Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
2001-03-30
2004-12-28
Scheiner, Laurie (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S324000, C514S002600
Reexamination Certificate
active
06835806
ABSTRACT:
BACKGROUND
Generally, stable water soluble purified polypeptides which are potent inhibitors of endothelial cell proliferation in-vivo may be useful in elucidating the mechanism by which angiogenesis is regulated. Specifically, stable water soluble polypeptide inhibitors of angiogenesis reduce the volume of animal tumors, including human tumors, in-vivo.
Evidence suggests that angiogenesis is essential for the growth and persistence of solid tumors. Folkman, 1989; Hori et al., 1991; Kim et al. 1993. To stimulate angiogenesis, tumors up regulate their production of a variety of angiogenic factors, including the fibroblast growth factors. Kandel et al., 1991. Studies suggest that proteins such as murine endostatin protein (184 residue protein derived from the cleavage of Type XVIII collagen expressed by hemanioendothelioma EOMA or obtained as an expression product from recombinant cells) may cause marked reduction of mouse tumors. U.S. Pat. No. 5,854,205, hereby incorporated by reference. Similarly, angiostatin protein (200 residue protein derived from the cleavage of plasminogen) has been shown to inhibit the metastasis of certain primary mouse tumors. http://www.ultranet.com/jkimball/BiologicalPages/A/Angiogenesis.html (Mar. 31, 2000), hereby incorporated by reference.
The potential use of the entire endostatin and angiostatin proteins as human drug therapies has been surrounded by enormous publicity and there appears to be a large commercial market for endostatin or angiostatin proteins, or other angiogenesis inhibitors as drugs. Because of the growing commercial, therapeutic, and research potential for angiogenesis inhibitors, numerous research studies have been conducted which disclose a variety of uses for the endostatin and angiostatin proteins. In spite of the numerous studies conducted with whole endostatin and angiostatin proteins, substantial problems remain unresolved with regard to providing angiogenesis inhibitors that can be commercialized, or used as drug therapies in animals or in humans, or as angiogenic compounds in research.
A significant problem with whole protein angiogenesis inhibitors, for example endostatin proteins or angiostatin proteins, can be that it may be difficult to predict what portion of the protein is biologically active. As disclosed by U.S. Pat. No. 5,854,205, hereby incorporated by reference, the endostatin protein is comprised of 184 residues with a molecular weight of about 20 kDa. Similarly, the angiostatin protein comprised of 200 residues has a molecular weight of about 21 kDa. With respect to such protein angiogenesis inhibitors, it is difficult to predict which residues encompassed by the primary structure of the protein may be responsible for the observed angiogenesis inhibition activity. One aspect of this difficulty may be that the biologically active portion of the protein may comprise discontinuous regions of the primary structure of the protein which must be held in a specific secondary or tertiary structure by the remaining portions of the protein molecule to acquire angiogenesis inhibition activity. A second aspect of this difficulty may be that the biologically active region of the protein molecule may be a continuous region of the primary structure of the protein which must be similarly held in a specific secondary or tertiary structure by the remaining portion of the protein molecule to acquire angiogenesis inhibition activity. A third aspect of this difficulty may be that there may be a plurality of biologically active regions encompassed by the primary sequence of the protein some of which may be discontinuous or some of which may be continuous. A fourth aspect of this difficulty may be that such plurality of biologically active regions encompassed by the primary sequence of the protein overlap one another and may not be independently excised from the primary sequence with out disabling the other biologically active regions. A fifth aspect of this difficulty may be that the region of a protein having angiogenesis inhibition activity may be discontinuous from the region of the protein which has an affinity for the target cell receptor. A sixth aspect of this difficulty may be that a portion of an angiogenic protein may apparently lack biological activity when assayed in-vitro but may acquire biological activity when assayed in-vivo. A seventh aspect of this difficulty may be that a portion of an angiogenic protein when chemically or enzymatically excised, or when identified and subsequently chemically synthesized, may not be biologically available to the target receptor in-vitro or in-vivo. This lack of biological availability may be due to insolubility of the compound, a binding affinity to surrounding substrates that is greater than to the target cell receptor, instability of the angiogenic compound with respect to cleavage, or with respect to modification of the peptide backbone, N-terminus, C-terminus, side chain, or other peptide or chemical moiety associated with the excised or chemically synthesized portion of the protein. Due to these, and a variety of other difficulties well known to those with skill in the art, assignment of angiogenesis inhibition activity to any specific biochemical structure, which may be a portion of a protein, such as endostatin or angiostatin, or any other molecule, may be unpredictable without an actual reduction to practice involving at least isolation, purification, and in-vitro and in-vivo assays to confirm biological activity of a particular compound. Subsequent characterization and identification of the chemical structure may further serve to differentiate biologically active compounds which in every other respect may seem similar.
Another significant problem with the commercial development of additional novel angiogenesis inhibitors may be that small polypeptides (primary sequences comprised of 65 or fewer amino acid residues) have not been shown to have angiogenesis inhibition activity. One aspect of this problem may be due to the failure to incorporate within the primary sequence of the polypeptide the essential residues which have an affinity for the target cell receptor. A second aspect of this problem may be the failure to incorporate within the primary sequence of the polypeptide the essential residues which comprise the region conferring angiogenesis inhibition activity to the polypeptide. A third aspect of this problem may be that in-vitro methods of assaying polypeptides for biological activity may fail to properly address the processing requirements of the polypeptide in a manner which in-vivo methods for assaying polypeptides do address.
Another significant difficulty with existing angiogenesis inhibitors may be that therapeutic results are difficult to replicate. With respect to endostatin protein, for example, it can be difficult to reproduce results which show endostatin protein dramatically shrinks tumors. Harvard Cancer Research Questioned, New York Times Company, Associated Press (1998); Ovarian Cancer Research Notebook http://www.slip.net/mcdavis/database/angio188.htm; National Cancer Institute Clarifies Role in Development of Endostatin,
Angiogenesis Weekly,
(Tuesday, Oct. 5, 1999), each hereby incorporated by reference.
Another significant problem with existing angiogenesis inhibitors may be stability. Some angiogenesis inhibitors have proven to be unstable in shipment, or during subsequent routine handling, or during routine use in research studies resulting in a partial or complete loss of biological activity. National Cancer Institute Clarifies Role in Development of Endostatin,
Angiogenesis Weekly,
(Tuesday, Oct. 5, 1999), hereby incorporated by reference.
Another significant problem with existing angiogenesis inhibitors may be that it is difficult to produce sufficient amounts for wide spread use either for research or for therapies. Recombinant unfolded liner endostatin protein, for example, is insoluble (as disclosed by U.S. Pat. No. 5,854,205, hereby incorporated by reference) and may be difficult to use in applications which require soluble protein
CR Miles P.C.
Miles Craig
Phoenix Pharmaceuticals, Inc.
Scheiner Laurie
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