Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-06-17
2001-03-20
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S252300, C435S254110, C536S023100, C536S023500, C530S350000
Reexamination Certificate
active
06204011
ABSTRACT:
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes a human receptor tyrosine kinase, KDR, which is expressed on human endothelial cells. This receptor is activated by VEGF and mediates a mitogenic signal. The present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding human KDR, a DNA fragment encoding the intracellular portion of KDR, a DNA fragment encoding the extracellular portion of KDR with or without a membrane anchor sequence, substantially purified forms of associated human KDR, and human mutant forms of KDR.
BACKGROUND OF THE INVENTION
Vascular endothelial cells form a luminal non-thrombogenic monolayer throughout the vascular system. Mitogens promote embryonic vascular development, growth, repair and angiogenesis in these cells. Angiogenesis involves the proteolytic degradation of the basement membrane on which endothelial cells reside followed by the subsequent chemotactic migration and mitosis of these cells to support sustained growth of a new capillary shoot. One class of mitogens selective for vascular endothelial cells include vascular endothelial growth factor (referred to as VEGF or VEGF-A) and the homologues placenta growth factor (PlGF), VEGF-B and VEGF-C.
Human VEGF exists as a glycosylated homodimer in one of four mature processed forms containing 206, 189 (see U.S. Pat. No. 5,240,848), 165 (see U.S. Pat. No. 5,332,671), and 121 (U.S. Pat. No. 5,332,671) amino acids, the most prevalent being the 165 amino acid form. The 206 amino acid and 189 amino acid forms of human VEGF each contain a highly basic 24-amino acid insert that promotes tight binding to heparin, and presumably, heparin proteoglycans on cellular surfaces and within extracellular matrices (Ferrara et al., 1991,
J. Cell. Biochem.
47:211-218).
Human PlGF is also a glycosylated homodimer which shares 46% homology with VEGF at the protein level. Differential splicing of human PlGF mRNA leads to either a 170 or 149 amino acid residue precursor, which are proteolytically processed to mature forms of 152 or 131 amino acid residues in length, respectively (Maglione et al., 1993,
Oncogene
8: 925-931; Bayne and Thomas, 1992, EPO Publication No. 0 506 477 A1; Hauser and Weich, 1993,
Growth Factors
9: 259-268).
VEGF-B has been isolated and characterized (Grimmond et al., 1996,
Genome Research
6: 124-131; Olofsson et al., 1996,
Proc. Natl. Acad. Sci. USA
93: 2576-2581). The full-length human cDNAs encode 188 and 207 amino acid residue precursors wherein the NH
2
terminal portions are proteolytically processed to mature forms 167 and 186 amino acid residues in length. Human VEGF-B expression was found predominantly in heart and skeletal muscle as a disulfide-linked homodimer. However, human VEGF-B may also form a heterodimer with VEGF (id. @ 2580).
VEGF-C has also been isolated and characterized (Joukov et al., 1996,
EMBO J.
15: 290-298). A cDNA encoding VEGF-C was obtained from a human prostatic adenocarcinoma cell line. A 32 kDa precursor protein is proteolytically processed to generate the mature 23 kDa form, which binds the receptor tyrosine kinase, Flt-4.
VEGF and its homologues impart activity by binding to vascular endothelial cell plasma membrane-spanning tyrosine kinase receptors which then activate an intracellular mitogenic signal. The KDR receptor family is the major tyrosine kinase receptor which transduces the mitogenic signal initiated by VEGF.
Shibuya et al. (1990,
Oncogene
5: 519-524) disclose a human receptor type tyrosine kinase gene fit, which comprises a 4.2 Kb open reading frame encoding a 1338 amino acid protein which comprises a glycosylated extracelluar domain, membrane spanning region and predicted tyrosine kinase domain.
Pajusola et al. (1992,
Cancer Res.
52: 5738-5743) disclose a human receptor type tyrosine kinase gene which, as noted above, binds human VEGF-C.
Vascular endothelial growth factor (VEGF) binds the high affinity membrane-spanning tyrosine kinase receptors KDR and Flt-1. Cell culture and gene knockout experiments indicate that each receptor contributes to different aspects of angiogenesis. KDR mediates the mitogenic function of VEGF whereas Flt-1 appears to modulate non-mitogenic functions such as those associated with cellular adhesion. Inhibiting KDR thus significantly diminishes the level of mitogenic VEGF activity.
Vascular growth in the retina leads to visual degeneration culminating in blindness. VEGF accounts for most of the angiogenic activity produced in or near the retina in diabetic retinopathy. Ocular VEGF mRNA and protein are elevated by conditions such as retinal vein occlusion in primates and decreased pO
2
levels in mice that lead to neovascularization. Intraocular injections of either anti-VEGF mono-clonal antibodies or VEGF receptor immunofusions inhibit ocular neovascularization in rodent and primate models. Regardless of the cause of induction of VEGF in human diabetic retinopathy, inhibition of ocular VEGF is useful in treating the disease.
Expression of VEGF is also significantly increased in hypoxic regions of animal and human tumors adjacent to areas of necrosis. Monoclonal and polyclonal anti-VEGF antibodies inhibit the growth of human tumors in nude mice. Although these same tumor cells continue to express VEGF in culture, the antibodies do not diminish their mitotic rate of most, if not all, tumor cells derived from cells other than vascular endothelial cells themselves. Thus tumor-derived VEGF does not function as an autocrine mitogenic factor for most tumors. Therefore, VEGF contributes to tumor growth in vivo by promoting angiogenesis through its paracrine vascular endothelial cell chemotactic and mitogenic activities. These monoclonal antibodies also inhibit the growth of typically less well vascularized human colon cancers in athymic mice and decrease the number of tumors arising from inoculated cells. Viral expression of a VEGF-binding construct of Flk-1, the mouse KDR receptor homologue, truncated to eliminate the cytoplasmic tyrosine kinase domains but retaining a membrane anchor, virtually abolishes the growth of a transplantable glioblastoma in mice presumably by the dominant negative mechanism of heterodimer formation with membrane-spanning endothelial cell VEGF receptors. Embryonic stem cells, which normally grow as solid tumors in nude mice, do not produce detectable tumors if both VEGF alleles are knocked out. Taken together, these data indicate the role of VEGF in the growth of solid tumors. KDR and Flt-1 are implicated in pathological neoangiogenesis, and inhibitors of these receptors are useful in the treatment of diseases in which neoangiogenesis is part of the overall pathology, e.g., diabetic retinal vascularization, various forms of cancer as well as forms of inflammation such as rheumatoid arthritis, psoriasis, contact dermatitis and hypersensitivity reaction.
Terman et al. (1991,
Oncogene
6: 1677-1683; 1992,
Biochem. Biophys. Res. Commun.
187: 1579-1586) disclose a full-length cDNA encoding a form of KDR. However, the Terman et al. disclosures do not identify a novel, optimal nucleic acid fragment encoding the human form of the receptor type tyrosine kinase gene, KDR. It will be advantageous to identify and isolate a human cDNA sequence encoding an optimized form of human KDR. A nucleic acid molecule expressing the human KDR protein will be useful in screening for compounds acting as a modulator of the protein kinase domain of this protein. Such a compound or compounds will be useful in modulating the mitogenic signal of VEGF and VEGF-related proteins on vascular endothelial cells. The KDR nucleic acid sequence may be also useful for gene therapy encoding a portion of the KDR protein that would contain functional ligand binding and membrane anchoring moieties but not tyrosine kinase activity. Either all or a portion of the KDR protein is also useful to scre
Kendall Richard L.
Mao Xianzhi
Tebben Andrew
Thomas Kenneth A.
Hand J. Mark
Merck & Co. , Inc.
Spector Lorraine
Tribble Jack L.
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