WO2013176667A2

WO2013176667A2 - Maspin-based treatment and prevention of cancer

Info

Publication number: WO2013176667A2
Application number: PCT/US2012/039165
Authority: WO
Inventors: Ming Zhang
Original assignee: Northwestern University
Priority date: 2012-05-23
Filing date: 2012-05-23
Publication date: 2013-11-28
Also published as: WO2013176667A3

Abstract

The present invention provides compositions and methods for the treatment of prevention of cancer through enhanced cell adhesion. In particular the present invention provides maspin-related peptides, and methods or use thereof, for the promotion of cell adhesion.

Description

MASPIN-BASED TREATMENT AND PREVENTION OF CANCER

FIELD OF THE INVENTION

BACKGROUND

Maspin is a non-inhibitory serine protease inhibitor (serpin) that was originally identified as a type II tumor suppressor protein in mammary epithelial cells (Zou et al. (1994) Science 263, 526-529; herein incorporated by reference in its entirety). One major tumor suppressor function of maspin is suppression of tumor cell motility, since it inhibits tumor cell migration/invasion in vitro and suppresses metastasis in mouse models (Zou et al. (1994) Science 263, 526-529; Abraham et al. (2003) J Urol 169, 1 157-1 161; Seftor et al. (1998)

Cancer Res 58, 5681-5685; Shi et al. (2001) Cancer Res 61, 6945-6951; Shi et al. (2002) Mol Ther 5, 755-761; Zhang et al. (1997) Mol Med 3, 49-59; Zhang et al. (2000) Oncogene 19, 6053-6058; herein incorporated by reference in their entireties). Several studies show that pericellular maspin inhibits cell motility by enhancing cell adhesion (Abraham et al. (2003) J Urol 169, 1 157-1161 ; Seftor et al. (1998) Cancer Res 58, 5681-5685; Ngamkitidechakul et al. (2001) Invest Ophthalmol Vis Sci 42, 3135-3141 ; Cella et al. (2006) Faseb J 20, 1510-1512; herein incorporated by reference in their entireties). In addition to its tumor suppressing functions, maspin is also essential for normal fetal development since

maspin knockout mice are embryonic lethal during the peri-implantation stage partially due to disrupted visceral endodermal cell adhesion (Gao et al. (2004) Development 131, 1479- 1489; herein incorporated by reference in its entirety).

Utilizing competition peptides and mutation analyses, two unique regions (amino acid residues 190-202 and 260-275) have been identified that are involved in facilitating the increased adhesion function of maspin (Endsley et al. J Biol Chem. 201 1 Jul

15;286(28):24599-607.; herein incorporated by reference in its entirety.). It has also been demonstrated that the urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) complex is required for the localization and adhesion function of maspin, and that maspin, uPAR, and βΐ integrin co-immunoprecipitate, suggesting a novel maspin-uPA-uPAR-βΙ integrin mega-complex that regulates mammary epithelial cell adhesion (Endsley et al. J Biol Chem. 2011 Jul 15;286(28):24599-607.; herein incorporated by reference in its entirety.).

SUMMARY OF THE INVENTION In some embodiments, the present invention provides a method for treating and/or preventing a disease or disorder by administering a maspin-related peptide or polypeptide to a sample (e.g., cells, tissue, etc.) or subject (e.g., human, non-human primate, rodent, etc.). In some embodiments, maspin-related peptides or polypeptides are pro-cell-adhesion peptides. In some embodiments, administration of maspin-related peptides or polypeptide enhances cell adhesion, inhibits cell motility, and/or suppresses metastasis. In some embodiments, enhancing cell adhesion results in reduced cell motility. In some embodiments, maspin- related peptides or polypeptides find use in the treatment and/or prevention of cancer, angiogenesis, tumor metastasis, and/or bone formation disorders (e.g., osteoporosis). In some embodiments, maspin-related peptides or polypeptides are administered to the cells, tissue, or subject comprising cancer cells, cells at risk of becoming cancerous, or cells at risk of metastasis.

In some embodiments, a maspin-related peptide or polypeptide comprises a region with 70% sequence identity (e.g., 70%... 80%... 90%... 95%... 98%... 99%) with

STANAKVKLSIP (SEQ ID NO: l). In some embodiments, a maspin-related peptide or polypeptide comprises a region with 70% sequence identity (e.g., 70%... 80%... 90%... 95%... 98%... 99%) with TANAEVKLSIPK (SEQ ID NO:2). In some embodiments, a maspin- related peptide or polypeptide comprises a region with 70% sequence identity (e.g., 70%... 80%... 90%... 95%... 98%... 99%) with STENAKVKLSIP (SEQ ID NO:3). In some embodiments, a maspin-related peptide or polypeptide comprises at least 70% sequence (e.g., 70%... 80%... 90%... 95%... 98%... 99%) identity with all or a portion of SEQ ID NO: 1, SEQ ID NO:2, and/or SEQ ID NO:3.

In some embodiments, a maspin-related peptide or polypeptide is at least 6 amino acids in length (e.g., 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids,, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids... 30 amino acids... 40 amino acids, or more). In some embodiments, a maspin-related peptide or polypeptide is 6-15 amino acids. In some embodiments, a maspin-related peptide or polypeptide is 8-12 amino acids. In some embodiments, a maspin-related peptide or polypeptide is 10-20 amino acids. In some embodiments, a maspin-related peptide or polypeptide is 10-30 amino acids.

In some embodiments, a maspin-related peptide or polypeptide is capable of physically associating with uPAR, βΐ integrin, or both. In some embodiments, the protein or peptide localizes at the cell surface upon administration to the cells, tissue, or a subject. In some embodiments, the protein or peptide co-localizes with the uPA/uPAR complex.

In some embodiments, the present invention provides a pharmaceutical composition comprising: (a) a peptide or polypeptide comprising a portion with at least 70% sequence identity with all or a portion of (e.g., >50% of, >60% of, >70% of, >80% of, >90% of) a maspin-related peptide (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or structurally, sequentially, or functionally similar peptides), and (b) a physiologically suitable buffer. In some embodiments, the peptide or polypeptide enhances cell adhesion when administered to cells, tissue, or a subject. In some embodiments, all or a portion of the peptide or polypeptide comprises at least 70% sequence identity with one or more of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:3. In some embodiments, the peptide or polypeptide comprises at least 70% sequence identity with one or more of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:3. In some embodiments, the peptide or polypeptide comprises at least 90% sequence identity with one or more of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:3. In some embodiments, the peptide or polypeptide is SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:3.

In some embodiments, the present invention provides a pharmaceutical composition comprising: (a) one or more peptides or polypeptides comprising: (i) a peptide with at least 70% sequence identity with SEQ ID NO: 1, (ii) a peptide with at least 70% sequence identity with SEQ ID NO:2, (iii) a peptide with at least 70% sequence identity with SEQ ID NO:3; and (b) a physiologically suitable buffer. In some embodiments, the pharmaceutical composition enhances cell adhesion, inhibits angiogenesis, inhibits metastasis, treats or prevents cancer, and/or treats or prevent bone formation disorders when administered to cells, tissue, or a subject.

In some embodiments, the present invention provides a method of treating or preventing cancer or metastasis comprising administering to a cell, tissue, or subject a composition comprising a protein or peptide comprising at least 70% sequence identity with a or a portion of one or more of: SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO:3.

In some embodiments the peptide and polypeptides described herein (e.g., comprising a sequence with 70% identity to all or a portion of one or more of SEQ ID NOS: l-3) find use in treating and/or preventing cancer, angiogenesis, and/or metastasis. In some embodiments, the present invention provides pharmaceutical compositions and methods of coadministering an effective amount of at least one conventional anticancer agent in addition to a peptide or polypeptide described herein (e.g., comprising a sequence with 70% identity to all or a portion of one or more of SEQ ID NOS: l-3) to a patient, such that cancer, angiogenesis, and/or metastasis is treated and/or prevented.

The present invention is not limited to treatment and/or prevention of cancer. The peptides provided herein also find use in treating other diseases and disorders. In certain embodiments, the present invention provides methods for treating bone formation disorders (e.g., by administration of peptides provided herein). In some embodiments, the methods comprise administering to a subject suffering from a bone formation disorder a composition comprising a protein or peptide comprising at least 70% sequence identity with a or a portion of one or more of: SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO:3. The methods are not limited to a particular type or severity of a bone formation disorder. An example of a bone formation disorder includes, but is not limited to, osteoporosis. The methods are not limited to treating a certain type of subject. In some embodiments, the subject is a rodent (e.g., mouse), while in other embodiments; the subject is a human being. In some embodiments, the composition is coadministered with, for example, an anti-osteoporosis agent (e.g., a hormone replacement therapy agent, a bisphosphonate (e.g., alendronate (e.g., FOSAMAX)), vitamin D, an androgen, a parathyroid hormone, a selective estrogen-receptor modulators, and a calcitonin-salmon). In certain embodiments, osteoblast cell proliferation is promoted by administering to a sample (or subject) comprising osteoblast cells a composition comprising a polypeptide or peptide comprising at least 70% sequence identity with a or a portion of one or more of: SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO:3.

In some embodiments, the present invention provides a composition comprising a peptide or polypeptide that comprises a portion with at least 70% sequence identity with all or a portion of one or more of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3 for use as a medicament. In some embodiments, the present invention provides a composition comprising a peptide or polypeptide that comprises a portion with at least 70% sequence identity with all or a portion of one or more of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3 in the treatment of cancer. In some embodiments, the present invention provides a composition comprising a peptide or polypeptide that comprises a portion with at least 70% sequence identity with all or a portion of one or more of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3in the treatment of a bone formation disorder. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the 3D structure of maspin. Inset (right panel) indicates that three key amino acids E201, K270, K268 are critical for maspin action. Figure 2 shows the effect of maspin peptides X and Y on HUVEC tube formation.

HUVECs were plated on MatriGel for tube formation assay. Various peptides were tested for the potency on tube angiogenensis. Peptide X and Y were shown to be highly potent compared to the G-Helix peptide or GST-maspin. DEFINITIONS

As used herein, the term "subject" refers to any human or animal (e.g., non-human primate, rodent, feline, canine, bovine, porcine, equine, etc.). For methods of treatment, a subject may be any human or animal subject having a neoplasia, such as cancer or precancer. For methods of prevention, the subject may be any human or animal subject who is at risk of metastasis or developing a cancer. The subject may be at risk due to exposure to carcinogenic agents; being genetically predisposed to disorders characterized by unwanted, rapid cell proliferation; being predisposed (e.g., genetically or otherwise) to increased cell motility; etc. The term "patient" is used herein to refer to a human subject receiving treatment for a disease, disorder, and or condition, or being administered compositions of the present invention.

As used herein, the term "effective amount" refers to the amount of a composition (e.g., maspin-derived or maspin-related protein or peptide) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

As used herein, the term "administration" refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., compositions of the present invention) to a subject (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplary routes of administration to the human body can be through the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like. As used herein, the terms "co-administration" and "co-administer" refer to the administration of at least two agent(s) (e.g., a combination maspin-derived or maspin-related proteins or peptides, a combination of oligonucleotides coding for a maspin-derived or maspin-related proteins or peptides, a maspin-related therapy and one or more other agents, etc.) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s).

As used herein, the term "pharmaceutical composition" refers to the combination of an active agent (e.g., maspin-derived or maspin-related protein or peptide) with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

The terms "pharmaceutically acceptable" or "pharmacologically acceptable," as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintigrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and

preservatives. For examples of carriers, stabilizers and adjuvants. (See e.g., Martin,

Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference).

As used herein, the term "pharmaceutically acceptable salt" refers to any salt (e.g., obtained by reaction with an acid or a base) of a compound of the present invention that is physiologically tolerated in the target subject (e.g., a mammalian subject, and/or in vivo or ex vivo, cells, tissues, or organs). "Salts" of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. DETAILED DESCRIPTION OF THE INVENTION

Tumor metastasis involves recognition, degradation, and migration through the surrounding extracellular matrix (ECM), a process intrinsically dependent on cell-ECM adhesion. Many different proteins (and/or protein complexes) are involved in promoting or inhibiting tumor metastasis. Maspin is a tumor suppressing protein that is abundantly produced in normal mammary luminal epithelial and myoepithelial cells (Zou et al. (1994) Science 263, 526-529; herein incorporated by reference in its entirety). Upon tumor progression, maspin expression is significantly reduced or lost in breast and prostate carcinoma cell lines and tissues (Zou et al. (1994) Science 263, 526-529; Seftor et al. (1998) Cancer Res 58, 5681-5685; Zhang et al. (1997) Mol Med 3, 49-59; Sheng et al. (1996) Proc Natl Acad Sci USA 93, 11669-1 1674; herein incorporated by reference in their entireties). One of the major tumor suppressing functions of maspin is its ability to inhibit tumor cell motility and invasiveness, which is partially mediated by enhancing cell adhesion. The region between amino acids residues 139-225 in maspin facilitates increased mammary luminal epithelial (MCF10A) cell adhesion (Cella et al. (2006) Faseb J20, 1510-1512; herein incorporated by reference in its entirety). Exogenous maspin treatment elicits similar anti-migratory effects seen in breast carcinoma cells overexpressing maspin cDNA (Seftor et al. (1998) Cancer Res 58, 5681- 5685; Sheng et al. (1996) Proc Natl Acad Sci USA 93, 1 1669-1 1674; herein incorporated by reference in their entireties). These findings indicate that both endogenous and exogenous maspin act similarly at an extracellular site or are transported into an intracellular site of action. Studies suggest that maspin is secreted or present/associated at the cell surface (Seftor et al. (1998) Cancer Res 58, 5681-5685; , Ngamkitidechakul et al. (2001) Invest Ophthalmol Vis Sci 42, 3135-3141 ; Cella et al. (2006) Faseb JIQ, 1510-1512; Bass et al. (2009) J Biol Chem 284, 27712-27720; Pemberton et al. (1997) J Histochem Cytochem 45, 1697-1706; Khalkhali-Ellis & Hendrix (2007) Cancer Res 67, 3535-3539; Sheng et al. (1996) Proc Natl Acad Sci USA 93, 1 1669-1 1674; Law et al. (2005) J Biol Chem 280, 22356-22364; herein incorporated by reference in their entireties). However, more recent data refutes these claims stating that maspin expression is absent from the surface of MCF 10A cells and not secreted through the classical secretion pathway (Teoh et al. (2010) J Biol Chem 285, 10862-10869; herein incorporated by reference in its entirety). Confocal microscopy has been used to demonstrate that maspin is present on MCF 10A cell surface and mediates adhesion (Cella et al. (2006) Faseb J20, 1510-1512).

Since expression of maspin in human coronary stromal cells is reduced to

undetectable levels in upon culturing (Ngamkitidechakul et al. (2001) Invest Ophthalmol Vis Sci 42, 3135-3141 ; herein incorporated by reference in its entirety), it was investigated whether maspin secretion is lost as a result of culturing conditions (Endsley et al. J Biol Chem. 2011 Jul 15;286(28):24599-607.; herein incorporated by reference in its entirety.). The detection of maspin protein, by Western immunoblot, is reduced in media obtained from high passage MCF 10A cells when compared to low passage cells. Additionally, no significant difference was detected between cellular maspin expressions in the high versus low passage MCF 1 OA cells. These data indicated that extracellular maspin detection is lost depending on culturing conditions; thereby providing an explaination for previous conflicting results.

Further experiments were conducted to determine the region involved in maspin- mediated cell adhesion (Endsley et al. J Biol Chem. 2011 Jul 15;286(28):24599-607.; herein incorporated by reference in its entirety.). Experiments indicated that peptides spanning maspin residues 181-202 and 190-21 1, but not 169- 189, were important in mediating cell adhesion. Moreover, a point mutation (E201K) within this region significantly reduced MCF 1 OA cell adhesion. Experiments indicate that the amino acids overlapping these peptides, amino acids 190-202 in maspin are integral for maspin-mediated cell adhesion. These amino acids are highly conserved between human, mouse, rat, and chicken species (84% sequence identity and 100% similarity), indicating their importance throughout evolution (Law et al. (2005) J Biol Chem 280, 22356-22364; incorporated herein by reference in its entirety). In some embodiments, the present invention provides compositions, kits, systems, and/or methods to treat or prevent cancer, metastatasis, or unusual cell motility. In some embodiments, the present invention enhances cell adhesion and/or prevents cell motility. In some embodiments, cell adhesion is enhanced in cells undergoing metastasis or at risk of entering metastasis. In some embodiments, cell adhesion is enhanced in neoplastic cells (e.g., cancerous, pre-cancerous). In some embodiments, compositions and methods are utilized in the treatment and/or prevention of: bladder cancer, lung cancer, breast cancer, melanoma, colon and rectal cancer, non-Hodgkin lymphoma, endometrial cancer, pancreatic cancer, kidney (renal cell) cancer, prostate cancer, leukemia, thyroid cancer, and/or metastasis thereof. In some embodiments, the present invention enhances cell adhesion and/or inhibits cell motility in non-neoplastic or non-cancerous cells.

In some embodiments, the present invention provides a pharmaceuticals, small molecules, peptides, proteins, polypeptides, nucleic acids, molecular complexes, etc. for the treatment or prevention of cancer (e.g., metastasis), enhancement of cell adhesion, and/or inhibition of cell motility. In some embodiments, the present invention provides

administration of a maspin-based peptide (e.g., peptide or polypeptide comprising: SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, derivatives thereof, etc.) to inhibit cell motility, tube formation, and/or enhance cell adhesion.

In some embodiments, the maspin-based peptides and/or polypeptides described herein effectively enhance cell adhesion, reduce cell motility, treat and/or prevent: cancer, angiogenesis, neoplasia, bone formation defects, etc. at unexpectedly low concentration. Experiments demonstrated that administration of a concentration as low as 1.0 μΜ are effective. In some embodiments, concentrations (e.g., administered concentration, local concentration, effective local concentration) of maspin-based peptides (e.g., comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or a variant or derivative thereof) as low as 0.01 μΜ, 0.02 μΜ, 0.05 μΜ, 0.1 μΜ, etc. are effective in enhancing cell adhesion, reducing cell motility, treating and/or preventing: cancer, angiogenesis, neoplasia, bone formation defects, etc.

In some embodiments, a peptide or polypeptide of the present invention can be prepared by methods known to those of ordinary skill in the art. For example, the claimed polypeptide can be synthesized using solid phase polypeptide synthesis techniques (e.g. Fmoc). Alternatively, the polypeptide can be synthesized using recombinant DNA technology (e.g., using bacterial or eukaryotic expression systems). Accordingly, to facilitate such methods, the present invention provides genetic vectors (e.g., plasmids) comprising a sequence encoding the inventive polypeptide, as well as host cells comprising such vectors. Furthermore, the invention provides the polypeptide produced via recombinant methods.

In some embodiments, the present invention provides administration of maspin-based (e.g., maspin-related, maspin-derived, etc.) compositions (e.g. maspin, maspin-based peptides, mimetics of maspin, nucleic acids encoding maspin-based peptides, etc.). In some embodiments, the present invention provides administration of polypeptides which inhibit cell motility (e.g. SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, combinations thereof, derivatives thereof, etc.). In some embodiments, the present invention provides

administration of nucleic acids which encode polypeptides (e.g. SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, , combinations thereof, derivatives thereof, etc.) which inhibit cell motility and/or enhance cell adhesion. In some embodiments, a polypeptide comprising or consisting of SEQ ID NO: 1 is administered. In some embodiments, a polypeptide comprising or consisting of SEQ ID NO:2 is administered. In some embodiments, a polypeptide comprising or consisting of SEQ ID NO:3 is administered. In some

embodiments, a polypeptide comprising a portion with at least 50% homology to SEQ ID NO: l, SEQ ID NO:2, and/or SEQ ID NO:3 is administered (e.g. at least 60% homology, at least 70% homology, at least 80% homology, at least 90% homology, at least 95% homology, at least 99% homology, etc.).

In some embodiments, administering a maspin-based peptide, nucleic acid, or a drug- like small molecule to a subject or cell inhibits pathways related to cell-motility, inhibits cell motility, and/or protects against undesired cell motility.

In some embodiments, peptide and polypeptides of the present invention are isolated and/or purified (or substantially isolated and/or substantially purified). Accordingly, the invention provides polypeptide in substantially isolated form. In some embodiments, polypeptides are isolated from other polypeptides as a result of solid phase protein synthesis, for example. Alternatively, polypeptides can be substantially isolated from other proteins after cell lysis from recombinant production. Standard methods of protein purification (e.g., HPLC) can be employed to substantially purify polypeptides. In some embodiments, the present invention provides a preparation of polypeptides in a number of formulations, depending on the desired use. For example, where the polypeptide is substantially isolated (or even nearly completely isolated from other proteins), it can be formulated in a suitable medium solution for storage (e.g., under refrigerated conditions or under frozen conditions). Such preparations may contain protective agents, such as buffers, preservatives,

cryprotectants (e.g., sugars such as trehalose), etc. The form of such preparations can be solutions, gels, etc., and the inventive polypeptide can, in some embodiments, be prepared in lyophilized form. Moreover, such preparations can include other desired agents, such as small molecules or even other polypeptides and proteins, if desired. Indeed, the invention provides such a preparation comprising a mixture of different embodiments of the inventive polypeptide (e.g., a plurality of polypeptide species as described herein).

In some embodiments, the present invention also provides a pharmaceutical composition comprising of one or more polypeptides (e.g., polypeptides comprising SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, mixtures thereof, derivatives thereof, mutants thereof, etc.) and a pharmaceutically acceptable carrier. Any carrier which can supply a polypeptide without destroying the vector within the carrier is a suitable carrier, and such carriers are well known in the art. The composition can be formulated for parenteral, oral, or topical administration. For example, a parenteral formulation could consist of a prompt or sustained release liquid preparation, dry powder, emulsion, suspension, or any other standard formulation. An oral formulation of the pharmaceutical composition could be, for example, a liquid solution, such as an effective amount of the composition dissolved in diluents (e.g., water, saline, juice, etc.), suspensions in an appropriate liquid, or suitable emulsions. An oral formulation could also be delivered in tablet form, and could include excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and

pharmacologically compatible excipients. A topical formulation could include compounds to enhance absorption or penetration of the active ingredient through the skin or other affected areas, such as dimethylsulfoxide and related analogs. The pharmaceutical composition could also be delivered topically using a transdermal device, such as a patch, which could include the composition in a suitable solvent system with an adhesive system, such as an acrylic emulsion, and a polyester patch. Compositions could be delivered via eye drops or other topical eye delivery method. Compositions may be delivered intraocularly, anywhere in the eye including, for example, the vitreous cavity, the anterior chamber, etc. Compositions may be delivered intravitrealy as is commonly done with intravitreal injections of Lucentis (ranabizumab), Avastin (bevazizumab), triamcinolone acetonide, antibiotics, etc.

Compositions may be administered using encapsulated cell technology (e.g. by Neurotech) in which genetically modified cells are engineered to produce and secrete compositions of the present invention (e.g. maspin-based proteins or peptides).

In some embodiments, the methods of the present invention are employed in vivo. In some embodiments, polypeptides are delivered to a human or animal subject in an amount and at a location sufficient to inhibit or attenuate cell motility or enhance cell adhesion within a population of cells (e.g., within desired tissue, within the patient, etc.). Polypeptide can be formulated into a suitable pharmaceutical composition (e.g., as described above or as otherwise known to those of ordinary skill in the art) for delivery into the subject. The delivery can be local (e.g., by injection or implantation within the desired tissue to be treated) or systemic (e.g., by intravenous or parenteral injection).

In some embodiments, the present invention provides a method for treating patients suffering from (or at risk of) neoplasia and in need of treatment (or preventative therapy). In some embodiments, a pharmaceutical composition comprising at least one polypeptide of the present invention is delivered to such a patient in an amount and at a location sufficient to treat the condition. In some embodiments, polypeptides of the present invention (or pharmaceutical composition comprising such) can be delivered to the patient systemically or locally, and it will be within the ordinary skill of the medical professional treating such patient to ascertain the most appropriate delivery route, time course, and dosage for treatment. It will be appreciated that application of the inventive method of treating a patient most preferably substantially alleviates or even eliminates such symptoms; however, as with many medical treatments, application of the inventive method is deemed successful if, during, following, or otherwise as a result of the inventive method, the symptoms of the disease or disorder in the patient subside to an ascertainable degree.

A pharmaceutical compound may be administered in the form of a composition which is formulated with a pharmaceutically acceptable carrier and optional excipients, adjuvants, etc. in accordance with good pharmaceutical practice. The maspin-based (e.g., comprising SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, mixtures thereof, derivatives thereof, mutants thereof, etc.) pharmaceutical composition may be in the form of a solid, semi-solid or liquid dosage form: such as powder, solution, elixir, syrup, suspension, cream, drops, paste and spray. As those skilled in the art would recognize, depending on the chosen route of administration (e.g. pill, injection, etc.), the composition form is determined. In general, it is preferred to use a unit dosage form of the inventive inhibitor in order to achieve an easy and accurate administration of the active pharmaceutical compound. In general, the

therapeutically effective pharmaceutical compound is present in such a dosage form at a concentration level ranging from about 0.5% to about 99% by weight of the total composition: i.e., in an amount sufficient to provide the desired unit dose. In some embodiments, the pharmaceutical composition may be administered in single or multiple doses. The particular route of administration and the dosage regimen will be determined by one of skill in keeping with the condition of the individual to be treated and said individual's response to the treatment. In some embodiments, a maspin-based pharmaceutical composition in a unit dosage form for administration to a subject, comprising a

pharmaceutical compound (e.g., comprising SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, mixtures thereof, derivatives thereof, mutants thereof, etc.) and one or more nontoxic pharmaceutically acceptable carriers, adjuvants or vehicles. The amount of the active ingredient that may be combined with such materials to produce a single dosage form will vary depending upon various factors, as indicated above. A variety of materials can be used as carriers, adjuvants and vehicles in the composition of the invention, as available in the pharmaceutical art. Injectable preparations, such as oleaginous solutions, suspensions or emulsions, may be formulated as known in the art, using suitable dispersing or wetting agents and suspending agents, as needed. The sterile injectable preparation may employ a nontoxic parenterally acceptable diluent or solvent such as sterile nonpyrogenic water or 1,3- butanediol. Among the other acceptable vehicles and solvents that may be employed are 5% dextrose injection, Ringer's injection and isotonic sodium chloride injection (as described in the USP/NF). In addition, sterile, fixed oils may be conventionally employed as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono-, di- or triglycerides. Fatty acids such as oleic acid can also be used in the preparation of injectable compositions.

In some embodiments, maspin-based compositions of the present invention are provided as part of a kit. In some embodiments, a kit of the present invention comprises one or more maspin-based compositions and/or maspin-based pharmaceutical compositions. In some embodiments, a kit comprises a maspin-based composition configured for coadministration with one or more additional compositions (e.g. pharmaceutical compositions). In some embodiments, one or more maspin-based compositions are co-administered with one or more other agents for effective enhancement of cell adhesion, inhibition of cell motility, and/or treatment or prevention of cancer or metastasis.

In some embodiments, maspin-related compositions are provided for the promotion of cell adhesion and/or inhibition of cell migration. However, the compositions and methods described herein are not limited by their particular application or field of use. In some embodiments, compositions and methods find use in the treatment and/or prevention of cancer, metastasis, or other neoplastic disorders of conditions. In some embodiments, compositions and methods find use in the treatment and/or prevention disorders associated with bone formation (e.g., osteoporosis), as is described for purified maspin in US Pat. App. 20090263363; herein incorporated by reference in its entirety. Various modification, recombination, and variation of the described features and embodiments will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although specific embodiments have been described, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes and embodiments that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

EXPERIMENTAL

Example 1

Anti-angiogenesis maspin peptides

Experiments were conducted during development of embodiments of the present invention to develop small maspin mimetic peptides for therapeutic treatment. Through maspin deletion and site-specific mutagenesis analysis, (SEE FIG. 1). Based on the unique region/sites within maspin that have been identified as being responsible for its binding to the cell surface and the mechanism of maspin-mediated signal transduction, competitive peptides were designed (e.g., SEQ ID NOS. 1-3) that act as antagonists and blocks wildtype maspin' s effect on cell adhesion. Two peptides, containing 15 and 22 amino acids respectively, are located in two different regions in primary sequence of maspin but are spatially very close to each other in the folded protein (SEE FIG. 1). Synthetic peptides were generated based on these petoide sequences, tested that contain the key amino acids in both the parental peptides. Amino acid residues were mutated, deleted, added (e.g., capped), etc. with the goal of making the peptides stable, suitable for administration to cells and/or subjects, and effective at low concentration (e.g., 0.10-1.0 uM). The length of the peptides was minimized (e.g., smaller than 12 amino acids) in order to confer stability to the peptides. Further, the ends were capped with protective moieties. Some (e.g., SEQ ΓΝ NOS: 1 and 2) resulting peptides (e.g., SEQ IN NOS: 1-3) were synthesized in high purity and tested in biological assays to determine their effectiveness in preventing angiogenesis (e.g., at low concentration).

Peptides were tested using a Human Umbilical Vein Endothelial Cells (HUVEC) Tube Formation Assay. This is one of the most widely used in vitro assays to model the reorganization stage of angiogenesis is the tube formation assay. The assay is employed to determine the ability of compounds to promote or inhibit tube formation. Inhibition of tube formation indicates that compounds are useful in the treatment of various diseases, such as cancer, where tumors stimulate new blood vessel formation to receive oxygen and nutrients in order to grow beyond a relatively small size.

HUVEC endothelial cells were used to test the effect of synthetic maspin peptides on HUVEC tube formation assay in cell culture systems. Random peptide was used as a negative control and maspin G-helix peptide as a positive control. G-Helix was previously found to be effective in inhibiting endothelial cell migration, thereby mimicking maspin effect against angiogenesis. G-Helix had a modest effect on HUVEC tube formation at the concentration of 10 uM. However, at 1.0 uM, G-helix was not effective. However, two of the newly identified peptides: STANAKVKLSIP (SEQ ID NO: 1) and TANAEVKLSIPK (SEQ ID NO:2) were highly effective in inhibiting tube formation at a low concentration of 1.0 uM (SEE FIG. 2).

Based on the experiments conducted during development of embodiments of the present invention, it is further contemplated the peptide ZSTENAKVKLSIP (Seq ID NO.3) is effective in enhancing cell adhesion and/or inhibits cell motility; inhibiting tube formation; associating with uPAR, βΐ integrin, or both; localizing at the cell surface; co-localizing with the uPA/uPAR complex; and/or treating cancer, metastasis, and/or bone formation disorders.

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All publications and patents mentioned in the present application and/or listed below are herein incorporated by reference in their entireties.

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Claims

CLAIMS What is claimed is:

1. A composition comprising a peptide or polypeptide that comprises a portion with at least 70% sequence identity with all or a portion of one or more of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3.

2. The composition of claim I, wherein said peptide or polypeptide is at least 6 amino acids in length.

3. The composition of claim 2, wherein said peptide or polypeptide is 8-20 amino acids in length.

4. The composition of claim 2, wherein said peptide or polypeptide comprises at least 90% sequence identity with all or a portion of one or more of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3.

5. The composition of claim 4, wherein said peptide or polypeptide comprises SEQ ID NO: l.

6. The composition of claim 4, wherein said peptide or polypeptide comprises SEQ ID NO:2.

7. The composition of claim 4, wherein said peptide or polypeptide comprises SEQ ID NO:3.

8. A pharmaceutical composition comprising: (a) one or more of: ((i) a peptide or polypeptide with at least 70% sequence identity with all or a portion of SEQ ID NO: 1, (ii) a peptide or polypeptide with at least 70% sequence identity with all or a portion of SEQ ID NO:2, (iii) a peptide or polypeptide with at least 70% sequence identity with all or a portion of SEQ ID NO:3; and (b) a physiologically suitable buffer.

9. A method of treating or preventing a disease or disorder comprising administering a composition of claim 1 to a cell, tissue, or subject.

10. The method of claim 9, wherein administering enhances cell adhesion, results in reduced cell motility, or both

1 1. The method of claim 9, wherein said cells, tissue, or subject comprises cancer cells, cells at risk of becoming cancerous, or cells at risk of metastasis.

12. The method of claim 11, wherein administering treats or prevents angiogenesis, cancer, and/or metastasis.

13. The method of claim 9, wherein said peptide or polypeptide localizes at the cell surface upon administration to said cells, tissue, or a subject.

14. The method of claim 9, wherein said peptide or polypeptide co-localizes with the uPA/uPAR complex.

15. A method of treating or preventing cancer or metastasis comprising administering to a cell, tissue, or subject a composition of claim 1, wherein administering said peptide or polypeptide enhances cell adhesion and/or inhibits cell motility.

16. A method of treating or preventing a bone formation disorder comprising administering to a cell, tissue, or subject a composition of claim 1.

17. The composition of one of claims 1-8 for use as a medicament.

18. The composition of one or claims 1-8 in the treatment of cancer.

19. The composition of one or claims 1-8 in the treatment of a bone formation disorder.