U.S. patent application number 10/705476 was filed with the patent office on 2004-09-09 for vascular endothelial growth factor d(vegf-d) antibodies and vectors, and methods of use.
This patent application is currently assigned to Ludwig Institute for Cancer Research. Invention is credited to Achen, Marc G., Alitalo, Kari, Stacker, Steven A., Wilks, Andrew F..
Application Number | 20040175730 10/705476 |
Document ID | / |
Family ID | 46149776 |
Filed Date | 2004-09-09 |
United States Patent
Application |
20040175730 |
Kind Code |
A1 |
Achen, Marc G. ; et
al. |
September 9, 2004 |
Vascular endothelial growth factor D(VEGF-D) antibodies and
vectors, and methods of use
Abstract
VEGF-D, a new member of the PDGF family of growth factors, which
among other things stimulates endothelial cell proliferation and
angiogenesis and increases vascular permeability, as well as
nucleotide sequences encoding it, methods for producing it,
antibodies and other antagonists to it, transfected or transformed
host cells for expressing it, pharmaceutical compositions
containing it, and uses thereof in medical and diagnostic
applications.
Inventors: |
Achen, Marc G.; (Fitzroy,
AU) ; Wilks, Andrew F.; (South Yarra, AU) ;
Stacker, Steven A.; (North Fitzroy, AU) ; Alitalo,
Kari; (Espoo, FI) |
Correspondence
Address: |
CROWELL & MORING LLP
INTELLECTUAL PROPERTY GROUP
P.O. BOX 14300
WASHINGTON
DC
20044-4300
US
|
Assignee: |
Ludwig Institute for Cancer
Research
New York
NY
Helsinki University Licensing Ltd., OY
Helsinki
|
Family ID: |
46149776 |
Appl. No.: |
10/705476 |
Filed: |
November 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10705476 |
Nov 12, 2003 |
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10161694 |
Jun 5, 2002 |
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10161694 |
Jun 5, 2002 |
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09296275 |
Apr 22, 1999 |
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6689580 |
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09296275 |
Apr 22, 1999 |
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08915795 |
Aug 21, 1997 |
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6235713 |
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Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 19/02 20180101;
A61P 43/00 20180101; A61P 9/00 20180101; A61P 35/04 20180101; A61K
38/00 20130101; C07K 14/52 20130101; A61P 9/10 20180101; A61P 7/00
20180101; A61P 9/08 20180101; A61P 17/06 20180101; A61P 17/02
20180101; C07K 14/475 20130101; A61P 35/00 20180101; A61P 27/02
20180101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 23, 1996 |
AU |
PO 1825 |
Nov 11, 1996 |
AU |
PO 3554 |
Feb 5, 1997 |
AU |
PO 4954 |
Jun 19, 1997 |
AU |
PO 7435 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a nucleic acid
sequence which encodes a polypeptide comprising a sequence of amino
acids substantially corresponding to the amino acid sequence set
out in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.8 or SEQ ID NO. 9, said
polypeptide having the ability to stimulate vascular permeability
or proliferation of endothelial cells, or a fragment or analogue
thereof which has the ability to stimulate at least one biological
activity selected from the group consisting of angiogenesis,
vascular permeability, endothelial cell proliferation,
differentiation, migration or survival, or which has the ability to
bind to endothelial cells, but is unable to stimulate at least one
of said biological activities.
2. A nucleic acid molecule according to claim 1, wherein said
nucleic acid molecule comprises a nucleic acid sequence which
encodes the amino acid sequence
Pro-xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys (SEQ ID
NO.2).
3. A nucleic acid molecule according to claim 1, wherein said
endothelial cells are selected from the group consisting of
vascular endothelial cells and lymphatic endothelial cells.
4. A nucleic acid molecule according to claim 1, which is a genomic
DNA.
5. A nucleic acid molecule according to claim 1, which is a
cDNA.
6. A nucleic acid molecule according to claim 5, which comprises
the nucleic acid sequence of SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.6,
SEQ ID NO.7, or a DNA sequence which hybridizes to one of the
foregoing sequences under stringent conditions.
7. A nucleic acid molecule according to claim 6, which comprises
the nucleic acid sequence of SEQ ID NO.4.
8. A nucleic acid molecule according to claim 1, which encodes a
polypeptide which has the ability to stimulate vascular
permeability or proliferation of endothelial cells.
9. A nucleic acid molecule according to claim 1, which encodes a
polypeptide comprising amino acid residues 64 through 172 of SEQ ID
NO:3 or amino acid residues 93 through 201 of SEQ ID NO:5.
10. A nucleic acid molecule according to claim 9, wherein said
polypeptide further comprises an affinity tag peptide sequence.
11. A nucleic acid molecule according to claim 1, which encodes a
polypeptide which has the ability to bind to endothelial cells but
is unable to stimulate endothelial cell proliferation.
12. A nucleic acid molecule according to claim 11, wherein said
endothelial cells are selected from the group consisting of
vascular endothelial cells and lymphatic endothelial cells.
13. A nucleic acid molecule according to claim 1, wherein said
nucleic acid molecule is a human DNA molecule.
14. A vector comprising a nucleic acid according to claim 1.
15. A host cell transformed or transformed with a vector according
to claim 14.
16. An isolated polypeptide which comprises a sequence of amino
acids substantially corresponding to the amino acid sequence set
out in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.8 or SEQ ID NO. 9, said
polypeptide having the ability to stimulate vascular permeability
or proliferation of endothelial cells, or a fragment or analogue
thereof which has the ability to stimulate at least one endothelial
cell biological activity selected from the group consisting of cell
proliferation, cell differentiation, cell migration, cell survival
and vascular permeability, or which has the ability to bind to
endothelial cells but is unable to stimulate at least one of said
biological activities.
17. A polypeptide according to claim 16, wherein said polypeptide
comprises the amino acid sequence
Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa- -Gly-Cys-Cys (SEQ ID
NO.2).
18. A polypeptide according to claim 16, wherein said endothelial
cells are selected from the group consisting of vascular
endothelial cells and lymphatic endothelial cells.
19. A polypeptide according to claim 16, which comprises a sequence
of amino acids substantially corresponding to SEQ ID NO:3 or SEQ ID
NO:5.
20. A polypeptide according to claim 19, which comprises a sequence
of amino acids substantially corresponding to SEQ ID NO 5.
21. A polypeptide according to claim 16, which has the ability to
stimulate proliferation of endothelial cells.
22. A polypeptide according to claim 16, which has the ability to
induce endothelial cell differentiation.
23. A polypeptide according to claim 16, which has the ability to
induce vascular permeability.
24. A polypeptide according to claim 16, comprising amino acid
residues 64 through 172 of SEQ ID NO:3 or 93 through 201 of SEQ ID
NO:5.
25. A polypeptide according to claim 24, further comprising an
affinity tag peptide sequence.
26. A polypeptide according to claim 16, which has the ability to
bind to endothelial cells but is unable to stimulate proliferation
of endothelial cells.
27. A polypeptide according to claim 26, wherein said endothelial
cells are selected from the group consisting of vascular
endothelial cells and lymphatic endothelial cells.
28. A polypeptide according to claim 16, wherein said polypeptide
is a human protein.
29. An antibody specifically reactive with a polypeptide according
to claim 16.
30. An antibody according to claim 29, wherein said antibody is a
polyclonal antibody.
31. An antibody according to claim 29, wherein said antibody is a
monoclonal antibody.
32. An antibody according to claim 29, wherein said antibody is
labelled with a detectable label.
33. A method of making a polypeptide according to claim 16, said
method comprising the steps of: culturing a host cell transformed
or transfected with a vector comprising a nucleic acid sequence
encoding said polypeptide operably associated with a promoter
sequence such that the nucleic acid sequence encoding said
polypeptide is expressed; and isolating said polypeptide from said
host cell or from a growth medium in which said host cell is
cultured.
34. A method of isolation of polypeptide according to claim 16,
said method comprising the step of exposing a cell which expresses
said polypeptide to heparin to facilitate release of the
polypeptide from the cell, and purifying the thus-released
polypeptide.
35. A method of making a vector capable of expressing a polypeptide
encoded by a nucleic acid molecule according to claim 1, said
method comprising inserting said nucleic acid molecule into a
vector in a position in which said nucleic acid molecule is
operatively connected with at least one promoter.
36. A vector comprising an anti-sense nucleotide sequence, said
anti-sense nucleotide sequence-being complementary to at least a
part of a VEGF-D genomic DNA sequence or a VEGF-D RNA sequence or a
cDNA sequence which encodes' VEGF-D or a fragment or analogue
thereof which promotes at least one bioactivity selected from
vascular permeability, proliferation of endothelial cells and
endothelial cell differentiation, whereby said vector can be used
to inhibit said at least one bioactivity.
37. A method of stimulating endothelial cell proliferation
comprising contacting endothelial cells with an effective
endothelial cell proliferation stimulating amount of a polypeptide
according to claim 16.
38. A method according to claim 37, wherein said endothelial cells
are selected from the group consisting of vascular endothelial
cells and lymphatic endothelial cells.
39. A method of stimulating at least one bioactivity selected from
endothelial cell proliferation, endothelial cell differentiation
and vascular permeability, in vivo in a mammal, said method
comprising administering to said mammal an effective bioactivity
stimulating amount of a polypeptide according to claim 16, which
has the ability to stimulate said at least one bioactivity.
40. A method according to claim 39, wherein said polypeptide
comprises amino acid residues 64 through 172 of SEQ ID NO:3 or
amino acid residues 93 through 201 of SEQ ID NO:5.
41. A method according to claim 39, wherein lymphatic vessel
endothelial cell proliferation is stimulated.
42. A method of stimulating at least one bioactivity selected from
angiogenesis and neovascularization in a mammal, said method
comprising the step of administering to said mammal an effective
angiogenesis or neovascularization stimulating amount of a
polypeptide according to claim 16, said polypeptide having the
ability to stimulate endothelial cell proliferation.
43. A method according to claim 42, wherein said polypeptide
comprises amino acid residues 64 through 172 of SEQ ID NO:3 or
amino acid residues 93 through 201 of SEQ ID NO:5.
44. A method according to claim 43, wherein said polypeptide
further comprises an affinity tag peptide sequence.
45. A method according claim 32, further comprising
co-administering at least one substance selected from the group
consisting of VEGF, VEGF-B, VEGF-C, PlGF, PDGF, FGF and
heparin.
46. A method of inhibiting a bioactivity selected from angiogenesis
and neovascularization in a mammal, said method comprising the step
of administering to said mammal an effective angiogenesis or
neovascularization inhibiting amount of a VEGF-D antagonist
according to claim 70.
47. A method according to claim 46, wherein said VEGF-D antagonist
comprises an antibody specific to VEGF-D.
48. A method according to claim 46, wherein said VEGF-D antagonist
comprises a polypeptide which binds to endothelial cells but which
is unable to stimulate at least one biological activity selected
from proliferation of endothelial cells, endothelial cell
differentiation and vascular permeability.
49. A method according to claim 48, wherein said endothelial cells
are selected from the group consisting of vascular endothelial
cells and lymphatic endothelial cells.
50. A method of inhibiting VEGF-D expression in a mammal comprising
the step of transforming target cells expressing VEGF-D with a
vector according to claim 36.
51. A pharmaceutical composition comprising a polypeptide according
to claim 16, and a pharmaceutically acceptable carrier or
adjuvant.
52. A pharmaceutical composition according to claim 51, further
comprising at least one substance selected from the group
consisting of VEGF, VEGF-B, VEGF-C, PlGF, PDGF, FGF and
heparin.
53. A pharmaceutical composition comprising an antibody according
to claim 29, and a pharmaceutically acceptable carrier or
adjuvant.
54. A pharmaceutical composition according to claim 53, wherein
said antibody is a monoclonal antibody.
55. A protein dimer comprising a first polypeptide according to
claim 16, and a second polypeptide.
56. A protein dimer according to claim 55, wherein said protein
dimer is a homodimer in which the second polypeptide is identical
to the first polypeptide.
57. A protein dimer according to claim 55, wherein said protein
dimer is a heterodimer in which the second polypeptide is selected
from VEGF, VEGF-B, VEGF-C, PlGF and PDGF.
58. A method of detecting a polypeptide according to claim 16 in a
biological sample, said method comprising the step of contacting
the sample with a reagent capable of binding said polypeptide, and
detecting the occurrence of binding of said reagent.
59. A method according to claim 58, wherein said reagent comprises
an antibody according to claim 29.
60. A method of modulating vascular permeability in a mammal, said
method comprising administering to said mammal an effective
vascular permeability modulating amount of a polypeptide according
to claim 16 or an antibody thereto.
61. A method according to claim 60, comprising administering to
said mammal a polypeptide according to claim 16, having the ability
to stimulate endothelial cell proliferation.
62. A method according to claim 60, comprising administering to
said mammal a polypeptide according to claim 16, which has the
ability to bind to endothelial cells, but which is unable to
stimulate endothelial cell proliferation.
63. A method of activation of at least one receptor selected from
the group consisting of VEGF receptor 2 and VEGF receptor 3, said
method comprising the step of exposing cells bearing said receptor
to an effective receptor activating dose of a polypeptide according
to claim 16.
64. A method according to claim 63, wherein said method is carried
out in vivo.
65. A method according to claim 63, wherein said method is carried
out in vitro.
66. A diagnostic or prognostic test kit comprising a specific
binding reagent for a polypeptide according to claim 16, and means
for detecting binding of said reagent.
67. A test kit according to claim 66, wherein said specific binding
reagent comprises an antibody to said polypeptide.
68. A diagnostic or prognostic test kit. comprising a pair of
primers specific to a nucleotide sequence according to claim 1,
operatively coupled to a polymerase, whereby said polymerase is
enabled to selectively amplify the nucleotide sequence from a DNA
sample.
69. A method of detecting aberrations in VEGF-D gene structure in a
test subject comprising the steps of: providing a DNA sample from
said test subject; contacting said sample with a set of primers
specific to a nucleotide sequence according to claim 1, operatively
coupled to a polymerase and selectively amplifying said nucleotide
sequence from said sample by polymerase chain reaction; and
comparing the nucleotide sequence of the amplified nucleotide
sequence from said sample with a nucleotide sequence as set forth
in SEQ ID NO:1 or SEQ ID NO:4.
70. A VEGF-D antagonist having the capability to inhibit at least
one biological activity induced by VEGF-D selected from vascular
permeability, endothelial cell proliferation and endothelial cell
differentiation, said antagonist binding to VEGF-D or to a VEGF-D
receptor, but being less able than VEGF-D to stimulate said at
least one biological activity.
71. A VEGF-D antagonist according to claim 70, wherein said
antagonist comprises an antibody which selectively binds
VEGF-D.
72. A VEGF-D antagonist according to claim 71, wherein said
antibody is a monoclonal antibody.
73. A VEGF-D antagonist according to claim 71, wherein said
antagonist comprises a VEGF-D polypeptide fragment or analogue
which binds to a VEGF-D receptor, but is less able to stimulate
said at least one biological activity.
74. A method of improving pulmonary blood circulation and/or gas
exchange in a mammal, said method comprising administering to said
mammal an effective blood circulation and/or gas exchange improving
amount of a polypeptide according to claim 16.
76. A method of treating fluid accumulation in the heart and/or
lung due to increases in vascular permeability in a mammal, said
method comprising administering to said mammal an effective
vascular permeability decreasing amount of an antagonist according
to claim 70.
76. A method of treating an intestinal malabsorption syndrome in a
patient suffering therefrom, said method comprising administering
to said patient an effective intestinal blood circulation and/or
vascular permeability increasing amount of a polypeptide according
to claim 16.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing dates of
the following copending U.S. Provisional Applications: Serial No.
60/023,751, filed Aug. 23, 1996; Serial No. 60/031,097, filed Nov.
14, 1996; Serial No. 60/038,814, filed Feb. 10, 1997; and Serial
No. 60/051,426, filed Jul. 1, 1997.
FIELD OF THE INVENTION
[0002] This invention relates to growth factors for endothelial
cells, and in particular to a novel vascular endothelial growth
factor, DNA encoding the factor, and to pharmaceutical and
diagnostic compositions and methods utilising or derived from the
factor.
BACKGROUND OF THE INVENTION
[0003] Angiogenesis is a fundamental process required for normal
growth and development of tissues, and involves the proliferation
of new capillaries from pre-existing blood vessels. Angiogenesis is
not only involved in embryonic development and normal tissue
growth, repair, and regeneration, but is also involved in the
female reproductive cycle, establishment and maintenance of
pregnancy, and in repair of wounds and fractures. In addition to
angiogenesis which takes place in the normal individual, angiogenic
events are involved in a number of pathological processes, notably
tumour growth and metastasis, and other conditions in which blood
vessel proliferation, especially of the microvascular system, is
increased, such as diabetic retinopathy, psoriasis and
arthropathies. Inhibition of angiogenesis is useful in preventing
or alleviating these pathological processes.
[0004] On the other hand, promotion of angiogenesis is desirable in
situations where vascularization is to be established or extended,
for example after tissue or organ transplantation, or to stimulate
establishment of collateral circulation in tissue infarction or
arterial stenosis, such as in coronary heart disease and
thromboangitis obliterans.
[0005] Because of the crucial role of angiogenesis in so many
physiological and pathological processes, factors involved in the
control of angiogenesis have been intensively investigated. A
number of growth factors have been shown to be involved in the
regulation of angiogenesis; these include fibroblast growth factors
(FGFs), platelet-derived growth factor (PDGF), transforming growth
factor .alpha. (TGF.alpha.), and hepatocyte growth factor (HGF).
See for example Folkman et al, "Angiogenesis", J. Biol. Chem., 1992
267 10931-10934 for a review.
[0006] It has been suggested that a particular family of
endothelial cell-specific growth factors and their corresponding
receptors is primarily responsible for stimulation of endothelial
cell growth and differentiation, and for certain functions of the
differentiated cells. These factors are members of the PDGF family,
and appear to act via endothelial receptor tyrosine kinases (RTKs).
Hitherto four vascular endothelial growth factor subtypes have been
identified. Vascular endothelial growth factor (VEGF), now known as
VEGF-A, has been isolated from several sources. VEGF-A shows highly
specific mitogenic activity against endothelial cells, and can
stimulate the whole sequence of events leading to angiogenesis. In
addition, it has strong chemoattractant activity towards monocytes,
can induce plasminogen activator and plasminogen activator
inhibitor in endothelial cells, and can also influence
microvascular permeability. Because of the latter activity, it is
also sometimes referred to as vascular permeability factor (VPF).
The isolation and properties of VEGF have been reviewed; see
Ferrara et al, "The Vascular Endothelial Growth Factor Family of
Polypeptides", J. Cellular Biochem., 1991 47 211-218 and Connolly,
"Vascular Permeability Factor: A Unique Regulator of Blood Vessel
Function", J. Cellular Biochem., 1991 47 219-223.
[0007] More recently, three further members of the VEGF family have
been identified. These are designated VEGF-B, described in
International Patent Application No. PCT/US96/02957 (WO 96/26736)
by Ludwig Institute for Cancer Research and The University of
Helsinki, VEGF-C, described in Joukov et al, The EMBO Journal, 1996
15 290-298, and VEGF2, described in International Patent
Application No. PCT/US94/05291 (WO 95/24473) by Human Genome
Sciences, Inc. VEGF-B has closely similar angiogenic and other
properties to those of VEGF, but is distributed and expressed in
tissues differently from VEGF. In particular, VEGF-B is very
strongly expressed in heart, and only weakly in lung, whereas the
reverse is the case for VEGF. This suggests that VEGF and VEGF-B,
despite the fact that they are co-expressed in many tissues, may
have functional differences.
[0008] VEGF-B was isolated using a yeast co-hybrid interaction trap
screening technique, screening for cellular proteins which might
interact with cellular retinoic acid-binding protein type I
(CRABP-I). Its isolation and characteristics are described in
detail in PCT/TS96/02597 and in Olofsson et al, Proc. Natl. Acad.
Sci., 1996 93 2576-2581.
[0009] VEGF-C was isolated from conditioned media of PC-3 prostate
adenocarcinoma cell line (CRL1435) by screening for ability of the
medium to produce tyrosine phosphorylation of the endothelial
cell-specific receptor tyrosine kinase Flt4, using cells
transfected to express Flt4. VEGF-C was purified using affinity
chromatography with recombinant Flt4, and was cloned from a PC-3
cDNA library. Its isolation and characteristics are described in
detail in Joukov et al, The EMBO Journal, 1996 15 290-298.
[0010] VEGF2 was isolated from a highly tumorgenic,
oestrogen-independent human breast cancer cell line. While this
molecule is stated to have about 22% homology to PDGF and 30%
homology to VEGF, the method of isolation of the gene encoding
VEGF2 is unclear, and no characterization of the biological
activity is disclosed.
[0011] Vascular endothelial growth factors appear to act by binding
to receptor tyrosine kinases of the PDGF-receptor family. Five
endothelial cell-specific receptor tyrosine kinases have been
identified, namely Flt-1 (VEGFR-1), KDR/Flk-1 (VEGFR-2), Flt4
(VEGFR-3), Tie and Tek/Tie-2. All of these have the intrinsic
tyrosine kinase activity which is necessary for signal
transduction. The essential, specific role in vasculogenesis and
angiogenesis of Flt-1, Flk-1, Tie and Tek/Tie-2 has been
demonstrated by targeted mutations inactivating these receptors in
mouse embryos. VEGFR-1 and VEGFR-2 bind VEGF with high affinity,
and VEGFR-1 also binds VEGF-B and placenta growth factor (PLGF).
VEGF-C has been shown to be the ligand for Flt4 (VEGFR-3), and also
activates VEGFR-2 (Joukov et al, 1996). A ligand for Tek/Tie-72 has
been described (International Patent Application No. PCT/US95/12935
(WO 96/11269) by Regeneron Pharmaceuticals, Inc.); however, the
ligand for Tie has not yet been identified.
[0012] The receptor Flt-4 is expressed in venous and lymphatic
endothelia in the fetus, and predominantly in lymphatic endothelia
in the adult (Kaipainen et al, Cancer Res, 1994 54 6571-6577; Proc
Natl. Acad. Sci. USA, 1995 92 3566-3570). It has been suggested
that VEGF-C may have a primary function in lymphatic endothelium,
and a secondary function in angiogenesis and permeability
regulation which is shared with VEGF (Joukov et al, 1996).
[0013] We have now isolated human cDNA encoding a novel protein of
the vascular endothelial growth factor family. The novel protein,
designated VEGF-D, has structural similarities to other members of
this family.
SUMMARY OF THE INVENTION
[0014] The invention generally provides an isolated novel growth
factor which has the ability to stimulate and/or enhance
proliferation or differentiation of endothelial cells, isolated DNA
sequences encoding the novel growth factor, and compositions useful
for diagnostic and/or therapeutic applications.
[0015] According to one aspect, the invention provides an isolated
and purified nucleic acid molecule which encodes a novel
polypeptide, designated VEGF-D, which is structurally homologous to
VEGF, VEGF-B and VEGF-C. In a preferred embodiment, the nucleic
acid molecule is a cDNA which comprises the sequence set out in SEQ
ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 7. This aspect
of the invention also encompasses DNA molecules of sequence such
that they hybridise under stringent conditions with DNA of SEQ ID
NO. 1, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 7. Preferably the
DNA molecule able to hybridise under stringent conditions encodes
the portion of VEGF-D from amino acid residue 93 to amino acid
residue 201, optionally operatively linked to a DNA sequence
encoding FLAG.TM. peptide.
[0016] Preferably the cDNA comprises the sequence set out in SEQ ID
NO. 4, SEQ ID NO. 6 or SEQ ID NO. 7, more preferably that of SEQ ID
NO. 4.
[0017] According to a second aspect, the invention provides a
polypeptide possessing the characteristic amino acid sequence:
[0018] Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys (SEQ ID
NO. 2),
[0019] said polypeptide having the ability to stimulate
proliferation of endothelial cells, and said polypeptide comprising
a sequence of amino acids substantially corresponding to the amino
acid sequence set out in SEQ ID NO. 3, or a fragment or analogue
thereof which has the ability to stimulate one or more of
endothelial cell proliferation, differentiation, migration or
survival.
[0020] These abilities are referred to herein as "biological
activities of VEGF-D" and can readily be tested by methods known in
the art. Preferably the polypeptide has the ability to stimulate
endothelial cell proliferation or differentiation, including, but
not limited to, proliferation or differentiation of vascular
endothelial cells and/or lymphatic endothelial cells.
[0021] More preferably the polypeptide has the sequence set out in
SEQ ID NO. 5, SEQ ID NO. 8 or SEQ ID NO. 9, and most preferably has
the sequence set out in SEQ ID NO. 5.
[0022] A preferred fragment of the polypeptide invention is the
portion of VEGF-D from amino acid residue 93 to amino acid residue
201, optionally linked to FLAG.TM. peptide. Where the fragment is
linked to FLAG.TM., the fragment is VEGFD.DELTA.N.DELTA.C, as
hereindefined.
[0023] Thus polypeptides comprising conservative substitutions,
insertions, or deletions, but which still retain the biological
activity of VEGF-D, are clearly to be understood to be within the
scope of the invention. The person skilled in the art will be well
aware of methods which can readily be used to generate such
polypeptides, for example the use of site-directed mutagenesis, or
specific enzymic cleavage and ligation. The skilled person will
also be aware that peptidomimetic compounds or compounds in which
one or more amino acid residues are replaced by a non-naturally
occurring amino acid or an amino acid analogue may retain the
required aspects of the biological activity of VEGF-D. Such
compounds can readily be made and tested by methods known in the
art, and are also within the scope of the invention.
[0024] In addition, variant forms of the VEGF-D polypeptide which
result from alternative splicing, as are known to occur with VEGF,
and naturally-occurring allelic variants of the nucleic acid
sequence encoding VEGF-D are encompassed within the scope of the
invention. Allelic variants are well known in the art, and
represent alternative forms or a nucleic acid sequence which
comprise substitution, deletion or addition of one or more
nucleotides, but which do not result in any substantial functional
alteration of the encoded polypeptide.
[0025] As used herein, the term "VEGF-D" collectively refers to the
polypeptides of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 8 and SEQ ID
NO. 9 and fragments or analogues thereof which have the biological
activity of VEGF-D as herein defined.
[0026] Such variant forms of VEGF-D can be prepared by targeting
non-essential regions of the VEGF-D polypeptide for modification.
These non-essential regions are expected to fall outside the
strongly-conserved regions indicated in the figures herein,
especially FIG. 2 and FIG. 10. In particular, the growth factors of
the PDGF family, including VEGF, are dimeric, and VEGF-B, VEGF-C,
PlGF, PDGF-A and PDGF-B show complete conservation of 8 cysteine
residues in the N-terminal domains, ie. the PDGF-like domains
(Olofsson et al, 1996; Joukov et al, 0.1996). These cysteines are
thought to be involved in intra- and inter-molecular disulphide
bonding. In addition there are further strongly, but not
completely, conserved cysteine residues in the C-terminal domains.
Loops 1, 2 and 3 of each subunit, which are formed by
intra-molecular disulphide bonding, are involved in binding to the
receptors for the PDGF/VEGF family of growth factors (Andersson et
al: Growth Factors, 1995 12 159-164) As shown herein, the cysteines
conserved in previously known members of the VEGF family are also
conserved in VEGF-D.
[0027] The person skilled in the art thus is well aware that these
cysteine residues should be preserved in any proposed variant form,
and that the active sites present in loops 1, 2 and 3 also should
be preserved. However, other regions of the molecule can be
expected to be of lesser importance for biological function, and
therefore offer suitable targets for modification. Modified
polypeptides can readily be tested for their ability to show the
biological activity of VEGF-D by routine activity assay procedures
such as cell proliferation tests.
[0028] It is contemplated that some modified VEGF-D polypeptides
will have the ability to bind to endothelial cells, ie. to VEGF-D
receptors, but will be unable to stimulate endothelial cell
proliferation, differentiation, migration or survival. These
modified polypeptides are expected to be able to act as competitive
or non-competitive inhibitors of VEGF-D, and to be useful in
situations where prevention or reduction of VEGF-D action is
desirable. Thus such receptor-binding but non-mitogenic,
non-differentiation inducing, non-migration inducing or
non-survival promoting variants of VEGF-D are also within the scope
of the invention, and are referred to herein as "receptor-binding
but otherwise inactive variants".
[0029] According to a third aspect, the invention provides a
purified and isolated nucleic acid encoding a polypeptide or
polypeptide fragment of the invention. The nucleic acid may be DNA,
genomic DNA, cDNA or RNA, and may be single-stranded or double
stranded. The nucleic acid may be isolated from a cell or tissue
source, or of recombinant or synthetic origin. Because of the
degeneracy of the genetic code, the person skilled in the art will
appreciate that many such coding sequences are possible, where each
sequence encodes the amino acid sequence shown in SEQ ID NO. 3, SEQ
ID NO. 5, SEQ ID NO. 8 or SEQ ID NO. 9, an active fragment or
analogue thereof, or a receptor-binding but otherwise inactive or
partially inactive variant thereof.
[0030] A fourth aspect of the invention provides vectors comprising
the cDNA of the invention or a nucleic acid according to the third
aspect of the invention, and host cells transformed or transfected
with nucleic acids or vectors of the invention. These cells are
particularly suitable for expression of the polypeptide of the
invention, and include insect cells such as Sf9 cells, obtainable
from the American Type Culture Collection (ATCC SRL-171),
transformed with a baculovirus vector, and the human embryo kidney
cell line 293EBNA transfected by a suitable expression plasmid.
Preferred vectors of the invention are expression vectors in which
a nucleic acid according to the invention is operatively connected
to one or more appropriate promoters and/or other control
sequences, such that appropriate host cells transformed or
transfected with the vectors are capable of expressing the
polypeptide of the invention. Other preferred vectors are those
suitable for transfection of mammalian cells, or for gene therapy,
such as adenovirus or retrovirus vectors or liposomes. A variety of
such vectors is known in the art.
[0031] The invention also provides a method of making a vector is
capable of expressing a polypeptide encoded by a nucleic acid
according to the invention, comprising the steps of operatively
connecting the nucleic acid to one or more appropriate promoters
and/or other control sequences, as described above.
[0032] The invention further provides a method of making a
polypeptide according to the invention, comprising the steps of
expressing a nucleic acid or vector of the invention in a host
cell, and isolating the polypeptide from the host cell or from the
host cell's growth medium. In one preferred embodiment of this
aspect of the invention, the expression vector further comprises a
sequence encoding an affinity tag, such as FLAG.TM. or
hexahistidine, in order to facilitate purification of the
polypeptide by affinity chromatography.
[0033] In yet a further aspect, the invention provides an antibody
specifically reactive with a polypeptide of the invention. This
aspect of the invention includes antibodies specific for the
variant forms, fragments and analogues of VEGF-D referred to above.
Such antibodies are useful as inhibitors or agonists of VEGF-D and
as diagnostic agents for detection and quantification of VEGF-D.
Polyclonal or monoclonal antibodies may be used. Monoclonal and
polyclonal antibodies can be raised against polypeptides of the
invention using standard methods in the art. For some purposes, for
example where a monoclonal antibody is to be used to inhibit
effects of VEGF-D in a clinical situation, it may be desirable to
use humanized or chimeric monoclonal antibodies. Methods for
producing these, including recombinant DNA methods, are also well
known in the art.
[0034] This aspect of the invention also includes an antibody which
recognises VEGF-D and which is suitably labelled.
[0035] Polypeptides or antibodies according to the invention may be
labelled with a detectable label, and utilised for diagnostic
purposes. Similarly, the thus-labelled polypeptide of the invention
may be used to identify its corresponding receptor in situ. The
polypeptide or antibody may be covalently or non-covalently coupled
to a suitable supermagnetic, paramagnetic, electron dense, ecogenic
or radioactive agent for imaging. For use in diagnostic assays,
radioactive or non-radioactive labels, the latter including enzyme
labels or labels of the biotin/avidin system, may be used.
[0036] Clinical applications of the invention include diagnostic
applications, acceleration of angiogenesis in wound healing, tissue
or organ transplantation, or to establish collateral circulation in
tissue infarction or arterial stenosis, such as coronary artery
disease, and inhibition of angiogenesis in the treatment of cancer
or of diabetic retinopathy. Quantitation of VEGF-D in cancer biopsy
specimens may be useful as an indicator of future metastatic
risk.
[0037] Inasmuch as VEGF-D is highly expressed in the lung, and it
also increases vascular permeability, it is relevant to a variety
of lung conditions. VEGF-D assays could be used in the diagnosis of
various lung disorders. VEGF-D could also be used in the treatment
of lung disorders to improve blood circulation in the lung and/or
gaseous exchange between the lungs and the blood stream. Similarly,
VEGF-D could be used to improve blood circulation to the heart and
O.sub.2 gas permeability in cases of cardiac insufficiency. In like
manner, VEGF-D could be used to improve blood flow and gaseous
exhange in chronic obstructive airway disease.
[0038] Conversely, VEGF-D antagonists (e.g. antibodies and/or
inhibitors) could be used to treat in conditions, such as
congestive heart failure, involving accumulations of fluid in, for
example, the lung resulting from increases in vascular
permeability, by exerting an offsetting effect on vascular
permeability in order to counteract the fluid accumulation.
[0039] VEGF-D is also expressed in the small intestine and colon,
and administrations of VEGF-D could be used to treat malabsorptive
syndromes in the intestinal tract as a result of its blood
circulation increasing and vascular permeabiltiy increasing
activities.
[0040] Thus the invention provides a method of stimulation of
angiogenesis and/or neovascularization in a mammal in need of such
treatment, comprising the step of administering an effective dose
of VEGF-D, or a fragment or analogue thereof which has the ability
to stimulate endothelial cell proliferation, to the mammal.
[0041] Optionally VEGF-D may be administered together with, or in
conjunction with, one or more of VEGF-A, VEGF-B, VEGF-C, PlGF,
PDGF, FGF and/or heparin.
[0042] Conversely the invention provides a method of inhibiting
angiogenesis and/or neovascularization in a mammal in need of such
treatment, comprising the step of administering an effective amount
of an antagonist of VEGF-D to the mammal. The antagonist may be any
agent that prevents the action of VEGF-D, either by preventing the
binding of VEGF-D to its corresponding receptor or the target cell,
or by preventing activation of the transducer of the signal from
the receptor to its cellular site of action. Suitable antagonists
include, but are not limited to, antibodies directed against
VEGF-D; competitive or non-competitive inhibitors of binding of
VEGF-D to the VEGF-D receptor, such as the receptor-binding but
non-mitogenic VEGF-D variants referred to above; and anti-sense
nucleotide sequences complementary to at least a part of the DNA
sequence encoding VEGF-D.
[0043] The invention also provides a method of detecting VEGF-D in
a biological sample, comprising the step of contacting the sample
with a reagent capable of binding VEGF-D, and detecting the
binding. Preferably the reagent capable of binding VEGF-D is an
antibody directed against VEGF-D, more preferably a monoclonal
antibody. In a preferred embodiment the binding and/or extent of
binding is detected by means of a detectable label; suitable labels
are discussed above.
[0044] Where VEGF-D or an antagonist is to be used for therapeutic
purposes, the dose and route of application will depend upon the
condition to be treated, and will be at the discretion of the
attending physician or veterinarian. Suitable routes include
subcutaneous, intramuscular or intravenous injection, topical
application, implants etc. Topical application of VEGF-D may be
used in a manner analogous to VEGF.
[0045] According to yet a further aspect, the invention provides
diagnostic/prognostic means typically in the form of test kits. For
example, in one embodiment of the invention there is provided a
diagnostic/prognostic test kit comprising an antibody to VEGF-D and
means for detecting, and more preferably evaluating, binding
between the antibody and VEGF-D. In one preferred embodiment of the
diagnostic/prognostic means according to the invention, either the
antibody or the VEGF-D is labelled with a detectable label, and
either the antibody or the VEGF-D is substrate-bound, such that the
VEGF-D-antibody interaction can be established by determining the
amount of label attached to the substrate following binding between
the antibody and the VEGF-D. In a particularly preferred embodiment
of the invention, the diagnostic/prognostic means may be provided
as a conventional ELISA kit.
[0046] In another alternative embodiment, the diagnostic/prognostic
means may comprise polymerase chain reaction means for establishing
the genomic sequence structure of a VEGF-D gene of a test
individual and comparing this sequence structure with that
disclosed in this application in order to detect any abnormalities,
with a view to establishing whether any aberrations in VEGF-D
expression are related to a given disease condition.
[0047] In accordance with a further aspect, the invention relates
to a method of detecting aberrations in VEGF-D gene structure in a
test subject which may be associated with a disease condition in
said test subject. This method comprises providing a DNA sample
from said test subject; contacting the DNA sample with a set of
primers specific to VEGF-D DNA operatively coupled to a polymerase
and selectively amplifying VEGF-D DNA from the sample by polymerase
chain reaction, and comparing the nucleotide sequence of the
amplified VEGF-D DNA from the sample with the nucleotide sequences
set forth in SEQ ID NO:1 or SEQ ID NO:4. The invention also
includes the provision of a test kit comprising a pair of primers
specific to VEGF-D DNA operatively coupled to a polymerase, whereby
said polymerase is enabled to selectively amplify VEGF-D DNA from a
DNA sample.
[0048] Another aspect of the invention concerns the provision of a
pharmaceutical composition comprising either VEGF-D polypeptide or
a fragment or analogue thereof which promotes proliferation of
endothelial cells, or an antibody thereto. Compositions which
comprise VEGF-D polypeptide may optionally further comprise one or
more of VEGF, VEGF-B and VEGF-C, and/or heparin.
[0049] In another aspect, the invention relates to a protein dimer
comprising VEGF-D polypeptide, particularly a disulphide-linked
dimer. The protein dimers of the invention include both homodimers
of VEGF-D polypeptide and heterodimers of VEGF-D and VEGF, VEGF-B,
VEGF-C, PlGF or PDGF.
[0050] According to a yet further aspect of the invention there is
provided a method for isolation of VEGF-D comprising the step of
exposing a cell which expresses VEGF-D to heparin to facilitate
release of VEGF-D from the cell, and purifying the thus-released
VEGF-D.
[0051] Another aspect of the invention involves providing a vector
comprising an anti-sense nucleotide sequence which is complementary
to at least a part of a DNA sequence which encodes VEGF-D or a
fragment or analogue thereof which promotes proliferation of
endothelial cells. According to a yet further aspect of the
invention such a vector comprising an anti-sense sequence may be
used to inhibit, or at least mitigate, VEGF-D expression. The use
of a vector of this type to inhibit VEGF-D expression is favoured
in instances where VEGF-D expression is associated with a disease,
for example where tumours produce VEGF-D in order to provide for
angiogenesis. Transformation of such tumour cells with a vector
containing an anti-sense nucleotide sequence would suppress or
retard angiogenesis, and so would inhibit or retard growth of the
tumour.
[0052] Polynucleotides of the invention such as those described
above, fragments of those polynucleotides, and variants of those
polynucleotides with sufficient similarity to the non-coding strand
of those polynucleotides to hybridise thereto under stringent
conditions all are useful for identifying, purifying, and isolating
polynucleotides encoding other, non-human, mammalian forms of
VEGF-D. Thus, such polynucleotide fragments and variants are
intended as aspects of the invention. Exemplary stringent
hybridisation conditions are as follows: hybridisation at
42.degree. C. in 5.times.SSC, 20 mM NaPO.sub.4, pH 6.8, 50%
formamide; and washing at 42.degree. C. in 0.2.times.SSC. Those
skilled in the art understand that it is desirable to vary these
conditions empirically based on the length and the GC nucleotide
base content of the sequences to be hybridised, and that formulae
for determining such variation exist. See for example Sambrook et
al, "Molecular Cloning: A Laboratory Manual", Second Edition, Cold
Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1989).
[0053] Moreover, purified and isolated polynucleotides encoding
other, non-human, mammalian VEGF-D forms also are aspects of the
invention, as are the polypeptides encoded thereby, and antibodies
that are specifically immunoreactive with the non-human VEGF-D
variants. Thus, the invention includes a purified and isolated
mammalian VEGF-D polypeptide, and also a purified and isolated
polynucleotide encoding such a polypeptide.
[0054] It will be clearly understood that nucleic acids and
polypeptides of the invention may be prepared by synthetic means or
by recombinant means, or may be purified from natural sources.
BRIEF DESCRIPTION OF THE FIGURES
[0055] FIG. 1 shows a comparison between the sequences of human
VEGF-D and human VEGF.sub.165 (FIG. 1a), human VEGF-B (FIG. 1b),
human VEGF-C (FIG. 1c) and human PlGF (FIG. 1d). The box indicates
residues which match those in human VEGF-D exactly;
[0056] FIG. 2 shows sequence alignments between the sequences of
human VEGF-D, human VEGF.sub.165, human VEGF-B, human VEGF-C and
human PlGF. The boxes indicate residues that match the VEGF-D
sequence exactly; and
[0057] FIG. 3 shows the amino acid sequence of human VEGF-D (SEQ ID
NO 3), as predicted from the cDNA sequence (SEQ ID NO 1). The boxes
indicate potential sites for N-linked glycosylation.
[0058] FIG. 4 shows the nucleotide sequence of a second cDNA
sequence encoding human VEGF-D (SEQ ID NO 4), isolated by
hybridisation from a commercial human lung cDNA library; this cDNA
contains the entire coding region for human VEGF-D;
[0059] FIG. 5 shows the amino acid sequence for human VEGF-D (SEQ
ID NO 5) deduced from the sequence of the cDNA of FIG. 4;
[0060] FIG. 6 shows the nucleotide sequence of cDNA encoding mouse
VEGF-D1 (SEQ ID NO 6), isolated by hybridisation screening for a
commercially-available mouse lung cDNA library;
[0061] FIG. 7 shows the nucleotide sequence of cDNA encoding mouse
VEGF-D2 (SEQ ID NO 7), isolated from the same library as in FIG.
6;
[0062] FIG. 8 shows the deduced amino acid sequences for mouse
VEGF-D1 (SEQ ID NO 8) and VEGF-D2 (SEQ ID NO 9);
[0063] FIG. 9 shows a comparison between the deduced amino acid
sequences of mouse VEGF-D1, mouse VEGF-D2 and human VEGF-D;
[0064] FIG. 10 shows sequence alignments between the amino acid
sequences of human VEGF-D, human VEGF.sub.165, human VEGF-B, human
VEGF-C and human PlGF; and
[0065] FIG. 11 shows the results of a bioassay in which conditioned
medium from COS cells expressing either VEGF-A or VEGF-D was tested
for ability to bind to the extracellular domain of a chimeric
receptor expressed in Ba/F3 cells.
[0066] FIG. 12 shows the results of immunoprecipitation and Western
blotting analysis of VEGF-D peptides
[0067] (A) pEFBOSVEGFDfullFLAG and pCDNA-1VEGF-A were transfected
into COS cells and biosynthetically labelled with
.sup.35S-cysteine/methionine for 4 hours. The supernatants from
these cultures were immunoprecipitated with either M2 gel or an
antiserum directed to VEGFrA coupled to protein A. Washed beads
were eluted with an equal volume of 2.times. SDS-PAGE sample buffer
and boiled. The samples were then resolved by 12% SDS-PAGE. Lanes
marked with an asterix (*) indicate where samples were reduced with
dithiothreitol and alkylated with iodoacetamide. Molecular weight
markers are indicated. fA and fB indicate the 43 kD and 25 kD
species immunoprecipitated by the M2 gel from the COS cells
expressing pEFBOSVEGFDfullFLAG.
[0068] (B) Western blotting analysis of purified
VEGFD.DELTA.N.DELTA.C. An aliquot of material eluted from the M2
affinity column (fraction #3, VEGFD.DELTA.N.DELTA.C) was combined
with 2.times. SDS-PAGE sample buffer and resolved on a 15% SDS-PAGE
gel. The proteins were then transferred to nitrocellulose membrane
and probed with either monoclonal antibody M2 or a control
isotype-matched antibody (Neg). Blots were developed using a goat
anti-mouse-HRP secondary antibody and chemiluminescence (ECL,
Amersham). Monomeric VEGFD.DELTA.N.DELTA.C is arrowed, as is the
putative dimeric form of this peptide (VEGFD.DELTA.N.DELTA.C").
Molecular weight markers are indicated.
[0069] FIG. 13 shows the results of analysis of
VEGFD.DELTA.N.DELTA.C protein using the VEGFR2 bioassay.
Recombinant VEGFD.DELTA.N.DELTA.C, and material purified by M2
affinity chromatography, was assessed using the VEGFR2 bioassay.
Bioassay cells (10.sup.4), washed to remove IL-3, were incubated
with aliquots of conditioned medium from VEGF-D transfected COS
cells, fraction #1 from the affinity column (void volume) or
fraction #3 from the affinity column (containing
VEGFD.DELTA.N.DELTA.C). All samples were tested at an initial
concentration of 20% (ie 1/5) followed by doubling dilutions. Cells
were allowed to incubate for 48 hours at 37.degree. C. in a
humidified atmosphere of 10% CO.sub.2. Cell proliferation was
quantitated by the addition of 1 .mu.Ci of .sup.3H-thymidine and
counting the amount incorporated over a period of 4 hours.
[0070] FIG. 14 shows stimulation of tyrosine phosphorylation of the
VEGFR3 receptor (Flt4) on NIH3T3 cells by culture supernatant from
HF cells infected with a recombinant baculovirus vector transformed
with VEGF-D.
[0071] FIG. 15 shows stimulation of tyrosine phosphorylation of the
VEGFR2 receptor (KDR) in PAE cells by culture supernatant prepared
as in FIG. 14.
[0072] FIG. 16 shows the mitogenic effect of VEGFD.DELTA.N.DELTA.C
on bovine aortic endothelial cells (BAEs). BAEs were treated with
fraction #3 containing VEGFD.DELTA.N.DELTA.C and, as positive
control, purified VEGF-A as described in the text. The result
obtained using medium without added growth factor is denoted Medium
Control.
DETAILED DESCRIPTION OF THE INVENTION
[0073] The invention will now be described in detail by reference
to the figures, and to the following non-limiting examples.
EXAMPLE 1
[0074] It has been speculated that no further members of the VEGF
family will be found, because there are no known orphan receptors
in the VEGFR family. Furthermore, we are not aware of any
suggestion in the prior art that other such family members would
exist.
[0075] A computer search of nucleic acid databases was carried out
incidentally to another project, using as search topics the amino
acid sequences of VEGF, VEGF-B, VEGF-C and PlGF. Several cDNA
sequences were identified by this search. One of these sequences,
GenBank Accession No. H24828, encoded a polypeptide which was
similar in structure to the cysteine-riched C-terminal region of
VEGF-C. This sequence was obtained from the database of expressed
sequence tags (dbEST), and for the purposes of this specification
is designated XPT. The XPT cDNA had been isolated from a human cDNA
library designated "Soares Breast 3NbHBst", which was constructed
using mRNA from an adult human female breast tissue. As far as can
be ascertained this was normal breast tissue. Sequencing of the XPT
DNA was performed pursuant to the Integrated Molecular Analysis of
Genome Expression Consortium (IMAGE Consortium), which solicits
cDNA libraries from laboratories around the world, arrays the cDNA
clones, and provides them to other organisations for
sequencing.
[0076] The XPT sequence shown in the database was 419 nucleotides
long, and encoded an amino acid sequence similar to the C-terminal
100 amino acids of VEGF-C, ie. approximately residues 250 to 350,
using the numbering system of Joukov et al (1996). Similarly
cysteine-rich regions are found in other proteins, which are
entirely unrelated in function to the VEGF family, for example the
secreted silk-like protein spl85 synthesized in the salivary glands
of the midge Chironomus tentans. This protein is encoded by the
gene BR3, located in a Balbiani ring, a tissue specific chromosome
"puff" found on polytene chromosomes in the midge salivary gland
(Dignam and Case: Gene, 1990 88 133-140; Paulsson et al, J. Mol.
Biol., 1990 211 331-349). It is stated in Joukov et al (1996) that
the sp185-like structural motif in VEGF-C may fold into an
independent domain, which is thought to be at least partially
cleaved off after biosynthesis, and that there is at least one
cysteine motif of the spl85 type in the C-terminal region of
VEGF.
[0077] FIG. 3 of Joukov et al shows that the last two-thirds of the
C-terminal cysteine-rich region of VEGF-C do not align with VEGF or
PlGF, and in fact could be considered a C-terminal extension of
VEGF-C which is not present in VEGF or PlGF. The sequence encoded
by XPT is similar to this extension. As the XPT cDNA was truncated
at its 5' end, it was not possible to deduce or predict any amino
acid sequence for regions N-terminal to the cysteine-rich domain.
Thus the portion of VEGF-C which is similar to the XPT-derived
sequence does not extend to regions of VEGF-C which are conserved
among other members of the VEGF family.
[0078] As described above, it was not possible to predict whether
the N-terminal region of the polypeptide encoded by a full-length
XPT nucleic acid (as distinct from the truncated XPT cDNA reported
in dbEST) would show any further homology to any member of the VEGF
family, in particular VEGF-C, which has a further N-terminal 250
amino acids. For example, the naturally-occurring protein encoded
by a full-length XPT nucleic acid could have been the human
homologue of the midge salivary gland protein. Alternatively, the
type of cysteine-rich motif encoded by truncated XPT cDNA could be
widely distributed among proteins, as are many structural domains.
For example, clusters of cysteine residues may be involved in metal
binding, formation of intramolecular disulphide bonds to promote
accurate protein folding, or formation of intermolecular disulphide
bonds for assembly of protein subunits into complexes (Dignam and
Chase, 1990). In order to determine whether the truncated XPT cDNA
was derived from sequences encoding a VEGF-related molecule, it was
necessary to isolate a much longer cDNA.
EXAMPLE 2
Cloning of cDNA Encodine VEGF-D
[0079] A sample of the XPT cDNA reported in dbEST was obtained from
the American Type Culture Collection, which is a registered
supplier of cDNA clones obtained by the IMAGE Consortium. The
identity of the XPT cDNA was confirmed by nucleotide sequencing,
using the dideoxy chain termination method (Sanger et al, Proc.
Natl. Acad. Sci. USA, 1977 74 5463-5467).
[0080] The XPT cDNA was used as a hybridisation probe to screen a
human breast cDNA library, which was obtained commercially from
Clontech. One positive clone was isolated, and this clone was then
sequenced on both strands. The nucleotide sequence was compiled,
and an open reading frame was identified. The nucleic acid sequence
is set out in SEQ ID NO. 1. The polypeptide encoded by this
sequence was designated VEGF-D, and its deduced amino acid
sequence, designated SEQ ID NO. 3, is set out in FIG. 3. In FIG. 3
putative sites of N-linked glycosylation, with the consensus
sequence N-X-S/T in which X is any amino acid, are indicated by the
boxes.
EXAMPLE 3
Characteristics of VEGF-D
[0081] The amino acid sequence of VEGF-D was compared with those of
human VEGF-A.sub.165, VEGF-B, VEGF-C and PlGF. These comparisons
are set out in FIGS. 1a to d respectively. The degree of sequence
homology was calculated, and if gaps in sequence introduced for the
purposes of alignment are not considered in the calculation, VEGF-D
is 31% identical to VEGF, 48% identical to VEGF-C, 28% identical to
VEGF-B and 32% identical to PlGF. Thus the most closely-related
protein identified was VEGF-C.
[0082] Computer searches of the GenBank, EMBL and SwissProt nucleic
acid databases did not reveal any protein sequences identical to
VEGF-D. As expected from the sequence alignment referred to above,
the most closely related protein found in these databases was
VEGF-C. Searches of dbEST were also performed, but did not reveal
any sequences encompassing the entire coding region of VEGF-D. The
sequence of VEGF-D is unrelated to that of Tie-2 ligand 1 as
disclosed in WO 96/11269.
[0083] It is important to bear in mind that the only homologies
detected were at the level of amino acid sequence. Thus it would
not have been possible to isolate the cDNA or gDNA encoding VEGF-D
by methods such as low-stringency hybridization with a nucleic acid
sequence encoding another member of the VEGF family.
[0084] VEGF-D appears to be most closely related to VEGF-C of all
the members of the VEGF family. Because the VEGF-D amino acid
sequence includes the cysteine-rich spl85-like motif which is found
in VEGF-C, the polypeptide of the invention may play an important
functional role in lymphatic endothelia. While we do not wish to be
bound by any proposed mechanism, it is thought that VEGF-C and
VEGF-D may constitute a silk-like matrix over which endothelial
cells can grow. Lymphatic vessels have no basement membrane, so the
silk-like matrix can form a basement membrane-like material. This
may be important in promoting cell growth and/or in cell
differentiation, and may be relevant to cancer, especially
metastasis, drug therapy, cancer prognosis, etc.
EXAMPLE 4
Biological Characteristics of VEGF-D
[0085] The cDNA sequence of VEGF-D was used to predict the deduced
amino acid sequence of VEGF-D, the biochemical characteristics of
the encoded polypeptide, including the numbers of strongly basic,
strongly acidic, hydrophobic and polar amino acids, the molecular
weight, the isoelectric point, the charge at pH 7, and the
compositional analysis of the whole protein. This analysis was
performed using the Protean protein analysis program, Version 1.20
(DATASTAR). These results are summarised in Tables 1 and 2 below.
Table 1 also shows the codon usage.
1TABLE 1 Translated DNA Sequence of VEGF-D contig x(1,978) With
Standard Genetic Code Molecular Weight 37056.60 Daltons 425 Amino
Acids 46 Strong Basic (+) Amino Acids (K,R) 41 Strong Acidic (-)
Amino Acids (D,E) 79 hydrophobic Amino Acids (A, I, L, F, W, V) 108
Polar Amino Acids (N, C, Q, S, T, Y) 7.792 Isoelectric Point 6.371
Charge at pH 7.0 Total number of bases translated is 978 % A =
28.73 [281] % G = 23.11 [226] % T = 23.21 [227] % C = 24.95 [244] %
Ambiguous = 0.00 [0] % A + T = 51.94 [508] % C + G = 48.06 [470]
Davis, Botstein, Roth Melting Temp .degree. C. 84.09 Wallace Temp
.degree. C. 3384.00 Codon usage: ccg ( ) 0 # ugc Cys (C) 14 # cuc
Leu (L) 6 # ucg Ser (S) uaa ( ) 0 # ugu Cys (C) 16 # cug Leu (L) 4
# ucu Ser (S) uag ( ) 0 # --- Cys (C) 30 # cuu Leu (L) 2 # --- Ser
(S) 3 --- ( ) 0 # caa Gln (Q) 1 # uua Leu (L) 1 # uga Ter (.) gca
Ala (A) 5 # cag Gln (Q) 11 # uug Leu (L) 5 # --- Ter (.) gcc Ala
(A) 4 # --- Gln (Q) 12 # --- Leu (L) 23 # aca Thr (T) gcg Ala (A) 1
# gaa Glu (E) 16 # aaa Lys (K) 13 # acc Thr (T) gcu Ala (A) 5 # gag
Glu (E) 12 # aag Lys (K) 10 # acg Thr (T) --- Ala (A) 15 # --- Glu
(E) 28 # --- Lys (K) 23 # acu Thr (T) aga Arg (R) 7 # gga Gly (G) 1
# aug Met (M) 6 # --- Thr (T) 2 agg Arg (R) 5 # ggc Gly (G) 2 # ---
Met (M) 6 # ugg Trp (W) cga Arg (R) 5 # ggg Gly (G) 3 # uuc Phe (F)
4 # --- Trp (W) cgc Arg (R) 4 # ggu Gly (G) 2 # uuu Phe (F) 8 # uac
Tyr (Y) cgg Arg (R) 1 # --- Gly (G) 8 # --- Phe (F) 12 # uau Tyr
(Y) cgu Arg (R) 1 # cac His (H) 7 # cca Pro (P) 9 # --- Tyr (Y) ---
Arg (R) 23 # cau His (H) 7 # ccc Pro (P) 6 # gua Val (V) aac Asn
(N) 5 # --- His (H) 14 # ccu Pro (P) 8 # guc Val (V) aau Asn (N) 4
# aua Ile (I) 2 # --- Pro (P) 23 # gug Val (V) --- Asn (N) 9 # auc
Ile (I) 6 # agc Ser (S) 6 # guu Val (V) gac Asp (D) 8 # auu Ile (I)
5 # agu Ser (S) 8 # --- Val (V) 1 qau Asp (D) 5 # --- Ile (I) 13 #
uca Ser (S) 5 # nnn ??? (X) gau Asp (D) 5 # --- Ile (I) 13 # uca
Ser (S) 5 # nnn ??? (X) --- Asp (D) 13 # cua Leu (L) 5 # ucc Ser
(S) 7 # TOTAL 32 Contig 2: Contig Length: 2379 bases Average
Length/Sequence: 354 bases Total Sequence Length: 4969 bases
[0086]
2TABLE 2 Predicted Structural Class of the Whole Protein: Delage
& Roux Modification of Nishikawa & Ooi 1987 Analysis Whole
Protein Molecular Weight 37056.60 m.w. Length 325 1 microgram =
26.986 pMoles Molar Extinction 30200 .+-. 5% coefficient 1 A(280) =
1.23 mg/ml Isoelectric Point 7.79 Charge at pH 7 6.37 Whole Protein
Composition Anayalsis Amino Acid(s) Number count % by weight % by
frequency Charged (RKHYCDE) 134 46.30 41.23 Acidic (DE) 41 13.79
12.62 Basic (KR) 46 17.65 14.15 Polar (NCQSTY) 108 30.08 33.23
Hydrophobic (AILFWV) 79 23.86 24.31 A Ala 15 2.88 4.62 C Cys 30
8.35 9.23 D Asp 13 4.04 4.00 E Glu 28 9.75 8.62 F Phe 12 4.77 3.69
G Gly 8 1.23 2.46 H His 14 5.18 4.31 I Ile 13 3.97 4.00 K Lys 23
7.96 7.08 L Leu 23 7.03 7.08 M Met 6 2.12 1.85 N Asn 9 2.77 2.77 P
Pro 23 6.08 7.08 Q Gln 12 4.15 3.69 R Arg 23 9.69 7.08 S Ser 33
7.76 10.15 T Thr 21 5.73 6.46 V Val 12 3.21 3.69 W Trp 4 2.01 1.23
Y Trp 3 1.32 0.92 B Asx 0 0.00 0.00 Z Glx 0 0.00 0.00 X Xxx 0 0.00
0.00 . Ter 0 0.00 0.00
[0087] This analysis predicts a molecular weight for the
unprocessed VEGF-D monomer of 37 kilodaltons (kD), compared to the
experimentally determined values (for the fully processes peptides)
of 20 to 27 kD for VEGF-A monomers, 21 kD for the VEGF-B monomer
and 23 kD for the VEGF-C monomer.
EXAMPLE 5
[0088] The original isolation of a cDNA for VEGF-D, described in
Example 2 involved hybridisation screening of a human breast cDNA
library. As only one cDNA clone for VEGF-D was thus isolated, it
was not possible to confirm the structure of the cDNA by comparison
with other independently isolated VEGF-D cDNAs. The work described
in this example, which involved isolation of additional human
VEGF-D cDNA clones, was carried out in order to confirm the
structure of human VEGF-D cDNA. In addition, mouse VEGF-D cDNA
clones were isolated.
[0089] Two cDNA libraries which had been obtained commercially from
Stratagene, one for human lung and one for mouse lung (catalogue
numbers 937210 and 936307, respectively) were used for
hybridisation screening with a VEGF-D cDNA probe. The probe, which
spanned from nucleotides 1817 to 2495 of the cDNA for human VEGF-D
described in Example 2, was generated by polymerase chain reaction
(PCR) using a plasmid containing the VEGF-D cDNA as template and
the following two oligonucleotides:
3 5'-GGGCTGCTTCTAGTTTGGAG, (SEQ ID NO. 10) and
5'-CACTCGCAACGATCTTCGTC. (SEQ ID NO. 11)
[0090] Approximately two million recombinant bacteriophage were
screened with this probe from each of the two cDNA libraries. Nine
human and six mouse cDNA clones for VEGF-D were subsequently
isolated.
[0091] Two of the nine human cDNA clones for VEGF-D were sequenced
completely using the dideoxy chain termination method (Sanger et
al, Proc. Natl. Acad. Sci. USA, 1977.74 5463-5467). The two cDNAs
contained the entire coding region for human VEGF-D, and were
identical except that one of the clones was five nucleotides longer
than the other at the 5'-terminus. The nucleotide sequence of the
shorter cDNA is shown in FIG. 4, and is designated SEQ ID NO. 4.
The amino acid sequence for human VEGF-D (hVEGF-D) deduced from
this cDNA was 354 residues long, and is shown in FIG. 5; this is
designated SEQ ID NO. 5. The sequences of the 5' regions of five of
the other human VEGF-D cDNA clones were also determined. For each
clone, the sequence that was characterized contained more than 100
nucleotides of DNA immediately downstream from the translation
start site of the coding region. In all cases, the sequences of
these regions were identical to corresponding regions of the human
VEGF-D cDNA shown in FIG. 4.
[0092] All six mouse cDNA clones for VEGF-D were sequenced
completely. Only two of the clones contained an entire coding
region for VEGF-D; the other clones were truncated. The nucleotide
sequences of the two clones with an entire coding region are
different, and encode amino acid sequences of different sizes. The
longer amino acid sequence is designated mVEGF-D1, and the shorter
sequence is designated mVEGF-D2. The nucleotide sequences of the
cDNAs encoding mVEGF-D1 and mVEGF-D2 are shown in FIGS. 6 and 7
respectively. The deduced amino acid sequences for mVEGF-D1 and
mVEGF-D2 are shown in FIG. 8. These sequences are respectively
designated SEQ ID NOS. 6, 7, 8 and 9. The differences between the
amino acid sequences are:
[0093] i) an insertion of five amino acids (DFSFE) after residue 30
in mVEGF-D1 in comparison to mVEGF-D2;
[0094] ii) complete divergence of the C-terminal ends after residue
317 in mVEGF-D1 and residue 312 in mVEGF-D2, which results in
mVEGF-D1 being considerably longer.
[0095] Three of the four truncated cDNAs for mouse VEGF-D encoded
the C-terminal region, but not the N-terminal 50 amino acids. All
three of these cDNAs encoded a C-terminal end for VEGF-D which is
identical to that for mVEGF-D2. The other truncated cDNA encoded
only the N-terminal half of VEGF-D. The amino acid sequence deduced
from this cDNA contained the five amino acids DFSFE immediately
after residue 30 found in mVEGF-D1, but not in mVEGF-D2.
[0096] As described above, the entire sequence of the human VEGF-D
cDNA clone reported in this example has been validated by
comparison with that for a second human clone. In addition, the
sequence of the 5' end of the coding region was found to be
identical in five other human VEGF-D cDNA clones. In contrast, the
sequence reported in Example 2 contained most of the coding region
for VEGF-D, but was incorrect near the 5'-end of this region. This
was probably because the VEGF-D cDNA was truncated near the 5'-end
of the coding region and at that point had been ligated with
another unidentified cDNA, and consequently the first 30 codons of
the true coding sequence for VEGF-D had been deleted and replaced
with a methionine residue. This methionine residue was defined as
the N-terminal amino acid of the VEF-D sequence presented in
Example 2.
[0097] The N-terminal regions of the deduced amino acid sequences
of mouse VEGF-D1 and VEGF-D2 are very similar to that deduced for
human VEGF-D (see FIG. 9). This also indicates that the correct
deduced amino acid sequence for human VEGF-D is reported in this
example. The N-terminal 25 amino acids of human VEGF-D form an
extremely hydrophobic region, which is consistent with the notion
that part of this region may be a signal sequence for protein
secretion. FIG. 10 shows the alignment of the human VEGF-D sequence
with the sequences of other members of the VEGF family of growth
factors, namely human VEGF.sub.165 (hVEGF.sub.165), human VEGF-B
(hVEGF-B), human VEGF-C (hVEGF-C) and human Placental Growth Factor
(hPlGF). When gaps in the alignments are ignored for the purposes
of calculation, human VEGF-D is found to be 31% identical in amino
acid sequence to human VEGF.sub.165, 28% identical to human VEGF-B,
48% identical to VEGF-C and 32% identical to human PlGF. Clearly
VEGF-C is the member of this family which is most closely related
to VEGF-D.
[0098] The differences in sequence for mouse VEGF-D1 and VEGF-D2
most probably arise from differential mRNA splicing. The C-terminal
41 amino acid residues of VEGF-D1 are deleted in VEGF-D2, and are
replaced with 9 residues which are not closely related to the
VEGF-D1 sequence. Therefore 4 cysteine residues present near the
C-terminus of VEGF-D1 are deleted in VEGF-D2. This change may alter
the tertiary or quaternary structures of the protein, or may affect
the localisation of the protein in the cell or the extracellular
environment. The C-terminal end of human VEGF-D resembles that of
mouse VEGF-D1, not mouse VEGF-D2. The small 5 amino acid insertion
after residue 30 in mouse VEGF-D1, which is not present in either
mouse VEGF-D2 or human VEGF-D, may influence proteolytic processing
of the protein.
[0099] VEGF-D is highly conserved between mouse and man.
Eighty-five percent of the amino acid residues of human VEGF-D are
identical in mouse VEGF-D1. This is likely to reflect conservation
of protein function. Putative functions for VEGF-D have been
proposed herein. Although we have not found alternative forms of
human VEGF-D cDNA, it is possible that the RNA splice variation
which gives rise to numerous forms of mRNA for mouse VEGF-D may
also occur in human tissues.
EXAMPLE 6
Expression of VEGF-D in COS Cells
[0100] A fragment of the human cDNA for VEGF-D, spanning from
nucleotide 1 to 1520 of the sequence shown in Figure 4 and
containing the entire coding region, was inserted into the
mammalian expression vector pcDNA1-amp. The vector was used to
transiently transfect COS cells by the DEAE-Dextran method as
described previously (Aruffo and Seed, 1987) and the resulting
conditioned cell culture media, collected after 7 days of
incubation, were concentrated using Amicon concentrators (Centricon
10 with a 10,000 molecular weight cut off) according to the
manufacturer. The plasmids used for transfections were the
expression construct for human VEGF-D and, as positive control, a
construct made by insertion of mouse VEGF-A cDNA into pcDNA1-amp.
The conditioned media were tested in two different bioassays, as
described below, and the results demonstrate that the COS cells did
in fact express and secrete biologically-active VEGF-D.
EXAMPLE 7
Bioassay for Capacity of VEGF-D to Bind to VEGF Receptor-2
[0101] As shown in Example 5, VEGF-D is closely related in primary
structure to other members of the VEGF family Most members of this
protein family are mitogenic and/or chemotactic for endothelial
cells (Keck et al, 1989; Leung et al, 1989; Joukov, et al, 1996;
Olofsson et al, 1996 In addition VEGF-A (previously known as VEGF),
the first member of the VEGF family to be described in the
literature, is a potent inducer of vascular permeability (Keck et
al, 1989). As protein structure is an important determinant of
protein function, it seemed likely that VEGF-D might also be
mitogenic for endothelial cells or induce vascular permeability.
Therefore human VEGF-D was tested in a bioassay for its capacity to
bind to VEGF receptor-2 (VEGFR2; also known as Flk-1), an
endothelial cell-specific receptor which, when activated by VEGF-A,
is thought to give rise to a mitogenic signal (Strawn et al,
1996).
[0102] A bioassay for detection of growth factors which bind to
VEGFR2 has been developed in the factor-dependent cell line Ba/F3,
and is described in our earlier patent application, No.
PCT/US95/16755. These cells grow in the presence of interleukin-3
(IL-3); however removal of this factor results in cell death within
48 hours. If another receptor capable of delivering a growth
stimulus is transfected into the Ba/F3 cells, the cells can be
rescued by the specific growth factor which activates that receptor
when the cells are grown in medium lacking IL-3. In the specific
case of receptor-type tyrosine kinases (eg. VEGFR2), chimeric
receptors containing the extracellular domain of the receptor
tyrosine kinase and the transmembrane and cytoplasmic domains of
the erythropoietin receptor (EpoR) can be utilised. In this case
stimulation with the ligand (eg. VEGF), which binds to the
extracellular domain of the chimeric receptor, results in
signalling via the EpoR cytoplasmic domain and subsequent rescue of
the cell line in growth medium lacking IL-3. The construction of
the chimeric receptor used in this study, consisting of the mouse
VEGFR2 extracellular domain and the mouse EpoR transmembrane and
cytoplasmic domains, and the bioassay itself are described
below.
[0103] Plasmid Construction
[0104] i) Construction of a Plasmid for Generating Chimeric
[0105] VEGFR2 Receptors
[0106] To obtain a plasmid construct with which DNA encoding the
extracellular domain of mouse VEGFR2 could easily be ligated with
DNA encoding other protein domains, site-directed mutagenesis was
used to generate a BglII restriction enzyme site at the position of
mouse VEGFR2 cDNA which encoded the junction of the extracellular
domain and the transmembrane domain. The full-length clone of the
mouse VEGFR2 cDNA described by Oelrichs et al (1993) was subcloned
into the mammalian expression vector pcDNA1-amp, using the BstXI
restriction enzyme site. Single stranded UTP+DNA was generated
using the M13 origin of replication, and this was used as a
template to generate mouse VEGFR2 cDNA containing the BglII site at
the desired position. The plasmid containing the altered VEGFR2
cDNA was designated pVEGFR2Bgl. DNA fragments encoding the
transmembrane and cytoplasmic domains of any receptor can be
inserted at the BglII site of pVEGFR2Bgl in order to generate
chimeric VEGFR2 receptors.
[0107] ii) Construction of VEGFR2/EpoR Chimeric Receptor
[0108] The mouse EpoR cDNA was subcloned into the expression vector
pCDNA1-amp, and single stranded DNA was generated as a template for
mutagenesis. A BglII restriction enzyme site was inserted into the
EpoR cDNA at the position encoding the junction of the
transmembrane and extracellular domains of the EpoR to allow direct
ligation of this DNA fragment to the modified cDNA encoding the
extracellular domain of VEGFR2 in pVEGFR2Bgl. In addition a BglII
site in the cytoplasmic domain of the EpoR was removed by a silent
single nucleotide substitution. The DNA fragment encoding the
transmembrane and cytoplasmic domains of EpoR was then used to
replace the portion of pVEGFR2Bgl encoding the transmembrane and
cytoplasmic domains of VEGFR2. Thus a single reading frame was
generated which encoded the chimeric receptor consisting of the
VEGFR2 extracellular domain and the EpoR transmembrane and
cytoplasmic domains.
[0109] The DNA fragment encoding the chimeric receptor was
subcloned into the expression vector pBOS, and co-transfected into
the Ba/F3 cell line with plasmid pgk-neo at a ratio of 1:20. Cells
expressing the VEGFR2-EpoR protein were selected by flow cytometry
analysis using a monoclonal antibody to the VEGFR2 extracellular
domain (MAb 4H3). This monoclonal antibody is described in
Australian Patent Application No. PM 3794 filed 10 Feb. 1994. Cell
lines expressing higher levels of VEGFR2-EpoR were selected by
growing the cells in 5 .mu.g/ml MAb 4H3 or 25 ng/ml of recombinant
VEGF. A cell line expressing high levels of VEGFR2-EpoR, designated
Ba/F3-NYK-EpoR, was used for the bioassay.
[0110] The Bioassay
[0111] The Ba/F3-NYK-EpoR cells described above were washed three
times in PBS to remove all IL-3 and resuspended at a concentration
of 1000 cells per 13.5 .mu.l of culture medium and 13.5 .mu.l was
aliquoted per well of a 60-well Terasaki plate. Conditioned media
from transfected COS cells were then diluted into the cell culture
medium. Cells expressing a chimeric receptor consisting of the
extracellular domain of the endothelial cell receptor Tie2 and the
transmembrane and cytoplasmic domains of EpoR were used as a
non-responding control cell line. Cells were incubated for 48-96
hours, during which the cells incubated in cell culture medium
alone had died and the relative survival/proliferation seen in the
other wells (ie in the presence of COS cell-conditioned media) was
scored by counting the viable cells present per well.
[0112] The conditioned medium from COS cells which had been
transiently transfected with expression plasmids was concentrated
30-fold and used in the VEGFR2 bioassay. Concentrated conditioned
medium from COS cells transfected with pcDNA1-amp was used as
negative control.
[0113] The results are shown in FIG. 11, with the percentage of
30-fold concentrated COS cell-conditioned medium in the incubation
medium (vol/vol) plotted versus the number of viable cells in the
well after 48 hours of incubation. Clearly the conditioned medium
containing either VEGF-A or VEGF-D was capable of promoting cell
survival in this assay, indicating that both proteins can bind to
and activate VEGFR2.
EXAMPLE 8
Vascular Permeability Assay
[0114] Human VEGF-D, prepared as in Example 6 and concentrated
30-fold, was tested in the Miles vascular permeability assay (Miles
and Miles, 1952) performed in anaesthetized guinea pigs
(albino/white, 300-400 g). Concentrated conditioned medium for COS
cells transfected with pcDNA1-amp was again used as a negative
control. Guinea pigs were anaesthetised with chloral-hydrate (3.6
g/100 ml; 0.1 ml per 10 g of body weight). The backs of the animals
were then carefully shaved with clippers. Animals were given an
intracardiac injection of Evans Blue dye (0.5% in MT PBS, 0.5 ml)
using a 23G needle, and were then injected intra-dermally with
100-150 .mu.l of concentrated COS cell-conditioned medium. After
15-20 min the animals were sacrificed and the layer of skin on the
back excised to expose the underlying blood vessels. For
quantitation, the area of each injection was excised and heated to
45.degree. C. in 2-5 ml of formamide. The resulting supernatants,
containing extravasated dye, were then examined
spectrophotometrically at 620 nm.
[0115] For animal 1, the absorbance at 620 nm arising from
injection of 30-fold concentrated VEGF-A conditioned medium was
0.178, that for the 30-fold concentrated VEGF-D conditioned medium
was 0.114, and that for 30-fold concentrated medium from cells
transfected with pcDNA1-amp was 0.004. For animal 2, the 30-fold
concentrated media were diluted 4-fold in cell culture medium
before intra-dermal injection. The absorbance at 620 nm for the
VEGF-A conditioned sample was 0.141, that for the VEGF-D
conditioned sample was 0.116 and that for a sample matched for
serum content as negative control was 0.017. The enhanced
extravasation of dye observed for both animals in the presence of
VEGF-A or VEGF-D demonstrated that both of these proteins strongly
induced vascular permeability.
[0116] The data described here indicate that VEGF-D is a secreted
protein which, like VEGF-A, binds to and activates VEGFR2 and can
induce vascular permeability.
EXAMPLE 9
Bioactivities of Internal VEGF-D Polypeptides
[0117] The deduced amino acid sequence for VEGF-D includes a
central region which is similar in sequence to all other members of
the VEGF family (approximately residues 101 to 196 of the human
VEGF-D amino acid sequence as shown in the alignment in FIG. 10).
Therefore, it was thought that the bioactive portion of VEGF-D
might reside in the conserved region. In order to test this
hypothesis, the biosynthesis of VEGF-D was studied, and the
conserved region of human VEGF-D was expressed in mammalian cells,
purified, and tested in bioassays as described below.
[0118] Plasmid Construction
[0119] A DNA fragment encoding the portion of human VEGF-D from
residue 93 to 201, ie. with N- and C-terminal regions removed, was
amplified by polymerase chain reaction with Pfu DNA polymerase,
using as template a plasmid comprising full-length human VEGF-D
cDNA. The amplified DNA fragment, the sequence of which was
confirmed by nucleotide sequencing, was then inserted into the
expression vector pEFBOSSFIAG to give rise to a plasmid designated
pEFBOSVEGFD.DELTA.N.DELTA.C. The pEFBOSSFLAG vector contains DNA
encoding the signal sequence for protein secretion from the
interleukin-3 (IL-3) gene and the FLAG.TM. octapeptide. The
FLAG.TM. octapeptide can be recognized by commercially available
antibodies such as the M2 monoclonal antibody (IBI/Kodak). The
VEGF-D PCR fragment was inserted into the vector such that the IL-3
signal sequence was immediately upstream from the FLAG.TM.
sequence, which was in turn immediately upstream from the VEGF-D
sequence. All three sequences were in the same reading frame, so
that translation of mRNA resulting from transfection of
pEFBOSVEGFD.DELTA.N.DELTA.C into mammalian cells would give rise to
a protein which would have the IL-3 signal sequence at its
N-terminus, followed by the FLAG.TM. octapeptide and the VEGF-D
sequence. Cleavage of the signal sequence and subsequent secretion
of the protein from the cell would give rise to a VEGF-D
polypeptide which is tagged with the FLAG.TM. octapeptide adjacent
to the N-terminus. This protein was designated
VEGFD.DELTA.N.DELTA.C.
[0120] In addition, a second plasmid was constructed, designated
pEFBOSVEGFDfullFLAG, in which the full-length coding sequence of
human VEGF-D was inserted into PEFBOSIFLAG such that the sequence
for the FLAG.TM. octapeptide was immediately downstream from, and
in the same reading frame as, the coding sequence of VEGF-D. The
plasmid PEFBOSIFLAG lacks the IL-3 signal sequence, so secretion of
the VEGF-D/FLAG fusion protein was driven by the signal sequence of
VEGF-D. pEFBOSVEGFDfullFLAG was designed to drive expression in
mammalian cells of full-length VEGF-D which was C-terminally tagged
with the FLAG.TM. octapeptide. This protein is designated
VEGFDfullFLAG, and is useful for the study of VEGF-D
biosynthesis.
[0121] Analysis of the Post-Translational Processing of VEGF-D
[0122] To examine whether the VEGF-D polypeptide is processed to
give a mature and fully active protein, pEFBOSVEGFDfullFLAG was
transiently transfected into COS cells (Aruffo and Seed, 1987).
Expression in COS cells followed by biosynthetic labeling with
.sup.35S-methionine/cysteine and immunoprecipitation with M2 gel
has demonstrated species of approximately 43 kD (fA) and 25 kD (fB)
(FIG. 12A). These bands are consistent with the notion that VEGF-D
is cleaved to give a C-terminal fragment (FLAG.TM. tagged) and an
internal peptide (corresponding approximately to the
VEGFD.DELTA.N.DELTA.C protein). Reduction of the immunoprecipitates
(M2*) gives some reduction of the fA band, indicating the potential
for disulphide linkage between the two fragments.
[0123] Expression and Purification of Internal VEGF-D
Polypeptide
[0124] Plasmid pEFBOSVEGFD.DELTA.N.DELTA.C was used to transiently
transfect COS cells by the DEAE-Dextran method as described
previously (Aruffo and Seed, 1987). The resulting conditioned cell
culture medium (approximately 150 ml), collected after 7 days of
incubation, was subjected to affinity chromatography using a resin
to which the M2 monoclonal antibody had been coupled. In brief, the
medium was run batch-wise over a 1 ml M2 antibody column for
approximately 4 hours at 4.degree. C. The column was then washed
extensively with 10 mM Tris-HCl, pH 8.0, 150 mM NaCl before elution
with free FLAGS peptide at 25 .mu.g/ml in the same buffer. The
resulting material was used for the bioassays described below.
[0125] In order to detect the purified VEGFD.DELTA.N.DELTA.C,
fractions eluted from the M2 affinity column were subjected to
Western blot analysis. Aliquots of the column fractions were
combined with 2.times.SDS-PAGE sample buffer, boiled and loaded
onto a 15% SDS polyacrylamide gel. The resolved fractions were
transferred to nitrocellulose membrane and non-specific binding
sites blocked by incubation in Tris/NaCl/Tween 20 (TST) and 10%
skim milk powder (BLOTTO). Membranes were then incubated with
monoclonal antibody M2 or control antibody at 3 .mu.g/ml for 2 h at
room temperature, followed by extensive washing in TST. Membranes
were then incubated with a secondary goat anti-mouse HRP-conjugated
antiserum for 1 h at room temperature, followed by washing in TST
buffer. Detection of the protein species was achieved using a
chemiluminescent reagent (ECL, Amersham) (FIG. 12B).
[0126] Under non-reducing conditions a species of molecular weight
approximately 23 kD (VEGFD.DELTA.N.DELTA.C) was detected by the M2
antibody. This is consistent with the predicted molecular weight
for this internal fragment (12,800) plus N-linked glycosylation;
VEGFD.DELTA.N.DELTA.C contains two potential N-linked glycosylation
sites. A species of approximately 40 kD was also detected, and may
represent a non-covalent dimer of the 23 kD protein
(VEGFD.DELTA.N.DELTA.C).
[0127] Bioassays
[0128] The bioassay for the capacity of polypeptides to bind to
VEGF receptor-2 is described in detail in Example 7. Aliquots of
fractions eluted from the M2 affinity column, containing the
VEGFD.DELTA.N.DELTA.C protein, were diluted in medium and tested in
the VEGFR2 bioassay as previously described. Fraction #3 from the
affinity column, which was shown to contain the purified
VEGFD.DELTA.N.DELTA.C protein (FIG. 12B), demonstrated a clear
ability to induce proliferation of the bioassay cell line to a
dilution of 1/100 of the purified fraction (FIG. 13). In
comparison, the void volume of the affinity column (fraction #1)
showed no activity, whereas the original VEGFD.DELTA.N.DELTA.C
conditioned medium gave only weak activity.
[0129] The vascular permeability assay (Miles and Miles, 1952) is
described in brief in example 8. Aliquots of purified
VEGFD.DELTA.N.DELTA.C, and samples of the void volume from the M2
affinity column (negative control) were combined with medium and
injected intradermally into the skin of guinea pigs. The regions of
skin at the sites of injections were excised, and extravasated dye
was eluted. The absorbance of the extravasated dye at 620 nm
arising from injection of purified VEGFD.DELTA.N.DELTA.C was
0.131.+-.0.009. In comparison, the value for absorbance arising
from injection of a sample of the void volume was 0.092.+-.0.020.
Therefore VEGFD.DELTA.N.DELTA.C induced vascular permeability, but
the effect was only marginal.
[0130] Due to its ability to bind to VEGFR2 and its lower induction
of vascular permeability compared to full length VEGF-D,
VEGF-D.DELTA.N.DELTA.C may be said to relatively decrease the
induction of vascular permeability by VEGF-D through competitive
inhibition. In this sense, the VEGF-D.DELTA.N.DELTA.C fragment may
be thought of as an antagonist for VEGF-D as regards the induction
of vascular permeability.
SUMMARY
[0131] Two factors have led us to explore internal fragments of
VEGF-D for enhanced activity. Firstly, it is the central region of
VEGF-D which exhibits amino acid homology with all other members of
the VEGF family. Secondly, proteolytic processing which gives rise
to internal bioactive polypeptides occurs for other growth factors
such as PDGF-BB. In addition, the activity seen with the full
length VEGF-D protein in COS cells was lower than for the
corresponding conditioned medium from VEGF-A transfected COS
cells.
[0132] It was predicted that the mature VEGF-D sequence would be
derived from a fragment contained within residues 92-205, with
cleavage at FAA{circumflex over ( )}TFY and IIRR{circumflex over (
)}SIQI. Immunoprecipitation analysis of VEGF-DfullFLAG expressed in
COS cells produced species consistent with the internal proteolytic
cleavage of the VEGF-D polypeptide at these sites. Therefore a
truncated form of VEGF-D, with the N- and C-terminal regions
removed (VEGFD.DELTA.N.DELTA.C), was produced and expressed in COS
cells. This protein was identified and purified using the M2
antibody. The VEGFD.DELTA.N.DELTA.C protein was also detected by
the A2 antibody, which recognizes a peptide within the 92-205
fragment of VEGF-D (not shown). VEGFD.DELTA.N.DELTA.C was evaluated
by the VEGFR2 bioassay and the Miles vascular permeability assay,
and shown to bind to and activate the VEGFR2 receptor in a bioassay
designed to detect cross-linking of the VEGFR2 extracellular
domain. Induction of vascular permeability by this polypeptide in a
Miles assay was at best marginal, in contrast to the effect of
VEGF-A.
EXAMPLE 10
VEGF-D Binds to and Activates VEGFR-3
[0133] The human VEGF-D cDNA was cloned into baculovirus shuttle
vectors for the production of reconmbinant VEGF-D. In addition to
baculoviral shuttle vectors, which contained the unmodified VEGF-D
cDNA (referred to as "full length VEGF-D") two baculoviral shuttle
vectors were assembled, in which the VEGF-D cDNA was modified in
the following ways.
[0134] In one construct (referred to as "full length
VEGF-D-H.sub.6") a C-terminal histidine tag was added. In the other
construct the putative N- and C-terminal propeptides were removed,
the melittin signal peptide was fused in-frame to the N-terminus,
and a histidine tag was added to the C-terminus of the remaining
VEGF homology domain (referred to as
".DELTA.N.DELTA.c-MELsp-VEGF-D-H.sub.6").
[0135] For each of the three constructs baculoviral clones of two
or three independent transfections were amplified. The supernatant
of High Five (HF) cells was harvested 48 h post infection with high
titre virus stocks. The supernatant was adjusted to pH 7 with NaOH
and diluted with one volume of D-MEM (0.2% FCS).
[0136] The samples were tested for their ability to stimulate
tyrosine phosphorylation of VEGFR-3 (Flt4 receptor) on NIH3T3
cells, as described by Joukov et al, 1996. The supernatant of
uninfected cells and the supernatant of cells infected with the
short splice variant of VEGF-C, which does not stimulate tyrosine
phosphorylation of VEGFR-3, were used as negative controls. VEGF-C
modified in the same way as .DELTA.N.DELTA.C-melSP-VEGF-D-H.sub.6
was used as positive control. The results are shown in FIG. 14.
[0137] The appearance of new bands at 125 and 195 kD indicates
phosphorylation, and hence activation, of the receptor.
EXAMPLE 11
VEGF-D Binds to and Activates VEGFR-2
[0138] Modified and unmodified human VEGF-D cDNA was cloned into
baculovirus shuttle vectors for the production of recombinant
VEGF-D as described in Example 10.
[0139] For each of the three constructs full length VEGF-D, full
length VEGF-D-H.sub.6, and .DELTA.N.DELTA.C-melSP-VEGF-D-H.sub.6,
baculoviral clones of two or three independent transfections were
amplified. The supernatant of High Five (HF) cells was harvested 48
hours post infection with high titre virus stocks. The supernatant
was adjusted to pH 7 with NaOH and diluted with one volume of D-MEM
(0.2% FCS).
[0140] The supernatants conditioned with the histidine-tagged
proteins were tested for their ability to stimulate tyrosine
phosphorylation of the KDR receptor according to Joukov et al,
1996. KDR is the human homologue of flk1 (VEGFR-2).
[0141] The supernatant of uninfected cells and the supernatant of
cells infected with the VEGF-C 156S mutant, which does not
stimulate KDR, were used as negative controls. VEGF.sub.165 and
VEGF-C modified in the same way as ANAC-melSP-VEGF-D-H.sub.6 were
used as positive controls. The results are shown in FIG. 15.
[0142] The appearance of a new band at approximately 210 kD
indicates phosphorylation, and hence activation, of the
receptor.
EXAMPLE 12
Analysis of VEGF-D Gene Expression
[0143] In order to characterise the pattern of VEGF-D gene
expression in the human and in mouse embryos, VEGF-D cDNAs were
used as hybridization probes for Northern blot analysis of
polyadenylated human RNA and for in situ hybridization analysis
with mouse embryos.
[0144] Gene Expression in the Adult Human
[0145] A 1.1 kb fragment of the human VEGF-D cDNA shown in FIG. 4
(SEQ ID NO. 4) spanning from the EcoRV site to the 3'-terminus
(nucleotides 911 to 2029) was labelled with [.alpha.-.sup.32P] DATP
using the Megaprime DNA labelling system (Amersham) according to
manufacturer's instructions. This probe was used to screen human
multiple tissue northern blots (Clontech) by hybridization, also
according to manufacturer's instructions. These blots contained
polyadenylated RNA obtained from tissues of adult humans who were
apparently free of disease. Autoradiography with the labelled blots
revealed that VEGF-D mRNA was most abundant in heart, lung and
skeletal muscle. VEGF-D mRNA was of intermediate abundance in
spleen, ovary, small intestine and colon, and was of low abundance
in kidney, pancreas, thymus, prostate and testis. No VEGF-D mRNA
was detected in RNA from brain, placenta, liver or peripheral blood
leukocytes. In most of the tissues where VEGF-D mRNA was detected
the size of the transcript was 2.3 kb. The only exception was
skeletal muscle, where two VEGF-D transcripts of 2.3 kb and 2.8 kb
were detected. In skeletal muscle the 2.3 kb transcript was more
abundant than the 2.8 kb transcript.
[0146] Gene Expression in Mouse Embryos
[0147] In order to generate an antisense RNA probe for mouse VEGF-D
mRNA, the mouse VEGF-D2 cDNA shown in FIG. 7 (SEQ ID NO. 7) was
inserted into the transcription vector pBluescriptIIKS+
(Stratagene). The resulting plasmid was digested to completion with
the restriction endonuclease FokI and then used as template for an
in vitro transcription reaction with T3 RNA polymerase. This
transcription reaction gave rise to an antisense RNA probe for
VEGF-D mRNA which was complementary in sequence to the region of
the VEGF-D2 cDNA (FIG. 7) from the 3'-terminus to the FokI cleavage
site closest to the 3'-terminus (nucleotides 1135 to 700). This
antisense RNA probe was hybridized at high stringency with
paraffin-embedded tissue sections generated from mouse embryos at
post-coital day 15.5. Hybridization and washing were essentially as
described previously (Achen et al., 1995).
[0148] After washing and drying, slides were exposed to
autoradiography film for six days.
[0149] Development of the autoradiography film revealed that VEGF-D
mRNA is localised in the developing lung of post-coital day 15.5
embryos. The signal for VEGF-D mRNA in the lung was strong and
highly specific. Control hybridizations with sense probe gave no
detectable background in lung or any other tissue.
SUMMARY
[0150] The VEGF-D gene is broadly expressed in the adult human, but
is certainly not ubiquitously expressed. Strongest expression was
detected in heart, lung and skeletal muscle. In mouse embryos at
post-coital day 15.5, strong and specific expression of the VEGF-D
gene was detected in the lung. These data suggest that VEGF-D may
play a role in lung development, and that expression of the VEGF-D
gene in lung persists in the adult, at least in humans. Expression
of the gene in other tissues in the adult human suggests that
VEGF-D may fulfill other functions in other adult tissues.
EXAMPLE 13
VEGF-D is MitoQenic for Endothelial Cells
[0151] Some members of the VEGF family of proteins, namely VEGF-A
(Leung et al, 1989) and VEGF-B (olofsson et al, 1996), are
mitogenic for endothelial cells. In order to test the mitogenic
capacity of VEGFD.DELTA.N.DELTA.C for endothelial cells, this
protein was expressed and purified by affinity chromatography as
described in Example 9. Fraction #3, eluted from the M2 affinity
column, which contained VEGFD.DELTA.N.DELTA.C, was diluted 1 in 10
in cell culture medium containing 5% serum and applied to bovine
aortic endothelial cells (BAEs) which had been propagated in medium
containing 10% serum. The BAEs had been seeded in 24-well dishes at
a density of 10,000 cells per well the day before addition of
VEGFD.DELTA.N.DELTA.C, and 3 days after addition of this
polypeptide the cells were dissociated with trypsin and counted.
Purified VEGF-A was included in the experiment as positive control.
Results are shown in FIG. 16. The addition of fraction #3 to the
cell culture medium led to a 2.4-fold increase in the number of
BAEs after 3 days of incubation, a result which was comparable to
that obtained with VEGF-A. Clearly VEGFD.DELTA.N.DELTA.C is
mitogenic for endothelial cells.
EXAMPLE 14
Localization of the VEGF-D Gene on Human Chromosomes
[0152] In order to generate hybridization probes for localization
of the VEGF-D gene on human chromosomes, a human genomic DNA clone
for VEGF-D was isolated from a human genomic DNA library
(Clontech). The genomic library was screened by hybridization with
the human VEGF-D cDNA shown in FIG. 4, using standard methods
(Sambrook et al., 1989). One of the clones thus isolated was shown
to contain part of the VEGF-D gene by hybridization to numerous
oligonucleotides which were derived in sequence from the human
VEGF-D cDNA. A region of the genomic clone, approximately 13 kb in
size, was purified from agarose gel, labelled by nick-translation
with biotin-14-dATP and hybridized in situ at a final concentration
of 20 ng/.mu.l to metaphases from two normal human males. The
fluorescence in situ hybridization (FISH) method was modified from
that previously described (Callen et al, 1990) in that chromosomes
were stained before analysis with propidium iodide (as
counterstain) and DAPI (for chromosome identification). Images of
metaphase preparations were captured by a cooled CCD camera, using
the CytoVision Ultra image collection and enhancement system
(Applied Imaging Int. Ltd.). FISH signals and the DAPI banding
pattern were merged for analysis.
[0153] Fifteen metaphases from the first normal male were examined
for fluorescent signal. Ten of the metaphases showed signal on one
chromatid (3 cells) or both chromatids (7 cells) of the X
chromosome in band p22.1. There was a total of 9 non-specific
background dots observed in these 15 metaphases. A similar result
was obtained from hybridization of the probe to 15 metaphases from
the second normal male, where signal was observed at Xp22.1 on one
chromatid in 7 cells and on both chromatids in 4 cells. In
conclusion, the human VEGF-D gene is located on the X chromosome in
band p22.1.
EXAMPLE 15
Localization of the Murine VEGF-D Gene on Mouse Chromosomes
[0154] The mouse chromosomal location of the VEGF-D gene was
determined by interspecific backcross analysis using progeny
generated by mating (C57BL/6J.times.Mus spretus) F1 females and
CB7BL/67 males as described previously (Copeland and Jenkins,
1991). This interspecific backcross mapping panel has been typed
for over 2400 loci that are well distributed among all the
autosomes as well as the X chromosome (Copeland and Jenkins, 1991).
C57BL/6J and M. spretus DNAs were digested with several enzymes and
analyzed by Southern blot hybridization for informative restriction
fragment length polymorphisms (RFLPS) using a 1.3 kb mouse VEGF-D
cDNA probe essentially as described (Jenkins et al. 1982).
Fragments of 7.1, 6.3, 4.7, 2.5 and 2.2 kb were detected in
TaqI-digested C57BL/6J DNA and major fragments of 7.1, 3.7, 2.7 and
2.2 kb were detected in TaqI-digested M. spretus DNA. The presence
or absence of the 3.7 and 2.7 TaqI M. spretus-specific fragments,
which cosegregated, was followed in backcross mice. The mapping
results indicated that the VEGF-D gene is located in the distal
region of the mouse X chromosome linked to Bik, DxPasI and Ptmb4.
Although 89 mice were analyzed for every marker, up to 133 mice
were typed for some pairs of markers. Each locus was analyzed in
pairwise combinations for recombination frequencies using the
additional data. The ratios of the total number of mice exhibiting
recombinant chromosomes to the total number of mice analyzed for
each pair of loci and the most likely gene order are:
centromere-Btk-14/121-Dx- PasI-3/99-VEGF-D-5/133-Ptmb4. The
recombination frequencies [expressed as genetic distances in
centiMorgans (cM).+-.the standard error], calculated using Map
Manager (version 2.6.5), are-Btk-11.6+/-2.9-DxPasI-3.0+/-1.7-VE-
GF-D-3.8+/-1.7-Ptmb4. A description of the probes and RFLPs for the
loci linked to the VEGF-D gene, including Btk, DxPasI and Ptmb4,
has been reported previously (Hacfliger et al., 1992; Holloway et
al., 1997).
[0155] We have compared our interspecific map of the X chromosome
with a composite mouse linkage map that reports the map location of
many uncloned mutations (provided from Mouse Genome Database, a
computerized database maintained at The Jackson Library, Bar
Harbor, Me.). The VEGF-D gene mapped in a region of the composite
map that lacks mouse mutations with a phenotype that might be
expected for an alteration in the locus for an endothelial cell
mitogen. The distal region of the mouse X-chromosome shares a
region of homology with the short arm of the human X chromosomes
(Mouse Genome Database). The placement of the VEGF-D gene in this
interval in mouse suggests that the human homolog will map to Xp22.
This is consistent with our FISH analysis which has localized the
human gene to Xp22.1.
[0156] Numerous disease states are caused by mutations in unknown
genes which have been mapped to Xp22.1 and the positions
immediately surrounding this region in the human. These disease
states include Kallmann syndrome, ocular albinism (Nettleship-Falls
type), ocular albinism and sensorineural deafness, Partington
syndrome, spondyloepiphyseal dysplasia (late), retinitis pigmentosa
15, gonadal dysgenesis (XY female type), Nance-Horan
cataract-dental syndrome, retinoschisis, Charcot-Marie-Tooth
disease, F-cell production, hypomagnesemia, keratosis follicularis
spinulosa decalvans, Coffin-Lowry syndrome, corneal dermoids,
hypophosphatemia, agammaglobulinemia, Aicardi symdrome, hereditary
hypophosphatemia II, mental retardation (non-dysmorphic), Opitz G
syndrome, pigment disorder (reticulate), dosage-sensitive sex
reversal, adrenal hypoplasia, retinitis pigmentosa-6, deafness 4
(congenital sensorineural) and Wilson-Turner syndrome. The
positions of the genes involved in these disease states are
documented in the OMIM gene map which is edited by Dr. Victor
McKusick and colleagues at Johns Hopkins University (USA).
[0157] Bioassays to Determine the Function of VEGF-D
[0158] Other assays are conducted to evaluate whether VEGF-D has
similar activities to VEGF in relation to endothelial cell
function, angiogenesis and wound healing. Further assays may also
be performed, depending on the results of receptor binding
distribution studies.
[0159] I. Assays of Endothelial Cell Function
[0160] a) Endothelial Cell Proliferation
[0161] Endothelial cell growth assays are performed by methods well
known in the art, eg. those of Ferrara & Henzel (1989),
Gospodarowicz et al (1989), and/or Claffey et al, Biochim. Biophys.
Acta, 1995 1246 1-9.
[0162] b) Cell Adhesion Assay
[0163] The effect of VEGF-D on adhesion of polmor-phonuclear
granulocytes to endothelial cells is tested.
[0164] c) Chemotaxis
[0165] The standard Boyden chamber chemotaxis assay is used to test
the effect of VEGF-D on chemotaxis.
[0166] d) Plasminogen Activator Assay
[0167] Endothelial cells are tested for the effect of VEGF-D on
plasminogen activator and plasminogen activator inhibitor
production, using the method of Pepper et al (1991).
[0168] e) Endothelial cell Migration assay
[0169] The ability of VEGF-D to stimulate endothelial cells to
migrate and form tubes is assayed as described in Montesano et al
(1986). Alternatively, the three-dimensional collagen gel assay
described by Joukov et al (1996) or a gelatinized membrane in a
modified Boyden chamber (Glaser et al, 1980) may be used.
[0170] II Angiogenesis Assay
[0171] The ability of VEGF-D to induce an angiogenic response in
chick clorioallantoic membrane is tested as described in Leung et
al (1989). Alternatively the rat cornea assay of Rastinejad et al
(1989) may be used; this is an accepted method for assay of in vivo
angiogenesis, and the results are readily transferrable to other in
vivo systems.
[0172] III Wound Healing
[0173] The ability of VEGF-D to stimulate wound healing is tested
in the most clinically relevant model available, as described in
Schilling et al (1959) and utilised by Hunt et al (1967).
[0174] IV The Haemopoietic System
[0175] A variety of in vitro and in vivo assays using specific cell
populations of the haemopoietic system are known in the art, and
are outlined below. In particular a variety of in vitro murine stem
cell assays using fluorescence-activated cell sorter purified cells
are particularly convenient:
[0176] a) Repopulating Stem Cells
[0177] These are cells capable of repopulating the bone marrow of
lethally irradiated mice, and have the Lin.sup.-, Rh.sup.h1,
Ly-6A/E.sup.+, c-kit.sup.+ phenotype. VEGF-D is tested on these
cells either alone, or by co-incubation with other factors,
followed by measurement of cellular proliferation by
.sup.3H-thymidine incorporation.
[0178] b) Late Stage Stem Cells
[0179] These are cells that have comparatively little bone marrow
repopulating ability, but can generate D13 CFU-S. These cells have
the Lin.sup.-, Rh.sup.h1, Ly-6A/E.sup.+, c-kit.sup.+ phenotype.
VEGF-D is incubated with these cells for a period of time, injected
into lethally irradiated recipients, and the number of D13 spleen
colonies enumerated.
[0180] c) Progenitor-Enriched Cells
[0181] These are cells that respond in vitro to single growth
factors and have the Lin.sup.-, Rh.sup.h1, Ly-6A/E.sup.+,
c-kit.sup.+ phenotype. This assay will show if VEGF-D can act
directly on haemopoietic progenitor cells. VEGF-D is incubated with
these cells in agar cultures, and the number of colonies present
after 7-14 days is counted.
[0182] V Atherosclerosis
[0183] Smooth muscle cells play a crucial role in the development
or initiation of atherosclerosis, requiring a change of their
phenotype from a contractile to a synthetic state. Macrophages,
endothelial cells, T lymphocytes and platelets all play a role in
the development of atherosclerotic plaques by influencing the
growth and phenotypic modulations of smooth muscle cell. An in
vitro assay using a modified Rose chamber in which different cell
types are seeded on to opposite coverslips measures the
proliferative rate and phenotypic modulations of smooth muscle
cells in a multicellular environment, and is used to assess the
effect of VEGF-D on smooth muscle cells.
[0184] VI Metastasis
[0185] The ability of VEGF-D to inhibit metastasis is assayed using
the Lewis lung carcinoma model, for example using the method of Cao
et al (1995).
[0186] VII VEGF-D in Other Cell Types
[0187] The effects of VEGF-D on proliferation, differentiation and
function of other cell types, such as liver cells, cardiac muscle
and other cells, endocrine cells and osteoblasts can readily be
assayed by methods known in the art, such as .sup.3H-thymidine
uptake by in vitro cultures. Expression of VEGF-D in these and
other tissues can be measured by techniques such as Northern
blotting and hybridization or by in situ hybridization.
[0188] VIII Construction of VEQF-D Variants and Analogues
[0189] VEGF-D is a member of the PDGF family of growth factors
which exhibits a high degree of homology to the other members of
the PDGF family. VEGF-D contains eight conserved cysteine residues
which are characteristic of this family of growth factors. These
conserved cysteine residues form intra-chain disulfide bonds which
produce the cysteine knot structure, and inter-chain disulfide
bonds that form the protein dimers which are characteristic of
members of the PDGF family of growth factors. VEGF-D will interact
with protein tyrosine kinase growth factor receptors.
[0190] In contrast to proteins where little or nothing is known
about the protein structure and active sites needed for receptor
binding and consequent activity, the design of active mutants of
VEGF-D is greatly facilitated by the fact that a great deal is
known about the active sites and important amino acids of the
members of the PDGF family of growth factors.
[0191] Published articles elucidating the structure/activity
relationships of members of the PDGF family of growth factors
include for PDGF: Oestman et al, J. Biol. Chem., 1991 266
10073-10077; Andersson et al, J. Biol. Chem., 1992 267 11260-1266;
Oefner et al, EMBO J., 1992 11 3921-3926; Flemming et al, Molecular
and Cell Biol., 1993 13 4066-4076 and Andersson et al, Growth
Factors, 1995 12 159-164; and for VEGF: Kim et al, Growth Factors,
1992 7 53-64; Potgens et al, J. Biol. Chem., 1994 269 32879-32885
and Claffey et al, Biochem. Biophys. Acta, 1995 1246 1-9. From
these publications it is apparent that because of the eight
conserved cysteine residues, the members of the PDGF family of
growth factors exhibit a characteristic knotted folding structure
and dimerization, which result in formation of three exposed loop
regions at each end of the dimerized molecule, at which the active
receptor binding sites can be expected to be located.
[0192] Based on this information, a person skilled in the
biotechnology arts can design VEGF-D mutants with a very high
probability of retaining VEGF-D activity by conserving the eight
cysteine residues responsible for the knotted folding arrangement
and for dimerization, and also by conserving, or making only
conservative amino acid substitutions in the likely receptor
sequences in the loop 1, loop 2 and loop 3 region of the protein
structure.
[0193] The formation of desired mutations at specifically targeted
sites in a protein structure is considered to be a standard
technique in the arsenal of the protein chemist (Kunkel et al,
Methods in Enzymol., 1987 154 367-382). Examples of such
site-directed mutagenesis with VEGF can be found in Potgens et al,
J. Biol. Chem., 1994 269 32879-32885 and Claffey et al, Biochim.
Biophys. Acta, 1995 1246 1-9. Indeed, site-directed mutagenesis is
so common that kits are commercially available to facilitate such
procedures (eg. Promega 1994-1995 Catalog., Pages 142-145).
[0194] The endothelial cell proliferating activity of VEGF-D
mutants can be readily confirmed by well established screening
procedures. For example, a procedure analogous to the endothelial
cell mitotic assay described by Claffey et al, (Biochim. Biophys.
Acta., 1995 1246 1-9) can be used. Similarly the effects of VEGF-D
on proliferation of other cell types, on cellular differentiation
and on human metastasis can be tested using methods which are well
known in the art.
[0195] It will be apparent to the person skilled in the art that
while the invention has been described in some detail for the
purposes of clarity and understanding, various modifications and
alterations to the embodiments and methods described herein may be
made without departing from the scope of the inventive concept
disclosed in this specification.
[0196] References cited herein are listed on the following pages,
and are incorporated herein by this reference.
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Sequence CWU 1
1
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