U.S. patent application number 12/148656 was filed with the patent office on 2008-10-23 for antibodies to pdgf-d.
Invention is credited to Kari Alitalo, Ulf Eriksson, Marko Uutela.
Application Number | 20080261888 12/148656 |
Document ID | / |
Family ID | 46281284 |
Filed Date | 2008-10-23 |
United States Patent
Application |
20080261888 |
Kind Code |
A1 |
Uutela; Marko ; et
al. |
October 23, 2008 |
Antibodies to PDGF-D
Abstract
PDGF-D, a new member of the PDGF/VEGF family of growth factors,
as well as the nucleotide sequence encoding it, methods for
producing it, antibodies and other antagonists to it, transfected
and transformed host cells expressing it, pharmaceutical
compositions containing it, and uses thereof in medical and
diagnostic applications, including methods for stimulating growth
of a connective tissue or healing a wound in a mammal, which
methods comprise administering to the mammal an effective amount of
PDGF-D polypeptides or polynucleotides encoding the PDGF-D
polypeptides.
Inventors: |
Uutela; Marko; (US) ;
Eriksson; Ulf; (US) ; Alitalo; Kari;
(US) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
666 FIFTH AVE
NEW YORK
NY
10103-3198
US
|
Family ID: |
46281284 |
Appl. No.: |
12/148656 |
Filed: |
April 21, 2008 |
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Current U.S.
Class: |
514/8.2 ;
435/375 |
Current CPC
Class: |
C12Q 2600/158 20130101;
A61P 43/00 20180101; C07K 14/49 20130101; A61K 38/00 20130101; C12Q
1/6886 20130101; C07K 16/22 20130101 |
Class at
Publication: |
514/12 ;
435/375 |
International
Class: |
A61K 38/00 20060101
A61K038/00; C12N 5/06 20060101 C12N005/06; A61P 43/00 20060101
A61P043/00 |
Claims
1-31. (canceled)
32. A method for stimulating proliferation of a cell which
expresses a PDGF-D receptor, comprising contacting said cell with a
polypeptide comprising the amino acid sequence of SEQ ID NO: 25, in
an amount sufficient to stimulate said proliferation.
33. The method of claim 32, wherein said polypeptide comprises SEQ
ID NO: 8.
34. The method of claim 32, wherein said cell is a vascular
endothelial cell, a lymphatic endothelial cell, a connective tissue
cell, a myofibroblast, or a glial cell.
35. The method of claim 32, wherein said stimulation of a cell
causes angiogenesis.
36. The method of claim 32, further comprising directly delivering
said polypeptide to a site where proliferation of a cell is
desired.
37. The method of claim 35, wherein said direct delivery comprises
injection.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
10/260,539, filed Oct. 1, 2003, which in turn is a
continuation-in-part of U.S. application Ser. No. 10/086,623, filed
Mar. 4, 2002, which is a continuation-in-part of U.S. application
Ser. No. 09/691,200, filed Oct. 19, 2000, now abandoned, which is a
continuation-in-part of U.S. application Ser. No. 09/438,046, filed
Nov. 10, 1999, now U.S. Pat. No. 6,706,687, and claims the benefit
of U.S. Provisional Application No. 60/107,852, filed Nov. 10,
1998; U.S. Provisional Application No. 60/113,997, filed Dec. 28,
1998; U.S. Provisional Application No. 60/150,604, filed Aug. 26,
1999; U.S. Provisional Application No. 60/157,108, filed Oct. 4,
1999; and U.S. Provisional Application No. 60/157,756, filed Oct.
5, 1999.
FIELD OF THE INVENTION
[0002] This invention relates to growth factors for cells
expressing receptors to a novel growth factor that include
endothelial cells, connective tissue cells (such as fibroblasts)
myofibroblasts and glial cells, and in particular to a novel
platelet-derived growth factor/vascular endothelial growth
factor-like growth factor, polynucleotide sequences encoding the
factor, and to pharmaceutical and diagnostic compositions and
methods utilizing the factor for stimulating connective tissue
growth or promoting wound healing.
BACKGROUND OF THE INVENTION
[0003] In the developing embryo, the primary vascular network is
established by in situ differentiation of mesodermal cells in a
process called vasculogenesis. It is believed that all subsequent
processes involving the generation of new vessels in the embryo and
neovascularization in adults, are governed by the sprouting or
splitting of new capillaries from the pre-existing vasculature in a
process called angiogenesis (Pepper et al., 1996, Enzyme &
Protein, 49:38-162; Breier et al., 1995, Dev. Dyn., 204:228-239;
Risau, 1997, Nature, 386:671-674). 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 tumor 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] The angiogenic process is highly complex and involves the
maintenance of the endothelial cells in the cell cycle, degradation
of the extracellular matrix, migration and invasion of the
surrounding tissue and finally, tube formation. The molecular
mechanisms underlying the complex angiogenic processes are far from
being understood.
[0006] 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., 1992, J Biol. Chem., 267:10931-10934
for a review.
[0007] It has been suggested that a particular family of
endothelial cell-specific growth factors, the vascular endothelial
growth factors (VEGFs), and their corresponding receptors are
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 primarily via endothelial receptor tyrosine
kinases (RTKs).
[0008] Eight different proteins have been identified in the PDGF
family, namely two PDGFs (A and B), VEGF and five members that are
closely related to VEGF. The five members closely related to VEGF
are: VEGF-B, described in International Patent Application
PCT/US96/02957 (WO 96/26736) which corresponds to U.S. Pat. No.
5,928,939, and in U.S. Pat. Nos. 5,840,693 and 5,607,918 to Ludwig
Institute for Cancer Research and The University of Helsinki;
VEGF-C or VEGF-2, described in Joukov et al., 1996, EMBO J.,
15:290-298 and Lee et al., 1996, Proc. Natl. Acad. Sci. USA,
93:1988-1992, and U.S. Pat. Nos. 5,932,540, 5,935,820 and
6,040,157; VEGF-D, described in International Patent Application
No. PCT/US97/14696 (WO 98/07832), and Achen et al., 1998, Proc.
Natl. Acad. Sci. USA, 95:548-553; the placenta growth factor
(PIGF), described in Maglione et al., 1991, Proc. Natl. Acad. Sci.
USA, 88:9267-9271; and VEGF3, described in International Patent
Application Nos. PCT/US95/07283 (WO 96/39421) and PCT/US99/18054
(WO 00/09148) by Human Genome Sciences, Inc. Each VEGF family
member has between 30% and 45% amino acid sequence identity with
VEGF. The VEGF family members share a VEGF homology domain which
contains the six cysteine residues which form the cysteine knot
motif. Functional characteristics of the VEGF family include
varying degrees of mitogenicity for endothelial cells, induction of
vascular permeability and angiogenic and lymphangiogenic
properties.
[0009] Vascular endothelial growth factor (VEGF) is a homodimeric
glycoprotein that has been isolated from several sources. VEGF
shows highly specific mitogenic activity for endothelial cells.
VEGF has important regulatory functions in the formation of new
blood vessels during embryonic vasculogenesis and in angiogenesis
during adult life (Carmeliet et al., 1996, Nature, 380:435-439;
Ferrara et al., 1996, Nature, 380:439-442; reviewed in Ferrara and
Davis-Smyth, 1997, Endocrine Rev., 18:4-25). The significance of
the role played by VEGF has been demonstrated in studies showing
that inactivation of a single VEGF allele results in embryonic
lethality due to failed development of the vasculature (Carmeliet
et al., 1996, Nature, 380:435-439; Ferrara et al., 1996, Nature,
380:439-442). In addition VEGF has strong chemoattractant activity
towards monocytes, can induce the plasminogen activator and the
plasminogen activator inhibitor in endothelial cells, and can also
induce microvascular permeability. Because of the latter activity,
it is sometimes referred to as vascular permeability factor (VPF).
The isolation and properties of VEGF have been reviewed; see
Ferrara et al., 1991, J. Cellular Biochem., 47:211-218 and
Connolly, J., 1991, Cellular Biochem., 47:219-223. Alterative mRNA
splicing of a single VEGF gene gives rise to five isoforms of
VEGF.
[0010] VEGF-B has 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.
[0011] VEGF-B was isolated using a yeast co-hybrid interaction trap
screening technique by screening for cellular proteins which might
interact with cellular retinoid acid-binding protein type I
(CRABP-I). Its isolation and characteristics are described in
detail in PCT/US96/02957 and in Olofsson et al., 1996, Proc. Natl.
Acad. Sci. USA, 93:2576-2581.
[0012] VEGF-C was isolated from conditioned media of the 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 VEGFR-3 (Flt4),
using cells transfected to express VEGFR-3. VEGF-C was purified
using affinity chromatography with recombinant VEGFR-3, and was
cloned from a PC-3 cDNA library. Its isolation and characteristics
are described in detail in Joukov et al., 1996, EMBO J,
15:290-298.
[0013] VEGF-D was isolated from a human breast cDNA library,
commercially available from Clontech, by screening with an
expressed sequence tag obtained from a human cDNA library
designated "Soares Breast 3NbHBst" as a hybridization probe (Achen
et al., 1998, Proc. Natl. Acad. Sci. USA, 95:548-553). Its
isolation and characteristics are described in detail in
International Patent Application No. PCT/US97/14696
(W098/07832).
[0014] The VEGF-D gene is broadly expressed in the adult human, but
is certainly not ubiquitously expressed. VEGF-D is strongly
expressed in heart, lung and skeletal muscle. Intermediate levels
of VEGF-D are expressed in spleen, ovary, small intestine and
colon, and a lower expression occurs in kidney, pancreas, thymus,
prostate and testis. No VEGF-D mRNA was detected in RNA from brain,
placenta, liver or peripheral blood leukocytes.
[0015] PIGF was isolated from a term placenta cDNA library. Its
isolation and characteristics are described in detail in Maglione
et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9267-9271. Presently
its biological function is not well understood.
[0016] VEGF3 was isolated from a cDNA library derived from colon
tissue. VEGF3 is stated to have about 36% identity and 66%
similarity to VEGF. The method of isolation of the gene encoding
VEGF3 is unclear and no characterization of the biological activity
is disclosed.
[0017] Similarity between two proteins is determined by comparing
the amino acid sequence and conserved amino acid substitutions of
one of the proteins to the sequence of the second protein, whereas
identity is determined without including the conserved amino acid
substitutions.
[0018] PDGF/VEGF family members act primarily by binding to
receptor tyrosine kinases. Five endothelial cell-specific receptor
tyrosine kinases have been identified, namely VEGFR-1 (Flt-1),
VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt4), 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 VEGFR-1, VEGFR-2, VEGFR-3, Tie
and Tek/Tie-2 has been demonstrated by targeted mutations
inactivating these receptors in mouse embryos.
[0019] The only receptor tyrosine kinases known to bind VEGFs are
VEGFR-1, VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 bind VEGF with
high affinity, and VEGFR-1 also binds VEGF-B and PIGF. VEGF-C has
been shown to be the ligand for VEGFR-3, and it also activates
VEGFR-2 (Joukov et al., 1996, The EMBO Journal, 15:290-298). VEGF-D
binds to both VEGFR-2 and VEGFR-3. A ligand for Tek/Tie-2 has been
described in International Patent Application No. PCT/US95/12935
(WO 96/11269) by Regeneron Pharmaceuticals, Inc. The ligand for Tie
has not yet been identified.
[0020] Recently, a novel 130-135 kDa VEGF isoform specific receptor
has been purified and cloned (Soker et al., 1998, Cell,
92:735-745). The VEGF receptor was found to specifically bind the
VEGF.sub.165 isoform via the exon 7 encoded sequence, which shows
weak affinity for heparin (Soker et al., 1998, Cell, 92:735-745).
Surprisingly, the receptor was shown to be identical to human
neuropilin-1 (NP-1), a receptor involved in early stage
neuromorphogenesis. PIGF-2 also appears to interact with NP-1
(Migdal et al., 1998, J Biol. Chem., 273:22272-22278).
[0021] VEGFR-1, VEGFR-2 and VEGFR-3 are expressed differently by
endothelial cells. Both VEGFR-1 and VEGFR-2 are expressed in blood
vessel endothelia (Oelrichs et al., 1992, Oncogene, 8:11-18;
Kaipainen et al., 1993, J Exp. Med., 178:2077-2088; Dumont et al.,
1995, Dev. Dyn., 203:80-92; Fong et al., 1996, Dev. Dyn., 207:1-10)
and VEGFR-3 is mostly expressed in the lymphatic endothelium of
adult tissues (Kaipainen et al., 1995, Proc. Natl. Acad. Sci. USA,
9:3566-3570). VEGFR-3 is also expressed in the blood vasculature
surrounding tumors.
[0022] Disruption of the VEGFR genes results in aberrant
development of the vasculature leading to embryonic lethality
around midgestation. Analysis of embryos carrying a completely
inactivated VEGFR-1 gene suggests that this receptor is required
for functional organization of the endothelium (Fong et al., 1995,
Nature, 376:66-70). However, deletion of the intracellular tyrosine
kinase domain of VEGFR-1 generates viable mice with a normal
vasculature (Hiratsuka et al., 1998, Proc. Natl. Acad. Sci. USA,
95:9349-9354). The reasons underlying these differences remain to
be explained but suggest that receptor signalling via the tyrosine
kinase is not required for the proper function of VEGFR-1. Analysis
of homozygous mice with inactivated alleles of VEGFR-2 suggests
that this receptor is required for endothelial cell proliferation,
hematopoesis and vasculogenesis (Shalaby et al., 1995, Nature,
376:62-66; Shalaby et al., 1997, Cell, 89:981-990). Inactivation of
VEGFR-3 results in cardiovascular failure due to abnormal
organization of the large vessels (Dumont et al., 1998, Science,
282:946-949).
[0023] Although VEGFR-1 is mainly expressed in endothelial cells
during development, it can also be found in hematopoetic precursor
cells during early stages of embryogenesis (Fong et al., 1995,
Nature, 376:66-70). It is also is expressed by most, if not all,
vessels in embryos (Breier et al., 1995, Dev. Dyn., 204:228-239;
Fong et al., 1996, Dev. Dyn., 207:1-10). In adults, monocytes and
macrophages also express this receptor (Barleon et al., 1996,
Blood, 87:3336-3343).
[0024] The receptor VEGFR-3 is widely expressed on endothelial
cells during early embryonic development, but as embryogenesis
proceeds, it becomes restricted to venous endothelium and then to
the lymphatic endothelium (Kaipainen et al., 1994, Cancer Res.,
54:6571-6577; Kaipainen et al., 1995, Proc. Natl. Acad. Sci. USA,
92:3566-3570). VEGFR-3 continues to be expressed on lymphatic
endothelial cells in adults. This receptor is essential for
vascular development during embryogenesis. Targeted inactivation of
both copies of the VEGFR-3 gene in mice resulted in defective blood
vessel formation characterized by abnormally organized large
vessels with defective lumens, leading to fluid accumulation in the
pericardial cavity and cardiovascular failure at post-coital day
9.5. On the basis of these findings it has been proposed that
VEGFR-3 is required for the maturation of primary vascular networks
into larger blood vessels. However, the role of VEGFR-3 in the
development of the lymphatic vasculature could not be studied in
these mice because the embryos died before the lymphatic system
emerged. Nevertheless it is assumed that VEGFR-3 plays a role in
development of the lymphatic vasculature and lymphangiogenesis
given its specific expression in lymphatic endothelial cells during
embryogenesis and adult life. This is supported by the finding that
ectopic expression of VEGF-C, a ligand for VEGFR-3, in the skin of
transgenic mice, resulted in lymphatic endothelial cell
proliferation and vessel enlargement in the dermis. Furthermore
this suggests 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, EMBO J., 15:290-298).
[0025] Some inhibitors of the VEGF/VEGF-receptor system have been
shown to prevent tumor growth via an anti-angiogenic mechanism; see
Kim et al., 1993, Nature, 362:841-844 and Saleh et al., 1996,
Cancer Res., 56:393-401.
[0026] As mentioned above, the VEGF family of growth factors are
members of the PDGF family. PDGF plays an important role in the
growth and/or motility of connective tissue cells, fibroblasts,
myofibroblasts and glial cells (Heldin et al., "Structure of
platelet-derived growth factor: Implications for functional
properties", 1993, Growth Factor, 8:245-252). In adults, PDGF
stimulates wound healing (Robson et al., 1992, Lancet, 339:23-25).
Structurally, PDGF isoforms are disulfide-bonded dimers of
homologous A- and B-polypeptide chains, arranged as homodimers
(PDGF-AA and PDGF-BB) or a heterodimer (PDGF-AB).
[0027] PDGF isoforms exert their effects on target cells by binding
to two structurally related receptor tyrosine kinases (RTKs). The
alpha-receptor binds both the A- and B-chains of PDGF, whereas the
beta-receptor binds only the B-chain. These two receptors are
expressed by many cell lines grown in vitro, and are mainly
expressed by mesenchymal cells in vivo. The PDGFs regulate cell
proliferation, cell survival and chemotaxis of many cell types in
vitro (reviewed in Heldin et al., 1998, Biochim Biophys Acta.,
1378:F79-113). In vivo, they exert their effects in a paracrine
mode since they often are expressed in epithelial (PDGF-A) or
endothelial cells (PDGF-B) in close apposition to the PDGFR
expressing mesenchyme. In tumor cells and in cell lines grown in
vitro, coexpression of the PDGFs and the receptors generate
autocrine loops which are important for cellular transformation
(Betsholtz et al., 1984, Cell, 39:447-57; Keating et al., 1990, J.
R. Coll Surg Edinb., 35:172-4). Overexpression of the PDGFs have
been observed in several pathological conditions, including
malignancies, arteriosclerosis, and fibroproliferative diseases
(reviewed in Heldin et al., 1996, The Molecular and Cellular
Biology of Wound Repair, New York: Plenum Press, 249-273).
[0028] The importance of the PDGFs as regulators of cell
proliferation and survival are well illustrated by recent gene
targeting studies in mice that have shown distinct physiological
roles for the PDGFs and their receptors despite the overlapping
ligand specificities of the PDGFRs. Homozygous null mutations for
either of the two PDGF ligands or the receptors are lethal.
Approximately 50% of the homozygous PDGF-A deficient mice have an
early lethal phenotype, while the surviving animals have a complex
postnatal phenotype with lung emphysema due to improper alveolar
septum formation because of a lack of alveolar myofibroblasts
(Bostrom et al., 1996, Cell, 85:863-873). The PDGF-A deficient mice
also have a dermal phenotype characterized by thin dermis,
misshapen hair follicles and thin hair (Karlsson et al., 1999,
Development, 126:2611-2). PDGF-A is also required for normal
development of oligodendrocytes and subsequent myelination of the
central nervous system (Fruttiger et al., 1999, Development,
126:457-67). The phenotype of PDGFR-alpha deficient mice is more
severe with early embryonic death at E10, incomplete cephalic
closure, impaired neural crest development, cardiovascular defects,
skeletal defects, and edemas (Soriano et al., 1997, Development,
124:2691-70). The PDGF-B and PDGFR-beta deficient mice develop
similar phenotypes that are characterized by renal, hematological
and cardiovascular abnormalities (Leveen et al., 1994, Genes Dev.,
8:1875-1887; Soriano et al., 1994, Genes Dev., 8:1888-96; Lindahl
et al., 1997, Science, 277:242-5; Lindahl, 1998, Development,
125:3313-2), where the renal and cardiovascular defects, at least
in part, are due to the lack of proper recruitment of mural cells
(vascular smooth muscle cells, pericytes or mesangial cells) to
blood vessels (Leveen et al., 1994, Genes Dev., 8:1875-1887;
Lindahl et al., 1997, Science, 277:242-5; Lindahl et al., 1998,
Development, 125:3313-2).
[0029] Most recently, an additional member of the PDGF/VEGF family
of growth factors was identified, PDGF-C. PDGF-C is described in
International Patent Application PCT/US99/22668 (WO 00/18212),
filed Sept. 30, 1999. PDGF-C has a two-domain structure not
previously recognized within this family of growth factors, a
N-terminal C1r/C1s/embryonic sea urchin protein Uegf/bone
morphogenetic protein 1 (CUB) domain, and a C-terminal PDGF/VEGF
homology domain (P/VHD). The structure of the P/VHD in PDGF-C shows
a low overall sequence identity with other PDGFNEGF homology
domains, although the eight invariant cysteine residues involved in
inter- and intra-molecular disulfide bond formation are present.
The cysteine spacing in the central, most conserved region of this
domain is different from other PDGF/VEGF domains, with an insertion
of three amino acid residues. Despite the fact that the insertion
occurs close to the loop 2 region which has been proposed to be
involved in receptor binding, it was shown that this domain of
PDGF-CC binds PDGFR-alpha with almost identical affinities as
homodimers of PDGF-A or -B chains. In addition, four extra cysteine
residues are present in this domain. Full length and truncated
PDGF-CC were found not to bind to VEGFR-1,-2 or -3, or to
PDGFR-beta.
[0030] PDGF-C requires proteolytic removal of the N-terminal CUB
domain for receptor binding and activation of the receptor. This
indicates that the CUB domains are likely to sterically block the
receptor binding epitopes of the unprocessed dimer. The in vitro
and in vivo proteolytically processed proteins are devoid of
N-terminal portions corresponding to more than 14-16 kDa as
determined from SDS-PAGE analysis which is consistent with a loss
of the 110 amino acid long CUB domain and a part of the hinge
region between the CUB and core domains that vary in length.
[0031] PDGF-C is not proteolytically processed during secretion in
transfected COS cells indicating that proteolytic removal of the
CUB domain occurs extracellularly, and not during secretion. This
is in contrast to PDGF-A and -B (Ostman et al., 1992, J. Cell.
Biol., 118:509-519) which appear to be processed intracellularly by
furin-like endoproteases (Nakayama et al., 1997, Biochem J.,
327:625-635).
[0032] Northern blots show PDGF-C mRNA in a variety of human
tissues, including heart, liver, kidney, pancreas and ovary.
[0033] In situ localization studies demonstrate expression of
PDGF-C in certain epithelial structures, and PDGFR-alpha in
adjacent mesenchyme, indicating the potential of paracrine
signaling in the developing embryo. PDGF-C expression seems
particularly abundant at sites of ongoing ductal morphogenesis,
indicating a role of the factor in connective tissue remodeling at
these sites. The expression pattern is distinct from that of PDGF-A
or PDGF-B indicating that the three growth factors have different
roles despite their similar PDGFR-alpha binding and signaling
activities. This is illustrated by the mouse embryonic kidney, in
which PDGF-C is expressed in early aggregates of metanephric
mesenchyme undergoing epithelial conversion, whereas PDGF-A is
expressed in more mature tubular structures, and PDGF-B by vascular
endothelial cells. PDGFR-alpha is expressed in the mesenchyme of
the kidney cortex, adjacent to the sites of PDGF-C expression,
indicating that this mesenchyme may be targeted specifically by
PDGF-C. Indeed, PDGFR-alpha -/- mouse embryos show an extensive
loss of the cortical mesenchyme adjacent to sites of PDGF-C
expression, not seen in PDGF-A -/- mice or in PDGF-A/B -/- mice,
indicating that PDGF-C has an essential role in the development of
kidney mesenchyme.
SUMMARY OF THE INVENTION
[0034] The invention generally provides an isolated novel growth
factor, PDGF-D, a polypeptide that has the ability to stimulate, or
enhance, or both, one or more of proliferation, differentiation,
growth, and motility of cells expressing a PDGF-D receptor. The
cells affected by the inventive growth factor include, but are not
limited to, endothelial cells, connective tissue cells,
myofibroblasts and glial cells. The invention also provides
isolated polynucleotide molecules encoding the novel growth factor,
and compositions useful for diagnostic and/or therapeutic
applications.
[0035] According to one aspect, the invention provides an isolated
nucleic acid molecule which comprises a polynucleotide sequence
having at least 85% identity, more preferably at least 90%, and
still more preferably at least 95% identity, and most preferably at
100% identity to at least nucleotides 1 to 600 of the sequence set
out SEQ ID NO:3, at least nucleotides 1 to 966 of the sequence set
out in SEQ ID NO:5, at least nucleotides 176 to 1285 of the
sequence set out in SEQ ID NO:7, at least nucleotides 935 to 1285
set out in SEQ ID NO:7, at least nucleotides 1 to 1110 of SEQ ID
NO:35, at least nucleotides 1-1092 of SEQ ID NO:37, or SEQ ID
NO:39. The sequence of at least nucleotides 1 to 600 of the
sequence set out in FIG. 3 (SEQ ID NO:3) or at least nucleotides 1
to 966 of the sequence set out in FIG. 5 (SEQ ID NO:5) encodes a
5'-truncated polypeptide, designated PDGF-D (formerly designated
"VEGF-G"), while at least nucleotides 176 to 1285 of the sequence
set out in FIG. 7 (SEQ ID NO:7) encodes a full-length PDGF-D. The
sequence of at least nucleotides 1 to 1110 of SEQ ID NO:35 encodes
a murine PDGF-D, while the sequence of at least nucleotides 1-1092
of SEQ ID NO:37 encodes an identical protein as SEQ ID NO:35 except
for a six amino acid residue gap (a.a. #42-47) from the region
between the signal sequence and the CUB domain (see below for
details) , and SEQ ID NO:39 a C-terminal truncated protein of the
polypeptide encoded by SEQ ID NO:35. The PDGF-D polynucleotide of
the invention can be a naked polynucleotide and/or in a vector or
liposome.
[0036] PDGF-D is structurally homologous to PDGF-A, PDGF-B, VEGF,
VEGF-B, VEGF-C and VEGF-D. The sequence of at least nucleotides 935
to 1285 set out in FIG. 7 (SEQ ID NO:7) encodes a portion of the
PDGF/VEGF homology domain, which is the bioactive fragment of
PDGF-D. This bioactive fragment would also be encoded by the
sequence of at least nucleotides 1 to 600 of the sequence set out
in FIG. 3 (SEQ ID NO:3) or at least nucleotides 1 to 966 of the
sequence set out in FIG. 5 (SEQ ID NO:5).
[0037] According to a second aspect, the PDGF-D polypeptide of the
invention has the ability to stimulate and/or enhance proliferation
and/or differentiation and/or growth and/or motility of cells
expressing a PDGF-D receptor including, but not limited to,
endothelial cells, connective tissue cells, myofibroblasts and
glial cells and comprises a sequence of amino acids having at least
85% identity, more preferably at least 90%, and still more
preferably at least 95% identity, and most preferably at 100%
identity to the amino acid sequence set out in SEQ ID NOs:4, 6, 8,
36, 38 or 40, or a fragment or analog thereof which has PDGF-D
activity.
[0038] A preferred fragment is a truncated form of PDGF-D
comprising a portion of the PDGF/VEGF homology domain (PVHD) of
PDGF-D. The portion of the PVHD is from residues 254-370 of FIG. 8
(SEQ ID NO:8) where the putative proteolytic processing site RKSK
starts at amino acid residue 254 (SEQ ID NO:8). However, the PVHD
extends toward the N terminus up to residue 234 of FIG. 8 (SEQ ID
NO:8). Herein the PVHD is defined as truncated PDGF-D. The
truncated PDGF-D is the putative activated form of PDGF-D.
[0039] Another preferred fragment is a truncated form of PDGF-D
comprising only the CUB domain, as exemplified by the sequence set
forth in SEQ ID NO:40. There may exist PDGF-D receptors, other than
PDGFR-beta, that bind to the unprocessed or un-cleaved factor
(CUB+PDGF-homology domain). The CUB domain alone may bind to these
receptors and would prevent activation of said receptors by
blocking the receptors from binding to un-cleaved factors.
[0040] As used in this application, percent sequence identity is
determined by using the alignment tool of "MEGALIGN" from the
Lasergene package (DNASTAR, Ltd. Abacus House, Manor Road, West
Ealing, London W130AS United Kingdom). The MEGALIGN is based on the
J. Hein method (Methods in Enzymology, 1990 183 626-645). The PAM
250 residue weight table is used with a gap penalty of eleven and a
gap length penalty of three and a K-tuple value of two in the
pairwise alignments. The alignment is then refined manually, and
the number of identities are estimated in the regions available for
a comparison.
[0041] Preferably the polypeptide has the ability to stimulate one
or more of proliferation, differentiation, motility, survival or
vascular permeability of cells expressing a PDGF-D receptor
including, but not limited to, vascular endothelial cells,
lymphatic endothelial cells, connective tissue cells (such as
fibroblasts), myofibroblasts and glial cells. Preferably the
polypeptide has the ability to stimulate wound healing. PDGF-D also
has antagonistic effects on cells. For example, an antagonistic
PDGF-D variant would be a partial PDGF-D molecule containing one
intact full-length chain and one processed chain as a
disulphide-linked dimer. In principle such a molecule would be
monovalent and bind to single PDGFR-beta receptors, but prevent
their dimerization thereby blocking signal transduction. These
antagonistic activities are also included in the biological
activities of PDGF-D. Collectively, both the stimulating and
antagonistic abilities are referred to hereinafter as "biological
activities of PDGF-D" and can be readily tested by methods known in
the art.
[0042] In another preferred aspect, the invention provides a
polypeptide comprising an amino acid sequence:
TABLE-US-00001 PXCLLVXRCGGNCGC (SEQ ID NO: 25)
which is unique to PDGF-D and differs from the other members of the
PDGF/VEGF family of growth factors because of the insertion of the
three amino acid residues (NCG) between the third and fourth
cysteines (see FIG. 9).
[0043] Polypeptides comprising conservative substitutions,
insertions, or deletions, but which still retain a biological
activity of PDGF-D are within the scope of the invention. Persons
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 enzymatic 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
analog may retain the required aspects of the biological activity
of PDGF-D. Such compounds can readily be made and tested for their
ability to show the biological activity of PDGF-D by routine
activity assay procedures such as the fibroblast proliferation
assay and are also within the scope of the invention.
[0044] In addition, possible variant forms of the PDGF-D
polypeptide which may result from alternative splicing, as are
known to occur with VEGF and VEGF-B, and naturally-occurring
allelic variants of the nucleic acid sequence encoding PDGF-D are
within the scope of the invention. Examples of such a variant
include the polypeptides set forth in SEQ ID NOs: 38 and 40.
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.
[0045] Such variant forms of PDGF-D can be prepared by targeting
non-essential regions of the PDGF-D polypeptide for modification.
These non-essential regions are expected to fall outside the
strongly-conserved regions indicated in FIG. 9 (SEQ ID NOs:8 and
32). In particular, the growth factors of the PDGF family,
including PDGF-D, are dimeric. PDGF-D differs slightly from VEGF,
VEGF-B, VEGF-C, VEGF-D, P1GF, PDGF-A and PDGF-B because it shows
complete conservation of only seven of the eight cysteine residues
in the PVHD (Olofsson et al., 1996, Proc. Natl Acad. Sci. USA,
93:2576-2581; Joukov et al., 1996, EMBO J, 15:290-298). These
cysteines are thought to be involved in intra- and inter-molecular
disulfide bonding. Loops 1, 2 and 3 of each subunit, which are
formed by intra-molecular disulfide bonding, are involved in
binding to the receptors for the PDGF/VEGF family of growth factors
(Andersson et al., 1995, Growth Factors, 12:159-164).
[0046] Persons ordinarily skilled in the art thus are well aware
that these cysteine residues generally should be preserved 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 PDGF-D by routine activity assay procedures such as the
fibroblast proliferation assay.
[0047] It is contemplated that some modified PDGF-D polypeptides
will have the ability to bind to PDGF-D receptors on cells
including, but not limited to, endothelial cells, connective tissue
cells, myofibroblasts and/or glial cells, but will be unable to
stimulate cell proliferation, differentiation, migration, motility
or survival or to induce vascular proliferation, connective tissue
development or wound healing. These modified polypeptides are
expected to be able to act as competitive or non-competitive
inhibitors of the PDGF-D polypeptides and growth factors of the
PDGF/VEGF family, and to be useful in situations where prevention
or reduction of the PDGF-D polypeptide or PDGF/VEGF family growth
factor action is desirable. Thus such receptor-binding but
non-mitogenic, non-differentiation inducing, non-migration
inducing, non-motility inducing, non-survival promoting,
non-connective tissue promoting, non-wound healing or non-vascular
proliferation inducing variants of the PDGF-D polypeptide are also
within the scope of the invention, and are referred to herein as
"receptor-binding but otherwise inactive variants." Because PDGF-D
forms a dimer in order to activate its only known receptor, it is
contemplated that one monomer comprises the receptor-binding but
otherwise inactive variant modified PDGF-D polypeptide and a second
monomer comprises a wild-type PDGF-D or a wild-type growth factor
of the PDGF/VEGF family. These dimers can bind to its corresponding
receptor but cannot induce downstream signaling.
[0048] It is also contemplated that there are other modified PDGF-D
polypeptides that can prevent binding of a wild-type PDGF-D or a
wild-type growth factor of the PDGF/VEGF family to its
corresponding receptor on cells including, but not limited to,
endothelial cells, connective tissue cells (such as fibroblasts),
myofibroblasts and/or glial cells. Thus these dimers will be unable
to stimulate endothelial cell proliferation, differentiation,
migration, survival, or induce vascular permeability, and/or
stimulate proliferation and/or differentiation and/or motility of
connective tissue cells, myofibroblasts or glial cells. These
modified polypeptides are expected to be able to act as competitive
or non-competitive inhibitors of the PDGF-D growth factor or a
growth factor of the PDGF/VEGF family, and to be useful in
situations where prevention or reduction of the PDGF-D growth
factor or PDGF/VEGF family growth factor action is desirable. Such
situations include the tissue remodeling that takes place during
invasion of tumor cells into a normal cell population by primary or
metastatic tumor formation. Thus such PDGF-D or PDGF/VEGF family
growth factor-binding but non-mitogenic, non-differentiation
inducing, non-migration inducing, non-motility inducing,
non-survival promoting, non-connective tissue promoting, non-wound
healing or non-vascular proliferation inducing variants of the
PDGF-D growth factor are also within the scope of the invention,
and are referred to herein as "the PDGF-D growth factor-dimer
forming but otherwise inactive or interfering variants."
[0049] An example of a PDGF-D growth factor-dimer forming but
otherwise inactive or interfering variant is where the PDGF-D has a
mutation which prevents cleavage of CUB domain from the protein. It
is further contemplated that a PDGF-D growth factor-dimer forming
but otherwise inactive or interfering variant could be made to
comprise a monomer, preferably a monomer whose own N-terminal CUB
domain has been removed (hereinafter a "CUB-removed monomer") of
VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF-A, PDGF-B, PDGF-C, PDGF-D or
P1GF linked to a CUB domain that has a mutation which prevents
cleavage of CUB domain from the protein. Dimers formed with the
above mentioned PDGF-D growth factor-dimer forming but otherwise
inactive or interfering variants and the monomers linked to the
mutant CUB domain would be unable to bind to their corresponding
receptors.
[0050] A variation on this contemplation would be to insert a
proteolytic site between a CUB-removed monomer of VEGF, VEGF-B,
VEGF-C, VEGF-D, PDGF-A, PDGF-B, PDGF-C, PDGF-D or P1GF and the
mutant CUB domain which is dimerized to a CUB-removed monomer of
VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF-A, PDGF-B, PDGF-C, PDGF-D or
P1GF. Addition of the specific protease(s) for this proteolytic
site would cleave the CUB domain and thereby release an activated
dimer that can then bind to its corresponding receptor. In this
way, a controlled release of an activated dimer is made
possible.
[0051] According to a third aspect, the invention provides a
purified and isolated nucleic acid encoding a polypeptide or
polypeptide fragment of the invention as defined above. 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 FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:6) or
FIG. 8 (SEQ ID NO:8), a bioactive fragment or analog thereof, a
receptor-binding but otherwise inactive or partially inactive
variant thereof or a PDGF-D dimer-forming but otherwise inactive or
interfering variants thereof.
[0052] A fourth aspect of the invention provides vectors comprising
the cDNA of the invention or a nucleic acid molecule according to
the third aspect of the invention, and host cells transformed or
transfected with nucleic acids molecules or vectors of the
invention. These may be eukaryotic or prokaryotic in origin. 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 293-EBNA 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 adenoviral-, vaccinia- or
retroviral-based vectors or liposomes. A variety of such vectors is
known in the art.
[0053] The invention also provides a method of making a vector
capable of expressing a polypeptide encoded by a nucleic acid
molecule according to the invention, comprising the steps of
operatively connecting the nucleic acid molecule to one or more
appropriate promoters and/or other control sequences, as described
above.
[0054] 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.
[0055] In yet a further aspect, the invention provides an antibody
specifically reactive with a polypeptide of the invention or a
fragment of the polypeptide. This aspect of the invention includes
antibodies specific for the variant forms, immunoreactive
fragments, analogs and recombinants of PDGF-D. Such antibodies are
useful as inhibitors or antagonists of PDGF-D and as diagnostic
agents for detecting and quantifying PDGF-D. Polyclonal or
monoclonal antibodies may be used. Monoclonal and polyclonal
antibodies can be raised against polypeptides of the invention or
fragment or analog thereof using standard methods in the art. In
addition the polypeptide can be linked to an epitope tag, such as
the FLAG.RTM. octapeptide (Sigma, St. Louis, Mo.), to assist in
affinity purification. For some purposes, for example where a
monoclonal antibody is to be used to inhibit effects of PDGF-D in a
clinical situation, it may be desirable to use humanized or
chimeric monoclonal antibodies. Such antibodies may be further
modified by addition of cytotoxic or cytostatic drug(s). Methods
for producing these, including recombinant DNA methods, are also
well known in the art.
[0056] This aspect of the invention also includes an antibody which
recognizes PDGF-D and is suitably labeled.
[0057] Polypeptides or antibodies according to the invention may be
labeled with a detectable label, and utilized for diagnostic
purposes. Similarly, the thus-labeled 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 may be used. Examples
of radioactive labels include a radioactive atom or group, such as
.sup.125I or .sup.32P. Examples of non-radioactive labels include
enzymatic labels, such as horseradish peroxidase or fluorimetric
labels, such as fluorescein-5-isothiocyanate (FITC). Labeling may
be direct or indirect, covalent or non-covalent.
[0058] Clinical applications of the invention include diagnostic
applications, acceleration of angiogenesis in tissue or organ
transplantation to promote graft growth and vascularization, or
stimulation of wound healing, or connective tissue development, 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 and inhibition of tissue remodeling that takes place
during invasion of tumor cells into a normal cell population by
primary or metastatic tumor formation. Quantitation of PDGF-D in
cancer biopsy specimens may be useful as an indicator of future
metastatic risk.
[0059] PDGF-D may also be relevant to a variety of lung conditions.
PDGF-D assays could be used in the diagnosis of various lung
disorders. PDGF-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, PDGF-D
could be used to improve blood circulation to the heart and O.sub.2
gas permeability in cases of cardiac insufficiency. In a like
manner, PDGF-D could be used to improve blood flow and gaseous
exchange in chronic obstructive airway diseases.
[0060] Thus the invention provides a method for stimulating
angiogenesis, lymphangiogenesis, neovascularization, connective
tissue development and/or wound healing in a mammal in need of such
treatment, comprising the step of administering an effective dose
of PDGF-D, or a fragment or an analog thereof which has the
biological activity of PDGF-D to the mammal. The PDGF-D
polypeptides may be administered either in the form of its
bioactive fragment (e.g. residues 254-370 of SEQ ID NO: 8), or in
the form of a full-length sequence which may be activated, e.g.
with a suitable protease, in situ. Alternatively, a nucleic acid
molecule coding for a bioactive PDGF-D polypeptide may be
administered, or a nucleic acid molecule coding for a full-length
PDGF-D polypeptide together with a nucleic acid molecule coding for
a suitable protease are administered together, preferably under the
control of regulatory elements suitable for regulation of their
respective expression. Optionally the PDGF-D, or fragment or analog
thereof may be administered together with, or in conjunction with,
one or more of VEGF, VEGF-B, VEGF-C, VEGF-D, P1 GF, PDGF-A, PDGF-B,
PDGF-C, FGF and/or heparin.
[0061] PDGF-D polypeptides may be directly delivered to the site of
interest where angiogenesis etc are desired. Numerous direct
polypeptide delivery methods are known and may be used. See e.g.
Talmadge, 1993, The pharmaceutics and delivery of therapeutic
polypeptides and proteins, Adv. Drug Del. Rev. 10:247-299. The
polypeptides may be administered orally. Although polypeptides are
generally known to have poor availability through oral
administration, various methods known in the art have been
developed to overcome this limitation. For example, biodegradable
polymeric matrices have been used for delivering proteins over a
desired period of time. For example, the use of biodegradable
poly(d, 1-lactic-co-glycolic acid) (PLGA) microspheres for the
delivery of peptides and proteins has been widely reported (Mehta
et al., 1996, Peptide containing microspheres from low molecular
weight and hydrophilic poly(d,l-lactide-co-glycolide), J. Control
Release 41:249-257; Chiba et al., 1997, Controlled protein delivery
from biodegradable tyrosine-containing poly(anhydride-co-imide)
microspheres. Biomaterials 18:893-901; Ravivarapu et al., 2000,
Polymer and microsphere blending to alter the release of a peptide
from PLGA microspheres, Eur. J. Pharm. Biopharm. 50:263-270).
[0062] Preferably, direct application of the polypeptides,
especially direct injection, may be used. Because wound-healing and
other conditions requiring enhanced angiogenesis typically require
local application of PDGF-D polypeptides and other growth factors
for only a limited time, direct injection, even frequent direct
injection of the polypeptides to the desired site(s) is acceptable
and is not likely to be very tedious or expensive and pose problems
such as poor patient acceptance. Methods of direct application of
polypeptides are well-known to those ordinarily skilled in the art,
and recent successes, strategies, and potentials of topical
application of PDGF-BB in improving healing were reviewed by Cupp
et al., 2002, Gene therapy, electroporation, and the future of
wound-healing therapies, Facial Plast. Surg. 18:53-57.
[0063] In another preferred embodiment, the therapeutic
polypeptides of the present invention may be delivered in the form
of nucleic acid molecules encoding the polypeptides. Many
established and well-known methods for gene delivery or gene
therapy may be used for administering genes or other nucleic acid
molecules encoding PDGF-D to the patient. See e.g. Rubany, 2001,
The future of human gene therapy, Mol. Aspects. Med. 22:113-42. A
single dose of naked DNA of VEGF and PDGF was used to treat rats
with cysteanmine-induced duodenal ulcers, and was shown to
significantly accelerate chronic duodenal ulcer healing, and
increase VEGF and PDGF levels in duodenal mucosa (Szabo et al.,
2001, Gene Expression and gene therapy in experimental duodena
ulceration, J. Physiol. Paris 95:325-335).
[0064] The polynucleotides encoding PDGF-D preferably are linked
operatively under the control of suitable promoter so that they are
expressed when taken up by the host cells. PDGF-D is a diffusible
protein, and as such it will exert its effects on cells directly
expressing the polypeptides, as well as on surrounding cells.
Accordingly, suitable promoters may be constitutive promoters such
as promoter and enhancer elements from cytomegalovirus (CMV), Rous
sarcoma virus (RSV), and SV40, and the rat beta-actin promoter.
Preferably, inducible or tissue specific promoters are used to
increase expression level, improve specificity and reduce side
effects. In this regard, suitable promoters include the keratin 5
(K5) promoter (Pierce et al., 1998, Oncogene 16: 1267-1276; Pierce
et al., 1998, Proc. Natl. Acad. Sci. USA 95:8858-8863), the Cyr61
promoter (inducible in granulation tissue during wound healing)
(Latinkic et al., 2001, Promoter function of the angiogenic inducer
Cyr61 gene in transgenic mice: tissue specificity, inducibility
during wound healing, and role of the serum response element,
Endocrinol. 142:2549-2557), and the FAP promoter (Neidermeyer et
al., 2001, Expression of the fibroblast activation protein during
mouse embryo development, Int. J. Dev. Biol. 45:445-447).
[0065] Suitable polynucleotides may also be delivered as nonviral
vectors, using methods well-known to those ordinarily skilled in
the art. See e.g. Brown et al., 2001, Gene delivery with synthetic
(non-viral) carriers, Int. J. Pharm. 229:1-21; and Pouton et al.,
1998, Key issues in non-viral gene delivery, Adv. Drug Deliv. Rev.
34:3-19.). Lipofection, liposome mediated gene transfer are
preferred (Romano et al., 1999, Gene transfer technology in
therapy: current applications and future goals. Stem Cells
17:191-202; Mountain, 2000, Gene therapy: the first decade. Trends.
Biotechnol. 18:119-28; Mhashilkar et al., 2001, Gene therapy:
Therapeutic approaches and implications. Biotechnol. Adv.
19:279-97; and Lasic, 1998, Novel applications of liposomes, Trends
Biotechnol. 16:307-21).
[0066] One of the simplest ideas for non-viral gene delivery is the
use of purified DNA in the form of plasmids. A naked polynucleotide
operatively coding for the polypeptide may be delivered, along with
a pharmaceutically acceptable carrier, directly to the desired
site, where the polynucleotide is taken up by the cells at the site
and expressed or otherwise exerts its therapeutic effects. This is
particularly preferred if transient expression of the gene is
desired. The transfer of naked DNA by physical means is well known,
by such means as gene guns and electroporation. See e.g. Spack et
al., 2001, Developing non-viral DNA delivery systems for cancer and
infectious disease, DDT 6:186-97. See also Cupp et al., 2002,
supra.
[0067] In general, RNA molecules will have more transient effects
than DNA molecules. The effects of the naked RNA molecules so
delivered last typically for less than about 20 days, usually less
than 10 days, and often less than 3 to 5 days. Delivery may be by
injection, spray, biolistic methods, and so on, depending on the
site.
[0068] In another embodiment, suitable polynucleotides may also be
delivered within viral vectors, which are known to have higher
transfection efficiency compared to nonviral vectors. See e.g.
Robbins et al., 1998, Viral vectors for gene therapy, Pharmacol.
Ther. 80:35-47; and Kay et al., 2001, Viral vectors for gene
therapy: the art of turning infectious agents into vehicles of
therapeutics, Nat. Med. 7:33-40. Suitable viral vectors include
those derived from retroviruses (including lentivirues) (see e.g.
Breithart et al., 1999, Ann. Plast. Surg. 43:632-9), especially the
Moloney murineleukemia virus and pseudotyped retroviruses (Chen et
al., 2001, Safety testing for replication-competent retrovirus
associated with gibbon apeleukemia virus-pseudotyped retroviral
vectors. Hum. Gene. Ther. 12:61-70); adenoviruses, especially the
third generation "gutless" adenoviral vector (Kochanek et al.,
2001, High-capacity "gutless" adenoviral vectors. Curr. Opin. Mol.
Ther. 3:454-63.); chimeric viruses that combine the advantages of
both retroviruses and adenoviruses (Reynolds et al., 1999, Chimeric
viral vectors-the best of both worlds? Mol. Med. Today 5:25-31,
1999); adeno-associated virus (Ponnazhagan et al., 2001,
Adeno-associated virus for gene therapy. Cancer Res., 61:6313-21;
and Monahan et al., 2000, Adeno-associated virus vectors for gene
therapy: more pros than cons? Mol. Med. Today, 6:433-40.); vaccinia
viruses (Peplinski, et al., 1998, Vaccinia virus for human gene
therapy. Surg. Oncol. Clin. N. Am., 7:575-588); and herpes simplex
virus (Latchman. 2001, Gene delivery and gene therapy with herpes
simplex virus-based vectors. Gene 264:1-9).
[0069] Adenoviral vectors are preferred. Chen et al. (2002) showed
that recombinant adenoviruses encoding the PDGF-A gene express and
secrete PDGF-A in vitro, and induce sustained down regulation of
PDGF.alpha.R encoded by the growth arrest specific (gas) gene (Am.
J. Physiol. Cell Physiol. 282:C538-44). Szabo et al., supra, used a
single dose of adenoviral vectors expressing VEGF and PDGF to treat
rats with cysteanmine-induced duodenal ulcers, and showed
significant acceleration of chronic duodenal ulcer healing, and
increased VEGF and PDGF levels in duodenal mucosa. Giannobile et
al., 2001, J. Periodontol. 72:815-23 showed that adenoviral vectors
expressing PDGF-A stimulated cementoblast DNA synthesis and
subsequent proliferation. Zhu et al., 2001, J. Dent. Res. 80:892-7
demonstrated that adenoviruses encoding PDGF-A enhanced mitogenic
and proliferative responses in osteoblasts, periodontal ligament
fibroblasts and gingival fibroblasts. See also Liechty et al.,
1999, Adenoviral mediated overexpression of PDGF-B corrects
ischemic impaired wound healing, J. Invest. Dermatol. 11
3:375-83.
[0070] The effects of vectors coding for PDGF-D polypeptides may
also be improved with matrix immobilization to enhance tissue
repair activity. Biocompatible matrices capable of immobilizing
adenoviral vectors have been successfully used in treating ischemic
excisional wounds. Specifically, collagen-formulated vectors
encoding PDGF-B, when delivered as subcutaneously implanted
sponges, have been shown to enhance granulation tissue deposition,
enhance epithelial area, and improve wound closure more effectively
than aqueous formulations of the same vectors. With longer time,
complete healing without excessive scar formation was achieved. In
comparison, aqueous formulations allowed vector seepage and led to
PDGF-induced hyperplasia in surrounding tissues but not in wound
beds. In addition, repeated applications of PDGF-BB proteins were
required for neotissue induction approaching equivalence to a
single application of collagen-immobilized vectors. (Doukas et al.,
2002, Hum. Gene Ther. 12:783-98). In the same study, Doukas et al.
also showed that vectors encoding fibroblast growth factor 2 or
vascular endothelial growth factor promoted primarily angiogenic
responses. Similar improvements were observed in dermal ulcer
wounds in the ears of young adult New Zealand white rabbits with
collagen embedded PDGF-B or PDGF-A DNA plasmids (Tyrone et al.,
2000, J. Surg. Res. 93:230-6); in soft tissue repair by enhancing
de novo tissue formation (Chandler et al., 2000, Mol. Ther.
2:153-60).
[0071] Other materials may also be used as sustained release
matrices for delivering vectors encoding PDGF genes. For example,
matrices of poly(lactide-co-glycolide) (PLG) were loaded with
plasmids and shown to release the plasmids over a period ranging
from days to months in vitro, and led to the transfection of large
numbers of cells. In vivo delivery enhanced matrix deposition and
blood vessel formation in the developing tissue (Shea et al., 1999,
Nat. Biotechnol. 17:551-4).
[0072] Another method of gene delivery uses fusigenic virosomes.
This approach combines some of the advantages of viral delivery
vectors with the safety and `simplicity` of the liposome to produce
fusigenic virosomes (Dzau et al., 1996, Fusigenic viral liposome
for gene therapy in cardiovascular diseases. Proc Natl. Acad. Sci.
USA 93:11421-25). Virosomes have been engineered by complexing the
membrane fusion proteins of haemagglutinating virus of Japan (HVJ,
which is also known as Sendai virus) with either liposomes that
already encapsulate plasmid DNA or oligodeoxynucleotides (ODN) for
antisense applications. The inherent ability of the viral proteins
in virosomes to cause fusion with cell membranes means that these
hybrid vectors can be very efficient at introducing their nucleic
acid to the target cell, resulting in good gene expression. Each
viral vector has a limit on the size of transgene that can be
incorporated into its genome; no such limit exists for virosome or
liposome technology. Genes of up to 100 kilobase pairs have been
delivered by fusigenic virosomes to cells both ex vivo and in
vivo.
[0073] A further embodiment of the invention utilizes DNA-ligand
conjugates for delivery of genes encoding the PDGF-polypeptides.
DNA-ligand conjugates have two main components: a DNA-binding
domain and a ligand for cell-surface receptors. The transgene can
therefore be guided specifically to the target cell, where it is
internalized via receptor-mediated endocytosis. Once the DNA-ligand
complex is in the endocytic pathway, the conjugate is likely to be
destroyed when the endosome fuses with a lysosome. To avoid this,
an adenovirus-derived domain may be incorporated into the
cell-surface receptor part of the ligand (Curiel et al., 1992,
High-efficiency gene transfer mediated by adenovirus coupled to
DNA-polylysine complexes, Hum. Gene Ther. 3:147-154). The
conjugates then have the same specificity as adenoviruses, binding
to a wide host-cell range; they also possess an adenovirus
characteristic that allows the conjugate to leave the endosome and
enter the cytoplasm (by a process known as endosomolysis) before
the endosome is destroyed by a lysosome.
[0074] According to another embodiment of the invention, suitable
host cells may be transformed with polynucleotides, preferably
vectors, more preferably viral vectors, encoding the PDGF-D
polypeptides of the invention, and the host cells expressing the
PDGF-D polypeptides may be introduced to a host animal in need of
wound healing or other treatment. Many methods of in vitro cell
transformation are known and well established in the art, including
CaPO.sub.4 transfection, which is a chemical method that has been
successfully used by molecular biologists for many years to
introduce transgenes into cells in vitro with a relatively good
efficiency (10%). Mathisen et at. showed that autoreactive memory
Th2 T cells can be genetically modified so that upon engagement of
self antigen they produce regenerative growth factors such as
PDGF-A capable of mediating tissue repair during autoimmune disease
(Mathisen et al., 1999, J. Autoimmun. 13:31-8
[0075] Conversely, PDGF-D antagonists (e.g. antibodies and/or
competitive or noncompetitive inhibitors of binding of PDGF-D in
both dimer formation and receptor binding) could be used to treat
conditions, such as congestive heart failure, involving
accumulation 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. Administrations of PDGF-D could be used to treat
malabsorptive syndromes in the intestinal tract, liver or kidneys
as a result of its blood circulation increasing and vascular
permeability increasing activities.
[0076] Thus, the invention provides a method of inhibiting
angiogenesis, lymphangiogenesis, neovascularization, connective
tissue development and/or wound healing in a mammal in need of such
treatment, comprising the step of administering an effective amount
of an antagonist of PDGF-D to the mammal. The antagonist may be any
agent that prevents the action of PDGF-D, either by preventing the
binding of PDGF-D to its corresponding receptor on the target cell,
or by preventing activation of the receptor, such as using
receptor-binding but otherwise inactive PDGF-D variants. Suitable
antagonists include, but are not limited to, antibodies directed
against PDGF-D; competitive or noncompetitive inhibitors of binding
of PDGF-D to the PDGF-D receptor(s), such as the receptor-binding
or PDGF-D dimer-forming but non-mitogenic PDGF-D variants referred
to above; and anti-sense nucleotide sequences as described below.
For example, a truncated PDGF.beta. receptor was shown to inhibit
thrombosis and neointima formation in an avian arterial injury
model (Ding et al., 2001, Thromb. Haemost. 86:914-22).
[0077] In one embodiment, an antagonist of PDGF-D is a negative
dominant mutant of a PDGF-D gene. This negative dominant mutant is
able to inhibit the expression of the native PDGF-D gene in the
appropriate tissue of the animal, thereby disrupting PDGF-D
activity. See e.g. Chen et al., 2002, Am. J. Physiol. Cell Physiol.
282:0538-44 (showing that dominant negative mutant of PDGF-A gene
disrupts PDGF activity).
[0078] A method is provided for determining agents that bind to an
activated truncated form of PDGF-D. The method comprises contacting
an activated truncated form of PDGF-D with a test agent and
monitoring binding by any suitable means. Potential binding agents
include proteins and other substances. The invention provides a
screening system for discovering agents that bind an activated
truncated form of PDGF-D. The screening system comprises preparing
an activated truncated form of PDGF-D, exposing the activated
truncated form of PDGF-D to a test agent, and quantifying the
binding of said agent to the activated truncated form of PDGF-D by
any suitable means. The inhibitory effects of a binding agent are
further determined by assaying the PDGF-D activities of the PDGF-D
polypeptides bound with the binding agent. Both in vivo and in
vitro assay methods may be used. Specifically, this screening
system is used to identify agents which inhibit the proteolytic
cleavage of the full length PDGF-D protein and thereby prevent the
release of the activated truncated form of PDGF-D. For this use,
the full length PDGF-D is generally preferred.
[0079] Use of this screening system provides a means to determine
compounds that may alter the biological function of PDGF-D. This
screening method may be adapted to large-scale, automated
procedures such as a PANDEX.RTM. (Baxter-Dade Diagnostics) system,
allowing for efficient high-volume screening of potential
therapeutic agents.
[0080] For this screening system, an activated truncated form of
PDGF-D or full length PDGF-D is prepared as described herein,
preferably using recombinant DNA technology. A test agent, e.g. a
compound or protein, is introduced into a reaction vessel
containing the activated truncated form of or full length PDGF-D.
Binding of the test agent to the activated truncated form of or
full length PDGF-D is determined by any suitable means which
include, but is not limited to, radioactively- or
chemically-labeling the test agent. Binding of the activated
truncated form of or full length PDGF-D may also be carried out by
a method disclosed in U.S. Pat. No. 5,585,277, which is
incorporated by reference. In this method, binding of the test
agent to the activated truncated form of or full length PDGF-D is
assessed by monitoring the ratio of folded protein to unfolded
protein. Examples of this monitoring can include, but are not
limited to, monitoring the sensitivity of the activated truncated
form of or full length PDGF-D to a protease, or amenability to
binding of the protein by a specific antibody against the folded
state of the protein.
[0081] Those of skill in the art will recognize that IC.sub.50
values are dependent on the selectivity of the agent tested. For
example, an agent with an IC.sub.50 which is less than 10 nM is
generally considered an excellent candidate for drug therapy.
However, an agent which has a lower affinity, but is selective for
a particular target, may be an even better candidate. Those skilled
in the art will recognize that any information regarding the
binding potential, inhibitory activity or selectivity of a
particular agent is useful toward the development of pharmaceutical
products.
[0082] Where PDGF-D or a PDGF-D antagonist is to be used for
therapeutic purposes, the dose(s) and route of administration will
depend upon the nature of the patient and condition to be treated,
and will be at the discretion of the attending physician or
veterinarian. Suitable routes include oral, subcutaneous,
intramuscular, intraperitoneal or intravenous injection,
parenteral, topical application, implants etc. Topical application
of PDGF-D may be used in a manner analogous to VEGF. Where used for
wound healing or other use in which enhanced angiogenesis is
advantageous, an effective amount of the truncated active form of
PDGF-D is administered to an organism in need thereof in a dose
between about 0.1 and 1000 .mu.g/kg body weight.
[0083] The PDGF-D or a PDGF-D antagonist may be employed in
combination with a suitable pharmaceutical carrier. The resulting
compositions comprise a therapeutically effective amount of PDGF-D
or a PDGF-D antagonist, and a pharmaceutically acceptable non-toxic
salt thereof, and a pharmaceutically acceptable solid or liquid
carrier or adjuvant. Examples of such a carrier or adjuvant
include, but are not limited to, saline, buffered saline, Ringer's
solution, mineral oil, talc, corn starch, gelatin, lactose,
sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium
phosphate, sodium chloride, alginic acid, dextrose, water,
glycerol, ethanol, thickeners, stabilizers, suspending agents and
combinations thereof. Such compositions may be in the form of
solutions, suspensions, tablets, capsules, creams, salves, elixirs,
syrups, wafers, ointments or other conventional forms. The
formulation should be constituted to suit the mode of
administration. Compositions which comprise PDGF-D may optionally
further comprise one or more of PDGF-A, PDGF-B, PDGF-C, VEGF,
VEGF-B, VEGF-C, VEGF-D, PIGF and/or heparin. Compositions
comprising PDGF-D will contain from about 0.1% to 90% by weight of
the active compound(s), and most generally from about 10% to
30%.
[0084] For intramuscular preparations, a sterile formulation,
preferably a suitable soluble salt form of the truncated active
form of PDGF-D, such as hydrochloride salt, can be dissolved and
administered in a pharmaceutical diluent such as pyrogen-free water
(distilled), physiological saline or 5% glucose solution. A
suitable insoluble form of the compound may be prepared and
administered as a suspension in an aqueous base or a
pharmaceutically acceptable oil base, e.g. an ester of a long chain
fatty acid such as ethyl oleate.
[0085] According to yet a further aspect, the invention provides
diagnostic/prognostic devices 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 PDGF-D and
a means for detecting, and more preferably evaluating, binding
between the antibody and PDGF-D. In one preferred embodiment of the
diagnostic/prognostic device according to the invention, a second
antibody (the secondary antibody) directed against antibodies of
the same isotype and animal source of the antibody directed against
PDGF-D (the primary antibody) is provided. The secondary antibody
is coupled directly or indirectly to a detectable label, and then
either an unlabeled primary antibody or PDGF-D is substrate-bound
so that the PDGF-D/primary antibody interaction can be established
by determining the amount of label bound to the substrate following
binding between the primary antibody and PDGF-D and the subsequent
binding of the labeled secondary antibody to the primary antibody.
In a particularly preferred embodiment of the invention, the
diagnostic/prognostic device may be provided as a conventional
enzyme-linked immunosorbent assay (ELISA) kit.
[0086] In another alternative embodiment, a diagnostic/prognostic
device may comprise polymerase chain reaction means for
establishing sequence differences of a PDGF-D 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 PDGF-D expression are
related to a given disease condition.
[0087] In addition, a diagnostic/prognostic device may comprise a
restriction length polymorphism (RFLP) generating means utilizing
restriction enzymes and genomic DNA from a test individual to
generate a pattern of DNA bands on a gel and comparing this pattern
with that disclosed in this application in order to detect any
abnormalities, with a view to establishing whether any aberrations
in PDGF-D expression are related to a given disease condition.
[0088] In accordance with a further aspect, the invention relates
to a method of detecting aberrations in PDGF-D gene structure in a
test subject which may be associated with a disease condition in
the test subject. This method comprises providing a DNA sample from
said test subject; contacting the DNA sample with a set of primers
specific to PDGF-D DNA operatively coupled to a polymerase and
selectively amplifying PDGF-D DNA from the sample by polymerase
chain reaction, and comparing the nucleotide sequence of the
amplified PDGF-D DNA from the sample with the nucleotide sequences
shown in FIG. 3 (SEQ ID NO:3), FIG. 5 (SEQ ID NO:5) or FIG. 7 (SEQ
ID NO:7). The invention also includes the provision of a test kit
comprising a pair of primers specific to PDGF-D DNA operatively
coupled to a polymerase, whereby said polymerase is enabled to
selectively amplify PDGF-D DNA from a DNA sample.
[0089] The invention also provides a method of detecting PDGF-D in
a biological sample, comprising the step of contacting the sample
with a reagent capable of binding PDGF-D, and detecting the
binding. Preferably the reagent capable of binding PDGF-D is an
antibody directed against PDGF-D, particularly 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.
[0090] In another aspect, the invention relates to a protein dimer
comprising the PDGF-D polypeptide, particularly a disulfide-linked
dimer. The protein dimers of the invention include both homodimers
of PDGF-D polypeptide and heterodimers of PDGF-D and VEGF, VEGF-B,
VEGF-C, VEGF-D, P1GF, PDGF-A, PDGF-B or PDGF-C.
[0091] According to a yet further aspect of the invention there is
provided a method for isolation of PDGF-D comprising the step of
exposing a cell which expresses PDGF-D to heparin to facilitate
release of PDGF-D from the cell, and purifying the thus-released
PDGF-D.
[0092] 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 PDGF-D or a
fragment or analog thereof that has the biological activity of
PDGF-D. In addition the anti-sense nucleotide sequence can be to
the promoter region of the PDGF-D gene or other non-coding region
of the gene which may be used to inhibit, or at least mitigate,
PDGF-D expression.
[0093] 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, PDGF-D expression. The use of a vector of this
type to inhibit PDGF-D expression is favored in instances where
PDGF-D expression is associated with a disease, for example where
tumors produce PDGF-D in order to provide for angiogenesis, or
tissue remodeling that takes place during invasion of tumor cells
into a normal cell population by primary or metastatic tumor
formation. Transformation of such tumor cells with a vector
containing an anti-sense nucleotide sequence would suppress or
retard angiogenesis, and so would inhibit or retard growth of the
tumor or tissue remodeling.
[0094] Another aspect of the invention relates to the discovery
that the full length PDGF-D protein is likely to be a latent growth
factor that needs to be activated by proteolytic processing to
release an active PDGF/VEGF homology domain. A putative proteolytic
site is found in residues 254-257 in the full length protein,
residues --RKSK-- (SEQ ID NO:9). This is a dibasic motif. The
--RKSK-- (SEQ ID NO:9) putative proteolytic site is also found in
PDGF-A, PDGF-B, VEGF-C and VEGF-D. In these four proteins, the
putative proteolytic site is also found just before the minimal
domain for the PDGF/VEGF homology domain. Together these facts
indicate that this is the proteolytic site.
[0095] Preferred proteases include, but are not limited, to
plasmin, Factor X and enterokinase. The N-terminal CUB domain may
function as an inhibitory domain which might be used to keep PDGF-D
in a latent form in some extracellular compartment and which is
removed by limited proteolysis when PDGF-D is needed.
[0096] According to this aspect of the invention, a method is
provided for producing an activated truncated form of PDGF-D or for
regulating receptor-binding specificity of PDGF-D. These methods
comprise the steps of expressing an expression vector comprising a
polynucleotide encoding a polypeptide having the biological
activity of PDGF-D and supplying a proteolytic amount of at least
one enzyme for processing the expressed polypeptide to generate the
activated truncated form of PDGF-D.
[0097] This aspect also includes a method for selectively
activating a polypeptide having a growth factor activity. This
method comprises the step expressing an expression vector
comprising a polynucleotide encoding a polypeptide having a growth
factor activity, a CUB domain and a proteolytic site between the
polypeptide and the CUB domain, and supplying a proteolytic amount
of at least one enzyme for processing the expressed polypeptide to
generate the activated polypeptide having a growth factor
activity.
[0098] In addition, this aspect includes the isolation of a nucleic
acid molecule which codes for a polypeptide having the biological
activity of PDGF-D and a polypeptide thereof which comprises a
proteolytic site having the amino acid sequence RKSK (SEQ ID NO:9)
or a structurally conserved amino acid sequence thereof.
[0099] Also this aspect includes an isolated dimer comprising an
activated monomer of PDGF-D and an activated monomer of VEGF,
VEGF-B, VEGF-C, VEGF-D, PDGF-D, PDGF-A, PDGF-B, PDGF-C or P1 GF
linked to a CUB domain, or alternatively, an activated monomer of
VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF-D, PDGF-A, PDGF-B or P1GF and an
activated monomer of PDGF-D linked to a CUB domain. The isolated
dimer may or may not include a proteolytic site between the
activated monomer and the CUB domain.
[0100] 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 hybridize thereto under stringent
conditions all are useful for identifying, purifying, and isolating
polynucleotides encoding other, non-human, mammalian forms of
PDGF-D. Thus, such polynucleotide fragments and variants are
intended as aspects of the invention. Exemplary stringent
hybridization conditions are as follows: hybridization at
42.degree. C. in 5X SSC, 20 mM NaPO.sub.4, pH 6.8, 50% formamide;
and washing at 42.degree. C. in 0.2X 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 hybridized, and that formulas for
determining such variation exist. See for example Sambrook et al.,
"Molecular Cloning: A Laboratory Manual", Second Edition, pages
9.47-9.51, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
(1989).
[0101] Moreover, purified and isolated polynucleotides encoding
other, non-human, mammalian PDGF-D forms also are aspects of the
invention, as are the polypeptides encoded thereby and antibodies
that are specifically immunoreactive with the non-human PDGF-D
variants. Thus, the invention includes a purified and isolated
mammalian PDGF-D polypeptide and also a purified and isolated
polynucleotide encoding such a polypeptide.
[0102] 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.
[0103] It will be clearly understood that for the purposes of this
specification the word "comprising" means "included but not limited
to." The corresponding meaning applies to the word "comprises."
BRIEF DESCRIPTION OF THE DRAWINGS
[0104] FIG. 1 (SEQ ID NO:1) shows a nucleotide sequence that
includes a cDNA sequence encoding the C-terminal part of human
PDGF-D (hPDGF-D). The nucleotides which encode for the partial
fragment of hPDGF-D are 1 to 198. The deduced partial amino acid
sequence of hPDGF-D (66 amino acid residues-SEQ ID NO:2) derived
from nucleotides 1 to 198 of FIG. 1 is shown in FIG. 2;
[0105] FIG. 3 (SEQ ID NO:3) shows an extended sequence of a partial
human cDNA encoding for the hPDGF-D. The translated cDNA sequence
is from nucleotide 1 to 600. The deduced partial amino acid
sequence of hPDGF-D (200 residues-SEQ ID NO:4) derived from
nucleotides 1 to 600 of FIG. 3 is shown in FIG. 4;
[0106] FIG. 5 shows a still further extended nucleotide sequence of
a partial human cDNA. The nucleotides which encode for the
5'-truncated full-length hPDGF-D are 1 to 966 (SEQ ID NO:5). The
deduced partial amino acid sequence of hPDGF-D (322 residues-SEQ ID
NO:6) derived from nucleotides 1 to 966 of FIG. 5 is shown in FIG.
6;
[0107] FIG. 7 (SEQ ID NO:7) shows the complete nucleotide sequence
of cDNA encoding a hPDGF-D(1116 bp) and the deduced amino acid
sequence of full-length hPDGF-D encoded thereby which consists of
370 amino acid residues (FIG. 8--SEQ ID NO:8);
[0108] FIG. 9 shows an amino acid sequence alignment of the hPDGF-D
with hPDGF-C (SEQ ID NOs:8 and 32, respectively);
[0109] FIG. 10 shows an amino acid sequence alignment of the
PDGF/VEGF-homology domain in hPDGF-D with several growth factors
belonging to the VEGF/PDGF family (SEQ ID NOs:10-18,
respectively);
[0110] FIG. 11 shows a phylogenetic tree of several growth factors
belonging to the VEGF/PDGF family;
[0111] FIG. 12 provides the amino acid sequence alignment of the
CUB domain present in hPDGF-D (SEQ ID NO:19) and other CUB domains
present in human bone morphogenic protein-1 (hBMP-1, 3 CUB domains
CUB1-3) (SEQ ID NOs:20-22, respectively) and in human neuropilin-1
(2 CUB domains) (SEQ ID NOs:23-24, respectively);
[0112] FIG. 13 shows the results of the SDS-PAGE analysis of human
recombinant PDGF-D under reducing (R) and non-reducing (NR)
conditions;
[0113] FIG. 14 shows the results of the immunoblot analysis of
full-length PDGF-D and PDGF-C under reducing and non-reducing
conditions employing affinity-purified rabbit antibodies to
full-length PDGF-D;
[0114] FIG. 15 provides that results of the relative expression
levels of PDGF-D (upper panel) and PDGF-B (lower panel) transcripts
in several human tissues as determined by Northern Blot
analysis;
[0115] FIG. 16 shows PDGF-D expression in the developing kidney of
a mouse embryo;
[0116] FIG. 17 shows a more detailed view of PDGF-D expression in
the developing kidney of a mouse embryo;
[0117] FIG. 18 shows a more detailed view of PDGF-D expression in
the developing kidney of a mouse embryo;
[0118] FIG. 19 shows that conditioned medium(CM)containing
plasmin-digested PDGF-D stimulates tyrosine phosphorylation of
PDGFR-beta in PAE-1 cells;
[0119] FIG. 20 provides a graphical representation of the results
of the competitive binding assay between plasmin-digested PDGF-D
and PDGF-BB homodimers for the PDGFRs-beta; and
[0120] FIG. 21 provides a graphical representation of the results
of the competitive binding assay between plasmin-digested PDGF-D
and PDGF-AA homodimers for the PDGFRs-alpha.
[0121] FIG. 22A shows a schematic representation of the PDGF-D
sequence of SEQ ID NO:35.
[0122] FIG. 22B shows a schematic representation of the PDGF-D
sequence variant of SEQ ID NO:37, which corresponds to FIG. 22A but
for 6 missing amino acid residues.
[0123] FIG. 22C shows a schematic representation of the PDGF-D
sequence variant of SEQ ID NO:39, which corresponds to FIG. 22A but
for 6 missing amino acid residues and the loss of a CUB domain in
this sequence variant.
[0124] FIG. 23 shows a schematic representation of the PDGF-D
sequence, noting the spliced region from exon 5 to exon 7, removal
of which yields the PDGF-D sequence variant of SEQ ID NO:39.
[0125] FIG. 24 shows SDS-PAGE analysis under reducing conditions of
human PDGF-DD formed from the core domain of factor Xa-digested
mutant full-length form of PDGF-D.
[0126] FIG. 25 shows the in vivo angiogenic activity of human
PDGF-DD and other PDGF isoforms in the mouse cornea pocket assay.
In FIG. 25A-E, arrows point to where PDGF protein-containing beads
were implanted.
[0127] FIG. 26 is a schematic diagram showing the K14-PDGF-D
construct (See Example 11).
[0128] FIG. 27 shows a comparison of PDGF-D expression between
K14-PDGF-D transgenic mouse (TG) and wild-type mouse (wt). Paraffin
embedded mouse skin samples were stained with anti-PDGF-D. For
experimental details, see Uutela et al., 2001, "Chromosomal
location, exon structure and vascular expression patterns of the
human PDGFC and PDGFD genes," Circulation 103:2242-2247.
[0129] FIG. 28 shows a comparison of granulation tissue staining
between K14-PDGF-D transgenic mouse (TG) and wild-type mouse (wt)
in wound areas after two days. The samples were stained with the
Van Gieson method which stains elastin. The amount of granulation
tissue is greater in PDGF-D positive mouse (TG).
[0130] FIG. 29 shows a comparison of granulation tissue staining
between K14-PDGF-D transgenic mouse (TG) and wild-type mouse (wt)
in wound areas after four days. The samples were stained with the
Van Gieson method which stains elastin. The amount of granulation
tissue is greater in PDGF-D positive mouse (TG).
[0131] FIG. 30 shows a comparison of granulation tissue staining
between K14-PDGF-D transgenic mouse (TG) and wild-type mouse (wt)
in wound areas after seven days. The samples were stained with the
Van Gieson method which stains elastin. The amount of granulation
tissue is greater in PDGF-D positive mouse (TG).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0132] FIG. 1 shows a nucleotide sequence of human cDNA which
encodes a C-terminal portion of a novel growth factor, referred to
herein as PDGF-D (formerly VEGF-G). PDGF-D is a new member of the
VEGF/PDGF family. The nucleotide sequence of FIG. 1 (SEQ ID NO:1)
was derived from a human EST sequence (id. A1488780) in the dbEST
database at the NCBI in Washington, DC. The nucleotides 1 to 198 of
the cDNA of FIG. 1 (SEQ ID NO:1) encodes a 66 amino acid
polypeptide (FIG. 2--SEQ ID NO:2) which shows some sequence
similarity to the known members of the VEGF/PDGF family.
[0133] The amino acid sequence of the polypeptide encoded by the
nucleotides 1 to 198 of the polynucleotide of FIG. 1 (SEQ ID NO: 1)
is shown in FIG. 2 (SEQ ID NO:2).
[0134] To generate more sequence information on human PDGF-D, a
human fetal lung .lamda.gt10 cDNA library was screened using a 327
bp polymerase chain reaction (PCR)-generated probe, based on the
originally identified EST sequence. The probe was generated from
DNA from a commercially available human fetal lung cDNA library
(Clontech) which was amplified by PCR using two primers derived
from the identified EST (AI488780). The primers were:
TABLE-US-00002 5'-GTCGTGGAACTGTCAACTGG (forward) (SEQ ID NO: 26)
and 5'-CTCAGCAACCACTTGTGTTC (reverse). (SEQ ID NO: 27)
The amplified 327 bp fragment was cloned into the pCR2.1 vector
(Invitrogen). Nucleotide sequencing verified that the insert
corresponded to the EST. The screen identified several positive
clones. The inserts from two of these clones, clones 5 and 8 were
subcloned into pBluescript and subjected to nucleotide sequencing
using internal or vector-specific primers. The nucleotide sequences
determined were identical in both clones and are shown in FIG. 3
(SEQ ID NO:3). The coding region of the 690 bp polynucleotide is
nucleotides 1-600 (SEQ ID NO:3) that encodes for a large portion of
hPDGF-D with the exception of the 5'-end. This portion of hPDGF-D
includes the bioactive fragment of hPDGF-D. The deduced partial
amino acid sequence of hPDGF-D (200 residues-SEQ ID NO:4) derived
from nucleotides I to 600 of FIG. 3 (SEQ ID NO:3) is shown in FIG.
4 (SEQ ID NO:4).
[0135] Extended nucleotide sequencing of the isolated human PDGF-D
cDNA clones from this human fetal lung cDNA library has provided
additional sequence. FIG. 5 (SEQ ID NO:5) shows a nucleotide
sequence of a partial human cDNA (1934 bp) that encodes hPDGF-D.
The coding region of the 1934 bp polynucleotide is nucleotides 1 to
966 that encodes for hPDGF-D except for the most 5'-end of the
polypeptide. The deduced partial amino acid sequence of hPDGF-D
(322 residues-SEQ ID NO:6) derived from nucleotides 1 to 966 of
FIG. 5 (SEQ ID NO:5) is shown in FIG. 6 (SEQ ID NO:6).
[0136] FIG. 7 (SEQ ID NO:7) shows a polynucleotide sequence of cDNA
encoding a full-length hPDGF-D. The region encoding PDGF-D is 1116
bp. The deduced amino acid sequence of full-length hPDGF-D is 370
amino acid residues (FIG. 8--SEQ ID NO:8).
[0137] The sequence for the 5' end of full-length PDGF-D was
obtained using Rapid Amplification of cDNA Ends (RACE) PCR, and
clones containing cDNA from the human heart (Marathon-ReadyTM cDNA,
Clontech, Cat# 7404-1). These cDNA clones have an adaptor sequence
attached to the 5' end of each clone, including a site for primer
called Adaptor Primer 1 (Clontech):
TABLE-US-00003 5' CCATCCTAATACGACTCACTATAGGGC 3'. (SEQ ID NO:
28)
This primer and a second primer:
TABLE-US-00004 'AGTGGGATCCGTTACTGATGGAGAGTTAT 3' (SEQ ID NO:
29)
were used to amplify the sequence found at the 5' end of PDGF-D. In
the PCR reaction a special polymerase mix was used (Advantage
<<GC cDNA PCR Kit, Clontech, Cat# K1907-1). The reaction mix
included (in microliters):
TABLE-US-00005 Adaptor Primer 1 Gene specific primers 1 each
Template (Human Heart cDNA) 5 GC-Melt (from the K1907-1 Kit) 5 5xGC
cDNA PCR Reaction Buffer 10 50x dNTP mix 1 Sterile H.sub.2O 27
Total 50
[0138] The 5' end of PDGF-D was amplified for 31 cycles, five
cycles consisted of 45 seconds denaturation at 94.degree. C. and
four minutes extension at 72.degree. C., five cycles consisted of
45 seconds denaturation at 94.degree. C. and four minutes extension
at 70.degree. C., and twenty-one cycles consisted of 45 seconds
denaturation at 94.degree. C. and four minutes extension at
68.degree. C. and an initial denaturation step at 94.degree. C. for
two minutes. From this PCR, an approximately 790 bp long product
was obtained. This product was run on a 1% agarose gel, purified
(QIAquick gel extraction Kit, Qiagen, Cat # 28706) from the gel,
cloned into a vector (TOPO TA Cloning Kit, Invitrogen) and
transformed into bacteria (E. Coli). Transformed bacteria were
plated, and incubated at 37.degree. C. overnight. Single colonies
were picked and grown in fresh media overnight. Plasmids were
prepared (QIAprep Spin Miniprep Kit, Qiagen, Cat# 27106) and
sequenced with the plasmid primers, T7 and M13R. The result of this
sequencing was that 312 bp of previously unknown PDGF-D sequence
was obtained. The rest of the sequence (478 bp) was identical with
previously obtained sequence from other PDGF-D cDNA clones.
[0139] Similar to PDGF-C, PDGF-D has a two domain structure with a
N-terminal CUB domain (residues 67-167, discussed below) and a
C-terminal PDGF/VEGF homology domain (residues 272-362, the core
domain). The overall amino acid sequence identity between PDGF-C
(SEQ ID NO:32) and PDGF-D (SEQ ID NO:8) is approximately 43% (FIG.
9). The similarities are highest in the distinct protein domains
while the N-terminal region, including the hydrophobic signal
sequence, and the hinge region between the two domains display
lower identities. A putative signal peptidase cleavage site was
identified between residues 22-23. Cleavage results in a protein of
348 residue with a calculated molecular mass (Mr) of 44,000. A
single putative site for N-linked glycosylation was identified in
the core domain of PDGF-D (residues 276-278).
[0140] FIG. 10 shows the amino acid sequence alignment of the
PDGF/VEGF-homology domain of PDGF-D (found in the C-terminal region
of the polypeptide) with the PDGF/VEGF-homology domains of
PDGF/VEGF family members, PDGF-C, PDGF-A, PDGF-B, VEGF.sub.165,
P1GF-2, VEGF-B.sub.167, VEGF-C and VEGF-D (SEQ ID NOs:10-18,
respectively). Gaps were introduced to optimize the alignment. This
alignment was generated using the MEGALIGN alignment tool based on
the method of J. Hein, (1990, Methods Enzymol. 183:626-45) The PAM
250 residue weight table is used with a gap penalty of eleven and a
gap length penalty of three and a K-tuple value of two in the
pairwise alignments. The alignment is then refined manually, and
the number of identities are estimated in the regions available for
a comparison.
[0141] The alignment shows that the core domain of PDGF-D displays
about a 50% identity to the corresponding domain in PDGF-C, and
about a 20-23% identity to the core domains in the classical PDGFs
and VEGFs. It also shows that, with two exceptions, PDGF-D has the
expected pattern of invariant cysteine residues, involved in inter-
and intra-disulfide bonding, a hallmark of members of this family.
The first exception occurs between cysteine 3 and 4. Normally these
two cysteines are spaced by 2 residues. However, similar to PDGF-C,
PDGF-D has an unique insertion of three additional amino acids
residues, NCG. In total, ten cysteine residues reside in the core
domain, including the extreme C-terminal region, suggesting a
unique arrangement of the cysteines in the disulfide-bonded PDGF-D
dimer. The second exception is that the invariant fifth cysteine
found in the other members of the PDGF/VEGF family is not conserved
in PDGF-D. This feature is unique to PDGF-D.
[0142] Based on the amino acid sequence alignments in FIG. 10, a
phylogenetic tree was constructed and is shown in FIG. 11. The data
show that the PDGF/VEGF homology domain of PDGF-D forms a subgroup
of the PDGFs together with PDGF-C.
Cub Domain
[0143] The N-terminal region of the partial PDGF-D amino acid
sequence of FIG. 12 (residues 53-170 of SEQ ID NO:8) has a second
distinct protein domain which is referred to as a CUB domain (Bork
and Beckmann, 1993, J. Mol. Biol. 231:539-545). This domain of
about 115 amino acids was originally identified in complement
factors C1r/C1s, but has recently been identified in several other
extracellular proteins including signaling molecules such as bone
morphogenic protein 1 (BMP-1) (Wozney et al., 1988, Science,
242:1528-1534) as well as in several receptor molecules such as
neuropilin-1 (NP-1) (Soker et al., 1998, Cell 92:735-745). The
functional roles of CUB domains are not clear but they may
participate in protein-protein interactions or in interactions with
carbohydrates including heparin sulfate proteoglycans. These
interactions may play a role in the proteolytic activation of
PDGF-D.
[0144] As shown in FIG. 12, the amino acid sequences from several
CUB-containing proteins were aligned. The results show that the
single CUB domain in human PDGF-D (SEQ ID NO: 19) displays a
significant identify with the most closely related CUB domains.
Sequences from human BMP-1, with 3 CUB domains (CUBs1-3) (SEQ ID
NOs: 20-22, respectively) and human neuropilin-1 with 2 CUB domains
(CUBs1-2) (SEQ ID NOs: 23-24, respectively) are shown. This
alignment was generated as described above.
EXAMPLE 1
Expression of Human PDGF-D in Baculovirus Infected Sf9 Cells
[0145] The portion of the cDNA encoding amino acid residues 24-370
of SEQ ID NO:8 was amplified by PCR using Taq DNA polymerase
(Biolabs). The forward primer used was 5'GATATCTAGAAGCAACCCCGCAGAGC
3' (SEQ ID NO:33). This primer includes a XbaI site (underlined)
for in frame cloning. The reverse primer used was 5'
GCTCGAATTCTAAATGGTGATGGTGATGATGTCGAGGTGGTCTTGA 3' (SEQ ID NO:34).
This primer includes an EcoRI site (underlined) and sequences
coding for a C-terminal 6X His tag preceded by an enterokinase
site. The PCR product was digested with XbaI and EcoRI and
subsequently cloned into the baculovirus expression vector,
pAcGP67A. Verification of the correct sequence of the cloned PCR
product was done by nucleotide sequencing. The expression vectors
were then co-transfected with BaculoGold linearized baculovirus DNA
into Sf9 insect cells according to the manufacturer's protocol
(Pharmingen). Recombined baculovirus were amplified several times
before beginning large scale protein production and protein
purification according to the manual (Pharmingen).
[0146] Sf9 cells, adapted to serum free medium, were infected with
recombinant baculovirus at a multiplicity of infection of about
seven. Media containing the recombinant proteins were harvested
four days after infection and were incubated with Ni-NTA-Agarose
beads(Qiagen). The beads were collected in a column and after
extensive washing with 50 mM sodium phosphate buffer pH 8,
containing 300 mM NaCl (the washing buffer), the bound proteins
were eluted with increasing concentrations of imidazole (from 100
mM to 500 mM) in the washing buffer. The eluted proteins were
analyzed by SDS-PAGE using 12.5% polyacrylamide gels under reducing
and non-reducing conditions.
[0147] FIG. 13 shows the results of the SDS-PAGE analysis of human
recombinant PDGF-D under reducing (R) and non-reducing (NR)
conditions. PDGF-D was visualized by staining with Coomassie
Brilliant Blue. FIG. 13 also shows that the recombinant PDGF-D
migrates as a 90 kDa species under non-reducing conditions and as a
55 kDa species under reducing conditions. This indicates that the
protein was expressed as a disulfide-linked homodimer.
EXAMPLE 2
Generation of Antibodies to Human PDGF-D
[0148] Rabbit antisera against full-length PDGF-DD and against a
synthetic peptide derived from the PDGF-D sequence (residues
254-272, amino acid sequence RKSKVDLDRLNDDAKRYSC of SEQ ID NO:36
were generated. These peptides were each conjugated to the carrier
protein keyhole limpet hemocyanin (KLH, Calbiochem) using
N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Pharmacia
Inc.) according to the instructions of the supplier. 200-300
micrograms of the conjugates in phosphate buffered saline (PBS)
were separately emulsified in Freunds Complete Adjuvant and
injected subcutaneously at multiple sites in rabbits. The rabbits
were boostered subcutaneously at biweekly intervals with the same
amount of the conjugates emulsified in Freunds Incomplete Adjuvant.
Blood was drawn and collected from the rabbits. The sera were
prepared using standard procedures known to those skilled in the
art. The antibodies to full-length PDGF-DD were affinity-purified
on a column of purified PDGF-DD coupled to CNBr-activated Sepharose
4B (Pharmacia).
[0149] As seen in FIG. 14, the antibodies did not cross-react with
PDGF-C in the immunoblot analysis. For immunoblotting analyses, the
proteins were electrotransferred onto Hybond filters for 45
minutes.
EXAMPLE 3
Expression of PDGF-D Transcripts
[0150] To investigate the tissue expression of PDGF-D in several
human tissues, a Northern blot was done using a commercial Multiple
Tissue Northern blot (MTN, Clontech). The blots were hybridized at
according to the instructions from the supplier using ExpressHyb
solution at 68.degree. C. for one hour (high stringency
conditions), and probed sequentially with a .sup.32P-labeled 327 bp
PCR-generated probe from the human fetal lung cDNA library (see
description above) and full-length PDGF-B cDNA. The blots were
subsequently washed at 50.degree. C. in 2X SSC with 0.05% SDS for
30 minutes and at 50.degree. C. in 0.1X SSC with 0.1% SDS for an
additional 40 minutes. The blots were then put on film and exposed
at -70.degree. C. As shown in FIG. 15, upper panel, the highest
expression of a major 4.4 kilobase (kb) transcript occurred in
heart, pancreas and ovary while lower expression levels were noted
in several other tissues including placenta, liver, kidney,
prostate, testis, small intestine, spleen and colon. No expression
was detected in brain, lung, or skeletal muscle. In comparison, the
3.5 kb PDGF-B transcript was abundantly expressed in heart and
placenta, whereas lower levels were observed in all other tissues
(FIG. 15, lower panel). Prominent co-expression of PDGF-D and
PDGF-B occurred in heart, pancreas and ovary.
EXAMPLE 4
Immunohistochemistry Localization of VEGF-D in Mouse Embryos
[0151] The spatial and temporal patterns of expression of the
PDGF-D protein in mouse embryos were determined by
immunohistochemistry using standard procedures and employing
affinity-purified rabbit antibodies to full-length PDGF-DD
generated in Example 2 on tissue sections of embryos during
midgestation (embryonic day (E) 14.5). The embryos were fixed in 4%
paraformaldehyde overnight at 4.degree. C. and processed for
cryosectioning. 14 .mu.m cryosections were used for the stainings.
Paraffin-embedded sections which were prepared by routine
procedures were also used. After sectioning, the slides were air
dried for one to three hours followed by a ten minute post fixation
with 4% paraformaldehyde. After washing 3.times.5 minutes with
phosphate buffered saline (PBS) containing 0.3% Triton X-100
(PBS-T), the slides were incubated in 0.3% H.sub.20.sub.2 in PBS-T
for 30 minutes to quench the endogenous peroxidase activity. This
was followed by washing 2.times.5 minutes with PBS-T and 2.times.5
minutes in PBS. Blocking of non-specific binding was done using 3%
bovine serum albumin (BSA) in PBS for 30 minutes. The slides were
incubated with the affinity purified antibody to human PDGF-DD (3-9
mg of Ig/ml) overnight at 4.degree. C. After washing, the slides
were incubated with the secondary Ig (goat anti-rabbit HRP, Vector
Laboratories) at a dilution of 1:200 for one hour. After washing,
the slides were incubated with the AB complex (Vector Laboratories)
for one hour and washed with Tris pH 7.4. Either
3,3'-diaminobenzidine tetrahydrochloride (DAB from SIGMA) or
3-amino-9-ethyl carbazole (AEC from Vector Laboratories) was used
for color development. The reaction was quenched by washing in
Tris-HCl buffer. In control experiments the antibodies were
preincubated with a 30.times. molar excess of full-length PDGF-DD.
This blocked the staining, while a similar preincubation with
full-length PDGF-CC did not affect the staining of the tissue
sections. The photomicrographs were taken using a Zeiss microscope
equipped with differential interference contrast optics.
[0152] Intense staining for PDGF-D was noted in the developing
heart, lung, kidney and some muscle derivatives. FIGS. 16-18 show
the staining of the embryonic kidney. Intense staining of the
highly vascularized fibrous capsule (fc) surrounding the kidney,
the adjacent adrenal gland (ag), and in the most peripheral aspect
of the metanephric mesenchyme (mm) of the cortex was observed
(FIGS. 16 and 17). Staining was also observed in cells located in
the basal aspect of the branching ureter (FIG. 18), while the
developing nephron, including the ureter buds, glomeruli (gl) and
Henle's loops, were negative. Previous analysis have shown that
PDGFR-beta is expressed by the metanephric mesenchyme and the
developing vascular smooth muscle cells and mesangial cells of the
developing renal cortex. In contrast, renal expression of PDGF-B is
restricted to endothelial cells (Lindahl, P. et al., 1998,
Development 125:3313-3322). The non-overlapping patterns of
expression of the two PDGFR-beta ligands suggests that PDGF-B and
PDGF-D provide distinct signals to PDGFR-beta expressing
perivascular cells. This differential localization indicates that
PDGF-D might have a paracrine role in the proliferation and/or
commitment of PDGFR-beta expressing perivascular progenitor cells
of the undifferentiated metanephric mesenchyme. In line with the
phenotype of PDGF-B deficient mice, PDGF-B may then provide
proliferative signals and spatial clues of the branching vascular
tree of the kidney, thus allowing proliferation and co-recruitment
of the PDGFR-beta expressing perivascular cells to form the
mesangium of the glomeruli, and the smooth muscle cells of the
efferent and afferent arterioles.
[0153] The expression of PDGF-D partially overlaps with the
expression of PDGF-C in the cortical area of the developing kidney.
The different receptor specificities of PDGF-C and PDGF-D and their
apparent inability to form heterodimers indicate that the two novel
PDGFs may provide distinct signals for migration and proliferation
for at least two different cell populations in the undifferentiated
metanephric mesenchyme; either interstitial cell progenitors
expressing PDGF alpha-receptor, or the PDGFR-beta expressing
perivascular progenitor cells.
[0154] The phenotypic differences in the kidneys of mice lacking
PDGFR-alpha and PDGF-A argue for a unique role of PDGF-C in the
formation of the renal mesenchyme. Interestingly, a comparison of
the PDGFR-beta and PDGF-B deficient mice have not revealed a
similar phenotypic discrepancy arguing for, at least partially,
redundant roles of PDGF-D and PDGF-B during early stages of kidney
development.
EXAMPLE 5
Receptor Binding Properties of PDGF-D with the VEGF Receptors
[0155] To assess the interactions between PDGF-D and the VEGF
receptors, truncated PDGF-D was tested for its capacity to bind to
soluble Ig-fusion proteins containing the extracellular domains of
human VEGFR-1, VEGFR-2 and VEGFR-3 (Olofsson et al., 1998, Proc.
Natl. Acad. Sci. USA 95:11709-11714). An expression vector encoding
the PDGF/VEGF homology domain of PDGF-D was generated in the vector
pSecTag (Invitrogen). The primers 5'-CCCAAGCTTGAAGATCTTGAGAATAT 3'
(forward) (SEQ ID NO:30) and 5'-TGCTCTAGATCGAGGTGGTCTT 3' (reverse)
(SEQ ID NO:31) were used to amplify a 429 bp fragment (nucleotides
556 to 966 in FIG. 5) (SEQ ID NO:5) encoding amino acid residues
186 to 322 of FIG. 6 (SEQ ID NO:6). The fragment was subsequently
cloned into a HindIII and XbaI digested expression vector. COS
cells were transfected with the expression vector encoding
truncated PDGF-D or a control vector using calcium phosphate
precipitation. The expressed polypeptide included a C-terminal
c-myc tag and a 6X His tag (both derived from the pSecTag
vector).
[0156] The Ig-fusion proteins, designated VEGFR-1-Ig, VEGFR-2-Ig
and VEGFR-3-Ig, were transiently expressed in human 293 EBNA cells.
All Ig-fusion proteins were human VEGFRs. Cells were incubated for
24 hours after transfection, washed with Dulbecco's Modified Eagle
Medium (DMEM) containing 0.2% bovine serum albumin (BSA) and
starved for 24 hours. The fusion proteins were then precipitated
from the clarified conditioned medium using protein A-Sepharose
beads (Pharmacia). The beads were combined with 100 microliters of
10X binding buffer (5% BSA, 0.2% Tween 20 and 10 .mu.g/ml heparin)
and 900 microliter of conditioned medium prepared from the COS
cells transfected with the expression vector for truncated PDGF-D
or the control vector. The cells were then metabolically labeled
with .sup.35S-cysteine and methionine (Promix, Amersham) for 4 to 6
hours. After 2.5 hours at room temperature, the Sepharose beads
were washed three times with binding buffer at 4.degree. C., once
with phosphate buffered saline (PBS) and boiled in SDS-PAGE buffer.
Labeled proteins that were bound to the Ig-fusion proteins were
analyzed by SDS-PAGE under reducing conditions. Radiolabeled
proteins were detected using a phosphorimager analyzer and/or on
film. In all these analyses, radiolabeled PDGF-D failed to show any
interaction with any of the VEGF receptors. These results indicate
that secreted truncated PDGF-D does not bind to VEGF receptors R1,
R2 and R3.
EXAMPLE 6
PDGFR-beta Phosphorylation
[0157] To test if PDGF-D causes increased phosphorylation of the
PDGFR-beta, full-length and plasmin-digested PDGF-D were tested for
their capacity to bind to the PDGFR-beta and stimulate increased
phosphorylation.
[0158] A plasmin-digested preparation of PDGF-DD was generated and
analyzed since it is known that plasmin-digestion of full-length
PDGF-CC releases the core domain and thus allow the ligand to
interact with the receptor. Full length PDGF-DD was digested with
plasmin in 20 mM Tris-HCl (pH 7.5) containing 1 mM CaCl.sub.2, 1 mM
MgCl.sub.2 and 0.01% Tween 20 for 1.5 to 4.5 hours at 37.degree. C.
using two to three units of plasmin (Sigma) per ml.
[0159] Analysis of the plasmin-digested preparation of PDGF-DD by
SDS-PAGE under reducing conditions showed two prominent bands of 28
kDa and 15 kDa. The 15 kDa band was identified as the core domain
due to its immunoreactivity in immunoblotting with a peptide
antiserum raised against a sequence of PDGF-D just N-terminal of
the first cysteine residue in the core domain.
[0160] Serum-starved porcine aortic endothelial-1 (PAE-1) cells
stably expressing the human PDGFR-beta (Eriksson et al., 1992, EMBO
J 11:543-550) were incubated on ice for 90 minutes with a solution
of conditioned media mixed with an equal volume of PBS supplemented
with 1 mg/ml BSA and 10 ng/ml of PDGF-BB, 300 ng/ml or 1200 ng/ml
of full length human PDGF-DD homodimers or 300 ng/ml or 1200 ng/ml
of digested PDGF-DD. The full length and digested PDGF-DD
homodimers were produced as described above. Sixty minutes after
the addition of the polypeptides, the cells were lysed in lysis
buffer (20 mM tris-HC1, pH 7.5, 0.5% Triton X-100, 0.5% deoxycholic
acid, 10 mM EDTA, 1 mM orthovanadate, 1 mM PMSF 1% Trasylol). The
PDGFR-beta were immunoprecipitated from cleared lysates with rabbit
antisera against the human PDGFR-beta (Eriksson et al., 1992,
supra). The precipitated receptors were applied to a SDS-PAGE gel.
After SDS gel electrophoresis, the precipitated receptors were
transferred to nitrocellulose filters, and the filters were probed
with anti-phosphotyrosine antibody PY-20, (Transduction
Laboratories). The filters were then incubated with horseradish
peroxidase-conjugated anti-mouse antibodies. Bound antibodies were
detected using enhanced chemiluminescence (ECL, Amersham Inc). The
filters were then stripped and reprobed with the PDGFR-beta rabbit
antisera, and the amount of receptors was determined by incubation
with horseradish peroxidase-conjugated anti-rabbit antibodies.
Bound antibodies were detected using enhanced chemiluminescence
(ECL, Amersham Inc). The probing of the filters with PDGFR-beta
antibodies confirmed that equal amounts of the receptor were
present in all lanes. Human recombinant PDGF-BB (100 ng/ml) and
untreated cells were included in the experiment as a control. FIG.
19 shows plasmin-digested PDGF-DD efficiently induced PDGFR-beta
tyrosine phosphorylation. Full-length PDGF-DD failed to induce
PDGFR-beta tyrosine phosphorylation. PDGF-BB was included in the
experiment as a positive control. This indicates that
plasmin-digested PDGF-D is a PDGFR-beta ligand/agonist.
EXAMPLE 7
Competitive Binding Assay
[0161] Next, full length and plasmin-digested PDGF-D were tested
for their capacity to bind to human PDGF alpha- and beta-receptors
by analyzing their abilities to compete with PDGF-BB for binding to
the PDGF receptors. The binding experiments were performed on
porcine aortic endothelial-1 (PAE-1) cells stably expressing the
human PDGF alpha- and beta-receptors, respectively (Eriksson et
al., 1992, supra). Binding experiments were performed essentially
as in Heldin et al. (1998, EMBO J. 7:1387-1393). Different
concentrations of human full-length and plasmin-digested PDGF-DD,
or human PDGF-BB were mixed with 5 ng/ml of .sup.125I-PDGF-BB in
binding buffer (PBS containing 1 mg/ml of bovine serum albumin).
Aliquots were incubated with the receptor expressing PAE-1 cells
plated in 24-well culture dishes on ice for 90 minutes. After three
washes with binding buffer, cell-bound .sup.125I-PDGF-BB or
.sup.125I-PDGF-AA was extracted by lysis of cells in 20 mM
Tris-HCl, p H 7.5, 10% glycerol, 1% Triton X-100. The amount of
cell bound radioactivity was determined in a gamma-counter. An
increasing excess of the unlabeled protein added to the incubations
competed efficiently with cell association of the radiolabeled
tracer.
[0162] FIG. 20 provides a graphical representation of results which
show that conditioned medium containing plasmin-digested PDGF-DD
competes for binding with PDGF-BB homodimers for the PDGFRs-beta,
while the full length protein did not. Compared to PDGF-BB,
plasmin-activated PDGF-DD appeared 10-12 fold less efficient as a
competitor; probably a result of suboptimal activation of the
recombinant protein in vitro by the protease. Control experiments
showed that plasmin present in the digested PDGF-DD fraction did
not affect the binding of .sup.125I-labelled PDGF-BB to the
PDGFR-.beta.-expressing cells. Both the full length and
plasmin-digested PDGF-DD proteins failed to compete for binding to
the PDGFR-alpha (FIG. 21).
[0163] These studies indicate that PDGF-DD is a PDGFR-beta-specific
agonist and that proteolytic processing releases the core domains
of PDGF-DD from the N-terminal CUB domains which is necessary for
unmasking the receptor-binding epitopes of the core domain similar
to the situation for PDGF-CC.
EXAMPLE 8
Determination of Alternative Splicing of Murine PDGF-D
[0164] Primers were designed for the amplification of the whole
coding area of murine PDGF-D by PCR from mouse heart cDNA
(Clontech). These primers were: 5'-CAAATGCAACGGCTCGTTT-3' (SEQ ID
NO:41) and 5'-GATATTTGCTTCTTCTTGCCATGG-3' (SEQ ID NO:42). PCR
reaction conditions were as follows: PCR Cycles: 94.degree. C. for
2 minutes, followed by 30 cycles: 94.degree. C. for 45 seconds,
62.degree. C. for 45 seconds, 72.degree. C. for 90 seconds, and
72.degree. C. for 7 minutes.
[0165] The expected product from this reaction was a 1.2 kb cDNA
fragment. However, the product was two bands, one approximately 1.2
kb and the other only 1.0 kb. These two products were checked in a
1% agarose gel, purified from the gel (QIAquick Gel Extraction Kit,
Qiagen, Cat # 28706), cloned into a vector (TOPO TA Cloning Kit,
Invitrogen), and transformed into E. Coli bacteria.
[0166] Transformed bacteria were plated and incubated at 37.degree.
C. overnight. The next morning some single colonies were picked and
grown in fresh medium overnight. Plasmids were prepared (QIAprep
Spin Miniprep Kit, Qiagen, Cat # 27106) and sequenced with plasmid
primers T7 and M13R, and also with mPDGF-D specific primers. The
results revealed three different types of murine PDGF-D cDNAs, one
being completely identical with the earlier mouse clones, depicted
in SEQ ID NO: 35.
[0167] The second clone was almost identical to the earlier mouse
sequence, however, it lacked six amino acid residues (aa 42-47)
from the region between the signal sequence and the CUB domain. The
second clone is depicted in SEQ ID NO:37. The third clone was
comprised of part of the earlier mouse sequence, lacking amino
acids 42-47 as in the second clone, and also lacking the
PDGF-homology domain. The third clone is depicted in SEQ ID NO:39.
The similarities and differences between regions of the three
clones are depicted in FIG. 22.
[0168] The surprising results show that at least two alternatively
spliced versions of the PDGF-D gene are transcribed into
polyadenylated RNA. The variant transcript structures suggest an
alternative splice acceptor site is used in exon two, producing a
variant protein lacking six amino acid residues (ESNHLT).
[0169] In addition to lacking the above noted six amino acid
residues, the third clone also lacks the PDGF-homology domain. This
is because of the skipping of exon six and the resulting
frameshift. This ends the open reading frame in a stop codon after
four additional amino acid residues (GIEV). As shown in detail in
FIG. 23, this splice variant only contains the amino terminal CUB
domain and could potentially provide an inhibitor of PDGF-D
functions. The potential inhibition function is because the
activation of full-length PDGF-D binding to the PDGFR-D requires
proteolytic removal of the CUB domain.
EXAMPLE 9
Generation of Recombinant Human PDGF-DD Core Domain
[0170] The process as described (Bergsten et al., 2001, Nat. Cell
Biol. 3:512-516) was followed to generate recombinant human PDGF-DD
core domain. Human PDGF-DD was expressed as a mutant full-length
form containing a factor Xa protease cleavage site that allowed the
generation of the active C-terminal fragment of the protein
(PDGF-homology domain) upon cleavage with factor Xa. The
recombinant protein has an extreme C-terminal His.sub.6-tag to
allow its purification on a nickel-containing resin. Following
purification, the protein solution was dialyzed against 0.1 M
acetic acid and lyophilized. SDS-PAGE analysis under reducing
conditions on the purified protein revealed that it migrated as a
homogenous 21 kDa species (FIG. 24). The purified protein was
lyophilized for storage.
EXAMPLE 10
Comparison of Angiogenic Activities of the Human PDGF-DD Core
Domain with Other PDGF Isoforms
[0171] The mouse corneal micropocket assay was performed according
to procedures described in Cao et al., 1998, Proc Natl Acad Sci USA
95:14389-94; Cao et al., 1999, Nature 398:381. Specifically,
lyophilized proteins were dissolved in phosphate buffer solutions
(PBS) and used to make protein bound polymer beads, as
described.
[0172] The beads were then implanted in mouse cornea. Male 5-6
week-old C57BI6/J mice were acclimated and caged in groups of six
or less. Animals were anaesthetized by injection of a mixture of
dormicum and hypnorm (1:1) before all procedures. Corneal
micropockets were created with a modified von Graefe cataract knife
in both eyes of each male 5-6-week-old C57BI6/J mouse. A
micropellet (0.35.times.0.35 mm) of sucrose aluminum sulfate (Bukh
Meditec, Copenhagen, Denmark) coated with slow-release hydron
polymer type NCC (IFN Sciences, New Brunswick, N.J.) containing
various amounts of homodimers of truncated PDGF-DD was surgically
implanted into each cornal pocket.
[0173] For comparison purposes corresponding amounts of PDGF-AA,
PDGF-AB, PDGF-BB, and PDGF-CC were similarly implanted into corneal
pockets of test mice. In each case, the pellet was positioned
0.6-0.8 mm from the corneal limbus. After implantation,
erythromycin/ophthalmic ointment was applied to each eye.
[0174] On day 5 after growth factor implantation, animals were
sacrificed with a lethal dose of CO.sub.2, and corneal
neovascularization was measured and photographed with a slit-lamp
stereomicroscope. In FIGS. 25 A-E, arrows point to the implanted
pellets. Vessel length and clock hours of circumferential
neovascularization were measured. Quantitation of corneal
neovascularization is presented as maximal vessel length (FIG.
25F), clock hours of circumferential neovascularization (FIG. 25G),
and area of neovascularization (FIG. 25H). Graphs to represent mean
values (.ANG. SEM) of 11-16 eyes (6-8 mice) in each group.
[0175] The corneal angiogenesis model is one of the most rigorous
mammalian angiogenesis models that requires a putative compound to
be sufficiently potent in order to induce neovascularization in the
corneal avascular tissue. Potent angiogenic factors including FGF-2
and VEGF have profound effects in this system.
[0176] The results are shown in FIG. 25. The assays were done using
PDGF-AA (FIG. 25A), PDGF-AB (FIG. 25B), PDGF-BB (FIG. 25C), PDGF-CC
(FIG. 25D), and PDGF-DD (FIG. 25C). FIGS. 25F-H show the
quantitative analysis of vessel length, clock hours, and vessel
areas (means.+-.SD, n=4-6).
[0177] The overall angiogenic response induced by PDGF-DD was
similar to that induced by other PDGF isoforms. The results again
clearly demonstrate that the truncated PDGF-D homodimer exhibits
marked angiogenic activity in vivo. In light of the foregoing test
results, which demonstrate the in vivo angiogenesis inducing
activity of PDGF-DD, treatments with PDGF-DD alone, or in
combination with other angiogenic factors such as VEGF family
members and FGFs, provide an attractive approach for therapeutic
angiogenesis of ischemic heart, brain and limb disorders.
EXAMPLE 11
PDGF-D Promotes Connective Tissue Growth During Wound Healing.
[0178] The healing of wounds is a complex process involving three
discreet but overlapping stages: inflammation, proliferation and
repair, and remodeling. Wound healing involves many growth factors,
some of which exert different effects on multiple cell types. PDGF
in general has been known to be active in all stages of the healing
process and to promote wound healing. It is synthesized in
significant quantities throughout the process by a number of
different cells, including platelets, macrophages, fibroblasts and
endothelial cells. Despite the critical role played by other growth
factors involved in wound healing (EGF, FGF, insulin-like growth
factors, and the TGFs), only PDGF has been shown to augment wound
healing in vivo (Steed, 1998, Am. J Surg. 176:205-255). In fact, as
early as 1991, PDGF-B was used for would healing purposes. See e.g.
Pierce et al., 1991,. J. Biol. Chem. 45:319-326 "Role of PDGF in
wound-healing" and Pierce et al., 1994, Tissue repair processes in
healing chronic pressure ulcers treated with recombinant PDGF-BB,
Am. J. Pathology 145:1399-1410. Nagai and Embil (2002) Expert Opin.
Biol. Ther. 2:211-218 reviewed the use of recombinantly produced
PDGF-B and concluded that it is safe, effective and easy to use in
the treatment for healing diabetic foot ulcers.
[0179] PDGF-B and PDGF-D share the same type of receptors. The
effects of PDGF-D on wound healing were investigated using
transgenic mice which overexpress PDGF-D in skin keratinocytes. The
human PDGF-D gene was cloned and operatively linked with the
keratin 14 promoter (K-14 promoter), which directs the expression
of the gene to the basal epithelial cells of the skin of transgenic
animals (Jeltsch et al., 1997, Hyperplasia of lymphatic vessels in
VEGF-C transgenic mice, Science 276:1423-1425; Detmar et al., 1998,
Increased microvascular density and enhanced leukocyte rolling and
adhesion in the skin of VEGF transgenic mice, J. Investigative
Dermatol. 111:1-6). A schematic diagram of the K14-PDGF-D construct
is depicted in FIG. 26.
[0180] Transgenic mice overexpressing PDGF-D in skin keratinocytes
were obtained. Four mice from the same transgenic litter were
tested postitive for PDGF-D and four negative. FIG. 27 shows a
comparison of PDGF-D expression between K14-PDGF-D transgenic mouse
(TG) and wild-type mouse (wt). Paraffin embedded mouse skin samples
were stained with anti-PDGF-D. For experimental details, see Uutela
et al., 2001, Chromosomal location, exon structure and vascular
expression patterns of the human PDGFC and PDGFD genes, Circulation
103:2242-2247, which is incorporated herein by reference in its
entirety.
[0181] Mice were then anesthesised
(ksylatsine+ketaminehydrochloride) and punchbiopsy wounds were made
to their flank skin (4 wounds with a diameter of 6 mm per mouse).
An analgesic was used to inhibit pain (buprenorfine).
[0182] One positive and one negative mouse were sacrificed two days
after the wounding, and as can be seen from FIG. 28, the amount of
granulation tissue in the wound area was considerably greater in
the PDGF-D positive mouse (TG) when compared with transgene
negative littermate (wt).
[0183] The next mice were sacrificed after 4 days. The amount of
the developing connective tissue was greater in PDGF-D expressing
mouse as shown by the van Gieson elastic connective tissue stain
(FIG. 29). A similar augmentation of connective tissue development
was seen in the transgenic mice sacrificed 7 and 10 days after
wounding (FIG. 30).
[0184] Because increased amount of elastic connective tissue
results in a greater tensile strength of the PDGF-D treated wounds,
these results indicate that PDGF-D enhances the wound repair
process, and that PDGF-D can be used as a valuable enhancer of
wound healing.
[0185] The ability of PDGF-D to stimulate wound healing is also
tested in the most clinically relevant model available, as
described in Schilling et al., 1959, Surgery 46:702-710 and
utilized by Hunt et al., 1967, Surgery 114:302-307.
Bioassays to Determine the Function of PDGF-D
[0186] Assays are conducted to evaluate whether PDGF-D has similar
activities to PDGF-A, PDGF-B, VEGF, VEGF-B, VEGF-C and/or VEGF-D in
relation to growth and/or motility of connective tissue cells,
fibroblasts, myofibroblasts and glial cells; to endothelial cell
function; to angiogenesis; and to wound healing. Further assays may
also be performed, depending on the results of receptor binding
distribution studies.
I. Mitogenicity of PDGF-D for Endothelial Cells
[0187] To test the mitogenic capacity of PDGF-D for endothelial
cells, the PDGF-D polypeptide is introduced into cell culture
medium containing 5% serum and applied to bovine aortic endothelial
cells (BAEs) propagated in medium containing 10% serum. The BAEs
are previously seeded in 24-well dishes at a density of 10,000
cells per well the day before addition of the PDGF-D. Three days
after addition of this polypeptide the cells are dissociated with
trypsin and counted. Purified VEGF is included in the experiment as
positive control.
II. Mitogenicity of PDGF-D for Fibroblasts
[0188] To test the mitogenic capacity of PDGF-D for fibroblasts,
different concentrations of truncated homodimers of PDGF-DD or
PDGF-AA (as control) are added to serum starved human foreskin
fibroblasts in the presence of 0.2 .quadrature.mCi [3H]thymidine.
The fibroblasts are then incubated for 24 hours with 1 ml of
serum-free medium supplemented with 1 mg/ml BSA. After
trichloroacetic acid (TCA) precipitation, the incorporation of
[3H]thymidine into DNA is determined using a beta-counter. The
assay is performed essentially as described in Mori et al., 1991,
J. Biol. Chem. 266:21158-21164.
III. Assays of Endothelial Cell Function
[0189] a) Endothelial cell proliferation
[0190] Endothelial cell growth assays are performed by methods well
known in the art, e.g. those of Ferrara & Henzel, 1989, Nature
380:439-443, Gospodarowicz et al., 1989, Proc. Natl. Acad. Sci. USA
86:7311-7315, and/or Claffey et al., 1995, Biochem. Biophys. Acta
1246:1-9. [0191] b) Cell adhesion assay
[0192] The effect of PDGF-D on adhesion of polymorphonuclear
granulocytes to endothelial cells is tested. [0193] c)
Chemotaxis
[0194] The standard Boyden chamber chemotaxis assay is used to test
the effect of PDGF-D on chemotaxis. [0195] d) Plasminogen activator
assay
[0196] Endothelial cells are tested for the effect of PDGF-D on
plasminogen activator and plasminogen activator inhibitor
production, using the method of Pepper et al., 1991, Biochem.
Biophys. Res. Commun. 181:902-906. [0197] e) Endothelial cell
Migration assay
[0198] The ability of PDGF-D to stimulate endothelial cells to
migrate and form tubes is assayed as described in Montesano et al.,
1986, Proc. Natl. Acad. Sci. USA 83:7297-7301. Alternatively, the
three-dimensional collagen gel assay described in Joukov et al.,
1996, EMBO J. 15:290-298 or a gelatinized membrane in a modified
Boyden chamber (Glaser et al., 1980, Nature 288:483-484) may be
used.
IV. Angiogenesis Assay
[0199] The ability of PDGF-D to induce an angiogenic response in
chick chorioallantoic membrane is tested as described in Leung et
al., 1989, Science, 246:1306-1309. Alternatively the rat cornea
assay of Rastinejad et al., 1989, Cell, 56:345-355 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.
V. The Hemopoietic System
[0200] A variety of in vitro and in vivo assays using specific cell
populations of the hemopoietic 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 to purified
cells are particularly convenient: [0201] a) Repopulating Stem
Cells
[0202] These are cells capable of repopulating the bone marrow of
lethally irradiated mice, and have the Lin.sup.-, Rh.sup.hl,
Ly-6A/E.sup.+, c-kit.sup.+ phenotype. PDGF-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. [0203] b) Late Stage Stem
Cells
[0204] 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.hl, Ly-6A/E.sup.+, c-kit.sup.+ phenotype.
PDGF-D is incubated with these cells for a period of time, injected
into lethally irradiated recipients, and the number of D13 spleen
colonies is enumerated. [0205] c) Progenitor-Enriched Cells
[0206] These are cells that respond in vitro to single growth
factors and have the Lin.sup.-, Rh.sup.hl, Ly-6A/E.sup.+,
c-kit.sup.+ phenotype. This assay will show if PDGF-D can act
directly on haemopoietic progenitor cells. PDGF-D is incubated with
these cells in agar cultures, and the number of colonies present
after 7-14 days is counted.
VI. Atherosclerosis
[0207] 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 cover slips measures the
proliferative rate and phenotypic modulations of smooth muscle
cells in a multicellular environment, and is used to assess the
effect of PDGF-D on smooth muscle cells.
VII. Metastasis
[0208] The ability of PDGF-D to inhibit metastasis is assayed using
the Lewis lung carcinoma model, for example using the method of Cao
et al., 1995, J. Exp. Med. 182:2069-2077.
VIII. Migration of Smooth Muscle Cells
[0209] The effects of the PDGF-D on the migration of smooth muscle
cells and other cells types can be assayed using the method of
Koyama et al., 1992, J. Biol. Chem. 267:22806-22812.
IX. Chemotaxis
[0210] The effects of the PDGF-D on chemotaxis of fibroblast,
monocytes, granulocytes and other cells can be assayed using the
method of Siegbahn et al., 1990, J. Clin. Invest. 85:916-920.
X. PDGF-D in Other Cell Types
[0211] The effects of PDGF-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.
XI. Construction of PDGF-D Variants and Analogues
[0212] PDGF-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. PDGF-D contains seven 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. PDGF-D interacts with
a protein tyrosine kinase growth factor receptor.
[0213] 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
PDGF-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.
[0214] Published articles elucidating the structure/activity
relationships of members of the PDGF family of growth factors
include for PDGF: Oestman et al., 1991, J. Biol Chem.,
266:10073-10077; Andersson et al., 1992, J. Biol. Chem.,
267:11260-1266; Oeffier et al., 1992, EMBO J., 11:3921-3926;
Flemming et al., 1993, Molecular and Cell Biol., 13:4066-4076 and
Andersson et al., 1995, Growth Factors, 12:159-164; and for VEGF:
Kim et al, 1992, Growth Factors, 7:53-64; Potgens et al., 1994, J.
Biol Chem., 269:32879-32885 and Claffey et al., 1995, Biochem.
Biophys. Acta, 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.
[0215] Based on this information, a person skilled in the
biotechnology arts can design PDGF-D mutants with a very high
probability of retaining PDGF-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.
[0216] As used herein, the term "conservative substitution" denotes
the replacement of an amino acid residue by another, biologically
similar residue. Examples of conservative substitutions include the
substitution of one hydrophobic residue such as isoleucine, valine,
leucine, alanine, cysteine, glycine, phenylalanine, proline,
tryptophan, tyrosine, norleucine or methionine for another, or the
substitution of one polar residue for another, such as the
substitution of arginine for lysine, glutamic acid for aspartic
acid, or glutamine for asparagine, and the like. Neutral
hydrophilic amino acids which can be substituted for one another
include asparagine, glutamine, serine and threonine. The term
"conservative substitution" also includes the use of a substituted
amino acid in place of an unsubstituted parent amino acid.
[0217] As such, it should be understood that in the context of the
present invention, a conservative substitution is recognized in the
art as a substitution of one amino acid for another amino acid that
has similar properties. Exemplary conservative substitutions are
set out in the following Table A from WO 97/09433.
TABLE-US-00006 TABLE A Conservative Substitutions I SIDE CHAIN
CHARACTERISTIC AMINO ACID Aliphatic Non-polar G A P I L V Polar -
uncharged C S T M N Q Polar - charged D E K R Aromatic H F W Y
Other N Q D E
[0218] Alternatively, conservative amino acids can be grouped as
described in Lehninger, Biochemistry, Second Edition; Worth
Publishers, Inc. NY:N.Y. (1975), pp.71-77 as set out in the
following Table B.
TABLE-US-00007 TABLE B Conservative Substitutions II SIDE CHAIN
CHARACTERISTIC AMINO ACID Non-polar (hydrophobic) A. Aliphatic: A L
I V P B. Aromatic: F W C. Sulfur-containing: M D. Borderline: G
Uncharged-polar A. Hydroxyl: S T Y B. Amides: N Q C. Sulfhydryl: C
D. Borderline: G Positively Charged (Basic): K R H Negatively
Charged (Acidic): D E
[0219] Exemplary conservative substitutions are set out in the
following Table C.
TABLE-US-00008 TABLE C Conservative Substitutions III Original
Residue Exemplary Substitution Ala (A) Val, Leu, Ile Arg (R) Lys,
Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q)
Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met,
Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met
(M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S)
Thr Thr (T) Ser Trp (W) Tyr, Phe Tyr (Y) Trp, Phe, Thr, Ser Val (V)
Ile, Leu, Met, Phe, Ala
[0220] If desired, the peptides of the invention can be modified,
for instance, by glycosylation, amidation, carboxylation, or
phosphorylation, or by the creation of acid addition salts, amides,
esters, in particular C-terminal esters, and N-acyl derivatives of
the peptides of the invention. The peptides also can be modified to
create peptide derivatives by forming covalent or noncovalent
complexes with other moieties. Covalently-bound complexes can be
prepared by linking the chemical moieties to functional groups on
the side chains of amino acids comprising the peptides, or at the
N- or C-terminus.
[0221] In particular, it is anticipated that the aforementioned
peptides can be conjugated to a reporter group, including, but not
limited to a radiolabel, a fluorescent label, an enzyme (e.g., that
catalyzes a calorimetric or fluorometric reaction), a substrate, a
solid matrix, or a carrier (e.g., biotin or avidin).
[0222] 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.,
1987, Methods in Enzymol. 154:367-382). Examples of such
site-directed mutagenesis with VEGF can be found in Potgens et al.,
1994, J. Biol. Chem. 269:32879-32885 and Claffey et al., 1995,
Biochem. Biophys. Acta, 1246:1-9. Indeed, site-directed mutagenesis
is so common that kits are commercially available to facilitate
such procedures (e.g. Promega 1994-1995 Catalog., Pages
142-145).
[0223] The connective tissue cell, fibroblast, myofibroblast and
glial cell growth and/or motility activity, the endothelial cell
proliferation activity, the angiogenesis activity and/or the wound
healing activity of PDGF-D mutants can be readily confirmed by
well-established routine screening procedures. For example, a
procedure analogous to the endothelial cell mitotic assay described
by Claffey et al., 1995, Biochem. Biophys. Acta. 1246:1-9) can be
used. Similarly the effects of PDGF-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.
[0224] The foregoing description and examples have been set forth
merely to illustrate the invention and are not intended to be
limiting. Since modifications of the disclosed embodiments
incorporating the spirit and substance of the invention may occur
to persons skilled in the art, the invention should be construed
broadly to include all variations falling within the scope of the
appended claims and equivalents thereof.
Sequence CWU 1
1
421360DNAHomo sapiens 1aattgtggct gtggaactgt caactggagg tcctgcacat
gcaattcagg gaaaaccgtg 60aaaaagtatc atgaggtatt acagtttgag cctggccaca
tcaagaggag gggtagagct 120aagaccatgg ctctagttga catccagttg
gatcaccatg aacgatgtga ttgtatctgc 180agctcaagac cacctcgata
agagaatgtg cacatcctta cattaagcct gaaagaacca 240ttagtttaag
gagggtgaga taagagaccc ttttcctacc agcaaccaga cttactacta
300gcctgcaatg caatgaacac aagtggttgc tgagtctcag ccttgctttg
ttaatgccat 360266PRTHomo sapiens 2Asn Cys Gly Cys Gly Thr Val Asn
Trp Arg Ser Cys Thr Cys Asn Ser1 5 10 15Gly Lys Thr Val Lys Lys Tyr
His Glu Val Leu Gln Phe Glu Pro Gly 20 25 30His Ile Lys Arg Arg Gly
Arg Ala Lys Thr Met Ala Leu Val Asp Ile 35 40 45Gln Leu Asp His His
Glu Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro 50 55 60Pro
Arg653690DNAHomo sapiens 3ggaagatttc caacccgcag cagcttcaga
gaccaactgg aatctgtcac aagctctgtt 60tcagggtatc cctataactc tccatcagta
acggatccca ctctgattgc ggatgctctg 120gacaaaaaaa ttgcagaatt
tgatacagtg gaagatctgc tcaagtactt caatccagag 180tcatggcaag
aagatcttga gaatatgtat ctggacaccc ctcggtatcg aggcaggtca
240taccatgacc ggaagtcaaa agttgacctg gataggctca atgatgatgc
caagcgttac 300agttgcactc ccaggaatta ctcggtcaat ataagagaag
agctgaagtt ggccaatgtg 360gtcttctttc cacgttgcct cctcgtgcag
cgctgtggag gaaattgtgg ctgtggaact 420gtcaaactgg agtcctgcac
atgcaattca gggaaaaccg tgaaaaagta tcatgaggta 480ttacagtttg
agcctggcca catcaagagg aggggtagag ctaagaccat ggctctagtt
540gacatccagt tggatcacca tgaacgatgc gattgtatct gcagctcaag
accacctcga 600taagagaatg tgcacatcct tacattaagc ctgaaagaac
ctttagttta aggagggtga 660gataagagac ccttttccta ccagcaaccc
6904200PRTHomo sapiens 4Gly Arg Phe Pro Thr Arg Ser Ser Phe Arg Asp
Gln Leu Glu Ser Val1 5 10 15Thr Ser Ser Val Ser Gly Tyr Pro Tyr Asn
Ser Pro Ser Val Thr Asp 20 25 30Pro Thr Leu Ile Ala Asp Ala Leu Asp
Lys Lys Ile Ala Glu Phe Asp 35 40 45Thr Val Glu Asp Leu Leu Lys Tyr
Phe Asn Pro Glu Ser Trp Gln Glu 50 55 60Asp Leu Glu Asn Met Tyr Leu
Asp Thr Pro Arg Tyr Arg Gly Arg Ser65 70 75 80Tyr His Asp Arg Lys
Ser Lys Val Asp Leu Asp Arg Leu Asn Asp Asp 85 90 95Ala Lys Arg Tyr
Ser Cys Thr Pro Arg Asn Tyr Ser Val Asn Ile Arg 100 105 110Glu Glu
Leu Lys Leu Ala Asn Val Val Phe Phe Pro Arg Cys Leu Leu 115 120
125Val Gln Arg Cys Gly Gly Asn Cys Gly Cys Gly Thr Val Lys Leu Glu
130 135 140Ser Cys Thr Cys Asn Ser Gly Lys Thr Val Lys Lys Tyr His
Glu Val145 150 155 160Leu Gln Phe Glu Pro Gly His Ile Lys Arg Arg
Gly Arg Ala Lys Thr 165 170 175Met Ala Leu Val Asp Ile Gln Leu Asp
His His Glu Arg Cys Asp Cys 180 185 190Ile Cys Ser Ser Arg Pro Pro
Arg 195 20051934DNAHomo sapiensCDS(1)..(966) 5ttg tac cga aga gat
gag acc atc cag gtg aaa gga aac ggc tac gtg 48Leu Tyr Arg Arg Asp
Glu Thr Ile Gln Val Lys Gly Asn Gly Tyr Val1 5 10 15cag agt cct aga
ttc ccg aac agc tac ccc agg aac ctg ctc ctg aca 96Gln Ser Pro Arg
Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr 20 25 30tgg cgg ctt
cac tct cag gag aat aca cgg ata cag cta gtg ttt gac 144Trp Arg Leu
His Ser Gln Glu Asn Thr Arg Ile Gln Leu Val Phe Asp 35 40 45aat cag
ttt gga tta gag gaa gca gaa aat gat atc tgt agg tat gat 192Asn Gln
Phe Gly Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp 50 55 60ttt
gtg gaa gtt gaa gat ata tcc gaa acc agt acc att att aga gga 240Phe
Val Glu Val Glu Asp Ile Ser Glu Thr Ser Thr Ile Ile Arg Gly65 70 75
80cga tgg tgt gga cac aag gaa gtt cct cca agg ata aaa tca aga acg
288Arg Trp Cys Gly His Lys Glu Val Pro Pro Arg Ile Lys Ser Arg Thr
85 90 95aac caa att aaa atc aca ttc aag tcc gat gac tac ttt gtg gct
aaa 336Asn Gln Ile Lys Ile Thr Phe Lys Ser Asp Asp Tyr Phe Val Ala
Lys 100 105 110cct gga ttc aag att tat tat tct ttg ctg gaa gat ttc
caa ccc gca 384Pro Gly Phe Lys Ile Tyr Tyr Ser Leu Leu Glu Asp Phe
Gln Pro Ala 115 120 125gca gct tca gag acc aac tgg gaa tct gtc aca
agc tct att tca ggg 432Ala Ala Ser Glu Thr Asn Trp Glu Ser Val Thr
Ser Ser Ile Ser Gly 130 135 140gta tcc tat aac tct cca tca gta acg
gat ccc act ctg att gcg gat 480Val Ser Tyr Asn Ser Pro Ser Val Thr
Asp Pro Thr Leu Ile Ala Asp145 150 155 160gct ctg gac aaa aaa att
gca gaa ttt gat aca gtg gaa gat ctg ctc 528Ala Leu Asp Lys Lys Ile
Ala Glu Phe Asp Thr Val Glu Asp Leu Leu 165 170 175aag tac ttc aat
cca gag tca tgg caa gaa gat ctt gag aat atg tat 576Lys Tyr Phe Asn
Pro Glu Ser Trp Gln Glu Asp Leu Glu Asn Met Tyr 180 185 190ctg gac
acc cct cgg tat cga ggc agg tca tac cat gac cgg aag tca 624Leu Asp
Thr Pro Arg Tyr Arg Gly Arg Ser Tyr His Asp Arg Lys Ser 195 200
205aaa gtt gac ctg gat agg ctc aat gat gat gcc aag cgt tac agt tgc
672Lys Val Asp Leu Asp Arg Leu Asn Asp Asp Ala Lys Arg Tyr Ser Cys
210 215 220act ccc agg aat tac tcg gtc aat ata aga gaa gag ctg aag
ttg gcc 720Thr Pro Arg Asn Tyr Ser Val Asn Ile Arg Glu Glu Leu Lys
Leu Ala225 230 235 240aat gtg gtc ttc ttt cca cgt tgc ctc ctc gtg
cag cgc tgt gga gga 768Asn Val Val Phe Phe Pro Arg Cys Leu Leu Val
Gln Arg Cys Gly Gly 245 250 255aat tgt ggc tgt gga act gtc aac tgg
agg tcc tgc aca tgc aat tca 816Asn Cys Gly Cys Gly Thr Val Asn Trp
Arg Ser Cys Thr Cys Asn Ser 260 265 270ggg aaa acc gtg aaa aag tat
cat gag gta tta cag ttt gag cct ggc 864Gly Lys Thr Val Lys Lys Tyr
His Glu Val Leu Gln Phe Glu Pro Gly 275 280 285cac atc aag agg agg
ggt aga gct aag acc atg gct cta gtt gac atc 912His Ile Lys Arg Arg
Gly Arg Ala Lys Thr Met Ala Leu Val Asp Ile 290 295 300cag ttg gat
cac cat gaa cga tgc gat tgt atc tgc agc tca aga cca 960Gln Leu Asp
His His Glu Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro305 310 315
320cct cga taagagaatg tgcacatcct tacattaagc ctgaaagaac ctttagttta
1016Pro Argaggagggtga gataagagac ccttttccta ccagcaacca aacttactac
tagcctgcaa 1076tgcaatgaac acaagtggtt gctgagtctc agccttgctt
tgttaatgcc atggcaagta 1136gaaaggtata tcatcaactt ctatacctaa
gaatatagga ttgcatttaa taatagtgtt 1196tgaggttata tatgcacaaa
cacacacaga aatatattca tgtctatgtg tatatagatc 1256aaatgttttt
tttggtatat ataaccaggt acaccagagc ttacatatgt ttgagttaga
1316ctcttaaaat cctttgccaa aataagggat ggtcaaatat atgaaacatg
tctttagaaa 1376atttaggaga taaatttatt tttaaatttt gaaacacaaa
acaattttga atcttgctct 1436cttaaagaaa gcatcttgta tattaaaaat
caaaagatga ggctttctta catatacatc 1496ttagttgatt attaaaaaag
gaaaaaggtt tccagagaaa aggccaatac ctaagcattt 1556tttccatgag
aagcactgca tacttaccta tgtggactgt aataacctgt ctccaaaacc
1616atgccataat aatataagtg ctttagaaat taaatcattg tgttttttat
gcattttgct 1676gaggcatcct tattcattta acacctatct caaaaactta
cttagaaggt tttttattat 1736agtcctacaa aagacaatgt ataagctgta
acagaatttt gaattgtttt tctttgcaaa 1796acccctccac aaaagcaaat
cctttcaaga atggcatggg cattctgtat gaacctttcc 1856agatggtgtt
cagtgaaaga tgtgggtagt tgagaactta aaaagtgaac attgaaacat
1916cgacgtaact ggaaaccg 19346322PRTHomo sapiens 6Leu Tyr Arg Arg
Asp Glu Thr Ile Gln Val Lys Gly Asn Gly Tyr Val1 5 10 15Gln Ser Pro
Arg Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr 20 25 30Trp Arg
Leu His Ser Gln Glu Asn Thr Arg Ile Gln Leu Val Phe Asp 35 40 45Asn
Gln Phe Gly Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp 50 55
60Phe Val Glu Val Glu Asp Ile Ser Glu Thr Ser Thr Ile Ile Arg Gly65
70 75 80Arg Trp Cys Gly His Lys Glu Val Pro Pro Arg Ile Lys Ser Arg
Thr 85 90 95Asn Gln Ile Lys Ile Thr Phe Lys Ser Asp Asp Tyr Phe Val
Ala Lys 100 105 110Pro Gly Phe Lys Ile Tyr Tyr Ser Leu Leu Glu Asp
Phe Gln Pro Ala 115 120 125Ala Ala Ser Glu Thr Asn Trp Glu Ser Val
Thr Ser Ser Ile Ser Gly 130 135 140Val Ser Tyr Asn Ser Pro Ser Val
Thr Asp Pro Thr Leu Ile Ala Asp145 150 155 160Ala Leu Asp Lys Lys
Ile Ala Glu Phe Asp Thr Val Glu Asp Leu Leu 165 170 175Lys Tyr Phe
Asn Pro Glu Ser Trp Gln Glu Asp Leu Glu Asn Met Tyr 180 185 190Leu
Asp Thr Pro Arg Tyr Arg Gly Arg Ser Tyr His Asp Arg Lys Ser 195 200
205Lys Val Asp Leu Asp Arg Leu Asn Asp Asp Ala Lys Arg Tyr Ser Cys
210 215 220Thr Pro Arg Asn Tyr Ser Val Asn Ile Arg Glu Glu Leu Lys
Leu Ala225 230 235 240Asn Val Val Phe Phe Pro Arg Cys Leu Leu Val
Gln Arg Cys Gly Gly 245 250 255Asn Cys Gly Cys Gly Thr Val Asn Trp
Arg Ser Cys Thr Cys Asn Ser 260 265 270Gly Lys Thr Val Lys Lys Tyr
His Glu Val Leu Gln Phe Glu Pro Gly 275 280 285His Ile Lys Arg Arg
Gly Arg Ala Lys Thr Met Ala Leu Val Asp Ile 290 295 300Gln Leu Asp
His His Glu Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro305 310 315
320Pro Arg72253DNAHomo sapiensCDS(176)..(1288) 7cgctcggaaa
gttcagcatg caggaagttt ggggagagct cggcgattag cacagcgacc 60cgggccagcg
cagggcgagc gcaggcggcg agagcgcagg gcggcgcggc gtcggtcccg
120ggagcagaac ccggcttttt cttggagcga cgctgtctct agtcgctgat cccaa atg
178 Met 1cac cgg ctc atc ttt gtc tac act cta atc tgc gca aac ttt
tgc agc 226His Arg Leu Ile Phe Val Tyr Thr Leu Ile Cys Ala Asn Phe
Cys Ser 5 10 15tgt cgg gac act tct gca acc ccg cag agc gca tcc atc
aaa gct ttg 274Cys Arg Asp Thr Ser Ala Thr Pro Gln Ser Ala Ser Ile
Lys Ala Leu 20 25 30cgc aac gcc aac ctc agg cga gat gag agc aat cac
ctc aca gac ttg 322Arg Asn Ala Asn Leu Arg Arg Asp Glu Ser Asn His
Leu Thr Asp Leu 35 40 45tac cga aga gat gag acc atc cag gtg aaa gga
aac ggc tac gtg cag 370Tyr Arg Arg Asp Glu Thr Ile Gln Val Lys Gly
Asn Gly Tyr Val Gln50 55 60 65agt cct aga ttc ccg aac agc tac ccc
agg aac ctg ctc ctg aca tgg 418Ser Pro Arg Phe Pro Asn Ser Tyr Pro
Arg Asn Leu Leu Leu Thr Trp 70 75 80cgg ctt cac tct cag gag aat aca
cgg ata cag cta gtg ttt gac aat 466Arg Leu His Ser Gln Glu Asn Thr
Arg Ile Gln Leu Val Phe Asp Asn 85 90 95cag ttt gga tta gag gaa gca
gaa aat gat atc tgt agg tat gat ttt 514Gln Phe Gly Leu Glu Glu Ala
Glu Asn Asp Ile Cys Arg Tyr Asp Phe 100 105 110gtg gaa gtt gaa gat
ata tcc gaa acc agt acc att att aga gga cga 562Val Glu Val Glu Asp
Ile Ser Glu Thr Ser Thr Ile Ile Arg Gly Arg 115 120 125tgg tgt gga
cac aag gaa gtt cct cca agg ata aaa tca aga acg aac 610Trp Cys Gly
His Lys Glu Val Pro Pro Arg Ile Lys Ser Arg Thr Asn130 135 140
145caa att aaa atc aca ttc aag tcc gat gac tac ttt gtg gct aaa cct
658Gln Ile Lys Ile Thr Phe Lys Ser Asp Asp Tyr Phe Val Ala Lys Pro
150 155 160gga ttc aag att tat tat tct ttg ctg gaa gat ttc caa ccc
gca gca 706Gly Phe Lys Ile Tyr Tyr Ser Leu Leu Glu Asp Phe Gln Pro
Ala Ala 165 170 175gct tca gag acc aac tgg gaa tct gtc aca agc tct
att tca ggg gta 754Ala Ser Glu Thr Asn Trp Glu Ser Val Thr Ser Ser
Ile Ser Gly Val 180 185 190tcc tat aac tct cca tca gta acg gat ccc
act ctg att gcg gat gct 802Ser Tyr Asn Ser Pro Ser Val Thr Asp Pro
Thr Leu Ile Ala Asp Ala 195 200 205ctg gac aaa aaa att gca gaa ttt
gat aca gtg gaa gat ctg ctc aag 850Leu Asp Lys Lys Ile Ala Glu Phe
Asp Thr Val Glu Asp Leu Leu Lys210 215 220 225tac ttc aat cca gag
tca tgg caa gaa gat ctt gag aat atg tat ctg 898Tyr Phe Asn Pro Glu
Ser Trp Gln Glu Asp Leu Glu Asn Met Tyr Leu 230 235 240gac acc cct
cgg tat cga ggc agg tca tac cat gac cgg aag tca aaa 946Asp Thr Pro
Arg Tyr Arg Gly Arg Ser Tyr His Asp Arg Lys Ser Lys 245 250 255gtt
gac ctg gat agg ctc aat gat gat gcc aag cgt tac agt tgc act 994Val
Asp Leu Asp Arg Leu Asn Asp Asp Ala Lys Arg Tyr Ser Cys Thr 260 265
270ccc agg aat tac tcg gtc aat ata aga gaa gag ctg aag ttg gcc aat
1042Pro Arg Asn Tyr Ser Val Asn Ile Arg Glu Glu Leu Lys Leu Ala Asn
275 280 285gtg gtc ttc ttt cca cgt tgc ctc ctc gtg cag cgc tgt gga
gga aat 1090Val Val Phe Phe Pro Arg Cys Leu Leu Val Gln Arg Cys Gly
Gly Asn290 295 300 305tgt ggc tgt gga act gtc aac tgg agg tcc tgc
aca tgc aat tca ggg 1138Cys Gly Cys Gly Thr Val Asn Trp Arg Ser Cys
Thr Cys Asn Ser Gly 310 315 320aaa acc gtg aaa aag tat cat gag gta
tta cag ttt gag cct ggc cac 1186Lys Thr Val Lys Lys Tyr His Glu Val
Leu Gln Phe Glu Pro Gly His 325 330 335atc aag agg agg ggt aga gct
aag acc atg gct cta gtt gac atc cag 1234Ile Lys Arg Arg Gly Arg Ala
Lys Thr Met Ala Leu Val Asp Ile Gln 340 345 350ttg gat cac cat gaa
cga tgc gat tgt atc tgc agc tca aga cca cct 1282Leu Asp His His Glu
Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro Pro 355 360 365cga taa
gagaatgtgc acatccttac attaagcctg aaagaacctt tagtttaagg
1338Arg370agggtgagat aagagaccct tttcctacca gcaaccaaac ttactactag
cctgcaatgc 1398aatgaacaca agtggttgct gagtctcagc cttgctttgt
taatgccatg gcaagtagaa 1458aggtatatca tcaacttcta tacctaagaa
tataggattg catttaataa tagtgtttga 1518ggttatatat gcacaaacac
acacagaaat atattcatgt ctatgtgtat atagatcaaa 1578tgtttttttt
ggtatatata accaggtaca ccagagctta catatgtttg agttagactc
1638ttaaaatcct ttgccaaaat aagggatggt caaatatatg aaacatgtct
ttagaaaatt 1698taggagataa atttattttt aaattttgaa acacaaaaca
attttgaatc ttgctctctt 1758aaagaaagca tcttgtatat taaaaatcaa
aagatgaggc tttcttacat atacatctta 1818gttgattatt aaaaaaggaa
aaaggtttcc agagaaaagg ccaataccta agcatttttt 1878ccatgagaag
cactgcatac ttacctatgt ggactgtaat aacctgtctc caaaaccatg
1938ccataataat ataagtgctt tagaaattaa atcattgtgt tttttatgca
ttttgctgag 1998gcatccttat tcatttaaca cctatctcaa aaacttactt
agaaggtttt ttattatagt 2058cctacaaaag acaatgtata agctgtaaca
gaattttgaa ttgtttttct ttgcaaaacc 2118cctccacaaa agcaaatcct
ttcaagaatg gcatgggcat tctgtatgaa cctttccaga 2178tggtgttcag
tgaaagatgt gggtagttga gaacttaaaa agtgaacatt gaaacatcga
2238cgtaactgga aaccg 22538370PRTHomo sapiens 8Met His Arg Leu Ile
Phe Val Tyr Thr Leu Ile Cys Ala Asn Phe Cys1 5 10 15Ser Cys Arg Asp
Thr Ser Ala Thr Pro Gln Ser Ala Ser Ile Lys Ala 20 25 30Leu Arg Asn
Ala Asn Leu Arg Arg Asp Glu Ser Asn His Leu Thr Asp 35 40 45Leu Tyr
Arg Arg Asp Glu Thr Ile Gln Val Lys Gly Asn Gly Tyr Val 50 55 60Gln
Ser Pro Arg Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr65 70 75
80Trp Arg Leu His Ser Gln Glu Asn Thr Arg Ile Gln Leu Val Phe Asp
85 90 95Asn Gln Phe Gly Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr
Asp 100 105 110Phe Val Glu Val Glu Asp Ile Ser Glu Thr Ser Thr Ile
Ile Arg Gly 115 120 125Arg Trp Cys Gly His Lys Glu Val Pro Pro Arg
Ile Lys Ser Arg Thr 130 135 140Asn Gln Ile Lys Ile Thr Phe Lys Ser
Asp Asp Tyr Phe Val Ala Lys145 150 155 160Pro Gly Phe Lys Ile Tyr
Tyr Ser Leu Leu Glu Asp Phe Gln Pro Ala 165 170 175Ala Ala Ser Glu
Thr Asn Trp Glu Ser Val Thr Ser Ser Ile Ser Gly 180 185 190Val
Ser Tyr Asn Ser Pro Ser Val Thr Asp Pro Thr Leu Ile Ala Asp 195 200
205Ala Leu Asp Lys Lys Ile Ala Glu Phe Asp Thr Val Glu Asp Leu Leu
210 215 220Lys Tyr Phe Asn Pro Glu Ser Trp Gln Glu Asp Leu Glu Asn
Met Tyr225 230 235 240Leu Asp Thr Pro Arg Tyr Arg Gly Arg Ser Tyr
His Asp Arg Lys Ser 245 250 255Lys Val Asp Leu Asp Arg Leu Asn Asp
Asp Ala Lys Arg Tyr Ser Cys 260 265 270Thr Pro Arg Asn Tyr Ser Val
Asn Ile Arg Glu Glu Leu Lys Leu Ala 275 280 285Asn Val Val Phe Phe
Pro Arg Cys Leu Leu Val Gln Arg Cys Gly Gly 290 295 300Asn Cys Gly
Cys Gly Thr Val Asn Trp Arg Ser Cys Thr Cys Asn Ser305 310 315
320Gly Lys Thr Val Lys Lys Tyr His Glu Val Leu Gln Phe Glu Pro Gly
325 330 335His Ile Lys Arg Arg Gly Arg Ala Lys Thr Met Ala Leu Val
Asp Ile 340 345 350Gln Leu Asp His His Glu Arg Cys Asp Cys Ile Cys
Ser Ser Arg Pro 355 360 365Pro Arg 37094PRTHomo
sapiensmisc_featureA putative proteolytic site found at residues
255-258 of SEQ ID NO8 (PDGF-D) 9Arg Lys Ser Lys11091PRTHomo
sapiensmisc_featurePDGF/VEGF-homology domain of PDGF-D 10Cys Thr
Pro Arg Asn Tyr Ser Val Asn Ile Arg Glu Glu Leu Lys Leu1 5 10 15Ala
Asn Val Val Phe Phe Pro Arg Cys Leu Leu Val Gln Arg Cys Gly 20 25
30Gly Asn Cys Gly Cys Gly Thr Val Lys Leu Glu Ser Cys Thr Cys Asn
35 40 45Ser Gly Lys Thr Val Lys Lys Tyr His Glu Val Leu Gln Phe Glu
Pro 50 55 60Gly His Ile Lys Arg Arg Gly Arg Ala Lys Thr Met Ala Leu
Val Asp65 70 75 80Ile Gln Leu Asp His His Glu Arg Cys Asp Cys 85
901188PRTHomo sapiensmisc_featurePDGF/VEGF-homology domain of
PDGF-C 11Cys Thr Pro Arg Asn Phe Ser Val Ser Ile Arg Glu Glu Leu
Lys Arg1 5 10 15Thr Asp Thr Ile Phe Trp Pro Gly Cys Leu Leu Val Lys
Arg Cys Gly 20 25 30Gly Asn Cys Ala Cys Cys Leu His Asn Cys Asn Glu
Cys Gln Cys Val 35 40 45Pro Ser Lys Val Thr Lys Lys Tyr His Glu Val
Leu Gln Leu Arg Pro 50 55 60Lys Thr Gly Val Arg Gly Leu His Lys Ser
Leu Thr Asp Val Ala Leu65 70 75 80Glu His His Glu Glu Cys Asp Cys
851284PRTHomo sapiensmisc_featurePDGF/VEGF-homology domain of
PDGF-A 12Cys Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln
Val Asp1 5 10 15Pro Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val
Glu Val Lys 20 25 30Arg Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys
Cys Gln Pro Ser 35 40 45Arg Val His His Arg Ser Val Lys Val Ala Lys
Val Glu Tyr Val Arg 50 55 60Lys Lys Pro Lys Leu Lys Glu Val Gln Val
Arg Leu Glu Glu His Leu65 70 75 80Glu Cys Ala Cys1384PRTHomo
sapiensmisc_featurePDGF/VEGF-homology domain of PDGF-B 13Cys Lys
Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp1 5 10 15Arg
Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln 20 25
30Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr
35 40 45Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val
Arg 50 55 60Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp
His Leu65 70 75 80Ala Cys Lys Cys1479PRTHomo
sapiensmisc_featurePDGF/VEGF-homology domain of VEGF-165 14Cys His
Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp1 5 10 15Glu
Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys 20 25
30Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu
35 40 45Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly
Gln 50 55 60His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu
Cys65 70 751579PRTHomo sapiensmisc_featurePDGF/VEGF-homology domain
of PlGF-2 15Cys Arg Ala Leu Glu Arg Leu Val Asp Val Val Ser Glu Tyr
Pro Ser1 5 10 15Glu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu
Leu Arg Cys 20 25 30Thr Gly Cys Cys Gly Asp Glu Asp Leu His Cys Val
Pro Val Glu Thr 35 40 45Ala Asn Val Thr Met Gln Leu Leu Lys Ile Arg
Ser Gly Asp Arg Pro 50 55 60Ser Tyr Val Glu Leu Thr Phe Ser Gln His
Val Arg Cys Glu Cys65 70 751678PRTHomo
sapiensmisc_featurePDGF/VEGF-homology domain of VEGF-B167 16Cys Gln
Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly1 5 10 15Thr
Val Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys 20 25
30Gly Gly Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln
35 40 45His Gln Val Arg Met Gln Ile Leu Met Ile Arg Tyr Pro Ser Ser
Gln 50 55 60Leu Gly Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu
Cys65 70 751781PRTHomo sapiensmisc_featurePDGF/VEGF-homology domain
of VEGF-C 17Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe
Gly Val1 5 10 15Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val
Tyr Arg Cys 20 25 30Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met
Asn Thr Ser Thr 35 40 45Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr
Val Pro Leu Ser Gln 50 55 60Gly Pro Lys Pro Val Thr Ile Ser Phe Ala
Asn His Thr Ser Cys Arg65 70 75 80Cys1881PRTHomo
sapiensmisc_featurePDGF/VEGF-homology domain of VEGF-D 18Cys Ser
Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys1 5 10 15Thr
Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys 20 25
30Gly Gly Cys Cys Asn Glu Glu Gly Val Met Cys Met Asn Thr Ser Thr
35 40 45Ser Tyr Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr
Ser 50 55 60Val Pro Glu Leu Val Pro Val Lys Ile Ala Asn His Thr Gly
Cys Lys65 70 75 80Cys19118PRTHomo sapiensmisc_featureCUB domain of
PDGF-D 19Asp Glu Thr Ile Gln Val Lys Gly Asn Gly Tyr Val Gln Ser
Pro Arg1 5 10 15Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr Trp
Arg Leu His 20 25 30Ser Gln Glu Asn Thr Arg Ile Gln Leu Val Phe Asp
Asn Gln Phe Gly 35 40 45Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr
Asp Phe Val Glu Val 50 55 60Glu Asp Ile Ser Glu Thr Ser Thr Ile Ile
Arg Gly Arg Trp Cys Gly65 70 75 80His Lys Glu Val Pro Pro Arg Ile
Lys Ser Arg Thr Asn Gln Ile Lys 85 90 95Ile Thr Phe Lys Ser Asp Asp
Tyr Phe Val Ala Lys Pro Gly Phe Lys 100 105 110Ile Tyr Tyr Ser Leu
Leu 11520113PRTHomo sapiensmisc_featureCUB domain 1 of BMP-1 20Cys
Gly Glu Thr Leu Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro Glu1 5 10
15Tyr Pro Asn Gly Tyr Ser Ala His Met His Cys Val Trp Arg Ile Ser
20 25 30Val Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp
Leu 35 40 45Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg
Asp Gly 50 55 60Phe Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe Cys Gly
Ser Lys Leu65 70 75 80Pro Glu Pro Ile Val Ser Thr Asp Ser Arg Leu
Trp Val Glu Phe Arg 85 90 95Ser Ser Ser Asn Trp Val Gly Lys Gly Phe
Phe Ala Val Tyr Glu Ala 100 105 110Ile21112PRTHomo
sapiensmisc_featureCUB domain 2 of BMP-1 21Cys Gly Gly Asp Val Lys
Lys Asp Tyr Gly His Ile Gln Ser Pro Asn1 5 10 15Tyr Pro Asp Asp Tyr
Arg Pro Ser Lys Val Cys Ile Trp Arg Ile Gln 20 25 30Val Ser Glu Gly
Phe His Val Gly Leu Thr Phe Gln Ser Phe Glu Ile 35 40 45Glu Arg Met
Asp Ser Cys Ala Tyr Asp Tyr Leu Glu Val Arg Asp Gly 50 55 60His Ser
Glu Ser Ser Thr Leu Ile Gly Arg Tyr Cys Gly Tyr Glu Lys65 70 75
80Pro Asp Asp Ile Lys Ser Thr Ser Ser Arg Leu Trp Leu Lys Phe Val
85 90 95Ser Asp Gly Ser Ile Asn Lys Ala Gly Phe Ala Val Asn Phe Phe
Lys 100 105 11022113PRTHomo sapiensmisc_featureCUB domain 3 of
BMP-1 22Cys Gly Gly Phe Leu Thr Lys Leu Asn Gly Ser Ile Thr Ser Pro
Gly1 5 10 15Trp Pro Lys Glu Tyr Pro Pro Asn Lys Asn Cys Ile Trp Gln
Leu Val 20 25 30Ala Pro Thr Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe
Phe Glu Thr 35 40 45Glu Gly Asn Asp Val Cys Lys Tyr Asp Phe Val Glu
Val Arg Ser Gly 50 55 60Leu Thr Ala Asp Ser Lys Leu His Gly Lys Phe
Cys Gly Ser Glu Lys65 70 75 80Pro Glu Val Ile Thr Ser Gln Tyr Asn
Asn Met Arg Val Glu Pro Lys 85 90 95Ser Asp Asn Thr Val Ser Lys Lys
Gly Phe Lys Ala His Phe Phe Ser 100 105 110Glu23113PRTHomo
sapiensmisc_featureCUB domain 1 of Neuropilin 23Gly Asp Thr Ile Lys
Ile Glu Ser Pro Gly Tyr Leu Thr Ser Pro Gly1 5 10 15Tyr Pro His Ser
Tyr His Pro Ser Glu Lys Cys Glu Trp Leu Ile Gln 20 25 30Ala Pro Asp
Pro Tyr Gln Arg Ile Met Ile Asn Phe Asn Pro His Phe 35 40 45Asp Leu
Glu Asp Arg Asp Cys Lys Tyr Asp Tyr Val Glu Val Phe Asp 50 55 60Gly
Glu Asn Glu Asn Gly His Phe Arg Gly Lys Phe Cys Gly Lys Ile65 70 75
80Ala Pro Pro Pro Val Val Ser Ser Gly Pro Phe Leu Phe Ile Lys Phe
85 90 95Val Ser Asp Tyr Glu Thr His Gly Ala Gly Phe Ser Ile Arg Tyr
Glu 100 105 110Ile24119PRTHomo sapiensmisc_featureCUB domain 2 of
Neuropilin 24Cys Ser Gln Asn Tyr Thr Thr Pro Ser Gly Val Ile Lys
Ser Pro Gly1 5 10 15Phe Pro Glu Lys Tyr Pro Asn Ser Leu Glu Cys Thr
Tyr Ile Val Phe 20 25 30Ala Pro Lys Met Ser Glu Ile Ile Leu Glu Phe
Glu Ser Phe Asp Leu 35 40 45Glu Pro Asp Ser Asn Pro Pro Gly Gly Met
Phe Cys Arg Tyr Asp Arg 50 55 60Leu Glu Ile Trp Asp Gly Phe Pro Asp
Val Gly Pro His Ile Gly Arg65 70 75 80Tyr Cys Gly Gln Lys Thr Pro
Gly Arg Ile Arg Ser Ser Ser Gly Ile 85 90 95Leu Ser Met Val Phe Tyr
Thr Asp Ser Ala Ile Ala Lys Glu Gly Phe 100 105 110Ser Ala Asn Tyr
Ser Val Leu 1152515PRTHomo sapiensMISC_FEATURE(2)..(2)can be any
amino acid residue 25Pro Xaa Cys Leu Leu Val Xaa Arg Cys Gly Gly
Asn Cys Gly Cys1 5 10 152620DNAArtificial SequenceDescription of
Artificial Sequence Forward PCR primer used to amplify a 327 bp DNA
fragment from a human fetal lung cDNA library 26gtcgtggaac
tgtcaactgg 202720DNAArtificial SequenceDescription of Artificial
Sequence Reverse PCR primer used to amplify a 327 bp DNA fragment
from a human fetal lung cDNA library 27ctcagcaacc acttgtgttc
202827DNAArtificial SequenceDescription of Artificial Sequence
Adaptor primer 1 (Clontech) used to amplify the sequence found at
the 5' end of PDGF-D 28ccatcctaat acgactcact atagggc
272929DNAArtificial SequenceDescription of Artificial Sequence
Adaptor primer 2 (Clontech) used to amplify the sequence found at
the 5' end of PDGF-D 29agtgggatcc gttactgatg gagagttat
293026DNAArtificial SequenceDescription of Artificial Sequence
Forward PCR primer used to amplify a 429 bp DNA fragment
(nucleotides 556 to 966 of SEQ ID NO 5) of PDGF-D 30cccaagcttg
aagatcttga gaatat 263122DNAArtificial SequenceDescription of
Artificial Sequence Reverse PCR primer used to amplify a 429 bp DNA
fragment (nucleotides 556 to 966 of SEQ ID NO 5) of PDGF-D
31tgctctagat cgaggtggtc tt 2232345PRTHomo sapiensmisc_featureAmino
acid sequence for PDGF-C 32Met Ser Leu Phe Gly Leu Leu Leu Val Thr
Ser Ala Leu Ala Gly Gln1 5 10 15Arg Arg Gly Thr Gln Ala Glu Ser Asn
Leu Ser Ser Lys Phe Gln Phe 20 25 30Ser Ser Asn Lys Glu Gln Asn Gly
Val Gln Asp Pro Gln His Glu Arg 35 40 45Ile Ile Thr Val Ser Thr Asn
Gly Ser Ile His Ser Pro Arg Phe Pro 50 55 60His Thr Tyr Pro Arg Asn
Thr Val Leu Val Trp Arg Leu Val Ala Val65 70 75 80Glu Glu Asn Val
Trp Ile Gln Leu Thr Phe Asp Glu Arg Phe Gly Leu 85 90 95Glu Asp Pro
Glu Asp Asp Ile Cys Lys Tyr Asp Phe Val Glu Val Glu 100 105 110Glu
Pro Ser Asp Gly Thr Ile Leu Gly Arg Trp Cys Gly Ser Gly Thr 115 120
125Val Pro Gly Lys Gln Ile Ser Lys Gly Asn Gln Ile Arg Ile Arg Phe
130 135 140Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gly Phe Cys Ile
His Tyr145 150 155 160Asn Ile Val Met Pro Gln Phe Thr Glu Ala Val
Ser Pro Ser Val Leu 165 170 175Pro Pro Ser Ala Leu Pro Leu Asp Leu
Leu Asn Asn Ala Ile Thr Ala 180 185 190Phe Ser Thr Leu Glu Asp Leu
Ile Arg Tyr Leu Glu Pro Glu Arg Trp 195 200 205Gln Leu Asp Leu Glu
Asp Leu Tyr Arg Pro Thr Trp Gln Leu Leu Gly 210 215 220Lys Ala Phe
Val Phe Gly Arg Lys Ser Arg Val Val Asp Leu Asn Leu225 230 235
240Leu Thr Glu Glu Val Arg Leu Tyr Ser Cys Thr Pro Arg Asn Phe Ser
245 250 255Val Ser Ile Arg Glu Glu Leu Lys Arg Thr Asp Thr Ile Phe
Trp Pro 260 265 270Gly Cys Leu Leu Val Lys Arg Cys Gly Gly Asn Cys
Ala Cys Cys Leu 275 280 285His Asn Cys Asn Glu Cys Gln Cys Val Pro
Ser Lys Val Thr Lys Lys 290 295 300Tyr His Glu Val Leu Gln Leu Arg
Pro Lys Thr Gly Val Arg Gly Leu305 310 315 320His Lys Ser Leu Thr
Asp Val Ala Leu Glu His His Glu Glu Cys Asp 325 330 335Cys Val Cys
Arg Gly Ser Thr Gly Gly 340 3453326DNAArtificial
SequenceDescription of Artificial Sequence Forward PCR primer for
the cDNA encoding amino acid residues 24-370 of SEQ ID NO8 (PDGF-D)
33gatatctaga agcaaccccg cagagc 263446DNAArtificial
SequenceDescription of Artificial Sequence Reverse PCR primer for
amplication of the cDNA encoding amino acid residues 24-370 of SEQ
ID NO8 (PDGF-D) 34gctcgaattc taaatggtga tggtgatgat gtcgaggtgg
tcttga 46351252DNAMurinae gen. sp. 35atgcaacggc tcgttttagt
ctccattctc ctgtgcgcga actttagctg ctatccggac 60acttttgcga ctccgcagag
agcatccatc aaagctttgc gcaatgccaa cctcaggaga 120gatgagagca
atcacctcac agacttgtac cagagagagg agaacattca ggtgacaagc
180aatggccatg tgcagagtcc tcgcttcccg aacagctacc caaggaacct
gcttctgaca 240tggtggctcc gttcccagga gaaaacacgg atacaactgt
cctttgacca tcaattcgga 300ctagaggaag cagaaaatga catttgtagg
tatgactttg tggaagttga agaagtctca 360gagagcagca ctgttgtcag
aggaagatgg tgtggccaca aggagatccc tccaaggata 420acgtcaagaa
caaaccagat
taaaatcaca tttaagtctg atgactactt tgtggcaaaa 480cctggattca
agatttatta ttcatttgtg gaagatttcc aaccggaagc agcctcagag
540accaactggg aatcagtcac aagctctttc tctggggtgt cctatcactc
tccatcaata 600acggacccca ctctcactgc tgatgccctg gacaaaactg
tcgcagaatt cgataccgtg 660gaagatctac ttaagcactt caatccagtg
tcttggcaag atgatctgga gaatttgtat 720ctggacaccc ctcattatag
aggcaggtca taccatgatc ggaagtccaa agtggacctg 780gacaggctca
atgatgatgt caagcgttac agttgcactc ccaggaatca ctctgtgaac
840ctcagggagg agctgaagct gaccaatgca gtcttcttcc cacgatgcct
cctcgtgcag 900cgctgtggtg gcaactgtgg ttgcggaact gtcaactgga
agtcctgcac atgcagctca 960gggaagacag tgaagaagta tcatgaggta
ttgaagtttg agcctggaca tttcaagaga 1020aggggcaaag ctaagaatat
ggctcttgtt gatatccagc tggatcatca tgagcgatgt 1080gactgtatct
gcagctcaag accacctcga taaaacacta tgcacatctg tactttgatt
1140atgaaaggac ctttaggtta caaaaaccct aagaagcttc taatctcagt
gcaatgaatg 1200catatggaaa tgttgctttg ttagtgccat ggcaagaaga
agcaaatatc at 125236370PRTMurinae gen. sp. 36Met Gln Arg Leu Val
Leu Val Ser Ile Leu Leu Cys Ala Asn Phe Ser1 5 10 15Cys Tyr Pro Asp
Thr Phe Ala Thr Pro Gln Arg Ala Ser Ile Lys Ala 20 25 30Leu Arg Asn
Ala Asn Leu Arg Arg Asp Glu Ser Asn His Leu Thr Asp 35 40 45Leu Tyr
Gln Arg Glu Glu Asn Ile Gln Val Thr Ser Asn Gly His Val 50 55 60Gln
Ser Pro Arg Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr65 70 75
80Trp Trp Leu Arg Ser Gln Glu Lys Thr Arg Ile Gln Leu Ser Phe Asp
85 90 95His Gln Phe Gly Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr
Asp 100 105 110Phe Val Glu Val Glu Glu Val Ser Glu Ser Ser Thr Val
Val Arg Gly 115 120 125Arg Trp Cys Gly His Lys Glu Ile Pro Pro Arg
Ile Thr Ser Arg Thr 130 135 140Asn Gln Ile Lys Ile Thr Phe Lys Ser
Asp Asp Tyr Phe Val Ala Lys145 150 155 160Pro Gly Phe Lys Ile Tyr
Tyr Ser Phe Val Glu Asp Phe Gln Pro Glu 165 170 175Ala Ala Ser Glu
Thr Asn Trp Glu Ser Val Thr Ser Ser Phe Ser Gly 180 185 190Val Ser
Tyr His Ser Pro Ser Ile Thr Asp Pro Thr Leu Thr Ala Asp 195 200
205Ala Leu Asp Lys Thr Val Ala Glu Phe Asp Thr Val Glu Asp Leu Leu
210 215 220Lys His Phe Asn Pro Val Ser Trp Gln Asp Asp Leu Glu Asn
Leu Tyr225 230 235 240Leu Asp Thr Pro His Tyr Arg Gly Arg Ser Tyr
His Asp Arg Lys Ser 245 250 255Lys Val Asp Leu Asp Arg Leu Asn Asp
Asp Val Lys Arg Tyr Ser Cys 260 265 270Thr Pro Arg Asn His Ser Val
Asn Leu Arg Glu Glu Leu Lys Leu Thr 275 280 285Asn Ala Val Phe Phe
Pro Arg Cys Leu Leu Val Gln Arg Cys Gly Gly 290 295 300Asn Cys Gly
Cys Gly Thr Val Asn Trp Lys Ser Cys Thr Cys Ser Ser305 310 315
320Gly Lys Thr Val Lys Lys Tyr His Glu Val Leu Lys Phe Glu Pro Gly
325 330 335His Phe Lys Arg Arg Gly Lys Ala Lys Asn Met Ala Leu Val
Asp Ile 340 345 350Gln Leu Asp His His Glu Arg Cys Asp Cys Ile Cys
Ser Ser Arg Pro 355 360 365Pro Arg 370371234DNAMurinae gen. sp.
37atgcaacggc tcgttttagt ctccattctc ctgtgcgcga actttagctg ctatccggac
60acttttgcga ctccgcagag agcatccatc aaagctttgc gcaatgccaa cctcaggaga
120gatgacttgt accagagaga ggagaacatt caggtgacaa gcaatggcca
tgtgcagagt 180cctcgcttcc cgaacagcta cccaaggaac ctgcttctga
catggtggct ccgttcccag 240gagaaaacac ggatacaact gtcctttgac
catcaattcg gactagagga agcagaaaat 300gacatttgta ggtatgactt
tgtggaagtt gaagaagtct cagagagcag cactgttgtc 360agaggaagat
ggtgtggcca caaggagatc cctccaagga taacgtcaag aacaaaccag
420attaaaatca catttaagtc tgatgactac tttgtggcaa aacctggatt
caagatttat 480tattcatttg tggaagattt ccaaccggaa gcagcctcag
agaccaactg ggaatcagtc 540acaagctctt tctctggggt gtcctatcac
tctccatcaa taacggaccc cactctcact 600gctgatgccc tggacaaaac
tgtcgcagaa ttcgataccg tggaagatct acttaagcac 660ttcaatccag
tgtcttggca agatgatctg gagaatttgt atctggacac ccctcattat
720agaggcaggt cataccatga tcggaagtcc aaagtggacc tggacaggct
caatgatgat 780gtcaagcgtt acagttgcac tcccaggaat cactctgtga
acctcaggga ggagctgaag 840ctgaccaatg cagtcttctt cccacgatgc
ctcctcgtgc agcgctgtgg tggcaactgt 900ggttgcggaa ctgtcaactg
gaagtcctgc acatgcagct cagggaagac agtgaagaag 960tatcatgagg
tattgaagtt tgagcctgga catttcaaga gaaggggcaa agctaagaat
1020atggctcttg ttgatatcca gctggatcat catgagcgat gtgactgtat
ctgcagctca 1080agaccacctc gataaaacac tatgcacatc tgtactttga
ttatgaaagg acctttaggt 1140tacaaaaacc ctaagaagct tctaatctca
gtgcaatgaa tgcatatgga aatgttgctt 1200tgttagtgcc atggcaagaa
gaagcaaata tcat 123438364PRTMurinae gen. sp. 38Met Gln Arg Leu Val
Leu Val Ser Ile Leu Leu Cys Ala Asn Phe Ser1 5 10 15Cys Tyr Pro Asp
Thr Phe Ala Thr Pro Gln Arg Ala Ser Ile Lys Ala 20 25 30Leu Arg Asn
Ala Asn Leu Arg Arg Asp Asp Leu Tyr Gln Arg Glu Glu 35 40 45Asn Ile
Gln Val Thr Ser Asn Gly His Val Gln Ser Pro Arg Phe Pro 50 55 60Asn
Ser Tyr Pro Arg Asn Leu Leu Leu Thr Trp Trp Leu Arg Ser Gln65 70 75
80Glu Lys Thr Arg Ile Gln Leu Ser Phe Asp His Gln Phe Gly Leu Glu
85 90 95Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp Phe Val Glu Val Glu
Glu 100 105 110Val Ser Glu Ser Ser Thr Val Val Arg Gly Arg Trp Cys
Gly His Lys 115 120 125Glu Ile Pro Pro Arg Ile Thr Ser Arg Thr Asn
Gln Ile Lys Ile Thr 130 135 140Phe Lys Ser Asp Asp Tyr Phe Val Ala
Lys Pro Gly Phe Lys Ile Tyr145 150 155 160Tyr Ser Phe Val Glu Asp
Phe Gln Pro Glu Ala Ala Ser Glu Thr Asn 165 170 175Trp Glu Ser Val
Thr Ser Ser Phe Ser Gly Val Ser Tyr His Ser Pro 180 185 190Ser Ile
Thr Asp Pro Thr Leu Thr Ala Asp Ala Leu Asp Lys Thr Val 195 200
205Ala Glu Phe Asp Thr Val Glu Asp Leu Leu Lys His Phe Asn Pro Val
210 215 220Ser Trp Gln Asp Asp Leu Glu Asn Leu Tyr Leu Asp Thr Pro
His Tyr225 230 235 240Arg Gly Arg Ser Tyr His Asp Arg Lys Ser Lys
Val Asp Leu Asp Arg 245 250 255Leu Asn Asp Asp Val Lys Arg Tyr Ser
Cys Thr Pro Arg Asn His Ser 260 265 270Val Asn Leu Arg Glu Glu Leu
Lys Leu Thr Asn Ala Val Phe Phe Pro 275 280 285Arg Cys Leu Leu Val
Gln Arg Cys Gly Gly Asn Cys Gly Cys Gly Thr 290 295 300Val Asn Trp
Lys Ser Cys Thr Cys Ser Ser Gly Lys Thr Val Lys Lys305 310 315
320Tyr His Glu Val Leu Lys Phe Glu Pro Gly His Phe Lys Arg Arg Gly
325 330 335Lys Ala Lys Asn Met Ala Leu Val Asp Ile Gln Leu Asp His
His Glu 340 345 350Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro Pro Arg
355 36039768DNAMurinae gen. sp. 39atgcaacggc tcgttttagt ctccattctc
ctgtgcgcga actttagctg ctatccggac 60acttttgcga ctccgcagag agcatccatc
aaagctttgc gcaatgccaa cctcaggaga 120gatgacttgt accagagaga
ggagaacatt caggtgacaa gcaatggcca tgtgcagagt 180cctcgcttcc
cgaacagcta cccaaggaac ctgcttctga catggtggct ccgttcccag
240gagaaaacac ggatacaact gtcctttgac catcaattcg gactagagga
agcagaaaat 300gacatttgta ggtatgactt tgtggaagtt gaagaagtct
cagagagcag cactgttgtc 360agaggaagat ggtgtggcca caaggagatc
cctccaagga taacgtcaag aacaaaccag 420attaaaatca catttaagtc
tgatgactac tttgtggcaa aacctggatt caagatttat 480tattcatttg
tggaagattt ccaaccggaa gcagcctcag agaccaactg ggaatcagtc
540acaagctctt tctctggggt gtcctatcac tctccatcaa taacggaccc
cactctcact 600gctgatgccc tggacaaaac tgtcgcagaa ttcgataccg
tggaagatct acttaagcac 660ttcaatccag tgtcttggca agatgatctg
gagaatttgt atctggacac ccctcattat 720agaggcaggt cataccatga
tcggaagtcc aaaggtattg aagtttga 76840255PRTMurinae gen. sp. 40Met
Gln Arg Leu Val Leu Val Ser Ile Leu Leu Cys Ala Asn Phe Ser1 5 10
15Cys Tyr Pro Asp Thr Phe Ala Thr Pro Gln Arg Ala Ser Ile Lys Ala
20 25 30Leu Arg Asn Ala Asn Leu Arg Arg Asp Asp Leu Tyr Gln Arg Glu
Glu 35 40 45Asn Ile Gln Val Thr Ser Asn Gly His Val Gln Ser Pro Arg
Phe Pro 50 55 60Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr Trp Trp Leu
Arg Ser Gln65 70 75 80Glu Lys Thr Arg Ile Gln Leu Ser Phe Asp His
Gln Phe Gly Leu Glu 85 90 95Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp
Phe Val Glu Val Glu Glu 100 105 110Val Ser Glu Ser Ser Thr Val Val
Arg Gly Arg Trp Cys Gly His Lys 115 120 125Glu Ile Pro Pro Arg Ile
Thr Ser Arg Thr Asn Gln Ile Lys Ile Thr 130 135 140Phe Lys Ser Asp
Asp Tyr Phe Val Ala Lys Pro Gly Phe Lys Ile Tyr145 150 155 160Tyr
Ser Phe Val Glu Asp Phe Gln Pro Glu Ala Ala Ser Glu Thr Asn 165 170
175Trp Glu Ser Val Thr Ser Ser Phe Ser Gly Val Ser Tyr His Ser Pro
180 185 190Ser Ile Thr Asp Pro Thr Leu Thr Ala Asp Ala Leu Asp Lys
Thr Val 195 200 205Ala Glu Phe Asp Thr Val Glu Asp Leu Leu Lys His
Phe Asn Pro Val 210 215 220Ser Trp Gln Asp Asp Leu Glu Asn Leu Tyr
Leu Asp Thr Pro His Tyr225 230 235 240Arg Gly Arg Ser Tyr His Asp
Arg Lys Ser Lys Gly Ile Glu Val 245 250 2554119DNAMurinae gen. sp.
41caaatgcaac ggctcgttt 194224DNAMurinae gen. sp. 42gatatttgct
tcttcttgcc atgg 24
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