JP2001527410A - Methods for eosinophil depletion and compositions therefor - Google Patents

Abstract

  (57) [Summary] A binding composition and various methods of using the binding composition are provided. In particular, methods are provided that include administering to an eosinophil an effective amount of a binding composition, alone or in combination with other compounds, thereby reducing the level of eosinophils in an individual. . Preferably, the binding composition is a monoclonal antibody specific for CCR3.

Description

DETAILED DESCRIPTION OF THE INVENTION              Methods for eosinophil depletion and compositions therefor                                Field of the invention   The present invention generally relates to a method of depleting eosinophil levels, and to the use thereof. And, more particularly, diseases associated with an increased population of eosinophils Or a method of treating a condition.                                Background of the Invention   Eosinophils are white blood cells of the granulocyte lineage. Parasitic as their normal function Prevention of parasitic infections, especially parasite-like infections. For example, J aneway et al. (1996)Immunobiology : The Immune System in Health and Disease , Second Edition, Garland Publishing, New York, NY; and Rich (1996)Clinical Immunology ： Principles and Practice  Mosby, St. See Louis, Mo. However, their accumulation in tissues (a condition called eosinophilia) is also Acidophilia, chronic pneumonia, allergic bronchopulmonary aspergillosis, Churg-S Including Traus syndrome, atopic dermatitis, and most notably asthma Associated with some abnormal or disease state. For example, Frigas et al. (1986)J . All ergy Clin. Immunol. 77: 527-537; Weller (1984)J . Allergy Clin. Immunol . 73: 1-10; and Frank et al. (1995)Samter's Immunological DiseasesFifth edition , I-II, Little, Brown, and Co., Boston, MA.   Current glucocorticoid steroids are used for allergic diseases (eg, asthma) Is a preferred therapeutic drug for treating the acute effects of But extended Steroid treatment is associated with many adverse side effects. For example, Goodman and Gil man (ed.)The Pharmacological Basis of Therapeutics Macmillan Publishing See Company, New York, NY. In addition, steroids can affect affected tissues Clearly affect the production or accumulation of granulocyte cells (eg, eosinophils) Absent. Thus, leading to treatment of the symptoms rather than the underlying cause.   More recently, eosinophilia associated with certain immune disorders has been reduced to interleukin-5 Has been shown to be treatable by administration of an antagonist to (IL-5) You. For example, Coffman et al. (1989)Science 245: 308-310; and Coffman et al., USA See Patent No. 5,096,704. IL-5 is a potent eosinophil differentiation factor. It also exerts effects on other immune cells (eg, B cells). However The antagonist may affect other cell types besides eosinophils.   Alternative or complementary apros for treating disorders associated with eosinophilia The availability of approaches has significant clinical utility. Good in the relevant organization Prevention of the production or accumulation of eosinophils will block the underlying causes of these disorders or diseases. Can lock.                                Summary of the Invention   The present invention relates to a receptor for eotaxin, an eosinophil chemokine. Method of depleting eosinophils by administering an effective amount of an antagonist I will provide a. We have observed that this leads to a reduction in the number of eosinophils. . The receptor, designated CCR3, is mainly expressed on eosinophils. Preferably The CCR3 antagonist is a monoclonal antibody or And binding compositions derived therefrom.   The present invention provides antibodies or binding compositions that specifically bind to the CCR3 receptor. Reduce the level of eosinophilia in the sample, and block ligand binding. Do not lock. In a preferred embodiment, the antibodies are monoclonal antibodies. Anti Sterile compositions comprising the body or binding composition, and a pharmaceutically acceptable carrier. Provided.   The present invention provides that an effective amount of an antibody or binding composition, alone or interleukin 5 (IL-5) or in combination with antagonists to steroids Ameliorate an eosinophil-related disease or disorder in an individual by administering Includes a method for solving the problem. In a preferred embodiment, the antibody is a monoclonal antibody is there. An effective amount of the antibody or binding composition is, for example, preferably Both at 500 μg / kg body weight; usually at least 1000 μg / kg body weight; typically less Both 5 mg / kg body weight; or usually at least 10 mg / kg body weight; and usually 100 m less than g / kg body weight; typically less than 50 mg / kg body weight; or usually less than 25 mg / kg body weight It is. Remission is associated with a decrease in eosinophil levels (eg, 10%, 20%, 30%, or Or 50% or more). Eosinophil-related diseases include pulmonary inflammation; dermatitis; or It can be hypereosinophilia. Antagonists to IL-5 are intact Clonal antibodies, binding fragments thereof, soluble receptors for IL-5, or IL -5 mutein.   An effective amount of an IL-5 monoclonal antibody, binding fragment thereof, or soluble The scepter is preferably at least 1 μg / kg body weight per week; Both 5 μg / kg body weight; or typically at least 10 μg / kg body weight; and general Less than 1000 μg / kg body weight; usually less than 500 μg / kg body weight; It is less than μg / kg body weight. An effective amount of mutein is preferably at least 100 μg / Time; usually, at least 500 μg / hour; typically, at least 1 mg / hour; Usually at least 3 mg / hour; and preferably less than 100 mg / hour; Less than 30 mg / hr; typically less than 10 mg / hr; or usually less than 6 mg / hr. Is full. An effective amount of a steroid is preferably at least 1 mg / day; At least 2 mg / day; or typically at least 5 mg / day; and usually Less than 100 mg / day; typically less than 50 mg / day; usually less than 20 mg / day; Preferably, it is less than 10 mg / day.   The invention further provides a method of detecting for the presence of eosinophils in a sample. . The method comprises the steps of contacting the antibody or binding composition with the sample, and Detecting the binding of the antibody or binding composition to the CCR3 receptor on eosinophils I do. In a preferred embodiment, the antibody is a monoclonal antibody and The body is detectably labeled.   Contacting the mixture with the antibody or binding composition and isolating a population of cells Isolating a population of cells expressing the CCR3 receptor from a mixture of cells Methods for doing so are also included. In a preferred embodiment, the antibodies are monoclonal Antibodies. Antibodies or binding compositions are detectably labeled with a fluorescent moiety, a radioactive moiety. Known or bound to magnetic beads. Cells are separated by a fluorescent cell analyzer (FACS) or by magnetic cell separation.Detailed description of the invention I. Introduction   The present invention relates to eotaxin, a receptor for eosinophil-specific chemokines, Based in part on the finding that it is predominantly expressed on the sphere. About 30 monoclonal antibodies From the entire panel of (mAbs), surprisingly, two mAbs were parasitized by parasites. Could significantly reduce eosinophil levels in mice but did not bind ligand to the receptor. Did not block if. Reduced levels can be caused by any of several mechanisms (eg, Complement fixation / depletion, or opsonization by macrophages). For example, Janeway et al. (1996)Immunobiology, Garland Publishing Inc., New Y See ork, NY.   Chemokines are seven transmembrane receptors linked to specific G proteins Chemotherapeutic agents that classically mediate leukocyte trafficking by binding to It is a subfamily of Itokine. Chemokines are the first two cysteines Divided into four groups based on primary sequence: CXC, CC, C, and newly discovered CX3C family. CXC and C chemokine families are neutrophils and lymphocytes Each has an advantage over the other. CC chemokines, macrophages, lymph It has a preferential effect on spheres and eosinophils. As an eosinophil-specific chemokine For example, RANTES, MCP-2, MCP-3, MCP-4, MIP-1α, and recently described eo Taxin. For example, Alam et al. (1993)J . Immunol 150: 3442-3448; W eber et al. (1995)J . Immunol.154: 4166-4172; Dihlnden et al. (1994)J . Exp. Med . 179: 751-756; Uguccioni et al. (1996)J . Exp. Med.183: 2379-2384; Rot et al. (1 992)J . Exp. Med.176: 1489-1495; and Garcia-Zepeda et al. (1996).J . Immuno l . 157: 5613-5626. II. Preparation of antibody; binding composition   The present invention relates to a CCR3 (preferably a mammal, such as a primate, human, cat, dog, Rat or mouse))). Offer. Antibodies may be in their naturally occurring (full length) form or in their recombinant form. State, polymorphism, allele, strain, or various variants, and Can be raised against various CCR3 proteins, including fragments of In particular, Tasty epitopes include those that are accessible from the extracellular space. Furthermore, anti The body can be in their natural (or active) form or their inactive (eg, It can be raised against the CCR3 protein, in both (denatured) forms. Anti-idi Otype antibodies can also be used.   Many immunogens have antibodies that specifically react with the CCR3 protein (eg, on cells). (Expressed). Recombinant protein is monochrome Preferred immunogens for the production of null or polyclonal antibodies. Proper offer Naturally occurring proteins from sources (eg, primates, rodents, etc.) It can also be used in either pure or impure form. For example, See GenBank and NCBI databases. For example, as described herein Synthetic peptides made using human CCR3 protein sequences also It can be used as an immunogen to produce antibodies against protein. Recombinant tamper Quality can be determined, for example, by Coligan et al. (Ed. 1995, and regular replacement)Current Protocols in Protein Science  John Wiley & Sons, New York, NY; and Ausubel et al. ( 1987, and regular supplements)Current Protocols in Molecular Biology, Green ne / Eukaryotic cells or prokaryotic cells as described in Wiley, New York, NY It can be expressed in cells and purified. Naturally folded material or metamorphosis The substances obtained can be used to raise antibodies, if appropriate. Monoc Either lonal or polyclonal antibodies, for example, measure proteins. For subsequent use in immunoassays to determine Can be generated for the law.   Methods for producing polyclonal antibodies are well known to those skilled in the art. Typically, The epidermis (preferably purified protein) is mixed with an adjuvant, and Animals are immunized with the mixture. The immune response of the animal to the immunogen preparation is tested Perform blood sampling and determine titer of reactivity to the desired CCR3 protein Monitored by For example, high titers of appropriate antibodies to immunogens can be obtained. Blood is collected from the animal, usually after repeated immunizations, and Serum is prepared. Enhance antibody reactivity to proteins, if desired Further fractionation of the antisera can be performed to achieve this. For example, Harlow and Lane; See Or Coligan. Isoform class, or specific desired properties (eg, , Which do not block ligand binding) can be selected. Exemption Plasmids can also be performed by other methods, such as, for example, DNA vector immunization. For example, Wang et al. (1997)Virology 228: 278-284.   Monoclonal antibodies can be obtained by various techniques well known to those skilled in the art. obtain. Typically, spleen cells from an animal immunized with the desired antigen are generally Immortalized by fusion with myeloma cells. Kohler and Misstein (1976)E ur . J. Immunol. See 6: 511-519. Another way to immortalize is Epsta Transformation with an invar virus, oncogene, or retrovirus, or Other methods known in the art are included. See, for example, Doyle et al. Periodical addendum)Cell and Tissue Culture : Laboratory Procedures, John Wiley a See nd Sons, New York, NY. Rolls arising from a single immortalized cell Knee is screened for the production of antibodies of specificity and affinity for the desired antigen. Monoclonal antibodies that are leaned and produced by such cells Yield can be enhanced by a variety of techniques, including injection into the peritoneal cavity of a vertebrate host. You. Alternatively, for example, Huse et al. (1989)Science Outlined by 246: 1275-1281 According to a general protocol, a DNA library derived from human B cells is By screening, the monoclonal antibody or its binding fragment The DNA sequence encoding the strain can be isolated.   Binding fragments and fragments for predetermined fragments of the CCR3 protein Antibodies or binding compositions, including single chain versions and Can be triggered by immunizing animals with conjugates of proteins and fragments . Monoclonal antibodies are prepared from cells secreting the desired antibody. these Antibodies are screened for binding to normal or defective CCR3 protein Or can be screened for eosinophil depletion activity. These things Clonal antibodies will usually have at least about 1 mM, more usually at least about 300 mM. μM, Typically at least about 10 μM, more typically at least about 30 μM, preferably Is at least about 10 μM, and more preferably at least about 3 μM or Good K_DTo join.   In some examples, various mammalian hosts (eg, mice, rodents, spirits) It is desirable to prepare monoclonal antibodies (mAbs) from It is. Descriptions of techniques for preparing such monoclonal antibodies include, for example, Stites et al. (Ed.)Basic and Clinical Immunology(4th edition) Lange Medical Publi cations, Los Altos, CA, and references cited herein; Harlow And Lane (1988)Antibodies : A Laboratory Manual CSH Press; Goding (198 6)Monoclonal Antibodies ： Principles and Practice(2nd edition) Academic Pres s, New York, NY; and, in particular, one method for producing monoclonal antibodies. Discussing, Kohler and Milstein (1975)Nature 256: found in 495-497 obtain. Summarized briefly, the method involves injecting an animal with an immunogen. The animal is then sacrificed and cells are harvested from its spleen. Then this Is fused with myeloma cells. The result is a hybrid that can be regenerated in vitro. Cells or "hybridomas". Then the hybridoma population Are screened to isolate individual clones. Each of these Secretes a single antibody species against the immunogen. In this manner, the individual Antibody species are generated by the immune system generated in response to specific sites recognized on immunogenic substrates. Product of a single, immortalized and cloned B cell You.   Other suitable techniques include libraries of antibodies in phage or similar vectors -Selection. For example, Huse et al. (1989) "Generation of a Large Comb" inatorial Library of the Immunoglobulin Repertoire in Phage Lambda ''Scie nce  246: 1275-1281; and Ward et al. (1989).Nature 341: See 544-546 . The polypeptides and antibodies of the present invention may be used with or without modification And include chimeric or humanized antibodies. Frequently, polypeptides and And antibodies provide detectable signals, either covalently or non-covalently Labeled by linking to the substance. A wide variety of labels and conjugation technologies are publicly available Knowledgeable and reported in detail in both scientific and patent literature Sa Have been. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors , A fluorescent moiety, a chemiluminescent moiety, magnetic particles and the like. Use of such signs U.S. Pat. Nos. 3,817,837; 3,850,752; Nos. 939,350; 3,996,345; 4,277,437; 4,275,149; and No. 4,366,241. Also, whether recombinant immunoglobulin can be produced (C abilly, US Patent No. 4,816,567; and Queen et al. (1989).Proc . Nat'l Acad. Sc i. USA  86: 10029-10033); or in transgenic mice. (Mendez et al. (1997)Nature Genetics 15: 146-156) .   Antibodies are only one form of a specific binding composition. Often similar uses Other binding compositions having, for example, a binding partner-binding partner format , Antibody-antigen interactions, or natural physiologically relevant proteins-proteins Interaction with the CCR3 receptor, either covalently or non-covalently. Molecules that bind with isomerism (eg, specifically associate with CCR3 receptor protein) Protein). The molecule can be a polymer or a chemical reagent . The functional analog may be a protein with a structural modification, or Structurally unrelated molecules (e.g., which interact with appropriate binding determinants Having a molecular shape). Antibody binding compounds (binding fragments) of the invention ) May have important diagnostic or therapeutic value. These are neutralizing Can be useful as a binding compound, and can be bound to a toxin or radionuclide. As a result, when the binding compound binds to the antigen, it expresses it (eg, Cells are killed). In addition, these binding compounds may be drugs or other The therapeutic agent can be attached either directly or indirectly by a linker, and Can result in drug targeting. III. Immunoassay   An immunoassay is for diagnosing a disease or disorder associated with eosinophilia. Or monitoring the level of eosinophil depletion in treated patients. And useful. Specific proteins are measured by various immunoassay methods Can be done. In general, for an overview of immunological and immunoassay procedures, see If Stites and Terr (1991)Basic and Clinical Immunology(7th edition) That. Furthermore, the immunoassay of the present invention can be carried out, for example, by using Maggio (1980)Enzyme Immunoassay  CRC Press, Boca Raton, Florida; Tijan (1985) "Practice and  Theory of Enzyme Immunoassays ''Laboratory Techniques in Biochemistry an d Molecular Biology , Elsevier Science Publishers B.V., Amsterdam; Harlow and LaneAntibodies , A Laboratory ManualIn detail It can take place in many of the forms just outlined. Chan (1987)Immunoassay : A Practical Guide  Academic Press, Orlando, FL; Price and Newman (1991) )Principles and Practice of Immunoassays Stockton Press, NY; and Ng o (1988 edition)Nonisotopic Immunoassays See also Plenum Press, NY.   Immunoassays for the measurement of CCR3 proteins or peptides are publicly available to those skilled in the art. It can be done by various methods known. Briefly, a probe for measuring protein Muno assays can be either competitive or non-competitive binding assays. Competition In a combined assay, the sample to be analyzed is the capture assay bound to the solid surface. Compete with labeled analyte for specific binding sites on the drug. Preferably, capture Capture reagents are antibodies that specifically react with the CCR3 protein produced as described above. You. The concentration of labeled analyte bound to the capture reagent is determined by the amount of free analyte present in the sample. Is inversely proportional to the amount of analyte.   In a competitive binding immunoassay, the CCR3 protein present in the sample Binding to a specific binding reagent (eg, an antibody that is specifically reactive with the CCR3 protein) Compete with the labeled protein for binding. The binding reagent is the unbound labeled protein Attached to solid surface to achieve separation of bound labeled protein from protein Can be done. Alternatively, competitive binding assays can be performed in the liquid phase, and Techniques known in the art can be used to bind labeled proteins from unbound labeled proteins. It can be used to separate substrates. After separation, the amount of labeled protein bound is determined. Is determined. The amount of protein present in the sample is determined by the amount of Is inversely proportional to   Alternatively, a homogeneous immunoassay in which a separation step is not required can be performed. This In these immunoassays, the label on the protein is transferred to its specific binding reagent. Is changed by the binding of the protein. This change in the labeled protein A decrease or increase in the signal emitted by the label results in a Measurement of the label at the end of the munoassay allows detection or quantification of the protein It works.   CCR3 protein is also determined by various noncompetitive immunoassay methods. obtain. For example, a two-site solid phase sandwich immunoassay is used. Can be In this type of assay, binding reagents for proteins (eg, Antibody) is bound to a solid support. A second protein binding reagent (again, Can be an antibody and bind to the protein at different sites). Tan Unbound label binding reagent depleted after binding occurs at both sites on the protein And the amount of labeled binding reagent bound to the solid phase is measured. Bound label binding The amount of reagent is directly proportional to the amount of protein in the sample.   Western blot analysis determines the presence of CCR3 protein in the sample. Can be used for Tissues where electrophoresis is suspected of containing proteins, for example Performed on samples. Electrophoresis to separate proteins, and appropriate After transfer of protein to a stable solid support (eg, nitrocellulose felter) , The solid support is incubated with an antibody that is reactive to the protein . This antibody can be labeled or can be linked to a secondary labeled antibody that binds to the primary antibody. Can be detected by incubation.   The immunoassay formats described above may use labeled assay components. The sign is Directly or indirectly to the desired components of the assay according to methods well known in the art. Can be combined. A wide variety of labels and methods can be used. Traditionally,^ThreeH,¹²⁵ I,³⁵S,¹⁴C, or³²A radioactive label incorporating P was used. Nonradiative activity As a sex label, labeled antibodies, fluorophores, chemiluminescent reagents, enzymes, and antibodies ( This can act as a specific binding pair member for the labeled ligand). Substances that match. The choice of label depends on the sensitivity required and the binding to the compound Ease of use, stability requirements, and available equipment. Various that can be used For a review of labeling or signal production systems, see US Pat. No. 4,391,904. checking ...   Antibodies that react with a particular protein are also measured by various immunoassays. Can be determined. Thus, modifications of the above procedure may result in the modification of various CCR3 antibodies or antibody preparations. It can be used to determine the amount or affinity. Antibodies by immunoassay technology For an overview of immunological and immunoassay procedures applicable to the measurement of For example, Stites and Terr (ed.)Basic and Clinical Immunology(7th edition) before Out; Maggio (ed.)Enzyme ImmunoassaySupra; and Harlow and LaneAntib odies , A Laboratory Manual See above.   Screening to assess binding and activity of mAbs and binding compositions involves: Including various methods. Binding is detectably labeled with an antibody or binding composition as described above. Can be assayed by knowledge. Cells that express the CCR3 receptor Incubated with the antibody or binding composition, and binding is Assayed by Precipitation Separation Equipment (FACS) analysis.   For assessing eosinophil depletion activity, laboratory animals (eg, mice) are preferred. Is induced, for example, by eosinophilia by infection with parasites. Eosinophil total Numbers are given before and at various time points after administration of large amounts of candidate mAb or binding composition. Done in Levels can be measured for various samples (eg, blood, serum, nasal or lung). Lavage), or in tissue biopsy staining. Successful mAbs or binding pairs Adults significantly reduce the level of circulating eosinophils.   Antibody evaluations are performed in other animals (eg, humans) using various methods. obtain. For example, a patient whose blood sample is suffering from an eosinophil-related disease or disorder From before and after treatment with the candidate mAb, and eosinophil counts were performed. It is. Antibodies can be expressed in eosinophils in in vitro cultures, e.g., by FACS, tissue staining. It can be used in a diagnostic setting to assess the extent of polycythemia. IV. Use   The present invention relates to diseases associated with eosinophilia (eg, pulmonary inflammation, dermatitis, etc.) It is useful for the treatment of. For example, Hardy et al. (1968)Ann . Intern. Med.68: 1220-12 See 29. Pulmonary inflammation includes, for example, asthma, chronic pneumonia, allergic rhinitis Allergic bronchopulmonary aspergillosis, hypereosinophilia, or Churg- S Includes Traus's syndrome. For example, Frank et al. (1995)Samter's Immunologic Diseases Fifth Edition, Volume I-II, Little, Brown and Co., Boston, MA; Coffman et al. (19 89)Science 245: 308-310; and Frick et al. (1988)J . Allergy Clin. Immuno l. 82: 199-225. As dermatological diseases, for example, atopic skin Dermatitis. For example, Uehara et al. (1990)Clin Exp . Dermatol.15: 264-26 See 6. The present invention relates to a method for treating eosinophilia, alone or separately. For example, interleukin-5 (IL-5) antagonists); Paired with other compounds used (eg, steroids such as glucocorticoids) It can be administered in combination.   Pharmaceutical or sterile compositions (including CCR3 antibodies or binding compositions thereof) To prepare, the antibody or binding composition is preferably pharmaceutically acceptable, which is inert. It is mixed with an acceptable carrier or excipient. The preparation of such a pharmaceutical composition is Are known in the art. For example,Remington's Pharmaceutical SciencesAnd AndUS . Pharmacopeia: National Formulary, Mack Publishing Company, Easton , PA (1984).   The antibody or binding composition is usually administered parenterally, preferably Or intravenously. Such protein or peptide antagonists Since these are immunogenic, they are preferably slowly administered over a conventional IV administration set. Or subcutaneous depot formulations (see, for example, Tomasi et al., US Pat. No. 4,732,863). (As taught thereby).   When administered parenterally, the antibody or fragment is pharmaceutically acceptable In injectable unit dose form (solution, suspension, emulsion) associated with a parenteral vehicle Prescribed. Such vehicles are preferably essentially non-toxic and And non-therapeutic. The antagonist may be water, saline, or various additives and And / or water, such as a buffered vehicle with or without diluent It can be administered in a soluble vehicle. Alternatively, a suspension such as a zinc suspension It can be prepared to include the tide. Such suspensions can be subcutaneously (SQ) or intramuscular (IM ) May be useful for injections. The ratio of antagonist to excipient may vary widely depending on where both are present. It can be varied over a range, or the combination is within an effective amount. antibody Is preferably purified substantially free of aggregates, other proteins, endotoxins, etc. For example, at a concentration of about 5 to 30 mg / ml, preferably 10 to 20 mg / ml. Is done. Preferably, the level of endotoxin is less than 2.5 EU / ml. For example, Avis et al. (Eds.) (1993)Pharmaceutical Dosage Forms ： Parenteral Medications2nd edition , Dekker, NY; Lieberman et al. (Eds.) (1990)Pharmaceutical Dosage Forms :Tabl ets, 2nd edition, Dekker, NY; Lieberman et al. (eds.) (1990); PharmaceuticalDosag e Forms : Disperse Systems  Dekker, NY; Fodor et al. (1991)Science 251 ： 767-77 3, Coligan (ed.)Current Protocols in ImmunologyHood et al.Immunology Benj amin / Cummings; Paul (ed.)Fundamental ImmunologyAcademic 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 Crystallogra phy See Academic Press, New York.   Antagonist selection and administration regimen will depend on the serum or tissue Metabolic rate, eosinophilia levels, antagonist immunogenicity, target eosinophil contact Several factors, including near capacity (eg, when serum-free eosinophils are blocked) Dependent. Preferably, the dosing regimen is consistent with the level of acceptable side effects. Maximize the amount of antagonist delivered to the individual. Therefore, the antago delivered The amount of nyst depends in part on the particular antagonist and the severity of the condition being treated I do. Guidance in choosing an appropriate dose provides guidance on the therapeutic use of the antibody. Can be found in the literature (eg, Bach et al., Chapter 22, Ferrone et al. (1985))Handbook of Monoclonal Antibodies  Noges Publications, Park Ridge, NJ; and Russe ll, pages 303-357, and Smith et al., 365-389, Haber et al. (1977).Antibodies in Human Diagnosis and Therapy Raven Press, New York, NY).   The determination of the appropriate dosage can be determined by the physician, e.g., influencing treatment. Parameters or factors known to the subject or expected to affect treatment This is done using the child. In general, the dose should be somewhat less than the optimal dose. Yes, and this is then compared to any negative side effects Or it may be increased by small increments until the optimal effect is achieved. Circulate Eosinophil levels can be measured by measures of inflammation symptoms (eg, inflammatory cytokines produced). Level) as well as important indicators when an effective dose is reached. Good Preferably, the CCR3 antibody or binding composition used is targeted for treatment. From the same species as the animal to be treated, thereby minimizing humoral responses to reagents. To   For antibodies or fragments thereof, which specifically bind to CCR3 The total weekly dose range is generally from about 10 μg / kg body weight to about 10 μg / kg. More generally from about 100 μg, typically from about 500 μg, more typically from about 1000 μg. g, preferably from about 5 mg, and more preferably from about 10 mg. . Generally, the range will be less than 100 mg per kilogram of body weight, preferably , Less than about 50 mg, and more preferably less than about 25 mg.   Antagonists of IL-5 activity (eg, antibodies, binding fragments, or soluble Weekly dose range from about 1 μg / kg body weight, preferably Or at least about 5 μg, and more preferably at least about 10 μg. You. Generally, the range will be less than about 1000 μg / kg body weight, preferably Less than about 500 μg, and more preferably less than about 100 μg. The dose depends on the desired treatment. Schedule that affects the location and can be over a shorter or longer period based on. In general, the range should be at least about 10 μg / kg body weight. To about 50 mg, preferably from about 100 μg to about 10 mg.   Other antagonists of IL-5 activity (eg, muteins) are also contemplated -5 mutein dose range per hour should be at least about 100 μg or more per hour, Generally at least about 500 μg, typically at least about 1 mg, and preferably Is in the range of at least 3 mg. Generally, the dose is about 100 mg per hour Less than about 30 mg, preferably less than about 10 mg, and And more preferably less than about 6 mg. The general range is at least per hour From about 1 μg to about 1000 μg, preferably from about 10 μg to about 500 μg.   The invention also relates to the use of CCR3 antibodies or binding compositions in known therapeutic agents (eg, (In particular, glucocorticoids). This It alleviates the symptoms associated with eosinophilia. One day of glucocorticoid Dosage is at least about 1 mg or more per day, generally at least about 2 mg. , And preferably in the range of at least about 5 mg. Generally, the dose is 1 day Less than about 100 mg per serving, typically less than about 50 mg, preferably less than about 20 mg, and more. More preferably, less than about 10 mg. Generally, the range is at least about From 1 mg to about 100 mg, preferably from about 2 mg to 50 mg.   The phrase “effective amount” is sufficient to ameliorate the symptoms or signs of an inflammatory condition. means. Representative mammalian hosts include mice, rats, cats, dogs, and Primates including humans are included. The effective amount for a particular patient will depend on the condition being treated. Condition, overall health of the patient, mode of administration, route and dose, and side effects May vary depending on factors such as the severity of the disease. When combined, the effective amount is , The ratio to the combination of components, and the effect is limited to the individual components only Absent.   An effective amount of the antagonist is typically at least about 10%; About 20%; preferably at least about 30%; or more preferably at least about 50 % Decrease symptoms. The invention is not described elsewhere herein (e.g., eosinophilia). In general descriptions for treating disorders associated with Reagents found for use in therapeutic applications are provided. For example, Berkow (ed.)The Me rck Manual of Diagnosis and Therapy , Merck & Co., Rahway, N.J .; Thorn et al. ,Harrison's Principles of Internal Medicine, McGraw-Hill, NY; Gilman et al. (1990 edition)Goodman and Gilman's : The Pharmacological Bases of Therapeutic s , Eighth Edition, Pergamon Press;Remington's Pharmaceutical Sciences, 17th edition ( 1990), Mack Publishing Co., Easton, Penn; Langer (1990)Science 249: 15 27-1533; andMerck IndexSee, Merck & Co., Rahway, New Jersey .   Antibodies to the CCR3 protein can be used to express cell populations expressing the CCR3 protein (eg, , Eosinophils) can be used for identification or classification. Classify such populations Methods for doing so are well known in the art. For example, Melamed et al. (1990)Flow Cytometr y and Sorting  Wiley-Liss, Inc., New York, NY; Shapiro (1988)Practical F low Cytometry  Liss, New York, NY; and Robinson et al. (1993).Handbook of Fl ow Cytometry Methods  See Wiley-Liss, New York, NY. CCR3 receptor ー A population of cells that express is also described, for example, in Bieva et al. (1989).Exp . Hematol.17: 914 -920; Hernebtub et al. (1990)Bioconj . Chem.1: 411-418; Vaccaro et al. (1990)Am . Biotechnol. Lab. Using magnetic beads as described in April 1990 It can be purified.   Using the assay method described above, the antibody or binding composition is It is useful in diagnosing disease states that result in elevated eosinophil counts. Labeled antibody Can also be used in analyzing eosinophil infiltration in tissues. Each CCR3 Antibodies raised against the protein also raise anti-idiotype antibodies. Useful for These are the various immunological conditions associated with the expression of each antigen. It is useful in detecting or diagnosing a condition.   The broad scope of the present invention is best understood by reference to the following examples, which are particularly illustrative. It is not intended that the present invention be limited to the embodiments described.                                  Example I. General method   Some standard methods of molecular biology are described, for example, in Maniatis et al. (1982)Molecu lar Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Sp ring Harbor Press; Sambrook et al. (1989)Molecular Cloning : A Laboratory Man ual Ausubel et al., (Second Edition) Volumes 1-3, CSH Press, NY;Biology, Greene Pub lishing Associates, Brooklyn, NY; Ausubel et al. (1987 and supplementary)Current Pro tocols in Molecular Biology , Greene / Wiley, New York; or Innis et al. (1990 Ed.)PCR Protocols : A Guide to Methods and Applications Academic Press, N .Y. Or referenced. A method for protein purification Ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, Methods such as crystallization are exemplified. See, for example, Ausubel et al. (1987 and periodic Duetscher (1990) "Guide to Protein Purification"in Methods i n Enzymology , Volume 182, and other volumes in this series; Manufacturer's literature on use (eg, Pharmacia, Piscataway, N.J. (Bio-Rad, Richmond, CA); and Coligan et al. (1995 and regular supplements)Curre nt Protocols in Protein Science , John Wiley & Sons, New York, NY thing. Depending on the combination of recombinant techniques, the appropriate segment (eg FLAG sequence, Or equivalents that can be fused via a protease-removable sequence) It is. For example, Hochuli (1989)Chemische Industrie 12: 69-70; Hochuli (1 990) "Purification of Recombinant Proteins with Metal Chelate Absorbent "Setlow (ed.)Genetic Engineering , Principle and Methods 12: 87-98, Plen um Press, N.Y .; and Crowe et al. (1992)QIAexpress : The High Level Expressi on & Protein Purification System  See QIAGEN, Inc., Chatsworth, CA When.   FACS analysis was performed by Melamed et al. (1990).Flow Cytometry and Sorting Wiley-Liss, In c., New York, NY; Shapiro (1988)Practical Flow Cytometry Liss, New York , NY; and Robinson et al. (1993).Handbook of Flow Cytometry Methods Wiley-L iss, New York, NY. 11. Production of cell lines expressing CCR3   Mammalian cells (eg, NIH3T3) can be electroporated or Transfected with kutamine (Gibco BRL, Gaithersburg, MD) For 2 weeks in medium supplemented with neomycin. 96-well resistant colonies Separated by FACS in plates and grown. Is RNA an individual clone? Isolated using RNAzol (Friendswood, TX) and RTPCR (eg, Ausube l et al. (1987 and supplementary)Current Protocols in Molecular Biology, Greene / Wile y, New York; Innis et al. (eds.) (1990)PCR Protocols : A Gulde to Methods and Applications  Academic Press, N.Y.), followed by treatment with DNase And analyzed.   Positive clones are described, for example, in Kelner et al. (1994).Science 266: 1395-1399 Ca as described⁺⁺Eota as a ligand for CCR3 in flow assays Subjected to further analysis using the toxin. Highest Ca⁺⁺Clones that show flow, Expanded for use in generating mAbs.   Other methods for assessing expression (eg, staining and FACS analysis, tissue staining, Zan analysis) can also be used. III. Monoclonal antibody   Lewis rats are dosed 10 times every 2 weeks for 8 weeks⁸Transfected with Y3 / CCR3 Cells were immunized intraperitoneally. Last immunization given intravenously (IV) through tail vein Was. Four days after the IV injection, the spleen was removed and fused with SP2 / 0 and NS1 cells . HAT-resistant hybridomas were purchased from Stem Cell Technologies (Vancouver). , BC) using the designed protocol. 10 days after HAT selection The resistant foci were transferred to 96-well plates and expanded for 3 days. Antigen containing supernatant Bodies were separated by FACS for binding to NIH3T3 / CCR3 transfectants. Was analyzed. 29 different CCR3 mAbs were produced.   In addition, some mAbs were sensitized with Aspergillus fumagatus in RAG-/-mice. Was used to classify CCR3-positive cells in the spleen from E. coli. Total of sorted cells Appeared to be eosinophils as determined by Wright Giemsa staining . IV. Depletion of circulating eosinophils   All mAbs were purchased from Zymed, Inc. (So. San Francisco, CA) based on the kit , And ICN (Aurora, OH) Ouchterlony-based kit for ELISA And isotyped as indicated. mAbs are derived from IgG2A, IgG2B, and IgG2C. It had a so-type. Two IgG2B mAbs (designated 6S2-19-4 and 6SH2-88) and Eosinophil depletion assays were performed using an IgG2A mAb (designated 6SH2-59). This None of these antibodies were able to block eotaxin binding to CCR3 . Balb / C mice were treated with 500 Nippostrongylus brassii to induce eosinophil pneumonia. It was injected subcutaneously with larvae of siliensis. 9 to 14 days after injection, 0.5 mg of 6S2-19-4 or Any additional dose of 6SH2-59 was administered IP. Eosinophil counts were collected from the tail vein The blood was analyzed before and after mAb administration by staining. 6SH2-59 is circulating Eosinophils were not significantly depleted, whereas 6S2-19-4 and 6SH2-88 Cells were significantly depleted. Interestingly, circulating eosinophil levels are low. It remained low for at least seven days. 6S2-19-4 was launched on April 30, 1997 Lican Type Culture Collection (ATCC; 12301 Parklawn Drive, Rockvill e, MD) with ATCC number HB12351. 6SH2-88 is , ATCC number so Deposited.   Analysis of human antibodies can be assessed in a similar manner. Biological samples (eg, Blood, tissue biopsy, lung or mucosal lavage, skin perforation) Obtained from an individual suffering from a disease or disorder associated with Eosinophil counting previously Perform as described in. MAbs that specifically bind to the human CCR3 receptor Can be used alone or with another compound (eg, an IL-5 antagonist). Or steroids). Effective treatment, remission of symptoms (Eg, alleviation of lung or skin inflammation) or at different times It is determined by measuring the eosinophil count in the biological sample.   All situations herein are based on each individual publication or publication incorporated by reference. Patent applications are described herein to the same extent, as if set forth in detail and individually. It has been incorporated as a reference.   As will be apparent to those skilled in the art, many modifications and variations of the present invention include: It can be done without departing from its spirit and scope. As described herein Certain embodiments are provided for illustrative purposes only, and the present invention Along the complete scope equal to or including such claims, the following claims And the present invention is provided herein by way of illustration. It is not limited by the particular embodiment shown.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 45/00 A61P 29/00 A61P 17/00 37/00 29/00 C07K 16/28 37/00 C12P 21 / 08 C07K 16/28 C12Q 1/02 C12P 21/08 G01N 33/53 K C12Q 1/02 C12N 15/00 A G01N 33/53 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY) , DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML) , MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) RU, TJ, TM), AL, AM, AU, AZ, BA, BB, BG, BR, BY, CA, CN, CZ, EE, GE, GW, HU, ID, IL, IS, JP, KG, KR, KZ, LC, LK, LR, LT, LV, MD, MG, MK, MN, MX, NO, NZ, PL, RO, RU, SG, SI, SK, SL, TJ, TM, TR, TT , UA, UZ, VN, YU (72) Inventor Coffman, Robert El. United States 94028, Port La Berry, Echo Lane 239

Claims (1)

[Claims] 1. An antibody or binding composition, which comprises:   a) specifically binds to the CCR3 receptor;   b) reduce the level of eosinophils in the sample; and   c) Does not block binding of the ligand to the receptor. 2. The antibody according to claim 1, wherein said antibody is a monoclonal antibody. 3. An antibody or binding composition according to claim 1, and a pharmaceutically acceptable carrier.     A sterile composition comprising: 4. A method of ameliorating a disease or disorder associated with eosinophils in an individual, the method comprising:     Administering an effective amount of the antibody or binding composition of claim 1.     Method. 5. Wherein the antibody or binding composition is administered in an effective combination with:     Method according to claim 4:     a) antagonists to interleukin-5 (IL-5); or     b) steroids. 6. 5. The method according to claim 4, wherein said antibody is a monoclonal antibody. 7. 5. The method of claim 4, wherein the effective amount of the CCR3 antibody or binding composition is:     the method of:     a) at least 500 μg / kg body weight per week;     b) at least 1000 μg / kg body weight per week;     c) at least 5 mg / kg body weight per week;     d) at least 10 mg / kg body weight per week;     e) less than 100 mg / kg body weight per week;     f) less than 50 mg / kg body weight per week; or     g) less than 25 mg / kg body weight per week. 8. 5. The method of claim 4, wherein said remission is accompanied by a decrease in eosinophil levels. 9. 6. The method of claim 5, wherein the antagonist is:     a) an intact monoclonal antibody;     b) a binding fragment of the monoclonal antibody;     c) a soluble receptor for IL-5; or     d) IL-5 mutein. 10. 10. The method of claim 9, wherein said antagonist is said compound.     Contact monoclonal antibody, binding fragment of said monoclonal antibody     Or the soluble receptor, and the amount of the antagonist is     Is the method:     a) at least 1 μg / kg body weight per week;     b) at least 5 μg / kg body weight per week;     c) at least 10 μg / kg body weight per week;     d) less than 1000 μg / kg body weight per week;     e) less than 500 μg / kg body weight per week; or     f) less than 100 μg / kg body weight per week. 11. 10. The method of claim 9, wherein the antagonist is the mutant.     Wherein the amount of the antagonist is:     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. 6. The method of claim 5, wherein the effective amount of the steroid is:     a) at least 1 mg / day;     b) at least 2 mg / day;     c) at least 5 mg / 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. 13. 5. The method of claim 4, wherein the disease associated with eosinophils is:     a) pulmonary inflammation;     b) dermatitis; or     c) Hypereosinophilia. 14． A method for detecting the presence of eosinophils in a sample, comprising:     :     a) contacting the antibody or the binding composition according to claim 1 with the sample;         About; and     b) detecting the binding of the antibody or binding composition to the eosinophils;     Including the method 15. 15. The method of claim 14, wherein the antibody or binding composition is a monoclonal antibody.     The described method. 16. 15. The method of claim 14, wherein the antibody or binding composition is detectably labeled.     the method of. 17． By isolating a population of cells that express the CCR3 receptor from a mixture of cells     The method comprises the following steps:     a) contacting the antibody or binding composition of claim 1 with the mixture.         ;and     b) isolating the population of cells;     A method comprising: 18. 18. The method according to claim 17, wherein said antibody is a monoclonal antibody. 19. 18. The method of claim 17, wherein the antibody or binding composition is detectably labeled.     the method of. 20. 20. The method of claim 19, wherein the antibody or binding composition is:     a) labeled with a fluorescent moiety;     b) labeled with a radioactive moiety; or     c) bound to magnetic beads. 21. 18. The method of claim 17, wherein said isolation is performed by:     a) Fluorescent cell analyzer / separator (FACS); or     b) Magnetic cell separation.