U.S. patent application number 12/258140 was filed with the patent office on 2009-12-24 for vascular endothelial growth factor-2.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to RALPH ALDERSON, Robert Melder, Viktor Roschke, Craig A. Rosen, Steven M. Ruben.
Application Number | 20090317394 12/258140 |
Document ID | / |
Family ID | 34842026 |
Filed Date | 2009-12-24 |
United States Patent
Application |
20090317394 |
Kind Code |
A1 |
ALDERSON; RALPH ; et
al. |
December 24, 2009 |
VASCULAR ENDOTHELIAL GROWTH FACTOR-2
Abstract
The present invention is directed to VEGF-2 polynucleotides and
polypeptides and methods of using such polynucleotides and
polypeptides. In particular, provided are methods of treating
retinal disorders with VEGF-2 polynucleotides and polypeptides.
Inventors: |
ALDERSON; RALPH;
(Gaithersburg, MD) ; Melder; Robert; (Boyds,
MD) ; Roschke; Viktor; (Rockville, MD) ;
Ruben; Steven M.; (Brookeville, MD) ; Rosen; Craig
A.; (Laytonsville, MD) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Human Genome Sciences, Inc.
|
Family ID: |
34842026 |
Appl. No.: |
12/258140 |
Filed: |
October 24, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11078507 |
Mar 14, 2005 |
7524501 |
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12258140 |
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09499468 |
Feb 7, 2000 |
7223724 |
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11078507 |
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60119179 |
Feb 8, 1999 |
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60119926 |
Feb 12, 1999 |
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60137796 |
Jun 3, 1999 |
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60171505 |
Dec 22, 1999 |
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Current U.S.
Class: |
424/139.1 ;
435/325; 530/387.3; 530/387.9; 530/391.3 |
Current CPC
Class: |
A61K 38/1866 20130101;
C07K 16/22 20130101; C12N 2799/026 20130101; C07K 2317/74 20130101;
Y10S 514/963 20130101; Y10S 514/964 20130101; A61K 48/00 20130101;
Y10S 514/969 20130101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 530/387.3; 530/391.3; 435/325 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; C12N 5/06 20060101
C12N005/06 |
Claims
1-18. (canceled)
19. An isolated antibody or fragment thereof that specifically
binds to a polypeptide consisting of amino acid residues +9 to +80
of SEQ ID NO: 2, wherein the antibody or fragment enhances a
biological activity of VEGF2.
20. The antibody or fragment of claim 19, wherein said antibody or
fragment enhances angiogenesis, neovascularization, vascular
permeability, or endothelial cell growth in a mammal.
21. The antibody or fragment of claim 19, wherein the antibody is
assigned ATCC Accession No. PTA-435.
22. The antibody or fragment of claim 19, wherein the antibody is
assigned ATCC Accession No. PTA-199.
23. The antibody or fragment of claim 19, wherein the antibody is
assigned ATCC Accession No. PTA-201.
24. The antibody or fragment of claim 19 which is a humanized
antibody.
25. The antibody or fragment of claim 19 which is a chimeric
antibody.
26. The antibody or fragment of claim 19 which is a polyclonal
antibody.
27. The antibody or fragment of claim 19 which is a monoclonal
antibody.
28. The antibody or fragment of claim 19 which is a single chain
antibody.
29. The antibody or fragment of claim 19 which is a Fab
fragment.
30. The antibody or fragment of claim 19, comprising a detectable
label.
31. The antibody or fragment of claim 30, wherein the label is
selected from the group consisting of: (a) an enzyme; (b) a
fluorescent compound; (c) a radioactive compound; and (d) a
luminescent compound.
32. An isolated cell that produces the antibody or fragment of
claim 19.
33. A hybridoma that produces the antibody or fragment of claim
19.
34. A composition comprising an antibody or fragment of claim 19
and a pharmaceutically acceptable carrier or excipient.
35. A method of enhancing a biological activity of VEGF2 in a
patient comprising administering an isolated antibody or fragment
that specifically binds to a polypeptide consisting of amino acid
residues +9 to +80 of SEQ ID NO: 2, wherein the antibody or
fragment enhances a biological activity of VEGF2.
36. The method of claim 35, wherein said antibody or fragment
enhances angiogenesis, neovascularization, vascular permeability,
or endothelial cell growth in a mammal.
37. A method of enhancing angiogenesis in a patient comprising
administering to the patient a therapeutically effective amount of
the antibody or fragment of claim 19.
38. A method of enhancing neovascularization in a patient
comprising administering to the patient a therapeutically effective
amount of the antibody or fragment of claim 19.
39. A method of enhancing vascular permeability in a patient
comprising administering to the patient a therapeutically effective
amount of the antibody or fragment of claim 19.
40. A method of enhancing endothelial cell growth in a patient
comprising administering to the patient a therapeutically effective
amount of the antibody or fragment of claim 19.
41. A method of treating a patient having an injury to or a
disorder of an eye comprising administering to the patient a
therapeutically effective amount of the antibody or fragment of
claim 19.
42. The method of claim 41, wherein the injury or disorder is
selected from the group consisting of: age-related macular
degeneration, diabetic retinopathy, peripheral vitreoretinopathies,
photic retinopathies, surgery-induced retinopathies, viral
retinopathies, ischemic retinopathies, retinal detachment and
traumatic retinopathy.
43. The method of claim 35, wherein the patient is an animal.
44. The method of claim 43, wherein the animal is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/499,468, filed Feb. 7, 2000, which claims benefit of U.S.
Provisional Application No. 60/119,179, filed on Feb. 8, 1999, U.S.
Provisional Application No. 60/119,926, filed Feb. 12, 1999, U.S.
Provisional Application No. 60/137,796, filed Jun. 3, 1999, and
U.S. Provisional Application No. 60/171,505, filed Dec. 22, 1999.
Each of the four aforementioned applications are hereby
incorporated by reference in their entireties.
REFERENCE TO SEQUENCE LISTING ON COMPACT DISC
[0002] This application refers to a "Sequence Listing" listed
below, which is provided as an electronic document on two identical
compact discs (CD-R), labeled "Copy 1" and "Copy 2." These compact
discs each contain the file "PF112U1D1 SeqList.txt" (created Feb.
25, 2005, byte size=52,861 bytes), which is hereby 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.
Additionally, the present invention relates to antibodies directed
to the polypeptides of the present invention. The present invention
also relates to the administration of vascular endothelial growth
factor 2 (VEGF-2) polynucleotides and polypeptides to treat
disorders of or injuries to photoreceptor cells.
[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 Sep. 30, 1992.
[0014] The Retina. The differentiated retina is composed of seven
cell types: sensory (rod and cone photoreceptors), glia (Muller
cells), and two types of neurons, intemeurons, (horizontal,
bipolar, and amacrine), and projection neurons (ganglion cells).
The development of the various cell types in the retina does not
occur synchronously with the majority of the cones, and ganglion
and horizontal cells developing before birth. In contrast,
differentiation of a majority of the rods, the main cell type in
the rat retina, occurs postnatally. Clonal analysis of the progeny
of retinal precursor cells has demonstrated that these progenitor
cells can produce various combinations of retinal cell types
indicating that at least some of the progenitors are
multipotential. Furthermore, findings from both in vivo and in
vitro studies suggest that the final phenotype of the cell is
largely lineage independent which suggest that the changing
microenvironment within the retina has a role in determining the
cellular potential of the progenitor cells as well as the
differentiated phenotype of the progeny.
[0015] In vitro, retinal cell proliferation and differentiation is
regulated by a variety of factors, for example, FGF-2, CNTF, LIF,
TGF, retinoic acid, and BDNF, as well as by extracellular matrix
and cell adhesion molecules, for example s-laminin. Yang and Cepko
(J. Neurosci. 16(19):6089-6099 (1996)) and more recently Wen et al.
(J. Biol. Chem. 273(4):2090-2097(1998)) have identified and
characterized the expression pattern of VEGFR-2/FLK-1, a member of
the VEGF receptor family. VEGFR transcripts are first detected at
E11.5 in association with the developing retinal vasculature and
with the central region of the neural retina (Yang and Cepko, J.
Neurosci. 16(19):6089-6099 (1996)). Although it is not known if the
two events are related, this developmental period is also marked by
the onset of ganglion cell development. By developmental day E15,
VEGFR-2 expression extends to the periphery of the retina
consistent with the outward gradient of retinal development.
VEGFR-2 expression was largely localized to the ventricular zone
during the perinatal period when neurogenesis is at its peak and a
large number of post-mitotic neurons are being formed.
[0016] The PDGF/VEGF superfamily currently includes 7 members. The
5 members of the VEGF sub-family bind to 4 different VEGF tyrosine
kinase receptors with distinct but overlapping specificities. VEGF,
a 34-36 kDa homodimeric glycoprotein that is the prototypic family
member, binds to VEGFR-1 and VEGFR-2. VEGF-B and VEGF-D bind only
to VEGFR-1 or VEGFR-3, respectively. While VEGF-C, VEGF-2, has the
highest affinity for VEGFR-3, it also binds with a lower affinity
to VEGFR-2. Once activated the VEGF receptors tyrosine
phosphorylate a number of proteins downstream in the signal
transduction pathway including phosphatidylinositol 3-kinase,
phospholipase C, GAP, and Nck.
[0017] The hereditary retinal degenerative diseases ("HRD
diseases") are a group of inherited conditions in which
progressive, bilateral degeneration of retinal structures leads to
loss of retinal function; these diseases include, for example,
age-related macular degeneration, a leading cause of visual
impairment in the elderly; Leber's congenital amaurosis, which
causes its victims to be born blind; and retinitis pigmentosa
("RP"). RP is the name given to those inherited retinopathies which
are characterized by loss of retinal photoreceptors (rods and
cones), with retinal electrical responses to light flashes (i.e.
electroretinograms, or "ERGs") that are reduced in amplitude. As
the disease progresses, patients show attenuated retinal
arterioles, and frequently show "bone spicule" pigmentation of the
retina and waxy pallor of the optic discs.
[0018] The incidence of RP in the United States is estimated to be
about 1:3500 births. Familial cases of RP usually present in
childhood with night blindness and loss of midperipheral visual
field due to the loss of rods in the peripheral retina. As the
condition progresses, contraction of the visual fields eventually
leads to blindness. Signs on fundus examination in advanced stages
include retinal vessel attenuation, intraretinal pigment in the
peripheral fundus, and waxy pallor of the optic disc. Patients have
abnormal light-evoked electrical responses from the retina (i.e.,
electroretinograms or ERGs), even in the early stages in the
absence of visible abnormalities on fundus examination.
Histopathologic studies have revealed widespread loss of
photoreceptors in advanced stages. Therefore, there is a need in
the art for methods of treating photoreceptor cell disorders and
injuries.
SUMMARY OF THE INVENTION
[0019] The polypeptides of the present invention have been
identified as a novel vascular endothelial growth factor based on
amino acid sequence homology to human VEGF.
[0020] 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.
[0021] In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules comprising
polynucleotides encoding full length or truncated VEGF-2
polypeptides having the amino acid sequences shown in SEQ ID NOS:2
or 4, respectively, or the amino acid sequences encoded by the cDNA
clones deposited in bacterial hosts as ATCC.TM. Deposit Number
97149 on May 12, 1995 or ATCC.TM. Deposit Number 75698 on Mar. 4,
1994.
[0022] The present invention also relates to biologically active
and diagnostically or therapeutically useful fragments, analogs,
and derivatives of VEGF-2.
[0023] 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.
[0024] In accordance with yet another aspect of the present
invention, there are provided antibodies against such polypeptides
and processes for producing such antibodies.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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 protect or stimulate growth
of photoreceptor cells.
[0029] In accordance with yet another aspect of the present
invention, there are provided antagonists to such polypeptides,
which may be used, for example, to inhibit or prevent photoreceptor
growth.
[0030] 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
[0031] FIGS. 1A-1E show the full length nucleotide (SEQ ID NO:1)
and the deduced amino acid (SEQ ID NO:2) sequence of VEGF-2. The
polypeptide comprises approximately 419 amino acid residues of
which approximately 23 represent the leader sequence. The standard
one letter abbreviations for amino acids are used. Sequencing was
performed using the Model 373 Automated DNA Sequencer (Applied
Biosystems, Inc.). Sequencing accuracy is predicted to be greater
than 97%.
[0032] FIGS. 2A-2D show the nucleotide (SEQ ID NO:3) and the
deduced amino acid (SEQ ID NO:4) sequence of a truncated,
biologically active form of VEGF-2. The polypeptide comprises
approximately 350 amino acid residues of which approximately the
first 24 amino acids represent the leader sequence.
[0033] FIGS. 3A-3B are an illustration of the amino acid sequence
homology between PDGFa (SEQ ID NO:5), PDGFb (SEQ ID NO:6), VEGF
(SEQ ID NO:7), and VEGF-2 (SEQ ID NO:4). The boxed areas indicate
the conserved sequences and the location of the eight conserved
cysteine residues.
[0034] FIG. 4 shows, in table-form, the percent homology between
PDGFa, PDGFb, VEGF, and VEGF-2.
[0035] FIG. 5 shows the presence of VEGF-2 mRNA in human breast
tumor cell lines.
[0036] FIG. 6 depicts the results of a Northern blot analysis of
VEGF-2 in human adult tissues.
[0037] FIG. 7 shows a photograph of an SDS-PAGE gel after in vitro
transcription, translation and electrophoresis of the polypeptide
of the present invention. Lane 1: .sup.14C and rainbow M. W.
marker; Lane 2: FGF control; Lane 3: VEGF-2 produced by M13-reverse
and forward primers; Lane 4: VEGF-2 produced by M13 reverse and
VEGF-F4 primers; Lane 5: VEGF-2 produced by M13 reverse and VEGF-F5
primers.
[0038] FIGS. 8A and 8B depict photographs of SDS-PAGE gels. VEGF-2
polypeptide was expressed in a baculovirus system consisting of Sf9
cells. Protein from the medium and cytoplasm of cells were analyzed
by SDS-PAGE under non-reducing (FIG. 8A) and reducing (FIG. 8B)
conditions.
[0039] 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.
[0040] FIG. 10 depicts a photograph of an SDS-PAGE gel. VEGF-2 was
purified from the medium supernatant and analyzed by SDS-PAGE in
the presence or absence of the reducing agent b-mercaptoethanol and
stained by Coomassie brilliant blue.
[0041] FIG. 11 depicts reverse phase HPLC analysis of purified
VEGF-2 using a RP-300 column (0.21.times.3 cm, Applied Biosystems,
Inc.). The column was equilibrated with 0.1% trifluoroacetic acid
(Solvent A) and the proteins eluted with a 7.5 min gradient from 0
to 60% Solvent B, composed of acetonitrile containing 0.07% TFA.
The protein elution was monitored by absorbance at 215 mn ("red"
line) and 280 nm ("blue" line). The percentage of Solvent B is
shown by the "green" line.
[0042] FIG. 12 shows a schematic representation of the pHE4-5
expression vector (SEQ ID NO:9) and the subcloned VEGF-2 cDNA
coding sequence. The locations of the kanamycin resistance marker
gene, the VEGF-2 coding sequence, the oriC sequence, and the lacIq
coding sequence are indicated.
[0043] FIG. 13 shows the nucleotide sequence of the regulatory
elements of the pHE promoter (SEQ ID NO:10). The two lac operator
sequences, the Shine-Delgamo sequence (S/D), and the terminal
HindIII and NdeI restriction sites (italicized) are indicated.
[0044] FIG. 14A-D shows that VEGF-2 treatment increases the level
of rhodopsin protein and the number of photoreceptor cells.
Dissociated retinal cells were prepared from P1 animals, plated at
a density of 425 cells/mm.sup.2 and treated with VEGF-2 (A and B)
or VEGF-2 (C and D). After 2 (open squares), 5 (solid squares), 7
(open circles), or 9 (solid squares) days, the total number of
cells in the cultures was estimated by measuring the calcein
emission. The cultures were then fixed and the levels of rhodopsin
protein quantitated by ELISA.
[0045] FIG. 15 shows that the number of rhodopsin immunopositive
cells increased as a function of VEGF-2 concentration. The retinal
cells were maintained in vitro for 8 days in the presence of either
VEGF-1 or VEGF-2. The cultures were then fixed and
immunohistochemically stained for rhodopsin.
[0046] FIG. 16A-C shows that VEGF-2 increases BrdU and [3H]
thymidine incorporation in retinal cultures in a developmentally
restricted manner. The cells were isolated from P1 animals and
plated at a density of 425 cells/mm.sup.2. The cultures were
initially treated 4 hours after plating with either VEGF or VEGF-2.
After 1, 2, or 3 days, the cultures were labeled for 4 hours with
BrdU. The cells were then fixed and processed for BrdU
immunohistochemistry.
[0047] FIG. 17A-B shows the loss of the response to VEGF-2 or
VEGF-1 as a function of the time lapsed between the isolation of
the cells and the initial addition of the factors. One set of
cultures was initially treated with factors 4 hours after plating
(9/0) and subsequently, additional sets were treated after 24 or 48
hours (8/1 or 7/2, respectively). After 9 days in culture, the
cells were fixed and the level of rhodopsin protein was quantitated
by ELISA assay.
[0048] FIG. 18A-C shows VEGF increases the number of Amacrine but
not Muller or Endothelial cells. Retinal cells were treated for 8
days with the indicated concentrations of VEGF-2. The cells were
then fixed and immunohistochemically stained for syntaxin (A),
analyzed for the level of high-affinity GABA uptake (B), or GFAP
(C).
[0049] FIG. 19A-C shows the effect of developmental age on the
response to VEGF-2. Retinal cells derived from E15 (A), E20 (B) or
P1 (C) animals were plated at a density of 212 (open squares), 318
(solid squares), or 425 cells/mm.sup.2. Four hours after plating,
the cultures were treated with the indicated concentrations of
VEGF-2. After 24 hours, the cultures were switched to serum-free
medium and the factors were added again. The cultures were then
labeled with [3H] thymidine after 48 hours.
[0050] FIG. 20A-B compares the response of retinal cells to VEGF-2
and other factors. The cultures were seeded at a density of 425
cells/mm.sup.2 and treated for 9 days. Panel A shows the total
number of cells in the cultures was estimated using calcein, while
panel B shows the level of rhodopsin protein determined by ELISA
assay.
[0051] FIG. 21A-C shows that CNTF inhibits the response of the
photoreceptor cell progenitors to VEGF-2. Retinal cultures were
treated 24 hours after plating with the indicated concentrations of
CNTF in the presence or absence of 150 ng/ml of VEGF-2. After 8
days in vitro, the amount of rhodopsin protein was quantitated (A)
and the total number of cells in the cultures was determined (B).
(C) To determine the effect of CNTF treatment on the early
proliferative response induced by VEGF-1, the cultures were treated
with the indicated concentrations of VEGF-2 in the presence or
absence of 100 ng/ml CNTF. After 48 hours, the cultures were
labeled for 4 hours with [3H] thymidine.
[0052] FIG. 22 shows the enhanced LEC proliferation in response to
VEGF-2 and antibody treatment.
[0053] FIG. 23 shows LEC proliferation in response to VEGF-2 and
VEGF-2:antibody combination.
[0054] FIG. 24 shows the epitope map for murine anti VEGF-2
monoclonal antibodies.
[0055] FIG. 25 shows the status of the murine VEGF-2 monoclonal
antibodies.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0056] In accordance with one aspect of the present invention,
there are provided isolated nucleic acid molecules comprising a
polynucleotide encoding a VEGF-2 polypeptide having the deduced
amino acid sequence of FIG. 1 (SEQ ID NO:2), which was determined
by sequencing a cloned cDNA. The nucleotide sequence shown in SEQ
ID NO:1 was obtained by sequencing a cDNA clone, which was
deposited on May 12, 1995 at the American Type Tissue Collection
(ATCC.TM.), 10801 University Boulevard, Manassas, Va. 20110-2209,
and given ATCC.TM. Deposit No. 97149.
[0057] In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules comprising a
polynucleotide encoding a truncated VEGF-2 polypeptide having the
deduced amino acid sequence of FIG. 2 (SEQ ID NO:4), which was
determined by sequencing a cloned cDNA. The nucleotide sequence
shown in SEQ ID NO:3 was obtained by sequencing a cDNA clone, which
was deposited on Mar. 4, 1994 at the American Type Tissue
Collection (ATCC.TM.), 10801 University Boulevard, Manassas, Va.
20110-2209, and given ATCC.TM. Deposit Number 75698.
[0058] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
[0059] A polynucleotide encoding a polypeptide of the present
invention may be obtained from early stage human embryo (week 8 to
9) osteoclastomas, adult heart or several breast cancer cell lines.
The polynucleotide of this invention was discovered in a cDNA
library derived from early stage human embryo week 9. It is
structurally related to the VEGF/PDGF family. It contains an open
reading frame encoding a protein of about 419 amino acid residues
of which approximately the first 23 amino acid residues are the
putative leader sequence such that the mature protein comprises 396
amino acids, and which protein exhibits the highest amino acid
sequence homology to human vascular endothelial growth factor (30%
identity), followed by PDGFa (24%) and PDGFb (22%). (See FIG. 4).
It is particularly important that all eight cysteines are conserved
within all four members of the family (see boxed areas of FIG. 3).
In addition, the signature for the PDGF/VEGF family,
PXCVXXXRCXGCCN, (SEQ ID NO:8) is conserved in VEGF-2 (see FIG. 3).
The homology between VEGF-2, VEGF and the two PDGFs is at the
protein sequence level. No nucleotide sequence homology can be
detected, and therefore, it would be difficult to isolate the
VEGF-2 through simple approaches such as low stringency
hybridization.
[0060] The VEGF-2 polypeptide of the present invention is meant to
include the full length polypeptide and polynucleotide sequence
which encodes for any leader sequences and for active fragments of
the full length polypeptide. Active fragments are meant to include
any portions of the full length amino acid sequence which have less
than the full 419 amino acids of the full length amino acid
sequence as shown in SEQ ID NO:2, but still contain the eight
cysteine residues shown conserved in FIG. 3 and that still have
VEGF-2 activity.
[0061] There are at least two alternatively spliced VEGF-2 MRNA
sequences present in normal tissues. The two bands in FIG. 7, lane
5 indicate the presence of the alternatively spliced mRNA encoding
the VEGF-2 polypeptide of the present invention.
[0062] The polynucleotide of the present invention may be in the
form of RNA or in the form of DNA, which DNA includes cDNA, genomic
DNA, and synthetic DNA. The DNA may be double-stranded or
single-stranded, and if single stranded may be the coding strand or
non-coding (anti-sense) strand. The coding sequence which encodes
the mature polypeptide may be identical to the coding sequence
shown in FIG. 1 or FIG. 2, or that of the deposited clones, or may
be a different coding sequence which, as a result of the redundancy
or degeneracy of the genetic code, encodes the same, mature
polypeptide as the DNA of FIG. 1, FIG. 2, or the deposited
cDNAs.
[0063] The polynucleotide which encodes for the mature polypeptide
of FIG. 1 or FIG. 2 or for the mature polypeptides encoded by the
deposited cDNAs may include: only the coding sequence for the
mature polypeptide; the coding sequence for the mature polypeptide
and additional coding sequences such as a leader or secretory
sequence or a proprotein sequence; the coding sequence for the
mature polypeptide (and optionally additional coding sequences) and
non-coding sequences, such as introns or non-coding sequence 5'
and/or 3' of the coding sequence for the mature polypeptide.
[0064] Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding sequences
for the polypeptide as well as a polynucleotide which includes
additional coding and/or non-coding sequences.
[0065] The present invention further relates to variants of the
hereinabove described polynucleotides which encode for fragments,
analogs, and derivatives of the polypeptide having the deduced
amino acid sequence of 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.
[0066] 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.
[0067] 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.
[0068] The present invention also includes polynucleotides, wherein
the coding sequence for the mature polypeptide may be fused in the
same reading frame to a polynucleotide which aids in expression and
secretion of a polypeptide from a host cell, for example, a leader
sequence which functions as a secretory sequence for controlling
transport of a polypeptide from the cell. The polypeptide having a
leader sequence is a preprotein and may have the leader sequence
cleaved by the host cell to form the mature form of the
polypeptide. The polynucleotides may also encode for a proprotein
which is the mature protein plus additional 5' amino acid residues.
A mature protein having a prosequence is a proprotein and is an
inactive form of the protein. Once the prosequence is cleaved an
active mature protein remains.
[0069] Thus, for example, the polynucleotide of the present
invention may encode for a mature protein, or for a protein having
a prosequence or for a protein having both a prosequence and
presequence (leader sequence).
[0070] The polynucleotides of the present invention may also have
the coding sequence fused in frame to a marker sequence which
allows for purification of the polypeptide of the present
invention. The marker sequence may be a hexa-histidine tag supplied
by a pQE-9 vector to provide for purification of the mature
polypeptide fused to the marker in the case of a bacterial host,
or, for example, the marker sequence may be a hemagglutinin (HA)
tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson, I., et al., Cell 37:767 (1984)).
[0071] Further embodiments of the invention include isolated
nucleic acid molecules comprising a polynucleotide having a
nucleotide sequence at least 95% identical, and more preferably at
least 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence
encoding the polypeptide having the amino acid sequence in SEQ ID
NO:2; (b) a nucleotide sequence encoding the polypeptide having the
amino acid sequence in SEQ ID NO:2, but lacking the N-terminal
methionine; (c) a nucleotide sequence encoding the polypeptide
having the amino acid sequence at positions from about 1 to about
396 in SEQ ID NO:2; (d) a nucleotide sequence encoding the
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC.TM. Deposit No. 97149; (e) a nucleotide
sequence encoding the mature VEGF-2 polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No. 97149; or (f) a nucleotide sequence complementary to
any of the nucleotide sequences in (a), (b), (c), (d), or (e).
[0072] Further embodiments of the invention include isolated
nucleic acid molecules comprising a polynucleotide having a
nucleotide sequence at least 95% identical, and more preferably at
least 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence
encoding the polypeptide having the amino acid sequence in SEQ ID
NO:4; (b) a nucleotide sequence encoding the polypeptide having the
amino acid sequence in SEQ ID NO:4, but lacking the N-terminal
methionine; (c) a nucleotide sequence encoding the polypeptide
having the amino acid sequence at positions from about 1 to about
326 in SEQ ID NO:4; (d) a nucleotide sequence encoding the
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC.TM. Deposit No. 75698; (e) a nucleotide
sequence encoding the mature VEGF-2 polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No. 75698; or (f) a nucleotide sequence complementary to
any of the nucleotide sequences in (a), (b), (c), (d), or (e).
[0073] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a VEGF-2 polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence encoding the VEGF-2 polypeptide. In other
words, to obtain a polynucleotide having a nucleotide sequence at
least 95% identical to a reference nucleotide sequence, up to 5% of
the nucleotides in the reference sequence may be deleted or
substituted with another nucleotide, or a number of nucleotides up
to 5% of the total nucleotides in the reference sequence may be
inserted into the reference sequence. These mutations of the
reference sequence may occur at the 5N or 3N terminal positions of
the reference nucleotide sequence or anywhere between those
terminal positions, interspersed either individually among
nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0074] As a practical matter, whether any particular nucleic acid
molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for
instance, the nucleotide sequence shown in SEQ ID NOS:1 or 3, or to
the nucleotides sequence of the deposited cDNA clone(s) can be
determined conventionally using known computer programs such as the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). Bestfit uses the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2: 482-489 (1981), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set, of course, such that
the percentage of identity is calculated over the full length of
the reference nucleotide sequence and that gaps in homology of up
to 5% of the total number of nucleotides in the reference sequence
are allowed.
[0075] The VEGF-2 variants may contain alterations in the coding
regions, non-coding regions, or both. Especially preferred are
polynucleotide variants containing alterations which produce silent
substitutions, additions, or deletions, but do not alter the
properties or activities of the encoded polypeptide. Nucleotide
variants produced by silent substitutions due to the degeneracy of
the genetic code are preferred. Moreover, variants in which 5-10,
1-5, or 1-2 amino acids are substituted, deleted, or added in any
combination are also preferred. VEGF-2 polynucleotide variants can
be produced for a variety of reasons, e.g., to optimize codon
expression for a particular host (change codons in the human mRNA
to those preferred by a bacterial host such as E. coli).
[0076] For example, site directed changes at the amino acid level
of VEGF-2 can be made by replacing a particular amino acid with a
conservative amino acid. Preferred conservative mutations include:
M1 replaced with A, G, I, L, S, T, or V; H2 replaced with K, or R;
S3 replaced with A, G, I, L, T, M, or V; L4 replaced with A, G, I,
S, T, M, or V; G5 replaced with A, I, L, S, T, M, or V; F6 replaced
with W, or Y; F7 replaced with W, or Y; S8 replaced with A, G, I,
L, T, M, or V; V9 replaced with A, G, I, L, S, T, or M; A10
replaced with G, I, L, S, T, M, or V; S12 replaced with A, G, I, L,
T, M, or V; L13 replaced with A, G, I, S, T, M, or V; L14 replaced
with A, G, I, S, T, M, or V; A15 replaced with G, I, L, S, T, M, or
V; A16 replaced with G, I, L, S, T, M, or V; A17 replaced with G,
I, L, S, T, M, or V; L18 replaced with A, G, I, S, T, M, or V; L19
replaced with A, G, I, S, T, M, or V; G21 replaced with A, I, L, S,
T, M, or V; R23 replaced with H, or K; E24 replaced with D; A25
replaced with G, I, L, S, T, M, or V; A27 replaced with G, I, L, S,
T, M, or V; A28 replaced with G, I, L, S, T, M, or V; A29 replaced
with G, I, L, S, T, M, or V; A30 replaced with G, I, L, S, T, M, or
V; A31 replaced with G, I, L, S, T, M, or V; F32 replaced with W,
or Y; E33 replaced with D; S34 replaced with A, G, I, L, T, M, or
V; G35 replaced with A, I, L, S, T, M, or V; L36 replaced with A,
G, I, S, T, M, or V; D37 replaced with E; L38 replaced with A, G,
I, S, T, M, or V; S39 replaced with A, G, I, L, T, M, or V; D40
replaced with E; A41 replaced with G, I, L, S, T, M, or V; E42
replaced with D; D44 replaced with E; A45 replaced with G, I, L, S,
T, M, or V; G46 replaced with A,I, L, S, T, M, or V; E47 replaced
with D; A48 replaced with G, I, L, S, T, M, or V; T49 replaced with
A, G, I, L, S, M, or V; A50 replaced with G, I, L, S, T, M, or V;
Y51 replaced with F, or W; A52 replaced with G, I, L, S, T, M, or
V; S53 replaced with A, G, I, L, T, M, or V; K54 replaced with H,
or R; D55 replaced with E; L56 replaced with A, G, I, S, T, M, or
V; E57 replaced with D; E58 replaced with D; Q59 replaced with N;
L60 replaced with A, G, I, S, T, M, or V; R61 replaced with H, or
K; S62 replaced with A, G, I, L, T, M, or V; V63 replaced with A,
G, I, L, S, T, or M; S64 replaced with A, G, I, L, T, M, or V; S65
replaced with A, G, I, L, T, M, or V; V66 replaced with A, G, I, L,
S, T, or M; D67 replaced with E; E68 replaced with D; L69 replaced
with A, G, I, S, T, M, or V; M70 replaced with A, G, I, L, S, T, or
V; T71 replaced with A, G, I, L, S, M, or V; V72 replaced with A,
G, I, L, S, T, or M; L73 replaced with A, G, I, S, T, M, or V; Y74
replaced with F, or W; E76 replaced with D; Y77 replaced with F, or
W; W78 replaced with F, or Y; K79 replaced with H, or R; M80
replaced with A, G, I, L, S, T, or V; Y81 replaced with F, or W;
K82 replaced with H, or R; Q84 replaced with N; L85 replaced with
A, G, I, S, T, M, or V; R86 replaced with H, or K; K87 replaced
with H, or R; G88 replaced with A, I, L, S, T, M, or V; G89
replaced with A, I, L, S, T, M, or V; W90 replaced with F, or Y;
Q91 replaced with N; H92 replaced with K, or R; N93 replaced with
Q; R94 replaced with H, or K; E95 replaced with D;Q96 replaced with
N; A97 replaced with G, I, L, S, T, M, or V; N98 replaced with Q;
L99 replaced with A, G, I, S, T, M, or V; N100 replaced with Q;
S101 replaced with A, G, I, L, T, M, or V; R102 replaced with H, or
K; T103 replaced with A, G, I, L, S, M, or V; E104 replaced with D;
E105 replaced with D;T106 replaced with A, G, I, L, S, M, or V;
I107 replaced with A, G, L, S, T, M, or V; K108 replaced with H, or
R; F109 replaced with W, or Y; A110 replaced with G, I, L, S, T, M,
or V; A111 replaced with G, I, L, S, T, M, or V; A112 replaced with
G, I, L, S, T, M, or V; H113 replaced with K, or R;Y114 replaced
with F, or W; N115 replaced with Q; T116 replaced with A, G, I, L,
S, M, or V; E117 replaced with D; I118 replaced with A, G, L, S, T,
M, or V; L119 replaced with A, G, I, S, T, M, or V; K120 replaced
with H, or R; S121 replaced with A, G, I, L, T, M, or V; I122
replaced with A, G, L, S, T, M, or V; D123 replaced with E; N124
replaced with Q; E125 replaced with D; W126 replaced with F, or Y;
R127 replaced with H, or K; K128 replaced with H, or R; T129
replaced with A, G, I, L, S, M, or V; Q130 replaced with N; M132
replaced with A, G, I, L, S, T, or V; R134 replaced with H, or K;
E135replaced with D; V136 replaced with A, G, I, L, S, T, or M;
I138 replaced with A, G, L, S, T, M, or V; D139 replaced with E;
V140 replaced with A, G, I, L, S, T, or M; G141 replaced with A, I,
L, S, T, M, or V; K142 replaced with H, or R; E143 replaced with D;
F144 replaced with W, or Y; G145 replaced with A, I, L, S, T, M, or
V; V146 replaced with A, G, I, L, S, T, or M; A147 replaced with G,
I, L, S, T, M, or V; T148 replaced with A, G, I, L, S, M, or V;
N149 replaced with Q; T150 replaced with A, G, I, L, S, M, or V;
F151 replaced with W, or Y; F152 replaced with W, or Y; K153
replaced with H, or R;V157 replaced with A, G, I, L, S, T, or M;
S158 replaced with A, G, I, L, T, M, or V; V159 replaced with A, G,
I, L, S, T, or M; Y160 replaced with F, or W; R161 replaced with H,
or K; G163 replaced with A, I, L, S, T, M, or V; G164 replaced with
A, I, L, S, T, M, or V; N167 replaced with Q; S168 replaced with A,
G, I, L, T, M, or V; E169 replaced with D; G170 replaced with A, I,
L, S, T, M, or V; L171 replaced with A, G, I, S, T, M, or V; Q172
replaced with N; M174 replaced with A, G, I, L, S, T, or V; N175
replaced with Q; T176 replaced with A, G, I, L, S, M, or V; S177
replaced with A, G, I, L, T, M, or V; T178 replaced with A, G, I,
L, S, M, or V; S179 replaced with A, G, I, L, T, M, or V; Y180
replaced with F, or W; L181 replaced with A, G, I, S, T, M, or V;
S182 replaced with A, G, I, L, T, M, or V; K183 replaced with H, or
R; T184 replaced with A, G, I, L, S, M, or V; L185 replaced with A,
G, I, S, T, M, or V; F186 replaced with W, or Y; E187 replaced with
D; I188 replaced with A, G, L, S, T, M, or V; T189 replaced with A,
G, I, L, S, M, or V; V190 replaced with A, G, I, L, S, T, or M;
L192 replaced with A, G, I, S, T, M, or V; S193 replaced with A, G,
I, L, T, M, or V; Q194 replaced with N; G195 replaced with A, I, L,
S, T, M, or V; K197 replaced with H, or R; V199 replaced with A, G,
I, L, S, T, or M; T200 replaced with A, G, I, L, S, M, or V; I201
replaced with A, G, L, S, T, M, or V; S202 replaced with A, G, I,
L, T, M, or V; F203 replaced with W, or Y; A204 replaced with G, I,
L, S, T, M, or V; N205 replaced with Q; H206 replaced with K, or R;
T207 replaced with A, G, I, L, S, M, or V; S208 replaced with A, G,
I, L, T, M, or V; R210 replaced with H, or K; M212 replaced with A,
G, I, L, S, T, or V; S213 replaced with A, G, I, L, T, M, or V;
K214 replaced with H, or R; L215 replaced with A, G, I, S, T, M, or
V; D216 replaced with E; V217 replaced with A, G, I, L, S, T, or M;
Y218 replaced with F, or W; R219 replaced with H, or K;Q220
replaced with N; V221 replaced with A, G, I, L, S, T, or M; H222
replaced with K, or R; S223 replaced with A, G, I, L, T, M, or V;
I224 replaced with A, G, L, S, T, M, or V; I225 replaced with A, G,
L, S, T, M, or V; R226 replaced with H, or K; R227 replaced with H,
or K; S228 replaced with A, G, I, L, T, M, or V; L229 replaced with
A, G, I, S, T, M, or V; A231 replaced with G, I, L, S, T, M, or V;
T232 replaced with A, G, I, L, S, M, or V; L233 replaced with A, G,
I, S, T, M, or V; Q235 replaced with N; Q237 replaced with N; A238
replaced with G, I, L, S, T, M, or V; A239 replaced with G, I, L,
S, T, M, or V; N240 replaced with Q; K241 replaced with H, or R;
T242 replaced with A, G, I, L, S, M, or V; T245 replaced with A, G,
I, L, S, M, or V;N246 replaced with Q; Y247 replaced with F, or W;
M248 replaced with A, G, I, L, S, T, or V; W249 replaced with F, or
Y; N250 replaced with Q; N251 replaced with Q; H252 replaced with
K, or R; 1253 replaced with A, G, L, S, T, M, or V; R255 replaced
with H, or K; L257 replaced with A, G, I, S, T, M, or V; A258
replaced with G, I, L, S, T, M, or V; Q259 replaced with N; E260
replaced with D; D261 replaced with E; F262 replaced with W, or Y;
M263 replaced with A, G, I, L, S, T, or V; F264 replaced with W, or
Y; S265 replaced with A, G, I, L, T, M, or V; S266 replaced with A,
G, I, L, T, M, or V;D267 replaced with E; A268 replaced with G, I,
L, S, T, M, or V; G269 replaced with A, I, L, S, T, M, or V; D270
replaced with E; D271 replaced with E;S272 replaced with A, G, I,
L, T, M, or V; T273 replaced with A, G, I, L, S, M, or V; D274
replaced with E; G275 replaced with A, I, L, S, T, M, or V;F276
replaced with W, or Y; H277 replaced with K, or R; D278 replaced
with E; I279 replaced with A, G, L, S, T, M, or V; G281 replaced
with A, I, L, S, T, M, or V; N283 replaced with Q; K284 replaced
with H, or R; E285 replaced with D; L286 replaced with A, G, I, S,
T, M, or V; D287 replaced with E; E288 replaced with D; E289
replaced with D; T290 replaced with A, G, I, L, S, M, or V; Q292
replaced with N; V294 replaced with A, G, I, L, S, T, or M; R296
replaced with H, or K; A297 replaced with G, I, L, S, T, M, or V;
G298 replaced with A, I, L, S, T, M, or V; L299 replaced with A, G,
I, S, T, M, or V; R300 replaced with H, or K; A302 replaced with G,
I, L, S, T, M, or V; S303 replaced with A, G, I, L, T, M, or V;
G305 replaced with A, I, L, S, T, M, or V; H307 replaced with K, or
R; K308 replaced with H, or R; E309 replaced with D; L310 replaced
with A, G, I, S, T, M, or V; D311 replaced with E;R312 replaced
with H, or K; N313 replaced with Q; S314 replaced with A, G, I, L,
T, M, or V; Q316 replaced with N; V318 replaced with A, G, I, L, S,
T, or M; K320 replaced with H, or R; N321 replaced with Q; K322
replaced with H, or R; L323 replaced with A, G, I, S, T, M, or V;
F324 replaced with W, or Y; S326 replaced with A, G, I, L, T, M, or
V; Q327 replaced with N; G329 replaced with A, I, L, S, T, M, or V;
A330 replaced with G, I, L, S, T, M, or V; N331 replaced with Q;
R332 replaced with H, or K; E333 replaced with D; F334 replaced
with W, or Y; D335 replaced with E; E336 replaced with D; N337
replaced with Q; T338 replaced with A, G, I, L, S, M, or V; Q340
replaced with N; V342 replaced with A, G, I, L, S, T, or M; K344
replaced with H, or R; R345 replaced with H, or K; T346 replaced
with A, G, I, L, S, M, or V; R349 replaced with H, or K; N350
replaced with Q; Q351 replaced with N; L353replaced with A, G, I,
S, T, M, or V; N354 replaced with Q; G356 replaced with A, I, L, S,
T, M, or V; K357 replaced with H, or R; A359 replaced with G, I, L,
S, T, M, or V; E361 replaced with D; T363 replaced with A, G, I, L,
S, M, or V; E364 replaced with D; S365 replaced with A, G, I, L, T,
M, or V; Q367 replaced with N; K368 replaced with H, or R; L370
replaced with A, G, I, S, T, M, or V; L371 replaced with A, G, I,
S, T, M, or V; K372 replaced with H, or R; G373 replaced with A, I,
L, S, T, M, or V; K374 replaced with H, or R; K375 replaced with H,
or R; F376 replaced with W, or Y; H377replaced with K, or R; H378
replaced with K, or R; Q379 replaced with N; T380 replaced with A,
G, I, L, S, M, or V; S382 replaced with A, G, I, L, T, M, or V;
Y384 replaced with F, or W; R385 replaced with H, or K; R386
replaced with H, or K; T389 replaced with A, G, I, L, S, M, or V;
N390 replaced with Q; R391 replaced with H, or K; Q392 replaced
with N; K393 replaced with H, or R; A394 replaced with G, I, L, S,
T, M, or V; E396 replaced with D; G398 replaced with A, I, L, S, T,
M, or V; F399 replaced with W, or Y; S400 replaced with A, G, I, L,
T, M, or V; Y401 replaced with F, or W; S402 replaced with A, G, I,
L, T, M, or V; E403 replaced with D; E404 replaced with D; V405
replaced with A, G, I, L, S, T, or M; R407 replaced with H, or K;
V409 replaced with A, G, I, L, S, T, or M; S411 replaced with A, G,
I, L, T, M, or V; Y412 replaced with F, or W; W413 replaced with F,
or Y; Q414 replaced with N; R415 replaced with H, or K; Q417
replaced with N; M418 replaced with A, G, I, L, S, T, or V; and/or
S419 replaced with A, G, I, L, T, M, or V of FIGS. 1A-1E.
[0077] The resulting constructs can be routinely screened for
activities or functions described throughout the specification and
known in the art. Preferably, the resulting constructs have an
increased and/or a decreased VEGF-2 activity or function, while the
remaining VEGF-2 activities or functions are maintained. More
preferably, the resulting constructs have more than one increased
and/or decreased VEGF-2 activity or function, while the remaining
VEGF-2 activities or functions are maintained.
[0078] Besides conservative amino acid substitution, variants of
VEGF-2 include (i) substitutions with one or more of the
non-conserved amino acid residues, where the substituted amino acid
residues may or may not be one encoded by the genetic code, or (ii)
substitution with one or more of amino acid residues having a
substituent group, or (iii) fusion of the mature polypeptide with
another compound, such as a compound to increase the stability
and/or solubility of the polypeptide (for example, polyethylene
glycol), or (iv) fusion of the polypeptide with additional amino
acids, such as, for example, an IgG Fc fusion region peptide, or
leader or secretory sequence, or a sequence facilitating
purification. Such variant polypeptides are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0079] For example, VEGF-2 polypeptide variants containing amino
acid substitutions of charged amino acids with other charged or
neutral amino acids may produce proteins with improved
characteristics, such as less aggregation. Aggregation of
pharmaceutical formulations both reduces activity and increases
clearance due to the aggregate's immunogenic activity. (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).)
For example, preferred non-conservative substitutions of VEGF-2
include: M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H2
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
S3 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L4 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G5 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; F6 replaced with D, E, H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; F7 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; S8 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; V9 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; A10 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; C11 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or P; S12 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L13 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A15
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A16 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A17 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L18 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; L19 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; P20 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or C; G21 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; P22 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; R23 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; E24 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A25 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P26 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A27 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; A28 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; A29 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; A30 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; A31 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; F32 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; E33 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; S34 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; G35 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L36 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D37
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; L38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S39
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D40 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A41
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E42 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P43
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; D44 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; A45 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G46 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E47
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; A48 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T49
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A50 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y51 replaced with D, E,
H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A52 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; S53 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; K54 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; D55 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L56 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; E57 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E58 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q59 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L60
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R61 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S62
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V63 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S64 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; S65 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; V66 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; D67 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; E68 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; L69 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; M70 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; T71 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; V72 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L73
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y74 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; P75
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; E76 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; Y77 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; W78 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; K79 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; M80 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; Y81 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T, M, V, P, or C; K82replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; C83 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q84 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L85 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; R86 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K87 replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G88 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G89 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; W90 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; Q91 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; H92 replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N93 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R94
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E95 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; Q96 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; A97 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; N98 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; L99 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; N100 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; S101 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; R102 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; T103 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; E104 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; E105 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; T106 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; I107 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; K108 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; F109 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or C; A110 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; A111 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H113
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
Y114 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; N 115 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; T116 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; E117 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; I118 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K120 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; S121 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I122 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D123
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; N124 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,
Y, P, or C; E125 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; W126 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; R127 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; K128 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; T129 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; Q130 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; C131 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; M132 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P133 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R134
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E135 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; V136 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
C137 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or P; I138 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D139 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; V140 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; G141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K142
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E143 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; F144 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C; G145 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; V146 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A147 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T148
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N149 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T150
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F151 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F152
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
K153 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; P154 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; P155 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; C156 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V157 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; S158 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; V159 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; Y160 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; R161 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; C162 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; G163 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; G164 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; C165 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; C166 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; N167 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S168 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E169 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G170 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L171 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; Q172 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C173 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; M174
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N175 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T176
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S177 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T178 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; S179 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; Y180 replaced with D, E, H, K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; L181 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; S182 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; K183 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; T184 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; L185 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; F186 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; E187 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; I188 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; T189 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V190 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P191
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; L192 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S193 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q194
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; G195 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P196
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; K197 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; P198 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or C; V199 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; T200 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; I201 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S202 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F203
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
A204 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N205
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; H206 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; T207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S208 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C209
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; R210 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; C211 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or P; M212 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; S213 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; K214 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; L215 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D216 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; V217 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; Y218 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; R219 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; Q220 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; V221 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; H222 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; S223 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; I224 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; I225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R226
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
R227 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; S228 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L229 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P230
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; A231 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T232 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L233
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P234 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
Q235 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; C236 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or P; Q237 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; A238 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; A239 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; N240 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; K241 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; T242 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; C243 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; P244 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; T245 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; N246 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; Y247 replaced with D, E,
H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; M248 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; W249 replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; N250 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N251 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; H252
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
I253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C254
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; R255 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; C256 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or P; L257 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; A258 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; Q259 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; E260 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; D261 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; F262 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; M263 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; F264 replaced with D, E, H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; S265 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; S266 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; D267 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; A268 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; G269 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; D270 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; D271 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; S272
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T273 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D274 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G275 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; F276 replaced with D,
E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; H277 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D278
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; I279 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C280
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P;G281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P282
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; N283 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; K284 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; E285 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; L286 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; D287 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; E288 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; E289 replaced with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T290 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; C291 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q292 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C293
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; V294 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
C295 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or P; R296 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; A297 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; G298 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L299 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R300
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
P301 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; A302 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S303 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C304
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; G305 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P306 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; H307 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; K308 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; E309 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; L310 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; D311 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; R312 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; N313 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S314 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; C315 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q316 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C317
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; V318 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
C319 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or P; K320 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; N321 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; K322 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; L323 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; F324 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T, M, V, P, or C; P325 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S326 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; Q327 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; C328 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; G329 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A330 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; N331 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R332 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E333 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F334
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
D335 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; E336 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; N337 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; T338 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; C339 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; Q340 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; C341 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V342 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; C343 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; K344
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
R345 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; T346 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
C347 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or P; P348 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or C; R349 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; N350 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; Q351 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P352 replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L353
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N354 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P355
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; G356 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K357 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; C358 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or P; A359 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; C360 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or P; E361 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; C362 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T363 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; E364 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S365 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; P366 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Q367 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K368 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C369
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; L370 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L371 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K372
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
G373 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K374
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
K375 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; F376 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; H377 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; H378 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; Q379 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; T380 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; C381 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or P; S382 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; C383 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; Y384 replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R385 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R386 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P387
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; C388 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or P; T389 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; N390 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; R391 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; Q392 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; K393 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; A394 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; C395 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; E396 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P397 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
G398 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F399
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
S400 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y401
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
S402 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E403
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; E404 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; V405 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; C406 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or P; R407 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; C408 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; V409 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; P410 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; S411 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; Y412 replaced with D, E, H, K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; W413 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; Q414 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R415 replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P416 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
Q417 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; M418 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
and/or S419 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
of
[0080] FIGS. 1A-1E.
[0081] The resulting constructs can be routinely screened for
activities or functions described throughout the specification and
known in the art. Preferably, the resulting constructs have an
increased and/or decreased VEGF-2 activity or function, while the
remaining VEGF-2 activities or functions are maintained. More
preferably, the resulting constructs have more than one increased
and/or decreased VEGF-2 activity or function, while the remaining
VEGF-2 activities or functions are maintained.
[0082] 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 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).
[0083] 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).
[0084] 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. et al., (1983) 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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) G 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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)). 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.
[0094] 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
[0095] 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.
[0096] In addition, protein engineering may be employed in order to
improve or alter one or more characteristics of native VEGF-2. The
deletion of carboxy terminal amino acids can enhance the activity
of proteins. One example is interferon gamma that shows up to ten
times higher activity by deleting ten amino acid residues from the
carboxy terminus of the protein (Dobeli et al., J. of Biotechnology
7:199-216 (1988)). Thus, one aspect of the invention is to provide
polypeptide analogs of VEGF-2 and nucleotide sequences encoding
such analogs that exhibit enhanced stability (e.g., when exposed to
typical pH, thermal conditions or other storage conditions)
relative to the native VEGF-2 polypeptide. Particularly, preferred
VEGF-2 polypeptides are shown below (numbering starts with the
first amino acid in the protein (Met) (FIG. 1 (SEQ ID NO:2)):
[0097] Ala (residue 24 25) to Ser (residue 419); Pro (25 26) to Ser
(419); Ala (26 27) to Ser (419); Ala (27 28) to Ser (419); Ala (28
29) to Ser (419); Ala (29 30) to Ser (419); Ala (30 31) to Ser
(419); Phe (31 32) to Ser (419); Glu (32 33) to Ser (419); Ser (33
34) to Ser (419); Gly (34 35) to Ser (419); Leu (35 36) to Ser
(419); Asp (36 37) to Ser (419); Leu (37 38) to (Ser (419); Ser (38
39) to Ser (419); Asp (39 40) to Ser (419); Ala (40 41) to Ser
(419); Glu (41 42) to Ser (419); Pro (42 43) to Ser (419); Asp (43
44) to Ser (419); Ala (44 45) to Ser (419); Gly (45 46) to Ser
(419); Glu (46 47) to Ser (419); Ala (47 48) to Ser (419); Thr (48
49) to Ser (419); Ala (49 50) to Ser (419); Tyr (50 51) to Ser
(419); Ser (52 53) to Ser (419); Asp (54 55) to Ser (419); Val (62
63) to Ser (419); Val (65 66) to Ser (419); Met(1), Glu (23 24), or
Ala (24 25) to Met (418); Met (1), Glu (23 24), or Ala (24 25) to
Gln (417); Met (1), Glu (23 24), or Ala (24 25) to Pro (416);
Met(1), Glu (23 24), or Ala (24 25) to Arg (415); Met(1), Glu (23
24), or Ala (24 25) to Gln (414); Met(1), Glu (23 24), or Ala (24
25) to Trp (413); Met(1), Glu (23 24), or Ala (24 25) to Tyr (412);
Met(1), Glu (23 24), or Ala (24 25) to Ser (411); Met(1), Glu (23
24), or Ala (24 25) to Pro (410); Met(1), Glu (23 24), or Ala (24
25) to Val (409); Met(1), Glu (23 24), or Ala (24 25) to Cys (408);
Met(1), Glu (23 24), or Ala (24 25) to Arg (407); Met(1), Glu (23
24), or Ala (24 25) to Cys (406); Met(1), Glu (23 24), or Ala (24
25) to Val (405); Met(1), Glu (23 24), or Ala (24 25) to Glu (404);
Met(1), Glu (23 24), or Ala (24 25) to Glu (403); Met(1), Glu (23
24), or Ala (24 25) to Ser (402); Met(1), Glu (23 24), or Ala (24
25) to Gly (398); Met(1), Glu (23 24), or Ala (24 25) to Pro (397);
Met(1), Glu (23 24), or Ala (24 25) to Lys (393); Met(1), Glu (23
24), or Ala (24 25) to Met(263); Met(1), Glu (23 24), or Ala (24
25) to Asp(311); Met(1), Glu (23 24), or Ala (24 25) to Pro (367
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); 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).
[0098] Preferred embodiments include the following deletion
mutants: Thr(103)-Arg(227); Glu(104)-Arg(227); Ala(112)-Arg (227);
Thr(103)-Ser(213); Glu(104)-Ser(213); Thr(103)-Leu(215);
Glu(47)-Ser(419); Met(1), Glu (23 24), or Ala (24 25)-Met(263);
Met(1), Glu (23 24), or Ala (24 25)-Asp(311); Met(1), Glu (23 24),
or Ala (24 25)-Pro (367 366); Met(1)-Ser(419); and Met(1)-Ser(228)
of (FIG. 1 (SEQ ID NO:2)).
[0099] 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.
[0100] Particularly, N-terminal deletions of the VEGF-2 polypeptide
can be described by the general formula m-396, where m is an
integer from -23 to 388, where m corresponds to the position of the
amino acid residue identified in SEQ ID NO:2. Preferably,
N-terminal deletions retain the conserved boxed area of FIG. 3
(PXCVXXXRCXGCCN)(SEQ ID NO: 8), and include polypeptides comprising
the amino acid sequence of residues: 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; I-99 to S-396; D-100 to S-396; N-101 to S-396; E-102 to
S-396; W-103 to S-396; R-104 to S-396; K-105 to S-396; T-106 to
S-396; Q-107 to S-396; C-108 to S-396; M-109 to S-396; P-110 to
S-396; R-111 to S-396; E-112 to S-396; V-113 to S-396; C-114 to
S-396; I-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. Also preferred are
polynucleotides encoding these N-terminal deletion mutants.
[0101] 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 I-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 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 I-202; E-1
to I-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
I-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 I-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. Also preferred are
polynucleotides encoding these C-terminal deletion mutants.
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.
[0102] 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.
[0103] 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.
[0104] Thus, in one aspect, N-terminal deletion mutants are
provided by the present invention. Such mutants include those
comprising the amino acid sequence shown in FIG. 1 (SEQ ID NO:2)
except for a deletion of at least the first 24 N-terminal amino
acid residues (i.e., a deletion of at least Met (1)-Glu (24)) but
not more than the first 115 N-terminal amino acid residues of FIG.
1 (SEQ ID NO:2). Alternatively, first 24 N-terminal amino acid
residues (i.e., a deletion of at least Met (1)-Glu (24)) but not
more than the first 103 N-terminal amino acid residues of FIG. 1
(SEQ ID NO:2), etc., etc.
[0105] In another aspect, C-terminal deletion mutants are provided
by the present invention. Such mutants include those comprising the
amino acid sequence shown in FIG. 1 (SEQ ID NO:2) except for a
deletion of at least the last C-terminal amino acid residue (Ser
(419)) but not more than the last 220 C-terminal amino acid
residues (i.e., a deletion of amino acid residues Val (199)-Ser
(419)) of FIG. 1 (SEQ ID NO:2). Alternatively, the deletion will
include at least the last C-terminal amino acid residue but not
more than the last 216 C-terminal amino acid residues of FIG. 1
(SEQ ID NO:2). Alternatively, the deletion will include at least
the last C-terminal amino acid residue but not more than the last
204 C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).
Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 192
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).
[0106] Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 156
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).
Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 108
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).
Alternatively, the deletion will include at least the last
C-terminal amino acid residues but not more than the last 52
C-terminal amino acid residues of FIG. 1 (SEQ ID NO:2).
[0107] 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.
[0108] 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).
[0109] 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.
[0110] Moreover, representative examples of VEGF-2 polynucleotide
fragments include, for example, fragments having a sequence from
about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250,
251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600,
651-700, 701-750, 751-800, 800-850, 851-900, 901-950, or 951 to the
end of SEQ ID NO:1 or the cDNA contained in the deposited clone. In
this context "about" includes the particularly recited ranges,
larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at
either terminus or at both termini. Preferably, these fragments
encode a polypeptide which has biological activity.
[0111] Fragments of the full length gene of the present invention
may be used as a hybridization probe for a cDNA library to isolate
the full length cDNA and to isolate other cDNAs which have a high
sequence similarity to the gene or similar biological activity.
Probes of this type preferably have at least 30 bases and may
contain, for example, 50 or more bases. The probe may also be used
to identify a cDNA clone corresponding to a full length transcript
and a genomic clone or clones that contain the complete gene
including regulatory and promoter regions, exons, and introns. An
example of a screen comprises isolating the coding region of the
gene by using the known DNA sequence to synthesize an
oligonucleotide probe. Labeled oligonucleotides having a sequence
complementary to that of the gene of the present invention are used
to screen a library of human cDNA, genomic DNA or MRNA to determine
which members of the library the probe hybridizes to.
[0112] A VEGF-2 "polynucleotide" also includes those
polynucleotides capable of hybridizing, under stringent
hybridization conditions, to sequences contained in SEQ ID NO:1 or
for instance, the cDNA clone(s) contained in ATCC.TM. Deposit Nos.
97149 or 75698, the complement thereof. "Stringent hybridization
conditions" refers to an overnight incubation at 42.degree. C. in a
solution comprising 50% formamide, 5.times. SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6),
5.times.Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65.degree. C.
[0113] Also contemplated are nucleic acid molecules that hybridize
to the VEGF-2 polynucleotides at lower stringency hybridization
conditions. Changes in the stringency of hybridization and signal
detection are primarily accomplished through the manipulation of
formamide concentration (lower percentages of formamide result in
lowered stringency); salt conditions, or temperature. For example,
lower stringency conditions include an overnight incubation at
37.degree. C. in a solution comprising 6.times.SSPE (20.times.SSPE
=3M NaCl; 0.2M NaH.sub.2PO.sub.4; 0.02M EDTA, pH 7.4), 0.5% SDS,
30% formamide, 100 .mu.g/ml salmon sperm blocking DNA; followed by
washes at 50.degree. C. with 1.times.SSPE, 0.1% SDS. In addition,
to achieve even lower stringency, washes performed following
stringent hybridization can be done at higher salt concentrations
(e.g. 5.times.SSC).
[0114] 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.
[0115] 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).
[0116] 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.
[0117] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNA or the nucleotide sequence as shown in SEQ ID
NO:1). Of course, a polynucleotide which hybridizes only to a poly
A sequence (such as the 3N terminal poly(A) tract of the VEGF-2
cDNA shown in SEQ ID NOS:1 or 3), or to a complementary stretch of
T (or U) resides, would not be included in a polynucleotide of the
invention used to hybridize to a portion of a nucleic acid of the
invention, since such a polynucleotide would hybridize to any
nucleic acid molecule containing a poly (A) stretch or the
complement thereof (e.g., practically any double-stranded cDNA
clone).
[0118] The present application is directed to nucleic acid
molecules at least 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in SEQ ID NOS:1 or 3 or to the nucleic
acid sequence of the deposited cDNA(s), irrespective of whether
they encode a polypeptide having VEGF-2 activity. This is because
even where a particular nucleic acid molecule does not encode a
polypeptide having VEGF-2 activity, one of skill in the art would
still know how to use the nucleic acid molecule, for instance, as a
hybridization probe or a polymerase chain reaction (PCR) primer.
Uses of the nucleic acid molecules of the present invention that do
not encode a polypeptide having VEGF-2 activity include, inter
alia, (1) isolating the VEGF-2 gene or allelic variants thereof in
a cDNA library; (2) in situ hybridization (e.g., "FISH") to
metaphase chromosomal spreads to provide precise chromosomal
location of the VEGF-2 gene, as described in Verma et al., Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York
(1988); and Northern Blot analysis for detecting VEGF-2 MRNA
expression in specific tissues. Preferred, however, are nucleic
acid molecules having sequences at least 95%, 96%, 97%, 98% or 99%
identical to a nucleic acid sequence shown in SEQ ID NOS:1 or 3 or
to a nucleic acid sequence of the deposited cDNA(s) which do, in
fact, encode a polypeptide having VEGF-2 protein activity. By "a
polypeptide having VEGF-2 activity" is intended polypeptides
exhibiting VEGF-2 activity in a particular biological assay. For
example, VEGF-2 protein activity can be measured using, for
example, mitogenic assays and endothelial cell migration assays.
See, e.g., Olofsson et al., Proc. Natl. Acad. Sci. USA 93:2576-2581
(1996) and Joukov et al., EMBO J. 5:290-298 (1996).
[0119] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid
sequence of the deposited cDNA(s) or the nucleic acid sequence
shown in SEQ ID NO:1 or SEQ ID NO:3 will encode a polypeptide
"having VEGF-2 protein activity." In fact, since degenerate
variants of these nucleotide sequences all encode the same
polypeptide, this will be clear to the skilled artisan even without
performing the above described comparison assay. It will be further
recognized in the art that, for such nucleic acid molecules that
are not degenerate variants, a reasonable number will also encode a
polypeptide having VEGF-2 protein activity. This is because the
skilled artisan is fully aware of amino acid substitutions that are
either less likely or not likely to significantly effect protein
function (e.g., replacing one aliphatic amino acid with a second
aliphatic amino acid).
[0120] 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.
[0121] 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.
[0122] "Identity" per se has an art-recognized meaning and can be
calculated using published techniques. (See, e.g.: (Computational
Molecular Biology, Lesk, A. M., ed., Oxford University Press, New
York, (1988); Biocomputing: Informatics and Genome Projects, Smith,
D. W., ed., Academic Press, New York, (1993); Computer Analysis of
Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds.,
Humana Press, New Jersey, (1994); Sequence Analysis in Molecular
Biology, von Heinje, G., Academic Press, (1987); and Sequence
Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New York, (1991).) While there exists a number of methods to
measure identity between two polynucleotide or polypeptide
sequences, the term "identity" is well known to skilled artisans.
(Carillo, H., and Lipton, D., SIAM J. Applied Math. 48:1073
(1988).) Methods commonly employed to determine identity or
similarity between two sequences include, but are not limited to,
those disclosed in "Guide to Huge Computers," Martin J. Bishop,
ed., Academic Press, San Diego, (1994), and Carillo, H., and
Lipton, D., SIAM J. Applied Math. 48:1073 (1988). Methods for
aligning polynucleotides or polypeptides are codified in computer
programs, including the GCG program package (Devereux, J., et al.,
Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA
(Atschul, S. F. et al., J. Molec. Biol. 215:403 (1990), Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711 (using the local homology algorithm of
Smith and Waterman, Advances in Applied Mathematics 2:482-489
(1981)). By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence of
the present invention, it is intended that the nucleotide sequence
of the polynucleotide is identical to the reference sequence except
that the polynucleotide sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleotide
sequence encoding the VEGF-2 polypeptide. In other words, to obtain
a polynucleotide having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. The query sequence may be an entire
sequence SEQ ID NO:1, the ORF (open reading frame), or any fragment
specified as described herein.
[0123] As a practical matter, whether any particular nucleic acid
molecule or polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99%
identical to a nucleotide sequence of the presence invention can be
determined conventionally using known computer programs. A
preferred method for determining the best overall match between a
query sequence (a sequence of the present invention) and a subject
sequence, also referred to as a global sequence alignment, can be
determined using the FASTDB computer program based on the algorithm
of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a
sequence alignment the query and subject sequences are both DNA
sequences. An RNA sequence can be compared by converting U's to
T's. The result of said global sequence alignment is in percent
identity. Preferred parameters used in a FASTDB alignment of DNA
sequences to calculate percent identity are: Matrix=Unitary,
k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization
Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty
0.05, Window Size=500 or the length of the subject nucleotide
sequence, whichever is shorter. 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.
[0124] 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.
[0125] 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.
[0126] 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 SEQ ID NOs:2 or 4 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.
[0127] 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.
[0128] 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
[0129] 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.
[0130] 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.
[0131] 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.
[0132] Preferred polypeptide fragments include the secreted VEGF-2
protein as well as the mature form. Further preferred polypeptide
fragments include the secreted VEGF-2 protein or the mature form
having a continuous series of deleted residues from the amino or
the carboxy terminus, or both. For example, any number of amino
acids, ranging from 1-60, can be deleted from the amino terminus of
either the secreted VEGF-2 polypeptide or the mature form.
Similarly, any number of amino acids, ranging from 1-30, can be
deleted from the carboxy terminus of the secreted VEGF-2 protein or
mature form. Furthermore, any combination of the above amino and
carboxy terminus deletions are preferred. Similarly, polynucleotide
fragments encoding these VEGF-2 polypeptide fragments are also
preferred.
[0133] Also preferred are VEGF-2 polypeptide and polynucleotide
fragments characterized by structural or functional domains, such
as fragments that comprise alpha-helix and alpha-helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and
turn-forming regions, coil and coil-forming regions, hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions,
substrate binding region, and high antigenic index regions.
Polypeptide fragments of SEQ ID NO:2 falling within conserved
domains are specifically contemplated by the present invention.
(See FIG. 2.) Moreover, polynucleotide fragments encoding these
domains are also contemplated.
[0134] Other preferred fragments are biologically active VEGF-2
fragments. Biologically active fragments are those exhibiting
activity similar, but not necessarily identical, to an activity of
the VEGF-2 polypeptide. The biological activity of the fragments
may include an improved desired activity, or a decreased
undesirable activity.
[0135] The polypeptides of the present invention may be recombinant
polypeptides, natural polypeptides, or synthetic polypeptides,
preferably recombinant polypeptides.
[0136] It will be recognized in the art that some amino acid
sequences of the VEGF-2 polypeptide can be varied without
significant effect of the structure or function of the protein. If
such differences in sequence are contemplated, it should be
remembered that there will be critical areas on the protein which
determine activity.
[0137] Thus, the invention further includes variations of the
VEGF-2 polypeptide which show substantial VEGF-2 polypeptide
activity or which include regions of VEGF-2 protein such as the
protein portions discussed below. Such mutants include deletions,
insertions, inversions, repeats, and type substitutions. As
indicated above, guidance concerning which amino acid changes are
likely to be phenotypically silent can be found in Bowie, J. U., et
al., "Deciphering the Message in Protein Sequences: Tolerance to
Amino Acid Substitutions," Science 247:1306-1310 (1990).
[0138] Thus, the fragments, derivatives, or analogs of the
polypeptides of FIG. 1 or 2, or that encoded by the deposited cDNAs
may be: (I) one in which one or more of the amino acid residues are
substituted with a conserved or non-conserved amino acid residue
(preferably a conserved amino acid residue) and such substituted
amino acid residue may or may not be one encoded by the genetic
code; or (ii) one in which one or more of the amino acid residues
includes a substituent group; or (iii) one in which the mature
polypeptide is fused with another compound, such as a compound to
increase the half-life of the polypeptide (for example,
polyethylene glycol); or (iv) one in which the additional amino
acids are fused to the mature polypeptide, such as a leader or
secretory sequence or a sequence which is employed for purification
of the mature polypeptide or a proprotein sequence; or (v) one in
which comprises fewer amino acid residues shown in SEQ ID NOS: 2 or
4, and retains the conserved motif and yet still retains activity
characteristics of the VEGF family of polypeptides. Such fragments,
derivatives, and analogs are deemed to be within the scope of those
skilled in the art from the teachings herein.
[0139] Of particular interest are substitutions of charged amino
acids with another charged amino acid and with neutral or
negatively charged amino acids. The latter results in proteins with
reduced positive charge to improve the characteristics of the
VEGF-2 protein. The prevention of aggregation is highly desirable.
Aggregation of proteins not only results in a loss of activity but
can also be problematic when preparing pharmaceutical formulations,
because they can be immunogenic. (Pinckard et al., Clin. Exp.
Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845
(1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993)).
[0140] The replacement of amino acids can also change the
selectivity of binding to cell surface receptors. Ostade et al.,
Nature 361:266-268 (1993) describes certain mutations resulting in
selective binding of TNF-a to only one of the two known types of
TNF receptors. Thus, the VEGF-2 of the present invention may
include one or more amino acid substitutions, deletions or
additions, either from natural mutations or human manipulation.
[0141] 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 Tables 1 and
2).
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
TABLE-US-00002 TABLE 2 Preferred Amino Acid Substitutions Original
Preferred Residue Substitutions Exemplary Substitutions Ala (A) Val
Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Lys;
Arg Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp
Gly (G) Pro Pro His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu;
Val; Met; Ala; Phe; norleucine Leu (L) Ile norleucine; Ile; Val;
Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile
Phe (F) Leu Leu; Val; Ile; Ala Pro (P) Gly Gly Ser (S) Thr Thr Thr
(T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V)
Leu Ile; Leu; Met; Phe; Ala; norleucine
[0142] 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.
[0143] Amino acids in the VEGF-2 protein of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as receptor binding or in
vitro, or in vitro proliferative activity. Sites that are critical
for ligand-receptor binding can also be determined by structural
analysis such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904
(1992) and de Vos et al. Science 255:306-312 (1992)).
[0144] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
[0145] 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.
[0146] In specific embodiments, the polynucleotides of the
invention are less than 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10
kb, or 7.5 kb in length. In a further embodiment, polynucleotides
of the invention comprise at least 15 contiguous nucleotides of
VEGF-2 coding sequence, but do not comprise all or a portion of any
VEGF-2 intron. In another embodiment, the nucleic acid comprising
VEGF-2 coding sequence does not contain coding sequences of a
genomic flanking gene (i.e., 5' or 3' to the VEGF-2 gene in the
genome).
[0147] 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. 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.
[0148] 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.
[0149] 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.
VEGF-2 Derivatives
[0150] The VEGF-2 wild type and analogs may be further modified to
contain additional chemical moieties not normally part of the
protein. Those derivatized moieties may improve the solubility, the
biological half life or absorption of the protein. The moieties may
also reduce or eliminate any desirable side effects of the proteins
and the like. an overview for those moieties can be found in
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed., Mack Publishing Co.,
Easton, Pa. (1990).
[0151] The chemical moieties most suitable for derivatization
include water soluble polymers. A water soluble polymer is
desirable because the protein to which it is attached does not
precipitate in an aqueous environment, such as a physiological
environment. Preferably, the polymer will be pharmaceutically
acceptable for the preparation of a therapeutic product or
composition. One skilled in the art will be able to select the
desired polymer based on such considerations as whether the
polymer/protein conjugate will be used therapeutically, and if so,
the desired dosage, circulation time, resistance to proteolysis,
and other considerations. The effectiveness of the derivatization
may be ascertained by administering the derivative, in the desired
form (i.e., by osmotic pump, or, more preferably, by injection or
infusion, or, further formulated for oral, pulmonary or other
delivery routes), and determining its effectiveness.
[0152] Suitable water soluble polymers include, but are not limited
to, polyethylene glycol (PEG), copolymers of ethylene
glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl
alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
propropylene glycol homopolymers, prolypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have advantages in manufacturing due to its
stability in water.
[0153] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight ranges from about 2 kDa to about 100 kDa for ease
in handling and manufacturing (the term "about" indicating that in
preparations of polyethylene glycol, some molecules will weigh
more, some less, than the stated molecular weight). Other sizes may
be used, depending on the desired therapeutic profile (e.g., the
duration of sustained release desired, the effects, if any on
biological activity, the ease in handling, the degree or lack of
antigenicity and other known effects of polyethylene glycol on a
therapeutic protein or variant).
[0154] The number of polymer molecules so attached may vary, and
one skilled in the art will be able to ascertain the effect on
function. One may mono-derivatize, or may provide for a di-, tri-,
tetra- or some combination of derivatization, with the same or
different chemical moieties (e.g., polymers, such as different
weights of polyethylene glycols). The proportion of polymer
molecules to protein (or peptide) molecules will vary, as will
their concentrations in the reaction mixture. In general, the
optimum ratio (in terms of efficiency of reaction in that there is
no excess unreacted protein or polymer) will be determined by
factors such as the desired degree of derivatization (e.g., mono,
di-, tri-, etc.), the molecular weight of the polymer selected,
whether the polymer is branched or unbranched, and the reaction
conditions.
[0155] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art. See for example, EP 0 401 384, the disclosure of which is
hereby incorporated by reference (coupling PEG to G-CSF), see also
Malik et al., Exp. Hematol 20:1028-1035 (1992) (reporting
pegylation of GM-CSF using tresyl chloride). For example,
polyethylene glycol may be covalently bound through amino acid
residues via a reactive group, such as, a free amino or carboxyl
group. Reactive groups are those to which an activated polyethylene
glycol molecule may be bound. The amino acid residues having a free
amino group may include lysine residues and the N-terminal amino
acid residue. Those having a free carboxyl group may include
aspartic acid residues, glutamic acid residues, and the C-terminal
amino acid residue. Sulfhydryl groups may also be used as a
reactive group for attaching the polyethylene glycol molecule(s).
For therapeutic purposes, attachment at an amino group, such as
attachment at the N-terminus or lysine group is preferred.
Attachment at residues important for receptor binding should be
avoided if receptor binding is desired.
[0156] One may specifically desire an N-terminal chemically
modified protein. Using polyethylene glycol as an illustration of
the present compositions, one may select from a variety of
polyethylene glycol molecules (by molecular weight, branching,
etc.), the proportion of polyethylene glycol molecules to protein
(or peptide) molecules in the reaction mix, the type of pegylation
reaction to be performed, and the method of obtaining the selected
N-terminally pegylated protein. The method of obtaining the
N-terminally pegylated preparation (i.e., separating this moiety
from other monopegylated moieties if necessary) may be by
purification of the N-terminally pegylated material from a
population of pegylated protein molecules. Selective N-terminal
chemical modification may be accomplished by reductive alkylation
which exploits differential reactivity of different types of
primary amino groups (lysine versus the N-terminal) available for
derivatization in a particular protein. Under the appropriate
reaction conditions, substantially selective derivatization of the
protein at the N-terminus with a carbonyl group containing polymer
is achieved. For example, one may selectively N-terminally pegylate
the protein by performing the reaction at a pH which allows one to
take advantage of the pKa differences between the epsilon-amino
group of the lysine residues and that of the alpha-amino group of
the N-terminal residue of the protein. By such selective
derivatization, attachment of a water soluble polymer to a protein
is controlled: the conjugation with the polymer takes place
predominantly at the N-terminus of the protein and no significant
modification of other reactive groups, such as the lysine side
chain amino groups, occurs. Using reductive alkylation, the water
soluble polymer may be of the type described above, and should have
a single reactive aldehyde for coupling to the protein.
Polyethylene glycol propionaldehyde, containing a single reactive
aldehyde, may be used.
[0157] The present invention contemplates use of derivatives which
are prokaryote-expressed VEGF-2, or variants thereof, linked to at
least one polyethylene glycol molecule, as well as use of VEGF-2,
or variants thereof, attached to one or more polyethylene glycol
molecules via an acyl or alkyl linkage.
[0158] Pegylation may be carried out by any of the pegylation
reactions known in the art. See, for example: Focus on Growth
Factors, 3 (2): 4-10 (1992); EP 0 154 316, the disclosure of which
is hereby incorporated by reference; EP 0 401 384; and the other
publications cited herein that relate to pegylation. The pegylation
may be carried out via an acylation reaction or an alkylation
reaction with a reactive polyethylene glycol molecule (or an
analogous reactive water-soluble polymer).
[0159] Pegylation by acylation generally involves reacting an
active ester derivative of polyethylene glycol with the VEGF-2
protein or variant. Any known or subsequently discovered reactive
PEG molecule may be used to carry out the pegylation of VEGF-2
protein or variant. A preferred activated PEG ester is PEG
esterified to N-hydroxysuccinimide. As used herein, "acylation" is
contemplated to include without limitation the following types of
linkages between the therapeutic protein and a water soluble
polymer such as PEG: amide, carbamate, urethane, and the like. See
Bioconjugate Chem. 5:133-140 (1994). Reaction conditions may be
selected from any of those known in the pegylation art or those
subsequently developed, but should avoid conditions of temperature,
solvent, and pH that would inactivate the VEGF-2 or variant to be
modified.
[0160] Pegylation by acylation will generally result in a
poly-pegylated VEGF-2 protein or variant. Preferably, the
connecting linkage will be an amide. Also preferably, the resulting
product will be substantially only (e.g., >95%) mono-, di- or
tri-pegylated. However, some species with higher degrees of
peglylation may be formed in amounts depending on the specific
reaction conditions used. If desired, more purified pegylated
species may be separated from the mixture, particularly unreacted
species, by standard purification techniques, including, among
others, dialysis, salting-out, ultrafiltration, ion-exchange
chromatography, gel filtration chromatography and
electrophoresis.
[0161] Pegylation by alkylation generally involves reacting a
terminal aldehyde derivative of PEG with the VEGF-2 protein or
variant in the presence of a reducing agent. Pegylation by
alkylation can also result in poly-pegylated VEGF-2 protein or
variant. In addition, one can manipulate the reaction conditions to
favor pegylation substantially only at the a-amino group of the
N-terminus of the VEGF-2 protein or variant (i.e., a mono-pegylated
protein). In either case of monopegylation or polypegylation, the
PEG groups are preferably attached to the protein via a --CH2-NH--
group. With particular reference to the --CH2- group, this type of
linkage is referred to herein as an "alkyl" linkage.
[0162] Derivatization via reductive alkylation to produce a
monopegylated product exploits differential reactivity of different
types of primary amino groups (lysine versus the N-terminal)
available for derivatization. The reaction is performed at a pH
which allows one to take advantage of the pKa differences between
the .epsilon.-amino groups of the lysine residues and that of the
.alpha.-amino group of the N-terminal residue of the protein. By
such selective derivatization, attachment of a water soluble
polymer that contains a reactive group such as an aldehyde, to a
protein is controlled: the conjugation with the polymer takes place
predominantly at the N-terminus of the protein and no significant
modification of other reactive groups, such as the lysine side
chain amino groups, occurs. In one important aspect, the present
invention contemplates use of a substantially homogeneous
preparation of monopolymer/VEGF-2 protein (or variant) conjugate
molecules (meaning VEGF-2 protein or variant to which a polymer
molecule has been attached substantially only (i.e., >95%) in a
single location). More specifically, if polyethylene glycol is
used, the present invention also encompasses use of pegylated
VEGF-2 protein or variant lacking possibly antigenic linking
groups, and having the polyethylene glycol molecule directly
coupled to the VEGF-2 protein or variant.
[0163] Thus, it is contemplated that VEGF-2 to be used in
accordance with the present invention may include pegylated VEGF-2
protein or variants, wherein the PEG group(s) is (are) attached via
acyl or alkyl groups. As discussed above, such products may be
mono-pegylated or poly-pegylated (e.g., containing 2-6, and
preferably 2-5, PEG groups). The PEG groups are generally attached
to the protein at the .alpha.- or .epsilon.-amino groups of amino
acids, but it is also contemplated that the PEG groups could be
attached to any amino group attached to the protein, which is
sufficiently reactive to become attached to a PEG group under
suitable reaction conditions.
[0164] The polymer molecules used in both the acylation and
alkylation approaches may be selected from among water soluble
polymers as described above. The polymer selected should be
modified to have a single reactive group, such as an active ester
for acylation or an aldehyde for alkylation, preferably, so that
the degree of polymerization may be controlled as provided for in
the present methods. An exemplary reactive PEG aldehyde is
polyethylene glycol propionaldehyde, which is water stable, or mono
C1-C10 alkoxy or aryloxy derivatives thereof (see, U.S. Pat. No.
5,252,714). The polymer may be branched or unbranched. For the
acylation reactions, the polymer(s) selected should have a single
reactive ester group. For the present reductive alkylation, the
polymer(s) selected should have a single reactive aldehyde group.
Generally, the water soluble polymer will not be selected from
naturally-occurring glycosyl residues since these are usually made
more conveniently by mammalian recombinant expression systems. The
polymer may be of any molecular weight, and may be branched or
unbranched.
[0165] A particularly preferred water-soluble polymer for use
herein is polyethylene glycol. As used herein, polyethylene glycol
is meant to encompass any of the forms of PEG that have been used
to derivatize other proteins, such as mono-(C1-C10) alkoxy- or
aryloxy-polyethylene glycol.
[0166] In general, chemical derivatization may be performed under
any suitable condition used to react a biologically active
substance with an activated polymer molecule. Methods for preparing
pegylated VEGF-2 protein or variant will generally comprise the
steps of (a) reacting a VEGF-2 protein or variant with polyethylene
glycol (such as a reactive ester or aldehyde derivative of PEG)
under conditions whereby the protein becomes attached to one or
more PEG groups, and (b) obtaining the reaction product(s). In
general, the optimal reaction conditions for the acylation
reactions will be determined case-by-case based on known parameters
and the desired result. For example, the larger the ratio of
PEG:protein, the greater the percentage of poly-pegylated
product.
[0167] Reductive alkylation to produce a substantially homogeneous
population of mono-polymer/VEGF-2 protein (or variant) conjugate
molecule will generally comprise the steps of: (a) reacting a
VEGF-2 protein or variant with a reactive PEG molecule under
reductive alkylation conditions, at a pH suitable to permit
selective modification of the a-amino group at the amino terminus
of said VEGF-2 protein or variant; and (b) obtaining the reaction
product(s).
[0168] For a substantially homogeneous population of
mono-polymer/VEGF-2 protein (or variant) conjugate molecules, the
reductive alkylation reaction conditions are those which permit the
selective attachment of the water soluble polymer moiety to the
N-terminus of VEGF-2 protein or variant. Such reaction conditions
generally provide for pKa differences between the lysine amino
groups and the .alpha.-amino group at the N-terminus (the pKa being
the pH at which 50% of the amino groups are protonated and 50% are
not). The pH also affects the ratio of polymer to protein to be
used. In general, if the pH is lower, a larger excess of polymer to
protein will be desired (i.e., the less reactive the N-terminal
.alpha.-amino group, the more polymer needed to achieve optimal
conditions). If the pH is higher, the polymer:protein ratio need
not be as large (i.e., more reactive groups are available, so fewer
polymer molecules are needed). For purposes of the present
invention, the pH will generally fall within the range of 3-9,
preferably 3-6.
[0169] Another important consideration is the molecular weight of
the polymer. In general, the higher the molecular weight of the
polymer, the fewer polymer molecules may be attached to the
protein. Similarly, branching of the polymer should be taken into
account when optimizing these parameters. Generally, the higher the
molecular weight (or the more branches) the higher the
polymer:protein ratio. In general, for the pegylation reactions
contemplated herein, the preferred average molecular weight is
about 2 kDa to about 100 kDa. The preferred average molecular
weight is about 5 kDa to about 50 kDa, particularly preferably
about 12 kDa to about 25 kDa. The ratio of water-soluble polymer to
VEGF-2 protein or variant will generally range from 1:1 to 100:1,
preferably (for polypegylation) 1:1 to 20:1 and (for
monopegylation) 1:1 to 5:1.
[0170] Using the conditions indicated above, reductive alkylation
will provide for selective attachment of the polymer to any VEGF-2
protein or variant having an .alpha.-amino group at the amino
terminus, and provide for a substantially homogenous preparation of
monopolymer/VEGF-2 protein (or variant) conjugate. The term
"monopolymer/VEGF-2 protein (or variant) conjugate" is used here to
mean a composition comprised of a single polymer molecule attached
to a molecule of VEGF-2 protein or VEGF-2 variant protein. The
monopolymer/VEGF-2 protein (or variant) conjugate preferably will
have a polymer molecule located at the N-terminus, but not on
lysine amino side groups. The preparation will preferably be
greater than 90% monopolymer/VEGF-2 protein (or variant) conjugate,
and more preferably greater than 95% monopolymer/VEGF-2 protein (or
variant) conjugate, with the remainder of observable molecules
being unreacted (i.e., protein lacking the polymer moiety).
[0171] For the present reductive alkylation, the reducing agent
should be stable in aqueous solution and preferably be able to
reduce only the Schiff base formed in the initial process of
reductive alkylation. Preferred reducing agents may be selected
from sodium borohydride, sodium cyanoborohydride, dimethylamine
borane, trimethylamine borane and pyridine borane. A particularly
preferred reducing agent is sodium cyanoborohydride. Other reaction
parameters, such as solvent, reaction times, temperatures, etc.,
and means of purification of products, can be determined
case-by-case based on the published information relating to
derivatization of proteins with water soluble polymers (see the
publications cited herein).
Epitopes and Antibodies
[0172] The present invention encompasses polypeptides comprising,
or alternatively consisting of, an epitope of the polypeptide
having an amino acid sequence of SEQ ID NOS:2 or 4, or an epitope
of the polypeptide sequence encoded by a polynucleotide sequence
contained in ATCC.TM. Deposit No: 97149 or 75698 or encoded by a
polynucleotide that hybridizes to the complement of the sequence of
SEQ ID NOS:1 or 3 or contained in ATCC.TM. Deposit No: 97149 or
75698 under stringent hybridization conditions or lower stringency
hybridization conditions as defined supra. The present invention
further encompasses polynucleotide sequences encoding an epitope of
a polypeptide sequence of the invention (such as, for example, the
sequence disclosed in SEQ ID NOS:1 or 3), polynucleotide sequences
of the complementary strand of a polynucleotide sequence encoding
an epitope of the invention, and polynucleotide sequences which
hybridize to the complementary strand under stringent hybridization
conditions or lower stringency hybridization conditions defined
supra.
[0173] Specific monoclonal antibodies have been raised against the
VEGF-2 protein (SEQ ID NO:2). These monoclonal antibodies have been
given the following designations: 12E2; 13A2; 15C2; 13D6; 13E6;
19A3; 8G11; 11A8, 15E10, 9B4, and 13G11. Monoclonal antibodies
15C2, 13D6, and 15E10 were deposited as a group on Jun. 8, 1999,
and given ATCC.TM. Deposit Number PTA-198. Monoclonal antibody 13D6
was also deposited by itself on Jul. 29, 1999, and given ATCC.TM.
Deposit Number PTA-435. Monoclonal antibodies 13A2, 13E6, and 9B4
were deposited as a group on Jun. 8, 1999, and given ATCC.TM.
Deposit Number PTA-199. Monoclonal antibodies 8G11, 12E2, and 13G11
were deposited as a group on Jun.8, 1999, and given ATCC.TM.
Deposit Number PTA-200. Monoclonal antibodies 11A8 and 19A3 were
deposited as a group on Jun. 8, 1999, and given ATCC.TM. Deposit
Number PTA-201. The antibodies deposited in a mixture can be
isolated based on their characteristics, such as epitope map
position, affinity, species as described in the Examples.
[0174] The epitopes to which the above listed monoclonal antibodies
have specificity have been mapped to the VEGF-2 protein (See FIG.
24). Furthermore, the status of each monoclonal antibody, such as
the relative affinity and ELISA and Western reactivity, have been
disclosed for each of the monoclonal antibodies (See FIG. 25).
[0175] The term "epitopes," as used herein, refers to portions of a
polypeptide having antigenic or immunogenic activity in an animal,
preferably a mammal, and most preferably in a human. In a preferred
embodiment, the present invention encompasses a polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An "immunogenic epitope," as used herein, is defined
as a portion of a protein that elicits an antibody response in an
animal, as determined by any method known in the art, for example,
by the methods for generating antibodies described infra. (See, for
example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002
(1983)). The term "antigenic epitope," as used herein, is defined
as a portion of a protein to which an antibody can
immunospecifically bind its antigen as determined by any method
well known in the art, for example, by the immunoassays described
herein. Immunospecific binding excludes non-specific binding but
does not necessarily exclude cross-reactivity with other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[0176] Fragments which function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci.
USA 82:5131-5135 (1985), further described in U.S. Pat. No.
4,631,211).
[0177] In the present invention, antigenic epitopes preferably
contain a sequence of at least 4, at least 5, at least 6, at least
7, more preferably at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
20, at least 25, at least 30, at least 40, at least 50, and, most
preferably, between about 15 to about 30 amino acids. Preferred
polypeptides comprising immunogenic or antigenic epitopes are at
least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, or 100 amino acid residues in length. Additional
non-exclusive preferred antigenic epitopes include the antigenic
epitopes disclosed herein, as well as portions thereof. Antigenic
epitopes are useful, for example, to raise antibodies, including
monoclonal antibodies, that specifically bind the epitope.
Preferred antigenic epitopes include the antigenic epitopes
disclosed herein, as well as any combination of two, three, four,
five or more of these antigenic epitopes. Antigenic epitopes can be
used as the target molecules in immunoassays. (See, for instance,
Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science
219:660-666 (1983)).
[0178] Similarly, immunogenic epitopes can be used, for example, to
induce antibodies according to methods well known in the art. (See,
for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al.,
J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes
include the immunogenic epitopes disclosed herein, as well as any
combination of two, three, four, five or more of these immunogenic
epitopes. The polypeptides comprising one or more immunogenic
epitopes may be presented for eliciting an antibody response
together with a carrier protein, such as an albumin, to an animal
system (such as rabbit or mouse), or, if the polypeptide is of
sufficient length (at least about 25 amino acids), the polypeptide
may be presented without a carrier. However, immunogenic epitopes
comprising as few as 8 to 10 amino acids have been shown to be
sufficient to raise antibodies capable of binding to, at the very
least, linear epitopes in a denatured polypeptide (e.g., in Western
blotting).
[0179] Epitope-bearing polypeptides of the present invention may be
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). If in vivo immunization is used,
animals may be immunized with free peptide; however, anti-peptide
antibody titer may be boosted by coupling the peptide to a
macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus toxoid. For instance, peptides containing cysteine residues
may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g of peptide or carrier protein
and Freund's adjuvant or any other adjuvant known for stimulating
an immune response. 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.
[0180] As one of skill in the art will appreciate, and as discussed
above, the polypeptides of the present invention comprising an
immunogenic or antigenic epitope can be fused to other polypeptide
sequences. For example, the polypeptides of the present invention
may be fused with the constant domain of immunoglobulins (IgA, IgE,
IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination
thereof and portions thereof) resulting in chimeric polypeptides.
Such fusion proteins may facilitate purification and may increase
half-life in vivo. This has been shown for chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. See, e.g., EP 394,827;
Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of
an antigen across the epithelial barrier to the immune system has
been demonstrated for antigens (e.g., insulin) conjugated to an
FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT
Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that
have a disulfide-linked dimeric structure due to the IgG portion
desulfide bonds have also been found to be more efficient in
binding and neutralizing other molecules than monomeric
polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the
above epitopes can also be recombined with a gene of interest as an
epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid
in detection and purification of the expressed polypeptide. For
example, a system described by Janknecht et al. allows for the
ready purification of non-denatured fusion proteins expressed in
human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA 88:8972-897). In this system, the gene of interest is subcloned
into a vaccinia recombination plasmid such that the open reading
frame of the gene is translationally fused to an amino-terminal tag
consisting of six histidine residues. The tag serves as a matrix
binding domain for the fusion protein. Extracts from cells infected
with the recombinant vaccinia virus are loaded onto Ni2+
nitriloacetic acid-agarose column and histidine-tagged proteins can
be selectively eluted with imidazole-containing buffers.
[0181] Additional fusion proteins of the invention may be generated
through the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling"). DNA shuffling may be employed to modulate the
activities of polypeptides of the invention, such methods can be
used to generate polypeptides with altered activity, as well as
agonists and antagonists of the polypeptides. See, generally, U.S.
Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and
5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson,
et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco,
Biotechniques 24(2):308-13 (1998) (each of these patents and
publications are hereby incorporated by reference in its entirety).
In one embodiment, alteration of polynucleotides corresponding to
SEQ ID NO:1 or 3 and the polypeptides encoded by these
polynucleotides may be achieved by DNA shuffling. DNA shuffling
involves the assembly of two or more DNA segments by homologous or
site-specific recombination to generate variation in the
polynucleotide sequence. In another embodiment, polynucleotides of
the invention, or the encoded polypeptides, may be altered by being
subjected to random mutagenesis by error-prone PCR, random
nucleotide insertion or other methods prior to recombination. In
another embodiment, one or more components, motifs, sections,
parts, domains, fragments, etc., of a polynucleotide encoding a
polypeptide of the invention may be recombined with one or more
components, motifs, sections, parts, domains, fragments, etc. of
one or more heterologous molecules.
Antibodies
[0182] Further polypeptides of the invention relate to antibodies
and T-cell antigen receptors (TCR) which immunospecifically bind a
polypeptide, polypeptide fragment, or variant of SEQ ID NOS:2 or 4,
and/or an epitope, of the present invention (as determined by
immunoassays well known in the art for assaying specific
antibody-antigen binding). Antibodies of the invention include, but
are not limited to, polyclonal, monoclonal, multispecific, human,
humanized or chimeric antibodies, single chain antibodies, Fab
fragments, F(ab') fragments, fragments produced by a Fab expression
library, anti-idiotypic (anti-Id) antibodies (including, e.g.,
anti-Id antibodies to antibodies of the invention), and
epitope-binding fragments of any of the above. The term "antibody,"
as used herein, refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site that
immunospecifically binds an antigen. The immunoglobulin molecules
of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA
and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or
subclass of immunoglobulin molecule.
[0183] Most preferably the antibodies are human antigen-binding
antibody fragments of the present invention and include, but are
not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv),
single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments
comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the
variable region(s) alone or in combination with the entirety or a
portion of the following: hinge region, CH1, CH2, and CH3 domains.
Also included in the invention are antigen-binding fragments also
comprising any combination of variable region(s) with a hinge
region, CH1, CH2, and CH3 domains. The antibodies of the invention
may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine (e.g., mouse and rat),
donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As
used herein, "human" antibodies include antibodies having the amino
acid sequence of a human immunoglobulin and include antibodies
isolated from human immunoglobulin libraries or from animals
transgenic for one or more human immunoglobulin and that do not
express endogenous immunoglobulins, as described infra and, for
example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0184] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for a
heterologous epitope, such as a heterologous polypeptide or solid
support material. See, e.g., PCT publications WO 93/17715; WO
92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Inmunol.
147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553
(1992).
[0185] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention which they recognize or specifically bind.
The epitope(s) or polypeptide portion(s) may be specified as
described herein, e.g., by N-terminal and C-terminal positions, by
size in contiguous amino acid residues, or listed in the Tables and
Figures. Preferred epitopes of the invention include: 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, as well as polynucleotides
that encode these epitopes. Antibodies which specifically bind any
epitope or polypeptide of the present invention may also be
excluded. Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0186] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of a polypeptide of
the present invention are included. Antibodies that bind
polypeptides with at least 95%, at least 90%, at least 85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%,
at least 55%, and at least 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
In specific embodiments, antibodies of the present invention
cross-react with murine, rat and/or rabbit homologs of human
proteins and the corresponding epitopes thereof. Antibodies that do
not bind polypeptides with less than 95%, less than 90%, less than
85%, less than 80%, less than 75%, less than 70%, less than 65%,
less than 60%, less than 55%, and less than 50% identity (as
calculated using methods known in the art and described herein) to
a polypeptide of the present invention are also included in the
present invention. In a specific embodiment, the above-described
cross-reactivity is with respect to any single specific antigenic
or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or
more of the specific antigenic and/or immunogenic polypeptides
disclosed herein. Further included in the present invention are
antibodies which bind polypeptides encoded by polynucleotides which
hybridize to a polynucleotide of the present invention under
stringent hybridization conditions (as described herein).
Antibodies of the present invention may also be described or
specified in terms of their binding affinity to a polypeptide of
the invention. Preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-2 M,
10.sup.-2 M, 5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M,
10.sup.-4 M, 5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M,
10.sup.-6M, 5.times.10.sup.-7 M, 10.sup.7 M, 5.times.10.sup.-8 M,
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M, 5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
.sup.10-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M.
[0187] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of the invention as
determined by any method known in the art for determining
competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively
inhibits binding to the epitope by at least 95%, at least 90%, at
least 85%, at least 80%, at least 75%, at least 70%, at least 60%,
or at least 50%.
[0188] Antibodies of the present invention may act as agonists or
antagonists of the polypeptides of the present invention. For
example, the present invention includes antibodies which disrupt
the receptor/ligand interactions with the polypeptides of the
invention either partially or fully. Preferably, antibodies of the
present invention bind an antigenic epitope disclosed herein, or a
portion thereof. The invention features both receptor-specific
antibodies and ligand-specific antibodies. The invention also
features receptor-specific antibodies which do not prevent ligand
binding but prevent receptor activation. Receptor activation (i.e.,
signaling) may be determined by techniques described herein or
otherwise known in the art. For example, receptor activation can be
determined by detecting the phosphorylation (e.g., tyrosine or
serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example,
as described supra). In specific embodiments, antibodies are
provided that inhibit ligand activity or receptor activity by at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 60%, or at least 50% of the activity in
absence of the antibody.
[0189] The invention also features receptor-specific antibodies
which both prevent ligand binding and receptor activation as well
as antibodies that recognize the receptor-ligand complex, and,
preferably, do not specifically recognize the unbound receptor or
the unbound ligand. Likewise, included in the invention are
neutralizing antibodies which bind the ligand and prevent binding
of the ligand to the receptor, as well as antibodies which bind the
ligand, thereby preventing receptor activation, but do not prevent
the ligand from binding the receptor. Further included in the
invention are antibodies which activate the receptor. These
antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the
ligand-mediated receptor activation, for example, by inducing
dimerization of the receptor. The antibodies may be specified as
agonists, antagonists or inverse agonists for biological activities
comprising the specific biological activities of the peptides of
the invention disclosed herein. The above antibody agonists can be
made using methods known in the art. See, e.g., PCT publication WO
96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood
92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678
(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et
al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci.
111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods
205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241
(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);
Taryman et al., Neuron 14(4):755-762 (1995); Muller et al.,
Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine
8(1):14-20 (1996) (which are all incorporated by reference herein
in their entireties).
[0190] Antibodies of the present invention may be used, for
example, but not limited to, to purify, detect, and target the
polypeptides of the present invention, including both in vitro and
in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and
quantitatively measuring levels of the polypeptides of the present
invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its
entirety).
[0191] As discussed in more detail below, the antibodies of the
present invention may be used either alone or in combination with
other compositions. The antibodies may further be recombinantly
fused to a heterologous polypeptide at the N- or C-terminus or
chemically conjugated (including covalently and non-covalently
conjugations) to polypeptides or other compositions. For example,
antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays and
effector molecules such as heterologous polypeptides, drugs,
radionuclides, or toxins. See, e.g., PCT publications WO 92/08495;
WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP
396,387.
[0192] The antibodies of the invention include derivatives that are
modified, i.e., by the covalent attachment of any type of molecule
to the antibody such that covalent attachment does not prevent the
antibody from generating an anti-idiotypic response. For example,
but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids.
[0193] The antibodies of the present invention may be generated by
any suitable method known in the art. Polyclonal antibodies to an
antigen-of-interest can be produced by various procedures well
known in the art. For example, a polypeptide of the invention can
be administered to various host animals including, but not limited
to, rabbits, mice, rats, etc. to induce the production of sera
containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and include but are not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
Such adjuvants are also well known in the art.
[0194] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0195] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art
and are discussed in detail in the Examples. In a non-limiting
example, mice can be immunized with a polypeptide of the invention
or a cell expressing such peptide. Once an immune response is
detected, e.g., antibodies specific for the antigen are detected in
the mouse serum, the mouse spleen is harvested and splenocytes
isolated. The splenocytes are then fused by well known techniques
to any suitable myeloma cells, for example cells from cell line
SP20 available from the ATCC.TM.. Hybridomas are selected and
cloned by limited dilution. The hybridoma clones are then assayed
by methods known in the art for cells that secrete antibodies
capable of binding a polypeptide of the invention. Ascites fluid,
which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0196] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention.
[0197] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[0198] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular embodiment,
such phage can be utilized to display antigen binding domains
expressed from a repertoire or combinatorial antibody library
(e.g., human or murine). Phage expressing an antigen binding domain
that binds the antigen of interest can be selected or identified
with antigen, e.g., using labeled antigen or antigen bound or
captured to a solid surface or bead. Phage used in these methods
are typically filamentous phage including fd and M13 binding
domains expressed from phage with Fab, Fv or disulfide stabilized
Fv antibody domains recombinantly fused to either the phage gene
III or gene VIII protein. Examples of phage display methods that
can be used to make the antibodies of the present invention include
those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50
(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et
al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology
57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT
publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743
and 5,969,108; each of which is incorporated herein by reference in
its entirety.
[0199] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties).
[0200] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816397, which are incorporated herein by reference in their
entirety. Humanized antibodies are antibody molecules from
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species and a framework regions from a human
immunoglobulin molecule. Often, framework residues in the human
framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089;
Riechmann et al., Nature 332:323 (1988), which are incorporated
herein by reference in their entireties.) Antibodies can be
humanized using a variety of techniques known in the art including,
for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967;
U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology
28(4/5):489-498 (1991); Studnicka et al., Protein Engineering
7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and
chain shuffling (U.S. Pat. No. 5,565,332).
[0201] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety.
[0202] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring which express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO
96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; and 5,939,598, which are incorporated by
reference herein in their entirety. In addition, companies such as
Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.)
can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described
above.
[0203] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/technology 12:899-903 (1988)).
[0204] Further, antibodies to the polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan & Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol.
147(8):2429-2438 (1991)). For example, antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of
a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes that "mimic" the polypeptide multimerization and/or
binding domain and, as a consequence, bind to and neutralize
polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of such anti-idiotypes can be used in therapeutic
regimens to neutralize polypeptide ligand. For example, such
anti-idiotypic antibodies can be used to bind a polypeptide of the
invention and/or to bind its ligands/receptors, and thereby block
its biological activity.
Polynucleotides Encoding Antibodies
[0205] The invention further provides polynucleotides comprising a
nucleotide sequence encoding an antibody of the invention and
fragments thereof. The invention also encompasses polynucleotides
that hybridize under stringent or lower stringency hybridization
conditions, e.g., as defined supra, to polynucleotides that encode
an antibody, preferably, that specifically binds to a polypeptide
of the invention, preferably, an antibody that binds to a
polypeptide having the amino acid sequence of SEQ ID NOS:2 or
4.
[0206] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0207] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0208] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley &
Sons, NY, which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[0209] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the complementarity determining regions
(CDRs) by methods that are well know in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described supra. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substitutions improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[0210] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984);
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0211] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science
242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can
be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. Techniques for the assembly of functional
Fv fragments in E. coli may also be used (Skerra et al., Science
242:1038-1041 (1988)).
Methods of Producing Antibodies
[0212] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques.
[0213] Recombinant expression of an antibody of the invention, or
fragment, derivative or analog thereof, (e.g., a heavy or light
chain of an antibody of the invention or a single chain antibody of
the invention), requires construction of an expression vector
containing a polynucleotide that encodes the antibody. Once a
polynucleotide encoding an antibody molecule or a heavy or light
chain of an antibody, or portion thereof (preferably containing the
heavy or light chain variable domain), of the invention has been
obtained, the vector for the production of the antibody molecule
may be produced by recombinant DNA technology using techniques well
known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding
nucleotide sequence are described herein. Methods which are well
known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
[0214] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding an antibody of the invention, or a heavy or light chain
thereof, or a single chain antibody of the invention, operably
linked to a heterologous promoter. In preferred embodiments for the
expression of double-chained antibodies, vectors encoding both the
heavy and light chains may be co-expressed in the host cell for
expression of the entire immunoglobulin molecule, as detailed
below.
[0215] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule, are used for the expression of
a recombinant antibody molecule. For example, mammalian cells such
as Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2 (1990)).
[0216] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5509 (1989)); and the like. pGEX vectors may also be used
to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to matrix glutathione-agarose beads followed by elution in
the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[0217] In an insect system, Autographa califomica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter).
[0218] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk,
Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
[0219] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
[0220] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[0221] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
1993, TIB TECH 11(5):155-215); and hygro, which confers resistance
to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods
commonly known in the art of recombinant DNA technology may be
routinely applied to select the desired recombinant clone, and such
methods are described, for example, in Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,
Dracopoli et al. (eds), Current Protocols in Human Genetics, John
Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981), which are incorporated by reference herein in their
entireties.
[0222] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
[0223] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes, and is capable of expressing, both heavy and light
chain polypeptides. In such situations, the light chain should be
placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.
Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy
and light chains may comprise cDNA or genomic DNA.
[0224] Once an antibody molecule of the invention has been produced
by an animal, chemically synthesized, or recombinantly expressed,
it may be purified by any method known in the art for purification
of an immunoglobulin molecule, for example, by chromatography
(e.g., ion exchange, affinity, particularly by affinity for the
specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. In
addition, the antibodies of the present invention or fragments
thereof can be fused to heterologous polypeptide sequences
described herein or otherwise known in the art, to facilitate
purification.
[0225] The present invention encompasses antibodies recombinantly
fused or chemically conjugated (including both covalently and
non-covalently conjugations) to a polypeptide (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention to generate
fusion proteins. The fusion does not necessarily need to be direct,
but may occur through linker sequences. The antibodies may be
specific for antigens other than polypeptides (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in vitro immunoassays and purification
methods using methods known in the art. See e.g., Harbor et al.,
supra, and PCT publication WO 93/21232; EP 439,095; Naramura et
al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981;
Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.
146:2446-2452(1991), which are incorporated by reference in their
entireties.
[0226] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the constant region, hinge region, CH1 domain, CH2
domain, and CH3 domain or any combination of whole domains or
portions thereof. The polypeptides may also be fused or conjugated
to the above antibody portions to form multimers. For example, Fc
portions fused to the polypeptides of the present invention can
form dimers through disulfide bonding between the Fc portions.
Higher multimeric forms can be made by fusing the polypeptides to
portions of IgA and IgM. Methods for fusing or conjugating the
polypeptides of the present invention to antibody portions are
known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929;
5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166;
PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc.
Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad.
Sci. USA 89:11337-11341(1992) (said references incorporated by
reference in their entireties).
[0227] As discussed, supra, the polypeptides corresponding to a
polypeptide, polypeptide fragment, or a variant of SEQ ID NOS:2 or
4 may be fused or conjugated to the above antibody portions to
increase the in vivo half life of the polypeptides or for use in
immunoassays using methods known in the art. Further, the
polypeptides corresponding to SEQ ID NOS:2 or 4 may be fused or
conjugated to the above antibody portions to facilitate
purification. 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 394,827; Traunecker et
al., Nature 331:84-86 (1988). The polypeptides of the present
invention fused or conjugated to an antibody having
disulfide-linked dimeric structures (due to the IgG) may 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)). 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 232,262). Alternatively, deleting
the Fc part after the fusion protein has been expressed, detected,
and purified, would be desired. For example, the Fc portion may
hinder therapy and diagnosis if the fusion protein is used as an
antigen for immunizations. In drug discovery, for example, human
proteins, such as hIL-5, have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58
(1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[0228] Moreover, the antibodies or fragments thereof of the present
invention can be fused to marker sequences, such as a peptide to
facilitate purification. 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. Other peptide tags
useful for purification include, but are not limited to, the "HA"
tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the
"flag" tag.
[0229] The present invention further encompasses antibodies or
fragments thereof conjugated to a diagnostic or therapeutic agent.
The antibodies can be used diagnostically to, for example, monitor
the development or progression of a tumor as part of a clinical
testing procedure to, e.g., determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. The detectable substance may be coupled or conjugated
either directly to the antibody (or fragment thereof) or
indirectly, through an intermediate (such as, for example, a linker
known in the art) using techniques known in the art. See, for
example, U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include 1251, 131I, 111In or 99Tc.
[0230] Further, an antibody or fragment thereof may be conjugated
to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0231] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, a-interferon, .beta.-interferon, nerve growth
factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International Publication No. WO 99/23105), a thrombotic agent or
an anti-angiogenic agent, e.g., angiostatin or endostatin; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophage colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0232] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0233] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982).
[0234] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[0235] An antibody, with or without a therapeutic moiety conjugated
to it, administered alone or in combination with cytotoxic
factor(s) and/or cytokine(s) can be used as a therapeutic.
Immunophenotyping
[0236] The antibodies of the invention may be utilized for
immunophenotyping of cell lines and biological samples. The
translation product of the gene of the present invention may be
useful as a cell specific marker, or more specifically as a
cellular marker that is differentially expressed at various stages
of differentiation and/or maturation of particular cell types.
Monoclonal antibodies directed against a specific epitope, or
combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be
utilized using monoclonal antibodies to screen for cellular
populations expressing the marker(s), and include magnetic
separation using antibody-coated magnetic beads, "panning" with
antibody attached to a solid matrix (i.e., plate), and flow
cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al.,
Cell, 96:737-49 (1999)).
[0237] These techniques allow for the screening of particular
populations of cells, such as might be found with hematological
malignancies (i.e. minimal residual disease (MRD) in acute leukemic
patients) and "non-self" cells in transplantations to prevent
Graft-versus-Host Disease (GVHD). Alternatively, these techniques
allow for the screening of hematopoietic stem and progenitor cells
capable of undergoing proliferation and/or differentiation, as
might be found in human umbilical cord blood.
Assays For Antibody Binding
[0238] The antibodies of the invention may be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include but are not limited to
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffuision precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety). Exemplary immunoassays are described briefly below (but
are not intended by way of limitation).
[0239] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g., pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.16.1.
[0240] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32P or 125I) diluted in blocking buffer, washing the membrane in
wash buffer, and detecting the presence of the antigen. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected and to reduce the
background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0241] ELISAs comprise preparing antigen, coating the well of a 96
well microtiter plate with the antigen, adding the antibody of
interest conjugated to a detectable compound such as an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) to
the well and incubating for a period of time, and detecting the
presence of the antigen. In ELISAs the antibody of interest does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes the antibody of interest)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the antibody
may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the
addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1.
[0242] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., 3H or 125I) with the antibody of interest in the
presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody of interest for a particular antigen and
the binding off-rates can be determined from the data by scatchard
plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is
incubated with antibody of interest conjugated to a labeled
compound (e.g., 3H or 125I) in the presence of increasing amounts
of an unlabeled second antibody.
Therapeutic Uses
[0243] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the disclosed diseases,
disorders, or conditions. Therapeutic compounds of the invention
include, but are not limited to, antibodies of the invention
(including fragments, analogs and derivatives thereof as described
herein) and nucleic acids encoding antibodies of the invention
(including fragments, analogs and derivatives thereof and
anti-idiotypic antibodies as described herein). The antibodies of
the invention can be used to treat, inhibit or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of a polypeptide of the invention, including, but not
limited to, any one or more of the diseases, disorders, or
conditions described herein. The treatment and/or prevention of
diseases, disorders, or conditions associated with aberrant
expression and/or activity of a polypeptide of the invention
includes, but is not limited to, alleviating symptoms associated
with those diseases, disorders or conditions. Antibodies of the
invention may be provided in pharmaceutically acceptable
compositions as known in the art or as described herein.
[0244] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[0245] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors
(such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to
increase the number or activity of effector cells which interact
with the antibodies.
[0246] The antibodies of the invention may be administered alone or
in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and
anti-tumor agents). Generally, administration of products of a
species origin or species reactivity (in the case of antibodies)
that is the same species as that of the patient is preferred. Thus,
in a preferred embodiment, human antibodies, fragments derivatives,
analogs, or nucleic acids, are administered to a human patient for
therapy or prophylaxis.
[0247] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of disorders
related to polynucleotides or polypeptides, including fragments
thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an affinity for polynucleotides or
polypeptides of the invention, including fragments thereof.
Preferred binding affinities include those with a dissociation
constant or Kd less than 5.times.10.sup.-2 M, 10.sup.-2 M,
5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M,
5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M,
5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M,
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M, 5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, and
10.sup.-15 M.
Gene Therapy
[0248] In a specific embodiment, nucleic acids comprising sequences
encoding antibodies or functional derivatives thereof, are
administered to treat, inhibit or prevent a disease or disorder
associated with aberrant expression and/or activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded protein that
mediates a therapeutic effect.
[0249] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0250] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990).
[0251] In a preferred aspect, the compound comprises nucleic acid
sequences encoding an antibody, said nucleic acid sequences being
part of expression vectors that express the antibody or fragments
or chimeric proteins or heavy or light chains thereof in a suitable
host. In particular, such nucleic acid sequences have promoters
operably linked to the antibody coding region, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, nucleic acid molecules are used in
which the antibody coding sequences and any other desired sequences
are flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the antibody encoding nucleic acids (Koller and
Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al., Nature 342:435-438 (1989). In specific embodiments, the
expressed antibody molecule is a single chain antibody;
alternatively, the nucleic acid sequences include sequences
encoding both the heavy and light chains, or fragments thereof, of
the antibody.
[0252] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0253] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438
(1989)).
[0254] In a specific embodiment, viral vectors that contains
nucleic acid sequences encoding an antibody of the invention are
used. For example, a retroviral vector can be used (see Miller et
al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors
contain the components necessary for the correct packaging of the
viral genome and integration into the host cell DNA. The nucleic
acid sequences encoding the antibody to be used in gene therapy are
cloned into one or more vectors, which facilitates delivery of the
gene into a patient. More detail about retroviral vectors can be
found in Boesen et al., Biotherapy 6:291-302 (1994), which
describes the use of a retroviral vector to deliver the mdr1 gene
to hematopoietic stem cells in order to make the stem cells more
resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., J. Clin.
Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994);
Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and
Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114
(1993).
[0255] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development 3:499-503 (1993) present a review of adenovirus-based
gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155
(1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT
Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783
(1995). In a preferred embodiment, adenovirus vectors are used.
[0256] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med.
204:289-300 (1993); U.S. Pat. No. 5,436,146).
[0257] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0258] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen
et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther.
29:69-92m (1985) and may be used in accordance with the present
invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[0259] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0260] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as Tlymphocytes, Blymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0261] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0262] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an antibody are introduced
into the cells such that they are expressible by the cells or their
progeny, and the recombinant cells are then administered in vivo
for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which
can be isolated and maintained in vitro can potentially be used in
accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985
(1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow
and Scott, Mayo Clinic Proc. 61:771 (1986)).
[0263] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity
[0264] The compounds or pharmaceutical compositions of the
invention are preferably tested in vitro, and then in vivo for the
desired therapeutic or prophylactic activity, prior to use in
humans. For example, in vitro assays to demonstrate the therapeutic
or prophylactic utility of a compound or pharmaceutical composition
include, the effect of a compound on a cell line or a patient
tissue sample. The effect of the compound or composition on the
cell line and/or tissue sample can be determined utilizing
techniques known to those of skill in the art including, but not
limited to, rosette formation assays and cell lysis assays. In
accordance with the invention, in vitro assays which can be used to
determine whether administration of a specific compound is
indicated, include in vitro cell culture assays in which a patient
tissue sample is grown in culture, and exposed to or otherwise
administered a compound, and the effect of such compound upon the
tissue sample is observed.
Therapeutic/Prophylactic Administration and Composition
[0265] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention,
preferably an antibody of the invention. In a preferred aspect, the
compound is substantially purified (e.g., substantially free from
substances that limit its effect or produce undesired
side-effects). The subject is preferably an animal, including but
not limited to animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably
human.
[0266] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid or an
immunoglobulin are described above; additional appropriate
formulations and routes of administration can be selected from
among those described herein below.
[0267] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compounds or
compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0268] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
absorb.
[0269] In another embodiment, the compound or composition can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein,
ibid., pp. 317-327; see generally ibid.)
[0270] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. In one embodiment, a
pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61
(1983); see also Levy et al., Science 228:190 (1985); During et
al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.
71:105 (1989)). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, i.e.,
the brain, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)).
[0271] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0272] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0273] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0274] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0275] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0276] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a polypeptide of the invention can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0277] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation.
[0278] 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.
Optionally 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.
Diagnosis and Imaging
[0279] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to a polypeptide of interest can be used
for diagnostic purposes to detect, diagnose, or monitor diseases,
disorders, and/or conditions associated with the aberrant
expression and/or activity of a polypeptide of the invention. The
invention provides for the detection of aberrant expression of a
polypeptide of interest, comprising (a) assaying the expression of
the polypeptide of interest in cells or body fluid of an individual
using one or more antibodies specific to the polypeptide interest
and (b) comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of aberrant expression.
[0280] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of a particular disorder. With
respect to cancer, the presence of a relatively high amount of
transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a
means for detecting the disease prior to the appearance of actual
clinical symptoms. A more definitive diagnosis of this type may
allow health professionals to employ preventative measures or
aggressive treatment earlier thereby preventing the development or
further progression of the cancer.
[0281] Antibodies of the invention can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, et
al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell.
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful
for detecting protein gene expression include immunoassays, such as
the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase;
radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur
(35S), tritium (3H), indium (112In), and technetium (99Tc);
luminescent labels, such as luminol; and fluorescent labels, such
as fluorescein and rhodamine, and biotin.
[0282] One aspect of the invention is the detection and diagnosis
of a disease or disorder associated with aberrant expression of a
polypeptide of interest in an animal, preferably a mammal and most
preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled
molecule which specifically binds to the polypeptide of interest;
b) waiting for a time interval following the administering for
permitting the labeled molecule to preferentially concentrate at
sites in the subject where the polypeptide is expressed (and for
unbound labeled molecule to be cleared to background level); c)
determining background level; and d) detecting the labeled molecule
in the subject, such that detection of labeled molecule above the
background level indicates that the subject has a particular
disease or disorder associated with aberrant expression of the
polypeptide of interest. Background level can be determined by
various methods including, comparing the amount of labeled molecule
detected to a standard value previously determined for a particular
system.
[0283] It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of 99 mTc. The labeled antibody or antibody fragment
will then preferentially accumulate at the location of cells which
contain the specific protein. In vivo tumor imaging is described in
S. W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled
Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes,
eds., Masson Publishing Inc. (1982).
[0284] Depending on several variables, including the type of label
used and the mode of administration, the time interval following
the administration for permitting the labeled molecule to
preferentially concentrate at sites in the subject and for unbound
labeled molecule to be cleared to background level is 6 to 48 hours
or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval following administration is 5 to 20 days or 5 to 10
days.
[0285] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
[0286] Presence of the labeled molecule can be detected in the
patient using methods known in the art for in vivo scanning. These
methods depend upon the type of label used. Skilled artisans will
be able to determine the appropriate method for detecting a
particular label. Methods and devices that may be used in the
diagnostic methods of the invention include, but are not limited
to, computed tomography (CT), whole body scan such as position
emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0287] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the molecule is labeled with a
fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is
detected in the patent using positron emission-tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI).
Kits
[0288] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises an antibody of
the invention, preferably a purified antibody, in one or more
containers. In a specific embodiment, the kits of the present
invention contain a substantially isolated polypeptide comprising
an epitope which is specifically immunoreactive with an antibody
included in the kit. Preferably, the kits of the present invention
further comprise a control antibody which does not react with the
polypeptide of interest. In another specific embodiment, the kits
of the present invention contain a means for detecting the binding
of an antibody to a polypeptide of interest (e.g., the antibody may
be conjugated to a detectable substrate such as a fluorescent
compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the
first antibody may be conjugated to a detectable substrate).
[0289] In another specific embodiment of the present invention, the
kit is a diagnostic kit for use in screening serum containing
antibodies specific against proliferative and/or cancerous
polynucleotides and polypeptides. Such a kit may include a control
antibody that does not react with the polypeptide of interest. Such
a kit may include a substantially isolated polypeptide antigen
comprising an epitope which is specifically immunoreactive with at
least one anti-polypeptide antigen antibody. Further, such a kit
includes means for detecting the binding of said antibody to the
antigen (e.g., the antibody may be conjugated to a fluorescent
compound such as fluorescein or rhodamine which can be detected by
flow cytometry). In specific embodiments, the kit may include a
recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to
a solid support.
[0290] In a more specific embodiment the detecting means of the
above-described kit includes a solid support to which said
polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can
be detected by binding of the said reporter-labeled antibody.
[0291] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0292] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or calorimetric substrate (Sigma, St.
Louis, Mo.).
[0293] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0294] Thus, the invention provides an assay system or kit for
carrying out this diagnostic method. The kit generally includes a
support with surface-bound recombinant antigens, and a
reporter-labeled anti-human antibody for detecting surface-bound
anti-antigen antibody.
[0295] Fusion Proteins
[0296] Any VEGF-2 polypeptide can be used to generate fusion
proteins. For example, the VEGF-2 polypeptide, when fused to a
second protein, can be used as an antigenic tag. Antibodies raised
against the VEGF-2 polypeptide can be used to indirectly detect the
second protein by binding to the VEGF-2. Moreover, because secreted
proteins target cellular locations based on trafficking signals,
the VEGF-2 polypeptides can be used as a targeting molecule once
fused to other proteins.
[0297] Examples of domains that can be fused to VEGF-2 polypeptides
include not only heterologous signal sequences, but also other
heterologous functional regions. The fusion does not necessarily
need to be direct, but may occur through linker sequences.
[0298] Moreover, fusion proteins may also be engineered to improve
characteristics of the VEGF-2 polypeptide. For instance, a region
of additional amino acids, particularly charged amino acids, may be
added to the N-terminus of the VEGF-2 polypeptide to improve
stability and persistence during purification from the host cell or
subsequent handling and storage. Also, peptide moieties may be
added to the VEGF-2 polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the VEGF-2
polypeptide. The addition of peptide moieties to facilitate
handling of polypeptides are familiar and routine techniques in the
art.
[0299] Moreover, VEGF-2 polypeptides, including fragments, and
specifically epitopes, can be combined with parts of the constant
domain of immunoglobulins (IgG), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and
show an increased half-life in vivo. One reported example describes
chimeric proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins. (EP A 394,827;
Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having
disulfide-linked dimeric structures (due to the IgG) can also be
more efficient in binding and neutralizing other molecules, than
the monomeric secreted protein or protein fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)
[0300] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)
discloses fusion proteins comprising various portions of constant
region of immunoglobulin molecules together with another human
protein or part thereof. In many cases, the Fc part in a fusion
protein is beneficial in therapy and diagnosis, and thus can result
in, for example, improved pharmacokinetic properties. (EP-A 0232
262.) Alternatively, deleting the Fc part after the fusion protein
has been expressed, detected, and purified, would be desired. For
example, the Fc portion may hinder therapy and diagnosis if the
fusion protein is used as an antigen for immunizations. In drug
discovery, for example, human proteins, such as hIL-5, have been
fused with Fc portions for the 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).)
[0301] Moreover, the VEGF-2 polypeptides can be fused to marker
sequences, such as a peptide which facilitates purification of
VEGF-2. In preferred embodiments, the marker amino acid sequence is
a hexa-histidine peptide, such as the tag provided in a pQE vector
(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among
others, many of which are commercially available. As described in
Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for
instance, hexa-histidine provides for convenient purification of
the fusion protein. Another peptide tag useful for purification,
the "HA" tag, corresponds to an epitope derived from the influenza
hemagglutinin protein. (Wilson et al., Cell 37:767 (1984).)
[0302] Thus, any of these above fusions can be engineered using the
VEGF-2 polynucleotides or the polypeptides.
Vectors and Host Cells
[0303] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of VEGF-2 polypeptides or peptides by
recombinant techniques.
[0304] Host cells are genetically engineered (transduced,
transformed, or transfected) with the vectors of this invention
which may be, for example, a cloning vector or an expression
vector. The vector may be, for example, in the form of a plasmid, a
viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants, or amplifying the
VEGF-2 genes of the invention. The culture conditions, such as
temperature, pH and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
skilled artisan.
[0305] 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.
[0306] 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.
[0307] 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.
[0308] 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.
[0309] 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.
[0310] 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; and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from
Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,
pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,
pMSG and pSVL available from Pharmacia. Other suitable vectors will
be readily apparent to the skilled artisan.
[0311] 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.
[0312] As summarized in FIGS. 16 and 17, components of the pHE4a
vector (SEQ ID NO:9) include: 1) a neomycinphosphotransferase gene
as a selection marker, 2) an E. coli origin of replication, 3) a T5
phage promoter sequence, 4) two lac operator sequences, 5) a
Shine-Delgarno sequence, 6) the lactose operon repressor gene
(lacIq) and 7) a multiple cloning site linker region. The origin of
replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.).
The promoter sequence and operator sequences were made
synthetically. Synthetic production of nucleic acid sequences is
well known in the art. CLONTECH 95/96 Catalog, pages 215-216,
CLONTECH, 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.
[0313] 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,
BaniHI, 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.
[0314] As noted above, the pHE4a vector contains a lacIq gene.
LacIq is an allele of the lacI gene which confers tight regulation
of the lac operator. Amann, E. et al., Gene 69:301-315 (1988);
Stark, M., Gene 51:255-267 (1987). The lacIq gene encodes a
repressor protein which binds to lac operator sequences and blocks
transcription of down-stream (i.e., 3') sequences. However, the
lacIq gene product dissociates from the lac operator in the
presence of either lactose or certain lactose analogs, e.g.,
isopropyl B-D-thiogalactopyranoside (IPTG). VEGF-2 thus is not
produced in appreciable quantities in uninduced host cells
containing the pHE4a vector. Induction of these host cells by the
addition of an agent such as IPTG, however, results in the
expression of the VEGF-2 coding sequence.
[0315] The promoter/operator sequences of the pHE4a vector (SEQ ID
NO:10) 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.
[0316] 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.
[0317] Among known bacterial promoters suitable for use in the
production of proteins of the present invention include the E. coli
lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter,
the lambda PR and PL promoters and the trp promoter. Suitable
eukaryotic promoters include the CMV immediate early promoter, the
HSV thymidine kinase promoter, the early and late SV40 promoters,
the promoters of retroviral LTRs, such as those of the Rous Sarcoma
Virus (RSV), and metallothionein promoters, such as the mouse
metallothionein-I promoter.
[0318] 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. 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:9).
[0319] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacI, lacZ, T3, T7,
gpt, lambda 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-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art. In a further embodiment, the present
invention relates to host cells containing the above-described
construct. The host cell can be a higher eukaryotic cell, such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell,
or the host cell can be 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)).
[0320] 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.
[0321] 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.
[0322] 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.
[0323] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), a-factor, acid phosphatase, or heat shock proteins,
among others. The heterologous structural sequence is assembled in
appropriate phase with translation initiation and termination
sequences, and preferably, a leader sequence capable of directing
secretion of translated protein into the periplasmic space or
extracellular medium. Optionally, the heterologous sequence can
encode a fusion protein including an N-terminal identification
peptide imparting desired characteristics, e.g., stabilization or
simplified purification of expressed recombinant product.
[0324] 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.
[0325] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC.TM. 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia 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.
[0326] 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.
[0327] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification. Microbial cells employed in
expression of proteins can be disrupted by any convenient method,
well known to those skilled in the art, including freeze-thaw
cycling, sonication, mechanical disruption, or use of cell lysing
agents.
[0328] 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.
[0329] In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly mammalian origin, that have been engineered to
delete or replace endogenous genetic material (e.g., VEGF-2
sequence), and/or to include genetic material (e.g., heterologous
promoters) that is operably associated with VEGF-2 sequence of the
invention, and which activates, alters, and/or amplifies endogenous
VEGF-2 polynucleotides. For example, techniques known in the art
may be used to operably associate heterologous control regions and
endogenous polynucleotide sequences (e.g. encoding VEGF-2) via
homologous recombination (see, e.g., U.S. Pat. No. 5,641,670,
issued Jun. 24, 1997; International Publication No. WO 96/29411,
published Sep. 26, 1996; International Publication No. WO 94/12650,
published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438
(1989), the disclosures of each of which are incorporated by
reference in their entireties).
[0330] The host cell can be a higher eukaryotic cell, such as a
mammalian cell (e.g., a human derived cell), or a lower eukaryotic
cell, such as a yeast cell, or the host cell can be a prokaryotic
cell, such as a bacterial cell. The host strain may be chosen which
modulates the expression of the inserted gene sequences, or
modifies and processes the gene product in the specific fashion
desired. Expression from certain promoters can be elevated in the
presence of certain inducers; thus expression of the genetically
engineered polypeptide may be controlled. Furthermore, different
host cells have characteristics and specific mechanisms for the
translational and post-translational processing and modification
(e.g., glycosylation, phosphorylation, cleavage) of proteins.
Appropriate cell lines can be chosen to ensure the desired
modifications and processing of the protein expressed.
[0331] The polypeptides can be recovered and purified from
recombinant cell cultures by methods used heretofore, including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. It is
preferred to have low concentrations (approximately 0.1-5 mM) of
calcium ion present during purification (Price et al., J. Biol.
Chem. 244:917 (1969)). Protein refolding steps can be used, as
necessary, in completing configuration of the mature protein.
Finally, high performance liquid chromatography (HPLC) can be
employed for final purification steps.
[0332] 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.
VEGF-2 Agonist and Antagonists
[0333] This invention is also related to a method of screening
compounds to identify those which are VEGF-2 agonists or
antagonists. An example of such a method takes advantage of the
ability of VEGF-2 to significantly stimulate the proliferation of
human endothelial cells in the presence of the comitogen Con A.
Endothelial cells are obtained and cultured in 96-well
flat-bottomed culture plates (Costar, Cambridge, Mass.) in a
reaction mixture supplemented with Con-A (Calbiochem, La Jolla,
Calif.). Con-A, polypeptides of the present invention and the
compound to be screened are added. After incubation at 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.
[0334] To assay for antagonists, the assay described above is
performed and the ability of the compound to inhibit
.sup.3[H]thymidine incorporation in the presence of VEGF-2
indicates that the compound is an antagonist to VEGF-2.
Alternatively, VEGF-2 antagonists may be detected by combining
VEGF-2 and a potential antagonist with membrane-bound VEGF-2
receptors or recombinant receptors under appropriate conditions for
a competitive inhibition assay. VEGF-2 can be labeled, such as by
radioactivity, such that the number of VEGF-2 molecules bound to
the receptor can determine the effectiveness of the potential
antagonist.
[0335] Alternatively, the response of a known second messenger
system following interaction of VEGF-2 and receptor would be
measured and compared in the presence or absence of the compound.
Such second messenger systems include but are not limited to, cAMP
guanylate cyclase, ion channels or phosphoinositide hydrolysis. In
another method, a mammalian cell or membrane preparation expressing
the VEGF-2 receptor is incubated with labeled VEGF-2 in the
presence of the compound. The ability of the compound to enhance or
block this interaction could then be measured.
[0336] Potential VEGF-2 antagonists include an antibody, or in some
cases, an oligonucleotide, which bind to the polypeptide and
effectively eliminate VEGF-2 function. Alternatively, a potential
antagonist may be a closely related protein which binds to VEGF-2
receptors, however, they are inactive forms of the polypeptide and
thereby prevent the action of VEGF-2. Examples of these antagonists
include a negative dominant mutant of the VEGF-2 polypeptide, for
example, one chain of the hetero-dimeric form of VEGF-2 may be
dominant and may be mutated such that biological activity is not
retained. An example of a negative dominant mutant includes
truncated versions of a dimeric VEGF-2 which is capable of
interacting with another dimer to form wild type VEGF-2, however,
the resulting homo-dimer is inactive and fails to exhibit
characteristic VEGF activity.
[0337] Another potential VEGF-2 antagonist is an antisense
construct prepared using antisense technology. Antisense technology
can be used to control gene expression through triple-helix
formation or antisense DNA or RNA, both of which methods are based
on binding of a polynucleotide to DNA or RNA. For example, the 5'
coding portion of the polynucleotide sequence, which encodes for
the mature polypeptides of the present invention, is used to design
an antisense RNA oligonucleotide of from about 10 to 40 base pairs
in length. A DNA oligonucleotide is designed to be complementary to
a region of the gene involved in transcription (triple helix--see
Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science
241:456 (1988); and Dervan et al., Science 251:1360 (1991)),
thereby preventing transcription and the production of VEGF-2. The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into the VEGF-2 polypeptide
(Antisense-Okano, J. Neurochem.56:560 (1991); Oligodeoxynucleotides
as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton,
Fla. (1988)). The oligonucleotides described above can also be
delivered to cells such that the antisense RNA or DNA may be
expressed in vivo to inhibit production of VEGF-2. Potential VEGF-2
antagonists also include small molecules which bind to and occupy
the active site of the polypeptide thereby making the catalytic
site inaccessible to substrate such that normal biological activity
is prevented. Examples of small molecules include but are not
limited to small peptides or peptide-like molecules.
[0338] Antisense oligonucleotide technology provides a novel
approach to the inhibition of gene expression (see generally,
Agrawal (1992) Trends in Biotech. 10:152; Wagner (1994) Nature
372:333-335; and Stein et al. (1993) Science 261:1004-1012). By
binding to the complementary nucleic acid sequence (the sense
strand), antisense oligonucleotide are able to inhibit splicing and
translation of RNA. In this way, antisense oligonucleotides are
able to inhibit protein expression. Antisense oligonucleotides have
also been shown to bind to genomic DNA, forming a triplex, and
inhibit transcription. Furthermore, a 17 mer base sequence
statistically occurs only once in the human genome, and thus
extremely precise targeting of specific sequences is possible with
such antisense oligonucleotides.
[0339] The antagonists may be employed to limit angiogenesis
necessary for solid tumor metastasis. The identification of VEGF-2
can be used for the generation of certain inhibitors of vascular
endothelial growth factor. Since angiogenesis and
neovascularization are essential steps in solid tumor growth,
inhibition of angiogenic activity of the vascular endothelial
growth factor is very useful to prevent the further growth, retard,
or even regress solid tumors. Although the level of expression of
VEGF-2 is extremely low in normal tissues including breast, it can
be found expressed at moderate levels in at least two breast tumor
cell lines that are derived from malignant tumors. It is,
therefore, possible that VEGF-2 is involved in tumor angiogenesis
and growth.
[0340] Gliomas are also a type of neoplasia which may be treated
with the antagonists of the present invention.
[0341] 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.
[0342] The antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as hereinafter
described.
[0343] 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.
[0344] 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.
[0345] The antagonist/inhibitors may be employed in a composition
with a pharmaceutically acceptable carrier, e.g., as hereinabove
described. Moreover, as shown in Example 9, antibodies specific for
VEGF-2 may be combined with VEGF-2 polypeptides to increase
endothelial cell response. Endothelial cells responding to a
combination of VEGF-2 polypeptide and VEGF-2 specific antibodies
include vascular or lymphatic vessels. The combination of VEGF-2
specific antibodies and VEGF-2 polypeptide may be used to treat
individuals in need of an increase in proliferation of endothelial
cells, such as angiogenesis and/or lymphangiogenesis, as described
throughout the specification.
Therapeutic Applications of VEGF-2
[0346] As used in the section below, "VEGF-2" is intended to refer
to the full-length and mature forms of VEGF-2 polynucleotides and
polypeptides described herein and to the VEGF-2 analogs,
derivatives, and mutant polynucleotides and polypeptides described
herein.
[0347] The VEGF-2 polypeptide of the present invention is a mitogen
for photoreceptor cells. As shown in FIGS. 12-15, VEGF-2 increases
cell number, cell survival, rhodopsin expression, and the number of
rhodopsin cells in retinal cultures.
[0348] Accordingly, VEGF-2 may be employed to treat disorders of
the eye, including injuries and diseases. These disorders include
angioid streaks, retinitis pigmentosa, Keam's Syndrome, pigment
pattern dystrophies, retinal perforations, retinitis,
chorioretinitis, cytomegalovirus retinitis, acute retinal necrosis
syndrome, central alveolar choroidal dystrophy, dominant drusen,
hereditary hemorrhagic macular dystrophy, North Carolina macular
dystrophy, pericentral choroidal dystrophy, adult foveomacular
dystrophy, benign concentric annular macular dystrophy, central
aureolar pigment epithelial dystrophy, congenital macular coloboma,
dominantly inherited cystoid macular edema, familial foveal
retinoschisis, fenestrated sheen macular dystrophy, progressive
foveal dystrophy, slowly progressive macular dystrophy, Sorsby's
pseudoinflammatory dystrophy, cone-rod dystrophy, progressive cone
dystrophy, Leber's congenital amaurosis, Goldman-Favre syndrome,
Bardet-Biedl syndrome, Bassen-Komzweig syndrome
(abetalipoproteinemia), Best disease (vitelliform dystrophy),
choroidemia, gyrate atrophy, congenital amaurosis, Refsum syndrome,
Stargardt disease and Usher syndrome. Other retinopathies that may
benefit from VEGF-2 administration include age-related macular
degeneration (dry and wet forms), diabetic retinopathy, peripheral
vitreoretinopathies, photic retinopathies, surgery-induced
retinopathies, viral retinopathies (such as HIV retinopathy related
to AIDS), ischemic retinopathies, retinal detachment and traumatic
retinopathy.
[0349] VEGF-2 may be administered along with other proteins which
are therapeutic for eye cells, including, but not limited to:
retinoic acid, mitogens such as insulin, insulin-like growth
factors, epidermal growth factor, vasoactive growth factor,
pituitary adenylate cyclase activating polypeptide and
somatostatin; neurotrophic factors such as glial cell line-derived
neurotrophic factor, brain derived neurotrophic factor,
neurotrophin-3, neurotrophin-4/5, neurotrophin-6, insulin-like
growth factor, ciliary neurotrophic factor, acidic and basic
fibroblast growth factors, fibroblast growth factor-5, transforming
growth factor-beta, and cocaine-amphetamine regulated transcript
(CART); and other growth factors such as epidermal growth factor,
leukemia inhibitory factor, interleukins, interferons, and colony
stimulating factors; as well as molecules and materials which are
the functional equivalents to these factors.
[0350] Additionally, 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.
Pharmaceutical Compositions
[0351] The VEGF-2 polypeptides and polynucleotides of the present
invention may be employed in combination with a suitable
pharmaceutical carrier to comprise a pharmaceutical composition.
Such compositions comprise a therapeutically effective amount of
the polypeptide, polynucleotide, agonist or antagonist and a
pharmaceutically acceptable carrier or excipient. Such a carrier
includes, but is not limited to, antioxidants, preservatives,
coloring, flavoring and diluting agents, emulsifying agents,
suspending agents, solvents, fillers, bulking agents, buffers,
delivery vehicles, diluents, excipients and/or pharmaceutical
adjuvants. The formulation should suit the mode of administration.
For example, suitable vehicles include saline, buffered saline,
dextrose, water, glycerol, ethanol, and combinations thereof.
[0352] The primary solvent in a vehicle may be either aqueous or
non-aqueous in nature. In addition, the vehicle may contain other
pharmaceutically-acceptable excipients for modifying or maintaining
the pH, osmolarity, viscosity, clarity, color, sterility,
stability, rate of dissolution, or odor of the formulation.
Similarly, the vehicle may contain still other
pharmaceutically-acceptable excipients for modifying or maintaining
the rate of release of VEGF-2, or for promoting the absorption or
penetration of VEGF-2 across the membranes of the eye. Such
excipients are those substances usually and customarily employed to
formulate dosages for parenteral administration in either unit dose
or multi-dose form.
[0353] Once the therapeutic composition has been formulated, it may
be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or dehydrated or lyophilized powder. Such
formulations may be stored either in a ready to use form or in a
form, e.g., lyophilized, requiring reconstitution prior to
administration.
[0354] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such containers can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, the polypeptides, agonists and
antagonists of the present invention may be employed in conjunction
with other therapeutic compounds.
[0355] The VEGF-2 polypeptide or polynucleotide may be administered
in pharmaceutical compositions in combination with one or more
pharmaceutically acceptable excipients. It will be understood that,
when administered to a human patient, the total daily usage of the
pharmaceutical compositions of the present invention will be
decided by the attending physician within the scope of sound
medical judgment. The specific therapeutically effective dose level
for any particular patient will depend upon a variety of factors
including the type and degree of the response to be achieved; the
specific composition an other agent, if any, employed; the age,
body weight, general health, sex and diet of the patient; the time
of administration, route of administration, and rate of excretion
of the composition; the duration of the treatment; drugs (such as a
chemotherapeutic agent) used in combination or coincidental with
the specific composition; and like factors well known in the
medical arts. Suitable formulations, known in the art, can be found
in Remington's Pharmaceutical Sciences (latest edition), Mack
Publishing Company, Easton, Pa.
[0356] The VEGF-2 composition to be used in the therapy will be
formulated and dosed in a fashion consistent with good medical
practice, taking into account the clinical condition of the
individual patient (especially the side effects of treatment with
VEGF-2 alone), the site of delivery of the VEGF-2 composition, the
method of administration, the scheduling of administration, and
other factors known to practitioners. The "effective amount" of
VEGF-2 for purposes herein is thus determined by such
considerations.
[0357] The pharmaceutical compositions may be administered in a
convenient manner such as by the oral, topical, intravenous,
intraperitoneal, intramuscular, intraarticular, subcutaneous,
intranasal, intratracheal, intraocular or intradermal routes. The
pharmaceutical compositions are administered in an amount which is
effective for treating and/or prophylaxis of the specific
indication. In most cases, the VEGF-2 dosage is from about 1
.mu.g/kg to about 30 mg/kg body weight daily, taking into account
the routes of administration, symptoms, etc. However, the dosage
can be as low as 0.001 .mu.g/kg. For example, in the specific case
of topical administration dosages are preferably administered from
about 0.01 .mu.g to 9 mg per cm.sup.2. In the case of intraocular
administration, dosages are preferably administered from about
0.001 .mu.g/ml to about 10 mg/ml, and more preferably from about
0.05 mg/ml to about 4 mg/ml.
[0358] As a general proposition, the total pharmaceutically
effective amount of the VEGF-2 administered parenterally per more
preferably dose will be in the range of about 1 .mu.g/kg/day to 100
mg/kg/day of patient body weight, although, as noted above, this
will be subject to therapeutic discretion. If given continuously,
the VEGF-2 is typically administered at a dose rate of about 1
.mu.g/kg/hour to about 50 .mu.g/kg/hour, either by 1-4 injections
per day or by continuous subcutaneous infusions, for example, using
a mini-pump. An intravenous bag solution or bottle solution may
also be employed.
[0359] VEGF-2 is also suitably administered by sustained-release
systems. Suitable examples of sustained-release compositions
include semi-permeable polymer matrices in the form of shaped
articles, e.g., films, or mirocapsules. Sustained-release matrices
include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U.
Sidman et al., Biopolymers 22:547-556 (1983)), poly(2-hydroxyethyl
methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277
(1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene
vinyl acetate (R. Langer et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
VEGF-2 compositions also include liposomally entrapped VEGF-2.
Liposomes containing VEGF-2 are prepared by methods known per se:
DE 3,218,121; Epstein, et al., Proc. Natl. Acad. Sci. USA
82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA
77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949;
EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045
and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the
small (about 200-800 Angstroms) unilamellar type in which the lipid
content is greater than about 30 mol. percent cholesterol, the
selected proportion being adjusted for the optimal VEGF-2
therapy.
[0360] For parenteral administration, in one embodiment, the VEGF-2
is formulated generally by mixing it at the desired degree of
purity, in a unit dosage injectable form (solution, suspension, or
emulsion), with a pharmaceutically acceptable carrier, i.e., one
that is non-toxic to recipients at the dosages and concentrations
employed and is compatible with other ingredients of the
formulation. For example, the formulation preferably does not
include oxidizing agents and other compounds that are known to be
deleterious to polypeptides.
[0361] In certain embodiments, VEGF-2 is administered orally.
VEGF-2 which is administered in this fashion may be encapsulated
and may be formulated with or without those carriers customarily
used in the compounding of solid dosage forms. The capsule may be
designed to release the active portion of the formulation at the
point in the gastrointestinal tract when bioavailability is
maximized and pre-systemic degradation is minimized. Additional
excipients may be included to facilitate absorption of VEGF-2.
Diluents, flavorings, low melting point waxes, vegetable oils,
lubricants, suspending agents, tablet disintegrating agents, and
binders may also be employed.
[0362] VEGF-2 may also be administered to the eye to treat
photoreceptor injuries, disorders and pathologies in animals and
humans as a drop, or within ointments, gels, liposomes,
microparticulates, or biocompatible polymer discs, pellets or
carried within contact lenses. The intraocular composition may also
contain a physiologically compatible ophthalmic vehicle as those
skilled in the art can select using conventional criteria. The
vehicles may be selected from the known ophthalmic vehicles which
include but are not limited to water, polyethers such as
polyethylene glycol 400, polyvinyls such as polyvinyl alcohol,
povidone, cellulose derivatives such as carboxymethylcellulose,
methylcellulose and hydroxypropyl methylcellulose, petroleum
derivatives such as mineral oil and white petrolatum, animal fats
such as lanolin, vegetable fats such as peanut oil, polymers of
acrylic acid such as carboxylpolymethylene gel, polysaccharides
such as dextrans and glycosaminoglycans such as sodium chloride and
potassium, chloride, zinc chloride and buffer such as sodium
bicarbonate or sodium lactate. High molecular weight molecules can
also be used. Physiologically compatible preservatives which do not
inactivate the VEGF-2 present in the composition include alcohols
such as chlorobutanol, benzalknonium chloride and EDTA, or any
other appropriate preservative known to those skilled in the
art.
[0363] For example, VEGF-2 may be administered directly
intraocularly from about 1 .mu.g/eye to about 1 mg/eye in a single
injection or in multiple injections. The formulation of topical
ophthalmic preparations, including ophthalmic solutions,
suspensions and ointments is well known to those skilled in the art
(see Remington's Pharmaceutical Sciences, 18th Edition, Chapter 86,
pages 1581-1592, Mack Publishing Company, 1990). Other modes of
administration are available, including intracameral injections
(which may be made directly into the anterior chamber or directly
into the vitreous chamber), subconjunctival injections and
retrobulbar injections, and methods and means for producing
ophthalmic preparations suitable for such modes of administration
are also well known. VEGF-2 may also be administered in the
subretinal space between the photoreceptor layer and retinal
pigmentosa epithelial layers.
[0364] As used herein, "extraocular" refers to the ocular surface
and the (external) space between the eyeball and the eyelid.
Examples of extraocular regions include the eyelid fomix or
cul-de-sac, the conjunctival surface and the corneal surface. This
location is external to all ocular tissue and an invasive procedure
is not required to access this region. Examples of extraocular
systems include inserts and "topically" applied drops, gels or
ointments which may be used to deliver therapeutic material to
these regions. Extraocular devices are generally easily removable,
even by the patient.
[0365] The following patents disclose extraocular systems which are
used to administer drugs to the extraocular regions. Higuchi et al.
disclose in U.S. Pat. Nos. 3,981,303, 3,986,510 and 3,995,635 a
biodegradable ocular insert which contains a drug. The insert can
be made in different shapes for retention in the cul-de-sac of the
eyeball, the extraocular space between the eyeball and the eyelid.
Several common biocompatible polymers are disclosed as suitable for
use in fabricating this device. These polymers include zinc
alginate, poly(lactic acid), poly(vinyl alcohol), poly(anhydrides)
and poly(glycolic acid). The patents also describe membrane coated
devices with reduced permeation to the drug and hollow chambers
holding the drug formulation.
[0366] U.S. Pat. No. 4,217,898, discloses microporous reservoirs
which are used for controlled drug delivery. These devices are
placed extraocularly in the ocular cul-de-sac. Among the polymer
systems of interest are poly(vinylchloride)-co-poly(vinyl
acetate)copolymers. Kaufinan discloses in U.S. Pat. Nos. 4,865,846
and 4,882,150 an ophthalmic drug delivery system which contains at
least one bio-erodible material or ointment carrier for the
conjunctival sac. The patent discloses polymer systems, such as
poly(lactide), poly(glycolide), poly(vinyl alcohol) and cross
linked collagen as suitable delivery systems.
[0367] In the presently described use of VEGF-2 of the treatment of
retinal disease or injury it is also advantageous that a topically
applied ophthalmic formulation include an agent to promote the
penetration or transport of the therapeutic agent into the eye.
Such agents are known in the art. For example, Ke et al., U.S. Pat.
No. 5,221,696 disclose the use of materials to enhance the
penetration of ophthalmic preparations through the cornea.
[0368] Intraocular systems are those systems which are suitable for
use in any tissue compartment within, between or around the tissue
layers of the eye itself. These locations include subconjunctival
(under the ocular mucous membrane adjacent to the eyeball), orbital
(behind the eyeball), and intracameral (within the chambers of the
eyeball itself). In contrast to extraocular systems, an invasive
procedure consisting of injection or implantation is required to
access these regions.
[0369] The following patents disclose intraocular devices. Wong,
U.S. Pat. No. 4,853,224, discloses microencapsulated drugs for
introduction into the chamber of the eye. Polymers which are used
in this system include polyesters and polyethers. Lee, U.S. Pat.
No. 4,863,457, discloses a biodegradable device which is surgically
implanted intraocularly for the sustained release of therapeutic
agents. The device is designed for surgical implantation under the
conjunctiva (mucous membrane of the eyeball). Krezancaki, U.S. Pat.
No. 4,188,373, discloses a pharmaceutical vehicle which gels at
human body temperature. This vehicle is an aqueous suspension of
the drug and gums or cellulose derived synthetic derivatives.
Haslam et al. disclose in U.S. Pat. Nos. 4,474,751 and 4,474,752 a
polymer-drug system which is liquid at room temperature and gels at
body temperature. Suitable polymers used in this system include
polyoxyethylene and polyoxypropylene. Davis et al. disclose in U.S.
Pat. No. 5,384,333 a biodegradable injectable drug delivery polymer
which provides long term drug release. The drug composition is made
up of a pharmaceutically active agent in a biodegradable polymer
matrix, where the polymer matrix is a solid at temperatures in the
range 20 EC to 37 EC, and is flowable at temperatures in the range
38 EC to 52 EC. The drug delivery polymer is not limited to the
delivery of soluble or liquid drug formulations. For example, the
polymer can be used as a matrix for stabilizing and retaining at
the site of injection drug-containing microspheres, liposomes or
other particulate-bound drugs.
[0370] A particularly suitable vehicle for intraocular injection is
sterile distilled water in which VEGF-2 is formulated as a sterile,
isotonic solution, properly preserved. Yet another ophthalmic
preparation may involve the formulation of VEGF-2 with an agent,
such as injectable microspheres or liposomes, that provides for the
slow or sustained release of the protein which may then be
delivered as a depot injection. Other suitable means for the
intraocular introduction of VEGF-2 includes implantable drug
delivery devices which contain VEGF-2.
[0371] The ophthalmic preparations of the present invention,
particularly topical preparations, may include other components,
for example ophthalmically acceptable preservatives, tonicity
agents, cosolvents, wetting agents, complexing agents, buffering
agents, antimicrobials, antioxidants and surfactants, as are well
known in the art. For example, suitable tonicity enhancing agents
include alkali metal halides (preferably sodium or potassium
chloride), mannitol, sorbitol and the like. Sufficient tonicity
enhancing agent is advantageously added so that the formulation to
be instilled into the eye is hypotonic or substantially isotonic.
Suitable preservatives include, but are not limited to,
benzalkonium chloride, thimerosal, phenethyl alcohol,
methylparaben, propylparaben, chlorhexidine, sorbic acid and the
like. Hydrogen peroxide may also be used as preservative. Suitable
cosolvents include, but are not limited to, glycerin, propylene
glycol and polyethylene glycol. Suitable complexing agents include
caffeine, polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting
agents include, but are not limited to, sorbitan esters,
polysorbates such as polysorbate 80, tromethamine, lecithin,
cholesterol, tyloxapol and the like. The buffers can be
conventional buffers such as borate, citrate, phosphate,
bicarbonate, or Tris-HCl.
[0372] The formulation components are present in concentration that
are acceptable to the extraocular or intraocular site of
administration. For example, buffers are used to maintain the
composition at physiological pH or at slightly lower pH, typically
within a pH range of from about 5 to about 8.
[0373] Additional formulation components may include materials
which provide for the prolonged ocular residence of the
extraocularly administered therapeutic agent so as to maximize the
topical contact and promote absorbtion. Suitable materials include
polymers or gel forming materials which provide for increased
viscosity of the ophthalmic preparation. Chitosan is a particularly
suitable material as an ocular release-rate controlling agent in
sustained release liquid ophthalmic drug formulations (see U.S.
Pat. No. 5,422,116, Yen, et. al.) The suitability of the
formulations of the instant invention for controlled release (e.g.,
sustained and prolonged delivery) of an ophthalmic treating agent
in the eye can be determined by various procedures known in the
art, e.g., as described in Journal of Controlled Release 6:367-373,
1987, as well as variations thereof.
[0374] Yet another ophthalmic preparation may involve an effective
quantity of VEGF-2 in a mixture with non-toxic ophthalmically
acceptable excipients which are suitable for the manufacture of
tablets. By dissolving the tablets in sterile water, or other
appropriate vehicle, ophthalmic solutions can be prepared in unit
dose form. Suitable excipients include, but are not limited to,
inert diluents, such as calcium carbonate, sodium carbonate or
bicarbonate, lactose, or calcium phosphate; or binding agents, such
as starch, gelatin, or acacia; or lubricating agents such as
magnesium stearate, stearic acid, or talc.
[0375] Generally, the formulations are prepared by contacting the
VEGF-2 uniformly and intimately with liquid carriers or finely
divided solid carriers or both. Then, if necessary, the product is
shaped into the desired formulation. Preferably the carrier is a
parenteral carrier, more preferably a solution that is isotonic
with the blood of the recipient. Examples of such carrier vehicles
include water, saline, Ringer's solution, and dextrose solution.
Non-aqueous vehicles such as fixed oils and ethyl oleate are also
useful herein, as well as liposomes. Suitable formulations, known
in the art, can be found in Remington's Pharmaceutical Sciences
(latest edition), Mack Publishing Company, Easton, Pa.
[0376] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, mannose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[0377] VEGF-2 is typically formulated in such vehicles at a
concentration of about 0.01 .mu.g/ml to 100 mg/ml, preferably 0.01
.mu.g/ml to 10 mg/ml, at a pH of about 3 to 8. It will be
understood that the use of certain of the foregoing excipients,
carriers, or stabilizers will result in the formation of VEGF-2
salts.
[0378] VEGF-2 to be used for therapeutic administration must be
sterile. Sterility is readily accomplished by filtration through
sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutic VEGF-2 compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0379] VEGF-2 ordinarily will be stored in unit or multi-dose
containers, for example, sealed ampules or vials, as an aqueous
solution or as a lyophilized formulation for reconstitution. As an
example of a lyophilized formulation, 10 ml vials are filled with 5
ml of sterile-filtered 1% (w/v) aqueous VEGF-2 solution, and the
resulting mixture is lyophilized. The infusion solution is prepared
by reconstituting the lyophilized VEGF-2 using bacteriostatic
Water-for-Injection.
Gene Therapy Methods
[0380] Another aspect of the present invention is to gene therapy
methods for treating disorders, diseases and conditions. The gene
therapy methods relate to the introduction of nucleic acid (DNA,
RNA and antisense DNA or RNA) sequences into an animal to achieve
expression of the VEGF-2 polypeptide of the present invention. This
method requires a polynucleotide which codes for a VEGF-2
polypeptide operatively linked to a promoter and any other genetic
elements necessary for the expression of the polypeptide by the
target tissue. Such gene therapy and delivery techniques are known
in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat.
Nos. 5,693,622, 5,705,151, 5,580,859; Tabata H. et al. (1997)
Cardiovasc. Res. 35(3):470-479, Chao, J et al. (1997) Pharmacol.
Res. 35(6):517-522, Wolff, J. A. (1997) Neuromuscul. Disord.
7(5):314-318, Schwartz, B. et al. (1996) Gene Ther. 3(5):405-411,
Tsurumi, Y. et al. (1996) Circulation 94(12):3281-3290
(incorporated herein by reference).
[0381] As discussed more fully below, the VEGF-2 polynucleotide
sequences preferably have a therapeutic effect after being taken up
by a cell. Examples of polynucleotides that are themselves
therapeutic are anti-sense DNA and RNA; DNA coding for an
anti-sense RNA; or DNA coding for tRNA or rRNA to replace defective
or deficient endogenous molecules. For example, a promoter may be
operably linked to a DNA sequence encoding for an antisense RNA.
The antisense RNA oligonucleotide hybridizes to the mRNA in vivo
and blocks translation of an mRNA molecule into a polypeptide
(Okano, J. Neurochem 56:560 (1991)). The antisense RNA must be of
sufficient length and complementarity to prevent translation of its
target mRNA.
[0382] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) comprising a promoter operably
linked to a VEGF-2 polynucleotide ex vivo, with the engineered
cells then being provided to a patient to be treated with the
polypeptide. Such methods are well-known in the art. For example,
see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216
(1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112
(1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994);
Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et
al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al.,
Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene
Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene
Therapy 3: 31-38 (1996)), which are herein incorporated by
reference. In one embodiment, the cells which are engineered are
photoreceptor cells. These engineered cells may be reintroduced
into the patient through direct injection to the tissue of origin,
the tissues surrounding the tissue of origin, veins or arteries, or
through catheter injection. In one embodiment, the engineered cells
are attached to the sclera to produce and release VEGF-2 protein
directly into the vitreous humor.
[0383] Photoreceptor cell transplantation studies designed to
replace defective or lost cells due to retinal disease or damage
have been performed successfully in animal models of retinal
degeneration (Silverman and Hughes, Invest. Ophthalmol. Vis. Sci.
30:1684-1690(1989); Gouras et al., Neuro-Ophthalmol. 10:165-176
(1990)). It is contemplated that photoreceptor cells may be
obtained from donor eyes and maintained in culture as described
herein. The cells would then be used as a source of purified
photoreceptors to be transplanted via the subretinal space into the
retina of patients suffering from retinal disease or damage. These
patients will be treated with immunosuppressive therapies to
eliminate immunological responses and rejection of the grafted
cells. The ex vivo donor retinas will be cultured in the presence
of VEGF-2, in order to enhance their growth and survival. The
patients that will receive photoreceptor cell transplants will be
treated with intravitreal VEGF-2 needed to promote the survival and
the maturation of the grafted photoreceptors.
[0384] As discussed in more detail below, the VEGF-2 polynucleotide
constructs can be delivered by any method that delivers injectable
materials to the cells of an animal, such as, injection into the
interstitial space of tissues (heart, muscle, skin, lung, liver,
and the like). The VEGF-2 polynucleotide constructs may be
delivered in a pharmaceutically acceptable liquid or aqueous
carrier.
[0385] In one embodiment, the VEGF-2 polynucleotide is delivered as
a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA
refers to sequences that are free from any delivery vehicle that
acts to assist, promote or facilitate entry into the cell,
including viral sequences, viral particles, liposome formulations,
lipofectin.TM. or precipitating agents and the like. However, the
VEGF-2 polynucleotides can also be delivered in liposome
formulations and lipofectin.TM. formulations and the like can be
prepared by methods well known to those skilled in the art. Such
methods are described, for example, in U.S. Pat. Nos. 5,593,972,
5,589,466, and 5,580,859, which are herein incorporated by
reference. U. S. Pat. No. 5,770,580 describes gene therapy methods
for delivery into the eye.
[0386] The VEGF-2 polynucleotide vector constructs used in the gene
therapy method are preferably constructs that will not integrate
into the host genome nor will they contain sequences that allow for
replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44,
pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL
available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2
available from Invitrogen. Other suitable vectors will be readily
apparent to the skilled artisan.
[0387] Any strong promoter known to those skilled in the art can be
used for driving the expression of VEGF-2 DNA. Suitable promoters
include adenoviral promoters, such as the adenoviral major late
promoter; or heterologous promoters, such as the cytomegalovirus
(CMV) promoter; the respiratory syncytial virus (RSV) promoter;
inducible promoters, such as the MMT promoter, the metallothionein
promoter; heat shock promoters; the albumin promoter; the ApoAI
promoter; human globin promoters; viral thymidine kinase promoters,
such as the Herpes Simplex thymidine kinase promoter; retroviral
LTRs; the b-actin promoter; and human growth hormone promoters. The
promoter also may be the native promoter for VEGF-2.
[0388] Unlike other gene therapy techniques, one major advantage of
introducing naked nucleic acid sequences into target cells is the
transitory nature of the polynucleotide synthesis in the cells.
Studies have shown that non-replicating DNA sequences can be
introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
[0389] 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. Especially preferred is
the eye. Interstitial space of the tissues comprises the
intercellular, fluid, mucopolysaccharide matrix among the reticular
fibers of organ tissues, elastic fibers in the walls of vessels or
chambers, collagen fibers of fibrous tissues, or that same matrix
within connective tissue ensheathing muscle cells or in the lacunae
of bone. It is similarly the space occupied by the plasma of the
circulation and the lymph fluid of the lymphatic channels. They may
be conveniently delivered by injection into the tissues comprising
these cells. They are preferably delivered to and expressed in
persistent, non-dividing cells which are differentiated, although
delivery and expression may be achieved in non-differentiated or
less completely differentiated cells, such as, for example, stem
cells of blood or skin fibroblasts. In vivo muscle cells are
particularly competent in their ability to take up and express
polynucleotides.
[0390] 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.
[0391] The preferred route of administration is by the parenteral
route of injection into the interstitial space of tissues,
especially the eye. 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. The naked polynucleotides are delivered by also be
delivered by topical administration and so-called "gene guns".
These delivery methods are known in the art.
[0392] The constructs may also be delivered with delivery vehicles
such as viral sequences, viral particles, liposome formulations,
lipofectin.TM., precipitating agents, etc. Such methods of delivery
are known in the art.
[0393] In certain embodiments, the VEGF-2 polynucleotide constructs
are complexed in a liposome preparation. Liposomal preparations for
use in the instant invention include cationic (positively charged),
anionic (negatively charged) and neutral preparations. However,
cationic liposomes are particularly preferred because a tight
charge complex can be formed between the cationic liposome and the
polyanionic nucleic acid. Cationic liposomes have been shown to
mediate intracellular delivery of plasmid DNA (Felgner et al.,
Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein
incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad.
Sci. USA (1989) 86:6077-6081, which is herein incorporated by
reference); and purified transcription factors (Debs et al., J.
Biol. Chem. (1990) 265:10189-10192, which is herein incorporated by
reference), in functional form.
[0394] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are particularly useful and are available under the trademark
Lipofectin.TM., from GIBCO BRL, Grand Island, N.Y. (See, also,
Felgner et al., Proc. Natl Acad. Sci. USA (1987) 84:7413-7416,
which is herein incorporated by reference). Other commercially
available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
[0395] Other cationic liposomes can be prepared from readily
available materials using techniques well known in the art. See,
e.g. PCT Publication No. WO 90/11092 (which is herein incorporated
by reference) for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)liposomes.
Preparation of DOTMA liposomes is explained in the literature, see,
e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417,
which is herein incorporated by reference. Similar methods can be
used to prepare liposomes from other cationic lipid materials.
[0396] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl, choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0397] For example, commercially dioleoylphosphatidyl choline
(DOPC), dioleoylphosphatidyl glycerol (DOPG), and
dioleoylphosphatidyl ethanolamine (DOPE) can be used in various
combinations to make conventional liposomes, with or without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be prepared by drying 50 mg each of DOPG and DOPC under a stream of
nitrogen gas into a sonication vial. The sample is placed under a
vacuum pump overnight and is hydrated the following day with
deionized water. The sample is then sonicated for 2 hours in a
capped vial, using a Heat Systems model 350 sonicator equipped with
an inverted cup (bath type) probe at the maximum setting while the
bath is circulated at 15 EC. Alternatively, negatively charged
vesicles can be prepared without sonication to produce
multilamellar vesicles or by extrusion through nucleopore membranes
to produce unilamellar vesicles of discrete size. Other methods are
known and available to those of skill in the art. The liposomes can
comprise multilamellar vesicles (MLVs), small unilamellar vesicles
(SUVs), or large unilamellar vesicles (LUVs), with SUVs being
preferred. The various liposome-nucleic acid complexes are prepared
using methods well known in the art. See, e.g., Straubinger et al.,
Methods of Immunology (1983), 101:512-527, which is herein
incorporated by reference. For example, MLVs containing nucleic
acid can be prepared by depositing a thin film of phospholipid on
the walls of a glass tube and subsequently hydrating with a
solution of the material to be encapsulated. SUVs are prepared by
extended sonication of MLVs to produce a homogeneous population of
unilamellar liposomes. The material to be entrapped is added to a
suspension of preformed MLVs and then sonicated.
[0398] When using liposomes containing cationic lipids, the dried
lipid film is resuspended in an appropriate solution such as
sterile water or an isotonic buffer solution such as 10 mM
Tris/NaCl, sonicated, and then the preformed liposomes are mixed
directly with the DNA. The liposome and DNA form a very stable
complex due to binding of the positively charged liposomes to the
cationic DNA. SUVs find use with small nucleic acid fragments. LUVs
are prepared by a number of methods, well known in the art.
Commonly used methods include Ca.sup.2+-EDTA chelation
(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483;
Wilson et al., Cell (1979) 17:77); ether injection (Deamer, D. and
Bangham, A., Biochim. Biophys. Acta (1976) 443:629; Ostro et al.,
Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., Proc.
Natl. Acad. Sci. USA (1979) 76:3348); detergent dialysis (Enoch, H.
and Strittmatter, P., Proc. Natl. Acad. Sci. USA (1979) 76:145);
and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem.
(1980) 255:10431; Szoka, F. and Papahadjopoulos, D., Proc. Natl.
Acad. Sci. USA (1978) 75:145; Schaefer-Ridder et al., Science
(1982) 215:166), which are herein incorporated by reference.
[0399] Generally, the ratio of DNA to liposomes will be from about
10:1 to about 1:10. Preferably, the ration will be from about 5:1
to about 1:5. More preferably, the ration will be about 3:1 to
about 1:3. Still more preferably, the ratio will be about 1:1.
[0400] U.S. Pat. No. 5,676,954 (which is herein incorporated by
reference) reports on the injection of genetic material, complexed
with cationic liposomes carriers, into mice. U.S. Pat. Nos.
4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622,
5,580,859, 5,703,055, and international publication no. WO 94/9469
(which are herein incorporated by reference) provide cationic
lipids for use in transfecting DNA into cells and mammals. U.S.
Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and
international publication no. WO 94/9469 (which are herein
incorporated by reference) provide methods for delivering
DNA-cationic lipid complexes to mammals.
[0401] In certain embodiments, cells are be engineered, ex vivo or
in vivo, using a retroviral particle containing RNA which comprises
a sequence encoding VEGF-2. Retroviruses from which the retroviral
plasmid vectors may be derived include, but are not limited to,
Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma
Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape
leukemia virus, human immunodeficiency virus, Myeloproliferative
Sarcoma Virus, and mammary tumor virus.
[0402] 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, .phi.-2, .phi.-AM, PA12, T19-14X,
VT-19-17-H2, .phi.CRE, .phi.CRIP, GP+E-86, GP+envAm12, and DAN cell
lines as described in Miller, Human Gene Therapy 1:5-14 (1990),
which is incorporated herein by reference in its entirety. The
vector may transduce the packaging cells through any means known in
the art. Such means include, but are not limited to,
electroporation, the use of liposomes, and CaPO.sub.4
precipitation. In one alternative, the retroviral plasmid vector
may be encapsulated into a liposome, or coupled to a lipid, and
then administered to a host.
[0403] The producer cell line generates infectious retroviral
vector particles which include polynucleotide encoding VEGF-2. Such
retroviral vector particles then may be employed, to transduce
eukaryotic cells, either in vitro or in vivo. The transduced
eukaryotic cells will express VEGF-2.
[0404] In certain other embodiments, cells are engineered, ex vivo
or in vivo, with VEGF-2 polynucleotide contained in an adenovirus
vector. Adenovirus can be manipulated such that it encodes and
expresses VEGF-2, and at the same time is inactivated in terms of
its ability to replicate in a normal lytic viral life cycle.
Adenovirus expression is achieved without integration of the viral
DNA into the host cell chromosome, thereby alleviating concerns
about insertional mutagenesis. Furthermore, adenoviruses have been
used as live enteric vaccines for many years with an excellent
safety profile (Schwartz, A. R. et al. (1974) Am. Rev. Respir. Dis.
109:233-238). Finally, adenovirus mediated gene transfer has been
demonstrated in a number of instances including transfer of
alpha-1-antitrypsin and CFTR to the lungs of cotton rats
(Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld et
al., (1992) Cell 68:143-155). Furthermore, extensive studies to
attempt to establish adenovirus as a causative agent in human
cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl.
Acad. Sci. USA 76:6606).
[0405] Suitable adenoviral vectors useful in the present invention
are described, for example, in Kozarsky and Wilson, Curr. Opin.
Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155
(1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature
365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein
incorporated by reference. For example, the adenovirus vector Ad2
is useful and can be grown in human 293 cells. These cells contain
the E1 region of adenovirus and constitutively express E1a and E1b,
which complement the defective adenoviruses by providing the
products of the genes deleted from the vector. In addition to Ad2,
other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also
useful in the present invention.
[0406] Preferably, the adenoviruses used in the present invention
are replication deficient. Replication deficient adenoviruses
require the aid of a helper virus and/or packaging cell line to
form infectious particles. The resulting virus is capable of
infecting cells and can express the VEGF-2 polynucleotide of
interest which is operably linked to a promoter, but cannot
replicate in most cells. Replication deficient adenoviruses may be
deleted in one or more of all or a portion of the following genes:
E1a, E1b, E3, E4, E2a, or L1 through L5.
[0407] In certain other embodiments, the cells are engineered, ex
vivo or in vivo, using an adeno-associated virus (AAV). AAVs are
naturally occurring defective viruses that require helper viruses
to produce infectious particles (Muzyczka, N., Curr. Topics in
Microbiol. Immunol. 158:97 (1992)). It is also one of the few
viruses that may integrate its DNA into non-dividing cells. Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate, but space for exogenous DNA is limited to about 4.5
kb. Methods for producing and using such AAVs are known in the art.
See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
[0408] For example, an appropriate AAV vector for use in the
present invention will include all the sequences necessary for DNA
replication, encapsidation, and host-cell integration. The VEGF-2
polynucleotide construct is inserted into the AAV vector using
standard cloning methods, such as those found in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press
(1989). The recombinant AAV vector is then transfected into
packaging cells which are infected with a helper virus, using any
standard technique, including lipofection, electroporation, calcium
phosphate precipitation, etc. Appropriate helper viruses include
adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes
viruses. Once the packaging cells are transfected and infected,
they will produce infectious AAV viral particles which contain the
VEGF-2 polynucleotide construct. These viral particles are then
used to transduce eukaryotic cells, either ex vivo or in vivo. The
transduced cells will contain the VEGF-2 polynucleotide construct
integrated into its genome, and will express VEGF-2.
[0409] Another method of gene therapy involves operably associating
heterologous control regions and endogenous polynucleotide
sequences (e.g. encoding VEGF-2) via homologous recombination (see,
e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International
Publication No. WO 96/29411, published Sep. 26, 1996; International
Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et
al., Nature 342:435-438 (1989). This method involves the activation
of a gene which is present in the target cells, but which is not
normally expressed in the cells, or is expressed at a lower level
than desired.
[0410] Polynucleotide constructs are made, using standard
techniques known in the art, which contain the promoter with
targeting sequences flanking the promoter. Suitable promoters are
described herein. The targeting sequence is sufficiently
complementary to an endogenous sequence to permit homologous
recombination of the promoter-targeting sequence with the
endogenous sequence. The targeting sequence will be sufficiently
near the 5' end of the VEGF-2 desired endogenous polynucleotide
sequence so the promoter will be operably linked to the endogenous
sequence upon homologous recombination.
[0411] The promoter and the targeting sequences can be amplified
using PCR. Preferably, the amplified promoter contains distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3'
end of the first targeting sequence contains the same restriction
enzyme site as the 5' end of the amplified promoter and the 5' end
of the second targeting sequence contains the same restriction site
as the 3' end of the amplified promoter. The amplified promoter and
targeting sequences are digested and ligated together.
[0412] The promoter-targeting sequence construct is delivered to
the cells, either as naked polynucleotide, or in conjunction with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, whole viruses, lipofection,
precipitating agents, etc., described in more detail above. The P
promoter-targeting sequence can be delivered by any method,
included direct needle injection, intravenous injection, topical
administration, catheter infusion, particle accelerators, etc. The
methods are described in more detail below.
[0413] The promoter-targeting sequence construct is taken up by
cells. Homologous recombination between the construct and the
endogenous sequence takes place, such that an endogenous VEGF-2
sequence is placed under the control of the promoter. The promoter
then drives the expression of the endogenous VEGF-2 sequence.
[0414] Preferably, the polynucleotide encoding VEGF-2 contains a
secretory signal sequence that facilitates secretion of the
protein. Typically, the signal sequence is positioned in the coding
region of the polynucleotide to be expressed towards or at the 5'
end of the coding region. The signal sequence may be homologous or
heterologous to the polynucleotide of interest and may be
homologous or heterologous to the cells to be transfected.
Additionally, the signal sequence may be chemically synthesized
using methods known in the art.
[0415] Any mode of administration of any of the above-described
polynucleotides constructs can be used so long as the mode results
in the expression of one or more molecules in an amount sufficient
to provide a therapeutic effect. This includes direct needle
injection, systemic injection, catheter infusion, biolistic
injectors, particle accelerators (i.e., "gene guns"), gelfoam
sponge depots, other commercially available depot materials,
osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid
(tablet or pill) pharmaceutical formulations, and decanting or
topical applications during surgery. For example, direct injection
of naked calcium phosphate-precipitated plasmid into rat liver and
rat spleen or a protein-coated plasmid into the portal vein has
resulted in gene expression of the foreign gene in the rat livers
(Kaneda et al., Science 243:375 (1989)).
[0416] A preferred method of local administration is by direct
injection. Preferably, a recombinant molecule of the present
invention complexed with a delivery vehicle is administered by
direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries
refers to injecting the composition centimeters and preferably,
millimeters within arteries.
[0417] Another method of local administration is to contact a
polynucleotide construct of the present invention in or around a
surgical wound. For example, a patient can undergo surgery and the
polynucleotide construct can be coated on the surface of tissue
inside the wound or the construct can be injected into areas of
tissue inside the wound.
[0418] Therapeutic compositions useful in systemic administration,
include recombinant molecules of the present invention complexed to
a targeted delivery vehicle of the present invention. Suitable
delivery vehicles for use with systemic administration comprise
liposomes comprising ligands for targeting the vehicle to a
particular site.
[0419] Preferred methods of systemic administration, include
intravenous injection, aerosol, oral and percutaneous (topical)
delivery. Intravenous injections can be performed using methods
standard in the art. Aerosol delivery can also be performed using
methods standard in the art (see, for example, Stribling et al.,
Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is
incorporated herein by reference). Oral delivery can be performed
by complexing a polynucleotide construct of the present invention
to a carrier capable of withstanding degradation by digestive
enzymes in the gut of an animal. Examples of such carriers, include
plastic capsules or tablets, such as those known in the art.
Topical delivery can be performed by mixing a polynucleotide
construct of the present invention with a lipophilic reagent (e.g.,
DMSO) that is capable of passing into the skin.
[0420] Determining an effective amount of substance to be delivered
can depend upon a number of factors including, for example, the
chemical structure and biological activity of the substance, the
age and weight of the animal, the precise condition requiring
treatment and its severity, and the route of administration. The
frequency of treatments depends upon a number of factors, such as
the amount of polynucleotide constructs administered per dose, as
well as the health and history of the subject. The precise amount,
number of doses, and timing of doses will be determined by the
attending physician or veterinarian.
[0421] Therapeutic compositions of the present invention can be
administered to any animal, preferably to mammals and birds.
Preferred mammals include humans, dogs, cats, mice, rats, rabbits
sheep, cattle, horses and pigs, with humans being particularly
preferred.
Nucleic Acid Utilities
[0422] VEGF-2 nucleic acid sequences and VEGF-2 polypeptides may
also be employed for in vitro purposes related to scientific
research, synthesis of DNA and manufacture of DNA vectors, and for
the production of diagnostics and therapeutics to treat human
disease. For example, VEGF-2 may be employed for in vitro culturing
of photoreceptor cells, where it is added to the conditional medium
in a concentration from 10 pg/ml to 10 ng/ml.
[0423] Fragments of the full length VEGF-2 gene may be used as a
hybridization probe for a CDNA library to isolate other genes which
have a high sequence similarity to the gene or similar biological
activity. Probes of this type generally have at least 50 base
pairs, although they may have a greater number of bases. The probe
may also be used to identify a cDNA clone corresponding to a full
length transcript and a genomic clone or clones that contain the
complete VEGF-2 gene including regulatory and promoter regions,
exons, and introns. An example of a screen comprises isolating the
coding region of the VEGF-2 gene by using the known DNA sequence to
synthesize an oligonucleotide probe. Labeled oligonucleotides
having a sequence complementary to that of the gene of the present
invention are used to screen a library of human cDNA, genomic DNA
or MRNA to determine which members of the library the probe
hybridizes to.
[0424] This invention provides methods for identification of VEGF-2
receptors. The gene encoding the receptor can be identified by
numerous methods known to those of skill in the art, for example,
ligand panning and FACS sorting (Coligan et al., Current Protocols
in Immun., 1(2), Chapter 5, (1991)). Preferably, expression cloning
is employed wherein polyadenylated RNA is prepared from a cell
responsive to VEGF-2, and a cDNA library created from this RNA is
divided into pools and used to transfect COS cells or other cells
that are not responsive to VEGF-2. Transfected cells which are
grown on glass slides are exposed to labeled VEGF-2. VEGF-2 can be
labeled by a variety of means including iodination or inclusion of
a recognition site for a site-specific protein kinase. Following
fixation and incubation, the slides are subjected to
autoradiographic analysis. Positive pools are identified and
sub-pools are prepared and retransfected using an iterative
sub-pooling and rescreening process, eventually yielding a single
clone that encodes the putative receptor.
[0425] As an alternative approach for receptor identification,
labeled VEGF-2 can be photoaffinity linked with cell membrane or
extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGE and exposed to X-ray
film. The labeled complex containing VEGF-2 is then excised,
resolved into peptide fragments, and subjected to protein
microsequencing. The amino acid sequence obtained from
microsequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the
gene encoding the putative receptor.
Examples
[0426] 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.
[0427] In order to facilitate understanding of the following
examples, certain frequently occurring methods and/or terms will be
described.
[0428] "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.
[0429] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 mg of plasmid
or DNA fragment is used with about 2 units of enzyme in about 20 Fl
of buffer solution. For the purpose of isolating DNA fragments for
plasmid construction, typically 5 to 50 mg of DNA are digested with
20 to 250 units of enzyme in a larger volume. Appropriate buffers
and substrate amounts for particular restriction enzymes are
specified by the manufacturer. Incubation times of about 1 hour at
37 EC are ordinarily used, but may vary in accordance with the
supplier's instructions. After digestion the reaction is
electrophoresed directly on a polyacrylamide gel to isolate the
desired fragment.
[0430] 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).
[0431] "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.
[0432] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Sambrook
et al., Molecular Cloning: A Laboratory Manual, Second Edition,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), p.
146). Unless otherwise provided, ligation may be accomplished using
known buffers and conditions with 10 units of T4 DNA ligase
("ligase") per 0.5 mg of approximately equimolar amounts of the DNA
fragments to be ligated.
[0433] 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 VEGF-2 in Human Tissues and Breast Cancer
Cell Lines
[0434] Northern blot analysis was carried out to examine the levels
of expression of VEGF-2 in human tissues and breast cancer cell
lines in human tissues. Total cellular RNA samples were isolated
with RNAzol.TM. B system (Biotecx Laboratories, Inc.). About 10 mg
of total RNA isolated from each breast tissue and cell line
specified was separated on 1% agarose gel and blotted onto a nylon
filter, (Sambrook et al., Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y. (1989)). The labeling reaction was done according to the
Stratagene Prime-It kit with 50 ng DNA fragment. The labeled DNA
was purified with a Select-G-50 column from 5 Prime/3 Prime, Inc
(Boulder, Colo.). The filter was then hybridized with a radioactive
labeled full length VEGF-2 gene at 1,000,000 cpm/ml in 0.5 M
NaPO.sub.4 and 7% SDS overnight at 65.degree. C. After washing
twice at room temperature and twice at 60.degree. C. with
0.5.times. SSC, 0.1% SDS, the filters were then exposed at
-70.degree. C. overnight with an intensifying screen. A message of
1.6 Kd was observed in 2 breast cancer cell lines. FIG. 5, lane #4
represents a very tumorigenic cell line that is estrogen
independent for growth.
[0435] Also, 10 mg of total RNA from 10 human adult tissues were
separated on an agarose gel and blotted onto a nylon filter. The
filter was then hybridized with radioactively labeled VEGF-2 probe
in 7% SDS, 0.5 M NaPO4, pH 7.2; 1% BSA overnight at 65.degree. C.
Following washing in 0.2.times. SSC at 65.degree. C., the filter
was exposed to film for 24 days at -70.degree. C. with intensifying
screen. See FIG. 6.
Example 2
Expression of the Truncated Form of VEGF-2 (SEQ ID NO:4) by In
Vitro Transcription and Translation
[0436] The VEGF-2 cDNA was transcribed and translated in vitro to
determine the size of the translatable polypeptide encoded by the
truncated form of VEGF-2 and a partial VEGF-2 cDNA. The two inserts
of VEGF-2 in the pBluescript SK vector were amplified by PCR with
three pairs of primers, 1) 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.
TABLE-US-00003 M13-2 reverse primer: 5'-ATGCTTCCGGCTCGTATG-3' (SEQ
ID NO: 11)
[0437] This sequence is located upstream of the 5' end of the
VEGF-2 cDNA insert in the pBluescript vector and is in an
anti-sense orientation as the cDNA.
[0438] A T3 promoter sequence is located between this primer and
the VEGF-2 cDNA.
TABLE-US-00004 M13-2 forward primer: 5'GGGTTTTCCCAGTCACGAC-3' (SEQ
ID NO: 12)
[0439] This sequence is located downstream of the 3' end of the
VEGF-2 cDNA insert in the pBluescript vector and is in an
anti-sense orientation as the cDNA insert.
TABLE-US-00005 VEGF primer F4: 5'-CCACATGGTTCAGGAAAGACA-3' (SEQ ID
NO: 13)
[0440] This sequence is located within the VEGF-2 cDNA in an
anti-sense orientation from bp 1259-1239, which is about 169 bp
away from the 3' end of the stop codon and about 266 bp before the
last nucleotide of the cDNA.
[0441] PCR reaction with all three pairs of primers produce
amplified products with T3 promoter sequence in front of the cDNA
insert. The first and third pairs of primers produce PCR products
that encode the polypeptide of VEGF-2 shown in SEQ ID NO:4. The
second pair of primers produce PCR product that misses 36 amino
acids coding sequence at the C-terminus of the VEGF-2
polypeptide.
[0442] Approximately 0.5 mg of PCR product from first pair of
primers, 1 mg from second pair of primers, 1 mg from third pair of
primers were used for in vitro transcription/translation. The in
vitro transcription/translation reaction was performed in a 25 Fl
of volume, using the T.sub.NTJ Coupled Reticulocyte Lysate Systems
(Promega, CAT #L4950). Specifically, the reaction contains 12.5 Fl
of T.sub.NT rabbit reticulocyte lysate 2 Fl of T.sub.NT reaction
buffer, 1 Fl of T3 polymerase, 1 Fl of 1 mM amino acid mixture
(minus methionine), 4 Fl of .sup.35S-methionine (>1000 Ci/mmol,
10 mCi/ml), 1 Fl of 40 U/.mu.l; RNasin ribonuclease inhibitor, 0.5
or 1 mg of PCR products. Nuclease-free H.sub.2O was added to bring
the volume to 25 Fl. The reaction was incubated at 30.degree. C.
for 2 hours. Five microliters of the reaction product was analyzed
on a 4-20% gradient SDS-PAGE gel. After fixing in 25% isopropanol
and 10% acetic acid, the gel was dried and exposed to an X-ray film
overnight at 70.degree. C.
[0443] As shown in FIG. 7, PCR products containing the truncated
VEGF-2 cDNA (i.e., as depicted in SEQ ID NO:3) and the cDNA missing
266 bp in the 3' un-translated region (3'-UTR) produced the same
length of translated products, whose molecular weights are
estimated to be 38-40 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 VEGF-2 Using the Baculovirus Expression
System
[0444] The DNA sequence encoding the VEGF-2 protein without 46
amino acids at the N-terminus, see ATCC.TM. No. 97149, was
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' sequences of the gene:
[0445] 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:14) and contains
a BamH1 restriction enzyme site (in bold) and 17 nucleotide
sequence complementary to the 5' sequence of VEGF-2 (nt.
150-166).
[0446] The 3' primer has the sequence GATC TCT AGA TTA GCT CAT TTG
TGG TCT (SEQ ID NO:15) and contains the cleavage site for the
restriction enzyme XbaI and 18 nucleotides complementary to the 3'
sequence of VEGF-2, including the stop codon and 15 nt sequence
before stop codon.
[0447] 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, VEGF-2 cDNA was cloned in frame with the signal sequence
of baculovirus gp67 gene and was located at the 3' end of the
signal sequence in the vector. This is designated
pAcGP67A-VEGF-2.
[0448] To clone VEGF-2 with the signal sequence of gp67 gene to the
pRG1 vector for expression, VEGF-2 with the signal sequence and
some upstream sequence were excised from the pAcGP67A-VEGF-2
plasmid at the Xho restriction endonuclease site located upstream
of the VEGF-2 cDNA and at the XbaI restriction endonuclease site by
XhoI and XbaI restriction enzyme. This fragment was separated from
the rest of vector on a 1% agarose gel and was purified using
"Geneclean.TM." kit. It was designated F2.
[0449] The PRG1 vector (modification of pVL941 vector) is used for
the expression of the VEGF-2 protein using the baculovirus
expression system (for review see: Summers, M. D. and Smith, G. E.,
"A Manual of Methods for Baculovirus Vectors and Insect Cell
Culture Procedures," Texas Agricultural Experimental Station
Bulletin No. 1555, (1987)). This expression vector contains the
strong polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by the recognition sites for
the restriction endonucleases BamH1, Sma1, XbaI, Bg1II and Asp718.
A site for restriction endonuclease Xho1 is located upstream of
BamH1 site. The sequence between Xho1 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).
[0450] 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.
[0451] Fragment F2 and the dephosphorylated plasmid V2 were ligated
with T4 DNA ligase. E. coli HB101 cells were then transformed and
bacteria identified that contained the plasmid (pBac gp67-VEGF-2)
with the VEGF-2 gene using the enzymes BamH1 and XbaI. The sequence
of the cloned fragment was confirmed by DNA sequencing.
[0452] 5 mg of the plasmid pBac gp67-VEGF-2 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)).
[0453] 1 mg of BaculoGold.TM.J virus DNA and 5 mg of the plasmid
pBac gp67-VEGF-2 were mixed in a sterile well of a microtiter plate
containing 50 ml of serum free Grace's medium (Life Technologies
Inc., Gaithersburg, Md.). Afterwards 10 ml Lipofectin.TM. plus 90
ml Grace's medium were added, mixed and incubated for 15 minutes at
room temperature. Then the transfection mixture was added dropwise
to the Sf9 insect cells (ATCC.TM. CRL 1711) seeded in a 35 mM
tissue culture plate with 1 ml Grace's medium without serum. The
plate was rocked back and forth to mix the newly added solution.
The plate was then incubated for 5 hours at 27.degree. C. After 5
hours the transfection solution was removed from the plate and 1 ml
of Grace's insect medium supplemented with 10% fetal calf serum was
added. The plate was put back into an incubator and cultivation
continued at 27.degree. C. for four days.
[0454] 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). 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.
[0455] Sf9 cells were grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells were infected with the recombinant
baculovirus V-gp67-VEGF-2 at a multiplicity of infection (MOI) of
1. Six hours later the medium was removed and replaced with SF900
II medium minus methionine and cysteine (Life Technologies Inc.,
Gaithersburg). 42 hours later 5 mCi of .sup.35S-methionine and 5
mCi .sup.35S cysteine (Amersham) were added. The cells were further
incubated for 16 hours before they were harvested by centrifugation
and the labeled proteins visualized by SDS-PAGE and
autoradiography.
[0456] 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.
[0457] The medium supernatant was also diluted 1:10 in 50 mM MES,
pH 5.8 and applied to an SP-650M column (1.0.times.6.6 cm,
Toyopearl) at a flow rate of 1 ml/min. Protein was eluted with step
gradients at 200, 300 and 500 mM NaCl. The VEGF-2 was obtained
using the elution at 500 mM. The eluate was analyzed by SDS-PAGE in
the presence or absence of reducing agent, b-mercaptoethanol and
stained by Coomassie Brilliant Blue. See FIG. 10.
Example 4
Expression of Recombinant VEGF-2 in COS Cells
[0458] The expression of plasmid, VEGF-2-HA is derived from a
vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of
replication, 2) ampicillin resistance gene, 3) E. coli replication
origin, 4) CMV promoter followed by a polylinker region, an SV40
intron and polyadenylation site. A DNA fragment encoding the entire
VEGF-2 precursor and a HA tag fused in frame to its 3' end was
cloned into the polylinker region of the vector, therefore, the
recombinant protein expression is directed under the CMV promoter.
The HA tag corresponds to an epitope derived from the influenza
hemagglutinin protein as previously described (Wilson et al., Cell
37:767 (1984)). The infusion of HA tag to the target protein allows
easy detection of the recombinant protein with an antibody that
recognizes the HA epitope.
[0459] The plasmid construction strategy is described as
follows:
[0460] The DNA sequence encoding VEGF-2, ATCC.TM. No. 97149, was
constructed by PCR using two primers: the 5' primer (CGC GGA TCC
ATG ACT GTA CTC TAC CCA) (SEQ ID NO:16) contains a BamH1 site
followed by 18 nucleotides of VEGF-2 coding sequence starting from
the initiation codon; the 3' sequence (CGC TCT AGA TCA AGC GTA GTC
TGG GAC GTC GTA TGG GTA CTC GAG GCT CAT TTG TGG TCT 3') (SEQ ID
NO:17) contains complementary sequences to an XbaI site, HA tag,
XhoI site, and the last 15 nucleotides of the VEGF-2 coding
sequence (not including the stop codon). Therefore, the PCR product
contains a BamHI site, coding sequence followed by an XhoI
restriction endonuclease site and HA tag fused in frame, a
translation termination stop codon next to the HA tag, and an XbaI
site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
were digested with 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 VEGF-2,
COS cells were transfected with the expression vector by
DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis,
Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory
Press, (1989)). The expression of the VEGF-2-HA protein was
detected by radiolabelling and immunoprecipitation method (E.
Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, (1988)). Cells were 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
Construction of Amino Terminal and Carboxy Terminal Deletion
Mutants
[0461] 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
[0462] 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:2) into the E. coli protein expression vector, pHE4,
two oligonucleotide primers complementary to the desired region of
VEGF-2 were synthesized with the following base sequence:
[0463] 5' Primer (Nde I/START and 18 nt of coding sequence):
TABLE-US-00006 (SEQ ID NO: 18) 5'-GCA GCA CAT ATG ACA GAA GAG ACT
ATA AAA-3'
[0464] 3' Primer (Asp718, STOP, and 15 nt of coding sequence):
TABLE-US-00007 (SEQ ID NO: 19) 5'-GCA GCA GGT ACC TCA CAG TTT AGA
CAT GCA-3'
[0465] The above described 5' primer (SEQ ID NO:18), incorporates
an NdeI restriction site and the above described 3' Primer (SEQ ID
NO:19), incorporates an Asp718 restriction site. The 5' primer (SEQ
ID NO:18) 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:19) 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.
[0466] The Polymerase Chain Reaction was performed using standard
conditions well known to those skilled in the art and the
nucleotide sequence for the mature VEGF-2 (aa 24-419 in SEQ ID
NO:2) as, for example, constructed in Example 3 as template. The
resulting amplicon was restriction digested with NdeI and Asp718
and subcloned into NdeI/Asp718 digested pHE4a expression
vector.
2. Construction of VEGF-2 T103-R227 in pHE4
[0467] 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:2) into the E. coli protein expression vector, pHE4,
two oligonucleotide primers complementary to the desired region of
VEGF-2 were synthesized with the following base sequence:
[0468] 5' Primer (Nde I/START and 18 nt of coding sequence):
TABLE-US-00008 (SEQ ID NO: 20) 5'-GCA GCA CAT ATG ACA GAA GAG ACT
ATA AAA-3'
[0469] 3' Primer (Asp 718, STOP, and 15 nt of coding sequence):
TABLE-US-00009 (SEQ ID NO: 21) 5'-GCA GCA GGT ACC TCA ACG TCT AAT
AAT GGA-3'
[0470] In the case of the above described primers, an NdeI or
Asp718 restriction site was incorporated he 5' primer and 3'
primer, respectively. The 5' primer (SEQ ID NO:20) 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.
[0471] The Polymerase Chain Reaction was performed using standard
conditions well known to those skilled in the art and the
nucleotide sequence for the mature VEGF-2 (aa 24-419 in SEQ ID
NO:2) as, for example, constructed in Example 3, as template. The
resulting amplicon was restriction digested with NdeI and Asp718
and subcloned into NdeI/Asp718 digested pHE4a protein expression
vector.
3. Construction of VEGF-2 T103-L215 in pA2GP
[0472] 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:2), 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.
[0473] 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).
[0474] The cDNA sequence encoding the VEGF-2 protein without 102
amino acids at the N-terminus and without 204 amino acids at the
C-terminus in FIG. 1, was amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the gene.
[0475] 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 5'-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.
[0476] 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, VEGF-2 cDNA representing the N-terminal and C-terminal
deleted VEGF-2 protein (amino acids 103-215 in FIG. 1 or SEQ ID
NO:2) was cloned in frame with the signal sequence of baculovirus
GP gene and was located at the 3' end of the signal sequence in the
vector. This is designated pA2GPVEGF-2.T103-L215.
4. Construction of VEGF-2 T103-R227 in pA2GP
[0477] The cDNA sequence encoding the VEGF-2 protein without 102
amino acids at the N-terminus and without 192 amino acids at the
C-terminus in FIG. 1 (i.e., amino acids 103-227 of SEQ ID NO:2) was
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' sequences of the gene.
[0478] 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 BaniHI
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 5'-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.
[0479] 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, VEGF-2 cDNA representing the N-terminal and C-terminal
deleted VEGF-2 protein (amino acids 103-227 in FIG. 1 or SEQ ID
NO:2) was cloned in frame with the signal sequence of baculovirus
GP gene and was located at the 3' end of the signal sequence in the
vector. This construct is designated pA2GPVEGF-2.T103-R227.
5. Construction of VEGF-2 in pC1
[0480] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular
and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple
cloning sites, e.g., with the restriction enzyme cleavage sites
BamHI, XbaI and Asp718, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3N intron, the
polyadenylation and termination signal of the rat preproinsulin
gene.
[0481] The vector pC1 is used for the expression of VEGF-2 protein.
Plasmid pC1 is a derivative of the plasmid pSV2-dhfr [ATCC.TM.
Accession No. 37146]. Both plasmids contain the mouse DHFR gene
under control of the SV40 early promoter. Chinese hamster ovary--or
other cells lacking dihydrofolate activity that are transfected
with these plasmids can be selected by growing the cells in a
selective medium (alpha minus MEM, Life Technologies) supplemented
with the chemotherapeutic agent methotrexate. The amplification of
the DHFR genes in cells resistant to methotrexate (MTX) has been
well documented (see, e.g., Alt, F. W., Kellems, R. M., Bertino, J.
R., and Schimke, R. T., 1978, J Biol. Chem. 253:1357-1370, Hamlin,
J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143,
Page, M. J. and Sydenham, M. A. 1991, Biotechnology 9:64-68). Cells
grown in increasing concentrations of MTX develop resistance to the
drug by overproducing the target enzyme, DHFR, as a result of
amplification of the DHFR gene. If a second gene is linked to the
DHFR gene it is usually co-amplified and over-expressed. It is
state of the art to develop cell lines carrying more than 1,000
copies of the genes. Subsequently, when the methotrexate is
withdrawn, cell lines contain the amplified gene integrated into
the chromosome(s).
[0482] Plasmid pC1 contains for the expression of the gene of
interest a strong promoter of the long terminal repeat (LTR) of the
Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular
Biology, March 1985:438-4470) plus a fragment isolated from the
enhancer of the immediate early gene of human cytomegalovirus (CMV)
(Boshart et al., Cell 41:521-530, 1985). Downstream of the promoter
are the following single restriction enzyme cleavage sites that
allow the integration of the genes: BamHI, Pvu11, 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 HTLVI.
For the polyadenylation of the mRNA other signals, e.g., from the
human growth hormone or globin genes can be used as well.
[0483] 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.
[0484] 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.
[0485] The DNA sequence encoding VEGF-2, ATCC.TM. Accession No.
97149, was constructed by PCR using two primers corresponding to
the 5' and 3' ends of the VEGF-2 gene: the 5' Primer (5'-GAT CGA
TCC ATC ATG CAC TCG CTG GGC TTC TTC TCT GTG GCG TGT TCT CTG CTC
G-3' (SEQ ID NO:26)) contains a Klenow-filled BamHI site and 40 nt
of VEGF-2 coding sequence starting from the initiation codon; the
3' primer (5'-GCA GGG TAC GGA TCC TAG ATT AGC TCA TTT GTG GTC
TTT-3' (SEQ ID NO:27)) contains a BamHI site and 16 nt of VEGF-2
coding sequence not including the stop codon.
[0486] The PCR amplified DNA fragment is isolated from a 1% agarose
gel as described above and then digested with the endonuclease
BaniHI and then purified again on a 1% agarose gel. The isolated
fragment and the dephosphorylated vector are then ligated with T4
DNA ligase. E. coli HB101 cells are then transformed and bacteria
identified that contained the plasmid pC1. The sequence and
orientation of the inserted gene is confirmed by DNA sequencing.
This construct is designated pC1VEGF-2.
6. Construction of pC4SigVEGF-2 T103-L215
[0487] Plasmid pC4Sig is plasmid pC4 (Accession No. 209646)
containing a human IgG Fc portion as well as a protein signal
sequence.
[0488] 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:2) into pC4Sig, two oligonucleotide primers
complementary to the desired region of VEGF-2 were synthesized with
the following base sequence:
[0489] 5' Primer (Bam HI and 26 nt of coding sequence):
TABLE-US-00010 (SEQ ID NO: 28) 5'-GCA GCA GGA TCC ACA GAA GAG ACT
ATA AAA TTT GCT GC-3'
[0490] 3' Primer (Xba I, STOP, and 15 nt of coding sequence):
TABLE-US-00011 (SEQ ID NO: 29) 5'-CGT CGT TCT AGA TCA CAG TTT AGA
CAT GCA TCG GCA G-3'
[0491] 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 BaniHI and XbaI and
subcloned into BamHI/XbaI digested pC4Sig vector.
7. Construction of pC4SigVEGF-2 T103-R227
[0492] 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:2) into pC4Sig, two oligonucleotide primers
complementary to the desired region of VEGF-2 were synthesized with
the following base sequence:
[0493] 5' Primer (Bam HI and 26 nt of coding sequence):
TABLE-US-00012 (SEQ ID NO: 30) 5'-GCA GCA GGA TCC ACA GAA GAG ACT
ATA AAA TTT GCT GC-3'
[0494] 3' Primer (Xba I, STOP, and 21 nt of coding sequence):
TABLE-US-00013 (SEQ ID NO: 31) 5'-GCA GCA TCT AGA TCA ACG TCT AAT
AAT GGA ATG AAC-3'
[0495] The Polymerase Chain Reaction was performed using standard
conditions well known to those skilled in the art and the
nucleotide sequence for the mature VEGF-2 (aa 24-419) as, for
example, constructed in Example 3, as template. The resulting
amplicon was restriction digested with BamHI and XbaI and subcloned
into BamHI/XbaI digested pC4Sig vector.
8. Construction of pC4VEGF-2 M1-M263
[0496] 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 BamiHI, 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.
[0497] In this illustrative example, the cloned DNA encoding the
C-terminal deleted VEGF-2 M1-M263 protein (amino acids 1-263 in
FIG. 1 or SEQ ID NO:2) is inserted into the plasmid vector pC4 to
express the C-terminal deleted VEGF-2 protein.
[0498] To permit Polymerase Chain Reaction directed amplification
and sub-cloning of VEGF-2 M1-M263 into the expression vector, pC4,
two oligonucleotide primers complementary to the desired region of
VEGF-2 were synthesized with the following base sequence:
TABLE-US-00014 5' Primer (SEQ ID NO: 32) 5'-GAC TGG ATC CGC CAC CAT
GCA CTC GCT GGG CTT CTT CTC-3' 3' Primer (SEQ ID NO: 33) 5'-GAC TGG
TAC CTT ATC ACA TAA AAT CTT CCT GAG CC-3'
[0499] 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.
[0500] 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
[0501] In this illustrative example, the cloned DNA encoding the
C-terminal deleted VEGF-2 Ml -D311 protein (amino acids 1-311 in
FIG. 1 or SEQ ID NO:2) is inserted into the plasmid vector pC4 to
express the C-terminal deleted VEGF-2 protein.
[0502] To permit Polymerase Chain Reaction directed amplification
and sub-cloning of VEGF-2 M1-D311 into the expression vector, pC4,
two oligonucleotide primers complementary to the desired region of
VEGF-2 were synthesized with the following base sequence:
TABLE-US-00015 5' Primer (SEQ ID NO: 34) 5'-GAC TGG ATC CGC CAC CAT
GCA CTC GCT GGG CTT CTT CTC-3' 3' Primer (SEQ ID NO: 35) 5'-GAC TGG
TAC CTT ATC AGT CTA GTT CTT TGT GGG G-3'
[0503] 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.
[0504] 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.
10. Construction of pC4VEGF-2 M1-Q367
[0505] In this illustrative example, the cloned DNA encoding the
C-terminal deleted VEGF-2 M1-D311 protein (amino acids 1-311 in SEQ
ID NO:2) is inserted into the plasmid vector pC4 to express the
C-terminal deleted VEGF-2 protein.
[0506] To permit Polymerase Chain Reaction directed amplification
and sub-cloning of VEGF-2 M1-D311 into the expression vector, pC4,
two oligonucleotide primers complementary to the desired region of
VEGF-2 were synthesized with the following base sequence:
TABLE-US-00016 5' Primer (SEQ ID NO: 36) 5'-GAC TGG ATC CGC CAC CAT
GCA CTC GCT GGG CTT CTT CTC-3' 3' Primer (SEQ ID NO: 37) 5'-GAC TGG
TAC CTC ATT ACT GTG GAC TTT CTG TAC ATT C-3'
[0507] 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.
[0508] 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-Q367.
Example 6
Method of Treatment Using Gene Therapy for Production of VEGF-2
Polypeptide--In Vivo
[0509] Suitable template DNA for production of mRNA coding for
VEGF-2 is prepared in accordance with a standard recombinant DNA
methodology. Sterile and endotoxin-free oligonucleotides are
diluted in Sterile and endotoxin-free oligonucleotides are diluted
in Balanced Salt Solution (BSS, Alcon, Fort Worth, Tex.) so as to
have the same pH and electrolyte concentration as the aqueous or
vitreous of the eye. Emalphor EC620 (2.5%, GAF Corp.) (Bursell et
al. (1993) J. Clin. Invest. 92:2872-2876), a petroleum product, is
added to change viscosity and aid in delivery properties. Doses to
achieve intravitreal concentrations ranging from 0.1 .mu.M-100
.mu.M are administered. The volume delivered is between 1 .mu.l and
1 ml depending on the volume of the eye.
[0510] The intubated patient is Anesthetized with fluorane. The
face and eyes are prepared with a betadine scrub and draped in the
usual sterile fashion. The sterile polynucleotide with vehicle is
injected with a 33 gauge needle on a sterile syringe at the
posterior limbus (pars plana) through full thickness sclera into
the vitreous. No closing suture is required unless there is
leakage. Antibiotic drops containing gentamicin or erythromycin
ointment is applied to the surface of the globe in the palpebral
fissure several times per day until there is complete wound
closure. The frequency of injection ranges from every other day to
once every 6 months or less, depending on the severity of the
disease process, the degree of intraocular inflammation, the
character of the vehicle (i.e., slow release characteristics), and
the tolerance of the eye to injections. Short and long term
follow-up check-ups for possible retinal detachment from the
injections are necessary.
[0511] The eye upon dilation is monitored for signs of
inflammation, infection, and photoreceptor growth by both an direct
and a indirect ophthalmoscope to view the retina and fundus.
Monitoring can be as frequent as every day in cases where premature
infants are threatened with retinal detachment. The frequency of
monitoring will diminish with resolution of disease.
[0512] The patient is treated weekly with intraocular injections of
polynucleotide resuspended in the appropriate vehicle (BSS,
Emanfour) at concentrations within the range of 0.1 to 100 .mu.M.
This treatment may be supplemented with systemic delivery of
polynucleotide (i.e., intravenous, subcutaneous, or intramuscular)
from 2 to 5 times per day to once a month.
Example 7
Method of Treatment Using Gene Therapy--Ex vivo Homologous
Recombination
[0513] Photoreceptor cells are obtained from a subject by biopsy.
The resulting tissue is placed in DMEM+10% fetal calf serum.
Exponentially growing or early stationary phase fibroblasts are
trypsinized and rinsed from the plastic surface with nutrient
medium. An aliquot of the cell suspension is removed for counting,
and the remaining cells are subjected to centrifugation. The
supernatant is aspirated and the pellet is resuspended in 5 ml of
electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl,
0.7 mM Na.sub.2 HPO.sub.4, 6 mM dextrose). The cells are
recentrifuged, the supernatant aspirated, and the cells resuspended
in electroporation buffer containing 1 mg/ml acetylated bovine
serum albumin. The final cell suspension contains approximately
3.times.10.sup.6 cells/ml. Electroporation should be performed
immediately following resuspension.
[0514] Plasmid DNA is prepared according to standard techniques. To
construct a plasmid for targeting to the VEGF-2 locus, plasmid pUC
18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMV
promoter is amplified by PCR with an XbaI site on the 5' end and a
BamHI site on the 3' end. Two VEGF-2 non-coding sequences are
amplified via PCR: one VEGF-2 non-coding sequence (VEGF-2 fragment
1) is amplified with a HindIII site at the 5' end and an Xba site
at the 3' end; the other VEGF-2 non-coding sequence (VEGF-2
fragment 2) is amplified with a BamHI site at the 5' end and a
HindIII site at the 3' end. The CMV promoter and VEGF-2 fragments
are digested with the appropriate enzymes (CMV promoter-XbaI and
BamHI; VEGF-2 fragment 1-XbaI; VEGF-2 fragment 2-BamHI) and ligated
together. The resulting ligation product is digested with HindIII,
and ligated with the HindIII-digested pUC18 plasmid.
[0515] Plasmid DNA is added to a sterile cuvette with a 0.4 cm
electrode gap (Bio-Rad). The final DNA concentration is generally
at least 120 .mu.g/ml. 0.5 ml of the cell suspension (containing
approximately 1.5..times.10.sup.6 cells) is then added to the
cuvette, and the cell suspension and DNA solutions are gently
mixed. Electroporation is performed with a Gene-Pulser apparatus
(Bio-Rad). Capacitance and voltage are set at 960 .mu.F and 250-300
V, respectively. As voltage increases, cell survival decreases, but
the percentage of surviving cells that stably incorporate the
introduced DNA into their genome increases dramatically. Given
these parameters, a pulse time of approximately 14-20 mSec should
be observed.
[0516] Electroporated cells are maintained at room temperature for
approximately 5 min, and the contents of the cuvette are then
gently removed with a sterile transfer pipette. The cells are added
directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf
serum) in a 10 cm dish and incubated at 37 EC. The following day,
the media is aspirated and replaced with 10 ml of fresh media and
incubated for a further 16-24 hours.
[0517] The engineered photoreceptor cells are then injected into
the host. The photoreceptor cells now produce the protein
product.
Example 8
VEGF-2 Activity on Retinal Cells
[0518] The retina has proven to be an advantageous experimental
model for studying the role of intrinsic and extrinsic factors in
the regulation of the development of neuronal and non-neuronal cell
types from a more primitive neuroepithelial cell. The
differentiated retina is composed of seven cell types: sensory (rod
and cone photoreceptors), glia (Mutller cells), and two types of
neurons, interneurons (horizontal, bipolar, and amacrine), and
projection neurons (ganglion cells) (for review see Dowling, 1987).
The development of the various cell types in the retina does not
occur synchronously with the majority of the cones, and ganglion
and horizontal cells developing before birth (for review see
Altshuler et al., 1991; Harris, 1991; Reh, 1991). In contrast,
differentiation of a majority of the rods, the main cell type in
the rat retina, occurs postnatally. Clonal analysis of the progeny
of retinal precursor cells has demonstrated that the progenitor
cells can produce various combinations of retinal cell types
indicating that the progenitors are either totipotent or
multipotent depending on the developmental age examined (Turner and
Cepko, 1987; Turner et al., 1990; Wetts and Fraser, 1998).
Furthermore, findings from both in vivo and in vitro studies
demonstrate that the final phenotype of the retinal cells is
largely lineage independent which suggest that the changing
microenvironment within the retina has a role in determining the
cellular potential of the progenitor cells as well as the
differentiated phenotype of the progeny (Watanabe and Raff, 1990,
1992; Harris, 1991; Reh, 1991; Ezzeddine et al. 1997).
[0519] In vitro, retinal cell proliferation and differentiation is
regulated by a variety of factors; for example, FGF-2 (Hicks and
Courtois, 1992), CNTF (Ezzeddine et al., 1997; Fuhrmann et al.,
1995), LIF (Ezzeddine et al., 1997), TGF_(Lilleen and Cepko, 1992),
retinoic acid (Kelly et al., 1994), and EGF (Lillien, 1995).
Recently, Yang and Cepko (1996) have identified and characterized
the expression pattern of VEGFR-2/FLK-1 in developing and adult
retina. VEGFR transcripts are first detected at E11.5 in
association with the developing retinal vasculature and with the
central region of the neural retina. By developmental day E15,
VEGFR-2 expression extends to the periphery of the retina
consistent with the outward gradient of retinal development (Young,
1985; LaVail et al., 1991). VEGFR-2 expression was largely
localized to the ventricular zone during the perinatal period when
neurogenesis is at its peak and a large number of post-mitotic
neurons are being formed.
[0520] As shown below, the major in vitro effect of VEGF is during
early development and involves the proliferation of multipotent
progenitor cells since the level of BrdU and the number of
photoreceptor and amacrine cells are increased. VEGF-2 enhanced the
proliferation of retinal cells derived from E15 embryos and the
magnitude of the response increased with age. The early
proliferative response to VEGF-2 administration was not effected by
CNTF. However, CNTF did inhibit the VEGF-2 induced increase in the
level of rhodopsin protein.
[0521] Experimental Procedures
[0522] Animals. Timed pregnant animals are obtained from Harlan
Sprague-Dawley (Indianapolis, Ind.). All animal related procedures
are conducted in strict compliance with approved institutional
protocols and in accordance with provisions for animal care and use
described in the Guide for the Care and Use of Laboratory Animals
(NIH publication No. 86-23,1985).
[0523] Retinal Cultures The retinal tissue are obtained from either
late embryonic or neonatal rats. The dissociated primary cells are
prepared by incubating the tissue in 0.25% trypsin for 6 min at
37.degree. C. Following the inactivation of the trypsin by a 5 min
incubation in growth medium (F12:Dulbecco's modified Eagle's medium
(DMEM) containing 1% fetal bovine serum, 1% hormonal supplements
(N2, Bottenstein, 1983), 1% glutamine and 0.5%
penicillin-streptomycin (10,000 units/ml and 10 mg/ml,
respectively, Gibco, Grand Island, N.Y.) containing 50 .mu.g/ml
deoxyribonuclease type I (Sigma, St. Louis, Mo.), the tissue
fragments are passed repeatedly through a Pasteur pipette with a
constricted tip of a diameter of approximately 1 mm. The
dissociated cells are collected by centrifugation (800.times. g, 5
min) and resuspended in growth medium. The cells are seeded in 96
well plates precoated with poly-L-lysine (50 .mu.g/ml, Sigma) and
laminin (10 .mu.g/ml, Gibco), at a density of 425 cells/mm.sup.2
unless stated otherwise. The cultures are gradually shifted to
growth medium without serum by changing one-half of the medium
every other day. The trophic factors are replenished with each
medium change.
[0524] Hippocampal Astrocytes Purified cultures of astrocytes are
prepared from rat hippocampi using a method previously described
(Greene et al., 1998).
[0525] Rhodopsin Immunohistochemistry and Cell Counting Procedures
For the immunohistochemical staining, the cultures are fixed
overnight in 4% paraformaldehyde containing 4% sucrose. For
rhodopsin and syntaxin staining, the cultures are permeabilized
with 0.05% saponin in PBS for 30 min. The non-specific IgG binding
is inhibited by incubating the cells in PBS containing 5% horse
serum and 2% BSA for 3 h at room temperature. The cultures are then
incubated overnight at 4.degree. C. with anti-rhodopsin (1:10,000,
Rho 4D2, Dr. Molday, University of British Columbia) or
anti-syntaxin (1:10,000, Sigma) diluted in PBS containing 5% horse
serum and 2% BSA (Molday, 1989). Following the removal of the
primary antibody, the cultures are incubated with a biotinylated
anti-mouse antibody (1:2,500) for 90 min. The
avidin-biotin-peroxidase complex, diluted 1:50 in PBS containing 5%
horse serum and 2% BSA, is then added for 60 minutes. To visualize
the bound peroxidase, diaminobenzidine is used at a final
concentration of 0.4 mg/ml in a 0.1M acetate buffer containing 2.5%
nickel sulfate. The number of immunopositive cells per well is
determined by counting the labeled cells in an area representing
11% of the total surface area of the well and then corrected for
the total surface area.
[0526] BrdU Immunohistochemistry The retinal cells are incubated
with BrdU for 4 h and subsequently, washed twice with PBS. Growth
medium is then added back to the cultures, which are then
maintained in vitro for various time intervals. At the end of the
incubation period, the cultures are fixed and immunohistochemically
stained for the incorporated BrdU as per manufacturer's (Boehringer
Mannheim, Indianapolis, Ind.) instructions.
[0527] Rhodopsin and GFAP ELISAs For the rhodopsin ELISA, the
cultures are rinsed with PBS and fixed overnight with 4%
paraformaldehyde containing 4% sucrose. The cultures are then
rinsed with PBS and permeabilized with 0.05% saponin in PBS for 30
min at room temperature. The non-specific protein binding sites are
suppressed by incubating the cells in PBS containing 5% horse serum
and 2% BSA (blocking buffer) for at least 3 h. The cultures are
incubated overnight with the mouse anti-rhodopsin antibody (diluted
1:500 in blocking buffer) and then subsequently, with goat
anti-mouse IgG conjugated to horseradish peroxidase (diluted
1:2,500) in blocking solution. The cultures are washed extensively
with PBS and then the substrate (3,3', 5,5'-tetramethylbenzidine)
is added to the wells and the plates were incubated in the dark for
60 min. The reaction is stopped by adding 2 M H.sub.2SO.sub.4 and
the amount of product formed is quantitated by measuring the
absorbance at 450 nm. The absorption of the reagent blank ranged
from 0.1 to 0.15 and is not subtracted from the indicated values.
The GFAP ELISA is conducted essentially as previously described
(Greene et al., 1998).
[0528] Cell survival assay based on Calcein AM measurements. At the
end of the incubation period the cultures were rinsed once with
Ham's F-12. The calcein AM was added to a final concentration of 2
.mu.M in 100 .mu.l of Ham's F-12 and the cultures were incubated
for 60 min at 37 degree C. At the end of the incubation period the
cultures were rinsed. The absorbance at 530 mn was determined on an
ELISA plate reader.
[0529] High-affinity GABA uptake The level of high-affinity GABA
uptake is determined as previously described (Greene et al.,
1998).
[0530] [.sup.3H]Thymidine Incorporation The cultures are treated
with trophic factors for 24 h and during the last 4 h, the cells
are labeled with [.sup.3H]thyrnidine, at a final concentration of
0.33 .mu.M (25 Ci/mmol, Amersham, Arlington Heights, Ill.). The
incorporated [.sup.3H]thymidine is precipitated with ice-cold 10%
trichloroacetic acid for 24 h. Subsequently, the cells are rinsed
with ice-cold water. Following lysis in 0.5 M NaOH, the lysates and
PBS rinses (500 .mu.l) are pooled, and counted.
[0531] Results
[0532] The regulatory role of VEGFs on photoreceptor cell
development is initially investigated using cultures derived from
postnatal day 1 (PN1) animals. Previous reports have demonstrated
that multipotent progenitors are present during this developmental
period and retain their capacity to differentiate into
photoreceptor cells as well as other retinal cell types in vitro
(Marrow et al., 1998). Treatment with VEGF-2 or VEGF-1 (R and D
Systems, Minneapolis, Minn.) induces a dose- and time-dependent
increase in the level of rhodopsin protein in the retinal cultures
(FIG. 14A). The time course of the VEGF-induced increase in
rhodopsin is relatively slow, consistent with the known
developmental profile of photoreceptor cells. After 5 days of
treatment, a 25-40% increase in rhodopsin protein is noted with 10
to 100 ng/ml of VEGF-2. However, by 7 to 9 days of treatment, these
same concentrations of VEGF-2 produced a 200-250% increase in
rhodopsin protein. Furthermore, at these later time points,
concentrations of VEGF as low as 1 ng/ml significantly increased
rhodopsin levels. Changes in the amount of rhodopsin may reflect
changes in the level of expression of the protein or changes in the
number of photoreceptor cells or both. To ascertain if VEGF
treatment effected the total number of retinal cells, the level of
emission of calcein AM is monitored. An approximate 25-50% increase
in calcein emission is observed after a 5-7 day treatment with at
least 10 ng/ml of VEGF-2 (FIG. 14B). A pronounced increase in the
basal level of calcein emission is noted in the retinal cultures
between 7 and 9 days indicating that retinal cell proliferation
that is independent of exogenous VEGF had increased by this later
time point. The presence of VEGF-2, even at concentrations as high
as 100 ng/ml, for 9 days did not further increase the number of
retinal cells. However, there is not a concomitant increase in the
basal level of rhodopsin protein after 9 days in culture suggesting
that the proliferation of other cell types accounts for the
increase in the level of calcein emission (FIG. 14A). Treatment
with VEGF-1 induced similar changes in the level of rhodopsin and
calcein emission to those described for VEGF-2 (FIGS. 14C and 14D,
respectively).
[0533] To determine if the increase in rhodopsin content and
calcein emission reflected an increase in the number of
photoreceptor cells, cultures are treated with VEGF-1 or VEGF-2 for
9 days and then immunohistochemically stained for rhodopsin. To
quantitate the effects of VEGF treatment, cell counts are made. The
number of rhodopsin immunopositive cells increased as a function of
concentration with the response having an EC50 value of 0.25 and
1.5 ng/ml for VEGF-1 and VEGF-2, respectively (FIG. 15). At a
saturating concentration of VEGF-2, a 2.4-fold increase in the
number of rods is observed. Furthermore, the response is stable in
the presence of concentrations of VEGF-2 as high as 100 ng/ml
suggesting that VEGF-2 does not readily induce a desensitization of
the biological response. The dose response observed with VEGF-1 is
similar to that obtained with VEGF-2 which is consistent with the
results from the rhodopsin ELISAs.
[0534] The mechanism by which VEGF-2 induces an increase in
photoreceptor cell number may involve an increase in the
proliferation of precursor cells, enhanced survival of
differentiated photoreceptor cells, and/or the redirection of the
rod lineage pathway. To investigate if VEGF-2 is mitogenic for
retinal cells, the cultures are treated with factors 4 h after
plating and then subsequently labeled with BrdU after 24, 48 or 72
h. At the end of the final labeling period (72 h), the cultures are
fixed and the incorporated BrdU is immunohistochemically detected.
A significant increase in the number of BrdU labeled cells is not
observe until after 48 h of treatment, when 10 ng/ml of VEGF-2 or
VEGF-1 induced a 2- to 3-fold increase (FIGS. 16A and 16B,
respectively). The EC50 value for the response is calculated as 1
ng/ml. The 48 h time point appeared near maximal since after an
incubation of 72 h, the level of BrdU incorporation had declined in
the VEGF treated cultures regardless of concentration. However,
this decline in the number of immunopositive cells is not
specifically related to VEGF administration. The basal level of
BrdU incorporation decreased from 1300 to 700 immunopositive cells
per well suggesting that a general loss in the proliferative
activity of the retinal progenitor cells is occurring during this
time period. In spite of the lower over-all proliferative activity
in the cultures at the later time points, VEGF administration still
resulted in a 2-fold increase in the number of BrdU labeled cells.
Similar results are obtained using [.sup.3H]thymidine incorporation
(FIG. 16C).
[0535] To further characterize a role for VEGF during the early in
vitro culture period, the effect that delaying the addition of the
factor to the cultures had on the level of rhodopsin protein is
examined. The initial addition of VEGF is made 4, 24, or 48 h after
plating and the cultures are subsequently maintained for 9 days and
then prepared for the rhodopsin ELISA. The loss of the response to
VEGF-2 or VEGF-1 as a function of the time lapsed between the
isolation of the cells and the initial addition of the factors is
depicted in FIGS. 17A and 17B, respectively. The addition of
factors within 4 h of the plating of the cells resulted in a 3-fold
increase in the level of rhodopsin. However, delaying the initial
treatment with VEGF by 24 or 48 h resulted in the reduction of the
maximal response by 28 and 43%, respectively. Furthermore, after a
delay of 48 h, only treatment with 100 ng/ml of VEGF-2 induced a
significant increase in rhodopsin content. Thus suggesting that the
proliferative effect that the VEGFs are having on retinal cells is
developmentally restricted and involves the proliferation of
photoreceptor progenitors.
[0536] The possibility that VEGF effects other retinal cell types
that are born postnatally e.g. amacrine and Muller cells, is also
investigated. The morphology of amacrine cells, identified on the
basis of their expression of syntaxin, is examined. (Data not
shown). Treatment with either VEGF induced a dose-dependent
increase in the number of syntaxin immunopositive cells with 10
ng/ml inducing a maximal increase of approximately 2.4-fold as
compared to the vehicle treated controls (FIG. 18A). In contrast to
the results with the rhodopsin ELISA and cell counts, 100 ng/ml of
VEGF-1 or -2 induced a smaller increase in the number of syntaxin
immunopositive cells than is observed with 10 ng/ml. To further
characterize the effect VEGF-2 treatment on the phenotype of
amacrine cells, the level of high-affinity GABA uptake is measured.
FIG. 18B depicts the dose-dependent increase in GABA uptake induced
by VEGF. The dose response curve is similar to that observed when
using the number of immunopositive syntaxin cells as the endpoint
with a significant increase and saturation in GABA uptake occurring
with 1 and 10 ng/ml of VEGF-2, respectively.
[0537] Muller glial cells are identified on the basis of their
expression of glial fibillary acidic protein (Bjorklund et al.,
1985). To determine if VEGF-2 has an effect on the number of Muller
cells or on the level of differentiation, the amount of GFAP
protein is measured by ELISA. After 7 days in culture, there is no
significance difference in the level of GFAP, when comparing
treatment with factors versus the vehicle control (FIG. 18C).
Furthermore, treatment with VEGF-2 did not increase the number of
endothelial cells, immunopositive cells, in the retinal cultures
(data not shown).
[0538] To further characterize the developmental pattern of the
VEGF response, retinal cells are isolated at different
developmental stages, and the mitogenic response to VEGF-2 is
quantitated after 48 h by labeling the cultures with
[.sup.3H]thymidine. In addition, as it has been noted previously
that the differentiation of photoreceptor cells in vitro is density
dependent, the effect that plating density has on the response to
VEGF is also investigated.
[0539] When the cultures are derived from E15 animals and plated at
a density of 212 cells/mm.sup.2, the basal level of
[.sup.3H]thymidine incorporation is 1589.+-.94 dpm/well and
treatment with VEGF-2 induced a maximal increase of 50% (FIG. 19A).
In contrast to the dose response observed with P1 cultures where
saturation occurs at 10 ng/ml, the proliferative response in the
E15 cultures saturates at a concentration of 1 ng/ml. Furthermore,
there is an inverse relationship between the plating density and
the mitogenic response to VEGF-2. At a density of 318
cells/mm.sup.2, a leftward shift in the dose response curve is
noted with concentrations higher than 1 ng/ml causing a
desensitization of the response. At the highest density tested (425
cells/mm.sup.2), the retinal cells are unresponsive to VEGF-2. It
is interesting to note that FGF-2 (10 ng/ml), which has a similar
biological activity as VEGF-2 in the P1 cultures (see below),
inhibited the proliferation in the E15 cultures by as much as 62%
in the higher density cultures (data not shown).
[0540] In cultures derived from E20 animals the basal level of
[.sup.3H]thymidine incorporation at a plating density of 212
cells/mm.sup.2 is 3361.+-.192 and the level of stimulation of
[.sup.3H]thymidine incorporation with VEGF-2 treatment is generally
greater, ranging up to 80-100%, at the lower plating densities
(FIG. 19B). There is still a trend toward VEGF-2 having less of an
effect in cultures plated at the highest density. However, the
inhibitory effect is much less pronounced. By P1, where the basal
level of [.sup.3H]thymidine incorporation is 478.+-.33, there is a
leftward shift in the dose response with saturation occurring at 10
ng/ml and the extent of the maximal increase is greater, in the
range of 300% (FIG. 19C). Furthermore, there is no discernible
effect of plating density on the response to VEGF-2.
[0541] To characterize more fully the responsiveness of the rod or
rod progenitor cells, the effect that EGF, FGF-2 or TGF.beta.-1 has
on the number of retinal cells and on the level of rhodopsin
protein is compared to that achieved with VEGF-2. EGF, a mitogen
for various cell types, induces a 31% increase in the number of
retinal cells with the response saturating at 1 ng/ml and remaining
stable up to 100 ng/ml (FIG. 20A). However, there is no concomitant
increase in the level of rhodopsin protein in the EGF treated
cultures (FIG. 20B). FGF-2 in a concentration range of 1-100 ng/ml
induces a small increase (13%) in the number of retinal cells.
Furthermore, FGF-2, which activates a number of the FGF receptors,
induces an increase in the level of rhodopsin protein. A 45%
increase in the level of rhodopsin is observed with concentrations
of FGF-2 as low as 1 ng/ml resulting in an EC50 value for the
response in the range of 0.5 ng/ml. Treatment with TGF.beta.-1
results in a decrease in both the number of retinal cells and the
level of rhodopsin protein. At a concentration of 0.1 ng/ml,
TGF.beta.-1 maximally decreased calcein expression and the level of
rhodopsin protein by 40 and 90%, respectively.
[0542] The results from the BrdU labeling experiments demonstrate
that VEGF-2 enhances the rate of proliferation of retinal
progenitor cells. Since the developmental pathway of photoreceptor
cells is thought to be lineage independent and thus under the
regulation of environmental factors (Ezzeddine ZD et al., 1997),
VEGF may also modulate photoreceptor cell development at additional
downstream sites. It has been determined previously that CNTF
inhibits the differentiation of photoreceptor cells relatively late
in their developmental pathway by redirecting their phenotype
toward the bipolar cell lineage. To investigate the potential
interaction of the two factors by co-treating retinal cultures with
VEGF-2 at a concentration that is saturating for the induction of
photoreceptor cells and various concentrations of CNTF. The
increase in rhodopsin protein induced by VEGF-2 is inhibited by
CNTF in a dose-dependent manner (FIG. 21A). The inhibitory response
had an IC50 value of 0.4 ng/ml and treatment with 100 ng/ml of CNTF
resulted in the complete inhibition of the VEGF-2 response.
However, treatment with CNTF did not alter the total number of
retinal cells in the cultures (FIG. 21B). To determine if the
inhibitory effect of CNTF is an early or late event, the effect
that co-administration of CNTF had on the increased level of
[.sup.3H]thymidine incorporation induced by VEGF-2 is tested. In
contrast to the previous results, the addition of CNTF did not
inhibit the VEGF induced proliferative response (FIG. 21C). These
findings further substantiate that these two factors regulate
photoreceptor cell development at different points in the lineage
pathway.
[0543] Discussion
[0544] The above experiments identify and characterize the effect
of VEGF-1 and VEGF-2 on retinal cells in vitro. Treatment with VEGF
in the sub-nanomolar range induces an increase in the number of
photoreceptor and amacrine cells as well as increases the level of
rhodopsin protein and high-affinity GABA uptake. Time course
studies demonstrate that VEGF induces a maximal increase in
[.sup.3H]thymidine incorporation within 48 h of its addition and
delaying the treatment of the cultures by 24-48 h results in the
loss of the proliferative and differentiation responses. The
mitogenic response was developmentally regulated with VEGF-2
inducing an increase in [.sup.3H]thymidine incorporation with cells
derived from E15, E20 and P1 animals. In comparison with members of
other trophic factor families, the response to treatment with
VEGF-2 and FGF-2 were similar in that both factors increased the
level of rhodopsin protein without inducing an increase in the
total number of cells after 9 days in culture. The
co-administration of CNTF with VEGF-2 resulted in the inhibition of
the VEGF induced increase in the level of rhodopsin but not in the
proliferative response.
[0545] The VEGF receptor family is currently composed of four
members (for review see Klagsbrun and D'Amore, 1996; Wen et al.,
1998). The receptors demonstrate distinct yet overlapping ligand
specificity. VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1) bind the various
forms of VEGF-1; while, VEGFR-2 and VEGFR-3 (Flt-4) bind VEGF-2.
Thus both VEGF-1 and VEGF-2 activate VEGFR-2 (Joukov et al., 1998)
and both ligands have similar biological activities in the retinal
cultures. Recently, Yang and Cepko (1996) described the
developmental expression pattern of VEGFR-2 in the retina. Extent
from the expected expression of the receptor on the newly forming
vasculature, receptors were also present on components of the
neural retina. This expression pattern is maintained during
development as the retina grows in a centripetal manner.
[0546] The effect of developmental age on the response of the
retinal progenitor cells to VEGF is consistent with the
developmental expression pattern of the receptor (Yang and Cepko,
1996). Mitogenic effects of VEGF, based on [.sup.3H]thymidine
incorporation studies, were noted at the earliest developmental
time point examined, E15, as well as at E20 and P1. The magnitude
of the proliferative effect increased with age reaching a peak by
P1. VEGF-2 is more efficacious on E15 and E20 cultures than at P1
since that response saturated at 1 as opposed to 10 ng/ml,
respectively. Furthermore, the basal level of proliferation in
vitro also changed with developmental age with the highest levels
observed at E17. The finding that the basal level of proliferation
was relatively low at E15 but increased 4-fold with a 2-fold
increase in cell density, a greater proportional increase than was
observed at the other developmental ages, suggests that endogenous
mitogens may underlie the desensitization that occurs with VEGF-2
treatment in the E15 cultures. Moreover, these data indicate that
increased levels of VEGF during early development may have a
negative impact on the differentiation of photoreceptor cells. The
influence of developmental age on the response of retinal
progenitor cells to other growth factors has also been observed
(Altshuler and Cepko, 1992). Lillien and Cepko (1992) reported that
the proliferative response of retinal cells in monolayer cultures
to FGF-1 and FGF-2 was greater at earlier gestational ages (e.g.
E15 and E18) and by E21 or P0 a rightward shift in the dose
response curve was apparent.
[0547] Previous studies in goldfish and frog have suggested that
amacrine cell development is regulated by cell-cell contact
(Negishi et al., 1982; Reh and Tully, 1986). More recently, the
importance of cell-cell contact for the in vitro development of
photoreceptor cells was also described by Wantabe and Raff (1990,
1992) in reaggregated cultures and then later by Altshuler and
Cepko (1992) with dissociated retinal cells plated in collagen
gels. In the former study, when E15 retinal cells were reaggregated
with a 50-fold excess of neonatal retinal cells, there was no
change in the developmental time when the rhodopsin immunopositive
cells were observed. However, there was a significant increase in
the proportion of the E15 cells that eventually differentiated into
photoreceptor cells. In the case of the monolayer cultures used in
this study, there is a dissociation between the VEGF-2 induced
early proliferative response and the later differentiation of
photoreceptor cells. For example, VEGF-2 increases
[.sup.3H]thymidine incorporation by 3-4-fold in cultures seeded at
densities as low as 212 cells/mm.sup.2 and treatment for 7 days
resulted in cell densities equivalent to those achieved at the
higher plating densities (e.g. 425 cells/mm.sup.2). However, there
was no detectable rhodopsin protein or immunopositive cells in
these cultures. These results suggest that there is not only a
critical cell-cell interaction necessary for the development of
photoreceptor cells but also a time frame during which the stimulus
produced via cell contact is probable necessary.
[0548] Comparing the time course of the VEGF-induced proliferation
to the developmental time course of the appearance of rhodopsin
protein indicates that there is an approximate 5 day lag between
the two events. The appearance of rhodopsin protein likely reflects
the induction of gene transcription since the two events have been
shown to be closely correlated (Treisman et al., 1988). This time
interval is similar to that observed by Morrow et al. (1998) in
vivo and in vitro studies when considering progenitor cells derived
from animals within an age range of E20 to P3. Furthermore between
5 and 9 days in vitro, we observed the greatest increase in the
level of rhodopsin protein and this time period is within the
postnatal developmental period (day 6-10) in vivo during which
there is a pronounced appearance of rhodopsin immunopositive cells
(Morrow et al., 1998). The correlation in these developmental time
windows suggests that although VEGF-2 induces the proliferation of
photoreceptor progenitor cells, it does not induce a significant
delay in the differentiation of photoreceptor cells. As might be
expected if the progenitor cells were prevented from leaving the
cell cycle.
[0549] In comparison to members of other trophic factor families,
the response to VEGF-2 resembled that of FGF-2 in that both factors
increased the level of rhodopsin protein while inducing relatively
small increases in the total number of retinal cells after 9 days
in vitro. In addition, a proliferative response, based on
[.sup.3H]thymidine incorporation and cell counts, to FGF-2 was
noted by Lillien and Cepko (1992) as late as P3 suggesting that
FGF-2 retains some mitogenic activity in postnatal cultures. In
contrast to our findings with VEGF-2, Fontaine et al. (1998)
demonstrated that FGF-2 also has a survival effect on photoreceptor
cells derived from P5 animals (data not shown). TGF.beta.-1
treatment resulted in a decrease in both the number of retinal
cells and the level of rhodopsin protein. Kimichi et al. (1988)
reported similar observations using human fetal retinal cultures
with the exception that maximal inhibition with the human cells
required 0.5 ng/ml of TGFB-1 as compared to the less than 0.1 ng/ml
required in the rodent cultures.
[0550] CNTF, a member of the neuropoietic family of cytokines, is
known to effect the development of photoreceptor cells in vitro and
in vivo and to enhance the survival of photoreceptor cells
following light-induced damage (Unoki and LaVail, 1994; Fuhrman et
al., 1995; Ezzeddine et al., 1997; Cayouette et al., 1998). In
contrast to CNTF, VEGF-2 did not rescue photoreceptor cells in the
constant light-induced damage model (LaVail et al., 1992; Wen et
al., 1995; R. Wen and R. Alderson, unpublished data). Treatment of
postnatal rat retinal explant cultures with CNTF results in an
increase in the number of cells expressing bipolar cell markers
with a loss in the population of cells expressing rhodopsin.
Analysis of the effect of CNTF on the fate of [.sup.3H]thymidine
labeled P0 retinal cells suggests that the cytokine does not induce
the proliferation or increase the survival of this cell population
(Ezzeddine et al., 1997). Furthermore, the initiation of the effect
of CNTF occurred at about the time that the cells became
post-mitotic and begin to express rhodopsin. These data are
consistent with the findings reported here demonstrating that CNTF
inhibits the VEGF-2 induced increase in rhodopsin protein observed
between 5 and 7 days in culture, but not its mitogenic activity
observed between 1 and 2 days.
[0551] During the course of development in the retina, oxygen
levels control the microarchitecture of retinal vessels that in
turn match the pattern of differentiation of retinal neurons
(Chan-Ling et al., 1990; Phelps, 1990). Stone et al. (1995) have
demonstrated that in the retina, astrocytes and microglia respond
to hypoxia by synthesizing and secreting VEGF which in turn induces
vessel formation. The studies reported here suggest that the early
differentiation events regulated by VEGF involve not only vessel
formation but also photoreceptor progenitor cell proliferation.
This ultimately may result in the coordinated development of
numerous cell types in the retina.
Example 9
Enhanced Response of Endothelial Cells to VEGF-2 and Antibody
Co-Treatment
[0552] Antibodies generated by HGS have been shown to bind to
VEGF-2 by ELISA assays, but are not thought to bind to the sites
involved in receptor interactions. Monoclonal 13D was mapped to an
epitope on the N-terminal side of the molecule and monoclonal 13A2
was mapped to an epitope on the C-terminal end. (See FIG. 24). The
polyclonal antibody recognizes a number of different sites but it
is not believed to bind to the segment of the active protein which
interacts with the receptor. These antibodies were used to
quantitatively determine stimulation or inhibition of the
proliferation of bovine lymphatic endothelial cells (LEC) by
co-treating with VEGF-2 and the above antibodies.
[0553] An alamar blue.TM. LEC proliferation assay was used, which
incorporates a fluorometric growth indicator based on detection of
metabolic activity. The Alamar blue.TM. is an oxidation-reduction
indicator that both fluoresces and changes color in response to
chemical reduction of growth medium resulting from cell growth. As
cells grow in culture, innate metabolic activity results in a
chemical reduction of the immediate surrounding environment.
Reduction related to growth causes the indicator to change from
oxidized (non-fluorescent blue) form to reduced (fluorescent red)
form. i.e. stimulated proliferation will produce a stronger signal
and inhibited proliferation will produce a weaker signal and the
total signal is proportional to the total number of cells.
[0554] The materials used for the alamar blue.TM. LEC proliferation
assay include: Alamar Blue.TM. (Biosource Cat #DAL1100); DMEM 10%
FBS+PennStrep+Glutamine+75 mg BBE+45 mg Heparin (Growth media);
DMEM 10% FBS+PennStrep+Glutamine (Starvation Media); DMEM 0.5%
FBS+PennStrep+Glutamine (sample dilution media); CytoFluor.TM.
Fluorescence reader; 96 well plate(s); and LEC cells.
[0555] The alamar blue.TM. assay was performed as follows. For
timing purposes it is best to seed the cells onto the 96 well
plate(s) on a Wednesday, change to starvation media on Thursday,
inoculate samples on Friday, incubate over the weekend then add
alamar blue.TM. incubate and read on Monday.
[0556] LEC cells were seeded in growth media at a density of 5000
cells/well of a 96 well plate and placed at 37.degree. C.
overnight. After the overnight incubation of the LEC cells, the
growth media was removed and replaced with starvation medium and
incubated for another 24 hours at 37.degree. C. After the second 24
hour incubation, the cells were inoculated with the appropriate
dilutions of protein sample(s) (prepared in DMEM+0.5% FBS) in
triplicate wells. Once the cells have been inoculated with the
samples the plate was placed back in the 37.degree. C. incubator
for three days.
[0557] After three days, 10 .mu.l of stock alamar blue.TM. was
added to each well and the plate was placed back in the 37.degree.
C. incubator for four hours. The plate was then read at 530 nm
excitation and 590 nm emission using the CytoFluor.TM. fluorescence
reader. Direct output is recorded in relative fluorescence
units.
[0558] The background level of activity was observed with the
starvation medium alone. This is compared to the output observed
from the positive control samples (VEGF-1 and/or bFGF) and the HGS
protein dilutions.
[0559] Three different antibody preparations made by HGS (2 mouse
monoclonals, 13A2, 13D6 and a rabbit polyclonal antibody) were
evaluated for their ability to modulate the response of LECs to
VEGF-2 mediated activation. It was previously determined that a
proliferative response of LECs could be observed at a concentration
of 1000 ng/ml of VEGF-2. Therefore, VEGF-2 samples at a
concentration 1000 ng/ml in DMEM were premixed with one of the
three different anti-VEGF-2 antibodies (10 .mu.g/ml) and used in
the alamar blue.TM. assay system to determine the influence on LEC
proliferation. Controls in the first experiment included VEGF-2
alone (1000 ng/ml), bFGF (10 ng/ml, positive control), IL-2
(irrelevant protein negative control) and starvation medium (assay
negative control). The repeat experiment also included antibody
alone (10 .mu.g/ml) as a negative control.
[0560] As shown in FIG. 22, VEGF-2 treatment of LECs at a
concentration of 1000 ng/ml resulted in a proliferative response
relative to the negative controls, which was consistent with
previous proliferative assays conducted with these cells.
Simultaneous treatment of LECs with VEGF-2 and monoclonal 13A2 did
not augment the proliferative response above the level achieved
with VEGF-2 alone. However, an enhanced proliferative response was
observed with the 13D6 monoclonal and to a lesser degree, with the
rabbit polyclonal antibody.
[0561] As shown in FIG. 23, the experiment was repeated under more
stringent conditions, using 1000 cells/well as an initial cell
concentration and included stimulation with the antibody alone in
order to control for possible direct effects of the antibodies on
the LECs. This experiment demonstrated augmentation of VEGF-2
mediated proliferation by the 13D6 and polyclonal antibodies above
the proliferative response observed with VEGF-2 or the antibodies
alone. As observed in the previous experiment, the 13A2 antibody
did not induce an augmented proliferative response.
[0562] These observations suggest that antibody mediated
crosslinking of VEGF-2 molecules bound to receptors (VEGFR2 or
VEGFR3) may induce receptor dimerization. Such a process may be
used to intensify the signaling resulting from the VEGF-2 binding
to its receptors.
Example 10
Mouse Immunization for Monoclonal Antibody Production
[0563] Animals are individually housed and received food and water
ad libitum. All manipulations are performed using aseptic
techniques. The experiments are 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.
[0564] Dilute concentration of protein in 350 .mu.ls of phosphate
buffered solution (PBS), or other neutral buffer, to a final
protein concentration of 0.43 mg/ml. With 0.35 mls. Freund's
Complete Adjuvant, emulsify the adjuvant and protein solution for a
period of ten minutes using two glass 3 cc syringes and a three way
disposable stopcock (Baxter Cat. No. 2C6240). To test emulsion for
quality, place 50 .mu.ls of the emulsion onto the surface of cold
water in a beaker. If the emulsion does not remain as an intact
white droplet, then further mixing is required.
[0565] Draw all of the emulsion into one syringe, and using a 27
gauge needle, inject mouse subcutaneously with a total of 200
.mu.ls of emulsion distributed among 4-8 sites including axillary
and inguinal areas, the back of the neck, and along the back.
[0566] Following two to three weeks, repeat the above injection
substituting Freund's Incomplete Adjuvant (as opposed to Freund's
Complete Adjuvant).
[0567] Following an additional two to three weeks, a third
injection is given as outlined above, making sure to use Freund's
Incomplete Adjuvant.
[0568] Ten to Fourteen days following the third injection, obtain
100-200 .mu.ls of blood from the mouse by tail vein bleed. Incubate
the blood at 37.degree. C. for 60 minutes, and then allow to cool
overnight at 4.degree. C. Following incubation at 4.degree. C.,
centrifuge the blood for ten minutes. Transfer the serum to a new
tube, and test for mouse serum titer. If titer is found to be low,
intraperitoneal (ip) injections can be given at biweekly intervals.
For ip injections, prepare 10-20 .mu.gs protein per mouse in a
volume of 200-400 .mu.ls of PBS per mouse. Using a 1 cc syringe and
a 26 gauge needle, inject the solution into the mouse. Do a second
tail bleed 10-14 days following injection, and retest the mouse
serum titer.
Example 11
Mouse Serum Titer ELISA
[0569] Coat the ELISA plate with 50 .mu.l/well of purified antigen
at 2 .mu.gs/ml PBS. Cover the ELISA plate with parafilm and
incubate at 4.degree. C. overnight in a humid chamber. Following
incubation, wash the plate four times with 200 .mu.l/well of PBS
per wash. Block with 3% BSA, 200 .mu.ls/well for 60 minutes at room
temperature. Shake out blocking solution.
[0570] Add serum samples in duplicate, 50 .mu.ls/well, at dilutions
of 10.sup.-2, 10.sup.-3, 10.sup.-4, 10.sup.-5, 10.sup.-6, and
10.sup.-7, diluted in PBS containing 0.1% BSA. Include blanks of
buffer as well as positive and negative control serum at the above
dilutions. Incubate at room temp for 1-2 hours. Wash with PBST (PBS
with 0.05% tween), 250 .mu.ls/well, four times.
[0571] Add 50 .mu.ls/well of Biotinylated Anti-Mouse IgG at a
concentration of 0.5 .mu.g/ml in PBST containing 0.1% BSA and 2%
Horse Serum. Incubate at room temperature for 30 to 60 minutes.
Wash plate four times with PBST.
[0572] Add 50 .mu.ls/well of ABC reagent (Vector Cat. No. PK-6100)
to the plate and incubate at room temperature for 30 minutes. Wash
plate six times with PBST.
[0573] Prepare substrate for ELISA detection by dissolving 1
tetramethylbenzidine Dihydrochloride (TMB) tablet (Sigma Cat. No.
T-3405) in 5 mls. of ddH.sub.2O. Add 5 mls. of 0.1M Phosphate
Citrate Buffer (25.7 mls of 0.2M dibasic sodium phosphate, 24.3 mls
of 0.1M citric acid monohydrate, pH 5.0). Add 2 .mu.ls of fresh 30%
hydrogen peroxide, vortex and use immediately.
[0574] Following incubation and washing of the plate, add 100
.mu.ls of substrate solution and incubate at room temperature for
approximately 15-30 minutes. Stop the reaction by adding 25
.mu.ls/well of 2M H.sub.2SO.sub.4, and read the plate at 450 nm
within 30 minutes versus the controls.
Example 12
Fusion Protocolfor Hybridoma Production
[0575] One week prior to the fusion step, make P3.times. growth
medium (1.times. DMEM 0% (Gibco Cat. No. 11965-019), 5-10% Fetal
Bovine Serum, 1.times. L-Glutamine (Biofluids Cat. No. 300), and
1.times. Sodium Pyruvate (Biofluids Cat. No. 333). Thaw a new vial
of P3.times. mouse myeloma cells into 1 well of a 6-well dish (see
thawing protocol, infra) and start expanding them in P3.times.
growth medium. If viability is good the next day transfer to a 100
mm dish. Cell density must not exceed 10.sup.6 cells/ml or greater.
Furthermore, membranes of these cells should not look granular. By
the day of the fusion procedure there should 6-8 plates at
5-8.times.10.sup.5 cells/ml. It is a good idea to test some
P3.times. cells in HAT medium. All cells should be dead within
around 4 days. If not then P3.times. cells should be grown in
P3.times. medium containing 15 ug/ml 8-azaguanine to eliminate
revertants.
[0576] Four days prior to the fusion procedure, the mouse should be
immunized with an ip injection of approximately 10 .mu.g of high
purity protein.
[0577] One day before the fusion, split the P3.times. cells and
feed them with fresh medium as needed so that cells will be healthy
and growing in log phase by the next day.
[0578] On the day of the fusion procedure, place 50 mls of
P3.times. media, PEG solution and HAT media (1.times. DMEM 0%, 20%
Fetal Bovine Serum, 4% Hybridoma Supplement-BM Condimed HI
(Boehringer-Mannheim), 1.times. L-Glutamine, 1.times. NEAA,
1.times. Sodium Pyruvate, 1.times. HAT (Sigma Cat. No. H0262),
1.times.0.05M 2ME, and 1.times. Penicillin-Streptomycin) in a
37.degree. C. water bath. Have available approximately 100 ml cold
DMEM 0%.
[0579] Check all P3.times. plates for possible contamination and to
assess health of cells. Resuspend cells from 4 plates or flasks and
combine in 50 ml tubes. Centrifuge at 200.times. G for 10 min.
Aspirate the supernatant. Resuspend each tube with 10 mls DMEM 0%
and pool. Count live cells using trypan blue viability stain
(viability should be greater than 90%). The total number of cells
should be 2-4.times.10.sup.7 cells. If there are not enough cells
then repeat the process with some more plates. Let cells sit at
ambient room temperature (ART) until needed in future step.
[0580] Prepare hood where spleen will be removed with: 70% EtOH,
sterile instruments including sieve and plunger, 2 petri dishes
containing 10 ml DMEM 0% and 15 ml centrifuge tubes (2).
[0581] The mouse is sacrificed, and the spleen is then harvested
from the carcass. Place the spleen in the petri dish containing
DMEM 0%. Place the sieve in the other dish containing 10 ml DMEM 0%
and cover with plate lid. Transfer the spleen to the sieve using a
sterile pair of foreceps, and using the syringes with needles,
tease the spleen apart so that cells spill out into the media.
Then, using the other plunger, gently squish the spleen through the
sieve. Avoid grinding the spleen organ tissue through the sieve as
this will result in heavy fibroblast growth.
[0582] Remove the sieve and transfer the spleen cell suspension to
a 15 ml centrifuge tube. Wash remaining cells from the dish with 5
mls DMEM 0%, and add to the tube. Allow the tube to sit for 5
minutes to allow large debris to settle to the bottom. Then
transfer the cell suspension, minus debris, to the second 15 ml
tube. Centrifuge the cells for 10 min. at 200.times. G. Aspirate
s/n and resuspend the spleen cells in 5 mls DMEM 0%. Add 5 more mls
of DMEM 0%, and transfer the entire volume to a 50 ml tube.
[0583] Remove 10 .mu.ls of the spleen cell suspension, and add to
500 .mu.ls of Trypan blue in order to count lymphocytes. (Note:
normally a spleen will consistently yield 10.sup.8
lymphocytes).
[0584] Fusion
[0585] To the 50 ml centrifuge tube containing the spleen cells add
sufficient P3.times. cells to make a lymphocyte to P3.times. cells
ratio of 5:1. (e.g. for 10.sup.8 lymphocytes you will need
2.times.10.sup.7 P3.times. cells). Bring the total volume up to
45-50 mls with DMEM 0%, and centrifuge at 200.times. G for 10
minutes. Prepare a transfer hood with a timer, warm PEG, warm
P3.times. media, and a beaker of water approximately 38-40.degree.
C. Aspirate all of the supernatant from the P3.times.-lymphocyte
pellet and attempt to loosen the pellet by flicking the tube. Place
the tube in the small water bath. Keep the fusion tube in the warm
water, and while gently shaking, add 1 ml of PEG dropwise over 1
minute. Then, let sit with occasional shaking for 1-2 minutes,
following which add 1 ml of P3.times. media dropwise over 1 minute.
Next, add 3 mls. of P3.times. media dropwise over 1 minute,
followed by the addition of 10 mls. of P3.times. media dropwise
over 1 minute.
[0586] Gently add P3.times. media to make the total volume 45 mls.
Allow the tube to sit for 10 minutes, then centrifuge at 200.times.
G for 10 minutes. Aspirate the supernatant and gently resuspend the
pellet in 5 mls or less of HAT medium. Transfer the cell suspension
to the bottle containing 400 mls of HAT medium and swirl to mix.
Pour some of the cell suspension into a sterile reservoir.
[0587] Plant cells in 96 well plates, 200 .mu.ls/well, using a 12
channel pipettor with filtered tips. Place plates in the incubator.
Monitor plates every day for hybridoma growth or contamination.
Allow plates to incubate for three days. The first feeding (medium
change) is done by around day 7 by aspirating off approximately
half the media in each well using the 8-position manifold and
replacing it with 100-150 .mu.ls/well HT medium. Feeding a week or
so before the first screening helps to dilute out any antibody
produced by the unfused lymphocyte cells which have been found to
continue producing antibody after 2 weeks in culture. Many or all
of the wells will be ready to be sampled for screening within 2
weeks after the fusion when the colony or colonies fill more than
half the well and the supernatant has changed color to a
orange/yellow.
Example 13
ELISA Screening of Mouse Hybridomas
[0588] To screen the mouse hybridomas, coat the ELISA plate
(Immulon 2 "U" bottom microtiter plate (Dynatech Cat. No.
011-010-3555)) with 50 .mu.ls/well of the antigen at 2 .mu.gs/ml
PBS. Cover the ELISA plate with plastic seal and incubate at
4.degree. C. overnight. Following incubation, wash the plate four
times with 200 .mu.ls/well of PBS per wash. Block with 3% BSA, 200
.mu.ls/well for 60 minutes at room temperature. Shake out blocking
solution.
[0589] Add hybridoma supernatants, 150 .mu.ls/well, into a Corning
96 well assay plate, then transfer 50 .mu.ls of each supernatant
from the Coming assay plate into the ELISA plate. Include blanks of
culture medium as well as positive and negative mouse serum
controls. Incubate at room temp for 1-2 hours, or overnight at
4.degree. C. Wash with PBST (PBS with 0.05% tween), 250
.mu.ls/well, four times.
[0590] Add 50 .mu.ls/well of Biotinylated Anti-Mouse IgG H+L, at a
concentration of 0.5 .mu.g/ml in PBST containing 0.1-0.3% BSA and
1% Horse Serum. Incubate at room temperature for 30 to 60 minutes.
Wash plate four times with PBST.
[0591] Add 50 .mu.ls/well of ABC reagent (Vector Cat. No. PK-6100)
to the plate and incubate at room temperature for 30 minutes. Wash
plate six times with PBST.
[0592] Prepare substrate for ELISA detection by dissolving 1
tetramethylbenzidine Dihydrochloride (TMB) tablet (Sigma Cat. No.
T-3405) in 5 mls. of ddH.sub.2O. Add 5 mls. of 0.1M Phosphate
Citrate Buffer (25.7 mls of 0.2M dibasic sodium phosphate, 24.3 mls
of 0.1M citric acid monohydrate, pH 5.0). Add 2 .mu.ls of fresh 30%
hydrogen peroxide, vortex and use immediately.
[0593] Following incubation and washing of the plate, add 100
.mu.ls of substrate solution and incubate at room temperature for
approximately 15-30 minutes. Stop the reaction by adding 25
.mu.ls/well of 2M H.sub.2SO.sub.4, and read the plate at 450 nm
within 30 minutes versus the controls.
Example 14
Testing Relative Affinity of Monoclonals Derivedfrom Culture
Supernatants
[0594] A. Determining Antigen Coating Concentration
[0595] Make approximately 1 ml of the antigen at a concentration of
4 .mu.gs/ml in PBS. Transfer to a microdilution tube. Place 0.5 ml
PBS in each of 9 microdilution tubes, then do serial dilutions of
1/2 by transferring 0.5 ml from tube to tube starting from the 4
.mu.gs/ml tube. You will now have tubes containing 4, 2, 1, 0.5,
0.25, 0.125, 0.06, 0.03, 0.015 and 0.0075 .mu.gs/ml. Coat a plate
with the above concentrations, 6 wells each, 50 .mu.l/well.
[0596] Cover and incubate over night at 4.degree. C. Following
incubation, wash the plate four times with 200 .mu.ls/well of PBS
per wash. Block with 3% BSA, 200 .mu.ls/well for 60 minutes at room
temperature. Shake out blocking solution.
[0597] Look at the titer curve of mouse serum which is positive to
the antigen. Determine the serum dilution which is just at the top
of the titration curve. Add the positive mouse serum at this
dilution in PBS containing 0.1% BSA, 50 .mu.ls/well, rows B-D,
columns 2-11. Include negative control serum at the above dilution
in rows E-G, columns 2-11. Incubate overnight at 4.degree. C.
Following incubation, subtract Negative Control Serum values from
Positive Control Serum values. Plot mean value (O.D. 450) against
antigen concentration on linear scale. Determine antigen coating
concentration which will give a submaximal O.D. This is the coating
concentration to use for the relative affinity assay.
[0598] B. Determining the Mouse IgG Concentration of the Hybridoma
Supernatant Sample Using the Boehringer Mannheim Biochemica Kit
"Mouse-IgG ELISA" (Cat. No. 1333 151)
[0599] Dilute the coating buffer concentrate 1/10 with ddH.sub.2O.
Ten to twenty mls. will be necessary. Obtain an aliquot of capture
antibody. Thaw 3 tubes of Post Coating Buffer Concentrate (blocking
solution). Twelve wells will be necessary for the standards and 4-6
wells for each supernatant to be tested. Calculate the number of
mls of diluted Capture Antibody necessary assuming 50 .mu.ls/well
of coating volume. Dilute Capture Antibody in the following
proportion:
25 u l Capture Ab 1 ml Coating Buff = X l Capture Ab # ml Coating
Buff ##EQU00001##
[0600] Coat Nunc plate with the solution and incubate 30 mins at
room temperature on a shaker. Dilute the concentrated Post Coating
Buffer 1/10 in ddH.sub.2O. Wash the plate with ELISA wash buffer
(0.9% NaCl, 0.1% Tweeen 20), and block with 200 .mu.ls/well of Post
Coating Buffer (Block solution) for 15 minutes at room
temperature.
[0601] Dilute the IgG standard into Post Coating Buffer (blocking
solution) to the following concentrations: 0.2, 0.1, 0.05, 0.025,
0.0125 and 0.00625 .mu.gs/ml in Post Coating Buffer.
[0602] Dilute supernatants into blocking buffer to make final
concentrations of 1/100 and 1/1000. Following the blocking step,
wash the plate and add 50 .mu.ls/well of diluted IgG standards and
diluted supernatants in duplicate. Incubate for 30 minutes at room
temperature on a shaker.
[0603] Dilute the conjugate solution into Post Coating Buffer
(block solution) according to the proportion below:
50 l Conjugate 1 ml Block sol = X l Conjugate # ml Block sol
##EQU00002##
[0604] Wash the plate and add 50 ul/well of conjugate. Incubate the
plate for 30 minutes at room temperature on shaker.
[0605] Dissolve 1 substrate tablet in 5 mls substrate buffer. Wash
the plate and add 50 .mu.ls/well of substrate. Incubate 30 minutes
at room temperature on a shaker and read at 405 nm.
[0606] C. Relative Affinity Assay
[0607] Coat appropriate ELISA plate(s) overnight at 4.degree. C
with the antigen concentration previously determined. Block plate
as above. Make 1/3 serial dilutions into PBS+0.1% BSA of test
supernatant.
[0608] Add 50 .mu.ls/well of the dilutions of the supernatant
sample, including the undiluted sample, to the ELISA plate(s) in
duplicates or triplicates. The positive control consists of a few
wells of the positive control mouse serum at the same concentration
as used for determining the antigen coating concentration. The
negative control consists of a few wells of the dilution buffer.
Cover and incubate overnight at 4.degree. C.
[0609] Plot the IgG concentration of each supernatant against the
mean-value (O.D. 450) on a 4 parameter curve fit. Supernatant
curves that are more to the left are the supernatants with the
highest affinities.
Example 15
Ascites Production in Mice
[0610] Hybridoma cells should be healthy and in log phase of growth
for ascites production. Transfer cells to a 15 ml. tube and count.
Determine the volume which contains 4.times.10.sup.6 cells,
transfer that volume to a second tube and centrifuge the cells.
Resuspend the pellet in 0.9 mls of HBSS (Hank's Balanced Salt
Solution) and transfer to an eppendorf tube.
[0611] Fill a 1 cc syringe with the cell suspension and inject mice
ip as follows: 0.2 cc per mouse if the original cell number was
4.times.10.sup.6 and 0.3 cc if the original cell number was
3.times.10.sup.6. When the abdomen is very distended and slightly
taught to the touch, like a balloon, (usually by day 9 or 10 but
sometimes as late as day 14) then it is time to "tap the
mouse".
[0612] A. Tapping:
[0613] Hold the mouse in your left hand and use an alcohol pad to
wipe off the area of the abdomen just above the mouse's left hind
leg. While holding the mouse above an open 15 ml centrifuge tube,
insert a 19 gauge needle into the abdomen. Ascites fluid should
immediately begin to drip out of the end of the needle into the
centrifuge tube An average mouse should yield 3-6 mls. of ascites
fluid.
[0614] B. Processing and Storage of Ascites:
[0615] Pool ascitic fluid collected from each mouse in the group
(all injected with the same hybridoma) and leave at room
temperature for 1-2 hours or place at 37.degree. C. for 15-30
minutes. Then place ascites at 4.degree. C. overnight to allow for
clot formation. Centrifuge clotted ascites for 10 minutes. Transfer
the liquid ascites to a 50 ml centrifuge tube, and store the tube
at -20.degree. C. Subsequent taps may be added to this 50 ml tube.
When all mice are sacrificed, the pooled ascites can be thawed,
respun, and aliquotted for long term storage at -20.degree. C. or
-70.degree. C. Ascites should be titered by ELISA.
Example 16
Protocolfor Freezing and Thawing Mouse Hybridoma and Myeloma
Cells
[0616] A. Freezing
[0617] Cells to be frozen down should be healthy, in log phase of
growth and at a concentration of roughly 5-8.times.10.sup.5
cells/ml. Resuspend cells from a 6 well plate or flask, transfer to
a 15 ml tube and count. Calculate the number of total cells and
divide by 1-3.times.10.sup.6 cells per vial to determine the number
of vials to be frozen down.
[0618] Pellet the cells at 200-300.times.G for 5-10 minutes.
Aspirate the supernatant from the pellet and resuspend in
sufficient cold freeze medium (50% FBS, 10% DMSO in DMEM; or Origen
DMSO Freeze Medium (IGEN), Fisher Cat. No. IG-50-0715) to achieve
the desired number of cells/vial per ml (cell densities should be
in a range from 5.times.10.sup.5 to 1.times.10.sup.7 cells/vial).
Immediately transfer the cell suspension to the cryovials, 1 ml per
vial, and place on ice. Transfer the vials to a controlled rate
freezer and place the freezer at -70.degree. C. for overnight.
After 24 hours transfer the vials to a liquid nitrogen tank or
-130.degree. C. freezer for long term storage.
[0619] B. Thawing
[0620] Add 10 ml cold media (e.g. P3.times. media) to 15 ml tube.
Retrieve cryovial of frozen cells and keep on dry ice until ready
to thaw. Thaw cells quickly in 37.degree. C. water bath. Hold vial
during thawing and keep shaking gently until there is just a small
bit of ice left in vial. Don't allow the contents to warm above
4.degree. C. Alcohol off the outside of the cryovial.
[0621] Using a sterile pasteur pipette and without touching the
edges of the cryovial, transfer the cell suspension in the vial to
the 10 mls. of cold media. Spin at 200-300.times.G for 5-10
minutes. Aspirate the supernatant and resuspend the pellet in 6
mls. of HT Cloning Media (supra). Transfer to 1 well of a 6 well
dish. Assess the viability after 24 hours. Viability should not be
less than 50%.
Example 17
In Vitro Assay for Angiogenic Protein Activity
[0622] The following assay is designed to detect angiogenic protein
activity, preferably VEGF-2 activity. For example, a chimeric
receptor is generated by fusing the nucleotides encoding for the
extracellular domain of the Flt-4 receptor (SEQ ID NO:38) (Galland
et al., Genomics 13 (2):475-478 (1992), which is herein
incorporated by reference in its entirety), to the nucleotides
encoding for the transmembrane domain and intracellular domain of
Flk-1 (SEQ ID NO:39) (Davis-Smyth et al., EMBO J 15(18):4919-4927
(1996), which is herein incorporated by reference in its entirety).
Thus, the chimeric receptor would include amino acids 1 to 775 of
SEQ ID NO:38, fused to amino acids 765 to 1356 of SEQ ID NO:39,
respectively.
[0623] Alternatively, the chimeric receptor may be designed as
outlined above, but would substitute the transmembrane and
intracellular domains of the erythropoietin receptor (EPOR) for the
transmembrane and intracellular domains of the Flk-1 receptor, as
discussed in Pacifici et al., JBC 269(3): 1571-1574 (1994), which
is herein incorporated by reference in its entirety (see
specifically FIG. 1).
[0624] The resulting DNA encoding for the chimeric receptor is
cloned into an appropriate mammalian, baculoviral, or bacterial
expression vector, such as, for example, pC4, pCDNA3, or pA2, as
discussed supra. Mammalian host cells that could be used for
expression of the chimeric receptor include NIH3T3 (supra), or the
pre-B cell line BaF3 (Achen et al., PNAS 95(2): 548-553 (1998),
which is herein incorporated by reference in its entirety).
[0625] To test for activity, the angiogenic protein can be brought
into contact with a cell line expressing the chimeric receptor, or
extracts thereof. Then, angiogenic protein binding to the chimeric
receptor can be detected by measuring any resulting signal
transduced by the chimeric receptor.
[0626] 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.
[0627] The entire disclosure of all publications (including
patents, patent applications, journal articles, laboratory manuals,
books, or other documents) cited herein are hereby incorporated by
reference.
[0628] Additionally, the entire specification, including the
Sequence Listing of U.S. application Ser. No. 09/107,997, filed
Jun. 30, 1998, and PCT Application No. US 99/05021 filed Mar. 10,
1999, are hereby incorporated by reference in their entirety.
Sequence CWU 1
1
3911674DNAhomo
sapiensCDS(12)..(1268)mat_peptide(81)..()sig_peptide(12)..(80)
1gtccttccac 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 -20 -15ctc
gcc gct gcg ctg ctc ccg ggt cct cgc gag gcg ccc gcc gcc gcc 98Leu
Ala Ala Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala-10 -5
-1 1 5gcc gcc ttc gag tcc gga ctc gac ctc tcg gac gcg gag ccc gac
gcg 146Ala Ala Phe Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp
Ala 10 15 20ggc gag gcc acg gct tat gca agc aaa gat ctg gag gag cag
tta cgg 194Gly Glu Ala Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln
Leu Arg 25 30 35tct gtg tcc agt gta gat gaa ctc atg act gta ctc tac
cca gaa tat 242Ser Val Ser Ser Val Asp Glu Leu Met Thr Val Leu Tyr
Pro Glu Tyr 40 45 50tgg aaa atg tac aag tgt cag cta agg aaa gga ggc
tgg caa cat aac 290Trp Lys Met Tyr Lys Cys Gln Leu Arg Lys Gly Gly
Trp Gln His Asn55 60 65 70aga gaa cag gcc aac ctc aac tca agg aca
gaa gag act ata aaa ttt 338Arg Glu Gln Ala Asn Leu Asn Ser Arg Thr
Glu Glu Thr Ile Lys Phe 75 80 85gct gca gca cat tat aat aca gag atc
ttg aaa agt att gat aat gag 386Ala Ala Ala His Tyr Asn Thr Glu Ile
Leu Lys Ser Ile Asp Asn Glu 90 95 100tgg aga aag act caa tgc atg
cca cgg gag gtg tgt ata gat gtg ggg 434Trp Arg Lys Thr Gln Cys Met
Pro Arg Glu Val Cys Ile Asp Val Gly 105 110 115aag gag ttt gga gtc
gcg aca aac acc ttc ttt aaa cct cca tgt gtg 482Lys Glu Phe Gly Val
Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val 120 125 130tcc gtc tac
aga tgt ggg ggt tgc tgc aat agt gag ggg ctg cag tgc 530Ser Val Tyr
Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys135 140 145
150atg aac acc agc acg agc tac ctc agc aag acg tta ttt gaa att aca
578Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr
155 160 165gtg cct ctc tct caa ggc ccc aaa cca gta aca atc agt ttt
gcc aat 626Val Pro Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe
Ala Asn 170 175 180cac act tcc tgc cga tgc atg tct aaa ctg gat gtt
tac aga caa gtt 674His Thr Ser Cys Arg Cys Met Ser Lys Leu Asp Val
Tyr Arg Gln Val 185 190 195cat tcc att att aga cgt tcc ctg cca gca
aca cta cca cag tgt cag 722His Ser Ile Ile Arg Arg Ser Leu Pro Ala
Thr Leu Pro Gln Cys Gln 200 205 210gca gcg aac aag acc tgc ccc acc
aat tac atg tgg aat aat cac atc 770Ala Ala Asn Lys Thr Cys Pro Thr
Asn Tyr Met Trp Asn Asn His Ile215 220 225 230tgc aga tgc ctg gct
cag gaa gat ttt atg ttt tcc tcg gat gct gga 818Cys Arg Cys Leu Ala
Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly 235 240 245gat gac tca
aca gat gga ttc cat gac atc tgt gga cca aac aag gag 866Asp Asp Ser
Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu 250 255 260ctg
gat gaa gag acc tgt cag tgt gtc tgc aga gcg ggg ctt cgg cct 914Leu
Asp Glu Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro 265 270
275gcc agc tgt gga ccc cac aaa gaa cta gac aga aac tca tgc cag tgt
962Ala Ser Cys Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys
280 285 290gtc tgt aaa aac aaa ctc ttc ccc agc caa tgt ggg gcc aac
cga gaa 1010Val Cys Lys Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn
Arg Glu295 300 305 310ttt gat gaa aac aca tgc cag tgt gta tgt aaa
aga acc tgc ccc aga 1058Phe Asp Glu Asn Thr Cys Gln Cys Val Cys Lys
Arg Thr Cys Pro Arg 315 320 325aat caa ccc cta aat cct gga aaa tgt
gcc tgt gaa tgt aca gaa agt 1106Asn Gln Pro Leu Asn Pro Gly Lys Cys
Ala Cys Glu Cys Thr Glu Ser 330 335 340cca cag aaa tgc ttg tta aaa
gga aag aag ttc cac cac caa aca tgc 1154Pro Gln Lys Cys Leu Leu Lys
Gly Lys Lys Phe His His Gln Thr Cys 345 350 355agc tgt tac aga cgg
cca tgt acg aac cgc cag aag gct tgt gag cca 1202Ser Cys Tyr Arg Arg
Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro 360 365 370gga ttt tca
tat agt gaa gaa gtg tgt cgt tgt gtc cct tca tat tgg 1250Gly Phe Ser
Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp375 380 385
390caa aga cca caa atg agc taagattgta ctgttttcca gttcatcgat 1298Gln
Arg Pro Gln Met Ser 395tttctattat ggaaaactgt gttgccacag tagaactgtc
tgtgaacaga gagacccttg 1358tgggtccatg ctaacaaaga caaaagtctg
tctttcctga accatgtgga taactttaca 1418gaaatggact ggagctcatc
tgcaaaaggc ctcttgtaaa gactggtttt ctgccaatga 1478ccaaacagcc
aagattttcc tcttgtgatt tctttaaaag aatgactata taatttattt
1538ccactaaaaa tattgtttct gcattcattt ttatagcaac aacaattggt
aaaactcact 1598gtgatcaata tttttatatc atgcaaaata tgtttaaaat
aaaatgaaaa ttgtatttat 1658aaaaaaaaaa aaaaaa 16742419PRThomo sapiens
2Met His Ser Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala
-20 -15 -10Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala
Ala Phe -5 -1 1 5Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp
Ala Gly Glu Ala10 15 20 25Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu
Gln Leu Arg Ser Val Ser 30 35 40Ser Val Asp Glu Leu Met Thr Val Leu
Tyr Pro Glu Tyr Trp Lys Met 45 50 55Tyr Lys Cys Gln Leu Arg Lys Gly
Gly Trp Gln His Asn Arg Glu Gln 60 65 70Ala Asn Leu Asn Ser Arg Thr
Glu Glu Thr Ile Lys Phe Ala Ala Ala 75 80 85His Tyr Asn Thr Glu Ile
Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys90 95 100 105Thr Gln Cys Met
Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe 110 115 120Gly Val
Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr 125 130
135Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr
140 145 150Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val
Pro Leu 155 160 165Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala
Asn His Thr Ser170 175 180 185Cys Arg Cys Met Ser Lys Leu Asp Val
Tyr Arg Gln Val His Ser Ile 190 195 200Ile Arg Arg Ser Leu Pro Ala
Thr Leu Pro Gln Cys Gln Ala Ala Asn 205 210 215Lys Thr Cys Pro Thr
Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys 220 225 230Leu Ala Gln
Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser 235 240 245Thr
Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu250 255
260 265Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser
Cys 270 275 280Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys
Val Cys Lys 285 290 295Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn
Arg Glu Phe Asp Glu 300 305 310Asn Thr Cys Gln Cys Val Cys Lys Arg
Thr Cys Pro Arg Asn Gln Pro 315 320 325Leu Asn Pro Gly Lys Cys Ala
Cys Glu Cys Thr Glu Ser Pro Gln Lys330 335 340 345Cys Leu Leu Lys
Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr 350 355 360Arg Arg
Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser 365 370
375Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Gln Arg Pro
380 385 390Gln Met Ser 39531526DNAhomo sapiens 3cgaggccacg
gcttatgcaa gcaaagatct ggaggagcag ttacggtctg tgtccagtgt 60agatgaactc
atgactgtac tctacccaga atattggaaa atgtacaagt gtcagctaag
120gaaaggaggc tggcaacata acagagaaca ggccaacctc aactcaagga
cagaagagac 180tataaaattt gctgcagcac attataatac agagatcttg
aaaagtattg ataatgagtg 240gagaaagact caatgcatgc cacgggaggt
gtgtatagat gtggggaagg agtttggagt 300cgcgacaaac accttcttta
aacctccatg tgtgtccgtc tacagatgtg ggggttgctg 360caatagtgag
gggctgcagt gcatgaacac cagcacgagc tacctcagca agacgttatt
420tgaaattaca gtgcctctct ctcaaggccc caaaccagta acaatcagtt
ttgccaatca 480cacttcctgc cgatgcatgt ctaaactgga tgtttacaga
caagttcatt ccattattag 540acgttccctg ccagcaacac taccacagtg
tcaggcagcg aacaagacct gccccaccaa 600ttacatgtgg aataatcaca
tctgcagatg cctggctcag gaagatttta tgttttcctc 660ggatgctgga
gatgactcaa cagatggatt ccatgacatc tgtggaccaa acaaggagct
720ggatgaagag acctgtcagt gtgtctgcag agcggggctt cggcctgcca
gctgtggacc 780ccacaaagaa ctagacagaa actcatgcca gtgtgtctgt
aaaaacaaac tcttccccag 840ccaatgtggg gccaaccgag aatttgatga
aaacacatgc cagtgtgtat gtaaaagaac 900ctgccccaga aatcaacccc
taaatcctgg aaaatgtgcc tgtgaatgta cagaaagtcc 960acagaaatgc
ttgttaaaag gaaagaagtt ccaccaccaa acatgcagct gttacagacg
1020gccatgtacg aaccgccaga aggcttgtga gccaggattt tcatatagtg
aagaagtgtg 1080tcgttgtgtc ccttcatatt ggcaaagacc acaaatgagc
taagattgta ctgttttcca 1140gttcatcgat tttctattat ggaaaactgt
gttgccacag tagaactgtc tgtgaacaga 1200gagacccttg tgggtccatg
ctaacaaaga caaaagtctg tctttcctga accatgtgga 1260taactttaca
gaaatggact ggagctcatc tgcaaaaggc ctcttgtaaa gactggtttt
1320ctgccaatga ccaaacagcc aagattttcc tcttgtgatt tctttaaaag
aatgactata 1380taatttattt ccactaaaaa tattgtttct gcattcattt
ttatagcaac aacaattggt 1440aaaactcact gtgatcaata tttttatatc
atgcaaaata tgtttaaaat aaaatgaaaa 1500ttgtatttat aaaaaaaaaa aaaaaa
15264350PRThomo sapiens 4Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys
Met Tyr Lys Cys Gln Leu1 5 10 15Arg Lys Gly Gly Trp Gln His Asn Arg
Glu Gln Ala Asn Leu Asn Ser 20 25 30Arg Thr Glu Glu Thr Ile Lys Phe
Ala Ala Ala His Tyr Asn Thr Glu 35 40 45Ile Leu Lys Ser Ile Asp Asn
Glu Trp Arg Lys Thr Gln Cys Met Pro 50 55 60Arg Glu Val Cys Ile Asp
Val Gly Lys Glu Phe Gly Val Ala Thr Asn65 70 75 80Thr Phe Phe Lys
Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly Cys 85 90 95Cys Asn Ser
Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Ser Tyr Leu 100 105 110Ser
Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro Lys 115 120
125Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg Cys Met Ser
130 135 140Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg Arg
Ser Leu145 150 155 160Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn
Lys Thr Cys Pro Thr 165 170 175Asn Tyr Met Trp Asn Asn His Ile Cys
Arg Cys Leu Ala Gln Glu Asp 180 185 190Phe Met Phe Ser Ser Asp Ala
Gly Asp Asp Ser Thr Asp Gly Phe His 195 200 205Asp Ile Cys Gly Pro
Asn Lys Glu Leu Asp Glu Glu Thr Cys Gln Cys 210 215 220Val Cys Arg
Ala Gly Leu Arg Pro Ala Ser Cys Gly Pro His Lys Glu225 230 235
240Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys Leu Phe Pro
245 250 255Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys
Gln Cys 260 265 270Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu
Asn Pro Gly Lys 275 280 285Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln
Lys Cys Leu Leu Lys Gly 290 295 300Lys Lys Phe His His Gln Thr Cys
Ser Cys Tyr Arg Arg Pro Cys Thr305 310 315 320Asn Arg Gln Lys Ala
Cys Glu Pro Gly Phe Ser Tyr Ser Glu Glu Val 325 330 335Cys Arg Cys
Val Pro Ser Tyr Trp Gln Arg Pro Gln Met Ser 340 345 3505196PRThomo
sapiens 5Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly Cys Gly Tyr
Leu Ala1 5 10 15His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val
Ile Glu Arg 20 25 30Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu
Gln Arg Leu Leu 35 40 45Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu
Asp Thr Ser Leu Arg 50 55 60Ala His Gly Val His Ala Thr Lys His Val
Pro Glu Lys Arg Pro Leu65 70 75 80Pro Ile Arg Arg Lys Arg Ser Ile
Glu Glu Ala Val Pro Ala Val Cys 85 90 95Lys Thr Arg Thr Val Ile Tyr
Glu Ile Pro Arg Ser Gln Val Asp Pro 100 105 110Thr Ser Ala Asn Phe
Leu Ile Trp Pro Pro Cys Val Glu Val Lys Arg 115 120 125Cys Thr Gly
Cys Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg 130 135 140Val
His His Arg Ser Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys145 150
155 160Lys Pro Lys Leu Lys Glu Val Gln Val Arg Leu Glu Glu His Leu
Glu 165 170 175Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg
Glu Glu Asp 180 185 190Thr Asp Val Arg 1956241PRThomo sapiens 6Met
Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg1 5 10
15Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met
20 25 30Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu
Leu 35 40 45His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu
Asn Met 50 55 60Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala
Arg Gly Arg65 70 75 80Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro
Ala Met Ile Ala Glu 85 90 95Cys Lys Thr Arg Thr Glu Val Phe Glu Ile
Ser Arg Arg Leu Ile Asp 100 105 110Arg Thr Asn Ala Asn Phe Leu Val
Trp Pro Pro Cys Val Glu Val Gln 115 120 125Arg Cys Ser Gly Cys Cys
Asn Asn Arg Asn Val Gln Cys Arg Pro Thr 130 135 140Gln Val Gln Leu
Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg145 150 155 160Lys
Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu 165 170
175Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser
180 185 190Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr
Arg Val 195 200 205Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys
Gly Lys His Arg 210 215 220Lys Phe Lys His Thr His Asp Lys Thr Ala
Leu Lys Glu Thr Leu Gly225 230 235 240Ala7232PRThomo sapiens 7Met
Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu1 5 10
15Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly
20 25 30Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr
Gln 35 40 45Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe
Gln Glu 50 55 60Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys
Val Pro Leu65 70 75 80Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly
Leu Glu Cys Val Pro 85 90 95Thr Glu Glu Ser Asn Ile Thr Met Gln Ile
Met Arg Ile Lys Pro His 100 105 110Gln Gly Gln His Ile Gly Glu Met
Ser Phe Leu Gln His Asn Lys Cys 115 120 125Glu Cys Arg Pro Lys Lys
Asp Arg Ala Arg Gln Glu Lys Lys Ser Val 130 135 140Arg Gly Lys Gly
Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr145 150 155 160Lys
Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp 165 170
175Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys
180 185 190His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys
Lys Asn 195 200 205Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu
Asn Glu Arg Thr 210 215 220Cys Arg Cys
Asp Lys Pro Arg Arg225 230814PRThomo sapiensSITE(2)..(2)Xaa is
equal to any amino acid found in a naturally occuring protein. 8Pro
Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys Asn1 5
1093974DNAExpression vector
pHEA4-5misc_feature(1)..(3974)Expression vector pHE4-5 9ggtacctaag
tgagtagggc gtccgatcga cggacgcctt ttttttgaat tcgtaatcat 60ggtcatagct
gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
120ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
acattaattg 180cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
gtgccagctg cattaatgaa 240tcggccaacg cgcggggaga ggcggtttgc
gtattgggcg ctcttccgct tcctcgctca 300ctgactcgct gcgctcggtc
gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360taatacggtt
atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
420agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
aggctccgcc 480cccctgacga gcatcacaaa aatcgacgct caagtcagag
gtggcgaaac ccgacaggac 540tataaagata ccaggcgttt ccccctggaa
gctccctcgt gcgctctcct gttccgaccc 600tgccgcttac cggatacctg
tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660gctcacgctg
taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
720acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
cttgagtcca 780acccggtaag acacgactta tcgccactgg cagcagccac
tggtaacagg attagcagag 840cgaggtatgt aggcggtgct acagagttct
tgaagtggtg gcctaactac ggctacacta 900gaagaacagt atttggtatc
tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960gtagctcttg
atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
1020agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
tctacggggt 1080ctgacgctca gtggaacgaa aactcacgtt aagggatttt
ggtcatgaga ttatcgtcga 1140caattcgcgc gcgaaggcga agcggcatgc
atttacgttg acaccatcga atggtgcaaa 1200acctttcgcg gtatggcatg
atagcgcccg gaagagagtc aattcagggt ggtgaatgtg 1260aaaccagtaa
cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
1320cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
ggaagcggcg 1380atggcggagc tgaattacat tcccaaccgc gtggcacaac
aactggcggg caaacagtcg 1440ttgctgattg gcgttgccac ctccagtctg
gccctgcacg cgccgtcgca aattgtcgcg 1500gcgattaaat ctcgcgccga
tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga 1560agcggcgtcg
aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
1620ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
tgcctgcact 1680aatgttccgg cgttatttct tgatgtctct gaccagacac
ccatcaacag tattattttc 1740tcccatgaag acggtacgcg actgggcgtg
gagcatctgg tcgcattggg tcaccagcaa 1800atcgcgctgt tagcgggccc
attaagttct gtctcggcgc gtctgcgtct ggctggctgg 1860cataaatatc
tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
1920gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
tcccactgcg 1980atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
gcgccattac cgagtccggg 2040ctgcgcgttg gtgcggatat ctcggtagtg
ggatacgacg ataccgaaga cagctcatgt 2100tatatcccgc cgttaaccac
catcaaacag gattttcgcc tgctggggca aaccagcgtg 2160gaccgcttgc
tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
2220tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
tccccgcgcg 2280ttggccgatt cattaatgca gctggcacga caggtttccc
gactggaaag cgggcagtga 2340gcgcaacgca attaatgtaa gttagcgcga
attgtcgacc aaagcggcca tcgtgcctcc 2400ccactcctgc agttcggggg
catggatgcg cggatagccg ctgctggttt cctggatgcc 2460gacggatttg
cactgccggt agaactccgc gaggtcgtcc agcctcaggc agcagctgaa
2520ccaactcgcg aggggatcga gcccggggtg ggcgaagaac tccagcatga
gatccccgcg 2580ctggaggatc atccagccgg cgtcccggaa aacgattccg
aagcccaacc tttcatagaa 2640ggcggcggtg gaatcgaaat ctcgtgatgg
caggttgggc gtcgcttggt cggtcatttc 2700gaaccccaga gtcccgctca
gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc 2760gaatcgggag
cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
2820tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
cacacccagc 2880cggccacagt cgatgaatcc agaaaagcgg ccattttcca
ccatgatatt cggcaagcag 2940gcatcgccat gggtcacgac gagatcctcg
ccgtcgggca tgcgcgcctt gagcctggcg 3000aacagttcgg ctggcgcgag
cccctgatgc tcttcgtcca gatcatcctg atcgacaaga 3060ccggcttcca
tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg
3120caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
ggatactttc 3180tcggcaggag caaggtgaga tgacaggaga tcctgccccg
gcacttcgcc caatagcagc 3240cagtcccttc ccgcttcagt gacaacgtcg
agcacagctg cgcaaggaac gcccgtcgtg 3300gccagccacg atagccgcgc
tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg 3360gtcttgacaa
aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag
3420cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
agcggccgga 3480gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg
atcctcatcc tgtctcttga 3540tcagatcttg atcccctgcg ccatcagatc
cttggcggca agaaagccat ccagtttact 3600ttgcagggct tcccaacctt
accagagggc gccccagctg gcaattccgg ttcgcttgct 3660gtccataaaa
ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt
3720ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt
catccggggt 3780cagcaccgtt tctgcggact ggctttctac gtgttccgct
tcctttagca gcccttgcgc 3840cctgagtgct tgcggcagcg tgaagcttaa
aaaactgcaa aaaatagttt gacttgtgag 3900cggataacaa ttaagatgta
cccaattgtg agcggataac aatttcacac attaaagagg 3960agaaattaca tatg
397410112DNApromoter consensus sequencepromoter(1)..(112)regulatory
elements of the pHE promotor two lac operator sequences,
Shine-Delgarno sequence, and terminal Hind III and Nde I
restriction sites 10aagcttaaaa aactgcaaaa aatagtttga cttgtgagcg
gataacaatt aagatgtacc 60caattgtgag cggataacaa tttcacacat taaagaggag
aaattacata tg 1121118DNAoligonucleotideprimer_bind(1)..(18)M13-2
reverse primer 11atgcttccgg ctcgtatg
181219DNAoligonucleotideprimer_bind(1)..(19)M13-2 forward primer
12gggttttccc agtcacgac
191321DNAoligonucleotideprimer_bind(1)..(21)VEGF-2 5' primer F4
13ccacatggtt caggaaagac a
211450DNAoligonucleotideprimer_bind(1)..(50)VEGF-2 5' primer
containing a BamH1 site 14tgtaatacga ctcactatag ggatcccgcc
atggaggcca cggcttatgc
501528DNAoligonucleotideprimer_bind(1)..(28)VEGF-2 3' primer
containing an XbaI cleavage site 15gatctctaga ttagctcatt tgtggtct
281627DNAoligonucleotideprimer_bind(1)..(27)VEGF-2 5' primer
containing a BamH1 site 16cgcggatcca tgactgtact ctaccca
271760DNAoligonucleotideprimer_bind(1)..(60)VEGF-2 3' primer
containing complementary sequences to XbaI, HA tag, and XhoI sites
17cgctctagat caagcgtagt ctgggacgtc gtatgggtac tcgaggctca tttgtggtct
601830DNAoligonucleotideprimer_bind(1)..(30)VEGF-2 5' primer
containing an NdeI site and start codon 18gcagcacata tgacagaaga
gactataaaa 301930DNAoligonucleotideprimer_bind(1)..(30)VEGF-2 3'
primer containing an Asp718 site and a stop codon 19gcagcaggta
cctcacagtt tagacatgca
302030DNAoligonucleotideprimer_bind(1)..(30)VEGF-2 5' primer
containing an NdeI site and a start codon 20gcagcacata tgacagaaga
gactataaaa 302130DNAoligonucleotideprimer_bind(1)..(30)VEGF-2 3'
primer containing an Asp718 site and a stop codon 21gcagcaggta
cctcaacgtc taataatgga
302230DNAoligonucleotideprimer_bind(1)..(30)VEGF-2 5' primer
containing a BamHI site and one nucleotide spacer 22gcagcaggat
cccacagaag agactataaa
302330DNAoligonucleotideprimer_bind(1)..(30)VEGF-2 3' primer
containing an XbaI site and a stop codon 23gcagcatcta gatcacagtt
tagacatgca 302439DNAoligonucleotideprimer_bind(1)..(39)VEGF-2 5'
primer containing a Bam HIsite and a one nucleotide spacer
24gcagcaggat cccacagaag agactataaa atttgctgc
392536DNAoligonucleotideprimer_bind(1)..(36)VEGF-2 3' primer
containing an XbaI site and a stop codon 25gcagcatcta gatcaacgtc
taataatgga atgaac
362655DNAoligonucleotideprimer_bind(1)..(55)VEGF-2 5' primer
containing a Klenow-filled BamHI site 26gatcgatcca tcatgcactc
gctgggcttc ttctctgtgg cgtgttctct gctcg
552739DNAoligonucleotideprimer_bind(1)..(39)VEGF-2 3' primer
containing a BamHI site with no stop codon 27gcagggtacg gatcctagat
tagctcattt gtggtcttt
392838DNAoligonucleotideprimer_bind(1)..(38)VEGF-2 5' primer
containing a BamHI site 28gcagcaggat ccacagaaga gactataaaa tttgctgc
382937DNAoligonucleotideprimer_bind(1)..(37)VEGF-2 3' primer
containing an XbaI site and a stop codon 29cgtcgttcta gatcacagtt
tagacatgca tcggcag
373038DNAoligonucleotideprimer_bind(1)..(38)VEGF-2 5' primer
containing a BamHI site 30gcagcaggat ccacagaaga gactataaaa tttgctgc
383136DNAoligonucleotideprimer_bind(1)..(36)VEGF-2 3' primer
containing an XbaI site and a stop codon 31gcagcatcta gatcaacgtc
taataatgga atgaac
363239DNAoligonucleotideprimer_bind(1)..(39)VEGF-2 5' primer
containing a BamHI site and a start codon 32gactggatcc gccaccatgc
actcgctggg cttcttctc
393335DNAoligonucleotideprimer_bind(1)..(35)VEGF-2 3' primer
containing an Asp718 site 33gactggtacc ttatcacata aaatcttcct gagcc
353439DNAoligonucleotideprimer_bind(1)..(39)VEGF-2 5' primer
containing a BamHI site and a start codon 34gactggatcc gccaccatgc
actcgctggg cttcttctc
393534DNAoligonucleotideprimer_bind(1)..(34)VEGF-2 3' primer
containing an Asp718 site and a stop codon 35gactggtacc ttatcagtct
agttctttgt gggg 343639DNAoligonucleotideprimer_bind(1)..(39)VEGF-2
5' primer containing a BamHI site and a start codon 36gactggatcc
gccaccatgc actcgctggg cttcttctc
393737DNAoligonucleotideprimer_bind(1)..(37)VEGF-2 3' primer
containing an Asp718 site and a stop codon 37gactggtacc tcattactgt
ggactttctg tacattc 37381298PRThomo sapiens 38Met Gln Arg Gly Ala
Ala Leu Cys Leu Arg Leu Trp Leu Cys Leu Gly1 5 10 15Leu Leu Asp Gly
Leu Val Ser Asp Tyr Ser Met Thr Pro Pro Thr Leu 20 25 30Asn Ile Thr
Glu Glu Ser His Val Ile Asp Thr Gly Asp Ser Leu Ser 35 40 45Ile Ser
Cys Arg Gly Gln His Pro Leu Glu Trp Ala Trp Pro Gly Ala 50 55 60Gln
Glu Ala Pro Ala Thr Gly Asp Lys Asp Ser Glu Asp Thr Gly Val65 70 75
80Val Arg Asp Cys Glu Gly Thr Asp Ala Arg Pro Tyr Cys Lys Val Leu
85 90 95Leu Leu His Glu Val His Ala Asn Asp Thr Gly Ser Tyr Val Cys
Tyr 100 105 110Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala
Ala Ser Ser 115 120 125Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe
Ile Asn Lys Pro Asp 130 135 140Thr Leu Leu Val Asn Arg Lys Asp Ala
Met Trp Val Pro Cys Leu Val145 150 155 160Ser Ile Pro Gly Leu Asn
Val Thr Leu Arg Ser Gln Ser Ser Val Leu 165 170 175Trp Pro Asp Gly
Gln Glu Val Val Trp Asp Asp Arg Arg Gly Met Leu 180 185 190Val Ser
Thr Pro Leu Leu His Asp Ala Leu Tyr Leu Gln Cys Glu Thr 195 200
205Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro Phe Leu Val His Ile
210 215 220Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu Pro Arg Lys
Ser Leu225 230 235 240Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn
Cys Thr Val Trp Ala 245 250 255Glu Phe Asn Ser Gly Val Thr Phe Asp
Trp Asp Tyr Pro Gly Lys Gln 260 265 270Ala Glu Arg Gly Lys Trp Val
Pro Glu Arg Arg Ser Gln Gln Thr His 275 280 285Thr Glu Leu Ser Ser
Ile Leu Thr Ile His Asn Val Ser Gln His Asp 290 295 300Leu Gly Ser
Tyr Val Cys Lys Ala Asn Asn Gly Ile Gln Arg Phe Arg305 310 315
320Glu Ser Thr Glu Val Ile Val His Glu Asn Pro Phe Ile Ser Val Glu
325 330 335Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala Gly Asp Glu
Leu Val 340 345 350Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro
Glu Phe Gln Trp 355 360 365Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg
His Ser Pro His Ala Leu 370 375 380Val Leu Lys Glu Val Thr Glu Ala
Ser Thr Gly Thr Tyr Thr Leu Ala385 390 395 400Leu Trp Asn Ser Ala
Ala Gly Leu Arg Arg Asn Ile Ser Leu Glu Leu 405 410 415Val Val Asn
Val Pro Pro Gln Ile His Glu Lys Glu Ala Ser Ser Pro 420 425 430Ser
Ile Tyr Ser Arg His Ser Arg Gln Ala Leu Thr Cys Thr Ala Tyr 435 440
445Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His Trp Arg Pro Trp Thr
450 455 460Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg Arg Arg Gln
Gln Gln465 470 475 480Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala
Val Thr Thr Gln Asp 485 490 495Ala Val Asn Pro Ile Glu Ser Leu Asp
Thr Trp Thr Glu Phe Val Glu 500 505 510Gly Lys Asn Lys Thr Val Ser
Lys Leu Val Ile Gln Asn Ala Asn Val 515 520 525Ser Ala Met Tyr Lys
Cys Val Val Ser Asn Lys Val Gly Gln Asp Glu 530 535 540Arg Leu Ile
Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Thr Ile545 550 555
560Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly Gln Pro Val Leu Leu
565 570 575Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His Leu Arg Trp
Tyr Arg 580 585 590Leu Asn Leu Ser Thr Leu His Asp Ala His Gly Asn
Pro Leu Leu Leu 595 600 605Asp Cys Lys Asn Val His Leu Phe Ala Thr
Pro Leu Ala Ala Ser Leu 610 615 620Glu Glu Val Ala Pro Gly Ala Arg
His Ala Thr Leu Ser Leu Ser Ile625 630 635 640Pro Arg Val Ala Pro
Glu His Glu Gly His Tyr Val Cys Glu Val Gln 645 650 655Asp Arg Arg
Ser His Asp Lys His Cys His Lys Lys Tyr Leu Ser Val 660 665 670Gln
Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu Leu 675 680
685Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys Leu Val Ala Gly Ala
690 695 700His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu Arg Leu Leu
Glu Glu705 710 715 720Lys Ser Gly Val Asp Leu Ala Asp Ser Asn Gln
Lys Leu Ser Ile Gln 725 730 735Arg Val Arg Glu Glu Asp Ala Gly Pro
Tyr Leu Cys Ser Val Cys Arg 740 745 750Pro Lys Gly Cys Val Asn Ser
Ser Ala Ser Val Ala Val Glu Gly Ser 755 760 765Glu Asp Lys Gly Ser
Met Glu Ile Val Ile Leu Val Gly Thr Gly Val 770 775 780Ile Ala Val
Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn Met785 790 795
800Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr Leu Ser Ile Ile
805 810 815Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln Cys Glu Tyr
Leu Ser 820 825 830Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu Arg
Leu His Leu Gly 835 840 845Arg Val Leu Gly Tyr Gly Ala Phe Gly Lys
Val Val Glu Ala Ser Ala 850 855 860Phe Gly Ile His Lys Gly Ser Ser
Cys Asp Thr Val Ala Val Lys Met865 870 875 880Leu Lys Glu Gly Ala
Thr Ala Ser Glu Gln Arg Ala Leu Met Ser Glu 885 890 895Leu Lys Ile
Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu 900 905 910Leu
Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu Met Val Ile Val Glu 915 920
925Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg Ala Lys Arg Asp
930 935 940Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu Gln Arg Gly
Arg Phe945 950 955 960Arg Ala Met Val Glu Leu Ala Arg Leu Asp Arg
Arg Arg Pro Gly Ser 965 970 975Ser Asp Arg Val Leu Phe Ala Arg Phe
Ser Lys Thr Glu Gly Gly Ala 980 985 990Arg Arg Ala Ser Pro Asp Gln
Glu Ala Glu
Asp Leu Trp Leu Ser Pro 995 1000 1005Leu Thr Met Glu Asp Leu Val
Cys Tyr Ser Phe Gln Val Ala Arg 1010 1015 1020Gly Met Glu Phe Leu
Ala Ser Arg Lys Cys Ile His Arg Asp Leu 1025 1030 1035Ala Ala Arg
Asn Ile Leu Leu Ser Glu Ser Asp Val Val Lys Ile 1040 1045 1050Cys
Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr 1055 1060
1065Val Arg Lys Gly Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro
1070 1075 1080Glu Ser Ile Phe Asp Lys Val Tyr Thr Thr Gln Ser Asp
Val Trp 1085 1090 1095Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser
Leu Gly Ala Ser 1100 1105 1110Pro Tyr Pro Gly Val Gln Ile Asn Glu
Glu Phe Cys Gln Arg Val 1115 1120 1125Arg Asp Gly Thr Arg Met Arg
Ala Pro Glu Leu Ala Thr Pro Ala 1130 1135 1140Ile Arg His Ile Met
Leu Asn Cys Trp Ser Gly Asp Pro Lys Ala 1145 1150 1155Arg Pro Ala
Phe Ser Asp Leu Val Glu Ile Leu Gly Asp Leu Leu 1160 1165 1170Gln
Gly Arg Gly Leu Gln Glu Glu Glu Glu Val Cys Met Ala Pro 1175 1180
1185Arg Ser Ser Gln Ser Ser Glu Glu Gly Ser Phe Ser Gln Val Ser
1190 1195 1200Thr Met Ala Leu His Ile Ala Gln Ala Asp Ala Glu Asp
Ser Pro 1205 1210 1215Pro Ser Leu Gln Arg His Ser Leu Ala Ala Arg
Tyr Tyr Asn Trp 1220 1225 1230Val Ser Phe Pro Gly Cys Leu Ala Arg
Gly Ala Glu Thr Arg Gly 1235 1240 1245Ser Ser Arg Met Lys Thr Phe
Glu Glu Phe Pro Met Thr Pro Thr 1250 1255 1260Thr Tyr Lys Gly Ser
Val Asp Asn Gln Thr Asp Ser Gly Met Val 1265 1270 1275Leu Ala Ser
Glu Glu Phe Glu Gln Ile Glu Ser Arg His Arg Gln 1280 1285 1290Glu
Ser Gly Phe Arg 129539758PRThomo sapiens 39Met Val Ser Tyr Trp Asp
Thr Gly Val Leu Leu Cys Ala Leu Leu Ser1 5 10 15Cys Leu Leu Leu Thr
Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30Glu Leu Ser Leu
Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45Leu His Leu
Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60Glu Met
Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala65 70 75
80Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr
85 90 95Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala
Val 100 105 110Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr
Ile Phe Ile 115 120 125Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr
Ser Glu Ile Pro Glu 130 135 140Ile Ile His Met Thr Glu Gly Arg Glu
Leu Val Ile Pro Cys Arg Val145 150 155 160Thr Ser Pro Asn Ile Thr
Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175Leu Ile Pro Asp
Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190Ile Ile
Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200
205Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
210 215 220Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg
Pro Val225 230 235 240Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn
Cys Thr Ala Thr Thr 245 250 255Pro Leu Asn Thr Arg Val Gln Met Thr
Trp Ser Tyr Pro Asp Glu Lys 260 265 270Asn Lys Arg Ala Ser Val Arg
Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285Ala Asn Ile Phe Tyr
Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300Asp Lys Gly
Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys305 310 315
320Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val
325 330 335Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys
Arg Ser 340 345 350Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser
Pro Glu Val Val 355 360 365Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu
Lys Ser Ala Arg Tyr Leu 370 375 380Thr Arg Gly Tyr Ser Leu Ile Ile
Lys Asp Val Thr Glu Glu Asp Ala385 390 395 400Gly Asn Tyr Thr Ile
Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415Asn Leu Thr
Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430Lys
Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440
445Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile
450 455 460Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala
Arg Cys465 470 475 480Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile
Leu Asp Ala Asp Ser 485 490 495Asn Met Gly Asn Arg Ile Glu Ser Ile
Thr Gln Arg Met Ala Ile Ile 500 505 510Glu Gly Lys Asn Lys Met Ala
Ser Thr Leu Val Val Ala Asp Ser Arg 515 520 525Ile Ser Gly Ile Tyr
Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540Gly Arg Asn
Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His545 550 555
560Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser
565 570 575Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile
Leu Leu 580 585 590Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile
Ser Lys Gln Lys 595 600 605Met Ala Ile Thr Lys Glu His Ser Ile Thr
Leu Asn Leu Thr Ile Met 610 615 620Asn Val Ser Leu Gln Asp Ser Gly
Thr Tyr Ala Cys Arg Ala Arg Asn625 630 635 640Val Tyr Thr Gly Glu
Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655Asp Gln Glu
Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val 660 665 670Ala
Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro 675 680
685Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu
690 695 700Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser Thr Leu Phe Ile
Glu Arg705 710 715 720Val Thr Glu Glu Asp Glu Gly Val Tyr His Cys
Lys Ala Thr Asn Gln 725 730 735Lys Gly Ser Val Glu Ser Ser Ala Tyr
Leu Thr Val Gln Gly Thr Ser 740 745 750Asp Lys Ser Asn Phe Glu
755
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