WO2007095583A2

WO2007095583A2 - Methods for treating cancer and sensitizing cancer cells using the serine protease inhibitor, maspin

Info

Publication number: WO2007095583A2
Application number: PCT/US2007/062150
Authority: WO
Inventors: Mary J. C. Hendrix; Zhila Khalkhali-Ellis
Original assignee: Children's Memorial Research Center
Priority date: 2006-02-14
Filing date: 2007-02-14
Publication date: 2007-08-23
Also published as: WO2007095583A3

Abstract

Disclosed are methods using the serine protease inhibitor, maspin, for (a) the treatment or prevention of cancer; (b) modulation of cellular cathepsin D levels; (c) sensitizing cancer cells to anti-cancer/anti-neoplastic agents. Also disclosed are methods for inhibiting degradation of extracellular matrix and treatment or prevention of cancer and metastasis. Further disclosed is an assay for determining the likelihood of developing metastatic cancer. Compositions useful in the methods of the invention are also disclosed.

Description

METHODS FOR TREATING CANCER AND SENSITIZING CANCER CELLS USING THE SERINE PROTEASE INHIBITOR, MASPIN

BACKGROUND OF THE INVENTION This application claims priority of U.S. application serial no. 60/773022, filed

February 14, 2006, the disclosure of which is incorporated herein by reference in its entirety. Field of the Invention

The present invention relates methods for treating cancer, for sensitizing cancer cells to cancer therapeutics, and for modulating cellular levels of cathepsin-D as well as pharmaceutical compositions useful in the methods. More specifically, the invention relates to methods for treating cancer, for sensitizing cancer cells to cancer therapeutics, and for modulating cellular and extra-cellular levels of cathepsin-D, comprising the administration of the serine protease inhibitor, maspin, and to pharmaceutical compositions comprising maspin and interferons, particularly interferon-gamma (IFN-γ). The invention also relates to a diagnostic and/or prognostic method for determining the likelihood that a cancer will metastasize comprising measuring serum levels or activity of maspin and/or cathepsin-D.

Description of the Related Art Maspin (mammary serine protease inhibitor) is a member of the serpin (serine p_roteases inhibitor) superfamily and has been identified as a tumor suppressor protein in human breast epithelial cells (Zou et al, Science, 263: 526-529 (1994)). Maspin is primarily a cytoplasmic protein, however it is sometimes secreted and can also localize to cellular compartments such as the nucleus or cell membrane (Khalkhali-Ellis and Hendrix, Amer.J. Pathol, 162: 411-1418 (2003); Pemberton, et al, J Histochem Cytochem., 45: 1697-706 (1997); Sheng, et al, Proc. Nat. Acad. Sci. USA, 93: 11669-11674 (1996); Maass, et al, Clin. Cancer Res., 7: 812-817 (2001)). Research has identified several roles for intercellular and membrane-associated maspin, such as inhibiting cell motility and invasion (Khalkhali-Ellis & Hendrix (2003); Sheng, et al, (1996); Seftor, et al, Cancer Res., 58: 5681-5685 (1998); Odero, et al, Cancer Biol and Therapy, 2: 404-405 (2003)), promoting apoptosis (Khalkhali- Ellis & Hendrix (2003); Liu, et al, Cancer Res., 64: 1703-11 (2004); Latha, et al, MoI Cell Biol, 25: 1737-1748 (2005)), repressing angiogenesis (Zhang, et al, Nature Med., 6: 196-199 (2000)) and as a signal transducer (Odero, et al (2003); Odero, et al, Biochem. Biophys. Res. Com., 295: 800-805 (2002)). Maspin is known to be secreted by mammary epithelial cells (Pemberton PA. et al, Histochem Cytochem. 1997; 45:1697-706; Khalkhali-Ellis Z et al, Amer.J. Pathol. (2003)162:1411-1418), however its function in the extracellular milieu has remained mostly unexplored. These observations demonstrate the diverse biological activity that maspin possesses. Nevertheless, the molecular mechanism(s) of maspin's action have remained largely unresolved.

The aspartyl lysosomal proteinase, cathepsin D is a well characterized lysosomal enzyme that is synthesized as pre-proenzyme in the endoplasmic reticulum (ER) of normal mammalian cells (Von Figura & Hasilik, Ann. Rev. Biochem., 55: 167-193 (1986)). The proenzyme localizes to the golgi network upon processing of signal peptide and N-linked glycosylation steps. The N-linked carbohydrate chain(s) are modified by two enzymes. The first enzyme is UDP-GIcNAc lysosomal enzyme N-acetyleglucosaminyl phosphotransferase (phosphotransferase), which transfers N-acetyleglucosamine-1 -phosphate to cathepsin D, forming diester intermediates. The second enzyme, N-acetyleglucosamine-1-phosphodiester- α-N-acetyleglucosaminidase, removes the N-acetyleglucosamine residues, which exposes Man-6-P monoesters (Man-6-P) for recognition by Man-6-P receptors (Man-6-PR) in the trans golgi network (TGN) and further transport to lysosomal compartment (Hasilik, et al, Biochem. Biophys. Res. Commun., 98: 761-767 (1981); Kornfeld & Mellman, Annu. Rev. Cell Biol, 5: 483-525 (1989)). The complex (Man-6-PR-cathepsin D) then dissociates to allow further processing of cathepsin D to the 44 kDa active intermediary form ("I-cathepsin D") and its final cleavage to two chain mature active enzyme ("M-cathepsin D" or typically "cathepsin D") in the lysosomes (Gieselmann, et al, J. Cell Biol, 97: 1-5 (1983); Cantor, et al, J.Biol. Chem., 267: 23349-23356 (1992)).

Transport of cathepsin D to lysosomes is not solely dependent on Man-6-PR(s), as Man-6-PR-independent trafficking has also been observed in certain cell types. Studies on I- Cell disease lymphoblasts have indicated that despite disease associated deficiency of phosphotransferase activity and thus lack of Man-6-P residues on cathepsin D, 45% of the synthesized cathepsin D is targeted to the lysosomal compartment (Glickman & Kornfeld, J. Cell Biol, 123: 99-108 (1993)). These studies have identified a polypeptide determinant in the carboxy lobe of cathepsin D (residues 188-256) that is sufficient to confer Man-6-P- independent targeting of cathepsin D to the lysosomes. In addition, several studies indicate that the precursor form of cathepsin D could be membrane bound independent of Man-6-P residues, suggesting an unrecognized lysosomal binding protein which could play a role in targeting (Mclntyre & Erickson, J Biol Chem. 266:15438-45 (1991); Rijinboutt, et al, J. Biol. Chem., 266: 23586-23592 (1991)). Under normal conditions less than 20% of cathepsin D is secreted (Dittmer, et al., J. Biol. Chem., 272: 852-858 (1997)). However, in certain disease states, such as cancer, cathepsin D is aberrantly secreted and can be excessively produced by invasive cancer cells (Sloane & Honn, Cancer Metast. Rev., 3: 249-263 (1984); Rochefort, H., et al. J. Cell Biochem., 35: 17-29 (1987)). For example, a number of reports have indicated that, in addition to an aberrant production, increased secretion of cathepsin D is a molecular signature of breast cancer cells (see, e.g., Capony, et al., Cancer Res., 49: 3904-3909 (1989)). The mechanism underlying aberrant production and secretion of cathepsin D in cancer is not resolved, but altered binding to Man-6-PR (Canopy, F., et al., Exp. Cell Res., 215: 154-163 (1994)), mutation in Man-6-PR (Byrd, et al., J. Biol. Chem., 274: 24408-24416 (1999)) or an imbalance in the pH gradient between cytoplasm and Golgi/TGN (Kokkonen, N., et al., J. Biol. Chem., 279: 39982-39988 (2004)) have been proposed as contributing mechanistic factors.

Every year in the United States, cancer accounts for a large amount of the total mortality and is among the leading causes of death. Despite the great number of anti- cancer/anti-neoplastic agents that are used in the clinic for cancer treatment, a need still exists for more diverse and effective therapies. Chemotherapy, the systemic administration of antineoplastic agents that travel throughout the body via the blood circulatory system, along with and often in conjunction with surgery and/or radiation treatment, has for years been widely utilized in the treatment of a wide variety of cancers. Unfortunately, the available chemotherapeutic drugs can fail patients because they kill many healthy cells and thus bring on serious side effects that limit the doses physicians can administer safely.

In particular, cancerous tumors are difficult to treat because they contain both proliferating and non-proliferating cancer cells. As a cancerous tumor grows, the vascular development often cannot keep pace with the rapid proliferation of malignant cell population. Consequently, solid cancerous tumor masses typically exhibit abnormal blood vessel networks which, unlike vessels in normal tissues, fail to provide adequate nutritional support to the cancerous tumor cells for optimal growth. In most cancerous, solid tumors, non- proliferating tumor cells constitute the majority of the total tumor cell population. Moreover, as a tumor grows in size the proportion of non-proliferating tumor cells also increases relative to rapidly proliferating cells. As most current anti-cancer agents target proliferating cells, the non-proliferating tumor cell population has been implicated as a major contributing factor in the failure of radiation or chemotherapy used alone or together to treat and/or cure neoplastic disease. As mentioned above, as a tumor grows in size, it typically becomes more refractory to most chemotherapies. Accordingly, many tumor eradication procedures include a debulking step to decrease the mass of the tumor prior to the administration of anti-neoplastic agents. However, debulking does not always result in tumor eradication, even when combined with powerful chemotherapeutic agents. Accordingly, there is a need in the art for new treatments that sensitize cancer cells that exhibit resistance to chemotherapeutic regimens and/or target both proliferating and non-proliferating cancer cells for the treatment of malignancy.

One example of an anti-cancer agent is a class of molecules known as the interferons. When administered to mammary and prostate epithelial cells, interferon-gamma (IFN-γ) can induce cell cycle arrest, apoptosis, and/or differentiation (see, e.g., Grunberg, et al., Tumour Biol, 21 : 211-23 (2000); Untergasser, et al, Prostate, (2005) Mar 30). IFN-γ has been proposed to reduce the tissue tensile modulus of solid tumors, thereby improving anti-cancer therapeutic approaches that target rapidly proliferating cells (Seed, et al, US Patent 6,719,977). Some of the anti-neoplastic action exhibited by IFN-γ may be attributable to its proposed function as an anti-fibrotic agent. Nevertheless, certain types of cancer can become refractory to therapeutic regimes, including those employing IFN-γ

Thus, there is a continuing need to develop agents and methods useful in preventing and treating cancer. Methods that can (a) target and/or sensitize cancer cells to chemotherapeutics; (b) provide increased efficacy of known chemotherapeutics against cancer; and (c) modulate cathepsin-D levels or activity in cancer cells would be particularly useful in the continuing fight against cancer.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for treating or preventing cancer comprising administering to a subject an amount of a maspin polypeptide and an amount of interferon-gamma (IFN-γ) effective to treat or prevent cancer.

In an aspect, the invention provides a method of sensitizing a cancer cell to a therapeutic agent comprising contacting the cancer cell with an amount of a maspin polypeptide effective to sensitize the cancer cell to the therapeutic agent.

In an aspect, the invention further provides a method of modulating levels or activity of cathepsin D in a cell comprising contacting the cell with an amount of a maspin polypeptide effective to modulate cathepsin D levels or activity in the cell.

In an aspect, the invention provides a method of modulating the amount of cathepsin D secreted from a cell comprising contacting the cell with an amount of a maspin polypeptide effective to modulate the amount of cathepsin D secreted from the cell. In one aspect, the invention relates to compositions comprising maspin, IFN-γ, and a pharmaceutically acceptable carrier or formulation agent.

In one aspect, the invention provides a method of inhibiting undesired angiogenesis in a mammal comprising administering a therapeutically effective amount of a composition as described herein. In this aspect, the invention also relates to a method of modulating angiogenesis in a mammal comprising administering a therapeutically effective amount of a composition as described herein. This aspect of the invention further relates to a method of inhibiting tumor growth characterized by undesired and/or unregulated angiogenesis in a mammal comprising administering a therapeutically effective amount of a composition as described herein. Additionally, this aspect relates to a method of treating cancer in a mammal comprising administering a therapeutically effective amount of a composition as described herein, either alone or in combination with one or more additional anti-cancer agent(s).

In one aspect, the invention provides an assay for determining the likelihood that a patient will develop metastatic cancer comprising: (a) identifying a patient at risk for developing metastatic cancer; (b) obtaining a serum sample from the patient; (c) measuring the level of cathepsin-D activity in the serum sample; and (d) determining from the measurement in step (c) the likelihood that the patient will develop metastatic cancer; wherein the likelihood that the patient will develop metastatic cancer is elevated when the level of cathepsin-D activity in the sample is more than about twice the level in a control sample In this method, the level of cathepsin-D activity that is measured is preferably pro-cathepsin-D activity. In a preferred aspect of this method, there is an elevated likelihood that the patient will develop metastatic cancer when the level of pro-cathepsin-D in the sample is more than about 4 times the level in a control sample.

In one aspect, the invention provides a method of treating cancer and preventing metastasis in a mammal by administering to the mammal a therapeutically effective amount of maspin alone or in combination with a therapeutically effective amount of one or more anti- cancer agent(s) .

In one aspect, the invention provides a method for preventing degradation of extracellular matrix surrounding a cancer cell by contacting the cancer cell with an effective amount of maspin or maspin in combination with a therapeutically effective amount of one or more anti-cancer agent(s). In another aspect, the invention provides a method of treating cancer and/or preventing metastasis by inhibiting protease activity of cathepsin-D and/or pro-cathepsin D alone or in combination with therapeutically effective amounts of one or more anti-cancer agent(s).

In still another aspect, the invention provides a method of preventing metastasis by administration of an effective amount of maspin or by inhibition of sufficient cathepsin D and/or pro-cathepsin D activity to prevent matrix degradation.

In one aspect, the invention provides a method of preventing degradation of the extracellular matrix by inhibiting protease levels or activity of cathepsin-D and/or pro- cathepsin D. The activity of the cathepsin D or pro-cathepsin D can be inhibited by administration of a therapeutically effective amount of either a maspin polypeptide or an aspartyl protease inhibitor.

Other embodiments of this invention will be readily apparent from the disclosure provided herewith. BRIEF DESCRIPTION OF THE FIGURES

Figures IA-B depicts the amino acid sequence of maspin (SEQ ID NO:2) and the coding portion of the maspin gene (SEQ ID NO:1).

Figure 2 depicts Subcellular distribution of cathepsin D and maspin in mammary epithelial cells determined by Western blot analysis. Each fraction was subjected to immunoprecipitation using antibodies to maspin or cathepsin D. Both normal mammary epithelial cell lines tested (HMEC 1330 and HMEpC) gave comparable results so only one is represented herein.

Figure 3 depicts maspin and cathepsin D profile of normal mammary epithelial and breast cancer cell lines. Subcellular fractions (A) light membrane fraction (including ER, plasma membrane and microsomes) (B) heavy membrane fraction (containing lysosomes, mitochondria and peroxisomes) and (C) cytosol of different cell lines prepared as described herein.

Figure 4 depicts the response of normal mammary epithelial cells to treatment with blocking antibody to maspin. Arrow indicates the increase in ~47 kDa ICD upon antibody treatment.

Figure 5 depicts changes in cell morphology (A&B); mRNA expression (C&D); and protein secretion (E) of cathepsin D and maspin of normal mammary epithelial cells following exposure to IFN-γ.

Figure 6 depicts the response of normal mammary epithelial cells and breast cancer cell lines to IFN-γ: (A) HMEpC, (B) MCF-7, (C) maspin-transfected MCF-7, (D) MDA-MB-231, and

(E) maspin-transfected MDA-MB-231.

Figures 7A-B depicts the amino acid sequence of cathepsin D (SEQ ID NO: 3) and DNA containing the coding portion of the cathepsin D gene (SEQ ID NO: 5). See, GenBank

Accession No: NM_001909 and Faust, et al, Proc. Natl. Acad. ScL USA, 85:4910-4914 (Aug.1985). The single arrowhead indicates the end of the signal sequence and the diamond indicates the end of the prosequence.

Figure 8 depicts endoglycosidase-H digestion of cathepsin D in the CM (A) and cytosolic compartment (B) of the studied cell lines.

Figure 9A depicts detection of Maspin in the conditioned media and in the matrix deposited by normal mammary epithelial cells (HMEC 1330), and its incorporation into a 3D Col matrix

(HMEC-conditioned matrix) by Western blot; 9B depicts immunohistochemical analysis and confirmation of the presence of maspin in the matrix; 9C depicts a western blot analysis detecting levels of procathepsin-D and maspin secreted by breast cancer cell liness MCF-7 and MDA-MB-231 after four days in Col I matrix culture. The conditioned media (CM) was concentrated (1Ox for MCF-7; 2x for MDA-MB-231) prior to analysis. 9D shows that the cathepsin-D detected in the CM is the pro-form (-52-54 kDa) while the form in the matrix is the intermediary (ICD) and mature active forms (MCD).

Figure 1OA depicts incorporation of recombinant maspin (r-maspin) into Col solution prior to the gelation process at concentrations ranging from 100-500ng/well of 96 well culture dish by western blot analysis or by (10B) immunohistochemical analysis. 1OC Culturing MDA-MB- 231 breast cancer cells on maspin-incorporated Col matrix is associated with a reduction in CD mRNA. 1OD shows quantitative estimation of degradation fluorescein conjugated Col I (DQ™ collagen) Figure 11 depicts detection of secreted pro-CD and recombinant maspin mutants by western blot analysis in matrix with cells (A) and after removal of cells (B).

DETAILED DESCRIPTION OF INVENTION

The section headings are used herein for organizational purposes only, and are not to be construed as in any way limiting the subject matter described. Standard techniques can be used for recombinant DNA molecule, protein, and antibody production, as well as for tissue culture and cell transformation. Enzymatic reactions and purification techniques are typically performed according to the manufacturer's specifications or as commonly accomplished in the art using conventional procedures such as those set forth in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), or as described herein. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

Definitions

As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: The term "maspin" or "maspin polypeptide" refers to the polypeptide set forth in

Figure 1 or biologically active fragments thereof as well as related polypeptides which include biologically active allelic variants, splice variants, derivatives, sequence variants (substitution, deletion, and/or insertion derivatives), fusion peptides and polypeptides, and interspecies homologs. Maspin can include additional terminal residues, for example, leader sequences, targeting sequences, amino terminal methionine, amino terminal methionine and lysine residues, and/or tag or fusion proteins sequences, which can depend on the manner in which it is prepared.

The term "biologically active" when used in relation to maspin, or a fragment thereof, refers to maspin or maspin polypeptide having at least one activity characteristic of maspin. Biologically active maspin or maspin polypeptide can have agonist, antagonist, or neutralizing or blocking activity with respect to at least one biological activity of maspin.

Unless noted otherwise, the term "cathepsin-D" or "CD" as used herein refers to any form of the aspartyl lysosomal proteinase cathepsin-D, such as pro-cathepsin-D, the active intermediate form of cathepsin-D, or the mature form of cathepsin-D. The term is also meant to include biologically active fragments thereof, active allelic variants, splice variants, sequence variants (insertion, deletion, and/or substitution derivatives) and orthologs. Specific reference is made herein to a particular form of cathepsin-D, when necessary.

The term "variants," as used herein, include those polypeptides wherein amino acid residues are inserted into, deleted from and/or substituted into the naturally occurring (or at least a known) amino acid sequence for the binding agent. Variants of the invention include fusion proteins as described below.

"Derivatives" include those binding agents that have been chemically modified in some manner distinct from insertion, deletion, or substitution variants. The term "naturally occurring" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to those which are found in nature and not modified by a human being.

The term "isolated" when used in relation to maspin refers to a compound that is free from at least one contaminating polypeptide or compound that is found in its natural environment, and preferably substantially free from any other contaminating mammalian polypeptides that would interfere with its therapeutic or diagnostic use.

The terms "effective amount" and "therapeutically effective amount" when used in relation to maspin, or fragments thereof, refers to an amount that is useful or necessary to support an observable change in the level of one or more biological activities of maspin. For example, the change may be either an increase or decrease in the level of maspin expression and/or activity.

By "sensitizing" or "sensitize" as used herein with respect to a cancer cell or a cell undergoing unregulated angiogenesis is meant that the cell is made more susceptible and/or responsive to one or more therapeutic treatments. For example, certain cancers that demonstrate a resistance (or have become 'refractory') to one or more anti-cancer therapies are "sensitized" to the same one or more anti-cancer therapies when the cancer has a decreased resistance to the therapy.

Fusion Partners of Maspin In one embodiment of the invention, maspin or biologically active fragments, variants, and derivatives thereof, can be fused at either the N-terminus or the C-terminus to one or more regions of another protein that confers increased stability and half-life in vivo. For example, when constructed together with a therapeutic protein such as maspin, domains of an Fc region of human IgG can provide longer half-life or incorporate such functions as Fc receptor binding, Protein A binding, complement fixation and perhaps even placental transfer. (Capon et ah, Nature, 337: 525-531 (1989)). Peptides and proteins fused to an Fc region have been found to exhibit a substantially greater half-life in vivo than the unfused counterpart. Also, a fusion to an Fc region allows for dimerization and/or multimerization of the fusion polypeptide. The Fc region may be a naturally occurring Fc region, or may be altered to improve certain qualities, such as therapeutic qualities, circulation time, decrease aggregation problems, and the like. Other examples known in the art include those wherein the Fc region, which may be human or another species, or may be synthetic, is fused to the N-terminus of CD30L to treat Hodgkin's Disease, anaplastic lymphoma and T-cell leukemia (U.S. Patent No. 5,480,981), the Fc region is fused to the TNF receptor to treat septic shock (Fisher et ah, NEngl JMed, 334: 1697-1702 (1996)), and the Fc region is fused to the Cd4 receptor to treat AIDS (Capon et al, Nature, 337: 525-31 (1989)).

There are various commercially available fusion protein expression systems that may be used in the present invention. Particularly useful systems include but are not limited to the glutathione-S-transferase (GST) system (Pharmacia), the maltose binding protein system (NEB, Beverley, MA), the FLAG system (IBI, New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA). These systems are capable of producing recombinant polypeptides bearing only a small number of additional amino acids, which are unlikely to affect the antigenic ability of the recombinant polypeptide. For example, both the FLAG system and the 6xHis system add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of the polypeptide to its native conformation. Another N-terminal fusion that is contemplated to be useful is the fusion of a Met-Lys dipeptide at the N-terminal region of the protein or peptides. Such a fusion may produce beneficial increases in protein expression or activity. Other fusion constructs including heterologous polypeptides with desired properties, e.g., an Ig constant region to prolong serum half- life or an antibody or fragment thereof for targeting also are contemplated. Other fusion systems produce polypeptide hybrids where it is desirable to excise the fusion partner from the desired polypeptide. In one embodiment, the fusion partner is linked to the recombinant maspin polypeptide by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are well known in the art, for example, sequences recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Beverley, MA). Variants of Maspin

Variants of maspin that fall within the scope of the invention include insertion, deletion, and/or substitution variants. In one aspect of the invention, insertion variants are provided wherein one or more amino acid residues supplement a maspin amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the maspin amino acid sequence. Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels. Insertion variants include maspin sequence(s) wherein one or more amino acid residues are added to a maspin amino acid sequence, or fragment thereof.

Variant sequences useful in the methods of the invention also include mature maspin sequences. Such maspin sequences have any leader or signal sequences removed; however the resulting protein has additional amino terminal residues as compared to wild-type maspin. The additional amino terminal residues can be derived from another protein, or can include one or more residues that are not identifiable as being derived from a specific protein. Maspin sequences with an additional methionine residue at position -1 (Met^" ^maspin) are contemplated, as are maspin sequences with additional methionine and lysine residues at positions -2 and -1 (Met^-Lys^-maspin). Variants of maspin having additional Met, Met-Lys, Lys residues (or one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.

The invention also embraces maspin variants having additional amino acid residues that arise from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide as part of glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at amino acid position -1 after cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated, including those wherein poly-histidine tags are incorporated into the amino acid sequence, generally at the carboxy and/or amino terminus of the sequence.

Insertional variants also include fusion proteins as described above, wherein the amino and/or carboxy termini of the maspin polypeptide is fused to another polypeptide, a fragment thereof, or amino acid sequences which are not generally recognized to be part of any specific protein sequence.

In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a maspin polypeptide are removed. Deletions can be effected at one or both termini of the maspin polypeptide, or from removal of one or more residues within the maspin amino acid sequence. Deletion variants necessarily include all fragments of a maspin polypeptide.

The invention also embraces polypeptide fragments of a maspin sequence wherein the fragments maintain the ability to (a) sensitize cancer cells to chemotherapeutic therapies; (b) interact with cathepsin D; (c) modulate cellular levels or activity of cathepsin D; and/or (d) have anti-neoplastic activity. Fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 or more consecutive amino acids of a maspin sequence are encompassed by the invention. Fragments of the maspin sequences useful with the invention (i.e., having the desired biological properties) can be prepared by any of the methods well known and routinely practiced in the art. Preferred maspin fragments comprise the contiguous amino acid sequence that interacts with, and binds to, cathepsin D. Another preferred embodiment comprises fusion proteins that comprise maspin fragments that bind cathepsin D.

In still another aspect, the methods of the invention provide for substitution variants of the maspin sequences described herein. Substitution variants are generally considered to be "similar" to the original polypeptide or to have a certain "percent identity" to the original polypeptide, and include those polypeptides wherein one or more amino acid residues of a polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature, however, the invention embraces substitutions that are also non-conservative.

Identity and similarity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo et al, SIAM J. Applied Math., 48:1073 (1988). Preferred methods to determine the relatedness or percent identity of two polypeptides are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al, Nucl. Acid. Res., 12:387 (1984); Genetics Computer Group, University of Wisconsin, Madison, WI, BLASTP, BLASTN, and FASTA (Altschul et al, J. MoI. Biol, 215:403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al NCB/NLM/NIH Bethesda, MD 20894; Altschul et al, supra (1990)). The well-known Smith Waterman algorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences may result in the matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, in certain embodiments, the selected alignment method (GAP program) will result in an alignment that spans at least ten percent of the full length of the target polypeptide being compared, i.e., at least 40 contiguous amino acids where sequences of at least 400 amino acids are being compared, 30 contiguous amino acids where sequences of at least 300 to about 400 amino acids are being compared, at least 20 contiguous amino acids where sequences of 200 to about 300 amino acids are being compared, and at least 10 contiguous amino acids where sequences of about 100 to 200 amino acids are being compared.

For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, WI), two polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the "matched span", as determined by the algorithm). In certain embodiments, a gap opening penalty (which is typically calculated as 3X the average diagonal; the "average diagonal" is the average of the diagonal of the comparison matrix being used; the "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see Dayhoff et al, Atlas of Protein Sequence and Structure, 5(3)(1978) for the PAM 250 comparison matrix; Henikoff et al, Proc. Natl. Acad. Sci USA, 89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also used by the algorithm.

In certain embodiments, the parameters for a polypeptide sequence comparison include the following: Algorithm: Needleman et ah, J. MoI. Biol, 48:443-453 (1970);

Comparison matrix: BLOSUM 62 from Henikoff et ah, supra (1992); Gap Penalty: 12 Gap Length Penalty: 4 Threshold of Similarity: 0

The GAP program may be useful with the above parameters. In certain embodiments, the aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.

In certain embodiments, the parameters for polynucleotide molecule sequence comparisons include the following:

Algorithm: Needleman et ah, supra (1970); Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3

The GAP program may also be useful with the above parameters. The aforementioned parameters are the default parameters for polynucleotide molecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices, thresholds of similarity, etc. may be used, including those set forth in the Program Manual, Wisconsin Package, Version 9, September, 1997. The particular choices to be made will be apparent to those of skill in the art and will depend on the specific comparison to be made, such as DNA-to-DNA, protein-to-protein, protein-to-DNA; and additionally, whether the comparison is between given pairs of sequences (in which case GAP or BestFit are generally preferred) or between one sequence and a large database of sequences (in which case FASTA or BLASTA are preferred). As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. The amino acids may have either L or D stereochemistry (except for GIy, which is neither L nor D) and the polypeptides and compositions of the present invention may comprise a combination of stereochemistries. However, the L stereochemistry is preferred. The invention also provides reverse molecules wherein the amino terminal to carboxy terminal sequence of the amino acids is reversed. For example, the reverse of a molecule having the normal sequence X1-X2-X3 would be X₃-X₂-X₁. The invention also provides retro- reverse molecules wherein, as above, the amino terminal to carboxy terminal sequence of amino acids is reversed and residues that are normally "L" enantiomers are altered to the "D" stereoisomer form. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids are also suitable components for polypeptides of the present invention. Examples of unconventional amino acids include, without limitation: aminoadipic acid, beta-alanine, beta-aminopropionic acid, aminobutyric acid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminoisobutyric acid, aminopimelic acid, diaminobutyric acid, desmosine, diaminopimelic acid, diaminopropionic acid, N-ethylglycine, N-ethylaspargine, hyroxylysine, allo-hydroxylysine, hydroxyproline, isodesmosine, allo- isoleucine, N-methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine, 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N- acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, σ-N-methylarginine, and other similar amino acids and amino acids (e.g., A- hydroxyproline).

Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".

Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.

Naturally occurring residues may be divided into classes based on common side chain properties: 1) hydrophobic: Met, Ala, VaI, Leu, He;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, GIn;

3) acidic: Asp, GIu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: GIy, Pro; and 6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule. In making such changes, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et ah, J. MoI. Biol., 157:105- 131 (1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included. In certain embodiments, those which are within ±1 are included, and in certain embodiments, those within ±0.5 are included. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those which are within ±1 are included, and in certain embodiments, those within ±0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as "epitopic core regions."

Exemplary amino acid substitutions are set forth in Table 1.

Table 1 Amino Acid Substitutions

A skilled artisan will be able to determine suitable variants of the polypeptide as set forth herein using well-known techniques. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure. Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of a polypeptide with respect to its three dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change may be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

A number of scientific publications have been devoted to the prediction of secondary structure. See, e.g., Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al, Biochemistry, 13(2):222-245 (1974); Chou et al, Biochemistry, 113(2):211-222 (1974); Chou et al, Adv. Enzymol Relat. Areas MoI Biol, 47:45-148 (1978); Chou et al, Ann. Rev. Biochem., 47:251-276 and Chou et al, Biophys. J., 26:367-384 (1979). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See Holm et al, Nucl Acid. Res., 27(l):244-247 (1999). It has been suggested (Brenner et al, Curr. Op. Struct. Biol, 7(3):369-376 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will become dramatically more accurate.

Additional methods of predicting secondary structure include "threading" (Jones, D.,

Curr. Opin. Struct. Biol, 7(3):377-87 (1997); Sippl et al, Structure, 4(1):15-19 (1996)),

"profile analysis" (Bowie et al, Science, 253:164-170 (1991); Gribskov et al, Meth. Enzym., 183:146-159 (1990); Gribskov et al, Proc. Nat. Acad. ScL, 84(13):4355-4358 (1987)), and

"evolutionary linkage" (See Holm, supra (1999), and Brenner, supra (1997)).

According to certain embodiments, amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (5) confer or modify other functional properties on such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). In certain embodiments, a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. , Nature, 354:105 (1991)).

The maspin polypeptide or peptide substitution variants may have up to about ten to twelve percent of the original amino acid sequence replaced.

Derivatives of Maspin

The methods of the invention also encompass derivatives of maspin polypeptides. Derivatives include maspin polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. Preferably, the modifications are covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic and inorganic moieties. Derivatives of the invention may be prepared to increase circulating half-life of a maspin polypeptide, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs. The invention further embraces derivative maspin sequences covalently modified to include one or more water soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol as described U.S. Patent Nos: 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337. Still other useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers. Methods of Making and Purifying Maspin

The maspin polypeptides used in the methods and compositions of the invention can be prepared by chemical synthesis in solution or on a solid support in accordance with conventional techniques. The current limit for solid phase synthesis is about 85-100 amino acids in length. However, chemical synthesis techniques can often be used to chemically ligate a series of smaller peptides to generate full length polypeptides. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co., (1984); Tarn et al., JAm Chem Soc, 105:6442, (1983); Merrifield, Science, 232:341-347, (1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1-284; Barany et al., Int. J. Peptide Protein Res., 30, 705-739 (1987); and U.S. Pat. No. 5,424,398), each incorporated herein by reference.

Solid phase peptide synthesis methods use a copoly(styrene-divinylbenzene) containing 0.1-1.0 mM amines/g polymer. These methods for peptide synthesis use butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxy-carbonyl(FMOC) protection of alpha- amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C-terminus of the peptide (See, Coligan et al., Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). On completion of chemical synthesis, the synthetic peptide can be deprotected to remove the t-BOC or FMOC amino acid blocking groups and cleaved from the polymer by treatment with acid at reduced temperature (e.g., liquid HF-10% anisole for about 0.25 to about 1 hour at 0⁰C). After evaporation of the reagents, the maspin peptides are extracted from the polymer with 1% acetic acid solution that is then lyophilized to yield the crude material. This can normally be purified by such techniques as gel filtration on Sephadex G- 15 using 5% acetic acid as a solvent. Lyophilization of appropriate fractions of the column will yield the homogeneous maspin peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid phase Edman degradation. Chemical synthesis of maspin and its derivatives, variants, and fragments thereof, permits incorporation of non-naturally occurring amino acids into the agent.

Recombinant DNA techniques are a convenient method for preparing maspin, or fragments thereof. A cDNA molecule encoding maspin or maspin fragment can be inserted into an expression vector, which can in turn be inserted into a host cell for subsequent expression and purification. It is understood that the cDNAs encoding maspin or fragments thereof may be modified to vary from the "original" cDNA (translated from the mRNA) to provide for codon degeneracy or to permit codon preference usage in various host cells.

Generally, a DNA molecule encoding maspin can be obtained using procedures described herein in the Examples, or synthesized using any standard method known in the art.

For example, see, Zou, Z. et al., Science, 263:526-529 (1994); Watanabe, M., et al., Human

Gene Therapy, 16:699-710 (2005); and Shi, H.Y., et al., MoI. Ther., 5:755-761 (2002), all incorporated by reference in their entirety.

A variety of expression vector/host systems may be utilized to contain and express the polynucleotide molecules encoding the maspin polypeptides useful in the invention. These systems include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.

Mammalian cells, for example VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells are useful in recombinant production of maspin and its fragments. Some exemplary protocols for the recombinant expression of maspin are described in Sheng, S. et al., J. Biol. Chem., 269:30988-93 (1994).

The term "expression vector" refers to a plasmid, phage, virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. An expression vector can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or sequence that encodes maspin, which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant maspin is expressed without a leader or transport sequence, it may include an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final maspin product.

For example, maspin may be recombinantly expressed in yeast using a commercially available expression system, e.g., the Pichia Expression System (Invitrogen, San Diego, CA), following the manufacturer's instructions. This system also relies on the pre-pro-alpha sequence to direct secretion, but transcription of the insert is driven by the alcohol oxidase (AOXl) promoter upon induction by methanol. The secreted maspin is purified from the yeast growth medium by, for example, the known methods used to purify the peptide from bacterial and mammalian cell supernatants.

Alternatively, the cDNA encoding maspin may be cloned into a baculovirus expression vector, such as pVL1393 (PharMingen, San Diego, CA). This vector can be used according to the manufacturer's directions (PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-free media and to produce recombinant protein. The subsequently produced maspin protein can be purified and concentrated from the media using a heparin-Sepharose column (Pharmacia).

Alternatively, the peptide may be expressed in an insect system. Insect systems for protein expression are well known to those of skill in the art. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The maspin coding sequence can be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the maspinwill render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses can be used to infect S. frugiperda cells or Trichoplusia larvae in which peptide is expressed (Smith et ah, J Virol 46: 584 (1983); Engelhard et ah, Proc Nat Acad Sci (USA) 91 : 3224-7 (1994)).

In another example, the DNA sequence encoding maspin can be amplified by PCR and cloned into an appropriate vector for example, pGEX-3X (Pharmacia). The pGEX vector is designed to produce a fusion protein comprising glutathione-S-transferase (GST), encoded by the vector, and a maspin protein encoded by a DNA fragment inserted into the vector's cloning site. The primers for the PCR can be generated to include for example, an appropriate cleavage site. Where the maspin fusion moiety is used solely to facilitate expression or is otherwise not desirable as an attachment to the peptide of interest, the maspin may then be cleaved from the GST portion of the fusion protein. The pGEX-3X/maspin construct is transformed into E. coli XL-I Blue cells (Stratagene, La Jolla CA), and individual transformants isolated and grown. Plasmid DNA from individual transformants can be purified and partially sequenced using an automated sequencer to confirm the presence of the maspin-encoding nucleic acid insert in the proper orientation. Expression of polynucleotides encoding maspin and fragments thereof using the recombinant systems described above may result in production of maspin or fragments thereof that must be "re-folded" (to properly create various tertiary structure) in order to be biologically active. Typical refolding procedures for proteins are well known in the art. Maspin made in bacterial cells may be produced as an insoluble inclusion body in the bacteria, can be purified as follows. Host cells can be sacrificed by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/ml lysozyme (Sigma, St. Louis, MO) for 15 minutes at room temperature. The lysate can be cleared by sonication, and cell debris can be pelleted by centrifugation for 10 minutes at 12,000 X g. The maspin- containing pellet can be resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 min. at 6000 X g. The pellet can be resuspended in standard phosphate buffered saline solution (PBS) free of Mg⁺⁺ and Ca⁺⁺. The protein can be further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al, supra). The gel can be soaked in 0.4 M KCl to visualize the protein, which can be excised and electroeluted in gel-running buffer lacking SDS. If the GST fusion protein is produced in bacteria, as a soluble protein, it can be purified using the GST Purification Module (Pharmacia).

Mammalian host systems for the expression of the recombinant protein are well known to those of skill in the art. Host cell strains can be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that can be useful in providing protein and/or fusion protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Different host cells such as CHO, HeLa, MDCK, 293, WI38, as well as hybridoma cell lines, and the like have specific cellular machinery and characteristic mechanisms for such post-translational activities and can be chosen to ensure the correct modification and processing of the introduced, foreign protein.

A number of selection systems can be used to recover the cells that have been transformed for recombinant protein production. Such selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for DHFR which confers resistance to methotrexate; gpt which confers resistance to mycophenolic acid; neo which confers resistance to the aminoglycoside G418 and confers resistance to chlorsulfuron; and hygro which that confers resistance to hygromycin. Additional selectable genes that may be useful include trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. Markers that give a visual indication for identification of trans formants include anthocyanins, β-glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.

Purification of Maspin

Typically, when maspin polypeptides are produced using any expression system, purification steps are necessary and/or desirable. General protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the polypeptide and non-polypeptide fractions. Having separated the maspin polypeptide from cell debris and other proteins, the polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Preparatory and analytical methods particularly suited to the purification of a maspin polypeptide are ion-exchange chromatography; affinity chromatography (e.g., for fusion systems, based on hydrophobicity/hydrophilicity, etc.); size- exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC).

Generally, "purified" will refer to a composition containing maspin that has been subjected to fractionation to remove various other components, and which substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which maspin forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition. Various methods for quantifying the degree of purification of maspin will be known to those of skill in the art in light of the present disclosure.

Various standard techniques suitable for use in maspin purification will be well known to those of skill in the art. These can include, for example, precipitation with ammonium sulphate, PEG, antibodies (immunoprecipitation) and the like or by heat denaturation, followed by centrifugation; chromatography steps such as affinity chromatography (e.g., Protein-A-Sepharose), ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified form of maspin.

Diseases The invention provides a method comprising administering maspin that is useful for the treatment of cancer and the clinical and/or pathological conditions associated with cancer. Agents that modulate angiogenesis, inhibit cancer proliferation, or that have other cellular activity, may be used in combination with other therapeutic agents to enhance their therapeutic effects or decrease potential side effects. In one aspect, the invention provides reagents and methods useful for treating diseases and conditions characterized by undesirable or aberrant levels of cathepsin D in a cell. Thus, the invention provides a method for modulating levels/amounts of cathepsin D in a cell comprising contacting a cell with an amount of maspin effective to modulate cathepsin D levels/amounts. In certain embodiments, the method of modulating levels of cathepsin D in a cell comprises contacting the cell with an amount of a maspin polypeptide effective to modulate cathepsin D levels or activity in the cell and in the extracellular environment by reducing the secretion of cathepsin D from the cell. In one embodiment, "modulating" is preferably "normalizing" such that cathepsin D levels or activity (or "amounts") are brought to within standard levels or activity for the particular cell. These diseases include cancer, and other diseases that are associated with unregulated angiogenesis and cellular proliferation.

In one aspect, the invention provides reagents and methods useful for treating diseases and conditions characterized by undesirable or aberrant levels of cathepsin D that are secreted from a cell. Thus, the invention provides a method for modulating levels/amounts of cathepsin D that are secreted from a cell comprising contacting a cell with an amount of maspin effective to modulate cathepsin D levels/amounts secreted from a cell. In certain embodiments, the method of modulating levels or activity of cathepsin D in a cell comprises contacting the cell with an amount of a maspin polypeptide effective to reduce the amount of cathepsin D that is secreted from the cell into the extracellular environment. In one embodiment the amount of secreted cathepsin D is reduced by inhibiting production of cathepsin D in the cell. In another embodiment the amount of secreted cathepsin D is reduced by inhibiting secretion of cathepsin D from the cell. In one embodiment, "modulating" is preferably "normalizing" such that cathepsin D levels (or "amounts") are brought to within standard levels for the particular cell and/or the extracellular environment. The present invention also provides methods of treating cancer in an animal, including humans, comprising administering to the animal an amount of a maspin polypeptide and an effective amount of an IFN-γ effective to inhibit or prevent cancer. The invention is further directed to methods of inhibiting cancer cell growth, including processes of cellular proliferation, invasiveness, and metastasis in biological systems. Methods of the invention include the combined use of a maspin polypeptide and IFN-γ, or compositions comprising a maspin polypeptide and IFN-γ in combination with a pharmaceutically acceptable carrier, as an inhibitor of cancer cell growth. Preferably, the methods are employed to inhibit or reduce cancer cell growth, invasiveness, metastasis, or tumor incidence in living animals, such as mammals. In certain embodiments of this aspect the combination of maspin and IFN-γ can be administered as separate agents, for example, by liposomal delivery into tumor vasculature of a nucleic acid molecule encoding a biologically active maspin polypeptide, followed by administration of an effective amount of IFN-γ. Such liposomal delivery systems are described in the art, for example in Li, Z., et al, Oncogene, 24:2008-2019 (2005), incorporated herein by reference.

The cancers treatable by methods of the present invention preferably occur in mammals. Mammals include, for example, humans and other primates, as well as pet or companion animals such as dogs and cats, laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, sheep, and cattle. Tumors or neoplasms include growths of tissue cells in which the multiplication of the cells is uncontrolled and progressive. Some such growths are benign, but others are termed malignant and may lead to death of the organism. Malignant neoplasms or cancers are distinguished from benign growths in that, in addition to exhibiting aggressive cellular proliferation, they may invade surrounding tissues and metastasize. Moreover, malignant neoplasms are characterized in that they show a greater loss of differentiation (greater dedifferentiation), and of their organization relative to one another and their surrounding tissues. This property is also called "anaplasia."

Neoplasms treatable by the present invention also include solid tumors, i.e., carcinomas and sarcomas. Carcinomas include those malignant neoplasms derived from epithelial cells that infiltrate (invade) the surrounding tissues and give rise to metastases. Adenocarcinomas are carcinomas derived from glandular tissue, or which form recognizable glandular structures. Another broad category or cancers includes sarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance like embryonic connective tissue. The invention also enables treatment of cancers of the myeloid or lymphoid systems, including leukemias, lymphomas and other cancers that typically do not present as a tumor mass, but are distributed in the vascular or lymphoreticular systems.

The type of cancer or tumor cells amenable to treatment according to the invention include, for example, any cells that derive from original parental cells that express maspin. Such cells may include, but are not limited to, ACTH-producing tumor, acute lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer, brain cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and non-small cell), malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, non-Hodgkin's lymphoma, osteosarcoma, penile cancer, prostate cancer, retinoblastoma, skin cancer, soft tissue sarcoma, squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer, trophoblastic neoplasms, uterine cancer, vaginal cancer, cancer of the vulva, and Wilms' tumor.

The invention is particularly illustrated herein in reference to treatment of certain types of experimentally defined cancers. In these illustrative treatments, standard state-of-the-art in vitro and in vivo models have been used. These methods can be used to identify agents that can be expected to be efficacious in in vivo treatment regimens. However, it will be understood that the method of the invention is not limited to the treatment of these tumor types, but extends to any solid tumor derived from any organ system. Cancers whose invasiveness or metastasis is associated with modulated expression or activity of either maspin or cathepsin D are especially susceptible to being inhibited or even induced to regress by means of the invention. Further, cancers that are typically treatable using IFN-γ, but that have become refractory to IFN-γ treatment are also susceptible to being inhibited or induced to regress by the methods of the invention.

The invention can also be practiced by including with a maspin polypeptide and IFN- γ, and/or another anti-cancer chemotherapeutic agent, such as any conventional chemotherapeutic agent. The combination of a maspin polypeptide and IFN-γ with such other agents can potentiate the chemotherapeutic protocol. Numerous chemotherapeutic protocols will present themselves in the mind of the skilled practitioner as being capable of incorporation into the method of the invention. Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products. For example, the compound of the invention can be administered with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisp latin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH). Other antineoplastic protocols include the use of a tetracycline compound with another treatment modality, e.g., surgery, radiation, etc., also referred to herein as "adjunct antineoplastic modalities." Thus, the method of the invention can be employed with such conventional regimens with the benefit of reducing side effects and enhancing efficacy.

The present invention thus provides compositions and methods useful for the treatment of a wide variety of cancers, including solid tumors and leukemias that express maspin in the original parental cells. Thus, some types of cancer that may be treated include, but are not limited to: adenocarcinoma of the breast, prostate, and colon; forms of bronchogenic carcinoma of the lung; myeloid; melanoma; hepatoma; neuroblastoma; papilloma; apudoma; choristoma; branchioma; malignant carcinoid syndrome; carcinoid heart disease; carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell); histiocytic disorders; leukemia; histiocytosis malignant; Hodgkin's disease; immunoproliferative small lung cell carcinoma; non-Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumors; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; dysgerminoma; hamartoma; mesenchymoma; mesonephroma; myosarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma; tophoblastic tumor. Further, the following types of cancers may also be treated: adenoma; cholangioma; cholesteatoma; cyclindroma; cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma; hepatoma; hidradenoma; islet cell tumor; Ley dig cell tumor; papilloma; Sertoli cell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma; myoma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma; paraganglioma nonchromaffin; angiokeratoma; angiolymphoid hyperplasia with eosinophilia; angioma sclerosing; angiomatosis; glomangioma; hemangioendothelioma; hemangioma; hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; rhabdomyosarcoma; sarcoma; neoplasms; nerofϊbromatosis; and cervical dysplasia.

Other aspects of the present invention include treating various retinopathies (including diabetic retinopathy and age-related macular degeneration) in which angiogenesis is involved, as well as disorders/diseases of the female reproductive tract such as endometriosis, uterine fibroids, and other such conditions associated with dysfunctional vascular proliferation

(including endometrial microvascular growth) during the female reproductive cycle.

Another aspect of present invention is the prevention of cancers utilizing the compositions and methods provided by the present invention. Such methods and reagents will comprise maspin, or fragments thereof.

Pharmaceutical Compositions

Pharmaceutical compositions comprising maspin are within the scope of the present invention. Such compositions comprise a therapeutically or prophylactically effective amount of maspin, or a fragment, variant, derivative or fusion thereof as described herein, in admixture with a pharmaceutically acceptable agent. In a preferred embodiment, pharmaceutical compositions comprise maspin or a fragment, variant, derivative or fusion thereof in admixture with an interferon, such as IFN-γ, and a pharmaceutically acceptable agent. In another embodiment, the composition further comprises an additional agent effective in the treatment of cancer and/or inhibiting undesired angiogenesis.

The pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfϊte); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counter ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides (preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990). The optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. See for example, Remington's Pharmaceutical Sciences, supra. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the maspin polypeptide and IFN-γ agents. The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefore. In one embodiment of the present invention, binding agent compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, the binding agent product may be formulated as a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for enteral delivery such as orally, aurally, opthalmically, rectally, or vaginally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at slightly lower pH, typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired maspin polypeptide and IFN-γ agents in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a binding agent is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which may then be delivered via a depot injection. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

In another aspect, pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions. In another embodiment, a pharmaceutical composition may be formulated for inhalation. For example, a binding agent may be formulated as a dry powder for inhalation. Polypeptide or nucleic acid molecule inhalation solutions may also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions may be nebulized. Pulmonary administration is further described in PCT Application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.

It is also contemplated that certain formulations may be administered orally. In one embodiment of the present invention, binding agent molecules that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the binding agent molecule. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

Pharmaceutical compositions for oral administration can also be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross- linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.

Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally also include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Another pharmaceutical composition may involve an effective quantity of binding agent in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or other appropriate vehicle, solutions can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving binding agent molecules in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCT/US93/00829 that describes controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al, J Biomed Mater Res, 15:167-277, (1981)) and (Langer et al, Chem Tech, 12:98-105(1982)), ethylene vinyl acetate (Langer et al, supra) or poly-D(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also include liposomes, which can be prepared by any of several methods known in the art. See e.g., Eppstein et al., Proc Natl Acad Sci (USA), 82:3688-3692 (1985); EP 36,676; EP 88,046; EP 143,949.

The pharmaceutical composition to be used for in vivo administration typically must be sterile. This may be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits for producing a single-dose administration unit. The kits may each contain both a first container having a dried protein (maspin) and a second container having an aqueous formulation. Also included within the scope of this invention are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).

An effective amount of a pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the binding agent molecule is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage of maspin will range from about 0.1 mg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the maspin dosage will range from 0.1 mg/kg up to about 100 mg/kg; or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to about 100 mg/kg.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The exact dosage will be determined in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.

The frequency of dosing will depend upon the pharmacokinetic parameters of the binding agent molecule in the formulation used. Typically, a composition is administered until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as multiple doses (at the same or different concentrations/dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely made. Appropriate dosages may be ascertained through use of appropriate dose-response data. The route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, intralesional routes, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal means, by sustained release systems or by implantation devices. Where desired, the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.

Alternatively or additionally, the composition may be administered locally via implantation of a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.

In some cases, it may be desirable to use pharmaceutical compositions in an ex vivo manner. In such instances, cells, tissues, or organs that have been removed from the patient are exposed to the pharmaceutical compositions after which the cells, tissues and/or organs are subsequently implanted back into the patient.

In other cases, a binding agent which is a polypeptide can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide. Such cells may be animal or human cells, and may be autologous, heterologous, or xenogeneic. Optionally, the cells may be immortalized. In order to decrease the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues. The encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.

Combination Therapy The effective maspin and IFN-γ agents of the invention can be utilized in combination with other therapeutic agents in the treatment of neoplastic pathologies. These other therapeutics include, for example radiation treatment, chemotherapy, and targeted therapies as described herein below. Additional combination therapies not specifically listed herein are also within the scope of the present invention. The invention thus includes administration of maspin to the same patient in combination with one or more additionally suitable agent(s), each being administered according to a regimen suitable for that medicament. This includes sequential and concurrent administration of maspin and one or more suitable agents. As used herein, the terms "concurrently administered" and "concurrent administration" encompass substantially simultaneous administration of maspin and one or more additionally suitable agents(s).

As used herein, the term, "non-concurrent" administration encompasses administering maspin according to the invention and one or more additionally suitable agent(s), at different times, in any order, whether overlapping or not. This includes, but is not limited to, sequential treatment (such as pretreatment, post-treatment, or overlapping treatment) with the components of the combination, as well as regimens in which the drugs are alternated, or wherein one component is administered long-term and the other(s) are administered intermittently. Components may be administered in the same or in separate compositions, and by the same or different routes of administration.

In certain embodiments, the combination therapy comprises maspin, in combination with at least one anti-angiogenic agent. Agents include, but are not limited to, in vitro synthetically prepared chemical compositions, antibodies, antigen binding regions, radionuclides, and combinations and conjugates thereof. In certain embodiments, an agent may act as an agonist, antagonist, alllosteric modulator, or toxin. In certain embodiments, an agent may act to inhibit or stimulate its target (e.g., receptor or enzyme activation or inhibition), and thereby promote cell death or arrest cell growth.

Chemotherapy treatment can employ anti-neoplastic agents including, for example, alkylating agents including: nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); ethylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2- chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; ppipodophylotoxins such as etoposide and teniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents including platinium coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N- methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p'- DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide.

Cancer therapies, which are administered with maspin, also include, but are not limited to, targeted therapies as described herein. Examples of targeted therapies include, but are not limited to, use of therapeutic antibodies. Exemplary therapeutic antibodies, include, but are not limited to, mouse, mouse-human chimeric, CDR-grafted, humanized and fully human antibodies, and synthetic antibodies, including, but not limited to, those selected by screening antibody libraries. Exemplary antibodies include, but are not limited to, those which bind to cell surface proteins Her2, CDC20, CDC33, mucin-like glycoprotein, and epidermal growth factor receptor (EGFR) present on tumor cells, and optionally induce a cytostatic and/or cytotoxic effect on tumor cells displaying these proteins. Exemplary antibodies also include HERCEPTIN™ (trastuzumab), which may be used to treat breast cancer and other forms of cancer, and RITUXAN™ (rituximab), ZEVALIN™ (ibritumomab tiuxetan), GLEEVEC™, and LYMPHOCIDE™ (epratuzumab), which may be used to treat non-Hodgkin's lymphoma and other forms of cancer. Certain exemplary antibodies also include ERBITUX™ (IMC-C225); IRESSA™ (ertinolib) ; BEXXAR™ (iodine 131 tositumomab); KDR (kinase domain receptor) inhibitors; anti-VEGF antibodies and antagonists (e.g., AVASTIN™ and VEGAF-TRAP); anti-VEGF receptor antibodies and antigen binding regions; anti-Ang-1 antibodies and antigen binding regions; antibodies to Tie-2 and other Ang-1 and Ang-2 receptors; Tie-2 ligands; antibodies against Tie-2 kinase inhibitors; and Campath^® (Alemtuzumab). In certain embodiments, cancer therapy agents are polypeptides which selectively induce apoptosis in tumor cells, including, but not limited to, TNF-related polypeptides, such as TRAIL (TNF Receptor Apoptosis-Inducing Ligand).

In certain embodiments, suitable cancer therapy agents are known to be anti- angiogenic. Certain such agents include, but are not limited to, IL-8; Campath™, B-FGF; FGF antagonists; Tek antagonists (Cerretti et al, U.S. Publication No. 2003/0162712; Cerretti et al., U.S. Patent No. 6,413,932, and Cerretti et al., U.S. Patent No. 6,521,424, each of which is incorporated herein by reference for any purpose); anti-TWEAK agents (which include, but are not limited to, antibodies and antigen binding regions); soluble TWEAK receptor antagonists (Wiley, U.S. Patent No. 6,727,225); ADAM distintegrins (or domains thereof to antagonize the binding of integrin to its ligands (Fanslow et al., U.S. Publication No. 2002/0042368); anti-eph receptor and anti-ephrin antibodies; antigen binding regions, or antagonists (U.S. Patent Nos. 5,981,245; 5,728,813; 5,969,110; 6,596,852; 6,232,447; 6,057,124 and patent family members thereof); anti-VEGF agents as described herein {e.g., antibodies or antigen binding regions that specifically bind VEGF, or soluble VEGF receptors or a ligand binding regions thereof) such as AVASTIN™ or VEGF-TRAP™, and anti-VEGF receptor agents {e.g., antibodies or antigen binding regions that specifically bind thereto), EGFR inhibitory agents {e.g., antibodies or antigen binding regions that specifically bind thereto) such as panitumumab, IRESSA™ (gefitinib), TARCEV A™ (erlotinib), anti-Ang-1 and anti- Ang-2 agents (e.g., antibodies or antigen binding regions specifically binding thereto or to their receptors, e.g., Tie-2/TEK), and anti-Tie-2 kinase inhibitory agents (e.g., antibodies or antigen binding regions that specifically bind and inhibit the activity of growth factors, such as antagonists of hepatocyte growth factor (HGF, also known as Scatter Factor), and antibodies or antigen binding regions that specifically bind its receptor "c-met"; anti-PDGF- BB antagonists; antibodies and antigen binding regions to PDGF-BB ligands; and PDGFR kinase inhibitors.

In certain embodiments, cancer therapy agents are angiogenesis inhibitors. Certain such inhibitors include, but are not limited to, SD-7784 (Pfizer, USA); cilengitide. (Merck KGaA, Germany, EPO 770622); pegaptanib octasodium, (Gilead Sciences, USA); Alphastatin, (BioActa, UK); M-PGA, (Celgene, USA, US 5712291); ilomastat, (Arriva, USA, US 5892112); semaxanib, (Pfizer, USA, US 5792783); vatalanib, (Novartis, Switzerland); 2- methoxyestradiol, (EntreMed, USA); TLC ELL- 12, (Elan, Ireland); anecortave acetate, (Alcon, USA); alpha-D148 Mab, (Amgen, USA); CEP-7055,(Cephalon, USA); anti-Vn Mab, (Crucell, Netherlands) DAC:antiangiogenic, (ConjuChem, Canada); Angiocidin, (InKine Pharmaceutical, USA); KM-2550, (Kyowa Hakko, Japan); SU-0879, (Pfizer, USA); CGP- 79787, (Novartis, Switzerland, EP 970070); ARGENT technology, (Ariad, USA); YIGSR- Stealth, (Johnson & Johnson, USA); fibrinogen-E fragment, (BioActa, UK); angiogenesis inhibitor, (Trigen, UK); TBC-1635, (Encysive Pharmaceuticals, USA); SC-236, (Pfizer, USA); ABT-567, (Abbott, USA); Metastatin, (EntreMed, USA); angiogenesis inhibitor, (Tripep, Sweden); maspin, (Sosei, Japan); 2-methoxyestradiol, (Oncology Sciences Corporation, USA); ER-68203-00, (IVAX, USA); Benefin, (Lane Labs, USA); Tz-93, (Tsumura, Japan); TAN-1120, (Takeda, Japan); FR-111142, (Fujisawa, Japan, JP 02233610); platelet factor 4, (RepliGen, USA, EP 407122); vascular endothelial growth factor antagonist, (Borean, Denmark); cancer therapy, (University of South Carolina, USA); bevacizumab (pINN), (Genentech, USA); angiogenesis inhibitors, (SUGEN, USA); XL 784, (Exelixis, USA); XL 647, (Exelixis, USA); MAb, alpha5beta3 integrin, second generation, (Applied Molecular Evolution, USA and Medlmmune, USA); gene therapy, retinopathy, (Oxford BioMedica, UK); enzastaurin hydrochloride (USAN), (Lilly, USA); CEP 7055, (Cephalon, USA and Sanofi-Synthelabo, France); BC 1, (Genoa Institute of Cancer Research, Italy); angiogenesis inhibitor, (Alchemia, Australia); VEGF antagonist, (Regeneron, USA); rBPI 21 and BPI-derived antiangiogenic, (XOMA, USA); PI 88, (Progen, Australia); cilengitide (pINN), (Merck KGaA; Munich Technical University, Germany, Scripps Clinic and Research Foundation, USA); cetuximab (INN), (Aventis, France); AVE 8062, (Ajinomoto, Japan); AS 1404, (Cancer Research Laboratory, New Zealand); SG 292 (Telios, USA); Endostatin, (Boston Childrens Hospital, USA); ATN 161, (Attenuon, USA); ANGIOSTATIN, (Boston Childrens Hospital, USA); 2-methoxyestradiol, (Boston Childrens Hospital, USA); ZD 6474, (AstraZeneca, UK); ZD 6126, (Angiogene Pharmaceuticals, UK); PPI 2458, (Praecis, USA); AZD 9935, (AstraZeneca, UK); AZD 2171, (AstraZeneca, UK); vatalanib (pINN), (Novartis, Switzerland and Schering AG, Germany); tissue factor pathway inhibitors, (EntreMed, USA); pegaptanib (Pinn), (Gilead Sciences, USA); xanthorrhizol, (Yonsei University, South Korea); vaccine, gene-based, VEGF-2, (Scripps Clinic and Research Foundation, USA); SPV5.2, (Supratek, Canada); SDX 103, (University of California at San Diego, USA); PX 478, (ProlX, USA); METASTATIN, (EntreMed, USA); troponin I, (Harvard University, USA); SU 6668, (SUGEN, USA); OXI 4503, (OXiGENE, USA); o-guanidines, ( Dimensional Pharmaceuticals, USA); motuporamine C, (British Columbia University, Canada); CDP 791, (Celltech Group, UK); atiprimod (pINN), (Glaxo SmithKline, UK); E 7820, (Eisai, Japan); CYC 381, (Harvard University, USA); AE 941, (Aeterna, Canada); vaccine, angiogenesis, (EntreMed, USA); urokinase plasminogen activator inhibitor, (Dendreon, USA); oglufanide (pINN), (Melmotte, USA); HIF-lalfa inhibitors, (Xenova, UK); CEP 5214, (Cephalon, USA); BAY RES 2622, (Bayer, Germany); Angiocidin, (InKine, USA); A6, (Angstrom, USA); KR 31372, (Korea Research Institute of Chemical Technology, South Korea); GW 2286, (GlaxoSmithKline, UK); EHT 0101, (ExonHit, France); CP 868596, (Pfizer, USA); CP 564959, (OSI, USA); CP 547632, (Pfizer, USA); 786034, (GlaxoSmithKline, UK); KRN 633, (Kirin Brewery, Japan); drug delivery system, intraocular, 2-methoxyestradiol, (EntreMed, USA); anginex, (Maastricht University, Netherlands, and Minnesota University, USA); ABT 510, (Abbott, USA); AAL 993, (Novartis, Switzerland); VEGI, (ProteomTech, USA); tumor necrosis factor-alpha inhibitors, (National Institute on Aging, USA); SU 11248, (Pfizer, USA and SUGEN USA); ABT 518, (Abbott, USA); YHl 6, (Yantai Rongchang, China); S-3APG , (Boston Childrens Hospital, USA and EntreMed, USA); MAb, KDR, (ImClone Systems, USA); MAb, alpha5 betal, (Protein Design, USA); KDR kinase inhibitor, (Celltech Group, UK, and Johnson & Johnson, USA); GFB 116, (South Florida University, USA and Yale University, USA); CS 706, (Sankyo, Japan); combretastatin A4 prodrug, (Arizona State University, USA); chondroitinase AC, (IBEX, Canada); BAY RES 2690, (Bayer, Germany); AGM 1470, (Harvard University, USA, Takeda, Japan, and TAP, USA); AG 13925, (Agouron, USA); Tetrathiomolybdate, (University of Michigan, USA); GCS 100, (Wayne State University, USA) CV 247, (Ivy Medical, UK); CKD 732, (Chong Kun Dang, South Korea); MAb, vascular endothelium growth factor, (Xenova, UK); irsogladine (INN), (Nippon Shinyaku, Japan); RG 13577, (Aventis, France); WX 360, (Wilex, Germany); squalamine (pINN), (Genaera, USA); RPI 4610, (Sirna, USA); cancer therapy, (Marinova, Australia); heparanase inhibitors, (InSight, Israel); KL 3106, (Ko Ion, South Korea); Honokiol, (Emory University, USA); ZK CDK, (Schering AG, Germany); ZK Angio, (Schering AG, Germany); ZK 229561, (Novartis, Switzerland, and Schering AG, Germany); XMP 300, (XOMA, USA); VGA 1102, (Taisho, Japan); VEGF receptor modulators, (Pharmacopeia, USA); VE-cadherin-2 antagonists , (ImClone Systems, USA); Vasostatin, (National Institutes of Health, USA);vaccine, FIk-I, (ImClone Systems, USA); TZ 93, (Tsumura, Japan); TumStatin, (Beth Israel Hospital, USA); truncated soluble FLT 1 (vascular endothelial growth factor receptor 1), (Merck & Co, USA); Tie-2 ligands, (Regeneron, USA); thrombospondin 1 inhibitor, (Allegheny Health, Education and Research Foundation, USA); ; 2- Benzenesulfonamide,4-(5-(4-chlorophenyl)-3-(trifluoromethyl)-lH-pyrazol-l-yl)-; Arriva; and C-Met. AVE 8062 ((2S)-2-amino-3-hydroxy-N-[2-methoxy-5-[(lZ)-2-(3,4,5- trimethoxyphenyl)ethenyl]p henyljpropanamide monohydrochloride); metelimumab (pINN)(immunoglobulin G4, anti-(human transforming growth factor .beta.l (human monoclonal CAT 192 .gamma.4-chain)), disulfide with human monoclonal CAT 192 .kappa.- chain dimer); Flt3 ligand; CD40 ligand; interleukin-2; interleukin-12; 4- IBB ligand; anti-4- IBB antibodies; TNF antagonists and TNF receptor antagonists including TNFR/Fc, TWEAK antagonists and TWEAK-R antagonists including TWEAK-R/Fc; TRAIL; VEGF antagonists including anti-VEGF antibodies; VEGF receptor (including VEGF-Rl and VEGF-R2, also known as Fltl and Flkl or KDR) antagonists; CD148 (also referred to as DEP-I, ECRTP, and PTPRJ, see Takahashi et al, J. Am. Soc. Nephrol. 10: 2135-45 (1999), hereby incorporated by reference for any purpose) agonists; thrombospondin 1 inhibitor, and inhibitors of one or both of Tie-2 or Tie-2 ligands (such as Ang-2). A number of inhibitors of Ang-2 are known in the art, including certain anti- Ang-2 antibodies described in published U.S. Patent Application No. 20030124129 (corresponding to PCT Application No. WO03/030833), and U.S. Patent No. 6,166,185, the contents of which are hereby incorporated by reference in their entirety. Additionally, Ang-2 peptibodies are also known in the art, and can be found in, for example, published U.S. Patent Application No. 20030229023 (corresponding to PCT Application No. WO03/057134), and published U.S. Patent Application No. 20030236193, the contents of which are hereby incorporated by reference in their entirety.

Certain cancer therapy agents include, but are not limited to: thalidomide and thalidomide analogues (N-(2,6-dioxo-3-piperidyl)phthalimide); tecogalan sodium (sulfated polysaccharide peptidoglycan); TAN 1120 (8-acetyl-7,8,9,10-tetrahydro-6,8,l l-trihydroxy-l- methoxy- 10- [[octahydro-5 -hydroxy-2-(2-hydroxypropyl)-4, 10-dimethylpyrano [3 ,4-d] - 1 ,3 ,6- dioxazocin-8-yl]oxy]-5,12-naphthacenedione); suradista (7,7'-[carbonylbis[imino(l-methyl- 1 H-pyrrole-4,2-diyl)carbonylimino( 1 -methyl- 1 H- pyrrole-4,2-diyl)carbonylimino]]bis- 1,3- naphthalenedisulfonic acid tetrasodium salt); SU 302; SU 301; SU 1498 ((E)-2-cyano-3-[4- hydroxy-3,5-bis(l-methylethyl)phenyl]-N-(3-phenylpropyl)-2-pro penamide); SU 1433 (4- (6,7-dimethyl-2-quinoxalinyl)-l,2-benzenediol); ST 1514; SR 25989;soluble Tie-2; SERM derivatives, Pharmos; semaxanib (pINN)(3-[(3,5-dimethyl-lH-pyrrol-2-yl)methylene]-l,3- dihydro-2H-indol-2-one); S 836; RG 8803; RESTIN; R 440 (3-(l -methyl- lH-indol-3-yl)-4- (l-methyl-6-nitro-lH-indol-3-yl)-lH-pyrrole-2,5-dione); R 123942 (l-[6-(l,2,4-thiadiazol-5- yl)-3-pyridazinyl]-N-[3-(trifluoromethyl)phenyl]-4-piperidinamine); prolyl hydroxylase inhibitor; progression elevated genes; prinomastat (INN) ((S)-2,2-dimethyl-4-[[p-(4- pyridyloxy)phenyl]sulphonyl]-3-thiomorpholinecarbohyd roxamic acid); NV 1030; NM 3 (8- hydroxy-6-methoxy-alpha-methyl-l-oxo-lH-2-benzopyran-3 -acetic acid); NF 681; NF 050; MIG; METH 2; METH 1; manassantin B (alpha-[l-[4-[5-[4-[2-(3,4-dimethoxyphenyl)-2- hydroxy- 1 -methylethoxy] -3 -methoxyphenyl]tetrahydro-3 ,4-dimethyl-2-furanyl] -2- methoxyphenoxy]ethyl]-l,3-benzodioxole-5-methanol); KDR monoclonal antibody; alpha5beta3 integrin monoclonal antibody; LY 290293 (2-amino-4-(3-pyridinyl)-4H- naphtho[l,2-b]pyran-3-carbonitrile); KP 0201448; KM 2550; integrin-specifϊc peptides; INGN 401; GYKI 66475; GYKI 66462; greenstatin (101-354-plasminogen (human)); gene therapy for rheumatoid arthritis, prostate cancer, ovarian cancer, glioma, endostatin, colorectal cancer, ATF BTPI, antiangiogenesis genes, angiogenesis inhibitor, or angiogenesis; gelatinase inhibitor, FR 111142 (4,5-dihydroxy-2-hexenoic acid 5-methoxy-4-[2-methyl-3-(3-methyl-2- butenyl)oxiranyl]-l-oxaspiro[2.5]oct-6-yl ester); forfenimex (pINN) (S)-alpha-amino-3- hydroxy-4-(hydroxymethyl)benzeneacetic acid); fibronectin antagonist (1-acetyl-L-pro IyI-L- histidyl-L-seryl-L-cysteinyl-L-aspartamide); fibroblast growth factor receptor inhibitor; fibroblast growth factor antagonist; FCE 27164 (7,7'-[carbonylbis[imino(l-methyl-lH- pyrrole-4,2-diyl)carbonylimino(l -methyl- IH- pyrrole -4,2-diyl)carbonylimino]]bis- 1,3,5- naphthalenetrisulfonic acid hexasodium salt); FCE 26752 (8,8'-[carbonylbis[imino(l-methyl- 1 H-pyrrole-4,2-diyl)carbonylimino( 1 -methyl- 1 H-pyrrole-4,2-diyl)carbonylimino]]bis- 1,3,6- naphthalenetrisulfonic acid); endothelial monocyte activating polypeptide II; VEGFR antisense oligonucleotide; anti-angiogenic and trophic factors; ANCHOR angiostatic agent; endostatin; DeI-I angiogenic protein; CT 3577; contortrostatin; CM 101; chondroitinase AC; CDP 845; CanStatin; BST 2002; BST 2001; BLS 0597; BIBF 1000; ARRESTIN; apomigren (1304-1388-type XV collagen (human gene COL15A1 alphal-chain precursor)); angioinhibin; aaATIII; A 36; 9alpha-fluoromedroxyprogesterone acetate ((6-alpha)-17- (acetyloxy)-9-fluoro-6-methyl-pregn-4-ene-3,20-dione); 2-methyl-2-phthalimidino-glutaric acid (2-(l,3-dihydro-l-oxo-2H-isoindol-2-yl)-2-methylpentanedioic acid); Yttrium 90 labelled monoclonal antibody BC-I; Semaxanib (3-(4,5-Dimethylpyrrol-2- ylmethylene)indolin-2-one)(C15 H14 N2 O); PI 88 (phosphomannopentaose sulfate); Alvocidib (4H- 1 -Benzopyran-4-one, 2-(2-chlorophenyl)-5 ,7-dihydroxy-8-(3-hydroxy- 1 - methyl-4-piperidinyl)- cis-(-)-) (C21 H20 Cl N 05); E 7820; SU 11248 (5-[3-Fluoro-2-oxo- l,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-lH- pyrrole-3-carboxylic acid (2- diethylaminoethyl)amide) (C22 H27 F N4 02); Squalamine (Cholestane-7,24-diol, 3-[[3-[(4- aminobutyl)aminopropyl] amino]-, 24-(hydrogen sulfate), (3.beta.,5.alpha.,7.alpha.)-) (C34 H65 N3 05 S); Eriochrome Black T; AGM 1470 (Carbamic acid, (chloroacetyl)-, 5-methoxy- 4-[2-methyl-3-(3-methyl-2- butenyl)oxiranyl] -l-oxaspiro[2,5] oct-6-yl ester, [3R-[3alpha, 4alpha(2R, 3R), 5beta, 6beta]]) (C19 H28 Cl N 06); AZD 9935; BIBF 1000; AZD 2171; ABT 828; KS-interleukin-2; Uteroglobin; A 6; NSC 639366 (l-[3-(Diethylamino)-2- hydroxypropylamino]-4-(oxyran-2- ylmethylamino)anthraquinone fumerate) (C24 H29 N3 04 . C4 H4 04); ISV 616; anti-ED-B fusion proteins; HUI 77; Troponin I; BC-I monoclonal antibody; SPV 5.2; ER 68203; CKD 731 (3-(3,4,5-Trimethoxyphenyl)-2(E)-propenoic acid (3R,4S,5S,6R)-4-[2(R)- methyl-3(R)-3(R)-(3-methyl-2-butenyl)oxiran-2-yl]-5-methoxy-l- oxaspiro[2.5]oct-6-yl ester) (C28 H38 08); IMC-ICl 1; aaATIII; SC 7; CM 101; Angiocol; Kringle 5; CKD 732 (3-[4-[2-(Dimethylamino)ethoxy]phenyl]-2(E)-propenoic acid)(C29 H41 N 06); U 995; Canstatin; SQ 885; CT 2584 (l-[l l-(Dodecylamino)-10-hydroxyundecyl]-3,7- dimethylxanthine)(C30 H55 N5 03); Salmosin; EMAP II; TX 1920 (l-(4-Methylpiperazino)- 2-(2-nitro-lH-l-imidazoyl)-l-ethanone) (ClO Hl 5 N5 03); Alpha-v Beta-x inhibitor; CHIR 11509 (N-(I -Propynyl)glycyl-[N-(2-naphthyl)]glycyl-[N-(carbamoylmethyl)] glycine bis(4- methoxyphenyl)methylamide)(C36 H37 N5 06); BST 2002; BST 2001; B 0829; FR 111142; 4,5-Dihydroxy-2(E)-hexenoic acid (3R,4S, 5S, 6R)-4-[l(R),2(R)-epoxy-l,5- dimethyl-4- hexenyl]-5-methoxy-l-oxaspiro[2.5]octan-6-yl ester (C22 H34 07); and kinase inhibitors including, but not limited to, N-(4-chlorophenyl)-4-(4-pyridinylmethyl)-l-phthalazinamine; 4- [4- [ [[ [4-chloro-3 -(trifluoromethyl)phenyl] amino]carbonyl] amino]phenoxy] -N-methyl-2- pyridinecarboxamide; N-[2-(diethylamino)ethyl]-5-[(5-fluoro- 1 ,2-dihydro-2-oxo-3H-indol-3- ylidene)methyl]-2,4-dimethyl-lH-pyrrole-3-carboxamide; 3-[(4-bromo-2,6- difluorophenyl)methoxy] -5 -[ [ [[4-( 1 -pyrrolidinyl)butyl] amino] carbonyljamino] -4- isothiazolecarboxamide; N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(l-methyl-4- piperidinyl)methoxy]-4-quinazolinamine; 3-[5,6,7,13-tetrahydro-9-[(l-methylethoxy)methyl]- 5-oxo-12H-indeno[2,l-a]pyrrolo[3,4-c]carbazol-12-yl]propyl ester N,N-dimethyl-glycine; N- [5-[[[5-(l , 1 -dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide; N- [3 -chloro-4- [(3 -fluorophenyl)methoxy]phenyl]-6- [5 - [[ [2-

(methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine; 4-[(4-Methyl-l- piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]- phenyljbenzamide; Λ/-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4- quinazolinamine; Λ/-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine; N-(3- ((((2R)-l-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-2-((3-(l,3-oxazol-5- yl)phenyl)amino)-3 -pyridinecarboxamide; 2-(((4-fluorophenyl)methyl)amino)-N-(3 -((((2R)- 1 -methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-3 -pyridinecarboxamide; N- [3 -(Azetidin-3 -ylmethoxy)-5 -trifluoromethyl-phenyl] -2-(4-fluoro-benzylamino)- nicotinamide; 6-fluoro-N-(4-( 1 -methylethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3 - pyridinecarboxamide; 2-((4-pyridinylmethyl)amino)-N-(3-(((2S)-2-pyrrolidinylmethyl)oxy)- 5 -(trifluoromethyl)phenyl)-3 -pyridinecarboxamide; N-(3 -( 1 , 1 -dimethylethyl)- 1 H-pyrazol-5 - yl)-2-((4-pyridinylmethyl)amino)-3 -pyridinecarboxamide; N-(3 ,3 -dimethyl-2,3 -dihydro- 1 - benzofuran-6-yl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide; N-(3-((((2S)- 1 - methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-2-((4-pyridinylmethyl)amino)- 3 -pyridinecarboxamide; 2-((4-pyridinylmethyl)amino)-N-(3 -((2-( 1 -pyrrolidiny l)ethyl)oxy)-4- (trifluoromethyl)phenyl)-3 -pyridinecarboxamide; N-(3 ,3 -dimethyl-2,3 -dihydro- 1 H-indol-6- yl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide; N-(4-(pentafluoroethyl)-3-(((2S)-2- pyrrolidinylmethyl)oxy)phenyl)-2-((4-pyridinylmethyl)amino)-3 -pyridinecarboxamide; N-(3 - ((3 -azetidinylmethyl)oxy)-5 -(trifluoromethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3 - pyridinecarboxamide; N-(3 -(4-piperidinyloxy)-5 -(trifluoromethyl)phenyl)-2-((2-(3 - pyridinyl)ethyl)amino)-3-pyridinecarboxamide; N-(4,4-dimethyl- 1,2,3, 4-tetrahydro- isoquinolin-7-yl)-2-(lH-indazol-6-ylamino)-nicotinamide; 2-(lH-indazol-6-ylamino)-N-[3-(l- methylpyrrolidin-2-ylmethoxy)-5 -trifluoromethyl-phenyl] -nicotinamide; N- [ 1 -(2- dimethylamino-acetyl)-3,3-dimethyl-2,3-dihydro-lH-indol-6-yl]-2-(lH-indazol-6-ylamino)- nicotinamide; 2-( 1 H-indazol-6-ylamino)-N- [3 -(pyrrolidin-2-ylmethoxy)-5 -trifluoromethyl- phenyl] -nicotinamide; N-(l-acetyl-3,3-dimethyl-2,3-dihydro-lH-indol-6-yl)-2-(lH-indazol-6- ylamino)-nicotinamide; N-(4,4-dimethyl- 1 -oxo- 1 ,2,3 ,4-tetrahydro-isoquinolin-7-yl)-2-(l H- indazol-6-ylamino)-nicotinamide; N-[4-(tert-butyl)-3-(3-piperidylpropyl)phenyl][2-(lH- indazol-6-ylamino)(3-pyridyl)]carboxamide; N-[5-(tert-butyl)isoxazol-3-yl][2-(lH-indazol-6- ylamino)(3-pyridyl)]carboxamide; and N-[4-(tert-butyl)phenyl][2-(lH-indazol-6-ylamino)(3- pyridyl)]carboxamide, and kinase inhibitors disclosed in U.S. Patent Nos. 6,258,812; 6,235,764; 6,630,500; 6,515,004; 6,713,485; 5,521,184; 5,770,599; 5,747,498; 5,990,141; U.S. Publication No. US20030105091; and Patent Cooperation Treaty publication nos. WOO 1/37820; WOO 1/32651; WO02/68406; WO02/66470; WO02/55501; WO04/05279; WO04/07481; WO04/07458; WO04/09784; WO02/59110; WO99/45009; WO98/35958; WO00/59509; WO99/61422; WO00/12089; and WO00/02871, each of which publications are hereby incorporated by reference for any purpose.

Combination therapy with growth factors can include cytokines, lymphokines, growth factors, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin. Other compositions can include known angiopoietins, for example Ang-1, -2, -4, -Y, and/or the human Ang-like polypeptide, and/or vascular endothelial growth factor (VEGF). Growth factors include angiogenin, bone morphogenic protein- 1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein- 10, bone morphogenic protein-11, bone morphogenic protein- 12, bone morphogenic protein- 13, bone morphogenic protein- 14, bone morphogenic protein- 15, bone morphogenic protein receptor-IA, bone morphogenic protein receptor IB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor, cytokine-induced neutrophil chemotactic factor- 1, cytokine- induced neutrophil, chemotactic factor-2, cytokine-induced neutrophil chemotactic factor-2, endothelial cell growth factor, endothelin-1, epidermal growth factor, epithelial-derived neutrophil attractant, fibroblast growth factor-4, fibroblast growth factor-5, fibroblast growth factor-6, fibroblast growth factor-7, fibroblast growth factor-8, fibroblast growth factor-8b, fibroblast growth factor-8c, fibroblast growth factor-9, fibroblast growth factor- 10, fibroblast growth factor acidic, fibroblast growth factor basic, glial cell line-derived neutrophic factor receptor- 1, glial cell line-derived neutrophic factor receptor-2, growth related protein, growth related protein-2, growth related protein -2, growth related protein-3, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin- like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin- like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor- 1, nerve growth factor nerve growth factor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growth factor-2, platelet- derived endothelial cell growth factor, platelet derived growth factor, platelet derived growth factor A chain, platelet derived growth factor AA, platelet derived growth factor AB, platelet derived growth factor B chain, platelet derived growth factor BB, platelet derived growth factor receptor- 1, platelet derived growth factor receptor-2, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor- 1, transforming growth factor-2, transforming growth factor-3, transforming growth factor- 1.2, transforming growth factor-4, transforming growth factor-5, latent transforming growth factor- 1, transforming growth factor- 1 binding protein I, transforming growth factor- 1 binding protein II, transforming growth factor- 1 binding protein III, tumor necrosis factor receptor type I (TNF-Rl), tumor necrosis factor receptor type II (TNF -R2), urokinase-type plasminogen activator receptor, vascular endothelial growth factor, and chimeric proteins and biologically or immunologically active fragments thereof.

In an embodiment, the combination therapy comprises administering maspin polypeptide and IFN-γ to a patient in combination with one or more suitable IL-I inhibitor. Inhibitors of IL-I include, but are not limited to, receptor-binding peptide fragments of IL-I, antibodies directed against IL-I or IL-I beta or IL-I receptor type I, and recombinant proteins comprising all or portions of receptors for IL-I or modified variants thereof, including genetically-modified muteins, multimeric forms and sustained-release formulations. Specific antagonists include IL-lra polypeptides, IL-I beta converting enzyme (ICE) inhibitors, antagonistic type I IL-I receptor antibodies, IL-I binding forms of type I IL-I receptor and type II IL-I receptor, antibodies to IL-I, including IL-I alpha and IL-I beta and other IL-I family members, and a therapeutic known as IL-I Trap (Regeneron). IL-lra polypeptides include the forms of IL-lra described in US Patent No. 5,075,222 and modified forms and variants including those described in U.S. 5,922,573, WO 91/17184, WO 92 16221, and WO 96 09323. IL-I beta converting enzyme (ICE) inhibitors include peptidyl and small molecule ICE inhibitors including those described in PCT patent applications WO 91/15577; WO 93/05071; WO 93/09135; WO 93/14777 and WO 93/16710; and European patent application 0 547 699. Non-peptidyl compounds include those described in PCT patent application WO 95/26958, U.S. Patent No. 5,552,400, U.S. Patent No. 6,121,266, and Dolle et ah, J. Med. Chem., 39, pp. 2438-2440 (1996). Additional ICE inhibitors are described in U.S. Pat. Nos. 6,162,790, 6,204,261, 6,136,787, 6,103,711, 6,025,147, 6,008,217, 5,973,111, 5,874,424, 5,847,135, 5,843,904, 5,756,466, 5,656,627, 5,716,929. IL-I binding forms of Type I IL-I receptor and type II IL-I receptor are described in U.S. Patent Nos. 4,968,607, 4,968,607, 5,081,228, Re 35,450, 5,319,071, and 5,350,683. Other suitable IL-I antagonists include, but are not limited to, peptides derived from IL-I that are capable of binding competitively to the IL-I signaling receptor, IL-I R type I. Additional guidance regarding certain IL-I (and other cytokine) antagonists can be found in U.S. Patent No. 6,472,179. Additionally, TNF inhibitors are suitable, and include, but are not limited to, receptor- binding peptide fragments of TNFα, antisense oligonucleotides or ribozymes that inhibit TNFα production, antibodies directed against TNFα, and recombinant proteins comprising all or portions of receptors for TNFα or modified variants thereof, including genetically- modified muteins, multimeric forms and sustained-release formulations. Also suitable are TACE (Tumor Necrosis Factor-α Converting Enzyme) inhibitors, such as TAPI (Immunex Corp.) and GW-3333X (Glaxo Wellcome Inc.). Also suitable are molecules that inhibit the formation of the IgA-αiAT complex, such as the peptides disclosed in EP 0 614 464 B, or antibodies against this complex. Additionally suitable molecules include, but are not limited to, TNFα-inhibiting disaccharides, sulfated derivatives of glucosamine, or other similar carbohydrates described in U.S. Patent No. 6,020,323. Further suitable molecules include peptide TNFα inhibitors disclosed in U.S. Patent Nos. 5,641,751 and 5,519,000, and the D- amino acid-containing peptides described in U.S. Patent No. 5,753,628. In addition, inhibitors of TNFα converting enzyme are also suitable. WO 01/03719 describes further additional agents which can be used in combination in accordance with the invention.

Still further suitable compounds include, but are not limited to, small molecules such as thalidomide or thalidomide analogs, pentoxifylline, or matrix metalloproteinase (MMP) inhibitors or other small molecules. Suitable MMP inhibitors for this purpose include, for example, those described in U.S. Patent Nos. 5,883,131, 5,863,949 and 5,861,510 as well as mercapto alkyl peptidyl compounds as described in U.S. Patent No. 5,872,146. Other small molecules capable of reducing TNFα production, include, for example, the molecules described in U.S. Patent Nos. 5,508,300, 5,596,013, and 5,563,143. Additional suitable small molecules include, but are not limited to, MMP inhibitors as described in U.S. Patent Nos. 5,747,514, and 5,691,382, as well as hydroxamic acid derivatives such as those described in U.S. Patent No. 5,821,262. Further suitable molecules include, for example, small molecules that inhibit phosphodiesterase IV and TNFα production, such as substituted oxime derivatives (WO 96/00215), quinoline sulfonamides (U.S. Patent No. 5,834,485), aryl furan derivatives (WO 99/18095) and heterobicyclic derivatives (WO 96/01825; GB 2 291 422 A). Also useful are thiazole derivatives that suppress TNFα and IFNγ (WO 99/15524), as well as xanthine derivatives that suppress TNFα and other proinflammatory cytokines (see, for example, U.S. Patent Nos. 5,118,500, 5,096,906 and 5,196,430). Additional small molecules useful for treating the hereindescribed conditions include those disclosed in U.S. Patent No. 5,547,979. Further examples of drugs and drug types which can be administered by combination therapy include, but are not limited to, antivirals, antibiotics, analgesics (e.g., acetaminophen, codeine, propoxyphene napsylate, oxycodone hydrochloride, hydrocodone bitartrate, tramadol), corticosteroids, antagonists of inflammatory cytokines, Disease-Modifying Anti- Rheumatic Drugs (DMARDs), Non-Steroidal Anti-Inflammatory drugs (NSAIDs), and Slow- Acting Anti-Rheumatic Drugs (SAARDs).

Exemplary Disease-Modifying Anti-Rheumatic Drugs (DMARDs) include, but are not limited to: Rheumatrex™ (methotrexate); Enbrel® (etanercept); Remicade® (infliximab); Humira (adalimumab); Segard® (afelimomab); Arava (leflunomide); Kineret (anakinra); Arava™ (leflunomide); D-penicillamine; Myochrysine; Plaquenil; Ridaura™ (auranofm); Solganal; lenercept (Hoffman-La Roche); CDP870 (Celltech); CDP571 (Celltech), as well as the antibodies described in EP 0 516 785 Bl, U.S. Patent No. 5,656,272, EP 0 492 448 Al; onercept (Serono; CAS reg. no. 199685-57-9); MRA (Chugai); Imuran™ (azathioprine); NFKB inhibitors; Cytoxan™ (cyclophosphamide); cyclosporine; hydroxychloroquine sulfate; minocycline; sulfasalazine; and gold compounds such as oral gold, gold sodium thiomalate and aurothioglucose.

Further suitable molecules include, for example, soluble TNFRs derived from the extracellular regions of TNFα receptor molecules other than the p55 and p75 TNFRs, such as for example the TNFR described in WO 99/04001, including TNFR-Ig's derived from this TNFR. Additional suitable TNFα inhibitors are suitable for use as described herein. These include the use not only of an antibody against TNFα or TNFR as described herein, but also a TNFα-derived peptide that can act as a competitive inhibitor of TNFα (such as those described in U.S. Patent No. 5,795,859 or U.S. Patent No. 6,107,273), TNFR-IgG fusion proteins, such as one containing the extracellular portion of the p55 TNFα receptor, a soluble TNFR other than an IgG fusion protein, or other molecules that reduce endogenous TNFα levels, such as inhibitors of the TNFα converting enzyme (see e.g., U.S. 5,594,106), or small molecules or TNFα inhibitors, a number of which are described herein.

With respect to antibodies to TNF, although dose will optimally be determined by an experienced healthcare provider in accordance with the specific needs of the patient in mind, one exemplary preferred dose range for an antibody against TNFα is 0.1 to 20 mg/kg, and more preferably is 1-10 mg/kg. Another preferred dose range for anti-TNFα antibody is 0.75 to 7.5 mg/kg of body weight. The present invention can also utilize a maspin polypeptide and IFN -γ and any of one or more Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). NSAIDs owe their antiinflammatory action, at least in part, to the inhibition of prostaglandin synthesis. Goodman and Gilman, The Pharmacological Basis of Therapeutics, MacMillan 7th Edition (1985). NSAIDs can be characterized into nine groups: (1) salicylic acid derivatives; (2) propionic acid derivatives; (3) acetic acid derivatives; (4) fenamic acid derivatives; (5) carboxylic acid derivatives; (6) butyric acid derivatives; (7) oxicams; (8) pyrazoles and (9) pyrazolones. Examples of NSAIDs include, but are not limited to: Anaprox™, Anaprox DS™ (naproxen sodium); Ansaid™ (flurbiprofen); Arthrotec™ (diclofenac sodium + misoprostil); Cataflam™/Voltaren™ (diclofenac potassium); Clinoril™ (sulindac); Daypro™ (oxaprozin); Disalcid™ (salsalate); Dolobid™ (diflunisal); EC Naprosyn™ (naproxen sodium); Feldene™ (piroxicam); Indocin , Indocin SR (indomethacin); Lodine , Lodine XL (etodolac);

Motrin (ibuprofen); Naprelan (naproxen); Naprosyn (naproxen); Orudis ,

(ketoprofen); Oruvail (ketoprofen); Relafen (nabumetone); Tolectin , (tolmetin sodium); Trilisate™ (choline magnesium trisalicylate); Cox-1 inhibitors; Cox-2 Inhibitors such as Vioxx™ (rofecoxib); Arcoxia^tm (etoricoxib), Celebrex™ (celecoxib); Mobic™ (meloxicam); Bextra™ (valdecoxib), Dynastat™ paracoxib sodium; Prexige™ (lumiracoxib), and nambumetone. Additional suitable NSAIDs, include, but are not limited to, the following: ε-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, anitrazafen, antrafenine, bendazac, bendazac lysinate, benzydamine, beprozin, broperamole, bucolome, bufezolac, ciproquazone, cloximate, dazidamine, deboxamet, detomidine, difenpiramide, difenpyramide, difisalamine, ditazol, emorfazone, fanetizole mesylate, fenflumizole, floctafenine, flumizole, flunixin, fluproquazone, fopirtoline, fosfosal, guaimesal, guaiazolene, isonixirn, lefetamine HCl, leflunomide, lofemizole, lotifazole, lysin clonixinate, meseclazone, nabumetone, nictindole, nimesulide, orgotein, orpanoxin, oxaceprolm, oxapadol, paranyline, perisoxal, perisoxal citrate, pifoxime, piproxen, pirazolac, pirfenidone, proquazone, proxazole, thielavin B, tiflamizole, timegadine, tolectin, tolpadol, tryptamid and those designated by company code number such as 480156S, AA861, AD 1590, AFP802, AFP860, AI77B, AP504, AU8001, BPPC, BW540C, CHINOIN 127, CNlOO, EB382, EL508, F1044, FK-506, GV3658, ITFl 82, KCNTEI6090, KME4, LA2851, MR714, MR897, MY309, ONO3144, PR823, PV102, PV108, R830, RS2131, SCR152, SH440, SIR133, SPAS510, SQ27239, ST281, SY6001, TA60, TAI-901 (4-benzoyl-l-indancarboxylic acid), TVX2706, U60257, UR2301 and WY41770. Structurally related NSAIDs having similar analgesic and anti-inflammatory properties to the NSAIDs are also encompassed by this group.

Suitable SAARDs or DMARDS include, but are not limited to: allocupreide sodium, auranofm, aurothioglucose, aurothioglycanide, azathioprine, brequinar sodium, bucillamine, calcium 3-aurothio-2-propanol-l -sulfonate, chlorambucil, chloroquine, clobuzarit, cuproxoline, cyclophosphamide, cyclosporin, dapsone, 15-deoxyspergualin, diacerein, glucosamine, gold salts (e.g., cycloquine gold salt, gold sodium thiomalate, gold sodium thiosulfate), hydroxychloroquine, hydroxyurea, kebuzone, levamisole, lobenzarit, melittin, 6- mercaptopurine, methotrexate, mizoribine, mycophenolate mofetil, myoral, nitrogen mustard, D-penicillamine, pyridinol imidazoles such as SKNF86002 and SB203580, rapamycin, thiols, thymopoietin and vincristine. Structurally related SAARDs or DMARDs having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

Inhibitors of kinases in signaling cascades are also suitable agents for combination with the maspin polypeptide and IFN-γ agents of the invention. These include, but are not limited to, agents that are capable of inhibiting P-38 (a.k.a., "RK" or "SAPK-2", Lee et al., Nature, 372:739 (1994). P-38 is described as a serine/threonine kinase (see Han et al., Biochimica Biophysica Acta, 1265:224-227 (1995). Inhibitors of P-38 have been shown to intervene between the extracellular stimulus and the secretion of IL-I and TNFα from the cell involves blocking signal transduction through inhibition of a kinase which lies on the signal pathway.

Additionally suitable are MK2 inhibitors, and tpl-2 inhibitors. Additionally, T-cell inhibitors are also suitable, including, for example, ctla-4, CsA, Fk-506, OX40, OX40R-Fc, OX40 antibody, OX40 ligand, OX40 ligand antibody, lck, and ZAP70. Also suitable are retinoids, including oral retinoids, as well as antagonists of TGF-/?. Further suitable agents for combination treatment with the maspin polypeptide and

IFN-γ agents include, for example, any of one or more salicylic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. Such salicylic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof comprise: acetaminosalol, aloxiprin, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, choline magnesium trisalicylate diflusinal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide O-acetic acid, salsalate and sulfasalazine. Structurally related salicylic acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. Additionally suitable agents include, for example propionic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The propionic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof comprise: alminoprofen, benoxaprofen, bucloxic acid, carprofen, dexindoprofen, fenoprofen, flunoxaprofen, fluprofen, flurbiprofen, furcloprofen, ibuprofen, ibuprofen aluminum, ibuproxam, indoprofen, isoprofen, ketoprofen, loxoprofen, miroprofen, naproxen, oxaprozin, piketoprofen, pimeprofen, pirprofen, pranoprofen, protizinic acid, pyridoxiprofen, suprofen, tiaprofenic acid and tioxaprofen. Structurally related propionic acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. Also suitable for use are acetic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The acetic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof comprise: acemetacin, alclofenac, amfenac, bufexamac, cinmetacin, clopirac, delmetacin, diclofenac sodium, etodolac, felbinac, fenclofenac, fenclorac, fenclozic acid, fentiazac, furofenac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, oxametacin, oxpinac, pimetacin, proglumetacin, sulindac, talmetacin, tiaramide, tiopinac, tolmetin, zidometacin and zomepirac. Structurally related acetic acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. Further suitable for use as described herein are fenamic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The fenamic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof comprise: enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, meclofenamate sodium, medofenamic acid, mefanamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid and ufenamate. Structurally related fenamic acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

Also suitable are carboxylic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof which can be used comprise: clidanac, diflunisal, flufenisal, inoridine, ketorolac and tinoridine. Structurally related carboxylic acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. Additionally suitable are butyric acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The butyric acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof comprise: bumadizon, butibufen, fenbufen and xenbucin. Structurally related butyric acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. Oxicams, prodrug esters or pharmaceutically acceptable salts thereof are also suitable. Oxicams, prodrug esters and pharmaceutically acceptable salts thereof comprise: droxicam, enolicam, isoxicam, piroxicam, sudoxicam, tenoxicam and 4-hydroxyl-l,2-benzothiazine 1,1 -dioxide 4-(N- phenyl)-carboxamide. Structurally related oxicams having similar analgesic and antiinflammatory properties are also intended to be encompassed by this group. Pyrazoles, prodrug esters or pharmaceutically acceptable salts thereof are also suitable. The pyrazoles, prodrug esters and pharmaceutically acceptable salts thereof which may be used comprise: difenamizole and epirizole. Structurally related pyrazoles having similar analgesic and antiinflammatory properties are also intended to be encompassed by this group. Furthermore, pyrazolones, prodrug esters or pharmaceutically acceptable salts thereof are suitable. The pyrazolones, prodrug esters and pharmaceutically acceptable salts thereof which may be used comprise: apazone, azapropazone, benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propylphenazone, ramifenazone, suxibuzone and thiazolinobutazone. Structurally related pyrazalones having similar analgesic and antiinflammatory properties are also intended to be encompassed by this group.

Also suitable are prodrug esters or pharmaceutically acceptable salts thereof for the treatment of TNF-mediated diseases. Corticosteroids, prodrug esters and pharmaceutically acceptable salts thereof include hydrocortisone and compounds which are derived from hydrocortisone, such as 21-acetoxy-pregnenolone, alclomerasone, algestone, amcinonide, beclomethasone, beta-methasone, betamethasone valerate, budesonide, chloroprednisone, clobetasol, clobetasol propionate, clobetasone, clobetasone butyrate, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacon, desonide, desoximerasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flumethasone pivalate, flunisolide, flucinolone acetonide, fluocinonide, fluorocinolone acetonide, fluocortin butyl, fluocortolone, fluorocortolone hexanoate, diflucortolone valerate, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandenolide, formocortal, halcinonide, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, hydrocortisone acetate, hydro-cortisone butyrate, hydrocortisone phosphate, hydrocortisone 21 -sodium succinate, hydrocortisone tebutate, mazipredone, medrysone, meprednisone, methylprednicolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 21-diedryaminoacetate, prednisolone sodium phosphate, prednisolone sodium succinate, prednisolone sodium 21-m-sulfobenzoate, prednisolone sodium 21-stearoglycolate, prednisolone tebutate, prednisolone 21 -trimethylacetate, prednisone, prednival, prednylidene, prednylidene 21-diethylaminoacetate, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide and triamcinolone hexacetonide. Structurally related corticosteroids having similar analgesic and antiinflammatory properties are also intended to be encompassed by this group.

Antimicrobials (and prodrug esters or pharmaceutically acceptable salts thereof) are also suitable for combination use as described herein. Suitable antimicrobials include, for example, ampicillin, amoxycillin, aureomicin, bacitracin, ceftazidime, ceftriaxone, cefotaxime, cephachlor, cephalexin, cephradine, ciprofloxacin, clavulanic acid, cloxacillin, dicloxacillan, erythromycin, flucloxacillan, gentamicin, gramicidin, methicillan, neomycin, oxacillan, penicillin and vancomycin. Structurally related antimicrobials having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

Additional suitable compounds include, but are not limited to: BN 50730; tenidap; E 5531; tiapafant PCA 4248; nimesulide; panavir; rolipram; RP 73401; peptide T; MDL 201.449A; (lR,3S)-Cis-l-[9-(2,6-diaminopurinyl)]-3-hydroxy-4-cyclopentene hydrochloride; (lR,3R)-trans-l-[9-(2,6-diamino)purine]-3-acetoxycyclopentane; (1R,3R)- trans-l-[9-adenyl)-3-azidocyclopentane hydrochloride and (lR,3R)-trans-l-[6-hydroxy-purin- 9-yl)-3-azidocyclopentane. It has been found that IL-4 can induce an inflammatory effect in some instances, such as in asthma, in which over-expression of IL-4 in the lungs causes epithelial cell hypertrophy and an accumulation of lymphocytes, eosinophils, and neutrophils. This response is representative of the main features of the proinflammatory response induced by other Th2 cytokines. As noted above, therefore, inhibitors of IL-4 are also useful in accordance with the invention. Additionally, it will be appreciated that certain immunosuppressant drugs can also be used in the treatment of arthritis, including, but not limited to, iNOS inhibitors, and 5- lipoxygenase inhibitors.

Ginger has been shown to have certain anti-inflammatory properties, and is therefore suitable for use as an anti-inflammatory agent in accordance with the invention, as is chondroitin.

In certain embodiments, maspin may be administered prior to, concurrent with, and subsequent to treatment with a cancer therapy agent. Exemplary cancer type are those that express maspin in the original parental cell, and may include, but are not limited to, breast cancer, colorectal cancer, gastric carcinoma, glioma, head and neck squamous cell carcinoma, hereditary and sporadic papillary renal carcinoma, leukemia, lymphoma, Li-Fraumeni syndrome, malignant pleural mesothelioma, melanoma, non-small cell lung carcinoma, osteosarcoma, prostate cancer, small cell lung cancer, synovial sarcoma, thyroid carcinoma, and transitional cell carcinoma of urinary bladder.

In certain embodiments, maspin and IFN-γ may be used with at least one additional therapeutic agent for the treatment of cancer. In certain embodiments, maspin and IFN-γ are used in conjunction with a therapeutically effective amount of an additional therapeutic agent. Exemplary therapeutic agents that may be administered with the maspin/IFN-γ combination include, but are not limited to, a member of the geldanamycin family of anisamycin antibiotics; a Pro-HGF; NK2; a c-Met peptide inhibitor; an antagonist of Grb2 Src homology 2; a Gabl modulator; dominant-negative Src; a von-Hippel-Landau inhibitor, including, but not limited to, wortmannin; P13 kinase inhibitors, other anti-receptor therapies, anti EGFR, a COX-2 inhibitor, Celebrex™, Vioxx™; a vascular endothelial growth factor (VEGF), a VEGF modulator, a fibroblast growth factor (FGF), an FGF modulator, an epidermal growth factor (EGF); an EGF modulator; a keratinocyte growth factor (KGF), a KGF-related molecule, a KGF modulator; a matrix metalloproteinase (MMP) modulator.

The following examples are intended for illustration purposes only, and should not be construed as limiting the scope of the invention in any way.

EXAMPLES

General Techniques Cell Culture: Normal human mammary epithelial cells N1330 HMEC and HMEpC were maintained in defined mammary epithelial cell medium (Biowittaker, Inc. Wakersville, MD, and Cell Applications Inc, San Diego, CA). MCF-7, MDA-MB-231 breast cancer cell lines and their Maspin gene transfected counterparts were maintained in RPMI containing 10% fetal calf serum (FCS). Cultures were determined to be mycoplasma free using the GeneProbe rapid detection system.

Subcellular Fractionation: Two different methods were employed to manufacture cell fractions. In one methods, cytosolic fractions were prepared according to the description in Khalkhali-Ellis & Hendrix, Amer.J. Pathol, 162: 1411-1418 (2003). Briefly, control and treated cells were lysed in buffer A (1OmM HEPES buffer pH 7.9, containing 1OmM NaCl, ImM DTT, 10% glycerol, 15mM MgCl₂, 0.2mM EDTA, 0.1%NP40, protease inhibitor cocktail (Roche Applied Bioscience, Indianapolis, IN) and ImM PMSF, subjected to 3 freeze- thaw cycles and centrifuged (450Ox g, 10 min) to yield a post-nuclear cytosolic fraction. This fraction was used for general screening and in IFN-γ experiments, as is indicated in the Figure legend(s).

The procedure described Krajewski et al, Cancer Res. 53: 4701-14 (1993) was used to generate ER, lysosomal, and plasma membrane fractions. These fractions were used to determine the subcellular distribution of maspin and the region of its association with cathepsin D. In brief, 70-80% confluent cultures were mechanically detached with a cell scraper and lysed in MES buffer (17mM morpholinoethane sulfonic acid, pH 7.4 containing 25OmM sucrose and 2.5mM EDTA and protease inhibitor cocktail [Roche Applied Bioscience, Indianapolis, IN], 1OmM NaF, 1OmM sodium orthovanadate, ImM PMSF) and subjected to three cycles of freeze thaw. The suspension was centrifuged at 50Ox g for 10 min to pellet the nuclear fraction. The supernatant was centrifuged at 10,000x g for 15 min to yield a fraction containing lysosomes (including mitochondria and peroxisomes). Further centrifugation of the supernatant at 150,00Ox g pelleted the ER, plasma membrane and microsomes, while the remaining supernatant contained cytosolic protein. The protein content of all fractions was determined using BCA reagent. The light and heavy fractions were tested for the specific enzyme markers (α-glucosidase II for light and β-hexoseaminidase for heavy membrane fraction) to ensure absence of any cross contamination. Equal amounts of cellular protein from various fractions were subjected to SDS-PAGE and Western blot analysis using specific monoclonal antibody to maspin and cathepsin D (BD Bioscience Pharmingen, San Diego, CA). The blots were stripped and re-probed with antibodies to specific subcellular fractions for loading control. The reaction products were visualized using the ECL chemiluminecent kit (Amersham).

Immunohistochemical Analysis: Col matrices were prepared in Lab-Tek Chamber Slide System (Nalge Nunc International, Naperville, IL) with or without the addition of r- Maspin. MDA-MB-231 breast cancer cells (or HMECs) were plated on these 3D matrices for 3-5 days. The matrix after the removal of MDA-MB-231 cells was fixed in ice-cold methanol, blocked and treated with antibodies to CD.

Maspin Protein Purification from CM and Cell Lysate: HMEC total cell lysate and/or 10x concentrated CM were treated with polyclonal antibody to Maspin, the antibody- antigen complex was precipitated with protein A Sepharose-coupled beads and washed repeatedly with PBS. The bound Maspin was eluted with low-pH glycine-HCl, and analyzed by SDS-PAGE prior to its use in matrix studies.

Quantitative PCR (Q-PCR): Total RNA was isolated from cells using Trizol RNA isolation reagent (Life Technologies, Inc.) according to manufacturer's specifications. Reverse transcription of the total RNA was performed in a Robocycler gradient 96 thermocycler (Stratagene, La Jolla, CA) using the Advantage PCR kit according to the manufacturer's instructions (Clontech, Palo Alto, CA). Q-PCR was performed on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) using TaqMan® gene expression human (Hs00184728_ml) maspin-specific primer/probe sets (Applied Biosystems). Briefly, 5 μl cDNA, 1.25 μl 2OX Gene Expression Assay Mix, and 12.5 μl 2X TaqMan® Universal PCR Master Mix in a total of 25 μl were amplified with the following thermocycler protocol: 1 cycle at 5O⁰C for 2 min; 1 cycle at 95⁰C for 10 min; and 40 cycles at 95⁰C for 15 seconds/60°C for 1 min. All data were analyzed with the Sequence Detection Software (version 1.2.3, Applied Biosystems). The expression of each target gene was normalized to an endogenous control gene (GAPDH: 4333764F; β-actin: 4333762F). Each experiment was repeated twice and each sample was performed in triplicate.

EXAMPLE 1: In vitro identification ofmaspin-cathepsin D interaction:

A yeast-two-hybrid assay was used to identify potential interaction between maspin and cathepsin D. All the materials were purchased from Clontech (Palo Alto, CA). DNA encoding full-length maspin (GenBank U04313, SEQ ID NO:1) was cloned into the fusion plasmid pGBKT7 according to the manufacturer's protocol and utilized as bait to screen a human mammary epithelium MATCHMAKER™ cDNA library which was expressed in the prey plasmid pGADT7. Bait and prey plasmids were transformed into the yeast strain AH 109 in the GAL4-based MATCHMAKER™ System 3 (Bailey, et al, J. Biol. Chem., 280: 34210- 17 (2005)). Positive clones were selected by their color, and ability to grow on SD-Ade/-His/- Leu/-Trp/X-α-Gal selection plates. Plasmids from positive clones were isolated using the Wizard Plus SV Miniprep following digestion with Lyticase. Prey plasmids were sequenced using primers for pGADT7 supplied by Clontech. The positive clones identified cathepsin D (Fig. 7A-7C, GenBank Accession NM 001909 (SEQ ID NO:3)) and few (four) other proteins as putative binding partners for maspin. The data identifies a sequence toward the C-terminus of cathepsin D as the maspin-binding region (at about residues 160-229, VDQNIFSFYL SRDPDAQPGG ELMLGGTDSK YYKGSLSYLN VTRKAYWQVH LDQVEVASGL TLCKEGCEAI (SEQ ID NO:4)). This region contains Asnl99 (underlined), which has been identified as one of the sites for N-linked glycosylation (Faust, et al., Proc. Nat. Acad. Sci., 82: 4910-4914 (1985)).

Maspin-cathepsin D interaction identified by the yeast-two-hybrid system was verified by co-immunoprecipitation on cellular fractions generated from from normal mammary epithelial cells. The fractions were separated into a cytosolic and two membrane fractions (the heavy fraction including mitochondria, lysosome, and peroxisomes; the light fraction including ER, plasma membrane and microsomes) membrane and cytosolic fractions (Krajewski, et al., Cancer Res. 53, 4701-4714 (1993)). Although the majority (>60%) of maspin was present in the cytosolic fraction, a considerable amount (>20%) was also detected in the light membrane. The lysosomal (heavy) fraction also contained >10% of total maspin (Fig. 2A). The Western Blot results showed maspin interacted mostly with the full length pro-cathepsin D both in the light and heavy fractions (Fig. 2B). In the cytosolic fraction, which contained the majority of cellular maspin and both the I-cathepsin D and M-cathepsin D, little to no interaction was identified using either anti-cathepsin D and/or anti-maspin (Fig. 2B).

EXAMPLE 2: Cathepsin D profile in breast cancer cells and its regulation by maspin:

Cellular fractionation of non-invasive breast cancer cells (MCF-7) and highly invasive, metastatic breast cancer cells (MDA-MB231) showed that membrane-associated cathepsin D appears often as a doublet of 52-53 kDa (Fig. 3A); in the heavy membrane fraction of both cell lines the I-cathepsin D ran as multiple bands of about 47-, 43-, and 42 kDa. The apparent molecular mass of M-cathepsin D was the same for all the cell lines tested (Fig. 3B). The cytosolic fractions also contained I-cathepsin D and M-cathepsin D (Fig. 3C). Upon trans fection of the MDA-MB-231 breast cancer cell line with a vector encoding maspin, higher amounts of the I-cathepsin D are found in the heavy membrane and cytosol compartments (see, Figs. 3B-C). For protocols and vector construction, see, e.g., Odero- Marah, V.A., et al., Cancer Biol. Ther., 2 :62-67 (2003), which is incorporated by reference in its entirety. In these transfected cells, maspin partitioned in the light and heavy membrane and cytosolic fractions similarly to what was observed in normal mammary epithelial cells (Figs. 3A-C).

SDS-PAGE showed that secreted cathepsin D from MCF-7 cells was only detectable after concentrating the CM (conditioned medium), appearing as a doublet at about 52 & 54 kDa. The MDA-MB-231 cells secreted readily detectable levels of the proenzyme with the same molecular mass as MCF-7 (Fig. 3D). Maspin transfection of MDA-MB231 cells resulted in decreased secretion of procathepsin D with no changes in its molecular mass (Fig. 3D)

To examine if observed changes in the secretion and sorting of cathepsin D in maspin- transfected MDA-MB-231 could be mimicked by exogenous addition of r-Maspin, cultures of MDA-MB231 (maspin deficient) were treated with r-Maspin (5 μg/ml) for 24 hrs. Exogenous addition of r-Maspin over the time period tested had little to no effect on the processing or secretion of cathepsin D. Examination of subcellular fractions and CM indicated the presence of the majority of exogenously added r-Maspin in the CM with a minor fraction detected in the cytosol. To assess the effect of antibody administration, maspin-expressing mammary epithelial cells were cultured for 24-48hrs in the presence of anti -maspin (maspin antibody, which has been shown to be a blocking, inhibitory antibody). Control cultures received equal amounts of a non-specific IgG isotype control. The conditioned medium and post-nuclear cytosolic fractions indicated an increase in the higher molecular mass I-cathepsin D associated with a decrease in secreted maspin and a comparable increase in secreted cathepsin D (Figs. 4A-B).

EXAMPLE 3: Response of normal mammary epithelial and breast cancer cells to administration ofinterferon-gamma (INF-y)

Administration of IFN-γ to mammary and prostate epithelial cells can result in cell cycle arrest, apoptosis, and/or differentiation. Maspin and cathepsin D may play a role in regulating these IFN-γ-induced epithelial cell responses.

Normal human mammary epithelial cells HMEpC (Cell Applications Inc, San Diego, CA) and HMEC 1330 (Biowhittaker, Inc. Wakersville, MD), were maintained in defined mammary epithelial cell basal medium provided by the cell line providers. MCF-7 and MDA-MB-231 breast cancer cells, as well as a stable GFP-maspin transfectant MDA-MB- 231 (which was generated as described in Odero, et al. , Cancer Biol, and Therapy, 2 :404-05 (2003)) were maintained in RPMI containing 10% fetal calf serum (FCS) and gentamicin (50mg/L). Cultures were determined to be mycoplasma free using the GeneProbe rapid detection system. INF-γ (from in house R&D Dept.) treatment (from 0.25 - 0.5 ng/mL, for 0- 96 hr.) of both HMEpC and 1330N1 cells induced a concentration dependent reduction in proliferation and changes in cell morphology (epithelial to more mesenchymal appearance, Figs. 5A-B). In normal primary mammary epithelial cells, IFN-γ treatment reduced the mRNA levels of cathepsin D by -50% at concentration of 5 ng/mL (Fig. 5C). At the protein level, an initial complete processing of the I-cathepsin D to M-cathepsin D was followed by a reduction of M-cathepsin D at higher concentrations (Fig. 6A) and a concentration dependent decrease in secreted cathepsin D (Fig. 5E). Contrary to the response of cathepsin D, there was about a two-fold increase in maspin mRNA in response to administration of IFN-γ at concentrations >1 ng/ml, and a concentration dependent increase in secreted maspin concentrations, which inversely correlated with changes in secreted cathepsin D (Figs. 5D-E).

Breast cancer cell lines tested were refractory to INF -γ with respect to proliferation and cathepsin D expression (either as the procathepsin D, the M-cathepsin D or the secreted cathepsin D, Figs. 6B-C). Transfection of these refractory cells with maspin sensitized the cells to INF -γ in a concentration dependent manner, increasing the 43 kDa I-cathepsin D at low concentrations, while decreasing it at higher concentrations (Fig. 6D). Both MCF-7 and MDA-MB-231 breast cancer cell lines are devoid of maspin, and IFN-γ treatment did not alter this phenotype. There was no detectable change in maspin secretion of the GFP-maspin transfected MDA-MB-231 cells.

EXAMPLE 4: Maspin secretion by normal mammary epithelial cells.

For media analysis, conditioned media (CM) were collected from -70% confluent N1330 HMEC (and/or HMEpC, hereafter referred to collectively as HMEC) cells 48h in culture, centrifuged to remove cell debris and used for Maspin detection as described below. For deposited matrix studies, -70% confluent cultures of HMECs were kept in culture for two weeks without passaging to allow matrix accumulation. The cells were then removed by treating the monolayer with 2OmM NH₄OH solution followed by several washes with PBS. Complete removal of the cells was confirmed by microscopic observation and the remaining matrix was scraped, taken up in electrophoresis sample buffer and boiled for 5min. CM (20- 30μl) and solublized deposited matrix were subjected to SDS-PAGE under reducing conditions, and analyzed by Western blot.

The data indicate that HMECs maintained over one to two weeks without passaging, deposit a significant amount of matrix (deposited matrix) comprised of laminin, fibronectin and actin (data not shown, or we need "supplemental Fig.1" from manuscript). As shown in Figure 9A, maspin is secreted by N1330 HMEC (and HMEpC), and is incorporated into the deposited matrix. Both normal mammary epithelial cell lines gave comparable results. In addition, when HMECs were plated on Col I matrices, the deposition of Maspin in the HMEC-conditioned Col I matrix was detected by Western blot and immunohistochemical approaches (Figure 9A &B). EXAMPLE 5: Maspin inhibits CD-mediated Col matrix degradation.

A solution of 2.5 mg/ml of rat tail collagen I (BD BioSience, Bedford, MA) and 1 mg/ml human Col IV (Sigma, St. Louis, MO) in acetic acid was used to prepare 3D Col matrices of 1-2 mm thickness in wells of 96 well culture dish. The collagen solution is neutraled with 20 mM HEPES buffer pH 7.4 and 0.1 M NaOH solution followed by incubation at 37⁰C. The gels were washed with PBS, and HMECs were plated on these Col matrices at 10⁴ cells/well. After 4-5 days in culture, the cells were removed by treatment with 2OmM NH₄OH solution followed by several washes with PBS. These Col matrices (referred to as HMEC-conditioned Col matrix) were either tested for maspin deposition, or were used to examine the response of breast cancer cells (MCF-7 and MDA-MB-231 cell lines) to signals deposited by HMECs. Breast cancer cells were plated at 10⁴ cells/ HMEC-conditioned Col matrices in 96 well culture dishes in RPMI media containing Mito+ serum supplement. After 5-7 days cancer cells were also removed from the Col matrices by treatment with 2OmM NH₄OH solution. The Col matrices were washed with PBS, boiled in electrophoresis sample buffer, (5 min), centrifuged at 12,000rpm (10 min), and subjected to SDS-PAGE and Western blot analysis using antibodies to Maspin, cathepsin D, β-actin, laminin, and fibronectin. Col matrices without cells, with HMEC, MDA-MB-231 (or MCF-7) cells alone served as controls.

In some experiments, recombinant maspin (r-maspin) or mutant forms of maspin, including lysine 346 mutated to histidine (346K-H), tyrosine 357 mutated to phenylalanine (357Y-F), and arginine 341 to alanine (341R-A) were incorporated into Col matrix at concentrations ranging from 100-500ng/well. MDA-MB-231 breast cancer cells were then plated on these gels at 50xl0³/well and analyzed as described above.

Western blot analysis of the CM and the Col matrices indicated the presence of pro- CD (52-54kDa) in the CM of all the wells tested (Fig. 9C). Interestingly, the majority of cathepsin-D associated with the Col matrix was in the intermediary (ICD, 48 and 43kDa) and mature active (MCD, 34kDa) forms (Fig 9D). Although the pro-CD levels in the CM were minimally divergent, both ICD and MCD levels in the HMEC-conditioned matrices were lower than that of control. As anticipated, Maspin was present in the HMEC-conditioned matrices, and it was also detected in the CM of the wells which contained these matrices, presumably released by the proteolytic degradation of the matrix (Fig.9C).

To confirm that changes in CD levels of MDA-MB-231 cells were solely due to Maspin and not other secreted products of HMEC, MDA-MB-231 cells were cultured on Col matrix containing r-Maspin at concentrations ranging from 100-500ng. A concentration dependent decrease in Col matrix associated CD was observed (Fig. 10A), substitution of Maspin with BSA in Col matrix had minimal effect on CD associated Col matrix (Fig. 10A). These observations were further supported by our immunohistochemical analysis of the control and Maspin-incorporated Col matrices following exposure to MDA-MB-231 cells, which revealed reduced deposition of CD in r-Maspin incorporated matrices compared to control (Fig. 10B).

Fluorescence experiments were employed to confirm that maspin can inhibit matrix degradation. The matrix solution was supplemented with fluorescein conjugated type I collagen (DQ collagen™, Molecular Probe, Eugene, Oregon) prior to gelation process and incorporating DQ collagen™ into the matrix. By incorporating this marker, collagen degradation by proteases release the quenched fragment which can be quantified by ELISA reader FluoStar Optima (BMG Labteck GmbH, Durham, NC) using 485μm emission and 530 μm excitation wavelengths. To confirm the involvement of CD in the degradation process, the experiments were performed in the presence and absence of the specific aspartyl protease inhibitor, pepstatin. Breast cancer cells were first incubated in the media containing pepstatin (200ng/ml) for 30min, then plated on the Col matrices in the presence of pepstatin for required time intervals. Quantifying the released fluorescent fragments by an ELISA reader indicated that maspin reduces the amount of fluororescent fragments in a concentration dependent manner (Fig. 10D). This data is consistent with the idea that maspin can inhibit extracellular matrix degradation induced by cathepsin-D and/or cancer cells.

EXAMPLE 6: Maspin-dependent modulation of cathepsin-D mRNA levels.

The effect of HMEC-conditioned matrix (or Maspin-incorporated matrix) on CD gene expression of breast cancer cell lines was examined by culturing MDA-MB-231 (and/or MCF-7) cells on pre-conditioned matrix for 0-4 days. Total RNA was isolated using Trizol RNA reagent (Life Technologies, Inc.) and reverse transcribed in a Robocycler gradient 96 thermocycler (Stratagene, La Jolla, CA) using the Advantage PCR kit (Clontech, Palo Alto, CA). Semiquantitative PCR was performed using CD-specific primers (Integrated DNA Technologies, Coralville, IA) (forward 5'- ATCGAT ACAT GC AGCCCTCC AGCCTTCTGC CG-3' (SEQ ID N0:XX) and reverse 5'- CTCGAGCCTA GAGGCGGGCA GCCTCGGCGA A-3' (SEQ ID NO:XX+1). The products were separated on a 1% agarose gel with GAPDH as loading control. RT-PCR analysis of MDA-MB-231 mRNA from cells cultured on control and maspin-incorporated matrices indicated the latter exerted a suppressive effect on CD gene expression (Fig.10C). When compared to the HMEC- deposited maspin in the Col matrix, the data indicates that a much higher concentration of r- Maspin was required to exert a similar inhibitory effect. However in matrix degradation experiments involving maspin purified from HMEC CM and recombinant maspin, each form showed similar inhibitory effect on MDA-MB231 cells (data not shown, or supplemental Fig. 10).

EXAMPLE 7: Activity ofmapsin mutants.

In some experiments, recombinant maspin (r-maspin) or its mutant forms (346K-H- maspin; 357Y-F-maspin; (341R-A-maspin) were incorporated into Col matrix at concentrations ranging from 100-500ng/well. MDA-MB-231 breast cancer cells were then plated on these maspin-incorporated gels at 50xl0³/well and analyzed as described above in Example 5. Immunohistochemical analysis of the control (no maspin) and maspin- incorporated Col matrices showed reduced amounts of CD in r-maspin incorporated matrices compared to control (Fig. 10B).

Recombinant maspins comprising mutations in the reactive center loop (34 IR-A- maspin; 346K-H-maspin) did not effectively bind to the Col matrix, and were recovered from the conditioned media (Fig. HB). This indicates that mapsin's reactive center loop could play a key role in its ability to inhibit matrix degradation and/or inhibit cathepsin-D and its activity. Nevertheless, all the mutated forms of maspin, including 357Y-F, resulted in reduced inhibitory effect (Fig. 1 IA).

Claims

ClaimsWe Claim:

1. A method for treating or preventing cancer in a subject comprising administering to the subject an amount of a maspin polypeptide and an amount of a chemotherapeutic agent effective to treat or prevent cancer.

2. A method according to claim 1, where the chemotherapeutic agent is interferon-gamma (IFN-γ).

3. The method of claim 2 wherein the maspin polypeptide and the IFN-γ are administered at the same time.

4. The method of claim 2 wherein the maspin polypeptide and the IFN-γ are administered at different times.

5. A method for treating or preventing cancer in a subject comprising administering to the subject:

(a) a composition comprising an amount effective to allow expression of a maspin polypeptide of a liposome, wherein the liposome comprises a DNA molecule encoding the maspin polypeptide; and

(b) an amount of a chemotherapeutic agent effective to treat or prevent cancer.

6. A method according to claim 5, wherein the chemotherapeutic agent is interferon-gamma .

7. The method of claim 1 or claim 5, wherein the maspin polypeptide comprises an amino acid sequence according to SEQ ID NO:2, or a functional fragment thereof.

8. The method of claim 1 or claim 5, further comprising administering an additional agent effective for treating or preventing cancer.

9. The method of claim 8, wherein the additional agent is effective for treating or preventing breast cancer.

10. The method of claim 1 or claim 5, wherein the cancer is selected from cancer types that express maspin polypeptide.

11. The method of claim 1 , wherein the cancer is characterized by increased levels of cathepsin-D and/or pro-cathepsin D production or secretion.

12. The method of claim 10, wherein the cancer is breast cancer.

13. The method of claim 1 , wherein the amount of IFN-γ administered to the subject is from about 5 xlO⁶ NIH units/day to about 8 x 10⁶ NIH units/day.

14. The method of claim 1 , wherein the cancer is resistant to standard therapeutic regimens.

15. A method of sensitizing a cancer cell to a therapeutic agent comprising contacting the cancer cell with an amount of a maspin polypeptide effective to sensitize the cancer cell to the therapeutic agent.

16 The method of claim 15, wherein the cancer cell is sensitized to IFN-γ.

17. A method of sensitizing a cancer cell to a therapeutic agent comprising contacting a cancer cell with an amount of a polynucleotide encoding a maspin polypeptide under conditions that allow for expression of the maspin polypeptide.

18. The method of claim 15, wherein the cancer cell is a carcinoma.

19. The method of claim 18, wherein the carcinoma is a carcinoma of the breast tissue.

20 The method of claim 15, wherein the cancer cell is characterized by increased levels of cathepsin-D and/or pro-cathepsin D production or secretion.

21. A method for modulating levels of cathepsin D in a cell comprising contacting the cell with an amount of a maspin polypeptide effective to modulate cathepsin D and/or pro- cathepsin D levels in the cell.

22. The method of claim 21 , wherein the levels of cathepsin D and/or pro- cathepsin D in the cell are normalized to standard levels for the cell.

22. The method of claim 21 , wherein the contacting is in vivo

23. The method of claim 21 , wherein the contacting is in vitro.

24. The method of claim 21 , wherein the amount of the maspin peptide is effective to decrease cathepsin D and/or pro-cathepsin D levels in the cell.

25. The method of claim 21 , wherein the amount of the maspin peptide is effective to reduce secretion of cathepsin D and/or pro-cathepsin D from the cell.

26. The method of claim 21 , wherein the amount of the maspin peptide is effective to decrease the amount of cathepsin D mRNA in the cell.

27. A composition comprising a maspin polypeptide, an IFN molecule, and a pharmaceutically acceptable carrier or formulation agent.

28. The composition of claim 27, wherein the IFN molecule comprises an IFN- γ sequence.

29. The composition of claim 27, wherein the maspin polypeptide comprises a sequence of SEQ ID NO:2.

30. A method of modulating unregulated angiogenesis in a mammal comprising administering to the mammal a therapeutically effective amount of a maspin polypeptide and a therapeutically effective amount of a chemotherapeutic to modulate unregulated angiogenesis.

31. The method of claim 30, wherein the chemotherapeutic is IFN-γ.

32. A method of preventing metastasis in a mammal comprising administering to the mammal a therapeutically effective amount of a maspin polypeptide and a therapeutically effective amount of an anti-cancer agent to modulate metastasis.

33. The method according to claim 32 wherein the anti-cancer agent is IFN-γ.

34. A method for simultaneously treating cancer and inhibiting metastasis in a mammal comprising administering to the mammal a therapeutically effective amount of a maspin polypeptide and a therapeutically effective amount of an anti-cancer agent.

35. The method according to claim 34 wherein the anti-cancer agent is IFN-γ.

36. A method of preventing or inhibiting extracellular matrix degradation in a mammal comprising administering to the mammal a therapeutically effective amount of a maspin polypeptide wherein the administration of maspin results in reduction in cathepsin-D and/or pro-cathepsin D levels.

37. A method of preventing or inhibiting extracellular matrix degradation in a mammal comprising administering to the mammal a therapeutically effective amount of a maspin polypeptide or an aspartyl protease inhibitor.

38. An assay for determining the likelihood that a patient will develop metastatic cancer comprising: a) identifying a patient at risk for developing metastatic cancer; b) obtaining a serum sample from the patient; c) measuring the level of cathepsin-D activity in the serum sample; d) determining from the measurement in step c) the likelihood that the patient will develop metastatic cancer; wherein there is an elevated likelihood that the patient will develop metastatic cancer when the level of cathepsin-D activity in the sample is more than about twice the level in a control sample.

39. The assay of claim 38 wherein the cathepsin-D is pro-cathepsin-D.

40. A method according to claim 39, where there is an elevated likelihood that the patient will develop metastatic cancer when the level of pro-cathepsin-D in the sample is more than about 4 times the level in a control sample.