Nucleic acids encoding vascular endothelial cell growth...

Details Nucleic acids encoding vascular endothelial cell growth... Nucleic acids encoding vascular endothelial cell growth...

1999-03-10

2002-09-24

Spector, Lorraine (Department: 1647)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S252330, C435S360000, C435S254200, C435S348000, C435S320100, C536S023510, C530S399000

Reexamination Certificate

active

06455283

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to polypeptides related to vascular endothelial cell growth factor (hereinafter sometimes referred to as VEGF) and bone morphogenetic protein 1 (hereinafter sometimes referred to as BMP1), termed herein as VEGF-E polypeptides, nucleic acids encoding therefor, methods for preparing VEGF-E, and methods, compositions, and assays utilizing VEGF-E.
BACKGROUND OF THE INVENTION
Various naturally occurring polypeptides reportedly induce the proliferation of endothelial cells. Among those polypeptides are the basic and acidic fibroblast growth factors (FGF) (Burgess and Maciag,
Annual Rev. Biochem.,
58: 575 (1989)), platelet-derived endothelial cell growth factor (PD-ECGF) (Ishikawa et al.,
Nature,
338: 557 (1989)), and vascular endothelial growth factor (VEGF). Leung et al.,
Science,
246: 1306 (1989); Ferrara and Henzel,
Biochem. Biophys. Res. Commun.,
161: 851 (1989); Tischer et al.,
Biochem. Biophys. Res. Commun.,
165: 1198 (1989); EP 471,754B granted Jul. 31, 1996.
The heparin-binding endothelial cell-growth factor, VEGF, was identified and purified from media conditioned by bovine pituitary follicular or folliculo-stellate cells several years ago. See Ferrara et al.,
Biophys. Res. Comm.,
161: 851 (1989). Media conditioned by cells transfected with the human VEGF (hVEGF) cDNA promoted the proliferation of capillary endothelial cells, whereas control cells did not. Leung et al.,
Science,
246: 1306 (1989). VEGF is a naturally occurring compound that is produced in follicular or folliculo-stellate cells (FC), a morphologically well-characterized population of granular cells. The FC are stellate cells that send cytoplasmic processes between secretory cells.
VEGF is expressed in a variety of tissues as multiple homodimeric isoforms (121, 165, 189 and 206 amino acids per monomer), also collectively referred to as hVEGF-related proteins, resulting from alternative RNA splicing. The 121-amino acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 116 and 159 in hVEGF. The 189-amino acid protein differs from hVEGF by virtue of the insertion of 24 amino acids at residue 116 in hVEGF, and apparently is identical to human vascular permeability factor (hVPF). The 206-amino acid protein differs from hVEGF by virtue of an insertion of 41 amino acids at residue 116 in hVEGF. Houck et al.,
Mol. Endocrin.,
5: 1806 (1991); Ferrara et al.,
J. Cell. Biochem.,
47: 211 (1991); Ferrara et al.,
Endocrine Reviews,
13: 18 (1992); Keck et al.,
Science,
246: 1309 (1989); Connolly et al.,
J. Biol. Chem.,
264: 20017 (1989); EP 370,989 published May 30, 1990. VEGF
121
is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The heparin-binding forms of VEGF can be cleaved in the carboxy terminus by plasmin to release (a) diffusible form(s) of VEGF. The amino acid sequence of the carboxy-terminal peptide identified after plasmin cleavage is Arg
110
-Ala
111
. Amino terminal “core” protein, VEGF (1-110), isolated as a homodimer, binds neutralizing monoclonal antibodies (4.6.1 and 2E3) and soluble forms of FMS-like tyrosine kinase (FLT-1), kinase domain region (KDR) and fetal liver kinase (FLK) receptors with similar affinity compared to the intact VEGF
165
homodimer.
As noted, VEGF contains two domains that are responsible respectively for binding to the KDR and FLT-1 receptors. These receptors exist only on endothelial (vascular) cells. As cells become depleted in oxygen, because of trauma and the like, VEGF production increases in such cells which then bind to the respective receptors in order to signal ultimate biological effect. The signal then increases vascular permeability and the cells divide and expand to form new vascular pathways—vasculogenesis and angiogenesis.
Thus, VEGF is useful for treating conditions in which a selected action on the vascular endothelial cells, in the absence of excessive tissue growth, is important, for example, diabetic ulcers and vascular injuries resulting from trauma such as subcutaneous wounds. Being a vascular (artery and venus) endothelial cell growth factor, VEGF restores cells that are damaged, a process referred to as vasculogenesis, and stimulates the formulation of new vessels, a process referred to as angiogenesis.
VEGF would also find use in the restoration of vasculature after a myocardial infarct, as well as other uses that can be deduced. In this regard, inhibitors of VEGF are sometimes desirable, particularly to mitigate processes such as angiogenesis and vasculogenesis in cancerous cells.
It is now well established that angiogenesis, which involves the formation of new blood vessels from preexisting endothelium, is implicated in the pathogenesis of a variety of disorders. These include solid tumors and metastasis, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovascular syndromes such as proliferative retinopathies, e.g., diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis. Folkman et al.,
J. Biol. Chem.,
267: 10931-10934 (1992); Klagsbrun et al.,
Annu. Rev. Physiol.,
53: 217-239 (1991); and Garner A, “Vascular diseases”, In:
Pathobiology of Ocular Disease. A Dynamic Approach
, Garner A, Klintworth G K, Eds., 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710.
In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and for providing nourishment to the growing solid tumor. Folkman et al.,
Nature,
339: 58 (1989). The neovascularization allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to the normal cells. Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors. Weidner et al.,
N Engl J Med,
324: 1-6 (1991); Horak et al.,
Lancet,
340: 1120-1124 (1992); Macchiarini et al.,
Lancet,
340: 145-146 (1992).
The search for positive regulators of angiogenesis has yielded many candidates, including aFGF, bFGF, TGF-&agr;, TGF-&bgr;, HGF, TNF-&agr;, angiogenin, IL-8, etc. Folkman et al.,
J.B.C.
, supra, and Klagsbrun et al., supra. The negative regulators so far identified include thrombospondin (Good et al.,
Proc. Natl. Acad. Sci. USA.,
87: 6624-6628 (1990)), the 16-kilodalton N-terminal fragment of prolactin (Clapp et al.,
Endocrinology,
133: 1292-1299 (1993)), angiostatin (O'Reilly et al.
Cell,
79: 315-328 (1994)), and endostatin. O'Reilly et al.,
Cell,
88: 277-285 (1996). Work done over the last several years has established the key role of VEGF, not only in stimulating vascular endothelial cell proliferation, but also in inducing vascular permeability and angiogenesis. Ferrara et al.,
Endocr. Rev.,
18: 4-25 (1997). The finding that the loss of even a single VEGF allele results in embryonic lethality points to an irreplaceable role played by this factor in the development and differentiation of the vascular system. Furthermore, VEGF has been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders. Ferrara et al.,
Endocr. Rev.
, supra. The VEGF mRNA is overexpressed by the majority of human tumors examined. Berkman et al.,
J Clin Invest,
91: 153-159 (1993); Brown et al.,
Human Pathol.,
26: 86-91 (1995); Brown et al.,
Cancer Res.,
53: 4727-4735 (1993); Mattern et al.,
Brit. J. Cancer,
73: 931-934 (1996); Dvorak et al.,
Am J. Pathol.,
146: 1029-1039 (1995).
Also, the concentration levels of VEGF in eye fluids are highly correlated to the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies. Aiello et al.,
N. Engl. J. Med.,
331: 1480-1487 (1994). Furthermore, recent studies have demonstrated the localization of VEGF in choroidal neovascular membranes in pat

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