Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• 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)].
• 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.
• HES hypereosinophilic syndrome
• 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 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.
• these biomarkers are useful predictors of treatment response.
• IgE interleukin
• eosinophilic inflammation or high levels of periostin a surrogate marker for interleukin (IL)-13 activity in asthma
• IL-13/IL-4RCC a surrogate marker for interleukin-13 activity in asthma
• 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)
• IL-13/IL-4RCC Wenzel, Pulm Pharmacol Ther 26, 710-715 (2013)
• 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.
• DPP4 dipeptidyl peptidase-4
• POSTN periostin
• 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.
• 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.
• 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.
• 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.
• 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.
• the methods disclosed above further comprise determining the level of DPP4 and/or POSTN in the one or more samples obtained from the patient.
• the methods disclosed above further comprise advising a healthcare provider to
• the patient has elevated blood eosinophils.
• the eosinophil-targeted therapeutic agent is a monoclonal antibody or an antigen-binding fragment thereof.
• the monoclonal antibody or antigen-binding fragment thereof specifically binds to IL-5 or IL-5R.
• the monoclonal antibody or antigen-binding fragment thereof that specifically binds to IL-5 is mepolizumab, reslizumab, or an antigen-binding fragment thereof.
• 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).
• 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.
• 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.
• VH heavy variable region
• VL light chain variable region
• 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.
• 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.
• the eosinophil-targeted therapeutic agent is a fusion protein or a conjugate comprising mepolizumab, reslizumab, or benralizumab, or an antigen-binding fragment thereof.
• the fusion protein or conjugate comprises at least one heterologous therapeutic moiety and/or a half-life enhancing moiety.
• the eosinophil-targeted therapeutic agent is a polynucleotide.
• the polynucleotide is a DNA or an RNA.
• the polynucleotide is (i) an mRNA or a combination thereof, or (ii) an antisense oligonucleotide or a combination thereof.
• the antisense oligonucleotide or combination thereof is ASM8, PXS1100, or PXS2200.
• the polynucleotide comprises at least a nucleotide analog.
• 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.
• 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.
• 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.
• the eosinophil-targeted therapeutic agent e.g., benralizumab/MEDI-563
• the eosinophilic disease or disorder is a pulmonary disease or disorder.
• the pulmonary disease or disorder is asthma or COPD.
• 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.
• the asthma is mild- to- moderate asthma (defined as GINA 1-3) or severe asthma (defined as GINA 4+).
• 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, eo
• 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.
• the one or more additional therapies further comprise a steroid, bronchodilator, or both.
• the steroid is fluticasone or budesonide.
• the bronchodilator is salbutamol or salmeterol.
• the one or more additional therapies are administered by inhalation, by oral administration, by injection, or by a combination thereof.
• inhalation administration is conducted using a metered dose inhaler (MDI) or a dry powder inhaler (DPI).
• MDI metered dose inhaler
• DPI dry powder inhaler
• the steroid is administered at a high dose.
• the steroid is an inhaled corticosteroid such as beclomethasone or mometasone.
• 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.
• the sample taken from the patient is blood serum.
• 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).
• 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.
• FENO Exhaled Nitric Oxide
• EUN Eosinophil/Lymphocyte and Eosinophil/Neutrophil
• WA% patients wall area percentage
• the patient's eosinophil count is > 300 eosinophils/ ⁇ .. In some aspects, > 300 eosinophils/ ⁇ . is set as a threshold for mild to moderate asthmatics. In some aspects, the patient's eosinophil count is > 400 eosinophils/ ⁇ .. In some aspects, > 400 eosinophils/ ⁇ . is set as a threshold for severe asthmatics. In some aspects, the patient's eosinophil count is > 150 eosinophils ⁇ !..
• > 150 eosinophils ⁇ ! is set as a threshold, for example, for patients undergoing treatment with an anti-IL5 antibody (e.g., mepolizumab).
• an anti-IL5 antibody e.g., mepolizumab.
• the patient's DPP4 level is measured in a DPP4 detection assay, wherein the DPP4 detection assay is an immunoassay.
• the patient's POSTN level is measured in a POSTN detection assay, wherein the POSTN detection assay is an immunoassay.
• 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.
• the multiplexed immunoassay is an EMD Millipore MILLIPLEXTM, Bio-Rad BIO-PLEXTM, Life Technologies NOVEX MULTIPLEXTM, Thermo Fisher Scientific LUMINEX®, PerkinElmer ALPHAPLEXTM, Affymetrix (eBioscience) PROCARTATM, or R&D Systems LUMINEX® assay.
• the POSTN detection assay is an assay disclosed in WO2015120171A1 or WO2015120185, both of which are herein incorporated by reference in their entireties.
• 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.
• other molecular biomarkers such as IL-22, IL-25, IL-33, TSLP, LCN2, CCL20, s
• the predetermined DPP4 or POSTN threshold levels are selected from
• the blood serum from a plurality of patients is a pooled sample or individual samples.
• 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.
• 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.
• 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.
• the AER reduction is at least about 15%, 20%, 25%, 30%, 35%,
• 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.
• 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.
• 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).
• 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.
• FIG. 3 shows the prevalence of subgroups DPP4 High and Low (A), and POSTN
• FIG. 4 shows the lack of correlation between serum DPP4 levels and serum
• 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.
• 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.
• 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.
• 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.
• ADCC antibody-dependent cell-mediated cytotoxicity
• 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.
• Serum samples collected in the Phase 2b trial of benralizumab 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.
• 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.
• 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.
• 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).
• POSTN periostin
• 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.
• an eosinophil-targeted therapeutic agent e.g., an anti-IL
• 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.
• the specific therapeutic agent is an eosinophil-targeted therapeutic agent, e.g., anti-IL-5R antibody such as benralizumab (MEDI-563).
• Th2 high Th2 (high Th2 defined, e.g., as IgE > 100 IU/mL and blood eosinophils > 0.14 x 10 9 /L); and/or,
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563); and/or,
• 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.
• 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.
• polynucleotide oligonucleotide
• nucleic acid nucleic acid molecule
• gene a polymer of nucleotides of any length, and ribonucleotides, deoxyribonucleo tides, analogs thereof, or mixtures thereof.
• 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.
• polypeptide polypeptide
• peptide protein
• polymers of amino acids of any length 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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).
• basic side chains e
• 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.
• 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).
• an electropositive side chain e.g., Arg, His or Lys
• an electronegative residue e.g., Glu or Asp
• substitutions can be readily identified by workers of ordinary skill.
• a substitution can be taken from any one of D-alanine, glycine, beta-alanine, L-cysteine and D-cysteine.
• a replacement can be any one of D-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine, ornithine, or D- ornithine.
• 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.
• a polar residue e.g
• 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.
• 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.
• B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
• BLASTN is used to compare nucleic acid sequences
• 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.
• 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.
• 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.
• sequence alignments are 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.
• the term "antibody” 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.
• VH variable domain of a heavy chain
• VL variable domain of a light chain
• 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).
• 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.
• 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.
• 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.
• 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%.
• 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.
• an antigen e.g., a target polysaccharide that they recognize or specifically bind.
• 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.”
• epitope 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.
• 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.
• epitope refers to a protein determinant (e.g., an amino acid sequence) of the IL-5.
• binding of an antibody disclosed to either IL-5R or IL-5 prevents the interaction of IL-5R with IL-5.
• 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.
• 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.
• 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.
• 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.
• 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%.
• 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 mm 3 .
• the term eosinophilic disease or disorder also encompasses eosinophilic inflammation. Blood levels over approximately 300 per mm 3 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).
• 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.
• HES hypereosinophilic syndrome
• EGPA eosinophilic granulomatosis and polyangitis
• EGPA formerly Churg-Strauss syndrome
• EGPA formerly Churg-Strauss syndrome
• the eosinophilic disease or disorder can be a pulmonary disease or disorder.
• 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.
• 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.
• COPD chronic obstructive pulmonary disease.
• COPD includes two main conditions: emphysema and chronic obstructive bronchitis.
• COPD includes two main conditions: emphysema and chronic obstructive bronchitis.
• COPD refers to COPD itself and also its subconditions chronic bronchitis and emphysema.
• IPF Idiopathic Pulmonary Fibrosis
• fibrosis 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.
• 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.
• 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;
• 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;
• Infectious diseases Parasitic infections (helminthiasis - worms), fungal infections and some other types of infections are associated with increased numbers of eosinophils;
• Blood disorders Hematologic disorders with increased levels of eosinophils include hypereosinophilic syndrome, leukemias, lymphomas, tumors, mastocytosis, and atheroembolic disease;
• Eosinophils have also been found to be increased or pathologically present in the following diseases or disorders:
• Pulmonary Conditions Drug/toxin- induced eosinophilic lung disease, Loeffler's syndrome, allergic bronchopulmonary aspergillosis, eosinophilic pneumonia, Churg-Strauss syndrome, eosinophilic granuloma, and pleural eosinophilia;
• 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;
• 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;
• Renal Diseases Eosinophiluria (eosinophils in the urine) associated with infections, interstitial nephritis, eosinophilic cystitis.
• exacerbation refers to a worsening of symptoms of an eosinophilic disease or disorder, relative to a patient's baseline condition.
• 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.
• exacerbation of asthma may be an abrupt increase in symptoms of shortness of breath and/or wheezing, and/or increase in production of sputum.
• 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma
• reducing the occurrence of the eosinophilic disease or disorder 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
• 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.
• the terms "treat,” “treating” or “treatment of” refer to both prophylactic and therapeutic treatment regimes.
• 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.
• 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.
• eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma
• subject or patient include any human or nonhuman animal.
• 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.
• 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).
• 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.
• 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).
• a subject is a naive subject.
• a naive subject is a subject that has not been administered a therapy, for example a therapeutic agent.
• 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).
• 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.
• a subject has received at least one therapeutically effective dose of oral or inhaled corticosteroids.
• inhalation administration is conducted using a metered dose inhaler (MDI) or a dry powder inhaler (DPI).
• MDI metered dose inhaler
• DPI dry powder inhaler
• the steroid is administered at a high dose.
• high dose when application to an inhaled corticosteroid can refer, for example, to a total daily dose of at least 500 ⁇ g of ICS (e.g., fluticasone) DPI or at least 440 ⁇ g ICS MDI.
• ICS e.g., fluticasone
• the high ICS total daily dose is at least about 300 ⁇ g, at least about 350 ⁇ g, at least about 400 ⁇ g, at least about 450 ⁇ g, at least about 500 ⁇ g, at least about 550 ⁇ g, at least about 600 ⁇ g, at least about 650 ⁇ g, at least about 700 ⁇ g, at least about 750 ⁇ g, at least about 800 ⁇ g, at least about 850 ⁇ g, at least about 900 ⁇ g, at least about 950 ⁇ g, or at least 1000 ⁇ g of ICS (e.g., fluticasone) DPI.
• ICS e.g., fluticasone
• the high ICS total daily dose is at least about 300 ⁇ g, at least about 350 ⁇ g, at least about 400 ⁇ g, at least about 450 ⁇ g, at least about 500 ⁇ g, at least about 550 ⁇ g, at least about 600 ⁇ g, at least about 650 ⁇ g, at least about 700 ⁇ g, at least about 750 ⁇ g, at least about 800 ⁇ g, at least about 850 ⁇ g, at least about 900 ⁇ g, at least about 950 ⁇ g, or at least 1000 ⁇ g of ICS (e.g., fluticasone) MPI.
• ICS e.g., fluticasone
• 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 ⁇ g fluticasone and about 21 ⁇ g salmeterol as MDI at a dose of 2 inhalations twice per day, or to about 500 ⁇ g fluticasone and about 50 ⁇ g 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.
• ICS inhaled corticosteroid
• a subject has received multiple therapeutically effective doses of oral or inhaled corticosteroids.
• a subject is a chronic oral corticosteroid (OCS) user.
• the subject has received a long-acting beta2-adrenergic agonist, e.g., salmeterol xinafoate.
• a synthetic glucocorticoid e.g., fluticasone propionate.
• the subject has received a combination of salmeterol xinafoate and fluticasone propionate (ADVAIR®).
• ADVAIR® a beta2- adrenergic bronchodilator, e.g., albuterol sulfate.
• 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma
• therapeutic agents e.g., eosinophil-targeted therapeutic agent
• instrumentation e.g., eosinophil-targeted therapeutic agent
• supportive measures e.g., eosinophil-targeted therapeutic agent
• 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.
• DPP4 High a high or increased DPP4 level
• DPP4 Low a low or decreased DPP4 level
• 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.
• 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.
• 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.
• DPP4 level e.g., the level of protein or nucleic acid
• 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.
• POSTN level e.g., the level of protein or nucleic acid
• therapeutic agent includes, e.g.
• 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.
• a therapeutic agent can also be a pro-drug, which metabolizes into the desired therapeutically active substance when administered to a subject.
• the therapeutic agent is a prophylactic agent.
• 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.
• the therapeutic agent is a small molecule drug.
• the agent is a corticosteroid.
• the agent can be a leukotriene modifier such as montelukast, zafirlukast or zileuton.
• the therapeutic agent can be a methylxanthine (e.g., theophylline) or a cromone (e.g., sodium cromolyn and nedocromil).
• the therapeutic agent can be a long-acting beta-2 agonist such as salmeterol, fomoterol, or indacaterol.
• the agent can be methotrexate or cyclosporin.
• 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.
• eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma.
• 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
• 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.
• tryptase kinase inhibitors e.g. , GW-45, GW-58, and geisseine
• phosphatidylinositide-3 ' (PD)-kinase inhibitors e.g. , calphostin C
• other kinase inhibitors e.g. , staurosporine
• C2a receptor antagonists including antibodies
• supportive respiratory therapy such as supplemental and mechanical ventilation.
• 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.
• 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.
• 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).
• 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).
• eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma
• a pulmonary disease or disorder such as asthma
• the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
• 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.
• a "sufficient amount” or “an amount sufficient to” achieve a particular result in a patient having an eosinophilic disease or disorder 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).
• a therapeutic agent e.g. , an anti- IL-5R such as benralizumab
• a therapeutic effect i.e. , by administration of a therapeutically effective amount
• sample 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.
• that sample is blood or a fraction thereof. Samples can be obtained by any means known in the art.
• a sample is a computed tomography (CT) scan of a patient's organ or tissue including, but not limited to the lungs.
• CT computed tomography
• 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.
• a blood sample can be fractionated into serum or into fractions containing particular types of cells.
• a sample can be a combination of samples from an individual, such as a combination of a tissue and fluid sample.
• 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.
• 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.
• 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).
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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,
• 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.
• 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.
• CT Computer Tomography
• tomographic images virtual 'slices'
• 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.
• 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.
• MDCT multidetector computed tomography
• HRCT high-resolution computed tomography
• PET positron emission tomography
• PET-CT positron emission tomography computed tomography
• SPECT single-photon emission computed tomography
• MRI magnetic resonance imaging
• CAT scan computed axial tomography
• computer- assisted tomography xenon ventilation computed tomography
• 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);
• DPP4 refers to the dipeptidyl peptidase IV protein (EC
• 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).
• 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.
• 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).
• variants e.g., the KIR, V7I, S437I, T557I, D663E variants known in the art
• derivatives thereof e.g., glycosylated or aglycosilated protein forms of the DPP4 protein, or otherwise chemically modified forms of the protein.
• 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.
• 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.
• POSTN 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).
• OSF-2 osteoblast-specific factor 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.
• 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).
• 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.
• 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.
• biomarker 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).
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).
• an anti-IL-5R antibody such as benralizumab (MEDI-563).
• biomarker encompasses both clinical markers and molecular biomarkers (biological markers).
• 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.
• 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.
• 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 nucle
• 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
• the DPP4 and POSTN molecular biomarkers disclosed herein also include fragments, variants, and derivatives thereof.
• 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.
• 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.
• 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma
• POSTN a biomarker
• additional clinical and/or molecular biomarkers such as a patient's eosinophil count.
• 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).
• 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.
• a cut-off approach based on absolute values e.g., sputum EOS% cutoff
• 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.
• 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.
• wall area refers to the cross-sectional area of a bronchial tube wall (e.g. segmental and subsegmental bronchi in the upper lobes).
• Wall area percentage 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).
• airway dimensions are measured from Computed Tomography (CT) imaging data of the lungs.
• CT Computed Tomography
• 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).
• 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.
• sCTLA-3 soluble CTLA-3; also known as Cytotoxic T-Lymphocyte- Associated serine Este
• 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, C
• 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),
• CST1 cystat
• the determination that a patient's DPP4 level is High 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.
• the determination that a patient's DPP4 level is Low 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.
• the determination that a patient's DPP4 level is High 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.
• the determination that a patient's DPP4 level is Low 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.
• the determination that a patient's POSTN level is High 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.
• the determination that a patient's POSTN level is Low 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.
• the determination that a patient's POSTN level is High 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.
• the determination that a patient's POSTN level is Low 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.
• the determination that a patient's blood eosinophil count is Low requires that the measured level of blood eosinophils is ⁇ 150 cells ⁇ L.
• the determination that a patient's blood eosinophil count is Low requires that the measured level of blood eosinophils is ⁇ 300 cells ⁇ L.
• the determination that a patient's blood eosinophil count is Low requires that the measured level of blood eosinophils is ⁇ 400 cells ⁇ L.
• 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/ ⁇ L.
• 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.
• Levels of DPP4, POSTN, and other biomarkers disclosed herein 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.
• analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, mass spectroscopy and the like
• immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunohistochemistry, affinity
• 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.
• RNA e.g. , mRNA
• 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.
• qPCR quantitative PCR
• "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.
• SAGE Serial analysis of gene expression
• RNA-Seq RNA-Seq
• 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).
• 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.).
• 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.
• DPP4, POSTN, and other molecular biomarkers disclosed herein can be detected and/or quantified in the biological sample using an immunoassay.
• 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.
• 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).
• 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.
• 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.
• ELISA enzyme-linked immunosorbent assay
• ECL sandwich electrochemil
• 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.
• 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.
• 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.
• a sample suspected of containing the molecular biomarker e.g., DPP4 or POSTN
• 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.
• 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.
• 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.
• an enzyme e.g. , horseradish peroxidase or alkaline phosphatase
• 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.
• 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.
• Immunoassays for detecting molecular biomarkers can be either competitive or noncompetitive.
• Noncompetitive immunoassays are assays in which the amount of captured analyte is directly measured.
• 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.
• a known amount of, for example, labeled molecular biomarker e.g., DPP4 or POSTN
• a capture agent e.g., DPP4 or POSTN
• 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.
• 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.
• 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.
• 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.
• 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.
• the measured expression level is an average or mean of several measurements of expression levels of the same sample.
• 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.
• 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.
• DPP4 detection assay refers to both quantitative and qualitative assays capable of detecting the presence or absence of DPP4 in a biological sample.
• DPP4 detection assays encompassed, e.g., immunoassays such as ELISA.
• POSTN detection assay refers to both quantitative and qualitative assays capable of detecting the presence or absence of POSTN in a biological sample.
• POSTN detection assays encompassed, e.g., immunoassays such as ELISA.
• POSTN detection assays are well known in the art.
• the POSTN detection assay is an immunoassay disclosed in WO2015120171A1 or WO2015120185, or a commercial POSTN assay.
• the DPP4 detection assay or the POSTN detection assay is a multiplexed immunoassay, for example, Bio-Rad BIO-PLEXTM, EMD Millipore MILLIPLEXTM, Life Technologies NOVEX MULTIPLEXTM, Thermo Fisher Scientific LUMINEX®, PerkinElmer ALPHAPLEXTM, Affymetrix (eBioscience) PROCARTATM, or R&D Systems LUMINEX® capable of detecting the expression levels of DPP4 and/or POSTN in a sample.
• a multiplexed immunoassay for example, Bio-Rad BIO-PLEXTM, EMD Millipore MILLIPLEXTM, Life Technologies NOVEX MULTIPLEXTM, Thermo Fisher Scientific LUMINEX®, PerkinElmer ALPHAPLEXTM, Affymetrix (eBioscience) PROCARTATM, or R&D Systems LUMINEX® capable of detecting the expression levels of DPP4 and/or POSTN in a sample
• the DPP4 detection assay is an assay included in TABLE 1, or a variant thereof.
• CD26 (Human) ELH- Biocat www.biocat.com/products/ELH-CD26-5-RB ELISA Kit CD26-5- RB
• the predetermined DPP4 or POSTN threshold level will be the particular LOD for such assay.
• 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.
• level 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.
• 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.
• a biological sample e.g. , blood serum or sputum
• normal level e.g. a biological sample
• 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.
• 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.
• 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.
• a biological sample e.g. , blood serum or sputum
• normal level e.g. a biological sample
• a control sample e.g. a control sample
• a specified threshold disclosed herein 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.
• 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.
• the normal level or range for DPP4, POSTN, and other biomarkers disclosed herein can be defined in accordance with standard practice.
• the level measured in a particular biological sample can be compared with level or range of levels determined in similar normal samples.
• 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).
• 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.
• 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.
• 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.
• the level of a molecular biomarker disclosed herein 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%,
• the level of a molecular biomarker disclosed herein 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%,
• a threshold level refers to a level of DPP4, POSTN, or any other biomarker disclosed herein which may be of interest for comparative purposes.
• 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.
• 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.
• samples are normalized by a common factor.
• body fluid samples are normalized by volume body fluid and cell-containing samples are normalized by protein content or cell count.
• 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).
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).
• 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”).
• a threshold level or alternatively herein a "predetermined threshold level” is a cutoff or threshold against which the measured expression level of a protein or nucleic acid is compared.
• 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).
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).
• 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).
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).
• an anti-IL-5R antibody such as benralizumab (MEDI-563).
• threshold levels e.g. , protein expression levels or gene expression levels
• 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.
• 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.
• 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.
• 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma
• 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma
• the predetermined DPP4 threshold level is about 376 ng/mL +/-
• the DPP4 threshold level can be between about 100 and about
• 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.
• 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).
• the predetermined DPP4 threshold level corresponds to the 1 st .
• 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.
• 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.
• 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.
• 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.
• 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma
• 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma
• the predetermined POSTN threshold level is about 26 ng/niL +/-
• POSTN protein in serum 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.
• the POSTN threshold level is between about 7 and 105 ng/niL.
• 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.
• 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).
• the predetermined POSTN threshold level corresponds to the 1 st .
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 for each respective biomarker.
• 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).
• 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.
• the measured expression level of a biomarker disclosed herein 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
• the measured expression level of a biomarker disclosed herein 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.
• a biomarker disclosed herein e.g. , DPP4 or POSTN
• 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.
• the predetermined threshold level of a biomarker disclosed herein is a biomarker
• 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.
• 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma.
• 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.
• the threshold level e.g. , a protein expression level or a gene expression level
• a biomarker disclosed herein e.g. , DPP4 or POSTN
• the patient's DPP4 and/or POSTN levels can be compared to one or more control levels.
• the test sample e.g. , a sample from a patient suffering from a pulmonary disease or disorder such as asthma
• the 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.
• 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.
• a biomarker expression level can be recorded in a patient's medical record.
• DPP4 and/or POSTN levels can be combined with other biomarkers such as eosinophil levels.
• a high level of eosinophils e.g., peripheral blood eosinophils
• 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).
• 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).
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma.
• a blood analyte to use as biomarker can be determined from a complete blood cell count (CBC) with differential.
• CBC with differential refers to complete blood cell count (CBC) with white blood cell (WBC) differentials.
• WBC white blood cell
• white blood cell includes, e.g., neutrophils, lymphocytes, monocytes, eosinophils, and basophils.
• EOS eosinophil
• EOS eosinophil
• white blood cell count 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).
• 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.
• 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.
• 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).
• the tube 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.
• samples can be analyzed manually.
• the level of eosinophils can be determined by flow cytometry.
• 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.
• 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.
• 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, ⁇ / ⁇ 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).
• 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.
• 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.
• 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.
• 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
• 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
• 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.
• 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.
• 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).
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as asthma
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).
• an anti-IL-5R antibody such as benralizum
• eosinophil-targeted therapeutic agent refers a therapeutic agent capable of decreasing eosinophil count in a patient in need thereof.
• the eosinophil-targeted therapeutic agent can also deplete basophil count.
• the eosinophil-targeted therapeutic agent is
• the eosinophil-targeted therapeutic agent can be a small molecule.
• 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.).
• the therapeutic agent is a biologic.
• 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.
• 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.
• 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.
• An eosinophil-targeted therapeutic agent of the present disclosure e.g. , an antibody specifically binding to IL-5R or IL-5
• 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%.
• the eosinophil-targeted therapeutic agent is a therapeutic agent capable of
• an eosinophil-targeted therapeutic agent is a compound included in TABLE 2.
• IL-5R IL-5R
• AB Benralizumab
• 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• the anti-IL-5R molecule or anti-IL-5 molecule is an antibody-drug conjugate (ADC).
• ADC antibody-drug conjugate
• 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.
• 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.
• the anti-IL5R antibody specifically binds to the alpha subunit of IL-
• 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.
• the eosinophil-targeted therapeutic agent can be an antibody directed against IL-5, e.g. , reslizumab, mepolizumab, and any combination thereof.
• 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.
• the in vivo depletion can be mediated by ADCC, CDC or antibody mediated phagocytosis.
• the therapeutic agent can be an anti-IL-5R antibody having ADCC activity.
• the therapeutic agent can be an anti-IL-5R antibody having increased ADCC activity.
• 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 ⁇ heavy chain (224-214')- disulfide with humanized mouse monoclonal MEDI-523 ⁇ 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.
• 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.
• 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.
• 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.
• VL light chain variable region
• 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.
• 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.
• 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.
• the eosinophil-targeted therapeutic agent is a polynucleotide, e.g., a DNA or an RNA.
• the polynucleotide is (i) an mRNA or a combination thereof, or (ii) an antisense oligonucleotide or a combination thereof.
• the polynucleotide comprises at least a nucleotide analog.
• 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.
• ASM8 is a combination of 2 oligonucleotides (TOP004 and TOP005).
• TOP004 is a 19-mer directed against the mRNA of the common ⁇ subunit of the IL-3, IL-5, and GM-CSF receptors.
• the eosinophil-targeted therapeutic agent can be an antibody directed against IL-13/IL-4a.
• the therapeutic agent can be AerovantTM (Aerovance), GSK-679586 (GSK), IMA-026 (Wyeth), or MILR1444A (Genentech).
• the eosinophil-targeted therapeutic agent is an antibody directed against the IL-2 receptor.
• 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.
• the eosinophil-targeted therapeutic agent can be an anti-IgE antibody.
• 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.
• the eosinophil-targeted therapeutic agent can be a recombinantly-produced cytokine.
• the eosinophil-targeted therapeutic agent can be interferon-alpha.
• Non-limiting examples of interferon-alpha therapeutics include PEGASYS® (PEGinterferon alfa-2a) and ALBUFERON®/ ZALBINTM (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- ⁇ , IL-6, or TGF- ⁇ .
• 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.
• 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.
• DPP4 and POSTN levels 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).
• a certain therapy for example, an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).
• 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).
• a certain therapy e.g., therapy with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).
• the mere determination that the DPP4 and/or POSTN is expressed below a predetermined threshold level or below a detectable levels 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).
• a certain therapy e.g., therapy with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).
• 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.
• GINA 1-3 moderate asthmatic or mild-to-moderate asthmatic
• GINA 4+ severe asthmatic
• the patients suffers from COPD.
• DPP4 levels or POSTN levels can be used alone.
• the DPP4 and/or POSTN levels can be combined with other molecular or clinical biomarkers, such as blood eosinophil count.
• 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.
• 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as mild-to- moderate asthma, or severe asthma
• therapeutic agents e.g. , benralizumab/MEDI-563
• 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).
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma
• additional biomarkers e.g., eosinophil count
• 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
• 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 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
• 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
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563)
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563)
• 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
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563)
• a patient diagnosed with an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as mild-to
• 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
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563)
• 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
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563)
• 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
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizum
• 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.
• POSTN e.g. , a protein expression level or a gene expression level
• DPP4 and/or POSTN levels e.g. , protein expression levels or gene expression levels
• 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.
• 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.
• 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.
• the presence of DPP4 and/or POSTN levels above or below a predetermined threshold level in a patient with an eosinophilic disease or disorder can be used in combination with one or more of clinical or molecular biomarkers specific for such disease.
• DPP4 and/or POSTN levels can be combined with measurements of other biomarker levels (e.g. , blood eosinophils).
• 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),
• an anti-IL-5R antibody such as benralizumab (MEDI-563)
• 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.
• DPP4 and/or POSTN levels e.g. , protein expression levels or gene expression levels
• DPP4 and/or POSTN levels e.g. , protein expression levels or gene expression levels
• POSTN levels e.g. , protein expression levels or gene expression levels
• 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).
• 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.
• 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.
• 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.
• This diagnosis can also be recorded in a patient medical record.
• a pulmonary disease e.g., asthma
• a specific eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is recorded in a medical record.
• MEDI-563 an anti-IL-5R antibody such as benralizumab
• 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.
• 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.
• 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.
• RFID radiofrequency identification
• 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.
• diagnosis means detecting a disease or determining the stage or degree of a disease.
• 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.
• 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.
• 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.
• 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.
• 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.
• prognosis 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.
• determining the prognosis refers to the process by which the skilled artisan can predict the course or outcome of a condition in a patient.
• prognosis does not refer to the ability to predict the course or outcome of a condition with 100% accuracy.
• 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.
• 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.
• 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.
• the disclosure includes methods of treating an eosinophilic disease or disorder
• 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.
• a sample is obtained from a patient and is submitted for measurement of the level of DPP4 and/or POSTN in the sample.
• 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma
• an eosinophil-targeted therapeutic agent e.g.
• Also provided is method of treating a patient having an eosinophilic disease or disorder 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or severe asthma
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563)
• 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 eo
• a method of treating a patient having an eosinophilic disease or disorder 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/
• an eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as ben
• 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.
• 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.
• 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.
• the molecular biomarker e.g., DPP4 or POSTN detection assay
• 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).
• 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).
• 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.
• 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.
• 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.
• test can be performed by a healthcare provider or a clinical laboratory as noted above
• 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.
• 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.
• an eosinophilic disease or disorder e.g., a pulmonary disease or disorder such as mild- to- moderate asthma or severe asthma
• POSTN level e.g. , protein expression level or gene expression level
• 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
• 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.
• a biomarker e.g., blood eosinophils
• a combination thereof e.g., protein expression level or gene expression level
• 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.
• DPP4 and/or POSTN level e.g., protein expression level or gene expression level
• methods of treatment contemplated herein 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.
• 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/m
• the antibody or antigen-binding fragment thereof can be given to achieve a concentration of antibody in serum from about 12.5 ng/ml to about 1000 ng/ml.
• concentration of antibody in serum from about 12.5 ng/ml to about 1000 ng/ml.
• methods of treatment contemplated herein 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.
• 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.
• the amount and interval are: 15-21 mg every 0.5-1.0 month; 55-
• 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.
• 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.
• 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.
• the eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is administered intravenously (IV).
• the eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) is administered subcutaneously (SC).
• 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.
• 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.
• 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,
• an eosinophil- targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563)
• an eosinophil- targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563)
• the therapeutic agent may be administered through any suitable means, compositions and routes known in the art.
• 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.
• 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.
• 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.
• the pharmaceutical composition is administered by an intravenous route.
• the pharmaceutical composition is administered by a subcutaneous route.
• 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.
• transdermal delivery and sustained release from implants are contemplated. 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.
• 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.
• the composition additionally comprises one or more physiologically active agents for combination therapy.
• 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.
• 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
• Neutral buffered saline or saline mixed with conspecific serum albumin are examples of appropriate diluents.
• 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.
• 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.
• an anti-IL-5R antibody such as benralizumab
• 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).
• 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).
• a surfactant such as a non-ionic surfactant like polysorbate (polysorbate 20 or 80) or poloxamers (poloxamer 1888)
• eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563)
• eosinophil-targeted therapeutic agent e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563)
• AER Acute Exacerbation Rate
• FEV1 Forced Expiratory Volume in one second
• the AER reduction after administration of an eosinophil-targeted therapeutic agent 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.
• 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
• the FEVi increase after administration of an eosinophil-targeted therapeutic agent 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.
• 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%
• the FEVi increase after administration of an eosinophil-targeted therapeutic agent 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.
• an anti-IL-5R antibody such as benralizumab
• the ACQ-6 change after administration of an eosinophil-targeted therapeutic agent 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.
• 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.
• 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.
• 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
• the reduction of eosinophils after administration of an eosinophil- targeted therapeutic agent 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.
• 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
• the reduction of basophils after administration of an eosinophil- targeted therapeutic agent 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.
• 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
• kits for detecting DPP4 and/or POSTN levels for example, through an immunoassay method.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• a kit provided according to this disclosure can also comprise brochures or instructions describing the process.
• DPP4 or POSTN detection immunoassays e.g., single or multiplexed assays to detect DPP4 or POSTN
• 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.
• 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.
• the assay can further include washing steps, blocking steps and incubation steps.
• 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.
• the term "instructions" can include the address of an internet site that provides the instructions.
• 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).
• POSTN levels e.g., full length or soluble DPP4 protein expression levels, POSTN expression levels, or gene expression levels for DPP4 or POSTN
• 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.
• 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,
• Standard names, aliases, etc. of proteins and genes designated by identifiers used throughout this application can be identified, for example, via Genecards
• 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,
• DPP4 and/or POSTN levels e.g., protein expression levels or gene expression levels
• 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).
• 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.
• FENO Exhaled Nitric Oxide
• EUN Eosinophil/Lymphocyte and Eosinophil/Neutrophil
• WA wall area percentage
• 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).
• 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.
• 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.
• 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.
• 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.
• protocols protocol variations, extensions, etc.
• Customized hardware might also be utilized and/or particular elements might be implemented in hardware, software or both.
• 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.
• 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.
• 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.
• 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.
• 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).
• the input device can include a gene expression profiling system including, e.g., a mass spectrometer, gene chip or array reader, and the like.
• 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.
• 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.
• 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.
• 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.
• an eosinophil-targeted therapeutic agent e.g., an antibody that specifically binds to IL-5R such as benralizumab, or an antigen binding fragment thereof
• 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).
• 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,
• code that executes a classification method that indicates, e.g., whether to administer an eosinophil-targeted therapeutic agent to a patient in need thereof.
• 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.
• a Ph2b study 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)
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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)).
• DPP4 and POSTN POSTN
• 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/ ⁇ , respectively). Groups were further segmented combining DPP4 High and Low or POSTN High and Low with Eosinophil High and Low (FIG. 3).
• Exacerbation rate reduction 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.
• 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).

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