Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-07-29
2004-07-13
Spector, Lorraine (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120
Reexamination Certificate
active
06762290
ABSTRACT:
FIELD OF THE INVENTION
Described herein are methods for identifying and preparing high affinity nucleic acid ligands that bind to a VEGF receptor. The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by EXponential Enrichment. This invention includes high affinity nucleic acids to a VEGF receptor. Further disclosed are RNA ligands to a VEGF receptor. Also included are oligonucleotides containing nucleotide derivatives modified at the 2′ position of the pyrimidines. Additionally disclosed are ligands to a VEGF receptor containing 2′-F modifications of the pyrimidines. This invention also includes high affinity nucleic acid inhibitors of VEGF signaling. The oligonucleotide ligands of the present invention are useful in any process in which binding of VEGF to a VEGF receptor is required. This includes, but is not limited to, their use as pharmaceuticals, diagnostics, imaging agents, and immunohistochemical reagents.
BACKGROUND OF THE INVENTION
Angiogenesis in Disease
The growth of new blood vessels from existing endothelium (angiogenesis) is tightly controlled in healthy adults by opposing effects of positive and negative regulators. Under certain pathological conditions, including proliferative retinopathies, rheumatoid arthritis, psoriasis and cancer, positive regulators prevail and angiogenesis contributes to disease progression (reviewed in Folkman (1995) Nature Med. 1:27-31). In cancer, the notion that angiogenesis represents the rate limiting step of tumor growth and metastasis (Folkman (1971) New Engl. J. Med. 285:1182-1186) is now supported by considerable experimental evidence (reviewed in Aznavoorian et al. (1993) Cancer 71:1368-1383; Fidler and Ellis (1994) Cell 79:185-188; Folkman (1990) J. Natl. Cancer Inst. 82:4-6). The quantity of blood vessels in tumor tissue is a strong negative prognostic indicator in breast cancer (Weidner et al. (1992) J. Natl. Cancer Inst. 84:1875-1887), prostate cancer (Weidner et al. (1993) Am. J. Pathol. 143:401-409), brain tumors (Li et al.(1994) Lancet 344:82-86), and melanoma (Foss et al.(1996) Cancer Res. 56:2900-2903).
VEGF Signaling in Angiogenesis
A number of angiogenic growth factors have been described to date among which vascular endothelial growth factor (VEGF) appears to play a key role as a positive regulator of physiological and pathological angiogenesis (reviewed in Brown et al. (1997) in Control of Angiogenesis (Goldberg and Rosen, eds.), Birkhauser, Basel: 233-269; Thomas (1996) J. Biol. Chem. 271:603-606; Neufeld et al. (1999) FASEB J. 13: 9-22). VEGF is a secreted disulfide-linked homodimer that selectively stimulates endothelial cells to proliferate, migrate, and produce matrix-degrading enzymes (Conn et al. (1990) Proc. Natl. Acad. Sci.
U.S.A. 87: 1323-1327; Ferrara and Henzel (1989) Biochem. Biophys. Res. Commun. 161: 851-858; Gospodarowicz et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 7311-7315; Pepper et al. (1991) Biochem. Biophys. Res. Commun. 181: 902-906; Unemori et al. (1992) J. Cell. Physiol. 153: 557-562), all of which are processes required for the formation of new vessels. In addition to being the only known endothelial cell specific mitogen, VEGF is unique among angiogenic growth factors in its ability to induce a transient increase in blood vessel permeability to macromolecules (hence its original and alternative name, vascular permeability factor) (Dvorak et al.(1979) J. Immunol. 122:166-174; Senger et al.(1983) Science 219:983-985; Senger et al.(1986) Cancer Res. 46:5629-5632). Increased vascular permeability and the resulting deposition of plasma proteins in the extravascular space assists the new vessel formation by providing a provisional matrix for the migration of endothelial cells (Dvorak et al.(1995) Am. J. Pathol. 146:1029-1039). Hyperpermeability is indeed a characteristic feature of new vessels, including those associated with tumors (Dvorak et al.(1995) Am. J. Pathol. 146:1029-1039). Furthermore, compensatory angiogenesis induced by tissue hypoxia is now known to be mediated by VEGF (Levy et al.(1996) J. Biol. Chem. 271:2746-2753); Shweiki et al. (1992) Nature 359:843-845).
VEGF is produced and secreted in varying amounts by virtually all tumor cells (Brown et al. (1997) in Control of Angiogenesis, ibid). Direct evidence that VEGF and its receptors contribute to tumor growth was recently obtained by a demonstration that the growth of human tumor xenografts in nude mice could be inhibited by neutralizing antibodies to VEGF (Kim et al. (1993) Nature 362:841-844), by the expression of dominant-negative VEGFR2 (Millauer et al. (1996) Cancer Res. 56:1615-1620; Millauer et al. (1994) Nature 367:576-579), by low molecular weight inhibitors of VEGF receptor inhibitors (Strawn et al. (1966) Cancer Res. 56:3540-3545), or by the expression of antisense sequence to VEGF mRNA (Saleh et al. (1996) Cancer Res. 56:393401). Importantly, the incidence of tumor metastases was also found to be dramatically reduced by VEGF antagonists (Asano et al. (1995) Cancer Res. 55, 5296-5301; Warren et al. (1995) J. Clin. Invest. 95: 1789-1797; Claffey et al. (1996) Cancer Res. 56:172-181; Melnyk et al. (1996) Cancer Res. 56, 921-924). Inhibitors of VEGF signaling may thus have broad clinical utility as anticancer agents. In addition to cancer, as noted above, other proliferative diseases characterized by excessive neovascularization such as psoriasis, age-related macular degeneration, diabetic retinopathy and rheumatoid arthritis could be treated with antagonists of VEGF signaling.
VEGF occurs in several forms (VEGF-121, VEGF-145, VEGF-165, VEGF-189, VEGF-206) as a result of alternative splicing of the VEGF gene that consists of eight exons (Houck et al. (1991) Mol. Endocrin. 5:1806-1814; Tischer et al. (1991) J. Biol. Chem. 266:11947-11954; Poltorak et al. (1997) J. Biol. Chem. 272: 7151-7158). The three smaller forms are diffusable while the larger two forms remain predominantly localized to the cell membrane as a consequence of their high affinity for heparin. VEGF-165 and VEGF-145 also bind to heparin (as a consequence of containing basic exon 7- and exon 6-encoded domains, respectively), albeit with somewhat lower affinity compared with VEGF-189 (that contains both exons 6 and 7). VEGF-165 appears to be the most abundant form in most tissues (Houck et al. (1991) Mol. Endocrinol., ibid; Carmeliet et al. (1999) Nature Med. 5: 495-502). VEGF-121, the only alternatively spliced form that does not bind to heparin, appears to have a somewhat lower affinity for the receptors (Gitay-Goren et al. (1996) J. Biol. Chem. 271:5519-5523) as well as lower mitogenic potency (Keyt et al. (1996) J. Biol. Chem. 271:7788-7795).
VEGF Receptors
Biological effects of VEGF are mediated by two homologous tyrosine kinase receptors, Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2) whose expression is highly restricted to cells of endothelial origin (de Vries et al. (1992) Science 255: 989-991; Millauer et al. (1993) Cell 72: 835-846; Terman et al. (1991) Oncogene 6: 519-524). Both receptors have an extracellular domain consisting of seven IgG-like domains, a transmembrane domain and an intracellular tyrosine kinase domain. The affinity of VEGFR1 for VEGF (K
d
=1-20 pM) is higher compared to that of VEGFR2 (K
d
=50-770 pM) (Brown et al. (1997) in Regulation of Angiogenesis, ibid; de Vries et al. (1992) Science 255, 989-991; Terman et al. (1992) Biochem. Biophys. Res. Commun. 187, 1579-1586). In human umbilical cord endothelial cells (HUVECs) in 2-dimensional culture, VEGFR2 is by far the more abundant receptor (Brown et al. (1997) in Regulation of Angiogenesis, ibid). In vivo, however, in quiescent endothelial cells, both receptors are expressed at low levels (Kremer et al. (1997) Cancer Res. 57, 3852-3859; Barleon et al. (1997) Cancer Res. 57: 5421-5425).
Both receptors are substantially upregulated when endothelial cells are activated by a variety of stimuli. Hypoxia, for example, induces an increase in expression of both VEGFR1 and VEGFR2 in endo
No affiliations
Murphy Joseph F.
Spector Lorraine
LandOfFree
High affinity vascular endothelial growth factor (VEGF)... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with High affinity vascular endothelial growth factor (VEGF)..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and High affinity vascular endothelial growth factor (VEGF)... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3252547