WO2006101535A1 - Inhibition of proteasome function to potentiate the proapoptotic and antitumor activity of cytokines - Google Patents

Abstract

Disclosed herein are methods of using proteasome inhibitors to enhance an antitumor activity of a cytokine on a tumor cell that responds to treatment with the cytokine. Also provided herein are pharmaceutical compositions that include at least one cytokine and at least one proteasome inhibitor.

Description

INHIBITION OF PROTEASOME FUNCTION TO POTENTIA TE THE PROAPOPTOTICAND ANTITUMOR ACTIVITY OF CYTOKINES

This application claims the benefit of U.S. Provisional Application No.

60/665,167 filed March 23, 2005, which is incorporated in its entirety by reference.

FIELD OF THE DISCLOSURE

This disclosure relates to methods of using a proteasome inhibitor to enhance an antitumor activity of a cytokine, and related compositions.

BACKGROUND

Cancer has long been a leading cause of mortality in the United States. Significant efforts have been and continue to be made to find new approaches for treating this constellation of diseases. Malignant tumors develop when a cell escapes from its normal growth regulatory mechanisms and proliferates in an uncontrolled fashion. Such tumor cells can metastasize to secondary sites if treatment of the primary tumor is either not complete or not initiated before substantial progression of the disease. Early diagnosis and effective treatment of malignant tumors is therefore beneficial for survival.

Existing therapeutic modalities that include surgery, radiotherapy and dose- intensive chemotherapy, are the methods of choice for treating subjects with metastatic cancer or subjects with diffuse cancers such as leukemias. However, radiotherapy can cause substantial damage to normal tissue in the treatment field, resulting in scarring and loss of function of the normal tissue, and secondary tumors, especially at higher radiation doses. Chemotherapy can provide a therapeutic benefit in many cancer subjects, but it often fails to treat the disease because cancer cells may become resistant to the chemotherapeutic agent. The proteasome, a large, multiprotein particle present in both the cytoplasm and the nucleus of all eukaryotic cells breaks down cellular proteins. The proteasome is composed of two functional components, a 2OS core catalytic complex and a 19S regulatory subunit, which together form a functional 26S proteasome. The hydrolytic protease activity resides in a channel at the center of the 2OS complex, which is formed from four stacked, multiprotein rings. The outer α subunit rings form a narrow channel that allows only denatured proteins to enter the catalytic chamber formed by the central β subunit rings (Groll et al, Nature 386:463-71, 1997; Lowe et al, Science 268:533-39, 1995; Stock ed/., Cold Spring Harb. Symp. Quant. Biol. 60:525-32, 1995). A large number of inhibitors of the proteasome are known, and consist mainly of peptides that are modified at the predicted site of protein hydrolysis with a reactive functional group capable of modifying the attacking nucleophile of the proteasome, either reversibly or irreversibly (Bogyo et al, Biopoly. 43:269-80, 1997).

Recent studies have demonstrated that the proteasome is a key regulator of the turnover of proteins that modulate the cell cycle and apoptosis, implicating the proteasome as a potential target for the treatment of cancerous tumors (King et al., Science 274:1652-59, 1996; Adams, J. Cancer Cell 5:417-21, 2004; Hideshima et al, Cancer Res. 61:3071-76, 2001).

Other antitumor treatments include the use of cytokines such as IL-2 or IL- 12. Unfortunately, some tumors do not respond well or at all to therapeutic administration of cytokines, and other tumors that are initially sensitive to such treatments can develop resistance to these drugs.

SUMMARY OF THE DISCLOSURE A method for enhancing an antitumor activity of cytokine has been identified and is described herein. The method includes administering to a subject having a neoplasm a therapeutically effective amount of the cytokine and a proteasome inhibitor, wherein the proteasome inhibitor is present in a sufficient amount to enhance the activity (such as a proapoptotic activity) of the cytokine, thereby enhancing the antitumor activity of the cytokine. In disclosed examples, the proteasome inhibitor is a proteasome inhibitor, such as bortezomib, that inhibits degradation of proteins via the ubiquitin-proteasome pathway or that inhibits the 26S proteasome complex, and the cytokine is an interferon-alpha (IFN-α), interferon-beta (IFN-β), interferon-gamma (IFN-γ), and/or tumor necrosis factor alpha (TNF-α) inducing cytokine, such as interleukin-2 or interleukin-12. In some disclosed embodiments, the proteasome inhibitor upregulates expression of a cellular receptor for an antiproliferative cytokine, such as a cellular receptor for IFN-α, IFN-β, IFN-γ, and/or TNF-α.

This disclosure also includes methods of treating a tumor by administering a therapeutically effective combination of a cytokine and a proteasome inhibitor, wherein the proteasome inhibitor increases sensitivity of the tumor to the cytokine treatment. The administered cytokine can include a cytokine that acts directly on the tumor or indirectly, for example, through a second cytokine or an intermediary (such as an immune cell on which the administered cytokine acts). This disclosure also provides pharmaceutical compositions that include at least one antitumor cytokine and at least one proteasome inhibitor, wherein the cytokine and the proteasome inhibitor are present in a therapeutically effective amount for the proteasome inhibitor to enhance an antitumor activity (such as a proapoptotic activity) of the cytokine. In disclosed examples, the cytokine is an IFN-α, IFN-β, IFN-γ, and/or TNF-α inducing cytokine, such as a interleukin-2 or interleukin-12, and the proteasome inhibitor is bortezomib.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1D illustrate that bortezomib inhibits proliferation and survival of murine TBJ and Neuro-2a neuroblastoma cells. TBJ and Neuro-2a tumor cells were incubated with varying concentrations of bortezomib. At day 2 after treatment, either cells were labeled with H³ [Thymidine] and incorporation of radioactivity determined by standard methods, or cells were stained with annexin-v and propidium iodide and analyzed by flow cytometry. Cells that stained negative for annexin-v/propidium iodide were considered viable. Bortezomib inhibited proliferation of both TBJ (FIG. IA) and Neuro-2a (FIG. IB) tumor cells. Data are the mean +S.D. (n=3). Bortezomib also decreased viability of both TBJ (FIG. 1C) and Neuro-2a (FIG. ID) neuroblastoma cells in a dose-dependent manner. The experiment is representative of at least three separate experiments. Data are the mean +S.E. (n=2). FIG. 2A-2E illustrate that bortezomib inhibits AKT phosphorylation (serine

473) (an important antiapoptotic molecule) and induces subcellular translocation of Bid- EGFP (an important antiapoptotic molecule) in TBJ and Neuro-2a neuroblastoma cells. Tumor cells were treated with different concentrations of bortezomib for 24 h. Equal amounts of total cellular proteins extracted from these samples were then analyzed by western blotting for total AKT and phosphorylated AKT (ser-473) protein levels (FIG. 2A). TBJ cells overexpressing Bid fused to green fluorescence protein (TBJ-Bid- EGFP) were treated with either bortezomib or medium alone for 22 h and subsequently analyzed by confocal microscopy for subcellular localization of Bid-EGFP. Bid-EGFP was distributed through out the cytosol in viable cells showing a diffuse pattern of GFP fluorescence (FIG. 2B and 2C). In contrast, in cells treated with bortezomib Bid-EGFP aggregated around the mitochondria resulting in a punctate pattern of GFP fluorescence (FIG. 2D and 2E). The images are representative of at least two separate experiments. FIG. 3A-3F illustrate that bortezomib induced increases in expression levels of TNF-Rl and IFN-γ-Rα in TBJ neuroblastoma cells was accompanied by enhanced sensitization to TNF-α+IFN-γ-mediated apoptosis. Tumor cells were treated with bortezomib for 24 h. Cells were then stained with antibodies directed against either Fas, TRAIL-R2, TNF-Rl or IFN-γ-Rα/β and the number of receptor-positive cells were determined by flow cytometry (FIG. 3 A and 3B). Results are representative of at least three separate experiments. Data are the mean +S.E. (n=2). In other experiments, TBJ and Neuro-2a neuroblastoma tumor cells were pretreated with varying concentrations of bortezomib for 4 h prior to treatment with IFN-γ (100 IU/ml)+TNF-α (50 ng/ml). Cells were then stained with annexin-v and propidium iodide and analyzed by flow cytometry. Bortezomib-enhanced the sensitivity of TBJ (FIG. 3C: day 2 after treatment), Neuro-2a neuroblastoma (FIG. 3D: day 1 after treatment), and murine endothelial EOMA (FIG. 3E: day 1 after treatment) cells to IFN-γ+TNF-α-mediated apoptosis resulting in decreased viability. The experiment is representative of at least three separate experiments. Data are the mean +S.E. (n=2). Bortezomib also increased surface expression of IFN-γ-Rα in endothelial EOMA cells (FIG. 3F). Cells were exposed to bortezomib (10 nM or 30 nM) for 4 h prior to treatment with IFN-γ (100 IU/ml)+TNF-α (50 ng/ml ) for another 18 h. Cells were then stained with antibodies directed against either IFN-γ-Rα or IFN-γ-Rβ as outlined herein. The experiment is representative of at least two separate experiments. Data are the mean +S.E. (n=2). FIG. 4 is a graph, showing that combined administration of IL-2 and bortezomib delayed primary SC-TBJ neuroblastoma tumor growth more effectively than either of the agents alone. Mice bearing well-established SC-TBJ tumors were treated with either IL-2 (50, 000 IU) or vehicle alone on days 6-10, 13-17, and 20-24 after tumor implantation as described herein. Bortezomib (0.8 mg/kg) or vehicle alone were given on days 7, 10, 14, 17, and 21 after tumor implantation where indicated. Administration of bortezomib in combination with IL-2 inhibited TBJ tumor growth more effectively over time (day 9-27 post tumor implantation) than either of the single agents alone (Jonckheere test for trend, p<=0.05 for combination of bortezomib+IL-2 compared to either single agent bortezomib or IL-2 alone or control mice treated with vehicle alone). FIG. 5A-5D illustrate that administration of bortezomib in combination with IL-

2 inhibits TBJ tumor metastases more effectively than either agent alone. Mice were injected intravenously with TBJ-RFP tumor cells. At day 5 after tumor implantation, mice were treated with either IL-2 (50,000 IU) or vehicle alone as indicated on days 5-9 and 12-15 after tumor injection. Bortezomib (0.8 mg/kg) or vehicle alone was administered as indicated on days 6, 9, and 13. Livers were resected at day 16 after tumor injection (day 11 of therapy) and examined by light and fluorescent microscopy. A greater inhibition of tumor disease burden was observed in liver of mice treated with bortezomib and IL-2 compared to mice treated with either bortezomib or IL-2 alone or control mice treated with vehicle alone (FIG. 5A). Higher magnification images of livers from mice that received the combination treatment (bortezomib+IL-2) showed greater decreases in gross tumor vascularity than is observed in mice treated with either single agent alone or control mice treated with vehicle alone (FIG. 5B, 5D: light images, FIG. 5C: fluorescent images). FIG. 6 illustrates that bortezomib enhances the anti-tumor effects of IL- 12 in an induced model of TBJ neuroblastoma tumor cell metastases. Liver metastases were induced by intrasplenic (IS) injection of RFP-TBJ tumor cells as outlined herein. Mice were given two weekly doses of IL-12 (0.1 μg) or vehicle alone on days 5, 8, and 12 post tumor implantation. Other cohorts of 10 mice were injected with bortezomib (0.8 mg/kg) with or without IL-12 on days 5, 8, and 12 after tumor implantation. Mice were sacrificed for liver imaging on day 13 after tumor implantation (day 8 post therapy).

FIG. 7A-7B illustrate that MG 132 induces apoptosis and decreases viability of TBJ (FIG. 7A) and Neuro-2a (FIG. 7B) neuroblastoma cells. Preadhered TBJ and Neuro-2a cells were treated with varying concentrations of MG-132 or medium alone and incubated at 37°C for a period of 3 days. Cells were then harvested, stained with Annexin-V FITC and propidium iodide and analyzed for apoptosis on a FACScan flow cytometer. The percentage of cells that stained negative for Annexin-V FITC and/or propidium iodide were considered viable.

FIG. 8 illustrates that MG-132 induces apoptosis and decreases viability of murine endothelial EOMA cells. Preadhered murine endothelial EOMA cells were treated with varying concentrations of MG- 132 or medium alone and incubated at 37°C for a period of 3 days. Cells were harvested, stained with Annexin-V FITC and propidium iodide and then analyzed for apoptosis on a FACScan flow cytometer. The percentage of cells that stained negative for Annexin-V FITC and/or propidium iodide were considered viable.

FIG. 9 illustrates that MGl 32 induces apoptosis in renal carcinoma RENCA cells. Preadhered renal carcinoma RENCA cells were treated with varying concentrations of MG-132 or medium alone and incubated at 37⁰C for a period of 3 days. Cells were harvested, stained with Annexin-V FITC analyzed for apoptosis on a FACScan flow cytometer.

FIG. 1 OA-I OB illustrate that MG-132 increases sensitivity of TBJ (FIG. 10A) and Neuro-2a (FIG. 10B) neuroblastoma cells to TNF-α+IFN-γ-mediated apoptosis. Preadhered TBJ or Neuro-2a cells were treated with either different concentrations of MG-132 or medium alone for 2 hours. Afterwards, cells were exposed to TNF-α (50 ng/ml)+ IFN-γ (100IU/ml) for an additional 22 hours. Cells were then harvested, stained with Annexin-V FITC and propidium iodide and analyzed for apoptosis on a FACScan flow cytometer. The percentage of cells that stained negative for Annexin-V FITC and/or propidium iodide were considered viable.

FIG. 11 illustrates that MG-132 increases expression of death receptor FAS in Neuro-2a neuroblastoma cells. Preadhered Neuro-2a cells were treated with either MG- 132 (300 nM) or medium alone for 2 hours. Cells were then exposed to TNF-α (50 ng/ml)+IFN-γ (lOOIU/ml) for an additional 22 or 46 hours. Cells were then harvested and stained with either PE-labeled hamster anti-mouse Fas antibody or appropriate isotype control antibody and then analyzed on a FACScan flow cytometer.

FIG. 12 illustrates that MG-132 decreases mitochondrial membrane potential and increases apoptosis of TBJ neuroblastoma cells. Preadhered TBJ cells were treated with either different concentrations of MG-132 or medium alone for 2 hours. Afterwards, cells were exposed to TNF-α (50 ng/ml)+IFN-γ (100 IU/ml) for an additional 22 hours. Cells were then harvested, stained with either Annexin-V FITC to analyze for apoptosis or a freshly prepared tetramethylrhodamine methyl ester (TMRM, 100 nM) solution to monitor for changes in mitochondrial membrane potential. Cells were immediately analyzed on a FACScan flow cytometer. TMRM rapidly accumulates in the mitochondria of live cells. Thus, live cells show high TMR-M staining and low Annexin-V binding. On the other hand, apoptotic cells show low TMRM staining (loss of mitochondrial membrane potential) and high Annexin-V binding.

DETAILED DESCRIPTION

/. Abbreviations

°C: degrees Celsius

GFP: green fluorescent protein h: hour

IFN: interferon

IL: interleukin i.p.: intraperitoneally

Ls.: intrasplenically i.v.: intravenously min: minute(s) ml: milliliter nM: nanomolar

PE: phycoerythrin

SC: subcutaneous

TBJ-EGFP-BID: TBJ cells that express BID fused to GFP

TBJ: a metastatic sub-clone of Neuro-2a neuroblastoma cells

TBJ-RFP: TBJ cells that express red fluorescent protein

TNF: tumor necrosis factor μg: microgram(s) μl: microliter(s)

//. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

AKT: A serine threonine kinase that regulates the survival of many cell types by inhibiting the actions of a number of proapoptotic proteins, such as glycogen synthase kinase 3 (GSK-3), BAD, caspase-9, and Forkhead transcription factors (all of which are suppressed upon phosphorylation by AKT).

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non- human mammals. Similarly, the term "subject" includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, horses, and cows. Antitumor: Having antineoplastic activity, for example inhibiting the development or progression of a tumor, such as a malignant tumor, including local tumor growth or recurrence or metastatic spread.

Apoptosis: A form of programmed cell death characterized by morphological changes of a dying cell that can include plasma membrane blebbing, nuclear and cytoplasmic shrinkage, and chromatin condensation. Cells that undergo apoptosis often fragment into membrane-bound apoptotic bodies that are readily phagocytosed and digested by macrophages or by neighboring cells without generating an inflammatory response. (See Zhu L. and Chun J., editors, Apoptosis detection and assay methods, Eaton Publishing Company/Bio Techniques Books Division, 1998). Cell death is accomplished by the activation of endonucleases that fragment the cell's nuclear DNA into internucleosomal fragments (see, for example, Zhang et al, Cell Res. 10:205-11, 2000). Due to the generation of such fragments, the DNA of apoptotic cells typically migrates as a ladder of 180-200 bp multimers on an agarose gel. Cancer or malignant neoplasm: A neoplasm that has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and which is capable of metastasis.

Cytokine: The term "cytokine" is used as a generic name for a diverse group of soluble proteins and peptides that act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. Some cytokines, such as interleukins (for example, IL-2, IL-12, IL-18, and IL-32), act primarily as proliferation factors, activating and expanding T and B lymphocytes. Activated lymphocytes in turn secrete additional cytokines (for example, IFN-α, IFN-β, IFN-γ, and TNF-α). Other cytokines, such as interferons (for example, IFN-α, IFN-β, and IFN-γ) and tumor necrosis factors (for example, TNF-α and TNF-β) act primarily as antiproliferative factors, inhibiting the proliferation of both normal and transformed cells. Such cytokines are referred to herein as "antiproliferative cytokines." Cytokines also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Cytokines include, but are not limited to, interleukins (for example, IL-2, IL-12, IL- 18, and IL-32), interferons (for example, IFN-α, IFN-β, and IFN-γ), and tumor necrosis factors (for example, TNF-α and TNF-β). Enhancing: Improving an outcome, for example, as measured by a change in a specific value, such as an increase or a decrease in a particular parameter of an activity of a cytokine associated with tumor development or growth. In one embodiment, enhancement refers to at least a 25%, 50%, 100% or greater than 100% increase in a particular parameter. In another embodiment, enhancement refers to at least a 25%, 50%, 100% or greater than 100% decrease in a particular parameter. In one specific, non-limiting example, enhancement of an antitumor activity of a cytokine refers to an increase in the ability of the cytokine to inhibit or treat a neoplasm, such as at least a 25%, 50%, 100%, or greater than 100% increase in the effectiveness of the cytokine for that purpose. Inhibiting or treating: With respect to disease (such as neoplasm or metastasis), either term includes improving the development, course or outcome of a disease, for example, by (i) preventing the disease, for example, causing the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (ii) restraining the disease, for example, arresting the development of the disease or its clinical symptoms, (iii) ameliorating the disease, for example, delaying onset of the clinical symptoms of the disease in a susceptible subject or a reduction in severity of some or all clinical symptoms of the disease, or (iv) relieving the disease, for example, causing regression of the disease or some or all of its clinical symptoms.

Interferon-alpha (IFN-α): At least 23 different variants of IFN-α are known. The individual proteins have molecular masses of between 19-26 kDa and consist of proteins with lengths of 156-166 and 172 amino acids. All IFN-α subtypes possess a common conserved sequence region between amino acid positions 115-151, while the amino-terminal ends are variable. Many IFN-α subtypes differ in their sequences at only one or two positions. Naturally occurring variants also include proteins truncated by 10 amino acids at the carboxy-terminal end. IFN-alpha forms are produced by monocytes/macrophages, lymphoblastoid cells, fibroblasts, and a number of different cell types following induction by viruses, nucleic acids, and glucocorticoid hormones. All known subtypes of IFN-α show the same antiviral, antiparasitic and antiproliferative activities in suitable bioassays, although they may differ in relative activities.

Interferon-beta (IFN-β): IFN-beta is a glycoprotein of 20 kDa and has a length of 166 amino acids, as well as homologs having the same activity. Glycosylation is not required for biological activity in vitro. IFN-beta is produced mainly by fibroblasts and some epithelial cell types. The synthesis of IFN-β can be induced by common inducers of interferons, including viruses, double-stranded RNA, and microorganisms. IFN-beta is also induced by some cytokines, such as TNF-α and IL-I . IFN- beta is involved in the regulation of nonspecific humoral immune responses and immune responses against viral infections. IFN-beta stimulates the activity of NK-cells and hence also antibody-dependent cytotoxicity.

Interferon-gamma (IFN-γ): A dimeric protein with subunits of 146 amino acids (as well as homologs having the same activity) produced mainly by T cells and natural killer cells activated by antigens, mitogens or alloantigens. The synthesis of IFN-γ is induced by, among other things, IL-2, IL- 12, IL-18, and IL-32. Interferon- gamma has antiviral and antiparasitic activities and also inhibits the proliferation of a number of normal and transformed cells. Interferon-gamma synergizes with TNF-α and TNF-β in inhibiting the proliferation of various cell types. Interleukin-2 (IL-2): A protein of 133 amino acids (15.4 kDa) with a slightly basic pi that does not display sequence homology to any other factors. Interleukin-2 is a growth factor for all subpopulations of T lymphocytes. It is an antigen-nonspecific proliferation factor for T cells that induces cell cycle progression in resting cells and thus allows clonal expansion of activated T lymphocytes. Interleukin-2 also promotes the proliferation of activated B cells. Due to its effects on T cells and B cells, IL-2 is a central regulator of immune responses. It also plays a role in anti-inflammatory reactions, in hematopoiesis and in tumor surveillance. Interleukin-2 stimulates the synthesis of IFN-γ in leukocytes and also induces the secretion of TNF-α and TNF-β. Interleukin-12 (IL-12): A heterodimeric 70 kDa glycoprotein consisting of a 40 kDa subunit and a 35 kDa subunit linked by disulfide bonds. Interleukin-12 is secreted by peripheral lymphocytes after induction. It is produced mainly by B cells and to a lesser extent by T cells. Interleukin-12 stimulates the synthesis of IFN-γ, IL-2 and TNF-α in leukocytes. Interleukin-12 synergizes with suboptimal amounts of IL-2 in promoting the proliferation of mononuclear cells in the peripheral blood and in promoting the generation of activated killer cells.

Interleukin-18 (IL-18): An 18 to 19 kD glycoprotein that has homology to IL- 1. Interleukin-18 is initially synthesized as an inactive precursor molecule lacking a signal peptide and is cleaved by IL-I converting enzyme to yield an active molecule. Interleukin-18 is produced during the acute immune response by a variety of immune and non-immune cells, including monocytes, macrophages and immature dendritic cells. Interleukin-18 is a potent inducer of IFN-γ production by T cells.

Interleukin-32 (IL-32): A recently discovered member of the cytokine family, IL-32 induces various cytokines, including TNF-α and IL-8 in monocytic cells. Interleukin-32 is induced in human peripheral lymphocyte cells after mitogen stimulation, in human epithelial cells by IFN-γ and in NK cells after exposure to the combination of IL-12 plus IL-18.

Isolated or purified: An "isolated" or "purified" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs. The term "isolated" or "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell. Preferably, a preparation is purified such that the biological component represents at least 50%, such as at least 70%, at least 90%, at least 95%, or greater of the total biological component content of the preparation. ,

Metastasis: The process by which malignant cells transfer from one organ or part of the body to a separate organ or part of the body. This term also refers to a growth of malignant cells distant from the site of the primary neoplasm from which the malignant cells arose.

Neoplasm: An abnormal growth of cells or tissue, particularly a new growth of cells or tissue in which the growth is uncontrolled and progressive. A tumor is an example of a neoplasm. Neural cell: A cell that is specialized to conduct nerve impulses. A neural cell is typically composed of a cell body containing the nucleus, several short branches (dendrites), and one long arm (the axon) with short branches along its length and at its end. Neural cells conduct signals that control the actions of other cells in the body, such as muscle cells. A neural cell can also be referred to as a nerve cell or a neuron. Neuroblastoma: The most common solid malignancy of childhood. Neuroblastoma remains responsible for significant childhood cancer-related morbidity and mortality. It is an embryonal malignancy of the postganglionic sympathetic nervous system and has remarkably diverse clinical and biologic characteristics and behavior. Some tumors undergo spontaneous regression or differentiation to a benign neoplasm, while others exhibit an extremely malignant phenotype with regional or disseminated disease that is resistant to intensive therapy.

Parenteral: Administered outside of the intestine, for example, not via the alimentary tract. Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for example. Parenteral administration is preferred for some chemical compounds to avoid degradation of the chemical compound in the gastrointestinal tract.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules, such as one or more cytokines or proteasome inhibitors and additional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Proteasome : A large, multiprotein particle present in both the cytoplasm and the nucleus of all eukaryotic cells that breaks down proteins. The proteasome is composed of two functional components, a 2OS core catalytic complex and a 19S regulatory subunit, which together form the functional "26S proteasome." Proteins that are to be degraded are marked with ubiquitin chains, which bind to a receptor on the 19S complex. Once recognized by the regulatory complex, the ubiquitin chain is removed and the protein denatured in preparation for degradation. The hydrolytic protease activity resides in a channel at the center of the 2OS complex, which is formed from four stacked, multiprotein rings. The outer α subunit rings form a narrow channel that allows only denatured proteins to enter the catalytic chamber formed by the central β subunit rings (Groll et al, Nature 386:463-71, 1997; Lowe et al, Science 268:533-39, 1995; Stock et al, Cold Spring Harb. Symp. Quant. Biol. 60:525-32, 1995).

Inside the catalytic chamber, proteins are surrounded by six protease-active sites (three on each β subunit ring). The proteasome protease functions similarly to serine proteases but is unique since it relies on a threonine residue in the active site. Proteins processed by the proteasome are reduced to small polypeptides 3 to 22 residues in length (Nussbaum et al., Proc. Natl. Acad. Sci. USA 95:12504-509, 1998).

Proteasome inhibitor: A chemical compound characterized by its ability to inhibit the breakdown of proteins by interfering with proteasomal function. Particular examples of proteasome inhibitors are peptidyl boronic acid ester and acid compounds. Exemplary proteasome inhibitors include, but are not limited to, iV-pyrazinecarbonyl-L- phenylalanine-L-leucineboronic acid (bortezomib or PS-341), carbobenzyloxy-L- leucyl-L-leucyl-L-leucinal (MG-132), carbobenzyloxy-L-leucyl-L-leucyl-L-norvalinal (MG-115), N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucyl boronic acid (MG-262), N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI; and its epoxide), N-acetyl- Leu-Leu-norleucinal (MG-101, ALLΝ or calpain inhibitor I), N-acetyl-Leu-Leu-Met (ALLM or calpain inhibitor II), N-tosyl-Lys chloromethyl ketone (TLCK), Λf-tosyl-Phe chloromethyl ketone (TPCK), pyrrolidine dithiocarbamate (PDTC), [2S,3S]-trans- epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (EST), epoxomicin, lactacystin, and pentoxyfilline (PTX). Proteasome inhibitors and their methods of use are discussed, for example, in

Vinitsky et al. (J. Biol. Chem. 269:29860-66, 1994), Figueiredo-Pereira et al. (J. Neurochem. 63:1578-81, 1994), Wojcik et al. {Eur. J. Cell Biol. 71:311-18, 1996), Sin et al. (Biorg. Med. Chem. Lett. 9:2283-88, 1999), Combaret et al. (MoI. Biol. Rep. 26:95-101, 1999), U.S. Patent No. 5,693,617, published International Application Nos. WO 2004/043374 and WO 92/12140, and published U.S. Patent Application No. US 2005/0025734, all of which are incorporated herein by reference.

Resistant to antitumor activity: Reduced therapeutic response of a neoplastic cell to treatment with an antineoplastic/antitumor agent, such as a cytokine.

Substantially higher: A significant increase in a specific value, such as an increase in the amount of a mRNA or protein (for example, AKT) expressed in a tumor cell, as compared to a non-tumor cell, or, an increase in the activity of a protein (for example, phosphorylated (activated) AKT). In one embodiment, substantially higher refers to at least a 25%, 50%, 100% or greater than 100% increase in a specific value. Substantially higher expression includes, but is not limited to, at least a 25% increase in the amount of AKT mRNA or protein in a cell as compared to a control, such as, but not limited to, at least a 30%, 50%, 75%, 100%, or greater than 100% increase of AKT mRNA or protein. Likewise, substantially higher activity of AKT includes, but is not limited to, at least a 25% increase in the amount of phosphorylated AKT in a cell as compared to a control, such as, but not limited to, at least a 30%, 50%, 75%, 100%, or greater than 100% increase of phosphorylated AKT.

Sympathetic nervous system: The part of the autonomic nervous system originating in the thoracic and lumbar regions of the spinal cord, and that in general inhibits or opposes the physiological effects of the parasympathetic nervous system. Sympathetic preganglionic neurons originate in the lateral horns of the 12 thoracic and the first 2 or 3 lumbar segments of the spinal cord. They pass into sympathetic ganglia which are organized into two chains that run parallel to and on either side of the spinal cord. Tumors of the sypathetic nervous system include adrenal neuroblastomas.

Therapeutically effective amount: A quantity of a specified agent, including a combination of agents, sufficient to achieve a desired effect in a subject being treated with that agent. For example, this may be the amount of a cytokine and a proteasome inhibitor necessary to inhibit a neoplasm or metastasis of a malignant cell. In particular examples, a therapeutically effective amount of an agent is an amount sufficient to effect the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for treating (including preventing) a neoplasm or inhibiting metastasis of a malignant cell will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition.

Therapeutically effective amounts can be determined, in one example, by in vitro assays or animal studies. When in vitro or animal assays are used, a dosage is administered to provide a target tissue concentration similar to that which has been shown to be effective in the in vitro or animal assays.

A therapeutically effective amount of a cytokine and a proteasome inhibitor may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the frequency of administration is dependent on the preparation applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the therapy or compound.

Tumor: A neoplasm that may be either malignant or non-malignant (benign) and includes both solid and non-solid tumors (such as hematologic malignancies). As used herein, this term also encompasses other cell types found in the tumor microenvironment, such as vascular endothelial cells, pericytes, fibroblasts and/or other stromal elements.

Tumor necrosis factor alpha (TNF-α): A non-glycosylated protein of 17 kDa and a length of 157 amino acids, as well as homologs having the same activity. Tumor necrosis factor alpha is secreted by macrophages, monocytes, neutrophils, T cells, and NK cells following their stimulation. Tumor necrosis factor alpha shows a wide spectrum of biological activities. Among other activities, it inhibits proliferation and causes cytolysis of many tumor cell lines in vitro. Sensitive cells die within hours after exposure to picomolar concentrations of the factor and this involves, at least in part, mitochondria-derived second messenger molecules serving as common mediators of TNF cytotoxic and gene-regulatory signaling pathways. Tumor necrosis factor alpha induces hemorrhagic necrosis of transplanted tumors. Within hours after injection, TNF-α leads to the destruction of small blood vessels within many malignant tumors. Tumor necrosis factor alpha also enhances phagocytosis and cytotoxicity in neutrophilic granulocytes and also modulates the expression of many other proteins, including, for example, Fos, Myc, IL-I, and IL-6.

Ubiquitin: A small protein present in all eukaryotic cells, and highly conserved from yeast to humans. The covalent modification of proteins with chains of ubiquitin constitutes a potent targeting signal leading to recognition and destruction by the 26S proteasomes. During covalent modification of target proteins, the carboxyl-terminal glycine residue of ubiquitin becomes covalently attached to the ε-amino groups of several lysine residues on the proteins.

As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Also, as used herein, the term "comprises" means "includes." Hence "comprising A or B" means including A, B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

III. Overview of Several Embodiments Provided herein in various embodiments is a method of treating a tumor by sufficiently concurrently administering a cytokine and a proteasome inhibitor to enhance an anti-tumor effect of the cytokine. Other embodiments include a method for enhancing an antitumor activity of a cytokine (such as an exogenous cytokine) on a tumor cell that responds to treatment with the cytokine. In one embodiment, the method includes exposing the tumor cell to a therapeutically effective amount of the cytokine and a proteasome inhibitor, wherein the proteasome inhibitor is present in a sufficient amount to enhance the activity (such as a proapoptotic activity) of the cytokine, thereby enhancing the antitumor activity of the cytokine. In a specific, non-limiting example, the proteasome inhibitor induces expression in the tumor cell of a cellular receptor for an antiproliferative cytokine, such as an IFN-α receptor, an IFN-β receptor, an IFN-γ receptor, and/or a TNF-α receptor. In another specific, non-limiting example, exposing the tumor cell to a therapeutically effective amount of a cytokine includes exposing the cell to a cytokine that induces expression of another cytokine, wherein the other cytokine is an antiproliferative cytokine. Exemplary cytokines include IL-2, IL-12, IL- 18, and/or IL-32. Exemplary antiproliferative cytokines include IFN-α, IFN-β, IFN-γ, and/or TNF-α. In yet another specific, non-limiting example, exposing the tumor cell to a therapeutically effective amount of a cytokine includes exposing the cell to an antiproliferative cytokine, such as IFN-α, IFN-β, IFN-γ, and/or TNF-α. In still another specific, non-limiting example, the tumor cell expresses substantially higher levels of activated AKT than a corresponding non-tumor cell.

In another specific example of the method, the tumor cell is resistant to the antitumor activity of the cytokine, such as reduced or absent responsiveness of the tumor cell to the antitumor activity of the cytokine which has developed following treatment with the cytokine. In yet another specific example of the method, the therapeutically effective amount of the cytokine in the presence of the proteasome inhibitor is lower than when the tumor cell is exposed to the cytokine alone, hi still another specific example of the provided method, the cytokines include, but are not limited to, IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, IL-12, IL-18, IL-32, and combinations thereof.

Particular examples of proteasome inhibitors are peptidyl boronic acid ester and acid compounds. Exemplary proteasome inhibitors include, but are not limited to, bortezomib, MG-132, MG-115, MG-262, PSI, MG-IOl, ALLM, TLCK, TPCK, PDTC, EST, epoxomicin, lactacystin, and PTX. In one embodiment, the proteasome inhibitor inhibits degradation of proteins via the ubiquitin-proteasome pathway, for example, by inhibiting an El, E2 or E3 ubiquitin ligase or a deubiquitinating enzyme. In a specific, non-limiting example, the proteasome inhibitor inhibits the 26S proteasome. In another embodiment, the tumor cell is a neural cell, hi a specific, non-limiting example, the neural tumor cell is a tumor cell of the sympathetic nervous system, such as a neuroblastoma. In a further embodiment, exposing the tumor cell to a therapeutically effective amount of the cytokine and proteasome inhibitor includes administering the therapeutically effective amount of the cytokine and proteasome inhibitor to a subject having a tumor.

A method for treating a tumor in a subject is also described herein. This method includes administering to a subject a therapeutically effective amount of a cytokine having an antitumor activity and a therapeutically effective amount of a proteasome inhibitor, thereby treating the tumor in the subject. In a specific, non-limiting example, the proteasome inhibitor induces expression in the tumor of a cellular receptor for an antiproliferative cytokine, such as an IFN-α receptor, an IFN-β receptor, an IFN-γ receptor, and/or a TNF-α receptor. In another specific, non-limiting example, administering to the subject a therapeutically effective amount of the cytokine includes administering to the subject a cytokine that induces expression of another cytokine, wherein the other cytokine is an antiproliferative cytokine. Exemplary cytokines include IL-2, IL-12, IL-18, and/or IL-32. Exemplary antiproliferative cytokines include IFN-α, IFN-β, IFN-γ, and/or TNF-α. In yet another specific, non-limiting example, administering to the subject a therapeutically effective amount of the cytokine includes administering to the subject an antiproliferative cytokine, such as IFN-α, IFN-β, IFN-γ, and/or TNF-α. In one embodiment, the proteasome inhibitor is administered prior to the administration of the cytokine, hi another embodiment, the proteasome inhibitor is administered after the administration of the cytokine. In still another embodiment, the proteasome inhibitor is administered simultaneously with the administration of the cytokine. In yet another embodiment, administration of the cytokine and the administration of the proteasome inhibitor (either sufficiently sequentially or concurrently) results in tumor cell death that is greater than tumor cell death from administration of either the cytokine or proteasome inhibitor in the absence of the other. In a specific, non-limiting example, tumor cell death is the result of apoptosis.

Also provided herein is a method for treating a neuroblastoma tumor in a subject, including administering to the subject a therapeutically effective amount of interleukin-12 and a therapeutically effective amount of a proteasome inhibitor, such as bortezomib, that inhibits degradation of proteins via the ubiquitin-proteasome pathway, thereby treating the tumor in the subject.

Pharmaceutical compositions are also disclosed that include a pharmaceutical carrier, at least one cytokine having an antitumor activity and at least one proteasome inhibitor, wherein the cytokine and the proteasome inhibitor are present in a therapeutically effective amount for the proteasome inhibitor to enhance an antitumor activity of the cytokine, hi a specific, non-limiting example, the administered cytokine induces expression of another cytokine, wherein the other cytokine is an antiproliferative cytokine, hi another specific, non-limiting example, the administered cytokine is itself an antiproliferative cytokine. Representative cytokines include, but are not limited to, IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, IL-12, IL-18, IL-32, and combinations thereof. Representative proteasome inhibitors include, but are not limited to, bortezomib, MG-132, MG-115, MG-262, PSI, MG-IOl, ALLM, TLCK, TPCK, PDTC, EST, epoxomicin, lactacystin, PTX, and combinations thereof.

IV. Tumors That Respond to Treatment With Cytokines Many tumors respond therapeutically to treatment with cytokines, such as cytokines that induce IFN-α, IFN-β, IFN-γ, and/or TNF-α. When administered alone or in combination, IFN-γ/TNF-α inducing-cytokines, including IL-2 (Rosenberg et ah, J. Exp. Med. 161:1169-88, 1985), IL-12 (Nastala et al., J. Immunol. 153:1697-1706, 1994; Wigginton et al., J. Natl. Cancer Inst. 88:38-43, 1996) and IL-18 (Wigginton et al., J. Immunol. 169:4467-74, 2002) mediate potent therapeutic effects in several preclinical tumor models. Mechanistically, the antitumor activity of IL-12 or IL-18 administered alone or in combination with IL-2 acts through the induction of endogenous IFN-γ production, and more specifically, the ability of tumor cells to respond to IFN-γ (Wigginton et al., J. Immunol. 169:4467-74, 2002; Coughlin et al, Immunity 9:25-34, 1998; Wigginton et al., J. Clin. Invest. 108:51-62, 2001). In turn, IFN-γ can directly sensitize tumor and/or endothelial cell populations to receptor-mediated apoptosis induced by Fas/Fas-L (Bernassola et al, Cell Death Differ. 6:652-60, 1999; Varela et al, J. Biol. Chem. 276:17779-87, 2001; Sayers et al, J. Immunol. 161:3957-65, 1998), TRAIL/TRAIL-R (Yang et al, Cancer Res. 63: 1122-29, 2003; Varela et al, J. Biol. Chem. 276:17779-87, 2001; Fulda and Debatin, Oncogene 21:2295-08, 2002) or TNF/TNF-R (Varela et al., J. Biol Chem. 276:17779-87, 2001; Fulda and Debatin, Oncogene 21:2295-08, 2002) in vitro, and the antitumor activity of IL-12 or IL-18 administered alone or in combination with IL-2 appears to be mediated by the Fas/Fas- L (Wigginton et al, J. Clin. Invest. 108:51-62, 2001; Sanford et al, Hum. Gene Ther. 12:1485-98, 2001) and/or TRAIL/TRAIL-R (Smyth et al, J. Exp. Med. 193:661-70, 2001) pathways in vivo. Similarly, a positive correlation exists between clinical response to IL-2 therapy and the sustained production of endogenous TNF-α (Blay et al, Cancer Res. 50:2371-74, 1990). Additionally, clinical responsiveness to IL-2 in patients has been shown to correlate with immunologic responsiveness as measured by IFN-γ production (Gollob et al, Clin. Cancer Res. 6:1678-92, 2000). Thus, the induction of downstream cytokines, including IFN-α, IFN-β, IFN-γ, and TNF-α, and the subsequent engagement of proapoptotic pathways are believed to be particularly important mechanisms that mediate the antitumor activity of cytokines such as IL-2, IL- 12 and IL-18, among others.

The FDA has approved IL-2 for the management of patients with metastatic renal cell carcinoma (Culliton, BJ, Nature 355:287, 1992) or melanoma (Rosenberg et al, J. Natl. Cancer Inst. 85:622-32, 1993), and interferon-α has been utilized broadly in these patients as well (Kuzmits et al, Oncology 42 Suppl 1 :26-32, 1985). More recently, potent IFN-γ/TNF-α inducing antitumor cytokines such as IL- 12 (Gollob et al, Clin. Cancer Res. 6:1678-92, 2000) and IL- 18 (Robertson et al., J. Clin. Oncol. 22:2553, 2004) also have been investigated in the clinical setting. The overall clinical efficacy of single cytokines has been modest to date, however, and the side effects associated with administration of high dose cytokines can be substantial. Furthermore, many tumors initiate a range of mechanisms that can actively subvert the host antitumor immune response. These mechanisms can include, for example, defects in proapoptotic genes as well as the overexpression/activity of prosurvival factors, such as AKT, among others. In light of these obstacles, and the complexity of mechanisms required to initiate, expand and maintain a productive antitumor immune response, successful approaches for the immunotherapy of tumors often involve rationally-designed combinations of agents with complementary mechanisms of action.

The present disclosure provides methods for enhancing an antitumor activity of a cytokine on a tumor cell that responds to treatment with the cytokine, including exposing the tumor cell to a therapeutically effective amount of the cytokine and a proteasome inhibitor, wherein the proteasome inhibitor is present in a sufficient amount to enhance the activity (such as a proapoptotic activity) of the cytokine, thereby enhancing the antitumor activity of the cytokine. As used herein, "a tumor cell that responds to treatment with the cytokine" includes tumors and tumor cells that respond to the cytokine alone or the cytokine in combination with another agent, such as a cytokine sensitizing agent (for example, a proteasome inhibitor) that allows the tumor cell (for example, a tumor cell resistant to the cytokine alone) to respond or enhances its response to the cytokine. Examples of such cytokines and the tumors they treat include IL-2: melanoma (Atkins MB, Semin. Oncol. 29:12-7, 2002; Rosenberg et al, Ann.

Surg. 210:474-84, 1989; Rosenberg SA, JAMA 271:907-13, 1994; Atkins et al., J. Clin. Oncol. 17:2105-16, 1999); renal cell carcinoma (Rosenberg SA, JAMA 271:907-13, 1994; Yang et al., J. CHn. Oncol. 21:3127-32, 2003); and acute myelogenous leukemia. IL-12: colon carcinoma, breast carcinoma, prostate carcinoma, pancreatic carcinoma, lung carcinoma, melanoma, renal cell carcinoma, and sarcoma (Trinchieri et al,

Cytokine and Growth Factor Reviews 13:155-68, 2002; Wigginton et al, J. Clin. Invest. 108:51-62, 2001; Nasu et al, Gene Ther. 6:338-49, 1999; Yoshida et al, Anticancer Res. 18:333-35, 1998; Brunda et al, Clin. Immunol. Immunopathol. 71:253-55, 1994). IL-18: lung (Wigginton et al, J. Immunol. 169:4467-74, 2002); neuroblastoma (Redlinger et al, J. Pediatr. Surg. 38:301-07, 2003); melanoma (Hashimoto et al., J. Immunol. 163:583-89, 1999); sarcoma (Osaki et al, J. Immunol. 160:1742-49, 1998); and renal cell carcinoma (Hara et al., J. Urol. ,165:2039-43, 2001).

In some embodiments of the present disclosure, the therapeutically effective amount of the cytokine in the presence of the proteasome inhibitor is lower than when the tumor cell is exposed to the cytokine alone. Particular examples of proteasome inhibitors are peptidyl boronic acid ester and acid compounds. A proteasome inhibitor can include bortezomib, MG-132, MG-115, MG-262, PSI, MG-101, ALLM, TLCK, TPCK, PDTC, EST, epoxomicin, lactacystin, and PTX. Inhibitors are used, for example, that upregulate expression of a cellular receptor, such as a cell surface receptor for a cytokine (such as an IFN-α receptor, an IFN-β receptor, an IFN-γ receptor, and/or a TNF-α receptor). Examples of such inhibitors are bortezomib, MG-132, MG-115, MG-262, PSI, MG-101, ALLM, TLCK, TPCK, PDTC, EST, epoxomicin, lactacystin, PTX, and combinations thereof. Additional proteasome inhibitors can be identified by their ability to inhibit proteasome activity. Various strategies for the identification of such inhibitors are well known in the art. For example, small molecule libraries, often comprising extracts from plants or more simple organisms, can be screened for their ability to inhibit specific protease types. Alternatively, a rational design approach can be applied using, for example, peptide or peptidomimetic compounds designed specifically to interact with the active site of a proteasome component (see, for example, published international application WO91/13904; Powers et ai, in Proteinase Inhibitors, Barrett et al. (eds.), Elsevier, pp 55-152, 1986). The inhibitors can be stable analogs of catalytic transition states, such as Z-Gly-Gly-Leu-H, which inhibits the chymotrypsin-like activity of the proteasome (see, for example, OrIo wski, M., Biochemistry 29:10289-97, 1990). A further example is found herein in Example 3.

In particular embodiments, the proteasome inhibitor is a peptidyl boronic acid ester or acid compound, such as N-pyrazinecarbonyl-L-phenylalanine-L-leucineboronic acid (bortezomib or PS-341) or N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucyl boronic acid (MG-262). In another embodiment, the proteasome inhibitor is a peptide aldehyde, such as carbobenzyloxy-L-leucyl-L-leucyl-L-leucinal (MG- 132), carbobenzyloxy-L-leucyl-L-leucyl-L-norvalinal (MG-115), 7V-benzyloxycarbonyl-Ile- Glu(O-t-butyl)-Ala-leucinal (PSI; and its epoxide), N-acetyl-Leu-Leu-norleucinal (MG- 101, ALLN or calpain inhibitor I), or iV-acetyl -Leu-Leu-Met (ALLM or calpain inhibitor II). In other embodiments, the proteasome inhibitor is a peptide alpha-keto ester, such as TV-tosyl-Lys chloromethyl ketone (TLCK) and N-tosyl-Phe chloromethyl ketone (TPCK). In still other embodiments, the proteasome inhibitor is a non-peptide inhibitor, such as pyrrolidine dithiocarbamate (PDTC), [2S,3S]-trans-epoxysuccinyl-L- leucylamido-3-methylbutane ethyl ester (EST), epoxomicin, lactacystin, and pentoxyfilline (PTX). Peptidyl boronic acid ester and acid compounds (for example, bortezomib and

MG-262) that function as proteasome inhibitors act as transition-state analogues for serine proteinases because the boron can accept the oxygen lone pair of the active site serine residue. Without being bound by theory, it seems likely that these compounds react similarly with the catalytic N-terminal threonine residue of the proteasome catalytic subunit. Peptidyl boronic acid ester and acid compounds act reversible inhibitors of the 26S proteasome.

Bortezomib induces apoptosis in several distinct human tumor cell types in vitro (Hideshima et al, Cancer Res. 61:3071-76, 2001; Yin et al, Oncogene 24:344-54, 2005), and possesses potent antitumor activity in several preclinical models of cancer, including prostate (Adams et al, Cancer Res. 59:2615-22, 1999), breast and lung (Teicher et al, Clin. Cancer Res. 5:2638-45, 1999) carcinoma. Mechanistically, bortezomib can enhance the expression of key cell cycle and proapoptotic molecules including p53 (An et al, Leukemia 14:1276-83, 2000), p27 (Hideshima et al, Cancer Res. 61 :3071-76, 2001), p21 (Yin et al, Oncogene 24:344-54, 2005), Fas/FasL (Mitsiades et al, Proc. Natl. Acad. ScL USA 99:14374-79, 2002), and TRAIL-R2 (Mitsiades et al, Proc. Natl. Acad. ScL USA 99:14374-79, 2002). Furthermore, bortezomib can block the activation of nuclear factor κ-B (NF-κB) (Cusack et al, Cancer Res. 61:3535-40, 2001) and abrogates the expression/activity of prosurvival factors such as c-FLIP (Mitsiades et al, Proc. Natl Acad. ScL USA 99:14374-79,

2002), Bcl-xL (Yin et al, Oncogene 24:344-54, 2005), Bcl-2 (Bold et al, J. Surg. Res. 100:11-17, 2001), c-IAP-2 (Mitsiades et al, Proc. Natl Acad. ScL USA 99:14374-79, 2002), and AKT (Dai et al, Blood 104:509-18, 2004).

The peptide aldehydes (for example, MG- 132, MG-115 and PSI) inhibit the proteasome's chymotrypsin-like activity in a potent but reversible manner. MG-101 and ALLM are cell-permeable inhibitors of calpain I, calpain II, cathepsin B, and cathepsin L. They also inhibit the proteasome. TLCK and TPCK are serine protease inhibitors that also inhibit the proteasome. PDTC is an antioxidant and also functions as a proteasome inhibitor. EST is a cell-permeable, irreversible inhibitor of cysteine proteases and the proteasome. Epoxomicin, originally isolated from a species of Actinomycetes, is cell-permeable, irreversible and a relatively selective proteasome inhibitor that inhibits the chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide- hydrolyzing activities of the proteasome. Lactacystin is a natural, irreversible, nonpeptide, cell permeable inhibitor that is more selective than peptide aldehydes but less selective than peptide boronates. PTX, a xanthine derivative which is widely as a haemorheological agent also functions as a proteasome inhibitor.

In addition to known proteasome inhibitors, the present disclosure is intended to encompass other molecules that can be routinely tested for their ability to inhibit proteasome activity and/or the ubiquitin-proteasome pathway, such as inhibiting an El, E2 or E3 ubiquitin ligase or a deubiquitinating enzyme. Various strategies for the identification of such inhibitors are well known in the art. For example, small molecule libraries, often comprising extracts from plants or more simple organisms, can be screened for their ability to inhibit specific protease types. Alternatively, a rational design approach can be applied using, for example, peptide or peptidomimetic compounds designed specifically to interact with the active site of a proteasome component (see, for example, published International Application No. WO91/13904; Powers et ah, in Proteinase Inhibitors, Barrett et a (eds.), Elsevier, pp 55-152, 1986, Garber, K, J. Nat. Cancer Inst. 97:166-67, 2005, all of which are incorporated herein by reference). The inhibitors can be stable analogs of catalytic transition states, such as Z- Gly-Gly-Leu-H, which inhibits the chymotrypsin-like activity of the proteasome (see, for example, Orlowski, M., Biochemistry 29:10289-97, 1990, which is incorporated herein by reference).

The proteasome that is to be inhibited by a proteasome inhibitor can be found in a subject, or contained in a variety of biological samples. For example, the proteasome can be contained in a histologic section of a specimen obtained by biopsy, cells obtained from body fluids or cells that are placed in or adapted to tissue culture. An isolated or purified proteasome is removed or separated from at least one component with which it is naturally associated. Therefore, an isolated proteasome can be contained in a subcellular fraction or extract prepared from cells containing proteasomes, such as a cytoplasmic lysate, a membrane preparation, a nuclear extract, or a crude or purified protein preparation. A sample containing a proteasome can be prepared by methods known in the art suitable for the particular format of the detection method. For example, biochemical methods such as precipitation and immunoaffinity methods can be used to isolate a proteasome from a cell. Procedures for preparing subcellular fractions, such as nuclear fractions and cell lysates, are well known to those of skill in the art, and include, for example, cell disruption followed by separation methods such as gradient centrifugation and biochemical purification methods.

V. Methods of Using Proteasome Inhibitors

The present disclosure includes methods for enhancing an antitumor activity of a cytokine on a tumor cell that responds to treatment with the cytokine, including exposing the tumor cell to a therapeutically effective amount of the cytokine and a proteasome inhibitor, wherein the proteasome inhibitor is present in a sufficient amount to enhance the activity (such as a proapoptotic activity) of the cytokine, thereby enhancing the antitumor activity of the cytokine. In some methods, a therapeutically effective amount of a cytokine and a proteasome inhibitor is administered to a subject to inhibit the development of or treat an existing neoplasm of an exposed body surface. Additional methods involve administering to a subject or contacting one or more malignant cells of a subject with a therapeutically effective amount of a cytokine and a proteasome inhibitor to inhibit metastasis of the malignant cells. Any living, multicellular, vertebrate organism capable of developing one or more neoplasms is contemplated as a subject for the disclosed methods. Thus, in particular examples, a subject of a disclosed method is a human or veterinary subject.

A therapeutically effective amount of a cytokine and a proteasome inhibitor can be used to treat or prevent, or inhibit metastasis from, any neoplasm. Non-limiting examples of neoplasms include tumors of the skin, such as squamous cell carcinoma, basal cell carcinoma, melanoma, skin appendage tumors, papilloma, cutaneous T-cell lymphoma (mycosis fungoides), apocrine carcinoma of the skin, or Merkel cell carcinoma, breast carcinomas, for example, lobular and duct carcinomas and other solid tumors, sarcomas and carcinomas of the lung, such as small cell carcinoma, large cell carcinoma, squamous carcinoma, adenocarcinoma, and mesothelioma of the lung, colorectal adenocarcinoma, stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma, such as serous cystadenocarcinoma and mucinous cystadenocarcinoma, and ovarian germ cell tumors, testicular carcinomas, germ cell tumors, pancreatic adenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma, bladder carcinoma, including transitional cell carcinoma, adenocarcinoma and squamous carcinoma, renal cell adenocarcinoma, endometrial carcinomas, including adenocarcinomas and mixed Mullerian tumors (carcinosarcomas), carcinomas of the endocervix, ectocervix and vagina, such as adenocarcinoma and squamous carcinoma, esophageal carcinoma, carcinomas of the nasopharynx and oropharynx, including squamous carcinoma and adenocarcinomas, salivary gland carcinomas, brain and central nervous system tumors, including tumors of glial, neuronal and meningeal origin, tumors of peripheral nerve, soft tissue sarcomas and sarcomas of bone and cartilage, and non-solid hematopoietic tumors, such as leukemias.

A therapeutically effective amount of a cytokine and a proteasome inhibitor can also be used to treat (for example, by inducing apoptosis) other cell types found in the tumor microenvironment, such as vascular endothelial cells, pericytes, fibroblasts and/or other stromal elements.

Particular examples of cytokines include interferon-alpha, interferon-beta, interferon-gamma, and/or tumor necrosis factor alpha inducing cytokines. Non-limiting examples of cytokines include interleukin-2, interleukin-12, interleukin-18, interleukin- 32, interferon-alpha, interferon-beta, interferon-gamma, tumor necrosis factor alpha, and combinations thereof.

Exemplary methods involve treating, preventing, or inhibiting metastasis of an neoplasm. Treatment of a neoplasm using a disclosed method can involve, for example, inhibiting the growth of the neoplasm, reducing the size of the neoplasm, inducing apoptosis of the neoplasm, or inhibiting metastasis of the neoplasm. Inhibiting the growth of a neoplasm conveys a wide-range of inhibitory effects that a treatment (for example, a cytokine and a proteasome inhibitor) can have on the initiation and growth of a neoplasm, for example, as compared to an untreated (or pre-treatment) neoplasm. Thus, inhibiting the growth of a neoplasm includes situations wherein an incidence of neoplasm is reduced or the normal growth rate of the neoplasm has slowed (for example, the number of neoplastic cells still increases over time, but not as rapidly as in a control neoplastic cell population), equals zero (for example, there is substantially no change in number of neoplastic cells in the population over time; for instance, neoplastic cell growth is approximately equal to cell death or quiescence in the same population), or becomes negative (for example, the number of neoplastic cells decreases over time; for instance, cell death exceeds cell growth or quiescence). A reduction in the size of a neoplasm can be determined using any methods or standard known to the ordinarily skilled artisan. In one embodiment, the decrease in one or more physical dimensions of a neoplasm (such as, diameter, volume, length, width, or weight), as compared to corresponding measurement(s) made at an earlier time point (such as pre-treatment or earlier in a course of treatment), can indicate a neoplasm size reduction.

There are several assays for measuring apoptosis. For example, apoptotic cell death can be characterized by cell shrinkage, membrane blebbing and chromatin condensation culminating in cell fragmentation. Cells undergoing apoptosis also display a characteristic pattern of internucleosomal DNA cleavage. Apoptosis can be measured in the presence or the absence of Fas-mediated signals. In another example, cytochrome C release from mitochondria during cell apoptosis can be detected (see, for example, Bossy-Wetzel et al, Methods in Enzymol. 322:235-42, 2000). Other assays include cytofluorometric quantitation of nuclear apoptosis induced in a cell-free system (see, for example, Lorenzo et al, Methods in Enzymol. 322:198-201, 2000), apoptotic nuclease assays (see, for example, Hughes, FM, Methods in Enzymol. 322:47-62, 2000), microscopic analysis of apoptotic cells by flow and laser scanning cytometry (see, for example, Darzynkiewicz et al. , Methods in Enzymol. 322: 18-39, 2000), annexin-

V/propidium iodide labeling (as provided herein in Example 1), transient transfection assays for cell death genes (see, for example, Miura et al. , Methods in Enzymol. 322:480-92, 2000), and assays that detect DNA cleavage (see, for example, Kauffman et al, Methods in Enzymol 322:3-15, 2000). Apoptosis can also be measured by TdT incorporation of labeled nucleotides into DNA strand breaks (TUNEL assay). This system is a fluorescent TUNEL assay that measures apoptotic DNA fragmentation by directly incorporating fluorescein- 12- dUTP at the 3'-OH DNA ends using Terminal Deoxynucleotidyl Transferase (TdT), which forms a polymeric tail. The fluorescein-dUTP-labeled DNAs from transfected cells are then visualized directly by fluorescence microscope or quantitated by flow cytometry.

Inhibiting metastasis of a neoplasm (or malignant cells thereof) conveys a wide-range of inhibitory effects that a treatment (for example, a cytokine and a proteasome inhibitor) can have on metastasis of such neoplasm (or malignant cells). For example, inhibiting metastasis may be considered relative to an untreated (that is, uninhibited or control) rate of metastasis of a particular malignant cell or population of malignant cells of interest. Thus, inhibiting metastasis includes situations wherein the metastatic rate of a cell or cell population has slowed (that is, the number metastatic cells decreases over time as compared to a control population), or is reduced to near zero (that is, there are substantially no metastatic cells in the population over time).

Toxicity and therapeutic efficacy of a treatment, such as a cytokine and a proteasome inhibitor, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the MIC50 (the lowest tested concentration that inhibits the growth of the population by at least 50%), LD₅₀ (the dose lethal to 50% of the population) and/or the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it may be expressed, for example, as the ratio LD₅₀/ED₅₀. A combination cytokine and proteasome inhibitor that exhibit large therapeutic indices are useful, as are combinations that exhibit toxic side effects.

However, in the case of combinations with toxic side effects, it can be helpful to design a delivery system that targets such combinations to the site of affliction to minimize potential damage to normal cells and, thereby, reduce side effects. VI. Pharmaceutical Compositions

This disclosure contemplates administering to a subject a cytokine and a proteasome inhibitor as a method for enhancing an antineoplastic activity of the cytokine. Any delivery system or treatment regimen that effectively treats or inhibits the development (including metastasis) of a neoplasm of interest can be used.

Accordingly, pharmaceutical compositions comprising at least one cytokine (such as IL-2, IL-12, IL-18, IL-32, IFN-γ, or TNF-α) and at least one proteasome inhibitor (such as bortezomib or MG-132) are also described herein. The cytokine and proteasome inhibitors are present in the composition in a therapeutically effective amount. Formulations for pharmaceutical compositions are well known in the art. For example, Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes exemplary formulations (and components thereof) suitable for pharmaceutical delivery of a cytokine and a proteasome inhibitor. Pharmaceutical compositions comprising at least one cytokine and at least one proteasome inhibitor can be formulated for use in human or veterinary medicine. Particular formulations of a disclosed pharmaceutical composition may depend, for example, on the mode of administration (for example, topical, oral or parenteral) and/or on the location of the neoplasm to be treated. In some embodiments, formulations include a pharmaceutically acceptable carrier in addition to at least one cytokine and at least one proteasome inhibitor. In other embodiments, other medicinal or pharmaceutical agents, for example, with similar, related or complementary effects on the affliction being treated (such as a neoplasm), can also be included as active ingredients in a pharmaceutical composition.

The pharmaceutical compositions comprising at least one cytokine and at least one proteasome inhibitor described herein may be formulated in a variety of ways depending, for example, on the mode of administration and/or on the location and type of neoplasm to be treated. For example, such pharmaceutical compositions may be formulated as a pharmaceutically acceptable salt of a disclosed cytokine and/or proteasome inhibitor. As another example, parenteral formulations may comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles, such as water, physiological saline other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients may include, for example, nonionic solubilizers, such as cremophor, or proteins, such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition to be administered may also contain non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. For example, in addition to injectable fluids, topical and oral formulations may be employed. Topical preparations may include eye drops, ointments, sprays and the like. The compositions can be applied onto an exposed body surface using any known or otherwise effective application technique including, but not limited to, the techniques of rubbing, brushing, painting, wiping, and stroking a composition onto the skin. When the pharmaceutical composition administered in a cutaneous or topical carrier or diluent, the carrier or diluent may be chosen from any known in the cosmetic or medical arts; for example, any gel cream, lotion, ointment, liquid or non liquid carrier, emulsifier, solvent, liquid diluent or other similar vehicle which does not exert deleterious effect on the skin or other living animal tissue. Other methods of administering the pharmaceutical compositions comprising at least one cytokine and at least one proteasome inhibitor described herein include parental or enteral routes, such as intrathecal, intradermal, intramuscular, intraperitoneal (i.p.), intravenous (i.v.), subcutaneous, intranasal, epidural, and oral routes. For solid compositions, conventional non-toxic solid carriers may include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

The pharmaceutical compositions can be administered by any convenient route, including, for example, infusion or bolus injection, absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, and the like), ophthalmic, nasal, and transdermal, and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce a pharmaceutical composition by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. Pulmonary administration can also be employed (for example, by an inhaler or nebulizer), for instance using a formulation containing an aerosolizing agent.

In a specific embodiment, it may be desirable to administer a pharmaceutical composition locally to an area in need of treatment (for example, to an area of the body with a solid tumor). This can be achieved by, for example, local or regional infusion or perfusion during surgery, topical application, injection, catheter, suppository, or implant (for example, implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like, hi one embodiment, administration can be by direct injection at the site (or former site) of a neoplasm that is to be treated, hi another embodiment, the pharmaceutical composition is delivered in a vesicle, such as liposomes (see, for example, Langer, Science 249: 1527-33, 1990 and Treat et ah, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, N. Y., pp. 353-65, 1989). In treatment of a cutaneous tumor, such as a melanoma, the drug can be applied directly to a cutaneous lesion.

In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system, hi one example, a pump can be used (see, e.g., Langer, Science 249:1527-33, 1990; Sefton, Crit. Rev. Biomed. Eng. 14:201-40, 1987; Buchwald et ah, Surgery 88:507-16, 1980; Saudek et ah, N. Engl. J. Med. 321:574-79, . 1989). In another example, polymeric materials can be used (see, for example, Levy et ah, Science 228:190-92, 1985; During et ah, Ann. Neurol. 25:351-56, 1989; Howard et ah, J. Neurosurg. 71:105-12, 1989). Other controlled release systems, such as those discussed by Langer (Science 249:1527-33, 1990), can also be used. The ingredients in various embodiments are supplied either separately or mixed together in unit dosage form, for example, in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, or suspensions, or as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the pharmaceutical composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to administration.

VII. Therapeutically Effective Amounts and Dosage Regimens

Therapeutic treatments can include a therapeutically effective amount of a cytokine and a proteasome inhibitor. Ideally, a therapeutically effective amount of an agent is an amount sufficient to effect the desired result (for example, inhibiting a neoplasm or metastasis of a malignant cell), without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for preventing or otherwise treating a neoplasm or inhibiting metastasis of a malignant cell will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. Effective amounts can be determined by standard clinical techniques.

For example, when administering a pharmaceutical composition comprising at least one cytokine and at least one proteasome inhibitor, the precise dose to be employed in the formulation will depend on the route of administration, and should be decided according to the judgment of the health care practitioner and each patient's circumstances. The concentration of an active ingredient (such as a cytokine and a proteasome inhibitor) in a topical composition (such as an ointment, cream, gel, or lotion) is typically from about 0.2% to about 1% (by weight relative to the total weight of the topical composition); for example, from about 0.3% to about 0.9%, from about 0.4% to about 0.8%, and from about 0.5% to about 0.7%. Within the ranges, higher concentrations allow a suitable dosage to be achieved while applying the lotion, ointment, gel, or cream in a lesser amount or with less frequency. In other embodiments, a dosage range for non-topical administration (such as oral administration, or intravenous or intraperitoneal injection) of a pharmaceutical composition containing at least one cytokine and at least one proteasome inhibitor is from about 0.1 to about 200 mg/kg body weight in single or divided doses; for example from about 1 to about 100 mg/kg, from about 2 to about 50 mg/kg, from about 3 to about 25 mg/kg, or from about 5 to about 10 mg/kg.

Acceptable dosages of the active ingredients (such as a cytokine and a proteasome inhibitor) of the pharmaceutical compositions of the present disclosure are, for example, dosages that achieve a target tissue concentration similar to that which produces the desired antiproliferative effect in vitro. Acceptable dosages of both cytokines and proteasome inhibitors are known in the art. It is anticipated that these known dosages can be used in combination to provide the superior antitumor effects of the present methods.

The pharmaceutical compositions of the present disclosure can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (for example, in which the loading dose is about two to five times the maintenance dose). In some embodiments, the dose is varied during the course of a treatment based on the condition of the subject being treated, the severity of the neoplasm, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art. In some embodiments long-term treatment with a disclosed pharmaceutical composition is contemplated, for instance in order to prevent reoccurrence of a neoplasm.

The subject matter of the present disclosure is further illustrated by the following non-limiting Examples. EXAMPLES

Example 1

Bortezomib inhibits proliferation and decreases the viability of TBJ and Neuro-2a neuroblastoma cell lines and the EOMA endothelial cell line This example demonstrates the ability of a proteasome inhibitor to inhibit proliferation and decease the viability of neuroblastoma cells.

Murine Neuro-2a neuroblastoma and EOMA hemangioendothelioma cell lines were purchased from ATCC (Rockville, MD). TBJ, a metastatic sub-clone of Neuro-

2a, and was provided by Dr. Moritz Ziegler (Children's Hospital, Boston, MA). TBJ and Neuro-2a neuroblastoma cells, syngeneic to A/J mice, were maintained via serial passage in vivo. TBJ cells transfected to overexpress red fluorescent protein (TBJ-RFP) were generated as described by Salcedo et al. (J. Immunol. 173:7170-82, 2004). TBJ cells engineered to overexpress a fusion construct encoding BID fused to green fluorescent protein (pd4EGFP-BID) were generated using the standard FuGENE method of transfection. Stably-transfected TBJ clones expressing high levels of BID-

EGFP (TBJ-BID-EGFP) were subsequently selected in the presence of geneticin.

Expression of BID-EGFP was confirmed by fluorescence microscopy, as well as western blot analysis of tumor cell lysates using a monoclonal rat anti-mouse BID antibody. Bortezomib (Millennium Pharmaceuticals, Cambridge, MA) was reconstituted according to the manufacturer's instructions and diluted in 0.9% normal saline prior to in vivo administration.

To investigate the effect of bortezomib on tumor cell proliferation, Neuro-2a and

TBJ tumor cells (1x10⁴ cells/well) were incubated in triplicate for 48 h in 96-well plates with various concentrations of bortezomib. Cells were pulsed with ³ [H] -thymidine (1 μCi/well) 16 h prior to harvest, and ³ [H] -thymidine incorporation was determined using standard techniques.

To investigate the effect of bortezomib on tumor cell apoptosis, pre-adhered TBJ or Neuro-2a neuroblastoma cells were cultured with various concentrations of bortezomib for 4 h followed by IFN-γ (murine, specific activity > 1x10 U/mg, Peprotech, Rocky Hill, NJ, 100 IU/ml)+TNF-α (recombinant murine, specific activity > lxlO⁷U/mg, Peprotech, Rocky Hill, NJ, 50 ng/ml), FasL (Alexis Biochemicals, San Diego, CA, 100 ng/ml), TRAIL (Alexis Biochemicals, San Diego, CA, 200 ng/ml) or medium alone for an additional 20 or 44 h. EOMA endothelial cells were treated similarly with bortezomib and incubated for another 18 h with IFN-γ+TNF-α. Cells were subsequently harvested, stained with annexin-V-FITC and propidium iodide, and analyzed for apoptosis/viability using a FACScan flow cytometer and CellQuest software (BD Biosciences, Mountain View, CA). TBJ and Neuro-2a murine neuroblastoma tumors are intrinsically-resistant to receptor-mediated apoptosis. More specifically, these cells demonstrate limited expression of death receptors including FAS and TRAIL-R2, and express high levels of phosphorylated AKT, a key anti-apoptotic, prosurvival factor (Khan et al., manuscript submitted). TBJ and Neuro-2a neuroblastoma cells were treated with bortezomib and the proliferative capacity of tumor cells was determined as described herein. At day 3 after treatment, the proliferation of TBJ and Neuro-2a was inhibited by 86% and 77%, respectively, compared to controls, even at very low (10 nM) concentrations of bortezomib (FIG. IA and IB). Bortezomib also induced apoptosis and consequently inhibited tumor cell viability (as assessed via annexin-v/propidium iodide staining) at concentrations as low as 10 nM (FIG. 1C and ID). Only 69+3% (10 nM) and 14.4+2% (50 nM) of TBJ cells were viable after two days of exposure to bortezomib, compared to 95+0.3% of untreated control cells. Similarly, only 67+0.4% (10 nM) and 26%+0.5 (50 nM) of Neuro-2a cells were viable after two days of exposure to bortezomib, compared to 86+3% of untreated control cells. Example 2

Bortezomib inhibits AKT phosphorylation and induces Bid translocation in murine neuroblastoma cells

This example demonstrates the ability of a proteasome inhibitor to inhibit AKT phosphorylation and induce BID translocation in neuroblastoma cells.

Bortezomib was able to induce marked apoptosis in both TBJ and Neuro-2a cells despite previous observations that these cells express high levels of phosphorylated AKT (active) and are intrinsically resistant to apoptosis. Consequently, the ability of bortezomib to inhibit the activity of AKT and/or induce the expression/activity of proapoptotic genes in these cells was investigated. TBJ and

Neuro-2a tumor cells were treated with bortezomib or vehicle control for 24 h and total protein lysates were extracted from cultured cells using standard techniques and analyzed by western blotting for AKT expression/phosphorylation. Briefly, protein concentrations were determined using the BCA protein assay reagent (Pierce, Rockford, IL). Equal amounts of protein boiled in sample buffer were separated on an 8% SDS- polyacrylamide gel and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Tween-20, and then probed with either polyclonal rabbit anti-mouse AKT (total AKT) or phospho-specific polyclonal rabbit anti-mouse AKT (ser-473) antibodies (Cell Signaling Technology, Beverly, MA). After washing, the membranes were incubated with HRP -conjugated goat anti-rabbit antibody (BIO-RAD, Hercules, CA) and the immunoreactive bands were visualized using the ECL Plus detection system from Amersham Biosciences (Buckinghamshire, UK).

Even at low nanomolar concentrations, bortezomib markedly inhibited the phosphorylation of AKT (serine-473) in both TBJ and Neuro-2a tumor cells, while the levels of total AKT remained essentially unaltered (FIG. 2A). Because phosphorylation of AKT on ser-473 is required for maximum AKT activity (Stokoe et al., Science 277:567-70, 1997; Bellacosa et al., Oncogene 17:313-25, 1998), this data suggested that bortezomib inhibited AKT function in both TBJ and Neuro-2a neuroblastoma cells. Bid is a proapoptotic molecule that is normally suppressed by activated AKT (Nesterov et al., J. Biol. Chem. 276:10767-74, 2001; Chen et al, Oncogene 20:6073-83, 2001). During immune/receptor-mediated apoptosis, cytoplasmic Bid is proteolytically cleaved (activated) to form truncated Bid (tBid). tBid then translocates to the mitochondria, where it contributes to mitochondria-dependent pathways for apoptosis (Luo et al, Cell 94:481-90, 1998).

To investigate whether the ability of bortezomib to inhibit AKT phosphorylation resulted in a corresponding enhancement of Bid activation and subcellular translocation, TBJ cells engineered to overexpress a fusion construct consisting of Bid linked to green fluorescence protein were generated (TBJ-Bid-EGFP). TBJ-Bid-EGFP cells (IxIO⁵) were grown overnight in 35 mm glass bottom micro well poly-d-lysine coated dishes (MatTek Cultureware, Ashland, MA). Old culture medium was removed and the cells were incubated with either fresh medium or with bortezomib (20 nM) for an additional 18 h. Cells were then analyzed for subcellular BID-EGFP localization using the Carl Zeiss LSM 410 (Germany) confocal laser-scanning microscope. A 488 nm Ar/Kr laser with a 500-550 nm band pass emission filter was used. The objective used was an LD- Apostigmat 2OX dry lens with a numerical aperture of 0.30. In control cells, a diffuse pattern of GFP expression was noted consistent with cytoplasmic localization of Bid- EGFP (FIG. 2B and 2C). hi marked contrast, treatment of these cells with bortezomib induced a striking punctuate pattern of GFP distribution that is consistent with cleavage (activation) and translocation of Bid from the cytosol to the mitochondria (FIG. 2D and 2E).

Example 3 Bortezomib upregulates IFN-γ and TNF-α receptor expression and sensitizes TBJ neuroblastoma cells to apoptosis induced by IFN-γ+TNF-α

This example demonstrates the ability of a proteasome inhibitor to upregulate IFN-γ and TNF-α receptor expression and sensitize TBJ neuroblastoma cells to apoptosis induced by IFN-γ+TNF-α. Although TBJ neuroblastoma cells express receptors for TNF-α, they do not express Fas and only express negligible amounts of TRAIL-R2. To investigate the impact of bortezomib on cell surface death receptor expression, TBJ or EOMA or cells were incubated with various concentrations of bortezomib (5-30 nM) or medium alone for 4 h. This was followed by incubation with IFN-γ (100 IU/ml)+TNF-α (50 ng/ml)+/- bortezomib or medium alone for an additional 18-20 hours. Cells were then harvested and stained with either PE-labeled hamster anti-mouse TRAIL-R2 (MD5-1, e- Bioscience, San Diego, CA), PE-labeled hamster anti-mouse Fas antibody (Jo-2, BD Pharmingen, San Diego, CA) or PE-labeled hamster anti-mouse IgG isotype control antibody (BD Pharmingen, San Diego, CA). The expression of cell surface TNF-RI or IFN-γ-R was also evaluated on TBJ cells cultured under similar conditions. Cells were labeled with hamster anti-mouse TNF-RI, IFN-γ-Rα or IFN-γ-Rβ antibodies or hamster anti-mouse IgG isotype control antibody, followed by staining with biotin-conjugated mouse anti-hamster IgG and PE-labeled streptavidin (BD Pharmingen, San Diego, CA). Cells were fixed in 1% paraformaldehyde in PBS and analyzed using a FACScan flow cytometer and CellQuest software (BD Biosciences, Mountain View, CA).

Treatment of TBJ cells with bortezomib (5 nM) for 24 h did not result in any changes in the cell surface expression of either Fas or TRAIL-R2 (FIG. 3A), but did increase the expression of TNF-RI (control= 11+5% vs. bortezomib= 44+20%). As IFN-γ can enhance the sensitivity of several malignant cell types to TNF-α in vitro (Varela et al., J. Biol. Chem. 276:17779-87, 2001; Fulda and Debatin, Oncogene 21:2295-08, 2002), the ability of bortezomib to modulate the expression of the IFN-γ receptor on TBJ cells was also investigated. The IFN-γ receptor is composed of a ligand binding subunit (IFN-γ-Rα) and a signaling subunit (IFN-γ-Rβ) (Aguet et al, Cell 55:273-80, 1988; Soh et al, Cell 76:793-802, 1994). TBJ cells express low levels of IFN-γ-Rβ, and expression of this subunit is not upregulated by treatment with bortezomib. In contrast, bortezomib markedly enhances cell surface expression of IFN- γ-R-α subunit on TBJ cells (control= 13+0.8% vs. bortezomib at 5 nM= 24+7% vs. bortezomib at 10 nM= 53+7%) (FIG. 3B).

Inhibitors of protein synthesis (cycloheximide) or small molecule inhibitors of PI3K/AKT (LY294002/SH5) can also sensitize TBJ or Neuro-2a cells to apoptosis induced by IFN-γ/TNF-α in vitro. As bortezomib both inhibits AKT activity in murine neuroblastoma cells and enhances cell surface expression of receptors for TNF-α and IFN-γ, the ability of bortezomib to sensitize TBJ or Neuro-2a tumor cells to apoptosis induced by IFN-γ+TNF-α was investigated. Cells were pretreated with bortezomib for 4 hours and then incubated with IFN-γ (100 IU/ml)+TNF-α (50 ng/ml) for up to a total of 48 h. Bortezomib increased the sensitivity of both TBJ and Neuro-2a neuroblastoma cells to IFN-γ+TNF-α-mediated apoptosis resulting in large decreases in tumor cell viability (annexin-v/propidium iodide negative cells) (FIG. 3C and 3D). Thus, at day 2 of combined treatment with bortezomib (5nM) and IFN-γ+TNF-α only 22% of TBJ cells were viable, compared to 95% in controls, and 63% versus 93% of cells treated with bortezomib or IFN-γ+TNF-α alone, respectively (FIG. 3C). Somewhat higher concentrations of bortezomib were required to sensitize Neuro-2a cells to IFN-γ+TNF- α-mediated apoptosis. At concentrations of 30 nM and above, bortezomib markedly enhanced the sensitivity of Neuro-2a cells to IFN-γ+TNF-α as early as day one after treatment (FIG. 3D). Only 17% of Neuro-2a cells were viable after combined treatment with bortezomib (50 nM) and IFN-γ+TNF-α, compared to 77% in controls, and 77% versus 75% of cells treated with either bortezomib or IFN-γ+TNF-α alone. In contrast, bortezomib did not increase the sensitivity of either TBJ or Neuro-2a cells to FasL or TRAIL-mediated apoptosis.

To investigate whether bortezomib could also modulate apoptosis in endothelial cell populations, EOMA, a murine microvascular endothelial cell line, was utilized.

Pre-exposure of EOMA cells to bortezomib markedly enhanced their sensitivity to IFN- γ+TNF-α-mediated apoptosis (FIG. 3E). Only 27% of EOMA cells were viable (annexin-v/propidium iodide negative) after combined treatment with bortezomib and IFN-γ+TNF-α, compared to 97±0.1% in controls, and 95% versus 80% of cells treated with either bortezomib or IFN-γ+TNF-α alone, respectively. Bortezomib also enhanced cell surface expression of IFN-γ-Rα on EOMA cells in vitro (FIG. 3F). These results demonstrate that bortezomib can increase receptor expression for IFN-γ in both tumor and endothelial cells and sensitize them to IFN-γ+TNF-α-mediated apoptosis.

Example 4

Bortezomib potentiates the antitumor activity of IL-2 in mice bearing well- established primary TBJ neuroblastoma tumors This example demonstrates the ability of a proteasome inhibitor to potentiate the antitumor activity of IL-2 in mice with well-established TBJ neuroblastoma tumors.

As bortezomib markedly enhanced the sensitivity of TBJ neuroblastoma tumor cells to TNF-α+IFN-γ-mediated apoptosis in vitro, the ability of bortezomib to potentiate the proapoptotic and overall antitumor activity of potent IFN-γ- and TNF-α- inducing cytokines (such as IL-2 or IL- 12) in vivo was investigated. Male A/J mice purchased from the Animal Production Area (Charles River, Frederick, MD) were generally used at 8-10 weeks of age. Cohorts of 10 A/J mice per group were used in all therapy studies. To establish subcutaneous (SC) primary tumors, mice were injected subcutaneously in the mid-flank with syngeneic murine TBJ neuroblastoma tumor cells (1.2x10⁶ cells/animal in 0.2 ml HBSS).

Mice bearing well-established day 6 SC-TBJ tumors were injected intraperitoneally (i.p.) each morning with IL-2 (recombinant human, Chiron Corporation, Emeryville, CA, 50,000 IU in 0.2 ml HBSS containing 0.1% homologous serum) or vehicle alone, on days 6-10, 13-17, and 20-24 post tumor implantation. Bortezomib (20 μg in 0.2 ml 0.9% normal saline) or vehicle alone was administered i.p. in the afternoon on days 7, 10, 14, 17, 21, and 24 post tumor implantation. Mice were monitored for tumor growth, and bi-directional tumor dimensions were determined using calipers. Estimated tumor volumes were then determined by calculating the product of the smallest measured tumor dimension squared multiplied by the largest measured tumor dimension.

The Jonckheere-Terpstra test for trend was used to compare tumor volumes among the respective control, bortezomib, IL-2, or bortezomib/IL-2 treatment groups in mice bearing subcutaneous TBJ tumors. All p values were considered significant at /K0.05.

Combined administration of IL-2 and bortezomib delayed progression of SC- TBJ tumors more effectively than either of the single agents alone (FIG. 4). A highly significant trend towards reduction in tumor volume was observed in mice treated with the combination of IL-2 and bortezomib compared to mice treated with either single agent alone or control mice treated with vehicles alone. This strong trend was observed as early as day 9 post tumor implantation (day 3 of therapy) (p= 0.009), and was sustained through day 27 post tumor implantation (day 21 of therapy) (p= 0.001).

Example 5

Bortezomib potentiates the antitumor activity of IL-2 and IL-12 in mice bearing established metastatic TBJ neuroblastoma tumors

This example demonstrates the ability of a proteasome inhibitor to potentiate the antitumor activity of IL-2 and IL- 12 in mice with established metastatic TBJ neuroblastoma tumors.

As combined administration of IL-2 and bortezomib delayed progression of SC- TBJ tumors more effectively than either of the single agents alone, the ability of bortezomib to potentiate the antitumor activity of cytokine therapy (for example, IL-2 or IL- 12) in a more therapeutically-challenging setting of induced metastatic disease was investigated. Male A/J mice purchased from the Animal Production Area (Charles River, Frederick, MD) were generally used at 8-10 weeks of age. To induce disseminated hepatic and/or pulmonary neuroblastoma metastases, mice were injected intravenously (i.v.) with TBJ-RFP cells (IxIO⁵ cells/animal in 0.2 ml HBSS) and metastatic rumors were allowed to become well-established for 5 days post tumor cell injection. In some studies, mice were injected intrasplenically (i.s.) with TBJ-RFP cells (2.5xlO⁵ cells/animal in 0.5 ml HBSS), and selective hepatic metastases were allowed to become well established for 5 days post tumor cell injection.

Mice bearing well-established day 5 i.v.-induced metastatic tumors were injected i.p. each morning with IL-2 (recombinant human, Chiron Corporation,

Emeryville, CA, 50,000 IU in 0.2 ml HBSS containing 0.1% homologous serum) or vehicle alone on days 5-9 and 12-15 post tumor implantation. Bortezomib was administered i.p. in the afternoon on days 6, 9 and 13 post tumor implantation. Mice were euthanized on day 16 post tumor implantation, and livers were resected individually and stored in cold PBS. The metastatic disease burden in each liver was imaged via conventional light and fluorescence microscopy. Macroscopic imaging was carried out on a slit fiber optic illuminated light table (Lightools Research, Encinitas, CA) and images were captured by a zoom lens equipped Nikon DXM 1200 digital camera. A Nikon SMZ800 stereomicroscope equipped with a mercury lamp and a Nikon DXMl 200 digital camera was used to collect low power (10-63X magnification) images. RFP fluorescence was induced by excitation at 540 nm and collected through a 590 nm filter.

Systemic administration of bortezomib in combination with IL-2 markedly inhibited the growth of induced hepatic TBJ-RFP metastases, and did so more effectively than either single agent alone (FIG. 5A-5D). Additionally, significant differences in the gross appearance of metastases in mice from the respective treatment groups were noted as well. In livers from control mice, tumor metastases were large and were grossly highly vascularized (FIG. 5D). On the other hand, tumor metastases in livers from mice treated with bortezomib alone showed reduction in tumor size with only peripheral vascularity. Tumor metastases in livers from mice treated with IL-2 alone showed only decreases in gross tumor vascularity. In contrast, livers from mice treated with bortezomib+IL-2 had the largest reduction in tumor size and had grossly apparent decreases in tumor vascularity. In similarly designed experiments, mice bearing well-established day 5 i.s.- induced metastatic tumors were injected i.p. each morning with IL- 12 (murine, specific activity >lxlθ⁷ U/mg, Peprotech, Rocky Hill, NJ, 0.1 μg in 0.2 ml PBS containing 0.1% homologous serum) or vehicle alone on days 5, 8 and 12 post tumor implantation. Bortezomib (20 μg in 0.2 ml 0.9% saline) or vehicle alone was delivered i.p. 5-6 h later the same day. Mice were euthanized on day 13 post tumor injection. The impact of therapy on metastatic disease burden in the liver was then evaluated using fluorescent imaging as described herein. Bortezomib strongly potentiated the antitumor activity of IL- 12 in mice bearing established hepatic TBJ-RFP metastases (FIG. 6). Although some reductions in overall metastatic disease burden was noted in livers of some mice treated with bortezomib or IL- 12 alone compared to control mice, the livers from mice treated with the combination of bortezomib and IL- 12 did not have any detectable metastases.

While this disclosure has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations of the preferred embodiments may be used and it is intended that the disclosure may be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications encompassed within the spirit and scope of the disclosure as defined by the claims below.

Claims

We claim:

1. A method for enhancing an antitumor activity of a cytokine on a tumor cell that responds to treatment with the cytokine, comprising exposing the tumor cell to a therapeutically effective amount of the cytokine and a proteasome inhibitor, wherein the proteasome inhibitor is present in a sufficient amount to enhance the activity of the cytokine, thereby enhancing the antitumor activity of the cytokine.

2. The method of claim 1 , wherein the proteasome inhibitor induces expression in the tumor cell of a cellular receptor for an antiproliferative cytokine.

3. The method of claim 2, wherein the cellular receptor comprises an interferon-alpha (IFN-α) receptor, an interferon-beta (IFN-β) receptor, an interferon- gamma (IFN-γ) receptor, and/or a tumor necrosis factor alpha (TNF-α) receptor.

4. The method of claim 1 , wherein exposing the tumor cell to a therapeutically effective amount of the cytokine comprises exposing the cell to a cytokine that induces expression of another cytokine, and wherein the other cytokine is an antiproliferative cytokine.

5. The method of claim 4, wherein the cytokine comprises interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-18 (IL-18), and/or interleukin-32 (IL-32).

6. The method of claim 4, wherein the antiproliferative cytokine comprises interferon-alpha (IFN-α), interferon-beta (IFN-β), interferon-gamma (IFN-γ), and/or tumor necrosis factor alpha (TNF-α). 7. The method of claim 1, wherein exposing the tumor cell to a therapeutically effective amount of the cytokine comprises exposing the cell to an antiproliferative cytokine.

8. The method of claim 7, wherein the antiproliferative cytokine comprises interferon-alpha (IFN-α), interferon-beta (IFN-β), interferon-gamma (IFN-γ), and/or tumor necrosis factor alpha (TNF-α).

10. The method of claim 1 , wherein the tumor cell expresses substantially higher levels of activated AKT than a corresponding non-tumor cell.

11. The method of claim 1 , wherein the tumor cell is resistant to the antitumor activity of the cytokine.

12. The method of claim 11 , wherein the resistance is reduced or absent responsiveness of the tumor cell to the antitumor activity of the cytokine.

13. The method of claim 1 , wherein the therapeutically effective amount of the cytokine in the presence of the proteasome inhibitor is lower than when the tumor cell is exposed to the cytokine alone.

14. The method of claim 1, wherein the cytokine is selected from the group consisting of interferon-alpha (IFN-α), interferon-beta (IFN-β), interferon-gamma (IFN- γ), tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), interleukin-12 (IL- 12), interleukin-18 (IL-18), interleukin-32 (IL-32), and combinations thereof.

15. The method of claim 1 , wherein the proteasome inhibitor inhibits degradation of proteins via ubiquitin-proteasome pathway. 16. The method of claim 15, wherein the proteasome inhibitor inhibits 26S proteasome.

17. The method of claim 1, wherein the proteasome inhibitor is bortezomib.

18. The method of claim 1, wherein the tumor cell is a neural cell.

19. The method of claim 18, wherein the neural tumor cell is a tumor cell of the sympathetic nervous system.

20. The method of claim 19, wherein the neural tumor cell is a neuroblastoma.

21. The method of claim 1 , wherein exposing the tumor cell to a therapeutically effective amount of the cytokine and proteasome inhibitor comprises administering the therapeutically effective amount of the cytokine and proteasome inhibitor to a subject having a tumor.

22. A method for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a cytokine having an antitumor activity and a therapeutically effective amount of a proteasome inhibitor, thereby treating the tumor in the subject.

23. The method of claim 22, wherein the proteasome inhibitor induces expression in the tumor of a cellular receptor for an antiproliferative cytokine.

24. The method of claim 23, wherein the cellular receptor comprises an interferon-alpha (IFN-α) receptor, an interferon-beta (IFN-β) receptor, an interferon- gamma (IFN-γ) receptor, and/or a tumor necrosis factor alpha (TNF-α) receptor. 25. The method of claim 22, wherein administering to the subject a therapeutically effective amount of the cytokine comprises administering to the subject a cytokine that induces expression of another cytokine, and wherein the other cytokine is an antiproliferative cytokine.

26. The method of claim 25, wherein the cytokine comprises interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-18 (IL-18), and/or interleukin-32 (IL-32).

27. The method of claim 25, wherein the antiproliferative cytokine comprises interferon-alpha (IFN-α), interferon-beta (IFN-β), interferon-gamma (IFN-γ), and/or tumor necrosis factor alpha (TNF-α).

28. The method of claim 22, wherein administering to the subject a therapeutically effective amount of the cytokine comprises administering to the subject an antiproliferative cytokine.

29. The method of claim 28, wherein the antiproliferative cytokine comprises interferon-alpha (IFN-α), interferon-beta (IFN-β), interferon-gamma (IFN-γ), and/or tumor necrosis factor alpha (TNF-α).

30. The method of claim 22, wherein the proteasome inhibitor is administered prior to the administration of the cytokine.

31. The method of claim 22, wherein the proteasome inhibitor is administered after the administration of the cytokine.

32. The method of claim 22, wherein the proteasome inhibitor is administered simultaneously with the administration of the cytokine. 33. The method of claim 22, wherein the administration of the cytokine and the administration of the proteasome inhibitor results in tumor cell death.

34. The method of claim 22, wherein the administration of the cytokine and the administration of the proteasome inhibitor results in apoptosis of tumor cells.

35. A method for treating a neuroblastoma tumor in a subject, comprising administering to the subject a therapeutically effective amount of interleukin-12 (IL-12) and a therapeutically effective amount of bortezomib, thereby treating the tumor in the subject.

36. A pharmaceutical composition, comprising a cytokine having an antitumor activity, a proteasome inhibitor and a pharmaceutically acceptable carrier, wherein the cytokine and the proteasome inhibitor are present in a therapeutically effective amount for the proteasome inhibitor to enhance an antitumor activity of the cytokine.

37. The pharmaceutical composition of claim 36, wherein the cytokine induces expression of another cytokine, and wherein the other cytokine is an antiproliferative cytokine.

38. The pharmaceutical composition of claim 36, wherein the cytokine is an antiproliferative cytokine.

39. The pharmaceutical composition of claim 36, wherein the cytokine is interleukin-12 (IL-12) and the proteasome inhibitor is bortezomib.