MXPA99010259A - Methods for eosinophil depletion and compositions therefor - Google Patents

Abstract

Binding compositions and various methods of use of the binding compositions are provided. In particular, a method is provided which comprises administering an effective amount of the binding composition, alone or in combination with other compounds, to eosinophils, thereby reducing the level of eosinophils in an individual. Preferably, the binding composition is a monoclonal antibody specific for CCR3.

Description

METHODS FOR THE DISSIPATION OF EOSINOFI OS AND COMPOSITIONS FOR THEMSELVES FIELD OF THE INVENTION The invention generally relates to methods for dissipating eosinophil levels and compositions used for such and, more particularly, to methods for treating diseases or conditions associated with elevated populations of eosinophils.

BACKGROUND OF THE INVENTION Eosinophils are white blood cells of granulocytic lineage. Its normal functions include fighting parasitic infections, particularly helminthic infections. See, for example, Janeway et al., (Eds. 1996) Immunobioloqy: The Immune System in Health and Disease, 2nd Ed., Garland Publishing, New York, NY; and Rich, (ed. 1996) Clinical Immunoloqy: Principles and Practice Mosby, St. Louis, Mo. However, accumulation in tissues, a condition referred to as eosinophilia, is also associated with several abnormal or morbid conditions, including hypereosinophilia, chronic pneumonia, allergic bronchopulmonary aspergillosis, Churg-Strauss syndrome, atopic dermatitis and most notably asthma. See, for example, Frigas et al., (1986) J. Allergy Clin. Immunol. 77: 527-537; Weller, (1984) J. Allerqy Clin. Immunol., 73: 1-10; and Frank et al., (eds. (1995) Samter's Immunoloqical Diseases, 5th Ed., vol. I-II, Little, Brown, and Co., Boston, MA.) Glucocorticoid steroids are commonly the preferred therapeutic agents for treating acute effects of allergic diseases, such as asthma, however, prolonged treatment with steroids is associated with harmful side effects, see, for example, Goodman and Gilman, (eds.) The Pharmacological Basis of Therapeutics, MacMillan Publishing Company, New York In addition, the spheroid does not appear to affect the production or accumulation of granulocytic cells, such as eosinophils, in affected tissues, leading to the treatment of symptoms rather than the adjacent cause.More recently, it has been shown that eosinophilia associated with certain immune disorders could be treated with the administration of an antagonist to interleukin-5 (IL-5) See, for example, Conffman et al., (1989) Science 24 5: 308-310; and the US patent. No. 5,096,704 of Coffman et al. IL-5 is a potent eosinophil differentiating factor, but it also exerts effects on other immune cells, for example, B cells. However, this antagonist can affect other cell types besides eosinophils. The availability of alternative or complementary approaches to the treatment of disorders associated with eosinophilia would have important clinical utility. Impediment to the production or accumulation of eosinophils in the relevant tissues can obstruct the underlying cause of these disorders or diseases.

BRIEF DESCRIPTION OF THE INVENTION The invention provides a method for dissolving eosinophils by administering an effective amount of an antagonist against the receptor for an eosinophil chemokine, eotaxin. Applicants have observed that this leads to a decrease in the number of eosinophils. The receptor, designated CCR3, is expressed predominantly in eosinophils. Preferably, antagonists to CCR3 are monoclonal antibodies or binding compositions derived therefrom by regular techniques. The present invention provides an antibody or a binding composition that specifically binds to a CCR3 receptor, produces the level of eosinophilia in a sample that does not obstruct the binding of ligands. In preferred embodiments, the antibody is a monoclonal antibody. A sterile composition comprising the antibody or binding composition and a pharmaceutically acceptable carrier is also provided. The invention encompasses a method for alleviating a disease or disorder related to eosinophils in an individual, by administering an effective amount of the antibody or the binding composition, either alone or in combination with an interleukin-5 (IL-5) antagonist or a steroid In preferred embodiments, the antibody is a monoclonal antibody. An effective amount of the antibody or binding composition is, for example, preferably at least 500 μm / kg body weight. Usually at least 1000 μg / kg of body weight; typically at least 5 mg / kg of body weight; and ordinarily less than 100 mg / kg body weight; typically less than 50 mg / kg of body weight; or usually less than 25 mg / kg of body weight per week. The relief is a reduction in the level of eosinophils, for example, 10%, 20%, 30% or 50% or more. Eosinophil-related disease can be a lung inflammation; a dermatitis or a hypereosinophilia. The IL-5 antagonist can be an intact monoclonal antibody, a binding fragment thereof, a soluble receptor for IL-5 or a mutein of IL-5. An effective amount of a monoclonal antibody to IL-5, binding fragment thereof or soluble receptor is preferably at least 1 μg / kg of body weight; usually at least 5 μg / kg of body weight; or typically at least 10 μg / kg of body weight; and generally less than 1000 μg / kg body weight; usually less than 500 μg / kg of body weight; or preferably less than 100 μg / kg of body weight per week. An effective amount of the mutein is preferably at least 100 μg / hour; usually at least 500 μg / hour; typically at least 1 mg / hour; and ordinally at least 3 mg / hour; and preferably less than 100 mg / hour; usually less than 30 mg / hour; typically less than 10 mg / hour; Or ordinarily less than 6 mg / hour. An effective amount of steroid is preferably at least 1 mg / day; usually at least 2 mg / day; or typically at least 5 mg / day; and is ordinarily less than 100 mg / day; typically less than 50 mg / day; usually less than 20 mg / day; or preferably less than 10 mg / day. The present invention further provides a method for detecting the presence of eosinophils in a sample, which comprises the steps of contacting the sample with the antibody or binding composition and detecting the binding of the antibody to the receptor binding composition. CCR3 in eosinophils. In preferred embodiments, the antibody is a monoclonal antibody and the antibody is detectably labeled. A method is also involved for isolating a population of cells expressing the CCR3 receptor from a mixture of cells by contacting the mixture with the antibody or the binding composition and isolating the cell population. In preferred embodiments, the antibody is a monoclonal antibody. The antibody or binding composition is detectably labeled with a fluorescent moiety, a radioactive moiety or coupled to a magnetic globule. Cells are isolated by Fluorescent Activated Cell Separation (SCAF) or by magnetic cell separation.

DETAILED DESCRIPTION OF THE INVENTION I. General The invention is based, in part, on the discovery that a receptor for a specific chemokine for eosinophils, eotaxin, is predominantly expressed in eosinophils. From an entire pattern of approximately 30 monoclonal antibodies (mAb) surprisingly two mAbs were able to significantly reduce the level of eosinophils in mice plagued by a helminth, but without obstructing the binding of ligands to the receptor. The reduced levels can be attributed to any of several mechanisms, for example, fixation / dissipation of complements or optimization by microfagos. See, for example, Janeway et al., (Eds. 1996) Immunobiology, Garland Publishing Inc., New York, NY. Chemokines are a subfamily of chemoattractant cytokines that classically mediate leukocyte trafficking, joining seven specific transmembrane binding receptors bound to protein G. Chemokines are divided into four groups based on the primary sequence of the first two cysteines: families CXC, CC, C and, the recently discovered, CX3C. The CXC and C chemokine families are predominantly effective in neutrophils and lymphocytes, respectively. CC chemokines are preferably effective on macrophages, lymphocytes and eosinophils. Eosinophil-specific chemokines include, for example, RANTES, MCP-2, MCP-3, MCP-4, MIP-1a and, the one just described, eotaxin. See, for example, Alam et al., (1993) J. Immunol 150: 3442-3448; Weber et al., (1995) J. Immunol. 154: 4166-4172; Dahinden et al., (1994) J. Exp. Med. 179: 751-756; Uguccioni et al., (1996) J. Exp. Med. 183: 2379-2384; Rot et al., (1992) J. Exp. Med. 176: 1489-1495; and García-Zepeda et al., (1996) J. Immunoi 157: 5613-5626.

II. Production of antibodies; Binding compositions The present invention provides the use of an antibody or a binding composition that specifically binds to CCR3, preferably mammalian, e.g., primate, human, cat, dog, rat or mouse. Antibodies can be cultured in various CCR3 proteins, including individual, polymorphic, allelic, strain or species variants, and fragments thereof, both in their naturally occurring (full-length) forms or in their recombinant forms. Particularly interesting epitopes include those accessible from the extracellular space. Additionally, antibodies of CCR3 proteins can be cultured either in their native (or active) forms or in inactive, for example, denatured forms. Anti-idiotypic antibodies can also be used. A number of immunogens can be selected to produce antibodies specifically reactive with CCR3 proteins, for example, as expressed in cells. The recombinant protein is a preferred immunogen for the production of monoclonal or polyclonal antibodies. Protein present in nature can also be used, from appropriate sources, for example primate, rodent, etc., either in pure or impure form. See, for example, the GenBank and NCBI databases. Synthetic peptides, which are made using, for example, the human CCR3 protein sequences described herein, can also be used as an immunogen for the production of antibodies to CCR3 proteins. The recombinant protein can be expressed and purified in eukaryotic and prokaryotic cells as described, for example, in Coligan et al., (Eds. 1995 and periodic supplements) Current Protocols in Protein Science John Wiley & Sons, New York, NY; and Ausubel et al., (eds, 1987 and periodic supplements) Current Protocols in Molecular Bioloqy. Greene / Wiley, New York, NY. Naturally folded or denatured material, as appropriate, may be used to produce antibodies. Monoclonal or polyclonal antibodies can be added, for example, for subsequent use in immunoassays to prevent the protein or for immunopurification methods. Methods for producing polyclonal antibodies are well known to those skilled in the art. Typically, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized with the mixture. The immune response of the animal to the preparation of the immunogen is monitored, taking test indentations and alternating the titre of the reactivity to the CCR3 protein of interest. For example, when appropriately high titers of antibody to the immunogen are obtained, usually after repeated immunizations, blood is collected from the animal and antisera are prepared. The further fractionation of the antisera can be done by enriching for antibodies reactive to the protein, if desired. See, for example, Harlow and Lane; or Coligan. It is possible to select classes of isotypes or antagonists with specific properties, for example by not obstructing the binding of ligands. Immunization can also be performed by other means, for example, immunization of DNA vectors. See, for example, Wang, and Bulls (1997) Viroloqy 228: 278-284. Monoclonal antibodies can be obtained, by various techniques familiar to those skilled in the art typically, cells of the spleen of an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell. See, Kohler and Milstein, (1976) Eur. J. Immunol. 6:51 1-519. Alternative methods of immortalization include transformation with Epstein Barr virus, oncogenes or retroviruses, or other methods known in the art. See, for example, Doyle et al., (Eds 1994 and periodic supplements) Cell and Tissue Culture: Laboratorv Procedures, John Wiley and Sons, New York, NY. The colonies originating from individual immortalized cells are screened for the production of antibodies of the desired specificity and affinity for the antigen and the yield of the monoclonal antibodies produced by such cells can be enhanced by various techniques, including injection into the cavity. peritoneal of a vertebrate host.

Alternatively, DNA sequences encoding a monoclonal antibody or a binding fragment thereof can be isolated by screening a DNA library of human B cells according to, for example, the general protocol described by Huse et al. (1989). 246: 1275-1281. Antibodies or binding compositions, including binding fragments and single chain versions against predetermined fragments of CCR3 proteins, can be cultured by immunizing animals with conjugated products of the carrier protein fragments, as described above. Monoclonal antibodies are prepared to paritr of cells that secrete the desired antibody. Antibodies can be screened to bind to normal or defective CRR3 protein or sifted for their eosinophil clearance ability. These monoclonal antibodies will usually bind at least a KD of about 1 mM, more usually at least about 300 μM, typically at least about 10 μM, more typically at least about 30 μM, preferably at least of about 10 μM and more preferably at least about 3 μM or better. In some cases, it is desirable to prepare monoclonal antibodies (mAbs) from several mammalian hosts, such as mice, rodents, primates, humans, etc. A description of the techniques for preparing such monoclonal antibodies can be found, for example, in Stites et al., (Eds.) Basic and Clinical Immunoloqy (4th ed.) Lange Medical Publications, Los Altos, Ca, and references cited therein.; Harlow and Lane, (1988) Antibodies: A Laboratorv Manual CSH Pres; Goding, (1986) Monoclonal Antibodies: Principies and Practice (2nd ed.) Academic Press, New York, NY; and particularly in Kohler and Milstein, (1975) Nature 256: 495-497, which discusses a method for generating monoclonal antibodies. Briefly summarized, this method involves injecting an animal with an immunogen. The animal is sacrificed again and cells are taken from its spleen, which are then fused with myeloma. The result is a hybrid cell or a "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate the individual clones, each of which secretes a single antibody species to the immunogen. In this manner, the individual antibody-derived species are the products of individual B cells immortalized and cloned the immune animal generated in response to a specific site recognized in the immunogenic substance. Other suitable techniques involve the selection of antibody libraries in phage or similar vectors. See, for example, Huse et al., (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246: 1275-1281; and Ward et al., (1989) Nature 341: 544-546. The polypeptides and antibodies of the present invention can be used with or without modification, including chimeric or humanized antibodies. Frequently, polypeptides and ligand antibodies will be labeled, either covalently or non-covalently, a substance that provides a detectable signal. An extensive variety of labels and conjugation techniques are known and extensively reported in both scientific and patent forms. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent portions, magnetic particles, and the like. Some patents that teach about the use of such labels include the patents of E.U.A.

Nos. 3,817,837; 3,850,752, 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulins can be produced. See Cabilly, patent of E.U.A. No. 4,816,567; and Queen and others, (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10033; or make them in transgenic mice. See Méndez and others, (1997) Nature Genetics 15: 146-156. The antibodies are merely one form of specific binding compositions. Other binding compositions, which will often have similar uses, include molecules that bind with specificity to the CCR3 receptor, for example, in a binding partner-binding partner fashion, antibody-antigen interaction, or in a natural, physiological interaction. relevant, between protein and protein, either covalent or non-covalent, for example, proteins that are specifically associated with CCR3 receptor protein. The molecule can be a polymer or a chemical reagent. A functional analog may be a protein with structural modifications or it may be a structurally unrelated molecule, for example, having a molecular configuration that interacts with the appropriate binding determinants. The antibody binding compounds, including binding fragments, of this invention may have significant diagnostic value. They may be useful as non-neutralizing binding compounds and toxins or radionuclides may be coupled so that, when the antigen-binding compound is bound, a cell expressing it, for example, on its surface is killed. In addition, various drug-binding compounds or other therapeutic agents can be conjugated, either directly or indirectly by means of a linker, and can affect the orientation of the drug. lll. Immunoassays Immunoassays are valuable in the diagnosis of a disease or disorder associated with eosinophilia or in monitoring the level of eosinophil dissipation in a treated patient. A particular protein can be measured by a variety of immunoassay methods. For a critical appreciation of immunological and immunoassay procedures in general, see, for example, Stites and Terr, (eds.1991) Basic and Clinical Immunoloqy (7th ed.). In addition, immunoassays of the present invention can be performed in many configurations, which are critically appreciated extensively, for example, in Maggio, (ed., 1980) Enzyme Immunoassay CRC Press, Boca Raton, Florida; Tijan, (1985) "Practice and Theory of Enzyme Immunoassays," Laboratorv Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V., Amsterdam; and Harlow and Lane Antibodies, A Laboratorv Manual, previously mentioned.

See also Chan (ed.1987) Immunoassay: A Practical Guide Academic Press, Orlando, FL; Price and Newman (eds. 1991) Principles and Practice of Immunoassavs Stockton Press, NY; and Ngo, (ed., 1988) Non-isotopic Immunoassays Plenum Press, NY. Immunoassays can be performed for the measurement of CCR3 proteins or peptides by a variety of methods known to those skilled in the art. In short, the immunoassays to measure the protein can be binding or competitive or non-competitive assays. In competitive binding assays, the sample to be analyzed competes with an analyte labeled for specific binding sites on a capture agent bound to a solid surface. Preferably, the capture agent is an antibody specifically reactive with CCR3 proteins produced as described above. The concentration of the labeled analyte bound to the capture agent is inversely proportional to the amount of free analyte present in the sample. In a competitive binding immunoassay, the CCR3 protein present in the sample competes with the tagged protein to bind to a specific binding agent, for example, an antibody specifically reactive with the CCR3 protein. The binding agent can be attached to a solid surface to effect the separation of the bound tagged protein from the unbound labeled protein. Alternatively, the competitive binding assay in the liquid phase can be conducted and a variety of methods known in the art can be used to separate the bound tagged protein from the unbound tagged protein. Following the separation, the amount of tagged protein bound is determined. The amount of protein present in the sample is inversely proportional to the amount of labeled protein binding. Alternatively, a homogeneous immunoassay can be performed in which a separation step is not needed. In these In immunoassays, the label on the protein is altered by binding the protein to its specific binding agent. This alteration in the tagged protein results in a decrease or an increase in the signal emitted by the tag, so that the measurement of the tag at the end of the immunoassay allows detection or quantification of the protein. The CCR3 proteins can also be determined by a variety of non-competitive immunoassay methods. For example, a solid phase, two-site intercalated immunoassay can be used. In this type of assay, a binding agent for the protein, for example an antibody, is fixed to a solid support. A second protein binding agent is labeled, which may also be an antibody and which binds the protein at a different site. After the binding at both sites has taken place on the protein, the unbound labeled binding agent is removed and the amount of labeled binding agent bound to the solid phase is measured. The amount of labeled labeling agent bound is directly proportional to the amount of protein in the sample.

Western blot analysis can be used to determine the presence of CCR3 proteins in a sample. The electrophoresis is carried out, for example, in a sample of tissue of which it suspects that the protein is contained. Following the electrophoresis to separate the protein and the transfer of the proteins to a suitable solid support, for example, a nitrocellulose filter, the solid support is incubated with an antibody reactive with the protein. This antibody can be labeled or alternatively detected by subsequent incubation with a labeled second antibody that binds to the primary antibody. The immunoassay formats described above can employ labeled test components. The label can be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. An extensive variety of labels and methods can be used. Traditionally, a radioactive label was used that incorporated 3H, 125l, 35S, 14C or 32P. Non-radioactive labels include identities that bind labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies that can serve as specific binding members for a labeled ligand. The choice of label depends on the sensitivity required, the ease of conjugation with the compound, the stability requirements and the available instrumentation. For a critical appreciation of various labeling or signal reproduction systems that can be used, see U.S. No. 4,391, 904.

Antibodies reactive with a particular protein can also be measured by a variety of immunoassay methods. Thus, modifications of the above procedures can be used to determine the amounts the affinities of various CCR3 antibodies or the preparation of antibodies. For a critical assessment of the immunological and immunoassay procedures applicable to the measurement of antibodies by immunoassay techniques, see, for example, Stites and Terr, (eds,) Basic and Clinical Immunology (7th ed.) Mentioned above; Maggio (ed.) Enzvme Immunoassay, previously mentioned; and Harlow and Lane Antibodies, A Laboratorv Manual, previously mentioned. The screens for evaluating the binding and activity of the mAbs and the binding compositions enclose a variety of methods. The binding can be assayed by detectably labeling the antibody or binding composition as described above. Cells expressing a CCR3 receptor are incubated with this antibody or this binding composition, and binding is assayed by fluorescence activated cell separation (SCAF) analysis. To evaluate the eosinophil dissipation ability, experimental animals, e.g., mice, are preferably induced to eosinophilia, for example by infection with a parasite. The eosinophil counts are made before administration and at various points in time after the administration of a bolus of mAb or the proposed binding composition. The levels are analyzed in several samples, for example, blood, serum, nasal or lung washes, or spotted tissue biopsy. A mAb or a satisfactory binding composition will significantly decrease the level of eosinophils in circulation. The evaluation of antibodies in other animals, for example humans, can be carried out using various methods. For example, blood samples are taken from patients suffering from a disease or disorder related to eosinophils, before and after treatment with a proposed mAb, and eosinophil counts are made. The antibodies can be used in a diagnostic context to assess the degree of eosinophilia, for example by SCAF, tissue staining, last in vitro.

IV. Uses The present invention is useful in the treatment of diseases associated with eosiphylia, for example pulmonary inflation, dermatitis, etc. See, for example, Hardy et al., (1968) Ann. Intern. Med. 68: 1220-1229. Pulmonary inflation includes, for example, asthma, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, hypereosinophilia or Churg-Strauss syndrome. See, for example, Frank et al., (Eds. 1995) Samter's Immunologic Diseases, 5a. Ed., Vols. l-l I, Little, Brown and Co., Boston, MA; Coffman et al., (1989) Science 245: 308-310; and Frick et al., (1988) J. Allerqy Clin. mmunol. 82: 199-225. Dermatological diseases include, for example, atopic dermatitis. See, for example, Uehara et al., (1990) Clin Exp. Dermatol. 15: 264-266. The present invention can be administered alone or in combination with another eosinophilia inhibitor, for example, interleukin-5 (IL-5) antagonists; or other compounds used for the treatment of symptoms, for example, steroids such as glucocorticoids. To prepare pharmaceutical or sterile compositions, including the CCR3 antibody or the binding composition thereof, the antibody is bound and mixed with the binding composition with a pharmaceutically acceptable carrier or excipient which is preferably inert. The preparation of such pharmaceutical compositions is known in the art.

See, for example, Remington's Pharmaceutical Sciences and U.S. Pharmacopoeia: National Formulary, Mack Publishing Company, Easton, PA (1984). The antibodies or binding compositions are usually administered parenterally, preferably intravenously. Since such protein or peptide antagonists can be immunogenic, they can preferably be administered slowly, either by conventional equipment for intravenous administration or from a subcutaneous reservoir, for example as taught by Tomasi et al., U.S. Pat. 4,732,863. When administered parenterally, the antibodies or fragments will be formulated in unit dose injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are preferably inherently non-toxic and non-therapeutic. The antagonist can be administered in aqueous vehicles, such as water, saline or vehicles regulated in their pH with various additives and / or diluting agents, or without them. Alternatively, a suspension, such as a zinc suspension, can be prepared to include the peptide. Such suspension may be useful by subcutaneous (SQ) or intramuscular (IM) injection. The proportion of antagonist and additive can be varied over a wide range, since both are present, or the combination is, in effective amounts. The antibody is preferably formulated in a purified form substantially free of aggregates, other proteins, endotoxins, and the like, concentrations, for example from about 5 to 30 mg / ml, preferably from 10 to 20 mg / ml. Preferably, the endotoxin levels are less than 2.5 EU / ml. See, for example, Avis et al. (Eds). (1993) Pharmaceutical Dosage Forms: Parenteral Medications 2a. ed., Dekker, NY; Lieberman et al., (Eds) (1990) Pharmaceutical Dosage Forms. Tablets 2a. ed., Dekker, NY; Lieberman et al. (Eds) (1990) Pharmaceutical Dosaqe Forms: Disperse Systems Dekker, NY; Fodor et al., (1991) Science 251: 767-773, Coligan (ed.) Current Protocols in Immunology; Benjamin / Cummings; Paul (ed) Fundamental nmunoloqy; Academic Press; Parce et al., (1989) Science 246: 243-247; Owicki et al., (1990) Proc. Nat'l Acad. Sci. USA 87: 4007-4011; and Blundell and Johnson (1976) Protein Crystalloqraphv, Academic Press, New York. The selection of a regimen of administration for an antagonist depends on several factors, including the percentage of serum or tissue turnover of the antagonist, the level of eosinophilia, the immunogenicity of the antagonist, the accessibility of the target eisinophils. (for example, if they have to block eosinophils that are not serum).

Preferably, a delivery regimen maximizes the amount of antagonist delivered to the patient, compatible with an acceptable level of side effects. According to the above, the amount of antagonist delivered depends in part on the particular antagonist and the severity of the condition being treated. Orientation is found in the selection of appropriate doses in the literature on therapeutic uses of antibodies, for example, in Bach et al., Chapter 22, in Ferrone et al., (Eds. 1985) Handbook of Monoclonal Antibodies Noges Publications, Park Ridge , NJ; and Russell, pgs. 303-357, and Smith et al., P. 365-389, in Haber et al., (Eds. 1977) Antibodies in Human Diagnosis and Therapy, Raven Press, New York, NY. The determination of the appropriate dose is made by the physician, for example, using parameters or factors known in the art that affect the treatment or that are predicted to affect the treatment. Generally, the dose begins with a quantity a little less than the optimal dose and is increased by small increments thereafter until the desired or optimum effect is achieved in relation to any negative side effects. Eosinophil levels in circulation would be important indicators of when an effective dose is reached, along with measures of the symptoms of inflammation, for example, the level of inflammatory cytokines produced. Preferably, a CCR3 antibody or a binding composition thereof that is used is derived from the same species of the animal targeted for treatment, thereby minimizing a humoral response to the reagent. Total weekly dose ranges for antibodies or fragments thereof, which specifically bind to CCR3, generally range from about 10 μg, more generally about 100 μg, typically about 500 μg, more typically about 1000 μg, preferably from about 5 mg, and more preferably about 10 mg per kilogram of body weight. Generally, the range will be less than 100 mg, preferably less than 50 mg and more preferably less than about 25 mg per kilogram of body weight. Weekly dose ranges for antagonists of IL-5 activity, eg, antibodies, binding fragments, soluble receptors, vary from about 1 μg, preferably at least about 5 μg and more preferably at least about 10 μg. μg, per kilogram of body weight. Generally, the range will be less than about 1000 μg, preferably less than about 500 μg and more preferably less than about 500 μg, and more preferably less than about 100 μg, per kilogram of body weight. Dosages are about a program that affects the desired treatment and can be periodic for a shorter or longer period. In general, the ranges will be at least about 10 μg, about 50 mg, preferably about 100 μg, to about 10 mg per kilogram of body weight. Other antagonists of IL-5 activity are also contemplated, for example, muteins. The hourly dose ranges for mutants against IL-5 vary from at least about OO μg, generally at least about 500 μg, typically at least about 1 mg, and preferably at least 3 mg per hour.

Generally, the dosage will be less than about 100 mg, typically less than about 30 mg, preferably less than about 10 mg, and more preferably less than 6 mg, per hour. The general ranges are given at least from about 1 μg to about 1000 μg, preferably from about 10 μg, to about 500 μg per hour. The present invention also provides for the administration of CCR3 antibodies or binding compositions in combination with known therapies, for example, steroids, particularly glucocorticoids, which alleviate the symptoms associated with eosinophilia. Daily dosages for the glucocorticoids will vary from at least about 1 mg, generally at least about 2 mg and preferably at least about 5 mg per day. Generally, the dosage will be less than about 100 mg, typically at least about 50 mg, preferably less than about 20 mg, and more preferably less than about 10 mg per day. In general, the ranges will be at least from about 1 mg to about 100 mg, preferably from about 2 mg.to 50 mg per day. The phrase "effective amount" means an amount sufficient to alleviate a symptom or signal of the inflammatory condition. Typical mammalian hosts include mice, rats, cats, dogs and primates, including humans. An effective amount for a particular patient may vary depending on factors such as the condition being treated, the general health of the patient, the route of the method and the dose of administration, and the severity of the side effects. When in combination, the effective amount is related to the combination of components and the effect is not limited to the individual components only. An effective amount of antagonist will typically decrease symptoms by at least about 10%; usually at least about 20%; preferably at least about 30%; more preferably at least about 50%. The present invention provides reagents will be useful in therapeutic applications as described elsewhere herein, for example, in the general description for treating disorders associated with eosinophilia. See, for example, Berkow, (ed). The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J .; Thorn, et al., Harrison 'Principies of Medical Medicine, McGraw Hill, NY; Gilman et al. (Eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8a. Ed., Pergamon Press; Remigton's Pharmaceutical Sciences, 17th ed. (1990) Mack Publishing Co., Easton, Penn; Langer (1990) Science 249: 1527-1533; and Merck Index, Merck & Co., Rahway, New Jersey. Antibodies can be used in CCR3 proteins for the identification or separation of populations of cells expressing the CCR3 protein, e.g., eosinophil. Methods for separating all populations are well known in the art. See, for example, Melamed, et al., (1990) Flow Cvtometrv and Sorting Wiley-Liss, Inc., New York, NY; Shapiro, (1988) Practical Flow Cvtometry Liss, New York, NY; and Robinson et al., (1993) Handbook of Flow Cvtometrv Methods Wiley-Liss, New York, NY. Cell populations expressing the CCR3 receptor can also be purified, using magnetic beads as described, for example, in Bieva et al., (1989) Exp. Hematol. 17: 914-920; Hemebtud et al., (1990) Bioconi. Chem. 1: 41 1-418; Vaccaro et al., (1990) Am. Biotechnol. Lab. April 1990. Using the assay methods described above, antibodies or binding compositions are useful in the diagnosis of morbid states that result in high numbers of eosinophils. Labeling antibodies can also be used in the analysis of tissue infiltration of eosinophils. The antibodies raised against each CCR3 protein will also be useful for culturing anti-idiotypic antibodies. These will be useful in the arrest or diagnosis during immunological conditions related to the expression of the respective antigens. The broad scope of this invention will be better understood with reference to the following examples, which are not intended to limit invention to the specific embodiments.

EXAMPLES I. General Methods Some regular methods of molecular biology are described or referenced, for example, in Maniatis, et al., (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press; Sambrook et al., (1989) Molecular Cloning, A Laboratory Manual, (2nd edition) vols. 1-3, CSH Press, NY; Ausubel et al., Biology, Greene Publishing Associates, Brooklyn, NY; Ausubel et al., (1987) and supplements) Current Protocols in Molecular Biology, Greene / Wiley, New York; or Innis et al., (1990 editions) PCR Protocols: A Guide to Methods and Applications Academic Press, N.Y. Methods for protein purification include methods such as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, for example, Ausubel et al., (1987 and periodic supplements); Deutscher (1990) -Guide to Protein Purification in methods in Enzymology, vol. 182 and others, volumes in this series; the manufacturer's literature on the use of protein purification products, for example, Pharmacia, Piscataway, N, J., or Bio-Rad, Richmond, CA; and Coligan et al., (1995 editions and periodic supplements) Current Protocols in Protein Science, John Wiley & Sons, New York, NY. The combination with recombinant proteins allows fusion to appropriate segments, for example, to a FLAG sequence or an equivalent that can be fused through the removable sequence and protease. See, for example, Hochuli (1989) Chemische Industrie 12: 69-70; Hochuli (1990) -Purification of Recombinant Proteins with Metal Chelate Absorbent- in Setlow (ed) Genetic Engineering, Principie and Methods 12: 87-98, Plenum Press, N.Y .; and Crowe et al., (1992) OTAexpress: The High Level Expression & Protein Purification System QIAGEN, Inc., Chatsworth, CA. The SCAF analyzes are described in Melamed et al., (1990) Flow Cytometry and Sorting Wiley-Liss, Inc., New York, NY; Shapiro (1988) Practical Flow Cytometry Liss, New York, NY; and Robinson et al., (1993) Handbook of flow Cytometry Methods Wiley-Liss, New York, NY.

II. Production Cell Lines Expressing CCR3 Mammalian cells, for example, NIH3T3, were transfected by electropuration or lipofectamine (Gibco BRL, Gaithersburg, MD) and selected in media supplemented with neomcine for two weeks. The resistant colonies were separated by SCAF in 96-well plates and allowed to proliferate. RNA was isolated from individual clones using ARNzol (Friendswood, TX) and analyzed using RTPCR. See for example, Ausubel et al., (1987 and supplements) Current Protocols in Molecular Biology, Greene / WNey, New York; Innis et al., (Eds.) (1990) PCR Protocols: A Guide to Methods and Applications Academic Press, N.Y., next to DNase treatment. Positive clones were subject to further analysis than an CA ++ flow assay as described, for example, in Kelner et al., (1994) Science 266: 1395-1399, using otaxin as a ligand for CCR3. The clone exhibiting the highest CA ++ flow was expanded for use in gene mAbs. Other methods of evaluating the expression can also be used, for example, staining and analysis of SCAF, tissue staining, Northern analysis, etc. lll. Monoclonal antibodies Lewis rats were immunized with 108 cells transfected with Y3 / CCR3, intraperitoneally every 2 weeks for 8 weeks. The final immunization was delivered intravenously (IV) through the caudal vein. 4 days after the IV injection, the spleen was excised and fused to SP2 / 0 and NS1 cells. HAT-resistant hybridomas were selected using a protocol designed by Stem Cell Technologies (Vancouver, BC). After 10 days of HAT selection, the resistant foci were transferred to 96-well plates and expanded for 3 days. Supernatants containing antibodies were analyzed by SCAF, to bind them to NIH3T3 / CCR3 transfectants. 29 different CCR3 mAbs were produced. Additionally, some of the mAbs are used to separate CCR3 positive cells into spleens of sensitized RAG - / - mice with Asperguillus fumagatus. All the separated cells were found to be eosinophils as determined by staining by Wright Giemsa staining.

V. Dispersion of eosinophils in circulation Isotypes of all mAbs were obtained as directed in an ELISA based kit from Zymed, Inc. (So. San Francisco, CA) and in a team based on Ouchterlony from ICN (Aurora, OH). The mAbs had isotypes of IgG2A, IgG2B, and IgG2C. Using the IgG2B mAbs, designated 6S2-19-4 and -6SH2-88, as well as an IgG2A mAb, designated 6SH2-59, eosinophil dissipation assays were performed. None of these antibodies was able to obstruct the binding of eotaxin to CCR3. Balb / C mice were injected subcutaneously with 500 larvae of Nippostrongylus brasiliensis in order to induce eosinophilic pneumonia. From 9 to 14 days after infection, a 0.5 mg bolus of either 6S2-19-4 or 6SH2-59 was administered IP. The eosinophil counts were analyzed before and after the administration of the mAbs, staining the blood extracted from the caudal vein. 6SH2-59 did not significantly dissipate circulating eosinophils, whereas 6S2-19-4 and 6SH2-88 significantly dispelled those cells. Interestingly, the level of eosinophils in circulation remained low for at least 7 days. 6S2-19-4 was deposited with American Type Culture Collection (ATCC; 12301 Parklawn Drive, Rockville, MD), on April 30, 1997, ATCC No. HB12351. 6SH2-88 was deposited with ATCC in, ATCC Do not. . The analysis of human antibodies can be assessed in a similar way. A biological sample is obtained, for example, blood, tissue biopsy samples, pulmonary or nasal lavage, puncture in the skin, of the individual suffering from a disease or a disorder associated with eosinophilia. The eosinophil counts are taken as previously described. A mAb, which specifically binds to the human CCR3 receptor, is administered alone or in combination with another compound, for example, an IL-5 antagonist or a steroid, to the individual. An effective amount is measured for symptom relief, for example, relief of pulmonary or dermal inflammation, or eosinophil counts are measured in time. All citations in the present reference are incorporated herein to the same degree as if each publication or individual patent application that is incorporated by reference is specifically indicated. Many modifications and variations of this invention can be made, without deviating from its spirit and scope as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of examples only and the invention is to be limited only by the terms of the appended claims, together with the full scope of equivalents to which such claims are authorized; and the invention is not limited by the specific embodiments that have been presented herein by way of example.

Claims (9)

NOVELTY OF THE INVENTION CLAIMS

1. - An antibody or a binding composition that: a) binds specifically to a CCR3 receptor; b) reduces the level of eosinophils in a sample; and c) does not obstruct the binding of ligands to said receptor.

2. The antibody according to claim 1, further characterized in that said antibody is a monoclonal antibody. 3- A sterile composition comprising the antibody or binding composition according to claim 1 and a pharmaceutically acceptable carrier. 4. The use of an antibody or a binding composition according to claim 1, for the preparation of a medicament for alleviating a disease or disorder related to eosinophils in an individual. 5. The use according to claim 4, wherein said antibody or said binding composition is administered in combination with: a) an antagonist to interleukin-5 (IL-5); or b) a steroid. 6. The use according to claim 4, wherein said antibody is a monoclonal antibody. 7. The use according to claim 4, wherein said CCR3 antibody or said binding composition provides the individual: a) at least 500 μg / kg of body weight per week; b) at least 1000 μg / kg of body weight per week; c) at least 5 mg / kg of body weight per week; d) at least 10 mg / kg of body weight per week; e) less than 100 mg / kg of body weight per week; f) less than 50 mg / kg of body weight per week; or g) expression at least less than 25 mg / kg of body weight per week. 8. The use according to claim 4, wherein said relief is accompanied by a reduction in the level of eosinophils. 9. The use according to claim 5, wherein said antagonist is: a) an intact monoclonal antibody; b) a binding fragment of said monoclonal antibody; c) a soluble receptor for IL-5; or d) an IL-5 mutein. 10. The use according to claim 9, wherein said antagonist of said intact monoclonal antibody, said binding fragment thereof or said soluble receptor; and said antagonist provides the individual: a) at least 1 μg / kg body weight per week; b) at least 5 μg / kg of body weight per week; c) at least 10 μg / kg of body weight per week; d) less than 1000 μg / kg of body weight per week; e) less than 500 μg / kg of body weight per week; f) less than 100 μg / kg of body weight per week. 1. The use according to claim 9, wherein said antagonist is said mutein and said antagonist provides the individual: a) at least 100 μg / hour; b) at least 500 μg / hour; c) at least 1 mg / hour; d) at least 3 mg / hour; e) less than 100 mg / hour; f) less than 30 mg / hour; g) less than 10 mg / hour; or h) less than 6 mg / hour. 12. The use according to claim 5, wherein said steroid provides the individual: a) at least 1 mg / day; b) at least 2 mg / day; c) at least 5 μm / day; d) less than 100 mg / day; e) less than 50 mg / day; f) less than 20 mg / day; or g) less than 10 mg / day. 1

3. The use according to claim 4, wherein said disease related to eosinophils is: a) a lung inflammation; b) a dermatitis, or c) a hypereosinophilia. 1

4. A method for detecting the presence of eosinophils in a sample, which comprises the steps of: a) contacting said sample with an antibody or a binding composition according to claim 1; and b) detecting the binding of said antibody or said binding composition to said eosinophils. 1

5. The method according to claim 14, further characterized in that said antibody or said binding composition is a monoclonal antibody. 1

6. The method according to claim 14, further characterized in that said antibody or said binding composition is detectably labeled. 1

7. A method for isolating a population of cells expressing a CCR3 receptor from a mixture of cells, which comprises the steps of: a) contacting said mixture with an antibody or a binding composition according to claim 1; and b) isolating said cell population. 1

8. The method according to claim 17, further characterized in that said antibody or said binding composition is a monoclonal antibody. 1

9. The method according to claim 17, further characterized in that said antibody or said binding composition is detectably labeled. 20. The method according to claim 19, further characterized in that said antibody or said binding composition is: a) labeled with a fluorescent portion; b) labeled with a radioactive portion, or c) coupled to a magnetic globule. 21. The method according to claim 17, further characterized in that said isolation is through: a) Fluorescent Activated Cell Separation (SCAF); or b) megnetic cell separation.