Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
1995-12-22
2001-07-03
Guzo, David (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C530S327000
Reexamination Certificate
active
06255452
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application, various references are referred to within parenthesis. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the sequence listing and the claims.
Rapid cell proliferation, in vivo, is responsible for a wide variety of conditions in mammals, including particularly humans, such as carcinomas, sarcomas, tumors, warts, papillomas, psoriasis and keloid scars. There is a continuing need for control and inhibition of such rapid cell proliferation in mammals, particularly human beings.
A number of growth factors which cause rapid cell proliferation are known. Such growth factors include transforming growth factor (TGF), nerve growth factor (NGF) and epidermal growth factor (EGF).
EGF is known to be a prototype for a family of cytokines which are recognized by Epidermal Growth Factor Receptor (EGFR) and which share general similarities in structure.
The present invention was examined in connection with EGF. However, it is anticipated that the present invention is applicable to cellular proliferation facilitated by any member of the EGF family.
EGF's roles in normal physiology and oncogenesis are not clear (1). However, it is known to target both epithelial and stromal cells and to stimulate epithelial growth (2, 3).
Cell activation in response to EGF is facilitated by a specific receptor that recognizes it. After binding EGF, the external domain of EGFR undergoes conformational changes (4), leading to phosphorylation of EGFR cytoplasmic domain. Cell activation follows.
Several types of inhibitors of EGF activity have been reported. Some such inhibitors are structurally unrelated to EGF or EGFR, such as cyclosporin A, interferon-&ggr;, chrysarobin and TGF&bgr; (5, 6). Prostaglandin and some anti-EGFR monoclonal antibodies and phorbol esters also are known to inhibit stimulation of certain target cells by EGF (6, 7, 8, 9). Several monoclonal anti-EGFR antibodies inhibit EGF-dependent growth of a human breast carcinoma cell line in vitro (10).
EGF-like proteins and peptides have also been used to inhibit growth stimulation of target cells by EGF. Small proteins that compete with EGF for EGFR, and mimic EGF activity on target cells have been identified in two human tumors (11). Engineered mutants of EGF are associated with decreased EGF-stimulated tyrosine kinase activity (12). It has been reported that a synthetic peptide encompassing the third disulfide loop of TGF&agr; inhibits EGFR-related growth of human mammary carcinoma cells, although proliferation stimulated by fibroblasts or platelet derived growth factors was unaltered (13).
Several years ago, there was described a protein that appeared to alter the ability of target cells to respond to EGF. Strayer, D. S. et al., Inhibition of Epidermal Growth Factor-Induced Cellular Proliferation, Am. J. Pathol. 128:203-209 (1987).
Heretofore, production and purification methods for, therapeutic uses of, and useful compositions containing, this protein, referred to herein as EGF inhibitor (EGFI) have not been available.
SUMMARY OF THE INVENTION
This invention provides an epidermal growth factor inhibitor protein capable of inhibiting epidermal growth factor-induced cellular proliferation.
This invention also provides a method of obtaining epidermal growth factor inhibitor from a host cell, comprising:
a) infecting the host cell with a virus;
b) disrupting the infected host cell wall;
c) removing cellular debris;
d) precipitating with methanol;
e) removing precipitates; and
f) collecting supernatant containing epidermal growth factor inhibitor.
This invention also provides a method of obtaining purified epidermal growth factor inhibitor from a sample, comprising:
a) loading the sample onto a reverse phase HPLC column;
b) eluting from the reverse phase HPLC column with a gradient which proceeds from about fifty percent methanol in aqueous solution to about ninety percent acetonitrile;
c) collecting a fraction having epidermal growth factor inhibiting activity;
d) electrophoresing the fraction in an electrophoresis medium; and
e) recovering protein having a molecular weight of about 34 kilodaltons from the electrophoresis medium, thereby obtaining purified epidermal growth factor inhibitor from the sample.
This invention also provides a method for treating a cell proliferative condition in a mammalian subject, comprising administering to the subject a therapeutically effective amount of an agent selected from the group consisting of epidermal growth factor inhibitor, TC4, and CDC25.
This invention also provides a pharmaceutical composition, comprising a therapeutically effective amount of an agent selected from the group consisting of epidermal growth factor inhibitor, TC4, and CDC25, and a pharmaceutically acceptable carrier.
This invention also provides a nucleic acid molecule comprising a first segment coding for epidermal growth factor inhibitor.
This invention also provides a vector for gene therapy, comprising:
a) a nucleic acid molecule having i) a transcription regulatory segment; and ii) a second segment coding for a polypeptide having at least fifty percent homology to a polypeptide selected from the group consisting of: the epidermal growth factor inhibitor protein of this invention; TC4; CDC25; an epidermal growth factor inhibitor fragment; a TC4 fragment; and a CDC25 fragment; under transcriptional control of the transcription regulatory sequence; and
b) a delivery vehicle for delivering the nucleic acid molecule.
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Guzo David
Kumar Nanda P. B. A.
McNichol, Jr. William J.
ReedSmith LLP
Thomas Jefferson University
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