Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing – Testing efficacy or toxicity of a compound or composition
Reexamination Certificate
1999-10-26
2002-04-23
Guzo, David (Department: 1636)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Testing efficacy or toxicity of a compound or composition
C424S093100, C424S093200, C424S093600, C424S009100, C435S320100, C435S325000
Reexamination Certificate
active
06375929
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods of gene therapy for inhibiting angiogenesis associated with tumor growth, inflammation, psoriasis, rheumatoid arthritis, hemangiomas, diabetic retinopathy, angiofibromas, and macular degeneration.
This invention also relates to animal models useful in the investigation of gene therapy-mediated inhibition of angiogenesis. The invention also relates to recombinant vectors which are useful in the disclosed gene therapy methods.
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 five mature processed forms containing 206, 189, 165, 145 and 121 amino acids, the most prevalent being the 165 amino acid form.
U.S. Pat. No. 5,240,848 discloses the nucleotide and amino acid sequence encoding the 189 amino acid form of human VEGF.
U.S. Pat. No. 5,332,671 discloses the nucleotide and amino acid sequence encoding the 165 amino acid form of human VEGF.
Charnock-Jones et al (1993,
Biol. Reproduction
48: 1120-1128) discloses the VEGF145 splice variant m RNA.
U.S. Pat. No. 5,194,596 discloses the nucleotide and amino acid sequence encoding the 121 amino acid form of human VEGF.
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). The VEGF
165
form binds heparin to a lesser extent while VEGF
121
does not bind heparin.
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 amino acid or 149 amino acid precursor, which are proteolytically processed to mature forms of 152 or 131 amino acids in length, respectively (Maglione, et al., 1993,
Oncogene
8: 925-931; Hauser and Weich, 1993,
Growth Factors
9: 259-268).
VEGF-B was recently isolated and characterized (Olofsson, et al., 1996,
Proc. Natl. Acad. Sci.
93: 2576-2581; Grimmond et al., 1996,
Genome Research
6: 124-131). The full length human cDNAs encode 188 and 207 amino acid precursors wherein the NH
2
terminal portions are proteolytically processed to mature forms 167 and 186 amino acids 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 recently 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-D was identified in an EST library, the full-length coding region was cloned and recognized to be most homologous to VEGF-C among the VEGF family amino acid sequences (Yamada, et al., 1997, Genomics 42:483-488). The human VEGF-D mRNA was shown to be expressed in lung and muscle.
VEGF and its homologies impart activity by binding to vascular endothelial cell plasma membrane-spanning tyrosine kinase receptors which then activates signal transduction and cellular signals. The Flt receptor family is a major tyrosine kinase receptor which binds VEGF with high affinity. At present the fit receptor family includes flt-1 (Shibuya, et al., 1990,
Oncogene
5: 519-524), KDR/flk-1(Terman, et al., 1991,
Oncogene
6: 1677-1683; Terman, et al., 1992,
Biochem. Biophys. Res. Commun.
187: 1579-1586), and flt-4 (Pajusola, et al., 1992,
Cancer Res.
52:5738-5743).
The involvement of VEGF in promoting tumor angiogenesis has spawned studies investigating possible antagonists of the process. Both polyclonal (Kondo, et al., 1993,
Biochem. Biophys. Res. Commun.
194: 1234-1241) and monoclonal (Kim, et al., 1992,
Growth Factors
7: 53-64; Kim, et al., 1993,
Nature
362: 841-844) antibodies raised against VEGF have been shown to suppress VEGF activity in vivo. Anti-VEGF antibody strategies to interdict angiogenesis and its attendant tumor are also addressed in Kim et al. (1993,
Nature
362: 841-844) and Asano et al. (1995,
Cancer Research
55: 5296-5301).
Kendall and Thomas (1993,
Proc. Natl. Acad. Sci.
90: 10705-10709) isolated and characterized a cDNA encoding a secreted soluble form of flt-1 from cultured human umbilical vein endothelial cells (HUVEC). The recombinant version of this protein was purified by binding to immobilized heparin. Isolated soluble flt-1 was shown to inhibit VEGF activity in vitro. No suggestion regarding gene transfer protocols were disclosed.
Millauer et al. (1994,
Nature
367: 576-579) disclose in vivo inhibition of tumor angiogenesis by expression of an artificially generated flk-1 mutant in which the intracellular kinase domain but not the membrane-spanning anchor was deleted. The authors do not forward any teaching or suggestion that a soluble form of a VEGF tyrosine kinase receptor would be useful in gene therapy applications.
Neovascularization of malignant tumors is an integral process contributing to solid tumor growth and neoplastic progression (Kondo et al., 1993,
Biochemical
&
Biophysical Research Communications
194: 1234-1241; Carrau et al., 1995,
Invasion
&
Metastasis
15: 197-202). In this context, several studies have demonstrated a positive correlation between neovascularization in malignant tumors and poor clinical outcomes (Volm et al., 1996,
Anticancer Research
16: 213-217; Toi et al., 1994, Japanese Journal of Cancer Research 85: 1045-1049; Shpitzer et al., 1996,
Archives of Otolaryngology—Head
&
Neck Surgery;
122: 865-868; Staibano et al., 1996,
Human Pathology
27: 695-700; Giatromanolaki et al., 1996,
J. of Pathology
179: 80-88). While the angiogenic process has several mediators, it appears that vascular endothelial growth factor (VEGF) may be a critical growth factor with respect to initiating the cascade of events stimulating new blood vessel formation in several tumor types (Toi et al., 1996,
Cancer
77: 1101-1106; Maeda et al., 1996,
Cancer
77: 858-63; Anan et al., 1996,
Surgery
119: 333-339).
Aiello et al. (1995, Proc. Natl. Acad. Sci. USA 92:10457-10461) disclose genetically engineered chimeric extracellular VEGF receptors to block angiogenesis in non-malignant cells.
Despite recent advances in identifying genes encoding ligands and receptors involved in angiogenesis, no gene therapy application has been forwarded which overcomes the deleterious effect this process has in promoting primary tumor growth and subsequent metastasis. The present invention addresses and meets this need.
SUMMARY OF THE INVENTION
The present invention relates to methods of gene therapy for inhibiting VEGF-induced angiogenesis associated with diseases and disorders including, but not limited to, solid tumor growth, tumor metastasis, inflammation, psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy, and macular degeneration. These disorders are related in that VEGF acts as a mitogen to stimulate local angiogenesis from vascular endothelial cells which in turns exacerbates the condition.
The present invention relates to gene transfer of a DNA vector and concomitant in vivo expression of a soluble form of a tyrosine receptor kinase (sVEGF-R) within the mammalian host which binds VE
Bett Andrew J.
Goldman Corey K.
Huckle William R.
Kendall Richard L.
Thomas, Jr. Kenneth A.
Guzo David
Hand J. Mark
Merck & Co. , Inc.
Tribble Jack L.
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