Note: Descriptions are shown in the official language in which they were submitted.
CA 02413012 2002-O1-03
This 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 polypeptide of the
present invention has been. ~:iclentified as a member of the
vascular endothelial growth factor family. More
particularly, the polypeptide of the present invention is
vascular endothelial growth factor 2, sometimes hereinafter
referred to as "V8GF2." The invention also relates to
inhibiting the action of such polypeptide.
The f orznation of new blood vessels, or angiogenesis, is
essential f or embryonic development, subsequent growth, and
tissue repair. Angiogenesis, however, is an essential part
of certain pathological conditions such as neoplasia, for
example, tumors and gliomas, and abnoru~al angiogenesis is
associated With other diseases such as inflammation,
10..
CA 02413012 2002-O1-03
rheumatoid arthritis, psoriasis, and diabetic retinopathy
(Folkman, J. and Klagsbrun, M., Science 235:442-447,(1967)).
Both acidic and basic fibroblast growth factor molecules
are mitagens for endothelial cells and other cell types.
Angiotropin and angiogenin can induce angiogenesis, although
their functions are unclear (Folkman, J., 1993, Cancer
Medicine pp. 153-170, Lea and Febiger Press). A highly
selective mitogen for vascular endothelial cells is vascular
endothelial growth factor or VHGF (Perrara, N., et al.,
Bndocr. Rev. 13:19-32, (1992)), 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 VBGF gene has been characterized and its
expression pattern in embryogenesis has been analyzed. A
persistent expression of VBGF 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 V$GF as a multifunctional regulator of endothelial
cell growth and differentiation (Breier, G. et a1.
Development, 114:521-532 (1992)).
VBGF is structurally related to the a and ~ chains of
platelet-derived growth factor (PDGF), a ~ttogen for
mesenchymal cells and placenta growth factor (PLGF), an
endothelial cell mitogen. These three proteins belong to the
same family and share a conserved motif. Bight cysteine
residues contributing to disulfide-bond formation are
strictly conserved in these proteins. Alternatively spliced
mRNAs have been identified for both V$GF, PLGF and PDGF and
these different splicing products differ in biological
activity and in receptor-binding specificity. VBGF and PDGF
function as homo-dimers or hetero-dimers and bind to
receptors which elicit intrinsic tyrosine kinase activity
following receptor dimerization.
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CA 02413012 2002-O1-03
VBGF has four different forn~s of 121, 165, 189 and 206
amino acids due to alternative splicing. V$GF121 and VSGF165
are soluble and are capable of promoting angiogenesis,
whereas VBGF189 and V'SGF206 are bound to heparin containing
proteoglycans in the cell surface. The temporal and spatial
expression of VgGF has been correlated with physiological
proliferation of the blood vessels (Gajdusek, C.M., and
Carbon, S.J., Cell Physiol., 139:570-579, (1989)); McNeil,
P.L., Muthukrishnan, L., Warder, 8., D'Amore, P.A., 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 VBGF121 or VSGF165 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)1. The inhibition of vBGF function by anti-VSGF
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 VBGF
receptor has been shown to inhibit growth of glioblastomas in
mice. . . _ -
Vascular permeability factor, 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. 8xp. Med., 176:1375-9 (1992).
The expression of VBGF is high in vascularized tissues,
(e. g., lung, heart, placenta and solid tumors) and correlates
with angiogenesis both temporally and spatially. VBGF has
also been shown to induce angiogenesis in vivo. Since
angiogenesis is essential for the repair of normal tissues,
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CA 02413012 2002-O1-03
especially vascular tissues, VSGF has been proposed for use '
in promoting vascular tissue repair (e.g., in
atherosclerosis).
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, VBGF, 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 hetero-dimer or homo-dimer. The protein is
a mazmnalian vascular endothelial cell mitogen and, as such,
is useful for the promotion of vascular development and
repair, as disclosed in 8urapean Patent Application No.
92302750.2, published September 30, 1992.
The polypeptides of the present invention have been
putatively identified as a novel vascular endothelial growth
factor based on amino acid sequence homology to human VBGF.
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.
In accordance with another aspect of the present
invention, there are provided isolated nucleic acid molecules
encoding the polypeptides of the present invention, including
r~RNAs , DNAs , cDNAs , genotnic DNA as wel l as biologically
active and diagnostically or therapeutically useful
fragments, analogs and derivatives thereof.
In accordance with still another aspect of the present
invention, there are provided processes for producing such
polypeptides by recombinant techniques comprising culturing
recombinant prokaryotic and/or eukaryotic host cells,
containing a nucleic acid sequence encoding a polypeptide of
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CA 02413012 2002-O1-03
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 is provided a process for utilizing such
polypeptide, or polynucleotide encoding such polypeptide for
therapeutic purposes, for example, to stimulate angiogenesis,
wound-healing, and to promote vascular tissue repair.
In accordance with yet another aspect of the present
invention, there are provided antibodies against 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 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.
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.
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The following drawings are illustrative of embodiments
of the invention and are not meant to li~ai.t the scope of the
invention as encompassed by the claims.
Fig. 1 shows the cDNA sequence and the corresponding
deduced amino acid sequence of the polypeptide of the present
invention. The standard one letter abbreviations for amino
acids are used. Sequencing was performed using 373 Automated
DPIA Sequences (Applied Biosystems, Inc,). Sequencing
accuracy is predicted to be greater than 97%.
Fig. 2 is an illustration of the amino acid sequence
homology between the polypeptide of the present invention and
other members of the human PDGF/VBGF family. The boxed areas
indicate the conserved sequences and the location of the
eight conserved cysteine residues.
Fig. 3 shows a photograph of a gel after in vitro
transcription, translation and electrophoresis of the
polypeptide of the present invention. Lane 1: "C and rainbow
M.W. marker; Lane 2: FGF control; Lane 3: VBGF2 produced by
M13-reverse and forward primers; Lane 4: VSGF2 produced by
M13 reverse and VBGF-F4 primers; Lane 5: VBGF2 produced by
M13 reverse and VSGF-F5 primers.
Fig. 4. vSGF2 polypeptide is expressed in a baculovirus
system consisting of Sf9 cells. Protein from the medium and
cytoplasm of cells were analyzed by SDS-PAGE under reducing
and non-reducing conditions.
Fig. 5. The medium from Sf9 cells infected with a
nucleic acid sequence of the present invention was
precipitated and the resuspended precipitate was analyzed by
SDS-PAG$ and was stained with coamassie brilliant blue_
Fig.' 6. VBGF2 was purified from the medium supernatant
and analyzed by SDS-PAGE in the presence or absence of the
reducing agent ~-mercaptoethanol and stained by coomassie
brilliant blue.
Fig. 7. Reverse phase HPLC analysis of purified VBGF2
using a RP-300 column (0.21 x 3 cm, Applied Biosystems,
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CA 02413012 2002-O1-03
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 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 Green line.
Fig. 8 illustrates the effect of partially-purified
VEGF2 protein on the growth of vascular endothelial cells in
comparison to basic fibroblast growth factor.
Fig. 9 illustrates the effect of purified V8GF2 protein
on the growth of vascular endothelial cells.
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).
In accordance with one aspect of the present invention,
there are provided isolated nucleic acid molecules
(polynucleotides) which encode for the mature polypeptides ,
having the deduced amino acid sequence of Figure l or for the
mature polypeptide encoded by the cDNA of the clone deposited
as ATCC Deposit No 97149 ion May 12, 1995 at the American
Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, U.S.A., or
for polypeptides which have fewer amino acid residues than those showing in
Figure 1.
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 VSGF/PDGF
family. VEGP2 contains an open reading frame encoding a
protein of 419 amino acid residues of which approximately the
first 23 amino acid residues are the putative leader sequence
such that the mature protein comprises 396 amino acids. and
which protein ex'tlibits the highest amino acid sequence
CA 02413012 2002-O1-03
homology to human vascular endothelial growth factor (30%
identity), followed by PDGFa (23%) and PDGFS (22%).
It is particularly important that all eight cysteines
are consezved within all four members of the family (see
boxed areas of Figure 2). In addition, the signature for the
PDGF/VEGF family, PXCVXXXRCXGCCN, (S8Q ID N0:3) is conserved
in VSGF2 (see Figure 2).
The VBGF2 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 S$Q ID No . 2 and Figure 2 , but still contain the eight
cysteine residues shown conserved in Figure 2 and such
fragments still contain VSGF2 activity.
There are at least two alternatively spliced VBGF2 mRNA
sequences present in normal tissues . The size of the two
VBGF2 mRNA sequences which correspond to the full-length and
truncated version respectively are shown in Figure 3, lane 5
shows two bands indicating the presence of the alternatively
spliced mRNA encoding the VBGF2 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 that of
the deposited clone or may be a different coding sequence
which coding sequence, as a result of the redundancy or
degeneracy of the genetic code, encodes the same mature
polypeptide as the DNA of Figure 1 or the deposited cDNA.
_g_
CA 02413012 2002-O1-03
The polynucleotide which encodes for the mature
polypeptide of Figure 1 or for the mature polypeptide encoded
by the deposited cDNA may include: only the coding sequence
for the mature polypeptide; the coding sequence for the
mature polypeptide (and optionally additional coding
sequence) and non-coding sequence, 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 polynucleotide which includes only coding
sequence for the polypeptide as well as a polynucleotide
which includes additional coding and/or non-coding sequence.
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 Figure 1 or the
polypeptide encoded by the cDNA of the deposited clone. 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 Figure 1 or
the same mature polypeptide encoded by the cDNA of the
deposited clone as well as variants of such polpnucleotides
which variants encode for a fragment, derivative or analog of
the polypeptide of Figure 1 or the polypeptide encoded by the
cDNA of the deposited clone. Such nucleotide variants
include deletion variants, substitution variants and addition
or insertion variants.
As riereinabove indicated, the polynucleotide may have a
coding sequence which is a naturally occurring allelic
variant of the coding sequence shown in Figure 1 or of the
coding sequence of the deposited clone. As down in the art,
an allelic variant is an alternate form of a polynucleotide
sequence which may have a substitution, deletion or addition
_g_
CA 02413012 2002-O1-03
of one or more nucleotides, which does not substantially
alter the function of the encoded polypeptide.
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 pQB-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 hemagglutinin protein
(Wilson, I., et al., Cell, 37:767 (1984)).
The tetzn ~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
s egment s ( exons ) .
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 promotor regions, exons, and introns. An
example of a screen comprises isolating the cooling region of
the gene'Y 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.
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CA 02413012 2002-O1-03
The present invention further relates to
polynucleotides which hybridize to the hereinabove-described
sequences if there is at least 70%, preferably at least 90%,
and more preferably at least 95% identity between the
sequences. The present invention particularly relates to
polynucleotides which hybridize under stringent conditions to
the hereinabove-described polynucleotides. As herein used,
the term "stringent conditions" means hybridization will
occur only if there is at least 95% and preferably at least
97% identity between the sequences. The polynucleotides
which hybridize to the hereinabove described polynucleotides
in a preferred embodiment encode polypeptides which either
retain substantially the same biological function or activity
as the mature polypeptide encoded by the cDNAs of Figure 1
(SBQ ID N0:1) or the deposited cD~1(s) .
Alternatively, the pol.ynucleotide may have at least 20
bases, preferably 30 bases, and more preferably at least 50
bases which hybridize to a polynucleotide of the present
invention and which has an identity thereto, as hereinabove
described, and which may or may not retain activity. For
example, such polynucleotides may be employed as probes for
the polynucleotide of S$Q ID NO:1, for example, for recovery
of the polynucleotide or as a diagnostic probe or as a PCR
primer . ' '
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% identity to a
polynucleotide which encodes the polypeptide of SSQ ID N0:2
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.
The deposits) referred to herein will be maintained
under the terms of the Budapest Treaty on the International
Recognition of the Deposit of Micro-organisms for purposes of
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CA 02413012 2002-O1-03
I'at~nt Pro;.~~d~.~c-e. 'I'1m~s<:, .:l~~a,:>it_; aTw pr~widPd m<rrel.y a:~
c,onv<-~ni.enrc~
to tt~nse c~C ::kill i;i tlm ,-artair3 arE~ not an admi:~sicw t_ti.at ti
~'i<y~o;~~.t. i: r-c ci~m~~~i un<.3c-i ~:t=ion 38.1 (Z1 ~~f th~,e Puter~t Act.
The sequence of the polynucleotides contained in the
deposited materials, as well. as the amino acid sequence of
the polypeptides encoded thereby, are
controlling in the event of any conflict
with any description of sequences herein. A license may be
required to make, use or sell the deposited materials, and
no such license is hereby granted.
The present invention further relates to a polypeptides
which have the deduced amino acid sequence of Figure 1 or
which has the amino acid sequence encoded by the deposited
cDNA, as well as fragments, analogs and derivatives of such
polypeptide.
The terms "fragment," "derivative" and "analog" when
referring to the polypeptide of Figure 1 or that encoded by
the deposited cDNA, means a polypeptide which retains the
conserved motif of VSGF proteins as shown in Figure 2 and
essentially the same biological function or activity.
The polypeptides of the present invention may be
recombinant polypeptides, natural polypeptides or synthetic
polypeptides, preferably recombinant polypeptides_
The fragment, derivative or analog of the-polypeptide
of Figure 1 or that encoded by the deposited cDNA 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 -lif a of the
polypeptide (for example, polyethylene glycol), or (iv) one
in which the additional amino acids are fused to the mature
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CA 02413012 2002-O1-03
polypeptide or (v) one in which comprises fewer amino acid
residues shown in SgQ ID No. 2 and retains the conserved
motif and yet still retains activity characteristic of the
VBGF 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.
The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
The tez~m "isolated" means that the material is removed
from its original environment (e. g., the natural environment
if it is naturally occurring). For example, a naturally-
occurring polynucleotide or polypeptide present in a living
animal is not isolated, but the same polynucleotide or
polypeptide, separated from some or all of the coexisting
materials in the natural system, is isolated. Such
polynucleotides could be part of a vector and/or such
polynucleotides or polypeptides could be part of a~
camposition, and still be isolated in that such vector or
composition is not part of its natural environment.
The polypeptides of the present invention include the
polypeptide of S8Q ID N0:2 tin particular the mature
polypeptide) as well as polypeptides which have at least 70%
similarity (preferably at'~least 70% identity) to the
polypeptide of SBQ ID N0:2 and more preferably at least 90%
similarity (more preferably at least 95% identity) to the
polypeptide of SBQ ID N0:2 and still more preferably at least
95% similarity (still more preferably at least 90% identity)
to the polypeptide of SBQ ID N0:2 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
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CA 02413012 2002-O1-03
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 present invention may be used to
synthesize full-length polynucleotides of the present
invention.
The present invention also relates to vectors which
include polynucleotides of the present invention, host cells
which are genetically engineered with vectors of the
invention and the production of polypeptides of the invention
by recombinant techniques.
Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this
invention which may be, for example, a cloning vector or an
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 transfoxznants or amplifying the VBGF2
genes of the present 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 ordinarily skilled artisan.
The polynucleotides of the present invention may be
employed for producing polypeptides by recombinant
techniques. Thus, for example, the polynucleotide may be
included in any one of a variety of expression vectors for
expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives
of SV40; bacterial plasmids; phage DNA; baculovirus; yeast
plasmids; vectors derived from combinations of plasmids and
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CA 02413012 2002-O1-03
phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox
virus, and pseudorabies. However, any other vector may be
used as 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 tar , the phage lambda PL
promoter and other promoters known to control expression of
genes in prokaryotic or eukaryotic cells or their viruses.
The expression vector also contains a ribosv~ne 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
one or more selectable marker genes to provide a phenotypic
trait for selection of transformed host cells such as
dihydrofolate reductase or neomycin resistance for eukaryotic
cell culture, or such as tetracycline or ampicillin
resistance in E, coli.
The vector containing the appropriate DNA sequence as
hereinabove 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.
As representative examples of appropriate hosts, there
may be mentioned: bacterial cells, such as B. coli,
Streptomvces, Salmonella tmhimurium; fungal cells, such as
yeast; insect cells such as Drosophila S2 and Snod~tera Sf9;
animal cells such as CHO, COS or Bowes melanoma;
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CA 02413012 2002-O1-03
adenoviruses; plant cells, etc. 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, 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: pQB70, pQB60, pQB-9 (Qiagen),
pBS, pDlO, phagescript, psiX174, pBluescript SK, pBSRS,
pNHBA, pNHl6a, pNHlBA, pNH46A (Stratagene); ptrc99a, pKK223-
3, pKR233-3, pDR540, pRITS (Phartnacia). Bukazyotic: pWLNBO,
pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG,
pSVL (Pharmacia). However, any other plasmid or vector may
be used as long as they are replicable and viable in the
host.
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 lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp.
Bukaryatic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from 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 constructs. The
host cell can be a higher eukaryotic cell, such as a
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CA 02413012 2002-O1-03
mammalian 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. Introduction of the construct into the host
cell can be effected by calcium phosphate transfection, D&AS-
Dextran mediated transfection, or electroporation. (Davis,
L., Dibner, M., Battey, I., Basic Methods in Molecular
Biology, (1986) ) .
The constructs in host cells can be used in a
conventional manner to produce 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 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
prokaryotic and eukaryotic hosts are described by Sambrook,
et al., Molecular Cloning: A Laborato~cy Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989).
Transcription of the DNA encoding the polypeptides of
the present invention by higher eukaryotes is increased by
inserting an enhancer sequence into the vector. Bnhancers
are cis-acting elements of DNA, usually about from 10 to 300
by that act on a promoter to increase its transcription.
Examples including the SV40 enhancer on the late side of the
replication origin by 100 to 270, a cytomegalovirus early
promoter~enhancer, the polyotaa enhancer on the late side of
the replication origin, and adenovirus enh~ancers.
Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transfornzation of the host cell, e.g., the ampicillin
resistance gene of 8. coli and S. cerevisiae TRP1 gene, and
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a promotes 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 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 dr 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. coli,
Bacillus subtilis, Salmonella ty~himurium and various species
within the genera Pseudomonas, Streptomyces, and
Staphylococcus, although others may also be e~uployed 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 example,
pKR223-3 (Pharinacia Fine Chemicals, Uppsala, Sweden) and G8M1
(Promega Biotec, Madison, WI, USA). These pBR322 ~backbone"
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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 induced 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.
Microbial cells employed in expression of proteins can
be disrupted by any convenient method, including freeze-thaw
cycling, sonication, mechanical disruption, or use of cell
lysing agents, such methods are well know to those skilled in
the art.
Various mammalian cell culture systems can also be
employed to express recrnabinant protein. Bxamples of
mammalian expression systems include the COS-7 lines of
monkey kidney fibroblasts, described by Gluzm,a~n, Cell, 23:175
(1981), and other cell lines capable of expressing a
compatible vector, for example, the C127, 3T3, CHO, HeLa and
BHR cell lines. Mammalian expression vectors will comprise
an origin of replication, a suitable promoter and enhancer,
and also any necessary - ribosome bind:tng sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the
SV40 splice, and polyadenylation sites may be used to provide
the required nontranscribed genetic elements.
Thev polypeptides can be recovered and purified from
recombinant cell cultures by methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or
cation exchange chromatography, phosphocellulose
chromatography, hydrophobic interaction chromatography,
affinity chromatography, hydroxylapatite chromatography and
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lectin chromatography. Protein refolding steps can be used,
as necessary, in completing configuration of the mature
protein. Finally, high perfozmance 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 or may be non-glycosylated.
Polypeptides of the invention may also include an initial
methionine amino acid residue_
As shown in Figures 8 and 9, the VBGF2 polypeptide of
SBQ ID No. 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 VBGF2 nucleic acid sequence
encoding this polypeptide wherein 20 ~Cg of RNA from several
human tissues were probed with "P-VSGF2, illustrates that
this protein is actively expressed in the heart and lung
which is further evidence of mitogenic activity.
Accordingly, VBGF2 may be employed ~o promote
angiogenesis, for example, to stimulate the growth of
transplanted tissue where coronary bypass surgery is
perfozmed. V8GF2 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 desired. VBGFZ may be employed to
treat full-thickness wounds such as dermal ulcers, including
pressure sores, venous ulcers, and diabetic ulcers. In
addition, vBGF2 may be employed to treat full-thickness burns
and injuries where a skin graft or flap is used to repair
such burns and injuries. VSGF2 may also be employed for use
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in plastic surgery, for example, for the repair of
lacerations from trauma and cuts in association with surgery.
Along these same lines, vBGF2 may be employed to induce
the growth of damaged bone, periodontium or ligament tissue.
V8GP2 may also be employed for regenerating supporting
tissues of the teeth, including cementum and periodontal
ligament, that have been damaged by disease and trauma.
Since angiogenesis is important in keeping wounds clean
and non-infected, VBGF2 may be employed in association with
surgery and following the repair of cuts . It may also be
employed for the treatment of abdominal wounds where there is
a high risk of infection.
VBGF2 may be employed for the promotion of
endothelialization in vascular graft surgery. In the case of
vascular grafts using either transplanted or synthetic
material, VBGF2 can be applied to the surface of the graft or
at the junction to promote the growth of vascular endothelial
cells_ VBGF2 may also be employed to repair damage of
myocardial tissue as a result of myocardial infarction.
VBGF2 may also be employed to repair the cardiac vascular
system after ischemia. VBGF2 taay also be employed to treat
damaged vascular tissue as a result of coronary artery
disease and peripheral and (s1S vascular disease.
VBGF2 may also be employed to coat artificiar~prostheses
or natural organs which are to be transplanted in the body to
minimize rejection of the transplanted material and to
stimulate vascularization of the transplanted materials.
v~8GF2 may also be employed for vascular tissue repair,
for example, that occurring during arteriosclerosis and
required following balloon angioplasty where vascular tissues
are damaged.
VBGF2 nucleic acid sequences and vBGF2 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
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treat human disease. For example, VBGF2 may be ec~loyed 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 VBGF2 gene may be used as
a hybridization probe for a cDNA library to isolate other
genes which have a high sequence similarity 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
genamic clone or clones that contain the complete VBGF2 gene
including regulatory and promotor regions, exons, and
introns. An example of a screen comprises isolating the
coding region of the VBGF2 gene by using the known DNA
sequence to synthesize an oligonucleotide probe. Labeled
oligonucleotides having a sequence couiplementaxy 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.
This invention provides methods for identification of
VSGF2 receptors. The gene encoding the receptor can be
identified by numerous methods known to those of skill in the
art, for example, ligand panning and FRCS 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
VBGF2, 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 VEGF2. Transfected cells which
are grown on glass slides are exposed to labeled VBGF2.
VBGF2 can be labeled by a variety of means including
iodination or inclusion of a recognition site for a site-
specific protein kinase. Following fixation a.nd incubation,
the slides are subjected to autoradiographic analysis.
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Positive pools are identified and sub-pools are 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 VBGF2 can be photoaffinity linked with cell membrane
or extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGB and exposed to X-
ray film. The labeled complex containing VBGF2 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.
This invention is also related to a method of screening
compounds to identify those which are VBGF2 agonists or
antagonists. An example of such a method takes advantage of
the ability of vSGF2 to significantly stimulate the
proliferation of human endothelial cells in the presence of
the comitogen Con A. 8ndothelial 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 37°C, cultures are pulsed with 1 ~Ci of
' [H] thymidine (5 Cijmmol; 1 Ci = 37 BGq; N8N) for a sufficient
time to incorporate the '[H) and harvested onto glass fiber
filters (Cambridge Technology, Watertown, MA). Mean '[H]-
thymidine incorporation (rpm) of triplicate cultures is
determined using a liquid scintillation counter (Beckman
Instruments, Irvine, CA). Significant '[H]thymidine
incorporation, as compared to a control assay where the
compound is excluded, indicates stimulation of endothelial
cell proliferation.
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To assay for antagonists, the assay described above is
performed and the ability of the compound to inhibit
' IH] thymidine incorporation in the presence of VBGF2 indicates
that the compound is an antagonist to VBGF2. Alternatively,
VSGF2 antagonists may be detected by combining VBGF2 and a
potential antagonist With membrane-bound VBGF2 receptors or
recombinant receptors under appropriate conditions for a
competitive inhibition assay. V8GF2 can be labeled, such as
by radioactivity, such that the number of VEGF2 molecules
bound to the receptor can determine the effectiveness of the
potential antagonist.
Alternatively, the response of a known second messenger
system following interaction of V8GF2 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 VBGF2 receptor is
incubated with labeled VBGF2 in the presence of the compound.
The ability of the compound to enhance or block this
interaction could then be measured.
Potential VBGF2 antagonists include an antibody, or in
some cases, an oligonucleotide, which bind to the polypeptide
and effectively eliminate VSGF2 function. Alternatively, a
potential antagonist may be a closely related protein which
binds to VSGF2 receptors, however, they are inactive forms of
the polypeptide and thereby prevent the action of tTBGF2.
Examples of these antagonists include a negative dominant
mutant of the VBGF2 polypeptide, for example, one chain of
the hetero-dimeric form of VBGF2 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 VBGF2 which is capable of interacting
with another dimer to form wild type VSGF2, however, the
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resulting homo-dimer is inactive and fails to exhibit
characteristic VEGF activity.
Another potential VBGF2 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 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 al., Nucl. Acids
Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988);
and Dervan et al., Science, 251: 1360 (1991)), thereby
preventing transcription and the production of VBGF2. The
antisense RNA oligonucleotide hybridizes to the mRNA in viva
and blocks translation of the mRNA molecule into the VSGP2
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
viva to inhibit production of VBGF2.
Potential VEGF2 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. $xamples
of small molecules include but are not limited to small
peptides or peptide-like molecules.
The antagonists may be employed to treat limit
angiogenesis necessary for solid tumor metastasis.
The mRNA encoding for VEGF2 is found to be expressed at
moderate levels in~at least two breast tumor cell lines which
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is indicative of the role of VBGF2 polypeptides in the
malignant phenotype. 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
inf lamination caused by increased vascular permeability. In
addition to these disorders, the antagonists may 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.
The V8GF2 polypeptides 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 includes but is not limited to saline, buffered
saline, dextrose, water, glycerol, 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 compounds.
The pharmaceutical co~apositions may be administered in
a convenient manner such as by the topical, intravenous,
intraperitoneal, intramuscular, intratumor, subcutaneous,
intranasal or intradermal. routes. The pharmaceutical
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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 ug/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 ~Cg/kg to about 1 mg/kg body
weight daily, taking into account the routes of
administration, symptoms, etc.
The VBGFZ 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."
Thus, for example, cells such as bone marrow cells may
be engineered with a polynucleotide (DNA or RNA) encoding for
the polypeptide ex vavo, 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 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 vi vv. 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 example, an adenovirus,
which may be used to engineer cells in vivo after combination
with a suitable delivery vehicle.
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Retroviruses from which the retroviral plasmid vector
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.
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.,
Biotechnicrues, Vol. 7, No. 9, 980-990 (1989), or any other
promoter (e. g., cellular promoters such as eukaiyotic
cellular promoters including, but not limited to, the
histone, pol III, and ~-actin promoters). Other viral
promoters which may be employed include, but are not limited
to, adenovirus promoters, thymidine kinase (TK) promoters,
and B19 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 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 hetorologous promoters,
such as the cytomegalovirus (Q~iV) 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 ~-actin promoter; and human growth hormone
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CA 02413012 2002-O1-03
promoters. The promoter also may be the native promoter
which controls the gene encoding the polypeptide.
The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. gxamples
of packaging cells which may be transfected include, but are
not limited to, the PS501, PA317, ~-2, ~-AM, PA12, T19-14X,
VT-19-i7-H2, ~CRB, ~LCRIP, GP+B-86, GP+envAml2, and DAN cell
lines as described in Miller, Human Gene Therapy, Vol. 1,
pgs. 5-14 (1990),
The vector may transduce the packaging
cells through any means known in the art. Such means
include, but are not limited to, electroporation, the use of
liposomes, and CaPO, precipitation. In one alternative, the
retroviral plasmid vector may be encapsulated into a
liposame, 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 vi yo. The transduced eukaryotic cells will
express the nucleic acid sequences) encoding the
polypeptide. 8ukaryotic cells which may be transduced
include, but are not limited to, embryonic stem cells,
embryonic carcinoma cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothelial cells, and bronchial epithelial cells.
This invention is also related to the use of the V8GF2 gene
as part of a diagnostic assay for detecting diseases or
susceptibility to diseases related to the presence of
mutations in VBGF2 nucleic acid sequences.
Individuals carrying mutations in the V$GF2 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
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autopsy rnaterial_ 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 VSGF2 can be used to identify and analyze vBGP2
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 VBGF2
RNA or alternatively, radiolabeled V8GF2 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 specific melting or partial
melting temperatures (see, e:g., Myers et aI-, Science,
230:1242 (I985)).
Sequence changes at specific locations may also be
revealed by nuclease protection assays, such as RNase and S1
protection ar the chemical cleavage method (e.g., Cotton et
al., PNAS, BSA, 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 (RPLP)) and Southern blotting of genomic DNA.
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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
fox detecting altered levels of tlFGF2 protein in various
tissues since an over-expression of the proteins compared to
nornial control tissue samples may detect the presence of a
disease or susceptibility to a disease, for example, abnormal
cellular differentiation. Assays used to detect levels of
vBGF2 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 BLISA assay (Coligan, et
al., Current Protocols in Immunology, 1(2), Chapter 6,
(1991)) initially comprises preparing an antibody specific to
the ~TSGF2 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 dish, that binds the proteins in the sample. Any
free protein binding sites on the dish axe then covered by
incubating with a non-specifi:c-protein, such as, bovine serum
albumen. Next, the monoclonal antibody is incubated in the
dish during which time the monoclonal antibodies attach to
any V8GF2 proteins attached to the polystyrene dish. All
unbound monoclonal antibody is washed out with buffer. The
reporter antibody linked to horseradish peroxidase is placed
in the'dish resulting in binding of the reporter antibody to
any monoclonal antibody bound to VBGF2. Unattached 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 VBGF2
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protein present in a given volume of patient sample when
compared against a standard currre.
A competition assay may be employed wherein antibodies
specif is to VEGF2 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 VSGFZ in the sample.
A "sandwich" assay is similar to an BLISA assay. In a
"sandwich" assay VBGF2 is passed over a solid support and
binds to antibody attached to a solid support. A second
antibody is then bound to the VBGF2. 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.
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 polymorphism's) axe 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 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 chramosomes. Only those
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CA 02413012 2002-O1-03
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 f ram 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 hybricli zation, 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 step. This
technique can be used with cDNA as short as 50 or 60 bases.
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. McRusick, Mendelian
Inheritance in Man (availatSle'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 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 same or all of the
affected individuals but not in any nozznal individuals, then
the mutation is likely to be the causative agent of the
disease.
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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 ) .
The polypeptides, their fragments or other derivatives,
or analogs thereof, or cells expressing them can be used as
an immunogen to produce antibodies thereto. These antibodies
can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chimeric, single chain,
and humanized antibodies, as well as Fab fragments, or the
product of an Fab expression library. Various procedures
known in the art may be used for the production of such
antibodies and fragments.
Antibodies generated against the polypeptide
corresponding to a sequence of the present invention can be
obtained by direct injection of the polypeptide into an
animal or by administering the polypeptide to an animal,
preferably a nonhuman. The antibody so obtained will then
bind the polypeptide itself. In this manner, even a sequence
encoding only a fragment of the polypeptide can be used to
generate antibodies binding the whole native polypeptide.
Such antibodies can then be' used to isolate the-polypeptide
from tissue expressing that polypeptide. Far preparation of
monoclonal antibodies, any technique which provides
antibodies produced by continuous cell line cultures can be
used. Examples include the hybridoma technique (Rohler and
Milstein, 1975, Nature, 256:495-497), the trioma technique,
the human B-cell hybridoma technique (Rozbor et al., 1983,
Immunology Today 4:72), and the BBV-hybridoma technique to
produce human monoclonal antibodies (Cole, et al., 1985, in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,
PP. 77-96).
-34-
CA 02413012 2002-O1-03
Techniques described for the production of single chain
antibodies (U. S. Patent 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products
of this invention. Also, transgenic mice may be used to
express humanized antibodies to immunogenic polypeptide
products of this invention.
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 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 ~g of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20 ~1
of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~Cg 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
-35-
CA 02413012 2002-O1-03
manufacturer. Incubation times of about 1 hour at 37°C 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).
"Oligonucleotides" refers 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
dephosphoxylated.
"Ligation" refers to the process of forming
phosphodiester bonds between two double stranded nucleic acid
fragments (Maniatis, T., et al., Id., p. 1461. Unless
otherwise provided, ligation may be accomplished using Down
buff ers and conditions with 10 units of T4 DNA ligase
("ligase") per 0.5 ug of approximately equimolar amounts of
the DNA fragments to be ligated.
Unless otherwise stated,' t'ransfornnation was ~5erformed as
described by the method of Graham, F . and Van der Bb, A. ,
Virology, 52:456-457 (1973).
Bxample 1
Expression pattern of VBGF2 in human tissues and breast
cancerrcell lines
Northern blot analysis was carried out to examine the
levels of expression of the VBGF2 gene in human tissues and
human breast cancer cell lines. Total cellular Rrm samples
were isolated with RNAzol'~' H system (Biotecx Laboratories,
Inc.). About 10 ~Cg of total RNA isolated from each breast
-36-
CA 02413012 2002-O1-03
tissue and cell line specified was separated on 1% agarose
gel and blotted onto a nylon filter, (Molecular Cloning,
Sambrook Fritsch, and Maniatis, Cold Spring Harbor Press,
1989). The labeling reaction was done according to the
Th,
Stratagene Cloning Systems, Inc., Prime-It kit with 50 ng DNA
fragment. The labeled DNA was purified with a Select-G-SO
column from 5 Prime -- 3 Prime, Inc, Boulder, CO, USA. The
filter was then hybridized with radioactively labeled full
length VEGF2 gene at 1,000,000 cpm/ml in 0.5 M NaPO,and 7 %
SDS overnight at 65°C. After washing 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.
Bxample 2
Cloning,, and expression of V8GF2 using. the baculovirus
expression system
The DNA sequence encoding the V8GP2 protein without 46
amino acids at the N-terminus, see ATCC ; 971~~ 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 (S~Q ID N0:4) and
contains a BamHl restriction enzyme site (in bold) and 17
nucleotide nucleotide sequence complementary to the 5'
sequence of VEGF2 (nt. 150-166).
The 3' primer has the sequence GATC TCT AGA TTA GCT CAT
TTG TGG TCT (S$Q ID N0:5) and contains the cleavage site for
the restriction enzyme Xbal and 18 nucleotides complementary
to the 3' sequence of V8GF2, including the stop codon and 15
nt sequence before stop codon.
The amplified sequences were isolated from a 1% agarose
Th1
gel using a cottmiercially available kit ("Geneclean, " BIO 101,
Inc., La Jolla, CA). The fragment was then digested with the
-37-
CA 02413012 2002-O1-03
endonuclease BamAl and Xbal and then purified again on a I%
agarose gel. This fragment was licrated to pAcGP67A
baculovirus transfer vector (PHaxmingen) at the BamHl and
XbaI sites. Through this ligation, VBGF2 eDNA was cloned in
frame with the signal sequence of baculovirus gp67 gene and
was located at the 3' end of the signal sequence in the
vector. This is designated pAcGP67A-VBGF2.
To clone V$GF2 with the signal sequence of gp67 gene to
the pRGi vector for expression, V$GF2 with the signal
sequence and some upstream sequence were excised from the
pAcGP67A-Y8GF2 plasmid at the Xho restriction endonuclease
site located upstream of the VgGF2 cDNA and at the Xbal
restriction endonuclease site by Xhol anal XbaI restriction
enzyme. This fragment was separated from the rest of vector
TM
on a 1% agarose gel and was purified using "Geneclean" kit.
It was designated F2_
The PRG1 vector (modification of pVL941 vector) is used
for the expression of the V$GF2 grotein using the baculovirus
expression system (for review seep Summers, M.D. and Smith,
G.B. 1987, A manual of methods fvr baculovirus vectors and
insect cell culture procedures, Texas Agricultural
$xperimental Station Bulletin No. 1555?. This expression
vector contains the strong polyhedrin promoter of the
Autographs californica nuclear polyhedrosis virus (Aci~R~7PV)
followed by the recognition sites far the restriction
endonucleases BamHl, Smal, Xbal, BglII and Asp718. A site
for restriction endonuclease Xhol is located upstream of
BamHl site. The sequence between Xhol and BamHI is the same
as that in PAcGp67A (static on tape) vector. The
polyadeilylation site of the simian virus (8V)40 is used for
efficient polyadenylation. For an easy selection of
recombinant virus the beta-galactosidase gene from $.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
-3B-
CA 02413012 2002-O1-03
viral sequences for the cell-mediated homologous
recombination of cotransfected wild-type viral DNA. Many
other baculovirus vectors could be used in place of pRGI such
as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D.,
Virology, 170:31-39).
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 1% agarose gel using the commercially
TM
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 AB101 cells were then
transformed and bacteria identified that contained the
plasmid (pBac gp67-V8GF2) with the VBGF2 gene using the
enzymes BamHl and Xbal. The sequence of the cloned fragment
was confirmed by DNA sequencing.
5 ug of the plasmid pHac gp67-V8GF2 was cotransfected
with 1.0 ~g of a commercially available linearized
baculovirus ("HaculoGold" baculovirus DNA", Pharmingen, San
Diego, CA.) using the lipofection method (Felgner et al.
Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
lug of HaculoGold° virus DNA and 5 ug of the plasmid
pBac gp67-vBGF2 were mixed in a sterile well of a microtiter
plate containing 50 u1 of serum free Grace's medium (Life
Technologies Inc., Gaithersburg, MD). Afterwards 10 ~.1
Lipof ectin plus 90 u1 Grace' s medium were added, mixed and
incubated for 15 minutes at zoom 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
-39-
CA 02413012 2002-O1-03
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 user's guide for insect cell culture and baculovirology
distributed by Life Technologies Inc., Gaithersburg, page 9-
20) .
Four days after the serial dilution, the vinzs was added
to the cells, blue stained plaques were picked with the tip
of an Bppendorf pipette. The agar containing the recombinant
T'Af
viruses was then resuspended in an Sppendorf tube containing
200 ~.1 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 seeded in 35 mm
dishes . Four days later the supernatants of these culture
dishes were harvested and then stored at 4°C.
Sf 9 cells were grown in Grace' s medium supplemented with
10% heat-inactivated FHS. The cells were infected with the
recombinant baculovizus V-gp67-V8GF2 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 ~eCi of 'SS-methionine and 5 uCi '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-PAGB and autoradiography.
Protein from the medium and cytoplasm of the Sf9 cells
was analyzed by SDS-PAG$ under reducing and non-reducing
conditions. See Figure 4. The medium was dialyzed against
50 mM M$S, pH 5.8. Precpitates were obtained after dialysis
and resuspended in 100 mM NaCitrate, pH 5Ø The resuspended
-40-
CA 02413012 2002-O1-03
precipitate was analyzed again by SDS-PAGE and was stained
with Coomassie Brilliant Blue. See Figure 5.
The medium supernatant was also diluted 1:10 in 50 mM
MES, pH 5.8 and applied to an SP-650M column (1.0 x 6.6 cm,
Toyopearl) at a flow rate of 1 ml/min. Protein was eluted
with step gradients at 200, 300 and 500 mM NaCl. The V'EGF2
was obtained using the elution at 500 mM. The eluate was
analyzed by SDS-PAGE in the presence or absence of reducing
agent, J3-mercaptoethanol and stained by Cooctunassie Brilliant
Blue. See Figure 6.
Example 3
Bxpression of Recombinant VEGF2 in COS cells
The expression of plasmid, VBGF2-HA is derived from a
vector pcDNAI'Amp (Invitrogen) containing: 1) SV40 origin of
replication, 2) ampicillin resistance gene, 3) E.coli
replication origin, 4) CMV promoter followed by a polylinker
region, an SV40 intron and polyadenylation site. A DNA
fragment encoding the entire V$GF2 precursor and a HA tag
fused in frame to its 3' end was 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 (I. Wilson, H.
Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner,
1984, 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 HA epitope.
The plasmid construction strategy is described as
follows:
The DNA sequence encoding V$GF2, ATCC # 97149 was
constructed by PCR using two primers: the 5' primer (CGC GGA
TCC ATG ACT GTA CTC TAC CCA) (SEQ ID N0:6) contains a BamHl
site followed by 18 nucleotides of VEGF2 coding sequence
starting from the initiation colon; the 3' sequence (CGC TCT
-41-
CA 02413012 2002-O1-03
AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CTC GAG GCT CAT
TTG TGG TCT 3') (SBQ ID N0:7) contains complementary
sequences to an XbaI site, HA tag, Xhol site, and the last 15
nucleotides of the VgGF2 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 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 BamH1 and XbaI restriction enzyme and
ligated. The ligation mixture was transformed into E. coli
strain SUR.B (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 VBGF2, COS cells were
transfected with the expression vector by D8A8-DSX'I'RAN method
(J. Sambrook, B. Fritsch, T. Maniatis, Molecular Cloning: A
Laboratory Manual, Cold Spring Laboratory Press, (1989)).
The expression of the VSGF2-HA protein was detected by
radiolabelling and immunoprecipitation method (B. Harlow, 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 NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC,
50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)).
Hoth cell lysate and culture media were precipitated with an
HA specific monoclonal antibody. Proteins precipitated were
analyzed on 15% SDS-PAGB gels.
Example 4
The effect of partially-purified VBGF2 protein on the mrowth
of vascular endothelial cells
-42-
CA 02413012 2002-O1-03
On day 1, human umbilical vein endothelial cells (HUV'BC)
were seeded at 2-5x10' cells/35 nun dish density in M199 medium
containing 4% fetal bovine serum (FBS), 16 units/ml heparin,
and 50 units/m1 endothelial cell growth supplements ($CGS,
Biotechnique, Inc.). On day 2, the medium was replaced with
M199 containing 10% FBS, 8 units/ml heparin. VBGP2 protein
of SgQ ID NO. 2 minus the initial 45 amino acid residues,
(VRGF) and basic FGF (bFGF) were added, at the concentration
shown . On days 4 t~ 6 , the medium was replaced . On day 8 ,
rM
cell number was determined with a Coulter Counter (See Figure
8) .
8xample 5
The effect of urified VBGF2 rotein on the rowth of
vascular endothelial cells
On day 1, human umbilical vein endothelial cells (HLTVBC)
were seeded at 2-5 x 10' cells/35 mm dish density in M199
medium containing 4% fetal bovine serum (FBS), 16 units/ml
heparin, 50 unitslml. endothelial cell growth supplements
(EGGS, Biotechnique, Inc.). On day 2, the medium was
replaced with M199 containing 10% FBS, 8 units/ml heparin. __
Purified V8GF2 protein of SBQ ID No. 2 minus initial 45 amino
acid residues was added to the medium at this point. On days
4 & 6, the medium was replaced with fresh -medium and
supplements. On day B, cell number was determined with a
!'M
Coulter Counter (See Figure 9).
Bxample 6
$xpression via Gene Theraxw
Fibrablasts 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 room
-43-
CA 02413012 2002-O1-03
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 in
culture, a monolayer of fibroblasts emerge. The monolayer is
trypsinized and scaled into larger flasks.
pNiV-7 (Kirsc~neier, P.T. et al, DNA, 7:219-25 (1988)
flanked by the long texmi.nal repeats of the Moloney marine
sarcoma virus, is digested with BcoRI 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 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
BcoRI site and the 3' primer further includes a HindIII site.
Bqual quantities of the Moloney marine sarcoma virus linear
backbone and the amplified BcoRI 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 ligatioir mixture is
used to transform bacteria HBI01, which are then plated onto
agar-containing kanamycin far the purpose of confirming that
the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+ams2 packaging cells are
grown in tissue culture to confluent density in Dulbecco's
Modified Bagles Medium (DMBM) with l0% 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).
-44-
CA 02413012 2002-O1-03
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
Tnf
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
TM
on cytodex 3 microcarrier beads. The fibroblasts now produce
the protein product.
Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, within the scope of the appended claims, the
invention rnay be practiced otherwise than as particularly .
described.
-45-
CA 02413012 2002-O1-03
_~L~~:~UENCE LIS 1'1N(
(2) GENERAL INFORMATION:
(i) APPLICAb7T: f-Iuman Genome Sciences, Inc.
(ii) TITLE OF INVENTION: Human Vascular Endothelial Growth
Fr~~c.tor 2
(iii) NUMBER OF SEC_ltJENCES: 10
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MBM & C0.
(B) STREET: P.O. f30X 8~J9, STATION F.
(C) CITY: OTTAWA
(D) PROVINCE: ONTAR10
(E) COUNTRY: CANADA
(E') POSTAL CODE: fSlf? 5P9
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE;: Flob~py disk
(B) COMPUTER: IBM F;. compatible ,,M
(C) OPERATING :SYSTEM: PC-DOS/MS-DOS
(D) SOf~'TWARE: ~'atentIni~'Ver. 2.0
(vi) CURRENT APPLICA'T'ION DATA:
(A) APPLICATION NUMBER: 2,224,093
(B) FILING DATE: 6--JUNE-1996
(C) CLASSIFICATION:
(vii) PRTOR APPLICATION DATA:
(A) APPT~ICATION NUMBER: 08/465,968
(B) FILING DA'Z'E: 6-JUNE-7_995
(C) CLASSIFICATION;
4(i
CA 02413012 2002-O1-03
(Viii) ATTORNEYiA~ENT I"~Ir'ORMF_~~IC?rv:
,A) N~.~tE: St~lAiTl, Mar~~~ a~:=
(B) RE~CISTR.'=.'~i'IC~J ?~?CTi~TF'~,Y,: 1Q~"2o
(Cj REFERETT~~E/DO~=YET ~lr.~i~~BER: 30c-1.27
(ix) TELECOMMUNICATION INF~:RMA'='ION:
(A) TELEPHONE: 613/5:7-0762
(F) TELEFAX: 61~~/563--1671
(2) INFORMATION FOR SEQ Iii NO:1:
SEQUENCE CHAP,ACTERISTICS:
(A) LENGTH: 1674
(B) TYPE: NUCLEIC ACID
(C) STRANDED:VES'.~ : SNGT~~
(D) TOPOLc.~GY: L.LNEAr
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCR~vTTON: SEQ ID N0:1:
GTCCTTCCAC CATGCACTCG CTGGGCTTCT TCt'C'T'G'TGGC GTGTTCTCTG CTCGCCGCTG 60
CGCTGCTCCC GGGTCCTCGC GAGGCGCCCG CCGCCGCCGC CGCCrTCGAG TCCGGACTCG 120
ACC'r'CTCC-GA CGCGGAGCCC GACGCGGGCG AGGCCACGGC TTATGCAAGC AAAGATCTGG 180
AGGAGCAGTT ACGGTCTGi'G TCCAGTGTAG ATGAACTCAT GACTGTAC~C TACCCAGAAT 240
AZ'rGGAAAAT GTACARGTGT CAGCTAAGGA A~ri~AGGL.TG GCAACATRAC AGAGAACAGG 300
CCAACC'!'CAA CTCAAC-GALA GAACs.AG3~CTA TP,A_RPTT'~'iiC TGC~~CAT TATAATACr~ 360
AGATCITGAA AAGTATTGAT AATGaG'~GGA GAAAGACTCA ATGCATGCCA CGvGAGGTGT 420
GTATACv~TGT GGGuAAGC~IG TTTGCZAGTCG CGACRAACAC CTTCTTTAAA CCTCCA2'GTG 4 8 0
TGTCCGT_CTA ~CAGATGTI~GC> GGTTGCTGCA ATAGTGAGC-~a GCTGCAGTGC ATGAACACCA 540
GCACGAGCTA CCTCAGCAAG AC~uTTATlTG AAATTACAGT GCCTCTCTCT CARGGCCCCA 600
AACCAGTAAC AATCAGT':Z'T GCCAATCA~ CTTCCTGCCG ATGCATGTCT AAAGTGGATG 660
TTTACAGACA AGTTCATTCC ATTATTAGAC GTTCCCTGCC AGCAACACTA CCACAGTGTC 720
AGGCAGCGAA CAAGACCTGC CCCACCAATT ACATGTGGAA TAATCACATC TGCAGATGCC 7B0
TGGCTCAGGA AGATTTTATG TTTTCCTC"uG ATGCTC~GAGA TGACTCAACA GATGGATTCC 840
ATGACATCI'G T~~GACCAAAC AAGG.~~uCI~GG AT~uAAGAGAC 1C~C-TCAGTGT GTCTGCAGAG 900
CwGGCTTCG GCCTGCCAGC TGi'~GACCCC ACAAAGAAGT AGA~F1C TCA'I~GCCAGT 960
GTGTCrGTAA AAAC~,AACTC TTCCCCAGCc AP.'t:~~hGrc;;~: C_AACCG~:GAA TT:"'~:ATGAAA
1020
~(!
CA 02413012 2002-O1-03
ACACATGCCA GTGTCTATG~ AAAAu~IACC=' GCCC'""~.~~-.AAA '=CAACCC:."!'A1080
AATCCTGuAA
AATGTGCCT'G TCAATG~_ACli G:wAU CuIC ACAaATGCTT C'~TA:LA:u,-Ga110
AT~GAp~TCC
ACu.CCAAAC ATGC;aGCTG" TACA~:.CC-GC CATriAC"uAA CCGCr".~G~;~,1200
GC'_rC
CA::.~u'1.~C ATA_AGTr'.sAk GAAC:TGrulC G'I:CCC ':iG~T1'..T'iC-C,1260
CCCAC
AAATr',AGC"':.A ACAil ACT GT'tiTCCr'1GT TCiaTCi:~.'~' ':~I'ATTATGG1320
AAAACI'GTGT
TG CCACAGTA GAACrG~'L'CTis TG~ACAG~ CACCC~'TGTG G "~'CCAT~Ci13
AACAAACACT. Fi
0
FAAGTCTG'T'C TTTCCTCAAC CATGTGGATA A..' i~ITrCAGA tlf.TGG.RIC~;1440
AGCI'CATCTG
CAt~AAGGCCT CTI'GT'AAAGh CTGC:T~ iTCT GCCAP.TGACC AAACr'~C-CCAA1s40
GATTTTCCTC
TTGTGATTTC TTTAA~A.~=. TGA'tI'ATATA A'~'1'TATTTCC ACTAr~r~AATA1560
TTGTTTCfGC
ATTCATTTTT ATAGCAACAA CAAT'T'Cx..'vTAA AACTCAC?'GT GATCAATATT1620
2'1TATATCr'~T
GCl.AAATATG TTTAAAATAA AAT~..A~1ATT C:"_'AT'i'TF':AR AAAT,AAAAAA1674
AAAA
,,, (2) INFORMATTON FOR S$Q ID N0:2:
( i ) SBQff~TCE CHARACTERISTICS
(A) LENGTH: 419 AMINO ACIDS
(B) TYP&: AMINO ACID
(C) STRF_NDEDNFsSS
(D) TOPOLOGY: LINEAR
( ii ) MOLBCULB T'fP8 : PROTEIN
(xi) SBQU&NCS DESCRIPTION: SgQ ID N0:2:
Met His 5er Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala
-45 -40 -3S
Ala Leu Leu P;o Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala~Phe
-30 -25 -20 -15
Glu Ser Gly Lsu Asg Leu Ser Asp Ala Glu Pra Asp A1a Gly Glu Ala
-10 -5 1
Thr Ala Tyr Ala 5er Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser
10 15
Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyz Trp Lys MeC
20 4 25 30
Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Aan Arg Glu Gln
35 50 ~5 50
A,la Asn Leu Aan Ser Arg Thr Glu Glu Thr Ile Lys Phe Aln Aln Ala
c5 60 65
His Tvr Asn Trz Glu Ile Leu Lys Ser Ile Aap Asn Glu Trp A.-g Lys
70 75 80
_~g_
CA 02413012 2002-O1-03
Thr Gln Cars Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe
85 90 95
Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro ors Val Ser Val Tyr
1D0 105 110
Arg Cys Gly Gly Cys Cps Asn Ser Glu Gly Leu Gln Cys Met Asn Thr
115 120 125 130
Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu
135 140 145
Ser Gln Gly Pro Lys Pro Vnl Thr Ile Ser Phe Ala Asn His Thr Ser
150 155 160
Cars Arg Cars Met Ser Lys Leu Asp Val Tyr Arg Gln Val His 5er Ile
165 170 175
Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cars Gln Ala Ala Asn
180 185 190
Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg C'ys
195 200 205 210
Leu Ala Gln G1u Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser
215 220 225
Thr Asp Gly Phe His Asp Ile Cars Gly Pro Asn Lys Glu Leu Asp Glu
230 235 240
Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Axg Pro Ala Ser Cys
245 250 255
Gly Pro His Lys Glu Leu Asp Arg Asn Sex Cys Gln Care Val Cys Lys
260 265 270
Asn Lys Leu Phe Pro Ser Gln Cps Gly Ale Asn Arg Glu Phe Asp Glu
275 280 285 290
Asn Thr Cps Gln Cys Val Cys Lys Arg Thr Cys Pro Rrg Asn Gln Pro
295 300 305
Leu Asn Pro Gly Lys Cps Ala Cps Glu Cys Thr Glu Ser Pro Gln Lys
310 315 320
Cps Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr
325 330 335
Arg Arg Pro Cars Thr Asn Arg Gln Lys Ala Cars Glu Pro Gly Phe Ser
340 345 350
Tyr Ser Glu Glu Val Cps Arg Cars Val Pro Ser Tyr Trp Gln Arg Pro
355 ' 360 365 370
Gln Met Ser
(2) INFORMATION FOR SBQ ID N0:3:
( i ) SBQURNCS CFiAR.ACT$RISTICS
(A) LHNGTH: 14 AMINO ACIDS
(B) TYPH: AMINO ACID
-49-
CA 02413012 2002-O1-03
(C) STRANDSDNBSS.
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPfi: PBPTIDfi
(xi) SBQUBrICB DESCRIPTION: SBQ ID N0:3:
Pro Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys Aszl
5 10
(2) INFORMATION FOR SBQ ID N0:4:
(i) SBQUSNCB (~iARACTBRISTICS
(A) LENGTH: 50 SASB PAIRS
(B) TYPE: NUCLEIC ACID
(C) STR.ANDBDNBSS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPB: oligonucleotide
(xi) SEQUENCE DESCRIPTION: S$Q ID N0:4:
TGTAATACGA CTCACTATAG GGATCCCGCC ATGGAGGCCA CGGCTTATGC 50
(2) INFORMATION FOR SBQ ID NO: S:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 28 BASE PAIRS
(B) TYPB: NUCLEIC ACID
(C) STR.ANDBDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLBCULS TYPE: oligonucleotide
(xi) SBQUBNCB DBSCRIPTION: S8Q ID N0:5:
GATCTCTAGA TTAGCTCATT TGTGGTCT 28
(2) INFORMATION FOR SBQ ID N0:6:
-50-
CA 02413012 2002-O1-03
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 HASB PAIRS
(H) TYPB: NUCLEIC ACID
C ) STR.ANDBDNBS S : S INGLB
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: oligonucleotide
(xi) SBQUBNCB DBSCRIPTION: SBQ ID N0:6:
CGCGGATCCA TGACTGTACT CTACCCA 27
(2) INFORMATION FOR SEQ ID N0:7:
( i ) SBQUBNCE C~~ARACTBRISTICS
(A) LENGTH: 60 HASB PAIRS
(H) TYPE: N1JCLBIC ACID
(C) STRANDBDNBSS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: oligonucleotide
(xi) SBQU~CB DESCRIPTION: SEQ ID N0:7:
CGCTCTAGAT CAAGCGTAGT CTGGGACGTC GTATGGGTAC TCGAGGCTCA TTTGTGGTCT 60
(2) INFORMATION FOR SBQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 196 AMINO ACIDS
(B) TYPB: AMINO ACID
(C) STRANDBDNBSS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SBQ ID N0:8:
Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly Cys Gly Tyr Leu
5 10 15
Ala His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val Ile
-S1-
CA 02413012 2002-O1-03
20 25 30
Glu Arg Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu Gln
35 40 45
Arg Leu Leu Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp
50 55' 60
Thr Ser Leu Arg Ala His Gly Val His Ala Thr Lys His Val Pro
65 70 75
Glu Lys Arg Pro Leu Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu
80 85 90
Ala Val Pro Ala Val Cys Lys Thr Arg Thr Val Ile Tyr Glu Ile
95 100 105
Pro Arg Ser Gln Val Asp Pro Thr Ser Ala Asn Phe Leu Ile Trp
110 115 120
Pro Pro Cys Val Glu Val Lys Arg Cys Thr Gly Cys Cys Asn Thr
125 130 135
Ser Ser Val Lys Cys Gln Pro 5er Arg Val His His Arg Ser Val
140 145 150
Lys Val Ala Lys val Glu Tyr Val Arg Lys Lys Pro Lys Leu Lys
155 160 165
Glu Val Gln Val Arg Leu Glu Glu His Leu Glu Cys Ala Cys Ala
170 175 180
Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp Thr Asp Val
185 190 195
Arg
(2) INFORMATION FOR SBQ ID N0:9:
( i ) SEQUBrICB C'~~ARACTBRISTICS
(A) LgNGTH; 241 AMINO ACIDS
(H) TYPE: AMINO ACID
(C) STRANDSDNBSS:
( D ) TOPOLOGY : LIN'SAR
(ii) MOLBCULB TYPE: PROTBIN
(xi) SBQUENCB DESCRIPTION: SEQ ID N0:9:
Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu
-52-
CA 02413012 2002-O1-03
5 10 15
Arg Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr
20 25 30
Glu Met Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln
35 40 45
Arg Leu Leu His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu
50 55 60
Asp Leu Asn Met Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser
65 70 75
Leu Ala Arg Gly Arg Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu
BO 85 90
Pro Ala Met Ile Ala Glu Cys Lys Thr Arg Thr Glu Val Phe Glu
95 100 105
Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Ala Asn Phe Leu Val
110 115 120
Trp Pro Pro Cys Va1 Glu Val Gln Arg C'ys Ser Gly Cys Cys Asn
125 130 135
Asn Arg Asn Val Gln C'ys Arg Pro Thr Gln Val Gln Leu Arg Pro
140 145 150
Val Gln Val Arg Lys Ile Glu Ile Val Arg Lys Lys Pro Ile Phe
155 160 165
Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu Ala Cys Lys Cps
170 175 180
Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser-Pro Gly Gly
18S 190 195
Ser Gln Glu Gln Arg Ala Lys Thr Pra Gln Thr Arg Val Thr Ile
200 205 210
Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg Lys
215 220 225
Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly
230 235 240
Ala
(2) INFORMATION FOR SBQ ID NO:10:
( i ) S$QLTBNCB C~iARACTBRISTICS
-53-
CA 02413012 2002-O1-03
t F.) L~,ivGTii : ~? 2 Ai~'i<NO ACI DS
(E) '~'YPE: ~MII~fO ACID
(C) SiRANCE~7~dESS:
:;D) TOPOLOC'~: LIT1E5R
(ii) MOLECULE TYPE: Pt~OTEIra
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Men Asn Phe Leu Leu Ssr Trp Val His Trp Ser Leu Ala Leu Leu Leu
i S 10 15
Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly
20 25 30
Gly Gly Gin Asn His sis GIu ~lal. Val Lys Fhe Met Asp Val Tyr G1n
35 40 45
Arg Ser Tyr Cy~~ His Pro Ile G1u Thr Leu Val Asp Ile Phe Gln Glu
50 55 so
Tyr Pro Asp Glu IIe Glu Tyr Il~- Phe Lys Pro Ser Cys Val Pro Leu
6S 70 ~S 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 I1e Gly Glu Met Ser Phe Leu Gln His Asn.Lys Cys
115 1'? 0 12 S
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 1S0 I55 160
Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp
165 17G 1i5
Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys
180 185 190
;his L:eu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn
1~5 200 20S
5:~
CA 02413012 2002-O1-03
Ar Leu Glu Lea Asn Glu Arg T~.
Thr Asp Ser Arg Crs Lys Ala _g GLn
220
210 '
Cys Arg Cys Asp Lys Pro Arg Arg
350
