least the last C-terminal amino acid residues but not more than the
last 108
C-terminal amino acid residues of Figure 1 (SEQ ID N0:18). Alternatively, the
deletion will include at least the last C-terminal amino acid residues but not
more than
the last 52 C-terminal amino acid residues of Figure 1 (SEQ ID N0:18).
In yet another aspect, also included by the present invention are deletion
mutants having amino acids deleted from both the N-terminal and C-terminal
residues.
Such mutants include all combinations of the N-teririirial deletion mutants
and C
terminal deletion mutants described above.
The term "gene" means the segment of DNA involved in producing a
polypeptide chain; it includes regions preceding and following the coding
region
(leader and trailer) as well as intervening sequences (introns) between
individual
coding segments (exons).
The present invention is further directed to fragments of the isolated nucleic
acid molecules described herein. By a fragment of an isolated nucleic acid
molecule
having the nucleotide sequence of the deposited cDNA(s) or the nucleotide
sequence
shown in SEQ 117 NO:1 or SEQ ID N0:3 is intended fragments at least about 15
nt,
and more preferably at least about 20 nt, still more preferably at least about
30 nt, and
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even more preferably, at least about 40 nt in length which are useful as
diagnostic
probes and primers as discussed herein. Of course, larger fragments of 50, 75,
100,
125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,
500,
525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875,
900,
925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225,
1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550,
1575, 1600, 1625, 1650 or 1674 nt in length are also useful according to the
present
invention as are fragments corresponding to most, if not all, of the
nucleotide
sequence of the deposited cDNA(s) or as shown in SEQ ID NO: I or SEQ B7 N0:3.
By a fragment at least 20 nt in length, for example, is intended fragments
which
include 20 or more contiguous bases from the nucleotide sequence of the
deposited
cDNA(s) or the nucleotide sequence as shown in SEQ ID NOS:1 or 3.
Moreover, representative examples of VEGF-2 polynucleotide fragments
include, for example, fragments having a sequence from about nucleotide number
I
50, 51-100, 101- I 50, 151-200, 20 I -250, 251-300, 301-350, 3 S 1-400, 401-
450,
451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-
950, or 951 to the end of SEQ ID NO:1 or the cDNA contained in the deposited
clone.
In this context "about" includes the particularly recited ranges, larger or
smaller by
several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Preferably,
these fragments encode a polypeptide which has biological activity.
Fragments of the full length gene of the present invention may be used as a
hybridization probe for a cDNA library to isolate the full length cDNA and to
isolate
other cDNAs which have a high sequence similarity to the gene or similar
biological
activity. Probes of this type preferably have at least 30 bases and may
contain, for
example, 50 or more bases. The probe may also be used to identify a cDNA clone
corresponding to a full length transcript and a genomic clone or clones that
contain the
complete gene including regulatory and promoter regions, exons, and introns.
An
example of a screen comprises isolating the coding region of the gene by using
the
known DNA sequence to synthesize an oligonucleotide probe. Labeled
oligonucleotides having a sequence complementary to that of the gene of the
present
invention are used to screen a library of human cDNA, genomic DNA or rnRNA to
determine which members of the library the probe hybridizes to.
A VEGF-2 "polynucleotide" also includes those polynucleotides capable of
hybridizing, under stringent hybridization conditions, to sequences contained
in SEQ
ID NO:1 or for instance, the cDNA clones) contained in ATCC Deposit Nos. 97149
or 75698, the complement thereof. "Stringent hybridization conditions" refers
to an
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overnight incubation at 42° C in a solution comprising 50% formamide,
5x SSC (750
mM NaCI, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x
Denhardt's solution, 10% dextran sulfate, and 20 p.g/ml denatured, sheared
salmon
sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.
Also contemplated are nucleic acid molecules that hybridize to the VEGF-2
polynucleotides at lower stringency hybridization conditions. Changes in the
stringency of hybridization and signal detection are primarily accomplished
through
the manipulation of formamide concentration (lower percentages of formamide
result
in lowered stringency); salt conditions, or temperature. For example, lower
~0 stringency conditions include an overnight incubation at 37°C in a
solution comprising
6X SSPE (20X SSPE = 3M NaCI; 0.2M NaH~PO,~; 0.02M EDTA, pH 7.4), 0.5%
SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes
at 50°C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower
stringency,
washes performed following stringent hybridization can be done at higher salt
concentrations (e.g. 5X SSC).
Note that variations in the above conditions may be accomplished through the
inclusion and/or substitution of alternate blocking reagents used to suppress
background in hybridization experiments. Typical blocking reagents include
Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and
commercially available proprietary formulations. The inclusion of specific
blocking
reagents may require modification of the hybridization conditions described
above,
due to problems with compatibility.
Of course, a polynucleotide which hybridizes only to polyA+ sequences (such
as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or
to a
complementary stretch of T (or U) residues, would not be included in the
definition of
'_:polynucleotide," since such a polynucleotide would hybridize to any nucleic
acid
molecule containing a poly (A) stretch or the complement thereof (e.g.,
practically any
double-stranded cDNA clone).
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is
intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15
nucleotides (nt), and more preferably at least about 20 nt, still more
preferably at least
about 30 nt, and even more preferably about 30-70 nt of the reference
polynucleotide.
These are useful as diagnostic probes and primers as discussed above and in
more
detail below.
By a portion of a polynucleotide of "at least 20 nt in length," for example,
is
intended 20 or more contiguous nucleotides from the nucleotide sequence of the
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reference polynucleotide (e.g., the deposited cDNA or the nucleotide sequence
as
shown in SEQ ID NO:1 ). Of course, a polynucleotide which hybridizes only to a
poly
A sequence (such as the 3N terminal poly(A) tract of the VEGF-2 cDNA shown in
SEQ ID NOS:1 or 3), or to a complementary stretch of T (or U) resides, would
not be
included in a polynucleotide of the invention used to hybridize to a portion
of a nucleic
acid of the invention, since such a polynucleotide would hybridize to any
nucleic acid
molecule containing a poly (A) stretch or the complement thereof (e.g.,
practically any
double-stranded cDNA clone).
The present application is directed to nucleic acid molecules at least 95%,
96°10, 97%, 98% or 99% identical to the nucleic acid sequence shown in
SEQ >D
NOS: l or 3 or to the nucleic acid sequence of the deposited cDNA(s),
irrespective of
whether they encode a polypeptide having VEGF-2 activity. This is because even
where a particular nucleic acid molecule does not encode a polypeptide having
VEGF-
2 activity, one of skill in the art would still know how to use the nucleic
acid
molecule, for instance, as a hybridization probe or a polymerase chain
reaction (PCR)
primer. Uses of the nucleic acid molecules of the present invention that do
not encode
a polypeptide having VEGF-2 activity include, inter alia, ( 1 ) isolating the
VEGF-2
gene or allelic variants thereof in a cDNA library; (2) in situ hybridization
(e.g.,
"FISH") to metaphase chromosomal spreads to provide precise chromosomal
location
2o of the VEGF-2 gene, as described in Verma et al., Human Chromosomes: A
Manual
of Basic Techniques, Pergamon Press, New York ( 1988); and Northern Blot
analysis
for detecting VEGF-2 mRNA expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least
95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence shown in SEQ ID
NOS:I or 3 or to a nucleic acid sequence of the deposited cDNA(s) which do, in
fact,
encode a polypeptide having VEGF-2 protein activity. By "a polypeptide having
VEGF-2 activity" is intended polypeptides exhibiting VEGF-2 activity in a
particular
biological assay. For example, VEGF-2 protein activity can be measured using,
for
example, mitogenic assays and endothelial cell migration assays. See, e. g. ,
Olofsson
3o et al., Proc. Natl. Acad. Sci. USA 93:2576-2581 ( 1996) and Joukov et al.,
EMBO J.
5:290-298 ( 1996).
Of course, due to the degeneracy of the genetic code, one of ordinary skill in
the art will immediately recognize that a large number of the nucleic acid
molecules
having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic
acid sequence of the deposited cDNA(s) or the nucleic acid sequence shown in
SEQ
ID NO:1 or SEQ ID N0:3 will encode a polypeptide "having VEGF-2 protein
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activity." In fact, since degenerate variants of these nucleotide sequences
all encode
the same polypeptide, this will be clear to the skilled artisan even without
performing
the above described comparison assay. It will be further recognized in the art
that, for
such nucleic acid molecules that are not degenerate variants, a reasonable
number will
also encode a polypeptide having VEGF-2 protein activity. This is because the
skilled
artisan is fully aware of amino acid substitutions that are either less likely
or not likely
to significantly effect protein function (e.g., replacing one aliphatic amino
acid with a
second aliphatic amino acid).
For example, guidance concerning how to make phenotypically silent amino
acid substitutions is provided in Bowie, J. U. et al., "Deciphering the
Message in
Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-
1310
( 1990), wherein the authors indicate that proteins are surprisingly tolerant
of anuno
acid substitutions.
Thus, the present invention is directed to polynucleotides having at least a
70%
identity, preferably at least 90% and more preferably at least a 95%, 96%,
97%, or
98% identity to a polynucleotide which encodes the polypeptides of SEQ ID
NOS:2 or
4, as well as fragments thereof, which fragments have at least 30 bases and
preferably
at least 50 bases and to polypeptides encoded by such polynucleotides.
"Identity" per se has an art-recognized meaning and can be calculated using
2o published techniques. (See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY,
Lesk, A.M., ed., Oxford University Press, New York, ( 1988); BIOCOMPUTING:
INFORMATICS AND GENOME PROJECTS, Smith, D.W., ed., Academic Press,
New York, ( 1993 ); COMPUTER ANALYSIS OF SEQUENCE DATA, PART I ,
Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, ( 1994);
SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic
Press, ( 1987); and SEQUENCE ANALYSIS PRIMER, Gribskov, M. and
Devereux, J., eds., M Stockton Press, New York, ( 1991 ).) While there exists
a
number of methods to measure identity between two polynucleotide or
polypeptide
sequences, the term "identity" is well known to skilled artisans. (Carillo,
H., and
Lipton, D., SIAM J. Applied Math . 48:1073 ( 1988).) Methods commonly employed
to determine identity or similarity between two sequences include, but are not
limited
to, those disclosed in "Guide to Huge Computers," Martin J. Bishop, ed.,
Academic
Press, San Diego, ( 1994), and Carillo, H., and Lipton, D., SIAM J. Applied
Math.
48:1073 (1988). Methods for aligning polynucleotides or polypeptides are
codified in
computer programs, including the GCG program package (Devereux, J., et al.,
Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul,
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S.F. et al., J. Molec. Biol. 215:403 (1990), Bestfit program (Wisconsin
Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group, University
Research Park, 575 Science Drive, Madison, WI 53711 (using the local homology
algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489
( 1981 )). By a polynucleotide having a nucleotide sequence at least, for
example, 95%
"identical" to a reference nucleotide sequence of the present invention, it is
intended
that the nucleotide sequence of the polynucleotide is identical to the
reference sequence
except that the polynucleotide sequence may include up to five point mutations
per
each 100 nucleotides of the reference nucleotide sequence encoding the VEGF-2
to polypeptide. In other words, to obtain a polynucleotide having a nucleotide
sequence
at least 95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides
in the reference sequence may be deleted or substituted with another
nucleotide, or a
number of nucleotides up to 5% of the total nucleotides in the reference
sequence may
be inserted into the reference sequence. The query sequence may be an entire
sequence SEQ ID NO:1, the ORF (open reading frame), or any fragment specified
as
described herein.
As a practical matter, whether any particular nucleic acid molecule or
polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a
nucleotide
sequence of the presence invention can be determined conventionally using
known
computer programs. A preferred method for determining the best overall match
between a query sequence (a sequence of the present invention) and a subject
sequence, also referred to as a global sequence alignment, can be determined
using the
FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App.
Biosci. 6:237-245 ( 1990)). In a sequence alignment the query and subject
sequences
are both DNA sequences. An RNA sequence can be compared by converting U's to
T's. The result of said global sequence alignment is in percent identity.
Preferred
parameters used in a FASTDB alignment of DNA sequences to calculate percent
identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining
Penalty=30,
Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty
0.05, Window Size=500 or the length of the subject nucleotide sequence,
whichever
is shorter.
If the subject sequence is shorter than the query sequence because of 5' or 3
'
deletions, not because of internal deletions, a manual correction must be made
to the
results. This is because the FASTDB program does not account for 5' and 3 '
truncations of the subject sequence when calculating percent identity. For
subject
sequences truncated at the 5' or 3' ends, relative to the query sequence, the
percent
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identity is corrected by calculating the number of bases of the query sequence
that are
S' and 3' of the subject sequence, which are not matched/aligned, as a percent
of the
total bases of the query sequence. Whether a nucleotide is matched/aligned is
determined by results of the FASTDB sequence alignment. This percentage is
then
subtracted from the percent identity, calculated by the above FASTDB program
using
the specified parameters, to arrive at a final percent identity score. This
corrected
score is what is used for the purposes of the present invention. Only bases
outside the
5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment,
which are not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the 5' end of
the subject
sequence and therefore, the FASTDB alignment does not show a matched/alignment
of the first 10 bases at 5' end. The 10 unpaired bases represent 10% of the
sequence
(number of bases at the 5' and 3' ends not matched/total number of bases in
the query
sequence} so 10% is subtracted from the percent identity score calculated by
the
FASTDB program. If the remaining 90 bases were perfectly matched the final
percent
identity would be 90%. In another example, a 90 base subject sequence is
compared
with a 100 base query sequence. This time the deletions are internal deletions
so that
there are no bases on the 5' or 3' of the subject sequence which are not
matched/aligned with the query. In this case the percent identity calculated .
by
FASTDB is not manually corrected. Once again, only bases 5' and 3' of the
subject
sequence which are not matched/aligned with the query sequence are manually
corrected for. No other manual corrections are to made for the purposes of the
present
invention.
By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a query amino acid sequence of the present invention, it is
intended that
the amino acid sequence of the subject polypeptide is identical to the query
sequence
except that the subject polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the query amino acid sequence. In
other
words, to obtain a polypeptide having an amino acid sequence at least 95%
identical to
a query amino acid sequence, up to 5% of the amino acid residues in the
subject
sequence may be inserted, deleted, (indels) or substituted with another amino
acid.
These alterations of the reference sequence may occur at the amino or carboxy
terminal
positions of the reference amino acid sequence or anywhere between those
terminal
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positions, interspersed either individually among residues in the reference
sequence or
in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%,
95°l0,
96%, 97%, 98% or 99% identical to, for instance, the amino acid sequences
shown in
Table 1 or to the amino acid sequence encoded by deposited DNA clone can be
determined conventionally using known computer programs. A preferred method
for
determining the best overall match between a query sequence (a sequence of the
present invention) and a subject sequence, also referred to as a global
sequence
alignment, can be determined using the FASTDB computer program based on the
to algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a
sequence
alignment the query and subject sequences are either both nucleotide sequences
or
both amino acid sequences. The result of said global sequence alignment is in
percent
identity. Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization
Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5,
Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid
sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence due to N- or C
terminal deletions, not because of internal deletions, a manual correction
must be made
2o to the results. This is because the FASTDB program does not account for N-
and C
terminal truncations of the subject sequence when calculating global percent
identity.
For subject sequences truncated at the N- and C-termini, relative to the query
sequence, the percent identity is corrected by calculating the number of
residues of the
query sequence that are N- and C-terminal of the subject sequence, which are
not
matched/aligned with a corresponding subject residue, as a percent of the
total bases
. , of the query sequence. Whether a residue is matched/aligned is determined
by results
of the FASTDB sequence alignment. This percentage is then subtracted from the
percent identity, calculated by the above FASTDB program using the specified
parameters, to arrive at a final percent identity score. This final percent
identity score
is what is used for the purposes of the present invention. Only residues to
the N- and
C-termini of the subject sequence, which are not matched/aligned with the
query
sequence, are considered for the purposes of manually adjusting the percent
identity
score. That is, only query residue positions outside the farthest N- and C-
terminal
residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100
residue query sequence to determine percent identity. The deletion occurs at
the N-
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terminus of the subject sequence and therefore, the FASTDB alignment does not
show
a matching/alignment of the first 10 residues at the N-terminus. The 10
unpaired
residues represent 10% of the sequence (number of residues at the N- and C-
termini
not matched/total number of residues in the query sequence) so 10% is
subtracted
from the percent identity score calculated by the FASTDB program. If the
remaining
90 residues were perfectly matched the final percent identity would be 90%. In
another example, a 90 residue subject sequence is compared with a 100 residue
query
sequence. This time the deletions are internal deletions so there are no
residues at the
N- or C-termini of the subject sequence which are not matched/aligned with the
query.
to In this case the percent identity calculated by FASTDB is not manually
corrected.
Once again, only residue positions outside the N- and C-terniinal ends of the
subject
sequence, as displayed in the FASTDB alignment, which are not matched/aligned
with the query sequence are manually corrected for. No other manual
corrections are
to made for the purposes of the present invention.
VEGF-2 Polypeptides
The present invention further relates to polypeptides which have the deduced
amino acid sequence of Figures 1 or 2, or which has the amino acid sequence
encoded by the deposited cDNAs, as well as fragments, analogs, and derivatives
of
2o such polypeptides.
The terms "fragment," "derivative" and "analog" when referring to the
polypeptide of Figures 1 or 2 or that encoded by the deposited cDNA, means a
polypeptide which retains the conserved motif of VEGF proteins as shown in
Figure 3
and essentially the same biological function or activity.
In the present invention, a "polypeptide fragment" refers to a short amino
acid
sequence contained in SEQ ID N0:2 or encoded by the cDNA contained in the
deposited clone. Protein fragments may be "free-standing," or comprised within
a
larger polypeptide of which the fragment forms a part or region, most
preferably as a
single continuous region. Representative examples of polypeptide fragments of
the
invention, include, for example, fragments from about amino acid number 1-20,
21-
40, 41-60, 61-80, 81-100, 102-120, 12 I -140, 141-160, 161-180, 181-200, 201-
220, 221-240, 241-260, 261-280, or 281 to the end of the coding region.
Moreover,
polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,
120,
130, 140, or 150 amino acids in length. In this context "about" includes the
particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1 )
amino acids,
at either extreme or at both extremes.
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Preferred polypeptide fragments include the secreted VEGF-2 protein as well
as the mature form. Further preferred polypeptide fragments include the
secreted
VEGF-2 protein or the mature form having a continuous series of deleted
residues
from the amino or the carboxy terminus, or both. For example, any number of
amino
acids, ranging from I-60, can be deleted from the amino terminus of either the
secreted VEGF-2 polypeptide or the mature form. Similarly, any number of amino
acids, ranging from I -30, can be deleted from the carboxy terminus of the
secreted
VEGF-2 protein or mature form. Furthermore, any combination of the above amino
and carboxy terminus deletions are preferred. Similarly, polynucleotide
fragments
l0 encoding these VEGF-2 polypeptide fragments are also preferred.
Also preferred are VEGF-2 polypeptide and polynucleotide fragments
characterized by structural or functional domains, such as fragments that
comprise
alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming
regions, turn and turn-forming regions, coil and coil-forming regions,
hydrophilic
~5 regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic
regions,
flexible regions, surface-forming regions, substrate binding region, and high
antigenic
index regions. Polypeptide fragments of SEQ ID N0:2 falling within conserved
domains are specifically contemplated by the present invention. (See Figure 2.
)
Moreover, polynucleotide fragments encoding these domains are also
contemplated.
2o Other preferred fragments are biologically active VEGF-2 fragments.
Biologically active fragments are those exhibiting activity similar, but not
necessarily
identical, to an activity of the VEGF-2 polypeptide. The biological activity
of the
fragments may include an improved desired activity, or a decreased undesirable
activity.
25 The polypeptides of the present invention may be recombinant polypeptides,
natural polypeptides, or synthetic polypeptides, preferably recombinant
polypeptides.
It will be recognized in the art that some amino acid sequences of the VEGF-2
polypeptide can be varied without significant effect of the structure or
function of the
protein. If such differences in sequence are contemplated, it should be
remembered
3o that there will be critical areas on the protein which determine activity.
Thus, the invention further includes variations of the VEGF-2 polypeptide
which show substantial VEGF-2 polypeptide activity or which include regions of
VEGF-2 protein such as the protein portions discussed below. Such mutants
include
deletions, insertions, inversions, repeats, and type substitutions. As
indicated above,
35 guidance concerning which amino acid changes are likely to be
phenotypically silent
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can be found in Bowie, J.U., et al., "Deciphering the Message in Protein
Sequences:
Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990).
Thus, the fragments, derivatives, or analogs of the polypeptides of Figures 1
or 2, or that encoded by the deposited cDNAs may be: (I) one in which one or
more of
the amino acid residues are substituted with a conserved or non-conserved
amino acid
residue (preferably a conserved amino acid residue) and such substituted amino
acid
residue may or may not be one encoded by the genetic code; or (ii) one in
which one
or more of the amino acid residues includes a substituent group; or (iii) one
in which
the mature polypeptide is fused with another compound, such as a compound to
increase the half-life of the polypeptide (for example, polyethylene glycol);
or (iv) one
in which the additional amino acids are fused to the mature polypeptide, such
as a
leader or secretory sequence or a sequence which is employed for purification
of the
mature polypeptide or a proprotein sequence; or (v) one in which comprises
fewer
amino acid residues shown in SEQ ID NOS: 2 or 4, and retains the conserved
motif
and yet still retains activity characteristics of the VEGF family of
polypeptides. Such
fragments, derivatives, and analogs are deemed to be within the scope of those
skilled
in the art from the teachings herein.
Of particular interest are substitutions of charged amino acids with another
charged amino acid and with neutral or negatively charged amino acids. The
latter
2o results in proteins with reduced positive charge to improve the
characteristics of the
VEGF-2 protein. The prevention of aggregation is highly desirable. Aggregation
of
proteins not only results in a loss of activity but can also be problematic
when
preparing pharmaceutical formulations, because they can be immunogenic.
(Pinckard
et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-
845
( 1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377
(1.993)). _V.
The replacement of amino acids can also change the selectivity of binding to
cell surface receptors. Ostade et al., Nature 361:266-268 ( 1993) describes
certain
mutations resulting in selective binding of TNF-a to only one of the two known
types
of TNF receptors. Thus, the VEGF-2 of the present invention may include one or
more amino acid substitutions, deletions or additions, either from natural
mutations or
human manipulation.
As indicated, changes are preferably of a minor nature, such as conservative
amino acid substitutions that do not significantly affect the folding or
activity of the
protein (see Table 1).
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TABLE 1. Conservative Amino Acid Substitutions
Tryptophan
Tyrosine
Hydrophobic Leucine
Isoleucine
Valine
polar ~ Glutamine
Asparagine
Basic Arginine
Lysine
Histidine
Acidic ~ Aspartic Acid
Glutamic Acid
Small Alanine
Serine
Threonine
Methionine
Glycine
Of course, the number of amino acid substitutions a skilled artisan would
make depends on many factors, including those described above. Generally
speaking, the number of substitutions for any given VEGF-2 polypeptide will
not be
more than 50, 40, 30, 25, 20, 15, 10, 5 or 3.
Amino acids in the VEGF-2 protein of the present invention that are essential
for function can be identified by methods known in the art, such as site-
directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science
0 244:1081-1085 (1989)). The latter procedure introduces single alanine
mutations at
every residue in the molecule. The resulting mutant molecules are then tested
for
biological activity such as receptor binding or in vitro, or in vitro
proliferative activity.
Sites that are critical for ligand-receptor binding can also be determined by
structural
analysis such as crystallization, nuclear magnetic resonance or photoaffinity
labeling
~ 5 (Smith et al., J. Mol. Biol. 224:899-904 ( 1992) and de Vos et al. Science
255:306-
312 ( 1992)).
The polypeptides and polynucleotides of the present invention are preferably
provided in an isolated form, and preferably are purified to homogeneity.
The term "isolated" means that the material is removed from its original
2o environment (e.g., the natural environment if it is naturally occurring).
For example,'a
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naturally-occurring polynucleotide or polypeptide present in a living animal
is not
isolated, but the same polynucleotide or DNA or polypeptide, separated from
some or
all of the coexisting materials in the natural system, is isolated. Such
polynucleotide
could be part of a vector and/or such polynucleotide or polypeptide could be
part of a
composition, and still be isolated in that such vector or composition is not
part of its
natural environment.
In specific embodiments, the polynucleotides of the invention are less than
300
kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb in length. In a further
embodiment, polynucleotides of the invention comprise at least 15 contiguous
t o nucleotides of VEGF-2 coding sequence, but do not comprise all or a
portion of any
VEGF-2 intron. In another embodiment, the nucleic acid comprising VEGF-2
coding
sequence does not contain coding sequences of a genomic flanking gene (i.e.,
5' or 3 '
to the VEGF-2 gene in the genome).
The polypeptides of the present invention include the polypeptides of SEQ )D
NOS:2 and 4 (in particular the mature polypeptide) as well as polypeptides
which have
at least 70% similarity (preferably at least 70% identity) to the polypeptides
of SEQ >D
NOS:2 and 4, and more preferably at least 90% similarity (more preferably at
least
95% identity) to the polypeptides of SEQ ID NOS:2 and 4, and still more
preferably at
least 95% similarity (still more preferably at least 90% identity) to the
polypeptides of
2o SEQ ID NOS:2 and 4 and also include portions of such polypeptides with such
portion of the polypeptide generally containing at least 30 amino acids and
more
preferably at least 50 amino acids.
As known in the art "similarity" between two polypeptides is determined by
comparing the amino acid sequence and its conserved amino acid substitutes of
one
polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be
employed for producing the corresponding full-length polypeptide by peptide
synthesis; therefore, the fragments may be employed as intermediates for
producing
the full-length polypeptides. Fragments or portions of the polynucleotides of
the
3o present invention may be used to synthesize full-length polynucleotides of
the present
invention.
The polypeptides of the present invention include the polypeptide encoded by
the deposited cDNA including the leader; the mature polypeptide encoded by the
deposited the cDNA minus the leader (i.e., the mature protein); a polypeptide
comprising amino acids about - 23 to about 396 in SEQ )D N0:2; a polypeptide
comprising amino acids about - 22 to about 396 in SEQ ID N0:2; a polypeptide
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comprising amino acids about 1 to about 396 in SEQ ID N0:2; as well as
polypeptides
which are at least 95% identical, and more preferably at least 96%, 97%, 98%
or
- 99% identical to the polypeptides described above and also include portions
of such
polypeptides with at least 30 amino acids and more preferably at least 50
amino acids.
Fusion Proteins
Any VEGF-2 polypeptide can be used to generate fusion proteins. For
example, the VEGF-2 polypeptide, when fused to a second protein, can be used
as an
antigenic tag. Antibodies raised against the VEGF-2 polypeptide can be used to
indirectly detect the second protein by binding to the VEGF-2. Moreover,
because
secreted proteins target cellular locations based on trafficking signals, the
VEGF-2
polypeptides can be used as a targeting molecule once fused to other proteins.
Examples of domains that can be fused to VEGF-2 polypeptides include not
only heterologous signal sequences, but also other heterologous functional
regions.
The fusion does not necessarily need to be direct, but may occur through
linker
sequences.
Moreover, fusion proteins may also be engineered to improve characteristics
of the VEGF-2 polypeptide. For instance, a region of additional amino acids,
particularly charged amino acids, may be added to the N-terminus of the VEGF-2
2o polypeptide to improve stability and persistence during purification from
the host cell
or subsequent handling and storage. Also, peptide moieties may be added to the
VEGF-2 polypeptide to facilitate purification. Such regions may be removed
prior to
final preparation of the VEGF-2 polypeptide. The addition of peptide moieties
to
facilitate handling of polypeptides are familiar and routine techniques in the
art.
Moreover, VEGF-2 polypeptides, including fragments, and specifically
epitopes, can be combined with parts of the constant domain of immunoglobulins
(IgG), resulting in chimeric polypeptides. These fusion proteins facilitate
purification
and show an increased half life in vivo. One reported example describes
chimeric
proteins consisting of the first two domains of the human CD4-polypeptide and
3o various domains of the constant regions of the heavy or light chains of
mammalian
immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).)
Fusion proteins having disulfide-linked dimeric structures (due to the IgG)
can also be
more efficient in binding and neutralizing other molecules, than the monomeric
secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem.
270:3958-3964 ( 1995).)
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Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion
proteins comprising various portions of constant region of immunoglobulin
molecules
together with another human protein or part thereof. In many cases, the Fc
part in a
fusion protein is beneficial in therapy and diagnosis, and thus can result in,
for
example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively,
deleting the Fc part after the fusion protein has been expressed, detected,
and purified,
would be desired. For example, the Fc portion may hinder therapy and diagnosis
if
the fusion protein is used as an antigen for immunizations. In drug discovery,
for
example, human proteins, such as hIL-5, have been fused with Fc portions for
the
l0 purpose of high-throughput screening assays to identify antagonists of hIL-
5. (See,
D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et
al., J.
Biol. Chem. 270:9459-9471 (1995).)
Moreover, the VEGF-2 polypeptides can be fused to marker sequences, such
as a peptide which facilitates purification of VEGF-2. In preferred
embodiments, the
marker amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a
pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among
others, many of which are commercially available. As described in Gentz et
al., Proc.
Natl. Acad. Sci. USA 86:821-824 ( 1989), for instance, hexa-histidine provides
for
convenient purification of the fusion protein. Another peptide tag useful for
2o purification, the "HA" tag, corresponds to an epitope derived from the
influenza
hemagglutinin protein. (Wilson et al., Cell 37:767 (1984).)
Thus, any of these above fusions can be engineered using the VEGF-2
polynucleotides or the polypeptides.
Biological Activities of VEGF-2
VEGF-2 polynucleotides and polypeptides can be used in assays to test for
one or more biological activities. If VEGF-2 polynucleotides and polypeptides
do
exhibit activity in a particular assay, it is likely that VEGF-2 may be
involved in the
diseases associated with the biological activity. Therefore, VEGF-2 could be
used to
3o treat the associated disease.
Immune Activity
VEGF-2 polypeptides or polynucleotides may be useful in treating deficiencies
or disorders of the immune system, by activating or inhibiting the
proliferation,
differentiation, or mobilization (chemotaxis) of immune cells. Immune cells
develop
through a process called hematopoiesis, producing myeloid (platelets, red
blood cells,
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neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from
pluripotent stem cells. The etiology of these immune deficiencies or disorders
may be
genetic, somatic, such as cancer or some autoimmune disorders, acquired (e.g.,
by
chemotherapy or toxins), or infectious. Moreover, VEGF-2 polynucleotides or
polypeptides can be used as a marker or detector of a particular immune system
disease or disorder.
VEGF-2 polynucleotides or polypeptides may be useful in treating or detecting
deficiencies or disorders of hematopoietic cells. VEGF-2 polypeptides or
polynucleotides could be used to increase differentiation and proliferation of
to hematopoietic cells, including the pluripotent stem cells, in an effort to
treat those
disorders associated with a decrease in certain (or many) types hematopoietic
cells.
Examples of immunologic deficiency syndromes include, but are not limited to:
blood
protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia)> ataxia
telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV
infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome,
lymphopenia, phagocyte bactericidal dysfunction, severe combined
immunodeficiency
(SCIDs)> Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or
hemoglobinuria.
Moreover, VEGF-2 polypeptides or polynucleotides can also be used to
modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot
2o formation). For example, by increasing hemostatic or thrombolytic activity,
VEGF-2
polynucleotides or polypeptides could be used to treat blood coagulation
disorders
(e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (e.g.
thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
Alternatively, VEGF-2 polynucleotides or polypeptides that can decrease
hemostatic
or thrombolytic activity could be used to inhibit or dissolve clotting,
important in the
treatment of heart attacks (infarction), strokes, or scarring:-
VEGF-2 polynucleotides or polypeptides may also be useful in treating or
detecting autoimmune disorders. Many autoimmune disorders result from
inappropriate recognition of self as foreign material by immune cells. This
inappropriate recognition results in an immune response leading to the
destruction of
the host tissue. Therefore, the administration of VEGF-2 polypeptides or
polynucleotides that can inhibit an immune response, particularly the
proliferation,
differentiation, or chemotaxis of T-cells, may be an effective therapy in
preventing
autoimmune disorders.
Examples of autoimmune disorders that can be treated or detected by VEGF-2
include, but are not limited to: Addison's Disease, hemolytic anemia,
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antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic
encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves'
Disease,
Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous
Pemphigoid,
Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff Man
Syndrome,
Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary
Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis,
and
autoimmune inflammatory eye disease.
Similarly, allergic reactions and conditions, such as asthma (particularly
allergic asthma) or other respiratory problems, may also be treated by VEGF-2
to polypeptides or polynucleotides. Moreover, VEGF-2 can be used to treat
anaphylaxis, hypersensitivity to an antigenic molecule, or blood group
incompatibility.
VEGF-2 polynucleotides or polypeptides may also be used to treat and/or
prevent organ rejection or graft-versus-host disease (GVHD). Organ rejection
occurs
~ 5 by host immune cell destruction of the transplanted tissue through an
immune
response. Similarly, an immune response is also involved in GVHD, but, in this
case, the foreign transplanted immune cells destroy the host tissues. The
administration of VEGF-2 polypeptides or polynucleotides that inhibits an
immune
response, particularly the proliferation, differentiation, or chemotaxis of T-
cells, may
2o be an effective therapy in preventing organ rejection or GVHD. Similarly,
VEGF-2 polypeptides or polynucleotides may also be used to modulate
inflammation.
For example, VEGF-2 polypeptides or polynucleotides may inhibit the
proliferation
and differentiation of cells involved in an inflammatory response. These
molecules
can be used to treat inflammatory conditions, both chronic and acute
conditions,
25 including inflammation associated with infection (e.g., septic shock,
sepsis, or
systenvc inflammatory response syndrome (SIRS)), ischemia-reperfusion injury,
endotoxin lethality, arthritis, complement-mediated hyperacute rejection,
nephritis,
cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's
disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)
Hyperproliferative Disorders
VEGF-2 polypeptides or polynucleotides can be used to treat or detect
hyperproliferative disorders, including neoplasms. VEGF-2 antagonist
polypeptides
or polynucleotides may inhibit the proliferation of the disorder through
direct or
indirect interactions. Alternatively, VEGF-2 antagonist polypeptides or
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polynucleotides may proliferate other cells which can inhibit the
hyperproliferative
disorder.
For example, by increasing an immune response, particularly increasing
antigenic qualities of the hyperproliferative disorder or by proliferating,
differentiating, or mobilizing T-cells, hyperproliferative disorders can be
treated. This
immune response may be increased by either enhancing an existing immune
response,
or by initiating a new immune response. Alternatively, decreasing an immune
response may also be a method of treating hyperproliferative disorders, such
as a
chemotherapeutic agent.
to Examples of hyperproliferative disorders that can be treated or detected by
VEGF-2 antagonist polynucleotides or polypeptides include, but are not limited
to
neoplasms located in the: abdomen, bone, breast, digestive system, liver,
pancreas,
peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles,
ovary,
thymus, thyroid), eye, head and neck, nervous (central and peripheral),
lymphatic
t 5 system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
Similarly, other hyperproliferative disorders can also be treated or detected
by
VEGF-2 antagonist polynucleotides or polypeptides. Examples of such
hyperproliferative disorders include, but are not limited to:
hypergammaglobulinemia,
lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary
2o Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease,
histiocytosis, and
any other hyperproliferative disease, besides neoplasia, located in an organ
system
listed above.
Infectious Disease
25 VEGF-2 polypeptides ar polynucleotides can be used to treat or detect
infectious agents. For example, by increasing the immune response,
particularly
increasing the proliferation and differentiation of B and/or T cells,
infectious diseases
may be treated. The immune response may be increased by either enhancing an
existing immune response, or by initiating a new immune response.
Alternatively,
3o VEGF-2 polypeptides or polynucleotides may also directly inhibit the
infectious agent,
without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or
symptoms that can be treated or detected by VEGF-2 polynucleotides or
polypeptides.
Examples of viruses, include, but are not limited to the following DNA and RNA
viral
35 families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae,
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Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes
Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,
Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae,
Parvoviridae,
Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g.,
Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g.,
Rubivirus). Viruses falling within these families can cause a variety of
diseases or
symptoms, including, but not limited to: arthritis, bronchiollitis,
encephalitis, eye
infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome,
hepatitis (A, B,
C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g.,
AIDS),
pneumonia, Burkitt's Lymphoma, chickenpox , hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually
transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. VEGF-
2
polypeptides or polynucleotides can be used to treat or detect any of these
symptoms
or diseases.
1 s Similarly, bacterial or fungal agents that can cause disease or symptoms
and
that can be treated or detected by VEGF-2 polynucleotides or polypeptides
include,
but not limited to, the following Gram-Negative and Gram-positive bacterial
families
and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia),
Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae,
2o Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis,
Campylobacter,
Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae
(Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter,
Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae (e.g.,
Acinetobacter, Gonorrhea, Menigococcal), Pasteurellacea Infections (e.g.,
25 Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae,
Chlamydiaceae, Syphilis, and Staphylococcal. These bacterial or fungal
families can
cause the following diseases or symptoms, including, but not limited to:
bacteremia,
endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis),
gingivitis,
opportunistic infections (e.g., AIDS related infections), paronychia,
prosthesis-related
30 infections, Reiter's Disease, respiratory tract infections, such as
Whooping Cough or
Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid
Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia,
Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus,
Botulism,
gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually
transmitted
35 diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia,
urinary tract
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infections, wound infections. VEGF-2 polypeptides or polynucleotides can be
used to
treat or detect any of these symptoms or diseases.
Moreover, parasitic agents causing disease or symptoms that can be treated or
detected by VEGF-2 polynucleotides or polypeptides include, but not limited
to, the
following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis,
Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis,
lxishmaniasis,
Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas. These parasites
can cause a variety of diseases or symptoms, including, but not limited to:
Scabies,
Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,
giardiasis), liver
1o disease, lung disease, opportunistic infections (e.g., AIDS related),
Malaria,
pregnancy complications, and toxoplasmosis. VEGF-2 polypeptides or
polynucleotides can be used to treat or detect any of these symptoms or
diseases.
Preferably, treatment using VEGF-2 polypeptides or polynucleotides could
either be by administering an effective amount of VEGF-2 polypeptide to the
patient,
~ 5 or by removing cells from the patient, supplying the cells with VEGF-2
polynucleotide, and returning the engineered cells to the patient (ex vivo
therapy).
Moreover, the VEGF-2 polypeptide or polynucleotide can be used as an antigen
in a
vaccine to raise an immune response against infectious disease.
2o Regeneration
VEGF-2 polynucleotides or poIypeptides can be used to differentiate,
proliferate, and attract cells, leading to the regeneration of tissues. (See,
Science
276:59-87 (1997).) The regeneration of tissues could be used to repair,
replace, or
protect tissue damaged by congenital defects, trauma (wounds, burns,
incisions, or
25 ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal
disease, liver
failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion
injury, or
systemic cytokine damage.
Tissues that could be regenerated using the present invention include organs
(e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal
30 or cardiac), vascular (including vascular endothelium), lymphatic
{including lymphatic
endothelium), nervous, hematopoietic, and skeletal (hone, cartilage, tendon,
and
ligament) tissue. Preferably, regeneration occurs without or decreased
scarring.
Regeneration also may include angiogenesis.
Moreover, VEGF-2 polynucleotides or polypeptides may increase regeneration
35 of tissues difficult to heal. For example, increased tendon/ligament
regeneration
would quicken recovery time after damage. VEGF-2 polynucleotides or
poIypeptides
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of the present invention could also be used prophylactically in an effort to
avoid
damage. Specific diseases that could be treated include of tendinitis, carpal
tunnel
syndrome, and other tendon or ligament defects. A further example of tissue
regeneration of non-healing wounds includes pressure ulcers, ulcers associated
with
vascular insufficiency, surgical, and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using VEGF-2
polynucleotides or polypeptides to proliferate and differentiate nerve cells.
Diseases
that could be treated using this method include central and peripheral nervous
system
diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal
cord
disorders, head trauma, cerebrovascular disease, and stoke). Specifically,
diseases
associated with peripheral nerve injuries, peripheral neuropathy (e.g.,
resulting from
chemotherapy or other medical therapies), localized neuropathies, and central
nervous
system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's
disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be
treated
using the VEGF-2 polynucleotides or polypeptides.
Chemotaxis
VEGF-2 polynucleotides or polypeptides may have chemotaxis activity. A
chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts,
2o neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells) to a
particular site in the body, such as inflammation, infection, or site of
hyperproliferation. The mobilized cells can then fight off and/or heal the
particular
trauma or abnormality.
VEGF-2 polynucleotides or polypeptides may increase chemotaxic activity of
particular cells. These chemotactic molecules can then be used to treat
inflammation, ,
infection, hyperproliferative disorders, or any immune system disorder by
increasing
the number of cells targeted to a particular location in the body. For
example,
chemotaxic molecules can be used to treat wounds and other trauma to tissues
by
attracting immune cells to the injured location. As a chemotactic molecule,
VEGF-2
3o could also attract fibroblasts, which can be used to treat wounds.
It is also contemplated that VEGF-2 polynucleotides or polypeptides may
inhibit chemotactic activity. These molecules could also be used to treat
disorders.
Thus, VEGF-2 polynucleotides or polypeptides could be used as an inhibitor of
chemotaxis.
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Binding Activity
VEGF-2 polypeptides may be used to screen for molecules that bind to VEGF-
2 or for molecules to which VEGF-2 binds. The binding of VEGF-2 and the
molecule may activate (agonist), increase, inhibit (antagonist), or decrease
activity of
the VEGF-2 or the molecule bound. Examples of such molecules include
antibodies,
oligonucleotides, proteins (e.g., receptors),or small molecules.
Preferably, the molecule is closely related to the natural ligand of VEGF-2,
e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural
or
functional mimetic. (See, Coligan et al., Current Protocols in Immunology
l0 I(2):Chapter 5 ( 1991 ).) Similarly, the molecule can be closely related to
the natural
receptor to which VEGF-2 binds (i.e., Flt-4), or at least, a fragment of the
receptor
capable of being bound by VEGF-2 (e.g., active site). In either case, the
molecule
can be rationally designed using known techniques. Preferably, the screening
for
these molecules involves producing appropriate cells which express VEGF-2,
either
as a secreted protein or on the cell membrane. Preferred cells include cells
from
mammals, yeast, Drosophila, or E. coli. Cells expressing VEGF-2(or cell
membrane
containing the expressed polypeptide) are then preferably contacted with a
test
compound potentially containing the molecule to observe binding, stimulation,
or
inhibition of activity of either VEGF-2 or the molecule.
2o The assay may simply test binding of a candidate compound toVEGF-2,
wherein binding is detected by a label, or in an assay involving competition
with a
labeled competitor. Further, the assay may test whether the candidate compound
results in a signal generated by binding to VEGF-2.
Alternatively, the assay can be carried out using cell-free preparations,
polypeptide/molecule affixed to a solid support, chemical libraries, or
natural product
mixtures. The assay may also simply comprise the steps of mixing a candidate
compound with a solution containing VEGF-2, measuring VEGF-2/molecule activity
or binding, and comparing the VEGF-2/molecule activity or binding to a
standard.
Preferably, an ELISA assay can measure VEGF-2 level or activity in a sample
(e.g., biological sample) using a monoclonal or polyclonal antibody. The
antibody
can measure VEGF-2 level or activity by either binding, directly or
indirectly, to
VEGF-2 or by competing with VEGF-2 for a substrate.
All of these above assays can be used as diagnostic or prognostic markers.
The molecules discovered using these assays can be used to treat disease or to
bring
about a particular result in a patient {e.g., blood vessel growth) by
activating or
inhibiting the VEGF-2/molecule. Moreover, the assays can discover agents which
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may inhibit or enhance the production of VEGF-2 from suitably manipulated
cells or
tissues.
Therefore, the invention includes a method of identifying compounds which
bind to VEGF-2 comprising the steps of: (a) incubating a candidate binding
compound with VEGF-2; and (b) determining if binding has occurred. Moreover,
the
invention includes a method of identifying agonists/antagonists comprising the
steps
of: (a) incubating a candidate compound with VEGF-2, (b) assaying a biological
activity , and (b) determining if a biological activity of VEGF-2 has been
altered.
to Other Activities
VEGF-2 polypeptides or polynucleotides may also increase or decrease the
differentiation or proliferation of embryonic stem cells, besides, as
discussed above,
hematopoietic lineage.
VEGF-2 polypeptides or polynucleotides may also be used to modulate
mammalian characteristics, such as body height, weight, hair color, eye color,
skin,
percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic
surgery).
Similarly, VEGF-2 polypeptides or polynucleotides may be used to modulate
mammalian metabolism affecting catabolism, anabolism, processing, utilization,
and
storage of energy.
2o VEGF-2 polypeptides or polynucleotides may be used to change a mammal's
mental state or physical state by influencing biorhythms, caricadic rhythms,
depression (including depressive disorders), tendency for violence, tolerance
for pain,
reproductive capabilities (preferably by Activin or Inhibin-like activity),
hormonal or
endocrine levels, appetite, libido, memory, stress, or other cognitive
qualities.
VEGF-2 polypeptides or polynucleotides may also be used as a food additive
or preservative, such as to increase or decrease storage capabilities, fat
content, lipid,
protein, carbohydrate, vitamins, minerals, cofactors or other nutritional
components.
Vectors, Host Cells, and Protein Production
3o The present invention also relates to recombinant vectors, which include
the
isolated nucleic acid molecules of the present invention, and to host cells
containing
the recombinant vectors, as well as to methods of making such vectors and host
cells
and for using them for production of VEGF-2 polypeptides or peptides by
recombinant techniques.
Host cells are genetically engineered (transduced, transformed, or
transfected)
with the vectors of this invention which may be, for example, a cloning vector
or an
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-49
expression vector. The vector may be, for example, in the form of a plasmid, a
viral
particle, a phage, etc. The engineered host cells can be cultured in
conventional
nutrient media modified as appropriate for activating promoters, selecting
transformants, or amplifying the VEGF-2 genes of the invention. The culture
conditions, such as temperature, pH and the like, are those previously used
with the
host cell selected for expression, and will be apparent to the skilled
artisan.
The polynucleotides of the present invention may be employed for producing
polypeptides by recombinant techniques. Thus, for example, the polynucleotide
sequence may be included in any one of a variety of expression vectors for
expressing
1 o a polypeptide. Such vectors include chromosomal, nonchromosomal and
synthetic
DNA sequences, e. g. , derivatives of S V40; bacterial plasmids; phage DNA;
yeast
plasmids; vectors derived from combinations of plasmids and phage DNA, viral
DNA
such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any
other
plasmid or vector may be used so long as it is replicable and viable in the
host.
The appropriate DNA sequence may be inserted into the vector by a variety of
procedures. In general, the DNA sequence is inserted into an appropriate
restriction
endonuclease sites) by procedures known in the art. Such procedures and others
are
deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an
appropriate expression control sequences) (promoter) to direct mRNA synthesis.
As
representative examples of such promoters, there may be mentioned: LTR or SV40
promoter, the E. coli. lac or trp, the phage lambda P~ promoter and other
promoters
known to control expression of genes in prokaryotic or eukaryotic cells or
their
viruses. The expression vector also contains a ribosome binding site for
translation
initiation and a transcription terminator. The vector may also include
appropriate
sequences for amplifying expression.
In addition, the expression vectors preferably contain at least one selectable
marker gene to provide a phenotypic trait for selection of transformed host
cells. Such
markers include dihydrofolate reductase (DHFR) or neomycin resistance for
3o eukaryotic cell culture, and tetracycline or ampicillin resistance for
culturing in E. coli
and other bacteria.
The vector containing the appropriate DNA sequence as herein above
described, as well as an appropriate promoter or control sequence, may be
employed
to transform an appropriate host to permit the host to express the protein.
Representative examples of appropriate hosts, include but are not limited to:
bacterial
cells, such as E. coli, Salmonella typhimurium, and Streptomyces; fungal
cells, such
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as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells
such as
CHO, COS, and Bowes melanoma; and plant cells. The selection of an appropriate
host is deemed to be within the scope of those skilled in the art from the
teachings
herein.
More particularly, the present invention also includes recombinant constructs
comprising one or more of the sequences as broadly described above. The
constructs
comprise a vector, such as a plasmid or viral vector, into which a sequence of
the
invention has been inserted, in a forward or reverse orientation. In a
preferred aspect
of this embodiment, the construct further comprises regulatory sequences,
including,
to for example, a promoter, operably linked to the sequence. Large numbers of
suitable
vectors and promoters are known to those of skill in the art, and are
commercially
available. The following vectors are provided by way of example - bacterial:
pQE'70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,
Bluescript vectors, pNHBA, pNH 16a, pNH 18A, pNH46A, available from
~ 5 Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from
Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44,
pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia. Other suitable vectors will be readily apparent to
the skilled
artisan.
2o In addition to the use of expression vectors in the practice of the present
invention, the present invention further includes novel expression vectors
comprising
operator and promoter elements operatively linked to nucleotide sequences
encoding a
protein of interest. One example of such a vector is pHE4a which is described
in
detail below.
25 As summarized in Figures 28 and 29, components of the pHE4a vector (SEQ
ID N0:16) include: 1 ) a neomycinphosphotransferase gene as a selection
marker, 2)
an E. coli origin of replication, 3) a TS phage promoter sequence, 4) two lac
operator
sequences, 5) a Shine-Delgarno sequence, 6) the lactose operon repressor gene
(lacIq) and 7) a multiple cloning site linker region. The origin of
replication (oriC) is
3o derived from pUCl9 (LTI, Gaithersburg, MD). The promoter sequence and
operator
sequences were made synthetically. Synthetic production of nucleic acid
sequences ~s
well known in the art. CL.ON7ECH 95/96 Catalog, pages 215-216, Ct,o»cH, 1020
East Meadow Circle, Palo Alto, CA 94303. The pHE4a vector was deposited with
the
ATCC on February 25, 1998, and given accession number 209645.
35 A nucleotide sequence encoding VEGF-2 (SEQ ID NO:1 ), is operatively
linked to the promoter and operator of pHE4a by restricting the vector with
NdeI and
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either XbaI, BamHI, XhoI, or Asp718, and isolating the larger fragment (the
multiple
cloning site region is about 310 nucleotides) on a gel. The nucleotide
sequence
encoding VEGF-2 (SEQ ID NO:1 ) having the appropriate restriction sites is
generated, for example, according to the PCR protocol described in Example 1,
using
PCR primers having restriction sites for NdeI (as the 5' primer) and either
XbaI,
BamHI, XhoI, or Asp718 (as the 3' primer). The PCR insert is gel purified and
restricted with compatible enzymes. The insert and vector are ligated
according to
standard protocols.
As noted above, the pHE4a vector contains a lacIq gene. LacIq is an allele of
I o the lacI gene which confers tight regulation of the lac operator. Amann,
E. et al.,
Gene 69: 301-315 ( 1988); Stark, M., Gene 51:255-267 ( 1987). The lacIq gene
encodes a repressor protein which binds to lac operator sequences and blocks
transcription of down-stream (i.e., 3') sequences. However, the lacIq gene
product
dissociates from the lac operator in the presence of either lactose or certain
lactose
analogs, e.g., isopropyl B-D-thiogalactopyranoside (IPTG).VEGF-2 thus is not
produced in appreciable quantities in uninduced host cells containing the
pHE4a
vector. Induction of these host cells by the addition of an agent such as
IPTG,
however, results in the expression of the VEGF-2 coding sequence.
The promoter/operator sequences of the pHE4a vector (SEQ >D N0:17)
comprise a T5 phage promoter and two lac operator sequences. One operator is
located 5' to the transcriptional start site and the other is located 3' to
the same site.
These operators, when present in combination with the lacIq gene product,
confer
tight repression of down-stream sequences in the absence of a lac operon
inducer,
e.g., IPTG. Expression of operatively linked sequences located down-stream
from
the lac operators may be induced by the addition of a lac operon inducer, such
as
IPTG. Binding of a lac inducer to the lacIq proteins results in their release
from the
lac operator sequences and the initiation of transcription of operatively
linked
sequences. Lac operon regulation of gene expression is reviewed in Devlin, T.,
TEX~IBOOK OF BIOCHEMISTRY WI7li CLIMCAL CORRELATIONS, 4th Edition (1997),
pages
802-807.
The pHE4 series of vectors contain all of the components of the pHE4a vectur
except for the VEGF-2 coding sequence. Features of the pHE4a vectors include
optimized synthetic T5 phage promoter, lac operator, and Shine-Delagarno.
sequences.
Further, these sequences are also optimally spaced so that expression of an
inserted
gene may be tightly regulated and high level of expression occurs upon
induction.
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Among known bacterial promoters suitable for use in the production of
proteins of the present invention include the E. toll lacI and lacZ promoters,
the T3
and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp
promoter. Suitable eukaryotic promoters include the CMV immediate early
promoter,
the HSV thymidine kinase promoter, the early and late SV40 promoters, the
promoters of retroviral LTRs, such as those of the Rous Sarcoma Virus (RSV),
and
metallothionein promoters, such as the mouse metallothionein-I promoter.
The pHE4a vector also contains a Shine-Delgarno sequence 5' to the AUG
initiation codon. Shine-Delgarno sequences are short sequences generally
located
1o about 10 nucleotides up-stream (i.e., 5') from the AUG initiation codon.
These
sequences essentially direct prokaryotic ribosomes to the AUG initiation
codon.
Thus, the present invention is also directed to expression vector useful for
the
production of the proteins of the present invention. This aspect of the
invention is
exemplified by the pHE4a vector (SEQ ID N0:16).
1 s Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with selectable
markers. Two
appropriate vectors are pKK232-8 and pCM7. Particular named bacterial
promoters
include lack lacZ, T3, T7, gpt, lambda PR, P~ and trp. Eukaryotic promoters
include
CMV immediate early, HS V thymidine kinase, early and late S V40, LTRs from
2o retrovirus, and mouse metallothionein-I. Selection of the appropriate
vector and
promoter is well within the level of ordinary skill in the art.
In a further embodiment, the present invention relates to host cells
containing
the above-described construct. The host cell can be a higher eukaryotic cell,
such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host
cell can be
25 a prokaryotic cell, such as a bacterial cell. Introduction of the construct
into the host
cell can be effected by calcium phosphate transfection, DEAF-Dextran mediated
transfection, electroporation, transduction, infection, or other methods
(Davis, L., et
al., Basic Methods in Molecular Biology ( 1986)).
The constructs in host cells can be used in a conventional manner to produce
30 the gene product encoded by the recombinant sequence. Alternatively, the
polypeptides of the invention can be synthetically produced by conventional
peptide
synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other
cells under the control of appropriate promoters. Cell-free translation
systems can also
35 be employed to produce such proteins using RNAs derived from the DNA
constructs
of the present invention. Appropriate cloning and expression vectors for use
with
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prokaryotic and eukaryotic hosts are described by Sambrook. et al., Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby
incorporated by
reference.
Transcription of a DNA encoding the polypeptides of the present invention by
higher eukaryotes is increased by inserting an enhancer sequence into the
vector.
Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp,
that act
on a promoter to increase its transcription. Examples include the SV40
enhancer on
the late side of the replication origin (bp 100 to 270), a cytomegalovirus
early
1 o promoter enhancer, a polyoma enhancer on the late side of the replication
origin, and
adenovirus enhancers.
Generally, recombinant expression vectors will include origins of replication
and selectable markers permitting transformation of the host cell, e.g., the
ampicillin
resistance gene of E. coli and S. cerevisiae TRP 1 gene, and a promoter
derived from a
highly-expressed gene to direct transcription of a downstream structural
sequence.
Such promoters can be derived from operons encoding glycolytic enzymes such as
3-
phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock
proteins,
among others. The heterologous structural sequence is assembled in appropriate
phase with translation initiation and termination sequences, and preferably, a
leader
2o sequence capable of directing secretion of translated protein into the
periplasmic space
or extracellular medium. Optionally, the heterologous sequence can encode a
fusion
protein including an N-terminal identification peptide imparting desired
characteristics,
e.g., stabilization or simplified purification of expressed recombinant
product.
Useful expression vectors for bacterial use are constructed by inserting a
structural DNA sequence encoding a desired protein together with suitable
translation
initiation and termination signals in operable reading phase with a functional
promoter.
The vector will comprise one or more phenotypic selectable markers and an
origin of
replication to ensure maintenance of the vector and to, if desirable, provide
amplification within the host. Suitable prokaryotic hosts for transformation
include E.
3o coli, Bacillus subtilis, Salmonella typhimurium and various species within
the genera
Pseudomonas, Streptomyces, and Staphylococcus, although others may also be
employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for
bacterial use can comprise a selectable marker and bacterial origin of
replication
derived from commercially available plasmids comprising genetic elements of
the well
known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for
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example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMI
(Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are
combined with an appropriate promoter and the structural sequence to be
expressed.
Following transformation of a suitable host strain and growth of the host
strain
to an appropriate cell density, the selected promoter is derepressed by
appropriate
means (e. g. , temperature shift or chemical induction) and cells are cultured
for an
additional period.
Cells are typically harvested by centrifugation, disrupted by physical or
chemical means, and the resulting crude extract retained for further
purification.
io Microbial cells employed in expression of proteins can be disrupted by any
convenient method, well known to those skilled in the art, including freeze-
thaw
cycling, sonication, mechanical disruption, or use of cell lysing agents.
Various mammalian cell culture systems can also be employed to express
recombinant protein. Examples of mammalian expression systems include the COS-
7
t 5 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (
1981 ), and
other cell lines capable of expressing a compatible vector, for example, the C
127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise
an origin of replication, a suitable promoter and enhancer, and also any
necessary
ribosome binding sites, polyadenylation site, splice donor and acceptor sites,
2o transcriptional termination sequences, and 5' flanking nontranscribed
sequences.
DNA sequences derived from the SV40 viral genome, for example, SV40 origin,
early promoter, enhancer, splice, and polyadenylation sites may be used to
provide the
required nontranscribed genetic elements.
In addition to encompassing host cells containing the vector constructs
25 discussed herein, the invention also encompasses primary, secondary, and
immortalized host cells of vertebrate origin, particularly mammalian origin,
that have
been engineered to delete or replace endogenous genetic material (e.g., VEGF-2
sequence), and/or to include genetic material (e.g., heterologous promoters)
that is
operably associated with VEGF-2 sequence of the invention, and which
activates,
30 alters, and/or amplifies endogenous VEGF-2 polynucleotides. For example,
techniques known in the art may be used to operably associate heterologous
control
regions and endogenous polynucleotide sequences (e.g. encoding VEGF-2) via
homologous recombination (see, e.g., U.S. Patent No. 5,641,670, issued June
24,
1997; International Publication No. WO 9b/29411, published September 26, 1996;
35 International Publication No. WO 94/12650, published August 4, 1994; Koller
et al.,
Proc. Natl. Acad. Sci. USA $6:8932-8935 (1989); and Zijlstra et al., Nature
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342:435-438 ( 1989), the disclosures of each of which are incorporated by
reference in
their entireties).
The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g.,
a
human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the
host cell
can be a prokaryotic cell, such as a bacterial cell. The host strain may be
chosen
which modulates the expression of the inserted gene sequences, or modifies and
processes the gene product in the specific fashion desired. Expression from
certain
promoters can be elevated in the presence of certain inducers; thus expression
of the
genetically engineered polypeptide may be controlled. Furthermore, different
host
cells have characteristics and specific mechanisms for the translational and
post-translational processing and modification (e.g., glycosylation,
phosphorylation,
cleavage) of proteins. Appropriate cell lines can be chosen to ensure the
desired
modifications and processing of the protein expressed.
The polypeptides can be recovered and purified from recombinant cell cultures
by methods used heretofore, including ammonium sulfate or ethanol
precipitation,
acid extraction, anion or cation exchange chromatography, phosphocellulose
chromatography, hydrophobic interaction chromatography, affinity
chromatography,
hydroxylapatite chromatography and lectin chromatography. It is preferred to
have
low concentrations (approximately 0.1-SmM) of calcium ion present during
purification (Price et al., J. Biol. Chem. 244:917 ( 1969)). Protein refolding
steps
can be used, as necessary, in completing configuration of the mature protein.
Finally,
high performance liquid chromatography (HPLC) can be employed for final
purification steps.
The polypeptides of the present invention may be a naturally purified product,
or a
product of chemical synthetic procedures, or produced by recombinant
techniques
from a prokaryotic or eukaryotic host (for example, by bacterial, yeast,
higher plant,
insect and mammalian cells in culture). Depending upon the host employed in a
recombinant production procedure, the polypeptides of the present invention
may be
glycosylated with mammalian or other eukaryotic carbohydrates or may be non-
glycosylated. Polypeptides of the invention may also include an initial
methionine
amino acid residue.
In addition, polypeptides of the invention can be chemically synthesized using
techniques known in the art (e.g_, see Creighton, 1983, Proteins: Structures
and
Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al.,
1984, Nature 310:105-111). For example, a peptide corresponding to a fragment
of
the VEGF-2 polypeptides of the invention can be synthesized by use of a
peptide
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-56
synthesizer. Furthermore, if desired, nonclassical amino acids or chemical
amino acid
analogs can be introduced as a substitution or addition into the VEGF-2
polynucleotide sequence. Non-classical amino acids include, but are not
limited to, to
the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino
isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx,
6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,
ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,
homocitrulline,
cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, b-
alanine, fluoro-amino acids, designer amino acids such as b-methyl amino
acids, Ca-
methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
The invention encompasses VEGF-2 polypeptides which are differentially
modified during or after translation, e.g., by glycosylation, acetylation,
phosphorylation, amidation, derivatization by known protecting/blocking
groups,
proteolytic cleavage, linkage to an antibody molecule or other cellular
ligand, etc. Any
of numerous chemical modifications may be carried out by known techniques,
including but not limited, to specific chemical cleavage by cyanogen bromide,
trypsin,
chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation,
reduction; metabolic synthesis in the presence of tunicamycin; etc.
2o Additional post-translational modifications encompassed by the invention
include, for example, e.g., N-linked or O-linked carbohydrate chains,
processing of
N-terminal or C-terminal ends), attachment of chemical moieties to the amino
acid
backbone, chemical modifications of N-linked or O-linked carbohydrate chains,
and
addition or deletion of an N-terminal methionine residue as a result of
procaryotic host
cell expression. The polypeptides may also be modified with a detectable
label, such
as an enzymatic, fluorescent, isotopic or affinity label to allow for
detection and
isolation of the protein.
Also provided by the invention are chemically modified derivatives of VEGF-2
which may provide additional advantages such as increased solubility,
stability and
circulating time of the polypeptide, or decreased immunogenicity (see U. S.
Patent
No. 4,179,337). The chemical moieties for derivitization may be selected from
water
soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol
copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The
polypeptides may be modified at random positions within the molecule, or at
predetermined positions within the molecule and may include one, two, three or
more
attached chemical moieties.
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The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 1 kDa and about 100 kDa (the term "about" indicating that in
preparations of
polyethylene glycol, some molecules will weigh more, some less, than the
stated
molecular weight) for ease in handling and manufacturing. Other sizes may be
used,
depending on the desired therapeutic profile (e.g., the duration of sustained
release
desired, the effects, if any on biological activity, the ease in handling, the
degree or
lack of antigenicity and other known effects of the polyethylene glycol to a
therapeutic
protein or analog).
The polyethylene glycol molecules (or other chemical moieties) should be
attached to the protein with consideration of effects on functional or
antigenic domains
of the protein. There are a number of attachment methods available to those
skilled in
the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to
G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting
pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol
may
be covalently bound through amino acid residues via a reactive group, such as,
a free
amino or carboxyl group. Reactive groups are those to which an activated
polyethylene glycol molecule may be bound. The amino acid residues having a
free
amino group may include lysine residues and the N-terminal amino acid
residues;
2o those having a free carboxyl group may include aspartic acid residues
glutamic acid
residues and the C-terminal amino acid residue. Sulfhydryl groups may also be
used
as a reactive group for attaching the polyethylene glycol molecules. Preferred
for
therapeutic purposes is attachment at an amino group, such as attachment at
the
N-terminus or lysine group.
One may specifically desire proteins chemically modified at the N-terminus.
Using polyethylene glycol as an illustration of the present cflmposition; one
may select
from a variety of polyethylene glycol molecules (by molecular weight,
branching,
etc.), the proportion of polyethylene glycol molecules to protein (or peptide)
molecules in the reaction mix, the type of pegylation reaction to be
performed, and the
3o method of obtaining the selected N-terminally pegylated protein. The method
of
obtaining the N-terminally pegylated preparation (i.e., separating this moiety
from
other monopegylated moieties if necessary) may be by purification of the N-
terminally
pegylated material from a population of pegylated protein molecules. Selective
proteins chemically modified at the N-terminus modification may be
accomplished by
reductive alkylation which exploits differential reactivity of different types
of primary
amino groups (lysine versus the N-terminal) available for derivatization in a
particular
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-S 8
protein. Under the appropriate reaction conditions, substantially selective
derivatization of the protein at the N-terminus with a carbonyl group
containing
polymer is achieved.
The VEGF-2 polypeptides of the invention may be in monomers or multimers
(i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the
present
invention relates to monomers and multimers of the VEGF-2 polypeptides of the
invention, their preparation, and compositions (preferably, pharmaceutical
compositions) containing them. In specific embodiments, the polypeptides of
the
invention are monomers, dimers, trimers or tetramers. In additional
embodiments, the
multimers of the invention are at least dimers, at least trimers, or at least
tetramers.
Multimers encompassed by the invention may be homomers or heteromers.
As used herein, the term homomer, refers to a multimer containing only VEGF-2
polypeptides of the invention (including VEGF-2 fragments, variants, splice
variants,
and fusion proteins, as described herein). These homomers may contain VEGF-2
polypeptides having identical or different amino acid sequences. In a specific
embodiment, a homomer of the invention is a multimer containing only VEGF-2
poIypeptides having an identical amino acid sequence. In another specific
embodiment, a homomer of the invention is a multimer containing VEGF-2
polypeptides having different amino acid sequences. In specific embodiments,
the
2o multimer of the invention is a homodimer (e.g., containing VEGF-2
polypeptides
having identical or different amino acid sequences) or a homotrimer (e.g.,
containing
VEGF-2 polypeptides having identical and/or different amino acid sequences).
In
additional embodiments, the homomeric multimer of the invention is at least a
homodimer, at least a homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing one or more
hetexologous. polypeptides (i.e., polypeptides of different proteins) in
addition to the
VEGF-2 polypeptides of the invention. In a specific embodiment, the multimer
of the
invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional
embodiments, the homomeric multimer of the invention is at least a homodimer,
at
3o Ieast a homotrimer, or at least a homotetramer.
Multimers of the invention may be the result of hydrophobic. hydrophilic.
ionic and/or covalent associations and/or may be indirectly linked, by for
example,
liposome formation. Thus, in one embodiment, multimers of the invention, such
as,
for example, homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of
the invention, such as, for example, heterotrimers or heterotetramers, are
formed
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when polypeptides of the invention contact antibodies to the polypeptides of
the
invention (including antibodies to the heterologous polypeptide sequence in a
fusion
protein of the invention) in solution. In other embodiments, multimers of the
invention are formed by covalent associations with and/or between the VEGF-2
polypeptides of the invention. Such covalent associations may involve one or
more
amino acid residues contained in the polypeptide sequence ( e.g., that recited
in SEQ
ID N0:2, or contained in the polypeptide encoded by the deposited clone.) In
one
instance, the covalent associations are cross-linking between cysteine
residues located
within the polypeptide sequences which interact in the native (i.e., naturally
occurring)
polypeptide. In another instance, the covalent associations are the
consequence of
chemical or recombinant manipulation. Alternatively, such covalent
associations may
involve one or more amino acid residues contained in the heterologous
polypeptide
sequence in a VEGF-2 fusion protein. In one example, covalent associations are
between the heterologous sequence contained in a fusion protein of the
invention (see,
e.g., US Patent Number 5,478,925). In a specific example, the covalent
associations
are between the heterologous sequence contained in a VEGF-2-Fc fusion protein
of
the invention (as described herein). In another specific example, covalent
associations
of fusion proteins of the invention are between heterologous polypeptide
sequence
from another TNF family ligand/receptor member that is capable of forming
covalently
associated multimers, such as for example, oseteoprotegerin (see, e.g.,
International
Publication No. WO 98/49305, the contents of which are herein incorporated by
reference in its entirety).
The multimers of the invention may be generated using chemical techniques
known in the art. For example, polypeptides desired to be contained in the
multimers
of the invention may be chemically cross-linked using linker molecules and
linker
. rrmolecule._~ength optimization techniques known in the-art-(see, e:g:, US
Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
Additionally, multimers of the invention may be generated using techniques
known in
the art to form one or more inter-molecule cross-links between the cysteine
residues
located within the sequence of the polypeptides desired to be contained in the
multimer
(see, e.g., US Patent Number 5,478.925. which is herein incorporated by
reference
in its entirety). Further, polypeptides of the invention may be routinely
modified by
the addition of cysteine or biotin to the C terminus or N-terminus of the
polypeptide
and techniques known in the art may be applied to generate multimers
containing one
or more of these modified polypeptides (see, e.g., US Patent Number 5,478,925,
which is herein incorporated by reference in its entirety). Additionally,
techniques
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-60-
known in the art may be applied to generate liposomes containing the
polypeptide
components desired to be contained in the multimer of the invention (see,
e.g., US
Patent Number 5,478,925, which is herein incorporated by reference in its
entirety).
Alternatively, multimers of the invention may be generated using genetic
engineering techniques known in the art. In one embodiment, polypeptides
contained
in multimers of the invention are produced recombinantly using fusion protein
technology described herein or otherwise known in the art (see, e.g., US
Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
In a
specific embodiment, polynucleotides coding for a homodimer of the invention
are
generated by ligating a polynucleotide sequence encoding a polypeptide of the
invention to a sequence encoding a linker polypeptide and then further to a
synthetic
polynucleotide encoding the translated product of the polypeptide in the
reverse
orientation from the original C-terminus to the N-terminus (lacking the leader
sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
reference in its entirety). In another embodiment, recombinant techniques
described
herein or otherwise known in the art are applied to generate recombinant
polypeptides
of the invention which contain a transmembrane domain (or hyrophobic or signal
peptide) and which can be incorporated by membrane reconstitution techniques
into
liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
2o reference in its entirety).
Therapeutic Uses
The VEGF-2 polypeptide of the present invention is a potent mitogen for
vascular and lymphatic endothelial cells. As shown in Figures 12 and 13, the
VEGF-2
polypeptide of SEQ ID N0:2, minus the initial 46 amino acids, is a potent
mitogen for
vascular endothelial cells and stimulates their growth and proliferation. The
results of
a Northern blot analysis performed for the VEGF-2 nucleic acid sequence
encoding
this polypeptide wherein 20 mg of RNA from several human tissues were probed
with
~zP-VEGF-2, illustrates that this protein is actively expressed in the heart
and lung
which is further evidence of mitogenic activity.
Accordingly, VEGF-2, or biologically active portions thereof, may be
employed to treat vascular trauma by promoting angiogenesis. For example, to
stimulate the growth of transplanted tissue where coronary bypass surgery is
performed. VEGF-2, or biologically active portions thereof, may also be
employed to
promote wound healing, particularly to re-vascularize damaged tissues or
stimulate
collateral blood flow during ischemia and where new capillary angiogenesis is
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-61
desired. VEGF-2, or biologically active portions thereof, may be employed to
treat
full-thickness wounds such as dermal ulcers, including pressure sores, venous
ulcers,
and diabetic ulcers. In addition, VEGF-2, or biologically active portions
thereof, may
be employed to treat full-thickness burns and injuries where a skin graft or
flap is used
to repair such burns and injuries. VEGF-2, or biologically active portions
thereof,
may also be employed for use in plastic surgery, for example, for the repair
of
lacerations, burns, or other trauma. In addition, VEGF-2 can be used to
promote
healing of wounds and injuries to the eye as well as to treat eye diseases.
Along these same lines, VEGF-2, or biologically active portions thereof, may
also be employed to induce the growth of damaged bone, periodontium or
ligament
tissue. VEGF-2, or biologically active portions thereof, may also be employed
for
regenerating supporting tissues of the teeth, including cementum and
periodontal
ligament, that have been damaged by, e.g., periodontal disease or trauma.
Since angiogenesis is important in keeping wounds clean and non-infected,
VEGF-2, or biologically active portions thereof, may be employed in
association with
surgery and following the repair of incisions and cuts. VEGF-2, or
biologically active
portions thereof, may also be employed for the treatment of abdominal wounds
where
there is a high risk of infection.
VEGF-2, or biologically active portions thereof, may be employed for the
2o promotion of endothelialization in vascular graft surgery. In the case of
vascular
grafts using either transplanted or synthetic material, VEGF-2, or
biologically active
portions thereof, can be applied to the surface of the graft or at the
junction to promote
the growth of vascular endothelial cells. VEGF-2, or biologically active
portions
thereof, may also be employed to repair damage of myocardial tissue as a
result of
myocardial infarction. VEGF-2, or biologically active portions thereof, may
also be
employed to repair the cardiac vascular system after ischemia. VEGF-2, or
biologically active portions thereof, may also be employed to treat damaged
vascular
tissue as a result of coronary artery disease and peripheral and CNS vascular
disease.
VEGF-2, or biologically active portions thereof, may also be employed to coat
artificial prostheses or natural organs which are to be transplanted in the
body to
minimize rejection of the transplanted material and to stimulate
vascularication of the
transplanted materials.
VEGF-2, or biologically active portions thereof, may also be employed for
vascular tissue repair of injuries resulting from trauma, for example, that
occurring
during arteriosclerosis and required following balloon angioplasty where
vascular
tissues are damaged.
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VEGF-2, or biologically active portions thereof, may also be used to treat
peripheral arterial disease. Accordingly, in a further aspect, there is
provided a
process for utilizing VEGF-2 polypeptides to treat peripheral arterial
disease.
Preferably, a VEGF-2 polypeptide is administered to an individual for the
purpose of
alleviating or treating peripheral arterial disease. Suitable doses,
formulations, and
administration routes are described below.
VEGF-2, or biologically active portions thereof, may also to promote the
endothelial function of lymphatic tissues and vessels, such as to treat the
loss of
lymphatic vessels, occlusions of lymphatic vessels, and lymphangiomas. VEGF-2
1 o may also be used to stimulate lymphocyte production.
VEGF-2, or biologically active portions thereof, may also be used to treat
hemangioma in newborns. Accordingly, in a further aspect, there is provided a
process for utilizing VEGF-2 polypeptides to treat hemangioma in newborns.
Preferably, a VEGF-2 polypeptide is administered to an individual for the
purpose of
t 5 alleviating or treating hemangioma in newborns. Suitable doses,
formulations, and
administration routes are described below.
VEGF-2, or biologically active portions thereof, may also be used to prevent
or treat abnormal retinal development in premature newborns. Accordingly, in a
further aspect, there is provided a process for utilizing VEGF-2 polypeptides
to treat
2o abnormal retinal development in premature newborns. Preferably, a VEGF-2
polypeptide is administered to an individual for the purpose of alleviating or
treating
abnormal retinal development in premature newborns. Suitable doses,
formulations,
and administration routes are described below.
VEGF-2, or biologically active portions thereof, may be used to treat primary
25 (idiopathic) lymphademas, including Milroy's disease and Lymphedema
praecox.
Accordingly, in a further aspect, there is provided a process for utilizing
VEGF-2
polypeptides to treat primary (idiopathic) lymphademas, including Milroy's
disease
and Lymphedema praecox. Preferably, a VEGF-2 polypeptide is administered to an
individual for the purpose of alleviating or treating primary (idiopathic)
lymphademas,
3o including Milroy's disease and Lymphedema praecox. VEGF-2 or biologically
active
portions thereof, may also be used to treat edema as well as to effect blood
pressure in
an animal. Suitable doses, formulations, and administration routes are
described
below.
VEGF-2, or biologically active portions thereof, may also be used to treat
35 secondary (obstructive) lifetimes including those that result from (I) the
removal of
lymph nodes and vessels, (ii) radiotherapy and surgery in the treatment of
cancer, and
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-63
(iii) trauma and infection. Accordingly, in a further aspect, there is
provided a
process for utilizing VEGF-2 polypeptides to treat secondary (obstructive)
lifetimes
including those that result from (I) the removal of lymph nodes and vessels,
(ii)
radiotherapy and surgery in the treatment of cancer, and (iii) trauma and
infection.
Preferably, a VEGF-2 polypeptide is administered to an individual for the
purpose of
secondary (obstructive) lifetimes including those that result from (I) the
removal of
lymph nodes and vessels, (ii) radiotherapy and surgery in the treatment of
cancer, and
(iii) trauma and infection. Suitable doses, formulations, and administration
routes are
described below.
l0 VEGF-2, or biologically active portions thereof, may also be used to treat
Kaposi's Sarcoma. Accordingly, in a further aspect, there is provided a
process for
utilizing VEGF-2 polypeptides to treat Kaposi's Sarcoma. Preferably, a VEGF-2
polypeptide is administered to an individual for the purpose of alleviating or
treating
Kaposi's Sarcoma. Suitable doses, formulations, and administration routes are
described below.
VEGF-2 antagonists can be used to treat cancer by inhibiting the angiogenesis
necessary to support cancer and tumor growth.
Cardiovascular Disorders
The present inventors have shown that VEGF-2 stimulates the growth of
vascular endothelial cells, stimulates endothelial cell migrati~n_ stimulates
angiogenesis in the CAM assay, decreases blood pressure in spontaneously
hypertensive rats, and increases blood flow to ischemic limbs in rabbits.
Accordingly, VEGF-2 polypeptides or polynucleotides encoding VEGF-2 may be
used to treat cardiovascular disorders, including peripheral artery disease,
such as
limb ischemia.
Cardiovascular disorders include cardiovascular abnormalities, such as arterio-
arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations,
congenital
heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart
defects
include aortic coaretation, cor triatriatum, coronary vessel anomalies,
crisscross heart,
dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger
complex,
hypoplastic left heart syndrome, levocardia, tetralogy of fallot,
transposition of great
vessels, double outlet right ventricle, tricuspid atresia, persistent truncus
arteriosus,
and heart septal defects, such as aortopulmonary septal defect, endocardial
cushion
defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal
defects.
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Cardiovascular disorders also include heart disease, such as arrhythmias,
carcinoid heart disease, high cardiac output, low cardiac output, cardiac
tamponade,
endocarditis {including bacterial), heart aneurysm, cardiac arrest, congestive
heart
failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart
hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right
ventricular hypertrophy, post-infarction heart rupture, ventricular septal
rupture, heart
valve diseases, myocardial diseases, myocardial ischemia, pericardial
effusion,
pericarditis (including constrictive and tuberculous), pneumopericardium,
postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease,
l0 ventricular dysfunction, hyperemia, cardiovascular pregnancy complications,
Scimitar
Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter,
bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block,
sinoatrial
block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-
type pre-excitation syndrome, Wolff Parkinson-White syndrome, sick sinus
syndrome, tachycardias, and ventricular fibrillation. Tachycardias include
paroxysmal
tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm,
atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,
ectopic functional
tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades
de
2o Pointes, and ventricular tachycardia.
Heart valve disease include aortic valve insufficiency, aortic valve stenosis,
hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve
prolapse,
mitral valve insufficiency, mitral valve stenosis, pulmonary atresia,
pulmonary valve
insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve
insufficiency, and tricuspid valve stenosis.
Myocardial diseases include alcoholic cardiomyopathy, congestive
cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis,
pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas
cardiomyopathy,
endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome,
myocardial
reperfusion injury, and myocarditis.
Myocardial ischemias include coronary disease, such a5 angina pectoris,
coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary
vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms,
angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic
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-65
edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome,
arterial
occlusive diseases, arteritis, enarteritis, polyarteritis nodosa,
cerebrovascular
disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis,
erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension,
hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-
occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion,
Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia
telangiectasia,
hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose
ulcer,
vasculitis, and venous insufficiency.
1 o Aneurysms include dissecting aneurysms, false aneurysms, infected
aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary
aneurysms, heart aneurysms, and iliac aneurysms.
Arterial occlusive diseases include arteriosclerosis, intermittent
claudication,
carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion,
Moyamoya
~ 5 disease, renal artery obstruction, retinal artery occlusion, and
thromboangiitis
obliterans.
Cerebrovascular disorders include carotid artery diseases, cerebral amyloid
angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral
arteriovenous malformation, cerebral artery diseases, cerebral embolism and
2o thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's
syndrome,
cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid
hemorrhage, cerebral infarction, cerebral ischemia (including transient),
subclavian
steal syndrome, periventricular leukomalacia, vascular headache, cluster
headache,
migraine, and vertebrobasilar insufficiency.
25 Embolisms include air embolisms, amniotic fluid embolisms, cholesterol
embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and
thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein
thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus
thrombosis,
Wallenberg's syndrome, and thrombophlebitis.
3o Ischemia includes cerebral ischemia, ischemic colitis, compartment
syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion
injuries, and peripheral limb ischemia. Vasculitis includes aortitis,
arteritis, Behcet's
Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,
thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch
purpura,
35 allergic cutaneous vasculitis, and Wegener's granulomatosis.
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VEGF-2 polypeptides or polynucleotides are especially effective for the
treatment of critical limb ischemia and coronary disease. As shown in Example
18,
administration of VEGF-2 polynucleotides and polypeptides to an experimentally
induced ischemia rabbit hindlimb restored blood pressure ratio, blood flow,
angiographic score, and capillary density.
VEGF-2 polypeptides may be administered using any method known in the
art, including, but not limited to, direct needle injection at the delivery
site,
intravenous injection, topical administration, catheter infusion, biolistic
injectors,
particle accelerators, gelfoam sponge depots, other commercially available
depot
1 o materials, osmotic pumps, oral or suppositorial solid pharmaceutical
formulations,
decanting or topical applications during surgery, aerosol delivery. Such
methods are
known in the art. VEGF-2 polypeptides may be administered as part of a
pharmaceutical composition, described in more detail below. Methods of
delivering
VEGF-2 polynucleotides are described in more detail below.
Gene Therapy Methods
Another aspect of the present invention is to gene therapy methods for
treating
disorders, diseases and conditions. The gene therapy methods relate to the
introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences
into
an animal to achieve expression of the VEGF-2 polypeptide of the present
invention.
This method requires a polynucleotide which codes for a VEGF-2 polypeptide
operatively linked to a promoter and any other genetic elements necessary for
the
expression of the polypeptide by the target tissue. Such gene therapy and
delivery
techniques are known in the art, see, for example, WO 90/11092, which is
herein
incorporated by reference.
Thus, for example, cells from a patient may be engineered with a
polynucleotide (DNA or RNA) comprising a promoter operably linked to a VEGF-2
polynucleotide ex vivo, with the engineered cells then being provided to a
patient to be
treated with the polypeptide. Such methods are well-known in the art. For
example,
3o see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 ( 1993);
Ferrantini, M. et
al., Cancer Research 53: 1107-1112 {1993); Ferrantini, M. et ul., J.
Immunulo~y
153: 4604-4615 ( 1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 ( 1995);
Ogura,
H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al.,
Human
Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy 4:1246-1255
(1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which
are
herein incorporated by reference. In one embodiment, the cells which are
engineered
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-67
are arterial cells. The arterial cells may be reintroduced into the patient
through direct
injection to the artery, the tissues surrounding the artery, or through
catheter injection.
As discussed in more detail below, the VEGF-2 polynucleotide constructs can
be delivered by any method that delivers injectable materials to the cells of
an animal,
such as, injection into the interstitial space of tissues (heart, muscle,
skin, lung, liver,
and the like). The VEGF-2 polynucleotide constructs may be delivered in a
pharmaceutically acceptable liquid or aqueous carrier.
In one embodiment, the VEGF-2 polynucleotide is delivered as a naked
polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to
sequences
l0 that are free from any delivery vehicle that acts to assist, promote or
facilitate entry
into the cell, including viral sequences, viral particles, liposome
formulations,
lipofectin or precipitating agents and the like. However, the VEGF-2
polynucleotides
can also be delivered in liposome formulations and lipofectin formulations and
the like
can be prepared by methods well known to those skilled in the art. Such
methods are
~5 described, for example, in U.S. Patent Nos. 5,593,972, 5,589>466, and
5,580,859,
which are herein incorporated by reference.
The VEGF-2 polynucleotide vector constructs used in the gene therapy
method are preferably constructs that will not integrate into the host genome
nor will
they contain sequences that allow for replication. Appropriate vectors include
20 pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; pSVK3,
pBPV, pMSG and pSVL available from Pharmacia; and pEFlNS, pcDNA3.1, and
pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily
apparent
to the skilled artisan.
Any strong promoter known to those skilled in the art can be used for driving
25 the expression of VEGF-2 DNA. Suitable promoters include adenoviral
promoters,
such as the adenoviral major late promoter; or heterologous promoters, such as
the
cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)
promoter;
inducible promoters, such as the MMT promoter, the metallothionein promoter;
heat
shock promoters; the albumin promoter; the ApoAI promoter; human globin
30 promoters; viral thymidine kinase promoters, such as the Herpes Simplex
thymidine
kinase promoter; retroviral LTRs; the b-actin promoter; and human growth
hormone
promoters. The promoter also may be the native promoter for VEGF-2.
Unlike other gene therapy techniques, one major advantage of introducing
naked nucleic acid sequences into target cells is the transitory nature of the
35 polynucleotide synthesis in the cells. Studies have shown that non-
replicating DNA
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-68
sequences can be introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
The VEGF-2 polynucleotide construct can be delivered to the interstitial space
of tissues within the an animal, including of muscle, skin, brain, lung,
liver, spleen,
bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney,
gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system,
eye, gland,
and connective tissue. Interstitial space of the tissues comprises the
intercellular, fluid,
mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic
fibers in
the walls of vessels or chambers, collagen fibers of fibrous tissues, or that
same matrix
1 o within connective tissue ensheathing muscle cells or in the lacunae of
bone. It is
similarly the space occupied by the plasma of the circulation and the lymph
fluid of the
lymphatic channels. Delivery to the interstitial space of muscle tissue is
preferred for
the reasons discussed below. They may be conveniently delivered by injection
into the
tissues comprising these cells. They are preferably delivered to and expressed
in
persistent, non-dividing cells which are differentiated, although delivery and
expression may be achieved in non-differentiated or less completely
differentiated cells,
such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle
cells are
particularly competent in their ability to take up and express
polynucleotides.
For the naked acid sequence injection, an effective dosage amount of DNA or
2o RNA will be in the range of from about 0.05 mg/kg body weight to about 50
mg/kg
body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20
mg/kg
and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the
artisan of ordinary skill will appreciate, this dosage will vary according to
the tissue site
of injection. The appropriate and effective dosage of nucleic acid sequence
can readily
be deterniined by those of ordinary skill in the art and may depend on the
condition
being treated and the route of administration.
The preferred route of administration is by the parenteral route of injection
into
the interstitial space of tissues. However, other parenteral routes may also
be used,
such as, inhalation of an aerosol formulation particularly for delivery to
lungs or
3o bronchial tissues, throat or mucous membranes of the nose. In addition,
naked
VEGF-2 DNA constructs can be delivered to arteries during angioplasty by the
catheter used in the procedure.
The naked polynucleotides are delivered by any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous
injection, topical administration, catheter infusion, and so-called "gene
guns". These
delivery methods are known in the art.
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As is evidenced by Example 18, naked VEGF-2 nucleic acid sequences can be
administered in vivo results in the successful expression of VEGF-2
polypeptide in
the femoral arteries of rabbits.
The constructs may also be delivered with delivery vehicles such as viral
sequences, viral particles, liposome formulations, lipofectin, precipitating
agents, etc.
Such methods of delivery are known in the art.
In certain embodiments, the VEGF-2 polynucleotide constructs are complexed
in a liposome preparation. Liposomal preparations for use in the instant
invention
include cationic (positively charged), anionic (negatively charged) and
neutral
1 o preparations. However, cationic liposomes are particularly preferred
because a tight
charge complex can be formed between the cationic liposome and the polyanionic
nucleic acid. Cationic liposomes have been shown to mediate intracellular
delivery of
plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA ( 1987) 84:7413-?416,
which is herein incorporated by reference); mRNA {Malone et al., Proc. Natl.
Acad.
Sci. USA (1989) 86:6077-6081, which is herein incorporated by reference); and
purified transcription factors (Debs et al., J. Biol. Chem. ( 1990) 265:10189-
10192,
which is herein incorporated by reference), in functional form.
Cationic liposomes are readily available. For example,
N[ 1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are
2o particularly useful and are available under the trademark Lipofectin, from
GIBCO
BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA
( 1987) 84:7413-7416, which is herein incorporated by reference). Other
commercially
available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
Other cationic liposomes can be prepared from readily available materials
using
techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092
(which is herein incorporated by reference) for a description of the synthesis
of
DOTAP ( 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation
of DOTMA liposomes is explained in the literature, see, e.g., P. Felgner et
al., Proc.
3o Natl. Acad. Sci. USA 84:7413-7417, which is herein incorporated by
reference.
Similar methods can be used to prepare liposomes from other cationic lipid
materials.
Similarly, anionic and neutral liposomes are readily available, such as from
Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using
readily
available materials. Such materials include phosphatidyl, choline,
cholesterol,
phosphatidyl ethanolarnine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol {DOPG), dioleoylphoshatidyl ethanolamine (DOPE),
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among others. These materials can also be mixed with the DC~TMA and DOTAP
starting materials in appropriate ratios. Methods for making liposomes using
these
materials are well known in the art.
For example, commercially dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine
(DOPE) can be used in various combinations to make conventional liposomes,
with or
without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be
prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas
into a sonication vial. The sample is placed under a vacuum pump overnight and
is
l0 hydrated the following day with deionized water. The sample is then
sonicated for 2
hours in a capped vial, using a Heat Systems model 350 sonicator equipped with
an
inverted cup (bath type) probe at the maximum setting while the bath is
circulated at
15EC. Alternatively, negatively charged vesicles can be prepared without
sonication to
produce multilamellar vesicles or by extrusion through nucleopore membranes to
produce unilamellar vesicles of discrete size. Other methods are known and
available
to those of skill in the art.
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar
vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being
preferred.
The various liposome-nucleic acid complexes are prepared using methods well
known
in the art. See, e.g., Straubinger et al., Methods of Immunology {1983),
101:512-527, which is herein incorporated by reference. For example, MLVs
containing nucleic acid can be prepared by depositing a thin film of
phospholipid on
the walls of a glass tube and subsequently hydrating with a solution of the
material to
be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a
homogeneous population of unilamellar liposomes. The material to be entrapped
is
added to a suspension of preformed MLVs and then sonicated. When using
liposomes
containing cationic lipids, the dried lipid film is resuspended in an
appropriate solution
such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCI,
sonicated, and then the preformed liposomes are mixed directly with the DNA.
The
liposome and DNA form a very stable complex due to binding of the positively
charged liposomes to the cationic DNA. SL'Vs find use with small nucleic acid
fragments. LUVs are prepared by a number of methods, well known in the art.
Commonly used methods include Ca'-+-EDTA chelation (Papahadjopoulos et al.,
Biochim. Biophys. Acta ( 1975) 394:483; Wilson et al., Cell ( 1979) 17:77);
ether
injection (Deamer, D. and Bangham, A., Biochim. Biophys. Acta ( 1976) 443:629;
Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al.,
Proc.
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Natl. Acad. Sci. USA ( 1979) 76:3348); detergent dialysis (Enoch, H. and
Strittmatter, P., Proc. Natl. Acad. Sci. USA ( 1979) 76:145); and reverse-
phase
evaporation (REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F.
and
Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA ( 1978) 75:145; Schaefer-
Ridder et
al., Science ( 1982) 215:166), which are herein incorporated by reference.
Generally, the ratio of DNA to liposomes will be from about 10:1 to about
1:10. Preferably, the ration will be from about 5:1 to about 1:5. More
preferably, the
ration will be about 3:1 to about 1:3. Still more preferably, the ratio will
be about 1:1.
U.S. Patent No. 5,676,954 (which is herein incorporated by reference)
1o reports on the injection of genetic material, complexed with cationic
liposomes
carriers, into mice. U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386,
5,459,127,
5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no.
WO
94/9469 (which are herein incorporated by reference) provide cationic lipids
for use in
transfecting DNA into cells and mammals. U.S. Patent Nos. 5,589,466,
5,693,622,
5,580,859, 5,703,055, and international publication no. WO 94/9469 (which are
herein incorporated by reference) provide methods for delivering DNA-cationic
lipid
complexes to mammals.
In certain embodiments, cells are be engineered, ex vivo or in vivo, using a
retroviral particle containing RNA which comprises a sequence encoding VEGF-2.
2o Retroviruses from which the retroviral plasmid vectors may be derived
include, but
are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous
sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia
virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and
mammary tumor virus.
The retroviral plasmid vector is employed to transduce packaging cell lines to
form producer cell lines. Examples of packaging cells which may be transfected
include, but are not limited to, the PE501, PA317, R-2, R-AM, PA 12, T 19-14X,
VT-
19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as
described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated
herein by reference in its entirety. The vector may transduce the packaging
cells
through any means known in the art. Such means include, but are not limited
tu,
electroporation, the use of liposomes, and CaP04 precipitation. In one
alternative, the
retroviral plasmid vector may be encapsulated into a liposome, or coupled to a
lipid,
and then administered to a host.
The producer cell line generates infectious retroviral vector particles which
include polynucleotide encoding VEGF-2. Such retroviral vector particles then
may
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be employed, to transduce eukaryotic cells, either in vitro or in vivo. The
transduced
eukaryotic cells will express VEGF-2.
In certain other embodiments, cells are engineered, ex vivo or in vivo, with
VEGF-2 polynucleotide contained in an adenovirus vector. Adenovirus can be
manipulated such that it encodes and expresses VEGF-2, and at the same time is
inactivated in terms of its ability to replicate in a normal lytic viral life
cycle.
Adenovirus expression is achieved without integration of the viral DNA into
the host
cell chromosome, thereby alleviating concerns about insertional mutagenesis.
Furthermore, adenoviruses have been used as live enteric vaccines for many
years
to with an excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev.
Respir.
Dis.109:233-238). Finally, adenovirus mediated gene transfer has been
demonstrated
in a number of instances including transfer of alpha-1-antitrypsin and CFTR to
the
lungs of cotton rats (Rosenfeld, M. A. et al. ( 1991 ) Science 252:431-434;
Rosenfeld
et al., (1992) Cell 68:143-155). Furthermore, extensive studies to attempt to
establish
~ 5 adenovirus as a causative agent in human cancer were uniformly negative
(Green, M.
et al. ( 1979) Proc. Natl. Acad. Sci. USA 76:6606).
Suitable adenoviral vectors useful in the present invention are described, for
example, in Kozarsky and Wilson, Curr. Opin. Genet. bevel. 3:499-503 ( 1993);
Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet.
Ther.
20 4:759-769 (1993); Yang et al., Nature Genet. 7:362-369 (1994); Wilson et
al.,
Nature 365:691-692 (1993); and U.S. Patent No. 5,652,224, which are herein
incorporated by reference. For example, the adenovirus vector Ad2 is useful
and can
be grown in human 293 cells. These cells contain the E 1 region of adenovirus
and
constitutively express Ela and Elb, which complement the defective
adenoviruses by
25 providing the products of the genes deleted from the vector. In addition to
Ad2, other
varieties of adenovirus (e.g., Ad3, AdS, and Ad7) are also useful in the
present
invention.
Preferably, the adenoviruses used in the present invention are replication
deficient. Replication deficient adenoviruses require the aid of a helper
virus and/or
30 packaging cell line to form infectious particles. The resulting virus is
capable of
infecting cells and can express a polynucleotide of interest which is uperably
linked to
a promoter, for example, the HARP promoter of the present invention, but
cannot
replicate in most cells. Replication deficient adenoviruses may be deleted in
one or
more of all or a portion of the following genes: Ela, Elb, E3, E4, E2a, or L1
through
35 L5.
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In certain other embodiments, the cells are engineered, ex vivo or in vivo,
using an adeno-associated virus (AAV). AAVs are naturally occurring defective
viruses that require helper viruses to produce infectious particles (Muzyczka,
N.,
Curr. Topics in Microbiol. Immunol. 158:97 ( 1992)). It is also one of the few
viruses
that may integrate its DNA into non-dividing cells. Vectors containing as
little as 300
base pairs of AAV can be packaged and can integrate, but space for exogenous
DNA
is limited to about 4.5 kb. Methods for producing and using such AAVs are
known in
the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
For example, an appropriate AAV vector for use in the present invention will
include all the sequences necessary for DNA replication, encapsidation, and
host-cell
integration. The VEGF-2 polynucleotide construct is inserted into the AAV
vector
using standard cloning methods, such as those found in Sambrook et al.,
Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press ( 1989). The
recombinant
~ 5 AAV vector is then transfected into packaging cells which are infected
with a helper
virus, using any standard technique, including lipofection, electroporation,
calcium
phosphate precipitation, etc. Appropriate helper viruses include adenoviruses,
cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging
cells are
transfected and infected, they will produce infectious AAV viral particles
which
contain the VEGF-2 polynucleotide construct. These viral particles are then
used to
transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells
will contain
the VEGF-2 polynucleotide construct integrated into its genome, and will
express
VEGF-2.
Another method of gene therapy involves operably associating heterologous
control regions and endogenous polynucleotide sequences (e.g. encoding VEGF-2)
via homologous recombination (see, e.g., U.S. Patent No. 5,641,670, issued
June
24, 1997; International Publication No. WO 96/29411, published September 26,
1996; International Publication No. WO 94/12650, published August 4, 1994;
Koller
et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al.,
Nature
342:435-438 ( 1989). This method involves the activation of a gene which is
present in
the target cells, but which is not normally expressed in the cells, or is
expressed at a
lower level than desired.
Polynucleotide constructs are made, using standard techniques known in the
art, which contain the promoter with targeting sequences flanking the
promoter.
Suitable promoters are described herein. The targeting sequence is
sufficiently
complementary to an endogenous sequence to permit homologous recombination of
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the promoter-targeting sequence with the endogenous sequence. The targeting
sequence will be sufficiently near the 5' end of the VEGF-2 desired endogenous
polynucleotide sequence so the promoter will be operably linked to the
endogenous
sequence upon homologous recombination.
The promoter and the targeting sequences can be amplified using PCR.
Preferably, the amplified promoter contains distinct restriction enzyme sites
on the 5'
and 3' ends. Preferably, the 3' end of the first targeting sequence contains
the same
restriction enzyme site as the 5' end of the amplified promoter and the 5' end
of the
second targeting sequence contains the same restriction site as the 3' end of
the
amplified promoter. The amplified promoter and targeting sequences are
digested and
ligated together.
The promoter-targeting sequence construct is delivered to the cells, either as
naked polynucleotide, or in conjunction with transfection-facilitating agents,
such as
liposomes, viral sequences, viral particles, whole viruses, lipofection,
precipitating
agents, etc., described in more detail above. The P promoter-targeting
sequence can
be delivered by any method, included direct needle injection, intravenous
injection,
topical administration, catheter infusion, particle accelerators, etc. The
methods are
described in more detail below.
The promoter-targeting sequence construct is taken up by cells. Homologous
2o recombination between the construct and the endogenous sequence takes
place, such
that an endogenous VEGF-2 sequence is placed under the control of the
promoter.
The promoter then drives the expression of the endogenous VEGF-2 sequence.
The polynucleotides encoding VEGF-2 may be administered along with other
polynucleotides encoding other angiongenic proteins. Angiogenic proteins
include,
but are not limited to, acidic and basic fibroblast growth factors, VEGF-1,
epidermal
growth factor alpha and beta, platelet-derived endothelial cell growth factor,
platelet-
derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor,
insulin
like growth factor, colony stimulating factor, macrophage colony stimulating
factor,
granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
Preferably, the polynucleotide encoding VEGF-2 contains a secretory signal
sequence that facilitates secretion of the protein. Typically, the signal
sequence is
positioned in the coding region of the polynucleotide to be expressed towards
or at the
5' end of the coding region. The signal sequence may be homologous or
heterologous
to the polynucleotide of interest and may be homologous or heterologous to the
cells
to be transfected. Additionally, the signal sequence may be chemically
synthesized
using methods known in the art.
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Any mode of administration of any of the above-described polynucleotides
constructs can be used so long as the mode results in the expression of one or
more
molecules in an amount sufficient to provide a therapeutic effect. This
includes direct
needle injection, systemic injection, catheter infusion, biolistic injectors,
particle
accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially
available
depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial
solid
(tablet or pill) pharmaceutical formulations, and decanting or topical
applications
during surgery. For example, direct injection of naked calcium phosphate-
precipitated
plasmid into rat liver and rat spleen or a protein-coated plasmid into the
portal vein has
t0 resulted in gene expression of the foreign gene in the rat livers (Kaneda
et al., Science
243:375 ( 1989)).
A preferred method of local administration is by direct injection. Preferably,
a
recombinant molecule of the present invention complexed with a delivery
vehicle is
administered by direct injection into or locally within the area of arteries.
~ s Administration of a composition locally within the area of arteries refers
to injecting
the composition centimeters and preferably, millimeters within arteries.
Another method of local administration is to contact a polynucleotide
construct
of the present invention in or around a surgical wound. For example, a patient
can
undergo surgery and the polynucleotide construct can be coated on the surface
of
20 tissue inside the wound or the construct can be injected into areas of
tissue inside the
wound.
Therapeutic compositions useful in systemic administration, include
recombinant molecules of the present invention complexed to a targeted
delivery
vehicle of the present invention. Suitable delivery vehicles for use with
systemic
25 administration comprise liposomes comprising ligands for targeting the
vehicle to a
particular site.
Preferred methods of systemic administration, include intravenous injection,
aerosol, oral and percutaneous (topical) delivery. Intravenous injections can
be
performed using methods standard in the art. Aerosol delivery can also be
performed
30 using methods standard in the art (see, for example, Stribling et al.,
Proc. Natl. Acad.
Sci. GSA 189:11277-11281, 1992, which is incorporated herein by reference).
Oral
delivery can be performed by complexing a polynucleotide construct of the
present
invention to a carrier capable of withstanding degradation by digestive
enzymes in the
gut of an animal. Examples of such carriers, include plastic capsules or
tablets, such
35 as those known in the art. Topical delivery can be performed by mixing a
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-76
polynucleotide construct of the present invention with a lipophilic reagent
(e.g.,
DMSO) that is capable of passing into the skin.
Determining an effective amount of substance to be delivered can depend upon
a number of factors including, for example, the chemical structure and
biological
activity of the substance, the age and weight of the animal, the precise
condition
requiring treatment and its severity, and the route of administration. The
frequency of
treatments depends upon a number of factors, such as the amount of
polynucleotide
constructs administered per dose, as well as the health and history of the
subject. The
precise amount, number of doses, and timing of doses will be determined by the
attending physician or veterinarian.
Therapeutic compositions of the present invention can be administered to any
animal, preferably to mammals and birds. Preferred mammals include humans,
dogs,
cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being
particularly
preferred.
Nucleic Acid Utilities
VEGF-2 nucleic acid sequences and VEGF-2 polypeptides may also be
employed for in vitro purposes related to scientific research, synthesis of
DNA and
manufacture of DNA vectors, and for the production of diagnostics and
therapeutics to
2o treat human disease. For example, VEGF-2 may be employed for in vitro
culturing of
vascular endothelial cells, where it is added to the conditional medium in a
concentration from 10 pg/ml to 10 ng/ml.
Fragments of the full length VEGF-2 gene may be used as a hybridization
probe for a cDNA library to isolate other genes which have a high sequence
similarity
2s to the gene or similar biological activity. Probes of this type generally
have at least 50
base pairs, although they may have a greater number of bases. The probe may
also be
used to identify a cDNA clone corresponding to a full length transcript and a
genomic
clone or clones that contain the complete VEGF-2 gene including regulatory and
promoter regions, exons, and introns. An example of a screen comprises
isolating the
30 coding region of the VEGF-2 gene by using the known DNA sequence to
synthesize
an oligonucleotide probe. Labeled oligonucleotides having a sequence
complementary
to that of the gene of the present invention are used to screen a library of
human
cDNA, genomic DNA or mRNA to determine which members of the library the probe
hybridizes to.
35 This invention provides methods for identification of VEGF-2 receptors. The
gene encoding the receptor can be identified by numerous methods known to
those of
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skill in the art, for example, ligand panning and FACS sorting (Coligan et
al.,Current
Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably, expression cloning
is
employed wherein polyadenylated RNA is prepared from a cell responsive to VEGF-
2, and a cDNA library created from this RNA is divided into pools and used to
transfect COS cells or other cells that are not responsive to VEGF-2.
Transfected cells
which are grown on glass slides are exposed to labeled VEGF-2. VEGF-2 can be
labeled by a variety of means including iodination or inclusion of a
recognition site for
a site-specific protein kinase. Following fixation and incubation, the slides
are
subjected to autoradiographic analysis. Positive pools are identified and sub-
pools are
to prepared and retransfected using an iterative sub-pooling and rescreening
process,
eventually yielding a single clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled VEGF-2 can be
photoaffinity linked with cell membrane or extract preparations that express
the
receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-
ray
film. The labeled complex containing VEGF-2 is then excised, resolved into
peptide
fragments, and subjected to protein microsequencing. The amino acid sequence
obtained from microsequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the gene encoding
the
putative receptor.
VEGF-2 Agonist and Antagonists
This invention is also related to a method of screening compounds to identify
those which are VEGF-2 agonists or antagonists. An example of such a method
takes
advantage of the ability of VEGF-2 to significantly stimulate the
proliferation of
human endothelial cells in the presence of the comitogen Con A. Endothelial
cells are
obtained and cultured in 96-well flat-bottomed culture plates (Costar,
Cambridge,
MA) in a reaction mixture supplemented with Con-A (Calbiochem, La Jolla, CA).
Con-A, polypeptides of the present invention and the compound to be screened
are
added. After incubation at 37EC, cultures are pulsed with 1 FCi of
;[H)thymidine (5
3o Ci/mmol; 1 Ci = 37 BGq; NEN) for a sufficient time to incorporate the ;[H]
and
harvested unto glass fiber filters (Cambridge Technology, Watertown, MA). Mean
[H]-thymidine incorporation (cpm) of triplicate cultures is determined using a
liquid
scintillation counter (Beckman Instruments, Irvine, CA). Significant
;[H]thynudine
incorporation, as compared to a control assay where the compound is excluded,
indicates stimulation of endothelial cell proliferation.
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_78_
To assay for antagonists, the assay described above is performed and the
ability of the compound to inhibit ;[H]thymidine incorporation in the presence
of
VEGF-2 indicates that the compound is an antagonist to VEGF-2. Alternatively,
VEGF-2 antagonists may be detected by combining VEGF-2 and a potential
antagonist with membrane-bound VEGF-2 receptors or recombinant receptors under
appropriate conditions for a competitive inhibition assay. VEGF-2 can be
labeled,
such as by radioactivity, such that the number of VEGF-2 molecules bound to
the
receptor can determine the effectiveness of the potential antagonist.
Alternatively, the response of a known second messenger system following
l0 interaction of VEGF-2 and receptor would be measured and compared in the
presence
or absence of the compound. Such second messenger systems include but are not
limited to, cAMP guanylate cyclase, ion channels or phosphoinositide
hydrolysis. In
another method, a mammalian cell or membrane preparation expressing the VEGF-2
receptor is incubated with labeled VEGF-2 in the presence of the compound. The
~ 5 ability of the compound to enhance or block this interaction could then be
measured.
Potential VEGF-2 antagonists include an antibody, or in some cases, an
oligonucleotide, which bind to the polypeptide and effectively eliminate VEGF-
2
function. Alternatively, a potential antagonist may be a closely related
protein which
binds to VEGF-2 receptors, however, they are inactive forms of the polypeptide
and
20 thereby prevent the action of VEGF-2. Examples of these antagonists include
a
negative dominant mutant of the VEGF-2 polypeptide, for example, one chain of
the
hetero-dimeric form of VEGF-2 may be dominant and may be mutated such that
biological activity is not retained. An example of a negative dominant mutant
includes
truncated versions of a dimeric VEGF-2 which is capable of interacting with
another
25 dimer to form wild type VEGF-2, however, the resulting homo-dimer is
inactive and
fails to exhibit characteristic VEGF activity.
Another potential VEGF-2 antagonist is an antisense construct prepared using
antisense technology. Antisense technology can be used to control gene
expression
through triple-helix formation or antisense DNA or RNA, both of which methods
are
30 based on binding of a polynucleotide to DNA or RNA. For example, the 5'
coding
portion of the polynucleotide sequence, which encodes for the mature
polypeptides of
the present invention, is used to design an antisense RNA oligonucleotide of
from
about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription (triple helix -
see Lee et
35 al., Nucl. Acids Res.6:3073 ( 1979); Cooney et al., Science 241:456 (
1988); and
Dervan et al., Science 251: I 360 ( 1991 )), thereby preventing transcription
and the
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production of VEGF-2. The antisense RNA oligonucleotide hybridizes to the mRNA
in vivo and blocks translation of the mRNA molecule into the VEGF-2
polypeptide
(Antisense - Okano, J. Neurochem.56:560 ( 1991 ); Oligodeoxynucleotides as
Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL ( 1988)).
The
oligonucleotides described above can also be delivered to cells such that the
antisense
RNA or DNA may be expressed in vivo to inhibit production of VEGF-2.
Potential VEGF-2 antagonists also include small molecules which bind to and
occupy the active site of the polypeptide thereby making the catalytic site
inaccessible
to substrate such that normal biological activity is prevented. Examples of
small
molecules include but are not limited to small peptides or peptide-like
molecules.
The antagonists may be employed to limit angiogenesis necessary for solid
tumor metastasis. The identification of VEGF-2 can be used for the generation
of
certain inhibitors of vascular endothelial growth factor. Since angiogenesis
and
neovascularization are essential steps in solid tumor growth, inhibition of
angiogenic
~ 5 activity of the vascular endothelial growth factor is very useful to
prevent the further
growth, retard, or even regress solid tumors. Although the level of expression
of
VEGF-2 is extremely low in normal tissues including breast, it can be found
expressed at moderate levels in at least two breast tumor cell lines that are
derived
from malignant tumors. It is, therefore, possible that VEGF-2 is involved in
tumor
2o angiogenesis and growth.
Gliomas are also a type of neoplasia which may be treated with the antagonists
of the present invention.
The antagonists may also be used to treat chronic inflammation caused by
increased vascular permeability. In addition to these disorders, the
antagonists may
25 also be employed to treat retinopathy associated with diabetes, rheumatoid
arthritis and
psoriasis.
The antagonists may be employed in a composition with a pharmaceutically
acceptable carrier, e.g., as hereinafter described.
30 Pharmaceutical compositions
The VEGF-2 polypeptides, polynucleotides and agonists and antagonists may
be employed in combination with a suitable pharmaceutical carrier. Such
compositions comprise a therapeutically effective amount of the polypeptide or
agonist
or antagonist, and a pharmaceutically acceptable carrier or excipient. Such a
carrier
35 includes but is not limited to saline, buffered saline, dextrose, water,
glycerol,
CA 02322748 2000-09-07
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-80
ethanol, and combinations thereof. The formulation should suit the mode of
administration.
The invention also provides a pharmaceutical pack or kit comprising one or
more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the invention. Associated with such containers) can be a
notice in
the form prescribed by a governmental agency regulating the manufacture, use
or sale
of pharmaceuticals or biological products, which notice reflects approval by
the
agency of manufacture, use or sale for human administration. In addition, the
pharmaceutical compositions may be employed in conjunction with other
therapeutic
to compounds.
The pharmaceutical compositions may be administered in a convenient manner
such as by the topical, intravenous, intraperitoneal, intramuscular,
intratumor,
subcutaneous, intranasal or intradermal routes. The pharmaceutical
compositions are
administered in an amount which is effective for treating and/or prophylaxis
of the
specific indication. In general, the pharmaceutical compositions are
administered in an
amount of at least about 10 mg/kg body weight and in most cases they will be
administered in an amount not in excess of about 8 mg/Kg body weight per day.
In
most cases, the dosage is from about 10 mg/kg to about 1 mglkg body weight
daily,
taking into account the routes of administration, symptoms, etc.
2o The VEGF-2 polypeptides, and agonists or antagonists which are polypeptides
may also be employed in accordance with the present invention by expression of
such
polypeptide in vivo, which is often referred to as "gene therapy," described
above.
Thus, for example, cells such as bone marrow cells may be engineered with a
polynucleotide (DNA or RNA) encoding for the polypeptide ex vivo, the
engineered
cells are then provided to a patient to be treated with the polypeptide. Such
methods
are well-known in the art. For example, cells may be engineered by procedures
known in the art by use of a retroviral particle containing RNA encoding the
polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in
3o vivo, for example, by procedures known in the art. As known in the art, a
producer
cell for producing a retroviral particle containing RNA encoding a polypeptide
of the
present invention may be administered to a patient for engineering cells in
vivo and
expression of the polypeptide in vivo. These and other methods for
administering a
polypeptide of the present invention by such methods should be apparent to
those
skilled in the art from the teachings of the present invention. For example,
the
expression vehicle for engineering cells may be other than a retroviral
particle, for
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example, an adenovirus, which may be used to engineer cells in vivo after
combination with a suitable delivery vehicle.
Retroviruses from which the retroviral plasmid vectors hereinabove mentioned
may be derived include, but are not limited to, Moloney Murine Leukemia Virus,
spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma
Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency
virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
In
one embodiment, the retroviral plasmid vector is derived from Moloney Murine
Leukemia Virus.
t 0 The vector includes one or more promoters. Suitable promoters which may be
employed include, but are not limited to, the retroviral LTR; the SV40
promoter; and
the human cytomegalovirus (CMV) promoter described in Miller et al.,
Biotechniques
7: 980-990 ( 1989), or any other promoter ( e. g. , cellular promoters such as
eukaryotic
cellular promoters including, but not limited to, the histone, pol III, and b-
actin
promoters). Other viral promoters which may be employed include, but are not
limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B 19
parvovirus promoters. The selection of a suitable promoter will be apparent to
those
skilled in the art from the teachings contained herein.
The nucleic acid sequence encoding the polypeptide of the present invention is
2o under the control of a suitable promoter. Suitable promoters which may be
employed
include, but are not limited to, adenoviral promoters, such as the adenoviral
major late
promoter; or heterologous promoters, such as the cytomegalovirus (CMV)
promoter;
the respiratory syncytial virus (RSV) promoter; inducible promoters, such as
the
MMT promoter, the metallothionein promoter; heat shock promoters; the albumin
promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase
promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral
LTRs
(including the modified retroviral LTRs hereinabove described); the b-actin
promoter;
and human growth hormone promoters. The promoter also may be the native
promoter which controls the gene encoding the polypeptide.
3o The retroviral plasmid vector is employed to transduce packaging cell lines
to
form producer cell lines. Examples of packaging cells which may be
transfecte~l
include, but are not limited to, the PE501, PA317, y-2, y-AM, PA 12, T 19-14X,
VT-
19-17-H2, yCRE, yCRIP, GP+E-86, GP+envAml2, and DAN cell lines as
described in Miller, Human Gene Therapy 1:5-14 ( 1990), which is incorporated
herein by reference in its entirety. The vector may transduce the packaging
cells
through any means known in the art. Such means include, but are not limited
to,
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electroporation, the use of liposomes, and CaPO,~ precipitation. In one
alternative, the
retroviral plasmid vector may be encapsulated into a liposome, or coupled to a
lipid,
and then administered to a host.
The producer cell line generates infectious retroviral vector particles which
include the nucleic acid sequences) encoding the polypeptides. Such retroviral
vector
particles then may be employed, to transduce eukaryotic cells, either in vitro
or in
vivo. The transduced eukaryotic cells will express the nucleic acid sequences)
encoding the polypeptide. Eukaryotic cells which may be transduced include,
but are
not limited to, embryonic stem cells, embryonic carcinoma cells, as well as
1o hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,
keratinocytes,
endothelial cells, and bronchial epithelial cells.
Diagnostic assays
This invention is also related to the use of the VEGF-2 gene as part of a
diagnostic assay for detecting diseases or susceptibility to diseases related
to the
presence of mutations in VEGF-2 nucleic acid sequences.
Individuals carrying mutations in the VEGF-2 gene may be detected at the
DNA level by a variety of techniques. Nucleic acids for diagnosis may be
obtained
from a patient's cells, such as from blood, urine, saliva, tissue biopsy and
autopsy
2o material. The genomic DNA may be used directly for detection or may be
amplified
enzymatically by using PCR (Saiki et al., Nature 324:163-166 (1986)) prior to
analysis. RNA or cDNA may also be used for the same purpose. As an example,
PCR primers complementary to the nucleic acid encoding VEGF-2 can be used to
identify and analyze VEGF-2 mutations. For example, deletions and insertions
can be
detected by a change in size of the amplified product in comparison to the
normal
genotype. Point mutations can be identified by hybridizing amplified DNA to
radiolabeled VEGF-2 RNA or alternatively, radiolabeled VEGF-2 antisense DNA
sequences. Perfectly matched sequences can be distinguished from mismatched
duplexes by RNase A digestion or by differences in melting temperatures.
Genetic testing based on DNA sequence differences may be achieved by
detection of alteration in electrophoretic mobility of DNA fragments in gels
with or
without denaturing agents. Small sequence deletions and insertions can be
visualized
by high resolution gel electrophoresis. DNA fragments of different sequences
may be
distinguished on denaturing formamide gradient gels in which the mobilities of
different DNA fragments are retarded in the gel at different positions
according to their
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specific melting or partial melting temperatures (see, e.g., Myers et al.,
Science
230:1242 (1985)).
Sequence changes at specific locations may also be revealed by nuclease
protection assays, such as RNase and S 1 protection or the chemical cleavage
method
(e.g., Cotton et al., PNAS, USA 85:4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods
such as hybridization, RNase protection, chemical cleavage, direct DNA
sequencing
or the use of restriction enzymes, (e. g. , Restriction Fragment Length
Polymorphisms
(RFLP)) and Southern blotting of genomic DNA.
l0 In addition to more conventional gel-electrophoresis and DNA sequencing,
mutations can also be detected by in situ analysis.
The present invention also relates to a diagnostic assay for detecting altered
levels of VEGF-2 protein in various tissues since an over-expression of the
proteins
compared to normal control tissue samples may detect the presence of a disease
or
~ 5 susceptibility to a disease, for example, abnormal cellular
differentiation. Assays used
to detect levels of VEGF-2 protein in a sample derived from a host are well-
known to
those of skill in the art and include radioimmunoassays, competitive-binding
assays,
Western Blot analysis, ELISA assays and "sandwich" assay. An ELISA assay
(Coligan et al., Current Protocols in Immunology 1 (2), Chapter 6, ( 1991 ))
initially
20 comprises preparing an antibody specific to the VEGF-2 antigen, preferably
a
monoclonal antibody. In addition a reporter antibody is prepared against the
monoclonal antibody. To the reporter antibody is attached a detectable reagent
such as
radioactivity, fluorescence or, in this example, a horseradish peroxidase
enzyme. A
sample is removed from a host and incubated on a solid support, e.g. a
polystyrene
25 dish, that binds the proteins in the sample. Any free protein binding sites
on the dish
are then covered by incubating with a non-specific protein, such as, bovine
serum
albumen. Next, the monoclonal antibody is incubated in the dish during which
time
the monoclonal antibodies attach to any VEGF-2 proteins attached to the
polystyrene
dish. All unbound monoclonal antibody is washed out with buffer. The reporter
30 antibody linked to horseradish peroxidase is placed in the dish resulting
in binding of
the reporter antibody to any monoclonal antibody bound to VEGF-2. LnattachC~i
reporter antibody is then washed out. Peroxidase substrates are then added to
the dish
and the amount of color developed in a given time period is a measurement of
the
amount of VEGF-2 protein present in a given volume of patient sample when
35 compared against a standard curve.
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A competition assay may be employed wherein antibodies specific to VEGF-2
are attached to a solid support. Polypeptides of the present invention are
then labeled,
for example, by radioactivity, and a sample derived from the host are passed
over the
solid support and the amount of label detected, for example by liquid
scintillation
chromatography, can be correlated to a quantity of VEGF-2 in the sample.
A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay
VEGF-2 is passed over a solid support and binds to antibody attached to a
solid
support. A second antibody is then bound to the VEGF-2. A third antibody which
is
labeled and specific to the second antibody is then passed over the solid
support and
binds to the second antibody and an amount can then be quantified.
Chromosome identification
' The sequences of the present invention are also valuable for chromosome
identification. The sequence is specifically targeted to and can hybridize
with a
particular location on an individual human chromosome. Moreover, there is a
current
need for identifying particular sites on the chromosome. Few chromosome
marking
reagents based on actual sequence data (repeat polymorphisrri s) are presently
available for marking chromosomal location. The mapping of DNAs to chromosomes
according to the present invention is an important first step in correlating
those
2o sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is used to
rapidly select primers that do not span more than one exon in the genomic DNA,
thus
complicating the amplification process. These primers are then used for PCR
screening of somatic cell hybrids containing individual human chromosomes.
Only
those hybrids containing the human gene corresponding to the primer will yield
an
amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular DNA to a particular chromosome. Using the present invention with
the same
oligonucleotide primers, sublocalization can be achieved with panels of
fragments
from specific chromosomes or pools of large genomic clones in an analogous
manner.
Other mapping strategies that can similarly be used to map to its chromosome
include
in situ hybridization, prescreening with labeled flow-sorted chromosomes and
preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase
chromosomal spread can be used to provide a precise chromosomal location in
one
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step. This technique can be used with probes from the cDNA as short as 50 or
60
base pairs. For a review of this technique, see Verma et al., Human
Chromosomes: a
Manual of Basic Techniques, Pergamon Press, New York ( 1988).
Once a sequence has been mapped to a precise chromosomal location, the
physical position of the sequence on the chromosome can be correlated with
genetic
map data. Such data are found, for example, in V. McKusick, Mendelian
Inheritance
in Man (available on line through Johns Hopkins University Welch Medical
Library).
The relationship between genes and diseases that have been mapped to the same
chromosomal region are then identified through linkage analysis (coinheritance
of
1o physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic
sequence between affected and unaffected individuals. If a mutation is
observed in
some or all of the affected individuals but not in any normal individuals,
then the
mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques,
a cDNA precisely localized to a chromosomal region associated with the disease
could
be one of between 50 and 500 potential causative genes. (This assumes 1
megabase
mapping resolution and one gene per 20 kb).
Comparison of affected and unaffected individuals generally involves first
looking for structural alterations in the chromosomes, such as deletions or
translocations that are visible from chromosome spreads or detectable using
PCR
based on that cDNA sequence. Ultimately, complete sequencing of genes from
several
individuals is required to confirm the presence of a mutation and to
distinguish
mutations from polymorphisms.
Antisense
The present invention is further directed to inhibiting VEGF-2 in vivo by the
use of antisense technology. Antisense technology can be used to control gene
expression through triple-helix formation or antisense DNA or RNA, both of
which
methods are based on binding of a polynucleotide to DNA or RNA. For example,
the
5' coding portion of the mature polynucleotide sequence, which encodes for the
polypeptide of the present invention, is used to design an antisense RNA
oligonucleotide of from 10 to 40 base pairs in length. A DNA oligonucleotide
is
designed to be complementary to a region of the gene involved in transcription
(triple
helix -- see Lee et al., Nucl. Acids Res. 6:3073 ( 1979); Cooney et al.,
Science
241:456 ( 1988); and Dervan et al. Science, 251:1360 ( 1991 ), thereby
preventing
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transcription and the production of VEGF-2. The antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of an mRNA molecule into
the
VEGF-2 (antisense -- Okano, J. Neurochem. 56:560 {1991); Oligodeoxynucleotides
as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (
1988)).
Alternatively, the oligonucleotides described above can be delivered to cells
by
procedures in the art such that the anti-sense RNA or DNA may be expressed in
vivo
to inhibit production of VEGF-2 in the manner described above.
Antisense constructs to VEGF-2, therefore, may inhibit the angiogenic activity
of the VEGF-2 and prevent the further growth or even regress solid tumors,
since
angiogenesis and neovascularization are essential steps in solid tumor growth.
These
antisense constructs may also be used to treat rheumatoid arthritis,
psoriasis, diabetic
retinopathy and Kaposi's sarcoma which are all characterized by abnormal
angiogenesis.
Epitope-Bearing Portions
In another aspect, the invention provides peptides and polypeptides
comprising epitope-bearing portions of the polypeptides of the present
invention.
These epitopes are immunogenic or antigenic epitopes of the polypeptides of
the
present invention. An "immunogenic epitope" is defined as a part of a protein
that
2o elicits an antibody response in vivo when the whole polypeptide of the
present
invention, or fragment thereof, is the immunogen. On the other hand, a region
of a
polypeptide to which an antibody can bind is defined as an "antigenic
determinant" or
"antigenic epitope." The number of in vivo immunogenic epitopes of a protein
generally is less than the number of antigenic epitopes. See, e.g., Geysen, et
al.
( 1983) Proc. Natl. Acad. Sci. USA 81:3998-4002. However, antibodies can be
made. to any antigenic epitope, regardless of whether it is an immunogenic
epitope, by
using methods such as phage display. See e.g., Petersen G. et al. ( 1995) Mol.
Gen.
Genet. 249:425-431. Therefore, included in the present invention are both
immunogenic epitopes and antigenic epitopes.
It is particularly pointed out that the immunogenic epitopes comprises
predicted critical amino acid residues determined by the Jameson-Wolf
analysis.
Thus, additional flanking residues on either the N-terminal, C-terminal, or
both N-
and C-terminal ends may be added to these sequences to generate an epitope-
bearing
polypeptide of the present invention. Therefore, the immunogenic epitopes may
include additional N-terminal or C-terminal amino acid residues. The
additional
flanking amino acid residues may be contiguous flanking N-terminal and/or C-
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-87_
terminal sequences from the polypeptides of the present invention,
heterologous
polypeptide sequences, or may include both contiguous flanking sequences from
the
polypeptides of the present invention and heterologous polypeptide sequences.
Polypeptides of the present invention comprising immunogenic or antigenic
epitopes are at least 7 amino acids residues in length. "At least" means that
a
polypeptide of the present invention comprising an immunogenic or antigenic
epitope
may be 7 amino acid residues in length or any integer between 7 amino acids
and the
number of amino acid residues of the full length polypeptides of the
invention.
Preferred polypeptides comprising immunogenic or antigenic epitopes are at
least 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100
amino acid
residues in length. However, it is pointed out that each and every integer
between 7
and the number of amino acid residues of the full length polypeptide are
included in
the present invention.
The immuno and antigenic epitope-bearing fragments may be specified by
~ 5 either the number of contiguous amino acid residues, as described above,
or further
specified by N-terminal and C-terminal positions of these fragments on the
amino acid
sequence of SEQ >D N0:2. Every combination of a N-terminal and C-terminal
position that a fragment of, for example, at least 7 or at least 15 contiguous
amino acid
residues in length could occupy on the amino acid sequence of SEQ ID N0:2 is
2o included in the invention. Again, "at least 7 contiguous amino acid
residues in length"
means 7 amino acid residues in length or any integer between 7 amino acids and
the
number of amino acid residues of the full length polypeptide of the present
invention.
Specifically, each and every integer between 7 and the number of amino acid
residues
of the full length polypeptide are included in the present invention.
25 Immunogenic and antigenic epitope-bearing polypeptides of the invention are
useful,--for. example, to make antibodies which specifically bind the
polypeptides of
the invention, and in immunoassays to detect the polypeptides of the present
invention. The antibodies are useful, for example, in affinity purification of
the
polypeptides of the present invention. The antibodies may also routinely be
used in a
30 variety of qualitative or quantitative immunoassays, specifically for the
polypeptides
of the present invention using methods known in the art. See, e.g.. Harlow et
al..
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press; 2nd Ed.
1988).
The epitope-bearing polypeptides of the present invention may be produced by
35 any conventional means for making polypeptides including synthetic and
recombinant
methods known in the art. For instance, epitope-bearing peptides may be
synthesized
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using known methods of chemical synthesis. For instance, Houghten has
described a
simple method for the synthesis of large numbers of peptides, such as 10-20
mgs of
248 individual and distinct 13 residue peptides representing single amino acid
variants
of a segment of the HA 1 polypeptide, all of which were prepared and
characterized
(by ELISA-type binding studies) in less than four weeks (Houghten, R. A. Proc.
Natl. Acad. Sci. USA 82:5131-5135 (1985)). This "Simultaneous Multiple Peptide
Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to
Houghten and coworkers ( 1986). In this procedure the individual resins for
the
solid-phase synthesis of various peptides are contained in separate solvent-
permeable
l0 packets, enabling the optimal use of the many identical repetitive steps
involved in
solid-phase methods. A completely manual procedure allows 500-1000 or more
syntheses to be conducted simultaneously (Houghten et al. ( 1985) Proc. Natl.
Acad.
Sci. 82:5131-5135 at 5134.
Epitope-bearing polypeptides of the present invention are used to induce
~ 5 antibodies according to methods well known in the art including, but not
limited to, in
vivo immunization, in vitro immunization, and phage display methods. See,
e.g.,
Sutcliffe, et al., supra; Wilson, et al., supra, and Bittle, et al. (1985) J.
Gen. Virol.
66:2347-2354. If in vivo immunization is used, animals may be immunized with
free
peptide; however, anti-peptide antibody titer may be boosted by coupling of
the
20 peptide to a macromolecular carrier, such as keyhole limpet hemacyanin
(KLH) or
tetanus toxoid. For instance, peptides containing cysteine residues may be
coupled to
a carrier using a linker such as -maleimidobenzoyl- N-hydroxysuccinimide ester
(MBS), while other peptides may be coupled to carriers using a more general
linking
agent such as glutaraldehyde. Animals such as rabbits, rats and mice are
immunized
25 with either free or carrier-coupled peptides, for instance, by
intraperitoneal and/or
intradermal injection of emulsions- containing about 100 pgs of peptide or
cari'ier
protein and Freund's adjuvant. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful titer of anti-
peptide
antibody which can be detected, for example, by ELISA assay using free peptide
30 adsorbed to a solid surface. The titer of anti-peptide antibodies in serum
from an
immunized animal may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and elution of the
selected
antibodies according to methods well known in the art.
As one of skill in the art will appreciate, and discussed above, the
polypeptides
35 of the present invention comprising an immunogenic or antigenic epitope can
be fused
to heterologous polypeptide sequences. For example, the polypeptides of the
present
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-89
invention may be fused with the constant domain of immunoglobulins (IgA, IgE,
IgG, IgM), or portions thereof (CH 1, CH2, CH3, any combination thereof
including
' both entire domains and portions thereof) resulting in chimeric
polypeptides. These
fusion proteins facilitate purification, and show an increased half life in
vivo. This
has been shown, e.g., for chimeric proteins consisting of the first two
domains of the
human CD4-polypeptide and various domains of the constant regions of the heavy
or
light chains of mammalian immunoglobulins. See, e.g., EPA 0,394,827;
Traunecker
et al. ( 1988) Nature 331:84-86. Fusion proteins that have a disulfide-linked
dimeric
structure due to the IgG portion can also be more efficient in binding and
neutralizing
other molecules than monomeric polypeptides or fragments thereof alone. See,
e.g.,
Fountoulakis et al. ( 1995) J. Biochem. 270:3958-3964. Nucleic acids encoding
the
above epitopes can also be recombined with a gene of interest as an epitope
tag to aid
in detection and purification of the expressed polypeptide.
~5 Antibodies
The present invention further relates to antibodies and T-cell antigen
receptors
(TCR) which specifically bind the polypeptides of the present invention. The
antibodies of the present invention include IgG (including IgG 1, IgG2, IgG3,
and
IgG4), IgA (including IgA 1 and IgA2), IgD, IgE, or IgM, and IgY. As used
herein,
20 the term "antibody" (Ab) is meant to include whole antibodies, including
single-chain
whole antibodies, and antigen-binding fragments thereof. Most preferably the
antibodies are human antigen binding antibody fragments of the present
invention
include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs
(scFv),
single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising
either a
25 V~ or V,, domain. The antibodies may be from any animal origin including
birds and
mxriomals. Preferably, the antibodies are human, marine, rabbit, goat, guinea
pig,
camel, horse, or chicken.
Antigen-binding antibody fragments, including single-chain antibodies, may
comprise the variable regions) alone or in combination with the entire or
partial of the
30 following: hinge region, CHI, CH2, and CH3 domains. Also included in the
invention are any combinations of variable regions) and hinge region, CHI,
CH2,
and CH3 domains. The present invention further includes chimeric, humanized,
and
human monoclonal and polyclonal antibodies which specifically bind the
polypeptides
of the present invention. The present invention further includes antibodies
which are
35 anti-idiotypic to the antibodies of the present invention.
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The antibodies of the present invention may be monospecific, bispecific,
trispecific or of greater multispecificity. Multispecific antibodies may be
specific for
different epitopes of a polypeptide of the present invention or may be
specific for both
a polypeptide of the present invention as well as for heterologous
compositions, such
as a heterologous polypeptide or solid support material. See, e.g., WO
93/17715; WO
92/08802; WO 91100360; WO 92/05?93; Tutt, A. et al. ( 1991 ) J. Immunol.
147:60-
69; US Patents 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648;
Kostelny,
S.A. et al. (1992) J. Immunol. 148:1547-1553.
Antibodies of the present invention may be described or specified in terms of
1 o the epitope(s) or portions) of a polypeptide of the present invention
which are
recognized or specifically bound by the antibody. The epitope(s) or
polypeptide
portions) may be specified as described herein, e. g. , by N-terminal and C-
terminal
positions, by size in contiguous amino acid residues, or listed in the Tables
and
Figures. Antibodies which specifically bind any epitope or polypeptide of the
present
~ 5 invention may also be excluded. Therefore, the present invention includes
antibodies
that specifically bind polypeptides of the present invention, and allows for
the
exclusion of the same.
Antibodies of the present invention may also be described or specified in
terms
of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or
2o homolog of the polypeptides of the present invention are included.
Antibodies that do
not bind polypeptides with less than 95%, less than 90%, less than 85%, less
than
80%, less than 75%, less than 70%, less than 65%, less than 60%, less than
55%,
and less than 50% identity (as calculated using methods known in the art and
described herein) to a polypeptide of the present invention are also included
in the
25 present invention. Further included in the present invention are antibodies
which only
bind polypeptides encoded by polynucleotides which hybridize to a
polynucleotide of
the present invention under stringent hybridization conditions (as described
herein).
Antibodies of the present invention may also be described or specified in
terms of their
binding affinity. Preferred binding affinities include those with a
dissociation constant
30 or Kd less than 5X 10-6M, 10-6M, 5X 10~'M, 10-'M, 5X I O~~M, 10-8M, 5X
10~9M, 10-
9M. 5X10''°M, 10''°M, 5X10-"M, 10-"M, 5X10~'zM. 10~'ZM. 5X10"M.
10~"M.
5X10-''~M, 10-'~M, 5X10~'SM, and 10-'SM.
Antibodies of the present invention have uses that include, but are not
limited
to, methods known in the art to purify, detect, and target the polypeptides of
the
35 present invention including both in vitro and in vivo diagnostic and
therapeutic
methods. For example, the antibodies have use in immunoassays for
qualitatively and
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quantitatively measuring levels of the polypeptides of the present invention
in
biological samples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY
MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by
reference in the entirety).
The antibodies of the present invention may be used either alone or in
combination with other compositions. The antibodies may further be
recombinantly
fused to a heterologous polypeptide at the N- or C-terminus or chemically
conjugated
(including covalently and non-covalently conjugations) to polypeptides or
other
compositions. For example, antibodies of the present invention may be
recombinantly
fused or conjugated to molecules useful as labels in detection assays and
effector
molecules such as heterologous polypeptides, drugs, or toxins. See, e. g. , WO
92/08495; WO 91/14438; WO 89/12624; US Patent 5,314,995; and EP 0 396 387.
The antibodies of the present invention may be prepared by any suitable method
known in the art. For example, a polypeptide of the present invention or an
antigenic
t 5 fragment thereof can be administered to an animal in order to induce the
production of
sera containing polyclonal antibodies. Monoclonal antibodies can be prepared
using a
wide of techniques known in the art including the use of hybridoma and
recombinant
technology. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in:
2o MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier,
N.Y., 1981) (said references incorporated by reference in their entireties).
Fab and
F(ab')2 fragments may be produced by proteolytic cleavage, using enzymes such
as
papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
Alternatively, antibodies of the present invention can be produced through the
25 application of recombinant DNA technology or through synthetic chemistry
using
meth9ds known in. the_art. For example, the antibodies of the-present
invention can be
prepared using various phage display methods known in the art. In phage
display
methods, functional antibody domains are displayed on the surface of a phage
particle
which carries polynucleotide sequences encoding them. Phage with a desired
binding
30 property are selected from a repertoire or combinatorial antibody library
(e.g. human
or murine) by selecting directly with antigen, typically antigen bound or
captured to a
solid surface or bead. Phage used in these methods are typically filamentous
phage
including fd and M 13 with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII protein.
Examples of
35 phage display methods that can be used to make the antibodies of the
present invention
include those disclosed in Brinkman U. et al. (1995) J. Immunol. Methods
182:41-
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50; Ames, R.S. et al. ( 1995) J. Immunol. Methods 184:177-186; Kettleborough,
C.A. et al. ( 1994) Eur. J. Immunol. 24:952-958; Persic, L. et al. ( 1997)
Gene 187 9-
- 18; Burton, D.R. et al. ( 1994) Advances in Immunology 57:191-280;
PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236; WO 95/15982; WO 95/20401; and US Patents 5,698,426, 5,223,409,
5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908,
5,516,637, 5,780,225, 5,658,727 and 5,733,743 (said references incorporated by
reference in their entireties).
As described in the above references, after phage selection, the antibody
coding regions from the phage can be isolated and used to generate whole
antibodies,
including human antibodies, or any other desired antigen binding fragment, and
expressed in any desired host including mammalian cells, insect cells, plant
cells,
yeast, and bacteria. For example, techniques to recombinantly produce Fab,
Fab' and
F(ab')2 fragments can also be employed using methods known in the art such as
those
disclosed in WO 92/22324; Mullinax, R.L. et al. ( 1992) BioTechniques
12(6):864-
869; and Sawai, H. et al. ( 1995) AJRI 34:26-34; and Better, M. et al. ( 1988)
Science
240:1041-1043 (said references incorporated by reference in their entireties).
Examples of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston
2o et al. (1991) Methods in Enzymology 203:46-88; Shu, L. et al. (1993) PNAS
90:7995-7999; and Skerra, A. et al. ( 1988) Science 240:1038-1040. For some
uses,
including in vivo use of antibodies in humans and in vitro detection assays,
it may be
preferable to use chimeric, humanized, or human antibodies. Methods for
producing
chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202
z5 ( 1985); Oi et al., BioTechniques 4:214 ( 1986); Gillies, S.D. et al. (
1989) J. Immunol.
Methods 125:191-202; and US Patent 5,807,715. Antibodies can be humanized
using a variety of techniques including CDR-grafting (EP 0 239 400; WO
91/09967;
US Patent 5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106;
EP 0
519 596; Padlan E.A., (1991) Molecular Immunology 28(4/5):489-498; Studnicka
30 G.M. et al. ( 1994) Protein Engineering 7(6):805-814; Roguska M.A. et al. (
1994)
PNAS 91:969-973), and chain shuffling (US Patent 5,565,332). Human antibodies
can be made by a variety of methods known in the art including phage display
methods described above. See also, US Patents 4,444,887, 4,716,111, 5,545,806,
and 5,814,318; and WO 98/46645 (said references incorporated by reference in
their
35 entireties).
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Further included in the present invention are antibodies recombinantly fused
or
chemically conjugated (including both covalently and non-covalently
conjugations) to
a polypeptide of the present invention. The antibodies may be specific for
antigens
other than polypeptides of the present invention. For example, antibodies may
be
used to target the polypeptides of the present invention to particular cell
types, either in
vitro or in vivo, by fusing or conjugating the polypeptides of the present
invention to
antibodies specific for particular cell surface receptors. Antibodies fused or
conjugated to the polypeptides of the present invention may also be used in in
vitro
immunoassays and purification methods using methods known in the art. See e.
g. ,
Harbor et al. supra and WO 93/21232; EP 0 439 095; Naramura, M. et al. ( 1994)
Immunol. Lett. 39:91-99; US Patent 5,474,981; Gillies, S.O. et al. ( 1992)
PNAS
89:1428-1432; Fell, H.P. et al. ( 1991 ) J. Immunol. 146:2446-2452 (said
references
incorporated by reference in their entireties).
The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other
than the variable regions. For example, the polypeptides of the present
invention may
be fused or conjugated to an antibody Fc region, or portion thereof. The
antibody
portion fused to a polypeptide of the present invention may comprise the hinge
region,
CH 1 domain, CH2 domain, and CH3 domain or any combination of whole domains
or portions thereof. The polypeptides of the present invention may be fused or
conjugated to the above antibody portions to increase the in vivo half life of
the
polypeptides or for use in immunoassays using methods known in the art. The
polypeptides may also be fused or conjugated to the above antibody portions to
form
multimers. For example, Fc portions fused to the polypeptides of the present
invention can form dimers through disulfide bonding between the Fc portions.
Higher multimeric forms can. be made by fusing the polypeptides to portions of
IgA
and IgM. Methods for fusing or conjugating the polypeptides of the present
invention
to antibody portions are known in the art. See e.g., US Patents 5,336,603,
5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367
166; WO 96/04388, WO 91/06570; Ashkenazi, A. et al. (1991) PNAS 88:10535-
10539: Zheng, X.X. et al. ( 1995) J. Immunol. 154:5590-5600; and Vil, H. et
al.
(1992) PNAS 89:11337-11341 (said references incorporated by reference in their
entireties).
The invention further relates to antibodies which act as agonists or
antagonists
of the polypeptides of the present invention. For example, the present
invention
includes antibodies which disrupt the receptor/ligand interactions with the
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polypeptides of the invention either partially or fully. Included are both
receptor-
specific antibodies and ligand-specific antibodies. Included are receptor-
specific
antibodies which do not prevent ligand binding but prevent receptor
activation.
Receptor activation (i.e., signaling) may be determined by techniques
described herein
or otherwise known in the art. Also include are receptor-specific antibodies
which
both prevent ligand binding and receptor activation. Likewise, included are
neutralizing antibodies which bind the ligand and prevent binding of the
ligand to the
receptor, as well as antibodies which bind the ligand, thereby preventing
receptor
activation, but do not prevent the ligand from binding the receptor. Further
included
are antibodies which activate the receptor. These antibodies may act as
agonists for
either all or less than all of the biological activities affected by ligand-
mediated receptor
activation. The antibodies may be specified as agonists or antagonists for
biological
activities comprising specific activities disclosed herein. The above antibody
agonists
can be made using methods known in the art. See e.g., WO 96/40281; US Patent
~5 5,811,097; Deng, B. et al. (1998) Blood 92(6):1981-1988; Chen, Z. et al.
(1998)
Cancer Res. 58( 16):3668-3678; Harrop, J.A. et al. ( 1998) J. Immunol. 161
(4):1786-
1794; Zhu, Z. et al. ( 1998) Cancer Res. 58( 15):3209-3214; Yoon, D.Y. et al.
( 1998)
J. Immunol. 160(7):3170-3179; Prat, M. et al. ( 1998) J. Cell. Sci. 111
(Pt2):237-247;
Pitard, V. et al. ( 1997) J. Immunol. Methods 205(2):177-190; Liautard, J. et
al.
20 ( 1997) Cytokinde 9(4):233-241; Carlson, N.G. et al. ( 1997) J. Biol. Chem.
272(17):11295-11301; Taryman, R.E. et al. (1995) Neuron 14(4):755-762; Muller,
Y.A. et al. (1998) Structure 6(9):1153-1167; Bartunek, P. et al. (1996)
Cytokine
8( 1 ):14-20 (said references incorporated by reference in their entireties).
Antibodies may further be used in an immunoassay to detect the presence of
25 tumors in certain individuals. Enzyme immunoassay can be performed from the
blood
sample of an individual. Elevated levels of VEGF-2 can be considered
diagnostic of
cancer.
Truncated versions of VEGF-2 can also be produced that are capable of
interacting with wild type VEGF-2 to form dimers that fail to activate
endothelial cell
30 growth, therefore inactivating the endogenous VEGF-2. Or, mutant forms of
VEGF-
2 form dimers themselves and occupy the ligand binding domain of the proper
tyrosine kinase receptors on the target cell surface, but fail to activate
cell growth.
Alternatively, antagonists to the polypeptides of the present invention may be
employed which bind to the receptors to which a polypeptide of the present
invention
35 normally binds. The antagonists may be closely related proteins such that
they
recognize and bind to the receptor sites of the natural protein, however, they
are
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inactive forms of the natural protein and thereby prevent the action of VEGF-2
since
receptor sites are occupied. In these ways, the action of the VEGF-2 is
prevented and
the antagonist/inhibitors may be used therapeutically as an anti-tumor drug by
occupying the receptor sites of tumors which are recognized by VEGF-2 or by
inactivating VEGF-2 itself. The antagonist/inhibitors may also be used to
prevent
inflammation due to the increased vascular permeability action of VEGF-2. The
antagonist/inhibitors may also be used to treat solid tumor growth, diabetic
retinopathy, psoriasis and rheumatoid arthritis.
The antagonist/inhibitors may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as hereinabove described.
The present invention will be further described with reference to the
following
examples; however, it is to be understood that the present invention is not
limited to
such examples. All parts or amounts, unless otherwise specified, are by
weight.
In order to facilitate understanding of the following examples, certain
frequently occurring methods and/or terms will be described.
"Plasmids" are designated by a lower case p preceded and/or followed by
capital letters and/or numbers. The starting plasmids herein are either
commercially
available, publicly available on an unrestricted basis, or can be constructed
from
available plasmids in accord with published procedures. In addition,
equivalent
2o plasmids to those described are known in the art and will be apparent to
the ordinarily
skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction
enzyme that acts only at certain sequences in the DNA. The various restriction
enzymes used herein are commercially available and their reaction conditions,
cofactors and other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 mg of pIasmid or DNA
fragment
is used with about 2 units of enzyme in about 20 Fl of buffer solution. For
the
purpose of isolating DNA fragments for plasmid construction, typically 5 to 50
mg of
DNA are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate
buffers and substrate amounts for particular restriction enzymes are specified
by the
manufacturer. Incubation times of about 1 hour at 37EC are ordinarily used,
but may
vary in accordance with the supplier's instructions. After digestion the
reaction is
electrophoresed directly on a polyacrylamide gel to isolate the desired
fragment.
Size separation of the cleaved fragments is performed using 8 percent
polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res. 8:4057
( 1980).
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"Oligonucleotides" refer to either a single stranded polydeoxynucleotide or
two
complementary polydeoxynucleotide strands, which may be chemically
synthesized.
Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate
to
another oligonucleotide without adding a phosphate with an ATP in the presence
of a
kinase. A synthetic oligonucleotide will ligate to a fragment that has not
been
dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two
double stranded nucleic acid fragments (Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1989), p. 146). Unless otherwise provided, ligation may be
accomplished using known buffers and conditions with 10 units of T4 DNA ligase
("ligase") per 0.5 mg of approximately equimolar amounts of the DNA fragments
to
be ligated.
Unless otherwise stated, transformation was performed as described by the
t5 method of Graham, F. and Van der Eb, A., Virology 52:456-457 (1973).
Examples
Example 1
Expression Pattern of VEGF-2 in Human Tissues and Breast Cancer
Cell Lines
. Northern blot analysis was carried out to examine the .levels of expression
of
VEGF-2 in human tissues and breast cancer cell lines in human tissues. Total
cellular
RNA samples were isolated with RNAzoI'~"' B system (Biotecx Laboratories,
Inc.).
3o About 10 mg of total RNA isolated from each breast tissue and cell line
specified was
separated on I % agarose gel and blotted onto a nylon filter, (Sambrook et
al.,
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. ( 1989)). The labeling reaction was done
according to the Stratagene Prime-It kit with 50 ng DNA fragment. The labeled
DNA
was purified with a Select-G-50 column from 5 Prime = 3 Prime, Inc (Boulder,
CO).
The filter was then hybridized with a radioactive labeled full length VEGF-2
gene at
1,000,000 cpm/ml in 0.5 M NaPO~ and 7 % SDS overnight at 65°C. After
washing
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twice at room temperature and twice at 60°C with 0.5 X SSC, 0.1 % SDS,
the filters
were then exposed at -70°C overnight with an intensifying screen. A
message of 1.6
Kd was observed in 2 breast cancer cell lines. Figure 5, lane #4 represents a
very
tumorigenic cell line that is estrogen independent for growth.
Also, 10 mg of total RNA from 10 human adult tissues were separated on an
agarose gel and blotted onto a nylon filter. The filter was then hybridized
with
radioactively labeled VEGF-2 probe in 7% SDS, 0.5 M NaP04, pH 7.2; 1 % BSA
overnight at 65°C. Following washing in 0.2 X SSC at 65°C, the
filter was exposed
to film for 24 days at -70°C with intensifying screen. See Figure 6.
Example 2
Expression of the Truncated Form of VEGF-2 (SEQ ID N0:4) by in
vitro Transcription and Translation
The VEGF-2 cDNA was transcribed and translated in vitro to determine the
size of the translatable polypeptide encoded by the truncated form of VEGF-2
and a
partial VEGF-2 cDNA. The two inserts of VEGF-2 in the pBluescript SK vector
were amplified by PCR with three pairs of primers, 1 ) M 13-reverse and
forward
2o primers; 2) M 13-reverse primer and VEGF primer F4; and 3) M 13-reverse
primer and
VEGF primer F5. The sequence of these primers are as follows.
M13-2 reverse primer: 5'-ATGCTTCCGGCTCGTATG-3' (SEQ m N0:9)
This sequence is located upstream of the 5' end of the VEGF-2 cDNA insert in
the
pBluescript vector and is in an anti-sense orientation as the cDNA. A T3
promoter
sequence is located between this primer and the VEGF-2 cDNA.
M13-2 forward primer: 5'GGGTTTTCCCAGTCACGAC-3' (SEQ ID NO:10)
This sequence is located downstream of the 3' end of the VEGF-2 cDNA insert in
the
pBluescript vector and is in an anti-sense orientation as the cDNA insert.
VEGF primer F4: 5'-CCACATGGTTCAGGAAAGACA-3' (SEQ ID NO:11)
3o This sequence is located within the VEGF-2 cDNA in an anti-sense
orientation from
by 1259-1239, which is about 169 by away from the 3' end of the stop codon and
about 266 by before the last nucleotide of the cDNA.
PCR reaction with all three pairs of primers produce amplified products with
T3 promoter sequence in front of the cDNA insert. The first and third pairs of
primers
produce PCR products that encode the polypeptide of VEGF-2 shown in SEQ m
N0:4. The second pair of primers produce PCR product that misses 36 amino
acids
coding sequence at the C-terminus of the VEGF-2 polypeptide.
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Approximately 0.5 mg of PCR product from first pair of primers, 1 mg from
second pair of primers, 1 mg from third pair of primers were used for in vitro
transcription/translation. The in vitro transcription/translation reaction was
performed
in a 25 Fl of volume, using the TNTJ Coupled Reticulocyte Lysate Systems
(Promega,
CAT# L4950). Specifically, the reaction contains 12.5 Fl of TNT rabbit
reticulocyte
lysate 2 Fl of TNT reaction buffer, 1 FI of T3 polymerase, 1 Fl of 1 mM amino
acid
mixture (minus methionine), 4 Fl of ;SS-methionine (>1000 Ci/mmol, 10 mCi/ml),
1
Fl of 40 U/p.l; RNasin ribonuclease inhibitor, 0.5 or 1 mg of PCR products.
Nuclease-free H,O was added to bring the volume to 25 Fl. The reaction was
1o incubated at 30°C for 2 hours. Five microliters of the reaction
product was analyzed
on a 4-20% gradient SDS-PAGE gel. After fixing in 25% isopropanol and 10%
acetic
acid, the gel was dried and exposed to an X-ray film overnight at 70°C.
As shown in Figure 7, PCR products containing the truncated VEGF-2 cDNA
(i.e., as depicted in SEQ 117 N0:3) and the cDNA missing 266 by in the 3' un
translated region (3'-UTR) produced the same length of translated products,
whose
molecular weights are estimated to be 38-40 dk (lanes 1 and 3). The cDNA
missing
all the 3'UTR and missing sequence encoding the C-terminal 36 amino acids was
translated into a polypeptide with an estimated molecular weight of 36-38 kd
(lane 2).
Example 3
Cloning and Expression of VEGF-2 Using the Baculovirus Expression
System
The DNA sequence encoding the VEGF-2 protein without 46 amino acids at
the N-terminus, see ATCC No. 97149, was amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence TGT AAT ACG ACT CAC TAT AGG GAT
CCC GCC ATG GAG GCC ACG GCT TAT GC (SEQ ID N0:12) and contains a
BamHl restriction enzyme site (in bold) and 17 nucleotide sequence
complementary to
3o the 5' sequence of VEGF-2 (nt. 150-166).
The 3' primer has the sequence GATC TCT AGA TTA GCT CAT TTG
TGG TCT (SEQ ID N0:13) and contains the cleavage site for the restriction
enzyme
XbaI and 18 nucleotides complementary to the 3' sequence of VEGF-2, including
the
stop codon and 15 nt sequence before stop codon.
The amplified sequences were isolated from a 1 % agarose gel using a
commercially available kit ("Geneclean," BIO 101, Inc., La Jolla, CA). The
fragment
was then digested with the endonuclease BamHl and XbaI and then purified again
on
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a 1 % agarose gel. This fragment was ligated to pAcGP67A baculovirus transfer
vector (Pharmingen) at the BamH 1 and XbaI sites. Through this ligation, VEGF-
2
cDNA was cloned in frame with the signal sequence of baculovirus gp67 gene and
was located at the 3' end of the signal sequence in the vector. This is
designated
s pAcGP67A-VEGF-2.
To clone VEGF-2 with the signal sequence of gp67 gene to the pRG 1 vector
for expression, VEGF-2 with the signal sequence and some upstream sequence
were
excised from the pAcGP67A-VEGF-2 plasmid at the Xho restriction endonuclease
site
located upstream of the VEGF-2 cDNA and at the XbaI restriction endonuclease
site
by XhoI and XbaI restriction enzyme. This fragment was separated from the rest
of
vector on a I % agarose gel and was purified using "Geneclean" kit. It was
designated
F2.
The PRGI vector (modification of pVL941 vector) is used for the expression
of the VEGF-2 protein using the baculovirus expression system (for review see:
Summers, M.D. and Smith, G.E., "A Manual of Methods for Baculovirus Vectors
and Insect Cell Culture Procedures," Texas Agricultural Experimental Station
Bulletin
No. 1555, ( 1987)). This expression vector contains the strong polyhedrin
promoter
of the Autographa californica nuclear polyhedrosis virus {AcMNPV) followed by
the
recognition sites for the restriction endonucleases BamHl, Smal, XbaI, BgIII
and
Asp718. A site for restriction endonuclease Xhol is located upstream of BamHl
site.
The sequence between Xho I and BamHI is the same as that in PAcGp67A (static
on
tape) vector. The polyadenylation site of the simian virus (SV)40 is used for
efficient
polyadenylation. For an.easy selection of recombinant virus the beta-
galactosidase
gene from E. coli is inserted in the same orientation as the polyhedrin
promoter
followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin
sequences are flanked at both sides by viral sequences for the cell-mediated
homologous recombination of cotransfected wild-type viral DNA. Many other
baculovirus vectors could be used in place of pRG 1 such as pAc373, pVL941 and
pAcIMI (Luckow, V.A. and Summers, M.D., Virology 170:31-39 (1989).
3o The plasmid was digested with the restriction enzymes XboI and XbaI and
then dephosphorylated using calf intestinal phosphatase by procedures known in
the
art. The DNA was then isolated from a I % agarose gel using the commercially
available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is
designated V2.
Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA
ligase. E. coli HB 101 cells were then transformed and bacteria identified
that
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contained the plasmid (pBac gp67-VEGF-2) with the VEGF-2 gene using the
enzymes BarnHl and XbaI. The sequence of the cloned fragment was confirmed by
DNA sequencing.
mg of the plasmid pBac gp67-VEGF-2 was cotransfected with 1.0 mg of a
5 commercially available linearized baculovirus ("BaculoGoldJ baculovirus
DNA",
Pharmingen, San Diego, CA.) using the lipofectin method (Felgner et al., Proc.
Natl.
Acad. Sci. USA 84:7413-7417 ( 1987)).
1 mg of BaculoGoldJ virus DNA and 5 mg of the piasmid pBac gp67-VEGF-2
were mixed in a sterile well of a microtiter plate containing 50 ml of serum
free
Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ml
Lipofectin plus 90 ml Grace's medium were added, mixed and incubated for 15
minutes at room temperature. Then the transfection mixture was added dropwise
to
the Sf9 insect cells (ATCC CRL 1711 ) seeded in a 35 mm tissue culture plate
with 1
ml Grace's medium without serum. The plate was rocked back and forth to mix
the
newly added solution. The plate was then incubated for 5 hours at 27°C.
After 5
hours the transfection solution was removed from the plate and 1 ml of Grace's
insect
medium supplemented with 10% fetal calf serum was added. The plate was put
back
into an incubator and cultivation continued at 27°C for four days.
After four days the supernatant was collected and a plaque assay performed
similar as described by Summers and Smith, supra. As a modification an agarose
gel
with "Blue Gal" (Life Technologies Inc., Gaithersburg) was used which allows
an
easy isolation of blue stained plaques. (A detailed description of a "plaque
assay" can
also be found in the usei s guide for insect cell culture and baculovirology
distributed
by Life Technologies Inc., Gaithersburg, page 9-10).
Four days after the serial dilution, the virus was added to the cells, blue
stained plaques were picked with the tip of an Eppendorf pipette. The agar
containing
the recombinant viruses was then resuspended in an Eppendorf tube containing
200
ml of Grace's medium. The agar was removed by a brief centrifugation and the
supernatant containing the recombinant baculovirus was used to infect Sf9
cells
3o seeded in 35 mm dishes. Four days later the supernatants of these culture
dishes were
harvested and then stored at 4°C.
Sf9 cells were grown in Grace's medium supplemented with 10% heat-
inactivated FBS. The cells were infected with the recombinant baculovirus V-
gp67-
VEGF-2 at a multiplicity of infection (MOI) of 1. Six hours later the medium
was
removed and replaced with SF900 II medium minus methionine and cysteine (Life
Technologies Inc., Gaithersburg). 42 hours later 5 mCi of ;SS-methionine and 5
mCi
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'SS cysteine (Amersham) were added. The cells were further incubated for 16
hours
before they were harvested by centrifugation and the labelled proteins
visualized by
SDS-PAGE and autoradiography.
Protein from the medium and cytoplasm of the Sf9 cells was analyzed by
SDS-PAGE under non-reducing and reducing conditions. See Figures 8A and 8B,
respectively. The medium was dialyzed against 50 mM MES, pH 5.8. Precpitates
were obtained after dialysis and resuspended in 100 mM NaCitrate, pH 5Ø The
resuspended precipitate was analyzed again by SDS-PAGE and was stained with
Coomassie Brilliant Blue. See Figure 9.
1 o The medium supernatant was also diluted 1:10 in 50 mM MES, pH 5.8 and
applied to an SP-650M column ( 1.0 x 6.6 cm, Toyopearl) at a flow rate of 1
ml/min.
Protein was eluted with step gradients at 200, 300 and 500 mM NaCI. The VEGF-2
was obtained using the elution at 500 mM. The eluate was analyzed by SDS-PAGE
in
the presence or absence of reducing agent, b-mercaptoethanol and stained by
t5 Coomassie Brilliant Blue. See Figure 10.
Example 4
Expression of Recombinant VEGF-2 in COS Cells
20 The expression of plasmid, VEGF-2-HA is derived from a vector
pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)
ampicillin
resistance gene, 3) E. coli replication origin, 4) CMV promoter followed by a
polylinker region, an SV40 intron and polyadenylation site. A DNA fragment
encoding the entire VEGF-2 precursor and a HA tag fused in frame to its 3' end
was
25 cloned into the polylinker region of the vector, therefore, the recombinant
protein
expression is directed under the CMV promoter. The HA tag corresponds to an
epitope derived from the influenza hemagglutinin protein as previously
described
(Wilson et al., Cell 37:767 ( 1984)). The infusion of HA tag to the target
protein
allows easy detection of the recombinant protein with an antibody that
recognizes the
3o HA epitope.
The plasmid construction strategy is described as follows:
The DNA sequence encoding VEGF-2, ATCC No. 97149, was constructed
by PCR using two primers: the 5' primer (CGC GGA TCC ATG ACT GTA CTC
TAC CCA) (SEQ ID N0:14) contains a BamH 1 site followed by 18 nucleotides of
35 VEGF-2 coding sequence starting from the initiation codon; the 3' sequence
(CGC
TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CTC GAG GCT
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CAT TTG TGG TCT 3') (SEQ ID NO:15) contains complementary sequences to an
XbaI site, HA tag, XhoI site, and the last 15 nucleotides of the VEGF-2 coding
sequence (not including the stop codon). Therefore, the PCR product contains a
BamHI site, coding sequence followed by an XhoI restriction endonuclease site
and
S HA tag fused in frame, a translation termination stop codon next to the HA
tag, and an
XbaI site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were
digested with BamH 1 and XbaI restriction enzyme and ligated. The ligation
mixture
was transformed into E. coli strain SURE {Stratagene Cloning Systems, La
Jolla, CA
92037) the transformed culture was plated on ampicillin media plates and
resistant
colonies were selected. Plasmid DNA was isolated from transformants and
examined
by restriction analysis for the presence of the correct fragment. For
expression of the
recombinant VEGF-2, COS cells were transfected with the expression vector by
DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular
Cloning: A Laboratory Manual, Cold Spring Laboratory Press, { 1989)). The
~ 5 expression of the VEGF-2-HA protein was detected by radiolabelling and
immunoprecipitation method (E. Harlow and D. Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, ( 1988)). Cells were labelled for
8
hours with ;SS-cysteine two days post transfection. Culture media was then
collected
and cells were lysed with detergent (RIPA buffer ( 150 mM NaCI, 1 % NP-40, 0.1
%
SDS, 1 % NP-40, 0.5% DOC; SOmM Tris, pH 7.5) (Wilson et al., Cell 37:767
{ 1984)). Both cell lysate and culture media were precipitated with an HA
specific
monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.
Example S
The Effect of Partially Purified VEGF-2 Protein on the Growth of
Vascular Endothelial Cells
On day l, human umbilical vein endothelial cells (HUVEC) were seeded at 2-
5x104 cells/35 mm dish density in M199 medium containing 4% fetal bovine serum
(FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth
supplements
(ECGS, Biotechnique, Inc.). On day 2, the medium was replaced with M199
containing 10% FBS, 8 units/ml heparin. VEGF-2 protein of SEQ 1D NO. 2 minus
the initial 45 amino acid residues, (VEGF) and basic FGF (bFGF) were added, at
the
concentration shown. On days 4 and 6, the medium was replaced. On day 8, cell
number was determined with a Coulter Counter (See Figure 12).
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Example 6
The Effect of Purified VEGF-2 Protein on the Growth of Vascular
. Endothelial Cells
On day l, human umbilical vein endothelial cells (HUVEC) were seeded at 2-5
x 10'~ cells/35 mm dish density in M 199 medium containing 4% fetal bovine
serum
(FBS), 16 units/ml heparin, 50 units/ml endothelial cell growth supplements
(ECGS,
Biotechnique, Inc.). On day 2, the medium was replaced with M199 containing
10%
FBS, 8 units/ml heparin. Purified VEGF-2 protein of SEQ ID N0:2 minus initial
45
1 o amino acid residues was added to the medium at this point. On days 4 and
6, the
medium was replaced with fresh medium and supplements. On day 8, cell number
was determined with a Coulter Counter (See Figure 13).
Example 7
Expression via Gene Therapy
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue
is
placed in tissue-culture medium and separated into small pieces. Small chunks
of the
tissue are placed on a wet surface of a tissue culture flask, approximately
ten pieces
are placed in each flask. The flask is turned upside down, closed tight and
left at
2o room temperature over night. After 24 hours at room temperature, the flask
is
inverted and the chunks of tissue remain fixed to the bottom of the flask and
fresh
media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is
added.
This is then incubated at 37°C for approximately one week. At this
time, fresh media
is added and subsequently changed every several days. After an additional two
weeks
25 in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized
and
scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al., DNA 7:219-225 ( 1988) flanked by the long
terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI
and
HindIII and subsequently treated with calf intestinal phosphatase. The linear
vector is
3o fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using
PCR primers which correspond to the 5' and 3' end sequences respectively. The
5 '
primer containing an EcoRI site and the 3' primer further includes a HindIII
site.
Equal quantities of the Moloney murine sarcoma virus linear backbone and the
35 amplified EcoRI and HindIII fragment are added together, in the presence of
T4 DNA
ligase. The resulting mixture is maintained under conditions appropriate for
ligation
of the two fragments. The ligation mixture is used to transform bacteria HB
101,
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which are then plated onto agar-containing kanamycin for the purpose of
confirnung
that the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+am 12 packaging cells are grown in tissue
culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with
10% calf serum (CS), penicillin and streptomycin. The MSV vector containing
the
gene is then added to the media and the packaging cells are transduced with
the vector.
The packaging cells now produce infectious viral particles containing the gene
(the
packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the
media is harvested from a 10 cm plate of confluent producer cells. The spent
media,
containing the infectious viral particles, is filtered through a millipore
filter to remove
detached producer cells and this media is then used to infect fibroblast
cells. Media is
removed from a sub-confluent plate of fibroblasts and quickly replaced with
the media
from the producer cells. This media is removed and replaced with fresh media.
If the
titer of virus is high, then virtually all fibroblasts will be infected and no
selection is
required. If the titer is very low, then it is necessary to use a retroviral
vector that has
a selectable marker, such as neo or his.
The engineered fibroblasts are then injected into the host, either alone or
after
having been grown to confluence on cytodex 3 microcarrier beads. The
fibroblasts
now produce the protein product.
Example 8
Expression of VEGF-2 mRNA in Human Fetal and Adult Tissues
Experimental Design
Northern blot analysis was carried out to examine the levels of expression of
VEGF-2 mRNA in human fetal and adult tissues. A cDNA probe containing the
entire
nucleotide sequence of the VEGF-2 protein was labeled with ;ZP using the
rediprime°
DNA labeling system (Amersham Life Science), according to the manufacturer's
instructions. After labeling, the probe was purified using a CHROMA SPIN-100*
column (Clontech Laboratories, Inc.), accurding to manufacturer's protocol
number
PT 1200-1. The purified labeled probe was then used to examine various human
tissues for VEGF-2 mRNA.
A Multiple Tissue Northern (MTN) blot containing various human tissues
(Fetal Kidney, Fetal Lung, Fetal Liver, Brain, Kidney, Lung, Liver, Spleen,
Thymus, Bone Marrow, Testes, Placenta, and Skeletal Muscle) was obtained from
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Clontech. The MTN blot was examined with the labeled probe using ExpressHyb*
hybridization solution (Clontech) according to manufacturer's protocol number
PT 1190-1. Following hybridization and washing, the blot was exposed to film
at
70°C overnight with an intensifying screen and developed according to
standard
procedures.
Results
Expression of VEGF-2 mRNA is abundant in vascular smooth muscle and
several highly vascularized tissues. VEGF-2 is expressed at significantly
higher
1o levels in tissues associated with hematopoetic or angiogenic activities,
i.e. fetal
kidney, fetal lung, bone marrow, placental, spleen and lung tissue. The
expression
level of VEGF-2 is low in adult kidney, fetal liver, adult liver, testes; and
is almost
undetectable in fetal brain, and adult brain (See Figure 14).
In primary cultured cells, the expression of VEGF-2 mRNA is abundant in
vascular smooth muscle cells and dermal fibroblast cells, but much lower in
human
umbilical vein endothelial cells (see Figure 15). This mRNA distribution
pattern is
very similar to that of VEGF.
Example 9
2o Construction of Amino terminal and carboxy terminal deletion mutants
In order to identify and analyze biologically active VEGF-2 polypeptides, a
panel of deletion mutants of VEGF-2 was constructed using the expression
vector
pHE4a.
1. Construction of VEGF-2 T103-L215 in pHE4
To permit Polymerase Chain Reaction directed amplification and sub-cloning
of VEGF-2 T103-L215 (amino acids 103 to 215 in Figure 1 or SEQ )D N0:18) into
the E. coli protein expression vector, pHE4, two oligonucleotide primers
complementary to the desired region of VEGF-2 were synthesized with the
following
base sequence:
5' Primer (Nde I/START and 18 nt of coding sequence):
5'-GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3' (SEQ >D NO:
19)
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3' Primer (Asp718, STOP, and 15 nt of coding sequence):
5'-GCA GCA GGT ACC TCA CAG TTT AGA CAT GCA-3' (SEQ ID NO:
20)
The above described 5' primer (SEQ ID NO: 19), incorporates an NdeI
restriction site and the above described 3' Primer (SEQ ID N0:20),
incorporates an
Asp718 restriction site. The 5' primer (SEQ ID N0:19) also contains an ATG
sequence adjacent and in frame with the VEGF-2 coding region to allow
translation of
the cloned fragment in E. coli, while the 3' primer (SEQ ID N0:20) contains
one stop
1o codon (preferentially utilized in E. coli) adjacent and in frame with the
VEGF-2
coding region which ensures correct translational termination in E. coli.
The Polymerase Chain Reaction was performed using standard conditions well
known to those skilled in the art and the nucleotide sequence for the mature
VEGF-2
(aa 24-419 in SEQ ID N0:18) as, for example, constructed in Example 3 as
template.
The resulting amplicon was restriction digested with NdeI and Asp718 and
subcloned
into NdeI/Asp718 digested pHE4a expression vector.
2. Construction of VEGF-2 T103-8227 in pHE4
To permit Polymerase Chain Reaction directed amplification and sub-cloning
2o of VEGF-2 T 103-8227 (amino acids 103 to 227 in Figure 1 or SEQ ID N0:18)
into
the E. coli protein expression vector, pHE4, two oligonucleotide primers
complementary to the desired region of VEGF-2 were synthesized with the
following
base sequence:
S' Primer (Nde I/START and 18 nt of coding sequence):
5'-GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3' (SEQ ID
N0:19)
3' Primer (Asp 718, STOP, and 15 nt of coding sequence):
5'-GCA GCA GGT ACC TCA ACG TCT AAT AAT GGA-3' (SEQ 1D
N0:21)
In the case of the above described primers, an NdeI or Asp718 restriction site
was incorporated he 5' primer and 3' primer, respectively. The 5' primer (SEQ
ID
N0:19) also contains an ATG sequence adjacent and in frame with the VEGF-2
coding region to allow translation of the cloned fragment in E. coli, while
the 3 '
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Primer {SEQ ID N0:21 ) contains one stop codon (preferentially utilized in E.
coli)
adjacent and in frame with the VEGF-2 coding region which ensures correct
translational termination in E. coli.
The Polymerise Chain Reaction was performed using standard conditions well
known to those skilled in the art and the nucleotide sequence for the mature
VEGF-2
(aa 24-419 in SEQ ID N0:18) as, for example, constructed in Example 3, as
template.
The resulting amplicon was restriction digested with NdeI and Asp718 and
subcloned
into NdeI/Asp718 digested pHE4a protein expression vector.
to 3. Construction of VEGF-2 T103-L215 in pA2GP
In this illustrative example, the plasmid shuttle vector pA2 GP is used to
insert
the cloned DNA encoding the N-terminal and C-terminal deleted VEGF-2 protein
(amino acids 103-215 in Figure 1 or SEQ ID N0:18), into a baculovirus to
express
the N-terminal and C-terminal deleted VEGF-2 protein, using a baculovirus
leader
~ 5 and standard methods as described in Summers et al., A Manual of Methods
for
Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural
Experimental Station Bulletin No. 1555 ( 1987). This expression vector
contains the
strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis
virus
(AcMNPVj followed by the secretory signal peptide (leader) of the baculovirus
gp67
2o protein and convenient restriction sites such as BamHI, Xba I and Asp718.
The
polyadenylation site of the simian virus 40 ("SV40") is used for efficient
polyadenylation. For easy selection of recombinant virus, the plasmid contains
the
beta-galactosidase gene from E. coli under control of a weak Drosophila
promoter in
the same orientation, followed by the polyadenylation signal of the polyhedrin
gene.
25 The inserted genes are flanked on both sides by viral sequences for cell-
mediated
homologous recombination with wild-type viral DNA to generate viable virus
that
expresses the cloned polynucleotide.
Many other baculovirus vectors could be used in place of the vector above,
such as pAc373, pVL941 and pAcIMI, as one skilled in the art would readily
3o appreciate, as long as the construct provides appropriately located signals
for
transcription, translation, secretion and the like, including a signal peptide
and an in-
frame AUG as required. Such vectors are described, for instance, in Luckow et
al.,
Virology 170:31-39 (1989).
The cDNA sequence encoding the VEGF-2 protein without 102 amino acids at
35 the N-terminus and without 204 amino acids at the C-terminus in Figure 1,
was
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amplified using PCR oligonucleotide primers corresponding to the 5' and 3 '
sequences of the gene.
The 5' primer has the sequence 5' -GCA GCA GGA TCC CAC AGA AGA
GAC TAT AAA- 3' (SEQ ID N0:22) containing the BamHI restriction enzyme site
(in bold) followed by 1 spacer nt to stay in-frame with the vector-supplied
signal
peptide, and 17 nt of coding sequence bases of VEGF-2 protein. The 3' primer
has
the sequence SN-GCA GCA TCT AGA TCA CAG TTT AGA CAT GCA-3' (SEQ
ID N0:23) containing the XbaI restriction site (in bold) followed by a stop
codon and
17 nucleotides complementary to the 3' coding sequence of VEGF-2.
1 o The amplified sequences were isolated from a I % agarose gel using a
commercially available kit {"Geneclean," BIO 101, Inc., La Jolla, CA). The
fragment
was then digested with the endonuclease BamH 1 and XbaI and then purified
again on
a 1 % agarose gel. This fragment was ligated to pA2 GP baculovirus transfer
vector
{Supplier) at the BamH 1 and Xbal sites. Through this ligation, VEGF-2 cDNA
representing the N-terminal and C-terminal deleted VEGF-2 protein (amino acids
103-
215 in Figure 1 or SEQ >D N0:18) was cloned in frame with the signal sequence
of
baculovirus GP gene and was located at the 3' end of the signal sequence in
the
vector. This is designated pA2GPVEGF-2.T103-L215.
4. Construction of VEGF-2 T103-8227 in pA2GP
The cDNA sequence encoding the VEGF-2 protein without 102 amino acids at
the N-terminus and without 192 amino acids at the C-terminus in Figure 1
(i.e., amino
acids 103-227 of SEQ ID N0:18) was amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the gene.
The 5'-GCA GCA GGA TCC CAC AGA AGA GAC TAT AAA ATT TGC
TGC-3' primer has the sequence (SEQ ID N0:24) containing the BamHI restriction
enzyme site (in bold) followed by 1 spacer nt to stay in-frame with the vector-
supplied
signal peptide, and 26 nt of coding sequence bases of VEGF-2 protein. The 3'
primer
has the sequence 5N-GCA GCA TCT AGA TCA ACG TCT AAT AAT GGA ATG
3o AAC-3' {SEQ ID N0:25) containing the XbaI restriction site (in bold)
followed by a
stop codon and 21 nucleotides complementary to the 3' coding sequence of VEGF-
2.
The amplified sequences were isolated from a 1 % agarose gel using a
commercially available kit ("Geneclean," BIO 101, Inc., La Jolla, CA). The
fragment
was then digested with the endonuclease BamHl and XbaI and then purified again
on
a 1 % agarose gel. This fragment was ligated to pA2 GP baculovirus transfer
vector
(Supplier) at the BamH l and XbaI sites. Through this ligation, VEGF-2 cDNA
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representing the N-terminal and C-terminal deleted VEGF-2 protein (amino acids
103-
227 in Figure 1 or SEQ >D N0:18) was cloned in frame with the signal sequence
of
baculovirus GP gene and was located at the 3' end of the signal sequence in
the
vector. This construct is designated pA2GPVEGF-2.T 103-8227.
5. Construction of VEGF-2 in pCl
The expression vectors pC 1 and pC4 contain the strong promoter (LTR) of the
Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447
(March,
1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530
I O ( 1985)). Multiple cloning sites, e.g., with the restriction enzyme
cleavage sites
BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The
vectors
contain in addition the 3N intron, the polyadenylation and termination signal
of the rat
preproinsulin gene.
The vector pCl is used for the expression of VEGF-2 protein. Plasmid pCl is
t5 a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both
plasmids
contain the mouse DHFR gene under control of the S V40 early promoter. Chinese
hamster ovary- or other cells lacking dihydrofolate activity that are
transfected with
these plasmids can be selected by growing the cells in a selective medium
(alpha
minus MEM, Life Technologies) supplemented with the chemotherapeutic agent
2o methotrexate. The amplification of the DHFR genes in cells resistant to
methotrexate
(MTX) has been well documented (see, e.g., Alt, F.W., Kellems, R.M., Bertino,
J.R., and Schimke, R.T., 1978, J. Biol. Chem. 253:1357-1370, Hamlin, J.L. and
Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143, Page, M.J. and Sydenham,
M.A. 1991, Biotechnology 9:64-68). Cells grown in increasing concentrations of
25 MTX develop resistance to the drug by overproducing the target enzyme,
DHFR, as a
result of amplification of the DHFR gene. If a second gene is linked to the
DHFR
gene it is usually co-amplified and over-expressed. It is state of the art to
develop cell
lines carrying more than 1,000 copies of the genes. Subsequently, when the
methotrexate is withdrawn, cell lines contain the amplified gene integrated
into the
3o chromosome(s).
Plasmid pC 1 contains for the expression of the gene of interest a strong
promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen,
et
al., Molecular and Cellular Biology, March 1985:438-4470) plus a fragment
isolated
from the enhancer of the immediate early gene of human cytomegalovirus (CMV)
35 (Boshart et al., Cell 41:521-530, 1985). Downstream of the promoter are the
following single restriction enzyme cleavage sites that allow the integration
of the
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genes: BamHI, Pvull, and Nrul. Behind these cloning sites the plasmid contains
translational stop codons in all three reading frames followed by the 3N
intron and the
polyadenylation site of the rat preproinsulin gene. Other high efficient
promoters can
also be used for the expression, e.g., the human b-actin promoter, the SV40
early or
late promoters or the long terminal repeats from other retroviruses, e.g., HIV
and
HTLVI. For the polyadenylation of the mRNA other signals, e.g., from the human
growth hormone or globin genes can be used as well.
Stable cell lines carrying a gene of interest integrated into the chromosomes
can also be selected upon co-transfection with a selectable marker such as
gpt, 6418
l0 or hygromycin. It is advantageous to use more than one selectable marker in
the
beginning, e.g., G418 plus methotrexate.
The plasmid pCl is digested with the restriction enzyme BamHI and then
dephosphorylated using calf intestinal phosphates by procedures known in the
art.
The vector is then isolated from a 1 % agarose gel.
~ s The DNA sequence encoding VEGF-2, ATCC Accession No. 97149, was
constructed by PCR using two primers corresponding to the S' and 3'ends of the
VEGF-2 gene: the S' Primer (5'-GAT CGA TCC ATC ATG CAC TCG CTG GGC
TTC TTC TCT GTG GCG TGT TCT CTG CTC G-3' (SEQ ID N0:26)) contains a
Klenow-filled BamHI site and 40 nt of VEGF-2 coding sequence starting from the
2o initiation codon; the 3' primer (5'-GCA GGG TAC GGA TCC TAG ATT AGC TCA
TTT GTG GTC TTT-3' (SEQ ID N0:27)) contains a BamHI site and 16 nt of VEGF-
2 coding sequence not including the stop codon.
The PCR amplified DNA fragment is isolated from a 1 % agarose gel as
described above and then digested with the endonuclease BamHI and then
purified
25 again on a 1 % agarose gel. The isolated fragment and the dephosphorylated
vector are
- -- then ligated with T4 DNA ligase. E. coli HB 101 cells are then
transformed and
bacteria identified that contained the plasmid pCl. The sequence and
orientation of the
inserted gene is confirmed by DNA sequencing. This construct is designated
pC 1 VEGF-2.
6. Construction of pC4SigVEGF-2 T103-L215
Plasmid pC4Sig is plasmid pC4 (Accession No. 209646)containing a human
IgG Fc portion as well as a protein signal sequence.
To permit Polymerase Chain Reaction directed amplification and sub-cloning
of VEGF-2 T103-L215 (amino acids 103 to 215 in Figure 1 or SEQ ID N0:18) into
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pC4Sig, two oligonucleotide primers complementary to the desired region of
VEGF-
2 were synthesized with the following base sequence:
5' Primer (Bam HI and 26 nt of coding sequence):
s 5'-GCA GCA GGA TCC ACA GAA GAG ACT ATA AAA TTT GCT GC-3'
(SEQ ID N0:34)
3' Primer (Xba I, STOP, and 15 nt of coding sequence):
5'-CGT CGT TCT AGA TCA CAG TTT AGA CAT GCA TCG GCA G-3'
(SEQ ID N0:35)
The Polymerase Chain Reaction was performed using standard conditions well
known to those skilled in the art and the nucleotide sequence for the mature
VEGF-2
(aa 24-419) as, for example, constructed in Example 3, as template. The
resulting
amplicon was restriction digested with BamHI and XbaI and subcloned into BamHI
/XbaI digested pC4Sig vector.
7. Construction of pC4SigVEGF-2 T103-8227
To permit Polymerase Chain Reaction directed amplification and sub-cloning
of VEGF-2 T103-L215 (amino acids 103 to 227 in Figure 1 or SEQ >D N0:18) into
pC4Sig, two oligonucleotide primers complementary to the desired region of
VEGF-
2 were synthesized with the following base sequence:
5' Primer (Bam HI and 26 nt of coding sequence):
5'-GCA GCA GGA TCC ACA GAA GAG ACT ATA AAA TTT GCT GC-3'
(SEQ ID NO: 34)
3' Primer (Xba I, STOP, and 21 nt of coding sequence):
5'-GCA GCA TCT AGA TCA ACG TCT AAT AAT GGA ATG AAC-3'
(SEQ ID N0:25)
The Polymerase Chain Reaction was performed using standard conditions well
known to those skilled in the art and the nucleotide sequence for the mature
VEGF-2
(aa 24-419) as, for example, constructed in Example 3, as template. The
resulting
amplicon was restriction digested with BamHI and XbaI and subcloned into BamHI
/XbaI digested pC4Sig vector.
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8. Construction of pC4VEGF-2 M1-M2b3
The expression vector pC4 contains the strong promoter (LTR) of the Rous
Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March,
1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530
( 1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage
sites
BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The
vector
contains in addition the 3N intron, the polyadenylation and termination signal
of the
rat preproinsulin gene.
In this illustrative example, the cloned DNA encoding the C-terminal deleted
~o VEGF-2 M1-M263 protein (amino acids 1-263 in Figure 1 or SEQ )D N0:18) is
inserted into the plasmid vector pC4 to express the C-terminal deleted VEGF-2
protein.
To permit Polymerise Chain Reaction directed amplification and sub-cloning
of VEGF-2 M1-M263 into the expression vector, pC4, two oligonucleotide primers
~ 5 complementary to the desired region of VEGF-2 were synthesized with the
following
base sequence:
5' Primer 5'-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT
CTT CTC-3' (SEQ ID N0:28)
3' Primer 5'-GAC TGG TAC CTT ATC ACA TAA AAT CTT CCT GAG
CC-3' (SEQ ID N0:29)
In the case of the above described S' primer, an BamHlrestriction site was
incorporated, while in the case of the 3' primer, an Asp718 restriction site
was
incorporated. The 5' primer also contains 6 nt, 20 nt of VEGF-2 coding
sequence,
and an ATG sequence adjacent and in frame with the VEGF-2 coding region to
allow
translation of the cloned fragment in E. coli, while the 3' primer contains 2
nt, 20 nt
of VEGF-2 coding sequence, and one stop codon (preferentially utilized in E.
coli)
3o adjacent and in frame with the VEGF-2 coding region which ensures correct
translational termination in E. coli.
The Polymerise Chain Reaction was performed using standard conditions well
known to those skilled in the art and the nucleotide sequence for the mature
VEGF-2
(aa 24-419) as constructed, for example, in Example 3 as template. The
resulting
amplicon was restriction digested with BamHl and Asp718 and subcloned into
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BamHl/Asp718 digested pC4 protein expression vector. This construct is
designated
pC4VEGF-2 Ml-M263.
9. Construction of pC4VEGF-2 M1-D311
In this illustrative example, the cloned DNA encoding the C-terminal deleted
VEGF-2 Ml-D311 protein (amino acids 1-311 in Figure 1 or SEQ ID N0:18) is
inserted into the plasmid vector pC4 to express the C-terminal deleted VEGF-2
protein.
To permit Polymerase Chain Reaction directed amplification and sub-cloning
of VEGF-2 Ml-D311 into the expression vector, pC4, two oligonucleotide primers
complementary to the desired region of VEGF-2 were synthesized with the
following
base sequence:
5' Primer 5'-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT
~ 5 CTT CTC-3' (SEQ ID N0:30)
3' Primer 5'-GAC TGG TAC CTT ATC AGT CTA GTT CTT TGT GGG G-
3' (SEQ ID N0:31 )
In the case of the above described 5' primer, an BamHlrestriction site was
incorporated, while in the case of the 3' primer, an Asp718 restriction site
was
incorporated. The 5' primer also contains 6 nt, 20 nt of VEGF-2 coding
sequence,
and an ATG sequence adjacent and in frame with the VEGF-2 coding region to
allow
translation of the cloned fragment in E. coli, while the 3' primer contains 2
nt, 20 nt
of VEGF-2 coding sequence, and one stop codon (preferentially utilized in E.
coli)
adjacent and in frame with the VEGF-2 coding region which ensures correct
translational termination in E. coli.
The Polymerase Chain Reaction was performed using standard conditions well
known to those skilled in the art and the nucleotide sequence for the mature
VEGF-2
(aa 24-419) as constructed, for example, in Example 3 as template. The
resulting
amplicon was restriction digested with BamH 1 and Asp718 and subcloned into
BamHl/Asp718 digested pC4 protein expression vector.
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10. Construction of pC4VEGF-2 M1-Q367
In this illustrative example, the cloned DNA encoding the C-terminal deleted
VEGF-2 M1-D311 protein (amino acids 1-311 in SEQ ID N0:18) is inserted into
the
plasmid vector pC4 to express the C-terminal deleted VEGF-2 protein.
To permit Polymerise Chain Reaction directed amplification and sub-cloning
of VEGF-2 Ml-D311 into the expression vector, pC4, two oligonucleotide primers
complementary to the desired region of VEGF-2 were synthesized with the
following
base sequence:
5' Primer 5'-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT
CTT CTC-3' (SEQ ID N0:32)
3' Primer 5'-GAC TGG TAC CTC ATT ACT GTG GAC TTT CTG TAC
ATT C-3' (SEQ ID N0:33)
In the case of the above described 5' primer, an BamHlrestriction site was
incorporated, while in the case of the 3' primer, an Asp718 restriction site
was
incorporated. The 5' primer also contains 6 nt, 20 nt of VEGF-2 coding
sequence,
and an ATG sequence adjacent and in frame with the VEGF-2 coding region to
allow
translation of the cloned fragment in E. coli, while the 3' primer contains 2
nt, 20 nt
of VEGF-2 coding sequence, and one stop codon (preferentially utilized in E.
coli)
adjacent and in frame with the VEGF-2 coding region which ensures correct
translational termination in E. coli.
The Polymerise Chain Reaction was performed using standard conditions well
known to those skilled in the art and the nucleotide sequence for the mature
VEGF-2
(aa 24-419) as constructed, for example, in Example 3 as template. The
resulting
amplicon was restriction digested with BamHl and Asp718 and subcloned into
BamHl/Asp718 digested pC4 protein expression vector. This construct is
designated
pC4VEGF-2 Ml-Q367.
Example 10
Transient Expression of VEGF-2 Protein in COS-7 Cells
Experimental Design
Expression of the VEGF-2-HA fusion protein from the construct made in
Example 4, for example, was detected by radiolabeling and immunoprecipitation,
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using methods described in, for example Harlow and colleagues (Antibodies: A
Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York ( 1988)). To this end, two days after transfection, the cells
were
labeled by incubation in media containing 35S-cysteine for 8 hours. The cells
and the
media were collected, and the cells were washed and then lysed with detergent
containing RIPA buffer: 150 mM NaCI, 1 % NP-40, 0.1 % SDS, 1 % NP-40, 0.5 %
DOC, 50 mM TRIS, pH 7.5, as described by Wilson and colleagues (supra).
Proteins were precipitated from the cell lysate and from the culture media
using an
HA-specific monoclonal antibody. The precipitated proteins then were analyzed
by
1o SDS-PAGE and autoradiography.
Results
As shown in Figure 16, cells transfected with pcDNA 1 VEGF-2HA secreted a
56 kd and a 30 kd protein. The 56 kd protein, but not the 30 kd protein, could
also be
detected in the cell lysate but is note detected in controls. This suggests
the 30 kd
protein is likely to result from cleavage of the 56 kd protein. Since the HA-
tag is vn
the C-terminus of VEGF-2, the 30 kd protein must represent the C-terminal
portion of
the cleaved protein, whereas the N-terminal portion of the cleaved protein
would not
be detected by immunoprecipitation. These data indicate that VEGF-2 protein
expressed in mammalian cells is secreted and processed.
Example 11
Stimulatory effect of VEGF-2 on proliferation of
vascular endothelial cells
Experimental Design
Expression of VEGF-2 is abundant in highly vascularized tissues. Therefore
the role of VEGF-2 in regulating proliferation of several types of endothelial
cells was
examined.
Endothelial cell proliferation assay
For evaluation of mitogenic activity of growth factors, the colorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-
tetrazolium) assay with the electron coupling reagent PMS (phenazine
methosulfate)
was performed (CellTiter 96 AQ, Promega). Cells were seeded in a 96-well plate
(5,000 cells/well) in 0.1 mL serum-supplemented medium and allowed to attach
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overnight. After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF,
VEGF,65 or VEGF-2 in 0.5% FBS) with or without Heparin (8 U/ml) were added to
wells for 48 hours. 20 mg of MTS/PMS mixture ( 1:0.05) were added per well and
allowed to incubate for 1 hour at 37°C before measuring the absorbance
at 490 nm in
an ELISA plate reader. Background absorbance from control wells (some media,
no
cells) was subtracted, and seven wells were performed in parallel for each
condition.
See, Leak et al. In Vitro Cell. Dev. Biol. 30A: 512-518 ( 1994)
Results
VEGF-2 stimulated proliferation of human umbilical vein endothelial cells
(HUVEC) and dermal microvascular endothelial cells slightly (Figures 17 and
18).
The stimulatory effect of VEGF-2 is more pronounced on proliferation of
endometrial
and microvascular endothelial cells (Figure 19). Endometrial endothelial cells
(HEEC)
demonstrated the greatest response to VEGF-2 (96% of the effect of VEGF on
microvascular endothelial cells). The response of microvascular endothelial
cells
(HMEC) to VEGF-2 was 73% compared to VEGF. The response of HUVEC and
BAEC (bovine aortic endothelial cells) to VEGF-2 was substantially lower at
10% and
7%, respectively. The activity of VEGF-2 protein has varied between different
purification runs with the stimulatory effect of certain batches on HUVEC
2o proliferation being significantly higher than that of other batches.
Example 12
Inhibition of PDGF-induced vascular smooth muscle cell proliferation
VEGF-2 expression is high in vascular smooth muscle cells. Smooth muscle
is an important therapeutic target for vascular diseases, such as restenosis.
To
evaluate the potential effects of VEGF-2 on smooth muscle cells, the effect of
VEGF-
2 on human aortic smooth muscle cell (HAoSMC) proliferation was examined.
3o Experimental Design
HAoSMC proliferation can be measured, for example, by BrdUrd
incorporation. Briefly, subconfluent, quiescent cells grown on the 4-chamber
slides
are transfected with CRP or FITC-labeled AT2-3LP. Then, the cells are pulsed
with
10% calf serum and 6 mg/ml BrdUrd. After 24 h, immunocytochemistry is
performed
by using BrdUrd Staining Kit (Zymed Laboratories). In brief, the cells are
incubated
with the biotinylated mouse anti-BrdUrd antibody at 4 °C for 2 h after
exposing to
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denaturing solution and then with the streptavidin-peroxidase and
diaminobenzidine.
After counterstaining with hematoxylin, the cells are mounted for microscopic
examination, and the BrdUrd-positive cells are counted. The BrdUrd index is
calculated as a percent of the BrdUrd-positive cells to the total cell number.
In
addition, the simultaneous detection of the BrdUrd staining (nucleus) and the
FITC
uptake (cytoplasm) is performed for individual cells by the concomitant use of
bright
field illumination and dark field-UV fluorescent illumination. See, Hayashida
et al.,
J. Biol. Chem. 6; 271 (36):21985-21992 ( 1996).
Results
VEGF-2 has an inhibitory effect on proliferation of vascular smooth muscle
cells induced by PDGF, but not by Fetal Bovine Serum (FBS) (Figure 20).
Example 13
Stimulation of endothelial cell migration
Endothelial cell migration is an important step involved in angiogenesis.
Experimental Design
This example will be used to explore the possibility that VEGF-2 may
stimulate lymphatic endothelial cell migration. Currently, there are no
published
reports of such a model. However, we will be adapting a model of vascular
endothelial cell migration for use with lymphatic endothelial cells
essentially as
follows:
Endothelial cell migration assays are performed using a 48 well
microchemotaxis chamber (Neuroprobe Inc., Cabin John, MD; Falk, W., Goodwin,
R. H. J., and Leonard, E. J. "A 48 well micro chemotaxis assembly for rapid
and
accurate measurement of leukocyte migration." J. Immunological Methods
1980;33:239-247). Polyvinylpyrrolidone-free polycarbonate filters with a pore
size of
8 um (Nucleopore Corp. Cambridge, MA) are coated with 0.1 % gelatin for at
least 6
3o hours at room temperature and dried under sterile air. Test substances are
diluted to
appropriate concentrations in M 199 supplemented with 0.25 % bovine serum
albumin
(BSA), and 25 ul of the final dilution is placed in the lower chamber of the
modified
Boyden apparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are
washed and trypsinized for the minimum time required to achieve cell
detachment.
After placing the filter between lower and upper chamber, 2.5 x 105 cells
suspended in
50 ul M 199 containing 1 % FBS are seeded in the upper compartment. The
apparatus
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is then incubated for 5 hours at 37°C in a humidified chamber with 5%
C02 to allow
cell migration. After the incubation period, the filter is removed and the
upper side of
the filter with the non-migrated cells is scraped with a rubber policeman. The
filters
are fixed with methanol and stained with a Giemsa solution (Diff Quick,
Baxter,
McGraw Park, IL). Migration is quantified by counting cells of three random
high-
power fields (40x) in each well, and all groups are performed in
quadruplicate.
Results
In an assay examining HUVEC migration using a 43-well microchemotaxis
chamber, VEGF-2 was able to stimulate migration of HUVEC (Figure 21).
Example 14
Stimulation of nitric oxide production by endothelial cells
Nitric oxide released by the vascular endothelium is believed to be a mediator
of vascular endothelium relaxation. VEGF-1 has been demonstrated to induce
nitric
oxide production by endothelial cells in response to VEGF-1. As a result, VEGF-
2
activity can be assayed by determining nitric oxide production by endothelial
cells in
response to VEGF-2.
Experimental Design
Nitric oxide is measured in 96-well plates of confluent microvascular
endothelial cells after 24 hours starvation and a subsequent 4 hr exposure to
various
levels of VEGF-1 and VEGF-2. Nitric oxide in the medium is determined by use
of
the Griess reagent to measure total nitrite after reduction of nitric oxide-
derived nitrate
by nitrate reductase. The effect of VEGF-2 on nitric oxide release was
examined on
HUVEC.
Briefly, NO release from cultured HUVEC monolayer was measured with a
NO-specific polarographic electrode connected to a NO meter (Iso-NO, World
Precision Instruments Inc.) ( 1049). Calibration of the NO elements was
performed
according to the following equation:
2KN0~+2KI+2H,S0~62N0+I,+2H~0+2K,S0,~
The standard calibration curve was obtained by adding graded concentrations of
KNO, (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) into the calibration
solution
containing KI and H~SO~. The specificity of the Iso-NO electrode to NO was
previously determined by measurement of NO from authentic NO gas ( 1050). The
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culture medium was removed and HUVECs were washed twice with Dulbecco's
phosphate buffered saline. The cells were then bathed in 5 ml of filtered
Krebs-
Henseleit solution in 6-well plates, and the cell plates were kept on a slide
warmer
(Lab Line Instruments Inc.) To maintain the temperature at 37°C. The NO
sensor
probe was inserted vertically into the wells, keeping the tip of the electrode
2 mm
under the surface of the solution, before addition of the different
conditions.
S-nitroso acetyl penicillamin (SNAP) was used as a positive control. The
amount of
released NO was expressed as picomoles per 1 x 106 endothelial cells. All
values
reported were means of four to six measurements in each group (number of cell
culture wells). See, Leak et al. Biochem. and Biophys. Res. Comm. 217:96-105
( 1995).
Results
VEGF-2 was capable of stimulating nitric oxide release on HUVEC (Figure
22) to a higher level than VEGF. This suggested that VEGF-2 may modify
vascular
permeability and vessel dilation.
Example I S
Effect of VEGF-2 on cord formation in angiogenesis
Another step in angiogenesis is cord formation, marked by differentiation of
endothelial cells. This bioassay measures the ability of microvascular
endothelial cells
to form capillary-like structures (hollow structures) when cultured in vitro.
Experimental Design
CADMEC (microvascular endothelial cells) are purchased from Cell
Applications, Inc. as proliferating (passage 2) cells and are cultured in Cell
Applications' CADMEC Growth Medium and used at passage 5. For the in vitro
angiogenesis assay, the wells of a 48-well cell culture plate are coated with
Cell
Applications' Attachment Factor Medium (200 ml/well) for 30 min. at
37°C.
3o CADMEC are seeded onto the coated wells at 7,500 cells/well and cultured
overnight
in Growth Medium. The Growth Medium is then replaced with 300 mg Cell
Applications' Chord Formation Medium containing control buffer or HGS protein
(0.1 to 100 ng/ml) and the cells are cultured for an additional 48 hr. The
numbers and
lengths of the capillary-like chords are quantitated through use of the
Boeckeler
VIA-170 video image analyzer. All assays are done in triplicate.
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Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.
b-esteradiol ( 1 ng/ml) is used as a negative control. The appropriate buffer
(without
protein} is also utilized as a control.
Results
It has been observed that VEGF-2 inhibits cord formation similar to IFNa
which also stimulates endothelial cell proliferation (Figure 23). This
inhibitory effect
may be a secondary effect of endothelial proliferation which is mutually
exclusive with
the cord formation process.
Example 16
A~:giogenic effect on chick chorioallantoic membrane
Chick chorioallantoic membrane (CAM) is a well-established system to
~ 5 examine angiogenesis. Blood vessel formation on CAM is easily visible and
quantifiable. The ability of VEGF-2 to stimulate angiogenesis in CAM was
examined.
Experimental Design
Embryos
Fertilized eggs of the White Leghorn chick (callus gallus) and the Japanese
qual (Coturnix coturnix} were incubated at 37.8°C and 80% humidity.
Differentiated
CAM of 16-day-old chick and 13-day-old qual embryos was studied with the
following methods.
CAM Assay
On Day 4 of development, a window was made into the egg shell of chick
eggs. The embryos were checked for normal development and the eggs sealed with
cellotape. They were further incubated until Day 13. Thermanox coverslips
(Nunc,
Naperville, IL) were cut into disks of about 5 mm in diameter. Sterile and
salt-free
3o growth factors were dissolved in distilled water and about 3.3 mg/ 5 ml was
pipetted
on the disks. After air-drying, the inverted disks were applied on CAM. After
3
days, the specimens were fixed in 3% glutaraldehyde and 2% formaldehyde and
rinsed in 0.12 M sodium cacodylate buffer. They were photographed with a
stereo
microscope (Wild M8] and embedded for semi- and ultrathin sectioning as
described
above. Controls were performed with carrier disks alone.
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Results
This data demonstrates that VEGF-2 can stimulate angiogenesis in the CAM
assay nine-fold compared to the untreated control. However, this stimulation
is only
45% of the level of VEGF stimulation (Figure 24).
Example 17
Angiogenesis assay using a Matrigel implant in mouse
Experimental Design
In order to establish an in vivo model for angiogenesis to test protein
activities, mice and rats have been implanted subcutaneously with
methylcellulose
disks containing either 20 mg of BSA (negative control) and 1 mg of bFGF and
0.5
mg of VEGF-1 (positive control).
It appeared as though the BSA disks contained little vascularization, while
the
positive control disks showed signs of vessel formation. At day 9, one mouse
showed a clear response to the bFGF.
Results
Both VEGF proteins appeared to enhance Matrigel cellularity by a factor of
2o approximately 2 by visual estimation.
An additional 30 mice were implanted with disks containing BSA, bFGF, and
varying amounts of VEGF-1, VEGF-2-B8, and VEGF-2-C4. Each mouse received
two identical disks, rather than one control and one experimental disk.
Samples of all the disks recovered were immunostamed wnn von
Willebrand's factor to detect for the presence of endothelial cells in the
disks, and
flk-l and flt-4 to distinguish between vascular and lymphatic endothelial
cells.
However, definitive histochemical analysis of neovascularization and
lymphangiogenesis could not be determined.
3o Example 18
Rescue of Ischemia in Rabbit Lower Limb Model
Experimental Design
To study the in vivo effects of VEGF-2 on ischemia, a rabbit hindlimb
ischemia model was created by surgical removal of one femoral arteries as
described
previously (Takeshita, S. et al., Am J. Pathol 147:1649-1660 (1995)). The
excision
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of the femoral artery results in retrograde propagation of thrombus and
occlusion of
the external iliac artery. Consequently, blood flow to the ischemic limb is
dependent
upon collateral vessels originating from the internal iliac artery (Takeshita,
S. et al.
Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days was allowed for
post-
s operative recovery of rabbits and development of endogenous collateral
vessels. At
day post-operatively (day 0), after performing a baseline angiogram, the
internal
iliac artery of the ischemic limb was transfected with 500 mg naked VEGF-2
expression plasmid by arterial gene transfer technology using a hydrogel-
coated
balloon catheter as described (Riessen, R. et al. Hum Gene Ther. 4:749-758
(1993);
to Leclerc, G. et al. J. Clin. Invest. 90: 936-944 ( 1992)). When VEGF-2 was
used in
the treatment, a single bolus of 500 mg VEGF-2 protein or control was
delivered into
the internal iliac artery of the ischemic limb over a period of I min. through
an
infusion catheter. On day 30, various parameters were measured in these
rabbits.
Results
Both VEGF-2 protein (Figure 25, top panels) and naked expression plasmid
(Figure 25, middle panels) were able to restore the following parameters in
the
ischemic limb. Restoration of blood flow, angiographic score seem to be
slightly
more by administration of 500 mg plasmid compared with by 500 mg protein
(Figure
25, bottom panels) The extent of the restoration is comparable with that by
VEGF in
separate experiments (data not shown). A vessel dilator was not able to
achieve the
same effect, suggesting that the blood flow restoration is not simply due to a
vascular
dilation effect.
a. BP ratio (Figure 25a)
The blood pressure ratio of systolic pressure of the ischemic limb to that of
normal limb.
2. Blood Flow and Flow Reserve (Figure 25b)
3o Resting FL: the blood flow during un-dilated condition
Max FL: the blood flow during fully dilated condition (also an indirect
measure of the blood vessel amount)
Flow Reserve is reflected by the ratio of max FL: resting FL.
3. Angiographic Score (Figure 25c)
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This is measured by the angiogram of collateral vessels. A score was
determined by the percentage of circles in an overlaying grid that with
crossing
opacified arteries divided by the total number m the rabbit thigh.
4. Capillary density (Figure 25d)
The number of collateral capillaries determined in light microscopic sections
taken from hindlimbs.
As discussed. VEGF-2 is processed to an N-terminal and a C-terminal
fragment which are co-purified. The N-terminal fragment contains the intact
putative
functional domain and may be responsible for the biologic activity.
Example 19
Effect of VEGF-2 on Vasodilation
As described above, VEGF-2 can stimulate NO release, a mediator of vascular
endothelium dilation. Since dilation of vascular endothelium is important in
reducing
blood pressure, the ability of VEGF-2 to affect the blood pressure in
spontaneously
hypertensive rats (SHR) was examined. VEGF-2 caused a dose-dependent decrease
2o in diastolic blood pressure (Figures 26a and b). There was a steady decline
in
diastolic blood pressure with increasing doses of VEGF-2 which attained
statistical
significance when a dose of 300mg/kg was administered. The changes observed at
this dose were not different than those seen with acetylcholine (O.Smg/kg).
Decreased
mean arterial pressure (MAP) was observed as well (Figure 26c and d). VEGF-2
(300 mg/kg) and acetylcholine reduced the MAP of these SHR animals to normal
levels.
Additionally, increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of the
B8, C5, and C4 preps of VEGF-2 were administered to 13-14 week old
spontaneously hypertensive rats (SHR). Data are expressed as the mean +/- SEM.
Statistical analysis was performed with a paired t-test and statistical
significance was
defined as p<0.05 vs. the response to buffer alone.
Studies with VEGF-2 (CS prep) revealed that although it significantly
decreased the blood pressure, the magnitude of the response was not as great
as that
seen with VEGF-2 (B8 prep) even when used at a dose of 900 mg/kg.
Studies with VEGF-2 (C4 preparation) revealed that this CHO expressed
protein preparation yielded similar results to that seen with CS (i.e.
statistically
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significant but of far less magnitude than seen with the B8 preparation) (see
Figures
26A-D).
As a control and since the C4 and CS batches of VEGF-2 yielded minor, but
statistically significant, changes in blood pressure, experiments were
performed
experiments with another CHO-expressed protein, M-CIF. Administration of M-CIF
at doses ranging from 10-900 mg/kg produced no significant changes in
diastolic
blood pressure. A minor statistically significant reduction in mean arterial
blood
pressure was observed at doses of 100 and 900 mg/kg but no dose response was
noted. These results suggest that the reductions in blood pressure observed
with the
l0 C4 and CS batches of VEGF-2 were specific, i.e. VEGF-2 related.
Example 20
Rat Ischemic Skin Flap Model
t 5 Experimental Design
The evaluation parameters include skin blood flow, skin temperature, and
factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction.
VEGF-2 expression, during the skin ischemia, is studied using in situ
hybridization.
The study in this model is divided into three parts as follows:
2o a) Ischemic skin
b) Ischemic skin wounds
c) Normal wounds
The experimental protocol includes:
a) Raising a 3x4 cm, single pedicle full-thickness random skin flap
25 (myocutaneous flap over the lower back of the animal).
b) An excisional wounding (4-6 mm in diameter) in the ischemic skin
(skin-flap).
c) Topical treatment with VEGF-2 of the excisional wounds {day 0, 1, 2, 3, 4
post-wounding) at the following various dosage ranges: 1 mg to 100 mg.
30 d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21 post-wounding
for histological, immunohistochemical, and in situ studies.
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Example 21
Peripheral Arterial Disease Model
Angiogenic therapy using VEGF-2 has been developed as a novel therapeutic
strategy to obtain restoration of blood flow around the ischemia in case of
peripheral
arterial diseases.
Experimental Design
The experimental protocol includes:
a) One side of the femoral artery is ligated to create ischemic muscle of
the hindlimb, the other side of hindlimb serves as a control.
b) VEGF-2 protein, in a dosage range of 20 mg - 500 mg, is delivered
intravenously
and/or intramuscularly 3 times (perhaps more) per week for 2-3 weeks.
c) The ischemic muscle tissue is collected after ligation of the femoral
artery at 1, 2, and 3 weeks for the analysis of VEGF-2 expression and
histology.
Biopsy is also performed on the other side of normal muscle of the
contralateral hindlimb.
Example 22
2o Isehemic Myocardial Disease Model
VEGF-2 is evaluated as a potent mitogen capable of stimulating the
development of collateral vessels, and restructuring new vessels after
coronary artery
occlusion. Alteration of VEGF-2 expression is investigated in situ.
Experimental Design
The experimental protocol includes:
a) The heart is exposed through a left-side thoracotomy in the rat.
Immediately, the
left coronary artery is occluded with a thin suture (6-0) and the thorax is
closed.
3o b) VEGF-2 protein, in a dosage range of 20 mg - 500 mg, is delivered
intravenously
and/or intramuscularly 3 times (perhaps more) per week for 2-4 week.
c) Thirty days after the surgery, the heart is removed and cross-sectioned
for morphometric and in situ analyzes.
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Example 23
Rat Corneal Wound Healing Model
This animal model shows the effect of VEGF-2 on neovascularization.
Experimental Design
The experimental protocol includes:
a) Making a 1-l.S mm long incision from the center of cornea into the stromal
layer.
b) Inserting a spatula below the lip of the incision facing the outer corner
of the eye.
io c) Making a pocket (its base is I-1.5 mm form the edge of the eye).
d) Positioning a pellet, containing SOmg - SOOmg VEGF-2, within the pocket.
e) VEGF-2 treatment can also be applied topically to the corneal wounds in a
dosage
range of 20mg - SOOmg (daily treatment for five days).
Example 24
Diabetic Mouse and Glucocorticoid-Impaired
Wound Healing Models
Experimental Design
2o The experimental protocol includes:
1. Diabetic db+1db+ mouse model.
To demonstrate that VEGF-2 accelerates the healing process, the genetically
diabetic mouse model of wound healing iss used. The full thickness wound
healing
model in the db+/db+ mouse is a well characterized, clinically relevant and
reproducible model of impaired wound healing. Healing of the diabetic wound is
dependent on formation of granulation tissue and re-epithelialization rather
than
contraction (Gartner, M.H. et al., J. Surg. Res. 52:389 ( 1992); Greenhalgh,
D.G. et
al., Am. J. Pathol. 136:1235 ( 1990)).
The diabetic animals have many of the characteristic features observed in Type
3o II diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to
their
normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have
a
single autosomal recessive mutation on chromusome 4 (db+) (Coleman et al.
Proc.
Natl. Acad. Sci. USA 77:283-293 ( 1982)). Animals show polyphagia, polydipsia
and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose,
increased
or normal insulin levels, and suppressed cell-mediated immunity (Mandel et
al., J.
Immunol. 120:1375 ( 1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51 (1
):1-7
( 1983); Leiter et al., Am. J. of Pathol. 114:46-55 ( 1985)). Peripheral
neuropathy,
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myocardial complications, and microvascular lesions, basement membrane
thickening
and glomerular filtration abnormalities have been described in these animals
(Norido,
F. et al., Exp. Neurol. 83(2):221-232 ( 1984); Robertson et al., Diabetes 29(1
):60-67
( 1980); Giacomelli et al., Lab Invest. 40(4):460-473 ( 1979); Coleman, D.L.,
Diabetes
31 (Suppl):1-6 (1982)). These homozygous diabetic mice develop hyperglycemia
that
is resistant to insulin analogous to human type II diabetes (Mandel et al., J.
Immunol.
120:1375-1377 (1978)).
The characteristics observed in these animals suggests that healing in this
model may be similar to the healing observed in human diabetes (Greenhalgh, et
al.,
t o Am. J. of Pathol. 136:1235-1246 ( 1990)).
Animals
Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic
(db+/+m) heterozygous littermates were used in this study (Jackson
Laboratories).
~ 5 The animals were purchased at 6 weeks of age and were 8 weeks old at the
beginning
of the study. Animals were individually housed and received food and water ad
libitum. All manipulations were performed using aseptic techniques. The
experiments were conducted according to the rules and guidelines of Human
Genome
Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines
for
20 the Care and Use of Laboratory Animals.
Surgical Wounding
Wounding protocol is performed according to previously reported methods
(Tsuboi, R. and Rifkin, D.B., J. Exp. Med. 172:245-251 (1990)). Briefly, on
the
25 day of wounding, animals are anesthetized with an intraperitoneal injection
of Avertin
(0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in
deionized
water. The dorsal region of the animal is shaved and the skin washed with 70%
ethanol solution and iodine. The surgical area is dried with sterile gauze
prior to
wounding. An 8 mm full-thickness wound is then created using a Keyes tissue
punch. Immediately following wounding, the surrounding skin is gently
stretched to
eliminate wound expansion. The wounds are left open for the duration of the
experiment. Application of the treatment is given topically for 5 consecutive
days
commencing on the day of wounding. Prior to treatment, wounds are gently
cleansed
with sterile saline and gauze sponges.
35 Wounds are visually examined and photographed at a fixed distance at the
day
of surgery and at two day intervals thereafter. Wound closure is determined by
daily
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measurement on days 1-5 and on day 8. Wounds are measured horizontally and
vertically using a calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by a
continuous
epithelium.
VEGF-2 is administered using at a range different doses of VEGF-2, from
4mg to 500mg per wound per day for 8 days in vehicle. Vehicle control groups
received 50mL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium
pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested
for
l0 histology and immunohistochemistry. Tissue specimens are placed in 10%
neutral
buffered formalin in tissue cassettes between biopsy sponges for further
processing.
Experimental Design
Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) were
~ 5 evaluated: 1 ) Vehicle placebo control, 2) VEGF-2 .
Measurement of Wound Area and Closure
Wound closure is analyzed by measuring the area in the vertical and horizontal
axis and obtaining the total square area of the wound. Contraction is then
estimated
20 by establishing the differences between the initial wound area (day 0) and
that of post
treatment (day 8). The wound area on day 1 was 64mm2, the corresponding size
of
the dermal punch. Calculations were made using the following formula:
[Open area on day 8] - [Open area on day 1 ] / [Open area on day 1 ]
Histvlvgy
Specimens are fixed in 10% buffered formalin and paraffin embedded blocks
are sectioned perpendicular to the wound surface (5mm) and cut using a
Reichert-Jung
microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-
sections of bisected wounds. Histologic examination of the wounds are used to
assess whether the healing process and the morphologic appearance of the
repaired
skin is altered by treatment with KGF-2. This assessment included verification
of the
presence of cell accumulation, inflammatory cells, capillaries, fibroblasts,
re-
epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J.
Pathol.
136:1235 ( 1990)). A calibrated lens micrometer is used by a blinded observer.
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Immunohistochemistry
Re-epithelialization
Tissue sections are stained immunohistochemically with a polyclonal rabbit
anti-human keratin antibody using ABC Elite detection system. Human skin is
used
as a positive tissue control while non-immune IgG is used as a negative
control.
Keratinocyte growth is determined by evaluating the extent of
reepithelialization of the
wound using a calibrated lens micrometer.
Cell Proliferation Marker
1 o Proliferating cell nuclear antigen/cyclin {PCNA) in skin specimens is
demonstrated by using anti-PCNA antibody ( 1:50) with an ABC Elite detection
system. Human colon cancer served as a positive tissue control and human brain
tissue is used as a negative tissue control. Each specimen included a section
with
omission of the primary antibody and substitution with non-immune mouse IgG.
Ranking of these sections is based on the extent of proliferation on a scale
of 0-8, the
lower side of the scale reflecting slight proliferation to the higher side
reflecting
intense proliferation.
Statistical Analysis
2o Experimental data are analyzed using an unpaired t test. A p value of <
0.05 is
considered significant.
B. Steroid Impaired Rat Model
The inhibition of wound healing by steroids has been well documented in
various in vitro and in vivo systems (Wahl, S.M. Glucocorticoids and Wound
healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-
302
( 1989); Wahl, S.M.et al., J. Immunol. I15: 476-481 ( 1975); Werb, Z. et al.,
J.
Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard wound healing by
inhibiting angiogenesis, decreasing vascular permeability ( Ebert, R.H., et
al., An.
3o Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen
synthesis
(Beck, L.S. et al., Growth Factors. 5: 295-304 (1991); Haynrs, B.F. et ul., J.
Clin. Invest. 61: 703-797 ( 1978)) and producing a transient reduction of
circulating
monocytes (Haynes, B.F., et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, S.
M.,
"Glucocorticoids and wound healing", In: Antiinflammatory Steroid Action:
Basic
and Clinical Aspects, Academic Press, New York, pp. 280-302 ( 1989)). The
systenuc administration of steroids to impaired wound healing is a well
establish
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phenomenon in rats (Beck, L.S. et al., Growth Factors. 5: 295-304 (1991);
Haynes, B.F., et al., J. Clin. Invest. 61: 703-797 ( 1978); Wahl, S. M.,
"Glucocorticoids and wound healing", In: Antiinflammatory Steroid Action:
Basic and
Clinical Aspects, Academic Press, New York, pp. 280-302 ( 1989); Pierce, G.F.
et
al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 ( 1989)).
To demonstrate that VEGF-2 can accelerate the healing process, the effects of
multiple topical applications of VEGF-2 on full thickness excisional skin
wounds in
rats in which healing has been impaired by the systemic administration of
methylprednisolone is assessed.
Animals
Young adult male Sprague Dawley rats weighing 250-300 g (Charles River
Laboratories) are used in this example. The animals are purchased at 8 weeks
of age
and were 9 weeks old at the beginning of the study. The healing response of
rats is
impaired by the systemic administration of methylprednisolone ( l7mg/kg/rat
intramuscularly) at the time of wounding. Animals are individually housed and
received food and water ad libitum. All manipulations are performed using
aseptic
techniques. This study is conducted according to the rules and guidelines of
Human
Genome Sciences, Inc. Institutional Animal Care and Use Committee and the
2o Guidelines for the Care and Use of Laboratory Animals.
Surgical Wounding
The wounding protocol is followed according to section A, above. On the
day of wounding, animals are anesthetized with an intramuscular injection of
ketamine
(50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved
and the
skin washed with 70% ethanol and iodine solutions. The surgical area is dried
with
sterile gauze prior to wounding. An 8 mm full-thickness wound is created using
a
Keyes tissue punch. The wounds are left open for the duration of the
experiment.
Applications of the testing materials are given topically once a day for 7
consecutive
3o days commencing on the day of wounding and subsequent to methylprednisolone
administration. Prior to treatment, wounds are gently cleansed with sterile
saline and
gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day
of wounding and at the end of treatment. Wound closure is determined by daily
measurement on days 1-S and on day 8 for Figure. Wounds are measured
horizontally
and vertically using a calibrated Jameson caliper. Wounds are considered
healed if
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granulation tissue was no longer visible and the wound is covered by a
continuous
epithelium.
VEGF-2 is administered using at a range different doses of VEGF-2, from
4mg to SOOmg per wound per day for 8 days in vehicle. Vehicle control groups
received SOmL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium
pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested
for
histology. Tissue specimens are placed in 10% neutral buffered formalin in
tissue
cassettes between biopsy sponges for further processing.
to
Experimental Design
Four groups of 10 animals each (5 with methylprednisolone and 5 without
glucocorticoid) were evaluated: 1) Untreated group 2) Vehicle placebo control
3)
VEGF-2 treated groups.
Measurement of Wound Area and Closure
Wound closure is analyzed by measuring the area in the vertical and horizontal
axis and obtaining the total area of the wound. Closure is then estimated by
establishing the differences between the initial wound area (day 0) and that
of post
2o treatment (day 8). The wound area on day 1 was 64mm', the corresponding
size of
the dermal punch. Calculations were made using the following formula:
[Open area on day 8] - [Open area on day 1 ) / [Open area on day 1 ]
Zs Histology
Specimens are fixed in 10% buffered formalin and paraffin embedded blocks
are sectioned perpendicular to the wound surface (Smm) and cut using an
Olympus
microtome. Routine hematoxylin-eosin (H&E) staining was performed on cross-
sections of bisected wounds. Histologic examination of the wounds allows
3o assessment of whether the healing process and the morphologic appearance of
the
repaired skin was improved by treatment with VEGF-2. A calibrated lens
micrometer
was used by a blinded observer to determine the distance of the wound gap.
Statistical Analysis
35 Experimental data are analyzed using an unpaired t test. A p value of <
0.05 is
considered significant.
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Example 25
Specific Peptide Fragments to Generate
VEGF-2 Monoclonal Antibodies
Four specific peptides (designated SP-40, SP-41, SP-42 and SP-43) have
been generated. These will be used to generate monoclonal antibodies to
analyze
VEGF-2 processing. The peptides are shown below:
1. "SP-40": MTVLYPEYWKMY (amino acids 70-81 in SEQ ID N0:18)
2. "SP-41": KSIDNEWRKTQSMPREV (amino acids 120-136( note C->S mutation
at position 13 I ) in SEQ ID NO: I 8)
3. "SP-42": MSKLDVYRQVHSIIRR (amino acids 212-227 in SEQ ID NO: 18)
4. "SP-43": MFSSDAGDDSTDGFHDI (amino acids 263-279 in SEQ ID NO: 18)
Example 26
Lymphadema Animal Model
The purpose of this experimental approach is to create an appropriate and
consistent lymphedema model for testing the therapeutic effects of VEGF-2 in
lymphangiogenesis and re-establishment of the lymphatic circulatory system in
the rat
hind limb. Effectiveness is measured by swelling volume of the affected limb,
quantification of the amount of lymphatic vasculature, total blood plasma
protein, and
histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more
importantly, the chronic progress of the edema is followed for up to 3-4
weeks.
Experimental Procedure
Prior to beginning surgery, blood sample was drawn for protein concentration
analysis. Male rats weighing approximately -350g are dosed with Pentobarbital.
Subsequently, the right legs were shaved from laiee to hip. The shaved area i5
swabbed with gauze soaked in 70% EtOH. Blood is drawn for serum total protein
testing. Circumference and volumetric measurements were made prior to
injecting dye
into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of
dorsal
paw). The intradermal dorsum of both right and left paws are injected with
0.05 ml of
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1 % Evan's Blue. Circumference and volumetric measurements are then made
following injection of dye into paws.
Using the knee joint as a landmark, a mid-leg inguinal incision is made
circumferentially allowing the femoral vessels to be located. Forceps and
hemostats
are used to dissect and separate the skin flaps. After locating the femoral
vessels, the
lymphatic vessel that runs along side and underneath the vessels) is located.
The
main lymphatic vessels in this area are then electrically coagulated or suture
ligated.
Using a microscope, muscles in back of the leg (near the semitendinosis and
adductors) are bluntly dissected. The popliteal lymph node is then located.
to The 2 proximal and 2 distal lymphatic vessels and distal blood supply of
the popliteal
node are then and ligated by suturing. The popliteal lymph node, and any
accompanying adipose tissue, is then removed by cutting connective tissues.
Care was taken to control any mild bleeding resulting from this procedure.
After lymphatics were occluded, the skin flaps are sealed by using liquid skin
~ 5 (Vetbond) (AJ Buck). The separated skin edges are sealed to the underlying
muscle
tissue while leaving a gap of ~0.5 cm around the leg. Skin also may be
anchored by
suturing to underlying muscle when necessary.
To avoid infection, animals are housed individually with mesh (no bedding).
Recovering animals were checked daily through the optimal edematous peak,
which
2o typically occurred by day 5-7. The plateau edematous peak was then
observed. To
evaluate the intensity of the lymhedema, we measured the circumference and
volumes
of 2 designated places on each paw before operation and daily for 7 days. The
effect
plasma proteins have on lymphedema and determined if protein analysis is a
useful
testing perimeter is also investigated. The weights of both control and
edematous
25 limbs are evaluated at 2 places. Analysis is performed in a blind manner.
Circumference Measurements: Under brief gas anesthetic to prevent limb
movement, a
cloth tape is used to measure limb circumference. Measurements are done at the
ankle
bone and dorsal paw by 2 different people then those 2 readings are averaged.
3o Readings are taken from both control and edematous limbs.
Volumetric Measurements: On the day of surgery, animals are anesthetized with
Pentobarbital and are tested prior to surgery. For daily volumetrics animals
are under
brief halothane anesthetic (rapid immobilization and quick recovery), both
legs are
35 shaved and equally marked using waterproof marker on legs. Legs are first
dipped in
water, then dipped into instrument to each marked level then measured by Buxco
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edema software(Chen/Victor). Data is recorded by one person, while the other
is
dipping the limb to marked area.
Blood-plasma protein measurements: Blood is drawn, spun, and serum separated
prior to surgery and then at conclusion for total protein and Ca2+ comparison.
Limb Weight Comparison: After drawing blood, the animal is prepared for tissue
collection. The limbs were amputated using a quillitine, then both
experimental and
control legs were cut at the ligature and weighed. A second weighing is done
as the
0 tibio-cacaneal joint was disarticulated and the foot was weighed.
Histological Preparations: The transverse muscle located behind the knee
(popliteal)
area is dissected and arranged in a metal mold, filled with freezeGel, dipped
into cold
methylbutane, placed into labeled sample bags at - 80EC until sectioning. Upon
sectioning, the muscle was observed under fluorescent microscopy for
lymphatics.
~ 5 Other immuno/histological methods are currently being evaluated.
Example 27
20 Method of Treatment Using Gene Therapy for Production
of VEGF-2 Polypeptide - In Vivo
Another aspect of the present invention is using in vivo gene therapy methods
to treat disorders, diseases and conditions. The gene therapy method relates
to the
25 introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA)
comprising VEGF-2 operably linked to a promoter into an animal to increase the
expression of VEGF-2. Such gene therapy and delivery techniques and methods
are
known in the art, see, for example, WO 90/11092, WU 98/11779; U.S. Patent Nos.
5693622, 57051 S 1, 5580859; Tabata H. et al. ( 1997) Cardiovasc. Res.
35(3):470-
30 479, Chao, J et al. ( 1997) Pharmacol. Res. 35(6):517-522, Wolff, J.A. (
1997)
Neuromuscul. Disord. 7(5):314-318, Schwartz, B. et al. ( 1996) Gene Ther.
3(5):405-411, Tsurumi, Y. et al. ( 1996) Circulation 94112 )_32R 1-X790 l
in~nrnnratPri
herein by reference).
The VEGF-2 polynucleotide constructs may be delivered by any method that
35 delivers injectable materials to the cells of an animal, such as, injection
into the
interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and
the like).
The VEGF-2 polynucleotide constructs may also be delivered directly into
arteries.
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The VEGF-2 polynucleotide constructs can be delivered in a pharmaceutically
acceptable liquid or aqueous carrier.
The term "naked" polynucleotide, DNA or RNA, refers to sequences that are
free from any delivery vehicle that acts to assist, promote, or facilitate
entry into the
cell, including viral sequences, viral particles, liposome formulations,
lipofectin or
precipitating agents and the like. However, the VEGF-2 polynucleotide may also
be
delivered in liposome formulations (such as those taught in Felgner P.L. et
al. ( 1995)
Ann. NYAcad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-
7)
which can be prepared by methods well known to those skilled in the art.
1o The VEGF-2 vector constructs used in the gene therapy method are preferably
constructs that will not integrate into the host genome nor will they contain
sequences
that allow for replication. Unlike other gene therapies techniques, one major
advantage
of introducing naked nucleic acid sequences into target cells is the
transitory nature of
the polynucleotide synthesis in the cells. Studies have shown that non-
replicating
DNA sequences can be introduced into cells to provide production of the
desired
polypeptide for periods of up to six months.
The VEGF-2 construct can be delivered to the interstitial space of tissues
within the an animal, including of muscle, skin, brain, lung, liver, spleen,
bone
marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
2o bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system,
eye, gland,
and connective tissue. Interstitial space of the tissues comprises the
intercellular fluid,
mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic
fibers in
the walls of vessels or chambers, collagen fibers of fibrous tissues, or that
same
matrix within connective tissue ensheathing muscle cells or in the lacunae of
bone. It
is similarly the space occupied by the plasma of the circulation and the lymph
fluid of
the lymphatic channels. They may be conveniently delivered by injection into
the
tissues comprising these cells. They are preferably delivered to and expressed
in
persistent, non-dividing cells which are differentiated, although delivery and
expression may be achieved in non-differentiated or less completely
differentiated
3o cells, such as, for example, stem cells of blood or skin fibroblasts.
Preferably, they
are delivered by direct injection into the artery.
For the naked polynucleotide injection, an effective dosage amount of DNA or
RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg
body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20
mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course,
as
the artisan of ordinary skill will appreciate, this dosage will vary according
to the
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tissue site of injection. The appropriate and effective dosage of nucleic acid
sequence
can readily be determined by those of ordinary skill in the art and may depend
on the
condition being treated and the route of administration. The preferred route
of
administration is by the parenteral route of injection into the interstitial
space of
tissues, or directly into arteries. However, other parenteral routes may also
be used,
such as, inhalation of an aerosol formulation particularly for delivery to
lungs or
bronchial tissues, throat or mucous membranes of the nose. In addition, naked
VEGF-2 constructs can be delivered to arteries during angioplasty by the
catheter used
in the procedure.
o The dose response effects of injected VEGF-2 polynucleotide construct in
arteries in vivo is determined as follows. Suitable template DNA for
production of
mRNA coding for VEGF-2 is prepared in accordance with a standard recombinant
DNA methodology. The template DNA, which may be either circular or linear, is
either used as naked DNA or complexed with liposomes. The arteries of rabbits
are
then injected with various amounts of the template DNA.
Hindlimb ischemia in rabbits is surgically induced, as described in Example
18. Immediately following this, five different sites in the adductor (2
sites), medial
large (2 sites), and semimembranous muscles ( 1 site) are injected directly
with
plasmid DNA encoding VEGF-2 using a 3m1 syringe and 2-gauge needle advanced
2o through a small skin incision. The skin is then closed using 4.0 nylon.
The ability to rescue hindlimb ischemia is determined by measuring the
number of capillaries in light microsopic sections taken from the treated
hindlimbs,
compared to ischemic hindlimbs from untreated rabbits, measurement of calf
blood
pressure, and infra-arterial Doppler guidewire measurement of flow velocity
(Takeshita et al., J. Clin. Invest. 93:662-670 (1994)). The results of the
above
experimentation in rabbits can be use to extrapolate proper dosages and other
treatment
parameters in humans and other animals using VEGF-2 polynucleotide naked DNA.
Example 28
Method of Treatment Using Gene Therapy - Ex Vivo
One method of gene therapy transplants fibroblasts, which are capable of
expressing VEGF-2 polypeptides, onto a patient. Generally, fibroblasts are
obtained
from a subject by skin biopsy. The resulting tissue is placed in tissue-
culture medium
and separated into small pieces. Small chunks of the tissue are placed on a
wet
surface of a tissue culture flask, approximately ten pieces are placed in each
flask.
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The flask is turned upside down, closed tight and left at room temperature
over night.
After 24 hours at room temperature, the flask is inverted and the chunks of
tissue
remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12
media, with
10% FBS, penicillin and streptomycin) is added. The flasks are then incubated
at 37
degree C for approximately one week.
At this time, fresh media is added and subsequently changed every several
days. After an additional two weeks in culture, a monolayer of fibroblasts
emerge.
The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al., DNA, 7:219-25 ( 1988)), flanked by the
long terminal repeats of the Moloney murine sarcoma virus, is digested with
EcoRI
and HindIII and subsequently treated with calf intestinal phosphatase. The
linear
vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding VEGF-2 can be amplified using PCR primers which
correspond to the 5' and 3' end sequences respectively as set forth in Example
1.
Preferably, the 5' primer contains an EcoRI site and the 3' primer includes a
HindIII
site. Equal quantities of the Moloney murine sarcoma virus linear backbone and
the
amplified EcoRI and HindIII fragment are added together, in the presence of T4
DNA
ligase. The resulting mixture is maintained under conditions appropriate for
ligation
of the two fragments. The ligation mixture is then used to transform bacteria
HB 1 O 1,
2o which are then plated onto agar containing kanamycin for the purpose of
confirming
that the vector contains properly inserted VEGF-2.
The amphotropic pA317 or GP+am 12 packaging cells are grown in tissue
culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with
10% calf serum (CS), penicillin and streptomycin. The MSV vector containing
the
VEGF-2 gene is then added to the media and the packaging cells transduced with
the
vector. The packaging. cells . now produce infectious -viral particles
containing the
VEGF-2 gene(the packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the
media is harvested from a 10 cm plate of confluent producer cells. The spent
media,
3o containing the infectious viral particles, is filtered through a millipore
filter to remove
detached producer cells and this media is then used to infect fibroblast
cells. Media is
removed from a sub-confluent plate of fibroblasts and quickly replaced with
the media
from the producer cells. This media is removed and replaced with fresh media.
If the
titer of virus is high, then virtually all fibroblasts will be infected and no
selection is
required. If the titer is very low, then it is necessary to use a retroviral
vector that has
a selectable marker, such as neo or his. Once the fibroblasts have been
efficiently
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infected, the fibroblasts are analyzed to determine whether VEGF-2 protein is
produced.
The engineered fibroblasts are then transplanted onto the host, either alone
or
after having been grown to confluence on cytodex 3 microcarrier beads
Example 29
to Method of Treatment Using Gene Therapy
Homologous Recombination
Another method of gene therapy according to the present invention involves
operably associating the endogenous VEGF-2 sequence with a promoter via
~ 5 homologous recombination as described, for example, in U.S. Patent No.
5,641,670,
issued June 24, 1997; International Publication No. WO 96/29411, published
September 26, 1996; International Publication No. WO 94/12650, published
August
4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA $6:8932-8935 ( 1989); and
Zijlstra
et al., Nature 342:435-438 (1989). This method involves the activation of a
gene
2o which is present in the target cells, but which is not expressed in the
cells, or is
expressed at a lower level than desired.
Polynucleotide constructs are made which contain a promoter and targeting
sequences, which are homologous to the 5' non-coding sequence of endogenous
VEGF-2, flanking the promoter. The targeting sequence will be sufficiently
near the
25 5' end of VEGF-2 so the promoter will be operably linked to the endogenous
sequence upon homologous recombination. The promoter and the targeting
sequences
can be amplified using PCR. Preferably, the amplified promoter contains
distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3' end of the
first
targeting sequence contains the same restriction enzyme site as the 5' end of
the
30 amplified promoter and the 5' end of the second targeting sequence contains
the same
restriction site as the 3' end of the amplified promoter.
The amplified promoter and the amplified targeting sequences are digested
with the appropriate restriction enzymes and subsequently treated with calf
intestinal
phosphatase. The digested promoter and digested targeting sequences are added
35 together in the presence of T4 DNA ligase. The resulting mixture is
maintained under
conditions appropriate for ligation of the two fragments. The construct is
size
fractionated on an agarose gel then purified by phenol extraction and ethanol
precipitation.
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In this Example, the polynucleotide constructs are administered as naked
polynucleotides via electroporation. However, the polynucleotide constructs
may also
be administered with transfection-facilitating agents, such as liposomes,
viral
sequences, viral particles, precipitating agents, etc. Such methods of
delivery are
known in the art.
Once the cells are transfected, homologous recombination will take place
which results in the promoter being operably linked to the endogenous VEGF-2
sequence. This results in the expression of VEGF-2 in the cell. Expression may
be
detected by immunological staining, or any other method known in the art.
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue
is
placed in DMEM + 10% fetal calf serum. Exponentially growing or early
stationary
phase fibroblasts are trypsinized and rinsed from the plastic surface with
nutrient
medium. An aliquot of the cell suspension is removed for counting, and the
remaining
cells are subjected to centrifugation. The supernatant is aspirated and the
pellet is
resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM
NaCI, 5 mM KCI, 0.7 mM Naz HP04, 6 mM dextrose). The cells are recentrifuged,
the supernatant aspirated, and the cells resuspended in electroporation buffer
containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension
contains approximately 3X 106 cells/ml. Electroporation should be performed
immediately following resuspension.
Plasmid DNA is prepared according to standard techniques. To construct a
plasmid for targeting to the VEGF-2 locus, plasmid pUC 18 (MBI Fermentas,
Amherst, NY) is digested with HindIII. The CMV promoter is amplified by PCR
with an XbaI site on the 5' end and a BamHI site on the 3'end. Two VEGF-2 non-
coding sequences are amplified via PCR: one VEGF-2 non-coding sequence (VEGF-2
fragment .l ) is amplified with a HindIII site at the 5' end and an Xba site
at the 3'end;
the other VEGF-2 non-coding sequence (VEGF-2 fragment 2) is amplified with a
BamHI site at the 5'end and a HindIII site at the 3'end. The CMV promoter and
VEGF-2 fragments are digested with the appropriate enzymes (CMV promoter -
XbaI
and BamHI; VEGF-2 fragment 1 - XbaI; VEGF-2 fragment 2 - BamHI) and ligated
together. The resulting ligation product is digested with HindIII, and ligated
with the
HindIII-digested pUCl8 plasmid.
Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap
(Bio-Rad). The final DNA concentration is generally at least 120 pg/ml. 0.5 ml
of the
cell suspension (containing approximately 1.S.X 106 cells) is then added to
the cuvette,
and the cell suspension and DNA solutions are gently mixed. Electroporation is
CA 02322748 2000-09-07
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-140-
performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are
set at
960 ~F and 250-300 V, respectively. As voltage increases, cell survival
decreases,
but the percentage of surviving cells that stably incorporate the introduced
DNA into
their genome increases dramatically. Given these parameters, a pulse time of
approximately 14-20 mSec should be observed.
Electroporated cells are maintained at room temperature for approximately 5
min, and the contents of the cuvette are then gently removed with a sterile
transfer
pipette. The cells are added directly to 10 ml of prewarmed nutrient media
(DMEM
with 15% calf serum) in a 10 cm dish and incubated at 37EC. The following day,
the
to media is aspirated and replaced with 10 ml of fresh media and incubated for
a further
16-24 hours.
The engineered fibroblasts are then injected into the host, either alone or
after
having been grown to confluence on cytodex 3 microcarrier beads. The
fibroblasts
now produce the protein product.
Example 30
VEGF-2 Transgenic Animals
The VEGF-2 polypeptides can also be expressed in transgenic animals.
2o Animals of any species, including, but not limited to, mice, rats, rabbits,
hamsters,
guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.
g. ,
baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
In a
specific embodiment, techniques described herein or otherwise known in the
art, are
used to express polypeptides of the invention in humans, as part of a gene
therapy
protocol.
_- - .-- .. Any technique known in the art may be used to-introduce the
transgene (i.e.;
polynucleotides of the invention) into animals to produce the founder lines of
transgenic animals. Such techniques include, but are not limited to,
pronuclear
microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698
(1994);
3o Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al.,
Biotechnology
(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (19891):
retrovirus mediated gene transfer into germ lines (Van der Putten et al.,
Proc. Natl.
Acad. Sci., USA 82:6148-6152 ( 1985)), blastocysts or embryos; gene targeting
in
embryonic stem cells (Thompson et al., Cell 56:313-321 ( 1989));
electroporation of
cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction
of the
polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al.,
Science
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-141
259:1745 {1993); introducing nucleic acid constructs into embryonic
pleuripotent stem
cells and transferring the stem cells back into the blastocyst; and sperm-
mediated gene
' transfer (Lavitrano et al., Cell 57:717-723 ( 1989); etc. For a review of
such
techniques, see Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-229
( 1989), which is incorporated by reference herein in its entirety.
Any technique known in the art may be used to produce transgenic clones
containing polynucleotides of the invention, for example, nuclear transfer
into
enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells
induced to
quiescence (Campell et al., Nature 380:64-b6 ( 1996); Wilmut et al., Nature
385:810
to 813 (1997)).
The present invention provides for transgenic animals that carry the transgene
in all their cells, as well as animals which carry the transgene in some, but
not all their
cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a
single
transgene or as multiple copies such as in concatamers, e.g., head-to-head
tandems or
head-to-tail tandems. The transgene may also be selectively introduced into
and
activated in a particular cell type by following, for example, the teaching of
Lasko et
al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 ( 1992)). The
regulatory
sequences required for such a cell-type specific activation will depend upon
the
particular cell type of interest, and will be apparent to those of skill in
the art. When it
is desired that the polynucleotide transgene be integrated into the
chromosomal site of
the endogenous gene, gene targeting is preferred.
Briefly, when such a technique is to be utilized, vectors containing some
nucleotide sequences homologous to the endogenous gene are designed for the
purpose of integrating, via homologous recombination with chromosomal
sequences,
into and disrupting the function of the nucleotide sequence of the endogenous
gene.
The transgene may also be . selectively introduced rote--a particular cell
type, thus
inactivating the endogenous gene in only that cell type, by following, for
example, the
teaching of Gu et al. {Gu et al., Science 265:103-106 (1994)). The regulatory
sequences required for such a cell-type specific inactivation will depend upon
the
3o particular cell type of interest, and will be apparent to those of skill in
the art.
Once transgenic animals have been generated, the expression of the
recombinant gene may be assayed utilizing standard techniques. Initial
screening may
be accomplished by Southern blot analysis or PCR techniques to analyze animal
tissues to verify that integration of the transgene has taken place. The level
of mRNA
expression of the transgene in the tissues of the transgenic animals may also
be
assessed using techniques which include, but are not limited to, Northern blot
analysis
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of tissue samples obtained from the animal, in situ hybridization analysis,
and reverse
transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may
also
be evaluated immunocytochemically or immunohistochemically using antibodies
specific for the transgene product.
Once the founder animals are produced, they may be bred, inbred, outbred, or
crossbred to produce colonies of the particular animal. Examples of such
breeding
strategies include, but are not limited to: outbreeding of founder animals
with more
than one integration site in order to establish separate lines; inbreeding of
separate
lines in order to produce compound transgenics that express the tra.nsgene at
higher
t0 levels because of the effects of additive expression of each transgene;
crossing of
heterozygous transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate the need
for
screening of animals by DNA analysis; crossing of separate homozygous lines to
produce compound heterozygous or homozygous lines; and breeding to place the
transgene on a distinct background that is appropriate for an experimental
model of
interest.
Transgenic animals of the invention have uses which include, but are not
limited to, animal model systems useful in elaborating the biological function
of
VEGF-2 polypeptides, studying conditions and/or disorders associated with
aberrant
2o VEGF-2 expression, and in screening for compounds effective in ameliorating
such
conditions and/or disorders.
Example 31
VEGF-2 Knock-Out Animals
.. .. .Endogenous VEGF-2 gene expression can also be-reduced by inactivating
or
"knocking out" the VEGF-2 gene and/or its promoter using targeted homologous
recombination. (E.g., see Smithies et al., Nature 317:230-234 (1985); Thomas &
Capecchi, Cell 51:503-512 ( 1987); Thompson et al., Cell 5:313-321 ( 1989);
each of
3o which is incorporated by reference herein in its entirety). For example, a
mutant, non-
functional polynucleotide of the invention (or a completely unrelated DNA
sequence)
flanked by DNA homologous to the endogenous polynucleotide sequence (either
the
coding regions or regulatory regions of the gene) can be used, with or without
a
selectable marker and/or a negative selectable marker, to transfect cells that
express
polypeptides of the invention in vivo. In another embodiment, techniques known
in
the art are used to generate knockouts in cells that contain, but do not
express the gene
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-143
of interest. Insertion of the DNA construct, via targeted homologous
recombination,
results in inactivation of the targeted gene. Such approaches are particularly
suited in
' research and agricultural fields where modifications to embryonic stem cells
can be
used to generate animal offspring with an inactive targeted gene (e.g., see
Thomas &
Capecchi 1987 and Thompson 1989, supra). However this approach can be
routinely
adapted for use in humans provided the recombinant DNA constructs are directly
administered or targeted to the required site in vivo using appropriate viral
vectors that
will be apparent to those of skill in the art.
In further embodiments of the invention, cells that are genetically engineered
to
to express the polypeptides of the invention, or alternatively, that are
genetically
engineered not to express the polypeptides of the invention (e.g., knockouts)
are
administered to a patient in vivo. Such cells may be obtained from the patient
(i.e.,
animal, including human) or an MHC compatible donor and can include, but are
not
limited to fibroblasts, bone marrow cells, blood cells (~, lymphocytes),
adipocytes,
~ 5 muscle cells, endothelial cells etc. The cells are genetically engineered
in vitro using
recombinant DNA techniques to introduce the coding sequence of polypeptides of
the
invention into the cells, or alternatively, to disrupt the coding sequence
and/or
endogenous regulatory sequence associated with the polypeptides of the
invention,
a _g_, by transduction (using viral vectors, and preferably vectors that
integrate the
2o transgene into the cell genome) or transfection procedures, including, but
not limited
to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes,
etc.
The coding sequence of the polypeptides of the invention can be placed under
the
control of a strong constitutive or inducible promoter or promoter/enhancer to
achieve
expression, and preferably secretion, of the VEGF-2 polypeptides. The
engineered
25 cells which express and preferably secrete the polypeptides of the
invention can be
introduced into the.patient systemically, e.g., in the ciret~lation, or
intraperitoneally.
Alternatively, the cells can be incorporated into a matrix and implanted in
the
body, ~, genetically engineered fibroblasts can be implanted as part of a skin
graft;
genetically engineered endothelial cells can be implanted as part of a
lymphatic or
3o vascular graft. (See, for example, Anderson et al. U.S. Patent No.
5,399,349; and
Mulligan & Wilson, U.S. Patent No. 5,460,959 each of which is incorporated by
reference herein in its entirety).
When the cells to be administered are non-autologous or non-MHC compatible
cells, they can be administered using well known techniques which prevent the
35 development of a host immune response against the introduced cells. For
example,
the cells may be introduced in an encapsulated form which, while allowing for
an
CA 02322748 2000-09-07
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-144
exchange of components with the immediate extracellular environment, does not
allow
the introduced cells to be recognized by the host immune system.
Knock-out animals of the invention have uses which include, but are not
limited to, animal model systems useful in elaborating the biological function
of
VEGF-2 polypeptides, studying conditions andlor disorders associated with
aberrant
VEGF-2 expression, and in screening for compounds effective in ameliorating
such
conditions and/or disorders.
Numerous modifications and variations of the present invention are possible in
to light of the above teachings and, therefore, within the scope of the
appended claims,
the invention may be practiced otherwise than as particularly described.
The entire disclosure of all publications (including patents, patent
applications,
journal articles, laboratory manuals, books, or other documents) cited herein
are
hereby incorporated by reference.
is
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Applicant's or agent's file pF112PCT3 Internauonala hcatio No.
referencenumber n~rn ~R
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Applicant's or agent's file pFi 12PCT3 International
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Human Genome Sciences, Inc.
(ii) TITLE OF INVENTION: VASCULAR ENDOTHELIAL GROWTH FACTOR 2
(iii) NUMBER OF SEQUENCES: 35
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: HUMAN GENOME SCIENCES, INC.
(B) STREET: 9410 KEY WEST AVENUE
(C) CITY: ROCKVILLE
(D) STATE: MARYLAND
(E) COUNTRY: USA
(F) ZIP: 20850
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
{A) APPLICATION NUM$ER: TO BE ASSIGNED
(B) FILING DATE: HEREWITH
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 09/042,105
(B) FILING DATE: 13-MAR-1998
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 09/107,997
(B) FILING DATE: 30-JUN-1998
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: MICHELE M. WALES
(B) REGISTRATION NUMBER:43,975
(C) REFERENCE/DOCKET NUMBER: PF112PCT3
(ix) TELECOMMUNICATION INF RMATION:
(A) TELEPHONE: (301)309-8504
(B) TELEFAX: (301)-309-8439
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-2-
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1674 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: sig~peptide
(B) LOCATION: 12..80
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 81..1268
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 12..1268
(xi) SEQID
SEQUENCE NO:1:
DESCRIPTION:
GTC CTTCCACC ATG T 50
CAC GTG
TCG GCG
CTG TGT
GGC TCT
TTC CTG
TTC r
TC Val
Met Ala
His Cys
Ser Ser
Leu Leu
Gly _15
Phe
Phe
Se
-23
-20
CTC GCCGCTGCG CTCCCG GGTCCTCGC GAGGCGCCC GCCGCCGCC 9g
Leu AlaAlaCTG LeuPro GlyProArg GluAlaPro AlaAlaAla
-10 Ala _5 1 5
Leu
GCC GCCTTCGAG GGACTC GACCTCTCG GACGCGGAG CCCGACGCG 146
Ala AlaPheTCC GlyLeu AspLeuSer AspAlaGlu ProAspAla
Glu 15 20
Ser
10
GGC GAGGCCACG TATGCA AGCAAAGAT CTGGAGGAG CAGTTACGG 194
Gly GluAlaGCT TyrAla SerLysAsp LeuGluGlu GlnLeuArg
25 Thr 30 35
Ala
TCT GTGTCCAGT GATGAA CTCATGACT GTACTCTAC CCAGAATAT 242
Ser ValSerGTA AspGlu LeuMetThr ValLeuTyr ProGluTyr
40 Ser 45 50
Val
TGG AAAATGTAC TGTCAG CTAAGGAAA GGAGGCTGG CAACATAAC 290
Trp LysMetAAG CysGln LeuArgLys GlyGlyTrp GlnHisAsn
55 Tyr 60 65 70
Lys
AGA GAACAGGCC CTCAAC TCAAGGACA GAAGAGACT ATAAAATTT 338
Arg GluGlnAAC LeuAsn SerArgThr GluGluThr IleLysPhe
Ala 80 85
Asn
75
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GCT GCA GCA CAT TAT AAT ACA GAG ATC TTG AAA AGT ATT GAT AAT GAG 386
Ala Ala Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu
90 95 100
TGG ACT CAA ATG CCACGGGAG GTGTGTATA GATGTGGGG 434
AGA TGC
AAG
Trp ArgLysThr GlnCysMet ProArgGlu ValCysIle AspValGly
105 110 115
AAG GAGTTTGGA GTCGCGACA AACACCTTC TTTAAACCT CCATGTGTG 482
Lys GluPheGly ValAlaThr AsnThrPhe PheLysPro ProCysVal
120 125 130
TCC GTCTACAGA TGTGGGGGT TGCTGCAAT AGTGAGGGG CTGCAGTGC 530
Ser ValTyrArg CysGlyGly CysCysAsn SerGluGly LeuGlnCys
135 140 145 150
ATG AACACCAGC ACGAGCTAC CTCAGCAAG ACGTTATTT GAAATTACA 578
Met AsnThrSer ThrSerTyr LeuSerLys ThrLeuPhe GluIleThr
155 160 165
GTG CCTCTCTCT CAAGGCCCC AAACCAGTA ACAATCAGT TTTGCCAAT 626
Val ProLeuSer GlnGlyPro LysProVal ThrIleSer PheAlaAsn
170 175 180
CAC ACTTCCTGC CGATGCATG TCTAAACTG GATGTTTAC AGACAAGTT 674
His ThrSerCys ArgCysMet SerLysLeu AspValTyr ArgGlnVal
185 190 195
CAT TCCATTATT AGACGTTCC CTGCCAGCA ACACTACCA CAGTGTCAG 722
His SerIleIle ArgArgSer LeuProAla ThrLeuPro GlnCysGln
200 205 210
GCA GCGAACAAG ACCTGCCCC ACCAATTAC ATGTGGAAT AATCACATC 770
Ala AlaAsnLys ThrCysPro ThrAsnTyr MetTrpAsn AsnHisIle
215 220 225 230
TGC AGATGCCTG GCTCAGGAA GATTTTATG TTTTCCTCG GATGCTGGA 818
Cys ArgCysLeu AlaGlnGlu AspPheMet PheSerSer AspAlaGly
235 240 245
GAT GACTCAACA GATGGATTC CATGACATC TGTGGACCA AACAAGGAG 866
Asp AspSerThr AspGlyPhe HisAspIle CysGlyPro AsnLysGlu
250 255 260
CTG GATGAAGAG ACCTGTCAG TGTGTCTGC AGAGCGGGG CTTCGGCCT 914
Leu AspGluGlu ThrCysGln CysValCys ArgAlaGly LeuArgPro
265 270 275
GCC AGCTGTGGA CCCCACAAA GAACTAGAC AGAAACTCA TGCCAGTGT 962
Ala SerCysGly ProHisLys GluLeuAsp ArgAsnSer CysGlnCys
280 285 290
GTC TGT AAC AAACTCTTC CCCAGCCAA TGTGGGGCC AACCGAGAA 1010
AAA
Val CysLysAsn LysLeuPhe ProSerGln CysGlyAla ArgGlu
Asn
295 300 305 310
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-4-
TTT GAT ACA CAG GTATGT AAA TGCCCC
GAA TGC TGT AGA AGA
AAC ACC
1058
Phe AspGluAsnThr CysGln ValCys LysAr Thr C
Cys
g ys ProArg
315 320 325
AAT CAACCCCTAAAT CCTGGA TGTGCC TGTGAATGT ACAGAAAGT
AAA
1106
Asn GlnProLeuAsn ProGly CysAla C GluC
Lys s
y ys ThrGluSer
330 335 340
CCA CAGAAATGCTTG TTAAAA AAGAAG TTCCACCAC CAAACAT
GGA
GC 1154
Pro GlnLysCysLeu LeuLys LysLys PheHisHi
Gly
s GlnThrCys
345 350 355
AGC TGTTACAGACGG CCATGT AACCGC CAGAAGGCT TGTGAG
ACG
CCA 1202
Ser CysTyrArgArg ProCys AsnArg GlnL Al C
Thr s
y a ys GluPro
360
365 370
GGA TTTTCATATAGT GAAGAA TGTCGT TGTGTCCCT TCATATTGG
GTG
1250
Gly PheSerTyrSer GluGlu CysArg CysValPr S
Val
o er TyrTrp
375
380 385
390
CAA CCACAAATG AGCTAAGATTGTA A CGAT
AGA CTGTTTTCC GTTCAT
1298
Gln ProGlnMet Ser
Arg
395
TTTCTATTAT GGAAAACTGT GTTGCCACAGTAGAACTGTC TGTGAACAGAGAGACCCTTG1358
TGGGTCCATG CTAACAAAGA CAAAAGTCTGTCTTTCCTGA ACCATGTGGATAACTTTACA1418
GAAATGGACT GGAGCTCATC TGCAAAAGGCCTCTTGTAAA GACTGGTTTTCTGCCAATGA1478
CCAAACAGCC AAGATTTTCC TCTTGTGATTTCTTTAAAAG AATGACTATATAATTTATTT1538
CCACTAAAAA TATTGTTTCT GCATTCATTTTTATAGCAAC AACAATTGGTAAAACTCACT1598
GTGATCAATA TTTTTATATC ATGCAAAATATGTTTAAAAT AAAATGAAAATTGTATTTAT1658
AAAAAAAAAA AAAAAA
1674
(2) INFORMATION FOR SEQ ID
N0:2:
(i) SEQUENCE CHARACTERISTI CS:
(A) LENGTH: 419 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met His Ser Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala
-23 -20 -15 -10
Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe
-5 1 5
Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala
15 20 25
Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser
30 35 40
Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met
45 50 55
Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln
60 65 70
Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala
75 80 85
His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys
90 95 100
105
Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe
110 115 120
Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr
125 130 135
Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr
140 145 150
Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu
155 160 165
Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser
170 175 180
185
Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile
190 195
200
Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn
205 210
215
Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys
220 225 230
Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser
235 240
245
Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu
250 255
260 265
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-6-
PCTNS99/05021
Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys
270 275
280
Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys
285 290
295
Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu
300 305
310
Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro
315 320
325
Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys
330 335
340 345
Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr
350 355
360
Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser
365 370
375
Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Gln Arg Pro
380 385
390
Gln Met Ser
395
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1526 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 71..142
(ix) FEATURE:
(A) NAME/KEY: mat-peptide
(B) LOCATION: 143..1120
(ix) FEATURE:
(A) NAME/KEY; CDS
(B) LOCATION: 71..1120
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
CGAGGCCACG GCTTATGCAA GCAAAGATCT GGAGGAGCAG TTACGGTCTG TGTCCAGTGT 60
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AGATGAACTC ATG ACT GTA CTC TAC CCA GAA TAT TGG AA
A ATG TAC AAG 109
Met Thr Val Leu T
r P
l
y
ro G
u Tyr Trp Lys Met Tyr Lys
-24 -20
-15
TGT CAG CTA AGG AAA GGA GGC TGG CAA CAT AAC AGA G
AA CAG GCC AAC 157
Cys Gln Leu Arg Lys Gly Gl
Tr
Gl
y
p
n His Asn Arg Glu Gln Ala Asn
_S
1 S
CTC AAC TCA AGG ACA GAA GAG ACT ATA AAA TTT GCT GCA G
CA CAT TAT 205
Leu Asn Ser Arg Thr Glu Glu Thr Il
e Lys Phe Ala Ala Ala His Tyr
10 15
AAT ACA GAG ATC TTG AAA AGT ATT GAT AAT GAG TGG A
GA AAG ACT CAA 253
Asn Thr Glu Ile Leu Lys Ser Il
A
e
sp Asn Glu Trp Arg Lys Thr Gln
30
TGC ATG CCA CGG GAG GTG TGT ATA GAT GTG GGG AAG GAG
TTT GGA GTC 301
Cys Met Pro Arg Glu Val Cys Ile As
V
l
p
a
Gly Lys Glu Phe Gly Val
45
GCG ACA AAC ACC TTC TTT AAA CCT CCA TGT GTG TCC GTC T
AC AGA TGT 349
Ala Thr Asn Thr Phe Phe Lys Pro P
ro Cys Val Ser Val Tyr Arg Cys
60 65
GGG GGT TGC TGC AAT AGT GAG GGG CTG CAG TGC ATG AAC ACC
AG
C ACG 3g7
Gly Gly Cys Cys Asn Ser Glu Gly Leu Gl
C
n
ys Met Asn Thr Ser Thr
70
75 BO 85
AGC TAC CTC AGC AAG ACG TTA TTT GAA ATT ACA GTG CCT
CTC TCT CAA 445
Ser Tyr Leu Ser Lys Thr Leu Phe Gl
u Ile Thr Val Pro Leu Ser Gln
90 95
100
GGC CCC AAA CCA GTA ACA ATC AGT TTT GCC AAT CAC ACT T
CC TGC CGA 493
Gly Pro Lys Pro Val Thr Ile Ser Ph
A
e
la Asn His Thr Ser Cys Arg
I05 110
115
TGC ATG TCT AAA CTG GAT GTT TAC AGA CAA GTT CAT TCC
ATT ATT AGA 541
Cys Met Ser Lys Leu Asp Val T
r A
l
y
rg G
n Val His Ser Ile Ile Arg
120 125
130
CGT TCC CTG CCA GCA ACA CTA CCA CAG TGT CAG GCA GCG
AAC AAG ACC 589
Arg Ser Leu Pro Ala Thr Leu Pro Gl
n Cys Gln Ala Ala Asn Lys Thr
135 140
145
TGC CCC ACC AAT TAC ATG TGG AAT AAT CAC ATC TGC A
GA TGC CTG GCT 637
Cys Pro Thr Asn Tyr Met Tr
As
A
p
n
sn His Ile Cys Arg Cys Leu Ala
150 155
160
165
CAG GAA GAT TTT ATG TTT TCC TCG GAT GCT GGA GAT G
AC TCA ACA GAT 685
Gln Glu Asp Phe Met Phe S
er Ser Asp Ala Gly Asp Asp Ser Thr Asp
170 175
180
GGA TTC CAT GAC ATC TGT GGA CCA AAC AAG GAG CTG GAT
GAA GAG ACC 733
Gly Phe His Asp Ile Cys Gly Pro A
sn Lys Glu Leu Asp Glu Glu Thr
185 190
19S
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TGT CAGTGTGTC TGCAGAGCG GGGCTTCGG CCTGCC AGCTGTGGA CCC 781
Cys GlnCysVal CysArgAla GlyLeuArg ProAla SerCysGly Pro
200 205 210
CAC AAAGAACTA GACAGAAAC TCATGCCAG TGTGTC TGTAAAAAC AAA 829
His LysGluLeu AspArgAsn SerCysGln CysVal CysLysAsn Lys
215 220 225
CTC TTCCCCAGC CAATGTGGG GCCAACCGA GAATTT GATGAAAAC ACA 877
Leu PheProSer GlnCysGly AlaAsnArg GluPhe AspGluAsn Thr
230 235 240 245
TGC CAGTGTGTA TGTAAAAGA ACCTGCCCC AGAAAT CAACCCCTA AAT 925
Cys GlnCysVal CysLysArg ThrCysPro ArgAsn GlnProLeu Asn
250 255 260
CCT GGAAAATGT GCCTGTGAA TGTACAGAA AGTCCA CAGAAATGC TTG 973
Pro GlyLysCys AlaCysGlu CysThrGlu SerPro GlnLysCys Leu
265 270 275
TTA AAAGGAAAG AAGTTCCAC CACCAAACA TGCAGC TGTTACAGA CGG 1021
Leu LysGlyLys LysPheHis HisGlnThr CysSer CysTyrArg Arg
280 285 290
CCA TGTACGAAC CGCCAGAAG GCTTGTGAG CCAGGA TTTTCATAT AGT 1069
Pro CysThrAsn ArgGlnLys AlaCysGlu ProGly PheSerTyr Ser
295 300 305
GAA GAAGTGTGT CGTTGTGTC CCTTCATAT TGGCAA AGACCACAA ATG 1117
Glu GluValCys ArgCysVal ProSerTyr TrpGln ArgProGln Met
310 315 320 325
AGC TAAGATTGTA CTATTAT GGAAAACTGT 1170
CTGTTTTCCA
GTTCATCGAT
TTT
Ser
GTTGCCACAGTAGAACTGTC TGTGAACAGAGAGACCCTTGTGGGTCCATG CTAACAAAGA1230
CAAAAGTCTGTCTTTCCTGA ACCATGTGGATAACTTTACAGAAATGGACT GGAGCTCATC1290
TGCAAAAGGCCTCTTGTAAA GACTGGTTTTCTGCCAATGACCAAACAGCC AAGATTTTCC1350
TCTTGTGATTTCTTTAAAAG AATGACTATATAATTTATTTCCACTAAAAA TATTGTTTCT1410
GCATTCATTTTTATAGCAAC AACAATTGGTAAAACTCACTGTGATCAATA TTTTTATATC1470
ATGCAAAATATGTTTAAAAT AAAATGAAAATTGTATTTATF~~AAAAAAAA AAAAAA 1526
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 350 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met Tyr Lys Cys Gln Leu
-24 -20 -15 -10
Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln Ala Asn Leu Asn Ser
-5 1 5
Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr Asn Thr Glu
15 20
Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln Cys Met Pro
25 30 35 40
Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Val Ala Thr Asn
45 50 55
Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly Cys
60 65 70
Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Ser Tyr Leu
75 80 85
Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro Lys
90 95 100
Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg Cys Met Ser
105 110 115 120
Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg Arg Ser Leu
125 130 135
Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr Cys Pro Thr
140 145 150
Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys Leu Ala Gln Glu Asp
155 160 165
Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser Thr Asp Gly Phe His
170 175 180
Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr Cys Gln Cys
185 190 195 200
Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys Gly Pro His Lys Glu
205 210 215
Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys Leu Phe Pro
220 225 230
Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln Cys
235 240 245
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Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro Gly Lys
250 255 260
Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys Cys Leu Leu Lys Gly
265 270 275 280
Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr Arg Arg Pro Cys Thr
285 290 295
Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser Tyr Ser Glu Glu Val
300 305 310
Cys Arg Cys Val Pro Ser Tyr Trp Gln Arg Pro Gln Met Ser
315 320 325
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 196 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly Cys Gly Tyr Leu Ala
1 5 10 15
His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val Ile Glu Arg
20 25 30
Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu Gln Arg Leu Leu
35 40 45
Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp Thr Ser Leu Arg
50 55 60
Ala His Gly Val His Ala Thr Lys His Val Pro Glu Lys Arg Pro Leu
65 70 75 80
Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Val Pro Ala Val Cys
85 90 95
Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro
100 105 110
Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys Arg
115 120 125
Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg
130 135 140
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Val His His Arg Ser Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys
145 150 155 160
Lys Pro Lys Leu Lys Glu Val Gln Val Arg Leu Glu Glu His Leu Glu
165 170 175
Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp
180 185 190
Thr Asp Val Arg
195
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 241 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg
1 5 10 15
Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met
20 25 30
Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu
35 40 45
His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met
50 55 60
Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg
65 70 75 80
Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu
85 90 95
Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp
100 105 110
Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln
115 120 125
Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr
130 135 140
Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg
145 150 155 160
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Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu
165 170 175
Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser
180 185 190
Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val
195 200 205
Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg
210 215 220
Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly
225 230 235 240
Ala
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 232 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu
1 5 10 15
Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly
20 25 30
Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln
35 40 45
Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu
50 55 60
Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu
65 70 75 80
Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro
85 90 95
Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His
100 105 110
Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys
115 120 125
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Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val
130 135 140
Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr
145 150 155 160
Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp
165 170 175
Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys
180 185 190
His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn
195 200 205
Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr
210 215 220
Cys Arg Cys Asp Lys Pro Arg Arg
225 230
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Pro Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys Asn
1 5 10
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
ATGCTTCCGG CTCGTATG 18
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGGTTTTCCC AGTCACGAC 18
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CCACATGGTT CAGGAAAGAC A 21
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
TGTAATACGA CTCACTATAG GGATCCCGCC ATGGAGGCCA CGGCTTATGC 50
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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-15-
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GATCTCTAGA TTAGCTCATT TGTGGTCT 28
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
CGCGGATCCA TGACTGTACT CTACCCA 27
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
CGCTCTAGAT CAAGCGTAGT CTGGGACGTC GTATGGGTAC TCGAGGCTCA TTTGTGGTCT 60
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3974 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
GGTACCTAAG TGAGTAGGGC GTCCGATCGA CGGACGCCTT TTTTTTGAAT TCGTAATCAT 60
GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG 120
CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG 180
CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA 240
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TCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCA 300
CTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGG 360
TAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCC 420
AGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC 480
CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGAC 540
TATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCC 600
TGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATA 660
GCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGC 720
ACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA 780
ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG 840
CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTA 900
GAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTG 960
GTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGC 1020
AGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGT 1080
CTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCGTCGA 1140
CAATTCGCGCGCGAAGGCGAAGCGGCATGCATTTACGTTGACACCATCGAATGGTGCAAA 1200
ACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTG 1260
AAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCC 1320
CGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCG 1380
ATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCG 1440
TTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCG 1500
GCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGA 1560
AGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGG 1620
CTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACT 1680
AATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTC 1740
TCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAA 1800
ATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGG 1860
CATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGT 1920
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GCCATGTCCG GTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCG 1980
ATGCTGGTTG CCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGG 2040
CTGCGCGTTG GTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGT 2100
TATATCCCGC CGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTG 2160
GACCGCTTGC TGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTC 2220
TCACTGGTGA AAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG 2280
TTGGCCGATT CATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 2340
GCGCAACGCA ATTAATGTAAGTTAGCGCGAATTGTCGACCAAAGCGGCCATCGTGCCTCC 2400
CCACTCCTGC AGTTCGGGGGCATGGATGCGCGGATAGCCGCTGCTGGTTTCCTGGATGCC 2460
GACGGATTTG CACTGCCGGTAGAACTCCGCGAGGTCGTCCAGCCTCAGGCAGCAGCTGAA 2520
CCAACTCGCG AGGGGATCGAGCCCGGGGTGGGCGAAGAACTCCAGCATGAGATCCCCGCG 2580
CTGGAGGATC ATCCAGCCGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAGAA 2640
GGCGGCGGTG GAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGTCATTTC 2700
GAACCCCAGA GTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGC 2760
GAATCGGGAG CGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGC 2820
TCTTCAGCAA TATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGC 2880
CGGCCACAGT CGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAG 2940
GCATCGCCAT GGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCG 3000
AACAGTTCGG CTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGA 3060
CCGGCTTCCA TCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGG 3120
CAGGTAGCCG GATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTC 3180
TCGGCAGGAG CAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGC 3240
CAGTCCCTTC CCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTG 3300
GCCAGCCACG ATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCG 3360
GTCTTGACAA AAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAG 3420
CAGCCGATTG TCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGA 3480
GAACCTGCGT GCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGA 3540
TCAGATCTTG ATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACT 3600
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TTGCAGGGCT TCCCAACCTT ACCAGAGGGC GCCCCAGCTG GCAATTCCGG TTCGCTTGCT 3660
GTCCATAAAA CCGCCCAGTC TAGCTATCGC CATGTAAGCC CACTGCAAGC TACCTGCTTT 3720
CTCTTTGCGC TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACATT CATCCGGGGT 3780
CAGCACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT TCCTTTAGCA GCCCTTGCGC 3840
CCTGAGTGCT TGCGGCAGCG TGAAGCTTAA AAAACTGCAA AAAATAGTTT GACTTGTGAG 3900
CGGATAACAA TTAAGATGTA CCCAATTGTG AGCGGATAAC AATTTCACAC ATTAAAGAGG 3960
AGAAATTACA TATG 3974
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 112 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
AAGCTTAAAA AACTGCAAAA AATAGTTTGA CTTGTGAGCG GATAACAATT AAGATGTACC 60
CAATTGTGAG CGGATAACAA TTTCACACAT TAAAGAGGAG AAATTACATA TG 112
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 419 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met His Ser Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala
1 5 10 15
Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe
20 25 30
Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala
35 40 45
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Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser
50 55 60
Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met
65 70 75 80
Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln
85 90 95
Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala
100 105 110
His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys
115 120 125
Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe
130 135 140
Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr
145 150 155 160
Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr
165 170 175
Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu
180 185 190
Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser
195 200 205
Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile
210 215 220
Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn
225 230 235 240
Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys
245 250 255
Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser
260 265 270
Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu
275 280 285
Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys
290 295 300
Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys
305 310 315 320
Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu
325 330 335
Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro
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340 345 350
Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys
355 360 365
Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr
370 375 380
Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser
385 390 395 400
Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Gln Arg Pro
405 410 415
Gln Met Ser
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
GCAGCACATA TGACAGAAGA GACTATAAAA 30
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
GCAGCAGGTA CCTCACAGTT TAGACATGCA 30
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
GCAGCAGGTA CCTCAACGTC TAATAATGGA 30
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
GCAGCAGGAT CCCACAGAAG AGACTATAAA 30
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
GCAGCATCTA GATCACAGTT TAGACATGCA 30
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
GCAGCAGGAT CCCACAGAAG AGACTATAAA ATTTGCTGC 39
(2) INFORMATION FOR SEQ ID N0:25:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
GCAGCATCTA GATCAACGTC TAATAATGGA ATGAAC 36
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
GATCGATCCA TCATGCACTC GCTGGGCTTC TTCTCTGTGG CGTGTTCTCT GCTCG 55
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
GCAGGGTACG GATCCTAGAT TAGCTCATTT GTGGTCTTT 39
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
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GACTGGATCC GCCACCATGC ACTCGCTGGG CTTCTTCTC 39
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
GACTGGTACC TTATCACATA AAATCTTCCT GAGCC 35
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
GACTGGATCC GCCACCATGC ACTCGCTGGG CTTCTTCTC 39
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
GACTGGTACC TTATCAGTCT AGTTCTTTGT GGGG 34
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
GACTGGATCC GCCACCATGC ACTCGCTGGG CTTCTTCTC 39
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
{C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
GACTGGTACC TCATTACTGT GGACTTTCTG TACATTC 37
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
GCAGCAGGAT CCACAGAAGA GACTATAAAA TTTGCTGC 38
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
CGTCGTTCTA GATCACAGTT TAGACATGCA TCGGCAG 37
