MXPA98009733A - Chlorine type 2 ligature proteins and methods for your - Google Patents

Abstract

The present invention relates to a method of using a novel protein that binds to chemokines (CBP TYPE 2) encoded by poxvirus and having amino acid sequence homology with the family of T1 proteins of the Shope fibroma virus against disease syndromes. associated with acute or chronic dysregulated inflammatory responses

Description

OUI OCINA TYPE 2 LIGATURE PROTEINS AND METHODS FOR USE BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates generally to the field of immunology and specifly to a qui ocin binding protein encoded by a variety of poxviruses and methods for their use. 2. Description of Related Art It has become increasingly clear that viruses living within higher order vertebrate cells must have evolved to specifly avoid the host's immunolog system (Gooding, L., Cell, 91: 5-7, 1992; Marrack, P. and Kappler, J., Cell, 7.6: 323-332, 1994; Smith, G., Trends in Micro.,, 82: 80-88, 1994). In fact, the survival of viruses depends on strategies that can evade, suppress, counteract or otherwise confuse the multiplicity of host responses to a foreign invader. The selection pressure conferred by the executing arms of the immune system can clearly be a powerful element of evolutionary pressure, and all eukaryotic viruses currently in existence contain impurities or remnants of their battles with the immune system, either as encoded proteins or as it is evidenced by its particular biolog survival strategies. Larger DNA viruses (ie, adenoviruses, herpes viruses, iridoviruses and poxviruses) specifly encode proteins that function to protect the virus from recognition and / or immune clearance by the infected host. Such "subversive" proteins are now providing information concerning the functional operations of the immune system, and it is feasible that many more discoveries of new members of this growing family will be identified in the future. In the 1980s, the term "virocin" was proposed to describe proteins encoded by viruses secreted from infected cells that work by mimicking extracellular signaling molecules such as cytokines or other secreted regulators important for the immune repertoire of the host (Kotwal, G and Moss, B., Na ture, 335: 176-178, 1988). Later, in the decade of the nineties, the term "viroceptor" was introduced to take into account the observation that some proteins encoded by viruses mimic important cellular receptors and work by diverting the host cytokines away from their normal receptors, thus interrupting the circuit immunolog in its earliest stages (Upton et al., Virology, 184: 370, 1991; Schreiber and McFadden, Virology, 204: 692-705, 1994). Recent studies on a particular poxvirus, the myxoma virus, have shown that the virus disrupts the immune system through a variety of strategies (McFadden and Graham, Seminars in Virology, 5. 4421-429, 1994). Myxoma virus is the infectious agent of a virulent systemic disease of domestic rabbits called myxomatosis. Originally described in the last century, myxoma was the first pathogen virus discovered for a laboratory animal and was the first viral agent ever deliberately introduced into the environment for the explicit purpose of erading pests. Since their release into the habitat of Australian and European feral rabbit populations more than 40 years ago, field strains of both rabbits and viruses have been subjected to evolutionary and selective pressures from each other that have resulted in a stable state enzooty in the inoculated areas (Fenner, F. and Ratcliffe, FN, "Myxomatosis", Cambridge University Press, London, 1965). Myxoma shares many of the biolog characteristics associated with other poxviruses, namely the cytoplasmic location of replion and a large double-stranded DNA genome (160 kilobases). Multiple lines of evidence inde that the myxoma, like all poxviruses, encodes multiple gene products whose function is to allow spreading and spread of the virus in a variety of host tissues. Some of these viral proteins counteract or specifly attack the development of the host's inflammatory response and acquired cellular immunity, and poxviruses in general have been a rich source of such immunomodulatory proteins (Turner, PC and Moyer, RW, Cur. Top, Microbiol, Imm., 163: 125-152, 1990; Buller, R.M.L. and Palumbo, G.J., Micro. Dev., 55: 80-122, 1991; Smith, G.L., J. Gen. Virol. , 94 _1725-1740, 1993; McFadden, G. (ed.), "Viroceptors, virokines and related immune modulators encoded by DNA viruses", R.G. Landes Co. , Austin, Texas, 1995). Examples of such immunomodulatory gene products include myxoma growth factor (MGF), which stimulates neighboring cells in a paracrine-like manner via the epidermal cell growth factor receptor (Upton et al., JL Virol., 61: 1271-12 '5, 1987; 186: 185-191, 1992; Opgenorth et al., Virol., 192: 701-708, 1992; Opgenorth et al., Virol., 66: 4720-4731, 1992); Serpt 1, a secreted glycoprotein with activity of the serine protease inhibitor, which prevents the development of the early inflammatory response (Upton et al., Virol., 179: 628-631, 1990; Lomas et al., JBC, 268: 516-521 , 1993; Macen et al., Virol., 195: 348-363, 1993); T2, a viral homolog secreted from the tumor cell necrosis factor receptor (TNF) superfamily, which binds to inhibits rabbit TNF (Smith et al., BBRC, 176: 335-342, 1991; Schreiber, M. et al. McFadden, G., supra, 1994; Upton et al., Supra, 1991); T7, a viral homolog secreted from the interferon receptor? cellular (Upton et al., Science, 258: 1369, 1992; Upton and McFadden, Methods in Molecular Genetics, 4: 383, 1994; Mossman et al., in: "Viroceptors, virokines and related immune modulators", p. 54, editor McFadden, RG Landers Co., 1995, and M11L, a surface receptor-like protein that interferes with the inflammatory response by an unknown mechanism (Opgenorth et al., Supra; Graham et al., Virol., 191: 112 -124, 1992.) Immunomodulatory proteins also include chemoattractant cytokines, called "chemokines." Chemokines are low molecular weight immune ligands that are chemoattractants for leukocytes, such as especially neutrophils, basophils, monocytes, and T cells. two main classes of chemokines containing both four conserved cysteine residues-two that form bisulfide bonds in the tertiary structure of the proteins.The class is designated CXC (where X is any amino acid), which includes 11-8, CTAP-III, gro / MGSA and ENA-78; and class β, designated C-C, which includes MCP-1, MlP-la and β, and normal expressed and secreted T proteins, regulated upon activation (RANTES). The designations of the classes are according to whether an intervening residue separates the first two cysteines in the motif. In general, most C-X-C chemokines are chemoattractants for neutrophils but not for monocytes, whereas C-C chemokines seem to attract monocytes but not neutrophils. Recently, a third group of chemokines, group "C", was designated by the discovery of a new protein called lymphotactin (Kelner et al., Science, 266: 1395-1933, 1994). It is believed that the chemokine family is critically important in the infiltration of lymphocytes and monocytes at sites of inflammation. It is highly probable that more viral genes will be discovered in unmodified laboratories. Not only do these and related gene products provide useful tools to cut the different weapons of the host's anti-viral defense mechanisms, but they can also provide new probes to identify novel elements of the cellular immunological repertoire and new classes of drugs to suppress the inflammation and dysregulation of the immune system. SUMMARY OF THE INVENTION The present invention describes a new family of soluble inhibitors specific to viruses for a class of cytokines that are involved in leukocyte chemotaxis and are collectively referred to as "chemokines". These proteins are designated chemokine type 2 binding proteins (CBP type 2) and are a family of poxvirus proteins related to the TI protein encoded by the Shope fibroma virus and the myxoma virus (SFV-T1). 2 and functionally related homologues are useful for the treatment of a variety of inflammatory disorders in which an excessive influx of leukocytes is associated with the pathogenic process.

Brief Description of the Drawings Figure 1 shows the sequence alignment of the known mierabros of the type 2 CBP family of the poxvirus proteins that bind to the chemotactic cytokines (chemokines). The RPV 35KDa sequence is incomplete. Figure 2 shows the nucleotide sequence of the myxoma Ti gene, which expresses a type 2 CBP. The nucleotide triplet within a frame is the stop codon for the adjacent T2 gene (TNF receptor homolog), the arrow denotes the cut-off site of the predicted signal peptide, and the underlined amino acids are the two predicted N-glycosylation sites for the IT protein (SEQ ID NO: 1 and SEQ ID NO: 2). Figure 3 shows that the 35KDa protein of a rabbit poxvirus (RPV), one of the members of the CBP type 2 family, is linked to members of the CC family of chemokines (MlP-lß, upper panel), and the family CXC (11-8, middle panel). In the two upper panels, the radiolabelled ligands are cross-linked to viral proteins secreted from control-infected BGMK cells (MOCK), or myxoma-infected cells (MYX), a myxoma-suppressing mutant T7 (MYX-T7), poxvirus from rabbit (RPV), and a 35KDa deletion mutant of RPV (RPV-35K-). Complexes cross-linked between the ligand and the viral proteins are indicated by the arrows.

Although the myxoma type 1 CBP protein (T7) is also linked to chemokines, the type 2 CBP protein (TI) is virtually the same size (about 35KDa), as shown in the lower panel. Both the TI (type 2) and T7 (type 1) proteins of the myxoma are bound to chemokines, but only the 35KDa protein of RPV (type 2) has this activity. Figure 4 shows that the vaccinia 35KDa secreted protein (Lister strain), but which is absent from the WR strain, binds to the MlP-lß chemokine similarly to the 35KDa secreted RPV protein. The arrow marks the complex between MlP-lβ and the 35KDa protein secreted from the BGMK cells infected with Lister-type vaccinia, but not the WR strain. Detailed Description of the Invention The findings of the present invention provide a new and important source of anti-immune proteins that have the potential to treat a wide range of immunopathological conditions associated with trafficking of lymphocytes and monocytes from the circulation to sites of tissues during inflammation and immunological responses to damage, infection and various disease states. The cloned and sequenced type 2 CBP genes are non-secreted homologs of the known chemokine receptors, all of which possess seven membrane-spanning domains (called "serpentines"), as described in recent reviews (Kelvin, DJ et al., JL eukocyte Biol., 54: 604-612, 1993, Murphy, PM, Ann. Rev. Imm., 12: 593-633, 1994, Horuk, R., Imm. Today, 15: 169-174, 1994, and Horuk, R., Trends in Pharm, Sci., 15 .: 159-165, 1994). Although some DNA viruses encode homologs of such serpentine receptors (Ahuja, SK et al., Imm. Today, 15: 281-287, 1994), including at least one candidate gene in a poxvirus (Massung, RF et al., Virology , 197: 511-528, 1994), the type 2 CBP of the present invention is not a member of this particular family of receptors. The exemplary chemokine type 2 binding protein (CBP type 2) of the invention is one of the proteins secreted from cells infected with the Shope fibroma virus and is encoded by the Ti open reading frame (Upton et al., Virology, 160: 20-30, 1987, and access No. P25946 of GenBank). This protein has a significant sequence similarity to the 35kDa proteins secreted from vaccinia (Copenhagen and Lister strains) and rabbit poxvirus. In addition, type 2 CBP proteins are distinct from the myxoma M-T7 protein that binds specifically to IFN-α. of rabbit, but not IFN-? of mouse or human (Mossman et al., J. Biol. Chem., 270: 3031-3038, 1995), but they are also linked to chemokines and are designated as a chemokine-binding protein type (previously denoted chemokine-binding protein-1 (CBP-1)). The term "chemokine binding protein" refers to a protein that binds to and inhibits one or more chemokines. A "chemokine" is a class of cytokines that are responsible for leukocyte chemotaxis. Class a of chemokines is designated CXC (where X is any amino acid), which includes interleukin-8 (Il-i), connective tissue activating protein III (CTAP-III), gro / MGSA (MGSA) with stimulating activity of the growth of melanocytes, the IFN-α protein (IP-10), neutrophil activating peptide 2 (NAP2), β-thromoboglobulin and the attractant-78 neutrophil epithelial derivative (ENA-78); and class β, designated CC, which includes gene-3 activation of T cells (TCA-3), monocyte chemoattractant proteins (MCP-1, 2 and 3), macrophage inflammatory proteins (MlP-la and ß), and normal expressed and secreted T proteins, regulated upon activation (RANTES). Other chemokines can be detected by methods commonly used in the art. For example, a molecule can be tested using the Boyden chamber, which is the preferred microchemotaxis assay system for in vitro research of chemoattractants. A series of cavities are formed in a block of Plexiglas, each cavity consisting of two chambers, upper and lower, which are separated by any of several types of porous filters, such as nitrocellulose and polycarbonate, for example. The cell of interest, for example peripheral blood mononuclear cells (PBMC), is added to the upper chamber of each cavity and the test substance, for example the guimo-tracer, is added to the lower chamber. If the cells in the upper chamber are attracted to the substance in the lower chamber, they will migrate together with the theoretical concentration gradient that exists in solution and will crawl through the pores of the filter and adhere to the underside of that filter. Polypeptides suspected to be members of the chemokine family can now be analyzed using the CBP of the invention. Accordingly, in one embodiment, the invention provides a method for analyzing and identifying novel chemokines comprising contacting free or matrix-bound CBP of the invention with a composition suspected of containing one or more chemokines and detecting the binding of the CBP to the composition. If desirable, various labels can be used as a means to detect the binding of CBP to a chemokine. Chemokines or CBP can be detectably labeled directly or indirectly, for example, with a fluoroscope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. Technicians in the art will know other suitable labels or will be able to determine the above using routine experimentation. In one embodiment, the invention provides a method for treating an immunopathological disorder in a subject, comprising administering to the subject a therapeutically effective amount of an anti-inflammatory protein characterized by having a molecular weight of about 30-40 kD, depending on the degree of glycosylation, as determined by reduced SDS-PAGE, having amino acid sequence homology with homolog SFV TI or RPV 35kDa and having the property of being secreted from infected cells. The term "anti-inflammatory" refers to the reduction or suppression of an inflammatory response. The glycosylated and secreted form of CBP type 2 of the invention has an apparent molecular weight of about 35-40 kD, as determined under reducing conditions on SDS-PAGE. In addition, the protein has homology with the secreted proteins SFV TI and RPV 35kDa. The term "homology" refers to the degree of identity between CBP type 2 and other members of the family at the amino acid level. Preferably, CBP type 2 has between 59 and 95% amino acid sequence homology with the SFV Ti protein. The requirement of homology is not strict, however, since CBP type 2 must retain the biological function of interacting with human chemokines. In other words, homology is sufficient as long as type 2 CBP binds to and inhibits guimokines. The invention includes a functional polypeptide, CBP type 2, and its functional fragments. As used herein, the term "functional polypeptide" refers to a polypeptide that possesses a biological function or activity that is identified by a defined functional assay and that is associated with a particular biological, morphological or phenotypic response.

Functional fragments of the CBP type 2 polypeptide include fragments of CBP type 2 as long as the activity of CBP type 2 remains (ie, binding to chemokines). Smaller peptides containing the biological activity of CBP type 2 are included in the invention. Such peptides can be tested for chemokine binding by methods commonly known to those skilled in the art, including methods described in the examples herein. The biological function can vary from a polypeptide fragment as well as an epitope which can bind an antibody molecule to a large polypeptide that is capable of participating in the induction or programming characteristic of phenotypic changes within a cell. A "functional polynucleotide" denotes a polynucleotide that encodes a functional polypeptide, as described herein. Minor modifications of the primary amino acid sequence of CBP type 2 can result in proteins having substantially equivalent activity compared to the CBP type 2 polypeptide described herein. Such modifications may be deliberate, such as by site-directed mutagenesis, or may be spontaneous. All polypeptides produced by these modifications are included herein as long as the activity of CBP type 2 is retained. In addition, the deletion of one or more amino acids can result in a modification of the structure of the resulting molecule without significantly altering its activity. This may lead to the development of a smaller active molecule, which may have a wider utility. For example, it is possible to remove terminal amino or carboxy amino acids that may not be required for CBP type 2 activity. The type 2 CBP polypeptide of the invention also includes conservative variations of the polypeptide sequence. The term "conservative variation", as used herein, denotes the replacement of an amino acid residue by another biologically similar residue. Examples of conservative variations include the substitution of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine by asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid with the proviso that the antibodies created by the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Examples of viral sources of CBP type 2 used in the method of the present invention include myxoma virus, vaccinia (Lister and Copenhagen strains), Shope's fibroma virus, rabbit pox virus and other mammalian pox virus, while type 2 CBP has the biological function of an a-laryan anti-inflammatory protein characterized by having a molecular weight of approximately 30-40 kD, depending on the degree of glycosylation having homology with SFV protein TI homolog, and having the biological function of this protein family. An immunopathological disorder treated by the method of the invention may be associated with the production of chemokines and the resultant accumulation of reactive leukocytes in affected tissues. The method comprises administering to the subject a therapeutically effective amount of CBP type 2. The term "immunopathological disorder" refers to any disease that involves the immune response or immunity in general. "Therapeutically effective", as used herein, refers to that amount of CBP type 2 that is sufficient to ameliorate the cause of the immunopathological disorder. "Improvement" refers to a relief of the deleterious effect of the disorder on the patient receiving the therapy. The subject of the invention is preferably a human being; however, it can be stated that any animal with an immunopathological disorder can be treated by the method of the invention, for example a SCID mouse grafted with human bone marrow (humanized SCID). Examples of immunopathological disorders that can be treated by the method of the invention include acquired immune deficiency disorder (AIDS), toxic shock syndrome, allograft rejection, arteriosclerotic plaque growth, responses to radiation and ultraviolet light, and disorders associated with the activation of T cells, B cells, macrophages, and other inflammatory leukocytes during the immune response and acute phase response and disorders associated with advanced cancer such as tumor necrosis factor-mediated cachexia. The invention provides a method of treating or alleviating an immunopathological disorder, including endotoxemia or septic shock (sepsis), or one or more of the symptoms of sepsis, which comprises administering to a subject exhibiting symptoms of sepsis or at risk of developing sepsis a therapeutically effective amount of CBP type 2. The term "relieve" refers to a reduction or attenuation of the symptoms of the disorder being treated. A patient who exhibits the symptoms of an immunopathological disorder may be treated with an antibiotic or an anti-viral agent in addition to treatment with type 2 PBC. Typical antibiotics include an aminoglycoside, such as gentamicin or a beta-lactam such as penicillin, or cephalosporin . Thus, a therapeutic method of the invention includes administering a therapeutically effective amount of CBP type 2 substantially simultaneously with the administration of a bactericidal amount of an antibiotic or a sufficient amount of an anti-viral compound. The term "bactericidal amount", as used herein, refers to an amount sufficient to achieve a concentration in the blood that eliminates the bacteria in the patient receiving the treatment. The bactericidal amount of an antibiotic generally recognized as safe for administration to a human is well known in the art, and as is known in the art, varies with the specific antibiotic and the type of bacterial infection that is being treated. Preferably, the administration of CBP type 2 occurs within about 48 hours, and preferably within about 2 to 8 hours, and most preferably substantially simultaneously with the administration of the antibiotic. The administration of a type 2 CBP in the method of the invention can also be used to alleviate post-re-perfusion injury. When treating arterial thrombosis, induction of re-perfusion by clot lysing agents such as tissue plasminogen activator (t-PA) is often associated with tissue damage. Such damage to tissues is believed to be mediated at least in part by leukocytes including, but not limited to, polymorphonuclear leukocytes (PMN). Therefore, the administration of type 2 CBP would block endothelial interactions with leukocytes or PMN, and thereby decrease or prevent post-re-perfusion injury. The administration of CBP type 2 is also useful for the prevention of new onset and growth of recurrent arteriosclerotic plaque after arterial injuries. Restenosis and new plaque development are thought to be exacerbated by the local inflammatory response to the inner layer of the arterial wall. The method of the invention is also useful for the treatment of inflammation due to allergic or auto-immunological disorders. Examples of allergic disorders include rhinitis, asthma, allergic atopic dermatitis, and food allergies. Examples of autoimmune disorders, where the immune system attacks the host's own tissues, include, but are not limited to, insulin-dependent diabetes mellitus type 1, inflammatory bowel disease, dermatitis, meningitis, thrombotic thrombocytopenic purpura, Sjögren's syndrome, encephalitis, uveitis, leukocyte adhesion deficiency, reu a-toide arthritis or other forms of immune arthritis, rheumatic fever, Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis, primary biliary cirrhosis , pemfiga, pemfigoide, necrotizing vasculitis, myasthenia gravis, multiple sclerosis, lupus erythematosus, polymyositis, sarcoidosis, granulomatosis, vasculitis, pernicious anemia, inflammatory CNS disorder, diseases mediated by the antigen-antibody complex, autoimmune hemolytic anemia, Hashimoto's thyroiditis , serious illness, habitual miscarriages, Reynard's syndrome, glomerulonephritis, dermatomyositis, chronic active hepatitis, celiac disease, auto-immune complications of AIDS, atrophic gastritis, ankylosing spondylitis, and Addison's disease. The method is also useful for treating non-malignant cell proliferative diseases or related to immune cells, acute respiratory disease syndrome (ARDS), ischemic heart disease, atherosclerosis, post-dialysis syndrome, leukemia, acquired immuno-logical deficiency syndrome , septic shock and other types of acute inflammation, and lipid histiocytosis. Essentially, any disorder that is etiologically related to the pro-inflammatory process and cell infiltration due to production that chemokine (for example, induction of expression of IL-8, MlP-la or ß) would be considered amenable to treatment. The method of the invention is also useful for the treatment of microbial infections. Many microbes, such as bacteria, rickettsia, various parasites and viruses, bind to vascular endothelium and leukocytes, and induce an inflammatory reaction that results in the production of interleukins, for example. In this manner, the type 2 CBP used in the method of the invention can be administered to a patient to prevent the inflammation associated with such infections. The dose ranges for administration of type 2 CBP of the invention are those large enough to produce the desired effect in which the symptoms of the immune response show some degree of suppression. The dosage should not be so large as to cause adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and degree of the disease in the patient, and may be determined by a person skilled in the art. The dosage can be adjusted by the individual doctor in the case of any contraindication. The dosage can vary from 10 pg to 100 pg per dose, in one or more doses administered daily, for one or several days. CBP type 2 is administered by any suitable means, including parenteral, subcutaneous, intrapulmonary, intra-arterial, intra-rectal, intra-muscular, intravenous, and intra-nasal administration. Parenteral infusions include intra-muscular, intra-venous, intra-arterial or intra-peritoneal administration. CBP type 2 can also be administered transdermally in the form of a subcutaneous slow release implant, for example, or orally in the form of capsules, powders or granules. CBP type 2 can also be administered by inhalation. For example, when used therapeutically for the treatment of an inflammatory disorder of the lungs, a preferred route of administration would be by means of a pulmonary aerosol. Pharmaceutically acceptable carrier preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's dextrose, or fixed oils. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include water, saline, dextrose, glycerol and ethanol, or combinations thereof. Intravenous vehicles include fluid and nutrient restorers, electrolyte restorers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as, for example, anti-microbial, antioxidants, chelating agents, and inert gases and the like may also be present. The invention also relates to a method for preparing a medicament or a pharmaceutical composition comprising the CBP type 2 of the invention, the medicament being used for therapy of an inflammatory reaction / undesirable immuno-logical response where the immunological response results in the production of chemokines that bind to the type 2 CBP of the present invention. The invention provides a pharmaceutical composition comprising at least one dose of a non-therapeutically effective amount of an anti-inflammatory protein having a molecular weight of about 30-40 kD, depending on the degree of glycosylation, having amino acid sequence homology with the homologue of the IT receptor interferon-? of myxoma, and having the biological function of the IT receptor homologue interferon-? of myxoma, in a pharmacological carrier. The invention provides any pharmaceutical preparations and compositions containing the type 2 CBP of the invention for use in the method of the invention. The form will vary depending on the administration route. For example, compositions for injection may be provided in the form of an ampule, each containing a quantity of unit dose, or in the form of a container containing multiple doses. Type 2 CBP can be formulated into the therapeutic composition as pharmaceutically neutralized salt forms. These include the acid addition salts which are formed with inorganic acids such as, for example, hydrochlorhydride or phosphoric acid, or organic acids such as acetic, oxalic, tartaric acids, and the like. The salts also include those formed of inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. Controlled delivery can be achieved by selecting appropriate macromolecules, for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylene vinyl acetate, methyl cellulose, carboxymethyl cellulose, prota ina sulfate, or lactide / glycolide copolymers. The rate of release of CBP type 2 can be controlled by altering the concentration of the macromolecule.

Another method for controlling the duration of the action comprises incorporating the type 2 CBP into particles of a polymeric substance such as polyesters, polyane acids, hydrogels, polylactide / glycolide copolymers, or ethylene vinyl acetate copolymers. Alternatively, it is possible to trap type 2 CBP in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example by the use of hydroxymethylcellulose or gelatin microcapsules or poly (methylmethacrylate) microcapsules, respectively, or in a colloidal medication delivery system. Colloidal dispersion systems include complexes of macromolecules, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, mycelia, mixed mycelia, and liposomes. The following examples are intended to illustrate but not limit the invention. Although typical of those that could be used, other methods known to those skilled in the art may alternatively be used. Example 1 Materials and Methods Rat Model of Spinal Injury-induced Atherosclerosis Sprague Dawley rats were induced to have a lesion mediated by balloon angioplasty of the right or left iliofemoral artery. A 1.5 mm USCI angioplasty balloon was advanced retrogradely to the artery via a cut and arteriotomy under general anesthesia with pentobarbitai (6.5 mg per 100 g of weight per intramuscular injection, Somnotrol, MTC Pharmaceuticals, Cambridge, Ontario, Canada). ). 500 pg of CBP (6 rats) or saline solution (6 rats) were administered by intra-arterial injection of CBP or the control solution into the distant lumen of the angioplasty balloon catheter upstream of the site of subsequent balloon-mediated damage. The balloon was then inflated to a pressure of 8 bar for 1.0 minutes. After angioplasty, the balloon was deflated and removed and the site of closed arteriotomy with local application of n-butyl cyanoacrylate monomer (Nexaband, Veterinary Products Laboratories, Phoenix, Arizona, United States). Each rat was maintained on a normal rat diet and was followed up for up to 4 weeks after surgery. At the end of the follow-up, the rats were sacrificed with 2.0 ml of euthanil per kg and the aorta was harvested for histological examination. Lesion-induced Atherosclerosis Rabbit Model Eight New Zealand white rabbits, fed cholesterol, are treated by balloon angioplasty of the distant abdominal aorta. All rabbits (New Zealand white strain) are fed a diet of 2% cholesterol in 10% peanut oil for 4 days / week, starting two weeks before the balloon injury. An angioplasty balloon catheter of 3-3.5 mm (ratio of balloon diameter to aortic diameter = 1: 1) is introduced via a cut in the femoral artery after anesthesia (40 mg / kg ketalene, 8 mg / kg of xylasene and 0.5 mg / kg of acepromazine by intramuscular injection). The balloon is inflated to a pressure of 8 bars in the distant abdominal aorta and advanced retrograde to the distant thoracic aorta. The balloon is advanced and removed 3 times under fluoroscopic control in each rabbit to ensure endothelial denudation. Contrast angiograms were recorded before and after trauma mediated by balloon angioplasty and at 4 weeks of follow-up. Heparin (400 units) is given immediately after obtaining femoral access to reduce thrombosis associated with the catheter. Purified CBP type 2, 500 pg per sample, is applied by infusion immediately after the balloon-mediated lesion in the distant abdominal aorta of 4 rabbits. A parallel infusion of saline solution is applied by infusion locally into the distant abdominal aorta in 4 rabbits. Each infusate is administered via the Wolinsky catheter in a total volume of 10 ml diluted in 0.9% sterile saline., immediately after the balloon-mediated injury. All infusions are via a 3.25 mm Wolinsky balloon (inflated to a final pressure of 6 + 1 bar for 2 minutes) in the abdominal aorta near the iliac bifurcation. The Wolinsky balloon is placed immediately above the iliac bifurcation under fluoroscopic control such that the perfusion balloon is routinely located 0.5 to 2.5 cm above the bifurcation and is designated as the primary infusion site. Secondary sites upstream are defined in the region about 2.5 cm proximal to the iliac bifurcation. In all the experiments, the infusions are administered via the Wolinsky catheter in a total volume of 10 ml diluted in 0.9% sterile saline immediately after the balloon-mediated lesion. All infusions are via a 3.25 mm Wolinsky balloon (inflated to a final pressure of 6 + 1 bar for 2 minutes) in the abdominal aorta near the iliac bifurcation. Histology and Morphine-rich Analysis Histological analysis is performed at the primary site of Wolinsky infusion in the distant abdominal aorta (rabbits) or the superior iliofemoral arterial branches (rat), representing the primary infusion site as defined by the placement original perfusion balloon. In rabbits, internal control sections are taken from a downstream site, not infused, near the iliac bifurcation (0.5 cm above the bifurcation at 0.5 cm below the bifurcation) and at an upstream site, not submitted to infusion (the upper abdominal aorta, 2.5-3.5 cm above the iliac bifurcation). The 1.5-2.5 cm area above the iliac bifurcation is considered a borderline area with potentially variable infusion doses due to balloon placement and therefore is not included in the analysis. In rats, primary balloon sites were used for histological determination for both rats treated with T-1 and rats infused with saline solution. Staining with hematoxylin and eosin from fixed specimens with formalin was carried out, as previously described. Briefly, each specimen was fixed in formalin buffered with 10% (v / v) sodium phosphate, processed, impregnated, embedded in paraffin and cut into 5 μm sections by means of a microtome. Sections of each specimen (a minimum of 2 sections per site) were then stained with hematoxylin and eosin and examined by light microscopy. Example 2 Clearance of Cytokines to a Novel Viral Protein Briefly, a variety of human cytokines were radiolabeled with 125 I, exposed to secreted proteins, harvested from control or poxvirus-infected BGMK cells, cross-linked, and then analyzed by SDS- PAGE regarding novel cytokine / protein complexes. The cross-linkage assay revealed what was clearly a novel viral specific protein that was linked to each of the human chemokines that were tested: IL-8 and MIP-lβ (Figure 2). Figure 2 (the two upper panels) shows gel mobility shift assays using iodinated ligands and tissue culture supernatants. Tissue culture supernatants (Sups) were prepared as follows: BGMK (baby green monkey kidney cells) were left untreated (mock) or infected with myxoma (MYX), myxoma T7 suppressant mutant (myx-T7") , rabbit pox (RPV) or a suppression of 35kDA of RPV at a multiplicity of infection (MOI) of 3. The secreted proteins were prepared by washing the monolayer three times with PBS and restocking with serum-free medium 4 hours after infection, these supernatants were then harvested 18 hours after infection (L) .Mock supernatants were prepared in the same way in the absence of virus.The supernatants were concentrated approximately 15 times using Amicon concentrators.The human chemokines IL-8 and MIP-Iß were labeled with 125I using iodine beads (Pierce) according to the manufacturer's protocols Mobility gel displacement assays were carried out as follows: 5 μl ligand or iodinated were ezcla-dos with 10 μl of SUP and left to settle at room temperature for two hours. Then 2 μl of the cross-linking chemical reagent l-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) (200 mM in 100 mM potassium phosphate, pH 7.5) were added for 15 minutes, followed by an additional 2 μl per 15 minutes. The reaction was then suddenly quenched by the addition of 2 μl of Tris-HCl (1.0 M, pH 7.5). The resulting mixture was analyzed using SDS-PAGE and autoradiography. The arrows indicate the displaced complexes. The lower panel indicates the Coomassie stained gels, showing the loss of the T7 protein in the infection with myx-T7"and the loss of the 35kDa protein in the infection with RPV-35k". Example 3 Analysis of the Efficacy of CBP Type 2 (TI) as an Anti-Restenosis Protein, as Shown in Balloon-Mediated Lesion of Anteriooplasty in Rat Femoral Arteries Inflammation is associated with the accelerated development of atherosclerotic plaque in the arterial wall. There is a high rate of plaque recurrence, or restenosis, after the use of balloon angioplasty and other related angioplasty devices designed to open clogged arteries. The accelerated growth of atherosclerotic plaque has also been reported under conditions that lead to arterial injury, viral infections, vasculitis, I have ocistenuria, diabetes mellitus, hypertension, hyperlipidiuria, smoking and disorders generated by immune complexes. Larger DNA viruses have developed mechanisms, such as inflammatory proteins, that allow the virus to proliferate in the host with reduced inhibition by the inflammatory and immunological defense mechanisms of the host. Type 2 CBP (T-1) was tested as a potential therapeutic agent for the prevention of plaque growth after angioplasty. It is believed that CBP type 2 acts both as a gamma receptor homolog of interferon and as a chemokine inhibitor. CBP type 2 was tested in animal models of atherosclerosis induced by injury and the results showed a significant reduction in plaque formation four weeks after infusion. The 35kDa protein, a type 2 CBP isolated from vaccinia virus, considerably reduced intimal hyperplasia (growth of atherosclerotic plaque) after balloon angioplasty injury after a single intravenous injection (Table 1). This shows that CBP type 2 can be used as an anti-inflammatory agent for treatment or prevention of disorders based on the immune system. 10 Sprague Dawley rats were induced to have the balloon lesion of the right iliofemoral artery under general anesthesia. A 1.5 mm angioplasty balloon was introduced via the femoral artery and the distant tip of the balloon was advanced to the iliac bifurcation. Immediately before inflating the balloon, 1.0 ml was injected into the artery either saline (5 rats) or increasing concentrations of the 35k protein of vaccinia, purified, at 50 pg (5 rats). The angioplasty balloon was then inflated at 6-8 atm for 2 minutes, deflated and withdrawn. The femoral artery was sealed with nexabanda at the puncture site after the catheter was removed. The rats were allowed to recover and were supervised for 4 weeks. At 4 weeks, the rats were sacrificed and the arteries harvested for histological determination. The intimate area was measured by morphometric analysis. A considerable reduction in intimal hyperplasia (plaque area) was detected after the infusion of the 35kDA protein compared to infusions of control saline (p ^ 0.0089) (Table 1) • Table 1 Example 4 CBP-II Inhibits Chemokine Ligation to Cellular Receptors of Human Moites The inhibition of MlP-la binding to a surface receptor in primary human monocytes and THP-1 cells by the vaccinia 35kDa protein (Lister strain) (Table 2) and M-Tl (Table 3), respectively, It was demonstrated according to the following protocol. (Note: Experiment 3, in Tables 1 and 2 used THP-1 cells, all other results were obtained from primary monocytes). Radiolabelled MlP-la with 125 I (25 μCi / ml) was obtained and an appropriate volume of radiolabeled (hot) MIP-loi was added such that each tube contained 50,000 cpm. As a control, control MIP-loi was added, unlabelled (cold) (400 ng) together with the hot MlP-la to demonstrate background ligation. To measure the inhibitory properties of M-Tl (CBP-2) 35kDa and M-T7 (CBP-1), variable doses of the inhibitor were added with the radiolabelled ligand and the samples were incubated at 37 ° C (5% of C02) for 30 minutes. Primary monocytes isolated from human blood and separated on a Percoll gradient were diluted in RPMI 1640 containing 1% BSA at a concentration of 1 x 10 7 cells / ml, and 200 μl of these cells were added to each reaction. Similar concentrations were used for THP-1 cells (monocytic cell line). The sample tubes were rotated for 1 hour at room temperature, at which time the cells were rotated at 13,000 rpm for 5 minutes. The supernatants were removed, and the cells washed with 800 μl of 10% sucrose solution. The cells were again rotated at 13,000 rpm for 10 minutes, and the supernatants removed. The radioactivity of the beads was then measured using a gamma counter. As a second set of controls, the ligation properties of H3-fMLP to its cellular receptor were also tested in the presence of the inhibitors (Table 4). The above protocol was followed, with the exception that a uniform volume of 1 μl was distributed to each tube, which resulted in counts of approximately 180,000 cpm per tube. Note that 35kDa and M-Tl both blocked quantitatively the binding of the chemokine to the cellular receptors of MIP-lo :.

Table 2 MIP-IOÍ Linked to Surface Note: Experiment 3 used THP-1 cells; Experiments 1 and 2 used primary monocytes. Table 3 MIP-IOÍ Linked to Surface Table 4 fMLP Linked to Surface Although the invention has been described with reference to the presently preferred embodiment, it should be understood that modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims (11)

1. CLAIMS 1. A method of treating an immunopathological disorder related to chemokine selected from the group consisting of microbial infection, malignancy and metastasis, asthma, coronary restenosis, autoimmune diseases, cirrhosis, endotoxemia, atherosclerosis, re-perfusion injury and inflammatory responses , in a subject, comprising administering to the subject a therapeutically effective amount of a protein that binds to chemokine having an amino acid sequence as set forth in SEQ ID NO: 2. The method of claim 1, wherein the Chemokine is a class chemokine or class β. 3. The method of claim 2, wherein the chemokine is selected from the group consisting of CTAP-III, gro / MGSA, ENA-78, MCP-1, interleukin-8, RANTES, MlP-la, MlP-lß, PF -4, IP-10 and NAP-2. 4. The method of claim 1, further comprising administering an antibiotic or anti-viral to the subject. 5. The method of claim 1, wherein the administration of the anti-inflammatory protein is at a dose of about 10 pg to 100 pg per administration. The method of claim 1, wherein the administration of the anti-inflammatory protein is selected from the group consisting of subcutaneous, intra-venous, intra-arterial, intra-muscular, intra-rectal and transdermal administration. 7. A pharmaceutical composition comprising at least one dose of an immunotherapeutically effective amount of an anti-inflammatory protein having an amino acid sequence as set forth in SEQ ID NO: 2 in a pharmaceutically acceptable carrier. The composition of claim 7, wherein the anti-inflammatory protein is a protein that binds to qimocin type 2. 9. The composition of claim 8, wherein the chemokine is a chemokine class OI or class ß. The composition of claim 9, wherein the chemokine is selected from the group consisting of CTAP-III, gro / MGSA, ENA-78, MCP-1, interleukin-8, RANTES, MlP-la, MlP-lß, PF -4, IP-10 and NAP-2. 11. A method for identification of a chemokine, the method comprising contacting a protein that binds to chemokine having an amino acid sequence as set forth in SEQ ID NO: 2, with a composition that is suspected to be a chemokine, and detect the ligation of the chemokine composition to the protein that binds to guimocine.