In Situ Analysis of Sputum-derived Cellular Targets After Xolair (Omalizumab).
Phase: PHASE4
Conditions: Asthma
Sponsor: NYU Langone Health

 
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Title:  In situ analysis of sputum-derived cellular targets after Xolair® (Omalizumab) 
Principle Investigator:  Joan Reibman, MD  
Version Date: 8/22/2017 
 
 
Background and significance - Study purpose and rationale:   
Omalizumab improves asthma control and quality of life, and reduces corticosteroid use and asthma 
exacerbations in severe-persistent allergic asthma 1. Recent studies also suggest efficacy in non-allergic 
asthma 2. Early understanding of mechanisms of action of omalizumab centered around its targeting of 
the IgE-Fc R1 (high affinity IgE receptor) complex and interruption of the allergic cascade. Recent 
studies in non-allergic asthma now suggest additional mechanisms involving regulation of innate 
immune responses 3, 4. These findings raise the exciting possibility that a larger population might benefit 
from the addition of omalizumab and reinforce the need to identify a potentially responsive population. 
Endotyping, or biologic phenotyping of patients with asthma, may help identify individuals with 
potential to respond to omalizumab and has potential to provide information on mechanisms of action 
of omalizumab. There is an unmet need for non-invasive sampling of innate immune pathways in 
asthma to improve our ability to endotype patients with asthma. 
We have recently developed a novel technique to enrich for rare sputum cells from induced sputum, 
including bronchial epithelial cells (sputum-derived human bronchial epithelial cells; sHBEC). We have 
shown increased Th2 cytokine gene expression in sHBEC from subjects with asthma, and now propose to 
use this technique to test the hypothesis that treatment with omalizumab in adults with asthma 
reduces bronchial epithelial cell-derived Th2 cytokine gene expression. We will test this hypothesis 
with two specific aims: 
Specific Aim 1. Using sHBEC from well-characterized patients with moderate-persistent asthma, we will 
test the hypothesis that treatment with omalizumab reduces mRNA for sHBEC Th2/Th17 targets.  
SA1.a Design and complete a randomized, controlled, 16 week study to obtain sputum-derived human 
bronchial epithelial cells.  
SA1.b. Compare levels of candidate Th2/Th17 supporting cytokines in sHBEC mRNA in patients with 
moderate persistent asthma treated with omalizumab. 
Specific Aim 2.  Using sHBEC from well-characterized patients with moderate-persistent asthma, we will 
test the hypothesis that treatment with omalizumab results in a novel gene “signature” in sHBEC. 
SA2.a. Identify omalizumab response genes in sHBEC to identify novel mRNA targets regulated by 
treatment of patients with moderate-persistent asthma with omalizumab in sHBEC mRNA (gene 
expression). 
SA2.b. Validate sHBEC omalizumab response gene “signature” using nanoString technology to generate 
gene signature for potential use for patient stratification.  

 
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Significance 
Current clinical indications for omalizumab include its use in those with elevated allergen-specific IgE to 
perennial allergens. However, the presence of heterogeneity of inflammatory pathways is increasingly 
being recognized in asthma and there is a need for biologic phenotyping (endotyping) to identify 
patients who will respond to targeted therapy. Little is known about downstream biologic targets of 
omalizumab in key cells associated with asthma. Downregulation of sputum eosinophils, bronchoscopy-
obtained tissue eosinophils, cells expressing the high-affinity Fc receptor for IgE and surface IL-4 staining 
cells, have been shown after omalizumab treatment 5. Recent studies suggest that blood-derived 
plasmacytoid dendritic cells (pDC) express high levels of the Fc R1 and that expression is reduced in 
both allergic and non-allergic asthmatic patients after treatment with omalizumab 2. In refractory atopic 
dermatitis, treatment with omalizumab reduces plasma levels of thymic stromal lymphopoietin (TSLP), 
OX40L, and TARC, all involved in Th2 polarization 6. The suggestion has also been made that individuals 
with elevations in FeNO, serum periostin, and blood eosinophils might have improved response to 
omalizumab, suggesting their use as biomarkers for phenotyping and providing clues for mechanism of 
action 7. 
These studies raise the exciting possibility that additional mechanisms of action of omalizumab can be 
identified that will lead to improved stratification of patients for treatment. Understanding the response 
of specific innate immune effector cells in the lung can provide clues to these questions. We now 
propose to address these questions using a non-invasive technique of rare cell isolation from induced 
sputum. 
The airway epithelium actively participates in the response to allergens as well as to infections and 
pollutants. Human bronchial epithelial cell (HBEC)-derived cytokines signal to downstream innate 
immune and effector cells, including the recently described type 2 innate lymphoid cells (ILC2s) and 
airway dendritic cells (DC). We, and others have described an airway microenvironment regulated by 
airway epithelial cells, with potential to promote the activation of downstream innate immune cells8, 9. 
HBEC-derived cytokines that promote a Th2 response include but are not limited to: thymic stromal 
lymphopoietin (TSLP), IL-33 and IL-25. Using in vitro studies, we described the human Th2-supporting 
HBEC-DC nexus in response to pollutants 10-12; upregulation of HBEC-derived cytokines by other stimuli 
has also been shown. This nexus is linked to the initiation and maintenance of asthma in animal models. 
Invasive human studies using bronchoscopy show upregulation of TSLP and other epithelial cell-derived 
mediators even in patients on high dose inhaled corticosteroids (ICS); the invasive nature of 
bronchoscopy limits its applicability for endotyping and therapeutic stratification and outcomes. 
Analysis of whole induced sputum is under development for endotyping for asthma 13. Although 
exciting, analysis of whole induced sputum provides limited information about specific cell responses, 
and low level or rare cell-specific alterations, particularly those of airway epithelial cells, can be 
masked. The isolation of discrete cell populations from induced sputum allows sampling of rare cells 

 
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from conducting airways, repeated sampling, and cell-specific detailed genomic evaluation. We recently 
developed a novel technique to simultaneously enrich  innate immune cells from sputum 14. This 
technique allows for in situ analyses of sputum-derived human bronchial epithelial cells (sHBEC). The 
non-invasive nature of the technique provides a unique tool for in vivo human studies.  
Innovation 
These studies will be the first to use non-invasive measures of a discrete cell population to examine 
the downstream effects of omalizumab treatment in the lung. Information derived from these studies 
will help clarify mechanisms of action of omalizumab and help identify potential tools for patient 
endotyping and stratification for therapeutic interventions.  
SA1.a Design and complete a randomized, parallel-group, double blind 16-week study to obtain 
sputum-derived human bronchial epithelial cells.  
Preliminary data 
The PI (Reibman) has extensive experience performing clinical trials as part of the American Lung 
Association Asthma Clinical Research Center (ALA ACRC), a multi-center consortium, as well as Phase III 
asthma studies. These studies have resulted in numerous publications, with selected publications noted 
here 15-18. The current study at the ALA ACRC is to examine step-down therapy in patients with 
moderate-persistent asthma and was designed by her site. In addition, the PI has developed a registry of 
asthma and control participants (New York University/Bellevue Asthma Registry), designed to study 
characteristics of severe asthma 19-22. 
Objectives:   
We propose a randomized, parallel-group, double blind 16-week study to assess the effect of 
omalizumab treatment on sHBEC expression of target and novel cytokines. 
The primary outcome measure will be: 
 
The effect of omalizumab on TSLP and IL-33 gene expression in sHBEC in moderate persistent 
asthma   
The secondary outcome measures will be: 
 
The effect of omalizumab on asthma control defined as symptom control (Asthma control test), 
spirometry, and measures of small airway dysfunction (Impulse oscillometry) 
Exploratory outcomes 
 
The effect of omalizumab on newly identified sHBEC targets (gene expression) and gene 
“signature” generation 
Study design:   
Inclusion Criteria 
 
Age 18-65 years old 

 
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 
Physician diagnosed asthma 
 
Lung function (one or more of the following documented in the 5 years before enrollment or 
demonstration during screening) 
o Bronchial hyperresponsiveness (BhR) confirmed by ≥ 12% improvement in FEV1 post 
bronchodilator within the previous 5 years, or 
o Methacholine PC20 < 16mg/dl within the previous 5 years 
 
Severity criteria 
o Moderate-persistent asthma defined by ATS23 
 
Asthma control  
o Partly or uncontrolled asthma according to GINA 2012 guidelines (at least three of the 
following features: daytime symptoms more than 2 times/week, limitation of activities, 
nocturnal symptoms, need for rescue inhaler > 2 times/week, FEV1 <80% predicted)24 
 
Stable use of moderate-high dose inhaled corticosteroids in previous 3 months (definition 
derived from GINA 2012 guidelines: e.g. fluticasone propionate >250 mcg/day, budesonide > 
400mcg/day)24 
 
Ability to perform induced sputum maneuvers 
 
Presence of elevated allergen IgE to any perennial aeroallergen 
 
FeNO > 19 ppb at V1 (Hanania et al. Am J Respir Crit Care Med; 187pp 804–811, 2013) 
Exclusion Criteria 
 
Age <18 or >65 years old 
 
Pulmonary function 
o FEV1 ≤ 70% predicted 
 
Any major chronic illness including but not limited to COPD, uncontrolled hypertension, 
coronary artery disease, bronchiectasis, congestive heart failure, stroke, cystic fibrosis, insulin-
dependent diabetes mellitus, renal failure, liver disorders, immunodeficiency state, or other 
condition that would interfere with participation in the study  
 
Current or > 10 pack a year pack-year tobacco use 
 
Any investigational study within previous 1 month 
 
Inability to perform baseline measurements 
 
Inability to contact by telephone 
 
Pregnancy at screening and failure to use double barrier pregnancy protection in woman of 
childbearing age 
 
Hypersensitivity reaction to omalizumab in the past 
 
Exceeds limits of dosing table (IgE <30 or 700 IU/ml) or body weight of <30 or > 150kg 
 
Systemic corticosteroids within the previous month 
 
Known malignant neoplasm 
 
Investigational and reference therapy: 
The investigational therapy will be omalizumab, dosed according to dosing and U.S. administration 
guidelines for omalizumab. Omalizumab will be dosed every 2-4 weeks based on the patient’s 
pretreatment serum IgE level (IU/mL) and initial visit body weight (kg). Omalizumab will be delivered as 
a subcutaneous injection. Standard safety precautions for dosing will be observed, including clinical 
observation after dosing, and provision of an epinephrine pen. Maintenance asthma treatment will 

 
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remain unchanged. 
 
Omalizumab will be supplied by Novartis Pharma as a sterile, freeze-dried preparation.  
 
Reference therapy will consist of sterile saline with a volume and injection frequency that would have 
been indicated based on patient’s serum IgE and body weight. Sterile saline will be delivered as a 
subcutaneous injection and will be supplied by Novartis Pharma. 
 
Treatment or reference therapy will be delivered for a 16-week period and then terminated. No 
adjustments will be made for dosing after initial dose determination. 
 
Study duration: 
This study will be a 16-week study intervention with a 2-week run-in, including screening for each 
patient. We would anticipate starting the study in March 2015 (after grant approval, IRB approval, 
finalization of study) and completion of the study within 12 months. 
 
Protocol and Study design: 
 
The study will be a randomized, placebo-controlled, double blind, 16-week intervention study. 
This will be an exploratory  study to show feasibility and suggest proof of concept.  Our 
enrollment goal is 30 subjects.   
 
Recruitment for the study will be performed with advertisements at Metro NY or AM NY, Craig’s 
list, subjects who have participated in previous studies and have consented to be contacted for 
future research studies and referral from Bellevue or NYU physicians who are part of the study 
team. The treating physician, who is part of the study team, will introduce the study to his/her 
patients and obtain permission for the study team to contact the patient to further discuss the 
study and see if he/she is interested in participating.   
 
Consenting Process:  All subjects for this study will be provided a consent form describing this study and 
providing sufficient information for subjects to make an informed decision about their participation in 
this study. The consent form will be provided by the PI in a room with privacy. After the subject has read 
the consent form, the subject will be able to ask any questions to enable their understanding of the 
protocol.   
 
 
Visits and procedures are diagramed below: 
 
 

 
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Briefly, participants will be recruited according to inclusion/exclusion criteria previously defined. 
Visit 1. During this visit, subjects will sign consent, be screened for eligibility including severity. Asthma 
control will be assessed using the Asthma Control Test (ACT), a 5 question survey.  We will also assess 
the ability of the subject to complete the study. A limited physical exam will be performed (a doctor will 
listen to heart and lungs), and vital signs will be measured.  Blood (40ml) will be obtained for baseline 
allergen-specific IgE measurements if they have not been measured in the previous 5 years, and 
peripheral eosinophils, and serum will be obtained and blood stored for potential subsequent analysis. 
We will measure exhaled breath (FeNO). Nitric oxide (NO) is a naturally occurring gas made in the lungs.  
Measurement of FeNO provides information about the level of inflammation (redness, swelling and 
irritation) in the lungs. We will ask the participant to seal their lips tightly on the mouthpiece of the 
FeNO device and inhale (breathe in) as much air as possible. They will then exhale (blow out) the air out 
of their lungs slowly over a 10-second period of time. This test may have to be done more than once to 
get a good measurement.  Breathing measurements will be performed by spirometry and impulse 
oscillometry (IOS) before and after albuterol. Spirometry will be performed after a nose clip is placed on 
the subject and then the subject will be asked to breathe hard and fast through a tube until reproducible 
measurements are obtained (up to 7 trials). We then administer a bronchodilator via a metered dose 
inhaler (albuterol sulfate; 360g) and repeat the spirometry measurement. IOS maneuvers will be 
performed during tidal breathing in the seated position, with a nose clip in place, and with support of the 
cheeks. A minimum of three trials, lasting 30 seconds, will be performed. This will also be done before 
and after the bronchodilator.  
V1	
V3	
Randomization	
V5	
V6	
V1a	
V4	
Cases	
V2/a	
V7/a	
FeNO 
ACT 
Spirometry/IO
S  
Sputum 
FeNO 
ACT 
Spirometry/IOS  
Sputum 
2 You need 
to start a 
different 
treatment 
for your 

 
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Visit 1a. This visit will only be necessary if subject does not meet criteria with either a history of 
bronchodilator reversibility, or a history of a positive methacholine study as defined in inclusion criteria. 
During this visit, a methacholine challenge test will be performed. Methacholine is an inhaled 
medication, which is used to diagnose airway hyper-reactivity, a characteristic of asthma. The subject 
will be asked to inhale a low dose of methacholine and then perform spirometry. We will slowly increase 
the dose and repeat spirometry until the subject shows a decline in spirometry value of 20% or until we 
have reached a defined dose of methacholine (16 mg).  If spirometry measurement is reduced as 
expected with asthma, or if subject develops asthma symptoms, the effect is temporary and can be 
reversed by taking albuterol. At this visit, if the patient is female and of childbearing age, a urine 
pregnancy test will be done before the methacholine challenge test is done.  
 
Visit 2. During this visit, we will obtain an interim asthma history, obtain vital signs, and perform a 
limited physical examination. We will measure breathing with a peak flow (a quick exhalation into a 
peak flow meter). We will perform a sputum induction. To do this, we will first measure baseline 
breathing with spirometry. We will then ask the subject to inhale albuterol sulfate (360 mcg) and use a 
standardized protocol, with consists of inhalation of nebulized hypertonic saline solution (3%, 
NOUVAG 2000 ultrasonic nebulizer; NOUVAG USA, Inc.) for 2- minute intervals for a total of 20 
minutes. Volunteers will be encouraged to cough and expectorate throughout the procedure. 
 
Visit 2a. This visit will occur within approximately 1 week after Visit 2. At this visit, we will review 
symptoms to make sure that there has not been an asthma exacerbation. We will then perform a 
repeat sputum induction to insure adequate sputum collection. We will also review information about 
omalizumab and provide education on the use of the epinephrine pen, which we provide to all 
patients who are getting omalizumab as a standard safety precaution. An epinephrine pen is a device 
that delivers epinephrine for emergency. This would only be needed to reverse an allergic reaction. 
We will also provide patients with an “Anaphylaxis” information sheet by the AAIA. At this visit, we will 
calculate the dose of omalizumab (or placebo) that will be needed based on weight and serum IgE 
level. Some patients, may require biweekly dosing of omalizumab, to accommodate this dosing (3a, 
4a, 5a, 6a &7a). 
 
Visit 3. Randomization Visit. This visit will be performed at least 1 day after V2a but performed at least 
2 weeks after V1 in order to allow confirmation of asthma control as a run-in.  This is a randomization 
(group assignment) visit and a study drug visit. At this visit, we will obtain an interim asthma history, 
measure asthma control (ACT), limited physical examination with a peak flow, and review 
inclusion/exclusion criteria. The subject will receive the first dose of omalizumab or placebo. The study 
drug will be injected into the arm with up to 3 subcutaneous injections (just under the skin).The number 
of injections will be determined by the amount of medication the patient needs. The subject will be 
observed in the CTSI for 2 hours after the injection of the study drug or placebo. This is the standard 
protocol for all patients who receive this medication.  
 
Visit 3a.  Study drug visit (for subjects who need biweekly doses). If the patient needs to receive 
omalizumab every two weeks, we will call the subject back for Visit 3a. At this visit we will again 
measure the vital signs. We will then administer an injection of the study drug or placebo. The subject 
will again be observed in the CTSI for 2 hours after the injection of the study drug or placebo. This is the 

 
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standard protocol for all patients who receive this medication, where a 2 hour observation is routine for 
the first 2 doses of the drug.  
 
Visit 4. Study drug visit. This visit will occur 4 weeks after V3 and will be a monitoring visit. At this visit, 
we will review the asthma history or if the patient has had any side effects since the first dose of 
omalizumab. We will assess asthma control using the ACT. We will perform a limited exam by checking 
vital signs and listening to the lungs. We will inject the study drug or placebo into the arm with a 
subcutaneous injection. We will observe the patient in the CTSI for 2 hours for patients on monthly 
dosing receiving their second dose or for 30 minutes for patients on biweekly dosing receiving their third 
dose. This is the standard protocol for all patients receiving this medication. 
 
Visit 4a (V4a).  Study drug visit (for subjects who need biweekly doses). This visit will occur 
approximately 2 weeks after V4 if the patient needs omalizumab every 2 weeks. At this visit we will 
again measure the vital signs and provide the subject with an injection of the study drug or placebo. We 
will observe the patient in the CTSI for 30 minutes. This is the standard protocol for all patients who 
receive this medication. 
 
Visit 5. This visit will occur 4 weeks after V4 (week 8 of study drug) and will be a monitoring visit. At this 
visit, we will again review the asthma history and whether the patient has had any side effects since the 
last dose of omalizumab. We will assess asthma control (ACT) and we will auscultate the lungs and check 
vital signs. Study drug or placebo will be injected subcutaneously. We will observe the patient in the CTSI 
for 30 minutes. This is the standard protocol for all patients who receive this medication. 
 
Visit 5a (V5a).  Study drug visit (for subjects who need biweekly doses). This visit will occur 
approximately 2 weeks after V5 if their need omalizumab every 2 weeks. At this visit we will again 
measure vital signs and administer an injection of the study drug or placebo. We will observe the patient 
in the CTSI for 30 minutes. 
 
Visit 6. This visit will occur 4 weeks after V5 (week 12 of study drug) and will be a monitoring visit. At 
this visit, we will again review the interim asthma history and whether the patient has had any side 
effects since the last dose of omalizumab. We will assess asthma control (ACT) and we will auscultate 
the lungs and check vital signs. Study drug or placebo will be injected subcutaneously. We will observe 
the patient in the CTSI for 30 minutes.  
 
Visit 6a (V6a).  Study drug visit (for subjects who need biweekly doses). This visit will occur 
approximately 2 weeks after V6. We will again review the asthma history and whether the patient has 
had any side effects since the last dose of omalizumab. We will assess asthma control (ACT) and we will 
auscultate the lungs and check vital signs. Study drug or placebo will be injected subcutaneously. We will 
observe the patient in the CTSI for 30 minutes. This is the standard protocol for all patients who receive 
this medication. 
 

 
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Visit 7. This visit will occur 1-3 weeks after the last dose of medication (V6 or V6a). At this time, subjects 
will be assessed for safety indices and asthma control. In addition, subjects will undergo measurement 
of FeNO, Spirometry and IOS. There will be no more administration of study drug.  
Sputum induction will be performed as previously described. Blood will be obtained from a vein from 
their arm or hand for laboratory tests (approximately  40 ml or  3 tablespoons).  
 
Visit 7a (V7a). Final sputum induction visit. This visit will occur approximately 1 week after Visit 7. At this 
visit, we will again review whether there have been any side effects. We will assess asthma control and 
we will auscultate the lungs and check vital signs. Sputum induction will be performed as previously 
described.  
Once the patient has completed that study, they will receive a letter containing their treatment 
assignment. 
Because of differences in reconstitution time and viscosity between omalizumab and placebo, the 
research pharmacy will reconstitute and the nurses will inject the drugs independent of the 
investigator. 
Initial comparison will be performed between the omalizumab group compared to control group 
with mean of the individual difference (i.e. change, expressed as the % of baseline) and the values 
observed at V7 of the two groups. 
We have elected to include patients with moderate or uncontrolled asthma as opposed to severe 
asthma. The goal of this study is based on sputum induction, and so for our pilot study, for reasons of 
safety and experience, we have elected to include patients with moderate or partly/uncontrolled 
asthma. In addition, we have included the ability to successfully undergo the induced sputum 
maneuver (ability to tolerate the procedure, ability to expectorate good quality sputum) as an inclusion 
criteria. We will keep track of the number of patients that we have excluded because of this criteria. 
Subjects will be stratified on a 1:1 basis. There are no requirements for assignment as a treatment or 
control subject. Subjects will remain on current therapy (inhaled corticosteroid, with or without long 
acting beta-agonist).  This treatment will remain stable during the study period.  Use of a short acting 
beta-agonist will be allowed.  Use of rescue prednisone during the study period will not be allowed, and 
subject will be discontinued from the study.  Subjects who fail initial screening can be re-screened.  
 
 
Study Treatment 
Description of Study Treatments:  treatments used in this study will include  
 
Omalizumab will be dosed according to dosing and U.S. administration guidelines for 
omalizumab. Omalizumab will be dosed every 2-4 weeks based on the patient’s pretreatment 
serum IgE level (IU/mL) and initial visit body weight (kg).  Omalizumab will be delivered as a 
subcutaneous injection. Standard safety precautions for dosing will be observed, including 
clinical observation after dosing, and provision of an epinephrine pen. Maintenance asthma 
treatment will remain unchanged. 
 
Placebo reference therapy will consistent of sterile saline with a volume and injection 

 
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frequency that would have been indicated based on patient’s serum IgE and body weight.  
Sterile saline will be delivered as a subcutaneous injection and will be supplied by Novartis 
Pharma. 
 
Pre-randomization Study Treatment: 
 
There will be no specific pre-randomization treatment  
 
Randomization Procedure 
 
Randomization will occur at visit 3 (V3).  This visit will be performed at least 1 day after V2 but 
performed at least 2 weeks after V1 in order to allow confirmation of asthma control as a run-
in. At this visit, inclusion/exclusion criteria will be reviewed and baseline spirometry will be 
performed. Subject will receive first dose of omalizumab or placebo.  Patients will be 
randomized in a 1:1 ratio to either omalizumab or placebo.  Omalizumab will be supplied by 
Novartis Pharma as a sterile, freeze-dried preparation. Randomization will be generated by a 
computer randomization program. The study team will be blind with the exception of one 
coordinator.  The research pharmacy will as well as one coordinator of the study team will be 
unblind and will maintain the randomization code linked to the subjects. 
 
 
Maintenance asthma treatment will remain unchanged throughout the sixteen week study 
 
Side Effects of Drugs: 
 
Omalizumab (Xolair®) is an anti-IgE antibody indicated for moderate to severe persistent 
asthma in patients with positive skin test or in vitro reactivity to a perennial aeroallergen and 
symptoms that are inadequately controlled with inhaled corticosteroids 
 
Potential serious side effect 
o Anaphylaxis  
o Malignancy 
o Cardiovascular side effects. A recent 5 year study of omalizumab revealed a higher 
incidence rate of overall cardiovascular and cerebrovascular serious adverse events in 
patients treated with omalizumab. This was an observational study and could not take 
into account possible baseline imbalances in cardiovascular risk factors and inability to 
adjust for possible risk factors. An additional analysis of shorter studies with a younger 
age population failed to show this increased risk. The FDA has required a black box 
warning. 
   
 
Common side effects 
o Injection site reaction 
o Viral infections 
o Upper respiratory tract infections 
o Sinusitis 
o Headache 
o Pharyngitis 
 

 
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 
Methacholine.  Methacholine is delivered during a methacholine challenge test. Some people 
have no reaction to this at all while for others, this test can reproduce some asthma symptoms. 
Some people have coughing or a tight sensation in their chest from breathing the 
methacholine, but it is usually mild. However, the reaction to methacholine can include severe 
bronchoconstriction (such as a severe asthma attack) and a reduction in breathing function. If a 
subject develops an asthma episode during this procedure or if lung function drops, we will 
stop the procedure and reverse the symptoms and lung function reduction with albuterol to 
reverse the effects of the methacholine. A physician involved in the study will provide this and 
the subject will be monitored until symptoms and breathing has returned to its baseline. If a 
subject is female and of childbearing age,  a pregnancy test will be performed before the 
methacholine test. 
 
 
Albuterol sulfate (bronchodilator). We will ask you to inhale albuterol sulfate, a bronchodilator, 
during some breathing tests and before your sputum induction. Albuterol sulfate is a medicine 
that is commonly used for all people with asthma to relax the muscles in the airways and open 
up the lungs. We will do this as a screening test to see if you have reversible airflow 
obstruction or to reverse any reduction in airflow that you may have experienced with 
methacholine or with the sputum induction. Inhalation of albuterol sulfate can make you feel 
jittery or make your heart beat faster than usual. 
  
Potential Benefits:  This study will provide a comprehensive assessment of response to omalizumab 
that will may improve the health of a subject and provide guidance about subsequent treatment. .  The 
providers will select appropriate patients who may benefit from treatment.  The results from this study 
should add to the guidance health care providers with regard to measuring response to omalizumab 
treatment more objectively. 
 
Handing of Drug:   
 
CTSI pharmacy as per their standard protocol will conduct drug accountability, storage, diluting 
and destruction. 
 
Unmasking:   
Participants and clinical center investigators will be double masked as to treatment assignment during 
the randomization phase.  The investigators believe that under most circumstances, if there is a 
potential adverse reaction to study drug, the drug can be discontinued without unmasking of 
participant or staff.  In the event of an emergency, unmasking can occur by reviewing pharmacy notes 
with drug assignment number on the outside and treatment. The study PI will have a 24-hour contact 
number. Instances of unmasking prior to the termination of the trial data collection will be reported 
to the Data Safety Monitoring Board (led by Dr. John Hay) Before the first subject is entered into the 
study, a member of the study team will review and discuss the requirements of the clinical study 
protocol and related documents with the investigational staff and also train them in any study-specific 
procedures and system(s) utilized. The principal investigator will ensure that appropriate training 
relevant to the study is given to all of these staff, and that any new information relevant to the 
performance of this study is forwarded to the staff involved. 
The principal investigator will maintain a record of all individuals involved in the study. 

 
12 
 
 
 
 
 Once a participant completes the study, the participant will receive a letter containing the 
treatment assignment. 
 
Safety and Asthma Control 
 
Routine asthma treatment 
 
All participants must have partially or uncontrolled asthma according to GINA 2012 guidelines (at 
least three of the following features: daytime symptoms more than 2 times/week, limitation of 
activities, nocturnal symptoms, need for rescue inhaler> 2 times/week, FEV1 <80% predicted)24.  
Participants cannot have received treatment with Xolair within the 6 months prior to enrollment. At 
the time of enrollment, if the participant has been on a stable treatment regimen using other 
asthma and allergy treatments, including SABAs, LABAs, ICS, combination ICS/LABA, leukotriene 
modifiers, theophylline preparations, antihistamines, immunotherapy (allergy shots), and or nasal 
steroids, the participant may continue these treatments during the course of the study. If a 
participant experiences an asthma exacerbation or other signs of significant worsening of their 
asthma, the participant will be referred to the primary care physician or asthma provider for re-
evaluation of their asthma regimen. If the asthma care provider is not available in a timely fashion, 
the clinical center physician will evaluate the participant’s regimen and make recommendations for 
treatment.   
 
Asthma Action Plan 
 
Participants will receive an asthma action plan summarizing the procedures for home care in the 
event of increase in asthma symptoms or a drop in peak flow. At visit 1 the study physician or 
his/her designee will give the participant a temporary asthma action plan sheet with instructions for 
its use. The “red”, “yellow” and “green” zones will be based on peak flow readings and symptoms at 
Visit 1. At Visit 3, upon randomization the PEF values from the diary cards will be reviewed and 
symptoms reviewed.  Zones will be defined as “green” (> 85% of baseline PEF),”yellow” (65—85% of 
baseline PEF), and “red” (less than 65% of baseline PEF). These personal cut-offs will be entered 
onto an Asthma Action Plan Card. Both the temporary asthma action plan given at visit 1 and the 
Asthma Action Plan Card given at visit 3 will instruct participants to seek medical attention 
immediately if they are experiencing a serious asthma attack. Participants who experience less 
serious asthma symptoms or a drop in peak flow into the yellow zone or red zone are instructed to 
take 2 puffs of their rescue inhaler and wait 15-20 minutes. If symptoms persist or peak flow does 
not return to the green zone, participants are instructed to take 2 more puffs of the inhaler and wait 
15-20 minutes up to 2 more times. If after a total of 3 administrations of the rescue inhaler, 
symptoms persist or peak flow is not in the green zone participants are instructed to seek medical 
care immediately. 
 
Response to Treatment Failures 
 
This study is not designed to evaluate clinical treatment effects. Participants who experience 
worsening asthma, defined as an increase in use of albuterol,  will be treated according to best 
medical judgment and continue follow-up in their same treatment group. However, should a 
participant require a rescue course of prednisone (systemic corticosteroids), emergency room visit, 
or hospitalization during the study period (V2 – 7), the participant will be withdrawn from the study. 

 
13 
 
 
 
This will not be considered an adverse event due to study drug, because both EPR3 and GINA 
guidelines, consider one exacerbation per patient per year an indication in and of itself of lack of 
asthma control. 
 
Serious Adverse Events 
 
A serious adverse event (SAE) is an adverse event that results in one of the following outcomes: 
anaphylaxis, death, a life-threatening adverse drug experience, inpatient hospitalization or 
prolongation of existing hospitalization, a persistent or significant disability/incapacity, or congenital 
anomaly/birth defect. Also, important medical events that may not result in death, be life-
threatening, or require hospitalization may be considered a serious adverse event when, based 
upon appropriate medical judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention (treatment) to prevent any of the outcomes previously listed in this 
definition. When an investigator or clinical center staff member becomes aware of an SAE, it will be 
reported to the study PI within 72 hours with follow up reporting until the event is terminated.  
 
Adverse events 
Definition of an AE: Any untoward medical occurrence in a subject administered a pharmaceutical 
product that does not necessarily have a causal relationship with the treatment. An AE can therefore be 
any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a medicinal (investigational) product, whether or not considered 
related to the investigational medicinal product. 
Investigational Medicinal Product (IMP) includes the drug under evaluation and the comparator drug(s) 
if specified as part of the research objective, given at any time during the study. Medical 
conditions/diseases present before starting the drug of interest are only considered adverse events if 
they worsen after starting the drug of interest. 
The occurrence of adverse events will be sought by non-directive questioning of the patient at each visit 
during the study. Adverse events also may be detected when they are volunteered by the patient during 
or between visits or through physical examination, laboratory test, or other assessments. All adverse 
events will be recorded in the study database including the following information: 
1. 
the severity grade (mild, moderate, severe)  
2. 
its relationship to the drug(s) of interest (suspected/not suspected) 
3. 
its duration (start and end dates or if continuing at final exam) 
4. 
whether it constitutes a serious adverse event (SAE) 
Serious adverse event reporting 
A SAE is any untoward medical occurrence that at any dose: 
- 
results in death, 
- 
is life-threatening, 
- 
requires inpatient hospitalization or prolongation of existing hospitalization, 

 
14 
 
 
 
- 
results in persistent or significant disability/incapacity, 
- 
is a congenital anomaly/birth defect,  
- 
is otherwise a significant medical event. 
This includes any SAEs likely to arise from the trial indication or progression of underlying/concomitant 
illness (es) (e.g. progression of cancer in oncology trials), unless specified in the protocol as study 
specific exemptions. 
Any SAE, irrespective of causality, occurring after the subject has provided informed consent and until 
four weeks after the subject has stopped study participation must be reported unless otherwise stated 
in the protocol. SAEs occurring after four weeks from ending study participation should only be reported 
if considered by the Investigator attributable to the exposure to the investigational drug(s) during the 
trial period. This includes the period in which the study protocol interferes with the standard medical 
treatment given to a subject, even if study treatment has not yet started (e.g. withdrawal of previous 
treatment during washout period, change in treatment to a fixed dose of concomitant medication). 
 
Timelines: All serious adverse events (SAEs) from interventional clinical trials must be reported by the 
sites to Sponsor within 24 hours of occurrence of the SAE. The timelines for investigator initiated trials 
reporting to Novartis will be done as per Third Party Study/Investigator Initiated Trial Agreement.   
Follow-up reports:  
SAEs will be followed until resolution or until it is judged to be permanent, and an assessment will be 
made at each visit (or more frequently, if necessary) of any changes in severity, the suspected 
relationship to the drug of interest, the interventions required to treat it, and the outcome.  
The Sponsor shall support Novartis in the following-up of all SAEs so that complete information is 
available to maintain patient safety and also as part of any commitments by Novartis to any Health 
authority OR specific Health authority follow-up requests for the product under investigation. 
Pregnancies 
To ensure patient safety, each pregnancy in a patient (or a patients partner) on study drug must be 
reported to Novartis within 24 hours of learning of its occurrence. The pregnancy will be followed up to 
determine outcome, including spontaneous or voluntary termination, details of the birth, and the 
presence or absence of any birth defects, congenital abnormalities, or maternal and/or newborn 
complications. 
 
 
Specific Procedures 
 
Spirometry 
To perform spirometry, a nose clip will be placed on the subject and then the subject will be asked to 
breathe hard and fast through a tube until reproducible measurements are obtained (up to 7 trials). We 
then administer a bronchodilator via a metered dose inhaler (albuterol sulfate; 360g) and repeat the 
spirometry measurement. Pre and post-bronchodilator (albuterol sulfate; 360g) spirometry will be 

 
15 
 
 
 
performed with a Vitalograph spirometer according to American Thoracic Society standards.92 Normal 
values for spirometry will be obtained from Hankinson et al.93 
 
Impulse oscillometry (IOS).  
IOS maneuvers  will be performed during tidal breathing in the seated position, with a nose clip in place, 
and with support of the cheeks. A minimum of three trials, lasting 30 seconds, will be performed. This 
will also be done before and after the bronchodilator. IOS maneuvers (Jaeger Impulse Oscillation System; 
Jaeger USA; Yorba Linda, CA) will be performed in accordance with published recommendations 94 and 
as we have previously described 95-97. Maneuvers will be performed during tidal breathing in the seated 
position, with a nose clip in place, and with support of the cheeks. A minimum of three trials, lasting 30 
seconds, will be performed. Only data from trials with constant tidal volume and coherence >0·70 at 5 
Hertz (Hz) and 0·85 at 10Hz will be analyzed. Data for analysis will include resistance measured at an 
oscillating frequency of 5Hz (R5) and frequency dependence of resistance (FDR) calculated as the 
difference between resistance at 5Hz and 20Hz (R5-20). FDR provides a measure of non-uniformity of 
airflow distribution, which may reflect regional functional abnormalities in the small airways or distal 
lung 98-100. 
 
Mechacholine challenge test.  
Methacholine is an inhaled medication, which is used to diagnose airway hyper-reactivity, which is a 
characteristic of asthma. The subject will be asked to inhale a low dose of this medication and then 
perform spirometry. We will slowly increase the dose and repeat spirometry until the subject shows a 
decline in spirometry value or until we have reached a defined dose.  If spirometry measurement is 
reduced as expected with asthma, or if subject develops asthma symptoms, the effect is temporary and 
can be reversed by taking albuterol. 
 
Sputum Induction and processing 
Sputum induction will be performed according to standard methods 25. Briefly, volunteers will inhale 
albuterol sulfate (360 mcg) and sputum will be induced by inhalation of nebulized hypertonic saline 
solution (3%, NOUVAG 2000 ultrasonic nebulizer; NOUVAG USA, Inc.) for 2- minute intervals for a total 
of 20 minutes. Volunteers will be encouraged to cough and expectorate throughout the procedure. 
Sputum will be weighed and treated (37oC, 15 minutes) with freshly prepared dithiothereitol (1% DTT; 
Sputolysin Reagent; Calbiochem, Germany)25. The sample will be filtered (70 micrometer nylon cell 
strainer; Becton Dickinson, USA) to remove mucous and debris, centrifuged (300 x g, 10 minutes) and 
the pellet resuspended in cell dissociation buffer (CDB; Sigma #C5914) with 10% fetal calf serum to 
achieve a concentration of 106 cells/ml. A total cell count (TCC) will be performed (hematocytometer), 
cell viability determined (trypan blue exclusion) and slides prepared (cytospin). Quality specimens (less 
than 50% squamous epithelial cells and > 90% cell viability) will be used and cell counts performed on 
400 non-squamous cells. Results of cell counts will be expressed as a percentage of the total non-
squamous cell count. 
 
Flow Cytometry  
Washed cells will be labeled with pre-titered, monoclonal antibodies (mAbs; 4°C, 30 min). Fresh cells 

 
16 
 
 
 
will be sorted without fixation immediately after antibody labeling and cells will be sorted on a 
FACSAria II (BD Biosciences) on the same day. Compensation for spillover and spectral overlap will be 
performed using CompBeads plus (anti-mouse IgG and anti-rat IgG; BD Biosciences) as well as the ArC 
Amine Reactive Compensation Bead Kit (Invitrogen). Cellular debris/dead cells will be excluded by 
LIVE/DEAD staining (LIVE/DEAD Fixable Blue Dead Cell Stain Kit; L23105; Invitrogen). Squamous 
epithelial cells (sqEPI), DC (plasmacytoid and myeloid DC), monocytes, macrophages, and B cells, will 
be differentiated using size discrimination and fluorescent labeling. 
 
RNA 
Total RNA will be isolated using the miRVana (Life Technology) and RNA will be transcribed using the 
RT2 First Strand Kit (SABiosciences, Frederick, MD) and quantitative PCR (qPCR) will be performed 
using a SYBR Green/ROX qPCR Master Mix and RT2 qPCR Primer Assay using specific primers. Levels of 
respective transcripts will be normalized to GAPDH transcript level as an internal control 
ΔCt(target)=Ct(target)−Ct(GAP-DH). TSLP primers will be designed to detect both the long and short 
splice variants of TSLP that have been described 50. 
 
 
FeNO 
Measurement of FeNO will be performed with a handheld NIOX MINO (Aerocrine; Stockholm, 
Sweden) according to a standard protocol.101 FeNO measurements, expressed in parts per billion 
(ppb), will be reported for an exhalation flow rate of 50 mL/s. 
 
Peripheral Eosinophils  
Complete blood counts with cell differential will be performed by an automated laboratory 
procedure. 
 
Allergen-specific IgE 
Measurements of total and allergen-specific serum IgE for allergens common to the Northeastern 
United States will be performed in a commercial laboratory (Pharmacia ImmunoCAP assay; Quest 
Diagnostics; Teterboro, NJ). Atopy will be defined as the presence of at least one elevated allergen-
specific IgE ( ≥ 0.35 kilo-international units (kU)/L). Perennial allergy will be defined as an elevated 
allergen-specific indoor allergen common to the Northeastern U.S.: cat dander, dog dander, blatella 
germanica (cockroach) or dermatophagoides pteronyssinus (house dust mite). Outdoor allergy will be 
defined as an elevated allergen-specific IgE to any of the common outdoor allergens: maple, 
ragweed, birch, elm, oak and ash. Molds include aspergillus fumigatus and alternaria alternata. 
 
 
 
Statistical Design 
Variables to be analyzed: 
The primary outcome is the change in TSLP, IL-33 and IL-25 gene expression in sHBEC in mild- 
moderate persistent asthma after 16 weeks of treatment of omalizumab or placebo. We will estimate 
the difference in the gene expression of the treatment group of omalizumab compared with the 

 
17 
 
 
 
placebo group and report the results of estimated mean and 95% confidence interval. The difference 
between two randomized groups will be compared using the two-group t-test if data are normally 
distributed, and the nonparametric Wilcoxon test will be used for skewed data. The secondary 
outcomes include the change sHBEC targets (gene expression array), asthma control defined as 
symptom control (Asthma control test), spirometry, and measures of small airway dysfunction 
(Impulse oscillometry). The comparisons of secondary outcomes will be conducted in similar way as 
the primary outcome. We will account for the issue of false positives due to multiple testing 
hypotheses by using a conservative alpha level of 0.01. The effect of omalizumab on newly identified 
sHBEC targets (gene expression array). This is an exploratory goal and analysis will be performed 
initially with cufflinks and subsequent analyses will be performed with the NYU Genome Technology 
Center and the NYU Bioinformatics Center. 
 
Sample size and power: This is an exploratory study with a treatment group and control group and 
the goal is to provide an estimate of theof the mean difference between two groups and proper 
confidence interval. Sample size of 15 each group can achieve a margin of error of 0.73 assuming that 
outcomes are standardized with standard deviation of 1. 
 
Safety: 
Before the first subject is entered into the study, a member of the study team will review and discuss 
the requirements of the clinical study protocol and related documents with the investigational staff and 
also train them in any study-specific procedures and system(s) utilized. The principal investigator will 
ensure that appropriate training relevant to the study is given to all of these staff, and that any new 
information relevant to the performance of this study is forwarded to the staff involved. 
The principal investigator will maintain a record of all individuals involved in the study 
During the study: 
 Confirm that facilities remain acceptable  
Confirm that the investigational team is adhering to the protocol, that data are being accurately and 
timely recorded in the data base, that biological samples are handled in accordance with the protocol 
and that study drug accountability checks are being performed  
Perform source data verification (a comparison of the data in the data base with the subject’s medical 
records at the hospital or practice, and other records relevant to the study) including verification of 
informed consent of participating subjects.   
Ensure withdrawal of informed consent to the use of the subject’s biological samples is reported and 
biological samples are identified and disposed of/destroyed accordingly, and the action is documented, 
and reported to the subject  representative will be available between visits if the investigator(s) or 
other staff at the center needs information and advice about the study conduct.  
We have chosen to include subjects with moderate asthma to reduce the potential for adverse events 
with sputum induction in patients with severe asthma. The main complication from sputum induction 
is the potential for bronchospasm following inhalation of hypertonic saline. Standard safety procedures 

 
18 
 
 
 
will be used for sputum induction, which include clinical assessment and spirometry prior to initiation 
of sputum induction. In addition, peak flow measurements are performed throughout the procedure 
and the procedure is terminated if there is a reduction in peak flow, or spirometry of >20% baseline. 
Albuterol sulfate is administered should the subject experience a drop in their peak flow or 
Spirometry during the sputum induction procedure and the patient is monitored to insure a return to 
90% of baseline spirometry. 
 
We will review adverse events at each visit including presence of an asthma exacerbation between 
visits. An exacerbation will be defined as the use of rescue systemic corticosteroid (po, IM, IV) or an 
urgent care visit or an emergency room visit. Should this occur, the subject will be discontinued from 
the study. 
 
Omalizumab will be injected at the CTSI and subjects will be monitored at the CTSI for 2 hours for the 
first 2 doses and then will be 30 minutes subsequent doses. Subjects will be provided a pamphlet 
describing symptoms of anaphylaxis and instructions for procedures should they experience these 
symptoms. Subjects will also be provided an epinephrine auto-injector for the possibility of a rare 
occurrence of anaphylaxis to omalizumab. Subjects will be monitored for adverse reactions during 
each visit while at the CTSI as per recommended drug delivery protocol. We will also monitor for 
infection or skin reaction at injection site. 
 
Urine pregnancy testing for women of childbearing age will be performed prior to methacholine 
challenge and randomization and before each injection of omalizumab. Should a subject become 
pregnant during the study, they will be terminated from the study. We will ask for information about 
the outcome of the pregnancy and the health of the baby. 
Adverse events will be collected at each visit after the screening visit and severe adverse events will be 
reported to the NYU IRB. Adverse events that are not considered severe will be noted in the subject’s 
study binder. 
A DSMB (Data Safety and Monitoring Board) will be created to review this study on a semiannual basis. 
The individuals in this board will include physicians, some of whom will be pulmonary specialists with 
experience in clinical trials. Information reviewed by the DSMB will include number of subjects screened 
and enrolled, any adverse events, and any study deviations. The DSMB will meet twice each year. The 
DSMP will write a short summary of their findings and elaborate on any AE or study deviations, and will 
make suggestion of improving study safety and efficacy.  This summary will be communicated to the IRB.  
The DSMB may recommend that a study be stopped if analysis of the data suggests that the 
experimental intervention appears harmful to the trial participants. This recommendation could be 
made on the basis of serious adverse events or side effects that are cropping up in those getting the 
experimental intervention. 
 
 
Confidentiality: 
To protect every individual against the loss of confidentiality, the information will be kept in locked 

 
19 
 
 
 
files and in secured databases. Paper source documents and consent forms will be stored in a locked 
filing cabinet in a locked room on the 6th floor of Bellevue hospital Room 9-13. This room has limited 
access. Data files will be stored in Red Cap on the NYU secure server. 
 
Samples will be kept and processed in Dr. Reibman’s laboratory in the Rom Environmental Lab. 
 
Only members who are IRB approved as investigators on this study or with IRB-approved protocols 
will be able to access these data and samples. Use of de-identified data will be allowed after review by 
the PI and coPIs who will act as a Research Oversight Committee and after a data-sharing plan has 
been approved. If any member requests this information, they will need to follow protocol and be 
added properly via IRB Application for Study Personnel change form. 
 
PHI information will be stripped from all patient documents and samples and replaced with a unique 
study subject identifier. There will be one master list that will be under the protection of Dr. Reibman, 
which will code the patient name to the assigned study number. The linking key will be kept by the PI 
or her designate. No genetic testing will be done on samples during the study to identify risk for other 
conditions.  
The ICF will incorporate wording that complies with relevant data protection and privacy legislation. 
The PI of the study will not provide individual genotype results to subjects, any insurance company, any 
employer, their family members, general physician or any other third party, unless required to do so by 
law. 
Extra precautions are taken to preserve confidentiality and prevent genetic data being linked to the 
identity of the subject. In exceptional circumstances, however, certain individuals might see both the 
genetic data and the personal identifiers of a subject. For example, in the case of a medical emergency, 
a ROC or an investigator might know a subject’s identity and also have access to his or her genetic data. 
Also regulatory authorities may require access to the relevant files, though the subject’s medical 
information and the genetic files would remain physically separate. 
 
 
Sample Storage for Future Use: 
We will save the blood and sputum samples in a freezer for up to 10 years for potential testing of 
additional markers of inflammation, which may include testing of RNA (part of a cell that builds proteins 
and contains genetic information). Since RNA is a product of DNA (is part of the cell that contains the 
instructions for how you look and function), measurements of RNA can be considered a type of genetic 
testing. However, these studies will not provide us with information about their risks for other genetic 
conditions or the ability to do genetic counseling. Storage for future research is not an optional 
component to this study as these analyses are an integral part of the study for testing of additional 
markers of inflammation. Samples will be stored in a freezer for future research in Dr. Reibman’s 
Laboratory (with Rom Environmental Lab). Samples will be labeled as described above with the study 
number and the unique subject identifier. Only the PI, IRB approved designates will have access to the 

 
20 
 
 
 
stored samples. The linking key will be kept by the PI or her designate. Subjects can contact the PI to 
withdraw samples from storage and future use.  
 
 
Costs to Subjects: 
The patient will not have to pay for any tests that are in this study or any procedures that are in this 
study. These include blood tests and breathing tests. These funds are provided to help support with time 
and travel associated with their participation. 
The cost of the study drug, the medical visits and laboratory tests related to the research part of the 
study will be paid by NovartisPharma.  
 
Please note that Novartis will not pay for medication such as bronchodilators or other asthma 
controllers that it may be taking for the asthma outside of this study 
 
Payments to Subjects: 
We will reimburse subjects 75 dollars for each visit that includes sputum induction (V2, 2a, 7 7a) and 
50 dollar compensation per visit for all other visits 
 
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