Methods of reducing eosinophil levels

Abstract

To provide safe and effective treatments for eosinophil mediated diseases and disorders.SOLUTION: The present invention provides a method of reducing the number of eosinophils in a human subject, comprising administration to the subject of an IL-5R binding molecule that comprises (a) a region that specifically binds to the IL-5R and (b) an immunoglobulin Fc region.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for reducing eosinophil levels in a human subject.

Background Art

[0002] Eosinophils are associated with various diseases including allergic diseases, and are thought to play an important role in the development of allergic diseases such as chronic bronchial asthma and atopic dermatitis [Adv. Immunol., 39, 177 (1986), Immunol. Today, 13, 501 (1992)]. In addition to the above diseases, eosinophils are also associated with diseases generally referred to as hypereosinophilic syndrome (HES) such as hypereosinophilia, eosinophilic enterogastritis, eosinophilic leukemia, eosinophilic granuloma, and Kimura disease [Ann. Intern. Med., 97, 78 (1982)].

[0003] Eosinophilic granuloma is an osteolytic and local non-tumorous sudden lesion, and is known to be clearly associated with tissue hypereosinophilia [U.S. Armed Forces Med. J., 2, 1085 (1951)]. According to the registration of bone tumor patients in Japan (1972 - 1984), 379 out of 404 bone tumor patients (93.8%) suffered from eosinophilic granuloma. Early eosinophilic granuloma mainly contains eosinophils and histiocytes, and advanced granuloma contains fibrosis or may progress to pulmonary fibrosis. Therefore, in addition to inflammatory diseases such as allergy, eosinophils may cause various other diseases.

[0004] Members of the cytokine family, interleukin-5 (hereinafter referred to as IL-5), interleukin-3 (hereinafter referred to as IL-3), and granulocyte macrophage colony-stimulating factor (hereinafter referred to as GM-CSF), are involved in the regulation of eosinophil differentiation, proliferation, and activation. Among these cytokines, IL-5 is known to act specifically on eosinophils and specifically induce terminal differentiation [Proc. Natl. Acad. Sci. U.S.A., 85, 2288 (1988)].

[0005] In vitro, IL-3 and / or GM-CSF can activate eosinophils or prolong their survival [J. Clin. Invest., 81, 1986 (1988)]. Furthermore, IL-3 and / or GM-CSF also act mainly on the induction of immature eosinophils from bone marrow stem cells [Blood, 76, 1956 (1990)]. In addition, chemokines such as eotaxin and RANTES (regulated on activation of normal T-cell expressed and secreted) induce the chemotaxis of eosinophils to the site of inflammation [Clin. Exp. Allergy, 26, 1005 (1996)]. Stem cell factor (hereinafter referred to as SCF) is involved in the accumulation of eosinophils in allergic bronchitis. In addition to IL-5, there are many factors that affect eosinophil function.

[0006] Eosinophils are divided into subgroups of normodense eosinophils and low-density eosinophils. Eosinophils have been shown to be activated into low-density eosinophils [Immunology, 47, 531(1982)]. Low-density eosinophils are also referred to as activated eosinophils. It has been reported that in the eosinophils of the peripheral blood of HES patients, in addition to quantitative changes, qualitative changes are occurring [Clin. Exp. Immunol., 24, 423(1976)]. Activated eosinophils have been associated with the severity of HES symptoms [Am. J. Cardiol., 52, 321(1983)]. In addition to HES patients, activated eosinophils have also been found in peripheral blood and bronchoalveolar lavage fluid (BALF) of patients with bronchial asthma [Am. Rev. Respir. Dis, 132, 981(1985)]. Various receptors, such as those for cytokines, are expressed in activated eosinophils (low-density eosinophils) [J. Immunol., 142, 4416(1989)]. Compared with normodense eosinophils, these low-density eosinophils show high sensitivity to IL-5 [Clin. Exp. Immunol., 85, 312(1991);J. Exp. Med., 172, 1347(1990)].

[0007] The above-mentioned activated eosinophils are also known to survive in vitro without cytokines that induce eosinophil differentiation and proliferation [J. Exp. Med., 170, 343(1989)]. Thus, the characteristics of activated eosinophils are similar to those of eosinophils infiltrating tissues such as the alveoli [Int. Arch. Allergy Immunol., 120, 91(1999)]. Although the detailed explanation of why activated eosinophils become cytokine-independent remains unknown, however, their degranulation and extended survival seem to be induced by various life-functional molecules other than IL-5.

[0008] Substances having inhibitory activity against cytokines or chemokines involved in eosinophil differentiation or proliferation are considered to be substances that inhibit eosinophil function. However, in many cases, these substances do not act on cytokine-independent eosinophils that are activated and infiltrated into the inflammatory region. Therefore, eosinophil-specific inhibition and induction of cell death of activated eosinophils are necessary to inhibit any eosinophil function. However, to date, there is no anti-inflammatory agent known to induce apoptosis of activated eosinophils.

Prior Art Documents

Non-Patent Documents

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Summary of the Invention

Problems to be Solved by the Invention

[0010] Currently, the treatment of patients with eosinophil-related diseases consists of steroid administration. However, steroid administration often has side effects. In particular, such treatment has several other problems, such as the possibility that the patient's pathological condition may return to its original state when steroid administration is interrupted, and that prolonged steroid administration can induce steroid resistance. Therefore, there is a need for a safe and effective treatment for eosinophil-mediated diseases and disorders.

Means for Solving the Problems

[0011] The present invention provides a method for reducing the number of eosinophils in a human subject, the method comprising administering to the patient an IL-5R binding molecule comprising (a) a region that specifically binds to IL-5R and (b) an immunoglobulin Fc region.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] As described above, without being bound by a particular hypothesis or theory, eosinophils are associated with the etiology of numerous diseases and disorders. Many of these diseases or disorders are characterized by an excess of eosinophils (hypereosinophilia) and are referred to as eosinophilic syndromes.

[0014] Non-limiting examples of diseases and disorders in which eosinophils play a role include asthma, immunoglobulin (IgE)-mediated food allergy, eosinophilic esophagitis (inflammation of the esophagus), inflammatory bowel disease, COPD, allergic colitis, gastroesophageal reflux, eosinophilic gastrointestinal disease (EGID), eosinophilic gastroenteritis, endomyocardial fibrosis, Reiter's endocarditis, Davis disease, idiopathic angioedema associated with hypereosinophilia, eosinophilia-myalgia syndrome / Spanish toxic oil syndrome, cirrhosis, dermatitis herpetiformis, bullous pemphigoid, Churg-Strauss syndrome, acute myeloid eosinophilic leukemia, acute lymphocytic eosinophilic leukemia, systemic mastocytosis with hypereosinophilia, allergic rhinitis, eczema, Wegener's granulomatosis, polyarteritis nodosa, eosinophilic fasciulitis, and rheumatoid arthritis.

[0015] Accordingly, the present invention provides a method for reducing the number of eosinophils in a human subject, the method comprising administering to the patient an IL-5R binding molecule comprising (a) a region that specifically binds to IL-5R and (b) an immunoglobulin Fc region.

[0016] In one embodiment, the present invention provides a method for reducing the number of eosinophils in a human subject, the method comprising administering to the patient an IL-5R binding molecule comprising (a) a region that specifically binds to IL-5R and (b) an immunoglobulin Fc region. In a specific embodiment, the method of the present invention reduces the number of eosinophils in blood, bone marrow, the gastrointestinal tract (e.g., esophagus, stomach, small intestine, and large intestine), or the lung. In another specific embodiment, the method of the present invention reduces the number of blood eosinophils. In a further specific embodiment, the method of the present invention reduces the number of lung eosinophils. In a specific embodiment, the method of the present invention reduces the number of eosinophil progenitor cells.

[0017] In another embodiment, the method of the present invention reduces the number of eosinophils by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99%. In a specific embodiment, the method of the present invention reduces the number of eosinophils below the limit of detection.

[0018] In another embodiment, the method of the present invention reduces the number of eosinophil progenitors by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99%. In a specific embodiment, the method of the present invention reduces the number of eosinophil progenitors below the limit of detection.

[0019] In a further embodiment, the method of the invention eliminates all detectable eosinophils after a single administration of the IL-5R binding molecule. In a specific embodiment, the single administration of the IL-5R binding molecule eliminates all detectable eosinophils for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 20 weeks, or at least about 25 weeks.

[0020] In a further embodiment, the method of the invention eliminates all detectable eosinophil precursors after a single administration of the IL-5R binding molecule. In a specific embodiment, the single administration of the IL-5R binding molecule eliminates all detectable eosinophil precursors for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 20 weeks, or at least about 25 weeks.

[0021] In a specific embodiment, the method of the invention comprises administering to a subject a single dose of 0.03 mg / kg of an IL-5R binding molecule comprising (a) a region that specifically binds to IL-5R and (b) an immunoglobulin Fc region, wherein administration of the IL-5R binding molecule results in at least about 99% reduction of eosinophils from the circulation of the subject, the reduction is complete by 24 hours after dosing, and the reduction persists for at least about 28 days after dosing.

[0022] In a specific embodiment, the method of the present invention comprises administering to a subject a single dose of 0.1 mg / kg of an IL-5R binding molecule comprising (a) a region that specifically binds to IL-5R and (b) an immunoglobulin Fc region, wherein administration of the IL-5R binding molecule results in a reduction of at least about 99% of eosinophils from the circulation of the subject, the reduction is complete by 24 hours after dosing, and the reduction persists for at least about 84 days after dosing.

[0023] In one embodiment, the IL-5R binding molecule of the present invention comprises a fusion protein. In certain embodiments, the fusion protein comprises a polypeptide region that specifically binds to IL-5R and further comprises an immunoglobulin Fc region. Non-limiting examples of polypeptide regions that specifically bind to IL-5R can be found in U.S. Patent Nos. 7,109,299 and 5,677,280, and U.S. Patent Application Publication No. 2006 / 0014680A1. In other embodiments, the polypeptide region that specifically binds to IL-5R is human IL-5 (see, e.g., Tanabi et al., Journal of Biological Chemistry, 1987, Vol. 262, No. 34, pp. 16580-16584), or a fragment, derivative, or variant thereof (see, e.g., U.S. Patent No. 6,465,616).

[0024] In one embodiment, the IL-5R binding molecule of the present invention comprises an antibody. The antibodies of the present invention include, but are not limited to, monoclonal antibodies, synthetic antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFvs) (including bispecific scFvs), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), and epitope-binding fragments of any of the above. In particular, the antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing antigen-binding sites that specifically bind to an antigen. The immunoglobulin molecules of the present invention can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or type of immunoglobulin molecule.

[0025] Antibodies useful in the present invention can be derived from any animal, including birds and mammals (e.g., but not limited to, humans, mice, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses, or chickens). In a specific embodiment, the antibody is a human or humanized monoclonal antibody.

[0026] Antibodies useful in the present invention can be monospecific, bispecific, trispecific or more multispecific. Multispecific antibodies can specifically bind to different epitopes of a polypeptide or can specifically bind to both a polypeptide and a heterologous epitope such as a heterologous polypeptide or a solid support. See, e.g., International Publication Nos. WO93 / 17715, WO92 / 08802, WO91 / 00360, and WO92 / 05793; Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; and Kostelny et al., 1992, J. Immunol. 148:1547-1553.

[0027] The antibodies useful in the present invention can be single-chain antibodies. The design and construction of single-chain antibodies are described in Marasco et al., 1993, Proc Natl Acad Sci 90:7889-7893.

[0028] Non-limiting examples of the antibodies of the present invention can be found in U.S. Patent Nos. 7,179,464, 6,538,111, 6,018,032, and U.S. Patent Application Publication Nos. 2004 / 0136996A1 and 2005 / 0226867A1.

[0029] In one embodiment, the IL-5R binding molecule of the present invention comprises an antibody. In a further embodiment, the IL-5R binding molecule of the present invention is an antibody comprising any one of the amino acid sequences of SEQ ID NOs: 1-4. In a specific embodiment, the IL-5R binding molecule of the present invention is an antibody comprising the amino acid sequences of SEQ ID NOs: 1 and 3. In a specific embodiment, the IL-5R binding molecule of the present invention is an antibody comprising the amino acid sequences of SEQ ID NOs: 2 and 4.

[0030] In one embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to the same epitope as MEDI-563. In a specific embodiment, the antibody is MEDI-563. In a more specific embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to the same epitope as MEDI-563 on the condition that the antibody is not MEDI-563.

[0031] In one embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to an epitope comprising residues 1-102 of SEQ ID NO: 5. In a specific embodiment, the antibody is MEDI-563. In a more specific embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to an epitope comprising residues 1-102 of SEQ ID NO: 5 on the condition that the antibody is not MEDI-563.

[0032] In one embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to an epitope comprising residues 40 to 67 of SEQ ID NO: 5. In a specific embodiment, the antibody is MEDI-563. In a more specific embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to an epitope comprising residues 40 to 67 of SEQ ID NO: 5, provided that the antibody is not MEDI-563.

[0033] In one embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to an epitope comprising residues 52 to 67 of SEQ ID NO: 5. In a specific embodiment, the antibody is MEDI-563. In a more specific embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to an epitope comprising residues 52 to 67 of SEQ ID NO: 5, provided that the antibody is not MEDI-563.

[0034] In one embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to an epitope comprising residue 61 of SEQ ID NO: 5. In a specific embodiment, the antibody is MEDI-563. In a more specific embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to an epitope comprising residue 61 of SEQ ID NO: 5, provided that the antibody is not MEDI-563.

[0035] In one embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to a primary antigen comprising residues 1 to 102 of SEQ ID NO: 5, but does not specifically bind to a secondary antigen comprising a variant of residues 1 to 102 of SEQ ID NO: 5, wherein the variant comprises an I61K substitution. In a specific embodiment, the antibody is MEDI-563. In a more specific embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to a primary antigen comprising residues 1 to 102 of SEQ ID NO: 5, but does not specifically bind to a secondary antigen comprising a variant of residues 1 to 102 of SEQ ID NO: 5, wherein the variant comprises an I61K substitution, provided that the antibody is not MEDI-563.

[0036] In one embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to a primary antigen comprising residues 40 to 67 of SEQ ID NO: 5, but does not specifically bind to a secondary antigen comprising a variant of residues 40 to 67 of SEQ ID NO: 5, and this variant comprises an I61K substitution. In a specific embodiment, the antibody is MEDI-563. In a more specific embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to a primary antigen comprising residues 40 to 67 of SEQ ID NO: 5, but does not specifically bind to a secondary antigen comprising a variant of residues 40 to 67 of SEQ ID NO: 5, and this variant comprises an I61K substitution, provided that the antibody is not MEDI-563.

[0037] In one embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to human IL-5Rα (SEQ ID NO: 5), but does not specifically bind to mutant human IL-5Rα (SEQ ID NO: 5) comprising an I61K substitution. In a specific embodiment, the antibody is MEDI-563. In a more specific embodiment, the IL-5R binding molecule of the present invention is an antibody that specifically binds to human IL-5Rα (SEQ ID NO: 5), but does not specifically bind to mutant human IL-5Rα (SEQ ID NO: 5) comprising an I61K substitution, provided that the antibody is not MEDI-563.

[0038] The present invention provides an IL-5R binding molecule with an increased effector function. Non-limiting examples of methods for increasing effector function can be found in U.S. Patent Nos. 5,624,821, 6,602,684, 7,029,872, U.S. Patent Application Publication Nos. 2006 / 0067930A1, 2005 / 0272128A1, 2005 / 0079605A1, 2005 / 0123546A1, 2004 / 0072290A1, 2006 / 0257399A1, 2004 / 0261148A1, 2007 / 0092521, 2006 / 0040325A1, and 2006 / 0039904A1, as well as International Patent Application Publication Nos. WO04 / 029207, WO03011878, WO05044859, WO06071856, and WO06071280.

[0039] Methods of engineering the Fc region of an antibody to modify effector function are known in the art (e.g., Koenig et al., U.S. Patent Application Publication No. 20040185045 and PCT Publication No. WO2004 / 016750, the entire disclosures of which are incorporated herein by reference, which describe engineering the Fc region to enhance binding affinity for FcγRIIB compared to binding affinity for FcγRIIA; also see PCT Publication No. WO99 / 58572 by Armour et al., PCT Publication No. WO99 / 51642 by Idusogie et al., and U.S. Patent No. 6,395,272 by Deo et al.). Methods of improving the Fc region to reduce binding affinity for FcγRIIB are also known in the art (e.g., Ravetch et al., U.S. Patent Application Publication No. 20010036459 and PCT Publication No. WO01 / 79299, the entire disclosures of which are incorporated herein by reference). Improved antibodies having mutant Fc regions with enhanced binding affinity for FcγRIIIA and / or FcγRIIA compared to the wild-type Fc region have also been described (e.g., PCT Publication No. WO2004 / 063351 by Stavenhagen et al., the entire disclosure of which is incorporated herein by reference).

[0040] Antibody effector functions can also be improved by producing antibodies with modified glycosylation patterns. For example, antibodies with modified glycosylation, such as afucosylated / hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with an increased bisecting GlcNAc structure, can be produced. Such modified glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such sugar modifications can be achieved, for example, by expressing the antibody in a host cell with a modified glycosylation machinery. Cells with modified glycosylation machinery have been reported in the art and can be used as host cells for producing antibodies with modified glycosylation by expressing the recombinant antibodies of the present invention. For example, European Patent No. 1,176,195 by Hanai et al. describes a cell line in which the FUT8 gene encoding fucosyltransferase is functionally disrupted, and antibodies expressed in such a cell line show hypofucosylation. PCT Publication No. WO03 / 035835 by Presta describes a mutant CHO cell line, Lec13 cells, with a reduced ability of fucose to bind to the Asn(297)-linked sugar, and antibodies expressed in the host cells are also hypofucosylated (see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication No. WO99 / 54342 by Umana et al. describes a cell line engineered to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), and antibodies expressed in the engineered cell line show an increased bisecting GlcNAc structure that results in an increase in the ADCC activity of the antibody (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

[0041] Methods for making antibodies with modified glycoforms are known in the art and include, but are not limited to, those described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S. Patent No. 6,602,684; U.S. Patent Application No. 10 / 277,370; U.S. Patent Application No. 10 / 113,929; PCT No. WO00 / 61739A1; PCT No. WO01 / 292246A1; PCT No. WO02 / 311140A1; PCT No. WO02 / 30954A1; Potillegent™ technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland). See, for example, WO00061739; EA 01229125; U.S. Patent Application Publication No. 20030115614; Okazaki et al., 2004, JMB, 336:1239-49. Antibodies with modified fucosylation patterns can also be prepared by post-translational removal of fucose (e.g., with a fucosidase enzyme).

[0042] The present invention provides antibodies and antibody fragments that specifically bind to IL-5R and have an extended in vivo half-life. In particular, the present invention provides antibodies and antibody fragments that have a half-life in a mammal (e.g., but not limited to, a human) that is longer than 3 days, longer than 7 days, longer than 10 days, longer than 15 days, longer than 25 days, longer than 30 days, longer than 35 days, longer than 40 days, longer than 45 days, longer than 2 months, longer than 3 months, longer than 4 months, or longer than 5 months.

[0043] In order to extend the serum circulation of antibodies (e.g., but not limited to, monoclonal antibodies and single-chain antibodies) or antibody fragments (e.g., but not limited to, Fab fragments) in vivo, an inert polymer such as high molecular weight polyethylene glycol (PEG) can be conjugated to the antibody (including its antibody fragments) either through site-specific conjugation of PEG to the N-terminus or C-terminus of the antibody, or via the ε-amino groups present on lysine residues, with or without a multifunctional linker. Linear or branched polymer derivatization that minimizes loss of biological activity will be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by molecular sieve or by ion exchange chromatography. The PEG-derivatized antibody (including its antibody fragments) can be tested for binding activity and in vivo efficacy using methods known to those skilled in the art, such as the immunoassays described herein.

[0044] Antibodies having an increased half-life in vivo can also be generated by introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) into the IgG constant domain, or its FcRn binding fragment (e.g., Fc or hinge Fc domain fragment). See, e.g., International Publication No. WO98 / 23289; International Publication No. WO97 / 34631; and U.S. Patent No. 6,277,375, each of which is hereby incorporated by reference in its entirety.

[0045] Furthermore, the antibody (including its antibody fragments) can be conjugated to albumin to produce an antibody (including its antibody fragments) that is more stable or has a longer half-life in vivo. This technique is well known in the art. See, e.g., International Publication No. WO93 / 15199, WO93 / 15200, and WO01 / 77137; and European Patent No. EP 413,622, each of which is hereby incorporated by reference in its entirety.

[0046] The present invention provides an IL-5R binding molecule that specifically binds to IL-5R, wherein the binding molecule is recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or a fragment of a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids). In particular, the present invention provides a formulation of a fusion protein comprising an antigen-binding fragment of an antibody described herein (e.g., but not limited to, Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, VH domain, VH CDR, VL domain or VL CDR) and a heterologous protein, polypeptide, or peptide. Methods for fusing or conjugating a protein, polypeptide, or peptide to an antibody (including fragments thereof) are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International Publication Nos. WO96 / 04388 and WO91 / 06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341 (the entire disclosures of which are incorporated herein by reference).

[0047] Additional fusion proteins can also be made by techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be utilized to modify the activity of the antibodies or fragments thereof of the present invention (e.g., but not limited to, antibodies or fragments thereof with higher affinity and lower dissociation rate). Generally, see U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313 (each of these patents and publications is hereby incorporated by reference in its entirety). The antibody (including its antibody fragment), or the encoded antibody or its fragment, can be modified by subjecting it to random mutagenesis by mutagenic PCR, random nucleotide insertion or other methods prior to recombination. The polynucleotide encoding the antibody (including its antibody fragment) can also be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

[0048] Furthermore, the antibody (including its antibody fragments) can be fused with a marker sequence such as a peptide to facilitate purification. The marker amino acid sequence can be, inter alia, a hexahistidine peptide such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine enables the facile purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag corresponding to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the "flag" tag.

[0049] In other embodiments, the antibody or fragment thereof of the invention is conjugated to a diagnostic or detectable substance. Such antibodies can be useful for monitoring or predicting the onset, development, progression and / or severity of a disease or disorder (e.g., but not limited to, an autoimmune disorder) as part of clinical trial procedures, such as determining the effectiveness of a particular therapy. Such diagnosis and detection can be accomplished by conjugating the antibody with various detectable substances including, but not limited to, various enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; bridging molecules such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent substances such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent substances such as, but not limited to, luminol; bioluminescent substances such as, but not limited to, luciferase, luciferin, and aequorin; radioactive substances such as, but not limited to, iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Sn; as well as positron-emitting metals used in various positron emission tomography, and detectable substances including, but not limited to, non-radioactive paramagnetic metal ions, by conjugating the antibody with the detectable substance.

[0050] Alternatively, as described in Segal U.S. Patent No. 4,676,980, which is hereby incorporated by reference in its entirety, an antibody can be conjugated with a secondary antibody to form an antibody heteroconjugate.

[0051] The therapeutic moiety or drug conjugated to the antigen of interest (e.g., IL-5R) or a fragment thereof should be selected to achieve the desired prophylactic or therapeutic effect(s) against a particular disease or disorder of a subject, e.g., a disease or disorder associated with or characterized by abnormal expression and / or activity of interferon-α polypeptide, a disease or disorder associated with or characterized by abnormal expression and / or activity of interferon-α receptor or one or more of its subunits, an autoimmune disease, an autoimmune disease, graft rejection, graft-versus-host disease, or one or more of its symptoms. A clinician or other medical professional should consider the nature of the disease, the severity of the disease, and the condition of the subject when determining what to conjugate to the antibody of interest, e.g., an antibody that specifically binds to interferon-α polypeptide or a fragment thereof.

[0052] Antibodies (including fragments thereof) that specifically bind to an antigen can be produced by any method known in the art of antibody synthesis, particularly by chemical synthesis or by recombinant expression techniques (see U.S. Patent Application Publication No. 2007 / 0014724A1).

[0053] Polyclonal antibodies specific for an antigen can be produced by a variety of techniques well known in the art. For example, a human antigen can be administered to a variety of host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the human antigen. A variety of adjuvants can be used to increase the immune response, depending on the host species, and include, but are not limited to, Freund's (complete and incomplete), gel-like minerals such as aluminum hydroxide, surfactants such as lysophosphatidylcholine, pluronic (registered trademark) polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

[0054] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma techniques known in the art and as taught in, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al.,: Monoclonal Antibodies and T Cell Hybridomas 563 681 (Elsevier, N.Y., 1981); and Harlow et al., Using Antibodies: A laboratory Manual, Cold Spring Harbor Laboratory Press (1999) (the foregoing references are incorporated by reference in their entirety). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, regardless of the method by which it is produced.

[0055] Methods for producing and screening specific antibodies using hybridoma technology are commonly used and well known in the art. Briefly, a mouse can be immunized with a non-mouse antigen, and when an immune response is detected, e.g., antibodies specific for the antigen are detected in mouse serum, the mouse spleen is harvested and splenocytes are isolated. The splenocytes are then fused, by well-known techniques, with cells from any suitable myeloma cell line, e.g., the cell line SP20 available from ATCC. The hybridomas are selected and cloned by limiting dilution. Further, the RIMMS (repetitive immunization multiple sites) technique can be used to immunize the animals (Kilpatrack et al., 1997, Hybridoma 16:381-9, which is incorporated herein by reference in its entirety). The hybridoma clones are then assayed, by methods known in the art, for cells that secrete antibodies capable of binding to the polypeptide of the invention. Ascites fluid, generally containing high levels of antibodies, can be produced by immunizing mice with the positive hybridoma clones.

[0056] The present invention provides a method for producing monoclonal antibodies, which comprises culturing hybridoma cells secreting the antibodies of the present invention, wherein the hybridoma cells are prepared by fusing splenocytes isolated from a mouse immunized with a non-mouse antigen with myeloma cells, and then screening the hybridomas generated by the fusion for hybridoma clones that secrete antibodies capable of binding to the antigen, and also provides the antibodies produced by this method.

[0057] Antibody fragments that recognize specific particular epitopes can be produced by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments of the present invention can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). The F(ab')2 fragment contains the variable region, the light chain constant region, and the CH1 domain of the heavy chain. Furthermore, the antibodies of the present invention can also be produced using various phage display methods known in the art.

[0058] In the phage display method, a functional antibody domain is exposed on the surface of phage particles carrying the polynucleotide sequence encoding it. In particular, the DNA sequences encoding the VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of the affected tissue). The DNA encoding the VH and VL domains is recombined by PCR with an scFv linker and cloned into a phagemid vector. The vector is electroporated into Escherichia coli (E. coli), and the E. coli is infected with a helper phage. The phages used in these methods are typically filamentous phages including fd and M13, and the VH and VL domains are usually fused by recombination with either phage gene III or gene VIII. Phages expressing an antigen-binding domain that binds to a specific antigen can be selected or identified using the antigen, e.g., a labeled antigen or an antigen bound or captured on a solid surface or beads.Examples of phage display methods that can be used to generate the antibodies of the present invention are Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; International Application No. PCT / GB91 / O1 134; International Publications WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, 5,969,108, 6,33,187, 5,824,520, and 5,702,892, each of which is incorporated herein by reference in its entirety.

[0059] As described in the above references, after phage selection, the antibody coding regions from the phage are isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragments, and can be expressed, for example, in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria as described below. Techniques for producing recombinant Fab, Fab', and F(ab')2 fragments are also available using methods known in the art, such as those disclosed in PCT Publication No. WO92 / 22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043 (the above references are incorporated by reference in their entirety).

[0060] To generate whole antibodies, the VH or VL nucleotide sequences of the scFv clones can be amplified using PCR primers that include the VH or VL nucleotide sequence, restriction sites, and flanking sequences to protect the restriction sites. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector that expresses a VH constant region, such as the human γ4 constant region, and the PCR-amplified VL domain can be cloned into a vector that expresses a VL constant region, such as the human κ or λ constant region. Vectors for expressing the VH or VL domains can include an EF-1α promoter, a secretion signal, a cloning site for the variable domain, a constant domain, and a selectable marker such as neomycin. The VH and VL domains can also be cloned into one vector that expresses the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-introduced into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line that expresses a full-length antibody, such as, but not limited to, IgG.

[0061] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be appropriate to use humanized or chimeric antibodies. Fully human antibodies and humanized antibodies are particularly desirable for the therapeutic treatment of human subjects. Human antibodies can be produced by various methods known in the art, including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and International Publications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741, each of which is incorporated herein by reference in its entirety.

[0062] Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, in addition to the human heavy and light chain genes, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously by introduction of the human immunoglobulin locus by homologous recombination. In particular, homozygous deletion of the JH region prevents production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous progeny that express human antibodies. Transgenic mice are immunized in the normal way with a selected antigen, e.g., all or part of the polypeptide of the present invention. Monoclonal antibodies against the antigen can be obtained from transgenic mice immunized using conventional hybridoma technology. The human immunoglobulin transgene carried by the transgenic mouse rearranges during B cell differentiation and subsequently undergoes class switching and somatic mutation. Thus, using such techniques, it is possible to produce IgG, IgA, IgM, and IgE antibodies useful for therapy. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65 93). For a detailed discussion of this technology for the production of human antibodies and human monoclonal antibodies and the procedures for the production of such antibodies, see, for example, International Publication Nos. WO98 / 24893, WO96 / 34096, and WO96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are hereby incorporated by reference in their entirety.In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can engage in providing human antibodies against selected antigens using techniques similar to those described above.

[0063] Chimeric antibodies are molecules in which different portions of the antibody are derived from different immunoglobulin molecules. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415, which are hereby incorporated by reference in their entirety.

[0064] A humanized antibody is an antibody, variant thereof, or fragment thereof that can bind to a given antigen and comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and CDRs having substantially the amino acid sequence of a non-human immunoglobulin. A humanized antibody comprises substantially all, or at least one, typically two variable domains (Fab, Fab', F(ab')2, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., the donor antibody) and all or substantially all of the framework regions are of human immunoglobulin consensus sequences. In one embodiment, a humanized antibody also comprises at least a portion of the immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. Ordinarily, an antibody contains both a light chain and at least the variable domain of the heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. A humanized antibody can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and isotypes including IgG1, IgG2, IgG3, and IgG4. Usually, the constant domain is a complement-binding constant domain where it is desirable for the humanized antibody to exhibit cytotoxic activity, and the class is typically IgG1. If such cytotoxic activity is not desired, the constant domain can be of the IgG2 class. A humanized antibody can contain sequences from multiple classes or isotypes, and the selection of specific constant domains to optimize desired effector functions is within the purview of one of ordinary skill in the art. The framework and CDR regions of a humanized antibody need not exactly correspond to the parent sequences. For example, a donor CDR or consensus framework can be mutated by substitution, insertion, or deletion of at least one residue such that the CDR or framework residue at that site does not correspond to either the consensus or the grafted antibody. However, such mutations will not be extensive. Usually, at least 75% of the residues of a humanized antibody correspond to those of the parent framework and CDR sequences, usually 90%, and greater than 95%.Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Patent No. 5,565,332), and, for example, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, WO93 17105, Tan et al., J. Immunol. 169:1119 25(2002), Caldas et al., Protein Eng. 13(5):353-60(2000), Morea et al., Methods 20(3):267 79(2000), Baca et al., J. Biol. Chem. 272(16):10678 84(1997), Roguska et al., Protein Eng. 9(10):895 904(1996), Couto et al., Cancer Res. 55(23 Supp):5973s 5977s(1995), Couto et al., Cancer Res. 55(8):1717 22(1995), Sandhu JS, Gene 150(2):409 10(1994), and Pedersen et al., J. Mol. Biol. 235(3):959 73(1994). Often, framework residues in the framework regions will be substituted with the corresponding residues from the CDR donor antibody in order to modify, preferably improve, antigen binding.These framework replacements are identified by methods well known in the art, e.g., but not limited to, modeling the interaction of CDRs with framework residues to identify framework residues important for antigen binding and sequence comparison to identify abnormal framework residues at specific positions (see, e.g., Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature 332:323, the entireties of each of which are incorporated herein by reference).

[0065] Single domain antibodies, e.g., antibodies lacking a light chain, can be produced by methods well known in the art. See, e.g., Riechmann et al., 1999, J. Immuno. 231:25-38; Nuttall et al., 2000, Curr. Pharm. Biotechnol. 1(3):253-263; Muylderman, 2001, J. Biotechnol. 74(4):277302; U.S. Patent No. 6,005,079; and International Publications WO94 / 04678, WO94 / 25591, and WO01 / 44301, the entireties of each of which are incorporated herein by reference.

[0066] Furthermore, antibodies that specifically bind to an antigen (e.g., IL-5R) can be used, in turn, to make anti-idiotype antibodies that "imitate" the antigen using techniques well known to those of skill in the art (see, e.g., Greenspan & Bona, 1989, FASEB J. 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438).

[0067] The recombinant expression of the antibodies of the present invention (e.g., the heavy or light chain of the antibody of the present invention or a fragment thereof or the single-chain antibody of the present invention) may require the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule, the heavy or light chain of an antibody, or a fragment thereof is obtained, a vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, a method for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody is described herein. Expression vectors containing an antibody coding sequence as well as appropriate transcriptional and translational regulatory signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, the present invention provides a replicable vector containing a nucleotide sequence encoding an antibody molecule of the present invention, the heavy or light chain of an antibody, the variable domain of the heavy or light chain of an antibody (including fragments thereof), or the heavy or light chain CDR, operably linked to a promoter. Such vectors may contain a nucleotide sequence encoding the constant region of the antibody molecule (see, for example, International Publication No. WO86 / 05807; International Publication No. WO89 / 01036; and U.S. Patent No. 5,122,464), and the variable domain of the antibody can be cloned into such vectors for expression of the entire heavy chain, entire light chain, or both the entire heavy and light chains.

[0068] Expression vectors are introduced into host cells by conventional techniques, and then the transfected cells are cultured by conventional techniques to produce the antibodies of the present invention. Thus, the present invention includes host cells containing a polynucleotide encoding an antibody of the present invention or a fragment thereof, or its heavy or light chain, or a fragment thereof, or a single-chain antibody of the present invention, operably linked to a heterologous promoter. In a specific embodiment for the expression of a bispecific antibody, vectors encoding both the heavy and light chains can be co-expressed in a host cell for the expression of the entire immunoglobulin molecule, as detailed below.

[0069] A variety of host expression vector systems can be utilized to express the antibody molecules of the present invention (see, e.g., U.S. Patent No. 5,807,715). Such host expression systems represent agents by which the desired coding sequences can be produced and subsequently purified, but also represent cells that can express the antibody molecules of the present invention in situ when transformed or transfected with the appropriate nucleotide coding sequences. These include microorganisms such as bacteria (e.g., but not limited to, Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., but not limited to, Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell lines infected with recombinant virus expression vectors (e.g., but not limited to, baculovirus) containing the antibody coding sequences; plant cell lines infected with recombinant virus expression vectors (e.g., but not limited to, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., but not limited to, Ti plasmid) containing the antibody coding sequences; or mammalian cell lines (e.g., but not limited to, COS, CHO, BHK, 293, NS0, and 3T3 cells) having recombinant expression constructs containing promoters derived from the genomes of mammalian cells (e.g., but not limited to, the metallothionein promoter) or from mammalian viruses (e.g., but not limited to, the adenovirus late promoter; the vaccinia virus 7.5K promoter), including, but not limited to, these. Bacterial cells such as Escherichia coli, and in particular eukaryotic cells for the expression of fully recombinant antibody molecules, are used for the expression of recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) combined with vectors such as the major intermediate early gene promoter element from human cytomegalovirus are effective expression systems for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio / Technology 8:2).In a specific embodiment, the expression of the nucleotide sequence encoding the antibody, derivative, analog, or fragment thereof of the present invention is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0070] In the bacterial system, depending on the use for the purpose of expressing an antibody molecule, many expression vectors can be advantageously selected. For example, when a large amount of such an antibody is produced for the preparation of a pharmaceutical composition of an antibody molecule, a vector that induces the expression of a high-level fusion protein product that can be easily purified would be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791) in which the antibody coding sequence can be individually ligated into the vector in-frame with the lac Z coding region so that a fusion protein is produced; the pIN vector (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509), etc. The pGEX vector can also be used to express a foreign polypeptide as a fusion protein with glutathione S-transferase (GST). Generally, such a fusion protein is soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST moiety.

[0071] In the insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody-encoding sequences can be individually cloned into non-essential regions of the virus (such as the polyhedrin gene) and placed under the control of an AcNPV promoter (such as the polyhedrin promoter).

[0072] In mammalian host cells, many virus-derived expression systems can be utilized. When an adenovirus is used as an expression vector, the antibody-encoding sequence of interest can be linked to an adenovirus transcription / translation regulatory complex, such as a late promoter and a tripartite leader sequence. This chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the virus genome (such as region E1 or E3) results in a recombinant virus that can survive in infected hosts and express antibody molecules (see, for example, Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals may also be required for efficient translation of the inserted antibody-encoding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in-frame with the desired coding sequence to ensure translation of the entire inserted fragment. These exogenous translation regulatory signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see, for example, Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0073] In addition, host cell lines can be selected that regulate the expression of the inserted array or modify and process the gene product in a desired specific manner. Such modifications (e.g., but not limited to, glycosylation) and processing (e.g., but not limited to, cleavage) of the protein product can be important for the function of the protein. Different host cells have unique and specific mechanisms for the post-translational processing and maturation of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein. To achieve this goal, eukaryotic host cells having cellular mechanisms for the proper processing, glycosylation, and phosphorylation of the primary transcript of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O as well as HsS78Bst cells.

[0074] Stable expression can be used for the long-term, high-yield production of recombinant proteins. For example, cell lines that stably express antibody molecules can be engineered. Instead of using an expression vector containing a viral origin of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. Following the introduction of foreign DNA, the engineered cells can be cultured in rich medium for 1-2 days and then switched to selective medium. The selectable marker within the recombinant plasmid confers resistance to the selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci (which can be cloned and expanded into cell lines). This method can be advantageously used to engineer cell lines that express antibody molecules. Such engineered cell lines can be particularly useful in the screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

[0075] In one embodiment, the cell line used to express the IL-5R binding molecule is a cell that does not fucosylate the Fc region of the IL-5R binding molecule. Non-limiting examples of these types of cells are found in U.S. Patent No. 6,946,292, as well as U.S. Patent Application Publication Nos. 2006 / 0078991A1, 2004 / 0110282A1, 2006 / 0024800A1, 2005 / 0216958A1, 2004 / 0132140, and 2004 / 0259150. In a specific embodiment, the IL-5R binding molecule is a humanized, non-fucosylated IgG1 anti-IL-5Rα chain monoclonal antibody. In a more specific embodiment, the antibody is MEDI-563 (also known as BIW-8405). In a more specific embodiment, the antibody is not MEDI-563.

[0076] A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes, which can be utilized in tk-, hgprt-, or aprt- cells respectively. Additionally, metabolic antagonist resistance can be used based on selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O 'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt that confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo that confers resistance to aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217: May, 1993, TIB TECH 11(5):155-215); and hygro that confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology can be generally applied to the selection of desired recombinant clones, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13 of Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, the entireties of which are hereby incorporated by reference.

[0077] The expression level of the antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When the marker of the vector system expressing the antibody is amplifiable, an increase in the level of the inhibitor present during the culture of the host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, the production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

[0078] The two expression vectors of the present invention, the first vector encoding the heavy-chain-derived polypeptide and the second vector encoding the light-chain-derived polypeptide, can be co-introduced into the host cell. The two vectors may contain the same selectable marker that allows for equal expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding and capable of expressing both the heavy and light chain polypeptides can be used. In such a situation, the light chain should be placed upstream of the heavy chain to avoid excessive toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2 197). The coding sequences of the heavy and light chains can include cDNA or genomic DNA.

[0079] When the antibody molecules of the present invention are produced by recombinant expression, they can be purified by any method known in the art for the purification of immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen after protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Further, the antibodies or fragments thereof of the present invention may be fused with the heterologous polypeptide sequences described herein or with other ones known in the art to facilitate purification.

[0080] Regarding the IL-5R binding molecules (such as antibodies, proteins, polypeptides, peptides, and fusion proteins) encompassed by the present invention, the amount thereof administered to a patient is typically from 0.0001 mg to 100 mg per kg of the patient's body weight. Preferably, the dosage administered to the patient is from 0.0001 mg to 20 mg, 0.0001 mg to 10 mg, 0.0001 mg to 5 mg, 0.0001 to 2 mg, 0.0001 to 1 mg, 0.0001 mg to 0.75 mg, 0.0001 mg to 0.5 mg, 0.0001 mg to 0.25 mg, 0.0001 to 0.15 mg, 0.0001 to 0.10 mg, 0.001 to 0.5 mg, 0.01 to 0.25 mg, or 0.01 to 0.10 mg per kg of the patient's body weight. Generally, human antibodies have a longer half-life in the human body compared to antibodies from other species due to the immune response to foreign polypeptides. Thus, lower dosages and less frequent administrations of human antibodies are often possible. Further, the dosage and frequency of administration of the antibodies or fragments thereof of the present invention can be decreased, for example, by enhancing the uptake and tissue penetration of the antibody by modifications such as lipidation.

[0081] In a specific embodiment, the dosage of the IL-5R binding molecule administered to prevent, treat, manage and / or alleviate a disease or one or more symptoms thereof in a patient is 150 μg or less, preferably 125 μg or less, 100 μg or less, 95 μg or less, 90 μg or less, 85 μg or less, 80 μg or less, 75 μg or less, 70 μg or less, 65 μg or less, 60 μg or less, 55 μg or less, 50 μg or less, 45 μg or less, 40 μg or less, 35 μg or less, 30 μg or less, 25 μg or less, 20 μg or less, 15 μg or less, 10 μg or less, 5 μg or less, 2.5 μg or less, 2 μg or less, 1.5 μg or less, 1 μg or less, 0.5 μg or less, or less than 0.5 μg per kg of the patient's body weight. In another embodiment, the dosage of the IL-5R binding molecule of the present invention administered to prevent, treat, manage and / or alleviate a hyperproliferative disease or one or more symptoms thereof in a patient is a unit dosage of 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0082] In other embodiments, a subject is administered one or more doses of an effective amount of one or more therapeutic agents of the invention, and the dose of this effective amount achieves a serum titer of at least 0.1 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 5 μg, at least 6 μg, at least 10 μg, at least 15 μg, at least 20 μg, at least 25 μg, at least 50 μg, at least 100 μg, at least 125 μg, at least 150 μg, at least 175 μg, at least 200 μg, at least 225 μg, at least 250 μg, at least 275 μg, at least 300 μg, at least 325 μg, at least 350 μg, at least 375 μg, or at least 400 μg per ml of the therapeutic agent of the invention. In yet another embodiment, a subject is administered a dose of an effective amount of one IL-5R binding molecule of the invention to achieve a serum titer of at least 0.1 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 5 μg, at least 6 μg, at least 10 μg, at least 15 μg, at least 20 μg, at least 25 μg, at least 50 μg, at least 100 μg, at least 125 μg, at least 150 μg, at least 175 μg, at least 200 μg, at least 225 μg, at least 250 μg, at least 275 μg, at least 300 μg, at least 325 μg, at least 350 μg, at least 375 μg, or at least 400 μg per ml of the IL-5R binding molecule, and subsequent doses of the effective amount of one or more IL-5R binding molecules of the invention are at least 0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, at least 2 μg / ml, at least 5 μg / ml, at least 6 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 175 μg / ml, at least 200 μg / ml, at least 225 μg / ml, at least 250 μg / ml, at least 275 μg / ml, at least 300 μg / ml, at least 325 μg / ml, at least 350 μg / ml, at least 375 μg / ml, or at least 400 μg It is administered to maintain a serum titer of / ml. According to these embodiments, the subject may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more subsequent doses.

[0083] In a specific embodiment, the present invention provides a method for preventing, treating, managing or alleviating an eosinophil-mediated disease or one or more symptoms thereof, the method comprising administering to a subject in need thereof one or more therapeutic agents (e.g., therapeutic or prophylactic agents), combination therapies, or compositions of the present invention at a dose of at least 10 μg, preferably at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, at least 100 μg, at least 105 μg, at least 110 μg, at least 115 μg, or at least 120 μg. In another embodiment, the present invention provides a method for preventing, treating, managing and / or alleviating an eosinophil-mediated disease or disorder or one or more symptoms thereof, the method comprising administering to a subject in need thereof one or more IL-5R binding molecules, combination therapies, or compositions of the present invention at a dose of at least 10 μg, preferably at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, at least 100 μg, at least 105 μg, at least 110 μg, at least 115 μg, or at least 120 μg, once every 3 days, preferably once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 10 days, once every 2 weeks, once every 3 weeks, or once a month.

[0084] The present invention provides a method for preventing, treating, managing, or precluding eosinophil-mediated disorders or diseases or one or more symptoms thereof, the method comprising: (a) administering to a subject in need thereof one or more doses of a prophylactically or therapeutically effective amount of one or more IL-5R binding molecules, combination therapies, or compositions of the present invention; and (b) monitoring the plasma level / concentration of the administered IL-5R binding molecule in the subject after administration of a defined number of doses of the therapeutic agent (e.g., therapeutic or prophylactic agent). Further preferably, the defined number of doses is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses of a prophylactically or therapeutically effective amount of one or more IL-5R binding molecules, compositions, or combination therapies of the present invention.

[0085] In a specific embodiment, the present invention provides a method for preventing, treating, managing and / or alleviating an eosinophil-mediated disorder or disease or one or more symptoms thereof, the method comprising: (a) administering to a subject in need thereof one or more therapeutic agents (e.g., a therapeutic or prophylactic agent) of the present invention at a dose of at least 10 μg (preferably at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, or at least 100 μg); and (b) administering one or more subsequent doses to the subject when the plasma level of the administered IL-5R binding molecule in the subject is less than 0.1 μg / ml, preferably less than 0.25 μg / ml, less than 0.5 μg / ml, less than 0.75 μg / ml, or less than 1 μg / ml. In another embodiment, the present invention provides a method for preventing, treating, managing and / or alleviating an eosinophil-mediated disorder or disease or one or more symptoms thereof, the method comprising: (a) administering to a subject in need thereof one or more IL-5R binding molecules of the present invention at one or more doses of at least 10 μg (preferably at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, or at least 100 μg); (b) monitoring the plasma level of the administered IL-5R binding molecule in the subject after administration of a certain number of doses; and (c) administering a subsequent dose of the IL-5R binding molecule of the present invention when the plasma level of the administered IL-5R binding molecule in the subject is less than 0.1 μg / ml, preferably less than 0.25 μg / ml, less than 0.5 μg / ml, less than 0.75 μg / ml, or less than 1 μg / ml.In certain embodiments, the fixed number of doses is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses of the effective amount of one or more IL-5R binding molecules of the invention.

[0086] Therapeutic agents (e.g., prophylactic or therapeutic agents) that have been or are currently used to prevent, treat, manage, and / or alleviate a proliferative disorder or one or more symptoms thereof other than the IL-5R binding molecules of the invention can be administered in combination with one or more IL-5R binding molecules according to the methods of the invention for treating, managing, preventing, and / or alleviating an eosinophil-mediated disorder or disease or one or more symptoms thereof. Preferably, the dosage of the prophylactic or therapeutic agent used in the combination therapy of the invention is lower than that which has been or is currently used to prevent, treat, manage, and / or alleviate an eosinophil-mediated disorder or disease or one or more symptoms thereof. Recommended dosages of agents currently used for the prevention, treatment, management, or alleviation of a proliferative disorder or one or more symptoms thereof can be obtained from any literature in the art and include, but are not limited to, Hardman et al., eds., 2001, Goodman & Gilman's The Pharmacological Basis Of Basis Of Therapeutics, 10th ed., Mc-Graw-Hill, New York; Physician's Desk Reference (PDR) 58th ed., 2004, Medical Economics Co., Inc., Montvale, NJ, the entire contents of which are incorporated herein by reference.

[0087] In various embodiments, a therapeutic agent (e.g., a prophylactic or therapeutic agent) is administered at intervals of less than 5 minutes, less than 30 minutes, 1 hour, about 1 hour, about 1 to about 2 hours, about 2 to about 3 hours, about 3 to about 4 hours, about 4 to about 5 hours, about 5 to about 6 hours, about 6 to about 7 hours, about 7 to about 8 hours, about 8 to about 9 hours, about 9 to about 10 hours, about 10 to about 11 hours, about 11 to about 12 hours, about 12 to 18 hours, 18 to 24 hours, 24 to 36 hours, 36 to 48 hours, 48 to 52 hours, 52 to 60 hours, 60 to 72 hours, 72 to 84 hours, 84 to 96 hours, or 96 to 120 hours. In other embodiments, two or more therapeutic agents are administered during the same consultation.

[0088] In some embodiments, one or more IL-5R binding molecules of the invention and one or more other therapeutic agents (e.g., prophylactic or therapeutic agents) are administered cyclically. Cyclic therapy involves administration of a first therapeutic agent (e.g., a first prophylactic or therapeutic agent) for a period, followed by administration of a second therapeutic agent (e.g., a second prophylactic or therapeutic agent) for a period, optionally followed by administration of a third therapeutic agent (e.g., prophylactic or therapeutic agent) for a period, etc., and repeating this cycle in order to reduce development of resistance to one therapy, to prevent or reduce side effects of one therapeutic agent, and / or to improve the effectiveness of the therapeutic agent.

[0089] In some embodiments, administration of the same IL-5R binding molecule of the invention can be repeated, with intervals between administrations of at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months. In other embodiments, administration of the same therapeutic agent (e.g., prophylactic or therapeutic agent) other than the IL-5R binding molecule of the invention can be repeated, with intervals between administrations of at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

[0090] In a specific embodiment, the IL-5R binding molecule is administered as a single intravenous dose of 0.03 mg / kg.

[0091] The present invention provides a method for preventing, treating, managing or preventing an eosinophil-mediated disorder or disease or one or more symptoms thereof, said method comprising: (a) administering to a subject in need thereof one or more doses of a prophylactically or therapeutically effective amount of one or more IL-5R binding molecules, combination therapies, or compositions of the present invention; and (b) monitoring at least one disease indicator or symptom in the subject before or after administration of one or more doses of said therapeutic agent (e.g., therapeutic or prophylactic agent).

[0092] In one embodiment, the subject has COPD.

[0093] In one embodiment, the subject has mild persistent or mild intermittent asthma as defined by the 2002 Expert Panel report of the NAEPP.

[0094] In one embodiment, the disease indicator or symptom in the subject is monitored before and after administration of a single dose of one or more IL-5R binding molecules. In another embodiment, the disease indicator or symptom in the subject is monitored before and after administration of multiple doses of one or more IL-5R binding molecules.

[0095] In one embodiment, the disease indicator or symptom is a self-assessed asthma symptom score. Non-limiting examples of asthma symptom scores are self-assessed scores recorded daily by the subject at home. The score categorizes asthma symptoms over the past 24 hours based on the severity of morning, evening, and daytime symptoms. The symptoms and assigned scores are set forth in Table 1. The maximum score for a day is 9 and the minimum is 0. The subject self-assesses and records on a continuous basis.

Table 1

[0096] In one embodiment, the subject has a pre - administration asthma symptom score X for one or more doses of one or more IL - 5R binding molecules and a post - administration asthma symptom score X - Y for one or more doses of one or more IL - 5R binding molecules, where X is 1, 2, 3, 4, 5, 6, 7, 8, or 9, Y is 1, 2, 3, 4, 5, 6, 7, 8, or 9, and the post - administration score is not less than 0.

[0097] In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is from 0 to 9. In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is from 0 to 3, 1 to 4, 2 to 5, 3 to 5, 4 to 7, 5 to 8, or 6 to 9. In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is 1, 2, 3, 4, 5, 6, 7, 8, or 9. In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is 1. In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is 2. In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is 3. In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is 4. In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is 5. In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is 6. In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is 7. In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is 8. In one embodiment, the pre - administration asthma symptom score of the subject for one or more doses of one or more IL - 5R binding molecules is 9.

[0098] In one embodiment, the subject has a wheezing symptom score between 0 and 9 after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a wheezing symptom score of 0-3, 1-4, 2-5, 3-5, 4-7, 5-8, or 6-9 after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a wheezing symptom score of 1, 2, 3, 4, 5, 6, 7, 8, or 9 after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a wheezing symptom score of 1 after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a wheezing symptom score of 2 after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a wheezing symptom score of 3 after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a wheezing symptom score of 4 after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a wheezing symptom score of 5 after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a wheezing symptom score of 6 after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a wheezing symptom score of 7 after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a wheezing symptom score of 8 after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a wheezing symptom score of 9 after administration of one or more doses of one or more IL-5R binding molecules.

[0099] In one embodiment, the asthma symptom score of the subject is lower after administration of one or more doses of one or more IL-5R binding molecules than before administration of one or more doses of one or more IL-5R binding molecules, where the post-administration score does not fall below 0. In a specific embodiment, the asthma symptom score decreases by 1 point. In a specific embodiment, the asthma symptom score decreases by 9 points. In a specific embodiment, the asthma symptom score decreases by 2 points. In a specific embodiment, the asthma symptom score decreases by 3 points. In a specific embodiment, the asthma symptom score decreases by 4 points. In a specific embodiment, the asthma symptom score decreases by 5 points. In a specific embodiment, the asthma symptom score decreases by 6 points. In a specific embodiment, the asthma symptom score decreases by 7 points. In a specific embodiment, the asthma symptom score decreases by 8 points. In a specific embodiment, the asthma symptom score decreases by at least 1 point. In a specific embodiment, the asthma symptom score decreases by at least 9 points. In a specific embodiment, the asthma symptom score decreases by at least 2 points. In a specific embodiment, the asthma symptom score decreases by at least 3 points. In a specific embodiment, the asthma symptom score decreases by at least 4 points. In a specific embodiment, the asthma symptom score decreases by at least 5 points. In a specific embodiment, the asthma symptom score decreases by at least 6 points. In a specific embodiment, the asthma symptom score decreases by at least 7 points. In a specific embodiment, the asthma symptom score decreases by at least 8 points.

[0100] In one embodiment, the disease indicator or symptom is fractional exhaled nitric oxide (FENO). FENO can be measured according to the combined recommendations of the European Respiratory Society and the American Thoracic Society (American Thoracic Society, European Respiratory Society. (2005) ATS / ERS Recommendations for Standardized Procedures for the Online and Offline Measurements of Exhaled Lower Respiratory Nitric Oxide and Nasal Nitric Oxide, 2005. Am J Respir Crit Care Med. 171:912-930). The FENO measurement can be performed using NIOX at a flow rate of 50 ml / s (ATS standard).

[0101] In one embodiment, the subject has a FENO between 20 and 500 ppb prior to administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO prior to administration of one or more doses of one or more IL-5R binding molecules that is 20 - 500 ppb, 20 - 400 ppb, 20 - 300 ppb, 20 - 200 ppb, 50 - 500 ppb, 100 - 500 ppb, 150 - 500 ppb, 200 - 500 ppb, 20 - 50 ppb, 50 - 100 ppb, 100 - 200 ppb, 200 - 300 ppb, 300 - 500 ppb. In one embodiment, the subject has a FENO prior to administration of one or more doses of one or more IL-5R binding molecules that is at least 50 ppb, at least 100 ppb, at least 150 ppb, at least 200 ppb, at least 250 ppb, at least 300 ppb, at least 350 ppb, at least 400 ppb. In one embodiment, the subject has a FENO of 50 ppb prior to administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of 100 ppb prior to administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of 150 ppb prior to administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of 200 ppb prior to administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of 250 ppb prior to administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of 300 ppb prior to administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of 350 ppb prior to administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of 400 ppb prior to administration of one or more doses of one or more IL-5R binding molecules.

[0102] In one embodiment, the subject has a FENO between 20 and 500 ppb after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of 20-500 ppb, 20-400 ppb, 20-300 ppb, 20-200 ppb, 50-500 ppb, 100-500 ppb, 150-500 ppb, 200-500 ppb, 20-50 ppb, 50-100 ppb, 100-200 ppb, 200-300 ppb, 300-500 ppb after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of at most 50 ppb, at most 100 ppb, at most 150 ppb, at most 200 ppb, at most 250 ppb, at most 300 ppb, at most 350 ppb, at most 400 ppb after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of at most 20 ppb after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of at most 50 ppb after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of at most 100 ppb after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of at most 150 ppb after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of at most 200 ppb after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of at most 250 ppb after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of at most 300 ppb after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of at most 350 ppb after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a FENO of at most 400 ppb after administration of one or more doses of one or more IL-5R binding molecules.

[0103] In one embodiment, the FENO of the subject is lower after administration of one or more doses of one or more IL-5R binding molecules than before administration of one or more doses of one or more IL-5R binding molecules, where the decrease in FENO does not fall below 0 ppb. In a specific embodiment, the FENO decreases by at least 50 ppb. In a specific embodiment, the FENO decreases by at least 100 ppb. In a specific embodiment, the FENO decreases by at least 150 ppb. In a specific embodiment, the FENO decreases by at least 200 ppb. In a specific embodiment, the FENO decreases by at least 250 ppb. In a specific embodiment, the FENO decreases by at least 300 ppb. In a specific embodiment, the FENO decreases by at least 10%. In a specific embodiment, the FENO decreases by at least 20%. In a specific embodiment, the FENO decreases by at least 30%. In a specific embodiment, the FENO decreases by at least 40%. In a specific embodiment, the FENO decreases by at least 50%. In a specific embodiment, the FENO decreases by at least 60%. In a specific embodiment, the FENO decreases by at least 70%. In a specific embodiment, the FENO decreases by at least 80%. In a specific embodiment, the FENO decreases by at least 90%. In a specific embodiment, the FENO decreases by 10%. In a specific embodiment, the FENO decreases by 20%. In a specific embodiment, the FENO decreases by 30%. In a specific embodiment, the FENO decreases by 40%. In a specific embodiment, the FENO decreases by 50%. In a specific embodiment, the FENO decreases by 60%. In a specific embodiment, the FENO decreases by 70%. In a specific embodiment, the FENO decreases by 80%. In a specific embodiment, the FENO decreases by 90%.

[0104] In one embodiment, the disease indicator or symptom is eosinophil cationic protein (ECP). Serum ECP levels can be evaluated using any method known to those skilled in the art, such as, but not limited to, ELISA assays, radioimmunoassays. Serum ECP levels can be measured by any one of commercially available assays.

[0105] In one embodiment, the subject has a serum ECP of 20 to 500 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of 20 to 200 ng / ml, 20 to 150 ng / ml, 20 to 100 ng / ml, 20 to 50 ng / ml, 30 to 200 ng / ml, 40 to 200 ng / ml, 50 to 200 ng / ml, 30 to 100 ng / ml, 30 to 80 ng / ml, 30 to 70 ng / ml, 20 to 80 ng / ml, 20 to 70 ng / ml, 20 to 60 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of at least 20 ng / ml, at least 30 ng / ml, at least 40 ng / ml, at least 50 ng / ml, at least 60 ng / ml, at least 100 ng / ml, at least 150 ng / ml, at least 200 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of 25 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of 30 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of 35 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of 40 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of 50 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of 60 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of 70 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of 80 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of 100 ng / ml before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a serum ECP of 150 ng / ml before administration of one or more doses of one or more IL-5R binding molecules.In one embodiment, the subject has a serum ECP of 200 ng / ml before administration of one or more doses of one or more IL-5R binding molecules.

[0106] In one embodiment, the serum ECP of the subject is not detectable after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the serum ECP of the subject is 1 - 500 ng / ml after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the serum ECP of the subject is 1 - 200 ng / ml, 1 - 150 ng / ml, 1 - 100 ng / ml, 1 - 50 ng / ml, 1 - 20 ng / ml, 10 - 200 ng / ml, 10 - 100 ng / ml, 10 - 50 ng / ml, 20 - 100 ng / ml, 20 - 50 ng / ml after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the serum ECP of the subject is at most 1 ng / ml, at most 5 ng / ml, at most 10 ng / ml, at most 20 ng / ml, at most 30 ng / ml, at most 50 ng / ml after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the serum ECP of the subject is at most 1 ng / ml after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the serum ECP of the subject is at most 5 ng / ml after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the serum ECP of the subject is at most 10 ng / ml after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the serum ECP of the subject is at most 15 ng / ml after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the serum ECP of the subject is at most 20 ng / ml after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the serum ECP of the subject is at most 25 ng / ml after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the serum ECP of the subject is at most 30 ng / ml after administration of one or more doses of one or more IL-5R binding molecules.

[0107] In one embodiment, the serum ECP of the subject is lower after administration of one or more doses of one or more IL-5R binding molecules than before administration of one or more doses of one or more IL-5R binding molecules, where the decrease in serum ECP does not reach less than 0 ng / ml. In a specific embodiment, the serum ECP decreases by at least 50 ng / ml. In a specific embodiment, the serum ECP decreases by at least 100 ng / ml. In a specific embodiment, the serum ECP decreases by at least 150 ng / ml. In a specific embodiment, the serum ECP decreases by at least 200 ng / ml. In a specific embodiment, the serum ECP decreases by at least 250 ng / ml. In a specific embodiment, the serum ECP decreases by at least 300 ng / ml. In a specific embodiment, the serum ECP decreases by at least 10%. In a specific embodiment, the serum ECP decreases by at least 20%. In a specific embodiment, the serum ECP decreases by at least 30%. In a specific embodiment, the serum ECP decreases by at least 40%. In a specific embodiment, the serum ECP decreases by at least 50%. In a specific embodiment, the serum ECP decreases by at least 60%. In a specific embodiment, the serum ECP decreases by at least 70%. In a specific embodiment, the serum ECP decreases by at least 80%. In a specific embodiment, the serum ECP decreases by at least 90%. In a specific embodiment, the serum ECP decreases by at least 95%. In a specific embodiment, the serum ECP decreases by 10%. In a specific embodiment, the serum ECP decreases by 20%. In a specific embodiment, the serum ECP decreases by 30%. In a specific embodiment, the serum ECP decreases by 40%. In a specific embodiment, the serum ECP decreases by 50%. In a specific embodiment, the serum ECP decreases by 60%. In a specific embodiment, the serum ECP decreases by 70%. In a specific embodiment, the serum ECP decreases by 80%. In a specific embodiment, the serum ECP decreases by 90%. In a specific embodiment, the serum ECP decreases by 95%. In a specific embodiment, the serum ECP decreases by 99%.

[0108] In one embodiment, the serum ECP is undetectable in the subject after administration of one or more doses of one or more IL-5R binding molecules. In a specific embodiment, the serum ECP level remains undetectable for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 20 weeks, or at least about 25 weeks.

[0109] In one embodiment, the disease indicator or symptom is a methacholine challenge test (MCT). The MCT can be performed in the presence of a physician skilled in the management of bronchospasm and with appropriate therapeutic agents readily available, in accordance with the American Thoracic Society (ATS) guidelines (Guidelines for Methacholine and Exercise Testing - 1999. (2000) Am J Respir Crit Care Med. 161:309 - 329). Briefly, the spirometer used is calibrated in accordance with the ATS guidelines. The nebulizer used must generate particles with an aerodynamic particle size (mass median aerodynamic diameter) (MMAD) of 1 - 4 microns and a flow of 0.13 ± 10% mL / min. Methacholine from an FDA - approved source is used and diluted with sterile component - known physiological saline. The inhalation exposure can be performed using either a 2 - minute breathing cycle or a 5 - breath dosimeter method as described in the reference publications. The concentration of methacholine is administered according to the investigator's practice but is within the range of 0.06 mg / dL to 25.0 mg / dL. FEV 1 is measured 30 and 90 seconds after the end of each dose, and the higher of the two values is recorded. Incremental concentrations are administered until FEV 1 is considered to have dropped by at least 20% from the baseline value. PC20 is the FEV from the baseline value 1It is the concentration of methacholine that leads to at least a 20% drop. After the end of the final dose, the subject may be given albuterol by a metered-dose inhaler or a nebulizer at the discretion of the treating physician.

[0110] In one embodiment, the subject has a PC20 of 0.06 to 25 mg / dL before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of 0.06 to 25 mg / dL, 0.1 to 10 mg / dL, 0.06 to 3 mg / dL, 0.06 to 2 mg / dL, 0.06 to 1 mg / dL, 0.1 to 3 mg / dL, 0.1 to 2 mg / dL, 0.1 to 1 mg / dL, 0.2 to 10 mg / dL, 0.5 to 10 mg / dL, 1 to 10 mg / dL, 0.1 to 5 mg / dL, 0.2 to 5 mg / dL, 0.5 to 5 mg / dL, 0.1 to 2 mg / dL, 0.2 to 2 mg / dL, 0.5 to 2 mg / dL, 0.06 to 0.1 mg / dL, 0.1 to 0.2 mg / dL, 0.2 to 0.5 mg / dL, 0.5 to 1 mg / dL, 1 to 2 mg / dL, 2 to 5 mg / dL, 5 to 10 mg / dL before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at most 0.1 mg / dL, at most 0.2 mg / dL, at most 0.4 mg / dL, at most 0.5 mg / dL, at most 1 mg / dL, at most 2 mg / dL, at most 5 mg / dL, at most 10 mg / dL before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of 10 mg / dL before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of 5 mg / dL before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of 2 mg / dL before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of 1 mg / dL before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of 0.5 mg / dL before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of 0.2 mg / dL before administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of 0.1 mg / dL before administration of one or more doses of one or more IL-5R binding molecules.

[0111] In one embodiment, the subject has a PC20 of 0.5 to 25 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of 1 to 25 mg / dL, 2 to 25 mg / dL, 5 to 25 mg / dL, 10 to 25 mg / dL, 1 to 10 mg / dL, 2 to 10 mg / dL, 2 to 10 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 1 mg / dL, at least 2 mg / dL, at least 5 mg / dL, at least 10 mg / dL, at least 20 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 0.2 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 0.3 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 0.4 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 0.5 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 0.7 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 1 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 2 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 5 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 10 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 20 mg / dL after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a PC20 of at least 25 mg / dL after administration of one or more doses of one or more IL-5R binding molecules.

[0112] In one embodiment, the PC20 of the subject is higher after administration of one or more doses of one or more IL-5R binding molecules than before administration of one or more doses of one or more IL-5R binding molecules. In a specific embodiment, the PC20 increases by at least 0.3 mg / dL. In a specific embodiment, the PC20 increases by at least 0.5 mg / dL. In a specific embodiment, the PC20 increases by at least 0.7 mg / dL. In a specific embodiment, the PC20 increases by at least 1 mg / dL. In a specific embodiment, the PC20 increases by at least 3 mg / dL. In a specific embodiment, the PC20 increases by at least 5 mg / dL. In a specific embodiment, the PC20 increases by at least 10 mg / dL. In a specific embodiment, the PC20 increases by at least 15 mg / dL. In a specific embodiment, the PC20 increases by at least 20 mg / dL. In a specific embodiment, the PC20 increases by at least 2-fold. In a specific embodiment, the PC20 increases by at least 4-fold. In a specific embodiment, the PC20 increases by at least 8-fold. In a specific embodiment, the PC20 increases by at least 10-fold. In a specific embodiment, the PC20 increases by at least 12-fold. In a specific embodiment, the PC20 increases by at least 15-fold. In a specific embodiment, the PC20 increases by at least 20-fold. In a specific embodiment, the PC20 increases by 2-fold. In a specific embodiment, the PC20 increases by 4-fold. In a specific embodiment, the PC20 increases by 8-fold. In a specific embodiment, the PC20 increases by 10-fold. In a specific embodiment, the PC20 increases by 15-fold. In a specific embodiment, the PC20 increases by 60%. In a specific embodiment, the PC20 increases by 20-fold.

[0113] In one embodiment, the disease indicator or symptom is the circulating eosinophil count. The circulating eosinophil count can be evaluated by any method known to those skilled in the art, such as, but not limited to, histology, flow cytometry. The circulating eosinophil count can be measured by any one of commercially available kits.

[0114] In one embodiment, the subject has a pre-dose circulating eosinophil count of 50 to 1000 cells / microliter for one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a pre-dose circulating eosinophil count of 50 to 1000 cells / microliter, 100 to 1000 cells / microliter, 150 to 1000 cells / microliter, 200 to 1000 cells / microliter, 250 to 1000 cells / microliter, 300 to 1000 cells / microliter, 400 to 1000 cells / microliter, 500 to 1000 cells / microliter, 50 to 500 cells / microliter, 100 to 500 cells / microliter, 100 to 400 cells / microliter, 150 to 500 cells / microliter, 200 to 500 cells / microliter for one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a pre-dose circulating eosinophil count of at least 50 cells / microliter, at least 100 cells / microliter, at least 150 cells / microliter, at least 200 cells / microliter, at least 250 cells / microliter, at least 300 cells / microliter, at least 400 cells / microliter, at least 500 cells / microliter for one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a pre-dose circulating eosinophil count of 50 cells / microliter for one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a pre-dose circulating eosinophil count of 100 cells / microliter for one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a pre-dose circulating eosinophil count of 150 cells / microliter for one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a pre-dose circulating eosinophil count of 200 cells / microliter for one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a pre-dose circulating eosinophil count of 250 cells / microliter for one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a pre-dose circulating eosinophil count of 300 cells / microliter for one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a pre-dose circulating eosinophil count of 350 cells / microliter for one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a pre-dose circulating eosinophil count of 400 cells / microliter for one or more doses of one or more IL-5R binding molecules.In one embodiment, the subject has a circulating eosinophil count of 500 cells / microliter prior to administration of one or more doses of one or more IL-5R binding molecules.

[0115] In one embodiment, the subject has a circulating eosinophil count of 1 to 400 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating eosinophil count of 1 to 200 cells / microliter, 1 to 100 cells / microliter, 1 to 50 cells / microliter, 1 to 40 cells / microliter, 10 to 200 cells / microliter, 10 to 100 cells / microliter, 10 to 40 cells / microliter, 20 to 200 cells / microliter, 20 to 100 cells / microliter, 20 to 50 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating eosinophil count of at most 1 cell / microliter, at most 5 cells / microliter, at most 10 cells / microliter, at most 20 cells / microliter, at most 30 cells / microliter, at most 40 cells / microliter, at most 50 cells / microliter, at most 60 cells / microliter, at most 80 cells / microliter, at most 100 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating eosinophil count of at most 1 cell / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating eosinophil count of at most 5 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating eosinophil count of at most 10 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating eosinophil count of at most 20 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating eosinophil count of at most 30 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating eosinophil count of at most 40 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating eosinophil count of at most 50 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating eosinophil count of at most 60 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules.In one embodiment, the subject has a maximum circulating eosinophil count of 80 cells / microliter or less after administration of one or more doses of one or more IL-5R binding molecules.

[0116] In one embodiment, the circulating eosinophil value of the subject is lower after administration of one or more doses of one or more IL-5R binding molecules than before administration of one or more doses of one or more IL-5R binding molecules, where the decrease in the circulating eosinophil value does not reach less than 0 cells / microliter. In a specific embodiment, the circulating eosinophil value decreases by at least 50 cells / microliter. In a specific embodiment, the circulating eosinophil value decreases by at least 100 cells / microliter. In a specific embodiment, the circulating eosinophil value decreases by at least 150 cells / microliter. In a specific embodiment, the circulating eosinophil value decreases by at least 200 cells / microliter. In a specific embodiment, the circulating eosinophil value decreases by at least 250 cells / microliter. In a specific embodiment, the circulating eosinophil value decreases by at least 300 cells / microliter. In a specific embodiment, the circulating eosinophil value decreases by at least 10%. In a specific embodiment, the circulating eosinophil value decreases by at least 20%. In a specific embodiment, the circulating eosinophil value decreases by at least 30%. In a specific embodiment, the circulating eosinophil value decreases by at least 40%. In a specific embodiment, the circulating eosinophil value decreases by at least 50%. In a specific embodiment, the circulating eosinophil value decreases by at least 60%. In a specific embodiment, the circulating eosinophil value decreases by at least 70%. In a specific embodiment, the circulating eosinophil value decreases by at least 80%. In a specific embodiment, the circulating eosinophil value decreases by at least 90%. In a specific embodiment, the circulating eosinophil value decreases by at least 95%. In a specific embodiment, the circulating eosinophil value decreases by at least 99%. In a specific embodiment, the circulating eosinophil value decreases by 10%. In a specific embodiment, the circulating eosinophil value decreases by 20%. In a specific embodiment, the circulating eosinophil value decreases by 30%. In a specific embodiment, the circulating eosinophil value decreases by 40%. In a specific embodiment, the circulating eosinophil value decreases by 50%. In a specific embodiment, the circulating eosinophil value decreases by 60%. In a specific embodiment, the circulating eosinophil value decreases by 70%. In a specific embodiment, the circulating eosinophil value decreases by 80%. In a specific embodiment, the circulating eosinophil value decreases by 90%. In a specific embodiment, the circulating eosinophil value decreases by 95%.In a specific embodiment, the circulating eosinophil count decreases by 99%.

[0117] In one embodiment, the circulating eosinophil count of the subject is undetectable after administration of one or more doses of one or more IL-5R binding molecules. In a specific embodiment, the circulating eosinophil count level remains undetectable for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 20 weeks, or at least about 25 weeks.

[0118] In one embodiment, the disease indicator or symptom is the % eosinophils in induced sputum. The % eosinophils in induced sputum can be evaluated by any method known to those skilled in the art, for example, but not limited to, the method described in Belda et al. (2000) Am J Respir Crit Care Med 161:475-478. The % eosinophils in induced sputum can be determined by any one of commercially available kits.

[0119] In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 0.1% to 10%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 0.1% to 2%, 0.1% to 5%, 0.5% to 2%, 0.5% to 5%, 0.5% to 10%, 1% to 2%, 1% to 5%, 1% to 10%, 2% to 5%, 2% to 10%, 3% to 5%, 3% to 10%, 1.5% to 5%, 2.5% to 5%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of at least 0.1%, at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 0.5%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 1%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 1.5%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 2%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 2.5%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 3%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 4%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 5%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 6%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 7%. In one embodiment, the subject has a % eosinophil in induced sputum before administration of one or more doses of one or more IL-5R binding molecules of 8%.In one embodiment, the subject has 9% eosinophils in induced sputum prior to administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has 10% eosinophils in induced sputum prior to administration of one or more doses of one or more IL-5R binding molecules.

[0120] In one embodiment, the subject has 0.1% to 5% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has 0.1% to 3%, 0.1% to 2%, 0.1% to 1.5%, 0.5% to 5%, 0.5% to 3%, 0.5% to 1%, 1% to 5%, 1% to 3%, 2% to 5%, 3% to 5%, 2.5% to 5% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has at most 1% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has at most 2% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has at most 3% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has at most 4% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has at most 5% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has at most 6% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has at most 7% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has at most 8% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has at most 9% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has at most 10% eosinophils in induced sputum after administration of one or more doses of one or more IL-5R binding molecules.

[0121] In one embodiment, the % eosinophils in the induced sputum of the subject are lower after administration of one or more doses of one or more IL-5R binding molecules than before administration of one or more doses of one or more IL-5R binding molecules, where the decrease in % eosinophils in the induced sputum does not fall below 0%. In a specific embodiment, the % eosinophils in the induced sputum are reduced by at least 10%. In a specific embodiment, the % eosinophils in the induced sputum are reduced by at least 9%. In a specific embodiment, the % eosinophils in the induced sputum are reduced by at least 8%. In a specific embodiment, the % eosinophils in the induced sputum are reduced by at least 6%. In a specific embodiment, the % eosinophils in the induced sputum are reduced by at least 5%. In a specific embodiment, the % eosinophils in the induced sputum are reduced by at least 4%. In a specific embodiment, the % eosinophils in the induced sputum are reduced by at least 3%. In a specific embodiment, the % eosinophils in the induced sputum are reduced by at least 2%. In a specific embodiment, the % eosinophils in the induced sputum are reduced by at least 1%.

[0122] In one embodiment, the subject has no detectable eosinophils in the induced sputum after administration of one or more doses of one or more IL-5R binding molecules. In a specific embodiment, the eosinophils in the induced sputum remain undetectable for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 20 weeks, or at least about 25 weeks.

[0123] In one embodiment, the disease indicator or symptom is the circulating basophil count. The circulating basophil count can be evaluated by any method known to those skilled in the art, for example, but not limited to, histology, flow cytometry. The circulating basophil count can be measured by any one of commercially available kits.

[0124] In one embodiment, the subject has a pre - dose circulating basophil count of 5 to 500 cells / microliter for one or more doses of one or more IL - 5R binding molecules. In one embodiment, the subject has a pre - dose circulating basophil count of 50 to 500 cells / microliter, 10 to 500 cells / microliter, 20 to 500 cells / microliter, 30 to 500 cells / microliter, 40 to 500 cells / microliter, 50 to 500 cells / microliter, 10 to 400 cells / microliter, 10 to 300 cells / microliter, 10 to 200 cells / microliter, 10 to 100 cells / microliter, 20 to 100 cells / microliter, 30 to 100 cells / microliter, 10 to 75 cells / microliter for one or more doses of one or more IL - 5R binding molecules. In one embodiment, the subject has a pre - dose circulating basophil count of at least 5 cells / microliter, at least 10 cells / microliter, at least 15 cells / microliter, at least 20 cells / microliter, at least 30 cells / microliter, at least 50 cells / microliter, at least 60 cells / microliter, at least 100 cells / microliter for one or more doses of one or more IL - 5R binding molecules. In one embodiment, the subject has a pre - dose circulating basophil count of 5 cells / microliter for one or more doses of one or more IL - 5R binding molecules. In one embodiment, the subject has a pre - dose circulating basophil count of 10 cells / microliter for one or more doses of one or more IL - 5R binding molecules. In one embodiment, the subject has a pre - dose circulating basophil count of 15 cells / microliter for one or more doses of one or more IL - 5R binding molecules. In one embodiment, the subject has a pre - dose circulating basophil count of 20 cells / microliter for one or more doses of one or more IL - 5R binding molecules. In one embodiment, the subject has a pre - dose circulating basophil count of 30 cells / microliter for one or more doses of one or more IL - 5R binding molecules. In one embodiment, the subject has a pre - dose circulating basophil count of 50 cells / microliter for one or more doses of one or more IL - 5R binding molecules. In one embodiment, the subject has a pre - dose circulating basophil count of 60 cells / microliter for one or more doses of one or more IL - 5R binding molecules. In one embodiment, the subject has a pre - dose circulating basophil count of 100 cells / microliter for one or more doses of one or more IL - 5R binding molecules.

[0125] In one embodiment, the subject has a circulating basophil value of 1 to 100 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating basophil value of 1 to 100 cells / microliter, 1 to 50 cells / microliter, 1 to 30 cells / microliter, 1 to 20 cells / microliter, 1 to 10 cells / microliter, 5 to 100 cells / microliter, 5 to 50 cells / microliter, 5 to 20 cells / microliter, 5 to 10 cells / microliter, 10 to 30 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating basophil value of at most 1 cell / microliter, at most 5 cells / microliter, at most 10 cells / microliter, at most 20 cells / microliter, at most 30 cells / microliter, at most 50 cells / microliter, at most 100 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating basophil value of at most 1 cell / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating basophil value of at most 5 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating basophil value of at most 10 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating basophil value of at most 20 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating basophil value of at most 30 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating basophil value of at most 40 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules. In one embodiment, the subject has a circulating basophil value of at most 50 cells / microliter after administration of one or more doses of one or more IL-5R binding molecules.

[0126] In one embodiment, the number of circulating basophils in a subject is lower after administration of one or more doses of one or more IL-5R binding molecules than before administration of one or more doses of one or more IL-5R binding molecules, where the decrease in the number of circulating basophils does not fall below 0 cells / microliter. In a specific embodiment, the number of circulating basophils decreases by at least 10 cells / microliter. In a specific embodiment, the number of circulating basophils decreases by at least 20 cells / microliter. In a specific embodiment, the number of circulating basophils decreases by at least 30 cells / microliter. In a specific embodiment, the number of circulating basophils decreases by at least 50 cells / microliter. In a specific embodiment, the number of circulating basophils decreases by at least 75 cells / microliter. In a specific embodiment, the number of circulating basophils decreases by at least 100 cells / microliter. In a specific embodiment, the number of circulating basophils decreases by at least 10%. In a specific embodiment, the number of circulating basophils decreases by at least 20%. In a specific embodiment, the number of circulating basophils decreases by at least 30%. In a specific embodiment, the number of circulating basophils decreases by at least 40%. In a specific embodiment, the number of circulating basophils decreases by at least 50%. In a specific embodiment, the number of circulating basophils decreases by at least 60%. In a specific embodiment, the number of circulating basophils decreases by at least 70%. In a specific embodiment, the number of circulating basophils decreases by at least 80%. In a specific embodiment, the number of circulating basophils decreases by at least 90%. In a specific embodiment, the number of circulating basophils decreases by at least 95%. In a specific embodiment, the number of circulating basophils decreases by at least 99%. In a specific embodiment, the number of circulating basophils decreases by 10%. In a specific embodiment, the number of circulating basophils decreases by 20%. In a specific embodiment, the number of circulating basophils decreases by 30%. In a specific embodiment, the number of circulating basophils decreases by 40%. In a specific embodiment, the number of circulating basophils decreases by 50%. In a specific embodiment, the number of circulating basophils decreases by 60%. In a specific embodiment, the number of circulating basophils decreases by 70%. In a specific embodiment, the number of circulating basophils decreases by 80%. In a specific embodiment, the number of circulating basophils decreases by 90%.In a specific embodiment, the circulating basophil numerical value decreases by 95%. In a specific embodiment, the circulating basophil numerical value decreases by 99%.

[0127] In one embodiment, the circulating basophil numerical value of the subject is not detectable after administration of one or more doses of one or more IL-5R binding molecules. In a specific embodiment, the circulating basophil numerical value level remains undetectable for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 20 weeks, or at least about 25 weeks.

[0128] (Specific embodiment) 1. A method for reducing the number of eosinophils in a human subject, comprising administering to the subject an IL-5R binding molecule comprising (a) a region that specifically binds to IL-5R and (b) an immunoglobulin Fc region.

[0129] 2. The method according to embodiment 1, wherein the IL-5R binding molecule is an antibody.

[0130] 3. The method according to embodiment 2, wherein the antibody is a monoclonal antibody.

[0131] 4. The method according to embodiment 3, wherein the antibody is a chimeric antibody.

[0132] 5. The method according to embodiment 3, wherein the antibody is a humanized antibody.

[0133] 6. The method according to embodiment 3, wherein the antibody is a human antibody.

[0134] 7. The method according to embodiment 1, wherein the region specifically binding to the IL-5R comprises the amino acid sequence of IL-5, or a fragment, variant, or derivative thereof.

[0135] 8. The method according to embodiment 7, wherein the region specifically binding to the IL-5R comprises a non-functional variant of IL-5.

[0136] 9. The method according to any one of embodiments 1 to 8, wherein the IL-5R binding molecule specifically binds to the IL-5Rα chain.

[0137] 10. The method according to embodiment 1, wherein the immunoglobulin Fc region is modified to enhance effector function.

[0138] 11. The method according to embodiment 1, wherein the immunoglobulin Fc region contains a reduced level of fucose.

[0139] 12. The method according to embodiment 11, wherein the immunoglobulin Fc region is fucose-free.

[0140] 13. The method according to embodiment 1, wherein the immunoglobulin Fc region contains amino acid substitutions that result in enhanced effector function.

[0141] 14. The method according to embodiment 1, wherein the amino acid substitutions include the following amino acid sequences in the Fc region: 332E, 239D, and 330L (numbered according to the EU index by Kabat).

[0142] 15. The method according to embodiment 1, wherein the decrease in eosinophils occurs in peripheral blood circulation.

[0143] 16. The method according to embodiment 1, wherein the number of eosinophils is reduced to a level of less than 50 eosinophils / mm 3 3.

[0144] 17. The method according to embodiment 1, wherein the decrease in eosinophils occurs within 48 hours of administration.

[0145] 18. The method according to embodiment 1, wherein the decrease in eosinophils occurs within 24 hours after administration.

[0146] 19. The method according to embodiment 1, wherein the decrease in eosinophils is reversible.

[0147] 20. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is at least about 25 eosinophils / mm 3 .

[0148] 21. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is at least about 50 eosinophils / mm 3 .

[0149] 22. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is at least about 75 eosinophils / mm 3 .

[0150] 23. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is at least about 100 eosinophils / mm 3 .

[0151] 24. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is at least about 125 eosinophils / mm 3 .

[0152] 25. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is at least about 150 eosinophils / mm 3 .

[0153] 26. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is at least about 175 eosinophils / mm 3 .

[0154] 27. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is at least about 200 eosinophils / mm 3 .

[0155] 28. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is at least about 225 eosinophils / mm3 The method according to Embodiment 1, which is as follows.

[0156] 29. The method according to Embodiment 1, wherein the decrease in the absolute eosinophil count after administration is at least about 250 eosinophils / mm 3 The method according to Embodiment 1, which is as follows.

[0157] 30. The method according to Embodiment 1, wherein the decrease in the absolute eosinophil count after administration is at least about 275 eosinophils / mm 3 The method according to Embodiment 1, which is as follows.

[0158] 31. The method according to Embodiment 1, wherein the decrease in the absolute eosinophil count after administration is at least about 300 eosinophils / mm 3 The method according to Embodiment 1, which is as follows.

[0159] 32. The method according to Embodiment 1, wherein the decrease in the absolute eosinophil count after administration is at least about 325 eosinophils / mm 3 The method according to Embodiment 1, which is as follows.

[0160] 33. The method according to Embodiment 1, wherein the decrease in the absolute eosinophil count after administration is at least about 350 eosinophils / mm 3 The method according to Embodiment 1, which is as follows.

[0161] 34. The method according to Embodiment 1, wherein the decrease in the absolute eosinophil count after administration is at least about 375 eosinophils / mm 3 The method according to Embodiment 1, which is as follows.

[0162] 35. The method according to Embodiment 1, wherein the decrease in the absolute eosinophil count after administration is at least about 400 eosinophils / mm 3 The method according to Embodiment 1, which is as follows.

[0163] 36. The method according to Embodiment 1, wherein the decrease in the absolute eosinophil count after administration is at least about 425 eosinophils / mm 3 The method according to Embodiment 1, which is as follows.

[0164] 37. The method according to Embodiment 1, wherein the decrease in the absolute eosinophil count after administration is at least about 450 eosinophils / mm 3 The method according to Embodiment 1, which is as follows.

[0165] 38. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is at least about 475 eosinophils / mm 3

[0166] 39. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is at least about 500 eosinophils / mm 3

[0167] 40. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is about 50 to about 500 eosinophils / mm 3

[0168] 41. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is about 75 to about 250 eosinophils / mm 3

[0169] 42. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is about 100 to about 200 eosinophils / mm 3

[0170] 43. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is about 50 to about 250 eosinophils / mm 3

[0171] 44. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is about 50 to about 200 eosinophils / mm 3

[0172] 45. The method according to embodiment 1, wherein the decrease in the absolute number of eosinophils after administration is about 50 to about 150 eosinophils / mm 3

[0173] 46. The method according to embodiment 1, wherein the absolute number of eosinophils after administration is less than about 100 eosinophils / mm 3

[0174] 47. The method according to embodiment 1, wherein the absolute number of eosinophils after administration is less than about 75 eosinophils / mm 3

[0175] ​​​​​​​​​​48. The method according to embodiment 1, wherein the absolute number of eosinophils after administration is less than about 50 eosinophils / mm. 3

[0176] 49. The method according to embodiment 1, wherein the absolute number of eosinophils after administration is less than about 25 eosinophils / mm. 3

[0177] 50. The method according to embodiment 1, wherein the absolute number of eosinophils in the subject before administration is about 50 to about 500 eosinophils / mm. 3

[0178] 51. The method according to embodiment 1, wherein the absolute number of eosinophils in the subject before administration is about 75 to about 475 eosinophils / mm. 3

[0179] 52. The method according to embodiment 1, wherein the absolute number of eosinophils in the subject before administration is about 75 to about 200 eosinophils / mm. 3

[0180] 53. The method according to embodiment 1, wherein the absolute number of eosinophils in the subject before administration is about 100 to about 200 eosinophils / mm. 3

[0181] 54. The method according to embodiment 1, wherein the absolute number of eosinophils in the subject before administration is about 25 eosinophils / mm. 3

[0182] 55. The method according to embodiment 1, wherein the absolute number of eosinophils in the subject before administration is about 50 eosinophils / mm. 3

[0183] 56. The method according to embodiment 1, wherein the absolute number of eosinophils in the subject before administration is about 75 eosinophils / mm. 3

[0184] 57. The method according to embodiment 1, wherein the absolute number of eosinophils in the subject before administration is about 100 eosinophils / mm. 3

[0185] 58. The absolute number of eosinophils in the subject before administration is about 125 eosinophils / mm 3 The method according to Embodiment 1, wherein the method is as described above.

[0186] 59. The absolute number of eosinophils in the subject before administration is about 150 eosinophils / mm 3 The method according to Embodiment 1, wherein the method is as described above.

[0187] 60. The absolute number of eosinophils in the subject before administration is about 175 eosinophils / mm 3 The method according to Embodiment 1, wherein the method is as described above.

[0188] 61. The absolute number of eosinophils in the subject before administration is about 200 eosinophils / mm 3 The method according to Embodiment 1, wherein the method is as described above.

[0189] 62. The absolute number of eosinophils in the subject before administration is about 225 eosinophils / mm 3 The method according to Embodiment 1, wherein the method is as described above.

[0190] 63. The absolute number of eosinophils in the subject before administration is about 250 eosinophils / mm 3 The method according to Embodiment 1, wherein the method is as described above.

[0191] 64. The absolute number of eosinophils in the subject before administration is about 275 eosinophils / mm 3 The method according to Embodiment 1, wherein the method is as described above.

[0192] 65. The absolute number of eosinophils in the subject before administration is about 300 eosinophils / mm 3 The method according to Embodiment 1, wherein the method is as described above.

[0193] 66. The absolute number of eosinophils in the subject before administration is about 325 eosinophils / mm 3 The method according to Embodiment 1, wherein the method is as described above.

[0194] 67. The absolute number of eosinophils in the subject before administration is about 350 eosinophils / mm 3 The method according to Embodiment 1, wherein the method is as described above.

[0195] 68. The absolute eosinophil count before administration to the subject is about 375 eosinophils / mm 3 The method according to embodiment 1, wherein the method is as described above.

[0196] 69. The absolute eosinophil count before administration to the subject is about 400 eosinophils / mm 3 The method according to embodiment 1, wherein the method is as described above.

[0197] 70. The absolute eosinophil count before administration to the subject is about 425 eosinophils / mm 3 The method according to embodiment 1, wherein the method is as described above.

[0198] 71. The absolute eosinophil count before administration to the subject is about 450 eosinophils / mm 3 The method according to embodiment 1, wherein the method is as described above.

[0199] 72. The absolute eosinophil count before administration to the subject is about 475 eosinophils / mm 3 The method according to embodiment 1, wherein the method is as described above.

[0200] 73. The absolute eosinophil count before administration to the subject is about 500 eosinophils / mm 3 The method according to embodiment 1, wherein the method is as described above.

[0201] 74. The absolute basophil count after administration to the subject decreases by at least about 5 basophils / mm 3 The method according to any one of embodiments 1 to 73, wherein the method is as described above.

[0202] 75. The absolute basophil count after administration to the subject decreases by at least about 10 basophils / mm 3 The method according to any one of embodiments 1 to 73, wherein the method is as described above.

[0203] 76. The absolute basophil count after administration to the subject decreases by at least about 15 basophils / mm 3 The method according to any one of embodiments 1 to 73, wherein the method is as described above.

[0204] 77. The absolute basophil count after administration to the subject decreases by at least about 20 basophils / mm 3The method according to any one of embodiments 1 to 73, which decreases.

[0205] 78. The absolute number of basophils in the subject after administration is at least about 25 basophils / mm 3 The method according to any one of embodiments 1 to 73, which decreases.

[0206] 79. The absolute number of basophils in the subject after administration is at least about 30 basophils / mm 3 The method according to any one of embodiments 1 to 73, which decreases.

[0207] 80. The absolute number of basophils in the subject after administration is at least about 35 basophils / mm 3 The method according to any one of embodiments 1 to 73, which decreases.

[0208] 81. The absolute number of basophils in the subject after administration is at least about 40 basophils / mm 3 The method according to any one of embodiments 1 to 73, which decreases.

[0209] 82. The absolute number of basophils in the subject after administration is at least about 45 basophils / mm 3 The method according to any one of embodiments 1 to 73, which decreases.

[0210] 83. The absolute number of basophils in the subject after administration is at least about 50 basophils / mm 3 The method according to any one of embodiments 1 to 73, which decreases.

[0211] 84. The absolute number of basophils in the subject after administration is at least about 55 basophils / mm 3 The method according to any one of embodiments 1 to 73, which decreases.

[0212] 85. The absolute number of basophils in the subject after administration is at least about 60 basophils / mm 3 The method according to any one of embodiments 1 to 73, which decreases.

[0213] 86. The absolute number of basophils in the subject after administration is at least about 65 basophils / mm 3 The method according to any one of embodiments 1 to 73, wherein the absolute number of basophils decreases.

[0214] 87. The absolute number of basophils in the subject after administration is at least about 70 basophils / mm 3 The method according to any one of embodiments 1 to 73, wherein the absolute number of basophils decreases.

[0215] 88. The absolute number of basophils in the subject after administration is from 0 to about 10 basophils / mm 3 The method according to any one of embodiments 1 to 73, wherein the absolute number of basophils is as such.

[0216] 89. The absolute number of basophils in the subject after administration is about 2 basophils / mm 3 The method according to any one of embodiments 1 to 73, wherein the absolute number of basophils is as such.

[0217] 90. The absolute number of basophils in the subject after administration is about 5 basophils / mm 3 The method according to any one of embodiments 1 to 73, wherein the absolute number of basophils is as such.

[0218] 91. The absolute number of basophils in the subject after administration is about 7 basophils / mm 3 The method according to any one of embodiments 1 to 73, wherein the absolute number of basophils is as such.

[0219] 92. The absolute number of basophils in the subject after administration is about 9 basophils / mm 3 The method according to any one of embodiments 1 to 73, wherein the absolute number of basophils is as such.

[0220] 93. The method according to any one of embodiments 1 to 73, wherein the decrease in basophils occurs within 48 hours after administration.

[0221] 94. The method according to any one of embodiments 1 to 73, wherein the decrease in basophils occurs within 24 hours after administration.

[0222] 95. The method according to any one of embodiments 1 to 94, wherein the IL-5R binding molecule is administered to the subject at a dose ranging from about 0.001 to about 100 mg / kg.

[0223] 96. The method according to embodiment 95, wherein the dose is about 0.03 mg / kg.

[0224] 97. The method according to embodiment 95, wherein the dose is 0.03 mg / kg.

[0225] 98. The method according to any one of embodiments 1 to 97, wherein the IL-5R binding molecule is administered parenterally.

[0226] 99. The method according to embodiment 98, wherein the IL-5R binding molecule is administered intravenously.

[0227] 100. The method according to any one of embodiments 1 to 99, wherein the IL-5R binding molecule is not MEDI-563.

[0228] 101. The method according to any one of embodiments 1 to 100, wherein the reduction of eosinophils results in a reduction of asthma symptoms.

[0229] 102. The method according to any one of embodiments 1 to 100, wherein the reduction of eosinophils results in a reduction of COPD symptoms.

Examples

[0230] The present invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only and the present invention should not be construed as being limited to these examples, but should be construed as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0231] Example 1 MEDI-563 (an anti-interleukin-5 receptor antibody) has good tolerance and induces reversible blood eosinopenia in mild asthmatic patients in a Phase I clinical trial Background: Eosinophils are thought to play an important role in the development of asthma. Interleukin-5 (IL-5) is a major cytokine in eosinophil biology, and the expression of its receptor (IL-5R) is generally limited to eosinophils, basophils, and mast cells. The suboptimal efficacy of IL-5-targeted therapy for asthma is due to an incomplete reduction of eosinophils in lung tissue. Complete pulmonary eosinophil depletion should provide further insight into the role of these cells in asthma and could represent a novel therapeutic strategy.

[0232] Objective: To evaluate the safety and biological activity of MEDI-563 (already known as BIW-8405), a humanized non-fucosylated IgG1 anti-IL-5Rα chain monoclonal antibody. MEDI-563 was developed by BioWa, Inc through proprietary Potelligent® technology that significantly enhances antibody-dependent cytotoxicity. MEDI-563 neutralizes IL-5 activity and reduces tissue eosinophils in preclinical models with an acceptable toxicological profile.

[0233] Methods: Six corticosteroid-naive subjects with mild asthma were enrolled in the first cohort of the BIW-8405-001 study, a non-blinded first-in-human trial using MEDI-563. Patients received a single intravenous dose of 0.03 mg / kg of MEDI-563 and were followed for 84 days.

[0234] Results: MEDI-563 was well tolerated and no serious adverse events were reported. All adverse events (AEs) were mild, and the most frequently reported AE was fatigue after dosing on the day of administration (3 / 6 subjects). Circulating eosinophils decreased below the limit of detection in all 6 subjects during the 24 - 48 hours after dosing (including the pre-dose mean value). This effect persisted for 8 - 12 weeks, and eosinophils became detectable in some subjects on day 58 after dosing and reached more than 70% of baseline levels in all subjects analyzed by day 84 after dosing. A similar trend occurred with circulating basophils. Although it may be related to the predicted mechanism of action of MEDI-563, neutrophil levels decreased slightly and transiently within 72 hours after dosing, reaching mild neutropenia levels in 2 / 6 subjects and resolving within 3 days. Administration of MEDI-563 was associated with an immediate (within 6 hours), mild (<10× baseline), and transient (less than 1 week) increase in serum C-reactive protein (2 / 6 subjects) and IL-6 (2 / 3).

[0235] Conclusion: Single intravenous administration of 0.03 mg / kg of MEDI-563 induces robust blood eosinopenia with an acceptable safety profile to date.

[0236] Example 2 Antibody-dependent cell-mediated cytotoxicity KC1333 effector cells (human NK cells overexpressing human FcgRIIIa and FceRIg) were co-incubated with target CTLL-2 cell line (mouse lymphoma genetically modified to overexpress human IL-5Ra) at an effector-to-target ratio of 5:1 in the presence of MEDI-563 antibody or control antibody for 4 hours. Antibody-mediated cytotoxicity was evaluated using the Calcein AM cell viability assay. The results are summarized in Figure 9A. Using a similar methodology, an additional control (fucosylated MEDI-563) was analyzed. The results are summarized in Figure 9B.

[0237] Example 3 Surface plasmon resonance evaluation of the equilibrium binding between MEDI-563 and IL-5R The soluble human IL-5Ra extracellular domain, without carrier, was obtained from a commercial source (R+D Systems). Using standard protocols, recombinant huIL-5Ra was directly immobilized on the sensor chip via amine coupling. The interaction between MEDI-563 and immobilized huIL-5Ra was evaluated over time by changes in refractive index, from which the values of k on , k off and K D were calculated using standard techniques. The results are summarized in Figure 10.

[0238] Example 4 Surface plasmon resonance evaluation of the equilibrium binding between MEDI-563 and FcγR Using standard protocols, MEDI-563 was directly immobilized on the sensor chip via amine coupling. The interaction between soluble human FcγR (MedImmune) and immobilized MEDI-563 was evaluated over time by changes in refractive index, from which the values of k on , k off and K D were calculated using standard techniques. The results are summarized in Figure 11.

[0239] Example 5 Immunohistochemical analysis of IL-5Rα The excised nasal polyp tissues were fixed with formaldehyde for 24 hours and embedded in paraffin. Serial sections were stained for human IL-5Ra, IL-9R, CCR3 and c-kit using commercially available polyclonal antibodies against IL-5R (R+D Systems, Santa Cruz Biotechnology) and standard techniques. Lung tissues derived from IL-9 transgenic mice or wild-type syngeneic FVB control mice were fixed with formaldehyde for 24 hours and embedded in paraffin. Sections were analyzed for the expression of IL-9R (pAb, Santa Cruz Biotechnology) and IL-5R (pAb, R+D Systems) using standard immunohistochemical techniques. The results are summarized in Figures 12 and 13.

[0240] Example 6 MEDI-563 binds to eosinophils in the whole blood of healthy donors Granulocytes were isolated from the whole human blood of normal donors by density gradient centrifugation. Directly labeled primary antibody reagents were used for the analysis of the expression of CD16 (FITC fluorochrome) and MEDI-563 F(ab)'2 (Alexa-647 fluorochrome). A cocktail of CD16-FITC plus MEDI-563-Alexa647 or CD16-FITC plus Alexa647-labeled isotype control antibody was added to the granulocyte preparation at 1 μg / 10 6 cells. After incubation on ice for 45 minutes, the cells were washed three times with cold saline, and cell surface antibody binding was evaluated by flow cytometry. Eosinophils that were negative for CD16 were analyzed. Binding of MEDI-563 and the isotype control antibody in the CD16-negative granulocyte population is represented. The results are summarized in Figure 15.

[0241] Example 7 IL-5Rα staining of mouse leukocytes by flow cytometry Leukocytes were isolated from the blood, bone marrow, lung, and spleen of IL-5 transgenic mice. The cell suspension was stained in PBS containing 1% FCS. To reduce non-specific binding, the cells were incubated with Fc Block (BD Biosciences) for 15 minutes before staining. The antibodies used were anti-mouse CCR3 (R&D systems), anti-mouse Siglec F (BD Biosciences), and anti-mouse IL-5R (H7). The cells were stained on ice for 30 minutes, washed twice, and fixed with cytofix buffer (BD Biosciences). Flow cytometry analysis was performed using an LSRII (Becton Dickinson) and FACS Diva software (Becton Dickinson). The results were analyzed using FlowJo Software (TreeStar Inc.). The results are summarized in Figures 16A and 16B.

[0242] Example 8 MEDI-563 reduces bone marrow-derived IL-5Ra-positive mononuclear cells Frozen bone marrow mononuclear cells (BM MNC; Lonza) were thawed, washed, seeded, and incubated at 37 °C for 2 hours. After incubation, non-adherent bone marrow mononuclear cells (NA BM MNC) were recovered from the plates. The ADCC assay was performed by co-incubating 100,000 NA BM MNC cells and 50,000 KC1333 effector cells / well in 200 ul of 10% FBS / RPMI 1640 in 96-well TC plates in the presence of 10 ug / ml of the MEDI-563 antibody for 18 hours. The negative control reaction was performed using an R347 aFuc isotype control antibody of irrelevant specificity. The KC1333 effector cells used in the ADCC assay were painted with CFDA SE. After 18 hours of incubation, the cells from each reaction were washed three times with warm medium and immunostained for flow cytometry. IL-5Ra-positive cells were detected by staining with the KM1257 primary antibody / PE-labeled goat anti-Mu IgG Fcg-specific secondary antibody. Control staining of the samples was performed using a combination of a 1A7 isotype-matched control primary antibody and a PE-labeled goat anti-Mu IgG Fcg-specific secondary antibody. Immunostaining and flow cytometry were performed using standard protocols. The number of IL-5Ra-positive cells remaining in the samples after ADCC was determined by counting the number of KM1257-positive cells in the lymphocyte gate. The immunostaining and flow cytometry procedures were calibrated using the CTLL-2 cell line expressing the human IL-5Ra transgene. MEDI-563-mediated ADCC substantially reduced all IL-5Ra-positive cells derived from the NA BM MNC samples. The results are summarized in Figures 17A and 17B.

[0243] Example 9 MEDI-563-mediated reduction of peripheral blood eosinophils Two cohorts of six subjects with mild asthma were enrolled in an open-label trial of MEDI-563. Subjects in cohorts 1 and 2 received a single intravenous dose of MEDI-563 at 0.03 mg / kg and 0.1 mg / kg, respectively, and their peripheral blood eosinophil levels were counted at screening, on day 0 before dosing, and at regular intervals and follow-up through day 84. Circulating eosinophils were detected by flow cytometry. Circulating eosinophils decreased below the limit of detection within 24 hours of dosing in all six subjects in both cohorts. MEDI-563-induced eosinopenia persisted for 8 to 12 weeks. In cohort 1 of five subjects in whom the 84-day study was completed after a single dose of 0.03 mg / kg of MEDI-563, eosinophils became detectable in one subject on day 58 and in three subjects on day 84, and the fifth subject did not have detectable circulating eosinophils on day 84. After a single dose of 0.1 mg / kg of MEDI-563, no subjects in cohort 2 had detectable circulating eosinophils on day 84. However, peripheral blood eosinophils were detectable in all six subjects in cohort 2 on subsequent follow-up examinations. The levels of peripheral blood eosinophils detected in cohorts 1 and 2 at various time intervals after a single dose of MEDI-563 are presented in FIGS. 18A and 18B.

[0244] Example 10 Immunohistochemical analysis of IL-5Rα Lung sections from healthy human subjects were stained with MEDI-563 using standard histochemical techniques. The results are summarized in FIG. 19. IL-5Rα-expressing cells appear black in the image.

[0245] Lung tissue samples obtained from bronchial or transbronchial biopsies of asthmatic patients were stained with MEDI-563 using standard histochemical techniques. The results are summarized in FIG. 20. IL-5Rα-expressing cells appear dark gray / black in the image.

[0246] Example 11 MEDI-563 efficiently targets isolated basophils and eosinophils in an in vitro ADCC assay Basophils and eosinophils were isolated from healthy donors using a commercially available kit (RoboSep NK / Eosinophil / Basophil Negative Selection Kit, Stem Cell Technologies, Vancouver, Canada). The expression of IL-5Rα in the isolated cells was determined by flow cytometry. The cells were stained with the MEDI-563 antibody or an isotype control antibody of irrelevant specificity according to a standard protocol. The immunostained cells were analyzed by flow cytometry. The staining profiles are shown in Figure 21. Both the isolated basophils and eosinophils showed MEDI-563 staining levels that were above those observed using the isotype control antibody. The staining pattern of a cell line expressing the human IL-5Rα / β transgene is shown as a positive control.

[0247] The activities of fucosylated and non-fucosylated MEDI-563 were determined in an in vitro ADCC assay using isolated eosinophils and autologous NK cells. Eosinophils and NK cells were isolated from healthy donors using a commercially available kit (RoboSep NK / Eosinophil / Basophil Negative Selection Kit, Stem Cell Technologies, Vancouver, Canada). The ADCC assay was performed using the isolated NK cells and eosinophils as effector and target cells, respectively, at a ratio of 5:1. The antibody concentrations tested were 10 -15 ~10 -7 M. Cytotoxicity was measured using flow cytometry based on the annexin V assay after 24 hours of incubation. The ADCC activity of non-fucosylated MEDI-563 was several orders of magnitude higher than that of the fucosylated MEDI-563 antibody. The EC 50 value of non-fucosylated MEDI-563 was 0.965 pM in this assay. The results of a representative experiment are shown in Figure 22.

[0248] The activity of afucosylated MEDI-563 was determined in an in vitro ADCC assay using isolated basophils and autologous NK cells. Basophils and NK cells were isolated from healthy donors using a commercially available kit (RoboSep NK / Eosinophil / Basophil Negative Selection Kit, Stem Cell Technologies, Vancouver, Canada). The ADCC assay was performed using the isolated NK cells and eosinophils as effector and target cells, respectively, at a ratio of 5:1. The antibody concentrations tested were 10 -15 ~10 -11 M. Cytotoxicity was measured by determining annexin V-positive cells by flow cytometry after 24 hours of incubation. The EC 50 value of afucosylated MEDI-563 was 0.561 pM in this assay. The results of a representative experiment are shown in Figure 23.

[0249] Example 12 Eosinophils do not release cytotoxic granules in a MEDI-563-mediated ADCC assay Degranulation of eosinophils exposed to MEDI-563-targeted ADCC was determined by measuring the release of EDN (eosinophil-derived neurotoxin) into the supernatant. The in vitro ADCC conditions used were the same as those described in Example 11. Eosinophils and NK or PBMC cells isolated from healthy donors were used as target and effector cells, respectively. The assay was performed using fucosylated MEDI-563, afucosylated MEDI-563 or afucosylated R347 isotype control antibody. Maximum degranulation was achieved by exposing eosinophils to 1% triton X-100, and the EDN concentration detected at maximum degranulation of the cells was higher than 220 ng / ml. The results of a representative experiment are shown in Figure 24. The level of EDN remained below 25 ng / ml (baseline) after MEDI-563-mediated ADCC. The MEDI-563 concentration (33 or 100 μg / ml) or the fucosylation status of the antibody did not significantly affect the level of degranulation.

[0250] Example 13 Epitope mapping of MEDI-563 MEDI-563 specifically binds to transgenic cells expressing the human IL-5Rα protein. MEDI-563 does not bind to cells expressing the mouse IL-5Rα protein. See FIGS. 25B and 26C. The amino acid sequences of the mouse and human IL5-Rα proteins are very similar. The epitope specificity of MEDI-563 was determined by analyzing the binding characteristics of MEDI-563 to a large panel of mouse-human chimeric IL-5Rα proteins (FIGS. 25-27). The experiments utilized transgenic cells expressing the chimeric IL-5Rα protein on the cell surface. The transgene constructs were made and expressed using standard molecular methods. The binding of antibodies to the chimeric IL-5Rα protein expressed on the surface of the transgenic cells was determined by flow cytometry. The fluorescence staining profiles are shown in FIGS. 25-27. "Polyclonal" and "MEDI-563" represent the staining profiles observed using the polyclonal anti-human IL-5Rα antibody and MEDI-563, respectively. MEDI-563 is specific for a single epitope of the human IL-5Rα protein, whereas the polyclonal antibody recognizes multiple epitopes of human IL-5Rα (FIGS. 25B and 26C). "Double staining" represents the fluorescence staining profile with respect to the polyclonal antibody (x-axis) and the MEDI-563 antibody (y-axis).

[0251] First, the epitope of MEDI-563 was mapped to the D1 region of the extracellular domain of IL-5Rα. IL-5Rα contains three extracellular domains (D1, D2, and D3), a transmembrane domain, and an intracellular domain (Figure 25A). Since MEDI-563 recognizes IL-5Rα on intact cells, its epitope must be located in one of the extracellular domains. To map the epitope of MEDI-563 to one of the three extracellular domains, transgenic cells expressing chimeric IL-5Rα proteins containing mouse and human extracellular domains were generated using standard molecular cloning methods. A schematic of the chimeric proteins tested is shown in Figure 25A. The “knockout” mutants were chimeric IL-5Rα proteins containing a single mouse extracellular domain in a different human background. The “knockin” mutants were chimeric IL-5Rα proteins containing a single human extracellular domain in a different mouse background.

[0252] Figures 25B - C show the results of representative experiments. Both MEDI - 563 and the polyclonal antibody stained transgenic cells expressing human IL - 5Rα protein, and neither antibody stained transgenic cells expressing mouse IL - 5Rα (Figure 25B). MEDI - 563 did not bind to transgenic cells expressing a chimeric IL - 5Rα transgene containing the mouse D1 and human D2 - D3 extracellular domains (Figure 25C; "knockout D1"). MEDI - 563 specifically bound to transgenic cells expressing a chimeric IL - 5Rα transgene containing the mouse D2 or D3 extracellular domain in a human background (Figure 25C; "knockout D2 or D3"). MEDI - 563 specifically bound to transgenic cells expressing a chimeric IL - 5Rα transgene containing the human D1 and mouse D2 - D3 extracellular domains (Figure 25D; "knockin D1"). MEDI - 563 did not bind to transgenic cells expressing mouse IL - 5Rα based on a chimeric transgene containing either the human D2 or D3 extracellular domain (Figure 25D; "knockin D2 or D3"). All cells expressing a chimeric IL - 5Rα protein containing at least one extracellular domain of the human protein were stained by the polyclonal anti - human IL - 5Rα antibody, indicating that the differences in the staining pattern of MEDI - 563 in transgenic cells are not due to differences in the expression levels of the chimeric proteins.

[0253] Second, the epitope of MEDI-563 was mapped to segment B of the D1 extracellular domain of human IL-5Rα (Figure 26). The D1 extracellular domain of IL-5Rα was divided into three segments (Figure 26A; segments A, B, and C). A series of human-mouse chimeric IL-5Rα transgenes containing various combinations of human and mouse segments of the D1 extracellular domain were generated, and the chimeric proteins used at this stage contained all human sequences outside the D1 extracellular domain. The “knockout” transgene was a chimeric IL-5Rα construct containing a single mouse segment of the D1 extracellular domain in another human background. The “knock-in” transgene was a chimeric IL-5Rα construct containing a single human segment of the D1 extracellular domain in a mouse D1-human D2-mouse D3-mouse TM background (Figure 26B). Figure 26C shows the results of a control experiment. MEDI-563 specifically recognized transgenic cells expressing (i) a human IL-5Rα transgene or (ii) a mouse IL-5Rα chimeric transgene containing the human D1 extracellular domain (“human IL-5Ra” and “knock-in D1”). MEDI-563 did not bind to transgenic cells expressing (i) a mouse IL-5Rα receptor transgene or (ii) a human chimeric IL-5Rα transgene containing the mouse D1 extracellular domain (“mouse IL-5Ra”, “knockout-D1”). Figures 26D and E show the results of representative mapping experiments. MEDI-563 did not bind to transgenic cells expressing a chimeric IL-5Rα transgene (“knockout B”) containing the mouse segment B of the D1 extracellular domain in another human background. MEDI-563 specifically bound to transgenic cells expressing a chimeric IL-5Rα transgene (“knockout A”, “knockout-C”) containing the mouse segment A or C of the D1 extracellular domain in a human background. Figure 26E shows an example of the results obtained with the knock-in construct.MEDI-563 specifically bound to transgenic cells expressing a chimeric IL-5Rα transgene (the "knock-in B") containing the human segment B of the D1 extracellular domain in a mouse D1-human D2-mouse D3-mouse TM background. MEDI-563 did not bind to transgenic cells expressing a chimeric IL-5Rα transgene (the "knock-in A or C") containing the human segment A or C of the D1 extracellular domain in a mouse D1-human D2-mouse D3-mouse TM background. All cells expressing the chimeric IL-5Rα protein were stained with a polyclonal anti-human IL-5Rα antibody, indicating that the difference in the staining pattern of MEDI-563 in transgenic cells was not due to differences in the expression levels of the chimeric proteins.

[0254] Third, the epitopes of MEDI-563 were mapped to specific amino acid residues within segment B1 of the D1 extracellular domain of human IL-5Rα. A series of IL-5Rα receptor mutants containing at least one mutant amino acid residue within segment B1 of the D1 extracellular domain were expressed in transgenic cells. The positions of the mutant residues were selected by comparing the mouse and human amino acid sequences. A schematic of the mutant proteins tested is shown in FIG. 27A. The "knockout" IL-5Rα mutants were mutant human proteins containing at least one substitution that exchanges a human residue for the corresponding mouse residue. For example, the "knockout DE" mutant was a human IL-5Rα protein containing the amino acid substitutions D56E and E58D. The "knockin" IL-5Rα mutants were chimeric proteins containing mouse D1, human D2, mouse D3, and mouse TM, and a mutant form of mouse segment B in which the mouse D1 domain had at least one substitution that exchanges a mouse residue for the corresponding human residue. For example, the "knockin DE" mutant was a chimeric IL-5Rα protein containing the mutant mouse segment B in a mouse D1-human D2-mouse D3-mouse TM background, where the mutant mouse segment B contained the amino acid substitutions E56D and D58E. FIG. 27B shows an example of the results obtained using the knockout constructs. MEDI-563 did not bind to transgenic cells expressing a mutant human IL-5Rα protein containing the amino acid substitutions K53Q, D56E, E58D, I61K ("knockout-KDEI"). MEDI-563 specifically bound to transgenic cells expressing a mutant human IL-5Rα protein containing the amino acid substitutions N40H, N42D, Q46H ("knockout-NNQ") or D56E, E58D ("knockout-DE") or N40H, N42D, D56E, E58D ("knockout-NNDE"). FIG. 27C shows an example of the results obtained using the knockin constructs.MEDI-563 specifically bound to transgenic cells expressing a mutant IL-5Rα protein containing the D1 mutant mouse segment B with amino acid substitutions of Q53K, E56D, D58E, and K61I (“knock-in - KDEI”). FIG. 27D shows an example of the results obtained using a knockout construct. MEDI-563 did not bind to transgenic cells expressing a mutant human IL-5Rα protein containing an amino acid substitution of I61K (“knockout - I61”). MEDI-563 specifically bound to transgenic cells expressing a mutant human IL-5Rα protein containing an amino acid substitution of K53Q (“knockout - K53”). (E) FIG. 27E shows an example of the results obtained using a knock-in construct. MEDI-563 specifically bound to transgenic cells expressing a mutant IL-5Rα protein containing the D1 mutant mouse segment B with an amino acid substitution of K61I (“knock-in - I61”). MEDI-563 did not bind to transgenic cells expressing a mutant IL-5Rα protein containing the D1 mutant mouse segment B with an amino acid substitution of Q53K (“knock-in - K53”). All cells expressing the chimeric IL-5Rα protein were stained with a polyclonal anti-human IL-5Rα antibody, indicating that the difference in the staining pattern of MEDI-563 in transgenic cells was not due to differences in the expression levels of the chimeric proteins.

[0255] Example 14 In vivo reduction of eosinophils from various tissues The inventors evaluated the effectiveness of the afucosylated anti-mouse IL-5Ra antibody (afuc H7) for selectively reducing eosinophils from various tissues in vivo in comparison with fucosylated H7 (fuc H7).

[0256] Methods: Monoclonal antibody H7: The variable region of H7 was grafted onto hIgG1 Fc. Fuc H7 was expressed in wild-type CHO cells, and afuc H7 was expressed in FUT8-deficient CHO cells.

[0257] Antibody affinity (KD): Affinity was measured using surface plasmon resonance technology.

[0258] Mice: IL-5 transgenic mice and BALB / c mice were used at 6 - 8 weeks of age.

[0259] Reduction of eosinophils in IL-5Tg mice: Mice were intraperitoneally administered with 0.01 - 10 mg / kg of afuc and fuc H7, and the number of eosinophils was analyzed 48 hours later.

[0260] Induction of allergic airway inflammation: BALB / c mice were sensitized to OVA in alum, and challenged with OVA on days 17 - 22. Mice were intraperitoneally administered with 0.1 mg / kg of afuc H7 on day 22, and analysis was performed 1 hour, 24 hours and 72 hours after the final challenge. This corresponded to 25, 48 and 96 hours after antibody treatment.

[0261] Isolation of leukocytes: (i) Blood: Blood was collected by cardiac puncture and stored in heparin tubes. Blood leukocytes were phenotypically analyzed using a Sysmex Hematology Analyser (Sysmex Corp.) or by flow cytometry.

[0262] (ii) Airway lumen: The airway was washed with 3 × 0.6 ml of PBS. The BAL sample was centrifuged at 1200 rpm, the supernatant was removed, and the cells were resuspended in RPMI. The cells were counted using a Coulter Z2 counter (Beckman-Coulter) and phenotypically analyzed by flow cytometry.

[0263] (iii) Lung tissue: One lobe of the lung was incubated in a digestion reagent (18 μg / ml of Liberase [Blenzyme2; Roche], 25 μg / ml of DNase [type 1; Roche]) in RPMI / 10% FCS at 37°C for 1 hour. The recovered cells were filtered through a 70 μm nylon sieve (Falcon), washed twice, counted, and phenotypically analyzed for BAL.

[0264] (iv) Bone marrow: The femurs were isolated from donor mice, and the bone marrow was flushed out using a syringe containing PBS (calcium / magnesium-free) with a 25-gauge needle attached. A single cell suspension was prepared by gently pipetting the bone marrow up and down in a syringe with a 22-gauge needle attached. The bone marrow cells were centrifuged at 1200 rpm for 5 minutes, washed twice with PBS without additives, resuspended in RPMI, counted, and phenotypically analyzed by flow cytometry.

[0265] (v) Spleen: The spleen was removed, and a single cell suspension was prepared using a 70-μm nylon mesh. The leukocytes were resuspended in RPMI, counted, and phenotypically analyzed for BAL.

[0266] Flow cytometry: Flow cytometry analysis was used to analyze the phenotype of the cells. The antibodies used were anti-mouse CD4, CD19, CD11b, Siglec-F, Gr-1, IL-5R, c-kit (BD Biosciences), FcεR1 (eBiosciences), and CCR-3 (R and D Systems) as well as their corresponding isotype controls. The samples were analyzed using an LSRII flow cytometer and FACS DIVA software (BD Biosciences). The results were further analyzed using FlowJo (TreeStar Corp.).

[0267] Identification of eosinophils: Eosinophils were identified by flow cytometry analysis as cells with high side scatter that stained positive for CCR3 and Siglec-F.

[0268] Results: IL-5R was selectively expressed by eosinophils in the bone marrow, blood, spleen, and lung tissues of IL-5Tg mice. The expression of IL-5R was restricted to eosinophils and was not detected in any other cell type, including mast cells or basophils. Anti-IL-5R antibody selectively reduced eosinophils in the spleen, lung tissues, and blood of IL-5Tg mice. Neither the non-fucosylated nor fucosylated anti-IL-5R reduced neutrophils (Gr-1hi); macrophages / monocytes (CD11b+); T cells (CD3+); or B cells (CD19+). Both afuc and fuc H7 reduced eosinophils in the spleen, lung tissues, and blood of IL-5Tg mice. In the bone marrow, no reduction was detected. Afuc H7 was more potent than fuc H7 in eosinophil depletion, particularly at low antibody doses.

[0269] Afuc H7 also selectively reduced eosinophils in the allergen challenge model. Afuc H7 reduced eosinophils in the airway lumen, lung tissues, blood, and bone marrow. The reduction was highest at all sites 72 hours after the final challenge (96 hours after antibody delivery).

[0270] Certain embodiments of the invention have been described above for purposes of illustration, but it will be apparent to those skilled in the art that many more modifications can be made without departing from the invention as set forth in the appended claims.

[0271] All publications, patents, and patent applications cited herein are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Further, U.S. Provisional Patent Application Nos. 60 / 924,422 (filed May 14, 2007), 60 / 924,832 (filed Jun. 1, 2007), 60 / 935,005 (filed Jul. 20, 2007), and 61 / 064,612 (filed Mar. 14, 2008) are hereby incorporated by reference in their entirety for all purposes.

[0272] SEQUENCE LISTING <110> AstraZeneca AB Kyowa Kirin Co., Ltd. <120> METHODS OF REDUCING EOSINOPHIL LEVELS <130> PA25-092 <150> US 60 / 924,422 <151> 2007-05-14 <150> US 60 / 924,832 <151> 2007-06-01 <150> US 60 / 935,005 <151> 2007-07-20 <150> US 61 / 064,612 <151> 2008-03-14 <160> 6 <170> PatentIn version 3.3 <210> 1 <211> 107 <212> PRT <213> Homo sapiens <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Thr Ser Glu Asp Ile Ile Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 2 <211> 214 <212> PRT <213> Homo sapiens <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Thr Ser Glu Asp Ile Ile Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 3 <211> 121 <212> PRT <213> Homo sapiens <400> 3 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Ile His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Ala Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Arg Phe 50 55 60 Lys Gly Lys Val Thr Ile Thr Ser Asp Arg Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Leu Cys 85 90 95 Gly Arg Glu Gly Ile Arg Tyr Tyr Gly Leu Leu Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 451 <212> PRT <213> Homo sapiens <400> 4 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Ile His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Ala Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Arg Phe 50 55 60 Lys Gly Lys Val Thr Ile Thr Ser Asp Arg Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Leu Cys 85 90 95 Gly Arg Glu Gly Ile Arg Tyr Tyr Gly Leu Leu Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 5 <211> 400 <212> PRT <213> Homo sapiens <400> 5 Asp Leu Leu Pro Asp Glu Lys Ile Ser Leu Leu Pro Pro Val Asn Phe 1 5 10 15 Thr Ile Lys Val Thr Gly Leu Ala Gln Val Leu Leu Gln Trp Lys Pro 20 25 30 Asn Pro Asp Gln Glu Gln Arg Asn Val Asn Leu Glu Tyr Gln Val Lys 35 40 45 Ile Asn Ala Pro Lys Glu Asp Asp Tyr Glu Thr Arg Ile Thr Glu Ser 50 55 60 Lys Cys Val Thr Ile Leu His Lys Gly Phe Ser Ala Ser Val Arg Thr 65 70 75 80 Ile Leu Gln Asn Asp His Ser Leu Leu Ala Ser Ser Trp Ala Ser Ala 85 90 95 Glu Leu His Ala Pro Pro Gly Ser Pro Gly Thr Ser Ile Val Asn Leu 100 105 110 Thr Cys Thr Thr Asn Thr Thr Glu Asp Asn Tyr Ser Arg Leu Arg Ser 115 120 125 Tyr Gln Val Ser Leu His Cys Thr Trp Leu Val Gly Thr Asp Ala Pro 130 135 140 Glu Asp Thr Gln Tyr Phe Leu Tyr Tyr Arg Tyr Gly Ser Trp Thr Glu 145 150 155 160 Glu Cys Gln Glu Tyr Ser Lys Asp Thr Leu Gly Arg Asn Ile Ala Cys 165 170 175 Trp Phe Pro Arg Thr Phe Ile Leu Ser Lys Gly Arg Asp Trp Leu Ala 180 185 190 Val Leu Val Asn Gly Ser Ser Lys His Ser Ala Ile Arg Pro Phe Asp 195 200 205 Gln Leu Phe Ala Leu His Ala Ile Asp Gln Ile Asn Pro Pro Leu Asn 210 215 220 Val Thr Ala Glu Ile Glu Gly Thr Arg Leu Ser Ile Gln Trp Glu Lys 225 230 235 240 Pro Val Ser Ala Phe Pro Ile His Cys Phe Asp Tyr Glu Val Lys Ile 245 250 255 His Asn Thr Arg Asn Gly Tyr Leu Gln Ile Glu Lys Leu Met Thr Asn 260 265 270 Ala Phe Ile Ser Ile Ile Asp Asp Leu Ser Lys Tyr Asp Val Gln Val 275 280 285 Arg Ala Ala Val Ser Ser Met Cys Arg Glu Ala Gly Leu Trp Ser Glu 290 295 300 Trp Ser Gln Pro Ile Tyr Val Gly Asn Asp Glu His Lys Pro Leu Arg 305 310 315 320 Glu Trp Phe Val Ile Val Ile Met Ala Thr Ile Cys Phe Ile Leu Leu 325 330 335 Ile Leu Ser Leu Ile Cys Lys Ile Cys His Leu Trp Ile Lys Leu Phe 340 345 350 Pro Pro Ile Pro Ala Pro Lys Ser Asn Ile Lys Asp Leu Phe Val Thr 355 360 365 Thr Asn Tyr Glu Lys Ala Gly Ser Ser Glu Thr Glu Ile Glu Val Ile 370 375 380 Cys Tyr Ile Glu Lys Pro Gly Val Glu Thr Leu Glu Asp Ser Val Phe 385 390 395 400 <210> 6 <211> 398 <212> PRT <213> Mus musculus <400> 6 Asp Leu Leu Asn His Lys Lys Phe Leu Leu Leu Pro Pro Val Asn Phe 1 5 10 15 Thr Ile Lys Ala Thr Gly Leu Ala Gln Val Leu Leu His Trp Asp Pro 20 25 30 Asn Pro Asp Gln Glu Gln Arg His Val Asp Leu Glu Tyr His Val Lys 35 40 45 Ile Asn Ala Pro Gln Glu Asp Glu Tyr Asp Thr Arg Lys Thr Glu Ser 50 55 60 Lys Cys Val Thr Pro Leu His Glu Gly Phe Ala Ala Ser Val Arg Thr 65 70 75 80 Ile Leu Lys Ser Ser His Thr Thr Leu Ala Ser Ser Trp Val Ser Ala 85 90 95 Glu Leu Lys Ala Pro Pro Gly Ser Pro Gly Thr Ser Val Thr Asn Leu 100 105 110 Thr Cys Thr Thr His Thr Val Val Ser Ser His Thr His Leu Arg Pro 115 120 125 Tyr Gln Val Ser Leu Arg Cys Thr Trp Leu Val Gly Lys Asp Ala Pro 130 135 140 Glu Asp Thr Gln Tyr Phe Leu Tyr Tyr Arg Phe Gly Val Leu Thr Glu 145 150 155 160 Lys Cys Gln Glu Tyr Ser Arg Asp Ala Leu Asn Arg Asn Thr Ala Cys 165 170 175 Trp Phe Pro Arg Thr Phe Ile Asn Ser Lys Gly Phe Glu Gln Leu Ala 180 185 190 Val His Ile Asn Gly Ser Ser Lys Arg Ala Ala Ile Lys Pro Phe Asp 195 200 205 Gln Leu Phe Ser Pro Leu Ala Ile Asp Gln Val Asn Pro Pro Arg Asn 210 215 220 Val Thr Val Glu Ile Glu Ser Asn Ser Leu Tyr Ile Gln Trp Glu Lys 225 230 235 240 Pro Leu Ser Ala Phe Pro Asp His Cys Phe Asn Tyr Glu Leu Lys Ile 245 250 255 Tyr Asn Thr Lys Asn Gly His Ile Gln Lys Glu Lys Leu Ile Ala Asn 260 265 270 Lys Phe Ile Ser Lys Ile Asp Asp Val Ser Thr Tyr Ser Ile Gln Val 275 280 285 Arg Ala Ala Val Ser Ser Pro Cys Arg Met Pro Gly Arg Trp Gly Glu 290 295 300 Trp Ser Gln Pro Ile Tyr Val Gly Lys Glu Arg Lys Ser Leu Val Glu 305 310 315 320 Trp His Leu Ile Val Leu Pro Thr Ala Ala Cys Phe Val Leu Leu Ile 325 330 335 Phe Ser Leu Ile Cys Arg Val Cys His Leu Trp Thr Arg Leu Phe Pro 340 345 350 Pro Val Pro Ala Pro Lys Ser Asn Ile Lys Asp Leu Pro Val Val Thr 355 360 365 Glu Tyr Glu Lys Pro Ser Asn Glu Thr Lys Ile Glu Val Val His Cys 370 375 380 Val Glu Glu Val Gly Phe Glu Val Met Gly Asn Ser Thr Phe 385 390 395

Claims

1. A method for reducing the number of eosinophils in a human subject, comprising administering to the subject an IL-5R binding molecule comprising (a) a region that specifically binds to IL-5R and (b) an immunoglobulin Fc region.

2. The method of claim 1 , wherein the IL-5R binding molecule is an antibody.

3. The method of claim 2 , wherein the antibody is a monoclonal antibody.

4. The method of claim 3 , wherein the antibody is a chimeric antibody.

5. The method of claim 3 , wherein the antibody is a humanized antibody.

6. The method of claim 3 , wherein the antibody is a human antibody.

7. The method of claim 1 , wherein the region that specifically binds to IL-5R comprises the amino acid sequence of IL-5, or a fragment, substitution, or derivative thereof.

8. The method of claim 7, wherein the region that specifically binds to IL-5R comprises a non-functional mutant of IL-5.

9. The method of any one of claims 1 to 8, wherein the IL-5R binding molecule specifically binds to the IL-5Rα chain.

10. The method of claim 1 , wherein the immunoglobulin Fc region is modified to increase effector function.

11. The method of claim 1, wherein the immunoglobulin Fc region comprises a reduced level of fucose.

12. The method of claim 11 , wherein the immunoglobulin Fc region does not contain fucose.

13. The method of claim 1 , wherein the immunoglobulin Fc region comprises an amino acid substitution that results in increased effector function.

14. 2. The method of claim 1, wherein the amino acid substitutions comprise inclusion of the following amino acid sequences in the Fc region: 332E, 239D and 330L (numbered according to the EU index according to Kabat).

15. The method of claim 1 , wherein the reduction in eosinophils occurs in the peripheral blood circulation.

16. Eosinophil count 50 eosinophils / mm 3 The method of claim 1, wherein the concentration of

17. 2. The method of claim 1, wherein the reduction in eosinophils occurs within 48 hours of administration.

18. 2. The method of claim 1, wherein the reduction in eosinophils occurs within 24 hours of administration.

19. The method of claim 1 , wherein the reduction in eosinophils is reversible.

20. A post-treatment reduction in absolute eosinophil count of at least about 25 eosinophils / mm 3 The method of claim 1, wherein

21. A post-treatment reduction in absolute eosinophil count of at least about 50 eosinophils / mm 3 The method of claim 1, wherein

22. A post-treatment reduction in absolute eosinophil count of at least about 75 eosinophils / mm 3 The method of claim 1, wherein

23. A post-treatment reduction in absolute eosinophil count of at least about 100 eosinophils / mm 3 The method of claim 1, wherein

24. A post-treatment reduction in absolute eosinophil count of at least about 125 eosinophils / mm 3 The method of claim 1, wherein

25. A post-treatment reduction in absolute eosinophil count of at least about 150 eosinophils / mm 3 The method of claim 1, wherein

26. A post-treatment reduction in absolute eosinophil count of at least about 175 eosinophils / mm 3 The method of claim 1, wherein

27. A post-treatment reduction in absolute eosinophil count of at least about 200 eosinophils / mm 3 The method of claim 1, wherein

28. A post-treatment reduction in absolute eosinophil count of at least about 225 eosinophils / mm 3 The method of claim 1, wherein

29. A post-treatment reduction in absolute eosinophil count of at least about 250 eosinophils / mm 3 The method of claim 1, wherein

30. A post-treatment reduction in absolute eosinophil count of at least about 275 eosinophils / mm 3 The method of claim 1, wherein

31. A post-treatment reduction in absolute eosinophil count of at least about 300 eosinophils / mm 3 The method of claim 1, wherein

32. A post-treatment reduction in absolute eosinophil count of at least about 325 eosinophils / mm 3 The method of claim 1, wherein

33. A post-treatment reduction in absolute eosinophil count of at least about 350 eosinophils / mm 3 The method of claim 1, wherein

34. A post-treatment reduction in absolute eosinophil count of at least about 375 eosinophils / mm 3 The method of claim 1, wherein

35. A post-treatment reduction in absolute eosinophil count of at least about 400 eosinophils / mm 3 The method of claim 1, wherein

36. A post-treatment reduction in absolute eosinophil count of at least about 425 eosinophils / mm 3 The method of claim 1, wherein

37. A post-treatment reduction in absolute eosinophil count of at least about 450 eosinophils / mm 3 The method of claim 1, wherein

38. A post-treatment reduction in absolute eosinophil count of at least about 475 eosinophils / mm 3 The method of claim 1, wherein

39. A post-treatment reduction in absolute eosinophil count of at least about 500 eosinophils / mm 3 The method of claim 1, wherein

40. Post-treatment reduction in absolute eosinophil count from about 50 to about 500 eosinophils / mm 3 The method of claim 1, wherein

41. Post-treatment reduction in absolute eosinophil count from about 75 to about 250 eosinophils / mm 3 The method of claim 1, wherein

42. Post-treatment reduction in absolute eosinophil count of about 100 to about 200 eosinophils / mm 3 The method of claim 1, wherein

43. Post-treatment reduction in absolute eosinophil count from about 50 to about 250 eosinophils / mm 3 The method of claim 1, wherein

44. Post-treatment reduction in absolute eosinophil count from about 50 to about 200 eosinophils / mm 3 The method of claim 1, wherein

45. Post-treatment reduction in absolute eosinophil count from about 50 to about 150 eosinophils / mm 3 The method of claim 1, wherein

46. Absolute eosinophil count after administration is approximately 100 eosinophils / mm 3 The method of claim 1 , wherein the

47. Absolute post-injection eosinophil counts of approximately 75 eosinophils / mm 3 The method of claim 1 , wherein the

48. Absolute eosinophil count after administration is approximately 50 eosinophils / mm 3 The method of claim 1 , wherein the

49. Absolute post-injection eosinophil counts of approximately 25 eosinophils / mm 3 The method of claim 1 , wherein the

50. The subject's pre-administration absolute eosinophil count is between about 50 and about 500 eosinophils / mm 3 The method of claim 1, wherein

51. The subject has a pre-administration absolute eosinophil count of about 75 to about 475 eosinophils / mm 3 The method of claim 1, wherein

52. The subject has a pre-administration absolute eosinophil count of about 75 to about 200 eosinophils / mm 3 The method of claim 1, wherein

53. The subject has a pre-administration absolute eosinophil count of about 100 to about 200 eosinophils / mm 3 The method of claim 1, wherein

54. The subject's pre-administration absolute eosinophil count is about 25 eosinophils / mm 3 The method of claim 1, wherein

55. The subject has a pre-administration absolute eosinophil count of about 50 eosinophils / mm 3 The method of claim 1, wherein

56. The subject's pre-administration absolute eosinophil count is about 75 eosinophils / mm 3 The method of claim 1, wherein

57. The subject has a pre-administration absolute eosinophil count of about 100 eosinophils / mm 3 The method of claim 1, wherein

58. The subject has a pre-administration absolute eosinophil count of about 125 eosinophils / mm 3 The method of claim 1, wherein

59. The subject has a pre-administration absolute eosinophil count of about 150 eosinophils / mm 3 The method of claim 1, wherein

60. The subject has a pre-administration absolute eosinophil count of about 175 eosinophils / mm 3 The method of claim 1, wherein

61. The subject has a pre-administration absolute eosinophil count of about 200 eosinophils / mm 3 The method of claim 1, wherein

62. The subject has a pre-administration absolute eosinophil count of about 225 eosinophils / mm 3 The method of claim 1, wherein

63. The subject has a pre-administration absolute eosinophil count of about 250 eosinophils / mm 3 The method of claim 1, wherein

64. The subject has a pre-administration absolute eosinophil count of about 275 eosinophils / mm 3 The method of claim 1, wherein

65. The subject has a pre-administration absolute eosinophil count of about 300 eosinophils / mm 3 The method of claim 1, wherein

66. The subject has a pre-administration absolute eosinophil count of about 325 eosinophils / mm 3 The method of claim 1, wherein

67. The subject has a pre-administration absolute eosinophil count of about 350 eosinophils / mm 3 The method of claim 1, wherein

68. The subject has a pre-administration absolute eosinophil count of about 375 eosinophils / mm 3 The method of claim 1, wherein

69. The subject has a pre-administration absolute eosinophil count of about 400 eosinophils / mm 3 The method of claim 1, wherein

70. The subject's pre-administration absolute eosinophil count is about 425 eosinophils / mm 3 The method of claim 1, wherein

71. The subject has a pre-administration absolute eosinophil count of about 450 eosinophils / mm 3 The method of claim 1, wherein

72. The subject has a pre-administration absolute eosinophil count of about 475 eosinophils / mm 3 The method of claim 1, wherein

73. The subject has a pre-administration absolute eosinophil count of about 500 eosinophils / mm 3 The method of claim 1, wherein

74. The subject has an absolute basophil count after administration of at least about 5 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

75. The subject has an absolute basophil count after administration of at least about 10 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

76. The subject has an absolute basophil count after administration of at least about 15 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

77. The subject has an absolute basophil count after administration of at least about 20 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

78. The subject has an absolute basophil count after administration of at least about 25 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

79. The subject has an absolute basophil count after administration of at least about 30 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

80. The subject has an absolute basophil count after administration of at least about 35 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

81. The subject has an absolute basophil count after administration of at least about 40 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

82. The subject has an absolute basophil count after administration of at least about 45 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

83. The subject has an absolute basophil count after administration of at least about 50 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

84. The subject has an absolute basophil count after administration of at least about 55 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

85. The subject has an absolute basophil count after administration of at least about 60 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

86. The subject has an absolute basophil count after administration of at least about 65 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

87. The subject has an absolute basophil count after administration of at least about 70 basophils / mm 3 The method of any one of claims 1 to 73, wherein the amount of

88. The subject has an absolute basophil count of 0 to about 10 basophils / mm 3 The method according to any one of claims 1 to 73, wherein

89. The subject has an absolute basophil count of about 2 basophils / mm 3 The method according to any one of claims 1 to 73, wherein

90. The subject has an absolute basophil count of about 5 basophils / mm 3 The method according to any one of claims 1 to 73, wherein

91. The subject has an absolute basophil count of about 7 basophils / mm 3 The method according to any one of claims 1 to 73, wherein

92. The subject has an absolute basophil count of about 9 basophils / mm 3 The method according to any one of claims 1 to 73, wherein

93. The method of any one of claims 1 to 73, wherein the reduction in basophils occurs within 48 hours after administration.

94. The method of any one of claims 1 to 73, wherein the reduction in basophils occurs within 24 hours after administration.

95. 95. The method of any one of claims 1 to 94, wherein the IL-5R binding molecule is administered to the subject at a dose of about 0.001 to about 100 mg / kg.

96. 96. The method of claim 95, wherein the dose is about 0.03 mg / kg.

97. 96. The method of claim 95, wherein the dose is 0.03 mg / kg.

98. The method of any one of claims 1 to 97, wherein the IL-5R binding molecule is administered parenterally.

99. 99. The method of claim 98, wherein the IL-5R binding molecule is administered intravenously.

100. 100. The method of any one of claims 1 to 99, wherein the IL-5R binding molecule is not MEDI-563.

101. The method of any one of claims 1 to 100, wherein the reduction in eosinophils results in a reduction in asthma symptoms.

102. The method of any one of claims 1 to 100, wherein the reduction in eosinophils results in a reduction in COPD symptoms.

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