Note: Descriptions are shown in the official language in which they were submitted.
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DIPEPTIDYL PEPTIDASE-4 AND PERIOSTIN AS PREDICTORS OF
CLINICAL RESPONSE TO EOSINOPHIL-TARGETED THERAPEUTIC
AGENTS IN EOSINOPHILIC DISEASES
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0001] The
content of the electronically submitted sequence listing in ASCII text file
(Name: DPP4_sequence listing_5T25_ascii.txt; Size: 52,739 bytes; and Date of
Creation:
November 4, 2015) filed with the application is incorporated herein by
reference in its
entirety.
B AC KGROUND
[0002]
Eosinophils are implicated in various diseases including allergic diseases,
and are
thought to play an important role in generating morbidity of allergic diseases
such as chronic
bronchial asthma and atopic dermatitis. Adv. Immunol., 39, 177(1986), Immunol.
Today, 13,
501(1992)1. In addition to the above diseases, eosinophils are also implicated
in diseases
generally referred to as hypereosinophilic syndrome (HES), such as
eosinophilia, eosinophilic
enterogastritis, eosinophilic leukemia, eosinophilic granuloma and Kimura's
disease. Ann.
Intern. Med., 97, 78 (1982).
[0003]
Bronchial asthma is a common persistent inflammatory disease of the lung
characterized by airways hyper-responsiveness, mucus overproduction, fibrosis,
and raised
serum IgE levels. Airways hyper-responsiveness (AHR) is the exaggerated
constriction of the
airways to non-specific stimuli such as cold air. Both AHR and mucus
overproduction are
thought to be responsible for the variable airway obstruction that leads to
the shortness of
breath characteristic of asthma attacks (exacerbations) and which is
responsible for the
mortality associated with this disease.
[0004]
Current British Thoracic Society (BTS) and Global Initiative for Asthma (GINA)
guidelines suggest a stepwise approach to the treatment of asthma (Society, B.
T., Thorax,
2003. 58 Suppl 1:1-94; GINA, Global Strategy for Asthma Management and
Prevention.
2002, National Institute of Health). Mild to moderate asthma can usually be
controlled by the
use of inhaled corticosteroids, in combination with beta-agonists or
leukotriene inhibitors.
However, due to the documented side effects of corticosteroids, patients tend
not to comply
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with the treatment regime, which reduces the effectiveness of treatment
(Milgrom, H. et al.
Ann Allergy Asthma Immunol, 2002. 88:429-31; Fish, L. and C. L. Lung, Ann
Allergy
Asthma Immunol, 2001. 86:24-30; Bender, B. G. J. Allergy Clin. Immunol, 2002.
109:S554-
9). Asthma presents significant heterogeneity in response to various
treatments, thereby
highlighting the need to develop more effective therapies for this disease or
identify
biomarkers that predict response to specific therapies.
[0005] The
terms "precision medicine," "personalized health care," or "targeted
therapeutics" are often used interchangeably to describe efforts geared to
tailor therapies for
patient subgroups with a diverse collection of molecular and clinical
characteristics, such as
asthma. Current treatment of asthma is dominated by inhaled and oral
corticosteroids in
combination with bronchodilators. Although very effective in most patients,
about 10 to 20%
of the patients with asthma remain poorly controlled with current standard of
care (Bousquet
et al., Allergy Clin Immunol 126, 926-938 (2010)). Recently, much progress has
been made
through the discovery of new biomarkers using "omics" approaches, linking
clinical
phenotypes with molecular biomarkers and referred to as asthma "endotypes"
(Anderson,
Lancet 372, 1107-1119 (2008); Lotvall et al. J Allergy Clin Immunol 127, 355-
360 (2011);
Wenzel, Pulm Pharmacol Ther 26, 710-715 (2013)).
[0006] In
some cases, these biomarkers are useful predictors of treatment response. For
example, patients with elevated IgE, eosinophilic inflammation or high levels
of periostin, a
surrogate marker for interleukin (IL)-13 activity in asthma, preferentially
respond to
monoclonal antibodies targeting immunoglobulins E (Di Domenico et al., Inflamm
Allergy
Drug Targets 10, 2-12 (2011)), IL-5/IL-5Roc (Castro et al., Lancet Respir Med
3, 355-366
(2015)), or IL-13/IL-4Roc (Wenzel, Pulm Pharmacol Ther 26, 710-715 (2013)),
respectively.
However, a better appreciation of the dynamic changes of endotypes over time
and with
treatment, associations with clinical phenotypes and ultimately the discovery
of new
endotypes, which allow for the development of new and tailored treatments for
patients
whose asthma remains poorly controlled, is needed (Holgate et al., Nat Rev
Drug Discov 14,
367-368 (2015)).
BRIEF SUMMARY
[0007] The
present disclosure provides a method of treating an eosinophilic disease or
disorder in a patient in need thereof, comprising administering an eosinophil-
targeted
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therapeutic agent to the patient if the patient is determined or identified to
have lower or
decreased dipeptidyl peptidase-4 (DPP4) and/or periostin (POSTN) levels in one
or more
samples taken from the patient compared to predetermined DPP4 or POSTN
threshold levels,
or compared to DPP4 or POSTN threshold levels in one or more control samples.
[0008] Also
provided are methods of treating a patient having an eosinophilic disease or
disorder comprising suspending or not initiating the administration of an
eosinophil-targeted
therapeutic agent to the patient if the patient is determined or identified to
have higher or
increased DPP4 and/or POSTN levels in one or more samples taken from the
patient
compared to predetermined DPP4 or POSTN threshold levels, or compared to DPP4
or
POSTN threshold levels in one or more control samples.
[0009] The
disclosure provides also methods of treating an eosinophilic disease or
disorder in a patient in need thereof, wherein the patient failed, was non-
responsive or
intolerant to treatment with a therapeutic agent comprising administering an
eosinophil-
targeted therapeutic agent to the patient if the patient is determined or
identified to have
lower or decreased DPP4 and/or POSTN levels in one or more samples taken from
the patient
compared to predetermined DPP4 or POSTN threshold levels, or compared to DPP4
or
POSTN threshold levels in one or more control samples.
[0010] Also
provided are methods of determining whether to treat a patient having an
eosinophilic disease or disorder with an eosinophil-targeted therapeutic
agent, comprising
determining to treat the patient if the patient is determined or identified to
have lower or
decreased DPP4 and/or POSTN levels in one or more samples taken from the
patient
compared to predetermined DPP4 or POSTN threshold levels, or compared to DPP4
or
POSTN threshold level in one or more control samples.
[0011] The
disclosure also provides methods of selecting a patient diagnosed with an
eosinophilic disease or disorder as a candidate for treatment with an
eosinophil-targeted
therapeutic agent, comprising selecting the patient for treatment if the
patient is determined or
identified to have lower or decreased DPP4 and/or POSTN levels in one or more
samples
taken from the patient compared to predetermined DPP4 or POSTN threshold
levels, or
compared to DPP4 or POSTN threshold levels in one or more control samples.
[0012] In
some aspects, the methods disclosed above further comprise measuring the
level of DPP4 and/or POSTN in one or more of the samples obtained from the
patient or
instructing a clinical laboratory or healthcare provider to measure the level
of DPP4 and/or
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POSTN in the sample and/or submitting the one or more samples obtained from
the patient to
a clinical laboratory or healthcare provider to measure the level of DPP4
and/or POSTN in
the sample. In some aspects, the methods disclosed above further comprise
determining the
level of DPP4 and/or POSTN in the one or more samples obtained from the
patient. In some
aspects, the methods disclosed above further comprise advising a healthcare
provider to
(a) administer an eosinophil-targeted therapeutic agent to the patient if the
patient is
determined or identified to have lower or decreased DPP4 and/or POSTN levels
in one or
more samples taken from the patient compared to predetermined DPP4 or POSTN
threshold
levels, or compared to DPP4 or POSTN threshold levels in one or more control
samples; or
(b) suspend, not initiate, or deny the administration of an eosinophil-
targeted therapeutic
agent to the patient if the patient is determined or identified to have higher
or increased DPP4
and/or POSTN levels in one or more samples taken from the patient compared to
predetermined DPP4 or POSTN threshold levels, or compared to DPP4 or POSTN
threshold
levels in one or more control samples. In some aspects, the patient has
elevated blood
eosinophils.
[0013] In
some aspects, the eosinophil-targeted therapeutic agent is a monoclonal
antibody or an antigen-binding fragment thereof. In some aspects, the
monoclonal antibody
or antigen-binding fragment thereof specifically binds to IL-5 or IL-5R. In
some aspects, the
monoclonal antibody or antigen-binding fragment thereof that specifically
binds to IL-5 is
mepolizumab, reslizumab, or an antigen-binding fragment thereof.
[0014] In
some aspects, the monoclonal antibody or antigen-binding fragment thereof
that specifically binds to IL-5R, specifically binds to the alpha subunit of
IL-5R (SEQ ID
NO: 4). In some aspects, the monoclonal antibody or antigen-binding fragment
thereof that
specifically binds to the alpha subunit of IL-5R is benralizumab (MEDI-563),
or an antigen-
binding fragment thereof. In some aspects, the monoclonal antibody or antigen-
binding
fragment thereof that specifically binds to the alpha subunit of IL-5R
comprises a heavy
variable region (VH) comprising, consisting, or consisting essentially of SEQ
ID NO:12
and/or a light chain variable region (VL) comprising, consisting, or
consisting essentially of
SEQ ID NO:13.
[0015] In
some aspects, the monoclonal antibody or antigen-binding fragment thereof
that specifically binds to the alpha subunit of IL-5R comprises one, two, or
three
complementarity determining regions selected from SEQ ID NOS: 14-16 and/or
one, two, or
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three complementarity determining regions selected from SEQ ID NOS: 17-19. In
some
aspects, the monoclonal antibody or antigen-binding fragment thereof that
specifically binds
to the alpha subunit of IL-5R comprises at least one, two, three, four, five
or six
complementarity determining regions selected from SEQ ID NOS: 14-19.
[0016] In
some aspects, the monoclonal antibody or antigen-binding fragment thereof
binds to the same epitope as mepolizumab, reslizumab, or benralizumab, or
competitively
inhibits binding of mepolizumab, reslizumab, or benralizumab to their
respective target
epitopes. In some aspects, the eosinophil-targeted therapeutic agent is a
fusion protein or a
conjugate comprising mepolizumab, reslizumab, or benralizumab, or an antigen-
binding
fragment thereof. In some aspects, the fusion protein or conjugate comprises
at least one
heterologous therapeutic moiety and/or a half-life enhancing moiety.
[0017] In
some aspects, the eosinophil-targeted therapeutic agent is a polynucleotide.
In
some aspects, the polynucleotide is a DNA or an RNA. In some aspects, the
polynucleotide is
(i) an mRNA or a combination thereof, or (ii) an antisense oligonucleotide or
a combination
thereof. In some aspects, the antisense oligonucleotide or combination thereof
is ASM8,
PXS1100, or PX52200. In some aspects, the polynucleotide comprises at least a
nucleotide
analog.
[0018] In
some aspects, the eosinophil-targeted therapeutic agent (e.g.,
benralizumab/MEDI-563) is administered at a fixed dose. In some aspects, the
fixed dose is
between 1 and 1,000 mg/dose. In some aspects, the eosinophil-targeted
therapeutic agent
(e.g., benralizumab/MEDI-563) is administered in two or more doses. In some
aspects, the
eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is
administered weekly,
biweekly or monthly. In some aspects, the eosinophil-targeted therapeutic
agent (e.g.,
benralizumab/MEDI-563) is administered at about 2 mg to about 100 mg per dose.
In some
aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-
563) is
administered at about 20 mg per dose, at about 30 mg per dose, or at about 100
mg per dose.
In some aspects, the eosinophil-targeted therapeutic agent (e.g.,
benralizumab/MEDI-563) is
administered once every four weeks to once every twelve weeks. In some
aspects, the
eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is
administered once
every four weeks. In some aspects, the eosinophil-targeted therapeutic agent
(e.g.,
benralizumab/MEDI-563) is administered once every eight weeks. In some
aspects, the
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eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is
administered once
every four weeks for twelve weeks and then once every eight weeks.
[0019] In
some aspects, the eosinophil-targeted therapeutic agent (e.g.,
benralizumab/MEDI-563) is administered orally, by inhalation, intravenously,
intramuscularly, subcutaneously, or a combination thereof.
[0020] In
some aspects, the eosinophilic disease or disorder is a pulmonary disease or
disorder. In some aspects, the pulmonary disease or disorder is asthma or
COPD. In some
aspects, the asthma is allergic asthma, atopic asthma, corticosteroid naive
asthma, chronic
asthma, corticosteroid resistant asthma, corticosteroid refractory asthma,
asthma due to
smoking, or asthma uncontrolled on corticosteroids. In some aspects, the
asthma is mild-to-
moderate asthma (defined as GINA 1-3) or severe asthma (defined as GINA 4+).
[0021] In
some aspects, the eosinophilic disease or disorder is chronic bronchitis,
chronic
eosinophilic pneumonia (CEP), nasal polyposis, atopic dermatitis (eczema),
eosinophilic
esophagitis, hypereosinophilic syndrome (HES), eosinophilic granulomatosis and
polyangitis
(Churg-Strauss syndrome), eosinophilic gastritis, eosinophilic enteritis,
eosinophilic colitis,
allergic rhinoconjunctivitis (hay fever), leukemia, lymphoma, mastocytosis,
atheroembolic
disease, hyper-IgE syndrome, Omenn's syndrome, thymoma, transplant rejections,
hypoadrenalism, bullous pemphigoid, pemphigus vulgaris, dermatitis
herpetiformis, drug-
induced lesions, urticaria, eosinophilic panniculitis, angioedema with
eosinophilia, Kimura's
disease, Shulman's syndrome, Well's syndrome, eosinophilic ulcer of the oral
mucosa,
eosinophilic pustular folliculitis, recurrent cutaneous necrotizing
eosinophilic vasculitis,
drug/toxin-induced eosinophilic lung disease, Loeffler's syndrome, allergic
bronchopulmonary aspergillosis, eosinophilic granuloma, pleural eosinophilia,
gastroesophageal reflux, parasitic infection, fungal infection, Helicobacter
pylori infection,
inflammatory bowel disease (ulcerative colitis and Crohn's disease), food
allergic disorder,
protein-induced enteropathy, protein-induced enterocolitis, allergic colitis,
celiac disease,
pemphigus vegetans, leiomyomatosis, connective tissue disorder, vasculitic
disorder, chronic
subdural hematoma, central nervous system infection, ventriculoperitoneal
shunts, congenital
heart condition (septal defects or aortic stenosis), eosinophiluria associated
with kidney
infection, interstitial nephritis, or eosinophilic cystitis.
[0022] In
some aspects, the patient has been treated either before, during, after, or
alternatively to the administration of an eosinophil-targeted therapeutic
agent (e.g.,
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benralizumab/MEDI-563) with one or more additional therapies for the treatment
of the
eosinophilic disease or disorder. In some aspects, the one or more additional
therapies further
comprise a steroid, bronchodilator, or both. In some aspects, the steroid is
fluticasone or
budesonide. In some aspects, the bronchodilator is salbutamol or salmeterol.
In some aspects,
the one or more additional therapies are administered by inhalation, by oral
administration,
by injection, or by a combination thereof. In some aspects, inhalation
administration is
conducted using a metered dose inhaler (MDI) or a dry powder inhaler (DPI). In
some
aspects, the steroid is administered at a high dose. In some aspects, the
steroid is an inhaled
corticosteroid such as beclomethasone or mometasone.
[0023] In
some aspects, the one or more samples taken from the patient and/or the one or
more control samples comprises one or more of whole blood, serum, plasma,
saliva, sputum,
bronchoalveolar lavage fluid, lung epithelial cells, urine, skin, nasal
polyps, or a combination
thereof. In some aspects, the sample taken from the patient is blood serum. In
some aspects,
the one or more control samples are (a) a sample or samples obtained from (i)
normal healthy
individuals, (ii) patients with a subset of asthma; (iii) asthma patients
naïve for corticosteroid
treatment; or asthma patients treated with corticosteroids; (b) a pre-
determined standard
amount of isolated DPP4 or POSTN; or, (c) any combination samples in (a) and
(b).
[0024] In
some aspects, the methods disclosed above further comprise determining,
submitting a sample taken from the patient for determination, or instructing a
clinical
laboratory to determine (i) the level of the patient's IgE levels, (ii) the
patient's eosinophil
count, (iii) the patient's Fraction of Exhaled Nitric Oxide (FENO), (iv) the
patient's
Eosinophil/Lymphocyte and Eosinophil/Neutrophil (ELEN) index, (v) the
patient's EOS
index, (vi) the patients wall area percentage (WA%) of subsegmental airways
from a CT scan
of the lungs, (vii) a combination of two or more thereof. See, e.g., Intl
Publ. No.
W02012158954, which is herein incorporated by reference in its entirety.
[0025] In
some aspects, the patient's eosinophil count is? 300 eosinophils/uL. In some
aspects, > 300 eosinophils/uL is set as a threshold for mild to moderate
asthmatics. In some
aspects, the patient's eosinophil count is > 400 eosinophils/uL. In some
aspects, > 400
eosinophils/uL is set as a threshold for severe asthmatics. In some aspects,
the patient's
eosinophil count is > 150 eosinophils/uL. In some aspects, > 150
eosinophils/uL is set as a
threshold, for example, for patients undergoing treatment with an anti-IL5
antibody (e.g.,
mepolizumab).
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[0026] In
some aspects, the patient's DPP4 level is measured in a DPP4 detection assay,
wherein the DPP4 detection assay is an immunoassay. In some aspects, the
patient's POSTN
level is measured in a POSTN detection assay, wherein the POSTN detection
assay is an
immunoassay.
[0027] In
some aspects, the DPP4 detection immunoassay is (i) the immunoassay
described in Example 2; or, (ii) an immunoassay disclosed in TABLE 1; or, (ii)
a multiplexed
immunoassay. In some aspects, the multiplexed immunoassay is an EMD Millipore
MILLIPLEXTM, Bio-Rad BIoPLExTM, Life Technologies NOVEX MULTIPLEXTm, Thermo
Fisher Scientific LumiNEx0, PerkinElmer ALPHAPLEXTM, Affymetrix (eBioscience)
PROCARTATm, or R&D Systems LumiNEx0 assay. In some aspects, the POSTN
detection
assay is an assay disclosed in W02015120171A1 or W02015120185, both of which
are
herein incorporated by reference in their entireties.
[0028] In
some aspects, the methods disclosed above further comprise determining,
submitting a sample taken from the patient for determination, or instructing a
clinical
laboratory to determine the expression level or activity of other molecular
biomarkers such as
IL-22, IL-25, IL-33, TSLP, LCN2, CCL20, sCTLA-3, sCD28, CCL5, CCL11, CCL22,
CST1, CCL26, CLCA1, CST2, PRR4, SERPINB2, CEACAM5, iNOS, SERPINB4, CST4,
PRB4, TPSD1, TPSG1, MFSD2, CPA3, GPR105, CDH26, GSN, C20RF32,
TRACH2000196 (TMEM71), DNAJC12, RGS13, SLC18A2, SERPINB10, SH3RF2,
FCER1B, RUNX2, PTGS1, ALOX15, or combinations thereof.
[0029] In
some aspects, the predetermined DPP4 or POSTN threshold levels are selected
from
(a) about the mean DPP4 level or POSTN level as measured in blood serum from a
plurality
of patients using an immunoassay;
(b) about the median DPP4 level or POSTN level as measured in blood serum from
a
plurality of patients using an immunoassay; and
(c) about the 1", 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, or 9th decile baseline
DPP4 level or POSTN level
as measured in blood serum from a plurality of patients using an immunoassay;
wherein the
patients are (i) normal healthy volunteers, (ii) patients with mild to
moderate-asthma, and/or
(iii) patients with an severe-asthma. In some aspects, the blood serum from a
plurality of
patients is a pooled sample or individual samples.
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[0030] In
some aspects, the predetermined DPP4 threshold level is at least about 103
ng/mL to at least about 867 ng/mL as measured using an immunoassay. In some
aspects, the
predetermined DPP4 threshold level is at least about 363 ng/mL as measured
using an
immunoassay. In some aspects, the predetermined DPP4 threshold level is at
least about 376
ng/mL as measured using an immunoassay. In some aspects, the immunoassay is
the DPP4
detection assay described in Example 2 or an DPP4 detection assay disclosed in
TABLE 1.
[0031] In
some aspects, the predetermined POSTN threshold is at least about 7 ng/mL to
at least about 104 ng/mL as measured using an immunoassay. In some aspects,
the
predetermined POSTN threshold level is at least about 23 ng/mL as measured
using an
immunoassay. In some aspects, the predetermined POSTN threshold level is at
least about 26
ng/mL as measured using an immunoassay. In some aspects, the POSTN immunoassay
is an
assay disclosed in W02015120171A1 or W02015120185, or a commercial POSTN
assay.
[0032] In
some aspects, administration of the eosinophil-targeted therapeutic agent
results
in (a) AER (Acute Exacerbation Rate) reduction; (b) FEV1 (Forced Expiratory
Volume in
one second) increase; (c) improved ACQ-6 (Asthma Control Questionnaire, 6-item
version)
value; or (d) a combination thereof.
[0033] In
some aspects, the AER reduction is at least about 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to
the AER baseline values observed in a population of patients treated with a
placebo. In some
aspects, the FEV1 increase is at least about 50, 75, 100, 125, 150, 175, 200,
225, 250, 275,
300, 325, 350, 400, 450 or 500 mL compared to the FEV1 baseline values
observed in a
population of patients treated with a placebo. In some aspects, the improved
ACQ-6 value is
a change from baseline of at least about -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -
0.9, -1, -1.1, or -1.2
compared to the ACQ-6 baseline values observed in a population of patients
treated with a
placebo.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0034] FIG.
1 shows the design of benralizumab Phase 2b study CP220 (NCT01238861)
in severe controlled asthma. ACQ-6, Asthma Control Questionnaire-6; AER,
annual
exacerbation rate (total observed exacerbations to week 52 divided by total
duration of
person-year follow-up); CBC, complete blood count with differential; FeNo,
fraction of
exhaled nitric oxide; FEV1, forced expiratory volume in 1 second; ICS, inhaled
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corticosteroids; ppb, parts per billion; SC, subcutaneous. The ELEN Index is a
mathematical
algorithm to predict sputum eosinophils from CBC data, as described in Khatry
et al. Am J
Respir Crit Care Med 189:A4257 (2014).
[0035] FIG.
2 is a table summarizing the data related to DPP4 and POSTN in serum
obtained in the CP220 study (NCT01238861). "DPP4 High" values (i.e., higher or
increased
DPP4 levels) are those equal or above the median. "POSTN High" values are
those equal or
above the median. "DPP4 Low" values (i.e., lower or decreased DPP4 levels) are
those below
the median. "POSTN Low" values are those below the median.
[0036] FIG.
3 shows the prevalence of subgroups DPP4 High and Low (A), and POSTN
High and Low (panel B) by blood eosinophil < or? 300 cells/uL.
[0037] FIG.
4 shows the lack of correlation between serum DPP4 levels and serum
POSTN levels in subjects participating in the CP220 study (panel A); that
serum POSTN
correlates with blood eosinophil numbers (panel B); and that serum DPP4 does
not correlate
with blood eosinophil number (panel C).
[0038] FIG.
5 shows a comparison of the effect of benralizumab on exacerbation rate
reduction for the combined 20 mg plus 100 mg dose groups vs placebo in DPP4
High and
Low sub-groups (panel A) and POSTN High and Low subgroups (panel B). * = P
<0.05.
Numbers above the bars represent n in placebo/benralizumab treatment groups.
Percentage
numbers at the foot of the x-axis represent prevalence of each sub-group.
[0039] FIG.
6 shows a comparison of the effect of benralizumab on FEN1 for the
combined 20 mg plus 100 mg dose groups vs placebo in DPP4 High and Low sub-
groups
(panel A) and POSTN High and Low subgroups (panel B). * = P <0.05. Numbers
above the
bars represent n in placebo/benralizumab treatment groups. Percentage numbers
at the foot of
the x-axis represent prevalence of each sub-group.
[0040] FIG.
7 shows a comparison of the effect of benralizumab on ACQ-6 for the
combined 20 mg plus 100 mg group vs placebo in DPP4 High and Low sub-groups
(panel A)
and POSTN High and Low subgroups (panel B). * = P <0.05. Numbers under the
bars
represent n in placebo/benralizumab treatment groups. Percentage numbers above
the labels
of the x-axis represent prevalence of each sub-group.
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DETAILED DESCRIPTION
[0041]
Eosinophils are a key effector cell in the pathology of asthma and more than
50%
of severe asthmatics are associated with persistent eosinophilic inflammation
despite
treatment with corticosteroids. IL-13, a Th2 cytokine produced by mast cells,
basophils and
eosinophils, contributes to key features of asthma through a number of
different mechanisms.
Benralizumab (MEDI-563), a humanized anti-IL-5Ra mAb that binds with high
affinity to
the alpha chain of the IL-5R to block IL-5 function, induces apoptosis of
eosinophils and
basophils through antibody-dependent cell-mediated cytotoxicity (ADCC) thereby
depleting
eosinophils and basophils.
[0042] In a
phase 2b clinical trial (CP220), benralizumab showed greatest efficacy to
reduce asthma exacerbations in patients with increased levels of blood
eosinophils and this is
currently being further evaluated in Phase III clinical trials. It is
recognized that there may be
overlap of severe asthma patients with eosinophilic inflammation as well as
increased IL-13
pathway activation. Therefore we sought to identify biomarkers that may be
more selective
in identifying patients who may respond to anti-eosinophil therapy, e.g.,
using benralizumab.
[0043] Serum
samples collected in the Phase 2b trial of benralizumab (CP220) were
analyzed to assess predictive utility of baseline DPP4 and POSTN levels on
reduction of
exacerbation rate in benralizumab treated subjects compared to placebo
treated.
[0044] Blood
eosinophils and POSTN were not correlated with serum DPP4
concentration. In contrast, serum POSTN correlated with blood eosinophil
numbers. When
segmenting patients in the benralizumab and placebo treated groups by high
levels of DPP4
or POSTN (greater than or equal to median) or low levels of DPP4 or POSTN
(less than the
median), the efficacy of benralizumab on reduction of exacerbations was
greatest in subjects
with low levels of serum DPP4. This effect with low levels of DPP4 was evident
in subjects
with both high (> 300 cells/micro liter) and low (<300 cells/micro liter)
blood eosinophil
levels. The effect with periostin was equivalent with high and low levels in
subjects with high
blood eosinophils but in subjects with low blood eosinophils the effect was
greatest in
subjects with low levels of periostin.
[0045] A key
observation in the present disclosure is that asthma patients with elevated
blood eosinophils who also have 1L13-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
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eosinophils. In addition, asthma patients who have low eosinophils and low
IL13-driven
disease as assessed by below median POSTN or DPP4 levels derive a benefit from
treatment
with benralizumab. In other words, it isn't eosinophilic disease alone that
determines
benralizumab efficacy and potentially that of anti-1L5 antibodies. Rather, it
is suggested that
IL-13 driven disease or lack thereof, assessed by the presence of low DPP4
and/or POSTN
levels that appears to be predictive of benralizumab efficacy.
[0046] The
present disclosure relates to the use of the DPP4 and/or periostin (POSTN)
levels as biomarkers for eosinophilic diseases or disorders, e.g., asthma (and
in particular
moderate and severe asthma). The disclosure provides, for example, methods for
diagnosing
and treating a subject having a eosinophilic disease or disorder comprising
administering an
eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as
benralizumab
(MEDI-563), to the patient if DPP4 or POSTN levels in one or more samples
taken from the
patient are (i) below the limit of detection of a DPP4 or POSTN detection
assay, (ii) below a
predetermined DPP4 or POSTN threshold level, or (ii) below the DPP4 or POSTN
level in
one or more control samples.
[0047] In
some aspects, the presence of DPP4 and/or POSTN levels (protein or RNA
expression levels) above or below predetermined DPP4 or POSTN threshold levels
in
samples (e.g., blood serum or sputum) obtained from a patient suffering from
an eosinophilic
disease or disorder (e.g., a pulmonary disease or disorder such as asthma) can
be used, e.g.,
(i) to determine whether the patient is eligible or non-eligible for treatment
with a specific
therapeutic agent, (ii) to determine whether a specific treatment should
commence, be
suspended, or be modified, (iii) to diagnose whether the disease or disorder
is treatable or not
treatable with a specific therapeutic agent, (iv) to prognosticate the outcome
of treatment of
the disease or disorder with a specific therapeutic agent, etc. In some
aspects, the specific
therapeutic agent is an eosinophil-targeted therapeutic agent, e.g., anti-IL-
5R antibody such
as benralizumab (MEDI-563).
[0048] In
other aspects, the presence of DPP4 and/or POSTN levels above or below
predetermined DPP4 or POSTN threshold levels in samples (e.g., blood serum or
sputum)
obtained from a patient suffering from an eosinophilic disease or disorder
(e.g., a pulmonary
disease or disorder such as asthma) in combination with
(i) one or more molecular biomarkers; and/or,
(ii) one or more clinical biomarkers; and/or,
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(iii) high eosinophil cell count (e.g., blood eosinophil count? 300 cells/uL);
and/or,
(iv) high Th2 (high Th2 defined, e.g., as IgE > 100 IU/mL and blood
eosinophils > 0.14 x
109/L); and/or,
(v) FEV1 reversibility to a short-acting 132 agonist, e.g.,? 12%; and/or,
(vi) wall area % (WA%) of subsegmental airways above, e.g., about 68% as
measured via
CT scan of the lungs; and/or,
(vii) combinations thereof,
can be used, e.g.,
(i) to determine whether a patient suffering an eosinophilic disease or
disorder (e.g., a
pulmonary disease or disorder such as asthma) is eligible or non-eligible for
a specific
treatment with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R
antibody such as
benralizumab (MEDI-563); and/or,
(ii) to determine whether a specific treatment should commence, be suspended,
or be
modified; and/or
(iii) to diagnose whether the disease or disorder is treatable or not
treatable with a specific
therapeutic agent; and/or
(iv) to prognosticate the outcome of treatment of the disease or disorder with
a specific
therapeutic agent.
[0049] In order that the present disclosure can be more readily understood,
certain terms
are first defined. Additional definitions are set forth throughout the
detailed description.
I. Definitions
[0050] In this specification and the appended claims, the singular forms
"a", an and
the include plural referents unless the context clearly dictates otherwise.
The terms "a" (or
"an"), as well as the terms one or more, and at least one can be used
interchangeably
herein.
[0051] Furthermore, "and/or" where used herein is to be taken as specific
disclosure of
each of the two specified features or components with or without the other.
Thus, the term
"and/or" as used in a phrase such as "A and/or B" herein is intended to
include "A and B," "A
or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a
phrase such as
"A, B, and/or C" is intended to encompass each of the following aspects: A, B,
and C; A, B,
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or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone);
and C
(alone).
[0052]
Wherever aspects are described herein with the language "comprising,"
otherwise
analogous aspects described in terms of "consisting or and/or "consisting
essentially or are
also provided.
[0053] The
term "about" as used in connection with a numerical value throughout the
specification and the claims denotes an interval of accuracy, familiar and
acceptable to a
person skilled in the art. In general, such interval of accuracy is 15 %.
[0054]
Unless defined otherwise, all technical and scientific terms used herein have
the
same meaning as commonly understood by one of ordinary skill in the art to
which this
disclosure is related. For example, the Concise Dictionary of Biomedicine and
Molecular
Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and
Molecular
Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of
Biochemistry And
Molecular Biology, Revised, 2000, Oxford University Press, provide one of
skill with a
general dictionary of many of the terms used in this disclosure.
[0055]
Units, prefixes, and symbols are denoted in their Systeme International de
Unites
(SI) accepted form.
[0056]
Numeric ranges are inclusive of the numbers defining the range. Where a range
of
values is recited, it is to be understood that each intervening integer value,
and each fraction
thereof, between the recited upper and lower limits of that range is also
specifically disclosed,
along with each subrange between such values. The upper and lower limits of
any range can
independently be included in or excluded from the range, and each range where
either,
neither or both limits are included is also encompassed within the invention.
Where a value is
explicitly recited, it is to be understood that values which are about the
same quantity or
amount as the recited value are also within the scope of the invention. Where
a combination
is disclosed, each subcombination of the elements of that combination is also
specifically
disclosed and is within the scope of the invention. Conversely, where
different elements or
groups of elements are individually disclosed, combinations thereof are also
disclosed. Where
any element of an invention is disclosed as having a plurality of
alternatives, examples of that
invention in which each alternative is excluded singly or in any combination
with the other
alternatives are also hereby disclosed; more than one element of an invention
can have such
exclusions, and all combinations of elements having such exclusions are hereby
disclosed.
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[0057] Amino
acids are referred to herein by either their commonly known three letter
symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical
Nomenclature Commission. Unless otherwise indicated, amino acid sequences are
written left
to right in amino to carboxy orientation.
[0058]
Nucleotides are referred to by their commonly accepted single-letter codes.
Unless
otherwise indicated, nucleic acids are written left to right in 5' to 3'
orientation. Nucleotides
are referred to herein by their commonly known one-letter symbols recommended
by the
IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents
adenine, C
represents cytosine, G represents guanine, T represents thymine, and U
represents uracil.
[0059] The
terms "polynucleotide," "oligonucleotide," "nucleic acid," "nucleic acid
molecule," and "gene" are used interchangeably herein to refer to polymers of
nucleotides of
any length, and ribonucleotides, deoxyribonucleotides, analogs thereof, or
mixtures thereof.
[0060] The
phrase "DNA sequence" refers to a contiguous nucleic acid sequence. The
sequence can be either single stranded or double stranded, DNA or RNA, but
double stranded
DNA sequences are preferable. The sequence can be an oligonucleotide of 6 to
20
nucleotides in length to a full length genomic sequence of thousands or
hundreds of
thousands of base pairs.
[0061] The
terms "polypeptide," "peptide," and "protein" are used interchangeably herein
to refer to polymers of amino acids of any length. The polymer can be linear
or branched, it
can comprise modified amino acids, and it can be interrupted by non-amino
acids. The terms
also encompass an amino acid polymer that has been modified naturally or by
intervention;
for example, disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation,
or any other manipulation or modification, such as conjugation with a labeling
component.
Also included within the definition are, for example, polypeptides containing
one or more
analogs of an amino acid (including, for example, unnatural amino acids such
as
homocysteine, omithine, p-acetylphenylalanine, D-amino acids, and creatine),
as well as
other modifications known in the art.
[0062] A
polypeptide, antibody, polynucleotide, vector, cell, or composition which is
"isolated" is a polypeptide, polynucleotide, or composition which is in a form
not found in
nature. Isolated polypeptides, polynucleotides, or compositions include those
which have
been purified to a degree that they are no longer in a form in which they are
found in nature.
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In some aspects, a polypeptide, polynucleotide, or composition which is
isolated is
substantially pure.
[0063] The
term "amino acid substitution" refers to replacing an amino acid residue
present in a parent sequence with another amino acid residue. An amino acid
can be
substituted in a parent sequence, for example, via chemical peptide synthesis
or through
recombinant methods known in the art. Amino acid substitutions also occur in
natural
variants.
[0064]
Accordingly, references to a "substitution at position X" or "substitution at
position X" refer to the substitution of an amino acid present at position X
with an alternative
amino acid residue. In some aspects, substitution patterns can described
according to the
schema AXY, wherein A is the single letter code corresponding to the amino
acid naturally
present at position X, and Y is the substituting amino acid residue. In other
aspects,
substitution patterns can described according to the schema XY, wherein Y is
the single letter
code corresponding to the amino acid residue substituting the amino acid
naturally present at
position X.
[0065] A
"conservative amino acid substitution" is one in which the amino acid residue
is
replaced with an amino acid residue having a similar side chain. Families of
amino acid
residues having similar side chains have been defined in the art, including
basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid),
uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine,
threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline,
phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine).
Thus, if an amino acid in a polypeptide is replaced with another amino acid
from the same
side chain family, the substitution is considered to be conservative. In
another aspect, a string
of amino acids can be conservatively replaced with a structurally similar
string that differs in
order and/or composition of side chain family members.
[0066] Non-
conservative substitutions include those in which (i) a residue having an
electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by,
an electronegative
residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is
substituted for, or by,
a hydrophobic residue (e.g., Ala, Leu, Ile, 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
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chain (e.g., Val, His, Ile or Trp) is substituted for, or by, one having a
smaller side chain (e.g.,
Ala, Ser) or no side chain (e.g., Gly).
[0067] Other
substitutions can be readily identified by workers of ordinary skill. For
example, for the amino acid alanine, a substitution can be taken from any one
of D-alanine,
glycine, beta-alanine, L-cysteine and D-cysteine. For lysine, a replacement
can be any one of
D-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine,
omithine, or D-
ornithine. Generally, substitutions in functionally important regions that can
be expected to
induce changes in the properties of isolated polypeptides are those in which
(i) a polar
residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic
residue, e.g.,
leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is
substituted for (or by)
any other residue; (iii) a residue having an electropositive side chain, e.g.,
lysine, arginine or
histidine, is substituted for (or by) a residue having an electronegative side
chain, e.g.,
glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain,
e.g., phenylalanine,
is substituted for (or by) one not having such a side chain, e.g., glycine.
The likelihood that
one of the foregoing non-conservative substitutions can alter functional
properties of the
protein is also correlated to the position of the substitution with respect to
functionally
important regions of the protein: some non-conservative substitutions can
accordingly have
little or no effect on biological properties.
[0068] The
term "percent sequence identity" between two polypeptide or polynucleotide
sequences refers to the number of identical matched positions shared by the
sequences over a
comparison window, taking into account additions or deletions (i.e., gaps)
that must be
introduced for optimal alignment of the two sequences. A matched position is
any position
where an identical nucleotide or amino acid is presented in both the target
and reference
sequence. Gaps presented in the target sequence are not counted since gaps are
not
nucleotides or amino acids. Likewise, gaps presented in the reference sequence
are not
counted since target sequence nucleotides or amino acids are counted, not
nucleotides or
amino acids from the reference sequence.
[0069] The
percentage of sequence identity is calculated by determining the number of
positions at which the identical amino-acid residue or nucleic acid base
occurs in both
sequences to yield the number of matched positions, dividing the number of
matched
positions by the total number of positions in the window of comparison and
multiplying the
result by 100 to yield the percentage of sequence identity. The comparison of
sequences and
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determination of percent sequence identity between two sequences can be
accomplished
using readily available software both for online use and for download.
Suitable software
programs are available from various sources, and for alignment of both protein
and
nucleotide sequences. One suitable program to determine percent sequence
identity is bl2seq,
part of the BLAST suite of program available from the U.S. government's
National Center for
Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov). Bl2seq
performs a
comparison between two sequences using either the BLASTN or BLASTP algorithm.
BLASTN is used to compare nucleic acid sequences, while BLASTP is used to
compare
amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher,
Water, or
Matcher, part of the EMBOSS suite of bioinformatics programs and also
available from the
European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.
[0070]
Different regions within a single polynucleotide or polypeptide target
sequence
that align with a polynucleotide or polypeptide reference sequence can each
have their own
percent sequence identity. It is noted that the percent sequence identity
value is rounded to
the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded
down to 80.1,
while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is
noted that the
length value will always be an integer.
[0071] In
certain aspects, the percentage identity "X" of a first amino acid sequence to
a
second sequence amino acid is calculated as 100 x (Y/Z), where Y is the number
of amino
acid residues scored as identical matches in the alignment of the first and
second sequences
(as aligned by visual inspection or a particular sequence alignment program)
and Z is the total
number of residues in the second sequence. If the length of a first sequence
is longer than the
second sequence, the percent identity of the first sequence to the second
sequence will be
higher than the percent identity of the second sequence to the first sequence.
[0072] One
skilled in the art will appreciate that the generation of a sequence alignment
for the calculation of a percent sequence identity is not limited to binary
sequence-sequence
comparisons exclusively driven by primary sequence data. Sequence alignments
can be
derived from multiple sequence alignments. One suitable program to generate
multiple
sequence alignments is ClustalW2, available from www.clustal.org. Another
suitable
program is MUSCLE, available from www.drive5.com/muscle/. ClustalW2 and MUSCLE
are alternatively available, e.g., from the EBI.
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[0073] As
used herein, the term "antibody" (or a fragment, variant, or derivative
thereof)
refers to at least the minimal portion of an antibody which is capable of
binding to antigen,
e.g., at least the variable domain of a heavy chain (VH) and the variable
domain of a light
chain (VL) in the context of a typical antibody produced by a B cell. Basic
antibody
structures in vertebrate systems are relatively well understood. See, e.g.,
Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988).
[0074]
Antibodies or antigen-binding fragments, variants, or derivatives thereof
include,
but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric
antibodies,
single chain antibodies, epitope-binding fragments, e.g., Fab, Fab and
F(ab')2, Fd, Fvs,
single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv),
fragments
comprising either a VL or VH domain, fragments produced by a Fab expression
library. ScFv
molecules are known in the art and are described, e.g., in US Pat. No.
5,892,019.
Immunoglobulin or antibody molecules encompassed by this disclosure can be of
any type
(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1 , IgG2, IgG3,
IgG4, IgAl and
IgA2) or subclass of immunoglobulin molecule.
[0075] By
"specifically binds," it is generally meant that an antibody or fragment,
variant,
or derivative thereof binds to an epitope via its antigen-binding domain, and
that the binding
entails some complementarity between the antigen binding domain and the
epitope.
According to this definition, an antibody is said to "specifically bind" to an
epitope when it
binds to that epitope via its antigen-binding domain more readily than it
would bind to a
random, unrelated epitope.
[0076] An
antibody or fragment, variant, or derivative thereof is said to competitively
inhibit binding of a reference antibody or antigen binding fragment to a given
epitope if it
preferentially binds to that epitope to the extent that it blocks, to some
degree, binding of the
reference antibody or antigen binding fragment to the epitope. Competitive
inhibition can be
determined by any method known in the art, for example, competition ELISA
assays. A
binding molecule can be said to competitively inhibit binding of the reference
antibody or
antigen-binding fragment to a given epitope by at least 90%, at least 85%, at
least 80%, at
least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least
50%.
[0077]
Antibodies or antigen-binding fragments, variants, or derivatives thereof
disclosed
herein can be described or specified in terms of the epitope(s) or portion(s)
of an antigen, e.g.,
a target polysaccharide that they recognize or specifically bind. For example,
the portion of
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the alpha subunit of human IL-5R that specifically interacts with the antigen-
binding domain
of an anti-IL-5R antibody such as benralizumab (MEDI-563) is an "epitope."
[0078] The
term "epitope" as used herein refers to an antigenic protein determinant
capable of binding to an antibody (e.g., an anti-IL-5R antibody) or a
fragment, variant, or
derivative thereof disclosed herein. In some aspects, the term epitope refers
to a protein
determinant (e.g., an amino acid sequence) of a subunit of the IL-5R protein,
e.g., the alpha
subunit of IL-5R. In some aspects, the term epitope refers to a protein
determinant (e.g., an
amino acid sequence) of the IL-5. In some aspects, binding of an antibody
disclosed to either
IL-5R or IL-5 prevents the interaction of IL-5R with IL-5.
[0079]
Epitopes usually consist of chemically active surface groupings of molecules
such
as amino acids or sugar side chains and usually have specific three
dimensional structural
characteristics, as well as specific charge characteristics. The part of an
antibody or binding
molecule that recognizes the epitope is called a paratope. The epitopes of
protein antigens are
divided into two categories, conformational epitopes and linear epitopes,
based on their
structure and interaction with the paratope. A conformational epitope is
composed of
discontinuous sections of the antigen's amino acid sequence. These epitopes
interact with the
paratope based on the 3-D surface features and shape or tertiary structure of
the antigen. By
contrast, linear epitopes interact with the paratope based on their primary
structure. A linear
epitope is formed by a continuous sequence of amino acids from the antigen.
[0080] The
term "antibody binding site refers to a region in the antigen (e.g., an amino
acid sequence of IL-5 or IL-5R) comprising a continuous or discontinuous site
(i.e., an
epitope) to which a complementary antibody specifically binds. Thus, the
antibody binding
site can contain additional areas in the antigen which are beyond the epitope
and which can
determine properties such as binding affinity and/or stability, or affect
properties such as
antigen enzymatic activity or dimerization. Accordingly, even if two
antibodies bind to the
same epitope within an antigen, if the antibody molecules establish distinct
intermolecular
contacts with amino acids outside of the epitope, such antibodies are
considered to bind to
distinct antibody binding sites.
[0081] An
antibody or fragment, variant, or derivative thereof is said to competitively
inhibit binding of a reference antibody or antigen binding fragment to a given
epitope if it
preferentially binds to that epitope to the extent that it blocks, to some
degree, binding of the
reference antibody or antigen binding fragment to the epitope. Competitive
inhibition can be
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determined by any method known in the art, for example, competition ELISA
assays. A
binding molecule can be said to competitively inhibit binding of the reference
antibody or
antigen-binding fragment to a given epitope by at least 90%, at least 85%, at
least 80%, at
least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least
50%, at least 45%, at
least 40%, at least 35%, at least 30%, at least 25%, or at least 20%.
[0082] As
used herein, the term "eosinophilic disease or disorder" refers to any disease
or
disorder characterized by an elevated level of eosinophils in blood, a tissue,
or an organ.
Normal levels in blood are on the order of 250 eosinophils per mm3. The term
eosinophilic
disease or disorder also encompasses eosinophilic inflammation. Blood levels
over
approximately 300 per mm3 are considered elevated. Examples of eosinophilic
diseases and
disorders include pulmonary diseases and disorders such as bronchial asthma,
chronic
bronchitis (in COPD), or chronic eosinophilic pneumonia (CEP). In addition,
the term
eosinophilic disease or disorder comprises diseases and conditions such as
nasal polyposis,
atopic dermatitis, eosinophilic esophagitis, hypereosinophilic syndrome (HES),
eosinophilic
granulomatosis and polyangitis (EGPA, formerly Churg-Strauss syndrome),
eosinophilic
gastritis, eosiophilic enteritis, eosinophilic colitis, etc. Normal and
abnormal (e.g., elevated)
eosinophil levels in the eosinophilic diseases or disorders disclosed herein
are known in the
art.
[0083] In
some aspects, the eosinophilic disease or disorder can be a pulmonary disease
or disorder. As used herein, the term "pulmonary disease or disorder" refers
to any pathology
affecting at least in part the lungs or respiratory system characterized by an
elevated level of
eosinophils. Non-limiting examples include asthma, idiopathic pulmonary
fibrosis (IPF),
chronic obstructive pulmonary disease (COPD), allergic rhinitis, or chronic
rhinosinusitis.
[0084] The
term "asthma" refers to diseases that present as reversible airflow
obstruction
and/or bronchial hyper-responsiveness that may or may not be associated with
underlying
inflammation. Examples of asthma include allergic asthma, atopic asthma,
corticosteroid
naive asthma, chronic asthma, corticosteroid resistant asthma, corticosteroid
refractory
asthma, asthma due to smoking, asthma uncontrolled on corticosteroids and
other asthmas as
mentioned, e.g., in the Expert Panel Report 3: Guidelines for the Diagnosis
and Management
of Asthma, National Asthma Education and Prevention Program (2007) ("NAEPP
Guidelines"), incorporated herein by reference in its entirety.
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[0085] The
term "COPD" as used herein refers to chronic obstructive pulmonary disease.
The term "COPD" includes two main conditions: emphysema and chronic
obstructive
bronchitis. Thus, in the broadest sense, the term COPD as used herein refers
to COPD itself
and also its subconditions chronic bronchitis and emphysema. The Global
Initiative for
Chronic Obstructive Lung Disease (GOLD) has classified 4 different stages of
COPD. GOLD
classification for COPD Stage 0: At Risk for COPD. Symptoms of chronic cough
and sputum
production may be present, but patients have normal spirometry readings. Stage
I: Mild
COPD. Characterized by FEVi >= 80%, FEV i/FVC < 70%. Patients may have or not
have
chronic cough and increased sputum production. Stage II: Moderate COPD.
Characterized by
a worsening of airflow (30% >= FENi > 80%). Patients with Stage II disease
often are
symptomatic, seek medical attention, and have shortness of breath with
exertion. Stage II has
2 subcategories: HA and JIB. IIA patients have a FEVi between 50% and 80%;
stage JIB
patient have a FEVi between 30% and 50%. Patients with FEVi below 50% are
especially
prone to acute exacerbations of disease. Stage III: Severe COPD. Characterized
by an FEV
below 30%. Patients are also included in stage III if they have respiratory
failure or right
heart failure. The quality of life is severely affected in these patients.
Acute exacerbations in
this patient population often require hospitalization and they are frequently
life threatening.
[0086] The
term "Idiopathic Pulmonary Fibrosis" (IPF) refers to a disease characterized
by progressive scarring, or fibrosis, of the lungs. It is a specific type of
interstitial lung
disease in which the alveoli gradually become replaced by fibrotic tissue.
With IPF,
progressive scarring causes the normally thin and pliable tissue to thicken
and become stiff,
making it more difficult for the lungs to expand, preventing oxygen from
readily getting into
the bloodstream. See, e.g., Am. J. Respir. Crit. Care Med. 2000. 161:646-664.
[0087] The
term eosinophilic disease or disorder also encompasses the diseases and
conditions disclosed below, which a characterized by the presence of high
eosinophil levels.
Normal and abnormal (e.g., elevated) eosinophil levels in the eosinophilic
diseases or
disorders disclosed below are also known in the art, or can be determined
using methods
known in the art.
[0088] High
eosinophil levels have been observed in the following diseases or disorders:
(i) Allergic disorders: Allergic disorders are classically characterized by
the presence of
eosinophils. Allergic rhinoconjunctivitis (hay fever) has increased levels of
eosinophils in the
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nasal mucosa. Asthma, after an exacerbation, shows increased numbers of
eosinophils in the
lung;
(ii) Drug reactions: Any drug / medicine has the potential to cause a
reaction. Some of these
reactions are allergic in nature, and eosinophils might be elevated in blood
or in tissues where
the drug is concentrated;
(iii) Infectious diseases: Parasitic infections (helminthiasis ¨ worms),
fungal infections and
some other types of infections are associated with increased numbers of
eosinophils;
(iv) Blood disorders: Hematologic disorders with increased levels of
eosinophils include
hypereosinophilic syndrome, leukemias, lymphomas, tumors, mastocytos is , and
atheroembolic disease;
(v) Immunologic disorders and reactions: Hyper-IgE syndrome, Omenn's syndrome,
thymomas, and transplant rejections are only a few types of conditions with
increased
numbers of eosinophils;
(vi) Endocrine disorders: Hypoadrenalism has been associated with increases in
the levels of
eosinophils in the blood.
[0089]
Eosinophils have also been found to be increased or pathologically present in
the
following diseases or disorders:
(i) Skin and Subcutaneous Disorders: Atopic dermatitis (eczema), bullous
pemphigoid,
pemphigus vulgaris, dermatitis herpetiformis, drug-induced lesions, urticaria,
eosinophilic
panniculitis, angioedema with eosinophilia, Kimura's disease, Shulman's
syndrome, Well's
syndrome, eosinophilic ulcer of the oral mucosa, eosinophilic pustular
folliculitis, and
recurrent cutaneous necrotizing eosinophilic vasculitis;
(ii) Pulmonary Conditions: Drug/toxin-induced eosinophilic lung disease,
Loeffler's
syndrome, allergic bronchopulmonary aspergillosis, eosinophilic pneumonia,
Churg-Strauss
syndrome, eosinophilic granuloma, and pleural eosinophilia;
(iii) Gastrointestinal Diseases: Gastroesophageal reflux, parasitic
infections, fungal
infections, Helicobacter pylori infections, inflammatory bowel disease
(ulcerative colitis and
Crohn's disease), food allergic disorders, protein-induced enteropathy and
protein-induced
enterocolitis, allergic colitis, celiac disease, pemphigus vegetans (MR) and
primary
eosinophilic esophagitis, gastroenteritis, and colitis. Rare tumors
(leiomyomatosis),
connective tissue disorders, and vasculitic disorders;
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(iv) Neurologic Disorders: Organizing chronic subdural hematoma membranes,
central
nervous system infections, ventriculoperitoneal shunts, and drug-induced
adverse reactions;
(v) Cardiac Conditions: Secondary to systemic disorders such as the
hypereosinophilic
syndrome or the Churg-Strauss syndrome, heart damage has been reported.
Certain
congenital heart conditions (septal defects, aortic stenosis) are associated
with increased
levels of eosinophils in the blood;
(vi) Renal Diseases: Eosinophiluria (eosinophils in the urine) associated with
infections,
interstitial nephritis, eosinophilic cystitis.
[0090] As
used herein, the term "exacerbation" refers to a worsening of symptoms of an
eosinophilic disease or disorder, relative to a patient's baseline condition.
In certain aspects,
an "asthma exacerbation" may be defined as an event in the natural course of
the disease
characterized by a change in the patient's baseline lung function, dyspnea,
cough, and/or
sputum that is beyond normal day-to-day variations, is acute in onset and may
warrant a
change in medication in a patient with underlying asthma. In certain
embodiments,
exacerbation of asthma may be an abrupt increase in symptoms of shortness of
breath and/or
wheezing, and/or increase in production of sputum.
[0091] As
used herein the terms "treat," "treatment, " or "treatment or (e.g., in the
phrase
"treating a patient having an eosinophilic disease or disorder") refers to (i)
reducing the
potential for an eosinophilic disease or disorder (e.g., a pulmonary disease
or disorder such as
asthma), (ii) reducing the occurrence of the eosinophilic disease or disorder,
(iii) reducing
the severity of the eosinophilic disease or disorder, preferably, to an extent
that the subject no
longer suffers discomfort and/or altered function due to it (for example, in
the case of asthma,
a relative reduction in asthma exacerbations when compared to untreated
patients), or (iv) a
combination thereof.
[0092] For
example, treating can refer to the ability of a therapy when administered to a
subject, to prevent an eosinophilic disease or disorder from occurring and/or
to cure or to
alleviate the eosinophilic disease or disorder's symptoms, signs, or causes.
Treating also
refers to mitigating or decreasing at least one clinical symptom and/or
inhibition or delay in
the progression of the condition and/or prevention or delay of the onset of a
disease or illness.
Thus, the terms "treat," "treating" or "treatment or (or grammatically
equivalent terms) refer
to both prophylactic and therapeutic treatment regimes.
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[0093] The
present disclosure provides methods and systems providing a therapeutic
benefit in the treatment of an eosinophilic disease or disorder (e.g., a
pulmonary disease or
disorder such as asthma). A therapeutic benefit is not necessarily a cure for
a particular
eosinophilic disease or disorder, but rather encompasses a result which most
typically
includes (i) alleviation of the eosinophilic disease or disorder or increased
survival, (ii)
elimination of the eosinophilic disease or disorder, reduction of a symptom
associate with the
eosinophilic disease or disorder, (iii) prevention or alleviation of a
secondary disease, (iv)
disorder or condition resulting from the occurrence of a primary eosinophilic
disease or
disorder, (v) prevention of the eosinophilic disease or disorder, or (v) a
combination thereof.
[0094] The
terms "subject" or "patient" as used herein refer to any subject, particularly
a
mammalian subject, for whom diagnosis, prognosis, or therapy of an
eosinophilic disease or
disorder (e.g., a pulmonary disease or disorder such as asthma) is desired. As
used herein, the
terms "subject" or "patient" include any human or nonhuman animal. The term
"nonhuman
animal" includes all vertebrates, e.g., mammals and non-mammals, such as
nonhuman
primates, sheep, dogs, cats, horses, cows, bears, chickens, amphibians,
reptiles, etc. As used
herein, phrases such as "a patient having an eosinophilic disease or disorder
" includes
subjects, such as mammalian subjects, that would benefit from the
administration of a
therapy, imaging or other diagnostic procedure, and/or preventive treatment
for that
eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such
as asthma). The
term "normal healthy volunteer" is equivalent to "healthy subject" or "healthy
volunteer." The
skilled artisan recognizes that the term "healthy volunteer" is generally
defined in United
States Pharmacopeia, p. 2645 (U.S. Pharmacopeial Convention, Inc., 28th ed.,
2005).
[0095] In
some aspects of the present disclosure, a subject is a naïve subject. A naïve
subject is a subject that has not been administered a therapy, for example a
therapeutic agent.
In some aspects, a naïve subject has not been treated with a therapeutic agent
prior to being
diagnosed as having an eosinophilic disease or disorder (e.g., a pulmonary
disease or disorder
such as asthma).
[0096] In
another aspect, a subject has received therapy and/or one or more doses of a
therapeutic agent (e.g., a therapeutic agent capable of modulating an
inflammatory response
associated with an eosinophilic disease or disorder) prior to being diagnosed
as having an
eosinophilic disease or disorder.
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[0097] In
some aspects, a subject has received at least one therapeutically effective
dose
of oral or inhaled corticosteroids. In some aspects, inhalation administration
is conducted
using a metered dose inhaler (MDI) or a dry powder inhaler (DPI). In some
aspects, the
steroid is administered at a high dose. The term high dose when application to
an inhaled
corticosteroid (ICS) can refer, for example, to a total daily dose of at least
500 pg of ICS
(e.g., fluticasone) DPI or at least 440 pg ICS MDI. In some aspects, the high
ICS total daily
dose is at least about 300 pg, at least about 350 pg, at least about 400 pg,
at least about 450
pg, at least about 500 pg, at least about 550 pg, at least about 600 pg, at
least about 650 pg, at
least about 700 pg, at least about 750 pg, at least about 800 pg, at least
about 850 pg, at least
about 900 pg, at least about 950 pg, or at least 1000 pg of ICS (e.g.,
fluticasone) DPI. In
some aspects, the high ICS total daily dose is at least about 300 pg, at least
about 350 pg, at
least about 400 pg, at least about 450 pg, at least about 500 pg, at least
about 550 pg, at least
about 600 pg, at least about 650 pg, at least about 700 pg, at least about 750
pg, at least about
800 pg, at least about 850 pg, at least about 900 pg, at least about 950 pg,
or at least 1000 pg
of ICS (e.g., fluticasone) MPI.
[0098] The
term "high dose" when application to an inhaled corticosteroid (ICS) (e.g.,
fluticasone) in combination treatments (e.g., with a bronchodilator such as
salmeterol) can
refer, for example, to about 230 pg fluticasone and about 21 pg salmeterol as
MDI at a dose
of 2 inhalations twice per day, or to about 500 pg fluticasone and about 50 pg
salmeterol as
single dose DPI. Concentrations of corticosteroids considered to be high-dose
alone as well
as in combination with other therapeutic agents are well known in the art.
[0099] In
some aspects, a subject has received multiple therapeutically effective doses
of
oral or inhaled corticosteroids. In some aspects, a subject is a chronic oral
corticosteroid
(OCS) user.
[0100] In
certain aspects the subject has received a long-acting beta2-adrenergic
agonist,
e.g., salmeterol xinafoate. In some aspects the subject has received a
synthetic glucocorticoid,
e.g., fluticasone propionate. In certain aspects the subject has received a
combination of
salmeterol xinafoate and fluticasone propionate (ADVAIRO). In certain aspects
the subject
has received a beta2-adrenergic bronchodilator, e.g., albuterol sulfate.
[0101] The
term "therapy" as used herein includes any means for curing, mitigating, or
preventing an eosinophilic disease or disorder (e.g., a pulmonary disease or
disorder such as
asthma), including, for example, therapeutic agents (e.g., eosinophil-targeted
therapeutic
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agent), instrumentation, supportive measures, and surgical or rehabilitative
procedures. In this
respect, the term therapy encompasses any protocol, method and/or therapeutic
or diagnostic
that can be used in prevention, management, treatment, and/or amelioration of
an
eosinophilic disease or disorder.
[0102] In
some aspects, the patient is considered to be positive for DPP4 or POSTN when
measurements for the biomarkers are above a predetermined threshold level. A
patient is
considered to have a high or increased DPP4 level (DPP4 High) when the
measured DPP4
level in a sample is above a predetermined threshold level or above the level
of DPP4 in one
or more controls samples. Conversely, a patient is considered to have a low or
decreased
DPP4 level (DPP4 Low) when the measured DPP4 level in a sample is below a
predetermined threshold level or below the level of DPP4 in or more control
samples.
[0103]
Similarly, a patient is considered to have a high or increased POSTN level
(POSTN High) when the measured POSTN level in a sample is above a
predetermined
threshold level or above the level of POSTN in one or more controls samples. A
patient is
considered to have a low or decreased POSTN level (POSTN Low) when the
measured
POSTN level in a sample is below a predetermined threshold level or below the
level of
POSTN in or more control samples.
[0104] A
person skilled in the art would appreciate that it is possible to combine
quantitative and qualitative measures. For example, the presence of DPP4
and/or POSTN in a
sample may be determined using a qualitative assays that merely detects the
presence or
absence of the biomarker above a certain limit of quantification for the
assay, whereas the
levels of other biomarkers may be determined based on a quantitative assay
showing that the
level of biomarker is above or below a certain predetermined threshold or
within a certain
range.
[0105] In
some aspects, a subject can be administered at least one therapeutically
effective dose of an eosinophil-targeted therapeutic agent if the subject's
DPP4 level (e.g., the
level of protein or nucleic acid) in a sample is below a predetermined DPP4
threshold level,
or if the DPP4 level is decreased relative to the DPP4 level in one or more
control samples. In
other aspects, a subject can be administered at least one therapeutically
effective dose of an
eosinophil-targeted therapeutic agent if the subject's POSTN level (e.g., the
level of protein or
nucleic acid) in a sample is below a predetermined POSTN threshold level, or
if the POSTN
level is decreased relative to the POSTN level in one or more control samples.
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[0106] The
term "therapeutic agent" as used herein also refers to any therapeutically
active substance that is administered to a subject having an eosinophilic
disease or disorder
(e.g., a pulmonary disease or disorder such as asthma) to produce a desired,
usually
beneficial, effect. The term therapeutic agent includes, e.g., classical low
molecular weight
therapeutic agents commonly referred to as small molecule drugs and biologics
including but
not limited to antibodies or active fragments thereof, peptides, lipids,
protein drugs, protein
conjugate drugs, enzymes, oligonucleotides, ribozymes, genetic material,
prions, virus,
bacteria, and eukaryotic cells.
[0107] A
therapeutic agent can also be a pro-drug, which metabolizes into the desired
therapeutically active substance when administered to a subject. In some
aspects, the
therapeutic agent is a prophylactic agent. In addition, a therapeutic agent
can be
pharmaceutically formulated. A therapeutic agent can also be or comprise a
radioactive
isotope or agent activated by some other form of energy such as light or
ultrasonic energy, or
by other circulating molecules that can be systemically administered.
[0108] In
one aspect, the therapeutic agent is a small molecule drug. In a specific
aspect,
the agent is a corticosteroid. In another aspect, the agent can be a
leukotriene modifier such as
montelukast, zafirlukast or zileuton. In a further aspect, the therapeutic
agent can be a
methylxanthine (e.g., theophylline) or a cromone (e.g., sodium cromolyn and
nedocromil). In
another aspect, the therapeutic agent can be a long-acting beta-2 agonist such
as salmeterol,
fomoterol, or indacaterol. In a further aspect, the agent can be methotrexate
or cyclosporin.
[0109] In
certain aspects, the therapeutic agent can be an agent used for preventing,
treating, managing, or ameliorating eosinophilic disease or disorder, e.g., a
pulmonary
disease or disorder such as asthma. Non-limiting examples of therapies for
asthma include
anti-cholinergics (e.g., ipratropium bromide and oxitropium bromide), beta-2
antagonists
(e.g., albuterol (PROVENTIL or VENTOLIN ), bitolterol (TOMALATE ), fenoterol,
formoterol, isoetharine, metaproterenol, pibuterol (MAXAIR ), salbutamol,
salbutamol
terbutaline, and salmeterol, terbutlaine (BRETHAIRE )), corticosteroids (e.g.,
prednisone,
beclomethasone dipropionate (VANCERIL or BECLOVENT ), triamcinolone acetonide
(AZMACORF ), flunisolide (AEROBID ), mometasone (NASONEXO, ASMANEXO) and
fluticasone propionate (FLOVENT )), leukotriene antagonists (e.g.,
montelukast, zafirlukast,
and zileuton), theophylline (THEO-DUR , UNIDUR tablets, and SLO-BID
Gyrocaps),
and salmeterol (SEREVENT ), cromolyn, and nedorchromil (INTAL and TILADE )),
IgE
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antagonists, IL-4 antagonists (including antibodies), IL-5 antagonists
(including antibodies),
PDE4 inhibitors, NF-Kappa-B inhibitors, IL-13 antagonists (including
antibodies), CpG,
CD23 antagonists, selectin antagonist (e.g., TBC 1269), mast cell protease
inhibitors (e.g.,
tryptase kinase inhibitors (e.g., GW-45, GW-58, and genisteine),
phosphatidylinositide-3'
(P13)-kinase inhibitors (e.g., calphostin C), and other kinase inhibitors
(e.g., staurosporine),
C2a receptor antagonists (including antibodies), and supportive respiratory
therapy, such as
supplemental and mechanical ventilation.
[0110] An
eosinophil-targeted therapeutic agent as used herein can be any "therapeutic
agent" as defined herein, which either directly or indirectly can (i) inhibit,
lessen, or
neutralize the activity of eosinophils in the patient, or (ii) inhibit,
reduce, or deplete
eosinophil levels in the patient, either systemically or in a specific tissue
or organ, or (iii)
reduce the half-life of eosinophils in the patient, or (iv) can prevent
exacerbation of
symptoms associated with elevated levels of eosinophils, (v) combinations
thereof.
[0111] In
some aspects of the present disclosure, an eosinophil-targeted therapeutic
agent
can comprise (i) an antibody targeting, e.g., IL-5R or IL-5, or an antigen
binding fragment
thereof; (ii) any of the therapeutic agents disclosed above (e.g.,
corticosteroids); or (iii) any
combinations thereof.
[0112] A
"therapeutically effective" amount as used herein is an amount of therapeutic
agent that provides some improvement or benefit to a subject having an
eosinophilic disease
or disorder (e.g., a pulmonary disease or disorder such as asthma). Thus, a
"therapeutically
effective" amount is an amount that provides some alleviation, mitigation,
and/or decrease in
at least one clinical symptom of the eosinophilic disease or disorder (e.g., a
pulmonary
disease or disorder such as asthma).
[0113]
Clinical symptoms associated with the eosinophilic disease or disorder (e.g.,
a
pulmonary disease or disorder such as asthma) can be treated by the methods
and systems of
the disclosure are well known to those skilled in the art. Further, those
skilled in the art will
appreciate that the therapeutic effects need not be complete or curative, as
long as some
benefit is provided to the subject. In some aspects, the term "therapeutically
effective" refers
to an amount of a therapeutic agent therapeutic agent that is capable of
altering biomarker
levels, e.g., DPP4 level and/or POSTN level and/or patient's eosinophil count
in a patient in
need thereof.
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[0114] As
used herein, a "sufficient amount" or an amount sufficient to achieve a
particular result in a patient having an eosinophilic disease or disorder
(e.g., a pulmonary
disease or disorder such as asthma) refers to an amount of a therapeutic agent
(e.g., an anti-
IL-5R such as benralizumab) that is effective to produce a desired effect,
which is optionally
a therapeutic effect (i.e., by administration of a therapeutically effective
amount). In some
aspects, such particular result is a reduction in the patient's eosinophil
count.
[0115] The
term "sample" as used herein includes any biological fluid or tissue, such as
whole blood, serum, sputum, saliva, or epithelial tissue (e.g., lung tissue)
obtained from a
subject. Samples include any biological fluid or tissue, such as whole blood,
serum, saliva,
urine, nasal secretions, sputum, bronchoalveolar lavage fluid, lung tissue,
peripheral blood
mononuclear cells, total white blood cells, lymph node cells, spleen cells,
tonsil cells, skin,
nasal polyps, lung epithelial cells, etc. In some specific aspects, that
sample is blood or a
fraction thereof. Samples can be obtained by any means known in the art.
[0116] In
some aspects, a sample is a computed tomography (CT) scan of a patient's
organ or tissue including, but not limited to the lungs. In some aspects, a
sample can be
derived by taking biological samples from a number of subjects and pooling
them or pooling
an aliquot of each subjects biological sample. The pooled sample can be
treated as a sample
from a single subject. The term sample also includes experimentally separated
fractions of all
of the preceding. For example, a blood sample can be fractionated into serum
or into fractions
containing particular types of cells. In some aspects, a sample can be a
combination of
samples from an individual, such as a combination of a tissue and fluid
sample.
[0117] As
used herein, the term "control", when used to characterize a subject, refers
to a
subject that is healthy or to a patient who has been diagnosed with a specific
disease other
than an eosinophilic disease or disorder. The term "control sample" refers to
one, or more
than one, biological samples obtained from a healthy subject or from a patient
diagnosed with
a disease other than an eosinophilic disease or disorder.
[0118] In
order to apply the methods and systems of the disclosure, samples from a
patient can be obtained before or after the administration of a therapy to
treat an eosinophilic
disease or disorder (e.g., a pulmonary disease or disorder such as asthma). In
some cases,
successive samples can be obtained from the patient after therapy has
commenced or after
therapy has ceased. Samples can, for example, be requested by a healthcare
provider (e.g., a
doctor) or healthcare benefits provider, obtained and/or processed by the same
or a different
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healthcare provider (e.g., a nurse, a hospital) or a clinical laboratory, and
after processing, the
results can be forwarded to the original healthcare provider or yet another
healthcare
provider, healthcare benefits provider or the patient.
[0119]
Similarly, the quantification of the expression level of a biomarker disclosed
herein, e.g., DPP4 and/or POSTN alone or in combination with the measurement
of at least
one additional biomarker, e.g., a patient's eosinophil count; comparisons
and/or ratios
between biomarker gene or protein expression levels; evaluation of the absence
or presence
of biomarkers; determination of biomarker levels with respect to a certain
threshold;
treatment decisions; or combinations thereof, can be performed by one or more
healthcare
providers, healthcare benefits providers, and/or clinical laboratories.
[0120] As
used herein, the term "healthcare provider" refers to individuals or
institutions
that directly interact and administer to living subjects, e.g., human
patients. Non-limiting
examples of healthcare providers include doctors, nurses, technicians,
therapist, pharmacists,
counselors, alternative medicine practitioners, medical facilities, doctor's
offices, hospitals,
emergency rooms, clinics, urgent care centers, alternative medicine
clinics/facilities, and any
other entity providing general and/or specialized treatment, assessment,
maintenance,
therapy, medication, and/or advice relating to all, or any portion of, a
patient's state of health,
including but not limited to general medical, specialized medical, surgical,
and/or any other
type of treatment, assessment, maintenance, therapy, medication and/or advice.
[0121] As
used herein, the term "clinical laboratory" refers to a facility for the
examination or processing of materials derived from a living subject, e.g., a
human being.
Non-limiting examples of processing include biological, biochemical,
serological, chemical,
immunohematological, hematological, biophysical, cytological, pathological,
genetic, or
other examination of materials derived from the human body for the purpose of
providing
information, e.g., for the diagnosis, prevention, or treatment of any disease
or impairment of,
or the assessment of the health of living subjects, e.g., human beings. These
examinations can
also include procedures to collect or otherwise obtain a sample, prepare,
determine, measure,
or otherwise describe the presence or absence of various substances in the
body of a living
subject, e.g., a human being, or a sample obtained from the body of a living
subject, e.g., a
human being.
[0122] As
used herein, the term "healthcare benefits provider" encompasses individual
parties, organizations, or groups providing, presenting, offering, paying for
in whole or in
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part, or being otherwise associated with giving a patient access to one or
more healthcare
benefits, benefit plans, health insurance, and/or healthcare expense account
programs.
[0123] In
some aspects, a healthcare provider can administer or instruct another
healthcare provider to administer a therapy to treat an eosinophilic disease
or disorder (e.g., a
pulmonary disease or disorder such as asthma). A healthcare provider can
implement or
instruct another healthcare provider or patient to perform the following
actions: obtain a
sample, process a sample, submit a sample, receive a sample, transfer a
sample, analyze or
measure a sample, quantify a sample, provide the results obtained after
analyzing/measuring/quantifying a sample, receive the results obtained after
analyzing/measuring/quantifying a sample, compare/score the results obtained
after
analyzing/measuring/quantifying one or more samples, provide the
comparison/score from
one or more samples, obtain the comparison/score from one or more samples,
administer a
therapy (e.g., administration of an eosinophil-targeted therapeutic agent such
as
benralizumab/MEDI-563), commence the administration of a therapy, cease the
administration of a therapy, continue the administration of a therapy,
temporarily interrupt the
administration of a therapy, increase the amount of an administered
therapeutic agent,
decrease the amount of an administered therapeutic agent, continue the
administration of an
amount of a therapeutic agent, increase the frequency of administration of a
therapeutic
agent, decrease the frequency of administration of a therapeutic agent,
maintain the same
dosing frequency on a therapeutic agent, replace a therapy or therapeutic
agent by at least
another therapy or therapeutic agent, combine a therapy or therapeutic agent
with at least
another therapy or additional therapeutic agent.
[0124] In
some aspects, a healthcare benefits provider can authorize or deny, for
example, collection of a sample, processing of a sample, submission of a
sample, receipt of a
sample, transfer of a sample, analysis or measurement a sample, quantification
a sample,
provision of results obtained after analyzing/measuring/quantifying a sample,
transfer of
results obtained after analyzing/measuring/quantifying a sample,
comparison/scoring of
results obtained after analyzing/measuring/quantifying one or more samples,
transfer of the
comparison/score from one or more samples, administration of a therapy or
therapeutic agent,
commencement of the administration of a therapy or therapeutic agent,
cessation of the
administration of a therapy or therapeutic agent, continuation of the
administration of a
therapy or therapeutic agent, temporary interruption of the administration of
a therapy or
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therapeutic agent, increase of the amount of administered therapeutic agent,
decrease of the
amount of administered therapeutic agent, continuation of the administration
of an amount of
a therapeutic agent, increase in the frequency of administration of a
therapeutic agent,
decrease in the frequency of administration of a therapeutic agent, maintain
the same dosing
frequency on a therapeutic agent, replace a therapy or therapeutic agent by at
least another
therapy or therapeutic agent, or combine a therapy or therapeutic agent with
at least another
therapy or additional therapeutic agent.
[0125] In addition a healthcare benefits provides can, e.g., authorize or
deny the
prescription of a therapy, authorize or deny coverage for therapy, authorize
or deny
reimbursement for the cost of therapy, determine or deny eligibility for
therapy, etc.
[0126] In some aspects, a clinical laboratory can, for example, collect or
obtain a sample,
process a sample, submit a sample, receive a sample, transfer a sample,
analyze or measure a
sample, quantify a sample, provide the results obtained after
analyzing/measuring/quantifying
a sample, receive the results obtained after analyzing/measuring/quantifying a
sample,
compare/score the results obtained after analyzing/measuring/quantifying one
or more
samples, provide the comparison/score from one or more samples, obtain the
comparison/score from one or more samples, or other related activities.
[0127] As used herein, the term "Computed Tomography" or "CT" refers to an
imaging
method using tomographic images (virtual 'slices') of specific areas of a
scanned organ, tissue
or object. Digital geometry processing is used to generate a three-dimensional
(3D) image of
the inside of an object or organ from a series of two-dimensional (2D)
radiographic images
taken around a single axis of rotation.
[0128] As used herein, the term "Computed Tomography scan" or "CT scan"
refers to the
production of tomographic images obtained using any method suitable including,
but not
limited to, x-rays, multidetector computed tomography (MDCT), high-resolution
computed
tomography (HRCT), positron emission tomography (PET), positron emission
tomography
computed tomography (PET-CT) single-photon emission computed tomography
(SPECT),
magnetic resonance imaging (MRI), computed axial tomography (CAT scan),
computer-
assisted tomography, xenon ventilation computed tomography, and hyperpolarized
gas lung
MRI ventilation imaging.
II. Low DPP4 and POSTN as Eosinophilic Disease Biomarkers
[0129] In general, the methods disclosed herein are based on
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(a) detecting changes in the levels of DPP4 and/or POSTN, alone or in
combination with the
detection of changes in the levels of one, two, three, or more biomarkers,
including, e.g.,
blood eosinophil count in patients with an eosinophilic disease or disorder
(e.g., a pulmonary
disease or disorder such as asthma);
(b) predict an increased clinical response to therapy with an eosinophil-
targeted therapeutic
agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) based on
detected
DPP4 and/or POSTN levels; and,
(c) administering an eosinophil-targeted therapeutic agent to the patient if
DPP4 and/or
POSTN levels, alone or in combination with other biomarkers, indicate that the
patient will
benefit from therapy with the eosinophil-targeted therapeutic agent. If DPP4
and/or POSTN
levels, alone or in combination with other biomarkers, indicate that the
patient will not
benefit from therapy with the eosinophil-targeted therapeutic agent, then
therapy could be
discontinued, temporarily suspended, modified (e.g., increasing dosage or
frequency of
doses), etc.
[0130] In
other words, specific levels of DPP4 and/or POSTN alone or in combination
with other molecular or clinical biomarkers (e.g., a patient's eosinophil
count) are correlated
with clinical efficacy of therapies and useful to predict clinical outcomes in
specific
populations of patients suffering from an eosinophilic disease or disorder
(e.g., a pulmonary
disease or disorder such as asthma).
[0131] The
term "DPP4" as used herein refers to the dipeptidyl peptidase IV protein (EC
3.4.14.5; Uniprot: P27487) encoded by the DPP4 gene (cDNA sequence corresponds
to SEQ
ID NO:3). DPP4 is also known as DPP-IV, adenosine deaminase complexing protein
2, or
CD26 (cluster of differentiation 26). DPP4 is related to attractin, FAP, DPP8
and DPP9.
DPP4 is a highly conserved multifunctional type II transmembrane glycoprotein,
which is
present both in circulation (plasma) and on the surface of several cell types,
including
epithelial, endothelial and lymphoid cells. Thus, DPP4 exists both in membrane
bound form
(SEQ ID NO:1) and soluble form (SEQ ID NO:2).
[0132] DPP4
is part of the serine protease family that is involved in T-cell co-
stimulation,
chemokine biology, type II diabetes, and tumor biology (Zhong et al.,
Atherosclerosis
2013;226:305-314). A role for DPP4 in inflammatory respiratory diseases like
asthma is
suggested by Giovannini-Chami (Giovannini-Chami et al., European Respiratory
Journal.
2012 May;39(5):1197-205), who found elevated DPP4 transcripts (and other Th2
signature
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genes) in the nasal epithelia of children with dust mite allergic rhinitis,
associated with
uncontrolled asthma. The term DPP4 also includes fragments, variants (e.g.,
the K 1R, V7I,
S437I, T557I, D663E variants known in the art), and derivatives thereof (e.g.,
glycosylated or
aglycosilated protein forms of the DPP4 protein, or otherwise chemically
modified forms of
the protein).
[0133] In
some aspects, in addition or alternatively to the determination of the level
of
DPP4, the methods disclosed herein can comprise determining, submitting a
sample taken
from the patient for determination, or instructing a clinical laboratory to
determine the
expression level or activity of periostin. The use of periostin as a biomarker
for pulmonary
diseases such as asthma has been disclosed, e.g., in Jia, et al., J Allergy
Clin. Immunol 2012
130:647-654; Takayama, et al., J Allergy Clin Immunol 2006 118:98-104; and PCT
Publ. No.
WO 2012/083132, each herein incorporated by reference in their entirety.
[0134] The
term "POSTN" as used herein refers to the osteoblast specific factor protein
periostin (Uniprot: Q15063; SEQ ID NO:20) encoded by the POSTN gene. POSTN is
also
known as osteoblast-specific factor 2 (OSF-2). POSTN functions as a ligand for
alpha-
V/beta-3 and alpha-V/beta-5 integrins to support adhesion and migration of
epithelial cells.
POSTN is a gla domain vitamin K dependent factor. The term POSTN also includes
fragments, variants (e.g., isoforms produced by alternative splicing), and
derivatives thereof
(e.g., glycosylated or aglycosilated protein forms of the protein, or
otherwise chemically
modified forms of the protein).
[0135] Seven
POSTN isoforms produced by alternative splicing are known in the art:
Isoform 1 (Uniprot: Q15063-1), also known as OSF-205, which is 836 amino acids
long;
Isoform 2 (Uniprot: Q15063-2), also known as OSF-2p1, 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: 1290F, D421V, T339I, or V814M.
[0136] In
some aspects, the DPP4 and POSTN biomarkers disclosed herein can be
combined with other biomarkers related to eosinophilic diseases or disorders,
e.g., a patient's
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eosinophil count, which in turn can be substituted or combined with one or
more molecular
biomarkers in inflammation pathways or clinical biomarkers known in the art.
[0137] The
term "biomarker" as used herein refers to a factor that is a distinctive
indicator of a biological process, biological event, and/or pathologic
condition, e.g., a
predictor of clinical response to treatment with an eosinophil-targeted
therapeutic agent, e.g.,
an anti-IL-5R antibody such as benralizumab (MEDI-563). As used herein, the
term
biomarker encompasses both clinical markers and molecular biomarkers
(biological markers).
Thus, in the context of the present disclosure, the term "biomarker"
encompasses, e.g.,
"biological biomarkers" or "molecular biomarkers" such as the DPP4 and POSTN
biomarkers
disclosed herein alone or in combination with molecular biomarkers linked to
the IL-13
pathway, molecular biomarkers linked to a specific eosinophilic disease or
disorder (e.g., a
pulmonary disease or disorder such as asthma), and combinations thereof. The
biological
markers disclosed herein also include the genes encoding those proteins (DNA
and/or RNA),
as well as metabolic products.
[0138] As
disclosed above, the term "biomarker" also encompasses "clinical biomarkers,"
also referred to as "clinical status markers," that can be predictive of
response to biological
therapies, for example, gender, age, concomitant drugs, smoking status, body
mass index
(BMI), etc. See, e.g., U.S. Publ. Nos.U520150065530, U520140141990,
U520130005596,
U520090233304, US 20140199709, U520130303398, and U520110212104, which are
herein incorporated by reference in their entireties.
[0139] The
DPP4 molecular biomarker disclosed herein also includes proteins or
fragments thereof having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%,
95%, 96%, 97%, 98% or 99% sequence identity to the wild type sequence of
either its
membrane-bound (SEQ ID NO:1) or soluble form (SEQ ID NO:2), and nucleic acids
having
at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
or 99% sequence identity to the respective wild type nucleic acid sequences
encoding the
membrane-bound or soluble form of DPP4.
[0140] The
POSTN molecular biomarker disclosed herein also includes proteins or
fragments thereof having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%,
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95%, 96%, 97%, 98% or 99% sequence identity to the wild type sequence POST
(SEQ ID
NO:20) or POSTN isoforms known in the art, and nucleic acids having at least
about 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence
identity to the wild type nucleic acid sequence SEQ ID NO:20 or POSTN isoforms
known in
the art.
[0141] The
DPP4 and POSTN molecular biomarkers disclosed herein also include
fragments, variants, and derivatives thereof. As used herein, a "variant"
biomarker contains at
least one amino acid sequence alteration as compared to the amino acid
sequence of the
corresponding wild-type polypeptide. An amino acid sequence alteration can be,
for example,
a substitution, a deletion, or an insertion of one or more amino acids,
preferably conservative
substitutions. A variant biomarker can have any combination of amino acid
substitutions,
deletions or insertions. In one aspect, a biomarker variant polypeptide can
have an integer
number of amino acid alterations such that its amino acid sequence shares at
least 60, 70, 80,
85, 90, 95, 97, 98, 99, 99.5 or 100% identity with the amino acid sequence of
the
corresponding wild-type polypeptide.
[0142] In
some aspects of the present disclosure, the methods disclosed herein can be
applied to an eosinophilic disease or disorder (e.g., a pulmonary disease or
disorder such as
asthma) exclusively using DPP4 as a biomarker, exclusively using POSTN as a
biomarker,
using a combination of DPP4 and POSTN, or optionally by incorporating
additional clinical
and/or molecular biomarkers such as a patient's eosinophil count.
[0143] In
some aspects, the methods disclosed herein can comprise using additional
biomarkers, e.g., the blood eosinophil cell count, the level of the patient's
IgE levels, pre- or
post-bronchodilator FEV1 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 FEV1).
[0144] The
presence of eosinophils above normal baseline level, e.g., in blood, can be
used as a biomarker in combination with DPP4 levels and/or POSTN levels.
Severe asthma
patients have frequent exacerbations and hospitalizations and account for over
half of the cost
of the disease and most of its mortality (Gaga et al., 2009). Inflammation, an
important
feature in severe asthma, exhibits different phenotypes that can be
characterized by
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persistence of varying degrees of eosinophilic and neutrophilic infiltration
(Balzar et al.,
2002). The presence of eosinophils in asthma has been well documented via
airway biopsy
studies. The clinical importance of eosinophils in asthma has been
demonstrated by the
observation of frequent asthma exacerbations in patients who have sputum
eosinophil counts
>3%. Moreover, clinical trials designed to adjust inhaled anti-inflammatory
therapy to
maintain sputum eosinophil counts to <3% have resulted in fewer asthma
exacerbations
(Green et al., 2002). Symptomatic asthmatics with recalcitrant sputum
eosinophilia on
standard therapy have also improved after monoclonal antibody therapy
(mepolizumab) that
depletes airway eosinophils (Nair et al., 2009; Haldar et al., 2009).
[0145] To
date the only accurate and reliable method to identify eosinophilic asthmatics
has been limited to procurement of induced sputum samples from patients
(Molfino, 2012).
The sputum induction procedure is a tedious and complex process that requires
skilled
technicians and equipment that are not readily available in clinical practice.
Even with these
shortcomings, induced sputum remains the standard for assessing the cellular
inflammatory
processes that occur in asthma (Lieberman, 2007). A panel convened from the
National
Institutes of Health and federal agencies to propose biomarkers to assess
disease progression
and response to treatment has recommended 2% eosinophils in sputum as the cut-
off for
classifying patients as sputum eosinophilic asthmatics (Szefler et al., 2012).
[0146] A cut-
off approach based on absolute values, e.g., sputum EOS% cutoff, is used as
the standard for prediction and classification in pulmonary diseases and
disorders such as
eosinophilic asthma. In one aspect, the EOS% cutoff point to classify a
patient as eosinophilic
is 2% or greater sputum eosinophils. EOS% cutoff points of 1%, 2%, 2.5%, and
3% sputum
eosinophils have been reported as discriminating between eosinophilic and non-
eosinophilic
patients. See, e.g., Green et al., 2002 and Jayaram et al., 2006. Belda et
al., 2000 showed that
the mean + 2 standard deviations for sputum EOS% in healthy subjects is 2.2%.
To date,
attempts to predict and classify eosinophilic asthma have investigated the
correlations of
individual measures (such as blood eosinophil counts and FEN0) with sputum
EOS%.
[0147] The
cut-offs disclosed above with respect to eosinophil count, i.e., percentage of
eosinophils in sputum or sputum EOS% of 1%, 2%, 2.2%, 2.5%, or 3% can be used
as
thresholds in the methods disclosed herein.
[0148] The
term "wall area as used herein refers to the cross-sectional area of a
bronchial tube wall (e.g. segmental and subsegmental bronchi in the upper
lobes). Wall area
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percentage (WA%) is calculated as follows: 100*wall area/(wall area + lumen
area). Tools to
measure wall area and wall area percentage are well known in the art. See,
e.g., Gupta et al., J
Allergy Clin Immunol. 133(3): 729-738 (2014); Gupta et al., Thorax. 65(9):775-
81 (2010).
In some aspects, airway dimensions are measured from Computed Tomography (CT)
imaging data of the lungs. Such imaging data can be processed, for example,
using
commercially available software such as VIDA Apollo (e.g., the Volumetric
Information
Display and Analysis (VIDA) Pulmonary Workstation, VIDA Diagnostics,
Coralville, Iowa).
[0149] In
other aspects, the methods disclosed herein can also comprise using additional
molecular biomarkers in combination with DPP4 and/or POSTN levels such as the
expression
level or activity of sCTLA-3 (soluble CTLA-3; also known as Cytotoxic T-
Lymphocyte-
Associated serine Esterase 3, granzyme A, or granzyme 1; Uniprot: P12544),
sCD28 (soluble
CD28; also known as cluster of differentiation 28 or Tp44; Uniprot: P10747),
CCL5
(chemokine C-C motif ligand 5; also known as RANTES; Uniprot: P13501), CCL11
(C-C
motif chemokine 11; also known as eosinophil chemotactic protein or eotaxin-1;
Uniprot:
P51671), CCL22 (C-C motif chemokine 22; Uniprot: 000626), or combinations
thereof.
These biomarkers have been disclosed in IL-13 mediated pulmonary diseases,
e.g., in Lun et
al., J. Clin. Immunol. 2007 27:430-437.
[0150] In
some aspects, the methods disclosed herein can also comprise using additional
molecular biomarkers in combination with DPP4 and/or POSTN levels such as the
expression
level or activity of CCL26, FZD5, DOK1, CST2, ZNF436, C20orf100, NAGS, CST1,
CDH13, HRH1 , TMEM132B , NTRK1, SLCO2A1, IgE, FETUB , KRT3 lIKRT34, C6orf138,
ATP5J, TUBAL3, JAM2, NOVA2, NOS2A, H535T4, GRM8, IL1R2, CTDSPL, CEP72,
L0C199800, LYPD1, DISP1, NKX1-2, C4orf38, LOXL4, PRKD1, PAM124B, GPR44,
HIGD1B, CLCA1, SEPT11, CYYR1, CD36, ALOX15, AADAC, ACTA1, ODC1,
DKFZp434F142, ACHE, CSF3, L0C100132552, C12orf27, ZNF331, GK5, DUSP1IDUSP4,
LRWD1, PGLYRP4, GUSBL2, CLGN, NR1I2, EST, LRRC37B, SAA4, SLC12A3,
TMEM45A, FLJ37464, MUC5B, CXCL6, GLRB, DKFp686K01114, FOLR1, TSPAN6,
AKR1C1, KIAA0232, PTP4A1, PCYT2, RHOV, PROS1, Cl lorf63, TCTN1, PIP5K1B,
OSBPL6, NSUM7, GJB7, IR52, or combinations thereof. These genes are part of
the Th-2
signature as disclosed in Choi et al. J. Immunol. 186(3):1861-9 (2011) and
Int'l Publ. No.
W02009124090, both of which are herein incorporated by reference in their
entireties.
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[0151] In
some aspects, the methods disclosed herein can also comprise using additional
molecular biomarkers in combination with DPP4 and/or POSTN levels such as the
expression
level or activity of cystatin-SN (CST1); chemokine (C-C motif) ligand 26
(CCL26); calcium-
activated chloride channel regulator (1CLCA1); cystatin-SA (CST2); proline-
rich protein 4
(PRR4); plasminogen activator inhibitor-2 (placental PAI), also known as
HsT1201, PAI,
PAI-2, PAI2 or PLANH2 (SERPINB2); carcinoembryonic antigen-related cell
adhesion
molecule 5 (CEACAM5) also known as cluster of differentiation 66 (CD66e);
inducible NOS
(iNOS, NOS2), serpin peptidase inhibitor, clade B (ovalbumin), member 4
(SERPINB4,
LEUPIN, PI11, SCCA-2, SCCA1, SCCA2), cystatin S (CST4), basic salivary proline-
rich
protein 4 (PRB4), tryptase Delta 11 (TPSD1), tryptase gamma 1 (TPSG1), major
facilitator
superfamily domain containing 2A protein (MFSD2), carboxypeptidase A3 (CPA3),
G-
Protein coupled receptor 105 (GPR105), cadherin 26 (CDH26), gelsolin (GSN),
cannabinoid
receptor interacting protein 11 (C20RF32), transmembrane protein 711
(TRACH2000196,
TMEM71), DnaJ (Hsp40) homolog, subfamily C, member 121 (DNAJC12), RGS13
(regulator of G-protein signaling 13), solute carrier family 18 member 2
(SLC18A2), serpin
peptidase inhibitor, clade B (ovalbumin), member 10 (SERPINB10), SH3 ring
finger 2
protein (SH3RF2), high affinity immunoglobulin epsilon receptor subunit beta
(FCER1B),
runt-related transcription factor 2 (RUNX2), prostaglandin-endoperoxide
synthase 1
(PTGS1), arachidonate 15-lipoxygenase (ALOX15), and combinations thereof.
[0152] In
some aspects, the determination that a patient's DPP4 level is High (higher or
increased) requires that the measured level of DPP4 is > about 376 ng/mL as
determined by a
DDP4 detection immunoassay, including the immunoassay disclosed in Example 2.
In some
aspects, the determination that a patient's DPP4 level is Low (lower or
decreased) requires
that the measured level of DPP4 is < about 376 ng/mL as determined by a DDP4
detection
immunoassay, including the immunoassay disclosed in Example 2.
[0153] In
some aspects, the determination that a patient's DPP4 level is High (higher or
increased) requires that the measured level of DPP4 is > about 363 ng/mL as
determined by a
DDP4 detection immunoassay, including the immunoassay disclosed in Example 2.
In some
aspects, the determination that a patient's DPP4 level is Low (lower or
decreased) requires
that the measured level of DPP4 is < about 363 ng/mL as determined by a DDP4
detection
immunoassay, including the immunoassay disclosed in Example 2.
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[0154] In
some aspects, the determination that a patient's POSTN level is High (higher
or
increased) requires that the measured level of POSTN is? about 25.8 ng/mL as
determined
by a POSTN detection immunoassay, including the POSTN immunoassay disclosed in
W02015120185. In some aspects, the determination that a patient's POSTN level
is Low
(lower or decreased) requires that the measured level of DPP4 is < about 25.8
ng/mL as
determined by a POSTN detection immunoassay, including the POSTN immunoassay
disclosed in W02015120185.
[0155] In
some aspects, the determination that a patient's POSTN level is High (higher
or
increased) requires that the measured level of POSTN is > about 23.5 ng/mL as
determined
by a POSTN detection immunoassay, including the POSTN immunoassay disclosed in
W02015120185. In some aspects, the determination that a patient's POSTN level
is Low
(lower or decreased) requires that the measured level of POSTN is < about 23.5
ng/mL as
determined by a POSTN detection immunoassay, including the POSTN immunoassay
disclosed in W02015120185.
[0156] In
some aspects, the determination that a patient's blood eosinophil count is
High
(higher or increased) requires that the measured level of blood eosinophils is
> 150 cells/pL.
In some aspects, the determination that a patient's blood eosinophil count is
Low (lower or
decreased) requires that the measured level of blood eosinophils is < 150
cells/pL.
[0157] In
some aspects, the determination that a patient's blood eosinophil count is
High
(higher or increased) requires that the measured level of blood eosinophils is
> 300 cells/pL.
In some aspects, the determination that a patient's blood eosinophil count is
Low (lower or
decreased) requires that the measured level of blood eosinophils is <300
cells/pL.
[0158] In
some aspects, the determination that a patient's blood eosinophil count is
High
(higher or increased) requires that the measured level of blood eosinophils is
> 400 cells/pL.
In some aspects, the determination that a patient's blood eosinophil count is
Low (lower or
decreased) requires that the measured level of blood eosinophils is <400
cells/pL.
[0159] In
some aspects, the determination that a patient's blood eosinophil count is
High
(higher or increased) requires that the measured level of blood eosinophils is
at least about
150 cells/pL, 160 cells/p L, 170 cells/pL, 180 cells/pL, 190 cells/pL, 200
cells/pL, 210
cells/pL, 220 cells/pL, 230 cells/pL, 240 cells/pL, 250 cells/pL, 260
cells/pL, 270 cells/pL,
280 cells/pL, 290 cells/pL, 300 cells/pL, 310 cells/pL, 320 cells/ L, 330
cells/pL, 340
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cells/pL, 350 cells/pL, 360 cells/pL, 370 cells/pL, 380 cells/pL, 390 cells/
L, or 400
cell s/p L.
[0160] In
some aspects, the determination that a patient's blood eosinophil count is Low
(lower or decreased) requires that the measured level of blood eosinophils is
at least below
150 cells/pL, 150 cells/p L, 160 cells/pL, 170 cells/pL, 180 cells/pL, 190
cells/pL, 200
cells/pL, 210 cells/pL, 220 cells/pL, 230 cells/pL, 240 cells/pL, 250
cells/pL, 260 cells/pL,
270 cells/pL, 280 cells/p L, 290 cells/pL, 300 cells/pL, 310 cells/ L, 320
cells/pL, 330
cells/pL, 340 cells/pL, 350 cells/pL, 360 cells/pL, 370 cells/ L, 380 cells/
L, 390 cells/ L,
or 400 cells/p L.
III. Determination of DPP4, POSTN, Eosinophil Count, and Other Biomarkers
[0161]
Levels of DPP4, POSTN, and other biomarkers disclosed herein (either their
expressed protein levels, or their respective nucleic acid levels, such as
mRNA levels) can be
detected and quantified by any of a number of methods well known to those of
skill in the art.
These methods include analytic biochemical methods such as electrophoresis,
capillary
electrophoresis, high performance liquid chromatography (HPLC), thin layer
chromatography
(TLC), hyperdiffusion chromatography, mass spectroscopy and the like, or
various
immunological methods such as fluid or gel precipitin reactions,
immunodiffusion (single or
double), immunohistochemistry, affinity chromatography, immunoelectrophoresis,
radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISAs), chemi-
luminescence immunoassay (CLIA), immunofluorescent assays, Western blotting,
and the
like.
[0162] In
some aspects, the method used to detect and/or quantify DPP4, POSTN, or
other molecular biomarkers disclosed herein comprises measuring the level,
concentration, or
amount of RNA, e.g., mRNA, encoded by the gene or gene segments in the sample.
Levels of
RNA, e.g., mRNA, may be measured by any technique known in the art, including
but not
limited to northern blotting or quantitative PCR (qPCR), including methods
such as reverse
transcription qPCR, real time qPCR, and end-point qPCR. Alternatively, "tag
based"
technologies, such as Serial analysis of gene expression (SAGE) and RNA-Seq,
may be
carried out to provide a relative measure of the cellular concentration of
different mRNAs.
[0163] In
some aspects, DPP4, POSTN, and other molecular biomarkers disclosed herein
can be detected and/or quantified in an electrophoretic polypeptide separation
(e.g., a 1- or 2-
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dimensional electrophoresis). Means of detecting polypeptides using
electrophoretic
techniques are well known to those skilled in the art (see generally, R.
Scopes (1982)
Polypeptide Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methods in
Enzymology
Vol. 182: Guide to Polypeptide Purification, Academic Press, Inc., N.Y.).
[0164] In
some aspects, a Western blot (immunoblot) analysis is used to detect and
quantify the presence or absence of DPP4, POSTN, or any other molecular
biomarkers
disclosed herein in the sample. This technique generally comprises separating
sample
polypeptides by gel electrophoresis on the basis of molecular weight,
transferring the
separated polypeptides to a suitable solid support (such as a nitrocellulose
filter, a nylon
filter, or derivatized nylon filter), and incubating the sample with
antibodies that specifically
bind the analyte. Antibodies that specifically bind to the analyte may be
directly labeled or
alternatively may be detected subsequently using labeled antibodies (e.g.,
labeled sheep anti-
mouse antibodies) that specifically bind to a domain of the primary antibody.
[0165] In
some aspects, DPP4, POSTN, and other molecular biomarkers disclosed herein
can be detected and/or quantified in the biological sample using an
immunoassay. For a
general review of immunoassays, see also Methods in Cell Biology Volume 37:
Antibodies in
Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic and
Clinical
Immunology 7th Edition, Stites & Ten, eds. (1991). See also, e.g., U.S. Patent
Application
Publication No. 2007/0212723 Al, Shang et al., Circulation Research 101: 1146-
1154
(2007); and International Patent Application Publication Nos. WO/2012/094651
and
WO/2010/129964.
[0166] In
some aspects, the immunoassay can use one or more antibodies or antigen
binding fragments thereof which recognize the molecular biomarker (e.g., human
DPP4 or
human POSTN). In some aspects, the immunoassay comprises a sandwich
immunoassay,
e.g., an enzyme-linked immunosorbent assay (ELISA) or a sandwich
electrochemiluminescent (ECL) assay, in which a first antibody or antigen-
binding fragment
thereof against the molecular biomarker (e.g., DPP4 or POSTN) is used as a
"capture"
antibody. The capture antibody is attached to a solid support, an antigen from
a sample or
standard is allowed to bind to the capture antibody, and then a second
antibody or antigen
binding fragment thereof against the same biomarker comprising a detectable
label, i.e., a
"detection" antibody, is added. The detection antibody can be detected either
by an enzymatic
reaction, an ECL reaction, radioactivity, or any other detection method known
in the art.
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[0167] In
some aspects, the immunoassay comprises the following steps: First, the
capture antibody or fragment thereof is allowed to bind to a solid support,
e.g., a multi-well
plate or other assay device known to those of ordinary skill in the art. The
capture antibody is
allowed to attach for a period of time, e.g., overnight, and then unbound
antibody is removed.
The plate can then be washed to remove any unbound capture antibody. The plate
can then be
treated with a blocking solution to allow non-specific protein to bind to any
unbound regions
of the solid support.
[0168]
Typical blocking solutions include an unrelated protein, e.g., nonfat dry milk
or
serum albumin. The plate can then again be washed to remove any unbound
blocking
solution. Next, a sample suspected of containing the molecular biomarker
(e.g., DPP4 or
POSTN) is added to the plate. Samples are typically serially diluted and
plated in duplicate or
triplicate. Controls, including standard amounts of biomarker (e.g., pure
recombinant DPP4
or pure recombinant POSTN) or a suitable fragment thereof and various negative
controls are
also included. The antigen is allowed to bind to the capture antibody for a
period of time, e.g.,
one hour at room temperature. Following incubation, the plate can then be
washed to remove
any unbound antigen.
[0169] Next,
a detection antibody is added. The detection antibody is typically an anti-
antibody that specifically binds to an epitope of the molecular biomarker
(e.g., DPP4 or
POSTN) epitope that is different from the epitope to which the capture
antibody binds. The
detection antibody can be labeled or unlabeled. Where the detection antibody
is unlabeled, an
addition step of addition a labeled secondary antibody will be required, as is
well known by
those of ordinary skill in the art.
[0170] The
detection antibody can be directly labeled with an enzyme, e.g., horseradish
peroxidase or alkaline phosphatase, or can be labeled with a tag that will
allow an enzyme to
bind. For example the detection antibody can be conjugated to biotin, and the
enzyme
attached in a subsequent step by allowing enzyme-conjugated streptavidin to
bind to the
biotin tag. Alternatively, the detection antibody can be conjugated to a
chemiluminescent,
fluorescent, or ECL tag. An example of the latter is a ruthenium chelate.
Following
incubation, the plate can then be washed to remove any unbound detection
antibody.
Detection of the detection antibody can be accomplished by methods that vary
based on the
type of detection antibody that is used.
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[0171] If
the detection antibody is tagged with biotin, then enzyme-conjugated
streptavidin is added, unbound streptavidin is washed away, and a substrate is
added which
provides a colorimetric reaction that can be read, e.g., on a
spectrophotometer. If the
detection antibody is conjugated to a ruthenium chelate, the plate is
subjected to electrical
current, and light emission is measured.
[0172]
Immunoassays for detecting molecular biomarkers (e.g., DPP4 or POSTN) can be
either competitive or noncompetitive. Noncompetitive immunoassays are assays
in which the
amount of captured analyte is directly measured. In competitive assays, the
amount of analyte
in the sample is measured indirectly by measuring the amount of an added
(exogenous)
labeled analyte displaced (or competed away) from a capture agent by the
analyte present in
the sample. In one competitive assay, a known amount of, for example, labeled
molecular
biomarker (e.g., DPP4 or POSTN) is added to the sample, and the sample is then
contacted
with a capture agent. The amount of labeled molecular biomarker (e.g., DPP4 or
POSTN)
bound to the antibody is inversely proportional to the concentration of
molecular biomarker
(e.g., DPP4 or POSTN) present in the sample.
[0173] In
some aspects, the method directly measures the level of DPP4, POSTN, or
other biomarkers disclosed herein, in a patient sample, where absolute levels
are calculated
by plotting the immunoassay results on a standard curve using, e.g., purified
full length DPP4
or POSTN, or a DPP4 or POSTN fragment (or a full length biomarker disclosed
herein or a
fragment thereof). The detected signal from the detection antibody can then be
quantitated
based on the various standards and controls included on the plate. By plotting
the results on a
standard curve, the absolute levels of DPP4, POTN, or any other biomarker
disclosed herein
in the test samples can be calculated, e.g., in pg or ng of biomarker per mL,
or pg or ng of
biomarker per mg of total protein.
[0174]
Detection assays for DPP4, POSTN, or other molecular biomarkers disclosed
herein can be scored (as positive or negative or quantity of analyte)
according to standard
methods well known to those of skill in the art. The particular method of
scoring will depend
on the assay format and choice of label. For example, a Western Blot assay can
be scored by
visualizing the colored product produced by the enzymatic label. A clearly
visible colored
band or spot at the correct molecular weight is scored as a positive result,
while the absence
of a clearly visible spot or band is scored as a negative. The intensity of
the band or spot can
provide a quantitative measure of analyte concentration.
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[0175] In
some aspects, the measured expression level of molecular biomarker (e.g.,
DPP4 or POSTN) represents an average expression level or a mean expression
level based on
more than one measurement of the expression level. In some aspects, the
measured
expression level is an average or mean of several measurements of expression
levels of the
same sample. In some aspects, the measured expression level is an average or
mean of
several measurements of expression levels of different samples containing the
same
components obtained from the same subject. In some aspects, the measured
expression level
is quantile normalized, as is done in RNA Seq techniques using techniques well
known by
those of ordinary skill in the art.
[0176] The
term "DPP4 detection assay" as used herein refers to both quantitative and
qualitative assays capable of detecting the presence or absence of DPP4 in a
biological
sample. The term DPP4 detection assays encompassed, e.g., immunoassays such as
ELISA.
[0177] The
term "POSTN detection assay" as used herein refers to both quantitative and
qualitative assays capable of detecting the presence or absence of POSTN in a
biological
sample. The term POSTN detection assays encompassed, e.g., immunoassays such
as ELISA.
POSTN detection assays are well known in the art. In some aspects, the POSTN
detection
assay is an immunoassay disclosed in W02015120171A1 or W02015120185, or a
commercial POSTN assay.
[0178] In
some aspects, the DPP4 detection assay or the POSTN detection assay is a
multiplexed immunoassay, for example, Bio-Rad BIOPLEXTM, EMD Millipore
MILLIPLEXTM, Life Technologies NOVEX MULTIPLEXTm, Thermo Fisher Scientific
LUMINEXO, PerkinElmer ALPHAPLEXTM, Affymetrix (eBioscience) PROCARTATm, or
R&D Systems LUMINEXO capable of detecting the expression levels of DPP4 and/or
POSTN in a sample.
[0179] In
some aspects, the DPP4 detection assay is an assay included in TABLE 1, or a
variant thereof.
TABLE 1: DPP4 immunoassays.
DPP4/CD26 Product Source Manufacturer's Information &
Instructions
Assays Code
Human DPP4 DC260 R&D Systems www.rndsystems.com/products/human-
dppiv-
Quantikine cd26-quantikine-elisa-kit_dc260
ELISA Kit
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CD26 Human ab119513 Abcam www.abcam.com/cd26-human-elisa-kit-
ELISA Kit ab119513.html
ELISA Kit for SEA884H USCN www.uscnk.com/uscn/ELISA-Kit-for-Human-
Dipeptidyl u Dipeptidyl-Peptidase-IV-DPP4-CD26-
451.htm
Peptidase IV
(DPP4)
Human CD26 ELH- RayBiotech www.raybiotech.com/human-dppiv-
cd26-elisa-kit-
ELISA CD26 for-serum-plasma-cell-culture-
supernatant-and-
urine.html
Human DPPIV GWB- Genway Biotech www.genwaybio.com/human-dppiv-
elisa-kit
ELISA Kit SKR222
Human sCD26 BMS235 Affymetrix www.ebioscience.com/human-scd26-
platinum-
Platinum ELISA eBioscience elisa-kit.htm
Human DPPIV / RAB0147 Sigma-Aldrich
www.sigmaaldrich.com/catalog/product/sigmakab
CD26 ELISA 0147?lang=en®ion=US
Kit
sCD26 ELISA 61- ALPCO www.alpco.com/store/scd26-elisa.html
C26HU-
E01
sCD26, ELISA MBS9303 MyBioSource
www.mybiosource.com/prods/ELISA-
Kit 923 Kit/Human/soluble-cell-adhesion-
molecule-26-
sCD26/sCD26/datasheet.php?products_id=930392
3
ELISA Kit for E0884h EIAab
www.eiaab.com/entries/detail/ELISA%20Kit/DPP
Human 4_HUMAN
Dipeptidyl
peptidase 4
DPP4 (Human) KA3335 Abnova
www.abnova.com/products/products_detail.asp?ca
ELISA Kit talog_id=KA3335
DPP4 (Human) KA0141 Abnova
www.abnova.com/products/products_detail.asp?ca
ELISA Kit talog_id=KA0141
Human EK0696 Boster www.bosterbio.com/human-cd26-dpp4-
picokine-
CD26/DPP4 elisa-kit-ek0696.html
PicoKine TM
ELISA Kit
DPP IV/CD26 K4801- Biovision www.biovision.com/dpp-iv-cd26-human-
elisa-kit-
(human) ELISA 100 7827.html
Kit
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CD26 (DPP4) ThermoFisher
www.thermofisher.com/order/catalog/product/EH
ELISA Kit, EHDPP4 Scientific DPP4
Human
Dipeptidyl 27789A Takara / Clontech
www.clontech.com/US/Products/Cell_Biology_an
Peptidase-4
d_Epigenetics/Metabolic_Diseases/DPP4
Detection¨
DPP4 ELISA
DPPIV/CD26 KA0141 Novus Biologicals www.novusbio.com/DPPIV-CD26-
ELISA-
ELISA Kit Kit_KA0141.html
DPP4/CD26 JP27789 IBL International
www.ibl-international.com/en_us/dpp4-cd26-elisa
ELISA
Human E-EL- ELabscience
www.elabscience.com/index.php/product/view/aid
DPP4/CD26 H0058 /646.jsp
(Dipeptidyl
Peptidase IV)
ELISA Kit
Human CD26/ 29030600 Eton Bioscience
www.etonbio.com/Products/ELISA%20Kit/produc
DPP4 ELISA Kit 15 t.php?sku=290306
CD26 (Human) ELH- Biocat www.biocat.com/products/ELH-CD26-5-
RB
ELISA Kit CD26-5-
RB
[0180] When
the limit of detection (LOD) of an assay is used as predetermined DPP4 or
POSTN threshold level, the predetermined DPP4 or POSTN threshold level will be
the
particular LOD for such assay.
[0181] When
the level of expression of DPP4 or POSTN in control subjects or in subjects
suffering from a certain eosinophilic disease or condition (or subpopulations
thereof) is used
as predetermined DPP4 or POSTN biomarker threshold level, the predetermined
DPP4 or
POSTN threshold level will be level of molecular biomarker reported for each
population
(e.g., healthy controls, patients with moderate asthma, or patients with
severe asthma) in the
manufacturer's instructions for the assay.
[0182] The
term "level" as applied to a biomarker disclosed herein, e.g., DPP4, POSTN,
or to a set of biomarkers disclosed herein refers to a measurement or
measurements made
using any analytical method for detecting presence/absence or expression/lack
of expression
of the biomarker or set of biomarkers (protein expression or gene expression)
in one or more
biological sample and that indicates the presence, absence, absolute amount or
concentration,
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relative amount or concentration, titer, expression level, ratio of measured
levels, or the like,
of, for, or corresponding to the biomarker or biomarkers in the one or
biological samples.
[0183] The
exact nature of the "value" or "level" depends on the specific designs and
components of the particular analytical method (e.g., immunoassays, mass
spectrometry
methods, in vivo molecular imaging, gene expression profiling, aptamer-based
assays, etc.)
employed to detect DPP4, POSTN, or another biomarker disclosed herein. See,
e.g., U.S.
Publ. No. 2010/00221752.
[0184] As
used herein with reference to DPP4, POSTN, and other biomarkers disclosed
herein, the terms "elevated," "increased," or "higher" as applied to a
biomarker level, refer to
a level in a biological sample (e.g., blood serum or sputum) that is higher
than the expression
level or range of the biomarker measured in a control sample ("normal level"),
or a specified
threshold disclosed herein. These thresholds include, e.g., about 363 ng/mL or
about 376
ng/mL for DPP4 serum protein as measured using a DPP4 detection assay,
including the
DPP4 immunoassay described in Example 2 or one of the DPP4 assays disclosed in
TABLE
1. These thresholds also include, e.g., about 23.5 ng/mL or about 25.8 ng/mL
for POSTN
serum protein as measured using a POSTN detection assay, including the POSTN
immunoassay described in W02015120185 or any POSTN assays known in the art.
[0185] As
used herein with reference to DPP4, POSTN, and other biomarkers disclosed
herein, the terms "reduced," "decreased" or "lower" as applied to a biomarker
level, refer to a
level in a biological sample (e.g., blood serum or sputum) that is lower than
the expression
level or range of the biomarker measured in a control sample ("normal level"),
or a specified
threshold disclosed herein. These thresholds include, e.g., about 363 ng/mL or
about 376
ng/mL for DPP4 serum protein as measured using a DPP4 detection assay,
including the
DPP4 immunoassay described in Example 2 or one of the DPP4 immunoassays
disclosed in
TABLE 1. These thresholds include also, e.g., about 23.5 ng/mL or about 25.8
ng/mL for
POSTN serum protein as measured using a POSTN detection assay, including the
POSTN
immunoassay described in W02015120185 or any POSTN assays known in the art.
[0186] The
normal level or range for DPP4, POSTN, and other biomarkers disclosed
herein can be defined in accordance with standard practice. Thus, the level
measured in a
particular biological sample can be compared with level or range of levels
determined in
similar normal samples. In this context, a normal sample or a control sample
would be, for
example, a sample obtained from an individual with no detectable symptoms of
an
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eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such
as asthma). Thus,
the level of, e.g., DPP4 or POSTN, is said to be a low level, lowered level,
reduced level,
decreased level, or grammatical variants thereof when the level of DPP4 or
POSTN is present
in the test sample at a lower level or range than in a normal sample, control
sample, or a
specific threshold level disclosed herein.
[0187]
Conversely, the level of, e.g., DPP4 or POSTN is said to be a high level,
higher
level, increased level, elevated level, or grammatical variants thereof when
the level of DPP4
or POSTN is present in the test sample at a higher level or range than in a
normal sample,
control sample, or a specific threshold level disclosed herein. These
thresholds include, e.g.,
about 363 ng/mL or about 376 ng/mL for blood serum DPP4 protein as measured
using a
DDP4 immunoassay, including the DPP4 immunoassay described in Example 2 or one
of the
DPP4 detection assays disclosed in TABLE 2. These thresholds include also,
e.g., about 23.5
ng/mL or about 25.8 ng/mL for POSTN serum protein as measured using a POSTN
detection
assay, including the POSTN immunoassay described in W02015120185 or any POSTN
assays known in the art.
[0188] In
some aspects, the level of a molecular biomarker disclosed herein (e.g., DPP4
or POSTN) is considered to be elevated or high if it is at least about 5%,
10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
100%,
105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%,
170%, 175%, 180%, 185%, 190%, 195%, 200%, 205%, 210%, 215%, 220%, 225%, 230%,
235%, 240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 295%,
300%, 305%, 310%, 315%, 320%, 325%, 330%, 335%, 340%, 345%, 350%, 355%, 360%,
365%, 370%, 375%, 380%, 385%, 390%, 395%, 400%, 405%, 410%, 415%, 420%, 425%,
420%, 435%, 440%, 445%, 450%, 455%, 460%, 465%, 470%, 475%, 480%, 485%, 490%,
495%, or 500% higher than a normal sample or control sample, or a specific
threshold level
disclosed herein.
[0189] In
some aspects, the level of a molecular biomarker disclosed herein (e.g., DPP4
or POSTN) is considered to be reduced or low if it is at least about 5%, 10%,
15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
100%,
105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%,
170%, 175%, 180%, 185%, 190%, 195%, 200%, 205%, 210%, 215%, 220%, 225%, 230%,
235%, 240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 295%,
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300%, 305%, 310%, 315%, 320%, 325%, 330%, 335%, 340%, 345%, 350%, 355%, 360%,
365%, 370%, 375%, 380%, 385%, 390%, 395%, 400%, 405%, 410%, 415%, 420%, 425%,
420%, 435%, 440%, 445%, 450%, 455%, 460%, 465%, 470%, 475%, 480%, 485%, 490%,
495%, or 500% lower than a normal sample or control sample, or a specific
threshold level
disclosed herein.
[0190] As
used herein, the term "threshold level" (or alternatively herein "threshold
value" or "predetermined threshold level") refers to a level of DPP4, POSTN,
or any other
biomarker disclosed herein which may be of interest for comparative purposes.
In some
aspects, a threshold level may be the expression level of a protein or nucleic
acid expressed
as an average of the level of the expression level of a protein or nucleic
acid from samples
taken from a control population of healthy (disease-free) subjects.
[0191] In
some aspects, the threshold level may be the level in the same subject at a
different time, e.g., before the present assay, such as the level determined
prior to the subject
developing the disease or prior to initiating therapy. In general, samples are
normalized by a
common factor. For example, body fluid samples are normalized by volume body
fluid and
cell-containing samples are normalized by protein content or cell count. In
another aspect, the
threshold level may also refer to the level of expression of the same
biomarker in a
corresponding control sample or control group of subjects which do not respond
to treatment,
e.g., with an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R
antibody such as
benralizumab (MEDI-563).
[0192] In
some aspects, the expression level of DPP4, POSTN, or any other biomarker
disclosed herein is compared to a threshold level (or alternatively herein a
"predetermined
threshold level"). Thus, as used herein, the term "threshold level" or
"predetermined threshold
level" is a cutoff or threshold against which the measured expression level of
a protein or
nucleic acid is compared.
[0193] Based
on comparison to known control samples, a "threshold level" for DPP4 or
any other biomarker disclosed herein can be determined, and test samples that
fall above or
below the biomarker's threshold levels indicate that the patient from whom the
sample was
obtained may or may not benefit from treatment with an eosinophil-targeted
therapeutic
agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).
[0194] For
example, DPP4 or POSTN levels above its threshold level in a sample would
indicate that the patient may not benefit from treatment with an eosinophil-
targeted
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therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-
563).
Conversely, samples with DPP4 or POSTN levels below its threshold level would
indicate
that the patient may benefit from treatment with an eosinophil-targeted
therapeutic agent,
e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563).
[0195] In
some aspects, threshold levels (e.g., protein expression levels or gene
expression levels) for DPP4, POSTN, or any other biomarker disclosed herein
can be
predetermined and matched as to the type of sample (e.g., serum, lung tissue,
sputum), the
type of eosinophilic disease or disorder (e.g., asthma, IPF, COPD), and in
some instances, the
assay used.
[0196] DPP4
levels quantified using the immunoassay described in Example 2, one of the
DPP4 detection immunoassays disclosed in TABLE 1, or a multiplex immunoassay
disclosed
above, from serum samples from a population of mild-to-moderate asthma
patients, wherein
the DPP4 levels are lower than 376 ng/mL indicate that the patients may have
increased
clinical responses to eosinophil-targeted therapeutic agent, e.g., an anti-IL-
5R antibody such
as benralizumab (MEDI-563). Accordingly, in some aspects of the methods
disclosed herein,
the predetermined DPP4 threshold level is about 376 ng/mL of expressed DPP4
protein in
blood serum as measured using the immunoassay described in Example 2.
[0197] POSTN
levels quantified using the immunoassay described in W02015120185, or
any POSTN detection known in the art, or a multiplex immunoassay disclosed
above, from
serum samples from a population of mild-to-moderate asthma patients, wherein
the POSTN
levels are lower than 25.8 ng/mL indicate that the patients may have increased
clinical
responses to eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R
antibody such as
benralizumab (MEDI-563). Accordingly, in some aspects of the methods disclosed
herein,
the predetermined POSTN threshold level is about 25.8 ng/mL of expressed POSTN
protein
in blood serum as measured using the immunoassay described in W02015120185.
[0198] In
some other aspects, the predetermined DPP4 threshold level is based on the
median biomarker level in serum measured from a plurality of patients having
an eosinophilic
disease or disorder (e.g., a pulmonary disease or disorder such as asthma) as
measured for
example according to the DPP4 immunoassay described in Example 2 or any of the
DPP4
immunoassays disclosed in TABLE 1.
[0199]
Accordingly, in some aspects, the predetermined threshold level of DPP4 is
about
the median DPP4 value in serum measured from a plurality of patients having an
eosinophilic
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disease or disorder (e.g., a pulmonary disease or disorder such as asthma) as
measured using
the DPP4 immunoassay described in Example 2 or any of the DPP4 immunoassays
disclosed
in TABLE 1.
[0200] In
some aspects, the predetermined DPP4 threshold level is about 376 ng/mL +/-
25 pg/mL of expressed DPP4 protein in serum as measured using the DPP4
immunoassay
described in Example 2.
[0201] In
some aspects, the DPP4 threshold level can be between about 100 and about
870 ng/mL. Accordingly, the DPP4 threshold level can be about 100, 110, 120,
130, 140,
150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,
300, 310, 320,
330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,
480, 490, 500,
510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,
660, 670, 680,
690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,
840, 850, 860, or
870 ng/mL.
[0202] In
some aspects a "low level of DPP4" is defined as a value below one of these
threshold levels, whereas a "high level of DPP4" is defined as a value equal
to or above the
same threshold level (i.e., if the threshold level was 250 ng/mL, a low level
of DPP4 would
be below 250 ng/mL, and a high level of DPP4 would be 250 ng/mL or above).
[0203] In
some aspects, the predetermined DPP4 threshold level corresponds to the 1",
2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th or 10th decile DPP4 baseline level of
expression of DPP4 in
serum of control patients as measured using the DPP4 immunoassay described in
Example 2.
In some aspects, the predetermined DPP4 threshold level corresponds to the 1",
2nd, 3rd, 4th,
5th, 6th, / -tth,
8th, 9th or 10th decile DPP4 baseline level of expression of DPP4 in serum of
mild-
to-moderate asthmatic patients as measured using the DPP4 immunoassay
described in
Example 2. In some aspects, the predetermined DPP4 threshold level corresponds
to the Pt,
2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th or 10th decile DPP4 baseline level of
expression of DPP4 in
serum of severe asthmatic patients as measured using the DPP4 immunoassay
described in
Example 2.
[0204] In
some aspects a "lower or decreased DPP4 level" is defined as a value below
one of these threshold levels, whereas a "higher or increased DPP4 level" is
defined as a
value equal to or above the same threshold level.
[0205] In
some other aspects, the predetermined POSTN threshold level is based on the
median biomarker level in serum measured from a plurality of patients having
an eosinophilic
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disease or disorder (e.g., a pulmonary disease or disorder such as asthma) as
measured for
example according to the POSTN immunoassay described in W02015120185, or any
POSTN detection known in the art.
[0206]
Accordingly, in some aspects, the predetermined threshold level of POSTN is
about the median POSTN value in serum measured from a plurality of patients
having an
eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such
as asthma) as
measured using the POSTN immunoassay described in W02015120185, or any POSTN
detection known in the art.
[0207] In
some aspects, the predetermined POSTN threshold level is about 26 ng/mL +/-
250 pg/mL of expressed POSTN protein in serum as measured using the POSTN
immunoassay described in W02015120185, or any POSTN detection known in the
art.
[0208] In
some aspects, the POSTN threshold level is between about 7 and 105 ng/mL.
Accordingly, the POSTN threshold level can be about 7, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100 OR 105 ng/mL.
[0209] In
some aspects a "low level of POSTN" is defined as a value below one of these
threshold levels, whereas a "high level of POSTN" is defined as a value equal
to or above the
same threshold level (i.e., if the threshold level was 250 ng/mL, a low level
of POSTN would
be below 250 ng/mL, and a high level of POSTN would be 250 ng/mL or above).
[0210] In
some aspects, the predetermined POSTN threshold level corresponds to the 1st,
2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th or 10th decile POSTN baseline level of
expression of POSTN in
serum of control patients as measured using the POSTN immunoassay described in
W02015120185, or any POSTN detection known in the art. In some aspects, the
predetermined POSTN threshold level corresponds to the 1st, 2nd, 3rd, 4th,
5th, 6th, 7th, 8th, 9th or
10th decile POSTN baseline level of expression of POSTN in serum of mild-to-
moderate
asthmatic patients as measured using the POSTN immunoassay described in
W02015120185. In some aspects, the predetermined POSTN threshold level
corresponds to
the 1st, 211d, 3rd, 4th, 5th, 6th, /-,th,
8th, 9th or 10th decile POSTN baseline level of expression of
POSTN in serum of severe asthmatic patients as measured using the POSTN
immunoassay
described in W02015120185.
[0211] In
some aspects a "lower or decreased POSTN level" is defined as a value below
one of these threshold levels, whereas a "higher or increased POSTN level" is
defined as a
value equal to or above the same threshold level.
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[0212] In
some aspects, the predetermined DPP4 or POSTN threshold level corresponds
to the LOD of the assay used, e.g., for DPP4, the immunoassay described in
Example 2 or the
LOD of any of the DPP4 assays of TABLE 2. In some aspects, a DPP4 or POSTN
detection
assay can be adapted to use as a LOD-based method by diluting a sample so the
new DPP4 or
POSTN threshold level corresponds to the LOD. For example, if the initial
predetermined
DPP4 threshold level in an undiluted sample was 350 ng/mL, and the LOD of the
DPP4
immunoassay was 50 ng/mL, the patient's samples could be diluted 1:7 (ratio
between DPP4
threshold and LOD) with a suitable buffer. After such dilution, only samples
having DPP4
levels above the predetermined DPP4 threshold level in an undiluted sample
would have
DPP4 levels above the LOD of the assay.
[0213] In
some aspects, the threshold level is the average or mean expression level
measured from samples obtained from healthy volunteers as reported in the
manufacturer's
manual of a commercial immunoassay used to detect the presence or absence of
DPP4,
POSTN, or other biomarkers that can be combined with DPP4 and/or POSTN, in a
sample.
[0214] In
some aspects, the expression level of molecular biomarkers disclosed herein
(e.g., DPP4 or POSTN) measured in the sample is above or below the threshold
level or
threshold value for each respective biomarker. In these aspects where the
expression level of
each biomarker disclosed herein (e.g., DPP4 or POSTN) measured in the sample
is above or
below the threshold level or threshold value, the expression level can
indicate that the patient
from whom the sample was taken (e.g., a mild-to-moderate asthmatic patient or
a severe
asthmatic patient) may benefit or not from treatment with an eosinophil-
targeted therapeutic
agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563). The
extent to which
the measured expression level is above or below the threshold level or
threshold value may
be to any extent.
[0215] In
exemplary aspects, the measured expression level of a biomarker disclosed
herein (e.g., DPP4 or POSTN) is at least or about 10% greater or lower than
the threshold
level, e.g., at least or about 15% greater or lower than the threshold level,
at least or about
20% greater or lower than the threshold level, at least or about 25% greater
or lower than the
threshold level, at least or about 30% greater or lower than the threshold
level, at least or
about 35% greater or lower than the threshold level, at least or about 40%
greater or lower
than the threshold level, at least or about 45% greater or lower than the
threshold level, at
least or about 50% greater or lower than the threshold level, at least or
about 55% greater or
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lower than the threshold level, at least or about 60% greater or lower than
the threshold level,
at least or about 65% greater or lower than the threshold level, at least or
about 70% greater
or lower than the threshold level, at least or about 75% greater or lower than
the threshold
level, at least or about 80% greater or lower than the threshold level, at
least or about 85%
greater or lower than the threshold level, at least or about 90% greater or
lower than the
threshold level, at least or about 95% greater or lower than the threshold
level.
[0216] In
exemplary aspects, the measured expression level of a biomarker disclosed
herein (e.g., DPP4 or POSTN) is at least 2-fold greater or lower than the
threshold level, at
least 3-fold greater or lower than the threshold level, at least 4-fold
greater or lower than the
threshold level, at least 5-fold greater or lower than the threshold level, at
least 6-fold greater
or lower than the threshold level, at least 7-fold greater or lower than the
threshold level, at
least 8-fold greater or lower than the threshold level, at least 9-fold
greater or lower than the
threshold level, or at least 10-fold greater or lower than the threshold
level.
[0217] As
discussed above, the level of a biomarker disclosed herein (e.g., DPP4 or
POSTN) can be determined using methods known in the art. A person skilled in
the art would
appreciate that in addition to the assays disclosed above, there are numerous
methods
available in the art that would allow the skilled artisan to determine
threshold levels as
described throughout this section, including, e.g., the DPP4 detection methods
and
immunoassays described in Example 2 and TABLE 1.
[0218] In
some aspects, the predetermined threshold level of a biomarker disclosed
herein
(e.g., DPP4 or POSTN) is defined with respect to a certain percentile value in
a population of
subjects (e.g., a plurality of normal healthy volunteers, or a plurality of
patients with an
eosinophilic disease or disorder, e.g., a pulmonary disease or disorder such
as asthma). In
some aspects, the plurality of patients with a pulmonary disease or disorder
is a plurality of
patients with mild-to-moderate asthma, or a plurality of patients with severe
asthma.
[0219] In
some aspects, the predetermined threshold level for DPP4 or POSTN
corresponds to the 10th, 15th, 20th, 25th, 30th, 35th, 40th,
45th, 50th, 50th, 55th, 60th, 65th, 70th, 75th,
80th, 85th, or 90th percentile in a DPP4 or POSTN protein expression
distribution in a plurality
of normal healthy volunteers, or a plurality of patients with an eosinophilic
disease or
disorder, e.g., a pulmonary disease or disorder such as asthma. In some
aspects, the plurality
of patients with a pulmonary disease or disorder is a plurality of patients
with mild-to-
moderate asthma, or a plurality of patients with severe asthma.
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[0220] The
threshold level (e.g., a protein expression level or a gene expression level)
of
a biomarker disclosed herein (e.g., DPP4 or POSTN) can vary based on the
nature of the
assay, e.g., the capture and detection antibodies used, the source, purity,
and composition of
the standard, and the like. In one aspect, instead of using an arbitrary
threshold level to
determine whether a patient can benefit from treatment with eosinophil-
targeted therapeutic
agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563), the
patient's DPP4
and/or POSTN levels can be compared to one or more control levels. According
to this
aspect, the test sample (e.g., a sample from a patient suffering from a
pulmonary disease or
disorder such as asthma) is compared to one or more control samples, e.g.,
samples taken
from normal healthy individuals, earlier samples taken from the same patient,
samples taken
from patients with a subset of the patient's disease (e.g., asthma or COPD), a
pre-determined
standard amount of isolated biomarker protein or gene or a fragment thereof,
or a
combination thereof.
[0221] The
results can be expressed as a ratio with the control samples to determine a
percent increase or a percent decrease in the patient's biomarker levels
(e.g., a protein
expression level or a gene expression level) compared to the control biomarker
levels. The
control sample can be a matched pair with the patient sample, e.g., one or
more of whole
blood if the patient sample is whole blood, serum if the patient sample is
serum, plasma if the
patient sample is plasma, saliva if the patient sample is saliva, urine if the
patient sample is
urine, sputum if the patient sample is sputum, bronchoalveolar lavage fluid if
the patient
sample is bronchoalveolar lavage fluid, lung tissue if the patient sample is
lung tissue. Once
determined, a biomarker expression level can be recorded in a patient's
medical record.
[0222] In
some aspects, as disclosed above, DPP4 and/or POSTN levels can be combined
with other biomarkers such as eosinophil levels. Accordingly, in some aspects,
a high level of
eosinophils (e.g., peripheral blood eosinophils) of at least about 150
cells/pL, at least about
300 cells/pL or at least about 400 cells/pL combined with DPP4 and/or POSTN
levels below
the threshold levels disclosed above, are predictive of positive clinical
response to an
eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as
benralizumab
(MEDI-563) in a patient having an eosinophilic disease or disorder (e.g., a
pulmonary disease
or disorder such as asthma).
[0223] In
some aspect, a blood analyte to use as biomarker, e.g., eosinophil level, can
be
determined from a complete blood cell count (CBC) with differential. The term
"CBC with
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differential" as used herein refers to complete blood cell count (CBC) with
white blood cell
(WBC) differentials. The term "white blood cell" includes, e.g., neutrophils,
lymphocytes,
monocytes, eosinophils, and basophils. The terms "eosinophil" and
"eosinophils" can be
abbreviated as "EOS". The term "white blood cell count" as used herein refers
to a count of
white blood cells from any sample, for example, a complete blood count (CBC)
with white
blood cell (WBC) differentials (CBC with differential). Obtaining a CBC with
differential
can be achieved using any suitable techniques available in the art, e.g., by
automated
hematology analyzer or hematology coulter counters (e.g., flow cytometry) or
by manually
counting cells (e.g., using a microscope). A CBC with differential is one of
the most widely
ordered clinical laboratory tests in the world.
[0224] In
some aspects, samples can analyzed using automated hematology analyzers, for
example, SIEMENS ADVIA 120; ABBOTT CELL DYN 3500; BECKMAN COULTER LH750;
SYSMEX X SERIES; HORIBA ABX, etc. In some aspects, to maximize the accuracy of
the
disclosed methods, readouts from automated hematology analyzers report
absolute eosinophil
counts to at least 2 digits (e.g., 150, 220, 340 cells/uL), and at least 3
digits for lymphocytes
and neutrophils (e.g., 1,530, 2,340, 3,410 cells/uL). In some aspects, prior
to analyzing the
blood sample, the tube should be inverted several times, e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10 or more
than 10 times, or according to manufacturer's instructions. In some aspects,
samples can be
analyzed manually.
[0225] In
some aspects, the level of eosinophils can be determined by flow cytometry. In
some aspects, the methods of the disclosure can comprise WBC, eosinophil
count, neutrophil
count, lymphocyte count, eosinophil precursor count, basophil precursor count,
Eotaxin-2
level and any combination or ratio thereof. In a further aspect, a method or
system of the
disclosure can comprise the blood eosinophil/WBC ratio, the blood
eosinophil/blood
lymphocyte ratio and the log of the blood eosinophil/blood neutrophil ratio.
[0226] A
method or system of the disclosure can comprise any one or any combination of
the following non-limiting examples of physiological biomarkers: AFEV1 post-
albuterol,
AFEN1 post-tiotropium bromide, FEV1, FEV/FVC, AAM/PM 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),
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previous sinus surgery, history of atopy, history of intubation, history of
aspirin sensitivity,
history of corticosteroid bursts during past 3 or 12 months, or history of ER
visits during past
3 years. A method or system of the disclosure can also comprise any one or any
combination
of the following parameters: gender, age, weight, race, height, or body mass
index (BMI).
[0227] In
some aspects, method or system of the disclosure can comprise a value
corresponding to the average of several measurements from multiple samples
collected at
different time intervals. Thus, in some aspects, multiple samples can be
collected at different
intervals, e.g., about 1 day, about 2 days, about 3 days, about 4 days, about
5 days, about 6
days, or about 7 days apart. In some aspects, the multiple samples can be
collected about 1
week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6
weeks, about 7
weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about
12 weeks
apart. In some aspects, multiple samples can be collected about 1 month, about
2 months,
about 3 months, about 4 months, about 5 months, about 6 months, about 7
months, about 8
months, about 9 months, about 10 months, about 11 months, or about 12 months
apart. In
some aspects, multiple samples can be collected more than 12 months apart. In
some cases,
more than 2 samples are averaged, for examples, 3 samples, 4 samples, 5
samples, 6 samples,
7 samples, 8 samples, 9 samples, 10 samples, or more than same samples. In
some aspects,
samples are collected are regular intervals. In other aspects, samples are not
collected at
regular intervals. In some cases, samples are collected in response to an
event, for example,
exacerbation of symptoms
[0228] In
some aspects, administration of an eosinophil-targeted therapeutic agent,
e.g.,
an anti-IL-5R antibody such as benralizumab (MEDI-563) according to the
methods
disclosed herein results in
(a) AER (Acute Exacerbation Rate) reduction;
(b) FEV1 (Forced Expiratory Volume in one second) increase;
(c) improved ACQ-6 (Asthma Control Questionnaire, 6-item version) results;
(d) improved AQLQ (Asthma Quality of Life Questionnaire) results;
(e) reduction of cytokines secreted by type 2 innate lymphocytes (ILC2);
(f) reduction of ILC2 levels,
(g) reduction of eosinophil and/or basophil levels, or,
(h) a combination thereof.
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[0229] In
some aspects, administration of an eosinophil-targeted therapeutic agent,
e.g.,
an anti-IL-5R antibody such as benralizumab (MEDI-563) according to the
methods
disclosed herein results in improvement in other methods of detecting health-
related quality
of life known in the art.
IV. Eosinophil-Targeted Therapeutic Agents
[0230] DPP4
and/or POSTN, combined with other clinical or molecular biomarkers
disclosed herein (e.g., blood eosinophil levels), and/or in combination with
other clinical or
molecular biomarkers known in the art can be used, for example, to determine
whether to
treat, select for treatment, monitor the treatment, or a begin, modify, or
cease the treatment of
a patient suffering from an eosinophilic disease or disorder (e.g., a
pulmonary disease or
disorder such as asthma) with an eosinophil-targeted therapeutic agent, e.g.,
an anti-IL-5R
antibody such as benralizumab (MEDI-563).
[0231] The
term "eosinophil-targeted therapeutic agent" as used herein refers a
therapeutic agent capable of decreasing eosinophil count in a patient in need
thereof. In some
aspects, the eosinophil-targeted therapeutic agent can also deplete basophil
count. In certain
aspects, the eosinophil-targeted therapeutic agent is
(i) a monoclonal antibody, or
(ii) an antigen-binding fragment thereof, or
(iii)a compound comprising an monoclonal antibody or an antigen-binding
thereof
comprising one or more additional therapeutic moieties, or
(iv) a combination thereof,
capable of decreasing eosinophil and/or basophil count in a patient in need
thereof.
[0232] In
other aspects, the eosinophil-targeted therapeutic agent can be a small
molecule. In some aspects, the eosinophil-targeted therapeutic agent is a
combination of
eosinophil-targeted therapeutic agents (e.g., a combination therapy comprising
at least one
antibody or fragment thereof and at least one small molecule, or at least an
antibody or
fragment thereof and at least one nucleic acid, or at least one nucleic acid
and at least one
small molecule, etc.).
[0233] In
another aspect, the therapeutic agent is a biologic. In certain aspects, the
biological is any substance made by a living organism or its products, a
substance made using
recombinant DNA technology, a nucleotide, a nucleotide analogue, an
oligonucleotide, an
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oligonucleotide analogue, a peptide, or a peptide analogue produced by any
means. In
specific aspects, a biologic can be an antibody or antibody fragment, an
antibody mimetic, a
soluble receptor polypeptide, a soluble receptor fusion polypeptide,
interleukin, interleukin
fusion polypeptide, antisense molecule, siRNA or miRNA.
[0234] In
some aspects, an eosinophil-targeted therapeutic agent of the present
disclosure
(e.g., an antibody specifically binding to IL-5R or IL-5) competitively
inhibits binding of a
reference molecule (e.g., a different antibody specifically binding to IL-5R
or IL-5) to a given
target site if it preferentially binds to that target site to the extent that
it blocks, to some
degree, binding of the reference molecule to the target site. Competitive
inhibition can be
determined by any method known in the art, for example, competition ELISA
assays.
[0235] An
eosinophil-targeted therapeutic agent of the present disclosure (e.g., an
antibody specifically binding to IL-5R or IL-5) can be said to competitively
inhibit binding of
the reference molecule to a given epitope by at least about 90%, at least
about 85%, at least
about 80%, at least about 75%, at least about 70%, at least about 65%, at
least about 60%, at
least about 55%, or at least 50%.
[0236] In
some aspects, the eosinophil-targeted therapeutic agent is a therapeutic agent
capable of
(i) inhibiting or decreasing levels of IL-5 or IL-5R,
(ii) inhibiting of decreasing the expression of a molecule interacting with IL-
5 or IL-5R,
(iii) increasing the expression of a molecule interacting with IL-5 or IL-5R,
(iv) inhibiting or decreasing the expression of a molecule upregulated by IL-5
or IL-5R,
(v) inhibiting or inhibiting the expression of a molecule interacting with a
molecule
upregulated by IL-5 or IL-5R, or
(vi) a combination thereof.
[0237] In
some aspects, an eosinophil-targeted therapeutic agent is a compound included
in TABLE 2.
TABLE 2: Potential eosinophil-targeted therapeutic agents.
Target Therapeutic agent (AB=antibody; NU=polynucleotide)
IL-5 Mepolizumab (AB), reslizumab (AB)
IL-5R, a subunit Benralizumab (AB)
IL-5R, 13 subunit gene ASM8 (NU), PXS 1100 (NU), PXS 2200 (NU)
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[0238]
Eosinophil-targeted therapeutic agent comprising the antibodies disclosed
above
refer not only to the intact immunoglobulins, but also to antigen-binding
fragments, variant,
or derivatives thereof, antibodies or fragments thereof that bind to the same
epitopes the
antibodies disclosed above, or an antibodies or fragments thereof that
competitively inhibit
binding of the antibodies disclosed above to their respective targets.
Antibodies (or fragments
thereof) that are identical or similar in amino acid sequence to the
antibodies disclosed in
TABLE 2, particularly in the variable regions, or in the CDRs thereof
(however, variations in
the constant regions are also contemplated) are contemplated. For example, in
one aspect, an
eosinophil-targeted therapeutic agent is a polypeptide having an amino acid
sequence that is
about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%,
about
98%, about 99% or 100% identical to that of the polypeptide sequence of an
antibody
disclosed in TABLE 2 (i.e., the heavy chain and/or the light of the antibody).
[0239] In
some aspects, the eosinophil-targeted therapeutic agent is an isolated antigen
binding protein targeting any of the antigens disclosed above, comprising at
least one, two,
three, four, or the six complementarity determining regions of the antibodies
disclosed above.
In various aspects, the eosinophil-targeted therapeutic agents disclosed
herein comprise
antigen binding fragments of antibodies, such as fragments of any of the
antagonist
antibodies referred to herein, e.g., antibodies specifically binding to IL-5
(SEQ ID NO: 9) or
IL-5R, for example, to the alpha subunit of IL-5R (SEQ ID NOS: 4-8,
corresponding to 5
isoforms of the subunit generated by alternative splicing). Such fragments
include, but are not
limited to Fab, Fab', Fab'-SH, Fv, scFv, F(ab1)2, nanobodies, and diabodies.
[0240] In
some aspects, the eosinophil-targeted therapeutic agent is a therapeutic agent
capable of specifically binding to IL-5R, e.g., an anti-IL-5R antibody such as
benralizumab
or an antigen binding fragment thereof, or an anti-IL-5R molecule comprising
benralizumab
or an antigen binding fragment thereof. In some aspects, the eosinophil-
targeted therapeutic
agent is a therapeutic agent capable of specifically binding to IL-5, e.g, an
anti-IL-5 antibody
such as reslizumab, mepolizumab, or an antigen binding fragment thereof, or an
anti-IL-5
molecule comprising reslizumab, mepolizumab, or an antigen binding fragment
thereof. In
some aspects, the anti-IL-5R molecule or anti-IL-5 molecule is an antibody-
drug conjugate
(ADC).
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[0241] In
one aspect, the eosinophil-targeted therapeutic agent can be an anti-cytokine
antibody or an anti-cytokine receptor antibody binding to a cytokine and its
receptors other
that IL-5 or IL-5R. In one aspect, the eosinophil-targeted therapeutic agent
can be an anti-IL-
or anti-IL5R antibody capable of preventing the signaling of IL-5 through the
IL-5 receptor.
[0242] In
one aspect, the anti-IL5R antibody specifically binds to the alpha subunit of
IL-
5R (SEQ ID NO: 4). Non-limiting examples of anti-human IL-5 antibodies are
reslizumab
and mepolizumab. Non-limiting examples of anti-human IL-5 receptor alpha
antibodies of
the disclosure can be found in U.S. Patent Nos. 7,179,464, 6,538,111,
6,018,032, and U.S.
Patent Application Publication Nos. 2004/0136996A1, 2005/0226867A1, or Intl
Publ. No.
WO 2008/143878. In one aspect, the eosinophil-targeted therapeutic agent can
be an
antibody directed against IL-5, e.g., reslizumab, mepolizumab, and any
combination thereof.
[0243]
Without being bound by a particular theory, the eosinophil-targeted
therapeutic
agent used according to the methods and systems described herein can be an
anti-IL-5R
antibody capable to mediate the in vivo depletion of eosinophils. In one
aspect, the in vivo
depletion can be mediated by ADCC, CDC or antibody mediated phagocytosis. In a
specific
aspect, the therapeutic agent can be an anti-IL-5R antibody having ADCC
activity.
[0244] In
another specific aspect, the therapeutic agent can be an anti-IL-5R antibody
having increased ADCC activity. A non-limiting example for an anti-IL-5R
antibody with
increased ADCC activity is benralizumab (also referred to herein as "MEDI-563"
as
described in Intl Publ. No. WO 2008/143878). Benralizumab is an immunoglobulin
G1
antibody comprising humanized mouse monoclonal MEDI-523 y 1 heavy chain (224-
214')-
disulfide with humanized mouse monoclonal MEDI-523 lc light chain, dimer (230-
230":233-
233")-bisdisulfide. See U.S. Pat. No. 8501176, U.S. Publ. No. U520150044204,
and Intl
Publ. No. W02015023504, all of which are herein incorporated by reference in
their
entireties.
[0245] In
another specific aspect, the therapeutic agent can be an anti-IL-5R antibody
that
binds the same epitope as benralizumab or competes with benralizumab for
binding to IL-5R.
The benralizumab epitope is described in Intl Publ. WO 2008/143878, the
disclosure of
which is hereby incorporated by reference in its entirety. In one aspect, the
anti-IL-5R
antibody or an antigen-binding fragment comprises a heavy chain variable
region (VH)
comprising, consisting, or consisting essentially of SEQ ID NO:12. In one
aspect, the anti-
IL-5R antibody or an antigen-binding fragment comprises a light chain variable
region (VL)
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comprising, consisting, or consisting essentially of SEQ ID NO:13. In some
aspects, the anti-
IL-5R antibody or an antigen-binding fragment comprises at least one, two,
three, four, five
or six complementarity determining regions selected from SEQ ID NOS: 14-19.
[0246] In
some aspects, the eosinophil-targeted therapeutic agent is a fusion protein or
a
conjugate comprising one of the monoclonal antibodies of TABLE 2 or an antigen-
binding
fragment thereof. In some aspects, the eosinophil-targeted therapeutic agent
is a fusion
protein or a conjugate comprising one of the monoclonal antibodies of any one
of claims 12-
18 or an antigen-binding fragment thereof. In some aspects, the fusion protein
or conjugate
comprises at least one heterologous therapeutic moiety and/or a half-life
enhancing moiety.
In some aspects, the term eosinophil-targeted therapeutic agent also
encompasses, for
example, antagonists of IL-5 or IL-5R such as aptamers, peptides, mRNAs, iRNA,
shRNAs,
or small molecule inhibitors.
[0247] In
some aspects, the eosinophil-targeted therapeutic agent is a polynucleotide,
e.g., a DNA or an RNA. In some aspects, the polynucleotide is (i) an mRNA or a
combination thereof, or (ii) an antisense oligonucleotide or a combination
thereof. In some
aspects, the polynucleotide comprises at least a nucleotide analog. In some
aspects, the
antisense oligonucleotide or combination thereof is an oligonucleotide
targeting the gene
encoding the beta subunit of IL-5R, e.g., ASM8, PXS1100, or PX52200. See,
e.g., Intl Publ.
WO 2006045202, which is herein incorporate by reference in its entirety. ASM8
is a
combination of 2 oligonucleotides (T0P004 and T0P005). TOP004 is a 19-mer
directed
against the mRNA of the common 13 subunit of the IL-3, IL-5, and GM-CSF
receptors.
[0248] In
one aspect, the eosinophil-targeted therapeutic agent can be an antibody
directed against IL-13/IL-4a. In a specific embodiment, the therapeutic agent
can be
AerovantTM (Aerovance), GS K-679586 (GSK), IMA-026 (Wyeth), or MILR1444A
(Genentech).
[0249] In a
further aspect, the eosinophil-targeted therapeutic agent is an antibody
directed against the IL-2 receptor. In a specific embodiment, the eosinophil-
targeted agent
can be daclizumab (ZENAPAx0). Daclizumab is a therapeutic humanized monoclonal
antibody to the alpha subunit of the IL-2 receptor of T cells. In another
aspect, the
eosinophil-targeted therapeutic agent can be an anti-IgE antibody. In a
specific embodiment,
the eosinophil-targeted therapeutic agent can be omalizumab (X0EAIRO).
Omalizumab is a
recombinant DNA-derived humanized IgGlk monoclonal antibody that selectively
binds to
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human immunoglobulin E (IgE). Omalizumab is FDA-approved to treat moderate to
severe
allergic asthmatics. It has not been specifically approved for treatment of
eosinophilic asthma
though some studies have demonstrated it decreases airway eosinophil numbers.
In another
aspect, the eosinophil-targeted therapeutic agent can be a recombinantly-
produced cytokine.
In a specific embodiment, the eosinophil-targeted therapeutic agent can be
interferon-alpha.
Non-limiting examples of interferon-alpha therapeutics include PEGASYSO
(PEGinterferon
alfa-2a) and ALBuFER0N0/ 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-113, IL-6, or
TGF-13.
[0250] In
certain aspects, the half-lives of antibodies used according to the methods
and
systems of the disclosure can be prolonged by methods known in the art. Such
prolongation
can in turn reduce the amount and/or frequency of dosing of the antibody
compositions.
Antibodies with improved in vivo half-lives and methods for preparing them are
disclosed in
U.S. Patent No. 6,277,375; and International Publication Nos. WO 98/23289 and
WO
97/3461.
V. DPP4 and POSTN Levels for Diagnosis and Treatment of Eosinophilic Diseases
[0251] DPP4
and POSTN are differentially expressed in subjects having an eosinophilic
disease or disorder, e.g., a pulmonary disease or disorder such as asthma.
DPP4 and POSTN
are present at different levels in samples from patients having mild-to-
moderate asthma with
respect to healthy controls, or in patients having severe asthma with respect
to healthy
controls.
[0252] The
differences in DPP4 and POSTN levels observed, e.g., in healthy individuals,
patients with mild-to-moderate asthma, and patients with severe asthma, can be
applied to
predicting clinical outcomes when the patients are treated with a certain
therapy, for example,
an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as
benralizumab
(MEDI-563). Thus, if a subject's DPP4 and/or POSTN levels are above a certain
threshold,
that subject would become a candidate for treatment with a certain therapy,
e.g., therapy with
an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as
benralizumab
(MEDI-563).
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[0253] In
some aspects, the mere determination that the DPP4 and/or POSTN is
expressed below a predetermined threshold level or below a detectable levels
(e.g., using a
diluted sample) would suffice to identify a subject as a candidate for
treatment with a certain
therapy, e.g., therapy with an eosinophil-targeted therapeutic agent, e.g., an
anti-IL-5R
antibody such as benralizumab (MEDI-563).
[0254] In
some aspects of the methods disclosed herein, the patient is a moderate
asthmatic or mild-to-moderate asthmatic (GINA 1-3). In other aspects of the
methods
disclosed herein, the patient is a severe asthmatic (GINA 4+). In other
aspects, the patients
suffers from COPD.
[0255] In
some aspects of the methods disclosed herein, DPP4 levels or POSTN levels
can be used alone. In other aspects, the DPP4 and/or POSTN levels can be
combined with
other molecular or clinical biomarkers, such as blood eosinophil count. In
other aspects,
additional biomarkers, e.g., other biomarkers indicative of inflammation or
clinical
biomarkers (e.g., age, smoker status, body mass index, etc) can be combined
with DPP4
and/or POSTN.
[0256] This
finding can be applied, for example, to devise new methods of determining
treatment (e.g., by selecting patients as candidates for a certain therapy),
methods of treating
an eosinophilic disease or disorder (e.g., a pulmonary disease or disorder
such as mild-to-
moderate asthma, or severe asthma), methods of monitoring efficacy of
therapeutic agents
(e.g., benralizumab/MEDI-563) to treat eosinophilic diseases or disorders, or
methods to
adjust formulations, dosage regimens, or routes of administration.
[0257] The
methods disclosed herein include prescribing, initiating, and/or altering
prophylaxis and/or treatment, e.g., for an eosinophilic disease or disorder
(e.g., a pulmonary
disease or disorder such as mild-to-moderate asthma or severe asthma), based
at least in part
on a subject's expression level of DPP4 and/or POSTN, alone or in combination
with one or
more additional biomarkers (e.g., eosinophil count).
[0258] The
present disclosure provides a method of determining whether to treat a patient
having an eosinophilic disease or disorder (e.g., a pulmonary disease or
disorder such as
mild-to-moderate asthma or severe asthma) with a therapeutic regimen
comprising the
administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-
5R antibody such
as benralizumab (MEDI-563) wherein the method comprises: (a) measuring or
instructing a
clinical laboratory to measure DPP4 and/or POSTN levels, and optionally levels
of additional
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biomarkers such as blood eosinophils in a sample taken from the patient, and
(b) treating or
instructing a healthcare provider to treat the patient, or suspending the
treatment, not
initiating the treatment, denying the treatment, or instructing a healthcare
provider to
suspend, not initiate, or deny the treatment with a therapeutic regimen
comprising the
administration of an eosinophil-targeted therapeutic agent if the patient is
determined to have
a higher or lower DPP4 and/or POSTN levels, and optionally levels of
additional biomarkers
such blood eosinophils in the sample compared to each biomarker predetermined
threshold
level or levels, or compared to each biomarker level or levels in one or more
control samples.
[0259] In
one aspect, the disclosure provides a method of determining whether to treat a
patient having an eosinophilic disease or disorder (e.g., a pulmonary disease
or disorder such
as mild-to-moderate asthma or severe asthma) with a therapeutic regimen
comprising the
administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-
5R antibody such
as benralizumab (MEDI-563) wherein the method comprises: (a) measuring or
instructing a
clinical laboratory to measure DPP4 and/or POSTN levels and optionally levels
of additional
biomarkers such as blood eosinophils in a sample taken from the patient, and
(b) treating or
instructing a healthcare provider to treat the patient with a therapeutic
regimen comprising
the administration of an eosinophil-targeted therapeutic agent if the patient
is determined to
have lower or decreased DPP4 and/or POSTN levels, and higher or increased
levels of at
least one optional additional biomarker such as eosinophil count in the sample
compared to a
predetermined biomarker threshold level or levels, or compared to a biomarker
level or levels
in one or more control samples.
[0260] In
one aspect, the disclosure provides a method of determining whether to treat a
patient having an eosinophilic disease or disorder (e.g., a pulmonary disease
or disorder such
as mild-to-moderate asthma or severe asthma) with a therapeutic regimen
comprising the
administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-
5R antibody such
as benralizumab (MEDI-563) wherein the method comprises (a) measuring or
instructing a
clinical laboratory to measure the DPP4 and/or POSTN levels and optionally
levels of
additional biomarkers such blood eosinophils in a sample taken from the
patient, and (b)
suspending the treatment, not initiating treatment, denying the treatment, or
instructing a
healthcare provider to suspend, not initiate, or deny the treatment of the
patient with a
therapeutic regimen comprising the administration of an eosinophil-targeted
therapeutic agent
to the patient if the patient is determined to have higher or increased DPP4
and/or POSTN
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levels, and lower or decreased levels of at least one optional additional
biomarker such as
blood eosinophils in the sample compared to a predetermined biomarker
threshold level or
levels, or compared to a biomarker level or levels in one or more control
samples.
[0261] Also
provided is a method of selecting a patient diagnosed with an eosinophilic
disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-
moderate asthma or
severe asthma) as a candidate for treatment with an eosinophil-targeted
therapeutic agent,
e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563), comprising (a)
measuring or
instructing a clinical laboratory to measure the DPP4 and/or POSTN levels and
optionally
levels of additional biomarkers such as blood eosinophils in a sample taken
from the patient,
and (b) treating or instructing a healthcare provider to treat the patient
with an eosinophil-
targeted therapeutic agent if the patient is determined to have lower or
decreased DPP4
and/or POSTN levels, and higher or increased levels of at least one optional
additional
biomarker such as blood eosinophils in the sample compared to a predetermined
threshold
level or levels, or compared to a biomarker level or levels in one or more
control samples.
[0262] Also
provided is method of selecting a patient diagnosed with an eosinophilic
disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-
moderate asthma or
severe asthma) as a candidate for treatment with an eosinophil-targeted
therapeutic agent,
e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563), comprising (a)
measuring or
instructing a clinical laboratory to measure the DPP4 and/or POSTN levels and
optionally
levels of additional biomarkers such as blood eosinophils in a sample taken
from the patient,
and (b) suspending the treatment, not initiating treatment, denying the
treatment, or
instructing a healthcare provider to suspend, not initiate, or deny the
treatment of the patient
with an eosinophil-targeted therapeutic agent to the patient if the patient is
determined to
have higher or increased DPP4 and/or POSTN levels, and lower or decreased
levels of at
least one optional additional biomarker such as blood eosinophils in the
sample compared to
a predetermined threshold level or levels, or compared to a biomarker level or
levels in one or
more control samples.
[0263] In
some aspects, the methods disclosed can entail ordering and/or performing one
or more additional assays. For example, if the levels of DPP4 and/or POSTN
(e.g., a protein
expression level or a gene expression level) are determined to be within a
normal range (i.e.,
not elevated), the DPP4 or POSTN detection assay may be repeated to rule out a
false
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negative result, and/or one or more additional DPP4 or POSTN detection assays
may be
performed to monitor the subject's status.
[0264]
Conversely, if the DPP4 and/or POSTN levels (e.g., protein expression levels
or
gene expression levels) are determined to be elevated, it may be desirable
repeat the DPP4 or
POSTN detection assay to rule out a false positive result.
[0265] In
some aspects, the predetermined DPP4 threshold level is at least about 363
ng/mL (median value in mild-to-moderate asthmatics), or at least about 376
ng/mL (mean
value in mild-to-moderate asthmatics) as measured in serum using a DPP4
detection assay
including, e.g., the immunoassay described in Example 2. Accordingly, DPP4
expression
levels equal or above those values are consider high or elevated, and DPP4
expression levels
below those values are considered low or reduced.
[0266] In
some aspects, the predetermined POSTN threshold level is at least about 23.5
ng/mL (median value in mild-to-moderate asthmatics), or at least about 25.8
ng/mL (mean
value in mild-to-moderate asthmatics) as measured in serum using a POSTN
detection assay
including, e.g., the immunoassay described in W02015120185. Accordingly, POSTN
expression levels equal or above those values are consider high or elevated,
and POSTN
expression levels below those values are considered low or reduced.
[0267] In
some aspects, the predetermined blood eosinophil level is 150 cells/pL. In
some
aspects, the predetermined blood eosinophil level is 300 cells/pL. In other
aspects, the blood
eosinophil threshold level is 400 cells/ L. Accordingly, blood eosinophil
levels equal or
above those values are consider high or elevated, and blood eosinophil levels
below those
values are considered low or reduced.
[0268] In
some aspects, the presence of DPP4 and/or POSTN levels above or below a
predetermined threshold level in a patient with an eosinophilic disease or
disorder (e.g., a
pulmonary disease or disorder such as mild-to-moderate asthma or severe
asthma) can be
used in combination with one or more of clinical or molecular biomarkers
specific for such
disease.
[0269] For
example, for patients with asthma, the measurement of DPP4 and/or POSTN
levels can be combined with measurements of other biomarker levels (e.g.,
blood
eosinophils). Accordingly, in one aspect, levels of DPP4 and/or POSTN can be
combined
with, e.g., blood eosinophils in any of the methods disclosed herein
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(i) to determine whether a patient suffering an pulmonary disease (e.g.,
asthma or COPD) is
eligible or non-eligible for a specific treatment or will respond to a
specific treatment with an
eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as
benralizumab
(MEDI-563),
(ii) to determine whether a specific treatment (e.g., with an eosinophil-
targeted therapeutic
agent) should commence, be suspended, or be modified,
(iii) to diagnose whether the disease (e.g., asthma or COPD) is treatable or
not treatable with
a specific therapeutic agent, or
(iv) to prognosticate or predict the outcome of treatment of the disease
(e.g., asthma or
COPD) with a specific therapeutic agent, etc.
[0270] A
person skilled in the art would understand that DPP4 and/or POSTN levels
(e.g., protein expression levels or gene expression levels) can be used
according to the
methods disclosed herein, including but not limited to treatment, diagnostic,
and monitoring
methods, as positive selectors, i.e., a specific action would be taken (e.g.,
treating a patient
having an eosinophilic disease or disorder such as asthma with an eosinophil-
targeted
therapeutic agent) if the DPP4 and/or POSTN levels (e.g., protein expression
levels or gene
expression levels) in a sample taken from the patient are below predetermined
DPP4 or
POSTN threshold levels, or are decreased relative to the DPP4 or POSTN levels
in one or
more control samples.
[0271] A
person skilled in the art would also understand that DPP4 and/or POSTN levels
(e.g., protein expression levels or gene expression levels) can be used
according to the
methods disclosed herein, including but not limited to treatment, diagnostic,
and monitoring
methods, as negative selectors, i.e., a specific action would not be taken
(e.g., treating a
patient having a pulmonary disease such as asthma with an eosinophil-targeted
therapeutic
agent) if the DPP4 and/or POSTN levels (e.g., protein expression levels or
gene expression
levels) in a sample taken from the patient are above predetermined DPP4 or
POSTN
threshold level, or are increased relative to the DPP4 or POSTN levels in one
or more control
samples.
[0272] In
one aspect, the disclosure includes methods, assays, and kits to facilitate a
determination by a healthcare provider, a healthcare benefits provider, or a
clinical laboratory
to as to whether a patient will benefit from treatment with an eosinophil-
targeted therapeutic
agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563). The
methods assays
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and kits provided herein also facilitate a determination by a healthcare
provider, a healthcare
benefits provider, or a clinical laboratory to as to whether a patient will
benefit from
treatment with any other eosinophil-targeted therapeutic agent known to those
of ordinary
skill in the art.
[0273] In
one aspect, the methods disclosed herein include making a diagnosis, which
may be a differential diagnosis, based at least in part on the level of DPP4
and/or POSTN of a
patient. In some aspects, the methods disclosed herein include informing the
subject of a
result of the DPP4 and/or POSTN detection assay and/or of a diagnosis based at
least in part
on the DPP4 and/or POSTN levels. The patient can be informed verbally, in
writing, and/or
electronically.
[0274] This
diagnosis can also be recorded in a patient medical record. For example, in
various aspects, the diagnosis of a pulmonary disease (e.g., asthma) treatable
with a specific
eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as
benralizumab
(MEDI-563) is recorded in a medical record.
[0275] The
term "medical record" or "patient medical record" refers to an account of a
patient's examination and/or treatment that typically includes one or more of
the following:
the patient's medical history and complaints, the physician's physical
findings, the results of
diagnostic tests and procedures, and patient medications and therapeutic
procedures. A
medical record is typically made by one or more physicians and/or physicians
assistants and
it is a written, transcribed or otherwise recorded record and/or history of
various illnesses or
injuries requiring medical care, and/or inoculations, and/or allergies, and/or
treatments,
and/or prognosis, and/or frequently health information about parents,
siblings, and/or
occupation. The record may be reviewed by a physician in diagnosing the
condition.
[0276] The
medical record can be in paper form and/or can be maintained in a computer-
readable medium. The medical record can be maintained by a laboratory,
physician's office, a
hospital, a healthcare maintenance organization, an insurance company, and/or
a personal
medical record website. In some aspects, a diagnosis, based at least in part
on the measured
DPP4 and/or POSTN levels, is recorded on or in a medical alert article such as
a card, a worn
article, and/or a radiofrequency identification (RFID) tag. As used herein,
the term "worn
article" refers to any article that can be worn on a subject's body,
including, but not limited to,
a tag, bracelet, necklace, arm band, or head band.
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[0277] As
used herein, the term "diagnosis" means detecting a disease or determining the
stage or degree of a disease. Usually, a diagnosis of a disease is based on
the evaluation of
one or more factors and/or symptoms that are indicative of the disease. That
is, a diagnosis
can be made based on the presence, absence or amount of a factor which is
indicative of
presence or absence of the disease or disorder. Each factor or symptom that is
considered to
be indicative for the diagnosis of a particular disease does not need be
exclusively related to
the particular disease, e.g. there may be differential diagnoses that can be
inferred from a
diagnostic factor or symptom. Likewise, there may be instances where a factor
or symptom
that is indicative of a particular disease is present in an individual that
does not have the
particular disease.
[0278] The
term "diagnosis" also encompasses determining the therapeutic effect of a
drug therapy, or predicting the pattern of response to a drug therapy. The
diagnostic methods
may be used independently, or in combination with other diagnosing and/or
staging methods
known in the medical arts for a particular disease.
[0279] As
used herein, the term "differential diagnosis" refers to the determination of
which of two or more diseases with similar symptoms is likely responsible for
a subject's
symptom(s), based on an analysis of the clinical data. The term is also used
to refer to the
determination of whether a patient is susceptible to treatment with an
eosinophil-targeted
therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-
563) depending
on whether the measured DPP4 levels in a patient sample are above a
predetermined
threshold level, or elevated relative to the level in one or more control
samples.
[0280] The
term "prognosis" as used herein refers to a prediction of the probable course
and outcome of a clinical condition or disease. A prognosis is usually made by
evaluating
factors or symptoms of a disease that are indicative of a favorable or
unfavorable course or
outcome of the disease. The phrase "determining the prognosis" as used herein
refers to the
process by which the skilled artisan can predict the course or outcome of a
condition in a
patient. The term "prognosis" does not refer to the ability to predict the
course or outcome of
a condition with 100% accuracy. Instead, the skilled artisan will understand
that the term
"prognosis" refers to an increased probability that a certain course or
outcome will occur; that
is, that a course or outcome is more likely to occur in a patient exhibiting a
given condition,
when compared to those individuals not exhibiting the condition.
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[0281] The
terms "favorable prognosis" and "positive prognosis," or "unfavorable
prognosis" and "negative prognosis" as used herein are relative terms for the
prediction of the
probable course and/or likely outcome of a condition or a disease. A favorable
or positive
prognosis predicts a better outcome for a condition than an unfavorable or
negative
prognosis. In a general sense, a "favorable prognosis" is an outcome that is
relatively better
than many other possible prognoses that could be associated with a particular
condition,
whereas an unfavorable prognosis predicts an outcome that is relatively worse
than many
other possible prognoses that could be associated with a particular condition.
Typical
examples of a favorable or positive prognosis include a better than average
remission rate, a
lower propensity for metastasis, a longer than expected life expectancy,
differentiation of a
benign process from a cancerous process, and the like.
[0282] The
disclosure includes methods of treating an eosinophilic disease or disorder
(e.g., a pulmonary disease or disorder such as mild-to-moderate asthma or
severe asthma) in a
subject based on the changes in expression of DPP4 and/or POSTN. The
disclosure provides
a method of treating a patient having an eosinophilic disease or disorder
(e.g., a pulmonary
disease or disorder such as mild-to-moderate asthma or severe asthma)
comprising:
administering an eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R
antibody such as
benralizumab (MEDI-563) to the patient if the patient is determined to have a
lower or
decreased DPP4 and/or POSTN level in one or more samples taken from the
patient
compared to predetermined DPP4 or POSTN threshold levels, or compared to the
DPP4 or
POSTN level in one or more control samples. In some aspects, a sample is
obtained from a
patient and is submitted for measurement of the level of DPP4 and/or POSTN in
the sample.
[0283] The
disclosure also provides a method of treating a patient having an eosinophilic
disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-
moderate asthma or
severe asthma) comprising: (a) submitting a sample taken from the patient for
measurement
of the DPP4 and/or POSTN level in the sample, and (b) administering an
eosinophil-targeted
therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-
563), to the
patient if the patient has a lower or decreased DPP4 and/or POSTN level in the
sample taken
compared to a predetermined DPP4 or POSTN threshold level, or compared to the
level of
DPP4 or POSTN in one or more control samples.
[0284] Also
provided is method of treating a patient having an eosinophilic disease or
disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate
asthma or severe
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asthma) comprising: (a) submitting a sample taken from the patient for
measurement of the
DPP4 and/or POSTN level in the sample, and (b) suspending or not initiating
the
administration of an eosinophil-targeted therapeutic agent, e.g., an anti-IL-
5R antibody such
as benralizumab (MEDI-563) to the patient if the patients has a higher or
increased DPP4
and/or POSTN level in the sample compared to a predetermined DPP4 or POSTN
threshold
level, or compared to the level of DPP4 or POSTN in one or more control
samples.
[0285] The
disclosure also provides a method of treating a patient having an eosinophilic
disease or disorder (e.g., a pulmonary disease or disorder such as mild-to-
moderate asthma or
severe asthma) comprising: (a) measuring the levels of DPP4 and/or POSTN in a
sample
obtained from the patient; (b) determining whether the level of DPP4 and/or
POSTN in the
sample is higher or increased, or lower or decreased compared to a
predetermined DPP4 or
POSTN threshold level, and, (c) advising a healthcare provider to administer
an eosinophil-
targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab
(MEDI-563) to
the patient if the patient is determined to have a lower or decreased DPP4
and/or POSTN
level in the sample compared to a predetermined DPP4 or POSTN threshold level,
or
compared to the DPP4 or POSTN level in one or more control samples; or to
suspend or deny
the administration of an eosinophil-targeted therapeutic agent to the patient
if the patient is
determined to have a higher or increased DPP4 and/or POST level in the sample
compared to
a predetermined DPP4 or POSTN threshold level, or compared to the DPP4 or
POSTN level
in one or more control samples.
[0286] Also
provided is a method of treating a patient having an eosinophilic disease or
disorder (e.g., a pulmonary disease or disorder such as mild-to-moderate
asthma or severe
asthma) comprising: (a) submitting a sample taken from a patient for
measurement of the
level of DPP4 and/or POSTN in a sample obtained from the patient, and (b)
administering an
eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as
benralizumab
(MEDI-563) to the patient if the patient is determined to have a lower or
decreased DPP4
and/or POSNT level in the sample compared to a predetermined DPP4 or POSTN
threshold
level, or compared to the DPP4 or POSTN level in one or more control samples;
or
suspending, not initiating, or denying the administration of an eosinophil-
targeted therapeutic
agent to the patient if the patient is determined to have a higher or
increased DPP4 and/or
POSTN level in the sample compared to a predetermined DPP4 or POSTN threshold
level, or
compared to the DPP4 or POSTN level in one or more control samples.
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[0287] In
some aspects, the patient's DPP4 and/or POSTN level is measured in an
immunoassay as described herein employing antibodies or antigen binding
fragments thereof
which recognize human DPP4, POSTN, or antigen-binding fragments, variants or
derivatives
thereof. In some aspects, the sample is obtained from the patient and is
submitted for
measurement of the level of DPP4 and/or POSTN in the sample, for example, to a
clinical
laboratory.
[0288] In
some aspects of the above treatment methods, the patient's DPP4 and/or
POSTN level (e.g., DNA or RNA level) is measured in an assay employing one or
more
oligonucleotide probes capable of specifically measuring the expression levels
of the DPP4
gene and/or the POSTN gene.
[0289] In
some aspects, the molecular biomarker (e.g., DPP4 or POSTN) detection assay
(e.g., an immunoassay) is performed on a sample obtained from the patient, by
the healthcare
professional treating the patient (e.g., using an immunoassay as described
herein including,
e.g., the DPP4 immunoassay described in Example 2 or the POSTN assay described
in
W02015120185, formulated as a "point of care" diagnostic kit).
[0290] In
some aspects, a sample is obtained from the patient and is submitted, e.g., to
a
clinical laboratory, for measurement of the DPP4 and/or POSTN level in the
sample
according to the healthcare professional's instructions (e.g., using an
immunoassay as
described herein including, e.g., the DPP4 immunoassay described in Example 2
or any of the
immunoassays disclosed in TABLE 1, or the POSTN assay described in
W02015120185).
[0291] In
some aspects, the clinical laboratory performing the assay will advise the
healthcare provide as to whether the patient can benefit from treatment with
an eosinophil-
targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab
(MEDI-563)
based on whether the patient's DPP4 and/or POSTN level is above a
predetermined DPP4 or
POSTN threshold value or is elevated relative to one or more control samples.
[0292] In
some aspects, this disclosure includes a method of treating a patient having
an
eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such
as mild-to-
moderate asthma or severe asthma) over a period of time, comprising: measuring
the level of
DPP4 and/or POSTN (e.g., protein expression level or gene expression level) in
a first sample
taken from the patient, or submitting a first sample taken from the patient
for measurement of
the level of DPP4 and/or POSTN in the sample, wherein the patient's DPP4
and/or POSTN
level is, for example, measured using immunoassays, including, e.g., the DPP4
immunoassay
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described in Example 2 or any of the DPP4 immunoassays disclosed in TABLE 1 or
the
POSTN assay described in W02015120185, and administering an eosinophil-
targeted
therapeutic agent to the patient, e.g., an anti-IL-5R antibody such as
benralizumab (MEDI-
563) if the patient's DPP4 and/or POSTN level in the first sample is below a
predetermined
DPP4 or POSTN threshold level, or is lowered relative to the DPP4 or POSTN
level in one or
more control samples. The test can be performed by a healthcare provider or a
clinical
laboratory as noted above.
[0293] In
some aspects, results of an immunoassay as provided herein can be submitted
to a healthcare benefits provider for determination of whether the patient's
insurance will
cover treatment with an eosinophil-targeted therapeutic agent.
[0294] In
some aspects, this disclosure includes a method of treating a patient having
an
eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such
as mild-to-
moderate asthma or severe asthma) comprising: measuring, e.g., in a clinical
laboratory, the
DPP4 and/or POSTN level (e.g., protein expression level or gene expression
level) and
eosinophil levels in a first sample obtained from a patient having an
eosinophilic disease or
disorder, e.g., a sample provided by a healthcare provider, wherein the
patient's DPP4 and/or
POSTN level in the first sample is, for example, measured in an immunoassay,
including,
e.g., the DPP4 immunoassay described in Example 2 or any of the DPP4
immunoassays
disclosed in TABLE 1, or the POSTN assay described in W02015120185;
determining
whether the patient's DPP4 and/or POSTN level in the first sample is below a
predetermined
DPP4 or POSTN threshold level, or is reduced relative to the DPP4 or POSTN
level in one or
more control samples; and advising a healthcare provider to administer an
eosinophil-targeted
therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-
563) to the
patient if the patient's DPP4 and/or POSTN level is below a predetermined DPP4
or POSTN
threshold level, or is decreased relative to the DPP4 or POSTN level in one or
more control
samples.
[0295] In
some aspects, a sample is obtained from the patient and is submitted, e.g., to
a
clinical laboratory, for measurement of the level of DPP4 and//or POSTN alone
or in
combination with the level of at least another biomarker, e.g., blood
eosinophils; or a
combination thereof (e.g., protein expression level or gene expression level)
in the sample,
e.g., using an immunoassay.
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[0296] In
some aspects, the clinical laboratory performing the assay will advise the
healthcare provide as to whether the patient can benefit from treatment with
an eosinophil-
targeted therapeutic agent based on whether the patient's DPP4 and/or POSTN
level (e.g.,
protein expression level or gene expression level) is below a predetermined
DPP4 or POSTN
threshold value or is low relative to one or more control samples.
[0297] In
some aspects, methods of treatment contemplated herein (e.g., for an
pulmonary disease or disorder such as asthma) comprise administering to the
subject an
eosinophil-targeted therapeutic agent in a sufficient amount and/or at
sufficient interval to
achieve and/or maintain a certain quantity of eosinophil-targeted therapeutic
agent per
volume of serum, using, for example, an assay as described herein.
[0298] For
example, an antibody or antigen-binding fragment thereof (e.g., targeting IL-5
or IL-5R) can be given to achieve at least about 15 ng/ml, 20 ng/ml, 25 ng/ml,
30 ng/ml, 35
ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/ml, 55 ng/ml, 60 ng/ml, 65 ng/ml, 70 ng/ml,
75 ng/ml, 80
ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, 100 ng/ml, 120 ng/ml, 140 ng/ml, 160
ng/ml, 180
ng/ml, 200 ng/ml, 220 ng/ml, 240 ng/ml, 260 ng/ml, 280 ng/ml, 300 ng/ml, 320
ng/ml, 340
ng/ml, 360 ng/ml, 380 ng/ml, 400 ng/ml, 420 ng/ml, 440 ng/ml, 460 ng/ml, 480
ng/ml, 500
ng/ml, 520 ng/ml, 540 ng/ml, 560 ng/ml, 580 ng/ml, 600 ng/ml, 620 ng/ml, 640
ng/ml, 660
ng/ml, 680 ng/ml, 700 ng/ml, 720 ng/ml, 740 ng/ml, 760 ng/ml, 780 ng/ml, 800
ng/ml, 820
ng/ml, 840 ng/ml, 860 ng/ml, 880 ng/ml, 900 ng/ml, 920 ng/ml, 940 ng/ml, 960
ng/ml, 980
ng/ml, or 1000 ng/ml in serum.
[0299] In a
further embodiment, the antibody or antigen-binding fragment thereof (e.g.,
targeting IL-5 or IL-5R) can be given to achieve a concentration of antibody
in serum from
about 12.5 ng/ml to about 1000 ng/ml. Those of skill in the art will
understand that the
amounts given here apply to a full-length antibody or immunoglobulin molecule;
if an
antigen binding fragment thereof is used, the absolute quantity will differ
from that given in a
manner that can be calculated based on the molecular weight of the fragment.
[0300] In
some aspects, methods of treatment contemplated herein, e.g., for an
eosinophilic disease or disorder (e.g., a pulmonary disease or disorder such
as mild-to-
moderate asthma or severe asthma) comprise administering to the subject an
eosinophil-
targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab
(MEDI-563) in
an amount and at an interval of: 15-54 mg every 0.5-1.5 months; 55-149 mg
every 1.5-4.5
months; 150-299 mg every 4-8 months; or 300-1100 mg every 4-12 months.
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[0301] In
some aspects, the amount and interval are: 15-21 mg every 0.5-1.0 month; 55-
70 mg every 1.5-3.0 months; 150-260 mg every 4-6 months; or 300-700 mg every 4-
8
months. In some aspects, the amount and interval are: 21 mg every month; 70 mg
every 3
months; 210 mg every 6 months; or 700 mg every 6 months. In some aspects, the
amount and
interval are: 210 mg every 3 months or 700 mg every 3 months. In some aspects,
the amount
and interval are: 210 mg every 1 month or 700 mg every 1 month.
[0302] . In
some aspects, the eosinophil-targeted therapeutic agent (e.g.,
benralizumab/MEDI-563) is administered at about 2 mg to about 100 mg per dose.
In some
aspects, the eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-
563) is
administered at about 20 mg per dose, at about 30 mg per dose, or at about 100
mg per dose.
In some aspects, the eosinophil-targeted therapeutic agent (e.g.,
benralizumab/MEDI-563) is
administered once every four weeks to once every twelve weeks. In some
aspects, the
eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is
administered once
every four weeks. In some aspects, the eosinophil-targeted therapeutic agent
(e.g.,
benralizumab/MEDI-563) is administered once every eight weeks. In some
aspects, the
eosinophil-targeted therapeutic agent (e.g., benralizumab/MEDI-563) is
administered once
every four weeks for twelve weeks and then once every eight weeks.
[0303] In
some aspects of the methods, the eosinophil-targeted therapeutic agent, e.g.,
an
anti-IL-5R antibody such as benralizumab (MEDI-563) is administered
intravenously (IV). In
some aspects of the methods, the eosinophil-targeted therapeutic agent, e.g.,
an anti-IL-5R
antibody such as benralizumab (MEDI-563) is administered subcutaneously (SC).
[0304] In
some aspects, the eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R
antibody such as benralizumab (MEDI-563) is administered in one or more fixed
doses. In
some aspects, the doses as administered every week, every two weeks, every
three weeks,
every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks,
every 9 weeks
every 10 weeks, or every 12 weeks. In some aspects, the dose comprises about 1
mg, 2 mg,
3mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50
mg, 60 mg,
70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg,
170 mg,
180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270
mg, 280
mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg,
380 mg,
390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480
mg, 490
mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg,
590 mg,
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600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690
mg, 700
mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg,
800 mg,
810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900
mg, 910
mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, or 1000
mg. In
some aspects, the dose is higher than 1000 mg.
[0305] In
some aspects, two, three, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49 or 50 doses are administered.
[0306] The
formulation, dosage regimen, and route of administration of an eosinophil-
targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab
(MEDI-563),
can be adjusted to provide an effective amount for an optimum therapeutic
response
according to the method disclosed herein. With regard to the administration of
an eosinophil-
targeted therapeutic agent, the therapeutic agent may be administered through
any suitable
means, compositions and routes known in the art. With regard to dosage
regiments, a single
bolus can be administered, several divided doses can be administered over time
or the dose
can be proportionally reduced or increased as indicated by the exigencies of
the therapeutic
situation.
[0307] The
eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R antibody such as
benralizumab (MEDI-563) may be administered by any suitable technique,
including but not
limited to, parenterally, topically, or by inhalation. If injected, the
pharmaceutical
composition can be administered, for example, via intra-articular,
intravenous, intramuscular,
intralesional, intraperitoneal or cutaneous routes (including intra-, trans-
or sub-dermal, and
subcutaneous), by bolus injection, or continuous infusion. In some aspects,
the
pharmaceutical composition is administered by an intravenous route. In some
aspects the
pharmaceutical composition is administered by a subcutaneous route.
[0308] In
further aspects, the compositions are administered by oral, buccal, rectal,
intratracheal, gastric, or intracranial routes. Localized administration, e.g.
at a site of disease
or injury is contemplated, for example, by enema or suppository for conditions
involving the
gastrointestinal tract. Also contemplated are transdermal delivery and
sustained release from
implants. Delivery by inhalation includes, for example, nasal or oral
inhalation, use of a
nebulizer, inhalation of the antagonist in aerosol form, and the like. Other
alternatives include
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eyedrops; oral preparations including pills, syrups, lozenges or chewing gum;
and topical
preparations such as lotions, gels, sprays, and ointments, prefilled syringes
and autoinjectors.
[0309]
Advantageously, the eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R
antibody such as benralizumab (MEDI-563) can be administered in the form of a
composition
comprising one or more additional components such as a physiologically
acceptable carrier,
excipient or diluent. Optionally, the composition additionally comprises one
or more
physiologically active agents for combination therapy.
[0310] A
pharmaceutical composition may comprise an eosinophil-targeted therapeutic
agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-563) together
with one or
more substances selected from the group consisting of a buffer, an antioxidant
such as
ascorbic acid, a low molecular weight polypeptide (such as those having fewer
than 10 amino
acids), a protein, an amino acid, a carbohydrate such as glucose, sucrose or
dextrins, a
chelating agent such as EDTA, glutathione, a stabilizer, and an excipient.
[0311]
Neutral buffered saline or saline mixed with conspecific serum albumin are
examples of appropriate diluents. In accordance with appropriate industry
standards,
preservatives such as benzyl alcohol may also be added. The composition may be
formulated
as a lyophilate using appropriate excipient solutions (e.g., sucrose) as
diluents.
[0312] In
some aspects, the eosinophil-targeted therapeutic agent, e.g., an anti-IL-5R
antibody such as benralizumab (MEDI-563) can be provided at a concentration of
about 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
105, 110, 115, 120,
125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or
200 mg/ml.
[0313]
Exemplary formulations useful for the present invention are those that include
a
glutamic acid, citric acid or acetic acid buffer at an appropriate pH, from
4.5 to 5.2, an
excipient such as sucrose, glycine, proline, glycerol, and/or sorbitol at an
appropriate
concentration such as 1 to 20% (w/v), and a surfactant such as a non-ionic
surfactant like
polysorbate (polysorbate 20 or 80) or poloxamers (poloxamer 1888) at an
appropriate
concentration of 0.001% - 0.1% (w/v). Such formulations are disclosed in US
Patent No.
6171586 and WIPO Published Applications Nos. W020100027766 and W02011088120.
In
some aspects, the formulations comprise sodium acetate, sucrose and
polysorbate 20.
[0314] In
some aspects, the administration of eosinophil-targeted therapeutic agent,
e.g.,
an anti-IL-5R antibody such as benralizumab (MEDI-563), to the patient results
in:
(a) AER (Acute Exacerbation Rate) reduction;
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(b) FEV1 (Forced Expiratory Volume in one second) increase;
(c) improved ACQ-6 (Asthma Control Questionnaire, 6-item version) results;
(d) improved AQLQ (Asthma Quality of Life Questionnaire) results;
(e) reduction of cytokines secreted by type 2 innate lymphocytes (ILC2);
(f) reduction of ILC2 levels,
(g) reduction in eosinophil and/or basophil count, or,
(h) a combination thereof.
[0315] In
some aspects, the AER reduction after administration of an eosinophil-targeted
therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-
563) is at least
about 5%, at least about 10%, at least about 15%, at least about 20%, at least
about 25%, at
least about 30%, at least about 35%, at least about 40%, at least about 45%,
at least about
50%, at least about 55%, or at least 60%, at least about 65%, at least about
70%, at least
about 75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, or
at least about 100%, compared to the AER observed in a population of patients
treated with a
placebo.
[0316] In
some aspects, the FEVi increase after administration of an eosinophil-targeted
therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-
563) is at least
about 3%, at least about 5%, at least about 7%, at least about 9%, at least
about 11%, at least
about 13%, at least about 15%, least about 17%, at least about 19%, at least
about 21%, at
least about 23%, at least about 25%, at least about 27%, at least about 29%,
at least about
31% , at least about 33%, or at least about 35% compared to the FEVi observed
in a
population of patients treated with a placebo.
[0317] In
some aspects, the FEVi increase after administration of an eosinophil-targeted
therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-
563) is at least
about 25 mL, at least about 50 mL, at least about 75 mL, at least about 100
mL, at least about
125 mL, at least about 150 mL, at least about 175 mL, least about 200 mL, at
least about 225
mL, at least about 250 mL, at least about 275 mL, at least about 300 mL, at
least about 325
mL, or at least about 350 mL compared to the FEVi observed in a population of
patients
treated with a placebo.
[0318] In
some aspects, the ACQ-6 change after administration of an eosinophil-targeted
therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab (MEDI-
563) is about -
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0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1, -1.1, or -1.2 compared to
the mean ACQ-6
observed in a population of patients treated with a placebo.
[0319] In some aspects, the reduction of cytokines secreted by type 2
innate lymphocytes
(ILC2) after administration of an eosinophil-targeted therapeutic agent, e.g.,
an anti-IL-5R
antibody such as benralizumab (MEDI-563) is at least about 5%, at least about
10%, at least
about 15%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at
least about 40%, at least about 45%, at least about 50%, at least about 55%,
at least about
60%, at least about 65%, at least about 70%, at least about 75%, at least
about 80%, at least
about 85%, at least about 90%, at least about 95% compared to the level of
cytokines secreted
by ILC2 observed in a population of patients treated with a placebo.
[0320] In some aspects, the reduction of eosinophils after administration
of an eosinophil-
targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab
(MEDI-563) is
at least about 5%, at least about 10%, at least about 15%, at least about 20%,
at least about
25%, at least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about 65%, at
least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least about 90%,
at least about
95% compared to the eosinophil count observed in a population of patients
treated with a
placebo.
[0321] In some aspects, the reduction of basophils after administration of
an eosinophil-
targeted therapeutic agent, e.g., an anti-IL-5R antibody such as benralizumab
(MEDI-563) is
at least about 5%, at least about 10%, at least about 15%, at least about 20%,
at least about
25%, at least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about 65%, at
least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least about 90%,
at least about
95% compared to the basophil count observed in a population of patients
treated with a
placebo.
VII. Kits for DPP4 or POSTN Detection and Quantification
[0322] This disclosure also provides kits for detecting DPP4 and/or POSTN
levels, for
example, through an immunoassay method. Such kits can comprise containers,
each with one
or more of the various reagents (e.g., in concentrated form) utilized in the
method, including,
for example, one or more anti-DPP4 antibodies (i.e., antibodies capable of
detecting DPP4).
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One or more anti-DPP4 or anti-POSTN antibodies, e.g., capture antibodies
capable of
detecting DPP4 or POSTN can be provided already attached to a solid support.
One or more
antibodies, e.g., detection antibodies, can be provided already conjugated to
a detectable
label, e.g., biotin or a ruthenium chelate.
[0323] The
kit can also provide reagents for coupling a detectable label to an antibody
(as
well as the label itself), buffers, and/or reagents and instrumentation to
support the practice of
the assays provided herein. In certain aspects, a labeled secondary antibody
can be provided
that binds to the detection antibody. A kit provided according to this
disclosure can further
comprise suitable containers, plates, and any other reagents or materials
necessary to practice
the assays provided herein.
[0324] In
some aspects, a kit comprises one or more nucleic acid probes (e.g.,
oligonucleotides comprising naturally occurring and/or chemically modified
nucleotide units)
capable of hybridizing a subsequence of the gene sequence of DPP4 or POSTN
under high
stringency conditions. In some aspects, one or more nucleic acid probes (e.g.,
oligonucleotides comprising naturally occurring and/or chemically modified
nucleotide units)
capable of hybridizing a subsequence of the gene sequence of DPP4 or POSTN
under high
stringency conditions are attached to a microarray chip.
[0325] A kit
provided according to this disclosure can also comprise brochures or
instructions describing the process. For DPP4 or POSTN detection immunoassays
(e.g.,
single or multiplexed assays to detect DPP4 or POSTN), and in particular
sandwich
immunoassays, e.g., an ELISA assay or an ECL assay, the sandwich immunoassay
process
comprises a first "capture" antibody (e.g., an antibody specifically binding
to DPP4 or
POSTN) or antigen-binding fragment thereof attached to a solid support, and a
second anti-
DPP4 "detection" antibody or antigen binding fragment thereof. The immunoassay
can be
performed by methods provided herein or methods well known and understood by
those of
ordinary skill in the art.
[0326] In
one aspect, the immunoassay comprises attaching a capture antibody or
fragment thereof to a solid support; applying the test sample or a control
sample, allowing
DPP4 or POSTN, if present in the sample, to bind to the capture antibody or
fragment
thereof; applying the detection antibody or fragment thereof, which can bind
to DPP4 or
POSTN already bound to the capture antibody or fragment thereof; and measuring
the
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amount of detection antibody or fragment thereof bound to DPP4 or POSTN. In
certain
aspects, the assay can further include washing steps, blocking steps and
incubation steps.
[0327] Test kits can include instructions for carrying out one or more DPP4
and/or
POSTN detection assays, e.g., immunoassays or nucleic acid detection assays to
detect the
presence/absence or levels of DPP4 or POSTN. Instructions included in the kits
can be
affixed to packaging material or can be included as a package insert. While
the instructions
are typically written or printed materials they are not limited to such. Any
medium capable of
storing such instructions and communicating them to an end user is
contemplated. Such
media include, but are not limited to, electronic storage media (e.g.,
magnetic discs, tapes,
cartridges, chips), optical media (e.g., CD ROM), and the like. As used
herein, the term
"instructions" can include the address of an internet site that provides the
instructions.
VIII. Companion Diagnostic System
[0328] The methods disclosed herein can be provided as a companion
diagnostic, for
example available via a web server, to inform the clinician or patient about
potential
treatment choices. The methods disclosed herein can comprise collecting or
otherwise
obtaining a biological sample and performing an analytical method to detect
and measure
DPP4 and/or POSTN levels (e.g., full length or soluble DPP4 protein expression
levels,
POSTN expression levels, or gene expression levels for DPP4 or POSTN) alone or
in
combination with other biomarkers (e.g., a panel a genes used to derive a gene
signature,
such as a Th-2 signature or the patient's eosinophil count).
[0329] Molecular biomarkers that can be combined with DPP4 and/or POSTN
include
IL-33, IL-25, TSLP, IL-22, CCL20, LCN2, sCTLA-3, sCD28, CCL5, CCL11, CCL22,
CCL26, FZD5, DOK1, CST2, ZNF436, C20orf100, NAGS, CST1, CDH13, HRH1,
TMEM132B, NTRK1, SLCO2A1, IgE, FETUB, KRT31IKRT34, C6orf138, ATP5J,
TUBAL3, JAM2, NOVA2, NOS2A, H535T4, GRM8, IL1R2, CTDSPL, CEP72,
L0C199800, LYPD1, DISP1, NKX1-2, C4orf38, LOXL4, PRKD1, PAM124B, GPR44,
HIGD1B, CLCA1, SEPT11, CYYR1, CD36, ALOX15, AADAC, ACTA1, ODC1,
DKFZp434F142, ACHE, CSF3, L0C100132552, C12orf27, ZNF331, GK5, DUSP1IDUSP4,
LRWD1, PGLYRP4, GUSBL2, CLGN, NR1I2, EST, LRRC37B, SAA4, SLC12A3,
TMEM45A, FLJ37464, MUC5B, CXCL6, GLRB, DKFp686K01114, FOLR1, TSPAN6,
AKR1C1, KIAA0232, PTP4A1, PCYT2, RHOV, PROS1, Cl lorf63, TCTN1, PIP5K1B,
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OSBPL6, NSUM7, GJB7, IRS2, or combinations thereof. See Lun et al., J. Clin.
Immunol.
27:430-437 (2007), Choi et alõ J. Immunol. 186(3):1861-9 (2011), and
W02009124090A1,
which are herein incorporated by reference in their entireties.
[0330]
Standard names, aliases, etc. of proteins and genes designated by identifiers
used
throughout this application (e.g., PIP5K1B) can be identified, for example,
via Genecards
(www.genecards.org) or Uniprot (www.uniprot.org).
[0331]
Molecular biomarkers that can be combined with DPP4 and/or POSTN include,
e.g., IL-33, IL-25, TSLP, IL-22, CCL20, LCN2, CST1, CCL26, CLCA1, CST2, PRR4,
SERPINB2, CEACAM5, iNOS, SERPINB4, CST4, PRB4, TPSD1, TPSG1, MFSD2, CPA3,
GPR105, CDH26, GSN, C20RF32, TRACH2000196 (TMEM71), DNAJC12, RGS13,
SLC18A2, SERPINB10, SH3RF2, FCER1B, RUNX2, PTGS1, ALOX15, and combinations
thereof.
[0332] DPP4
and/or POSTN levels (e.g., protein expression levels or gene expression
levels) or normalized scores derived from measured DPP4 and/or POSTN levels
can be used
alone (e.g., for treatment, diagnostic, prognostic, or monitoring purposes),
or in combination
with levels or normalized scores derived from other biomarkers (e.g., a panel
a genes used to
derive a gene signature, such as a Th-2 signature or the patient's eosinophil
count).
[0333] These
scores can also be combined with scores corresponding, for example, to (i)
the level of the patient's IgE levels, (ii) the patient's eosinophil or
basophil count, (iii) the
patient's Fraction of Exhaled Nitric Oxide (FEN0), (iv) the patient's
Eosinophil/Lymphocyte
and Eosinophil/Neutrophil (ELEN) index, (v) the patient's EOS index, (vi) the
patient's IgE
levels, (vii), pre- or post-bronchodilator FEV1, FVC measurements or
reversibility, (viii) wall
area percentage (WA%) of subsegmental airways from CT scan of the lungs, or
(ix) a
combination of two or more thereof, to yield a diagnostic score.
[0334] In
this approach, the diagnostic score may be a single number determined from the
sum of all the marker calculations that is compared to a preset DPP4 or POSTN
threshold
value that is an indication of the presence or absence of disease (or of a
certain disease status,
e.g., moderate or severe asthma). Or the diagnostic score may be a series of
bars that each
represent a biomarker value and the pattern of the responses may be compared
to a pre-set
pattern for determination of the presence or absence of disease.
[0335] At
least some aspects of the methods described herein, due to the complexity of
the calculations involved, a method comprising the use of DPP4 and/or POSTN
levels (e.g.,
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levels in blood serum) as a biomarker disclosed herein can be implemented with
the use of a
computer. In some aspects, the computer system comprises hardware elements
that are
electrically coupled via bus, including a processor, input device, output
device, storage
device, computer-readable storage media reader, communications system,
processing
acceleration (e.g., DSP or special-purpose processors), and memory. The
computer-readable
storage media reader can be further coupled to computer-readable storage
media, the
combination comprehensively representing remote, local, fixed and/or removable
storage
devices plus storage media, memory, etc. for temporarily and/or more
permanently
containing computer-readable information, which can include storage device,
memory and/or
any other such accessible system resource.
[0336] A
single architecture might be utilized to implement one or more servers that
can
be further configured in accordance with currently desirable protocols,
protocol variations,
extensions, etc. However, it will be apparent to those skilled in the art that
embodiments may
well be utilized in accordance with more specific application requirements.
Customized
hardware might also be utilized and/or particular elements might be
implemented in
hardware, software or both. Further, while connection to other computing
devices such as
network input/output devices (not shown) may be employed, it is to be
understood that wired,
wireless, modem, and/or other connection or connections to other computing
devices might
also be utilized.
[0337] In
one aspect, the system further comprises one or more devices for providing
input data to the one or more processors. The system further comprises a
memory for storing
a data set of ranked data elements. In another aspect, the device for
providing input data
comprises a detector for detecting the characteristic of the data element,
e.g., such as a
fluorescent plate reader, mass spectrometer, or gene chip reader.
[0338] The
system additionally may comprise a database management system. User
requests or queries can be formatted in an appropriate language understood by
the database
management system that processes the query to extract the relevant information
from the
database of training sets. The system may be connectable to a network to which
a network
server and one or more clients are connected. The network may be a local area
network
(LAN) or a wide area network (WAN), as is known in the art. Preferably, the
server includes
the hardware necessary for running computer program products (e.g., software)
to access
database data for processing user requests. The system can be in communication
with an
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input device for providing data regarding data elements to the system (e.g.,
expression
values). In one aspect, the input device can include a gene expression
profiling system
including, e.g., a mass spectrometer, gene chip or array reader, and the like.
[0339] Some
aspects described herein can be implemented so as to include a computer
program product. A computer program product may include a computer readable
medium
having computer readable program code embodied in the medium for causing an
application
program to execute on a computer with a database. As used herein, a "computer
program
product" refers to an organized set of instructions in the form of natural or
programming
language statements that are contained on a physical media of any nature
(e.g., written,
electronic, magnetic, optical or otherwise) and that may be used with a
computer or other
automated data processing system. Such programming language statements, when
executed
by a computer or data processing system, cause the computer or data processing
system to act
in accordance with the particular content of the statements.
[0340]
Computer program products include without limitation: programs in source and
object code and/or test or data libraries embedded in a computer readable
medium.
Furthermore, the computer program product that enables a computer system or
data
processing equipment device to act in pre-selected ways may be provided in a
number of
forms, including, but not limited to, original source code, assembly code,
object code,
machine language, encrypted or compressed versions of the foregoing and any
and all
equivalents.
[0341] In
one aspect, a computer program product is provided to implement the
treatment, diagnostic, prognostic, or monitoring methods disclosed herein, for
example, to
determine whether to administer an eosinophil-targeted therapeutic agent
(e.g., an antibody
that specifically binds to IL-5R such as benralizumab, or an antigen binding
fragment
thereof) to a patient in need thereof if the levels of DPP4 and/or POSTN in
one or more
samples taken from the patient are lower or decreased compared to
predetermined DPP4 or
POSTN threshold levels, or compared to DPP4 or POSTN levels in one or more
control
samples.
[0342] The
computer program product includes a computer readable medium embodying
program code executable by a processor of a computing device or system, the
program code
comprising:
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(a) code that retrieves data attributed to a biological sample from a subject,
wherein the data
comprises DPP4 and/or POSTN level data (or data otherwise derived from DPP4
and/or
POSTN level values) alone or combination with values corresponding to other
biomarkers in
the biological sample (e.g., a panel a genes used to derive a gene signature,
such as a Th-2
signature). These values can also be combined with values corresponding, for
example, to (i)
the level of the patient's IgE levels, (ii) the patient's eosinophil or
basophil count, (iii) the
patient's Fraction of Exhaled Nitric Oxide (FEN0), (iv) the patient's
Eosinophil/Lymphocyte
and Eosinophil/Neutrophil (ELEN) index, (v) the patient's EOS index, (vi) wall
area
percentage (WA%) of subsegmental airways from CT scan data of the lungs, (vii)
the
patient's IgE levels, (viii), pre- or post-bronchodilator FEV1, FVC
measurements or
reversibility, or (ix) a combination of two or more thereof; and,
(b) code that executes a classification method that indicates, e.g., whether
to administer an
eosinophil-targeted therapeutic agent to a patient in need thereof.
[0343] While
various aspects have been described as methods or apparatuses, it should be
understood that aspects can be implemented through code coupled with a
computer, e.g.,
code resident on a computer or accessible by the computer. For example,
software and
databases could be utilized to implement many of the methods discussed above.
Thus, in
addition to aspects accomplished by hardware, it is also noted that these
aspects can be
accomplished through the use of an article of manufacture comprised of a
computer usable
medium having a computer readable program code embodied therein, which causes
the
enablement of the functions disclosed in this description. Therefore, it is
desired that aspects
also be considered protected by this patent in their program code means as
well.
[0344]
Furthermore, some aspects can be code stored in a computer-readable memory of
virtually any kind including, without limitation, RAM, ROM, magnetic media,
optical media,
or magneto-optical media. Even more generally, some aspects could be
implemented in
software, or in hardware, or any combination thereof including, but not
limited to, software
running on a general purpose processor, microcode, PLAs, or ASICs.
[0345] It is
also envisioned that some aspects could be accomplished as computer signals
embodied in a carrier wave, as well as signals (e.g., electrical and optical)
propagated through
a transmission medium. Thus, the various types of information discussed above
could be
formatted in a structure, such as a data structure, and transmitted as an
electrical signal
through a transmission medium or stored on a computer readable medium.
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TABLE 3: Description of Sequences
SEQ ID Sequence Description SEQ ID Sequence Description
NO NO
1 DPP4, full, protein 11 Benralizumab Light chain (LC)
2 DPP4, soluble, protein 12 Benralizumab VH
3 DPP4, full, cDNA 13 Benralizumab VL
4 IL-5R alpha subunit, Isoform 1 14 Benralizumab VH-CDR1
IL-5R alpha subunit, Isoform 2 15 Benralizumab VH-CDR2
6 IL-5R alpha subunit, Isoform 3 16 Benralizumab VH-CDR3
7 IL-5R alpha subunit, Isoform 4 17 Benralizumab VL-CDR1
8 IL-5R alpha subunit, Isoform 5 18 Benralizumab VL-CDR2
9 Interleukin 5 (IL-5) 19 Benralizumab VL-CDR2
Benralizumab Heavy chain (HC) 20 Perios tin
Examples
Example 1
A Phase 2b, Dose-ranging Study to Evaluate the Efficacy and Safety of
Benralizumab
(MEDI-563) in Adults with Uncontrolled Asthma
[0346] A Ph2b
study (CP220) was conducted with benralizumab (MEDI-563) in a
population of moderate-to-severe, uncontrolled asthmatics (FIG. 1). Post hoc
exploratory
analyses were conducted with two data sets (i) using samples collected at
baseline (week 1
day 1 predose) that were measured for POSTN and DPP4 levels and (ii) using
samples
collected at week -1 (screening) and week 40 that were. DPP4 was measured
using an R&D
Systems DPP4 detection kit. POSTN was measured using a periostin detection
method
developed as a companion diagnostic for tralokinumab (see W02015120185 which
is herein
incorporated by reference in its entirety)
[0347] POSTN is
a downstream activation marker of IL-13 pathway activation and is
being developed as a potential biomarker to identify patients who may respond
to anti-IL-13
therapy. It is also used as a potential marker for identifying patients with
evidence of raised
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Th2 pathway activation. DPP4 is also a downstream activation marker of IL-13
pathway
activation.
[0348]
Patient population was composed of uncontrolled asthmatics on medium or high
dose inhaled corticosteroids and long acting (3 agonists. The study included
609 randomized
subjects. 324 in the EOS+ group (PBO= 80; benralizumab 2 mg = 81; 20 mg = 81
and 100
mg = 82). 285 in the EOS- group (PBO = 143; benralizumab 100 mg = 142). The
study was
double blind, placebo controlled, in moderate-severe asthma patients
evaluating 3 doses of
benralizumab vs placebo in subjects with high blood eosinophil levels (as
determined by
ELEN index) and evaluating one dose vs placebo in subjects with low blood
eosinophil levels
(as determined by ELEN index).
[0349] In
order to understand the patient populations that may respond to treatment with
benralizumab, it was proposed to segment the CP220 patient population into
other known
patient sub-groups by biomarkers or other clinical characteristics to gain a
greater
understanding of other potential responder groups. For samples measured in
Dataset A (i.e.
those where POSTN and DPP4 were measured in samples collected at week 1 day
1), the
following exploratory objectives were planned (i) evaluate the relationship of
blood
eosinophil levels with serum POSTN levels and serum DPP4 levels at baseline
(the
exploratory outcome variables were blood eosinophils, serum POSTN, and serum
DPP4); (ii)
evaluate the efficacy of benralizumab by baseline POSTN on exacerbations, lung
function
and symptoms (the exploratory outcome variables were serum periostin, annual
exacerbation
rate, FEV1, and ACQ); (iii) evaluate the efficacy of benralizumab by baseline
DPP4 on
exacerbations, lung function and symptoms (the exploratory outcome variables
were serum
DPP4, annual exacerbation rate, FEV1, and ACQ); (iv) evaluate the efficacy of
benralizumab
by baseline POSTN together with baseline eosinophil level on exacerbations,
lung function
and symptoms (the exploratory outcome variables were blood eosinophils, serum
periostin,
annual exacerbation rate, FEV1, and ACQ); and (v) evaluate the efficacy of
benralizumab by
baseline DPP4 together with baseline eosinophil level on exacerbations, lung
function and
symptoms (the exploratory outcome variables were blood eosinophils, serum
DPP4, annual
exacerbation rate, FEV1, and ACQ).
[0350] For
samples measured in data set B (i.e. those where POSTN and DPP4 were
measured in samples collected at week -1 and week 40), the following
objectives were
planned (i) evaluate the effect of treatment with benralizumab on serum
periostin levels in
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mITT population, by baseline blood eosinophils and by baseline POSTN levels
(the
exploratory outcome variables were blood eosinophils and serum POSTN); and
(ii) evaluate
the effect of treatment with benralizumab on serum DPP4 levels in mITT
population, by
baseline blood eosinophils and by baseline DPP4 levels (the exploratory
outcome variables
were blood eosinophils and serum DPP4).
[0351] For evaluating treatment effect of benralizumab on efficacy
endpoints the
following analyses were conducted (i) evaluation by placebo (combined E0S+ &
EOS ¨),
2mg, 20mg and 100mg (combined E0S+ & EOS ¨) evaluating any dose response
effect; and
(ii) evaluation by placebo (combined E0S+ & EOS ¨), pooled group of 20 mg and
100 mg
dose groups (E0S+ and E0S-).
[0352] For any sub-group analyses referring to cut-offs of certain
biomarkers: eosinophil
high population is defined as blood eosinophils >300 cells/pL and eosinophil
low population
is defined as blood eosinophils <300 cells/pL. POSTN high and DPP4 high is
defined as
those subjects with levels > median and POSTN low and DPP4 low is defined as
those
subjects with levels < median.
[0353] The current and predicted outputs for Dataset A are the following:
(a) A table of descriptive statistics of median, mean, standard deviation and
range of periostin
and DPP4 in mITT population was prepared to evaluate POSTN and DPP4 levels in
study
population and in other clinical sub-groups of interest, (FIG. 2).
(b) A table of descriptive statistics of median, mean, standard deviation and
range of POSTN
and DPP4 in reversible and non-reversible and ICS medium and ICS high and in
blood
eosinophil >300 and <300 cells/pL sub-populations will be prepared.
(c) A table of the demographics and clinical characteristics of subjects by
POSTN high,
POSTN low, DPP4 high and DPP4 low will be prepared to show the association of
DPP4 or
POSTN which particular clinical characteristics or treatment when divided into
the sub-
groups. The demographic and clinical characteristics to use in the table are
age, gender, BMI,
FEV1(L), FEV1 (% predicted), Reversibility, ACQ, ICS dose, OCS dose, number of
asthma
exacerbations in the previous 12 months (from the asthma history
questionnaire), asthma
symptom diary card score, serum IgE, and blood eosinophil count.
(d) A plot of correlation of baseline serum POSTN vs baseline DPP4 will be
prepared to
show the relationship between POSTN and DPP4 in the study population at
baseline.
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(e) A plot of correlation of baseline serum POSTN with baseline blood
eosinophil will be
prepared to show the relationship between POSTN and blood eosinophils in the
study
population at baseline.
(f) A plot of correlation of baseline serum DPP4 with baseline blood
eosinophils will be
prepared to show any relationship between DPP4 and blood eosinophils in the
study
population at baseline.
(g) A graphic of the overlap of blood eosinophils >300, <300 cells/pL with
POSTN high and
POSTN low subjects by mITT, lung function reversible subjects, lung function
non-
reversible subjects, ICS high subjects and ICS medium subjects was prepared to
evaluate
overlap between different sub-groups in the study population.
(h) A graphic of the overlap of blood eosinophils >300, <300 cells/pL with
DPP4 high and
DPP4 low subjects by mITT, lung function reversible subjects, lung function
non-reversible
subjects, ICS high subjects and ICS medium subjects was prepared to evaluate
overlap
between different sub-groups in the study population.
(i) Plots showing the effect of benralizumab on (i) exacerbate rate by
baseline serum POSTN
by treatment group, (ii) change in FEV1 by baseline serum POSTN by treatment
group, (iii)
change in ACQ by baseline serum POSTN by treatment group, (iv) exacerbate rate
by
baseline serum DPP4 by treatment group, (v) change in FEV1 by baseline serum
DPP4 by
treatment group, (vi) change in ACQ by baseline serum DPP4 by treatment group,
(vii)
exacerbation rate by baseline serum POSTN with baseline blood eosinophils by
treatment
group, (viii) exacerbation rate by baseline serum DPP4 with baseline blood
eosinophils by
treatment group, (ix) change in FEV1 by baseline serum POSTN with baseline
blood
eosinophils by treatment group, (x) change in ACQ by baseline serum POSTN with
baseline
blood eosinophils by treatment group, (xi) change in FEV1 by baseline serum
DPP4 with
baseline blood eosinophils by treatment group, and (xii) change in ACQ by
baseline serum
DPP4 with baseline blood eosinophils by treatment group, will be prepared.
(j) To evaluate the effect of benralizumab on efficacy endpoints in specific
sub-populations,
plots of effect of benralizumab in subjects with high or low baseline serum
POSTN on
exacerbation rate, change in FEV1 from baseline and change in ACQ from
baseline were
prepared for the following sub-groups:
(i) POSTN high and POSTN low (mITT only),
(ii) POSTN high and POSTN low in subjects with reversible lung function,
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(iii) Each of the above group in POSTN high plus eosinophils >300 and <
300cells/pL
and POSTN low plus eosinophils >300 and < 300cells/pL sub-groups.
(k) To evaluate the effect of benralizumab on efficacy endpoints in specific
sub-populations,
plots of effect of benralizumab in subjects with high or low baseline serum
POSTN on
exacerbation rate, change in FEV1 from baseline and change in ACQ from
baseline will be
prepared for the following sub-groups:
(i) POSTN high and POSTN low in subjects with non-reversible lung function,
(ii) POSTN high and POSTN low in subjects on high dose ICS,
(iii) POSTN high and POSTN low in subjects on medium dose ICS,
(iv) POSTN high and POSTN low in subjects who are not on chronic OCS,
(v) POSTN high and POSTN low in subjects who are not on chronic OCS and
>12%
reversible at baseline,
(vi) Each of the above group in POSTN high plus eosinophils >300 and <
300cells/pL
and POSTN low plus eosinophils >300 and < 300cells/pL sub-groups.
(1) To evaluate the effect of benralizumab on efficacy endpoints in specific
sub-populations,
plots of effect of benralizumab in subjects with high or low baseline serum
DPP4 on
exacerbation rate, change in FEV1 from baseline and change in ACQ from
baseline were
prepared for the following sub-groups:
(i) DPP4 high and DPP4 low (mITT only),
(ii) DPP4 high and DPP4 low in subjects with reversible lung function,
(iii) Each of the above in DPP4 high plus eosinophils >300 and <
300cells/pL
and DPP4 low plus eosinophils >300 and < 300cells/pL.
[0354] To
evaluate the effect of benralizumab on efficacy endpoints in specific sub-
populations, plots of the effect of benralizumab in subjects with high or low
baseline serum
DPP4 on exacerbation rate, change in FEV1 from baseline and change in ACQ from
baseline
will be prepared for the following sub-groups:
(i) DPP4 high and DPP4 low in subjects with non-reversible lung function,
(ii) DPP4 high and DPP4 low in subjects on high dose ICS,
(iii) DPP4 high and DPP4 low in subjects on medium dose ICS,
(iv) DPP4 high and DPP4 low in subjects who are not on chronic OCS,
(v) DPP4 high and DPP4 low in subjects who are not on chronic OCS and
>=12% reversible at baseline,
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(vi) Each
of the above in DPP4 high plus eosinophils >300 and < 300ce11s/pL
and DPP4 low plus eosinophils >300 and < 300ce11s/pL.
[0355] The predicted outputs for Dataset B are the following:
(a) Descriptive statistics showing POSTN levels in study population and in
other clinical sub-
groups of interest at baseline and post treatment with benralizumab will be
presented in a
table of descriptive statistics of median, mean, standard deviation and range
of POSTN at
week -1 and week 40 in mITT population.
(b) Descriptive statistics showing DPP4 levels in study population and in
other clinical sub-
groups of interest at baseline and post treatment with benralizumab will be
presented in a
table of descriptive statistics of median, mean, standard deviation and range
of DPP4 at week
-1 and week 40 in mITT population.
(c) A plot of change in baseline POSTN vs baseline blood eosinophil level by
treatment
group (i.e. after benralizumab treatment and placebo) will be prepared to show
the effect of
benralizumab on serum POSTN level by increasing baseline blood eosinophil
level.
(d) A plot of change in baseline DPP4 vs baseline blood eosinophil level by
treatment group
(i.e. after benralizumab treatment and placebo) will be prepared to show the
effect of
benralizumab on serum DPP4 level by increasing baseline blood eosinophil
level.
(e) A plot of effect of benralizumab on POSTN levels from week -1 to week 40
in the
following populations (i) mITT, (ii) blood eosinophils >300 and <300 cells/pL,
(iii) FEV1
reversible and non-reversible subjects, (iv) ICS high and ICS medium, will be
prepared to
show the effect of treatment with benralizumab on serum POSTN levels by study
population
and by sub-groups.
(f) A plot of the effect of benralizumab on DPP4 levels from week -1 to week
40 in the
following populations (i) mITT, (ii) blood eosinophils >300 and <300 cells/pL,
(iii) FEV1
reversible and non-reversible subjects, and (iv) ICS high and ICS medium, will
be prepared
to show the effect of treatment with benralizumab on serum DPP4 levels by
study population
and by sub-groups.
Example 2
DPP4 ELISA Immunoassay
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[0356] The
DPP4 detection assay disclosed herein is a quantitative ELISA-based
immunoassay. A mouse monoclonal antibody specific for human DPP4 was pre-
coated onto
a microplate (R&D Systems, Cat #DC260). One hundred microliters of assay
diluent were
first added to wells of microplates followed by addition of 50 pL of
standards, controls and
1:50 diluted serum samples. The plates were incubated for 2 hours 15 minutes
at room
temperature to allow DPP4 to bind to the capture antibody on the plates.
[0357] To
remove unbound materials plates were then washed 4 times with 1X Wash
buffer (MedImmune) using the plate washer. Plates were then incubated with 200
pL of the
detection antibody (polyclonal anti-DPP4 antibody-HRP conjugate, R&D Systems,
Cat
#DC260) for 2 hours 15 minutes at room temperature. After that, the plates
were washed
and incubated with 200 pl of a chromogenic HRP substrate, hydrogen
peroxide/tetramethylbenzidine (R&D Systems, Cat #DC260) for 30 minutes 5
minutes at
room temperature in the place protected from light.
[0358] The
enzyme reaction was stopped by the addition of 50 pL stop solution (2N
sulfuric acid). Within 30 minutes after stopping of the reaction plates were
read on
SpectraMaxPlus 384 Microplate Spectrophotometer (Molecular Devices) to measure
the
optical density at 450 nm. The intensity of the color generated was directly
proportional to
the amount of bound DPP4. The DPP4 concentrations in samples and controls were
interpolated from the standard curve of recombinant human DPP4, which was run
on each
plate. The quadratic model was used for curve fitting. The derived DPP4
concentrations were
adjusted for the initial sample dilution.
[0359] The
quantifiable range was 16 ng/mL to 1000 ng/mL of DPP4 in 100% serum.
The assay was reproducible, precise and specific for DPP4. The parallelism
between
recombinant standard and endogenous serum DPP4 has been demonstrated. The
assay has
acceptable matrix interference, 3 freeze-thaw cycles stability and 1.5 years
longitudinal
stability at -80 C.
Example 3
Baseline High Blood Eosinophils or Serum Biomarkers of Low IL-13 Pathway
Activation Predict Exacerbation Rate Reduction by Benralizumab in Moderate-to-
Severe Asthmatics
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[0360]
Benralizumab, a humanized monoclonal antibody that selectively depletes
eosinophils and basophils through enhanced antibody dependent cellular
cytotoxicity,
reduced exacerbations in a Ph2b study (NCT01238861, see FIG. 1) in moderate-to-
severe
asthmatics with eosinophilic inflammation (Castro et al., Lancet Respir Med.
2: 878-90
(2014)). Dipeptidyl peptidase (DPP4) and POSTN (POSTN) are upregulated by IL-
13 and
are potential biomarkers of response to anti-IL-13 therapy (Brightling et al.,
Lancet Respir
Med. 3: 692-701 (2015)). We explored the ability of baseline levels of serum
DPP4 and
POSTN to predict exacerbation rate reduction by benralizumab in the Ph2 study.
[0361] Serum
DPP4 was measured using the immunoassay of Example 2 and POSTN
using an immunoassay from Abbott Diagnostics. Asthma subjects were segmented
into
subgroups of DPP4 or POSTN High or Low, (defined as serum concentration > or <
median,
respectively) or Eosinophil High or Low (defined by baseline blood eosinophils
> or < 300
cells/pL, respectively). Groups were further segmented combining DPP4 High and
Low or
POSTN High and Low with Eosinophil High and Low (FIG. 3).
[0362] The
effect of benralizumab (combined 20 mg and 100 mg s.c. treatment arms) was
evaluated compared to placebo on exacerbation rate in each subgroup.
[0363]
Median serum DPP4 and POSTN concentrations were 363.5 ng/mL (ranging from
103.3 ng/mL to 867.5 ng/mL) and 23.6 ng/mL (ranging from 7.5 ng/mL to 104.1
ng/mL)
respectively. See FIG. 2.
[0364] Serum
DPP4 did not correlate with blood eosinophils (correlation co-efficient =
0.05, P = 0.22) or serum POSTN (correlation co-efficient = 0.03, P=0.48).
Serum POSTN
correlated with blood eosinophils (correlation co-efficient = 0.33, P<0.001)
(FIG. 4).
[0365] High
eosinophils, DPP4 low and POSTN low significantly predicted exacerbation
rate reduction with benralizumab treatment. In the eosinophil high subgroup,
exacerbation
rate reduction was independent of POSTN level. For the eosinophil low
subgroup, POSTN
low showed numerically greater exacerbation rate reduction than POSTN high.
DPP-4 low
was predictive of exacerbation rate reduction in eosinophil high and
identified numerically
greater exacerbation rate reduction in the eosinophil low subgroup FIGS. 5, 6
and 7. See also
TABLE 4.
TABLE 4. Effect of benralizumab (combined 20 mg and 100 mg s.c. treatment
arms) on
exacerbation rate reduction in biomarker defined subsets.
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Asthma exacerbation rate P value
reduction % (95% C.I.)
N (placebo/benralizumab)
48
EOS high 0.01
83/167
9
EOS low 0.687
139/135
28
POS TN high 0.157
99/148
POSTN low 0.025
116/137
EOS high and 47
0.054
POS TN high 49/98
EOS high and 53
0.066
POSTN low 33/60
EOS low and -8
0.81
POS TN high 50/49
EOS low and 40
0.157
POSTN low 83/77
11
DPP4 high 0.701
106/141
DPP4 low 0.008
107/141
EOS high and DPP4 11
0.799
high 39/84
EOS high and DPP4 57
0.007
low 43/72
EOS low and DPP4 -1
0.99
high 67/56
EOS low and DPP4 31
0.247
low 64/69
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[0366]
Exacerbation rate reduction (ERR) was performed by Poisson regression model
with baseline ICS status as a covariate with the log of follow-up time as the
offset term.
Overdispers ion correction was given by the Pearson' s chi-square statistic
divided by the
degrees of freedom and applied to standard errors and likelihood ratio
statistics to be adjusted
accordingly.
[0367]
CONCLUSIONS: High baseline blood eosinophil concentrations predicted
benralizumab efficacy, and, effects were independent of POSTN in this
subgroup. Below-
median baseline DPP-4 concentrations indicative of low IL-13 pathway
activation predicted
benralizumab efficacy and may identify patients with low blood eosinophil
concentrations
responsive to benralizumab. The biomarker of IL-13 pathway activation, DPP4,
may identify
patient subsets who benefit from anti¨IL-5R/eosinophil-depleting benralizumab
(DPP4 low)
and IL-13¨targeting tralokinumab (DPP4 high)..
TABLE 5. List of Abbreviations
ACQ-6 Asthma Control Questionnaire (6-item version)
AER asthma exacerbation rate
AHR airway hyperresponsiveness
CBC complete blood count with differential
DPI dry powder inhaler
EOS Eosinophils
FeNo fraction of exhaled nitric oxide
FEY forced expiratory volume in one second
IC50 half-maximal inhibitory concentration
ICS inhaled corticosteroids
IgE immunoglobulin E
MDI metered dose inhaler
OCS oral corticosteroid(s)
POSTN Perios tin
SC Subcutaneous
Th2 T helper type 2
***
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[0368] It is
to be appreciated that the Detailed Description section, and not the Summary
and Abstract sections, is intended to be used to interpret the claims. The
Summary and
Abstract sections may set forth one or more but not all exemplary embodiments
of the present
invention as contemplated by the inventor(s), and thus, are not intended to
limit the present
invention and the appended claims in any way.
[0369] The
present invention has been described above with the aid of functional building
blocks illustrating the implementation of specified functions and
relationships thereof. The
boundaries of these functional building blocks have been arbitrarily defined
herein for the
convenience of the description. Alternate boundaries can be defined so long as
the specified
functions and relationships thereof are appropriately performed.
[0370] The
foregoing description of the specific embodiments will so fully reveal the
general nature of the invention that others can, by applying knowledge within
the skill of the
art, readily modify and/or adapt for various applications such specific
embodiments, without
undue experimentation, without departing from the general concept of the
present invention.
Therefore, such adaptations and modifications are intended to be within the
meaning and
range of equivalents of the disclosed embodiments, based on the teaching and
guidance
presented herein. It is to be understood that the phraseology or terminology
herein is for the
purpose of description and not of limitation, such that the terminology or
phraseology of the
present specification is to be interpreted by the skilled artisan in light of
the teachings and
guidance.
[0371] The
breadth and scope of the present invention should not be limited by any of the
above-described exemplary embodiments, but should be defined only in
accordance with the
following claims and their equivalents.
[0372] All
publications, patents, patent applications, and/or other documents cited in
this
application are incorporated by reference in their entirety for all purposes
to the same extent
as if each individual publication, patent, patent application, and/or other
document were
individually indicated to be incorporated by reference for all purposes.
