EP3371225A2 - Dipeptidyl peptidase-4 and periostin as predictors of clinical response to eosinophil-targeted therapeutic agents in eosinophilic diseases - Google Patents

Abstract

The present disclosure relates to the use of protein and/or gene expression levels of dipeptidyl peptidase-4 (DPP4/CD26) and periostin (POSTN) as peripheral biomarkers for eosinophilic diseases or disorders, e.g., moderate to severe asthma. Levels of the DPP4 and/or POSTN above or below predetermined DPP4 or POSTN threshold levels can be used, e.g., (i) to determine a patient's eligibility for a certain treatment with an IL-5R antagonist, e.g., an anti-IL- 5R antibody such as benralizumab (MEDI-563), (ii) to determine whether a certain treatment of an eosinophilic disease or disorder with a specific IL-5R antagonist should commence, be suspended, or be modified, (iii) to diagnose whether an eosinophilic disease or disorder is treatable or not treatable with a specific IL-5R antagonist, (iv) to prognosticate the outcome of treating an eosinophilic disease or disorder with a specific IL-5R antagonist, etc.

Description

DIPEPTIDYL PEPTIDASE-4 AND PERIOSTIN AS PREDICTORS OF CLINICAL RESPONSE TO EOSINOPHIL-TARGETED THERAPEUTIC

AGENTS IN EOSINOPHILIC DISEASES

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The content of the electronically submitted sequence listing in ASCII text file

(Name: DPP4_sequence listing_ST25_ascii.txt; Size: 52,739 bytes; and Date of Creation: November 4, 2015) filed with the application is incorporated herein by reference in its entirety.

BACKGROUND

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

[0003] Bronchial asthma is a common persistent inflammatory disease of the lung characterized by airways hyper-responsiveness, mucus overproduction, fibrosis, and raised serum IgE levels. Airways hyper-responsiveness (AHR) is the exaggerated constriction of the airways to non-specific stimuli such as cold air. Both AHR and mucus overproduction are thought to be responsible for the variable airway obstruction that leads to the shortness of breath characteristic of asthma attacks (exacerbations) and which is responsible for the mortality associated with this disease.

[0004] Current British Thoracic Society (BTS) and Global Initiative for Asthma (GINA) guidelines suggest a stepwise approach to the treatment of asthma (Society, B. T., Thorax, 2003. 58 Suppl 1:1-94; GINA, Global Strategy for Asthma Management and Prevention. 2002, National Institute of Health). Mild to moderate asthma can usually be controlled by the use of inhaled corticosteroids, in combination with beta-agonists or leukotriene inhibitors. However, due to the documented side effects of corticosteroids, patients tend not to comply with the treatment regime, which reduces the effectiveness of treatment (Milgrom, H. et al. Ann Allergy Asthma Immunol, 2002. 88:429-31; Fish, L. and C. L. Lung, Ann Allergy Asthma Immunol, 2001. 86:24-30; Bender, B. G. J. Allergy Clin. Immunol, 2002. 109:S554- 9). Asthma presents significant heterogeneity in response to various treatments, thereby highlighting the need to develop more effective therapies for this disease or identify biomarkers that predict response to specific therapies.

[0005] The terms "precision medicine," "personalized health care," or "targeted therapeutics" are often used interchangeably to describe efforts geared to tailor therapies for patient subgroups with a diverse collection of molecular and clinical characteristics, such as asthma. Current treatment of asthma is dominated by inhaled and oral corticosteroids in combination with bronchodilators. Although very effective in most patients, about 10 to 20% of the patients with asthma remain poorly controlled with current standard of care (Bousquet et al., Allergy Clin Immunol 126, 926-938 (2010)). Recently, much progress has been made through the discovery of new biomarkers using "omics" approaches, linking clinical phenotypes with molecular biomarkers and referred to as asthma "endotypes" (Anderson, Lancet 372, 1107-1119 (2008); Lotvall et al. J Allergy Clin Immunol 127, 355-360 (2011); Wenzel, Pulm Pharmacol Ther 26, 710-715 (2013)).

[0006] In some cases, these biomarkers are useful predictors of treatment response. For example, patients with elevated IgE, eosinophilic inflammation or high levels of periostin, a surrogate marker for interleukin (IL)-13 activity in asthma, preferentially respond to monoclonal antibodies targeting immunoglobulins E (Di Domenico et al., Inflamm Allergy Drug Targets 10, 2-12 (2011)), IL-5/IL-5RCC (Castro et al., Lancet Respir Med 3, 355-366 (2015)), or IL-13/IL-4RCC (Wenzel, Pulm Pharmacol Ther 26, 710-715 (2013)), respectively. However, a better appreciation of the dynamic changes of endotypes over time and with treatment, associations with clinical phenotypes and ultimately the discovery of new endotypes, which allow for the development of new and tailored treatments for patients whose asthma remains poorly controlled, is needed (Holgate et al., Nat Rev Drug Discov 14, 367-368 (2015)).

BRIEF SUMMARY

[0007] The present disclosure provides a method of treating an eosinophilic disease or disorder in a patient in need thereof, comprising administering an eosinophil-targeted therapeutic agent to the patient if the patient is determined or identified to have lower or decreased dipeptidyl peptidase-4 (DPP4) and/or periostin (POSTN) levels in one or more samples taken from the patient compared to predetermined DPP4 or POSTN threshold levels, or compared to DPP4 or POSTN threshold levels in one or more control samples.

[0008] Also provided are methods of treating a patient having an eosinophilic disease or disorder comprising suspending or not initiating the administration of an eosinophil-targeted therapeutic agent to the patient if the patient is determined or identified to have higher or increased DPP4 and/or POSTN levels in one or more samples taken from the patient compared to predetermined DPP4 or POSTN threshold levels, or compared to DPP4 or POSTN threshold levels in one or more control samples.

[0009] The disclosure provides also methods of treating an eosinophilic disease or disorder in a patient in need thereof, wherein the patient failed, was non-responsive or intolerant to treatment with a therapeutic agent comprising administering an eosinophil- targeted therapeutic agent to the patient if the patient is determined or identified to have lower or decreased DPP4 and/or POSTN levels in one or more samples taken from the patient compared to predetermined DPP4 or POSTN threshold levels, or compared to DPP4 or POSTN threshold levels in one or more control samples.

[0010] Also provided are methods of determining whether to treat a patient having an eosinophilic disease or disorder with an eosinophil-targeted therapeutic agent, comprising determining to treat the patient if the patient is determined or identified to have lower or decreased DPP4 and/or POSTN levels in one or more samples taken from the patient compared to predetermined DPP4 or POSTN threshold levels, or compared to DPP4 or POSTN threshold level in one or more control samples.

[0011] The disclosure also provides methods of selecting a patient diagnosed with an eosinophilic disease or disorder as a candidate for treatment with an eosinophil-targeted therapeutic agent, comprising selecting the patient for treatment if the patient is determined or identified to have lower or decreased DPP4 and/or POSTN levels in one or more samples taken from the patient compared to predetermined DPP4 or POSTN threshold levels, or compared to DPP4 or POSTN threshold levels in one or more control samples.

[0012] In some aspects, the methods disclosed above further comprise measuring the level of DPP4 and/or POSTN in one or more of the samples obtained from the patient or instructing a clinical laboratory or healthcare provider to measure the level of DPP4 and/or POSTN in the sample and/or submitting the one or more samples obtained from the patient to a clinical laboratory or healthcare provider to measure the level of DPP4 and/or POSTN in the sample. In some aspects, the methods disclosed above further comprise determining the level of DPP4 and/or POSTN in the one or more samples obtained from the patient. In some aspects, the methods disclosed above further comprise advising a healthcare provider to

(a) administer an eosinophil-targeted therapeutic agent to the patient if the patient is determined or identified to have lower or decreased DPP4 and/or POSTN levels in one or more samples taken from the patient compared to predetermined DPP4 or POSTN threshold levels, or compared to DPP4 or POSTN threshold levels in one or more control samples; or

(b) suspend, not initiate, or deny the administration of an eosinophil-targeted therapeutic agent to the patient if the patient is determined or identified to have higher or increased DPP4 and/or POSTN levels in one or more samples taken from the patient compared to predetermined DPP4 or POSTN threshold levels, or compared to DPP4 or POSTN threshold levels in one or more control samples. In some aspects, the patient has elevated blood eosinophils.

[0013] In some aspects, the eosinophil-targeted therapeutic agent is a monoclonal antibody or an antigen-binding fragment thereof. In some aspects, the monoclonal antibody or antigen-binding fragment thereof specifically binds to IL-5 or IL-5R. In some aspects, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to IL-5 is mepolizumab, reslizumab, or an antigen-binding fragment thereof.

[0014] In some aspects, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to IL-5R, specifically binds to the alpha subunit of IL-5R (SEQ ID NO: 4). In some aspects, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to the alpha subunit of IL-5R is benralizumab (MEDI-563), or an antigen- binding fragment thereof. In some aspects, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to the alpha subunit of IL-5R comprises a heavy variable region (VH) comprising, consisting, or consisting essentially of SEQ ID NO: 12 and/or a light chain variable region (VL) comprising, consisting, or consisting essentially of SEQ ID NO: 13.

[0015] In some aspects, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to the alpha subunit of IL-5R comprises one, two, or three complementarity determining regions selected from SEQ ID NOS: 14-16 and/or one, two, or three complementarity determining regions selected from SEQ ID NOS: 17-19. In some aspects, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to the alpha subunit of IL-5R comprises at least one, two, three, four, five or six complementarity determining regions selected from SEQ ID NOS: 14-19.

[0016] In some aspects, the monoclonal antibody or antigen-binding fragment thereof binds to the same epitope as mepolizumab, reslizumab, or benralizumab, or competitively inhibits binding of mepolizumab, reslizumab, or benralizumab to their respective target epitopes. In some aspects, the eosinophil-targeted therapeutic agent is a fusion protein or a conjugate comprising mepolizumab, reslizumab, or benralizumab, or an antigen-binding fragment thereof. In some aspects, the fusion protein or conjugate comprises at least one heterologous therapeutic moiety and/or a half-life enhancing moiety.

[0017] In some aspects, the eosinophil-targeted therapeutic agent is a polynucleotide. In some aspects, the polynucleotide is a DNA or an RNA. In some aspects, the polynucleotide is (i) an mRNA or a combination thereof, or (ii) an antisense oligonucleotide or a combination thereof. In some aspects, the antisense oligonucleotide or combination thereof is ASM8, PXS1100, or PXS2200. In some aspects, the polynucleotide comprises at least a nucleotide analog.

[0018] In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered at a fixed dose. In some aspects, the fixed dose is between 1 and 1,000 mg/dose. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered in two or more doses. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered weekly, biweekly or monthly. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered at about 2 mg to about 100 mg per dose. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered at about 20 mg per dose, at about 30 mg per dose, or at about 100 mg per dose. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered once every four weeks to once every twelve weeks. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered once every four weeks. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered once every eight weeks. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered once every four weeks for twelve weeks and then once every eight weeks.

[0019] In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered orally, by inhalation, intravenously, intramuscularly, subcutaneously, or a combination thereof.

[0020] In some aspects, the eosinophilic disease or disorder is a pulmonary disease or disorder. In some aspects, the pulmonary disease or disorder is asthma or COPD. In some aspects, the asthma is allergic asthma, atopic asthma, corticosteroid naive asthma, chronic asthma, corticosteroid resistant asthma, corticosteroid refractory asthma, asthma due to smoking, or asthma uncontrolled on corticosteroids. In some aspects, the asthma is mild- to- moderate asthma (defined as GINA 1-3) or severe asthma (defined as GINA 4+).

[0021] In some aspects, the eosinophilic disease or disorder is chronic bronchitis, chronic eosinophilic pneumonia (CEP), nasal polyposis, atopic dermatitis (eczema), eosinophilic esophagitis, hypereosinophilic syndrome (HES), eosinophilic granulomatosis and polyangitis (Churg-Strauss syndrome), eosinophilic gastritis, eosinophilic enteritis, eosinophilic colitis, allergic rhinoconjunctivitis (hay fever), leukemia, lymphoma, mastocytosis, atheroembolic disease, hyper-IgE syndrome, Omenn's syndrome, thymoma, transplant rejections, hypoadrenalism, bullous pemphigoid, pemphigus vulgaris, dermatitis herpetiformis, drug- induced lesions, urticaria, eosinophilic panniculitis, angioedema with eosinophilia, Kimura's disease, Shulman's syndrome, Well's syndrome, eosinophilic ulcer of the oral mucosa, eosinophilic pustular folliculitis, recurrent cutaneous necrotizing eosinophilic vasculitis, drug/toxin-induced eosinophilic lung disease, Loeffler's syndrome, allergic bronchopulmonary aspergillosis, eosinophilic granuloma, pleural eosinophilia, gastroesophageal reflux, parasitic infection, fungal infection, Helicobacter pylori infection, inflammatory bowel disease (ulcerative colitis and Crohn's disease), food allergic disorder, protein-induced enteropathy, protein-induced enterocolitis, allergic colitis, celiac disease, pemphigus vegetans, leiomyomatosis, connective tissue disorder, vasculitic disorder, chronic subdural hematoma, central nervous system infection, ventriculoperitoneal shunts, congenital heart condition (septal defects or aortic stenosis), eosinophiluria associated with kidney infection, interstitial nephritis, or eosinophilic cystitis.

[0022] In some aspects, the patient has been treated either before, during, after, or alternatively to the administration of an eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) with one or more additional therapies for the treatment of the eosinophilic disease or disorder. In some aspects, the one or more additional therapies further comprise a steroid, bronchodilator, or both. In some aspects, the steroid is fluticasone or budesonide. In some aspects, the bronchodilator is salbutamol or salmeterol. In some aspects, the one or more additional therapies are administered by inhalation, by oral administration, by injection, or by a combination thereof. In some aspects, inhalation administration is conducted using a metered dose inhaler (MDI) or a dry powder inhaler (DPI). In some aspects, the steroid is administered at a high dose. In some aspects, the steroid is an inhaled corticosteroid such as beclomethasone or mometasone.

[0023] In some aspects, the one or more samples taken from the patient and/or the one or more control samples comprises one or more of whole blood, serum, plasma, saliva, sputum, bronchoalveolar lavage fluid, lung epithelial cells, urine, skin, nasal polyps, or a combination thereof. In some aspects, the sample taken from the patient is blood serum. In some aspects, the one or more control samples are (a) a sample or samples obtained from (i) normal healthy individuals, (ii) patients with a subset of asthma; (iii) asthma patients naive for corticosteroid treatment; or asthma patients treated with corticosteroids; (b) a pre-determined standard amount of isolated DPP4 or POSTN; or, (c) any combination samples in (a) and (b).

[0024] In some aspects, the methods disclosed above further comprise determining, submitting a sample taken from the patient for determination, or instructing a clinical laboratory to determine (i) the level of the patient's IgE levels, (ii) the patient's eosinophil count, (iii) the patient's Fraction of Exhaled Nitric Oxide (FENO), (iv) the patient's Eosinophil/Lymphocyte and Eosinophil/Neutrophil (ELEN) index, (v) the patient's EOS index, (vi) the patients wall area percentage (WA%) of subsegmental airways from a CT scan of the lungs, (vii) a combination of two or more thereof. See, e.g., Int'l Publ. No. WO2012158954, which is herein incorporated by reference in its entirety.

[0025] In some aspects, the patient's eosinophil count is > 300 eosinophils/μΐ.. In some aspects, > 300 eosinophils/μΐ. is set as a threshold for mild to moderate asthmatics. In some aspects, the patient's eosinophil count is > 400 eosinophils/μΐ.. In some aspects, > 400 eosinophils/μΐ. is set as a threshold for severe asthmatics. In some aspects, the patient's eosinophil count is > 150 eosinophils^!.. In some aspects, > 150 eosinophils^!, is set as a threshold, for example, for patients undergoing treatment with an anti-IL5 antibody (e.g., mepolizumab). [0026] In some aspects, the patient's DPP4 level is measured in a DPP4 detection assay, wherein the DPP4 detection assay is an immunoassay. In some aspects, the patient's POSTN level is measured in a POSTN detection assay, wherein the POSTN detection assay is an immunoassay.

[0027] In some aspects, the DPP4 detection immunoassay is (i) the immunoassay described in Example 2; or, (ii) an immunoassay disclosed in TABLE 1; or, (ii) a multiplexed immunoassay. In some aspects, the multiplexed immunoassay is an EMD Millipore MILLIPLEX™, Bio-Rad BIO-PLEX™, Life Technologies NOVEX MULTIPLEX™, Thermo Fisher Scientific LUMINEX®, PerkinElmer ALPHAPLEX™, Affymetrix (eBioscience) PROCARTA™, or R&D Systems LUMINEX® assay. In some aspects, the POSTN detection assay is an assay disclosed in WO2015120171A1 or WO2015120185, both of which are herein incorporated by reference in their entireties.

[0028] In some aspects, the methods disclosed above further comprise determining, submitting a sample taken from the patient for determination, or instructing a clinical laboratory to determine the expression level or activity of other molecular biomarkers such as IL-22, IL-25, IL-33, TSLP, LCN2, CCL20, sCTLA-3, sCD28, CCL5, CCL11, CCL22, CST1, CCL26, CLCA1, CST2, PRR4, SERPINB2, CEACAM5, iNOS, SERPINB4, CST4, PRB4, TPSD1, TPSG1, MFSD2, CPA3, GPR105, CDH26, GSN, C20RF32, TRACH2000196 (TMEM71), DNAJC12, RGS13, SLC18A2, SERPINB10, SH3RF2, FCER1B, RUNX2, PTGS1, ALOX15, or combinations thereof.

[0029] In some aspects, the predetermined DPP4 or POSTN threshold levels are selected from

(a) about the mean DPP4 level or POSTN level as measured in blood serum from a plurality of patients using an immunoassay;

(b) about the median DPP4 level or POSTN level as measured in blood serum from a plurality of patients using an immunoassay; and

(c) about the 1^st, 2^nd, 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, or 9^th decile baseline DPP4 level or POSTN level as measured in blood serum from a plurality of patients using an immunoassay; wherein the patients are (i) normal healthy volunteers, (ii) patients with mild to moderate-asthma, and/or (iii) patients with an severe- asthma. In some aspects, the blood serum from a plurality of patients is a pooled sample or individual samples. [0030] In some aspects, the predetermined DPP4 threshold level is at least about 103 ng/mL to at least about 867 ng/mL as measured using an immunoassay. In some aspects, the predetermined DPP4 threshold level is at least about 363 ng/mL as measured using an immunoassay. In some aspects, the predetermined DPP4 threshold level is at least about 376 ng/mL as measured using an immunoassay. In some aspects, the immunoassay is the DPP4 detection assay described in Example 2 or an DPP4 detection assay disclosed in TABLE 1.

[0031] In some aspects, the predetermined POSTN threshold is at least about 7 ng/mL to at least about 104 ng/mL as measured using an immunoassay. In some aspects, the predetermined POSTN threshold level is at least about 23 ng/mL as measured using an immunoassay. In some aspects, the predetermined POSTN threshold level is at least about 26 ng/mL as measured using an immunoassay. In some aspects, the POSTN immunoassay is an assay disclosed in WO2015120171A1 or WO2015120185, or a commercial POSTN assay.

[0032] In some aspects, administration of the eosinophil-targeted therapeutic agent results in (a) AER (Acute Exacerbation Rate) reduction; (b) FEV1 (Forced Expiratory Volume in one second) increase; (c) improved ACQ-6 (Asthma Control Questionnaire, 6-item version) value; or (d) a combination thereof.

[0033] In some aspects, the AER reduction is at least about 15%, 20%, 25%, 30%, 35%,

40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the AER baseline values observed in a population of patients treated with a placebo. In some aspects, the FEV1 increase is at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400, 450 or 500 mL compared to the FEV1 baseline values observed in a population of patients treated with a placebo. In some aspects, the improved ACQ-6 value is a change from baseline of at least about -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1, -1.1, or -1.2 compared to the ACQ-6 baseline values observed in a population of patients treated with a placebo.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0034] FIG. 1 shows the design of benralizumab Phase 2b study CP220 (NCT01238861) in severe controlled asthma. ACQ-6, Asthma Control Questionnaire-6; AER, annual exacerbation rate (total observed exacerbations to week 52 divided by total duration of person- year follow-up); CBC, complete blood count with differential; FeNo, fraction of exhaled nitric oxide; FEVi, forced expiratory volume in 1 second; ICS, inhaled corticosteroids; ppb, parts per billion; SC, subcutaneous. The ELEN Index is a mathematical algorithm to predict sputum eosinophils from CBC data, as described in Khatry et al. Am J Respir Crit Care Med 189:A4257 (2014).

[0035] FIG. 2 is a table summarizing the data related to DPP4 and POSTN in serum obtained in the CP220 study (NCT01238861). "DPP4 High" values (i.e., higher or increased DPP4 levels) are those equal or above the median. "POSTN High" values are those equal or above the median. "DPP4 Low" values (i.e., lower or decreased DPP4 levels) are those below the median. "POSTN Low" values are those below the median.

[0036] FIG. 3 shows the prevalence of subgroups DPP4 High and Low (A), and POSTN

High and Low (panel B) by blood eosinophil < or > 300 cells/uL.

[0037] FIG. 4 shows the lack of correlation between serum DPP4 levels and serum

POSTN levels in subjects participating in the CP220 study (panel A); that serum POSTN correlates with blood eosinophil numbers (panel B); and that serum DPP4 does not correlate with blood eosinophil number (panel C).

[0038] FIG. 5 shows a comparison of the effect of benralizumab on exacerbation rate reduction for the combined 20 mg plus 100 mg dose groups vs placebo in DPP4 High and Low sub-groups (panel A) and POSTN High and Low subgroups (panel B). * = P <0.05. Numbers above the bars represent n in placebo/benralizumab treatment groups. Percentage numbers at the foot of the x-axis represent prevalence of each sub-group.

[0039] FIG. 6 shows a comparison of the effect of benralizumab on FEVl for the combined 20 mg plus 100 mg dose groups vs placebo in DPP4 High and Low sub-groups (panel A) and POSTN High and Low subgroups (panel B). * = P <0.05. Numbers above the bars represent n in placebo/benralizumab treatment groups. Percentage numbers at the foot of the x-axis represent prevalence of each sub-group.

[0040] FIG. 7 shows a comparison of the effect of benralizumab on ACQ-6 for the combined 20 mg plus 100 mg group vs placebo in DPP4 High and Low sub-groups (panel A) and POSTN High and Low subgroups (panel B). * = P <0.05. Numbers under the bars represent n in placebo/benralizumab treatment groups. Percentage numbers above the labels of the x-axis represent prevalence of each sub-group. DETAILED DESCRIPTION

[0041] Eosinophils are a key effector cell in the pathology of asthma and more than 50% of severe asthmatics are associated with persistent eosinophilic inflammation despite treatment with corticosteroids. IL-13, a Th2 cytokine produced by mast cells, basophils and eosinophils, contributes to key features of asthma through a number of different mechanisms. Benralizumab (MEDI-563), a humanized anti-IL-5Ra mAb that binds with high affinity to the alpha chain of the IL-5R to block IL-5 function, induces apoptosis of eosinophils and basophils through antibody-dependent cell-mediated cytotoxicity (ADCC) thereby depleting eosinophils and basophils.

[0042] In a phase 2b clinical trial (CP220), benralizumab showed greatest efficacy to reduce asthma exacerbations in patients with increased levels of blood eosinophils and this is currently being further evaluated in Phase III clinical trials. It is recognized that there may be overlap of severe asthma patients with eosinophilic inflammation as well as increased IL-13 pathway activation. Therefore we sought to identify biomarkers that may be more selective in identifying patients who may respond to anti-eosinophil therapy, e.g., using benralizumab.

[0043] Serum samples collected in the Phase 2b trial of benralizumab (CP220) were analyzed to assess predictive utility of baseline DPP4 and POSTN levels on reduction of exacerbation rate in benralizumab treated subjects compared to placebo treated.

[0044] Blood eosinophils and POSTN were not correlated with serum DPP4 concentration. In contrast, serum POSTN correlated with blood eosinophil numbers. When segmenting patients in the benralizumab and placebo treated groups by high levels of DPP4 or POSTN (greater than or equal to median) or low levels of DPP4 or POSTN (less than the median), the efficacy of benralizumab on reduction of exacerbations was greatest in subjects with low levels of serum DPP4. This effect with low levels of DPP4 was evident in subjects with both high (> 300 cells/micro liter) and low (<300 cells/micro liter) blood eosinophil levels. The effect with periostin was equivalent with high and low levels in subjects with high blood eosinophils but in subjects with low blood eosinophils the effect was greatest in subjects with low levels of periostin.

[0045] A key observation in the present disclosure is that asthma patients with elevated blood eosinophils who also have IL13-driven disease as assessed by above median POSTN or DPP4 levels derive less benefit from benralizumab. This reduced efficacy is more apparent in DPP4 high than POSTN high, which may be explained by the correlations of POSTN with eosinophils. In addition, asthma patients who have low eosinophils and low IL 13 -driven disease as assessed by below median POSTN or DPP4 levels derive a benefit from treatment with benralizumab. In other words, it isn't eosinophilic disease alone that determines benralizumab efficacy and potentially that of anti-IL5 antibodies. Rather, it is suggested that IL-13 driven disease or lack thereof, assessed by the presence of low DPP4 and/or POSTN levels that appears to be predictive of benralizumab efficacy.

[0046] The present disclosure relates to the use of the DPP4 and/or periostin (POSTN) levels as biomarkers for eosinophilic diseases or disorders, e.g., asthma (and in particular moderate and severe asthma). The disclosure provides, for example, methods for diagnosing and treating a subject having a eosinophilic disease or disorder comprising administering an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563), to the patient if DPP4 or POSTN levels in one or more samples taken from the patient are (i) below the limit of detection of a DPP4 or POSTN detection assay, (ii) below a predetermined DPP4 or POSTN threshold level, or (ii) below the DPP4 or POSTN level in one or more control samples.

[0047] In some aspects, the presence of DPP4 and/or POSTN levels (protein or RNA expression levels) above or below predetermined DPP4 or POSTN threshold levels in samples (e.g., blood serum or sputum) obtained from a patient suffering from an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) can be used, e.g., (i) to determine whether the patient is eligible or non-eligible for treatment with a specific therapeutic agent, (ii) to determine whether a specific treatment should commence, be suspended, or be modified, (iii) to diagnose whether the disease or disorder is treatable or not treatable with a specific therapeutic agent, (iv) to prognosticate the outcome of treatment of the disease or disorder with a specific therapeutic agent, etc. In some aspects, the specific therapeutic agent is an eosinophil-targeted therapeutic agent, e.g., anti-IL-5R antibody such as benralizumab (MEDI-563).

[0048] In other aspects, the presence of DPP4 and/or POSTN levels above or below predetermined DPP4 or POSTN threshold levels in samples (e.g., blood serum or sputum) obtained from a patient suffering from an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) in combination with

(i) one or more molecular biomarkers; and/or,

(ii) one or more clinical biomarkers; and/or, (iii) high eosinophil cell count (e.g., blood eosinophil count > 300 cells^L); and/or,

(iv) high Th2 (high Th2 defined, e.g., as IgE > 100 IU/mL and blood eosinophils > 0.14 x 10⁹/L); and/or,

(v) FEV1 reversibility to a short-acting β2 agonist, e.g., > 12%; and/or,

(vi) wall area % (WA%) of subsegmental airways above, e.g., about 68% as measured via CT scan of the lungs; and/or,

(vii) combinations thereof,

can be used, e.g.,

(i) to determine whether a patient suffering an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) is eligible or non-eligible for a specific treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563); and/or,

(ii) to determine whether a specific treatment should commence, be suspended, or be modified; and/or

(iii) to diagnose whether the disease or disorder is treatable or not treatable with a specific therapeutic agent; and/or

(iv) to prognosticate the outcome of treatment of the disease or disorder with a specific therapeutic agent.

[0049] In order that the present disclosure can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

I. Definitions

[0050] In this specification and the appended claims, the singular forms "a", "an" and

"the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein.

[0051] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0052] Wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of" and/or "consisting essentially of" are also provided.

[0053] The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + 15 %.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0055] Units, prefixes, and symbols are denoted in their Systeme International de Unites

(SI) accepted form.

[0056] Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the invention. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the invention. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the invention. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of an invention is disclosed as having a plurality of alternatives, examples of that invention in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an invention can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed. [0057] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

[0058] Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Nucleotides are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0059] The terms "polynucleotide," "oligonucleotide," "nucleic acid," "nucleic acid molecule," and "gene" are used interchangeably herein to refer to polymers of nucleotides of any length, and ribonucleotides, deoxyribonucleo tides, analogs thereof, or mixtures thereof.

[0060] The phrase "DNA sequence" refers to a contiguous nucleic acid sequence. The sequence can be either single stranded or double stranded, DNA or RNA, but double stranded DNA sequences are preferable. The sequence can be an oligonucleotide of 6 to 20 nucleotides in length to a full length genomic sequence of thousands or hundreds of thousands of base pairs.

[0061] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

[0062] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is

"isolated" is a polypeptide, polynucleotide, or composition which is in a form not found in nature. Isolated polypeptides, polynucleotides, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, a polypeptide, polynucleotide, or composition which is isolated is substantially pure.

[0063] The term "amino acid substitution" refers to replacing an amino acid residue present in a parent sequence with another amino acid residue. An amino acid can be substituted in a parent sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. Amino acid substitutions also occur in natural variants.

[0064] Accordingly, references to a "substitution at position X" or "substitution at position X" refer to the substitution of an amino acid present at position X with an alternative amino acid residue. In some aspects, substitution patterns can described according to the schema AXY, wherein A is the single letter code corresponding to the amino acid naturally present at position X, and Y is the substituting amino acid residue. In other aspects, substitution patterns can described according to the schema XY, wherein Y is the single letter code corresponding to the amino acid residue substituting the amino acid naturally present at position X.

[0065] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another aspect, a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and/or composition of side chain family members.

[0066] Non-conservative substitutions include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, He, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, He or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).

[0067] Other substitutions can be readily identified by workers of ordinary skill. For example, for the amino acid alanine, a substitution can be taken from any one of D-alanine, glycine, beta-alanine, L-cysteine and D-cysteine. For lysine, a replacement can be any one of D-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine, ornithine, or D- ornithine. Generally, substitutions in functionally important regions that can be expected to induce changes in the properties of isolated polypeptides are those in which (i) a polar residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is substituted for (or by) any other residue; (iii) a residue having an electropositive side chain, e.g., lysine, arginine or histidine, is substituted for (or by) a residue having an electronegative side chain, e.g., glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side chain, e.g., glycine. The likelihood that one of the foregoing non-conservative substitutions can alter functional properties of the protein is also correlated to the position of the substitution with respect to functionally important regions of the protein: some non-conservative substitutions can accordingly have little or no effect on biological properties.

[0068] The term "percent sequence identity" between two polypeptide or polynucleotide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence.

[0069] The percentage of sequence identity is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using readily available software both for online use and for download. Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.

[0070] Different regions within a single polynucleotide or polypeptide target sequence that align with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.

[0071] In certain aspects, the percentage identity "X" of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0072] One skilled in the art will appreciate that the generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequence- sequence comparisons exclusively driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One suitable program to generate multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com/muscle/. ClustalW2 and MUSCLE are alternatively available, e.g., from the EBI. [0073] As used herein, the term "antibody" (or a fragment, variant, or derivative thereof) refers to at least the minimal portion of an antibody which is capable of binding to antigen, e.g., at least the variable domain of a heavy chain (VH) and the variable domain of a light chain (VL) in the context of a typical antibody produced by a B cell. Basic antibody structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0074] Antibodies or antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, e.g., in US Pat. No. 5,892,019. Immunoglobulin or antibody molecules encompassed by this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0075] By "specifically binds," it is generally meant that an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.

[0076] An antibody or fragment, variant, or derivative thereof is said to competitively inhibit binding of a reference antibody or antigen binding fragment to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody or antigen binding fragment to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. A binding molecule can be said to competitively inhibit binding of the reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50%.

[0077] Antibodies or antigen-binding fragments, variants, or derivatives thereof disclosed herein can be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g., a target polysaccharide that they recognize or specifically bind. For example, the portion of the alpha subunit of human IL-5R that specifically interacts with the antigen-binding domain of an anti-IL-5R antibody such as benralizumab (MED 1-563) is an "epitope."

[0078] The term "epitope" as used herein refers to an antigenic protein determinant capable of binding to an antibody (e.g., an anti-IL-5R antibody) or a fragment, variant, or derivative thereof disclosed herein. In some aspects, the term epitope refers to a protein determinant (e.g., an amino acid sequence) of a subunit of the IL-5R protein, e.g., the alpha subunit of IL-5R. In some aspects, the term epitope refers to a protein determinant (e.g., an amino acid sequence) of the IL-5. In some aspects, binding of an antibody disclosed to either IL-5R or IL-5 prevents the interaction of IL-5R with IL-5.

[0079] Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. The part of an antibody or binding molecule that recognizes the epitope is called a paratope. The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. A conformational epitope is composed of discontinuous sections of the antigen's amino acid sequence. These epitopes interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. By contrast, linear epitopes interact with the paratope based on their primary structure. A linear epitope is formed by a continuous sequence of amino acids from the antigen.

[0080] The term "antibody binding site" refers to a region in the antigen (e.g., an amino acid sequence of IL-5 or IL-5R) comprising a continuous or discontinuous site (i.e., an epitope) to which a complementary antibody specifically binds. Thus, the antibody binding site can contain additional areas in the antigen which are beyond the epitope and which can determine properties such as binding affinity and/or stability, or affect properties such as antigen enzymatic activity or dimerization. Accordingly, even if two antibodies bind to the same epitope within an antigen, if the antibody molecules establish distinct intermolecular contacts with amino acids outside of the epitope, such antibodies are considered to bind to distinct antibody binding sites.

[0081] An antibody or fragment, variant, or derivative thereof is said to competitively inhibit binding of a reference antibody or antigen binding fragment to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody or antigen binding fragment to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. A binding molecule can be said to competitively inhibit binding of the reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, or at least 20%.

[0082] As used herein, the term "eosinophilic disease or disorder" refers to any disease or disorder characterized by an elevated level of eosinophils in blood, a tissue, or an organ. Normal levels in blood are on the order of 250 eosinophils per mm³. The term eosinophilic disease or disorder also encompasses eosinophilic inflammation. Blood levels over approximately 300 per mm³ are considered elevated. Examples of eosinophilic diseases and disorders include pulmonary diseases and disorders such as bronchial asthma, chronic bronchitis (in COPD), or chronic eosinophilic pneumonia (CEP). In addition, the term eosinophilic disease or disorder comprises diseases and conditions such as nasal polyposis, atopic dermatitis, eosinophilic esophagitis, hypereosinophilic syndrome (HES), eosinophilic granulomatosis and polyangitis (EGPA, formerly Churg-Strauss syndrome), eosinophilic gastritis, eosiophilic enteritis, eosinophilic colitis, etc. Normal and abnormal (e.g., elevated) eosinophil levels in the eosinophilic diseases or disorders disclosed herein are known in the art.

[0083] In some aspects, the eosinophilic disease or disorder can be a pulmonary disease or disorder. As used herein, the term "pulmonary disease or disorder" refers to any pathology affecting at least in part the lungs or respiratory system characterized by an elevated level of eosinophils. Non-limiting examples include asthma, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), allergic rhinitis, or chronic rhinosinusitis.

[0084] The term "asthma" refers to diseases that present as reversible airflow obstruction and/or bronchial hyper-responsiveness that may or may not be associated with underlying inflammation. Examples of asthma include allergic asthma, atopic asthma, corticosteroid naive asthma, chronic asthma, corticosteroid resistant asthma, corticosteroid refractory asthma, asthma due to smoking, asthma uncontrolled on corticosteroids and other asthmas as mentioned, e.g., in the Expert Panel Report 3: Guidelines for the Diagnosis and Management of Asthma, National Asthma Education and Prevention Program (2007) ("NAEPP Guidelines"), incorporated herein by reference in its entirety. [0085] The term "COPD" as used herein refers to chronic obstructive pulmonary disease.

The term "COPD" includes two main conditions: emphysema and chronic obstructive bronchitis. Thus, in the broadest sense, the term COPD as used herein refers to COPD itself and also its subconditions chronic bronchitis and emphysema. The Global Initiative for Chronic Obstructive Lung Disease (GOLD) has classified 4 different stages of COPD. GOLD classification for COPD Stage 0: At Risk for COPD. Symptoms of chronic cough and sputum production may be present, but patients have normal spirometry readings. Stage I: Mild COPD. Characterized by FEVi >= 80%, FEVJFVC < 70%. Patients may have or not have chronic cough and increased sputum production. Stage II: Moderate COPD. Characterized by a worsening of airflow (30% >= FEVi > 80%). Patients with Stage II disease often are symptomatic, seek medical attention, and have shortness of breath with exertion. Stage II has 2 subcategories: IIA and IIB. IIA patients have a FEVi between 50% and 80%; stage IIB patient have a FEVi between 30% and 50%. Patients with FEVi below 50% are especially prone to acute exacerbations of disease. Stage III: Severe COPD. Characterized by an FEVi below 30%. Patients are also included in stage III if they have respiratory failure or right heart failure. The quality of life is severely affected in these patients. Acute exacerbations in this patient population often require hospitalization and they are frequently life threatening.

[0086] The term "Idiopathic Pulmonary Fibrosis" (IPF) refers to a disease characterized by progressive scarring, or fibrosis, of the lungs. It is a specific type of interstitial lung disease in which the alveoli gradually become replaced by fibrotic tissue. With IPF, progressive scarring causes the normally thin and pliable tissue to thicken and become stiff, making it more difficult for the lungs to expand, preventing oxygen from readily getting into the bloodstream. See, e.g., Am. J. Respir. Crit. Care Med. 2000. 161 :646-664.

[0087] The term eosinophilic disease or disorder also encompasses the diseases and conditions disclosed below, which a characterized by the presence of high eosinophil levels. Normal and abnormal (e.g., elevated) eosinophil levels in the eosinophilic diseases or disorders disclosed below are also known in the art, or can be determined using methods known in the art.

[0088] High eosinophil levels have been observed in the following diseases or disorders:

(i) Allergic disorders: Allergic disorders are classically characterized by the presence of eosinophils. Allergic rhinoconjunctivitis (hay fever) has increased levels of eosinophils in the nasal mucosa. Asthma, after an exacerbation, shows increased numbers of eosinophils in the lung;

(ii) Drug reactions: Any drug / medicine has the potential to cause a reaction. Some of these reactions are allergic in nature, and eosinophils might be elevated in blood or in tissues where the drug is concentrated;

(iii) Infectious diseases: Parasitic infections (helminthiasis - worms), fungal infections and some other types of infections are associated with increased numbers of eosinophils;

(iv) Blood disorders: Hematologic disorders with increased levels of eosinophils include hypereosinophilic syndrome, leukemias, lymphomas, tumors, mastocytosis, and atheroembolic disease;

(v) Immunologic disorders and reactions: Hyper-IgE syndrome, Omenn's syndrome, thymomas, and transplant rejections are only a few types of conditions with increased numbers of eosinophils;

(vi) Endocrine disorders: Hypoadrenalism has been associated with increases in the levels of eosinophils in the blood.

9] Eosinophils have also been found to be increased or pathologically present in the following diseases or disorders:

(i) Skin and Subcutaneous Disorders: Atopic dermatitis (eczema), bullous pemphigoid, pemphigus vulgaris, dermatitis herpetiformis, drug-induced lesions, urticaria, eosinophilic panniculitis, angioedema with eosinophilia, Kimura's disease, Shulman's syndrome, Well's syndrome, eosinophilic ulcer of the oral mucosa, eosinophilic pustular folliculitis, and recurrent cutaneous necrotizing eosinophilic vasculitis;

(ii) Pulmonary Conditions: Drug/toxin- induced eosinophilic lung disease, Loeffler's syndrome, allergic bronchopulmonary aspergillosis, eosinophilic pneumonia, Churg-Strauss syndrome, eosinophilic granuloma, and pleural eosinophilia;

(iii) Gastrointestinal Diseases: Gastroesophageal reflux, parasitic infections, fungal infections, Helicobacter pylori infections, inflammatory bowel disease (ulcerative colitis and Crohn's disease), food allergic disorders, protein-induced enteropathy and protein-induced enterocolitis, allergic colitis, celiac disease, pemphigus vegetans (MR) and primary eosinophilic esophagitis, gastroenteritis, and colitis. Rare tumors (leiomyomatosis), connective tissue disorders, and vasculitic disorders; (iv) Neurologic Disorders: Organizing chronic subdural hematoma membranes, central nervous system infections, ventriculoperitoneal shunts, and drug-induced adverse reactions;

(v) Cardiac Conditions: Secondary to systemic disorders such as the hypereosinophilic syndrome or the Churg-Strauss syndrome, heart damage has been reported. Certain congenital heart conditions (septal defects, aortic stenosis) are associated with increased levels of eosinophils in the blood;

(vi) Renal Diseases: Eosinophiluria (eosinophils in the urine) associated with infections, interstitial nephritis, eosinophilic cystitis.

[0090] As used herein, the term "exacerbation" refers to a worsening of symptoms of an eosinophilic disease or disorder, relative to a patient's baseline condition. In certain aspects, an "asthma exacerbation" may be defined as an event in the natural course of the disease characterized by a change in the patient's baseline lung function, dyspnea, cough, and/or sputum that is beyond normal day-to-day variations, is acute in onset and may warrant a change in medication in a patient with underlying asthma. In certain embodiments, exacerbation of asthma may be an abrupt increase in symptoms of shortness of breath and/or wheezing, and/or increase in production of sputum.

[0091] As used herein the terms "treat," "treatment, " or "treatment of" (e.g. , in the phrase

"treating a patient having an eosinophilic disease or disorder") refers to (i) reducing the potential for an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma), (ii) reducing the occurrence of the eosinophilic disease or disorder, (iii) reducing the severity of the eosinophilic disease or disorder, preferably, to an extent that the subject no longer suffers discomfort and/or altered function due to it (for example, in the case of asthma, a relative reduction in asthma exacerbations when compared to untreated patients), or (iv) a combination thereof.

[0092] For example, treating can refer to the ability of a therapy when administered to a subject, to prevent an eosinophilic disease or disorder from occurring and/or to cure or to alleviate the eosinophilic disease or disorder's symptoms, signs, or causes. Treating also refers to mitigating or decreasing at least one clinical symptom and/or inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness. Thus, the terms "treat," "treating" or "treatment of" (or grammatically equivalent terms) refer to both prophylactic and therapeutic treatment regimes. [0093] The present disclosure provides methods and systems providing a therapeutic benefit in the treatment of an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma). A therapeutic benefit is not necessarily a cure for a particular eosinophilic disease or disorder, but rather encompasses a result which most typically includes (i) alleviation of the eosinophilic disease or disorder or increased survival, (ii) elimination of the eosinophilic disease or disorder, reduction of a symptom associate with the eosinophilic disease or disorder, (iii) prevention or alleviation of a secondary disease, (iv) disorder or condition resulting from the occurrence of a primary eosinophilic disease or disorder, (v) prevention of the eosinophilic disease or disorder, or (v) a combination thereof.

[0094] The terms "subject" or "patient" as used herein refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy of an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) is desired. As used herein, the terms "subject" or "patient" include any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g. , mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, bears, chickens, amphibians, reptiles, etc. As used herein, phrases such as "a patient having an eosinophilic disease or disorder " includes subjects, such as mammalian subjects, that would benefit from the administration of a therapy, imaging or other diagnostic procedure, and/or preventive treatment for that eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma). The term "normal healthy volunteer" is equivalent to "healthy subject" or "healthy volunteer." The skilled artisan recognizes that the term "healthy volunteer" is generally defined in United States Pharmacopeia, p. 2645 (U.S. Pharmacopeial Convention, Inc., 28th ed., 2005).

[0095] In some aspects of the present disclosure, a subject is a naive subject. A naive subject is a subject that has not been administered a therapy, for example a therapeutic agent. In some aspects, a naive subject has not been treated with a therapeutic agent prior to being diagnosed as having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma).

[0096] In another aspect, a subject has received therapy and/or one or more doses of a therapeutic agent (e.g., a therapeutic agent capable of modulating an inflammatory response associated with an eosinophilic disease or disorder) prior to being diagnosed as having an eosinophilic disease or disorder. [0097] In some aspects, a subject has received at least one therapeutically effective dose of oral or inhaled corticosteroids. In some aspects, inhalation administration is conducted using a metered dose inhaler (MDI) or a dry powder inhaler (DPI). In some aspects, the steroid is administered at a high dose. The term high dose when application to an inhaled corticosteroid (ICS) can refer, for example, to a total daily dose of at least 500 μg of ICS (e.g., fluticasone) DPI or at least 440 μg ICS MDI. In some aspects, the high ICS total daily dose is at least about 300 μg, at least about 350 μg, at least about 400 μg, at least about 450 μg, at least about 500 μg, at least about 550 μg, at least about 600 μg, at least about 650 μg, at least about 700 μg, at least about 750 μg, at least about 800 μg, at least about 850 μg, at least about 900 μg, at least about 950 μg, or at least 1000 μg of ICS (e.g., fluticasone) DPI. In some aspects, the high ICS total daily dose is at least about 300 μg, at least about 350 μg, at least about 400 μg, at least about 450 μg, at least about 500 μg, at least about 550 μg, at least about 600 μg, at least about 650 μg, at least about 700 μg, at least about 750 μg, at least about 800 μg, at least about 850 μg, at least about 900 μg, at least about 950 μg, or at least 1000 μg of ICS (e.g., fluticasone) MPI.

[0098] The term "high dose" when application to an inhaled corticosteroid (ICS) (e.g., fluticasone) in combination treatments (e.g., with a bronchodilator such as salmeterol) can refer, for example, to about 230 μg fluticasone and about 21 μg salmeterol as MDI at a dose of 2 inhalations twice per day, or to about 500 μg fluticasone and about 50 μg salmeterol as single dose DPI. Concentrations of corticosteroids considered to be high-dose alone as well as in combination with other therapeutic agents are well known in the art.

[0099] In some aspects, a subject has received multiple therapeutically effective doses of oral or inhaled corticosteroids. In some aspects, a subject is a chronic oral corticosteroid (OCS) user.

[0100] In certain aspects the subject has received a long-acting beta2-adrenergic agonist, e.g., salmeterol xinafoate. In some aspects the subject has received a synthetic glucocorticoid, e.g., fluticasone propionate. In certain aspects the subject has received a combination of salmeterol xinafoate and fluticasone propionate (ADVAIR®). In certain aspects the subject has received a beta2- adrenergic bronchodilator, e.g., albuterol sulfate.

[0101] The term "therapy" as used herein includes any means for curing, mitigating, or preventing an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma), including, for example, therapeutic agents (e.g., eosinophil-targeted therapeutic agent), instrumentation, supportive measures, and surgical or rehabilitative procedures. In this respect, the term therapy encompasses any protocol, method and/or therapeutic or diagnostic that can be used in prevention, management, treatment, and/or amelioration of an eosinophilic disease or disorder.

[0102] In some aspects, the patient is considered to be positive for DPP4 or POSTN when measurements for the biomarkers are above a predetermined threshold level. A patient is considered to have a high or increased DPP4 level (DPP4 High) when the measured DPP4 level in a sample is above a predetermined threshold level or above the level of DPP4 in one or more controls samples. Conversely, a patient is considered to have a low or decreased DPP4 level (DPP4 Low) when the measured DPP4 level in a sample is below a predetermined threshold level or below the level of DPP4 in or more control samples.

[0103] Similarly, a patient is considered to have a high or increased POSTN level

(POSTN High) when the measured POSTN level in a sample is above a predetermined threshold level or above the level of POSTN in one or more controls samples. A patient is considered to have a low or decreased POSTN level (POSTN Low) when the measured POSTN level in a sample is below a predetermined threshold level or below the level of POSTN in or more control samples.

[0104] A person skilled in the art would appreciate that it is possible to combine quantitative and qualitative measures. For example, the presence of DPP4 and/or POSTN in a sample may be determined using a qualitative assays that merely detects the presence or absence of the biomarker above a certain limit of quantification for the assay, whereas the levels of other biomarkers may be determined based on a quantitative assay showing that the level of biomarker is above or below a certain predetermined threshold or within a certain range.

[0105] In some aspects, a subject can be administered at least one therapeutically effective dose of an eosinophil-targeted therapeutic agent if the subject's DPP4 level (e.g., the level of protein or nucleic acid) in a sample is below a predetermined DPP4 threshold level, or if the DPP4 level is decreased relative to the DPP4 level in one or more control samples. In other aspects, a subject can be administered at least one therapeutically effective dose of an eosinophil-targeted therapeutic agent if the subject's POSTN level (e.g., the level of protein or nucleic acid) in a sample is below a predetermined POSTN threshold level, or if the POSTN level is decreased relative to the POSTN level in one or more control samples. [0106] The term "therapeutic agent" as used herein also refers to any therapeutically active substance that is administered to a subject having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) to produce a desired, usually beneficial, effect. The term therapeutic agent includes, e.g. , classical low molecular weight therapeutic agents commonly referred to as small molecule drugs and biologies including but not limited to antibodies or active fragments thereof, peptides, lipids, protein drugs, protein conjugate drugs, enzymes, oligonucleotides, ribozymes, genetic material, prions, virus, bacteria, and eukaryotic cells.

[0107] A therapeutic agent can also be a pro-drug, which metabolizes into the desired therapeutically active substance when administered to a subject. In some aspects, the therapeutic agent is a prophylactic agent. In addition, a therapeutic agent can be pharmaceutically formulated. A therapeutic agent can also be or comprise a radioactive isotope or agent activated by some other form of energy such as light or ultrasonic energy, or by other circulating molecules that can be systemically administered.

[0108] In one aspect, the therapeutic agent is a small molecule drug. In a specific aspect, the agent is a corticosteroid. In another aspect, the agent can be a leukotriene modifier such as montelukast, zafirlukast or zileuton. In a further aspect, the therapeutic agent can be a methylxanthine (e.g., theophylline) or a cromone (e.g., sodium cromolyn and nedocromil). In another aspect, the therapeutic agent can be a long-acting beta-2 agonist such as salmeterol, fomoterol, or indacaterol. In a further aspect, the agent can be methotrexate or cyclosporin.

[0109] In certain aspects, the therapeutic agent can be an agent used for preventing, treating, managing, or ameliorating eosinophilic disease or disorder, e.g., a pulmonary disease or disorder such as asthma. Non-limiting examples of therapies for asthma include anti-cholinergics (e.g. , ipratropium bromide and oxitropium bromide), beta-2 antagonists (e.g. , albuterol (PROVENTIL^® or VENTOLIN^®), bitolterol (TOM AL ATE^®), fenoterol, formoterol, isoetharine, metaproterenol, pibuterol (MAXAIR^®), salbutamol, salbutamol terbutaline, and salmeterol, terbutlaine (BRETHAIRE^®)), corticosteroids (e.g. , prednisone, beclomethasone dipropionate (VANCERIL^® or BECLOVENT^®), triamcinolone acetonide (AZMACORF^®), flunisolide (AEROBID^®), mometasone (NASONEX®, ASMANEX®) and fluticasone propionate (FLO VENT^®)), leukotriene antagonists (e.g. , montelukast, zafirlukast, and zileuton), theophylline (THEO-DUR^®, UNIDUR^® tablets, and SLO-BID^® Gyrocaps), and salmeterol (SEREVENT^®), cromolyn, and nedorchromil (INTAL^® and TILADE^®)), IgE antagonists, IL-4 antagonists (including antibodies), IL-5 antagonists (including antibodies), PDE4 inhibitors, NF-Kappa-B inhibitors, IL-13 antagonists (including antibodies), CpG, CD23 antagonists, selectin antagonist (e.g. , TBC 1269), mast cell protease inhibitors (e.g. , tryptase kinase inhibitors (e.g. , GW-45, GW-58, and genisteine), phosphatidylinositide-3 ' (PD)-kinase inhibitors (e.g. , calphostin C), and other kinase inhibitors (e.g. , staurosporine), C2a receptor antagonists (including antibodies), and supportive respiratory therapy, such as supplemental and mechanical ventilation.

[0110] An eosinophil-targeted therapeutic agent as used herein can be any "therapeutic agent" as defined herein, which either directly or indirectly can (i) inhibit, lessen, or neutralize the activity of eosinophils in the patient, or (ii) inhibit, reduce, or deplete eosinophil levels in the patient, either systemically or in a specific tissue or organ, or (iii) reduce the half-life of eosinophils in the patient, or (iv) can prevent exacerbation of symptoms associated with elevated levels of eosinophils, (v) combinations thereof.

[0111] In some aspects of the present disclosure, an eosinophil-targeted therapeutic agent can comprise (i) an antibody targeting, e.g., IL-5R or IL-5, or an antigen binding fragment thereof; (ii) any of the therapeutic agents disclosed above (e.g., corticosteroids); or (iii) any combinations thereof.

[0112] A "therapeutically effective" amount as used herein is an amount of therapeutic agent that provides some improvement or benefit to a subject having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma). Thus, a "therapeutically effective" amount is an amount that provides some alleviation, mitigation, and/or decrease in at least one clinical symptom of the eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma).

[0113] Clinical symptoms associated with the eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) can be treated by the methods and systems of the disclosure are well known to those skilled in the art. Further, those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. In some aspects, the term "therapeutically effective" refers to an amount of a therapeutic agent therapeutic agent that is capable of altering biomarker levels, e.g., DPP4 level and/or POSTN level and/or patient's eosinophil count in a patient in need thereof. [0114] As used herein, a "sufficient amount" or "an amount sufficient to" achieve a particular result in a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) refers to an amount of a therapeutic agent (e.g. , an anti- IL-5R such as benralizumab) that is effective to produce a desired effect, which is optionally a therapeutic effect (i.e. , by administration of a therapeutically effective amount). In some aspects, such particular result is a reduction in the patient's eosinophil count.

[0115] The term "sample" as used herein includes any biological fluid or tissue, such as whole blood, serum, sputum, saliva, or epithelial tissue (e.g., lung tissue) obtained from a subject. Samples include any biological fluid or tissue, such as whole blood, serum, saliva, urine, nasal secretions, sputum, bronchoalveolar lavage fluid, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, skin, nasal polyps, lung epithelial cells, etc. In some specific aspects, that sample is blood or a fraction thereof. Samples can be obtained by any means known in the art.

[0116] In some aspects, a sample is a computed tomography (CT) scan of a patient's organ or tissue including, but not limited to the lungs. In some aspects, a sample can be derived by taking biological samples from a number of subjects and pooling them or pooling an aliquot of each subjects' biological sample. The pooled sample can be treated as a sample from a single subject. The term sample also includes experimentally separated fractions of all of the preceding. For example, a blood sample can be fractionated into serum or into fractions containing particular types of cells. In some aspects, a sample can be a combination of samples from an individual, such as a combination of a tissue and fluid sample.

[0117] As used herein, the term "control", when used to characterize a subject, refers to a subject that is healthy or to a patient who has been diagnosed with a specific disease other than an eosinophilic disease or disorder. The term "control sample" refers to one, or more than one, biological samples obtained from a healthy subject or from a patient diagnosed with a disease other than an eosinophilic disease or disorder.

[0118] In order to apply the methods and systems of the disclosure, samples from a patient can be obtained before or after the administration of a therapy to treat an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma). In some cases, successive samples can be obtained from the patient after therapy has commenced or after therapy has ceased. Samples can, for example, be requested by a healthcare provider (e.g. , a doctor) or healthcare benefits provider, obtained and/or processed by the same or a different healthcare provider (e.g. , a nurse, a hospital) or a clinical laboratory, and after processing, the results can be forwarded to the original healthcare provider or yet another healthcare provider, healthcare benefits provider or the patient.

[0119] Similarly, the quantification of the expression level of a biomarker disclosed herein, e.g., DPP4 and/or POSTN alone or in combination with the measurement of at least one additional biomarker, e.g., a patient's eosinophil count; comparisons and/or ratios between biomarker gene or protein expression levels; evaluation of the absence or presence of biomarkers; determination of biomarker levels with respect to a certain threshold; treatment decisions; or combinations thereof, can be performed by one or more healthcare providers, healthcare benefits providers, and/or clinical laboratories.

[0120] As used herein, the term "healthcare provider" refers to individuals or institutions that directly interact and administer to living subjects, e.g. , human patients. Non-limiting examples of healthcare providers include doctors, nurses, technicians, therapist, pharmacists, counselors, alternative medicine practitioners, medical facilities, doctor's offices, hospitals, emergency rooms, clinics, urgent care centers, alternative medicine clinics/facilities, and any other entity providing general and/or specialized treatment, assessment, maintenance, therapy, medication, and/or advice relating to all, or any portion of, a patient's state of health, including but not limited to general medical, specialized medical, surgical, and/or any other type of treatment, assessment, maintenance, therapy, medication and/or advice.

[0121] As used herein, the term "clinical laboratory" refers to a facility for the examination or processing of materials derived from a living subject, e.g. , a human being. Non-limiting examples of processing include biological, biochemical, serological, chemical, immunohematological, hematological, biophysical, cytological, pathological, genetic, or other examination of materials derived from the human body for the purpose of providing information, e.g. , for the diagnosis, prevention, or treatment of any disease or impairment of, or the assessment of the health of living subjects, e.g. , human beings. These examinations can also include procedures to collect or otherwise obtain a sample, prepare, determine, measure, or otherwise describe the presence or absence of various substances in the body of a living subject, e.g. , a human being, or a sample obtained from the body of a living subject, e.g. , a human being.

[0122] As used herein, the term "healthcare benefits provider" encompasses individual parties, organizations, or groups providing, presenting, offering, paying for in whole or in part, or being otherwise associated with giving a patient access to one or more healthcare benefits, benefit plans, health insurance, and/or healthcare expense account programs.

[0123] In some aspects, a healthcare provider can administer or instruct another healthcare provider to administer a therapy to treat an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma). A healthcare provider can implement or instruct another healthcare provider or patient to perform the following actions: obtain a sample, process a sample, submit a sample, receive a sample, transfer a sample, analyze or measure a sample, quantify a sample, provide the results obtained after analyzing/measuring/quantifying a sample, receive the results obtained after analyzing/measuring/quantifying a sample, compare/score the results obtained after analyzing/measuring/quantifying one or more samples, provide the comparison/score from one or more samples, obtain the comparison/score from one or more samples, administer a therapy (e.g. , administration of an eosinophil-targeted therapeutic agent such as benralizumab/MEDI-563), commence the administration of a therapy, cease the administration of a therapy, continue the administration of a therapy, temporarily interrupt the administration of a therapy, increase the amount of an administered therapeutic agent, decrease the amount of an administered therapeutic agent, continue the administration of an amount of a therapeutic agent, increase the frequency of administration of a therapeutic agent, decrease the frequency of administration of a therapeutic agent, maintain the same dosing frequency on a therapeutic agent, replace a therapy or therapeutic agent by at least another therapy or therapeutic agent, combine a therapy or therapeutic agent with at least another therapy or additional therapeutic agent.

[0124] In some aspects, a healthcare benefits provider can authorize or deny, for example, collection of a sample, processing of a sample, submission of a sample, receipt of a sample, transfer of a sample, analysis or measurement a sample, quantification a sample, provision of results obtained after analyzing/measuring/quantifying a sample, transfer of results obtained after analyzing/measuring/quantifying a sample, comparison/scoring of results obtained after analyzing/measuring/quantifying one or more samples, transfer of the comparison/score from one or more samples, administration of a therapy or therapeutic agent, commencement of the administration of a therapy or therapeutic agent, cessation of the administration of a therapy or therapeutic agent, continuation of the administration of a therapy or therapeutic agent, temporary interruption of the administration of a therapy or therapeutic agent, increase of the amount of administered therapeutic agent, decrease of the amount of administered therapeutic agent, continuation of the administration of an amount of a therapeutic agent, increase in the frequency of administration of a therapeutic agent, decrease in the frequency of administration of a therapeutic agent, maintain the same dosing frequency on a therapeutic agent, replace a therapy or therapeutic agent by at least another therapy or therapeutic agent, or combine a therapy or therapeutic agent with at least another therapy or additional therapeutic agent.

[0125] In addition a healthcare benefits provides can, e.g. , authorize or deny the prescription of a therapy, authorize or deny coverage for therapy, authorize or deny reimbursement for the cost of therapy, determine or deny eligibility for therapy, etc.

[0126] In some aspects, a clinical laboratory can, for example, collect or obtain a sample, process a sample, submit a sample, receive a sample, transfer a sample, analyze or measure a sample, quantify a sample, provide the results obtained after analyzing/measuring/quantifying a sample, receive the results obtained after analyzing/measuring/quantifying a sample, compare/score the results obtained after analyzing/measuring/quantifying one or more samples, provide the comparison/score from one or more samples, obtain the comparison/score from one or more samples, or other related activities.

[0127] As used herein, the term "Computed Tomography" or "CT" refers to an imaging method using tomographic images (virtual 'slices') of specific areas of a scanned organ, tissue or object. Digital geometry processing is used to generate a three-dimensional (3D) image of the inside of an object or organ from a series of two-dimensional (2D) radiographic images taken around a single axis of rotation.

[0128] As used herein, the term "Computed Tomography scan" or "CT scan" refers to the production of tomographic images obtained using any method suitable including, but not limited to, x-rays, multidetector computed tomography (MDCT), high-resolution computed tomography (HRCT), positron emission tomography (PET), positron emission tomography computed tomography (PET-CT) single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), computed axial tomography (CAT scan), computer- assisted tomography, xenon ventilation computed tomography, and hyperpolarized gas lung MRI ventilation imaging.

II. Low DPP4 and POSTN as Eosinophilic Disease Biomarkers

[0129] In general, the methods disclosed herein are based on (a) detecting changes in the levels of DPP4 and/or POSTN, alone or in combination with the detection of changes in the levels of one, two, three, or more biomarkers, including, e.g., blood eosinophil count in patients with an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma);

(b) predict an increased clinical response to therapy with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) based on detected DPP4 and/or POSTN levels; and,

(c) administering an eosinophil-targeted therapeutic agent to the patient if DPP4 and/or POSTN levels, alone or in combination with other biomarkers, indicate that the patient will benefit from therapy with the eosinophil-targeted therapeutic agent. If DPP4 and/or POSTN levels, alone or in combination with other biomarkers, indicate that the patient will not benefit from therapy with the eosinophil-targeted therapeutic agent, then therapy could be discontinued, temporarily suspended, modified (e.g., increasing dosage or frequency of doses), etc.

[0130] In other words, specific levels of DPP4 and/or POSTN alone or in combination with other molecular or clinical biomarkers (e.g., a patient's eosinophil count) are correlated with clinical efficacy of therapies and useful to predict clinical outcomes in specific populations of patients suffering from an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma).

[0131] The term "DPP4" as used herein refers to the dipeptidyl peptidase IV protein (EC

3.4.14.5; Uniprot: P27487) encoded by the DPP4 gene (cDNA sequence corresponds to SEQ ID NO:3). DPP4 is also known as DPP-IV, adenosine deaminase complexing protein 2, or CD26 (cluster of differentiation 26). DPP4 is related to attractin, FAP, DPP8 and DPP9. DPP4 is a highly conserved multifunctional type II transmembrane glycoprotein, which is present both in circulation (plasma) and on the surface of several cell types, including epithelial, endothelial and lymphoid cells. Thus, DPP4 exists both in membrane bound form (SEQ ID NO:l) and soluble form (SEQ ID NO:2).

[0132] DPP4 is part of the serine protease family that is involved in T-cell co-stimulation, chemokine biology, type II diabetes, and tumor biology (Zhong et al., Atherosclerosis 2013;226:305-314). A role for DPP4 in inflammatory respiratory diseases like asthma is suggested by Giovannini-Chami (Giovannini-Chami et al., European Respiratory Journal. 2012 May;39(5): 1197-205), who found elevated DPP4 transcripts (and other Th2 signature genes) in the nasal epithelia of children with dust mite allergic rhinitis, associated with uncontrolled asthma. The term DPP4 also includes fragments, variants (e.g., the KIR, V7I, S437I, T557I, D663E variants known in the art), and derivatives thereof (e.g., glycosylated or aglycosilated protein forms of the DPP4 protein, or otherwise chemically modified forms of the protein).

[0133] In some aspects, in addition or alternatively to the determination of the level of

DPP4, the methods disclosed herein can comprise determining, submitting a sample taken from the patient for determination, or instructing a clinical laboratory to determine the expression level or activity of periostin. The use of periostin as a biomarker for pulmonary diseases such as asthma has been disclosed, e.g., in Jia, et al., J Allergy Clin. Immunol 2012 130:647-654; Takayama, et al., J Allergy Clin Immunol 2006 118:98-104; and PCT Publ. No. WO 2012/083132, each herein incorporated by reference in their entirety.

[0134] The term "POSTN" as used herein refers to the osteoblast specific factor protein periostin (Uniprot: Q15063; SEQ ID NO:20) encoded by the POSTN gene. POSTN is also known as osteoblast-specific factor 2 (OSF-2). POSTN functions as a ligand for alpha- V/beta-3 and alpha- V/beta-5 integrins to support adhesion and migration of epithelial cells. POSTN is a gla domain vitamin K dependent factor. The term POSTN also includes fragments, variants (e.g., isoforms produced by alternative splicing), and derivatives thereof (e.g., glycosylated or aglycosilated protein forms of the protein, or otherwise chemically modified forms of the protein).

[0135] Seven POSTN isoforms produced by alternative splicing are known in the art:

Isoform 1 (Uniprot: Q15063-1), also known as OSF-20S, which is 836 amino acids long; Isoform 2 (Uniprot: Q15063-2), also known as OSF-2pl, which is 779 amino acids long; Isoform 3 (Uniprot: Q15063-3), which is 781 amino acids long; Isoform 4 (Uniprot: Q15063- 4), which is 751 amino acids long; Isoform 5 (Uniprot: Q15063-5), which is 809 amino acids long; Isoform 6 (Uniprot: Q15063-6), which is 749 amino acids long; and Isoform 7 (Uniprot: Q15063-7), which is 721 amino acids long. Known POSTN variants include those with any of the following sequence differences with respect to the canonical Isoform- 1 sequence: I290F, D421V, T339I, or V814M.

[0136] In some aspects, the DPP4 and POSTN biomarkers disclosed herein can be combined with other biomarkers related to eosinophilic diseases or disorders, e.g., a patient's eosinophil count, which in turn can be substituted or combined with one or more molecular biomarkers in inflammation pathways or clinical biomarkers known in the art.

[0137] The term "biomarker" as used herein refers to a factor that is a distinctive indicator of a biological process, biological event, and/or pathologic condition, e.g. , a predictor of clinical response to treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563). As used herein, the term biomarker encompasses both clinical markers and molecular biomarkers (biological markers). Thus, in the context of the present disclosure, the term "biomarker" encompasses, e.g., "biological biomarkers" or "molecular biomarkers" such as the DPP4 and POSTN biomarkers disclosed herein alone or in combination with molecular biomarkers linked to the IL-13 pathway, molecular biomarkers linked to a specific eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma), and combinations thereof. The biological markers disclosed herein also include the genes encoding those proteins (DNA and/or RNA), as well as metabolic products.

[0138] As disclosed above, the term "biomarker" also encompasses "clinical biomarkers," also referred to as "clinical status markers," that can be predictive of response to biological therapies, for example, gender, age, concomitant drugs, smoking status, body mass index (BMI), etc. See, e.g. , U.S. Publ. Nos.US20150065530, US20140141990, US20130005596, US20090233304, US 20140199709, US20130303398, and US20110212104, which are herein incorporated by reference in their entireties.

[0139] The DPP4 molecular biomarker disclosed herein also includes proteins or fragments thereof having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the wild type sequence of either its membrane -bound (SEQ ID NO: l) or soluble form (SEQ ID NO:2), and nucleic acids having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the respective wild type nucleic acid sequences encoding the membrane -bound or soluble form of DPP4.

[0140] The POSTN molecular biomarker disclosed herein also includes proteins or fragments thereof having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the wild type sequence POST (SEQ ID NO:20) or POSTN isoforms known in the art, and nucleic acids having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the wild type nucleic acid sequence SEQ ID NO:20 or POSTN isoforms known in the art.

[0141] The DPP4 and POSTN molecular biomarkers disclosed herein also include fragments, variants, and derivatives thereof. As used herein, a "variant" biomarker contains at least one amino acid sequence alteration as compared to the amino acid sequence of the corresponding wild-type polypeptide. An amino acid sequence alteration can be, for example, a substitution, a deletion, or an insertion of one or more amino acids, preferably conservative substitutions. A variant biomarker can have any combination of amino acid substitutions, deletions or insertions. In one aspect, a biomarker variant polypeptide can have an integer number of amino acid alterations such that its amino acid sequence shares at least 60, 70, 80, 85, 90, 95, 97, 98, 99, 99.5 or 100% identity with the amino acid sequence of the corresponding wild-type polypeptide.

[0142] In some aspects of the present disclosure, the methods disclosed herein can be applied to an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) exclusively using DPP4 as a biomarker, exclusively using POSTN as a biomarker, using a combination of DPP4 and POSTN, or optionally by incorporating additional clinical and/or molecular biomarkers such as a patient's eosinophil count.

[0143] In some aspects, the methods disclosed herein can comprise using additional biomarkers, e.g., the blood eosinophil cell count, the level of the patient's IgE levels, pre- or post-bronchodilator FEVl reversibility, the wall area percentage (WA%) of subsegmental airways from CT scan data of the lungs, or combinations thereof. Wall Area %(WA%) as determined using a CT scan of the lungs of subsegmental airways (WA%) can be used to predict treatment response (for example, improvements in airway resistant and/or FEVi).

[0144] The presence of eosinophils above normal baseline level, e.g., in blood, can be used as a biomarker in combination with DPP4 levels and/or POSTN levels. Severe asthma patients have frequent exacerbations and hospitalizations and account for over half of the cost of the disease and most of its mortality (Gaga et al., 2009). Inflammation, an important feature in severe asthma, exhibits different phenotypes that can be characterized by persistence of varying degrees of eosinophilic and neutrophilic infiltration (Balzar et al., 2002). The presence of eosinophils in asthma has been well documented via airway biopsy studies. The clinical importance of eosinophils in asthma has been demonstrated by the observation of frequent asthma exacerbations in patients who have sputum eosinophil counts >3 %. Moreover, clinical trials designed to adjust inhaled anti-inflammatory therapy to maintain sputum eosinophil counts to <3% have resulted in fewer asthma exacerbations (Green et al., 2002). Symptomatic asthmatics with recalcitrant sputum eosinophilia on standard therapy have also improved after monoclonal antibody therapy (mepolizumab) that depletes airway eosinophils (Nair et al., 2009; Haldar et al., 2009).

[0145] To date the only accurate and reliable method to identify eosinophilic asthmatics has been limited to procurement of induced sputum samples from patients (Molfino, 2012). The sputum induction procedure is a tedious and complex process that requires skilled technicians and equipment that are not readily available in clinical practice. Even with these shortcomings, induced sputum remains the standard for assessing the cellular inflammatory processes that occur in asthma (Lieberman, 2007). A panel convened from the National Institutes of Health and federal agencies to propose biomarkers to assess disease progression and response to treatment has recommended 2% eosinophils in sputum as the cut-off for classifying patients as sputum eosinophilic asthmatics (Szefler et al., 2012).

[0146] A cut-off approach based on absolute values, e.g., sputum EOS% cutoff, is used as the standard for prediction and classification in pulmonary diseases and disorders such as eosinophilic asthma. In one aspect, the EOS% cutoff point to classify a patient as eosinophilic is 2% or greater sputum eosinophils. EOS% cutoff points of 1%, 2%, 2.5%, and 3 % sputum eosinophils have been reported as discriminating between eosinophilic and non-eosinophilic patients. See, e.g., Green et al., 2002 and Jayaram et al., 2006. Belda et al., 2000 showed that the mean + 2 standard deviations for sputum EOS% in healthy subjects is 2.2%. To date, attempts to predict and classify eosinophilic asthma have investigated the correlations of individual measures (such as blood eosinophil counts and FENO) with sputum EOS%.

[0147] The cut-offs disclosed above with respect to eosinophil count, i.e., percentage of eosinophils in sputum or sputum EOS% of 1%, 2%, 2.2%, 2.5%, or 3% can be used as thresholds in the methods disclosed herein.

[0148] The term "wall area" as used herein refers to the cross-sectional area of a bronchial tube wall (e.g. segmental and subsegmental bronchi in the upper lobes). Wall area percentage (WA%) is calculated as follows: 100*wall area/(wall area + lumen area). Tools to measure wall area and wall area percentage are well known in the art. See, e.g., Gupta et al., J Allergy Clin Immunol. 133(3): 729-738 (2014); Gupta et al, Thorax. 65(9):775-81 (2010). In some aspects, airway dimensions are measured from Computed Tomography (CT) imaging data of the lungs. Such imaging data can be processed, for example, using commercially available software such as VIDA Apollo (e.g., the Volumetric Information Display and Analysis (VIDA) Pulmonary Workstation, VIDA Diagnostics, Coralville, Iowa).

[0149] In other aspects, the methods disclosed herein can also comprise using additional molecular biomarkers in combination with DPP4 and/or POSTN levels such as the expression level or activity of sCTLA-3 (soluble CTLA-3; also known as Cytotoxic T-Lymphocyte- Associated serine Esterase 3, granzyme A, or granzyme 1 ; Uniprot: P12544), sCD28 (soluble CD28; also known as cluster of differentiation 28 or Tp44; Uniprot: P10747), CCL5 (chemokine C-C motif ligand 5; also known as RANTES; Uniprot: P13501), CCL11 (C-C motif chemokine 11 ; also known as eosinophil chemotactic protein or eotaxin-1; Uniprot: P51671), CCL22 (C-C motif chemokine 22; Uniprot: 000626), or combinations thereof. These biomarkers have been disclosed in IL-13 mediated pulmonary diseases, e.g., in Lun et al., J. Clin. Immunol. 2007 27:430-437.

[0150] In some aspects, the methods disclosed herein can also comprise using additional molecular biomarkers in combination with DPP4 and/or POSTN levels such as the expression level or activity of CCL26, FZD5, DOK1, CST2, ZNF436, C20orfl00, NAGS, CST1, CDH13, HRH1, TMEM132B, NTRK1, SLC02A1, IgE, FETUB, KRT31IKRT34, C6orfl38, ATP5J, TUBAL3, JAM2, NOVA2, NOS2A, HS3ST4, GRM8, IL1R2, CTDSPL, CEP72, LOC199800, LYPD1, DISP1, NKX1-2, C4orf38, LOXL4, PRKD1, PAM124B, GPR44, HIGD1B, CLCA1, SEPT11, CYYR1, CD36, ALOX15, A AD AC, ACTA1, ODC1, DKFZp434F142, ACHE, CSF3, LOC100132552, C12orf27, ZNF331, GK5, DUSP1IDUSP4, LRWD1, PGLYRP4, GUSBL2, CLGN, NR1I2, EST, LRRC37B, SAA4, SLC12A3, TMEM45A, FLJ37464, MUC5B, CXCL6, GLRB, DKFp686K01114, FOLR1, TSPAN6, AKR1C1, KIAA0232, PTP4A1, PCYT2, RHOV, PROS1, Cl lorf63, TCTN1, PIP5K1B, OSBPL6, NSUM7, GJB7, IRS2, or combinations thereof. These genes are part of the Th-2 signature as disclosed in Choi et al. J. Immunol. 186(3): 1861-9 (2011) and Int'l Publ. No. WO2009124090, both of which are herein incorporated by reference in their entireties. [0151] In some aspects, the methods disclosed herein can also comprise using additional molecular biomarkers in combination with DPP4 and/or POSTN levels such as the expression level or activity of cystatin-SN (CST1); chemokine (C-C motif) ligand 26 (CCL26); calcium- activated chloride channel regulator (1CLCA1); cystatin-SA (CST2); proline -rich protein 4 (PRR4); plasminogen activator inhibitor-2 (placental PAI), also known as HsT1201, PAI, PAI-2, PAI2 or PLANH2 (SERPINB2); carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) also known as cluster of differentiation 66 (CD66e); inducible NOS (iNOS, NOS2), serpin peptidase inhibitor, clade B (ovalbumin), member 4 (SERPINB4, LEUPIN, Pil l, SCCA-2, SCCA1, SCCA2), cystatin S (CST4), basic salivary proline-rich protein 4 (PRB4), tryptase Delta 11 (TPSDl), tryptase gamma 1 (TPSGl), major facilitator superfamily domain containing 2A protein (MFSD2), carboxypeptidase A3 (CPA3), G- Protein coupled receptor 105 (GPR105), cadherin 26 (CDH26), gelsolin (GSN), cannabinoid receptor interacting protein 11 (C20RF32), transmembrane protein 711 (TRACH2000196, TMEM71), DnaJ (Hsp40) homolog, subfamily C, member 121 (DNAJC12), RGS13 (regulator of G-protein signaling 13), solute carrier family 18 member 2 (SLC18A2), serpin peptidase inhibitor, clade B (ovalbumin), member 10 (SERPINB10), SH3 ring finger 2 protein (SH3RF2), high affinity immunoglobulin epsilon receptor subunit beta (FCER1B), runt-related transcription factor 2 (RUNX2), prostaglandin-endoperoxide synthase 1 (PTGS1), arachidonate 15-lipoxygenase (ALOX15), and combinations thereof.

[0152] In some aspects, the determination that a patient's DPP4 level is High (higher or increased) requires that the measured level of DPP4 is > about 376 ng/mL as determined by a DDP4 detection immunoassay, including the immunoassay disclosed in Example 2. In some aspects, the determination that a patient's DPP4 level is Low (lower or decreased) requires that the measured level of DPP4 is < about 376 ng/mL as determined by a DDP4 detection immunoassay, including the immunoassay disclosed in Example 2.

[0153] In some aspects, the determination that a patient's DPP4 level is High (higher or increased) requires that the measured level of DPP4 is > about 363 ng/mL as determined by a DDP4 detection immunoassay, including the immunoassay disclosed in Example 2. In some aspects, the determination that a patient's DPP4 level is Low (lower or decreased) requires that the measured level of DPP4 is < about 363 ng/mL as determined by a DDP4 detection immunoassay, including the immunoassay disclosed in Example 2. [0154] In some aspects, the determination that a patient's POSTN level is High (higher or increased) requires that the measured level of POSTN is > about 25.8 ng/niL as determined by a POSTN detection immunoassay, including the POSTN immunoassay disclosed in WO2015120185. In some aspects, the determination that a patient's POSTN level is Low (lower or decreased) requires that the measured level of DPP4 is < about 25.8 ng/mL as determined by a POSTN detection immunoassay, including the POSTN immunoassay disclosed in WO2015120185.

[0155] In some aspects, the determination that a patient's POSTN level is High (higher or increased) requires that the measured level of POSTN is > about 23.5 ng/mL as determined by a POSTN detection immunoassay, including the POSTN immunoassay disclosed in WO2015120185. In some aspects, the determination that a patient's POSTN level is Low (lower or decreased) requires that the measured level of POSTN is < about 23.5 ng/mL as determined by a POSTN detection immunoassay, including the POSTN immunoassay disclosed in WO2015120185.

[0156] In some aspects, the determination that a patient's blood eosinophil count is High

(higher or increased) requires that the measured level of blood eosinophils is > 150 cells^L. In some aspects, the determination that a patient's blood eosinophil count is Low (lower or decreased) requires that the measured level of blood eosinophils is < 150 cells^L.

[0157] In some aspects, the determination that a patient's blood eosinophil count is High

(higher or increased) requires that the measured level of blood eosinophils is > 300 cells^L. In some aspects, the determination that a patient's blood eosinophil count is Low (lower or decreased) requires that the measured level of blood eosinophils is < 300 cells^L.

[0158] In some aspects, the determination that a patient's blood eosinophil count is High

(higher or increased) requires that the measured level of blood eosinophils is > 400 cells^L. In some aspects, the determination that a patient's blood eosinophil count is Low (lower or decreased) requires that the measured level of blood eosinophils is < 400 cells^L.

[0159] In some aspects, the determination that a patient's blood eosinophil count is High

(higher or increased) requires that the measured level of blood eosinophils is at least about 150 cells^L, 160 cells^L, 170 cells^L, 180 cells^L, 190 cells^L, 200 cells^L, 210 cells^L, 220 cells^L, 230 cells^L, 240 cells^L, 250 cells^L, 260 cells^L, 270 cells^L, 280 cells^L, 290 cells^L, 300 cells^L, 310 cells^L, 320 cells^L, 330 cells^L, 340 cells^L, 350 cells^L, 360 cells^L, 370 cells^L, 380 ceUs/ L, 390 cells^L, or 400 cel\s/μL.

[0160] In some aspects, the determination that a patient's blood eosinophil count is Low

(lower or decreased) requires that the measured level of blood eosinophils is at least below 150 cells^L, 150 cells^L, 160 cells^L, 170 cells^L, 180 cells^L, 190 cells^L, 200 cells^L, 210 cells^L, 220 cells^L, 230 cells^L, 240 cells^L, 250 cells^L, 260 cells^L, 270 cells^L, 280 cells^L, 290 cells^L, 300 cells^L, 310 cells^L, 320 cells^L, 330 cells^L, 340 cells^L, 350 cells^L, 360 cells^L, 370 cells^L, 380 cells^L, 390 cells^L, or 400 cells^L.

III. Determination of DPP4, POSTN, Eosinophil Count, and Other Biomarkers

[0161] Levels of DPP4, POSTN, and other biomarkers disclosed herein (either their expressed protein levels, or their respective nucleic acid levels, such as mRNA levels) can be detected and quantified by any of a number of methods well known to those of skill in the art. These methods include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, mass spectroscopy and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunohistochemistry, affinity chromatography, Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISAs), chemi- luminescence immunoassay (CLIA), immunofluorescent assays, Western blotting, and the like.

[0162] In some aspects, the method used to detect and/or quantify DPP4, POSTN, or other molecular biomarkers disclosed herein comprises measuring the level, concentration, or amount of RNA, e.g. , mRNA, encoded by the gene or gene segments in the sample. Levels of RNA, e.g. , mRNA, may be measured by any technique known in the art, including but not limited to northern blotting or quantitative PCR (qPCR), including methods such as reverse transcription qPCR, real time qPCR, and end-point qPCR. Alternatively, "tag based" technologies, such as Serial analysis of gene expression (SAGE) and RNA-Seq, may be carried out to provide a relative measure of the cellular concentration of different mRNAs.

[0163] In some aspects, DPP4, POSTN, and other molecular biomarkers disclosed herein can be detected and/or quantified in an electrophoretic polypeptide separation (e.g. , a 1- or 2- dimensional electrophoresis). Means of detecting polypeptides using electrophoretic techniques are well known to those skilled in the art (see generally, R. Scopes (1982) Polypeptide Purification, Springer- Verlag, N.Y.; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to Polypeptide Purification, Academic Press, Inc., N.Y.).

[0164] In some aspects, a Western blot (immunoblot) analysis is used to detect and quantify the presence or absence of DPP4, POSTN, or any other molecular biomarkers disclosed herein in the sample. This technique generally comprises separating sample polypeptides by gel electrophoresis on the basis of molecular weight, transferring the separated polypeptides to a suitable solid support (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with antibodies that specifically bind the analyte. Antibodies that specifically bind to the analyte may be directly labeled or alternatively may be detected subsequently using labeled antibodies (e.g., labeled sheep anti- mouse antibodies) that specifically bind to a domain of the primary antibody.

[0165] In some aspects, DPP4, POSTN, and other molecular biomarkers disclosed herein can be detected and/or quantified in the biological sample using an immunoassay. For a general review of immunoassays, see also Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, eds. (1991). See also, e.g. , U.S. Patent Application Publication No. 2007/0212723 Al, Shang et al., Circulation Research 101 : 1146-1154 (2007); and International Patent Application Publication Nos. WO/2012/094651 and WO/2010/129964.

[0166] In some aspects, the immunoassay can use one or more antibodies or antigen binding fragments thereof which recognize the molecular biomarker (e.g., human DPP4 or human POSTN). In some aspects, the immunoassay comprises a sandwich immunoassay, e.g. , an enzyme-linked immunosorbent assay (ELISA) or a sandwich electrochemiluminescent (ECL) assay, in which a first antibody or antigen-binding fragment thereof against the molecular biomarker (e.g., DPP4 or POSTN) is used as a "capture" antibody. The capture antibody is attached to a solid support, an antigen from a sample or standard is allowed to bind to the capture antibody, and then a second antibody or antigen binding fragment thereof against the same biomarker comprising a detectable label, i.e., a "detection" antibody, is added. The detection antibody can be detected either by an enzymatic reaction, an ECL reaction, radioactivity, or any other detection method known in the art. [0167] In some aspects, the immunoassay comprises the following steps: First, the capture antibody or fragment thereof is allowed to bind to a solid support, e.g. , a multi-well plate or other assay device known to those of ordinary skill in the art. The capture antibody is allowed to attach for a period of time, e.g. , overnight, and then unbound antibody is removed. The plate can then be washed to remove any unbound capture antibody. The plate can then be treated with a blocking solution to allow non-specific protein to bind to any unbound regions of the solid support.

[0168] Typical blocking solutions include an unrelated protein, e.g. , nonfat dry milk or serum albumin. The plate can then again be washed to remove any unbound blocking solution. Next, a sample suspected of containing the molecular biomarker (e.g., DPP4 or POSTN) is added to the plate. Samples are typically serially diluted and plated in duplicate or triplicate. Controls, including standard amounts of biomarker (e.g. , pure recombinant DPP4 or pure recombinant POSTN) or a suitable fragment thereof and various negative controls are also included. The antigen is allowed to bind to the capture antibody for a period of time, e.g. , one hour at room temperature. Following incubation, the plate can then be washed to remove any unbound antigen.

[0169] Next, a detection antibody is added. The detection antibody is typically an anti- antibody that specifically binds to an epitope of the molecular biomarker (e.g., DPP4 or POSTN) epitope that is different from the epitope to which the capture antibody binds. The detection antibody can be labeled or unlabeled. Where the detection antibody is unlabeled, an addition step of addition a labeled secondary antibody will be required, as is well known by those of ordinary skill in the art.

[0170] The detection antibody can be directly labeled with an enzyme, e.g. , horseradish peroxidase or alkaline phosphatase, or can be labeled with a tag that will allow an enzyme to bind. For example the detection antibody can be conjugated to biotin, and the enzyme attached in a subsequent step by allowing enzyme-conjugated streptavidin to bind to the biotin tag. Alternatively, the detection antibody can be conjugated to a chemiluminescent, fluorescent, or ECL tag. An example of the latter is a ruthenium chelate. Following incubation, the plate can then be washed to remove any unbound detection antibody. Detection of the detection antibody can be accomplished by methods that vary based on the type of detection antibody that is used. [0171] If the detection antibody is tagged with biotin, then enzyme-conjugated streptavidin is added, unbound streptavidin is washed away, and a substrate is added which provides a colorimetric reaction that can be read, e.g. , on a spectrophotometer. If the detection antibody is conjugated to a ruthenium chelate, the plate is subjected to electrical current, and light emission is measured.

[0172] Immunoassays for detecting molecular biomarkers (e.g., DPP4 or POSTN) can be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte is directly measured. In competitive assays, the amount of analyte in the sample is measured indirectly by measuring the amount of an added (exogenous) labeled analyte displaced (or competed away) from a capture agent by the analyte present in the sample. In one competitive assay, a known amount of, for example, labeled molecular biomarker (e.g., DPP4 or POSTN) is added to the sample, and the sample is then contacted with a capture agent. The amount of labeled molecular biomarker (e.g., DPP4 or POSTN) bound to the antibody is inversely proportional to the concentration of molecular biomarker (e.g., DPP4 or POSTN) present in the sample.

[0173] In some aspects, the method directly measures the level of DPP4, POSTN, or other biomarkers disclosed herein, in a patient sample, where absolute levels are calculated by plotting the immunoassay results on a standard curve using, e.g., purified full length DPP4 or POSTN, or a DPP4 or POSTN fragment (or a full length biomarker disclosed herein or a fragment thereof). The detected signal from the detection antibody can then be quantitated based on the various standards and controls included on the plate. By plotting the results on a standard curve, the absolute levels of DPP4, POTN, or any other biomarker disclosed herein in the test samples can be calculated, e.g. , in pg or ng of biomarker per mL, or pg or ng of biomarker per mg of total protein.

[0174] Detection assays for DPP4, POSTN, or other molecular biomarkers disclosed herein can be scored (as positive or negative or quantity of analyte) according to standard methods well known to those of skill in the art. The particular method of scoring will depend on the assay format and choice of label. For example, a Western Blot assay can be scored by visualizing the colored product produced by the enzymatic label. A clearly visible colored band or spot at the correct molecular weight is scored as a positive result, while the absence of a clearly visible spot or band is scored as a negative. The intensity of the band or spot can provide a quantitative measure of analyte concentration. [0175] In some aspects, the measured expression level of molecular biomarker (e.g.,

DPP4 or POSTN) represents an average expression level or a mean expression level based on more than one measurement of the expression level. In some aspects, the measured expression level is an average or mean of several measurements of expression levels of the same sample. In some aspects, the measured expression level is an average or mean of several measurements of expression levels of different samples containing the same components obtained from the same subject. In some aspects, the measured expression level is quantile normalized, as is done in RNA Seq techniques using techniques well known by those of ordinary skill in the art.

[0176] The term "DPP4 detection assay" as used herein refers to both quantitative and qualitative assays capable of detecting the presence or absence of DPP4 in a biological sample. The term DPP4 detection assays encompassed, e.g., immunoassays such as ELISA.

[0177] The term "POSTN detection assay" as used herein refers to both quantitative and qualitative assays capable of detecting the presence or absence of POSTN in a biological sample. The term POSTN detection assays encompassed, e.g., immunoassays such as ELISA. POSTN detection assays are well known in the art. In some aspects, the POSTN detection assay is an immunoassay disclosed in WO2015120171A1 or WO2015120185, or a commercial POSTN assay.

[0178] In some aspects, the DPP4 detection assay or the POSTN detection assay is a multiplexed immunoassay, for example, Bio-Rad BIO-PLEX™, EMD Millipore MILLIPLEX™, Life Technologies NOVEX MULTIPLEX™, Thermo Fisher Scientific LUMINEX®, PerkinElmer ALPHAPLEX™, Affymetrix (eBioscience) PROCARTA™, or R&D Systems LUMINEX® capable of detecting the expression levels of DPP4 and/or POSTN in a sample.

[0179] In some aspects, the DPP4 detection assay is an assay included in TABLE 1, or a variant thereof.

TABLE 1: DPP4 immunoassays.

DPP4/CD26 Product Source Manufacturer's Information & Instructions

Assays Code

Human DPP4 DC260 R&D Systems www.rndsystems.com/products/human-dppiv-

Quantikine cd26-quantikine-elisa-kit_dc260

ELISA Kit CD26 Human abl 19513 Abeam www.abcam.com/cd26-human-elisa-kit- ELISA Kit abl l9513.html

ELISA Kit for SEA884H USCN www.uscnk.com/uscn/ELISA-Kit-for-Human- Dipeptidyl u Dipeptidyl-Peptidase-IV-DPP4-CD26-451.htm Peptidase IV

(DPP4)

Human CD26 ELH- RayBiotech www.raybiotech.com/human-dppiv-cd26-elisa-kit- ELISA CD26 for-serum-plasma-cell-culture-supernatant-and- urine.html

Human DPPIV GWB- Genway Biotech www.genwaybio.com/human-dppiv-elisa-kit ELISA Kit SKR222

Human sCD26 BMS235 Affymetrix www.ebioscience.com/human-scd26-platinum- Platinum ELISA eBioscience elisa-kit.htm

Human DPPIV / RAB0147 Sigma-Aldrich www.sigmaaldrich.com/catalog/product/sigma/rab CD26 ELISA 0147 ?lang=en&region=US

Kit

sCD26 ELISA 61- ALPCO www.alpco.com/store/scd26-elisa.html

C26HU- E01

sCD26, ELISA MBS9303 MyBioSource www.mybiosource.com/prods/ELISA- Kit 923 Kit/Human/soluble-cell-adhesion-molecule-26- sCD26/sCD26/datasheet.php?products_id=930392

3

ELISA Kit for E0884h EIAab www.eiaab.com/entries/detail/ELISA%20Kit/DPP Human 4_HUMAN

Dipeptidyl

peptidase 4

DPP4 (Human) KA3335 Abnova www.abnova.com/products/products_detail.asp7ca ELISA Kit talog_id=KA3335

DPP4 (Human) KA0141 Abnova www.abnova.com/products/products_detail.asp7ca ELISA Kit talog_id=KA0141

Human EK0696 Boster www.bosterbio.com/human-cd26-dpp4-picokine- CD26/DPP4 elisa-kit-ek0696.html

PicoKine™

ELISA Kit

DPP IV/CD26 K4801- Biovision www.biovision.com/dpp-iv-cd26-human-elisa-kit- (human) ELISA 100 7827.html

Kit CD26 (DPP4) ThermoFisher www.thermofisher.com/order/catalog/product/EH ELISA Kit, EHDPP4 Scientific DPP4

Human

Dipeptidyl 27789A Takara / Clontech www.clontech.com/US/Products/Cell_Biology_an Peptidase-4 d_Epigenetics/Metabolic_Diseases/DPP4 Detection—

DPP4 ELISA

DPPIV/CD26 KA0141 Novus Biologicals www.novusbio.com/DPPIV-CD26-ELISA- ELISA Kit Kit_KA0141.html

DPP4/CD26 JP27789 IBL International www.ibl-international.com/en_us/dpp4-cd26-elisa ELISA

Human E-EL- ELabscience www.elabscience.com/index.php/product/view/aid DPP4/CD26 H0058 /646.jsp

(Dipeptidyl

Peptidase IV)

ELISA Kit

Human CD26/ 29030600 Eton Bioscience www.etonbio.com/Products/ELISA%20Kit/produc DPP4 ELISA Kit 15 t.php?sku=290306

CD26 (Human) ELH- Biocat www.biocat.com/products/ELH-CD26-5-RB ELISA Kit CD26-5- RB

[0180] When the limit of detection (LOD) of an assay is used as predetermined DPP4 or

POSTN threshold level, the predetermined DPP4 or POSTN threshold level will be the particular LOD for such assay.

[0181] When the level of expression of DPP4 or POSTN in control subjects or in subjects suffering from a certain eosinophilic disease or condition (or subpopulations thereof) is used as predetermined DPP4 or POSTN biomarker threshold level, the predetermined DPP4 or POSTN threshold level will be level of molecular biomarker reported for each population (e.g., healthy controls, patients with moderate asthma, or patients with severe asthma) in the manufacturer's instructions for the assay.

[0182] The term "level" as applied to a biomarker disclosed herein, e.g., DPP4, POSTN, or to a set of biomarkers disclosed herein refers to a measurement or measurements made using any analytical method for detecting presence/absence or expression/lack of expression of the biomarker or set of biomarkers (protein expression or gene expression) in one or more biological sample and that indicates the presence, absence, absolute amount or concentration, relative amount or concentration, titer, expression level, ratio of measured levels, or the like, of, for, or corresponding to the biomarker or biomarkers in the one or biological samples.

[0183] The exact nature of the "value" or "level" depends on the specific designs and components of the particular analytical method (e.g. , immunoassays, mass spectrometry methods, in vivo molecular imaging, gene expression profiling, aptamer-based assays, etc.) employed to detect DPP4, POSTN, or another biomarker disclosed herein. See, e.g. , U.S. Publ. No. 2010/00221752.

[0184] As used herein with reference to DPP4, POSTN, and other biomarkers disclosed herein, the terms "elevated," "increased," or "higher" as applied to a biomarker level, refer to a level in a biological sample (e.g. , blood serum or sputum) that is higher than the expression level or range of the biomarker measured in a control sample ("normal level"), or a specified threshold disclosed herein. These thresholds include, e.g. , about 363 ng/mL or about 376 ng/mL for DPP4 serum protein as measured using a DPP4 detection assay, including the DPP4 immunoassay described in Example 2 or one of the DPP4 assays disclosed in TABLE 1. These thresholds also include, e.g. , about 23.5 ng/mL or about 25.8 ng/mL for POSTN serum protein as measured using a POSTN detection assay, including the POSTN immunoassay described in WO2015120185 or any POSTN assays known in the art.

[0185] As used herein with reference to DPP4, POSTN, and other biomarkers disclosed herein, the terms "reduced," "decreased" or "lower" as applied to a biomarker level, refer to a level in a biological sample (e.g. , blood serum or sputum) that is lower than the expression level or range of the biomarker measured in a control sample ("normal level"), or a specified threshold disclosed herein. These thresholds include, e.g. , about 363 ng/mL or about 376 ng/mL for DPP4 serum protein as measured using a DPP4 detection assay, including the DPP4 immunoassay described in Example 2 or one of the DPP4 immunoassays disclosed in TABLE 1. These thresholds include also, e.g. , about 23.5 ng/mL or about 25.8 ng/mL for POSTN serum protein as measured using a POSTN detection assay, including the POSTN immunoassay described in WO2015120185 or any POSTN assays known in the art.

[0186] The normal level or range for DPP4, POSTN, and other biomarkers disclosed herein can be defined in accordance with standard practice. Thus, the level measured in a particular biological sample can be compared with level or range of levels determined in similar normal samples. In this context, a normal sample or a control sample would be, for example, a sample obtained from an individual with no detectable symptoms of an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma). Thus, the level of, e.g., DPP4 or POSTN, is said to be a low level, lowered level, reduced level, decreased level, or grammatical variants thereof when the level of DPP4 or POSTN is present in the test sample at a lower level or range than in a normal sample, control sample, or a specific threshold level disclosed herein.

[0187] Conversely, the level of, e.g., DPP4 or POSTN is said to be a high level, higher level, increased level, elevated level, or grammatical variants thereof when the level of DPP4 or POSTN is present in the test sample at a higher level or range than in a normal sample, control sample, or a specific threshold level disclosed herein. These thresholds include, e.g. , about 363 ng/mL or about 376 ng/mL for blood serum DPP4 protein as measured using a DDP4 immunoassay, including the DPP4 immunoassay described in Example 2 or one of the DPP4 detection assays disclosed in TABLE 2. These thresholds include also, e.g. , about 23.5 ng/mL or about 25.8 ng/mL for POSTN serum protein as measured using a POSTN detection assay, including the POSTN immunoassay described in WO2015120185 or any POSTN assays known in the art.

[0188] In some aspects, the level of a molecular biomarker disclosed herein (e.g., DPP4 or POSTN) is considered to be elevated or high if it is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 205%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 295%, 300%, 305%, 310%, 315%, 320%, 325%, 330%, 335%, 340%, 345%, 350%, 355%, 360%, 365%, 370%, 375%, 380%, 385%, 390%, 395%, 400%, 405%, 410%, 415%, 420%, 425%, 420%, 435%, 440%, 445%, 450%, 455%, 460%, 465%, 470%, 475%, 480%, 485%, 490%, 495%, or 500% higher than a normal sample or control sample, or a specific threshold level disclosed herein.

[0189] In some aspects, the level of a molecular biomarker disclosed herein (e.g., DPP4 or POSTN) is considered to be reduced or low if it is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 205%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 295%, 300%, 305%, 310%, 315%, 320%, 325%, 330%, 335%, 340%, 345%, 350%, 355%, 360%, 365%, 370%, 375%, 380%, 385%, 390%, 395%, 400%, 405%, 410%, 415%, 420%, 425%, 420%, 435%, 440%, 445%, 450%, 455%, 460%, 465%, 470%, 475%, 480%, 485%, 490%, 495%, or 500% lower than a normal sample or control sample, or a specific threshold level disclosed herein.

[0190] As used herein, the term "threshold level" (or alternatively herein "threshold value" or "predetermined threshold level") refers to a level of DPP4, POSTN, or any other biomarker disclosed herein which may be of interest for comparative purposes. In some aspects, a threshold level may be the expression level of a protein or nucleic acid expressed as an average of the level of the expression level of a protein or nucleic acid from samples taken from a control population of healthy (disease-free) subjects.

[0191] In some aspects, the threshold level may be the level in the same subject at a different time, e.g. , before the present assay, such as the level determined prior to the subject developing the disease or prior to initiating therapy. In general, samples are normalized by a common factor. For example, body fluid samples are normalized by volume body fluid and cell-containing samples are normalized by protein content or cell count. In another aspect, the threshold level may also refer to the level of expression of the same biomarker in a corresponding control sample or control group of subjects which do not respond to treatment, e.g., with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).

[0192] In some aspects, the expression level of DPP4, POSTN, or any other biomarker disclosed herein is compared to a threshold level (or alternatively herein a "predetermined threshold level"). Thus, as used herein, the term "threshold level" or "predetermined threshold level" is a cutoff or threshold against which the measured expression level of a protein or nucleic acid is compared.

[0193] Based on comparison to known control samples, a "threshold level" for DPP4 or any other biomarker disclosed herein can be determined, and test samples that fall above or below the biomarker' s threshold levels indicate that the patient from whom the sample was obtained may or may not benefit from treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).

[0194] For example, DPP4 or POSTN levels above its threshold level in a sample would indicate that the patient may not benefit from treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563). Conversely, samples with DPP4 or POSTN levels below its threshold level would indicate that the patient may benefit from treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).

[0195] In some aspects, threshold levels (e.g. , protein expression levels or gene expression levels) for DPP4, POSTN, or any other biomarker disclosed herein can be predetermined and matched as to the type of sample (e.g. , serum, lung tissue, sputum), the type of eosinophilic disease or disorder (e.g. , asthma, IPF, COPD), and in some instances, the assay used.

[0196] DPP4 levels quantified using the immunoassay described in Example 2, one of the

DPP4 detection immunoassays disclosed in TABLE 1, or a multiplex immunoassay disclosed above, from serum samples from a population of mild-to-moderate asthma patients, wherein the DPP4 levels are lower than 376 ng/mL indicate that the patients may have increased clinical responses to eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563). Accordingly, in some aspects of the methods disclosed herein, the predetermined DPP4 threshold level is about 376 ng/mL of expressed DPP4 protein in blood serum as measured using the immunoassay described in Example 2.

[0197] POSTN levels quantified using the immunoassay described in WO2015120185, or any POSTN detection known in the art, or a multiplex immunoassay disclosed above, from serum samples from a population of mild-to-moderate asthma patients, wherein the POSTN levels are lower than 25.8 ng/mL indicate that the patients may have increased clinical responses to eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563). Accordingly, in some aspects of the methods disclosed herein, the predetermined POSTN threshold level is about 25.8 ng/mL of expressed POSTN protein in blood serum as measured using the immunoassay described in WO2015120185.

[0198] In some other aspects, the predetermined DPP4 threshold level is based on the median biomarker level in serum measured from a plurality of patients having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) as measured for example according to the DPP4 immunoassay described in Example 2 or any of the DPP4 immunoassays disclosed in TABLE 1.

[0199] Accordingly, in some aspects, the predetermined threshold level of DPP4 is about the median DPP4 value in serum measured from a plurality of patients having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) as measured using the DPP4 immunoassay described in Example 2 or any of the DPP4 immunoassays disclosed in TABLE 1.

[0200] In some aspects, the predetermined DPP4 threshold level is about 376 ng/mL +/-

25 pg/mL of expressed DPP4 protein in serum as measured using the DPP4 immunoassay described in Example 2.

[0201] In some aspects, the DPP4 threshold level can be between about 100 and about

870 ng/mL. Accordingly, the DPP4 threshold level can be about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, or 870 ng/mL.

[0202] In some aspects a "low level of DPP4" is defined as a value below one of these threshold levels, whereas a "high level of DPP4" is defined as a value equal to or above the same threshold level (i.e., if the threshold level was 250 ng/mL, a low level of DPP4 would be below 250 ng/mL, and a high level of DPP4 would be 250 ng/mL or above).

[0203] In some aspects, the predetermined DPP4 threshold level corresponds to the 1^st,

2^nd, 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th or 10^th decile DPP4 baseline level of expression of DPP4 in serum of control patients as measured using the DPP4 immunoassay described in Example 2. In some aspects, the predetermined DPP4 threshold level corresponds to the 1^st, 2^nd, 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th or 10^th decile DPP4 baseline level of expression of DPP4 in serum of mild- to-moderate asthmatic patients as measured using the DPP4 immunoassay described in Example 2. In some aspects, the predetermined DPP4 threshold level corresponds to the 1^st, 2^nd, 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th or 10^th decile DPP4 baseline level of expression of DPP4 in serum of severe asthmatic patients as measured using the DPP4 immunoassay described in Example 2.

[0204] In some aspects a "lower or decreased DPP4 level" is defined as a value below one of these threshold levels, whereas a "higher or increased DPP4 level" is defined as a value equal to or above the same threshold level.

[0205] In some other aspects, the predetermined POSTN threshold level is based on the median biomarker level in serum measured from a plurality of patients having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) as measured for example according to the POSTN immunoassay described in WO2015120185, or any POSTN detection known in the art.

[0206] Accordingly, in some aspects, the predetermined threshold level of POSTN is about the median POSTN value in serum measured from a plurality of patients having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) as measured using the POSTN immunoassay described in WO2015120185, or any POSTN detection known in the art.

[0207] In some aspects, the predetermined POSTN threshold level is about 26 ng/niL +/-

250 pg/mL of expressed POSTN protein in serum as measured using the POSTN immunoassay described in WO2015120185, or any POSTN detection known in the art.

[0208] In some aspects, the POSTN threshold level is between about 7 and 105 ng/niL.

Accordingly, the POSTN threshold level can be about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 OR 105 ng/niL.

[0209] In some aspects a "low level of POSTN" is defined as a value below one of these threshold levels, whereas a "high level of POSTN" is defined as a value equal to or above the same threshold level (i.e., if the threshold level was 250 ng/niL, a low level of POSTN would be below 250 ng/mL, and a high level of POSTN would be 250 ng/niL or above).

[0210] In some aspects, the predetermined POSTN threshold level corresponds to the 1^st,

2^nd, 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th or 10^th decile POSTN baseline level of expression of POSTN in serum of control patients as measured using the POSTN immunoassay described in WO2015120185, or any POSTN detection known in the art. In some aspects, the predetermined POSTN threshold level corresponds to the 1^st, 2^nd, 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th or 10^th decile POSTN baseline level of expression of POSTN in serum of mild-to-moderate asthmatic patients as measured using the POSTN immunoassay described in WO2015120185. In some aspects, the predetermined POSTN threshold level corresponds to the 1^st, 2^nd, 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th or 10^th decile POSTN baseline level of expression of POSTN in serum of severe asthmatic patients as measured using the POSTN immunoassay described in WO2015120185.

[0211] In some aspects a "lower or decreased POSTN level" is defined as a value below one of these threshold levels, whereas a "higher or increased POSTN level" is defined as a value equal to or above the same threshold level. [0212] In some aspects, the predetermined DPP4 or POSTN threshold level corresponds to the LOD of the assay used, e.g., for DPP4, the immunoassay described in Example 2 or the LOD of any of the DPP4 assays of TABLE 2. In some aspects, a DPP4 or POSTN detection assay can be adapted to use as a LOD-based method by diluting a sample so the new DPP4 or POSTN threshold level corresponds to the LOD. For example, if the initial predetermined DPP4 threshold level in an undiluted sample was 350 ng/mL, and the LOD of the DPP4 immunoassay was 50 ng/mL, the patient's samples could be diluted 1 :7 (ratio between DPP4 threshold and LOD) with a suitable buffer. After such dilution, only samples having DPP4 levels above the predetermined DPP4 threshold level in an undiluted sample would have DPP4 levels above the LOD of the assay.

[0213] In some aspects, the threshold level is the average or mean expression level measured from samples obtained from healthy volunteers as reported in the manufacturer's manual of a commercial immunoassay used to detect the presence or absence of DPP4, POSTN, or other biomarkers that can be combined with DPP4 and/or POSTN, in a sample.

[0214] In some aspects, the expression level of molecular biomarkers disclosed herein

(e.g. , DPP4 or POSTN) measured in the sample is above or below the threshold level or threshold value for each respective biomarker. In these aspects where the expression level of each biomarker disclosed herein (e.g. , DPP4 or POSTN) measured in the sample is above or below the threshold level or threshold value, the expression level can indicate that the patient from whom the sample was taken (e.g., a mild-to-moderate asthmatic patient or a severe asthmatic patient) may benefit or not from treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563). The extent to which the measured expression level is above or below the threshold level or threshold value may be to any extent.

[0215] In exemplary aspects, the measured expression level of a biomarker disclosed herein (e.g. , DPP4 or POSTN) is at least or about 10% greater or lower than the threshold level, e.g. , at least or about 15% greater or lower than the threshold level, at least or about 20% greater or lower than the threshold level, at least or about 25% greater or lower than the threshold level, at least or about 30% greater or lower than the threshold level, at least or about 35% greater or lower than the threshold level, at least or about 40% greater or lower than the threshold level, at least or about 45% greater or lower than the threshold level, at least or about 50% greater or lower than the threshold level, at least or about 55% greater or lower than the threshold level, at least or about 60% greater or lower than the threshold level, at least or about 65% greater or lower than the threshold level, at least or about 70% greater or lower than the threshold level, at least or about 75% greater or lower than the threshold level, at least or about 80% greater or lower than the threshold level, at least or about 85% greater or lower than the threshold level, at least or about 90% greater or lower than the threshold level, at least or about 95% greater or lower than the threshold level.

[0216] In exemplary aspects, the measured expression level of a biomarker disclosed herein (e.g. , DPP4 or POSTN) is at least 2-fold greater or lower than the threshold level, at least 3 -fold greater or lower than the threshold level, at least 4-fold greater or lower than the threshold level, at least 5-fold greater or lower than the threshold level, at least 6-fold greater or lower than the threshold level, at least 7-fold greater or lower than the threshold level, at least 8-fold greater or lower than the threshold level, at least 9-fold greater or lower than the threshold level, or at least 10-fold greater or lower than the threshold level.

[0217] As discussed above, the level of a biomarker disclosed herein (e.g. , DPP4 or

POSTN) can be determined using methods known in the art. A person skilled in the art would appreciate that in addition to the assays disclosed above, there are numerous methods available in the art that would allow the skilled artisan to determine threshold levels as described throughout this section, including, e.g., the DPP4 detection methods and immunoassays described in Example 2 and TABLE 1.

[0218] In some aspects, the predetermined threshold level of a biomarker disclosed herein

(e.g. , DPP4 or POSTN) is defined with respect to a certain percentile value in a population of subjects (e.g. , a plurality of normal healthy volunteers, or a plurality of patients with an eosinophilic disease or disorder, e.g., a pulmonary disease or disorder such as asthma). In some aspects, the plurality of patients with a pulmonary disease or disorder is a plurality of patients with mild-to-moderate asthma, or a plurality of patients with severe asthma.

[0219] In some aspects, the predetermined threshold level for DPP4 or POSTN corresponds to the 10^th, 15^th, 20^th, 25^th, 30^th, 35^th, 40^th, 45^th, 50^th, 50^th, 55^th, 60^th, 65^th, 70^th, 75^th, 80^th, 85^th, or 90^th percentile in a DPP4 or POSTN protein expression distribution in a plurality of normal healthy volunteers, or a plurality of patients with an eosinophilic disease or disorder, e.g., a pulmonary disease or disorder such as asthma. In some aspects, the plurality of patients with a pulmonary disease or disorder is a plurality of patients with mild-to- moderate asthma, or a plurality of patients with severe asthma. [0220] The threshold level (e.g. , a protein expression level or a gene expression level) of a biomarker disclosed herein (e.g. , DPP4 or POSTN) can vary based on the nature of the assay, e.g. , the capture and detection antibodies used, the source, purity, and composition of the standard, and the like. In one aspect, instead of using an arbitrary threshold level to determine whether a patient can benefit from treatment with eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563), the patient's DPP4 and/or POSTN levels can be compared to one or more control levels. According to this aspect, the test sample (e.g. , a sample from a patient suffering from a pulmonary disease or disorder such as asthma) is compared to one or more control samples, e.g. , samples taken from normal healthy individuals, earlier samples taken from the same patient, samples taken from patients with a subset of the patient's disease (e.g. , asthma or COPD), a pre-determined standard amount of isolated biomarker protein or gene or a fragment thereof, or a combination thereof.

[0221] The results can be expressed as a ratio with the control samples to determine a percent increase or a percent decrease in the patient's biomarker levels (e.g. , a protein expression level or a gene expression level) compared to the control biomarker levels. The control sample can be a matched pair with the patient sample, e.g. , one or more of whole blood if the patient sample is whole blood, serum if the patient sample is serum, plasma if the patient sample is plasma, saliva if the patient sample is saliva, urine if the patient sample is urine, sputum if the patient sample is sputum, bronchoalveolar lavage fluid if the patient sample is bronchoalveolar lavage fluid, lung tissue if the patient sample is lung tissue. Once determined, a biomarker expression level can be recorded in a patient's medical record.

[0222] In some aspects, as disclosed above, DPP4 and/or POSTN levels can be combined with other biomarkers such as eosinophil levels. Accordingly, in some aspects, a high level of eosinophils (e.g., peripheral blood eosinophils) of at least about 150 cells^L, at least about 300 cells^L or at least about 400 cells^L combined with DPP4 and/or POSTN levels below the threshold levels disclosed above, are predictive of positive clinical response to an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) in a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma).

[0223] In some aspect, a blood analyte to use as biomarker, e.g., eosinophil level, can be determined from a complete blood cell count (CBC) with differential. The term "CBC with differential" as used herein refers to complete blood cell count (CBC) with white blood cell (WBC) differentials. The term "white blood cell" includes, e.g., neutrophils, lymphocytes, monocytes, eosinophils, and basophils. The terms "eosinophil" and "eosinophils" can be abbreviated as "EOS". The term "white blood cell count" as used herein refers to a count of white blood cells from any sample, for example, a complete blood count (CBC) with white blood cell (WBC) differentials (CBC with differential). Obtaining a CBC with differential can be achieved using any suitable techniques available in the art, e.g., by automated hematology analyzer or hematology coulter counters (e.g., flow cytometry) or by manually counting cells (e.g., using a microscope). A CBC with differential is one of the most widely ordered clinical laboratory tests in the world.

[0224] In some aspects, samples can analyzed using automated hematology analyzers, for example, SIEMENS ADVIA 120; ABBOTT CELL DYN 3500; BECKMAN COULTER LH750; SYSMEX X SERIES ; HORIBA ABX, etc. In some aspects, to maximize the accuracy of the disclosed methods, readouts from automated hematology analyzers report absolute eosinophil counts to at least 2 digits (e.g. , 150, 220, 340 cells^L), and at least 3 digits for lymphocytes and neutrophils (e.g. , 1,530, 2,340, 3,410 cells^L). In some aspects, prior to analyzing the blood sample, the tube should be inverted several times, e.g. , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times, or according to manufacturer's instructions. In some aspects, samples can be analyzed manually.

[0225] In some aspects, the level of eosinophils can be determined by flow cytometry. In some aspects, the methods of the disclosure can comprise WBC, eosinophil count, neutrophil count, lymphocyte count, eosinophil precursor count, basophil precursor count, Eotaxin-2 level and any combination or ratio thereof. In a further aspect, a method or system of the disclosure can comprise the blood eosinophil/WBC ratio, the blood eosinophil/blood lymphocyte ratio and the log of the blood eosinophil/blood neutrophil ratio.

[0226] A method or system of the disclosure can comprise any one or any combination of the following non-limiting examples of physiological biomarkers: AFEVl post-albuterol, AFEVl post-tiotropium bromide, FEV1, FEV/FVC, ΔΑΜ/ΡΜ PEF variation, and FENO- These biomarkers are known in the art and can be determined following standard medical protocols. A method or system of the disclosure can also comprise any one or any combination of patient symptom biomarkers, such as, but not limited to, ACQ score, AQLQ score, Berlin Questionnaire (sleep apnea screen), Borg Score (assessment of dyspnea), previous sinus surgery, history of atopy, history of intubation, history of aspirin sensitivity, history of corticosteroid bursts during past 3 or 12 months, or history of ER visits during past 3 years. A method or system of the disclosure can also comprise any one or any combination of the following parameters: gender, age, weight, race, height, or body mass index (BMI).

[0227] In some aspects, method or system of the disclosure can comprise a value corresponding to the average of several measurements from multiple samples collected at different time intervals. Thus, in some aspects, multiple samples can be collected at different intervals, e.g., about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days apart. In some aspects, the multiple samples can be collected about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks apart. In some aspects, multiple samples can be collected about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months apart. In some aspects, multiple samples can be collected more than 12 months apart. In some cases, more than 2 samples are averaged, for examples, 3 samples, 4 samples, 5 samples, 6 samples, 7 samples, 8 samples, 9 samples, 10 samples, or more than same samples. In some aspects, samples are collected are regular intervals. In other aspects, samples are not collected at regular intervals. In some cases, samples are collected in response to an event, for example, exacerbation of symptoms

[0228] In some aspects, administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) according to the methods disclosed herein results in

(a) AER (Acute Exacerbation Rate) reduction;

(b) FEV1 (Forced Expiratory Volume in one second) increase;

(c) improved ACQ-6 (Asthma Control Questionnaire, 6-item version) results;

(d) improved AQLQ (Asthma Quality of Life Questionnaire) results;

(e) reduction of cytokines secreted by type 2 innate lymphocytes (ILC2);

(f) reduction of ILC2 levels,

(g) reduction of eosinophil and/or basophil levels, or,

(h) a combination thereof. [0229] In some aspects, administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) according to the methods disclosed herein results in improvement in other methods of detecting health-related quality of life known in the art.

IV. Eosinophil-Targeted Therapeutic Agents

[0230] DPP4 and/or POSTN, combined with other clinical or molecular biomarkers disclosed herein (e.g., blood eosinophil levels), and/or in combination with other clinical or molecular biomarkers known in the art can be used, for example, to determine whether to treat, select for treatment, monitor the treatment, or a begin, modify, or cease the treatment of a patient suffering from an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as asthma) with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).

[0231] The term "eosinophil-targeted therapeutic agent" as used herein refers a therapeutic agent capable of decreasing eosinophil count in a patient in need thereof. In some aspects, the eosinophil-targeted therapeutic agent can also deplete basophil count. In certain aspects, the eosinophil-targeted therapeutic agent is

(i) a monoclonal antibody, or

(ii) an antigen-binding fragment thereof, or

(iii) a compound comprising an monoclonal antibody or an antigen-binding thereof comprising one or more additional therapeutic moieties, or

(iv) a combination thereof,

capable of decreasing eosinophil and/or basophil count in a patient in need thereof.

[0232] In other aspects, the eosinophil-targeted therapeutic agent can be a small molecule. In some aspects, the eosinophil-targeted therapeutic agent is a combination of eosinophil-targeted therapeutic agents (e.g., a combination therapy comprising at least one antibody or fragment thereof and at least one small molecule, or at least an antibody or fragment thereof and at least one nucleic acid, or at least one nucleic acid and at least one small molecule, etc.).

[0233] In another aspect, the therapeutic agent is a biologic. In certain aspects, the biological is any substance made by a living organism or its products, a substance made using recombinant DNA technology, a nucleotide, a nucleotide analogue, an oligonucleotide, an oligonucleotide analogue, a peptide, or a peptide analogue produced by any means. In specific aspects, a biologic can be an antibody or antibody fragment, an antibody mimetic, a soluble receptor polypeptide, a soluble receptor fusion polypeptide, interleukin, interleukin fusion polypeptide, antisense molecule, siRNA or miRNA.

[0234] In some aspects, an eosinophil-targeted therapeutic agent of the present disclosure

(e.g. , an antibody specifically binding to IL-5R or IL-5) competitively inhibits binding of a reference molecule (e.g. , a different antibody specifically binding to IL-5R or IL-5) to a given target site if it preferentially binds to that target site to the extent that it blocks, to some degree, binding of the reference molecule to the target site. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays.

[0235] An eosinophil-targeted therapeutic agent of the present disclosure (e.g. , an antibody specifically binding to IL-5R or IL-5) can be said to competitively inhibit binding of the reference molecule to a given epitope by at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, or at least 50%.

[0236] In some aspects, the eosinophil-targeted therapeutic agent is a therapeutic agent capable of

(i) inhibiting or decreasing levels of IL-5 or IL-5R,

(ii) inhibiting of decreasing the expression of a molecule interacting with IL-5 or IL-5R,

(iii) increasing the expression of a molecule interacting with IL-5 or IL-5R,

(iv) inhibiting or decreasing the expression of a molecule upregulated by IL-5 or IL-5R,

(v) inhibiting or inhibiting the expression of a molecule interacting with a molecule upregulated by IL-5 or IL-5R, or

(vi) a combination thereof.

[0237] In some aspects, an eosinophil-targeted therapeutic agent is a compound included in TABLE 2.

TABLE 2: Potential eosinophil-targeted therapeutic agents.

Target Therapeutic agent (AB=antibody; NU=polynucleotide)

IL-5 Mepolizumab (AB), reslizumab (AB)

IL-5R, a subunit Benralizumab (AB)

IL-5R, β subunit gene ASM8 (NU), PXS 1100 (NU), PXS 2200 (NU) [0238] Eosinophil-targeted therapeutic agent comprising the antibodies disclosed above refer not only to the intact immunoglobulins, but also to antigen-binding fragments, variant, or derivatives thereof, antibodies or fragments thereof that bind to the same epitopes the antibodies disclosed above, or an antibodies or fragments thereof that competitively inhibit binding of the antibodies disclosed above to their respective targets. Antibodies (or fragments thereof) that are identical or similar in amino acid sequence to the antibodies disclosed in TABLE 2, particularly in the variable regions, or in the CDRs thereof (however, variations in the constant regions are also contemplated) are contemplated. For example, in one aspect, an eosinophil-targeted therapeutic agent is a polypeptide having an amino acid sequence that is about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 98%, about 99% or 100% identical to that of the polypeptide sequence of an antibody disclosed in TABLE 2 (i.e., the heavy chain and/or the light of the antibody).

[0239] In some aspects, the eosinophil-targeted therapeutic agent is an isolated antigen binding protein targeting any of the antigens disclosed above, comprising at least one, two, three, four, or the six complementarity determining regions of the antibodies disclosed above. In various aspects, the eosinophil-targeted therapeutic agents disclosed herein comprise antigen binding fragments of antibodies, such as fragments of any of the antagonist antibodies referred to herein, e.g., antibodies specifically binding to IL-5 (SEQ ID NO: 9) or IL-5R, for example, to the alpha subunit of IL-5R (SEQ ID NOS: 4-8, corresponding to 5 isoforms of the subunit generated by alternative splicing). Such fragments include, but are not limited to Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, nanobodies, and diabodies.

[0240] In some aspects, the eosinophil-targeted therapeutic agent is a therapeutic agent capable of specifically binding to IL-5R, e.g., an anti-IL-5R antibody such as benralizumab or an antigen binding fragment thereof, or an anti-IL-5R molecule comprising benralizumab or an antigen binding fragment thereof. In some aspects, the eosinophil-targeted therapeutic agent is a therapeutic agent capable of specifically binding to IL-5, e.g, an anti-IL-5 antibody such as reslizumab, mepolizumab, or an antigen binding fragment thereof, or an anti-IL-5 molecule comprising reslizumab, mepolizumab, or an antigen binding fragment thereof. In some aspects, the anti-IL-5R molecule or anti-IL-5 molecule is an antibody-drug conjugate (ADC). [0241] In one aspect, the eosinophil-targeted therapeutic agent can be an anti-cytokine antibody or an anti-cytokine receptor antibody binding to a cytokine and its receptors other that IL-5 or IL-5R. In one aspect, the eosinophil-targeted therapeutic agent can be an anti-IL- 5 or anti-IL5R antibody capable of preventing the signaling of IL-5 through the IL-5 receptor.

[0242] In one aspect, the anti-IL5R antibody specifically binds to the alpha subunit of IL-

5R (SEQ ID NO: 4). Non-limiting examples of anti-human IL-5 antibodies are reslizumab and mepolizumab. Non-limiting examples of anti-human IL-5 receptor alpha antibodies of the disclosure can be found in U.S. Patent Nos. 7,179,464, 6,538,111, 6,018,032, and U.S. Patent Application Publication Nos. 2004/0136996A1, 2005/0226867A1, or Int'l Publ. No. WO 2008/143878. In one aspect, the eosinophil-targeted therapeutic agent can be an antibody directed against IL-5, e.g. , reslizumab, mepolizumab, and any combination thereof.

[0243] Without being bound by a particular theory, the eosinophil-targeted therapeutic agent used according to the methods and systems described herein can be an anti-IL-5R antibody capable to mediate the in vivo depletion of eosinophils. In one aspect, the in vivo depletion can be mediated by ADCC, CDC or antibody mediated phagocytosis. In a specific aspect, the therapeutic agent can be an anti-IL-5R antibody having ADCC activity.

[0244] In another specific aspect, the therapeutic agent can be an anti-IL-5R antibody having increased ADCC activity. A non-limiting example for an anti-IL-5R antibody with increased ADCC activity is benralizumab (also referred to herein as "MEDI-563" as described in Int'l Publ. No. WO 2008/143878). Benralizumab is an immunoglobulin Gl antibody comprising humanized mouse monoclonal MEDI-523 γΐ heavy chain (224-214')- disulfide with humanized mouse monoclonal MEDI-523 κ light chain, dimer (230-230":233- 233")-bisdisulfide. See U.S. Pat. No. 8501176, U.S. Publ. No. US20150044204, and Int'l Publ. No. WO2015023504, all of which are herein incorporated by reference in their entireties.

[0245] In another specific aspect, the therapeutic agent can be an anti-IL-5R antibody that binds the same epitope as benralizumab or competes with benralizumab for binding to IL-5R. The benralizumab epitope is described in Int'l Publ. WO 2008/143878, the disclosure of which is hereby incorporated by reference in its entirety. In one aspect, the anti-IL-5R antibody or an antigen-binding fragment comprises a heavy chain variable region (VH) comprising, consisting, or consisting essentially of SEQ ID NO: 12. In one aspect, the anti- IL-5R antibody or an antigen-binding fragment comprises a light chain variable region (VL) comprising, consisting, or consisting essentially of SEQ ID NO: 13. In some aspects, the anti- IL-5R antibody or an antigen-binding fragment comprises at least one, two, three, four, five or six complementarity determining regions selected from SEQ ID NOS: 14-19.

[0246] In some aspects, the eosinophil-targeted therapeutic agent is a fusion protein or a conjugate comprising one of the monoclonal antibodies of TABLE 2 or an antigen-binding fragment thereof. In some aspects, the eosinophil-targeted therapeutic agent is a fusion protein or a conjugate comprising one of the monoclonal antibodies of any one of claims 12- 18 or an antigen-binding fragment thereof. In some aspects, the fusion protein or conjugate comprises at least one heterologous therapeutic moiety and/or a half-life enhancing moiety. In some aspects, the term eosinophil-targeted therapeutic agent also encompasses, for example, antagonists of IL-5 or IL-5R such as aptamers, peptides, mRNAs, iRNA, shRNAs, or small molecule inhibitors.

[0247] In some aspects, the eosinophil-targeted therapeutic agent is a polynucleotide, e.g., a DNA or an RNA. In some aspects, the polynucleotide is (i) an mRNA or a combination thereof, or (ii) an antisense oligonucleotide or a combination thereof. In some aspects, the polynucleotide comprises at least a nucleotide analog. In some aspects, the antisense oligonucleotide or combination thereof is an oligonucleotide targeting the gene encoding the beta subunit of IL-5R, e.g., ASM8, PXS1100, or PXS2200. See, e.g., Int'l Publ. WO 2006045202, which is herein incorporate by reference in its entirety. ASM8 is a combination of 2 oligonucleotides (TOP004 and TOP005). TOP004 is a 19-mer directed against the mRNA of the common β subunit of the IL-3, IL-5, and GM-CSF receptors.

[0248] In one aspect, the eosinophil-targeted therapeutic agent can be an antibody directed against IL-13/IL-4a. In a specific embodiment, the therapeutic agent can be Aerovant™ (Aerovance), GSK-679586 (GSK), IMA-026 (Wyeth), or MILR1444A (Genentech).

[0249] In a further aspect, the eosinophil-targeted therapeutic agent is an antibody directed against the IL-2 receptor. In a specific embodiment, the eosinophil-targeted agent can be daclizumab (ZENAPAX®). Daclizumab is a therapeutic humanized monoclonal antibody to the alpha subunit of the IL-2 receptor of T cells. In another aspect, the eosinophil-targeted therapeutic agent can be an anti-IgE antibody. In a specific embodiment, the eosinophil-targeted therapeutic agent can be omalizumab (XOLAIR®). Omalizumab is a recombinant DNA-derived humanized IgGlk monoclonal antibody that selectively binds to human immunoglobulin E (IgE). Omalizumab is FDA-approved to treat moderate to severe allergic asthmatics. It has not been specifically approved for treatment of eosinophilic asthma though some studies have demonstrated it decreases airway eosinophil numbers. In another aspect, the eosinophil-targeted therapeutic agent can be a recombinantly-produced cytokine. In a specific embodiment, the eosinophil-targeted therapeutic agent can be interferon-alpha. Non-limiting examples of interferon-alpha therapeutics include PEGASYS® (PEGinterferon alfa-2a) and ALBUFERON®/ ZALBIN™ (albinterferon alfa-2b). The eosinophil-targeted therapeutic agents of the present disclosure can also be used in combination with one or more antagonists (e.g. antibodies) of other cytokines associated with inflammation, including but not limited to, IL-13, IL-17A, IL-17F, IL-25, IL-33, TNF-a, IL-Ιβ, IL-6, or TGF-β.

[0250] In certain aspects, the half-lives of antibodies used according to the methods and systems of the disclosure can be prolonged by methods known in the art. Such prolongation can in turn reduce the amount and/or frequency of dosing of the antibody compositions. Antibodies with improved in vivo half-lives and methods for preparing them are disclosed in U.S. Patent No. 6,277,375; and International Publication Nos. WO 98/23289 and WO 97/3461.

V. DPP4 and POSTN Levels for Diagnosis and Treatment of Eosinophilic Diseases

[0251] DPP4 and POSTN are differentially expressed in subjects having an eosinophilic disease or disorder, e.g., a pulmonary disease or disorder such as asthma. DPP4 and POSTN are present at different levels in samples from patients having mild-to-moderate asthma with respect to healthy controls, or in patients having severe asthma with respect to healthy controls.

[0252] The differences in DPP4 and POSTN levels observed, e.g., in healthy individuals, patients with mild-to-moderate asthma, and patients with severe asthma, can be applied to predicting clinical outcomes when the patients are treated with a certain therapy, for example, an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563). Thus, if a subject's DPP4 and/or POSTN levels are above a certain threshold, that subject would become a candidate for treatment with a certain therapy, e.g., therapy with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563). [0253] In some aspects, the mere determination that the DPP4 and/or POSTN is expressed below a predetermined threshold level or below a detectable levels (e.g., using a diluted sample) would suffice to identify a subject as a candidate for treatment with a certain therapy, e.g., therapy with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).

[0254] In some aspects of the methods disclosed herein, the patient is a moderate asthmatic or mild-to-moderate asthmatic (GINA 1-3). In other aspects of the methods disclosed herein, the patient is a severe asthmatic (GINA 4+). In other aspects, the patients suffers from COPD.

[0255] In some aspects of the methods disclosed herein, DPP4 levels or POSTN levels can be used alone. In other aspects, the DPP4 and/or POSTN levels can be combined with other molecular or clinical biomarkers, such as blood eosinophil count. In other aspects, additional biomarkers, e.g., other biomarkers indicative of inflammation or clinical biomarkers (e.g., age, smoker status, body mass index, etc) can be combined with DPP4 and/or POSTN.

[0256] This finding can be applied, for example, to devise new methods of determining treatment (e.g. , by selecting patients as candidates for a certain therapy), methods of treating an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to- moderate asthma, or severe asthma), methods of monitoring efficacy of therapeutic agents (e.g. , benralizumab/MEDI-563) to treat eosinophilic diseases or disorders, or methods to adjust formulations, dosage regimens, or routes of administration.

[0257] The methods disclosed herein include prescribing, initiating, and/or altering prophylaxis and/or treatment, e.g. , for an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma), based at least in part on a subject's expression level of DPP4 and/or POSTN, alone or in combination with one or more additional biomarkers (e.g., eosinophil count).

[0258] The present disclosure provides a method of determining whether to treat a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) with a therapeutic regimen comprising the administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) wherein the method comprises: (a) measuring or instructing a clinical laboratory to measure DPP4 and/or POSTN levels, and optionally levels of additional biomarkers such as blood eosinophils in a sample taken from the patient, and (b) treating or instructing a healthcare provider to treat the patient, or suspending the treatment, not initiating the treatment, denying the treatment, or instructing a healthcare provider to suspend, not initiate, or deny the treatment with a therapeutic regimen comprising the administration of an eosinophil-targeted therapeutic agent if the patient is determined to have a higher or lower DPP4 and/or POSTN levels, and optionally levels of additional biomarkers such blood eosinophils in the sample compared to each biomarker predetermined threshold level or levels, or compared to each biomarker level or levels in one or more control samples.

[0259] In one aspect, the disclosure provides a method of determining whether to treat a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) with a therapeutic regimen comprising the administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) wherein the method comprises: (a) measuring or instructing a clinical laboratory to measure DPP4 and/or POSTN levels and optionally levels of additional biomarkers such as blood eosinophils in a sample taken from the patient, and (b) treating or instructing a healthcare provider to treat the patient with a therapeutic regimen comprising the administration of an eosinophil-targeted therapeutic agent if the patient is determined to have lower or decreased DPP4 and/or POSTN levels, and higher or increased levels of at least one optional additional biomarker such as eosinophil count in the sample compared to a predetermined biomarker threshold level or levels, or compared to a biomarker level or levels in one or more control samples.

[0260] In one aspect, the disclosure provides a method of determining whether to treat a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) with a therapeutic regimen comprising the administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) wherein the method comprises (a) measuring or instructing a clinical laboratory to measure the DPP4 and/or POSTN levels and optionally levels of additional biomarkers such blood eosinophils in a sample taken from the patient, and (b) suspending the treatment, not initiating treatment, denying the treatment, or instructing a healthcare provider to suspend, not initiate, or deny the treatment of the patient with a therapeutic regimen comprising the administration of an eosinophil-targeted therapeutic agent to the patient if the patient is determined to have higher or increased DPP4 and/or POSTN levels, and lower or decreased levels of at least one optional additional biomarker such as blood eosinophils in the sample compared to a predetermined biomarker threshold level or levels, or compared to a biomarker level or levels in one or more control samples.

[0261] Also provided is a method of selecting a patient diagnosed with an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) as a candidate for treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563), comprising (a) measuring or instructing a clinical laboratory to measure the DPP4 and/or POSTN levels and optionally levels of additional biomarkers such as blood eosinophils in a sample taken from the patient, and (b) treating or instructing a healthcare provider to treat the patient with an eosinophil- targeted therapeutic agent if the patient is determined to have lower or decreased DPP4 and/or POSTN levels, and higher or increased levels of at least one optional additional biomarker such as blood eosinophils in the sample compared to a predetermined threshold level or levels, or compared to a biomarker level or levels in one or more control samples.

[0262] Also provided is method of selecting a patient diagnosed with an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) as a candidate for treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563), comprising (a) measuring or instructing a clinical laboratory to measure the DPP4 and/or POSTN levels and optionally levels of additional biomarkers such as blood eosinophils in a sample taken from the patient, and (b) suspending the treatment, not initiating treatment, denying the treatment, or instructing a healthcare provider to suspend, not initiate, or deny the treatment of the patient with an eosinophil-targeted therapeutic agent to the patient if the patient is determined to have higher or increased DPP4 and/or POSTN levels, and lower or decreased levels of at least one optional additional biomarker such as blood eosinophils in the sample compared to a predetermined threshold level or levels, or compared to a biomarker level or levels in one or more control samples.

[0263] In some aspects, the methods disclosed can entail ordering and/or performing one or more additional assays. For example, if the levels of DPP4 and/or POSTN (e.g. , a protein expression level or a gene expression level) are determined to be within a normal range (i.e., not elevated), the DPP4 or POSTN detection assay may be repeated to rule out a false negative result, and/or one or more additional DPP4 or POSTN detection assays may be performed to monitor the subject's status.

[0264] Conversely, if the DPP4 and/or POSTN levels (e.g. , protein expression levels or gene expression levels) are determined to be elevated, it may be desirable repeat the DPP4 or POSTN detection assay to rule out a false positive result.

[0265] In some aspects, the predetermined DPP4 threshold level is at least about 363 ng/niL (median value in mild-to-moderate asthmatics), or at least about 376 ng/mL (mean value in mild-to-moderate asthmatics) as measured in serum using a DPP4 detection assay including, e.g., the immunoassay described in Example 2. Accordingly, DPP4 expression levels equal or above those values are consider high or elevated, and DPP4 expression levels below those values are considered low or reduced.

[0266] In some aspects, the predetermined POSTN threshold level is at least about 23.5 ng/mL (median value in mild-to-moderate asthmatics), or at least about 25.8 ng/mL (mean value in mild-to-moderate asthmatics) as measured in serum using a POSTN detection assay including, e.g., the immunoassay described in WO2015120185. Accordingly, POSTN expression levels equal or above those values are consider high or elevated, and POSTN expression levels below those values are considered low or reduced.

[0267] In some aspects, the predetermined blood eosinophil level is 150 cells^L. In some aspects, the predetermined blood eosinophil level is 300 cells^L. In other aspects, the blood eosinophil threshold level is 400 cells^L. Accordingly, blood eosinophil levels equal or above those values are consider high or elevated, and blood eosinophil levels below those values are considered low or reduced.

[0268] In some aspects, the presence of DPP4 and/or POSTN levels above or below a predetermined threshold level in a patient with an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) can be used in combination with one or more of clinical or molecular biomarkers specific for such disease.

[0269] For example, for patients with asthma, the measurement of DPP4 and/or POSTN levels can be combined with measurements of other biomarker levels (e.g. , blood eosinophils). Accordingly, in one aspect, levels of DPP4 and/or POSTN can be combined with, e.g., blood eosinophils in any of the methods disclosed herein (i) to determine whether a patient suffering an pulmonary disease (e.g., asthma or COPD) is eligible or non-eligible for a specific treatment or will respond to a specific treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563),

(ii) to determine whether a specific treatment (e.g., with an eosinophil-targeted therapeutic agent) should commence, be suspended, or be modified,

(iii) to diagnose whether the disease (e.g., asthma or COPD) is treatable or not treatable with a specific therapeutic agent, or

(iv) to prognosticate or predict the outcome of treatment of the disease (e.g., asthma or COPD) with a specific therapeutic agent, etc.

[0270] A person skilled in the art would understand that DPP4 and/or POSTN levels

(e.g. , protein expression levels or gene expression levels) can be used according to the methods disclosed herein, including but not limited to treatment, diagnostic, and monitoring methods, as positive selectors, i.e. , a specific action would be taken (e.g. , treating a patient having an eosinophilic disease or disorder such as asthma with an eosinophil-targeted therapeutic agent) if the DPP4 and/or POSTN levels (e.g. , protein expression levels or gene expression levels) in a sample taken from the patient are below predetermined DPP4 or POSTN threshold levels, or are decreased relative to the DPP4 or POSTN levels in one or more control samples.

[0271] A person skilled in the art would also understand that DPP4 and/or POSTN levels

(e.g. , protein expression levels or gene expression levels) can be used according to the methods disclosed herein, including but not limited to treatment, diagnostic, and monitoring methods, as negative selectors, i.e. , a specific action would not be taken (e.g. , treating a patient having a pulmonary disease such as asthma with an eosinophil-targeted therapeutic agent) if the DPP4 and/or POSTN levels (e.g. , protein expression levels or gene expression levels) in a sample taken from the patient are above predetermined DPP4 or POSTN threshold level, or are increased relative to the DPP4 or POSTN levels in one or more control samples.

[0272] In one aspect, the disclosure includes methods, assays, and kits to facilitate a determination by a healthcare provider, a healthcare benefits provider, or a clinical laboratory to as to whether a patient will benefit from treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563). The methods assays and kits provided herein also facilitate a determination by a healthcare provider, a healthcare benefits provider, or a clinical laboratory to as to whether a patient will benefit from treatment with any other eosinophil-targeted therapeutic agent known to those of ordinary skill in the art.

[0273] In one aspect, the methods disclosed herein include making a diagnosis, which may be a differential diagnosis, based at least in part on the level of DPP4 and/or POSTN of a patient. In some aspects, the methods disclosed herein include informing the subject of a result of the DPP4 and/or POSTN detection assay and/or of a diagnosis based at least in part on the DPP4 and/or POSTN levels. The patient can be informed verbally, in writing, and/or electronically.

[0274] This diagnosis can also be recorded in a patient medical record. For example, in various aspects, the diagnosis of a pulmonary disease (e.g., asthma) treatable with a specific eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is recorded in a medical record.

[0275] The term "medical record" or "patient medical record" refers to an account of a patient's examination and/or treatment that typically includes one or more of the following: the patient's medical history and complaints, the physician's physical findings, the results of diagnostic tests and procedures, and patient medications and therapeutic procedures. A medical record is typically made by one or more physicians and/or physicians' assistants and it is a written, transcribed or otherwise recorded record and/or history of various illnesses or injuries requiring medical care, and/or inoculations, and/or allergies, and/or treatments, and/or prognosis, and/or frequently health information about parents, siblings, and/or occupation. The record may be reviewed by a physician in diagnosing the condition.

[0276] The medical record can be in paper form and/or can be maintained in a computer- readable medium. The medical record can be maintained by a laboratory, physician's office, a hospital, a healthcare maintenance organization, an insurance company, and/or a personal medical record website. In some aspects, a diagnosis, based at least in part on the measured DPP4 and/or POSTN levels, is recorded on or in a medical alert article such as a card, a worn article, and/or a radiofrequency identification (RFID) tag. As used herein, the term "worn article" refers to any article that can be worn on a subject's body, including, but not limited to, a tag, bracelet, necklace, arm band, or head band. [0277] As used herein, the term "diagnosis" means detecting a disease or determining the stage or degree of a disease. Usually, a diagnosis of a disease is based on the evaluation of one or more factors and/or symptoms that are indicative of the disease. That is, a diagnosis can be made based on the presence, absence or amount of a factor which is indicative of presence or absence of the disease or disorder. Each factor or symptom that is considered to be indicative for the diagnosis of a particular disease does not need be exclusively related to the particular disease, e.g. there may be differential diagnoses that can be inferred from a diagnostic factor or symptom. Likewise, there may be instances where a factor or symptom that is indicative of a particular disease is present in an individual that does not have the particular disease.

[0278] The term "diagnosis" also encompasses determining the therapeutic effect of a drug therapy, or predicting the pattern of response to a drug therapy. The diagnostic methods may be used independently, or in combination with other diagnosing and/or staging methods known in the medical arts for a particular disease.

[0279] As used herein, the term "differential diagnosis" refers to the determination of which of two or more diseases with similar symptoms is likely responsible for a subject's symptom(s), based on an analysis of the clinical data. The term is also used to refer to the determination of whether a patient is susceptible to treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) depending on whether the measured DPP4 levels in a patient sample are above a predetermined threshold level, or elevated relative to the level in one or more control samples.

[0280] The term "prognosis" as used herein refers to a prediction of the probable course and outcome of a clinical condition or disease. A prognosis is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease. The phrase "determining the prognosis" as used herein refers to the process by which the skilled artisan can predict the course or outcome of a condition in a patient. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy. Instead, the skilled artisan will understand that the term "prognosis" refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition. [0281] The terms "favorable prognosis" and "positive prognosis," or "unfavorable prognosis" and "negative prognosis" as used herein are relative terms for the prediction of the probable course and/or likely outcome of a condition or a disease. A favorable or positive prognosis predicts a better outcome for a condition than an unfavorable or negative prognosis. In a general sense, a "favorable prognosis" is an outcome that is relatively better than many other possible prognoses that could be associated with a particular condition, whereas an unfavorable prognosis predicts an outcome that is relatively worse than many other possible prognoses that could be associated with a particular condition. Typical examples of a favorable or positive prognosis include a better than average remission rate, a lower propensity for metastasis, a longer than expected life expectancy, differentiation of a benign process from a cancerous process, and the like.

[0282] The disclosure includes methods of treating an eosinophilic disease or disorder

(e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) in a subject based on the changes in expression of DPP4 and/or POSTN. The disclosure provides a method of treating a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) comprising: administering an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) to the patient if the patient is determined to have a lower or decreased DPP4 and/or POSTN level in one or more samples taken from the patient compared to predetermined DPP4 or POSTN threshold levels, or compared to the DPP4 or POSTN level in one or more control samples. In some aspects, a sample is obtained from a patient and is submitted for measurement of the level of DPP4 and/or POSTN in the sample.

[0283] The disclosure also provides a method of treating a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) comprising: (a) submitting a sample taken from the patient for measurement of the DPP4 and/or POSTN level in the sample, and (b) administering an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563), to the patient if the patient has a lower or decreased DPP4 and/or POSTN level in the sample taken compared to a predetermined DPP4 or POSTN threshold level, or compared to the level of DPP4 or POSTN in one or more control samples.

[0284] Also provided is method of treating a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) comprising: (a) submitting a sample taken from the patient for measurement of the DPP4 and/or POSTN level in the sample, and (b) suspending or not initiating the administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) to the patient if the patients has a higher or increased DPP4 and/or POSTN level in the sample compared to a predetermined DPP4 or POSTN threshold level, or compared to the level of DPP4 or POSTN in one or more control samples.

[0285] The disclosure also provides a method of treating a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) comprising: (a) measuring the levels of DPP4 and/or POSTN in a sample obtained from the patient; (b) determining whether the level of DPP4 and/or POSTN in the sample is higher or increased, or lower or decreased compared to a predetermined DPP4 or POSTN threshold level, and, (c) advising a healthcare provider to administer an eosinophil- targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) to the patient if the patient is determined to have a lower or decreased DPP4 and/or POSTN level in the sample compared to a predetermined DPP4 or POSTN threshold level, or compared to the DPP4 or POSTN level in one or more control samples; or to suspend or deny the administration of an eosinophil-targeted therapeutic agent to the patient if the patient is determined to have a higher or increased DPP4 and/or POST level in the sample compared to a predetermined DPP4 or POSTN threshold level, or compared to the DPP4 or POSTN level in one or more control samples.

[0286] Also provided is a method of treating a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma) comprising: (a) submitting a sample taken from a patient for measurement of the level of DPP4 and/or POSTN in a sample obtained from the patient, and (b) administering an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) to the patient if the patient is determined to have a lower or decreased DPP4 and/or POSNT level in the sample compared to a predetermined DPP4 or POSTN threshold level, or compared to the DPP4 or POSTN level in one or more control samples; or suspending, not initiating, or denying the administration of an eosinophil-targeted therapeutic agent to the patient if the patient is determined to have a higher or increased DPP4 and/or POSTN level in the sample compared to a predetermined DPP4 or POSTN threshold level, or compared to the DPP4 or POSTN level in one or more control samples. [0287] In some aspects, the patient's DPP4 and/or POSTN level is measured in an immunoassay as described herein employing antibodies or antigen binding fragments thereof which recognize human DPP4, POSTN, or antigen-binding fragments, variants or derivatives thereof. In some aspects, the sample is obtained from the patient and is submitted for measurement of the level of DPP4 and/or POSTN in the sample, for example, to a clinical laboratory.

[0288] In some aspects of the above treatment methods, the patient's DPP4 and/or

POSTN level (e.g. , DNA or RNA level) is measured in an assay employing one or more oligonucleotide probes capable of specifically measuring the expression levels of the DPP4 gene and/or the POSTN gene.

[0289] In some aspects, the molecular biomarker (e.g., DPP4 or POSTN) detection assay

(e.g. , an immunoassay) is performed on a sample obtained from the patient, by the healthcare professional treating the patient (e.g. , using an immunoassay as described herein including, e.g., the DPP4 immunoassay described in Example 2 or the POSTN assay described in WO2015120185, formulated as a "point of care" diagnostic kit).

[0290] In some aspects, a sample is obtained from the patient and is submitted, e.g. , to a clinical laboratory, for measurement of the DPP4 and/or POSTN level in the sample according to the healthcare professional's instructions (e.g. , using an immunoassay as described herein including, e.g., the DPP4 immunoassay described in Example 2 or any of the immunoassays disclosed in TABLE 1, or the POSTN assay described in WO2015120185).

[0291] In some aspects, the clinical laboratory performing the assay will advise the healthcare provide as to whether the patient can benefit from treatment with an eosinophil- targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) based on whether the patient's DPP4 and/or POSTN level is above a predetermined DPP4 or POSTN threshold value or is elevated relative to one or more control samples.

[0292] In some aspects, this disclosure includes a method of treating a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild- to- moderate asthma or severe asthma) over a period of time, comprising: measuring the level of DPP4 and/or POSTN (e.g. , protein expression level or gene expression level) in a first sample taken from the patient, or submitting a first sample taken from the patient for measurement of the level of DPP4 and/or POSTN in the sample, wherein the patient's DPP4 and/or POSTN level is, for example, measured using immunoassays, including, e.g., the DPP4 immunoassay described in Example 2 or any of the DPP4 immunoassays disclosed in TABLE 1 or the POSTN assay described in WO2015120185, and administering an eosinophil-targeted therapeutic agent to the patient, e.g., an anti-IL-5R antibody such as benralizumab (MEDI- 563) if the patient's DPP4 and/or POSTN level in the first sample is below a predetermined DPP4 or POSTN threshold level, or is lowered relative to the DPP4 or POSTN level in one or more control samples. The test can be performed by a healthcare provider or a clinical laboratory as noted above.

[0293] In some aspects, results of an immunoassay as provided herein can be submitted to a healthcare benefits provider for determination of whether the patient's insurance will cover treatment with an eosinophil-targeted therapeutic agent.

[0294] In some aspects, this disclosure includes a method of treating a patient having an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild- to- moderate asthma or severe asthma) comprising: measuring, e.g. , in a clinical laboratory, the DPP4 and/or POSTN level (e.g. , protein expression level or gene expression level) and eosinophil levels in a first sample obtained from a patient having an eosinophilic disease or disorder, e.g. , a sample provided by a healthcare provider, wherein the patient's DPP4 and/or POSTN level in the first sample is, for example, measured in an immunoassay, including, e.g., the DPP4 immunoassay described in Example 2 or any of the DPP4 immunoassays disclosed in TABLE 1, or the POSTN assay described in WO2015120185; determining whether the patient's DPP4 and/or POSTN level in the first sample is below a predetermined DPP4 or POSTN threshold level, or is reduced relative to the DPP4 or POSTN level in one or more control samples; and advising a healthcare provider to administer an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) to the patient if the patient's DPP4 and/or POSTN level is below a predetermined DPP4 or POSTN threshold level, or is decreased relative to the DPP4 or POSTN level in one or more control samples.

[0295] In some aspects, a sample is obtained from the patient and is submitted, e.g., to a clinical laboratory, for measurement of the level of DPP4 and//or POSTN alone or in combination with the level of at least another biomarker, e.g., blood eosinophils; or a combination thereof (e.g., protein expression level or gene expression level) in the sample, e.g., using an immunoassay. [0296] In some aspects, the clinical laboratory performing the assay will advise the healthcare provide as to whether the patient can benefit from treatment with an eosinophil- targeted therapeutic agent based on whether the patient's DPP4 and/or POSTN level (e.g., protein expression level or gene expression level) is below a predetermined DPP4 or POSTN threshold value or is low relative to one or more control samples.

[0297] In some aspects, methods of treatment contemplated herein (e.g., for an pulmonary disease or disorder such as asthma) comprise administering to the subject an eosinophil-targeted therapeutic agent in a sufficient amount and/or at sufficient interval to achieve and/or maintain a certain quantity of eosinophil-targeted therapeutic agent per volume of serum, using, for example, an assay as described herein.

[0298] For example, an antibody or antigen-binding fragment thereof (e.g., targeting IL-5 or IL-5R) can be given to achieve at least about 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/ml, 55 ng/ml, 60 ng/ml, 65 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, 100 ng/ml, 120 ng/ml, 140 ng/ml, 160 ng/ml, 180 ng/ml, 200 ng/ml, 220 ng/ml, 240 ng/ml, 260 ng/ml, 280 ng/ml, 300 ng/ml, 320 ng/ml, 340 ng/ml, 360 ng/ml, 380 ng/ml, 400 ng/ml, 420 ng/ml, 440 ng/ml, 460 ng/ml, 480 ng/ml, 500 ng/ml, 520 ng/ml, 540 ng/ml, 560 ng/ml, 580 ng/ml, 600 ng/ml, 620 ng/ml, 640 ng/ml, 660 ng/ml, 680 ng/ml, 700 ng/ml, 720 ng/ml, 740 ng/ml, 760 ng/ml, 780 ng/ml, 800 ng/ml, 820 ng/ml, 840 ng/ml, 860 ng/ml, 880 ng/ml, 900 ng/ml, 920 ng/ml, 940 ng/ml, 960 ng/ml, 980 ng/ml, or 1000 ng/ml in serum.

[0299] In a further embodiment, the antibody or antigen-binding fragment thereof (e.g., targeting IL-5 or IL-5R) can be given to achieve a concentration of antibody in serum from about 12.5 ng/ml to about 1000 ng/ml. Those of skill in the art will understand that the amounts given here apply to a full-length antibody or immunoglobulin molecule; if an antigen binding fragment thereof is used, the absolute quantity will differ from that given in a manner that can be calculated based on the molecular weight of the fragment.

[0300] In some aspects, methods of treatment contemplated herein, e.g., for an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such as mild- to- moderate asthma or severe asthma) comprise administering to the subject an eosinophil- targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) in an amount and at an interval of: 15-54 mg every 0.5-1.5 months; 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months; or 300-1100 mg every 4-12 months. [0301] In some aspects, the amount and interval are: 15-21 mg every 0.5-1.0 month; 55-

70 mg every 1.5-3.0 months; 150-260 mg every 4-6 months; or 300-700 mg every 4-8 months. In some aspects, the amount and interval are: 21 mg every month; 70 mg every 3 months; 210 mg every 6 months; or 700 mg every 6 months. In some aspects, the amount and interval are: 210 mg every 3 months or 700 mg every 3 months. In some aspects, the amount and interval are: 210 mg every 1 month or 700 mg every 1 month.

[0302] . In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered at about 2 mg to about 100 mg per dose. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered at about 20 mg per dose, at about 30 mg per dose, or at about 100 mg per dose. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered once every four weeks to once every twelve weeks. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered once every four weeks. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered once every eight weeks. In some aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is administered once every four weeks for twelve weeks and then once every eight weeks.

[0303] In some aspects of the methods, the eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is administered intravenously (IV). In some aspects of the methods, the eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is administered subcutaneously (SC).

[0304] In some aspects, the eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is administered in one or more fixed doses. In some aspects, the doses as administered every week, every two weeks, every three weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks every 10 weeks, or every 12 weeks. In some aspects, the dose comprises about 1 mg, 2 mg, 3mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg , 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, or 1000 mg. In some aspects, the dose is higher than 1000 mg.

[0305] In some aspects, two, three, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,

20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 doses are administered.

[0306] The formulation, dosage regimen, and route of administration of an eosinophil- targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563), can be adjusted to provide an effective amount for an optimum therapeutic response according to the method disclosed herein. With regard to the administration of an eosinophil- targeted therapeutic agent, the therapeutic agent may be administered through any suitable means, compositions and routes known in the art. With regard to dosage regiments, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0307] The eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) may be administered by any suitable technique, including but not limited to, parenterally, topically, or by inhalation. If injected, the pharmaceutical composition can be administered, for example, via intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or cutaneous routes (including intra-, trans- or sub-dermal, and subcutaneous), by bolus injection, or continuous infusion. In some aspects, the pharmaceutical composition is administered by an intravenous route. In some aspects the pharmaceutical composition is administered by a subcutaneous route.

[0308] In further aspects, the compositions are administered by oral, buccal, rectal, intratracheal, gastric, or intracranial routes. Localized administration, e.g. at a site of disease or injury is contemplated, for example, by enema or suppository for conditions involving the gastrointestinal tract. Also contemplated are transdermal delivery and sustained release from implants. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of the antagonist in aerosol form, and the like. Other alternatives include eyedrops; oral preparations including pills, syrups, lozenges or chewing gum; and topical preparations such as lotions, gels, sprays, and ointments, prefilled syringes and autoinjectors.

[0309] Advantageously, the eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) can be administered in the form of a composition comprising one or more additional components such as a physiologically acceptable carrier, excipient or diluent. Optionally, the composition additionally comprises one or more physiologically active agents for combination therapy.

[0310] A pharmaceutical composition may comprise an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) together with one or more substances selected from the group consisting of a buffer, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (such as those having fewer than 10 amino acids), a protein, an amino acid, a carbohydrate such as glucose, sucrose or dextrins, a chelating agent such as EDTA, glutathione, a stabilizer, and an excipient.

[0311] Neutral buffered saline or saline mixed with conspecific serum albumin are examples of appropriate diluents. In accordance with appropriate industry standards, preservatives such as benzyl alcohol may also be added. The composition may be formulated as a lyophilate using appropriate excipient solutions (e.g. , sucrose) as diluents.

[0312] In some aspects, the eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) can be provided at a concentration of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 mg/ml.

[0313] Exemplary formulations useful for the present invention are those that include a glutamic acid, citric acid or acetic acid buffer at an appropriate pH, from 4.5 to 5.2, an excipient such as sucrose, glycine, proline, glycerol, and/or sorbitol at an appropriate concentration such as 1 to 20% (w/v), and a surfactant such as a non-ionic surfactant like polysorbate (polysorbate 20 or 80) or poloxamers (poloxamer 1888) at an appropriate concentration of 0.001% - 0.1% (w/v). Such formulations are disclosed in US Patent No. 6171586 and WIPO Published Applications Nos. WO20100027766 and WO2011088120. In some aspects, the formulations comprise sodium acetate, sucrose and polysorbate 20.

[0314] In some aspects, the administration of eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563), to the patient results in:

(a) AER (Acute Exacerbation Rate) reduction; (b) FEV1 (Forced Expiratory Volume in one second) increase;

(c) improved ACQ-6 (Asthma Control Questionnaire, 6-item version) results;

(d) improved AQLQ (Asthma Quality of Life Questionnaire) results;

(e) reduction of cytokines secreted by type 2 innate lymphocytes (ILC2);

(f) reduction of ILC2 levels,

(g) reduction in eosinophil and/or basophil count, or,

(h) a combination thereof.

[0315] In some aspects, the AER reduction after administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, compared to the AER observed in a population of patients treated with a placebo.

[0316] In some aspects, the FEVi increase after administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is at least about 3%, at least about 5%, at least about 7%, at least about 9%, at least about 11%, at least about 13%, at least about 15%, least about 17%, at least about 19%, at least about 21%, at least about 23%, at least about 25%, at least about 27%, at least about 29%, at least about 31% , at least about 33%, or at least about 35% compared to the FEVi observed in a population of patients treated with a placebo.

[0317] In some aspects, the FEVi increase after administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is at least about 25 mL, at least about 50 mL, at least about 75 mL, at least about 100 mL, at least about 125 mL, at least about 150 mL, at least about 175 mL, least about 200 mL, at least about 225 mL, at least about 250 mL, at least about 275 mL, at least about 300 mL, at least about 325 mL, or at least about 350 mL compared to the FEVi observed in a population of patients treated with a placebo.

[0318] In some aspects, the ACQ-6 change after administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is about - 0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1, -1.1, or -1.2 compared to the mean ACQ-6 observed in a population of patients treated with a placebo.

[0319] In some aspects, the reduction of cytokines secreted by type 2 innate lymphocytes

(ILC2) after administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% compared to the level of cytokines secreted by ILC2 observed in a population of patients treated with a placebo.

[0320] In some aspects, the reduction of eosinophils after administration of an eosinophil- targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% compared to the eosinophil count observed in a population of patients treated with a placebo.

[0321] In some aspects, the reduction of basophils after administration of an eosinophil- targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% compared to the basophil count observed in a population of patients treated with a placebo.

VII. Kits for DPP4 or POSTN Detection and Quantification

[0322] This disclosure also provides kits for detecting DPP4 and/or POSTN levels, for example, through an immunoassay method. Such kits can comprise containers, each with one or more of the various reagents (e.g., in concentrated form) utilized in the method, including, for example, one or more anti-DPP4 antibodies (i.e., antibodies capable of detecting DPP4). One or more anti-DPP4 or anti-POSTN antibodies, e.g., capture antibodies capable of detecting DPP4 or POSTN can be provided already attached to a solid support. One or more antibodies, e.g., detection antibodies, can be provided already conjugated to a detectable label, e.g., biotin or a ruthenium chelate.

[0323] The kit can also provide reagents for coupling a detectable label to an antibody (as well as the label itself), buffers, and/or reagents and instrumentation to support the practice of the assays provided herein. In certain aspects, a labeled secondary antibody can be provided that binds to the detection antibody. A kit provided according to this disclosure can further comprise suitable containers, plates, and any other reagents or materials necessary to practice the assays provided herein.

[0324] In some aspects, a kit comprises one or more nucleic acid probes (e.g., oligonucleotides comprising naturally occurring and/or chemically modified nucleotide units) capable of hybridizing a subsequence of the gene sequence of DPP4 or POSTN under high stringency conditions. In some aspects, one or more nucleic acid probes (e.g., oligonucleotides comprising naturally occurring and/or chemically modified nucleotide units) capable of hybridizing a subsequence of the gene sequence of DPP4 or POSTN under high stringency conditions are attached to a microarray chip.

[0325] A kit provided according to this disclosure can also comprise brochures or instructions describing the process. For DPP4 or POSTN detection immunoassays (e.g., single or multiplexed assays to detect DPP4 or POSTN), and in particular sandwich immunoassays, e.g., an ELISA assay or an ECL assay, the sandwich immunoassay process comprises a first "capture" antibody (e.g., an antibody specifically binding to DPP4 or POSTN) or antigen-binding fragment thereof attached to a solid support, and a second anti- DPP4 "detection" antibody or antigen binding fragment thereof. The immunoassay can be performed by methods provided herein or methods well known and understood by those of ordinary skill in the art.

[0326] In one aspect, the immunoassay comprises attaching a capture antibody or fragment thereof to a solid support; applying the test sample or a control sample, allowing DPP4 or POSTN, if present in the sample, to bind to the capture antibody or fragment thereof; applying the detection antibody or fragment thereof, which can bind to DPP4 or POSTN already bound to the capture antibody or fragment thereof; and measuring the amount of detection antibody or fragment thereof bound to DPP4 or POSTN. In certain aspects, the assay can further include washing steps, blocking steps and incubation steps.

[0327] Test kits can include instructions for carrying out one or more DPP4 and/or

POSTN detection assays, e.g., immunoassays or nucleic acid detection assays to detect the presence/absence or levels of DPP4 or POSTN. Instructions included in the kits 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. 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.

VIII. Companion Diagnostic System

[0328] The methods disclosed herein can be provided as a companion diagnostic, for example available via a web server, to inform the clinician or patient about potential treatment choices. The methods disclosed herein can comprise collecting or otherwise obtaining a biological sample and performing an analytical method to detect and measure DPP4 and/or POSTN levels (e.g., full length or soluble DPP4 protein expression levels, POSTN expression levels, or gene expression levels for DPP4 or POSTN) alone or in combination with other biomarkers (e.g., a panel a genes used to derive a gene signature, such as a Th-2 signature or the patient's eosinophil count).

[0329] Molecular biomarkers that can be combined with DPP4 and/or POSTN include

IL-33, IL-25, TSLP, IL-22, CCL20, LCN2, sCTLA-3, sCD28, CCL5, CCL11, CCL22, CCL26, FZD5, DOK1, CST2, ZNF436, C20orfl00, NAGS, CST1, CDH13, HRH1, TMEM132B, NTRK1, SLC02A1, IgE, FETUB, KRT31IKRT34, C6orfl38, ATP5J, TUBAL3, JAM2, NOVA2, NOS2A, HS3ST4, GRM8, IL1R2, CTDSPL, CEP72, LOC199800, LYPD1, DISP1, NKX1-2, C4orf38, LOXL4, PRKD1, PAM124B, GPR44, HIGD1B, CLCA1, SEPT11, CYYR1, CD36, ALOX15, A AD AC, ACTA1, ODC1, DKFZp434F142, ACHE, CSF3, LOC100132552, C12orf27, ZNF331, GK5, DUSP1IDUSP4, LRWD1, PGLYRP4, GUSBL2, CLGN, NR1I2, EST, LRRC37B, SAA4, SLC12A3, TMEM45A, FLJ37464, MUC5B, CXCL6, GLRB, DKFp686K01114, FOLR1, TSPAN6, AKR1C1, KIAA0232, PTP4A1, PCYT2, RHOV, PROS1, Cl lorf63, TCTN1, PIP5K1B, OSBPL6, NSUM7, GJB7, IRS2, or combinations thereof. See Lun et al, J. Clin. Immunol.

27:430-437 (2007), Choi et al„ J. Immunol. 186(3): 1861-9 (2011), and WO2009124090A1, which are herein incorporated by reference in their entireties.

[0330] Standard names, aliases, etc. of proteins and genes designated by identifiers used throughout this application (e.g., PIP5K1B) can be identified, for example, via Genecards

(www.genecards.org) or Uniprot (www.uniprot.org).

[0331] Molecular biomarkers that can be combined with DPP4 and/or POSTN include, e.g., IL-33, IL-25, TSLP, IL-22, CCL20, LCN2, CST1, CCL26, CLCA1, CST2, PRR4,

SERPINB2, CEACAM5, iNOS, SERPINB4, CST4, PRB4, TPSD1, TPSG1, MFSD2, CPA3,

GPR105, CDH26, GSN, C20RF32, TRACH2000196 (TMEM71), DNAJC12, RGS13,

SLC18A2, SERPINB 10, SH3RF2, FCER1B, RUNX2, PTGS1, ALOX15, and combinations thereof.

[0332] DPP4 and/or POSTN levels (e.g., protein expression levels or gene expression levels) or normalized scores derived from measured DPP4 and/or POSTN levels can be used alone (e.g., for treatment, diagnostic, prognostic, or monitoring purposes), or in combination with levels or normalized scores derived from other biomarkers (e.g., a panel a genes used to derive a gene signature, such as a Th-2 signature or the patient's eosinophil count).

[0333] These scores can also be combined with scores corresponding, for example, to (i) the level of the patient's IgE levels, (ii) the patient's eosinophil or basophil count, (iii) the patient's Fraction of Exhaled Nitric Oxide (FENO), (iv) the patient's Eosinophil/Lymphocyte and Eosinophil/Neutrophil (ELEN) index, (v) the patient's EOS index, (vi) the patient's IgE levels, (vii), pre- or post-bronchodilator FEV1, FVC measurements or reversibility, (viii) wall area percentage (WA%) of subsegmental airways from CT scan of the lungs, or (ix) a combination of two or more thereof, to yield a diagnostic score.

[0334] In this approach, the diagnostic score may be a single number determined from the sum of all the marker calculations that is compared to a preset DPP4 or POSTN threshold value that is an indication of the presence or absence of disease (or of a certain disease status, e.g., moderate or severe asthma). Or the diagnostic score may be a series of bars that each represent a biomarker value and the pattern of the responses may be compared to a pre-set pattern for determination of the presence or absence of disease.

[0335] At least some aspects of the methods described herein, due to the complexity of the calculations involved, a method comprising the use of DPP4 and/or POSTN levels (e.g., levels in blood serum) as a biomarker disclosed herein can be implemented with the use of a computer. In some aspects, the computer system comprises hardware elements that are electrically coupled via bus, including a processor, input device, output device, storage device, computer-readable storage media reader, communications system, processing acceleration (e.g., DSP or special-purpose processors), and memory. The computer-readable storage media reader can be further coupled to computer-readable storage media, the combination comprehensively representing remote, local, fixed and/or removable storage devices plus storage media, memory, etc. for temporarily and/or more permanently containing computer-readable information, which can include storage device, memory and/or any other such accessible system resource.

[0336] A single architecture might be utilized to implement one or more servers that can be further configured in accordance with currently desirable protocols, protocol variations, extensions, etc. However, it will be apparent to those skilled in the art that embodiments may well be utilized in accordance with more specific application requirements. Customized hardware might also be utilized and/or particular elements might be implemented in hardware, software or both. Further, while connection to other computing devices such as network input/output devices (not shown) may be employed, it is to be understood that wired, wireless, modem, and/or other connection or connections to other computing devices might also be utilized.

[0337] In one aspect, the system further comprises one or more devices for providing input data to the one or more processors. The system further comprises a memory for storing a data set of ranked data elements. In another aspect, the device for providing input data comprises a detector for detecting the characteristic of the data element, e.g., such as a fluorescent plate reader, mass spectrometer, or gene chip reader.

[0338] The system additionally may comprise a database management system. User requests or queries can be formatted in an appropriate language understood by the database management system that processes the query to extract the relevant information from the database of training sets. The system may be connectable to a network to which a network server and one or more clients are connected. The network may be a local area network (LAN) or a wide area network (WAN), as is known in the art. Preferably, the server includes the hardware necessary for running computer program products (e.g., software) to access database data for processing user requests. The system can be in communication with an input device for providing data regarding data elements to the system (e.g., expression values). In one aspect, the input device can include a gene expression profiling system including, e.g., a mass spectrometer, gene chip or array reader, and the like.

[0339] Some aspects described herein can be implemented so as to include a computer program product. A computer program product may include a computer readable medium having computer readable program code embodied in the medium for causing an application program to execute on a computer with a database. As used herein, a "computer program product" refers to an organized set of instructions in the form of natural or programming language statements that are contained on a physical media of any nature (e.g., written, electronic, magnetic, optical or otherwise) and that may be used with a computer or other automated data processing system. Such programming language statements, when executed by a computer or data processing system, cause the computer or data processing system to act in accordance with the particular content of the statements.

[0340] Computer program products include without limitation: programs in source and object code and/or test or data libraries embedded in a computer readable medium. Furthermore, the computer program product that enables a computer system or data processing equipment device to act in pre-selected ways may be provided in a number of forms, including, but not limited to, original source code, assembly code, object code, machine language, encrypted or compressed versions of the foregoing and any and all equivalents.

[0341] In one aspect, a computer program product is provided to implement the treatment, diagnostic, prognostic, or monitoring methods disclosed herein, for example, to determine whether to administer an eosinophil-targeted therapeutic agent (e.g., an antibody that specifically binds to IL-5R such as benralizumab, or an antigen binding fragment thereof) to a patient in need thereof if the levels of DPP4 and/or POSTN in one or more samples taken from the patient are lower or decreased compared to predetermined DPP4 or POSTN threshold levels, or compared to DPP4 or POSTN levels in one or more control samples.

[0342] The computer program product includes a computer readable medium embodying program code executable by a processor of a computing device or system, the program code comprising: (a) code that retrieves data attributed to a biological sample from a subject, wherein the data comprises DPP4 and/or POSTN level data (or data otherwise derived from DPP4 and/or POSTN level values) alone or combination with values corresponding to other biomarkers in the biological sample (e.g., a panel a genes used to derive a gene signature, such as a Th-2 signature). These values can also be combined with values corresponding, for example, to (i) the level of the patient's IgE levels, (ii) the patient's eosinophil or basophil count, (iii) the patient's Fraction of Exhaled Nitric Oxide (FENO), (iv) the patient's Eosinophil/Lymphocyte and Eosinophil/Neutrophil (ELEN) index, (v) the patient's EOS index, (vi) wall area percentage (WA%) of subsegmental airways from CT scan data of the lungs, (vii) the patient's IgE levels, (viii), pre- or post-bronchodilator FEV1, FVC measurements or reversibility, or (ix) a combination of two or more thereof; and,

(b) code that executes a classification method that indicates, e.g., whether to administer an eosinophil-targeted therapeutic agent to a patient in need thereof.

[0343] While various aspects have been described as methods or apparatuses, it should be understood that aspects can be implemented through code coupled with a computer, e.g., code resident on a computer or accessible by the computer. For example, software and databases could be utilized to implement many of the methods discussed above. Thus, in addition to aspects accomplished by hardware, it is also noted that these aspects can be accomplished through the use of an article of manufacture comprised of a computer usable medium having a computer readable program code embodied therein, which causes the enablement of the functions disclosed in this description. Therefore, it is desired that aspects also be considered protected by this patent in their program code means as well.

[0344] Furthermore, some aspects can be code stored in a computer-readable memory of virtually any kind including, without limitation, RAM, ROM, magnetic media, optical media, or magneto-optical media. Even more generally, some aspects could be implemented in software, or in hardware, or any combination thereof including, but not limited to, software running on a general purpose processor, microcode, PLAs, or ASICs.

[0345] It is also envisioned that some aspects could be accomplished as computer signals embodied in a carrier wave, as well as signals (e.g., electrical and optical) propagated through a transmission medium. Thus, the various types of information discussed above could be formatted in a structure, such as a data structure, and transmitted as an electrical signal through a transmission medium or stored on a computer readable medium. TABLE 3: Description of Sequences

Examples

Example 1

A Phase 2b, Dose-ranging Study to Evaluate the Efficacy and Safety of Benralizumab

(MEDI-563) in Adults with Uncontrolled Asthma

[0346] A Ph2b study (CP220) was conducted with benralizumab (MEDI-563) in a population of moderate-to-severe, uncontrolled asthmatics (FIG. 1). Post hoc exploratory analyses were conducted with two data sets (i) using samples collected at baseline (week 1 day 1 predose) that were measured for POSTN and DPP4 levels and (ii) using samples collected at week -1 (screening) and week 40 that were. DPP4 was measured using an R&D Systems DPP4 detection kit. POSTN was measured using a periostin detection method developed as a companion diagnostic for tralokinumab (see WO2015120185 which is herein incorporated by reference in its entirety)

[0347] POSTN is a downstream activation marker of IL-13 pathway activation and is being developed as a potential biomarker to identify patients who may respond to anti-IL-13 therapy. It is also used as a potential marker for identifying patients with evidence of raised Th2 pathway activation. DPP4 is also a downstream activation marker of IL-13 pathway activation.

[0348] Patient population was composed of uncontrolled asthmatics on medium or high dose inhaled corticosteroids and long acting β agonists. The study included 609 randomized subjects. 324 in the EOS+ group (PBO= 80; benralizumab 2 mg = 81; 20 mg = 81 and 100 mg = 82). 285 in the EOS- group (PBO = 143; benralizumab 100 mg = 142). The study was double blind, placebo controlled, in moderate- severe asthma patients evaluating 3 doses of benralizumab vs placebo in subjects with high blood eosinophil levels (as determined by ELEN index) and evaluating one dose vs placebo in subjects with low blood eosinophil levels (as determined by ELEN index).

[0349] In order to understand the patient populations that may respond to treatment with benralizumab, it was proposed to segment the CP220 patient population into other known patient sub-groups by biomarkers or other clinical characteristics to gain a greater understanding of other potential responder groups. For samples measured in Dataset A (i.e. those where POSTN and DPP4 were measured in samples collected at week 1 day 1), the following exploratory objectives were planned (i) evaluate the relationship of blood eosinophil levels with serum POSTN levels and serum DPP4 levels at baseline (the exploratory outcome variables were blood eosinophils, serum POSTN, and serum DPP4); (ii) evaluate the efficacy of benralizumab by baseline POSTN on exacerbations, lung function and symptoms (the exploratory outcome variables were serum periostin, annual exacerbation rate, FEV1, and ACQ); (iii) evaluate the efficacy of benralizumab by baseline DPP4 on exacerbations, lung function and symptoms (the exploratory outcome variables were serum DPP4, annual exacerbation rate, FEV1, and ACQ); (iv) evaluate the efficacy of benralizumab by baseline POSTN together with baseline eosinophil level on exacerbations, lung function and symptoms (the exploratory outcome variables were blood eosinophils, serum periostin, annual exacerbation rate, FEV1, and ACQ); and (v) evaluate the efficacy of benralizumab by baseline DPP4 together with baseline eosinophil level on exacerbations, lung function and symptoms (the exploratory outcome variables were blood eosinophils, serum DPP4, annual exacerbation rate, FEV1, and ACQ).

[0350] For samples measured in data set B (i.e. those where POSTN and DPP4 were measured in samples collected at week -1 and week 40), the following objectives were planned (i) evaluate the effect of treatment with benralizumab on serum periostin levels in mlTT population, by baseline blood eosinophils and by baseline POSTN levels (the exploratory outcome variables were blood eosinophils and serum POSTN); and (ii) evaluate the effect of treatment with benralizumab on serum DPP4 levels in mlTT population, by baseline blood eosinophils and by baseline DPP4 levels (the exploratory outcome variables were blood eosinophils and serum DPP4).

[0351] For evaluating treatment effect of benralizumab on efficacy endpoints the following analyses were conducted (i) evaluation by placebo (combined EOS+ & EOS -), 2mg, 20mg and lOOmg (combined EOS+ & EOS -) evaluating any dose response effect; and (ii) evaluation by placebo (combined EOS+ & EOS -), pooled group of 20 mg and 100 mg dose groups (EOS+ and EOS-).

[0352] For any sub-group analyses referring to cut-offs of certain biomarkers: eosinophil high population is defined as blood eosinophils >300 cells^L and eosinophil low population is defined as blood eosinophils <300 cells^L. POSTN high and DPP4 high is defined as those subjects with levels > median and POSTN low and DPP4 low is defined as those subjects with levels < median.

[0353] The current and predicted outputs for Dataset A are the following:

(a) A table of descriptive statistics of median, mean, standard deviation and range of periostin and DPP4 in mlTT population was prepared to evaluate POSTN and DPP4 levels in study population and in other clinical sub-groups of interest, (FIG. 2).

(b) A table of descriptive statistics of median, mean, standard deviation and range of POSTN and DPP4 in reversible and non-reversible and ICS medium and ICS high and in blood eosinophil >300 and <300 cells^L sub-populations will be prepared.

(c) A table of the demographics and clinical characteristics of subjects by POSTN high, POSTN low, DPP4 high and DPP4 low will be prepared to show the association of DPP4 or POSTN which particular clinical characteristics or treatment when divided into the subgroups. The demographic and clinical characteristics to use in the table are age, gender, BMI, FEV1(L), FEVl (% predicted), Reversibility, ACQ, ICS dose, OCS dose, number of asthma exacerbations in the previous 12 months (from the asthma history questionnaire), asthma symptom diary card score, serum IgE, and blood eosinophil count.

(d) A plot of correlation of baseline serum POSTN vs baseline DPP4 will be prepared to show the relationship between POSTN and DPP4 in the study population at baseline. (e) A plot of correlation of baseline serum POSTN with baseline blood eosinophil will be prepared to show the relationship between POSTN and blood eosinophils in the study population at baseline.

(f) A plot of correlation of baseline serum DPP4 with baseline blood eosinophils will be prepared to show any relationship between DPP4 and blood eosinophils in the study population at baseline.

(g) A graphic of the overlap of blood eosinophils >300, <300 cells^L with POSTN high and POSTN low subjects by mITT, lung function reversible subjects, lung function nonreversible subjects, ICS high subjects and ICS medium subjects was prepared to evaluate overlap between different sub-groups in the study population.

(h) A graphic of the overlap of blood eosinophils >300, <300 cells^L with DPP4 high and DPP4 low subjects by mITT, lung function reversible subjects, lung function non-reversible subjects, ICS high subjects and ICS medium subjects was prepared to evaluate overlap between different sub-groups in the study population.

(i) Plots showing the effect of benralizumab on (i) exacerbate rate by baseline serum POSTN by treatment group, (ii) change in FEVl by baseline serum POSTN by treatment group, (iii) change in ACQ by baseline serum POSTN by treatment group, (iv) exacerbate rate by baseline serum DPP4 by treatment group, (v) change in FEVl by baseline serum DPP4 by treatment group, (vi) change in ACQ by baseline serum DPP4 by treatment group, (vii) exacerbation rate by baseline serum POSTN with baseline blood eosinophils by treatment group, (viii) exacerbation rate by baseline serum DPP4 with baseline blood eosinophils by treatment group, (ix) change in FEVl by baseline serum POSTN with baseline blood eosinophils by treatment group, (x) change in ACQ by baseline serum POSTN with baseline blood eosinophils by treatment group, (xi) change in FEVl by baseline serum DPP4 with baseline blood eosinophils by treatment group, and (xii) change in ACQ by baseline serum DPP4 with baseline blood eosinophils by treatment group, will be prepared.

(j) To evaluate the effect of benralizumab on efficacy endpoints in specific sub-populations, plots of effect of benralizumab in subjects with high or low baseline serum POSTN on exacerbation rate, change in FEVl from baseline and change in ACQ from baseline were prepared for the following sub-groups:

(i) POSTN high and POSTN low (mITT only),

(ii) POSTN high and POSTN low in subjects with reversible lung function, (iii) Each of the above group in POSTN high plus eosinophils >300 and < 300cells^L and POSTN low plus eosinophils >300 and < 300cells^L sub-groups.

(k) To evaluate the effect of benralizumab on efficacy endpoints in specific sub-populations, plots of effect of benralizumab in subjects with high or low baseline serum POSTN on exacerbation rate, change in FEV1 from baseline and change in ACQ from baseline will be prepared for the following sub-groups:

(i) POSTN high and POSTN low in subjects with non-reversible lung function,

(ii) POSTN high and POSTN low in subjects on high dose ICS,

(iii) POSTN high and POSTN low in subjects on medium dose ICS,

(iv) POSTN high and POSTN low in subjects who are not on chronic OCS,

(v) POSTN high and POSTN low in subjects who are not on chronic OCS and >12% reversible at baseline,

(vi) Each of the above group in POSTN high plus eosinophils >300 and < 300cells^L and POSTN low plus eosinophils >300 and < 300cells^L sub-groups.

(1) To evaluate the effect of benralizumab on efficacy endpoints in specific sub-populations, plots of effect of benralizumab in subjects with high or low baseline serum DPP4 on exacerbation rate, change in FEV1 from baseline and change in ACQ from baseline were prepared for the following sub-groups:

(i) DPP4 high and DPP4 low (mITT only),

(ii) DPP4 high and DPP4 low in subjects with reversible lung function,

(iii) Each of the above in DPP4 high plus eosinophils >300 and < 300cells^L and DPP4 low plus eosinophils >300 and < 300cells^L.

4] To evaluate the effect of benralizumab on efficacy endpoints in specific sub- populations, plots of the effect of benralizumab in subjects with high or low baseline serum DPP4 on exacerbation rate, change in FEV1 from baseline and change in ACQ from baseline will be prepared for the following sub-groups:

(i) DPP4 high and DPP4 low in subjects with non-reversible lung function,

(ii) DPP4 high and DPP4 low in subjects on high dose ICS,

(iii) DPP4 high and DPP4 low in subjects on medium dose ICS,

(iv) DPP4 high and DPP4 low in subjects who are not on chronic OCS,

(v) DPP4 high and DPP4 low in subjects who are not on chronic OCS and >=12% reversible at baseline, (vi) Each of the above in DPP4 high plus eosinophils >300 and < 300cells^L and DPP4 low plus eosinophils >300 and < 300cells^L.

5] The predicted outputs for Dataset B are the following:

(a) Descriptive statistics showing POSTN levels in study population and in other clinical subgroups of interest at baseline and post treatment with benralizumab will be presented in a table of descriptive statistics of median, mean, standard deviation and range of POSTN at week - 1 and week 40 in mITT population.

(b) Descriptive statistics showing DPP4 levels in study population and in other clinical subgroups of interest at baseline and post treatment with benralizumab will be presented in a table of descriptive statistics of median, mean, standard deviation and range of DPP4 at week - 1 and week 40 in mITT population.

(c) A plot of change in baseline POSTN vs baseline blood eosinophil level by treatment group (i.e. after benralizumab treatment and placebo) will be prepared to show the effect of benralizumab on serum POSTN level by increasing baseline blood eosinophil level.

(d) A plot of change in baseline DPP4 vs baseline blood eosinophil level by treatment group (i.e. after benralizumab treatment and placebo) will be prepared to show the effect of benralizumab on serum DPP4 level by increasing baseline blood eosinophil level.

(e) A plot of effect of benralizumab on POSTN levels from week -1 to week 40 in the following populations (i) mITT, (ii) blood eosinophils >300 and <300 cells^L, (iii) FEV1 reversible and non-reversible subjects, (iv) ICS high and ICS medium, will be prepared to show the effect of treatment with benralizumab on serum POSTN levels by study population and by sub-groups.

(f) A plot of the effect of benralizumab on DPP4 levels from week -1 to week 40 in the following populations (i) mITT, (ii) blood eosinophils >300 and <300 cells/ 'μί, (iii) FEV1 reversible and non-reversible subjects, and (iv) ICS high and ICS medium, will be prepared to show the effect of treatment with benralizumab on serum DPP4 levels by study population and by sub-groups.

Example 2

DPP4 ELISA Immunoassay [0356] The DPP4 detection assay disclosed herein is a quantitative ELISA-based immunoassay. A mouse monoclonal antibody specific for human DPP4 was pre-coated onto a microplate (R&D Systems, Cat #DC260). One hundred microliters of assay diluent were first added to wells of microplates followed by addition of 50 of standards, controls and 1:50 diluted serum samples. The plates were incubated for 2 hours ± 15 minutes at room temperature to allow DPP4 to bind to the capture antibody on the plates.

[0357] To remove unbound materials plates were then washed 4 times with IX Wash buffer (Medlmmune) using the plate washer. Plates were then incubated with 200 of the detection antibody (polyclonal anti-DPP4 antibody-HRP conjugate, R&D Systems, Cat #DC260) for 2 hours ± 15 minutes at room temperature. After that, the plates were washed and incubated with 200 μΐ of a chromogenic HRP substrate, hydrogen peroxide/tetramethylbenzidine (R&D Systems, Cat #DC260) for 30 minutes ± 5 minutes at room temperature in the place protected from light.

[0358] The enzyme reaction was stopped by the addition of 50 stop solution (2N sulfuric acid). Within 30 minutes after stopping of the reaction plates were read on SpectraMaxPlus 384 Microplate Spectrophotometer (Molecular Devices) to measure the optical density at 450 nm. The intensity of the color generated was directly proportional to the amount of bound DPP4. The DPP4 concentrations in samples and controls were interpolated from the standard curve of recombinant human DPP4, which was run on each plate. The quadratic model was used for curve fitting. The derived DPP4 concentrations were adjusted for the initial sample dilution.

[0359] The quantifiable range was 16 ng/niL to 1000 ng/niL of DPP4 in 100% serum.

The assay was reproducible, precise and specific for DPP4. The parallelism between recombinant standard and endogenous serum DPP4 has been demonstrated. The assay has acceptable matrix interference, 3 freeze-thaw cycles stability and 1.5 years longitudinal stability at -80°C.

Example 3

Baseline High Blood Eosinophils or Serum Biomarkers of Low IL-13 Pathway Activation Predict Exacerbation Rate Reduction by Benralizumab in Moderate-to-

Severe Asthmatics [0360] Benralizumab, a humanized monoclonal antibody that selectively depletes eosinophils and basophils through enhanced antibody dependent cellular cytotoxicity, reduced exacerbations in a Ph2b study (NCT01238861, see FIG. 1) in moderate-to- severe asthmatics with eosinophilic inflammation (Castro et al., Lancet Respir Med. 2: 878-90 (2014)). Dipeptidyl peptidase (DPP4) and POSTN (POSTN) are upregulated by IL-13 and are potential biomarkers of response to anti-IL-13 therapy (Brightling et al., Lancet Respir Med. 3: 692-701 (2015)). We explored the ability of baseline levels of serum DPP4 and POSTN to predict exacerbation rate reduction by benralizumab in the Ph2 study.

[0361] Serum DPP4 was measured using the immunoassay of Example 2 and POSTN using an immunoassay from Abbott Diagnostics. Asthma subjects were segmented into subgroups of DPP4 or POSTN High or Low, (defined as serum concentration > or < median, respectively) or Eosinophil High or Low (defined by baseline blood eosinophils > or < 300 cells/ μί, respectively). Groups were further segmented combining DPP4 High and Low or POSTN High and Low with Eosinophil High and Low (FIG. 3).

[0362] The effect of benralizumab (combined 20 mg and 100 mg s.c. treatment arms) was evaluated compared to placebo on exacerbation rate in each subgroup.

[0363] Median serum DPP4 and POSTN concentrations were 363.5 ng/niL (ranging from

103.3 ng/niL to 867.5 ng/niL) and 23.6 ng/niL (ranging from 7.5 ng/mL to 104.1 ng/niL) respectively. See FIG. 2.

[0364] Serum DPP4 did not correlate with blood eosinophils (correlation co-efficient =

0.05, P = 0.22) or serum POSTN (correlation co-efficient = 0.03, P=0.48). Serum POSTN correlated with blood eosinophils (correlation co-efficient = 0.33, P<0.001) (FIG. 4).

[0365] High eosinophils, DPP4 low and POSTN low significantly predicted exacerbation rate reduction with benralizumab treatment. In the eosinophil high subgroup, exacerbation rate reduction was independent of POSTN level. For the eosinophil low subgroup, POSTN low showed numerically greater exacerbation rate reduction than POSTN high. DPP-4 low was predictive of exacerbation rate reduction in eosinophil high and identified numerically greater exacerbation rate reduction in the eosinophil low subgroup FIGS. 5, 6 and 7. See also TABLE 4.

TABLE 4. Effect of benralizumab (combined 20 mg and 100 mg s.c. treatment arms) on exacerbation rate reduction in biomarker defined subsets. Asthma exacerbation rate P value reduction % (95% C.I.)

N (placebo/benralizumab)

48

EOS high 0.01

83/167

9

EOS low 0.687

139/135

28

POSTN high 0.157

99/148

45

POSTN low 0.025

116/137

OS high and 47

0.054 POSTN high 49/98

OS high and 53

0.066 POSTN low 33/60

EOS low and -8

0.81 POSTN high 50/49

EOS low and 40

0.157 POSTN low 83/77

11

DPP4 high 0.701

106/141

45

DPP4 low 0.008

107/141

high and DPP4 11

0.799 high 39/84

high and DPP4 57

0.007 low 43/72

S low and DPP4 -1

0.99 high 67/56

S low and DPP4 31

0.247 low 64/69 [0366] Exacerbation rate reduction (ERR) was performed by Poisson regression model with baseline ICS status as a covariate with the log of follow-up time as the offset term. Overdispersion correction was given by the Pearson's chi-square statistic divided by the degrees of freedom and applied to standard errors and likelihood ratio statistics to be adjusted accordingly.

[0367] CONCLUSIONS: High baseline blood eosinophil concentrations predicted benralizumab efficacy, and, effects were independent of POSTN in this subgroup. Below- median baseline DPP-4 concentrations indicative of low IL-13 pathway activation predicted benralizumab efficacy and may identify patients with low blood eosinophil concentrations responsive to benralizumab. The biomarker of IL-13 pathway activation, DPP4, may identify patient subsets who benefit from anti-IL-5R/eosinophil-depleting benralizumab (DPP4 low) and IL-13-targeting tralokinumab (DPP4 high)..

TABLE 5. List of Abbreviations

*** [0368] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0369] The present invention has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0370] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0371] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0372] All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method of treating an eosinophilic disease or disorder in a patient, comprising administering an eosinophil-targeted therapeutic agent to the patient if the patient is determined or identified to have a lower or decreased dipeptidyl peptidase-4 (DPP4) level in one or more samples taken from the patient compared to a predetermined DPP4 threshold level, or compared to a DPP4 threshold level in one or more control samples.

2. A method of treating a patient having an eosinophilic disease or disorder comprising suspending or not initiating the administration of an eosinophil-targeted therapeutic agent to the patient if the patient is determined or identified to have a higher or increased DPP4 level in one or more samples taken from the patient compared to predetermined DPP4 threshold level, or compared to a DPP4 threshold level in one or more control samples.

3. A method of treating an eosinophilic disease or disorder in a patient, wherein the patient failed, was non-responsive or intolerant to treatment with a therapeutic agent comprising administering an eosinophil-targeted therapeutic agent to the patient if the patient is determined or identified to have a lower or decreased DPP4 level in one or more samples taken from the patient compared to a predetermined DPP4 threshold level, or compared to a DPP4 threshold level in one or more control samples.

4. A method of determining whether to treat a patient having an eosinophilic disease or disorder with an eosinophil-targeted therapeutic agent, comprising determining to treat the patient if the patient is determined or identified to have a lower or decreased DPP4 level in one or more samples taken from the patient compared to a predetermined DPP4 threshold level, or compared to a DPP4 threshold level in one or more control samples.

5. A method of selecting a patient diagnosed with an eosinophilic disease or disorder as a candidate for treatment with an eosinophil-targeted therapeutic agent, comprising selecting the patient for treatment if the patient is determined or identified to have a lower or decreased DPP4 levels in one or more samples taken from the patient compared to a predetermined DPP4 threshold level, or compared to a DPP4 threshold level in one or more control samples.

6. The method according to any one of claims 1 to 5, further comprising measuring the level of DPP4 in one or more of the samples obtained from the patient or instructing a clinical laboratory or healthcare provider to measure the level of DPP4 in the sample and/or submitting the one or more samples obtained from the patient to a clinical laboratory or healthcare provider to measure the level of DPP4 in the sample.

7. The method according to any one of claims 1 to 6, further comprising determining the level of DPP4 in the one or more samples obtained from the patient.

8. The method according to any one of claims 1 to 7 further comprising advising a healthcare provider to

(a) administer an eosinophil-targeted therapeutic agent to the patient if the patient is determined or identified to have a lower or decreased DPP4 level in one or more samples taken from the patient compared to a predetermined DPP4 threshold level, or compared to a DPP4 threshold level in one or more control samples; or

(b) suspend, not initiate, or deny the administration of an eosinophil-targeted therapeutic agent to the patient if the patient is determined or identified to have a higher or increased DPP4 level in one or more samples taken from the patient compared to a predetermined DPP4 threshold level, or compared to a DPP4 threshold level in one or more control samples.

9. The method according to any one of claims 1 to 8, wherein the eosinophil-targeted therapeutic agent is a monoclonal antibody or an antigen-binding fragment thereof.

10. The method according to claim 9, wherein the monoclonal antibody or antigen- binding fragment thereof specifically binds to IL-5 or IL-5R.

11. The method according to claim 10, wherein the monoclonal antibody or antigen- binding fragment thereof that specifically binds to IL-5 is mepolizumab, reslizumab, or an antigen-binding fragment thereof.

12. The method according to claim 10, wherein the monoclonal antibody or antigen- binding fragment thereof that specifically binds to IL-5R, specifically binds to the alpha subunit of IL-5R (SEQ ID NO: 4).

13. The method according to claim 12, wherein the monoclonal antibody or antigen- binding fragment thereof that specifically binds to the alpha subunit of IL-5R is benralizumab, or an antigen-binding fragment thereof.

14. The method according to claim 124, wherein the monoclonal antibody or antigen- binding fragment thereof that specifically binds to the alpha subunit of IL-5R comprises a heavy chain variable region (VH) comprising or consisting of SEQ ID NO: 12 and/or a light chain variable region (VL) comprising or consisting of SEQ ID NO: 13.

15. The method according to claim 12, wherein the monoclonal antibody or antigen- binding fragment thereof that specifically binds to the alpha subunit of IL-5R comprises one, two, or three complementarity determining regions selected from SEQ ID NOS: 14-16 and/or one, two, or three complementarity determining regions selected from SEQ ID NOS: 17-19.

16. The method according to claim 12, wherein the monoclonal antibody or antigen- binding fragment thereof that specifically binds to the alpha subunit of IL-5R comprises at least one, two, three, four, five or six complementarity determining regions selected from SEQ ID NOS: 14-19.

17. The method according to claim 9, wherein the monoclonal antibody or antigen- binding fragment thereof binds to the same epitope as one of the monoclonal antibodies or antigen-binding fragment thereof of any one of claims 12-18 or competitively inhibits binding of one of the monoclonal antibodies or antigen-binding fragment thereof of any one of claims 12- 18, or both.

18. The method according to any one of claims 1 to 17, wherein the eosinophil- targeted therapeutic agent is a fusion protein comprising one of the monoclonal antibodies of any one of claims 12-19 or an antigen-binding fragment thereof.

19. The method according to any one of claims 1 to 17, wherein the eosinophil- targeted therapeutic agent is a conjugate comprising one of the monoclonal antibodies of any one of claims 12-19 or an antigen-binding fragment thereof.

20. The method according to claim 19, wherein fusion protein or conjugate comprises at least one heterologous therapeutic moiety and/or a half-life enhancing moiety.

21. The method according to any one of claims 1 to 8, wherein the eosinophil-targeted therapeutic agent is a polynucleotide.

22. The method according to claim 21, wherein the polynucleotide is a DNA or anRNA.

23. The method according to claim 21, wherein the polynucleotide is (i) an mRNA or a combination thereof, or (ii) an antisense oligonucleotide or a combination thereof.

24. The method according to claim 23, wherein the antisense oligonucleotide or combination thereof is ASM8, PXS1100, or PXS2200.

25. The method according to any one of claims 21 to 24, wherein the polynucleotide comprises at least a nucleotide analog.

26. The method according to any one of claims 1 to 25, wherein the eosinophil- targeted therapeutic agent is administered at a fixed dose.

27. The method according to claim 26, wherein the fixed dose is between 1 and 1000 mg/dose.

28. The method according to any one of claims 1 to 27, wherein the eosinophil- targeted therapeutic agent is administered in two or more doses.

29. The method according to any one of claims 1 to 28, wherein the eosinophil- targeted therapeutic agent is administered week, biweekly, bimonthly, and quarterly.

30. The method according to any one of claims 1 to 29, wherein the eosinophil- targeted therapeutic agent is administered orally, by inhalation, intravenously, intramuscularly, subcutaneously, or a combination thereof.

31. The method according to any one of claims 1 to 30, wherein the eosinophilic disease or disorder is a pulmonary disease or disorder.

32. The method according to claim 31, wherein the pulmonary disease or disorder is asthma.

33. The method according to claim 32, wherein the asthma is allergic asthma, atopic asthma, corticosteroid naive asthma, chronic asthma, corticosteroid resistant asthma, corticosteroid refractory asthma, asthma due to smoking, or asthma uncontrolled on corticosteroids.

34. The method according to claim 32, wherein the asthma is mild-moderate asthma or severe asthma.

35. The method according to any one of claims 1 to 30, wherein the eosinophilic disease or disorder is chronic bronchitis, chronic eosinophilic pneumonia (CEP), nasal polyposis, atopic dermatitis (eczema), eosinophilic esophagitis, hypereosinophilic syndrome (HES), eosinophilic granulomatosis and polyangitis (Churg-Strauss syndrome), eosinophilic gastritis, eosiophilic enteritis, eosinophilic colitis, allergic rhinoconjunctivitis (hay fever), leukemia, lymphoma, mastocytosis, atheroembolic disease, hyper-IgE syndrome, Omenn's syndrome, thymoma, transplant rejections, hypoadrenalism, bullous pemphigoid, pemphigus vulgaris, dermatitis herpetiformis, drug-induced lesions, urticaria, eosinophilic panniculitis, angioedema with eosinophilia, Kimura's disease, Shulman's syndrome, Well's syndrome, eosinophilic ulcer of the oral mucosa, eosinophilic pustular folliculitis, recurrent cutaneous necrotizing eosinophilic vasculitis, drug/toxin-induced eosinophilic lung disease, Loeffler's syndrome, allergic bronchopulmonary aspergillosis, eosinophilic granuloma, pleural eosinophilia, gastroesophageal reflux, parasitic infection, fungal infection, Helicobacter pylori infection, inflammatory bowel disease (ulcerative colitis and Crohn's disease), food allergic disorder, protein-induced enteropathy, protein-induced enterocolitis, allergic colitis, celiac disease, pemphigus vegetans, leiomyomatosis, connective tissue disorder, vasculitic disorder, chronic subdural hematoma, central nervous system infection, ventriculoperitoneal shunts, congenital heart condition (septal defects or aortic stenosis), eosinophiluria associated with kidney infection, interstitial nephritis, or eosinophilic cystitis.

36. The method according to any one of claims 1 to 35, wherein the patient has been treated either before, during, after, or alternatively to the administration of an eosinophil-targeted therapeutic agent with one or more additional therapies for the treatment of the eosinophilic disease or disorder.

37. The method according to claim 36, wherein the one or more additional therapies further comprises a steroid, bronchodilator, or both.

38. The method according to claim 37, wherein the steroid is fluticasone, budesonide, mometasone, or beclomethasone.

39. The method according to claim 37, wherein the bronchodilator is salbutamol or salmeterol.

40. The method according to claim 36, wherein the one or more additional therapies are administered by inhalation, by oral administration, by injection, or by a combination thereof.

41. The method according to claim 40, wherein inhalation administration is conducted using a metered dose inhaler (MDI) or a dry powder inhaler (DPI).

42. The method according to claim 37, wherein the steroid is administered at a high dose.

43. The method according to any one of claims 1 to 42, wherein the one or more samples taken from the patient and/or the one or more control samples comprises one or more of whole blood, serum, plasma, saliva, sputum, bronchoalveolar lavage fluid, lung epithelial cells, urine, skin, nasal polyps, or a combination thereof.

44. The method according to claim 43, wherein the sample taken from the patient is blood serum.

45. The method according to any one of claims 1 to 44, wherein the one or more control samples are

(a) a sample or samples obtained from

(i) normal healthy individuals;

(ii) patients with a subset of asthma;

(iii) asthma patients naive for corticosteroid treatment; or asthma patients treated with corticosteroids;

(b) a pre-determined standard amount of isolated DPP4; or,

(c) any combination samples in (a) and (b).

46. The method according to any one of claims 1 to 45, further comprising determining, submitting a sample taken from the patient for determination, or instructing a clinical laboratory to determine

(i) the level of the patient's IgE levels,

(ii) the patient' s eosinophil and/or basophile count,

(iii) the patient's Fraction of Exhaled Nitric Oxide (FENO),

(iv) the patient's Eosinophil/Lymphocyte and Eosinophil/Neutrophil (ELEN) index,

(v) the patient's EOS index,

(vi) the patients wall area percentage (WA%) of subsegmental airways from a CT scan of the lungs,

(vii) a combination of two or more thereof.

47. The method according to claim 46, wherein the patient's eosinophil count is >150 eosinophils^!., > 300 eosinophils^!., or > 400 eosinophils/μ!..

48. The method according to claim 48, wherein the patient's eosinophil count is between 150 and 400 eosinophils^!..

49. The method according to any one of claims 1 to 48, wherein the patient's DPP4 level is measured in a DPP4 detection assay, wherein the DPP4 detection assay is an immunoassay.

50. The method according to claim 51, wherein the immunoassay is:

(i) the immunoassay described in Example 2; or,

(ii) an immunoassay disclosed in TABLE 1; or,

(ii) a multiplexed immunoassay.

51. The method according to claim 50, wherein the multiplexed immunoassay is an EMD Millipore MILLIPLEX™, Bio-Rad BIO-PLEX™, Life Technologies NOVEX MULTIPLEX™, Thermo Fisher Scientific LUMINEX®, PerkinElmer ALPHAPLEX™, Affymetrix (eBioscience) PROCARTA™, or R&D Systems LUMINEX® assay.

52. The method according to any one of claims 1 to 51, further comprising determining, submitting a sample taken from the patient for determination, or instructing a clinical laboratory to determine the expression level or activity of periostin, IL-22, IL-25, IL-33, TSLP, LCN2, CCL20, sCTLA-3, sCD28, CCL5, CCL11, CCL22, CST1, CCL26, CLCA1, CST2, PRR4, SERPINB2, CEACAM5, iNOS, SERPINB4, CST4, PRB4, TPSDl, TPSGl, MFSD2, CPA3, GPR105, CDH26, GSN, C20RF32, TRACH2000196 (TMEM71), DNAJC12, RGS13, SLC18A2, SERPINB10, SH3RF2, FCER1B, RUNX2, PTGS1, ALOX15, or combinations thereof.

53. The method according to any one of claims 1 to 52, wherein the predetermined DPP4 threshold level is selected from

(a) about the mean DPP4 level as measured in blood serum from a plurality of patients using an immunoassay; (b) about the median DPP4 level as measured in blood serum from a plurality of patients using an immunoassay; and

(c) about the 1^st, 2^nd, 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, or 9^th decile baseline DPP4 level as measured in blood serum from a plurality of patients using an immunoassay;

wherein the patients are

(i) normal healthy volunteers,

(ii) patients with mild to moderate- asthma, and/or

(iii) patients with an severe-asthma.

54. The method according to claim 53, wherein the blood serum from a plurality of patients is a pooled sample or individual samples.

55. The method according to any one of claims 1 to 54, wherein the predetermined DPP4 threshold level is at least about 103 ng/mL to at least about 867 ng/mL as measured using an immunoassay.

56. The method according to any one of claims 1 to 54, wherein the predetermined DPP4 threshold level is at least about 363 ng/mL as measured using an immunoassay.

57. The method according to any one of claims 1 to 54, wherein the predetermined DPP4 threshold level is at least about 376 ng/mL as measured using an immunoassay.

58. The method according to any one of claims 55 to 57, wherein the immunoassay is the DPP4 detection assay described in Example 2 or a DPP4 detection assay disclosed in TABLE 1.

59. The method according to any one of claims 1 to 58, wherein administration of the eosinophil-targeted therapeutic agent results in

(a) AER (Acute Exacerbation Rate) reduction;

(b) FEV1 (Forced Expiratory Volume in one second) increase;

(c) improved ACQ-6 (Asthma Control Questionnaire, 6-item version) value; or

(d) a combination thereof.

60. The method according to claim 59, wherein the AER reduction is at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the AER baseline values observed in a population of patients treated with a placebo.

61. The method according to claim 59, wherein the FEVl increase is at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mL compared to the FEVl baseline values observed in a population of patients treated with a placebo.

62. The method according to claim 59, wherein the improved ACQ-6 value is a change from baseline of at least about -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1, -1.1, or -1.2 compared to the ACQ-6 baseline values observed in a population of patients treated with a placebo.