NZ724413B2

NZ724413B2 - Methods of reducing basophil levels

Info

Publication number: NZ724413B2
Application number: NZ724413A
Authority: NZ
Inventors: Masamichi Koike; George L Spitalny; Alistair Wheeler; Barbara White
Original assignee: Astrazeneca Ab; Kyowa Kirin Co Ltd
Priority date: 2007-05-14
Filing date: 2008-05-14
Publication date: 2021-06-29

Abstract

Disclosed is the use of a monoclonal, chimeric, humanised or human antibody that binds IL-5R in the manufacture of a medicament for treating or preventing an basophil mediated disease or disorder in a human subject, wherein said antibody is parenterally administered to said subject between about 0.001 to about 100 mg/kg, and wherein said antibody comprises an immunoglobulin Fc region comprising no fucose, and wherein the administration of the antibody reduces the number of peripheral blood basophils from the human subject’s circulation. 01 to about 100 mg/kg, and wherein said antibody comprises an immunoglobulin Fc region comprising no fucose, and wherein the administration of the antibody reduces the number of peripheral blood basophils from the human subject’s circulation.

Description

Patents Form No. 5 N.Z. No. 724413 Divided out of Application No. 705746, itself divided our of No.614901, itself divided out of No. 599278, itself divided out of No. 581142 NEW ZEALAND s Act 1953 COMPLETE SPECIFICATION METHODS OF REDUCING EOSINOPHIL LEVELS We, MedImmune, LLC a company of the United States of a of One Medimmune Way Gaithersburg, MD 20878, UNITED STATES OF AMERICA; and Biowa, Inc., a company of the United States of America of 9420 Athena Circle, La Jolla, CA 92037, United States of America do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be ularly described in and by the following statement:- (followed by page 1A) METHODS OF REDUCING BASOPHIL LEVELS The present application is a divisional application divided out of New Zealand Patent Application No. 705746, itself d out of NZ 614901, itself divided out of NZ 599278, itself d out of NZ 581142.

FIELD OF THE INVENTION The present invention relates to methods of reducing basophil levels in human subjects.

(Followed by page 2) cell expressed and secreted), induce the chemotaxis of eosinophils to inﬂamed site [Clin.

Exp. Allergy, 26, 1005 (1996)]. Stem cell factors after referred to as SCF) are involved in the accumulation ofeosinophils in allergic bronchitis. In addition to lL-S, there are many factors ing function of eosinophils. phils are divided into subgroups, normodense eosinophils and hypodense eosinophils. Eosinophils have been shown to be hypodense eosinophils upon activation [Immunology, 47, 531 (1982)]. Hypodense eosinophils are also referred to as activated eosinophils. it has been reported that a ative change occurs in addition to a tative change in eosinophils in the peripheral blood ofan HES patients [Clin. Exp. lmmunol., 24, 423 (1976)]. Activated eosinophils have been implicated in the severity ofHES symptom [Am. J. Cardiol., 52, 321 (1983)]. Aside from HES patients, activated eosinophils have been also found in the peripheral blood, and in bronchoalveolar lavage ﬂuid (BALF) ofa patient with bronchial asthma [Am. Rev. Respir. Dis, 132, 981 (1985)]. Various receptors, such as those of cytokines, are expressed on activated eosinophils (hypodense phils) [1. lmmunol., 142, 4416 (1989)]. Compared to normodense eosinophils, these hypodense eosinophils show elevated sensitivities against lL-5 [Clin. Exp. lmmunol., 85, 312 (1991); J.

Exp. Med, 172, 1347 (1990)].

The above-mentioned ted eosinophils are also known to survive in vitro without the cytokines ng in the differentiation and proliferation of eosinophils [1. Exp. Med., 170, 89)]. Thus, the properties ofactivated eosinophils are r to those of phils, which inﬁltrate tissues, such as alveoli [Int. Arch. Allergy lmmunol., 120, 91 ]. A detailed explanation of why activated eosinophils become ne-independent remains unknown, however, their degranulation and prolonged survival are likely to be induced by various vital functional molecules other than lL-S.

Substances having inhibition activity on cytokines or chemokines that are involved in the differentiation or proliferation ofeosinophils have been considered as agents that inhibit the eosinophil ons. However, in most cases these agents do not act on cytokine- independent eosinophils that have been activated and inﬁltrated into inﬂamed areas. Hence, eosinophil-specific tion and the induction ofcellular death of activated eosinophils are necessary to inhibit the functions of any eosinophil. However, no anti-inflammatory agent, so far, has been known to induce apoptosis of activated eosinophils. tly, treatment for patients with eosinophilic diseases consists of administration of steroids. However, steroid administration is often associated with side effects.

Specifically, the treatment has some other ms, such that patient's pathological condition may return to the original state when steroid administration is discontinued, and prolonged steroid administration may induce steroid resistance.

SUMMARY OF THE INVENTION The invention provides for the use of a onal, chimeric, humanized or human antibody that binds IL-5R in the manufacture of a medicament for treating or preventing a basophil mediated disease or disorder in a human subject. In ular the dy comprises an immunoglobulin Fc region comprising no fucose. The antibody may be parenterally administered to said subject at n about 0.001 to about 100 mg/kg and reduce the number of peripheral blood ils from the human subject’s circulation.

In certain embodiments the number of peripheral blood basophils is reduced by about 5 to about 70 basophils/mm3 by 24 hours post-administration. There may be a post-administration reduction in absolute basophil count of at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, or at least about 70 basophils/mm3. In one embodiment the number of peripheral blood basophils from the human subject’s circulation is d to a level that is less than 9 basophils/mm3. In certain embodiments the reduction is reversible.

The subject’s pre-administration absolute basophil count may be between about 5 and about 500 basophils/mm3, for example about 5, about 10, about 15, about 20, about 25, about 30, about 50, about 60, about 70, about 100, about 200, about 300, about 400, or about 500 basophils/mm3.

In certain ments the disease or disorder mediated by basophils is asthma. For example, the reduction in peripheral blood ils leads to a reduction in asthma ms.

In an embodiment the dy specifically binds to the IL-5R α chain. In certain embodiments the antibody may be MEDI-563 or r antibody which binds to IL-5R.

The invention will be described in further detail below. Certain embodiments described herein for completeness may form the subject of related applications, including New Zealand Patent No 614901.

BRIEF DESCRIPTION OF THE FIGURES For the purpose of rating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings. (followed by 3A) wed by 4) Figure 4. Minimal increase in serum lL-6. Measurement ofthe lL-6 cytokine in patient cohort l is summarized. The y-axis izes lL-6 levels (pg/ml) and x-axis summarizes time (in hours).

Figure 5. Variable decrease ofcirculating neutrophils. Neutrophil levels in patient cohort l were measured and are summarized in both panels.

Figure 6. Variable decrease ofcirculating lymphocytes. Lymphocyte levels in patient cohort l were measured and are summarized in both panels.

Figure 7. tent and modest reduction of %NK at Day I. NK cell levels in patient cohort 1 were measured prior to treatment, at day 1 dministration, and at day 28 post- administration.

IS Figure 8. Reduced FENO in subjects with higher baseline. The fraction of exhaled nitric oxide was measured in patient cohort 1. This assay is a noninvasive ement oflung inﬂammation, with the data indicating a trend towards reduction in inﬂammation.

Figure 9. In vitro cytotoxicity assay: MEDl-563 was assayed in an in vitro cytoxicity assay ed to a control antibody that does not bind lL-SR (A) and also to the additional control of fucosylated MEDl-563 (B). KCl333 or cells were used in a 5:1 ratio against CTLL2 target cells. Cytotoxicity was measured at 4 hours. The Y axis measures percent cytotoxicity and the X axis is the concentration ofantibody.

Figure 10. 63 binding to rhulL-SRoc: binding afﬁnity ofMEDl-563 to recombinant human lL-5R0t was measured by surface plasmon resonance in three te experiments and is summarized in this ﬁgure.

Figure 11. MEDl-563 binding to rhchyRs: g afﬁnity ofMEDl-563 to recombinant human FcyRs of several different lots was measured as compared to a isotype-matched fucosylated control antibody and is summarized in this ﬁgure. Note that MEDl-563 binds with 5—10 fold higher y to hchlella and mchleV.

Figure 12. lL-5Roc sion in the lL-9tg mouse lung was analyzed via immunohistochemistry and is visualized in this ﬁgure.

Figure 13. lL-SROL expression in nasal polyps was analyzed via immunohistochemistry using MEDl-563 and is visualized in this ﬁgure. MEDI—563 stains all eosinophils in nasal Figure 14. Minimal Transient Neutropenia in Subjects: absolute neutrophil counts were taken for subjects in cohort l and are summarized in this ﬁgure. The Y-axis izes neutrophil counts (neutrophils/mm3) and the X-axis summarizes time in days.

Figure 15. MEDl—563 Binds to Eosinophils in Whole Blood of Healthy Donors: flow cytometry analysis was performed on whole blood samples as described in Example 6 herein.

The three panels of data, particularly the third panel entitled "MEDl-563 Binds Eos," demonstrates by FACS that MEDl-563 binds to phils.

Figure 16. FACS Analysis of ytes from lL—5 Transgenic Mice: ﬂow cytometry analysis was performed on leukocytes from lL-5 transgenic mice as described in Example 7.

Figure 16A summarizes FACS analysis of SiglecF+CCR3+ eosinophils. Figure 168 trates that all eosinophils (SiglecF+CCR3jl~) in the bone marrow, , blood and lung express IL-SRa+ using anti-IL—SRu mAb H7.

Figure 17. MEDl-563 depletes lL—5Ra positive mononuclear cells from bone marrow in an in vitro ADCC assay. Isolated non-adherent bone marrow mononuclear cells were exposed to MEDl-563, or e control antibody (R347), in the presence ofCFSE stained effector cells. lL—5Ra positive cells were visualized by KM1257 antibody/ PE conjugated goat anti- Mu lgG. Control staining of samples was done using the 1A7 e control antibody/ PE ated goat anti-Mu lgG. Staining proﬁle ofthe sample cell populations following MEDl-563 or R347 mediated depletion is displayed as KMI257/PE vs. CFSE or lA7/PE vs.

CFSE dot plots. A comparison ofthe KM 1257/PE vs. CFSE dot plots obtained for MEDI- 563 and R347 treated samples reveals that MEDl-563 mediated ADCC depletes substantially all lL-SRa positive cells from the sample.

Figure 18. 63 reversibly depletes peripheral blood eosinophils in mild asthmatics.

Six volunteers with mild atopic asthma received a single lV dose of (A) 0.03 mg/kg or (B) 0.1 mg/kg MEDl-563. Peripheral blood eosinophils were enumerated by ﬂow cytometry at screening, on day 0 prior to dosing, and at regular intervals up to day 84 and at follow-up.

The y-axis summarizes phil counts (eosinophils/mm3) and x-axis summarizes time (in days). Rapid reduction of eosinophils in the periphery was observed by 24 hours post— administration. The MEDl-563 induced eosinopenia was reversible.

Figure 19. lL-SRoc is expressed on all phils in normal human lung as analyzed via immunohistochemistry using MEDl-563 and visualized in this ﬁgure.

Figure 20. lL-5Roc is expressed on all eosinophils in lung biopsies from asthmatic human patients as analyzed via immunohistochemistry using MEDl-563 and visualized in this ﬁgure.

Figure 21. IL-5R0t expression by y basophils and eosinophils isolated from healthy donors was analyzed via ﬂow cytometry. Staining proﬁles obtained using the MEDl 563 anti-lL5Ralpha antibody and an isotype l dy of irrelevant speciﬁcity are shown, CTLLh5r cells (lL-5Ralpha/beta transfected tumor cells) served as a positive control.

Figure 22. In vitro antibody dependent cell-mediated cytotoxicity (ADCC) assay: The activity of afucosylated and fucosylated MEDl-563 was compared in an in vitro ADCC assay. ed primary NK cells and eosinophils were used as effector and target cells, tively, at a 5:1 ratio. The assay was performed in the presence ofl ng/ml human lL—2.

Cell death was ed by ﬂow cytometry based on Annexin V staining. The Y and X axes display percent maximum cytotoxicity and antibody concentration, respectively. The EC50 value for the afucosylated 63 antibody was 0.965 pM.

Figure 23. In vitro antibody dependent cell—mediated cytotoxicity (ADCC) assay: The activity osylated MEDI-563 was analyzed in an in vitro ADCC assay. Isolated primary NK cells and basophils were used as effector and target cells, respectively. The Y and X axes y percent maximum cytotoxicity and antibody concentration, respectively.

The EC50 value for the afucosylated MEDl-563 antibody was 0.56] prl in this assay.

Figure 24. Eosinophil degranulation in an in vitro antibody dependent cell-mediated cytotoxicity (ADCC) assay: EDN (Eosinophil Derived Neurotoxin) release by eosinophils in an in vitro ADCC assay using various levels of fucosylated (MEDl-563F) and afucosylated (MEDl-563) anti-lLSRalpha antibody was analyzed. The assay ed freshly isolated eosinophils and NK or PBMC cells as target and effector cells, respectively. Maximum eosinophil degranulation detected in response to treatment with 1% Triton X-100 is shown for comparison.

Figure 25. MEDl-563 specifically binds an epitope within the D1 domain ofthe extracellular region of human lL-SRalpa. Antibody binding to transgenic cells transiently expressing chimeric lL-SRalpha ns was ascertained by flow cytometry. Fluorescent staining s are shown. “Polyclonal” and “MEDl-563” denotes staining es observed using a polyclonal anti—human lL-SRalpha and MEDI-563, respectively, antibodies.

“Dual staining” denotes the ﬂuorescent staining e for the “polyclonal” (x axis) and MEDl-563 (y axis) dies. (A) A series of human-mouse chimeric lL-5Ralpha transgenes were expressed transiently. -out” transgenes were chimeric IL-S Ralpha constructs comprising a single mouse extracellular domain in an otherwise human background. -in” transgenes were chimeric IL-SRalpha constructs comprising a single human extracellular domain in an ise mouse background. (B) MEDl—563 specifically bound transgenic cells expressing human lL-5Ralpha. MEDl-563 did not bind transgenic cells expressing mouse IL-SRalpha. (C) MEDl-563 did not bind transgenic cells expressing a chimeric lL-5Ralpha transgene comprising mouse D1 and human D2-D3 extracellular domains (“knock—out D1”). MEDl-563 ically bound transgenic cells expressing a chimeric lL-SRalpha transgene comprising mouse D2 or D3 extracellular s in a human ound (“knock-out D2 or D3”). (D) MEDl-563 specifically bound transgenic cells expressing a chimeric lL-SRalpha transgene comprising human Dl and mouse D2-D3 extracellular domains (“knock-in Dl”). MEDl-563 did not bind transgenic cells expressing a mouse lL-5 Ralpha based chimeric transgene sing either the human D2 or D3 extracellular domain (“knock-in D2 or D3”).

Figure 26. MEDl-563 specifically binds an epitope within Segment B ofthe Dl ellular domain of human lL-SRaIpa. Antibody binding to transgenic cells expressing a chimeric lL-SRalpha protein was ascertained by ﬂow cytometry. Fluorescent staining profiles are shown. “Polyclonal” and “MEDl-563” s staining profiles observed using a polyclonal anti-human lL-SRalpha and MEDl-563, respectively, antibodies. “Dual staining” denotes the ﬂuorescent staining proﬁle for the polyclonal (x axis) and MEDl-563 (y axis) antibodies. (A) The amino acid sequence ofthe Dl extracellular domain of mouse lL- 5Ralpha is 75% identical to that ofthe human lL-SRalpha protein. The Dl extracellular domain oflL-SRalpha was divided into Segments A, B and C. The human and mouse lL— 5Ralpha amino acid sequences shown are residues 1-102 of SEQ lD NO: 5 and 6 , respectively. (B) A series of human-mouse chimeric lL-5Ralpha transgenes were expressed transiently. “Knock-out” transgenes were chimeric lL-5 Ralpha constructs comprising a single mouse Segment ofthe Dl extracellular domain in an otherwise human background.

“Knock-in” transgenes were chimeric lpha constructs sing a single human Segment ofthe Dl extracellular domain in an mouse Dl-human D2-mouse D3-mouse TM background. (C) MEDl-563 speciﬁcally recognized transgenic cells expressing (i) a human lL—5Ralpha transgene or (ii) a mouse lL-SRalpha chimeric transgene sing a human Dl extracellular domain (:knock-in Dl”). MEDI-563 did not bind transgenic cells expressing (i) mouse lL-SRalpha or transgene or (ii) a human chimeric lL-SRalpha transgene sing a mouse Dl extracellular domain. (D) MEDl-563 did not bind transgenic cells a ic lL-5Ralpha transgene sing a mouse Segment B ofthe Dl . expressing extracellular domain in an otherwise human background (“knock-out B”). MEDl—563 speciﬁcally bound transgenic cells sing a chimeric lL-5Ralpha transgene comprising mouse Segment A or C ofthe Dl extracellular domains in a human background (“knock-out A or C”). (E) MEDl-563 speciﬁcally bound enic cells expressing a chimeric lL- SRalpha transgene comprising a human Segment B ofthe Dl extracellular domain in a mouse Dl-human D2-mouse D3-mouse TM background k-in B”). MEDl-563 did not bind transgenic cells expressing a chimeric lpha transgene that comprised a human Segment A or C in an mouse an D2-mouse D3-mouse TM background (“knock-in A or C”).

Figure 27. MEDl-563 cally binds an epitope of human lL-5Ralpha comprising amino acid residue lle6l ofthe Dl extracellular domain. dy binding to transgenic cells expressing a variant lL-S Ralpha protein was ascertained by ﬂow cytometry. Fluorescent staining s are shown. “Polyclonal” and “MEDl-563” denotes staining proﬁles observed using a onal anti-human lL-5Ralpha and MEDl-563, respectively, antibodies.

“Dual staining” denotes the ﬂuorescent staining proﬁle for the polyclonal (x axis) and MEDI- 563 (y axis) antibodies. (A) Residues 50-61 ofthe Dl extracellular domain of human lL- SRalpha are shown (residues 40-61 of SEQ ID NO:5). Residues shown in italics are different in the corresponding region ofthe mouse lL—SRalpha n. A series Ralpha receptor variants comprising at least one mutant amino acid residue were expressed in transgenic cells. The “knock-out” lL-SRalpha variants were mutant human proteins comprising at least one substitution exchanging a human residue for the ponding mouse residue. For example, the “knock-out DE” variant is a human lL-SRaIpha protein comprising the D56E and E58D amino acid substitutions. The “knock-in” lL-5 Ralpha variants were chimeric proteins sing the mouse D1, human D2, mouse D3 and mouse TM domains wherein the mouse DI domain comprised a mutant Segment B having at least one substitution exchanging a mouse residue for the corresponding human residue. For example, the “knock-in DE” variant was a chimeric IL—5 Ralpha protein comprising a mutant mouse Segment B wherein the mutant mouse segment B comprised the E56D and D58E amino acid substitutions. (B) MEDl-563 did not bind transgenic cells expressing a mutant human lL- a n comprising the K53Q, D56E, E58D, 161K amino acid tutions (“knock out-KDEl”). MEDl-563 cally binds to transgenic cells expressing a mutant human lL- 5Ralpha protein comprising the N40H, N42D, Q46H (“knock out-NNQ”) or D56E, E58D k out-DE”), or N40H, N42D, D56E, E58D (“knock out-NNDE”) amino acid substitutions. (C) MEDl—563 specifically bound transgenic cells expressing a chimeric IL- a protein comprising a mutant mouse Segment B wherein the mutant mouse Segment B comprised the Q53K, E56D, D58E, K61] amino acid substitutions (“knock in-KDEI”). (D) MEDl-563 did not bind transgenic cells expressing a mutant human lL-5Ralpha protein comprising the l6lK amino acid substitution k l”). MEDl-563 specifically binds to transgenic cells expressing a mutant human lL-SRalpha n comprising the K53Q (“knock out—K53”) amino acid substitution. (E) MEDl-563 specifically bound transgenic cells expressing a chimeric lL-SRalpha protein comprising a mutant mouse Segment B wherein the mutant mouse Segment B comprised the K61] amino acid substitution (“knock in-l6l”). MEDI—563 did not bind transgenic cells expressing a chimeric lL-S Ralpha protein comprising a mutant mouse Segment B n the mutant mouse Segment B comprised the Q53K amino acid substitution (“knock in-K53”).

Figure 28. ic anti-mouse lL-SRa (H7) binding to murine FcyRs: binding affinity of chimeric anti—mouse lL-5 Ra (H7) to recombinant murine FcyRs was measured as compared to an isotype-matched fucosylated control antibody and is summarized in this figure.

Dissociation constants are shown (nM). Measurements were done by surface plasmon resonance.

Figure 29. (A) Eosinophils were identiﬁed by ﬂow cytometric analysis as cells with high side scatter that stained positively for CCR3 and Siglec-F. (B) lL-SR was selectively expressed by eosinophils in bone , blood, spleen and lung tissue oflL-STg mice.

Figure 30. Both afuc and fuc H7 depleted eosinophils in spleen (A), lung tissue (A) and blood (B) oflL-STg mice. No ion was ed in the bone marrow (B). Afuc H7 was more potent at removing eosinophils compared with fuc H7, ally at lower antibody doses. Data are expressed as meaniSEM, n=6-8mice/group, p<0.05 antibody treated compared with Control lgG treated, Mann-Whitney U test.

Figure 31. Afuc H7 also depletes eosinophils in an allergen nge model. Afuc H7 ed eosinophils in the airway lumen, lung , blood and bone . Depletion was highest in all compartments 72h after the ﬁnal challenge (96h after antibody delivery).

Data are expressed as meaniSEM, n=6 mice/group, 5 antibody treated compared with Control lgG treated, Mann-Whitney U test.

DETAILED DESCRIPTION OF THE INVENTION As discussed herein above and not being bound by a particular hypothesis or theory, eosinophils have been implicated in the pathogenesis of numerous diseases and disorders.

Many of these diseases or disorders are characterized by an overabundance of eosinophils (eosinoph-ilia), and are termed hypereosinophilic syndromes.

Nonlimiting examples ofdiseases and disorders in which eosinophils play a role are: asthma, immunoglobulin (lgE)-mediated food allergy, eosinophilic esophagitis (inﬂammation ofthe esophagus), inﬂammatory bowel disease, COPD, allergic colitis, astro-esophageal reﬂux, eosinophilic gastrointestinal e (EGlD), eosinophilic gastroenteritis, endomyocardial ﬁbrosis, Loefﬂer’s endocarditis, Davies disease, Episodic Angioedema Associated With Eosinophilia, Eosinophilia—Myalgia Syndrome/Spanish Toxic Oil me, liver sis, itis herpetiformis, Bullous pemphigoid, Churg-Strauss syndrome, Acute myelogenous eosinophilic leukemia, Acute lymphocytic eosinophilic leukemia, ic mastocytosis with eosinophilia, Allergic rhinitis, Eczema, Wegener’s granulomatosis, Polyarteritis nodosa, Eosinophilic fasiculitis, and Rheumatoid arthritis.

Accordingly, the invention provides a method of reducing the numbers ofeosinophils in a human subject comprising administration to said patient an lL-SR binding molecule that comprises (a) a region that speciﬁcally binds to the lL-5R and (b) an globulin Fc In one embodiment, the invention provides methods of reducing the number of eosinophils in a human subject comprising administration to said patient an lL-SR binding molecule that ses (a) a region that speciﬁcally binds to the lL-SR and (b) an immunoglobulin Fc . In a speciﬁc embodiment, a method ofthe invention reduces the number ofeosinophils in blood, bone marrow, gastrointestinal tract (e.g., esophagus, stomach, small intestine and colon), or lung. In another speciﬁc embodiment, a method of the invention reduces the number of blood eosinophils. In a further c embodiment, a method of the invention reduces the number oflung eosinophils. In a speciﬁc embodiment, a method ofthe invention reduces the number of eosinophil precursor cells.

In another embodiment, a method ofthe ion 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, a method of the invention s the number ofeosinophils below the limit ofdetection.

In another embodiment, a method ofthe invention reduces the number of eosinophil precursors 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%. ln a c embodiment, a method ofthe invention reduces the number of eosinophil precursors below the limit of detection.

In a further embodiment, a method ofthe invention eliminates all detectable eosinophils following a single administration ofan lL-SR binding molecule. In a speciﬁc embodiment, a single administration of an lL-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 weeks, or at least about 25 weeks.

In a further embodiment, a method ofthe invention eliminates all detectable phil precursors following a single administration ofan lL-SR binding molecule. In a speciﬁc embodiment, a single administration ofan lL~5R binding molecule ates 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 l0 weeks, at least about 12 weeks, at least about l4 weeks, at least about 16 weeks, at least about 20 weeks, or at least about 25 weeks.

In a speciﬁc embodiment, method ofthe invention comprises the stration to a subject a single dose of 0.03 mg/kg of an lL-5R binding molecule that comprises (a) a region that speciﬁcally binds to the IL-5R and (b) an immunoglobulin Fc region, wherein the administration ofthe IL-SR binding le leads to depletion of at least about 99% of eosinophils from the subject’s circulation, wherein the depletion is complete by 24 hrs after dosing, and wherein the depletion lasts for at least about 28 days after .

In a speciﬁc embodiment, method ofthe invention comprises the administration to a subject a single dose of 0.] mg/kg of an lL-5R binding molecule that comprises (a) a region that speciﬁcally binds to the lL-SR and (b) an globulin Fc region, wherein the administration ofthe lL-5R binding molecule leads to depletion of at least about 99% of eosinophils from the subject’s circulation, wherein the depletion is complete by 24 hrs after , and n the depletion lasts for at least about 84 days after dosing.

In one embodiment, the lL-SR binding molecules ofthe t invention include fusion proteins. In n embodiments, the fusion proteins comprise a polypeptide region that speciﬁcally binds to the lL-SR, and further comprise an immunoglobulin Fc region.

Nonlimiting examples ofa polypeptide region that speciﬁcally bind to the lL-5R can be found in US. Patent Nos. 7,109,299 and 5,677,280, US. Patent Application Publication No. 014680 A1. In other embodiments, the polypeptide region that speciﬁcally binds to the lL-SR is human lL-5 (see, for example, Tanabi et al., Journal ofBiological Chemistry, 1987, Vol. 262, No. 34, pp. l6580-l6584), or fragments, derivatives or variants thereof(see, for example, US. Patent No. 6,465,6l6).

In one embodiment, the lL-5R binding molecules ofthe present ion comprise antibodies. Antibodies ofthe t invention include, but are not limited to, monoclonal antibodies, synthetic antibodies, multispeciﬁc antibodies (including bi-speciﬁc antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv) (including bi-speciﬁc , single chain antibodies, Fab fragments, F(ab’) fragments, disulﬁde-linked Fvs (dev), and epitope-binding fragments ofany ofthe above. In particular, antibodies ofthe present invention include immunoglobulin molecules and immunologically active portions noglobulin molecules, i.e., molecules that contain an antigen binding site that speciﬁcally bind to an antigen. The immunoglobulin molecules ofthe invention can be ofany type (e.g., lgG, lgE, lgM, lgD, IgA and ng), class (e.g., lgGl, 1gG2, lgG3, lgG4, lgAl and 1gA2) or subclass ofimmunoglobulin molecule.

The antibodies useful in the present invention may be from any animal origin including birds and s (for example, but not limited to, human, , donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken). 1n speciﬁc embodiments, the antibodies are human or humanized monoclonal antibodies.

The antibodies useful in the present invention may be monospeciﬁc, bispeciﬁc, trispeciﬁc or of greater multispeciﬁcity. Multispeciﬁc antibodies may speciﬁcally bind to different epitopes ofa polypeptide or may speciﬁcally bind to both a polypeptide as well a heterologous epitope, such as a logous polypeptide or solid support material. See, e.g., International Publication Nos. WO 93/17715, WO 92/08802, WO 60, and WO 92/05793; Tutt, et al., 1991, J. lmmunol. 147:60-69; US. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 920, and 5,601,819; and Kostelny et al., 1992, J. lmmunol. 148:1547-1553.

The antibodies useful in the present invention can be single-chain antibodies. The design and construction ofa single-chain antibody is described in o et a1, 1993, Proc Natl Acad Sci 90:7889-7893.

Nonlimiting examples of antibodies ofthe invention can be found in US. Patent Nos. 7,179,464, l l 1, 6,018,032, and US. Patent Application Publication Nos. 2004/0136996A1, 2005/0226867A 1.

In one embodiment, the lL-5R binding molecules ofthe present invention comprise antibodies. In a further embodiment, an 1L-5R binding molecule ofthe present invention is an antibody comprising any one ofthe amino acid sequence of SEQ ID NO: 1-4. In a speciﬁc embodiment, an lL-SR g molecule ofthe present invention is an antibody comprising the amino acid sequence of SEQ ID NO: 1 and 3. In a speciﬁc embodiment, an lL-SR binding molecule ofthe present ion is an antibody comprising the amino acid sequence ofSEQ ID NO: 2 and 4.

In one embodiment, an lL-SR binding molecule ofthe t invention is an dy that speciﬁcally binds to the same epitope as MEDl-563. In a speciﬁc embodiment, the antibody is MEDl-563. In a further speciﬁc embodiment, an lL-SR binding molecule of the present ion is an antibody that speciﬁcally binds to the same epitope as MEDl-563 provided that the antibody is not MEDI-563.

In one embodiment, an IL-SR binding molecule ofthe 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 further speciﬁc embodiment, an IL- 5R binding molecule ofthe present invention is an antibody that speciﬁcally binds to an epitope comprising residues 1-102 of SEQ ID NO:5 provided that the dy is not MEDI- 563.

In one embodiment, an IL—5R binding le ofthe present invention is an I0 antibody that speciﬁcally binds to an epitope comprising residues 40-67 of SEQ ID NO:5. In a c embodiment, the dy is MEDI-563. In a further c embodiment, an IL- 5R binding molecule ofthe present invention is an antibody that cally binds to an epitope comprising residues 40-67 of SEQ ID NO:5 provided that the antibody is not MEDI- 563.

IS In one embodiment, an IL-5R binding molecule ofthe t invention is an antibody that speciﬁcally binds to an epitope comprising residues 52-67 of SEQ ID NO:5. In a c embodiment, the antibody is MEDI-563. In a further speciﬁc ment, an IL- 5R binding molecule ofthe present invention is an antibody that speciﬁcally binds to an epitope sing residues 52-67 of SEQ ID NO:5 provided that the antibody is not MEDI- 563.

In one embodiment, an IL-5R binding molecule ofthe 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 further speciﬁc ment, an IL-5R binding molecule ofthe present invention is an antibody that speciﬁcally binds to an epitope comprising residue 6| of SEQ ID NO:5 provided that the antibody is not MEDI-563.

In one embodiment, an IL-SR g molecule ofthe present invention is an antibody that speciﬁcally binds to a ﬁrst antigen comprising residues 1-102 of SEQ ID NO:5 but does not speciﬁcally bind to a second antigen comprising a variant of residues 1-102 of SEQ ID NO:5 wherein the variant comprises the I6IK substitution. In a speciﬁc embodiment, the antibody is MEDI-563. In a further speciﬁc embodiment, an IL-5R binding molecule ofthe present invention is an antibody that speciﬁcally binds to a ﬁrst antigen comprising residues l-I02 of SEQ ID NO:5 but does not speciﬁcally bind to a second antigen comprising a variant of es 1-102 ofSEQ ID NO:5 wherein the variant comprises the I6IK substitution, provided that the antibody is not MEDl-563.

In one embodiment, an lL-5R binding le ofthe present invention is an antibody that speciﬁcally binds to a ﬁrst antigen comprising residues 40-67 of SEQ ID N025 but does not speciﬁcally bind to a second antigen comprising a variant of residues 40—67 of SEQ 1D N0:5 wherein the variant comprises the 161K substitution. In a speciﬁc embodiment, the dy is MEDl-563. In a further speciﬁc embodiment, an lL—5R binding molecule ofthe present invention is an antibody that speciﬁcally binds to a ﬁrst antigen comprising residues 40—67 of SEQ ID N0:5 but does not speciﬁcally bind to a second antigen comprising a variant of residues 40-67 of SEQ ID N025 wherein the t ses the 161 K substitution, provided that the antibody is not 63.

In one embodiment, an lL-5R g molecule ofthe present invention is an antibody that speciﬁcally binds to human IL-5Ralpha (SEQ ID N025) but does not speciﬁcally bind to mutant human lL-5Ralpha (SEQ ID N025) comprising the 161K substitution. In a speciﬁc embodiment, the antibodyis MEDl-563. In a further speciﬁc embodiment, an lL-5R binding molecule ofthe present invention is an dy that cally binds to human lL-5Ralpha (SEQ ID N0:5) but does not speciﬁcally bind to mutant human lL-5 Ralpha (SEQ ID N0z5) comprising the 161 K substitution, provided that the antibody is not MEDl-563.

The present invention provides lL-5R binding molecules with increased effector function. Nonlimiting examples ofmethods for increasing effector function can be found in US. Patent Nos. 5,624,821, 6,602,684, 7,029,872, US. Patent ation Publication Nos. 2006/0067930A1, 2005/0272128A1, 2005/0079605A1, 2005/0123546A1, 2004/0072290A1, 2006/0257399Al, 2004/0261 148A], 2007/0092521, 2006/0040325A1, and 2006/0039904Al, and ational Patent Application Publication Nos. W0 04/029207, W00301 1878, W005044859, W0 06071856, and W0 06071280.

Methods ofengineering Fc regions of antibodies so as to alter effector functions are known in the art (e.g., US. Patent Publication No. 20040185045 and PCT Publication No.

W0 2004/016750, both to Koenig et al., which describe altering the Fc region to enhance the binding afﬁnity for FclelB as compared with the g afﬁnity for FClelA; see, also, PCT Publication Nos. W0 99/58572 to Armour et al., W0 99/51642 to ldusogie et al., and US. 6,395,272 to Deo et al.; the disclosures of which are incorporated herein in their entireties). Methods fying the Fc region to decrease binding y to FclelB are also known in the art (e.g., US. Patent ation No. 20010036459 and PCT Publication No. W0 01/79299, both to Ravetch et al., the sures of which are incorporated herein in their entireties). Modiﬁed antibodies having t Fc regions with enhanced binding afﬁnity for FclellA and/or FclelA as ed with a wildtype Fc region have also been described (e.g., PCT Publication Nos. , to Stavenhagen et al., the disclosure ofwhich is orated herein in its entirety).

Antibody effector function may also be modiﬁed through the generation of antibodies with d glycosylation patterns. For e, an antibody can be made that has an altered type of glycosylation, such as an afucosylated/hypofucosylated antibody having d amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.

Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modiﬁcations can be accomplished by, for example, m expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered ylation machinery have been described in the art and can be used as host cells in which to express recombinant dies ofthe invention to thereby produce an antibody with d glycosylation. For example, EP 1,176,195 by Hanai ct a1. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line t hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn(297)—linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et a1. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N- acetylglucosaminyltransferase lll (GnTlll)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et a1. (1999) Nat. Biotech. 17: 1 76-180).

Methods for generating antibodies with altered orms are known in the art, and e but are not limited to those described in Umana et al, 1999, Nat. Biotechnol 17:176- 180; Davies et al., 20017 Biotechnol Bioeng 742288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) US. Pat. No. 6,602,684; US. Ser. No. 10/277,370; US. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A l; PCT W0 02/31 1 140A 1; PCT WO 02/30954A1; PotillegentTM technology , lnc. Princeton, NJ); GlycoMAbTM glycosylation ering technology (GLYCART biotechnology AG, Zurich, Switzerland). See, e.g., WO 00061739; 9125; US 20030115614;Okazaki etal., 2004, JMB, 336: 1239-49. Antibodies with altered fucosylation pattern may also be ed by post-translational removal of fucose (e.g. with a dase enzyme), The present invention provides for antibodies and antibody nts that speciﬁcally bind to lL-5R which have an extended ife in vivo. In particular, the present invention provides antibodies and antibody fragments which have a half-life in a mammal (for example, but not limited to, a human), of greater than 3 days, greater than 7 days, greater than 10 days, greater than 15 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, r than 2 months, r than 3 months, greater than 4 months, or greater than 5 months.

To prolong the serum circulation of antibodies (for example, but not limited to, monoclonal antibodies and single chain dies) or antibody fragments (for example, but not limited to, Fab fragments) in vivo, for example, inert polymer molecules such as high molecular weight polyethyleneglycol (PEG) can be attached to the antibodies ding antibody fragments thereof) with or without a multifunctional linker either through site- specific conjugation ofthe PEG to the N— or inus ofthe antibodies or via epsilon- amino groups present on lysine residues. Linear or branched polymer derivatization that I5 results in minimal loss of biological activity will be used. The degree ofconjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be ted from antibody-PEG conjugates by size-exclusion or by change chromatography. PEG-derivatized antibodies ding antibody fragments thereof) can be tested for binding ty as well as for in vivo efficacy using methods known to those of skill in the art, for e, by immunoassays described herein.

Antibodies having an increased half-life in vivo can also be generated ucing one or more amino acid modiﬁcations (i.e., substitutions, insertions or deletions) into an lgG constant domain, or FcRn binding fragment thereof (e.g., PC or hinge Fc domain fragment).

See, e.g., International Publication No. WO 98/23289; international Publication No. WO 97/3463 1; and US. Patent No. 6,277,375, each ofwhich is incorporated herein by reference in its entirety.

Further, antibodies ding antibody fragments thereof) can be ated to . albumin in order to make the antibody (including antibody fragment thereof) more stable in vivo or have a longer fe in vivo. The techniques are well known in the art, see e.g., International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP 4l3, 622, all of which are incorporated herein by reference.

The present invention provides lL-SR binding molecules that specifically bind to [L- 5R, where the binding molecules are recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous n or polypeptide (or fragment ofa 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) to generate fusion proteins. In particular, the invention provides formulations of fusion proteins comprising an antigen-binding fragment of an antibody described herein (for e, but not limited to, a Fab fragment, Fd fragment, Fv nt, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein, ptide, or peptide.

Methods for fusing or conjugating proteins, polypeptides, or peptides to an antibody (including antibody nt thereof) are known in the art. See, e.g., US. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,1 12,946; European Patent Nos.

EP 307,434 and EP 367,166; International Publication Nos. WO 96/04388 and WO 70; Ashkenazi etal., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J. lmmunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:1 1337- 1 1341 (said references are incorporated herein by reference in their entireties).

Additional fusion proteins may be generated through the techniques of gene—shufﬂing, motif-shufﬂing, exon-shuffling, and/or codon—shuffling (collectively referred to as “DNA shuffling”). DNA shufﬂing may be employed to alter the ties bodies ofthe invention or fragments thereof (for example, but not limited to, antibodies or fragments thereof with higher afﬁnities and lower dissociation rates). See, generally, US. 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. 76-82; Hansson, et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308- 313 (each ofthese patents and publications are hereby incorporated by reference in its entirety).

Antibodies ding antibody fragments thereof), or the encoded dies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. A polynucleotide encoding an antibody (including dy nt thereof) thereof may be recombined with one or more ents, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Moreover, the antibodies (including antibody fragments thereof) can be fused to marker sequences, such as a peptide to facilitate purification. The marker amino acid sequence may be a hexa-histidine peptide, such as the tag provided in a pQE vector (QlAGEN, lnc., 9259 Eton , Chatsworth, CA, 9131 1), among others, many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient ation of the fusion protein. Other e tags useful for purification include, but are not d to, the hemagglutinin (“HA”) tag, which corresponds to an epitope d from the inﬂuenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the “ﬂag” tag. ln other embodiments, dies ofthe present invention or fragments f conjugated to a diagnostic or detectable agent. Such antibodies can be useful for ring or prognosing the onset, development, progression and/or severity ofa disease or disorder (for example, but not limited to, an autoimmune disorder) as part ofa clinical testing procedure, such as determining the efficacy ofa particular therapy. Such diagnosis and detection can accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidinlbiotin and avidin/biotin; ﬂuorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, rotriazinylamine IS ﬂuorescein, dansyl chloride or rythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, rase, luciferin, and aequorin; radioactive materials, such as, but not limited to, iodine (13 l l, l25l, 1231, and 12”,), carbon (14C), sulfur (35$), tritium (3H), indium (l lSln, l 131n, l 12ln, and lllln,), technetium (99Tc), thallium ), gallium (686a, 6703), palladium (l03Pd), molybdenum , xenon (l33Xe), fluorine (18F), 153Sm, l77Lu, 159Gd, l49Pm, l40La, 175Yb, I66Ho, 90Y, 47Sc, l86Re, 188Re,142 Pr, lOSRh, 97Ru, 68Ge, 57Co, 65Zn, SSSr, 32F, l53Gd, l69Yb, 5lCr, 54Mn, 758e, 113Sn, and ll7Sn; and positron emitting metals using various positron emission tomographies, and noradioactive paramagnetic metal ions.

Alternatively, an antibody can be conjugated to a second antibody to form an antibody conjugate as described by Segal in US. Patent No. 980, which is incorporated herein by reference in its entirety.

The therapeutic moiety or drug conjugated to an antigen ofinterest (e.g. lL-5 R) or fragment thereofshould be chosen to achieve the desired prophylactic or therapeutic effect(s) for a particular e or disorder, for example, a disease or disorder associated with or terized by aberrant expression and/or activity of an interferon alpha polypeptide, a e or disorder associated with or characterized by aberrant expression and/or activity of the interferon alpha receptor or one or more subunits thereof, an autoimmune disease, an autoimmune disease, transplant rejection, graﬁ versus host disease, or one or more symptoms thereof, in a subject. A clinician or other medical personnel should consider the following when deciding on what to conjugate to an antibody of interest, for example, an antibody that speciﬁcally binds to an interferon alpha polypeptide or nt f: the, nature ofthe disease, the severity ofthe disease, and the ion ofthe subject.

The antibodies (including antibody fragments thereof) that speciﬁcally bind to an antigen can be produced by any method known in the art for the synthesis ofantibodies, in particular, by al synthesis or by recombinant expression techniques (see, US Patent Publication 2007/00l4724A l ).

Polyclonal antibodies speciﬁc for an antigen can be produced by various procedures nown in the art. For e, a human antigen can be administered to various 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. Various adjuvants may be used to increase the immunological response, depending on the host s, and include but are not limited to, Freund’s ete and incomplete), l gels such as aluminum hydroxide, surface active substances such as lysolecithin, ic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be ed using a wide variety oftechniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T Cell Hybridomas 563 68l ier, N.Y., I981), and Harlow et al., Using Antibodies: A laboratory Manual, Cold Spring Harbor Laboratory Press (I999) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not d to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, yotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for speciﬁc antibodies using hybridoma technology are routine and well known in the art. Briefly, mice can be immunized with a non-murine antigen and once an immune se is detected, e.g., antibodies c for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are ed and cloned by limited dilution. Additionally, a RlMMS (repetitive immunization multiple sites) technique can be used to immunize an animal track et al., 1997, Hybridoma l6:38l-9, 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 a polypeptide ofthe invention. Ascites ﬂuid, which generally contains high levels of antibodies, can be generated by zing mice with positive oma .

The present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an l0 antibody ofthe invention wherein the hybridoma is generated by fusing splenocytes isolated from a mouse zed with a non-murine antigen with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind to the antigen.

Antibody fragments which recognize speciﬁc particular epitopes may be generated by any que known to those of skill in the art. For e, Fab and F(ab’)2 nts of the invention may be produced by proteolytic cleavage ofimmunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab’)2 fragments).

F(ab’)2 fragments contain the variable region, the light chain constant region and the CHI domain ofthe heavy chain. r, the antibodies ofthe present invention can also be generated using various phage y methods known in the art.

In phage y methods, functional dy domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences ng VH and VL domains are ied from animal cDNA libraries (e.g., human-or murine cDNA ies of affected tissues). The DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated in E. coli and the E. coli is infected with helper phage.

Phage used in these methods are typically filamentous phage including fd and M13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene Vlll. Phage expressing an antigen binding domain that binds to a particular antigen can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid e or bead. Examples of phage display methods that can be used to make the antibodies ofthe present invention include those disclosed in Brinkman et al., 1995, J.

Immunol. Methods 182:4l-50; Ames et al., 1995, J. Immunol. Methods l84zl77-l86; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in lmmunology 572191-280; International application No.

PCT/GB9l/Ol 134; International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 36, WO 95/15982, WO 95/20401, and WO97/l3844; and US. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, ,821,047, 5,571,698, 908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, 108, 6,33,187, 5,824,520, and 5,702,892; each h is incorporated herein by reference in its . entirety.

As described in the above references, after phage selection, the antibody coding s from the phage can be isolated and used to generate whole antibodies, including human dies, or any other desired antigen binding fragment, and sed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below. Techniques to recombinantly produce Fab, Fab’ and F(ab’)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864—869; Sawai et al., 1995, AR] 34:26-34; and Better et al., 1988, Science 240:1041-1043 (said references incorporated by reference in their entireties).

To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a ﬂanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv . Utilizing cloning ques known to those of skill in the art, the PCR ampliﬁed VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 4 nt region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. The vectors for expressing the VH or VL domains may comprise an EF—la promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin. The VH and VL domains may also cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then nsfected into cell lines to generate stable or ent cell lines that express ength antibodies, for example, but not limited to, lgG, using techniques known to those ofskill in the art.

For some uses, including in vivo use ofantibodies in humans and in vitro ion assays, it may be appropriate to use humanized antibodies or chimeric antibodies.

Completely human antibodies and humanized antibodies are particularly ble for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also US. Patent Nos. 4,444,887 and 4,716,].1 I; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO98/l6654, WO 96/34096, WO 96/33735, and WO 9l/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous globulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant , and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the “-1 region prevents endogenous antibody tion. The modiﬁed embryonic stem cells are expanded and microinjected into blastocysts to produce ic mice. The chimeric mice are then be bred to produce homozygous offspring which express human, antibodies.

The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion ofa polypeptide ofthe invention. Monoclonal antibodies directed t the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and uently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to e therapeutically useful lgG, lgA, lgM and lgE antibodies. For an overview ofthis technology for producing human antibodies, see g and Huszar (1995, Int. Rev. lmmunol. 13:65 93). For a detailed discussion of this technology for ing human antibodies and human monoclonal antibodies and protocols for ing such dies, see, e.g., International Publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and US. Patent Nos. 5,413,923, ,625,126, 5,633,425, 5,569,825, 5,66l,0|6, 5,545,806, 5,814,318, and 598, which are incorporated by reference herein in their entirety. In addition, companies suchas Abgenix, lnc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to e human antibodies directed against a selected antigen using technology r to that described above.

A chimeric antibody is a molecule in which different portions ofthe antibody are derived from different immunoglobulin molecules. Methods for ing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et a1., 1989, J. Immunol. Methods 125: 191-202; and US.

Patent Nos. 5,807,715, 4,816,567, 4,8 16397, and 6,331,415, which are incorporated herein by nce in their ty.

A humanized antibody is an antibody or its variant or fragment thereof which is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence ofa human immunoglobulin and a CDR having substantially the amino acid sequence ofa non-human immunoglobulin. A zed antibody comprises substantially all of at least one, and lly two, le domains (Fab, Fab’, F(ab’)2, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those ofa non human immunoglobulin (i.e., donor antibody) and all or substantially all ofthe ork regions are those ofa human immunoglobulin consensus ce. In one embodiment, a humanized antibody also comprises at least a n of an immunoglobulin constant region (Fc), typically that ofa human immunoglobulin. Ordinarily, the antibody will contain both the light chain as well as at least the variable domain ofa heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions ofthe heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including lgM, lgG, lgD, lgA and lgE, and any isotype, including lgGI, lgGZ, IgG3 and lgG4. y the constant domain is a complement ﬁxing constant domain where it is desired that the humanized antibody exhibit cytotoxic activity, and the class is typically lgGl. Where such cytotoxic activity is not desirable, the constant domain may be ofthe lgG2 class. The humanized antibody may comprise sequences from more than one class or isotype, and selecting ular constant domains to optimize d effector ons is within the ordinary skill in the art. The framework and CDR regions ofa humanized antibody need not correspond precisely to the al sequences, e.g., the donor CDR or the consensus framework may be nized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the import antibody. Such mutations, however, will not be extensive. y, at least 75% ofthe humanized antibody residues will correspond to those ofthe parental framework and CDR sequences, more often 90%, and r than 95%. Humanized antibody can be produced using variety oftechniques known in the art, including but not limited to, CDR- grafting (European Patent No. EP 239,400; International publication No. WO 91/09967; and US. 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):805-8l4; and Roguska et al., 1994, PNAS 912969-973), chain shufﬂing (US. Patent No. 5,565,332), and techniques disclosed in, e.g., US. Pat. No. 213, US. Pat. No. 5,766,886, WO 9317105, Tan et al., J. lmmunol. 169:1 l 19 25 (2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods (3):267 79 (2000), Baca et al., J. Biol. Chem. 272(16):]0678 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):]717 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 residue from the CDR donor antibody to alter, ably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, but not limited to, by modeling ofthe interactions ofthe CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to fy unusual framework residues at particular positions (see, e.g., Queen et al., US. Patent No. 5,585,089; and Riechmann et al., 1988, Nature 332:323, which are incorporated herein by reference in their entireties).

Single domain dies, for example, dies g the light chains, can be produced by methods well-known in the art. See Riechmann et al., 1999, J. lmmuno. 231:25- 38; Nuttall et al., 2000, Curr. Pharm. Biotechnol. 1(3):253-263; Muylderman, 2001, J.

Biotechnol. 277302; US. Patent No. 6,005,079; and International Publication Nos. WO 94/04678, WO 94/25591, and WO 01/44301, each ofwhich is incorporated herein by reference in its entirety. r, the antibodies that specifically bind to an antigen (e.g. lL-5R) can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” an antigen using techniques well known to those skilled in the art. (See, e.g., pan & Bona, 1989, FASEB J. 7(5):437- 444; and Nissinoff, 1991, J. Immunol. :2429-2438).

Recombinant expression ofan antibody ofthe invention (e.g., a heavy or light chain ofan antibody ofthe invention or a fragment thereof or a single chain antibody ofthe invention) may require construction of an expression vector ning a polynucleotide that encodes the antibody. Once a cleotide encoding an antibody molecule, heavy or light chain of an antibody, or fragment thereof has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well-known in the art. Thus, methods for preparing a protein by sing a polynucleotide ning an antibody encoding nucleotide ce are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable s comprising a nucleotide sequence ng an antibody molecule ofthe invention, a heavy or light chain ofan antibody, 3 heavy or light chain variable domain ofan antibody (including antibody nt thereof), or a heavy or light chain CDR, operably linked to a promoter. Such vectors may include the tide sequence encoding the constant region ofthe antibody molecule (see, e.g., international Publication No. WO 07; International Publication No. WO 89/01036; and US. Patent No. 5,122,464) and the variable domain ofthe antibody may be cloned into such a vector for expression ofthe entire heavy, the entire light chain, or both the entire heavy and light chains.

The expression vector is erred to a host cell by conventional techniques and the transfected cells are then ed by conventional ques to produce an antibody ofthe invention. Thus, the invention es host cells containing a polynucleotide encoding an dy of the invention or nts thereof, or a heavy or light chain thereof, or fragment thereof, or a single chain antibody ofthe invention, operably linked to a heterologous promoter. ln c embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for sion 2O ofthe entire globulin molecule, as detailed below.

A y of host-expression vector systems may be utilized to express the antibody molecules ofthe invention (see, e.g., US. Patent No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences ofinterest may be produced and subsequently puriﬁed, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include but are not limited to microorganisms such as bacteria (for example, but not limited to, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (for example, but not limited to, Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell s infected with recombinant virus expression vectors (for example, but not limited to, baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (for example, but not limited to, cauliﬂower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with inant plasmid expression vectors (for example, but not limited to, Ti plasmid) containing antibody coding sequences; or mammalian cell systems (for example, but not limited to, C08, CHO, BHK, 293, N80, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, but not limited to, metallothionein promoter) or from mammalian viruses (for example, but not d to, the adenovirus late promoter; the vaccinia virus 7.5K promoter). Bacterial cells such as Escherichia coli, and eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression ofa recombinant antibody le. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene ; and Cockett et al., 1990, Bio/Technology 8:2). In a c embodiment, the - expression of nucleotide ces encoding antibodies ofthe invention, derivative, analog, or fragment thereof is regulated by a constitutive promoter, inducible promoter or tissue speciﬁc er. ln bacterial systems, a number of expression vectors may be advantageously ed depending upon the use intended for the antibody molecule being expressed. For e, when a large quantity of such an antibody is to be produced, for the tion of pharmaceutical compositions of an antibody le, vectors which direct the expression of high levels of fusion protein products that are readily puriﬁed may be desirable. Such vectors e, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO l2:l79l), in which the dy coding ce may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; plN vectors (lnouye & lnouye, 1985, Nucleic Acids Res. l3:3lOl-3109; Van Heeke & Schuster, 1989, J.

Biol. Chem. 24:5503-5509); and the like. pGEX s may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (CST). In general, such fusion proteins are soluble and can easily be puriﬁed from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to e thrombin or factor Xa protease cleavage sites so that 3O the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in tera frugiperda cells.

The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) ofthe virus and placed under control of an AcN PV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based sion systems may be utilized.

In cases where an adenovirus is used as an expression vector, the antibody coding sequence ofinterest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a sential region ofthe viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, IO I984, Proc. Natl. Acad. Sci. USA 8 1:355—359). Specific tion signals may also be required for efﬁcient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame ofthe desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be ofa y of origins, both natural and synthetic. The efficiency of sion may be enhanced by the inclusion of appropriate transcription enhancer ts, transcription terminators, etc. (see, e.g., Bittner et al., I987, s in Enzymol. 153251—544).

In on, a host cell strain may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in the specific fashion-r desired. Such modiﬁcations (for e, but not limited to, glycosylation) and processing (for example, but not d to, ge) of n products may be important for the function ofthe protein. Different host cells have teristic and specific mechanisms for the post—translational processing and modiﬁcation of proteins and gene products.

Appropriate cell lines or host systems can be chosen to ensure the correct cation and - processing ofthe foreign n expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation ofthe gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, BT483, H5578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL703O and HsS78Bst cells.

For long-term, high-yield production of recombinant proteins, stable expression is may be used. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate sion control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction ofthe foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid intotheir chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.

This method may ageously be used to engineer cell lines which s the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

In one ment, the cell line used to express the IL-SR binding molecule is a cell that does not fucosylate the Fc region ofthe IL-5R binding molecule. Nonlimiting examples of these types of cells are found in US. Patent No. 6,946,292, and US. Patent Application Publication Nos. 2006/007899IAI, 2004/0110282A1, 2006/0024800AI, 2005/02 I 6958A I 2004/0132140, and 2004/0259150. In a specific embodiment, the lL-5R binding molecule is I5 a humanized, afucosylated IgGI anti-IL-5R 0t chain monoclonal antibody. In a further speciﬁc ment, the antibody is MEDI—563 (also known as BIW-8405). In yet a further specific embodiment, the antibody is not MEDI-563.

A number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., I977, Cell ), hypoxanthineguanine phosphoribosyltransferase lska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48202), and adenine phosphoribosyltransferase (Lowy et al., I980, Cell 22:8—17) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite ance can be used as the basis of selection for the following genes: dhfr, which confers ance to methotrexate (Wigler et al., I980, Natl. Acad. Sci. USA ; O’Hare et al., I981, Proc.

Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to enolic acid (Mulligan & Berg, I98], Proc. Natl. Acad. Sci. USA 782072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, I991, Biotherapy 3:87-95; Tolstoshev, I993, Ann. Rev. Pharmacol. Toxicol. -596; Mulligan, I993, e 260:926-932; and Morgan and Anderson, I993, Ann. Rev. Biochem. 62: I9l-2I7; May, I993, TIB TECH l l(5):l55-2 15); and hygro, which confers resistance to hygromycin (Santerre et al., I984, Gene 30: I47). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the d recombinant clone, and such methods are bed, for e, 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 in rs l2 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol.

Biol. 150:], which are incorporated by reference herein in their entireties.

The expression levels of an 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 a marker in the vector system expressing antibody is ampliﬁable, se in the level ofinhibitor present in culture of host cell will increase the number of copies ofthe marker gene. Since the ampliﬁed region is ated with the antibody gene, tion ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell may be co—transfected with two expression vectors ofthe invention, the ﬁrst vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which s, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, 1986, Nature ; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2 197). The coding sequences for the heavy and light chains may comprise cDNA or c DNA.

Once an antibody molecule ofthe ion has been produced by recombinant expression, it may be puriﬁed by any method known in the art for puriﬁcation of an immunoglobulin molecule, for example, by chromatography (e.g., ion ge, afﬁnity, particularly by affinity for the speciﬁc antigen after Protein A, and sizing column chromatography), centrifugation, differential lity, or by any other standard technique for the puriﬁcation of proteins. Further, the antibodies ofthe present invention or fragments f may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate puriﬁcation.

For the lL-SR g molecules (e.g. antibodies, proteins, ptides, peptides and fusion proteins) encompassed by the invention, the dosage administered to a patient is typically 0.000] mg/kg to 100 mg/kg ofthe patient’s body weight. Preferably, the dosage administered to a patient is between 0.000] mg/kg and 20 mg/kg, 0.000l mg/kg and IQ mg/kg, 0.000] mg/kg and 5 mg/kg, 0.000l and 2 mg/kg, 0.000l and | mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg ofthe patient’s body weight. lly, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and ncy of administration of antibodies of the ion or fragments thereofmay be reduced by enhancing uptake and tissue penetration of the antibodies by modifications such as, for example, tion.

In a specific embodiment, the dosage oflL-5R binding molecule administered to prevent, treat, manage, and/or ameliorate a disease or one or more symptoms thereof in a patient is 150 ug/kg or less, preferably 125 ug/kg or less, 100 ug/kg or less, 95 pig/kg or less, 90 ug/kg or less, 85 ug/kg or less, 80 ug/kg or less, 75 ug/kg or less, 70 pig/kg or less, 65 ug/kg or less, 60 ug/kg or less, 55 ug/kg or less, 50 ug/kg or less, 45 ug/kg or less, 40 ug/kg or less, 35 ug/kg or less, 30 ug/kg or less, 25 ug/kg or less, 20 ug/kg or less, 15 ug/kg or less, 10 ug/kg or less, 5 ug/kg or less, 2.5 ug/kg or less, 2 ug/kg or less, 1.5 ug/kg or less, 1 ug/kg or less, 0.5 ug/kg or less, or 0.5 ug/kg or less ofa patient’s body . In another embodiment, the dosage ofthe 1L-5R binding molecules ofthe invention administered to prevent, treat, manage, and/or ameliorate a hyperproliferative disease, or one or more symptoms thereofin a patient is a unit dose of 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 2O 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 7m g, 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 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

In other embodiments, a subject is administered one or more doses of an ive amount of one or therapies of the invention, wherein the dose of an effective amount achieves a serum titer of at least 0.1 ug/ml, at least 0.5 ug/ml, at least 1 ug/ml, at least 2 ug/ml, at least ug/ml, at least 6 ug/ml, at least 10 ug/ml, at least 15 ug/ml, at least 20 ug/ml, at least 25 ug/ml, at least 50 ug/ml, at least 100 pg/ml, at least 125 ug/ml, at least 150 ug/ml, at least 175 ug/ml, at least 200 ug/ml, at least 225 ug/ml, at least 250 ug/ml, at least 275 ug/ml, at least 300 ug/ml, at least 325 ug/ml, at least 350 pg/ml, at least 375 ug/ml, or at least 400 ug/ml of the therapies ofthe invention. In yet other embodiments, a subject is administered a dose of an effective amount ofone ofthe lL-5R binding le ofthe invention to achieve a serum . titer of at least 0.1 ug/ml, at least 0.5 ug/ml, at least 1 ug/ml, at least, 2 ug/ml, at least 5 ug/ml, at least 6 ug/ml, at least 10 ug/ml, at least 15 ug/ml, at least 20 ug/ml, at least 25 pg/ml, at least 50 pg/ml, at least 100 pg/ml, at least 125 pg/ml, at least 150 pg/ml, at least 175 pg/ml, at least 200 pg/ml, at least 225 pg/ml, at least 250 pg/ml, at least 275 pg/ml, at least 300 pg/ml, at least 325 pg/ml, at least 350 pg/ml, at least 375 pg/ml, or at least 400 pg/ml of the lL-5R binding molecule and a uent dose of an ive amount of one or more 1L- 5R binding molecule ofthe invention is administered to maintain a serum titer ofat least 0.1 pg/ml, 0.5 pg/ml, 1 pg/ml, at least, 2 pg/ml, at least 5 pg/ml, at least 6 pg/ml, at least 10 pg/ml, at least 15 pg/ml, at least 20 pg/ml, at least 25 pg/ml, at least 50 pg/ml, at least 100 pg/ml, at least 125 pg/ml, at least 150 pg/ml, at least 175 pg/ml, at least 200 pg/ml, at least 225 pg/ml, at least 250 pg/ml, at least 275 pg/ml, at least 300 pg/ml, at least 325 pg/ml, at least 350 pg/ml, at least 375 pg/ml, or at least 400 pg/ml. In accordance with these embodiments, a subject may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, l 1, 12 or more subsequent doses.

In a speciﬁc embodiment, the invention provides methods of preventing, treating, managing, or ameliorating an eosinophil mediated disease or one or more symptoms thereof, said method comprising administering to a subject in need thereofa dose of at least 10 pg, preferably at least 15 pg, at least 20 pg, at least 25 pg, at least 30 pg, at least 35 pg, at least 40 pg, at least 45 pg, at least 50 pg, at least 55 pg, at least 60 pg, at least 65 pg, at least 70 pg, at least 75 pg, at least 80 pg, at least 85 pg, at least 90 pg, at least 95 pg, at least 100 pg, at least 105 pg, at least l 10 pg, at least 1 15 pg, or at least 120 pg of one or more therapies (e.g., therapeutic or prophylactic agents), ation ies, or itions ofthe ion.

In another embodiment, the invention provides a method of preventing, treating, managing, and/or ameliorating an eosinophil mediated disease or disorder or one or more symptoms thereof, said methods comprising administering to a subject in need thereofa dose of at least pg, preferably at least 15 pg, at least 20 pg, at least 25 pg, at least 30 pg, at least 35 pg, at least 40 pg, at least 45 pg, at least 50 pg, at least 55 pg, at least 60 pg, at least 65 pg, at least 70 pg, at least 75 pg, at least 80 pg, at least 85 pg, at least 90 pg, at least 95 pg, at least 100 pg, at least 105 pg, at least 1 10 pg, at least 1 15 pg, or at least 120 pg ofone or more lL-5R binding molecules, combination therapies, or compositions ofthe invention 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 two weeks, once every three weeks, or once a month.

The present invention provides methods of preventing, treating, managing, or preventing an eosinophil mediated disorder or e or one or more ms thereof, said method comprising: (a) administering to a subject in need thereofone or more doses ofa prophylactically or therapeutically effective amount ofone or more lL-SR binding molecules, combination therapies, or compositions ofthe invention; and (b) monitoring the plasma level/concentration ofthe said stered 1L-5R binding molecules in said subject after administration ofa certain number of doses ofthe said therapies (e.g., therapeutic or prophylactic ). Moreover, preferably, said n number of doses is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 doses ofa prophylactically or therapeutically ive amount one or more 1L-5R binding molecules, compositions, or combination therapies ofthe invention.

In a speciﬁc embodiment, the ion provides a method of preventing, ng, managing, and/or ameliorating an eosinophil mediated disorder or disease or one or more symptoms thereof, said method comprising: (a) stering to a subject in need thereofa dose ofat least 10 ug (preferably at least 15 ug, at least 20 ug, at least 25 ug, at least 30 ug, at least 35 ug, at least 40 ug, at least 45 ug, at least 50 ug, at least 55 ug, at least 60 ug, at least 65 ug, at least 70 ug, at least 75 ug, at least 80 ug, at least 85 ug, at least 90 ug, at least 95 ug, or at least 100 ug) of one or more ies (e.g., therapeutic or prophylactic agents) ofthe invention; and (b) administering one or more subsequent doses to said subject when the plasma level ofthe 1L-5R binding molecule administered in said subject is less than 0.1 ug/ml, preferably less than 0.25 ug/ml, less than 0.5 ug/ml, less than 0.75 ug/ml, or less than 1 ug/ml. In another embodiment, the invention provides a method of preventing, treating, managing, and/or ameliorating 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 at least 10 ug (preferably at least 15 ug, at least 20 ug, at least 25 ug, at least ug, at least 35 ug, at least 40 ug, at least 45 ug, at least 50 ug, at least 55 ug, at least 60 ug, at least 65 ug, at least 70 ug, at least 75 ug, at least 80 ug, at least 85 ug, at least 90 ug, at least 95 pg, or at least 100 ug) of one or more 1L-5R binding molecules ofthe invention; (b) monitoring the plasma level ofthe administered lL-5R binding molecules in said subject after the administration ofa certain number ofdoses; and (c) administering a subsequent dose of 1L—5R binding molecules ofthe ion when the plasma level ofthe administered lL-SR binding molecule in said subject is less than 0.1 ug/ml, preferably less than 0.25 ug/ml, less .than 0.5 ug/ml, less than 0.75 ug/ml, or less than 1 ug/ml. In certain embodiments, said certain number ofdoses is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, l 1, or 12 doses of an effective amount ofone or more lL-SR binding molecules ofthe invention.

Therapies (e.g., prophylactic or eutic agents), other than the lL-5R g molecules ofthe invention, which have been or are currently being used to prevent, treat, manage, and/or ameliorate a hyperproliferative disease or one or more ms thereof can be administered in combination with one or more lL-SR binding molecules according to the methods ofthe invention to treat, manage, prevent, and/or ameliorate an eosinophil mediated disorder or disease or one or more symptoms thereof. Preferably, the dosages of prophylactic or therapeutic agents used in combination therapies ofthe invention are lower than those which have been or are currently being used to prevent, treat, manage, and/or ameliorate an eosinophil mediated disorder or disease or one or more symptoms thereof. The recommended dosages of agents currently used for the prevention, treatment, management, or amelioration ofa hyperproliferative disease or one or more ms thereof can be obtained from any reference in the art including, but not limited to, Hardman et al., eds., 2001, Goodman & Gilman’s The Pharmacological Basis Of Basis OfTherapeutics, 10th ed., Mc- Graw-Hill, New York; Physician’s Desk nce (PDR) 58th ed., 2004, Medical Economics Co., lnc., le, NJ, which are incorporated herein by reference in its entirety.

In various embodiments, the ies (e.g., prophylactic or therapeutic agents) are administered less than 5 minutes apart, less than 30 s apart, 1 hour apart, at about 1 hour apart, at about I to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 1 1 hours apart, at about 1 1 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours part.

In other embodiments, two or more therapies are administered within the same patient visit.

In certain embodiments, one or more lL-SR g molecules ofthe ion and one or more other therapies (e.g., prophylactic or eutic agents) are cyclically administered. Cycling therapy involves the administration ofa ﬁrst therapy (e.g., a first prophylactic or therapeutic agent) for a period oftime, followed by the administration ofa second therapy (e.g., a second prophylactic or therapeutic agent) for a period , optionally, followed by the administration ofa third therapy (e.g., prophylactic or therapeutic agent) for a period oftime and so forth, and repeating this sequential administration, i.e., the cycle in order to reduce the development of resistance to one ofthe therapies, to avoid or reduce the side s ofone ofthe therapies, and/or to improve the efficacy of the therapies.

In certain embodiments, the administration ofthe same lL-SR binding molecule ofthe invention may be repeated and the strations may be separated by at least 1 day, 2 days, 3 days, 5 days, IO days, l5 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months. In other embodiments, the administration ofthe same therapy (e.g., prophylactic or therapeutic agent) other than an lL-5R binding molecule of the invention may be repeated and the administration may be separated by at least 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 a specific embodiment, the lL-SR binding molecule is stered as a single enous dose of 0.03 mg/kg.

The present invention provides methods of preventing, ng, managing, or preventing an eosinophil mediated er or disease or one or more symptoms thereof, said method comprising: (a) administering to a subject in need thereof one or more doses ofa prophylactically or eutically effective amount of one or more lL-SR binding molecules, combination ies, or compositions of the invention; and (b) monitoring at least one disease indicator or symptom in the subject prior to and following the administration of one or more doses ofsaid therapies (e.g., therapeutic or prophylactic agents). ln one embodiment, the subject suffers from COPD.

In one embodiment, the subject suffers from mild persistent or mild intermittent asthma as deﬁned by the by the 2002 Expert Panel report 0fthe NAEPP.

In one embodiment, the disease indicator or symptom in the subject is monitored prior to and following the administration ofa single dose ofone or more lL-5R binding molecules.

In another embodiment, the disease indicator or symptom in the t is monitored prior to and following the administration of multiple doses ofone or more lL-5R g molecules.

In one embodiment, the disease tor or symptom is a self-assessed Asthma Symptom Score. A non—limiting e of an Asthma Symptom Score is a self—assessed score ed daily by the subject at home. The score grades asthma symptoms for the past 24 hours, based on the severity of morning, nocturnal, and daytime symptoms. The symptoms and assigned scores are described in Table l. The daily maximum score is 9, minimum is 0. Subjects ssess and record on a continuous basis.

Table l. Asthma Symptom Score key; Nocturnal lasts from going to'bed until awakening in the morning. Morning lasts from ing until I hour after awakening.

Daytime begins 1 hour after awakening and ends at bedtime.

- Nocturnal symptoms nI did not wake up because of breathing problems.

I awoke once because of my breathing problems, but did not use my rescue medication.

I awoke once e of my breathing problems, but my rescue medication lled my symptoms.

I awoke more than once because of my breathing problems, but my rescue medication controlled my symptoms.

I had difﬁculty sleeping because of my breathing problems even though I used my rescue tions.

Morning symptoms Symptoms caused some discomfort; at times limiting strenuous activity Symptoms caused moderate discomfort; at times limiting routine activity Symptoms occurred at rest, caused marked discomfort, and usually d routine activity In one embodiment, a subject has an Asthma Symptom Score ofX prior to the administration ofthe one or more doses of one or more lL-SR binding les and an Asthma Symptom Score-ofX-Y following the stration ofthe one or more doses of one or more lL-SR binding molecules, wherein X is l, 2, 3, 4, 5, 6, 7, 8, or 9, wherein Y is l, 2, 3, 4, 5, 6, 7, 8, or 9, and wherein the post-administration Score is never below 0.

In" one embodiment, a subject has an Asthma m ScOre of between 0 and 9 prior to the administration of the one or more doses ofone or more lL-SR binding molecules.

In one embodiment, a subject has an Asthma Symptom Score between 0 and 3, between I and 4, between 2 and 5, between 3 and 5, between 4 and 7, between 5 and 8 or between 6 and 9 prior to the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one ment, a subject has an Asthma Symptom Score of l, 2, 3, 4, 5, 6, 7, 8 or 9 prior to the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a subject has an Asthma Symptom Score of l prior to the administration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a t has an Asthma Symptom Score of2 prior to the administration ofthe one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has an Asthma Symptom Score of3 prior to the administration of the one or more doses of one or more IL-5R binding molecules. In one embodiment, a subject has an Asthma Symptom Score of4 prior to the administration of the one or more doses of one or more IL- 5R binding molecules. In one embodiment, a subject has an Asthma Symptom Score of5 prior to the stration ofthe one or more doses of one or more IL-SR binding molecules.

IO In one embodiment, a subject has an Asthma Symptom Score of6 prior to the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a t has an Asthma Symptom Score of7 prior to the administration of the one or more doses of one or more IL-SR g molecules. In one embodiment, a-subject has an Asthma Symptom Score of 8 prior to the administration ofthe one or more doses of one or more IL- 5R binding molecules. In one embodiment, a t has an Asthma Symptom Score of9 prior to the administration ofthe one or more doses ofone or more IL-SR binding molecules.

In one embodiment, a subject has an Asthma Symptom Score of between 0 and 9 following the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has an Asthma Symptom Score between 0 and 3, between I and 4, between 2 and 5, between 3 and 5, between 4 and 7, between 5 and 8 or between 6 and 9 following the stration ofthe one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has an Asthma Symptom Score of I, 2, 3, 4, 5, 6, 7, 8 or 9 following the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a t has an Asthma Symptom Score of I following the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a subject has an Asthma Symptom Score of2 following the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one ment, a subject has an Asthma Symptom Score of3 ing the administration of the one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has an Asthma Symptom Score of4 following the stration ofthe one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has an Asthma Symptom Score of5 ing the administration of the one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has an Asthma Symptom Score of6 following the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has an Asthma Symptom Score of7 following the stration ofthe one or more doses ofone or more lL-5R binding molecules. ln one embodiment, a subject has an Asthma Symptom Score of8 following the administration of the one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has an Asthma Symptom Score of9 following the administrationofthe one or more doses of one or more lL-SR binding molecules.

In one embodiment, the Asthma Symptom Score ofa subject is lower following the administration of one or more doses of one or more lL-5R binding les than prior to the administration of one or more doses ofone or more lL-5R binding molecules wherein the post-\administration Score is never lower than 0. In a c embodiment, the Asthma Symptom Score is I point lower. In a speciﬁc embodiment, the Asthma Symptom Score is 9 point lower. In a speciﬁc embodiment, the Asthma Symptom Score is 2 point lower. In a speciﬁc embodiment, the Asthma Symptom Score is 3 point lower. In a speciﬁc embodiment, the Asthma m Score is 4 point lower. In a speciﬁc embodiment, the Asthma Symptom I5 Score is 5 point lower. In a c embodiment, the Asthma Symptom Score is 6 point lower. In a speciﬁc embodiment, the Asthma m Score is 7 point lower. In a speciﬁc embodiment, the Asthma Symptom Score is 8 point lower. ln a-speciﬁc embodiment, the Asthma Symptom Score is at least 1 point lower. In a speciﬁc embodiment, the Asthma Symptom Score is at least 9 point lower. In a speciﬁc embodiment, the Asthma Symptom Score is at least 2 point lower. In a speciﬁc embodiment, the Asthma Symptom Score is at least 3 point lower. ln a speciﬁc embodiment, the Asthma Symptom Score is at least 4 point lower. In a speciﬁc embodiment, the Asthma Symptom Score is at least 5 point lower. In a speciﬁc embodiment, the Asthma Symptom Score is at least 6 point lower. In a c embodiment, the Asthma Symptom Score is at least 7 point lower. In a speciﬁc embodiment, the Asthma Symptom Score is at least 8 point lower.

In one embodiment, the disease tor or symptom is Fractional Exhaled Nitric Oxide (FENO ). FENO may be measured according to the combined recommendations of the European Respiratory Society and the American Thoracic Society (American ic Society, European Respiratory Society. (2005) ATS/ERS Recommendations for Standardized Procedures for the Online and Ofﬂine Measurements of Exhaled Lower atory Nitric Oxide and Nasal Nitric Oxide, 2005. Am J Respir Crit Care Med. 171: 912-930). The FENO measurements may be performed using the NlOX at a 50 ml/s flow rate (ATS standard).

In one ment, a subject has a FENO of between 20 and 500 ppb prior to the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a FENO between 20 and 500 ppb, between 20 and 400 ppb, between 20 and 300 ppb, between 20 and 200 ppb, between 50 and 500 ppb, between I00 and 500 ppb, between 150 and 500 ppb, between 200 and 500 ppb, between 20 and 50 ppb, between 50 and 100 ppb, between 100 and 200 ppb, between 200 and 300 ppb, between 300 and 500 ppb prior to the administration ofthe one or more doses of one or more IL-SR g molecules. In one embodiment, a subject has a FENO ofat least 50 ppb, at least I00 ppb, at least 150 ppb, at least 200 ppb, at least 250 ppb, at least 300 ppb, at least 350 ppb, IO at least 400 ppb prior to the stration ofthe one or more doses of one or more IL-SR binding les. In one embodiment, a subject has a FENO of 50 ppb prior to the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a FENO of 100 ppb prior to the administration of the one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a FENO of I50 ppb prior to the administration ofthe one or more dosesof one or'more lL-SR binding molecules. In one embodiment, a subject has a FENO of200 ppb prior to the administration ofthe one or more doses ofone or more IL-5R binding molecules. In one embodiment, a subject has a FENO of 250 ppb prior to the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a FENO of 300 ppb prior to the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a FENO of350 ppb prior to the stration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a subject has a FENO of400 ppb prior to the administration ofthe one or more doses ofone or more IL-SR binding les.

In one embodiment, a subject has a FENO of between 20 and 500 ppb following the administration ofthe one or more doses of one or more IL-SR binding les. In one ment, a subject has a FENO between 20 and 500 ppb, between 20 and 400 ppb, between 20 and 300 ppb, between 20 and 200 ppb, between 50 and 500 ppb, between IOO and 500 ppb, between 150 and 500 ppb, between 200 and 500 ppb, between 20 and 50 ppb, between 50 and IOO ppb, n IOO and 200 ppb, between 200 and 300 ppb, between 300 and 500 ppb following the administration ofthe one or more doses of one or more IL-SR binding les. In one embodiment, a subject has a FENO of at most 50 ppb, at most IOO 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 following the administration ofthe one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has a FENO of at most 20 ppb following the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one ment, a subject has a FENO ofat most 50 ppb following the administration ofthe one or more doses ofone or more IL-5R binding molecules. In one embodiment, a subject has a FENO ofat most 100 ppb following the administration ofthe one or more doses of one or more IL-5R binding molecules. In one embodiment, a subject has a FENO ofat most 150 ppb following the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a FENO ofat most 200 ppb following the administration ofthe one or more doses ofone or more IL-SR g IO les. In one embodiment, a subject has a FENO of at most 250 ppb following the administration of the one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has a FENO of at most 300 ppb ing the administration ofthe one or more doses of one or more IL-5R binding molecules.. In one embodiment, a subject has a FENO of at most 350 ppb following the administration ofthe one or more doses ofone or more lL-SR g les. In one ment, a subject has a FENO of at most 400 ppb ing the administration ofthe one or more doses ofone or more IL-SR binding molecules.

In one embodiment, the FENO ofa subject is lower following the administration of one or more doses ofone or more IL-5R binding molecules than prior to the administration of one or more doses of one or more IL-SR binding molecules, wherein the FENO is never below 0 ppb. In a speciﬁc embodiment, the FENO is at least 50 ppb lower. In a speciﬁc embodiment, the FENO is at least 100 ppb lower. In a speciﬁc embodiment, the FENO is at least 150 ppb lower. In a speciﬁc ment, the FENO is at least 200 ppb lower. In a speciﬁc embodiment, the FENO is at least 250 ppb lower. In a speciﬁc embodiment, the FENO is at least 300 ppb lower. In a speciﬁc embodiment, the FENO is at least l0% lower.

In a speciﬁc embodiment, the FENO is at least 20% lower. In a speciﬁc embodiment, the FENO is at least 30% lower. In a speciﬁc embodiment, the FENO is at least 40% lower. In a speciﬁc embodiment, the FENO is at least 50% lower. In a speciﬁc embodiment, the FENO is at least 60% lower. In a speciﬁc embodiment, the FENO is at least 70% lower. In a speciﬁc embodiment, the FENO is at least 80% lower. In a speciﬁc embodiment, the FENO is at least 90% lower. In a speciﬁc embodiment, the FENO is 10% lower. In a c embodiment, the FENO is 20% lower. In a speciﬁc embodiment, the FENO is 30% lower.

In a speciﬁc embodiment, the FENO is 40% lower. In a speciﬁc ment, the FENO is 50% lower. In a speciﬁc embodiment, the FENO is 60% lower. In a speciﬁc embodiment, the FENO is 70% lower. In a speciﬁc embodiment, the FENO is 80% lower. In a speciﬁc embodiment, the FENO is 90% lower.

In one embodiment, the disease indicator or symptom is phil Cationic Protein (ECP). Serum ECP levels may be assessed using any methods known to one of skill in the art, for e, but not limited to ELISA assay, radioimmunoassay. Serum ECP levels may be measured by any one of the cially available assays.

In one embodiment, a t has a serum ECP of between 20 and 500 ng/ml prior to the stration ofthe one or more doses ofone or more IL—SR binding molecules. In one embodiment, a subject has a serum ECP between 20 and 200 ng/ml, between 20 and IO 150 ng/ml, between 20 and 100 ng/ml, between 20 and 50 ng/ml, between 30 and 200 ng/ml, between 40 and 200 ng/ml, between 50 and 200 ng/ml, between 30 and 100 ng/ml, between and 80 ng/ml, between 30 and 70 ng/ml, between 20 and 80 ng/ml, between 20 and 70 ng/ml, between 20 and 60 ng/ml prior to the administration ofthe one or more doses of one or more IL—SR binding molecules. In one embodiment, a subject has a serum ECP ofat . 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 I00 ng/ml, at least 150 ng/ml, at least 200 ng/ml prior to the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a serum ECIS of 25 ng/ml prior to the administration ofthe one or more doses of one or more IL-SR binding molecules. In one embodiment, a t has a serum ECP of 30 ng/ml prior to the stration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a serum ECP of35 ng/ml prior to the administration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a serum ECP of 40 ng/ml prior to the administration of the one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has a serum ECP of 50 ng/ml prior to the stration ofthe one or more doses ofone or more IL-SR binding molecules.

In one embodiment, a subject has a serum ECP of 60 ng/ml prior to the administration ofthe one or more doses of one or more IL-SR binding molecules. In one embodiment, a‘subject has a serum ECP of 70 ng/ml prior to the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a t has a serum ECP of 80 ng/ml prior to the administration ofthe one or more doses ofone or more IL-SR binding molecules.

In one embodiment, a subject has a serum ECP of 100 ng/ml prior to the administration ofthe one or more doses of one or more IL-5R binding molecules. In one embodiment, a subject has a serum ECP of 150 ng/ml prior to the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one ment, a subject has a serum ECP of200 ng/ml prior to the administration of the one or more doses of one or more IL-SR binding molecules.

In one embodiment, a subject has no detectable serum ECP ing the administration ofthe one or more doses ofone or more IL-5R binding molecules. In one embodiment, a subject has a serum ECP of between I and 500 ng/ml following the administration ofthe one or more doses of one or more lL-5R binding molecules. In one embodiment, a subject has a serum ECP between I and 200 ng/ml, between I and 150 ng/ml, between I and 100 ng/ml, between I and 50 ng/ml, between I and 20 ng/ml, between IO and 200 ng/ml, n 10 and 100 ng/ml, between 10 and 50 ng/ml, between 20 and 100 ng/ml, IO between 20 and 50 ng/ml following the stration ofthe one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has a serum ECP ofat most I ng/ml, at most 5 ng/ml, at most IO ng/ml, at most 20 ng/ml, at most 30 ng/ml, at most 50 ng/ml following the administration ofthe one or more doses ofone or more IL-5R binding molecules. In one embodiment, a subject has a serum ECP of at most 1 ng/ml following the stration ofthe one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has a serum ECP of at most 5 ng/ml following the administration of the one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a serum ECP of at most IO ng/ml following the stration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a t has a serum ECP ofat most 15 ng/ml following the administration ofthe one or more doses ofone or more lL—SR binding molecules. In one embodiment, a subject has a serum ECP ofat most ng/ml ing the administration ofthe one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has a serum ECP of at most 25 ng/ml following the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a serum ECP of at most 30 ng/ml following the administration of the one or more doses ofone or more IL—SR binding molecules.

In one embodiment, the serum ECP ofa subject is lower following the administration of one or more doses of one or more IL-SR binding molecules than prior to the administration ofone or more doses ofone or more IL-SR binding les, wherein the serum ECP is never below 0 ng/ml. In a speciﬁc embodiment, the serum ECP is at least 50 ng/ml lower. In. a c embodiment, the serum ECP is at least 100 ng/ml lower. In a speciﬁc embodiment, the serum ECP is at least 150 ng/ml lower. In a speciﬁc embodiment, the serum ECP is at least 200 ng/ml lower. In a speciﬁc embodiment, the serum ECP is at least 250 ng/ml lower.

In a speciﬁc embodiment, the serum ECP is at least 300 ng/ml lower. In a speciﬁc embodiment, the serum ECP is at least 10% lower. In a speciﬁc embodiment, the serum ECP is at least 20% lower. In a speciﬁc embodiment, the serum ECP is at least 30% lower. In a speciﬁc embodiment, the serum ECP is at least 40% lower. In a speciﬁc ment, the serum ECP is at least 50% lower. In a speciﬁc embodiment, the serum ECP is at least 60% lower. In a speciﬁc embodiment, the serum ECP is at least 70% lower. In a speciﬁc embodiment, the serum ECP is at least 80% lower. In a speciﬁc embodiment, the serum ECP is at least 90% lower. In a speciﬁc embodiment, the serum ECP is at least 95% lower. In a speciﬁc embodiment, the serum ECP is l0% lower. In a speciﬁc embodiment, the serum ECP is 20% lower. In a speciﬁc embodiment, the serum ECP is 30% lower. In a speciﬁc IO embodiment, the serum ECP is 40% lower. In a speciﬁc embodiment, the serum ECP is 50% lower. In a speciﬁc embodiment, the serum ECP is 60% lower. In a speciﬁc embodiment, the serum ECP is 70% lower. In a speciﬁc embodiment, the serum ECP is 80% lower. In a speciﬁc embodiment, theserum ECP is 90% lower.. In a speciﬁc embodiment, the serum ECP is 95% lower. In a speciﬁc embodiment, the serum ECP is 99% lower.

In one ment, a t has no detectable serum ECP following the stration ofthe one or more doses ofone or more IL-SR binding molecules. In a speciﬁc embodiment, the serum ECP level remains undetectable for at least about I 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 IO 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.

In one embodiment, the disease indicator or symptom is Methacholine Challenge Test (MCT) MCT may be performed according to American Thoracic y (ATS) guidelines (Guidelines for Methacholine and Exercise Testing — 1999. (2000) Am J Respir Crit Care Med. 161:309-329) in the presence ofa ian who is experienced in the management of bronchospasm and with appropriate therapeutic agents immediately available. Brieﬂy, the spirometer used is calibrated according to the guidelines ofthe ATS. The nebulizer used must produce a particle size with mass median aerodynamic er (MMAD) of l-4 microns and ﬂow of 0.13il0% mL/min. Methacholine from an FDA ed source is used and diluted with sterile normal saline. Inhalation nge may be conducted using either 2 minutes l breathing or the ﬁve-breath dosimeter method as described in the referenced publication. Concentrations of methacholine are administered according to the established practice ofthe investigator, but within the range of0.06 mg/dL to 25.0 mg/dL.

FEV. is measured 30 and 90 seconds after tion of each dose and the higher ofthe two values recorded. sing concentrations is administered until the FEV. has been seen to fall at least 20% from the ne value. PC20 is the concentration of methacholine that leads to at least 20% fall in FEVI from the baseline value. After tion ofthe ﬁnal dose the subject may be given albuterol by metered-dose inhaler or nebulizer at the discretion of the Principal Investigator.

In one embodiment, a subject has a PC20 of between 0.06 and 25 mg/dL prior to the IO administration ofthe one or more doses ofone or more lL-5R binding molecules. ln one embodiment, a subject has a PC20 between 0.06 and 25 mg/dL, between 0.1 and 10 mg/dL, between 0.06 and 3 mg/dL, between 0.06 and 2 mg/dL, between 0.06 and 1 mg/dL, between” 0.1 and 3 mg/dL, between 0.1 and 2 mg/dL, between 0.1 and l mg/dL, between 0.2 and mg/dL, between 0.5 and IO mg/dL, between I and 10 mg/dL, between 0.1 and 5 mg/dL, between 0.2 and 5 mg/dL, n 0.5 and 5 mg/dL, between 0.1 and 2 mg/dL, n 0.2 and 2 mg/dL, between 0.5 and 2 mg/dL, between 0.06 and 0.1 mg/dL, between 0.1 and 0.2 mg/dL, between 0.2 and 0.5 mg/dL, between 0.5 and l mg/dL, between 1 and 2 mg/dL, between 2 and 5 mg/dL, between 5 and 10 mg/dL prior to the administration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a PC20 ofat 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 prior to the administration ofthe one or more doses ofone or more lL-SR binding molecules. ln one embodiment, a subject has a PC20 of IO mg/dL prior to the administration ofthe one or more doses ofone or more lL—5R binding molecules. In one ment, a subject has a PC20 of 5 mg/dL prior to the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a subject has a PC20 of2 mg/dL prior to the administration ofthe one or more doses of one or more lL-SR binding les. In one embodiment, a subject has a PC20 of l mg/dL prior to the administration ofthe one or more doses of one or more lL-SR g molecules. In one embodiment, a subject has a PC20 of 0.5 mg/dL prior to the administration ofthe one or more doses of one or more lL-5R binding les. In one embodiment, a subject has a PC20 of 0.2 mg/dL prior to the administration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a PC20 of 0.1 mg/dL prior to the administration ofthe one or more doses of one or more lL-SR binding molecules.

‘In one embodiment, a subject has a PC20 of between 0.5 and 25 mg/dL following the administration ofthe one or more doses ofone or more lL-SR g molecules. In one embodiment, a subject has a PC20 between 1 and 25 mg/dL, between 2 and 25 mg/dL, between 5 and 25 mg/dL, between 10 and 25 mg/dL, between I and 10 mg/dL, n 2 and mg/dL, between 2 and 10 mg/dL following the administration ofthe one or more doses of one or more lL-5R binding molecules. In one embodiment, a subject has a PC20 of at least I mg/dL, at least 2 mg/dL,-at least 5 mg/dL, at least 10 mg/dL, at least 20 mg/dL following the administration ofthe one or more doses of one or more IL-5R binding molecules. In one embodiment, a subject has a PC20 ofat least 0.2 mg/dL following the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a PC20 of at least 0.3 mg/dL ing the administration of the one or more doses of one or more lL-5R binding molecules. In one embodiment, a t has a PC20 ofat least 0.4 mg/dL following the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a PC20 of at least 0.5 mg/dL following the stration ofthe one or more doses ofone or more IL-5R binding molecules. In one embodiment, a subject has a PC20 of at least 0.7 mg/dL following the administration ofthe one or more doses ofone or more lL—5R binding molecules. In one embodiment, a subject has a PC20 of at least I mg/dL following the administration ofthe one or more doses of one or more lL-5R binding molecules. In one embodiment, a subject has a PC20 of at least 2 mg/dL ing the administration ofthe one or more doses of one or more IL-5R binding molecules. In one embodiment, a subject has a PC20 of at least 5 mg/dL following the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a PC20 of at least 10 mg/dL following the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one ment, a subject has a PC20 of at least 20 mg/dL following the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a PC20 of at least mg/dL following the administration ofthe one or more doses ofone or more lL-5R g molecules.

In one embodiment, the PC20 ofa subject is higher following the administration of one or more doses ofone or more lL-5R binding molecules than prior to the administration of one or more doses of one or more lL-5R binding molecules. In a ic ment, the PC20 is at least 0.3 mg/dL higher. In a c embodiment, the PC20 is at least 0.5 mg/dL higher. In a speciﬁc embodiment, the PC20 is at least 0.7 mg/dL higher. In a speciﬁc ment, the PC20 is at least 1 mg/dL higher. In a speciﬁc ment, the PC20 is at least 3 mg/dL higher. In a speciﬁc embodiment, the PC20 is at least 5 mg/dL higher. In a speciﬁc embodiment, the PC20 is at least 10 mg/dL higher. In a speciﬁc embodiment, the PC20 is at least 15 mg/dL higher. In a speciﬁc embodiment, the PC20 is at least 20 mg/dL higher. In a speciﬁc embodiment, the PC20 is at least 2 fold higher. In a speciﬁc embodiment, the PC20 is at least 4 fold higher. In a speciﬁc embodiment, the PC20 is at least 8 fold higher. In a speciﬁc embodiment, the PC20 is at least 10 higher. In a specific ment, the PC20 is at least 12 fold higher. In a speciﬁc embodiment, the PC20 is at least 15 fold higher. In a c embodiment, the PC20 is at least 20 fold higher. In a speciﬁc embodiment, the PC20 is 2 fold higher. In a speciﬁc embodiment, the PC20 is 4 fold higher. In a c embodiment, the PC20 is 8 fold higher. In a speciﬁc embodiment, the PC20 is IO fold higher. In a speciﬁc embodiment, the PC20 is 15 fold . In a speciﬁc embodiment, the PC20 is 60% higher. In a speciﬁc embodiment, the PC20 is 20 fold higher.

In one embodiment, the e indicator or symptom is circulating eosinophil count. ating eosinophil count may be assessed using any methods known to one of skill in the art, for e, but not limited to histology, ﬂow cytometry. Circulating eosinophil count may be ed by any one ofthe commercially available kits.

In one embodiment, a subject has a circulating eosinophil count of n 50 and 1000 cells/microL prior to the administration ofthe one or more doses ofone or more lL—5R binding molecules. In one embodiment, a subject has a circulating eosinophil count between 50and 1000 cells/microL, n 100 and 1000 cells/microL, between 150 and 1000 cells/microL, between 200 and 1000 cells/microL, between 250 and 1000 microL, between 300 and 1000 cells/microL, between 400 and 1000 cells/microL, between 500 and 1000 cells/microL, n 50 and 500 cells/microL, between 100 and 500 cells/microL, between 100 and 400 cells/microL, between 150 and 500 cells/microL, between 200 and 500 cells/microL prior to the administration ofthe one or more doses of one or more IL-SR binding molecules. In one embodiment, a t has a circulating eosinophil count ofat least 50 cells/microL, at least 100 cells/microL, at least 150 cells/microL, at least 200 cells/microL, at least 250 cells/microL, at least 300 cells/microL, at least 400 cells/microL, at least 500 cells/microL prior to the administration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a circulating eosinophil count of 50 cells/microL prior to the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a subject has a circulating eosinophil count of 100 cells/microL prior to the administration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a circulating eosinophil count of 150 cells/microL prior to the stration ofthe one or more doses ofone or more lL-SR binding les. In one embodiment, a subject has a circulating eosinophil count of200 cells/microL prior to the administration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a circulating eosinophil count of250 cells/microL prior to the administration ofthe one or more doses ofone or more lL—SR g molecules. In one embodiment, a subject has a circulating eosinophil count of300 cells/microL prior to the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a t has a circulating eosinophil count of350 cells/microL prior to the administration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a - circulating eosinophil count of400 cells/microL prior to the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a subject has a circulating eosinophil count of 500 cells/microL prior to the stration ofthe one or more doses ofone or more lL-SR g molecules. ln one ment, a subject has a circulating eosinophil count of n I and 400 microL following the administration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a circulating eosinophil count between 1 and 200 cells/microL, between I and IOO cells/microL, between I and 50 cells/microL, between 1 and 40 cells/microL, between 10 and 200 cells/microL, between l0 and 100 cells/microL, between 10 and 40 cells/microL, between 20 and 200 cells/microL, n 20 and 100 cells/microL, between 20 and 50 cells/microL following the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one ment, a subject has a ating eosinophil count ofat most I cells/microL, at most cells/microL, at most 10 cells/microL, at most 20 cells/microL, at most 30 cells/microL, at most 40 cells/microL, at most 50 cells/microL, at most 60 cells/microL, at most 80 cells/microL, at most 100 cells/microL ing the administration ofthe one or more doses ofone or more lL-SR binding molecules. ‘In one embodiment, a subject has a circulating eosinophil count of at most I cells/microL following the stration ofthe one , or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a circulating eosinophil count of at most 5 cells/microL following the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a subject has a circulating eosinophil count ofat most 10 cells/microL following the administration ofthe one or more doses ofone or more lL-5R g les. In one embodiment, a subject has a circulating eosinophil count of at most 20 cells/microL following the administration of the one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject has a circulating eosinophil count ofat most 30 cells/microL following the stration ofthe one or more doses of one or more IL—5R binding molecules. In one embodiment, a subject has a circulating eosinophil count ofat most 40 cells/microL following the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a circulating eosinophil count ofat most 50 cells/microL ing the stration of the one or more doses of one or more IL-5R IO binding molecules. In one embodiment, a subject has a circulating eosinophil count of at most 60 cells/microL following the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one embodiment, a subject'has a circulating eosinophil count of at most 80 cells/microL following the administration ofthe one or more doses ofone or more IL-5R binding molecules.

In one embodiment, the circulating eosinophil count ofa subject is lower following the administration of one or more doses of one or more lL-5R binding molecules than prior to the stration ofone or more doses ofone or more lL-5R binding molecules, wherein the circulating eosinophil count is never below 0 cells/microL. In a ic embodiment, the circulating eosinophil count is at least 50 cells/microL lower. In a speciﬁc embodiment, the circulating eosinophil count is at least 100 cells/microL lower. In a speciﬁc embodiment, the circulating eosinophil count is at least 150 cells/microL lower. In a speciﬁc embodiment, the circulating eosinophil count is at least 200 cells/microL lower. In a speciﬁc embodiment, the circulating eosinophil count is at least 250 cells/microL lower. In a speciﬁc ment, the circulating eosinophil count is at least 300 microL lower. In a speciﬁc embodiment, the circulating eosinophil count is at least 10% lower. In a specific embodiment, the circulating eosinophil count is at least 20% lower. In a speciﬁc embodiment, the circulating phil count is at least 30% lower. In a c embodiment, the circulating eosinophil count is at least 40% lower. In a speciﬁc embodiment, the circulating eosinophil count is at least 50% lower. In a speciﬁc embodiment, the circulating eosinophil count is at least 60% lower. In a speciﬁc embodiment, the circulating eosinophil count is at least 70% lower. In a speciﬁc embodiment, the circulating phil count is at least 80% lower. In a speciﬁc embodiment, the circulating eosinophil count is at least 90% lower. In a speciﬁc embodiment, the circulating eosinophil count is at least 95% lower. In a speciﬁc ment, the circulating eosinophil count is at least 99% lower. In a speciﬁc embodiment, the circulating eosinophil count is 10% lower. In a speciﬁc embodiment, the circulating eosinophil count is 20% lower. In a speciﬁc embodiment, the ating eosinophil count is 30% lower. 1n 3 speciﬁc embodiment, the circulating eosinophil count is 40% lower. In a speciﬁc ment, the circulating eosinophil count is 50% lower. In a speciﬁc embodiment, the circulating eosinophil count is 60% lower. In a speciﬁc ment, the circulating phil count is 70% lower. In a speciﬁc embodiment, the circulating eosinophil count is 80% lower. In a speciﬁc embodiment, the circulating eosinophil count is 90% lower. In a speciﬁc embodiment, the circulating eosinophil count is 95% lower. In a speciﬁc embodiment, the circulating eosinophil count is 99% lower.

In one embodiment, a subject has no detectable ating eosinophil count following the administration ofthe one or more doses ofone or more lL-SR 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.

In one embodiment, the disease indicator or symptom is % eosinophil in induced sputum. % phil in induced sputum may be assessed using any methods known to one of skill in the art, for example, but not limited to the methods described in Belda et al. (2000) Am JRespir Crit Care Med 161 :475-478. % eosinophil in induced sputum may be determined by any one ofthe commercially ble kits.

In one ment, a subject has a % eosinophil in d sputum of between 0.1% and 10% prior to the administration ofthe one or more doses ofone or more 1L-5R binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum between 0.1% and 2%, between 0.1% and 5%, between 0.5% and 2%, between 0.5% and 5%, between 0.5% and 10%, between 1% and 2%, between 1% and 5%, between 1% and 10%, between 2% and 5%, n 2% and 10%, between 3% and 5%, between 3% and 10%, between 1.5% and 5%, between 2.5% and 5%, prior to the administration ofthe one or more doses of one or more 1L-5R binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum 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% prior to the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a t has a % eosinophil in induced sputum of 0.5% prior to the administration of the one or more doses of one or more IL-5R binding molecules.

In one embodiment, a subject has a % eosinophil in induced sputum of 1% prior to the administration ofthe one or more doses of one or more IL-5R binding molecules. In one ment, a subject has a % eosinophil in induced sputum of 1.5% prior to the administration ofthe one or more doses of one or more IL-5R binding molecules. In one embodiment, a t has a % eosinophil in induced sputum of 2% prior to the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum of2.5% prior to the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum of 3% prior to the administration ofthe one or more doses ofone or more IL-5R g molecules. In one embodiment, a subject has a % phil in induced sputum of 4% prior to the administration ofthe one or more doses of one or more lL-5R binding molecules. In one ment, a subject has a % eosinophil in induced sputum of 5% prior to the administration ofthe one or more doses of one or more IL-5R binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum of 6% prior to the administration ofthe one or more doses ofone or more lL—5R binding molecules. In one embodiment, a t has a % phil in induced sputum of 7% prior to the administration ofthe one or more doses ofone or more IL-5R binding les. In one embodiment, a subject has a % eosinophil in induced sputum of 8% prior to the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum of 9% prior to the stration ofthe one or more doses ofone or more lL—5R binding molecules. In one embodiment, a subject has a % eosinophil in d sputum of 10% prior to the administration ofthe one or more doses of one or more IL-5R binding molecules.

In one ment, a subject has a % eosinophil in induced sputum of between 0.1% and 5% following the administration ofthe one or more doses of one or more IL-5R binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum between 0.1% and 3%, between 0.1% and 2%, between 0.1% and 1.5%, between 0.5% and 5%, between 0.5% and 3%, between 0.5% and 1%, between 1% and 5%, between 1% and 3%, between 2% and 5%, between 3% and 5%, between 2.5% and 5% following the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum of 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 |0% following the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a % phil in induced sputum of at most 1% following the stration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum of at most 2% following the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one ment, a subject has a % eosinophil in induced sputum of at most 3% following the administration ofthe one or more doses of one or more. lL-SR binding . molecules. In one embodiment, a subject has a % eosinophil in induced sputum of at most 4% following the administration ofthe one or more doses ofone or more IL-SR binding molecules. In one ment, a t has a % eosinophil in induced sputum of at most % following the administration ofthe one or more doses of one or more lL—SR binding les. In one embodiment, a subject has a % eosinophil in induced sputum of at most 6% following the administration ofthe one or more doses of one or more lL-SR g molecules. In one embodiment, a subject has a % eosinophil in induced sputum ofat most 7% following the administration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum of at most 8% following the administration ofthe one or more doses of one or more lL-SR binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum ofat most 9% following the administration ofthe one or more doses of one or more IL-SR binding molecules. In one embodiment, a subject has a % eosinophil in induced sputum ofat most % following the administration ofthe one or more doses of one or more lL-5R binding molecules.

In one embodiment, the % eosinophil in d sputum ofa subject is lower ing the administration ofone or more doses of one or more lL—SR binding les than prior to the administration ofone or more doses of one or more lL-SR binding molecules, wherein the % eosinophil in induced sputum is never below 0%. In a speciﬁc embodiment, the % eosinophil in induced sputum is by at least 10% lower. In a speciﬁc embodiment, the % eosinophil in induced sputum is by at least 9% lower. In a speciﬁc embodiment, the % eosinophil in induced sputum is by at least 8% lower. In a speciﬁc embodiment, the % eosinophil in induced sputum is by at least 6% lower. ln a speciﬁc embodiment, the % eosinophil in induced sputum is by at least 5% lower. In a speciﬁc embodiment, the % eosinophil in induced sputum is by at least 4% lower. In a c embodiment, the % eosinophil in induced sputum is by at least 3% lower. In a speciﬁc embodiment, the % eosinophil in induced sputum is by at least 2% lower. In a speciﬁc embodiment, the % eosinophil in induced sputum is by at least 1% lower.

In one embodiment, a subject has no detectable eosinophil in induced sputum following the administration ofthe one or more doses ofone or more lL-5R binding molecules. In a speciﬁc embodiment, the eosinophils in 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 leastabout 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.

In one embodiment, the disease tor or symptom is circulating il count.

Circulating basophil count may be assessed using any methods known to one of skill in the art, for example, but not limited to histology, ﬂow cytometry. Circulating basophil count may be measured by any one ofthe commercially available kits. in one embodiment, a subject has a circulating basophil count of between 5 and 500 cells/microL prior to the administration ofthe one or more doses of one or more lL-SR binding molecules: In one embodiment, a subject has a circulating basophil count between 50 and 500 cells/microL, between 10 and 500 cells/microL, between 20 and 500 cells/microL, n 30 and 500 cells/microL, n 40 and 500 cells/microL, n 50 and 500 cells/microL, between 10 and 400 microL, between 10 and 300 cells/microL, between IO and 200 cells/microL, between 10 and 100 cells/microL, between 20 and 100 cells/microL, n 30 and 100 cells/microL, between 10 and 75 cells/microL prior to the administration ofthe one or more doses of one or more lL-5R binding molecules. In one embodiment, a subject has a circulating basophil count of at least cells/microL, at least 10 cells/microL, at least 15 cells/microL, at least 20 cells/microL, at least 30 cells/microL, at least 50 cells/microL, at least 60 cells/microL, at least 100 cells/microL prior to the administration ofthe one or more doses ofone or more lL-5R binding les. In one embodiment, a subject has a circulating basophil count of 5 cells/microL prior to the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a t has a ating basophil count of cells/microL prior to the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a ating basophil count of cells/microL prior to the administration ofthe one or more doses of one or more 1L-5R binding molecules. In one embodiment, a subject has a circulating il count of cells/microL prior to the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a subject has a circulating basophil count of cells/microL prior to the administration ofthe one or more doses of one or more lL—5R binding les. In one embodiment, a subject has a circulating basophil count of 50 cells/microL prior to the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a t has a circulating basophil count of 60 cells/microL prior to the administration ofthe one or more doses of one or more lL-5R binding molecules. In one ment, a subject has a circulating basophil count of 100 cells/microL prior to the administration ofthe one or more doses of one or more IL-SR binding molecules.

In one embodiment, a subject has a circulating basophil count of between 1 and 100 cells/microL following the administration ofthe one or more doses of one or more lL-SR binding les. In one embodiment, a t has a circulating basophil count between 1 and 100 cells/microL, between I and 50 microL, between I and 30 cells/microL, between I and 20 cells/microL, between 1 and 10 cells/microL, n 5 and . 100 cells/microL, between 5 and 50 cells/microL, between 5 and 20 cells/microL, between 5 and IQ cells/microL, between 10 and 30 cells/microL following the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a circulating basophil count of at most 1 cells/microL, at most 5 cells/microL, at most microL, at most 20 cells/microL, at most 30 microL, at most 50 cells/microL, at most 100 cells/microL following the administration ofthe one or more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a circulating basophil count ofat most l cells/microL following the administration ofthe one or more doses ofone or more lL- 5R binding molecules. In one embodiment, a subject has a circulating basophil count ofat most 5 cells/microL ing the administration ofthe one or more doses of one or more IL- 5R binding molecules. In one embodiment, a subject has a circulating basophil count of at most 10 cells/microL following the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a subject has a circulating basophil count ofat most 20 cells/microL following the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a subject has a circulating il count ofat most 30 microL following the stration ofthe oneor more doses ofone or more lL-5R binding molecules. In one embodiment, a subject has a circulating basophil count ofat most 40 cells/microL following the administration ofthe one or more doses ofone or more lL-SR binding molecules. In one embodiment, a subject has a circulating basophil count ofat most 50 cells/microL following the stration ofthe one or more doses ofone or more, lL-5R g molecules.

In one embodiment, the circulating basophil count ofa subject is lower following the administration ofone or more doses ofone or more lL-5R binding les than prior to the administration ofone or more doses ofone or more lL-5R binding molecules, wherein the circulating basophil count is never below 0 cells/microL. In a speciﬁc embodiment, the circulating basophil count is at least 10 cells/microL lower. In a speciﬁc embodiment, the ating basophil count is at least 20 cells/microL lower. In a speciﬁc embodiment, the circulating basophil count is at least 30 cells/microL lower. In a speciﬁc embodiment, the circulating basophil count is at least 50 cells/microL lower. In a speciﬁc embodiment, the circulating basophil count is at least 75 cells/microL lower. In a speciﬁc embodiment, the circulating basophil count is at least 100 cells/microL lower. In a speciﬁc embodiment, the circulating basophil count is at least 10% lower. In a speciﬁc ment, the circulating basophil count is at least 20% lower. In a speciﬁc embodiment, the circulating basophil count is at least 30% lower. In a speciﬁc embodiment, the circulating basophil count is at least 40% lower. In a speciﬁc embodiment, the circulating basophil count is at least 50% lower. In a speciﬁc embodiment, the circulating basophil count is at least 60% lower. In a c embodiment, the circulating basophil count is at least 70% lower. In a speciﬁc embodiment, the circulating basophil count is at least 80% lower. In a speciﬁc embodiment, the circulating basophil count is at least 90% lower. In a speciﬁc ment, the circulating basophil count is at least 95% lower. In a speciﬁc embodiment, the circulating basophil count is at least 99% lower. In a c embodiment, the circulating basophil count is 10% lower. In a speciﬁc embodiment, the circulating basophil count is 20% lower. In a speciﬁc embodiment, thecirculating basophil count is 30% lower. In a c embodiment, the circulating basophil count is 40% lower. In a speciﬁc embodiment, the circulating basophil count is 50% lower. In a speciﬁc embodiment, the circulating basophil count is 60% lower.

In a speciﬁc embodiment, the ating basophil count is 70% lower. In a speciﬁc embodiment, the circulating il count is 80% lower. In a c embodiment, the circulating basophil count is 90% lower. In a speciﬁc embodiment, the circulating basophil count is 95% lower. In a speciﬁc embodiment, the circulating basophil count is 99% lower.

In one embodiment, a subject has no detectable ating basophil count following the administration ofthe one or more doses ofone or more lL-5R g molecules. In a c embodiment, the circulating basophil 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 l0 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.

SPECIFIC EMBODIMENTS l. A method of reducing the s of eosinophils in a human t comprising administration to said subject an lL-SR binding molecule that comprises (a) a region that cally binds to the IL-SR and (b) an immunoglobulin Fc region. 2. The method ofembodiment 1, wherein said IL-SR binding molecule is an anﬁbody. 3. The method of embodiment 2, wherein said antibody is a onal antibody. 4. The method of embodiment 3, wherein said antibody is a chimeric antibody.

. The method ofembodiment 3, n said antibody is a humanized antibody. 6. The method ofembodiment 3, wherein said antibody is a human antibody. 7. The method of embodiment 1, wherein said region that speciﬁcally binds to the lL-SR comprises the amino acid sequence of lL-5, or fragments, substitutions, or derivatives thereof. 8. The method ofembodiment 7, wherein said region that specifically binds to the lL-5R comprises a nonfunctional variant of lL-5. 9. The method of any of embodiments 1-8, wherein said lL—SR binding molecule specifically binds to the lL-SR ? chain.

. The method of embodiment I, wherein said immunoglobulin Fc region is altered in a manner that increases effector function.

, II. The method ofembodiment I, wherein said globulin Fc region comprises reduced levels of fucose. 12. The method of embodiment l I, wherein said immunoglobulin Fc region ses no fucose. 13. The method of embodiment 1, wherein said immunoglobulin Fc region comprises amino acid substitutions that yield increased effector function.

I4. The method ofembodiment I, wherein said amino acid substitutions comprise the inclusion ofthe following amino acid sequences in the Fc region: 332E, 239D and 330L, as numbered by the EU index as set forth in Kabat.

. The method ofembodiment I, wherein said reduction in eosinophils occurs in the peripheral blood circulation. 16. The method of embodiment I, wherein the numbers of eosinophils are reduced to a level that is less than 50 eosinophils/mm3. l7. The method of embodiment I, wherein the reduction of eosinophils takes place within the ﬁrst 48 hours after stration. 18. The method of embodiment I, wherein the ion of phils takes place within the ﬁrst 24 hours after administration. 19. The method of embodiment I, n the reduction ofeosinophils is reversible.

.. The method of embodiment I, wherein there is a post-administration reduction . in absolute eosinophil count of at least about 25 eosinophils/mm3.

IS 2 l. The method of embodiment I, n there is a post-administration reduction in absolute eosinophil count ofat least about 50 eosinophils/mm3. 22. The method ofembodiment I, wherein there is a post-administration reduction in absolute eosinophil count ofat least about 75 eosinophils/mm3. 23. The method ofembodiment 1, n there is a post-administration reduction in absolute eosinophil count ofat least about 100 eosinophils/mm3. 24. The method ofembodiment I, wherein there is a post-administration reduction in absolute eosinophil count ofat least about 125 eosinophils/mm3.

. The method of embodiment I, n there is a dministration ion in absolute eosinophil count ofat least about 150 eosinophils/mm3. 26. The method ofembodiment 1, wherein there is a post-administration reduction in absolute eosinophil count ofat least about 175 eosinophils/1111113. 27. The method ofembodiment 1, n there is a post-administration reduction in absolute eosinophil count of at least about 200 eosinophils/mm3. 28. The method of embodiment I, wherein there is a post-administration reduction in absolute eosinophil count of at least about 225 phils/mm3. 29. The method ofembodiment I, wherein there is a post-administration reduction in absolute eosinophil count of at least about 250 eosinophils/mm3.

. The method of embodiment 1, wherein there is a post-administration reduction in absolute eosinophil count ofat least about 275 eosinophils/mm3. 3 l. The method ofembodiment I, wherein there is a dministration reduction in absolute eosinophil count of at least about 300 eosinophils/mm3. 32. The method of embodiment 1, wherein there is a post-administration reduction in absolute eosinophil count ofat least about 325 eosinophils/mm3. . 33. The method of embodiment I, wherein there is a post-administration reduction in absolute eosinophil count of atleast about 350 eosinophils/mm3. 34. The method of embodiment 1, wherein there is a post-administration reduction in absolute eosinophil count of at least about 375 eosinophils/mm3.

. The method of embodiment 1, wherein there is a post—administration ion in te eosinophil count of at least about 400 eosinophils/mm3. 36. The method of embodiment 1, wherein there is a dministration reduction in te eosinophil count of at least about 425 eosinophils/mm3. 37. The method of embodiment I, wherein there is a post-administration. reduction in absolute eosinophil count of at least about 450 eosinophils/mm3. 38. The method of embodiment 1, wherein there is a post-administration reduction in absolute eosinophil count of at least about 475 eosinophils/mm3. 39. The method ofembodiment 1, wherein there is a post-administration reduction in absolute eosinophil count of at least about 500 eosinophils/mm3. 40.‘ Themethod of ment 1, wherein there is a post-administration reduction in absolute eosinophil count of between and including about 50 to about 500 eosinophils/mm3. 4 l. The method of embodiment 1, wherein there is a post-administration reduction in te eosinophil count of between and ing about 75 to about 250 eosinophils/mm3. 42. The method ofembodiment I, wherein there is a dministration reduction in absolute eosinophil count of between and including about 100 to about 200 eosinophils/mm3. 43. The method diment I, wherein there is a post-administration reduction in absolute eosinophil count of between and including about 50 to about 250 eosinophils/mm3. 44. The method of embodiment I, wherein there is a post-administration reduction in absolute eosinophil count of between and including about 50 to about 200 eosinophils/mm3. 45. The method of embodiment I, wherein there is a post-administration reduction in absolute eosinophil count of n and including about 50 to about 150 eosinophils/mm3. 46. The method of embodiment I, wherein the absolute eosinophil count post- administration is less than about 100 eosinophils/mm3. 47. The method of embodiment 1, wherein the te eosinophil count post- administration is less than about 75 eosinophiIs/mm3. 48. The method of ment 1, n the absolute eosinophil count post-A administration is less than about 50 eosinophils/mm3. 49. The method ofembodiment I, wherein the absolute eosinophil count post- administration is less than about 25 eosinophils/mm3. 50. The method of embodiment 1, wherein said t's pre-administration absolute eosinophil count is n about 50 and about 500 eosinophils/mm3. 51. The method of embodiment ], wherein said t's pre-administration absolute eosinophil count is between about 75 and about 475 eosinophiIs/mm3. 52. The method of embodiment 1, wherein said subject's pre-administration absolute . eosinophil count is between about 75 and about 200 eosinophils/mm3. 53. The method of embodiment I, wherein said subject's pre-administration absolute eosinophil count is between about 100 and about 200 eosinophiIs/mm3. 54. The method of embodiment I, wherein said subject's ministration absolute eosinophil count is about 25 eosinophils/mm3. 55. The method ofembodiment I, wherein said subject's pre-administration absolute eosinophil count is about 50 eosinophils/mm3. 56. The method of embodiment 1, n said subject's pre-administration te eosinophil count is about 75 eosinophils/mm3. 57. The method ofembodiment I, wherein said subject's pre-administration te eosinophil count is about 100 eosinophils/mm3. 58. The method of embodiment 1, wherein said subject's pre-administration absolute eosinophil count is about l25 eosinophils/mm3. 59. The method of embodiment I, wherein said subject's pre-administration absolute eosinophil count is about 150 eosinophils/mmS. 60. The method of embodiment I, wherein said subject's pre-administration absolute eosinophil count is about I75 eosinophils/mm3. ‘61. The method of embodiment I, wherein said subject's pre-administration te phil count is about 200 eosinophils/mm3. 62. The method of embodiment I, wherein said subject's pre-administration absolute eosinophil count is about 225 eosinophils/mm3.

VI 63. The method of embodiment 1, wherein said t's pre-administration absolute eosinophil count is about 250 eosinophils/mm3. 64. The method of embodiment I, wherein said subject's pre-administration absolute phil count is about 275 eosinophils/mm3. 65. The method of embodiment 1, wherein said subject's pre-administration absolute eosinophil count is about 300 phils/mm3. 66. The method of embodiment 1, wherein said subject's pre-administration absolute eosinophil count is about 325 eosinophils/mm3. 67. The method of embodiment 1, wherein said subject's pre-administration absolute eosinophil count is about 350 eosinophils/mm3. 68. The method of ment l, wherein said subject's pre-administration absolute eosinophil count is about 375 eosinophils/mm3. 69. The method of ment I, wherein said subject's pre—administration absolute eosinophil count is about 400 eosinophils/mm3. 70. The method of embodiment I, wherein said subject's pre-administration absolute eosinophil count is about 425 eosinophils/mm3. 71. The method of embodiment 1, wherein said subject's pre—administration absolute eosinophil count is about 450 eosinophils/mm3. 72. The method of embodiment 1, wherein said subject's pre-administration absolute eosinophil count is about 475 eosinophils/mm3. 73. The method ofembodiment 1, wherein said subject's ministration absolute eosinophil count is about-500 eosinophils/mm3. 74. The method of any of embodiments l-73, wherein said t's post- administration absolute basophil count is reduced by at least about 5 basophils/mm3. 75. The method of any of embodiments .1-73, wherein said t's post- administration absolute basophil count is reduced by at least about 10 basophils/mm3. 76. The method of any of embodiments 1-73, wherein said subject's post- administration absolute basophil count is reduced by at least about 15 basophils/mm3. 77. The method of any of embodiments [-73, wherein said subject's post- administration absolute basophil count is reduced by at least about 20 basophils/mm3. 78. The method ofany of ments l-73, wherein said subject's post- administration absolute basophil count is reduced by at least about 25 basophils/mm3. 79. The method of any of embodiments 1-73, wherein said subject's post- administration absolute basophil count is d by at least about 30 basophils/mm3. 80. The method of any of embodiments l—73, wherein said t's post- administration absolute basophil count is reduced by at least about 35 basophils/mm3. 81. The method ofany of embodiments 1-73, wherein said subject's post- administration absolute basophil count is reduced by at least about 40 basophils/mm3. 82. The method ofany ofembodiments 1-73, wherein said subject's post- administration absolute il count is d by at least about 45 basophils/mm3. 83. The method of any of embodiments 1-73, wherein said subject's post- administration absolute basophil count is reduced by at least about 50 ils/mm3. 84. The method of any of embodiments 1-73, wherein said subject's post- administration absolute basophil count is reduced by at least about 55 basophils/mm3. 85. The method of any of embodiments 1-73, wherein saidsubject's post- administration absolute basophil count is reduced by at least about 60 basophils/mm3. 86. The method of any ofembodiments 1-73, wherein said subject's post- administration absolute basophil count is reduced by at least about 65 basophils/mm3. 87. The method ofany of embodiments 1-73, wherein said subject's post- stration absolute basophil count is reduced by at least about 70 basophils/mm3. 88. The method of any of embodiments 1-73, wherein said subject's post- administration absolute basophil count is between 0 and about I0 basophils/mm3. 89. The method ofany diments 1-73, n said subject's post- administration absolute basophil count is about 2 basophils/mm3. 90. The method ofany of embodiments l-73, n said subject's post- administration absolute basophil count is about 5 basophils/mm3. 91. The method ofany of embodiments 1-73, wherein said subject's post- administration absolute basophil count is aboUt 7 basophils/mm3. 92. The method ofany of embodiments l-73, wherein said subject's post- administration te basophil count is about 9 basophils/mm3. 93. The method of any of embodiments l-73, wherein the basophil reduction occurs within 48 hours post-administration. 94. The method of any of embodiments l-73, wherein the basophil reduction occurs within 24 hours post-administration. 95. The method of any diments [-94, wherein said lL-SR binding molecule is administered to said subject at a dose ranging from between about 0.00] to about 100 mg/kg. 96. The method ofembodiment 95= wherein said dose is about 0.03 mg/kg. 97. The method ofembodiment 95, wherein said dose is 0.03 mg/kg. 98. The method of any ofembodiments 1-97, wherein said lL-SR binding le is administered erally. 99. The method ofembodiment 98, wherein said lL-SR binding molecule is stered intravenously. 100. The method of any of ments l-99, with the proviso that the-lL-5R binding molecule is not MEDl-563. 101. The method of any of embodiments 1-100, wherein said reduction in eosinophils leads to a reduction in asthma symptoms. 102. The method of any of embodiments 1-100, wherein said reduction in phils leads to a reduction in COPD symptoms.

EXAMPLES The invention is now described with reference to the following examples. These examples are provided for the purpose ofillustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all ions which become evident as a result ofthe teachings ed herein.

EXAMPLE 1 MEDI-563, AN ANTI-INTERLEUKIN—S-RECEPTOR ANTIBODY, IS WELL TOLERATED AND INDUCES REVERSIBLE BLOOD EOSINOPENIA IN MILD ASTHMATICS IN A PHASE I TRIAL Background: Eosinophils are believed to play a key role in the pathogenesis ofasthma.

Interleukin-5 (IL-5) is a major cytokine in eosinophil biology, and expression ofits receptor (IL-5R) is largely restricted to eosinophils, basophils, and mast cells. The suboptimal efficacy of lLtargeted therapies in asthma has been attributed to an incomplete depletion ofeosinophils in lung tissue. Complete lung phil depletion should provide onal insight into the role ofthese cells in asthma and could represent a novel therapeutic strategy.

Objectives: To assess the safety and biological activity ofMEDl-563 (previously known as BlW'—8405), a humanized afucosylated lgGl anti-lL-SR alpha chain monoclonal antibody.

MEDl-563 was developed by BioWa, lnc. through proprietary igent® technology that significantly enhances antibody-dependent cellular cytotoxicity. MEDl-563 neutralizes lL-S activity and depletes tissue eosinophils in pre-clinical models with an acceptable toxicology Methods: Six subjects with mild asthma and absence of corticosteroid therapy were enrolled in the first cohort of study BlW001, an open—label ﬁrst-in-human study with MEDl- 563. The patients received a single intravenous dose of 0.03 mg/kg MEDl-563and were followed for 84 days.

Results: 63 was well tolerated, and no serious adverse events were reported. All adverse events (AE5) were mild, and the most frequently reported AE was fatigue on the dosing day post administration (3/6 subjects). Circulating eosinophils sed below detection limits within 24-48 hours of dosing in all 6 subjects (will include mean value prior to ). This effect lasted for 8-12 weeks, and phils became detectable in some subjects at Day 58 post dosing and reached 270% of baseline levels by Day 84 post dosing in all subjects analyzed. Circulating basophils followed a similar trend. Possibly linked to the ed mechanism of action ofMEDl—563, neutrophil levels experienced a slight and transient decrease within 72 hours post dosing, reaching mild neutropenia levels in 2/6 subjects that resolved within 3 days. MEDl-563 administration was associated with immediate (within 6 hrs), modest (<l Ox baseline) and transient (<l week duration) increases in serum C-reactive protein (2/6 ts) and lL-6 (2/3). sions: A single 0.03 mg/kg lV dose of MEDl-563 induces a robust blood eosinopenia, with anacceptable safety proﬁle to date.

EXAMPLE 2 Antibody Dependent Cell-Mediated Cytotoxicity KCI333 effector cells (human NK cell overexpressing human chRllla and Fceng) were co— incubated for 4 hours with target CTLL-2 cell line (mouse lymphoma genetically modiﬁed to overexpress human lL-5 Ra) at a ratio of5 effectorszl target, in the presence of MEDI-563 or control antibody. dy mediated xicity was assessed using a Calcein AM cell viability assay. Results are summarized in Figure 9A. Using a similar methodology, a further control (fucosylated MEDl-563) was analyzed. Results are summarized in Figure 9B. l0 EXAMPLE 3 Surface Plasmon Resonance Evaluation of Equilibrium Binding of MEDI-563 t0 IL—5R Carrier-free, soluble human lL-5 Ra extracellular domain was obtained from a commercial source (R+D Systems). The recombinant hulL-SRa was directly immobilized to a sensor chip through amine es using a standard protocol. The interaction of MEDI-563 with immobilized hulL-5Ra over time was evaluated by the change in refractive index, from which k0", km, and KD values were calculated using rd techniques. Results are summarized in Figure 10.

EXAMPLE 4 Surface n Resonance Evaluation of Equilibrium Binding of MEDI-563 t0 FcyRs MEDl-563 was directly immobilized to a sensor chip through amine linkages using a rd protocol. The interactions of soluble human FcyRs (Medlmmune) with immobilized 63 over time were ted by change in refractive index, from which k0", kom and KD values were calculated using standard techniques. Results are summarized in Figure l l. ' 3O EXAMPLE 5 IL-SRa Immunohistochemistry Resected nasal polyp tissue was ﬁxed in formaldehyde for 24 hours and embedded in parafﬁn. Consecutive sections were stained for human lL-SRa, lL-9R, CCR3, and c-kit using commercially available lL-SR directed polyclonal antibodies (R+D Systems, Santa Cruz Biotechnology) using standard techniques. Lung tissue from lL-9 transgenic mice or wild type strain matched FVB control mice were ﬁxed in formaldehyde for 24 hours and embedded in parafﬁn. Sections were analyzed for lL-9R (pAb, Santa Cruz Biotechnology) and lL-SR (pAb, R+D Systems) expression using standard immunohistochemistry techniques. Results are summarized in Figures l2 and I3.

EXAMPLE 6 Medi-563 Binds to Eosinophils in Whole Blood of Healthy Donors ocytes were isolated from human whole blood of normal donors by density gradient IS centrifugation. Directly labeled primary antibody reagents were used for the analysis of CD16 (FlTC ﬂuorochrome) and MEDl-563 F(Ab)'2 -647 fluorochrome) sion.

A cocktail ofCDl6-FlTC plus MEDlAlexa647, or CDl6-FlTC plus an Alexa647- labeled isotype control dy, were added to the granulocyte preparation at l ram per l0"6 cells. After incubation for 45 minutes on ice, cells were washed three times in cold saline, and cell surface antibody g was assessed by flow cytometry. Eosinophils, - which are negative for CD16, were analyzed. The g ofMEDl-563 versus the isotype control antibody in egative granulocyte population is expressed. Results are summarized in Figure l5.

EXAMPLE 7 Mouse yte IL—5Ra Staining by Flow Cytometry Leukocytes were isolated from blood, bone marrow, lungs and spleen oflL-S transgenic mice. Cell suspensions were stained in PBS containing 1% FCS. To reduce nonspeciﬁc binding, cells were incubated with Fc Block (BD Biosciences) for IS min before ng.

The antibodies used were anti—mouse CCR3 (R&D systems), anti—mouse Siglec F (BD Biosciences) and anti-mouse lL-SR (H7). Cells were stained for 30 min on ice, washed twice, and ﬁxed in cytoﬁx buffer (BD ences). Flow cytometric is was performed using a LSRll (Becton Dickinson) with FACS Diva software (Becton Dickinson). Results were analyzed using FlowJo Software (TreeStar lnc.). Results are summarized in Figures 16A and 168.

EXAMPLE 8 Medi-563 Depletes IL-5Ra Positive Mononuclear Cells From Bone Marrow Frozen bone marrow mononuclear cells (BM MNC; Lonza) were thawed, washed, plated, and incubated for 2 hrs at 37°C. Non-adherent bone marrow mononuclear cells (NA BM MNC) were collected from the plates following incubation. ADCC assay was performed by coincubating for 18 hrs 100,000 NA BM MNC and 50,000 KCI333 effector cells per well in 200 ul l0% FBS/RPMl 1640 in 96 well TC plate in the presenceof 10 ug/ml Medi-563 antibody. Negative control ons were performed using the R347 aFuc isotype control IS antibody of irrelevant specificity. The KC 1 333 effector cells used in the ADCC assay were d with CFDA SE. Following the 18 hr incubation, cells from each reaction were washed three times with warm medium and immunostained for flow cytometry. lL-5 Ra ve cells were detected by KM1257 primary antibody/ PE conjugated goat anti-Mu lgG ch specific secondary antibody ng. Control staining of samples was done with the 1A7 isotype matched control primary dy in combination with the PE conjugated goat anti- Mu lgG ch specific secondary antibody. lmmunostaining and flow cytometry was med using rd protocols. The number oflL—SRa positive cells remaining in a sample following ADCC was ascertained by counting the number of KM1257 ve cells in a lymphocyte gate. The immunostaining and ﬂow try procedures were calibrated using a CTLL-2 cell line expressing a human lL-5Ra transgene. MEDl-563 mediated ADCC depleted substantially all lL—SRa ve cells from the NA BM MNC samples. Results are summarized in Figures 17A and 178.

EXAMPLE 9 MEDI-563 Mediated Depletion of Peripheral Blood Eosinophils Two cohorts of six subjects with mild asthma were enrolled in an open-label study of MEDI- 563. The subjects ofcohort l and 2 received a single intravenous dose of0.03 mg/kg and 0.] mg/kg MEDl-563, respectively, and their peripheral blood eosinophil levels were enumerated at screening, on day 0 prior to dosing, and at regular intervals up to day 84 and at follow-up.

Circulating eosinophils were detected by ﬂow cytometry. Circulating eosinophils decreased below the limit ofdetection within 24 hours ofdosing in all 6 subjects of both cohorts. The MEDl-563 induced eosinopenia lasted for 8-12 weeks. In cohort 1, following the administration ofa single dose of 0.03 mg/kg MEDI-563, ofthe ﬁve subjects that completed the 84 day study, eosinophils became detectable in 1 t at day 58, in 3 ts at day 84; the fifth subject had no detectable circulating eosinophils at day 84. In cohort 2, following the administration ofa single dose of 0.l mg/kg MEDl-563, none ofthe subjects had detectable circulating eosinophils at day 84. Peripheral blood eosinophils were detectable, however, in all six subjects of cohort 2 at a subsequent follow-up examination.

Peripheral blood eosinophils levels detected in cohorts l and 2 at various time intervals following the administration ofa single dose of MEDl-563 is presented in Figures 18A and 18B.

EXAMPLE 10 IL-5R0t Immunohistochemistry Lung sections from a healthy human subject were d with MEDl-563 using standard histochemical techniques. s are ized in Figure 19. lL-5 Ralpha expressing cells appear black in the image.

Lung tissue samples obtained from bronchial or transbronchial biopsy of asthmatic patients were stained with MEDl-563 using standard histochemical techniques. Results are summarized in Figure 20. lpha expressing cells appear dark grey/black in the image. e 11 MEDI-563 efﬁciently targets ed basophils and eosinophils in an in vitro ADCC assay.

Basophils and phils were isolated from healthy donors with a commercially available kit (RoboSep NK/EosinophiI/Basophil Negative Selection Kit, Stem Cell Technologies, Vancouver, Canada). lL-SRalpha expression ofthe isolated cells were ascertained by ﬂow cytometry. Cells were stained by MEDl-563 antibody or an isotype control antibody of vant specificity following standard protocols. lmmunostained cells were analyzed by ﬂow try. Staining proﬁles are shown in Figure 2 1. Both the isolated basophils and eosinophils displayed a MEDl-563 staining level above that of observed with the isotype l antibody. Staining pattern ofa cell line expressing a human lL-5Ralpha/beta transgene is shown as a positive control.

The activity of fucosylated and afucosylated MEDl-563 was ascertained in an in vitro ADCC assay using isolated eosinophils and autologous NK cells. phils and NK cells were isolated from healthy donors using cially available kits (RoboSep NK/Eosinophil/Basophil Negative Selection Kit, Stem Cell Technologies, Vancouver, Canada). The ADCC assay was performed with isolated NK cells and eosinophils as effectors and target cells at a 5:1 ratio. dy concentrations assayed range from 10‘'5 to '7 M. Cytotoxicity was measured after 24 hrs ofincubation using a flow cytometry based Annexin V assay. ‘The ADCC activity of afucosylated MEDI-563 was several orders of magnitude higher than that ofthe fucosylated MEDl-563 antibody. The ECSO value of afucosylated MEDl-563 was 0.965pM in this assay. The results ofa entative ment are shown in Figure 22.

The ty of afucosylated MEDl-563 was ascertained in an in vitro ADCC assay using isolated basophils and autologous NK cells. Basophils and NK cells were isolated from healthy donors using cially available kits (RoboSep NK/Eosinophil/Basophil Negative Selection Kit, Stem Cell Technologies, Vancouver, Canada). The ADCC assay was performed with isolated NK cells and eosinophils as effectors and target cells at a 5:l ratio.

Antibody trations assayed range from 10"5 to 10‘” M. Cytotoxicity was measured after 24 hrs of incubation by determining Annexin V positive cells by ﬂow cytometry. The EC50 value ofafucosylated 63 was 0.561 pM in this assay. The s ofa representative experiment are shown in Figure 23.

Example 12 Eosinophils do not release cytotoxic granules in Medi-563 mediated ADCC assay.

Degranulation of eosinophils exposed to MEDl-563 targeted ADCC was ascertained by measuring EDN (eosinophil derived neurotoxin) release into the supernatant. In vitro ADCC conditions used were similar to that of described in Example I l. Eosinophils and NK or PBMC cells isolated from healthy donors were used as target and effector cells, respectively.

Assays were performed using fucosylated MEDl-563, ylated 63 or the afucosylated R347 isotyope control antibody. Maximum degranulation was achieved by exposing the eosinophils to 1% triton X-IOO; EDN concentration >220 ng/ml were detected upon maximum degranulation of the cells. The results ofa representative experiment are shown in Figure 24. EDN levels remained below 25 ng/ml (baseline) following MEDl-563 mediated ADCC. MEDl-563 concentration (33 or 100 ug/ml) or the fucosylation status of the antibody did not signiﬁcantly affect degranulation levels.

Example 13 MEDI-563 epitope mapping.

MEDl—563 cally binds to transgenic cells expressing the human lL-5Ralpha protein. MEDl-563 does not bind to cells expressing a mouse lL—SRalpha protein. See s 25B and 26C. The amino acid sequence of mouse and human lLS-Ralpha proteins are highly similar. The epitope speciﬁcity of MEDl-563 was determined by analyzing the binding characteristics ofMEDl-563 to a large panel of mouse-human chimeric IL-5Ralpha proteins (Figures 25-27). The experiments utilized transgenic cells expressing the chimeric lL-SRalpha proteins on their cell surface. Transgene constructs were generated and expressed using standard molecular methods. Antibody binding to a chimeric lpha protein expressed on the surface of transgenic cells was ascertained by ﬂow try.

Fluorescent ng s are shown in Figures 25-27. “Polyclonal” and “MEDl—563” denotes staining s observed using a polyclonal anti-human lL—5Ralpha antibody and 2O MEDl-563, respectively. While MEDl-563 is speciﬁc for a single epitope ofthe human lL- 5Ralpha protein, the polyclonal antibody izes multiple epitopes of human lpha (Figures 258 and 26C). “Dual staining” denotes the ﬂuorescent staining proﬁle for the polyclonal (x axis) and MEDl-563 (y axis) antibodies.

First, the MEDl-563 epitope was mapped to the D1 region ofthe extracellular domain Ralpha. lL-5Ralpha comprises 3 extracellular domains (DI, D2 and D3), a transmembrane domain and an ellular domain (Figure 25A). e MEDl-563 recognizes lL-5Ralpha on intact cells, its epitope must be d in one ofthe extracellular domains. To map the MEDl-563 epitope to one ofthe three extracellular domains: transgenic cells expressing chimeric lL-SRalpha’proteins comprising mouse and human extracellular domains were generated using standard molecular g methods. A schematic representation ofthe chimeric proteins tested are shown in Figure 25A. “Knock-out” variants were chimeric lL—SRalpha proteins comprising a single mouse extracellular domain in an otherwise human background. “Knock-in” variants were chimeric lL-S Ralpha proteins comprising a single human extracellular domain in an ise mouse background.

Figure 25B-C shows the result ofa entative experiment. Both MEDl-563 and the polyclonal antibody stained transgenic cells expressing the human lL-5Ralpha protein; neither antibody stained transgenic cells expressing mouse lL-SRalpha (Figure 258). MEDl- 563 did not bind transgenic cells expressing a chimeric lL-5Ralpha transgene comprising mouse D1 and human D2-D3 extracellular domains (Figure 25C; “knock-out Dl”). MEDl- 563 ically bound transgenic cells expressing a chimeric lL-5Ralpha transgene comprising mouse D2 or D3 extracellular domains in a human background (Figure 25C; IO “knock-out D2 or D3”). MEDl-563 speciﬁcally bound enic cells expressing a chimeric lL-5Ralpha transgene comprising human D1 and mouse D2-D3 extracellular domains (Figure 25D; “knock-in Dl.”). MEDl-563 did not bind to transgenic cells expressing a mouse lL- 5Ralpha based ic ene comprising either the human D2 or D3 extracellular domain (Figure 25D; “knock-in D2 or D3”). All cells sing a chimeric lL-SRalpha protein comprising at least one extracellular domain ofthe human protein were stained by the polyclonal anti-human lL-5Ralpha antibody showing that the difference in MEDl—563 staining pattern among the transgenic cells was not due to a difference in chimeric protein expression level.

Second, the MEDl-563 epitope was mapped to t B ofthe Dl extracellular domain of human lL-5Ralpa (Figure 26). The D] extracellular domain oflL—5Ralpha was divided into three segments e 26A; Segment A, B and C). A series ofhuman-mouse chimeric lL-5Ralpha transgenes comprising various combinations n and mouse Segments of the D1 extracellular domain were generated; the chimeric proteins used at this stage comprised all human sequences outside the D] extracellular domain. “Knock-out” transgenes were chimeric lL-5Ralpha ucts comprising a single mouse Segment ofthe Dl extracellular domain in an ise human background. “Knock-in” transgenes were ic lL-5Ralpha constructs comprising a single human Segment ofthe Dl extracellular domain in a mouse Dl-human D2-mouse se TM background (Figure 268). Figure 26C shows the result ofa control experiment. MEDl-563 ically recognized transgenic cells expressing (i) a human lpha transgene or (ii) a mouse lL-SRalpha chimeric transgene comprising a human Dl extracellular domain ( “human lL-5Ra” and “knock-in Dl”~). MEDl-563 did not bind transgenic cells expressing (i) mouse lL-SRalpha receptor transgene or (ii) a human chimeric lL-5Ralpha ene comprising a mouse Dl extracellular domain (“mouse lL-SRa”, “knock out-D1”). Figure 26D and E shows the result ofa representative g experiment. MEDl-563 did not bind to transgenic cells expressing a chimeric lL-5Ralpha transgene comprising a mouse Segment B ofthe D1 extracellular domain in an otherwise human ound (“knock-out B”). MEDl-563 speciﬁcally bound transgenic cells expressing a chimeric lpha transgene comprising mouse Segment A or C ofthe Dl extracellular domains in a human background (“knock-out A”, “knock out-C”). Figure 26E'shows an example of results obtained with the knock in constructs. MEDl-563 speciﬁcally bound transgenic cells sing a chimeric lL-SRalpha transgene comprising human Segment B ofthe Dl extracellular domain in a mouse Dl - human D2-mouse D3—mouse TM background (“knock-in B”). MEDl-563 did not bind IO transgenic cells expressing a chimeric lL-S Ralpha ene comprising a human Segment A or C ofthe Dl extracellular domain in a mouse Dl-human D2-mouse D3-mouse TM ound (“knock:in A or C”). All cells sing a chimeric lL-SRalpha protein were stained by the polyclonal anti-human IL-SRalpha antibody showing that the difference in MEDl-563 staining pattern among the transgenic cells was not due to a difference in chimeric protein expression level.

Third, the MEDI-563 epitope was mapped to particular amino acid residues within Segment Bl ofthe Dl extracellular domain of human lL~5Ralpha. A series oflL-SRalpha receptor variants comprising at least one mutant amino acid residue within Segment Bl ofthe D1 extracellular domain were expressed in transgenic cells. The position of mutant residues were selected by comparing the mouse and human amino acid sequence. A schematic of the variant proteins tested are shown in Figure 27A. The “knock-out” lL-SRalpha variants were mutant human proteins comprising at least one substitution exchanging a human e for the ponding mouse residue. For example, the “knock-out DE” variant was a human IL- a protein comprising the D56E and E58D amino acid substitutions. The -in” lL-5Ralpha variants were chimeric proteins comprising mouse D], human D2, mouse D3 and mouse TM wherein the mouse D1 domain comprised a mutant version of mouse Segment B having at least one substitution exchanging a mouse residue for the corresponding human residue. For e, the “knock-in DE” variant was a chimeric lL-5Ralpha n comprising mutant mouse Segment B in a mouse Dl-human D2-mouse D3-mouse TM background wherein the mutant mouse Segment B comprised the E56D and D58E amino acid tutions. Figure 27B shows an example of the results obtained using the knock out constructs. MEDl-563 did not bind transgenic cells sing a mutant human lL-5Ralpha n comprising the K53Q, D56E, E58D, l6] K amino acid substitutions (“knock out- KDEI”). MEDl-563 speciﬁcally bound transgenic cells expressing a mutant human IL— 5Ralpha protein comprising the N40H, N42D, Q46H (“knock out-NNQ”) or D56E, E58D (“knock out-DE”), or N40H, N42D, D56E, E58D (“knock out-NNDE”) amino acid substitutions. Figure 27C shows an example ofthe results ed using the knock in constructs. MEDl-563 speciﬁcally bound transgenic cells expressing a variant lL-5Ralpha protein comprising a mutant mouse Segment B ofDl having the Q53K, E56D, D58E, K611 amino acid substitutions (“knock in-KDEI”). Figure 27D shows an example ofthe results obtained using the knock out constructs. MEDl-563 did not bind transgenic cells sing a mutant human IL-SRalpha protein sing the 161K amino acid substitution (“knock out-161”). MEDl-563 speciﬁcally bound transgenic cells expressing a mutant human IL- 5Ralpha protein comprising the K53Q (“knock out-K53”) amino acid substitution. (E) Figure 27E shows an example of the results obtained using the knock in constructs. MEDI- 563 speciﬁcally bound transgenic cells expressing a variant lL-SRalpha n comprising a mutant mouse Segment B ofDl having the K611 amino acid substitution ck in-l6l”).

MED1-563 did not bind transgenic cells expressing a variant lL-5Ralpha protein comprising a mutant mouse Segment B ofDl having the Q53K amino acid substitution (“knock in-K53”).

All cells expressing a chimeric lL-SRalpha protein were stained by the polyclonal anti-human lL-5Ralpha dy showing that the difference in MEDl-563 staining pattern among the transgenic cells was not due to a difference in chimeric protein expression level.

Example 14 In vivo depletion of eosinophils from various tissues.

We assessed the y of an afucosylated anti-mouselL-S Ra antibody (afuc H7) to selectively deplete eosinophils from various tissues in vivo in comparison with fucosylated H7 (fuc H7).

Methods: Monoclonal antibody H7: The variable regions of H7 were grafted onto hlgGl Fc. Fuc H7 was expressed in wild-type CHO cells and afuc H7 in CHO cells deﬁcient in FUT8. dy afﬁnities (KD): Afﬁnities were measured using surface n nce technology.

Mice: IL—5 transgenic mice, and BALB/c mice were used at 6-8 weeks of age. ion of eosinophils in IL-5Tg mice: Mice were dosed with 0mg/kg afuc and fuc H7 i.p. and eosinophil numbers were analyzed 48h later.

Induction of allergic airway inﬂammation: BALB/c mice were sensitized to OVA in alum and nged with OVA on days l7-22. Mice were dosed with 0.1mg/kg afuc H7 i.p. on day 22 and analysis was performed lhr, 24hrs and 72hrs after the ﬁnal challenge.

This ponded to 25, 48 and 96hrs post-antibody treatment.

Isolation of leukocytes: 1‘) Blood Blood was collected by cardiac puncture and kept in heparinised tubes. Blood leukocytes were phenotyped using a Sysmex Hematology Analyser (Sysmex Corp.), or by ﬂow cytometry. it) Airway lumen s were lavaged with 3X0.6ml PBS. BAL samples were centrifuged at 1200rpm, supernatants were removed, and cells resuspended in RPMI. Cells were counted using a Coulter 22 counter an-Coulter), and phenotyped by ﬂow cytometry. iii) Lung tissue One lobe oflung was incubated at 37°C for l h in digest t(18 ug/ml Liberase [Blenzyme 2; Roche], 25 ug/ml DNase [type I; Roche]) in RPMl/lO%FCS.

The recovered cells were ﬁltered through a 70-um nylon sieve n), washed twice, and counted and yped as for BAL. iv) Bone marrow Femurs from donor mice were isolated, and the marrow was ﬂushed out with a syringe attached to a 25-gauge needle containing PBS (without calcium/magnesium). Single cell sions were prepared by ﬂushing the marrow gently up and down in a syringe attached to a 22-gauge needle. The bone marrow cells were centrifuged at 1200 rpm for 5 minutes, washed twice with PBS without additives, resuspended in RPMI, counted and phenotyped by flow cytometry. v) Spleen Spleens were removed and single cell suspensions were prepared using 70-um nylon sieves. Leukocytes were resuspended in RPMI, counted and phenotyped as for BAL.

Flow cytometry Cells were phenotyped using flow cytometric analysis. dies used were anti-mouse CD4, CDl9, CD1 lb, Siglec-F, Gr-l, lL-SR, c—kit (BD Biosciences), FceRl (eBiosciences), and CCR-3 (R and D Systems), and their relevant isotype controls.

Samples were analyzed using an LSRll flow cytometer and FACS DIVA software (BD Biosciences), Results were further analyzed using FlowJo (TreeStar Corp.) Identiﬁcation of eosinophils: Eosinophils were identiﬁed by flow cytometric analysis as cells with high side scatter that stained positively for CCR3 and -F.

Results: lL-5R was selectively expressed by eosinophils in bone marrow, blood, spleen and lung tissue of lL-5Tg mice. lL-5R expression was restricted to eosinophils and was not detected on any other cell type, ing mast cells or basophils. Anti-lL-SR antibody selectively es eosinophils in spleen, lung tissue and blood oflL-STg mice.

Neither afucosylated nor fucosylated anti-lL-SR ed: Neutrophils (Gr-lhi); hages/monocytes (CD1 1b+); T cells (CD3+); B cells (CD19+). Both afuc and fuc H7 depleted eosinophils in spleen, lung tissue and blood Tg mice. No depletion was detected in the bone marrow. Afuc H7 was more potent at removing eosinophils compared with fuc H7, especially at lower antibody doses.

Afuc H7 also selectively depletes eosinOphils in an allergen challenge model. Afuc H7 depleted eosinophils in the airway lumen, lung tissue, blood and bone marrow.

Depletion was highest in all compartments 72h after the ﬁnal nge (96h after antibody delivery).

Whereas, particular embodiments ofthe invention have been described above for purposes of description, it will be appreciated by those skilled in the art that numerous variations ofthe details may be made without departing from the invention as described in the appended claims.

All publications, patents and patent applications mentioned in this speciﬁcation are herein orated by reference into the speciﬁcation to the same extent as if each individual publication, patent or patent application was cally and individually indicated to be orated herein by reference. In addition, US. Provisional Application Nos 60/924,422, ﬁled May 14, 2007, 60/924,832, ﬁled June I, 2007, 60/935,005, ﬁled July 20, 2007, and 61/064,612, ﬁled March 14, 2008, are hereby incorporated by reference in their entirety for all purposes.

Claims

What we claim is:

1. The use of a monoclonal, chimeric, humanized or human antibody that binds IL- 5R in the manufacture of a medicament for treating or preventing a basophil mediated e or disorder in a human subject, wherein said treating or preventing comprises administering the antibody parenterally to said t at between about 0.001 to about 100 mg/kg, wherein said antibody comprises an immunoglobulin Fc region comprising no fucose, and wherein the administration of the antibody reduces the number of peripheral blood basophils from the human subject’s ation.

2. The use of claim 1, wherein said antibody specifically binds to the IL-5R α chain.

3. The use of claim 1 or 2, n the reduction of eral blood basophils is reversible.

4. The use of any one of claims 1 to 3, n the reduction of peripheral blood basophils is by about 5 to about 70 basophils/mm3 by 24 hours post-administration

5. The use of any one of claims 1 to 4, wherein the administration of the antibody reduces the number of peripheral blood basophils from the human subject’s circulation to a level that is less than 9 basophils/mm3.

6. The use of any one of claims 1 to 5, wherein there is a dministration reduction in absolute basophil count of at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, or at least about 70 basophils/mm3.

7. The use of any one of claims 1 to 6, wherein said subject's pre-administration absolute basophil count is between about 5 and about 500 basophils/mm3.

8. The use of any one of claims 1 to 7, wherein said subject's pre-administration absolute basophil count is about 5, about 10, about 15, about 20, about 25, about 30, about 50, about 60, about 70, about 100, about 200, about 300, about 400, or about 500 basophils/mm3.

9. The use of any one of claims 1 to 8, wherein the disease or disorder mediated by basophils is asthma.

10. The use of claim 9, wherein said reduction in peripheral blood basophils leads to a reduction in asthma symptoms.

11. The use of any one of claims 1 to 10, with the proviso that the IL-5R binding antibody is not MEDI-563.

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