AU1642000A - Chemokine binding protein and methods of use therefor - Google Patents

Description

1

AUSTRALIA

Patents Act 1990 The John P Robarts Research Institute TRUE COPY COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Chemokine binding protein and methods of use therefor The following statement is a full description of this invention including the best method of performing it known to us:- CHEMOKINE BINDING PROTEIN AND METHODS OF USE THEREFOR BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates generally to the field of immunology and specifically to a chemokine binding protein encoded by a variety of poxviruses and methods of use therefor.

2. Description of RelatedArt It is becoming increasingly clear that viruses which make their living within cells of higher-order vertebrates must have evolved to specifically avoid the host immune system (Gooding, Cell, 21:5-7, 1992; Marrack, P. and Kappler, Cell, 76:323-332, 1994; Smith, Trends in Micro., 82:80-88, 1994). In fact, virus survival is dependent upon strategies which can evade, suppress, counteract, or otherwise confound the myriad of host responses to a foreign 15 invader. The selection pressure conferred by the effector arms of the immune system can clearly be a powerful element of evolutionary pressure, and all eukaryotic viruses existing today contain imprints or remnants of their battles with the immune system, either as encoded proteins or as evidenced by their particular biological survival strategies.

The larger DNA viruses the adenoviruses, herpesviruses, iridoviruses and poxviruses) specifically encode proteins that function to protect the virus from immune recognition and/or clearance by the infected host. Such "subversive" viral proteins are now providing information concerning the functional operations of the immune system, and it is likely that many more discoveries of new members of this growing family will be identified in the future.

In the 1980's the term "virokinc" was proposed to describe virus-encoded proteins secreted from infected cells which function by mimicking extracellular signaling molecules such as cytokines or other secreted regulators important for the host immune repertoire (Kotwal, G. and Moss, Nature. 335:176-178, 1988). Later, in the 1990's the term "viroceptor" was introduced to account for the observation that some virus encoded proteins that mimic important cellular receptors and function by diverting host cytokines away from their normal 10 receptors, thus interrupting the immune circuitry at its earliest stages (Upton, .i et al., Virology, 184:370, 1991; Schreiber and McFadden, Virology, 204:692- 705, 1994).

"ee ~Recent studies on a particular poxvirus, myxoma virus, have shown that the virus disrupts the immune system by a variety of strategies (McFadden and Graham, Seminars in Virology, 5:421-429, 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 Svirus pathogen discovered for a laboratory animal and was the first viral agent ever deliberately introduced into the environment for the explicit purpose of 20 pest eradication. Since its release into the Australian and European feral rabbit populations more than 40 years ago, the field strains of both the rabbit and virus have been subjected to mutual evolutionary and selective pressures that have resulted in a steady-state enzootic in the inoculated areas (Fenner, F. and Ratcliffe, "Myxomatosis", Cambridge University Press. London, 1965).

Myxoma shares many of the biologic features associated with other poxviruses, namely cytoplasmic location of replication and a large double stranded

DNA

genome (160 kilobases). Multiple lines of evidence indicate that myxoma, like all poxviruses, encodes multiple gene products whose function is to permit the spread and propagation of the virus in a variety of host tissues. Some of these viral proteins specifically counteract or subvert the development of the host inflammatory response and acquired cellular immunity, and poxviruses in general have been a rich source of such immunomodulatory proteins (Turner, and Moyer, Cur. Top. Microbiol. Imm., 163:125-152, 1990; Buller, and Palumbo, Micro. Dev., 55:80-122, 1991; Smith, Gen.

Virol., 94:1725-1740, 1993; McFadden, "Viroceptors, virokines and related immune modulators encoded by DNA viruses", R.G. Landes Co., Austin Texas, 1995).

Examples of such irnmunomodulatory gene products include myxoma growth factor (MGF), which stimulates neighboring cells in a paracrine-like fashion via w" the cellular epidermal growth factor receptor (Upton, et al., J. Virol., 61:1271- 1275, 1987; Opgenorth, et al., Virol., 186:185-191, 1992; Opgenorth, et al..

Virol., 192:701-708, 1992; Opgenorth, el al., J Virol., 66:4720-4731, 1992); Serp 1, a secreted glycoprotein with serine protease inhibitor activity, that prevents 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 secreted viral homologue of the cellular tumor necrosis factor (TNF) receptor superfamily, that binds and inhibits rabbit TNF (Smith, et al., BBRC, 176:335-342, 1991; Schreiber, M. and McFadden, G., supra, 1994; Upton, et al., supra, 1991); T7, a secreted viral homologue of the cellular interferon-y receptor, that binds and inhibits rabbit interferon-y (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: 41-54 Ed. McFadden, R.G. Landers, Co., 1995); and M 1L, a surface receptor-like protein that interferes within the inflammatory response by an unknown mechanism (Opgenorth, et al., supra; Graham, et al., Virol, 191:112-124, 1992); Immunomodulatory proteins also include chemotactic cytokines, called "chemokines". Chemokines are small molecular weight immune ligands which are chemoattractants for leukocytes, such as especially neutrophils, basophils, monocytes and T cells. There are two major classes of chemokines which both contain four conserved cysteine residues which form disulfide bonds in the tertiary structure of the proteins. The a class is designated C-X-C (where X is any amino acid), which includes IL-8, CTAP-III, gro/MGSA and ENA-78; and 10 the p class, designated C-C, which includes MCP-1, MIP-la and P, and regulated on activation, normal T expressed and secreted protein (RANTES).

The designations of the classes are according to whether an intervening residue spaces the first two cysteines in the motif. In general, most C-X-C chemokines oo are chemoattractants for neutrophils but not monocytes, whereas C-C chemokines appear to attract monocytes but not neutrophils. Recently, a third group of chemokines, the group, was designated by the discovery of a new protein called lymphotactin (Kelner, et al.. Science, 266:1395-1933, 1994). The chemokine family is believed to be critically important in the infiltration of lymphocytes and monocytes into sites of inflammation.

It is highly likely that more immunomodulatory viral genes remain to be discovered. Not only will these and related gene products provide useful tools to dissect out the different arms of the host antiviral defense mechanisms, but they may also provide new probes to identify novel elements of the cellular immune repertoire and new classes of drugs to suppress inflammation and dysregulation of the immune system.

SUMMARY OF THE INVENTION The present invention describes an unexpectedly discovered novel soluble virus-specific inhibitor for a class of cytokines which are involved in leukocyte chemotaxis and are collectively referred to as "chemokines". The protein of the invention is a Type 1 "chemokine binding protein" (CBP-1) and is a gene product of the T7 gene of myxoma virus, which previously had been shown to encode the interferon-y (IFN-y) receptor homolog. However, in sharp contrast to the extreme specificity of the T7 gene product for the rabbit ligand (IFN-y), the CBP-1 of the invention binds very well to mouse and human chemokines. The CBP-1 and related homologs encoded by other poxviruses is useful for treatment of a variety of inflammatory disorders in which excessive influx of leukocytes is associated with the pathogenic process.

S* BRIEF DESCRIPTION OF THE DRAWINGS FIGURE IA shows an SDS-PAGE of 11 2 1 -labelled IL-8, RANTES and MIP- I after exposure to poxviral-secreted proteins. FIGURE lB3 shows an SDS-PAGE of T' 2 -MIP-1p in nwxoma or ectromelia virus-infected cells.

FIGURE 2 shows an SDS-PAGE of l1'-Iabelled RANTES as a ligand for myxoma secreted proteins and competition with unlabelled RANTES, MIP- I P MIP-ict, IL-8 and MCP-l.

FIGURE 3A and 313 shows an SDS-PAGE of 11 2 1 -labelled MIP- IP as a ligand for poxviral secreted proteins and competition with unlabelled MIP- 1J, IFN-y.

MCAF, MIP-ict, RANTES and IL-8. FIGURE 3A shows myxoma, vaccinia, and SFV. FIGURE 3B shows myxoma, cow pox, rabbit pox, ectromelia, and

SFV.

FIGURE 4 shows an SDS-PAGE of I1 2 1-labelled IL-8 as a ligand for myxoma secreted proteins and competition with unlabelled MIP-l1r, IFN-y, MCAF, and :15 MIP- I a.

FIGURE 5 shows a Western blot that displays T7 protein following interaction with a variety of cytokines (IL-I P, ML-2, M1-3, IL-4, IL-6, IL-7, 11-8, IFNcz, and IFN-y).

FIGURE 6 shows a Western blot that displays T7 protein following interaction with a variety of chemokines (RANTES, MCP-l, MCP-3, IL-8, PF4, IP-1O, NAP-2, MGSA and lymphotactin).

FIGURE 7 shows an SDS-PAGE of total proteins from myxoma infected BGMK cells, semi-purified proteins after mono Q fractionation, and purified CBP-I (T7) protein after superdex 75 gel filtration.

FIGURE 8 shows binding of human RANTES chemokine to purified/partiallypurified CBP-I. Silver stain analysis shows a cross-linked complex (CBP-I Rantes) of approximately 47kD when either purified or partially purified CBP-I was incubated with RANTES (Lanes 2 and no gel mobility shifted complex was observed in the absence of RANTES (Lanes 1 and 3).

FIGURE 9 shows the binding of human RANTES chemokine to partiallyo. 10 purified CBP-I. Silver stain analysis revealed a cross-linked binding complex (CBP-I+ Rantes) of approximately 47kD when CBP-I is incubated with biotinylated Rantes (Lanes and this binding can be titrated out by decreasing amounts of the unlabelled chemokine ligand.

o FIGURE 10A shows plaque thickness/depth (mm) of atherosclerotic plaque in :an arterial injury rat model 1 month after CBP-I (T7) treatment.

FIGURE 10B and 10C show plaque area (mm2) of atherosclerotic plaque in an arterial injury rat model 1 month after CBP-I (T7) treatment.

FIGURE 11 shows plaque thickness (mm2) of atherosclerotic plaque in an arterial injury rabbit model 1 month after CBP-I (T7) treatment.

-8- DETAILED DESCRIPTION OF THE INVENTION The findings of the present invention provide an important new source of antiimmune proteins which have the potential to treat a wide range of immunopathological conditions associated with the trafficking of lymphocytes and monocytes from the circulation to tissue sites during inflammation and immune responses to damage, infection and various disease states.

The exemplary Type I chemokine binding protein (CBP-I) of the invention is the major secreted protein from cells infected with myxoma virus and is encoded by the M-T7 open reading frame (Upton, el al., Science, 258:1369, 1992; and GenBank Accession No: M81919; SEQ ID NO:1 and SEQ ID NO:2).

This protein has significant sequence similarity to the human and mouse receptors for interferon gamma (IFN-y). Further, the myxoma M-T7 protein specifically binds rabbit IFN-y, but not mouse or human IFN-y (Mossman, et al, J. Biol. Chem., 270:3031-3038, 1995).

15 The term "chemokine binding protein" refers to a protein which binds to and inhibits one or more chemokines. A "chemokine" is a class of cytokines which are responsible for leukocyte chemotaxis. The a class of chemokines is designated C-X-C (where X is any amino acid), which includes interleukin-8 connective tissue activating protein III (CTAP-III), melanocyte growth 20 stimulatory activity (MGSA) gro/MGSA, IFN-y inducible protein neutrophil activating peptide 2 (NAP2), P-thromboglobulin and epithelialderived neutrophil attractant-78 (ENA-78); and the p class, designated C-C, which includes T-cell activation gene-3 (TCA-3), monocyte chemotactic proteins (MCP-1, 2, and macrophage inflammatory proteins (MIP-lca and and regulated on activation, normal T expressed and secreted protein

(RANTES).

Other chemokines can be detected by methods commonly used in the art. For example, a molecule may be tested using the Boyden chamber, which is the preferred microchemotaxis assay system for m vitro investigation of chemoattractant substances. A series of wells is formed into a plexiglass block, each well consisting of two chambers, upper and lower, which are separated by any one of several types of porous filters, such as nitrocellulose and polycarbonate, for example. The cell of interest, for example peripheral blood mononuclear cells (PBMC) are added to the top chamber of each well and the test substance, the chemoattractant, is added to the bottom chamber. If the cells in the top chamber are attracted to the substance in the bottom chamber, they will migrate along the theoretical concentration gradient which exists in solution and crawl through the pores of the filter and adhere to the bottom side of that filter.

Polypeptides suspected of being members of the chemokine family can now be screened using the CBP-I of the invention. Therefore, in one embodiment, the invention provides a method for screening and identifying novel chemokines comprsing contacting free or matrix-bound CBP-I of the invention with a composition suspected of containing one or more chemokines and detecting binding of the CBP-I to the composition. Methods for detecting binding of the CBP-I to the composition (chemokine) will be known to those of skill in the art and include those described in the EXAMPLES herein.

If desirable, various labels can be used as means for detecting binding of CBP-I to a chemokine. Chemokines or the CBP-I can be directly or indirectly detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. Those of ordinary skill in the art will know of other suitable labels or will be able to ascertain such, using routine experimentation.

In another 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 as having a molecular weight of approximately 30-40 kD, depending on the extent of glycosylation, as determined by reduced SDS- PAGE, having amino acid sequence homology with myxoma T7 interferon-y receptor homolog, and having the biological function of myxoma T7 interferony receptor homolog. The term "anti-inflammatory" refers to reduction or suppression of an inflammatory response.

10 The glycosylated and secreted form of the exemplary CBP-I of the invention has an apparent molecular weight of approximately 38 kD as determined under reducing conditions on an SDS-PAGE. In addition, the protein has homology with the myxoma T7 IFN-y receptor homolog. The term "homology" refers to the extent of identity between the CBP-I and the viral IFN-y receptor at the amino acid level. Preferably, the CBP-I has between 50-95% amino acid sequence homology with the myxoma T7 IFN-y receptor. The homology requirement is not stringent, however, the CBP-I must retain the biological function of the myxoma T7 IFN-y receptor. In other words, the homology is sufficient as long as the CBP-I binds and inhibits chemokines.

20 The invention includes a functional polypeptide, CBP-I, and functional fragments thereof. As used herein, the term "functional polypeptide" refers to a polypeptide which possesses a biological function or activity which is identified through a defined functional assay and which is associated with a particular biologic, morphologic, or phenotypic response. Functional fragments of the CBP-I polypeptide, include fragments of CBP-I as long as the activity of CBP-I remains binding to chemokines). Smaller peptides containing the biological activity of CBP-I are included in the invention. Such peptides can -1 1- -jIbe assayed for binding to chemokines by methods commonly known to those of skill in the art, including methods described in the EXAMPLES herein. The biological function can vary from a polypeptide fragment as small as an epitope to which an antibody molecule can bind to a large polypeptide which is capable of participating in the characteristic induction or programming of phenotypic changes within a cell. A "functional polynucleotide" denotes a polynucleotide which encodes a functional polypeptide as described herein.

Minor modifications of the CBP-I primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the CBP-I polypeptide described herein. Such modifications may be deliberate, as by sitedirected mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the activity of CBP-I is retained. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its activity. This can lead to the development of a smaller active molecule which would have broader utility. For example, it is possible to remove amino or carboxy terminal amino acids which may not be required for CBP-I activity.

The CBP-I polypeptide of the invention also includes conservative variations 20 of the polypeptide sequence. The term "conservative variation" as used herein S" denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one 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 for aspartic acids, or glutamine for 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 provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

Examples of viral sources of the CBP-I used in the method of the present invention include myxoma virus, cowpox, shope fibroma virus, ectromelia, rabbitpox and other mammalian pox viruses, as long as the CBP-I has the biological function of an anti-inflammatory protein characterized as having a molecular weight of approximately 30-40 kD, depending on extent of glycosylation having homology with myxoma T7 interferon-y receptor homolog, and having the biological function of myxoma T7 interferon-y 10 receptor homolog.

*0 An immunopathological disorder treated by the method of the invention may be associated with production of chemokines and resultant accumulation of reactive leukocytes at afflicted tissues. The method comprises administering to the subject a therapeutically effective amount of CBP-I. The term 15 "immunopathological disorder" refers to any disease which involves the immune response or immunity in general. "Therapeutically effective" as used herein, refers to that amount of CBP-I that is of sufficient quantity to ameliorate the cause of the immunopathological disorder. "Ameliorate" refers to a lessening of the detrimental effect of the disorder in the patient receiving the 20 therapy. The subject of the invention is preferably a human, however, it can be envisioned 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 which can be treated by the method of the invention include acquired immunodeficiency disorder (AIDS), toxic shock syndrome, allograft rejection, artherosclerotic plaque growth, ultraviolet and radiation responses, and disorders associated with the activation ofT cells, B cells, macrophages, and -13other inflammatory leukocytes during the immune response and the acute phase response and disorders associated with advanced cancer such as tumor necrosis factor-mediated cachexia.

The invention provides a method of treating or ameliorating an immunopathological disorder including endotoxemia or septic shock (sepsis), or one or more of the symptoms of sepsis comprising administering to a subject displaying symptoms of sepsis or at risk for developing sepsis, a therapeutically effective amount of CBP-I. The term "ameliorate" refers to a decrease or lessening of the symptoms of the disorder being treated.

10 A patient who exhibits the symptoms of an immunopathological disorder may be treated with an antibiotic or antiviral agent in addition to the treatment with CBP-I. Typical antibiotics include an aminoglycoside, such as gentamycin or a beta-lactam such as penicillin, or cephalosporin. Therefore, a therapeutic method of the invention includes administering a therapeutically effective 15 amount of CBP-I substantially simultaneously with administration of a bactericidal amount of an antibiotic or sufficient amount of an anti-viral compound.

The term "bactericidal amount" as used herein refers to an amount sufficient to achieve a bacteria-killing blood concentration in the patient receiving the treatment. The bactericidal amount of 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 being treated. Preferably, administration of CBP-I occurs within about 48 hours and preferably within about 2-8 hours, and most preferably, substantially concurrently with administration of the antibiotic.

-14- Administration of a CBP-I in the method of the invention may also be used for ameliorating post-reperfusion injury. When treating arterial thrombosis, induction of reperfusion by clot lysing agents such as tissue plasminogen activator (t-PA) is often associated with tissue damage. Such tissue damage is thought to be mediated at least in part by leukocytes including but not limited to polymorphonuclear leukocytes (PMN). Therefore administration of the CBP-I would block leukocyte or PMN-endothelial interactions, and thereby diminish or prevent post-reperfusion injury. Administration of CBP-I is also useful for prevention of new onset and recurrent atherosclerotic plaque growth after arterial injury. Restenosis and new growth of plaque is believed to be exacerbated by the local inflammatory response to the internal layer of the Sartery wall.

The method of the invention is also useful for treatment of inflammation due to allergic or autoimmune disorders. Examples of allergic disorders include 15 allergic rhinitis, asthma, 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, type 1 insulin-dependent diabetes mellitus, inflammatory bowel disease, dermatitis, meningitis, thrombotic thrombocytopenic purpura, Sjogren's syndrome, encephalitis, uveitis, leukocyte adhesion 20 deficiency, rheumatoid and other forms of immune arthritis, rheumatic fever, Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis, primary biliary cirrhosis, pemphigus, pemphigoid, necrotizing vasculitis, myasthenia gravis, multiple sclerosis, lupus erythematosus, polymyositis, sarcoidosis, granulomatosis, vasculitis, pernicious anemia, CNS inflammatory disorder, antigen-antibody complex mediated diseases, autoimmune hemolytic anemia, Hashimoto's thyroiditis, Graves disease, habitual spontaneous abortions, Reynard's syndrome, glomerulonephritis, dermatomyositis, chronic active hepatitis, celiac disease, autoimmune complications of AIDS, atrophic gastritis, ankylosing spondylitis and Addison's disease.

The method is also useful in treating non-malignant or immunological-related cell-proliferative diseases such as psoriasis, pemphigus vulgaris, Behcet's syndrome, acute respiratory distress syndrome (ARDS), ischemic heart disease, atherosclerosis, post-dialysis syndrome, leukemia, acquired immune deficiency syndrome, septic shock and other types of acute inflammation, and lipid histiocytosis. Essentially, any disorder which is etiologically linked to the proinflammatory process and cellular infiltration due to chemokine production 10 induction of IL-8, MIP-lc or 3 expression) would be considered susceptible 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 15 inflammatory reaction resulting in production of interleukins for example. Thus, the CBP-I used in the method of the invention may be administered to a patient to prevent inflammation associated with such infections.

The dosage ranges for the administration of the CBP-I 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 unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary from about 10 pg to 100 ug per dosage, in one or more dose administrations daily, for one or several days.

The CBP-I is administered by any suitable means, including parenteral, subcutaneous, intrapulmonary, intraareterial, intrarectal, intramuscular, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, or intraperitoneal administration. CBP-I may also be administered transdermally in the form of a slow-release subcutaneous implant for example, or orally in the form of capsules, powders or granules. CBP-I can also be administered by inhalation. For example, when used therapeutically for 10 treatment of an inflammatory disorder of the lungs, a preferred route of administration would be by 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, 15 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, or fixed oils. The active 20 therapeutic ingredient is often mixed with excipients which 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 replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

The invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the CBP-I of the invention, the medicament being used for therapy of an undesirable immune response/inflamunatory reaction wherein the immune response results in production of chemokines which bind to the CBP-I of the present invention.

The invention provides a pharmaceutical composition comprising at least one dose of an immunotherapeutically effective amount of an anti-inflammatory protein having a molecular weight of approximately 30-40 kD, depending on the extent of glycosylation, having amino acid sequence homology with the 10 myxoma T7 interferon-y receptor homolog, and having the biological function of the myxoma T7 interferon-y receptor homolog, in a pharmacological carrier.

The invention provides any pharmaceutical preparations and compositions containing the CBP-1 of the invention for use in the method of the invention.

S The form will vary depending upon the route of administration. For example, 15 compositions for injection can be provided in the form of an ampule, each containing a unit dose amount, or in the form of a container containing multiple doses.

CBP-I can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. These include the acid addition salts which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric and the like.

Salts also include those formed from 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.

-18- Controlled delivery may be achieved by selecting appropriate macromolccules, for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers. The rate of release of the CBP-I may be controlled by altering the concentration of the macromolecule.

Another method for controlling the duration of action comprises incorporating the CBP-I into particles of a polymeric substance such as polyesters, polyamino acids, hydrogels, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers. Alternatively, it is possible to entrap CBP-I in microcapsules 10 prepared, for example, by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatinmicrocapsules or poly(methylmethacrolate) microcapsules, respectively, or in .a colloid drug delivery system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based 15 systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

9* The following examples are intended to illustrate but not limit the invention.

o While they are typical of those that might be used, other procedures known to **those skilled in the art may alternatively be used.

0 EXAMPLE 1 MATERIALS AND METHODS Rat model of injury induced atherosclerosis: Nine Sprague Dawley rats had balloon angioplasty mediated injury of the left iliofemoral artery. A 1.5mm USCI angioplasty balloon was advanced retrograde into the artery via cut down and arteriotomy under general pentobarbitol anesthetic (6.5mg per 100g weight by i.m. injection, Soinnotrol, MTC Pharmaceuticals, Cambridge, Ontario). 5 00pg of CBP-I (6 rats) or saline rats) was given by intra-artenal injection of the CBP-I or control solution into the distal lumen of the angioplasty balloon catheter upstream from the site of subsequent balloon mediated damage. The balloon was then inflated to 8 bars pressure for 1.0 minutes. After angioplasty the balloon was deflated and with drawn and the arteriotomy site closed with local application of n-butyl cyanoacrylate monomer (Nexaband, Veterinary Products Laboratories, Phoenix, Arizona). Each rat was maintained on a normal rat diet and was followed up for 4 weeks post surgery. At follow up the rats were sacrificed with euthanyl per kg and the aorta was harvested for histological examination.

Rabbit model of injury induced atherosclerosis: Seven cholesterol fed New Zealand white rabbits had balloon angioplasty of the distal abdominal aorta. All rabbits (strain New Zealand white) were fed 2% 15 cholesterol in 10% peanut oil diet for 4 days/week, beginning 2 weeks before balloon injury. A 3-3.5mm angioplasty balloon catheter (2 1:1 ratio of balloon to aorta diameter) was introduced via femoral arterial cut down following anesthetic (40mg/kg ketalean, 8mg/kg xylazene, and 0.5mg/kg acepromazine by intramuscular injection). The balloon was inflated to 8 bars pressure in the 20 distal abdominal aorta and advanced retrograde to the distal thoracic aorta. The balloon was advanced and withdrawn 3 times under flouroscopic control in each rabbit to ensure endothelial denudation. Contrast angiograms were recorded prior to and after balloon angioplasty mediated trauma and at 4 weeks follow-up. Heparin (400 units) was given immediately after obtaining femoral access to decrease catheter associated thrombosis.

Purified CBP-I (T7) protein, 500pg per sample, was infused immediately after balloon mediated injury in the distal abdominal aorta of 4 rabbits. A parallel infusion of saline was infused locally into the distal abdominal aorta in 3 rabbits. Each infusate was administered via Wolinsky catheter in a total volume diluted in sterile 0.9% saline immediately following balloon mediated injury. All infusions were via a 3.25mm Wolinsky balloon (inflated to a final pressure of 6±1 bars for 2 minuets) in the abdominal aorta proximal to the iliac bifurcation. The Wolinsky balloon was positioned immediately above the iliac bifurcation under flouroscopic control such that the perfusion balloon was routinely located from 0.5 2.5 cm above the bifurcation and designated as the primary infusion site. Upstream secondary sites were defined in the region above 2.5cm proximal to the iliac bifurcation. In all experiments, infusates were administered via Wolinsky catheter in a total volume of 10 ml diluted in sterile 0.9% saline immediately following balloon mediated injury. All infusions were via a 3.25mm Wolinsky balloon (inflated to a final pressure of 61 bars for 2 minutes) in the abdominal aorta proximal to the iliac bifurcation.

15 CBP-I Protein isolation and purification: Myxoma T-7 protein (CBP-I) was isolated and purified as described in EXAMPLE 4 herein.

Histology and morphometric analysis: Histological analysis was performed at the primary site of Wolinsky infusion 20 in the distal abdominal aorta (rabbits) or upper iliofemoral arterial branches (rat) representing the primary infusion site as defined by the original positioning of the perfusion balloon. In rabbits, internal control sections were taken from a downstream, non-infused site near the iliac bifurcation (0.5cm above the bifurcation to 0.5cm below the bifurcation). and in upstream, non-infused site (the upper abdominal aorta, 2.5cm-3.5cm above the iliac bifurcation). The area from 1.5-2.5cm above the iliac bifurcation was considered a border zone with potentially variable infusion doses due to balloon placement and was therefore -21not included in this analysis. In rats the primary balloon sites for both T-7 treated and saline infused rats were used for histological assessment.

Hematoxylin and eosin staining of formalin fixed specimens was performed as previously described. Briefly, each specimen was fixed in 10% sodium phosphate buffered formalin, processed, impregnated, embedded in paraffin and cut into Sm sections by microtome as has been previously described. Sections from each specimen (a minimum of 2 sections per site) were then stained with hematoxylin and eosin and examined by light microscopy.

Schwartzman reaction: New Zealand White female rabbits weighing 3kg are injected with lipopolysaccharide (LPS) of the E. coli serotype 01111:B4 (Sigma) and CBP-I (T7) protein which had been purified to homogeneity using column chromatography. Eight intradermal injections (0.1 ml each) of 50-100g LPS in the presence and absence of 0.1-1.0Og CBP-I was applied to the back of the rabbit; there are 4 injection sites on each side, separated by about 2.5 cm. 24h later 100lg of LPS is administered to the rabbit intravenously in the marginal ear vein. About 4-6h after the intravenous injection necrotic inflammation developed at the sites of intradermal injection. As soon as the inflammation was significant, the rabbit was sacrificed by a lethal injection of euthanol. The size and redness of the lesions are assessed, and tissue samples were collected.

EXAMPLE 2 BINDING OF CYTOKINES TO A NOVEL VIRAL PROTEIN Briefly, a variety of human cytokines were radiolabelled with 2'I, exposed to the secreted proteins harvested from control or poxvirus-infected BGMK cells, cross-linked, and then analyzed by SDS-PAGE for novel cytokine/protein complexes.

Surprisingly, cross-linking assay uncovered what was clearly a novel viral-specific protein that bound to each of the three human chemokincs that was tested: 11-8, RANTES and MIP-1P (FIGURE FIGURE IA shows gel mobility shift assays using iodinated ligands and tissue culture supernatants.

Tissue culture supematants (Sups) were prepared as follows: BGMK (Baby Green Monkey Kidney Cells) were infected with Myxoma (MYX), vaccinia virus or Shope fibroma virus (SFV) at a multiplicity of infection (MOI) of 3. To collect early Sups the infected monolayer was washed three times with phosphate buffered saline (PBS) and replenished with serum free medium which was then collected 4h post infection Late Sups were prepared by washing the monolayer three times with PBS and replenishing it with serum free medium at 4h post infection; these Sups were then collected at 18h post infection Mock Sups were prepared in the same way in the absence of virus. Sups were concentrated approximately 15 fold using Amicon concentrators. The human chemokines IL-8, RANTES, and MIP-1 were labeled with using iodobeads (Pierce) according to the manufacturer's protocols.

The gel mobility shift assays were performed as follows: 5pl of iodinated ligand was mixed with 10 il SUP and allowed to sit at room temperature for 2 20 hours. then 2 pl of the chemical cross linking reagent 1-ethyl-3-(3i dimethylaminopropyl)-carbodiimide (EDC) (200mM in 100mM potassium phosphate, pH 7.5) was added for 15 minutes, followed by an additional 2 pl for 15 minutes. The reaction was then quenched by the addition of 2 pl of Tris- HC1 (l.OM, pH The resulting mixture was analyzed using SDS-PAGE and autoradiography. The arrows indicate the shifted complexes.

FIGURE 1B shows an experiment as described in FIGURE 1A, however, iodinated MIP-13 was reacted with mock, MYX or ectromelia (ECT) Sups.

The data show that cells infected with Shope fibroma virus (SFV) or myxoma (MYX) secreted a novel protein that created a cross-linked species (marked with arrow) of approximately 50kD, indicating that the unknown species which bound to the 12kD ligands was about 38kD. Importantly, the novel complex was not detected in supernatants from control cells that were mock infected or cells that had been infected with vaccinia virus a poxvirus from a different genus (orthopoxvirus) from that of SFV or myxoma (leporipoxvirus).

FIGURE 2 shows gel mobility shift assays using RANTES with competitors. lodinated RANTES was mixed with 0, 1, 10, and 100 fold molar 10 excess of unlabelled human RANTES, MIP-lp, MIP-I a, IL-8, or MCAF. This mixture was then reacted with mock or myxoma early Sups as described in Figure 1A and analyzed using SDS-PAGE and autoradiography. The figure shows self competition with RANTES as well as cross competition with MIP- .13. MIP-la, IL-8, and MCAF. The arrows indicate the shifted complexes.

o In FIGURE 3A, iodinated MIP-1p was mixed with 0, 1, 10, and 100 fold molar -excess of unlabelled human MIP-10, IFNy, MCAF, MIP-la, RANTES or IL-8.

This mixture was then reacted with mock or myxoma early Sups as described in Figure IA and analyzed using SDS-PAGE and autoradiography. The figure shows self competition with MIP- 10 as well as cross competition with IFNy, MCAF, MIP-la, RANTES and IL-8. The arrows indicate the shifted complexes.

In FIGURE 3B, iodinated MIP-10 was mixed with Sups from mock, MYX, Cow Pox Virus (CPV), rabbit Pox Virus (RPV) ECT, SFV, or VV using SDS- PAGE and autoradiography. The arrows indicate the shifted complexes and the free ligand.

In FIGURE 4, iodinated IL-8 was mixed with 0, 1, 10, and 100 fold molar excess of unlabelled human MIP-1P, MIP-la, MCAF, or IFNy. This mixture was then reacted with mock or myxoma early Sups as described in Figure IA and analyzed using SDS-PAGE and autoradiography. The figure shows self competition with IL-8 as well as cross competition with MIP-la, MCAF, and IFNy. The arrows indicate the shifted complexes.

As shown in FIGURE 2, using '"I-labelled RANTES as a ligand, binding was able to be competed with a variety of cold chemokine competitors, including RANTES, MIP-lp, MIP-la and MCP-1, but not IL-8. Similarly, using 10 ':'I-MIP-lp as the labelled ligand, competition was observed with unlabelled MIP-1p, MCAF, MIP-la and RANTES, but not IL-8 or human IFN-y (FIGURE However, when labelled IL-8 was used as the ligand, competition was observed with cold 11-8 as well as MIP-la, MIP-lp, MCAF but not IFN-y (FIGURE 4).

15 It is interesting that the C-X-C and C-C chemokines have this curious pattern of cross-competitions, but it is possible that it relates to the various affinity constants of these different human chemokines with the viral protein. The g apparent wide spectrum of chemokine binding to a viral 38kDa secreted protein species suggested that this protein might be a generalized inhibitor of many 20 human chemokines, and thus prevent chemotaxis of a wide variety of leukocytes.

In FIGURE 5, T7 protein (CBP-I) was purified (see Example mixed with the indicated cytokines, cross-linked and anlyzed by SDS-PAGE, followed by Western blotting with anti-T7 antibody. Of the cytokines tested, only human IL- 8 and rabbit IFN-y formed high molecular weight complexes with T7, indicating the specificity of the binding.

In FIGURE 6, a spectrum of representative chemokines from all three major classes (CXC, CC and C) were tested for binding to T7 protein (CBP-I), as described in FIGURE 5. All chemokines tested bound T7 protein with comparable avidity.

EXAMPLE 3 PURIFICATION OF CBP-I To purify CBP-1, secreted proteins from myxoma infected cells were concentrated, fractionated by MonoQ chromatography and then size filtration chromatography. Figure 7 shows the purification of CBP-1 to homogeneity 10 from supematants of Myxoma virus infected cells. Briefly, supernatants from overnight myxoma virus infected Baby Green Monkey Kidney (BGMK) cells were harvested, centrifuged at 10,000 RPM for 1 hour, and concentrated fold using a stirred Ultrafiltration Cell (Amicon). Virus-free concentrated myxoma supernatants (Sups) were dialysed in 20mM bis-Tris pH 6.0 (Sigma) and stored at 4 0 C prior to purification (Lane CBP-I was purified to homogeneity from myxoma supernatants by a 2-step purification procedure S. using Fast Protein Liquid Chromatography (FPLC). Briefly, 5 mls of myxoma superatants were loaded onto a MonoQ HR 5/5 (Pharmacia) anion exchange column pre-equilibriated with low ionic strength start buffer (20 mM bis-Tris 20 pH Proteins were eluted off the column by increasing the eluting buffer (1M NaCI 20mM bis-Tris pH 6.0) to 500mM NaCI in a step-gradient. Protein fractions were collected, resolved by SDS-PAGE, and analyzed by silver staining. Analysis of proteins that were eluted between 150-200mM NaCI (fractions 21-27) revealed a prominent band of approximately 37,000 MW (CBP-I) and an unknown contaminating protein equivalent to the size of bovine serum albumin (Lane Pooled MonoQ Fractions #21-27 were subsequently loaded onto a HiLoad 16/60 Superdex 75 gel filtration column (Pharmacia) and eluted at a flow rate of 0.5 m/min using 20mM bis-Tris pH 6.0. Fractions from the equivalent of 2 column bed volumes were collected, resolved by SDS- PAGE and analyzed by silver staining. Fractions #26-31 collected from the Superdex 75 flow-through volume, revealed a single protein species of approximately 37 kD corresponding to purified CBP-I (Lane 3).

The final CBP-I product (FIGURE 7) was a 38 kDa glycosylated protein that co-purified with a smaller component (35 kDa) which appeared to be an under-glycosylated variant of CBP-I.

1 pg of purified or partially purified CBP-I was incubated with or without 1 I 10 recombinant human RANTES for 2 hours at room temperature After incubation, the proteins were cross-linked by the addition of EDC (Sigma) to 40mM (final concentration) at room temperature for 30 minutes and quenched by the addition of one-tenth volume IM Tris (pH7.5). SDS-loading buffer was added to the mixtures, the samples were boiled for 3 minutes, subjected to SDS- 15 PAGE,.and detected by silver straining. Silver stain analysis revealed a crosslinked complex (CBP-I RANTES) of approximately 47kD when either purified or partially purified CBP-I was incubated with RANTES (Lanes 2 and however no gel mobility shifted complex was observed in the absence of Rantes (Lanes 1 and 3).

When the partially purified MonoQ alone) or fully purified CBP-I were tested in standard cross linking assays with human RANTES, the appropriate shifted 1:1 complex of CBP-I/chemokines was detected, as predicted if the binding activity was a property conferred by CBP-I alone (FIGURE 8 and 9).

Figure 9 shows binding of human Rantes Chemokine to partially-purified CBP- I. 1ug of partially purified CBP-I was incubated without (Lane 1) or with increasing amounts (Lane 2-6) of recombinant human Rantes for 2 hours at room temperature. After incubation, the proteins were cross-linked by the addition of EDC (Sigma) to 40mM (final concentration) at RT for 30 minutes and quenched by the addition of one-tenth volume 1M tris (ph SDSloading buffer was added to the mixtures, the samples were boiled for 3 minutes, subjected to SDS-PAGE, and detected by silver staining. Silver stain analysis revealed a cross-linked binding complex (CBP-I+ Rantes) of approximately 47kD when CBP-I is incubated with Rantes (Lanes and this binding can be titrated out by decreasing amounts of the chemokine ligand. A single contaminating band of approximately 66kD that appears is an unknown protein that co-fractionates with CBP-I during MonoQ chromatography, but ~which is removed by Superdex 75 chromatography.

EXAMPLE 4 ANALYSIS OF THE EFFICACY OF CBP-I (T-7) AS AN ANTI-RESTENOSIS PROTEIN AS SHOWN 15 IN ANGIOPLASTY BALLOON MEDIATED INJURY IN RAT FEMORAL ARTERIES Inflammation has been associated with accelerated atherosclerotic plaque development in the arterial wall. There is a high rate of plaque recurrence, restenosis, after the use of balloon angioplasty and other related angioplasty devices designed to open occluded arteries. Accelerated atherosclerotic plaque growth also has been reported under conditions leading to arterial injury, viral infections, vasculitis, homocystinuria, diabetes melitis, hypertension, hyperlipideuria, smoking and immune complex generated disorders. The larger DNA viruses have evolved mechanisms, anti-inflammatory proteins, that allow the virus to proliferate in the host with decreased inhibition by the host immune and inflammatory defense mechanisms. These examples demonstrate the use of viral proteins as potential anti-inflammatory agents for the treatment of or prevention of immune based disorders. CBP-I was tested as a potential therapeutic agent for the prevention of plaque growth after angioplasty. CBP-I has been reported to act as an interferon gamma receptor homologue and as a chemokine inhibitor. CBP-I was tested in 2 animal models of injury induced atherosclerosis (rat and rabbit) and the results show a significant decrease in plaque formation 4 weeks after infusion.

There was a significant decrease in plaque growth after CBP-I infusion on comparison with saline infusion. (FIGURE 10A-C) In the rat model there was a mean plaque area of 0.005±0.002mm 2 (FIGURE 10B, C) and a mean plaque thickness of 8.33±4.01m (FIGURE 10A) at 4 weeks follow up after CBP-I 10 infusion (p(0.0003). With saline infusion the plaque area was 0.036±0.006mnm and the plaque thickness was 62±7.351m at 4 weeks follow up (p(0.0001). this represents a 7 fold decrease in plaque area and in plaque thickness with CBP-I infusion. The decrease in plaque development was seen at 4 weeks follow up after only a single infusion of CBP-I immediately prior to balloon mediated 15 injury. The visible lesions consisted predominately of smooth muscle cellular proliferative changes characteristic of the rat arterial injury model (FIGURE In the rabbit model there was also a significant decrease in plaque area and thickness on comparison with the saline treated controls. At 4 weeks follow up 20 there was a mean plaque thickness of 30±21.6pm after CBP-I infusion and 600±200pm after saline infusion In this case the plaque observed was the fibrous and fatty foam cell plaque commonly seen in the cholesterol fed rabbit models (FIGURE 11).

Examination of the use of a viral anti-inflammatory protein in 2 models of injury induced atherosclerosis (rabbit and rat). In both models a significant decrease in subsequent plaque formation was detectable on histological -29analysis. In each case only a single infusion of the protein was given immediately following balloon injury.

EXAMPLE SCHWARTZMAN

REACTION

One of the classic examples of necrotizing inflammation is the Schwartzman reaction, in which lipopolysaccharide (LPS) is introduced first into rabbit skin and then, 24 hours later, followed by a second intravenous dose of the same LPS. Within hours after the second LPS injection, infiltrating macrophages induce a reproducible necrotizing response at the site of the primary injection 10 which is highly reproducible and readily quantified. The ability of CBP-I to nhibit macrophage influx and activation at the primary injection site was examined.

LPS was injected intradermally on the back of a rabbit in the presence or absence of purified T7 protein and 24h later an intravenous injection of LPS was administered. Inflammation quickly appears at the sites of intradermal injection, and the animals were euthanized and data was collected.

TABLE 1 Schwartzman Reaction in Rabbits LPS Ii&) lesion LES plus CBP-1 (17) (JIM) lesion (intradermal) (intradermal) I 0(LPS)+1I(T7) 50(LPS)+ I (T7) 100 -H--100(LPS)+0.5(T7)

+H-

100 I0(LPS)+0.i(n7)

H

t0 =No reaction to l i i indicates the degree of inflammnation from minimal to extreme to to The lesions were graded as follows: raised I1-10mmru in diameter, red, raised 1-2rrm 10-1I5mm in diameter, :intensley red, 2-3mm raised more than 15mmn in diameter, intensley red So 15 with a dark haemoragic center, 2-3mm raised The LPS (100 jig) lesions were haemorrhagic and swollen, whereas sin injected with LPS (100gjg) plus CBP-I (T7) Protein (ijig) was slightly red and raised.

When a dose of 50 jig of LPS was used I ±g of CBP-I completely inhibited all visible signs of the Schwartzman reaction. CBP-1 alone injected intradernially, followed by the intravenous LPS injection, induced no inflammation. Bovine serum albumin (1.0 jg) injected with 100gjg UPS, followed by the intravenous LPS injection, was not able to inhibit inflammation. These experiments demonstrate that purified CBP-I protein was able to protect the rabbit from the localized Schwartzman reaction.

As T7 has been shown to bind both lENy and chemokines such as 1L8 and RANTES, the involvement of these cytokines in the Schwartzman reaction is of Uiterest. The Schwartzman reaction is complex involving the cytokines IL- 12, IFNy, TNFa(Ozmen ct J rExp. Med,180:907.915, 1994.) and lL8 (Harada, et a. Imi. Immunol., 5:681-690, 1993). For example, it has been shown that neutralizing antibodies to either 1FNy(Billiau et al-, Euro. J Immunol., 17:1851-1854, 1987; Heremans, J hmnunol__ 138:4175-4 179, 1987) or 11-8 (Harada et supra.) block or inhibit the Schwartzman reaction. Thus, the inhibitory effect of CBP-I on the Schwartzmnan reaction in rabbits could be due to it ability to bind IFNy, or chemokines, or both. As CBP-I shows species specificity for IFNy but not chernokine binding, these experiments will be repeated in rats in the attempt to distinguish between the IFNy and chcmokine binding activity of T7 in this model of inflammnation.

SUMMARY:

The cloned and sequenced myxoma CBP-l gene, which is not a secreted homologue of the known chemokine receptors, which all possess seven membrane-spanning domains (and are called "serpentines") and are described in numerous recent reviews (Kelvin, et J. Leukocyte Biol., 54:604-6 12, 1993;, Murphy, Ann. Rev. 1mm., 12:593-633, 1994; Horuk., 1mm.

Today., 169-174, 1994; and Horuk, Trends inPharm. Sc., 15:159-165, 1994). Although some DNA viruses do encode homologues of such serpentine receptors (Ahuja, et., 1mm. Today 15:28 1-287, 1994), including at least one gene candidate in a poxvirus (Massung, el Virology, 197:511-528, -32- 1994), the CBP-1 of the present invention is not a member of this particular receptor family. Thus, CBP-I represents a new class of anti-inflammatory protein that acts by presumably modulating a spectrum ofchemokines in treated tissues.

Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

*e SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: THE GOVERNORS OF THE UNIVERSITY OF ALBERTA (ii) TITLE OF INVENTION: CHEMOKINE BINDING PROTEIN AND METHODS OF USE THEREFOR (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Fish Richardson (13) STREET: 4225 Executive Square, Suite 1400 (Cl CITY: La Jolla STATE: California COUNTRY: USA ZIP: 9203' 9 15 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible 9*99 OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release l1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 19-APR-1996

CLASSIFICATION:

.(viii) ATTORNEY/AGENT INFORMATION: NAME: Haile, Ph.D., Lisa A., REGISTRATION NUMBER: 38,347 REFERENCE/DOCKET NUMBER: 07254/034W01 (ix) TELECCOUNICATION INFORMATION: TELEPHONE: (619) 678-5070 TELEFAX: (619) 678-5099 -34- INFORMATION FOR SEQ ID) NO:l: (W SEQUENCE CHARACTERISTICS: LENGTH: 1677 base pairs TYPE: nucleic acid STRANDEtJNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (genormic) (vii) IMMEDIATE SOURCE: CLONE: CBP-1 (ix) FE.ATURE: NAME/KEY: CDs LOCATION: 455.. 1243 SEQUENCE DESCRIPTION., SEQ ID NO:1: GGATCCATCG AGACGGCGTC CCGGACGTCA CGGACTTCGT TCAGAAACTA TCCGGAGGTA CATGGACGAA GGTGAACGAh CTGTCCGTCC CCAAGGCGAG CGTTACG4GCG ATCGTCTATA 120 AAGAGAGGTT GTACTGCGTAL GGGGGGCTGG TGGATCGAIA CGCTCCAACG AACGAAGTTA 160 ***TCCGTTACAG G.GACGACACG AACGAGTGGG A ATACGTGGG ATCTACGAAG ATCGAACGAG 240 CCGGTTCCGT GGGGTGTGTG TACAACGACG AGCTCTACGT CTTCGGAGGA ACGGATACGT 300 .TTACGTCCGA GCGATACA\AC GGAGTCATTT GGAAACGAGC GAACGACGTC TCCTGTCACT 360 TCGCCACCAT GAACGCGGCG TACGCCACCT ACCTCGAGCT GTAGAAACGT TTTTATAACT 420 GAAAAAGTAT CCTAAAATA GAGTAATACT cAAG ATG GAC GGG AGA CTG GTG 472 Met Asp Gly Arg Leu Val 1 TTT CTC CTC GCG TCG CTC GCT ATC GTC TCC GAC GCC GTA CGC CTT ACG 520 Phe Leu Leu Ala Ser Leu Ala Ile Val Ser Asp Ala Val. Arg Leu Thr is TCC TAC GAC TTA AAC ACA TTC GTT ACG TGG CM GAC G.AT GGA TAC ACC 568 Ser Tyr Asp Leu Asn Thr Phe Val Thr Trp Gin Asp Asp Gly Tyr Thr 30 TAC AAC GTC ACT ATT AAA CCG TAT ACC ACG GCT Tyr Asn Val Se- Ile Lys Pro Tyx Thr Thr Ala ACG TGG ATC AAT GTG Thr Trp Ile Asfl Val TGT GAA TGG C TCT TCT AGC Cys Glu Trp Ala Set Sex Set TGC AAC GTA TCT CTC GCC Cys Axn Vai Ser Leu Ala CTA CAA TAC Leu Gin Tyr GAT TTG GAC GTC GIG TCT TCC CCC AGA CTG ACC CCC CIT GGT Asp Leu Asp Val Val Ser Trp Ala Arg Leu Thr Axg Val Cly ACA C.AA TAC AG? CTC CAA CCG ACG; TCT GCC CIG GCT CGC Thr Ciu Tyr Set Leu Glu Pro Thr Cys Ala Val Ala Axg TTC TCT CCA Phe Set Pro 100

S

S

*5 S 9*

S.

S

S S

S

S

S

CCG GAG GTA Pro Ciu Val 105 CAA CTC GTA Gin Leu Val AGA ACA Arg Thr 110 GGT ACC AGC GTA GAA CrC TTA CIT Gly Thr Spar Val Giu Val Leu Val 115 AGA CAC Arg His 120 CCC GTC GTC TAT CTA CCC CCC CAG CAA GIG TCC GTC TAC GGA Pro Val Val Tyr Leu Aeg Cly Gin Clu Val Set Val Tyr Gly 125 130

CAT

20 His 135 TCA TTC TGC GAC Sex Phe Cys Asp GAC TTC CCC TAT AMA ACG ATC TIC CTG TTC Asp Phe Gly Tyr Lys Thr Ile Phe Leu Phe TCG AAG AAT AN. CGA CC GAG TAC GTC GTA CCC GGC CGA TAT Set Lys Asn Lys Aeg Ala Glu Tyr Val Vai Pro Giy Arg Tyr 25 AAC GTA GAG Asn Val Glu TCT CGT Cys Argj 170 TIC TCC AIC GAT TCC CAN GAA Phe Set Ile Asp Sex Gin Glu 175 AGT CTA TGT C Ser Val Cys Ala 180 1000 ACG CC CIT Thr Ala Val 185 CTT ACG TAC GGT GAC ACT TAT CCI TCC GAXG C CCI CTC Leu Thr Tyr Gly Asp Ser Tyr Arg Sex Glu Ala Cly Val 190 195 GAG GTC Clu Val 200 TCC GTT CCC GAA Cys Val Pro Glu CC AAG, ACA Ala Lys Arg CMA GTC Clu Val 210 AGT CCC TAC ATC Sex Pro Tyr Ile 1096 GTG AAA AAG TCG TCC GAC CTG GAA TAC GTC AAA CGT GCC ATA CAC AAC Val Lys Lys Sex Sex Asp Leu Glu Tyr Val Lys Arg Ala Ile His Asn 215 220 -225 230 GAA TAC CGA CTC GAC ACC TCC TCC GAG GGA CGC AGA TTG GAG GAA CTG Glu Tyr Arg Leiu Asp Thr Ser Ser Glu Gly Arg Arg Leu Glu Glu Leu 235 240 245 TAT CTA ACG GTC GCC TCC ATG TTT GAA CGT CTC GTG GAA GAT GTC TTC Tyr Leu Thr Val Ala Ser Met Phe Glu Arg Leu Val Glu Asp Val Phe 250 255 260 GAA TAATCGAAAT ATAAATAATG TAGTTTTTGT ATCGGAATCA TGGAACGTAC Glu 1144 1192 1240 1293 I I *1 4I** 0@ I

I

I

III.

1* I I *1

*III

I

I

.1I I

I

CCTGGTAAGT

GTCGTTCACG

15 TAACGGGAGT

CACCGTGCTC

TCTACAATCC

TGCGGATTAC

AGAGACGTAT

GCTGAACTCC

AGCGTCCTAC

AAAAGG.ACCC

TTCTTGGACA G~CGGTACCAT GAGCGACATC ACCCTCGTCG CGGGGGAGAC GCGCATCGAC TGATTTTATC CGTCCATCG GATTACTTCT ATCGTCTGTT TTTGAGGTAC cGGATIACGAT CACGTTGGAT kC:GGACGATG GCGTCCTTCG CGCTACATGT ACACGGGATh rAGCAAC-ATA CGAGACCGTA CCGTALGAGGA ATTATCGTAT TGGCGGACTA CCTGGGTATA ACGAAACTGG TGAAAGAGTG ATGGTAAGTC GAGTGGACCC GACGAACTGC GTATCCGCT TCCAGTTTGC CACATAGAGG, ATTTAAAACG AAACCTCAAT ACGTTCTTAC CCGAACT^CTT CGAGGGGCGT TTACGAAATT GGATACGGAC GAAGCGGTCG TGGTTCTACG GAGATCGTCG ACAGACGGTT TGTGCTTAAG GCTATTCTAG ATTGGGTGCG AAACGCATCG AGCGGATAAA GACGCTGTCC GCGO 1353 1413 1473 1533 1593 1653 133 1773 1833 1877 INE'ORKATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 263 anuino acids TYPE: antino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Asp Gly Arg Leu Val Phe Leu Leu Ala Ser Thr Ser Tyr Asp Leu Asn 25 Asp Ala Val.

Gin Asp Asp Ala Thr Txp Ser Leu Ala Axg Leu Gly Tyr Ile Asn Leu Ala Ile Val Ser Thr Phe Val. Thr Trp Lys Pro Tyr Thr Thr Thr Tyr Asn Val Cys Glu Val Ser Ilie Trp Ala Ser Cys Asn Val 0 ost 0 Leu Gin Tyr Gly Gly Tyr Asp Leu Asp Val Val. Txp, Ala Pro Thr Arg Leu Cys Ala Arg Val Thr Glu Tyr Sex Leu Glu Val. Ala Axg Ser Val. Glu 115 Giu Val Sex Ser Pro Pro Giu Leu Val Axg Gin Val Cys Leu Val Arg Thr Gly Thr 110 Val. Tyr Leu Arg Gly Gin 125 Asp Tyr Asp Phe Gly Tyr Val Tyr Gly 130 Lys Thr Ile Phe Leu Gly Arg Tyr Gin Glu sex Val 180 Axg Ser Glu Ala 195 Giu Val. Sex Pro 210 Cys Asp 165 Cys Ala Gly Val His Sex Phe 135 Sex Lys Aan Asn Val Glu Thr Ala Val Glu Val Cys Lys Arg 155 Cy3 Arg 170 Leu Thr Glu Tyr Val Phe Sex Ile Tyr Gly Val Pro Glu Asp Ser Tyr 190 Ala Lys Axg Glu Tyr Val 200 Tyr Ile Val. Lys Lys Sex Ser Asp 215 220 His Asn Glu Tyr Axg Leu Asp Thr 230 235 Arg Ala Ile Sex Ser Giu Gly 240 38A Arg Arg Leu. Glu Glu Leu Tyr Leu Thr Val Ala Ser Met Phe Glu Arg 245 250 255 Leu Val Glu Asp Val Phe Glu 260 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS Length: 1877 base pairs Type: Nucleic acid Strandedness:single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE: CLONE: CBP-l (ix) FEATURE: NAME/KEY: CDS LOCATION: 455. .1243 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 GGATCCATCG AGACGGCGTC CCGGACGTCA CGG.ACTTCGT TCAGAAACTA. TCCGGAGGTA CATGGACGAA GGTGAACGAA CTGTCCGTCC CCAAGGCGAG CGTTACGGCG ATCGTCTATA AAGAGAGGTT GTACTGCGTA GGGGGGCTGG TGGATCGATA CGCTCCAACG AACGAAGTTA TCCGTTACAG GGACGACACG AACGAGTGGG AATACGTGGG ATCTACGAAG ATCGAACGAG GCGGTTCCGT GGGGTGTGTG TACAACGACG AGCTCTACGT CTTCGGAGGA ACGGATACGT TTACGTCCGA GCGATACAAC GGAGTCATTT GGAAACGAGC GAACGACGTC TCCTGTCACT TCGCCACCAT GAACGCGGCG TACGCCACCT ACCTCGAGCT GTAGAAACGT TTTTATAACT GAAAAAGTAT CCTAAAAATA. GAGTAATACT CAAG ATG GAC GGG AGA CTG GTG Met Asp Gly Arg Leu Val 1 38B TTT CTC CTC GCG TCG CTC GCT Phe Leu Leu Ala Ser Leu Ala TTA AAC ACA TTC Leu Asn Thr Phe ATC GTC TCC GAC GCC GTA CGC CTT ACG Ile Val Ser Asp Ala Val Arg Leu Thr 1s TCC TAC GAC Ser Tyr Asp TAC AAC GTC Tyr Asn Val GTT ACG Vai Thr TGG CAA GAC GAT GGA TAC ACC Trp Gin Asp Asp Giy Tyr Thr AGT ATT AAA CCG TAT ACG ACG Ser Ile Lys Pro Tyr Thr Thr 45 GCT ACG Ala Thr TGG ATC AAT GTG Trp Ile Asn Val

TGT

Cys GAA TGG GCG TCT Giu Trp Ala Ser AGC TGC AAC GTA Ser Cys Asn Vai CTC GCC CTA CAA Leu Ala Leu Gin GAT TTG GAC GTC Asp Leu Asp Val TCT TGG GCC AGA Ser Trp Ala Arg ACC CGG GTT GGT Thr Arg Val Giy AAA TAC Lys Tyr ACA GAA TAC Thr Giu Tyr CCG GAG GTA Pro Giu Val 105 CTG GAA CCG ACG Leu Giu Pro Thr GCC GTG GCT CGG Ala Val Ala Arg TTC TCT CCA Phe Ser Pro 100 CAA CTC GTA AGA Gin Leu Val Arg GGT ACC AGC GTA GAA GTC TTA GTT Gly Thr Ser Val Glu Val Leu Vai 115 AGA CAC Arg His 120 CAT TCA His Ser 135 CCC. GTC GTG TAT Pro Val Val Tyr CGG GGG CAG GAA Arg Gly Gin Giu

GTG

Val 130 TCC GTC TAC GGA Ser Val Tyr Gly TTC TGC GAG Phe Cys Asp GAC TTC GGG TAT Asp Phe Giy Tyr ACG ATC TTC CTG Thr Ile Phe Leu TCG AAG AAT AAA Ser Lys Asn Lys

CGA

Arg 155 GCG GAG TAC GTC Ala Giu Tyr Vai CCC GGC CGA TAT Pro Gly Arg Tyr TGC GAC Cys Asp 165 AAC GTA GAG TGT CGT TTC TCC ATC Asn Val Giu Cys Arg Phe Ser Ilie TCC CAA GAA Ser Gin Glu TAT CGT TCC Tyr Arg Ser ACG GCG GTT Thr Ala Val 185 CTT ACG TAC Leu Thr Tyr GGT GAC AGT Gly Asp Ser 190 AGT GTA TGT GCT Ser Val Cys Ala 180 GAG GCG GGT GTG Giu Ala Gly Val 195 AGT CCC TAC ATC Ser Pro Tyr Ile 1000 1048 GAG GTC Giu Val 200 TGC GTT CCC GAA Cys Val Pro Glu CTC GCG AAG AGA GAA GTC Leu Ala Lys Arg Giu Val 205 210 1096 38C GTG AAA AAG TGG TCC GAG GTG GAA TAG GTC AAA CGT Val Lys Lys Ser Ser Asp Leu Glu Tyr Val Lys Arg 215 220 225 GAA TAG CGA CTC GAG AGG TGG TGG GAG GGA CGG AGA Glu Tyr Arg Leu Asp Thr Ser Ser Glu Gly Arg Arg 235 240 TAT GTA AGG GTG GCG TGG ATG TTT GAA GGT CTG GTG Tyr Leu Thr Val Ala Ser Met Phe Glu Arg Leu Val 250 255 GAA TAATGGAAAT ATAAATAATG TAGTTTTTGT ATCGGAATCA Glu GGC ATA GAG AAG Ala Ile His Asn 230 TTG GAG GAA GTG Leu Glu Glu Leu 245 GAA GAT GTG TTG Glu Asp Val Phe 260

TGGAAGGTAG

1144 1192 1240 1293 4 4*

GGTGGTAAGT

20 GTGGTTGAG

TAACGGGAGT

GAGGGTGGTC

TGTAGAATGG

TGCGGATTAC

AGAGAGGTAT

GGTGAAGTGG

AGCGTGGTAG

AAAAGGAGGG

TTGTTGGAGA

GCGATGGAG

TTTGAGGTAG

GGTACATGT

ATTATGGTAT

ATGGTAAGTC

CAGATAGAGG

GGAGGGGGGT

GAGATCGTCG

AAAGGCATG

GGGGTAGGAT

TGATTTTATC

GGGATAGGAT

AGAGGGGATA

TGGCGGAGTA

GAGTGGAGC

ATTTAAAAG

TTAGGAAATT

AGAGAGGGTT

AGCGGATAAA

GAGCGACATG

GGTGGATTCG

GACGTTGGAT

GAGGAACATA

GGTGGGTATA

GAGGAACTGGC

AAAGGTCAAT

GGATAGGGAG

TGTGCTTAAG

GAGGCTGTGG

ACGGTCGTG

GATTACTTGT

ACGGACGATG

GGAGACCGTA

ACGAAPACTGG

GTATCCGCTT

AGGTTCTTAG

GAAGCGGTCG

GGTATTCTAG

GCGG

GGGGGGAGAG

ATCGTGTGTT

GCGTCGTTG

GGGTAGAGGA

TGAAAGAGTG

TGCAGTTTGC

GGGAAGTGTT

TGGTTGTAG

ATTGGGTGCG

1353 1413 1473 1533 1593 1653 1713 1773 1833 1877 4

Claims (12)

1. 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 as: having a molecular weight of approximately 30-40 kD; having amino acid sequence homology with myxoma T7 interferon-y receptor homolog; and having the biological function of myxoma T7 interferon-y receptor homolog.

2. The method of claim 1, wherein the anti-inflammatory protein is a chemokine binding protein.

3. The method of claim 2, wherein the chemokine is a class a or a class P chemokine.

4. The method of claim 3, wherein the chemokine is selected from the group consisting of CTAP-III, gro/MGSA, ENA-78, MCP-1, interleukin-8, RANTES, MIP-la, and MIP-1p.

The method of claim 1, wherein the immunopathological disorder is selected from the group consisting of microbial infection, malignancy and metastasis, asthma, coronary restenosis, autoimmune diseases, cirrhosis, endotoxemia, atherosclerosis, and reperfusion injury.

6. The method of claim 1, further including administering an antibiotic or antiviral to the subject.

7. The method of claim 1, wherein the administering of anti- inflammatory protein is at a dosage from about lOpg to 100[tg per administration.

8. The method of claim 1, wherein the administering of anti- inflammatory protein is selected from the group consisting of subcutaneous, intravenous, intraarterial, intramuscular, intrarectal and transdermal.

9. A pharmaceutical composition comprising at least one dose of an immunotherapeutically effective amount of an anti-inflammatory protein having a molecular weight of approximately 30-40 kD; having amino acid homology with the myxoma T7 interferon-y receptor homolog; and having the biological function of the myxoma T7 interferon-y receptor homolog, in a pharmacological carrier.

The composition of claim 9, wherein the anti-inflammatory protein is a Type I chemokine binding protein.

11. The composition of claim 10, wherein the chemokine is a class a or a class p chemokine.

12. The composition of claim 11, wherein the chemokine is selected from 15 the group consisting of CTAP-III, gro/MGSA, ENA-78, MCP-1, interleukin-8, RANTES, MIP-la, and MIP-13. Dated this fifteenth day of February 2000. THE JOHN P ROBARTS RESEARCH INSTITUTE Patent Attorneys for the Applicant: F B RICE CO F B RICE &CO