CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application no. 61/397, 174, filed June 7, 2010, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for treating eosinophilic disorders, including asthma and atopy, by administering antagonists of IL-4 and IL- 13, such as mutant human interleukin-4 (IL-4) muteins.

BACKGROUND OF THE INVENTION

[0003] Interleukin-4 (IL-4) and Interleukin- 13 (IL- 13) are pleiotropic cytokines with a broad spectrum of biological effects on several target cells important in the pathogenesis of atopy and asthma. IL-4 is increasingly appreciated as the pivotal cytokine initiating the "Th2-type" inflammatory response forming the underling milieu necessary for the development of atopy and asthma. IL-4 effects include activation, proliferation and differentiation of T and B cells. During proliferation of B-lymphocytes, IL-4 acts as a differentiation factor by regulating class switching from IgG to the IgE thus, encouraging the development of allergic reactions. IL- 13 is now appreciated as the more probable downstream effector cytokine. IL-13 dominant effects include induction of airways hyperresponsiveness (AHR) and goblet cell hyperplasia, both cardinal features of asthma. However, there is considerable redundancy in the effects of these two cytokines.

[0004] The redundancy in effects associated with the binding and signaling of these two cytokines can be explained by their sharing of common receptors. The IL-4 receptor alpha chain (IL-4Ra) has two binding partners with which it can associate and signal. IL-4Ra polypeptide associates with the cytokine common receptor gamma chain (yc) to form the type 1 IL-4R heterodimer. IL-4Ra polypeptide can also form a heterodimer with the IL-13 receptor alpha 1 chain to create the type 2 IL- 4R (aka IL-13R). IL-4 activates both the type 1 and type 2 receptors, whereas IL-13 only activates the type 2 receptor heterodimer. Both receptors, when activated, signal through the transcription factor signal transducer and activator of transcription 6 (STAT6). Although IL-4 may uniquely initiate the T-helper 2 (Th2) pathway, since only type 1 receptors are localized to T lymphocytes, IL- 13 may be both more abundant and more potent.

[0005] Certain antagonistic and partially antagonistic properties have been observed in human IL-4 (hIL-4) mutant proteins in which the amino acid(s) occurring naturally in the wild type at one or more of positions 120, 121 , 122, 123, 124, 125, 126, 127 or 128 have been replaced with one or more natural amino acids.

SUMMARY OF THE INVENTION

[0006] The invention includes a method for treating a subject for an eosinophilic disorder, wherein the subject has elevated eosinophil levels. The method entails administering to the subject a therapeutically effective amount of an antagonist of IL-4 and IL-13 (IL-4/IL-13 antagonist). In particular embodiments, the subject has elevated eosinophil levels in blood or sputum. In illustrative embodiments, the subject has a blood eosinophil level of greater than about 300, or about 350, eosinophils per mm ³ . Such subjects can have atopy and/or asthma.

[0007] The invention also includes a method for treating a subject for asthma.

The method entails administering to the subject an antagonist of IL-4 and IL- 13 (IL- 4/IL-13 antagonist) twice daily by inhalation in doses of at least 10 mg. In certain embodiments, the IL-4/IL-13 antagonist is administered for a period of at least 6 months.

[0008] In each of these methods, the subject can have moderate to severe asthma, e.g., GINA level 4 or 5 asthmas. In addition, or alternatively, the subject can have adult-onset asthma. In each method, the antagonist can reduce one or more of the following: incidence of asthma exacerbations, time to first exacerbation after initiating treatment, night awakenings, severity of asthma symptoms, and impact of asthma on daily activities. In either method, if the subject was being treated with an inhaled corticosteroid (ICS), a long-acting beta agonist (LABA) or both, when treatment with the IL-4/IL-13 antagonist was initiated, the subject can, in some embodiments, be withdrawn from the ICS and/or the LABA after initiation of treatment with the IL-4/IL-13 antagonist.

[0009] In either method, the IL-4/IL-13 antagonist can be an antisense antagonist, an RNAi antagonist, or an antibody. The antagonist can also be a mutant human IL-4 protein including the amino acid sequence of wild-type hIL-4 with modifications, wherein a first modification is replacement of one or more of the amino acids occurring in the wild-type hIL-4 protein at positions 121 , 124, or 125 with another natural amino acid. In particular embodiments, the first modification includes amino acid substitutions R121 D and Y 124D, numbered in accordance with wild-type hIL-4. In certain embodiments, the mutant human IL-4 protein additionally includes a second modification selected from the group consisting of:

the modification of the C-terminus therein;

the deletion of one or more potential glycosylation sites therein;

the coupling of the protein to a non-protein polymer, and any combination thereof.

In some embodiments, the mutant human IL-4 protein additionally includes an N- terminal methionine. In particular embodiments, the antagonist be a modified IL-4 mutein receptor antagonist that includes the following:

the substitution of each of the amino acids occurring in the wild-type human IL-4 protein at positions 121 and 124 with different amino acids;

the substitution of the threonine occurring in the wild-type human IL 4 protein at position 13 with a different amino acid;

the substitution of the asparagine occurring in the wild-type human IL 4 protein at position 38 with a cysteine; and

a non-protein polymer covalently attached to the substituted cysteine at position 38. For example, the modified IL-4 mutein receptor antagonist can include amino acid substitutions T13D, N38C, R121 D, and Y 124D, numbered in accordance with wild-type hIL-4, with polyethylene glycol (PEG) attached to the substituted cysteine at position 38. In particular embodiments, any of the above-described mutant human IL-4 proteins can have at least 95 percent, or at least 99 percent, sequence identity with the wild-type human IL-4 protein.

[0010] In each method, the antagonist can be administered systemically or locally. In certain embodiments, the antagonist is administered by inhalation. In variations of these embodiments, the antagonist is administered to the lungs of a subject via inhalation and enters systemic circulation of the subject. In an illustrative embodiment, the antagonist is aerosolized prior to administration. For example the antagonist can be nebulized as a liquid or aerosolized as a dry powder prior to administration. In certain embodiments of the first method, the antagonist is administered twice per day. In particular embodiments, that antagonist is mutant human IL-4 protein, and a therapeutically effective amount of the mutant human IL-4 protein is at least 20 mg per day. In some embodiments, the antagonist is administered in a pharmaceutical composition including a pharmaceutically acceptable carrier selected from the group consisting of lactate, citrate, and sucrose buffer.

[0011] Where the antagonist is a mutant human IL-4 protein, this protein can be conjugated to a non-protein polymer. The polymer can be hydrophilic (e.g., polyvinylpyrrolidone) or hydrophobic (e.g., polyethylene glycol).

[0012] In certain embodiments of either method, a mutant human IL-4 protein can co-administered with a therapeutically effective amount of an additional agent that mitigates a symptom of an eosinophilic disorder (e.g., atopy or asthma).

[0013] Another aspect of the invention is a method of determining whether a subject having an atopic or inflammatory disorder is a candidate for treatment with an antagonist of IL-4 and IL-13 (IL-4/IL-13 antagonist). In a first embodiment, the method entails

determining whether the subject has an elevated level of eosinophils; selecting an IL-4 IL-13 antagonist for use in treating the subject; and recording the eosinophil level and an indication of the selected IL- 4 IL- 13 antagonist in a patient medical record. [0014] In a second embodiment, a method of determining whether a subject having an atopic or inflammatory disorder is a candidate for treatment with an antagonist of IL-4 and IL-13 (IL-4 IL-13 antagonist) entails

determining whether the subject has:

an elevated level of eosinophils; and/or

the major allele in one or more single nucleotide

polymorphisms (SNPs) in the IL-RA gene selected from the group consisting of rs8832, rs 1029489, rs3024585, rs3024622, and rs4787956; and

recording the eosinophil level or the presence of the major allele in one or more of said SNP(s) in a patient medical record. An elevated level of eosinophils and/or the major allele in one or more of said SNPs indicates that the subject is a candidate for treatment with an IL-4 IL- 13 antagonist. A variation of this second embodiment additionally includes selecting an IL-4/IL-13 antagonist for use in treating the subject, and recording the selected IL-4/IL-13 antagonist in the patient medical record.

[0015] In preferred embodiments of the method of determining whether a subject having an atopic or inflammatory disorder is a candidate for treatment with an IL-4/IL-13 antagonist, the subject is human. The subject can also have atopy and/or asthma. In particular embodiments, the subject can have moderate to severe asthma, e.g., GI A level 4 or 5 asthmas. In addition, or alternatively, the subject can have adult-onset asthma. Variations of the first and second embodiments of this method can include administering the IL-4 IL-13 antagonist to a subject having:

an elevated level of eosinophils; and/or

the major allele in one or more single nucleotide

polymorphisms (SNPs) in the IL-RA gene selected from the group consisting of rs8832, rsl 029489, rs3024585, rs3024622, and rs4787956.

[0016] In certain embodiments of the method of determining whether a subject having an atopic or inflammatory disorder is a candidate for treatment with an IL-4/IL-13 antagonist, the eosinophil level is determined. For example, the eosinophil levels in blood and/or sputum can be determined. In illustrative embodiments, the subject has a blood eosinophil level of greater than about 300, or greater than about 350, eosinophils per mm ³ .

[0017] Alternatively, or in addition, the presence of the major allele in one or more of the above-mentioned SNPs is determined. In certain embodiments, both alleles are determined in one of more of these SNPs. The determination that the subject is homozygous for the major allele in one or more of these SNPs, in some embodiments, provides a stronger indication that the subject is a candidate for treatment with an IL-4/IL-13 antagonist than if the subject were heterozygous. In specific embodiments, the presence of the major allele in rs8832 and/or rsl 029489 is determined. In a preferred embodiment, the presence of the major allele in rs8832 is determined. In various embodiments, the presence of the major allele in at least two, or three, of these SNPs is determined.

[0018] The presence of a major allele in any of the above-mentioned SNPs can be determined by assaying a nucleic acid in a sample from the subject. In some embodiments, the presence of the major allele is determined by hybridizing the sample nucleic acid to a probe that specifically hybridizes to the major allele. For example, the probe can be a member of a plurality of probes that forms an array of probes. In certain embodiments, the presence of the major allele is determined using a nucleic acid amplification reaction.

[0019] The invention also provides pharmaceutical composition, wherein the composition includes:

a therapeutically effective amount of any of the mutant human IL-4 proteins described above; and

a therapeutically effective amount of an additional agent that is useful for mitigating a symptom of an eosinophic disorder.

[0020] Another aspect of the invention is a kit that includes:

at least one unit dosage form including a therapeutically effective amount of any of the mutant human IL-4 proteins described above; and at least one unit dosage form including a therapeutically effective amount of an additional agent that is useful for mitigating a symptom of eosinophilic disease.

[0021] In either the composition or the kit, in some embodiments, the therapeutically effective amount of the mutant human IL-4 protein is at least 10 mg. In either the composition or the kit, the additional agent can be one that mitigates a symptom of atopy and/or asthma. In certain embodiments, the composition or kit does not include a corticosteroid (CS) or the composition or kit does not include a long-acting beta agonist (LABA), or the composition or kit does not include either a CS or an LABA.

[0022] Another aspect of the invention is a kit for determining the presence of the major allele in one or more single nucleotide polymorphisms (SNPs) in the IL-RA gene selected from the group consisting of rs8832, rs 1029489, rs3024585, rs3024622, and rs4787956. In certain embodiments, the kit includes a container containing one or more probes and/or primers that specifically hybridize under stringent conditions to a nucleic acid including the major allele in one or more SNPs selected from the group consisting of rs8832, rsl029489, rs3024585, rs3024622, and rs478795. The kit can, optionally, include instructional materials teaching that the determination the major allele in the one or more SNPs in a nucleic acid sample from a subject indicates that the subject is a candidate for treatment with an IL-4 IL-13 antagonist.

[0023] In specific embodiments, the kit includes one or more probes and/or primers that specifically hybridize under stringent conditions to a nucleic acid including the major allele in one or more SNPs selected from the group consisting of rs8832 and rs 1029489. A preferred kit includes one or more probes and/or primers that specifically hybridize under stringent conditions to a nucleic acid including the major allele in rs8832. In other various embodiments, the kit includes at least two probes and/or primers that specifically hybridize under stringent conditions to nucleic aci(s) including the major allele in at least two, or a least three, of the above- mentioned SNPs. In illustrative embodiments, the kit includes one or more probes that are members of a plurality of probes that forms an array of probes. In certain embodiments, the kit includes one or more probes and/or primers for use in nucleic acid amplification reaction. [0024] The invention also provides a labeled nucleic acid that specifically hybridizes under stringent conditions to a nucleic acid including one or more single nucleotide polymorphisms (SNPs) in the IL-RA gene selected from the group consisting of rs8832, rsl 029489, rs3024585, rs3024622, and rs4787956. In particular embodiments, the nucleic acid specifically hybridizes under stringent conditions to a nucleic acid including one or more SNPs selected from the group consisting of rs8832 and rs 1029489. In a specific embodiment, the nucleic acid specifically hybridizes under stringent conditions to a nucleic acid including the SNP rs8832.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Incidence of exacerbation in eosinophilic

population (> 0.35 K/cumm).

[0026] Figure 2A-B: Effect of Aerovant on asthma symptom score for night awakenings (eosinophil >0.35 /cu mm population). A: asthma symptom score: weekly average; B: asthma symptom score: change from baseline at visit 8.

[0027] Figure 3A-B: Effect of Aerovant on asthma symptom score for severity (eosinophil >0.35K/cu mm population). A: asthma symptom score:

weekly average; B: asthma symptom score: change from baseline at visit 8.

[0028] Figure 4A-B: Effect of Aerovant on asthma symptom score for daily activities (eosinophil >0.35K/cu mm population). A: asthma symptom score: weekly average; B: asthma symptom score: change from baseline at visit 8.

[0029] Figure 5: IL-4RA non-Hispanic white LD.

[0030] Figure 6: IL-4RA/rs8832 and asthma exacerbations dose response relationship. Subjects with the rs8832 GG genotype demonstrated a significant dose- dependent reduction (placebo/1 mg/3mg/10mg) in exacerbations. There was no dose- dependent relationship with exacerbations for subjects with the AG/AA genotypes.

[0031] Figure 7: IL-4RA/rs 1029489 and asthma exacerbations dose response relationship. Subjects with the rs 1029489 GG genotype demonstrated a significant dose-dependent reduction (placebo/1 mg/3mg/10mg) in exacerbations. There was no dose-dependent relationship with exacerbations for subjects with the AG/AA genotypes. IL-4RA/rs 1029489 is highly correlated with rs8832 (r2=0.75).

Significant dose-response relationships were also observed for subjects homozygous for the major allele in rs3024585, rs3024622, and rs4787956 (P=0.03).

[0032] Figure 8: IL-4RA/rs8832 genotype and asthma exacerbations by treatment assignment. IL-4RA/rs8832 is significantly associated with exacerbations at the 10 mg pitrakinra level and borderline associated at the 3 mg level; NS in l mg and placebo group. The common rs8832 GG genotype was associated with reduced exacerbations.

[0033] Figure 9: IL-4RA polymorphisms associated with exacerbations. IL- 4RA polymorphisms are significantly associated with exacerbations in subjects randomized to 3 and 10 mg pitrakinra; NS in placebo group. For all SNPs except rsl 1 10470, subjects homozygous for the major allele were less likely to exacerbate.

[0034] Figure 10: IL-4RA/rs8832 GG genotype and time to asthma exacerbation. Time to exacerbation Kaplan-Meier plot for subjects with rs8832 GG genotype. There was a significant difference in time to exacerbation for subjects randomized to 10 mg pitrakinra, compared to placebo (log-rank test P=0.02). Number of exacerbations by treatment assignment indicated.

DETAILED DESCRIPTION

[0035] The present invention is based on the finding that antagonists of IL-4 and IL-13 are useful for treating disorders characterized by elevated eosinophil levels, including moderate to severe asthma. Thus, the present invention discloses methods and compositions for treating such disorders, in some embodiments, with

therapeutically effective amounts of mutant human IL-4 proteins.

Definitions

[0036] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0037] As used herein and in the appended claims, the singular forms "a,"

"an," and "the" include plural reference unless the context clearly dictates otherwise. [0038] The term "eosinophilic disorder" refers to any disorder characterized by an elevated level of eosinophils. Eosinophil levels can be conveniently measured in a sputum or blood sample. Normal levels in blood are on the order of 250 eosinophils per mm ³ . Blood levels over 300 per mm ³ , and especially over 350 per mm ³ , are considered elevated. Examples of eosinophilic disorders include eosinophilic asthma and typical atopic diseases or allergic dermatitis including contact dermatitis, atopic dermatitis (i.e., eczema), psoriasis, seborrheic dermatitis, and the like. Churg-Strauss syndrome (also known as "Allergic granulomatosis") is an serious, life-threatening eosinophilic disorder characterized by medium and small vessel autoimmune vasculitis, leading to necrosis. It involves mainly the blood vessels of the lungs (manifesting as a severe type of asthma), gastrointestinal system, and peripheral nerves, but also affects the heart, skin, and kidneys.

[0039] The term "asthma" is used herein to generally describe a chronic respiratory disease, often arising from allergies, that is characterized by sudden, recurring attacks of labored breathing, chest constriction, and coughing. In a typical asthmatic reaction, IgE antibodies predominantly attach to mast cells that lie in the lung interstitium in close association with the bronchioles and small bronchi. An antigen entering the airway will thus react with the mast cell-antibody complex, causing release of several substances, including, but not limited to interleukin cytokines, chemokines and arachidonic acid-derived mediators, resulting in bronchoconstriction, airway hyperreactivity, excessive mucus secretion, and airway inflammation. Thus, in certain embodiments of the invention, the treatment of asthma may include the treatment of airway hyperreactivity and/or the treatment of lung inflammation.

[0040] "Level 4 asthma" and "level 5 asthma" refer to levels as defined by the

Global Initiative for Asthma (GI A) Guidelines.

[0041] The term "adult-onset asthma" refers to asthma beginning at age 20 or after.

[0042] The term "antigen" as used herein refers to any substance that when introduced into the body stimulates the production of an antibody. Antigens include insect, animal and plant proteins, toxins, bacteria, foreign blood cells, and the cells of transplanted organs. "Allergens" refer to any substances that cause an allergic immune reaction in a subject. Typically, allergens are from foods, plants, insects, or animals that inflame the airway and cause mucus production and bronchoconstriction.

[0043] The term "subject" as used herein refers to any individual or patient on which the subject methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus, other animals, including mammals, such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and non-human primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0044] The term "therapeutically effective amount" or "effective amount" means the amount of a compound or pharmaceutical composition that will elicit the biological or medical response in a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor, or other clinician.

[0045] As used herein, the term "agonist" refers to an agent or analog that binds productively to a receptor and activates it.

[0046] The term "antagonist" refers to an agent that binds to a receptor but does not provoke the normal biological response and blocks or partially blocks the activity of the agonist.

[0047] As used herein, "wild type IL-4" or "wtIL-4" and equivalents thereof are used interchangeably and refer to human Interleukin-4, native or recombinant, having the 129 normally occurring amino acid sequence of native human IL-4, as disclosed in U.S. Pat. No. 5,017,691 , which is incorporated herein by reference.

Further, the human IL-4 receptor antagonists described herein may have various insertions and/or deletions and/or couplings to a non-protein polymer, and are numbered in accordance with the wtIL-4. Accordingly, one skilled in the art will appreciate that the normally occurring amino acids at positions, for example, 121 (arginine), 124 (tyrosine), and/or 125 (serine), may be shifted in the mutein.

Similarly, an insertion of a cysteine residue at amino acid position(s), for example, 38, 102, and/or 104 may be shifted in the mutein. However, the location of the shifted Ser (S), Arg (R), Tyr (Y) or inserted Cys (C) can be determined by inspection and correlation of the flanking amino acids with those flanking Ser, Arg, Tyr, or Cys in wtIL-4.

[0048] As used herein, the terms "mutant human IL-4 protein," "human IL-4 receptor antagonist," "mhIL-4," "IL-4 mutein," "IL-4 antagonist," and equivalents thereof are used interchangeably. These polypeptides and functional fragments thereof refer to polypeptides wherein specific amino acid substitutions to the mature human IL-4 protein have been made. These polypeptides include the mIL-4 compositions of the present invention, which are administered to a subject in need of treatment for an eosinophilic disorder. In particular, the mhIL-4 described herein includes a replacement of one or more of the amino acids occurring in the wild-type hIL-4 protein at positions 121 , 124, or 125 with another natural amino acid, e.g., the R121 D/Y124D pair of substitutions ("IL-4RA"). Such modifications of hIL-4 and of mhIL-4 are described in U.S. Patent Publication No. 20070212308 (published September 13, 2007), U.S. Patent Publication No. 20090010874 (published Jan. 8, 2009), International Publication No. WO/2009/009775 (published Jan. 15, 2009), International Publication No. WO 2009065007 (published May 22, 2009), the entire contents of both of which are incorporated herein by reference, in particular, for their description of mhIL-4 and pharmaceutical compositions including mhIL-4.

[0049] As used with respect to sequences, the term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms").

[0050] Identity of two nucleic acids or two polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in COMPUTATIONAL MOLECULAR BIOLOGY, (Lesk, A.M., ed.), 1988, Oxford University Press, New York; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, (Smith, D.W., ed.), 1993, Academic Press, New York; COMPUTER ANALYSIS OF SEQUENCE DATA, Part 1 , (Griffin, A.M., and Griffin, H.G., eds.), 1994, Humana Press, New Jersey; von Heinje, G., SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, 1987, Academic Press; SEQUENCE ANALYSIS PRIMER, (Gribskov, M. and Devereux, J., eds.), 1991 , M. Stockton Press, New York; Carillo et al, 1988, SIAM J. Applied Math., 48: 1073; and Durbin et al, 1998, BIOLOGICAL SEQUENCE ANALYSIS, Cambridge University Press.

[0051] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al, 1984, Nucl. Acid. Res., 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and FASTA (Altschul et al, 1990, J. Mol Biol, 215:403-410). The

BLASTX program is publicly available from the National Center for Biotechnology Information (NCB1) and other sources (BLAST Manual, Altschul et al

NCB/NLM/NIH Bethesda, MD 20894; Altschul et al, 1990, supra). The well-known Smith Waterman algorithm may also be used to determine identity.

[0052] Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, in certain embodiments, the selected alignment method (GAP program) will result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide.

[0053] For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, WI), two polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the "matched span", as determined by the algorithm). In certain embodiments, a gap opening penalty (which is calculated as three-times the average diagonal; where the "average diagonal" is the average of the diagonal of the comparison matrix being used; the "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually one-tenth of the gap opening penalty), as well as a comparison matrix such as PAM250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see Dayhoff et al, 1978, Atlas of Protein Sequence and Structure, 5:345-352 for the PAM 250 comparison matrix; Henikoff et al, 1992, Proc. Natl. Acad. Sci USA, 89: 10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

[0054] In certain embodiments, the parameters for a polypeptide sequence comparison include the following:

Algorithm: Needleman et al, 1970, J. Mol Biol, 48:443-453;

Comparison matrix: BLOSUM 62 from Henikoff et al, 1992, supra;

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program may be useful with the above parameters. In certain embodiments, the aforementioned parameters are the default parameters for polypeptide

comparisons (along with no penalty for end gaps) using the GAP algorithm.

[0055] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See IMMUNOLOGY— A SYNTHESIS, 2nd Edition, (E. S. Golub and D. R. Gren, Eds.), Sinauer Associates: Sunderland, MA, 1991 , incorporated herein by reference for any purpose. Stereoisomers (e.g., D- amino acids) of the twenty conventional amino acids; unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides described herein. Examples of unconventional amino acids include: 4- hydroxyproline, γ-carboxyglutamate, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O- phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, σ-Ν-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl- terminal direction, in accordance with standard usage and convention.

[0056] As used herein, a "functional fragment" is a polypeptide which has IL-

4 antagonistic activity, including smaller peptides. These and other aspects of mhIL-4 are described in U.S. Pat. Nos. 6,335,426; 6,313,272; and 6,028, 176, the entire contents of which are incorporated herein by reference.

[0057] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics". See Fauchere, 1986, Adv. Drug Res. 15:29; Veber & Freidinger, 1985, TINS p.392; and Evans et al, 1987, J. Med. Chem. 30: 1229, which are incorporated herein by reference for their descriptions of peptide mimetics. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce a similar therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from: -CH ₂ -.NH-, -CH ₂ -S-, -CH ₂ - CH ₂ -, -CH=CH-(cis and trans), -COCH ₂ -, -CH(OH)CH ₂ -, and -CH ₂ SO-, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type {e.g., D-lysine in place of L-lysine) may be used in certain embodiments to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo & Gierasch, \ 992, Ann. Rev. Biochem. 61 :387, incorporated herein by reference for any purpose); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

[0058] As used herein, "mutein" refers to any protein arising as a result of a natural mutation or a site-directed amino acid substitution to any protein created by a person skilled in the art.

[0059] "Glycosylation" refers to the addition of glycosyl groups to a protein to form a glycoprotein. As such, the term includes both naturally occurring

glycosylation and synthetic glycosylation, such as the linking of a carbohydrate skeleton to the side chain of an asparagine residue ("N-glycosylation") or the coupling of a sugar, preferably N-acetylgalactosamine, galactose or xylose to serine, threonine,

4-hydroxyproline, or 5 -hydroxy lysine (O-glycosylation). [0060] As used herein, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0061] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50- 70 kD). The N-terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0062] Antibodies exist as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' _2i a dimer of Fab which itself is a light chain joined to VH-CH 1 by a disulfide bond. The F(ab)' ₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab') ₂ dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (See, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term "antibody", as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Preferred antibodies include single chain antibodies, more preferably single chain Fv (scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. [0063] The term a "primary particle size" is defined for the purposes of the present application as the size of the particle as measured by various techniques such as laser diffraction, scanning electron microscopy, and sedimentation.

[0064] The term "aerodynamic" is defined for the purposes of the present application as the diameter of a sphere of unit density which has the same settling velocity in air as the aerosol particle being measured. Aerodynamic diameter is measured by a cascade impactor.

[0065] The term "mass median aerodynamic diameter" or "MMAD" is defined as the median of the distribution of mass with respect to aerodynamic diameter. The median aerodynamic diameter and the geometric standard deviation are used to describe the particle size distribution of an aerosol, based on the mass and size of the particles. According to such a description, fifty percent of the particles by mass will be smaller than the median aerodynamic diameter, and fifty percent of the particles will be larger than the median aerodynamic diameter.

[0066] The term a "powder" is defined for the purposes of the present application as a solid substance formulated as finely divided solid particles that are smaller than about 10 micrometers in dimension, such as a solid substance formulated as finely divided dry solid particles that are smaller than about 6 micrometers in dimension.

[0067] The term "glass transition temperature" is defined for the purposes of the present application as an approximate midpoint in the temperature range at which a reversible change occurs in a substance when it is heated to a certain temperature and undergoes a transition from glassy condition to elastomeric condition. Glass transition (Tg) is determined using differential scanning calorimetry (DSC). The definition of Tg is always arbitrary and there is no present international convention.

[0068] A "polymorphism" or "polymorphic site" is a locus at which nucleotide sequence divergence occurs. Illustrative markers have at least two alleles. A polymorphic site may be as small as one base pair. The allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form, but is referred to herein as the "major" allele. Diploid organisms may be homozygous or heterozygous for allelic forms. A diallelic polymorphism has two forms. A triallelic polymorphism has three forms.

[0069] A "single nucleotide polymorphism" (SNP) occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations). A SNP usually arises due to substitution of one nucleotide for another at the polymorphic site. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine by a pyrimidine or vice versa.

[0070] In particular embodiments, hybridizations are carried out under stringent hybridization conditions. The phrase "stringent hybridization conditions" generally refers to a temperature in a range from about 5°C to about 20°C or 25°C below than the melting temperature (T _m ) for a specific sequence at a defined ionic strength and pH. As used herein, the T _m is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the T _m of nucleic acids are well known in the art (see, e.g., Berger and Kimmel ( 1987) METHODS IN ENZYMOLOGY, VOL.152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., VOLS. 1 -3, Cold Spring Harbor Laboratory), both incorporated herein by reference). As indicated by standard references, a simple estimate of the T _m value may be calculated by the equation: T _m =81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The melting temperature of a hybrid (and thus the conditions for stringent hybridization) is affected by various factors such as the length and nature (DNA, RNA, base composition) of the primer or probe and nature of the target nucleic acid (DNA, RNA, base composition, present in solution or immobilized, and the like), as well as the concentration of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and are discussed in standard references in the art. Illustrative stringent conditions suitable for achieving specific hybridization of most sequences are: a temperature of at least about 60°C and a salt concentration of about 0.2 molar at pH7.

Treatment Method In General

[0071] In certain embodiments of the invention, a human IL-4 mutein receptor antagonist is useful for treating various conditions associated with one of the pleiotropic effects of IL-4 and IL-13. For instance, antagonists of IL-4 and IL- 13 are useful in treating conditions exacerbated by IL-4 and IL- 13 production including asthma, atopy (allergy), or other inflammatory response-related conditions. Some uses of the human IL-4 mutein receptor antagonists are described in U.S. Pat. Nos. 6, 130,318 and 7,404,957 and in U.S. Publication No. 20070009479 (published Jan. 1 1, 2007), U.S. Patent Publication No. 20070212308 (published September 13, 2007), and International Publication No. WO/2009/009775 (published Jan. 15, 2009), all three of which are incorporated herein by reference in their entirety, and specifically for their description of uses of human IL-4 mutein receptor antagonists.

[0072] In particular embodiments, IL-4/IL-13 antagonists are useful for treating disorders characterized by elevated eosinophils, such as eosinophilic asthma. Patients suffering from this form of asthma generally have moderate to severe disease, which, in many cases, cannot be adequately controlled using conventional therapies, such as inhaled corticosteroids (ICSs), long-acting beta agonists (LABAs), or both.

[0073] The subject of the method can be any organism that exhibits (i.e., is experiencing or at risk for) an allergic response, such as an eosinophilic disorder. Examples of suitable subjects include research animals or pets, such as mice, rats, guinea pigs, rabbits, cats, dogs, as well as monkeys and other primates, and humans. IL-4/IL-13 Antagonists

[0074] Any agent capable of antagonizing IL-4 and IL-13 can be employed in the methods of the invention. IL-4/IL-13 antagonists can act to prevent production of one or more key proteins related to IL-4/IL-13 signaling or can inhibit the activity of such proteins. For example, an antisense or RNAi antagonist can target the IL-4 receptor alpha chain (IL-4Roc) protein necessary for the biological activities IL-4 and IL-13. Examples of agents that inhibit IL-4/IL-13 activity include antibodies, e.g., directed against IL-4Ra, and muteins, e.g., IL-4 muteins.

Antisense Antagonists

[0075] An "antisense sequence or antisense polynucleotide" is a

polynucleotide that is complementary to the target polynucleotide sequence or a subsequence thereof. In particular embodiments, the methods described herein employ antisense molecules useful for inhibiting binding of the target polynucleotide. Suitable antisense molecules include oligonucleotides and oligonucleotide analogs that are hybridizable with the target polynucleotide of interest. Such oligonucleotides include, for example, polynucleotides formed from naturally-occurring bases and/or cyclofuranosyl groups joined by native phosphodiester bonds. The term

"oligonucleotide" encompasses moieties that function similarly to oligonucleotides, but that have non-naturally occurring portions. Thus, oligonucleotides may have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur-containing species that are known for use in the art. In accordance with some preferred embodiments, at least one of the phosphodiester bonds of the oligonucleotide has been substituted with a structure which functions to enhance the ability of the compositions to penetrate into the region of cells where the target polynucleotide whose activity is to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short-chain alkyl or cycloalkyl structures. In accordance with other preferred embodiments, the phosphodiester bonds are substituted with structures that are, at once, substantially non-ionic and non-chiral, or with structures that are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in the practice of the invention.

[0076] In an exemplary embodiment, the internucleotide phosphodiester linkage is replaced with a peptide linkage. Such peptide polynucleotides tend to show improved stability, penetrate the cell more easily, and show enhanced affinity for their target. Methods of making peptide polynucleotides are known to those of skill in the art (see, e.g., U.S. Patent Nos: 6,015,887, 6,015,710, 5,986,053, 5,977,296, 5,902,786, 5,864,010, 5,786,461 , 5,773,571 , 5,766,855, 5,736,336, 5,719,262, and 5,714,331 ).

[0077] Oligonucleotides useful in the antisense methods of the invention may also include one or more modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be employed. Similarly, the furanosyl portions of the nucleotide subunits may also be modified, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2'-0- alkyl- and 2'-halogen-substituted nucleotides. Some specific examples of

modifications at the 2' position of sugar moieties which are useful in the present invention are: OH, SH, SCH ₃ , F, OCH ₃ , OCN, 0(CH ₂ )[n]NH ₂ or 0(CH ₂ )[n]CH ₃ , where n is from 1 to about 10, and other substituents having similar properties.

[0078] All such analogs can be used in the antisense methods of the invention so long as the analogs function effectively to hybridize with the target polynucleotide of interest and inhibit its function.

[0079] Antisense oligonucleotides in accordance with this invention preferably comprise from about 3 to about 50 subunits (i.e., bases in unmodified polynucleotides). It is more preferred that such oligonucleotides and analogs comprise from about 8 to about 25 subunits and still more preferred to have from about 12 to about 25 subunits. The oligonucleotides used in accordance with this invention can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors (e.g., Applied Biosystems).

[0080] Antisense oligonucleotides of the invention can be synthesized, formulated, and administered to cells, tissues, or organisms in accordance with standard practice.

[0081] An illustrative antisense antagonist that can be used in the methods described herein is AIR645, which antagonizes the action of the IL-4 receptor alpha chain (IL-4Ra) protein necessary for the biological activities of IL-4 and IL-13 (produced by Altair Therapeutics, Inc.). See, e.g., U.S. Patent No. 7,507,810 (issued March 24, 2009), which is incorporated by reference herein in its entirety and specifically for its description of antisense molecules targeting IL-4R . R Ai Antagonists

[0082] Another approach to reducing the level of target polynucleotides entails RNA interference (RNAi). RNAi, also termed post-transcriptional gene silencing (PTGS), refers to a mechanism by which double-stranded (sense strand) RNA (dsRNA) specifically blocks expression of its homologous gene when injected, or otherwise introduced into cells. This approach is based on the observation that injection of antisense or sense RNA strands into C. elegans cells resulted in gene- specific inactivation (Guo and Kempheus (1995) Cell 81 : 61 1 -620). While gene inactivation by the antisense strand was expected, gene silencing by the sense strand was unexpected. Surprisingly, it was determined that the gene-specific inactivation was actually due to trace amounts of contaminating dsRNA (Fire et al. (1998) Nature 391 : 806-81 1).

[0083] Since then, this mode of post-transcriptional gene silencing has been demonstrated in a wide variety of organisms: plants, flies, trypanosomes, planaria, hydra, zebrafish, and mice (Zamore et al. (2000) Cell 101 : 25-33; Gura (2000) Nature 404: 804-808). RNAi activity has been associated with functions as disparate as transposon-silencing, anti-viral defense mechanisms, and gene regulation (Grant (1999) Cell 96: 303-306).

[0084] It has been shown that dsRNA is cleaved by a nuclease into 21 -23- nucleotide fragments. These fragments, in turn, target the homologous region of their corresponding mRNA, hybridize, and result in a double-stranded substrate for a nuclease that degrades it into fragments of the same size (Hammond et al. (2000) Nature 404:293-298; Zamore et al. (2000) Cell 101 :25-33). Although typically employed to target coding RNA (mRNA), this strategy is equally applicable to non- coding RNA.

[0085] dsRNA can be formulated and administered to cells, tissues, or organisms in accordance with standard practice. In certain embodiments, dsRNA can be synthesized using one or more vectors designed to transcribe the two

complementary RNA strands that hybridize to form the dsRNA. These may be introduced into host cells using any of the techniques known in the art for this purpose. [0086] In an illustrative embodiment, RNAi can be used to inhibit production of of the 1L-4 receptor alpha chain (IL-4oc) protein necessary for the biological activities of IL-4 and IL-13.

Antibody Antagonists

[0087] Antibody antagonists can also be employed in the methods of the invention. For example, an antibody that targets the IL-4 receptor alpha chain (IL-4a) can interfere with its function sufficiently to inhibit the IL-4/IL-13 signaling pathways. Amgen's AMG 3 17 is an IL-4R antagonist of this type. AMG-317 and related antibodies are disclosed in U.S. Ser. No. 05/01 1,8176, U.S. Ser. No.

05/01 1 ,2694 and in Clinical Trials Gov. Identifier: NCT00436670.

IL-4 Mutein Receptor Antagonists

[0088] The methods of the invention can also employ hIL-4 muteins that antagonize human IL-4 and/or the human IL-13 by interfering with the binding of these two interleukins to the type 1 and type 2 IL-4R. Antagonists to IL-4 have been reported in the literature. Mutants of IL-4 that function as antagonists include the IL- 4 antagonist mutein IL-4/Y124D (Kruse, N., Tony, H. P., Sebald, W., Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid

replacement, Embo J. 1 1 :3237-44, 1992) and a double mutein IL-4[R121D Y124D] (Tony, H., et al., Design of Human Interleukin-4 Antagonists in Inhibiting

Interleukin-4-dependent and Interleukin-13-dependent responses in T-cells and B- cells with high efficiency, Eur. J. Biochem. 225:659-664 (1994)). The single mutein is a substitution of tyrosine by aspartic acid at position 124 in the D-helix. The double mutein is a substitution of arginine by aspartic cid at position 121 , and of tyrosine by aspartic acid at position 124 in the D-helix, as disclosed in U.S. Pat. Nos. 6,313,272 and 6,028,176, incorporated herein by reference. Variations in this section of the D helix positively correlate with changes in interactions at the second binding region of the IL-4RA chain. The hIL-4 muteins used in certain embodiments of the methods described herein include replacement(s) at positions 121 , 124, and/or 125 and may include other modifications. In illustrative embodiments, a mutant human IL-4 protein of the invention includes the amino acid sequence of wild-type hIL-4 with modifications, wherein a first modification is replacement of one or more of the amino acids occurring in the wild-type hIL-4 protein at positions 121 , 124 or 125 with another natural amino acid, and further optionally comprising an N-terminal methionine. In specific embodiments, the mutant protein includes a first modification of the protein that includes substitutions R121D and Y124D (IL-4RA), numbered in accordance with the wild-type hIL-4.

[0089] As provided herein, further modification of the mutein may include one or more of the following: the N terminus and/or C terminus of the molecule being modified, glycosylation sites which are present in the molecule being partially or completely deleted, the coupling of the protein to a non-protein polymer, such as polyethylene glycol, and/or at least one amino acid substitution selected from the group consisting of substitutions at positions 13, 16, 81, and 89. In certain embodiments, such modifications can be carried out in order to increase the stability of the hIL-4 muteins, in order to extend the biological half life, or in order to facilitate the preparation and purification process.

[0090] In one embodiment, the mutein has an N-terminus modification that is an insertion of an amino acid, at amino acid position +2. In another embodiment, the mutein has a C-terminus modification that is a deletion of at least one, at least two, at least three, at least four and at least five amino acids. However, deletions of greater than five amino acids from the C-terminus may affect the activity of the mutein.

Activity of the mutein from any of the modifications mentioned above and herein can be determined by using any of the methods described previously in related applications and/or patents, and methods described herein, e.g., the Bimolecular Interaction Analysis (BIA) and proliferative assays as described in U.S. Patent No. 7,404,957, which is incorporated herein by reference for this disclosure.

[0091] Muteins useful in the methods of the invention may further include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of the parent polypeptide. In certain embodiments, muteins comprise a greater or a lesser number of N-linked

glycosylation sites than the native protein. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created.

[0092] Additional muteins include cysteine variants wherein one or more cysteine residues are added, deleted, or substituted for another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine variants may be useful when proteins must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. In one embodiment, cysteine variants will have fewer cysteine residues than the native protein, and an even number of cysteines to minimize interactions resulting from unpaired cysteines. In another embodiment, the cysteine variants will permit site-specific coupling of at least one non-protein polymer, such as a polyethylene glycol (PEG) molecule, to the mutein.

[0093] Muteins useful in the invention can include additional amino acid substitutions that enable the site-specific coupling of at least one non-protein polymer, such as polypropylene glycol, polyoxyalkylene, or polyethylene glycol (PEG) molecule to the mutein. Site-specific coupling of PEG, for example, allows the generation of a modified mutein which possesses the benefits of a polyethylene- glycosylated (PEGylated) molecule, namely increased plasma half life (e.g., at least 2 to 10-fold greater, or 10 to 100-fold greater than that of unmodified IL-4RA) while maintaining greater potency over non-specific PEGylation strategies such as N- terminal and lysine side-chain PEGylation. Methods providing for efficient

PEGylation are described in U.S. Patent No. 7,404,957 which is incorporated herein by reference. The IL-4 mutein must be purified properly to allow efficient

PEGylation. Purification is also described in this patent.

[0094] In one embodiment, the mutein is coupled to a non-protein polymer at any of various amino acid residues, in particular, at positions 28, 36, 37, 38, 102, 104, 105 or 106. The amino acid positions are numbered according to the wild type IL-

4 (i.e. human interleukin-4) amino acid sequence (see U.S. Patent No. 5,017,691 which is incorporated herein by reference). Non-protein polymers include, for example polyethylene glycol, polypropylene glycol or polyoxyalkylenes, as described in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301 , 144; 4,670,417; 4,791 , 192 or

4, 179,337, the entire contents of which are incorporated herein by reference.

[0095] In particular embodiments, specific sites of amino acid substitution of hIL-4 are selected to ensure proper folding of the polypeptide following expression. In specific embodiments, IL-4 mutein receptor antagonists that have been modified, e.g., by PEGylation, bind to IL-4 and IL-13 receptors with an affinity loss not greater than 100-fold relative to that of unmodified IL-4RA. In specific embodiments, modified IL-4 mutein receptor antagonists inhibit IL-4- and IL- 13 -mediated activity with a loss of potency not greater than 10-fold relative to that of unmodified IL-4RA. In addition, in specific embodiments, modified IL-4 mutein receptor antagonists possess a plasma half-life which is at least 2 to 10-fold greater than that of unmodified IL-4RA.

[0096] In particular embodiments, a modified IL-4 mutein receptor antagonist can be employed in the treatment method described herein. "Modified IL-4 mutein receptor antagonists," as used herein, includes the IL-4RA mutein described in US Pat. Nos. 6,028, 176; 6,313,272; and 7,785,580 (hereby incorporated by reference in their entirety), with additional amino acid substitutions at one or more positions of the mature IL-4 protein. Exemplary triple muteins include, but are not limited to a substitution of arginine by aspartic acid at position 121 , of tyrosine by aspartic acid at position 124, and of serine by aspartic acid at position 125 in the D-helix; and a substitution of threonine by aspartic acid at position 13, of arginine by aspartic acid at position 121 , and of tyrosine by aspartic acid at position 124 in the D-helix. In one embodiment, the triple muteins further comprise an N-terminal methionine.

Variations in this section of the D helix positively correlate with changes in interactions at the second binding region. The modified IL-4 mutein receptor antagonist may further include one or more substitutions wherein said substitutions enable the site-specific coupling of at least one non-protein polymer, such as polypropylene glycol, polyoxyalkylene, or polyethylene glycol (PEG) molecule to the mutein. Site-specific coupling of PEG, for example, allows the generation of a modified mutein which possesses the benefits of a polyethylene-glycosylated

(PEGylated) molecule, namely increased plasma half life and decreased immunogenicity while maintaining greater potency over non-specific PEGylation strategies such as N-terminal and lysine side-chain PEGylation.

[0097] A number of modified IL-4 mutein receptor antagonists with the characteristics described above have been identified and are described in U.S. Patent No. 7,785,580, issued August 31, 2010 to Pan et al., which is hereby incorporated by reference in its entirety and for its description of modified IL-4 mutein receptor antagonists and their production. Illustrative antagonists described therein have the polypeptide sequences shown in Table 1 (SEQ ID NOS: 1 -10).

[0098] Polynucleotides that encode the illustrative modified IL-4 mutein receptor antagonists of Table 1 are shown in Table 2 (SEQ ID NOs: l 1 -19).

[0099] A specific, illustrative modified IL-4 mutein receptor antagonist includes the following:

(1 ) substitution of each of the amino acids occurring in the wild-type human IL-4 protein at positions 121 and 124 with different amino acids;

(2) substitution of the threonine occurring in the wild-type human IL 4 protein at position 13 with a different amino acid;

(3) substitution of the asparagine occurring in the wild-type human IL 4 protein at position 38 with a cysteine; and

(4) a non-protein polymer covalently attached to the substituted cysteine at position 38. For example, the modified IL-4 mutein receptor antagonist can have a substitution of threonine by aspartic acid at position 13, a substitution of cysteine by an asparagine at position 38, a substitution of arginine by aspartic acid at position 121 , a substitution of tyrosine by aspartic acid at position 124, and polyethylene glycol attached to the substituted cysteine at position 38.

[0100] Furthermore, one of skill in the art will be able to determine suitable additional modifications using well-known techniques. As such, the skilled artisan may identify ( 1) suitable areas of the polypeptide that may be changed without destroying activity by targeting regions not believed to be important for activity (see Kreitman et al. (1994) Biochemistry 33: 1 1637-1 1644, incorporated herein by reference); (2) residues and portions of the polypeptides that are conserved among similar polypeptides; and (3) areas that may be important for biological activity or for structure that can still be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0101] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of a polypeptide with respect to its three dimensional structure. In certain embodiments, one skilled in the art may choose to not make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change can be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

[0102] A number of scientific publications have been devoted to the prediction of secondary structure. See Moult, 1996, Curr. Op. in Biotech. Ί-Λ22 Π; Chou et al, 1974, Biochemistry 13:222-245; Chou et al, 1974, Biochemistry 1 13:21 1- 222; Chou et al, 1978, Adv. Enzymol Relat. Areas Mol. Biol 47:45-148; Chou et al, 1979, Ann. Rev. Biochem. 47:251 -276; and Chou et al, 1979, Biophys. J. 26:367-384. Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins that have a sequence identity of greater than about 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural database has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See Holm et al, 1999, Nucl. Acid. Res. 27:244-247. It has been suggested (Brenner et al, 1997, Curr. Op. Struct. Biol. 7:369-376) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of 5 structures have been resolved, structural prediction will become dramatically more accurate.

[0103] Further modificatioins include, but are not limited to, mutations such as substitutions, additions, deletions, or any combination thereof, and are typically produced by site-directed mutagenesis using one or more mutagenic

oligonucleotide(s) according to methods known in the art (see, for example,

Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 3rd Ed, 2001 , Cold Spring Harbor, N.Y. and Berger and immel, METHODS IN

ENZYMOLOG Y, Volume 152, Guide to Molecular Cloning Techniques, 1987,

Academic Press, Inc., San Diego, CA, which are incorporated herein by reference).

[0104] Amino acid substitutions can include those substitutions that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (5) confer or modify other physicochemical or functional properties on such

polypeptides. According to certain embodiments, single or multiple amino acid substitutions (and in some cases, conservative amino acid substitutions) may be made in the naturally occurring sequence (e.g., in the portion of the polypeptide outside the domain(s) forming intermolecular contacts).

[0105] A conservative amino acid substitution typically does not substantially change the structural characteristics of the nucleotide sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the nucleotide sequence, or disrupt other types of secondary structure that characterizes the nucleotide sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in PROTEINS, STRUCTURES AND MOLECULAR PRINCIPLES, (Creighton, Ed.), 1984, W. H. Freeman and Company, New York; INTRODUCTION TO PROTEIN STRUCTURE (C. Branden and J. Tooze, eds.), 1991, Garland Publishing, New York, N.Y.; and Thornton et al., 1991, Nature 354: 105, each of which are incorporated herein by reference.

[0106] Peptide analogs, e.g., "peptide mimetics" or "peptidomimetics," analogous to those of the IL-14 mutein receptor antagonists described above can be employed in the methods described herein. Suitable peptide analogs can be developed, for example, with the aid of computerized molecular modeling. In certain embodiments, suitable peptide analogs have one or more peptide linkages optionally replaced by a linkage selected from: -CH2-NH-, -CH2-S-, -CH2-CH2-, -CH=CH-(cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used in certain embodiments to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo & Gierasch, 1992, Ann. Rev. Biochem. 61 :387, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

[0107] A number of IL-4 mutein receptor antagonists with the characteristics described above have been identified in U.S. Patent No. 7,404,957, by screening candidates with the above assays. In one embodiment, a non-protein polymer (e.g., polyethylene glycol) is coupled to at least amino acid residue positions 38, 102, and/or 104.

[0108] The above polypeptide variants are illustrative of the types of human IL-4 polypeptides to be used in the methods claimed herein, but are not exhaustive of the types of variations of the claimed invention which may be embodied by the invention. Derivatives of the above polypeptide which fit the criteria of the claims should also be considered. All of the polypeptides and functional fragments thereof can be screened for efficacy following the methods taught herein and in the examples.

Production of IL-4 Mutein Receptor Antagonists

[0109] Modified IL-4 mutein receptor antagonists can be produced using any method capable of producing polypeptides having the desired amino acid sequence. Typically, recombinant expression will be the most convenient method.

Polynucleotides Encoding Modified IL-4 Mutein Receptor

Antagonists

[0110] Recombinant expression requires polynucleotides encoding IL-4 mutein receptor antagonists. These polynucleotides can be used, for example, to produce quantities of the antagonists for therapeutic use. Methods of constructing and expressing degenerative DNA sequences capable of expressing the same amino acid sequence as a given polynucleotide sequence are known in the art.

[0111] A polynucleotide of the invention can be readily obtained in a variety of ways including, without limitation, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA. Such methods are well known and include those set forth in Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) and/or Current Protocols in Molecular Biology (Ausubel et al, eds., Green Publishers Inc. and Wiley and Sons 1994).

[0112] Polynucleotides of the invention present in a host cell can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be isolated from cells using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated polynucleotides encoding antagonists of the invention. For example, restriction enzymes and probes can be used to isolate polynucleotides which encode the antagonists. Preferably, isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.

[0113] One method for obtaining a suitable nucleic acid sequence is the polymerase chain reaction (PCR). In this method, cDNA is prepared from

poly(A)+RNA or total R A using the enzyme reverse transcriptase. Two primers, typically complementary to two separate regions of a IL-4 mutein receptor antagonist cDNA, are then added to the cDNA along with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the two primers.

[0114] Another means of preparing a nucleic acid molecule of the invention is chemical synthesis using methods well known to the skilled artisan such as those described by Engels et al, 1989, Angew. Chem. Intl. Ed. 28:716-34. These methods include, inter alia, the phosphotriester, phosphoramidite, and H-phosphonate methods for nucleic acid synthesis. A preferred method for such chemical synthesis is polymer-supported synthesis using standard phosphoramidite chemistry. Typically, the DNA will be several hundred nucleotides in length. Nucleic acids larger than about 100 nucleotides can be synthesized as several fragments using these methods. The fragments can then be ligated together.

Vectors

[0115] A polynucleotide can be incorporated into a vector for propagation and/or expression in a host cell. Such vectors typically contain a replication sequence capable of effecting replication of the vector in a suitable host cell (i.e., an origin of replication) as well as sequences encoding a selectable marker, such as an antibiotic resistance gene. Upon transformation of a suitable host, the vector can replicate and function independently of the host genome or integrate into the host genome. Vector design depends, among other things, on the intended use and host cell for the vector, and the design of a vector of the invention for a particular use and host cell is within the level of skill in the art.

[0116] If the vector is intended for expression of a polypeptide, the vector includes one or more control sequences capable of effecting and/or enhancing the expression of an operably linked polypeptide coding sequence. Control sequences that are suitable for expression in prokaryotes, for example, include a promoter sequence, an operator sequence, and a ribosome binding site. Control sequences for expression in eukaryotic cells include a promoter, an enhancer, and a transcription termination sequence (i.e., a polyadenylation signal).

[0117] An expression vector according to the invention can also include other sequences, such as, for example, nucleic acid sequences encoding a signal sequence or an amplifiable gene. A signal sequence can direct the secretion of a polypeptide fused thereto from a cell expressing the protein. In the expression vector, nucleic acid encoding a signal sequence is linked to a polypeptide coding sequence so as to preserve the reading frame of the polypeptide coding sequence. The nucleic acid sequence encoding a human IL-4 mutein may or may not be linked to a nucleic acid sequence that encodes a signal sequence. Such signal sequence, if present, should be one recognized by the cell chosen for expression of the IL-4 mutein. It may be prokaryotic, eukaryotic, or a combination of the two. It may also be the signal sequence of native IL-4. The inclusion of a signal sequence depends on whether it is desired to secrete the IL-4 mutein from the recombinant cells in which it is made. If the chosen cells are prokaryotic, it generally is preferred that the nucleic acid sequence not encode a signal sequence but include an N-terminal methionine to direct expression. If the chosen cells are eukaryotic, it generally is preferred that a signal sequence be encoded and most preferably that the wild-type IL-4 signal sequence be used, as disclosed in U.S. Pat. No. 6,028,176, incorporated herein by reference for this disclosure. The inclusion in a vector of a gene complementing an auxotrophic deficiency in the chosen host cell allows for the selection of host cells transformed with the vector.

[0118] Vectors are typically produced by linking desired elements by ligation at convenient restriction sites. If such sites do not exist, suitable sites can be introduced by standard mutagenesis (e.g., site-directed or cassette mutagenesis) or synthetic oligonucleotide adaptors or linkers can be used in accordance with conventional practice.

Host Cells

[0119] A wide variety of host cells are available for propagation and/or expression of vectors. Examples include prokaryotic cells (such as E. coli and strains of Bacillus, Pseudomonas, and other bacteria), yeast or other fungal cells (including S. cerevesiae and P. pastoris), insect cells, plant cells, and phage, as well as higher eukaryotic cells (such as human embryonic kidney cells and other mammalian cells).

[0120] A vector can be introduced into a host cell by any convenient method, which will vary depending on the vector-host system employed. Generally, a vector is introduced into a host cell by transformation (also known as "transfection") or infection with a virus (e.g., phage) bearing the vector. If the host cell is a prokaryotic cell (or other cell having a cell wall), convenient transformation methods include the calcium treatment method described by Cohen, et al. (1972) Proc. Natl. Acad. Sci., USA, 69:21 10-14. If a prokaryotic cell is used as the host and the vector is a phagemid vector, the vector can be introduced into the host cell by infection. Yeast cells can be transformed using polyethylene glycol, for example, as taught by Hinnen (1978) Proc. Natl. Acad. Sci, USA, 75: 1929-33. Mammalian cells are conveniently transformed using the calcium phosphate precipitation method described by Graham, et al. (1978) Virology, 52:546 and by Gorman, et al. (1990) DNA and Prot. Eng.

Tech., 2:3-10. However, other known methods for introducing DNA into host cells, such as nuclear injection, electroporation, and protoplast fusion also are acceptable for use in the invention.

Recombinant Production Methods

[0121] Host cells transformed with expression vectors can be used to express the polypeptides encoded by the polynucleotides of the invention. Expression entails culturing the host cells under conditions suitable for cell growth and expression and recovering the expressed polypeptides from a cell lysate or, if the polypeptides are secreted, from the culture medium. In particular, the culture medium contains appropriate nutrients and growth factors for the host cell employed. The nutrients and growth factors are, in many cases, well known or can be readily determined empirically by those skilled in the art. Suitable culture conditions for mammalian host cells, for instance, are described in Mammalian Cell Culture (Mather ed., Plenum Press 1984) and in Barnes and Sato (1980) Cell 22:649.

[0122] In addition, the culture conditions should allow transcription, translation, and protein transport between cellular compartments. Factors that affect these processes are well-known and include, for example, DNA/RNA copy number; factors that stabilize DNA; nutrients, supplements, and transcriptional inducers or repressors present in the culture medium; temperature, pH and osmolality of the culture; and cell density. The adjustment of these factors to promote expression in a particular vector-host cell system is within the level of skill in the art. Principles and practical techniques for maximizing the productivity of in vitro mammalian cell cultures, for example, can be found in Mammalian Cell Biotechnology: a Practical Approach (Butler ed., IRL Press (1991).

[0123] Any of a number of well-known techniques for large- or small-scale production of proteins can be employed in expressing the polypeptides of the invention. These include, but are not limited to, the use of a shaken flask, a fluidized bed bioreactor, a roller bottle culture system, and a stirred tank bioreactor system. Cell culture can be carried out in a batch, fed-batch, or continuous mode.

[0124] Methods for recovery of recombinant proteins produced as described above are well-known and vary depending on the expression system employed. A polypeptide including a signal sequence can be recovered from the culture medium or the periplasm. Polypeptides can also be expressed intracellularly and recovered from cell lysates.

[0125] The expressed polypeptides can be purified from culture medium or a cell lysate by any method capable of separating the polypeptide from one or more components of the host cell or culture medium. Typically, the polypeptide is separated from host cell and/or culture medium components that would interfere with the intended use of the polypeptide. As a first step, the culture medium or cell lysate can be centrifuged or filtered to remove cellular debris. The supernatant is then typically concentrated or diluted to a desired volume or diafiltered into a suitable buffer to condition the preparation for further purification. The polypeptide can then be further purified using well-known techniques. The technique chosen will vary depending on the properties of the expressed polypeptide.

[0126] In certain embodiments, an additional aspect of this invention is provided in the method with which the protein is expressed and refolded. The IL-4 mutein is preferably purified so as to allow efficient PEGylation. When the mutein is refolded in the presence of a sulfhydryl protecting agent, a covalent disulfide bond is formed between the IL-4 mutein's free cysteine and the protecting agent. In contrast, the use of the sulfhydryl protecting agent dithiothreitol (DTT), which oxidizes to form a stable disulfide bond, will not form a covalent bond with the IL-4 mutein's free cysteine, thus leaving its sulfydryl group free to react with the PEG maleimide reagent. IL-4 muteins purified after refolding in the presence of a sulfhydryl protecting agent can react with the PEG reagent if treated with DTT, but a mixture of monoPEGylated and multiPEGylated products are generated, suggesting that existing IL-4 cysteines are also PEGylated. PEGylation of existing cysteines would lead to misfolded products that are inactive.

Confirmation of Therapeutic Utility of Modified IL-4 Mutein Receptor Antagonists

[0127] To assess the therapeutic utility of a particular IL-4 mutein receptor antagonists in allergic therapy, e.g., treatment of an eosinophilic disorder, the antagonist can be tested in vitro, e.g., in receptor binding and cell proliferation assays.

[0128] The K _d IL-4 mutein receptor antagonists to the IL-4 receptor can be assayed using any method known in the art, including technologies such as real-time Bimolecular Interaction Analysis (BIA), as described in U.S. Patent No. 7,404,957. BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

[0129] Data suggests that IL-4RA binds to the IL-4 receptor a chain with similar on and off rates as wtIL-4, which inhibits assembly of either yc (type 1 ) or IL-13Rα (type 2) into receptor complexes that signal downstream events (see Table 3). Thus, IL-4RA blocks recruitment of either IL-13Rα1 or yc to form a stable heterodimeric complex with the IL-4 receptor a chain (A.L. Andrews et al., ATS 2004).

[0130] In a BIAcore™ assay, IL-4 mutein receptor antagonists of the present invention specifically bind to the human IL-4 receptor with a preferred K _d in the range of from about 0.1 nM to about 10 μΜ. Other embodiments of the present invention bind to human IL-4 receptor with a K _d of approximately 0.5 nM to about 1.0 μΜ. Still other embodiments of the present invention bind to human IL-4 receptor with a K _d of approximately 1.0 nM to about 100 nM.

[0131] The capacity of IL-4 mutein receptor antagonists to inhibit the proliferative response of immune cells can be assessed using proliferative assays, as described in U.S. Patent No. 7,404,957, and this capacity expressed as an Inhibitory Concentration 50% (IC ₅₀ ). IL-4 mutein receptor antagonists of the present invention, as envisioned, will bind to human IL-4 receptor and neutralize its capacity to promote immune cell proliferation with a IC50 ranging from about 0.1 nM to about 10 μΜ. Other human antagonists bind IL-4 receptor and neutralize its immune cell proliferation capacity with an IC50 ranging from approximately 0.5 nM to 1 μΜ, with still other antagonists of this invention binding and inhibiting IL-4 receptor with an IC50 of approximately 1.0 nM to about 100 nM.

Co-administration of IL-4 Mutein Receptor Antagonists with Additional Agents

[0132] In a particular embodiment of the method, IL-4 mutein receptor antagonist is co-administered with an additional agent that is useful for mitigating a symptom of an eosinophilic disorder. In this embodiment, the amount of additional agent administered is sufficient to produce a beneficial effect (e.g., mitigation of an eosinophilic disorder) in the subject when co-administered with the selected IL-4 mutein receptor antagonist. [0133] Any additional agent that mitigates a symptom of the eosinophilic disorder being treated (e.g., eosinophilic asthma or allergic dermatitis, such as eczema) and is tolerated by the subject can be employed in the method of the invention. The additional agent can be one that acts by the same, or a different, mechanism than the IL-4 mutein receptor antagonist with which it is co-administered. Examples of additional agents suitable for use in this embodiment include steroids, such as inhaled corticosteroids, for treatment of asthma, and corticosteroid creams, for treatment of allergic dermatitis.

[0134] Inhaled corticosteroids (ICSs) that are used to treat asthma include, e.g, beclomethasone, budesonide, ciclesonide, flunisolide, fluticasone, mometasone, and triamcinolone. Also used alone or in combination with ICSs are bronchodilators, such as short-acting beta agonists, long-acting beta agonists (LABAs), and anticholinergics. Exemplary bronchodilators include pirbuterol (MAXAIR), epinephrine (PRIMATENE), salbutamol, also called "albuterol" (PROVENTIL and VENTOLIN), salmeterol (SEREVENT), levosalbutamol, also called "levalbuterol" (XOPENEX), clenbuterol (SPIROPENT), and formoterol. Exemplary ICS LABA combinations include budesonide and formoterol (SYMBICORT) and fluticasone and salmeterol (ADVAIR). The IL-4 mutein receptor antagonists described herein can be coadministered with any of these or similar agents. It is also contemplated that, in certain embodiments, a subject may be withdrawn from such drug after the subject begins to respond to treatment with an IL-4 mutein receptor antagonist.

[0135] Corticosteroid creams are sometimes prescribed to decrease the inflammatory reaction in the skin. These may be mild-, medium-, or high-potency corticosteroid creams depending upon the severity of the symptoms. If itching is severe, oral antihistamines may be prescribed. To control itching, the sedative type antihistamine drugs, such as, e.g., diphenhydramine (Benadryl), hydroxyzine (Atarax, Vistaril), and cyproheptadine) can be helpful. In some cases, a short course of oral corticosteroids (such as prednisone) is prescribed to control an acute outbreak of eczema, although their long-term use is discouraged in the treatment of this non life- threatening condition because of unpleasant and potentially harmful side effects. The oral immunosuppressant drug cyclosporine has also been used to treat some cases of eczema. Ultraviolet light therapy (phototherapy) is another treatment option. In addition, two topical (cream) medications have been approved by the U.S. FDA for the treatment of eczema: tacrolimus (PROTOPIC) and pimecrolimus (ELIDEL).

Pharmaceutical Compositions

[0136] The active agents described are typically combined with a

pharmaceutically acceptable carrier (excipient), such as are described in Remington's Pharmaceutical Sciences (1980) 16th editions, Osol, ed., 1980. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). A pharmaceutically acceptable carrier suitable for use in the invention is non-toxic to cells, tissues, or subjects at the dosages employed, and can include a buffer (such as a phosphate buffer, citrate buffer, and buffers made from other organic acids), an antioxidant (e.g., ascorbic acid), a low-molecular weight (less than about 10 residues) peptide, a polypeptide (such as serum albumin, gelatin, and an immunoglobulin), a hydrophilic polymer (such as polyvinylpyrrolidone), an amino acid (such as glycine, glutamine, asparagine, arginine, and/or lysine), a monosaccharide, a disaccharide, and/or other carbohydrates (including glucose, mannose, and dextrins), a chelating agent (e.g., ethylenediaminetetratacetic acid

[EDTA]), a sugar alcohol (such as mannitol and sorbitol), a salt-forming counterion (e.g., sodium), and/or an anionic surfactant (such as Tween™, Pluronics™, and PEG). In one embodiment, the pharmaceutically acceptable carrier is an aqueous pH- buffered solution.

[0137] Other pharmaceutically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would appreciate that the choice of pharmaceutically acceptable carrier(s), including a physiologically acceptable compound depends, for example, on the route of administration of the active agent(s) and on the particular physio-chemical characteristics of the active agent(s).

[0138] Pharmaceutical compositions of the invention can be stored in any standard form, including, e.g., an aqueous solution or a lyophilized cake. Such compositions are typically sterile when administered to subjects. Sterilization of an aqueous solution is readily accomplished by filtration through a sterile filtration membrane. If the composition is stored in lyophilized form, the composition can be filtered before or after lyophilization and reconstitution. Dry Powder Aerosol Formulation

[0139] According to embodiments of the invention, there are provided pharmaceutical dispersible dry powder compositions exhibiting good temperature and structural stability, including resistance to moisture and aggregation. The

compositions include a therapeutic agent comprising a human interleukin-4 mutein (mhIL-4) receptor antagonist, as described above.

[0140] In some embodiments, any compositions described above, in addition to mhIL-4 described above may further include a buffer, such as a citrate, an acetate, a lactate, a tartarate, a succinate, or a maleate, and a stabilizing agent, such as a carbohydrate, e.g., sucrose, mannitol, or trehalose, or a magnesium salt, e.g., magnesium sulfate. In some embodiments, any composition, whether it does or does not include a buffer and/or a stabilizing agent, may further comprise, in addition to mhIL-4 described above, an excipient selected from a group consisting of an amino acid, e.g., leucine, or a poly(amino acid).

[0141] A variety of embodiments can generally characterize and illustrate the features of the instant invention. In one embodiment, there is provided a dispersible powder composition suitable for inhalation by a patient in need thereof, the composition including a therapeutic agent comprising a human interleukin-4 mutein (mhIL-4) receptor antagonist, as described above, wherein a glass transition temperature of the composition is at least 50°C higher than a storage temperature at which the composition is stored, and wherein the composition retains at least 80 % of the original specific activity after the composition is stored at the storage temperature over a period of three months.

[0142] Compositions of any embodiment discussed above can have a glass transition temperature of at least 75°C higher than the storage temperature (i.e., the temperature at which the composition is stored), which may be room temperature or below, e.g., between about 2°C and 8°C or, alternatively, a storage temperature between about 2°C and 8°C during a first portion of the storage period and room temperature during a second portion of the storage period). For example, the glass transition temperature can be at least 100°C higher than the storage temperature. After the total storage period of at least two years, compositions of any embodiment discussed above can retain at least 95 % of the original specific activity after the expiration of a total storage period, for example retaining at least 98 % of the original specific activity.

[0143] The mass concentration of the therapeutically active material in such compositions can be between about 10 % and about 98 %, such as between about 10 % and about 75 %, for example, between about 10 % and about 60 %.

[0144] Compositions of any embodiment discussed above can have a moisture content between about 1 % and about 10 %, such as between about 1 % and about 5 %, for example, between about 1 % and about 3 %.

[0145] Compositions of any embodiment discussed above can have a degree of aggregation of about 3 % or less after the expiration of a total storage period of at least two years, such as about 1 % or less, or about 0 %.

[0146] Compositions of any embodiment discussed above have the degree of oxidation, relative to the drug substance, of the therapeutically active material after the expiration of a total storage period of about 5 % or less, wherein the total storage period is at least two years. For example, such degree of oxidation may be about 3 % or less, or about 2 %.

[0147] Compositions of any embodiment discussed above can be a powder formed by particles having the mean diameter of less than about 10 μπι, for example between about 2 μιη and about 6 μηη, such as between about 2 μιη and about 4 μτη.

[0148] Compositions of any embodiment discussed above can include particles having the geometric standard deviation in particle size of between about 1 and 3, for example, between about 1.5 and about 2.5.

[0149] Compositions of any embodiment discussed above can provide an emitted dose, when inhaled by the patient, that is about 70 mass % or higher, such as about 80 mass % or higher, for example, about 90 mass % or higher. [0150] Compositions of any embodiment discussed above can provide, when inhaled by a patient, a deposited fraction of the particles having a diameter not exceeding about 5 μπι that is between about 25 and about 60 mass %, such as between about 40 and about 60 mass %, for example, between about 50 and about 60 mass %.

[0151] Compositions of any embodiment discussed above have the pH value that is between about 3 and 6, such as between about 4 and 5.

[0152] Compositions of any embodiment discussed above have a nominal dose of the active substance between about 0.3 and 30 mg, for example, between about 1 and 20 mg, such as between about 3 and 15 mg.

[0153] Compositions of any embodiment discussed above may be prepared by freeze drying, spray drying, and freeze spray drying, and may further optionally include milling or lyophilization with milling. In one preferred procedure, a solution is prepared having a mass concentration of solids between about 1 and 5% solids.

[0154] The solution is then directed through a nozzle that is set at a specific pressure and temperature to create droplets. The droplets enter a chamber established at a specified temperature to dry. The dry particles of a specific size range are collected in a cyclone. This powder is then filled into a primary container at a specified fill weight. The primary container can be any suitable container that provides for storage at a specified fill weight and provides for release of the material contained therein into an inhaler. A particularly preferred embodiment of the primary storage container is a capsule which can be punctured or broken after it has been inserted into an inhaler. Those having ordinary skill in the art can determine the pressure and temperature to be used to form the droplets as well as the drying temperature.

[0155] Further details of dry powder aerosol formulations useful in the methods described herein can be found in International Publication No.

WO/2009/009775 (published Jan. 15, 2009), which is incorporated by reference herein in its entirety and specifically for its description of such formulations. Administration

[0156] The active agents identified herein are useful for parenteral (e.g., subcutaneously, intravenously, intra-arterially, intramuscularly, intraperitoneal ly, intradermally), nasal (or otherwise inhaled), oral, sublingual, rectal, topical, or local administration, such as by aerosol (e.g., nebulization, dry powder or metered dose inhalation), or transdermally, for prophylactic and/or therapeutic treatment of one or more of the pathologies/indications described herein (e.g., to mitigate one or more symptoms of an allergic response, such as an eosinophilic disorder).

[0157] In illustrative embodiments, a IL-4 mutein receptor antagonist can be administered parenterally (e.g., subcutaneously) no more than about twice per week, once per week, every two weeks, every three weeks, once per month, or once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 months. For example, about 10 mg/kg can be administered no more than about once per week; about 20 mg kg can be administered no more than about every two weeks; and about 40 mg/kg can be administered no more than about once per month.

[0158] In various embodiments, the active agents described herein can be administered orally, in which case delivery can be enhanced by the use of protective excipients. This is typically accomplished either by complexing the active agent(s) with a composition to render them resistant to acidic and enzymatic hydrolysis or by packaging the agents in an appropriately resistant carrier, e.g. a liposome. Means of protecting agents for oral delivery are well known in the art (see, e.g., U.S. Patent No. 5,391,377).

[0159] Elevated serum half-life can be maintained by the use of sustained- release "packaging" systems. Such sustained release systems are well known to those of skill in the art (see, e.g., Tracy ( 1998) Biotechnol. Prog. 14: 108; Johnson et al. (1996), Nature Med. 2: 795 ; Herbert et al. (1998), Pharmaceut. Res. 15, 357).

[0160] The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectibles, implantable sustained-release formulations, lipid complexes, etc. In another embodiment, one or more components of a solution can be provided as a "concentrate," e.g., in a storage container (e.g., in a premeasured volume) ready for dilution or in a soluble capsule ready for addition to a volume of water.

[0161] In certain embodiments, one or more active agents described herein are administered alone or in combination with other therapeutics in implantable (e.g., subcutaneous) matrices, termed "depot formulations."

[0162] In particular embodiments, one or more drugs (e.g., the active agents described herein) are embedded in various matrix materials for sustained release. Drugs embedded, for example, in polymer beads or in polymer wafers have several advantages. First, most systems allow slow release of the drug, thus creating a continuous dosing of the body with small levels of drug. This typically prevents side effects associated with high burst levels of normal injected or pill-based drugs.

Secondly, since these polymers can be made to release over hours to months, the therapeutic span of the drug is markedly increased. Often, by mixing different ratios of the same polymer components, polymers of different degradation rates can be made, allowing remarkable flexibility depending on the agent being used. A long rate of drug release is beneficial for people who might have trouble staying on regular dosage, such as the elderly, but also represents an ease of use improvement that everyone can appreciate. Most polymers can be made to degrade and be cleared by the body over time, so they will not remain in the body after the therapeutic interval.

[0163] Another advantage of polymer-based drug delivery is that the polymers often can stabilize or solubilize proteins, peptides, and other large molecules that would otherwise be unusable as medications. Finally, many drug/polymer mixes can be placed directly in the disease area, allowing specific targeting of the medication where it is needed without losing drug to the "first pass" effect.

[0164] A wide variety of approaches to designing depot formulations that provide sustained release of an active agent are known and are suitable for use in the invention. Generally, the components of such formulations are biocompatible and may be biodegradable. Biocompatible polymeric materials have been used extensively in therapeutic drug delivery and medical implant applications to effect a localized and sustained release. See Leong et al., "Polymeric Controlled Drug Delivery," Advanced Drug Delivery Rev., 1 : 199-233 (1987); Langer, "New Methods of Drug Delivery," Science, 249: 1527-33 (1990); Chien et al., Novel Drug Delivery Systems (1982). Such delivery systems offer the potential of enhanced therapeutic efficacy and reduced overall toxicity.

[0165] Examples of classes of synthetic polymers that have been studied as possible solid biodegradable materials include polyesters (Pitt et al., "Biodegradable Drug Delivery Systems Based on Aliphatic Polyesters: Applications to Contraceptives and Narcotic Antagonists," Controlled Release of Bioactive Materials, 19-44 (Richard Baker ed., 1980); poly(amino acids) and pseudo-poly(amino acids) (Pulapura et al. "Trends in the Development of Bioresorbable Polymers for Medical Applications," J. Biomaterials Appl., 6: 1 , 216-50 (1992); polyurethanes (Bruin et al., "Biodegradable Lysine Diisocyanate-based Poly(Glycolide-co-.epsilon. Caprolactone)-Urethane Network in Artificial Skin," Biomaterials, 1 1 :4, 291 -95 (1990); polyorthoesters (Heller et al., "Release of Norethindrone from Poly(Ortho Esters)," Polymer

Engineering Sci., 21 : 1 1 , 727-31 ( 1981); and polyanhydrides (Leong et al.,

"Polyanhydrides for Controlled Release of Bioactive Agents," Biomaterials 7:5, 364- 71 (1986).

[0166] Thus, for example, the active agent(s) can be incorporated into a biocompatible polymeric composition and formed into the desired shape outside the body. This solid implant is then typically inserted into the body of the subject through an incision. Alternatively, small discrete particles composed of these polymeric compositions can be injected into the body, e.g., using a syringe. In an exemplary embodiment, the active agent(s) can be encapsulated in microspheres of poly (D,L- lactide) polymer suspended in a diluent of water, mannitol, carboxymethyl-cellulose, and polysorbate 80. The polylactide polymer is gradually metabolized to carbon dioxide and water, releasing the active agent(s) into the system.

[0167] In yet another approach, depot formulations can be injected via syringe as a liquid polymeric composition. Liquid polymeric compositions useful for biodegradable controlled release drug delivery systems are described, e.g., in U.S. Patent Nos. 4,938,763; 5,702,716; 5,744, 153; 5,990, 194; and 5,324,519. After injection in a liquid state or, alternatively, as a solution, the composition coagulates into a solid. [0168] One type of polymeric composition suitable for this application includes a nonreactive thermoplastic polymer or copolymer dissolved in a body fluid- dispersible solvent. This polymeric solution is placed into the body where the polymer congeals or precipitates and solidifies upon the dissipation or diffusion of the solvent into the surrounding body tissues. See, e.g., Dunn et al., U.S. Patent Nos. 5,278,201 ; 5,278,202; and 5,340,849 (disclosing a thermoplastic drug delivery system in which a solid, linear-chain, biodegradable polymer or copolymer is dissolved in a solvent to form a liquid solution).

[0169] The active agent(s) can also be adsorbed onto a membrane, such as a silastic membrane, which can be implanted, as described in International Publication No. WO 91/04014. Other exemplary implantable sustained release systems include, but are not limited to Re-Gel®, SQ2Gel®, and Oligosphere® by MacroMed, ProLease® and Medisorb® by Alkermes, Paclimer® and Gliadel® Wafer by Guilford pharmaceuticals, the Duros implant by Alza, acoustic biSpheres by Point Biomedical, the Intelsite capsule by Scintipharma, Inc., and the like.

Inhalation of Dry Powder Aerosol Formulation

[0170] In some embodiments, a dry powder aerosol formulation can be administered using an inhaler device. An illustrative inhaler device includes an inhaler body defining a recess for holding therein a capsule containing the dispersible powder composition of any embodiment discussed above, and a nosepiece communicating with said capsule, wherein the inhaler device further includes perforating means associated with the inhaler body and adapted to perforate said capsule to allow an outside air flow to be mixed with the dispersible powder composition for inhalation through said nosepiece. Such devices are designed to ensure that when the properly formulated dry powder composition is inhaled by the patient, the emitted dose of the composition is about 70 mass % or higher. In some embodiments, the perforating means in the inhaler devices comprise one or more perforating needles for transversely sliding against the biasing of resilient elements and operating between an abutment element, rigid with the inhaler body and a corresponding operating push-button element, each perforating needle having a contour including a beveled tip, for facilitating a perforation of a coating of the capsule.

[0171] In further embodiments, the nosepiece in the inhaler devices is movable with respect to the inhaler body to provide at least two operating condition, the two operating conditions comprising an open condition in which the recess for the capsule is accessible to engage therein a new capsule or to withdraw therefrom a used capsule, and a closed use condition in which said inhaler nosepiece is snap locked. The nosepiece may be further locked in its closure position by a snap locking means including a hook portion of a flange of the nosepiece, having a corresponding ridge formed inside a latching seat formed in the inhaler body. In further embodiments, the flange of the inhaler nosepiece may include a peg which is engageable in a hole formed in the inhaler body. In further embodiments, the hole may define a longitudinal slot adapted to allow a transversal tooth of said peg to pass through the slot, and the hole includes a bottom annular recess adapted to allow the tooth to slide in, thereby allowing said peg to be engaged in said hole. In further embodiments, the pin may be rotatable in the hole and the nosepiece is rotatable with respect to the inhaler body. In further embodiments, the recess for the capsule of the inhaler body of the device may communicate with the outside through a perforated plate or grid provided in the inhaler nosepiece at the flange and is adapted to separate the capsule recess from a duct of the nosepiece, the capsule recess having a bottom

communicating with the outside through one or more air inlet holes.

[0172] Further details of inhalers suitable for use in the methods described herein can be found in International Publication No. WO/2009/009775 (published Jan. 15, 2009), which is incorporated by reference herein in its entirety and specifically for its description of such inhalers.

Co-administration with an Additional Agent

[0173] Modified IL-4 mutein receptor antagonist compounds can be coadministered with additional agents that mitigate a symptom of an eosinophilic disorder. Co-administration of an additional agent can be useful, for example, to control symptoms of an eosinophilic disorder prior to the point at which the IL-4 mutein receptor antagonists begins to reduce such symptoms. More specifically, in certain embodiments, there will be a delay between the administration of the antagonist and the time at which the subject experiences diminution of symptoms. Treatment with an additional agent can provide relief from symptoms during this period. In particular embodiments, treatment with the additional agent can be initiated before, at the same time as, or after treatment with the antagonist. Treatment with the additional agent can be continued or discontinued, if it does not provide additional therapeutic benefit beyond that of the antagonist. In illustrative embodiments, treatment with an additional agent is initiated before, or at the same time as, the initiation of treatment with the antagonist, and the additional agent is administered for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, or about 2 months from the time treatment with the antagonist is initiated.

[0174] Additional agents can be administered by a route that is the same as, or different from, the route of administration of the IL-4 mutein receptor antagonist. Where possible, it is generally desirable to administer these agents by the same route of administration, preferably in the same composition. However, differences in pharmacodynamics, pharmacokinetics, or other considerations may dictate the coadministration of IL-4 mutein receptor antagonist compound and additional agent in separate compositions. Additional agents can be administered according to standard practice.

Dose

[0175] In therapeutic applications, the compositions of this invention are administered, for example, to a subject experiencing, or at risk for, an allergic response, such as an eosinophilic disorder, to mitigate at least one symptom of this response. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend upon the severity of the condition and the general state of the subject's health. Single or multiple doses of the compositions may be administered depending on the dosage and frequency as required and tolerated by the subject. In any event, the composition should provide a sufficient quantity of the active agent(s) of the composition(s) of this invention to effectively treat the condition. [0176] The concentration of active agent(s) can vary widely and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs. In accordance with standard practice, the clinician can titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Generally, the clinician begins with a low dose and increases the dosage until the desired therapeutic effect is achieved. Starting doses for a given active agent can, for example be extrapolated from in vitro and/or animal data.

[0177] In particular embodiments, concentrations of IL-4 mutein receptor antagonists will typically be selected to provide dosages of about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 0.7 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg kg, about 35 mg/kg, about 40 mg/kg about 45 mg/kg, about 50 mg/kg and sometimes higher. For example, lower dosages can be employed with more frequent dosing, and higher dosages can be employed with more infrequent dosing. It will be appreciated that such dosages may be varied to optimize a therapeutic regimen in a particular subject or group of subjects, and thus any of these values can represent the upper or lower limit of a suitable dosage range according to the invention.

[0178] In illustrative embodiments, a typical dosage of IL-4RA will be about

0.1 to 1 mg/kg. For example, for administration of IL-4 RA, an approximate dosage by aerosol inhalation would be about 10 mg to 60 mg. Approximate dosages include, but are not limited to, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, or about 60 mg, to a subject, with dosages administered one, two, or more times per day or week (including a dosage falling within any range bounded by any of these values). In another illustrative example, an approximate dosage for administration of IL-4RA by subcutaneous injection includes, but is not limited to, about 25 mg. Treatment by administration of IL-4RA may span days, weeks, years, or continue indefinitely, as symptoms persist.

[0179] In embodiments of the method in which an additional agent that mitigates a symptom of an eosinophilic disorder is co-administered with the IL-4 mutein receptor antagonist, suitable doses of additional agents are known and can be adjusted by the clinician for co-administration with an IL-4 mutein receptor antagonist described herein.

[0180] The foregoing compositions and administration methods are intended to be illustrative and not limiting. It will be appreciated that, using the teaching provided herein, other suitable compositions and modes of administration can be readily devised.

Monitoring of Response

[0181] The immune response of a subject being treated as described herein can be monitored by making periodic, e.g., daily observations of one or more existing symptoms of an eosinophilic disorder and/or plasma levels of IgE before and after dosing. When those subjects receiving the drug have a pronounced reduction in the immune response, they can, in certain embodiments, be taken off the IL-4 mutein receptor antagonist and monitored for the length of remission. Administration of the antagonist to subjects is expected to reduce or eliminate early and/or latent immune responses.

[0182] Administration of the antagonist to human subjects should produce a reduced immune response, as compared to that in other animals, because fewer antibodies against the antagonists are expected. Fewer antibodies to the IL-4 mutein receptor antagonist and its epitopes imply that there are fewer inhibiting substances or agents binding to the antagonist. Fewer inhibiting substances and agents binding directly or indirectly to the antagonist allows the drug to bind to the IL-4 receptor and thus inhibit the IL-4- and IL-13-induced response and inactivating the cascade of downstream events, e.g. release of various interleukins, chemokines, and

chemoattractants involved in an immune response.

Methods for Identifying Candidates for Treatment with an IL-4/IL-13

Antagonist

[0183] In certain embodiments, the invention provides a method of determining whether a subject is a candidate for treatment with an antagonist of IL-4 and IL- 13 (IL-4 IL-13 antagonist). Typically, the subject is one suffering from an atopic or inflammatory disorder. The method can entail determining whether the subject has an elevated level of eosinophils, which indicates that the subject is a candidate for treatment with an IL-4/IL-13 antagonist. Alternatively, or in addition, the method can entail determining whether the subject has the major allele in one or more single nucleotide polymorphisms (SNPs) in the IL-RA gene selected from the group consisting of rs8832, rsl 029489, rs3024585, rs3024622, and rs4787956. These SNPs are identified with respect to the human genome. The major alleles in these SNPs are shown in Table 23 and the surrounding sequences are shown in Table 24 (see Example 15). The presence of the major allele in one or more of these SNP(s) indicates that the subject is a candidate for treatment with an IL-4/IL-13 antagonist.

[0184] Generally, the method includes recording the eosinophil level or the presence of the major allele in one or more of said SNP(s) in a patient medical record. In some embodiments, the method additionally entails selecting an IL-4/IL-13 antagonist for use in treating the subject, which is typically recorded in the patient medical record. In particular embodiments, the method can include administering the IL-4/IL-13 antagonist to a subject having an elevated level of eosinophils and/or the major allele in one or more single nucleotide polymorphisms (SNPs) in the IL-RA gene selected from the group consisting of rs8832, rsl 029489, rs3024585, rs3024622, and rs4787956. It is understood that certain steps of the method may be performed indirectly, that is, a clinician may order the test to determine the eosinophil level and/or the presence of major alleles at SNPs, but not perform the test. Similarly, the clinician will generally read the test result, and record it in the patient medical record, and in certain embodiments, prescribe an IL-4/IL-13 antagonist, but may not perform the actual administration, which may, in some cases, be performed by the subject. The method of the invention is intended to encompass the actions of the clinician in ordering the test and prescribing the antagonist, even though others may carry out the test and/or administer the antagonist.

Measurement of Eosinophil Levels

[0185] Eosinophil levels can be measured using standard methods and any convenient sample, such as a sputum or blood sample. Normal levels in blood are on the order of 250 eosinophils per mm . Blood levels over 300 per mm , and especially over 350 per mm ³ , are considered elevated.

Measurement of SNP Alleles

[0186] The methods of the invention include determining the whether one copy of the major allele is present in a given SNP, i.e., whether the subject is heterozygous for the major allele in that SNP or, in certain embodiments, determining whether two copies of the major allele are present in the SNP, i.e., whether the subject is homozygous for the major allele. In certain embodiments, the determination that the subject is homozygous for the major allele in one or more of the SNPs (e.g., rs8832) provides a stronger indication that the subject is a candidate for treatment with an IL-4/IL-13 antagonist than if the subject were heterozygous. In various embodiments, the presence of one of both major alleles in at ^' least two, three, four, or all five of the SNPs is determined. In some embodiments, a haplotype is determined, i.e., the genotype of one allele in two, three, four, or all five of the SNPs. In certain embodiments, the determination that the subject has a haplotype including the major allele in two, three, four, or all five of the SNPs provides a successively stronger indication that the subject is a candidate for treatment with an IL-4/IL-13 antagonist.

Target Molecules

[0187] The SNPs described herein can be most conveniently detected by detecting SNP DNAs and/or SNP RNAs, i.e., SNP nucleic acids. The nucleic acid is generally found in or derived from a biological sample. The term "biological sample," as used herein, refers to a sample obtained from a subject or from

components (e.g., cells) of a subject. The sample may be of any biological tissue or fluid. Biological samples may also include organs or sections of tissues such as frozen sections taken for histological purposes. Sputum and blood provide particularly convenient samples for detecting SNP nucleic acids.

[0188] The nucleic acid (e.g., genomic DNA, RNA, nucleic acid derived from

RNA, etc.) is, in certain embodiments, isolated from the sample according to any of a number of methods well known to those of skill in the art. For example, methods of isolation and purification of nucleic acids are described in detail in by Tijssen ed., (1993) Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid

Preparation, Elsevier, N.Y. and Tijssen ed.

[0189] Frequently, it is desirable to amplify the nucleic acid sample prior to assaying for SNP alleles. Methods of amplifying nucleic acids are well known to those of skill in the art and include, but are not limited to polymerase chain reaction (PCR, see. e.g, Innis, et al, (1990) PCR Protocols. A guide to Methods and

Application. Academic Press, Inc. San Diego,), ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 1 17, transcription amplification ( woh et al. (1989) Proc. Natl. Acad. Sci. USA $6: 1 173), self-sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.).

Hybridization-Based Assays

[0190] Using the SNP sequences provided herein, detecting and/or quantifying the SNP alleles can be routinely accomplished using nucleic acid hybridization techniques (see, e.g., Sambrook et al. supra). For example, one method for evaluating the presence, absence, or quantity of SNP genomic DNA or reverse- transcribed cDNA involves a Southern Blot. Alternatively, SNP RNA can be detected/quantified in a Northern blot. SNP alleles can also be detecting using in situ hybridization.

[0191] In certain embodiments, SNP allele can be detected and/or quantified in an array-based hybridization format. Arrays are a multiplicity of different "probe" or "target" nucleic acids (or other compounds) attached to one or more surfaces (e.g., solid, membrane, or gel). In a preferred embodiment, the multiplicity of nucleic acids (or other moieties) is attached to a single contiguous surface or to a multiplicity of surfaces juxtaposed to each other. In an array format a large number of different hybridization reactions can be run essentially "in parallel." This provides rapid, essentially simultaneous, evaluation of a number of hybridizations in a single "experiment." Methods of performing hybridization reactions in array based formats are well known to those of skill in the art (see, e.g., Pastinen (1997) Genome Res. 7: 606-614; Jackson (1996) Nature Biotechnology 14: 1685; Chee (1995) Science 274: 610; WO 96/17958, Pinkel et al. (1998) Nature Genetics 20: 207-21 1 ).

[0192] Arrays, particularly nucleic acid arrays can be produced according to a wide variety of methods well known to those of skill in the art. For example, in a simple embodiment, "low density" arrays can simply be produced by spotting (e.g. by hand using a pipette) different nucleic acids at different locations on a solid support (e.g. a glass surface, a membrane, etc.). This simple spotting, approach has been automated to produce high density spotted arrays {see, e.g., U.S. Patent No:

5,807,522). This patent describes the use of an automated system that taps a microcapillary against a surface to deposit a small volume of a biological sample. The process is repeated to generate high density arrays. Arrays can also be produced using oligonucleotide synthesis technology. Thus, for example, U.S. Patent No. 5, 143,854 and PCT Patent Publication Nos. WO 90/15070 and 92/10092 teach the use of light-directed combinatorial synthesis of high density oligonucleotide arrays.

Synthesis of high-density arrays is also described in U.S. Patents 5,744,305,

5,800,992 and 5,445,934.

[0193] A wide variety of other nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Such assay formats are generally described in Hames and Higgins (1985) Nucleic Acid Hybridization, A Practical Approach, IRL Press; Gall and Pardue (1969) Proc. Natl. Acad. Sci. USA 63: 378-383; and John et al. (1969) Nature 223: 582-587.

[0194] Sandwich assays are commercially useful hybridization assays for detecting or isolating nucleic acid sequences. Such assays utilize a "capture" nucleic acid covalently immobilized to a solid support and a labeled "signal" nucleic acid in solution. The sample will provide the target nucleic acid. The capture nucleic acid and signal nucleic acid probe hybridize with the target nucleic acid to form a sandwich hybridization complex. To be most effective, the signal nucleic acid should not hybridize with the capture nucleic acid. Amplification-Based Assays

[0195] In other embodiments, amplification-based assays can be used to detect/measure the SNP allele. In such amplification-based assays, the target nucleic acid sequences act as template(s) in amplification reaction(s) (e.g. Polymerase Chain Reaction (PCR) or reverse-transcription PCR (RT-PCR)). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template (e.g., SNP) in the original sample.

[0196] Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). One approach, for example, involves simultaneously co-amplifying a known quantity of a control sequence using the same primers as those used to amplify the target. This provides an internal standard that may be used to calibrate the PCR reaction.

[0197] Amplification typically relies on the use of primers. The term

"primer" refers to an oligonucleotide that is capable of hybridizing (also termed "annealing") with a nucleic acid and serving as an initiation site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but primers are typically at least 7 nucleotides long and, more typically range from 10 to 30 nucleotides, or even more typically from 15 to 30 nucleotides, in length. Other primers can be somewhat longer, e.g., 30 to 50 nucleotides long. In this context, "primer length" refers to the portion of an oligonucleotide or nucleic acid that hybridizes to a complementary "target" sequence and primes nucleotide synthesis. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template. The term "primer site" or "primer binding site" refers to the segment of the target nucleic acid to which a primer hybridizes.

[0198] SNP allele detection can be carried out by allele-specific amplification, in which the amplification reaction produces an amplicon only if a specific allele is present. Alternatively, both alleles can be amplified and the individual alleles identified during or after the amplification reaction on the basis of a distinguishable characteristic of the amplicon (e.g., length or melting temperature), the use of differently labeled primers for each allele, or the use of a nucleic acid probe (e.g., a TaqMan probe). Optimization of Hybridization/Annealing Conditions

[0199] Nucleic acid hybridization simply involves providing a denatured probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing. The same is true for annealing of a primer to a target in an amplification reaction. It is generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids, or in the addition of chemical agents, or the raising of the pH. Under low stringency conditions (e.g., low temperature and/or high salt and/or high target concentration) hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed sequences are not perfectly complementary. Thus specificity of hybridization is reduced at lower stringency. Conversely, at higher stringency (e.g., higher temperature or lower salt) successful hybridization requires fewer mismatches. For SNP allele detection, the stringency is preferably sufficiently high to distinguish the alleles.

[0200] Methods of optimizing hybridization conditions are well known to those of skill in the art (see, e.g., Tijssen (1993) Laboratory Techniques in

Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, Elsevier, N.Y.). Labeling and Detection of Nucleic Acids

[0201] Probes used herein for detection of SNP alleles can be full length or less than the full length of the SNP allele sequences given herein. Shorter probes can be empirically tested for specificity. Preferred probes are sufficiently long so as to specifically hybridize with the SNP target nucleic acid(s) under stringent conditions. The preferred size range is from about 20 bases to the length of the SNP sequence, more preferably from about 30 bases to the length of the SNP sequence, and most preferably from about 40 bases to the length of the SNP sequence.

[0202] The probes, or in some cases, primers are typically labeled, with a detectable label. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like, see, e.g., Molecular Probes, Eugene, Oregon, USA), radiolabels (e.g., ³ H, ¹²⁵ I, ³⁵ S, ^{l 4} C, or ³² P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold (e.g., gold particles in the 40 -80 nm diameter size range scatter green light with high efficiency) or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;

4,277,437; 4,275, 149; and 4,366,241.

[0203] The label may be added to the target (sample) nucleic acid(s) prior to, or after the hybridization. So called "direct" labels are detectable labels that are directly attached to or incorporated into the target (sample) nucleic acid prior to hybridization. In contrast, so called "indirect labels" are joined to the hybrid duplex after hybridization. Often, the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization. Thus, for example, the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin bearing hybrid duplexes providing a label that is easily detected. For a detailed review of methods of labeling nucleic acids and detecting labeled hybridized nucleic acids see Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y., (1993)).

Kits

[0204] The invention also provides kits useful in practicing the methods of the invention. In one embodiment, a kit of the invention includes a IL-4 mutein receptor antagonist in a suitable container. In a variation of this embodiment, the IL-4 mutein receptor antagonist is formulated in a pharmaceutically acceptable carrier. The kit preferably includes instructions for administering the antagonist to a subject to treat an eosinophilic disorder. In an illustrative embodiment, the instructions are directed to the treatment of asthma.

[0205] In some embodiments, there are provided kits comprising the dispersible powder composition of any embodiment discussed above, an inhaling means for inhalation by the patient, and a storage means for storing the composition, the storage means comprising a primary container and a secondary storage container, and optionally further including a label affixed to the storage means and providing the patient with instructions for use, with the further proviso that the primary capsule is adapted to fit the inhaling means. In further embodiments, the composition used with the kit is in form of capsules, each capsule containing one dose of the composition. Further details of such kits can be found in International Publication No.

WO/2009/009775 (published Jan. 15, 2009), which is incorporated by reference herein in its entirety and specifically for its description of such kits.

[0206] The invention also provides kits for determining the presence of the major allele in one or more single nucleotide polymorphisms (SNPs) in the IL-RA gene. In specific embodiments, the SNP is one or more of rs8832, rs 1029489, rs3024585, rs3024622, and rs4787956. In certain embodiments, the kit can include one or more probes and/or primers that specifically hybridize under stringent conditions to a nucleic acid including the major allele in one or more of these SNPs. In various embodiments, the kit can include at least two, three, four, or five probes and/or primers that each specifically hybridize under stringent conditions to nucleic acid(s) comprising the major allele in at least two, three, four, or five SNPs, respectively. In illustrative embodiments, the kit can include one or more probes that are members of an array of probes. For amplification-based assays, the kit can include primers and/or probes for use in a nucleic acid amplification reaction.

Furthermore, the kit can, optionally, include instructional materials teaching that the determination the major allele in said one or more SNPs in a nucleic acid sample from a subject indicates that the subject is a candidate for treatment with an antagonist of IL-4 and IL- 13 (IL-4/IL- 13 antagonist).

[0207] Instructions included in kits of the invention can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include the address of an internet site that provides the instructions.

[0208] The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. The following examples are intended to illustrate but not limit the invention.

EXAMPLES

EXAMPLE 1

Fermentation of E. coli

[0209] Cells containing genes for the production of muteins were grown in LB medium (10 g Bacto tryptone, 5 g Yeast extract, 10 g NaCI per liter, pH 7,5) until OD600 reached 0.8-1 .0. Expression was induced by addition of IPTG to a final concentration of 0.5 mM and incubation continued for 5 hours. Cells were harvested by centrifugation. E.coli transformants expressing hIL4 mutant proteins were cultured as described in U.S. Patent No. 6, 130,318. Briefly, E.coli were fermented in LB nutrient solution of the following composition: Bacto tryptone 10 g/1, Bacto yeast extract 5 g/1, and sodium chloride 10 g/1. The constituents were dissolved in deionized water, which was sterilized at 121 °C for 20 min. Prior to inoculation, an antibiotic which was suitable for selecting the transformants (e.g., 100 mg/1 Na ampicillin or 50 mg/1 kanamycin sulphate depending on the selection marker used in the vector) was added to the nutrient solution under sterile conditions. Strain stocks of all the E.coli transformants were laid down by taking 2 ml aliquots of a preliminary culture and storing them in liquid nitrogen. The preliminary culture fermentations were carried out in 1 ltr. shaking flasks which contained 200 ml of LB nutrient solution. The nutrient solution was inoculated with a strain stock or with a single colony from an LB agar plate. The cultures were incubated at 30°C for 12-18 hrs. while being shaken continuously.

[0210] The main culture fermentations were carried out in LB nutrient solution using 10 liter stirred tank fermenters. The nutrient solution was inoculated with 1 -5% by vol. of a preliminary culture, with the biomass being centrifuged out of the preliminary culture and resuspended in fresh LB medium prior to the inoculation. The fermentation conditions for the 10 liter main culture were as follows: 37°C, stirrer revolution rate 500 rpm, aeration rate 0.5 vvma.

[0211] In order to monitor the growth of the biomass, sterile samples were removed from the culture broth at intervals of approx. 1 hr., and their optical density was determined at 600 nm (OD600). The cultures were induced when an OD600 of 0.8-1.2 had been reached. Induction took place as follows, IPTG induction: Sterile addition of isopropyl-P-D-thio-galactopyranoside (IPTG) to a concentration of 0.4 mM. The induction time was typically 4-8 hrs.

[0212] After the fermentation had finished (6-14 hrs.), the contents of the fermenter were cooled down to 10-15°C, and the bacterial cells were harvested using standard centrifugation techniques (e.g., bucket centrifuge). The cell mass which was obtained after centrifugation was temporarily stored, where appropriate, in the frozen state. The product was worked up from the biomass which had been obtained in this way.

EXAMPLE 2

Expression of Interleukin 4 Mutant Proteins in E. coli Using Inducible

Promoters [0213] Surprisingly, a custom made vector system and a E.coli-codon optimized gene of the IL-4 mutein has demonstrated that bacteria transformed with said plasmid according to U.S. Patent No. 6,506,590 gives expression rates, plasmid and expression stability values many times higher than those observed after transforming the identical hosts with plasmids known in the art.

[0214] The E.coli phage T5 promoter together with two lac operator sequences is derived from the pQE30 plasmid (Qiagen) belonging to the pDS family of plasmids (Bujard et al., Methods Enzymol. 155, 416-433, 1987; and Stuber et al., Immunological Methods, I. Lefkovits and B. Pernis, eds., Academic Press, Inc., Vol. IV, 121 -152, 1990).

[0215] The ribosomal binding site (rbs) is derived from the region upstream from gene 10 of the phage T7 (T7 g 10 leader). Gene 10 of phage T7 codes for the coat protein, which is the major protein expressed after T7 infection. The T7 gl O rbs was obtained from the vector .pET-9a (Studier et al., Methods Enzymol. 185, 60-89, 1990). The T7 gl O leader spans a region of about 100 bp (Olins et al., Gene 227-235, 1988). In the final expression construct the region upstream of the Xbal site is deleted. The T7 gl O leader sequence now spans 42 bp and harbors one base exchange from G to A in position 3638 of the preferred plasmid.

[0216] As an effective measure of synonymous codon usage bias, the codon adaptation index (CAI) can be useful for predicting the level of expression of a given gene (Sharp et al., Nucleic Acids Res. 15, 1281 -1295, 1987; and Apeler et al., Eur. J.

Biochem. 247, 890-895, 1997). The CAI is calculated as the geometric mean of relative synonymous codon usage (RSCU) values corresponding to each of the codons used in a gene, divided by the maximum possible CAI for a gene of the same amino acid composition. RSCU values for each codon are calculated from very highly expressed genes of a particular organism, e.g., E.coli, and represent the observed frequency of a codon divided by the frequency expected under the assumption of equal usage of the synonymous codons for an amino acid. Highly expressed genes, e.g., genes encoding ribosomal proteins, have generally high CAI values.gtoreq.0.46. Poorly expressed genes like lad and trpR in E. coli have low CAI values. ltoreq.0.3.

The calculated E.coli CAI value for the natural IL-4 sequence is 0.733. This means that the natural gene should be well-suited for high level expression in E.coli. Nevetheless a synthetic gene with optimal E.coli codon usage (CAI value=l ) has the potential to further increase the expression level. Therefore synthetic IL-4 and IL-4 mutein genes were designed and cloned.

[0217] A T7 DNA fragment containing the transcription terminator Τφ is derived from the vector pET-9a (Studier et al., Methods Enzymol. 185, 60-89, 1990). Transcriptional terminators determine the points where the mRNA-RNA polymerase- DNA complex dissociates, thereby ending transcription. The presence of a transcriptional terminator at the end of a highly expressed gene has several advantages: they minimize sequestering of RNA polymerase that might be engaged in unnecessary transcription, they restrict the mRNA length to the minimal, thus limiting energy expense, as strong transcription may interfere with the origin of replication, a transcriptional terminator increases plasmid stability due to copy number maintenance (Balbas and Bolivar, Methods Enzymol. 185, 14-37, 1990).

[0218] The kan resistance gene is derived from the vector pET-9a (Studier et al., Methods Enzymol. 185, 60-89, 1990). Originally, this is the kan gene of Tn903 from the vector pUC4 ISS (Barany, Gene 37, 1 1 1 -123, 1985). In the preferred plasmid the kan gene and the IL-4 and IL-4 mutein gene have opposite orientations, so there should not be an increase in kan gene product after induction due to read- through transcription from the T5 promoter. Kanamycin was chosen as selective marker because it is the preferred antibiotic for GMP-purposes. In addition, kan gene based vectors are more stable than ampicillin resistant (bla) plasmids. Ampicillin selection tends to be lost in cultures as the drug is degraded by the secreted β- lactamase enzyme. The mode of bacterial resistance to kanamycin relies upon an aminogly-coside phosphotransferase that inactivates the antibiotic.

[0219] Controlled gene expression is absolutely necessary for the set-up of a stable plasmid system, particularly if the protein of interest is deleterious to the host cell. The preferred plasmid uses a lac-based inducible system consisting of a lac repressor gene (lacl) and two synthetic lac operator sequences fused downstream to the E.coli phage T5 promoter. The lacl.sup.q promoter and the lacl structural gene were isolated from the vector pTrc99A (Amann et al., Gene 69, 301 -315, 1988).

I.sup.q is a promoter mutation which leads to overproduction of the lacl repressor.

The wild-type lac repressor is a tetrameric molecule comprising four identical subunits of 360 amino acids each. The lac repressor tetramer is a dimer of two functional dimers. The four subunits are held together by a four-helix bundle formed from residues 340-360. Due to the isolation of the lad gene from the vector pTrc99A by a Narl cut the residues beyond amino acid 331 are deleted and 10 amino acids not normally encoded in the lad gene are added. It is known that mutations or deletions that occur in the C-terminal part of lad, beyond amino acid 329, result in functional dimers that appear phenotypically similar to the wild-type repressor (Pace et al., TIBS 22, 334-339, 1997).

[0220] The origin of replication (ori) of the preferred plasmid is derived from the vector pET-9a, the ori of which originates from pBR322. The preferred plasmid therefore carries the pMBI (ColEl) replicon. Plasmids with this replicon are multicopy plasmids that replicate in a 'relaxed' fashion. A minimum of 15-20 copies of plasmid are maintained in each bacterial cell under normal growth conditions. The actual number for the preferred plasmid is within this range. Replication of the ColEl-type ori is initiated by a 555-nucleotide RNA transcript, RNA II, which forms a persistent hybrid with its template DNA near the ori. The RNA II-DNA hybrid is then cleaved by RNase H at the ori to yield a free 3ΌΗ that serves as a primer for DNA polymerase I. This priming of DNA synthesis is negatively regulated by RNA I, a 108-nucleotide RNA molecule complementary to the 5' end of RNA II.

Interaction of the antisense RNA I with RNA II causes a conformational change in RNA II that inhibits binding of RNA II to the template DNA and consequently prevents the initiation of plasmid DNA synthesis. The binding between RNAs I and II is enhanced by a small protein of 63 amino acids (the Rop protein, Repressor of primer), which is encoded by a gene located 400 nucleotides downstream from the origin of replication (Sambrook et al., Molecular Cloning, Cold Spring Harbor, 1989). Deletion of the rop gene leads to an increase in copy number and due to a gene dosage effect to enhanced expression levels of the plasmid encoded heterologous gene. This observation was also made for the IL-4 expression vectors tested. But it turned out that rop-plasmids are instable and lost very rapidly during fermentation under non- selective conditions. Therefore, the replicon of the preferred plasmid contains the rop gene to ensure high plasmid stability. The preferred plasmid lacks the mob gene that is required for mobilization and is therefore incapable of directing its own conjugal transfer from one bacterium to another.

EXAMPLE 3

Preparation of an IL-4 Mutant Protein [0221] Cell disruption and isolation of the inclusion bodies: 25g of E.coli moist mass from Example 1 were taken up in 200 ml of buffer (0.1 M phosphate buffer, pH 7.3, 0.1% Triton, 1 mM EDTA, 1 μg/ml pepstatin) and disrupted by sonication (Branson B 15 sonifier). The inclusion bodies, which contain the product, were isolated by centrifugation (35,000 x g, 20 min) and washed in disruption buffer which additionally contained 4M urea.

[0222] The washed inclusion bodies were solubilized in 125 ml of buffer (0.2

M Tris, pH 8.1 , 8M guanidine hydrochloride). 4g of sodium sulphite and 2g of potassium tetrathionate were added and the reaction mixture was stirred for 2 h. Undissolved constituents were removed by centrifugation (35,000 x g, 20 min) after the reaction had finished. The supernatant was loaded onto a gel filtration column (Sephacryl S-300 HR, Pharmacia, l Ox 90 cm) and subjected to gel filtration in PBS buffer containing 6M guanidine hydrochloride at a flow rate of 280 ml/h. Product- containing fractions were identified by means of SDS-PAGE and combined.

[0223] β-Mercaptoethanol (final concentration 15 mM) was added in order to reduce the molecules. Following a two-hour incubation at room temperature, the mixture was diluted 5 times with water and dialyzed against buffer (3 mM NaH ₂ P0 ₄ , 7 mM Na ₂ HP0 ₄ , 2 mM KCl, 120 mM NaCl) for 3-4 days. The dialyzed material was adjusted to pH 5.0 with acetic acid and its conductivity was decreased to .ltoreq.10 mS/cm by adding water. 50 ml of CM Sepharose-FF (Pharmacia), which was equilibrated with 25 mM ammonium acetate, pH 5.0, were added to the mixture while stirring. Unbound material was filtered off and the gel was used to fill a column. The product was eluted with a linear gradient from 0 to 1 M NaCl in 25 mM ammonium acetate, pH 5.0, at a flow rate of 300 ml h. Product containing fractions were identified by SDS-PAGE or by analytical RP chromatography.

[0224] The pool of CM sepharose was loaded on to a Vydac C-4 column (l x

25 cm, 10 μιη) which was equilibrated with 0.1 % TFA and eluted with an increasing gradient of acetonitrile. Fractions which contained the pure product were combined and lyophilized.

EXAMPLE 4

Preparation of Formulations [0225] Four formulations, each comprising the active ingredient mIL-4, were prepared by combining the components into a solution. Subsequent tests and evaluations discussed in the examples that follow refer to them as Formulations 1 -4, as shown in Table 4.

EXAMPLE 5

Method of Making Dry Powder

[0226] The spray drying parameters used to manufacture formulations 1 -4 summarized in Table 5.

EXAMPLE 6

Studies of Mass of Active Component in Composition and Aggregation of the

Protein

[0227] The content and purity of each formulation was determined by analyzing approximately 20 mg of powder by a RP-HPLC assay method. Three determinations were performed for each powder and the percent by weight was calculated. Table 6 shows the assay results for each formulation. The results are consistent with the theoretical values.

[0228] The greatest deviation was observed in Formulation 4 where the measured concentration was approximately 8% lower than the expected

concentration. The degradation profile was characterized using a reverse phase HPLC degradation method. No noticeable signs of degradation were observed for the spray dried powders other than for Formulation 4 which showed an additional peak indicating aggregation at about 7.6 minutes.

SDS PAGE (Aggregation of the Protein)

[0229] Samples were incubated with the anionic detergent sodium dodecyl sulfate. The proteins were separated under non-reducing conditions in a

polyacrylamide gel with defined pore size (e.g. ExcelGel SDS, 15% Polyacrylamid, Pharmacia). The separation is proportional to the molecular weight of the proteins. After staining with Coomassie Blue, the gels were scanned (e.g. Scanner JX-330, Sharp) and the number of the individual bands determined. The SDS PAGE evaluation of the protein was conducted for all 4 formulations. A faint band was observed in spray dried Formulation 4 suggesting some minimal protein

decomposition. Moisture Content

[0230] The moisture content of each formulation was determined using the

Karl Fisher titration method. Approximately 10 mg of each formulation was dissolved in 10 mL of Karl Fisher reagent. Samples were analyzed by injecting 1 mL of sample solution into the coulometric cell. Typically three samples were analyzed for each formulation and sample solutions were injected in triplicate. Blanks consisting of the Karl Fisher reagent were used. Table 7 provides the data regarding the moisture content of each formulation.

EXAMPLE 7

Evaluation of Biological Activity TF1 Functionality Bioassay

[0231] The proliferative response of TF-1 cells to IL-4 or IL-13 was used to assess the functional antagonist activity of mIL-4. The TF-1 line was derived from a non-adherent erythroleukemia and is used extensively as a model system because the cells proliferate in response to a number of inflammatory cytokines including IL-4 and IL-13. TF- 1 cells were cultured with and without IL-4, IL-13 and mIL-4. The concentration of mIL-4(log nM) versus the mean relative fluorescence units is plotted and data extracted to determine 50% antagonist effect. The EC50 of mIL-4(50% inhibitory effect) for IL-4 and IL13 is reported as a mean and the 95% confidence interval is reported.

[0232] The results of the TF1 functionality bio-assay are presented in Table 8.

The results indicate there was no loss of activity in the spray dry powder samples due to spray drying.

EXAMPLE 8

Glass Transition Temperature Measurements

[0233] Glass transition temperatures were measured using the method of differential scanning calorimetry (DSC), which was performed using a TA

Instruments DSC2920 apparatus using an N ₂ flow rate of 10°C per minute. In a range between about 75°C and 125°C, there were changes in the heat flow indicating glass transition temperatures (50 degrees above the storage temperature). The results indicate that the formulations 1 and 2 differ in their thermal behavior because a change in the baseline is observed around 140°C in the formulations with sucrose.

EXAMPLE 9

Determining Primary Particle Size Distribution by Laser Diffraction [0234] The geometric particle size distribution of the spray dried formulations was determined using a wet dispersion, laser diffraction method. The Malvern Mastersizer 2000 was used in combination with the Hydro2000S wet cell.

[0235] Samples were prepared by weighing approximately 10 to 25 mg of formulation into a 20 mL glass vial. Ten milliliters of dispersant was added to produce a suspension. Samples were sonicated using a probe sonicator at 10% amplitude (40 watts) for 2 minutes to promote particle dispersion. Three samples were prepared for each formulation. The samples were then analyzed using the Malvern Mastersizer 2000 at the settings shown in Table 9.

[0236] The results of the measurements are summarized in Table 10, for Formulations 1 through 4. Only marginal differences in particle size distribution were observed despite the differences in formulation composition. Median diameters ranged from 2.3 to 2.8 μιτι. Mean D90 values ranged from 4.4 μπι to 4.9 μπι. Mean SPAN values, a measure of the width of the particle distribution, were less than or equal to 1.4 indicating the particles were relatively monodisperse.

EXAMPLE 10

Determining Aerodynamic Particle Size Distribution by Next Generation

Impactor

[0237] The aerodynamic performance of each formulation was determined using the Next Generation Impactor (NGI). For aerosolization of the formulation, the Plastiape RS01 Model 7, a low resistance capsule device was used. Formulations were filled into size 3 hydroxypropylmethylcellulose (HPMC) capsules from Shinogi.

[0238] Nominally, about 5 mg of formulation was weighed into each capsule.

Capsule filling and weighing was performed in a conditioned glove box (less than 5 % relative humidity, at a temperature of between 15°C and 25°C). Capsules were loaded into the device, pierced, and sampled into the NGI immediately after preparation.

[0239] The flow rate corresponding to a 4 kPa pressure drop was determined using a volumetric flow meter. A modified adapter connecting the inhaler to the USP IP allowed for a direct measurement of the pressure drop. NGIs were setup without a pre-separator. A Copley Critical Flow Controller was setup to draw 4 liters of air through the device during testing. NGI samples were analyzed using the RP HPLC assay method. An air flow rate of approximately 100 L/min was required to produce a 4 kPa pressure drop across the inhaler device. This flow rate was used in all NGI tests.

[0240] Table 1 1 lists the performance parameters calculated for these experiments. The mean fine particle dose (FPD) varied due to the differences in mlL- 4 concentration amongst the formulations. When normalized as fine particle fraction (FPF), Formulation 2 had the highest FPF at 96 %, followed by Formulations 1 and 3 at 85 % and 83 %, respectively. Mean MMADs approximated the D50 values determined by Malvern (see Table 9). These results indicate that Formulations 1 , 2 and 4, in combination with the inhaler device, are appropriate for inhalation delivery.

EXAMPLE 11

Determining Emitted and Fine Particle Dose

[0241] The data for the emitted mass and fine particle dose (deposition) results are summarized in Table 12 using different fill weights for Formulation 1 manufactured at two different primary particle sizes (Batch 1 with a mean particle size of 4.8 μm and Batch 2 with a mean particle size of 3.3 μm Data for the emitted mass and fine particle dose (deposition) results are summarized in Table 13 using different fill weights for Formulation 2 manufactured at two different primary particle sizes (Batch 1 with a mean particle size of 2.9 μm and Batch 2 with a mean particle size of 4.2 μm )). As can be seen in the results, the fill weight impacts both the emitted mass and the fine particle fraction.

EXAMPLE 12

Studies of Storage Conditions and Stability

[0242] Four formulations were evaluated for their physical and chemical properties after being spray dried under similar conditions. All formulations had geometric particle size distributions, as determined by laser diffraction, satisfactory for inhalation delivery. Mean particle size diameter (D50) values ranged from 2.3 to 2.8 μm and mean 90 ^th percentile diameter (D90) values ranged from 4.4 to 4.9 μιη. The moisture content of all formulations was less than 2% by mass. However, the moisture absorption profile for formulation 3 showed a distinct weight loss at 30-40% RH indicating crystallization. The formulation was eliminated from further testing. [0243] The aerodynamic performance of formulations 1 , 2 and 4 appeared suitable for inhalation delivery. However, chemical analysis of formulation 4 indicated the presence of an unknown peak by RP-HPLC. Thus, formulations 1 and 2 were considered for 3 month stability testing at 5°C and 30°C/65% RH.

[0244] The stability data results in Table 14 pertain to a new batch of dry powder similar to Formulation 2, i.e., 75% mIL-4, 15% sucrose and 10% citrate, pH 5.0.

EXAMPLE 13

Kit Contents and Packaging

[0245] Dynamic Vapor Sorption (DVS) was performed on the spray dried material to evaluate moisture uptake. The samples were analyzed using a SMS DVS 2000 system ramping from 0 to 90% RH with a dM/dT value of 0.001%. Lyophilized mIL-4 was also characterized for reference. The dynamic vapor sorption (DVS) studies of the four spray-dried formulations of IL-4indicate that the moisture uptake properties of formulation 1 , 2 and 4 are consistent to each other and to mIL-4 lyophilized cake. These show an increase in moisture with increasing relative humidity and no other moisture induced events.

[0246] Formulation 3 shows a distinct weight loss event at 30-40 % RH which can be attributed to a crystallization and potential instability for this formulation. Tables 15 and 16 provide summaries of DVS data.

[0247] The moisture uptake of these formulations requires that the packaging configuration used must minimize moisture ingress to the product. Improper packaging and moisture ingress of these dry powders resulted in instability.

[0248] Tables 17 and 18 show stability data for bulk powder and filled capsules from formulations 1 and 2 packaged in foil overwrap and stored at 5°C, 25°/60% RH and 40°C/75% RH for 12 weeks. Also, for each formulation a set of capsule samples were stored in a glass vial but left exposed (no overwrap, no dessicant) to 25°C and 60% relative humidity for 8 weeks. In both formulations, bulk powder and capsules over the period of 12 weeks stored in foil overwrap showed a substantial increase in moisture demonstrating that the foil overwrap did not protect the drug product from moisture ingress. The data shown for RP-HPLC and SDS PAGE is characteristic of moisture induced degradation. Capsule samples stored unprotected for 8 weeks showed the largest increase in moisture and pronounced degradation. With improved packaging, there was no evidence of increasing moisture and no protein degradation under the accelerated storage condition (30°C/65% RH) over the same timeframe.

EXAMPLE 14

Clinical Trial: Use of an IL-4 Mutant Protein Dry Powder Aerosol Formulation

(Aerovant) to Treat Asthma in Humans

Abstract

[0249] Results from a Phase 2b clinical trial that show the inhaled dry powder formulation of Aerovant (pitrakinra inhalation powder) is effective in a pre-defined subset of patients with eosinophilic asthma. Aerovant is a dry powder for inhalation formulation for a recombinant human IL-4 variant having R 121 D and Y 124D amino acid substitutions (numbered in accordance with wild-type human IL-4) that is a potent inhibitor of both IL-4 and IL-13 activity.

[0250] Data from a 534-patient, double-blind, randomized, placebo- controlled, dose-ranging study, showed a 37% reduction (p < 0.004) in the incidence of asthma exacerbations in patients on Aerovant 10 mg twice daily treatment compared to placebo in the subset of patients (n = 125) with eosinophilic asthma, as measured by elevated blood eosinophils. Time to exacerbation and asthma symptom scores also improved significantly compared to placebo in the same patient population. In the broader study population of moderate to severe asthmatics (eosinophilic plus non-eosinophilic asthmatics), there was no statistically significant difference between Aerovant and placebo, although Aerovant 10 mg (highest dose) did show trends towards drug effect. Overall, Aerovant was safe and well-tolerated in this study.

[0251] The data demonstrate that by inhibiting both IL-4 and IL-13, Aerovant can safely and effectively mitigate the Th2 pathway inflammation associated with high eosinophilic activity, which often leads to asthma attacks. A significant number of patients suffering from eosinophilic asthma do not respond well to current treatment with inhaled corticosteroids in combination with long-acting beta agonists. Approximately 1 million people in the U.S. and 3.5 million people worldwide suffer from eosinophilic asthma, for which there are limited treatment alternatives.

Aerovant provides these patients with a non-steroidal treatment option that shows benefit after only 12 weeks of dosing, delivered as an inhaled dry powder, as are the most widely-used asthma medications. [0252] The clinical trial was conducted at 75 sites in the United States and

Europe. In the study, patients with moderate to severe asthma who were inadequately controlled on the combination of inhaled corticosteroids (ICS) and long-acting beta agonists (LAB A) were assigned to receive one of three doses (1 mg, 3 mg or 10 mg) of Aerovant or placebo by inhalation twice daily for 12 weeks. Consecutive visits from Visit 2 to Visit 8 were 2 weeks apart. During this time, the standard ICS and LABA therapies were gradually withdrawn. The trial is the largest clinical study to date of an IL-4/IL-13 inhibitor, a compound that targets a root cause of the inflammatory cascade in asthma.

[0253] Eosinophilic asthma, which can be diagnosed with a simple blood or sputum test in patients with clinical symptoms and signs of asthma, is a type of airway inflammation associated with increased levels of eosinophils, a type of white blood cell. Patients living with eosinophilic asthma may experience changes in their airways, impaired lung function, more frequent asthma exacerbations and life- threatening asthma attacks.

Trial Protocol

[0254] The phase 2b trial protocol followed is given below.

Weeks 1-4; Run-in Period

[0255] Patients were switched to fluticasone + salmeterol and the doses maintained during the run-in period.

Weeks 5-8: Stabilization Period

[0256] Aerovant (doses 1, 3, 10 mg) or placebo twice daily were added to fluticasone + salmeterol.

Weeks 9-16: Treatment Reduction Period

[0257] Salmeterol was removed completely at beginning of treatment reduction period. The fluticasone dose was halved after 2 weeks and eliminated after 4 weeks for patients receiving < 500mcg fluticasone per day at the beginning of the study. The fluticasone dose was halved after 2 weeks and after 4 weeks and eliminated completely after 6 weeks for patients receiving > 500mcg fluticasone per day at the beginning of the study.

Week 17: Follow-up

[0258] Follow-up was carried out 1 week after the last dose of Aerovant. Primary Endpoint:

[0259] The primary endpoint was asthma exacerbation incidence as compared to placebo.

Patient Population

[0260] The study design and patient population mimicked those for anti-lgE, anti-TNFalpha, and inhaled steroid clinical Ph 2/3 pivotal trials. The patient population consisted of asthma patients:

(1) likely to exacerbate on withdrawal of steroids;

(2) with moderate to severe asthma on moderate-high dose inhaled corticosteroids (ICS);

(3) with 1 exacerbation in the past 2 years; and

(4) < 20 on ACT, symptomatic, borderline "controlled" asthma

Inclusion Criteria

[0261] The inclusion criteria where as follows:

(1) male or female >18yrs with history of asthma and not fully

controlled on current asthma therapy;

(2) GINA-defined mod-persistent asthma;

(3) moderate-high doses of ICS/long-acting beta agonist (LABA) in form of combination therapy;

(4) 1 exacerbation in previous 2 years;

(5) FEV 1 : 50-95% predicted;

(6) > 12% bronchodilator reversibility; (7) < 20 on ACT™;and

(8) not pregnant, non-smoker, stable for 4 weeks, and willing to give consent.

Exclusion Criteria

[0262] The exclusion criteria where as follows:

(1 ) respiratory diagnosis other than asthma (e.g. COPD);

(2) OCS or anti-lgE treatment within 4 wks of screen or LT antagonist treatment within one week of screen;

(3) intubated for asthma in past 5 years;

(4) pregnant or lactating female;

(5) clinical history that precludes steroid reduction;

(6) use of drugs that would interfere in study;

(7) alcohol or substance abuse, excessive alcohol consumption;

(8) has taken AER 001 previously;

(9) participation in a trial within 12 wks of randomization; and

(10) cannot reliably communicate.

Primary Endpoint of Trial: Exacerbation Incidence

[0263] Exacerbation was defined by any one of the following:

(1 ) morning PEF > 30% below baseline for > 2 consecutive days and > 6 additional reliever occasions (1 puff = 1 reliever occasion) in a

24-hour period over baseline for > 2 consecutive days; or

(2) deterioration of asthma requiring treatment with oral corticosteroids; or

(3) deterioration of asthma requiring treatment increase > 4 times the baseline dose of inhaled corticosteroids; or

(4) deterioration of asthma requiring hospitalization; or (5) in the opinion of the Investigator, the patient's condition was deteriorating significantly to the point of considering it an asthma exacerbation.

Note: A patient could experience only one exacerbation of their asthma while on this study. Once exacerbated, the patient was withdrawn and considered "complete" as their condition had achieved the primary endpoint.

Daily Diary of Symptoms

[0264] Patients were required to keep a diary of asthma symptoms twice daily via an electronic recording device. The purpose of the diary was to record the impact of asthma on daily activities. The patients were also required to perform spirometry testing at home and to record the results. The scores shown below were used. For the purposes of this study, "AM Questions" were those defined as being answered by the patient in the morning upon waking, i.e., referring to the previous -12 hours or overnight symptoms. "PM Questions" were defined as those questions answered by the patient in the evening before retiring, i.e., referring to the previous ~12 hours or during the day when the patient was awake.

AM QUESTIONS:

1. How many puffs of rescue inhaler did you use last night?

[Patient typed in the number of puffs]

2. How many times did you awaken due to asthma last night?

0 Did not awaken due to asthma

1 Awoke once due to asthma

2 Awoke twice due to asthma

3 Awoke three times due to asthma

4 Was not able to sleep at all due to my asthma

3. Did you take your study drug this morning?

0 No

1 Yes PM QUESTIONS;

1. How many puffs of rescue inhaler did you use today?

[Patient typed in the number of puffs]

2. Describe your asthma symptoms today.

0 No noticeable symptoms of asthma

1 Mild asthma symptoms, barely noticeable

2 Asthma symptoms with exercise/exertion

3 Asthma symptoms with mild exertion

4 Almost constant difficulty breathing due to my asthma

3. Were your daily activities affected by your asthma today?

0 No effect of asthma on my daily activities

1 Activity was normal and only mildly affected by my asthma

2 Activity was normal but moderately affected by my asthma

3 Asthma symptoms limited my activity to a significant degree

4 Asthma symptoms severely restricted my daily activities

4. Did you take your study drug this evening?

0 No

1 Yes

Demographics

[0265] Table 19 shows the demographic characteristics of the global, eosinophilic asthma and non-eosinophilic asthma populations. Table 20 shows the demographic characteristics of the eosinophilic asthma population (>350 cells/mm ³ ) in the phase 2b study.

EXAMPLE 15

Interleukin 4 receptor polymorphisms predict therapeutic pitrakinra treatment response in moderate to severe asthma

Abstract

[0267] Rationale: This is the first large pharmacogenetic investigation of the inflammatory interleukins 4/13 (IL-4/-13) pathway in patients with uncontrolled, moderate to severe asthma. We hypothesized that genetic variants in the IL-4 receptor a gene (IL-4RA) would contribute to treatment response in a phase 2 randomized, placebo-controlled efficacy trial of pitrakinra (1 , 3 and 10 mg doses), a recombinant IL-4 mutein protein which antagonizes the IL-4 receptor, inhibiting the IL-4/-13 pathway (Clinicaltrials.gov NCT00801853).

[0268] Methods: Non-Hispanic whites (n=407) in the intent-to-treat population were genotyped for IL-4RA single-nucleotide polymorphisms (SNPs). All polymorphisms tested for association (n=19) with the primary endpoint of asthma exacerbations had a minor allele frequency >5% and were in Hardy- Weinberg equilibrium. Additive SNP pharmacogenetic associations were tested using stratified contingency tables or regression models adjusted for geographic region with a SNP by treatment interaction term.

[0269] Results: Consistent pharmacogenetic associations with asthma exacerbations were observed for correlated tagging SNPs rs8832 and rs 1029489 in the IL-4RA 3' untranslated and proximal gene region. There was a significant pitrakinra dose response trend in treatment outcome for asthma exacerbation in subjects with the major allele in IL-4RA/rs8832 (P=0.009), IL-4RA/rs 1029489 (P=0.005) and IL-4RA variants rs3024585, rs3024622, and rs4787956 (P=0.03). For example, individuals homozygous for the common IL-4RA/rs8832 GG genotype had 25%, 16%, 12%, and 3% frequency of exacerbations in the placebo, 1 mg, 3 mg, and 10 mg treatment groups respectively.

[0270] Conclusions: IL-4 receptor variation interacts with pitrakinra treatment responses, resulting in a dose-dependent reduction in exacerbations in subjects with moderate to severe asthma. Summary of clinical trial results

[0271] Data for this pharmacogenetic analysis was obtained from a double- blind, randomized, placebo-controlled, multicenter trial of the efficacy and safety of inhaled pitrakinra, a recombinant form of 1L-4 and competitive antagonist of IL-4 IL- 13, in 534 intent-to-treat subjects with moderate to severe asthma who had at least one exacerbation in the past two years (Clinicaltrials.gov NCT00801853).

[0272] Subjects were run-in for 4 weeks on fluticasone and salmeterol, then randomized to 1 mg (n=132), 3 mg (n=137), 10 mg (n=128) pitrakinra or placebo (n=137) for a 12-week treatment period. Subjects were stabilized on pitrakinra as corticosteroids or long-acting beta agonists were withdrawn.

[0273] Incident exacerbation was defined by one of the following: a morning peak flow of > 30% below baseline for > 2 consecutive days and > 6 additional uses of reliever medication in a 24-hour period over baseline for > 2 consecutive days; or deterioration of asthma requiring treatment with oral corticosteroids; or deterioration of asthma requiring treatment increase > 4 times the baseline dose of inhaled corticosteroids; or deterioration of asthma requiring hospitalization; or in the opinion of the investigator, the patient's condition had deteriorated significantly enough be considered an asthma exacerbation.

[0274] No statistically significant changes were observed for primary endpoint of incident asthma exacerbations or secondary endpoints in the overall study population.

[0275] Subjects with blood eosinophilia > 350 cells/mm ³ (n=125) showed statistically significant changes favoring active pitrakinra for incidence of asthma exacerbations (p=0.004, 10 mg vs placebo), time to exacerbation (p=0.008, 10mg vs placebo), and for asthma symptom scores.

Methods

Pharmacogenetic study population

[0276] The study population consisted of 407 non-Hispanic whites from the pitrakinra intent-to-treat population (Table 22).

Genotvping

[0277] Genomic DNA was isolated by PPD Central Labs.

[0278] Interleukin 4 receptor alpha gene (IL-4RA) single nucleotide polymorphisms (SNPs) were genotyped using Sequenom MassARRAY iPLEX platform in the laboratory of Dr. R.E. Slager at Wake Forest Center for Genomics and Personalized Medicine Research.

[0279] IL-4RA SNPs tested for association (n=19) had a minor allele frequency >5% and were in Hardy- Weinberg equilibrium (Table 23).

Statistical methods

[0280] Additive SNP associations with the primary endpoint incident exacerbation were evaluated using contingency tables stratified by treatment assignment or regression models adjusted for geographic region.

[0281] To assess dose response, treatment group by exacerbation status contingency tables were developed, stratified by genotype (heterozygotes and homozygous recessive subjects combined), testing for trend.

[0282] Kaplan-Meier plots with a log-rank test for significance were developed to evaluate differences in time to exacerbation by treatment assignment, stratified by genotype.

[0283] Analysis was performed using Plink v l .07, Haploview v4.2, and

SPSSv l 8. Hypothesis

[0284] The primary hypothesis for this analysis is that variation in IL-4RA would predict reductions in asthma exacerbations in participants randomized to pitrakinra therapy. Results

[0285] The results of the analysis are shown in Figures 5-10. Table 24

(below) shows IL-4RA sliding window haplotypes from the 3' end and proximal gene region with frequencies > 10% for the 3 and 10 mg pitrakinra treatment levels. P values and odds ratios (OR) are included for haplotype association with asthma exacerbations, adjusted for geographic region in logistic regression models. An OR > 1 indicates a haplotype is associated with increased exacerbations.

Summary

[0286] Seven IL-4RA SNPs were associated with a significant (additive) difference in exacerbation frequency in subjects randomized to 3 and 10 mg pitrakinra; except for rsl 1 10470, subjects homozygous for most major allele were less likely to exacerbate. Correlated tagging SNPs rs8832 and rsl029489 in the IL-4RA 3' untranslated and proximal gene region were most consistently associated with exacerbations and secondary outcomes. There was a dose-dependent

(placebo/1 mg/3mg/10mg pitrakinra) reduction in asthma exacerbations in subjects with the major allele in rs8832 (25%, 16%, 12%, and 3% frequency of exacerbations, respectively; P=0.009), rsl 029489 (P=0.005) and rs3024585, rs3024622, and rs4787956 (P=0.03). There was a dose-related increased time to exacerbation in subjects with the rs8832 GG genotype (P=0.02; log rank test l Omg vs placebo). There was a dose-response relationship between global asthma symptoms scores including asthma-related night awakenings in subjects with the rs8832 GG genotype (P=0.02). After correction for an estimated 10 effective SNP tests (r2<0.5), rsl 029489 remained nominally associated with pitrakinra dose response (P=0.05). This pharmacogenetic analysis reveals a group of non-Hispanic white subjects with moderate to severe asthma by genotype who responded significantly better to pitrakinra therapy than the overall study population.

References

[0287] Wenzel et al., 2007 AJCCRM 175: 570-6

[0288] Wenzel et al., 2010 ERS meeting abstract

[0289] Slager RE et al., 2010 JACI 126: 875-8