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
2000-06-07
2004-09-28
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069600, C435S483000
Reexamination Certificate
active
06797488
ABSTRACT:
BACKGROUND OF THE INVENTION
The prognosis for metastatic cancer remains highly unfavorable. Despite advances in radiation therapy and chemotherapy, the long term survival of treated patients has shown only marginal improvement over the past few decades. The lack of significant treatment options available for metastatic cancers emphasizes the need to focus on the development of novel therapeutic strategies. In this regard, targeting tumor vasculature of solid tumors has recently shown promising results in several animal model systems (Baillie et al. (1995)
Br. J. Cancer
72:257-67; Bicknell, R. (1994)
Ann. Oncol.
5 (Suppl.) 4:45-50; Fan et al. (1995)
Trends Pharmacol. Sci.
16:57-66, Thorpe, P. E. and Burrows, F. J. (1995)
Breast Cancer Res. Treat.
36:237-51; Burrows, F. J. and Thorpe, P. E. (1994)
Pharmacol. Ther.
64:155-74). In a nude mouse model, for instance, introduction of a wild type VHL gene into 786-0 cells, a RCC tumor cell line, inhibited tumor growth (Iliopoulos et al. (1995)
Nat. Med.
1:822-26) and angiogenesis.
The growth of solid tumors beyond a few mm
3
depends on the formation of new blood vessels (Folknan, J. (1971)
N. Engl. J. Med.
285:1182-86). Numerous studies have shown that both primary tumor and metastatic growth are angiogenesis-dependent (Folkman, J. (1971)
N. Engl. J. Med.
285:1182-86; Folkman, J. (1972)
Ann. Surg.
175:409-16; Folknan, J. and Shing, Y. (1992)
J. Biol. Chem.
267:10931-34; Folkman, J. (1996)
Sci. Am.
275:150-54). A number of angiogenesis inhibitors have been identified. Certain ones, such as platelet factor-4 (Maione et al. (1990)
Science
247:77-79; Gupta et al. (1995)
Proc. Natl. Acad. Sci.
(
USA
) 92:7799-7803), interferon, interferon-inducible protein-10, and PEX (Angiolillo et al. (1995)
J. Exp. Med.
182:155-62; Stricter et al. (1995)
Biochem. Biophys. Res. Commun.
210:51-57; Brooks et al. (1998)
Cell
92:391-400), are not “associated with tumors,” whereas two others, angiostatin and endostatin, are “tumor-associated” (O'Reilly et al. (1994)
Cell
79:315-28; O'Reilly et al. (1997)
Cell
88:277-85). Angiostatin, a potent endogenous inhibitor of angiogenesis generated by tumor-infiltrating macrophages that upregulate matrix metalloelastase (Dong et al. (1997)
Cell
88:801-10), inhibits the growth of a wide variety of primary and metastatic tumors (Lannutti et al. (1997)
Cancer Res.
57:5277-80; O'Reilly et al. (1994)
Cold Spring Harb. Symp. Quant. Biol.
59:471-82; O'Reilly, M. S., (1997)
Exs.
79:273-94; Sim et al. (1997)
Cancer Res.
57:1329-34; Wu et al. (1997)
Biochem. Biophys. Res. Commun.
236:651-54). Recently, O'Reilly, et al. ((1997)
Cell
88:277-85) have isolated endostatin, an angiogenesis inhibitor from a murine hemangioendothelioma cell line (EOMA). Circulating levels of a fragment of human endostatin have been detected in patients with chronic renal insufficiency with no detectable tumor (Wu et al. (1997)
Biochem. Biophys. Res. Commun.
236:651-54).
The amino terminal sequence of endostatin corresponds to the carboxy terminal portion of collagen XVIII. Endostatin is a specific inhibitor of endothelial proliferation and angiogenesis. Systemic administration of non-refolded precipitated protein expressed in
Escherichia coli
caused growth regression of Lewis lung carcinoma, T241 fibrosarcoma, B16 melanoma and EOMA cells (O'Reilly et al. (1997)
Cell
88:277-85)in a xenograft model. Moreover, no drug resistance was noted in three of the tumor types studied. Repeated cycles of administration with endostatin have been reported to result in tumor dormancy (Boehm et al.(1997)
Nature
390:404-407).
The results from these angiostatin and endostatin studies open new avenues for treatment of cancer and provide promising routes for overcoming the drug resistance often seen during chemotherapy. However, in all of these investigations, a non-refolded precipitated form of the inhibitor protein was administered in the form of a suspension to tumor bearing animals. In addition, large amounts of protein were required to cause tumor regression and to lead to tumor dormancy. As pointed out by Kerbel ((1997)
Nature
390:335-36), oral drug equivalents of these proteins are needed. Mechanistic investigations could be undertaken if recombinant forms of these proteins were available in soluble form. Moreover, initial testing could be done in vitro with soluble protein before studying its efficacy under in vivo conditions.
Furthermore, there have been reports that despite the great promise held by these proteins, evaluation of their clinical potential is stymied due to difficulties in producing enough of the protein to test, and inconsistent test results regarding their anti-angiogenic properties, e.g., anti-angiogenic activity (King, R. T.,
Wall Street J
., Page 1, November 12 (1998); Leffe, D. N.,
BioWorld Today,
9:1, October 20 (1998)). There clearly exists at the present time a great need for a reproducible method of producing soluble forms of anti-angiogenic proteins with sufficient biological activity to be clinically effective and to reliably produce these proteins in high yields without sacrificing such critical activity.
SUMMARY OF THE INVENTION
The present invention relates to the discovery of a reproducible method of producing anti-angiogenic proteins with biological activity sufficient to be clinically effective. As described herein, the anti-angiogenic proteins encompassed by the present methods are reproducibly produced in high yields (for example from 10 to 20 mg/liter of culture medium. Importantly, the anti-angiogenic proteins produced by the methods described herein retain high biological activity.
Anti-angiogenic proteins are well-known to those of skill in the art. For example, angiostatin, endostatin, the 16 kD prolactin fragment, RNAisn, TNP470, 2-methoxy estradiol and heparin, to name a few. As used herein, the term “anti-angiogenic protein(s)” encompass not only intact proteins, but mutants (e.g., with amino acid residues added, deleted or altered), fragments (e.g., specifically with amino acids deleted), derivatives (e.g., modified proteins or peptides, for example to reduce protease degradation) and fusion proteins wherein the fusion proteins comprise a combination of two or more known anti-angiogenic proteins (e.g., angiostatin and endostatin, or biologically active fragments of angiostatin and endostatin), or an anti-angiogenic protein in combination with a targeting agent (e.g., endostatin with epidermal growth factor (EGF) or RGD peptides), or an anti-angiogenic protein in combination with an immunoglobulin molecule (e.g., endostatin and IgG, specifically with the Fc portion removed). The term “fusion protein” as used herein can also encompass additional components for e.g., delivering a chemotherapeutic agent, wherein a polynucleotide encoding the chemotherapeutic agent is linked to the polynucleotide encoding the anti-angiogenic protein. Fusion proteins can also encompass multimers of the anti-angiogenic protein, e.g., a dimer or trimer of endostatin.
It is also to be recognized that any of the anti-angiogenic proteins described herein can be post-translationally modified to encompass targeting moieties (e.g., vascular endotheleial growth factor (VEGF) or chemotherapeutic agents, such as ricin, or radioisotopes. Additional post-translational modifications can include multimerization, e.g., by chemical cross-linking using techniques well-know to those of skill in the art. However, for brevity, the term “anti-angiogenic protein” is used herein without specifically referring to mutant, derivatives, fragments and fusion proteins.
Encompassed by the present invention are methods of producing a biologically active anti-angiogenic protein, or a biologically active mutant, fragment, derivative or fusion protein thereof, using a prokaryotic or eukaryotic expression system. Specifically encompassed is the use of a yeast expression system, more specifically the
Pichia pastoris
yeast expression system.
The method steps involve inserting an isolated
Beth Israel Deaconess Medical Center
Gyure Barbara A.
Ketter James
Palmer & Dodge LLP
Williams Kathleen
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