AU2006235254A1 - Use of FRIL proteins for reducing the production of pro-inflammatory cytokines - Google Patents

Description

WO 2006/110577 PCT/US2006/013149 METHODS AND COMPOSITIONS FOR REDUCING THE PRODUCTION OF PRO-INFLAMMATORY CYTOKINES BACKGROUND 5 Field The invention relates to methods and compositions for reducing the production of pro-inflammatory cytokines. More specifically, the invention relates to methods of treatment of disorders characterized by pathologic or deleterious production of pro inflammatory cytokines using proteins which are members of the FLT3-receptor 10 interacting ligand (FRIL) family of progenitor cell preservation factors. Background Inflammation is a physiological response by the body to restore and maintain homeostasis following insults such as infections and tissue injury (Kuby, J., Immunology 15 , Second Edition, (W.H. Freeman and Company, New York)). Most of the immune cells responsible for the body's defense are located in the blood, and inflammation is the . means by which these cells leave the blood and enter the tissues around the infected or injured site. The inflammatory response provides early protection by restricting the tissue damage to the site of the infection or tissue injury. 20 Secreted cytokine proteins provide signals between immune cells to coordinate the inflammatory response. Cytokines are a group of low molecular weight regulatory proteins secreted in response to inducing stimuli by cells such as lymphocytes, monocytes/macrophages, mast cells, eosinophils, and endothelial cells lining blood vessels. There are four major categories of cytokines: interferons (e.g., IFN-a, IFN-p, 25 IFN-y), colony stimulating factors (e.g., G-CSF, M-CSF, GM-CSF), tumor necrosis factors (e.g., TNF-a, TNF-P), and interleukins (e.g., IL-4, IL-5, IL-10). Cytokines can exert their effect through the bloodstream on distant target cells (endocrine), on target cells adjacent to those that produce them (paracrine), or on the same cell that produces the cytokine (autocrine). Physiologically, cytokines appear to exert their most important 30 effects in a paracrine/autocrine manner (Kuby, J. supra). 1 WO 2006/110577 PCT/US2006/013149 Defects in the complex regulatory networks governing tne expression or cytokines have been implicated in several diseases. Specifically, deregulation of pro-inflammatory cytokines (such as IL-la, IL-1p, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, TNF-a, and IFN-y) have been implicated in diseases such as psoriasis, septic shock, periodontal disease, 5 rheumatoid arthritis, inflammatory bowel disease, toxic shock syndrome, inflammatory sequelae of viral infections, eczema, autoimmune disorders, and neurodegenerative diseases. In view of the large number of diseases that result from deregulation in the levels of pro-inflammatory cytokines, there is a need in the art to discover new compositions and methods for treating conditions involving undesired or pathological 10 levels of pro-inflammatory cytokines. The FRIL family of proteins was previously identified and described as mannose binding plant lectins having the ability to preserve progenitor cells by inhibiting proliferation and/or differentiation (e.g., Moore et al. (1997), Blood 90, Suppl. 1, 308 (abstract); Mo et al. (1999), Glycobiology 9:173-179; Colucci et al. (1999), Proc. Natl. 15 Acad. Sci. USA 96:646-650; Moore et al. (2000), Biochim. Biophys. Acta 25027:1-9). Prior to the present disclosure, however, the FRIL proteins were not known to have any activity or utility with respect to treating or alleviating conditions characterized by pathologic or deleterious production of pro-inflammatory cytokines. 20 BRIEF SUMMARY The present invention depends, in part, upon the discovery that certain lectins are useful in reducing undesired or pathological levels of one or more pro-inflammatory cytokines. In particular, the invention relates to the discovery that FRIL proteins reduce the production of pro-inflammatory cytokines by cells that have been exposed to inducers 25 of pro-inflammatory cytokines. Thus, the FRIL proteins of the invention can selectively reduce the levels of pro-inflammatory cytokines and, therefore, they can be used in treatments for patients diagnosed with disorders characterized by pathologic or deleterious production of a pro-inflammatory cytokine. Thus, in one aspect, the invention provides a method for reducing the levels 30 and/or production of a pro-inflammatory cytokine in a mammal in need thereof by administering an effective amount of a FRIL protein to the mammal. In some 2 WO 2006/110577 PCT/US2006/013149 embodiments, the pro-inflammatory cytokine is selected from me group consisting or IFN-y, IL-6, TNF-a, and combinations thereof. In a specific embodiment, the mammal is a human patient. In some embodiments, the patient has been diagnosed with a condition selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, 5 lupus, Crohn's disease, amyotrophic lateral sclerosis, atherosclerosis, septic shock, inflammatory bowel disease, endotoxemia, and graft-versus-host disease. In another aspect, the invention provides a method for treating a patient diagnosed with a condition characterized by undesired or pathological levels of a pro-inflammatory cytokine by administering a therapeutically effective amount of a FRIL protein to the 10 patient, wherein the administration of the FRIL protein reduces the undesired or pathological levels of the pro-inflammatory cytokine. In some embodiments, the patient has been diagnosed with a condition selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, lupus, Crohn's disease, amyotrophic lateral sclerosis, atherosclerosis, septic shock, inflammatory bowel disease, endotoxemia, and 15 graft-versus-host disease. In another aspect, the invention provides a method for treating a pro-inflammatory cytokine-mediated disorder in a cell by administering a therapeutically effective amount of a FRIL protein to the patient. In yet another aspect, the invention provides a method for treating inflammation 20 by administering a therapeutically effective amount of a FRIL protein to the patient. In some embodiments, the inflammation is chronic inflammation, or acute inflammation. In some specific embodiments, the inflammation is associated with a condition selected from the group consisting of psoriasis, eczema, dermatitis, and arthritis. In another aspect, the invention provides a method for treating a patient diagnosed 25 with an autoimmune disease by administering a therapeutically effective amount of a FRIL protein to the patient, wherein the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby treating the patient with the autoimmune disease. In another aspect, the invention provides a method for treating a patient diagnosed 30 with psoriasis by administering a therapeutically effective amount of a FRIL protein to 3 WO 2006/110577 PCT/US2006/013149 the patient, wherein the administration of the FRIL protein reduces me levels ot a pro inflammatory cytokine in the patient, thereby treating the patient with psoriasis. In another aspect, the invention provides a method for treating a patient diagnosed with multiple sclerosis by administering a therapeutically effective amount of a FRIL 5 protein to the patient, wherein the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby treating the patient with multiple sclerosis. In another aspect, the invention provides a method for treating a patient diagnosed with rheumatoid arthritis by administering a therapeutically effective amount of a FRIL 10 protein to the patient, wherein the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby treating the patient with rheumatoid arthritis. In yet another aspect, the invention provides a method for suppressing rejection to a graft in a patient by administering a therapeutically effective amount of a FRIL 15 molecule to the patient prior to, at the same time as, or subsequent to the graft, wherein the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby suppressing rejection by the patient of the graft. In some embodiments the graft is an allograft or a xenograft. In a further aspect, the invention provides a method for treating mucositis by 20 administering a therapeutically effective amount of a FRIL protein to the patient. In some embodiments, the mucositis is selected from the group consisting of radiation-induced mucositis, chemotherapy-induced mucositis, and infection-induced mucositis. In another aspect, the invention provides a composition comprising a FRIL protein and a second agent. In some embodiments, the second agent is selected from the group 25 consisting of an IL-1 receptor antagonist, a soluble cytokine receptor that binds IL-2, a soluble cytokine receptor that binds IL-4, a soluble cytokine receptor that binds IL-6, a soluble cytokine receptor that binds IL-7, a soluble cytokine receptor that binds IFN-y, a soluble cytokine receptor that binds TNF-a, a soluble cytokine receptor that binds TNF-p, a soluble cytokine receptor that binds LIF, an endogenous anti-endotoxin antibody, and 30 any combinations thereof. 4 WO 2006/110577 PCT/US2006/013149 In some embodiments of any of the foregoing aspects, the FRIL protein can be selected from the group consisting of a native FRIL protein and a recombinant FRIL protein. In specific embodiments, the native FRIL protein is selected from the group consisting of a native Dl-FRIL protein, a native Pv-FRIL protein, and a native Yam-FRIL 5 protein. In some specific embodiments, the FRIL protein is a mature FRIL protein lacking an N-terminal leader sequence. In some specific embodiments, the FRIL protein corresponds to an amino acid sequence included in at least one of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 6, or SEQ ID NO.: 8. In some embodiments, the FRIL protein is expressed from a nucleic acid including SEQ ID NO.: 1, SEQ ID NO.:5, or SEQ ID 10 NO.:7. In another aspect, the invention provides for the use of a FRIL protein in the manufacture of a medicament for reducing the levels and/or production of a pro inflammatory cytokine. In some embodiments, the FRIL protein corresponds to an amino acid sequence included in at least one of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 15 6, or SEQ ID NO.: 8. In some embodiments, the FRIL protein is expressed from a nucleic acid including SEQ ID NO.:1, SEQ ID NO.:5, or SEQ ID NO.:7. In some embodiments, the FRIL protein is a native FRIL protein or a recombinant FRIL protein. In certain embodiments, the pro-inflammatory cytokine is selected from the group consisting of IFN-y, IL-6, TNF-a, and combinations thereof. 20 In another aspect, the invention provides for the use of a FRIL protein in the manufacture of a medicament for the treatment of a condition characterized by undesired or pathological levels of a pro-inflammatory cytokine. In some embodiments, the condition characterized by undesired or pathological levels of a pro-inflammatory cytokine is selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid 25 arthritis, lupus, Crohn's disease, amyotrophic lateral sclerosis, atherosclerosis, septic shock, inflammatory bowel disease, endotoxemia, and graft-versus-host disease. In some embodiments, the FRIL protein corresponds to an amino acid sequence included in at least one of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 6, or SEQ ID NO.: 8. In some embodiments, the FRIL protein is expressed from a nucleic acid including SEQ ID NO.: 1, 30 SEQ ID NO.:5, or SEQ ID NO.:7. In some embodiments, the FRIL protein is a native FRIL protein or a recombinant FRIL protein. In certain embodiments, the pro 5 WO 2006/110577 PCT/US2006/013149 inflammatory cytokine is selected from the group consisting of IFN-y, IL-6, TNF-a, and combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS 5 The following figures are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims. Figure 1 is a schematic representation of a whole blood assay to detect the effect of various stimuli on cytokine levels (e.g., effect of addition of LPS with or without FRIL on IL-6 levels). 10 Figure 2 is a schematic representation of a bar graph showing the reduction of IL 6 (pg/mL) in the supernatants of human blood cultures from donor 1, 24 hours after stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl FRIL. Figure 3 is a schematic representation of a bar graph showing the reduction of 15 IFN-y (pg/mL) in the supernatants of human blood cultures from donor 1, 24 hours after stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl FRIL. Figure 4 is a schematic representation of a bar graph showing the reduction of IFN-y (pg/mL) in the supernatants of human blood cultures from donor 2, 24 hours after 20 stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl FRIL. Figure 5 is a schematic representation of a bar graph showing the reduction of IFN-y (pg/mL) in the supernatants of human blood cultures from donor 3, 24 hours after stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl 25 FRIL. Figure 6 is a schematic representation of a bar graph showing the reduction of IFN-y (pg/mL) in the supernatants of human blood cultures from donor 4, 24 hours after stimulation with heat-killed Staphylococcus epidermidis or LPS in the presence or absence of Dl-FRIL. 30 Figure 7 is a schematic representation of a bar graph showing the reduction of IFN-y (pg/mL) in the lysate of human blood cultures from donor 4, 48 hours after 6 WO 2006/110577 PCT/US2006/013149 stimulation with heat-killed Staphylococcus epidermidis or LPS in the presence or absence of D-FRIL. Figure 8 is a schematic representation of a bar graph showing the reduction of IFN-y (pg/mL) in the supernatant of human blood cultures from donor 5, 24 hours after 5 stimulation with heat-killed Staphylococcus epidermidis or LPS in the presence or absence of Di-FRIL. Figure 9 is a schematic representation of a bar graph showing the reduction of IFN-y (pg/mL) in the supernatant of human blood cultures from donor 7, 24 hours after stimulation with LPS in the presence or absence of Dl-FRIL. 10 Figure 10 is a schematic representation of a bar graph showing the reduction of IL-l a (pg/mL) in the lysate of human blood cultures from donor 1, 24 hours after stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl FRIL. Figure 11 is a schematic representation of a bar graph showing the reduction of 15 TNF-a (pg/mL) in the supernatant of human blood cultures from donor 1, 24 hours after stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl FRIL. Figure 12 is a schematic representation of a bar graph showing the reduction of TNF-a (pg/mL) in the supernatant of human blood cultures from donor 3, 24 hours after 20 stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl FRIL. Figure 13 is a schematic representation of a bar graph showing the reduction of TNF-a (pg/mL) in the supernatant of human blood cultures from donor 4, 24 hours after stimulation with heat-killed Staphylococcus epidermidis or LPS in the presence or 25 absence of Dl-FRIL. Figure 14 is a schematic representation of a bar graph showing the reduction of IFN-y (pg/mL) in the supernatant of human blood cultures from donors 8 and 9, 24 hours after stimulation with IL- 12 and IL- 18 in the presence or absence of DI-FRIL. Figure 15 is a schematic representation of a bar graph showing the reduction of 30 IFN-y (pg/mL) in the supernatant of human blood cultures from donors 8 and 9, 48 hours after stimulation with IL- 12 and IL-18 in the presence or absence of Dl-FRIL. 7 WO 2006/110577 PCT/US2006/013149 Figure 16 is a schematic representation of a bar graph showing the reduction of IFN-y (pg/mL) in the lysate of human blood cultures from donors 8 and 9, 48 hours after stimulation with IL- 12 and IL- 18 in the presence or absence of D1-FRIL. 5 DETAILED DESCRIPTION The patent, scientific, and medical publications referred to herein establish knowledge that was available to those of ordinary skill in the art at the time the invention was made. The entire disclosures of the issued U.S. patents, published, and pending patent applications, and other references cited herein, are hereby incorporated by 10 reference. Definitions All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in 15 the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent or later-developed techniques which would be apparent to one of skill in the art. In addition, in order to more clearly and concisely describe the subject matter which is the invention, the following definitions are provided for certain terms 20 which are used in the specification and appended claims. As used herein, the term "native FRIL-protein" means a FRIL protein isolated from a legume in which the protein is naturally expressed. As used herein, the term "recombinant FRIL-protein" means a FRIL protein isolated from an organism in which the protein is expressed by a recombinant gene 25 including, without limitation, bacteria, yeast, plant, or animal cells which have been transfected with a recombinant construct encoding the FRIL protein. A recombinant FRIL protein can have an amino acid sequence identical to a native FRIL protein, or can have an amino acid sequence including one or more amino acid insertions, deletions, and/or substitutions including, without limitation, N-terminal additions or deletions, C 30 terminal additions or deletions, and chimeric proteins. 8 WO 2006/110577 PCT/US2006/013149 As used herein, the term "FRIL-protein," without further modification, means any native FRIL protein or recombinant FRIL protein. As used herein, the term "inflammation," or "inflammatory response" means a non-specific immune response that occurs in reaction to a bodily injury. 5 As used herein, the term "pro-inflammatory cytokine" means a cytokine that is capable of promoting, inducing, or increasing inflammation in a responsive tissue. Non limiting examples of such cytokines include IL-la, IL-i I3, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, IL-15, IL-16, IL-17, IL-18, TNF-a, TGF-p, IFN-a, and IFN-y. As used herein with respect to amino acid sequences, the term "percent identity" 10 and "sequence identity" means a measure of the degree of similarity of two sequences based upon an alignment of the sequences which maximizes identity and which is a function of the number of identical nucleotides or residues, the number of total nucleotides or residues, and the presence and length of gaps in the sequence alignment. A variety of algorithms and computer programs are available for determining sequence 15 identity using standard parameters, for example, Gapped BLAST or PSI-BLAST (Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402), BLAST (Altschul et al. (1990), J. Mol. Biol. 215:403 -410), and Smith-Waterman (Smith et al. (1981), J. Mol. Biol. 147:195-197). As used herein, percent identity is based upon the default values for the BLAST algorithms. 20 As used herein with respect to protein preparations, the term "substantially pure" means a preparation which contains at least 60% (by dry weight) of the protein of interest, exclusive of the weight of other intentionally included compounds. In some embodiments, the preparation is at least 75%, at least 90%, or at least 99%, by dry weight the protein of interest, exclusive of the weight of other intentionally included compounds. 25 Purity can be measured by any appropriate method, e.g., column chromatography, gel electrophoresis, or HPLC analysis. If a preparation intentionally includes two or more different proteins of the invention, a "substantially pure" preparation means a preparation in which the total dry weight of the proteins of the invention is at least 60% of the total dry weight, exclusive of the weight of other intentionally included compounds. For such 30 preparations containing two or more proteins of the invention, the total weight of the proteins of the invention can be at least 75%, at least 90%, or at least 99%, of the total dry 9 WO 2006/110577 PCT/US2006/013149 weight of the preparation, exclusive of the weight of other intentionally included compounds. Thus, if the proteins of the invention are mixed with one or more other proteins (e.g., serum albumin) or compounds (e.g., diluents, detergents, excipients, salts, polysaccharides, sugars, lipids) for purposes of administration, stability, storage, and the 5 like, the weight of such other proteins or compounds is ignored in the calculation of the purity of the preparation. As used herein, the term "therapeutically effective amount" means the total amount of each active component of a pharmaceutical composition or method that is sufficient to show a meaningful patient benefit (e.g., a statistically significant decrease in 10 the production of a pro-inflammatory cytokine). When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. As used herein, the term "reduce" means to cause a decrease of at least 5% in a 15 specified characteristic, such as a level or concentration of a molecule in the circulation or in a specified tissue, or a rate of cell proliferation (i.e., cell reproduction) or survival, relative to a baseline level or a level which would have been expected in the absence of a specified treatment. As used herein, the term "inhibiting/reducing inflammation," means to reduce the 20 level of one or more pro-inflammatory cytokines in a mammal suffering from an inflammatory response, thereby reducing the damage to the cells, tissues, and/or organs affected by the inflammation. As used herein, the term "suppressing rejection," means inhibiting a graft recipient's immune response which would otherwise occur, e.g., in response to the 25 introduction of a non-self MHC antigen into the recipient. Thus, suppressing rejection of a graft refers to an inhibition of the immune system of the recipient to a graft of a donor compared to a situation where a method of the invention was not used. As used herein, the term "statistically significant" means having a probability of less than 10% under the relevant null hypothesis (i.e., p < 0.1). 30 As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values 10 WO 2006/110577 PCT/US2006/013149 within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an 5 example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1, or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values > 0 and ; 2 if the variable is inherently continuous. As used herein, unless specifically indicated otherwise, the word "or" is used in 10 the inclusive sense of "and/or" and not the exclusive sense of "either/or." General Considerations The present invention depends, in part, upon the discovery that certain lectins are useful in reducing undesired or pathological levels of one or more pro-inflammatory 15 cytokines. In particular, the invention relates to the discovery that FRIL proteins can reduce the production of pro-inflammatory cytokines by cells that have been exposed to inducers of pro-inflammatory cytokines. Thus, the FRIL proteins of the invention can selectively reduce the levels of pro-inflammatory cytokines and, therefore, they can be used in treatments for patients diagnosed with disorders characterized by pathologic or 20 deleterious production of a pro-inflammatory cytokine. FRIL Proteins The FRIL proteins are mannose/glucose-specific legume lectins which were initially identified as having the ability to preserve progenitor cells, in the sense of 25 inhibiting differentiation, with or without inducing proliferation, and were referred to as "pylartin" (see, e.g., U.S. Pat. No. 6,084,060). The proteins were also shown in a biological assay to stimulate the proliferation of NIH 3T3 cells transfected with the flk2/Flt3 receptor but not untransfected cells and, therefore, were designated as Flt3 Receptor Interacting Lectins (FRIL) (See, e.g., Moore et al. (1997), Blood 90, Suppl. 1, 30 308 (abstract); Mo et al. (1999), Glycobiology 9:173-179; Colucci et al. (1999), Proc. Natl. Acad. Sci. USA 96:646-650; Moore et al. (2000), Biochim. Biophys. Acta 25027:1 11 WO 2006/110577 PCT/US2006/013149 9). However, because the ability to bind the Flt3 receptor has not been shown to be necessary to the present utility of the proteins, the designation FRIL is to be understood as the historical name of the proteins, and not as a functional requirement. The first FRIL protein was identified in the hyacinth bean (Dolichos lab lab), but 5 FRIL proteins have now been identified in other legumes (tribe Phaseoleae) including, without limitation, Phaseolus vulgaris, Sphenostylis stenocaipa, Cicer arietinum, Phaseolus acutifolius, Phaseolus lunatus, Vigna sinensis, and Voandzeia subterranea. Native FRIL proteins useful in the invention include, but are not limited to, the FRIL proteins of Dolichos lab lab ("Dl-FRIL"), Phaseolus vulgaris ("Pv-FRIL"), Phaseolus 10 acutifolius ("Pa-FRIL"), and Sphenostylis stenocarpa ("Yam-FRIL"). The native FRIL proteins are expressed as heterodimers of a and p chains and have calculated molecular weights of approximately 15-20 kD for the a chain and approximately 12-20 kD for the P chain. The a and p chains are initially expressed as a single polypeptide but are subsequently cleaved. The proteins also appear to possess N 15 linked glycosylation sites. The amino acid sequence of one Dl-FRIL protein is provided in SEQ ID NO.: 2. The sequence begins with a 22 amino acid leader sequence which is cleaved from the mature protein. Residues 23-145 constitute the P chain, and residues 146-286 constitute the a chain. In the mature native protein, the C-terminus is often truncated to varying 20 degrees, including deletions of the last 14 residues. The protein of SEQ ID NO.: 2 is based on the sequence of Colucci et al. (1999), Proc. Natl. Acad. Sci. USA 96:646-650, but with several changes based on subsequent data. Kotlarczyk et al. (2002), UBEP 2002 Ninth Annual Undergraduate Research Symposium, Arizona State University, Abstract #35 described an additional FRIL protein from Dolichos lab lab having the amino acid 25 sequence provided in SEQ ID NO.: 3. The relation of this protein to the native Dl-FRIL protein of Colucci et al. (1999) is unknown. The protein of SEQ ID NO.: 3 begins with an 8 amino acid leader sequence derived from an immunoglobulin kappa chain and is not part of the native protein. Residues 9 to approximately 129-135 constitute the p chain, and the residues from approximately 130-136 to 276 constitute the a chain. As before, 30 the C-terminus can be truncated to varying degrees, including deletions of the last approximately 14 residues. Gowda et al. (1994), J. Biol. Chem. 269:18789-18793, 12 WO 2006/110577 PCT/US2006/013149 described a FRIL-like protein from Dolichos lab lab having the amino acid sequence provided in SEQ ID NO.: 4. The relation of this protein to the native Di-FRIL protein is unknown. The amino acid sequence of one Pv-FRIL protein is provided in SEQ ID NO.: 6. 5 The sequence begins with a 22 amino acid leader sequence which is cleaved from the mature protein. Residues 23-145 constitute the p chain, and residues 146-301 constitute the a chain. In the mature native protein, the C-terminus is often truncated to varying degrees. The amino acid sequence of one Yam-FRIL protein is provided in SEQ ID NO.: 8. 10 In the case of each of the FRIL proteins, various N-terminal or C-terminal deletions or additions, as well as internal insertions, deletions, and substitutions, can be made without affecting biological activity. Such variants can be produced by routine methods known to those of skill in the art, and can be tested for therapeutic utility without undue experimentation. For example, the variants can be tested using the whole blood 15 cell assay described in Example 1. Those variants or newly identified FRIL family proteins that reduce pro-inflammatory cytokine production in this assay are considered useful for the methods and compositions of the present invention. In addition to native FRIL proteins, the methods of the present invention can utilize recombinant FRIL proteins. A recombinant FRIL protein can have an amino acid 20 sequence identical to a native FRIL protein, or can have an amino acid sequence including one or more amino acid insertions, deletions, and/or substitutions. For example, the N-terminal leader sequence of a native FRIL protein can be deleted or can be replaced with an alternative leader sequence. The C-terminal sequences can also be truncated or replaced. Fusion proteins can also be produced, adding purification tags or 25 epitopes (e.g., poly-His tag, Flag tag, c-myc epitope, HA tag, MBP epitope, GST epitope), or targeting sequences (e.g., ligands for cell surface receptors or immunoglobulin domains). Internal substitutions, deletions, and insertions are also possible. In some embodiments, the recombinant proteins are chimeric sequences produced by intermingling the sequences of two or more native FRIL proteins. Depending upon the 30 hosts in which such recombinant FRIL proteins are expressed, the recombinant proteins can also differ from native FRIL proteins due to differences in post-translational 13 WO 2006/110577 PCT/US2006/013149 processing, such as cleavage of the a and P chains, removal of N-terminal leader sequences, and/or C-terminal truncation or degradation. Descriptions of many recombinant FRIL variants can be found in, for example, U.S. Pat. No. 6,310,195, PCT International Publication No. WO 98/59038, and PCT 5 International Publication No. WO 01/49851, the entire disclosures of which are hereby incorporated by reference. As a general matter, recombinant FRIL proteins, including chimeric proteins, can be produced which have at least 45% amino acid sequence identity, at least 55% amino acid sequence identity, at least 65% amino acid sequence identity, at least 75% amino acid sequence identity, at least 85% amino acid sequence 10 identity, at least 90% amino acid sequence identity or at least 95% amino acid sequence identity with a native FRIL protein. In certain embodiments, a recombinant FRIL protein can have at least 85%-95% amino acid sequence identity with a native FRIL protein (e.g., SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 6, SEQ ID NO.: 8). In certain other embodiments, a recombinant FRIL protein can have at least 85%-95% amino acid 15 sequence identity with a native FRIL protein (e.g., SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 6, SEQ ID NO.: 8), wherein the recombinant FRIL protein reduces the level of a pro-inflammatory cytokine (e.g., by the assay described in Examplel). Amino acid sequence identity and nucleic acid sequence identity between two proteins or two nucleic acid molecules can be measured according to standard methods (see, e.g., Pearson and 20 Lipman (1988), Proc. Natl. Acad. Sci. USA 85:2444-2448; George et al., in Macromolecular Sequencing and Synthesis, Selected Methods and Applications, pps. 127-149, Alan R. Liss, Inc. 1988; Feng and Doolittle (1987), Journal ofMolecular Evolution 25:351-360; and the BLAST programs of the National Center for Biotechnology, National Library of Medicine, Bethesda, MD). 25 Any FRIL protein, whether native or recombinant, which is capable of reducing the levels of a pro-inflammatory cytokine produced by cells that have been exposed to an inducer of pro-inflammatory cytokines is considered useful in the invention. FRIL proteins can be tested for their ability to reduce the levels of pro-inflammatory cytokines using simple in vitro assays such as that described below in the examples. 30 14 WO 2006/110577 PCT/US2006/013149 Purification of FRIL proteins FRIL proteins are readily purified using standard techniques. Methods for purifying proteins are known in the art and include, without limitation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoprecipitation, 5 immunosorption, high performance liquid chromatography (HPLC), size-exclusion chromatography (SEC), immunoaffinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, or a combination of any of these methods. These and other suitable methods are described, e.g., in Marston (1987), in DNA Cloning, Glover, ed., Volume III, IRL Press Ltd., Oxford; Marston and Hartley (1990), in Guide to 10 Protein Purification, Deutscher, ed., Academic Press, San Diego; Laemmli (1970), Nature 227:680-685; Ausubel et al. (1999), Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY; and U.S. Pat. No. 6,084,060. A FRIL family member molecule can also be purified by binding to mannose, which can be coupled to a solid support (e.g., a sepharose bead). 15 Purification of a FRIL protein from a legume (e.g., Dolichos lab lab, Phaseolus vulgaris) can be achieved rapidly and inexpensively. For example, FRIL proteins can be purified from extracts of ground legumes by mannose-affinity chromatography, or by ovalbumin affinity chromatography. FRIL proteins are relatively abundant in legumes. For example, D1-FRIL can account for approximately 0.02% of the mass of hyacinth 20 beans. Purified FRIL proteins can also be made by recombinant methods. Thus, a FRIL protein can be produced by introducing a nucleic acid sequence encoding the FRIL protein into any appropriate host cell type including bacterial (e.g., E. coli), yeast (e.g., S. cerevisiae), plant (e.g., Arabidopsis, Lemna, tobacco, or corn), insect (e.g., Drosophila), 25 or mammalian cells (e.g., CHO cells), by recombinant techniques well-known to those with skill in the art. For example, a FRIL protein-encoding nucleic acid sequence can be inserted into a baculovirus vector which can be used to generate recombinant baculovirus particles. Insect cells (e.g., Sf9 cells) transduced with the recombinant baculovirus will express the FRIL protein. Following lysis, the FRIL protein can be purified. 30 Alternatively, recombinant FRIL proteins can be produced in dicotyledonous plants, such as Nicotiana tabacus or Arabidopsis thaliana. For example, Arabidopsis 15 WO 2006/110577 PCT/US2006/013149 plants can be transformed using a strain of Agrobacterium tumefaciens carrying a nucleic acid molecule encoding a FRIL protein. Methods for making vectors for producing Agrobacteriuin with a desired nucleic acid molecule are known in the art (see, e.g., McBride and Summerfelt (1990), Plant Mol Biol. 14(2):269-276; U.S. Pat. No. 5 4,940,838, and U.S. Pat. No. 5,464,763). The FRIL protein can be purified from the transformed plant by standard methods (see, e.g., Ausubel et al., supra). Nucleic acid sequences encoding a FRIL protein include, without limitation, any sequence encoding the proteins of SEQ ID NOs.: 2, 3, 6, or 8, including the nucleic acid sequences of SEQ ID NOs.: 1, 5, or 7. In addition, nucleic acid sequences can be 10 designed and produced encoding any of the recombinant FRIL variants described herein. Methods for Treatment FRIL proteins reduce the levels of several pro-inflammatory cytokines. Thus, the FRIL proteins of the present invention are useful in treating or alleviating disorders 15 characterized by pathologic or deleterious production of a pro-inflammatory cytokine. Such treatment could be beneficial for treating acute and chronic conditions in which inflammation is key to the pathogenesis of the diseases including, but not limited to, psoriasis, eczema, dermatitis, and/or arthritis. Thus, the present invention provides a method for preventing or reducing inflammation, or treating diseases characterized by 20 inflammation, comprising the administration of a therapeutically effective amount of a FRIL protein. The FRIL proteins of the present invention have uses in the prevention and/or treatment of inflammatory conditions. For example, because FRIL proteins can reduce production of cytokines by cells involved in an inflammatory response, these proteins can 25 be used to prevent and/or treat chronic and acute inflammatory conditions. Such inflammatory conditions include, but are not limited to, inflammation associated with infection (e.g., septic shock, toxic shock syndrome, sepsis, systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine- or chemokine-induced 30 lung injury, inflammatory bowel disease, Crohn's disease, over-production of cytokines (e.g., IFN-y, IL-6, TNF-a), respiratory disorders (e.g., asthma, allergy); gastrointestinal 16 WO 2006/110577 PCT/US2006/013149 disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric cancer, ovarian cancer, lung cancer, bladder cancer, liver cancer, breast cancer, cervical cancer, lymphoid cancers, cardiac myxoma, myeloma, plasmacytoma); CNS disorders (e.g., multiple sclerosis; ischemic brain injury and/or stroke; traumatic brain injury; neurodegenerative 5 disorders, Parkinson's disease, Alzheimer's disease, AIDS-related dementia, prion disease); cardiovascular disorders (e.g., atherosclerosis, myocarditis, cardiovascular disease, cardiopulmonary bypass complications); autoimmune disorders (e.g., Addison's Disease, hemolytic anemia, anti-phospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, 10 multiple sclerosis, myasthenia gravis, neuritis, ophthalmia, bullous pemphigoid, pemphigus, polyendocrinopathies, purpura, Reiter's disease, stiff-man syndrome, autoimmune thyroiditis, systemic lupus erythematosus, autoimmune pulmonary inflammation, Guillain-Barre syndrome, insulin-dependent diabetes mellitus, autoimmune inflammatory eye disease), as well as many additional diseases, conditions, and disorders 15 that are characterized by inflammation (e.g., hepatitis, gout, trauma, pancreatitis, sarcoidosis, dermatitis, mucositis (e.g., infection-induced, chemotherapy-induced, radiation therapy-induced), renal ischemia-reperfusion injury, uveal inflammation, pleurisy, stroke, and allogeneic and xenogeneic transplant rejection. Because inflammation is a fundamental defense mechanism, inflammatory 20 disorders can affect virtually any tissue of the body. Accordingly, the FRIL proteins of the invention have uses in the treatment of tissue-specific inflammatory disorders including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, 25 endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis, mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis, pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis, 30 scleritis, sclerochoroiditis, scrotitis, sinusitis, sponylitis, steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis. 17 WO 2006/110577 PCT/US2006/013149 Inflammation can also be a life-threatening complication of severe physical trauma (e.g., traumatic head injury), burns, cardiopulmonary bypass surgery, renal ischemia-reperfusion, and organ transplant surgery. FRIL proteins can be administered to subjects in these situations who are in need of a treatment that reduces inflammation. 5 In addition, chronic inflammation increases the risk of cancer (Wiseman and Halliwell (1996), Biochem. J. 313:17-29). For example, patients with inflammatory bowel disease are at higher risk of developing gastrointestinal cancer (Lewis et al., Gastroenterol. Clin. North Amer. 28(2):459-77 (1999)), while lung cancer has been linked to chemical-induced lung inflammation (Emmendoerffer et al. (2000), Toxicol. 10 Lett. 112-113: 185-191). Thus, FRIL can be administered to patients that are at a risk of cancer due to chronic inflammation. In one aspect, the invention features a method for treating or reducing inflammation in a mammal by administering a pharmaceutical composition including a therapeutically effective amount of a FRIL protein. In one embodiment, the inflammation 15 is characterized by deregulated levels of a pro-inflammatory cytokine. In some specific embodiments, the pro-inflammatory cytokine is selected from the group consisting of IFN-y, IL-6 and TNF-a. In another aspect, the invention features a method for treating a mammal diagnosed with a disorder characterized by undesired or pathological levels of a pro 20 inflammatory cytokine by administering to the mammal a pharmaceutical composition including a therapeutically effective amount of a FRIL protein to reduce the levels of the pro-inflammatory cytokine. In some specific embodiments, the pro-inflammatory cytokine is selected from the group consisting of IFN-y, IL-6 and TNF-at. In some embodiments, the disorder is selected from the group consisting of psoriasis, multiple 25 sclerosis, mucositis, endotoxemia, inflammatory bowel disease, and rheumatoid arthritis. In one embodiment, the mucositis is selected from the group consisting of radiation induced mucositis, chemotherapy-induced mucositis, and infection-induced mucositis. In another aspect, the invention provides a method for treating a mammal diagnosed with an autoimmune disease or a neurodegenerative disease by administering 30 to the mammal a pharmaceutical composition including a therapeutically effective amount of a FRIL protein to reduce the levels of the pro-inflammatory cytokine. In one 18 WO 2006/110577 PCT/US2006/013149 embodiment of this aspect, the method is directed to treatment or a mammal diagnosed with an autoimmune disease selected from the group consisting of multiple sclerosis, rheumatoid arthritis, juvenile arthritis, myocarditis, colitis, nephritis, scleroderma, tendonitis, lupus, chronic inflammatory demyelinating polyneuropathy, psoriasis, and 5 autoimmune hepatitis. In a specific embodiment, the autoimmune disease is psoriasis. In another specific embodiment, the autoimmune disease is multiple sclerosis. In yet another specific embodiment, the autoimmune disease is rheumatoid arthritis. In another embodiment of this aspect, the method is directed to treatment of a mammal diagnosed with a neurodegenerative disease selected from the group consisting of Alzheimer's 10 disease, and Parkinson's disease. In another aspect, the invention features a method for treating a mammal diagnosed with a cancer by administering to the mammal a pharmaceutical composition including a therapeutically effective amount of a FRIL protein to reduce the progression of the cancer. Abnormalities in the production of cytokines or their receptors have been 15 associated with several types of cancer. In one embodiment of this aspect of the invention, the cancer is selected from the group consisting of a cardiac myxoma, a myeloma, a plasmacytoma, a cervical cancer, a bladder cancer, and a lymphoid cancer. In a specific embodiment, the cancer is a cardiac myxoma. In another specific embodiment, the cancer is a myeloma. In a further specific embodiment, the cancer is a lymphoid 20 cancer. In a further aspect, the invention provides a method for suppressing rejection of a graft, or increasing tolerance to the graft, in a mammal undergoing transplantation. Specifically, administration of the FRIL proteins of the present invention to the mammal prior to, at the same time as, or subsequent to transplantation reduces the levels of pro 25 inflammatory cytokines thereby permitting the graft (i.e., autograft, graft from an MHC matched donor, allograft, xenograft) to be accepted by the mammal. The mammal can be a human patient or, in some embodiments, the mammal can be a non-human primate, laboratory animal (e.g., mouse, rat, rabbit, hamster), a livestock or breeding animal (e.g., horse, sheep, cow, pig, goat), or a pet (e.g., cat, dog). 30 In another aspect, the invention provides a method for determining whether a subject (e.g., a human patient) diagnosed with a condition characterized by undesired or 19 WO 2006/110577 PCT/US2006/013149 pathological levels of a pro-inflammatory cytokine will benefit from treatment with a pharmaceutical composition including a FRIL protein. This method includes contacting a cell from diseased tissue of the subject with a FRIL protein and determining whether the FRIL protein reduces the production of pro-inflammatory cytokines (e.g., by comparison 5 to an extrinsic standard or to an untreated control sample of cells from the diseased tissue of the same subject). Typically, a FRIL protein is chosen which is known to be effective against the category of cancer from which the patient is suffering. If it is determined that the cancer is FRIL-sensitive, the patient can undergo treatment with the FRIL protein. Any route of administration can be employed which is suitable to the particular 10 formulation chosen for the FRIL protein pharmaceutical composition including, without limitation, parenteral routes such as intravenous, intra-arterial, intra-muscular, subcutaneous, intraperitoneal, intranasal, intrapulmonary, intrarectal, and intravaginal. Oral administration can also be employed for certain formulations. The pharmaceutical preparations can be administered locally to an affected area or can be administered 15 systemically. The exact amount of a FRIL protein which will constitute a therapeutically effective amount will depend upon the activity of the FRIL protein selected, the nature of the cytokine-related disorder to be treated, the extent of the cytokine-related disorder to be treated, and the age, weight, and general health of the subject, as well as the use of any 20 combination therapy. As a general matter, when administered systemically, a therapeutically effective amount can be in the range of 500 ng/kg (i.e., 500 ng of the FRIL protein per kg total body weight of the subject) to 100 mg/kg per day. In some embodiments, a therapeutically effective amount is in the range of 1 pig/kg to 50 mg/kg per day, or 5 pg/kg to 25 mg/kg per day. 25 Administration of a FRIL protein can begin before the subject is symptomatic, upon diagnosis of the disease, or after the disease has progressed. For example, a FRIL protein can be administered prophylactically to a subject that is considered to be at a risk of developing a condition characterized by undesired or pathological levels of a pro inflammatory cytokine. Alternatively, a human patient newly diagnosed with a cytokine 30 related disorder (e.g., by virtue of high levels of a pro-inflammatory cytokine) who has yet to exhibit characteristic symptoms of the disorder, can be treated with a FRIL protein 20 WO 2006/110577 PCT/US2006/013149 as a first line of therapy. Or, in some embodiments, the FRIL protein can be administered as an adjuvant therapy in combination with other, standard treatments for the disease/disorder being treated. For example, FRIL may be given as an adjuvant therapy for the treatment of a patient diagnosed with rheumatoid arthritis, multiple sclerosis, or 5 psoriasis. Combination Therapy Targeting one specific cytokine or inflammatory mediator may not be sufficient to treat a patient with a disease involving cytokine deregulation. Accordingly, in one aspect 10 of the invention, the method comprises administering a therapeutic amount of a FRIL protein and a second agent. The "second agent" is any substance that is capable of alleviating the condition being treated. In one embodiment, the second agent is an inhibitor of the production or activity of one or more cytokines. Non-limiting examples of such a second agent include natural cytokine antagonists such as IL-1 receptor 15 antagonist, soluble cytokine receptors for IL-2, IL-4, IL-6, IL-7, IFN-y, TNF-a, TNF-p, and LIF, and endogenous anti-endotoxin antibody (EndoCAb); and antibodies that block the ability of pro-inflammatory cytokines to bind their cognate receptors. In another embodiment, the second agent is a drug that is used to treat inflammatory diseases selected from the group consisting of non-steroidal anti-inflammatory drugs, anti-malarial 20 drugs (e.g., hydroxychloroquine), corticosteroids, methotrexate, sulfasalazine, penicillamine, cyclophosphamide, and cyclosporine. In another embodiment, the second agent is any drug that is known to be useful to treat the specific disease in the patient. For example, if the patient has psoriasis, the patient can be treated with a FRIL protein and a known treatment for psoriasis including, but not limited to, AMEVIVE*, ENBREL*, 25 HUMIRA*, RAPTIVA*, and REMICADE*. Alternatively, if the patient has multiple sclerosis, the patient can be treated with a FRIL protein and a known treatment for multiple sclerosis including, but not limited to, COPAXONE*, BETASERON*, AVONEX*, NOVANTRONE*, and REBIF®. Furthermore, if the patient has rheumatoid arthritis, the patient can be treated with a FRIL protein and a treatment for rheumatoid 30 arthritis including, but not limited to, RITUXAN*, ZEVALIN*, AND BEXXAR*. 21 WO 2006/110577 PCT/US2006/013149 It is to be understood that the FRIL protein can be administered to the patient prior to, at the same time as, or subsequent to the second agent. In one embodiment, a substantially pure FRIL protein can be formulated in combinations with other pharmaceuticals or therapeutics useful in the treatment of inflammatory disorders. For 5 example, the FRIL protein can be combined with natural cytokine antagonists such as IL 1 receptor antagonist, soluble cytokine receptors for IL-2, IL-4, IL-6, IL-7, IFN-y, TNF-a, TNF-P, and LIF, and endogenous anti-endotoxin antibody (EndoCAb). Pharmaceutical Preparations 10 In another aspect, the invention provides pharmaceutical preparations including a substantially pure FRIL protein for use in the treatment of a condition characterized by undesirable levels of a pro-inflammatory cytokine or the manufacture of a medicament for use in such treatments. The pharmaceutical preparations can include a FRIL protein in dry form (e.g., 15 lyophilized alone or with a stabilizer) or in liquid solutions or suspensions (e.g., in a pharmaceutically acceptable carrier or diluent). Pharmaceutically acceptable carriers for parenteral administration of liquids include, without limitation, water, buffered saline, polyols (e.g., glycerol), polyalkylene glycols (e.g., propylene glycol, liquid polyethylene glycol), vegetable oils, hydrogenated napthalenes, or suitable mixtures thereof. The FRIL 20 proteins can also be formulated with buffers or excipients. In some embodiments, the FRIL proteins are formulated in sustained-release particles or implantable devices. For example, such particles or devices can be formed from biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, polyoxyethylene-poloxypropylene copolymers, ethylene-vinyl acetate copolymers, and 25 the like, to control the release of the FRIL protein. Other potentially useful parenteral delivery systems include osmotic pumps, implantable infusion systems, and liposomes. In one embodiment, the FRIL proteins are delivered to a patient using DUROS* Implant or ALZAMER* Depot technology (Alza Corporation). Methods for formulating pharmaceutical preparations can be found, for example, 30 in Remington's Pharmaceutical Sciences (18th edition), ed. A. Gennaro (1990), Mack Publishing Company, Easton, PA. 22 WO 2006/110577 PCT/US2006/013149 Kits In one aspect, the invention provides a kit comprising, in a suitable container, a therapeutically effective amount of one or more substantially pure FRIL proteins and a second agent. The FRIL protein and the second agent may be in separate containers or 5 formulated together in one container. The components of the kit may be provided in a liquid solution(s), and/or as a dried powder(s). When the components are provided in liquid solution, the liquid solution is a sterile solution. When the reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent (e.g., water or buffered saline). The solvent may be provided as part of 10 the kit. The following examples are intended to further illustrate certain preferred embodiments of the invention and are not limiting in nature. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents 15 are considered to be within the scope of this invention and the appended claims. Example 1 Whole Blood Assay for Detecting Cytokines Whole blood from nine healthy human volunteers was collected and stored in 20 separate sterile heparinized tubes. A portion of the blood from each of these subjects was diluted 1:4 in a 4 mL polypropylene tube containing medium that either contained or lacked stimulants (e.g., E.coli lipopolysaccharide (LPS) or heat-killed Staphylococcus epidermidis), and which contained or lacked a FRIL protein (e.g., D1-FRIL). The mixture was incubated at 37'C for 24 or 48 hrs. At the end of 24 and 48 hours, the supernatant 25 was isolated and tested using commercially available cytokine assay kits. In addition, whole blood cells from the tubes were lysed to measure total cytokine levels in the lysate. A schematic representation of this procedure is depicted in Fig. 1. 30 23 WO 2006/110577 PCT/US2006/013149 Example 2 Reduction of IL-6 in Human Whole Blood Cultures Stimulated by Staphylococcus epidermidis Human whole blood cultures were stimulated with heat-killed Staphylococcus 5 epidermidis (S. epi) in the presence or absence of DI-FRIL (1 ig, 10 Rg or 100 pjg) and these mixtures were incubated at 37'C. Supernatants from these cultures were isolated 24 hours after the incubation and assayed for levels of secreted IL-6. Supernatants of cultures treated only with S. epi had high levels of IL-6 (see, Fig. 2). In contrast, supernatants of cultures treated only with FRIL (100 ng, 1 jig, or 10 pg) 10 had only background levels of IL-6. Significantly, supernatants of cultures treated with S. epi and DI-FRIL (10 pg or 100 jig) showed an approximately 5-20 fold reduction in the levels of secreted IL-6 relative to cultures treated only with S. epi. These levels of IL-6 were lower than those found in a control culture containing S. epi and IL-18 binding protein (IL- 1 8BP), a known inhibitor of cytokine production. 15 Accordingly, this experiment showed that FRIL can reduce the levels of the pro inflammatory cytokine IL-6 under conditions which normally lead to production of high levels of this cytokine. Example 3 20 Reduction of IFN-y in Human Whole Blood Cultures Stimulated by Staphylococcus epidermidis Human whole blood cultures were stimulated with heat-killed S. epi in the presence or absence of Dl-FRIL (1 pg, 10 jg, or 100 pg), and these mixtures were incubated at 37"C. Supernatants from these cultures were isolated 24 hours after the 25 incubation and assayed for levels of secreted IFN-y. Supernatants of cultures treated only with S. epi had high levels of IFN-y (see, Figs. 3-6). In contrast, supernatants of cultures treated only with FRIL (100 ng, 1 jig or 10 jg) had only background levels of IFN-y. Supernatants of cultures treated with S. epi and Dl-FRIL (10 pg or 100 pg) showed a significant reduction in the levels of secreted 30 IFN-y relative to cultures treated only with S. epi. These levels of IFN-y were comparable to those found in a control culture containing S. epi and IL-18BP. 24 WO 2006/110577 PCT/US2006/013149 Thus, this experiment showed that FRIL can reduce the levels of the pro inflammatory cytokine IFN-y under conditions which normally lead to high levels of this cytokine. 5 Example 4 Reduction of IFN-y in Human Whole Blood Cultures Stimulated by Staphylococcus epidermidis or LPS Human whole blood cultures were stimulated with heat-killed S. epi in the presence or absence of Dl-FRIL (1 pg, 10 [tg or 100 [g), and these mixtures were 10 incubated at 37 0 C. Similarly, human whole blood cultures also were stimulated in separate experiments with LPS in the presence or absence of DL-FRIL (1 ptg or 10 jig). Supernatants from these cultures were isolated 24 hours after the incubation and assayed for levels of secreted IFN-y. In addition, lysates of the whole blood cells were prepared after 48 hours of incubation and assayed for levels of IFN-y. 15 As in Examples 2 and 3, supernatants of cultures treated only with S. epi or LPS had high levels of IFN-y (see, Figs. 6-9), while supernatants of cultures treated only with FRIL (1 pg or 10 pig) had only background levels of IFN-y. In striking contrast, supernatants of cultures treated with both S. epi or LPS and DL-FRIL (10 jig) showed a significant reduction in the levels of secreted IFN-y. These levels of IFN-y were 20 comparable to those found in a control culture containing S. epi or LPS and IL-18BP. Lysates collected at 48 hours showed the same pattern of IFN-y expression as seen with the supernatants at 24 hours (see, Fig. 7). In summary, these experiments showed that FRIL can reduce the levels of the pro inflammatory cytokine IFN-y when blood cells are treated with either S. epi or LPS, both 25 well-known inducers of pro-inflammatory cytokines. Example 5 Reduction of IL-la in Human Whole Blood Cultures Stimulated by Staphylococcus epidermidis 30 Human whole blood cultures were stimulated with heat-killed S. epi in the presence or absence of Dl-FRIL (1 jig, 10 jig or 100 jig), and these mixtures were 25 WO 2006/110577 PCT/US2006/013149 incubated at 37 0 C. Lysates from these cultures were isolated 24 hours after the incubation and assayed for levels of IL- I a. Lysates of cultures treated only with S. epi had high levels of IL-1 a (see, Fig. 10), whereas supernatants of cultures treated only with FRIL (100 ng, 1 pg or 10 pg) had only 5 background levels of IL-la. Supernatants of cultures treated with S. epi and Dl-FRIL (1 ptg, 10 pg, or 100 pg) showed no significant reduction in the levels of IL-la. Thus, this experiment showed that FRIL does not reduce IL-la to any significant level under conditions which normally lead to production of high levels of this cytokine. 10 Example 6 Reduction of TNF-a in Human Whole Blood Cultures Stimulated by Staphylococcus epidermidis Human whole blood cultures were stimulated with heat-killed S. epi in the presence or absence of Dl-FRIL (1 jg, 10 pg or 100 pg), and these mixtures were 15 incubated at 37*C. Supernatants from these cultures were isolated 24 hours after the incubation and assayed for levels of secreted TNF-a. Supernatants of cultures treated only with S. epi had high levels of TNF-a (see, Figs. 11-12), whereas supernatants of cultures treated only with FRIL (100 ng, 1 pg, or 10 pg) had only background levels of TNF-a. Supernatants of cultures treated with S. epi 20 and D1-FRIL (10 jg) showed a relatively small reduction in the levels of secreted TNF-a. These levels of TNF-a were lower than those found in a control culture containing S. epi and IL-18BP. Thus, this experiment showed that FRIL can reduce the levels of the pro inflammatory cytokine TNF-a under conditions which normally lead to production of 25 high levels of this cytokine. Example 7 TNF-a Expression in Human Whole Blood Cultures Stimulated by LPS Human whole blood cultures were stimulated with LPS in the presence or absence 30 of Dl-FRIL (1 pg or 10 pg), and these mixtures were incubated at 37"C. Supernatants 26 WO 2006/110577 PCT/US2006/013149 from these cultures were isolated 24 hours after the incubation and assayed for levels of secreted TNF-a. Supernatants of cultures treated only with LPS had roughly the same levels of TNF-a as supernatants of cultures treated with LPS and Dl-FRIL (1 gg or 10 jpg) (see, 5 Fig. 13). The levels of TNF-a were slightly lower than those found in a control culture containing S. epi and IL-1 8BP. These experiments indicate that FRIL had a relatively small effect on LPS mediated TNF-a secretion. 10 Example 8 Reduction of IFN-y levels in Human Whole Blood Cultures Stimulated with IL-12 and IL-18 Human whole blood cultures were stimulated with IL-12 and IL-18 in the presence or absence of Dl-FRIL (1 Rg or 10 pg), and these mixtures were incubated at 15 37'C for 24 and 48 hours. Supernatants from these cultures were isolated 24 and 48 hours after the incubation and assayed for levels of secreted IFN-y. In addition, lysates of the whole blood cells were prepared after 48 hours of incubation and assayed for levels of IFN-y. Supernatants of cultures treated only with IL- 12 and IL- 18 produced high levels of 20 IFN-y (see, Figs. 14-15) at both the 24 and 48 hour timepoints. In striking contrast, supernatants of cultures treated with IL-12 and IL-18, and Dl-FRIL (1 jg and 10 jig) showed a significant reduction in the levels of secreted IFN-y at both the 24 and 48 hour timepoints. Lysates collected at 48 hours recapitulated the same pattern of IFN-y expression 25 as seen with the supernatants at 24 and 48 hours (see, Fig. 16). In summary, these experiments showed that FRIL can reduce the levels of the pro inflammatory cytokine IFN-y in blood cells that are treated with the combination of IL-12 and IL-18. 30 Example 9 Reduction of Experimental Allergic Encephalomyelitis by Treatment with FRIL 27 WO 2006/110577 PCT/US2006/013149 Pro-inflammatory cytokines are involved in the pathogenesis of experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The development of demyelinating lesions in EAE or MS is the result of a complex chain of events that involves recognition of specific antigen, T cell activation, recruitment of 5 nonspecific cells to the lesion, release of numerous cytokines and inflammatory mediators by resident glial cells and infiltrating cells, which in turn leads to demyelination and CNS damage. Given that FRIL can reduce the levels of pro-inflammatory cytokines, FRIL is tested as a therapeutic agent for the amelioration of the autoimmune demyelinatory disease in EAE. 10 Female Lewis rats (Charles River Breeding Laboratories, Wilmington, Mass., USA) are induced to develop EAE by injecting intradermally 50 pg of myelin basic protein (MBP) per animal emulsified in complete Freund's adjuvant (CFA) into the medial footpad of each hind leg on day 1 followed by a booster injection on the 7 day under ether anesthesia. Clinical symptoms in these rats are followed and the signs of 15 EAE are scored as: (0) normal; (1) piloerection; (2) loss in tail tonicity; (3) hind leg paralysis; (4) paraplegia; and (5) moribund. One group of rats is injected with FRIL up to 4 times starting on the first day of immunization and continuing daily for the duration of the study. As controls, one group of rats is induced for EAE but left untreated (positive control group), and another group 20 of animals is not induced for EAE and used as the negative control group. Treatment with FRIL will reduce the development of EAE disease in the Lewis female rats. Expression of IFN-y and iNOS (a known inducer of EAE) in brain sections of all groups of rats is studied. In contrast to the rats with EAE, the FRIL treated rats will show low levels of IFN- y and iNOS in the central nervous system. 25 Example 10 Suppression of Graft Rejection in a Patient by Administration of FRIL A patient who is to undergo a transplantation of an organ, a tissue, or a group of cells from an allogeneic or xenogeneic donor is prepared for this procedure with 30 immunosuppressive therapy. The immunosuppressive therapy depletes the patient's immune cells facilitating the transplant of the graft. At the time of immunosuppressive 28 WO 2006/110577 PCT/US2006/013149 therapy, or shortly thereafter, the patient may be administered FRIL. If the immunosuppressive therapy is performed prior to transplantation, FRIL may be administered to the patient prior to (e.g., 3-5 days), at the same time as, or subsequent to the transplant procedure. FRIL is administered up to four times daily at a dosage of 0.05 5 mg/kg to 5 mg/kg. FRIL treatment is continued 5 to10 days following transplantation. In some instances the FRIL treatment is extended until 30 to 60 days after transplantation of the graft. Patients treated with FRIL will show decreased rejection of the transplanted grafts compared to patients that were not administered FRIL. 10 Example 11 Reduction of Indomethacin-Induced Intestinal Mucositis by FRIL Rats are administered FRIL (i.v. or i.p., up to 4 doses daily at a dosage of 0.05 mg/kg to 5 mg/kg) 1-7 days before indomethacin (7-9 mg/kg) is administered 15 subcutaneously. As controls, rats are either administered buffer (negative control) or repifermin (positive control) in place of FRIL. On the fourth day after administration of indomethacin, intestinal ulceration in the rats is determined using standard methods. Rats treated with FRIL will show significantly reduced ulceration compared to rats treated with buffer, and comparable protection to rats treated with repifermin. 20 Example 12 Reduction of Cyclophosphamide-Induced Bladder Mucositis by FRIL Rats are administered FRIL (i.v. or i.p., up to 4 doses daily at a dosage of 0.05 mg/kg to 5 mg/kg) a day before being provided with cyclophosphamide (150, 200 or 250 25 mg/kg, intraperitoneally). As controls, rats are either administered buffer (negative control) or repifermin (positive control) in place of FRIL. The day after cyclophosphamide treatment, the efficacy of FRIL in reducing bladder mucositis is assessed by (i) measuring bladder capacity; (ii) measuring bladder net weight; and (iii) determining the bladder histological score based on histology. 29 WO 2006/110577 PCT/US2006/013149 Rats treated with FRIL 24 hours prior to cyclophosphamide administration will show significantly reduced cystitis compared to rats treated with vehicle, and comparable protection to rats treated with repifermin. 5 Example 13 Prophylactic Activity of FRIL in Sub-Lethal Endotoxic Shock in Mice Mice are administered FRIL (i.v. or i.p., up to 4 doses daily at a dosage of 0.05 mg/kg to 5 mg/kg) 10 minutes before treating them with LPS (2 pg/mouse, intraperitoneally). As controls, rats are either administered buffer or HSA (negative 10 controls), or repifermin (positive control) in place of FRIL. Whole blood of these mice are then assayed 90 and 180 hours after LPS treatment for levels of pro-inflammatory cytokines (e.g., TNF-a, IFN-y, IL-6) using commercially available cytokine assay kits. FRIL-treated mice will show reduced levels of pro-inflammatory cytokines compared to the negative controls and comparable levels of pro-inflammatory cytokines 15 as repifermin. Example 14 Anti-Inflammatory Effect of FRIL on Platelet Activating Factor (PAF)-Induced Edema Groups of four Lewis rats are injected subcutaneously in the foot pad of the right hind paw with PAF alone, PAF and LPS, PAF and KGF-2, and PAF and FRIL. The 20 dosage of FRIL is 0.05 mg/kg to 5 mg/kg. The left hind paw of each of these groups of rats is injected with an equal volume of buffer to use as a parallel control. Paw volume is a measure of edema and is quantified immediately before or 30 and 90 minutes after injection using a plethysmograph, and the percent change in paw volume is calculated. Right hind paws injected with PAF alone will show a significant increase in paw 25 volume 30 minutes post injection, while left hind paws receiving buffer will show little sign of edema. However, when FRIL is administered together with PAF, there will be a substantial reduction in paw volume compared with PAF-alone challenged paws. 30 WO 2006/110577 PCT/US2006/013149 Example 15 Use of FRIL to Reduce Cutaneous Inflammation FRIL proteins are used to test whether they attenuate the progression of contact dermatitis using a tetradecanoylphorbol acetate (TPA)-induced cutaneous inflammation 5 model in mice. The use of the female BALB/c and male Swiss Webster mice in experimental cutaneous inflammation are well-characterized and reproducible models of contact dermatitis. These strains of mice have been shown to develop a long-lasting inflammatory response, following topical application of TPA, which is comprised of local hemodynamics, vascular permeability and local migration of leukocytes, and these 10 pathological changes are similar to those of human dermatitis (Rao et al., Inflammation 17(6):723, 1993; Rao et al., J. Lipid Mediators Cell Signalling 10:213, 1994). Groups of mice receive either vehicle or FRIL intraperitoneally, sub-cutaneously, or intravenously 60 min. after the topical application of TPA (4 pg/ear), which is applied as a solution in acetone (200 pg/ml), 10 pl each to the inner and outer surface of ear. The 15 control group receives 20 pl of acetone as a topical application. Four hours following the application of TPA, increase in ear thickness is measured and ears are excised for histology. To determine vascular permeability in response to TPA, mice are intravenously injected through tail veins with Evans blue (300 mg/kg) at selected times after topical application of TPA and are sacrificed 15 min. thereafter. Ears are excised 20 and removed then extracted into dimethylformamide and centrifuged. Absorbance readings are spectrophotometrically measured at 590 nm. FRIL will be found to inhibit inflammation mediated by TPA. Example 16 25 Dextran Sulfate Sodium-Induced Colitis: Mouse Model of Inflammatory Bowel Disease In this model of colitis, an acute inflammation of the colon is produced by administration of dextran sulfate sodium (DSS) as a 5% solution in tap water. DSS induced colitis is characterized by histological events and an influx of neutrophils, macrophages, and mediators of inflammation similar to those observed with human 30 inflammatory bowel diseases (IBD). Several drugs known to be useful for treating IBD, such as corticosteroids and 5-ASA, are known to have activity in this model. 31 WO 2006/110577 PCT/US2006/013149 One hundred test animals (female, 6 week old Swiss Webster mice, 18-30 g) are divided into ten groups. Each animal is dosed daily (i.p. or i.v.) with either FRIL (dosage of 0.05 mg/kg to 5 mg/kg, up to four times daily), or vehicle, starting on Day 0. Beginning on Day 1, acute colon inflammation is induced by the administration ad 5 libitum in drinking water of dextran sulfate sodium (DSS) as a 5% solution in tap water (10 mL/mouse/day for 5-6 days), with no other fluid source for animals in the DSS arm of the study. Filtered tap water is available ad libitum except for animals receiving 5% DSS as the sole source of fluid. After four days, signs of acute disease will occur with the loss of weight, diarrhea, and bloody stools. Histological changes will include initial 10 shortening of the crypts, then areas of separation of the crypts and the muscularis mucosae in the absence of destructive inflammatory filtrate. After five days, pathological changes will become confluent with the appearance of erosions and early hyperplastic epithelium. Inflammation scores will be high with neutrophils, lymphocytes, and plasma cells in the lamina propria, but sparing the epithelium. 15 FRIL is evaluated for prophylactic activity, and FRIL given after the disease state is established is also evaluated for therapeutic activity. Test animals are weighed daily from Day 0 to Day 8, or until completion of the study. The total duration of the study with DSS arm of the study varies depending on the time for progression of colitis. The condition of the test animals and consistency of stools 20 is noted. At the conclusion of the study, test animals are euthanized (CO 2 ), a midline incision is made, and a stool sample is obtained. The sample is placed on a slide and tested for occult blood (Quic-Cultm, Laboratory Diagnostics Co., Morganville, N.J.). Occult blood is determined by placing two drops of the reagent onto the sample and observing any color change. Occult blood presence is graded using a scoring protocol 25 assigning a score of 0 for no color; 1 for a very light blue color (+/-) forming in >30 seconds; 2 for a blue color developing in 30 seconds or more (+); 3 for a change in color occurring in less than 30 seconds (++); and 4 for gross blood observable on the slide. The colon is gently stretched and the length from the colon-cecal junction to the end of the distal rectum is measured to the nearest 0.1 cm. 30 The data will show that FRIL provides significant protection from the inflammatory responses to DSS-induced colitis. 32 WO 2006/110577 PCT/US2006/013149 Example 17 Effect of FRIL on Pro-Inflammatory Cytokine Production by Keratinocytes UVB light is a potent stimulus for pro-inflammatory cytokine release by 5 keratinocytes. Reduction in the levels of such cytokines is important in treating psoriasis. Keratinocytes are UVB irradiated before and/or after FRIL treatment (1 ptg to 100 ptg). As a control, keratinocytes are treated with buffer alone. The levels of IFN-y, IL-6, IL-la, and TNF-a are determined by quantitative two site enzyme immunoassay using commercially available ELISA kits for these cytokines. 10 FRIL treatment will reduce the levels of one or more of these pro-inflammatory cytokines compared to the buffer control. 33

Claims (27)

1. A method for reducing the levels and/or production of a pro-inflammatory cytokine in a mammal in need thereof, comprising administering an effective amount of a FRIL protein to the mammal, wherein the administration of the FRIL protein reduces the 5 production of the pro-inflammatory cytokine in the mammal.

2. The method of claim 1, wherein the pro-inflammatory cytokine is selected from the group consisting of IFN-y, IL-6, and combinations thereof. 10

3. The method of claim 1, wherein the mammal is a human patient.

4. The method of claim 3, wherein the patient has been diagnosed with a condition selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, lupus, Crohn's disease, amyotrophic lateral sclerosis, atherosclerosis, septic shock, 15 inflammatory bowel disease, endotoxemia, and graft-versus-host disease.

5. The method of claim 1, wherein the FRIL protein is a native FRIL protein or a recombinant FRIL protein. 20

6. The method of claim 5, wherein the native FRIL protein is selected from the group consisting of a native Dl-FRIL protein, a native Pv-FRIL protein, and a native Yam-FRIL protein.

7. The method of claim 6, wherein the amino acid sequence of said FRIL protein 25 corresponds to an amino acid sequence included in at least one of SEQ ID NOS: 2, 3, 6, or 8.

8. A method for treating a patient diagnosed with a condition characterized by undesired or pathological levels of a pro-inflammatory cytokine, comprising 30 administering a therapeutically effective amount of a FRIL protein to the patient, wherein the administration of the FRIL protein reduces the undesired or pathological levels of the 34 WO 2006/110577 PCT/US2006/013149 pro-inflammatory cytokine.

9. The method of claim 8, wherein the patient has been diagnosed with a condition selected from the group consisting of multiple sclerosis, rheumatoid arthritis, psoriasis, 5 lupus, Crohn's disease, amyotrophic lateral sclerosis, atherosclerosis, septic shock, inflammatory bowel disease, endotoxemia, and graft-versus-host disease.

10. The method of claim 8, wherein the FRIL protein is a native FRIL protein or a recombinant FRIL protein. 10

11. The method of claim 10, wherein the native FRIL protein is selected from the group consisting of a native Dl-FRIL protein, a native Pv-FRIL protein, and a native Yam-FRIL protein. 15

12. The method of claim 11, wherein the amino acid sequence of said FRIL protein corresponds to an amino acid sequence included in at least one of SEQ ID NOS: 2, 3, 6, or 8.

13. A method for treating a patient diagnosed with an autoimmune disease, 20 comprising administering a therapeutically effective amount of a FRIL protein to the patient, wherein the administration of the FRIL protein reduces the levels of a pro inflammatory cytokine in the patient, thereby treating the patient with the autoimmune disease. 25

14. A method for treating a patient diagnosed with psoriasis, comprising administering a therapeutically effective amount of a FRIL protein to the patient, wherein the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby treating the patient with psoriasis. 30

15. A method for treating a patient diagnosed with multiple sclerosis, comprising administering a therapeutically effective amount of a FRIL protein to the patient, wherein 35 WO 2006/110577 PCT/US2006/013149 the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby treating the patient with multiple sclerosis.

16. A method for treating a patient diagnosed with rheumatoid arthritis, comprising 5 administering a therapeutically effective amount of a FRIL protein to the patient, wherein the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby treating the patient with rheumatoid arthritis.

17. A method for suppressing rejection to a graft in a patient, comprising 10 administering a therapeutically effective amount of a FRIL molecule to the patient prior to, at the same time as, or subsequent to the graft, wherein the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby suppressing rejection by the patient of the graft. 15

18. The method of claim 17, wherein the graft is an allograft or a xenograft.

19. A composition comprising a FRIL protein and a second agent, wherein the second agent is selected from the group consisting of an IL-I receptor antagonist, a soluble cytokine receptor that binds IL-2, a soluble cytokine receptor that binds IL-4, a soluble 20 cytokine receptor that binds IL-6, a soluble cytokine receptor that binds IL-7, a soluble cytokine receptor that binds IFN-y, a soluble cytokine receptor that binds TNF-a, a soluble cytokine receptor that binds TNF-3, a soluble cytokine receptor that binds LIF, an endogenous anti-endotoxin antibody, and any combinations thereof. 25

20. A method for treating a pro-inflammatory cytokine-mediated disorder in a cell, comprising administering a therapeutically effective amount of a FRIL protein to the patient.

21. A method for treating inflammation, comprising administering a therapeutically 30 effective amount of a FRIL protein to the patient. 36 WO 2006/110577 PCT/US2006/013149

22. The method of claim 21, wherein the inflammation is chronic or acute inflammation. 5

23. The method of claim 21, wherein the inflammation is the result of a condition, wherein the condition is selected from the group consisting of psoriasis, eczema, dermatitis, arthritis, and combinations thereof.

24. A method for treating mucositis, comprising administering a therapeutically 10 effective amount of a FRIL protein to the patient.

25. The method of claim 24, wherein the mucositis is selected from the group consisting of radiation-induced mucositis, chemotherapy-induced mucositis, and infection-induced mucositis. 15

26. Use of a FRIL protein in the manufacture of a medicament for the treatment of a condition characterized by undesired or pathological levels of a pro-inflammatory cytokine. 20

27. Use of a FRIL protein in the manufacture of a medicament for reducing the levels and/or production of a pro-inflammatory cytokine. 37