U.S. patent application number 11/233119 was filed with the patent office on 2006-02-02 for vascular endothelial growth factor 2.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Liang Cao, Jing-Shan Hu, Craig A. Rosen.
Application Number | 20060025331 11/233119 |
Document ID | / |
Family ID | 35784569 |
Filed Date | 2006-02-02 |
United States Patent
Application |
20060025331 |
Kind Code |
A1 |
Hu; Jing-Shan ; et
al. |
February 2, 2006 |
Vascular endothelial growth factor 2
Abstract
Disclosed are human VEGF2 polypeptides, biologically active,
diagnostically or therapeutically useful fragments, analogs, or
derivatives thereof, and DNA (RNA) encoding such VEGF2
polypeptides. Also provided are procedures for producing such
polypeptides by recombinant techniques and antibodies and
antagonists against such polypeptides. Such polypeptides 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: |
Hu; Jing-Shan; (Mountain
View, CA) ; Rosen; Craig A.; (Laytonsville, MD)
; Cao; Liang; (Bethesda, 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: |
35784569 |
Appl. No.: |
11/233119 |
Filed: |
September 23, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09107997 |
Jun 30, 1998 |
|
|
|
11233119 |
Sep 23, 2005 |
|
|
|
09042105 |
Mar 13, 1998 |
6040157 |
|
|
09107997 |
Jun 30, 1998 |
|
|
|
08999811 |
Dec 24, 1997 |
5932540 |
|
|
09042105 |
Mar 13, 1998 |
|
|
|
08207550 |
Mar 8, 1994 |
|
|
|
08999811 |
Dec 24, 1997 |
|
|
|
08465968 |
Jun 6, 1995 |
6608182 |
|
|
08999811 |
Dec 24, 1997 |
|
|
|
08207550 |
Mar 8, 1994 |
|
|
|
08465968 |
Jun 6, 1995 |
|
|
|
Current U.S.
Class: |
514/8.1 ;
424/145.1; 514/19.4 |
Current CPC
Class: |
A61K 38/1866 20130101;
A61K 38/179 20130101; A61K 2039/505 20130101; C07K 16/22
20130101 |
Class at
Publication: |
514/002 ;
424/145.1 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 39/395 20060101 A61K039/395 |
Claims
1. A method of treating a mammal having breast cancer characterized
by endothelial cells that express Flt4 tyrosine kinase (Flt4),
comprising a step of administering to said mammal a composition,
said composition comprising an inhibitor of binding between Flt4
ligand protein and Flt4 expressed in cells of said organism,
thereby inhibiting Flt4 function, wherein the inhibitor comprises a
member selected from the group consisting of: (a) an anti-Flt4
antibody or a polypeptide comprising an antigen binding fragment
thereof; (b) an anti-VEGF-C antibody or a polypeptide comprising an
antigen binding fragment thereof; (c) an anti-VEGF-D antibody or a
polypeptide comprising an antigen binding fragment thereof; and (d)
a soluble polypeptide comprising a fragment of Flt4, wherein the
polypeptide and the fragment are capable of binding to human VEGF-C
(SEQ ID NO: 2).
2. A method according to claim 1, wherein the mammal is human.
3. A method according to claim 2, comprising a screening step
preceding the administering step, wherein the screening step
comprises screening a human to identify breast cancer characterized
by endothelial cells expressing FIt4; and wherein the administering
step comprises administering the composition to a human identified
by the screening step as having breast cancer characterized by
endothelial cells expressing Flt4.
4. A method according to any one of claims 2-3 wherein the
inhibitor comprises a member selected from the group consisting of:
(a) an anti-Flt4 antibody or a polypeptide comprising an antigen
binding fragment thereof; and (b) a soluble polypeptide comprising
a fragment of Flt4, wherein the polypeptide and the fragment are
capable of binding to human VEGF-C (SEQ ID NO: 2).
5. A method according to claim 1, wherein the inhibitor further
comprises an anti-neoplastic agent conjugated to the antibody or
polypeptide.
6. A method according to claim 1, wherein the composition further
comprises a pharmaceutically acceptable diluent, adjuvant, or
carrier.
7. A method of treating a human having breast cancer characterized
by endothehlial cells that express Flt4 tyrosine kinase (Flt4),
comprising a step of administering to said human a composition,
said composition comprising an inhibitor of binding between FIt4
ligand protein and FIt4 expressed in cells of said human, thereby
inhibiting Flt4 function, wherein the inhibitor comprises a
polypeptide comprising an FIt4 binding fragment of human
prepro-VEGF-C (SEQ ID NO: 2) or human prepro-VEGF-D conjugated to
an antineoplastic agent.
8. A method according to claim 7, comprising a screening step
preceding the administering step, wherein the screening step
comprises screening a human to identify breast cancer characterized
by endothelial cells expressing Flt4; and wherein the administering
step comprises administering the composition to a human identified
by the screening step as having breast cancer characterized by
endothelial cells expressing Flt4.
9. A method of treating a mammal having breast cancer characterized
by endothelial cells that express a tyrosine kinase receptor
capable of binding VEGF-C, comprising a step of administering to
said mammal a composition, said composition comprising an inhibitor
of binding between VEGF-C and the receptor expressed in cells of
said organism, thereby inhibiting receptor function, wherein the
inhibitor comprises a member selected from the group consisting of:
(a) an anti-VEGF-C antibody or a polypeptide comprising an antigen
binding fragment thereof; and (b) a soluble polypeptide comprising
a fragment of the receptor, wherein the polypeptide and the
fragment are capable of binding to human VEGF-C (SEQ ID NO: 2).
10. A method according to claim 9, wherein the mammal is human.
11. A method according to claim 10, comprising a screening step
preceding the administering step, wherein the screening step
comprises screening a human to identify breast cancer characterized
by endothelial cells expressing the receptor; and wherein the
administering step comprises administering the composition to a
human identified by the screening step as having breast cancer
characterized by endothelial cells expressing the receptor.
12. A method according to any one of claims 10-11 wherein the
inhibitor comprises a soluble polypeptide comprising a fragment of
the receptor, wherein the polypeptide and the fragment are capable
of binding to human VEGF-C (SEQ ID NO: 2).
13. A method according to claim 9, wherein the inhibitor further
comprises an anti-neoplastic agent conjugated to the antibody or
polypeptide.
14. A method according to claim 9, wherein the composition further
comprises a pharmaceutically acceptable diluent, adjuvant, or
carrier.
15. A method of treating a human having breast cancer characterized
by endothelial cells that express a tyrosine kinase receptor
capable of binding VEGF-C, comprising a step of administering to
said human a composition, said composition comprising an inhibitor
of binding between VEGF-C and the receptor expressed in cells of
said human, thereby inhibiting receptor function, wherein the
inhibitor comprises a polypeptide comprising a receptor binding
fragment of human prepro-VEGF-C (SEQ ID NO: 2) conjugated to an
antineoplastic agent.
16. A method according to claim 15, comprising a screening step
preceding the administering step, wherein the screening step
comprises screening a human to identify breast cancer characterized
by endothelial cells expressing the receptor; and wherein the
administering step comprises administering the composition to a
human identified by the screening step as having breast cancer
characterized by endothelial cells expressing the receptor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 09/107,997, filed Jun. 30, 1998, which is herein
incorporated by reference, which is a continuation-in-part of U.S.
application Ser. No. 09/042,105, filed Mar. 13, 1998 (now U.S. Pat.
No. 6,040,157), which is herein incorporated by reference, which is
a continuation-in-part of U.S. application Ser. No. 08/999,911
filed Dec. 24, 1997, now issued as U.S. Pat. No. 5,932,540, which
is herein incorporated by reference; and which is a
continuation-in-part of both 08/465,968, filed Jun. 6, 1995 (now
U.S. Pat. No. 6,608,182), which is herein incorporated by
reference; and U.S. application Ser. No. 08/207,550, filed Mar. 8,
1994 (now abandoned); said 08/465,968 is a continuation-in-part of
said 08/207,550, which is also herein incorporated by
reference.
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 I" and "Copy 2." These compact
discs each contain the file "PF112P4D1 SeqList.txt" (38,866 bytes,
created on Jul. 20, 2005), which is incorporated by reference in
its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] 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 (VEGF2). The invention also
relates to inhibiting the action of such polypeptides.
[0005] 2. Related Art
[0006] 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)).
[0007] 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).
[0008] 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)).
[0009] 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.
[0010] 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
VEGF206 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.
[0011] Vascular permeability factor (VPF) has also been found to be
responsible for persistent microvascular hyperpermeability to
plasma proteins even after the cessation of injury, which is a
characteristic feature of normal wound healing. This suggests that
VPF is an important factor in wound healing. Brown, L. F. et al.,
J. Exp. Med. 176:1375-1379 (1992).
[0012] 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).
[0013] 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 Sept. 30, 1992.
SUMMARY OF THE INVENTION
[0014] 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.
[0015] 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.
[0016] In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules comprising
polynucleotides encoding full length or truncated VEGF2
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.
[0017] The present invention also relates to biologically active
and diagnostically or therapeutically useful fragments, analogs,
and derivatives of VEGF2.
[0018] 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.
[0019] 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.
[0020] In accordance with yet another aspect of the present
invention, there are provided antibodies against such polypeptides
and processes for producing such polypeptides.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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
[0026] 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.
[0027] FIGS. 1A-1E show the full length nucleotide (SEQ ID NO:1)
and the deduced amino acid (SEQ ID NO:2) sequence of VEGF2. 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%.
[0028] 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 VEGF2. The polypeptide comprises
approximately 350 amino acid residues of which approximately the
first 24 amino acids represent the leader sequence.
[0029] 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 VEGF2 (SEQ ID NO:4). The boxed areas indicate
the conserved sequences and the location of the eight conserved
cysteine residues.
[0030] FIG. 4 shows, in table-form, the percent homology between
PDGFa, PDGFb, VEGF, and VEGF2.
[0031] FIG. 5 shows the presence of VEGF2 mRNA in human breast
tumor cell lines.
[0032] FIG. 6 depicts the results of a Northern blot analysis of
VEGF2 in human adult tissues.
[0033] 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: VEGF2 produced by M13-reverse and
forward primers; Lane 4: VEGF2 produced by M13 reverse and VEGF-F4
primers; Lane 5: VEGF2 produced by M13 reverse and VEGF-F5
primers.
[0034] FIGS. 8A and 8B depict photographs of SDS-PAGE gels. VEGF2
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.
[0035] 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.
[0036] FIG. 10 depicts a photograph of an SDS-PAGE gel. VEGF2 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.
[0037] FIG. 11 depicts reverse phase HPLC analysis of purified
VEGF2 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.
[0038] FIG. 12 is a bar graph illustrating the effect of
partially-purified VEGF2 protein on the growth of vascular
endothelial cells in comparison to basic fibroblast growth
factor.
[0039] FIG. 13 is a bar graph illustrating the effect of purified
VEGF2 protein on the growth of vascular endothelial cells.
[0040] FIG. 14 depicts expression of VEGF2 mRNA in human fetal and
adult tissues.
[0041] FIG. 15 depicts expression of VEGF2 mRNA in human primary
culture cells.
[0042] FIG. 16 depicts transient expression of VEGF2 protein in
COS-7 cells.
[0043] FIG. 17 depicts VEGF2 stimulated proliferation of human
umbilical vein endothelial cells (HUVEC).
[0044] FIG. 18 depicts VEGF2 stimulated proliferation of dermal
microvascular endothelial cells.
[0045] FIG. 19 depicts the stimulatory effect of VEGF2 on
proliferation of microvascular, umbilical cord, endometrial, and
bovine aortic endothelial cells.
[0046] FIG. 20 depicts inhibition of PDGF-induced vascular (human
aortic) smooth muscle cell proliferation.
[0047] FIG. 21 depicts stimulation of migration of HUVEC and bovine
microvascular endothelial cells (BMEC) by VEGF-2.
[0048] FIG. 22 depicts stimulation of nitric oxide release of HUVEC
by VEGF-2 and VEGF-1.
[0049] FIG. 23 depicts inhibition of cord formation of
microvascular endothelial cells (CADMEC) by VEGF-2.
[0050] FIG. 24 depicts stimulation of angiogenesis by VEGF, VEGF-2,
and bFGF in the CAM assay.
[0051] FIGS. 25A-25D depict restoration of certain parameters in
the ischemic limb by VEGF2 protein (FIG. 25, top panels) and naked
expression plasmid (FIG. 25, middle panels): BP ratio (FIG. 25a);
Blood Flow and Flow Reserve (FIG. 25b); Angiographic Score (FIG.
25c); Capillary density (FIG. 25d).
[0052] FIGS. 26A-26G depict the ability of VEGF2 to affect the
diastolic blood pressure in spontaneously hypertensive rats
(SHR).
[0053] FIGS. 26a and b depict the dose-dependent decrease in
diastolic blood pressure achieved with VEGF2. (FIGS. 26c and d
depict the decreased mean arterial pressure (MAP) observed with
VEGF2. 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.
[0054] FIG. 27 depicts inhibition of VEGF-2N=and VEGF-2-induced
proliferation.
[0055] 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.
[0056] 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.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0057] In accordance with one aspect of the present invention,
there are provided isolated nucleic acid molecules comprising a
polynucleotide encoding a VEGF2 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.
[0058] In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules comprising a
polynucleotide encoding a truncated VEGF2 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.
[0059] The deposited strains are maintained under the terms of the
Budapest Treaty and will be made available to a patent office
signatory to the Budapest Treaty.
[0060] 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.
[0061] 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 PDGFNEGF family, PXCVXXXRCXGCCN,
(SEQ ID NO: 8) is conserved in VEGF2 (see FIG. 3). The homology
between VEGF2, 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 VEGF2 through
simple approaches such as low stringency hybridization.
[0062] The VEGF2 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
VEGF2 activity.
[0063] There are at least two alternatively spliced VEGF2 mRNA
sequences present in normal tissues. The two bands in FIG. 7, lane
5 indicate the presence of the alternatively spliced mRNA encoding
the VEGF2 polypeptide of the present invention.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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 FIG. 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.
[0068] Thus, the present invention includes polynucleotides
encoding the same mature polypeptide as shown in FIG. 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 FIG. 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.
[0069] As hereinabove indicated, the polynucleotide may have a
coding sequence which is a naturally occurring allelic variant of
the coding sequence shown in FIG. 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.
[0070] 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.
[0071] 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).
[0072] 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)).
[0073] 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 VEGF2 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).
[0074] 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 VEGF2 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).
[0075] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a VEGF2 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 VEGF2 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.
[0076] 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.
[0077] 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).
[0078] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. These immunogenic epitopes are believed to be confined
to a few loci on the molecule. On the other hand, a region of a
protein molecule to which an antibody can bind is defined as an
"antigenic epitope." The number of immunogenic epitopes of a
protein generally is less than the number of antigenic epitopes.
See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA
81:3998-4002 (1983).
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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 which 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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)).
[0088] 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.
[0089] 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
[0090] 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.
[0091] 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.
[0092] Particularly preferred VEGF-2 polypeptides are shown below
(numbering starts with the first amino acid in the protein (Met)
(FIG. 1 (SEQ ID NO:18)): 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); Glu (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).
[0093] 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:18)).
[0094] 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.
[0095] Particularly, N-terminal deletions of the VEGF-2 polypeptide
can be described by the general formula m.sup.-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: A-2 to S-396; P-3
to S-396; A-4 to S-396; A-5 to S-396; A-6 to S-396; A-7 to S-396;
A-8 to S-396; F-9 to S-396; E-10 to S-396; S-11 to S-396; G-12 to
S-396; L-13 to S-396; D-14 to S-396; L-15 to S-396; S-16 to S-396;
D-17 to S-396; A-18 to S-396; E-19 to S-396; P-20 to S-396; D-21 to
S-396; A-22 to S-396; G-23 to S-396; E-24 to S-396; A-25 to S-396;
T-26 to S-396; A-27 to S-396; Y-28 to S-396; A-29 to S-396; S-30 to
S-396; K-31 to S-396; D-32 to S-396; L-33 to S-396; E-34 to S-396;
E-35 to S-396; Q-36 to S-396; L-37 to S-396; R-38 to S-396; S-39 to
S-396; V-40 to S-396; S-41 to S-396; S-42 to S-396; V-43 to S-396;
D-44 to S-396; E-45 to S-396; L-46 to S-396; M-47 to S-396; T-48 to
S-396; V-49 to S-396; L-50 to S-396; Y-51 to S-396; P-52 to S-396;
E-53 to S-396; Y-54 to S-396; W-55 to S-396; K-56 to S-396; M-57 to
S-396; Y-58 to S-396; K-59 to S-396; C-60 to S-396; Q-61 to S-396;
L-62 to S-396; R-63 to S-396; K-64 to S-396; G-65 to S-396; G-66 to
S-396; W-67 to S-396; Q-68 to S-396; H-69 to S-396; N-70 to S-396;
R-71 to S-396; E-72 to S-396; Q-73 to S-396; A-74 to S-396; N-75 to
S-396; L-76 to S-396; N-77 to S-396; S-78 to S-396; R-79 to S-396;
T-80 to S-396; E-81 to S-396; E-82 to S-396; T-83 to S-396; I-84 to
S-396; K-85 to S-396; F-86 to S-396; A-87 to S-396; A-88 to S-396;
A-89 to S-396; H-90 to S-396; Y-91 to S-396; N-92 to S-396; T-93 to
S-396; E-94 to S-396; I-95 to S-396; L-96 to S-396; K-97 to S-396;
S-98 to S-396; 1-99 to S-396; D-100 to S-396; N-101 to S-396; E-102
to S-396; W-103 to S-396; R-104 to S-396; K-105 to S-396; T-106 to
S-396; Q-107 to S-396; C-108 to S-396; M-109 to S-396; P-110 to
S-396; R-111 to S-396; E-112 to S-396; V-113 to S-396; C-114 to
S-396; 1-115 to S-396; D-116 to S-396; V-117 to S-396; G-118 to
S-396; K-119 to S-396; E-120 to S-396; F-121 to S-396; G-122 to
S-396; V-123 to S-396; A-124 to S-396; T-125 to S-396; N-126 to
S-396; T-127 to S-396; F-128 to S-396; F-129 to S-396; K-130 to
S-396; P-131 to S-396 of SEQ ID NO:2.
[0096] 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: E-1 to M-395; E-1 to Q-394;
E-1 to P-393; E-1 to R-392; E-1 to Q-391; E-1 to W-390; E-1 to
Y-389; E-1 to S-388; E-1 to P-387; E-1 to V-386; E-1 to C-385; E-1
to R-384; E-1 to C-383; E-1 to V-382; E-1 to E-381; E-1 to E-380;
E-1 to S-379; E-1 to Y-378; E-1 to S-377; E-1 to F-376; E-1 to
G-375; E-1 to P-374; E-1 to E-373; E-1 to C-372; E-1 to A-371; E-1
to K-370; E-1 to Q-369; E-1 to R-368; E-1 to N-367; E-1 to T-366;
E-1 to C-365; E-1 to P-364; E-1 to R-363; E-1 to R-362; E-1 to
Y-361; E-1 to C-360; E-1 to S-359; E-1 to C-358; E-1 to T-357; E-1
to Q-356; E-1 to H-355; E-1 to H-354; E-1 to F-353; E-1 to K-352;
E-1 to K-351; E-1 to G-350; E-1 to K-349; E-1 to L-348; E-1 to
L-347; E-1 to C-346; E-1 to K-345; E-1 to Q-344; E-1 to P-343; E-1
to S-342; E-1 to E-341; E-1 to T-340; E-1 to C-339; E-1 to E-338;
E-1 to C-337; E-1 to A-336; E-1 to C-335; E-1 to K-334; E-1 to
G-333; E-1 to P-332; E-1 to N-331; E-1 to L-330; E-1 to P-329; E-1
to Q-328; E-1 to N-327; E-1 to R-326; E-1 to P-325; E-1 to C-324;
E-1 to T-323; E-1 to R-322; E-1 to K-321; E-1 to C-320; E-1 to
V-319; E-1 to C-318; E-1 to Q-317; E-1 to C-316; E-1 to T-315; E-1
to N-314; E-1 to E-313; E-1 to D-312; E-1 to F-311; E-1 to E-310;
E-1 to R-309; E-1 to N-308; E-1 to A-307; E-1 to G-306; E-1 to
C-305; E-1 to Q-304; E-1 to S-303; E-1 to P-302; E-1 to F-301; E-1
to L-300; E-1 to K-299; E-1 to N-298; E-1 to K-297; E-1 to C-296;
E-1 to V-295; E-1 to C-294; E-1 to Q-293; E-1 to C-292; E-1 to
S-291; E-1 to N-290; E-1 to R-289; E-1 to D-288; E-1 to L-287; E-1
to E-286; E-1 to K-285; E-1 to H-284; E-1 to P-283; E-1 to G-282;
E-1 to C-281; E-1 to S-280; E-1 to A-279; E-1 to P-278; E-1 to
R-277; E-1 to L-276; E-1 to G-275; E-1 to A-274; E-1 to R-273; E-1
to C-272; E-1 to V-271; E-1 to C-270; E-1 to Q-269; E-1 to C-268;
E-1 to T-267; E-1 to E-266; E-1 to E-265; E-1 to D-264; E-1 to
L-263; E-1 to E-262; E-1 to K-261; E-1 to N-260; E-1 to P-259; E-1
to G-258; E-1 to C-257; E-1 to 1-256; E-1 to D-255; E-1 to H-254;
E-1 to F-253; E-1 to G-252; E-1 to D-251; E-1 to T-250; E-1 to
S-249; E-1 to D-248; E-1 to D-247; E-1 to G-246; E-1 to A-245; E-1
to D-244; E-1 to S-243; E-1 to S-242; E-1 to F-241; E-1 to M-240;
E-1 to F-239; E-1 to D-238; E-1 to E-237; E-1 to Q-236; E-1 to
A-235; E-1 to L-234; E-1 to C-233; E-1 to R-232; E-1 to C-231; E-1
to 1-230; E-1 to H-229; E-1 to N-228; E-1 to N-227; E-1 to W-226;
E-1 to M-225; E-1 to Y-224; E-1 to N-223; E-1 to T-222; E-1 to
P-221; E-1 to C-220; E-1 to T-219; E-1 to K-218; E-1 to N-217; E-1
to A-216; E-1 to A-215; E-1 to Q-214; E-1 to C-213; E-1 to Q-212;
E-1 to P-211; E-1 to L-210; E-1 to T-209; E-1 to A-208; E-1 to
P-207; E-1 to L-206; E-1 to S-205; E-1 to R-204; E-1 to R-203; E-1
to 1-202; E-1 to 1-201; E-1 to S-200; E-1 to H-199; E-1 to V-198;
E-1 to Q-197; E-1 to R-196; E-1 to Y-195; E-1 to V-194; E-1 to
D-193; E-1 to L-192; E-1 to K-191; E-1 to S-190; E-1 to M-189; E-1
to C-188; E-1 to R-187; E-1 to C-186; E-1 to S-185; E-1 to T-184;
E-1 to H-183; E-1 to N-182; E-1 to A-181; E-1 to F-180; E-1 to
S-179; E-1 to 1-178; E-1 to T-177; E-1 to V-176; E-1 to P-175; E-1
to K-174; E-1 to P-173; E-1 to G-172; E-1 to Q-171; E-1 to S-170;
E-1 to L-169; E-1 to P-168; E-1 to V-167; E-1 to T-166; E-1 to
1-165; E-1 to E-164; E-1 to F-163; E-1 to L-162; E-1 to T-161; E-1
to K-160; E-1 to S-159; E-1 to L-158; E-1 to Y-157; E-1 to S-156;
E-1 to T-155; E-1 to S-154; E-1 to T-153; E-1 to N-152; E-1 to
M-151; E-1 to C-150; E-1 to Q-149; E-1 to L-148; E-1 to G-147; E-1
to E-146; E-1 to S-145; E-1 to N-144; of SEQ ID NO:2. Preferably,
any of the above listed N- or C-terminal deletions can be combined
to produce a N- and C-terminal deleted VEGF-2 polypeptide, which
retains the conserved box domain.
[0097] 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.
[0098] 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:1 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.
[0099] 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:18)
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:18). Alternatively, first 24 N-terminal amino acid
residues (i.e., a deletion of at least Met (1)-Glu (24)) but not
more than the first 103 N-terminal amino acid residues of FIG. 1
(SEQ ID NO:18), etc, etc.
[0100] 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:18) except for a
deletion of at least the last C-terminal amino acid residue (Ser
(419)) but not more than the last 220 C-terminal amino acid
residues (i.e., a deletion of amino acid residues Val (199)-Ser
(419)) of FIG. 1 (SEQ ID NO:18). Alternatively, the deletion will
include at least the last C-terminal amino acid residue but not
more than the last 216 C-terminal amino acid residues of FIG. 1
(SEQ ID NO:18). Alternatively, the deletion will include at least
the last C-terminal amino acid residue but not more than the last
204 C-terminal amino acid residues of FIG. 1 (SEQ ID NO:18).
Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 192
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:18).
Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 156
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:18).
Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 108
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:18).
Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 52
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:18).
[0101] 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.
[0102] 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).
[0103] 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.
[0104] Moreover, representative examples of VEGF2 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.
[0105] Fragments of the full length gene of the present invention
may be used as a hybridization probe for a cDNA library to isolate
the full length cDNA and to isolate other cDNAs which have a high
sequence similarity to the gene or similar biological activity.
Probes of this type preferably have at least 30 bases and may
contain, for example, 50 or more bases. The probe may also be used
to identify a cDNA clone corresponding to a full length transcript
and a genomic clone or clones that contain the complete gene
including regulatory and promotor regions, exons, and introns. An
example of a screen comprises isolating the 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.
[0106] A VEGF2 "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 NaCl, 75 mM sodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured, sheared salmon sperm DNA,
followed by washing the filters in 0.1.times. SSC at about
65.degree. C.
[0107] Also contemplated are nucleic acid molecules that hybridize
to the VEGF2 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).
[0108] 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.
[0109] 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).
[0110] 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.
[0111] 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 VEGF2 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).
[0112] 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 VEGF2 activity. This is because
even where a particular nucleic acid molecule does not encode a
polypeptide having VEGF2 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 VEGF2 activity include, inter alia,
(1) isolating the VEGF2 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
VEGF2 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 VEGF2 mRNA expression in
specific tissues.
[0113] 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 VEGF2 protein activity. By "a polypeptide having
VEGF2 activity" is intended polypeptides exhibiting VEGF2 activity
in a particular biological assay. For example, VEGF2 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).
[0114] 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 VEGF2 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 VEGF2 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).
[0115] 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.
[0116] 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.
[0117] "Identity" per se bas 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 (1984) 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).)
[0118] 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 VEGF2 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.
[0119] 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. (1990) 6:237-245). 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.
[0120] 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.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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
[0126] The present invention further relates to polypeptides which
have the deduced amino acid sequence of FIG. 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.
[0127] The terms "fragment," "derivative" and "analog" when
referring to the polypeptide of FIG. 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.
[0128] 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.
[0129] Preferred polypeptide fragments include the secreted VEGF2
protein as well as the mature form. Further preferred polypeptide
fragments include the secreted VEGF2 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 VEGF2 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 VEGF2 protein or
mature form. Furthermore, any combination of the above amino and
carboxy terminus deletions are preferred. Similarly, polynucleotide
fragments encoding these VEGF2 polypeptide fragments are also
preferred.
[0130] Also preferred are VEGF2 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.
[0131] Other preferred fragments are biologically active VEGF2
fragments. Biologically active fragments are those exhibiting
activity similar, but not necessarily identical, to an activity of
the VEGF2 polypeptide. The biological activity of the fragments may
include an improved desired activity, or a decreased undesirable
activity.
[0132] The polypeptides of the present invention may be recombinant
polypeptides, natural polypeptides, or synthetic polypeptides,
preferably recombinant polypeptides.
[0133] It will be recognized in the art that some amino acid
sequences of the VEGF2 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.
[0134] Thus, the invention further includes variations of the VEGF2
polypeptide which show substantial VEGF2 polypeptide activity or
which include regions of VEGF2 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).
[0135] Thus, the fragments, derivatives, or analogs of the
polypeptides of FIG. 1 or 2, or that encoded by the deposited cDNAs
may be: (1) 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.
[0136] 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 VEGF2
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)).
[0137] 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 VEGF2 of the present invention may include
one or more amino acid substitutions, deletions or additions,
either from natural mutations or human manipulation.
[0138] 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).
[0139] TABLE 1. Conservative Amino Acid Substitutions
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
[0140] 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.
[0141] Amino acids in the VEGF2 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)).
[0142] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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
[0148] Any VEGF2 polypeptide can be used to generate fusion
proteins. For example, the VEGF2 polypeptide, when fused to a
second protein, can be used as an antigenic tag. Antibodies raised
against the VEGF2 polypeptide can be used to indirectly detect the
second protein by binding to the VEGF2. Moreover, because secreted
proteins target cellular locations based on trafficking signals,
the VEGF2 polypeptides can be used as a targeting molecule once
fused to other proteins.
[0149] Examples of domains that can be fused to VEGF2 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.
[0150] Moreover, fusion proteins may also be engineered to improve
characteristics of the VEGF2 polypeptide. For instance, a region of
additional amino acids, particularly charged amino acids, may be
added to the N-terminus of the VEGF2 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 VEGF2 polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the VEGF2
polypeptide. The addition of peptide moieties to facilitate
handling of polypeptides are familiar and routine techniques in the
art.
[0151] Moreover, VEGF2 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).)
[0152] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)
discloses fusion proteins comprising various portions of constant
region of immunoglobulin molecules together with another human
protein or part thereof. In many cases, the Fc part in a fusion
protein is beneficial in therapy and diagnosis, and thus can result
in, for example, improved pharmacokinetic properties. (EP-A 0232
262.) Alternatively, deleting the Fc part after the fusion protein
has been expressed, detected, and purified, would be desired. For
example, the Fe 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 Fe 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).)
[0153] Moreover, the VEGF2 polypeptides can be fused to marker
sequences, such as a peptide which facilitates purification of
VEGF2. 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).)
[0154] Thus, any of these above fusions can be engineered using the
VEGF2 polynucleotides or the polypeptides.
Biological Activities of VEGF2
[0155] VEGF2 polynucleotides and polypeptides can be used in assays
to test for one or more biological activities. If VEGF2
polynucleotides and polypeptides do exhibit activity in a
particular assay, it is likely that VEGF2 may be involved in the
diseases associated with the biological activity. Therefore, VEGF2
could be used to treat the associated disease.
[0156] Immune Activity
[0157] VEGF2 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, VEGF2 polynucleotides or polypeptides can be used as a
marker or detector of a particular immune system disease or
disorder.
[0158] VEGF2 polynucleotides or polypeptides may be useful in
treating or detecting deficiencies or disorders of hematopoietic
cells. VEGF2 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, HW infection,
HTLV-BLV infection, leukocyte adhesion deficiency syndrome,
lymphopenia, phagocyte bactericidal dysfunction, severe combined
immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia,
thrombocytopenia, or hemoglobinuria.
[0159] Moreover, VEGF2 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, VEGF2 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, VEGF2 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.
[0160] VEGF2 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 VEGF2 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.
[0161] Examples of autoimmune disorders that can be treated or
detected by VEGF2 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.
[0162] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated by VEGF2 polypeptides or polynucleotides. Moreover,
VEGF2 can be used to treat anaphylaxis, hypersensitivity to an
antigenic molecule, or blood group incompatibility.
[0163] VEGF2 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 VEGF2 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, VEGF2
polypeptides or polynucleotides may also be used to modulate
inflammation. For example, VEGF2 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.)
[0164] Hyperproliferative Disorders
[0165] VEGF2 polypeptides or polynucleotides can be used to treat
or detect hyperproliferative disorders, including neoplasms. VEGF2
antagonist polypeptides or polynucleotides may inhibit the
proliferation of the disorder through direct or indirect
interactions. Alternatively, VEGF2 antagonist polypeptides or
polynucleotides may proliferate other cells which can inhibit the
hyperproliferative disorder.
[0166] 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.
[0167] Examples of hyperproliferative disorders that can be treated
or detected by VEGF2 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.
[0168] Similarly, other hyperproliferative disorders can also be
treated or detected by VEGF2 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.
[0169] Infectious Disease
[0170] VEGF2 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, VEGF2 polypeptides or polynucleotides may
also directly inhibit the infectious agent, without necessarily
eliciting an immune response.
[0171] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated or detected by VEGF2
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, Poxyiridae (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, Burkin'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. VEGF2 polypeptides or
polynucleotides can be used to treat or detect any of these
symptoms or diseases.
[0172] Similarly, bacterial or fungal agents that can cause disease
or symptoms and that can be treated or detected by VEGF2
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, Chiamydia, 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. VEGF2 polypeptides or polynucleotides can be used to
treat or detect any of these symptoms or diseases.
[0173] Moreover, parasitic agents causing disease or symptoms that
can be treated or detected by VEGF2 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.
VEGF2 polypeptides or polynucleotides can be used to treat or
detect any of these symptoms or diseases.
[0174] Preferably, treatment using VEGF2 polypeptides or
polynucleotides could either be by administering an effective
amount of VEGF2 polypeptide to the patient, or by removing cells
from the patient, supplying the cells with VEGF2 polynucleotide,
and returning the engineered cells to the patient (ex vivo
therapy). Moreover, the VEGF2 polypeptide or polynucleotide can be
used as an antigen in a vaccine to raise an immune response against
infectious disease.
[0175] Regeneration
[0176] VEGF2 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.
[0177] 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.
[0178] Moreover, VEGF2 polynucleotides or polypeptides may increase
regeneration of tissues difficult to heal. For example, increased
tendon/ligament regeneration would quicken recovery time after
damage. VEGF2 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.
[0179] Similarly, nerve and brain tissue could also be regenerated
by using VEGF2 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 VEGF2 polynucleotides or
polypeptides.
[0180] Chemotaxis
[0181] VEGF2 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.
[0182] VEGF2 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, VEGF2 could also
attract fibroblasts, which can be used to treat wounds.
[0183] It is also contemplated that VEGF2 polynucleotides or
polypeptides may inhibit chemotactic activity. These molecules
could also be used to treat disorders. Thus, VEGF2 polynucleotides
or polypeptides could be used as an inhibitor of chemotaxis.
[0184] Binding Activity
[0185] VEGF2 polypeptides may be used to screen for molecules that
bind to VEGF2 or for molecules to which VEGF2 binds. The binding of
VEGF2 and the molecule may activate (agonist), increase, inhibit
(antagonist), or decrease activity of the VEGF2 or the molecule
bound. Examples of such molecules include antibodies,
oligonucleotides, proteins (e.g., receptors), or small
molecules.
[0186] Preferably, the molecule is closely related to the natural
ligand of VEGF2, 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 VEGF2 binds (i.e., Flt-4), or at least, a
fragment of the receptor capable of being bound by VEGF2 (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
VEGF2, either as a secreted protein or on the cell membrane.
Preferred cells include cells from mammals, yeast, Drosophila, or
E. coli. Cells expressing VEGF2(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 VEGF2 or the
molecule.
[0187] The assay may simply test binding of a candidate compound
toVEGF2, 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 VEGF2.
[0188] 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 VEGF2, measuring VEGF2/molecule activity or
binding, and comparing the VEGF2/molecule activity or binding to a
standard.
[0189] Preferably, an ELISA assay can measure VEGF2 level or
activity in a sample (e.g., biological sample) using a monoclonal
or polyclonal antibody. The antibody can measure VEGF2 level or
activity by either binding, directly or indirectly, to VEGF2 or by
competing with VEGF2 for a substrate.
[0190] 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
VEGF2/molecule. Moreover, the assays can discover agents which may
inhibit or enhance the production of VEGF2 from suitably
manipulated cells or tissues.
[0191] Therefore, the invention includes a method of identifying
compounds which bind to VEGF2 comprising the steps of: (a)
incubating a candidate binding compound with VEGF2; 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 VEGF2, (b)
assaying a biological activity, and (b) determining if a biological
activity of VEGF2 has been altered.
[0192] Other Activities
[0193] VEGF2 polypeptides or polynucleotides may also increase or
decrease the differentiation or proliferation of embryonic stem
cells, besides, as discussed above, hematopoietic lineage.
[0194] VEGF2 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,
VEGF2 polypeptides or polynucleotides may be used to modulate
mammalian metabolism affecting catabolism, anabolism, processing,
utilization, and storage of energy.
[0195] VEGF2 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.
[0196] VEGF2 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 and Host Cells
[0197] 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 VEGF2 polypeptides or peptides by
recombinant techniques.
[0198] 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
VEGF2 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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 tinker 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.
[0207] 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.
[0208] As noted above, the pHE4a vector contains a lacIq gene.
Laclq is an allele of the lad 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.
[0209] The promoter/operator sequences of the pHE4a vector (SEQ ID
NO:17) comprise a T5 phage promoter and two lac operator sequences.
One operator is located 5' to the transcriptional start site and
the other is located 3' to the same site. These operators, when
present in combination with the lacIq gene product, confer tight
repression of down-stream sequences in the absence of a lac operon
inducer, e.g., IPTG. Expression of operatively linked sequences
located down-stream from the lac operators may be induced by the
addition of a lac operon inducer, such as IPTG. Binding of a lac
inducer to the lacIq proteins results in their release from the lac
operator sequences and the initiation of transcription of
operatively linked sequences. Lac operon regulation of gene
expression is reviewed in Devlin, T., TEXTBOOK OF BIOCHEMISTRY WITH
CLINICAL CORRELATIONS, 4th Edition (1997), pages 802-807.
[0210] 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-Delagamo 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.
[0211] Among known bacterial promoters suitable for use in the
production of proteins of the present invention include the E. coli
lad 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-1 promoter.
[0212] The pHE4a vector also contains a Shine-Delgarno sequence 5'
to the AUG initiation codon. Shine-Delgamo 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.
[0213] 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).
[0214] 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 lac, lacZ, T3, T7,
gpt, lambda P.sub.R, P.sub.L and trp. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40,
LTRs from retrovirus, and mouse metallothionein-1. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art.
[0215] 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)).
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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 TRPI 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.
[0220] 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.
[0221] 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 (ATCCT.TM. 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia 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.
[0222] 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.
[0223] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0224] 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.
[0225] 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.
[0226] 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,
hydroxyapatite 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.
[0227] 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.
Therapeutic Uses
[0228] 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 VEGF2 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 VEGF2
nucleic acid sequence encoding this polypeptide wherein 20 .mu.g of
RNA from several human tissues were probed with .sup.32P-VEGF2,
illustrates that this protein is actively expressed in the heart
and lung which is further evidence of mitogenic activity.
[0229] Accordingly, VEGF2, 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. VEGF2, 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. VEGF2, 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, VEGF2, 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. VEGF2, 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.
[0230] Along these same lines, VEGF2, or biologically active
portions thereof, may also be employed to induce the growth of
damaged bone, periodontium or ligament tissue. VEGF2, 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.,
periodental disease or trauma.
[0231] Since angiogenesis is important in keeping wounds clean and
non-infected, VEGF2, or biologically active portions thereof, may
be employed in association with surgery and following the repair of
incisions and cuts. VEGF2, or biologically active portions thereof,
may also be employed for the treatment of abdominal wounds where
there is a high risk of infection.
[0232] VEGF2, 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, VEGF2, 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.
VEGF2, or biologically active portions thereof, may also be
employed to repair damage of myocardial tissue as a result of
myocardial infarction. VEGF2, or biologically active portions
thereof, may also be employed to repair the cardiac vascular system
after ischemia. VEGF2, 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.
[0233] VEGF2, 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.
[0234] VEGF2, 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.
[0235] VEGF2, 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 VEGF2
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.
[0236] VEGF2, 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.
[0237] VEGF2, 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 VEGF2
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.
[0238] VEGF2, 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 VEGF2 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.
[0239] VEGF2, 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 VEGF2 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.
[0240] VEGF2, or biologically active portions thereof, may also be
used to treat secondary (obstructive) lyphademas 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 VEGF2 polypeptides to treat
secondary (obstructive) lyphademas 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) lyphademas 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.
[0241] VEGF2, 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 VEGF2 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.
[0242] VEGF-2 antagonists can be used to treat cancer by inhibiting
the angiogenesis necessary to support cancer and tumor growth.
Gene Therapy Methods
[0243] Another aspect of the present invention is to 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) sequences into an animal to
achieve expression of the VEGF-2 polypeptide of the present
invention. This method requires 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, WO90/11092, which is herein
incorporated by reference.
[0244] 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 are delivered
in a pharmaceutically acceptable liquid or aqueous carrier.
[0245] 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.
[0246] 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. Any strong promoter known to those skilled in the art
can be used for driving the expression of VEGF-2 DNA. 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.
[0247] 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.
[0248] 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.
[0249] 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.
[0250] 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.
Nucleic Acid Utilities
[0251] VEGF2 nucleic acid sequences and VEGF2 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, VEGF2 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.
[0252] Fragments of the full length VEGF2 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 VEGF2 gene including regulatory and promotor regions,
exons, and introns. An example of a screen comprises isolating the
coding region of the VEGF2 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.
[0253] This invention provides methods for identification of VEGF2
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 VEGF2, and a cDNA library created from this RNA is
divided into pools and used to transfect COS cells or other cells
that are not responsive to VEGF2. Transfected cells which are grown
on glass slides are exposed to labeled VEGF2. VEGF2 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.
[0254] As an alternative approach for receptor identification,
labeled VEGF2 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 VEGF2 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
[0255] This invention is also related to a method of screening
compounds to identify those which are VEGF2 agonists or
antagonists. An example of such a method takes advantage of the
ability of VEGF2 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 37EC,
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.
[0256] 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 VEGF2 indicates
that the compound is an antagonist to VEGF2. Alternatively, VEGF2
antagonists may be detected by combining VEGF2 and a potential
antagonist with membrane-bound VEGF2 receptors or recombinant
receptors under appropriate conditions for a competitive inhibition
assay. VEGF2 can be labeled, such as by radioactivity, such that
the number of VEGF2 molecules bound to the receptor can determine
the effectiveness of the potential antagonist.
[0257] Alternatively, the response of a known second messenger
system following interaction of VEGF2 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 VEGF2 receptor is incubated with labeled VEGF2 in the presence
of the compound. The ability of the compound to enhance or block
this interaction could then be measured.
[0258] Potential VEGF2 antagonists include an antibody, or in some
cases, an oligonucleotide, which bind to the polypeptide and
effectively eliminate VEGF2 function. Alternatively, a potential
antagonist may be a closely related protein which binds to VEGF2
receptors, however, they are inactive forms of the polypeptide and
thereby prevent the action of VEGF2. Examples of these antagonists
include a negative dominant mutant of the VEGF2 polypeptide, for
example, one chain of the hetero-dimeric form of VEGF2 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 VEGF2 which is capable of
interacting with another dimer to form wild type VEGF2, however,
the resulting homo-dimer is inactive and fails to exhibit
characteristic VEGF activity.
[0259] Another potential VEGF2 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 VEGF2. The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into the VEGF2 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 VEGF2.
[0260] Potential VEGF2 antagonists also include small molecules
which bind to and occupy the active site of the polypeptide thereby
making the catalytic site inaccessible to substrate such that
normal biological activity is prevented. Examples of small
molecules include but are not limited to small peptides or
peptide-like molecules.
[0261] The antagonists may be employed to limit angiogenesis
necessary for solid tumor metastasis. The identification of VEGF2
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
VEGF2 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 VEGF2 is involved in tumor angiogenesis
and growth.
[0262] Gliomas are also a type of neoplasia which may be treated
with the antagonists of the present invention.
[0263] 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.
[0264] The antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as hereinafter
described.
[0265] The VEGF2 polypeptides and agonists and antagonists may be
employed in combination with a suitable pharmaceutical carrier.
Such compositions comprise a therapeutically effective amount of
the polypeptide or agonist or antagonist, and a pharmaceutically
acceptable carrier or excipient. Such a carrier includes but is not
limited to saline, buffered saline, dextrose, water, glycerol,
ethanol, and combinations thereof. The formulation should suit the
mode of administration.
[0266] 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.
[0267] 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 101 .mu.g/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 .mu.g/kg
to about 1 mg/kg body weight daily, taking into account the routes
of administration, symptoms, etc.
[0268] The VEGF2 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."
[0269] 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.
[0270] 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.
[0271] 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, Mycloproliferative Sarcoma
Virus, and mammary tumor virus. In one embodiment, the retroviral
plasmid vector is derived from Moloney Murine Leukemia Virus.
[0272] 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.
[0273] 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.
[0274] 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, T19-14.times.,
VT-19-17-H2, yCRE, yCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, Huinan 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.
[0275] 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.
[0276] This invention is also related to the use of the VEGF2 gene
as part of a diagnostic assay for detecting diseases or
susceptibility to diseases related to the presence of mutations in
VEGF2 nucleic acid sequences.
[0277] Individuals carrying mutations in the VEGF2 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 VEGF2 can be used to identify and analyze
VEGF2 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 VEGF2 RNA or
alternatively, radiolabeled VEGF2 antisense DNA sequences.
Perfectly matched sequences can be distinguished from mismatched
duplexes by RNase A digestion or by differences in melting
temperatures.
[0278] 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)).
[0279] 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)).
[0280] 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.
[0281] In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations can also be detected by in situ analysis.
[0282] The present invention also relates to a diagnostic assay for
detecting altered levels of VEGF2 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 VEGF2 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 VEGF2 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 VEGF2 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 VEGF2. 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 VEGF2 protein present in a given
volume of patient sample when compared against a standard
curve.
[0283] A competition assay may be employed wherein antibodies
specific to VEGF2 are attached to a solid support. Polypeptides of
the present invention are then labeled, for example, by
radioactivity, and a sample derived from the host are passed over
the solid support and the amount of label detected, for example by
liquid scintillation chromatography, can be correlated to a
quantity of VEGF2 in the sample.
[0284] A "sandwich" assay is similar to an ELISA assay. In a
"sandwich" assay VEGF2 is passed over a solid support and binds to
antibody attached to a solid support. A second antibody is then
bound to the VEGF2. 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.
[0285] 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.
[0286] 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.
[0287] 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.
[0288] 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).
[0289] 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).
[0290] 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.
[0291] 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).
[0292] 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.
[0293] The present invention is further directed to inhibiting
VEGF2 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 VEGF2. The antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of an mRNA
molecule into the VEGF2 (antisense--Okano, J. Neurochem. 56:560
(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, Fla. (1988)).
[0294] 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 VEGF2 in the manner described above.
[0295] Antisense constructs to VEGF2, therefore, may inhibit the
angiogenic activity of the VEGF2 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.
[0296] The polypeptides, their fragments or other derivatives, or
analogs thereof, or cells expressing them can be used as an
immunogen to produce antibodies thereto. These antibodies can be,
for example, polyclonal or monoclonal antibodies. The present
invention also includes chimeric, single chain, and humanized
antibodies, as well as Fab fragments, or the product of an Fab
expression library. Various procedures known in the art may be used
for the production of such antibodies and fragments.
[0297] Antibodies generated against the polypeptide corresponding
to a sequence of the present invention can be obtained by direct
injection of the polypeptide into an animal or by administering the
polypeptide to an animal, preferably a nonhuman. The antibody so
obtained will then bind the polypeptide itself. In this manner,
even a sequence encoding only a fragment of the polypeptide can be
used to generate antibodies binding the whole native polypeptide.
Such antibodies can then be used to isolate the polypeptide from
tissue expressing that polypeptide.
[0298] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, Nature 256:495-497 (1975)), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., Immunology Today
4:72 (1983)), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., in Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, Inc. (1985), pp. 77-96).
[0299] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention.
[0300] Neutralization antibodies can be identified and applied to
mask the vascular endothelial growth factor, and that has been
shown in mice model systems against VEGF. VEGF2 can also be
inactivated by certain dominant negative mutants within the gene
itself. It is known that both PDGFa and b form either heterodimers
or homodimers, and VEGF forms homodimers. Similar interaction
between VEGF2 could be expected. These antibodies therefore may be
used to block the angiogenic activity of VEGF2 and retard the
growth of solid tumors. These antibodies may also be used to treat
inflammation caused by the increased vascular permeability which
results from the presence of VEGF2.
[0301] These 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 VEGF2 can be considered diagnostic
of cancer.
[0302] The present invention is also directed to
antagonist/inhibitors of the polypeptides of the present invention.
The antagonist/inhibitors are those which inhibit or eliminate the
function of the polypeptide.
[0303] Thus, for example, antagonists bind to a polypeptide of the
present invention and inhibit or eliminate its function. The
antagonist, for example, could be an antibody against the
polypeptide which binds to the polypeptide or, in some cases, an
oligonucleotide. An example of an inhibitor is a small molecule
which binds to and occupies the catalytic 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.
[0304] Truncated versions of VEGF2 can also be produced that are
capable of interacting with wild type VEGF2 to form dimers that
fail to activate endothelial cell growth, therefore inactivating
the endogenous VEGF2. Or, mutant forms of VEGF2 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.
[0305] 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 VEGF2 since receptor sites are occupied. In
these ways, the action of the VEGF2 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 VEGF2 or by inactivating VEGF2 itself. The antagonist/inhibitors
may also be used to prevent inflammation due to the increased
vascular permeability action of VEGF2. The antagonist/inhibitors
may also be used to treat solid tumor growth, diabetic retinopathy,
psoriasis and rheumatoid arthritis.
[0306] The antagonist/inhibitors may be employed in a composition
with a pharmaceutically acceptable carrier, e.g., as hereinabove
described.
[0307] 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.
[0308] In order to facilitate understanding of the following
examples, certain frequently occurring methods and/or terms will be
described.
[0309] "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.
[0310] "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 .mu.g 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 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37EC are ordinarily used, but may vary in
accordance with the supplier's instructions. After digestion the
reaction is electrophoresed directly on a polyacrylamide gel to
isolate the desired fragment.
[0311] 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).
[0312] "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.
[0313] "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 .mu.g of approximately equimolar amounts of the
DNA fragments to be ligated.
[0314] Unless otherwise stated, transformation was performed as
described by the method of Graham, F. and Van der Eb, A., Virology
52:456-457 (1973).
EXAMPLE 1
Expression Pattern of VEGF2 in Human Tissues and Breast Cancer Cell
Lines
[0315] Northern blot analysis was carried out to examine the levels
of expression of VEGF2 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
.mu.g 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 VEGF2 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 Kb was observed in 2 breast cancer cell lines. FIG. 5, lane #4
represents a very tumorigenic cell line that is estrogen
independent for growth.
[0316] Also, 10 .mu.g 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 VEGF2 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 VEGF2 (SEQ ID NO:4) by in vitro
Transcription and Translation
[0317] The VEGF2 cDNA was transcribed and translated in vitro to
determine the size of the translatable polypeptide encoded by the
truncated form of VEGF2 and a partial VEGF2 cDNA. The two inserts
of VEGF2 in the 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.
[0318] M13-2 reverse primer: [0319] 5'-ATGCTTCCGGCTCGTATG-3' (SEQ
ID NO: 9) This sequence is located upstream of the 5' end of the
VEGF2 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 VEGF2 cDNA.
[0320] M13-2 forward primer: [0321] 5'GGGTTTTCCCAGTCACGAC-3' (SEQ
ID NO:10) This sequence is located downstream of the 3' end of the
VEGF2 cDNA insert in the pBLUESCRIPT.TM. vector and is in an
anti-sense orientation as the cDNA insert.
[0322] VEGF primer F4: [0323] 5'-CCACATGGTTCAGGAAAGACA-3' (SEQ ID
NO:11) This sequence is located within the VEGF2 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.
[0324] 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 VEGF2 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 VEGF2
polypeptide.
[0325] Approximately 0.5 .mu.g of PCR product from first pair of
primers, 1 .mu.g from second pair of primers, 1 .mu.g from third
pair of primers were used for in vitro transcription/translation.
The in vitro transcription/translation reaction was performed in a
25 Fl of volume, using the TNTJ Coupled Reticulocyte Lysate Systems
(PROMEGA.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 .mu.g 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.
[0326] As shown in FIG. 7, PCR products containing the truncated
VEGF2 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 Kd (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 VEGF2 Using the Baculovirus Expression
System
[0327] The DNA sequence encoding the VEGF2 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:
[0328] 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 BamH1 restriction enzyme site (in bold) and 17 nucleotide
nucleotide sequence complementary to the 5' sequence of VEGF2 (nt.
150-166).
[0329] 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 VEGF2, including the stop codon and 15 nt sequence
before stop codon.
[0330] 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 BamH1 and XbaI and then purified again on a 1% agarose
gel. This fragment was ligated to pAcGP67A baculovirus transfer
vector (Pharmingen) at the BamH1 and XbaI sites. Through this
ligation, VEGF2 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-VEGF2.
[0331] To clone VEGF2 with the signal sequence of gp67 gene to the
pRG1 vector for expression, VEGF2 with the signal sequence and some
upstream sequence were excised from the pAcGP67A-VEGF2 plasmid at
the Xho restriction endonuclease site located upstream of the VEGF2
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.
[0332] The PRG1 vector (modification of pVL941 vector) is used for
the expression of the VEGF2 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 BamH1, Sma1, XbaI, BglII and Asp718.
A site for restriction endonuclease XhoI is located upstream of
BamH1 site. The sequence between XhoI and BamH1 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 pAcIM1
(Luckow, V. A. and Summers, M. D., Virology 170:31-39 (1989).
[0333] 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.
[0334] 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-VEGF2)
with the VEGF2 gene using the enzymes BamH1 and XbaI. The sequence
of the cloned fragment was confirmed by DNA sequencing.
[0335] 5 mg of the plasmid pBac gp67-VEGF2 was cotransfected with
1.0 mg of a commercially available linearized baculovirus
("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)).
[0336] 1 mg of BACULOGOLD.TM.J virus DNA and 5 mg of the plasmid
pBac gp67-VEGF2 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.
[0337] 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).
[0338] 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.
[0339] Sf9 cells were grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells were infected with the recombinant
baculovirus V-gp67-VEGF2 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 labeled proteins visualized by SDS-PAGE and
autoradiography.
[0340] 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. Precipitates were obtained after dialysis and
resuspended in 100 mM Na Citrate, pH 5.0. The resuspended
precipitate was analyzed again by SDS-PAGE and was stained with
Coomassie Brilliant Blue. See FIG. 9.
[0341] 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 VEGF2 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 Coommassie Brilliant Blue. See FIG. 10.
EXAMPLE 4
Expression of Recombinant VEGF2 in COS Cells
[0342] The expression of plasmid, VEGF2-HA is derived from a vector
pcDNAL/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 VEGF2
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.
[0343] The plasmid construction strategy is described as
follows:
[0344] The DNA sequence encoding VEGF2, 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 BamH1 site
followed by 18 nucleotides of VEGF2 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 VEGF2 coding sequence
(not including the stop codon). Therefore, the PCR product contains
a BamHI site, coding sequence followed by an XhoI restriction
endonuclease site and HA tag fused in frame, a translation
termination stop codon next to the HA tag, and an XbaI site. The
PCR amplified DNA fragment and the vector, pcDNAI/Amp, were
digested with BamH1 and XbaI restriction enzyme and ligated. The
ligation mixture was transformed into E. coli strain SURE
(Stratagene 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 VEGF2,
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 VEGF2-HA protein was detected
by radiolabeling and immunoprecipitation method (E. Harlow and D.
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, (1988)). Cells were labeled 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 VEGF2 Protein on the Growth of
Vascular Endothelial Cells
[0345] 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. VEGF2 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 VEGF2 Protein on the Growth of Vascular
Endothelial Cells
[0346] 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. PLurified VEGF2 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
[0347] 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.
[0348] 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.
[0349] 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.
[0350] 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).
[0351] 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.
[0352] 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 VEGF2 mRNA in Human Fetal and Adult Tissues
Experimental Design
[0353] Northern blot analysis was carried out to examine the levels
of expression of VEGF2 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 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.
[0354] 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
[0355] Expression of VEGF2 mRNA is abundant in vascular smooth
muscle and several highly vascularized tissues. VEGF2 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 VEGF2 is low in adult kidney, fetal liver,
adult liver, testes; and is almost undetectable in fetal brain, and
adult brain (See FIG. 14).
[0356] 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
[0357] 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
[0358] 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 VEGF2 were synthesized with the following base
sequence:
[0359] 5' Primer (Nde I/START and 18 nt of coding sequence):
TABLE-US-00002 5'-GCA GCA CAT ATG ACA GAA GAG ACT (SEQ ID NO: 19)
ATA AAA-3'
[0360] 3' Primer (Asp718, STOP, and 15 nt of coding sequence):
TABLE-US-00003 5'-GCA GCA GGT ACC TCA CAG TTT AGA (SEQ ID NO: 20)
CAT GCA-3'
[0361] 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.
[0362] 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:18) as, for example, constructed in Example 3 as template. The
resulting amplicon was restriction digested with NdeI and Asp718
and subcloned into NdeI/Asp718 digested pHE4a expression
vector.
2. Construction of VEGF-2 T103-R227 in pHE4
[0363] 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 VEGF2 were synthesized with the following base
sequence:
[0364] 5' Primer (Nde USTART and 18 nt of coding sequence):
TABLE-US-00004 5'-GCA GCA CAT ATG ACA GAA GAG ACT (SEQ ID NO: 19)
ATA AAA-3'
[0365] 3' Primer (Asp 718, STOP, and 15 nt of coding sequence):
TABLE-US-00005 5'-GCA GCA GGT ACC TCA ACG TCT AAT (SEQ ID NO: 21)
AAT GGA-3'
[0366] In the case of the above described primers, an NdeI or
Asp718 restriction site was incorporated the 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.
[0367] 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:18) as, for example, constructed in Example 3, as template. The
resulting amplicon was restriction digested with NdeI and Asp718
and subcloned into NdeI/Asp718 digested pHE4a protein expression
vector.
3. Construction of VEGF-2 T103-L215 in pA2GP
[0368] 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, Xba I and Asp718. The polyadenylation site of the simian
virus 40 ("SV40") is used for efficient polyadenylation. For easy
selection of recombinant virus, the plasmid contains the
beta-galactosidase gene from E. coli under control of a weak
Drosophila promoter in the same orientation, followed by the
polyadenylation signal of the polyhedrin gene. 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.
[0369] 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).
[0370] The cDNA sequence encoding theVEGF-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.
[0371] 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.
[0372] 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 BamH1 and XbaI and then purified again on a 1% agarose
gel. This fragment was ligated to pA2 GP baculovirus transfer
vector (Supplier) at the BamH1 and XbaI sites. Through this
ligation, VEGF2 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 pA2GPVEGF2.T103-L215.
4. Construction of VEGF-2 T103-R227 in pA2GP
[0373] The cDNA sequence encoding theVEGF-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:18)
was amplified using PCR oligonucleotide primers corresponding to
the 5' and 3' sequences of the gene.
[0374] 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.
[0375] 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 BamH1 and XbaI and then purified again on a 1% agarose
gel. This fragment was ligated to pA2 GP baculovirus transfer
vector (Supplier) at the BamH1 and XbaI sites. Through this
ligation, VEGF2 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 pA2GPVEGF2.T103-R227.
5. Construction of VEGF-2 in pC1
[0376] The expression vectors pCI 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.
[0377] The vector pCI is used for the expression of VEGF-2 protein.
Plasmid pCI 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, Ufe 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 M a, C. 1990, Biochem. et Biophys. Acta, 1097:107-143,
Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol. 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).
[0378] 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: BamH1, PvuII, and Nru1. 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 HTLV1.
For the polyadenylation of the mRNA other signals, e.g., from the
human growth hormone or globin genes can be used as well.
[0379] 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.
[0380] 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.
[0381] The DNA sequence encoding VEGF-2, ATCC.TM. Accession No.
97149, was constructed by PCR using two primers coressponding 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 TfT 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.
[0382] 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. coliHB101 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 pCIVEGF-2.
6. Construction of pC4SigVEGF-2 T103-L215
[0383] Plasmid pC4Sig is plasmid pC4 (Accession No.
209646)containing a human IgG Fc portion as well as a protein
signal sequence.
[0384] 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 VEGF2 were synthesized with
the following base sequence:
[0385] 5' Primer (Bam H1 and 26 nt of coding sequence):
TABLE-US-00006 5'-GCA GCA GGA TCC ACA GAA GAG ACT SEQ ID NO: 34 ATA
AAA TTT GCT GC-3'
[0386] 3' Primer (Xba 1, STOP, and 15 nt of coding sequence):
TABLE-US-00007 5'-CGT CGT TCT AGA TCA CAG TTT AGA SEQ ID NO: 35 CAT
GCA TCG GCA G-3'
[0387] 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
[0388] 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 VEGF2 were synthesized with
the following base sequence:
[0389] 5' Primer (Bam H1 and 26 nt of coding sequence):
TABLE-US-00008 5'-GCA GCA GGA TCC ACA GAA GAG ACT SEQ ID NO: 34 ATA
AAA TTT GCT GC-3'
[0390] 3' Primer (Xba 1, STOP, and 21 nt of coding sequence):
TABLE-US-00009 5'-GCA GCA TCT AGA TCA ACG TCT AAT SEQ ID NO: 25 AAT
GGA ATG AAC-3'
[0391] 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 BamH1 and XbaI and subcloned
into BamH1/IXbaI digested pC4Sig vector.
8. Construction of pC4VEGF-2 M1-M263
[0392] 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.
[0393] 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:18) is inserted into the plasmid vector pC4 to
express the C-terminal deleted VEGF-2 protein.
[0394] 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
VEGF2 were synthesized with the following base sequence:
TABLE-US-00010 5' Primer 5'-GAC TGG ATC CGC CAC CAT GCA CTC (SEQ ID
NO: 28) GCT GGG CTT CTT CTC-3' 3' Primer 5'-GAC TGG TAC CTT ATC ACA
TAA AAT (SEQ ID NO: 29) CTT CCT GAG CC-3'
[0395] In the case of the above described 5' primer, an
BamHlrestriction site was incorporated, while in the case of the 3'
primer, an Asp718 restriction site was incorporated. The 5' primer
also contains 6 nt, 20 nt of VEGF-2 coding sequence, and an ATG
sequence adjacent and in frame with the VEGF-2 coding region to
allow translation of the cloned fragment in E. coli, while the 3'
primer contains 2 nt, 20 nt of VEGF-2 coding sequence, and one stop
codon (preferentially utilized in E. coli) adjacent and in frame
with the VEGF-2 coding region which ensures correct translational
termination in E. coli.
[0396] 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 BamH1 and Asp718 and
subcloned into BamH1/Asp718 digested pC4 protein expression vector.
This construct is designated pC4VEGF-2 M1-M263.
9. Construction of pC4VEGF-2 M1-D311
[0397] 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:18) is inserted into the plasmid vector pC4 to
express the C-terminal deleted VEGF-2 protein.
[0398] 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
VEGF2 were synthesized with the following base sequence:
TABLE-US-00011 5' Primer 5'-GAC TGG ATC CGC CAC CAT GCA CTC (SEQ ID
NO: 30) GCT GGG CTT CTT CTC-3' 3' Primer 5'-GAC TGG TAC CTT ATC AGT
CTA GTT (SEQ ID NO: 31) CTT TGT GGG G-3'
[0399] In the case of the above described 5' primer, an BamH1
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.
[0400] 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 BamH1 and Asp718 and
subcloned into BamH1/Asp718 digested pC4 protein expression
vector.
[0401] 10. Construction of pC4VEGF-2 M1-Q367
[0402] 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:18) is inserted into the plasmid vector pC4 to express the
C-terminal deleted VEGF-2 protein.
[0403] 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
VEGF2 were synthesized with the following base sequence:
TABLE-US-00012 5' Primer 5'-GAC TGG ATC CGC CAC CAT GCA CTC (SEQ ID
NO: 32) GCT GGG CTT CTT CTC-3' 3' Primer 5'-GAC TGG TAC CTC ATT ACT
GTG GAC (SEQ ID NO: 33) TTT CTG TAC ATT C-3'
[0404] In the case of the above described 5' primer, an BamH1
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.
[0405] 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 24419) 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 COS7 Cells
Experimental Design
[0406] 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
[0407] 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 VEGF2 on proliferation of vascular
endothelial cells
Experimental Design
[0408] Expression of VEGF2 is abundant in highly vascularized
tissues. Therefore the role of VEGF2 in regulating proliferation of
several types of endothelial cells was examined.
Endothelial Cell Proliferation Assay
[0409] For evaluation of mitogenic activity of growth factors, the
colorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
).sub.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, VEFG.sub.165 or VEFG-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
[0410] VEGF2 stimulated proliferation of human umbilical vein
endothelial cells (HUVEC) and dermal microvascular endothelial
cells slightly (FIGS. 17 and 18). The stimulatory effect of VEGF2
is more pronounced on proliferation of endometrial and
microvascular endothelial cells (FIG. 19). Endometrial endothetial
cells (HEEC) demonstrated the greatest response to VEGF2 (96% of
the effect of VEGF on microvascular endothelial cells). The
response of microvascular endothetial cells (HMEC) to VEGF2 was 73%
compared to VEGF. The response of HUVEC and BAEC (bovine aortic
endothelial cells) to VEGF2 was substantially lower at 10% and 7%,
respectively. The activity of VEGF2 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
[0411] VEGF2 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
VEGF2 on smooth muscle cells, the effect of VEGF2 on human aortic
smooth muscle cell (HAOSMC) proliferation was examined.
Experimental Design
[0412] 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% caff 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-LW fluorescent
illumination. See, Hayashida et al., J. Biol. Chem. 6;
271(36):21985-21992 (1996).
Results
[0413] VEGF2 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
[0414] Endothelial cell migration is an important step involved in
angiogenesis.
Experimental Design
[0415] 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:
[0416] 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% CO.sub.2 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, 1L). 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
[0417] In an assay examining HUVEC migration using a 43-well
microchemotaxis chamber, VEGF2 was able to stimulate migration of
HUVEC (FIG. 21).
EXAMPLE 14
Stimulation of Nitric Oxide Production by Endothelial Cells
[0418] 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
[0419] 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 VEGF2 on
nitric oxide release was examined on HUVEC.
[0420] 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+2 KI+2H.sub.2SO.sub.4 6 2
NO+I.sub.2+2H.sub.2O+2 K.sub.2SO.sub.4 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 K.sub.1 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 Une 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 1x10.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
[0421] VEGF-2 was capable of stimulating nitric oxide release on
HUVEC (FIG. 22) to a higher level than VEGF. This suggested that
VEGF2 may modify vascular permeability and vessel dilation.
EXAMPLE 15
Effect of VEGF-2 on Cord Formation in Angiogenesis
[0422] 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
[0423] 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.
[0424] 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
[0425] It has been observed that VEGF2 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
[0426] 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 VEGF2 to stimulate
angiogenesis in CAM was examined.
Experimental Design
[0427] Embryos
[0428] 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
[0429] 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
[0430] This data demonstrates that VEGF2 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.TM. Implant in Mouse
Experimental Design
[0431] 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 VEFG-1
(positive control).
[0432] 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
[0433] Both VEGF proteins appeared to enhance MATRIGEL.TM.
cellularity by a factor of approximately 2 by visual
estimation.
[0434] 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.
[0435] 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
[0436] To study the in vivo effects of VEGF2 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 VEGF2 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 VEGF2 was used in the treatment,
a single bolus of 500 mg VEGF2 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
[0437] Both VEGF2 protein (FIG. 25, top panels) and naked
expression plasmid (FIG. 25, middle panels) were able to restore
the following parameters in the ischemic limb. Restoration of blood
flow, angiographic score seem to be slightly more by administration
of 500 mg plasmid compared with by 500 mg protein (FIG. 25, bottom
panels) The extent of the restoration is comparable with that by
VEGF in separate experiments (data not shown). A vessel dilator was
not able to achieve the same effect, suggesting that the blood flow
restoration is not simply due to a vascular dilation effect.
[0438] a. BP ratio (FIG. 25a) [0439] The blood pressure ratio of
systolic pressure of the ischemic limb to that of normal limb.
[0440] 2. Blood Flow and Flow Reserve (FIG. 25b) [0441] Resting FL:
the blood flow during un-dilated condition [0442] Max FL: the blood
flow during fully dilated condition (also an indirect measure of
the blood vessel amount) [0443] Flow Reserve is reflected by the
ratio of max FL: resting FL.
[0444] 3. Angiographic Score (FIG. 25c) [0445] 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.
[0446] 4. Capillary density (FIG. 25d) [0447] The number of
collateral capillaries determined in light microscopic sections
taken from hindlimbs.
[0448] As discussed, VEGF2 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 VEGF2 on Vasodilation
[0449] As described above, VEGF2 can stimulate NO release, a
mediator of vascular endothelium dilation. Since dilation of
vascular endothelium is important in reducing blood pressure, the
ability of VEGF2 to affect the blood pressure in spontaneously
hypertensive rats (SHR) was examined. VEGF2 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
VEGF2 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 (FIGS. 26c and
d). VEGF2 (300 mg/kg) and acetylcholine reduced the MAP of these
SHR animals to normal levels.
[0450] 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.
[0451] 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.
[0452] 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).
[0453] 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
[0454] 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.
[0455] The study in this model is divided into three parts as
follows: [0456] a) Ischemic skin [0457] b) Ischemic skin wounds
[0458] c) Normal wounds
[0459] The experimental protocol includes: [0460] a) Raising a
3.times.4 cm, single pedicle full-thickness random skin flap
(myocutaneous flap over the lower back of the animal). [0461] b) An
excisional wounding (4-6 mm in diameter) in the ischemic skin
(skin-flap). [0462] 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. [0463] d)
Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21
post-wounding for histological, immunohistochemical, and in situ
studies.
EXAMPLE 22
Peripheral Arterial Disease Model
[0464] 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
[0465] The experimental protocol includes: [0466] 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. [0467] 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. [0468] 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 23
Ischemic Myocardial Disease Model
[0469] 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
[0470] The experimental protocol includes: [0471] 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. [0472] 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 24 weeks. [0473] c) Thirty days
after the surgery, the heart is removed and cross-sectioned for
morphometric and in situ analyzes.
EXAMPLE 24
Rat Corneal Wound Healing Model
[0474] This animal model shows the effect of VEGF-2 on
neovascularization.
Experimental Design
[0475] The experimental protocol includes: [0476] a) Making a 1-1.5
mm long incision from the center of cornea into the stromal layer.
[0477] b) Inserting a spatula below the lip of the incision facing
the outer corner of the eye. [0478] c) Making a pocket (its base is
1-1.5 mm form the edge of the eye). [0479] d) Positioning a pellet,
containing 50 mg-500 mg VEGF-2, within the pocket. [0480] 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 25
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
[0480] Experimental Design
[0481] The experimental protocol includes:
[0482] 1. Diabetic db+/db+mouse model.
[0483] 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)).
[0484] 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 1 diabetes (Mandel
et al., J. Immunol. 120:1375-1377 (1978)).
[0485] 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
[0486] Genetically diabetic female C57BUKsJ (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
[0487] 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.
[0488] 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.
[0489] 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 50mL of vehicle
solution.
[0490] 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
[0491] 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
[0492] 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 mm2, 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
[0493] 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
[0494] Re-epithelialization
[0495] 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
[0496] 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
[0497] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
B. Steroid Impaired Rat Model
[0498] 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
Anti-inflammatory 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
Anti-inflammatory 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)).
[0499] 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
[0500] 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
[0501] 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.
[0502] 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.
[0503] 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 50mL of vehicle
solution.
[0504] 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
[0505] 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
[0506] 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
[0507] 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
[0508] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
EXAMPLE 26
Specific Peptide Fragments to Generate VEGF-2 Monoclonal
Antibodies
[0509] 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-00013 1. "SP-40": MTVLYPEYWKMY (amino
acids 70-81 in SEQ ID NO: 18) 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 27
[0510] Lymphadema Animal Model
[0511] 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 34 weeks.
Experimental Procedure
[0512] 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.
[0513] 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.
[0514] Using a microscope, muscles in back of the leg (near the
semitendinosis and adductors) are bluntly dissected. The popliteal
lymph node is then located. 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.
[0515] 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) (AJ Buck). The separated skin
edges are sealed to the underlying muscle tissue while leaving a
gap of -0.5 cm around the leg. Skin also may be anchored by
suturing to underlying muscle when necessary.
[0516] 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.
[0517] 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.
[0518] 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.
[0519] Blood-plasma protein measurements: Blood is drawn, spun, and
serum separated prior to surgery and then at conclusion for total
protein and Ca2+ comparison.
[0520] 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.
[0521] 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 -80C until sectioning. Upon
sectioning, the muscle was observed under fluorescent microscopy
for lymphatics. Other immuno/histological methods are currently
being evaluated.
[0522] 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.
Sequence CWU 1
1
35 1 1674 DNA homo sapiens CDS (12)..(1268) mat_peptide (81)..()
sig_peptide (12)..(80) 1 gtccttccac c atg cac tcg ctg ggc ttc ttc
tct gtg gcg tgt tct ctg 50 Met His Ser Leu Gly Phe Phe Ser Val Ala
Cys Ser Leu -23 -20 -15 ctc gcc gct gcg ctg ctc ccg ggt cct cgc gag
gcg ccc gcc gcc gcc 98 Leu Ala Ala Ala Leu Leu Pro Gly Pro Arg Glu
Ala Pro Ala Ala Ala -10 -5 1 5 gcc gcc ttc gag tcc gga ctc gac ctc
tcg gac gcg gag ccc gac gcg 146 Ala Ala Phe Glu Ser Gly Leu Asp Leu
Ser Asp Ala Glu Pro Asp Ala 10 15 20 ggc gag gcc acg gct tat gca
agc aaa gat ctg gag gag cag tta cgg 194 Gly Glu Ala Thr Ala Tyr Ala
Ser Lys Asp Leu Glu Glu Gln Leu Arg 25 30 35 tct gtg tcc agt gta
gat gaa ctc atg act gta ctc tac cca gaa tat 242 Ser Val Ser Ser Val
Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr 40 45 50 tgg aaa atg
tac aag tgt cag cta agg aaa gga ggc tgg caa cat aac 290 Trp Lys Met
Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn 55 60 65 70 aga
gaa cag gcc aac ctc aac tca agg aca gaa gag act ata aaa ttt 338 Arg
Glu Gln Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe 75 80
85 gct gca gca cat tat aat aca gag atc ttg aaa agt att gat aat gag
386 Ala Ala Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu
90 95 100 tgg aga aag act caa tgc atg cca cgg gag gtg tgt ata gat
gtg ggg 434 Trp Arg Lys Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp
Val Gly 105 110 115 aag gag ttt gga gtc gcg aca aac acc ttc ttt aaa
cct cca tgt gtg 482 Lys Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys
Pro Pro Cys Val 120 125 130 tcc gtc tac aga tgt ggg ggt tgc tgc aat
agt gag ggg ctg cag tgc 530 Ser Val Tyr Arg Cys Gly Gly Cys Cys Asn
Ser Glu Gly Leu Gln Cys 135 140 145 150 atg aac acc agc acg agc tac
ctc agc aag acg tta ttt gaa att aca 578 Met Asn Thr Ser Thr Ser Tyr
Leu Ser Lys Thr Leu Phe Glu Ile Thr 155 160 165 gtg cct ctc tct caa
ggc ccc aaa cca gta aca atc agt ttt gcc aat 626 Val Pro Leu Ser Gln
Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn 170 175 180 cac act tcc
tgc cga tgc atg tct aaa ctg gat gtt tac aga caa gtt 674 His Thr Ser
Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val 185 190 195 cat
tcc att att aga cgt tcc ctg cca gca aca cta cca cag tgt cag 722 His
Ser Ile Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln 200 205
210 gca gcg aac aag acc tgc ccc acc aat tac atg tgg aat aat cac atc
770 Ala Ala Asn Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile
215 220 225 230 tgc aga tgc ctg gct cag gaa gat ttt atg ttt tcc tcg
gat gct gga 818 Cys Arg Cys Leu Ala Gln Glu Asp Phe Met Phe Ser Ser
Asp Ala Gly 235 240 245 gat gac tca aca gat gga ttc cat gac atc tgt
gga cca aac aag gag 866 Asp Asp Ser Thr Asp Gly Phe His Asp Ile Cys
Gly Pro Asn Lys Glu 250 255 260 ctg gat gaa gag acc tgt cag tgt gtc
tgc aga gcg ggg ctt cgg cct 914 Leu Asp Glu Glu Thr Cys Gln Cys Val
Cys Arg Ala Gly Leu Arg Pro 265 270 275 gcc agc tgt gga ccc cac aaa
gaa cta gac aga aac tca tgc cag tgt 962 Ala Ser Cys Gly Pro His Lys
Glu Leu Asp Arg Asn Ser Cys Gln Cys 280 285 290 gtc tgt aaa aac aaa
ctc ttc ccc agc caa tgt ggg gcc aac cga gaa 1010 Val Cys Lys Asn
Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu 295 300 305 310 ttt
gat gaa aac aca tgc cag tgt gta tgt aaa aga acc tgc ccc aga 1058
Phe Asp Glu Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg 315
320 325 aat caa ccc cta aat cct gga aaa tgt gcc tgt gaa tgt aca gaa
agt 1106 Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr
Glu Ser 330 335 340 cca cag aaa tgc ttg tta aaa gga aag aag ttc cac
cac caa aca tgc 1154 Pro Gln Lys Cys Leu Leu Lys Gly Lys Lys Phe
His His Gln Thr Cys 345 350 355 agc tgt tac aga cgg cca tgt acg aac
cgc cag aag gct tgt gag cca 1202 Ser Cys Tyr Arg Arg Pro Cys Thr
Asn Arg Gln Lys Ala Cys Glu Pro 360 365 370 gga ttt tca tat agt gaa
gaa gtg tgt cgt tgt gtc cct tca tat tgg 1250 Gly Phe Ser Tyr Ser
Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp 375 380 385 390 caa aga
cca caa atg agc taagattgta ctgttttcca gttcatcgat 1298 Gln Arg Pro
Gln Met Ser 395 tttctattat ggaaaactgt gttgccacag tagaactgtc
tgtgaacaga gagacccttg 1358 tgggtccatg ctaacaaaga caaaagtctg
tctttcctga accatgtgga taactttaca 1418 gaaatggact ggagctcatc
tgcaaaaggc ctcttgtaaa gactggtttt ctgccaatga 1478 ccaaacagcc
aagattttcc tcttgtgatt tctttaaaag aatgactata taatttattt 1538
ccactaaaaa tattgtttct gcattcattt ttatagcaac aacaattggt aaaactcact
1598 gtgatcaata tttttatatc atgcaaaata tgtttaaaat aaaatgaaaa
ttgtatttat 1658 aaaaaaaaaa aaaaaa 1674 2 419 PRT homo sapiens 2 Met
His Ser Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala -23 -20
-15 -10 Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala
Phe -5 1 5 Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly
Glu Ala 10 15 20 25 Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu
Arg Ser Val Ser 30 35 40 Ser Val Asp Glu Leu Met Thr Val Leu Tyr
Pro Glu Tyr Trp Lys Met 45 50 55 Tyr Lys Cys Gln Leu Arg Lys Gly
Gly Trp Gln His Asn Arg Glu Gln 60 65 70 Ala Asn Leu Asn Ser Arg
Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala 75 80 85 His Tyr Asn Thr
Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys 90 95 100 105 Thr
Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe 110 115
120 Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr
125 130 135 Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met
Asn Thr 140 145 150 Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile
Thr Val Pro Leu 155 160 165 Ser Gln Gly Pro Lys Pro Val Thr Ile Ser
Phe Ala Asn His Thr Ser 170 175 180 185 Cys Arg Cys Met Ser Lys Leu
Asp Val Tyr Arg Gln Val His Ser Ile 190 195 200 Ile Arg Arg Ser Leu
Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn 205 210 215 Lys Thr Cys
Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys 220 225 230 Leu
Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser 235 240
245 Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu
250 255 260 265 Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro
Ala Ser Cys 270 275 280 Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys
Gln Cys Val Cys Lys 285 290 295 Asn Lys Leu Phe Pro Ser Gln Cys Gly
Ala Asn Arg Glu Phe Asp Glu 300 305 310 Asn Thr Cys Gln Cys Val Cys
Lys Arg Thr Cys Pro Arg Asn Gln Pro 315 320 325 Leu Asn Pro Gly Lys
Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys 330 335 340 345 Cys Leu
Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr 350 355 360
Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser 365
370 375 Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Gln Arg
Pro 380 385 390 Gln Met Ser 395 3 1526 DNA homo sapiens 3
cgaggccacg gcttatgcaa gcaaagatct ggaggagcag ttacggtctg tgtccagtgt
60 agatgaactc atg act gta ctc tac cca gaa tat tgg aaa atg tac aag
109 Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met Tyr Lys -24 -20 -15
tgt cag cta agg aaa gga ggc tgg caa cat aac aga gaa cag gcc aac 157
Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln Ala Asn -10
-5 1 5 ctc aac tca agg aca gaa gag act ata aaa ttt gct gca gca cat
tat 205 Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala His
Tyr 10 15 20 aat aca gag atc ttg aaa agt att gat aat gag tgg aga
aag act caa 253 Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg
Lys Thr Gln 25 30 35 tgc atg cca cgg gag gtg tgt ata gat gtg ggg
aag gag ttt gga gtc 301 Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly
Lys Glu Phe Gly Val 40 45 50 gcg aca aac acc ttc ttt aaa cct cca
tgt gtg tcc gtc tac aga tgt 349 Ala Thr Asn Thr Phe Phe Lys Pro Pro
Cys Val Ser Val Tyr Arg Cys 55 60 65 ggg ggt tgc tgc aat agt gag
ggg ctg cag tgc atg aac acc agc acg 397 Gly Gly Cys Cys Asn Ser Glu
Gly Leu Gln Cys Met Asn Thr Ser Thr 70 75 80 85 agc tac ctc agc aag
acg tta ttt gaa att aca gtg cct ctc tct caa 445 Ser Tyr Leu Ser Lys
Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln 90 95 100 ggc ccc aaa
cca gta aca atc agt ttt gcc aat cac act tcc tgc cga 493 Gly Pro Lys
Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg 105 110 115 tgc
atg tct aaa ctg gat gtt tac aga caa gtt cat tcc att att aga 541 Cys
Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg 120 125
130 cgt tcc ctg cca gca aca cta cca cag tgt cag gca gcg aac aag acc
589 Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr
135 140 145 tgc ccc acc aat tac atg tgg aat aat cac atc tgc aga tgc
ctg gct 637 Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys
Leu Ala 150 155 160 165 cag gaa gat ttt atg ttt tcc tcg gat gct gga
gat gac tca aca gat 685 Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly
Asp Asp Ser Thr Asp 170 175 180 gga ttc cat gac atc tgt gga cca aac
aag gag ctg gat gaa gag acc 733 Gly Phe His Asp Ile Cys Gly Pro Asn
Lys Glu Leu Asp Glu Glu Thr 185 190 195 tgt cag tgt gtc tgc aga gcg
ggg ctt cgg cct gcc agc tgt gga ccc 781 Cys Gln Cys Val Cys Arg Ala
Gly Leu Arg Pro Ala Ser Cys Gly Pro 200 205 210 cac aaa gaa cta gac
aga aac tca tgc cag tgt gtc tgt aaa aac aaa 829 His Lys Glu Leu Asp
Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys 215 220 225 ctc ttc ccc
agc caa tgt ggg gcc aac cga gaa ttt gat gaa aac aca 877 Leu Phe Pro
Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr 230 235 240 245
tgc cag tgt gta tgt aaa aga acc tgc ccc aga aat caa ccc cta aat 925
Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn 250
255 260 cct gga aaa tgt gcc tgt gaa tgt aca gaa agt cca cag aaa tgc
ttg 973 Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys Cys
Leu 265 270 275 tta aaa gga aag aag ttc cac cac caa aca tgc agc tgt
tac aga cgg 1021 Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser
Cys Tyr Arg Arg 280 285 290 cca tgt acg aac cgc cag aag gct tgt gag
cca gga ttt tca tat agt 1069 Pro Cys Thr Asn Arg Gln Lys Ala Cys
Glu Pro Gly Phe Ser Tyr Ser 295 300 305 gaa gaa gtg tgt cgt tgt gtc
cct tca tat tgg caa aga cca caa atg 1117 Glu Glu Val Cys Arg Cys
Val Pro Ser Tyr Trp Gln Arg Pro Gln Met 310 315 320 325 agc
taagattgta ctgttttcca gttcatcgat tttctattat ggaaaactgt 1170 Ser
gttgccacag tagaactgtc tgtgaacaga gagacccttg tgggtccatg ctaacaaaga
1230 caaaagtctg tctttcctga accatgtgga taactttaca gaaatggact
ggagctcatc 1290 tgcaaaaggc ctcttgtaaa gactggtttt ctgccaatga
ccaaacagcc aagattttcc 1350 tcttgtgatt tctttaaaag aatgactata
taatttattt ccactaaaaa tattgtttct 1410 gcattcattt ttatagcaac
aacaattggt aaaactcact gtgatcaata tttttatatc 1470 atgcaaaata
tgtttaaaat aaaatgaaaa ttgtatttat aaaaaaaaaa aaaaaa 1526 4 350 PRT
homo sapiens 4 Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met Tyr Lys
Cys Gln Leu -24 -20 -15 -10 Arg Lys Gly Gly Trp Gln His Asn Arg Glu
Gln Ala Asn Leu Asn Ser -5 1 5 Arg Thr Glu Glu Thr Ile Lys Phe Ala
Ala Ala His Tyr Asn Thr Glu 10 15 20 Ile Leu Lys Ser Ile Asp Asn
Glu Trp Arg Lys Thr Gln Cys Met Pro 25 30 35 40 Arg Glu Val Cys Ile
Asp Val Gly Lys Glu Phe Gly Val Ala Thr Asn 45 50 55 Thr Phe Phe
Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly Cys 60 65 70 Cys
Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Ser Tyr Leu 75 80
85 Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro Lys
90 95 100 Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg Cys
Met Ser 105 110 115 120 Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile
Ile Arg Arg Ser Leu 125 130 135 Pro Ala Thr Leu Pro Gln Cys Gln Ala
Ala Asn Lys Thr Cys Pro Thr 140 145 150 Asn Tyr Met Trp Asn Asn His
Ile Cys Arg Cys Leu Ala Gln Glu Asp 155 160 165 Phe Met Phe Ser Ser
Asp Ala Gly Asp Asp Ser Thr Asp Gly Phe His 170 175 180 Asp Ile Cys
Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr Cys Gln Cys 185 190 195 200
Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys Gly Pro His Lys Glu 205
210 215 Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys Leu Phe
Pro 220 225 230 Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr
Cys Gln Cys 235 240 245 Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro
Leu Asn Pro Gly Lys 250 255 260 Cys Ala Cys Glu Cys Thr Glu Ser Pro
Gln Lys Cys Leu Leu Lys Gly 265 270 275 280 Lys Lys Phe His His Gln
Thr Cys Ser Cys Tyr Arg Arg Pro Cys Thr 285 290 295 Asn Arg Gln Lys
Ala Cys Glu Pro Gly Phe Ser Tyr Ser Glu Glu Val 300 305 310 Cys Arg
Cys Val Pro Ser Tyr Trp Gln Arg Pro Gln Met Ser 315 320 325 5 196
PRT homo sapiens 5 Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly Cys
Gly Tyr Leu Ala 1 5 10 15 His Val Leu Ala Glu Glu Ala Glu Ile Pro
Arg Glu Val Ile Glu Arg 20 25 30 Leu Ala Arg Ser Gln Ile His Ser
Ile Arg Asp Leu Gln Arg Leu Leu 35 40 45 Glu Ile Asp Ser Val Gly
Ser Glu Asp Ser Leu Asp Thr Ser Leu Arg 50 55 60 Ala His Gly Val
His Ala Thr Lys His Val Pro Glu Lys Arg Pro Leu 65 70 75 80 Pro Ile
Arg Arg Lys Arg Ser Ile Glu Glu Ala Val Pro Ala Val Cys 85 90 95
Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro 100
105 110 Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys
Arg 115 120 125 Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys Gln
Pro Ser Arg 130 135 140 Val His His Arg Ser Val Lys Val Ala Lys Val
Glu Tyr Val Arg Lys 145 150 155 160 Lys Pro Lys Leu Lys Glu Val Gln
Val Arg Leu Glu Glu His Leu Glu 165 170 175 Cys Ala Cys Ala Thr Thr
Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp 180 185 190 Thr Asp Val Arg
195 6 241 PRT homo sapiens 6 Met Asn Arg Cys Trp Ala Leu Phe Leu
Ser Leu Cys Cys Tyr Leu Arg 1 5 10 15 Leu Val Ser Ala Glu Gly Asp
Pro Ile Pro Glu Glu Leu Tyr Glu Met 20 25 30 Leu Ser Asp His Ser
Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu 35 40 45 His Gly Asp
Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met
50 55 60 Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg
Gly Arg 65 70 75 80 Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala
Met Ile Ala Glu 85 90 95 Cys Lys Thr Arg Thr Glu Val Phe Glu Ile
Ser Arg Arg Leu Ile Asp 100 105 110 Arg Thr Asn Ala Asn Phe Leu Val
Trp Pro Pro Cys Val Glu Val Gln 115 120 125 Arg Cys Ser Gly Cys Cys
Asn Asn Arg Asn Val Gln Cys Arg Pro Thr 130 135 140 Gln Val Gln Leu
Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg 145 150 155 160 Lys
Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu 165 170
175 Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser
180 185 190 Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr
Arg Val 195 200 205 Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys
Gly Lys His Arg 210 215 220 Lys Phe Lys His Thr His Asp Lys Thr Ala
Leu Lys Glu Thr Leu Gly 225 230 235 240 Ala 7 232 PRT homo sapiens
7 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1
5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu
Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp
Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val
Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe
Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys
Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn
Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln
His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu
Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val 130 135
140 Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr
145 150 155 160 Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu
Met Pro Trp 165 170 175 Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys
Ser Glu Arg Arg Lys 180 185 190 His Leu Phe Val Gln Asp Pro Gln Thr
Cys Lys Cys Ser Cys Lys Asn 195 200 205 Thr Asp Ser Arg Cys Lys Ala
Arg Gln Leu Glu Leu Asn Glu Arg Thr 210 215 220 Cys Arg Cys Asp Lys
Pro Arg Arg 225 230 8 14 PRT homo sapiens SITE (2)..(2) Xaa is
equal to any amino acid found in a naturally occuring protein. 8
Pro Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys Asn 1 5 10 9 18
DNA oligonucleotide 9 atgcttccgg ctcgtatg 18 10 19 DNA
oligonucleotide 10 gggttttccc agtcacgac 19 11 21 DNA
oligonucleotide 11 ccacatggtt caggaaagac a 21 12 50 DNA
oligonucleotide 12 tgtaatacga ctcactatag ggatcccgcc atggaggcca
cggcttatgc 50 13 28 DNA oligonucleotide 13 gatctctaga ttagctcatt
tgtggtct 28 14 27 DNA oligonucleotide 14 cgcggatcca tgactgtact
ctaccca 27 15 60 DNA oligonucleotide 15 cgctctagat caagcgtagt
ctgggacgtc gtatgggtac tcgaggctca tttgtggtct 60 16 3974 DNA
Expression vector pHEA4-5 misc_feature (1)..(3974) Expression
vector pHE4-5 16 ggtacctaag tgagtagggc gtccgatcga cggacgcctt
ttttttgaat tcgtaatcat 60 ggtcatagct gtttcctgtg tgaaattgtt
atccgctcac aattccacac aacatacgag 120 ccggaagcat aaagtgtaaa
gcctggggtg cctaatgagt gagctaactc acattaattg 180 cgttgcgctc
actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 240
tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
300 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
tcaaaggcgg 360 taatacggtt atccacagaa tcaggggata acgcaggaaa
gaacatgtga gcaaaaggcc 420 agcaaaaggc caggaaccgt aaaaaggccg
cgttgctggc gtttttccat aggctccgcc 480 cccctgacga gcatcacaaa
aatcgacgct caagtcagag gtggcgaaac ccgacaggac 540 tataaagata
ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 600
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
660 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
ggctgtgtgc 720 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
taactatcgt cttgagtcca 780 acccggtaag acacgactta tcgccactgg
cagcagccac tggtaacagg attagcagag 840 cgaggtatgt aggcggtgct
acagagttct tgaagtggtg gcctaactac ggctacacta 900 gaagaacagt
atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
1020 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
tctacggggt 1080 ctgacgctca gtggaacgaa aactcacgtt aagggatttt
ggtcatgaga ttatcgtcga 1140 caattcgcgc gcgaaggcga agcggcatgc
atttacgttg acaccatcga atggtgcaaa 1200 acctttcgcg gtatggcatg
atagcgcccg gaagagagtc aattcagggt ggtgaatgtg 1260 aaaccagtaa
cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc 1320
cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
1380 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
caaacagtcg 1440 ttgctgattg gcgttgccac ctccagtctg gccctgcacg
cgccgtcgca aattgtcgcg 1500 gcgattaaat ctcgcgccga tcaactgggt
gccagcgtgg tggtgtcgat ggtagaacga 1560 agcggcgtcg aagcctgtaa
agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg 1620 ctgatcatta
actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact 1680
aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
1740 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
tcaccagcaa 1800 atcgcgctgt tagcgggccc attaagttct gtctcggcgc
gtctgcgtct ggctggctgg 1860 cataaatatc tcactcgcaa tcaaattcag
ccgatagcgg aacgggaagg cgactggagt 1920 gccatgtccg gttttcaaca
aaccatgcaa atgctgaatg agggcatcgt tcccactgcg 1980 atgctggttg
ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg 2040
ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
2100 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
aaccagcgtg 2160 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
gcaatcagct gttgcccgtc 2220 tcactggtga aaagaaaaac caccctggcg
cccaatacgc aaaccgcctc tccccgcgcg 2280 ttggccgatt cattaatgca
gctggcacga caggtttccc gactggaaag cgggcagtga 2340 gcgcaacgca
attaatgtaa gttagcgcga attgtcgacc aaagcggcca tcgtgcctcc 2400
ccactcctgc agttcggggg catggatgcg cggatagccg ctgctggttt cctggatgcc
2460 gacggatttg cactgccggt agaactccgc gaggtcgtcc agcctcaggc
agcagctgaa 2520 ccaactcgcg aggggatcga gcccggggtg ggcgaagaac
tccagcatga gatccccgcg 2580 ctggaggatc atccagccgg cgtcccggaa
aacgattccg aagcccaacc tttcatagaa 2640 ggcggcggtg gaatcgaaat
ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc 2700 gaaccccaga
gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc 2760
gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
2820 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
cacacccagc 2880 cggccacagt cgatgaatcc agaaaagcgg ccattttcca
ccatgatatt cggcaagcag 2940 gcatcgccat gggtcacgac gagatcctcg
ccgtcgggca tgcgcgcctt gagcctggcg 3000 aacagttcgg ctggcgcgag
cccctgatgc tcttcgtcca gatcatcctg atcgacaaga 3060 ccggcttcca
tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg 3120
caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc
3180 tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
caatagcagc 3240 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg
cgcaaggaac gcccgtcgtg 3300 gccagccacg atagccgcgc tgcctcgtcc
tgcagttcat tcagggcacc ggacaggtcg 3360 gtcttgacaa aaagaaccgg
gcgcccctgc gctgacagcc ggaacacggc ggcatcagag 3420 cagccgattg
tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga 3480
gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
3540 tcagatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat
ccagtttact 3600 ttgcagggct tcccaacctt accagagggc gccccagctg
gcaattccgg ttcgcttgct 3660 gtccataaaa ccgcccagtc tagctatcgc
catgtaagcc cactgcaagc tacctgcttt 3720 ctctttgcgc ttgcgttttc
ccttgtccag atagcccagt agctgacatt catccggggt 3780 cagcaccgtt
tctgcggact ggctttctac gtgttccgct tcctttagca gcccttgcgc 3840
cctgagtgct tgcggcagcg tgaagcttaa aaaactgcaa aaaatagttt gacttgtgag
3900 cggataacaa ttaagatgta cccaattgtg agcggataac aatttcacac
attaaagagg 3960 agaaattaca tatg 3974 17 112 DNA promoter consensus
sequence promoter (1)..(112) regulatory elements of the pHE
promotor two lac operator sequences, Shine-Delgarno sequence, and
terminal Hind III and Nde I restriction sites 17 aagcttaaaa
aactgcaaaa aatagtttga cttgtgagcg gataacaatt aagatgtacc 60
caattgtgag cggataacaa tttcacacat taaagaggag aaattacata tg 112 18
419 PRT homo sapiens 18 Met His Ser Leu Gly Phe Phe Ser Val Ala Cys
Ser Leu Leu Ala Ala 1 5 10 15 Ala Leu Leu Pro Gly Pro Arg Glu Ala
Pro Ala Ala Ala Ala Ala Phe 20 25 30 Glu Ser Gly Leu Asp Leu Ser
Asp Ala Glu Pro Asp Ala Gly Glu Ala 35 40 45 Thr Ala Tyr Ala Ser
Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser 50 55 60 Ser Val Asp
Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met 65 70 75 80 Tyr
Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln 85 90
95 Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala
100 105 110 His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp
Arg Lys 115 120 125 Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val
Gly Lys Glu Phe 130 135 140 Gly Val Ala Thr Asn Thr Phe Phe Lys Pro
Pro Cys Val Ser Val Tyr 145 150 155 160 Arg Cys Gly Gly Cys Cys Asn
Ser Glu Gly Leu Gln Cys Met Asn Thr 165 170 175 Ser Thr Ser Tyr Leu
Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu 180 185 190 Ser Gln Gly
Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser 195 200 205 Cys
Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile 210 215
220 Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn
225 230 235 240 Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile
Cys Arg Cys 245 250 255 Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp
Ala Gly Asp Asp Ser 260 265 270 Thr Asp Gly Phe His Asp Ile Cys Gly
Pro Asn Lys Glu Leu Asp Glu 275 280 285 Glu Thr Cys Gln Cys Val Cys
Arg Ala Gly Leu Arg Pro Ala Ser Cys 290 295 300 Gly Pro His Lys Glu
Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys 305 310 315 320 Asn Lys
Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu 325 330 335
Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro 340
345 350 Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln
Lys 355 360 365 Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys
Ser Cys Tyr 370 375 380 Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys
Glu Pro Gly Phe Ser 385 390 395 400 Tyr Ser Glu Glu Val Cys Arg Cys
Val Pro Ser Tyr Trp Gln Arg Pro 405 410 415 Gln Met Ser 19 30 DNA
oligonucleotide 19 gcagcacata tgacagaaga gactataaaa 30 20 30 DNA
oligonucleotide 20 gcagcaggta cctcacagtt tagacatgca 30 21 30 DNA
oligonucleotide 21 gcagcaggta cctcaacgtc taataatgga 30 22 30 DNA
oligonucleotide 22 gcagcaggat cccacagaag agactataaa 30 23 30 DNA
oligonucleotide 23 gcagcatcta gatcacagtt tagacatgca 30 24 39 DNA
oligonucleotide 24 gcagcaggat cccacagaag agactataaa atttgctgc 39 25
36 DNA oligonucleotide 25 gcagcatcta gatcaacgtc taataatgga atgaac
36 26 55 DNA oligonucleotide 26 gatcgatcca tcatgcactc gctgggcttc
ttctctgtgg cgtgttctct gctcg 55 27 39 DNA oligonucleotide 27
gcagggtacg gatcctagat tagctcattt gtggtcttt 39 28 39 DNA
oligonucleotide 28 gactggatcc gccaccatgc actcgctggg cttcttctc 39 29
35 DNA oligonucleotide 29 gactggtacc ttatcacata aaatcttcct gagcc 35
30 39 DNA oligonucleotide 30 gactggatcc gccaccatgc actcgctggg
cttcttctc 39 31 34 DNA oligonucleotide 31 gactggtacc ttatcagtct
agttctttgt gggg 34 32 39 DNA oligonucleotide 32 gactggatcc
gccaccatgc actcgctggg cttcttctc 39 33 37 DNA oligonucleotide 33
gactggtacc tcattactgt ggactttctg tacattc 37 34 38 DNA
oligonucleotide 34 gcagcaggat ccacagaaga gactataaaa tttgctgc 38 35
37 DNA oligonucleotide 35 cgtcgttcta gatcacagtt tagacatgca tcggcag
37
* * * * *