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Q3 sparc deletion mutant and uses thereof

Q3 sparc deletion mutant and uses thereof description/claims

This patent application is a divisional of copending U.S. patent application Ser. No. 11/356,829, filed Feb. 17, 2006, which is hereby incorporated by reference in its entirety. This patent application claims the benefit of U.S. Provisional Patent Application No. 60/654,261 filed on Feb. 18, 2005, which is hereby incorporated by reference in its entirety.

This invention relates to methods for treating cancer, other diseases involving abnormal proliferative, hyperplastic, remodeling, and inflammatory activity in tissues and organs using antibody or other suitable ligands recognizing SPARC. The invention also relates to mutant SPARC polypeptides and nucleic acids and methods of their use, as well as methods for targeting, methods for imaging, and methods for determining the response of mammalian tumors to anti-SPARC therapy.

Secreted protein acidic and rich in cysteine (also known as osteonectin, BM40, or SPARC) (hereafter “SPARC”), is a matrix-associated protein that elicits changes in cell shape, inhibits cell-cycle progression, and influences the synthesis of extracellular matrix (Bradshaw et al., Proc. Nat. Acad. Sci. USA 100: 6045-6050 (2003)). The murine SPARC gene was cloned in 1986 (Mason et al., EMBO J. 5: 1465-1472 (1986)) and a full-length human SPARC cDNA was cloned and sequenced in 1987 (Swaroop et al., Genomics 2: 37-47 (1988)). SPARC expression is developmentally regulated, and is predominantly expressed in tissues undergoing remodeling during normal development or in response to injury. For example, high levels of SPARC protein are expressed in developing bones and teeth (see, e.g., Lane et al., FASEB J., 8, 163 173 (1994); Yan & Sage, J. Histochem. Cytochem. 47:1495-1505 (1999)).

SPARC is upregulated in several aggressive cancers, but is absent in the corresponding inormal tissues (Porter et al., J. Histochem. Cytochem., 43, 791 (1995)). SPARC expression is induced among a variety of tumors (e.g., bladder, liver, ovary, kidney, gut, and breast). In bladder cancer, for example, SPARC expression has been associated with advanced carcinoma. Invasive bladder tumors of stage T2 or greater have been shown to express higher levels of SPARC relative to bladder tumors of stage T1 (or less superficial tumors), and poorer prognosis (see, e.g., Yamanaka et al., J. Urology, 166, 2495 2499 (2001)). In meningiomas, SPARC expression has been associated only with invasive tumors (see, e.g., Rempel et al., Clincal Cancer Res., 5, 237 241 (1999)). SPARC expression also has been detected in 74.5% of in situ invasive breast carcinoma lesions (see, e.g., Bellahcene, et al., Am. J. Pathol., 146, 95 100 (1995)), and 54.2% of infiltrating ductal carcinoma of the breast (see, e.g., Kim et al., J. Korean Med. Sci., 13, 652 657 (1998)). SPARC expression also has been associated with frequent microcalcification in breast cancer (see, e.g., Bellahcene et al., supra), suggesting that SPARC expression may be responsible for the affinity of breast metastases for the bone.

Surprisingly, SPARC has also been shown to have anti-tumor activity in some systems. SPARC is a potent cell cycle inhibitor that arrests cells in mid-G (Yan & Sage, J. Histochem. Cytochem. 47:1495-1505 (1999)) and the inducible expression of SPARC has been shown to inhibit breast cancer cell proliferation in an in vitro model system (Dhanesuan et al., Breast Cancer Res. Treat. 75:73-85 (2002)). Similarly, exogenous SPARC can reduce the proliferation of both HOSE (human ovarian surface epithelial) and ovarian cancer cells in a concentration-dependent manner. In addition, SPARC induces apoptosis in ovarian cancer cells. Further evidence for SPARC receptors present on cells such as ovarian epithelial cells has been report. It has been proposed that the binding of SPARC to its receptor is likely to trigger tissue-specific signaling pathways that mediate its tumor suppressing functions (Yiu et al., Am. J. Pathol. 159:609-622 (2001)). Purified SPARC has also been reported to potently inhibit angiogenesis and significantly impair neuroblastoma tumor growth in an in vivo xenograft model system (Chlenski et al., Cancer Res. 62:7357-7363 (2002)).

SPARC also plays a role in non-neoplastic proliferative diseases. Mesangial cell proliferation is a characteristic feature of many glomerular diseases and often precedes extracellular matrix expansion and glomerulosclerosis. In a model of experimental mesangioproliferative glomerulonephritis, SPARC mRNA was increased 5-fold by day 7 and was identified in the mesangium by in situ hybridization. However, recombinant SPARC or a synthetic SPARC peptide inhibited platelet-derived-growth-factor-induced mesangial cell DNA synthesis in vitro (Pichlier et al., Am. J. Pathol. 148(4): 1153-67 (1996)). Similarly, while renal enlargement, due to hyperplasia, hypertrophy, and increase inter-cellular matrix, is a characteristic feature of diabetes in humans, kidney SPARC mRNA levels fell in diabetic animals. In addition, the onset of diabetes-related kidney growth is associated with a reduction in SPARC mRNA and protein (Gilbert et al., Kidney Int. 48(4):1216-25 (1995)).

SPARC has been implicated in the pathogenesis of atherosclerotic lesions. Plasma SPARC levels are elevated in patients with coronary artery disease (Masahiko et al., Obesity Res. 9:388-393 (2001)). The proliferation of vascular smooth muscle cells in the arterial intima plays a central role in the pathogenesis of atherosclerosis. SPARC is expressed in vascular smooth muscle cells and macrophages associated with atherosclerotic lesions. In addition, SPARC has been hypothesized to regulate the action of platelet-derived growth factor during vascular injury (Masahiko et al., Obesity Res. 9:388-393 (2001); Raines et al., Proc. Natl. Acad. Sci. USA 89:1281-1285 (1992)). A stimulating effect of SPARC on endothelial PAI-1 production has been reported at the site of vascular injury (Hasselaar et al., J. Biol. Chem. 266:13178-13184 (1991)) and has been postulated to accelerate atherosclerosis (Masahiko et al., Obesity Res. 9:388-393 (2001)).

SPARC has affinity for a wide variety of ligands including cations (e.g., Ca 2+, Cu 2+, Fe 2+), growth factors (e.g., platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF)), extracellular matrix (ECM) proteins (e.g., collagen IV and collagen IX, vitronectin, and thrombospondin 1), endothelial cells, platelets, albumin, and hydroxyapaptite (see, e.g., Lane et al., FASEB J., 8, 163 173 (1994); Yan & Sage, J. Histochem. Cytochem. 47:1495-1505 (1999)). SPARC is also known to bind albumin (see, e.g., Schnitzer, J. Biol. Chem., 269, 6072 (1994)).

Antibody therapy is an effective method for controlling disease wherein a specific protein marker can be identified. Examples include Avastin (anti-VEGF antibody), Rituxan (anti-CD20 antibody), and Remicade (anti-TNF antibody). As such, antibody against SPARC represent an important therapeutic agent for treating human and other mammalian tumors, or other proliferative, hyperplastic, remodeling, and inflammatory disorders, that express the SPARC protein.

Accordingly, there is a need for novel forms of SPARC and antibodies reactive with such novel forms of SPARC. The present invention provides for such novel SPARC polypeptides, nucleic acids which encode such novel SPARC polypeptides, and methods of use of such novel SPARC polypeptides and nucleic acids. The invention additionally provides for antibodies against SPARC polypeptides.

The glutamine corresponding to amino acid 20 in SEQ ID NO: 1 (the sequence of the unprocessed primary translation product) corresponds to the glutamine at amino acid position 3 in the mature protein (the polypeptide without the 17 amino acid SPARC leader sequence) (the “Q3” glutamine). The invention provides for an isolated SPARC polypeptide comprising an amino acid sequence wherein the glutamine corresponding to amino acid 20 in SEQ ID NO: 1 is deleted (hereafter “Q3 SPARC deletion mutant polypeptide”). In particular, the invention provides for an isolated human Q3 SPARC deletion mutant polypeptide. The invention also provides for an isolated SPARC polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2, which corresponds to a mature Q3 SPARC deletion mutant polypeptide. The invention further provides for an isolated nucleic acid molecule encoding a Q3 SPARC deletion mutant polypeptide. In particular, the invention provides for an isolated nucleic acid molecule encoding human Q3 SPARC deletion mutant polypeptide. Isolated nucleic acids encompassed by the invention include, but are not limited to, nucleic acids comprising the sequence of SEQ ID NO: 3.

The invention also provides for a vector comprising a nucleic acid molecule encoding a Q3 SPARC deletion mutant polypeptide including, but not limited to, wherein the vector further comprises a promoter controlling the expression of the Q3 SPARC deletion mutant polypeptide encoding nucleic acid sequences. In addition, the invention provides for a cell comprising a nucleic acid molecule encoding a Q3 SPARC deletion mutant polypeptide, wherein the cell is either a prokaryotic cell or a eukaryotic cell. The invention further provides for a method of making a Q3 SPARC deletion mutant polypeptide of comprising: (a) transforming cells with a nucleic acid encoding a Q3 SPARC deletion mutant polypeptide; (b) inducing the expression of the Q3 SPARC deletion mutant polypeptide by the transformed cells, and (c) purifying the Q3 SPARC deletion mutant polypeptide.

In another embodiment the invention provides for composition comprising a Q3 SPARC deletion mutant polypeptide or a nucleic acid encoding a Q3 SPARC deletion mutant polypeptide and a pharmaceutically acceptable carrier and method of treating a disease comprising administering the Q3 SPARC deletion mutant polypeptide and carrier. Such a method can be used in accordance with the invention to treat a disease including, without limitation, wherein the disease is a cell proliferative disease (e.g., cancer, benign tumor, atherosclerosis, vascular restenosis). The invention also provides for a method of sensitizing a disease comprising administering a Q3 SPARC deletion mutant polypeptide including wherein the disease is a cell proliferative disease (e.g., cancer, benign tumor, atherosclerosis, vascular restenosis). In addition, the invention provides for compositions comprising a Q3 SPARC deletion mutant polypeptide, wherein the polypeptide is coupled or conjugated to a therapeutic or diagnostic agent, such as, e.g., a radioisotope or radioinuclide, drug, polypeptide or toxin. Alternatively, the Q3 SPARC deletion mutant polypeptide can be coupled or conjugated to molecule which stabilizes the Q3 SPARC deletion mutant polypeptide in vivo, such as, e.g., a polyethylene glycol. The Q3 SPARC deletion mutant polypeptide or nucleic acid encoding a Q3 SPARC deletion mutant polypeptide and pharmaceutically acceptable carrier can be administered through any suitable route including, without limitation, intravenous, intraperitoneal, intratumoral, or inhalational.

In one embodiment the invention provides for antibody or fragment thereof with recognition for a SPARC polypeptide, in particular for a Q3 SPARC deletion mutant polypeptide, and a pharmaceutically acceptable carrier. Such an antibody with recognition for a SPARC polypeptide, can be used in accordance with the invention to, e.g., mediate complement activation and/or cell mediated cytotoxicity against the tumor or other proliferative disease.

In yet another embodiment, the invention provides for a composition comprising a therapeutic agent coupled to an antibody or fragment thereof with recognition for a SPARC polypeptide, in particular for a Q3 SPARC deletion mutant polypeptide, and a pharmaceutically acceptable carrier including, without limitation, wherein said antibody or fragments thereof are humanized and include monovalent Fab′, divalent Fab2, scfv, diabody, or a chimera (hereafter collectively an “anti-Q3 SPARC deletion mutant antibody.”) The antibody prepared in accordance with the invention can be monoclonal or polyclonal, manufactured in non-human animals, and be humanized. In addition, the invention provides for an anti-SPARC antibody, in particular, an anti-Q3 SPARC deletion mutant antibody, coupled or conjugated to a therapeutic agent, wherein the therapeutic agent is a chemotherapeutic drug, radionuclide, or peptide. Suitable therapeutic agents also include therapeutic agents that are biological molecules such as, e.g, tTF and TNF.

The Q3 SPARC deletion mutant polypeptide or anti-SPARC antibody, such as, e.g., anti-Q3 SPARC deletion mutant antibody, and pharmaceutically acceptable carrier can be administered through any suitable route including, without limitation, intravenous, intraperitoneal, intratumoral, or inhalational. Accordingly, the invention provides for a method for delivering a therapeutic agent to a disease site, such as, e.g., a tumor in a mammal, which method comprises administering to the mammal a therapeutically effective amount of a pharmaceutical composition, wherein the therapeutic agent comprises a chemotherapeutic agent or radioactive agent coupled to an pharmaceutically acceptable carrier and a pharmaceutically acceptable carrier. Suitable tumors that can be treated in accordance with the invention include, without limitation, tumors located in the bladder, liver, ovary, kidney, gut, brain, or breast of a human or non-human animal.

The invention additionally provides for a method for delivering a diagnostic agent to a disease site, such as, e.g., a tumor in a mammal, which method comprises administering to the mammal a diagnostically effective amount of a pharmaceutical composition. Suitable such compositions include, without limitation, wherein the composition comprises a diagnostic agent coupled to an anti-SPARC antibody, such as, e.g., an anti-Q3 SPARC deletion mutant antibody, and a pharmaceutically acceptable carrier. Suitable diagnostic agents include, without limitation, radioactive agents, MRI contrast agents, X-Ray contrast agents, ultrasound contrast agents, and PET contrast agents.

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