Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
2000-02-18
2004-08-31
Horlick, Kenneth R. (Department: 1637)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C435S007100, C435S183000, C530S350000, C536S023100
Reexamination Certificate
active
06784285
ABSTRACT:
The present invention discloses the isolation of a key portion of the catalytic kinase region of vascular endothelial growth factor receptor 2 or VEGFR-2 through cloning, sequencing and x-ray crystallography. Also disclosed is the deletion of various amino acid residues from an area of the catalytic region called the kinase insert domain (KID). The resulting polypeptide retains comparable in vitro kinase activity to that of the wild-type KID and is not necessary for the catalytic activity of the polypeptide, and more importantly, allows complete crystallization of the protein such that it may be characterized by X-ray crystallography. The present invention further discloses x-ray crystallography data useful for identification and construction of therapeutic compounds in the treatment of various disease conditions associated with VEGFR-2.
BACKGROUND OF THE INVENTION
Many physiological events including embryogenesis, organ development, estrus, and wound healing require vascular growth and remodeling (Folkman et al., (1992)
J. Biol. Chem
. 267, 10931-10934; Risau, W. (1995)
FASEB J
. 9, 926-933.). In addition to these beneficial processes, angiogenesis is also involved in the proliferation of disease states such as tumor growth, metastasis, psoriasis, rheumatoid arthritis, macular degeneration and retinopathy (Pepper, M. S., (1996)
Vasc. Med
. 1, 259-266; Kuiper et al., (1998)
Pharmacol. Res
. 37, 1-16, 1998; Kumar and Fidler, (1998)
In Vivo
18, 27-34; Szekanecz et al., (1998)
J. Investig. Med
. 46, 27-41; Tolentino and Adamis, (1988)
Int. Ophthalmol. Clin
. 38, 77-94. Of the signaling pathways known to influence vascular formation, these involving vascular endothelial growth factor (VEGF) have been shown to be essential and selective for vascular endothelial cells (Dvorak et al., (1995)
Am. J. Path
. 146, 1029-1039; Thomas, K., (1996)
J. Biol. Chem
. 271, 603-606; Ferrara N. and Davis-Smyth, (1997)
Endocrine Rev
. 18, 4-25). The therapeutic potential of inhibiting the VEGF pathway has been directly demonstrated by anti-VEGF monoclonal antibodies which were active against a variety of human tumors (Borgström et al, (1996)
Cancer Res
. 56, 4032-4039) and ischemic retinal disease (Adamis et al., (1996)
Arch. Ophthalmol
. 114, 66-71).
Normal vasculogenesis and angiogenesis play important roles in a variety of physiological processes such as embryonic development, wound healing, organ regeneration and female reproductive processes such as follicle development in the corpus luteum during ovulation and placental growth after pregnancy (Folkman & Shing, 1992). Uncontrolled vasculogenesis and/or angiogenesis has been associated with diseases, such as diabetes, as well as malignant solid tumors that rely on vascularization for growth. Klagsburn & Soker, (1993)
Current Biology
3(10):699-702; Folkham, (1991)
J. Natl., Cancer Inst
. 82:4-6; Weidner, et al., (1991)
New Engl. J. Med
. 324:1-5.
Several polypeptides with in vitro endothelial cell growth promoting activity have been identified. Examples include acidic and basic fibroblastic growth factor (FGF), vascular endothelial growth factor (VEGF)and placental growth factor. Unlike FGF, VEGF has recently been reported to be an endothelial cell specific mitogen (Ferrara & Henzel, (1989)
Biochem. Biophys. Res. Comm
. 161:851-858; Vaisman et al., (1990)
J. Biol. Chem
. 265:19461-19566).
Thus, identification of the specific receptors to which VEGF binds is important to understanding of the regulation of endothelial cell proliferation. Two structurally related tyrosine kinases have been identified to bind VEGF with high affinity: the flt-1 receptor (Shibuya et al., (1990)
Oncogene
5:519-524; De Vries et al., (1992)
Science
255:989-991) and the KDR/FLK-1 receptor, discussed herein. Consequently, it had been surmised that RTKs may have a role in the modulation and regulation of endothelial cell proliferation.
Recent disclosures, such as information set forth in U.S. patent application Ser. Nos. 08/193,829, 08/038,596 and 07/975,750, strongly suggest that VEGF is not only responsible for endothelial cell proliferation, but also is the prime regulator of normal and pathological angiogenesis. See generally, Klagsburn & Soker, (1993)
Current Biology
3:699-702; Houck, et al., (1992)
J. Biol. Chem
267:26031-26037.
VEGF is a homodimeric cytokine that is expressed in at least four splice-variant forms of 121-206 residues (Ferrara and Davis-Smyth, 1997). Vascular endothelial cells express at least two high-affinity receptors for VEGF: VEGF-R1/Flt-1 and VEGFR-2/KDR. VEGF-R1 and VEGFR-2 are receptor tyrosine kinases each comprised of an extracellular domain that contains 7 immunoglobulin-like segments and binds VEGF, a short membrane spanning region, and a cytosolic domain possessing tyrosine kinase activity. The kinase domain directly follows the extracellular and juxtamembrane regions and is itself followed by another domain (post-kinase domain), which may function in binding of other proteins for signal transduction. These two receptors appear to have different signaling pathways and functions with VEGFR-2 being of primary importance in mitosis of endothelial cells (Waltenberger et al., (1994)
J. Biol. Chem
. 269, 26988-26995; Seetharm et al., (1995)
Oncogene
10, 135-147; Shalaby et al., (1995)
Nature
376, 576-579).
Both FGF and VEGF are potent angiogenic factors which induce formation of new capillary blood vessels. Transfection of human breast carcinoma cell line MCF-7 with FGF resulted in cell lines that form progressively growing and metastatic tumors when injected (s.c.) into nude mice. FGF may play a critical role in progression of breast tumors to an estrogen-independent, anti-estrogen resistant metastatic phenotype (McLeskey et al., (1993)
Cancer Res
. 53: 2168-2177). Breast tumor cells exhibited increased neovascularization, increased spontaneous metastasis and more rapid growth in vivo than did the non-transfected tumors. FGF has been shown to be transforming in NIH-3T3 cells and implicated in tumorigenesis and metastasis of mouse mammary tumors. FGF overexpression conferred a tumorigenic phenotype on a human adrenal carcinoma cell line suggesting that FGF's may also play a role in the transformation of epithelial cells. Polyclonal neutralizing antibodies to FGF inhibited tumor growth in Balb/c nude mice transplanted with K1000 cells (transfected with the leader sequence of bFGF) which form tumors in these mice (Hori et al., (1991)
Cancer Res
. 51: 6180-9184).
Due to the role of FGF in neovascularization, tumorigenesis and metastasis, there is a need in the art for FGF inhibitors as potent anti-cancer agents that exert their anti-FGF activity by preventing intracellular signaling of FGF.
VEGF, by contrast, is an endothelial cell-specific mitogen and an angiogenesis inducer that is released by a variety of tumor cells and expressed in human tumor cells in situ. Unlike FGF, transfection of cell lines with a cDNA sequence encoding VEGF, did not promote transformation, but did facilitate tumor growth in vivo (Ferrara, N., and Davis-Smyth, T. (1997)). Furthermore, administration of a polyclonal antibody which neutralized VEGF also inhibited growth of human rhabdomyosarcoma, glioblastoma multiforme and leiomyosarcoma cell lines in nude mice (Kim et al., (1993)
Nature
362: 841-843).
In view of the importance of receptor tyrosine kinases (RTKs) to the control, regulation and modulation of endothelial cell proliferation and potentially vasculogenesis and/or angiogenesis, many attempts have been made to identify RTK “inhibitors” using a variety of approaches, including the use of mutant ligands (U.S. Pat. No. 4,966,849), soluble receptors and antibodies (Application No. WO 94/10202; Kendall & Thomas, (1994)
Proc. Natl. Acad. Sci
. 90:10705-09; Kim, et al., 1993), RNA ligands (Jellinek, et al., (1994)
Biochemistry
3:10450-56), protein kinase C inhibitors (Schuchter, et al., (1991)
Cancer Res
. 51:682-687); Takano, et al., (1993)
Mol. Bio. Cell
4:358A; Kinsella, et al., (1992)
Exp. Cell Res
. 199:56-62; Wright
Appelt Krzysztof
Gehring Michael R.
Kan Chen-Chen
McTigue Michele A.
Mroczkowski Barbara
Agouron Pharmaceuticals , Inc.
Horlick Kenneth R.
Kim Young J.
Prodnuk Stephen D.
Richardson Peter
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