U.S. patent application number 11/249422 was filed with the patent office on 2006-03-16 for vascular endothelial growth factor 2.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Timothy A. Coleman.
Application Number | 20060057117 11/249422 |
Document ID | / |
Family ID | 22835805 |
Filed Date | 2006-03-16 |
United States Patent
Application |
20060057117 |
Kind Code |
A1 |
Coleman; Timothy A. |
March 16, 2006 |
Vascular endothelial growth factor 2
Abstract
Disclosed are human VEGF-2 polypeptides, biologically active,
diagnostically or therapeutically useful fragments, analogs, or
derivatives thereof, and DNA(RNA) encoding such VEGF-2
polypeptides. Also provided are procedures for producing such
polypeptides by recombinant techniques and antibodies and
antagonists against such polypeptides. Such polypeptides and
polynucleotides may be used therapeutically for stimulating wound
healing and for vascular tissue repair. Also provided are methods
of using the antibodies and antagonists to inhibit tumor
angiogenesis and thus tumor growth, inflammation, diabetic
retinopathy, rheumatoid arthritis, and psoriasis.
Inventors: |
Coleman; Timothy A.;
(Derwood, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
22835805 |
Appl. No.: |
11/249422 |
Filed: |
October 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09921143 |
Aug 3, 2001 |
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11249422 |
Oct 14, 2005 |
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60223276 |
Aug 4, 2000 |
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Current U.S.
Class: |
424/93.2 ;
514/44R |
Current CPC
Class: |
A61P 31/12 20180101;
A61P 9/06 20180101; A61P 29/00 20180101; A61P 37/08 20180101; A61P
37/06 20180101; A61P 27/02 20180101; A61K 48/00 20130101; A61P
17/06 20180101; A61P 9/10 20180101; A01K 2217/05 20130101; A61P
9/00 20180101; A61P 35/00 20180101; C07K 14/52 20130101; A61P 31/10
20180101; A61P 31/04 20180101; A61P 33/00 20180101; A01K 2217/075
20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/093.2 ;
514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Claims
1. A method of stimulating angiogenesis in a mammal, comprising: i.
introducing a first replication-deficient adenovirus vector
comprising a polynucleotide sequence encoding VEGF-B.sub.167 or a
fragment or conservative substitution thereof to said mammal; and
ii. introducing a second replication-deficient adenovirus vector
comprising a polynucleotide sequence encoding VEGF-A or a fragment
or conservative substitution thereof to said mammal, wherein said
first replication-deficient adenovirus vector and said second
replication-deficient adenovirus vector are directly delivered to a
site in said mammal where there is at least one living cell
selected from the group consisting of endothelial cells and cells
proximate to endothelial cells of said mammal, and wherein density
of PECAM-1 positive vessels is increased in said site when compared
to a site untreated or treated with VEGF-B.sub.167 alone or VEGF-A
alone.
2. The method according to claim 1, wherein said at least one
living cell is a vascular cell.
3. The method according to claim 1, wherein said endothelial cells
are microvascular endothelial cells.
4. The method according to claim 1, wherein said endothelial cells
are aortic endothelial cells.
5. The method according to claim 1, wherein said mammal is
murine.
6. The method according to claim 1, wherein said mammal is
human.
7. The method according to claim 1, wherein 10.sup.7 to 10.sup.13
of vector particles of each adenovirus vector are introduced.
8. The method according to claim 1, wherein expression of the
polynucleotide sequence in the first or second vector is driven by
a CMV promoter.
9. A method of stimulating angiogenesis in a mammal, comprising: i.
introducing a first replication-deficient adenovirus vector
comprising a polynucleotide sequence encoding VEGF-B.sub.167 or a
fragment or conservative substitution thereof to said mammal; and
ii. introducing a second replication-deficient adenovirus vector
comprising a polynucleotide sequence encoding VEGF-C or a fragment
or conservative substitution thereof to said mammal, wherein said
first replication-deficient adenovirus vector and said second
replication-deficient adenovirus vector are directly delivered to a
site in said mammal where there is at least one living cell
selected from the group consisting of endothelial cells and cells
proximate to endothelial cells of said mammal, and wherein density
of PECAM-1 positive vessels is increased in said site when compared
to a site untreated or treated with VEGF-B.sub.167 alone or VEGF-C
alone.
10. The method according to claim 9, wherein said at least one
living cell is a vascular cell.
11. The method according to claim 9, wherein said endothelial cells
are microvascular endothelial cells.
12. The method according to claim 9, wherein said endothelial cells
are aortic endothelial cells.
13. The method according to claim 9, wherein said mammal is
murine.
14. The method according to claim 9, wherein said mammal is
human.
15. The method according to claim 9, wherein 10.sup.7 to 10.sup.13
of vector particles of each adenovirus vector are introduced.
16. The method according to claim 9, wherein expression of the
polynucleotide sequence in the first or second vector is driven by
a CMV promoter.
17. A method of stimulating angiogenesis in a mammal, comprising:
i. introducing a first replication-deficient adenovirus vector
comprising a polynucleotide sequence encoding VEGF-B.sub.167 or a
fragment or conservative substitution thereof to said mammal; and
ii. introducing a second replication-deficient adenovirus vector
comprising a polynucleotide sequence encoding VEGF-D or a fragment
or conservative substitution thereof to said mammal, wherein said
first replication-deficient adenovirus vector and said second
replication-deficient adenovirus vector are directly delivered to a
site in said mammal where there is at least one living cell
selected from the group consisting of endothelial cells and cells
proximate to endothelial cells of said mammal, and wherein density
of PECAM-1 positive vessels is increased in said site when compared
to a site untreated or treated with VEGF-B.sub.167 alone or VEGF-D
alone.
18. The method according to claim 17, wherein said at least one
living cell is a vascular cell.
19. The method according to claim 17, wherein said endothelial
cells are microvascular endothelial cells.
20. The method according to claim 17, wherein said endothelial
cells are aortic endothelial cells.
21. The method according to claim 17, wherein said mammal is
murine.
22. The method according to claim 17, wherein said mammal is
human.
23. The method according to claim 1, wherein 10.sup.7 to 10.sup.13
of vector particles of each adenovirus vector are introduced.
24. The method according to claim 1, wherein expression of the
polynucleotide sequence in the first or second vector is driven by
a CMV promoter.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 09/921,143 filed Aug. 3, 2001 (allowed), which claims benefit
of 35 U.S.C. section 119(e) based on copending U.S. Provisional
Application Ser. No. 60/223,276, filed Aug. 4, 2000. Each of the
above-listed applications is herein incorporated by reference in
its entirety.
REFERENCE TO SEQUENCE LISTING ON COMPACT DISC
[0002] This application refers to a "Sequence Listing" listed
below, which is provided as an electronic document on two identical
compact discs (CD-R), labeled "Copy 1" and "Copy 2." These compact
discs each contain the file "PF112P6C1 Sequence Listing.txt"
(created Oct. 12, 2005, byte size=37,046 bytes), which is hereby
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] The present invention relates to newly identified
polynucleotides, polypeptides encoded by such polynucleotides, the
use of such polynucleotides and polypeptides, as well as the
production of such polynucleotides and polypeptides. The
polypeptides of the present invention have been identified as
members of the vascular endothelial growth factor family. More
particularly, the polypeptides of the present invention are human
vascular endothelial growth factor 2 (VEGF-2). The invention also
relates to inhibiting the action of such polypeptides.
[0004] The formation of new blood vessels, or angiogenesis, is
essential for embryonic development, subsequent growth, and tissue
repair. Angiogenesis is also an essential part of certain
pathological conditions, such as neoplasia (i.e., tumors and
gliomas). Abnormal angiogenesis is associated with other diseases
such as inflammation, rheumatoid arthritis, psoriasis, and diabetic
retinopathy (Folkman, J. and Klagsbrun, M., Science
235:442-447(1987)).
[0005] Both acidic and basic fibroblast growth factor molecules are
mitogens for endothelial cells and other cell types. Angiotropin
and angiogenin can induce angiogenesis, although their functions
are unclear (Folkman, J., Cancer Medicine, Lea and Febiger Press,
pp. 153-170 (1993)). A highly selective mitogen for vascular
endothelial cells is vascular endothelial growth factor or VEGF
(Ferrara, N. et al., Endocr. Rev. 13:19-32 (1992)), which is also
known as vascular permeability factor (VPF).
[0006] Vascular endothelial growth factor is a secreted angiogenic
mitogen whose target cell specificity appears to be restricted to
vascular endothelial cells. The murine VEGF gene has been
characterized and its expression pattern in embryogenesis has been
analyzed. A persistent expression of VEGF was observed in
epithelial cells adjacent to fenestrated endothelium, e.g., in
choroid plexus and kidney glomeruli. The data was consistent with a
role of VEGF as a multifunctional regulator of endothelial cell
growth and differentiation (Breier, G. et al., Development
114:521-532 (1992)).
[0007] VEGF shares sequence homology with human platelet-derived
growth factors, PDGFa and PDGFb (Leung, D. W., et al., Science
246:1306-1309, (1989)). The extent of homology is about 21% and
23%, respectively. Eight cysteine residues contributing to
disulfide-bond formation are strictly conserved in these proteins.
Although they are similar, there are specific differences between
VEGF and PDGF. While PDGF is a major growth factor for connective
tissue, VEGF is highly specific for endothelial cells.
Alternatively spliced mRNAs have been identified for both VEGF,
PLGF, and PDGF and these different splicing products differ in
biological activity and in receptor-binding specificity. VEGF and
PDGF function as homo-dimers or hetero-dimers and bind to receptors
which elicit intrinsic tyrosine kinase activity following receptor
dimerization.
[0008] VEGF has four different forms of 121, 165, 189 and 206 amino
acids due to alternative splicing. VEGF121 and VEGF165 are soluble
and are capable of promoting angiogenesis, whereas VEGF189 and
VEGF-206 are bound to heparin containing proteoglycans in the cell
surface. The temporal and spatial expression of VEGF has been
correlated with physiological proliferation of the blood vessels
(Gajdusek, C. M., and Carbon, S. J., Cell Physiol.139:570-579
(1989); McNeil, P. L., et al., J. Cell. Biol. 109:811-822 (1989)).
Its high affinity binding sites are localized only on endothelial
cells in tissue sections (Jakeman, L. B., et al., Clin. Invest.
89:244-253 (1989)). The factor can be isolated from pituitary cells
and several tumor cell lines, and has been implicated in some human
gliomas (Plate, K. H., Nature 359:845-848 (1992)). Interestingly,
expression of VEGF121 or VEGF165 confers on Chinese hamster ovary
cells the ability to form tumors in nude mice (Ferrara, N. et al.,
J. Clin. Invest. 91:160-170 (1993)). The inhibition of VEGF
function by anti-VEGF monoclonal antibodies was shown to inhibit
tumor growth in immune-deficient mice (Kim, K. J., Nature
362:841-844 (1993)). Further, a dominant-negative mutant of the
VEGF receptor has been shown to inhibit growth of glioblastomas in
mice.
[0009] Vascular permeability factor (VPF) has also been found to be
responsible for persistent microvascular hyperpermeability to
plasma proteins even after the cessation of injury, which is a
characteristic feature of normal wound healing. This suggests that
VPF is an important factor in wound healing. Brown, L. F. et al.,
J. Exp. Med. 76:1375-1379 (1992).
[0010] The expression of VEGF is high in vascularized tissues,
(e.g., lung, heart, placenta and solid tumors) and correlates with
angiogenesis both temporally and spatially. VEGF has also been
shown to induce angiogenesis in vivo. Since angiogenesis is
essential for the repair of normal tissues, especially vascular
tissues, VEGF has been proposed for use in promoting vascular
tissue repair (e.g., in atherosclerosis).
[0011] U.S. Pat. No. 5,073,492, issued Dec. 17, 1991 to Chen et
al., discloses a method for synergistically enhancing endothelial
cell growth in an appropriate environment which comprises adding to
the environment, VEGF, effectors and serum-derived factor. Also,
vascular endothelial cell growth factor C sub-unit DNA has been
prepared by polymerase chain reaction techniques. The DNA encodes a
protein that may exist as either a heterodimer or homodimer. The
protein is a mammalian vascular endothelial cell mitogen and, as
such, is useful for the promotion of vascular development and
repair, as disclosed in European Patent Application No. 92302750.2,
published Sep. 30, 1992.
SUMMARY OF THE INVENTION
[0012] 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 VEGF.
[0013] 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.
[0014] In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules comprising
polynucleotides encoding full length or truncated VEGF-2
polypeptides having the amino acid sequences shown in SEQ ID NOS:2
or 4, respectively, or the amino acid sequences encoded by the cDNA
clones deposited in bacterial hosts as ATCC.TM. Deposit Number
97149 on May 12, 1995 or ATCC.TM. Deposit Number 75698 on Mar. 4,
1994.
[0015] The present invention also relates to biologically active
and diagnostically or therapeutically useful fragments, analogs,
and derivatives of VEGF-2.
[0016] 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 the present
invention, under conditions promoting expression of said proteins
and subsequent recovery of said proteins.
[0017] In accordance with yet a further aspect of the present
invention, there are provided processes for utilizing such
polypeptides, or polynucleotides encoding such polypeptides for
therapeutic purposes, for example, to stimulate angiogenesis,
wound-healing, growth of damaged bone and tissue, and to promote
vascular tissue repair. In particular, there are provided processes
for utilizing such polypeptides, or polynucleotides encoding such
polypeptides, for treatment of peripheral artery disease, such as
critical limb ischemia and coronary disease.
[0018] In accordance with yet another aspect of the present
invention, there are provided antibodies against such polypeptides
and processes for producing such polypeptides.
[0019] In accordance with yet another aspect of the present
invention, there are provided antagonists to such polypeptides,
which may be used to inhibit the action of such polypeptides, for
example, to prevent tumor angiogenesis and thus inhibit the growth
of tumors, to treat diabetic retinopathy, inflammation, rheumatoid
arthritis and psoriasis.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] These and other aspects of the present invention should be
apparent to those skilled in the art from the teachings herein.
BRIEF DESCRIPTION OF THE FIGURES
[0024] The following drawings are illustrative of embodiments of
the invention and are not meant to limit the scope of the invention
as encompassed by the claims.
[0025] FIGS. 1A-1E show the full length nucleotide (SEQ ID NO:1)
and the deduced amino acid (SEQ ID NO:2) sequence of VEGF-2. The
polypeptide comprises approximately 419 amino acid residues of
which approximately 23 represent the leader sequence. The standard
one letter abbreviations for amino acids are used. Sequencing was
performed using the Model 373 Automated DNA Sequencer (Applied
Biosystems, Inc.). Sequencing accuracy is predicted to be greater
than 97%.
[0026] FIGS. 2A-2D show the nucleotide (SEQ ID NO:3) and the
deduced amino acid (SEQ ID NO:4) sequence of a truncated,
biologically active form of VEGF-2. The polypeptide comprises
approximately 350 amino acid residues of which approximately the
first 24 amino acids represent the leader sequence.
[0027] FIGS. 3A-3B are an illustration of the amino acid sequence
homology between PDGFa (SEQ ID NO:5), PDGFb (SEQ ID NO:6), VEGF
(SEQ ID NO:7), and VEGF-2 (SEQ ID NO:4). The boxed areas indicate
the conserved sequences and the location of the eight conserved
cysteine residues.
[0028] FIG. 4 shows, in table-form, the percent homology between
PDGFa, PDGFb, VEGF, and VEGF-2.
[0029] FIG. 5 shows the presence of VEGF-2 mRNA in human breast
tumor cell lines.
[0030] FIG. 6 depicts the results of a Northern blot analysis of
VEGF-2 in human adult tissues.
[0031] FIG. 7 shows a photograph of an SDS-PAGE gel after in vitro
transcription, translation and electrophoresis of the polypeptide
of the present invention. Lane 1: .sup.14C and rainbow M.W. marker;
Lane 2: FGF control; Lane 3: VEGF-2 produced by M13-reverse and
forward primers; Lane 4: VEGF-2 produced by M13 reverse and VEGF-F4
primers; Lane 5: VEGF-2 produced by M13 reverse and VEGF-F5
primers.
[0032] FIGS. 8A and 8B depict photographs of SDS-PAGE gels. VEGF-2
polypeptide was expressed in a baculovirus system consisting of Sf9
cells. Protein from the medium and cytoplasm of cells were analyzed
by SDS-PAGE under non-reducing (FIG. 8A) and reducing (FIG. 8B)
conditions.
[0033] FIG. 9 depicts a photograph of an SDS-PAGE gel. The medium
from Sf9 cells infected with a nucleic acid sequence of the present
invention was precipitated. The resuspended precipitate was
analyzed by SDS-PAGE and stained with Coomassie brilliant blue.
[0034] FIG. 10 depicts a photograph of an SDS-PAGE gel. VEGF-2 was
purified from the medium supernatant and analyzed by SDS-PAGE in
the presence or absence of the reducing agent b-mercaptoethanol and
stained by Coomassie brilliant blue.
[0035] FIG. 11 depicts reverse phase HPLC analysis of purified
VEGF-2 using a RP-300 column (0.21.times.3 cm. Applied Biosystems,
Inc.). The column was equilibrated with 0.1% trifluoroacetic acid
(Solvent A) and the proteins eluted with a 7.5 min gradient from 0
to 60% Solvent B, composed of acetonitrile containing 0.07% TFA.
The protein elution was monitored by absorbance at 215 nm ("red"
line) and 280 nm ("blue" line). The percentage of Solvent B is
shown by the "green" line.
[0036] FIG. 12 is a bar graph illustrating the effect of
partially-purified VEGF-2 protein on the growth of vascular
endothelial cells in comparison to basic fibroblast growth
factor.
[0037] FIG. 13 is a bar graph illustrating the effect of purified
VEGF-2 protein on the growth of vascular endothelial cells.
[0038] FIG. 14 depicts expression of VEGF-2 mRNA in human fetal and
adult tissues.
[0039] FIG. 15 depicts expression of VEGF-2 mRNA in human primary
culture cells.
[0040] FIG. 16 depicts transient expression of VEGF-2 protein in
COS-7 cells.
[0041] FIG. 17 depicts VEGF-2 stimulated proliferation of human
umbilical vein endothelial cells (HUVEC).
[0042] FIG. 18 depicts VEGF-2 stimulated proliferation of dermal
microvascular endothelial cells.
[0043] FIG. 19 depicts the stimulatory effect of VEGF-2 on
proliferation of microvascular, umbilical cord, endometrial, and
bovine aortic endothelial cells.
[0044] FIG. 20 depicts inhibition of PDGF-induced vascular (human
aortic) smooth muscle cell proliferation.
[0045] FIG. 21 depicts stimulation of migration of HUVEC and bovine
microvascular endothelial cells (BMEC) by VEGF-2.
[0046] FIG. 22 depicts stimulation of nitric oxide release of HUVEC
by VEGF-2 and VEGF-1.
[0047] FIG. 23 depicts inhibition of cord formation of
microvascular endothelial cells (CADMEC) by VEGF-2.
[0048] FIG. 24 depicts stimulation of angiogenesis by VEGF, VEGF-2,
and bFGF in the CAM assay.
[0049] FIGS. 25A-25O depict restoration of certain parameters in
the ischemic limb by VEGF2 protein (FIGS. 25A, C, D, E, H, I, J, L,
M, O) and naked expression plasmid (FIGS. 25B, C, F, G, H, I, K, L,
M, O): BP ratio (FIGS. 25A-25C); Blood Flow and Flow Reserve (FIGS.
25D-25I); Angiographic Score (FIGS. 25J-25L); Capillary density
(FIGS. 25M-25O).
[0050] FIGS. 26A-26G depict the ability of VEGF2 to affect the
diastolic blood pressure in spontaneously hypertensive rats (SHR).
FIGS. 26a and b depict the dose-dependent decrease in diastolic
blood pressure achieved with VEGF-2. (FIGS. 26c and d depict the
decreased mean arterial pressure (MAP) observed with VEGF-2. Panel
E shows the effect of increasing doses of VEGF-2 on the mean
arterial pressure (MAP) of SHR rats. Panel F shows the effect of
VEGF-2 on the diastolic pressure of SHR rats. Panel G shows the
effect of VEGF-2 on the diastolic blood pressure of SHR rats.
[0051] FIG. 27 depicts inhibition of VEGF-2N= and VEGF-2-induced
proliferation.
[0052] FIG. 28 shows a schematic representation of the pHE4a
expression vector (SEQ ID NO:16). The locations of the kanamycin
resistance marker gene, the multiple cloning site linker region,
the oriC sequence, and the lacIq coding sequence are indicated.
[0053] FIG. 29 shows the nucleotide sequence of the regulatory
elements of the pHE4a promoter (SEQ ID NO:17). The two lac operator
sequences, the Shine-Delgarno sequence (S/D), and the terminal
HindIII and NdeI restriction sites (italicized) are indicated.
[0054] FIG. 30 shows a schematic representation of the pVGI.1
expression vector construct containing a polynucleotide encoding
VEGF-2.
[0055] FIG. 31A-U shows the nucleotide sequence of the pVGI.1
vector construct containing the VEGF-2 insert (SEQ ID NO:36).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0056] In accordance with one aspect of the present invention,
there are provided isolated nucleic acid molecules comprising a
polynucleotide encoding a VEGF-2 polypeptide having the deduced
amino acid sequence of FIG. 1 (SEQ ID NO:2), which was determined
by sequencing a cloned cDNA. The nucleotide sequence shown in SEQ
ID NO:1 was obtained by sequencing a cDNA clone, which was
deposited on May 12, 1995 at the American Type Tissue Collection
(ATCC.TM.), 10801 University Boulevard, Manassas, Va. 20110-2209,
and given ATCC.TM. Deposit No. 97149.
[0057] In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules comprising a
polynucleotide encoding a truncated VEGF-2 polypeptide having the
deduced amino acid sequence of FIG. 2 (SEQ ID NO:4), which was
determined by sequencing a cloned cDNA. The nucleotide sequence
shown in SEQ ID NO:3 was obtained by sequencing a cDNA clone, which
was deposited on Mar. 4, 1994 at the American Type Tissue
Collection (ATCC.TM.), 10801 University Boulevard, Manassas, Va.
20110-2209, and given ATCC.TM. Deposit Number 75698.
[0058] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
[0059] A polynucleotide encoding a polypeptide of the present
invention may be obtained from early stage human embryo (week 8 to
9) osteoclastomas, adult heart or several breast cancer cell lines.
The polynucleotide of this invention was discovered in a cDNA
library derived from early stage human embryo week 9. It is
structurally related to the VEGF/PDGF family. It contains an open
reading frame encoding a protein of about 419 amino acid residues
of which approximately the first 23 amino acid residues are the
putative leader sequence such that the mature protein comprises 396
amino acids, and which protein exhibits the highest amino acid
sequence homology to human vascular endothelial growth factor (30%
identity), followed by PDGFa (24%) and PDGFb (22%). (See FIG. 4).
It is particularly important that all eight cysteines are conserved
within all four members of the family (see boxed areas of FIG. 3).
In addition, the signature for the PDGF/VEGF family,
PXCVXXXRCXGCCN, (SEQ ID NO:8) is conserved in VEGF-2 (see FIG. 3).
The homology between VEGF-2, VEGF and the two PDGFs is at the
protein sequence level. No nucleotide sequence homology can be
detected, and therefore, it would be difficult to isolate the
VEGF-2 through simple approaches such as low stringency
hybridization.
[0060] The VEGF-2 polypeptide of the present invention is meant to
include the full length polypeptide and polynucleotide sequence
which encodes for any leader sequences and for active fragments of
the full length polypeptide. Active fragments are meant to include
any portions of the full length amino acid sequence which have less
than the full 419 amino acids of the full length amino acid
sequence as shown in SEQ ID NO:2, but still contain the eight
cysteine residues shown conserved in FIG. 3 and that still have
VEGF-2 activity.
[0061] There are at least two alternatively spliced VEGF-2 mRNA
sequences present in normal tissues. The two bands in FIG. 7, lane
5 indicate the presence of the alternatively spliced mRNA encoding
the VEGF-2 polypeptide of the present invention.
[0062] 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 FIG. 1 or FIG. 2, or that of the deposited clones, or may
be a different coding sequence which, as a result of the redundancy
or degeneracy of the genetic code, encodes the same, mature
polypeptide as the DNA of FIG. 1, FIG. 2, or the deposited
cDNAs.
[0063] The polynucleotide which encodes for the mature polypeptide
of FIG. 1 or FIG. 2 or for the mature polypeptides encoded by the
deposited cDNAs may include: only the coding sequence for the
mature polypeptide; the coding sequence for the mature polypeptide
and additional coding sequences such as a leader or secretory
sequence or a proprotein sequence; the coding sequence for the
mature polypeptide (and optionally additional coding sequences) and
non-coding sequences, such as introns or non-coding sequence 5'
and/or 3' of the coding sequence for the mature polypeptide.
[0064] Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding sequences
for the polypeptide as well as a polynucleotide which includes
additional coding and/or non-coding sequences.
[0065] 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 FIGS. 1 or 2, or the polypeptide encoded by
the cDNA of the deposited clones. The variant of the polynucleotide
may be a naturally occurring allelic variant of the polynucleotide
or a non-naturally occurring variant of the polynucleotide.
[0066] Thus, the present invention includes polynucleotides
encoding the same mature polypeptide as shown in FIGS. 1 or 2 or
the same mature polypeptide encoded by the cDNA of the deposited
clones as well as variants of such polynucleotides which variants
encode for a fragment, derivative, or analog of the polypeptides of
FIGS. 1 or 2, or the polypeptide encoded by the cDNA of the
deposited clones. Such nucleotide variants include deletion
variants, substitution variants, and addition or insertion
variants.
[0067] As hereinabove indicated, the polynucleotide may have a
coding sequence which is a naturally occurring allelic variant of
the coding sequence shown in FIGS. 1 or 2, or of the coding
sequence of the deposited clones. As known in the art, an allelic
variant is an alternate form of a polynucleotide sequence which
have a substitution, deletion or addition of one or more
nucleotides, which does not substantially alter the function of the
encoded polypeptide.
[0068] The present invention also includes polynucleotides, wherein
the coding sequence for the mature polypeptide may be fused in the
same reading frame to a polynucleotide which aids in expression and
secretion of a polypeptide from a host cell, for example, a leader
sequence which functions as a secretory sequence for controlling
transport of a polypeptide from the cell. The polypeptide having a
leader sequence is a preprotein and may have the leader sequence
cleaved by the host cell to form the mature form of the
polypeptide. The polynucleotides may also encode for a proprotein
which is the mature protein plus additional 5' amino acid residues.
A mature protein having a prosequence is a proprotein and is an
inactive form of the protein. Once the prosequence is cleaved an
active mature protein remains.
[0069] Thus, for example, the polynucleotide of the present
invention may encode for a mature protein, or for a protein having
a prosequence or for a protein having both a prosequence and
presequence (leader sequence).
[0070] The polynucleotides of the present invention may also have
the coding sequence fused in frame to a marker sequence which
allows for purification of the polypeptide of the present
invention. The marker sequence may be a hexa-histidine tag supplied
by a pQE-9 vector to provide for purification of the mature
polypeptide fused to the marker in the case of a bacterial host,
or, for example, the marker sequence may be a hemagglutinin (HA)
tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson, I., et al., Cell 37:767 (1984)).
[0071] Further embodiments of the invention include isolated
nucleic acid molecules comprising a polynucleotide having a
nucleotide sequence at least 95% identical, and more preferably at
least 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence
encoding the polypeptide having the amino acid sequence in SEQ ID
NO:2; (b) a nucleotide sequence encoding the polypeptide having the
amino acid sequence in SEQ ID NO:2, but lacking the N-terminal
methionine; (c) a nucleotide sequence encoding the polypeptide
having the amino acid sequence at positions from about 1 to about
396 in SEQ ID NO:2; (d) a nucleotide sequence encoding the
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC.TM. Deposit No.97149; (e) a nucleotide
sequence encoding the mature VEGF-2 polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No.97149; or (f) a nucleotide sequence complementary to any
of the nucleotide sequences in (a), (b), (c), (d), or (e).
[0072] Further embodiments of the invention include isolated
nucleic acid molecules comprising a polynucleotide having a
nucleotide sequence at least 95% identical, and more preferably at
least 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence
encoding the polypeptide having the amino acid sequence in SEQ ID
NO:4; (b) a nucleotide sequence encoding the polypeptide having the
amino acid sequence in SEQ ID NO:4, but lacking the N-terminal
methionine; (c) a nucleotide sequence encoding the polypeptide
having the amino acid sequence at positions from about 1 to about
326 in SEQ ID NO:4; (d) a nucleotide sequence encoding the
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC.TM. Deposit No.75698; (e) a nucleotide
sequence encoding the mature VEGF-2 polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No.75698; or (f) a nucleotide sequence complementary to any
of the nucleotide sequences in (a), (b), (c), (d), or (e).
[0073] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a VEGF-2 polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence encoding the VEGF-2 polypeptide. In other
words, to obtain a polynucleotide having a nucleotide sequence at
least 95% identical to a reference nucleotide sequence, up to 5% of
the nucleotides in the reference sequence may be deleted or
substituted with another nucleotide, or a number of nucleotides up
to 5% of the total nucleotides in the reference sequence may be
inserted into the reference sequence. These mutations of the
reference sequence may occur at the 5N or 3N terminal positions of
the reference nucleotide sequence or anywhere between those
terminal positions, interspersed either individually among
nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0074] As a practical matter, whether any particular nucleic acid
molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for
instance, the nucleotide sequence shown in SEQ ID NOS:1 or 3, or to
the nucleotides sequence of the deposited cDNA clone(s) can be
determined conventionally using known computer programs such as the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). Bestfit uses the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2: 482-489 (1981), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set, of course, such that
the percentage of identity is calculated over the full length of
the reference nucleotide sequence and that gaps in homology of up
to 5% of the total number of nucleotides in the reference sequence
are allowed.
[0075] As described in detail below, the polypeptides of the
present invention can be used to raise polyclonal and monoclonal
antibodies, which are useful in diagnostic assays for detecting
VEGF-2 protein expression as described below or as agonists and
antagonists capable of enhancing or inhibiting VEGF-2 protein
function. Further, such polypeptides can be used in the yeast
two-hybrid system to "capture" VEGF-2 protein binding proteins
which are also candidate agonist and antagonist according to the
present invention. The yeast two hybrid system is described in
Fields and Song, Nature 340:245-246 (1989).
[0076] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. As to the selection of peptides or polypeptides
bearing an antigenic epitope (i.e., that contain a region of a
protein molecule to which an antibody can bind), it is well known
in that art that relatively short synthetic peptides that mimic
part of a protein sequence are routinely capable of eliciting an
antiserum that reacts with the partially mimicked protein. See, for
instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner,
R. A. (1983) Antibodies that react with predetermined sites on
proteins. Science 219:660-666. Peptides capable of eliciting
protein-reactive sera are frequently represented in the primary
sequence of a protein, can be characterized by a set of simple
chemical rules, and are confined neither to immunodominant regions
of intact proteins (i.e., immunogenic epitopes) nor to the amino or
carboxyl terminals. Peptides that are extremely hydrophobic and
those of six or fewer residues generally are ineffective at
inducing antibodies that bind to the mimicked protein; longer,
soluble peptides, especially those containing proline residues,
usually are effective. Sutcliffe et al., supra, at 661. For
instance, 18 of 20 peptides designed according to these guidelines,
containing 8-39 residues covering 75% of the sequence of the
influenza virus hemagglutinin HA1 polypeptide chain, induced
antibodies that reacted with the HA1 protein or intact virus; and
12/12 peptides from the MuLV polymerase and 18/18 from the rabies
glycoprotein induced antibodies that precipitated the respective
proteins.
[0077] Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. Thus, a high proportion of hybridomas obtained by
fusion of spleen cells from donors immunized with an antigen
epitope-bearing peptide generally secrete antibody reactive with
the native protein. Sutcliffe et al., supra, at 663. The antibodies
raised by antigenic epitope-bearing peptides or polypeptides are
useful to detect the mimicked protein, and antibodies to different
peptides may be used for tracking the fate of various regions of a
protein precursor which undergoes post-translational processing.
The peptides and anti-peptide antibodies may be used in a variety
of qualitative or quantitative assays for the mimicked protein, for
instance in competition assays since it has been shown that even
short peptides (e.g., about 9 amino acids) can bind and displace
the larger peptides in immunoprecipitation assays. See, for
instance, Wilson et al., Cell 37:767-778 (1984) at 777. The
anti-peptide antibodies of the invention also are useful for
purification of the mimicked protein, for instance, by adsorption
chromatography using methods well known in the art.
[0078] Antigenic epitope-bearing peptides and polypeptides of the
invention designed according to the above guidelines preferably
contain a sequence of at least seven, more preferably at least nine
and most preferably between about 15 to about 30 amino acids
contained within the amino acid sequence of a polypeptide of the
invention. However, peptides or polypeptides comprising a larger
portion of an amino acid sequence of a polypeptide of the
invention, containing about 30, 40, 50, 60, 70, 80, 90, 100, or 150
amino acids, or any length up to and including the entire amino
acid sequence of a polypeptide of the invention, also are
considered epitope-bearing peptides or polypeptides of the
invention and also are useful for inducing antibodies that react
with the mimicked protein. Preferably, the amino acid sequence of
the epitope-bearing peptide is selected to provide substantial
solubility in aqueous solvents (i.e., the sequence includes
relatively hydrophilic residues and highly hydrophobic sequences
are preferably avoided); and sequences containing proline residues
are particularly preferred.
[0079] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate VEGF-2-specific antibodies include the
following: a polypeptide comprising amino acid residues from about
leu-37 to about glu-45 in SEQ ID NO:2, from about Tyr-58 to about
Gly-66 in SEQ ID NO:2, from about Gln-73 to about Glu-81 in SEQ ID
NO:2, from about Asp-100 to about Cys-108 in SEQ ID NO:2, from
about Gly-140 to about Leu-148 in SEQ ID NO:2, from about Pro-168
to about Val-176 in SEQ ID NO:2, from about His-183 to about
Lys-191 in SEQ ID NO:2, from about Ile-201 to about Thr-209 in SEQ
ID NO:2, from about Ala-216 to about Tyr-224 in SEQ ID NO:2, from
about Asp-244 to about His-254 in SEQ ID NO:2, from about Gly-258
to about Glu-266 in SEQ ID NO:2, from about Cys-272 to about
Ser-280 in SEQ ID NO:2, from about Pro-283 to about Ser-291 in SEQ
ID NO:2, from about Cys-296 to about Gln-304 in SEQ ID NO:2, from
about Ala-307 to about Cys-316 in SEQ ID NO:2, from about Val-319
to about Cys-335 in SEQ ID NO:2, from about Cys-339 to about
Leu-347 in SEQ ID NO:2, from about Cys-360 to about Glu-373 in SEQ
ID NO:2, from about Tyr-378 to about Val-386 in SEQ ID NO:2, and
from about Ser-388 to about Ser-396 in SEQ ID NO:2. These
polypeptide fragments have been determined to bear antigenic
epitopes of the VEGF-2 protein by the analysis of the Jameson-Wolf
antigenic index.
[0080] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means for making
peptides or polypeptides including recombinant means using nucleic
acid molecules of the invention. For instance, a short
epitope-bearing amino acid sequence may be fused to a larger
polypeptide that acts as a carrier during recombinant production
and purification, as well as during immunization to produce
anti-peptide antibodies. Epitope-bearing peptides also may be
synthesized using known methods of chemical synthesis. For
instance, Houghten has described a simple method for synthesis of
large numbers of peptides, such as 10-20 mg of 248 different 13
residue peptides representing single amino acid variants of a
segment of the HA1 polypeptide which were prepared and
characterized (by ELISA-type binding studies) in less than four
weeks. Houghten, R. A. (1985) General method for the rapid
solid-phase synthesis of large numbers of peptides: specificity of
antigen-antibody interaction at the level of individual amino
acids. Proc. Natl. Acad Sci. USA 82:5131-5135. This "Simultaneous
Multiple Peptide Synthesis (SMPS)" process is further described in
U.S. Pat. No. 4,631,211 to Houghten et al. (1986). In this
procedure the individual resins for the solid-phase synthesis of
various peptides are contained in separate solvent-permeable
packets, enabling the optimal use of the many identical repetitive
steps involved in solid-phase methods. A completely manual
procedure allows 500-1000 or more syntheses to be conducted
simultaneously. Houghten et al., supra, at 5134.
[0081] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art. See, for instance, Sutcliffe et al., supra; Wilson et al.,
supra; Chow, M. et al., Proc. Natl. Acad Sci. USA 82:910-914; and
Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985).
Generally, animals may be immunized with free peptide; however,
anti-peptide antibody titer may be boosted by coupling of the
peptide to a macromolecular carrier, such as keyhole limpet
hemacyanin (KLH) or tetanus toxoid. For instance, peptides
containing cysteine may be coupled to carrier using a linker such
as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carrier using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 mg peptide or carrier protein and
Freund's adjuvant. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0082] Immunogenic epitope-bearing peptides of the invention, i.e.,
those parts of a protein that elicit an antibody response when the
whole protein is the immunogen, are identified according to methods
known in the art. For instance, Geysen et al., supra, discloses a
procedure for rapid concurrent synthesis on solid supports of
hundreds of peptides of sufficient purity to react in an
enzyme-linked immunosorbent assay. Interaction of synthesized
peptides with antibodies is then easily detected without removing
them from the support. In this manner a peptide bearing an
immunogenic epitope of a desired protein may be identified
routinely by one of ordinary skill in the art. For instance, the
immunologically important epitope in the coat protein of
foot-and-mouth disease virus was located by Geysen et al. with a
resolution of seven amino acids by synthesis of an overlapping set
of all 208 possible hexapeptides covering the entire 213 amino acid
sequence of the protein. Then, a complete replacement set of
peptides in which all 20 amino acids were substituted in turn at
every position within the epitope were synthesized, and the
particular amino acids conferring specificity for the reaction with
antibody were determined. Thus, peptide analogs of the
epitope-bearing peptides of the invention can be made routinely by
this method. U.S. Pat. No. 4,708,781 to Geysen (1987) further
describes this method of identifying a peptide bearing an
immunogenic epitope of a desired protein.
[0083] Further still, U.S. Pat. No. 5,194,392 to Geysen (1990)
describes a general method of detecting or determining the sequence
of monomers (amino acids or other compounds) which is a topological
equivalent of the epitope (i.e., a Amimotope) which is
complementary to a particular paratope (antigen binding site) of an
antibody of interest. More generally, U.S. Pat. No. 4,433,092 to
Geysen (1989) describes a method of detecting or determining a
sequence of monomers which is a topographical equivalent of a
ligand which is complementary to the ligand binding site of a
particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971
to Houghten, R. A. et al. (1996) on Peralkylated Oligopeptide
Mixtures discloses linear C.sub.1-C.sub.7-alkyl peralkylated
oligopeptides and sets and libraries of such peptides, as well as
methods for using such oligopeptide sets and libraries for
determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus,
non-peptide analogs of the epitope-bearing peptides of the
invention also can be made routinely by these methods.
[0084] As one of skill in the art will appreciate, VEGF-2
polypeptides of the present invention and the epitope-bearing
fragments thereof described above can be combined with parts of the
constant domain of immunoglobulins (IgG), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and
show an increased half-life in vivo. This has been shown, e.g., for
chimeric proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins (EPA 394,827;
Traunecker et al., Nature 331:84-86 (1988)).
[0085] In accordance with the present invention, novel variants of
VEGF-2 are also described. These can be produced by deleting or
substituting one or more amino acids of VEGF-2. Natural mutations
are called allelic variations. Allelic variations can be silent (no
change in the encoded polypeptide) or may have altered amino acid
sequence.
[0086] In order to attempt to improve or alter the characteristics
of native VEGF-2, protein engineering may be employed. Recombinant
DNA technology known to those skilled in the art can be used to
create novel polypeptides. Muteins and deletions can show, e.g.,
enhanced activity or increased stability. In addition, they could
be purified in higher yield and show better solubility at least
under certain purification and storage conditions. Set forth below
are examples of mutations that can be constructed.
Amino Terminal and Carboxy Terminal Deletions
[0087] Furthermore, VEGF-2 appears to be proteolytically cleaved
upon expression resulting in polypeptide fragments of the following
sizes when run on a SDS-PAGE gel (sizes are approximate) (See,
FIGS. 6-8, for example): 80, 59, 45, 43, 41, 40, 39, 38, 37, 36,
31, 29, 21, and 15 kDa. These polypeptide fragments are the result
of proteolytic cleavage at both the N-terminal and C-terminal
portions of the protein. These proteolytically generated fragments
appears to have activity, particularly the 21 kDa fragment.
[0088] In addition, protein engineering may be employed in order to
improve or alter one or more characteristics of native VEGF-2. The
deletion of carboxyterminal amino acids can enhance the activity of
proteins. One example is interferon gamma that shows up to ten
times higher activity by deleting ten amino acid residues from the
carboxy terminus of the protein (Dobeli et al., J. of Biotechnology
7:199-216 (1988)). Thus, one aspect of the invention is to provide
polypeptide analogs of VEGF-2 and nucleotide sequences encoding
such analogs that exhibit enhanced stability (e.g., when exposed to
typical pH, thermal conditions or other storage conditions)
relative to the native VEGF-2 polypeptide.
[0089] Particular preferred VEGF-2 polypeptides are shown below
(numbering starts with the first amino acid in the protein (Met)
FIG. 1 (SEQ ID NO:2)): Ala (residue 25) to Ser (residue 419); Pro
(26) to Ser (419); Ala (27) to Ser (419); Ala (28) to Ser (419);
Ala (29) to Ser (419); Ala (30) to Ser (419); Ala (31) to Ser
(419); Phe (32) to Ser (419); Blu (33) to Ser (419); Ser (34) to
Ser (419); Gly (35) to Ser (419); Leu (36) to Ser (419); Asp (37)
to Ser (419); Leu (38) to (Ser (419); Ser (39) to Ser (419); Asp
(40) to Ser (419); Ala (41) to Ser (419); Glu (42) to Ser (419);
Pro (43) to Ser (419); Asp (44) to Ser (419); Ala (45) to Ser
(419); Gly (46) to Ser (419); Glu (47) to Ser (419); Ala (48) to
Ser (419); Thr (49) to Ser (419); Ala (50) to Ser (419); Tyr (51)
to Ser (419); Ser (53) to Ser (419); Asp (55) to Ser (419); Val
(63) to Ser (419); Val (66) to Ser (419); Met(1), Glu (24), or Ala
(25) to Met (418); Met (1), Glu (24), or Ala (25) to Gln (417); Met
(1), Glu (24), or Ala (25) to Pro (416); Met(1), Glu (24), or Ala
(25) to Arg (415); Met(1), Glu (24), or Ala (25) to Gln (414);
Met(1), Glu (24), or Ala (25) to Trp (413); Met(1), Glu (24), or
Ala (25) to Tyr (412); Met(1), Glu (24), or Ala (25) to Ser (411);
Met(1), Glu (24), or Ala (25) to Pro (410); Met(1), Glu (24), or
Ala (25) to Val (409); Met(1), Glu (24), or Ala (25) to Cys (408);
Met(1), Glu (24), or Ala (25) to Arg (407); Met(1), Glu (24), or
Ala (25) to Cys (406); Met(1), Glu (24), or Ala (25) to Val (405);
Met(1), Glu (24), or Ala (25) to Glu (404); Met(1), Glu (24), or
Ala (25) to Glu (403); Met(1), Glu (24), or Ala (25) to Ser (402);
Met(1), Glu (24), or Ala (25) to Gly (398); Met(1), Glu (24), or
Ala (25) to Pro (397); Met(1), Glu (24), or Ala (25) to Lys (393);
Met(1), Glu (24), or Ala (25) to Met(263); Met(1), Glu (24), or Ala
(25) to Asp(311); Met(1), Glu (24), or Ala (25) to Pro (366);
Met(1) to Ser (419); Met(1) to Ser(228); Glu(47) to Ser(419);
Ala(111) to Lys(214); Ala(112) to Lys(214); His(113) to Lys(214);
Tyr(114) to Lys(214); Asn(115) to Lys(214); Thr(116) to Lys(214);
Thr(103) to Leu(215); Glu(104) to Leu(215); Glu(105) to Leu(215);
Thr(106) to Leu(215); Ile(107) to Leu(215); Lys(108) to Leu(215);
Phe(109) to Leu(215); Ala(110) to Leu(215); Ala(111) to Leu(215);
Ala(112) to Leu(215); His(113) to Leu(215); Tyr(114) to Leu(215);
Asn(115) to Leu(215); Thr(116) to Leu(215); Thr(103) to Ser(228);
Glu(104) to Ser(228); Glu(105) to Ser(228); Thr(106) to Ser(228);
Ile(107) to Ser (228); Lys(108) to Ser(228); Phe(109) to Ser(228);
Ala(110) to Ser(228); Ala(111) to Ser(228); Ala(112) to Ser(228);
His(113) to Ser(228); Tyr(114) to Ser(228); Asn(115) to Ser(228);
Thr(116) to Ser(228); Thr(103) to Leu(229); Glu(104) to Leu(229);
Thr(103) to Arg(227); Glu(104) to Arg(227); Glu(105) to Arg (227);
Thr(106) to Arg (227); Ile(107) to Arg (227); Lys(108) to Arg
(227); Phe(109) to Arg (227); Ala(110) to Arg (227); Ala(111) to
Arg (227); Ala(112) to Arg (227); His(113) to Arg (227); Tyr(114)
to Arg (227); Asn(115) to Arg (227); Thr(116) to Arg (227);
Thr(103) to Ser(213); Glu(104) to Ser(213); Glu(105) to Ser(213);
Thr(106) to Ser(213); Ile(107) to Ser(213); Lys(108) to Ser(213);
Phe(109) to Ser(213); Ala(110) to Ser(213); Ala(111) to Ser(213);
Ala(112) to Ser(213); His(113) to Ser(213); Tyr(114) to Ser(213);
Asn(115) to Ser(213); Thr(116) to Ser(213); Thr(103) to Lys(214);
Glu(104) to Lys(214); Glu(105) to Lys(214); Thr(106) to Lys(214);
Ile(107) to Lys(214); Lys(108) to Lys(214); Phe(109) to Lys(214);
Ala(110) to Lys(214); Glu(105) to Leu(229); Thr(106) to Leu(229);
Ile(107) to Leu(229); Lys(108) to Leu(229); Phe(109) to Leu(229);
Ala(110) to Leu(229); Ala(111) to Leu(229); Ala(112) to Leu(229);
His(113) to Leu(229); Tyr(114) to Leu(229); Asn(115) to Leu(229);
Thr(116) to Leu(229).
[0090] Preferred embodiments include the following deletion
mutants: Thr(103)-Arg(227); Glu(104)-Arg(227); Ala(112)-Arg (227);
Thr(103)-Ser(213); Glu(104)-Ser(213); Thr(103)-Leu(215);
Glu(47)-Ser(419); Met(1), Glu (24), or Ala (25)-Met(263); Met(1),
Glu (24), or Ala (25)-Asp(311); Met(1), Glu (24), or Ala (25)-Pro
(366); Met(1)-Ser(419); and Met(1)-Ser(228) of (FIG. 1 (SEQ ID
NO:2)).
[0091] Also included by the present invention are deletion mutants
having amino acids deleted from both the NB terminus and the
C-terminus. Such mutants include all combinations of the N-terminal
deletion mutants and C-terminal deletion mutants described above.
Those combinations can be made using recombinant techniques known
to those skilled in the art.
[0092] Particularly, N-terminal deletions of the VEGF-2 polypeptide
can be described by the general formula m-396, where m is an
integer from -23 to 388, where m corresponds to the position of the
amino acid residue identified in SEQ ID NO:2. Preferably,
N-terminal deletions retain the conserved boxed area of FIG. 3
(PXCVXXXRCXGCCN)(SEQ ID NO: 8), and include polypeptides comprising
the amino acid sequence of residues: N-terminal deletions of the
polypeptide of the invention shown as SEQ ID NO:2 include
polypeptides comprising the amino acid sequence of residues: : E-24
to S-419; A-25 to S-419; P-26 to S-419; A-27 to S-419; A-28 to
S-419; A-29 to S-419; A-30 to S-419; A-31 to S-419; F-32 to S-419;
E-33 to S-419; S-34 to S-419; G-35 to S-419; L-36 to S-419; D-37 to
S-419; L-38 to S-419; S-39 to S-419; D-40 to S-419; A-41 to S-419;
E-42 to S-419; P-43 to S-419; D-44 to S-419; A-45 to S-419; G-46 to
S-419; E-47 to S-419; A-48 to S-419; A-49 to S-419; A-50 to S-419;
Y-51 to S-419; A-52 to S-419; S-53 to S-419; K-54 to S-419; D-55 to
S-419; L-56 to S-419; E-57 to S-419; E-58 to S-419; Q-59 to S-419;
L-60 to S-419; R-61 to S-419; S-62 to S-419; V-63 to S-419; S-64 to
S-419; S-65 to S-419; V-66 to S-419; D-67 to S-419; E-68 to S-419;
L-69 to S-419; M-70 to S-419; T-71 to S-419; V-72 to S-419; L-73 to
S-419; Y-74 to S-419; P-75 to S-419; E-76 to S-419; Y-77 to S-419;
W-78 to S-419; K-79 to S-419; M-80 to S-419; Y-81 to S-419; K-82 to
S-419; C-83 to S-419; Q-84 to S-419; L-85 to S-419; R-86 to S-419;
K-87 to S-419; G-88 to S-419; G-89 to S-149; W-90 to S-419; Q-91 to
S-419; H-92 to S-419; N-93 to S-419; R-94 to S-419; E-95 to S-419;
Q-96 to S-419; A-97 to S-419; N-98 to S-419; L-99 to S-419; N-100
to S-419; S-101 to S-419; R-102 to S-419; T-103 to S-419; E-104 to
S-419; E-105 to S-419; T-106 to S-419; I-107 to S-419; K-108 to
S-419; F-109 to S-419; A-110 to S-419; A-111 to S-419; A-112 to
S-419; H-113 to S419; Y-114 to S-419; N-115 to S-419; T-116 to
S-419; E-117 to S-419; I-118 to S-419; L-119 to S-419; K-120 to
S-419; S-121 to S-419; I-122 to S-419; D-123 to S-419; N-124 to
S-419; E-125 to S-419; S-126 to S-419; R-127 to S-419; K-128 to
S-419; T-129 to S-419; Q-130 to S-419; C-131 to S-419; M-132 to
S-419; P-133 to S-419; R-134 to S-419; E-135 to S-419; V-136 to
S-419; C-137 to S-419; I-138 to S-419; D-139 to S-419; V-140 to
S-419; G-141 to S-419; K-142 to S-419; E-143 to S-419; F-144 to
S-419; G-145 to S-419; V-146 to S-419; A-147 to S-419; T-148 to
S-419 N-149 to S-419; T-150 to S-419; F-151 to S-419; F-152 to
S-419; K-153 to S-419; P-154 to S-419; P-155 to S-419; C-156 to
S-419; V-157 to S-419; S-158 to S-419; V-159 to S-419; Y-160 to
S-419; R-161 to S-419; C-162 to S-419; G-163 to S-419; G-164 to
S-419; C-165 to S-419; C-166 to S-419; N-167 to S-419; S-168 to
S-419; E-169 to S-419; G-170 to S-419; L-171 to S-419; Q-172 to
S-419; C-173 to S-419; M-174 to S-419; N-175 to S-419; T-176 to
S-419; S-177 to S-419; T-178 to S-419; S-179 to S-419; Y-180 to
S-419; L-181 to S-419; S-182 to S-419; K-183 to S-419; T-184 to
S-419; L-185 to S-419; F-186 to S-149; E-187 to S-149; I-188 to
S-419; T-189 to S-419; V-190 to S-419; P-191 to S-419; L-192 to
S-419; S-193 to S-419; Q-194 to S-419; G-195 to S-419; P-196 to
S-419; K-197 to S-419; P-198 to S-419; V-199 to S-419; T-200 to
S-419; I-201 to S-419; S-202 to S-419; F-203 to S-419; A-204 to
S-419; N-205 to S-419; H-206 to S-419; T-207 to S-419; S-208 to
S-419; C-209 to S-419; R-210 to S-419; C-211 to S-419; M-212 to
S-419;S-213 to S-419; K-214 to S-419; L-215 to S-419; D-216 to
S-419; V-217 to S-419; Y-218 to S-419; R-219 to S-419; Q-220 to
S-419 V-221 to S-419; H-222 to S-419; S-223 to S-419; I-224 to
S-419; I-225 to S-419; R-226 to S-419; R-227 to S-419; S-228 to
S-419; L-229 to S-419; P-230 to S-419; A-231 to S-419; T-232 to
S-419; L-233 to S-419; P-234 to S-419; Q-235 to S-419; C-236 to
S-419; Q-237 to S-419; A-238 to S-419; A-239 to S-419; N-240 to
S-419; K-241 to S-419; T-242 to S-419; C-243 to S-419; P-244 to
S-419; T-245 to S-419; N-246 to S-419; Y-247 to S-419; M-248 to
S-419; W-249 to S-419; N-250 to S-419; N-251 to S-419; H-252 to
S-419; I-253 to S-419; C-254 to S-419; R-255 to S-419; C-256 to
S-419; L-257 to S-419; A-258 to S-419; Q-259 to S-419; E-260 to
S-419; D-261 to S-419; F-262 to S-419; M-263 to S-419; F-264 to
S-419; S-265 to S-419; S-266 to S-419; D-267 to S-419; A-268 to
S-419; G-269 to G-419; D-270 to S-419; D-271 to S-419; S-272 to
S-419; T-273 to S-419; D-274 to S-419; G-275 to S-419; F-276 to
S-419; H-277 to S-419; D-278 to S-419; I-279 to S-419; C-280 to
S-419; G-281 to S-419; P-282 to S-419; N-283 to S-419; K-284 to
S-419; E-285 to S-419; L-286 to S-419; D-287 to S-419; E-288 to
S-419; E-289 to S-419; T-290 to S-419; C-291 to S-419; Q-292 to
S-419; C-293 to S-419; V-294 to S-419; C-295 to S-419; R-296 to
S-419; A-297 to S-419; G-298 to S-419; L-299 to S-419; R-300 to
S-419; P-301 to S-419; A-302 to S-419; S-303 to S-419; C-304 to
S-419; G-305 to S-419; P-306 to S-419; H-307 to S-419; K-308 to
S-419; E-309 to S-419; L-310 to S-419; D-311 to S-419; R-312 to
S-419; N- 313 to S-419; S-314 to S-419; C-315 to S-419; Q-316 to
S-419; C-317 to S-419; V-318 to S-419; C-319 to S-419; K-320 to
S-419; N-321 to S-419; K-322 to S-419; L-323 to S-419; F-324 to
S-419; P-325 to S-419; S-326 to S-419; Q-327 to S-419; C-328 to
S419; G-329 to S-419; A-330 to S-419; N-331 to S-419; R-332 to
S-419; E-333 to S-419; F-334 to S-419; D-335 to S-419; E-336 to
S-419; N-337 to S-419; T-338 to S-419; C-339 to S-419; Q-340 to
S-419; C-341 to S-419; V-342 to S-419; C-343 to S-419; K-344 to
S-419; R-345 to S-419; T-346 to S-419; C-347 to S-419; P-348 to
S-419; R-349 to S-419; N-350 to S-419; Q-351 to S-419; P-352 to
S-419; L-353 to S-419; N-354 to S-419; P-355 to S-419; G-356 to
S-419; K-357 to S-419; C358 to S-419; A-359 to S-419; C-360 to
S-419; E-361 to S-419; C-362 to S-419; T-363 to S-419; E-364 to
S-419; S-365 to S-419; P-366 to S-419; Q-367 to S-419; K-368 to
S-419; S-369 to S-419; L-370 to S-419; L-371 to S-419; K-372 to
S-419; G-373 to S-419; K-374 to S-419; K-375 to S-419; F-376 to
S-419; H-377 to S-419; H-378 to S-419; Q-379 to S-419; T-380 to
S-419; C-381 to S-419; S-382 to S-419; C-383 to S-419; Y-384 to
S-419; R-385 to S-419; R-386 to S-419; P-387 to S-419; C-388 to
S-419; T-389 to S-419; N-390 to S-419; R-391 to S-419; Q-392 to
S-419; K-393 to S-419; A-394 to S-419; C-395 to S-419; E-396 to
S-419; P-397 to S-419; G-398 to S-419; F-399 to S-419; S-400 to
S-419; Y-401 to S-419; S-402 to S-419; E-403 to S-419; E-404 to
S-419; V-405 to S-419; C-406 to S-419; R-407 to S-419; C-408 to
S-419; V-409 to S-419; P-410 to S-419; S-411 to S-419; Y-412 to
S-419; S-413 to S-419; Q-414 to S-419 of SEQ ID NO:2. One preferred
embodiment comprises amino acids S-223 to S-419 of SEQ ID NO:2.
Also preferred are polynucleotides encoding these polypeptides.
[0093] Moreover, C-terminal deletions of the VEGF-2 polypeptide can
also be described by the general formula -23-n, where n is an
integer from -15 to 395 where n corresponds to the position of
amino acid residue identified in SEQ ID NO:2. Preferably,
C-terminal deletions retain the conserved boxed area of FIG. 3
(PXCVXXXRCXGCCN)(SEQ ID NO:8), and include polypeptides comprising
the amino acid sequence of residues: Likewise, C-terminal deletions
of the polypeptide of the invention shown as SEQ ID NO:2 include
polypeptides comprising the amino acid sequence of residues: E-24
to M-418; E-24 to Q-417; E-24 to P-416; E-24 to R-415; E-24 to
Q-414; E-24 to W-413; E-24 to Y-412; E-24 to S-411; E-24 to P-410;
E-24 to V-409; E-24 to C-408; E-24 to R-407; E-24 to C-406; E-24 to
V-405; E-24 E-404; E-24 to E-403; E-24 to S-402; E-24 to Y-401;
E-24 to S-400; E-24 to F-399; E-24 to G-398; E-24 to P-397; E-24 to
E-396; E-24 to C-395; E-24 to A-394; E-24 to K-393; E-24 to Q-392;
E-24 to R-391; E-24 to N-390; E-24 to T-389; E-24 to C-388; E-24 to
P-387; E-24 to R-386; E-24 to R-385; E-24 to Y-384; E-24 to C-383;
E-24 to S-382; E-24 to C-381; E-24 to T-380; E-24 to Q-379; E-24 to
H-378; E-24 to H-377; E-24 to F-376; E-24 to K-375; E-24 to K-374;
E-24 to G-373; E-24 to K-372; E-24 to L-371; E-24 to L-370; E-24 to
C-369; E-24 to K-368; E-24 to Q-367; E-24 to P-366; E-24 to S-365;
E-24 to E-364; E-24 to T-363; E-24 to C-362; E-24 to E-361; E-24 to
C-360; E-24 to A-359; E-24 to C-358; E-24 to K-357; E-24 to G-356;
E-24 to P-355; E-24 to N-354; E-24 to L-353; E-24 to P-352; E-24 to
Q-351; E-24 to N-350; E-24 to R-349; E-24 to P-348; E-24 to C-347;
E-24 to T-346; E-24 to R-345; E-24 to K-344; E-24 to C-343; E-24 to
V-342; E-24 to C-341; E-24 to Q-340; E-24 to C-339; E-24 to T-338;
E-24 to N-337; E-24 to E-336; E-24 to D-335; E-24 to F-334; E-24 to
E-333; E-24 to R-332; E-24 to N-331; E-24 to A-330; E-24 to G-329;
E-24 to C-328; E-24 to Q-327; E-24 to S-326; E-24 to P-325; E-24 to
F-324; E-24 to L-323; E-24 to K-322; E-24 to N-321; E-24 to K-320;
E-24 to C-319; E-24 to V-318; E-24 to C-317; E-24 to Q-316; E-24 to
C-315; E-24 to S-314; E-24 to N-313; E-24 to R-312; E-24 to D-311;
E-24 to L-310; E-24 to E-309; E-24 to K-308; E-24 to H-307; E-24 to
P-306; E-24 to G-305; E-24 to C-304; E-24 to S-303; E-24 to A-302;
E-24 to P-301; E-24 to R-300; E-24 to L-299; E-24 to G-298; E-24 to
A-297; E-24 to R-296; E-24 to C-295; E-24 to V-294; E-24 to C-293;
E-24 to Q-292; E-24 to C-291; E-24 to T-290; E-24 to E-289; E-24 to
E-288; E-24 to D-287; E-24 to L-286; E-24 to E-285; E-24 to K-284;
E-24 to N-283; E-24 to P-282; E-24 to G-281; E-24 to C-280; E-24 to
1-279; E-24 to D-278; E-24 to H-277; E-24 to F-276; E-24 to G-275;
E-24 to D-274; E-24 to T-273; E-24 to S-272; E-24 to D-271; E-24 to
D-270; E-24 to G-269; E-24 to A-268; E-24 to D-267; E-24 to S-266;
E-24 to S-265; E-24 to F-264; E-24 to M-263; E-24 to F-262; E-24 to
D-261; E-24 to E-260; E-24 to Q-259; E-24 to A-258; E-24 to L-257;
E-24 to C-256; E-24 to R-255; E-24 to C-254; E-24 to I-253; E-24 to
H-252; E-24 to N-251; E-24 to N-250; E-24 to W-249; E-24 to M-248;
E-24 to Y-247; E-24 to N-246; E-24 to T-245; E-24 to P-244; E-24 to
C-243; E-24 to T-242; E-24 to K-241; E-24 to N-240; E-24 to A-239;
E-24 to A-238; E-24 to Q-237; E-24 to C-236; E-24 to Q-235; E-24 to
P-234; E-24 to L-233; E-24 to T-232; E-24 to A-231; E-24 to P-230;
E-24 to L-229; E-24 to S-228; E-24 to R-227; E-24 to R-226; E-24 to
I-225; E-24 to I-224; E-24 to S-223; E-24 to H-222; E-24 to V-221;
E-24 to Q-220; E-24 to R-219; E-24 to Y-218; E-24 to V-217; E-24 to
D-216; E-24 to L-215; E-24 to K-214; E-24 to S-213; E-24 to M-212;
E-24 to C-211; E-24 to R-210; E-24 to C-209; E-24 to S-208; E-24 to
T-207; E-24 to H-206; E-24 to N-205; E-24 to A-204; E-24 to F-203;
E-24 to S-202; E-24 to I-201; E-24 to T-200; E-24 to V-199; E-24 to
P-198; E-24 to K-197; E-24 to P-196; E-24 to G-195; E-24 to Q-194;
E-24 to S-193; E-24 to L-192; E-24 to P-191; E-24 to V-190; E-24 to
T-189; E-24 to I-188; E-24 to E-187; E-24 to F-186; E-24 to L-185;
E-24 to T-184; E-24 to K-183; E-24 to S-182; E-24 to L-181; E-24 to
Y-180; E-24 to S-179; E-24 to T-178; E-24 to S-177; E-24 to T-176;
E-24 to N-175; E-24 to M-174; E-24 to C-173; E-24 to Q-172; E-24 to
L-171; E-24 to G-170; E-24 to E-169; E-24 to S-168; E-24 to N-167;
E-24 to C-166; E-24 to C-165; E-24 to G-164; E-24 to G-163; E-24 to
C-162; E-24 to R-161; E-24 to Y-160; E-24 to V-159; E-24 to S-158;
E-24 to V-157; E-24 to C-156; E-24 to P-155; E-24 to P-154; E-24 to
K-153; E-24 to F-152; E-24 to F-151; E-24 to T-150; E-24 to N-149;
E-24 to T-148; E-24 to A-147; E-24 to V-146; E-24 to G-145; E-24 to
F-144; E-24 to E-143; E-24 to K-142; E-24 to G-141; E-24 to V-140;
E-24 to D-139; E-24 to I-138; E-24 to C-137; E-24 to V-136; E-24 to
E-135; E-24 to R-134; E-24 to P-133; E-24 to M-132; E-24 to C-131;
E-24 to Q-130; E-24 to T-129; E-24 to K-128; E-24 to R-127; E-24 to
W-126; E-24 to E-125; E-24 to N-124; E-24 to D-123; E-24 to I-122;
E-24 to S-121; E-24 to K-120; E-24 to L-119; E-24 to I-118; E-24 to
E-117; E-24 to T-116; E-24 to N-115; E-24 to Y-114; E-24 to H-113;
E-24 to A-112; E-24 to A-111; E-24 to A-110; E-24 to F-109; E-24 to
K-108; E-24 to I-107; E-24 to T-106; E-24 to E-105; E-24 to E-104;
E-24 to T-103; E-24 to R-102; E-24 to S-101; E-24 to N-100; E-24 to
L-99; E-24 to N-98; E-24 to A-97; E-24 to Q-96; E-24 to E-95; E-24
to R-94; E-24 to N-93; E-24 to H-92; E-24 to Q-91; E-24 to W-90;
E-24 to G-89; E-24 to G-88; E-24 to K-87; E-24 to R-86; E-24 to
L-85; E-24 to Q-84; E-24 to C-83; E-24 to K-82; E-24 to Y-81; E-24
to M-80; E-24 to K-79; E-24 to W-78; E-24 to Y-77; E-24 to E-76;
E-24 to P-75; E-24 to Y-74; E-24 to L-73; E-24 to V-72; E-24 to
T-71; E-24 to M-70; E-24 to L-69; E-24 to E-68; E-24 to D-67; E-24
to V-66; E-24 to S-65; E-24 to S-64; E-24 to V-63; E-24 to S-62;
E-24 to R-61; E-24 to L-60; E-24 to Q-59; E-24 to E-58; E-24 to
E-57; E-24 to L-56; E-24 to D-55; E-24 to K-54; E-24 to S-53; E-24
to A-52; E-24 to Y-51; E-24 to A-50; E-24 to T-49; E-24 to A-48;
E-24 to E-47; E-24 to G-46; E-24 to A-45; E-24 to D-44; E-24 to
P-43; E-24 to E-42; E-24 to A-41; E-24 to D-40; E-24 to S-39; E-24
to L-38; E-24 to D-37; E-24 to L-36; E-24 to G-35; E-24 to S-34;
E-24 to E-33; E-24 to F-32; E-24 to A-31; E-24 to A-30 of SEQ ID
No:2. Also preferred are polynucleotides encoding these
polypeptides.
[0094] Moreover, the invention also provides polypeptides having
one or more amino acids deleted from both the amino and the
carboxyl termini, which may be described generally as having
residues m-n of SEQ ID NO:2, where n and m are integers as
described above.
[0095] Likewise, also preferred are C-terminal deletions of the
VEGF-2 polypeptide of the invention shown as SEQ ID NO:2 which
include polypeptides comprising the amino acid sequence of
residues: F-32 to M418; F-32 to Q-417; F-32 to P-416; F-32 to
R-415; F-32 to Q-414; F-32 to W-413; F-32 to Y-412; F-32 to S-411;
F-32 to P-410; F-32 to V-409; F-32to C-408; F-32 to R-407; F-32 to
C-406; F-32 to V-405; F-32 to E-404; F-32 to E-403; F-32 to S-402;
F-32 to Y-401; F-32 to S-400; F-32 to F-399; F-32 to G-398; F-32 to
P-397; F-32 to E-396; F-32 to C-395; F-32 to A-394; F-32 to K-393;
F-24 to Q-392; F-32 to R-391; F-32 to N-390; F-32 to T-389; F-32 to
C-388; F-32 to P-387; F-32 to R-386; F-32 to R-385; F-32 to Y-384;
F-32 to C-383; F-32 to S-382; F-32 to C-381; F-32 to T-380; F-32 to
Q-379; F-32 to H-378; F-32 to H-377; F-32 to F-376; F-32 to K-375;
F-32 to K-374; F-32 to G-373; F-32 to K-372; F-32 to L-371; F-32 to
L-370; F-32 to C-369; F-32 to K-368; F-32 to Q-367; F-32 to P-366;
F-32 to S-365; F-32 to E-364; F-32 to T-363; F-32 to C-362; F-32 to
E-361; F-32 to C-360; F-32 to A-359; F-32 to C-358; F-32 to K-357;
F-32 to G-356; F-32 to P-355; F-32 to N-354; F-32 to L-353; F-32
P-352; F-32 to Q-351; F-32 to N-350; F-32 to R-349; F-32 to P-348;
F-32 to C-347; F-32 to T-346; F-32 to R-345; F-32 to K-344; F-32 to
C-343; F-32 to V-342; F-32 to C-341; F-32 to Q-340; F-32 to C-339;
F-32 to T-338; F-32 to N-337; F-32 to E-336; F-32 to D-335; F-32 to
F-334; F-32 to E-333; F-32 to R-332; F-32 to N-331; F-32 to A-330;
F-32 to G-329; F-32 to C-328; F-32 to Q-327; F-32 to S-326; F-32 to
P-325; F-32 to F-324; F-32 to L-323; F-32 to K-322; F-32 to N-321;
F-32 to K-320; F-32 to C-319; F-32 to V-318; F-32 to C-317; F-32 to
Q-316; F-32 to C-315; F-32 to S-314; F-32 to N-313; F-32 to R-312;
F-32 to D-311; F-32 to L-310; F-32 to E-309; F-32 to K-308; F-32 to
H-307; F-32 to P-306; F-32 to G-305; F-32 to C-304; F-32 to S-303;
F-32 to A-302; F-32 to P-301; F-32 to R-300; F-32 to L-299; F-32 to
G-298; F-32 to A-297; F-32 to R-296; F-32 to C-295; F-32 to V-294;
F-32 to C-293; F-32 to Q-292; F-32 to C-291; F-32 to T-290; F-32 to
E-289; F-32 to E-288; F-32 to D-287; F-32 to L-286; F-32 to E-285;
F-32 to K-284; F-32 to N-283; F-32 to P-282; F-32 to G-281; F-32 to
C-280; F-32 to I-279; F-32 to D-278; F-32 to H-277; F-32 to F-276;
F-32 to G-275; F-32 to D-274; F-32 to T-273; F-32 to S-272; F-32 to
D-271; F-32 to D-270; F-32 to G-269; F-32 to A-268; F-32 to D-267;
F-32 to S-266; F-32 to S-265; F-32 to F-264; F-32 to M-263; F-32 to
F-262; F-32 to D-261; F-32 to E-260; F-32 to Q-259; F-32 to A-258;
F-32 to L-257; F-32 to C-256; F-32 to R-255; F-32 to C-254; F-32 to
I-253; F-32 to H-252; F-32 to N-251; F-32 to N-250; F-32 to W-249;
F-32 to M-248; F-32 to Y-247; F-32 to N-246; F-32 to T-245; F-32 to
P-244; F-32 to C-243; F-32 to T-242; F-32 to K-241; F-32 to N-240;
F-32 to A-239; F-32 to A-238; F-32 to Q-237; F-32 to C-236; F-32 to
Q-235; F-32 to P-234; F-32 to L-233; F-32 to T-232; F-32 to A-231;
F-32 to P-230; F-32 to L-229; F-32 to S-228; F-32 to R-227; F-32 to
R-226; F-32 to I-225; F-32 to I-224; F-32 to S-223; F-32 to H-222;
F-32 to V-221; F-32 to Q-220; F-32 to R-219; F-32 to Y-218; F-32 to
V-217; F-32 to D-216; F-32 to L-215; F-32 to K-214; F-32 to S-213;
F-32 to M-212; F-32 to C-211; F-32 to R-210; F-32 to C-209; F-32 to
S-208; F-32 to T-207; F-32 to H-206; F-32 to N-205; F-32 to A-204;
F-32 to F-203; F-32 to S-202; F-32 to I-201; F-32 to T-200; F-32 to
V-199; F-32 to P-198; F-32 to K-197; F-32 to P-196; F-32 to G-195;
F-32 to Q-194; F-32 to S-193; F-32 to L-192; F-32 to P-191; F-32 to
V-190; F-32 to T-189; F-32 to I-188; F-32 to E-187; F-32 to F-186;
F-32 to L-185; F-32 to T-184; F-32 to K-183; F-32 to S-182; F-32 to
L-181; F-32 to Y-180; F-32 to S-179; F-32 to T-178; F-32 to S-177;
F-32 to T-176; F-32 to N-175; F-32 to M-174; F-32 to C-173; F-32 to
Q-172; F-32 to L-171; F-32 to G-170; F-32 to E-169; F-32 to S-168;
F-32 to N-167; F-32 to C-166; F-32 to C-165; F-32 to G-164; F-32 to
G-163; F-32 to C-162; F-32 to R-161; F-32 to Y-160; F-32 to V-159;
F-32 to S-158; F-32 to V-157; F-32 to C-156; F-32 to P-155; F-32 to
P-154; F-32 to K-153; F-32 to F-152; F-32 to F-151; F-32 to T-150;
F-32 to N-149; F-32 to T-148; F-32 to A-147; F-32 to V-146; F-32 to
G-145; F-32 to F-144; F-32 to E-143; F-32 to K-142; F-32 to G-141;
F-32 to V-140; F-32 to D-139; F-32 to I-138; F-32 to C-137; F-32 to
V-136; F-32 to E-135; F-32 to R-134; F-32 to P-133; F-32 to M-132;
F-32 to C-131; F-32 to Q-130; F-32 to T-129; F-32 to K-128; F-32 to
R-127; F-32 to W-126; F-32 to E-125; F-32 to N-124; F-32 to D-123;
F-32 to I-122; F-32 to S-121; F-32 to K-120; F-32 to L-119; F-32 to
I-118; F-32 to E-117; F-32 to T-116; F-32 to N-115; F-32 to Y-114;
F-32 to H-113; F-32 to A-112; F-32 to A-111; F-32 to A-110; F-32 to
F-109; F-32 to K-108; F-32 to I-107; F-32 to T-106; F-32 to E-105;
F-32 to E-104; F-32 to T-103; F-32 to R-102; F-32 to S-101; F-32 to
N-100; F-32 to L-99; F-32 to N-98; F-32 to A-97; F-32 to Q-96; F-32
to E-95; F-32 to R-94; F-32 to N-93; F-32 to H-92; F-32 to Q-91;
F-32 to W-90; F-32 to G-89; F-32 to G-88; F-32 to K-87; F-32 to
R-86; F-32 to L-85; F-32 to Q-84; F-32 to C-83; F-32 to K-82; F-32
to Y-81; F-32 to M-80; F-32 to K-79; F-32 to W-78; F-32 to Y-77;
F-32 to E-76; F-32 to P-75; F-32 to Y-74; F-32 to L-73; F-32 to
V-72; F-32 to T-71; F-32 to M-70; F-32 to L-69; F-32 to E-68; F-32
to D-67; F-32 to V-66; F-32 to S-65; F-32 to S-64; F-32 to V-63;
F-32 to S-62; F-32 to R-61; F-32 to L-60; F-32 to Q-59; F-32 to
E-58; F-32 to E-57; F-32 to L-56; F-32 to D-55; F-32 to K-54; F-32
to S-53; F-32 to A-52; F-32 to Y-51; F-32 to A-50; F-32 to T-49;
F-32 to A-48; F-32 to E-47; F-32 to G-46; F-32 to A-45; F-32 to
D-44; F-32 to P-43; F-32 to E-42; F-32 to A-41; F-32 to D-40; F-32
to S-39; F-32 to L-38; of SEQ ID NO:2. Specifically preferred is
the polypeptide fragment comprising amino acid residues F-32 to
R-226 of SEQ ID NO:2, as well as polynucleotides encoding this
polypeptide. This F-32 to R-226 of SEQ ID NO:2 polypeptide
preferably is associated with a S-228 to S-419 of SEQ ID NO:2
polypeptide. Association may be through disulfide, covalent or
noncovalent interactions, by linkage via a linker (e.g. serine,
glycine, proline linkages), or by an antibody
[0096] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO: I and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1660 of SEQ ID NO:1, b is an integer
of 15 to 1674, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:1, and where the b is
greater than or equal to a+14.
[0097] Thus, in one aspect, N-terminal deletion mutants are
provided by the present invention. Such mutants include those
comprising the amino acid sequence shown in FIG. 1 (SEQ ID NO:2)
except for a deletion of at least the first 24 N-terminal amino
acid residues (i.e., a deletion of at least Met (1)-Glu (24)) but
not more than the first 115 N-terminal amino acid residues of FIG.
1 (SEQ ID NO:2). Alternatively, first 24 N-terminal amino acid
residues (i.e., a deletion of at least Met (1)-Glu (24)) but not
more than the first 103 N-terminal amino acid residues of FIG. 1
(SEQ ID NO:2), etc.
[0098] In another aspect, C-terminal deletion mutants are provided
by the present invention. Such mutants include those comprising the
amino acid sequence shown in FIG. 1 (SEQ ID NO:2) except for a
deletion of at least the last C-terminal amino acid residue (Ser
(419)) but not more than the last 220 C-terminal amino acid
residues (i.e., a deletion of amino acid residues Val (199)-Ser
(419)) of FIG. 1 (SEQ ID NO:2). Alternatively, the deletion will
include at least the last C-terminal amino acid residue but not
more than the last 216 C-terminal amino acid residues of FIG. 1
(SEQ ID NO:2). Alternatively, the deletion will include at least
the last C-terminal amino acid residue but not more than the last
204 C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).
Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 192
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).
Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 156
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).
Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 108
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).
Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 52
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).
[0099] In yet another aspect, also included by the present
invention are deletion mutants having amino acids deleted from both
the N-terminal and C-terminal residues. Such mutants include all
combinations of the N-terminal deletion mutants and C-terminal
deletion mutants described above.
[0100] 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).
[0101] The present invention is further directed to fragments of
the isolated nucleic acid molecules described herein. By a fragment
of an isolated nucleic acid molecule having the nucleotide sequence
of the deposited cDNA(s) or the nucleotide sequence shown in SEQ ID
NO:1 or SEQ ID NO:3 is intended fragments at least about 15 nt, and
more preferably at least about 20 nt, still more preferably at
least about 30 nt, and even more preferably, at least about 40 nt
in length which are useful as diagnostic probes and primers as
discussed herein. Of course, larger fragments of 50, 75, 100, 125,
150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,
475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775,
800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075,
1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350,
1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625,
1650 or 1674 nt in length are also useful according to the present
invention as are fragments corresponding to most, if not all, of
the nucleotide sequence of the deposited cDNA(s) or as shown in SEQ
ID NO:1 or SEQ ID NO:3. By a fragment at least 20 nt in length, for
example, is intended fragments which include 20 or more contiguous
bases from the nucleotide sequence of the deposited cDNA(s) or the
nucleotide sequence as shown in SEQ ID NOS:1 or 3.
[0102] Moreover, representative examples of VEGF-2 polynucleotide
fragments include, for example, fragments having a sequence from
about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250,
251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600,
651-700, 701-750, 751-800, 800-850, 851-900, 901-950, or 951 to the
end of SEQ ID NO:1 or the cDNA contained in the deposited clone. In
this context "about" includes the particularly recited ranges,
larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at
either terminus or at both termini. Preferably, these fragments
encode a polypeptide which has biological activity.
[0103] Fragments of the full length gene of the present invention
may be used as a hybridization probe for a cDNA library to isolate
the full length cDNA and to isolate other cDNAs which have a high
sequence similarity to the gene or similar biological activity.
Probes of this type preferably have at least 30 bases and may
contain, for example, 50 or more bases. The probe may also be used
to identify a cDNA clone corresponding to a full length transcript
and a genomic clone or clones that contain the complete gene
including regulatory and promoter regions, exons, and introns. An
example of a screen comprises isolating the coding region of the
gene by using the known DNA sequence to synthesize an
oligonucleotide probe. Labeled oligonucleotides having a sequence
complementary to that of the gene of the present invention are used
to screen a library of human cDNA, genomic DNA or mRNA to determine
which members of the library the probe hybridizes to.
[0104] A VEGF-2 "polynucleotide" also includes those
polynucleotides capable of hybridizing, under stringent
hybridization conditions, to sequences contained in SEQ ID NO:1 or
for instance, the cDNA clone(s) contained in ATCC.TM. Deposit Nos.
97149 or 75698, the complement thereof. "Stringent hybridization
conditions" refers to an overnight incubation at 42.degree. C. in a
solution comprising 50% formamide, 5.times.SSC (750 mM NaCI, 75 mM
sodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65.degree. C.
[0105] Also contemplated are nucleic acid molecules that hybridize
to the VEGF-2 polynucleotides at lower stringency hybridization
conditions. Changes in the stringency of hybridization and signal
detection are primarily accomplished through the manipulation of
formamide concentration (lower percentages of formamide result in
lowered stringency); salt conditions, or temperature. For example,
lower stringency conditions include an overnight incubation at
37.degree. C. in a solution comprising 6.times. SSPE (20.times.
SSPE=3M NaCl; 0.2M NaH.sub.2PO.sub.4; 0.02M EDTA, pH 7.4), 0.5%
SDS, 30% formamide, 100 .mu.g/ml salmon sperm blocking DNA;
followed by washes at 50.degree. C. with 1.times. SSPE, 0.1% SDS.
In addition, to achieve even lower stringency, washes performed
following stringent hybridization can be done at higher salt
concentrations (e.g. 5.times.SSC).
[0106] Note that variations in the above conditions may be
accomplished through the inclusion and/or substitution of alternate
blocking reagents used to suppress background in hybridization
experiments. Typical blocking reagents include Denhardt's reagent,
BLOTTO, heparin, denatured salmon sperm DNA, and commercially
available proprietary formulations. The inclusion of specific
blocking reagents may require modification of the hybridization
conditions described above, due to problems with compatibility.
[0107] Of course, a polynucleotide which hybridizes only to polyA+
sequences (such as any 3' terminal polyA+ tract of a cDNA shown in
the sequence listing), or to a complementary stretch of T (or U)
residues, would not be included in the definition of
"polynucleotide," since such a polynucleotide would hybridize to
any nucleic acid molecule containing a poly (A) stretch or the
complement thereof (e.g., practically any double-stranded cDNA
clone).
[0108] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 nt of the
reference polynucleotide. These are useful as diagnostic probes and
primers as discussed above and in more detail below.
[0109] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNA or the nucleotide sequence as shown in SEQ ID
NO:1). Of course, a polynucleotide which hybridizes only to a poly
A sequence (such as the 3N terminal poly(A) tract of the VEGF-2
cDNA shown in SEQ ID NOS:1 or 3), or to a complementary stretch of
T (or U) resides, would not be included in a polynucleotide of the
invention used to hybridize to a portion of a nucleic acid of the
invention, since such a polynucleotide would hybridize to any
nucleic acid molecule containing a poly (A) stretch or the
complement thereof (e.g., practically any double-stranded cDNA
clone).
[0110] The present application is directed to nucleic acid
molecules at least 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in SEQ ID NOS:1 or 3 or to the nucleic
acid sequence of the deposited cDNA(s), irrespective of whether
they encode a polypeptide having VEGF-2 activity. This is because
even where a particular nucleic acid molecule does not encode a
polypeptide having VEGF-2 activity, one of skill in the art would
still know how to use the nucleic acid molecule, for instance, as a
hybridization probe or a polymerase chain reaction (PCR) primer.
Uses of the nucleic acid molecules of the present invention that do
not encode a polypeptide having VEGF-2 activity include, inter
alia, (1) isolating the VEGF-2 gene or allelic variants thereof in
a cDNA library; (2) in situ hybridization (e.g., "FISH") to
metaphase chromosomal spreads to provide precise chromosomal
location of the VEGF-2 gene, as described in Verma et al., Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York
(1988); and Northern Blot analysis for detecting VEGF-2 mRNA
expression in specific tissues.
[0111] Preferred, however, are nucleic acid molecules having
sequences at least 95%, 96%, 97%, 98% or 99% identical to a nucleic
acid sequence shown in SEQ ID NOS:1 or 3 or to a nucleic acid
sequence of the deposited cDNA(s) which do, in fact, encode a
polypeptide having VEGF-2 protein activity. By "a polypeptide
having VEGF-2 activity" is intended polypeptides exhibiting VEGF-2
activity in a particular biological assay. For example, VEGF-2
protein activity can be measured using, for example, mitogenic
assays and endothelial cell migration assays. See, e.g., Olofsson
et al., Proc. Natl. Acad. Sci. USA 93:2576-2581 (1996) and Joukov
et al., EMBO J. 5:290-298 (1996).
[0112] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid
sequence of the deposited cDNA(s) or the nucleic acid sequence
shown in SEQ ID NO:1 or SEQ ID NO:3 will encode a polypeptide
"having VEGF-2 protein activity." In fact, since degenerate
variants of these nucleotide sequences all encode the same
polypeptide, this will be clear to the skilled artisan even without
performing the above described comparison assay. It will be further
recognized in the art that, for such nucleic acid molecules that
are not degenerate variants, a reasonable number will also encode a
polypeptide having VEGF-2 protein activity. This is because the
skilled artisan is fully aware of amino acid substitutions that are
either less likely or not likely to significantly effect protein
function (e.g., replacing one aliphatic amino acid with a second
aliphatic amino acid).
[0113] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in Bowie, J. U. et al.,
"Deciphering the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990), wherein the
authors indicate that proteins are surprisingly tolerant of amino
acid substitutions.
[0114] Thus, the present invention is directed to polynucleotides
having at least a 70% identity, preferably at least 90% and more
preferably at least a 95%, 96%, 97%, or 98% identity to a
polynucleotide which encodes the polypeptides of SEQ ID NOS:2 or 4,
as well as fragments thereof, which fragments have at least 30
bases and preferably at least 50 bases and to polypeptides encoded
by such polynucleotides.
[0115] "Identity" per se has an art-recognized meaning and can be
calculated using published techniques. (See, e.g.: (COMPUTATIONAL
MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New
York, (1988); BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith,
D. W., ed., Academic Press, New York, (1993); COMPUTER ANALYSIS OF
SEQUENCE DATA, PART 1, Griffin, A. M., and Griffin, H. G., eds.,
Humana Press, New Jersey, (1994); SEQUENCE ANALYSIS IN MOLECULAR
BIOLOGY, von Heinje, G., Academic Press, (1987); and SEQUENCE
ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New York, (1991).) While there exists a number of methods to
measure identity between two polynucleotide or polypeptide
sequences, the term "identity" is well known to skilled artisans.
(Carillo, H., and Lipton, D., SIAM J. Applied Math. 48:1073
(1988).) Methods commonly employed to determine identity or
similarity between two sequences include, but are not limited to,
those disclosed in "Guide to Huge Computers," Martin J. Bishop,
ed., Academic Press, San Diego, (1994), and Carillo, H., and
Lipton, D., SIAM J. Applied Math. 48:1073 (1988). Methods for
aligning polynucleotides or polypeptides are codified in computer
programs, including the GCG program package (Devereux, J., et al.,
Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA
(Atschul, S. F. et al., J. Molec. Biol. 215:403 (1990), Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711 (using the local homology algorithm of
Smith and Waterman, Advances in Applied Mathematics 2:482-489
(1981)). By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence of
the present invention, it is intended that the nucleotide sequence
of the polynucleotide is identical to the reference sequence except
that the polynucleotide sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleotide
sequence encoding the VEGF-2 polypeptide. In other words, to obtain
a polynucleotide having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. The query sequence may be an entire
sequence SEQ ID NO:1, the ORF (open reading frame), or any fragment
specified as described herein.
[0116] As a practical matter, whether any particular nucleic acid
molecule or polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99%
identical to a nucleotide sequence of the presence invention can be
determined conventionally using known computer programs. A
preferred method for determining the best overall match between a
query sequence (a sequence of the present invention) and a subject
sequence, also referred to as a global sequence alignment, can be
determined using the FASTDB computer program based on the algorithm
of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a
sequence alignment the query and subject sequences are both DNA
sequences. An RNA sequence can be compared by converting U's to
T's. The result of said global sequence alignment is in percent
identity. Preferred parameters used in a FASTDB alignment of DNA
sequences to calculate percent identity are: Matrix=Unitary,
k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization
Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty
0.05, Window Size=500 or the length of the subject nucleotide
sequence, whichever is shorter.
[0117] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only bases outside the 5' and 3' bases of the
subject sequence, as displayed by the FASTDB alignment, which are
not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
[0118] For example, a 90 base subject sequence is aligned to a 100
base query sequence to determine percent identity. The deletions
occur at the 5' end of the subject sequence and therefore, the
FASTDB alignment does not show a matched/alignment of the first 10
bases at 5' end. The 10 unpaired bases represent 10% of the
sequence (number of bases at the 5' and 3' ends not matched/total
number of bases in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject
sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the
5' or 3' of the subject sequence which are not matched/aligned with
the query. In this case the percent identity calculated by FASTDB
is not manually corrected. Once again, only bases 5' and 3' of the
subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are to made for the purposes of the present invention.
[0119] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence of the
present invention, it is intended that the amino acid sequence of
the subject polypeptide is identical to the query sequence except
that the subject polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the query amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a query amino acid
sequence, up to 5% of the amino acid residues in the subject
sequence may be inserted, deleted, (indels) or substituted with
another amino acid. These alterations of the reference sequence may
occur at the amino or carboxy terminal positions of the reference
amino acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0120] As a practical matter, whether any particular polypeptide is
at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the amino acid sequences shown in Table 1 or to the amino acid
sequence encoded by deposited DNA clone can be determined
conventionally using known computer programs. A preferred method
for determining the best overall match between a query sequence (a
sequence of the present invention) and a subject sequence, also
referred to as a global sequence alignment, can be determined using
the FASTDB computer program based on the algorithm of Brutlag et
al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence alignment
the query and subject sequences are either both nucleotide
sequences or both amino acid sequences. The result of said global
sequence alignment is in percent identity. Preferred parameters
used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2,
Mismatch Penalty=1, Joining Penalty=20, Randomization Group
Length=0, Cutoff Score=1, Window Size=sequence length, Gap
Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of
the subject amino acid sequence, whichever is shorter.
[0121] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N- and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the query sequence, the percent identity is corrected
by calculating the number of residues of the query sequence that
are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence.
[0122] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C- termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are to made for the purposes of the
present invention.
VEGF-2 Polypeptides
[0123] The present invention further relates to polypeptides which
have the deduced amino acid sequence of FIGS. 1 or 2, or which has
the amino acid sequence encoded by the deposited cDNAs, as well as
fragments, analogs, and derivatives of such polypeptides.
[0124] The terms "fragment," "derivative" and "analog" when
referring to the polypeptide of FIGS. 1 or 2 or that encoded by the
deposited cDNA, means a polypeptide which retains the conserved
motif of VEGF proteins as shown in FIG. 3 and essentially the same
biological function or activity.
[0125] In the present invention, a "polypeptide fragment" refers to
a short amino acid sequence contained in SEQ ID NO:2 or encoded by
the cDNA contained in the deposited clone. Protein fragments may be
"free-standing," or comprised within a larger polypeptide of which
the fragment forms a part or region, most preferably as a single
continuous region. Representative examples of polypeptide fragments
of the invention, include, for example, fragments from about amino
acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140,
141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, or
281 to the end of the coding region. Moreover, polypeptide
fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,
120, 130, 140, or 150 amino acids in length. In this context
"about" includes the particularly recited ranges, larger or smaller
by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at
both extremes.
[0126] Preferred polypeptide fragments include the secreted VEGF-2
protein as well as the mature form. Further preferred polypeptide
fragments include the secreted VEGF-2 protein or the mature form
having a continuous series of deleted residues from the amino or
the carboxy terminus, or both. For example, any number of amino
acids, ranging from 1-60, can be deleted from the amino terminus of
either the secreted VEGF-2 polypeptide or the mature form.
Similarly, any number of amino acids, ranging from 1-30, can be
deleted from the carboxy terminus of the secreted VEGF-2 protein or
mature form. Furthermore, any combination of the above amino and
carboxy terminus deletions are preferred. Similarly, polynucleotide
fragments encoding these VEGF-2 polypeptide fragments are also
preferred.
[0127] Also preferred are VEGF-2 polypeptide and polynucleotide
fragments characterized by structural or functional domains, such
as fragments that comprise alpha-helix and alpha-helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and
turn-forming regions, coil and coil-forming regions, hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions,
substrate binding region, and high antigenic index regions.
Polypeptide fragments of SEQ ID NO:2 falling within conserved
domains are specifically contemplated by the present invention.
(See FIG. 2.) Moreover, polynucleotide fragments encoding these
domains are also contemplated.
[0128] Other preferred fragments are biologically active VEGF-2
fragments. Biologically active fragments are those exhibiting
activity similar, but not necessarily identical, to an activity of
the VEGF-2 polypeptide. The biological activity of the fragments
may include an improved desired activity, or a decreased
undesirable activity.
[0129] The polypeptides of the present invention may be recombinant
polypeptides, natural polypeptides, or synthetic polypeptides,
preferably recombinant polypeptides.
[0130] It will be recognized in the art that some amino acid
sequences of the VEGF-2 polypeptide can be varied without
significant effect of the structure or function of the protein. If
such differences in sequence are contemplated, it should be
remembered that there will be critical areas on the protein which
determine activity.
[0131] Thus, the invention further includes variations of the
VEGF-2 polypeptide which show substantial VEGF-2 polypeptide
activity or which include regions of VEGF-2 protein such as the
protein portions discussed below. Such mutants include deletions,
insertions, inversions, repeats, and type substitutions. As
indicated above, guidance concerning which amino acid changes are
likely to be phenotypically silent can be found in Bowie, J. U., et
al., "Deciphering the Message in Protein Sequences: Tolerance to
Amino Acid Substitutions," Science 247:1306-1310 (1990).
[0132] Thus, the fragments, derivatives, or analogs of the
polypeptides of FIGS. 1 or 2, or that encoded by the deposited
cDNAs may be: (I) one in which one or more of the amino acid
residues are substituted with a conserved or non-conserved amino
acid residue (preferably a conserved amino acid residue) and such
substituted amino acid residue may or may not be one encoded by the
genetic code; or (ii) one in which one or more of the amino acid
residues includes a substituent group; or (iii) one in which the
mature polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol); or (iv) one in which the additional amino
acids are fused to the mature polypeptide, such as a leader or
secretory sequence or a sequence which is employed for purification
of the mature polypeptide or a proprotein sequence; or (v) one in
which comprises fewer amino acid residues shown in SEQ ID NOS: 2 or
4, and retains the conserved motif and yet still retains activity
characteristics of the VEGF family of polypeptides. Such fragments,
derivatives, and analogs are deemed to be within the scope of those
skilled in the art from the teachings herein.
[0133] Of particular interest are substitutions of charged amino
acids with another charged amino acid and with neutral or
negatively charged amino acids. The latter results in proteins with
reduced positive charge to improve the characteristics of the
VEGF-2 protein. The prevention of aggregation is highly desirable.
Aggregation of proteins not only results in a loss of activity but
can also be problematic when preparing pharmaceutical formulations,
because they can be immunogenic. (Pinckard et al., Clin. Exp.
Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845
(1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993)).
[0134] The replacement of amino acids can also change the
selectivity of binding to cell surface receptors. Ostade et al.,
Nature 361:266-268 (1993) describes certain mutations resulting in
selective binding of TNF-a to only one of the two known types of
TNF receptors. Thus, the VEGF-2 of the present invention may
include one or more amino acid substitutions, deletions or
additions, either from natural mutations or human manipulation.
[0135] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 1).
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0136] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of substitutions
for any given VEGF-2 polypeptide will not be more than 50,40, 30,
25, 20, 15, 10, 5 or 3.
[0137] Amino acids in the VEGF-2 protein of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as receptor binding or in
vitro, or in vitro proliferative activity. Sites that are critical
for ligand-receptor binding can also be determined by structural
analysis such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904
(1992) and de Vos et al. Science 255:306-312 (1992)).
[0138] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
[0139] The term "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 DNA or polypeptide,
separated from some or all of the coexisting materials in the
natural system, is isolated. Such polynucleotide could be part of a
vector and/or such polynucleotide or polypeptide could be part of a
composition, and still be isolated in that such vector or
composition is not part of its natural environment.
[0140] In specific embodiments, the polynucleotides of the
invention are less than 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10
kb, or 7.5 kb in length. In a further embodiment, polynucleotides
of the invention comprise at least 15 contiguous nucleotides of
VEGF-2 coding sequence, but do not comprise all or a portion of any
VEGF-2 intron. In another embodiment, the nucleic acid comprising
VEGF-2 coding sequence does not contain coding sequences of a
genomic flanking gene (i.e., 5' or 3' to the VEGF-2 gene in the
genome).
[0141] The polypeptides of the present invention include the
polypeptides of SEQ ID NOS:2 and 4 (in particular the mature
polypeptide) as well as polypeptides which have at least 70%
similarity (preferably at least 70% identity) to the polypeptides
of SEQ ID NOS:2 and 4, and more preferably at least 90% similarity
(more preferably at least 95% identity) to the polypeptides of SEQ
ID NOS:2 and 4, and still more preferably at least 95% similarity
(still more preferably at least 90% identity) to the polypeptides
of SEQ ID NOS:2 and 4 and also include portions of such
polypeptides with such portion of the polypeptide generally
containing at least 30 amino acids and more preferably at least 50
amino acids.
[0142] As known in the art "similarity" between two polypeptides is
determined by comparing the amino acid sequence and its conserved
amino acid substitutes of one polypeptide to the sequence of a
second polypeptide.
[0143] 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.
[0144] The polypeptides of the present invention include the
polypeptide encoded by the deposited cDNA including the leader; the
mature polypeptide encoded by the deposited the cDNA minus the
leader (i.e., the mature protein); a polypeptide comprising amino
acids about -23 to about 396 in SEQ ID NO:2; a polypeptide
comprising amino acids about -22 to about 396 in SEQ ID NO:2; a
polypeptide comprising amino acids about 1 to about 396 in SEQ ID
NO:2; as well as polypeptides which are at least 95% identical, and
more preferably at least 96%, 97%, 98% or 99% identical to the
polypeptides described above and also include portions of such
polypeptides with at least 30 amino acids and more preferably at
least 50 amino acids.
Fusion Proteins
[0145] Any VEGF-2 polypeptide can be used to generate fusion
proteins. For example, the VEGF-2 polypeptide, when fused to a
second protein, can be used as an antigenic tag. Antibodies raised
against the VEGF-2 polypeptide can be used to indirectly detect the
second protein by binding to the VEGF-2. Moreover, because secreted
proteins target cellular locations based on trafficking signals,
the VEGF-2 polypeptides can be used as a targeting molecule once
fused to other proteins.
[0146] Examples of domains that can be fused to VEGF-2 polypeptides
include not only heterologous signal sequences, but also other
heterologous functional regions. The fusion does not necessarily
need to be direct, but may occur through linker sequences.
[0147] Moreover, fusion proteins may also be engineered to improve
characteristics of the VEGF-2 polypeptide. For instance, a region
of additional amino acids, particularly charged amino acids, may be
added to the N-terminus of the VEGF-2 polypeptide to improve
stability and persistence during purification from the host cell or
subsequent handling and storage. Also, peptide moieties may be
added to the VEGF-2 polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the VEGF-2
polypeptide. The addition of peptide moieties to facilitate
handling of polypeptides are familiar and routine techniques in the
art.
[0148] Moreover, VEGF-2 polypeptides, including fragments, and
specifically epitopes, can be combined with parts of the constant
domain of immunoglobulins (IgG), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and
show an increased half-life in vivo. One reported example describes
chimeric proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins. (EP A 394,827;
Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having
disulfide-linked dimeric structures (due to the IgG) can also be
more efficient in binding and neutralizing other molecules, than
the monomeric secreted protein or protein fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)
[0149] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)
discloses fusion proteins comprising various portions of constant
region of imunoglobulin molecules together with another human
protein or part thereof. In many cases, the Fc part in a fusion
protein is beneficial in therapy and diagnosis, and thus can result
in, for example, improved pharmacokinetic properties. (EP-A 0232
262.) Alternatively, deleting the Fc part after the fusion protein
has been expressed, detected, and purified, would be desired. For
example, the Fc portion may hinder therapy and diagnosis if the
fusion protein is used as an antigen for immunizations. In drug
discovery, for example, human proteins, such as hIL-5, have been
fused with Fc portions for the purpose of high-throughput screening
assays to identify antagonists of hIL-5. (See, D. Bennett et al.,
J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J.
Biol. Chem. 270:9459-9471 (1995).)
[0150] Moreover, the VEGF-2 polypeptides can be fused to marker
sequences, such as a peptide which facilitates purification of
VEGF-2. In preferred embodiments, the marker amino acid sequence is
a hexa-histidine peptide, such as the tag provided in a pQE vector
(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among
others, many of which are commercially available. As described in
Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for
instance, hexa-histidine provides for convenient purification of
the fusion protein. Another peptide tag useful for purification,
the "HA" tag, corresponds to an epitope derived from the influenza
hemagglutinin protein. (Wilson et al., Cell 37:767 (1984).)
[0151] Thus, any of these above fusions can be engineered using the
VEGF-2 polynucleotides or the polypeptides.
Biological Activities of VEGF-2
[0152] VEGF-2 polynucleotides and polypeptides can be used in
assays to test for one or more biological activities. If VEGF-2
polynucleotides and polypeptides do exhibit activity in a
particular assay, it is likely that VEGF-2 may be involved in the
diseases associated with the biological activity. Therefore, VEGF-2
could be used to treat the associated disease.
Immune Activity
[0153] VEGF-2 polypeptides or polynucleotides may be useful in
treating deficiencies or disorders of the immune system, by
activating or inhibiting the proliferation, differentiation, or
mobilization (chemotaxis) of immune cells. Immune cells develop
through a process called hematopoiesis, producing myeloid
(platelets, red blood cells, neutrophils, and macrophages) and
lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
The etiology of these immune deficiencies or disorders may be
genetic, somatic, such as cancer or some autoimmune disorders,
acquired (e.g., by chemotherapy or toxins), or infectious.
Moreover, VEGF-2 polynucleotides or polypeptides can be used as a
marker or detector of a particular immune system disease or
disorder.
[0154] VEGF-2 polynucleotides or polypeptides may be useful in
treating or detecting deficiencies or disorders of hematopoietic
cells. VEGF-2 polypeptides or polynucleotides could be used to
increase differentiation and proliferation of hematopoietic cells,
including the pluripotent stem cells, in an effort to treat those
disorders associated with a decrease in certain (or many) types
hematopoietic cells. Examples of immunologic deficiency syndromes
include, but are not limited to: blood protein disorders (e.g.
agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia,
common variable immunodeficiency, Digeorge Syndrome, HIV infection,
HTLV-BLV infection, leukocyte adhesion deficiency syndrome,
lymphopenia, phagocyte bactericidal dysfunction, severe combined
immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia,
thrombocytopenia, or hemoglobinuria.
[0155] Moreover, VEGF-2 polypeptides or polynucleotides can also be
used to modulate hemostatic (the stopping of bleeding) or
thrombolytic activity (clot formation). For example, by increasing
hemostatic or thrombolytic activity, VEGF-2 polynucleotides or
polypeptides could be used to treat blood coagulation disorders
(e.g., afibrinogenemia, factor deficiencies), blood platelet
disorders (e.g. thrombocytopenia), or wounds resulting from trauma,
surgery, or other causes. Alternatively, VEGF-2 polynucleotides or
polypeptides that can decrease hemostatic or thrombolytic activity
could be used to inhibit or dissolve clotting, important in the
treatment of heart attacks (infarction), strokes, or scarring.
[0156] VEGF-2 polynucleotides or polypeptides may also be useful in
treating or detecting autoimmune disorders. Many autoimmune
disorders result from inappropriate recognition of self as foreign
material by immune cells. This inappropriate recognition results in
an immune response leading to the destruction of the host tissue.
Therefore, the administration of VEGF-2 polypeptides or
polynucleotides that can inhibit an immune response, particularly
the proliferation, differentiation, or chemotaxis of T-cells, may
be an effective therapy in preventing autoimmune disorders.
[0157] Examples of autoimmune disorders that can be treated or
detected by VEGF-2 include, but are not limited to: Addison's
Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid
arthritis, dermatitis, allergic encephalomyelitis,
glomerulonephritis, Goodpasture's Syndrome, Graves' Disease,
Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia,
Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura,
Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis,
Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,
Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and
autoimmune inflammatory eye disease.
[0158] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated by VEGF-2 polypeptides or polynucleotides.
Moreover, VEGF-2 can be used to treat anaphylaxis, hypersensitivity
to an antigenic molecule, or blood group incompatibility.
[0159] VEGF-2 polynucleotides or polypeptides may also be used to
treat and/or prevent organ rejection or graft-versus-host disease
(GVHD). Organ rejection occurs by host immune cell destruction of
the transplanted tissue through an immune response. Similarly, an
immune response is also involved in GVHD, but, in this case, the
foreign transplanted immune cells destroy the host tissues. The
administration of VEGF-2 polypeptides or polynucleotides that
inhibits an immune response, particularly the proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing organ rejection or GVHD. Similarly, VEGF-2
polypeptides or polynucleotides may also be used to modulate
inflammation. For example, VEGF-2 polypeptides or polynucleotides
may inhibit the proliferation and differentiation of cells involved
in an inflammatory response. These molecules can be used to treat
inflammatory conditions, both chronic and acute conditions,
including inflammation associated with infection (e.g., septic
shock, sepsis, or systemic inflammatory response syndrome (SIRS)),
ischemia-reperfusion injury, endotoxin lethality, arthritis,
complement-mediated hyperacute rejection, nephritis, cytokine or
chemokine induced lung injury, inflammatory bowel disease, Crohn's
disease, or resulting from over production of cytokines (e.g., TNF
or IL-1.)
Hyperproliferative Disorders
[0160] VEGF-2 polypeptides or polynucleotides can be used to treat
or detect hyperproliferative disorders, including neoplasms. VEGF-2
antagonist polypeptides or polynucleotides may inhibit the
proliferation of the disorder through direct or indirect
interactions. Alternatively, VEGF-2 antagonist polypeptides or
polynucleotides may proliferate other cells which can inhibit the
hyperproliferative disorder.
[0161] For example, by increasing an immune response, particularly
increasing antigenic qualities of the hyperproliferative disorder
or by proliferating, differentiating, or mobilizing T-cells,
hyperproliferative disorders can be treated. This immune response
may be increased by either enhancing an existing immune response,
or by initiating a new immune response. Alternatively, decreasing
an immune response may also be a method of treating
hyperproliferative disorders, such as a chemotherapeutic agent.
[0162] Examples of hyperproliferative disorders that can be treated
or detected by VEGF-2 antagonist polynucleotides or polypeptides
include, but are not limited to neoplasms located in the: abdomen,
bone, breast, digestive system, liver, pancreas, peritoneum,
endocrine glands (adrenal, parathyroid, pituitary, testicles,
ovary, thymus, thyroid), eye, head and neck, nervous (central and
peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,
thoracic, and urogenital.
[0163] Similarly, other hyperproliferative disorders can also be
treated or detected by VEGF-2 antagonist polynucleotides or
polypeptides. Examples of such hyperproliferative disorders
include, but are not limited to: hypergammaglobulinemia,
lymphoproliferative disorders, paraproteinemias, purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,
Gaucher's Disease, histiocytosis, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
Infectious Disease
[0164] VEGF-2 polypeptides or polynucleotides can be used to treat
or detect infectious agents. For example, by increasing the immune
response, particularly increasing the proliferation and
differentiation of B and/or T cells, infectious diseases may be
treated. The immune response may be increased by either enhancing
an existing immune response, or by initiating a new immune
response. Alternatively, VEGF-2 polypeptides or polynucleotides may
also directly inhibit the infectious agent, without necessarily
eliciting an immune response.
[0165] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated or detected by VEGF-2
polynucleotides or polypeptides. Examples of viruses, include, but
are not limited to the following DNA and RNA viral families:
Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae,
Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as,
Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus
(e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),
Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae,
Picornaviridae, Poxviridae (such as Smallpox or Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,
Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling
within these families can cause a variety of diseases or symptoms,
including, but not limited to: arthritis, bronchiollitis,
encephalitis, eye infections (e.g., conjunctivitis, keratitis),
chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active,
Delta), meningitis, opportunistic infections (e.g., AIDS),
pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever,
Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio,
leukemia, Rubella, sexually transmitted diseases, skin diseases
(e.g., Kaposi's, warts), and viremia. VEGF-2 polypeptides or
polynucleotides can be used to treat or detect any of these
symptoms or diseases.
[0166] Similarly, bacterial or fungal agents that can cause disease
or symptoms and that can be treated or detected by VEGF-2
polynucleotides or polypeptides include, but not limited to, the
following Gram-Negative and Gram-positive bacterial families and
fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,
Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax,
Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia,
Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis,
Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella,
Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter,
Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,
Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal),
Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus,
Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis,
and Staphylococcal. These bacterial or fungal families can cause
the following diseases or symptoms, including, but not limited to:
bacteremia, endocarditis, eye infections (conjunctivitis,
tuberculosis, uveitis), gingivitis, opportunistic infections (e.g.,
AIDS related infections), paronychia, prosthesis-related
infections, Reiter's Disease, respiratory tract infections, such as
Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch
Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid,
pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria,
Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene,
tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually
transmitted diseases, skin diseases (e.g., cellulitis,
dermatocycoses), toxemia, urinary tract infections, wound
infections. VEGF-2 polypeptides or polynucleotides can be used to
treat or detect any of these symptoms or diseases.
[0167] Moreover, parasitic agents causing disease or symptoms that
can be treated or detected by VEGF-2 polynucleotides or
polypeptides include, but not limited to, the following families:
Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis,
Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis,
Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and
Trichomonas. These parasites can cause a variety of diseases or
symptoms, including, but not limited to: Scabies, Trombiculiasis,
eye infections, intestinal disease (e.g., dysentery, giardiasis),
liver disease, lung disease, opportunistic infections (e.g., AIDS
related), Malaria, pregnancy complications, and toxoplasmosis.
VEGF-2 polypeptides or polynucleotides can be used to treat or
detect any of these symptoms or diseases.
[0168] Preferably, treatment using VEGF-2 polypeptides or
polynucleotides could either be by administering an effective
amount of VEGF-2 polypeptide to the patient, or by removing cells
from the patient, supplying the cells with VEGF-2 polynucleotide,
and returning the engineered cells to the patient (ex vivo
therapy). Moreover, the VEGF-2 polypeptide or polynucleotide can be
used as an antigen in a vaccine to raise an immune response against
infectious disease.
Regeneration
[0169] VEGF-2 polynucleotides or polypeptides can be used to
differentiate, proliferate, and attract cells, leading to the
regeneration of tissues. (See, Science 276:59-87 (1997).) The
regeneration of tissues could be used to repair, replace, or
protect tissue damaged by congenital defects, trauma (wounds,
burns, incisions, or ulcers), age, disease (e.g. osteoporosis,
osteocarthritis, periodontal disease, liver failure), surgery,
including cosmetic plastic surgery, fibrosis, reperfusion injury,
or systemic cytokine damage.
[0170] Tissues that could be regenerated using the present
invention include organs (e.g., pancreas, liver, intestine, kidney,
skin, endothelium), muscle (smooth, skeletal or cardiac), vascular
(including vascular endothelium), lymphatic (including lymphatic
endothelium), nervous, hematopoietic, and skeletal (bone,
cartilage, tendon, and ligament) tissue. Preferably, regeneration
occurs without or decreased scarring. Regeneration also may include
angiogenesis.
[0171] Moreover, VEGF-2 polynucleotides or polypeptides may
increase regeneration of tissues difficult to heal. For example,
increased tendon/ligament regeneration would quicken recovery time
after damage. VEGF-2 polynucleotides or polypeptides of the present
invention could also be used prophylactically in an effort to avoid
damage. Specific diseases that could be treated include of
tendinitis, carpal tunnel syndrome, and other tendon or ligament
defects. A further example of tissue regeneration of non-healing
wounds includes pressure ulcers, ulcers associated with vascular
insufficiency, surgical, and traumatic wounds.
[0172] Similarly, nerve and brain tissue could also be regenerated
by using VEGF-2 polynucleotides or polypeptides to proliferate and
differentiate nerve cells. Diseases that could be treated using
this method include central and peripheral nervous system diseases,
neuropathies, or mechanical and traumatic disorders (e.g., spinal
cord disorders, head trauma, cerebrovascular disease, and stoke).
Specifically, diseases associated with peripheral nerve injuries,
peripheral neuropathy (e.g., resulting from chemotherapy or other
medical therapies), localized neuropathies, and central nervous
system diseases (e.g., Alzheimer's disease, Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager
syndrome), could all be treated using the VEGF-2 polynucleotides or
polypeptides.
Chemotaxis
[0173] VEGF-2 polynucleotides or polypeptides may have chemotaxis
activity. A chemotaxic molecule attracts or mobilizes cells (e.g.,
monocytes, fibroblasts, neutrophils, T-cells, mast cells,
eosinophils, epithelial and/or endothelial cells) to a particular
site in the body, such as inflammation, infection, or site of
hyperproliferation. The mobilized cells can then fight off and/or
heal the particular trauma or abnormality.
[0174] VEGF-2 polynucleotides or polypeptides may increase
chemotaxic activity of particular cells. These chemotactic
molecules can then be used to treat inflammation, infection,
hyperproliferative disorders, or any immune system disorder by
increasing the number of cells targeted to a particular location in
the body. For example, chemotaxic molecules can be used to treat
wounds and other trauma to tissues by attracting immune cells to
the injured location. As a chemotactic molecule, VEGF-2 could also
attract fibroblasts, which can be used to treat wounds.
[0175] It is also contemplated that VEGF-2 polynucleotides or
polypeptides may inhibit chemotactic activity. These molecules
could also be used to treat disorders. Thus, VEGF-2 polynucleotides
or polypeptides could be used as an inhibitor of chemotaxis.
Binding Activity
[0176] VEGF-2 polypeptides may be used to screen for molecules that
bind to VEGF-2 or for molecules to which VEGF-2 binds. The binding
of VEGF-2 and the molecule may activate (agonist), increase,
inhibit (antagonist), or decrease activity of the VEGF-2 or the
molecule bound. Examples of such molecules include antibodies,
oligonucleotides, proteins (e.g., receptors),or small
molecules.
[0177] Preferably, the molecule is closely related to the natural
ligand of VEGF-2, e.g., a fragment of the ligand, or a natural
substrate, a ligand, a structural or functional mimetic. (See,
Coligan et al., Current Protocols in Immunology 1(2):Chapter 5
(1991).) Similarly, the molecule can be closely related to the
natural receptor to which VEGF-2 binds (i.e., Flt-4), or at least,
a fragment of the receptor capable of being bound by VEGF-2 (e.g.,
active site). In either case, the molecule can be rationally
designed using known techniques. Preferably, the screening for
these molecules involves producing appropriate cells which express
VEGF-2, either as a secreted protein or on the cell membrane.
Preferred cells include cells from mammals, yeast, Drosophila, or
E. coli. Cells expressing VEGF-2 (or cell membrane containing the
expressed polypeptide) are then preferably contacted with a test
compound potentially containing the molecule to observe binding,
stimulation, or inhibition of activity of either VEGF-2 or the
molecule.
[0178] The assay may simply test binding of a candidate compound to
VEGF-2, wherein binding is detected by a label, or in an assay
involving competition with a labeled competitor. Further, the assay
may test whether the candidate compound results in a signal
generated by binding to VEGF-2.
[0179] Alternatively, the assay can be carried out using cell-free
preparations, polypeptide/molecule affixed to a solid support,
chemical libraries, or natural product mixtures. The assay may also
simply comprise the steps of mixing a candidate compound with a
solution containing VEGF-2, measuring VEGF-2/molecule activity or
binding, and comparing the VEGF-2/molecule activity or binding to a
standard.
[0180] Preferably, an ELISA assay can measure VEGF-2 level or
activity in a sample (e.g., biological sample) using a monoclonal
or polyclonal antibody. The antibody can measure VEGF-2 level or
activity by either binding, directly or indirectly, to VEGF-2 or by
competing with VEGF-2 for a substrate.
[0181] All of these above assays can be used as diagnostic or
prognostic markers. The molecules discovered using these assays can
be used to treat disease or to bring about a particular result in a
patient (e.g., blood vessel growth) by activating or inhibiting the
VEGF-2/molecule. Moreover, the assays can discover agents which may
inhibit or enhance the production of VEGF-2 from suitably
manipulated cells or tissues.
[0182] Therefore, the invention includes a method of identifying
compounds which bind to VEGF-2 comprising the steps of: (a)
incubating a candidate binding compound with VEGF-2; and (b)
determining if binding has occurred. Moreover, the invention
includes a method of identifying agonists/antagonists comprising
the steps of: (a) incubating a candidate compound with VEGF-2, (b)
assaying a biological activity, and (b) determining if a biological
activity of VEGF-2 has been altered.
Other Activities
[0183] VEGF-2 polypeptides or polynucleotides may also increase or
decrease the differentiation or proliferation of embryonic stem
cells, besides, as discussed above, hematopoietic lineage.
[0184] VEGF-2 polypeptides or polynucleotides may also be used to
modulate mammalian characteristics, such as body height, weight,
hair color, eye color, skin, percentage of adipose tissue,
pigmentation, size, and shape (e.g., cosmetic surgery). Similarly,
VEGF-2 polypeptides or polynucleotides may be used to modulate
mammalian metabolism affecting catabolism, anabolism, processing,
utilization, and storage of energy.
[0185] VEGF-2 polypeptides or polynucleotides may be used to change
a mammal's mental state or physical state by influencing
biorhythms, caricadic rhythms, depression (including depressive
disorders), tendency for violence, tolerance for pain, reproductive
capabilities (preferably by Activin or Inhibin-like activity),
hormonal or endocrine levels, appetite, libido, memory, stress, or
other cognitive qualities.
[0186] VEGF-2 polypeptides or polynucleotides may also be used as a
food additive or preservative, such as to increase or decrease
storage capabilities, fat content, lipid, protein, carbohydrate,
vitamins, minerals, cofactors or other nutritional components.
Vectors, Host Cells, and Protein Production
[0187] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of VEGF-2 polypeptides or peptides by
recombinant techniques.
[0188] Host cells are genetically engineered (transduced,
transformed, or transfected) with the vectors of this invention
which may be, for example, a cloning vector or an expression
vector. The vector may be, for example, in the form of a plasmid, a
viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants, or amplifying the
VEGF-2 genes of the invention. The culture conditions, such as
temperature, pH and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
skilled artisan.
[0189] The polynucleotides of the present invention may be employed
for producing polypeptides by recombinant techniques. Thus, for
example, the polynucleotide sequence may be included in any one of
a variety of expression vectors for expressing a polypeptide. Such
vectors include chromosomal, nonchromosomal and synthetic DNA
sequences, e.g., derivatives of SV40; bacterial plasmids; phage
DNA; yeast plasmids; vectors derived from combinations of plasmids
and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox
virus, and pseudorabies. However, any other plasmid or vector may
be used so long as it is replicable and viable in the host.
[0190] 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 site(s) by
procedures known in the art. Such procedures and others are deemed
to be within the scope of those skilled in the art.
[0191] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct mRNA synthesis. As representative examples of such
promoters, there may be mentioned: LTR or SV40 promoter, the E.
coli. lac or trp, the phage lambda P.sub.L promoter and other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses. The expression vector also
contains a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
[0192] In addition, the expression vectors preferably contain at
least one selectable marker gene to provide a phenotypic trait for
selection of transformed host cells. Such markers include
dihydrofolate reductase (DHFR) or neomycin resistance for
eukaryotic cell culture, and tetracycline or ampicillin resistance
for culturing in E. coli and other bacteria.
[0193] The vector containing the appropriate DNA sequence as herein
above described, as well as an appropriate promoter or control
sequence, may be employed to transform an appropriate host to
permit the host to express the protein. Representative examples of
appropriate hosts, include but are not limited to: bacterial cells,
such as E. coli, Salmonella typhimurium, and Streptomyces; fungal
cells, such as yeast; insect cells, such as Drosophila S2 and
Spodoptera Sf9; animal cells such as CHO, COS, and Bowes melanoma;
and plant cells. The selection of an appropriate host is deemed to
be within the scope of those skilled in the art from the teachings
herein.
[0194] 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: pQE70, pQE60 and pQE-9, available from
Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors,
pNH8A, pNH16a, pNH18A, pNH46A, available from STRATAGENE.TM.; and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from
PHARMACIA.TM.. Among preferred eukaryotic vectors are pWLNEO,
pSV2CAT, pOG44, pXT1 and pSG available from STRATAGENE.TM.; and
pSVK3, pBPV, pMSG and pSVL available from PHARMACIA.TM.. Other
suitable vectors will be readily apparent to the skilled
artisan.
[0195] In addition to the use of expression vectors in the practice
of the present invention, the present invention further includes
novel expression vectors comprising operator and promoter elements
operatively linked to nucleotide sequences encoding a protein of
interest. One example of such a vector is pHE4a which is described
in detail below.
[0196] As summarized in FIGS. 28 and 29, components of the pHE4a
vector (SEQ ID NO:16) include: 1) a neomycinphosphotransferase gene
as a selection marker, 2) an E. coli origin of replication, 3) a T5
phage promoter sequence, 4) two lac operator sequences, 5) a
Shine-Delgarno sequence, 6) the lactose operon repressor gene
(lacIq) and 7) a multiple cloning site linker region. The origin of
replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.).
The promoter sequence and operator sequences were made
synthetically. Synthetic production of nucleic acid sequences is
well known in the art. CLONTECH.TM. 95/96 Catalog, pages 215-216,
CLONTECH.TM., 1020 East Meadow Circle, Palo Alto, Calif. 94303. The
pHE4a vector was deposited with the ATCC.TM. on Feb. 25, 1998, and
given accession number 209645.
[0197] A nucleotide sequence encoding VEGF-2 (SEQ ID NO:1), is
operatively linked to the promoter and operator of pHE4a by
restricting the vector with NdeI and either XbaI, BamHI, XhoI, or
Asp718, and isolating the larger fragment (the multiple cloning
site region is about 310 nucleotides) on a gel. The nucleotide
sequence encoding VEGF-2 (SEQ ID NO:1) having the appropriate
restriction sites is generated, for example, according to the PCR
protocol described in Example 1, using PCR primers having
restriction sites for NdeI (as the 5' primer) and either XbaI,
BamHI, XhoI, or Asp718 (as the 3' primer). The PCR insert is gel
purified and restricted with compatible enzymes. The insert and
vector are ligated according to standard protocols.
[0198] As noted above, the pHE4a vector contains a LacIq gene.
LacIq is an allele of the lacI gene which confers tight regulation
of the lac operator. Amann, E. et al., Gene 69:301-315 (1988);
Stark, M., Gene 51:255-267 (1987). The lacIq gene encodes a
repressor protein which binds to lac operator sequences and blocks
transcription of down-stream (i.e., 3') sequences. However, the
lacIq gene product dissociates from the lac operator in the
presence of either lactose or certain lactose analogs, e.g.,
isopropyl B-D-thiogalactopyranoside (IPTG). VEGF-2 thus is not
produced in appreciable quantities in uninduced host cells
containing the pHE4a vector. Induction of these host cells by the
addition of an agent such as IPTG, however, results in the
expression of the VEGF-2 coding sequence.
[0199] The promoter/operator sequences of the pHE4a vector (SEQ ID
NO: 17) comprise a T5 phage promoter and two operator sequences.
One operator is located 5' to the transcriptional start site and
the other is located 3' to the same site. These operators, when
present in combination with the LacIq gene product, confer tight
repression of down-stream sequences in the absence of a lac operon
inducer, e.g., IPTG. Expression of operatively linked sequences
located down-stream from the lac operators may be induced by the
addition of a lac operon inducer, such as IPTG. Binding of a lac
inducer to the lacIq proteins results in their release from the lac
operator sequences and the initiation of transcription of
operatively linked sequences. Lac operon regulation of gene
expression is reviewed in Devlin, T., TEXTBOOK OF BIOCHEMISTRY WITH
CLINICAL CORRELATIONS, 4th Edition (1997), pages 802-807.
[0200] The pHE4 series of vectors contain all of the components of
the pHE4a vector except for the VEGF-2 coding sequence. Features of
the pHE4a vectors include optimized synthetic T5 phage promoter,
lac operator, and Shine-Delagarno sequences. Further, these
sequences are also optimally spaced so that expression of an
inserted gene may be tightly regulated and high level of expression
occurs upon induction.
[0201] Among known bacterial promoters suitable for use in the
production of proteins of the present invention include the E. coli
lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter,
the lambda PR and PL promoters and the trp promoter. Suitable
eukaryotic promoters include the CMV immediate early promoter, the
HSV thymidine kinase promoter, the early and late SV40 promoters,
the promoters of retroviral LTRs, such as those of the Rous Sarcoma
Virus (RSV), and metallothionein promoters, such as the mouse
metallothionein-I promoter.
[0202] The pHE4a vector also contains a Shine-Delgarno sequence 5'
to the AUG initiation codon. Shine-Delgarno sequences are short
sequences generally located about 10 nucleotides up-stream (i.e.,
5') from the AUG initiation codon. These sequences essentially
direct prokaryotic ribosomes to the AUG initiation codon.
[0203] Thus, the present invention is also directed to expression
vector useful for the production of the proteins of the present
invention. This aspect of the invention is exemplified by the pHE4a
vector (SEQ ID NO: 16).
[0204] 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, T-7,
gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV
immediate early, HSV thymidine kinase, early and late SV40, LTRs
from retrovirus, and mouse metallothionein-l. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art.
[0205] In a further embodiment, the present invention relates to
host cells containing the above-described construct. The host cell
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection,
electroporation, transduction, infection, or other methods (Davis,
L., et al., Basic Methods in Molecular Biology (1986)).
[0206] 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.
[0207] 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 Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1989), the disclosure of which is hereby incorporated by
reference.
[0208] Transcription of a DNA encoding the polypeptides of the
present invention by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp, that act on a
promoter to increase its transcription. Examples include the SV40
enhancer on the late side of the replication origin (bp 100 to
270), a cytomegalovirus early promoter enhancer, a polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers.
[0209] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), a-factor, acid phosphatase, or heat shock proteins,
among others. The heterologous structural sequence is assembled in
appropriate phase with translation initiation and termination
sequences, and preferably, a leader sequence capable of directing
secretion of translated protein into the periplasmic space or
extracellular medium. Optionally, the heterologous sequence can
encode a fusion protein including an N-terminal identification
peptide imparting desired characteristics, e.g., stabilization or
simplified purification of expressed recombinant product.
[0210] 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 typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0211] 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.TM. 37017). Such commercial
vectors include, for example, pKK223-3 (PHARMACIA.TM. Fine
Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison,
Wis., USA). These pBR322 "backbone" sections are combined with an
appropriate promoter and the structural sequence to be
expressed.
[0212] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is derepressed by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period.
[0213] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0214] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, well known to those skilled in
the art, including freeze-thaw cycling, sonication, mechanical
disruption, or use of cell lysing agents.
[0215] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40 viral
genome, for example, SV40 origin, early promoter, enhancer, splice,
and polyadenylation sites may be used to provide the required
nontranscribed genetic elements.
[0216] In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly mammalian origin, that have been engineered to
delete or replace endogenous genetic material (e.g., VEGF-2
sequence), and/or to include genetic material (e.g., heterologous
promoters) that is operably associated with VEGF-2 sequence of the
invention, and which activates, alters, and/or amplifies endogenous
VEGF-2 polynucleotides. For example, techniques known in the art
may be used to operably associate heterologous control regions and
endogenous polynucleotide sequences (e.g. encoding VEGF-2) via
homologous recombination (see, e.g., U.S. Pat. No. 5,641,670,
issued Jun. 24, 1997; International Publication No. WO 96/29411,
published Sep. 26, 1996; International Publication No. WO 94/12650,
published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438
(1989), the disclosures of each of which are incorporated by
reference in their entireties).
[0217] The host cell can be a higher eukaryotic cell, such as a
mammalian cell (e.g., a human derived cell), or a lower eukaryotic
cell, such as a yeast cell, or the host cell can be a prokaryotic
cell, such as a bacterial cell. The host strain may be chosen which
modulates the expression of the inserted gene sequences, or
modifies and processes the gene product in the specific fashion
desired. Expression from certain promoters can be elevated in the
presence of certain inducers; thus expression of the genetically
engineered polypeptide may be controlled. Furthermore, different
host cells have characteristics and specific mechanisms for the
translational and post-translational processing and modification
(e.g., glycosylation, phosphorylation, cleavage) of proteins.
Appropriate cell lines can be chosen to ensure the desired
modifications and processing of the protein expressed.
[0218] The polypeptides can be recovered and purified from
recombinant cell cultures by methods used heretofore, including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. It is
preferred to have low concentrations (approximately 0.1-5 mM) of
calcium ion present during purification (Price et al., J. Biol.
Chem. 244:917 (1969)). Protein refolding steps can be used, as
necessary, in completing configuration of the mature protein.
Finally, high performance liquid chromatography (HPLC) can be
employed for final purification steps.
[0219] The polypeptides of the present invention may be a naturally
purified product, or a product of chemical synthetic procedures, or
produced by recombinant techniques from a prokaryotic or eukaryotic
host (for example, by bacterial, yeast, higher plant, insect and
mammalian cells in culture). Depending upon the host employed in a
recombinant production procedure, the polypeptides of the present
invention may be glycosylated with mammalian or other eukaryotic
carbohydrates or may be non-glycosylated. Polypeptides of the
invention may also include an initial methionine amino acid
residue.
[0220] In addition, polypeptides of the invention can be chemically
synthesized using techniques known in the art (e.g., see Creighton,
1983, Proteins: Structures and Molecular Principles, W.H. Freeman
& Co., N.Y., and Hunkapiller, M., et al., 1984, Nature
310:105-111). For example, a peptide corresponding to a fragment of
the VEGF-2 polypeptides of the invention can be synthesized by use
of a peptide synthesizer. Furthermore, if desired, nonclassical
amino acids or chemical amino acid analogs can be introduced as a
substitution or addition into the VEGF-2 polynucleotide sequence.
Non-classical amino acids include, but are not limited to, to the
D-isomers of the common amino acids, 2,4-diaminobutyric acid,
a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric
acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric
acid, 3-amino propionic acid, omithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino
acids such as b-methyl amino acids, Ca-methyl amino acids,
Na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0221] The invention encompasses VEGF-2 polypeptides which are
differentially modified during or after translation, e.g., by
glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc. Any of numerous chemical modifications may be carried out by
known techniques, including but not limited, to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.4; acetylation, formylation, oxidation,
reduction; metabolic synthesis in the presence of tunicamycin;
etc.
[0222] Additional post-translational modifications encompassed by
the invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of procaryotic host cell expression. The polypeptides may
also be modified with a detectable label, such as an enzymatic,
fluorescent, isotopic or affinity label to allow for detection and
isolation of the protein.
[0223] Also provided by the invention are chemically modified
derivatives of VEGF-2 which may provide additional advantages such
as increased solubility, stability and circulating time of the
polypeptide, or decreased immunogenicity (see U.S. Pat. No.
4,179,337). The chemical moieties for derivitization may be
selected from water soluble polymers such as polyethylene glycol,
ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the
molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[0224] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about I kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a therapeutic protein or analog).
[0225] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.
20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound
through amino acid residues via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated polyethylene glycol molecule may be bound. The amino acid
residues having a free amino group may include lysine residues and
the N-terminal amino acid residues; those having a free carboxyl
group may include aspartic acid residues glutamic acid residues and
the C-terminal amino acid residue. Sulfhydryl groups may also be
used as a reactive group for attaching the polyethylene glycol
molecules. Preferred for therapeutic purposes is attachment at an
amino group, such as attachment at the N-terminus or lysine
group.
[0226] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration of the
present composition, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein (or peptide)
molecules in the reaction mix, the type of pegylation reaction to
be performed, and the method of obtaining the selected N-terminally
pegylated protein. The method of obtaining the N-terminally
pegylated preparation (i.e., separating this moiety from other
monopegylated moieties if necessary) may be by purification of the
N-terminally pegylated material from a population of pegylated
protein molecules. Selective proteins chemically modified at the
N-terminus modification may be accomplished by reductive alkylation
which exploits differential reactivity of different types of
primary amino groups (lysine versus the N-terminal) available for
derivatization in a particular protein. Under the appropriate
reaction conditions, substantially selective derivatization of the
protein at the N-terminus with a carbonyl group containing polymer
is achieved.
[0227] The VEGF-2 polypeptides of the invention may be in monomers
or multimers (i.e., dimers, trimers, tetramers and higher
multimers). Accordingly, the present invention relates to monomers
and multimers of the VEGF-2 polypeptides of the invention, their
preparation, and compositions (preferably, pharmaceutical
compositions) containing them. In specific embodiments, the
polypeptides of the invention are monomers, dimers, trimers or
tetramers. In additional embodiments, the multimers of the
invention are at least dimers, at least trimers, or at least
tetramers.
[0228] Multimers encompassed by the invention may be homomers or
heteromers. As used herein, the term homomer, refers to a multimer
containing only VEGF-2 polypeptides of the invention (including
VEGF-2 fragments, variants, splice variants, and fusion proteins,
as described herein). These homomers may contain VEGF-2
polypeptides having identical or different amino acid sequences. In
a specific embodiment, a homomer of the invention is a multimer
containing only VEGF-2 polypeptides having an identical amino acid
sequence. In another specific embodiment, a homomer of the
invention is a multimer containing VEGF-2 polypeptides having
different amino acid sequences. In specific embodiments, the
multimer of the invention is a homodimer (e.g., containing VEGF-2
polypeptides having identical or different amino acid sequences) or
a homotrimer (e.g., containing VEGF-2 polypeptides having identical
and/or different amino acid sequences). In additional embodiments,
the homomeric multimer of the invention is at least a homodimer, at
least a homotrimer, or at least a homotetramer.
[0229] As used herein, the term heteromer refers to a multimer
containing one or more heterologous polypeptides (i.e.,
polypeptides of different proteins) in addition to the VEGF-2
polypeptides of the invention. In a specific embodiment, the
multimer of the invention is a heterodimer, a heterotrimer, or a
heterotetramer. In additional embodiments, the homomeric multimer
of the invention is at least a homodimer, at least a homotrimer, or
at least a homotetramer.
[0230] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic and/or covalent associations and/or may be
indirectly linked, by for example, liposome formation. Thus, in one
embodiment, multimers of the invention, such as, for example,
homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of the invention, such as, for example,
heterotrimers or heterotetramers, are formed when polypeptides of
the invention contact antibodies to the polypeptides of the
invention (including antibodies to the heterologous polypeptide
sequence in a fusion protein of the invention) in solution. In
other embodiments, multimers of the invention are formed by
covalent associations with and/or between the VEGF-2 polypeptides
of the invention. Such covalent associations may involve one or
more amino acid residues contained in the polypeptide sequence (
e.g., that recited in SEQ ID NO:2, or contained in the polypeptide
encoded by the deposited clone.) In one instance, the covalent
associations are cross-linking between cysteine residues located
within the polypeptide sequences which interact in the native
(i.e., naturally occurring) polypeptide. In another instance, the
covalent associations are the consequence of chemical or
recombinant manipulation. Alternatively, such covalent associations
may involve one or more amino acid residues contained in the
heterologous polypeptide sequence in a VEGF-2 fusion protein. In
one example, covalent associations are between the heterologous
sequence contained in a fusion protein of the invention (see, e.g.,
U.S. Pat. No. 5,478,925). In a specific example, the covalent
associations are between the heterologous sequence contained in a
VEGF-2-Fc fusion protein of the invention (as described herein). In
another specific example, covalent associations of fusion proteins
of the invention are between heterologous polypeptide sequence from
another TNF family ligand/receptor member that is capable of
forming covalently associated multimers, such as for example,
oseteoprotegerin (see, e.g., International Publication No. WO
98/49305, the contents of which are herein incorporated by
reference in its entirety).
[0231] The multimers of the invention may be generated using
chemical techniques known in the art. For example, polypeptides
desired to be contained in the multimers of the invention may be
chemically cross-linked using linker molecules and linker molecule
length optimization techniques known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). Additionally, multimers of the invention may be
generated using techniques known in the art to form one or more
inter-molecule cross-links between the cysteine residues located
within the sequence of the polypeptides desired to be contained in
the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Further, polypeptides
of the invention may be routinely modified by the addition of
cysteine or biotin to the C terminus or N-terminus of the
polypeptide and techniques known in the art may be applied to
generate multimers containing one or more of these modified
polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Additionally,
techniques known in the art may be applied to generate liposomes
containing the polypeptide components desired to be contained in
the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925,
which is herein incorporated by reference in its entirety).
[0232] Alternatively, multimers of the invention may be generated
using genetic engineering techniques known in the art. In one
embodiment, polypeptides contained in multimers of the invention
are produced recombinantly using fusion protein technology
described herein or otherwise known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In a specific embodiment, polynucleotides coding for
a homodimer of the invention are generated by ligating a
polynucleotide sequence encoding a polypeptide of the invention to
a sequence encoding a linker polypeptide and then further to a
synthetic polynucleotide encoding the translated product of the
polypeptide in the reverse orientation from the original C-terminus
to the N-terminus (lacking the leader sequence) (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In another embodiment, recombinant techniques
described herein or otherwise known in the art are applied to
generate recombinant polypeptides of the invention which contain a
transmembrane domain (or hyrophobic or signal peptide) and which
can be incorporated by membrane reconstitution techniques into
liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety).
Therapeutic Uses
[0233] The VEGF-2 polypeptide of the present invention is a potent
mitogen for vascular and lymphatic endothelial cells. As shown in
FIGS. 12 and 13, the VEGF-2 polypeptide of SEQ 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 VEGF-2
nucleic acid sequence encoding this polypeptide wherein 20 mg of
RNA from several human tissues were probed with .sup.32P-VEGF-2,
illustrates that this protein is actively expressed in the heart
and lung which is further evidence of mitogenic activity.
[0234] Accordingly, VEGF-2, or biologically active portions
thereof, may be employed to treat vascular trauma by promoting
angiogenesis. For example, to stimulate the growth of transplanted
tissue where coronary bypass surgery is performed. VEGF-2, or
biologically active portions thereof, may also be employed to
promote wound healing, particularly to re-vascularize damaged
tissues or stimulate collateral blood flow during ischemia and
where new capillary angiogenesis is desired. VEGF-2, or
biologically active portions thereof, may be employed to treat
full-thickness wounds such as dermal ulcers, including pressure
sores, venous ulcers, and diabetic ulcers. In addition, VEGF-2, or
biologically active portions thereof, may be employed to treat
full-thickness burns and injuries where a skin graft or flap is
used to repair such burns and injuries. VEGF-2, or biologically
active portions thereof, may also be employed for use in plastic
surgery, for example, for the repair of lacerations, burns, or
other trauma. In addition, VEGF-2 can be used to promote healing of
wounds and injuries to the eye as well as to treat eye
diseases.
[0235] Along these same lines, VEGF-2, or biologically active
portions thereof, may also be employed to induce the growth of
damaged bone, periodontium or ligament tissue. VEGF-2, or
biologically active portions thereof, may also be employed for
regenerating supporting tissues of the teeth, including cementum
and periodontal ligament, that have been damaged by, e.g.,
periodontal disease or trauma.
[0236] Since angiogenesis is important in keeping wounds clean and
non-infected, VEGF-2, or biologically active portions thereof, may
be employed in association with surgery and following the repair of
incisions and cuts. VEGF-2, or biologically active portions
thereof, may also be employed for the treatment of abdominal wounds
where there is a high risk of infection.
[0237] VEGF-2, or biologically active portions thereof, may be
employed for the promotion of endothelialization in vascular graft
surgery. In the case of vascular grafts using either transplanted
or synthetic material, VEGF-2, or biologically active portions
thereof, can be applied to the surface of the graft or at the
junction to promote the growth of vascular endothelial cells.
VEGF-2, or biologically active portions thereof, may also be
employed to repair damage of myocardial tissue as a result of
myocardial infarction. VEGF-2, or biologically active portions
thereof, may also be employed to repair the cardiac vascular system
after ischemia. VEGF-2, or biologically active portions thereof,
may also be employed to treat damaged vascular tissue as a result
of coronary artery disease and peripheral and CNS vascular
disease.
[0238] VEGF-2, or biologically active portions thereof, may also be
employed to coat artificial prostheses or natural organs which are
to be transplanted in the body to minimize rejection of the
transplanted material and to stimulate vascularization of the
transplanted materials.
[0239] VEGF-2, or biologically active portions thereof, may also be
employed for vascular tissue repair of injuries resulting from
trauma, for example, that occurring during arteriosclerosis and
required following balloon angioplasty where vascular tissues are
damaged.
[0240] VEGF-2, or biologically active portions thereof, may also be
used to treat peripheral arterial disease. Accordingly, in a
further aspect, there is provided a process for utilizing VEGF-2
polypeptides to treat peripheral arterial disease. Preferably, a
VEGF-2 polypeptide is administered to an individual for the purpose
of alleviating or treating peripheral arterial disease. Suitable
doses, formulations, and administration routes are described
below.
[0241] VEGF-2, or biologically active portions thereof, may also to
promote the endothelial function of lymphatic tissues and vessels,
such as to treat the loss of lymphatic vessels, occlusions of
lymphatic vessels, and lymphangiomas. VEGF-2 may also be used to
stimulate lymphocyte production.
[0242] VEGF-2, or biologically active portions thereof, may also be
used to treat hemangioma in newborns. Accordingly, in a further
aspect, there is provided a process for utilizing VEGF-2
polypeptides to treat hemangioma in newborns. Preferably, a VEGF-2
polypeptide is administered to an individual for the purpose of
alleviating or treating hemangioma in newborns. Suitable doses,
formulations, and administration routes are described below.
[0243] VEGF-2, or biologically active portions thereof, may also be
used to prevent or treat abnormal retinal development in premature
newborns. Accordingly, in a further aspect, there is provided a
process for utilizing VEGF-2 polypeptides to treat abnormal retinal
development in premature newborns. Preferably, a VEGF-2 polypeptide
is administered to an individual for the purpose of alleviating or
treating abnormal retinal development in premature newborns.
Suitable doses, formulations, and administration routes are
described below.
[0244] VEGF-2, or biologically active portions thereof, may be used
to treat primary (idiopathic) lymphademas, including Milroy's
disease and Lymphedema praecox. Accordingly, in a further aspect,
there is provided a process for utilizing VEGF-2 polypeptides to
treat primary (idiopathic) lymphademas, including Milroy's disease
and Lymphedema praecox. Preferably, a VEGF-2 polypeptide is
administered to an individual for the purpose of alleviating or
treating primary (idiopathic) lymphademas, including Milroy's
disease and Lymphedema praecox. VEGF-2 or biologically active
portions thereof, may also be used to treat edema as well as to
effect blood pressure in an animal. Suitable doses, formulations,
and administration routes are described below.
[0245] VEGF-2, or biologically active portions thereof, may also be
used to treat secondary (obstructive) lifetimes including those
that result from (I) the removal of lymph nodes and vessels, (ii)
radiotherapy and surgery in the treatment of cancer, and (iii)
trauma and infection. Accordingly, in a further aspect, there is
provided a process for utilizing VEGF-2 polypeptides to treat
secondary (obstructive) lifetimes including those that result from
(I) the removal of lymph nodes and vessels, (ii) radiotherapy and
surgery in the treatment of cancer, and (iii) trauma and infection.
Preferably, a VEGF-2 polypeptide is administered to an individual
for the purpose of secondary (obstructive) lifetimes including
those that result from (I) the removal of lymph nodes and vessels,
(ii) radiotherapy and surgery in the treatment of cancer, and (iii)
trauma and infection. Suitable doses, formulations, and
administration routes are described below.
[0246] VEGF-2, or biologically active portions thereof, may also be
used to treat Kaposi's Sarcoma. Accordingly, in a further aspect,
there is provided a process for utilizing VEGF-2 polypeptides to
treat Kaposi's Sarcoma. Preferably, a VEGF-2 polypeptide is
administered to an individual for the purpose of alleviating or
treating Kaposi's Sarcoma. Suitable doses, formulations, and
administration routes are described below.
[0247] VEGF-2 antagonists can be used to treat cancer by inhibiting
the angiogenesis necessary to support cancer and tumor growth.
Cardiovascular Disorders
[0248] The present inventors have shown that VEGF-2 stimulates the
growth of vascular endothelial cells, stimulates endothelial cell
migration, stimulates angiogenesis in the CAM assay, decreases
blood pressure in spontaneously hypertensive rats, and increases
blood flow to ischemic limbs in rabbits. Accordingly, VEGF-2
polypeptides or polynucleotides encoding VEGF-2 may be used to
treat cardiovascular disorders, including peripheral artery
disease, such as limb ischemia.
[0249] Cardiovascular disorders include cardiovascular
abnormalities, such as arterio-arterial fistula, arteriovenous
fistula, cerebral arteriovenous malformations, congenital heart
defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart
defects include aortic coarctation, cor triatriatum, coronary
vessel anomalies, crisscross heart, dextrocardia, patent ductus
arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic
left heart syndrome, levocardia, tetralogy of fallot, transposition
of great vessels, double outlet right ventricle, tricuspid atresia,
persistent truncus arteriosus, and heart septal defects, such as
aortopulmonary septal defect, endocardial cushion defects,
Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal
defects.
[0250] Cardiovascular disorders also include heart disease, such as
arrhythmias, carcinoid heart disease, high cardiac output, low
cardiac output, cardiac tamponade, endocarditis (including
bacterial), heart aneurysm, cardiac arrest, congestive heart
failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac
edema, heart hypertrophy, congestive cardiomyopathy, left
ventricular hypertrophy, right ventricular hypertrophy,
post-infarction heart rupture, ventricular septal rupture, heart
valve diseases, myocardial diseases, myocardial ischemia,
pericardial effusion, pericarditis (including constrictive and
tuberculous), pneumopericardium, postpericardiotomy syndrome,
pulmonary heart disease, rheumatic heart disease, ventricular
dysfunction, hyperemia, cardiovascular pregnancy complications,
Scimitar Syndrome, cardiovascular syphilis, and cardiovascular
tuberculosis.
[0251] Arrhythmias include sinus arrhythmia, atrial fibrillation,
atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome,
bundle-branch block, sinoatrial block, long QT syndrome,
parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type
pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus
syndrome, tachycardias, and ventricular fibrillation. Tachycardias
include paroxysmal tachycardia, supraventricular tachycardia,
accelerated idioventricular rhythm, atrioventricular nodal reentry
tachycardia, ectopic atrial tachycardia, ectopic junctional
tachycardia, sinoatrial nodal reentry tachycardia, sinus
tachycardia, Torsades de Pointes, and ventricular tachycardia.
[0252] Heart valve disease include aortic valve insufficiency,
aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral
valve prolapse, tricuspid valve prolapse, mitral valve
insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary
valve insufficiency, pulmonary valve stenosis, tricuspid atresia,
tricuspid valve insufficiency, and tricuspid valve stenosis.
[0253] Myocardial diseases include alcoholic cardiomyopathy,
congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic
subvalvular stenosis, pulmonary subvalvular stenosis, restrictive
cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis,
endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion
injury, and myocarditis.
[0254] Myocardial ischemias include coronary disease, such as
angina pectoris, coronary aneurysm, coronary arteriosclerosis,
coronary thrombosis, coronary vasospasm, myocardial infarction and
myocardial stunning.
[0255] Cardiovascular diseases also include vascular diseases such
as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,
Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome,
Sturge-Weber Syndrome, angioneurotic edema, aortic diseases,
Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive diseases, arteritis, enarteritis, polyarteritis nodosa,
cerebrovascular disorders, diabetic angiopathies, diabetic
retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids,
hepatic veno-occlusive disease, hypertension, hypotension,
ischemia, peripheral vascular diseases, phlebitis, pulmonary
veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal
vein occlusion, Scimitar syndrome, superior vena cava syndrome,
telangiectasia, atacia telangiectasia, hereditary hemorrhagic
telangiectasia, varicocele, varicose veins, varicose ulcer,
vasculitis, and venous insufficiency.
[0256] Aneurysms include dissecting aneurysms, false aneurysms,
infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral
aneurysms, coronary aneurysms, heart aneurysms, and iliac
aneurysms.
[0257] Arterial occlusive diseases include arteriosclerosis,
intermittent claudication, carotid stenosis, fibromuscular
dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal
artery obstruction, retinal artery occlusion, and thromboangiitis
obliterans.
[0258] Cerebrovascular disorders include carotid artery diseases,
cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,
cerebral arteriosclerosis, cerebral arteriovenous malformation,
cerebral artery diseases, cerebral embolism and thrombosis, carotid
artery thrombosis, sinus thrombosis, Wallenberg's syndrome,
cerebral hemorrhage, epidural hematoma, subdural hematoma,
subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia
(including transient), subclavian steal syndrome, periventricular
leukomalacia, vascular headache, cluster headache, migraine, and
vertebrobasilar insufficiency.
[0259] Embolisms include air embolisms, amniotic fluid embolisms,
cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary
embolisms, and thromoboembolisms. Thrombosis include coronary
thrombosis, hepatic vein thrombosis, retinal vein occlusion,
carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome,
and thrombophlebitis.
[0260] Ischemia includes cerebral ischemia, ischemic colitis,
compartment syndromes, anterior compartment syndrome, myocardial
ischemia, reperfusion injuries, and peripheral limb ischemia.
Vasculitis includes aortitis, arteritis, Behcet's Syndrome,
Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,
thromboangiitis obliterans, hypersensitivity vasculitis,
Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and
Wegener's granulomatosis.
[0261] VEGF-2 polypeptides or polynucleotides are especially
effective for the treatment of critical limb ischemia and coronary
disease. As shown in Example 18, administration of VEGF-2
polynucleotides and polypeptides to an experimentally induced
ischemia rabbit hindlimb restored blood pressure ratio, blood flow,
angiographic score, and capillary density.
[0262] VEGF-2 polypeptides may be administered using any method
known in the art, including, but not limited to, direct needle
injection at the delivery site, intravenous injection, topical
administration, catheter infusion, biolistic injectors, particle
accelerators, gelfoam sponge depots, other commercially available
depot materials, osmotic pumps, oral or suppositorial solid
pharmaceutical formulations, decanting or topical applications
during surgery, aerosol delivery. Such methods are known in the
art. VEGF-2 polypeptides may be administered as part of a
pharmaceutical composition, described in more detail below. Methods
of delivering VEGF-2 polynucleotides are described in more detail
below.
Gene Therapy Methods
[0263] Another aspect of the present invention is to gene therapy
methods for treating disorders, diseases and conditions. The gene
therapy methods relate to the introduction of nucleic acid (DNA,
RNA and antisense DNA or RNA) sequences into an animal to achieve
expression of the VEGF-2 polypeptide of the present invention. This
method requires a polynucleotide which codes for a VEGF-2
polypeptide operatively linked to a promoter and any other genetic
elements necessary for the expression of the polypeptide by the
target tissue. Such gene therapy and delivery techniques are known
in the art, see, for example, WO 90/11092, which is herein
incorporated by reference.
[0264] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) comprising a promoter operably
linked to a VEGF-2 polynucleotide ex vivo, with the engineered
cells then being provided to a patient to be treated with the
polypeptide. Such methods are well-known in the art. For example,
see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216
(1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112
(1993); Ferrantini, M. et al. J. Immunology 153: 4604-4615 (1994);
Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et
al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al.,
Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene
Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene
Therapy 3: 31-38 (1996)), which are herein incorporated by
reference. In one embodiment, the cells which are engineered are
arterial cells. The arterial cells may be reintroduced into the
patient through direct injection to the artery, the tissues
surrounding the artery, or through catheter injection.
[0265] As discussed in more detail below, the VEGF-2 polynucleotide
constructs can be delivered by any method that delivers injectable
materials to the cells of an animal, such as, injection into the
interstitial space of tissues (heart, muscle, skin, lung, liver,
and the like). The VEGF-2 polynucleotide constructs may be
delivered in a pharmaceutically acceptable liquid or aqueous
carrier.
[0266] In one embodiment, the VEGF-2 polynucleotide is delivered as
a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA
refers to sequences that are free from any delivery vehicle that
acts to assist, promote or facilitate entry into the cell,
including viral sequences, viral particles, liposome formulations,
LIPOFECTIN.TM. or precipitating agents and the like. However, the
VEGF-2 polynucleotides can also be delivered in liposome
formulations and LIPOFECTIN.TM. formulations and the like can be
prepared by methods well known to those skilled in the art. Such
methods are described, for example, in U.S. Pat. Nos. 5,593,972,
5,589,466, and 5,580,859, which are herein incorporated by
reference.
[0267] The VEGF-2 polynucleotide vector constructs used in the gene
therapy method are preferably constructs that will not integrate
into the host genome nor will they contain sequences that allow for
replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44,
pXT1 and pSG available from STRATAGENE.TM.; pSVK3, pBPV, pMSG and
pSVL available from PHARMACIA.TM.; and pEF1/V5, pcDNA3.1, pRc/CMV2
available from Invitrogen, and the vector containing the VEGF-2
polynucleotide, pVGI.1, deposited as ATCC.TM. Deposit Number
PTA-2185. Other suitable vectors will be readily apparent to the
skilled artisan.
[0268] Any strong promoter known to those skilled in the art can be
used for driving the expression of VEGF-2 DNA. Suitable promoters
include adenoviral promoters, such as the adenoviral major late
promoter; or heterologous promoters, such as the cytomegalovirus
(CMV) promoter; the respiratory syncytial virus (RSV) promoter;
inducible promoters, such as the MMT promoter, the metallothionein
promoter; heat shock promoters; the albumin promoter; the ApoAI
promoter; human globin promoters; viral thymidine kinase promoters,
such as the Herpes Simplex thymidine kinase promoter; retroviral
LTRs; the b-actin promoter; and human growth hormone promoters. The
promoter also may be the native promoter for VEGF-2.
[0269] Unlike other gene therapy techniques, one major advantage of
introducing naked nucleic acid sequences into target cells is the
transitory nature of the polynucleotide synthesis in the cells.
Studies have shown that non-replicating DNA sequences can be
introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
[0270] The VEGF-2 polynucleotide construct can be delivered to the
interstitial space of tissues within the an animal, including of
muscle, skin, brain, lung, liver, spleen, bone marrow, thymus,
heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, eye, gland, and connective tissue. Interstitial space of
the tissues comprises the intercellular, fluid, mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers
in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that same matrix within connective tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space
occupied by the plasma of the circulation and the lymph fluid of
the lymphatic channels. Delivery to the interstitial space of
muscle tissue is preferred for the reasons discussed below. They
may be conveniently delivered by injection into the tissues
comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such
as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells are particularly competent in their ability to take up
and express polynucleotides.
[0271] For the naked acid sequence injection, an effective dosage
amount of DNA or RNA will be in the range of from about 0.05 mg/kg
body weight to about 50 mg/kg body weight. Preferably the dosage
will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration.
[0272] For myocardium delivery, multiple doses of pVGI.1 (VEGF-2)
can be administered to a patient, at various dose levels such as,
for example, 200, 800, and 2000 .mu.g. One way to deliver the dose
could be through direct injection into the myocardium using, for
example, a minimally invasive thoracotomy. If necessary, multiple
injection sites can be selected according to the areas of ischemia
identified by a baseline myocardial profusion study such as SPECT
(single photon emission computed tomography) imaging.
[0273] For limb delivery, multiple doses of pVGI.1 (VEGF-2) can be
administered to the limb of a patient, at various dose levels such
as, for example, 2, 4, and 8 mg. One way to deliver the dose could
be through intramuscular injection.
[0274] The preferred route of administration is by the parenteral
route of injection into the interstitial space of tissues. However,
other parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
VEGF-2 DNA constructs can be delivered to arteries during
angioplasty by the catheter used in the procedure.
[0275] The naked polynucleotides are delivered by any method known
in the art, including, but not limited to, direct needle injection
at the delivery site, intravenous injection, topical
administration, catheter infusion, and so-called "gene guns". These
delivery methods are known in the art.
[0276] As is evidenced by Example 18, naked VEGF-2 nucleic acid
sequences can be administered in vivo results in the successful
expression of VEGF-2 polypeptide in the femoral arteries of
rabbits.
[0277] The constructs may also be delivered with delivery vehicles
such as viral sequences, viral particles, liposome formulations,
LIPOFECTIN.TM., precipitating agents, etc. Such methods of delivery
are known in the art.
[0278] In certain embodiments, the VEGF-2 polynucleotide constructs
are complexed in a liposome preparation. Liposomal preparations for
use in the instant invention include cationic (positively charged),
anionic (negatively charged) and neutral preparations. However,
cationic liposomes are particularly preferred because a tight
charge complex can be formed between the cationic liposome and the
polyanionic nucleic acid. Cationic liposomes have been shown to
mediate intracellular delivery of plasmid DNA (Felgner et al.,
Proc. Natl. Acad Sci. USA (1987) 84:7413-7416, which is herein
incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad
Sci. USA (1989) 86:6077-6081, which is herein incorporated by
reference); and purified transcription factors (Debs et al., J.
Biol. Chem. (1990) 265:10189-10192, which is herein incorporated by
reference), in functional form.
[0279] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are particularly useful and are available under the trademark
LIPOFECTIN.TM., from GIBCO BRL, Grand Island, N.Y. (See, also,
Felgner et al., Proc. Natl Acad. Sci. USA (1987) 84:7413-7416,
which is herein incorporated by reference). Other commercially
available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
[0280] Other cationic liposomes can be prepared from readily
available materials using techniques well known in the art. See,
e.g. PCT Publication No. WO 90/11092 (which is herein incorporated
by reference) for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)liposomes.
Preparation of DOTMA liposomes is explained in the literature, see,
e.g., P. Felgner et al., Proc. Natl. Acad Sci. USA 84:7413-7417,
which is herein incorporated by reference. Similar methods can be
used to prepare liposomes from other cationic lipid materials.
[0281] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl, choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0282] For example, commercially dioleoylphosphatidyl choline
(DOPC), dioleoylphosphatidyl glycerol (DOPG), and
dioleoylphosphatidyl ethanolamine (DOPE) can be used in various
combinations to make conventional liposomes, with or without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be prepared by drying 50 mg each of DOPG and DOPC under a stream of
nitrogen gas into a sonication vial. The sample is placed under a
vacuum pump overnight and is hydrated the following day with
deionized water. The sample is then sonicated for 2 hours in a
capped vial, using a Heat Systems model 350 sonicator equipped with
an inverted cup (bath type) probe at the maximum setting while the
bath is circulated at 15 EC. Alternatively, negatively charged
vesicles can be prepared without sonication to produce
multilamellar vesicles or by extrusion through nucleopore membranes
to produce unilamellar vesicles of discrete size. Other methods are
known and available to those of skill in the art.
[0283] The liposomes can comprise multilamellar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs), with SUVs being preferred. The various liposome-nucleic
acid complexes are prepared using methods well known in the art.
See, e.g., Straubinger et al., Methods of Immunology (1983),
101:512-527, which is herein incorporated by reference. For
example, MLVs containing nucleic acid can be prepared by depositing
a thin film of phospholipid on the walls of a glass tube and
subsequently hydrating with a solution of the material to be
encapsulated. SUVs are prepared by extended sonication of MLVs to
produce a homogeneous population of unilamellar liposomes. The
material to be entrapped is added to a suspension of preformed MLVs
and then sonicated. When using liposomes containing cationic
lipids, the dried lipid film is resuspended in an appropriate
solution such as sterile water or an isotonic buffer solution such
as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are
mixed directly with the DNA. The liposome and DNA form a very
stable complex due to binding of the positively charged liposomes
to the cationic DNA. SUVs find use with small nucleic acid
fragments. LUVs are prepared by a number of methods, well known in
the art. Commonly used methods include Ca.sup.2+-EDTA chelation
(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483;
Wilson et al., Cell (1979) 17:77); ether injection (Deamer, D. and
Banghamn, A., Biochim. Biophys. Acta (1976) 443:629; Ostro et al.,
Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., Proc.
Natl. Acad. Sci. USA (1979) 76:3348); detergent dialysis (Enoch, H.
and Strittmatter, P., Proc. Natl. Acad. Sci. USA (1979) 76:145);
and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem.
(1980) 255:10431; Szoka, F. and Papahadjopoulos, D., Proc. Natl.
Acad. Sci. USA (1978) 75:145; Schaefer-Ridder et al., Science
(1982) 215:166), which are herein incorporated by reference.
[0284] Generally, the ratio of DNA to liposomes will be from about
10:1 to about 1:10. Preferably, the ration will be from about 5:1
to about 1:5. More preferably, the ration will be about 3:1 to
about 1:3. Still more preferably, the ratio will be about 1:1.
[0285] U.S. Pat. No. 5,676,954 (which is herein incorporated by
reference) reports on the injection of genetic material, complexed
with cationic liposomes carriers, into mice. U.S. Pat. Nos.
4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622,
5,580,859, 5,703,055, and international publication no. WO 94/9469
(which are herein incorporated by reference) provide cationic
lipids for use in transfecting DNA into cells and mammals. U.S.
Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and
international publication no. WO 94/9469 (which are herein
incorporated by reference) provide methods for delivering
DNA-cationic lipid complexes to mammals.
[0286] In certain embodiments, cells are be engineered, ex vivo or
in vivo, using a retroviral particle containing RNA which comprises
a sequence encoding VEGF-2. Retroviruses from which the retroviral
plasmid vectors may be derived include, but are not limited to,
Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma
Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape
leukemia virus, human immunodeficiency virus, Myeloproliferative
Sarcoma Virus, and mammary tumor virus.
[0287] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X,
VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, Human Gene Therapy 1:5-14 (1990), which is
incorporated herein by reference in its entirety. The vector may
transduce the packaging cells through any means known in the art.
Such means include, but are not limited to, electroporation, the
use of liposomes, and CaPO.sub.4 precipitation. In one alternative,
the retroviral plasmid vector may be encapsulated into a liposome,
or coupled to a lipid, and then administered to a host.
[0288] The producer cell line generates infectious retroviral
vector particles which include polynucleotide encoding VEGF-2. Such
retroviral vector particles then may be employed, to transduce
eukaryotic cells, either in vitro or in vivo. The transduced
eukaryotic cells will express VEGF-2.
[0289] In certain other embodiments, cells are engineered, ex vivo
or in vivo, with VEGF-2 polynucleotide contained in an adenovirus
vector. Adenovirus can be manipulated such that it encodes and
expresses VEGF-2, and at the same time is inactivated in terms of
its ability to replicate in a normal lytic viral life cycle.
Adenovirus expression is achieved without integration of the viral
DNA into the host cell chromosome, thereby alleviating concerns
about insertional mutagenesis. Furthermore, adenoviruses have been
used as live enteric vaccines for many years with an excellent
safety profile (Schwartz, A. R. et al. (1974) Am. Rev. Respir. Dis.
109:233-238). Finally, adenovirus mediated gene transfer has been
demonstrated in a number of instances including transfer of
alpha-1-antitrypsin and CFTR to the lungs of cotton rats
(Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld et
al., (1992) Cell 68:143-155). Furthermore, extensive studies to
attempt to establish adenovirus as a causative agent in human
cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl.
Acad Sci. USA 76:6606).
[0290] Suitable adenoviral vectors useful in the present invention
are described, for example, in Kozarsky and Wilson, Curr. Opin.
Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155
(1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993);
Yang et al, Nature Genet. 7:362-369 (1994); Wilson et al., Nature
365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein
incorporated by reference. For example, the adenovirus vector Ad2
is useful and can be grown in human 293 cells. These cells contain
the E1 region of adenovirus and constitutively express E1a and E1b,
which complement the defective adenoviruses by providing the
products of the genes deleted from the vector. In addition to Ad2,
other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also
useful in the present invention.
[0291] Preferably, the adenoviruses used in the present invention
are replication deficient. Replication deficient adenoviruses
require the aid of a helper virus and/or packaging cell line to
form infectious particles. The resulting virus is capable of
infecting cells and can express a polynucleotide of interest which
is operably linked to a promoter, for example, the HARP promoter of
the present invention, but cannot replicate in most cells.
Replication deficient adenoviruses may be deleted in one or more of
all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or
L1 through L5.
[0292] In certain other embodiments, the cells are engineered, ex
vivo or in vivo, using an adeno-associated virus (AAV). AAVs are
naturally occurring defective viruses that require helper viruses
to produce infectious particles (Muzyczka, N., Curr. Topics in
Microbiol. Immunol. 158:97 (1992)). It is also one of the few
viruses that may integrate its DNA into non-dividing cells. Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate, but space for exogenous DNA is limited to about 4.5
kb. Methods for producing and using such AAVs are known in the art.
See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
[0293] For example, an appropriate AAV vector for use in the
present invention will include all the sequences necessary for DNA
replication, encapsidation, and host-cell integration. The VEGF-2
polynucleotide construct is inserted into the AAV vector using
standard cloning methods, such as those found in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press
(1989). The recombinant AAV vector is then transfected into
packaging cells which are infected with a helper virus, using any
standard technique, including lipofection, electroporation, calcium
phosphate precipitation, etc. Appropriate helper viruses include
adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes
viruses. Once the packaging cells are transfected and infected,
they will produce infectious AAV viral particles which contain the
VEGF-2 polynucleotide construct. These viral particles are then
used to transduce eukaryotic cells, either ex vivo or in vivo. The
transduced cells will contain the VEGF-2 polynucleotide construct
integrated into its genome, and will express VEGF-2.
[0294] Another method of gene therapy involves operably associating
heterologous control regions and endogenous polynucleotide
sequences (e.g. encoding VEGF-2) via homologous recombination (see,
e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International
Publication No. WO 96/29411, published Sep. 26, 1996; International
Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,
Proc. Natl. Acad Sci. USA 86:8932-8935 (1989); and Zijlstra et al.,
Nature 342:435-438 (1989). This method involves the activation of a
gene which is present in the target cells, but which is not
normally expressed in the cells, or is expressed at a lower level
than desired.
[0295] Polynucleotide constructs are made, using standard
techniques known in the art, which contain the promoter with
targeting sequences flanking the promoter. Suitable promoters are
described herein. The targeting sequence is sufficiently
complementary to an endogenous sequence to permit homologous
recombination of the promoter-targeting sequence with the
endogenous sequence. The targeting sequence will be sufficiently
near the 5' end of the VEGF-2 desired endogenous polynucleotide
sequence so the promoter will be operably linked to the endogenous
sequence upon homologous recombination.
[0296] The promoter and the targeting sequences can be amplified
using PCR. Preferably, the amplified promoter contains distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3'
end of the first targeting sequence contains the same restriction
enzyme site as the 5' end of the amplified promoter and the 5' end
of the second targeting sequence contains the same restriction site
as the 3' end of the amplified promoter. The amplified promoter and
targeting sequences are digested and ligated together.
[0297] The promoter-targeting sequence construct is delivered to
the cells, either as naked polynucleotide, or in conjunction with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, whole viruses, lipofection,
precipitating agents, etc., described in more detail above. The P
promoter-targeting sequence can be delivered by any method,
included direct needle injection, intravenous injection, topical
administration, catheter infusion, particle accelerators, etc. The
methods are described in more detail below.
[0298] The promoter-targeting sequence construct is taken up by
cells. Homologous recombination between the construct and the
endogenous sequence takes place, such that an endogenous VEGF-2
sequence is placed under the control of the promoter. The promoter
then drives the expression of the endogenous VEGF-2 sequence.
[0299] The polynucleotides encoding VEGF-2 may be administered
along with other polynucleotides encoding other angiongenic
proteins. Angiogenic proteins include, but are not limited to,
acidic and basic fibroblast growth factors, VEGF-1, epidermal
growth factor alpha and beta, platelet-derived endothelial cell
growth factor, platelet-derived growth factor, tumor necrosis
factor alpha, hepatocyte growth factor, insulin like growth factor,
colony stimulating factor, macrophage colony stimulating factor,
granulocyte/macrophage colony stimulating factor, and nitric oxide
synthase.
[0300] Preferably, the polynucleotide encoding VEGF-2 contains a
secretory signal sequence that facilitates secretion of the
protein. Typically, the signal sequence is positioned in the coding
region of the polynucleotide to be expressed towards or at the 5'
end of the coding region. The signal sequence may be homologous or
heterologous to the polynucleotide of interest and may be
homologous or heterologous to the cells to be transfected.
Additionally, the signal sequence may be chemically synthesized
using methods known in the art.
[0301] Any mode of administration of any of the above-described
polynucleotides constructs can be used so long as the mode results
in the expression of one or more molecules in an amount sufficient
to provide a therapeutic effect. This includes direct needle
injection, systemic injection, catheter infusion, biolistic
injectors, particle accelerators (i.e., "gene guns"), gelfoam
sponge depots, other commercially available depot materials,
osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid
(tablet or pill) pharmaceutical formulations, and decanting or
topical applications during surgery. For example, direct injection
of naked calcium phosphate-precipitated plasmid into rat liver and
rat spleen or a protein-coated plasmid into the portal vein has
resulted in gene expression of the foreign gene in the rat livers
(Kaneda et al., Science 243:375 (1989)).
[0302] A preferred method of local administration is by direct
injection. Preferably, a recombinant molecule of the present
invention complexed with a delivery vehicle is administered by
direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries
refers to injecting the composition centimeters and preferably,
millimeters within arteries.
[0303] Another method of local administration is to contact a
polynucleotide construct of the present invention in or around a
surgical wound. For example, a patient can undergo surgery and the
polynucleotide construct can be coated on the surface of tissue
inside the wound or the construct can be injected into areas of
tissue inside the wound.
[0304] Therapeutic compositions useful in systemic administration,
include recombinant molecules of the present invention complexed to
a targeted delivery vehicle of the present invention. Suitable
delivery vehicles for use with systemic administration comprise
liposomes comprising ligands for targeting the vehicle to a
particular site.
[0305] Preferred methods of systemic administration, include
intravenous injection, aerosol, oral and percutaneous (topical)
delivery. Intravenous injections can be performed using methods
standard in the art. Aerosol delivery can also be performed using
methods standard in the art (see, for example, Stribling et al.,
Proc. Natl. Acad Sci. USA 189:11277-11281, 1992, which is
incorporated herein by reference). Oral delivery can be performed
by complexing a polynucleotide construct of the present invention
to a carrier capable of withstanding degradation by digestive
enzymes in the gut of an animal. Examples of such carriers, include
plastic capsules or tablets, such as those known in the art.
Topical delivery can be performed by mixing a polynucleotide
construct of the present invention with a lipophilic reagent (e.g.,
DMSO) that is capable of passing into the skin.
[0306] Determining an effective amount of substance to be delivered
can depend upon a number of factors including, for example, the
chemical structure and biological activity of the substance, the
age and weight of the animal, the precise condition requiring
treatment and its severity, and the route of administration. The
frequency of treatments depends upon a number of factors, such as
the amount of polynucleotide constructs administered per dose, as
well as the health and history of the subject. The precise amount,
number of doses, and timing of doses will be determined by the
attending physician or veterinarian.
[0307] Therapeutic compositions of the present invention can be
administered to any animal, preferably to mammals and birds.
Preferred mammals include humans, dogs, cats, mice, rats, rabbits
sheep, cattle, horses and pigs, with humans being particularly
preferred.
Nucleic Acid Utilities
[0308] VEGF-2 nucleic acid sequences and VEGF-2 polypeptides may
also be employed for in vitro purposes related to scientific
research, synthesis of DNA and manufacture of DNA vectors, and for
the production of diagnostics and therapeutics to treat human
disease. For example, VEGF-2 may be employed for in vitro culturing
of vascular endothelial cells, where it is added to the conditional
medium in a concentration from 10 pg/ml to 10 ng/ml.
[0309] Fragments of the full length VEGF-2 gene may be used as a
hybridization probe for a cDNA library to isolate other genes which
have a high sequence similarity to the gene or similar biological
activity. Probes of this type generally have at least 50 base
pairs, although they may have a greater number of bases. The probe
may also be used to identify a cDNA clone corresponding to a full
length transcript and a genomic clone or clones that contain the
complete VEGF-2 gene including regulatory and promoter regions,
exons, and introns. An example of a screen comprises isolating the
coding region of the VEGF-2 gene by using the known DNA sequence to
synthesize an oligonucleotide probe. Labeled oligonucleotides
having a sequence complementary to that of the gene of the present
invention are used to screen a library of human cDNA, genomic DNA
or mRNA to determine which members of the library the probe
hybridizes to.
[0310] This invention provides methods for identification of VEGF-2
receptors. The gene encoding the receptor can be identified by
numerous methods known to those of skill in the art, for example,
ligand panning and FACS sorting (Coligan et al., Current Protocols
in Immun., 1(2), Chapter 5, (1991)). Preferably, expression cloning
is employed wherein polyadenylated RNA is prepared from a cell
responsive to VEGF-2, and a cDNA library created from this RNA is
divided into pools and used to transfect COS cells or other cells
that are not responsive to VEGF-2. Transfected cells which are
grown on glass slides are exposed to labeled VEGF-2. VEGF-2 can be
labeled by a variety of means including iodination or inclusion of
a recognition site for a site-specific protein kinase. Following
fixation and incubation, the slides are subjected to
autoradiographic analysis. Positive pools are identified and
sub-pools are prepared and retransfected using an iterative
sub-pooling and rescreening process, eventually yielding a single
clone that encodes the putative receptor.
[0311] As an alternative approach for receptor identification,
labeled VEGF-2 can be photoaffinity linked with cell membrane or
extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGE and exposed to X-ray
film. The labeled complex containing VEGF-2 is then excised,
resolved into peptide fragments, and subjected to protein
microsequencing. The amino acid sequence obtained from
microsequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the
gene encoding the putative receptor.
VEGF-2 Agonist and Antagonists
[0312] This invention is also related to a method of screening
compounds to identify those which are VEGF-2 agonists or
antagonists. An example of such a method takes advantage of the
ability of VEGF-2 to significantly stimulate the proliferation of
human endothelial cells in the presence of the comitogen Con A.
Endothelial cells are obtained and cultured in 96-well
flat-bottomed culture plates (Costar, Cambridge, Mass.) in a
reaction mixture supplemented with Con-A (Calbiochem, La Jolla,
Calif.). Con-A, polypeptides of the present invention and the
compound to be screened are added. After incubation at 37 EC,
cultures are pulsed with 1 FCi of .sup.3[H]thymidine (5 Ci/mmol; 1
Ci=37 BGq; NEN) for a sufficient time to incorporate the .sup.3[H]
and harvested onto glass fiber filters (Cambridge Technology,
Watertown, Mass.). Mean .sup.3[H]-thymidine incorporation (cpm) of
triplicate cultures is determined using a liquid scintillation
counter (Beckman Instruments, Irvine, Calif.). Significant
.sup.3[H]thymidine incorporation, as compared to a control assay
where the compound is excluded, indicates stimulation of
endothelial cell proliferation.
[0313] To assay for antagonists, the assay described above is
performed and the ability of the compound to inhibit
.sup.3[H]thymidine incorporation in the presence of VEGF-2
indicates that the compound is an antagonist to VEGF-2.
Alternatively, VEGF-2 antagonists may be detected by combining
VEGF-2 and a potential antagonist with membrane-bound VEGF-2
receptors or recombinant receptors under appropriate conditions for
a competitive inhibition assay. VEGF-2 can be labeled, such as by
radioactivity, such that the number of VEGF-2 molecules bound to
the receptor can determine the effectiveness of the potential
antagonist.
[0314] Alternatively, the response of a known second messenger
system following interaction of VEGF-2 and receptor would be
measured and compared in the presence or absence of the compound.
Such second messenger systems include but are not limited to, cAMP
guanylate cyclase, ion channels or phosphoinositide hydrolysis. In
another method, a mammalian cell or membrane preparation expressing
the VEGF-2 receptor is incubated with labeled VEGF-2 in the
presence of the compound. The ability of the compound to enhance or
block this interaction could then be measured.
[0315] Potential VEGF-2 antagonists include an antibody, or in some
cases, an oligonucleotide, which bind to the polypeptide and
effectively eliminate VEGF-2 function. Alternatively, a potential
antagonist may be a closely related protein which binds to VEGF-2
receptors, however, they are inactive forms of the polypeptide and
thereby prevent the action of VEGF-2. Examples of these antagonists
include a negative dominant mutant of the VEGF-2 polypeptide, for
example, one chain of the hetero-dimeric form of VEGF-2 may be
dominant and may be mutated such that biological activity is not
retained. An example of a negative dominant mutant includes
truncated versions of a dimeric VEGF-2 which is capable of
interacting with another dimer to form wild type VEGF-2, however,
the resulting homo-dimer is inactive and fails to exhibit
characteristic VEGF activity.
[0316] Another potential VEGF-2 antagonist is an antisense
construct prepared using antisense technology. Antisense technology
can be used to control gene expression through triple-helix
formation or antisense DNA or RNA, both of which methods are 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 VEGF-2. The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into the VEGF-2 polypeptide
(Antisense--Okano, J. Neurochem. 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988)). The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of VEGF-2.
[0317] Potential VEGF-2 antagonists also include small molecules
which bind to and occupy the active site of the polypeptide thereby
making the catalytic site inaccessible to substrate such that
normal biological activity is prevented. Examples of small
molecules include but are not limited to small peptides or
peptide-like molecules.
[0318] The antagonists may be employed to limit angiogenesis
necessary for solid tumor metastasis. The identification of VEGF-2
can be used for the generation of certain inhibitors of vascular
endothelial growth factor. Since angiogenesis and
neovascularization are essential steps in solid tumor growth,
inhibition of angiogenic activity of the vascular endothelial
growth factor is very useful to prevent the further growth, retard,
or even regress solid tumors. Although the level of expression of
VEGF-2 is extremely low in normal tissues including breast, it can
be found expressed at moderate levels in at least two breast tumor
cell lines that are derived from malignant tumors. It is,
therefore, possible that VEGF-2 is involved in tumor angiogenesis
and growth.
[0319] Gliomas are also a type of neoplasia which may be treated
with the antagonists of the present invention.
[0320] The antagonists may also be used to treat chronic
inflammation 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.
[0321] The antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as hereinafter
described.
Pharmaceutical Compositions
[0322] The VEGF-2 polypeptides, polynucleotides and agonists and
antagonists may be employed in combination with a suitable
pharmaceutical carrier. Such compositions comprise a
therapeutically effective amount of the polypeptide or agonist or
antagonist, and a pharmaceutically acceptable carrier or excipient.
Such a carrier includes but is not limited to saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations
thereof. The formulation should suit the mode of
administration.
[0323] 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 container(s) 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.
[0324] The pharmaceutical compositions may be administered in a
convenient manner such as by the topical, intravenous,
intraperitoneal, intramuscular, intratumor, subcutaneous,
intranasal or intradermal routes. The pharmaceutical compositions
are administered in an amount which is effective for treating
and/or prophylaxis of the specific indication. In general, the
pharmaceutical compositions are administered in an amount of at
least about 10 mg/kg body weight and in most cases they will be
administered in an amount not in excess of about 8 mg/Kg body
weight per day. In most cases, the dosage is from about 10 mg/kg to
about 1 mg/kg body weight daily, taking into account the routes of
administration, symptoms, etc.
[0325] The VEGF-2 polypeptides, and agonists or antagonists which
are polypeptides may also be employed in accordance with the
present invention by expression of such polypeptide in vivo, which
is often referred to as "gene therapy," described above.
[0326] Thus, for example, cells such as bone marrow cells may be
engineered with a polynucleotide (DNA or RNA) encoding for the
polypeptide ex vivo, the engineered cells are then provided to a
patient to be treated with the polypeptide. Such methods are
well-known in the art. For example, cells may be engineered by
procedures known in the art by use of a retroviral particle
containing RNA encoding the polypeptide of the present
invention.
[0327] 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 vivo. These and other
methods for administering a polypeptide of the present invention by
such methods should be apparent to those skilled in the art from
the teachings of the present invention. For example, the expression
vehicle for engineering cells may be other than a retroviral
particle, for example, an adenovirus, which may be used to engineer
cells in vivo after combination with a suitable delivery
vehicle.
[0328] Retroviruses from which the retroviral plasmid vectors
hereinabove mentioned may be derived include, but are not limited
to, Moloney Murine Leukemia Virus, spleen necrosis virus,
retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,
avian leukosis virus, gibbon ape leukemia virus, human
immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma
Virus, and mammary tumor virus. In one embodiment, the retroviral
plasmid vector is derived from Moloney Murine Leukemia Virus.
[0329] The vector includes one or more promoters. Suitable
promoters which may be employed include, but are not limited to,
the retroviral LTR; the SV40 promoter; and the human
cytomegalovirus (CMV) promoter described in Miller et al.,
Biotechniques 7:980-990 (1989), or any other promoter (e.g.,
cellular promoters such as eukaryotic cellular promoters including,
but not limited to, the histone, pol III, and b-actin promoters).
Other viral promoters which may be employed include, but are not
limited to, adenovirus promoters, thymidine kinase (TK) promoters,
and B19 parvovirus promoters. The selection of a suitable promoter
will be apparent to those skilled in the art from the teachings
contained herein.
[0330] 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 heterologous promoters, such as the cytomegalovirus
(CMV) promoter; the respiratory syncytial virus (RSV) promoter;
inducible promoters, such as the MMT promoter, the metallothionein
promoter; heat shock promoters; the albumin promoter; the ApoAI
promoter; human globin promoters; viral thymidine kinase promoters,
such as the Herpes Simplex thymidine kinase promoter; retroviral
LTRs (including the modified retroviral LTRs hereinabove
described); the b-actin promoter; and human growth hormone
promoters. The promoter also may be the native promoter which
controls the gene encoding the polypeptide.
[0331] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, y-2, y-AM, PA12, T19-14X,
VT-19-17-H2, yCRE, yCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, Human Gene Therapy 1:5-14 (1990), which is
incorporated herein by reference in its entirety. The vector may
transduce the packaging cells through any means known in the art.
Such means include, but are not limited to, electroporation, the
use of liposomes, and CaPO.sub.4 precipitation. In one alternative,
the retroviral plasmid vector may be encapsulated into a liposome,
or coupled to a lipid, and then administered to a host.
[0332] The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the polypeptides. Such retroviral vector particles then
may be employed, to transduce eukaryotic cells, either in vitro or
in vivo. The transduced eukaryotic cells will express the nucleic
acid sequence(s) encoding the polypeptide. Eukaryotic 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.
Diagnostic Assays
[0333] This invention is also related to the use of the VEGF-2 gene
as part of a diagnostic assay for detecting diseases or
susceptibility to diseases related to the presence of mutations in
VEGF-2 nucleic acid sequences.
[0334] Individuals carrying mutations in the VEGF-2 gene may be
detected at the DNA level by a variety of techniques. Nucleic acids
for diagnosis may be obtained from a patient's cells, such as from
blood, urine, saliva, tissue biopsy and autopsy material. The
genomic DNA may be used directly for detection or may be amplified
enzymatically by using PCR (Saiki et al., Nature 324:163-166
(1986)) prior to analysis. RNA or cDNA may also be used for the
same purpose. As an example, PCR primers complementary to the
nucleic acid encoding VEGF-2 can be used to identify and analyze
VEGF-2 mutations. For example, deletions and insertions can be
detected by a change in size of the amplified product in comparison
to the normal genotype. Point mutations can be identified by
hybridizing amplified DNA to radiolabeled VEGF-2 RNA or
alternatively, radiolabeled VEGF-2 antisense DNA sequences.
Perfectly matched sequences can be distinguished from mismatched
duplexes by RNase A digestion or by differences in melting
temperatures.
[0335] 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 al., Science 230:1242 (1985)).
[0336] Sequence changes at specific locations may also be revealed
by nuclease protection assays, such as RNase and S1 protection or
the chemical cleavage method (e.g., Cotton et al., PNAS, USA
85:4397-4401 (1985)).
[0337] Thus, the detection of a specific DNA sequence may be
achieved by methods such as hybridization, RNase protection,
chemical cleavage, direct DNA sequencing or the use of restriction
enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP))
and Southern blotting of genomic DNA.
[0338] In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations can also be detected by in situ analysis.
[0339] The present invention also relates to a diagnostic assay for
detecting altered levels of VEGF-2 protein in various tissues since
an over-expression of the proteins compared to normal control
tissue samples may detect the presence of a disease or
susceptibility to a disease, for example, abnormal cellular
differentiation. Assays used to detect levels of VEGF-2 protein in
a sample derived from a host are well-known to those of skill in
the art and include radioimmunoassays, competitive-binding assays,
Western Blot analysis, ELISA assays and "sandwich" assay. An ELISA
assay (Coligan et al., Current Protocols in Immunology 1(2),
Chapter 6, (1991)) initially comprises preparing an antibody
specific to the VEGF-2 antigen, preferably a monoclonal antibody.
In addition a reporter antibody is prepared against the monoclonal
antibody. To the reporter antibody is attached a detectable reagent
such as radioactivity, fluorescence or, in this example, a
horseradish peroxidase enzyme. A sample is removed from a host and
incubated on a solid support, e.g. a polystyrene dish, that binds
the proteins in the sample. Any free protein binding sites on the
dish are then covered by incubating with a non-specific protein,
such as, bovine serum albumen. Next, the monoclonal antibody is
incubated in the dish during which time the monoclonal antibodies
attach to any VEGF-2 proteins attached to the polystyrene dish. All
unbound monoclonal antibody is washed out with buffer. The reporter
antibody linked to horseradish peroxidase is placed in the dish
resulting in binding of the reporter antibody to any monoclonal
antibody bound to VEGF-2. 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 VEGF-2 protein present in a given
volume of patient sample when compared against a standard
curve.
[0340] A competition assay may be employed wherein antibodies
specific to VEGF-2 are attached to a solid support. Polypeptides of
the present invention are then labeled, for example, by
radioactivity, and a sample derived from the host are passed over
the solid support and the amount of label detected, for example by
liquid scintillation chromatography, can be correlated to a
quantity of VEGF-2 in the sample.
[0341] A "sandwich" assay is similar to an ELISA assay. In a
"sandwich" assay VEGF-2 is passed over a solid support and binds to
antibody attached to a solid support. A second antibody is then
bound to the VEGF-2. A third antibody which is labeled and specific
to the second antibody is then passed over the solid support and
binds to the second antibody and an amount can then be
quantified.
Chromosome Identification
[0342] 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) are presently
available for marking chromosomal location. The mapping of DNAs to
chromosomes according to the present invention is an important
first step in correlating those sequences with genes associated
with disease.
[0343] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis
of the cDNA is used to rapidly select primers that do not span more
than one exon in the genomic DNA, thus complicating the
amplification process. These primers are then used for PCR
screening of somatic cell hybrids containing individual human
chromosomes. Only those hybrids containing the human gene
corresponding to the primer will yield an amplified fragment.
[0344] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of fragments from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0345] 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
probes from the cDNA as short as 50 or 60 base pairs. For a review
of this technique, see Verma et al., Human Chromosomes: a Manual of
Basic Techniques, Pergamon Press, New York (1988).
[0346] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man (available on
line through Johns Hopkins University Welch Medical Library). The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0347] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0348] 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).
[0349] Comparison of affected and unaffected individuals generally
involves first looking for structural alterations in the
chromosomes, such as deletions or translocations that are visible
from chromosome spreads or detectable using PCR based on that cDNA
sequence. Ultimately, complete sequencing of genes from several
individuals is required to confirm the presence of a mutation and
to distinguish mutations from polymorphisms.
Antisense
[0350] The present invention is further directed to inhibiting
VEGF-2 in vivo by the use of antisense technology. Antisense
technology can be used to control gene expression through
triple-helix formation or antisense DNA or RNA, both of which
methods are based on binding of a polynucleotide to DNA or RNA. For
example, the 5' coding portion of the mature polynucleotide
sequence, which encodes for the polypeptide of the present
invention, is used to design an antisense RNA oligonucleotide of
from 10 to 40 base pairs in length. A DNA oligonucleotide is
designed to be complementary to a region of the gene involved in
transcription (triple helix--see Lee et al., Nucl. Acids Res.
6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et
al. Science, 251:1360 (1991), thereby preventing transcription and
the production of VEGF-2. The antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of an mRNA
molecule into the VEGF-2 (antisense--Okano, J. Neurochem. 56:560
(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, Fla. (1988)).
[0351] Alternatively, the oligonucleotides described above can be
delivered to cells by procedures in the art such that the
anti-sense RNA or DNA may be expressed in vivo to inhibit
production of VEGF-2 in the manner described above.
[0352] Antisense constructs to VEGF-2, therefore, may inhibit the
angiogenic activity of the VEGF-2 and prevent the further growth or
even regress solid tumors, since angiogenesis and
neovascularization are essential steps in solid tumor growth. These
antisense constructs may also be used to treat rheumatoid
arthritis, psoriasis, diabetic retinopathy and Kaposi's sarcoma
which are all characterized by abnormal angiogenesis.
Epitope-Bearing Portions
[0353] In another aspect, the invention provides peptides and
polypeptides comprising epitope-bearing portions of the
polypeptides of the present invention. These epitopes are
immunogenic or antigenic epitopes of the polypeptides of the
present invention. An "immunogenic epitope" is defined as a part of
a protein that elicits an antibody response in vivo when the whole
polypeptide of the present invention, or fragment thereof, is the
immunogen. On the other hand, a region of a polypeptide to which an
antibody can bind is defined as an "antigenic determinant" or
"antigenic epitope." The number of in vivo immunogenic epitopes of
a protein generally is less than the number of antigenic epitopes.
See, e.g., Geysen, et al. (1983) Proc. Natl. Acad. Sci. USA
81:3998-4002. However, antibodies can be made to any antigenic
epitope, regardless of whether it is an immunogenic epitope, by
using methods such as phage display. See e.g., Petersen G. et al.
(1995) Mol. Gen. Genet. 249:425-431. Therefore, included in the
present invention are both immunogenic epitopes and antigenic
epitopes.
[0354] It is particularly pointed out that the immunogenic epitopes
comprises predicted critical amino acid residues determined by the
Jameson-Wolf analysis. Thus, additional flanking residues on either
the N-terminal, C-terminal, or both N- and C-terminal ends may be
added to these sequences to generate an epitope-bearing polypeptide
of the present invention. Therefore, the immunogenic epitopes may
include additional N-terminal or C-terminal amino acid residues.
The additional flanking amino acid residues may be contiguous
flanking N-terminal and/or C-terminal sequences from the
polypeptides of the present invention, heterologous polypeptide
sequences, or may include both contiguous flanking sequences from
the polypeptides of the present invention and heterologous
polypeptide sequences.
[0355] Polypeptides of the present invention comprising immunogenic
or antigenic epitopes are at least 7 amino acids residues in
length. "At least" means that a polypeptide of the present
invention comprising an immunogenic or antigenic epitope may be 7
amino acid residues in length or any integer between 7 amino acids
and the number of amino acid residues of the full length
polypeptides of the invention. Preferred polypeptides comprising
immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino
acid residues in length. However, it is pointed out that each and
every integer between 7 and the number of amino acid residues of
the full length polypeptide are included in the present
invention.
[0356] The immuno and antigenic epitope-bearing fragments may be
specified by either the number of contiguous amino acid residues,
as described above, or further specified by N-terminal and
C-terminal positions of these fragments on the amino acid sequence
of SEQ ID NO:2. Every combination of a N-terminal and C-terminal
position that a fragment of, for example, at least 7 or at least 15
contiguous amino acid residues in length could occupy on the amino
acid sequence of SEQ ID NO:2 is included in the invention. Again,
"at least 7 contiguous amino acid residues in length" means 7 amino
acid residues in length or any integer between 7 amino acids and
the number of amino acid residues of the full length polypeptide of
the present invention. Specifically, each and every integer between
7 and the number of amino acid residues of the full length
polypeptide are included in the present invention.
[0357] Immunogenic and antigenic epitope-bearing polypeptides of
the invention are useful, for example, to make antibodies which
specifically bind the polypeptides of the invention, and in
immunoassays to detect the polypeptides of the present invention.
The antibodies are useful, for example, in affinity purification of
the polypeptides of the present invention. The antibodies may also
routinely be used in a variety of qualitative or quantitative
immunoassays, specifically for the polypeptides of the present
invention using methods known in the art. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press; 2nd Ed. 1988).
[0358] The epitope-bearing polypeptides of the present invention
may be produced by any conventional means for making polypeptides
including synthetic and recombinant methods known in the art. For
instance, epitope-bearing peptides may be synthesized using known
methods of chemical synthesis. For instance, Houghten has described
a simple method for the synthesis of large numbers of peptides,
such as 10-20 mgs of 248 individual and distinct 13 residue
peptides representing single amino acid variants of a segment of
the HA1 polypeptide, all of which were prepared and characterized
(by ELISA-type binding studies) in less than four weeks (Houghten,
R. A. Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985)). This
"Simultaneous Multiple Peptide Synthesis (SMPS)" process is further
described in U.S. Pat. No. 4,631,211 to Houghten and coworkers
(1986). In this procedure the individual resins for the solid-phase
synthesis of various peptides are contained in separate
solvent-permeable packets, enabling the optimal use of the many
identical repetitive steps involved in solid-phase methods. A
completely manual procedure allows 500-1000 or more syntheses to be
conducted simultaneously (Houghten et al. (1985) Proc. Natl. Acad.
Sci. 82:5131-5135 at 5134.
[0359] Epitope-bearing polypeptides of the present invention are
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe, et
al., supra; Wilson, et al., supra, and Bittle, et al. (1985) J.
Gen. Virol. 66:2347-2354. If in vivo immunization is used, animals
may be immunized with free peptide; however, anti-peptide antibody
titer may be boosted by coupling of the peptide to a macromolecular
carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
For instance, peptides containing cysteine residues may be coupled
to a carrier using a linker such as
-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.gs of peptide or carrier protein
and Freund's adjuvant. Several booster injections may be needed,
for instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0360] As one of skill in the art will appreciate, and discussed
above, the polypeptides of the present invention comprising an
immunogenic or antigenic epitope can be fused to heterologous
polypeptide sequences. For example, the polypeptides of the present
invention may be fused with the constant domain of immunoglobulins
(IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, any
combination thereof including both entire domains and portions
thereof) resulting in chimeric polypeptides. These fusion proteins
facilitate purification, and show an increased half-life in vivo.
This has been shown, e.g., for chimeric proteins consisting of the
first two domains of the human CD4-polypeptide and various domains
of the constant regions of the heavy or light chains of mammalian
immunoglobulins. See, e.g., EPA 0,394,827; Traunecker et al. (1988)
Nature 331:84-86. Fusion proteins that have a disulfide-linked
dimeric structure due to the IgG portion can also be more efficient
in binding and neutralizing other molecules than monomeric
polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al. (1995) J. Biochem. 270:3958-3964. Nucleic acids encoding the
above epitopes can also be recombined with a gene of interest as an
epitope tag to aid in detection and purification of the expressed
polypeptide.
Antibodies
[0361] The present invention further relates to antibodies and
T-cell antigen receptors (TCR) which specifically bind the
polypeptides of the present invention. The antibodies of the
present invention include IgG (including IgG1, IgG2, IgG3, and
IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. As
used herein, the term "antibody" (Ab) is meant to include whole
antibodies, including single-chain whole antibodies, and
antigen-binding fragments thereof. Most preferably the antibodies
are human antigen binding antibody fragments of the present
invention include, but are not limited to, Fab, Fab' and F(ab')2,
Fd, single-chain Fvs (scFv), single-chain antibodies,
disulfide-linked Fvs (sdFv) and fragments comprising either a
V.sub.L or V.sub.H domain. The antibodies may be from any animal
origin including birds and mammals. Preferably, the antibodies are
human, murine, rabbit, goat, guinea pig, camel, horse, or
chicken.
[0362] Antigen-binding antibody fragments, including single-chain
antibodies, may comprise the variable region(s) alone or in
combination with the entire or partial of the following: hinge
region, CH1, CH2, and CH3 domains. Also included in the invention
are any combinations of variable region(s) and hinge region, CH1,
CH2, and CH3 domains. The present invention further includes
chimeric, humanized, and human monoclonal and polyclonal antibodies
which specifically bind the polypeptides of the present invention.
The present invention further includes antibodies which are
anti-idiotypic to the antibodies of the present invention.
[0363] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for heterologous
compositions, such as a heterologous polypeptide or solid support
material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO
92/05793; Tutt, A. et al. (1991) J. Immunol. 147:60-69; U.S. Pat.
Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648;
Kostelny, S. A. et al. (1992) J. Immunol. 148:1547-1553.
[0364] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention which are recognized or specifically bound
by the antibody. The epitope(s) or polypeptide portion(s) may be
specified as described herein, e.g., by N-terminal and C-terminal
positions, by size in contiguous amino acid residues, or listed in
the Tables and Figures. Antibodies which specifically bind any
epitope or polypeptide of the present invention may also be
excluded. Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0365] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of the polypeptides
of the present invention are included. Antibodies that do not bind
polypeptides with less than 95%, less than 90%, less than 85%, less
than 80%, less than 75%, less than 70%, less than 65%, less than
60%, less than 55%, and less than 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
Further included in the present invention are antibodies which only
bind polypeptides encoded by polynucleotides which hybridize to a
polynucleotide of the present invention under stringent
hybridization conditions (as described herein). Antibodies of the
present invention may also be described or specified in terms of
their binding affinity. Preferred binding affinities include those
with a dissociation constant or Kd less than 5.times.10.sup.-6M,
10.sup.-6M, 5.times.10.sup.-7M, 10.sup.-7M, 5.times.10.sup.-8M,
10.sup.-8M, 5.times.10.sup.-9M, 10.sup.-9M, 5.times.10.sup.-10M,
10.sup.-10M, 5.times.10.sup.-11M, 10.sup.-11M, 5.times.10.sup.-12M,
10.sup.-12M, 5.times.10.sup.-13M, 10.sup.-13M, 5.times.10.sup.-14M,
10.sup.-14M, 5.times.10.sup.-15M, and 10.sup.-15M.
[0366] Antibodies of the present invention have uses that include,
but are not limited to, methods known in the art to purify, detect,
and target the polypeptides of the present invention including both
in vitro and in vivo diagnostic and therapeutic methods. For
example, the antibodies have use in immunoassays for qualitatively
and quantitatively measuring levels of the polypeptides of the
present invention in biological samples. See, e.g., Harlow et al.,
ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference in the
entirety).
[0367] The antibodies of the present invention may be used either
alone or in combination with other compositions. The antibodies may
further be recombinantly fused to a heterologous polypeptide at the
N- or C-terminus or chemically conjugated (including covalently and
non-covalently conjugations) to polypeptides or other compositions.
For example, antibodies of the present invention may be
recombinantly fused or conjugated to molecules useful as labels in
detection assays and effector molecules such as heterologous
polypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO
91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 0 396
387.
[0368] The antibodies of the present invention may be prepared by
any suitable method known in the art. For example, a polypeptide of
the present invention or an antigenic fragment thereof can be
administered to an animal in order to induce the production of sera
containing polyclonal antibodies. Monoclonal antibodies can be
prepared using a wide of techniques known in the art including the
use of hybridoma and recombinant technology. See, e.g., Harlow et
al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: MONOCLONAL
ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981)
(said references incorporated by reference in their entireties).
Fab and F(ab')2 fragments may be produced by proteolytic cleavage,
using enzymes such as papain (to produce Fab fragments) or pepsin
(to produce F(ab')2 fragments).
[0369] Alternatively, antibodies of the present invention can be
produced through the application of recombinant DNA technology or
through synthetic chemistry using methods known in the art. For
example, the antibodies of the present invention can be prepared
using various phage display methods known in the art. In phage
display methods, functional antibody domains are displayed on the
surface of a phage particle which carries polynucleotide sequences
encoding them. Phage with a desired binding property are selected
from a repertoire or combinatorial antibody library (e.g. human or
murine) by selecting directly with antigen, typically antigen bound
or captured to a solid surface or bead. Phage used in these methods
are typically filamentous phage including fd and M13 with Fab, Fv
or disulfide stabilized Fv antibody domains recombinantly fused to
either the phage gene III or gene VIII protein. Examples of phage
display methods that can be used to make the antibodies of the
present invention include those disclosed in Brinkman U. et al.
(1995) J. Immunol. Methods 182:41-50; Ames, R. S. et al. (1995) J.
Immunol. Methods 184:177-186; Kettleborough, C. A. et al. (1994)
Eur. J. Immunol. 24:952-958; Persic, L. et al. (1997) Gene 187
9-18; Burton, D. R. et al. (1994) Advances in Immunology
57:191-280; PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047;
WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat.
Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,
5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,
5,658,727 and 5,733,743 (said references incorporated by reference
in their entireties).
[0370] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host including mammalian cells, insect cells, plant cells,
yeast, and bacteria. For example, techniques to recombinantly
produce Fab, Fab' and F(ab')2 fragments can also be employed using
methods known in the art such as those disclosed in WO 92/22324;
Mullinax, R. L. et al. (1992) BioTechniques 12(6):864-869; and
Sawai, H. et al. (1995) AJRI 34:26-34; and Better, M. et al. (1988)
Science 240:1041-1043 (said references incorporated by reference in
their entireties).
[0371] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al. (1991) Methods in
Enzymology 203:46-88; Shu, L. et al. (1993) PNAS 90:7995-7999; and
Skerra, A. et al. (1988) Science 240:1038-1040. For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies, S. D. et al. (1989) J.
Immunol. Methods 125:191-202; and U.S. Pat. No. 5,807,715.
Antibodies can be humanized using a variety of techniques including
CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101;
and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519
596; Padlan E. A., (1991) Molecular Immunology 28(4/5):489-498;
Studnicka G. M. et al. (1994) Protein Engineering 7(6):805-814;
Roguska M. A. et al. (1994) PNAS 91:969-973), and chain shuffling
(U.S. Pat. No. 5,565,332). Human antibodies can be made by a
variety of methods known in the art including phage display methods
described above. See also, U.S. Pat. Nos. 4,444,887, 4,716,111,
5,545,806, and 5,814,318; and WO 98/46645 (said references
incorporated by reference in their entireties).
[0372] Further included in the present invention are antibodies
recombinantly fused or chemically conjugated (including both
covalently and non-covalently conjugations) to a polypeptide of the
present invention. The antibodies may be specific for antigens
other than polypeptides of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al. supra and WO 93/21232; EP 0 439 095; Naramura, M. et
al. (1994) Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981;
Gillies, S. O. et al. (1992) PNAS 89:1428-1432; Fell, H. P. et al.
(1991) J. Immunol. 146:2446-2452 (said references incorporated by
reference in their entireties).
[0373] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the hinge region, CH1 domain, CH2 domain, and CH3
domain or any combination of whole domains or portions thereof. The
polypeptides of the present invention may be fused or conjugated to
the above antibody portions to increase the in vivo half life of
the polypeptides or for use in immunoassays using methods known in
the art. The polypeptides may also be fused or conjugated to the
above antibody portions to form multimers. For example, Fc portions
fused to the polypeptides of the present invention can form dimers
through disulfide bonding between the Fc portions. Higher
multimeric forms can be made by fusing the polypeptides to portions
of IgA and IgM. Methods for fusing or conjugating the polypeptides
of the present invention to antibody portions are known in the art.
See e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,
5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO
96/04388, WO 91/06570; Ashkenazi, A. et al. (1991) PNAS
88:10535-10539; Zheng, X. X. et al. (1995) J. Immunol.
154:5590-5600; and Vil, H. et al. (1992) PNAS 89:1137-11341 (said
references incorporated by reference in their entireties).
[0374] The invention further relates to antibodies which act as
agonists or antagonists of the polypeptides of the present
invention. For example, the present invention includes antibodies
which disrupt the receptor/ligand interactions with the
polypeptides of the invention either partially or fully. Included
are both receptor-specific antibodies and ligand-specific
antibodies. Included are receptor-specific antibodies which do not
prevent ligand binding but prevent receptor activation. Receptor
activation (i.e., signaling) may be determined by techniques
described herein or otherwise known in the art. Also include are
receptor-specific antibodies which both prevent ligand binding and
receptor activation. Likewise, included are neutralizing antibodies
which bind the ligand and prevent binding of the ligand to the
receptor, as well as antibodies which bind the ligand, thereby
preventing receptor activation, but do not prevent the ligand from
binding the receptor. Further included are antibodies which
activate the receptor. These antibodies may act as agonists for
either all or less than all of the biological activities affected
by ligand-mediated receptor activation. The antibodies may be
specified as agonists or antagonists for biological activities
comprising specific activities disclosed herein. The above antibody
agonists can be made using methods known in the art. See e.g., WO
96/40281; U.S. Pat. No. 5,811,097; Deng, B. et al. (1998) Blood
92(6):1981-1988; Chen, Z. et al. (1998) Cancer Res.
58(16):3668-3678; Harrop, J. A. et al. (1998) J. Immunol.
161(4):1786-1794; Zhu, Z. et al. (1998) Cancer Res.
58(15):3209-3214; Yoon, D. Y. et al. (1998) J. Immunol.
160(7):3170-3179; Prat, M. et al. (1998) J. Cell. Sci.
111(Pt2):237-247; Pitard, V. et al. (1997) J. Immunol. Methods
205(2):177-190; Liautard, J. et al. (1997) Cytokinde 9(4):233-241;
Carlson, N. G. et al. (1997) J. Biol. Chem. 272(17):11295-11301;
Taryman, R. E. et al. (1995) Neuron 14(4):755-762; Muller, Y. A. et
al. (1998) Structure 6(9):1153-1167; Bartunek, P. et al. (1996)
Cytokine 8(1):14-20 (said references incorporated by reference in
their entireties).
[0375] Antibodies may further be used in an immunoassay to detect
the presence of tumors in certain individuals. Enzyme immunoassay
can be performed from the blood sample of an individual. Elevated
levels of VEGF-2 can be considered diagnostic of cancer.
[0376] Truncated versions of VEGF-2 can also be produced that are
capable of interacting with wild type VEGF-2 to form dimers that
fail to activate endothelial cell growth, therefore inactivating
the endogenous VEGF-2. Or, mutant forms of VEGF-2 form dimers
themselves and occupy the ligand binding domain of the proper
tyrosine kinase receptors on the target cell surface, but fail to
activate cell growth.
[0377] Alternatively, antagonists to the polypeptides of the
present invention may be employed which bind to the receptors to
which a polypeptide of the present invention normally binds. The
antagonists may be closely related proteins such that they
recognize and bind to the receptor sites of the natural protein,
however, they are inactive forms of the natural protein and thereby
prevent the action of VEGF-2 since receptor sites are occupied. In
these ways, the action of the VEGF-2 is prevented and the
antagonist/inhibitors may be used therapeutically as an anti-tumor
drug by occupying the receptor sites of tumors which are recognized
by VEGF-2 or by inactivating VEGF-2 itself. The
antagonist/inhibitors may also be used to prevent inflammation due
to the increased vascular permeability action of VEGF-2. The
antagonist/inhibitors may also be used to treat solid tumor growth,
diabetic retinopathy, psoriasis and rheumatoid arthritis.
[0378] The antagonist/inhibitors may be employed in a composition
with a pharmaceutically acceptable carrier, e.g., as hereinabove
described.
[0379] 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.
[0380] In order to facilitate understanding of the following
examples, certain frequently occurring methods and/or terms will be
described.
[0381] "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.
[0382] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 mg of plasmid
or DNA fragment is used with about 2 units of enzyme in about 20 Fl
of buffer solution. For the purpose of isolating DNA fragments for
plasmid construction, typically 5 to 50 mg of DNA are digested with
20 to 250 units of enzyme in a larger volume. Appropriate buffers
and substrate amounts for particular restriction enzymes are
specified by the manufacturer. Incubation times of about 1 hour at
37 EC 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.
[0383] 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).
[0384] "Oligonucleotides" refer to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands, which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with an ATP in
the presence of a kinase. A synthetic oligonucleotide will ligate
to a fragment that has not been dephosphorylated.
[0385] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Sambrook
et al., Molecular Cloning: A Laboratory Manual, Second Edition,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), p.
146). Unless otherwise provided, ligation may be accomplished using
known buffers and conditions with 10 units of T4 DNA ligase
("ligase") per 0.5 mg of approximately equimolar amounts of the DNA
fragments to be ligated.
[0386] Unless otherwise stated, transformation was performed as
described by the method of Graham, F. and Van der Eb, A., Virology
52:456-457 (1973).
EXAMPLES
Example 1
Expression Pattern of VEGF-2 in Human Tissues and Breast Cancer
Cell Lines
[0387] Northern blot analysis was carried out to examine the levels
of expression of VEGF-2 in human tissues and breast cancer cell
lines in human tissues. Total cellular RNA samples were isolated
with RNAzol.TM. B system (Biotecx Laboratories, Inc.). About 10 mg
of total RNA isolated from each breast tissue and cell line
specified was separated on 1% agarose gel and blotted onto a nylon
filter, (Sambrook et al., Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y. (1989)). The labeling reaction was done according to the
STRATAGENE.TM. Prime-It kit with 50 ng DNA fragment. The labeled
DNA was purified with a Select-G-50 column from 5 Prime/3 Prime,
Inc (Boulder, Colo.). The filter was then hybridized with a
radioactive labeled full length VEGF-2 gene at 1,000,000 cpm/ml in
0.5 M NaPO.sub.4 and 7% SDS overnight at 65.degree. C. After
washing twice at room temperature and twice at 60.degree. C. with
0.5.times.SSC, 0.1% SDS, the filters were then exposed at
-70.degree. C. overnight with an intensifying screen. A message of
1.6 Kd was observed in 2 breast cancer cell lines. FIG. 5, lane #4
represents a very tumorigenic cell line that is estrogen
independent for growth.
[0388] Also, 10 mg of total RNA from 10 human adult tissues were
separated on an agarose gel and blotted onto a nylon filter. The
filter was then hybridized with radioactively labeled VEGF-2 probe
in 7% SDS, 0.5 M NaPO4, pH 7.2; 1% BSA overnight at 65.degree. C.
Following washing in 0.2.times.SSC at 65.degree. C., the filter was
exposed to film for 24 days at -70.degree. C. with intensifying
screen. See FIG. 6.
Example 2
Expression of the Truncated Form of VEGF-2 (SEQ ID NO:4) by in
vitro Transcription and Translation
[0389] The VEGF-2 cDNA was transcribed and translated in vitro to
determine the size of the translatable polypeptide encoded by the
truncated form of VEGF-2 and a partial VEGF-2 cDNA. The two inserts
of VEGF-2 in the pBLUESCRIPT.TM.pBLUESCRIPT.TM. SK vector were
amplified by PCR with three pairs of primers, 1) M13-reverse and
forward primers; 2) M13-reverse primer and VEGF primer F4; and 3)
M13-reverse primer and VEGF primer F5. The sequence of these
primers are as follows.
[0390] M13-2 reverse primer: 5'-ATGCTTCCGGCTCGTATG-3' (SEQ ID NO:9)
This sequence is located upstream of the 5' end of the VEGF-2 cDNA
insert in the pBLUESCRIPT.TM. vector and is in an anti-sense
orientation as the cDNA. A T3 promoter sequence is located between
this primer and the VEGF-2 cDNA. TABLE-US-00002 M13-2 forward
primer: 5'GGGTTTTCCCAGTCACGAC-3' (SEQ ID NO:10)
[0391] This sequence is located downstream of the 3' end of the
VEGF-2 cDNA insert in the pBLUESCRIPT.TM. vector and is in an
anti-sense orientation as the cDNA insert. TABLE-US-00003 VEGF
primer F4: 5'-CCACATGGTTCAGGAAAGACA-3' (SEQ ID NO:11)
[0392] This sequence is located within the VEGF-2 cDNA in an
anti-sense orientation from bp 1259-1239, which is about 169 bp
away from the 3' end of the stop codon and about 266 bp before the
last nucleotide of the cDNA.
[0393] PCR reaction with all three pairs of primers produce
amplified products with T3 promoter sequence in front of the cDNA
insert. The first and third pairs of primers produce PCR products
that encode the polypeptide of VEGF-2 shown in SEQ ID NO:4. The
second pair of primers produce PCR product that misses 36 amino
acids coding sequence at the C-terminus of the VEGF-2
polypeptide.
[0394] Approximately 0.5 mg of PCR product from first pair of
primers, 1 mg from second pair of primers, 1 mg from third pair of
primers were used for in vitro transcription/translation. The in
vitro transcription/translation reaction was performed in a 25 Fl
of volume, using the T.sub.NTJ Coupled Reticulocyte Lysate Systems
(PROMEGA.TM., CAT# L4950). Specifically, the reaction contains 12.5
Fl of T.sub.NT rabbit reticulocyte lysate 2 Fl of T.sub.NT reaction
buffer, 1 Fl of T3 polymerase, 1 Fl of 1 mM amino acid mixture
(minus methionine), 4 Fl of .sup.35S-methionine (>1000 Ci/mmol,
10 mCi/ml), 1 Fl of 40 U/.mu.l; RNasin ribonuclease inhibitor, 0.5
or 1 mg of PCR products. Nuclease-free H.sub.2O was added to bring
the volume to 25 Fl. The reaction was incubated at 30.degree. C.
for 2 hours. Five microliters of the reaction product was analyzed
on a 4-20% gradient SDS-PAGE gel. After fixing in 25% isopropanol
and 10% acetic acid, the gel was dried and exposed to an X-ray film
overnight at 70.degree. C.
[0395] As shown in FIG. 7, PCR products containing the truncated
VEGF-2 cDNA (i.e., as depicted in SEQ ID NO:3) and the cDNA missing
266 bp in the 3' un-translated region (3'-UTR) produced the same
length of translated products, whose molecular weights are
estimated to be 38-40 dk (lanes 1 and 3). The cDNA missing all the
3'UTR and missing sequence encoding the C-terminal 36 amino acids
was translated into a polypeptide with an estimated molecular
weight of 36-38 kd (lane 2).
Example 3
Cloning and Expression of VEGF-2 Using the Baculovirus Expression
System
[0396] The DNA sequence encoding the VEGF-2 protein without 46
amino acids at the N-terminus, see ATCC.TM. No. 97149, was
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' sequences of the gene:
[0397] The 5' primer has the sequence TGT AAT ACG ACT CAC TAT AGG
GAT CCC GCC ATG GAG GCC ACG GCT TAT GC (SEQ ID NO:12) and contains
a BamHI restriction enzyme site (in bold) and 17 nucleotide
sequence complementary to the 5' sequence of VEGF-2 (nt.
150-166).
[0398] The 3' primer has the sequence GATC TCT AGA TTA GCT CAT TTG
TGG TCT (SEQ ID NO: 13) and contains the cleavage site for the
restriction enzyme XbaI and 18 nucleotides complementary to the 3'
sequence of VEGF-2, including the stop codon and 15 nt sequence
before stop codon.
[0399] The amplified sequences were isolated from a 1% agarose gel
using a commercially available kit ("GENECLEAN.TM.," BIO 101, Inc.,
La Jolla, Calif.). The fragment was then digested with the
endonuclease BamHI and XbaI and then purified again on a 1% agarose
gel. This fragment was ligated to pAcGP67A baculovirus transfer
vector (Pharmingen) at the BamHI and XbaI sites. Through this
ligation, VEGF-2 cDNA was cloned in frame with the signal sequence
of baculovirus gp67 gene and was located at the 3' end of the
signal sequence in the vector. This is designated
pAcGP67A-VEGF-2.
[0400] To clone VEGF-2 with the signal sequence of gp67 gene to the
pRG1 vector for expression, VEGF-2 with the signal sequence and
some upstream sequence were excised from the pAcGP67A-VEGF-2
plasmid at the Xho restriction endonuclease site located upstream
of the VEGF-2 cDNA and at the XbaI restriction endonuclease site by
XhoI and XbaI restriction enzyme. This fragment was separated from
the rest of vector on a 1% agarose gel and was purified using
"GENECLEAN.TM." kit. It was designated F2.
[0401] The PRG1 vector (modification of pVL941 vector) is used for
the expression of the VEGF-2 protein using the baculovirus
expression system (for review see: Summers, M. D. and Smith, G. E.,
"A Manual of Methods for Baculovirus Vectors and Insect Cell
Culture Procedures," Texas Agricultural Experimental Station
Bulletin No. 1555, (1987)). This expression vector contains the
strong polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by the recognition sites for
the restriction endonucleases BamHI, SmaI, XbaI, BgIII and Asp718.
A site for restriction endonuclease XhoI is located upstream of
BamHI site. The sequence between XhoI and BamHI is the same as that
in PAcGp67A (static on tape) vector. The polyadenylation site of
the simian virus (SV)40 is used for efficient polyadenylation. For
an easy selection of recombinant virus the beta-galactosidase gene
from E. coli is inserted in the same orientation as the polyhedrin
promoter followed by the polyadenylation signal of the polyhedrin
gene. The polyhedrin sequences are flanked at both sides by viral
sequences for the cell-mediated homologous recombination of
cotransfected wild-type viral DNA. Many other baculovirus vectors
could be used in place of pRG1 such as pAc373, pVL941 and pAcIMI
(Luckow, V. A. and Summers, M. D., Virology 170:31-39 (1989).
[0402] 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 available kit
("GENECLEAN.TM." BIO 101 Inc., La Jolla, Calif.). This vector DNA
is designated V2.
[0403] Fragment F2 and the dephosphorylated plasmid V2 were ligated
with T4 DNA ligase. E. coli HB101 cells were then transformed and
bacteria identified that contained the plasmid (pBac gp67-VEGF-2)
with the VEGF-2 gene using the enzymes BamHI and XbaI. The sequence
of the cloned fragment was confirmed by DNA sequencing.
[0404] 5 mg of the plasmid pBac gp67-VEGF-2 was cotransfected with
1.0 mg of a commercially available linearized baculovirus
("BACULOGOLD.TM.BACULOGOLD.TM.J baculovirus DNA", Pharmingen, San
Diego, Calif.) using the LIPOFECTIN.TM. method (Felgner et al.,
Proc. Natl Acad. Sci. USA 84:7413-7417 (1987)).
[0405] 1 mg of BACULOGOLD.TM.J virus DNA and 5 mg of the plasmid
pBac gp67-VEGF-2 were mixed in a sterile well of a microtiter plate
containing 50 ml of serum free Grace's medium (Life Technologies
Inc., Gaithersburg, Md.). Afterwards 10 ml LIPOFECTIN.TM. plus 90
ml Grace's medium were added, mixed and incubated for 15 minutes at
room temperature. Then the transfection mixture was added dropwise
to the Sf9 insect cells (ATCC.TM. 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.degree. C. After 5
hours the transfection solution was removed from the plate and 1 ml
of Grace's insect medium supplemented with 10% fetal calf serum was
added. The plate was put back into an incubator and cultivation
continued at 27.degree. C. for four days.
[0406] 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 9-10).
[0407] Four days after the serial dilution, the virus was added to
the cells, blue stained plaques were picked with the tip of an
Eppendorf pipette. The agar containing the recombinant viruses was
then resuspended in an Eppendorf tube containing 200 ml of Grace's
medium. The agar was removed by a brief centrifugation and the
supernatant containing the recombinant baculovirus was used to
infect Sf9 cells seeded in 35 mm dishes. Four days later the
supernatants of these culture dishes were harvested and then stored
at 4.degree. C.
[0408] Sf9 cells were grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells were infected with the recombinant
baculovirus V-gp67-VEGF-2 at a multiplicity of infection (MOI) of
1. Six hours later the medium was removed and replaced with SF900
II medium minus methionine and cysteine (Life Technologies Inc.,
Gaithersburg). 42 hours later 5 mCi of .sup.35S-methionine and 5
mCi .sup.35S cysteine (Amersham) were added. The cells were further
incubated for 16 hours before they were harvested by centrifugation
and the labelled proteins visualized by SDS-PAGE and
autoradiography.
[0409] Protein from the medium and cytoplasm of the Sf9 cells was
analyzed by SDS-PAGE under non-reducing and reducing conditions.
See FIGS. 8A and 8B, respectively. The medium was dialyzed against
50 mM MES, pH 5.8. Precpitates were obtained after dialysis and
resuspended in 100 mM NaCitrate, pH 5.0. The resuspended
precipitate was analyzed again by SDS-PAGE and was stained with
Coomassie Brilliant Blue. See FIG. 9.
[0410] The medium supernatant was also diluted 1:10 in 50 mM MES,
pH 5.8 and applied to an SP-650M column (1.0.times.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 VEGF-2 was obtained
using the elution at 500 mM. The eluate was analyzed by SDS-PAGE in
the presence or absence of reducing agent, b-mercaptoethanol and
stained by Coomassie Brilliant Blue. See FIG. 10.
Example 4
Expression of Recombinant VEGF-2 in COS Cells
[0411] The expression of plasmid, VEGF-2-HA is derived from a
vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of
replication, 2) ampicillin resistance gene, 3) E. coli replication
origin, 4) CMV promoter followed by a polylinker region, an SV40
intron and polyadenylation site. A DNA fragment encoding the entire
VEGF-2 precursor and a HA tag fused in frame to its 3' end was
cloned into the polylinker region of the vector, therefore, the
recombinant protein expression is directed under the CMV promoter.
The HA tag corresponds to an epitope derived from the influenza
hemagglutinin protein as previously described (Wilson et al., Cell
37:767 (1984)). The infusion of HA tag to the target protein allows
easy detection of the recombinant protein with an antibody that
recognizes the HA epitope.
[0412] The plasmid construction strategy is described as
follows:
[0413] The DNA sequence encoding VEGF-2, ATCC.TM. No. 97149, was
constructed by PCR using two primers: the 5' primer (CGC GGA TCC
ATG ACT GTA CTC TAC CCA) (SEQ ID NO:14) contains a BamHI site
followed by 18 nucleotides of VEGF-2 coding sequence starting from
the initiation codon; the 3' sequence (CGC TCT AGA TCA AGC GTA GTC
TGG GAC GTC GTA TGG GTA CTC GAG GCT CAT TTG TGG TCT 3') (SEQ ID
NO:15) contains complementary sequences to an XbaI site, HA tag,
XhoI site, and the last 15 nucleotides of the VEGF-2 coding
sequence (not including the stop codon). Therefore, the PCR product
contains a BamHI site, coding sequence followed by an XhoI
restriction endonuclease site and 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 BamHI and XbaI restriction enzyme and ligated.
The ligation mixture was transformed into E. coli strain SURE
(STRATAGENE.TM. Cloning Systems, La Jolla, Calif. 92037) the
transformed culture was plated on ampicillin media plates and
resistant colonies were selected. Plasmid DNA was isolated from
transformants and examined by restriction analysis for the presence
of the correct fragment. For expression of the recombinant VEGF-2,
COS cells were transfected with the expression vector by
DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis,
Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory
Press, (1989)). The expression of the VEGF-2-HA protein was
detected by radiolabelling and immunoprecipitation method (E.
Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours
with .sup.35S-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, 50 mM Tris,
pH 7.5) (Wilson et al., Cell 37:767 (1984)). Both cell lysate and
culture media were precipitated with an HA specific monoclonal
antibody. Proteins precipitated were analyzed on 15% SDS-PAGE
gels.
Example 5
The Effect of Partially Purified VEGF-2 Protein on the Growth of
Vascular Endothelial Cells
[0414] On day 1, human umbilical vein endothelial cells (HUVEC)
were seeded at 2-5.times.10.sup.4 cells/35 mm dish density in M199
medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin,
and 50 units/ml endothelial cell growth supplements (ECGS,
Biotechnique, Inc.). On day 2, the medium was replaced with M199
containing 10% FBS, 8 units/ml heparin. VEGF-2 protein of SEQ ID
NO. 2 minus the initial 45 amino acid residues, (VEGF) and basic
FGF (bFGF) were added, at the concentration shown. On days 4 and 6,
the medium was replaced. On day 8, cell number was determined with
a Coulter Counter (See FIG. 12).
Example 6
The Effect of Purified VEGF-2 Protein on the Growth of Vascular
Endothelial Cells
[0415] On day 1, human umbilical vein endothelial cells (HUVEC)
were seeded at 2-5.times.10.sup.4 cells/35 mm dish density in M199
medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin,
50 units/ml endothelial cell growth supplements (ECGS,
Biotechnique, Inc.). On day 2, the medium was replaced with M199
containing 10% FBS, 8 units/ml heparin. Purified VEGF-2 protein of
SEQ ID NO:2 minus initial 45 amino acid residues was added to the
medium at this point. On days 4 and 6, the medium was replaced with
fresh medium and supplements. On day 8, cell number was determined
with a Coulter Counter (See FIG. 13).
Example 7
Expression via Gene Therapy
[0416] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in tissue-culture medium and separated
into small pieces. Small chunks of the tissue are placed on a wet
surface of a tissue culture flask, approximately ten pieces are
placed in each flask. The flask is turned upside down, closed tight
and left at room temperature over night. After 24 hours at room
temperature, the flask is inverted and the chunks of tissue remain
fixed to the bottom of the flask and fresh media (e.g., Ham's F12
media, with 10% FBS, penicillin and streptomycin, is added. This is
then incubated at 37.degree. C. for approximately one week. At this
time, fresh media is added and subsequently changed every several
days. After an additional two weeks in culture, a monolayer of
fibroblasts emerge. The monolayer is trypsinized and scaled into
larger flasks.
[0417] pMV-7 (Kirschmeier, P. T. et al., DNA 7:219-225 (1988)
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[0418] The cDNA encoding a polypeptide of the present invention is
amplified using PCR primers which correspond to the 5' and 3' end
sequences respectively. The 5' primer containing an EcoRI site and
the 3' primer further includes a HindIII site. Equal quantities of
the Moloney murine sarcoma virus linear backbone and the amplified
EcoRI and HindIII fragment are added together, in the presence of
T4 DNA ligase. The resulting mixture is maintained under conditions
appropriate for ligation of the two fragments. The ligation mixture
is used to transform bacteria HB101, which are then plated onto
agar-containing kanamycin for the purpose of confirming that the
vector had the gene of interest properly inserted.
[0419] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the gene is then added to
the media and the packaging cells are transduced with the vector.
The packaging cells now produce infectious viral particles
containing the gene (the packaging cells are now referred to as
producer cells).
[0420] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his.
[0421] The engineered fibroblasts are then injected into the host,
either alone or after having been grown to confluence on cytodex 3
microcarrier beads. The fibroblasts now produce the protein
product.
Example 8
Expression of VEGF-2 mRNA in Human Fetal and Adult Tissues
Experimental Design
[0422] Northern blot analysis was carried out to examine the levels
of expression of VEGF-2 mRNA in human fetal and adult tissues. A
cDNA probe containing the entire nucleotide sequence of the VEGF-2
protein was labeled with .sup.32P using the REDIPRIME.TM. DNA
labeling system (Amersham Life Science), according to the
manufacturer's instructions. After labeling, the probe was purified
using a CHROMA SPIN-100* column (CLONTECH.TM. Laboratories, Inc.),
according to manufacturer's protocol number PT1200-1. The purified
labeled probe was then used to examine various human tissues for
VEGF-2 mRNA.
[0423] A Multiple Tissue Northern (MTN) blot containing various
human tissues (Fetal Kidney, Fetal Lung, Fetal Liver, Brain,
Kidney, Lung, Liver, Spleen, Thymus, Bone Marrow, Testes, Placenta,
and Skeletal Muscle) was obtained from CLONTECH.TM.. The MTN blot
was examined with the labeled probe using EXPRESSHYB.TM.
hybridization solution (CLONTECH.TM.) according to manufacturer's
protocol number PT1190-1. Following hybridization and washing, the
blot was exposed to film at -70.degree. C. overnight with an
intensifying screen and developed according to standard
procedures.
Results
[0424] Expression of VEGF-2 mRNA is abundant in vascular smooth
muscle and several highly vascularized tissues. VEGF-2 is expressed
at significantly higher levels in tissues associated with
hematopoetic or angiogenic activities, i.e. fetal kidney, fetal
lung, bone marrow, placental, spleen and lung tissue. The
expression level of VEGF-2 is low in adult kidney, fetal liver,
adult liver, testes; and is almost undetectable in fetal brain, and
adult brain (See FIG. 14).
[0425] In primary cultured cells, the expression of VEGF-2 mRNA is
abundant in vascular smooth muscle cells and dermal fibroblast
cells, but much lower in human umbilical vein endothelial cells
(see FIG. 15). This mRNA distribution pattern is very similar to
that of VEGF.
Example 9
Construction of Amino Terminal and Carboxy Terminal Deletion
Mutants
[0426] In order to identify and analyze biologically active VEGF-2
polypeptides, a panel of deletion mutants of VEGF-2 was constructed
using the expression vector pHE4a.
1. Construction of VEGF-2 T103-L215 in pHE4
[0427] To permit Polymerase Chain Reaction directed amplification
and sub-cloning of VEGF-2 T103-L215 (amino acids 103 to 215 in FIG.
1 or SEQ ID NO:18) into the E. coli protein expression vector,
pHE4, two oligonucleotide primers complementary to the desired
region of VEGF-2 were synthesized with the following base sequence:
TABLE-US-00004 5' Primer (Nde I/START and 18 nt of coding
sequence): 5'-GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3' (SEQ ID
NO:19) 3' Primer (Asp718, STOP, and 15 nt of coding sequence):
5'-GCA GCA GGT ACC TCA CAG TTT AGA CAT GCA-3' (SEQ ID NO:20)
[0428] The above described 5' primer (SEQ ID NO: 19), incorporates
an NdeI restriction site and the above described 3' Primer (SEQ ID
NO:20), incorporates an Asp718 restriction site. The 5' primer (SEQ
ID NO:19) also contains an ATG sequence adjacent and in frame with
the VEGF-2 coding region to allow translation of the cloned
fragment in E. coli, while the 3' primer (SEQ ID NO:20) contains
one stop codon (preferentially utilized in E. coli) adjacent and in
frame with the VEGF-2 coding region which ensures correct
translational termination in E. coli.
[0429] The Polymerase Chain Reaction was performed using standard
conditions well known to those skilled in the art and the
nucleotide sequence for the mature VEGF-2 (aa 24-419 in SEQ ID
NO:2) as, for example, constructed in Example 3 as template. The
resulting amplicon was restriction digested with NdeI and Asp718
and subcloned into NdeI/Asp718 digested pHE4a expression
vector.
2. Construction of VEGF-2 T103-R227 in pHE4
[0430] To permit Polymerase Chain Reaction directed amplification
and sub-cloning of VEGF-2 T103-R227 (amino acids 103 to 227 in FIG.
1 or SEQ ID NO:18) into the E. coli protein expression vector,
pHE4, two oligonucleotide primers complementary to the desired
region of VEGF-2 were synthesized with the following base sequence:
TABLE-US-00005 5' Primer (Nde I/START and 18 nt of coding
sequence): 5'-GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3' (SEQ ID
NO:19) 3' Primer (Asp 718, STOP, and 15 nt of coding sequence):
5'-GCA GCA GGT ACC TCA ACG TCT AAT AAT GGA-3' (SEQ ID NO:21)
[0431] In the case of the above described primers, an NdeI or
Asp718 restriction site was incorporated he 5' primer and 3'
primer, respectively. The 5' primer (SEQ ID NO:19) also contains an
ATG sequence adjacent and in frame with the VEGF-2 coding region to
allow translation of the cloned fragment in E. coli, while the 3'
Primer (SEQ ID NO:21) contains one stop codon (preferentially
utilized in E. coli) adjacent and in frame with the VEGF-2 coding
region which ensures correct translational termination in E.
coli.
[0432] The Polymerase Chain Reaction was performed using standard
conditions well known to those skilled in the art and the
nucleotide sequence for the mature VEGF-2 (aa 24-419 in SEQ ID
NO:2) as, for example, constructed in Example 3, as template. The
resulting amplicon was restriction digested with NdeI and Asp718
and subcloned into NdeI/Asp718 digested pHE4a protein expression
vector.
3. Construction of VEGF-2 T103-L215 in pA2GP
[0433] In this illustrative example, the plasmid shuttle vector pA2
GP is used to insert the cloned DNA encoding the N-terminal and
C-terminal deleted VEGF-2 protein (amino acids 103-215 in FIG. 1 or
SEQ ID NO: 18), into a baculovirus to express the N-terminal and
C-terminal deleted VEGF-2 protein, using a baculovirus leader and
standard methods as described in Summers et al., A Manual of
Methods for Baculovirus Vectors and Insect Cell Culture Procedures,
Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
This expression vector contains the strong polyhedrin promoter of
the Autographa californica nuclear polyhedrosis virus (AcMNPV)
followed by the secretory signal peptide (leader) of the
baculovirus gp67 protein and convenient restriction sites such as
BamHI, XbaI and Asp718. The polyadenylation site of the simian
virus 40 ("SV40") is used for efficient polyadenylation. For easy
selection of recombinant virus, the plasmid contains the
beta-galactosidase gene from E. coli under control of a weak
Drosophila promoter in the same orientation, followed by the
polyadenylation signal of the polyhedrin gene. The inserted genes
are flanked on both sides by viral sequences for cell-mediated
homologous recombination with wild-type viral DNA to generate
viable virus that expresses the cloned polynucleotide.
[0434] Many other baculovirus vectors could be used in place of the
vector above, such as pAc373, pVL941 and pAcIM1, as one skilled in
the art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an in-frame
AUG as required. Such vectors are described, for instance, in
Luckow et al., Virology 170:31-39 (1989).
[0435] The cDNA sequence encoding the VEGF-2 protein without 102
amino acids at the N-terminus and without 204 amino acids at the
C-terminus in FIG. 1, was amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the gene.
[0436] The 5' primer has the sequence 5'-GCA GCA GGA TCC CAC AGA
AGA GAC TAT AAA-3' (SEQ ID NO:22) containing the BamHI restriction
enzyme site (in bold) followed by 1 spacer nt to stay in-frame with
the vector-supplied signal peptide, and 17 nt of coding sequence
bases of VEGF-2 protein. The 3' primer has the sequence 5N-GCA GCA
TCT AGA TCA CAG TTT AGA CAT GCA-3' (SEQ ID NO:23) containing the
XbaI restriction site (in bold) followed by a stop codon and 17
nucleotides complementary to the 3' coding sequence of VEGF-2.
[0437] The amplified sequences were isolated from a 1% agarose gel
using a commercially available kit ("GENECLEAN.TM.," BIO 101, Inc.,
La Jolla, Calif.). The fragment was then digested with the
endonuclease BamHI and XbaI and then purified again on a 1% agarose
gel. This fragment was ligated to pA2 GP baculovirus transfer
vector (Supplier) at the BamHI and XbaI sites. Through this
ligation, VEGF-2 cDNA representing the N-terminal and C-terminal
deleted VEGF-2 protein (amino acids 103-215 in FIG. 1 or SEQ ID NO:
18) was cloned in frame with the signal sequence of baculovirus GP
gene and was located at the 3' end of the signal sequence in the
vector. This is designated pA2GPVEGF-2.T103-L215.
4. Construction of VEGF-2 T103-R227 in pA2GP
[0438] The cDNA sequence encoding the VEGF-2 protein without 102
amino acids at the N-terminus and without 192 amino acids at the
C-terminus in FIG. 1 (i.e., amino acids 103-227 of SEQ ID NO:2) was
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' sequences of the gene.
[0439] The 5'-GCA GCA GGA TCC CAC AGA AGA GAC TAT AAA ATT TGC
TGC-3' primer has the sequence (SEQ ID NO:24) containing the BamHI
restriction enzyme site (in bold) followed by 1 spacer nt to stay
in-frame with the vector-supplied signal peptide, and 26 nt of
coding sequence bases of VEGF-2 protein. The 3' primer has the
sequence 5N-GCA GCA TCT AGA TCA ACG TCT AAT AAT GGA ATG AAC-3' (SEQ
ID NO:25) containing the XbaI restriction site (in bold) followed
by a stop codon and 21 nucleotides complementary to the 3' coding
sequence of VEGF-2.
[0440] The amplified sequences were isolated from a 1% agarose gel
using a commercially available kit ("GENECLEAN.TM.," BIO 101, Inc.,
La Jolla, Calif.). The fragment was then digested with the
endonuclease BamHI and XbaI and then purified again on a 1% agarose
gel. This fragment was ligated to pA2 GP baculovirus transfer
vector (Supplier) at the BamHI and XbaI sites. Through this
ligation, VEGF-2 cDNA representing the N-terminal and C-terminal
deleted VEGF-2 protein (amino acids 103-227 in FIG. 1 or SEQ ID
NO:18) was cloned in frame with the signal sequence of baculovirus
GP gene and was located at the 3' end of the signal sequence in the
vector. This construct is designated pA2GPVEGF-2.T103-R227.
5. Construction of VEGF-2 in pC1
[0441] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular
and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple
cloning sites, e.g., with the restriction enzyme cleavage sites
BamHI, XbaI and Asp718, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3N intron, the
polyadenylation and termination signal of the rat preproinsulin
gene.
[0442] The vector pC1 is used for the expression of VEGF-2 protein.
Plasmid pC1 is a derivative of the plasmid pSV2-dhfr [ATCC.TM.
Accession No. 37146]. Both plasmids contain the mouse DHFR gene
under control of the SV40 early promoter. Chinese hamster ovary--or
other cells lacking dihydrofolate activity that are transfected
with these plasmids can be selected by growing the cells in a
selective medium (alpha minus MEM, Life Technologies) supplemented
with the chemotherapeutic agent methotrexate. The amplification of
the DHFR genes in cells resistant to methotrexate (MTX) has been
well documented (see, e.g., Alt, F. W., Kellems, R. M., Bertino, J.
R., and Schimke, R. T., 1978, J. Biol. Chem. 253:1357-1370, Hamlin,
J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143,
Page, M. J. and Sydenham, M. A. 1991, Biotechnology 9:64-68). Cells
grown in increasing concentrations of MTX develop resistance to the
drug by overproducing the target enzyme, DHFR, as a result of
amplification of the DHFR gene. If a second gene is linked to the
DHFR gene it is usually co-amplified and over-expressed. It is
state of the art to develop cell lines carrying more than 1,000
copies of the genes. Subsequently, when the methotrexate is
withdrawn, cell lines contain the amplified gene integrated into
the chromosome(s).
[0443] Plasmid pC1 contains for the expression of the gene of
interest a strong promoter of the long terminal repeat (LTR) of the
Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular
Biology, March 1985:438-4470) plus a fragment isolated from the
enhancer of the immediate early gene of human cytomegalovirus (CMV)
(Boshart et al., Cell 41:521-530, 1985). Downstream of the promoter
are the following single restriction enzyme cleavage sites that
allow the integration of the genes: BamHI, Pvull, and Nrul. Behind
these cloning sites the plasmid contains translational stop codons
in all three reading frames followed by the 3N intron and the
polyadenylation site of the rat preproinsulin gene. Other high
efficient promoters can also be used for the expression, e.g., the
human b-actin promoter, the SV40 early or late promoters or the
long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
For the polyadenylation of the mRNA other signals, e.g., from the
human growth hormone or globin genes can be used as well.
[0444] Stable cell lines carrying a gene of interest integrated
into the chromosomes can also be selected upon co-transfection with
a selectable marker such as gpt, G418 or hygromycin. It is
advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
[0445] The plasmid pC1 is digested with the restriction enzyme
BamHI and then dephosphorylated using calf intestinal phosphates by
procedures known in the art. The vector is then isolated from a 1%
agarose gel.
[0446] The DNA sequence encoding VEGF-2, ATCC.TM. Accession No.
97149, was constructed by PCR using two primers corresponding to
the 5' and 3'ends of the VEGF-2 gene: the 5' Primer (5'-GAT CGA TCC
ATC ATG CAC TCG CTG GGC TTC TTC TCT GTG GCG TGT TCT CTG CTC G-3'
(SEQ ID NO:26)) contains a Klenow-filled BamHI site and 40 nt of
VEGF-2 coding sequence starting from the initiation codon; the 3'
primer (5'-GCA GGG TAC GGA TCC TAG ATT AGC TCA TTT GTG GTC TTT-3'
(SEQ ID NO:27)) contains a BamHI site and 16 nt of VEGF-2 coding
sequence not including the stop codon.
[0447] The PCR amplified DNA fragment is isolated from a 1% agarose
gel as described above and then digested with the endonuclease
BamHI and then purified again on a 1% agarose gel. The isolated
fragment and the dephosphorylated vector are then ligated with T4
DNA ligase. E. coli HB101 cells are then transformed and bacteria
identified that contained the plasmid pC1. The sequence and
orientation of the inserted gene is confirmed by DNA sequencing.
This construct is designated pC1VEGF-2.
6. Construction of pC4SigVEGF-2 T103-L215
[0448] Plasmid pC4Sig is plasmid pC4 (Accession No. 209646)
containing a human IgG Fc portion as well as a protein signal
sequence.
[0449] To permit Polymerase Chain Reaction directed amplification
and sub-cloning of VEGF-2 T103-L215 (amino acids 103 to 215 in FIG.
1 or SEQ ID NO: 18) into pC4Sig, two oligonucleotide primers
complementary to the desired region of VEGF-2 were synthesized with
the following base sequence: TABLE-US-00006 5' Primer (Bam HI and
26 nt of coding sequence): 5'-GCA GCA GGA TCC ACA GAA GAG ACT ATA
AAA TTT GCT GC-3' (SEQ ID NO:34) 3' Primer (Xba I, STOP, and 15 nt
of coding sequence): 5'-CGT CGT TCT AGA TCA CAG TTT AGA CAT GCA TCG
GCA G-3' (SEQ ID NO:35)
[0450] The Polymerase Chain Reaction was performed using standard
conditions well known to those skilled in the art and the
nucleotide sequence for the mature VEGF-2 (aa 24-419) as, for
example, constructed in Example 3, as template. The resulting
amplicon was restriction digested with BamHI and XbaI and subcloned
into BamHI/XbaI digested pC4Sig vector.
7. Construction of pC4SigVEGF-2 T103-R227
[0451] To permit Polymerase Chain Reaction directed amplification
and sub-cloning of VEGF-2 T103-L215 (amino acids 103 to 227 in FIG.
1 or SEQ ID NO: 18) into pC4Sig, two oligonucleotide primers
complementary to the desired region of VEGF-2 were synthesized with
the following base sequence: TABLE-US-00007 5' Primer (Bam HI and
26 nt of coding sequence): 5'-GCA GCA GGA TCC ACA GAA GAG ACT ATA
AAA TTT GCT GC-3' (SEQ ID NO:34) 3' Primer (Xba I, STOP, and 21 nt
of coding sequence): 5'-GCA GCA TCT AGA TCA ACG TCT AAT AAT GGA ATG
AAC-3' (SEQ ID NO:25)
[0452] The Polymerase Chain Reaction was performed using standard
conditions well known to those skilled in the art and the
nucleotide sequence for the mature VEGF-2 (aa 24-419) as, for
example, constructed in Example 3, as template. The resulting
amplicon was restriction digested with BamHI and XbaI and subcloned
into BamHI/XbaI digested pC4Sig vector.
8. Construction of pC4 VEGF-2 M1-M263
[0453] The expression vector pC4 contains the strong promoter (LTR)
of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular
Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer
(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites,
e.g., with the restriction enzyme cleavage sites BamHI, XbaI and
Asp718, facilitate the cloning of the gene of interest. The vector
contains in addition the 3N intron, the polyadenylation and
termination signal of the rat preproinsulin gene.
[0454] In this illustrative example, the cloned DNA encoding the
C-terminal deleted VEGF-2 M1-M263 protein (amino acids 1-263 in
FIG. 1 or SEQ ID NO:2) is inserted into the plasmid vector pC4 to
express the C-terminal deleted VEGF-2 protein.
[0455] To permit Polymerase Chain Reaction directed amplification
and sub-cloning of VEGF-2 M1-M263 into the expression vector, pC4,
two oligonucleotide primers complementary to the desired region of
VEGF-2 were synthesized with the following base sequence:
TABLE-US-00008 5' Primer 5'-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG
CTT CTT CTC-3' (SEQ ID NO:28) 3' Primer 5'-GAC TGG TAC CTT ATC ACA
TAA AAT CTT CCT GAG CC-3' (SEQ ID NO:29)
[0456] In the case of the above described 5' primer, an BamHI
restriction site was incorporated, while in the case of the 3'
primer, an Asp718 restriction site was incorporated. The 5' primer
also contains 6 nt, 20 nt of VEGF-2 coding sequence, and an ATG
sequence adjacent and in frame with the VEGF-2 coding region to
allow translation of the cloned fragment in E. coli, while the 3'
primer contains 2 nt, 20 nt of VEGF-2 coding sequence, and one stop
codon (preferentially utilized in E. coli) adjacent and in frame
with the VEGF-2 coding region which ensures correct translational
termination in E. coli.
[0457] The Polymerase Chain Reaction was performed using standard
conditions well known to those skilled in the art and the
nucleotide sequence for the mature VEGF-2 (aa 24-419) as
constructed, for example, in Example 3 as template. The resulting
amplicon was restriction digested with BamHI and Asp718 and
subcloned into BamHI/Asp718 digested pC4 protein expression vector.
This construct is designated pC4VEGF-2 M1-M263.
9. Construction of pC4VEGF-2 M1-D311
[0458] In this illustrative example, the cloned DNA encoding the
C-terminal deleted VEGF-2 M1-D311 protein (amino acids 1-311 in SEQ
ID NO:2) is inserted into the plasmid vector pC4 to express the
C-terminal deleted VEGF-2 protein.
[0459] To permit Polymerase Chain Reaction directed amplification
and sub-cloning of VEGF-2 M1-D311 into the expression vector, pC4,
two oligonucleotide primers complementary to the desired region of
VEGF-2 were synthesized with the following base sequence:
TABLE-US-00009 5' Primer 5'-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG
CTT CTT CTC-3' (SEQ ID NO:30) 3' Primer 5'-GAC TGG TAC CTT ATC AGT
CTA GTT CTT TGT GGG G-3' (SEQ ID NO:31)
[0460] In the case of the above described 5' primer, an BamHI
restriction site was incorporated, while in the case of the 3'
primer, an Asp718 restriction site was incorporated. The 5' primer
also contains 6 nt, 20 nt of VEGF-2 coding sequence, and an ATG
sequence adjacent and in frame with the VEGF-2 coding region to
allow translation of the cloned fragment in E. coli, while the 3'
primer contains 2 nt, 20 nt of VEGF-2 coding sequence, and one stop
codon (preferentially utilized in E. coli) adjacent and in frame
with the VEGF-2 coding region which ensures correct translational
termination in E. coli.
[0461] The Polymerase Chain Reaction was performed using standard
conditions well known to those skilled in the art and the
nucleotide sequence for the mature VEGF-2 (aa 24-419) as
constructed, for example, in Example 3 as template. The resulting
amplicon was restriction digested with BamHI and Asp718 and
subcloned into BamHI/Asp718 digested pC4 protein expression
vector.
10. Construction of pC4VEGF-2 M1-Q367
[0462] In this illustrative example, the cloned DNA encoding the
C-terminal deleted VEGF-2 M1-D311 protein (amino acids 1-311 in
FIG. 1 or SEQ ID NO:2) is inserted into the plasmid vector pC4 to
express the C-terminal deleted VEGF-2 protein.
[0463] To permit Polymerase Chain Reaction directed amplification
and sub-cloning of VEGF-2 M1-D311 into the expression vector, pC4,
two oligonucleotide primers complementary to the desired region of
VEGF-2 were synthesized with the following base sequence:
TABLE-US-00010 5' Primer 5'-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG
CTT CTT CTC-3' (SEQ ID NO:32) 3' Primer 5'-GAC TGG TAC CTC ATT ACT
GTG GAC TTT CTG TAC ATT C-3' (SEQ ID NO:33)
[0464] In the case of the above described 5' primer, a BamHI
restriction site was incorporated, while in the case of the 3'
primer, an Asp718 restriction site was incorporated. The 5' primer
also contains 6 nt, 20 nt of VEGF-2 coding sequence, and an ATG
sequence adjacent and in frame with the VEGF-2 coding region to
allow translation of the cloned fragment in E. coli, while the 3'
primer contains 2 nt, 20 nt of VEGF-2 coding sequence, and one stop
codon (preferentially utilized in E. coli) adjacent and in frame
with the VEGF-2 coding region which ensures correct translational
termination in E. coli.
[0465] The Polymerase Chain Reaction was performed using standard
conditions well known to those skilled in the art and the
nucleotide sequence for the mature VEGF-2 (aa 24-419) as
constructed, for example, in Example 3 as template. The resulting
amplicon was restriction digested with BamHI and Asp718 and
subcloned into BamHI/Asp718 digested pC4 protein expression vector.
This construct is designated pC4VEGF-2 M1-Q367.
Example 10
Transient Expression of VEGF-2 Protein in COS-7 Cells
Experimental Design
[0466] Expression of the VEGF-2-HA fusion protein from the
construct made in Example 4, for example, was detected by
radiolabeling and immunoprecipitation, using methods described in,
for example Harlow and colleagues (Antibodies: A Laboratory Manual,
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1988)). To this end, two days after transfection, the cells
were labeled by incubation in media containing 35S-cysteine for 8
hours. The cells and the media were collected, and the cells were
washed and then lysed with detergent-containing RIPA buffer: 150 mM
NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5,
as described by Wilson and colleagues (supra). Proteins were
precipitated from the cell lysate and from the culture media using
an HA-specific monoclonal antibody. The precipitated proteins then
were analyzed by SDS-PAGE and autoradiography.
Results
[0467] As shown in FIG. 16, cells transfected with pcDNA1 VEGF-2HA
secreted a 56 kd and a 30 kd protein. The 56 kd protein, but not
the 30 kd protein, could also be detected in the cell lysate but is
note detected in controls. This suggests the 30 kd protein is
likely to result from cleavage of the 56 kd protein. Since the
HA-tag is on the C-terminus of VEGF-2, the 30 kd protein must
represent the C-terminal portion of the cleaved protein, whereas
the N-terminal portion of the cleaved protein would not be detected
by immunoprecipitation. These data indicate that VEGF-2 protein
expressed in mammalian cells is secreted and processed.
Example 11
Stimulatory effect of VEGF-2 on Proliferation of Vascular
Endothelial Cells
Experimental Design
[0468] Expression of VEGF-2 is abundant in highly vascularized
tissues. Therefore the role of VEGF-2 in regulating proliferation
of several types of endothelial cells was examined.
Endothelial Cell Proliferation Assay
[0469] For evaluation of mitogenic activity of growth factors, the
colorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)2H-tetrazolium) assay with the electron coupling reagent PMS
(phenazine methosulfate) was performed (CellTiter 96 AQ,
PROMEGA.TM.). Cells were seeded in a 96-well plate (5,000
cells/well) in 0.1 mL serum-supplemented medium and allowed to
attach overnight. After serum-starvation for 12 hours in 0.5% FBS,
conditions (bFGF, VEGF.sub.165 or VEGF-2 in 0.5% FBS) with or
without Heparin (8 U/ml) were added to wells for 48 hours. 20 mg of
MTS/PMS mixture (1:0.05) were added per well and allowed to
incubate for 1 hour at 37.degree. C. before measuring the
absorbance at 490 nm in an ELISA plate reader. Background
absorbance from control wells (some media, no cells) was
subtracted, and seven wells were performed in parallel for each
condition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518
(1994)
Results
[0470] VEGF-2 stimulated proliferation of human umbilical vein
endothelial cells (HUVEC) and dermal microvascular endothelial
cells slightly (FIGS. 17 and 18). The stimulatory effect of VEGF-2
is more pronounced on proliferation of endometrial and
microvascular endothelial cells (FIG. 19). Endometrial endothelial
cells (HEEC) demonstrated the greatest response to VEGF-2 (96% of
the effect of VEGF on microvascular endothelial cells). The
response of microvascular endothelial cells (HMEC) to VEGF-2 was
73% compared to VEGF. The response of HUVEC and BAEC (bovine aortic
endothelial cells) to VEGF-2 was substantially lower at 10% and 7%,
respectively. The activity of VEGF-2 protein has varied between
different purification runs with the stimulatory effect of certain
batches on HUVEC proliferation being significantly higher than that
of other batches.
Example 12
Inhibition of PDGF-Induced Vascular Smooth Muscle Cell
Proliferation
[0471] VEGF-2 expression is high in vascular smooth muscle cells.
Smooth muscle is an important therapeutic target for vascular
diseases, such as restenosis. To evaluate the potential effects of
VEGF-2 on smooth muscle cells, the effect of VEGF-2 on human aortic
smooth muscle cell (HAoSMC) proliferation was examined.
Experimental Design
[0472] HAoSMC proliferation can be measured, for example, by BrdUrd
incorporation. Briefly, subconfluent, quiescent cells grown on the
4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP.
Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd.
After 24 h, immunocytochemistry is performed by using BrdUrd
Staining Kit (Zymed Laboratories). In brief, the cells are
incubated with the biotinylated mouse anti-BrdUrd antibody at 4
.degree. C. for 2 h after exposing to denaturing solution and then
with the streptavidin-peroxidase and diaminobenzidine. After
counterstaining with hematoxylin, the cells are mounted for
microscopic examination, and the BrdUrd-positive cells are counted.
The BrdUrd index is calculated as a percent of the BrdUrd-positive
cells to the total cell number. In addition, the simultaneous
detection of the BrdUrd staining (nucleus) and the FITC uptake
(cytoplasm) is performed for individual cells by the concomitant
use of bright field illumination and dark field-UV fluorescent
illumination. See, Hayashida et al., J. Biol. Chem.
6;271(36):21985-21992 (1996).
Results
[0473] VEGF-2 has an inhibitory effect on proliferation of vascular
smooth muscle cells induced by PDGF, but not by Fetal Bovine Serum
(FBS) (FIG. 20).
Example 13
Stimulation of Endothelial Cell Migration
[0474] Endothelial cell migration is an important step involved in
angiogenesis.
Experimental Design
[0475] This example will be used to explore the possibility that
VEGF-2 may stimulate lymphatic endothelial cell migration.
Currently, there are no published reports of such a model. However,
we will be adapting a model of vascular endothelial cell migration
for use with lymphatic endothelial cells essentially as
follows:
[0476] Endothelial cell migration assays are performed using a 48
well microchemotaxis chamber (Neuroprobe Inc., Cabin John, M D;
Falk, W., Goodwin, R. H. J., and Leonard, E. J. "A 48 well micro
chemotaxis assembly for rapid and accurate measurement of leukocyte
migration." J. Immunological Methods 1980;33:239-247).
Polyvinylpyrrolidone-free polycarbonate filters with a pore size of
8 um (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1%
gelatin for at least 6 hours at room temperature and dried under
sterile air. Test substances are diluted to appropriate
concentrations in M199 supplemented with 0.25% bovine serum albumin
(BSA), and 25 .mu.l of the final dilution is placed in the lower
chamber of the modified Boyden apparatus. Subconfluent, early
passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for
the minimum time required to achieve cell detachment. After placing
the filter between lower and upper chamber, 2.5.times.10.sup.5
cells suspended in 50 .mu.l M199 containing 1% FBS are seeded in
the upper compartment. The apparatus is then incubated for 5 hours
at 37.degree. C. in a humidified chamber with 5% CO2 to allow cell
migration. After the incubation period, the filter is removed and
the upper side of the filter with the non-migrated cells is scraped
with a rubber policeman. The filters are fixed with methanol and
stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park,
Ill.). Migration is quantified by counting cells of three random
high-power fields (40.times.) in each well, and all groups are
performed in quadruplicate.
Results
[0477] In an assay examining HUVEC migration using a 43-well
microchemotaxis chamber, VEGF-2 was able to stimulate migration of
HUVEC (FIG. 21).
Example 14
Stimulation of Nitric Oxide Production by Endothelial Cells
[0478] Nitric oxide released by the vascular endothelium is
believed to be a mediator of vascular endothelium relaxation.
VEGF-1 has been demonstrated to induce nitric oxide production by
endothelial cells in response to VEGF-1. As a result, VEGF-2
activity can be assayed by determining nitric oxide production by
endothelial cells in response to VEGF-2.
Experimental Design
[0479] Nitric oxide is measured in 96-well plates of confluent
microvascular endothelial cells after 24 hours starvation and a
subsequent 4 hr exposure to various levels of VEGF-1 and VEGF-2.
Nitric oxide in the medium is determined by use of the Griess
reagent to measure total nitrite after reduction of nitric
oxide-derived nitrate by nitrate reductase. The effect of VEGF-2 on
nitric oxide release was examined on HUVEC.
[0480] Briefly, NO release from cultured HUVEC monolayer was
measured with a NO-specific polarographic electrode connected to a
NO meter (Iso-NO, World Precision Instruments Inc.) (1049).
Calibration of the NO elements was performed according to the
following equation: 2
KNO.sub.2+2KI+2H.sub.2SO.sub.462NO+I.sub.2+2H.sub.2O+2K.sub.2SO.sub.4
[0481] The standard calibration curve was obtained by adding graded
concentrations of KNO.sub.2 (0, 5, 10, 25, 50, 100, 250, and 500
nmol/L) into the calibration solution containing KI and
H.sub.2SO.sub.4. The specificity of the Iso-NO electrode to NO was
previously determined by measurement of NO from authentic NO gas
(1050). The culture medium was removed and HUVECs were washed twice
with Dulbecco's phosphate buffered saline. The cells were then
bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well
plates, and the cell plates were kept on a slide warmer (Lab Line
Instruments Inc.) To maintain the temperature at 37.degree. C. The
NO sensor probe was inserted vertically into the wells, keeping the
tip of the electrode 2 mm under the surface of the solution, before
addition of the different conditions. S-nitroso acetyl penicillamin
(SNAP) was used as a positive control. The amount of released NO
was expressed as picomoles per 1.times.10.sup.6 endothelial cells.
All values reported were means of four to six measurements in each
group (number of cell culture wells). See, Leak et al. Biochem. and
Biophys. Res. Comm. 217:96-105 (1995).
Results
[0482] VEGF-2 was capable of stimulating nitric oxide release on
HUVEC (FIG. 22) to a higher level than VEGF. This suggested that
VEGF-2 may modify vascular permeability and vessel dilation.
Example 15
Effect of VEGF-2 on Cord Formation in Angiogenesis
[0483] Another step in angiogenesis is cord formation, marked by
differentiation of endothelial cells. This bioassay measures the
ability of microvascular endothelial cells to form capillary-like
structures (hollow structures) when cultured in vitro.
Experimental Design
[0484] CADMEC (microvascular endothelial cells) are purchased from
Cell Applications, Inc. as proliferating (passage 2) cells and are
cultured in Cell Applications' CADMEC Growth Medium and used at
passage 5. For the in vitro angiogenesis assay, the wells of a
48-well cell culture plate are coated with Cell Applications'
Attachment Factor Medium (200 ml/well) for 30 min. at 37.degree. C.
CADMEC are seeded onto the coated wells at 7,500 cells/well and
cultured overnight in Growth Medium. The Growth Medium is then
replaced with 300 mg Cell Applications' Chord Formation Medium
containing control buffer or HGS protein (0.1 to 100 ng/ml) and the
cells are cultured for an additional 48 hr. The numbers and lengths
of the capillary-like chords are quantitated through use of the
Boeckeler VIA-170 video image analyzer. All assays are done in
triplicate.
[0485] Commercial (R&D) VEGF (50 ng/ml) is used as a positive
control. b-esteradiol (1 ng/ml) is used as a negative control. The
appropriate buffer (without protein) is also utilized as a
control.
Results
[0486] It has been observed that VEGF-2 inhibits cord formation
similar to IFNa which also stimulates endothelial cell
proliferation (FIG. 23). This inhibitory effect may be a secondary
effect of endothelial proliferation which is mutually exclusive
with the cord formation process.
Example 16
Angiogenic Effect on Chick Chorioallantoic Membrane
[0487] Chick chorioallantoic membrane (CAM) is a well-established
system to examine angiogenesis. Blood vessel formation on CAM is
easily visible and quantifiable. The ability of VEGF-2 to stimulate
angiogenesis in CAM was examined.
Experimental Design
Embryos
[0488] Fertilized eggs of the White Leghorn chick (Gallus gallus)
and the Japanese quail (Coturnix coturnix) were incubated at
37.8.degree. C. and 80% humidity. Differentiated CAM of 16-day-old
chick and 13-day-old quail embryos was studied with the following
methods.
CAM Assay
[0489] On Day 4 of development, a window was made into the egg
shell of chick eggs. The embryos were checked for normal
development and the eggs sealed with cellotape. They were further
incubated until Day 13. THERMANOX.TM. coverslips (Nunc, Naperville,
Ill.) were cut into disks of about 5 mm in diameter. Sterile and
salt-free growth factors were dissolved in distilled water and
about 3.3 mg/5 ml was pipetted on the disks. After air-drying, the
inverted disks were applied on CAM. After 3 days, the specimens
were fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in
0.12 M sodium cacodylate buffer. They were photographed with a
stereo microscope [Wild M8] and embedded for semi- and ultrathin
sectioning as described above. Controls were performed with carrier
disks alone.
Results
[0490] This data demonstrates that VEGF-2 can stimulate
angiogenesis in the CAM assay nine-fold compared to the untreated
control. However, this stimulation is only 45% of the level of VEGF
stimulation (FIG. 24).
Example 17
Angiogenesis Assay Using a Matrigel Implant in Mouse
Experimental Design
[0491] In order to establish an in vivo model for angiogenesis to
test protein activities, mice and rats have been implanted
subcutaneously with methylcellulose disks containing either 20 mg
of BSA (negative control) and 1 mg of bFGF and 0.5 mg of VEGF-1
(positive control).
[0492] It appeared as though the BSA disks contained little
vascularization, while the positive control disks showed signs of
vessel formation. At day 9, one mouse showed a clear response to
the bFGF.
Results
[0493] Both VEGF proteins appeared to enhance MATRIGEL.TM.
cellularity by a factor of approximately 2 by visual
estimation.
[0494] An additional 30 mice were implanted with disks containing
BSA, bFGF, and varying amounts of VEGF-1, VEGF-2-B8, and VEGF-2-C4.
Each mouse received two identical disks, rather than one control
and one experimental disk.
[0495] Samples of all the disks recovered were immunostained with
Von Willebrand's factor to detect for the presence of endothelial
cells in the disks, and flk-1 and flt-4 to distinguish between
vascular and lymphatic endothelial cells. However, definitive
histochemical analysis of neovascularization and lymphangiogenesis
could not be determined.
Example 18
Rescue of Ischemia in Rabbit Lower Limb Model
Experimental Design
[0496] To study the in vivo effects of VEGF-2 on ischemia, a rabbit
hindlimb ischemia model was created by surgical removal of one
femoral arteries as described previously (Takeshita, S. et al., Am
J. Pathol 147:1649-1660 (1995)). The excision of the femoral artery
results in retrograde propagation of thrombus and occlusion of the
external iliac artery. Consequently, blood flow to the ischemic
limb is dependent upon collateral vessels originating from the
internal iliac artery (Takeshita, S. et al. Am J. Pathol
147:1649-1660 (1995)). An interval of 10 days was allowed for
post-operative recovery of rabbits and development of endogenous
collateral vessels. At 10 day post-operatively (day 0), after
performing a baseline angiogram, the internal iliac artery of the
ischemic limb was transfected with 500 mg naked VEGF-2 expression
plasmid by arterial gene transfer technology using a
hydrogel-coated balloon catheter as described (Riessen, R. et al.
Hum Gene Ther. 4:749-758 (1993); Leclerc, G. et al. J. Clin.
Invest. 90: 936-944 (1992)). When VEGF-2 was used in the treatment,
a single bolus of 500 mg VEGF-2 protein or control was delivered
into the internal iliac artery of the ischemic limb over a period
of 1 min. through an infusion catheter. On day 30, various
parameters were measured in these rabbits.
Results
[0497] Both VEGF-2 protein (FIGS. 25A, C, D, E, H, I, J, L, M, O)
and naked expression plasmid (FIGS. 25B, C, F H, I, K, L, M, O)
were able to restore the following parameters in the ischemic limb.
Restoration of blood flow, angiographic score seem to be slightly
more by administration of 500 mg plasmid compared with by 500 mg
protein (FIGS. 25H, I, L). The extent of the restoration is
comparable with that by VEGF in separate experiments (data not
shown). A vessel dilator was not able to achieve the same effect,
suggesting that the blood flow restoration is not simply due to a
vascular dilation effect.
A. BP Ratio (FIGS. 25A-25C)
[0498] The blood pressure ratio of systolic pressure of the
ischemic limb to that of normal limb.
B. Blood Flow and Flow Reserve (FIGS. 25D-25I)
[0499] Resting FL: the blood flow during un-dilated condition
[0500] Max FL: the blood flow during fully dilated condition (also
an indirect measure of the blood vessel amount)
[0501] Flow Reserve is reflected by the ratio of max FL:resting
FL.
C. Angiographic Score (FIGS. 25J-25L)
[0502] This is measured by the angiogram of collateral vessels. A
score was determined by the percentage of circles in an overlaying
grid that with crossing opacified arteries divided by the total
number m the rabbit thigh.
D. Capillary Density (FIGS. 25M-25O)
[0503] The number of collateral capillaries determined in light
microscopic sections taken from hindlimbs.
[0504] As discussed, VEGF-2 is processed to an N-terminal and a
C-terminal fragment which are co-purified. The N-terminal fragment
contains the intact putative functional domain and may be
responsible for the biologic activity.
Example 19
Effect of VEGF-2 on Vasodilation
[0505] As described above, VEGF-2 can stimulate NO release, a
mediator of vascular endothelium dilation. Since dilation of
vascular endothelium is important in reducing blood pressure, the
ability of VEGF-2 to affect the blood pressure in spontaneously
hypertensive rats (SHR) was examined. VEGF-2 caused a
dose-dependent decrease in diastolic blood pressure (FIGS. 26a and
b). There was a steady decline in diastolic blood pressure with
increasing doses of VEGF-2 which attained statistical significance
when a dose of 300 mg/kg was administered. The changes observed at
this dose were not different than those seen with acetylcholine
(0.5 mg/kg). Decreased mean arterial pressure (MAP) was observed as
well (FIG. 26c and d). VEGF-2 (300 mg/kg) and acetylcholine reduced
the MAP of these SHR animals to normal levels.
[0506] Additionally, increasing doses (0, 10, 30, 100, 300, and 900
mg/kg) of the B8, C5, and C4 preps of VEGF-2 were administered to
13-14 week old spontaneously hypertensive rats (SHR). Data are
expressed as the mean .+-.SEM. Statistical analysis was performed
with a paired t-test and statistical significance was defined as
p<0.05 vs. the response to buffer alone.
[0507] Studies with VEGF-2 (C5 prep) revealed that although it
significantly decreased the blood pressure, the magnitude of the
response was not as great as that seen with VEGF-2 (B8 prep) even
when used at a dose of 900 mg/kg.
[0508] Studies with VEGF-2 (C4 preparation) revealed that this CHO
expressed protein preparation yielded similar results to that seen
with C5 (i.e. statistically significant but of far less magnitude
than seen with the B8. preparation) (see FIGS. 26A-D).
[0509] As a control and since the C4 and C5 batches of VEGF-2
yielded minor, but statistically significant, changes in blood
pressure, experiments were performed experiments with another
CHO-expressed protein, M-CIF. Administration of M-CIF at doses
ranging from 10-900 mg/kg produced no significant changes in
diastolic blood pressure. A minor statistically significant
reduction in mean arterial blood pressure was observed at doses of
100 and 900 mg/kg but no dose response was noted. These results
suggest that the reductions in blood pressure observed with the C4
and C5 batches of VEGF-2 were specific, i.e. VEGF-2 related.
Example 20
Rat Ischemic Skin Flap Model
Experimental Design
[0510] The evaluation parameters include skin blood flow, skin
temperature, and factor VIII immunohistochemistry or endothelial
alkaline phosphatase reaction. VEGF-2 expression, during the skin
ischemia, is studied using in situ hybridization.
[0511] The study in this model is divided into three parts as
follows: [0512] a) Ischemic skin [0513] b) Ischemic skin wounds
[0514] c) Normal wounds
[0515] The experimental protocol includes: [0516] a) Raising a
3.times.4 cm, single pedicle full-thickness random skin flap
(myocutaneous flap over the lower back of the animal). [0517] b) An
excisional wounding (4-6 mm in diameter) in the ischemic skin
(skin-flap). [0518] c) Topical treatment with VEGF-2 of the
excisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the
following various dosage ranges: 1 mg to 100 mg. [0519] d)
Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21
post-wounding for histological, immunohistochemical, and in situ
studies.
Example 21
Peripheral Arterial Disease Model
[0520] Angiogenic therapy using VEGF-2 has been developed as a
novel therapeutic strategy to obtain restoration of blood flow
around the ischemia in case of peripheral arterial diseases.
Experimental Design
[0521] The experimental protocol includes: [0522] a) One side of
the femoral artery is ligated to create ischemic muscle of the
hindlimb, the other side of hindlimb serves as a control. [0523] b)
VEGF-2 protein, in a dosage range of 20 mg -500 mg, is delivered
intravenously and/or intramuscularly 3 times (perhaps more) per
week for 2-3 weeks. [0524] c) The ischemic muscle tissue is
collected after ligation of the femoral artery at 1, 2, and 3 weeks
for the analysis of VEGF-2 expression and histology. Biopsy is also
performed on the other side of normal muscle of the contralateral
hindlimb.
Example 22
Ischemic Myocardial Disease Model
[0525] VEGF-2 is evaluated as a potent mitogen capable of
stimulating the development of collateral vessels, and
restructuring new vessels after coronary artery occlusion.
Alteration of VEGF-2 expression is investigated in situ.
Experimental Design
[0526] The experimental protocol includes: [0527] a) The heart is
exposed through a left-side thoracotomy in the rat. Immediately,
the left coronary artery is occluded with a thin suture (6-0) and
the thorax is closed. [0528] b) VEGF-2 protein, in a dosage range
of 20 mg -500 mg, is delivered intravenously and/or intramuscularly
3 times (perhaps more) per week for 2-4 weeks. [0529] c) Thirty
days after the surgery, the heart is removed and cross-sectioned
for morphometric and in situ analyzes.
Example 23
Rat Corneal Wound Healing Model
[0530] This animal model shows the effect of VEGF-2 on
neovascularization.
Experimental Design
[0531] The experimental protocol includes: [0532] a) Making a 1-1.5
mm long incision from the center of cornea into the stromal layer.
[0533] b) Inserting a spatula below the lip of the incision facing
the outer corner of the eye. [0534] c) Making a pocket (its base is
1-1.5 mm form the edge of the eye). [0535] d) Positioning a pellet,
containing 50 mg -500mg VEGF-2, within the pocket. [0536] e) VEGF-2
treatment can also be applied topically to the corneal wounds in a
dosage range of 20 mg-500 mg (daily treatment for five days).
Example 24
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
[0536] Experimental Design
[0537] The experimental protocol includes:
1. Diabetic db+/db+ Mouse Model.
[0538] To demonstrate that VEGF-2 accelerates the healing process,
the genetically diabetic mouse model of wound healing is used. The
full thickness wound healing model in the db+/db+ mouse is a well
characterized, clinically relevant and reproducible model of
impaired wound healing. Healing of the diabetic wound is dependent
on formation of granulation tissue and re-epithelialization rather
than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389
(1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235
(1990)).
[0539] The diabetic animals have many of the characteristic
features observed in Type II diabetes mellitus. Homozygous
(db+/db+) mice are obese in comparison to their normal heterozygous
(db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single
autosomal recessive mutation on chromosome 4 (db+) (Coleman et al.
Proc. Natl. Acad Sci. USA 77:283-293 (1982)). Animals show
polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+)
have elevated blood glucose, increased or normal insulin levels,
and suppressed cell-mediated immunity (Mandel et al., J. Immunol.
120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.
51(1):1-7 (1983); Leiter et al, Am. J. of Pathol. 114:46-55
(1985)). Peripheral neuropathy, myocardial complications, and
microvascular lesions, basement membrane thickening and glomerular
filtration abnormalities have been described in these animals
(Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et
al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest.
40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6
(1982)). These homozygous diabetic mice develop hyperglycemia that
is resistant to insulin analogous to human type II diabetes (Mandel
et al., J. Immunol. 120:1375-1377 (1978)).
[0540] The characteristics observed in these animals suggests that
healing in this model may be similar to the healing observed in
human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246
(1990)).
Animals
[0541] Genetically diabetic female C57BL/KsJ (db+/db+) mice and
their non-diabetic (db+/+m) heterozygous littermates were used in
this study (Jackson Laboratories). The animals were purchased at 6
weeks of age and were 8 weeks old at the beginning of the study.
Animals were individually housed and received food and water ad
libitum. All manipulations were performed using aseptic techniques.
The experiments were conducted according to the rules and
guidelines of Human Genome Sciences, Inc. Institutional Animal Care
and Use Committee and the Guidelines for the Care and Use of
Laboratory Animals.
Surgical Wounding
[0542] Wounding protocol is performed according to previously
reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med.
172:245-251 (1990)). Briefly, on the day of wounding, animals are
anesthetized with an intraperitoneal injection of Avertin (0.01
mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in
deionized water. The dorsal region of the animal is shaved and the
skin washed with 70% ethanol solution and iodine. The surgical area
is dried with sterile gauze prior to wounding. An 8 mm
full-thickness wound is then created using a Keyes tissue punch.
Immediately following wounding, the surrounding skin is gently
stretched to eliminate wound expansion. The wounds are left open
for the duration of the experiment. Application of the treatment is
given topically for 5 consecutive days commencing on the day of
wounding. Prior to treatment, wounds are gently cleansed with
sterile saline and gauze sponges.
[0543] Wounds are visually examined and photographed at a fixed
distance at the day of surgery and at two day intervals thereafter.
Wound closure is determined by daily measurement on days 1-5 and on
day 8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by
a continuous epithelium.
[0544] VEGF-2 is administered using at a range different doses of
VEGF-2, from 4 mg to 500 mg per wound per day for 8 days in
vehicle. Vehicle control groups received 50 mL of vehicle
solution.
[0545] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology and
immunohistochemistry. Tissue specimens are placed in 10% neutral
buffered formalin in tissue cassettes between biopsy sponges for
further processing.
Experimental Design
[0546] Three groups of 10 animals each (5 diabetic and 5
non-diabetic controls) were evaluated: 1) Vehicle placebo control,
2) VEGF-2.
Measurement of Wound Area and Closure
[0547] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total square area of
the wound. Contraction is then estimated by establishing the
differences between the initial wound area (day 0) and that of post
treatment (day 8). The wound area on day 1 was 64 mm.sup.2, the
corresponding size of the dermal punch. Calculations were made
using the following formula: [Open area on day 8]-[Open area on day
1]/[Open area on day 1] Histology
[0548] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using a Reichert-Jung microtome. Routine
hematoxylin-eosin (H&E) staining is performed on cross-sections
of bisected wounds. Histologic examination of the wounds are used
to assess whether the healing process and the morphologic
appearance of the repaired skin is altered by treatment with KGF-2.
This assessment included verification of the presence of cell
accumulation, inflammatory cells, capillaries, fibroblasts,
re-epithelialization and epidermal maturity (Greenhalgh, D. G. et
al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer
is used by a blinded observer.
Immunohistochemistry
Re-Epithelialization
[0549] Tissue sections are stained immunohistochemically with a
polyclonal rabbit anti-human keratin antibody using ABC Elite
detection system. Human skin is used as a positive tissue control
while non-immune IgG is used as a negative control. Keratinocyte
growth is determined by evaluating the extent of
reepithelialization of the wound using a calibrated lens
micrometer.
Cell Proliferation Marker
[0550] Proliferating cell nuclear antigen/cyclin (PCNA) in skin
specimens is demonstrated by using anti-PCNA antibody (1:50) with
an ABC Elite detection system. Human colon cancer served as a
positive tissue control and human brain tissue is used as a
negative tissue control. Each specimen included a section with
omission of the primary antibody and substitution with non-immune
mouse IgG. Ranking of these sections is based on the extent of
proliferation on a scale of 0-8, the lower side of the scale
reflecting slight proliferation to the higher side reflecting
intense proliferation.
Statistical Analysis
[0551] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
B. Steroid Impaired Rat Model
[0552] The inhibition of wound healing by steroids has been well
documented in various in vitro and in vivo systems (Wahl, S. M.
Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid
Action: Basic and Clinical Aspects. 280-302 (1989); Wahl, S. M. et
al., J. Immunol. 115: 476-481 (1975); Werb, Z. et al., J. Exp. Med.
147:1684-1694 (1978)). Glucocorticoids retard wound healing by
inhibiting angiogenesis, decreasing vascular permeability (Ebert,
R. H., et al., An. Intern. Med. 37:701-705 (1952)), fibroblast
proliferation, and collagen synthesis (Beck, L. S. et al., Growth
Factors. 5: 295-304 (1991); Haynes, B. F. et al., J. Clin. Invest.
61: 703-797 (1978)) and producing a transient reduction of
circulating monocytes (Haynes, B. F., et al., J. Clin. Invest. 61:
703-797 (1978); Wahl, S. M., "Glucocorticoids and wound healing",
In: Antiinflammatory Steroid Action: Basic and Clinical Aspects,
Academic Press, New York, pp. 280-302 (1989)). The systemic
administration of steroids to impaired wound healing is a well
establish phenomenon in rats (Beck, L. S. et al., Growth Factors.
5: 295-304 (1991); Haynes, B. F., et al., J. Clin. Invest. 61:
703-797 (1978); Wahl, S. M., "Glucocorticoids and wound healing",
In: Antiinflammatory Steroid Action: Basic and Clinical Aspects,
Academic Press, New York, pp. 280-302 (1989); Pierce, G. F. et al.,
Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).
[0553] To demonstrate that VEGF-2 can accelerate the healing
process, the effects of multiple topical applications of VEGF-2 on
full thickness excisional skin wounds in rats in which healing has
been impaired by the systemic administration of methylprednisolone
is assessed.
Animals
[0554] Young adult male Sprague Dawley rats weighing 250-300 g
(Charles River Laboratories) are used in this example. The animals
are purchased at 8 weeks of age and were 9 weeks old at the
beginning of the study. The healing response of rats is impaired by
the systemic administration of methylprednisolone (17 mg/kg/rat
intramuscularly) at the time of wounding. Animals are individually
housed and received food and water ad libitum. All manipulations
are performed using aseptic techniques. This study is conducted
according to the rules and guidelines of Human Genome Sciences,
Inc. Institutional Animal Care and Use Committee and the Guidelines
for the Care and Use of Laboratory Animals.
Surgical Wounding
[0555] The wounding protocol is followed according to section A,
above. On the day of wounding, animals are anesthetized with an
intramuscular injection of ketamine (50 mg/kg) and xylazine (5
mg/kg). The dorsal region of the animal is shaved and the skin
washed with 70% ethanol and iodine solutions. The surgical area is
dried with sterile gauze prior to wounding. An 8 mm full-thickness
wound is created using a Keyes tissue punch. The wounds are left
open for the duration of the experiment. Applications of the
testing materials are given topically once a day for 7 consecutive
days commencing on the day of wounding and subsequent to
methylprednisolone administration. Prior to treatment, wounds are
gently cleansed with sterile saline and gauze sponges.
[0556] Wounds are visually examined and photographed at a fixed
distance at the day of wounding and at the end of treatment. Wound
closure is determined by daily measurement on days 1-5 and on day 8
for Figure. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue was no longer visible and the wound is covered
by a continuous epithelium.
[0557] VEGF-2 is administered using at a range different doses of
VEGF-2, from 4 mg to 500 mg per wound per day for 8 days in
vehicle. Vehicle control groups received 50 mL of vehicle
solution.
[0558] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology. Tissue specimens
are placed in 10% neutral buffered formalin in tissue cassettes
between biopsy sponges for further processing.
Experimental Design
[0559] Four groups of 10 animals each (5 with methylprednisolone
and 5 without glucocorticoid) were evaluated: 1) Untreated group 2)
Vehicle placebo control 3) VEGF-2 treated groups.
Measurement of Wound Area and Closure
[0560] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total area of the
wound. Closure is then estimated by establishing the differences
between the initial wound area (day 0) and that of post treatment
(day 8). The wound area on day 1 was 64 mm.sup.2, the corresponding
size of the dermal punch. Calculations were made using the
following formula: [Open area on day 8]-[Open area on day 1]/[Open
area on day 1] Histology
[0561] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using an Olympus microtome. Routine hematoxylin-eosin
(H&E) staining was performed on cross-sections of bisected
wounds. Histologic examination of the wounds allows assessment of
whether the healing process and the morphologic appearance of the
repaired skin was improved by treatment with VEGF-2. A calibrated
lens micrometer was used by a blinded observer to determine the
distance of the wound gap.
Statistical Analysis
[0562] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
Example 25
Specific Peptide Fragments to Generate VEGF-2 Monoclonal
Antibodies
[0563] Four specific peptides (designated SP-40, SP-41, SP-42 and
SP-43) have been generated. These will be used to generate
monoclonal antibodies to analyze VEGF-2 processing. The peptides
are shown below: TABLE-US-00011 1. "SP-40": 1. "SP-40":
MTVLYPEYWKMY (amino acids 70-81 in SEQ ID NO:2) 2. "SP-41":
KSIDNEWRKTQSMPREV (amino acids 120-136 (note C -> S mutation at
position 131) in SEQ ID NO:18) 3. "SP-42": MSKLDVYRQVHSIIRR (amino
acids 212-227 in SEQ ID NO:18) 4. "SP-43": MFSSDAGDDSTDGFHDI (amino
acids 263-279 in SEQ ID NO:18)
Example 26
Lymphadema Animal Model
[0564] The purpose of this experimental approach is to create an
appropriate and consistent lymphedema model for testing the
therapeutic effects of VEGF-2 in lymphangiogenesis and
re-establishment of the lymphatic circulatory system in the rat
hind limb. Effectiveness is measured by swelling volume of the
affected limb, quantification of the amount of lymphatic
vasculature, total blood plasma protein, and histopathology. Acute
lymphedema is observed for 7-10 days. Perhaps more importantly, the
chronic progress of the edema is followed for up to 3-4 weeks.
Experimental Procedure
[0565] Prior to beginning surgery, blood sample was drawn for
protein concentration analysis. Male rats weighing approximately
.about.350 g are dosed with Pentobarbital. Subsequently, the right
legs were shaved from knee to hip. The shaved area is swabbed with
gauze soaked in 70% EtOH. Blood is drawn for serum total protein
testing. Circumference and volumetric measurements were made prior
to injecting dye into paws after marking 2 measurement levels (0.5
cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of
both right and left paws are injected with 0.05 ml of 1% Evan's
Blue. Circumference and volumetric measurements are then made
following injection of dye into paws.
[0566] Using the knee joint as a landmark, a mid-leg inguinal
incision is made circumferentially allowing the femoral vessels to
be located. Forceps and hemostats are used to dissect and separate
the skin flaps. After locating the femoral vessels, the lymphatic
vessel that runs along side and underneath the vessel(s) is
located. The main lymphatic vessels in this area are then
electrically coagulated or suture ligated.
[0567] Using a microscope, muscles in back of the leg (near the
semitendinosis and adductors) are bluntly dissected. The popliteal
lymph node is then located.
[0568] The 2 proximal and 2 distal lymphatic vessels and distal
blood supply of the popliteal node are then and ligated by
suturing. The popliteal lymph node, and any accompanying adipose
tissue, is then removed by cutting connective tissues.
[0569] Care was taken to control any mild bleeding resulting from
this procedure. After lymphatics were occluded, the skin flaps are
sealed by using liquid skin (Vetbond) (A J Buck). The separated
skin edges are sealed to the underlying muscle tissue while leaving
a gap of .about.0.5 cm around the leg. Skin also may be anchored by
suturing to underlying muscle when necessary.
[0570] To avoid infection, animals are housed individually with
mesh (no bedding). Recovering animals were checked daily through
the optimal edematous peak, which typically occurred by day 5-7.
The plateau edematous peak was then observed. To evaluate the
intensity of the lymhedema, we measured the circumference and
volumes of 2 designated places on each paw before operation and
daily for 7 days. The effect plasma proteins have on lymphedema and
determined if protein analysis is a useful testing perimeter is
also investigated. The weights of both control and edematous limbs
are evaluated at 2 places. Analysis is performed in a blind
manner.
[0571] Circumference Measurements: Under brief gas anesthetic to
prevent limb movement, a cloth tape is used to measure limb
circumference. Measurements are done at the ankle bone and dorsal
paw by 2 different people then those 2 readings are averaged.
Readings are taken from both control and edematous limbs.
[0572] Volumetric Measurements: On the day of surgery, animals are
anesthetized with Pentobarbital and are tested prior to surgery.
For daily volumetrics animals are under brief halothane anesthetic
(rapid immobilization and quick recovery), both legs are shaved and
equally marked using waterproof marker on legs. Legs are first
dipped in water, then dipped into instrument to each marked level
then measured by Buxco edema software(Chen/Victor). Data is
recorded by one person, while the other is dipping the limb to
marked area.
[0573] Blood-plasma protein measurements: Blood is drawn, spun, and
serum separated prior to surgery and then at conclusion for total
protein and Ca2+ comparison.
[0574] Limb Weight Comparison: After drawing blood, the animal is
prepared for tissue collection. The limbs were amputated using a
quillitine, then both experimental and control legs were cut at the
ligature and weighed. A second weighing is done as the
tibio-cacaneal joint was disarticulated and the foot was
weighed.
[0575] Histological Preparations: The transverse muscle located
behind the knee (popliteal) area is dissected and arranged in a
metal mold, filled with freezeGel, dipped into cold methylbutane,
placed into labeled sample bags at -80 EC until sectioning. Upon
sectioning, the muscle was observed under fluorescent microscopy
for lymphatics. Other immuno/histological methods are currently
being evaluated.
Example 27
Method of Treatment Using Gene Therapy for Production of VEGF-2
Polypeptide--In Vivo
[0576] Another aspect of the present invention is using in vivo
gene therapy methods to treat disorders, diseases and conditions.
The gene therapy method relates to the introduction of naked
nucleic acid (DNA, RNA, and antisense DNA or RNA) comprising VEGF-2
operably linked to a promoter into an animal to increase the
expression of VEGF-2. Such gene therapy and delivery techniques and
methods are known in the art, see, for example, WO 90/11092, WO
98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Tabata H.
et al. (1997) Cardiovasc. Res. 35(3):470-479, Chao, J et al. (1997)
Pharmacol. Res. 35(6):517-522, Wolff, J. A. (1997) Neuromuscul.
Disord. 7(5):314-318, Schwartz, B. et al. (1996) Gene Ther.
3(5):405-41 1, Tsurumi, Y. et al. (1996) Circulation
94(12):3281-3290 (incorporated herein by reference).
[0577] The VEGF-2 polynucleotide constructs may be delivered by any
method that delivers injectable materials to the cells of an
animal, such as, injection into the interstitial space of tissues
(heart, muscle, skin, lung, liver, intestine and the like). The
VEGF-2 polynucleotide constructs may also be delivered directly
into arteries. The VEGF-2 polynucleotide constructs can be
delivered in a pharmaceutically acceptable liquid or aqueous
carrier.
[0578] The term "naked" polynucleotide, DNA or RNA, refers to
sequences that are free from any delivery vehicle that acts to
assist, promote, or facilitate entry into the cell, including viral
sequences, viral particles, liposome formulations, LIPOFECTIN.TM.
or precipitating agents and the like. However, the VEGF-2
polynucleotide may also be delivered in liposome formulations (such
as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci.
772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7)
which can be prepared by methods well known to those skilled in the
art.
[0579] The VEGF-2 vector constructs used in the gene therapy method
are preferably constructs that will not integrate into the host
genome nor will they contain sequences that allow for replication.
Unlike other gene therapies techniques, one major advantage of
introducing naked nucleic acid sequences into target cells is the
transitory nature of the polynucleotide synthesis in the cells.
Studies have shown that non-replicating DNA sequences can be
introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
[0580] Preferably, the VEGF-2 construct will comprise a VEGF-2
polynucleotide operably inserted into the pVGI.1 plasmid, as
illustrated in FIG. 30. The pVGI.I plasmid construct, the sequence
of which is shown in FIG. 31A-G, was deposited on Jul. 3, 2000 at
the American Type Tissue Collection (ATCC.TM.), 10801 University
Boulevard, Manassas, Va. 20110-2209, and given ATCC.TM. Deposit
Number PTA-2185. This VEGF-2 polynucleotide vector construct can be
delivered to tissues, by methods know in the art and described
above, preferably by direct injection using naked polynucleotide,
for therapeutic applications. Such uses include the promotion of
angiogenesis in the treatment of a number of diseases and
conditions, as described in "Therapeutic Uses" above, and elsewhere
herein. Preferred uses of this construct include the treatment of
critical limb ischemia and coronary artery disease.
[0581] The VEGF-2 construct can be delivered to the interstitial
space of tissues within the an animal, including of muscle, skin,
brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,
blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,
intestine, testis, ovary, uterus, rectum, nervous system, eye,
gland, and connective tissue. Interstitial space of the tissues
comprises the intercellular fluid, mucopolysaccharide matrix among
the reticular fibers of organ tissues, elastic fibers in the walls
of vessels or chambers, collagen fibers of fibrous tissues, or that
same matrix within connective tissue ensheathing muscle cells or in
the lacunae of bone. It is similarly the space occupied by the
plasma of the circulation and the lymph fluid of the lymphatic
channels. They may be conveniently delivered by injection into the
tissues comprising these cells. They are preferably delivered to
and expressed in persistent, non-dividing cells which are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such
as, for example, stem cells of blood or skin fibroblasts.
Preferably, they are delivered by direct injection into the
artery.
[0582] For the naked polynucleotide injection, an effective dosage
amount of DNA or RNA will be in the range of from about 0.05 g/kg
body weight to about 50 mg/kg body weight. Preferably the dosage
will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration. The
preferred route of administration is by the parenteral route of
injection into the interstitial space of tissues, or directly into
arteries. However, other parenteral routes may also be used, such
as, inhalation of an aerosol formulation particularly for delivery
to lungs or bronchial tissues, throat or mucous membranes of the
nose. In addition, naked VEGF-2 constructs can be delivered to
arteries during angioplasty by the catheter used in the
procedure.
[0583] The dose response effects of injected VEGF-2 polynucleotide
construct in arteries in vivo is determined as follows. Suitable
template DNA for production of mRNA coding for VEGF-2 is prepared
in accordance with a standard recombinant DNA methodology. The
template DNA, which may be either circular or linear, is either
used as naked DNA or complexed with liposomes. The arteries of
rabbits are then injected with various amounts of the template
DNA.
[0584] Hindlimb ischemia in rabbits is surgically induced, as
described in Example 18. Immediately following this, five different
sites in the adductor (2 sites), medial large (2 sites), and
semimembranous muscles (1 site) are injected directly with plasmid
DNA encoding VEGF-2 using a 3 ml syringe and 2-gauge needle
advanced through a small skin incision. The skin is then closed
using 4.0 nylon.
[0585] The ability to rescue hindlimb ischemia is determined by
measuring the number of capillaries in light microsopic sections
taken from the treated hindlimbs, compared to ischemic hindlimbs
from untreated rabbits, measurement of calf blood pressure, and
intra-arterial Doppler guidewire measurement of flow velocity
(Takeshita et al., J. Clin. Invest. 93:662-670 (1994)). The results
of the above experimentation in rabbits can be use to extrapolate
proper dosages and other treatment parameters in humans and other
animals using VEGF-2 polynucleotide naked DNA.
Example 28
Method of Treatment Using Gene Therapy--Ex Vivo
[0586] One method of gene therapy transplants fibroblasts, which
are capable of expressing VEGF-2 polypeptides, onto a patient.
Generally, fibroblasts are obtained from a subject by skin biopsy.
The resulting tissue is placed in tissue-culture medium and
separated into small pieces. Small chunks of the tissue are placed
on a wet surface of a tissue culture flask, approximately ten
pieces are placed in each flask. The flask is turned upside down,
closed tight and left at room temperature over night. After 24
hours at room temperature, the flask is inverted and the chunks of
tissue remain fixed to the bottom of the flask and fresh media
(e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin)
is added. The flasks are then incubated at 37 degree C. for
approximately one week.
[0587] 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.
[0588] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)),
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[0589] The cDNA encoding VEGF-2 can be amplified using PCR primers
which correspond to the 5' and 3' end sequences respectively as set
forth in Example 1. Preferably, the 5' primer contains an EcoRI
site and the 3' primer includes a HindIII site. Equal quantities of
the Moloney murine sarcoma virus linear backbone and the amplified
EcoRI and HindIII fragment are added together, in the presence of
T4 DNA ligase. The resulting mixture is maintained under conditions
appropriate for ligation of the two fragments. The ligation mixture
is then used to transform bacteria HB101, which are then plated
onto agar containing kanamycin for the purpose of confirming that
the vector contains properly inserted VEGF-2.
[0590] The amphotropic pA317 or GP+am2 packaging cells are grown in
tissue culture to confluent density in Dulbecco's Modified Eagles
Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the VEGF-2 gene is then
added to the media and the packaging cells transduced with the
vector. The packaging cells now produce infectious viral particles
containing the VEGF-2 gene (the packaging cells are now referred to
as producer cells).
[0591] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his. Once the
fibroblasts have been efficiently infected, the fibroblasts are
analyzed to determine whether VEGF-2 protein is produced.
[0592] The engineered fibroblasts are then transplanted onto the
host, either alone or after having been grown to confluence on
cytodex 3 microcarrier beads
Example 29
Method of Treatment Using Gene Therapy Homologous Recombination
[0593] Another method of gene therapy according to the present
invention involves operably associating the endogenous VEGF-2
sequence with a promoter via homologous recombination as described,
for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;
International Publication No. WO 96/2941 1, published Sep. 26,
1996; International Publication No. WO 94/12650, published Aug. 4,
1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935
(1989); and Zijlstra et al., Nature 342:435-438 (1989). This method
involves the activation of a gene which is present in the target
cells, but which is not expressed in the cells, or is expressed at
a lower level than desired.
[0594] Polynucleotide constructs are made which contain a promoter
and targeting sequences, which are homologous to the 5' non-coding
sequence of endogenous VEGF-2, flanking the promoter. The targeting
sequence will be sufficiently near the 5' end of VEGF-2 so the
promoter will be operably linked to the endogenous sequence upon
homologous recombination. The promoter and the targeting sequences
can be amplified using PCR. Preferably, the amplified promoter
contains distinct restriction enzyme sites on the 5' and 3' ends.
Preferably, the 3' end of the first targeting sequence contains the
same rest enzyme site as the 5' end of the amplified promoter and
the 5' end of the second targeting sequence contains the same
restriction site as the 3' end of the amplified promoter.
[0595] The amplified promoter and the amplified targeting sequences
are digested with the appropriate restriction enzymes and
subsequently treated with calf intestinal phosphatase. The digested
promoter and digested targeting sequences are added together in the
presence of T4 DNA ligase. The resulting mixture is maintained
under conditions appropriate for ligation of the two fragments. The
construct is size fractionated on an agarose gel then purified by
phenol extraction and ethanol precipitation.
[0596] In this Example, the polynucleotide constructs are
administered as naked polynucleotides via electroporation. However,
the polynucleotide constructs may also be administered with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, precipitating agents, etc. Such methods
of delivery are known in the art.
[0597] Once the cells are transfected, homologous recombination
will take place which results in the promoter being operably linked
to the endogenous VEGF-2 sequence. This results in the expression
of VEGF-2 in the cell. Expression may be detected by immunological
staining, or any other method known in the art. Fibroblasts are
obtained from a subject by skin biopsy. The resulting tissue is
placed in DMEM +10% fetal calf serum. Exponentially growing or
early stationary phase fibroblasts are trypsinized and rinsed from
the plastic surface with nutrient medium. An aliquot of the cell
suspension is removed for counting, and the remaining cells are
subjected to centrifugation. The supernatant is aspirated and the
pellet is resuspended in 5 ml of electroporation buffer (20 mM
HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na.sub.2HPO.sub.4, 6 mM
dextrose). The cells are recentrifuged, the supernatant aspirated,
and the cells resuspended in electroporation buffer containing 1
mg/ml acetylated bovine serum albumin. The final cell suspension
contains approximately 3.times.10.sup.6 cells/ml. Electroporation
should be performed immediately following resuspension.
[0598] Plasmid DNA is prepared according to standard techniques. To
construct a plasmid for targeting to the VEGF-2 locus, plasmid
pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The
CMV promoter is amplified by PCR with an XbaI site on the 5' end
and a BamHI site on the 3'end. Two VEGF-2 non-coding sequences are
amplified via PCR: one VEGF-2 non-coding sequence (VEGF-2 fragment
1) is amplified with a HindIII site at the 5' end and an Xba site
at the 3'end; the other VEGF-2 non-coding sequence (VEGF-2 fragment
2) is amplified with a BamHI site at the 5'end and a site at the
3'end. The CMV promoter and VEGF-2 fragments are digested with the
appropriate enzymes (CMV promoter--XbaI and BamHI; VEGF-2 fragment
1--XbaI; VEGF-2 fragment 2-BamHI) and ligated together. The
resulting ligation product is digested with HindIII, and ligated
with the HindIII-digested pUC18 plasmid.
[0599] Plasmid DNA is added to a sterile cuvette with a 0.4 cm
electrode gap (Bio-Rad). The final DNA concentration is generally
at least 120 .mu.g/ml. 0.5 ml of the cell suspension (containing
approximately 1.5.times.10.sup.6 cells) is then added to the
cuvette, and the cell suspension and DNA solutions are gently
mixed. Electroporation is performed with a Gene-Pulser apparatus
(Bio-Rad). Capacitance and voltage are set at 960 .mu.F and 250-300
V, respectively. As voltage increases, cell survival decreases, but
the percentage of surviving cells that stably incorporate the
introduced DNA into their genome increases dramatically. Given
these parameters, a pulse time of approximately 14-20 mSec should
be observed.
[0600] Electroporated cells are maintained at room temperature for
approximately 5 min, and the contents of the cuvette are then
gently removed with a sterile transfer pipette. The cells are added
directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf
serum) in a 10 cm dish and incubated at 37.degree. C. The following
day, the media is aspirated and replaced with 10 ml of fresh media
and incubated for a further 16-24 hours.
[0601] The engineered fibroblasts are then injected into the host,
either alone or after having been grown to confluence on cytodex 3
microcarrier beads. The fibroblasts now produce the protein
product.
Example 30
VEGF-2 Transgenic Animals
[0602] The VEGF-2 polypeptides can also be expressed in transgenic
animals. Animals of any species, including, but not limited to,
mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs,
goats, sheep, cows and non-human primates, e.g., baboons, monkeys,
and chimpanzees may be used to generate transgenic animals. In a
specific embodiment, techniques described herein or otherwise known
in the art, are used to express polypeptides of the invention in
humans, as part of a gene therapy protocol.
[0603] Any technique known in the art may be used to introduce the
transgene (i.e., polynucleotides of the invention) into animals to
produce the founder lines of transgenic animals. Such techniques
include, but are not limited to, pronuclear microinjection
(Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994);
Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et
al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S.
Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into
germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA
82:6148-6152 (1985)), blastocysts or embryos; gene targeting in
embryonic stem cells (Thompson et al., Cell 56:313-321 (1989));
electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.
3:1803-1814 (1983)); introduction of the polynucleotides of the
invention using a gene gun (see, e.g., Ulmer et al., Science
259:1745 (1993); introducing nucleic acid constructs into embryonic
pleuripotent stem cells and transferring the stem cells back into
the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,
Cell 57:717-723 (1989); etc. For a review of such techniques, see
Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989),
which is incorporated by reference herein in its entirety.
[0604] Any technique known in the art may be used to produce
transgenic clones containing polynucleotides of the invention, for
example, nuclear transfer into enucleated oocytes of nuclei from
cultured embryonic, fetal, or adult cells induced to quiescence
(Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-813 (1997)).
[0605] The present invention provides for transgenic animals that
carry the transgene in all their cells, as well as animals which
carry the transgene in some, but not all their cells, i.e., mosaic
animals or chimeric. The transgene may be integrated as a single
transgene or as multiple copies such as in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. The transgene may
also be selectively introduced into and activated in a particular
cell type by following, for example, the teaching of Lasko et al.
(Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The
regulatory sequences required for such a cell-type specific
activation will depend upon the particular cell type of interest,
and will be apparent to those of skill in the art. When it is
desired that the polynucleotide transgene be integrated into the
chromosomal site of the endogenous gene, gene targeting is
preferred.
[0606] Briefly, when such a technique is to be utilized, vectors
containing some nucleotide sequences homologous to the endogenous
gene are designed for the purpose of integrating, via homologous
recombination with chromosomal sequences, into and disrupting the
function of the nucleotide sequence of the endogenous gene. The
transgene may also be selectively introduced into a particular cell
type, thus inactivating the endogenous gene in only that cell type,
by following, for example, the teaching of Gu et al. (Gu et al.,
Science 265:103-106 (1994)). The regulatory sequences required for
such a cell-type specific inactivation will depend upon the
particular cell type of interest, and will be apparent to those of
skill in the art.
[0607] Once transgenic animals have been generated, the expression
of the recombinant gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to verify that
integration of the transgene has taken place. The level of mRNA
expression of the transgene in the tissues of the transgenic
animals may also be assessed using techniques which include, but
are not limited to, Northern blot analysis of tissue samples
obtained from the animal, in situ hybridization analysis, and
reverse transcriptase-PCR (rt-PCR). Samples of transgenic
gene-expressing tissue may also be evaluated immunocytochemically
or immunohistochemically using antibodies specific for the
transgene product.
[0608] Once the founder animals are produced, they may be bred,
inbred, outbred, or crossbred to produce colonies of the particular
animal. Examples of such breeding strategies include, but are not
limited to: outbreeding of founder animals with more than one
integration site in order to establish separate lines; inbreeding
of separate lines in order to produce compound transgenics that
express the transgene at higher levels because of the effects of
additive expression of each transgene; crossing of heterozygous
transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate
the need for screening of animals by DNA analysis; crossing of
separate homozygous lines to produce compound heterozygous or
homozygous lines; and breeding to place the transgene on a distinct
background that is appropriate for an experimental model of
interest.
[0609] Transgenic animals of the invention have uses which include,
but are not limited to, animal model systems useful in elaborating
the biological function of VEGF-2 polypeptides, studying conditions
and/or disorders associated with aberrant VEGF-2 expression, and in
screening for compounds effective in ameliorating such conditions
and/or disorders.
Example 31
VEGF-2 Knock-Out Animals
[0610] Endogenous VEGF-2 gene expression can also be reduced by
inactivating or "knocking out" the VEGF-2 gene and/or its promoter
using targeted homologous recombination. (E.g., see Smithies et
al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell
51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of
which is incorporated by reference herein in its entirety). For
example, a mutant, non-functional polynucleotide of the invention
(or a completely unrelated DNA sequence) flanked by DNA homologous
to the endogenous polynucleotide sequence (either the coding
regions or regulatory regions of the gene) can be used, with or
without a selectable marker and/or a negative selectable marker, to
transfect cells that express polypeptides of the invention in vivo.
In another embodiment, techniques known in the art are used to
generate knockouts in cells that contain, but do not express the
gene of interest. Insertion of the DNA construct, via targeted
homologous recombination, results in inactivation of the targeted
gene. Such approaches are particularly suited in research and
agricultural fields where modifications to embryonic stem cells can
be used to generate animal offspring with an inactive targeted gene
(e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra).
However this approach can be routinely adapted for use in humans
provided the recombinant DNA constructs are directly administered
or targeted to the required site in vivo using appropriate viral
vectors that will be apparent to those of skill in the art.
[0611] In further embodiments of the invention, cells that are
genetically engineered to express the polypeptides of the
invention, or alternatively, that are genetically engineered not to
express the polypeptides of the invention (e.g., knockouts) are
administered to a patient in vivo. Such cells may be obtained from
the patient (i.e., animal, including human) or an MHC compatible
donor and can include, but are not limited to fibroblasts, bone
marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle
cells, endothelial cells etc. The cells are genetically engineered
in vitro using recombinant DNA techniques to introduce the coding
sequence of polypeptides of the invention into the cells, or
alternatively, to disrupt the coding sequence and/or endogenous
regulatory sequence associated with the polypeptides of the
invention, e.g., by transduction (using viral vectors, and
preferably vectors that integrate the transgene into the cell
genome) or transfection procedures, including, but not limited to,
the use of plasmids, cosmids, YACs, naked DNA, electroporation,
liposomes, etc. The coding sequence of the polypeptides of the
invention can be placed under the control of a strong constitutive
or inducible promoter or promoter/enhancer to achieve expression,
and preferably secretion, of the VEGF-2 polypeptides. The
engineered cells which express and preferably secrete the
polypeptides of the invention can be introduced into the patient
systemically, e.g., in the circulation, or intraperitoneally.
[0612] Alternatively, the cells can be incorporated into a matrix
and implanted in the body, e.g., genetically engineered fibroblasts
can be implanted as part of a skin graft; genetically engineered
endothelial cells can be implanted as part of a lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Pat. No.
5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each
of which is incorporated by reference herein in its entirety).
[0613] When the cells to be administered are non-autologous or
non-MHC compatible cells, they can be administered using well known
techniques which prevent the development of a host immune response
against the introduced cells. For example, the cells may be
introduced in an encapsulated form which, while allowing for an
exchange of components with the immediate extracellular
environment, does not allow the introduced cells to be recognized
by the host immune system.
[0614] Knock-out animals of the invention have uses which include,
but are not limited to, animal model systems useful in elaborating
the biological function of VEGF-2 polypeptides, studying conditions
and/or disorders associated with aberrant VEGF-2 expression, and in
screening for compounds effective in ameliorating such conditions
and/or disorders.
[0615] 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
may be practiced otherwise than as particularly described.
[0616] The entire disclosure of all publications (including
patents, patent applications, journal articles, laboratory manuals,
books, or other documents) cited herein are hereby incorporated by
reference.
[0617] Additionally, the Sequence Listing submitted in Provisional
Application Ser. No. 60/223,276, filed Aug. 4, 2000, whether in
computer, microfiche, CD-R, and/or paper forms, is hereby
incorporated by reference in its entirety.
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